UNIVERSITY OF CALIFORNIA, IRVINE

Characterization of -40, a Novel -Associated

DISSERTATION

submitted in partial satisfaction of the requirements for the degree of

DOCTOR OF PHILOSOPHY

in Biomedical Sciences

by

Jovani Catalán-Dibene

Dissertation Committee: Professor Albert Zlotnik, Chair Professor Francesco Tombola Professor Craig M. Walsh

2018

Parts of Chapter 1 © 2018 Journal of Cytokine and Research, Mary Ann Liebert, Inc., New Rochelle, NY. Figure 1.2 by Kurt Buchmann, licensed under Creative Commons Attribution Parts of Chapter 2 © 2017 Journal of Immunology, http://www.jimmunol.org/content/early/2017/10/04/jimmunol.1700534 All other materials © 2018 Jovani Catalán-Dibene DEDICATION A

Mi Padre, César Catalán Martínez.

Por inculcarme el amor a la Biología y por demostrarme qué es el amor incondicional

“Quien no conoce nada, no ama nada. Quien no puede hacer nada, no comprende nada. Quien nada comprende, nada vale. Pero quien comprende también ama, observa, ve... Cuanto mayor es el conocimiento inherente a una cosa, más grande es el amor... Quien cree que todas las frutas maduran al mismo tiempo que las fresas nada sabe acerca de las uvas.” — Paracelso

To My Father, Cesar Catalan Martinez.

For instilling in me the love for Biology and for showing me what unconditional love is

“He who knows nothing, loves nothing. He who can do nothing understands nothing. He who understands nothing is worthless. But he who understands also loves, notices, sees... The more knowledge is inherent in a thing, the greater the love... Anyone who imagines that all fruits ripen at the same time as the strawberries knows nothing about grapes.” — Paracelsus

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TABLE OF CONTENTS Page

LIST OF FIGURES vi

LIST OF TABLES viii

ABREVIATIONS ix

ACKNOWLEDGEMENTS xii

CURRICULUM VITAE xiv

ABSTRACT OF THE DISSERTATION xvi

CHAPTER 1: Introduction to Cytokine and B Cell Biology 1

1.1 , THE MESSENGERS OF THE IMMUNE SYSTEM 2

1.2 CYTOKINES AND B CELL BIOLOGY 6

B cells as cytokine producers 8

1.3 CYTOKINES IN DISEASE AND THERAPY 13

Interleukin 1 13

Tumor necrosis factor α 14

Interleukin 17A 16

Interleukin 6 17

Colony stimulation factors 18

Future directions in cytokine therapy 19

1.4 -40 21

The search for new cytokines 21

Interleukin 30 23

Interleukin 31 25 26

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Interleukin 32

Interleukin 33 27

Interleukin 34 30

Interleukin 35 32

Interleukin 36 35

Interleukin 37 37

Interleukin 38 39

Interleukin 39 40

Future perspectives on cytokine discovery 41

1.5 REFERENCES 44

CHAPTER 2: Identification of IL-40, a Novel B Cell–Associated Cytokine 62

2.1 INTRODUCTION 63

2.2 MATERIALS AND METHODS 65

2.3 RESULTS 71 C17orf99 encodes a novel cytokine 71

C17orf99 encodes a secreted produced by activated B cells 74

IL-40 expression is induced upon the onset of lactation in the mammary gland. 78

An Il40-/- mouse exhibits B cell abnormalities 79

B cell populations are altered in the BM of Il40-/- mice 82

Il40-/- mice exhibit reduced levels of IgA in mucosal and exhibit

GALT abnormalities. 85

An Il40-/- mouse exhibits microbiome abnormalities 89 Human IL-40 is expressed by activated human B cells and human B cell lymphomas. 90

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2.3 DISCUSSION AND CONCLUSIONS 92

2.4 REFERENCES 97

CHAPTER 3: IL-40 and Its Potential Role in Disease 101

3.1 INTRODUCTION 102

3.2 MATERIALS AND METHODS 107

3.3 RESULTS 109

IL-40 plays a role in the modulation of commensal induced IgA in the gut 109

IL-40 in EAE 112

IL-40 role in SLE-like disease 115

3.3 DISCUSSION AND CONCLUSIONS 116

3.4 REFERENCES 121

CHAPTER 4: Summary and General Conclusions 126

4.1 SUMMARY AND FUTURE DIRECTIONS 127

Discovery, mammalian functions, IgA and GALT homeostasis 127

Salmonella infection 128

B cell development 130

B40 cells 131

Clinical implications of IL-40 133

4.2 CONCLUSIONS 135

4.3 REFERENCES 136

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LIST OF FIGURES

Page Figure 1.1 Role of cytokines in the regulation of the immune 4 system.

Figure 1.2 Evolution of the immune system 6

Figure 1.3 B effector cell polarization 12

Figure 2.1 C17orf99 encodes a novel cytokine (interleukin 40) 72

Figure 2.2 IL-40 expression in mouse and human 73

Figure 2.3 IL-40 is expressed by Lin-CD45- in the BM. 75

Figure 2.4 IL-40 production is induced upon B cell activation 77

Figure 2.5 IL-40 expression is induced in the mammary gland 79 upon the onset of lactation and the Il40-/- mouse exhibits decreased IgA levels in milk

Figure 2.6 B cell populations are altered in the Il40-/- mouse. 81

Figure 2.7 IL-40 is highly expressed in BM stroma and is 83 required for normal B cell homeostasis

Figure 2.8 Total cells and B cells are reduced in the BM of 84 Il40-/- mouse

Figure 2.9 Il40-/- mice exhibit defects in GALT B cell 87 populations

Figure 2.10 B cells from IL-40 wt and Il40-/- mice were measured 88 by in vitro stimulation and flow cytometry assays for their relative ability to undergo CSR to the indicated isotypes and plasmablast/plasma cell differentiation.

Figure 2.11 The microbiome of Il40-/- mouse is altered. 90

Figure 2.12 Human B cells produce IL-40. 91

Figure 3.1 Bacterial burden of Salmonella after challenge 111

Figure 3.2 IgA normalizes after Salmonella challenge 112

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Figure 3.3 IL-40 ablation may not play a role during EAE 114 induced by MOG35-55 immunization

Figure 3.4 Dihybrid crossbreeding of Il40-/+Faslpr/wt mice does 116 not produce a double mutant

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LIST OF TABLES

Page Table I Influence of cytokines in class switch recombination 8

Table II Cytokine related drugs developed and approved for 20 clinical use

Table III Summarized characteristics of new cytokines 42

Table IV qRT-PCR oligonucleotides used in this study 66

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ABBREVIATIONS

Th - T helper cells IL – CSF – Colony stimulation factors IFN – TGF – Tumor TNF – Ig – Immunoglobulin BM – HSCS – Hematopoietic stem cells IL-7R – receptor Pro-B cell – Progenitor B cell Pre-B cell – Precursor B cell BCR – B cell receptor CSR – Class switch recombination mAB – RA – Rheumatoid arthritis SLE – Systemic lupus erythematosus MS – Multiple sclerosis Bregs – B regulatory cells EAE – Experimental autoimmune encephalomyelitis Be cells – B effector cells Th1 – Type 1 helper Th2 – Type 2 helper T cell sIL-6R – Soluble IL-6 receptor TD1 – Type 1 diabetes TD2 – Type 2 diabetes IL-1RA – IL-1 receptor antagonist TRAPS – Tumor necrosis factor receptor associated periodic syndrome

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HIDS – Hyperimmunoglobulin D syndrome FMF – Familial Mediterranean Fever TACE – TNFα converting enzyme CD – Crohn’s disease UC – Ulcerative colitis ANA – Antinuclear antibodies dsDNA – Double stranded DNA ACL – Anticardiolipin antibodies AS – Ankylosing spondylitis CNS – Central nervous system PA – Psoriatic arthritis HS – Hidradenitis suppurativa JIA – Juvenile idiopathic arthritis IL-17R – receptor IBD – Inflammatory bowel disease FDA – US Food and Drug Administration PASI – Psoriasis area severity index CRS – Cytokine release syndrome CAR-T cell – Chimeric antigen receptor T cell TCR – T cell receptor G-CSF – Granulocyte colony stimulation factor PHA – Phytohemagglutinin Con A – Concanavalin A LPS – Lipopolysaccharide LAF – Lymphocyte activating factor EBV – Epstein-Barr Virus Ebi3 – EBV induced 3 IL-27p28 – IL-27 p28 subunit TLR – Toll-like receptor STAT1 – Signal transducer and activator of 1

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CLF-1 – Cytokine-like factor 1 PCSLC – stem-like cells OSMRbeta – receptor beta SEB – Staphylococcal enterotoxin B PBMCs – peripheral mononuclear cells NF-HEV – Nuclear factor in high endothelial venules HEVs – High endothelial venules PAMPs – Pathogen-associated molecular patterns HMVEC-LBI – Human microvascular endothelial cells from blood vessels ILC2s – Group 2 innate lymphoid cells CSF-1 – Colony stimulating factor 1 receptor nTregs – Natural regulatory T cells iTregs – Induced regulatory T cells EAU – Experimental autoimmune uveitis DITRA – Deficiency of receptor antagonist GPP – Generalized pustular psoriasis DSS – Dextran sulphate IL-37tg – IL-37 transgenic mouse GWAS – -wide association studies BIGE – Body Index of C17orf99 – 17 open reading frame 99 PPs – Peyer’s patches FAS – First apoptotic signal receptor Lpr – Lymphoproliferation

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ACKNOWLEDGMENTS

I want to acknowledge my advisor and mentor Dr. Albert Zlotnik for his guidance and effort put into turning me in to an immunologist. It is an honor to be called one of your pupils (now I have immunologist “pedigree”) and I really hope to make you proud with my future scientific adventures. I will remember your science and life teachings forever ¡Gracias Doc! I would like to express my gratitude to my committee members Dr. Francesco Tombola and Dr. Craig Walsh. Their advice and comments to make my work better are greatly appreciated. I would also like to thank Dr. Eric Pearlman and Dr. Manuella Raffatellu for their support and advice during my advancement. I want to thank my laboratory mates José Luis, Amanda, Monica, Irina, Marce and Gerardo for making the time spent in the lab more fun and for all your ideas, protocols, lunches, stories, support, laughs, etcetera, etcetera. Thank you! To the friends I made at UCI, Alborz, Christian Jenna, Marti, Vlad, Sarah, Honyin, Laura, Laura, Ben and Chris, for technical support, for helping me in the lab, for being part of my research, for the tacos and the moral support. Thank you! Thanks to the administrative staff of the Department of Physiology and Biophysics, Duke, Janita and Anthony for helping us to focus on science and not bureaucracy. I want to also acknowledge Dr. Armando Villalta, for being there for me as a friend and as a scientist. I hope to still get your advice when I am no longer here. Gracias doctor. I am especially thankful to Dr. Raffatellu, for giving the opportunity to work with her lab and for all the encouragement and scientific advice; as well as members of the Raffatellu Lab for being my second home lab. Thanks to my friend Dr. Araceli Perez-Lopez who should be part of my committee. Thanks for all your help and support in the lab, for dropping your research to help me (and saying its nothing and work late because you helped me), for all the lunches and long talks about my project, for teaching me B cell biology and a little bit of host-pathogen interactions. I could have not finished this work without you. Gracias por ser mi amiga Chely. Thanks to my family. To my father for being my hero, the person I aspire to be, I hope to find a way to inspire the love for knowledge, like you do to me and my brothers. To my mom for teaching me about love and empathy, about life and strength, about what it feels like to have a wonderful mother. To my brothers for sharing your lives with me to make want to make you proud, for being there with me and for me. To my uncle Pachy and aunt Rosy who I am lucky to have as second parents; thanks for being there for me, for treating me as your son and giving me my little sisters. Los amo a todos.

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I also want to thank my friends from Mexico German, Valentin, Luis y Diego for not forgetting about me and for supporting me during my stay here. I would also like to thank my country Mexico and the US that funded my education through CONACYT/ UC/MEXUS. Especial thanks to Veronica and Jasmin for being helpful and always there to support us, becarios. I also thank my wonderful in laws and brothers in law. They have been a source of support since the beginning of my career, specially my mother in law who is always there for us and the first to offer help and encouraging words. Finally, Adriana thanks for being a role model, the scientist I look up the most, the love of my life, thanks for being everything to me. Thanks for loving me and for putting up with my stubborn ways with love. For caring for me and for helping realize when and where I need to evolve, I owe you much more than this work. I hope I keep earning your love and respect forever. Te amo.

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CURRICULUM VITAE

Education PhD program: (2013-2018) University of California, Irvine. Department of Physiology and Biophysics, School of Medicine. • PhD project: “Identification of interleukin 40, a novel B cell cytokine”, advisor: Dr. Albert Zlotnik.

Master’s degree: (2009-2012) Graduate program in Molecular Ecology and Biotechnology, School of Marine Sciences, UABC. • Thesis: (2012) “Characterization of the rodent population at Valle de las Palmas as carriers for Coccidioides immitis”. Center for Scientific Research and Higher Education of Ensenada (CICESE), Department of Microbiology, advisor: Dr. Meritxell Riquelme Perez. • Research residency: (2011), Department of Medical Microbiology and Immunology, University of California Davis, CA, USA, advisors: Dr. Suzanne M. Johnson and Dr. Demosthenes Pappagianis.

Bachelor’s degree: 2003-2008. Bachelor of Science, Biology, Autonomous University of Baja California (UABC), School of Sciences, Ensenada, Baja California, Mexico. • Thesis (2008): “Localization of the general permease in Neurospora crassa” • Social Service: (2003-2004) managed the herpetarium of the School of sciences bioterium. • Internship: (2007) “Characterization of the human growth hormone” advisor: Dr. Hugo Barrera Saldaña, School of Medicine of the Autonomous University of Nuevo Leon. XVII Summer of scientific research - Mexican Academy of Sciences. • Professional internship: (2007-2009) advisor: Dr. Meritxell Riquelme Perez.

Publications Catalán-Dibene J, McIntyre LM and Zlornik A, 2018. Interleukin 30 to 40. Journal of Cytokine and Interferon Research. In press. Catalán-Dibene J, Vazquez M, Van L, Nuccio SP, Karimzadeh A, Kastenshmidt JM, Villalta SA, Ushach I, White CA, Pone E, Burkhardt A, Casali P, Raffatellu M, Hernandez M, Hevezi PA and Zlotnik A. 2017. Identification of interleukin 40, a novel B cell-associated cytokine. Journal of Immunology 199: 3326-3335. Sun S, Payer B, Namekawa S, An JY, Press W, Catalán-Dibene J, Sunwoo, and Lee JT. 2015. Xist imprinting is promoted by the hemizygous (unpaired) state in the male germ line. Proceedings of the Natural Academy of Sciences. 112: 14415-14422.

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Vazquez M, Catalán-Dibene J and Zlotnik A. 2015. B cells responses and cytokine production are regulated by their immune microenvironment. Cytokine. 74:318-26. Catalán-Dibene J, Johnson SM, Eaton R, Romero-Olivares AL, Baptista-Rosas RC, Pappagianis D and Riquelme M. 2014. Detection of coccidioidal antibodies in serum of a small rodent community in Baja California, Mexico. Fungal Biology, 118:330-9. Baptista-Rosas RC, Riquelme M, Muñiz-Salazar R, Catalán-Dibene J, Zimbrón- Hernández MA, Meza A, Cardenas-Bautista D, Arreola-Cruz AA, Radilla-Chavez P, Salas-Vargas SD, 2012. Seroprevalence of antibodies against Coccidioides spp. In Tuberculosis patients in Ensenada, Baja California. (Spanish). Enfermedades Infecciosas y Microbiología, 34: 69-73. Baptista-Rosas RC, Catalán-Dibene J, Romero-Olivares AL, Hinojosa A and Riquelme M, 2012. Molecular detection of Coccidioides spp. from environmental samples in Baja California: linking Valley Fever to soil and climate conditions. Fungal Ecology. 5: 177-190.

Honors and Awards • UCMEXUS-Conacyt Graduate Student Scholarship. PhD Studies in a foreign country. (2013-2018) • Grant. Complement Scholarship for Mexicans studying in foreign countries. Secretaría de Educación Publica (2013) • Travel Grant American Association of Immunology for the IMMUNOLOGY 2017 conference in Washington, D.C., USA (May 12th to 16th).

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ABSTRACT OF THE DISSERTATION

Identification of IL-40, a Novel B Cell–Associated Cytokine By

Jovani Catalán-Dibene

Doctor of Philosophy in Biomedical Sciences

University of California, Irvine, 2018

Professor Albert Zlotnik, Chair

We describe a novel B cell-associated cytokine, encoded by an uncharacterized gene

(C17orf99), whose expression is induced in B cells upon activation. C17orf99 is only present in mammalian , and it encodes a small (~27kDa) secreted protein unrelated to other cytokine families, suggesting a function in mammalian immune responses. Accordingly, C17orf99 expression is induced in the mammary gland upon the onset of lactation, and a C17orf99-/- mouse exhibits reduced levels of IgA in the serum, gut, feces, and lactating mammary gland. C17orf99-/- mice have smaller and fewer

Peyer’s patches and lower numbers of IgA secreting cells. The microbiome of C17orf99-

/- mice exhibits altered composition, likely a consequence of the reduced levels of IgA in the gut. While naïve B cells can express C17orf99 upon activation, their production increases following culture with various cytokines, including IL4 and TGF-β, suggesting that differentiation can result in the expansion of C17orf99-producing B cells during some immune responses. Taken together, these observations indicate that C17orf99 encodes

xvi a novel B cell-associated cytokine, which we have called Interleukin-40, that plays an important role in the development of humoral immune responses. Importantly, IL-40 is also expressed by human activated B cells and by several human B cell lymphomas. The latter observation suggests that it may play a role in the pathogenesis of certain human diseases.

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CHAPTER 1 INTRODUCTION TO CYTOKINE AND B CELL BIOLOGY

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1.1 CYTOKINES, THE MESSENGERS OF THE IMMUNE RESPONSE

The main function of the immune system is to protect the body from foreign (infection, toxins, etc.) and domestic (autoimmune diseases, sterile , cancer, etc.) insults. The immune system carries its functions through highly specialized cells divided in two effector systems, the innate immune system and the adaptive system. The innate immune system is the first line of defense and can carry out its effector function without antigen presentation. It is composed of complement , barrier tissues () and hematopoietic cells (dendritic cells, , , neutrophils, basophils, eosinophils, natural killer cells, natural killer T cells and mast cells). The epithelium produces a wide variety of defenses and also represent a physical barrier to the outside world (1); while hematopoietic cells such as macrophages and neutrophils are regularly the first cells to respond to an invading pathogen and thus they are initiators of the second line of defense of the adaptive immune system that initiate the recruitment and activation of cells of the adaptive immune system, that get activated through antigen processing and presentation (2-4). The adaptive immune system, in contrast, needs antigen presentation and “training” to develop a highly specific and potent immune response (3) and it is comprised of T and B lymphocytes. T cells are responsible for cytotoxic activity specialized in eliminating intracellular infections, as well as directing the immune response as T helper (Th) cells (5, 6); B cells (which can develop into plasma cells) are responsible for the production of antibodies and are paramount for the development of long lasting immunity (7). Together, they confer immune memory in response to specific antigens, a hallmark of adaptive immunity (8). The development and

2 control of all the phases necessary for mounting innate and adaptive immune responses include cells of each of these systems but they are all regulated by cytokines (9-11).

Cytokines are small secreted proteins produced by cells of the immune system, usually upon cell activation. Cytokines are orchestrators of immune responses, since they coordinate the inflammatory response by inducing maturation, polarization, proliferation, survival and trafficking of immune cells (Figure 1) (12). They accomplish these tasks by activating specific gene expression pathways in responding cells (13). The regulation of gene expression by cytokines is complex and often involves checkpoints and multiple steps to ensure that the right immune response is elicited (14). From the evolutionary perspective, cytokines evolved from superfamilies that include interleukins (IL), colony- stimulating factors (CSF), interferons (IFN), transforming growth factors (TGF), Tumor

Necrosis Factors (TNF) and ; many of these exhibit overlapping, synergic or antagonistic functions.

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Figure 1.1. Role of cytokines in the regulation of the immune system. Cytokines can induce immune cells to polarize into effector cells that defend the body against pathogens (Inflammation) or to suppress inflammation and/or induce tolerance (Immune regulation). They also play an important role in tissue homeostasis by controlling trafficking of immune and somatic cells to other tissues and the maintenance of hematopoiesis (Tissue homeostasis).

It is often overlooked that “simple” lifeforms such as flies (class Insecta) and early vertebrates such as jawless fish (class Agnata) are already capable of mounting effective immune responses (15-17). Pluricellular Protista and Invertebrates were the first organisms capable of mounting innate immune responses (around 542 million years ago), while the earliest adaptive immune responses first appeared in jawless fish (around 500 million year ago) and has continued its evolution to date (Figure 2) (17). Cytokines were needed to manage these newly acquired defensive abilities, so as the defensive capabilities of organisms grew, the diversity of cytokines also grew. The evolutionary conservation of these cytokines is such that several mammalian cytokines are capable to elicit immune responses in worms, mollusk and insects. While Fish express ortholog cytokines of IL-1, IL-6, IL-17A, IL-10, IL-21 and TGF-β (17, 18). There is evidence that

4 cytokines evolved from intracellular proteins in invertebrates, which can only mount innate immune responses (10). Early cytokines acted mainly as transcription factors, an example of this is IL-33 (19). The diversity and functions of cytokines evolved as evolutionary pressure on pluricellular organisms by microorganisms, parasites and viruses grew.

Additionally, the evolving organisms needed to control its own newly developed weapons turning against itself, further promoting diversification of immune components. In general, most gene families arose through evolution from an original ancestral gene through gene duplication and the resulting offspring then specialized, giving rise to a family of genes that encode structurally related proteins that developed specialized functions (20).

The chemokines are an excellent example of this process, but it also applies to other cytokine families. Among the interleukins, IL-2 is evolutionarily related to IL-15 and to IL-

21, while IL-4 and IL-13 are the result of a gene duplication (indeed, their genes are still located adjacent to each other in the ) (16). There are several genes of the IL-10 family, but some of the largest interleukin superfamilies include IL-1 and IL-12, with several members each (16). In fact, among the latest discovered interleukins (IL-30 to IL-40) there are several IL-1 superfamily members (IL-33, IL-36, IL-37, and IL-38) as well as several new members of the IL-12 family (IL-30, IL-35, and IL-39).

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Figure 1.2. Evolution of the immune system. The inner painting shows representative animals of different organisms of the animal kingdom (Animalia). The rounded square shows the immune mechanism and the brackets indicate the animal groups where a specific immune mechanism evolved. Years are presented in million years (mya). The outer squares show the approximate geological period where the immune mechanisms appeared. First published in (17).

1.2 CYTOKINES AND B CELL BIOLOGY

B cells are the effector cells responsible for immunoglobulin (Ig) secretion in the organism, and thus the main drivers of the humoral immunity, a function of the adaptive immune response (21). They develop and mature in the bone marrow (BM) and continue their development and polarization in the periphery. These processes are tightly regulated by

6 cytokines (22). B cells develop from hematopoietic stem cells (HSCs) present in the bone marrow (23) that become committed to the B cell lineage. In the case of B cells, IL-7 drives HSC differentiation to the B cell lineage and is produced by specialized stromal cells in the BM (24, 25). In fact, IL-7 receptor (IL-7R) is specifically expressed by early stages of B cell precursors in the bone marrow (26). TGF-β also plays a role in early B cell development by inhibiting the expression of IL-7 by stromal cells and by directly inhibiting the proliferation of Progenitor and Precursor-B cells (Pro- and Pre-B cell respectively) (27, 28). The maturation stages of B cells in the BM are marked by the rearrangement of the V, D and J immunoglobulin segments of the heavy chain and the V and J segments of the chain of the B cell receptor (BCR) culminating in a fully functional IgM BCR (22). Once the BCR is formed the Immature B cell exits the BM and continues maturation in the germinal centers of the or lymph nodes. Immature B cells are able to switch Ig class depending on the cytokines present in the microenvironment and help from CD4+ T helper cells. (29, 30). An immature B cell is capable of class switching to any of the known antibody classes: IgM, IgG1, IgG2a, IgG2b,

IgG3 IgA and IgE. Each of these immunoglobulin classes has different functions (31).

Outside of the BM, cytokines play a role in B cell activation, survival and antibody class switch recombination (CSR). IL-4 is one of the most important cytokines. IL-4 was first described as B cell growth factor and plays a role in the secretion of IgG1 and IgE (32,

33) . IL-4 is produced by Th2 cells and it is necessary for effective B cell proliferation and survival (34). IL-6 is another important pro-. It was originally named

B cell stimulation factor 2, because it is capable of increasing the secretion of IgM and

IgG (35). IFNγ, another cytokine found in the periphery, was originally described as an

7 antiviral cytokine, but it has a variety of other functions including the ability to regulate antibody production of immunoglobulins by B cells, inhibiting the production IgM, IgG1 and IgE but inducing the secretion of IgG2a and IgG3 (which can neutralize viruses) (31,

36). Finally, TGF-β induces CSR to IgA while inhibiting the induction of IgM, IgG2a, IgG3

(37). Table I summarizes the effect of several cytokines in CSR and their ability to promote production of the different antibody classes.

After B cells encounter the required signals to become a fully mature B cell it has three possible fates: 1) release antibodies and cytokines and die after losing the ability to respond to survival signals and cytokines; 2) following successful clonal selection

(positive selection), B cells producing high affinity antibodies are selected to keep dividing and become long lived memory B cells; 3) B cells with the highest affinity for antigens will develop into long lived plasma cells (LPCs). These processes occur in the germinal centers of the spleen and lymph nodes (38-40). All these cells can produce cytokines to further improve and regulate immune responses (41, 42).

Table I. Influence of cytokines in class switch recombination

Inmunoglobulin class Cytokine IgM IgG3 IgG1 IgG2b IgG2a IgA IgE Il-4 Inhibits Inhibits Induces Inhibits Induces IL-5 Enhances IL-6 Induces IFNγ Inhibits Induces Inhibits Induces Inhibits TGF-β Inhibits Inhibits Induces Induces

B cells as cytokine producers

As discussed above, B cells are mostly thought of as antibody secreting cells and for many years B cell research was shaped by this notion. However, since the late 2000s

8 thanks to the disseminated use of Rituximab, an anti-human CD20 monoclonal antibody

(mAB) developed for the treatment of non-Hodgkin lymphoma, there is a growing body of evidence that points to a role of B cells in autoimmunity that is independent of their ability to produce antibodies (43, 44). Rituximab is a chimeric antibody that targets CD20 expressing cells. In the B cell lineage, CD20 is expressed as early as the late Pro-B cell stages up until the memory B cell stage (45). Administration of Rituximab in vivo therefore leads to severe depletion of most CD-20-expressing B cells. In autoimmune disease,

Rituximab was first tested in rheumatoid arthritis (RA) patients because RA pathology is characterized by the presence of activated B cells in the inflamed synovium as well as circulating autoantibodies that target multiple self-antigens (46). Rituximab was shown to have a dramatic beneficial effect in the resolution of RA, especially in combination with methotrexate (47, 48). Surprisingly, elimination of B cells does not cause autoantibody secretion to disappear (because LPCs do not express CD20). This was also true for systemic lupus erythematosus (SLE) patients treated with Rituximab (49, 50). These observations lead to the conclusion that the therapeutic effect of B cell depletion observed in RA patients must be mediated by antibody independent effector functions carried by B cells.

In parallel to these observations, in recent years it has become clear that B cells are capable of secreting significant quantities of some cytokines (51). -α (LTα) and TNF-α produced by B cells play an important role in the organization of the germinal center in lymph nodes and the spleen (52, 53). In RA, activated B cells have been found to form active lymphoid ectopic structures in non-lymphoid tissues driven by LTα and

TNF-α (54). These ectopic lymphoid structures are capable of presenting autoantigens to

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T cells and induce pathogenic T cell activation (54). Recently a possible mechanism for

Rituximab therapy in multiple sclerosis (MS) has been proposed. Li and collaborators identified a B cell subpopulation in MS patients capable of secreting GM-CSF, which in turn can activate pathogenic macrophages in vitro (55).

The most researched B cell subpopulation with cytokine driven functions are B regulatory cells (Bregs). The role of B cells as immune suppressive cells was discovered by Wolf and collaborators when exploring the role of B cells in experimental autoimmune encephalomyelitis (EAE), a murine model of MS (56). In this study, they found that B cell deficient B10.PL (I-Au) mice have greater variation in disease onset, disease severity and are unable to recover from EAE pathology (56). Later the expression and secretion of IL-

10 by B cells was reported in colitis, EAE and RA, were they play a central role (57-59).

Bregs are capable of secreting TGF-β and IL-10 when their BCR engages autoantigens

(58). Interestingly plasma cells are also capable of producing IL-10 and another newly discovered anti-inflammatory cytokine, IL-35 (60). IL-10 and TGF-β work by downregulating the expression of pro-inflammatory cytokines which in turn is mediated by inhibition of the pro-inflammatory transcription nuclear factor κB (NF-κB) or antagonizing it in the case of TGF-β (61, 62).

In fact, B cells (like T cells) require polarization with activating agents and cytokines in order to become active cytokine producers. Given the characteristics of activated B cells,

Lund and collaborators coined the term effector B cell (Be cells) (51). B cells can also be primed for Type 1 (pro-inflammatory) and Type 2 (humoral) responses and called them

Be1 and Be2 cells respectively to mirror Type 1 (Th1) and Type 2 (Th2) helper CD4+ T cell subsets (42). Not surprisingly, Be1 and Be2 can be found under the same

10 inflammatory environments that their T cell equivalents, Be1/Th1 in infections that induce

Type 1 responses and Be2/Th2 in infections that induce Type 2 responses (Figure 3)

(63).

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Figure 1.3. B effector cell polarization. B cells can secrete cytokines in response to T cell help and cytokine stimulation. B cells can be polarized into B effector 1 cells (Be1) which mirror Type 1 T-helper cells, B effector cells 2 (Be2) which mirror Type 2 T-helper cells 2 and B regulatory cells (Breg) which mirror T regulatory cells.

1.3 CYTOKINES IN DISEASE AND THERAPY

Due to the pivotal role that cytokines play during inflammation it was evident that they are perfect targets for developing blocking or administration therapies. Surprisingly, there are not many treatments available involving cytokines. This could be due to the complexity of cytokine regulation. Many cytokines are pleiotropic, meaning that they can elicit different responses in different cells, sometimes even antagonistic. A good example of this is IL-

6, which can elicit pro-inflammatory and anti-inflammatory responses depending on the signaling mechanism used, membrane bound IL-6 receptor or soluble IL-6R (sIL-6R).

Only few cells express the membrane bound receptor which triggers anti-inflammatory responses. Conversely, all cells express gp130 a membrane protein that interacts with sIL-6R to initiate a pro-inflammatory response (64). This complexity represents a challenge in developing effective cytokine-centered therapies. With the discovery of IFNs and their effects in vitro on viral infected cells and immune cell stimulation it was believed that they would be the perfect antiviral drugs (65), nevertheless they never proved effective for those purposes in humans (66). Given the processes that cytokines can regulate, uncontrolled expression of certain cytokines can lead to autoimmune disease and cancer (67). It is in these pathologies that cytokine-based therapies have proven most effective (67, 68).

Interleukin 1

In autoimmune diseases, pro-inflammatory, anti-inflammatory cytokines and chemokines play important roles. IL-1 family members represent mostly pro-inflammatory cytokines; in combination with other inflammatory cytokines, such as TNF-α and IL-6, induce cartilage destruction in RA (67, 69). IL-1β is upregulated in patients with RA compared

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with patients that are on remission. It is also known to promote the activity of proteolytic enzymes that drive the destruction of cartilage in the joints (70, 71). Furthermore, IL-1 also induces autoimmune attacks on β cells by T cells in type 1 diabetes (T1D) and it has also been shown that the NLRP3 inflammasome (which activates inactive intracellular IL-

1β) is upregulated in type 2 diabetes (T2D), contributing to β cell death and scaling inflammation in the pancreas (72, 73). Furthermore, IL-1β has been implicated in uveitis, pericarditis, gout, Sjögren’s syndrome, and interstitial lung disease, among others (70,

74-77). In some of these cases IL-1β blocking therapies have proven successful (70).

The most common treatment to date is the use of (brand name Kineret) a recombinant human IL-1 receptor antagonist (IL-1RA), marketed by Swedish Orphan

Biovitrium AB pharmaceutical. Anakinra has proven moderately effective in the treatment of RA as a second line of treatment or in combination with other drugs such as methotrexate (78-80). (brand name Ilaris) developed by Novartis is an anti- human IL-1β mAB originally intended for the treatment of RA, but its effectiveness has been marginal (81, 82). Recently it has been approved for three rare autoimmune diseases Tumor Necrosis Factor Receptor Associated Periodic Syndrome (TRAPS),

Hyperimmunoglobulin D syndrome (HIDS) and Familial Mediterranean Fever (FMF); IL-

1β (83-85).

Tumor necrosis factor α

TNF-α, a dual pro-inflammatory/anti-inflammatory cytokine, is one of the most prominent cytokines in autoinflammatory diseases (86). TNF-α is produced first as membrane bound cytokine and is released after cleavage by a TNF-α converting enzyme (TACE). Both forms signal through two receptors with different affinities and effects (87). Due to TNF-

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α’s effect on mucosal epithelia permeability, it has a prominent role in the pathogenesis of Crohn’s disease (CD) and ulcerative colitis (UC) (88, 89). It has also been found to participate in the pathogenesis of SLE, where surprisingly, patients undergoing TNF-α blockade for RA can develop autoimmune pathologies such as the production of antinuclear antibodies (ANAs), anti-double stranded DNA (dsDNA) and anticardiolipin antibodies (ACL) that can lead to SLE (90). In the context of SLE, TNF-α decreases the autoantigen presentation derived of apoptosis by acting directly on B cells as an inhibitor

(activated B cells are also capable of TNF-α production); while at the same time inducing tissue destruction via apoptosis and immune cell activation (91-93). This is echoed in

T1D, where TNF-α has a dual role, preventing the induction of autoantibodies that attack

β cells and promoting inflammation at the same time (94, 95). Furthermore, some patients undergoing TNF blockade treatment for autoimmune diseases such as ankylosing spondylitis (AS), RA, psoriasis and CD and UC can show signs of demyelination in the central nervous system (CNS) a hallmark of MS (96, 97). In fact, the use of TNF-α blockade in MS proved to be counterproductive in humans by exacerbating the inflammation in the CNS (98).

Adalimumab (trade name Humira) and Infliximab (trade name Remicade), developed and marketed by AbbVie Inc. and Johnson & Johnson respectively, are the most commonly used anti-TNF-α mAB in the world (99) designed to block TNFa effects in vivo. In fact,

Adalimumab was the number one of the top selling drugs in the world until 2016 (when its US patent expired) with Infliximab in 5th position (100). Both mABs are prescribed for

RA, psoriathic arthritis (PA), AS, CD, UC, psoriasis, hidradenitis suppurativa (HS) and juvenile idiopathic arthritis (JIA) alone or in combination with drugs such as methotrexate,

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in the case of RA it doubles the patient response rate of methotrexate (101). These mAbs therefore represent some of the most successful drugs for treatment of autoimmune diseases and underscore the pivotal role that TNFa plays in these diseases. Ironically, their exact mechanism of action in these diseases is not well understood, even though they are very effective and widely used.

Interleukin 17A

IL-17A is pro-inflammatory cytokine discovered in 1993 by Rouvier and collaborators

(102). IL-17A signals through IL-17 receptor (IL-17R), which is comprised of subunits IL-

17RA, IL-17RB, IL-17RC, IL-17D and IL-17E (103). IL-17 was one of the first cytokines to break the Th1/Th2 paradigm set by Mosmann and Coffman in 1989 (104) and consolidated a new cell linage that produces high quantities of IL-17 but no IFNγ or IL-4

(Th1 and Th2 cytokines resplectively), named Th17 cells (105). IL-17 is important for the defense against extracellular pathogens such as Fungi through the recruitment and activation of neutrophils (82, 106, 107). Also, IL-17A expression synergizes with TNF-α and IL-1 and plays a particularly important role in barrier tissues, many epithelial cell express IL-17R (108, 109). IL-17 stimulation of epithelial cells leads to upregulation of IL-

6, TNF-α, CXCL8 and matrix metalloproteases (108, 110). Upregulation of IL-17A in several autoimmune diseases was quickly reported in disease such as psoriasis, RA, MS

(111) and inflammatory bowel disease (IBD) (112-114). Blocking therapy for IL-17 was quickly developed in the early 2000s. The first anti IL-17A blocking antibodies were developed soon after the upregulation of IL-22 and IL-23 was discovered in several autoimmune diseases which are produced by Th17 cells (115-117). One of the first blocking antibodies developed and published with exceptional results in the treatment of

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RA, psoriasis and uveitis (UV) was AIN457 later branded as (trade name

Cosentyx) by Novartis. Secukinumab is a fully human mAB antibody targeting IL-17A

(118). As of 2018 it has been approved by the US Food and Drug Administration (FDA) for the treatment of plaque psoriasis, PA, and AS (118). Secukinumab is very effective in the treatment of all spectrums of severity in psoriasis, inducing rapid reduction (~4 weeks) of 50-75% of the psoriasis area and severity index (PASI) with some of the patients achieving PASI scores of 90-100%, outperforming anti-IL-23 mAB

(Centocor/Janssen biotech) (118, 119). IL-17A blockade provokes susceptibility to upper respiratory tract infections, nevertheless the adverse effects measured after 3 years of treatment are comparable to alternative treatments but with a significant increase in quality of life (118, 120).

Interleukin 6

IL-6 is a pleiotropic cytokine with pro-inflammatory and anti-inflammatory properties. As mentioned before, IL-6R can be expressed in two forms soluble and membrane bound.

In both cases IL-6 signaling requires gp130 coreceptor to signal, but gp130 is capable of interact with soluble IL-6/IL-6R complex by itself (121). While membrane bound IL-6R is only expressed in hepatocytes, monocytes, macrophages and lymphocytes, the expression of gp130 is expressed in all cells, eliciting different responses depending on the signaling pathway engaged (121). IL-6 is a potent mediator of inflammation and IgG humoral responses (122, 123). Also, IL-6 acts directly on hepatocytes and is an activator of the acute phase response in the and thus is one of the main drivers of cytokine release syndrome (CRS) (124, 125). CRS is the most prominent adverse outcome of the newly developed Chimeric Antigen Receptor T cell (CAR-T cell) therapy for cancer (126,

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127). CAR-T cells are genetically engineered to express specific T cell receptors (TCRs) with high specificity to tumor antigens (128).Tociluzimab (trade name Actemra), a mAB that targets IL-6, is very effective in the treatment of CRS (126, 127, 129, 130). Developed by Hoffman-La Roche/Chugai pharmaceutical company was initially developed for

Castleman’s disease and JIA, both characterized by the upregulation of IL-6 (131).

Although tociluzimab is approved by the FDA for RA, JIA and Castleman’s disease its use has been more effective in the treatment of CRS and has found a niche use in combination with CAR-T cell therapies.

Colony stimulation factors

CSFs are cytokines responsible of hematopoiesis in the bone marrow. Among CSFs and granulocyte colony stimulation factor (G-CSF) are the most used hematopoietic cytokines for the treatment of disease (66). They are particular in their clinical application in that they are among the only cytokines that are administered (via injection) as exogenous recombinant cytokines and are synthetized in vitro (132-134).

Erythropoietin is marketed as Epoietin (trade name Epogen), developed by Amgen and used for the treatment of anemia provoked by chronic renal failure and cancer chemotherapy (132, 135). G-CSF is used to treat febrile neutropenia caused by myelosuppressive chemotherapy that leads to a myriad of complication such as infection susceptibility (136). The most popular recombinant G-CSF is (trade name

Neupogen) also developed by Amgen and it is used to combat neutropenia caused by chemotherapy or induced neutropenia due to organ transplantation (134, 137).

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Future directions in cytokine therapy

The development of successful therapies that target cytokines has increased the interest in the discovery and functional characterization of novel cytokines and their potential role in human disease. In this section we reviewed the most successfully targeted cytokines that have led to new treatments for cytokine-driven pathologies (summarized in Table II).

Given the intricate regulation of cytokines, harnessing their functions could be problematic. Nevertheless, various therapies have proven very successful, especially, in the treatment of autoimmune disease where novel biologics that target several cytokines have been developed. These successes have led to wide interest on the identification of novel cytokine targets. As a result, there are many new cytokines whose biology is a developing story. We will review some of these novel cytokines in this section and we will also predict that there are still some cytokines left to discover, a subject that we will touch on the next section.

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Table II. Cytokine related drugs developed and approved for clinical use.

Drug Trade name Type Target Diseases (FDA approved) Rheumatoid arthritis (RA), Cryopirin-Associated Recombinant Anakinra Kineret IL-1β Periodic Syndrome/Neonatal Onset Multisystem IL-1RA Inflammatory Disease (CAPS/NOMID) CAPS, Tumor Necrosis Factor Receptor Associated Periodic Syndrome (TRAPS), Hyperimmunoglobulin D Canakinumab Ilaris mAB IL-1β syndrome (HIDS) and Familial Mediterranean Fever (FMF) RA, psoriasis, psoriatic arthritis (PA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis Adalimumab Humira mAB TNF-α (CD), hidradenitis suppurativa (HS), juvenile idiopathic arthritis (JIA) Infliximab Remicade mAB TNF-α RA, psoriasis, PA, AS, CD, HS, JIA Secukinumab Cosentyx mAB IL-17A Psoriasis, PA, AS

20 Ustekinumab Stelara mAB IL-23 Psoriasis, PA

Cytokine release syndrome (CSR), Castleman's disease, Tociluzimab Actemra mAB IL-6 RA, JIA Megakaryocytes- Recombinant Epoietin Epogen erythroid progenitor Anemia Erythropoietin cells Recombinant Filgrastim Neupogen HSCs Neutropenia G-CSF

1.4 INTERLEUKIN 30-40

The search for new cytokines

From a functional standpoint, the first mention of cytokines as effector molecules came from ancient philosophers who wrote about “factors” existing in pus that led to fever, swelling and the production of more pus (10). It was until the 1970s when the first soluble factor with a specific immune function was described by Gery and Waksman (138), by stimulating splenocytes and thymocytes with phytohemagglutinin (PHA), concanavalin A

(Con A) or lipopolysaccharide (LPS) and using “active” supernatants to stimulate proliferation of lymphocytes(138). They named the factor “lymphocyte activating factor”

(LAF) and later was purified and characterized as “T cell growth factor” by Mier and Gallo

(138, 139).

The original cytokines were named “Interleukin 1” and “”, which received their interleukin designation at the Second International Conference that was held in Interlaken, Switzerland in 1979. Since then, the number of interleukins has grown significantly. However, even though they are molecules of extreme importance in immunology, their numbers have made it difficult even for immunologists to keep pace with the discovery and characterization of these cytokines. Indeed, among them, there are some that have become ‘superstars’ of immunology (for example, IL17A), while others languish in relative obscurity (IL-11) as judged by the number of scientific papers documenting studies with these cytokines. By the onset of the XXI century, the number of interleukins reached IL-30 and has kept on increasing. There are currently 40 interleukins named. Consequently, the last 10 are among the ones least studied.

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The growing use of gene arrays and other gene expression analysis methods have also helped expand the universe of interleukins. A certain interleukin may become ‘popular’ through key studies published in the literature, but gene expression studies do not depend on the ‘popularity’ of a particular cytokine. They will faithfully reflect the levels of expression of a particular transcript in a given cell/tissue independently of its popularity in the literature. We expect that many of the interleukins we discuss in this review will show up in gene array studies which will help understand their functions and become better known. Personally, we found this exercise revealing, as it became clear that several of these molecules exhibit very interesting functions which strongly suggest that they may be important potential targets for future therapeutics. In fact, anti-cytokine (or anti- ) antibodies already represent important therapeutics (anti-TNF-α, anti-

IL17A, anti-IL-23).

The human genome contains an estimated 10% of genes encoding secreted proteins, so we can assume that there may be several new cytokines which are yet to be identified.

Within the IL-30 to IL-40 cohort, some of them do not share homology with other cytokine families, these are represented by IL-31, IL-32, IL-34 and IL-40. These latter interleukins are generally more difficult to identify (because they do not share structural/genetic similarities to other cytokines) so they were generally identified through screening of genomic databases or other bioinformatics-based screening methods.

Interestingly, the discovery of a new cytokine used to be a highly significant event in immunology. However, several of the novel cytokines described herein have not received significant attention when they were first reported. This is likely due to a phenomenon of

‘interleukin fatigue’, that is, a belief that cytokine biology is mostly a done deal and not an

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‘up and coming’ topic in immunology. This may be related to the fact that most cytokines were discovered by an older generation of immunologists, and cytokine discovery and characterization is not a very popular topic among young investigators. Another problem is that even when a new cytokine is discovered, there are no reagents available. This situation underscores the difficulty of studying the biology of new interleukins. However, despite these difficulties, it is apparent that the discovery of these new interleukins will open many new fields of research based on their very interesting biology and new potential targets for therapy. In this section I would like to summarize the finding and characteristics of the latest cytokines described.

Interleukin 30

IL-30, represented by the p28 subunit of IL-27, is part of the IL-12 cytokine family. IL-27, discovered by Pflanz et al (140), is a heterodimer composed of Epstein- Barr virus (EBV)- induced gene 3 (Ebi3) subunit non-covalently associated with the smaller p28 subunit (IL-

27p28) (140). IL-30, the p28 subunit of IL-27, remains functional even in the absence of

Ebi3 (141-143). family cytokines have emerged as critical regulators of immunity that regulate both pro-inflammatory (IL-12 and IL-23) and anti-inflammatory (IL-

27, IL-35, and IL-39) immune cell responses through their influence on T and B cell fates.

The IL-12 family is unique in having the only heterodimeric cytokines, this promiscuous pairing of alpha and beta chains is speculated to contribute to the dual role of inflammatory and anti-inflammatory cytokines (144).

IL-27 was initially thought to be a pro-inflammatory cytokine (145), but is now considered an immunoregulatory cytokine (146-148). IL-27 is secreted by activated antigen- presenting cells such as macrophages and dendritic cells (140, 149). Toll-like receptor

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(TLR) agonists such as LPS or CpG oligodeoxynucleotides and IFNα treatment of murine and human monocytes results in the production of IL-30. Once secreted, IL-27 binds to

IL-27R alpha and the T cell cytokine receptor signaling through WSX-1 subunits and gp130 protein to include signal transducer and activator of transcription 1 (STAT1) and

STAT3 in monocytes (149, 150). Recent studies have suggested that IL-30 may also signal through WSX-1 and gp130, but it may require a binding partner such as cytokine- like factor (CLF-1) or soluble IL-6 receptor (sIL-6Ra) (141, 143, 151).

Much more information is known about IL-27 than IL-30, however, they have been suggested to share overlapping roles. IL-27 has been shown to play an anti-inflammatory role in models of autoimmunity such as autoimmune encephalomyelitis and collagen- induced arthritis (152, 153). IL-27 downregulates the immune response by inhibiting IL-2 and IL-17A expression, while promoting IL-10 production (154-158). Additional studies have shown that IL-30 exhibits an anti-inflammatory role similar to IL-27. IL-27 can suppress IL-17A production, albeit at a much lower level than IL-27 (159).

Recent studies have identified IL-30 as a critical regulator of breast and prostate cancer metastasis (160, 161). IL-30 is expressed in triple-negative HER2+ breast cancer tumors.

In addition, expression of IL-30 was correlated with disease stage and re-occurrence.

Mice injected with triple-negative HER2+ breast cancer cells treated with IL-30 displayed increased proliferation and vascular dissemination of breast cancer cells. Silencing of

STAT1/3 signaling, downstream targets of IL-30, prevented the expression of CXCL1,

CSF1, IL-1β, IL-6, IL-8, PTGS2/COX2, and Myc, which are known to promote tumor growth and metastasis (160). Expression of IL-30 has also been identified in prostate cancer cells and cancer infiltrating leukocytes (140). IL-30 expression is correlated with

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grade and stage of disease in prostate cancer patients (162). Sorrentino and colleagues found that IL-30 secreted by human and murine prostate cancer stem-like cells (PCSLC) had significant autocrine and paracrine affects which supported PCSLC viability, self- renewal, and tumorgenicity (161). Therefore, they concluded that IL-30 could be a potential target for development of therapeutic monoclonal antibodies for cancer treatment.

Interleukin 31

IL-31 was reported in 2004 by Dillon et al (163). It is a member of the IL-6 family of cytokines, a four-helix bundle cytokine which is produced by Th2 cells upon activation. It signals through a receptor composed of two chains, an IL-31 receptor A and oncostatin

M receptor (OSMRbeta). The receptor is expressed constitutively in epithelial cells but is induced upon activation in monocytes. IL-31 seems to mediate pruritus, as this is an evident phenotype in IL-31 transgenic mice, which also develop alopecia and lesions.

These observations strongly suggest that IL-31 is involved in dermatitis present in allergic diseases.

Sonkoly et al. (164) showed that IL-31 is overexpressed in pruritic skin.

Highest levels were observed in prurigo nodularis, one of the most pruritic forms of chronic skin inflammation. In vivo, staphylococcal enterotoxin B (SEB) (a super-antigen targeting

Vβ8) induced IL-31 expression in atopic individuals, and in vitro SEB induced IL-31 production in leukocytes. IL-31 receptor A showed the most abundant expression in dorsal root ganglia, the site where the cell bodies of cutaneous sensory neurons reside.

Cells expressing IL-31RA mRNA were detected in the dorsal root and trigeminal ganglia,

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and these cells also expressed OSMRbeta, but the expression of these receptor chains appear at different times during development (165).

Expression of IL-31 correlates with the enhanced expression of IL-4 and IL-13 (two other

Th2 cytokines) in atopic dermatitis and atopic contact dermatitis (166). However, IL-31 mRNA was not observed increased in psoriasis. These observations strongly suggested that IL-31 is associated with itching, nevertheless, the situation may be more complex since other studies have found IL-31 levels strongly increased in Atopic Dermatitis but they do not correlate with the intensity of the disease according to the SCORAD severity score (167). However, support for the concept that IL-31 is a pivotal cytokine regulating pruritus comes from studies on contact dermatitis with Il-31-/- mice which exhibit reduced scratching frequency and duration during hapten-induced contact dermatitis (168).

Eosinophils can also be potent sources of IL-31 (169), and anti-IL-31 antibodies ameliorate scratching behavior in a mouse model of atopic dermatitis (170).

Interleukin 32

Kim et al. (171) reported the identification of a novel cytokine produced by a cell line

(human lung carcinoma A549) transfected with an IL-18 receptor β chain. This rendered the cell responsive to IL-18 and it produced several cytokines, including a novel one they called IL-32. It is expressed by human peripheral blood mononuclear cells (PBMCs) upon activation, induced in human epithelial cells by IFNγ, and in NK cells by IL-12 plus IL-18.

In fact, it was originally called NK Transcript 4. Its expression pattern mirrors other inflammatory cytokines so it is not surprising that it also is capable of inducing the production of other cytokines like TNFα, IL-1β, IL-6 and various CXC chemokines in various cells. There are various isoforms of IL-32, but the gamma isoform has been

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reported to be the most active (172). IL-32 signals through p38 MAPK and NF-κВ pathways, and has been implicated in inflammatory disorders, inflammatory bowel disease, and influenza virus A infections. It has also been associated with several autoimmune diseases such as rheumatoid arthritis, ulcerative colitis and Crohn’s disease

(173). However, the specific role of this pro-inflammatory cytokine in these diseases is not yet known. It has also been implicated in the latent stage of tuberculosis (174).

IL-32 is likely to have both intracellular and extracellular functions (175). It could be involved in resistance against intracellular pathogens (176). Intracellular IL-32 may be involved in regulation of cell activation and cell death. IL-32 also exhibits angiogenic properties (177).

IL-32 has also been implicated in cancer progression. For example, IL-32 promotes the production of IL-2 by bone marrow cells which in turn may promote the proliferation of multiple myeloma cells (178). IL-32 expression has been associated with hypoxia in multiple myeloma cells, and high expression is associated with poor survival and bone loss (179).

Given that there are various isoforms of IL-32, it represents a situation where each isoform may have different expression patterns, properties and functions. We can expect future more reports where specific isoforms of IL-32 will be functionally characterized.

Interleukin 33

IL-33 is a member of the Interleukin 1 (IL-1) superfamily. IL-33 induces Th2 responses in

T cells, mast cells, basophils and eosinophils. Previously known as nuclear factor in high endothelial venules (NF-HEV) it was discovered in high endothelial cells. Baekkevold et

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al (180) discovered IL-33 by using molecular and bioinformatic approaches to study genes preferentially expressed by HEV endothelial cells, although it was found to be also expressed in human lymphoid organs (tonsils, lymph nodes and Peyer’s patches) and in single cells of the T cell zones of lymph nodes. Nevertheless, its function was uncertain, and it was pointed out as a possible transcription factor in charge of the regulation of expression after cytokine signaling in HEV cells.

In 2005 researchers at Merck Research Labs showed that IL-33 signals through a ST2 receptor, a previously orphan IL-1 receptor family member, which is expressed in human and murine Th2 cells, mast cells, eosinophils and basophils and it is involved in Th2 responses (181, 182). Using a pull-down method with biotinylated IL-33 to capture the

ST2 receptor and a GFP-NF-κВ reporter transfected cell line Shmidtz et al proved that IL-

33 was binding and signaling through ST2 (Shmidtz and others 2005). Naturally, stimulation with IL-33 drives the expression of Th2 cytokines, IL-4, IL-5, and IL-13 and leads to an increase in the secretion of IgE, IgA and eosinophilia in vivo (182);(183). The connection of IL-33 with asthma and other Th2 related diseases was immediately drawn.

Using microarrays, the group that originally reported IL-33, described that it is constitutively expressed in lymphoid tissues, epithelial cells, fibroblasts, mucosal tissues, tumor cells and vascular tissues where its expression is particularly high (184). The same group previously demonstrated that IL-33 has transcriptional regulation properties and that it can be located in the nucleus of high endothelial venules cells (HEVs), drawing a parallel to IL-1α (182). IL-1α, another member of the IL-1 family that shares structural and functional characteristics with IL-33, such as the long 100 amino acid residues at the

N-terminus and the presence in high concentrations as a precursor in the intracellular

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compartment of expressing cells, rather than mature secreted forms due to the lack of signal peptide (185). The maturation and activity of IL-1 family members IL-1α, and IL-1β starts after the release from intracellular compartments mediated by mechanical or infection-related cell death, effectively making them representatives of molecules known as alarmins (186). Alarmins are the endogenous equivalent of pathogen-associated molecular patterns (PAMPs) and are released during non-programmed cell death. Once released alarmins can signal immune system cells such as macrophages and dendritic cells to initiate a cellular immune response to resolve inflammation (186). Given that IL-

33 is constitutively expressed in healthy barrier tissues, its similarities with other members of the IL-1 family of alarmins led the Girard group to conclude that IL-33 is an alarmin.

Asthma leads to chronic inflammation and persistent airway remodeling in the

(187). IL-33 is upregulated in bronchoalveolar lavage fluid from asthma patients and a

SNP polymorphism in ST2 is strongly associated with asthma in different human populations (188) (189). Yagami and collaborators showed that IL-33 acts directly in lung endothelial and epithelial cells but not fibroblast or muscle cells by inducing the expression of CXCL8, having a direct effect in controlling neutrophil recruitment to the lungs and thus regulating inflammation in the lungs (183). Furthermore in vitro stimulation of human microvascular endothelial cells from lung blood vessels (HMVEC-LBI) with IL-

33 also led to an increase of IL-6 and CCL2 secretion (183). From analysis of these data, we can conclude that IL-33 plays a major role in lung inflammation and that the IL-33/SP2 signaling axis is important in the pathogenesis of asthma and allergies.

Recently IL-33 has also been implicated in the regulation of immune responses in the gut.

It has been reported that IL-33 is upregulated in patients with inflammatory bowel disease

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(IBD) (190-192). Colonic T regulatory cells (Tregs) and group 2 innate lymphoid cells

(ILC2s) are known to respond to IL-33 in the gut, reviewed in (193). Furthermore, IL-33 has been shown to be a key cytokine in the maturation, proliferation and function of colonic Tregs, with an antagonistic activity to IL-23, a key inflammatory molecule of the gut (193, 194). From the research done in the last 15 years we can conclude that IL-33 has emerged as a key regulatory cytokine in barrier tissues, making it an important target for inhibition therapy in autoimmune diseases such as IBD, rheumatoid arthritis (195), asthma, and allergies.

We should note that a recent study on the ancestral origins of the Interleukin-1 family concluded that IL-33 and IL-18 have poor sequence similarity and no chromosomal evidence of common ancestry with the IL-1β cluster and therefore should not be included in the IL-1 ligand ancestral family (196). This observation has important functional implications. IL-33 and IL-18 therefore represent genes derived from an ancestral gene that also was the ancestor of the IL-1β cluster. However, since this split occurred a long time (millions of years) ago, each of these genes (IL-1β cluster, IL-18 and IL-33) has had a long time to evolve independently ever since. For this reason, we can predict that each of these genes should exhibit very different functional properties. In contrast, other IL-1 family members derived directly from the IL-1β cluster in more recent evolutionary times should exhibit similar functions to other members of the IL-1β family cluster.

Interleukin 34

IL-34 is a cytokine that also binds the colony stimulating factor-1 receptor

(CSF-1R). Its discovery was a ‘tour de force’. Lin et al (197) performed a functional screening of the extracellular proteome. This was represented by a comprehensive set of

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recombinant secreted proteins and the extracellular domains of transmembrane proteins.

Each protein was tested in several functional assays. One of the secreted proteins, designated IL-34, stimulated viability. They also reported that IL-34 binds CSF-

1. However, it quickly became apparent that IL-34 and M-CSF did not exhibit identical biological activities. They differ in their capacity to induce the production of chemokines like CCL2 and CCL24 in primary macrophages. Interestingly, IL-34 and M-CSF do not share , but some antibodies against CSF-1R can block the binding of one ligand but not another. This suggests that these ligands bind different parts of CSF-

1R, suggesting that they can mediate different bioactivities and signal transduction pathways.

Both M-CSF and IL-34 mediate osteoclastogenesis (198) in combination with RANKL.

This suggests a potential therapeutic role in osteoporosis.

IL-34 is produced by in the skin and neurons in the . IL-34-/- mice displayed a marked reduction of Langerhans cells and microglia, whereas monocytes, dermal and lymphoid tissue macrophages were unaffected. Langerhans cell survival is dependent on IL-34, whereas microglia and their yolk sac precursors develop independently of IL-34 but rely on it for their maintenance in the adult brain (199).

There have been other intriguing studies that have implicated IL-34. For example, Bezie et al reported that IL-34 is specifically produced by T regulatory cells and mediates transplant tolerance (200). In a rat cardiac allograft model, treatment of rats with IL-34 promoted allograft tolerance that was mediated by induction of Tregs. Treatment of human macrophages with IL-34 greatly expanded CD8+ and CD4+ FoxP3+ Tregs, cells that exhibited greater suppressive potential than non-IL-34 expanded Tregs.

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In addition, IL-34 is known to influence the phenotype of myeloid lineage cells that express

CSF-1R. For example, it has been reported to inhibit acute rejection of rat liver transplantation by inducing the polarization of Kupffer cells to the M2 phenotype (201).

Similarly, it has been reported to be expressed at the fetal-maternal interface and to induce immunoregulatory macrophages of a decidual phenotype (202).

IL-34 levels in serum have been reported in various diseases including systemic lupus erythematosus (203), periodontal disease (204), patients experiencing acute rejection following liver transplantation (205), development of liver fibrosis in patients with Hepatitis

B (206) and rheumatoid arthritis (207). The potential role of IL-34 in these diseases remains to be clarified.

Finally, IL-34 may play a role in both cancer and fibrosis. High co-expression of IL-34 and

M-CSF correlates with tumor progression and poor survival in lung cancer (208). It also has been implicated in colon cancer (209), and it may play a role in the functional polarization of tumor-associated macrophages (210). It has been reported to promote fibrocyte proliferation, suggesting that it is a pro-fibrotic factor (211).

We conclude that IL-34 has strong potential to be a target for development of therapeutic antibodies.

Interleukin 35

Interleukin 35 (IL-35) was named a decade ago (i.e. 2007) independently by two labs,

Niedbala (212) and Collision (213), but it was first described in 1997 (213, 214). IL-35 has gained much interest for its role in strongly inhibiting immune cell function, with prospects in development of therapeutics for autoimmune disease. As part of the IL-12 family, IL-35

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consists of a heterodimeric glycoprotein formed with disulfide linked IL-12alpha chain

(p35) and IL-27 b chain Ebi3 (215). Collision et al originally reported that IL-35 was produced by populations of -derived natural regulatory T cells (nTregs) and was required for their maximal suppressive activity (213). Regulatory Bregs have also been reported to produce anti-inflammatory cytokines including IL-10 and IL-35 (216), (60). Like other IL-12 cytokine family members, IL-35R binds to activate STAT proteins (217).

Binding of IL-35 to IL-35R activates STAT1 and STAT4 in T cells (216). However, in B cells IL-35 signaling mediates STAT1 and STAT3 (60).

IL-35 has gained much interest for its suppressive role. Ebi3 is a downstream target of

FoxP3, restricting its expression to the Treg lineage. IL-35 has been shown to prevent

Th1 and Th17 proliferation by arresting T cells in the G1 phase of cell division (218).

Although IL-35 is able to block T cell proliferation, it does not promote the conversion of

Th1 cells to Tregs as IFNγ is a strong negative regulator of Ebi3 and P35 transcription

(144). In Th2 cells, IL-35 blocks Th2 differentiation by repressing GATA3 and IL-4 expression. Additionally, IL-35, like TGF-β and IL-10, can induce regulatory T cells

(iTregs). Recent studies have focused on the role of IL-35 in autoimmunity. Numerous studies have demonstrated that IL-35 plays a protective role in mouse models of human autoimmune disease such as, EAE, experimental autoimmune uveitis (EAU), encephalitis, allergic airway model, diabetes, and colitis (213, 219-222).

Recently, Bregs have also been identified as potent producers of IL-35 (223) Bregs are believed to mediate their immunosuppressive capabilities through the secretion of IL-10 upon stimulation of toll-like receptor agonists CD40L and IL-21 (224). Recent reports have suggested that IL-35 in addition to IL-10, contributes to Breg suppressive function. Bregs

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have been shown to be capable of producing IL-35 (219), (60). Addition of rIL-35 results in selective proliferation of CD19+CD5+B220lo which display enhanced secretion of IL-10 and IL-35. Similarly, rIL-35 inhibits B220hi B cell proliferation (219). IL-35 produced by

Bregs acts in an autocrine manner to stimulate the IL-35R on Bregs and also in a paracrine fashion to promote the generation of induced Tregs (225).

Alternatively, recent studies have suggested that IL-35 plays a protective role in tumor immune escape. Ebi3 overexpression has been reported in Hodgkin’s lymphoma (226), and lung cancer cells (227). In addition, tumors expressing IL-35 have an increase in myeloid derived suppressors cells (MDSCs), which are known to be immune suppressive and inhibit cytotoxic T cell responses (228).

While IL-35 may be an intriguing cytokine to treat autoimmune diseases, recombinant IL-

35 is difficult to obtain, thus limiting immunology research and its potential production as a therapeutic. IL35 is not secreted as a disulfide-linked heterodimer, therefore only about

4% of secreted Ebi3 is co-precipitated with IL-12p35 (214). Although approaches to generate rIL-35 in mouse models and attempts to covalently link Ebi3 and IL-12p35 have been made (60, 213, 229).

While the immune inhibitory role of IL-35 has been well characterized in mouse models, the immunosuppressive effects of IL-35 in humans is modest (230). However, Seyeri et al reported that human Tregs produced IL-35 under strong stimulation (231). Thus, the role of IL-35 in human Tregs still warrants future investigation.

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Interleukin 36

IL-36 is a very interesting group of cytokines, derived from a member of the IL-1 family.

In humans, they exist as 4 distinct secreted proteins, previously orphan IL-1 family ligands; IL-36α, IL-36β, IL-36γ and the receptor antagonist IL-36ra. All of them require post-transcriptional processing to be functional and bind the IL-36 receptor (IL-36R) (232).

Due to the pro-inflammatory nature of all the IL-36 members and the similarity of the responses they elicit signaling through the IL-36R they were designated under the IL-36 name in 2010 (233).

The first member of the IL-36 cytokine group termed IL1HY1 (now IL-36ra) was discovered in 1999 by Mulero and collaborators, using a cDNA screening derived from human liver and spleen. The cDNA caught the authors’ attention due to the relatively high homology (65%) to IL-1ra (234). The next year four different groups would publish the discovery of new members of the IL-1 family, (235-238). The function of these cytokines would not become apparent until their receptor was identified, IL-36R, formerly known as

IL-1 receptor-related protein 2 (IL-1rp2). Debets and collaborators reported that IL-36α elicited NF-κВ signaling through IL-1Rrp2 and was strongly inhibited by IL-36ra (239).

Interestingly, they also found high expression of IL-36α, IL-36ra and IL-36R in skin and a significant upregulation in lesional psoriasis. It was later found that all the proposed members of the IL-36 cytokine subfamily signal through IL36R and activate the NF-κВ,

MAPKs, JNK and ERK1/2 kinase cascade which leads to CXCL8, TNF-α, IL-17A, IL-23 and IL-6 secretion. Interestingly, IL-36ra functions as a direct antagonist of the other IL-

36 members (232, 240, 241).

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IL-36R is mainly expressed in T cells and is especially predominant in naïve CD4 T cells

(242, 243). Stimulation with IL-36 induced naïve T cell proliferation and enhanced IL-2 secretion. Furthermore, IL-36β along with IL-12 promote Th1 polarization and plays an important role against Mycobacterium infections (242). IL-36 cytokines are mainly expressed in skin keratinocytes, myeloid cells, Langerhans cells and mucosal epithelium

(243-245). This expression pattern and the responding cells indicate that the IL-36 cytokines are likely very important in skin homeostasis and defense. As such, studies with an IL-36α-/- mouse showed that IL-36A (but not IL-36β or IL36γ) is required to induce and maintain psoriasis-like disease in a mouse model of psoriasis (246). Interestingly, the same group found that IL-36α acts in a self-amplifying loop with IL-1α, which is also required in order to maintain skin inflammation during psoriatic lesion flares (246).

Ongoing clinical studies are currently evaluating the potential efficacy of IL-36R blockage using a therapeutic monoclonal antibody in psoriasis. A hint of the potential use of an IL-

36R blocking monoclonal antibody came from an unlikely source, a rare human disease caused by a loss of function mutation on the IL36RA gene, termed Deficiency of

Interleukin 36 receptor antagonist (DITRA) syndrome (247). Patients with DITRA syndrome develop generalized pustular psoriasis (GPP) and some have been successfully treated with anakinra, a recombinant human IL-1RA originally developed for the treatment of RA (248-250). There is no certainty that the administration of IL-36R blocking antibody will help resolve skin inflammation in autoimmunity, but the concept is supported by a wide body of research that suggests that it will be effective in these indications. In fact, there are currently at least three active clinical trials testing the efficacy of a humanized anti-IL-36R antibody (251).

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In recent years it has been observed that IL-36 family members, especially IL-36α, are upregulated during IBD pathogenesis (252). Walsh and collaborators found that the expression of IL-36α is upregulated in human patients with ulcerative colitis and mouse models of sterile inflammation. The inflammation was significantly reduced in a dextran sulphate (DSS) colitis model using IL-36r-/- mice. Furthermore, other studies indicate that

IL-36 cytokines are also necessary for effective tissue repair in the gut (253, 254). These studies on the role of IL-36 family on IBD further improve the case for the development of

IL-36R blocking therapies and highlights the need of further research on the role of IL-36 cytokines in the homeostasis of the gut.

Interleukin 37

Another member of the IL-1 cytokine family, originally known as IL-1F7, shares structural similarities with IL-18 and can bind to the IL-18 receptor (IL-18Rα) although at much lower affinity (255, 256). It has 5 isoforms (IL-37a, b, c, d and e) and each isoform expression is tissue specific (257, 258). IL-37 was first described as an IL-1 family member (which was identified using the same approach that led to the identification of other IL-1 family members) through bioinformatics analyses of the human genome for uncharacterized genes that share similarity and/or share chromosomal location with other known IL-1 family members. In fact, IL-37 was described along with the previously discussed IL-36 cytokines simultaneously by three different groups using similar approaches (235, 237,

255). Dunn and collaborators reported that IL-37 could bind to IL-18R suggesting a function similar or parallel to IL-18 (255). IL-36 is expressed in various tissues, but its expression is particularly high in thymus, testis and uterus as well as macrophages, DCs,

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tonsillar B cells and plasma cells (259, 260). IL-37 is also expressed in gut and skin epithelial cancers (260).

Remarkably, IL-37 is an anti-inflammatory cytokine. It was until 2010, almost 10 years after the first report by Nold and collaborators who found that the expression of IL-37 by epithelium and macrophages suppressed almost completely the expression of several pro-inflammatory cytokines (mainly IL-1β, IL-6 and TNF-α) and chemokines (CCL2,

CXCL2 and CXCL8) in vitro and protected mice against sepsis induced by LPS in vivo

(256). They also found that IL-37 forms a complex with SMAD3, an important transcription factor in the TGF-β pathway (261) and is necessary for IL-37 driven responses. Taken together, these observations indicated that IL-37 was likely to be a novel anti- inflammatory cytokine.

Most of the effects of IL-37 in metabolic, autoimmune and infectious diseases have been tested in several metabolic, autoimmune and infectious models using a transgenic mouse that overexpresses human IL-37 (IL37-tg) (256). In these experiments, IL-37 has proven to have a protective effect on tissue damage and mediates a reduction in the secretion of pro-inflammatory cytokines in several models including colitis, Rheumatoid Arthritis, allergy and fungal pulmonary infections (260, 262-265). Additionally, IL-37 appears to play a role in the regulation of fat metabolism, aortic disease and aging (266-268).

Examination of the research done with IL-37 in mouse models of metabolic, autoimmune and infectious diseases indicates that IL-37 is important in homeostasis and as an immune regulator. While involvement of IL-37 has also been reported in human disease

(269-272), there are currently no human clinical trials evaluating the effects of exogenous administration on humans. IL-37 seems as a potential candidate for supplementation

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therapy in conjunction with blocking therapy (the most likely candidates would be monoclonal antibodies).

Interleukin 38

IL-38 is another IL-1 family protein discovered in the early 2000s along with IL-36 and IL-

37. The IL-38 gene was identified and termed IL-1HY2 by Lin and collaborators in 2001.

They reported that IL-1HY2 shares sequence 52% and structural similarity to IL-1ra, and also significant similarity to IL-36ra (273, 274). The IL-38 gene is located in chromosome

2, along with other members of the IL-1 family (275) indicating that it is part of recent evolutionary gene duplication. IL-38 is expressed in skin, fetal liver, placenta, brain, thymus and tonsils (273-275). Remarkably, years later van de Veerdonk and collaborators reported that IL-38 is an accessory IL-36 receptor antagonist that binds to the IL-36 receptor to decrease secretion of IL-22 and IL-17 after stimulation with heat- killed Candida albicans in memory T cells. It also reduced the production of CXCL8 by

PBMC induced by LPS, similar to the canonical IL-36ra (276). The confirmation of IL-38 binding to IL-36R came by using immobilized extracellular domains of the IL-1 family receptors to screen receptor binding and IL-38 and IL-36ra bound to the extracellular domain of IL-36 (276). Additionally, PBMCs treated with IL-38 siRNA produced up to 28- fold more pro-inflammatory cytokines (including IL-36, CCL2 and APRIL), suggesting that

IL-38 exhibits an antagonist function (277).

Several genome-wide association studies (GWAS) studies have associated IL-38 with human diseases where other IL-1 family cluster alleles have been implicated including ankylosing spondylitis, RA and cardiovascular disease (278-281). IL-38 has been reported to be increased in patients with SLE and its levels directly correlate with an

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increased risk of renal lupus and central nervous system lupus (277). It has also been found that IL-38 is increased in patients with hidradenitis suppurativa, hepatitis B, gestational diabetes mellitus, asthma, lung fibrosis and lung adenocarcinoma (282-287).

Interestingly, in Hepatitis B, IL-38 expression appears to be a marker of good prognosis

(patients with high levels of IL-38 were more likely to respond to therapy). In the case of asthma and cancer it appears to be the opposite, in asthma it correlated with lower numbers of circulating Tregs and in cancer it was associated with higher expression of

PD-L1 by the tumors (284, 286, 287). The function and role of IL-38 has not been extensively explored and is one of the IL-1 family members for which less information is available. However, the data available strongly suggests that it is an attractive candidate to be a potential target for human diseases where the IL-36/IL-36R axis has been implicated.

Interleukin 39

In 2016, Wang et al described the most recently discovered member of the IL-12 cytokine family, Interleukin 39 (IL-39) (288). IL-39 is a heterodimer composed of the IL-23p19 alpha subunit and Ebi3 beta subunit. IL-39 was found to be secreted as a heterodimer by activated B cells, similar to IL-35. Following LPS stimulation, IL-39 was significantly upregulated in B cells. In addition, p19 and Ebi3 mRNA was found to be expressed by

DCs and macrophages (288). Similarly to IL-35, IL-39 has been shown to mediate inflammatory responses though activation of STAT1/STAT3 (289).

SLE is an autoimmune disease in which B cells increase the production of autoantibodies

(290). Wang and colleagues found that activated B cells in lupus-prone mice secreted IL-

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39. In addition, administration of IL-39 in vitro and in vivo induced the differentiation and/or expansion of neutrophils which contribute to disease (288, 291). Knock down of p19 or

Ebi3 reduced lupus severity in mice. Therefore, targeting IL-39 may be used as a potential target for treating SLE.

Future perspectives on cytokine discovery

We have summarized the characteristics of the last 10 cytokines in Table III (an in-depth review of the latest one IL-40 will be done in the next chapters). It is apparent from the information already available on these proteins that their potential functional role(s) in both innate and adaptive immune responses and their potential role in human disease are vast. Still, it is obvious that the research focusing on these novel cytokines is still in early stages. As an example, a literature search on publications mentioning IL-10 in the

US National Library of Medicine of the National Institutes of Health (pubmed.gov) yields

56,241 publications, while a similar search for IL-35, one of the most “popular” new cytokines, yields only 409 results. This suggests that there are many novel fields of research to be defined based on the physiology of these novel cytokines.

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Table III. Summarized characteristics of new cytokines.

Main human Other Gene Chromosome Immune cells In-depth Cytokine Receptor Functions expression Producing cells Potential role in disease names Superfamily location responding Reviews tissues1 Placenta, Bone Monocytes, p28, Suppression of IL-17A Marrow, Liver, IL-30 IL-12 16p12.1 IL-27Rα Macrophages, T cells Prostate Cancer, Breast Cancer (42) IL-27p282 production, Anti-Inflammatory Stomach, Skin, DCs Testis Bone Marrow, Eosinophils Epithelial cells, Pruritic Atopic Dermatitis, Airway hypersensitivity, IL-31 None IL-6 12q24.31 IL-31RA/OSMRβ Pruritus, Pro-Inflammatory (292) Tonsil, Spleen, Skin CD4+ T cells Monocytes Inflammatory Bowel Disease

Monocytes, Angiogenesis, IL-2 Appendix, Lung, Monocytes, Cancer, Tuberculosis, Influenza virus A infection, T cells, IL-32 NK4 None 16p13.3 Unknown production in bone marrow, Duodenum, Small Macrophages, Bone Rheumatoid Arthritis, Ulcerative Colitis, Chron's disease, (293); (294) NK cells, Pro-Inflammatory Intestine Marrow stroma Chronic Obstructive Pulmonary Disease epithelial cells Skin, Stomach, Endothelial cells, Cervix, T cells, Mast cells, NF-HEV, Epithelial cells, IL-33 IL-1 9p24.1 ST2 Alarmin, Pro-Inflammatory Endometrium, Eosinophils, Basophils, Asthma, Inflammatory Bowel Disease (295) IL-1F11 Fibroblast, Nasopharynx, ILC2s Mucosal epithelium Bronchus Keratinocytes, Macrophages, Neurons, Monocytes, Osteoporosis, Lupus, Periodontal disease, Hepatitis B, CSF-1R, PTP-ζ, Myeloid cell proliferation, Tregs, IL-34 C16orf77 None 16q22.1 Spleen3, Skin3 Langerhans cells, Rheumatoid Arthritis, Transplant rejection, Lung Cancer, (296) CD138 Anti-Inflammatory Monocytes, Microglia, Kupffer cells, Fibrosis Macrophages, DCs DCs IL-12Rβ2/IL- Prevents Th1 and Th17 cell 3q25.33 12Rβ2; proliferation, Induce Tregs, Multiple Sclerosis, Diabetes, Colitis, Hodgkin lymphoma, IL-35 None IL-12 (IL12A); Placenta, Spleen3, Macrophages, T cells (297) gp130/gp130; Treg/Breg polarization, Anti- Bregs, Lung cancer 19p13.3 (EBI3) IL-12Rβ2/gp130 Inflammatory Keratinocytes, IL-36α FIL1ε/IL1F6, Mucosal epithelium, Epithelial cells, IL-36β FIL1η/IL1F8, Th1 responses, Pro- Skin, Esophagus, Monocytes, Macrophages, DCs, T DITRA4, Lesional Psoriasis, Tuberculosis, Inflammatory IL-1 2q14.1 IL-36R (245)

42 IL-36γ IL1F9, Inflammatory Tonsil Macrophages, cells, B cells, Plasma Bowel Disease IL-36RA FIL1δ/IL1F5 Langerhans cells, cells + CD4 T cells, Epithelial cells, Epithelial cells, Keratinocytes, Fibroblasts, Systemic Lupus Erythematosus, Rheumatoid Arthritis, IL-37 FIL1ζ, IL-1F7 IL-1 2q14.1 IL-18Rα Anti-Inflammatory Skin Monocytes, Monocytes, Inflammatory Bowel Disease, Ankylosing spondylitis, (258) NK cells, Macrophages, DCs, T Graves’ disease, HIV infection, Coronary Artery Disease B cells cells Rheumatoid Arthritis, Atherosclerosis, Cardiovascular Epithelial cells, IL-1F10, IL-1R; IL-36R; Blocking of IL-36, Anti- Epithelial cells, disease, Systemic Lupus Erythematosus, Hidradenitis IL-38 IL-1 2q14.1 Skin Macrophages, DCs, B (274), (298) FIL1θ IL-1RAPL1 Inflammatory B cells suppurativa, Hepatitis B, Diabetes, Asthma, Fibrosis, cells, Plasma cells Lung cancer Macrophages, 12q13.3 (P19); IL-39 None IL-12 Unknown Pro-Inflammatory Spleen DCs, Neutrophils Lupus None 19p13.3 (EBI3) B cells Involved in IgA production, B Bone Marrow, Fetal B cells, IL-40 C17orf99 None 17q25.3 Unknown cell homeostasis and B cells B cell lymphoma None Liver Bone marrow stroma development 1 Data from protein atlas of human expression (https://www.proteinatlas.org) (299) 2 IL-30 is a subunit of IL-27 but exhibits activity by itself 3 RNA expression 4 Deficiency of Interleukin 36 receptor antagonist is a disease caused by the lack of functional IL-36ra

One of the biggest challenges following the identification of a novel cytokine is to start defining its biology. Usually one of the first steps is the production of a knockout mouse.

However, identifying a phenotype of a new knockout mouse is not easy either. These considerations make this type of research (searching for novel cytokines) a very challenging project. Importantly, evolutionary considerations can suggest functional roles.

Even chromosomal location can provide important clues about the function of a particular gene (20).

The discovery of cytokines in the late XX and early XXI centuries was supported by advances in molecular biology techniques and the development of gene databases and bioinformatics. Many of the cytokines, including several in this section, were found because they belong to cytokine super-families and share similarities to previously known cytokines. In our experience, the process that led us to the identification of IL-40, which is encoded by a previously uncharacterized gene for which no information was available, all the way to the identification of its role in IgA production and B cell development, has been difficult. A key question is whether there remain many other cytokines to be identified. Ten percent of the human genome is estimated to encode secreted proteins, suggesting that there may be more. Given the popularity of databases and gene arrays, and novel techniques like RNAseq and single cell sequencing, we may predict that cytokines produced by relatively rare cell subsets may yet be identified. If so, we would predict that their genes will not related to any of the known cytokine families.

In the next chapter we will talk about the discovery of IL-40, a novel B cell cytokine. The discovery of a cytokine can be exciting and daunting at the same time. After identifying

IL-40 in silico we had a few clues and tools to explore what IL-40 could be doing in the

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immune system. As you will be able to see the roles of IL-40 in the immune system started to unfold as the analysis of the IL-40 knockout mouse (Il40-/-) proceeded. In this thesis we only describe the beginning of the research of IL-40 and, due to our results, we anticipate that in years to come more exciting new research about IL-40 will be published.

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277. Rudloff I, et al. (2015) Brief Report: Interleukin-38 Exerts Antiinflammatory Functions and Is Associated With Disease Activity in Systemic Lupus Erythematosus. Arthritis Rheumatol 67(12):3219-3225. 278. Chou CT, et al. (2006) Replication of association of IL1 gene complex members with ankylosing spondylitis in Taiwanese Chinese. Ann Rheum Dis 65(8):1106- 1109. 279. Rahman P, et al. (2006) Association between the interleukin-1 family gene cluster and psoriatic arthritis. Arthritis Rheum 54(7):2321-2325. 280. Jung MY, et al. (2010) The interleukin-1 family gene polymorphisms in Korean patients with rheumatoid arthritis. Scand J Rheumatol 39(3):190-196. 281. Lopez-Mejias R, et al. (2016) Influence of elevated-CRP level-related polymorphisms in non-rheumatic Caucasians on the risk of subclinical atherosclerosis and cardiovascular disease in rheumatoid arthritis. Sci Rep 6:31979. 282. Hessam S, et al. (2018) Interleukin-36 in hidradenitis suppurativa: evidence for a distinctive proinflammatory role and a key factor in the development of an inflammatory loop. Br J Dermatol 178(3):761-767. 283. Yu Z, et al. (2017) IL-37 and 38 signalling in gestational diabetes. J Reprod Immunol 124:8-14. 284. Wang HJ, Jiang YF, Wang XR, Zhang ML, & Gao PJ (2016) Elevated serum interleukin-38 level at baseline predicts virological response in telbivudine-treated patients with chronic hepatitis B. World J Gastroenterol 22(18):4529-4537. 285. Tominaga M, et al. (2017) Overexpression of IL-38 protein in anticancer drug- induced lung injury and acute exacerbation of idiopathic pulmonary fibrosis. Respir Investig 55(5):293-299. 286. Takada K, et al. (2017) Clinical implications of the novel cytokine IL-38 expressed in lung adenocarcinoma: Possible association with PD-L1 expression. PLoS One 12(7):e0181598. 287. Chu M, et al. (2016) Aberrant Expression of Novel Cytokine IL-38 and Regulatory T Lymphocytes in Childhood Asthma. Molecules 21(7). 288. Wang X, et al. (2016) A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice. Eur J Immunol 46(6):1343-1350. 289. Floss DM, et al. (2017) IL-6/IL-12 Cytokine Receptor Shuffling of Extra- and Intracellular Domains Reveals Canonical STAT Activation via Synthetic IL-35 and IL-39 Signaling. Sci Rep 7(1):15172. 290. Oku K & Atsumi T (2018) Systemic Lupus Erythematosus: nothing stale her infinite variety. Mod Rheumatol:1-20. 291. Wang X, et al. (2016) Interleukin (IL)-39 [IL-23p19/Epstein-Barr virus-induced 3 (Ebi3)] induces differentiation/expansion of neutrophils in lupus-prone mice. Clin Exp Immunol 186(2):144-156. 292. Zhang Q, Putheti P, Zhou Q, Liu Q, & Gao W (2008) Structures and biological functions of IL-31 and IL-31 receptors. Cytokine Growth Factor Rev 19(5-6):347- 356. 293. Khawar MB, Abbasi MH, & Sheikh N (2016) IL-32: A Novel Pluripotent Inflammatory Interleukin, towards Gastric Inflammation, Gastric Cancer, and Chronic Rhino Sinusitis. Mediators Inflamm 2016:8413768.

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CHAPTER 2

IDENTIFICATION OF IL-40, A NOVEL B CELL–ASSOCIATED CYTOKINE

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2.1 INTRODUCTION

Cytokines are small secreted proteins that play an essential role in host defense, inflammation, the development of the immune system and in immune responses.

Cytokines exert their effects by binding specific receptors on the membrane of target cells.

The elucidation of cytokine receptor/ligand pairs has furthered our understanding of the mechanisms through which cytokines regulate the development of immune responses

(1). Cytokine genes likely arose through gene duplication from ancient precursors (2), and therefore exhibit common structural features in their sequences that reflect their common evolutionary origins. This characteristic has facilitated the identification of all the members that belong to a particular cytokine superfamily (3). It follows that, if any cytokines remain to be discovered, they are not likely to be members of any known cytokine family.

We sought to identify novel immune system-associated genes encoding either secreted or transmembrane proteins. To this end, we analyzed a comprehensive database of human gene expression (BIGE) that includes 105 different human tissues or cells (4, 5).

These analyses led to the identification of 35 poorly characterized genes predicted to encode either transmembrane or secreted proteins expressed by either leukocytes or immune system-associated organs. We have reported three of these novel genes including Isthmin 1, TSPAN33 and Meteorin-like (6-8). Here, we report an uncharacterized gene (C17orf99: Chromosome 17 open reading frame 99), that encodes a small secreted protein (~27kDa) that is produced by B cells upon activation. These characteristics strongly suggest that C17orf99 encodes a novel B cell-expressed cytokine. We therefore predicted that C17orf99 would have effects in the immune system.

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To test this hypothesis, we obtained and analyzed a mouse with a targeted deletion of

C17orf99. Phenotypic analysis of this mouse indicates that it exhibits altered B cell numbers, low levels of IgA in the serum, mammary gland and feces, and an altered microbiome. We conclude that C17orf99 encodes a novel B cell derived cytokine.

Recently, a new member of the IL-12 family has been identified and named IL-39 (9, 10).

We therefore have named the novel cytokine encoded by C17orf99 interleukin-40 (IL-40)

(11); in this chapter, we demonstrate that IL-40 is a novel cytokine involved in the regulation of humoral immunity.

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2.2 MATERIALS AND METHODS

Cells. The human B cell line 2E2 (derived from a Burkitt’s lymphoma) has been described

(12). The human T cell line Jurkat was obtained from the ATCC (American Type Culture

Collection, Manassas, VA). The human B cell lymphoma cell lines have been described previously (16) and were a generous gift from Dr. David Fruman (University of California,

Irvine, CA).The murine cell line A20-2J has been described (13) and was a kind gift of Dr.

P. Marrack (National Jewish Health, Denver, CO). Human peripheral blood B cells were purified by flow cytometry (>95%).

B40 cell differentiation. Mouse spleen B cells were isolated using Pan B cell isolation (Stemcell Technologies, Vancouver, Canada). B cells were plated (5x106 cells/well) in

24 well plates and stimulated with anti-CD40 Ab (clone 3/23, Biolegend) and anti-IgM

(F(ab')2-Goat anti-Mouse IgM, Thermo-Fisher, MA) (5µg/mL) alone, or in presence of IL-

4, TGF-β, (both at 10ng/mL, Biolegend) or IL-21 (30 ng/mL, Biolegend) for 2 days. Cells were lysed and processed using Trizol reagent (Invitrogen, CA). qRT-PCR. Human cDNAs were obtained from Clontech (Mountain View, CA) and PBMCs from Sanguine Biosciences (Sherman Oaks, CA). RNA was isolated from human cell lines/cells or tissues using the QiagenRNeasy® kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). cDNA reaction performed using the QuantiTect®

Reverse Transcription (Qiagen). qPCR was performed using the Roche LightCycler® 480

Real-Time PCR system with probes designed to detect CD19 (B cell marker), C17orf99 and GAPDH (housekeeping gene) (Roche, Pleasanton, CA) (Table IV). The mouse gene homolog of human C17orf99 is 6030468B19.

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Table IV. qRT-PCR oligonucleotides used in this study

Mice. All animal protocols were approved by the Institutional Animal Care and Use

Committee (IACUC), of the University of California, Irvine. MRL/MpJ-Faslpr/J mice (Stock number 000485) were obtained from the Jackson Laboratory (Bar Harbor, ME). C57BL/6J

(stock number 000664) were obtained from the Jackson Laboratory. Il40-/- mice

(B6/129S5-6030468B19Rik-/-) were obtained from the Knockout mouse repository

(KOMP), (UC Davis, Davis, CA). The B6/129S5-6030468B19Rik-/- mice obtained were backcrossed to C57BL/6J (stock number 000664) at least 5 generations. Wild Type (WT) mice used as controls were littermates from het/het pairings that also yielded homozygous Il40-/- mice. Serum samples were obtained through sub-mandibular cheek bleed as described (14). Fecal samples were collected as described (15). Breast milk samples were obtained by collecting the stomach contents of neonate mice. Briefly, stomach contents were centrifuged at max speed for 15 minutes and supernatants were collected. Genotyping was done by PCR usin primers IL40 Wt Fwd (5’-GGG GGC AAA

ATT CAC CTG TT-3’), IL40 Wt Rev (5’-TAC CGG GAC TAC TGG GGA GAA-3’), IL40

KO Fwd (5’-CAG TCT GCC CTT GTC CAC TC -3’) and IL40 KO Rev (5’-GCA GCG CAT

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CGC CTT CTA TC-3’). Amplicons expected are 420 bp for Wt and 320 bp for Il40-/- mice, both for Heterozygotes.

ELISA. ELISA assays were performed according to each manufacturer’s protocols

(Ebioscience, San Diego CA). Plates were read at 450nm. For fecal IgA ELISA plates

(Sigma Aldrich, St Louis, MO) were coated with anti-IgA mAb (clone RMA-1) (BioLegend,

San Diego CA) at 2µg/ml. 5% milk in PBS-T was used as blocking buffer. Plates were washed 3 times with PBS-T after each step. AP conjugated anti-mouse IgA (Southern

Bio, Birmingham, AL) used as secondary antibodies for 1 h at room temperature and washed 3 times with PBS-T. SureBlue substrate (KPL Gaithersburg, MD) was added, reaction was stopped with 2N H2SO4. Plates were read at 630nm.

Western Blot. In order to verify that IL-40 is secreted, human IL40 cDNA was cloned from human 2E2 B cells, a Burkitt’s lymphoma model of B cell activation and differentiation(12), and inserted into pTT5 vector(16) resulting in a recombinant gene encoding a fusion protein with a C-terminal 8x Histidine (HIS) tag. HEK 293 cells were transiently transfected with the pTT5-IL40 construct, (or empty vector as a control), and day 1 and day 3 supernatants were collected, concentrated in an anti-HIS column

(GenScript), and analyzed for the presence of recombinant IL-40 protein by Western blot

(using anti-His antibody)(Bio-Rad).

Flow cytometry. Peyer’s patches were isolated from the small intestines of WT and IL40-

/- mice. Peyer’s patch B cells were stained with phycoerythrin (PE)-Labeled anti-mouse

CD45R (B220), rat mAb (RA3-6B2, eBioscience) and FITC-labeled PNA (E-Y

Laboratories, San Mateo, CA), 7AAD (BioLegend), and APC-labeled IgM (BioLegend)

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and analyzed by flow cytometry using BD FACSCalibur (BD Biosciences San Jose CA) using Flowjo data analysis software (TreeStar Inc., Ashland OR).

Bone marrow cells were isolated from femurs of C57BL/6J and IL40-/- mice. The cells were isolated by flushing cells out with a 25G needle with FACS buffer (PBS 5% HI

FBS). The cells were stained with monoclonal conjugates, as follows; PE anti-mouse

CD45 conjugate (30-F11; Biolegend, CA, USA), FITC anti-mouse CD11b (M1/70;

Biolegend), BV421 anti-mouse B220 (RA3-6B2; Biolegend), anti-mouse CD51 (RMV-7,

Biolegend) and Fixable Viability dye eFluor 760 (eBioscience) in FACS buffer for 20 minutes and sorted using a FACSaria Fusion sorter (BD Bioscience). The data were analyzed with FlowJo V10.0.8 software (TreeStar Inc.).

Bone Marrow B cell subsets. Mice of 8 weeks were used for FACS analyses of the bone marrow. Single bone marrow cell suspensions were stained using anti-mouse antibodies against B220 (RA3-6B2, eBioscience, CA, USA), CD19 (1D3, eBioscience), CD43 (S7,

BD bioscience, NJ, USA), IgM (RMM-1, Biolegend, CA, USA), IgD (IA6-2, Biolegend), cKit (2B8, eBioscience), Flk2 (A2F10, eBioscience), Ter119 (Ter119, eBioscience) and

B220 (6B2, eBioscience). The gating was done as described in (17, 18).

Splenic B cell subsets analyses. from mice of 8 weeks of age were used for analysis. Single splenocyte suspension were stained with anti-mouse antibodies

(Biolegend, CA, USA) anti CD19 (HIB19), anti-CD93 (AA4.1), anti IgD (IA6-2), anti IgM

(RMM-1), anti CD21/35 (CR2/CR1) and anti CD23 (B3B4). The gating strategy was done as described by Allman and Pillai (19).

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Class Switch Recombination Assay. This assay was performed as described in (20,

21). Briefly, B cells were purified from splenocyte suspensions using a MojoSort pan B cell isolation kit (Biolegend) following the manufacturer’s protocol. B cells were cultured at 50x103/ml in RPMI supplemented with 10% and 50 μM BME, and stimulated with

LPS (10 µg/ml) for CSR to IgG3. Additionally, B cells were stimulated with LPS (10

μg/ml) and with the following cytokines and reagents: IL-4 (5 ng/ml) for CSR to IgG1;

TGF-β (5 ng/ml) for CSR to IgG2b; IL-4 (5 ng/ml), IL-5 (5 ng/ml), anti-IgD/dextran (10 ng/ml) and TGF-β for CSR to IgA. Cells were harvested by centrifugation at 500XG, stained with antibodies to B220, IgM, IgG1, IgG2b, IgG3, IgA and CD138, and analyzed by flow cytometry as described above.

Statistical analyses. We utilized the unpaired Students T test for all experiments.

Differences with p < 0.05 were considered statistically significant and are labeled as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Error bars represent Mean plus/minus SEM.

Microbiome Analyses. DNA extracted from fecal samples was amplified by PCR of 16S rDNA (V4 region) with primers 515F and 806R modified by addition of barcodes for multiplexing, then sequenced on an Illumina MiSeq system (UC Davis HMSB Facility).

Sequences were processed and analyzed by employing the QIIME (22) pipeline v1.9.1 with default settings, except as noted. In brief: paired-end sequences were joined, quality filtered, and chimera filtered (usearch61 option, RDP gold database); operational taxonomic units (OTUs) were picked de novo (‘pick_otus’ options: enable_rev_strand_match True, otu_picking_method usearch61) at 97% similarity, employing the SILVA rRNA gene database v123 (23, 24) (align_seqs:template_fp

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core_alignment_SILVA123.fasta; ‘filter_alignment’ options: allowed_gap_frac 0.80, entropy_threshold 0.10, suppress_lane_mask_filter True); taxonomy was assigned with the RDP classifier (‘assign_taxonomy’ options: assignment_method rdp, confidence 0.8, rdp_max_memory 24000, reference_seqs_fp 97_otus_16S.fasta, id_to_taxonomy_fp consensus_taxonomy_7_levels.txt). Samples were rarefied to 10,000 reads, then alpha

(Shannon index) and beta (unweighted and weighted UniFrac) diversity were assessed via QIIME. Prism 7 software (Graphpad) was utilized for statistical analyses (Mann-

Whitney-U).

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2.3 RESULTS

C17orf99 encodes a novel cytokine

Analysis of the BIGE database led to the identification of several poorly characterized genes encoding either transmembrane or secreted proteins and expressed by cells or organs of the immune system. Further bioinformatics analyses led to the identification of an uncharacterized gene annotated as C17orf99 (Chromosome 17 open reading frame

99) that encodes a small secreted protein (~27KDa). Other than its identification as one of 472 genes reported in a large study aimed at the identification of poorly characterized or unknown human genes encoding either transmembrane or secreted proteins(25), and a previous gene survey predicting that C17orf99 encodes a small secreted protein(26), there is currently no information on C17orf99 in the literature. The BIGE database (which consists of microarray data) indicates that C17orf99 is expressed in fetal liver and bone marrow as well as activated B cells (Figure 2.1a). C17orf99 encodes a small secreted protein of 265 amino acids (including a 20 amino acid signal peptide) predicting a mature protein of ~27 kDa (Figure 2.1b). We confirmed these data in human tissues by end-point

PCR (Figure 2.1c). Since C17orf99 is currently an unannotated gene of unknown function, we performed further bioinformatics analyses and BLAST searches, specifically looking for shared synteny among different genomes. These analyses revealed that

C17orf99 is only present in mammalian genomes (Figure 2.1d). To confirm that it encodes a secreted protein, we transfected the predicted human C17orf99 open reading frame with a HIS tag into HEK293 cells. Supernatants were analyzed for the presence of recombinant C17orf99 protein using an anti-His antibody. A band between 23 and 30kDa

(consistent with a ~27kDa protein) was detected in cell cultures transfected with the

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pTT5V5H8-C17orf99 construct, (Figure 2.1e), confirming that C17orf99 encodes a secreted protein. We also confirmed the expression of C17orf99 mouse and human tissues by qPCR (Figure 2.2).

Figure 2.1. C17orf99 encodes a novel cytokine (interleukin 40). (a) C17orf99 expression in normal human tissues and immune cells from the BIGE (Body Index of Gene Expression) database. IL-40 is expressed in fetal liver, bone marrow, and peripheral blood B cells activated with anti-CD40 and IL4. (b) C17orf99 amino acid sequence showing the predicted signal peptide (underlined). (c) RT-PCR of C17orf99 showing a positive band in human fetal liver (FL) and bone marrow (BM) but not in Thymus (T) or lung (L) (which were used as controls), one experiment out of three independent experiments shown. (d) Clustal Omega analyses of the C17orf99amino acid sequence in 10 selected mammalian species. C17orf99 homologs exhibit a minimum sequence homology with human C17orf99 (at the amino acid level) of 55% and a maximum E value of 3 x e-70). (e) Western blot of supernatant from day 1 (D1) or day 3 (D3) of cultures of HEK293 cells transfected with IL-40pTT5V5H8-C17orf99-HIS vector. Supernatants were concentrated in an anti-His column before being run in western blots with anti-HIS antibody. Representative of two independent experiments; (M): molecular weight ladder.

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Figure 2.2. IL-40 expression in mouse and human. IL-40 expression is conserved in human and mice (72% homology at the amino acid level). (a) Human and mouse primary protein structure alignment. (b) qPCR expression pattern of human tissues. (c) qPCR expression pattern of mouse tissues.

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C17orf99 encodes a secreted protein produced by activated B cells.

The expression of C17orf99 in bone marrow and fetal liver suggested a hematopoietic function. We initially hypothesized that it would be expressed by stromal cells, since these cells express many hematopoietic cytokines (27). We analyzed various freshly harvested mouse bone marrow cell populations for expression of the mouse homolog of human C17orf99 (6030468B19Rik) and observed that its expression decreased rapidly following culture in vitro. By 48 h following culture the expression of C17orf99 was undetectable. We then FACS-sorted various bone marrow cell populations and performed q-PCR for C17orf99. As shown in Figure 2.3, the main source of C17orf99 expression in the bone marrow is Lin-CD45- cells (representing mostly stromal cells), while very low expression was detected in the CD45-CD45+ compartment (that comprises mostly precursor and immature lymphocytes), and no detectable expression was observed in

Lin+CD45+ cells or any cell expressing CD45 (that represent myeloid, B cells, T cell precursors and DCs).

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Figure 2.3. IL-40 is expressed by Lin-CD45- in the BM. BM cells were sorted using leukocyte marker CD45, precursor cell marker Kit and B cell precursor marker IL-7r (a) or CD45 and Lineage (Lin) markers (Gr-1, F4/80, CD11b, CD3 and CD19) (b). qRT-PCR was performed on the sorted cells measuring the expression levels of IL-40. Representative of 3 separate experiments.

The BIGE database indicates that besides bone marrow and fetal liver, activated (with anti-CD40 and IL4) human peripheral blood B cells also express C17orf99 (Figure 2.1a).

We therefore studied the expression of C17orf99 by B cells. As shown in Figure 2.4a, activation of the mouse B cell lymphoma A20-2J (13) with CD40L and IL-4 induced strong

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expression of C17orf99. Spleen B cells from Wild Type (WT) C57BL/6 mice activated with anti-CD40 and IL-4 also express C17orf99 (Figure 2.4b). We next tested several activating stimuli on these cells, including various cytokines, and observed that B cells require several stimuli for optimal expression of C17orf99. These include a B cell receptor stimulus (anti-IgM), anti-CD40, and several cytokines. Interestingly, TGF-β strongly potentiates the expression of C17orf99 by activated B cells and the strongest expression was observed when B cells were activated with aCD40+anti-IgM+IL-4+TGF-β (Figure

2.4b). Taken together, the characteristics of C17orf99 (encoding a small (~27 kDa) secreted protein, expressed upon activation by B cells) strongly suggests that C17orf99 encodes a novel B cell-derived cytokine that we have called interleukin 40 (IL-40) (11), a nomenclature we will use in this study.

Since we observed the upregulation of Il40 in response of TGF-β we wanted to know if

IL-40 is produced by Breg cells. Bregs can secrete IL-10 and TGF-β in response to CD40,

BCR crosslinking, IL-4 and IL-21 stimulation in vitro (28). We stimulated B cells from spleen in the presence or absence of IL-21 and measured the expression of IL-40 and IL-

10. Surprisingly we found that IL-21 downregulated the expression of IL-40 while TGF-β inhibited the expression of IL-10 (Figure 2.4c). Several cytokines and immunogens (such as LPS) were tested before with the only treatment able to upregulate the expression of

IL-40 is shown in Figure 2.4b. Taking these results together we propose that IL-40 is secreted by a specialized effector B cell subset that we call B40 cells.

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Figure 2.4. IL-40 production is induced upon B cell activation. (a) IL-40 production by mouse A20-2J cell line stimulated with anti-CD40 mAb and IL-4 for 24h, representative of 2 independent experiments (Mean +/-SEM) (** p < 0.01) (qRT-PCR). (b) qRT-PCR analysis of Il-40 expression in mouse spleen B cells stimulated with anti-CD40 Ab and anti-IgM (5µg/mL) alone, or in presence of IL-4, or TGF-β, (all at 10ng/mL) for 2 days, fold expression compared to resting cells, representative of three independent experiments (Mean+/-SEM)(*p<0.05, **p<0.01). (c) qRT-PCR analysis of Il-40 expression in mouse spleen B cells stimulated with anti-CD40 Ab and anti-IgM (5µg/mL) alone, or in presence of IL-4 (10ng/mL), TGF-β, (10ng/mL) or IL-21 (30 ng/mL) for 2 days. Representative of three independent experiments.

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IL-40 expression is induced upon the onset of lactation in the mammary gland.

IL-40 is 72% conserved at the amino acid level between mouse and human (Figure 2.2a) and exhibits a similar expression profile between the two species (Figures 2.2b; human and 2.2c; mouse). As shown in Figure 2.1d, C17orf99 is present only in mammalian genomes, and is expressed by activated B cells (Figure 2.5). We therefore hypothesized that it may be involved in mammalian-specific functions such as lactation. To test this hypothesis, we measured the expression of Il40 in virgin, pregnant, 1 week post-natal

(PN), and 3 week PN mammary glands of WT C57BL/6 mice. As shown in Figure 2.5a,

Il40 expression is induced upon the onset of lactation and correlates with the onset of IgA production in the mammary gland (Figure 2.5b).

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Figure 2.5. IL-40 expression is induced in the mammary gland upon the onset of lactation and the Il40-/- mouse exhibits decreased IgA levels in milk. (a) qRT-PCR analysis of Il-40 expression in WT virgin, pregnant, 1-week post-natal (PN) lactating, and 3-week PN lactating mouse mammary glands (Mean+/-SEM) (*p<0.02, **p<0.001). (b) qRT-PCR analysis of IgA expression in WT virgin, pregnant, 1 week post-natal (PN), and 3-week PN (Mean+/-SEM) (*p<0.029). (c) Total number of T or B cells in spleens from WT or Il40- /- mice (n=5 per group, (Mean+/- SEM) (*p<0.038) Shown is a representative experiment (out of three).

An Il40-/- mouse exhibits B cell abnormalities

To further explore the role of IL-40 in the immune response, we obtained a mouse with a targeted deletion of the mouse homolog of C17orf99 (6030468B19Rik), which has been described as part of a large mouse knockout database (25), and that results in complete absence of IL-40 expression. This Il40-/- mouse is viable and reproduces normally. We hypothesized that this mouse would exhibit B cell abnormalities. The Il40-/- mouse has less B cells in the spleen compared to WT (Figures 2.6a) indicating that the absence of

IL-40 affects B cell homeostasis. To obtain more information about the effects of IL-40 in

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splenic B cells we analyzed transitional and marginal zone B cells from Il40-/-mice, by flow cytometry and observed a significant reduction in the number of transitional, follicular and total marginal zone B cells (Figure 2.6b). These observations confirm that IL-40 is involved in peripheral B cell homeostasis.

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Figure 2.6. B cell populations are altered in the Il40-/- mouse. (a) Total B cells (CD19+) in the WT or Il40-/- mice. (b) B cell subpopulation analysis in the spleen. Transitional B cells (T1: CD23−CD93+IgMhighIgD−/lowCD21/35−/low; T2: CD23+CD93+IgMhighIgDhighCD21/35low; T3: CD23+CD93+IgMlowIgDhighCD21/35low), Total Follicular B cells (Follicular: CD23+CD93−CD21/35int.), Follicular 1 (Fol 1: CD23+CD93−IgMlowIgDhighCD21/35int) and Follicular 2 (Fol 2: CD23+CD93−/lowsIgMhighsIgDhighCD21/35int), Total Marginal zone B cells (MZ total: CD93−/lowIgMhighCD21/35high), Marginal zone (MZ: CD23−CD93−IgMhighIgDlowCD21/35high) and Marginal Zone Precursors (MZP: CD23+CD93−/lowIgMhighIgDhighCD21/35high) all of the cells were CD19+. Bars represent +/- SEM, n=4 per group. Representative of two independent experiments. *p=<0.05, **p=<0.002.

B cell populations are altered in the BM of Il40-/- mice

The Il40-/- mouse exhibits a deficiency of B220+ cells in the bone marrow. B220 is a B cell

lineage marker that is expressed by B cell progenitors (pre-B cells) during development

in the mouse bone marrow (29). B220+ cells account for approximately 20% of the cells

in the bone marrow of normal adult mice (30). We stained for the stromal and progenitor

cells (CD45-) and leukocytes (CD45+), which were in turn divided into the B cell

compartment (CD45+B220+), myeloid compartment (CD45+CD11b+) and T cell

progenitors (CD45+CD11b-)(31, 32). We observed a significant reduction in the number

of B220+ cells in the bone marrow of Il40-/- mice (Figures 2.7a and 2.7b) which include

precursor and mature B cells in the BM. These data suggest that IL-40 is involved in the

development of B cells. Because BM expresses IL-40 (Figure 2.1a), we tried to further

82 identify the cells that produce IL-40. We had observed that CD45- bone marrow cells

express IL-40 (Figure 2.3). The CD45+ population contains B cells precursors (Pro-B,

Pre-B cells), while the CD45- lineage negative (CD3, Ly-6G/Ly-6C, CD11b, B220 and Ter-

119) population represents mostly stromal cells. We sought to subdivide the CD45-

population by the expression of Lin markers and CD51 (αV integrin) (33). As shown in

Figure 2.7c, IL-40 is strongly expressed by CD45-Lin-CD51- population, which contains

mostly mature stromal cells (33). We also analyzed the B cell precursor populations in

the BM and found reduced numbers of pre-B cells (Figure 2.7d and Figure 2.8). Taken

together, the fact that some BM stromal cells produce IL-40, whereas there are alterations

in the B cell precursor compartment in the BM, strongly suggests that IL-40 plays a role

in B cell development.

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Figure 2.7. IL-40 is highly expressed in BM stroma and is required for normal B cell homeostasis. (a) Bone marrow staining of different cell compartments: stromal cells (CD45-), leukocytes (CD45+), the B cell compartment (CD45+B220+), myeloid compartment (CD45+CD11b+) and T cell progenitors (CD45+CD11b-). Shown is a representative plot of n=5 per group and is representative of two independent experiments. (b) Total cell count for each compartment in c. Results shown are representative of two independent experiments. (c) qPCR of sorted stromal populations in the bone marrow. Whole bone marrow expression (BM), stromal precursors (CD45-CD51+) and mature stroma (CD45-CD51-). Relative expression compared to whole bone marrow (BM). Data are representative of two independent experiments. (d) Number of pre-B cells (CD19+, B220+, IgM-, IgD-, CD43-) in BM of WT and Il40-/- mice. *p < 0.05.

Figure 2.8. Total cells and B cells are reduced in the BM of Il40-/-mouse (a) Gating strategy for analyses of B cell populations in the bone marrow. (b) Total number of cells + + and B cell populations in the bone marrow of WT and Il40-/- mice. B cells (CD19 ,B220 ), + + + + + + - - + Pre-Pro B cells (B220 , IL7R , Flk2 , Ly6D ), Pro-B (CD19 , B220 , IgM , IgD , CD43 ), + + + - + Immature B cells (CD19 , B220 , IgM , IgD ), and mature recirculating B cells (CD19 , + + + B220 , IgM , IgD ). Bars represent mean +/- SEM, n=4 per group, *p < 0.05. Representative of two independent experiments.

We have also analyzed hematopoietic stem cell populations in the bone marrow (short term hematopoietic stem cells, long term hematopoietic stem cells, common lymphoid

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progenitors, common lymphoid progenitors, B lymphocyte progenitors’ common myeloid progenitors, granulocyte-macrophage progenitors and megakaryocyte and erythrocyte progenitors) and found no significant alteration in the Il40-/- mice bone marrow compared to WT mice (Data not shown). These data suggest that IL40 does not affect hematopoiesis.

Il40-/- mice exhibit reduced levels of IgA in mucosal secretions and exhibit GALT abnormalities.

Lactation triggers the onset of IgA production in the mammary gland. Several genes have been implicated in IgA production in the mammary gland. For example, a CCR10-/- mouse has no IgA in the milk, because the CCL28/CCR10 chemokine axis mediates the recruitment of IgA plasmablasts to the lactating mammary gland (34). We therefore explored whether IgA production is affected by the absence of IL-40. To this end, we analyzed gut associated lymphoid tissue (GALT), which contains a large number of immune cells and is a preferred site of IgA responses (35), in Il40-/- or WT mice. Peyer’s

Patches (PPs) are GALTs that participate in immune surveillance of the digestive tract, and contain both germinal center B cells and IgA-secreting plasma cells (35). The Il40-/- mouse has fewer and smaller PP than WT mice (Figures 2.9a) (36, 37). Since IgA is the main immunoglobulin produced at this site (36-38), we measured total IgA switched cells

(IgA+) and found a 5-fold reduction in total IgA positive cells (Figures 2.9b, 2.9c and

2.9d). Taken together, these data indicate that IL-40 participates in the development of

IgA responses in the GALT, especially in the PP. Given these results, we next measured

IgA levels in the serum, feces or milk of WT or Il40-/- mice and observed a significant reduction in IgA levels in the Il40-/- mouse (Figures 2.9d-g). The lower levels of IgA in

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feces of the Il40-/- mouse correlate with defects observed in the Peyer’s Patches (Figures

2.9a-d). Serum levels of IgM and total IgG were unaffected (data not shown). We then hypothesized that the defects in IgA production may be due to defects in class-switch recombination (20). To test this, we performed a class switch recombination assay but did not detect differences when using cells from Il40-/- or WT mice, suggesting that this mechanism is not affected by lack of IL40. (Figure 2.10). Based on these observations, we conclude that IL-40 is involved in the development/control of humoral immune responses, especially those involving IgA production.

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Figure 2.9. Il40-/- mice exhibit defects in GALT B cell populations. (a) Total counts of Peyer’s patches (PP) from WT or Il40-/- mice (Mean+/- SEM) (n = 7) (*p<0.0102). (b) Flow cytometry analysis of germinal center B cells from PP showing lower numbers of IgA+ B cells. (c) Total number of IgA+ cells from (b) (x10-5) (****p<0.01). (d) Serum IgA levels in Il40-/- mice compared to WT controls as measured by sandwich ELISA (n=8) (Mean+/- SEM) (*p<0.0114). (e) Measurements of total IgA in fecal pellets by sandwich ELISA of WT vs Il40-/-mice (Mean+/-SEM) (n=10) (***p<0.006) are representative of 3 independent experiments, each performed with 5 mice per group. (f) Measurement of total IgA in breast milk by sandwich ELISA of WT vs Il40-/-mice, (Mean+/- SEM) (WT n = 5; Il40-/- n = 6) (****p<0.0001).

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Figure 2.10. B cells from IL-40 wt and IL-40 -/- mice were measured by in vitro stimulation and flow cytometry assays for their relative ability to undergo CSR to the indicated isotypes and plasmablast/plasma cell differentiation. B cells were stimulated for 4 days with LPS and the following cytokines inducing isotype-specific CSR: IL-4, IL-5, TGF-β and anti-IgD/dextran for IgA (a), IL-4 for CSR to IgG1 (b); TGF-β anti-IgD/dextran for CSR to IgG2b (c); LPS alone to induce IgG3 (d). B cells stimulated with LPS and IL-4 for 4 days were measured by flow cytometry for the percentage of IgG1+ CD138+ class-switched plasmablasts/plasma cells (E). Panels on the right of each set of plots indicate results from triplicate experiments.

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An Il40-/- mouse exhibits microbiome abnormalities

The gut is one of the major producers of IgA of the body (39, 40). IgA is an important regulator of commensal bacteria in the gut and controls infections in mucosal secretions

(40). We hypothesized that the low levels of IgA measured in Il40-/- mice could be affecting the composition of the intestinal microbiome. To test this hypothesis, we collected fecal samples from WT and Il40-/- mice to perform total DNA extraction and 16S sequencing.

Eubacterial diversity was reduced in the Il40-/- mouse, and both the community composition and structure were substantially different by the absence of IL-40 expression

(Figures 2.11a-b). At the phylum level (Figure 2.11c), we observed a significant reduction of Firmicutes in Il40-/- mice relative to WT, with a concomitant increase in

Bacteriodetes. Deeper analysis of the affected phyla revealed significantly altered families and genera in the Il40-/- mouse. Among the Bacteroidetes, the uncultured Bacteroidales family S24-7(41) was significantly increased (mean = WT 36.99%, Il40-/- 70.02%; **). For the Firmicutes, the family Lachnospiraceae was the most prominent among those significantly reduced (WT 45.54%, Il40-/- 11.93%; **), reflecting decreases in genera such as Blautia (WT 0.48%, Il40-/- 0.02%; **), Coprococcus 1 (WT 0.58%, Il40-/- 0.07%; **),

Roseburia (WT 2.37%, Il40-/- 0.13%; **), and UCG-001 (WT 11.08%, Il40-/- 0.22%; **).

There were also significant changes in the phylum Proteobacteria, where the genus

Desulfovibrio was not detected in three Il40-/- mice (WT 0.62%, Il40-/- 0.01%; **), while the genus Bilophila was detected in all Il40-/- mice, but not in WT mice (WT 0%, Il40-/- 0.06%;

**). These data strongly suggest that the lower levels of IgA in the gut of Il40-/- mice affect the diversity of their microbiome.

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Figure 2.11. The microbiome of Il40-/- mouse is altered. (a) Alpha diversity of fecal samples from WT or IL40-/- mice. (b) Beta diversity of WT or IL40-/- mice. (c) Relative abundances in percentage of different microbial Phyla represented in WT or IL40-/- mice. n=5 per group (*p<0.05).

Human IL-40 is expressed by activated human B cells and human B cell lymphomas.

Human peripheral blood B cells activated with anti-CD40 Ab plus IL-4 for 24 hours, showed strong up-regulation of expression of human C17orf99/Il40 (Figure 2.12a). We also tested various human non-Hodgkin B cell lymphoma lines (42) for IL-40 expression by qRT-PCR and observed that several of them (OCI-Ly1, HL-2 and Val) produce IL-40 constitutively (Figure 2.12b). The latter observation suggests that IL-40 may play a role in the pathogenesis of human lymphomas (43). Interestingly, the Il40+ lymphomas also

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express the B cell activation antigen TSPAN33 (8), indicating that they exhibit an

‘activated’ B cell phenotype (43). Taken together, these results indicate that C17orf99 encodes a novel B cell-derived cytokine (interleukin 40) that has significant effects in the immune system.

Figure 2.12. Human B cells produce IL-40. (a) qRT-PCR analysis of IL-40 expression in human resting and activated human B cells. Cells were activated for 24h with anti-CD40 Ab + IL-4, representative of three independent experiments (Mean+/-SEM) (***p<0.0005). (b) IL-40 expression pattern in several human diffuse large B-cell lymphoma cell lines (qPCR) (representative experiment out of 3 shown).

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2.3 DISCUSSION AND CONCLUSIONS

We initially sought to identify novel immune system associated genes. To this end, we analyzed a comprehensive database of human gene expression (BIGE) for genes expressed by immune system-associated organs including tonsil, lymph nodes, spleen, thymus, bone marrow and fetal liver. This analysis yielded 35 poorly characterized genes predicted to encode either transmembrane or secreted proteins, many of which are completely uncharacterized and were only annotated in the genome through their chromosomal location. For most of these, there is yet, no information linking them to the immune system. We have reported three of these genes: Isthmin 1, a large secreted protein originally described in the brain of Xenopus, but which in mammals is expressed by NK, NKT and Th17 cells (7), Tetraspanin 33 (TSPAN33), a membrane protein which represents a novel biomarker of activated B cells (8), and Meteorin-Like (METRNL/IL-

39)(44), a secreted protein related to a known neurotrophic growth factor (Meteorin) that is expressed by activated macrophages. In the present study, we focused on C17orf99, because it is predicted to encode a small secreted protein (~27 kDa) and is expressed by activated B cells, fetal liver and bone marrow. Further analyses indicate that the cellular source of mouse C17orf99 in the bone marrow is CD45- cells, which are enriched in stromal cells. This is not surprising since these cells are known to produce many cytokines

(27)). Mouse C17orf99-producing cells in the bone marrow are further enriched in the lineage-CD45-CD51- bone marrow population (Figure 2.7c). Interestingly, CD45-CD51+ cells include stromal cell precursors that support the development of bone and cartilage, while CD45-CD51- cells include mature stromal cells that support hematopoiesis and lymphopoiesis (33). These observations strongly suggest that mouse C17orf99 is

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produced by stromal cells that participate in lymphopoiesis. Along with this observation, we have observed reduced numbers of B220+ cells in the bone marrow of C17orf99-/- mice (Figure 2.7b), reflecting slightly reduced numbers of Pro and Pre-B cell precursors in the bone marrow (Figure 2.8). Taken together, these observations suggest that IL40 plays a role in B cell development in the bone marrow. However, further studies are necessary to confirm this hypothesis. Conversely, we have not detected abnormalities in hematologic precursors in C17orf99-/- mice suggesting that C17orf99 is mainly linked to

B cell development and function.

Data from the BIGE database indicate that C17orf99 is expressed by human peripheral blood B cells activated with anti-CD40 and IL-4 (Figure 2.1a). We confirmed the latter observation with the murine B cell lymphoma A20-2J (Figure 2.4a) and mouse spleen B cells (Figure 2B). The fact that C17orf99 encodes a small secreted protein (~27kDa) and that its expression is inducible in B cells upon activation, strongly suggests that C17orf99 encodes a novel cytokine. Recently, a new member of the IL-12 family has been identified and named IL-39(44, 45). We therefore have named the product of C17orf99 Interleukin

40 (IL-40) since that is the next number in the interleukin sequence (11).

Cytokines are very important molecules in the immune system. It is therefore relevant to ask why C17orf99/Il40 has not yet been identified as a cytokine. Several factors may account for this. These include: a) It is expressed by a limited number of tissues (bone marrow and fetal liver); b) It does not belong to any recognized cytokine family (structural features would have facilitated its identification) and c) Its main producers in the periphery

(B cells) are not known to be prolific cytokine producers (46). On this last point, we should note that recently, some B cell subsets have achieved notoriety precisely through their

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ability to differentiate into cytokine producers. An excellent example are the B10 regulatory cells (which produce IL-10) and are believed to be involved in autoimmunity

(47). On the other hand, C17orf99 has been recognized to encode a secreted protein

(26), and Tang et al (25) included it in a library of knockout mice that encode transmembrane or secreted proteins.

B cells require several signals for optimal expression of IL-40. One of these involves the

B cell receptor (represented by the anti-IgM stimulus), and anti-CD40 Ab, or CD40L. The second signal is represented by cytokines like IL-4, which induces B cell activation and proliferation. Interestingly, among several cytokines tested, we found that TGF-β potentiates the expression of Il40 by activated B cells (Figure 2.4b). The ability of cytokines like IL-4 and TGF-β to potentiate IL-40 production by B cells suggests that optimal IL-40 expression by B cells may require a specific differentiation mechanism (46) to achieve optimal production. These “B40” cells could develop in vivo under certain conditions. In support of this, other B cell subsets have been described that require a specific differentiation mechanism in order to become strong cytokine producers. For example, IL21 is required for B10 cells to develop in vitro (28). Similarly, an IL-40- producing subset of cells (B40 cells) may be associated with specific immune responses or human diseases.

As represented in a phylogram (Figure 2.1d), C17orf99 is only present in mammalian genomes. This observation led us to hypothesize that IL-40 may have a mammalian- specific function. Importantly, IL-40 expression is induced upon the onset of lactation, and it correlates with the expression of IgA in the milk. Further support for a role in IgA responses comes from the phenotype of an Il40-/- mouse which shows a reduction in size

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and numbers of Peyer’s patches, in IgA+ B cells, in IgA levels, as well as significant differences in its microbiota. The reduction in serum immunoglobulins is reminiscent of the phenotype of the B cell activating factor BAFF-/- mouse (48), which exhibits a severe reduction in serum immunoglobulins; however, unlike the Il40-/- mouse, the BAFF-/- mouse only exhibits a moderate reduction in IgA levels. Ablation of another TNF family member,

APRIL, also resulted in reduction of IgA serum levels (49). The inability of the Il40-/- mouse to produce normal levels of IgA suggests that the ablation of Il40 may be altering a mechanism that specifically affects this B cell subpopulation. Furthermore, the changes observed in the microbiome of the Il40-/- mouse are likely a consequence of the altered

IgA production in mucosal tissues (50). Conversely, the number of lymphocytes in the

Peyer’s Patches and other GALT is influenced by microbial colonization (51), reflecting common evolutionary mechanisms. Taken together, these observations suggest an important role for IL-40 in either the proliferation or differentiation of IgA-committed B cells.

Another interesting IL-40-linked cytokine is TGF-β, which is also involved in IgA responses (52), and, as we show in Figure 2.4b, potentiates IL-40 production by B cells.

This raised the possibility that abnormal production of TGF-β in the Il40-/- mouse could account for the lower IgA levels observed in this mouse. However, we have tested the capacity of activated T cells from Il40-/- mice to produce TGF-β and it appears normal.

Activated human peripheral blood B cells also produce IL-40 (Figure 8a). Importantly, some human B cell lymphomas can produce IL-40 constitutively (Figure 2.12b). B cell ablation therapies (i.e. anti-CD-20 mAbs) have shown significant therapeutic benefit in several autoimmune diseases including multiple sclerosis (53), and rheumatoid arthritis

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(54) . Yet, the mechanism(s) through which B cells participate in the pathology of these diseases remains undefined (46), although it is unlikely to be due to autoantibodies because the ablation of B cells (for example, using rituximab (55)) does not affect plasma cells or autoantibody titers. An emerging concept is that B cell derived cytokines (28, 47) may affect the development of autoimmunity. The latter observations suggest that IL-40, as a B cell-associated novel cytokine, is an excellent candidate to be involved in the pathology of human diseases, including autoimmune diseases (56) and lymphomas (57), where activated B40 cells may play an important role in their pathogenesis (46).

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2.4 REFERENCES

1. Lunney JK (1998) Cytokines orchestrating the immune response. Rev Sci Tech 17(1):84-94. 2. Zlotnik A & Yoshie O (2012) The chemokine superfamily revisited. Immunity 36(5):705-716. 3. Clavel G, Thiolat A, & Boissier MC (2013) Interleukin newcomers creating new numbers in rheumatology: IL-34 to IL-38. Joint Bone Spine 80(5):449-453. 4. Lee J, Hever A, Willhite D, Zlotnik A, & Hevezi P (2005) Effects of RNA degradation on gene expression analysis of human postmortem tissues. FASEB J 19(10):1356- 1358. 5. Roth RB, et al. (2006) Gene expression analyses reveal molecular relationships among 20 regions of the human CNS. Neurogenetics 7(2):67-80. 6. Ushach I, et al. (2015) METEORIN-LIKE is a cytokine associated with barrier tissues and alternatively activated macrophages. Clin Immunol 156(2):119-127. 7. Valle-Rios R, et al. (2014) Isthmin 1 Is a Secreted Protein Expressed in Skin, Mucosal Tissues, and NK, NKT, and Th17 Cells. J Interferon Cytokine Res. 8. Luu VP, et al. (2013) TSPAN33 is a novel marker of activated and malignant B cells. Clin Immunol 149(3):388-399. 9. Wang X, et al. (2016) Interleukin (IL)-39 [IL-23p19/Epstein-Barr virus-induced 3 (Ebi3)] induces differentiation/expansion of neutrophils in lupus-prone mice. Clin Exp Immunol 186(2):144-156. 10. Wang X, et al. (2016) A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice. Eur J Immunol 46(6):1343-1350. 11. Anonymous (1997) Nomenclature for secreted regulatory proteins of the immune system (interleukins): update. IUIS/WHO Standing Committee on Interleukin Designation. Bull World Health Organ 75(2):175. 12. Xu Z, et al. (2010) 14-3-3 adaptor proteins recruit AID to 5'-AGCT-3'-rich switch regions for class switch recombination. Nature structural & molecular biology 17(9):1124-1135. 13. Jones B, Tite JP, & Janeway CA, Jr. (1986) Different phenotypic variants of the mouse B cell tumor A20/2J are selected by antigen- and mitogen-triggered cytotoxicity of L3T4-positive, I-A-restricted T cell clones. J Immunol 136(1):348- 356. 14. Golde WT, Gollobin P, & Rodriguez LL (2005) A rapid, simple, and humane method for submandibular bleeding of mice using a lancet. Lab Anim (NY) 34(9):39-43. 15. Carvalho JJ, et al. (2012) Non-invasive monitoring of immunization progress in mice via IgG from feces. In Vivo 26(1):63-69. 16. Loignon M, et al. (2008) Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells. BMC biotechnology 8:65. 17. Inlay MA, et al. (2009) Ly6d marks the earliest stage of B-cell specification and identifies the branchpoint between B-cell and T-cell development. Genes Dev 23(20):2376-2381.

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18. Inlay MA, et al. (2014) Identification of multipotent progenitors that emerge prior to hematopoietic stem cells in embryonic development. Stem Cell Reports 2(4):457- 472. 19. Allman D & Pillai S (2008) Peripheral B cell subsets. Curr Opin Immunol 20(2):149- 157. 20. Pone EJ, Xu Z, White CA, Zan H, & Casali P (2012) B cell TLRs and induction of immunoglobulin class-switch DNA recombination. Front Biosci (Landmark Ed) 17:2594-2615. 21. Xu Z, Zan H, Pone EJ, Mai T, & Casali P (2012) Immunoglobulin class-switch DNA recombination: induction, targeting and beyond. Nat Rev Immunol 12(7):517-531. 22. Caporaso JG, et al. (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7(5):335-336. 23. Quast C, et al. (2013) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41(Database issue):D590-596. 24. Yilmaz P, et al. (2014) The SILVA and "All-species Living Tree Project (LTP)" taxonomic frameworks. Nucleic Acids Res 42(Database issue):D643-648. 25. Tang T, et al. (2010) A mouse knockout library for secreted and transmembrane proteins. Nat Biotechnol 28(7):749-755. 26. Clark HF, et al. (2003) The secreted protein discovery initiative (SPDI), a large- scale effort to identify novel human secreted and transmembrane proteins: a bioinformatics assessment. Genome Res 13(10):2265-2270. 27. Sison EA & Brown P (2011) The bone marrow microenvironment and leukemia: biology and therapeutic targeting. Expert Rev Hematol 4(3):271-283. 28. Yoshizaki A, et al. (2012) Regulatory B cells control T-cell autoimmunity through IL-21-dependent cognate interactions. Nature 491(7423):264-268. 29. Baumgarth N (2004) B-cell immunophenotyping. Methods Cell Biol 75:643-662. 30. Cimmino L, et al. (2015) TET1 is a tumor suppressor of hematopoietic malignancy. Nat Immunol 16(6):653-662. 31. Nagasawa T (2006) Microenvironmental niches in the bone marrow required for B-cell development. Nat Rev Immunol 6(2):107-116. 32. Kincade PW, et al. (2002) Lymphoid lineage cells in adult murine bone marrow diverge from those of other blood cells at an early, hormone-sensitive stage. Semin Immunol 14(6):385-394. 33. Chan CK, et al. (2015) Identification and specification of the mouse skeletal stem cell. Cell 160(1-2):285-298. 34. Morteau O, et al. (2008) An indispensable role for the CCR10 in IgA antibody-secreting cell accumulation. J Immunol 181(9):6309-6315. 35. Cerutti A (2008) The regulation of IgA class switching. Nature reviews. Immunology 8(6):421-434. 36. Park SR, et al. (2009) HoxC4 binds to the promoter of the cytidine deaminase AID gene to induce AID expression, class-switch DNA recombination and somatic hypermutation. Nat Immunol 10(5):540-550. 37. Tezuka H, et al. (2007) Regulation of IgA production by naturally occurring TNF/iNOS-producing dendritic cells. Nature 448(7156):929-933.

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38. Pone EJ, et al. (2012) BCR-signalling synergizes with TLR-signalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-kappaB pathway. Nat Commun 3:767. 39. Lindner C, et al. (2012) Age, microbiota, and T cells shape diverse individual IgA repertoires in the intestine. J Exp Med 209(2):365-377. 40. Macpherson AJ, McCoy KD, Johansen FE, & Brandtzaeg P (2008) The immune geography of IgA induction and function. Mucosal Immunol 1(1):11-22. 41. Ormerod KL, et al. (2016) Genomic characterization of the uncultured Bacteroidales family S24-7 inhabiting the guts of homeothermic animals. Microbiome 4(1):36. 42. Mehra S, Messner H, Minden M, & Chaganti RS (2002) Molecular cytogenetic characterization of non-Hodgkin lymphoma cell lines. Genes Cancer 33(3):225-234. 43. Alizadeh AA, et al. (2000) Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403(6769):503-511. 44. Ushach I, et al. (2015) Meteorin-Like is a cytokine associated with barrier tissues and alternatively activated macrophages. Clin Immunol 156(2):119-127. 45. Bensen JT, Dawson PA, Mychaleckyj JC, & Bowden DW (2001) Identification of a novel human cytokine gene in the interleukin gene cluster on chromosome 2q12- 14. J Interferon Cytokine Res 21(11):899-904. 46. Luu VP, Vazquez MI, & Zlotnik A (2014) B cells participate in tolerance and autoimmunity through cytokine production. Autoimmunity 47(1):1-12. 47. Bouaziz JD, Yanaba K, & Tedder TF (2008) Regulatory B cells as inhibitors of immune responses and inflammation. Immunol Rev 224:201-214. 48. Schiemann B, et al. (2001) An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway. Science 293(5537):2111-2114. 49. Castigli E, et al. (2004) Impaired IgA class switching in APRIL-deficient mice. Proc Natl Acad Sci U S A 101(11):3903-3908. 50. Mueller C & Macpherson AJ (2006) Layers of mutualism with commensal bacteria protect us from intestinal inflammation. Gut 55(2):276-284. 51. Yamanaka T, et al. (2003) Microbial colonization drives lymphocyte accumulation and differentiation in the follicle-associated epithelium of Peyer's patches. J Immunol 170(2):816-822. 52. Ingman WV & Robertson SA (2008) Mammary gland development in transforming growth factor beta1 null mutant mice: systemic and epithelial effects. Biology of reproduction 79(4):711-717. 53. Bar-Or A, et al. (2008) Rituximab in relapsing-remitting multiple sclerosis: a 72- week, open-label, phase I trial. Ann Neurol 63(3):395-400. 54. Wang D, Li Y, Liu Y, & Shi G (2014) The use of biologic therapies in the treatment of rheumatoid arthritis. Curr Pharm Biotechnol 15(6):542-548. 55. Reddy V, Dahal LN, Cragg MS, & Leandro M (2016) Optimising B-cell depletion in autoimmune disease: is obinutuzumab the answer? Drug Discov Today 21(8):1330-1338. 56. Youinou P, Taher TE, Pers JO, Mageed RA, & Renaudineau Y (2009) B lymphocyte cytokines and rheumatic autoimmune disease. Arthritis Rheum 60(7):1873-1880.

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57. Mahadevan D & Fisher RI (2011) Novel therapeutics for aggressive non-Hodgkin's lymphoma. J Clin Oncol 29(14):1876-1884.

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CHAPTER 3

IL-40 and its potential role in disease

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3.1 INTRODUCTION In Chapter 2 we observed the potential role of IL-40 in disease (Figure 2.12), we decided to further explore the role of IL-40 in disease. As discussed before, B cells are more than immunoglobulin-producing cells for the immune response. B cells also take part in the regulation of immune response, such as, antigen presentation and the regulation of cellular immunity through the secretion of cytokines (1, 2). The potential of B cells in immune response regulation was proven by the significant effects observed upon B cell depletion using the anti-CD20 antibody rituximab (2). It has been observed that differentiated plasma cells (which do not express CD20) can resist B cell depletion targeting CD20, thus the autoantibody production does not decline after treatment.

However, when targeting CD19 the specific IgM and IgG1 responses can be nullified; in both cases the immune response of T cells is affected by the depletion of B cells (3-8).

The patient’s reaction to depletion therapy cannot be predicted, since depending on the autoimmune disease, it can either aggravate or alleviate the disease (6, 9, 10). This wide effect caused by B cell depletion can be explained by the positive feedback interaction between T cells and B cells in the lymph nodes where B cells can present autoantigens to T cells which in turn “stimulate” B cells to produce specific autoantibodies and secrete pro-inflammatory cytokines (11, 12). Recent studies have shown that the ablation of IL-

6-producing B cells ameliorate the disease in experimental encephalomyelitis (EAE), a murine model of multiple sclerosis (MS)(11). Barr and colleagues found that B cells are a major source of IL-6 in mice and that IL-6-producing B cell supernatant have an enhanced proliferative effect on T cells in vitro (11). The accumulating evidence over the last years suggests that B cells are more than an immunoglobulin-producing machine. The capacity of B cells to participate in antigen presentation, cytokine production, as well as regulatory

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functions (2), strongly suggests that they represent important targets of research for the control and diagnosis of autoimmune disease.

The ability of B cells to produce cytokines has been known since the early 1970’s. B cells can secrete proinflammatory cytokines, growth factors, and immunosuppressive cytokines at similar levels of other cytokine-producing cells (1, 13). Cytokines produced by B cells are important, especially to regulate the interaction between B cells and regulatory T cells (Tregs), in some cases, they act as direct activators of T cells or as indirect activators through macrophages. In addition, they can regulate macrophage activity during pathogenesis (14, 15). The role of Tregs in the regulation of immune system is essential, nevertheless the inability to produce Tregs in vitro with full regulating activity in vivo (16) makes the study of B cell/-Treg interactions an important case study to explain how B cells-cytokine production and repertoire help in the development of pathogenic T cells and Tregs in autoimmune pathogenesis. This notion is further supported by the multiple reports of altered cytokine patterns in many autoimmune diseases (1).

In 1974 an immune suppressive role for B cells was first identified (17). Almost 30 years later the mechanism of action of regulatory B cells (Bregs) was discovered to be the ability to produce IL-10 in a murine model of chronic intestinal inflammation (18). IL-10 is a known regulatory cytokine first discovered in populations of T helper cells (Th) with the ability to suppress the production of proinflammatory cytokines, such as, , IL-2, and tumor necrosis factor (19) and its ability to co-stimulate the proliferation of thymocytes (20). In fact, it was originally recognized that it was a product of certain B cell lymphomas (21, 22).It is better known for its ability to inhibit the production of certain

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cytokines, especially IFN, although its mechanism of action was mapped to its ability to inhibit macrophages (20).

Nevertheless, we now know that IL-10 exerts its activity not only in the adaptive response, but in the innate immune system as well. The mechanism of action of IL-10 has been extensively reviewed in the context of autoimmune disease and is considered a master regulator of autoimmune response (23-26). IL-10 is capable of reducing T cell activation through the downregulation of MHC-II and co-stimulatory molecules, as well as suppressing antigen presentation by dendritic cells further reducing the activation of T cells and B cells (24). In the case of MS B cells, they are known to amplify the pathogenic autoimmune response, producing high levels of pro-inflammatory cytokines while producing low levels IL-10 (27). Interestingly, this reduced level of IL-10 is shared by other autoimmune diseases such as colitis and arthritis (18, 28). It has also been found that during relapses in patients with MS, triggered by infection through the activation of TLRs, the B cell populations present an abnormal cytokine response characterized by increased lymphotoxin to IL-10 ratios which can be reverted by B cell depletion (29).

B cells also play an important role in the pathogenesis of RA, as indicated by the rate of success of B cell depletion treatment in RA patients (12). One of the mayor pathogenic agents of RA, the receptor activator of nuclear factor B ligand (RANKL), is highly expressed by B cells in patients with RA (30). RANKL expression is induced by proinflammatory cytokines (TNF, IL-6, IL-1) and the expression of RANKL drives bone destruction in RA (30, 31). In addition to RANKL, B cells in the synovial fluid of RA patients have been found to produce IL-6 and (LTβ) which haven show to exacerbate inflammation of the joint tissue(32, 33).

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Atherosclerosis is a disease where autoimmunity plays a role in the establishment of the disease. Early chronic inflammation sets the stage for atherosclerosis development and both the adaptive and the innate immune response play a role in its pathogenesis (34). B cells are known to infiltrate to the atherosclerotic lesions and form follicle-like structures within the lesion, comprised of populations of follicular dendritic cells, macrophages and

CD3+ T cells (35). As pointed by Hamze and collaborators (2013), B cells can have contradictory roles in atherosclerosis. It has been observed that the treatment of atherosclerosis prone mice (apoE-/- model) by B cell transfer has a protective effect (36).

However, it has been found that while ablation of CD20+ B cells ameliorates the disease progression in mice, the selective transfer of B2 B cells exacerbates the disease by

>300% (37). These data hints at the possible regulatory role and that B cells are carrying and effector function regardless of antibody production. Indeed, it was later reported that mature B2 B cells express proinflammatory cytokines (IL-6, GM-CSF, and TNF) and can induce polarization of other effector immune cells (1).

B cells are also involved in the development of Diabetes, a metabolic disease. Obesity- provoked glucose intolerance and insulin resistance are characterized by the presence of proinflammatory T cells and macrophages in visceral adipose tissue (VAT). B cells are known to infiltrate in diet-induced obese (DIO) mice with insulin resistance, but mice lacking B cells are protected against insulin resistance (38). Winer et al showed that those

VAT infiltrating B cells were producing elevated quantities of IgG2 antibodies and promoting pro-inflammatory cytokine production by T cells. However, it has also been found that patients with T2D produce lower levels of IL-10 and higher levels of CXCL8.

These patients also present a slightly elevated presence of TLR in the membrane of B

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cells, which could be one of the causes of abnormal levels of cytokines (39). In the case of TD1, B cells support the proliferation and survival of pathogenic CD8+ T cells and in the absence of B cells, the cytotoxic T cells were found apoptotic and unable to survive

(40).

During infection B cells can respond to PAMPs and release cytokines and antibodies to combat infection. B cells express different TLRs and can be upregulated in response to

BCR crosslinking (41, 42). Although expression levels and functions vary between mouse and humans, both human and murine B cells engaging them lead to CSR, proliferation, migration and differentiation (41, 43). This process is especially important in mucosal tissues since it is constantly colonized by microorganisms. It has been found that mucosal

B cells in contact with Polyinosinic:polycytidylic acid (Poly I:C), a synthetic analog to dsRNA, can induce CSR to IgA in the presence of B cell-activating factor of the TNF family (BAFF)(44). B cells arrive to the gut and receive cues from the environment in mucosal-associated lymphoid mucosal tissue (MALT) which includes PPs and mesenteric lymph nodes (MLNs). In mucosal tissues IgA is the most secreted class of immunoglobulin (45). IgA secretion does not lead to inflammation and contributes to the homeostasis of the mucosal epithelium, with a pivotal role in gut homeostasis (46). There are two types of IgA secreted in the gut, high affinity and low affinity IgA. The high affinity is induced by pathogen’s PAMPs and toxins and is T cell help dependent (carried out in

GALTs); whereas low affinity IgA is induced by commensal pathogens is produced independent of T cell help from B cells in the intestinal lumen (47). Low affinity IgA is important in the structuration and control of the commensal microbiota, nevertheless both

Low and High affinity IgA have been shown to have small roles in both aspects of mucosal

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defense and homeostasis (46). Recently it was reported by the Flavell lab that colitogenic bacteria is highly coated by IgA in patients with IBD (48). The IgA coating the colitogenic bacteria is believed to be high affinity IgA. This interplay between the control infection and the control of commensal bacteria remarks the complex roles that B cells can play in pathogenesis.

Understanding the role B cells in the pathogenesis of a given disease is a precondition for the development of effective treatments. Since the depletion of B cells can often have contradictory outcomes it is important to study how B cells are contributing to pathogenesis. Is it through the production of autoantibodies or through an effector pathway mediated by cytokine production? IL-40 could influence disease, through any of these mechanisms. Therefore, we decided to test the influence of IL-40 using the Il40-/- mouse as a tool. We sought to test Il40-/- mice in murine models of disease where B cells have significant role; microbiome-dependent Salmonella gut infection (IgA can also play a role), EAE for MS-like disease and mutant (Faslpr/lpr) mice which develop autoimmune lymphoproliferative disease and are widely considered an SLE-like model.

3.2 MATERIALS AND METHODS

Bacterial Strains and Growth Conditions. IR715 is a fully virulent, nalidixic acid resistant derivative of Salmonella enterica serovar Typhimurium wild-type isolate ATCC

14028. ΔaroA is a mutant strain of S. enterica serovar Typhimurium lacking aroA protein involved in the synthesis of aromatic amino acids. The lack of aroA renders S. enterica

Thyphimurium ΔaroA strain highly attenuated (49). All strains were grown aerobically at

37°C in Luria-Bertani broth supplemented with nalidixic acid.

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Mouse oral immunization with Salmonella. Il40-/- mice or WT littermates were immunized by oral inoculation with 1x109 Colony Forming Units (CFUs) of S. enterica serovar Typhimurium ΔaroA. Mice were boosted 7 days later with 1x109 CFUs of the same Salmonella strain.

Challenge with full virulent Salmonella. Il40-/- mice or WT littermates were challenged with the full virulent S. enterica serovar Typhimurium strain IR715 15 days post immunization. To do so, mice were treated with streptomycin (1g per kg of mouse weight in sterile water) intragastrically 1 day prior to infection and the following day mice were infected orally with full virulent S. Typhimurium (1× 109 cfu/mouse). Fecal pellets were collected at 24, 48 and 72 hours post-infection to monitor bacterial burden. Mice were euthanized 96 hours after challenge, and spleen, mesenteric lymph nodes, terminal ileum, colon contents and fecal pellets were collected to determine bacterial burden.

Serial 10-fold dilutions were plated for enumerating bacterial CFUs on agar plates containing nalidixic acid.

Measurement of total IgA in feces. Fresh fecal samples were collected before and after immunization, and 96 hours post challenge. Fecal pellets were weighed and resuspended in 1 mL of sterile PBS containing a protease cocktail inhibitor (Roche) and incubated at room temperature shaking for 30 minutes. Samples were centrifuged at 7,200x g for 10 minutes and the supernatants were collected to measure total IgA using an ELISA kit (Bio-Legend). Data was normalized using the weight of the sample.

Induction of EAE and clinical scoring. Age-matched (8 weeks) male C57BL/6 mice and

Il40-/- were immunized by subcutaneous injections with 100l of emulsion containing 100g pf

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myelin oligodendrocyte glycoprotein MOG35-55 (MEVGWYRSPFSRVVHLYRNGK-COOH;

Thermo Fisher) in PBS, with complete Freund’s adjuvant containing 200g Mycobacterium tuberculosis H37Ra (DIFCO Laboratories). Clinical evaluation was performed every 2 days double-blind and based on the following scoring system; 0, asymptomatic; 0.5, ruffled fur; 1, flaccid tail; 2, hind limb paresis; 2.5, partial hind limb paralysis; 3, hind limb paralysis; 4, hind limb and forelimb paralysis; 5, moribund. Mice were sacrificed via inhalation of a lethal dose of isoflurane and cardiac perfusion with PBS was performed at defined time points post-transplant for tissue harvesting and analysis.

Il40-/-Faslpr/lpr mouse generation. C57B/6J Faslpr/lpr males (carrying both alleles of Fas receptor lymphoproliferation=lpr autosomal recessive mutant) were purchased from

Jackson Laboratory (Bar Harbor, Maine, USA) and crossbred with female Il40-/- mice

(bred at our UCI’s mouse facilities). The double heterozygous F1 generation (Il40-

/+Faslpr/wt) were crossbred to obtain Il40-/-Faslpr/lpr mice. Mice were validated for Faslpr/lpr by genotyping using standard multiplex primer PCR with common primer oIMR1678 (5’ GTA

AAT AAT TGT GCT TCG TCA G 3’), mutant primer oIMR1679 (5’ TAG AAA GGT GCA

CGG GTG TG 3’) and WT primer oIMR1680 (5’ CAA ATC TAG GCA TTA ACA GTG 3’).

The amplicons were analyzed by electrophoresis. Mutant amplicon size is 217

(bp); Heterozygote is 179 and 217 bp and WT is 179 bp. Il40-/- mutation was genotyped as described in chapter 2.

3.3 RESULTS

IL-40 plays a role in the modulation of commensal induced IgA in the gut

The Il40-/- mice exhibit a shift in the taxonomical composition of the gut microbiota (Figure

2.11) and reduction of IgA production in the intestinal mucosa under homeostatic

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conditions (Figure 2.9). Both, the microbiota and IgA confer protection to the host against enteric pathogens (50), therefore we tested whether IL-40 plays a role in the host defense using the enteric pathogen Salmonella.

In homeostatic conditions, the gut microbiota provides effective colonization resistance to

Salmonella (50). We infected WT mice and Il40-/- littermates with Salmonella enterica serovar Typhimurium infection and mice were euthanized at 96 hours post-infection to determine the bacterial burden in the feces, cecum content, mesenteric lymph nodes and spleen. We did not find any difference in the bacterial burden between WT and Il40-/- mice

(Figure 3.1 white dots). These data indicate that the changes of the gut microbiota in absence of IL-40 do not have an impact in colonization resistance during the acute infection with Salmonella. Because Il40-/- mice also have a defect on IgA production in the intestinal mucosa, we evaluated whether the absence of IL-40 might have an impact on the induction of a secondary immune response against Salmonella. To this end we immunized WT and Il40-/- mice with an avirulent Salmonella strain (Salmonella ΔaroA) and 15 post-immunization we challenged the mice with the full virulent Salmonella strain

IR715. Immunized WT mice was protected against the challenge with full virulent

Salmonella since bacterial burden was reduced in the gut, MLN and spleen. We found that immunized Il40-/- mice was also protected after challenge with full virulent Salmonella, we observed similar bacterial burden in WT and IL-40 deficient mice after immunization

(Figure 3.1, black dots). These findings suggest that IL-40 does not participate in the induction of an adaptative immune response against the enteric pathogen Salmonella.

However, when we measured the levels of IgA in the gut, during the primary infection and after the challenge with full virulent Salmonella in immunized mice, we found that IgA

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levels are restored in IL-40 deficient mice upon acute infection (Figure 3.2). Remarkably,

IgA levels are similar in WT and Il40-/- mice when were immunized and challenged with full virulent Salmonella (Figure 3.2).; this finding correlates with what we observe in terms of bacterial burden. These results suggest that IL-40 may play a role in the regulation of commensal induced IgA (low affinity IgA) as opposed to pathogen induced IgA (high affinity IgA) secreted in the gut under pathogenic bacterial attacks.

Figure 3.1. Bacterial burden of Salmonella after challenge. (a) Bacterial burden in WT and Il41-/- measured in fecal samples (FS) at 72 hours (h) post infection in logarithmic colony forming units (Log CFU) and normalized to fecal sample weight. Representative of two separate experiments. Bars represent mean, *p<0.05 and **p<0.01. (b) Bacterial burden in WT or Il40-/- spleen, mesenteric lymph nodes (MLN) and cecum content (CC). Representative of two separate experiments. Bars represent mean, **p<0.001. Each dot represents an individual mouse.

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Figure 3.2. IgA normalizes after Salmonella challenge in Il40-/- mice. IgA levels were measured before and after immunization and/or challenge by ELISA. Representative of two separate experiments. Each dot represents an individual mouse. *p<0.05 and, **p<0.01, ***p<0.001.

IL-40 in EAE

Considering the role of B cells in MS and knowing that Il40-/- mice have reduced numbers of circulating mature B cells, we decided to test if IL-40 is important for the amelioration or exacerbation of EAE pathology. To test this, we immunized WT and Il40-/- mice with

MOG35-55. On our first experiment we observed a significantly delayed onset of disease but no significant differences in disease severity (not shown). Surprisingly in a second experiment we found no significative difference between in EAE score or onset of the disease (Figure 3.3a) and no significant differences in the number of cells, other than B cells, in the draining cervical lymph nodes (cLN) and spleen at the peak of the disease

(day 35) (Figure 3.3b and c). The fact that even with reduced numbers of B cells we did

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not see any significant change in the clinical scores or in the onset of disease is vexing however we did notice that none of the individuals tested surpassed score 3 in the clinical score scale and thus we obtained a low overall average score per group. Taking these data together we can conclude that IL-40 does not play a role in EAE pathogenesis in the mouse immunized with MOG35-55.

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Figure 3.3. IL-40 ablation does not play a role during EAE induced by MOG35-55 immunization. (a) Clinical scores over time in days of WT and Il40-/- mice immunized with MOG35-55. (b) Total cell numbers in cervical lymph node (cLN) 35 days post immunization. (c) Total cell numbers in spleen 35 days post immunization. CD45+ cells (CD45+), conventional DCs (cDCs), Natural Killer (NK), plasmacytoid DCs (pDCs), CD8+ T cells (CD8+) and CD4+ T cells (CD4+). Bars represent +/- SEM, n= 5 mice per group, *p<0.05.

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IL-40 role in SLE-like disease

After observing the B cell deficiencies in Il40-/- mice and the importance of B cell effector function we decided to test whether IL-40 participates in the pathogenesis of lymphoproliferative autoimmune disease. First apoptosis antigen receptor (FAS) mutations lead to a series T cell and B cell homeostasis disruptions that leads to the secretion of autoreactive antibodies, lymphoproliferation of pathogenic T cells (CD4-CD8-

CD3+ T cells or DN T cells), pathogenic B cell activation and nephritis, all present during

SLE pathogenesis (51).

To test this out we sought to cross the Il40-/- mice with a FAS lymphoproliferation mutant mouse (C57B/6J Faslpr/lpr) to produce a double mutant cross (Il40-/-Faslpr/lpr). We sought to measure hallmarks of autoimmune disease such as the presence of anti dsDNA antibodies in serum and lymphoproliferation of DN T cells in lymph nodes and spleen characteristic of Faslpr/lpr mice strains (52). Nevertheless, we did not obtain Il40-/-Faslpr/lpr mice after crossing heterozygous mice for both genes (Figure 3.4). These results strongly suggest that the disruption of both genes results in an embryonic lethal phenotype.

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Figure 3.4. Dihybrid crossbreeding of Il40-/+Faslpr/wt mice does not produce a double mutant. Punnet squares predicting allele combinations in a dihybrid Het/Het crossbreeding event (Il40-/+Faslpr/wt x Il40-/+Faslpr/wt) depicted as AaBb x AaBb. The table shows the theorical predicted frequencies and the expected probabilities of obtaining the multiple genotypes. The litter genotype and Litter % column represent the number obtained after crossbreeding.

3.3 DISCUSSION AND CONCLUSIONS

Non-typhoidal Salmonella (NTS) infections, including S. enterica serovar Typhimurium, account for a significant share of food-borne around the world (53). In the typical cases of NTS diarrhea, the pathogen begins to grow in the gut and disease symptoms manifest eight to 24h after consumption of contaminated food or water. Elimination of S.

Typhimurium from the gut lumen involves two mechanisms. The first one acts during acute disease and is mediated by inflammatory response and the second involves the

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formation of a protective IgA antibody response. The high-affinity IgA is secreted into the gut lumen, where it binds to the pathogen surface and blocks further pathogen access to the mucosal surface (54) and/or accelerates pathogen clearance from the gut lumen (50).

Protection mediated by IgA occurs in an O-antigen-specific fashion a carbohydrate epitope exposed in the surface of Salmonella (50, 55). Also, it should be mentioned that the IgA-mediated S. Typhimurium removal is enhanced by the microbiota (50). The immune response against Salmonella in humans is driven by the adaptive immune response, mainly Th1, Th17 and B cells (56). B cells are dispensable for the for primary infections with Salmonella but are indispensable for subsequent challenges (57). In fact,

B cell deficient mouse are susceptible to Salmonella infection due to impaired Th1 responses, underscoring the importance that the effector role that B cells may play in immunity against Salmonella (57). The main humoral response in the gut is represented by IgA. B cells (and plasma cells) in the gut secrete large amounts of IgA. Hybridomas engrafted in mice producing a clone of anti-O antigen IgA (Sal4) can prevent Salmonella infections in vivo (55, 58). IgA also is one the main regulators of normal microbiota in the gut which is important to control infection (59). We hypothesized that IL-40 could play a role in the during Salmonella infection due to the IgA deficiency (Figure 2.6), the disrupted microbiome (Figure 2.11) and B cell deficiency (Figure 2.9) in the Il40-/- mice, since these three factors can play a role during Salmonella infections. Interestingly, we did not see any difference in disease severity, clearance or bacterial burden (Figure 3.1), but we did observe a change in the secretion of IgA after challenge in the Il40-/- mice after challenge with virulent Salmonella which could explain why we do not see any difference in disease severity (Figure 3.2). Nevertheless, we do not know what type of IgA is being secreted

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after challenge, but we hypothesize that high affinity IgA likely represents the type increased after challenge while low affinity IgA is the one decreased constitutively in the

Il40-/- mice. This hypothesis is supported by fact that immunization did protect mice against colonization (Figure 3.1a). This model therefore suggests that IL-40 plays a role in the induction of low affinity IgA but not in high affinity.

B cells have a long history in EAE pathogenesis (60). In fact, a novel anti-CD20 monoclonal antibody has recently been approved for treatment of human multiple sclerosis (60). Autoreactive antibodies targeting myelin and axon structures are a hallmark of EAE and MS pathology. Effector B cells act as APCs to autoreactive T cells and coordinate the formation of ectopic lymphoid organs that promote pathogenesis in

EAE (23, 61, 62). Additionally, mice lacking B cells have shown to have increased variability in disease onset, clinical scores and impaired recovery in EAE (63). The generalized B cell deficiency in the Il40-/- made hypothesize for a role of IL-40 in EAE.

Notwithstanding, Il41-/- mice did not behave differently to WT mice in any of the parameters measured despite significantly reduced B cell numbers in cLN and spleen

(Figure 3.3). We believe that the immunogen selected for the induction of EAE could be a factor. MOG35-55 peptide induces EAE independently of B cell effector activity, whereas full length oligodendrocyte glycoprotein (MOG) is unable to induce pathogenesis in the absence of mature B cells (64). Unique autoantibodies in plasma of mice immunized against MOG but not MOG35-55 have been found (65). Thus, we cannot eliminate the possibility that IL-40 may be playing a role in the development of B cell responses in the presence of MOG protein. Taken these data together, we can conclude that IL-40 does not play a role in EAE pathogenesis induced by MOG35-55 peptide, but there is still the

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possibility that it may play a role when the (alternate) peptide would be used. Future studies should aim to answer this question.

Finally, we wanted to explore the role of IL-40 in Fas lymphoproliferation allele (Faslpr/lpr) mice, a murine model that recapitulates some of the SLE and autoimmune lymphoproliferative syndrome (ALPS) hallmarks (66). Faslpr/lpr mice have a mutant allele

Similar to MS the presence of autoantibodies is a hallmark of the pathogenesis in SLE and in Faslpr/lpr mice (67, 68). Fas induces apoptosis of autoreactive T cells and B cells through the binding of its ligand (FasL) (69-71). In B cell biology, expressed in

CD4+ T cells induces apoptosis of germinal center B cells undergoing selection for non- reactive or autoreactive BCRs (69, 72). Disruption of the Fas/FasL interaction enables the proliferation of autoreactive T cells, double negative T cells (CD4- CD8- CD3+ T cells;

DN T cells) and autoreactive B cells (69, 73). Due to the B cell defects present in the Il40-

/- mice and previous reports of B cell deficient mice being protected from autoimmune disease (74), we decided to cross our Il40-/- mice (C57BL/6J background) with a

C57BL/6J Faslpr/lpr mice to determine the role of IL-40 in autoimmune lymphoproliferative disease. Nevertheless, we were unable to obtain a double mutant (Il40-/-Faslpr/lpr) mice after crossing Il40-/+Faslpr with Il40-/+Faslpr/wt individuals. The chromosomic location of the

Fas allele and the Il40 allele did not interfere with the crossing ( and chromosome 11 respectively). None of the pups obtained (n=48) were double negative suggesting that the combination of mutant alleles are likely embryonic lethal. We hypothesize that the lack of Il40-/-Faslpr/lpr after heterozygous crossing could be due to an unknown role of IL-40 during hematopoiesis in fetal liver, were IL-40 is highly expressed

(Figure 2.1a) while FAS plays a role in the development of HCSs (75). We do not yet

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know what functions IL-40 could be playing in early hematopoiesis in the fetal liver, but the generalized B cell deficiency and the role of other cytokines (76) in hematopoiesis suggests that IL-40 could be important in the fetal liver. Furthermore, FAS is barely expressed by B cells in the periphery and the BM, but its expression is strongly upregulated in the presence of CD40 ligand (CD40L) (77). The balance of CD40/CD40L and Fas/FasL is necessary to shutdown T cell-dependent B cell responses; CD4+ T cell expressed CD40L inhibits apoptosis mediated by Fas/FasL as inflammation winds down expression of CD40L by CD4+ T cells diminishes inducing apoptosis on B cells highly expressing Fas (78). Interestingly, in the absence of T cell help, B cells can go into CSR and develop unchecked high affinity antibodies which could be autoreactive, the deletion of these cells is also mediated by Fas/FasL and thus Fas is an important mechanism of peripheral tolerance for B cells (79). These studies indicate the importance of Fas in the regulation of B cell lymphopoiesis, but Fas deficiencies can alter hematopoiesis since earlier stages, leading to a uncontrolled migration and proliferation of immature hematopoietic cells in the fetal liver and BM in adult mice with the lpr phenotype (80)This suggest that the IL-40 could have a major role in the control of hematopoiesis in the fetal liver and bone marrow that in combination with the defects created by the lpr phenotype make the mutations embryonic lethal. Further studies on the role of IL-40 in hematopoiesis in the fetal liver are necessary to confirm this.

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58. Amarasinghe JJ, D'Hondt RE, Waters CM, & Mantis NJ (2013) Exposure of Salmonella enterica Serovar typhimurium to a protective monoclonal IgA triggers exopolysaccharide production via a diguanylate cyclase-dependent pathway. Infect Immun 81(3):653-664. 59. Donaldson GP, et al. (2018) Gut microbiota utilize immunoglobulin A for mucosal colonization. Science 360(6390):795-800. 60. Fillatreau S, Gray D, & Anderton SM (2008) Not always the bad guys: B cells as regulators of autoimmune pathology. Nat Rev Immunol 8(5):391-397. 61. McLaughlin KA & Wucherpfennig KW (2008) B cells and autoantibodies in the pathogenesis of multiple sclerosis and related inflammatory demyelinating diseases. Advances in immunology 98:121-149. 62. Robinson AP, Harp CT, Noronha A, & Miller SD (2014) The experimental autoimmune encephalomyelitis (EAE) model of MS: utility for understanding disease pathophysiology and treatment. Handb Clin Neurol 122:173-189. 63. Wolf SD, Dittel BN, Hardardottir F, & Janeway CA, Jr. (1996) Experimental autoimmune encephalomyelitis induction in genetically B cell-deficient mice. J Exp Med 184(6):2271-2278. 64. Lyons JA, San M, Happ MP, & Cross AH (1999) B cells are critical to induction of experimental allergic encephalomyelitis by protein but not by a short encephalitogenic peptide. Eur J Immunol 29(11):3432-3439. 65. Liu G, Muili KA, Agashe VV, & Lyons JA (2012) Unique B cell responses in B cell- dependent and B cell-independent EAE. Autoimmunity 45(3):199-209. 66. Boggio E, et al. (2014) IL-17 protects T cells from apoptosis and contributes to development of ALPS-like phenotypes. Blood 123(8):1178-1186. 67. Arbuckle MR, et al. (2003) Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med 349(16):1526-1533. 68. Izui S, et al. (1984) Induction of various autoantibodies by mutant gene lpr in several strains of mice. J Immunol 133(1):227-233. 69. Hao Z, et al. (2008) Fas receptor expression in germinal-center B cells is essential for T and B lymphocyte homeostasis. Immunity 29(4):615-627. 70. Waring P & Mullbacher A (1999) Cell death induced by the Fas/Fas ligand pathway and its role in pathology. Immunol Cell Biol 77(4):312-317. 71. Butt D, et al. (2015) FAS Inactivation Releases Unconventional Germinal Center B Cells that Escape Antigen Control and Drive IgE and Autoantibody Production. Immunity 42(5):890-902. 72. Koncz G & Hueber AO (2012) The Fas/CD95 Receptor Regulates the Death of Autoreactive B Cells and the Selection of Antigen-Specific B Cells. Front Immunol 3:207. 73. Hammond DM, et al. (1993) Double-negative T cells from MRL-lpr/lpr mice mediate cytolytic activity when triggered through adhesion molecules and constitutively express perforin gene. J Exp Med 178(6):2225-2230. 74. Shlomchik MJ, Madaio MP, Ni D, Trounstein M, & Huszar D (1994) The role of B cells in lpr/lpr-induced autoimmunity. J Exp Med 180(4):1295-1306. 75. Barcena A, Muench MO, Song KS, Ohkubo T, & Harrison MR (1999) Role of CD95/Fas and its ligand in the regulation of the growth of human CD34(++)CD38(- ) fetal liver cells. Exp Hematol 27(9):1428-1439.

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CHAPTER 4

Summary and General Conclusions

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4.1 SUMMARY AND FUTURE DIRECTIONS

Discovery, mammalian functions, IgA secretion and GALT tissue homeostasis

Interleukin 40 is the latest cytokine to be discovered (1). The discovery of IL-40 followed a different approach than other recently discovered cytokines. The gene encoding IL-40 is annotated in the human genome as C17orf99 and it is mainly expressed in fetal liver, bone marrow and activated B cells. It encodes a small secreted protein (27kDa) of 265 amino acids including a 20 amino acid signal peptide. We became interested in this gene while screening the Body Index of Gene Expression (BIGE) database (2) for uncharacterized genes related to the immune system. C17orf99 was identified as a gene of interest because of its expression pattern indicated that it was expressed in certain immune system organs (fetal liver, bone marrow), and the presence of a signal peptide predicted that it encoded a small secreted protein. It is not structurally related to any other cytokine family suggesting that it has unique evolutionary history. An estimated 10% of the human genome encodes secreted proteins. This human ‘secretome’ is very important because it contains many proteins that regulate a variety of physiological processes. In the case of the immune system, cytokines are pivotal molecules that regulate the development of cells of the immune system as well as development of immune responses. Furthermore, C17orf99 had been identified as a potential gene of interest on a wide screen of uncharacterized secreted genes (3). Bioinformatics analyses revealed that C17orf99 gene orthologs are only present in mammals, leading us to hypothesize that the function of C17orf99 should be related to a mammalian-specific immune function.

That hypothesis proved correct when we measured immunoglobulin (Ig) levels in the milk of lactating Il40-/- mice. There was a significant decrease in the concentration of IgA in the

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milk of Il40-/- mice and we also found that IL-40 expression is normally induced in the mammary gland prior to the onset of IgA production. In fact, we found a general IgA deficiency in Il40-/- mice (not only in the mammary gland). Interestingly, the gut of these mice is affected by the absence of IL-40, since we found a lower number (and smaller size) of Peyer’s patches (PPs) which could be explained by a significant decrease in the number of IgA+ B cells in the PPs as a well as a dysregulated microbiota.

Salmonella infection

The immunization experiments with Salmonella shed light on the potential mechanism that IL-40 carries in the gut. The first experiment performed was in an acute model of

Salmonella infection that involves pretreatment with antibiotics to thin out the microbiome which allows for a severe gut colonization. In this model, IgA is not involved because the infection kills the mice very quickly (less than 1 week). In our experience, other knockout mice are highly susceptible, namely CCL28-/- mice (data not published), indicating that

CCL28 is important in recruiting immune cells to the gut that are required to resist

Salmonella in this model. In contrast, the Il40-/- mouse is resistant to Salmonella in this model indicating that IL-40 is not involved in recruiting the cells that mediate the resistance to Salmonella during acute infection.

However, in another infection model that involves previous immunization, infection with virulent Salmonella (R715 strain) induced an increase of IgA in the gut of Il40-/- mice rescuing the reduced IgA phenotype observed in uninfected Il40-/- mice. This is an interesting observation, which suggests that IL-40 is important in maintaining the homeostasis of IgA responses, however, during active immunization IL-40 does not seem to participate in IgA responses. This indicates that it is possible that the mechanism of

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production of homeostatic (low affinity) vs ‘immunizing’ (high affinity) IgA are different. To test this hypothesis, it is necessary to study the characteristics of IgA found in the gut of

Il40-/- mice during infection and compare it to ‘homeostatic’ IgA. To further confirm this observations, it would be interesting to challenge Il40-/- mice with other gut pathogens.

One candidate is Citrobacter rodentium, whose colonization depends on effector B cell function but not on IgA secretion (4). C. rodentia infection would help us determine two things; 1) whether the IgA secretion can be rescued by inflammation generated by another gut pathogen and 2) whether the altered GALT phenotype observed in Il40-/- mice increases the susceptibility to C. rodentia infection. Another good candidate is the protozoan Giardia muris whose colonization control is dependent on anti-Giardia IgA secretion (5). The importance of IgA in this model was observed when mice deprived of

CD4+ T cells were unable to produce IgA to neutralize G. muris and, thus clearance of infection was impaired (6). By infecting Il40-/- mice with G muris we can infer the type of

IgA being secreted, if we observe the same IgA secretion rescue observed with

Salmonella and a normal clearance of G. muris would confirm that highly specific IgA is not controlled by IL-40. The easiest and more direct approach would be to perform IgA-

SEQ on the IgA secreted after exposure to Salmonella and compare it against naturally secreted IgA of uninfected Il40-/- mice. IgA-SEQ is a technique developed by Palm and collaborators (7) to sort bacteria based on the level of IgA coating on colonic bacteria (low to high level coating) using FACS. Fecal pellets are homogenized and stained with anti-

IgA antibody and sorted by FACS. Using this technique would also allow us to sort bacteria into different groups (depending on the IgA coating level) and perform rRNA sequencing to identify the bacteria being coated. By doing this we can compare

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uninfected and infected Il40-/- bacterial populations based on their IgA coating allowing us to infer what kind of IgA is being increased after Salmonella infection.

B cell development

The IgA deficiency observed in the Il40-/- mice indicates that IL-40 is required for normal

B cell function in the periphery. Thus, we decided to explore a possible function of IL-40 in the BM, where B cells originate. We hypothesized that B cell development in the BM is impaired in Il40-/- mice. We first observed that mice have reduced numbers of mature B cells as well as of Pre-B cells in the bone marrow. Furthermore, the cells producing IL-40 in the bone marrow are stromal cells, probably a subtype involved in lymphopoiesis.

Following these results, we wanted to know whether B cells in the periphery were also affected in the Il40-/- mice. In fact, all B cell populations (transitional, follicular, and marginal zone) in the spleen were significantly reduced in Il40-/- when compared to WT mice, confirming that Il-40 plays a role in B cell maturation in the bone marrow and in the periphery. We do not know the exact mechanism that IL-40 is playing in the development of B cells, but it is remarkable that we see its effects in the BM and in the periphery. A good way to approach the role of IL-40 in this regard would be to generate chimeric mice

(8). By transplanting Il40-/- BM cells to a WT mouse we can assess if B cell derived IL-40 versus stromal derived IL-40 is important for B cell maturation in the BM and in the periphery. After 4-6 weeks post-transplant we could screen B cell populations in the BM and the spleen to determine if B cells remain affected in the periphery. If they remain affected that would mean that B cell derived IL-40 (or from another source) is needed for normal B cell maturation. A chimeric mouse would also allow us to observe whether GALT deficiencies and IgA general deficiency is dependent on B cell derived IL-40 or whether

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there are other cell types that can produce IL-40 that is required for IgA secretion. Another subset of B cell biology that remains unexplored is the development of IgA+ plasma cells, it is possible that IL-40 is also important for the maturation or proliferation of IgA secreting cells. Measuring the presence of plasma cells and LPCs in the bone marrow and in the periphery would open another area of research for IL-40

B40 cells

We found that IL-40 can be produced by spleen B cells upon activation with anti-IgM, anti-

CD40 and IL-4 but that their ability to produce IL-40 increases significantly if the B cells are polarized in vitro with TGF-β. Therefore, TGF-β is necessary to have increased expression of IL-40 by activated B cells and is also necessary for IgA class switching in naïve B cells (9). In analogy to the IL-10 producing B cells I have previously discussed, we have named this TGF-β polarized, IL-40 expressing B cells, “B40 cells”. We hypothesize that B40 cells are present in tissues where IgA response develop. This raised a very important question, namely, are B10 cells the same as B40 cells? To clarify this issue, we sought to rule out the possibility of Breg/B10 cells were also producing IL-40.

To this end, we differentiated B cells in vitro under conditions that favor B10 polarization using IL-21, the cytokine that drives B10 cell polarization and IL-10 upregulation (10). In parallel, we differentiated B cells to the B40 lineage and finally tested both B10 and B40 polarized cells for IL-10 and IL-40 production. We found that IL-21 as described induces

B10 cells as judged by their capacity to produce IL-10, while TGF induces B40 cells which lose their capacity to make IL-10 as well. These observations confirm that B40 cells are indeed a novel polarization stage of B cells that likely have important clinical implications.

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There are two B cell lineages that can be polarized into effector cells, B1 cells and B2 cells, both present in the spleen (11). B1 cells (also called innate-like B cells) are responsible for most of the secretion of natural (low affinity) IgM and IgA in a T cell independent fashion (12, 13). B1 cells are present in large numbers in the pleural and peritoneal cavities, as well as mucosal tissues but low overall in lymphoid tissues (12).

The B2 B cell lineage represents most of the B cells found on the lymphoid tissues and are responsible for T cell dependent high affinity antibody production, although the MZ B cell subset can produce low affinity IgM and IgA independently of T cells (13). Both lineages are able to turn into effector Bregs cells given the right stimuli (11). For this reason, we tested B cells from the spleen which houses a mix of different subsets of B1 and B2 cells for our polarization studies, but each B1 and B2 subset has specific functions. Thus, sorting these cells and stimulating specific subsets of B cells would tell us which B cell subsets are able to secrete IL-40 or if all of them can do it, helping us pinpoint the specific niches were IL-40 is needed for B cell development and IgA production. Other cells that can be a potential source of IL-40 are plasma cells, plasma cells are known to be cytokine producers although cytokine repertoire in plasma cells is limited, they are known to secrete cytokines that increase antibody production, such as

TGF-β and IL-10 (14, 15). Furthermore, identifying specific populations that produce IL-

40 could help us discover subsets that could express the unknown IL-40 receptor.

Finding the receptor would give important information about IL-40’s mechanism. We already have taken steps towards this goal using RNA sequencing. We stimulated human

PBMCs with recombinant human IL-40. We observed that IL-40 is able to induce expression changes in B cells, T cells and Monocytes in combination with other

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stimulatory signals (αCD40 + IL-4; αCD3 + αCD28 and LPS) or by itself (data not shown).

Using the sequencing database, we aim to identify the membrane proteins that are upregulated in response to IL-40 to pinpoint the receptor. Alternatively, once an antibody is developed we could use immune precipitation followed by mass spectrometry to identify the possible receptor. We believe that since, IL-40 is likely an autocrine cytokine, B cells are good candidates to express the receptor.

Clinical implications of IL-40

We observed that some human B cell lymphoma cell lines (OCI-Ly1) constitutively express IL-40, suggesting a potential role for IL-40 in the pathogenesis of human B cell associated diseases. Many cytokines have been implicated in the prognosis of cancer.

Elevated or decrease levels of cytokines can be markers for prognosis, for example high serum levels of IL-6 are associated with increased probability of metastasis in colorectal cancer (16) but in other cancers it correlates with better chances of survival (17). In the case of IL-40 we do not know whether IL-40 has direct role in the pathogenesis of B cell lymphomas. To study this, we propose to study the IL-40 expression in samples from

Hodgkin’s and non-Hodgkin’s lymphoma patients and analyze whether IL-40 is associated with worse prognosis of the disease.

In recent years, the importance of B cells in autoimmunity has become evident. Gene expression analyses have documented that activated B cells are present in autoimmune disease lesions like affected joint synovium in rheumatoid arthritis (18). Rituximab (an antibody against CD20, a marker of human B cells) has proven efficacious and has been approved by the FDA for treatment of several autoimmune diseases (19). Recently, another anti-CD20 antibody has been approved for multiple sclerosis (20). These

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developments point to a pivotal role of B cells in autoimmunity. Given that the effectiveness of these treatments depends on their ability to eliminate B cells and not plasma cells (since the latter do not express CD20), their mechanism of action does not depend on the elimination of autoimmune antibodies, but rather, on the elimination of effector functions directly mediated by B cells. The most likely effector function therefore would be cytokine production. From this perspective, the identification of a novel B cell associated cytokine is a very interesting development (21). Importantly, we have also identified a novel effector B cell subset, the B40 cells, that are defined by their ability to express IL-40 following polarization with TGF-β. We hypothesize that in the future, we should learn more about the potential role of B40 cells in human disease. As B40 cells become better characterized at the molecular level, other biomarkers will be identified that may serve diagnostic purposes in either human autoimmune diseases where B cells are suspected to play a role as well as B cell lymphomas. As we have reviewed, activated effector B cells are now recognized as major players in the pathogenesis of several human autoimmune diseases and we therefore predict that IL-40, as a B cell associated cytokine, has the potential to be involved in the pathogenesis of some human diseases.

We consider that gut autoimmune diseases where TGF-β is highly expressed may be likely to contain B40 cells (22). I am confident that in the next few years future studies will document associations of IL-40 with autoimmune mechanisms.

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4.2 CONCLUSIONS

This work summarizes the information we know about IL-40 to date. A previously uncharacterized gene (C17ORF99) encodes IL-40 which represents an important cytokine for B cell biology.

Certainly, our research on IL-40 has left us with many more questions than answers, but we have a new field of research.

In conclusion:

• IL-40 is a novel cytokine produced by B cells upon activation

• IL-40 is essential for efficient IgA production and normal GALT tissue development.

o IL-40 likely plays a role in the production of high affinity IgA in the gut.

• IL-40 participates in B cell ontogeny in the bone marrow and spleen and is

produced by special cells in the bone marrow stroma.

• In the periphery, IL-40 is likely produced by a B cell subset polarized by TGF-β,

that we have designated B40 cells.

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