Increased cathepsin S plays an important role in ocular surface manifestations in Sjögren’s syndrome

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Content
INCREASED CATHEPSIN S PLAYS AN IMPORTANT ROLE IN
OCULAR SURFACE MANIFESTATIONS IN SJÖGREN’S SYNDROME

By

Wannita Klinngam

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
PHARMACEUTICAL SCIENCES

August 2019

Copyright 2019 Wannita Klinngam

i

Dedication
This dissertation is dedicated to my beloved family especially to my parents, brother, sister,
and nephews who have supported and trusted that I can handle and overcome adversity in my life.
In addition, this dissertation is dedicated to the Royal Thai Government that has supported my
financial expenses during the entirety of my 6 years studying in the States. I hope that I can use all
my knowledge and experiences that I have obtained during my study here in the States to develop
future scientific research in Thailand.

ii

Acknowledgements
Firstly, I never would have been able to graduate without the useful guidance and kind
assistance from my best PI, Dr. Sarah F. Hamm-Alvarez. During my 5 years’ experience in her
lab, since I was as a master’s student, she has always given me excellent suggestions, understood
and sympathized with my struggles, and provided me with a conducive environment for doing
research. It is an honor to be a graduate student under her advice and obtain more knowledge and
experience from her. Secondly, I would like to deeply thank my committee member, Dr. Curtis
Okamoto for providing me with biological knowledge and Dr. Jianming Xie for giving me the
knowledge about immunology.
More importantly, I would like to thank all my current and previous lab members. I could
not finish my experiments without the help from Dr. Maria Edman-Woolcott who is like a mentor
to me, Srikanth Reddy Janga who always helped with my many difficult experiments and gave so
many useful advice, Francie Yarber who is the best lab manager forever, Christina Fu who helped
me resolve difficult experimental problems that I encountered, Yaping Ju who helped me with my
experiments and listened to my problems, Changrim Lee, Hao Guo, and Shruti Kakan who were
always willing to help me whenever I needed. Also, I thank Dr. Zhen Meng and Dr. Mihir Shah,
who were previous lab members, and Dr. Dandan Wang, the pathologist from LAC+USC Medical
Center for their support, guidance, and help with histological toxicity.
Finally, I would like to thank my family who believed in my abilities and allowed me to
pursue my degree and continued education in the States without worrying about them. Also, I
would like to thank all my friends especially David Wong who provided the support,
encouragement, and technical expertise to get me through it all.

iii

Table of Contents
Dedication ................................................................................................................................. i
Acknowledgements ................................................................................................................. ii
Table of Contents ................................................................................................................... iii
List of Tables ......................................................................................................................... vii
List of Figures ....................................................................................................................... viii
Abbreviations ......................................................................................................................... xi
Abstract .....................................................................................................................................1
Chapter 1: Introduction ..........................................................................................................3
1.1 The ocular surface system........................................................................................................... 3
1.2 The lacrimal functional unit (LFU) ............................................................................................. 4
1.3 Sjögren’s Syndrome (SS) and dry eye disease ........................................................................... 5
1.4 Non-Obese Diabetic (NOD) mouse: A murine model of SS .................................................... 10
1.5 Cathepsin S (CTSS) and its functions ...................................................................................... 11
1.6 Cathepsin S (CTSS) as a novel biomarker of SS ..................................................................... 13
1.7 CTSS inhibitor as a therapeutic target of autoimmune diseases ............................................... 14
1.8 Goals ....................................................................................................................................... 16
Chapter 2: CTSS induces the pro-inflammatory cytokines, proteases, and protease-
activated receptor-2 (PAR-2) expression in human corneal epithelial cells .....................18
2.1 Acute CTSS exposure significantly increases IL-1β, IL-8, IL-6, and TNF-α gene and
protein expression in cell culture medium and cell lysates of HCE T-cells ................................... 18
2.2 Chronic CTSS exposure significantly increases MMP-9 gene expression ............................... 23
2.3 Chronic CTSS exposure significantly increases PAR-2 gene and protein expression .............. 24
2.4 CTSS exposure significantly increases CTSS gene and protein expression at 8- and 24-hours 27
2.5 Acute exposure to heat-inactivated CTSS does not induce pro-inflammatory cytokines ........28
Chapter 3: CTSS-dependent activation of PAR-2 activation is involved in induction of
Pro-inflammatory cytokines and proteases in human corneal epithelial cells .................30
3.1 Transfection efficiency of PAR-2 siRNA in HCE T-cells ........................................................ 31
3.2 The efficiency of PAR-2 knockdown by using human PAR-2 siRNA in HCE T-cells ............. 31
3.3 The effect of CTSS activation of PAR-2 on the increase in pro-inflammatory cytokines and
Proteases at 4 and 24 hours of recombinant CTSS treatment ......................................................... 34
3.4 An initial PAR-2 dependent increase in TNF-α after acute CTSS exposure may drive IL-6 and
IL-1β gene expression in HCE-T cells ............................................................................................ 37
iv

Table of Contents
Chapter 4: Systemic administration of the CTSS inhibitor (Z-FL-COCHO) given
intraperitoneally reduces LG inflammation and increases tear flow in a Mouse model of
SS .............................................................................................................................................40
4.1 Identification of i.p. doses of Z-FL ........................................................................................... 41
4.2 Intraperitoneal Z-FL reduces CTSS activity in tears, LG, and spleen lysates in male NOD
mice ................................................................................................................................................ 42
4.3 Intraperitoneal Z-FL decreases LG lymphocytic infiltration and increases stimulated tear
Secretion in male NOD mice .......................................................................................................... 43
4.4 Intraperitoneal Z-FL reduces the CD3+ T-cell population and pan-macrophage abundance in
lymphocytic infiltrates in parallel with MHC II (H2-Ab1) gene expression in LG ......................... 45
4.5 Intraperitoneal Z-FL does not affect the expression of other inflammation-associated genes in
LG of male NOD mice ................................................................................................................... 46
4.6 Intraperitoneal Z-FL does not elicit gross systemic toxicity at the dose evaluated ................... 49
4.7 Comparison between Z-FL (Millipore) and Z-FL-hydrate (Adooq Bioscience) ....................... 52
4.8 Intraperitoneal Z-FL-hydrate reduces CTSS activity in LG and spleen lysates, but not in tears in
Male NOD mice ............................................................................................................................. 54
4.9 Intraperitoneal Z-FL-hydrate decreases LG lymphocytic infiltration and increases stimulated
tear secretion in male NOD mice .................................................................................................... 58
4.10 Intraperitoneal Z-FL-hydrate improves basal tear secretion, but not corneal integrity in male
NOD mice ...................................................................................................................................... 59
4.11 Intraperitoneal Z-FL-hydrate decreases IFN-  gene expression in LG in male NOD mice ..... 60
Chapter 5: The effect of topical administration of the CTSS inhibitor (Z-FL-COCHO)
on LG inflammation in a mouse model of SS ......................................................................61
5.1 Identification of topical doses of Z-FL in vitro ......................................................................... 61
5.2 Identification of topical doses of Z-FL in vivo .......................................................................... 62
5.3 Topical Z-FL reduces CTSS activity in tears, not in LG and spleen lysates in male NOD
mice ................................................................................................................................................ 63
5.4 Topical Z-FL does not affect the LG lymphocytic infiltration and improvement of stimulated
Tear secretion in male NOD mice .................................................................................................. 64
5.5 Topical Z-FL does not affect CD3+ T-cell population in areas of LG lymphocytic infiltration
in male NOD mice .......................................................................................................................... 65
5.6 Topical Z-FL reduces macrophage abundance in lymphocytic infiltrates in parallel with
Reduced MHC II (H2-Ab1) gene expression in LG ........................................................................ 66
v

Table of Contents
5.7 Topical Z-FL does not affect B220+ positive B-cell abundance within the LG lymphocytic
infiltrates ........................................................................................................................................ 68
5.8 Topical Z-FL significantly reduces gene expression of CTSS, TNF-α, IFN-  and PAR-2 in LG
of male NOD mice.......................................................................................................................... 69
5.9 Topical Z-FL attenuates the reduction of basal tear secretion associated with disease
progression but has no measurable effect on corneal surface integrity in male NOD mice ............ 70
5.10 Topical Z-FL does not elicit gross systemic toxicity at the dose evaluated ............................ 71
Chapter 6: Materials and methods .......................................................................................73
6.1 Reagents ................................................................................................................................... 73
6.2 Mice.......................................................................................................................................... 74
6.3 Cell culture ............................................................................................................................... 75
6.4 Calculation of human recombinant CTSS dosage for treatment of human corneal epithelial cells
(HCE T-cells) ................................................................................................................................. 75
6.5 SiRNA transfection................................................................................................................... 76
6.6 Transfection efficiency ............................................................................................................. 76
6.7 Treatment of HCE T-cells with recombinant human CTSS ...................................................... 77
6.8 Cell viability after 24 hours of CTSS treatment in HCE T-cells ............................................... 77
6.9 Multiplex assay and ELISA methods for measurement of pro-inflammatory cytokines in HCE-
T cell medium and lysates .............................................................................................................. 78
6.10 ELISA method for measurement of CTSS in HCE-T cell lysates ........................................... 79
6.11 ELISA method for measurement of PAR-2 in HCE-T cell lysates ......................................... 79
6.12 Multiplex assay and ELISA methods for measurement of pro-inflammatory cytokines, CTSS,
and MMP-9 .................................................................................................................................... 79
6.13 CTSS activity in HCE-T cell lysates ....................................................................................... 80
6.14 Immunofluorescence of PAR-2 in HCE-T cells ...................................................................... 81
6.15 Western Blotting for PAR-2 ................................................................................................... 81
6.16 Z-FL preparation for i.p. and topical administration ............................................................... 82
6.17 CTSS activity assays in Z-FL preparation .............................................................................. 82
6.18 CTSS activity assays in tears, LG, and spleen of male NOD mice ......................................... 83
6.19 In vivo studies for determining Z-FL toxicity in i.p. administration ....................................... 83
6.20 In vitro and in vivo studies for determining Z-FL toxicity in topical administration ............... 84
6.21 Administration of Z-FL by i.p. injection in male NOD mice ................................................... 84
vi

Table of Contents
6.22 Administration of Z-FL by topical eye drops in male NOD mice ........................................... 85
6.23 Quantification of LG lymphocytic infiltration by hematoxylin-eosin staining........................ 85
6.24 LG immunofluorescence and quantitative analysis of immune cells in LG lymphocytic
infiltration ....................................................................................................................................... 86
6.25 Corneal fluorescein staining ................................................................................................... 87
6.26 Basal tear collection ................................................................................................................ 87
6.27 Stimulated tear secretion ......................................................................................................... 87
6.28 Gene expression in HCE T-cells and in mouse LG lysates .................................................... 88
6.29 Histotoxicological evaluation ................................................................................................. 89
6.30 Statistics ................................................................................................................................. 89
Chapter 7: Discussion ............................................................................................................90
Chapter 8: Future Perspectives ..........................................................................................102
References………………………………………………………………………………….105

vii

List of Tables
Table 1: Decreased pro-inflammatory cytokines and proteases gene expression in human
corneal epithelial cells transfected with PAR-2 siRNA after 4 and 24 hours of CTSS treatment ....... 35
Table 2: Pro-inflammatory cytokines and proteases protein expression in cell culture medium and
cell lysates of human corneal epithelial cells transfected with PAR-2 siRNA after 4 and 24 hours of
CTSS treatment .................................................................................................................................. 36
Table 3: Histopathological report on kidney, spleen, and liver of male NOD mice after 2 weeks of
Z-FL i.p. injection .............................................................................................................................. 51
Table 4: Histopathological report on kidney, spleen, and liver of male NOD mice after 2 weeks of
Z-FL-hydrate i.p. injection ................................................................................................................. 57
Table 5: Histopathological report on kidney, spleen, and liver of male NOD mice after 6 weeks of
topical Z-FL eyedrop .......................................................................................................................... 72

viii

List of Figures
Figure 1: Schematic diagram of ocular surface system........................................................................ 3
Figure 2: Schematic diagram of the lacrimal functional unit (LFU) .................................................... 5
Figure 3: Diagram of the current concepts of SS pathogenesis............................................................ 8
Figure 4: Role of cathepsin S in MHC class II-associated invariant chain processing and
Peptide loading ................................................................................................................................... 12
Figure 5: Structure of CTSS inhibitors .............................................................................................. 16
Figure 6: Cell viability measured by MTT assay after 24 hours of CTSS treatment in a
human corneal epithelial cell line (HCE T-cells) compared to untreated cells ................................... 19
Figure 7: CTSS increases IL-1β, IL-8, IL-6, and TNF-α gene expression after 2- and 4-hours
of treatment in a human corneal epithelial cell line (HCE T-cells) ..................................................... 21
Figure 8: CTSS significantly increases IL-8, IL-6, TNF-α, and IL-1β protein expression in
cell culture medium and cell lysates from human corneal epithelial cell line (HCE T-cells) at
2, 4, and 8 hours of exposure .............................................................................................................. 22
Figure 9: CTSS s increases MMP-9 gene expression after 24 hours in human corneal
epithelial cells ..................................................................................................................................... 24
Figure 10: CTSS increases PAR-2 gene and protein expression after 24 hours in human
Corneal epithelial cells ....................................................................................................................... 26
Figure 11: CTSS exposure in human corneal epithelial cells increases cellular CTSS gene
and protein expression after 8- and 24-hours ...................................................................................... 28
Figure 12: CTSS activity is required for early induction of pro-inflammatory cytokines in
Human corneal epithelial cells............................................................................................................ 29
Figure 13: Transfection efficiency of siRNA using BLOCK-iT Fluorescent Oligo labeled
with FITC in human corneal epithelial cells after 48 hours of incubation ......................................... 32
Figure 14: PAR-2 gene and protein expression after 48 hours of PAR-2 or scrambled siRNA
transfection in human corneal epithelial cells ..................................................................................... 33
Figure 15: Experimental design to study the effect of CTSS activation of PAR-2 on the
increase in pro-inflammatory cytokines (IL-8, IL-6, TNF-α, and IL-1β) and proteases (CTSS
and MMP-9) at 4 and 24 hours of recombinant CTSS treatment ........................................................ 34
Figure 16: Gene expression of pro-inflammatory cytokines (IL-8, IL-6, TNF-α, and IL-1β)
after 15 minutes, 1 hour, and 2 hours of CTSS treatment in human corneal epithelial cells
(HCE T-cells) .................................................................................................................................... 39
Figure 17: The structure of Z-FL-COCHO (Z-FL) ............................................................................ 41
ix

List of Figures
Figure 18: There were no gross differences in weights of liver, spleen, or kidneys after Z-FL i.p.
injection .............................................................................................................................................. 42
Figure 19: Intraperitoneal Z-FL reduces CTSS activity in tears, LG, and spleen lysates ................... 43
Figure 20: Intraperitoneal Z-FL decreases lymphocytic infiltration of the LG and increases
stimulated tear secretion ..................................................................................................................... 44
Figure 21: Intraperitoneal Z-FL reduces CD3+ T-cells and pan-macrophage abundance in
Lymphocytic infiltrates in parallel with reduced MHC II (H2-Ab1) gene expression in LG .............. 47
Figure 22: Intraperitoneal Z-FL does not alter B220+ positive B-cell abundance within the LG ...... 48
Figure 23: Intraperitoneal Z-FL does not affect Ctss, Tnf, Ifng, and F2rl1 gene expression in LG of
male NOD mice .................................................................................................................................. 49
Figure 24: The structure of Z-FL-hydrate (from Adooq Bioscience) ................................................ 52
Figure 25: The Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) from Z-FL
(Millipore) vs Z-FL-hydrate (Adooq Bioscience) ............................................................................... 53
Figure 26: The half maximal inhibitory concentration (IC
50
) and the CTSS inhibitory potential in
mouse spleen lysate of Z-FL and Z-FL-hydrate ................................................................................. 54
Figure 27: No significant weight changes in liver, spleen, and kidney in male NOD mice treated i.p.
with 15 mg/kg Z-FL-hydrate or vehicle were detected ....................................................................... 55
Figure 28: Intraperitoneal Z-FL-hydrate reduces CTSS activity in LG and spleen lysates, but not in
tears .................................................................................................................................................... 56
Figure 29: Intraperitoneal Z-FL-hydrate decreases lymphocytic infiltration of the LG and increases
stimulated tear secretion ..................................................................................................................... 58
Figure 30: Intraperitoneal Z-FL-hydrate improves basal tear secretion, but not corneal integrity in
male NOD mice .................................................................................................................................. 59
Figure 31: Intraperitoneal Z-FL-hydrate decreases Ifng gene expression in LG in male NOD mice . 60
Figure 32: Z-FL does not reduce cell viability or cause cell death in vitro in human corneal epithelial
(HCE-T) cells .................................................................................................................................... 62
Figure 33: No difference in basal tear secretion or corneal surface integrity were detected after 1
week of topical Z-FL ......................................................................................................................... 63
Figure 34: Topical instillation of Z-FL reduces CTSS activity locally in tears, but not in LG or spleen
lysates ................................................................................................................................................. 64
Figure 35: Topical instillation of Z-FL does not affect LG lymphocytic infiltration and stimulated
tear secretion ...................................................................................................................................... 65
x

List of Figures
Figure 36: Topical instillation of Z-FL does not affect CD3+ T-cells within areas of lymphocytic
infiltration in LG ................................................................................................................................ 66
Figure 37: Topical instillation of Z-FL reduces expression of MHC II (H2-Ab1) in LG and pan-
macrophage abundance in LG lymphocytic infiltrates........................................................................ 67
Figure 38: Topical instillation of Z-FL does not alter B220+ positive B-cell abundance within the LG
lymphocytic infiltrates ........................................................................................................................ 68
Figure 39: Topical instillation of Z-FL significantly reduces gene expression of Ctss, Tnf, Ifng, and
F2rl1 .................................................................................................................................................. 69
Figure 40: Topical instillation of Z-FL attenuates the reduction of basal tear secretion but has no
measurable effect on corneal surface integrity associated with disease progression ........................... 70
Figure 41: The possible cell signaling pathway induced by acute exposure to 4 hours of CTSS in
human corneal epithelial cells contributing to increased release of pro-inflammatory cytokines and
MMP-9 that may contribute to ocular surface inflammation .............................................................. 92
Figure 42: The chemical structure of PAR-2 antagonists .................................................................. 95

xi

Abbreviations
LFU Lacrimal functional unit
LG Lacrimal gland
CNS Central nervous system
ADDE Aqueous tear-deficient dry eye
EDE Evaporative dry eye
SS Sjögren’s Syndrome
pSS Primary Sjögren’s Syndrome
sSS Secondary Sjögren’s Syndrome
SG Salivary gland
HLA Human leucocyte antigen
IFN Interferon
CD Cluster of differentiation
ANA Antinuclear antibodies
RA Rheumatoid arthritis
SLE Systemic lupus erythematosus
NOD Non-Obese Diabetic
IL Interleukin
TNF Tumor necrosis factor
KC/GRO Keratinocyte chemoattractant/human growth-regulated oncogene
CTSS Cathepsin S
APC Antigen-presenting cells
DC Dendritic cells
ER Endoplasmic reticulum
MHC Major histocompatibility complex
CLIP Class II-associated invariant chain peptides
HLA-DM Human leukocyte antigen DM
ECM Extracellular matrix
HCE-T Human corneal epithelial cell line
PAR-2 Protease-activated receptor-2
i.p. Intraperitoneal injection
TGF Transforming growth factor
xii

RFU Relative fluorescence unit

Abbreviations
SEM Estimated standard error of the mean
MMP Matrix metalloproteinase
ERK Extracellular-signal-regulated kinase
PKA Protein kinase A
siRNA Small interfering ribonucleic acid
FITC Fluorescein isothiocyanate
MAPK Mitogen-activated protein kinase
PI3K Phosphoinositide 3-kinase
Z-FL Z-FL-COCHO
MTD Maximum tolerated dose
PEG Poly (ethylene glycol)
PBS Phosphate-buffered saline
DMSO Dimethyl sulfoxide
RP-HPLC Reverse-phase high-performance liquid chromatography
KSFM Keratinocyte-SFM
EthD-1 Ethidium Homodimer-1
H&E Hematoxylin and eosin
SD Standard variation
RT Reverse transcriptase
EGF Epidermal growth factor
BPE Bovine pituitary extract
EDTA Ethylenediaminetetraacetic acid
NMWL Nominal molecular weight limit
BSA Bovine serum albumin
O.C.T Optimum cutting temperature
cAMP Cyclic adenosine monophosphate
CIITA Class II transactivator
H2-Ab1 Histocompatibility 2, class II antigen A, beta 1
LDALT Lacrimal duct-associated lymphoid tissue

xiii

1

Abstract
Highly increased cathepsin S (CTSS) is found in Sjögren’s syndrome (SS) patient tears and
in the tears and LG of the NOD male mouse, a murine model of SS. SS is a systemic autoimmune
disease associated with lacrimal gland (LG) and salivary gland (SG) inflammation which leads to
dry eye and dry mouth. In the first part of my dissertation (Chapter 2-3) that was already published
in International Journal of Molecular Sciences (Klinngam et al., 2018), I demonstrated utilizing
recombinant human CTSS at the same elevated enzymatic activity seen in SS patient tears, that
exposure of cultured human corneal epithelial cells (HCE-T cells) to CTSS elicits acute and
chronic inflammatory responses. Specifically, CTSS can promote an acute increase in pro-
inflammatory cytokine gene and protein expression, including IL-8, IL-6, TNF-α, and IL-1β, and
also induce later increases in MMP-9 gene expression, gene and protein expression of CTSS itself,
in this in vitro model system. One possible mechanism underlying these CTSS-induced effects
may involve CTSS activation of protease-activated receptor-2 (PAR-2), as demonstrated by the
inhibition some effects in HCE-T cells transfected with PAR-2 siRNA before exposure to acute
and chronic CTSS. In PAR-2 siRNA transfected HCE-T cells exposed to CTSS, the increased
expression of IL-6, IL-1β, TNF-α, and MMP-9 in cell culture medium was reduced in response to
acute CTSS exposure. On the contrary, the increased IL-8 protein expression in both cell culture
medium and cell lysates did not change in PAR-2 siRNA transfected cells after acute CTSS
treatment. These results suggest that CTSS activation of PAR-2 plays an important role in acute
induction of IL-6, IL-1β, TNF-α, and MMP-9 secretion while, the increased IL-8 expression might
be through a PAR-2 independent pathway. Additionally, I found that gene expression of 4 pro-
inflammatory cytokines (IL-8, IL-6, TNF-α, and IL-1β) and 2 proteases (MMP-9 and CTSS) after
chronic CTSS exposure in PAR-2 siRNA transfected cells were decreased relative to scrambled
2

siRNA transfected cells suggesting that chronic CTSS activation of PAR-2 and/or the early
increased inflammatory mediators may affect the late induction of these inflammatory responses.
CTSS appears to play an important role in ocular surface and LG inflammation in SS. In
the second part of my dissertation (Chapter 4-5), I utilized the peptide-based inhibitor, Z-FL-
COCHO (Z-FL) administered to 14-15 weeks male NOD mice by systemic intraperitoneal (i.p.)
injection for 2 weeks or topically as twice daily eyedrops for 6 weeks to explore CTSS’s utility as
a therapeutic target for mitigating ocular manifestations of SS. Systemic intraperitoneal (i.p.)
injection for 2 weeks significantly reduced CTSS activity in tears, LG and spleen, significantly
reduced total lymphocytic infiltration into LG and T cell and pan-macrophage abundance within
lymphocytic infiltrates, and significantly increased stimulated tear secretion. Topical
administration of Z-FL to a different cohort of 14-15 week male NOD mice for 6 weeks
significantly reduced only tear CTSS while not affecting LG and spleen CTSS and attenuated the
disease-progression related reduction of basal tear secretion, while not significantly impacting
lymphocytic infiltration of the LG. In addition, I also repeat i.p. study with another form of Z-FL,
Z-FL-hydrate, with the equipotent dose with Z-FL. The results still showed the reduction of CTSS
activity in LG and spleen lysates, reduced LG lymphocytic infiltration area, and increased
stimulated tear secretion. These findings suggest that CTSS inhibitors administered either topically
or systemically can mitigate aspects of the ocular manifestations of SS. In conclusion, CTSS
appears to mediate aspects of ocular surface inflammation and to represent a potential therapeutic
target in SS.

3

Chapter 1: Introduction
1.1 The ocular surface system
The ocular surface system is composed of the cornea, conjunctiva, lacrimal gland,
accessory lacrimal glands, and meibomian gland, and their tears and connective tissue matrices,
the eyelashes, and nasolacrimal duct (Gipson, 2007). All of these components are functionally
connected by continuous epithelium derived from the surface ectoderm and regulated by
innervation, endocrine, vascular and immune system (Figure 1) (Gipson, 2007). The primary
function of the ocular surface system is to provide, protect and maintain a smooth refractive surface
by producing components of a refractive tear film (Gipson, 2007). The tear film consists of 3
components: the lipid component which is produced by meibomian glands, the aqueous
components which is produced by the lacrimal gland and the accessory lacrimal glands, and the
mucus component which is the product of the conjunctiva and cornea (Paulsen et al., 2003).

Figure 1. Schematic diagram of ocular surface system. (A) shows the continuous ocular surface
epithelium (pink) and the regions contributing to composition of the tear film (blue). (B) shows
how these components of the ocular surface system are functionally integrated (Gipson, 2007).
4

1.2 The Lacrimal functional unit (LFU)
The lacrimal functional unit (LFU) which is one part of the ocular surface system consists
of the lacrimal gland (LG), accessory lacrimal glands, ocular surface which includes cornea,
conjunctiva, and meibomian glands and lids, and the interconnecting innervation (Gipson, 2007).
The major function of the LFU is to control the tear film component in order to preserve the
integrity of the tear film, the transparency of the cornea, and the quality of the image projected into
the retina (Lemp, 2007). Any damages or diseases which occur in the LFU can lead to
destabilization of the tear film and create ocular surface disease associated with dry eye (Lemp,
2007). The LG is a part of the LFU which plays an important role in aqueous tear production which
consists of water, electrolytes, and proteins. 80% of the LG is acinar epithelial cells and the
remaining 20% is composed of ducts, nerves, myoepithelial cells, leukocytes, and connective
tissue (Edman et al., 2010). The appropriate amount and composition of LG fluid is essential for
healthy ocular surface. As a result, tear secretion in the LG is highly regulated (Edman et al., 2010).
The neural regulation is one parts of the lacrimal functional unit that controls LG tear secretion
(Dartt, 2004). Any stimulations that occur in cornea and conjunctiva can send a signal to stimulate
afferent sensory neurons that send signal to the central nervous system (CNS) for activation the
efferent parasympathetic and sympathetic nerves that innervate LG acinar and ductal cells (Dartt,
2009). The activated nerves can stimulate the LG to secrete tear into the ocular surface via the
ductal system, then, drains into the lacrimal drainage system (Figure 2) (Dartt, 2009).
Not only LG but also cornea and conjunctiva can secrete tears. Sensory nerves which are
abundant in cornea and conjunctiva can regulate conjunctival goblet cells, stratified squamous
cells, and corneal epithelial cells to secrete mucins, proteins, electrolytes, and water to the tear
5

film. In addition, the alterations of tear secretion such as tear hyperosmolarity also affect functional
and structural of corneal nerve (Hirata et al., 2015).

Figure 2. Schematic diagram of the lacrimal functional unit (LFU) (Dartt, 2009).
1.3 Sjögren’s Syndrome (SS) and dry eye disease
As mention above, dysfunction of the LFU can alter tear composition leading to dry eye.
Dry eye is defined as a multifactorial disease resulting in discomfort, visual disturbance, and tear
film instability that can affect ocular surface inflammation (Lemp, 2007). Dry eye is classified into
2 classes: aqueous tear-deficient dry eye (ADDE), which is associated with a failure of LG fluid
secretion, and evaporative dry eye (EDE), which is due to excessive water loss from ocular surface
with normal LG function. ADDE is categorized into 2 subtypes: Sjögren’s Syndrome (SS), which
is characterized by an autoimmune process, and non-Sjögren’s Syndrome (non-SS) which is not
6

related to autoimmune diseases and includes conditions associated with age-related dry eye, LG
duct obstruction and others (Lemp, 2007).
SS is a one of the most prevalent autoimmune diseases which is mostly found in women
with a 9:1 prevalence (Rodrigues and Soares, 2017). The clinical presentation of SS can vary from
the local involvement of lymphocytic infiltration of exocrine glands which are LG and salivary
glands (SG) leading to dry eyes and mouth into systemic involvement affecting multiple systems
such as musculoskeletal system, nervous system, lungs, kidney skin, and blood vessels (Saraux et
al., 2016). SS is divided into 2 subgroups which are primary SS (pSS) and secondary SS (sSS)
which is associated with other autoimmune diseases especially systemic lupus erythematosus (15-
36%), rheumatoid arthritis (20-32%), and systemic sclerosis (11-24%) (Stefanski et al., 2017).
Normally, SS is considered as a chronic, non-life-threatening disease but it can become a more
serious disease with excess mortality in SS patients with B cell lymphoma, interstitial lung disease,
renal failure, cryoglobulinaemic vasculitis, and cardiovascular diseases (Brito-Zeron et al., 2016).
Lymphocytic infiltration of the LG and release of pro-inflammatory cytokines can affect
the ocular surface by impairing the neural network responsible for modulating tear secretion,
resulting in reduced tear flow and production of inflammatory and proteolytic tears (Hyon et al.,
2007; Stern and Pflugfelder, 2004). One of the consequences of inflammation of the ocular surface
in SS is enhanced apoptosis and autophagy of the cells of the ocular surface system (Byun et al.,
2017). A recent study has shown that increased autophagy marker expression in tears and
conjunctival epithelium have positive correlations with ocular surface damage (Byun et al., 2017).
The corneal epithelium is a part of the ocular surface system greatly affected by development and
progression of SS. The corneal epithelium of SS patients is poorly lubricated, and its irregular
surface can enhance tear film instability. SS patient corneal epithelium also exhibits evidence of
7

impaired corneal epithelial barrier function that can be detected by corneal fluorescein staining
(Stern and Pflugfelder, 2004). SS patients who have suffered from dry eye for more than 10 years
show higher amounts of corneal fluorescein staining, suggesting that ocular surface damage is
associated with SS progression (Hyon et al., 2007). The reduction of tear volume, tear film
instability, and ocular surface inflammation in SS patients are all associated with significant patient
discomfort and impaired vision due to decreased corneal sensation and neural damage from
inflammation (Foulks et al., 2015b; Hyon et al., 2007). SS patients are at higher risk for sight-
threatening ocular manifestations such as corneal ulceration, corneal perforation, cicatrizing
conjunctivitis, uveitis, optic neuritis, scleritis, or retinal vasculitis (Foulks et al., 2015a).
The etiology of SS is still unclear. There are several concepts of the pathogenesis
underlying SS including the genetic predisposition, especially in human leucocyte antigen (HLA),
which is associated with antigen presentation, gene encoding molecules involved in IFN type I and
II, NF- B, lymphocyte trafficking, and antibody-processing signaling pathways (Cruz-Tapias et
al., 2012; Lessard et al., 2013). Viral infections such as Epstein-Barr virus (EBV), human
herpesvirus 6 (HHV6), human T cell lymphotropic virus 1 (HTLV1), and hepatitis C virus (HCV)
are considered as factors that can promote the induction of autoantibodies associated with SS
pathogenesis (Biberfeld et al., 1988; Brito-Zeron et al., 2015; Kivity et al., 2014). Sex hormones
are implicated in SS pathogenesis because SS usually occurs in women after menopause. Some
studies have found that removal of sex hormone by ovariectomy in an SS mouse model can
accelerate the symptoms of disease while giving sex hormone replacement to ovariectomized mice
can prevent LG inflammation (Mostafa et al., 2012). These suggested that sex hormones might be
involved with SS. The other current theories about SS pathogenesis are about autoimmune
epithelitis which proposes that epithelial cells are central regulators of autoimmune responses by
8

acting as atypical antigen presenting cells associated with autoantibody complexes which are
found on the surface of apoptotic cells (Brito-Zeron et al., 2016; Stefanski et al., 2017). Altogether,
multiple factors related to SS pathogenesis can alter both the innate and the adaptive immune
system resulting in the initiation of diseases and creating immune responses by activation of CD4
+

T cells and B cells which play important role in autoantibody production, formation of ectopic
germinal center-like structures to the malignant transformation to Non-Hodgkin lymphoma (NHL)
(Figure 3) (Stefanski et al., 2017).

Figure 3. Diagram of the current concepts of SS pathogenesis (Stefanski et al., 2017)
9

The current diagnostic evaluations of SS are non-specific and impractical for routine
diagnosis. For example, the Schirmer’s test, which is used to determine tear production, cornea
fluorescein staining, which is used to visualize corneal and conjunctival lesions associated with
dry eye, and parotid sialography and salivary gland scintigraphy, which are used to measure
salivation, are not of sufficient specificity (Stefanski et al., 2017). Immunofluorescence for
antinuclear antibodies (ANA) such as anti-SSA, and anti-SSB are non-specific to SS because
patients with other diseases such as Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus
(SLE), and polymyositis (Burbelo et al., 2009; Stefanski et al., 2017) may express these factors. A
salivary gland biopsy, which is used to measure lymphocytic infiltrates in exocrine glandular
tissue, is considered as the gold standard for diagnostic SS, but results should be interpreted
carefully if patients lack clinical manifestations (Brito-Zeron et al., 2016). Therefore, the
development of specific biomarkers for SS is essential for early diagnosis and treatment that can
prevent the serious complications and mortality of SS patients.
Symptomatic treatment of SS is usually limited to the treatment of sicca symptoms. The
first baseline treatments for dry eye therapy are topical tear substitutes and lubrication. Artificial
tears with viscous agents can increase the residence time of tears on the ocular surface (Foulks et
al., 2015b). Anti-inflammatory therapy such as topical corticosteroids, cyclosporin (Restasis
®
),
and omega 3 essential fatty acids can be used in patients with moderate or severe ocular surface
disease (Foulks et al., 2015b; Saraux et al., 2016). However, there are some adverse effects from
using these anti-inflammatory agents in long term exposure (Marsh, 1999; Schultz, 2014).
Secretogogue therapy such as oral pilocarpine and cevimeline can be used to stimulate tear and
saliva secretion, however, there are some common adverse effects including sweating, flushing,
urgent urination, and gastrointestinal discomfort (Brito-Zeron et al., 2016). Punctal occlusion,
10

which can block the tear drainage system leading to increased residence time of ocular surface
tears, is used for dry eye treatment, however, there are no prospective-controlled clinical reports
to strongly support the effect of punctal occlusion on SS associated dry eye (Foulks et al., 2015b).
Therefore, identification of potential therapeutic targets involved in the specific
immunopathological mechanisms of SS is necessary for SS treatment.
1.4 Non-Obese Diabetic (NOD) mouse: A murine model of SS
Mouse models of SS are valuable tool to understand pathological changes during disease
progression and to identify efficacy of therapeutics. The non-Obese Diabetic (NOD) mouse is one
of the spontaneous animal models of SS that naturally develops an autoimmune condition
resembling SS (Jonsson et al., 2007). NOD mice are derived from the outbred Jcl/ICR mice that
develop insulin-dependent diabetes mellitus (IDDM) and SS-like autoimmune exocrinopathy
independently (Bolstad and Jonsson, 2002; Park et al., 2015). Lymphocytic infiltration in NOD
mice were detected in SG and LG leading to sialadenitis and dacryoadenitis (Hu et al., 1992; Janga
et al., 2018). In addition, NOD mice exhibit several autoantibodies which are usually common in
SS patients such as ANA, anti-SSA/Ro, anti-SSB/La and also some increased inflammatory
cytokines such as IL-10, IFN- , IL-7, IL-17, IL-11, and IL-18 in serum and saliva (Delaleu et al.,
2008; Hu et al., 1992). TNF-α, IL-6, IL-1β, IL-10, IL-2, IFN- , KC/GRO, and major
histocompatibility complex class II (MHC-II) expression in LG of NOD mice were increased
relative to BALB/c healthy controls (Janga et al., 2018; Li et al., 2010; Meng et al., 2017). The
pattern of disease progression is different between female and male NOD mice. Male NOD mice
establish significantly severe LG inflammation, but less SG inflammation at 12-24 weeks old
(Janga et al., 2018; Ju et al., 2018). In addition, male NOD mice with 12 weeks of age also show
the reduction of stimulated tear secretion compared to BALB/c mice (Ju et al., 2018). On the
11

contrary, female NOD mice show SG infiltration by 12 weeks of age, but lesser LG inflammation
even by 20 weeks of age (Hunger et al., 1998). A possible reason that male NOD mice show the
high incidence of spontaneous autoimmune dacryoadenitis might be dependent on sex steroids
especially testosterone (Takahashi et al., 1997). Since we have focused on the pathological changes
in LG that contribute to ocular surface inflammation underlying SS-associated dry eye, the male
NOD mouse is chosen as an animal model of SS-associated dry eye.
1.5 Cathepsin S (CTSS) and its functions
Cathepsin S (CTSS) is one of the 11 cysteine cathepsins which is highly expressed in the
spleen, professional antigen-presenting cells (APC) including B lymphocytes, macrophages,
dendritic cells (DC), and non-professional APC such as epithelial cells (Bania et al., 2003; Riese
et al., 1996). CTSS is mainly synthesized on membrane-bound ribosomes as an N-glycosylated
precursor and is transferred into endoplasmic reticulum (ER) and subjected to post-translational
modification in the Golgi network (Kirschke, 2013). CTSS is synthesized as a zymogen that needs
to be cleaved at the N-terminus by other proteases or autocatalytic process at pH 4.5 to become
the active form (Kirschke, 2013). CTSS is a single chain, non-glycosylated papain-like protease
that is active over the range of pH 5.0-8.0 but its optimum activity is at pH 5.5 and 6.5 (Kirschke
and Wiederanders, 1994). Cells releasing CTSS might provide 2 important conditions for CTSS
activation which includes an acidic environment and preventing the interaction of secreted CTSS
with its inhibitors which are found in all tissues and body fluids (Gupta et al., 2008).
The well-established function of CTSS is related to lysosomal protein degradation which is
essential for cellular homeostasis and differentiation (Stoka et al., 2016). In addition, CTSS is also
associated with MHC class II-antigen presentation. The newly synthesized MHC class II is
composed of α/β heterodimers which are assembled with invariant chain (Ii) to form a αβ-Ii
12

nanomeric complex that can prevent premature association of MHC class II molecules with
endogenous polypeptides. This complex is delivered to endosomes in which Ii can be cleaved by
CTSS creating class II-associated invariant chain peptides (CLIP), which is the smaller fragment
of Ii that remains in the peptide-binding groove of MHC class II. After that, CLIP is replaced with
the MHC class II-like molecule, which is HLA-DM (in human) or H-2DM (in mice), allowing
MHC class II to be associated with antigen that is transported to plasma membrane for presentation
of antigen to CD4
+
T-cells (Chang et al., 2007; Shi et al., 1999) (Figure 4).

Figure 4. Role of cathepsin S in MHC class II-associated invariant chain processing and
peptide loading (Chang et al., 2007).
In addition, CTSS also plays an important role in extracellular matrix (ECM) degradation.
ECM is a dynamic structure that can be remodeled through post-translational modification by
13

enzymatic and non-enzymatic processes (Fonovic and Turk, 2014). CTSS uses ECM as a substrate
leading to ECM remodeling. ECM is functionally divided into 2 subtypes; interstitial matrix and
basement membrane (Frantz et al., 2010). There are mainly 2 types of ECM components:
proteoglycans and fibrous proteins, which include collagen, elastin, and fibronectin (Fonovic and
Turk, 2014). CTSS is one of the cysteine proteases that can degrade these ECM components and
dysfunction of CTSS activity and its expression can contribute to ECM-linked diseases such as
osteoarthritis, RA, cardiovascular diseases, cancer, and lung fibrosis (Fonovic and Turk, 2014).
1.6 Cathepsin S (CTSS) as a novel biomarker of SS
Our lab has focused on the relationship between CTSS and SS in both the NOD mouse, a
murine model for SS, and in SS patients. In NOD mice, we have found that CTSS activity was
highly increased in tears of male NOD mice at 8 weeks of age and reached a maximum by 24
weeks old compared to BALB/c control mice (Janga et al., 2018; Ju et al., 2018; Li et al., 2010).
Also, CTSS activity, gene, and protein expression in LG of male NOD mice at 8 weeks old were
statistically significantly increased and continue increasing until 24 weeks of age relative to
BALB/c mice (Janga et al., 2018). Interestingly, we have also seen the change of CTSS distribution
in LG of male NOD mice by immunofluorescence (IF) at different ages of mice. For example, at
8 weeks old male NOD mice, CTSS was abundantly detected in basolateral lysosomal vesicles but
when mice reached to 12-24 weeks of age, CTSS was distributed near the apical membrane and
lumen, suggesting that enriched-CTSS vesicles were ready to be secreted at the apical membrane
and into tears (Janga et al., 2018). The increased CTSS in tear and LG in the male NOD mice
occurred in parallel with the increase of LG proinflammatory cytokines and LG lymphocytic
infiltration, and also the reduction of tear secretion during the development of autoimmune
14

dacryoadenitis, suggesting that CTSS might be an initiator of ocular surface manifestation in SS
animal model.
In clinical studies, CTSS activity in tears of SS patients was significantly higher than in
patients with RA, other autoimmune diseases, non-autoimmune dry eye diseases, or healthy
controls (Edman et al., 2018; Hamm-Alvarez et al., 2014). Our recent study has demonstrated that
increased tear CTSS might directly or indirectly affect the quality of tears in SS patients by
degradation of other tear proteins such as cystatin C, an endogenous CTSS inhibitor, lactoferrin,
which has antibacterial action and anti-inflammatory activities in tear film, and secretory IgA,
which is the main component of mucosal defense in tear (Edman et al., 2018). These results also
have reconfirmed that CTSS can be a novel biomarker of SS that can use to differentiate SS patient
from other autoimmune or non-autoimmune dry eye patients. In addition, CTSS might be one of
the initiators contributing to pathological changes in ocular surface and LG during the development
of SS disease. Importantly, CTSS can be a therapeutic target for alleviation of ocular inflammation
and manifestation in SS-associated dry eye.
1.7 CTSS inhibitor as a therapeutic target of autoimmune diseases
Several studies have previously explored the ability of CTSS inhibitors to prevent
autoantigen presentation and autoimmunity. For example, Clik60, which is an epoxysuccinate
peptide derivative, exhibited strong specificity for CTSS at a concentration of 10
-7
Molar (M),
while there is no inhibition of cathepsin B, L, K, and C observed at 10
-6
M (Katunuma et al., 1999).
Its structure was shown in Figure 5A. Clik60 can impair autoantigen-specific T cell responses in
mouse salivary gland cells and also blocked SG and LG lymphocytic infiltration, decreased the
induction of aautoantibody production in serum of murine model for SS (Saegusa et al., 2002).
15

RO5459072 is another competitive inhibitor of the active site of CTSS and its nitrile
function allows covalent reversible inhibition of CTSS. It has an IC 50 of 0.1 nM and 0.3 nM to
human and murine CTSS respectively and no sub-micromolar inhibition was detected to other
cathepsins including Cathepsin L, B, K, and F except for cathepsin V with an IC 50 of 700 nM (Tato
et al., 2017). Its structure is shown below in Figure 5B (National Institutes of Health, 2018). Some
studies have shown that its oral administration attenuated systemic autoimmunity in MRL-(Fas)lpr
mice, an animal model of systemic lupus erythematosus (Tato et al., 2017). RO5461111 is another
reversible competitive CTSS inhibitor which has an IC 50 of 0.4 nM and 0.5 nM to human and
murine CTSS respectively (Rupanagudi et al., 2015). Its structure is shown below in Figure 5C
(Rupanagudi et al., 2015). Its oral administration improves lupus nephritis in MRL-(Fas)lpr mice
and also in CKO, another model of the lupus phenotype (Kim et al., 2017; Rupanagudi et al.,
2015), and reduce albuminuria, glomerulosclerosis and retinopathy in delayed treatment of type 2
diabetic db/db mice (Kumar Vr et al., 2016). MV026031 is another CTSS inhibitor which has a K i
of 47 nM and exhibits a binding constant of 22 nM to human and murine CTSS respectively (Zhao
et al., 2014). Its structure is shown in Figure 5D (Barlow et al., 2015). Oral administration of
MV026031 can suppress formalin-induced endogenous CTSS activity leading to hyperalgesia in
mice (Zhao et al., 2014).
Additionally, there are several CTSS inhibitors that are in phase I and II clinical trials for
treatment of autoimmune diseases. For example, LY3000328 (www.clinicaltrials.gov, Identifier:
NCT01515358), which is in phase I clinical trial, is used for abdominal aortic aneurysm treatment
(Payne et al., 2014). RWJ-445380 (www.clinicaltrials.gov, Identifier: NCT00425321) for RA
treatment and RO5459072 (www.clinicaltrials.gov, Identifier: NCT02701985) for SS treatment
16

are in phase II clinical trial (Theron et al., 2017). It suggests that utility of CTSS as a therapeutic
target for mitigating the ocular manifestations of SS.

Figure 5. Structure of CTSS inhibitors (A) Clik60; (B) RO5459072; (C) RO5461111;
(D) MV026031
1.8 Goals
In this study, I hypothesized that CTSS might affect ocular surface inflammation in SS
patients. I utilized human corneal epithelial cell line (HCE-T cells) treated with human
recombinant CTSS at the same enzymatic activity level as we have seen in SS patients tears. I
investigated whether there are any changes in pro-inflammatory cytokines and related proteases
after cells treated with CTSS. Also, I investigated how CTSS alters inflammatory responses in
ocular surface epithelial cells by focusing on role of protease-activated receptor-2 (PAR-2), which
is discussed in detail later. Additionally, I studied whether using a CTSS inhibitor can alleviate
ocular and LG inflammation and improve tear secretion in male NOD mice which is a murine
17

model of SS. In this study, mice were administered the inhibitor by 2 routes: intraperitoneal
injection (i.p.) and topical eye drops and therapeutic effects from each administration were
evaluated after CTSS inhibitor treatment.
Some parts of my dissertation including some information and data in Chapter 1, 2, 3, 6,
and 7 were from my original article which was already published in International Journal of
Molecular Sciences (Klinngam et al., 2018).

18

Chapter 2: CTSS induces pro-inflammatory cytokines, proteases, and protease-activated
receptor-2 (PAR-2) expression in human corneal epithelial cells
As mentioned in Chapter 1, CTSS is associated with inflammation and autoimmune
responses in other diseases. Our studies have shown that CTSS is highly expressed in tears and
LG of male NOD mice and in tears of SS patients (Edman et al., 2018; Janga et al., 2018; Li et al.,
2010). My overall hypothesis is that elevated tear fluid CTSS activity in SS patients might affect
corneal and ocular surface homeostasis. In this study, I investigated the effects of CTSS on the
expression of pro-inflammatory cytokines, proteases, and protease-activated receptor-2 (PAR-2)
in human corneal epithelial cell lines (HCE-T cells). HCE-T cells are derived from primary
cultured human corneal epithelial cells transfected with a recombinant SV40-adenovirus vector
and cloned 3 times to obtain a continuously growing cell line (Araki-Sasaki et al., 1995). HCE T-
cells showed similar characteristics to normal corneal epithelial cells in culture including a cobble-
stone-like appearance, desmosome formation and microvillar development, expression of cornea-
specific 64 kD cytokeratin, and multilayer differentiation under an air-liquid interface culture
(Araki-Sasaki et al., 1995). Therefore, I utilized cultured human corneal epithelial cells treated
with human recombinant CTSS at activity levels approximating those found in SS patient tears,
and measured changes in downstream proteins of interest. Most data and information in this
chapter come from my published paper (Klinngam et al., 2018).
2.1 Acute CTSS exposure significantly increases IL-1β, IL-8, IL-6, and TNF-α gene and
protein expression in cell culture medium and cell lysates of HCE T-cells
There are some literature studying the imbalance of cytokines in conjunctival epithelium
and tears of SS patients. IL-1α, IL-6, IL-8, TNF-α, and TGF-β1 expression was elevated in
conjunctival epithelium of SS patients relative to healthy controls (Pflugfelder et al., 2009). Other
19

studies also have shown that IL-1, IL-6, IL-8, TNF-α, IFN-  and IL-17 are highly expressed in SS
patient tears relative to non-SS patients (D'Souza and Tong, 2014; Liu et al., 2017; Solomon et al.,
2001; Yoon et al., 2007) suggesting that elevated pro-inflammatory cytokines may be related to
disease progression in SS patients. In this study, I hypothesized that tear CTSS may directly
increase pro-inflammatory cytokine expression in HCE T-cells.
To investigate whether CTSS affects pro-inflammatory cytokine gene expression, HCE-T
cells were treated with recombinant human CTSS at an enzymatic activity comparable to that
detected at the 90
th
– 95
th
percentile (18,000 Relative Fluorescence Units or RFU per 500 µl of cell
medium) in SS patient tears (Edman et al., 2018). Cells were treated with human recombinant
CTSS for 2, 4, 8, and 24 hours. The cell viability of cells treated with recombinant CTSS for 24
hours measured by the MTT assay was 97.3 ± 5.2% (mean ± SEM) relative to untreated cells
(Figure 6).

Figure 6. Cell viability measured by MTT assay after 24 hours of CTSS treatment in a human
corneal epithelial cell line (HCE T-cells) compared to untreated cells. The amount of CTSS
added corresponded to an activity level found in the 90
th
– 95
th
percentile of SS patients (18,000
RFU, added to 500 µl of cell medium) (n = 3 samples/group, data are represented as mean ± SEM,
and a two-tailed, unpaired Student’s t-test was used to compare treated to untreated cells.
20

Gene expression of pro-inflammatory cytokines of interest (IL-1β, IL-8, IL-6, and TNF-α)
was measured and compared to untreated cells at 4 different time points. The results showed that
CTSS can enhance IL-1β, IL-8, IL-6, and TNF-α gene expression after 2 to 4 hours which is
considered as an acute exposure (Figure 7A – D). IL-1β gene expression was significantly
increased after 2 and 24 hours of CTSS treatment (Figure 7A). IL-8 and IL-6 gene expression
began to increase after 2 hours and reached the highest expression after 4 hours of CTSS treatment
(Figure 7B, C). Additionally, CTSS significantly increased TNF-α gene expression after 2 hours
of treatment (Figure 7D).
To confirm whether CTSS affected protein expression comparably to gene expression, the
Pro-inflammatory Panel 1 (human) Multiplex assay kit (MSD
®
, Rockville, MD), which allows
quantitation of up to 10 pro-inflammatory cytokines in the same sample, was used to analyze the
protein expression of pro-inflammatory cytokines in cell culture medium and cell lysates in HCE
cells treated with CTSS for 2, 4, 8, and 24 hours, compared to untreated cells. Protein expression
results corresponded with gene expression data, showing that CTSS increased IL-8, IL-6, and
TNF-α protein expression in both cell culture medium and cell lysates at 2, 4, and 8 hours of
treatment (Figure 8A – F). Although IL-1β protein expression in cell medium was not increased,
IL-1β protein expression in cell lysates was still induced after 2 and 4 hours of CTSS treatment
(Figure 8G, H). In addition, CTSS increased IL-1β gene expression in cell lysates and IL-6 protein
expression in cell culture medium after cells were treated with CTSS for 24 hours, suggesting that
there might be a later phase of cytokine responsiveness to chronic exposure to this protease.

21

Figure 7. CTSS increases IL- 1β, IL-8, IL-6, and TNF- α gene expression after 2- and 4-hours
of treatment in a human corneal epithelial cell line (HCE-T cells) (A) IL-1β gene expression;
(B) IL-8 gene expression; (C) IL-6 gene expression; (D) TNF-α gene expression. The amount of
CTSS added corresponded to an activity level found in the 90
th
– 95
th
percentile of SS patients
(18,000 RFU, added to 500 µl of cell medium). Expression of genes of interest were normalized
to expression of the endogenous gene, GAPDH (n = 3 samples/group, * P ≤ 0.05, ** P ≤ 0.01, ***
P ≤ 0.001, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple
comparison was used to compare treated to untreated cells).

22

Figure 8. CTSS significantly increases IL-8, IL-6, TNF-α, IL-1β protein expression in cell
culture medium and cell lysates from human corneal epithelial cells (HCE-T cells) at 2, 4,
and 8 hours of exposure. (A) IL-8 protein expression in cell culture medium; (B) IL-8 protein
expression in cell lysates; (C) IL-6 protein expression in cell culture medium; (D) IL-6 protein
expression in cell lysates; (E) TNF-α protein expression in cell culture medium; (F) TNF-α protein
expression in cell lysates; (G) IL-1β protein expression in cell culture medium; (H) IL-1β protein
expression in cell lysate. The amount of CTSS added corresponded to an activity level found in
the 90
th
– 95
th
percentile of SS patients (18,000 RFU, added to 500 µl of cell medium). Expression
of proteins of interest were normalized to total protein concentration in either cell culture medium
or lysates. a = individual ELISA kit (n = 3 samples/group, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001,
data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison
was used to compare treated to untreated cells).
23

2.2 Chronic CTSS exposure significantly increases MMP-9 gene expression
Matrix metalloproteinase 9 (MMP-9) is a protease responsible for remodeling ECM, and
is associated with pathology in diverse inflammatory diseases including arthritis, cardiovascular
diseases, pulmonary diseases, cancer, SLE, and SS (Ram et al., 2006). MMP-9 protein expression
and enzymatic activity are elevated in LG of female MRL/lpr and male NOD mice, murine models
of SS (Aluri et al., 2015). MMP-9 activity is also highly increased in SS patient tears when
compared to healthy controls (Solomon et al., 2001), suggesting that increased MMP-9 may be
related to ocular surface pathogenesis. Additionally, the relationship between MMP-9 and pro-
inflammatory cytokines have been reported. For instance, increased IL-6, IL-8, IL-1β, and TNF-α
can induce MMP-9 expression in malignant non-Hodgkin’s lymphoma, human neutrophils, and
corneal epithelial cells respectively (Chakrabarti and Patel, 2005; Gordon et al., 2009;
Kossakowska et al., 1999; Li et al., 2001). MMP-9 itself may elicit expression of IL-1β, TNF-α,
and TGF-β which may then perpetuate the inflammatory cycle (Chotikavanich et al., 2009; Stern
and Pflugfelder, 2004). Therefore, it is possible CTSS and/or early CTSS-induced pro-
inflammatory cytokines might affect MMP-9 expression.
To investigate whether CTSS can induce MMP-9 expression in corneal epithelial cells,
possibly enhancing extracellular matrix degradation and ocular surface inflammation, gene
expression was analyzed in HCE-T cells without and with treatment with CTSS for 2, 4, 8, and 24
hours. The results show that MMP-9 gene expression is significantly elevated after 24 hours of
CTSS relative to levels in untreated cells (Figure 9).

24

Figure 9. CTSS increases MMP-9 gene expression after 24 hours in human corneal epithelial
cells. MMP-9 gene expression in HCE-T cells without and with CTSS. The amount of CTSS added
corresponded to an activity level found in the 90
th
– 95
th
percentile of SS patients (18,000 RFU,
added to 500 µl of cell medium). MMP-9 gene expression was normalized to expression of the
endogenous gene, GAPDH (n = 3 samples/group, ** P ≤ 0.01, data are represented as mean ± SEM
and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to
untreated cells).
2.3 Chronic CTSS exposure significantly increases PAR-2 gene and protein expression
Protease–activated receptor-2 (PAR-2) is a G-protein-coupled receptor activated by
specific proteases such as trypsin, mast cell tryptase, factor Xa and VIIa, and other serine proteases.
PAR-2 is activated through cleavage of its extracellular domain generating a tethered ligand which
induces downstream intracellular signaling pathways (Yau et al., 2013). PAR-2 is expressed in a
variety of cells such as keratinocytes (Lee et al., 2010), endothelial cells (Nikolic-Paterson, 2016),
fibroblasts (Gruber et al., 2004), neurons (Vergnolle et al., 2001), immune and inflammatory cells
(Ramelli et al., 2010) and also in epithelial cells such as lung, gastrointestinal tract and corneal
cells (Dulon et al., 2003; Iablokov et al., 2014; Lang et al., 2003). Recent studies have
25

demonstrated that activated PAR-2 is associated with inflammatory process contributing to
respiratory, gastrointestinal, metabolic, cardiovascular, and neurological diseases (Ramachandran
et al., 2012). CTSS can also cleave PAR-2 near the N-terminus at a site distinct from trypsin,
exposing a novel tethered ligand and leading to release of inflammatory mediators linked to several
pathological conditions including hyperexcitability of nociceptive neurons promoting neurogenic
inflammation and neuropathic pain (Zhao et al., 2014), endothelial cell injury (Kumar Vr et al.,
2016; Tato et al., 2017), visceral hyperalgesia during colitis (Cattaruzza et al., 2011), induction of
chronic atopic dermatitis (Kim et al., 2012), and activation of liver tumor-initiating cells associated
with hepatocellular carcinoma (Lee et al., 2014).
To investigate whether CTSS affects PAR-2 expression in human corneal epithelial cells,
HCE T-cells were treated with human recombinant CTSS for 2, 4, 8, and 24 hours. Then, gene and
protein expression of PAR-2 was measured and compared to untreated cells. We found that PAR-
2 gene and protein expression were significantly increased after 24 hours of CTSS treatment
relative to untreated cells (Figure 10A-C). PAR-2 gene expression was increased by 2-fold
(Figure 10A), which corresponded to a 1.7-fold increased PAR-2 protein expression measured by
ELISA (Figure 10B). In addition, when PAR-2 protein expression was evaluated using
immunofluorescence, CTSS-treated cells showed a higher intensity of PAR-2
immunofluorescence relative to untreated cells (Figure 10C). These findings suggest CTSS can
increase PAR-2 expression at both the gene and protein expression after chronic CTSS exposure
(at 24 hours of CTSS treatment).

26

Figure 10. CTSS increases PAR-2 gene and protein expression after 24 hours in human
corneal epithelial cells. (A) PAR-2 gene expression in HCE-T cells without and with CTSS. PAR-
2 gene expression was normalized to expression of the endogenous gene, GAPDH (n = 3
samples/group and one-way ANOVA with Dunnett’s multiple comparison was used to compare
treated to untreated cells); (B) PAR-2 protein expression measured by using the human PAR-2
ELISA assay in HCE-T cell lysates without and with CTSS. PAR-2 protein expression was
normalized to total protein in lysates (n = 4 samples/group and a two-tailed, unpaired Student’s t-
test was used to compare treated to untreated cells); (C) HCE-T cells treated without and with
CTSS for 24 hours and fixed and processed using primary and secondary antibodies to detect PAR-
2 by indirect immunofluorescence. The amount of CTSS added corresponded to an activity level
found in the 90
th
– 95
th
percentile of SS patients (18,000 RFU, added to 500 µl of cell medium). *
P ≤ 0.05, ** P ≤ 0.01 and data are represented as mean ± SEM.
27

2.4 CTSS exposure significantly increases CTSS gene and protein expression at 8- and 24-
hours
Some studies have demonstrated that intracellular CTSS can also affect expression of pro-
inflammatory cytokines. For example, CTSS-overexpressing mice showed higher expression of
Th2-type cytokines such as IL-4 and IL-10, and also increased levels of Th1-type cytokines such
as TNF-α, IFN- , and IL-1β (Kim et al., 2012). CTSS from splenic dendritic cells induced IL-6
production in response to systemic exposure to lipopolysaccharide (Dekita et al., 2017). Both
extracellular and intracellular CTSS can also activate IL-36 , a newly discovered IL-1 family
member, and can induce the secretion of IL-8 in a human keratinocyte cell line (Ainscough et al.,
2017). Our findings of later or sustained elevations in some pro-inflammatory cytokines and
MMP-9 after 24 hours of CTSS treatment suggested that there might be pathways that stimulate
their later induction, possibly a positive feedback loop involving increased CTSS expression itself
after 24 hours.
To prove my hypothesis, CTSS gene and protein expression were analyzed after 2, 4, 8,
and 24 hours of CTSS treatment. The results showed that CTSS gene expression was increased
after cells were treated with recombinant CTSS for 24 hours (Figure 11A). CTSS protein
expression in cell lysates measured using the human CTSS ELISA kit also revealed that the protein
was increased after 8 hours of CTSS treatment (Figure 11B). In addition, CTSS activity in cell
lysates was significantly increased after 24 hours of CTSS treatment, relative to untreated cells
(Figure 11C). These data suggest that CTSS exposure induces its own gene and protein
expression, in a way that may promote a positive feedback loop which contributes further to a
cycle of induction of pro-inflammatory cytokines as has been reported in other systems.

28

Figure 11. CTSS exposure in human corneal epithelial cells increases cellular CTSS gene and
protein expression after 8- and 24-hours (A) CTSS gene expression in HCE-T cells without and
with recombinant CTSS. CTSS gene expression was normalized to expression of the endogenous
gene, GAPDH. one-way ANOVA with Dunnett’s multiple comparison was used to compare
treated to untreated cells; (B) CTSS protein expression measured by using the human CTSS ELISA
assay in HCE-T cell lysates without and with recombinant CTSS treatment at different time points.
CTSS protein expression was normalized to total protein in lysates and a one-way ANOVA with
Dunnett’s multiple comparison was used to compare treated to untreated cells; (C) Enzymatic
CTSS activity after 24 hours in HCE-T cells without and with recombinant CTSS. Enzymatic
CTSS activity was normalized to total protein in lysates and a two-tailed, unpaired Student’s t-test
was used to compare treated to untreated cells. (n = 3 samples/group, * P ≤ 0.05, ** P ≤ 0.01, data
are represented as mean ± SEM). The amount of CTSS added corresponded to an activity level
found in the 90
th
– 95
th
percentile of SS patients (18,000 RFU, added to 500 µl of cell medium).
2.5 Acute exposure to heat-inactivated CTSS does not induce pro-inflammatory cytokines
As mentioned in Figure 7, 4 hours of CTSS treatment was sufficient to induce maximal
pro-inflammatory cytokine gene and protein expression, especially IL-8 and IL-6. To investigate
whether CTSS activity was critical for this early induction of pro-inflammatory cytokine
expression, heat-inactivated CTSS was used to treat HCE T-cells for 4 hours, and the gene
expression of IL-8 and IL-6 after 4 hours treatment was measured and compared to HCE -T cells
that were treated with active CTSS under the same conditions. Recombinant human CTSS was
inactivated by heating at 90
°
C for 30 minutes, resulting in reduction of its activity from 364 RFU/µl
to 1 RFU/µl. IL-8 gene expression in cells treated with heat-inactivated CTSS was not significantly
different relative to untreated cells, but was significantly reduced relative to levels in cells treated
29

with active CTSS (Figure 12A). Similarly, there was not a significant difference in IL-6 gene
expression between untreated cells and cells exposed to heat-inactivated CTSS, but a statistically
significant increase in IL-6 gene expression in cells treated with active CTSS versus cells treated
with heat-inactivated CTSS or untreated cells (Figure 12B). These results suggest that CTSS
activity is essential for the early induction of pro-inflammatory cytokines, especially IL-8 and IL-
6.

Figure 12. CTSS activity is required for early induction of pro-inflammatory cytokines in
human corneal epithelial cells. (A) IL-8 gene expression in HCE-T cells without (untreated),
with heat-inactivated CTSS, and with active CTSS; (B) IL-6 gene expression in HCE-T cells
without (untreated), with heat-inactivated CTSS, and with active CTSS. IL-8 and IL-6 gene
expression were normalized to expression of the endogenous gene, GAPDH (n = 3 samples/group,
* P ≤ 0.05, ** P ≤ 0.01, data are represented as mean ± SEM, and one-way ANOVA with Tukey’s
multiple comparison was used to compare cells within different CTSS treatments. The amount of
CTSS added corresponded to an activity level found in the 90
th
– 95
th
percentile of SS patients
(18,000 RFU, added to 500 µl of cell medium). Heat inactivation was by heating at 90
°
C for 30
minutes.
30

Chapter 3: CTSS-dependent activation of PAR-2 is involved in induction of pro-
inflammatory cytokines and proteases in human corneal epithelial cells
PAR-2 has different protease cleavage sites, resulting in distinct intracellular signaling
pathways. For example, trypsin cleaves PAR-2 at R
36
and S
37
leading to mobilization of
intracellular Ca
2+
, activation of ERK1/2, and β-arrestin recruitment inducing PAR-2 endocytosis,
while CTSS cleaves PAR-2 at E
56
and T
57
contributing to adenylyl cyclase and PKA mechanisms
(Zhao et al., 2014). Several studies primarily conducted with serine proteases such as trypsin, mast
cell tryptase, bacterial proteases, arginine proteases, and elastase link activated PAR-2 to induction
of pro-inflammatory cytokines. For example, activated PAR-2 leads to the release of IL-6 and IL-
8 in oral epithelial cells, keratinocytes, sebocytes, and corneal epithelial cells (Ishikawa et al.,
2009; Lee et al., 2015; Lourbakos et al., 2001; Tripathi and Alizadeh, 2014), and also stimulates
mRNA expression of IL-1 and TNF-α in keratinocytes, endothelial cells, and sebocytes (Lee et al.,
2015; Lee et al., 2010; Napoli et al., 2000; Park et al., 2011). PAR-2 activation by trypsin increases
MMP-9 expression and enzymatic activity in airway epithelial cells and keratinocyte (Lee et al.,
2010; Vliagoftis et al., 2000). However, there is no any studies about how CTSS-activated PAR-2
affects inflammatory responses in corneal epithelial cells. As mentioned in Chapter 2, we found
that CTSS can induce PAR-2 gene and protein expression after CTSS chronic exposure. Therefore,
it might be possible that tear CTSS may induce inflammatory responses, which are related to the
increased pro-inflammatory cytokines and proteases, in corneal epithelial cells through activation
of corneal epithelial PAR-2. Again, most data and information in this chapter come from my
published paper (Klinngam et al., 2018).

31

To study whether activation of PAR-2 by CTSS is responsible for the induction of pro-
inflammatory cytokines and proteases associated with exposure of cultured corneal epithelial cells
to CTSS, human PAR-2 siRNA was used to reduce cellular PAR-2 expression before treatments
with recombinant CTSS. Several concentrations of siRNA and durations of incubation were tested
to find the optimal condition for siRNA transfection. We found that 25 mol of PAR-2 siRNA for
48 hours was the best condition, showing the highest transfection efficiency and the efficiency of
PAR-2 knockdown using human PAR-2 siRNA in HCE-T cells.
3.1 Transfection efficiency of PAR-2 siRNA in HCE T-cells
The transfection efficiency of siRNA in HCE-T cells was determined using the BLOCK-
iT Fluorescent Oligo labeled with FITC. The BLOCK-iT Fluorescent Oligo is a fluorescein-
labeled double-stranded RNA with the same length, charge, and configuration as a standard
siRNA. It is used to indicate the transfection efficiency of siRNA (Liu et al., 2016). In this study,
the transfection efficiency of 25 mol of siRNA transfected in HCE T-cells for 48 hours measured
by immunofluorescence and flow cytometry was 95.4% (Figure 13A-C).
3.2 The efficiency of PAR-2 knockdown by using human PAR-2 siRNA in HCE T-cells
The efficiency of PAR-2 knockdown using PAR-2 siRNA was evaluated by analysis of
PAR-2 gene and protein expression. The results showed that after HCE-T cells were transfected
with 25 pmol of PAR-2 siRNA for 48 hours, PAR-2 gene expression was reduced approximately
77% relative to scrambled siRNA-transfected cells which showed no reduction in gene expression
(Figure 14A). The PAR-2 band migrates from 44-55 kDa. In our study, we observed a principal
immunoreactive band at 55 kDa (Figure 14B). The PAR-2 band intensity in cells transfected with
scrambled and PAR-2 siRNA was normalized to the band intensity of GAPDH, used as a loading
32

control. We found that the PAR-2 band intensity in cells transfected with PAR-2 siRNA showed
a 42% reduction compared to cells transfected with scrambled siRNA (Figure 14C) which
corresponded with ELISA results that showed a 45% reduction in cells transfected with PAR-2
siRNA relative to cells transfected with scrambled siRNA (Figure 14D). This suggests that
although the mRNA is markedly reduced, that cellular PAR-2 protein may turn over more slowly
within the experimental time frame of 48 hours siRNA treatment.

Figure 13. Transfection efficiency of siRNA using BLOCK-iT Fluorescent Oligo labeled with
FITC in human corneal epithelial cells after 48 hours of incubation. (A) Cellular distribution
of BLOCK-iT Fluorescent Oligo labeled with FITC in transfected HCE-T cells compared to
untransfected cells; (B) The flow cytometry histogram from transfected cells compared to
untransfected cells. The black bar represents the ranged gate in which 99.1% of untreated cells
were determined as FITC negative and 0.9% of untransfected cells were determined as FITC
positive; (C) Transfection efficiency measure by flow cytometry in transfected HCE-T cells
relative to untransfected cells (n = 3 samples/group, **** P ≤ 0.0001, data represented as mean ±
SEM, and a two-tailed, unpaired Student’s t-test was used to compare transfected to untransfected
cells).
33

Figure 14. PAR-2 gene and protein expression after 48 hours of PAR-2 or scrambled siRNA
transfection in human corneal epithelial cells. (A) PAR-2 gene expression in HCE-T cells
transfected with PAR-2 or scrambled siRNA. PAR-2 gene expression was normalized to
expression of the endogenous gene, GAPDH (n = 3 samples/group; (B) PAR-2 bands measured by
Western Blotting in lysates from human corneal epithelial cells transfected with PAR-2 or
scrambled siRNA; (C) PAR-2 band intensity in human corneal epithelial cells transfected with
PAR-2 or scrambled siRNA. The intensity signal of PAR-2 band was normalized to the band
intensity of GAPDH and designated as 100% for scrambled siRNA-treated cells (n = 5
samples/group); (D) PAR-2 protein expression in HCE-T cells transfected with PAR-2 or
scrambled siRNA as determined by ELISA. PAR-2 protein expression was normalized to total
protein in lysates (n = 3 samples in PAR-2 siRNA transfected and n = 2 in scrambled siRNA
transfected, * P ≤ 0.05, *** P ≤ 0.001 data are represented as mean ± SEM, and a two-tailed,
unpaired Student’s t-test was used to compare PAR-2 siRNA transfected to scrambled siRNA
transfected cells).
34

3.3 The effect of CTSS activation of PAR-2 on the increase in pro-inflammatory cytokines
and proteases at 4 and 24 hours of recombinant CTSS treatment
To study the effect of CTSS activation of PAR-2 on the expression of pro-inflammatory
cytokines and proteases, cells, which were already transfected with either PAR-2 or scrambled
siRNA treatment, were exposed to recombinant CTSS to 4 or 24 hours, representing acute and
chronic exposures respectively (Figure 15). Gene and protein expression of pro-inflammatory
cytokines of interest (IL-1β, IL-8, IL-6, TNF-α) and proteases (CTSS and MMP-9) were measured
according to the experimental protocol shown in Figure 15. However, protein expression of CTSS
in cell medium was not measured because recombinant CTSS treatments can interfere with
measurement of CTSS released from the cells.

Figure 15. Experimental design to study the effect of CTSS activation of PAR-2 on the
increase in pro-inflammatory cytokines (IL-8, IL-6, TNF-α, and IL-1β) and proteases (CTSS
and MMP-9) at 4 and 24 hours of recombinant CTSS treatment.

35

Gene and protein expression of pro-inflammatory cytokines and proteases in HCE T-cells
transfected with PAR-2 siRNA after 4 and 24 hours of CTSS treatment were shown in Table 1
and Table 2 respectively.
Table 1. Decreased pro-inflammatory cytokine and protease gene expression in human
corneal epithelial cells transfected with PAR-2 siRNA after 4 and 24 hours of CTSS
treatment.
Gene
expression
Relative Expression
4 hours of CTSS treatment 24 hours of CTSS treatment
Scrambled
siRNA
PAR-2
siRNA
P value
Scrambled
siRNA
PAR-2
siRNA
P value
IL-8 1.007 ± 0.084 1.109 ± 0.130 Ns 1.029 ± 0.180 0.348 ± 0.057 *
IL-6 1.007 ± 0.085 0.525 ± 0.079 * 1.000 ± 0.021 0.090 ± 0.007 ****
IL- 1β 1.187 ± 0.493 0.546 ± 0.153 ns 1.032 ± 0.183 0.222 ± 0.068 *
TNF- α 1.048 ± 0.224 0.382 ± 0.083 * 1.007 ± 0.087 0.267 ± 0.040 **
CTSS 1.012 ± 0.115 1.156 ± 0.263 ns 1.017 ± 0.131 0.289 ± 0.059 **
MMP-9 1.050 ± 0.243 0.723 ± 0.112 ns 1.011 ± 0.104 0.099 ± 0.034 **

The amount of CTSS added corresponded to an activity level found in the 90
th
– 95
th
percentile
of SS patients (18,000 RFU, added to 700 µl of cell medium). n = 3 samples/group, * P ≤ 0.05, **
P ≤ 0.01, **** P ≤ 0.0001, ns = not significant, data are represented as mean ± SEM, and a two-
tailed, unpaired Student’s t-test was used to compare PAR-2 siRNA transfected to scrambled
siRNA transfected cells.

36

Table 2. Pro-inflammatory cytokine and protease protein expression in cell culture medium
and cell lysates of human corneal epithelial cells transfected with PAR-2 siRNA after 4 and
24 hours of CTSS treatment.
Protein
expression
Protein expression (pg/mg protein)
4 hours of CTSS treatment 24 hours of CTSS treatment
Scrambled
siRNA
PAR-2
siRNA
P value
Scrambled
siRNA
PAR-2
siRNA
P value
Cell culture medium
IL-8 18759 ± 61.6 19800 ± 472.2 ns 15254 ± 445 20377 ± 289.2 ns
IL-6
a
79003 ± 12649 26608 ± 9138 * 42730 ± 2075 17810 ± 844.4 ***
IL-1β
a
1.240 ± 0.1052 0.738 ± 0.095 * 1.826 ± 0.235 1.302 ± 0.072 ns
TNF-α 105.8 ± 7.221 43.52 ± 1.785 ** 44.64 ± 2.173 22.09 ± 0.662 ***
MMP-9 10834 ± 881 5613 ± 48.90 ** 34666 ± 836 19702 ± 3355 *
Cell lysates
IL-8 147.5 ± 16.61 115.7 ± 10.92 ns 108.8 ± 18.13 113.8 ± 21.64 ns
IL-6 8.784 ± 0.560 3.862 ± 1.133 * 3.155 ± 0.746 1.548 ± 0.231 ns
IL-1β 138.0 ± 7.153 94.75 ± 15.88 ns 62.99 ± 11.64 36.33 ± 7.632 ns
TNF-α 0.836 ± 0.056 0.778 ± 0.145 ns 0.794 ± 0.039 0.669 ± 0.117 ns
CTSS 25.16 ± 5.543 33.53 ± 6.185 ns 35.60 ± 6.494 34.30 ± 8.386 ns
MMP-9 6160 ± 329.0 3322 ± 309.5 ** 10628 ± 1137 5980 ± 1390 ns

The amount of CTSS added corresponded to an activity level found in the 90
th
– 95
th
percentile
of SS patients (18,000 RFU, added to 700 µl of cell medium). n = 3 samples/group, * P ≤ 0.05, **
P ≤ 0.01, *** P ≤ 0.001, ns = not significant, a = individual ELISA kit, data are represented as
mean ± SEM, and a two-tailed, unpaired Student’s t-test was used to compare PAR-2 siRNA
transfected to scrambled siRNA transfected cells. CTSS protein expression in cell culture medium
was not measured because we could not distinguish added recombinant CTSS from that released
from the cells.
The gene expression results demonstrated that only IL-6 and TNF-α gene expression in
cells transfected with PAR-2 siRNA were reduced relative to scrambled siRNA transfected cells
at 4 hours of CTSS treatment, while all of 4 proinflammatory cytokines (IL-8, IL-6, TNF-α, IL-1β)
and proteases (MMP-9 and CTSS) gene expression were decreased in PAR-2 siRNA transfected
cells after 24 hours of CTSS treatment relative to scrambled siRNA transfected cells (Table 1).
For protein expression results, after 4 hours of CTSS treatment, PAR-2 siRNA transfected cells
showed reduced secretion of IL-6, TNF-α and IL-1β into culture medium relative to scrambled
siRNA transfected cells, with the effect persisting in culture medium for IL-6 and TNF-α at 24
37

hours of CTSS treatment. IL-6 was the only pro-inflammatory cytokine with protein levels in
lysates that showed a reduction at 4 hours of CTSS treatment after PAR-2 knockdown. These
changes all occurred at times independent of CTSS-induced changes in PAR-2 protein expression,
suggesting they are mediated under normal conditions by existing plasma membrane PAR-2 in the
absence of any CTSS induction (Table 2). On the contrary, there was no significant changes in
IL-8 secretion to cell culture medium or in cell lysates was seen at either 4-and 24-hours treatment
relative to scrambled siRNA-transfected cells (Table 2). PAR-2 siRNA-transfected cells showed
the decrease of MMP-9 secretion into culture medium at both 4 and 24 hours, and the decrease of
MMP-9 protein expression in cell lysates at 4 hours of CTSS treatment. All these findings suggest
that PAR-2, which is known to be activated by CTSS, is important for the early CTSS-dependent
induction of IL-6 and TNF-α gene expression (Table 1), the increased acute CTSS-dependent
secretion of IL-6, IL-1β, TNF- α and MMP-9 protein (Table 2), and possibly the later CTSS-
dependent changes in gene expression of many of these factors (Table 1).
3.4 An initial PAR-2 dependent increase in TNF-α after acute CTSS exposure may drive IL-
6 and IL-1β gene expression in HCE T-cells
Some literature has shown that TNF-α can induce IL-6 and IL-1β expression via the TNFRI
receptor and p38 mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase
(PI3K)/Akt and nuclear factor (NF)- B pathways (Turner et al., 2007). To investigate whether an
early CTSS-mediated and PAR-2 dependent increase in TNF-α could theoretically drive PAR-2
independent elevations in IL-6 and IL-1β, we explored the time course of gene expression of pro-
inflammatory cytokines from Figure 7 at 15 minutes, 1 hour and 2 hours of CTSS treatment
relative to untreated cells. Figures 7 and 8 show that changes in gene and protein expression of
these cytokines occur in parallel under these conditions. Figure 16 shows that only TNF-α gene
38

expression was increased after 15 minutes of CTSS treatment relative to untreated cells, while
expression of the other pro-inflammatory cytokines was increased only after 2 hours of CTSS
treatment. Notably, induction of TNF-α gene transcription after exposure to certain stimuli such as
lipopolysaccharide or receptor for IgE (FcεRI) is considered as an immediate early gene which
does not require de novo synthesis (Falvo et al., 2010; Pelletier et al., 1998). NF- B is one of the
transcription factors that regulate TNF-α mediate early gene (Pelletier et al., 1998). Therefore, the
early induction of TNF-α gene expression after 15-minute CTSS treatment might be related to NF-
B transcription factors. Since increased expression of TNF-α temporally precedes that of IL-6
and IL-1β and it is PAR-2 dependent, it is therefore possible that it may drive the downstream
changes in IL-6 and IL-1β which may be PAR-2 independent but TNF-α-dependent.

39

Figure 16. Gene expression of pro-inflammatory cytokines (IL-8, IL-6, TNF- α, and IL- 1β)
after 15 minutes, 1 hour, and 2 hours of CTSS treatment in human corneal epithelial cells
(HCE T-cells). The amount of CTSS added corresponded to an activity level found in the 90
th
–
95
th
percentile of SS patients (18,000 RFU, added to 500 µl of cell medium). Expression of genes
of interest was normalized to expression of the endogenous gene, GAPDH (n = 3 samples/group,
* P ≤ 0.05, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple
comparison was used to compare treated to untreated cells).

40

Chapter 4: Systemic administration of the CTSS inhibitor (Z-FL-COCHO) given
intraperitoneally reduces LG inflammation and increases tear flow in a mouse model of SS
CTSS is highly increased in SS patient tears and in tears and inflamed LG of male NOD
mice, a murine model of SS. In addition, the results from Chapter 2 and 3 also suggest that the
highly increased CTSS characteristic of SS patient tears may induce pro-inflammatory cytokines
including IL-8, IL-6, TNF-α, and IL-1β, as well as MMP-9 and CTSS itself. These inflammatory
mediators may create a cycle of escalating inflammation on the ocular surface of SS patients. A
possible mechanism that might be involved with these inflammatory inductions is CTSS activation
of PAR-2 in ocular surface. In this Chapter 4, I hypothesized that if elevated CTSS is actively
involved in disease progression in either tear production or in the LG inflammation, then the
inhibition of its activity in these environments would reduce autoimmune dacryoadenitis and
improve tear flow in male NOD mouse which is an animal model of SS.
To test this hypothesis, we used the commercially available CTSS inhibitor, Z-FL-COCHO
(Z-FL), administered by intraperitoneal (i.p.) injection. Z-FL is a synthetic peptide-based CTSS
inhibitor composed of phenylalanine and leucine. Its structure was shown in Figure 17. It exhibits
slow binding and reversible kinetics with 400-fold more selectivity for CTSS (Ki = 0.185 nM)
than for cathepsin B (Ki = 76 nM) (Walker et al., 2000). Z-FL has previously been administered
i.p. in a mouse model for treatment of CD
+
4 T-cell-associated neuropathic pain (Zhang et al., 2014)
and also in an experimental mouse model of liver injury for treatment of natural killer T cells-
associated hepatitis (de Mingo Pulido et al., 2018). In addition, Z-FL has been used as a novel
synergistic reagent to induce cell sensitivity to anti-cancer drugs in vitro and in vivo by inducing
endoplasmic reticulum stress and cell apoptosis (Seo et al., 2017; Seo et al., 2018).
41

Figure 17. The structure of Z-FL-COCHO (Z-FL) with molecular weight 424.5 g/mol
(Walker et al., 2000)

4.1 Identification of i.p. dose of Z-FL
To estimate the Maximum Tolerated Dose (MTD) for Z-FL administered i.p., a dose-
escalation study was conducted with Z-FL dissolved in vehicle (10% DMSO + 40% PEG 300 +
50% sterile PBS) at 0.25, 0.5, 1.0, 2.0 and 4.0 mg/kg body weight. This vehicle was chosen from
several pilot formulations as non-toxic and best able to dissolve the hydrophobic agent; 4.0 mg/kg
was the highest dose that this vehicle could dissolve. Z-FL concentrations at each dose were 140
µM, 290 µM, 580 µM, 1.1 mM and 2.3 mM, respectively, determined using reverse-phase high-
performance liquid chromatography (RP-HPLC). 12 Male BALB/c mice at 14 weeks old were
divided into 6 groups, 2 mice/group, and were injected with volumes of 100-120 µl, depending
upon mouse body weight, every other day for 2 weeks. During this protocol, there was no any
mice that lost more than 15% of their starting body weight, which was the threshold set for toxicity.
At the end of the treatment, no notable differences in body weights nor in the weight or appearance
of spleen, liver or kidney in any treatment group were seen (Figure 18). Because no signs of
toxicity were observed at any dose, the highest evaluated dose of 4 mg/kg was considered as the
MTD, while 1 mg/kg was selected as an intermediate dose for a therapeutic i.p. injection protocol.
42

Figure 18. There were no gross differences in liver, spleen, or kidney weight after Z-FL i.p
injection. Z-FL was injected i.p. at the indicated doses in mg/kg body weight every other day for
2 weeks starting at 14 weeks of age in BALB/c mice. (n = 2 mice/group, data represent mean ±
SD).
4.2 Intraperitoneal Z-FL reduces CTSS activity in tears, LG and spleen lysates in male NOD
mice
Z-FL was administered i.p., every other day for 2 weeks, to 14-15 week male NOD mice,
at which time autoimmune dacryoadenitis is typically established (Li et al., 2010). As NOD male
mice can develop type 1 diabetes with a usual onset between 16-24 weeks of age (Bao et al., 2002),
blood glucose was measured before and after treatment. Mice with blood glucose levels > 250
mg/dl were considered to have developed diabetes during the study and were excluded (Ize-
Ludlow et al., 2011; King, 2012; Morris et al., 2011). No mice in the i.p. cohort had blood glucose
levels > 250 mg/dl at the conclusion of the study, so the number of mice in each of these cohorts
remained 10.
To investigate how Z-FL i.p. administration affected CTSS activity, we examined CTSS
activity in tears, LG and spleen. CTSS activity in tears from mice treated with 4 mg/kg of Z-FL
i.p. was reduced by 3-fold relative to levels in mice treated with 1 mg/kg of Z-FL (P = 0.04) and
by 2.5-fold relative to levels in mice treated with vehicle (P = 0.02) (Figure 19A). For CTSS
activity in LG lysates, 4 mg/kg Z-FL treated mice showed less CTSS activity (2-fold) relative to
43

CTSS activity in the vehicle-treated group (P = 0.046) (Figure 19B). Additionally, 4 mg/kg Z-FL
also significantly reduced CTSS activity in mouse spleen lysates relative to levels in mice treated
with vehicle (3.2- fold, P = 0.003) (Figure 19C). 1 mg/kg Z-FL treatment did not reduce CTSS
activity in tears, LG or spleen. (Figure 19A-C).

Figure 19. Intraperitoneal Z-FL reduces CTSS activity in tears, LG lysates, and spleen
lysates. 14-15 week old male NOD mice were treated every other day for 2 weeks with i.p. Z-FL
at 1, 4 mg/kg body weight. (A) Tears were assayed for CTSS activity, which showed 4 mg/kg Z-
FL significantly reduced activity relative to 1 mg/kg and vehicle (P = 0.04 and P = 0.02
respectively); (B) LG lysates were assayed for CTSS activity, which showed that only Z-FL at 4
mg/kg reduced activity relative to vehicle (P = 0.046); (C) Spleen lysates were assayed for activity,
and only 4 mg/kg reduced activity relative to vehicle (P = 0.003). N = 7 mice/group, data are
represented as mean ± SD; a one-way ANOVA with Tukey’s multiple comparison was used to
compare treatments.
4.3 Intraperitoneal Z-FL decreases LG lymphocytic infiltration and increases stimulated
tear secretion in male NOD mice
To investigate the effect of systemic Z-FL administration on the reduction of LG
inflammation, the percentage of LG area infiltrated by lymphocytes was determined by
hematoxylin and eosin (H&E staining). 4 mg/kg of Z-FL given i.p. reduced LG inflammation by
48% relative to vehicle (P = 0.04), while 1 mg/kg did not reduce LG infiltration compared to
vehicle. (Figure 20A). Representative images of LG cross sections from mice exposed to i.p. are
shown in Figure 20C-E. Additionally, to investigate whether Z-FL administered systemically
44

could improve tear secretion, stimulated tear secretion was measured at study conclusion by adding
topical carbachol to the exposed LG. 4 mg/kg of Z-FL i.p. increased stimulated tear secretion
relative to 1 mg/kg Z-FL and vehicle (P = 0.04 and P = 0.049, respectively), while 1 mg/kg Z-FL
had no effect (Figure 20B).

Figure 20. Intraperitoneal Z-FL decreases lymphocytic infiltration of the LG and increases
stimulated tear secretion. 14-15 week old male NOD mice were treated every other day for 2
weeks with i.p. Z-FL at 1, 4 mg/kg body weight. (A) The percentage of LG area occupied by
lymphocytic infiltration was significantly reduced by 4 mg/kg Z-FL compared to vehicle alone (n
= 10 mice/group, P = 0.04); (B) Carbachol-stimulated tear secretion was assessed, and 4 mg/kg of
Z-FL significantly increased tear production relative to vehicle or 1 mg/kg treatment groups (n =
7 mice/group, P = 0.049, P = 0.04 respectively). Data are represented as mean ± SD, a one-way
ANOVA with Tukey’s multiple comparison was used to compare between treatment groups; (C-
E) Representative hematoxylin/eosin stained section of LG are provided from mice treated with
(C) vehicle; (D) 1 mg/kg Z-FL; (E) 4 mg/kg Z-FL. Scale bar = 400 µm.
45

4.4 Intraperitoneal Z-FL reduces CD3+ T-cell and pan-macrophage abundance in
lymphocytic infiltrates in parallel with reduced MHC II (H2-Ab1) gene expression in LG
Our previous work has shown that the immune cell infiltrates in the LG of male NOD mice
at 18 weeks include a variety of T-cells, macrophages, and B-cells, as well as smaller populations
of other cells such as neutrophilic and eosinophilic granulocytes (Schenke-Layland et al., 2010).
To investigate how Z-FL, administered i.p. affected the major immune cell populations, we
specifically evaluated changes in total T-cells, pan-macrophages and B-cells. Utilizing CD3 as a
T-cell marker, which detects T cells at all stages of development, we measured the density of CD3+
T-cells as the number of CD3+cells/total area of LG lymphocytic infiltration in each treatment.
The density of CD3+ T-cells in LG from mice given 4 mg/kg of Z-FL i.p. was significantly reduced
relative to those in LG from mice treated with vehicle (P = 0.02) (Figure 21A). Representative
images depicting CD3+ immunostaining in LG sections from mice exposed to i.p. are shown in
Figure 21D.
To investigate whether i.p. Z-FL affected the number of pan-macrophages in the
lymphocytic infiltrates, CD68 immunofluorescence was used to detect macrophages in mouse LG
lymphocytic infiltrates and reported as the percentage of CD68+ labelling area normalized to the
total area of lymphocytic infiltrates in each image field. As shown in Figure 21B, 4 mg/kg of i.p.
Z-FL decreased macrophages in the lymphocytic infiltrates relative to 1 mg/kg (P = 0.05) and
vehicle (P = 0.006) respectively. Representative images of the CD68+ macrophages in each
treatment group are shown in Figure 21E. Comparable analysis of the effects of i.p. Z-FL using
an antibody to B220, a pan-B-cell marker, showed no significant differences in B cell in
lymphocytic infiltrates among any mouse treatment groups (Figure 22A). Representative images
of the B220+ cells in each treatment group are shown in Figure 22B.
46

CTSS cleaves Ii, which is required for MHC II to present antigen to T-cells (Riese et al.,
1996). MHC II is typically expressed in professional antigen presenting cells such as macrophages
and dendritic cells (DC), as well as B lymphocytes (Mantegazza et al., 2013). H2-Ab1 gene
expression in LG of 4 mg/kg Z-FL treated mice was significantly reduced relative to LG treated
with 1 mg/kg Z-FL and vehicle (by 40%, P = 0.007 and by 35%, P = 0.03, respectively) (Figure
21C). This reduction of H2-Ab1 gene expression paralleled the reduction in macrophage content
within the LG with i.p. Z-FL treatments, but not the reduction in total lymphocytic infiltrates
(Figure 20) nor T-cell infiltrates (Figure 21A).
4.5 Intraperitoneal Z-FL does not affect the expression of other inflammation-associated
genes in LG of male NOD mice.
Our previous work found that not only MHC II but also CTSS, TNF-α, and IFN-  were
significantly increased in NOD mouse LG during development of autoimmune dacryoadenitis (Li
et al., 2010; Meng et al., 2017). In addition, according to Chapter 2, CTSS increases TNF-α and
PAR-2 gene and protein expression in cultured human corneal epithelial cells, suggesting that its
activity can initiate ocular surface inflammation. We analyzed whether these additional CTSS-
associated genes were affected in LG in mice treated with Z-FL i.p. The gene expression results
showed that expression of other genes linked to CTSS activity including CTSS itself, TNF-α, IFN-
, and PAR-2 were unchanged by i.p. Z-FL treatment at either dose (Figure 23A-D).

47

Figure 21. Intraperitoneal Z-FL reduces CD3+ T-cells and pan-macrophage abundance in
lymphocytic infiltrates in parallel with reduced MHC II (H2-Ab1) gene expression in LG. 14-
15 week old male NOD mice were treated every other day for 2 weeks with i.p. Z-FL at 1, 4 mg/kg
body weight. (A) LG were assessed for density of CD3+ T-cells in areas of lymphocytic
infiltration, and the group treated with 4 mg/kg Z-FL had significantly lower than vehicle alone (n
= 3 mice/group, P = 0.02); (B) LG were assessed for the percentage of CD68+ cells in areas of
lymphocytic infiltration, and the group treated with 4 mg/kg Z-FL had significantly fewer pan-
macrophages compared to vehicle alone or 1 mg/kg of Z-FL (n = 3 mice/group, P = 0.006, P =
0.05 respectively); (C) LG homogenates were assessed for H2-Ab1 gene expression normalized to
endogenous Gapdh, and only 4 mg/kg Z-FL significantly reduced expression relative to vehicle or
1 mg/kg of Z-FL (n= 7 mice/group, P = 0.03, P = 0.007 respectively). Data represent mean ± SD,
a one-way ANOVA with Tukey’s multiple comparison was used to compare between mouse
groups. (D) Representative images of CD3+ T-cell immunostaining in areas of lymphocytic
infiltration from treatment groups in (A). CD3 (Green) labels total T-cells while DAPI labels nuclei
(blue); (E) Representative LG images of CD68+ pan-macrophage immunostaining in areas of
lymphocytic infiltration from treatment groups in (B). CD68 (Green) labels pan-macrophages and
DAPI (Blue) labels nuclei. Scale bar = 10 µm.
48

Figure 22. Intraperitoneal Z-FL does not alter B220+ positive B-cell abundance within the
LG. 14-15 week old male NOD mice were treated every other day for 2 weeks with i.p. Z-FL at
1, 4 mg/kg body weight. (A) LG were assessed for density of B220+ positive B-cells in areas of
lymphocytic infiltration (number of cells/mm
2
). Both doses of systemic Z-FL treatment showed
no difference from vehicle.; (B) Representative images of B220+ B cell immunostaining in areas
of lymphocytic infiltration from treatments in (A). B220 (Red) is used as a pan-B-cell marker,
while DAPI (blue) labels nuclei. Scale bar = 10 µm. (n = 3 mice/group, data represent mean ± SD,
a one-way ANOVA was used to compare between mouse groups.
49

Figure 23. Intraperitoneal Z-FL does not affect Ctss, Tnf, Ifng, and F2rl1 gene expression in
LG of male NOD mice. 14-15 week old male NOD mice were treated every other day for 2 weeks
with i.p. Z-FL at 1, 4 mg/kg body weight. Gene expression levels of (A) Ctss; (B) Tnf; (C) Ifng;
(D) F2rl1. Expression of genes of interest was normalized to expression of the endogenous gene,
Gapdh. N = 7 mice/group, data represent mean ± SD, and a one-way ANOVA was used to compare
between mouse groups.
4.6 Intraperitoneal Z-FL does not elicit gross systemic toxicity at the dose evaluated
To confirm that i.p. administration of the CTSS inhibitor, Z-FL, did not cause toxicity, the
spleen, liver, and kidney of treated mice were evaluated for tissue toxicity by a trained pathologist
following all treatments. The histopathological report on kidney, spleen, and liver of male NOD
mice after 2 weeks of Z-FL i.p. injection was shown in Table 3. 6 different mice showed either
50

kidney or liver findings when given Z-FL i.p.. Only 1 mouse from the 4 mg/kg Z-FL treatment
group showed both kidney and liver findings. Fisher’s exact test using SPSS (IBM, Armonk, NY)
was used to determine whether there was a statistical association between the kidney findings vs
mouse treatment groups or liver findings vs. mouse treatment groups. The data showed that there
was not any statistical association between kidney or liver findings vs. mouse treatment groups.
Also, the mild diffuse vacuolization of the tubular epithelial cells that was found in mice exposed
to 1 mg/kg and 4 mg/kg of Z-FL given i.p. is normally present in the tubules of male mice (Seely
and Brix, 2019). There was no notable difference in the number and/or size of vacuoles compared
to vehicle-treated mice suggesting that drug does not elicit kidney abnormalities. Also, the focal
cytoplasmic swelling and vacuolization noted in one vehicle-treated mouse, two mice treated with
1 mg/kg Z-FL i.p., and three mice treated with 4 mg/kg Z-FL i.p. reflect nonspecific changes in
liver cells reflective of factors such as ischemia, or changes in the diet or metabolic condition of
the mice (Thoolen et al., 2010).

51

Table 3: Histopathological report on kidney, spleen, and liver of male NOD mice after 2 weeks of Z-FL i.p. injection

Organ Vehicle 1 mg/kg of Z-FL 4 mg/kg of Z-FL
Kidney H&E stained sections displayed kidney
with a well-defined cortex and medulla.
Numerous glomeruli were identified in the
cortex. The renal medulla with loops of
Henle and collecting ducts of Bellini were
identified. No histopathologic abnormality
was identified in the kidney.

H&E stained sections displayed kidney with a
well-defined cortex and medulla. Numerous
glomeruli were identified in the cortex. The
renal medulla with loops of Henle and
collecting ducts of Bellini were identified. One
case showed mild diffuse vacuolization of the
tubular epithelial cells. No histopathologic
abnormality was otherwise identified in the
kidney.

H&E stained sections displayed kidney with
a well-defined cortex and medulla.
Numerous glomeruli were identified in the
cortex. The renal medulla with loops of
Henle and collecting ducts of Bellini were
identified. One case showed mild diffuse
vacuolization of the tubular epithelial cells.
No histopathologic abnormality was
otherwise identified in the kidney.
Spleen H&E stained sections displayed white and
red pulp of the parenchyma and septa are
seen. Germinal centers and surrounding
lymphocytes are identified in the white
pulp area. Reticular fibers, reticulocytes,
macrophages, hematopoietic cells and
venous sinuses are seen in the red pulp
area. These structures are essentially
unremarkable.

H&E stained sections displayed white and red
pulp of the parenchyma and septa are seen.
Germinal centers and surrounding lymphocytes
are identified in the white pulp area. Reticular
fibers, reticulocytes, macrophages,
hematopoietic cells and venous sinuses are
seen in the red pulp area. These structures are
essentially unremarkable
H&E stained sections displayed white and
red pulp of the parenchyma and septa are
seen. Germinal centers and surrounding
lymphocytes are identified in the white pulp
area. Reticular fibers, reticulocytes,
macrophages, hematopoietic cells and
venous sinuses are seen in the red pulp area.
These structures are essentially unremarkable

Liver The H&E sections displayed liver
parenchyma with hepatocytes and
sinusoids with Kupffer cells. Several
hepatocytes were binucleated. Focal mild
(1 case) cytoplasmic swelling and
vacuolization was noted. Morphology of
hepatocytes is otherwise unremarkable.
Portal triads with bile duct, hepatic artery
and portal vein were identified and were
essentially unremarkable.

The H&E sections displayed liver parenchyma
with hepatocytes and sinusoids with Kupffer
cells. Several hepatocytes were binucleated.
Focal mild (1 case) to moderate (1 case)
cytoplasmic swelling and vacuolization was
noted. Morphology of hepatocytes is otherwise
unremarkable. Portal triads with bile duct,
hepatic artery and portal vein were identified
and were essentially unremarkable.

The H&E sections displayed liver
parenchyma with hepatocytes and sinusoids
with Kupffer cells. Several hepatocytes were
binucleated. Focal mild (2 cases) to moderate
(1 case) cytoplasmic swelling and
vacuolization was noted. Morphology of
hepatocytes is otherwise unremarkable.
Portal triads with bile duct, hepatic artery
and portal vein were identified and were
essentially unremarkable.

52

4.7 Comparison between Z-FL (Millipore) and Z-FL-hydrate (Adooq Bioscience)
According to i.p. Z-FL results, I found that systemic Z-FL i.p. injection, every other day,
for 2 weeks significantly reduced CTSS activity in tears, LG and spleen, significantly reduced total
lymphocytic infiltration into LG and T cell and pan-macrophage abundance within lymphocytic
infiltrates, and significantly increased stimulated tear secretion. However, as this was the first
cohort study that I performed, I did not secure measurements on some endpoints following i.p. Z-
FL on basal tear secretion, corneal integrity, and systemic autoimmunity in male NOD mice.
Therefore, I repeated the systemic Z-FL study again to validate initial findings in a second cohort
and to obtain these additional measures. Unfortunately, the original supplier (Millipore,
Burlington, MA) of Z-FL (MW=424.5 g/mol) discontinued manufacture of the product after my
first study. According to Z-FL’s chemical structure (Figure 17), it has 2 chiral centers, which
result from the linkage of two L-amino acids. Z-FL includes a carboxy-terminal aldehyde, which
is easily hydrated. We could not find another supplier of Z-FL; however, we were able to obtain
Z-FL-hydrate (MW=442.5 g/mol) from a second supplier (Adooq Bioscience, Irvine, CA, Figure
24).

Figure 24. The structure of Z-FL-hydrate (from Adooq Bioscience, molecular weight = 442.5
g/mol). The increased molecular weight reflects a hydrate of the carboxyterminal aldehyde
(Blue square)
53

We performed quality-control RP-HPLC, which determined that both Z-FL and Z-FL-
hydrate do in fact elute at different retention times (Figure 25). We collected the main peaks from
each HPLC eluate and confirmed that both are potent inhibitors of CTSS with 96.51% and 96.26%
inhibition from Z-FL and Z-FL-hydrate, respectively.

Figure 25. The Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) from
Z-FL (Millipore) vs Z-FL-hydrate (Adooq Bioscience). To confirm relative equivalence of these
two forms of Z-FL, Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) was
used to evaluate both in comparison to vehicle alone (25% DMSO + 75% Acetonitrile). Z-FL
showed a retention time at 8.03 min, while Z-FL-hydrate showed retention time at 10.32 min. Both
peaks indicated by the black arrow were collected and confirmed to have an inhibitory effect on
12.5 nM human recombinant CTSS with 96.51% and 96.26% from Z-FL and Z-FL-hydrate,
respectively

Also, inhibition studies against recombinant human CTSS suggested that both forms had
equivalence of the half maximal inhibitory concentration (IC50) with values of 172.5 ± 32.7 nM
and 261.9 ± 59.3 nM for the batches of Z-FL and Z-FL-hydrate respectively (Figure 26A).
However, in a mouse spleen lysate at a dose when Z-FL was able to effectively inhibit most CTSS,
54

Z-FL-hydrate was 3.5-less effective (Figure 26B). Based on this information, the i.p. repeat mouse
study of the Z-FL-hydrate was performed at an increased dose of 15 mg/kg to obtain roughly an
equipotent inhibitory CTSS effect relative to Z-FL i.p.

Figure 26. The half maximal inhibitory concentration (IC50) and the CTSS inhibitory
potential in mouse spleen lysate of Z-FL and Z-FL-hydrate. (A) The IC
50
of both forms of Z-
FL toward human recombinant CTSS. The IC 50 were generally equivalent, with values of 172.5 ±
32.7 nM and 261.9 ± 59.3 nM for the batches of Z-FL and Z-FL-hydrate respectively; (B) The
CTSS inhibitory potential of Z-FL and Z-FL-hydrate at 20 µM in spleen lysates obtained from 16-
week male NOD mice. Z-FL-hydrate showed less potent CTSS inhibition in spleen lysates
compared to Z-FL (n = 4, P = 0.0007, data represent mean ± SD, a two-tailed, unpaired Student’s
t-test was used to compare between groups).

4.8 Intraperitoneal Z-FL-hydrate reduces CTSS activity in LG and spleen lysates, but not in
tears in male NOD mice
At a dose of 15 mg/kg of Z-FL-hydrate, no mouse showed significant weight changes of
body weight and internal organs (Figure 27A-D) or evidence of any gross systemic toxicity (Table
4). With this Z-FL-hydrate experiment, I replicated many measurements that performed in the
previous Z-FL i.p. studies. Just like Z-FL, Z-FL-hydrate reduced CTSS activity relative to vehicle
alone in LG and spleen lysates (Figure 28). A trend to decreased tear CTSS was seen, but this was
not significant. This difference in the magnitude of the effect of Z-FL-hydrate relative to the effect
55

seen with Z-FL (Millipore) may reflect its slightly reduced inhibitory potential and, alternatively,
variations in the degree of SS-like symptoms reflected by the higher absolute CTSS activity levels
in tears of this particular cohort of NOD mice.

Figure 27. No significant weight changes in body weight, liver, spleen, and kidneys in male
NOD mice treated i.p. with 15 mg/kg Z-FL-hydrate or vehicle were detected. (A) Body
weight; (B) Liver weight; (C) Spleen weight; (D) Kidney weight. N = 13 mice/group, data
represent mean ± SD, a two-tailed, paired Student’s t-test was used to compare body weight at
pretreatment and after 2 weeks of treatment in each mouse group in (A), and a two-tailed, unpaired
Student’s t-test was used to compare internal weight organs between mouse groups in (B-D).

56

Figure 28. Intraperitoneal Z-FL-hydrate reduces CTSS activity in LG and spleen lysates,
but not in tears. 14-15 week old male NOD mice were treated every other day for 2 weeks with
i.p. Z-FL-hydrate at 15 mg/kg body weight. CTSS activity in tears (n = 7 and 9 mice in vehicle
and drug groups, respectively), LG (n = 8 and 9 mice in vehicle and drug groups respectively, P
= 0.04) and spleen lysate (n = 8 and 9 mice in vehicle and drug group, respectively, P = 0.03).
Data represent mean ± SD, a two-tailed, unpaired Student’s t-test was used to compare between
mouse groups

57

Table 4. The Histopathological report in kidney, spleen, and liver of male NOD mice after 2 weeks of 15 mg/kg Z-FL-hydrate
i.p. injection
Organ 15 mg/kg of Z-FL Vehicle
Kidney H&E stained sections displayed kidney with a well-defined cortex
and medulla. Numerous glomeruli were identified in the cortex. The
renal medulla with loops of Henle and collecting ducts of Bellini
were identified. One case showed mild focal vacuolization of the
tubular epithelial cell. No histopathologic abnormality was otherwise
identified in the kidney.

H&E stained sections displayed kidney with a well-defined cortex and
medulla. Numerous glomeruli were identified in the cortex. The renal
medulla with loops of Henle and collecting ducts of Bellini were
identified. One case showed mild focal vacuolization of the tubular
epithelial cells and focal lymphocytes infiltration. No histopathologic
abnormality was otherwise identified in the kidney
Spleen H&E stained sections displayed white and red pulp of the
parenchyma and septa are seen. Germinal centers and surrounding
lymphocytes are identified in the white pulp area. Reticular fibers,
reticulocytes, macrophages, hematopoietic cells and venous sinuses
are seen in the red pulp area. There structures are essentially
unremarkable.

H&E stained sections displayed white and red pulp of the parenchyma
and septa are seen. Germinal centers and surrounding lymphocytes are
identified in the white pulp area. Reticular fibers, reticulocytes,
macrophages, hematopoietic cells and venous sinuses are seen in the red
pulp area. There structures are essentially unremarkable.

Liver The H&E sections displayed liver parenchyma with hepatocytes and
sinusoids with Kupffer cells. Several hepatocytes were binucleated.
Focal mild (2 cases) to moderate (2 case) cytoplasmic swelling and
vacuolization was noted. Morphology of hepatocytes is otherwise
unremarkable. Portal triads with bile duct, hepatic artery and portal
vein were identified and were essentially unremarkable.

The H&E sections displayed liver parenchyma with hepatocytes and
sinusoids with Kupffer cells. Several hepatocytes were binucleated.
Focal mild (3 cases) to moderate (2 case) cytoplasmic swelling and
vacuolization was noted. Morphology of hepatocytes is otherwise
unremarkable. Portal triads with bile duct, hepatic artery and portal
vein were identified and were essentially unremarkable.

58

4.9 Intraperitoneal Z-FL-hydrate decreases LG lymphocytic infiltration and increases
stimulated tear secretion in male NOD mice
Like Z-FL, 15 mg/kg of Z-FL-hydrate also significantly reduced LG lymphocytic
infiltration area compared to vehicle (P = 0.04) (Figure 29A). Representative images of LG cross
sections from mice exposed to i.p. are shown in Figure 29C-D. Additionally, 15 mg/kg of Z-FL-
hydrate increased stimulated tear secretion relative to vehicle (P = 0.011) (Figure 29B).

Figure 29. Intraperitoneal Z-FL-hydrate decreases lymphocytic infiltration of the LG and
increases stimulated tear secretion. 14-15 week old male NOD mice were treated every other
day for 2 weeks with i.p. Z-FL-hydrate at 15 mg/kg body weight. (A) The percentage of LG area
occupied by lymphocytic infiltration in male NOD mice treated i.p. with 15 mg/kg Z-FL-hydrate
or vehicle (n = 13 mice/group, P = 0.04); (B) Stimulated tear secretion in male NOD mice treated
with 15 mg/kg of Z-FL-hydrate i.p. relative to those treated with vehicle (n = 7 and 9 mice in
vehicle and drug groups, respectively, P = 0.011); (C-D) Representative hematoxylin/eosin stained
section of LG are provided from mice treated with (C) vehicle; (D) 15 mg/kg Z-FL-hydrate. Scale
bar = 400 µm. Data represent mean ± SD, a two-tailed, unpaired Student’s t-test was used to
compare between mouse groups
59

4.10 Intraperitoneal Z-FL-hydrate improves basal tear secretion, but not corneal integrity
in male NOD mice
The improvement of tear secretion in Z-FL-hydrate treated mice was confirmed by basal
tear secretion measured by a thread test. 15 mg/kg of Z-FL-hydrate treated mice showed a higher
basal tear secretion after 2 weeks of Z-FL treatment. On the contrary, there was not statistically
significant difference in basal tear secretion in vehicle-treated mice (Figure 30A). However, Z-
FL-hydrate did not improve ocular surface integrity as measured by corneal fluorescein staining
(Figure 30B).

Figure 30. Intraperitoneal Z-FL-hydrate improves basal tear secretion, but not corneal
integrity in male NOD mice. 14-15 week old male NOD mice were treated every other day for 2
weeks with i.p. Z-FL-hydrate at 15 mg/kg body weight. (A) Basal tear secretion measured by
thread test at pretreatment and 2 weeks after treatment of male NOD mice with 15 mg/kg Z-FL-
hydrate i.p relative to vehicle (n = 13 mice/group, P = 0.03 between pretreatment vs 2 weeks in
drug group); (B) Corneal fluorescein staining at pretreatment and 2 weeks after treatment of 15
mg/kg Z-FL-hydrate i.p relative to vehicle (n = 13 mice/group). Data represent mean ± SD, a two-
tailed, paired Student’s t-test was used to compare before and after treatments in each group.

60

4.11 Intraperitoneal Z-FL-hydrate decreases Ifng gene expression in LG in male NOD mice
Z-FL-hydrate showed the reduction of Ifng gene expression in LG relative to vehicle
(Figure 31A), but no changes in other genes related to CTSS including H2-Ab1, Ctss, Tnf, and
F2rl1 gene expression (Figure 31B-E). The difference in effect with Z-FL might be related to the
potency of CTSS inhibition in the LG or to the extent of disease developments between these two
mouse cohorts.

Figure 31. Intraperitoneal Z-FL-hydrate decreases Ifng gene expression in LG in male NOD
mice. 14-15 week old male NOD mice were treated every other day for 2 weeks with i.p. Z-FL-
hydrate at 15 mg/kg body weight. Gene expression levels of (A) Ifng; (B) H2-Ab1; (C) Ctss; (D)
Tnf; (E) F2rl1. Expression of genes of interest was normalized to expression of the endogenous
gene, Gapdh. N = 6 mice/group, data represent mean ± SD, and a two-tailed, unpaired Student’s
t-test was used to compare between mouse groups
61

Chapter 5: The effect of topical administration of the CTSS inhibitor (Z-FL-COCHO) on
LG inflammation in a mouse model of SS
In Chapter 4, I showed that Z-FL and an equipotent dose of Z-FL-hydrate given i.p. can
reduce LG inflammation and increase tear flow in male NOD mice. In this chapter, I focus on
using Z-FL (Millipore) as a topical eyedrop treatment in 14-15 week old male NOD mice. To
mimic the time course required for topical regimens such as Restasis
®
and rapamycin eyedrops,
twice a day administration over 6 weeks of treatment was selected for topical administration (Shah
et al., 2017). At the conclusion of this study, when Z-FL became unavailable I was unable to
repeat topical administration using Z-FL-hydrate from Adooq Bioscience as in Chapter 4, because
the reduced potency of the Z-FL-hydrate limited its formulation at an equipotent CTSS inhibitory
dose to Z-FL (Millipore) in PBS. Therefore, the results in this Chapter were performed only with
Z-FL from Millipore.
5.1 Identification of topical doses of Z-FL in vitro
To provide an initial estimate of the dose of topical Z-FL that would not elicit corneal
epithelial cell toxicity, cell viability and cytotoxicity were assessed in human corneal epithelial
cell line (HCE-T cells) using 20, 100, and 200 µM of Z-FL in Keratinocyte-SFM (KSFM) medium.
Cells treated with KSFM only served as a positive control for live cells, while cells treated by 0.1%
saponin served as a positive control for dead cells. Cell viability measured as green Calcein AM
fluorescence intensity is shown in Figure 32A. All intensities of green Calcein AM were
normalized to that seen in only KSFM treated cells. The cell viability showed no significant
differences at any dose of Z-FL relative to cells treated with only KSFM. On the contrary, there
was a significant difference between saponin-treated cells and all doses of Z-FL treatment
suggesting that Z-FL treatment did not affect cell viability. Cell death measured by red Ethidium
62

Homodimer-1 (EthD-1) fluorescence intensity is shown in Figure 32B. All intensities were
normalized to that seen for saponin-treated cells. While saponin elicited marked cell death, there
was a statistically significant difference between Z-FL-treated cells at all doses and saponin-treated
cells, while, there was no significant difference between KSFM-treated cells and all doses of Z-
FL treatment, suggesting that Z-FL did not cause cell death.

Figure 32. Z-FL does not reduce cell viability or cause cell death in vitro in human corneal
epithelial (HCE-T) cells. (A) The percentage of cell viability at all doses tested of Z-FL is plotted
relative to that seen with KSFM treatment (cultured medium) and saponin treatment. P = 0.04 (20
µM vs saponin), P = 0.02 (100 µM vs saponin), P = 0.046 (200 µM vs saponin), and P = 0.007
(KSFM vs saponin); (B) The percentage of cell death at all doses of Z-FL tested is plotted relative
to saponin treatment, and KSFM treatment (cultured medium). P = 0.002 (20 µM vs saponin), P
= 0.002 (100 µM vs saponin), P = 0.004 (200 µM vs saponin), and P = 0.005 (KSFM vs saponin).
Cells treated with KSFM were positive controls for live cells, while cells treated with saponin were
positive controls for dead cells. N = 3 wells/group, data shown as mean ± SD, and a one-way
ANOVA with Tukey’s multiple comparison was used to compare between treatment groups).
5.2 Identification of topical doses of Z-FL in vivo
To estimate the optimal dose of Z-FL eye drops that were not associated with gross toxicity,
a 1-week pilot eye drop study was performed in 14-week male NOD mice. These same doses of
20, 100, and 200 µM Z-FL were administered to mouse eyes twice daily in sterile PBS every day
for 1 week (3 mice/group). Basal tear secretion measurements and corneal fluorescein staining
were measured before and after treatment and the results showed no changes among any treatment
63

groups at any doses of Z-FL relative to vehicle (Figure 33A-B). Thus, 200 µM of Z-FL in sterile
PBS, the highest dose in this pilot study, appeared appropriate for therapeutic Z-FL eye drop
administration in male NOD mice.

Figure 33. No differences in basal tear secretion or corneal surface integrity were detected
after 1 week of topical Z-FL. 14-15 week old male NOD mice were treated twice a day for 1
week with 20, 100, and 200 µM of topical Z-FL. (A) Basal tear secretion was measured by thread
test before and after 1 week of treatment; (B) Corneal fluorescein staining before and after 1 week
of topical Z-FL given to male NOD mice as in (A). (n = 3 mice/group, data represent mean ± SD
and a two-tailed, paired Student’s t-test was used to compare before and after treatments in each
group).
5.3 Topical Z-FL reduces CTSS activity in tears, not in LG and spleen lysates in male NOD
mice.
Blood glucose was measured at pretreatment, 3 weeks, and 6 weeks after treatment. Mice
that had blood glucose levels more than 250 mg/dl at the conclusion of the study were excluded
because of considered as diabetic (Ize-Ludlow et al., 2011; King, 2012; Morris et al., 2011). At
the end of the treatment, 2 mice from the vehicle (PBS) group and 1 mouse from the Z-FL group,
were excluded at the conclusion of treatments leading to cohorts of 13 mice for the vehicle group
and 14 mice for 200 µM Z-FL group.
Inhibition of tear CTSS activity by topical Z-FL also showed a reduction of CTSS activity
by 2.4-fold relative to mice treated with vehicle (Figure 34A), but showed no inhibitory potential
64

against CTSS levels in either LG or spleen (Figure 34B-C). These findings suggest that 200 µM
of topical Z-FL affects only local CTSS activity on the ocular surface.

Figure 34. Topical instillation of Z-FL reduces CTSS activity locally in tears, but not in LG
or spleen lysates. 14-15 week old male NOD mice were treated twice a day for 6 weeks with 200
µM Z-FL. (A) Tear samples were obtained, CTSS activity was assessed, and Z-FL-treatment
significantly decreased activity relative to the vehicle alone (P = 0.04); (B) LG lysates were
prepared, CTSS activity was assessed, and no differences were observed upon Z-FL-treatment;
(C) Spleen lysates were prepared, CTSS activity was assessed, and no differences were observed
upon Z-FL treatment. N = 8 mice/group, data are represented as mean ± SD, and a two-tailed,
unpaired Student’s t-test was used to compare between mouse groups.
5.4 Topical Z-FL does not affect the LG lymphocytic infiltration and improvement of
stimulated tear secretion in male NOD mice.
There was no effect on LG inflammation measured by mice treated with 200 µM of topical
Z-FL-treated compared to vehicle (Figure 35A). Representative images of LG cross sections from
mice exposed to topical Z-FL treatments are shown in Figure 35C-D. Also, no effect on stimulated
tear secretion in mice treated with topical Z-FL eye drop were seen as shown in Figure 35B.

65

Figure 35. Topical instillation of Z-FL does not affect LG lymphocytic infiltration and
stimulated tear secretion. 14-15 week old male NOD mice were treated twice a day for 6 weeks
with 200 µM Z-FL. (A) LG were obtained and assessed for area occupied by lymphocytic
infiltration. Topical treatment with Z-FL was no different from vehicle. (n = 13, 14 for vehicle,
drug groups respectively); (B) Carbachol-stimulated tear secretion was assessed and topical
treatment with Z-FL was no different from vehicle (n = 8 mice/group); (C-D) Representative
hematoxylin/eosin stained section of LG are provided from mice treated with (C) vehicle; (D) 200
µM. Scale bar = 400 µm, data represent mean ± SD, a two-tailed, unpaired Student’s t-test was
used to compare between treatment groups.
5.5 topical Z-FL does not affect the CD3+ T-cell population in areas of LG lymphocytic
infiltration.
In contrast to systemic Z-FL, topical Z-FL did not reduce the density of CD3+ T-cells in
LG lymphocytic infiltrates relative to vehicle (Figure 36A). Representative images depicting
CD3+ T cell immunostaining in LG sections from mice exposed to topical Z-FL treatments are
shown in Figure 36B.
66

Figure 36. Topical instillation of Z-FL does not affect CD3+ T-cells within areas of
lymphocytic infiltration in LG. 14-15 week old male NOD mice were treated twice a day for 6
weeks with 200 µM Z-FL. (A) LG were assessed for density of CD3+ positive T-cells in areas of
lymphocytic infiltration (number of cells/mm
2
). Topical treatment with Z-FL was no different from
vehicle. (B) Representative images of CD3+ positive T cell immunostaining in areas of
lymphocytic infiltration from treatment groups in (A). CD3+ (Green) is used as total T-cells
marker, while DAPI (blue) labels nuclei. N = 3 mice/group, Scale bar = 10 µm, data represent
mean ± SD and a two-tailed, unpaired Student’s t-test was used to compare between treatment
groups.
5.6 Topical Z-FL reduces macrophage abundance in lymphocytic infiltrates in parallel with
reduced MHC II (H2-Ab1) gene expression in LG.
Gene expression results revealed a 70% reduction of H2-Ab1 gene expression in LG from
Z-FL topically-treated mice compared to vehicle (Figure 37A). Also, 200 µM of topical Z-FL
67

reduced CD68+ pan-macrophages and DC relative to vehicle (Figure 37B, representative images
in Figure 37C). These results support the hypothesis that reduced macrophage composition of
lymphocytic infiltrates may be directly responsible for reduced MHC II expression.

Figure 37. Topical instillation of Z-FL reduces expression of MHC II (H2-Ab1) in LG and
pan-macrophage abundance in LG lymphocytic infiltrates. 14-15 week old male NOD mice
were treated twice a day for 6 weeks with 200 µM Z-FL. (A) LG lysates were prepared and
assessed for H2-Ab1 gene expression. Treatment with Z-FL significantly reduced gene expression
relative to vehicle (n = 7 /group, P = 0.04). Expression of H2-Ab1 was normalized to endogenous
Gapdh; (B) LG were obtained, sectioned, assessed by immunofluorescence for CD68, and
quantified. The percentage of CD68+ cells in areas of lymphocytic infiltration were significantly
lower upon treatment with Z-FL compared to vehicle alone (n = 3 /groups, P = 0.04); (C)
Representative LG images of CD68+ pan-macrophage immunostaining in areas of lymphocytic
infiltration quantified in (B). CD68 (Green) labels pan-macrophages and DAPI (Blue) labels
nuclei. Scale bar = 10 µm. Data represent mean ± SD. A two-tailed, unpaired Student’s t-test was
used to compare treatments.
68

5.7 Topical Z-FL does not affect B220+ positive B-cell abundance within the LG lymphocytic
infiltrates
Comparable analysis of the effects of i.p. Z-FL, an antibody to B220 was used to detect a
pan-B-cell marker in LG lymphocytic infiltration. However, no significant differences in B cells
in lymphocytic infiltrates among treatment groups were seen (Figure 38A). Representative images
of the B220+ cells in each group are shown in Figure 38B.

Figure 38. Topical instillation of Z-FL does not alter B220+ positive B-cell abundance within
the LG. 14-15 week old male NOD mice were treated twice a day for 6 weeks with 200 µM Z-
FL. (A) LG were assessed for density of B220+ positive B-cells in areas of lymphocytic infiltration
(number of cells/mm
2
). Topical treatment with Z-FL was no different from vehicle.; (B)
Representative images of B220+ B cell immunostaining in areas of lymphocytic infiltration from
treatments in (A). B220 (Red) is used as a pan-B-cell marker, while DAPI (blue) labels nuclei.
Scale bar = 10 µm. N = 3 mice/group, data represent mean ± SD, a two-tailed, unpaired Student’s
t-test was used to compare between treatment groups.
69

5.8 Topical Z-FL significantly reduces gene expression of CTSS, TNF- α, IFN- , and PAR-2
in LG.
I analyzed whether other CTSS-associated inflammatory genes were affected in LG in mice
treated with topical Z-FL. Exposure of male NOD mice to Z-FL topical eye drops for 6 weeks
significantly decreased LG gene expression of Ctss, Tnf, Ifng, and F2rl1 relative to mice treated
with vehicle (Figure 39A-D).

Figure 39. Topical instillation of Z-FL significantly reduces gene expression of Ctss, Tnf, Ifng,
and F2rl1. 14-15 week old male NOD mice were treated twice a day for 6 weeks with 200 µM Z-
FL. LG lysates were obtained and assayed for gene expression levels of (A) Ctss (P = 0.01); (B)
Tnf (P = 0.01); (C) Ifng (P = 0.03); (D) F2rl1 (P = 0.02). Expression of genes of interest was
normalized to expression of the endogenous gene, Gapdh. N = 7 mice/group, data represent mean
± SD. A two-tailed, unpaired Student’s t-test was used to compare between mouse groups.

70

5.9 Topical Z-FL attenuates the reduction of basal tear secretion associated with disease
progression but has no measurable effect on corneal surface integrity in male NOD mice.
To investigate whether topical Z-FL improved basal tear secretion and ocular surface
integrity, a thread test and corneal fluorescein staining were performed at the beginning, middle,
and the end of treatments. The mice given Z-FL eye drops did not show a significant change in
basal tear secretion across this time period. On the contrary, mice treated with vehicle showed a
reduction in basal tear secretion after 6 weeks of treatment (Figure 40A). These findings suggest
that topical Z-FL may suppress the reduction of basal tear secretion associated with disease
progression that otherwise continues to develop over the 6-week treatment period in the vehicle-
treated NOD mice. Z-FL eye drops did not affect the corneal staining score across this time period
(Figure 40B).

Figure 40. Topical instillation of Z-FL attenuates the reduction of basal tear secretion but
has no measurable effect on corneal surface integrity associated with disease progression.
14-15 week old male NOD mice were treated twice a day for 6 weeks with 200 µM Z-FL. (A)
Basal tear secretion measured by thread test at pretreatment, 3 weeks, and 6 weeks after treatment
was assessed. Vehicle showed the reduction of basal tear secretion after 6-week of treatment (P =
0.02), while topical treatment with Z-FL attenuated the reduction of tear secretion; (B) Corneal
fluorescein staining at pretreatment, 3 weeks, and 6 weeks after treatment was assessed. Topical
71

treatment with Z-FL was no different from vehicle (n = 14 mice for Z-FL and 13 mice for vehicle,
data represent mean ± SD, a repeated measures ANOVA with the Tukey’s multiple comparison
was used to compare within the same treatment group at different time points, and a two-tailed,
unpaired Student’s t-test was used to compare 2 different mouse groups at the same time points).
5.10 Topical Z-FL does not elicit gross systemic toxicity at the dose evaluated
No significant systemic changes were observed in the histological appearance of spleen,
liver, and kidneys from mice treated with 200 µM of Z-FL topical eye drops relative to vehicle
(Table 5). However, 1 mouse treated with topical Z-FL eye drops showed cytoplasmic swelling
and vacuolization and lymphocytic infiltration in the liver, a finding typical of primary SS patients
(Matsumoto et al., 2005).

72

Table 5: Histopathological report of kidney, spleen, and liver of male NOD mice after 6 weeks of topical Z-FL eyedrop
treatment

Organ Vehicle 200 µM of Z-FL
Kidney H&E stained sections displayed kidney with a well-defined cortex
and medulla. Numerous glomeruli were identified in the cortex.
The renal medulla with loops of Henle and collecting ducts of
Bellini were identified. No histopathologic abnormality was
otherwise identified in the kidney.

H&E stained sections displayed kidney with a well-defined cortex and
medulla. Numerous glomeruli were identified in the cortex. The renal
medulla with loops of Henle and collecting ducts of Bellini were
identified. No histopathologic abnormality was otherwise identified in
the kidney.
Spleen H&E stained sections displayed white and red pulp of the
parenchyma and septa are seen. Germinal centres and surrounding
lymphocytes are identified in the white pulp area. Reticular fibres,
reticulocytes, macrophages, hematopoietic cells and venous sinuses
are seen in the red pulp area. These structures are essentially
unremarkable.

H&E stained sections displayed white and red pulp of the parenchyma
and septa are seen. Germinal centres and surrounding lymphocytes are
identified in the white pulp area. Reticular fibres, reticulocytes,
macrophages, hematopoietic cells and venous sinuses are seen in the
red pulp area. There structures are essentially unremarkable.
Liver The H&E sections displayed liver parenchyma with hepatocytes
and sinusoids with Kupffer cells. Several hepatocytes were
binucleated. Morphology of hepatocytes is generally unremarkable.
Portal triads with bile duct, hepatic artery and portal vein were
identified and were essentially unremarkable.

The H&E sections displayed liver parenchyma with hepatocytes and
sinusoids with Kupffer cells. Several hepatocytes were binucleated.
Focal mild (1 cases) cytoplasmic swelling and vacuolisation with
lymphocyte infiltration was noted. Morphology of hepatocytes is
otherwise unremarkable. Portal triads with bile duct, hepatic artery and
portal vein were identified and were essentially unremarkable.
73

Chapter 6: Materials and Methods
6.1 Reagents
Human recombinant CTSS and the Cathepsin S Activity Assay Kit were purchased from
Biovision (Milpitas, CA). The RNase
®
plus mini kit, Hs_F2RL1_5 FlexiTube siRNA and AllStar
Negative Control siRNA were from Qiagen (Germantown, MD). The RT kit for reverse
transcriptase, human IL-1β (Hs00174097_m1), human IL-8 (Hs00174103_m1), human IL-6
(Hs00985639_m1), human TNF-α (Hs00174128_m1), human MMP-9 (Hs00234579_m1), human
CTSS (Hs00175407_m1), human PAR-2 (Hs00608346_m1) and human GAPDH
(Hs02786624_g1), mouse CTSS (Mm01255859_m1), mouse TNF-α (Mm00443258_m1), mouse
IFN-  (Mm0081778_m1), mouse H2-Ab1 (Mm00439216_m1), mouse F2Rl1
(Mm00433160_m1), mouse GAPDH (Mm99999915_g1), and master mix were purchased from
Applied Biosystems (Grand Island, NY). The CytoSelect
TM
Cell viability and cytotoxicity assay
kit was from Cell Biolabs, Inc. (San Diego, CA). 0.25% Trypsin-EDTA was from Mediatech, Inc.
(Manassas, VA). The human V-plex pro-inflammatory cytokine panel 1 kit was from Meso Scale
Discovery (MSD
®
) (Rockville, MD). The human IL-6 and IL-1β ELISA kits, lipofectamine
RNAiMAX transfection reagent, Opti-Mem Reduced Serum Medium, the BLOCK-IT
TM

Fluorescent Oligo labeled with FITC, the mouse anti-GAPDH monoclonal antibody (MA5-
15738), Micro BCA reagents, KSFM and supplements, gentamicin, heat-inactivated fetal bovine
serum, anti-CD68 monoclonal antibody (14-0681-82), AF568 goat anti-rat antibody (A11077),
DAPI (62248) were from Thermo Fisher Scientific (Rockford, IL). The human IL-8 ELISA kit
was from Innovative Research (Novi, MI). The human PAR-2 ELISA kit was purchased from
Cloud-Clone Corp. (Katy, TX) and the human Magnetic Luminex Assay Plex to measure protein
expression of IL-1β, IL-8, IL-6, TNF-α, MMP-9, and CTSS was purchased from R&D Systems
74

(Minneapolis, MN). The anti-PAR-2 polyclonal antibody (ab128628), the rabbit anti-PAR-2
monoclonal antibody (ab180953) and, the anti-B220 monoclonal antibody (ab64100) were from
Abcam (Cambridge, MA). FITC-CD3 antibody (17A2) was from Biolegend (San Diego, CA).
Alexa Fluor
®
488 donkey anti-rabbit secondary antibody (A-21206), rhodamine-phalloidin
(A22287), and ProLong
®
Gold Antifade Mounting Medium were from Invitrogen (Carlsbad, CA).
IRDye
®
800CW Goat anti-rabbit (926-32211) and IRDye
®
680RD Donkey anti-mouse (925-
68072) were purchased from LICOR (Lincoln, NE). FITC donkey anti-rat antibody (712-095-150)
was from Jackson ImmunoResearch (West Grove, PA). Z-FL-COCHO (Z-FL), DMSO, Bovine
serum albumin (BSA), carbamylcholine, and CHAPS were purchased from Sigma–Aldrich (St.
Louis, MO), while Tris-HCL, sodium chloride, and acetone were from J.T. Baker (Center Valley,
PA). Z-FL-hydrate was from Adooq Bioscience (Irvine, CA). PEG 300 was from Spectrum
(Gardena, CA) and sterile PBS was from VWR (Solon, OH). Ketamine (Ketaject
®
) was from
Phoenix (St. Joseph, MO). Xylazine (AnaSed
®
) and FUL-GLO
®
fluorescein sodium strips were
from Akorn (Lake Forest, IL). Isoflurane (Fluriso
®
) was from VetOne (Boise, ID). Carbachol was
from Alfa Aesar (Haverhill, MA). Free Style Lite
®
test strips were from Abbott Diabetes Care
(Alameda, CA). ZoneQuick
®
Phenol Red threads were from Showa Yakuhin Kako Co., Ltd.
(Tokyo, Japan). Microcapillary tubes were from Drummond (Broomall, PA). Bio-Rad protein
assay dye reagent concentrate was from Bio-Rad (Hercules, CA).
6.2 Mice
Male NOD mice (NOD/MrkTac) used with Z-FL (Millipore) i.p. and topical administration
were bred in-house from the breeding pairs purchased from Taconic (Hudson, NY) and male NOD
mice (NOD/ShitLtJ # 001976) used with Z-FL-hydrate (Adooq Bioscience) i.p. administration
were from The Jackson Laboratory (Sacramento, CA). Male BALB/c mice (000651) were also
75

purchased from The Jackson Laboratory (Sacramento, CA). Animal use was in compliance with
policies approved by the University of Southern California Institutional Animal Care and Use
Committee.
6.3 Cell culture
A human corneal epithelial cell line, HCE-T cells, transformed with Simian virus 40-
Adeno vector (Araki-Sasaki et al., 1995), was obtained from the RIKEN Cell Bank, Japan
(RCB2280). Medium for cell culture was prepared according to Cell Bank instructions and
consisted of 250 ml of keratinocyte-SFM (KSFM) supplemented with 40 µl of human recombinant
EGF (rEGF), 1 ml of Bovine Pituitary Extract (BPE), and 30 µl of gentamycin. Cells were cultured
to 80 – 90% confluence and split with 0.25% Trypsin-EDTA. 10% of heat-inactivated fetal bovine
serum in sterile PBS was used to quench trypsin digestion. All cells utilized were from passage 4
– 8.
6.4 Calculation of human recombinant CTSS dosage for treatment of human corneal
epithelial cells
Human recombinant CTSS at the same activity level detected in the 90th – 95th percentile
in tears of SS patients (Hamm-Alvarez et al., 2014) and as used for other in vitro biochemical
studies on SS tear properties (Edman et al., 2018) were used to treat HCE T-cell. The 90th – 95th
percentile of enzymatic CTSS activity corresponded to 18,000 RFU (Relative Fluorescence Units)
or 300 RFU per minute. Because the specific activity of the recombinant human enzyme varied by
lot and also with storage time, a standard curve to determine the Relative Fluorescence Unit (RFU)
and the concentration of recombinant human CTSS to achieve this activity was conducted for each
experiment using the CTSS activity assay kit. Recombinant human CTSS was diluted into 5
concentrations: 2.5, 5.0, 12.5, 25, and 50 nM. 50 µl from each concentration was added into 2
76

wells of a 96-well plate. 50 µl of CTSS reaction buffer and 2 µl of substrate (Z-VVR-AFC) were
added into each well. Then, 2 µl of CTSS inhibitor was added into one of the two wells as a
negative control. The plate was incubated at 37°C for 1 hour and the fluorescence intensity read
using a microplate spectrofluorometer (SpectraMax Gemini Plate Reader, Molecular Devices,
Sunnyvale, CA) with 400/505 nm excitation/emission filters. The concentration of human
recombinant CTSS that showed enzymatic activity at 18,000 RFU or 300 RFU per minute was
used to treat HCE T-cells in 500 µl of cell medium in a 12-well plate and in 700 µl of cell medium
in a 6-well plate.
6.5 siRNA transfection
Cells were seeded in 6-well plates with KSFM with supplements but without gentamycin
which can cause cell death during siRNA transfection. Cells at 50% - 60% confluency were
transfected with PAR-2 siRNA or scrambled siRNA which is a non-specific random sequence
siRNA used as a negative control. 5 µl of Lipofectamine RNAiMAX reagent was diluted with 120
µl of Opti-Mem medium to make 125 µl of diluted Lipofectamine reagent, while 25 pmol of
siRNA was diluted with Opti-Mem medium to make a total volume equal to 125 µl of diluted
siRNA. These mixtures were mixed (ratio 1:1) and incubated at room temperature for 10 minutes.
After that, the 250 µl of siRNA-lipid complex was added to the cells and incubated at 37°C for 48
hours. After 48 hours, PAR-2 gene and protein expression were analyzed to analyze the extent of
inhibition of PAR-2 expression in cells transfected with PAR-2 siRNA relative to cells transfected
with scrambled siRNA as described below.
6.6 Transfection efficiency
Transfection efficiency of siRNA was determined with the BLOCK-iT Fluorescent Oligo
labeled with FITC as previously reported (Liu et al., 2016). Cells were transfected with 25 pmol
77

of BLOCK-iT fluorescent Oligo and 5 µl of Lipofectamine RNAiMAX reagent as described
above. After 48 hours, cells were washed with PBS and transfection efficiency was semi-
quantitatively determined by confocal fluorescence microscopy using a ZEISS LSM 800 confocal
microscope (Carl Zeiss, Thornwood, NY). For quantitative measurements, cells were detached
using Trypsin-EDTA and suspended in 700 µl of PBS. Flow cytometry using a BD LSRFortessa
TM
X-20 (BD Biosciences, San Jose, CA) was used to detect the FITC-positive HCE-T cells.
6.7 Treatment of HCE T-cells with recombinant human CTSS
HCE T-cells were seeded in KSFM medium supplemented with BPE, rEGF, and
gentamycin as described in 12-well plates. Cells were starved for 16-18 hours with KSFM medium
without any additives. Human recombinant CTSS was added to cells at the dose corresponding to
18,000 RFU per 500 µl of cell medium as described above and incubated for 15 minutes, 1, 2, 4,
8, and 24 hours at 37°C. Gene expression of various pro-inflammatory cytokines and other markers
were evaluated under each treatment condition as described below.
For treatment of cells with heat-inactivated CTSS, active recombinant CTSS was heat-
inactivated for 30 minutes at 90°C and the loss of activity was confirmed using the CTSS activity
assay kit. HCE T-cells were then treated with an equivalent starting activity of active versus heat-
inactivated CTSS for 4 hours. For treatment of siRNA-transfected cells with CTSS, cells which
were already transfected with siRNA was treated with human recombinant CTSS at the dose
corresponding to 18,000 RFU per 700 µl of cell medium for 4 and 24 hours. After 4 and 24 hours
of CTSS treatment, IL8, IL-6, TNF-α, and IL-1β, CTSS and MMP-9 gene and protein expression
were measured as described below.

78

6.8 HCE-T cell viability after 24 hours of CTSS treatment
Cell viability after 24 hours of CTSS treatment was measured using MTT colorimetric
detection with reagents provided with the CytoSelect
TM
Cell viability and cytotoxicity assay kit.
Cells were seeded in a 24-well plate and treated according to the manufacturer’s protocol. Briefly,
250 µl of KSFM medium and 25 µl of the MTT reagent was added into each well. Then, the plate
was incubated for 2 hours at 37°C until the purple precipitate was clearly visible. 250 µl of the
provided detergent solution was added into each well and then, the solution was gently mixed by
pipetting. After that, the plate was incubated at room temperature for 2 hours and absorbance was
measured at 570 nm using a SpectraMax iD3 (Molecular Devices, San Jose, CA).
6.9 Multiplex assay and ELISA methods for measurement of pro-inflammatory cytokines in
HCE-T cell medium and lysates
For measurement of pro-inflammatory cytokines expression in cell culture medium, the
cell medium was collected and then centrifuged at 500 g for 10 minutes (Z 216 MK-2 High
Capacity Refrigerated Microcentrifuge, Edison, NJ). Supernatants were kept on ice and
concentrated by centrifugal filters (regenerated cellulose 10,000 NMWL). The total protein
concentration in the culture medium was measured using the Bio-Rad assay.
For measurement of protein content in lysates, cells were lysed with lysis buffer (150 mM
NaCl, 20 nM Tris base pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100) (Meng et al., 2017)
containing protease inhibitor cocktail as previously described (Li et al., 2010) and were also
incubated on ice for 30 minutes with vortexing every 10 minutes. Samples were centrifuged at
14,000 g for 15 minutes at 4°C. Supernatants were collected and concentrated using centrifugal
filters. The total protein concentration in cell lysates was measured using the BCA assay. The
manufacturer’s protocol for the MSD
®
MULTI-SPOT Assay System was followed to analyze the
79

expression of 10 pro-inflammatory cytokines. The multiplex plate was read with an MSD
®
Sector
Imager 2400A Imaging System. A standard curve for each cytokine was created. Because the
concentration of IL-6 and IL-8 in cell medium exceeded the limitation of the multiplex kit, IL-6
and IL-8 protein expression in cell medium were measured separately using human ELISA kits
according to the protocols provided and read using a SpectraMax iD3 (Molecular Devices, San
Jose, CA).
6.10 ELISA method for measurement of CTSS in HCE-T cell lysates
Cells were washed twice with PBS, then scraped gently into PBS for centrifugation at 1,000
g for 5 minutes. The supernatant was removed and the cell pellets were resuspended with lysis
buffer (without protease inhibitor cocktail). Then, cell lysates were prepared using the same
method as for measurement of pro-inflammatory cytokines. The human CTSS ELISA kit with the
protocol provided were used and the plate was read using a SpectraMax iD3 (Molecular Devices,
San Jose, CA). The total protein concentration in cell lysates was measured using the BCA assay.
6.11 ELISA method for measurement PAR-2 in HCE-T cell lysates
Cell lysates were prepared by lysing cells with lysis buffer containing protease inhibitor
cocktail as previously described (Li et al., 2010) and were also incubated on ice for 30 minutes
with vortexing every 10 minutes. Samples were centrifugated at 1,500 g for 10 minutes at 4°C.
Supernatants were collected and concentrated using centrifugal filters. The human PAR-2 ELISA
kit and the protocol provided were used and the plate was read using a SpectraMax iD3 (Molecular
Devices, San Jose, CA). The total protein concentration in cell lysates was measured using the
BCA assay.
80

6.12 Multiplex assay and ELISA methods for measurement of pro-inflammatory cytokines,
CTSS, and MMP-9 in siRNA transfected HCE-T cells
For cells treated with siRNA and then exposed without and with CTSS, total CTSS, MMP-
9, IL-6, IL-8, IL-1β, and TNF-α were quantified in both media and cell lysates using a Human
Magnetic Luminex Assay, a bead-based multiplex ELISA kit (LXSAHM; R&D systems,
Minneapolis, MN) according to the manufacture’s protocol. Data from the multiplex ELISA was
acquired on a validated and calibrated Bio-Plex Suspension Array 200 system (MAPTM
Technology; Austin, TX) and analyzed using Bio-Plex Manager 6.1 software. IL-6 and IL-1β
protein expression in cell medium from cells treated with siRNA and exposed without and with
CTSS were measured separately using human ELISA kits according to the protocol provided and
read using SpectraMax iD3 (Molecular Devices, San Jose, CA) because their range exceeded that
of the other components measured by multiplex ELISA. The protein value acquired for each
analyte was normalized to total protein concentration. Biorad was used to measured total protein
concentration in cell medium, while BCA was used to measured total protein concentration in cell
lysate.
6.13 CTSS activity in HCE-T cell lysates
Cell medium was removed, and cells were washed twice with PBS, then scraped gently
into PBS for centrifugation at 1,000 g for 5 minutes. The supernatant was removed, and cell lysates
were prepared in the cell lysis buffer provided in the CTSS assay kit and incubated on ice for 30
minutes. Cell lysates were vortexed every 10 minutes and then, centrifuged at 14,000 g for 15
minutes at 4°C Supernatants were concentrated using centrifugal filters. CTSS activity in cell
lysates was determined with the CTSS activity assay kits described above, according to the
manufacturer’s instructions, and the enzymatic reaction was incubated at 37°C for 1 hour. The
81

quantity of the resulting fluorescent products was measured in a microplate spectrofluorometer
(SpectraMax Gemini Plate Reader, Molecular Devices, Sunnyvale, CA) with 400/505 nm
excitation/emission filters. Activity of CTSS was measured as relative fluorescence units. Total
protein concentration in cell lysates was measured using the BCA protein assay and plates were
read using a Tecan GENios Plus Microplate Reader with absorbance set to 595 nm. The unit of
total protein concentration was µg/µl.
6.14 Immunofluorescence of PAR-2 in HCE T-cells
HCE T- cells were seeded on coverslips in 12-well plates. Then, cells were washed with
PBS and incubated with acetone at -20°C for 10 minutes. After that, cells were washed with PBS
and gently shaken with 1% BSA (in PBS) for 1 hour at room temperature. Cells were then
incubated with rabbit anti-PAR-2 polyclonal antibody (ab128628) at 1:50 dilution in BSA for 1
hour at 37°C. After that, cells were washed with PBS for 5 minutes, 3 times. Rhodamine-phalloidin
was then added at 1:200 dilution, DAPI at 1:2000 dilution, along with Alexa Fluor
®
488 donkey
anti-rabbit secondary antibody at 1:100 dilution and incubated for 1 hour at 37°C. Samples were
mounted using ProLong
®
Gold Antifade mounting medium and imaged by confocal fluorescence
microscopy using a ZEISS LSM 800 confocal microscope (Carl Zeiss, Thornwood, NY).
6.15 Western Blotting for PAR-2
Cells were removed by scraping in CHAPS lysis solution (30 mM CHAPS in TBS/ 20 mM
Tris HCl, 150 mM NaCl, pH 8.0). Cells were further lysed by passing through a 23-gauge needle
10 to 15 times. Cell lysates were left at 4°C for 1 hour. Post-incubation, insoluble debris was
removed from the lysate by centrifugation at 15,000 g at 4°C for 5 minutes. The supernatants were
collected and concentrated using centrifugal filters. The protein concentration of the concentrated
lysates was measured using the BCA assay. Lysate samples were left in a reducing dye containing
82

β-mercaptoethanol for 1 hour at room temperature. Precast 10% PAGEr™ EX Gels (Lonza,
Rockland, ME) were used for resolution of samples. 40 µg of lysate/ β-mercaptoethanol /dye mix
were loaded into each well, and run at 80V, 4°C for 2 hours. Proteins were then transferred to
nitrocellulose membranes with the iBlot 2 dry blotting system (ThermoFisher Scientific, Rockford,
IL). Membranes were incubated at room temperature in fluorescent blocking buffer (Rockland
Immunochemicals Inc, Limerick, PA). After blocking, membranes were gently shaken overnight
in blocking buffer containing primary PAR-2 antibody at a 1:750 dilution (and/or 1:1000 dilution
for GAPDH) at 4°C. On the second day, membranes were washed 3 times, each 5 minutes, with
0.2% Tween containing Tris-buffered saline. Membranes were then incubated in secondary
antibody containing blocking buffer (1:2000 dilution) at room temperature, for 1 hour. After
washing with 0.2% Tween containing Tris-buffered saline again as above, membranes were
imaged with an Odyssey Licor imaging system (LI-COR Biotechnology, Lincoln, NE).
6.16 Z-FL preparation for i.p. and topical administration
For Z-FL i.p. formulation, 4 mg/kg (2.3 mM) or 1 mg/kg (1.1 mM) of Z-FL was dissolved
in 10% DMSO + 40% PEG 300 + 50% sterile PBS. For Z-FL-hydrate i.p. formulation, 15 mg/kg
(8.6 mM) of Z-FL-hydrate was dissolved in 10% DMSO + 40% PEG300 + 50% sterile PBS. For
eyedrop formulation, 200 µM of Z-FL was dissolved in sterile PBS. A Branson 2510 Ultrasonic
Sonicator (Danbury, CT) was used to dissolve Z-FL using 40 Hz for 1-2 hours at 4
o
C until the
solution was clear. Formulations were prepared for use within a week and Z-FL concentrations
were measured relative to a standard curve determined by RP-HPLC using a C4 column (150 ×
4.6 mm, particle size 5 µm, YMC CO., LTD., Allentown, PA). The mobile phase was comprised
of 10% H 2O and 90% acetonitrile and sample was eluted at a flow rate of 1 ml/min and subjected
to UV detection at 206 nm under isocratic conditions.
83

6.17 CTSS activity assays in Z-FL preparation
For each Z-FL preparation, the level of CTSS inhibition was measured using the CTSS
activity assay kit. The Z-FL preparation was diluted with vehicle into 5 serial dilutions: undiluted,
1:10, 1:50, 1:100, and 1:500. The CTSS activity assay was performed in a 96-well plate. 2 µl of
12.5 nM of an active human recombinant CTSS, 2 µl of CTSS substrate (Z-VVR-AFC), and 98 µl
of CTSS reaction buffer was added into each well. 2 µl of Z-FL at each dilution was added into
each well. For the negative control, 2 µl of vehicle, and 2 µl of CTSS reaction buffer (without Z-
FL inhibitor) were added into each well. The inhibitory potential against human recombinant
CTSS in each Z-FL preparation was compared to that in the negative control wells.
6.18 CTSS activity assays in tears, LG, and spleen of male NOD mice
For CTSS activity in tears, LG, and spleen, activity was measured as described previously
(Meng et al., 2016; Shah et al., 2017). Briefly, for tear CTSS analysis, stimulated tears were diluted
to 200 µl with CTSS reaction buffer and equally divided into 2 wells on a 96-well plate. For LG
and spleen lysate preparation, LG and spleen were homogenized in 300 µl of CTSS lysis buffer in
a Beadbug
TM
prefilled tube with 1.0 mm Zirconium beads (Sigma-Aldrich, St. Louis, MO). The
homogenate was centrifuged at 10,000 x g for 10 minutes at 4
o
C. 40 µl of supernatant was collected
and diluted with 60 µl of lysis buffer and 100 µl of reaction buffer, then, this mixture was equally
divided into 2 wells on 96-well plate. 2 µl of substrate was added into each well and 2 µl of CTSS
inhibitor was added into one of the 2 wells as a negative control.
6.19 In vivo studies for determining Z-FL toxicity in i.p. administration
To estimate the MTD of Z-FL for i.p., 14-week male BALB/c mice were divided into 6
groups (2 mice/group). Mice were given the Z-FL in mg/kg body weight every other day for 2
weeks: 0.25, 0.50, 1.0, 2.0, 4.0. Vehicle groups served as controls. Body weight of mice were
84

monitored before each injection. At the end of the study, body weight and weights and appearances
of internal organs (spleen, liver, and kidney) were recorded.
6.20 In vitro and in vivo studies for determining Z-FL toxicity in topical administration
Ocular toxicity was evaluated in vitro using HCE-T cells. 20, 100 and 200 µM of Z-FL
were dissolved in KSFM medium containing human recombinant EGF, Bovine Pituitary Extract
and gentamicin. HCE T-cells at 80% confluency were treated with Z-FL doses for 15 min and then
Z-FL was removed. After 8 hours, cells were again exposed to Z-FL for 15 minutes and then
incubated in Z-FL-free KSFM medium for 16 hours, with the regimen reflecting twice-daily
eyedrops. 24 hours after initial Z-FL exposure, cell viability and cytotoxicity were analyzed using
the CytoSelect
TM
Cell viability and cytotoxicity assay kit. Green Calcein AM detection measured
cell viability, while EthD-1 detection measured cell toxicity. Measurements utilized a BioTek
Synergy H1 Hybrid Multi-Mode Microplate Reader (Winooski, VT) with scanning area mode to
measure the fluorescence intensity from the whole area in each well. KFSM-treated cells served
as live cell controls, while 0.1% saponin-treated cells were dead cell controls.
Male NOD mice were used to assess in vivo toxicity. Z-FL was administered topically
twice daily to 15 male NOD in 4 treatment groups of 3 mice each: 20, 100, and 200 µM Z-FL,
vehicle (sterile PBS). Basal tear production and corneal fluorescein staining were measured before
and after treatments.
6.21 Administration of Z-FL and Z-FL-hydrate i.p. injection in male NOD mice
30 male NOD mice were divided into 3 groups: 4 mg/kg body weight, 1 mg/kg body
weight, and vehicle (10% DMSO + 40% PEG 300 + 50% sterile PBS). Mice were injected every
other day for 2 weeks and body weight of each mouse was monitored at the time of each injection.
Blood glucose was recorded before treatments and after the last dose in peripheral blood collected
85

by tail nick using Free Style Lite
®
test strips. Mice with blood glucose >250 mg/dl were considered
diabetic and excluded (Morris et al., 2011). At the end of the treatment, mice were euthanized by
i.p. with 50 - 60 mg/kg ketamine and 5 - 10 mg/kg xylazine, followed by cervical dislocation after
stimulated tear collection. After euthanasia, one LG from each mouse was isolated for histology
and fixed in 10% neutral-buffered formalin overnight at 4
o
C and then, 70% ethanol. 3 randomized
mice/group were selected for use of the other LG for immunofluorescence. The other LGs from
the remaining mice were divided into 2 parts: ½ LG was used for mRNA extraction and the
remaining ½ LG was analyzed for CTSS activity. For Z-FL-hydrate i.p. studies, the same
procedure was repeated as for Z-FL i.p. except 30 male NOD mice were equally divided into 2
groups: 15 mg/kg body weight of Z-FL-hydrate and vehicle. Mice were injected every other day
for 2 weeks. After 2 weeks of treatment, 2 mice/group were excluded because of diabetes. As a
result, the number of mice in each group was 13 mice.
6.22 Administration of Z-FL by topical eyedrops in male NOD mice
30 male NOD mice were given eyedrops twice daily every day for 6 weeks. Mice were
divided into 2 groups (15 mice/group): 200 µM of Z-FL or vehicle (sterile PBS). Basal tear
secretion, corneal fluorescein staining and blood glucose were recorded before treatment, 3 weeks
and 6 weeks. At the end of treatments, mice were euthanized and LGs were collected, divided and
processed as described above for i.p.
6.23 Quantification of LG lymphocytic infiltration by hematoxylin-eosin staining
LG histology and quantification of lymphocytic infiltration was previously described
(Janga et al., 2018). Briefly, LG were stained with hematoxylin-eosin (H&E) staining according
to standard procedure and photographed by using Nikon 80i microscope (Melville, NY) equipped
with digital camera. 3 non-consecutive LG sections (at 25%, 50%, and 75% of the whole LG) were
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imaged and calculated lymphocytic infiltration area by a blinded reviewer using mage J software
(National Institutes of Health, http://imagej.nih.gov/ij). The percentage of LG lymphocytic
infiltration area from each mouse was reported as an average.
6.24 LG immunofluorescence and quantitative analysis of immune cells in LG lymphocytic
infiltration
LG from 3 randomized mice/group were prepared for immunofluorescence staining.
According to a standard protocol, LG was fixed in 4% paraformaldehyde for 2 hours and then
immersed in 30% sucrose at 4
o
C overnight. LG was embedded in O.C.T. compound and frozen in
dry ice. The block was cryosectioned at 5 µm thickness and mounted on glass slides (Meng et al.,
2017). The section was quenched with 50 mM ammonium chloride in PBS for 5 minutes and
permeabilized with 0.1 triton X-100 in PBS for 10 minutes. After that, the section was blocked
with 1% BSA in PBS at room temperature for 1 hour and incubated with a primary antibody at the
optimal ratio at 37
o
C for 1 hour: FITC-CD3 antibody and B220 antibody were used at 1:50, while
CD68 antibody was used at 1:100. Then, the tissue was incubated with secondary antibodies at
37
o
C for 1 hour. AF568 goat anti-rat at 1:100 dilution was used to detect B220, while FITC donkey
anti-rat at 1:100 dilution was used to detect CD68. DAPI at 1:2000 dilution was used to detect
nuclei. Samples were imaged using a ZEISS LSM 800 confocal microscope (Carl Zeiss,
Thornwood, NY).
Quantification of positively stained cells within areas of lymphocytic infiltration and the
total area of the lymphocytic infiltrates used Image J software (National Institutes of Health,
http://imagej.nih.gov/ij). 3 tissue sections/mouse were imaged for CD68 staining and reported as
an average per mouse. 1 section/mouse was imaged for CD3 and B220 staining. In each section, 8
images were randomly acquired, and results averaged for each section. CD3+ T-cells and B220+
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B-cells were calculated as the density of cells of interest in the total area of lymphocytic infiltration
in each field (number of cell/mm
2
). CD68+ macrophages were calculated as the percentage of
CD68+ labelling area normalized to total area occupied by lymphocytic infiltration in each field.
6.25 Corneal fluorescein staining
Mice were anesthetized with isoflurane and 1 µl of fluorescein sodium (FUL-GLO) was
applied to both eyes (Shah et al., 2017). A Cobalt blue light was used for illumination and the
ocular surface photographed using an Excelis HD Microscope Camera with an 11.6-inch AU-600-
HDS attached to a Unitron Z-8 series stereo microscope (Microscope Central, Feasterville, PA).
Images were acquired from both eyes and graded by a blinded reviewer as previously described
(Shah et al., 2017). Briefly, the cornea was divided into 5 areas and the intensity of fluorescein
puncta in each area was graded from 0 to 3 and summed. Corneal staining scores from both eyes
are reported as an average.
6.26 Basal tear collection
The mice were anesthetized using isoflurane and phenol red thread was inserted gently
under the lower of eyelid for 10 seconds. When the yellow thread (acidic) contacts with tears, it
turns to a light red color which can be measured as the length of wetting of the thread in millimeters
using a loupe with a millimeter scale (Ted Pella, CA). Basal tear secretion was measured from
both eyes and reported as an average.
6.27 Stimulated tear secretion
Both sides of LG from each mouse were exposed to 3 µl of 50 µM of carbachol. The eye
was washed with PBS before adding carbachol. After stimulating LG, tears from both eyes were
collected by using 2 µl microcapillary tubes which were placed at the tear meniscus in the medial
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canthus for 5 minutes (Shah et al., 2017). Repeated stimulation was performed 3 times, 5 minutes
per each time. The volume of collected tears was recorded from both eyes and reported as an
average. Then, collected tears was used for CTSS activity in tears as described above.
6.28 Gene expression in HCE-T cells and in mouse LG lysates
RNA was prepared from HCE-T cells using the RNeasy
®
Plus Mini Kit, while RNA from
mouse LG lysates was isolated by using the RNeasy Plus Universal Mini Kit. Reverse transcription
reactions from both HCE-T cells and mouse LG were performed with 1 µg of total RNA per 50 µl
of reaction volume which was composed of TaqMan
®
Reverse Transcription Reagents to obtain
cDNA from RNA using the GeneAmp
®
PCR System 9700 (Applied Biosystems, Grand Island,
NY) with incubation at 25°C for 10 minutes, then, at 48°C for 30 minutes, and finally terminated
by incubation at 95°C for 5 minutes. Real time quantitative polymerase chain reaction (qPCR)
from HCE-T cells and mouse LG were conducted using an ABI 7900HT Fast Real-Time PCR
System according to published procedures (Meng et al., 2017). Briefly, samples were preheated at
95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minutes. For
HCE-T cell samples, human GAPDH (Hs02786624_g1) was run as the internal control and human
IL-1β (Hs00174097_m1), IL-8 (Hs00174103_m1), IL-6 (Hs00985639_m1), TNF-α
(Hs00174128_m1), MMP-9 (Hs00234579_m1), CTSS (Hs00175407_m1), and PAR-2
(Hs00608346_m1) primers were used to measure gene expression in HCE-T cells. For mouse LG
samples, mouse GAPDH (Mm99999915_g1) was used as an endogenous gene and mouse CTSS
(Mm01255859_m1), TNF-α (Mm00443258_m1), IFN-  (Mm0081778_m1), H2-Ab1
(Mm00439216_m1), and PAR-2 (Mm00433160_m1) were analyzed as genes of interest. The
reaction conditions and calculation methods were as described previously (Meng et al., 2017).

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6.29 Histotoxicological evaluation
The spleen, liver, and kidney from each mouse were collected to compare appearances and
weights after treatments. Organs from 5 mice/group were randomly selected for evaluation of
histological toxicity by hematoxylin-eosin (H&E) staining and all sections analyzed by a blinded
trained pathologist.
6.30 Statistics
All statistical analyses were performed using Graphpad Prism software (Graphpad, San
Diego, CA). A two-tailed, unpaired Student’s t-test was used to compare between 2 independent
groups. A two-tailed, paired Student’s t-test was used to compare within the same sample before
and after treatment. Differences in 3 or more independent groups were compared using one-way
ANOVA. Tukey’s multiple comparison was used for all possible pairwise comparisons, while
Dunnette’s multiple comparison was used for comparing one control group to each of the other
experimental groups (Liu, 1997). A repeated measures ANOVA with Tukey’s multiple
comparison was used to compare the difference within the same sample at 3 different time points.
P ≤ 0.05 was considered as a significant difference.

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Chapter 7: Discussion
Sjögren’s syndrome (SS) is a chronic inflammatory autoimmune disease attacking LG and
salivary gland functions (Stefanski et al., 2017). One of the hallmarks of SS is the infiltration of
inflammatory mediators which are recapitulated in LG and salivary glands of SS mouse models,
and also in glands of SS patients (Jonsson et al., 2006; Li et al., 2010; Rodrigues and Soares, 2017).
These mediators are also found in tears and conjunctival epithelium (Pflugfelder et al., 2009), and
their release from the inflamed LG may impact the ocular surface. Cathepsin S (CTSS) is a
lysosomal cysteine protease implicated in antigen presentation leading to inflammation and
associated with extracellular matrix degradation. CTSS is expressed at increased levels in the LG
and tears of male NOD mice, a murine model of SS, which corresponds to the increased enzymatic
activity of CTSS found in SS patients tears (Edman et al., 2018; Janga et al., 2018). In this study,
I demonstrate that activity of CTSS at the same enzymatic levels as found in SS patient tears, can
acutely stimulate pro-inflammatory gene and protein expression including IL-8, IL-6, TNF-α, and
IL-1β in a human corneal epithelial cell line (HCE-T cells). These finding suggest that tear and
ocular surface tissue cytokines may not necessarily originate in the LG in SS but may be elicited
via CTSS-mediated processes in ocular surface epithelia.
As mentioned in Chapter 3, protease-activated receptor-2 (PAR-2) can be activated by
CTSS contributing to several inflammatory responses. Here, I implicate PAR-2 as a potential
mediator of acute CTSS - induced ocular surface inflammatory responses. In HCE-T cells
transfected with PAR-2 siRNA but not scrambled siRNA and followed by acute exposure to 4
hours of CTSS treatment, IL-6, TNF-α, IL-1β, and MMP-9 secretion into cell culture medium were
significantly reduced, corresponding with decreased IL-6 and TNF-α gene expression and IL-6
and MMP-9 protein expression in cell lysates. This effect occurred prior to the CTSS-induced
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increase in PAR-2 protein expression at 24 hours, suggesting it is mediated by PAR-2 already
present on or recruited to the cell surface. These results demonstrate that CTSS activation of PAR-
2 may play a possible role in priming of acute inflammatory responses. The possible cell signaling
pathway induced by acute CTSS exposure (after 4 hours of CTSS treatment) in HCE-T cells
contributing to increased pro-inflammatory cytokines and MMP-9 is shown in Figure 29.
However, there was no change of IL-8 gene and protein expression in cell medium and cell lysates
in cells transfected with PAR-2 siRNA at 4 hours of CTSS treatment relative to scrambled siRNA-
transfected cells. These data suggest that the acute phase of IL-8 release induced by CTSS after 4
hours treatment was not reduced by the partial knockdown of PAR-2 suggesting that increased IL-
8 by CTSS is PAR-2 independent (Figure 41). One possible alternative signaling mechanism for
stimulation of IL-8 is through IL-36 , a member of the IL-1 family (Dinarello et al., 2010)
abundant in epithelia, neurons, glia, dendritic cells, and macrophages. IL-36γ is highly increased
in blood and salivary glands of primary SS patients and correlated with IL-17A and IL-22 serum
levels (Ding et al., 2018). A recent study has shown that CTSS cleavage of IL-36 induces
secretion of IL-8 in human keratinocytes (Ainscough et al., 2017).

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Figure 41. The possible cell signaling pathways induced by acute exposure to 4 hours of
CTSS in human corneal epithelial cells contributing to increased release of pro-
inflammatory cytokines and MMP-9 that may contribute to ocular surface inflammation.
(A) The effects of acute CTSS on increased pro-inflammatory cytokines and MMP-9 mediated
by PAR-2; (B) Acute CTSS induces IL-8 through a PAR-2- independent pathway. An one-way
black solid arrow ( ) represents a proposed inducible pathway, an one-way black dashed arrow
( ) represents a possible one-way inducible pathway, a two-way red dashed arrow ( ) represents
a possible two-way inducible pathway and a solid line with blunt end ( ) represents a negative
feedback pathway.
According to time course studies of CTSS treatment in HCE-T cells (Figure 16), early
CTSS-mediated and PAR-2-dependent increases in TNF-α after 15 minutes of exposure could
subsequently contribute through PAR-2 independent mechanisms to elevated IL-6 and IL-1β
(Figure 41, solid arrows). In addition, the effects elicited by acute CTSS exposure on pro-
inflammatory cytokines and MMP-9 may be interrelated. Previous studies have shown that both
IL-1β and TNF-α can upregulate MMP-9 in corneal epithelial cells, again through the NF- B and
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p38 pathways or by upregulating other mediators that regulate MMP-9 (Gordon et al., 2009; Li et
al., 2001). Furthermore, IL-6 can induce MMP-9 expression in human malignant non-Hodgkin’s
lymphomas (Kossakowska et al., 1999), and IL-8 can induce MMP-9 expression in human
neutrophils through activation of the CXCR2 receptor (Chakrabarti and Patel, 2005). Additionally,
MMP-9 can itself stimulate IL-1β and TNF-α, possibly creating a positive feedback cycle
accelerating ocular surface inflammation (Schonbeck et al., 1998; Stern and Pflugfelder, 2004).
However, this study cannot currently distinguish whether the acute phase increase in IL-6, IL-1β
and MMP-9 that appears to be PAR-2-dependent are direct or influenced by changes in TNF-α as
well as cross-talk between pathways as indicated (Figure 41, dash arrows).
According to the gene and protein expression results after chronic exposure of CTSS in
PAR-2 siRNA transfected cells (Table 1-2), IL-6, TNF-α, and MMP-9 release were reduced,
continuing the trend at 4 hours. Since the expression of these effectors is interrelated, it is unclear
whether the more sustained effects on IL-6, TNF-α, and MMP-9 are truly PAR-2 dependent or
whether the effect at 24 hours is related to accumulated changes from the acute burst. No changes
in protein content in cell lysates of any CTSS-sensitive proteins were observed by 24 hours in
CTSS-treated cells with PAR-2 siRNA relative to scrambled siRNA (Table 2). However, CTSS-
dependent upregulation of gene expression of all proteins of interest examined at 24 hours was
significantly reduced by siRNA to PAR-2 (Table 1), suggesting that it may play a role in the cycle
of chronic inflammation, perhaps mediated one of the acute phase pro-inflammatory cytokines.
Not only MMP-9 but also CTSS itself, which is increased after 24 hours of CTSS exposure,
can create a positive feedback loop possibly leading to more severe ocular surface inflammation.
As mentioned in Chapter 2 (Figure 7-8), after an acute increase in IL-6, IL-8, and IL-1β gene
expression at 2 and 4 hours of CTSS exposure, this elevation was reduced by 8 hours but was then
94

again increased after 24 hours, suggestive of a cyclical spiking of inflammatory responses. Finally,
CTSS induces PAR-2 expression after 24 hours of exposure in HCE-T cells, suggesting its
upregulation as a chronic consequence of CTSS exposure. This later CTSS-induced stimulation of
PAR-2 protein synthesis may be required to replenish cellular PAR-2 after its initial cleavage and
activation during acute CTSS treatment, thus restoring PAR-2 stores in the plasma membrane. In
contrast TNF-α gene and protein expression gradually decreased after the initial peak at 2 hours of
CTSS treatment, suggesting the possibility of early induction of other proteins which may inhibit
TNF-α such as IL-6 (Charousos et al., 1995) (Figure 41, solid line with blunt end).
Theses finding suggests that highly increased CTSS which is found in SS patients tears
plays an important role in alteration of pro-inflammatory cytokines such as IL-8, IL-6, TNF-α, and
IL-1β and also in proteases which include MMP-9 and CTSS itself. These inflammatory mediators
can create inflammation in ocular surface of SS patients and a possible mechanism related to these
acute inflammatory responses is activation of PAR-2 in the ocular surface. Recent studies have
suggested the potential of PAR-2 inhibitors as anti-inflammatory therapies. The GB83 inhibitor
(IC 50 2 µM) is derived from the non-peptidic PAR-2 agonist and leads to complete inhibition of
intracellular calcium influx induced by trypsin-activated PAR-2 (Barry et al., 2010). Its structure
is shown in Figure 42A. A combination of GB83 with RO5461111, which is a CTSS inhibitor
with IC 50 0.4 nM and 0.5 nM toward human and murine CTSS, respectively, reduced markers of
diabetic nephropathy and diabetic retinopathy in type 2 diabetic (db/db) mice (Kumar Vr et al.,
2016). The GB88 inhibitor (IC 50 1 µM), is a biased PAR-2 antagonist that selectively inhibits PAR-
2/G q/11 /Ca
2+
/PKC signaling in vivo, while being an agonist in activating three other PAR-2
activated pathways (cAMP, ERK, Rho) in vitro (Suen et al., 2014). GB88 is thought to inhibit both
trypsin, elastase, and CTSS cleavage. Its structure is shown in Figure 42B. One study has shown
95

that GB88 inhibited PAR-2 activation of nociceptor signaling leading to abolish extracellular
CTSS-induced edema and attenuate stimulated mechanical and thermal hyperalgesia in mice (Lieu
et al., 2016). Another study about GB88 has demonstrated that either using GB88 or another CTSS
inhibitor, MV026031 (K i 47 nM and 22 nM affinity toward human and murine CTSS,
respectively), can suppress formalin-induced endogenous CTSS activity leading to hyperalgesia
in mice (Zhao et al., 2014). While the GB88 is a biased antagonist, I-191 is a novel full PAR-2
antagonist with no agonist activity (pIC 50 6.7 ± 0.1 toward bovine trypsin) (Figure 42C). It can
inhibit phosphorylation of ERK1/2, RhoA activation, and forskolin-stimulated cAMP
accumulation induced by biased GB88 (Jiang et al., 2018). I-191 also potently inhibited PAR-2
induced expression of inflammatory cytokines such as IL-8, decreased cytokine-triggered cleavage
of caspase 3 and caspase 8, and inhibited cell migration in human colon adenocarcinoma grade II
cell line and human breast adenocarcinoma cells (Jiang et al., 2018).

Figure 42. The chemical structure of PAR-2 antagonists (Jiang et al., 2018; Yau et al., 2013)
96

As mentioned in Chapter 1, several studies have investigated the effects of CTSS
inhibitors on autoimmune presentation and autoimmune disease treatments. Also, there are several
CTSS inhibitors that have been studied in phase I and II clinical trials for abdominal aortic
aneurysm, rheumatoid arthritis, and SS (Gupta et al., 2008; Payne et al., 2014; Theron et al., 2017).
These studies suggest that CTSS inhibitors have therapeutic potential for autoimmune diseases,
including SS. In this study, I used a CTSS inhibitor, Z-FL from Millipore (Burlington, MA)
administered systemically (i.p.), every other day for 2 weeks and twice-daily topically (eyedrops)
for 6 weeks in 14-15 weeks male NOD mice, a model of autoimmune dacryoadenitis in SS. These
results showed that i.p. Z-FL significantly reduced tears, spleen and LG CTSS activity. Inhibition
of tear CTSS may be either by reduction of activity in the secreting tissue, the LG, and/or by direct
access of inhibitor to tears. With i.p. administration, total lymphocytic infiltration and CD3+ T cell
infiltration were both significantly reduced in LG. These findings are consistent with previous
reports of the inhibitory effect of RO5461111, another CTSS inhibitor, which reduced CD3+ T
cells cells in the spleen of the SLE mouse model (Rupanagudi et al., 2015). Clik60, another CTSS
inhibitor previously given i.p. in an SS mouse model, showed a decrease in autoantigen-specific
T-cell proliferation in regional lymph node cells of treated mice (Saegusa et al., 2002). It is possible
that systemic CTSS inhibitor directly affects immune cells in the LG or alternatively affects
immune cells in lymph nodes including dendritic cells which can present autoantigen to
recirculating naïve T-cells, generating T-memory cells that migrate from blood into LG (Mackay
et al., 1990; Mikulowska-Mennis et al., 2001). In addition, improved stimulated tear flow was also
seen for treatment with i.p. Z-FL, an effect that may be due to the reduction of specific infiltrating
immune cells into the LG, e.g., T cells. Other studies have suggested that suppression of LG
97

lymphocytic infiltration is associated with improved tear flow (Ma et al., 2014; Nishiyama et al.,
2007; Shah et al., 2017; Tsubota et al., 2001).
Previous studies have shown that H2-Ab1 gene expression is upregulated in the LG of male
NOD mice, in parallel with increased LG CTSS gene expression and increased LG and tear CTSS
activity (Janga et al., 2018; Meng et al., 2017). CTSS can also induce class II transactivator
(CIITA), Ii, and MHC II in response to IFN-  (Chan et al., 2010; Maubach et al., 2007; Meng et
al., 2017). Reduction of H2-Ab1 gene expression in the LG by i.p. Z-FL may reflect a specific
change in a subpopulation of cells within the LG such as macrophages or DC which are particularly
enriched in MHC II and reduced by Z-FL. Another CTSS inhibitor (RO5459072) reduced
macrophage inflammatory responses, as demonstrated by the downregulation of MHC II and
reduction of pro-inflammatory cytokines in human monocyte-derived macrophages (Thanei et al.,
2017). The possible mechanism of MHC II and pan-macrophage reduction in my systemic Z-FL
i.p. study might be related to the systemic access of Z-FL to the draining lymph node.
Subperitoneal lymphatic lacunae, which are abundant in the diaphragm, play an important role in
lymphatic absorption from the peritoneal cavity (Abu-hijleh et al., 1995). Peritoneal fluid enters
the lymphatic lacunae via special channels in the diaphragm. The regional lymph drainage from
the diaphragm is predominantly to mediastinal lymph nodes by way of retrosternal lymphatic
trunks, the thoracic duct and other smaller lymph channels being only secondary pathways (Abu-
hijleh et al., 1995). It is possible that Z-FL i.p. might gain access to lymphatic drainage in the
diaphragm, reducing or modulating the immune cell populations. Future studies on the effects of
Z-FL given i.p. on immune cells in draining lymph nodes versus the LG may elucidate the
mechanism of these effects.
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Surprisingly, i.p. Z-FL did not affect LG gene expression of Tnf and Ifng, which are both
implicated in SS pathogenesis and are highly expressed in LG and SG (Dorner et al., 1995; Jabs
et al., 2004; Kang et al., 2011; Meng et al., 2017), nor of Ctss and F2rl1 gene expression, which
can be induced by CTSS (Chapters 2-3). Previous studies have suggested that the activity and
protein expression of IFN-  and TNF-α in SG and peripheral blood are up-regulated even in the
absence of lymphocytic infiltrates in SG, suggesting that these cytokines and some CTSS-related
genes are not correlated directly with lymphocytic infiltration (Moutsopoulos et al., 2008; van
Woerkom et al., 2005).
In the topical Z-FL administration cohort, only tear CTSS activity was reduced, and not
CTSS levels in LG and spleen. Inhibition of tear CTSS in this cohort is thus likely a local effect,
occurring directly on the ocular surface, and not through inhibition of activity in the originating
secreting gland, the LG. As expected, topical Z-FL did not affect LG lymphocytic infiltration,
CD3+ T-cells in lymphocytic infiltrates, nor improve stimulated tear secretion associated with
disease progression. These findings suggest the lack of effect of topical Z-FL on what may be a
systemically-driven infiltration primarily sensitive to modulation of systemic (spleen, LG and/or
circulating) CTSS levels with topical administration of inhibitor insufficient to achieve enough a
concentration which affects target cells and organs. However, topical Z-FL showed reduced H2-
Ab1 gene expression in the LG in parallel with reduced pan-macrophage and DC abundance in LG
lymphocytic infiltrates. Since topical Z-FL had no effect on bulk lymphocytic infiltration of LG,
the reduced H2-Ab1 gene expression in topical Z-FL supports the hypothesis that reduced
macrophage composition of lymphocytic infiltrates may be directly responsible for reduced MHC
II expression. The possible mechanism of MHC-II and pan-macrophage reduction in LG of mice
treated with topical Z-FL might be related to modulation of lacrimal duct-associated lymphoid
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tissue (LDALT), which is located near the nasolacrimal duct and densely supplied with lymphatics,
and which plays an important role in antigen-specific responses to ocular immunization in mice
(Lohrberg et al., 2018). A recent study has observed lymphoid aggregates in the nasolacrimal duct.
These aggregates can directly access the lymphatics in LDALT, which serve as a primary
recognition site for antigens, and then, carry antigens to cervical lymph nodes (Lohrberg et al.,
2018). The nasolacrimal drainage of tears containing topical Z-FL to LDALT and then the cervical
lymph nodes, could provide sufficient CTSS inhibition to impair macrophage activity in these
tissues, and reduce MHC II expression, thus accounting for the reduced number of total infiltrating
macrophages in the NOD mouse LG. Future studies on the pharmacokinetics of topical Z-FL in
the regional lymph nodes may reveal the exact mechanism of action of Z-FL.
Topical Z-FL reduced LG gene expression of Tnf, Ifng, Ctss, and F2rl1. These findings
suggest that topical Z-FL can elicit some anti-inflammatory effects in the LG that do not require
systemic administration. These changes may be elicited through inhibitory effects on the
production of cytokines by immune cells in draining lymph nodes that supply particular immune
subpopulations to the LG. Given the effects of CTSS activity on corneal epithelial cell cytokine
signaling (Chapter 2-3), changes in CTSS tear activity may also directly affect tear cytokines,
suppressing factors that hyperactivate nerves providing a constant stimulus to the LG (Zoukhri,
2006). Overstimulation or irritation of the ocular surface can activate afferent sensory nerves that
can activate efferent parasympathetic and sympathetic nerves supplying the LG to stimulate
abnormal protein secretion (Dartt, 2009). Some studies have shown that increased production of
pro-inflammatory cytokines in tears and LG can affect the function of both the afferent and efferent
neural reflex arcs, shutting down communication between the ocular surface and the LG (Zoukhri,
2006). Also, it is possible that the prolonged exposure of ocular surface and draining lymph nodes
100

to CTSS inhibition by topical Z-FL is required to obtain the additional suppression of
proinflammatory cytokines and other effectors of disease that did not occur with i.p. Z-FL. Future
studies on the effects of Z-FL and other CTSS inhibitors administered topically and i.p. on cytokine
expression in parallel with more extensive bioavailability measures will provide further
information regarding possible differences in mechanisms of action.
In addition, topical Z-FL can attenuate loss of basal tear flow seen over 6-weeks in the
NOD mouse cohort. I did not conduct this analysis in the initial i.p. study, and the original Z-FL
used is no longer available from the vendor, Millipore. Alternatively, I found the other vendor,
Adooq Bioscience, which provides Z-FL in hydrate form. Both 2 forms showed the different
retention time and different CTSS inhibitory potency in mouse spleen lysate (Figure 25-26). Z-
FL-hydrate was 3.5-less effective than Z-FL, as a result, I needed to increase the Z-FL-hydrate i.p.
dose to 15 mg/kg body weight. Using this formulation in a second i.p. study, the basal tear secretion
was increased in mice treated with Z-FL-hydrate i.p. (Figure 30). Also, Z-FL-hydrate significantly
inhibited LG and spleen CTSS, while trending towards inhibition of tear CTSS (Figure 28). Like
Z-FL, Z-FL-hydrate significantly reduced lymphocytic infiltration of the LG and increased
stimulated and basal tear flow (Figure 29). These Z-FL-hydrate results supports my hypothesis
that inhibition of CTSS activity with a CTSS inhibitor (Z-FL or Z-FL-hydrate), reduces LG
inflammation and improves tear secretion in a murine model of autoimmune dacryoadenitis when
disease is established, indicating the potential for CTSS inhibition as a strategy for disease
treatment rather than prevention.
In conclusion, highly increased CTSS, which has previously been shown in tears of SS
patients and in male NOD mice, an animal model of SS, can increase the expression of pro-
inflammatory cytokines (IL-8, IL-6, TNF-α, and IL-1β) and proteases (MMP-9 and CTSS itself)
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partially through PAR-2 in human corneal epithelial cells, potentially contributing to a cycle of
escalating inflammation on the ocular surface of SS patients. In addition, CTSS can represent as a
potential therapeutic target for the treatment of SS. Here, I demonstrated that inhibition of CTSS
using a model CTSS inhibitor, Z-FL, reduced LG inflammation and increased tear flow in male
NOD mice when given through two routes of administration. These findings implicate CTSS as
an effector of autoimmune dacryoadenitis, representing a viable therapeutic target for systemic
and local administration in treatment of SS-associated dry eye.

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Chapter 8: Future Perspectives
In this study, I demonstrated that CTSS, found in SS patient tears and in male NOD mouse
tears and LG, can induce pro-inflammatory cytokines, particularly IL-8, IL-6, TNF-α, and IL-1β
after acute exposure, and can increase MMP-9 and CTSS itself after chronic exposure. In addition,
I found that PAR-2 activated by CTSS plays an important role in acute induction of IL-6, TNF-α,
IL-1β, and MMP-9 secretion and also in chronic induction of pro-inflammatory cytokines and
protease gene expression. These finding suggest that CTSS activation of PAR-2 may be a new
therapeutic target for alleviation of ocular surface inflammation in SS patients.
As mentioned in Chapter 7, there are several studies using PAR-2 antagonists for
reduction of inflammatory responses. However, most commercially available PAR-2 antagonists
such as GB83, AZ-8838, AZ-3451, FSLLRY-NH
2.
and ENMD 547 are specific for its trypsin
cleavage site. GB88 is the only PAR-2 antagonist that can prevent both trypsin and CTSS cleavage;
however, this inhibitor can be a biased antagonist which selectively inhibits PAR-2/G q/11
/Ca
2+
/PKC signaling in vivo, while being an agonist by activating three other PAR-2 activated
pathways (cAMP, ERK, Rho) in vitro (Suen et al., 2014). However, other studies have shown that
activated PAR-2 cleaved by CTSS stimulates intracellular G
αs
-mediated cAMP and PKA (Zhao et
al., 2014). It suggests that GB88 specially inhibits CTSS activation of PAR-2 might crosstalk with
the trypsin-activated signaling pathway. I-191, which was discovered by Dr. Fairlie’s group at
University of Queensland, is the newest full PAR-2 antagonist with no agonist activity (Jiang et
al., 2018). However, there are no reports on the specificity of I-191 toward the CTSS cleavage site.
Therefore, further studies on the effect of PAR-2 antagonists which are specific to the CTSS
cleavage site, with or without using CTSS inhibitor, on ocular surface inflammation in human
corneal epithelial cells or even in male NOD mice will be of interest in future.
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As mentioned in Chapter 1, there are several CTSS inhibitors which have been in pre-
clinical and clinical phase I and II trials for treatment of rheumatoid arthritis, asthma, multiple
sclerosis, atherosclerosis, SS, neuropathic pain, and cancer (Gupta et al., 2008). The CTSS
inhibitor, Z-FL or Z-FL-hydrate, that I used in this study showed reduction of LG inflammation
and improvement of tear flow by i.p. administration. However, topical Z-FL administration
showed less efficiency in the murine model of SS. In this study, I did not conduct extensive
pharmacokinetics studies of systemic and topical Z-FL in LG and draining lymph nodes, nor
evaluate the effects of Z-FL in both administration modalities on immune cells and cytokine
expression in the draining lymph nodes versus the LG which can help clarify the possible
mechanisms of action of Z-FL. Future studies of the effects of CTSS inhibitors on autoimmune
dacryoadenitis that explore these questions would be valuable.
For clinical applications, synthetic CTSS inhibitors require high potency, selectivity,
acceptable pharmacokinetic, pharmacodynamics, and low toxicity. The compound should be able
to reach the targets in the appreciate tissues without creating side effects to other irrelevant organs.
One of the possible side effects of CTSS inhibitors might be impairment of wound-healing-
associated microvessel growth which was observed in CTSS-deficient mice (Shi et al., 2003).
Angiogenesis requires local degradation of endothelial basement membranes, followed by
penetration of ECM in the perivascular interstitium. Proteases including MMP-9, serine proteases,
and CTSS are responsible for this capillary growth development (Shi et al., 2003). Inhibition of
CTSS reduces invasion of collagen type I and IV, contributing to defective microvessel
development during wound repair (Shi et al., 2003). Therefore, development of the new targeted
drug delivery system of CTSS inhibitors such as liposomes or nanocarriers might be useful for
increasing drug efficacy and reducing undesirable drug side effects.
104

Another challenge of pharmaceutical development of CTSS inhibitors is elusive target
validation and pharmacodynamic effects in the clinic (Lee-Dutra et al., 2011; Theron et al., 2017).
Currently, therapeutic targeting of CTSS has focused on the cleavage of a 10-kDa fragment of Ii
called lip10. CTSS cleaves Ii, mediating lip 10, to form CLIP. Inhibition of CTSS results in the
intracellular accumulation of lip10 (Theron et al., 2017). However, detection of lip1, which is
usually measured by Western Blotting, is difficult in the clinical setting because of large sample
sizes and low throughput (Theron et al., 2017). Therefore, the development of target validation
and better pharmacodynamic biomarker analysis for CTSS inhibitors might be important for future
clinical efficacy studies.

105

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Asset Metadata

Creator Klinngam, Wannita (author)

Core Title Increased cathepsin S plays an important role in ocular surface manifestations in Sjögren’s syndrome

Contributor Electronically uploaded by the author (provenance)

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 07/25/2019

Defense Date 06/05/2019

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cathepsin S,dry eye,lacrimal glands,non-obese diabetic mouse,OAI-PMH Harvest,ocular surface,pro-inflammatory cytokines,protease-activated receptor-2,proteases,Sjögren’s syndrome,Z-FL-COCHO

Format application/pdf (imt)

Language English

Advisor Hamm-Alvarez, Sarah (committee chair), Okamoto, Curtis (committee member), Xie, Jianming (committee member)

Creator Email klinngam@usc.edu,peepoptang_19@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-191845

Unique identifier UC11663024

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Legacy Identifier etd-KlinngamWa-7622.pdf

Dmrecord 191845

Document Type Dissertation

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Abstract (if available)

Abstract Highly increased cathepsin S (CTSS) is found in Sjögren’s syndrome (SS) patient tears and in the tears and LG of the NOD male mouse, a murine model of SS. SS is a systemic autoimmune disease associated with lacrimal gland (LG) and salivary gland (SG) inflammation which leads to dry eye and dry mouth. In the first part of my dissertation (Chapter 2-3) that was already published in International Journal of Molecular Sciences (Klinngam et al., 2018), I demonstrated utilizing recombinant human CTSS at the same elevated enzymatic activity seen in SS patient tears, that exposure of cultured human corneal epithelial cells (HCE-T cells) to CTSS elicits acute and chronic inflammatory responses. Specifically, CTSS can promote an acute increase in pro-inflammatory cytokine gene and protein expression, including IL-8, IL-6, TNF-α, and IL-1β, and also induce later increases in MMP-9 gene expression, gene and protein expression of CTSS itself, in this in vitro model system. One possible mechanism underlying these CTSS-induced effects may involve CTSS activation of protease-activated receptor-2 (PAR-2), as demonstrated by the inhibition some effects in HCE-T cells transfected with PAR-2 siRNA before exposure to acute and chronic CTSS. In PAR-2 siRNA transfected HCE-T cells exposed to CTSS, the increased expression of IL-6, IL-1β, TNF-α, and MMP-9 in cell culture medium was reduced in response to acute CTSS exposure. On the contrary, the increased IL-8 protein expression in both cell culture medium and cell lysates did not change in PAR-2 siRNA transfected cells after acute CTSS treatment. These results suggest that CTSS activation of PAR-2 plays an important role in acute induction of IL-6, IL-1β, TNF-α, and MMP-9 secretion while, the increased IL-8 expression might be through a PAR-2 independent pathway. Additionally, I found that gene expression of 4 pro-inflammatory cytokines (IL-8, IL-6, TNF-α, and IL-1β) and 2 proteases (MMP-9 and CTSS) after chronic CTSS exposure in PAR-2 siRNA transfected cells were decreased relative to scrambled siRNA transfected cells suggesting that chronic CTSS activation of PAR-2 and/or the early increased inflammatory mediators may affect the late induction of these inflammatory responses. ❧ CTSS appears to play an important role in ocular surface and LG inflammation in SS. In the second part of my dissertation (Chapter 4-5), I utilized the peptide-based inhibitor, Z-FL-COCHO (Z-FL) administered to 14-15 weeks male NOD mice by systemic intraperitoneal (i.p.) injection for 2 weeks or topically as twice daily eyedrops for 6 weeks to explore CTSS’s utility as a therapeutic target for mitigating ocular manifestations of SS. Systemic intraperitoneal (i.p.) injection for 2 weeks significantly reduced CTSS activity in tears, LG and spleen, significantly reduced total lymphocytic infiltration into LG and T cell and pan-macrophage abundance within lymphocytic infiltrates, and significantly increased stimulated tear secretion. Topical administration of Z-FL to a different cohort of 14-15 week male NOD mice for 6 weeks significantly reduced only tear CTSS while not affecting LG and spleen CTSS and attenuated the disease-progression related reduction of basal tear secretion, while not significantly impacting lymphocytic infiltration of the LG. In addition, I also repeat i.p. study with another form of Z-FL, Z-FL-hydrate, with the equipotent dose with Z-FL. The results still showed the reduction of CTSS activity in LG and spleen lysates, reduced LG lymphocytic infiltration area, and increased stimulated tear secretion. These findings suggest that CTSS inhibitors administered either topically or systemically can mitigate aspects of the ocular manifestations of SS. In conclusion, CTSS appears to mediate aspects of ocular surface inflammation and to represent a potential therapeutic target in SS.