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PD-1 is a regulator of ILC2-mediated airway hyperresponsiveness and inflammation

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PD-1 is a regulator of ILC2-mediated airway hyperresponsiveness and inflammation

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Content PD-1 is a Regulator of ILC2-mediated Airway Hyperresponsiveness and Inflammation By Richard Lo A Thesis Presented to the Faculty of THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE in Molecular Microbiology and Immunology May 2018 2 TABLE OF CONTENTS ABBREVIATIONS .................................................................................................................... 4 LIST OF FIGURES .................................................................................................................... 5 CHAPTER 1: INTRODUCTION ................................................................................................ 6 1.1. Asthma ............................................................................................................................. 6 1.2. Pathophysiology of Asthma .............................................................................................. 8 1.3. Diagnosis and Treatment of Asthma ................................................................................. 9 1.4. The Discovery, Characterization, and Role of Type 2 Innate Lymphoid Cells in Asthma 10 1.5. The Role of Programmed Cell Death-1 in Immune Regulation ....................................... 14 CHAPTER 2: HYPOTHESIS ................................................................................................... 17 CHAPTER 3: MATERIALS AND METHODS ........................................................................ 19 3.1. Animals .......................................................................................................................... 19 3.2. Isolation of Murine Pulmonary ILC2s ............................................................................ 19 3.2.1. In vivo ILC2 Activation by Cytokine Treatment....................................................... 19 3.2.2. Single Cell Suspension Preparation .......................................................................... 19 3.2.3. Fluorescence-Activated Cell Sorting (FACS) for ILC2 Isolation .............................. 20 3.3. Cell Culture Conditions .................................................................................................. 21 3.4. Assessment of PD-1 Expression on Murine ILC2s .......................................................... 22 3.5. Murine Asthma Model ................................................................................................... 23 3.5.1. Murine Model for Asthma ....................................................................................... 23 3.5.2. Adoptive Transfer of Activated ILC2s ..................................................................... 23 3.5.3. PD-1 Neutralization Model ...................................................................................... 24 3.5.4. Measurement of Airway Hyperresponsiveness (AHR) ............................................. 24 3.5.5. Analysis of Bronchoalveolar Lavage (BAL) Fluid ................................................... 24 3.6. Assessment of Cytokine Production................................................................................ 26 3 3.6.1. Measurement of ex vivo Cytokine Secretion via Enzyme-linked Immunosorbent Assay (ELISA) ........................................................................................................ 26 3.6.2. Measurement of ex vivo Cytokine Secretion via Luminex ® ...................................... 27 3.6.3. Analysis of Intracellular Cytokine Production .......................................................... 27 3.7. Transcriptome Analysis of Murine ILC2s ....................................................................... 28 3.8. Statistical Analysis ......................................................................................................... 28 CHAPTER 4: RESULTS .......................................................................................................... 29 4.1. Murine ILC2s Express PD-1 ........................................................................................... 29 4.2. PD-1 −/− Mice Have Increased ILC2 Numbers in the Lungs ............................................. 30 4.3. Lack of PD-1 Enhances Cytokine Production by Murine ILC2s...................................... 31 4.4. Lack of PD-1 Increases Lung Eosinophil Counts ............................................................ 33 4.5. Lack of PD-1 in ILC2s Modulates Transcription Factors and Signals Related to Inflammation .................................................................................................................. 34 4.6. PD-1 Deficiency Exacerbates IL-33-induced AHR ......................................................... 35 4.7. Adoptively Transferred PD-1 −/− ILC2s Enhance IL-33-induced AHR ............................. 37 4.8. Blocking PD-1 Exacerbates IL-33-induced AHR and Lung Inflammation in Rag2 −/− Mice ............................................................................................................................... 39 CHAPTER 5: DISCUSSION .................................................................................................... 41 CHAPTER 6: FUTURE DIRECTIONS .................................................................................... 47 REFERENCES ......................................................................................................................... 49 4 ABBREVIATIONS Ab Antibody aILC2 Activated ILC2 AHR Airway Hyperresponsiveness BAL Bronchoalveolar Lavage CD Cluster of Differentiation cDyn Dynamic Compliance ELISA Enzyme-linked Immunosorbent Assay FACS Fluorescence-activated Cell Sorting GATA3 GATA-binding Protein 3 GM-CSF Granulocyte-macrophage Colony-stimulating Factor ICOS Inducible T cell Costimulator IL Interleukin ILC Innate Lymphoid Cell ILC2 Type 2 Innate Lymphoid Cell i.n. Intranasal i.p. Intraperitoneal KLRG1 Killer Cell Lectin-like Receptor G1 nILC2 Naïve ILC2 PBS Phosphate Buffered Saline PD-1 Programmed Cell Death Protein 1 PD-L1 Programmed Cell Death Ligand 1 PD-L1 Programmed Cell Death Ligand 2 Rag2 Recombination Activating Gene 2 TH2 Type 2 Helper T Cell TSLP Thymic Stromal Lymphopoietin WT Wild-type gc Gamma-chain 5 LIST OF FIGURES Figure 1: Current Asthma Prevalence by Percent in the United States in 2015 Figure 2: Innate Lymphoid Cell Development Figure 3: The Role of Lung ILC2s in Asthma Figure 4: PD-1 Pathway-mediated Inhibition of Effector T Cell Responses Figure 5: Activators and Regulators of ILC2s Figure 6: FACS Gating Strategy for Murine ILC2s Figure 7: Murine Model for PD-1 Expression Figure 8: Murine Asthma Model Figure 9: FACS Gating Strategy for Eosinophils Figure 10: PD-1 Expression is Upregulated on Murine ILC2s Upon IL-33 Stimulation Figure 11: The Lack of PD-1 Increases ILC2 Counts in the Lungs Figure 12: The Lack of PD-1 Enhances Type 2 Cytokine Production by ILC2s Figure 13: Lung Eosinophil Counts Increase in the Absence of PD-1 Figure 14: PD-1 Modulates the Expression of ILC2 Transcription Factors and Signals Figure 15: IL-33-induced AHR is Exacerbated in the Absence of PD-1 Figure 16: Adoptively Transferred PD-1 −/− ILC2s Exacerbate IL-33-induced AHR Figure 17: Blocking PD-1 in Rag2 −/− Mice Exacerbates IL-33-induced AHR & Inflammation Figure 18: PD-1 Agonists as a Potential Therapeutic for ILC2-mediated Allergic Asthma 6 CHAPTER 1 INTRODUCTION 1.1. Asthma Asthma is a chronic immunological disease of the lung airways. It is characterized by persistent airway inflammation, airway hyperresponsiveness (AHR), and airflow obstruction that can contribute to permanent airway remodeling and damage 1 . Symptoms of asthma include coughing, shortness of breath, wheezing, difficulty breathing, and chest tightness or pain 2 . The World Health Organization (WHO) estimates that over 300 million individuals worldwide suffer from asthma 3 . In the United States alone, at least 25 million individuals have asthma. Asthma affects individuals of all age groups, genders, ethnicities, and socioeconomic statuses; however, certain groups are more likely to develop asthma than others (Figure 1). Asthma is the most common chronic disease among children 4 . One in every 12 children develop asthma, while asthma affects one in every 13 adults in the United States 5 . Asthma prevalence is greater among females than in males. Furthermore, black or multi-race adults are more likely to develop asthma than white and Hispanic adults. Adults with an income below the federal poverty threshold are more likely to have asthma as well 5 . 7 Asthma can disrupt everyday life, especially when symptoms exacerbate. The Centers for Disease Control and Prevention (CDC) reports that approximately one in every two children miss at least a day of school, while one in every three adults miss at least one work day each year because of asthma. With proper care and education, asthma can be managed to limit its disruption of daily life; however, as healthcare costs continue to rise at staggering rates, not all are able to access the resources needed to manage their asthma. This is especially problematic for those of low socioeconomic statuses 2 . Without proper access, the likelihood of dying from asthma is much greater. With over 3,000 deaths occurring from asthma each year in the United States, this number can be drastically decreased if the resources to manage asthma are accessible to all 5 . Figure 1: Current Asthma Prevalence by Percent in the United States in 2015 One in every 12 (8.4% of total) children in the U.S. have asthma, while approximately one in every 13 (7.6%) adults have asthma. Females (9.1%) are more likely to have asthma compared to males (6.5%). The prevalence of asthma is the greatest among blacks (10.3%), followed by whites (7.8%), and Hispanics (6.6%) (Adapted from the National Health Interview Survey of the National Center for Health Statistics, Centers for Disease Control and Prevention). Children Adult 0 2 4 6 8 10 12 Age % Prevalence Male Female Gender White Black Hispanic Race/Ethnicity 8.4% 7.6% 6.5% 9.1% 7.8% 10.3% 6.6% Source: National Health Interview Survey, National Center for Health Statistics, Centers for Disease Control and Prevention 8 1.2. Pathophysiology of Asthma Asthma has been classically considered to be driven by the type 2 T helper (TH2) cell response, which is responsible for allergic inflammation, helminth expulsion, and tissue repair 6 . Inhaled allergens that reach the airway epithelium activate epithelial cells to secrete cytokines, such as thymic stromal lymphopoietin (TSLP), interleukin (IL)-25, and IL-33. TSLP and select chemoattractants of the CC chemokine family (e.g. CCL19, CCL20, and CCL27) mediate the recruitment and maturation of dendritic cells (DCs) from the bone marrow to the mucosal epithelium 7 . The mature DCs then take up allergens that breached the epithelium, process them into small peptides, and traffic to local lymph nodes to present the processed antigens to the T cell receptors (TCRs) of naïve cluster of differentiation 4 + (CD4 + ) T cells. In the presence of IL-4, transcription factors (TFs)—signal transducer and activator of transcription 6 (STAT6) and GATA-binding protein 3 (GATA3)—are activated to mediate the differentiation of naïve CD4 + T cells into TH2 cells 7 . Activated TH2 cells secrete type 2 cytokines (e.g. IL-4, IL-5, IL-9, and IL-13) that are responsible for the pathogenicity of asthma. TH2 cell-derived IL-4 promotes the production of immunoglobulin E (IgE). IgE binds to high-affinity IgE receptors, FcεRI, on granulocytes— eosinophils, basophils, and mast cells—to aid in granulocyte activation. Activated granulocytes release inflammatory mediators (e.g. histamine, kinins, and proteases) that lead to bronchospasms, increased vascular permeability, and inflammatory cell recruitment 6 . IL-5 promotes eosinophil maturation and survival, while IL-9 promotes mast cell hyperplasia 7 . IL-13 promotes goblet cell- mediated mucus production in the lumen and directly affects the airway smooth muscles, leading to AHR 8 . 9 1.3. Diagnosis and Treatment of Asthma Guidelines have been set by the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health (NIH) regarding the recommended methods of asthma diagnosis 1 . Clinicians first consider patients’ medical history by evaluating several factors: symptoms and their patterns, reported known allergies, current medications, family history of asthma or allergies, and employment history. Physical examinations of the upper respiratory tract, chest, and skin are also recommended for diagnosis. Physical indications of asthma include a hyper-expanded thorax, wheezing during normal breathing (or during forced exhalation), and atopic dermatitis. There is currently no cure for asthma, so available pharmacologic therapies only aid the control of asthma symptoms. Asthma medications are generally classed as long-term or short-term drugs. Long-term control medications are taken daily to attenuate airway inflammation and prevent asthmatic symptoms from arising. Inhaled corticosteroids are generally most effective, but they may sometimes be combined with other long-term control medications, such as immunomodulators and long-acting beta2-agonists. Even with long-term medications, asthmatic symptoms can still be exacerbated; therefore, short-term control medications are used to acutely relieve airflow obstruction. They do not attenuate airway inflammation and are not used in lieu of long-term drugs. Anticholinergics and systemic corticosteroids can be used, but short-acting beta2- agonists are the first-line drugs for quick relief. 10 1.4. The Discovery, Characterization, and Role of Type 2 Innate Lymphoid Cells in Asthma Asthma was once considered to be a simple TH2 cell-driven disorder that primarily functions through adaptive immunity; however, the discovery and characterization of type 2 innate lymphoid cells (ILC2s) have revealed that asthma actually involves both innate and adaptive immune cells. In the early 2000s, it was first discovered that IL-25 induces type 2 cytokine production by non-B and non-T cells, leading to eosinophilia, epithelial cell hyperplasia, mucus hypersecretion, and AHR 9,10 . IL-25 did not induce significant amounts of type 2 cytokines in Rag2-deficient gamma-chain-deficient (Rag2 −/− gc −/− ) mice, which suggested that non-B and non-T cell sources of type 2 cytokines require the common gamma chain 11 . Nearly a decade later, these cells were classified as ILC2s 12 . ILC2s are part of the cytokine-producing innate lymphoid cell (ILC) family that are of lymphoid morphology, but lack antigen-specific receptors characteristic of B and T lymphocytes 13 . ILCs are classified into three major subtypes—ILC1, ILC2, and ILC3—that mirror the three subtypes of CD4 + T helper cells—TH1, TH2, and TH17—in regard to the master transcription factors that regulate their development and the cells’ cytokine production profiles 14 . Like TH2 cells, ILC2s secrete type 2 cytokines that include: IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF) 7,13,14 . ILC2s can be found in the adipose tissue, blood, gastrointestinal tract, lungs, oral mucosa, secondary lymphoid organs, and skin of humans and mice 14 . Like all ILCs and lymphocytes, ILC2s arise from common lymphoid progenitors (CLPs) derived from hematopoietic stem cells in the bone marrow (Figure 2). CLPs develop into common helper innate lymphoid cell progenitors 11 (CHILPs) in the presence of TFs: GATA3 and T cell factor 1 (TCF1). The expression of the transcriptional inhibitor, inhibitor of DNA binding 2 (Id2), then directs the differentiation of CHILPs to ILC precursors (ILCPs). Lastly, ILCPs develop into ILC2 precursors (ILC2Ps) and then ILC2s upon the expression of critical TFs—GATA3 and retinoid-related orphan receptor alpha (RORα)—along with other TFs, like growth factor independence 1 (GFI1), B cell lymphoma/leukemia 11B (Bcl11b), and ETS1 transcription factors 14,15 . GATA3 is crucial because it is also required for the production of type 2 cytokines and the maintenance of ILC2 characteristics. In human ILC2s, silencing of the Gata3 gene attenuated ILC2-responsiveness to epithelial cell-derived cytokines 16 . Although GATA3 is also crucial for TH2 cell differentiation, RORα is uniquely required for ILC2 differentiation in the immune system. RORα directs ILC2 proliferation and effector function; without RORα, ILC2s fail to develop 13 . Figure 2: Innate Lymphoid Cell Development Like T cells, innate lymphoid cells (ILCs) commonly derive from common lymphoid progenitors (CLPs) that originate from hematopoietic stem cells (HSCs) in the bone marrow. CLPs develop into common helper ILC progenitors (CHILPs) and then to ILC progenitors (ILCPs), before differentiating into specific types of ILCs. The three types of ILCs resemble the three types of CD4 + helper T cells. CHILP Thymocyte ILCP ILC2P ILC1 T-bet T H 2 GATA3 ILC2 RORα GATA3 ILC3 RORγt T H 17 RORγt IFNγ IL-17 IL-22 IFNγ IL-17 IL-22 IL-4 IL-5 IL-13 IL-4 IL-5 IL-13 CLP HSC T H 1 T-bet 12 There is no specific cell surface marker for ILCs; therefore, ILCs are currently defined by the lack of expression of identifiable lineage markers of other immune cells (B220, CD11b, CD11c, CD3, FCεRI, Gr-1, and Ter119 for mice; CD1a, CD3, CD14, CD19, CD20, CD56, CD123, and CD235a for humans) and the expression of the leukocyte common antigen, CD45 13,14 . Furthermore, murine ILC2s are defined by the expression of the IL-33 receptor (ST2), IL-25 receptor (IL-17RB), inducible T cell costimulator (ICOS), killer cell lectin-like receptor G1 (KLRG1), IL-2 receptor-α (CD25), IL-7 receptor-α (CD127), and Thy1 (CD90). With the exception of CD90, human ILC2s express the same markers with the addition of CD161, CCR6, and prostaglandin D2 receptor 2 (CRTH2) 14 . In murine models of allergic asthma, lung exposure to IL-25, IL-33, TSLP, and allergens (e.g. house dust mite, protease papain, and Alternaria alternata) directly activate pulmonary ILC2s 17 . Activated ILC2s rapidly secrete type 2 cytokines—notably IL-5 and IL-13—that lead to eosinophilia, AHR, and airway inflammation (Figure 3). Because ILC2s are potent sources of type 2 cytokines, they can elicit characteristics of allergic asthma in a T cell-independent manner 18 . 13 In addition to having a role in the pathogenesis of asthma in an innate manner, ILC2s can also influence the induction of adaptive immunity. ILC2-secreted IL-13 promotes pulmonary DC activation and migration from the lymph nodes for the differentiation of TH2 cells from naïve CD4 + T cells. IL-13 also mediates the recruitment of TH2 cells to the lungs for the induction of memory TH2 cells by pulmonary DCs 14 . In addition to indirectly activating naïve CD4 + T cells, certain subsets of ILC2s also directly activate naïve T cells by expressing IL-4, IL-13, major histocompatibility complex (MHC)-II, and costimulatory molecules 14,17,18 . In addition to interacting with T cells with IL-13, ILC2-secreted IL-5 can interact with B cells to promote IgE antibody production 19 . Figure 3: The Role of Lung ILC2s in Asthma Inhaled allergens that reach the airway epithelium trigger the release of IL-25, IL-33, and TSLP. These cytokines directly activate lung ILC2s, which will potently secrete type 2 cytokines. IL-5 leads to the activation and recruitment of lung eosinophils. IL-13 can lead to changes, such as airway smooth muscle contraction and goblet cell-mediated mucus hypersecretion. The effects of IL-5 and IL-13 consequently promote airway hyperresponsiveness and inflammation. 14 Human ILC2s appear to have similar roles in the immunopathology of asthma. In comparison with healthy individuals, asthmatics tend to have an increased expression of IL-25, IL-33, or TSLP in their lungs that coincides with a greater number of ILC2s in the blood and sputum 14,20 . Furthermore, the number of IL-5 + and IL-13 + ILC2s are higher in the sputum of severe asthmatics. Ex vivo stimulation of peripheral blood mononuclear cells of asthmatic individuals resulted in greater number of ILC2s—as well as IL-5 and IL-13 production—compared to that of healthy individuals 14,17 . Because of its role in humans, ILC2s may be a potential therapeutic target for the treatment of asthma. 1.5. The Role of Programmed Cell Death-1 in Immune Regulation The programmed cell death-1 (PD-1) protein is an inhibitory receptor of the CD28 immunoglobulin superfamily. It was first discovered in 1992 as a gene upregulated in the programmed cell death of murine T cell hybridomas 21 . Despite its name, PD-1 has since been understood to have a physiological role unrelated to that of cell death 22 . PD-1 has two binding partners: programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2). PD-1 is expressed early in thymic development on CD4 − CD8 − thymocytes at low levels, but it is not expressed on mature resting cells 23,24 . Its expression is induced upon activation of various cells, including B cells, macrophages, DCs, T cells, and natural killer T (NKT) cells 25 . PD- L1 is constitutively expressed on resting B cells, DCs, macrophages, bone marrow-derived mast cells, T cells, and non-hematopoietic cells. PD-L1 expression is further upregulated upon cell activation 25 . In contrast to PD-L1, PD-L2 expression is only inducible on activated DCs, macrophages, bone marrow-derived mast cells, and select B cell subtypes 26 . Unlike PD-1 and PD- L1, PD-L2 is not expressed on T cells 24 . 15 PD-1 signaling functions as an immune checkpoint: the interaction between PD-1 and its ligands delivers co-inhibitory signals in effector T cells that prevent autoimmunity (Figure 4). Additionally, the PD-1-mediated suppression of T cell proliferation will direct the augmentation of peripheral tolerance for the maintenance of immune homeostasis 27 . PD-1’s role in tolerance was first suggested when PD-1 deficient (PD-1 −/− ) C57BL/6 mice spontaneously developed lupus-like glomerulonephritis and arthritis, while PD-1 −/− BALB/c mice developed autoimmune dilated cardiomyopathy 28,29 . Figure 4: PD-1 Pathway-mediated Inhibition of Effector T Cell Responses When PD-L1 or PD-L2 of antigen presenting cells (APCs) binds to the PD-1 receptor on T cells, the PD-1 pathway will inhibit activation signals of the T cell receptor (TCR) complex and CD28 costimulatory receptor; therefore, PD-1 inhibits the survival, proliferation, and cytokine production of effector helper and cytotoxic T cells. This suppression prevents autoimmunity and maintains peripheral tolerance. 16 Upon ligation, PD-1’s suppressive role is mediated by its recruitment of the Src homology- 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to its cytoplasmic domain. SHP-2 dephosphorylates molecules, like CD3ζ and ζ-associated protein of 70 kDa (ZAP-70), part of the TCR signaling complex; this consequently inhibits the Ras-MEK-ERK pathway 26 . SHP-2 also inhibits the phosphoinositide 3-kinase (PI3K)-Akt-mTOR pathway by inhibiting the phosphorylation and activation of phosphatase and tensin homolog (PTEN) tumor suppressor protein, which normally activates PI3K 22 . The PI3K pathway can also be inhibited by SHP-2- mediated dephosphorylation of the CD28 co-receptor 30 . By inhibiting the Ras-MEK-ERK and PI3K-Akt-mTOR pathways, PD-1 signaling consequently inhibits the survival, proliferation, and cytokine production of helper and cytotoxic T cells 31 . Furthermore, the subsequent inhibition of Akt in the PI3K-Akt-mTOR pathway allows for the production of the transforming growth factor- beta (TGF-β) cytokine. TGF-β induces the expression of the forkhead box P3 (Foxp3) transcription factor, which is responsible for the conversion of naïve T cells to induced regulatory T cells (iTregs), thus mediating the induction of peripheral tolerance 32 . Foxp3 + iTregs highly express PD-1 and PD- L1 for sustained immune tolerance 26 . Although the lack of PD-1 can spontaneously lead to autoimmune diseases, high expression of PD-1 and/or its ligands can aberrantly lead to chronic microbial infections and tumor growth in cancer 28,29,33 . Given its implications, the PD-1 pathway has become a popular target in therapeutics, especially with the success of PD-1 and PD-L1 blocking antibodies in cancer immunotherapy. 17 CHAPTER 2 HYPOTHESIS Asthma is a complex disease that disrupts the daily lives of millions of individuals throughout the world. Although asthma was once considered to be a simple TH2-driven disease of the adaptive immune response, it has become clear that innate immunity plays a significant role in asthma pathophysiology. This was brought into the forefront with the discovery of ILC2s. ILC2s mirror TH2 cells and provide an innate source of type 2 cytokines that are responsible for eosinophilic airway inflammation, AHR, and airflow obstruction—characteristics of allergic asthma. ILC2s are like other immune cells in that they express positive and negative regulatory surface molecules that maintain ILC2 function and homeostasis. ILC2s were recently shown to express inducible T cell costimulator (ICOS) and its ligand, ICOS-Ligand (ICOS-L), which provide the co-stimulatory signaling necessary for the survival and function of ILC2s 34 . Furthermore, the killer cell lectin-like receptor G1 (KLRG1) co-inhibitory molecule has also been found to be expressed on ILC2s and is necessary for the attenuation of ILC2-mediated inflammatory responses 35,36 . The PD-1 pathway is crucial for immune homeostasis and its involvement in adaptive immunity has been extensively studied; however, little is known about the PD-1 pathway in innate immunity. Although the roles of other regulatory molecules—ICOS and KLRG1—on ILC2s have been implicated, the role of PD-1 on ILC2s has yet to be clarified. PD-1 is highly expressed on innate lymphoid cell progenitors (ILCPs) in the bone marrow, serving as an early marker for ILC2 development 37,38 . With this in mind, we hypothesized that PD-1 may be expressed on mature ILC2s and have a tolerogenic role like that observed on effector T cells. 18 In this study, we investigated the expression of PD-1 on ILC2s—at steady state and after stimulation—and identified PD-1’s functional significance regarding the regulation of ILC2- mediated asthma in murine models (Figure 5). Exploring PD-1’s role on lung ILC2s may lead to the discovery and design of novel treatments to better control ILC2-mediated allergic asthma. Figure 5: Activators and Regulators of ILC2s ILC2s can be activated by cytokines, such as IL-25, IL-33, and TSLP. In addition to other cytokines that are required for ILC2 function and homeostasis, inducible T cell costimulator (ICOS) and killer cell lectin-like receptor G1 (KLRG1) were recently discovered as positive and negative regulators of ILC2s, respectively. In this study, we investigated the presence and role of PD-1 on murine ILC2s in the lungs. ST2 TSLPR IL-9R IL-7R IL-4R IL-17RB ILC2 KLRG1 ICOS ICOS-L E-cadherin IL-33 IL-25 TSLP IL-7 IL-9 IL-4 PD-1? Type 2 Cytokine Secretion IL-2R IL-2 ? 19 CHAPTER 3 MATERIALS AND METHODS 3.1. Animals All mice were housed and used in accordance with the guidelines set by the Institutional Animal Care and Use Committee of University of Southern California (USC). Wild-type (WT) BALB/cByJ, Rag2-deficient (Rag2 −/− ), and Rag2-deficient gamma-chain-deficient (Rag2 −/− gc −/− ) mice of BALB/c background mice were purchased from Jackson Laboratory (Bar Harbor, ME) and bred in our facility at USC. PD-1-deficient (PD-1 −/− ) mice of BALB/c background were kindly provided by Dr. Arlene Sharpe (Harvard Medical School, Boston, MA) and bred in our facility at the USC. 6-8-week-old age-matched female mice were used in all studies. 3.2. Isolation of Murine Pulmonary ILC2s 3.2.1. In vivo ILC2 Activation by Cytokine Treatment Mice were given intranasal (i.n.) treatments of recombinant mouse interleukin-33 (IL-33) (Biolegend ® , San Diego, CA) cytokine (0.5 µg/50 µL) once per day for three consecutive days. All mice were put under general anesthesia with 150 µL of ketamine xylazine cocktail prior to i.n. treatment. IL-33 treatment activated lung ILC2s in vivo, resulting in the phenotype of activated ILC2s (aILC2s). ILC2s not activated in vivo by IL-33 were naïve ILC2s (nILC2s). 3.2.2. Single Cell Suspension Preparation Lungs were surgically removed and minced, using fine surgical scissors, in a sterile environment and were incubated in type IV collagenase (1.6 mg/mL; Worthington Biochemicals, Lakewood, NJ) at 37°C for 60 minutes. The digested lung fragments were then pressed through a 20 70 µm nylon cell strainer, using the rubber end of a sterile 10 mL syringe plunger, to create a single cell suspension. To terminate the enzymatic reaction of collagenase, the cells were washed with 1x phosphate buffered saline (PBS) by centrifugation at 400x g for 7 minutes at 4°C. The cell pellet was resuspended in 1x red blood cell (RBC) lysis (Biolegend ® , San Diego, CA) and incubated at room temperature (RT) for 5 minutes to lyse the RBCs. The cells were then washed and centrifuged—at 400x g for 7 minutes at 4°C—with 1x PBS to terminate the chemical reaction. The remaining pellet was then further prepared for flow cytometry. 3.2.3. Fluorescence-Activated Cell Sorting (FACS) for ILC2 Isolation The single cell suspension was stained with a cocktail of biotinylated and fluorochrome- conjugated antibodies in a two-step staining process. The cells were first stained with cell surface markers: phycoerythrin/cyanine-7 (PE/Cy7) CD127, allophycocyanin/Cy7 (APC/Cy7) CD45, peridinin-chlorophyll-protein complex-eFluor ® 710 (PerCP-eFluor ® 710) ST2, and biotinylated lineage markers: gdTCR, Ly-6G/Ly-6C (Gr-1), Ter119, CD11b, CD11c, CD45R/B220, CD3e, and Fc Block; this is referred to as the primary stain. The PerCP-eFluor ® 710 ST2 and biotinylated gdTCR antibodies were sourced from eBioscience ™ (Thermo Fisher Scientific, Waltham, MA), while the remaining antibodies were sourced from Biolegend ® (San Diego, CA). The stained cells were incubated for 25 minutes at 4°C in 0.5% bovine serum albumin (BSA)/PBS staining buffer and then washed at 400x g for 7 minutes at 4°C. A secondary stain of fluorescein isothiocyanate (FITC) streptavidin (Biolegend ® , San Diego, CA) was added and the stained cells were incubated and washed under the same conditions as the previous staining step. Following the final wash, the cells were filtered through an 80 µm nylon mesh filter. The resulting stained single cell suspension, ready for fluorescence-activated cell sorting (FACS), was sorted on a 5-color BD FACSAria Ô III flow cytometer (BD Biosciences, Mountain 21 View, CA) for the isolation of purified ILC2s. Stained cells were also analyzed by FACS on an 8- color BD FACSCanto Ô II flow cytometer (BD Biosciences, Mountain View, CA). Murine ILC2s were identified as Lineage - , CD45 + , CD127 + , and ST2 + (Figure 6). ILC2s were quantified using CountBright Ô absolute counting beads (Life Technologies, Eugene, OR). All cells were sorted with a purity greater than 90%. FACS data in this study were analyzed with the FlowJo software (Ashland, OR). 3.3. Cell Culture Conditions All cells in this study were cultured (5 x 10 3 cells/well) in 96-well round-bottom plates with Gibco ™ Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA) that was supplemented with 10% fetal bovine serum (FBS), 2% antibiotics (penicillin and streptomycin), and 0.05 mM b-mercaptoethanol. The ILC2s were maintained in a 37°C incubator with 5% CO2. All ILC2s were cultured in the presence of recombinant mouse IL- Figure 6: FACS Gating Strategy for Murine ILC2s To identify murine ILC2s by FACS, the stained single cell suspension is gated for lymphocytes, single cells, lineage - , CD45 + , CD127 + , and ST2 + . The lineage markers are CD3ε, B220/CD45R, Gr-1, CD11b, CD11c, Ter119, TCR-γδ and FCεRI. CD45 SSC-A CD11c Siglec-F CD45 Lineage ST2 CD127 22 2 (10 ng/mL), IL-7 (10 ng/mL), and/or IL-33 (10 ng/mL). Naïve ILC2s stimulated ex vivo by IL- 33 became activated in culture. ILC2s activated in vivo were cultured with IL-2 and IL-7 only. 3.4. Assessment of PD-1 Expression on Murine ILC2s Naïve ILC2s (5 x 10 3 cells/well) of female BALB/cByJ mice were purified and cultured in recombinant mouse IL-2 (10 ng/mL), IL-7 (10 ng/mL), and IL-33 (10 ng/mL). ILC2s were analyzed for PD-1 expression after four time-points in culture: 0, 12, 24, and 48 hours. nILC2s assessed after 0 hours remained naïve, while ILC2s assessed after 12, 24, and 48 hours were activated ex vivo. After each respective time-point, ILC2s were stained with Brilliant Violet 421 ™ anti-PD-1 (Biolegend ® , San Diego, CA) and incubated at 4°C for 15 minutes. Cells were then washed, and the expression of PD-1 was assessed by FACS (Figure 7). + IL-2 + IL-7 + IL-33 Stain ILC2s for PD-1 Analyze ILC2s Naïve ILC2s Activated ILC2s (aILC2s) (nILC2s) nILC2s 48 Hr. 24 Hr. 12 Hr. 0 Hr. A B C D 1 2 3 4 5 6 Figure (__): Murine Asthma Model Figure 7: Murine Model for PD-1 Expression Naïve ILC2s (5 x 10 3 cells/well) of female 6-8-week-old BALB/cByJ mice were purified and cultured for 0, 12, 24, and 48 hours. 0-hour ILC2s remained naïve; 12-, 24-, and 48-hour ILC2s were activated ex vivo by IL-33 in culture. After each time-point, ILC2s were removed from culture, stained for PD-1, and analyzed for PD-1 expression by FACS. 23 3.5. Murine Asthma Model 3.5.1. Murine Model for Asthma Cohorts of female 6-8-week-old WT and PD-1 −/− mice were treated with three consecutive days of IL-33 (0.5 µg/50 µL) or 1x PBS (50 µL) i.n., as a control (Figure 8). All mice were put under general anesthesia prior to each i.n. treatment. 24 hours following the last i.n., the mice were put under general anesthesia for the assessment of lung tissue, airway hyperresponsiveness, and/or bronchoalveolar lavage fluid. 3.5.2. Adoptive Transfer of Activated ILC2s Pulmonary ILC2s from female WT and PD-1 −/− mice were activated after five consecutive days of IL-33 i.n. (0.5 µg/50 µL) and their aILC2s were purified by FACS. The aILC2s (2.5 x 10 4 ILC2s/mouse) were then resuspended in 1x PBS and adoptively transferred to 6-8-week old female Rag2 −/− gc −/− mice via intravenous tail injection (150 µL/mouse). The recipient Rag2 −/− gc −/− mice then received three consecutive days of i.n. with either IL-33 (1 µg/50 µL) or 1x PBS (50 µL). On the fourth day, airway hyperresponsiveness was assessed. i.n. IL-33 or PBS Day 1 2 3 4 WT PD-1 –/– Lung Dissection Figure 8: Murine Asthma Model Female 6-8-week-old WT and PD-1 −/− mice (n = 3-5) were treated with i.n. IL-33 (0.5 µg/50µL per mouse) or 1x PBS (50 µL) for three consecutive days. On the fourth day, the lungs of the mice were surgically removed and processed for analysis. 24 3.5.3. PD-1 Neutralization Model Cohorts of Rag2 −/− mice were treated intraperitoneally (i.p.) with an anti-PD-1 (αPD-1) blocking antibody (500 µg/mouse; clone: 29F.1A12; BioXcell, West Lebanon, NH) or a rat IgG2a κ isotype control (500 µg/mouse) on day one. Mice were then i.n. treated with IL-33 (0.5 µg/50 µL) or 1x PBS (50 µL) for the following three days. On the fifth day, airway hyperresponsiveness and bronchoalveolar lavage fluid cells were assessed. 3.5.4. Measurement of Airway Hyperresponsiveness (AHR) Mice were fully anesthetized (300 µL/mouse) for the measurement of airway hyperresponsiveness (AHR) by lung plethysmography, 24 hours following the last i.n. treatment. Lung function was assessed based on the measurement of lung resistance and dynamic compliance (cDyn) in restrained, tracheostomized, and mechanically ventilated mice using the Buxco Ò FinePointe Series Resistance and Compliance (RC) System (Data Sciences International, St. Paul, MN). The Buxco Ò FinePointe Series RC System challenged mice with an aerosolized bronchoconstrictor, methacholine (Sigma-Aldrich, St. Louis, MO), through an in-line nebulizer with increasing concentrations: 2.5, 5, 10, 20, and 40 mg/mL of methacholine. 3.5.5. Analysis of Bronchoalveolar Lavage (BAL) Fluid Upon completion of lung plethysmography, the mice were removed from the Buxco Ò FinePointe Series RC System and bronchoalveolar lavage (BAL) was performed. A blunt 19-gauge needle was inserted into the mice’s trachea and a 1 mL syringe, containing 1x PBS, was attached for the infusion of PBS to wash the lower respiratory tract, including the trachea, primary bronchi, and lungs. This was repeated two more times for the collection of BAL fluid cells in a total of 3 mL 1x PBS. 25 The cells collected in the BAL fluid were centrifuged at 400x g for 7 minutes at 4°C and the supernatant was removed. The cells were then stained with fluorochrome-conjugated antibodies and incubated for 25 minutes at 4°C. The antibodies used for BAL staining were: FITC CD45R/B220, PE Siglec-F, PerCP-Cy5 CD3, APC Gr-1, eFluor450 CD11b, APC-Cy7 CD11c, PE-Cy7 CD45, and Fc-Block. eFluor450 CD11b was purchased from eBioscience ™ (Thermo Fisher Scientific, Waltham, MA), while PE Siglec-F was purchased from BD Biosciences (Mountain View, CA). The remaining antibodies were sourced from Biolegend ® (San Diego, CA). Following the incubation period, the cells were washed with 1x PBS and resuspended with CountBright Ô absolute counting beads (Life Technologies, Eugene, OR) for acquisition on a BD FACSCanto Ô II (BD Biosciences, Mountain View, CA). Eosinophils were identified as CD45 + , Siglec-F + , and CD11c - (Figure 9). As previously mentioned, FACS data was analyzed with FlowJo (Ashland, OR). Figure 9: FACS Gating Strategy for Eosinophils Eosinophils can be identified by FACS as CD45 + , Siglec-F + , and CD11c - . CD45 SSC-A CD11c Siglec-F CD45 Lineage ST2 CD127 26 3.6. Assessment of Cytokine Production 3.6.1. Measurement of ex vivo Cytokine Secretion via Enzyme-linked Immunosorbent Assay (ELISA) Activated ILC2s of WT and PD-1 −/− mice were purified by FACS and cultured for 24 and 48 hours in IL-2 and IL-7. After each time-point, the culture supernatant was collected and analyzed by enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of type 2 cytokines secreted by the WT and PD-1 −/− aILC2s. Cytokine production was assessed using the Affymetrix ELISA Ready-Set-Go! ® IL-5 and IL-13 kits (Thermo Fisher Scientific, Waltham, MA). The ELISA kits were used in accordance with the protocols provided by the manufacturers. 96-well ELISA plates (BD Falcon ™ , Bedford, MA) were first coated with 100 µL/well of capture antibody (Ab)—specific for the cytokines being measured—diluted to 1x in coating buffer; the plates were then incubated overnight at 4°C. The following day, the plates were washed three times with 0.05% Tween Ò 20/PBS (VWR International, Radnor, PA) wash buffer. Each well was then blocked with 200 µL of 1x ELISA/ELISPOT diluent and the plate was incubated on a plate shaker at room temperature (RT) for 1 hour. The plate was then washed several times with wash buffer, followed by the loading of the cytokine standards at 100 µL/well. Serial dilutions of the cytokine standard were made, resulting in eight standard concentrations ranging from 500 pg/mL to 3.9 pg/mL. The supernatants of the samples were concurrently loaded onto the ELISA plates and were diluted in 1x ELISA/ELISPOT diluent for a total well volume of 100 µL. Once the standards and samples were loaded, the plates were incubated on a plate shaker at RT for 2 hours. Another wash was performed and 1x detection Ab was added to the plate at 100 µL/well. After incubating on a plate shaker for 1 hour at RT, the plates were washed and loaded with 100 µL/well 27 of 1x avidin-horseradish peroxidase (HRP), followed by a 30-minute incubation on a plate shaker at RT. A final wash was then performed and the plates were loaded with 100 µL/well of 1x 3,3’,5,5’-tetramethylbenzidine (TMB) substrate solution. Following a 15-incubation period at RT, 50 µL/well of stop solution (1M H3PO4) was added to halt the chromogenic reaction. The plates were then read on a DTX 880 Multimode Detector (Beckman Coulter, Brea, CA) microplate reader at 450 nm. 3.6.2. Measurement of ex vivo Cytokine Secretion via Luminex ® Activated ILC2s of WT and PD-1 −/− mice were purified by FACS and cultured for 24 and 48 hours in IL-2 and IL-7. After each time-point, the culture supernatant was collected for the detection and quantification of type 2 cytokines. In addition to ELISAs, the Luminex ® (R&D Systems, Minneapolis, MN) bead-based multiplex assay was also used. Supernatant samples were first preserved at -20°C and then sent to a third-party for Luminex ® analysis. 3.6.3. Analysis of Intracellular Cytokine Production Intracellular cytokine production was assessed with the BD Cytofix/Cytoperm ™ Plus (BD Biosciences, Mountain View, CA) kit. The kit was used in accordance with the protocols provided by the manufacturer. Using the murine asthma model, lungs of WT and PD-1 −/− mice were processed into a single cell suspension, which was treated with RBC lysis. The remaining cells were incubated in 10% FBS/RPMI, containing phorbol 12-myristate-13-acetate (PMA), ionomycin, and BD GolgiPlug ™ , for 4 hours at 37°C. PMA and ionomycin stimulate cytokine-producing cells, while the BD GolgiPlug ™ inhibits intracellular protein transport and disrupts protein secretion. After incubation, the cells were washed and incubated in the primary staining cocktail for ILC2s, as previously mentioned. Following another wash, the cells were incubated in 250 µL of 28 fixation/permeabilization solution for 30 minutes at 4°C. The cells were then washed twice in BD Perm/Wash ™ buffer, stained with FITC streptavidin, PE IL-5 (Biolegend ® , San Diego, CA), and eFluor ® 450 IL-13 (eBioscience ™ , Thermo Fisher Scientific, Waltham, MA)—all diluted in BD Perm/Wash ™ buffer—and incubated for another 30 minutes at 4°C. The stained cells were washed a final time in BD Perm/Wash ™ buffer. Lastly, CountBright Ô beads were added to quantify IL- 5 + IL-13 + ILC2s by FACS. 3.7. Transcriptome Analysis of Murine ILC2s ILC2s of WT and PD-1 −/− mice were activated in vivo and the aILC2s (5 x 10 5 cells) were purified by FACS. Ribonucleic acid (RNA) extraction of the ILC2s was then performed using the RNeasy Micro Kit (Qiagen ® , Germantown, MD). The RNeasy kit was used in accordance with the protocols provided by the manufacturers. The purified total RNA was then set to a third-party for transcriptome analysis using NanoString nCounter ® technology (Seattle, WA). 3.8. Statistical Analysis All data are expressed as mean ± standard error of the mean (SEM). Comparisons between study groups were analyzed by Student’s t-tests. P values of <0.05 were considered to be statistically significant: *p < 0.05, **p < 0.01, ***p < 0.001. Statistical analyses were performed using the GraphPad Prism 7 software (La Jolla, CA). 29 CHAPTER 4 RESULTS 4.1. Murine ILC2s Express PD-1 The expression of PD-1 has been widely reported on various cells, such as B and T cells, upon activation; however, the expression of PD-1 on ILC2s have yet to be determined. In this study, we first examined the extent of PD-1 expression on murine nILC2s and aILC2s by FACS (Figure 7). Murine ILC2s are identified as lineage - , CD45 + , CD127 + , and ST2 + (Figure 6). We found that freshly isolated naïve ILC2s express low levels of PD-1 (Figure 10A). The expression of PD-1 was significantly upregulated (~ 4.7-fold) by ILC2s as early as 12 hours after ex vivo stimulation with IL-33. PD-1 expression remained upregulated up to 48 hours following initial stimulation (Figure 10B). These findings suggest that murine ILC2s express PD-1—albeit at low levels—at steady state and that PD-1 expression is inducible on ILC2s by IL-33 stimulation. Figure 10: PD-1 Expression is Upregulated on Murine ILC2s Upon IL-33 Stimulation nILC2s (5 x 10 3 cells/well) were purified by FACS and cultured in IL-2, IL-7, and IL-33 for 0, 12, 24, or 48 hours. ILC2s were then stained for PD-1 and analyzed by FACS. nILC2s analyzed after 0 hours remained unstimulated; ILC2s analyzed after 12, 24, and 48 hours were stimulated by IL- 33 in culture and became aILC2s. A) Representative histogram of FACS analysis of PD-1 expression on ILC2s at the indicated time points. B) Bar graph reflecting the median fluorescence intensity (MFI) of PD-1 expression by purified murine ILC2s at the indicated time points after culture. Data are expressed as means ± SEM (n=3). *p < 0.05, **p < 0.01. PD-1 Count Isotype Control 0 Hour 12 Hour 24 Hour 48 Hour A B 30 4.2. PD-1 −/− Mice Have Increased ILC2 Numbers in the Lungs Since PD-1 is expressed on ILC2s, we next examined if PD-1 has an effect on the homeostasis of lung ILC2s. To do so, we used FACS to assess the number of lung ILC2s of WT and PD-1 −/− mice treated with i.n. IL-33 or PBS (Figure 8). At steady state, the number of naïve ILC2s was 1.9-fold higher in the lungs of PD-1 −/− than the WT mice (Figure 11A). Both PD-1 −/− and WT mice had increased ILC2 counts upon activation by IL-33; however, the number of activated ILC2s observed in PD-1 −/− mice was approximately 2.5-fold greater than that observed in the IL-33-treated WT mice (Figure 11B). These results suggest that PD-1 suppresses the number of lung ILC2s at steady state and when activated. WT PD-1 -/- 0 1 2 3 4 # of Naïve ILC2s in Lungs (x10 4 ) ** WT PD-1 -/- 0 2 4 6 8 10 # of Activated ILC2s in Lungs (x10 4 ) *** A B Figure 11: The Lack of PD-1 Increases ILC2 Counts in the Lungs Female 6-8-week-old WT and PD-1 −/− mice were given three consecutive days of IL-33 or PBS i.n. Total ILC2 count in the lungs were measured by FACS. A) Total number of naïve ILC2s in lungs. B) Total number of activated ILC2s in lungs. Data are expressed as means ± SEM (n=4). **p < 0.01, ***p < 0.001. 31 4.3. Lack of PD-1 Enhances Cytokine Production by Murine ILC2s Because we observed a greater number of ILC2s in the lungs of PD-1 −/− mice compared to that of WT mice, we examined if PD-1 deficiency leads to an increase in type 2 cytokine secretion by ILC2s. To do so, we measured the ex vivo production of type 2 cytokines by WT and PD-1 −/− aILC2s after 24 and 48 hours in culture. Cytokine production was first measured with ELISA. PD-1 −/− aILC2s had a 68% and 58% increase in IL-5 production after 24 and 48 hours, respectively, when compared to the WT aILC2s (Figure 12A). Similarly, PD-1 −/− aILC2s secreted 84% and 85% more IL-13 after 24 and 48 hours, respectively, compared to the WT (Figure 12B). When measured by Luminex ® , PD-1 −/− aILC2s also showed an increase in IL-5 secretion by 80% and 15% after 24 and 48 hours, respectively (Figure 12C). The Luminex ® assay was not optimized for IL-13; therefore, IL-13 could not be quantified. The Luminex ® assay did indicate that PD-1 −/− aILC2s had an increase in granulocyte-macrophage colony-stimulating factor (GM- CSF) secretion by 105% after 24 hours and a 38% increase after 48 hours (Figure 12D). Both the ELISA and Luminex ® results showed that PD-1 −/− aILC2s secreted more type 2 cytokines ex vivo, indicating that PD-1 suppresses ILC2 secretion of type 2 cytokines. To confirm that ILC2s produce more type 2 cytokines in the absence of PD-1 in vivo, we quantified the number of IL-5 and IL-13 producing (IL-5 + IL-13 + ) ILC2s of WT and PD-1 −/− mice using intracellular staining. At steady state, PBS-treated PD-1 −/− mice had approximately 60% more IL-5 + IL-13 + naïve ILC2s than the PBS-treated WT (Figure 12E). Furthermore, IL-33-treated PD-1 −/− mice had 180% more IL-5 + IL-13 + activated ILC2s when compared with their IL-33- treated counterpart (Figure 12F). These results again suggest that PD-1 suppresses ILC2s’ ability to produce type 2 cytokines, both at steady state and when activated. 32 Figure 12: The Lack of PD-1 Enhances Type 2 Cytokine Production by ILC2s A-D) Activated ILC2s (5 x 10 3 cells/well) from the lungs of WT and PD-1 −/− mice were purified by FACS and cultured with IL-2 and IL-7. The culture supernatants were retrieved after 24 and 48 hours for cytokine measurement. Measurement of (A) IL-5 and (B) IL-13 levels by ELISA. Measurement of (C) IL-5 and (D) GM-CSF by Luminex ® . E-F) Female 6-8-week old WT and PD-1 −/− mice were given three days of i.n. IL-33 or PBS. Lung ILC2 were intracellularly stained for IL-5 and IL-13 and analyzed by FACS. The total count of IL-5 + IL-13 + (E) naïve ILC2s (nILC2s) and (F) activated ILC2s (aILC2s). Data are expressed as means ± SEM (n=5). *p < 0.05, **p < 0.01, ***p < 0.001. 24 48 0 5 10 15 20 Hours IL-5 (x10 2 pg/mL) WT PD-1 -/- ** * A 24 48 0 2 4 6 8 10 Hours IL-13 (x10 2 pg/mL) WT PD-1 -/- * * B 24 48 0 1 2 3 4 Hours IL-5 (x10 3 pg/mL) WT PD-1 -/- *** C 24 48 0 2 4 6 8 10 Hours GM-CSF (x10 2 pg/mL) WT PD-1 -/- *** ** D E WT PD-1 -/- 0 1 2 3 4 5 # of IL-5 + IL-13 + nILC2s in Lungs (x10 2 ) ** F WT PD-1 -/- 0 1 2 3 4 5 # of IL-5 + IL-13 + aILC2s in Lungs (x10 3 ) * 33 4.4. Lack of PD-1 Increases Lung Eosinophil Counts Since our immunodetection assays indicated that PD-1 −/− aILC2s secreted significantly more IL-5 and GM-CSF than the WT aILC2s, we next assessed whether PD-1 −/− mice have more lung eosinophils. PD-1 −/− and WT mice were i.n. treated with three-consecutive days of IL-33 or PBS and lung eosinophil counts were then measured by FACS (Figure 8). Eosinophils can be identified as CD45 + , Siglec-F + , CD11c − (Figure 9). FACS analysis showed that PD-1 −/− mice had a 5-fold increase in eosinophils in their lungs, compared to the WT mice, when treated with PBS. Both groups exhibited increased eosinophil counts when treated with IL-33; however, PD-1 −/− mice had a 1.5-fold increase in lung eosinophils when compared to the WT (Figure 13). The increase in lung eosinophils in the absence of PD-1 suggests that PD-1 inhibits ILC2 secretion of IL-5 and GM-CSF, thus hindering the recruitment, activation, and survival of lung eosinophils. Figure 13: Lung Eosinophil Counts Increase in the Absence of PD-1 Female 6-8-week-old WT and PD-1 −/− mice were given three consecutive days of i.n. with IL-33 or PBS. The lungs were processed the following day and analyzed for total eosinophil count by flow cytometry. Data are expressed as means ± SEM (n=4). *p < 0.05. 0 5 10 15 20 25 Eosinophil Count in Lungs (x10 4 ) WT * PD-1 -/- * IL-33 + 34 4.5. Lack of PD-1 in ILC2s Modulates Transcription Factors and Signals Related to Inflammation To determine changes in the gene expression of transcription factors and signals related to lung inflammation in the absence of PD-1, ribonucleic acid (RNA) was extracted from purified aILC2s from PD-1 −/− and WT mice and the samples were sent out for transcriptome analysis. Transcriptome analysis revealed that the expression of over 200 genes were modulated by PD-1 on ILC2s (Figure 14). Included were genes of transcription factors, such as GATA3: there was a 28% increase in Gata3 expression in the absence of PD-1. Furthermore, the gene expression of type 2 cytokines was upregulated in our PD-1 −/− samples. IL-5 gene expression was upregulated by 75%, while the expression of the GM-CSF gene, csf2, was increased by 67%. Although IL-13 gene expression did get upregulated, there was only a 27% increase. These results suggest that PD- 1 downregulates the gene expression of key transcription factors and cytokines of ILC2s. 35 4.6. PD-1 Deficiency Exacerbates IL-33-induced AHR To assess whether PD-1 has a role in the regulation of ILC2-mediated airway hyperresponsiveness (AHR) in vivo, cohorts of WT and PD-1 −/− mice were treated with three consecutive days of i.n. IL-33 or PBS (Figure 8). On the following fourth day, AHR was assessed based on the measurement of lung resistance and dynamic compliance (cDyn) in response to increasing concentrations of a nebulized bronchoconstrictor, methacholine. Figure 14: PD-1 Modulates the Expression of ILC2 Transcription Factors and Signals aILC2s (5 x 10 5 cells) of PD-1 −/− and WT mice were first cultured and then extracted for RNA. Transcriptome analysis of the RNA samples were performed by NanoString. The heat map depicts over 200 genes whose expression was modulated in the presence (left) or absence (right) of PD-1 (n=2). 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A NC12 IL-13 IL-5 GM-CSF GATA3 Control PD-1 −/− 36 Upon methacholine challenge, lung resistance was greater among the IL-33-treated groups when compared to their PBS-treated counterparts; however, IL-33-treated PD-1 −/− mice had significantly greater resistance than the IL-33-treated WT mice. (Figure 15A). Consistent with our lung resistance findings, the airways of the IL-33-treated groups were much less compliant compared to that of the PBS-treated groups (Figure 15B). Not only did the IL-33-treated PD-1 −/− mice have more lung resistance, but they also showed significantly less cDyn than the IL-33- treated WT mice. These results indicate that PD-1 suppresses IL-33-induced AHR. Figure 15: IL-33-Induced AHR is Exacerbated in the Absence of PD-1 Female 6-8-week-old PD-1 −/− and WT mice were given three consecutive days of IL-33 or PBS i.n. and AHR was assessed on the following fourth day. A) Airway resistance measured in response to increasing concentrations of methacholine. B) Dynamic compliance (cDyn) measured in response to increasing concentrations of methacholine. Data are expressed as means ± SEM (n=5). *p < 0.05. A B 0 2.5 5 10 20 0 5 10 15 Methacholine (mg.ml -1 ) Resistance (cmH 2 O.ml -1 .s -1 ) WT + PBS WT + IL-33 PD-1 -/- + PBS PD-1 -/- + IL-33 * 0 2.5 5 10 20 0.01 0.02 0.03 0.04 0 Methacholine (mg.ml -1 ) cDyn (ml.cmH 2 O -1 ) WT + PBS WT + IL-33 PD-1 -/- + PBS PD-1 -/- + IL-33 * 37 4.7. Adoptively Transferred PD-1 −/− ILC2s Enhance IL-33-induced AHR To demonstrate that PD-1 deficiency on ILC2s leads to enhanced IL-33-induced AHR and to eliminate the possibility that the observed effects were due to bystander immune cells, we adoptively transferred purified aILC2s from WT and PD-1 −/− mice into immunodeficient Rag2 −/− gc −/− mice. The Rag2 −/− gc −/− mice then received three consecutive of i.n. IL-33 or PBS. On the fourth day, their lung function was evaluated (Figure 16A). Upon methacholine challenge, no significant difference in lung resistance was observed among the PBS-treated groups. Again, lung resistance was enhanced by i.n. IL-33 treatment; however, the IL-33-treated Rag2 −/− gc −/− mice that had adoptively transferred PD-1 −/− ILC2s had significantly greater AHR at the 40 mg/mL dose of methacholine compared to the Il-33-treated WT ILC2 recipients (Figure 16B). Although there was a drastic decline in airway cDyn due to IL- 33 administration, no further decrease in cDyn was observed in the PD-1 −/− ILC2 recipient group (Figure 16C). Taken these data together, PD-1 suppresses ILC2-mediated AHR that is induced by IL-33. 38 A B C Figure 16: Adoptively Transferred PD-1 −/− ILC2s Exacerbate IL-33-induced AHR 2.5 x 10 4 aILC2s from WT and PD-1 −/− mice were adoptively transferred to each female 6-8-week- old Rag2 −/− gc −/− mice, which were then treated with three-consecutive days of IL-33 (1 µg/mouse) or PBS. A) Schematic diagram of adoptive transfer murine asthma model used in this study. B) Maximum resistance measured with increasing concentration of methacholine. C) Dynamic compliance (cDyn) measured with increasing concentration of methacholine. Data are expressed as mean ± SEM (n=4). *p < 0.05. 39 4.8. Blocking PD-1 Exacerbates IL-33-induced AHR and Lung Inflammation in Rag2 −/− Mice We next examined whether blocking PD-1 would lead to the same results as observed in the PD-1 −/− mice. To do so, we first i.p. treated Rag2 −/− mice with an αPD-1 Ab or a rat IgG2a κ isotype control, followed by three consecutive days of i.n. IL-33 or PBS, and AHR assessment on the fifth day (Figure 17A). As previously observed, there was no difference in lung resistance among the PBS-treated groups—regardless of ⍺PD-1 Ab treatment—and IL-33 administration induced AHR; however, IL-33-treated mice that received the blocking Ab had even greater lung resistance than the IL-33-treated control mice (Figure 17B). Since IL-33-induced AHR was exacerbated due to the blockage of PD-1 in the Rag2 −/− mice, these results again suggest that PD- 1 suppresses ILC2-mediated AHR elicited by IL-33. Since we observed an enhancement of IL-33-induced AHR in the Rag2 −/− mice that received the ⍺PD-1 Ab, we also examined if these mice had greater eosinophil-driven lung inflammation. After assessing AHR, BAL was performed; BAL fluid cells were then stained and analyzed for eosinophils (Figure 9). BAL fluid analysis revealed that eosinophil count increased in response to IL-33, but BAL eosinophil count was even greater (~ 1.2-fold) when treated with both IL-33 and ⍺PD-1 (Figure 17C). Blocking PD-1 exacerbated AHR and enhanced eosinophil counts in the BAL fluid, suggesting that PD-1 inhibits ILC2-mediated AHR and eosinophil-driven airway inflammation in response to IL-33. 40 A B C 0 5 10 20 40 0 2 4 6 8 10 Methacholine (mg.ml -1 ) Resistance (cmH 2 O.ml -1 .s -1 ) Control + PBS Control + IL-33 !PD-1 + IL-33 !PD-1 + PBS PBS IL-33 !PD-1 + IL-33 0 5 10 15 20 25 Eosinophil Count in BAL (x10 4 ) Figure 17: Blocking PD-1 in Rag2 −/− Mice Exacerbates IL-33-induced AHR & Inflammation Female 6-8-week-old Rag2 −/− mice were first i.p. treated with either an ⍺PD-1 antibody (500 µg/mouse) or rat IgG2a κ isotype control (500 µg/mouse). After antibody treatment, mice were i.n. treated with three consecutive days of IL-33 (0.5 µg/mouse) or PBS. A) Schematic diagram of murine asthma model used in this study. B) Average resistance measured with increasing concentrations of methacholine. C) Total eosinophil count in the BAL fluid. Data are expressed as means ± SEM (n=3). 41 CHAPTER 5 DISCUSSION The phenotype and pathogenesis of allergic asthma was once considered to be driven only by TH2 cells and their production of type 2 cytokines, but it is now understood that ILC2s are the primary drivers of type 2 cytokine production and allergic asthma. While it has been suggested that PD-1 is expressed on ILCPs during the development of ILC2s in the bone marrow, our study is the first to demonstrate that mature murine ILC2s express PD-1 in the lungs 37,38 . Furthermore, we are the first to report that PD-1 has a negative regulatory role on ILC2s in the context of ILC2- mediated AHR and inflammation in murine asthma models. We first showed in this study that murine ILC2s express PD-1. At steady state, PD-1 was expressed at low levels on naïve ILC2s. This contrasts naïve T cells, which do not express PD-1 at steady state 24 . In response to the epithelium-derived cytokine, IL-33, the expression of PD-1 was significantly upregulated, which suggests that PD-1 expression is inducible on ILC2s upon activation by IL-33. This is similar to the inducible expression of PD-1 on activated T cells 26 . The kinetics of PD-1 expression on ILC2s also resemble that of KLRG1 on ILC2s 35,36 . Although ILC2s can be activated by IL-25, IL-33, and TSLP, an IL-33-based model was used for ILC2 activation in our studies because IL-33 has been shown to be a more potent activator of ILC2s and more strongly induces ILC2-mediated AHR and airway inflammation 34,39,40 . Because murine ILC2s express PD-1, the functional role of PD-1 on ILC2s was assessed. We observed that PD-1 −/− mice had significantly greater naïve ILC2 counts in the lung, as compared to the PD-1-expressing WT mice, at steady state. This suggests that PD-1 may be expressed at basal levels in the airways to keep naïve ILC2s in check. When treated with IL-33, lung ILC2 numbers increased in both WT and PD-1 −/− mice due to ILC2 activation, as expected. 42 Nonetheless, the PD-1 −/− mice still had significantly more activated ILC2s in the lung. Activated T cells highly express PD-1 to help shut down the effector T cell response and prevent further activation of additional effector T cells in a negative feedback loop 24 . PD-1 may play a similar role on activated ILC2s as well. PD-1 may normally keep ILC2 numbers in check, but the absence of PD-1 allows lung ILC2s to increase unsuppressed at steady state and when activated by IL-33. Given the increased number of lung ILC2s in the absence of PD-1, we assessed whether PD-1 −/− aILC2s secreted more type 2 cytokines than the WT ex vivo. IL-5 and IL-13 ELISAs detected significantly more IL-5 and IL-13 in the supernatant of PD-1 −/− aILC2s than in the WT aILC2s, both after 24 and 48 hours in culture. Although IL-13 was not detectable at quantifiable levels via Luminex ® , significantly greater levels of IL-5 were secreted by PD-1 −/− aILC2s, matching our ELISA results. Furthermore, Luminex ® indicated that PD-1 −/− aILC2s also secreted significantly more GM-CSF than WT aILC2s. ILC2s do secrete other type 2 cytokines, such as IL-4, but quantifiable changes in cytokine secretion were not detectable by our immunodetection assays. This is likely due to the effects of IL-33 stimulation, as ILC2s activated by TSLP have been shown to potently produce IL-4 41 . Given these results together, aILC2s more potently produce type 2 cytokines in the absence of PD-1. These data suggest that PD-1 expression is upregulated on activated ILC2s to suppress ILC2 production of type 2 cytokines. To confirm our ex vivo findings, we analyzed the in vivo production of type 2 cytokines by intracellular staining. PD-1 −/− mice had more IL-5 and IL-13 cytokine-expressing ILC2 subsets in the lungs than the WT mice at steady state. Both cohorts treated with IL-33 had increased IL-5 + IL- 13 + ILC2s, which was expected since activated ILC2s are potent sources of IL-5 and IL-13 42 . When compared to the IL-33-treated WT mice, the IL-33-treated PD-1 −/− mice had significantly more IL-5 + IL-13 + aILC2s. In congruence with our ELISA and Luminex ® findings, we confirmed 43 that type 2 cytokine production is enhanced in the absence of PD-1 on ILC2s. These results collectively suggest that PD-1 may negatively regulate ILC2 production of type 2 cytokines, both at steady state and when stimulated by IL-33. Eosinophilia is hallmark of allergic asthma driven by IL-5, which potently activates eosinophils and recruits them to the lungs 17,43 . Additionally, GM-CSF also promotes the production, activation, and survival of eosinophils, which in turn contribute to allergic airway inflammation in murine models 44 . Since IL-5 and GM-CSF secretion was significantly enhanced in the absence of PD-1, we assessed the lungs for eosinophils. At basal levels, eosinophils typically reside in the gastrointestinal tract and are rarely localized in the lung 36,45 . Low counts of lung eosinophils were congruently observed in the PBS-treated WT mice; however, this was not observed among the PBS-treated PD-1 −/− mice. At basal levels, PD-1 −/− mice had significantly more lung eosinophils than their WT counterpart. Although IL-33 stimulation increased eosinophil count in both cohorts, the count remained significantly greater in the PD-1 −/− mice. The lack of PD-1 enhances the number of lung eosinophils, presumably due to the increased production and secretion of IL-5 and GM-CSF by PD-1 −/− ILC2s. This effect is further amplified upon IL-33- induced ILC2 activation. These results suggest that the PD-1 suppresses the production of IL-5 and GM-CSF to prevent the activation, recruitment, and/or survival of lung eosinophils—factors that contribute to the number of eosinophils present in the lungs. Our findings thus far have revealed that the absence of PD-1 is associated with the significant increase in lung ILC2s as well as the enhanced production of type 2 cytokines; therefore, we assessed whether PD-1 influences the expression of ILC2-related genes. Transcriptome analysis revealed that the expression of the GATA3 gene, Gata3, is upregulated in the absence of PD-1. This explains our previous findings that PD-1 −/− mice had more lung ILC2s, 44 since GATA3 is one of the main transcription factors required for ILC2 differentiation and maintenance 46 . Without PD-1, high expression of GATA3 drives the differentiation of ILC2s; therefore, more ILC2s are generated and maintained in the lungs. In addition to GATA3, the genes of IL-5, IL-13, and GM-CSF were upregulated among PD-1 −/− ILC2s. This demonstrates why a significant increase in type 2 cytokine production was observed in ILC2s lacking PD-1. Given these results from transcriptome analysis, PD-1 inhibits the transcription and subsequent expression of transcription factors and cytokines associated with ILC2 homeostasis and function in the lungs. Based on our findings, PD-1 has a negative regulatory role on the function and homeostasis of ILC2s, like that of KLRG1. We next assessed whether the absence of PD-1 leads to phenotypic changes, such as enhanced airway hyperresponsiveness. Lung plethysmography of WT and PD- 1 −/− mice revealed that PBS-treated groups showed no difference in lung resistance; however, IL- 33-treated PD-1 −/− mice exhibited greater lung resistance compared to that of the IL-33-treated WT group. Additionally, the IL-33-treated PD-1 −/− mice exhibited significantly less cDyn than their IL-33-treated counterpart. These results indicate that the absence of PD-1 exacerbates IL-33- induced AHR. This is in agreement with our finding that PD-1 −/− ILC2s have enhanced IL-13 secretion, since IL-13 can directly affect airway epithelium and smooth muscles to elicit AHR 17 . Since PD-1 suppresses the production of IL-13, it may have the downstream effect of preventing the exacerbation of IL-33-induced AHR. Despite these findings, the WT and PD-1 −/− mice used in the previous study inherently have other cells, like TH2 cells, that may contribute to AHR with the production of type 2 cytokines; therefore, we wanted to confirm that the increase in AHR was mediated by ILC2s and not due to bystander immune cells. To do so, we purified aILC2s from PD-1 −/− and WT mice and adoptively 45 transferred the aILC2s into Rag2 −/− gc −/− mice. Rag2 −/− gc −/− mice that received PD-1 −/− ILC2s had greater IL-33-induced AHR than that of the WT ILC2-recipient group. Since Rag2 −/− gc −/− mice not only lack B and T cells, but also NK cells and ILCs, the observed changes in AHR were representative of the ILC2s that we had adoptively transferred into these immunocompromised mice. This confirms that the enhanced IL-33-induced AHR we observed in the absence of PD-1 was mediated by ILC2s. Lastly, we wanted to determine if blocking PD-1 elicits similar effects as when PD-1 was genetically deleted in our PD-1 −/− mice. Using Rag2 −/− mice, we observed that blocking PD-1 resulted in greater IL-33-induced AHR when compared to the cohort receiving the isotype control. Again, this confirms that PD-1 has a suppressive role in ILC2-mediated AHR. We also examined whether blocking PD-1 leads to enhanced airway inflammation. Although IL-33 treatment led to greater BAL fluid eosinophils, mice treated with both IL-33 and αPD-1 had even greater levels of BAL eosinophils. This suggests that airway inflammation is driven by eosinophilia in the absence of PD-1 signaling on ILC2s. This is consistent with our previous findings, as we had observed a significant increase in IL-5 and GM-CSF secretion by PD-1 −/− ILC2s. This also correlated with the increase in lung eosinophil counts observed in our PD-1 −/− mice. In conclusion, our study reveals that the lack of PD-1 enhances lung ILC2 and eosinophil counts, associated with increased GATA3 and type 2 cytokine gene expression. Furthermore, we show that blocking or genetically deleting PD-1 exacerbates IL-33-induced AHR and lung inflammation that is due to increased type 2 cytokine production. These results collectively demonstrate that PD-1 suppresses lung ILC2 homeostasis and function, thus hindering ILC2- mediated cytokine production. As a result of reduced IL-5 and GM-CSF secretion, PD-1 subsequently prevents the activation and recruitment of lung eosinophils that would typically drive 46 eosinophilic lung inflammation. Furthermore, the reduction of IL-13 secretion prevents the exacerbation of IL-33-induced AHR. Our findings indicate that PD-1 plays an important role in the regulation of ILC2-mediated allergic asthma in murine models. Although our findings have yet to be confirmed in humans, PD- 1 may play a similar role on human ILC2s as well. It may be possible that patients with severe allergic asthma may have a lack of or dysfunctional expression of PD-1 on their lung ILC2s, which could consequently drive the pathogenesis of their asthma. If that may be the case, PD-1 could potentially become a therapeutic target for the treatment of ILC2-mediated allergic asthma in the future. 47 CHAPTER 6 FUTURE DIRECTIONS I strongly believe that our study has provided novel data that furthers the research of PD-1 in the context of innate immunity and ILC2-mediated allergic asthma. Nonetheless, the findings are proof-of-concept and more work must be done before we can begin to consider targeting PD- 1 as a clinical therapy to alleviate allergic asthma. First, we would like to investigate the molecular mechanism of PD-1 signaling in murine ILC2s. PD-1 inhibits T cell effector homeostasis and function by suppressing the signaling of the T cell receptor (TCR) and the CD28 costimulatory molecule 26,30 . As a result, pathways such as the Ras-MEK-ERK and PI3K-Akt-mTOR pathways are inhibited. Although our results indicate that PD-1 also has a negative regulatory role on ILC2s, the signaling mechanism has not been elucidated yet. ILC2s do not express antigen-specific receptors, such as TCRs, nor do they express CD28; therefore, the inhibitory signaling of PD-1 is likely mediated by different pathways than that observed in T cells. Next, we would like to utilize a PD-1 agonist antibody in our murine asthma model. Since our results suggest that the absence of PD-1 signaling exacerbates ILC2-mediated allergic asthma, we intend to assess whether a PD-1 agonist could reverse the effects we had observed among the mice that had PD-1 blocked or genetically deleted. Furthermore, we would like to determine if an agonist could also attenuate the IL-33-induced AHR and lung inflammation—by stimulating the PD-1 pathway—in PD-1-expressing WT mice. Since this project has been focused on PD-1 in the context of murine models, we hope to move onto human models in the future. Murine ILC2s express PD-1, but we do not know if PD-1 is expressed on human ILC2s and if the kinetics of PD-1 expression on human ILC2s mirror that 48 of murine ILC2s. If PD-1 is expressed on human ILC2s, we will examine if PD-1 has a similar role in the suppression of ILC2-mediated allergic asthma in humans. Although humans are far more complex than our murine asthma models, it is our hope that our findings in this study will provide the groundwork for a better understanding of this immunological disease. This may pave the way towards the development of a novel immunotherapy, such as an agonist antibody, that specifically targets PD-1 on ILC2s to suppress ILC2-mediated allergic asthma (Figure 18). ILC2 PD-1 PD-1 Agonist ILC2 Production of Type 2 Cytokines Airway Hyperresponsiveness & Lung Inflammation X X X Figure 18: PD-1 Agonists as a Potential Therapeutic for ILC2-mediated Allergic Asthma Although more research is necessary, PD-1 agonists may serve as a novel potential therapeutic to treat patients with ILC2-mediated allergic asthma. Strengthening the PD-1 pathway signaling could suppress type 2 cytokine secretion, thus preventing the exacerbation of AHR and eosinophilic airway inflammation. 49 REFERENCES 1. Expert Panel Report 3 (EPR-3): Guidelines for the Diagnosis and Management of Asthma; Summary Report 2007. Journal of Allergy and Clinical Immunology 120, S94-S138 (2007). 2. 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Abstract (if available)

Abstract Asthma is a chronic inflammatory disease of the lung airways that is characterized by airway inflammation, airway hyperresponsiveness (AHR), and reversible airflow obstruction. Type 2 innate lymphoid cells (ILC2s) are potent producers of type 2 cytokines that drive the pathogenicity of asthma upon exposure to inhaled allergens. Here, we show that murine ILC2s express the programmed cell death 1 (PD-1) receptor at steady state and when activated. We reveal that the lack of PD-1 significantly increases pulmonary ILC2 counts and the production of type 2 cytokines by ILC2s. Furthermore, the lack of or blocking of PD-1 on ILC2s exacerbates interleukin (IL)-33-induced AHR and airway inflammation. Collectively, we suggest that PD-1 inhibits ILC2 function and homeostasis

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Asset Metadata

Creator Lo, Richard (author)

Core Title PD-1 is a regulator of ILC2-mediated airway hyperresponsiveness and inflammation

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 03/30/2020

Defense Date 03/12/2018

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

Tag airway hyperresponsiveness,asthma,OAI-PMH Harvest,programmed cell death 1,type 2 innate lymphoid cells

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Akbari, Omid (committee chair), Epstein, Alan (committee member), Yuan, Weiming (committee member)

Creator Email lorichar@usc.edu,rich.lo.1993@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-489688

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