University of Southern California Dissertations and Theses

The histone methyltransferase SMYD5 plays a role in regulating plasma-cell antibody production and macrophage inflammatory cytokine secretion in vivo

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The histone methyltransferase SMYD5 plays a role in regulating plasma-cell antibody production and macrophage inflammatory cytokine secretion in vivo

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Content 1

The Histone Methyltransferase SMYD5 Plays a Role in Regulating
Plasma-Cell Antibody Production and
Macrophage Inflammatory Cytokine Secretion in vivo

By
Suzi Sanchez

A Dissertation Presented to the Faculty of the
UNIVERSITY OF SOUTHERN CALIFORNIA GRADUATE SCHOOL
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR of PHILOSOPHY
(GENETICS, MOLECULAR, AND CELLULAR BIOLOGY)
December 2014

2

Dedications
I would like to dedicate this dissertation to my family; especially to my mother and father for all
of the encouragement and support they have bestowed upon me throughout my educational
journey. Your unconditional love provided me with the ability to believe in myself in the face of
opposition. Without you I would not have achieved this goal, and for that, I can’t thank you
enough. I would also like to thank my husband Istvan Zambori, for being a friend and an
inspiration.

3

Acknowledgements
I would like to thank all of my committee members, Dr. Si-Yi Chen, Dr. Minnie
McMillan, Dr. Andre Ouellette, Dr. Omid Akbari, and Dr. Weiming Yuan for the academic
support and advice they have provided to me throughout the last four years. I am very grateful to
have met such an inspirational and kind group of individuals. All of you truly make the
University of Southern California feel like a community for me. I would also like to say a very
special thanks to my mentor Dr. Si-Yi Chen, who has made it possible for me to grow as a
scientist.
Secondly, I want to thank all of my previous colleagues, whom I now have the privilege
to consider very close friends. Thanks you for making me feel welcome and appreciated in the
lab. I also want to thank all of you for training me and passing on your knowledge during our
years of working together. I want to thank Vijayalakshmi Nandakumar for always being a great
friend and the voice or reason, Haejung Won for always making me laugh, Peter Yates for
making the mundane more interesting, and Lindsey Jones, for always making time for
conversation, paper editing, and being a great friend.
Funding
This work was supported by grants from the National Institute of Health [R01CA090427,
AI084811, CA116677 and AI068472 to S.Y.C.] and [CA100841 and AI08185 to X.F.H.] and the
Leukemia & Lymphoma Society SCOR Award to S.Y.C.

4

List of Tables
Table 1. List of B-cell primers 118
Table 2. List of M1 and M2 macrophage primers 119

5

List of Figures Chapter I

Figure 1.1A. Histone modifications of H3 and H4 ........................................................... 13
Figure 1.1B. Models depicting the relationship between DNA methylation, histone
deacetylation, and histone methylation .............................................................................13

Figure 1.2. Schematic representation of the five mammalian SMYD5 proteins... ............15
Figure 1.3. Tissue specific expression of human SMYD5 mRNA ................................... 19
Figure 1.4. Tissue specific expression of murine SMYD5 mRNA ..................................20
Figure 1.5. Expression of SMYD subfamily in murine macrophages .............................. 21
Figure 1.6. The HMT SMYD5 is a negative regulator of inflammatory
response genes .................................................................................................................. 22

Figure 1.7. SMYD5 trimethylates H4-K20 at TLR-4 responsive promoters ................... 23
Figure 1.8. Control of proinflammatory gene programs by regulated trimethylation
and demethylation of histone H4-K20 ...............................................................................24

Figure 1.9. Epigenetic marks and elements, and their functions in the
antibody response.............................................................................................................. 30

Figure 1.10. Epigenetic regulation of inflammatory cytokine gene loci
in macrophages ................................................................................................................33

Figure 1.11. IL-21 is a master regulator of B-cell differentiation .....................................38
Figure 1.12. Macrophage lineages, ontogeny, and contribution to populations
of resident tissue macrophages ......................................................................................... 39

Figure 1.13. Distinct macrophage subsets regulate inflammation and wound healing .... 43

6

List of Figures Chapter II

Figure 2.1. SMYD5 mutagenesis cassette ......................................................................... 54
Figure 2.2. Confirmation of SMYD5 genetic inactivation ................................................. 55
Figure 2.3. SMYD5 is dispensable for B-cell and T-cell development .............................. 56
Figure 2.4. SMYD5 is dispensable for marginal zone and follicular
B-cell development ..............................................................................................................57

Figure 2.5. Reduced sera Ig levels in response to TD-antigen in vivo ............................... 59
Figure 2.6. Reduced IgG1 secretion from SMYD5-KO splenocytes .................................60
Figure 2.7. SMYD5 deficient mice show decreased plasma cell numbers ..........................61
Figure 2.8. In vitro analysis of PC differentiation in SMYD5 deficient B-cells ................ 63

Figure 2.9. In vitro analysis of Ab secretion from SMYD5 deficient B-cells .....................64

Figure 2.10. Decreased germinal center B-cells in spleens of SMYD5-KO mice ............. 65

Figure 2.11. Decreased CD19
+
and B220
+
B-cell numbers in vivo .....................................66

Figure 2.12. Decreased proliferation in GC B-cells in the absence of SMYD5 in vivo ......67

Figure 2.13. SMYD5 plays a role in CD4
+
T
FH
cell proliferation in vivo............................69

Figure 2.14. Lack of SMYD5 results in CD4
+
T-cell proliferation changes .......................70

Figure 2.15. SMYD5 deficient CD138
+
cells show sustained expression of Bcl6
and Pax5 and decreased expression of IRF-4, Xbp1, and Blimp-1 .................................. 72-73

Figure 2.16. SMYD5 does not play a role in B-cell cytokine regulation ......................... 74-75

7

List of Figures Chapter III

Figure 3.1. SMYD5 plays a role in repressing systemic LPS-induced inflammatory
responses in vivo ...............................................................................................................90

Figure 3.2. SMYD5 deficient peritoneal cavity macrophages show increased
inflammatory cytokine production in response to LPS challenge in vivo ................... 91-92

Figure 3.3. SMYD5 is not important for activation marker expression ............................93

Figure 3.4. Lack of SMYD5 leads to spontaneous increases in peritoneal cavity
monocytes/macrophages in vivo ........................................................................................94
.
Figure 3.5. Lack of SMYD5 leads to increased elicited peritoneal cavity
monocytes/macrophages in response to LPS in vivo .........................................................95
.
Figure 3.6. Lack of SMYD5 leads to increased Ly6C
+
inflammatory peritoneal
cavity monocytes in vivo ...................................................................................................96

Figure 3.7. SMYD5 is not essential in splenic monocyte/macrophage responses....... 97-98

Figure 3.8. Cytokine expression within splenocytes of SMYD5 deficient
mice ...................................................................................................................................99
.
Figure 3.9. Increased inflammatory cytokines from SMYD5
-/-
BMDCs and BMMS .....101

Figure 3.10. Decreased M2 gene expression in M-BMMs derived from
SMYD5
-/-
mice ......................................................................................................... 102-103

Figure 3.11. Role of SMYD5 in GM-BMM M1 polarization in response to
Salmonella typhimurium ................................................................................................. 104

Figure 3.12. Increased cytokine secretion and bacterial killing in ST infected
SMYD5-KO GM-BMMs .................................................................................................105

8

Table of Contents
Dedications......................................................................................................................... 2

Acknowledgements ........................................................................................................... 3

List of Tables .....................................................................................................................4

List of Figures ................................................................................................................ 5-7

CHAPTER I. Epigenetics and Immunological Regulation ......................................... 10
Epigenetics and Gene Regulation ......................................................................................11
Epigenetic Gene Regulation by SMYD Family Proteins ................................................. 14
Biological Significance of Histone Methylation .............................................................. 25
The Function of B-Cells in Adaptive Immunity ................................................................35
Biological Function of Macrophages .................................................................................39

CHAPTER II. The Histone Methyltransferase SMYD5 Plays a Role in
Regulating Plasma Cell Differentiation and Antibody Production ............................ 44

Abstract ............................................................................................................................ 45

Introduction .......................................................................................................................46

Materials and Methods .......................................................................................................49

Results and Figures ............................................................................................................54

Discussion ..........................................................................................................................76

9

CHAPTER III. Increased Peritoneal Cavity Macrophages and Systemic
Sensitivity to LPS in the Absence of the Histone Methyltransferase SMYD5 .......... 81

Abstract ...........................................................................................................................82

Introduction ...................................................................................................................... 83

Materials and Methods ......................................................................................................86

Results and Figures .......................................................................................................... 90

Discussion .......................................................................................................................106

CHAPTER IV: Conclusions and Future Studies .......................................................110

Related Publications by the Author .............................................................................120

References ......................................................................................................................121

10

Chapter I

Epigenetics and Immunological Regulation

11

Epigenetics and Immunological Regulation

Epigenetics and Gene Regulation
At least three epigenetic processes work in concert to control higher order chromatin
structure in the mammalian cell nucleus: DNA methylation, histone modifications, and ATP-
dependent chromatin remodeling [1]. Histones are subject to more than 100 post-translational
modifications, including methylation, acetylation, phosphorylation, sumoylation, ADP-
ribosylation, citrullination, and ubiquitination [2, 3]. Modifications such as phosphorylation and
acetylation are reversible, on the other hand, methylation is a more stable modification involved
in creating a heritable cellular memory of a specific transcriptional program [4]. According to the
“Histone Code Hypothesis” histones act as signaling platforms, integrating upstream signaling
pathways with gene regulation at the transcriptional level.
Both arginine and lysine residues can be methylated at multiple positions on histones H3
and H4 (Figure 1.1 A). Lysine methylation is associated with both repression and activation of
transcription [5]. Specifically, methylation at lysine 4 (H3-K4), lysine 36 (H3-K36) and lysine 79
(H3-K79) of histone H3 activates transcription; whereas methylation at lysine 9 (H3-K9) and
lysine 27 (H3-K27) of histone H3, and methylation at lysine 20 of histone H4 (H4-K20) are
epigenetic marks of a repressed chromatin state [6]. The level of specificity is heightened by the
number of lysines added to a particular residue, which range from mono-, di-, or tri- methylation.
Histone modifications at independent sites are known to work in tandem with other types of
modifications located across the genome, and such interplay can bring about unique biological
consequences.
The various biological effects of histone methylation are likely due to the action of
recruited proteins which contain binding domains that recognize methylated lysines. For
12

example, three groups have reported that the chromodomain of HP1/Swi6p recognizes H3-K9
methylated by SUV39H1/Clr4, and that this association leads to heterochromatin formation and
gene silencing [7]. Recent evidence also indicates that H3-K4 methylation recruits the chromatin
remodeling factor CHD1 [8]. Nevertheless, a single histone modification is not an indicator of
whether a gene will be expressed or repressed, since the chromatin state may change if other
modifications are simultaneously present. For instance, H3-K9 methylation can also activate
transcription in the presence of H3-K27 in combination with H4-K20 methylation [1]. It is now
known that histone methylation and DNA methylation may occur in concert, and many histone
methyl transferases (HMTs) contain methyl-CpG binding domains. Zhang and Reinberg [1]
propose 3 possible models to explain how DNA and histone methylation work together to control
chromatin structure and gene transcription (Figure 1.1 B).

13

14

Epigenetic Gene Regulation by SMYD Family Proteins

The SMYD5 protein is part of the SET and MYND (SMYD) domain containing family
of methyltransferases. This family of proteins contains a SET domain that is split into two
segments, with an MYND zinc finger domain located between them, followed by a cysteine-rich
post SET domain (Figure 1.2) [9]. The MYND domain is found in a large number of proteins
with important roles in normal development and cancers, and has been shown to mediate
protein–protein interactions, mainly in the context of transcriptional regulation [9]. Based on
phylogenetic analysis the SMYD subfamily can be divided into nine orthology groups, named
SMYD1–SMYD5, SUV4-20, SETD7, SETD8 and MLL5. It is believed that the SMYD1–
SMYD5 genes originated from an ancestral gene in the common ancestor of vertebrates and
invertebrates [10].
This family of proteins are important in gene regulation due to enzymatic action of their
SET domain, which catalyzes methylation. A large body of evidence suggests that histone lysine
methylation plays a critical role in regulating transcription, X chromosome inactivation, DNA
methylation, and chromatin structure [11, 12]. It is known that SMYD1 and SMYD3 catalyze
lysine-4 methylation on histone H3 (H3-K4), while SMYD2 catalyzes lysine-36 methylation on
histone H3 (H3-K36) [13]. Additionally, SMYD2 methylates lysine residues present on non-
histone proteins [14]. SMYD5 catalyzes H4-K20me3 on promoters of multiple TLR4 target
genes through its association with a larger molecular complex called the NcoR corepressor
complex [15]. To date, a thorough characterization of the catalytic activity of SMYD4 has not
been published.

15

In humans SMYD1, SMYD2, and SMYD3 share a high degree of sequence homology,
and all 5 SMYD proteins contain both the SET and MYND domains. SMYD1, SMYD2, and
SMYD3 share a C- terminal protein interaction tetratrico peptide repeat (TPR) domain, which is
absent in SMYD5. Using a combination of immunoprecipitation coupled with high throughput
mass spectrometry, protein interaction networks have been proposed for human SMYD 2, 3, and
5 [14]. Previous studies report that these three proteins have overlapping and unique binding
partners, and these interactions govern their localization and functions. It was found that SMYD2
and SMYD3 share up to 14 interacting partners, while only 6 interactors were identified for
SMYD5 [14]. To date multiple reports have been published regarding the various biological
functions of the SMYD proteins.

16

Studies on SMYD1 have identified this protein as being critical for differentiation of
cardiomyocytes and heart morphogenesis in developing mouse embryos [16]. SMYD1 is the
main binding partner of the muscle specific transcription factor skNAC in the developing heart,
and binds via the MYND domain to the PXLXP domain of this target proteins [17]. In zebrafish
simultaneous knockdown of Smyd1a and Smyd1b resulted in a complete disruption of myofibril
organization in skeletal muscles, however, individual knockdown of each gene copy did not
result in any visible defects [18]. Additionally, SMYD1 functions as a histone deacetylase
(HDAC) dependent transcriptional repressor [8].
SMYD2 has been shown to interact with an HDAC called Sin3A, and this complex is
associated with transcriptional repression, despite the known H3-K36 methylation specificity of
SMYD2 – which is usually associated with transcriptional activation[19]. Further studies showed
that SMYD2 regulates transcription through its association with the RNA helicase HELZ and
RNA polymerase II [20]. Interestingly, this protein can also methylate non-histone targets, and
SMYD2 has the capacity to act as an oncogene by eliminating the tumor supressor function of
p53 by methylating p53-K370 [21]. SMYD2 also regulates the association between the
transcriptional repressor L3MBTL1 and the cell cycle regulator retinoblastoma tumor suppressor
(RB) through its methylation of RB-K860, which allows RB to bind L3MBTL1 [22]. Besides its’
role in the nucleus, SMYD2 is also functional in the cytoplasm of cardiomyocytes and skeletal
muscle myocytes. In skeletcal muscle mycoytes SMYD2 serves a protective function by
ensuring proper sarcomeric organzation. Here SMYD2 methylates the chaperone Hsp90, thus
promoting the interaction of a SMYD2-methyl-Hsp90 complex with the N2A-domain of titin
[18]. In zebra fish Smyd2-knockdown using an antisense oligonucleotide morpholino approach
strongly impaired cardiac performance [18].
17

SMYD3 forms part of the RNA polymerase II complex as it also associates with the
RNA helicase HELZ. Furthermore, SMYD3 can directly bind DNA [23]. Of biological
significance, SMYD3 is overexpressed in most hepatocellular (HCC) and colorectal carcinomas
(CRC), and it is upregulated in proliferating breast cancer cells [23]. However, mechanistically it
is unknown what role this protein plays in tumorigenesis. Recently it was reported that in mouse
models of K-Ras driven cancers, SMYD3 methylates a lysine residue (K260) on MAP3K2, a
kinase that when methylated potentiates cellular signaling through the MEK–ERK mitogen-
activated protein-kinase pathway [24].
SMYD4 has not been extensively studied, and little is known about its binding partners
or histone substrate. However, it has been reported that SMYD4 may serve a tumor suppressor
role in mammary carcinogenesis. Using an in vitro loxP-Cre system to inactivate SMYD4 in a
non-tumorigenic mouse mammary epithelial cell line NOG8, it was found that decreased
SMYD4 resulted in malignant transformation. Ectopic expression in nude mice resulted in tumor
formation. Furthermore, microarray analysis revealed that platelet-derived growth factor receptor
alpha polypeptide (Pdgfr-α) was highly expressed in tumor cells compared with normal NOG8
cells following knockdown of Smyd4 [25]. Additionally, a SMYD4 homologue has been
identified in drosophila, termed dSmyd4, and it is reported to be involved in adult fly muscle
development. Genetic and biochemical studies revealed that dSmyd4 interacts selectively with
Ebi, a component of the dHDAC3/SMRTER co-repressor complex. During embryogenesis
dSmyd4 is expressed throughout the mesoderm, and RNAi knockdown of dSmyd4 results in a
lethality rate of 80% during the final stage of Drosophila development [26].
To date, there is only one published study characterizing the biochemical function of
SMYD5. In 2012, Stender et al, reported that SMYD5 catalyzes tri-methylation of lysine 20 on
18

H4 [15]. In humans Smyd5 is expressed in multiple organs (Figure 1.3). Additionally, the
Eurexpress Transcriptome Atlas Database has reported that based on RNA in-situ hybridization
studies Smyd5 was found to be expressed in the Haemolyphoid system (spleen and thymus
primordium) in the developing mouse embryo. A comparison of SMYD family gene expression
levels (bioGPS.org) from various adult mouse tissues shows that Smyd5 is highly expressed in
embryonic stem cells, macrophages, and multiple hematopoeitic cell lineages relative to
Smyd1,2,3, and 4 (Figure 1.4). Published data reports that the SMYD family of proteins are all
expressed in primary mouse macrophages, with Smyd5 and Smyd3 showing the highest
expression levels [15] (Figure 1.5).

19

20

21

Furthermore, the Stender et al paper showed that knockdown of SMYD5 using an siRNA
approach results in inreased expression of the TLR4 target genes: ILα, IL1β, TNF, Ccl4, and
Cxcl10 in thioglycollate elicited primary mouse macrophages,following stimulation with a
purified LPS; Kdo2 lipid A (KLA) (Figure 1.6 A). Using a luciferase reporter assay they also
found that over expression of SMYD5 leads to repression of a KLA-activated TNF reporter gene
construct, however, transfection with a mutant SMYD5 lacking HMT activity reversed this
transcriptional repression (Figure 1.6 B).

22

Using a histone methylation in vitro assay the authors determined that SMYD5
specifically catalyzes trimethylation of H4-K20, a repressive histone mark. Furthermore, they
found that siRNA knockdown of SMYD5 resulted in a drastic decrease of H4-K20me3 at these
promoters, but did not affect the total amount of H-4 present (Figure 1.7A). In order to determine
if SMYD5 localized to these promoters, a SMYD5-biotin fusion protein was constructed and
used for streptavidin-based ChIP assays to detect enrichment for SMYD5 on the Tnf and Cxcl10
promoters under basal conditions. They reported that SMYD5 was indeed detected at both sites,
and that activation of macrophages with KLA resulted in a large decrease of SMYD5 occupancy
at these sites (Figure 1.7 B). Additionally, this paper reports that SMYD5 co-immunoprecipitates
as part of a larger molecular complex called NCoR, and shRNA-mediated knockdown of
23

NCoR leads to loss of SMYD5 localization to the Tnf and Cxcl10 promoters in RAW
264.7 macrophages. Interestingly, they also identified a novel H4-K20me3 demethylase called
PHF2, which catalyzes the removal of H4-K20me3 at many of the promoters targeted by
SMYD5 upon KLA activation. Data from this study culminated in the proposal of a mechanistic
model describing negative regulation of TLR-4 pro-inflammatory genes by the HMT SMYD5
(Figure 1.8). Clearly research indicates that SMYD proteins are important in epigenetic gene
regulation. However, although they share the SET/MYND domains each has a unique
architecture, and further research is needed to detrmine their biological function.

24

25

Biological significance of histone methylation
Studies on the biological function of histone methylation report that epigenetic gene
regulation plays a major role in immune responses, warranting further investigation of lysine
methyl transferases (KMTs) through the use of in vivo models. H4-K20 methylation is
significantly important for the maintenance of genome integrity, and processes such as DNA
damage repair, DNA replication, and chromatin compaction. Multiple KMTs have been
characterized, and genetic inactivation of SET8/PR-Set7 enzymes that catalyze monomethylation
of H4-K20, or SUV4-20H1 and SUV4-20H2 which catalyze di and tri methylation, results in
genomic instability [27]. In mammalian cells the majority of H4 methylation is found on the N-
terminal region of lysine 20, furthermore, this epigenetic mark is evolutionarily conserved from
yeast to humans. K20 methylation can exist in the mono, di, and tri states, and each of these
states results in a different biological outcome. Research shows that H4-K20me1 and H4-
K20me2 are involved in DNA replication and DNA damage repair, whereas trimethylated H4-
K20 (H4-K20me3) is a hallmark of silenced heterochromatic regions and transcriptional
repression [28-31] . H4-K20 methylation is just one of many possible histone modifications that
result in abnormal cellular activity. Currently, there is a consensus in the scientific community
that an in depth knowledge of epigenetic mechanisms may be essential in developing new
therapeutic approaches to combat autoimmune diseases, hematological disorders, and organ
transplantation rejection [32].
Innate and adaptive immune responses are orchestrated by highly specialized cells of
hematopoietic origin, which are influenced by the microenvironment in which they are
differentiated and activated. For example, CD4
+
T-cells give rise to multiple flexible lineages
such as: Th17, induced regulatory T (iTreg) cells, Th1, Th2, and memory T-cells. Studies in
26

humans have revealed that lineage determining events giving rise to Th1 and Th2 cells are
epigenetic in nature. Epigenetic histone marks regulating Th1/Th2 cell fate decisions are
deposited across the IF N γ locus by multiple transcription factors. These include (STAT4 and T-
bet) for Th1, or (STAT6 and GATA-3) for Th2. The IF N γ gene displays acetylation of H4
(AcH4) and H3-K4me3 in Th1 cells, this exists in combination with H3-K27 di- and tri-
methylation in Th2 cells [33, 34]. During the formation of memory T-cells multiple epigenetic
marks regulate expression of the transcription factors T-bet and eomesodermin, the cytokines IL-
2 and IFN-γ, and the molecules: CD70, CD40L, ITGAL, PRF and CCR6, which together are
essential for controlling function and transcriptional profiles [35].
DNA methylation and histone methylation also play a major role in B-cell lineage
differentiation and in the maturation of the antibody response to foreign antigens. When mature
B-cells encounter foreign antigens in secondary lymphoid organs they undergo differentiation
giving rise to antibody producing plasma cells and memory B-cells [36]. In non-active resting B-
cells the Ig heavy chain (Igh) locus exists in a closed chromatin state with little accessibility to
transcription factors, and this area is marked with H3-K9me3 and H3-K27me3 promoting the
maintenance of this B-cell state [37, 38]. Furthermore, resting B-cells undergo V
H
DJ
H
-Cμ
transcription giving rise to the expression of the B-cell receptor (BCR), which is comprised of
the Igμ and Ig δ heavy chains. Chromatin in the transcribed Igh V
H
DJ
H
(V, variable; D, diversity;
J, joining) regions is characterized by the presence of DNA hypomethylation [39].
The differentiation of B-cells into proliferating hyper-mutating cells undergoing class
switch recombination (CSR) involves genome wide DNA-hypomethylation and multiple histone
modifications. Both somatic hypermutation (SHM) and class switch recombination (CSR) are
27

largely controlled by epigenetic gene regulation of the multiple factors involved in these
processes, as well as epigenetic control of the chromatin state of the V
H
DJ
H
DNA segment.
Somatic hypermutation (SHM) is critical to the antibody response and involves insertion of point
mutations in Ig V(D)J DNA, which gives rise to antibodies with higher affinity for particular
antigens. SHM involves the coordinated recruitment and enzymatic activities of multiple factors
at V(D)J DNA to facilitate transcription of this region. Research shows that both DNA
methylation and histone modifications work in concert during this process. Specifically, DNA
demethylation facilitates SHM targeting by promoting H3-K9ac/K14ac, H4-K8ac, and H3-
K4me3 histone modifications which in turn promote an open chromatin state allowing access to
error prone DNA polymerases, error prone translesion DNA synthesis (TLS) polymerase, and
activation induced deaminase (AID) [40].
CSR is also mediated by the action of activation induced deaminase (AID) and multiple
DNA polymerases and repair enzymes [41]. To initiate this process AID deaminates
deoxycytidines (dCs) leaving deoxyuracils (dUs) in their place and leading to DNA mismatches.
The result is the creation of DNA lesions due to the DNA repair process leading to either point
mutations in SHM, or double strand breaks in the CSR process [42]. By excising intervening
DNA between upstream donor (Sµ) and downstream acceptor (S) regions, CSR juxtaposes the
expressed V
H
DJ
H
DNA segment to a new C
H
exon cluster, thereby changing the C
H
chain and
effector functions of an antibody [41]. Histone modifications target the CSR machinery to the Sµ
and S sites. Research shows that the Sµ region is in a constitutively open chromatin state and is
rich in activating histone marks such as : H2BK5ac, H3-K9ac/K14ac, H3-K27ac, H4-K8ac, H3-
K4me3, and H3-K36me3 [43]. Induction of CSR involves primary T-dependent stimuli in the
form of CD40 engagement, or T-independent stimuli in the form of TLR activation and BCR
28

engagement. Secondary stimuli in the form of the cytokine interleukin (IL)-4, transforming
growth factor (TGF)-β, and interferon (IFN)-γ are also needed in mice [41]. Primary stimuli can
induce histone modifying enzymes which open up the chromatin in the S region by removing
repressive methylation in the form of H3-K9me3 and H3-K27me3 [37]. The importance of
methylation in general is exemplified by the presence of defective CSR following inhibition of
the H3-K4 KMTs: mixed-lineage leukemia (MLL)/Su(var) 3-9 factors, Enhancer of zeste, and
trithorax domain protein 1 (SET1) [3].
Plasma cells and memory B-cells are terminally differentiated cells arising mainly from
germinal center B-cells; furthermore, these cells have generally already undergone CSR and
SHM. The stimuli that promote the lineage differentiation of B-cells are not fully understood, but
research shows that many of these processes are also regulated by epigenetic mechanisms. Up-
regulation of the Prdm1 gene encoding for the master regulator Blimp-1 is associated with
increased histone acetylation, which is thought to be the result of Bcl-6 downregulation, since
Bcl-6 binds and recruits HDACs to the Blimp-1 promoter region [44]. Blimp-1 promotes and
maintains plasma cells by in turn repressing Bcl6, Pax5, Spib, and c-Myc [45]. Blimp-1 has been
shown to repress activation of these genes by recruiting G9a and H3-K9 KMTs to the Pax5 and
Spib promoter regions [46]. In memory B-cells epigenetic information is passed down from
precursor cells, and new marks are also acquired depending on the activation state of these cells.
When activated by antigen re-encounter memory B-cells can re-initiate CSR and SHM, as well
as differentiate into plasma cells. The genes needed for these changes are said to be in a ‘poised’
state because they are characterized by the presence of open chromatin marked by both active
and repressive histone marks [47].
29

Additionally, altered patterns of histone modifications combined with changes in DNA
methylation within B-cells can interact to promote neoplastic transformation and the
development of cancer. Aberrant expression patterns of tumor suppressors or oncogenes can
give rise to tumor cells [48]. Furthermore, changes in the expression of histone modifying
enzymes themselves can also lead to cellular transformation, due to the downstream effects on
the target genes of these enzymes [49]. For example, EZH2 the HMT component of the
polycomb repressor complex (PRC) 2 which catalyzes H3-K27me3, has been implicated in large
B-cell lymphoma and follicular lymphoma [50]. This example is just one of many published
works highlighting the role of histone methylation in normal B-cell function and in the antibody
response (Figure 1.9).

30

Figure 1.9 Epigenetic marks and elements, and their functions in the
antibody response
Adopted from: Trends in Immunology, Volume 34, Issue 9, September 2013, Pages
460–470
31

Epigenetic mechanisms not only regulate responses from cells in the adaptive immune
system branch such as B-cell and T-cells, they also play a major role in regulating the innate
immune response. For example, following organ transplantation dendritic cells (DCs) have the
ability to induce an inflammatory response and activate other cell types such as T-lymphocytes
and natural killer cells. Conversely, DCs can also induce tolerance and suppress the recipient’s
immune response. Studies have shown that the presence of active H3-K4me3 and repressive H3-
K27me3 histone marks at specific gene loci controls the differentiation and function of activated
or tolerized monocyte derived DCs [51].
Although there is extensive literature on the epigenetic regulation of DCs, for purposes of
this dissertation a more detailed discussion of macrophages will be presented. Macrophages are
phagocytic cells which exist in multiple polarized states, each serving a specific function at a
specific anatomical location. Macrophage polarization refers to the programming of genes by
specific signaling factors giving rise to differing functional phenotypes. Specific environmental
factors such as cytokines and microbial pathogens induce macrophage polarization, however,
epigenetic mechanisms modulate and transmit these signals into changes in gene expression
patterns within these cells allowing for plasticity and re-programming. Among the most well
characterized phenotypes are the classical M1 and alternative M2 macrophages, which are
thought to represent opposites on a functional spectrum [52]. In general M1 macrophages
function in host defense against bacterial pathogens and in inflammatory responses, while M2
cells are essential in tissue homeostasis and the resolution of inflammation to prevent tissue
damage [53].

32

In the field of macrophage biology a key concept is that genes important for the
polarization of macrophages in response to external stimuli exist in three broad states
characterized by condensed chromatin , partially open chromatin in which genes are poised for
activation, and lastly an actively transcribing state [54]. In the first state, the presence of
repressive H3-K9me3 and H3-K27me3 methylation, results in a closed chromatin conformation
denying DNA accessibility to transcription factors. Secondly, there is a poised state characterized
by the presence of activating histone marks H3-K4me3 and H3-K9, 14-Ac, where chromatin
exists in a partially open conformation. Transcription at poised genes is restrained by
simultaneous presence of repressive histone marks such as H3-K9me3 and H3-K27me3, and
bound corepressor complexes. Additionally, RNA Pol II is present at the promoter regions of
many of these poised genes; however, transcriptional activation requires additional signals and
ATP-dependent nucleosome remodeling. Lastly there is a third state, characterized by active
histone marks, an open chromatin configuration, and ongoing transcription (Figure 1.10.) [55].
Toll like receptor (TLR) signaling leads to M1 activation identified by the expression of
inflammatory cytokines such as TNF, IL-1β, IL-6, IL12p40, and the chemokine CXC ligand
(CXCL)10 [55]. In the absence of TLR signals these genes are repressed by factors such as Bcl-
6, and nuclear receptors that recruit corepressor complexes that contain histone deacetylases
(HDACs) and demethylases. Chromatin immunoprecipitation sequencing (ChIP-seq) studies on
macrophages have shown that Bcl-6 is involved in regulating nearly a third of TLR4 targets, and
that 90% of the Bcl-6 cistrome is collapsed following TLR4 activation by LPS [56]. Repressed
inflammatory gene loci also contain the negative histone marks H3-K9me3, H3-
K27me3, and H4-K20me3 [15]. TLR signaling activates transcription via activation of mitogen-
activated protein kinases (MAPKs), NF-κB, and IRFs. Furthermore, there is removal of Bcl-6
33

from gene loci, the activation of demethylases, removal of repressive histone methylation, and
ATP-dependent nucleosome remodeling.

34

Chromatin remodeling facilitates recruitment of signaling transcription factors such as
NF-κB, an increase in positive histone marks such as H3S10-P, H4-Ac, and H3-K4me3, as well
as release of poised polymerase II [57]. Although less is known about the signaling cascades and
epigenetic regulation of M2 alternative macrophage activation, research has shown that the
histone demethylase JMJD3 plays a major role in this process. ChIPseq analysis revealed that
most M2 genes are not directly targeted by JMJD3, however, this molecule can remove negative
H3-K27me3 marks at the Irf4 gene locus, one of the major transcription factors promoting the
M2 phenotype [58].
Recent advances have been made in identifying and developing new therapeutic
approaches that suppress harmful M1 activation and chronic inflammation. For example,
compounds that can suppress Myc expression have proven useful in treatment of Myc-driven
cancers such as acute myeloid leukemia (AML) and multiple myeloma [59]. Additionally,
compounds that can suppress histone demethylases have shown promise in suppressing
inflammatory cytokine gene expression. HDAC inhibitors have also been shown to suppress
many of the same inflammatory genes and have shown efficacy in a Phase I trial of juvenile
inflammatory arthritis [60]. Although much progress has been made in elucidating the vast
molecular interplay between epigenetic modifying enzymes and cellular function, many
questions remain to be answered in order to advance the development of new therapeutic
approaches to treat inflammatory diseases.

35

The Function of B-Cells in Adaptive Immunity
B-cells play an important role in both the humoral and cellular immune responses
through the production of soluble factors such as antibodies and cytokines, and through cellular
interactions with antigen presenting cells. There are multiple types of mature B-cells such as:
follicular I and II (FoBI and FoBII), marginal zone (MZ), and B1 cells. These cells are produced
from fetal liver before birth, and bone marrow at later stages [61]. Mature B-cells undergo
differentiation in response to specific stimuli becoming effector, regulatory, memory, and plasma
cells (PCs). Early on during development in the BM pre-B-cells and BCR expressing cells
undergo checkpoints whereby auto-reactive B-cells undergo negative selection to prevent
autoimmune disorders [62]. Furthermore, early transitional (T1) B-cells that are trafficked to the
spleen undergo another round of negative selection by peripheral tolerance mechanisms. T1 cells
that pass this checkpoint differentiate into late transitional (T2) cells expressing CD23
+
,
IgD
+
,CD21
int
, and CD95
+
. These cells then undergo positive selection by the concerted function
of the BCR and B-cell-activating factor receptor (BAFF-R) to become mature MZ, FoBI, and
FoBII B-cells [63].
The generation of effector B-cells is regulated by their interactions with cytokines,
pathogens, and other cells of the immune system such as T-cells. B-cells play a role in the type 1
and 2 immune responses that are associated with Th1/Th2 cells, by secreting cytokines. These
cells are termed Be1 and Be2 cells based on the secretion of IFNγ, IL-2, and IL-12 by Be1 cells,
and IL-4, IL-5, and IL-10 by Be2 cells [64]. Certain subsets also display immune suppressive
function. B10 B-cells, FoB-cells, and innate-like B1a cells have all been shown to secrete the
anti-inflammatory cytokine IL-10. Regulatory B-cells have been identified in multiple murine
disease models, and it is believed that the regulatory function of these cells is exerted through
36

their ability to secrete the cytokines IL-10 and TGF-β and/or by their ability to interact with
pathogenic T-cells to dampen harmful immune responses [65]. There are also innate-like cells
called MZ B-cells which can elicit an innate immune response due to the fact that they express
polyreactive BCRs and TLRs. These cells express TLRs at similar levels to DCs and
macrophages. The dual recognition by BCR and TLR of the pathogen encoded conserved
molecular patterns allow these cells to respond to bacterial pathogens and act at the front-line of
defense before the activation of the adaptive branch [66].
B-cells are also recognized as professional antigen presenting (APCs) cells, which can
activate CD4 T-cells through the presentation of antigen digestion products via their MHC II
molecules. Antigen recognition by naïve B-cells occurs in the secondary lymphoid organ
follicles and results in the activation of signaling cascades and de novo gene expression. Small
antigens reach B-cells through lymphatic fluid or blood, while larger antigens are presented to B-
cells via macrophages or DCs [67]. Upon antigen internalization B-cells can initiate the
formation of germinal centers (GCs) through successful engagement of CD4 T-cells at the T and
B cell zones within the follicles, or they can undergo T-independent activation. Activated B-cells
undergo differentiation into PCs. Long lived PCs arise from the interaction of B-cells with T
follicular helper (TFH) cells within follicles, while shorter lived extrafollicular PCs arise in a T-
independent (TI) manner. GC B-cells have the capacity to differentiate into antibody secreting
PCs or memory B-cells which provide long lived immunity. As discussed above, these events
involve substantial changes in gene expression that are mediated by epigenetic mechanisms.
Some of the major transcription factors involved in regulating PC differentiation are BCL-6, B-
lymphocyte induced maturation protein (BLIMP)-1, PAX5, X-box-binding protein-1 (XBP-1),
and IFN-induced regulatory factor 4 (IRF4) [68]. These PCs are in turn able to protect our bodies
37

in multiple ways such as; secretion of antibodies which can prevent pathogens from entering or
damaging cells by binding to them, removal of pathogens by macrophages by coating the
pathogen, and they trigger destruction of pathogens by stimulating other immune responses such
as the complement pathway [67].
TI responses are mediated through interactions with professional APCs such as
macrophages and DCs. These cells activate B-cells by delivering multiple sequential signals;
signal 1 (antigen), signal 2 (co-stimulation), and signal 3 (polarizing signals mediated by soluble
or membrane-bound factors) [69]. Additionally, cytokines have the ability to promote B cell
proliferation, differentiation, survival, class switching, antibody secretion, and various other
biological functions. For example, studies have shown that IL-4 and IL-13 co-operate in both
mice and humans to regulate Ig class switching, especially to IgE. Studies on IL-4 deficient mice
have shown that it is essential for IgE production, while IL-13 transgenic mice, exhibited
substantially increased levels of serum IgE, even in the absence of IL-4 [70]. Furthermore,
several studies have emerged showing that IL-21 is a major regulator of B-cell activation,
proliferation, PC differentiation, and Ab-secretion in both mice and humans [71]. In addition to
those mentioned above, there are multiple in vitro and in vivo studies to substantiate the role of
multiple cytokines in B cell biology (Figure 1.11). It is now well established that PCs and
memory B-cells arise from mature B-cells due to the effects of different environmental stimuli
on these cells, however, the most essential function of these cells if that they serve to protect our
immune systems from harmful encounters.

38

39

Biological Function of Macrophages
Macrophages are evolutionarily conserved phagocytic cells which arise from two distinct
pathways. Recent studies have shown that adult mouse tissues have two populations of
macrophages; those that arise during embryonic development, and those that have differentiated
from circulating monocytes [72, 73]. Early on in gestation (embryonic day [E6.5–E8.5]),
primitive hematopoiesis of restricted progenitor cells located in the yolk sac give rise to red
blood cells and non-HSC macrophages. At days (E8.5–E10.5), definitive hematopoietic stem
cells (HSCs) emerge and migrate to the fetal liver, the site of hematopoiesis until the perinatal
period, at which time the BM becomes the major site of hematopoietic development (Figure
1.12) [74].

40

Within mice there exists a monocyte-macrophage dendritic cell (DC) progenitor (MDP)
giving rise to both DCs and monocytes. Furthermore, two subsets of monocytes have been
described; the first is the classical Ly6c
hi
monocytes, which appear to be directly descended from
Ly6c
+
monocyte progenitors, and secondly the Ly6c
lo
non-classical monocytes, which
differentiate from Ly6c
hi
monocytes through an Nr4a1-dependent transcriptional program [75,
76]. These monocytes serve a patrolling function in the vasculature and in tissues. The non-
classical Ly6c
lo
cells travel along the endothelium of blood vessels and clear damaged
endothelial cells, while the classical monocytes patrol extravascular tissues to pick up antigens
for transport to draining lymph nodes without themselves differentiating into macrophages or
DCs, which was previously thought to occur once monocytes exited the circulation [77, 78].
Under inflammatory conditions the classical Ly6c
hi
monocytes differentiate into macrophages.
Macrophages differ from monocytes in a functional manner. Experimental murine models that
lack transcription factors that are essential in macrophage development display increased
perinatal mortality, reduced postnatal survival, stunted growth, and defective development of
vasculature [79, 80]. Additionally, macrophages are involved in a multitude of other functions in
the adult body such as: antimicrobial defense, antitumor immune responses, metabolism, obesity,
allergy and asthma, tumorigenesis, autoimmunity, atherosclerosis, fibrosis and wound healing,
clearing cellular debris, tissue homeostasis, antigen presentation, and recruitment of other
immune cells [81].
In general, macrophage precursors are released into the circulation when they exit the
BM, these cells then extravasate through the endothelium into tissues where they serve the major
role of replenishing the pool of macrophages and DCs [81]. These subsets are usually defined by
their function and location. To date several subsets have been described which include:
41

classically activated macrophages M1, alternatively activated macrophages M2, 'regulatory'
macrophages which secrete large amounts of interleukin-10 (IL-10), tumor associated
macrophages (TAMs), marginal zone splenic macrophages, and myeloid-derived suppressor
cells (MDSCs) [82, 83]. Nevertheless, research shows that many of these phenotypes overlap and
that macrophages have a high degree of plasticity and represent a spectrum of activated
phenotypes which change based on stimuli [82].
The classical M1 and alternative M2 macrophages have been extensively studied and
characterized and will be the focus of this dissertation. In response to tissue damage or microbial
infection M1 cells mount a pro-inflammatory response and secrete mediators such as: tumor
necrosis factor (TNF), IL-1, IL-12, IL-6, and IL-23. These mediators drive multiple
antimicrobial mechanisms and influence the polarization of T
H
1 and T
H
17 cells, which also
participate in the inflammatory response. These activated macrophages produce reactive oxygen
and nitrogen intermediates, including NO and superoxide, that are highly toxic for
microorganisms, however, excessive inflammation and oxidative bursts can also damage cells
and lead to chronic disease states [84, 85]. When tissues are initially damaged M1 cells begin the
clearance of microbial pathogens by secreting multiple factors mentioned above. Secondly
secretion of matrix metalloproteinases (MMPs) such as MMP2 and MMP9 helps to degrade the
ECM, facilitating the recruitment of inflammatory cells to the site of tissue injury. The
inflammatory response can be heightened by recruiting large numbers of T helper 17 (T
H
17)
cells and neutrophils, leading to substantial tissue damage [81]. Several of the cytokines secreted
by M1 cells have been identified as important mediators in chronic inflammatory and
autoimmune diseases, including Crohn's disease, rheumatoid arthritis, multiple sclerosis, and
autoimmune hepatitis [86, 87].
42

Conversely, alternatively activated M2 cells are able to control excessive inflammatory
responses and promote wound healing in order to prevent tissue damage. These cells have the
ability to antagonize the M1 response, and interestingly M1 cells can transition into M2 cells
depending on the microenvironmental milieu. M2 cells promote wound healing and tissue repair
by producing transforming growth factor-β1 (TGFβ1) and PDGF, which functions by promoting
fibroblast differentiation into myofibroblasts, by enhancing expression of metalloproteinase
inhibitors that block the degradation of extracellular matrix (ECM), and by directly stimulating
the synthesis of interstitial fibrillar collagens in myofibroblasts [88, 89]. Additionally, M2
macrophages express immunoregulatory proteins such as IL-10, resistin-like molecule-α (
FIZZ1), chitinase-like proteins, and arginase 1 (ARG1) which have been shown to decrease the
magnitude and duration of inflammatory responses (Figure 1.13) [58]. Regulatory macrophages
also play an immune suppressive role during the final stages of wound healing by suppressing T
cell proliferation, as well as preventing the development of excessive fibrosis by limiting
collagen synthesis (Figure 1.13) [81].

43

44

CHAPTER II

The Histone Methyltransferase SMYD5 Plays a Role in
Regulating Plasma-Cell Differentiation and Antibody Production

45

Chapter II- Abstract

The Histone Methyltransferase SMYD5 Plays a Role in Regulating Plasma Cell
Differentiation and Antibody Production
Germinal center (GC) B-cells give rise to both memory B-cells and antibody-producing
plasma cells (PCs). The generation of T-dependent long-lived antibodies requires B-cell and T
follicular helper (TFH) cell interactions, and subsequent changes in gene expression within both
cell types. In recent years extensive progress has been made in understanding the gene regulatory
programs that control plasma cell differentiation, however, the role of DNA modifying proteins
in this process remains unclear. In this study we used KO-first mice to investigate the role of the
H4-K20me3 histone methyltransferase (HMT) SMYD5 in the terminal differentiation of B-cells
into PCs and in T-dependent antibody responses. SMYD5 KO mice displayed normal B-cell and
T-cell development, and baseline serum antibody levels were unperturbed. Conversely, we report
that SMYD5 KO mice showed a decrease in plasma cell development and serum antibody levels
compared with B6 controls when challenged with NPKLH in vivo. We also observed that PCs
from immunized KO mice had decreased expression of IRF4, Xbp-1, and Blimp-1 and sustained
expression of Pax5 and Bcl6 compared to B6 controls. In vitro analysis showed that isolated B-
cells could normally differentiate into PCs despite a lack of SMYD5; however, antibody
secretion per cell was affected both in vivo and in vitro, indicating an intrinsic role for SMYD5
within B-cells. Further analysis of GC B-cells and TFH-cells indicated that extrinsic cell
interactions may also be contributing to the phenotype observed in vivo. These results provide
new insights into the role of histone modifying proteins in humoral immunity.

46

Introduction
During the humoral response GCs are the sites of B cell-T cell interactions [90], memory
B-cell formation, and plasma cell differentiation [91]. The germinal center reaction is a dynamic
process where B-cells continuously migrate from distinct areas known as light and dark zones
where they undergo multiple rounds of proliferation, somatic hypermutation, class switch
recombination (CSR), and affinity-based selection [90, 92, 93]. Furthermore, PCs and memory
B-cells arising from GCs are believed to develop along alternative routes, although little is
known about the factors that drive the fate choices of B-cells to differentiation into quiescent
memory B-cells versus antibody secreting PCs. Because lineage differentiation and cellular
function is tightly controlled at the transcriptional level, determining the role of epigenetic
regulation in normal immune function has become crucial.
Epigenetics refers to heritable changes that occur at the DNA level resulting in regulation
of gene expression patterns without changes at the sequence level. One layer of epigenetics
involves the alteration of nucleosomes through post translational modifications targeted to the N-
terminal amino acid tails of these proteins [94]. Particularly, histone methylation can lead to
changes in chromatin structural conformation and thus DNA accessibility [94, 95]. Deposition of
specific epigenetic marks is essential for proper immune function. For example, in human CD4
+

T-cells, genome wide ChIP-Seq experiments have revealed that activation-inducible genes are
marked by specific epigenetic signatures [96]. Additionally, hematopoietic stem cells undergo
extensive chromatin changes upon differentiation [97] . In recent years, studies have shown that
the activities of multiple lysine methyltransferases (KMTs) play crucial roles in regulating gene
expression, cell cycle, immune function, and cellular differentiation [15, 94, 95, 98, 99].
47

Previous studies have characterized many of the cell intrinsic and extrinsic factors that
regulate B-cell terminal differentiation. At the transcriptional level, B-cells and plasma cells are
maintained by the influences of two opposing groups of transcription factors. Those that are
believed to promote the B-cell phenotype include Pax5, Bach2, and Bcl6 [68]. Conversely, Irf4,
Blimp1, and Xbp1 are believed to promote the differentiation of B-cells into PCs [68]. At both
the cellular and transcriptional levels, CD4
+
germinal center T follicular helper(GC TFH) cells
are the specialized providers of B-cell help [100] and are important for the formation and
maintenance of the germinal center reaction and B-cell selection [100, 101]. The GC TFH cell
precursors, which are in a less polarized state and are referred to as pre-TFH cells are typically
located at the T:B border where they prime B-cells and trigger their proliferation [102]. In vivo
TFH cells have been isolated as conjugates linked to B-cells undergoing CSR. In this case IL-4
and IFN-γ cytokine secretion by T-cells had direct influences on the isotype and affinity of
antibodies produced by the interacting B-cells [103]. Furthermore, IL-21 secreted by TFH cells
is required for normal plasma cell formation in the spleen and bone marrow of mice challenged
with protein antigens [104]. At the transcription level IL-21 induces plasma cell differentiation
via induction of Blimp-1 expression in a STAT3 dependent manner [105, 106].
The SMYD (SET and MYND domain) family of (KMTs) are a group of five proteins that
are involved in a multitude of cellular processes, such as cardiogenesis, myofibril organization,
cell cycle regulation, inflammatory responses, and gene regulation have been linked to various
forms of cancer [9, 14, 15, 21, 25, 107-111]. SMYD5 is a unique member of this family because
it lacks the C-terminal tetratrico peptide repeat (TPR) domain found in the other four family
members [14]. SMYD5 has been characterized as a retinoic acid response gene and contains
multiple RXR/RAR binding elements within its promoter [112]. In vitro analysis has shown that
48

SMYD5 catalyzes trimethylation of lysine 20 on histone H4 (H4-K20me3) by association with
the co-repressor NcoR complex, leading to repression of inflammatory genes within
macrophages [15]. Nevertheless, the in vivo role of SMYD5 in regulating immune responses has
not been explored. Here we used SMYD5

deficient mice to study the role of this protein in the B-
cell adaptive immune response. The data presented in this study identifies a role for SMYD5 in
the indirect regulation of PC differentiation and in regulating antibody secretion.

49

Materials and Methods
Mice and immunizations
SMYD5 KO-first (Smyd5
tm1a/tm1a
) mice were created by the insertion of a mutant cassette within
an intronic region of the open reading frame. This cassette carries a 3’polyadenylation
termination signal leading to the early truncation of transcripts and subsequent degradation of
mRNA. Mice were purchased from the Welcome Trust Sanger Institute (Hinxton, UK). All mice
were bred in a pathogen-free barrier facility (USC) and all experiments were approved and
performed in accordance to the regulations of the University of Southern California Institutional
Animal Care and Use Committee. SMYD5
-/-
mice and aged matched control mice, 8-12 weeks
old, were immunized intraperitoneally (i.p.) with 100 µg of NP-keyhole limpet hemocyanin (NP-
KLH) precipitated with alum in a 1:1 ratio. To induce a recall response, mice were immunized
with the same antigens at least 6 weeks after the initial immunization.
Flow cytometry and BrdU analysis
Single-cell suspensions of bone marrow (BM), draining lymph nodes (dLNs), and spleen were
prepared by mechanically disrupting tissues through a 40 μm cell strainer (BD Falcon). Red
blood cells were removed using cell lysis solution (BD Biosciences) according to manufacturer’s
instructions. All cells were first incubated for 20 min at 4° C with CD16/CD32 Fc-blocking
antibody (2.4G2), in flow cytometry buffer (1x PBS, 1-2% FBS), followed by incubation with an
array of antibodies conjugated to the following fluorophores: fluorescein isothiocyanate (FITC),
phycoerythrin (PE), peridinine chlorophyll protein complex–cyanine 5.5 (PerCP-Cy5.5),
phycoerythrin-indotricarbocyanine (PE-Cy7), allophycocyanin (APC), or allophycocyanin-
indotricarbocyanine (APC-Cy7). For each staining procedure between 0.2 - 1.0 x10
6
events were
50

collected for analysis. The following antibodies were purchased from BD Biosciences: CD16/32
(2.4G2), CD38 (90/CD38), B220 (RA3-6B2), Gr-1 (RB6-8C5), CD138 (281-2), IgG1 (X56),
CD3 (145-2C11), TCRβ chain (H57-597), GL-7 (GL-7), Fas (Jo2), CD19 (SJ25C1), IgM (G20-
127), IgG1 (G17-1), PD-1 (J43), CXCR5 (2G8), and CD8a (53-6.7). The following antibodies
were purchased from eBioscience: IgD (11-26C), CD19 (1D3), CD44 (RM4-5), and CD150
(9D-1). The following antibodies were purchased from BioLegend: CD23 (B3B4), IgM (331.12),
and CD4 (L3T4); except NP (Biosearch Technologies INC, Novato, CA). All were matched to
isotype controls. Data were collected on a FACSCanto II (BD) and analyzed with FlowJo
software (TreeStar). For BrdU labeling, mice were given intraperitoneal injections of 2 mg BrdU
(Sigma-Aldrich) 3 hours before being euthanized. BrdU incorporation was assessed using a
BrdU flow kit (BD Pharmingen) according to the manufacturer’s instructions.
Intracellular staining
Single cell suspensions from spleens were prepared as described above. Cells were stained with
surface antibodies and fixed for 20 min at 4° C using (BD Cytofix/Cytoperm). Cells were then
washed 2x using (BD Perm/Wash), followed by 30 min incubation in Perm/Wash buffer at 4° C
to permeabilize the cellular membrane. Cells were then stained with fluorescent-conjugated
antibodies IL-21( FFA21), IL-10 (JES5-16E3), and IL-6(MP5-20F3) for 30 min at 4° C,
followed by a washing step in FACS buffer (1x PBS with 2% FBS) before analysis.

B-cell cultures and cell isolation
Splenic B-cells were isolated through positive selection of B220-expressing cells using magnetic
cell separation with the MACS system (Miltenyi Biotech) according to the manufacturer’s
instructions. Splenic CD138
+
, CD45R(B220)
low/–
, CD19
low/–
antibody-secreting plasma cells

51

were isolated using a plasma cell isolation kit (Miltenyi Biotech). For in vitro plasma cell
differentiation assays, cells were cultured and stimulated as described in [113]. For in vitro B-
cell cytokine secretion assays isolated B-cells were plated at 1x10
^6
cells per well and stimulated
with LPS (5ug/ml), and F(ab’)2 fragment goat anti-mouse IgM μ chain specific (5ug/ml)
purchased from (Jackson Immuno Research), in the presence of GolgiPlug 1:1000 (BD
Biosciences). Cells were then used for intracellular staining as described above.

ELISA and ELISPOT assay
Ig concentrations from supernatant and sera were determined by Enzyme-Linked Immuno-
Sorbent Assay (ELISA). To measure the relative amounts of NP-specific serum antibodies,
polystyrene high binding plates (VWR) were coated with 25 μg/ml NP-(25)-BSA (Biosearch
Technology) overnight at 4° C. Biotinylated secondary antibodies were used to detect each
isotype. Plates were washed to remove unbound secondary antibodies and then incubated with
streptavidin-HRP (Sigma-Aldrich), followed by TMB substrate reagent (BD). To standardize and
quantify relative amounts of NP-specific Ig responses, all experimental samples were diluted
1:10,000 and compared with a standardized dilution of pooled serum obtained from immunized
B6 mice. For all other ELISA assays concentrations were determine by comparison to a standard
curve using mouse reference serum (Bethyl Laboratories; RS10-101). ELISPOT assays for
detection of NP-specific and all other Ig isotypes were performed using 96-well multiscreen
membrane filtration plates (Millipore) coated with 20 μg/ml NP(25)-BSA, or 4ug/ml of anti-
mouse antibodies overnight at 4° C. Wells were washed with 1x PBS and blocked in 2% BSA
solution. Cells were seeded at a concentration of 1.0X10
6
or 1-2.0x10
5
cells per well in triplicate
and incubated for >18h at 37° C in 5% CO
2
. Biotinylated secondary antibodies (2ug/ml)
52

followed by streptavidin HRP were then added to each well respectively. Plates were washed in
(0.1% tween/ PBS) and developed using 3-amino-9-ethylcarbazole (Sigma-Aldrich). Wells in
triplicate were then scored using a Zeiss KS ELISPOT Reader.
Quantitative PCR
Quantitative reverse transcriptase PCR (qRT-PCR) was performed as described previously [114].
Total RNA from MACS sorted cells was purified using RNeasy Microkit (Qiagen) according to
the manufacturer’s instructions. The SuperScript III First-Strand Synthesis kit (Invitrogen) was
used for cDNA preparation. A SYBR Green PCR kit (Bio-Rad) was used for quantitative PCR
and results were quantified with the ICycler IQ (Bio-Rad). Expression levels of all genes
analyzed were quantified and normalized to expression of the housekeeping gene Gapdh. In
some cases gene expression levels in WT-cells were set at a value of one and used as internal
controls. Sequences of all primer are available upon request.
Western blotting
For western blot analysis total proteins were isolated from mouse bone marrow using a NE-PER
nuclear and cytoplasmic extraction kit according to manufacturer’s protocol (Thermo Scientific).
Nuclear protein isolates were subjected to SDS-PAGE. Western blotting was performed using
standard techniques. Membranes were probed with rabbit anti-mouse SMYD5 antibody
(antibodies-online.com, Atlanta, GA). Goat anti-rabbit (IRDye 800 CW) fluorescently-
conjugated secondary antibodies were used to detect primary antibodies. Membranes were
imaged using the Odyssey infrared imaging system (LI-COR).

53

Thymidine incorporation assay
CD4
+
T-cells were isolated from spleens using magnetic cell sorting as described above. 96 well
plates were coated with anti-CD3ε mAb – (BD 55323) overnight at varying concentrations.
Plates were washed and cells were seeded in triplicate at a density of 1x10
5
cells in 200ul for 48
hours in RPMI complete media at 37° C in 5% CO
2.
At the end of the incubation period media
was replaced with media containing 3H-Thymidine, 0.1 μCi/100 μl to each well for an additional
16 hours. Plates were read using a scintillator counter.

Statistics
Groups of three to six sex-matched and age-matched mice were used for statistical analysis for
each experiment. P values were calculated using two-tailed Student's T-test. For all statistical
analysis α=0.05, and all p-values outside of this cut off were considered non-significant.

54

Results and Figures
Phenotypic characterization of the SMYD5 targeted mouse line
Smyd5 gene targeting was carried out as part of the International Knockout Mouse Consortium
effort to achieve genetic inactivation of a multitude of murine genes. Using (EPD0027_5_A02)
embryonic stem cells on a C57BL/6N genetic background the targeted SMYD5
tm1a (EUCOMM)Wtsi
allele contains a gene-trap DNA cassette within an intronic region (Fig 2.1).

The targeted allele is predicted to generate a truncated transcript due to the early poly-A site at
the 3’ end of the cassette and the splice acceptor site at the 5’end. The allele contains Frt sites
flanking the cassette to allow for a conditional knockout configuration upon crossing these mice
with flippase expressing mice. LoxP sites flank the gene’s exonic region to allow tissue specific
disruption of the gene. Mice no longer carrying the cassette are reverted to a traditional lox-Cre
system.
55

Our qRT-PCR analysis of bone marrow cDNA from B6 and SMYD5
-/-
mice showed that
the presence of the cassette successfully decreased the transcript levels of the gene (Fig 2.2 A).
Our gel electrophoresis DNA genotyping results confirm the presence of the cassette region in
the SMYD5 knockout (-/-)

mice and heterozygous (+/-) mice (Mutant band), but not in the DNA
of (+/+) B6 mice (Fig 2.2 B). Western blot analysis from BM protein lysates showed that
SMYD5 protein was dramatically decreased in the SMYD5
-/-
mice compared to B6 controls (Fig
2.2 C).

Phenotypically, mice lacking SMYD5 resembled age-matched B6 control mice; however,
homozygous breeding of these mice typically resulted in reduced litter sizes of 3-5 pups (data not
shown). In the spleen, draining lymph nodes, and BM we found no significant difference
between the percentages of CD4
+
, CD8
+
T-cells, or B220
+
, CD19
+
B-cells (Fig. 2.3 A-C).

56

Similarly, we found that transitional, follicular, marginal zone, and IgM
+
IgD
+
mature B-
cells were present at equivalent proportions in the spleens of SMYD5
-/-
and B6 mice (Fig. 2.4 A-
C). Furthermore, total cell numbers in the spleen and bone marrow of age-matched mice showed
no significant changes (Fig. 2.4 D). We also confirmed expression of SMYD5 in both CD4
+
T-
cells and B220
+
B-cells isolated from spleen under basal conditions (Fig. 2.4 E).
57

58

Altered antibody secretion and PC formation in response to TD-antigen in SMYD5
deficient mice in vivo
In order to investigate B-cell function under conditions of antigen challenge, we
immunized SMYD5
-/-
and B6 age-matched mice and assessed sera Ig levels as well as PC
formation at multiple time points. Blood was collected from tails of immunized mice at baseline,
8, 14, and 42 days. We performed ELISA to determine the arbitrary titer values for NP-specific
IgG1, IgM, IgG2b, and IgG3. We found that there was a decrease in NP
25
- specific IgG1
antibody production at 8 days, and a decrease in high affinity NP
5
- specific IgG1at 42 days (Fig
2.5 A). We also observed a decrease in IgG2b and IgG3 at 42 days (Fig 2.5 B). Our ELISA data
suggested that a lack of SMYD5 may be affecting levels of lower affinity NP
25
-IgG1 only during
early points of the response. In order to address this we assessed the ability of splenocytes from
SMYD5
-/-
and B6 control mice to secrete IgG1on a per cell basis using an ELISPOT assay 6
weeks post immunization and following a boost immunization. We found that there was a
decrease in NP
25
-IgG1 spots produced from splenocytes, but not BM cells at 47 days, of
SMYD5
-/-
mice (Fig 2.6 A, B). Taken together, the ELISA and ELLISPOT data suggests that
lack of SMYD5 is influencing antibody production by both short lived and longer lived ASCs.

59

60

The decrease in NP-specific Ig levels from mouse

sera prompted us to investigate
whether SMYD5
-/-
mice were able to normally form plasma cells in vivo. We used FACS
analysis to determine the percentage of PCs present in the spleens, dLNs, and BM of immunized
mice. Although both the SMYD5
-/-
and the control mice were able to form PCs, we observed a
statistically significant decrease in the B220
lo/neg
CD138
+
frequency and cell number in the
spleens and dLNs of KO mice at the 8 day time point (Fig 2.7 A). However, this decrease was
not apparent in the BM (Fig 2.7B).

61

62

SMYD5 deficient B-cells show normal PC differentiation but decreased antibody
production in vitro
To further investigate whether defective plasma cell differentiation was due to a lack of
SMYD5 within B-cells or due to extrinsic effects exerted on B-cells via signaling molecules or
direct T-cell interactions, we performed an in vitro PC differentiation assay. We sorted B220
+
B-
cells from spleens of naïve SMYD5
-/-
and B6 mice and cultured the cells in the presence of LPS
or LPS plus IL-4 for 4 days. Using flow cytometry we assessed the cells for expression of
CD138 and down regulation of B220. We found that SMYD5
-/-
B-cells were able to form plasma
cells normally under both conditions, and showed an increase in PC marker expression relative
to the B6 control cells (Fig 2.8 A, B). However, naïve B220
+
cells from spleens of KO mice
showed an initial decrease in CD138 expression, albeit not statistically significant, which is in
accordance to what we observed in whole spleen and dLNs of immunized mice (Fig 2.8 C).

We then sought to determine whether strong doses of LPS would have similar effects on
antibody secretion. B220 cells from spleens were isolated and cultured under the same conditions
stated above. After 4 days cells were harvested and transferred to ELISPOT plates to determine
their ability to secrete IgG1, IgM, IgG3, and IgG2b. We observed that SMYD5 deficient B-cells
cultured in media only control conditions produced less spots of each Ig isotype, contrary to what
we observed for baseline Ig levels in vivo (Fig 2.9 A,B). Similarly, when stimulated with LPS
SMYD5
-/-
B-cells displayed decreased IgG3 and IgG2b secretion (Fig 2.9 B), but we did not
observe these changes in the presence of LPS + IL-4 (Fig 2.9 A).

63

64

65

Decreased proliferation in the germinal centers of SMYD5
-/-
mice in vivo
The ability of SMYD5
-/-
B-cells to differentiate into PCs normally in the presence of
strong LPS stimulation in vitro, suggested that external influences or general changes in
proliferation may be causing the observed PC decrease in vivo. We therefore investigated GC B-
cells in the spleens of immunized mice. We found that at 8 and 14 days post NPKLH challenge
CD19
+
IgD
-
Fas
+
GL7
+
GC B-cell frequencies and cell numbers were decreased in SMYD5
-/-
mice
relative to B6 controls (Fig 2.10 A, B). Additionally we observed a decrease in the total percent
of CD19
+
(Fig 2.11 A) and B220+ (Fig 2.11 B, C) B-cells in KO mice post immunization,
contrary to what we found in naïve mice.

66

67

We further investigated the ability of the GC B-cells from both groups to proliferate using BrdU
incorporation. We found that SMYD5
-/-
GC B-cells had decreased BrdU incorporation at 8 days
post immunization, cumulatively, these data suggested that there may be a more general defect in
lymphocyte proliferation (Fig 2.12A, B).

68

Reduced T-follicular helper cell frequency and proliferation in SMYD5 deficient mice
Once GCs are formed, specialized B-cell helpers known as TFH cells regulate the
differentiation of plasma cells and memory B-cells through multiple T and B-cell interactions
[115-117]. Because we only observed reduced plasma cell development in vivo, we reasoned that
this could be a secondary affect resulting from changes in accessory cells. We therefore
investigated TFH cell development and cytokine secretion in vivo. SMYD5
-/-
and B6 mice were
immunized with NPKLH and the frequency of TFH cells at 8 days was assessed via flow
cytometry. In our analysis TFH cells were designated as CD4
+
CXCR5
+
PD-1
+
and GC TFH cells
as CD4
+
CXCR5
++
PD-1
++
. We found that there was a small (<10%) decrease in the total cell
frequency of TFH cells, and a larger significant decrease in the GC TFH cell population in the
SMYD5
-/-
mice

compared to the B6

mice (Fig 2.13 A-B). To determine if TFH cells in SMYD5
-/-
mice were functionally normal, we examined

cytokine production and proliferation in
immunized mice. Since IL-21 is reported to have a role in regulating gene expression and fate
choices of GC B-cells [104, 115], we assessed intracellular IL-21 production. Our data indicates
that SMYD5
-/-
GC TFH cells produce less IL-21 per cell compared to B6 mice one week after
immunization (Fig 2.13 C). We performed BrdU analysis by measuring incorporation of the
thymidine analog 5-bromodeoxyuridine (BrdU) in TFH cells in vivo. We found that there was a
decrease in the proliferative capacity (BrdU
+
) of splenic TFH cells from SMYD5
-/-
mice
compared to B6 mice (Fig 2.13D).

69

We also observed a general decrease in the percent of CD4
+
T-cells in the spleen one week
following immunization (Fig 2.14 A). Additionally, we found that splenic CD4
+
T-cells from
SMYD5 deficient mice had a reduced proliferative capacity compared to B6 controls when
activated in vitro with anti-CD3 and anti- CD28 mAb, based on a thymidine incorporation assay
(Fig 2.14 B). This data suggests that the decrease observed in TFH cell frequency may arise from
an underlying decrease in proliferation of all CD4
+
activated T-cells.

70

71

SMYD5 deficient CD138
+
cells show sustained expression of Bcl6 and Pax5 and decreased
expression of IRF-4, Xbp1, and Blimp-1
B-cells and plasma cells have unique transcriptomes that regulate the differentiation from
a B-cell to an antibody-producing plasma cell. Many of the transcription factors involved have
previously been characterized [68]; thus, we wanted to determine the relative expression pattern
of these genes. We sorted CD138
+
B220
-
plasma cells and B220
+
B-cell populations from
immunized mice for qRT-PCR analysis. Expression levels of all genes were normalized to
Gapdh, and normal expression levels for each gene measured in the B6 control mice were set to
a value of one. We found that in B220
+
cells Pax5 (>8 fold) and Bcl6 (~1.5 fold) were increased
in the KO mice, yet IRF-4, Blimp-1, and Xbp-1 showed no statistically significant change
between the two groups (Fig 2.15A). In the CD138
+
B220
-
PC population we found that
expression of the plasma cell repressors [118] Bcl-6 (~3.0 fold) and Pax5 (~6.0 fold) remained
increased in knockout mice. Conversely, we found that IRF-4 (~2.5 fold), Blimp-1 (~1.6 fold),
and the Xbp-1 (~9.0 fold) were significantly decreased in the KO CD138
+
cells compared to
expression levels in B6 control mice (Fig 2.15B).

72

73

74

SMYD5 is not important for IL-10 and IL-6 cytokine production by B-cells
Since previous studies reported that SMYD5 is a negative regulator of cytokine
production in macrophages (11), we wanted to investigate cytokine production in SMYD5
deficient B-cells in vitro. Using flow cytometry analysis we assessed intracellular levels of
cytokines. We found that isolated B220
+
cells from SMYD5 KO and B6 control mice produced
equivalent amounts of intracellular IL-10 upon TLR4 (LPS) and BCR (αIgM) stimulation
(Figure 2.16A). We observed similar results for IL-6 production upon LPS stimulation (Figure
2.16B). However, according to our QPCR analysis we did observe a small increase in IL-10 and
IL-6 mRNA levels in the SMYD5 deficient B-cells compared to controls (Figure 2.16C).

75

76

Discussion
Previous studies reported that SMYD5 contains retinoid X response elements (RXREs)
within its promoter region [112], and that SMYD5 expression is up-regulated in the presence of
retinoic acid [119]. It is known that retinoic acid (RA) can potentiate the production of antibodies
in normal adult animals in response to T-dependent antigens in vivo [120], making RA target
genes interesting candidates for studies on B-cell immune function. Using ChIP coupled with
mass spectrometry analysis, a protein interaction network for SMYD5 has been predicted [14].
Among the identified binding partners is the RUVBL2 protein. Furthermore, chemical germ line
mutagenesis in mice revealed that RUVBL2 is required for T-cell development and optimal T-
cell-dependent antibody responses in vivo [121]. However, whether SMYD5 HMT activity is
important for the function of RUVBL2 is a question for future study. Recently, SMYD5 was
shown to epigenetically regulate the expression of Toll-like receptor 4 (TLR4) inflammatory
cytokine target genes in macrophages [15], additionally, we observed changes in cytokine levels
in sera from LPS challenged mice (Figure 3.1). Prompted by these findings we investigated the
function of SMYD5 in the humoral immune response to a T-dependent antigen (NPKLH) in
vivo.
To our knowledge this study is the first to report that SMYD5 plays a role in regulating
B-cell function. We observed a decrease in serum Ig levels in KO mice at multiple time points
post immunization, and this change was concomitant with a decrease in the total number of
plasma cells present in peripheral tissues. Our in vitro analysis of isolated B-cells showed that in
the absence of T-cell help B-cells could normally differentiate into ASCs, albeit, the SMYD5
deficient B-cells showed decreased antibody secretion (Figure 2.9). CD138
+
B220
-
cells isolated
from spleens of immunized KO mice exhibited sustained expression of known PC repressors
77

relative to WT controls (Figure 2.15) Nevertheless, future mechanistic studies are needed to
clearly elucidate the role of SMYD5 in this transcriptional network. Because we did not observe
defects in PC formation in vitro, it is likely that both cell intrinsic and extrinsic forces are
resulting in the phenotype observed in vivo.
Upon immunization of SMYD5 KO and B6 age matched control mice with NP-KLH we
observed a decrease in NP-specific serum antibody titers. Multiple Ig subtypes were decreased
after 6 weeks, including high affinity IgG1 titers. Conversely, low affinity IgG1 titers were only
decreased in the KO mice at 8 days and this change was no longer detectable at 14 or 42 days. In
contrast to our ELISA data the KO mice produced fewer low affinity NP
25
-IgG1-secreting cells
at 6 weeks compared to controls as assessed by ELISPOT, indicating that other factors could be
masking this change within the serum. Interestingly, we found that baseline antibody levels were
equivalent between KO and B6 mice, which suggested that SMYD5 function within B-cells may
be important during times of cell activation. In our study we observed that SMYD5 is quickly
up-regulated in B-cells during LPS activation, and in vivo changes in B-cell function became
apparent only after NPKLH challenge.
A critical finding was that SMYD5-deficient mice developed less splenic and dLN
B220
neg
CD138
+
PCs when immunized. Furthermore, we found that there were decreases in both
B-cell and T-cell proliferation in KO mice. Our qRT-PCR analysis provides supportive evidence
as to why we observed a decrease in CD138
+
PCs in vivo, since sustained expression of B-cell
genes would inhibit or delay PC differentiation. On the other hand, transcription factors that
promote and facilitate the plasma cell phenotype such as Blimp-1 [122], IRF-4 [123], and Xbp-1
[124-126] were not up-regulated in a coordinated fashion with CD138 up-regulation or B220
down regulation.
78

Our qRT-PCR data showed a striking decrease in the transcription levels of Xbp-1 (~ 9
fold) in CD138
+
cells isolated from SMYD5
-/-
mice compared to B6 mice upon antigen
challenge. Previous reports using mouse lymphoid chimeras deficient in XBP-1 have shown that
XBP-1 is required for the generation of fully functional PCs and normal antibody production, but
is dispensable for the development of B and T lymphocytes [126]. Other studies found that XBP-
1 is sufficient to drive the production and secretion of Ig proteins in an in vitro plasma cell
differentiation system [127]. Interestingly, recent studies reported that CD138
+
B220
-
cells can
fully differentiate into plasma cells when XBP-1-deficient B-cells are treated with LPS and IL-4
in vitro, and that normal PC formation is observed in conditional Xbp1
fl/fl
Cd19
Cre/+
mice in vivo.
However, in the absence of secreted XBP-1, there was an average decrease in Ig production per
cell [128]. Our in vitro data is reminiscent of these latter findings. We observed that in the
absence of SMYD5, CD138 up-regulation and B220 down-regulation is not affected in the
presence of LPS or LPS + IL-4 stimulation, but that antibody production is decreased.
Our data shows that CD138
-
B-cells show an increase in Pax5 and Bcl6 expression in
immunized mice, indicating that these genes may be de-repressed by the lack of SMYD5 early
on. As plasma cells mature they down-regulate expression of B220 and up-regulate CD138,
which is concurrent with decreased expression of Pax5 and Bcl6 under normal conditions [68]. A
lack of down-regulation of repressors in SMYD5-deficient B-cells may indicate that this
molecule is important in the formation of fully functional and differentiated plasma cells.
Previous studies have shown that Pax5 and Blimp-1 form a double-negative feedback loop, while
Blimp-1 and IRF4 form a double positive feedback loop [129]. Because we observed changes in
multiple genes that makeup this network, it is likely that SMYD5 is acting upstream. One
79

possibility is a defect in upstream Stat3 signaling, which is linked to decreased IL-21 production
by TFH cells [105].
Previous studies using Stat3
fl/fl
CD19
Cre/+
conditional KO mice reported a similar
phenotype in TNP-Chicken Gamma Globulin (CGG) immunized mice. These Stat3 deficient
mice exhibited normal B-cell development, normal GC formation, and equivalent baseline serum
antibody levels, which sharply declined in the KO mice only following immunization.
Additionally, purified B-cells from Stat3
fl/fl
CD19
Cre/+
(KO) mice were able to form equivalent
numbers of ASCs when cultured and stimulated in vitro compared to B6 controls. Conversely,
there was a sharp decrease in ASC formation in vivo in the KO mice [106]. Similar to these
findings, we observed that SMYD5
-/-
B220
+
B-cells stimulated with LPS and LPS with IL-4 were
able to form B220
neg
CD138
+
cells in vitro. Surprisingly, we found an increase in the percent of
PCs formed from SMYD5-deficient B-cells compared to WT-cells in vitro. However, in vivo
alternative pathways become activated in response to NPKLH, independent of TLR4, producing
a distinct phenotype.
Multiple in vivo signals are integrated to promote B-cell survival and differentiation, and
among these are cytokine signals derived from TFH cells [100, 104, 130]. Several studies have
shown that cytokine-secreting TFH cells influence GC B-cell survival, plasma cell formation,
immunoglobulin class switching, and antibody responses [103, 104, 116, 131]. These findings
prompted us to investigate the development and function of TFH cells in SMYD5
-/-
mice. We
found that the total frequency of GC TFH cells was decreased at 8 days post immunization in
SMYD5 KO mice; however, pre-TFH cells were not affected. Overall SMYD5-deficient CD4
+

T-cells showed a decrease in proliferation both in vitro and in vivo. We also found there was a
decrease, although not statistically significant, in the level of intracellular IL-21 produced in the
80

KO mice compared to B6 controls. Hence, SMYD5 may play a non-redundant role in B and T-
cell function, and thus influence the phenotype resulting from the interaction of these cells.
Besides their role as ASCs, B-cells can also regulate immunity through the production of
cytokines. It has been shown that cytokine production by B-cells can alter T-cell responses [64,
132]. In particular IL-10 producing B-cells have been shown to serve a protective function by
suppressing immune responses [65], while IL-6 producing B-cells can contribute to
inflammation [132]. We assessed the ability of isolated B-cells to respond to BCR and TLR
stimulation in vitro. However, from our data we conclude that SMYD5 does not play a role in
regulating cytokines within B-cells.
Although further experiments are needed to reconcile the discrepancies in the data, we
favor the explanation that SMYD5 plays an intrinsic role in regulating antibody production,
although extrinsic cell interactions are likely influencing PC numbers. Taken together, the data
presented in this study suggests that SMYD5 functions in optimizing the intensity of the B-cell
response in vivo. Furthermore, our study adds to the body of growing knowledge regarding the
epigenetic regulation of immune function.

81

CHAPTER III

Increased Peritoneal Cavity Macrophages and Systemic
Sensitivity to LPS in the Absence of the
Histone Methyltransferase SMYD5

82

Chapter III- Abstract
Increased peritoneal cavity macrophages and systemic sensitivity to LPS in the absence of
the histone methyl transferase SMYD5
The SMYD subfamily of histone methyl transferases are all expressed within primary
mouse macrophages. A previous study reported that siRNA knockdown of SMYD5 results in
increased transcription of inflammatory genes in vitro, warranting further investigation into the
role of SMYD5 in macrophage function and polarization in vivo. In this study we evaluated the
systemic effects of TLR4 stimulation using a SMYD5-KO murine model. We observed that KO
mice exhibited a spontaneous increase in the peritoneal cavity (PerC) monocyte/macrophage
populations at baseline, and this increase persisted following LPS challenge by intraperitoneal
injection. Peritoneal cavity macrophages also produced more intracellular cytokines, and the KO
mice had increased pro-inflammatory serum cytokine levels. In assessing macrophage
polarization in vitro, both bone marrow derived macrophages (BMMs) and dendritic cells
(BMDCs) from KO mice secreted increased cytokine levels compared to controls. Quantitative-
PCR analysis of M2-polarized BMMs showed that lack of SMYD5 resulted in decreased
expression of alternative (M2) genes. We also determined the ability of BMMs to respond to
Salmonella typhimurium infection in vitro, and observed increased cytokine production, initial
bacterial uptake, and killing rate by the KO cells compared to controls. Our study reports an in
vivo role for SMYD5 in the regulation of macrophage polarization and migration.

83

Introduction
The mononuclear phagocytic system consists of hematopoietic stem cell derived myeloid
lineage monocytes, macrophages, and dendritic cells [81]. In response to microbial encounters
and immunological signals macrophages and neutrophils become activated and aid in regulating
the adaptive immune response. Macrophages express an array of receptors allowing them to
sense and respond to microbial components, cytokines, lineage-determining growth factors, B
cell products, and glucocorticoids [133]. These cells have multiple functions including:
phagocytosis, endocytosis, secretion of cytokines, microbial killing, tissue maintenance, antigen
presentation, and regulation of inflammation [52, 81, 134-136]. Macrophages are diverse and
exhibit a spectrum of activation and phenotypic states depending on stimuli and tissue location
[53, 137]. Specialized tissue resident macrophages can be found in bone, lung, liver, spleen, gut,
lymph nodes, and brain [138-143].
Multiple macrophage subsets have been described; however, due to the large amount of
overlap in surface marker expression they are often difficult to characterize [144]. Among the
characterized subsets are the classically activated (M1), alternatively activated (M2), regulatory
macrophages, tumor-associated macrophages, and myeloid-derived suppressor cells [137, 145-
147]. For purposes of this study we will focus on the M1 and M2 macrophages. The M1 and M2
polarized states depend on the effects of selective stimuli on expression of macrophage markers,
and the M2 subset can be further sub-divided into M2a, M2b, and M2c [148]. Classical M1 cells
are those that respond to INF-γ plus LPS or INF-γ plus TNF, while alternative M2 cells respond
to IL-4 and IL-13 [M2a], IL-10 and glucocorticoids [M2c], and immune complexes together with
TLR ligands [M2b] [137]. In general the stimuli associated with M1 macrophages are grouped
84

according to their ability to produce a pro-inflammatory immune response, while the M2 stimuli
are associated with immune suppression and restoration of tissue homeostasis [149].
Recent studies have focused on the epigenetic mechanisms and transcription factor
regulation underlying the polarization and functional states of macrophages. Epigenetic changes
can alter the expression of information encoded in DNA at the sequence level. These alterations
include methylation, acetylation, phosphorylation, ubiquitination, and sumoylation [148]. For
example, during myeloid lineage differentiation the transcription factor PU.1 is essential to
induce and maintain macrophage differentiation [150-152]. Additional transcription factors
known to play crucial roles in differentiation and function are the IFN regulatory factors IRF8,
IRF4, IRF5, and CCAAT/enhancer-binding protein-[beta] (C/EBP-β) [58, 153, 154]. The
demethylase Jmjd3which erases lysine 27 trimethylation on histone 3, has been shown to bind to
promoters of NF-κB dependent genes which are known to promote the M1 macrophage state
[155].
In general, changes in histone methylation result in alterations of chromatin structure and
gene transcription. Active transcriptional states are associated with [histone 3 lysine 4
trimethylation (H3-K4me3) and (H3-K4me1)]; as well as acetylation of [(H3-K9, 14-Ac), H4-
K5, 8, 12, 16-Ac), and (H3-K27-Ac)] [54]. Conversely transcriptional repression is associated
with [trimethylation of histone 3 on lysine 9 (H3-K9me3), (H3-K27me3), (H3-K79me3), and
(H4-K20me3)] [15, 54, 156-159]. Recently it was reported that SMYD5 in association with the
NcoR complex can bind to promoters of TLR4-responsive genes and catalyze H4-K20me3 at
these sites. In response to TLR activation SMYD5 is dissociated from the DNA and removal of
H4-K20me3 is removed by the action of PHF2 [15].
85

Although multiple macrophage types have been identified, understanding the
mechanisms controlling the activation and effector states of these cells is complicated by the
incredible diversity of microenvironments in which they exist, and differences between the in
vitro and in vivo effects of specific stimuli. In this study we wanted to investigate the systemic
effects of TLR4 activation in SMYD5-KO mice, in regards to PerC monocyte/macrophage
adhesion and migration, cytokine secretion, and M1 versus M2 polarization.

86

Materials and Methods
Mice and immunizations
SMYD5 KO-first (Smyd5
tm1a/tm1a
) mice were purchased from the Welcome Trust Sanger Institute
(Hinxton, UK). All mice were bred in a pathogen-free barrier facility (USC) and all experiments
were approved and performed in accordance to the regulations of the University of Southern
California Institutional Animal Care and Use Committee. SMYD5
-/-
mice and aged matched
SMYD5
+/+
control mice, 8-12 weeks old, were immunized intraperitoneally (i.p.) with 100 µg of
LPS unless otherwise stated.
ELISA
Cytokine concentrations from supernatant and sera were determined by Enzyme-Linked
Immuno-Sorbent Assay (ELISA). For measuring (TNF-α), (IL6), (IL-1β), and (IL-10) all kits
were purchased form (BD, San Diego, CA), and all assays were performed according to
manufacturer’s instructions. Wells were washed with (0.05% Tween 20 in 1x PBS), blocked in
(1% BSA/1x PBS solution), and diluted in (0. 1% BSA, 0.05% Tween 20 in TBS). Polystyrene
high binding plates were used for all assays (VWR). Blood was collected from tail (0-h) or heart
puncture (4-h) for all assays.
Macrophage invasion assays
The invasion assays were performed as described in [160]. For all assays GM-BMMs were used
at a concentration of (3-4x10
5
) per well. A MOI of 10:1 (Salmonella typhimurium) was used.
Infected cells were lysed with 1ml of 0.1% Triton X-114 for 10 minutes. We diluted the lysed
samples 10x and 100x with 10mM PIPES pH 7.4 and plated 50ul using a spiral plater (
Autoplater Model 4000) on 100mm LB agar plates.
87

Generation and activation of GM-BMMS, M-BMMs, and BMDCs
BMDCs were derived as in [161]. Mice were euthanized by CO
2
asphyxiation and tibias and
femurs were removed. The bones were sterilized by immersion in 70% ethanol for 10 min in a 35
mm Petri dish. All other work was performed inside a biosafety hood to avoid contamination of
the cell cultures. Bones were flushed with 1 ml of RPMI medium (without serum but with
antibiotics) using a sterile syringe. Cells were washed 2x in a 15 ml centrifuge tube at 1,100
RPM in a refrigerated centrifuge (4°C). Cells were resuspended in 2 ml of RBC lysis buffer and
incubate for 5 min at room temperature in order to eliminate red blood cells. Cells were washed
again and resuspended in 13 ml of RPMI with 10% FBS. Cells were counted and adjusted to a
concentration of 2 x 10
5
cells/ml with RPMI 10% FBS, and GM-CSF was added to the cultures
(20 ng/ml final concentration). 10 ml of this suspension was plated in 10 cm Petri dish, and
cultured in a CO
2
incubator (37°C, 5% CO
2
). Three days later, another 10 ml of RPMI 10%FBS
with 20 ng/ml of GM-CSF was added to each of the prepared plates. Another three days later 10
ml of cell suspension are recovered from each Petri dish, centrifuged and resuspended in the
same volume of media .Cells are then returned to the Petri dish. Cells are cultured for 2
additional days. After 8 days in culture loosely adherent cells are recovered by washing, these
cells are BMDCs.
GM-BMMS and M-BMMs were generated similarly. BM cells were extracted in the same
fashion and cultured in RPMI, supplemented with, 10% FBS, and recombinant M-CSF
(30ng/ml), or GM-CSF (30ng/ml). Cells were cultured in non treated Petri dishes for 5 days. On
day 5, wash cells twice with 5 ml of saline solution at room temperature and scrape the cells off
the dish with a cell scraper, transfer to a sterile tube and count. There should be about 5 ×10
6
per
88

dish (10
7
per bone) [162]. For all assays (100ng/ml) of LPS was used for stimulation, unless
otherwise stated. M-BMMS were stimulated with IL-4 (20 ng/ml) for M2 studies.

Purification of peritoneal macrophages
Peritoneal cells were obtained by lavage with 3% sodium citrate. Red blood cells were lysed
using RBC lysis buffer (BD Biosciences). Cells were plated in 24-well plates in DMEM with
10% FBS, 1% pen/strep, 1% l-glutamine, for indicated time points with indicated stimuli for
each assay.

Flow Cytometry
PerC cells were passed through 70 μm BD Falcon cell strainer (BioLegend, San Diego, CA,
USA). All cells were first incubated for 20 min at 4° C with CD16/CD32 Fc-blocking antibody
(2.4G2), in flow cytometry buffer (1x PBS, 1-2% FBS), followed by incubation for 30 min at 4°
C with an array of antibodies: CD11b-APC-Cy7 (M1/70) from Biolegend, F4/80-Pe-Cy7 (Clone
BM8) from Invitrogen (Carlsbad, CA, USA), TNF PE (MP6-XT22), IL-6 (MP5-20F3), CD86
FITC (B7-2), CD80 APC (B7-1) from BD Bioscience. Flow cytometry was performed using BD
FACS Canto (BD Bioscience, San Diego, CA, USA) and data were analyzed with FlowJo
software (TreeStar, Ashland, OR, USA).

Intracellular staining
Cells were stained with surface antibodies and fixed for 20 min at 4° C using (BD
Cytofix/Cytoperm). Cells were then washed 2x using (BD Perm/Wash), followed by 30 min
incubation in Perm/Wash buffer at 4° C to permeabilize the cellular membrane. Cells were then
89

stained for 30 min at 4° C, followed by a washing step in FACS buffer (1x PBS with 2% FBS)
before analysis.
Quantitative PCR
Quantitative reverse transcriptase PCR (qRT-PCR) was performed as described previously [114].
Total RNA from cells was purified using RNeasy Microkit (Qiagen) according to the
manufacturer’s instructions. The SuperScript III First-Strand Synthesis kit (Invitrogen) was used
for cDNA preparation. A SYBR Green PCR kit (Bio-Rad) was used for quantitative PCR and
results were quantified with the ICycler IQ (Bio-Rad). Expression levels of all genes analyzed
were quantified and normalized to expression of the housekeeping gene Gapdh. Sequences of all
primer are available upon request.
Statistics
Groups of 3-4 sex-matched and age-matched mice were used for statistical analysis for each
experiment. P values were calculated using two-tailed Student's T-test. For all statistical analysis
α=0.05, and all p-values outside of this cut off were considered non-significant (NS).

90

Results and Figures
SMYD5 plays a role in repressing systemic LPS-induced inflammatory responses in vivo
In order to determine if SMYD5 affects the systemic response to LPS, SMYD5
-/-
and
wild-type (WT) mice were challenged with LPS by intraperitoneal (i.p.) injection and bled at
various time points for serum collection. The levels of tumor necrosis factor-α (TNF-α),
interleukin-6 (IL6), interleukin-1β (IL-1β), and interleukin-10 (IL-10) were assessed by ELISA.
We found that all pro-inflammatory cytokines were increased in the SMYD5-KO mice compared
to the WT control mice (Fig 3.1 A-D).These results suggest that SMYD5-KO mice may be more
susceptible to increased levels of inflammation.

91

SMYD5 deficient PerC macrophages show increased inflammatory cytokine production in
response to LPS challenge in vivo
It was previously reported that siRNA knockdown of Smyd5 within thioglycollate-elicited
macrophages resulted in increased mRNA expression of TLR4 target genes in the presence of
Kdo2 lipid A (KLA) [15], providing rationale for testing the effects of SMYD5 deficiency on
cytokine protein levels in vivo. SMYD5-KO and WT control mice were challenged with LPS for
4 hours, and intracellular staining of PerC cells was performed to detect TNF and IL-6.
Compared to WT mice, we detected 7-fold and 3.1-fold increases in the number of KO mouse
PerC cells expressing TNF-α (Fig 3.2 A-B) and IL-6 (Fig 3.2 C-D), respectively.

92

For both cytokines the mean fluorescence intensity (MFI) showed a similar trend,
indicating higher levels of expression per cell (Fig 3.2 E-F). To determine if elevated cytokine
levels correlated with increased activation marker expression, we stained the cells for CD80 and
CD86, however, no significant differences were detected between the two groups (Fig 3.3 A).
Additionally, we observed that the percentage of CD11b
+
cells was comparable between the two
groups when other markers were excluded from the staining panel (Fig 3.3 B).

93

94

Lack of SMYD5 leads to increased resident and elicited PerC monocytes/macrophages in
response to LPS in vivo
We observed that genetic inactivation of SMYD5 resulted in spontaneous accumulation
of macrophages within the PerC (Fig 3.4 A-C), mimicking induced peritonitis.

95

This finding prompted us to further investigate the migration/adhesion responses within these
mice. We administered LPS or PBS i.p. into WT and KO age- and sex-matched mice and used
flow cytometry analysis to quantify the percent and cell numbers of monocytes/macrophages at 4
h post injection. We found that the SMYD5-KO mice had ~2-fold more F4/80
hi
CD11b
hi
cells, but
not F4/80
INT
CD11b
INT-cells
, at 4 hours post LPS administration (Fig 3.5 A-C). Thus, although
genetic deficiency of SMYD5 leads to increased recruitment of monocytes to the PerC in the
absence of induced peritonitis, it does not affect efflux of LPS activated cells, since we observed
a ~5-fold decrease in F4/80
hi
CD11b
hi
macrophages in both the KO and WT mice at 4 hours.
96

Including the Ly6C marker in our staining panel revealed that among the F4/80
+
population the
KO mice displayed an increase in the CD11b
hi
Ly6C
hi/INT
population, and a decrease in the
CD11b
INT
Ly6C
hi/INT
monocyte population (Fig 3.6 A). However, the KO mice displayed an
overall increase in the percent of CD11b
INT
Ly6C
hi/INT-cells
(Fig 3.6 B).

97

During acute inflammation monocytes enter multiple tissues of the body, including the
spleen. Since we had observed a spontaneous recruitment of monocytes/macrophages into the
PerC of SMYD-KO mice in the absence of activation, we wanted to determine if similar changes
were present in splenocytes. We found that the percentage of CD11b
+
Ly6C
+
and F4/80
+
Ly6C
+

monocytes [163], and F4/80
+
CD11b
+
macrophages [58] were equivalent in spleens of WT and
KO mice at baseline (Fig 3.7 A- B). Similarly, LPS challenged mice showed no change in the
percent of CD11b
+
MHCII
+
activated macrophages (Fig 3.7 C) or CD11c
+
MHCII
+
dendritic cells
(Fig 3.7 D) within spleens of WT and KO mice.

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We also investigated splenic intracellular cytokine levels at 4 hours post LPS challenge in
vivo, and found that there was a statistically significant increase in the percentage of
CD11b
+
F4/80
+
IL-6
+
splenocytes, however, the increase in CD11b
+
F4/80
+
TNF
+
splenocytes was
much smaller (Fig 3.8 A-B). Additionally, we tested cytokine secretion in vitro. We isolated
CD11b
+
splenocytes from naïve mice and stimulating these cells in culture with LPS in the
presence of a protein transport inhibitor (GolgiPlug). We observed that both WT and SMYD5-
99

KO CD11b
+
could normally

respond to LPS stimulation

and that there was no notable change in
the amount of intracellular TNF produced by the cells after 4 hours of stimulation (Figure 3.8 C-
D).

100

Increased inflammatory cytokines from SMYD5
-/-
BMDCs and GM-BMMs
Because differences between in vivo and in vitro micro-environmental stimuli can affect
cell activation, we wanted to assess whether bone marrow derived myeloid lineage cells from
SMYD5
-/-
mice showed any functional changes when compared to cells derived from WT mice.
Previous studies using multiple mouse models have shown that macrophages and dendritic
cells (CD11c
+
MHCII
+
, Mo-DCs) are derived from the same monocyte precursors [164]. In vitro
activation of BMDCs with LPS leads to secretion of the same pro-inflammatory cytokines
produced by classical macrophages [165]. We cultured BM cells from KO and WT mice in the
presence of GM-CSF, which induces both M1 GM-BMM and BMDC lineage differentiation
[161]. GM-BMMs and BMDCs were stimulated with LPS and culture media was assayed for
secreted cytokines at 3 h. Cells derived from SMYD5
-/-
BM cells secreted more IL-12p70 and
TNF-α in both cells types (Fig 3.9 A, C), however, at 3 h only BMDCs had increased IL-6
secretion, indicating that the in vitro kinetics of IL-6 regulation in the absence of SMYD5 may
differ between cell types (Fig 3.9 B).

101

102

Decreased gene expression in M2-polarized SMYD5
-/-
M-BMMs
Culture of murine BM cells with M-CSF has been reported to induce differentiation of
M-BMMs, with a functionally distinct M2 macrophage phenotype [166]. To assess whether
SMYD5 plays a role in regulating the activation status of this sub-set, we treated M-BMMs with
IL-4, which can induce expression of the M2 genes: arginase I, mannose receptor (MR), Ym1
(chitinase-like lectin), and Fizz1(resistin-like secreted protein) [137]. We found expression of
Ym1 and Fizz1 genes was impaired in KO M-BMMs relative to controls (Fig 3.10 A). In
contrast, expression of IL-1β and TNF-α in response to TLR4 ligand stimulation was increased
in the SMYD5
-/-
M-BMMs (Fig 3.10 B). Our data indicate that SMYD5 is involved in IL-4
responses; however, these changes were not associated with IL-4R changes (Fig 3.10 C).

103

Role of SMYD5 in GM-BMM M1 polarization in response to Salmonella typhimurium
infection

To assess the functional relevance of our findings, we investigated the role of SMYD5 in
an in vitro model of macrophage infection by the intracellular pathogen Salmonella typhimurium
(ST). GM-BMMs from WT and KO mice were infected at a multiplicity of infection of 10:1 with
ST to assess macrophage killing ability and cytokine production. Our QPCR analysis of TNF-α,
IL-1β, and IL-6 mRNA expression levels showed that only TNF-α expression in the KO GM-
104

BMMs was increased significantly at 60 minutes after initial infection, denoted as (0-h) to
indicate that gentamycin was not yet added to the culture medium (Fig 3.11 A).

105

We also found that secreted levels of TNF-α (0-h) and IL-6 (2-h) were increased in the KO GM-
BMMS compared to controls cells (Fig 3.12 A, B). Additionally, at 0-h the KO GM-BMMs
showed an initial increased ability to internalize the bacteria, but had reached equivalent levels of
infection as the WT by 2 hours, indicating an increased killing rate (Fig 3.12 C). This increased
up-take coincided with the increase in TNF-α secretion and transcription that we observed from
our QPCR and ELISA assays.

106

Discussion

In vivo macrophage phenotype is heterogeneous, with many subtypes co-existing to
maintain balance and proper immune function. LPS is a TLR4 ligand and is known to induce
activation of classical M1 macrophages, which have the potential to cause tissue damage under
conditions of excessive inflammation [54]. Alternatively, M2-like macrophages function in the
resolution of inflammation and prevent tissue damage through the production of anti-
inflammatory factors such as IL-10 [137]. We observed that SMYD5-KO mice challenged with
LPS in vivo had increased serum pro-inflammatory cytokine levels, a sign of elevated systemic
inflammation. We also observed that serum IL-10 levels were increased in the KO mice
compared to controls. In this case perhaps increased production of pro-inflammatory cytokines
throughout the body led to increased IL-10 production as a protective response, since IL-10 is
elevated during M2-macrophage responses to limit inflammation related damage.

We also observed that in the absence of external activation there was a spontaneous
increase in the percentage and cell numbers of macrophages present in the peritoneal cavity
(PerC) of the SMYD5-KO mice, compared to controls. These data suggested that the KO mice
had either increased trafficking of monocytes to the PerC, or that the differentiation of
monocytes into macrophages was more readily triggered in these mice, possibly due to increased
sensitivity to environmental influences otherwise well tolerated in normal wild type mice. The
presence of altered macrophage populations in the PerC prompted us to examine whether there
were also changes in the ability of activated cells to migrate out of this site.

107

In response to injury, infection, or inflammation monocytes must adhere to blood vessels
to migrate and infiltrate tissues. This process is chemokine-induced and is regulated by multiple
factors including: Nox4 [167], MKP-1 [168], and CCR2 [169]. During the resolution phase of
inflammation monocyte derived macrophages efflux out of the PerC to the blood stream and
lymphatics [170]. Macrophage adhesion/migration out of the peritoneum can be measured by
inducing peritonitis via thioglycollate administration followed by LPS activation [171]. This
method induces monocyte infiltration into the PerC over the course of several days, and
subsequent antigen activation induces macrophage adherence to the mesothelial lining of the
PerC, or efflux to the lymphatics system. In our model, we tested whether the accumulated
macrophages present under basal conditions in the KO mice showed similar patterns of efflux to
the WT control mice after LPS challenge. Interestingly we observed that both the SMYD5-KO
mice and the control mice had an approximately 5-fold decrease in the percent of macrophages
in the PerC at 4 hours post i.p. LPS injection. We therefore concluded that SMYD5 might not be
important in the adhesion of macrophages to the PerC cavity or their ability to efflux out to
external sites. More likely, lack of SMYD5 may play a role in sensitizing cells to the initial
activating stimuli causing increased monocyte migration to the PerC.

Additionally, we investigated the regulation of cytokine production in SMYD5 deficient
macrophages, since no previous studies have reported on the role of SMYD5 in vivo. In 2012
Stender et al, reported that SMYD5 epigenetically regulates gene transcription by depositing H4-
K20me3 at Tnf and Cxcl10 promoter regions. In this study the authors show that H4-K20me3
causes transcriptional repression by SMYD5 at the Tnf locus, however, the affect of decreased
levels of SMYD5 on protein levels was not investigated. Furthermore, this work employed the
108

use of siRNA technology to reduce levels of SMYD5 within an in vitro system. Our data
corroborate the finding that decreased SMYD5 leads to increased TNF levels, however, here we
report increased protein levels of both TNF and IL-6 within an in vivo model of SMYD5
deficient macrophages. Additionally we report that SMYD5 also regulates pro-inflammatory
cytokine secretion within BM derived dendritic cells. Using BMDCs derived from SMYD5-KO
BM cells, we found that TLR4 stimulation resulted in increased cytokine secretion in both GM-
BMMS and BMDCs, suggesting that SMYD5 may serve a similar function in multiple myeloid
lineage cells. However, we did not determine if SMYD5 directly binds to the promoter regions
of these genes in vivo in either cell type, a question that would be interesting to address in future
studies.
Because we had observed an increase in IL-10 serum cytokine levels in vivo, we
suspected that SMYD5 knockout might promote changes in transcriptional profiles within other
macrophage subsets. A dichotomy has been proposed for macrophage activation represented by
M1 and alternatively activated M2 macrophages [137]. Macrophage colony stimulating factor
(M-CSF) is a tyrosine kinase transmembrane receptor that is classified as an M2 stimulus [172,
173]. Normally, IL-10 binds to IL-10 receptor (IL-10R) leading to subsequent activation of
STAT3 and inhibition of pro-inflammatory cytokines [174]. However, in this case we observed
simultaneous up-regulation of IL-10 and TNF following LPS challenge. In order to determine if
decreased SMYD5 had any effect on activation of M2 macrophages, we used M-CSF and IL-4 to
differentiate and activateM2-like macrophages in vitro. We found that SMYD5-KO M2 cells had
decreased expression of alternative genes such as: arginase 1, YM1, mannose receptor (MR), and
Fizz 1 compared to controls. Furthermore, we found that the decrease in M2 activation
phenotype was not due to altered IL4 receptor (IL-4R) expression; however, this does not
109

exclude the possibility that IL-4 signaling may be the underlying cause, a question that should be
addressed in future studies.
Functionally, one of the main properties of macrophages is microbial killing, we
therefore wanted to examine whether the absence of SMYD5 would affect the ability of GM-
BMMs to recognize and kill the intracellular pathogen Salmonella enterica serovar
Typhimurium (ST) in vitro. This bacterial strain is linked to localized gastroenteritis in humans
but elicits a systemic infection in mice [160]. We tested the ability of ST to enter and survive
within murine BM derived macrophages from WT and KO mice. Initial infection of cells was
performed at an MOI of 10:1, and subsequent cell counts of surviving intracellular bacteria were
performed at 60 minutes (o-h), 3 hrs (2-h) and 7 hrs (6-h) post infection. Interestingly we
observed that initially the KO-GM-BMMs had taken up a larger amount of ST, 4x10
4
(WT)
versus 6x10
4
(KO), as assessed by a colony forming unit (CFU) assay. By 3 hrs the amount of
intracellular bacteria within the WT and KO macrophages had reached equivalent levels
according to CFU counts, indicating that the KO cells had an increased ability to kill the
bacterium. However, it is possible that there was increased apoptosis of the KO BMMs due to
infection, which should be determined by live dead staining in future experiments. We also
assessed whether there were differences in cytokine secretion between the infected cells. We
observed similar results to what we had observed when cells were activated with LPS, where KO
BMMs displayed increased TNF and IL-6 cytokine secretion. In this case the increased levels of
Tnf transcription and secretion coincided with the initial increase in bacterial up-take observed in
the CFU assay. Cumulatively, our data indicate that genetic inactivation of SMYD5 leads to
increased M1 macrophage activation and effector functions in response to microbial agents both
in vitro and in vivo.
110

Chapter IV
Conclusions and Future Studies

111

Conclusions and Future Studies
In recent years epigenetic studies have come to the forefront of basic biological research.
Published studies regarding the function of epigenetic regulation of vast numbers of genes have
contributed greatly to our understanding of normal immune function and disease states. Our
study is the first to characterize the biological function of the histone methyltransferase SMYD5
within B-cells and primary macrophages. Although this study does not provide detailed
mechanistic data regarding the interacting partners or gene targets of SMYD5, it does lay the
groundwork for future studies by identifying a role for this protein in the innate and adaptive
branches of the immune response within an in vivo knockout murine system.
In chapter two we report that SMYD5 plays a role in the formation of PCs and in the
secretion of antibodies. We found that in the absence of SMYD5, mice challenged with the TD-
antigen NP-KLH display a decreased number of PCs and serum Ig levels. We also report that in
vitro PC formation is unaltered in the SMYD5-KO B-cells when differentiation is driven by the
T-independent (TI) antigen LPS. Furthermore, our data suggests that expression patterns of
Smyd5 influence expression of other well characterized B-cell differentiation genes, contributing
to the general knowledge base of this gene network. This work encourages further confirmatory
and mechanistic studies; and there are multiple experiments that would serve to clarify some of
the implications of the data reported here.
For example, in order to confirm the conclusion that PC differentiation is only affected in
vivo, we could differentiate isolated naïve B-cells from WT and KO mice with a T-dependent
antigen such as CD40L +IL-4+IL-5, or alternatively, we could also cross-link the BCR using
αIgM. Since we used LPS to drive differentiation in our in vitro model, it remains unclear if the
112

differences we observed in vivo are due to differences in receptor stimulation, since in vivo
studies were conducted using NP-KLH as the activator. Additionally, we could immunize the
mice with NP-Ficoll to induce a T-independent response in vivo allowing us to assess PC
differentiation and antibody production under these conditions. Repeating the in vitro PC
differentiation assay using CD40L + IL-4 stimulation would also allow us to further determine if
there are defects in class switch recombination, which would strengthen the conclusions of our
study. This experiment would be particularly important since we did report that there was a large
decrease in NP
5
-IgG1 levels at the six-week time point, indicating a defect in affinity maturation
due to the absence of SMYD5.
Furthermore, additional experiments are needed in order to confirm that SMYD5 is
important for optimal antibody responses. Our FACS analysis shows that PC numbers are
affected at 8 days post immunization with NP-KLH; however, our ELISPOT analysis was
performed at the 6 week time point. ELISPOT assays should be performed at multiple time
points following in vivo immunization. FACS analysis of PCs should also be performed at both
early and late time points for comparison with ELISPOT results. Our in vitro analysis of
antibody secretion was performed by differentiating naïve B-cells into PCs by the use of LPS or
LPS + IL-4 stimulation, which we then used for ELISPOT assays. We found that only IgG3 and
IgG2b levels were decreased in the KO cells compared to WT controls. Switching to IgG3 or
IgG2b is division dependent and CFSE analysis of these cells would allow us to determine if cell
cycle changes are contributing to this phenotype.
Our data also indicated that there is a GC B cell defect and that it is likely due to changes
in proliferation of both B-cells and TFH CD4
+
T-cells. Future studies should include a more
thorough examination of general proliferation of lymphocytes within these mice through BrdU
113

analysis, or cell cycle analysis. Furthermore, GC B-cells should be sorted and the expression of
Bcl6 should determined, since our qPCR data shows that SMYD5-KO B220
+
B-cells show an
increased level of Bcl6 expression (Figure 2.15), which contradicts the decrease in GC B-cells
reported, according to published data. Intracellular Bcl6 staining would help resolve this
discrepancy. Furthermore, detailed qPCR analysis of GC B-cells may provide a more thorough
explanation as to why there may be decreased numbers of PCs in these mice, and whether these
changes are associated with defects in differentiation pathways. Also, serum from NP-KLH
immunized mice should be tested for cytokines such as IL-21 and IL-6, which play a crucial role
in GC B cell proliferation and TFH

differentiation.
Furthermore, because of the extensive interplay between GC B-cells and TFH

cells it is
difficult to determine if SMYD5 deficiency causes independent changes within each cell type. In
order to address this issue adoptive transfer experiments should be conducted, in which TFH
cells from WT immunized mice are transferred into SMYD5-KO mice to determine if these cells
can reverse the B-cell defects observed. The in vivo antibody response can then be assessed in
these mice to determine if normal TFH cells would correct the defect in antibody secretion
observed in serum. The activation status and cytokine secretion of CD4
+
T-cells from KO mice
should also be assessed by looking at CD25 expression and IL-2 production in vivo, since
changes in T-cell activation can also affect B-cell activation.
Lastly our qPCR analysis of isolated PCs from immunized mice showed that multiple B-
cell transcription factors displayed altered expression patterns in the KO mice compared to
controls. However, our study does not provide any mechanistic data as to where in this well
characterized pathway SMYD5 may be important. Published data show that SMYD5 catalyzes
H4-K20me3, an epigenetic mark associated with gene silencing and heterochromatin formation.
114

It is also known that IL-21 induces phosphorylation of STAT3 which binds to a regulatory
region of the Blimp1 gene in a complex that contains and requires Irf4 [175]. Based on our data it
is likely that SMYD5 is regulating expression of some factor that is upstream of Irf4 since
expression is decreased in this gene as well as its downstream targets (Figure 2.15). In vitro
analysis of STAT3 phosphorylation and signaling could also be conducted on isolated SMYD5
deficient B-cells in the context of IL-21 stimulation. ChIP qPCR analysis or Irf4 and
transcription factors upstream of this gene would allow us to determine at what loci SMYD5 is
localizing within B-cells. The same assay could be used to locate which B cell genes, if any,
display H4-K20me3, since this is the only epigenetic mark known to be associated with SMYD5.
It would also be beneficial to determine in what subset of cells SMYD5 is most highly expressed
under the stimulation conditions described above. Our data show that SMYD5 is up-regulated in
B220
+
cells upon LPS stimulation, however, this analysis should be extended to include,
activated B-cells, GC B-cells, and PCs following T-independent and T-dependent stimulation ex
vivo by sorting each cell type post immunization.
Chapter three of this dissertation focused on the role of SMYD5 within primary
macrophages. Here we evaluated the role of SMYD5 in the regulation of LPS-induced pro-
inflammatory cytokine production and in vitro macrophage polarization. We show that PerC
macrophages from KO mice display increased IL-6 and TNF production following LPS
challenge via intracellular staining. These results can be further substantiated by stimulating ex
vivo sorted F4/80
+
Cd11b
+
thioglycollate elicited macrophages from the PerC with LPS +IFNγ
(M1 activation) and KLA (M1 activation), in vitro. Cytokine expression and secretion could be
assessed by using qPCR analysis, intracellular staining, and ELISA assays.
115

Moreover, a more complete analysis of the disease relevance of our in vivo findings
should be conducted. For example, we reported that serum pro-inflammatory cytokine levels
were increased in the KO mice compared to controls. However, comparative analysis using a
model of LPS induced septic shock would show if the KO mice are more susceptible to the
systemic effects of exaggerated inflammation. It would be necessary to assess physiological
parameters such as changes in weight, hypothermia, and survival time following a lethal dose of
LPS. We have also shown that in vitro infection of SMYD5 deficient BMMs with Salmonella
typhimurium (ST) results in increased IL-6 and TNF production and in vitro bacterial killing.
However, these experiments would be more relevant within an in vivo setting, where mice are
infected with ST in order to evaluate serum cytokine levels and well as CFU counts within
multiple organs. The expression levels of NRAMP should also be measured, as this gene is
influenced by genetic background and has been extensively found to lead to artifacts in terms of
Salmonella typhimurium killing.
Mechanistically, it would also be useful to know if SMYD5 directly associates with the
IL-6 or Tnf promoter regions under basal conditions and following LPS challenge or ST infection
in vivo. This experiment would be of importance since previous studies using macrophage cell
lines and siRNA knockdown of SMYD5 reported that this molecule directly regulates Tnf
expression and that binding of SMYD5 leads to changes in histone methylation. These findings
have not been investigated in vivo, or under different experimental conditions. Using ChIP qPCR
experiments would allow us to answer many of these questions, and to further the body of
knowledge regarding the epigenetic regulation of SMYD5 gene targets. Ultimately
understanding how SMYD5 epigenetically regulates inflammatory genes could serve useful for
the development of new methods to manipulate the onset and duration of inflammation.
116

We also found that IL-4 activated M-CSF cultured BM cells showed changes in the
expression patterns of well-characterized M2 macrophage genes. This raises the question as to
whether SMYD5 is involved in M2 macrophage polarization in vivo. Other studies have shown
that the transcription factors STAT6 and peroxisome proliferator-activated receptor-γ (PPARγ)
are involved in polarization of M2 macrophages [54]. It would be interesting to determine if
SMYD5 deficiency leads to changes in the expression of alternative genes within macrophages
under conditions that promote M2 polarization. In vivo, administration of chitin, a polymerized
sugar and a structural component of helminths, arthropods and fungi [176], recruits macrophages
with the M2 phenotype to the site of administration. FACS analysis could be used to determine if
chitin-elicited M2 macrophages are recruited to the PerC at comparable levels within the
SMYD5-KO mice compared to controls. These M2 cells could also be isolated and mRNA
expression of hallmark genes such as: Arg1, Ym1, Fizz1 and MR could be determined [58]. It
would also be useful to determine if chitin administration has any indirect effects on the
polarization of M1 macrophages in vivo, since altered M2 phenotypes could possibly influence
other subtypes.
Other than what we have reported here regarding macrophage function, to our knowledge
we are the first group to identify a role for SMYD5 within BMDCs. Our ELISA assays showed
that LPS activated DCs from SMYD5-KO BM cells are able to secrete increased levels of
inflammatory cytokines compared to controls. However, because this is a novel finding
additional experiments would be needed in order to characterize the role of SMYD5 within DCs,
in vivo. DC development should be assessed in vivo by FACS analysis in naïve and antigen
challenged mice. Adoptive transfer experiments using SMYD5-KO DCs could also allow us to
determine if this molecule plays an intrinsic role in DC function. Both DCs and macrophages
117

secrete many of the same cytokines that can influence the inflammatory response and de-
regulation of genes involved in this response can lead to the development of diseases. Although
the research presented here raises many unanswered questions, our experiments identify SMYD5
as a potential target gene in the treatment of inflammatory conditions.

118

119

120

Related Publication by Author

Sanchez, S., L. Jones, V.L. Nandakumar, H.J. Won, and S.-Y. Chen. 2014. The histone
methyltransferase SMYD5 plays a role in regulating plasma cell differentiation and
antibody production. (submitted to BMC Immunology, under revision)

Sanchez, S., H. Jiang, L. Jones, H.J. Won, and S.-Y. Chen. 2014. Increased peritoneal cavity
macrophages and systemic sensitivity to LPS in the absence of the histone methyl
transferase SMYD5. (submitted to JI cutting edge, under revision)

Haejung Won, Vijayalakshmi Nandakumar, Peter Yates, Suzi Sanchez, Lindsey Jones, Xue F
Huang, and Si-Yi Chen. “Dendritic cell lineage specification driven by the histone
deubiquitinase MYSM1.” (submitted. Under revision, Blood)

Haejung Won, Yuchia Chou, Vijayalakshmi Nandakumar, Peter Yates, Suzi Sanchez, Lindsey
Jones, Xue F Huang, and Si-Yi Chen. Histone H2A deubiquitinase MYSM1 controls DC
function. (under preparation)

121

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Abstract (if available)

Abstract Chapter III: Abstract ❧ Increased peritoneal cavity macrophages and systemic sensitivity to LPS in the absence of the histone methyl transferase SMYD5 ❧ The SMYD subfamily of histone methyl transferases are all expressed within primary mouse macrophages. A previous study reported that siRNA knockdown of SMYD5 results in increased transcription of inflammatory genes in vitro, warranting further investigation into the role of SMYD5 in macrophage function and polarization in vivo. In this study we evaluated the systemic effects of TLR4 stimulation using a SMYD5-KO murine model. We observed that KO mice exhibited a spontaneous increase in the peritoneal cavity (PerC) monocyte/macrophage populations at baseline, and this increase persisted following LPS challenge by intraperitoneal injection. Peritoneal cavity macrophages also produced more intracellular cytokines, and the KO mice had increased pro-inflammatory serum cytokine levels. In assessing macrophage polarization in vitro, both bone marrow derived macrophages (BMMs) and dendritic cells (BMDCs) from KO mice secreted increased cytokine levels compared to controls. Quantitative-PCR analysis of M2-polarized BMMs showed that lack of SMYD5 resulted in decreased expression of alternative (M2) genes. We also determined the ability of BMMs to respond to Salmonella typhimurium infection in vitro, and observed increased cytokine production, initial bacterial uptake, and killing rate by the KO cells compared to controls. Our study reports an in vivo role for SMYD5 in the regulation of macrophage polarization and migration.

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Asset Metadata

Creator Sanchez, Suzi (author)

Core Title The histone methyltransferase SMYD5 plays a role in regulating plasma-cell antibody production and macrophage inflammatory cytokine secretion in vivo

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 09/25/2014

Defense Date 09/02/2014

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

Tag cytokines,histone methyltransferase,Inflammation,macrophages,OAI-PMH Harvest,SMYD5

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Chen, Si-Yi (committee chair), Akbari, Omid (committee member), McMillan, Minnie (committee member), Ouellette, Andre J. (committee member), Yuan, Weiming (committee member)

Creator Email Dr_suzis@yahoo.com,suzisanc@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-484782

Unique identifier UC11287185

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Legacy Identifier etd-SanchezSuz-2990.pdf

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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...

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