Beendigung Der Entzündungsreaktion Durch Interleukin-9 Sezernierende

Beendigung der Entz¨undungsreaktion durch Interleukin-9 sezernierende angeborene Lymphozyten

Der Naturwissenschaftlichen Fakult¨at der Friedrich-Alexander-Universit¨at Erlangen-Nurnberg¨ zur Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von Simon Rauber Als Dissertation genehmigt

von der Naturwissenschaftlichen Fakult¨at der Friedrich-Alexander-Universit¨at Erlangen-Nurnberg¨

Tag der mundlichen¨ Prufung:¨ 02. 05. 2919

Vorsitzender des Promotionsorgans: Prof. Dr. Georg Kreimer Gutachter: Prof. Dr. Steﬀen Backert Prof. Dr. Georg Schett Resolution of inﬂammation by interleukin-9 producing innate lymphoid cells

To the Faculty of Natural Sciences of the Friedrich-Alexander-University Erlangen-Nuremberg for the obtainment of the academic degree doctor rerum naturalium (Dr. rer. nat.)

submitted by Simon Rauber Approved by the Faculty of Natural Sciences of the Friedrich-Alexander-University Erlangen-Nuremberg

Date of oral examination: 02. 05. 2019

Chairman of examination board: Prof. Dr. Georg Kreimer Referees: Prof. Dr. Steﬀen Backert Prof. Dr. Georg Schett Table of contents

1 Deutsche Kurzfassung1

2 English abstract3

3 Introduction 5 3.1 Innate lymphoid cells and the bridge between primary and adaptive immune response6 3.1.1 The discovery ...... 7 3.1.2 The classification of innate lymphoid cells and their integration into the immune system ...... 11 3.1.3 Are innate lymphoid cells only innate mirrors or fully-fledged immune cells . . 12 3.2 The interleukin-9 ...... 15 3.2.1 The cellular source of interleukin-9 ...... 15 3.2.2 The immune-modulatory capacities of interleukin-9 ...... 17 3.2.2.1 Interleukin-9 in infection, tumour and allergy ...... 18 3.2.2.2 Interleukin-9 in autoimmune diseases ...... 18 3.3 Rheumatoid arthritis ...... 19 3.3.1 Molecular and cellular pathways in rheumatoid arthritis ...... 20 3.3.1.1 Auto-antibodies ...... 20 3.3.1.2 Alterations of the synovial joint micro-architecture in early and in clinically manifested rheumatoid arthritis ...... 21 3.3.1.3 T cells in rheumatoid arthritis ...... 23 3.3.1.4 The pro-inflammatory cyotkine cascades in rheumatoid arthritis . . 23

I Table of contents

3.3.2 Current therapies ...... 24 3.3.2.1 Biological disease modifying anti-rheumatic drugs that block cytokine pathways ...... 25 3.3.2.2 Other pathways targeted by disease modifying anti-rheumatic drugs 26 3.3.2.3 Antibody dependant cell mediated cytotoxicity induced by disease modifying anti-rheumatic drugs ...... 26 3.3.2.4 Are disease modifying anti-rheumatic drugs pro-resolving? . . . . . 27 3.4 Aim and rationale of the study ...... 27

4 Material and methods 29 4.1 Instruments, equipment, reagents, kits, buﬀers and media ...... 29 4.1.1 Auxiliary equipments ...... 30 4.1.2 Inorganic and organic chemicals ...... 31 4.1.3 Kits and other reagents ...... 33 4.1.4 Buﬀers and media for cell culture and lymphocyte isolations ...... 33 4.2 Software ...... 34 4.3 Patients ...... 34 4.3.1 Ethical statement ...... 35 4.3.2 Blood donors ...... 35 4.3.3 Donors of synovial biopsies ...... 35 4.4 Animals ...... 36 4.4.1 Genotypting ...... 36 4.4.2 Ethical statement ...... 38 4.4.3 Animal models ...... 38 4.4.3.1 Antigen induced arthritis ...... 38 4.4.3.2 Serum transfer induced arthritis ...... 39 4.4.3.3 Gouty arthritis ...... 40 4.4.3.4 Hydrodynamic gene transfer ...... 40

II Table of contents

4.5 Flow cytometry and cell sorting ...... 41 4.5.1 Sample preparation ...... 42 4.5.1.1 Secondary lymphoid organs ...... 42 4.5.1.2 Joints ...... 43 4.5.1.3 Blood ...... 44 4.5.2 Antibody staining ...... 44 4.6 T cell polarisation ...... 46 4.7 Restimulation of sorted ILC2s and T cells ...... 47 4.7.1 Culture of type 2 innate lymphoid cells ...... 47 4.7.2 Restimulation of sorted regulatory T cells ...... 47 4.7.3 Suppression assay ...... 49 4.8 Enzyme-linked immunosorbent assays ...... 50 4.9 Histology and microscopy ...... 50 4.9.1 Sample preparation ...... 50 4.9.2 Thin section cutting ...... 50 4.9.3 Deparaﬃnization and rehydration ...... 51 4.9.4 Conventional histological stainings ...... 51 4.9.4.1 Haematoxylin and eosin staining ...... 51 4.9.4.2 Safranin O staining ...... 51 4.9.4.3 Detection of tartrate-resistant acetic phosphatase ...... 52 4.9.5 Immunoﬂuorescence staining ...... 52 4.9.5.1 Epitope retrieval ...... 52 4.9.5.2 Antibody staining ...... 52 4.10 X-ray microtomography ...... 53 4.11 Semi-quantitative real-time expression analysis ...... 54 4.11.1 Primers ...... 54 4.12 Statistical analysis ...... 54

5 Results 56 5.1 Interleukin-9 deﬁciency leads to chronic inﬂammation in antigen induced arthritis . . 56

III Table of contents

5.2 Interleukin-9 limits chronic inflammation in the serum transfer induced arthritis . . . 61 5.3 Alterations of the cytokine signature in chronic arthritis ...... 62 5.4 Regulatory T cells are impaired in interleukin-9 deficient mice ...... 66 5.5 Type 2 innate lymphoid cells are the main producers of interleukin-9 in the arthritic joint ...... 69 5.6 Type 2 innate lymphoid cells significantly rely on interleukin-9 ...... 71 5.7 Type 2 innate lymphoid cells reinforce regulatory T cells ...... 74 5.8 Innate lymphoid cells as a potential diagnostic biomarker in human rheumatoid arthritis 77

6 Discussion 82 6.1 Skewing the innate compartment by interleukin-9 to a pro-resolving milieu in arthritis 82 6.2 Mast cell - regulatory T cell cooperation during inﬂammation control ...... 85 6.3 The balance of type 2 and type 3 innate lymphoid cells in arthritis ...... 87 6.4 Regulatory T cells in rheumatoid arthritis ...... 88 6.5 Co-stimulation of regulatory T cells and the interaction with type 2 innate lymphoid cells ...... 89

7 Conclusion and outlook 92

8 References 93

9 Appendix 122 9.1 List of acronyms ...... I 9.2 List of ﬁgures ...... V 9.3 List of tables ...... VII 9.4 Acknowledgments ...... VIII

IV 1 Deutsche Kurzfassung

Die Aktivierung der körpereigenen Selbstverteidigungsmechanismen, auch Entzundung¨ genannt, ist ein streng kontrollierter physiologischer Prozess. Eine aus der Balance geratene Entzundungsreaktion¨ kann zu einer unkontrollierten Attacke gegen den eigenen Körper fuhren.¨ Man spricht dann von einer sogenannten Autoimmunerkrankung. Autoimmunerkrankungen verlaufen zumeist chronisch und durch die persistierende Entzundungsreaktion¨ wird mit der Zeit das betroffene Gewebe zerstört. Dadurch wird dessen physiologische Funktionsfähigkeit beeinträchtigt und geht letztlich verloren. Rheumatoide Arthritis ist eine prototypische chronische Autoimmunerkrankung des Menschen, die etwa 1 % der weltweiten Bevölkerung betrifft. Es ist daher nicht nur von größter klinischer Bedeutung, sondern auch im sozio-ökonomischem Interesse, die Mechanismen, die dieser Erkrankung zugrunde liegen, zu verstehen. Ziel ist es daher, das Wissen um die Krankheitsmechanismen zu vertiefen und ein besseres Verständnis dafur¨ zu entwickeln, warum die Entzundung¨ persistiert und wie sie beendet werden kann. Dies erlaubte einerseits die Entwicklung neuer diagnostischer Biomarker, andererseits wäre auch die Entwicklung komplett neuer Therapieansätze möglich. In dieser Studie wurde dargelegt, wie das Zytokin Interleukin-9 (IL-9) durch die Aktivierung von Typ 2 angeborenen Lymphozyten (engl. type 2 innate lymphoid cells, ILC2s) die Beendigung einer chronischen Entzundungsreaktion¨ einleiten kann. IL-9 agierte dabei als ein autokriner Faktor, der sowohl von ILC2s produziert wurde, als auch proliferativ und aktivierend auf diese wirkte. Aktivierte ILC2s exprimierten Rezeptorliganden, die wiederum regulatorische T Zellen aktivieren konnten. Diese Liganden (-L) waren ICOS-L und GITR-L (engl. fur¨ inducible T cell co-stimulator und glucocorticoid-induced tumour necrosis factor-related). Regulatorische T Zellen wurden also durch die ILC2-vermittelte Aktivierung in die Lage versetzt, die Beendigung der persistierenden

Entzundungsreaktion¨ einzuleiten. IL-9 konnte somit als zentraler Schalter in der Entzundungskontrolle¨ etabliert werden. Im Maus-

1 modell konnte gezeigt werden, dass das Fehlen dieses Zytokins zu einer chronisch persistierenden

Arthritis fuhrte.¨ Umgekehrt konnte der Gentransfer von Il9, als therapeutischer Ansatz, die Been- digung der Entzundungsreaktion¨ einleiten. Im Menschen reicherten sich IL-9 sezernierende ILC2s besonders in den Gelenken derjenigen Patienten an, die sich in einer Phase klinisch unauffälliger rheumatoiden Arthritis befanden, also in sogenannter Remission waren. Da die Anzahl der ILC2s im Blut mit der Aktivität der Erkrankung korrelierte, könnte die Anzahl der zirkulierenden ILC2s zukunftig¨ als diagnostischer Biomarker genutzt werden. Schlussendlich ist es nicht nur fur¨ Autoimmunerkrankungen, sondern auch bei chronischen Infek- tionserkrankungen von zentraler Bedeutung die Mechanismen der chronischen Entzundungsreaktion¨ zu entschlusseln¨ und Wege zu finden, diese zu beenden. Aktuell zielen anti-rheumatische Therapien lediglich darauf ab, die entzundungsf¨ ördernden Signalwege zu blockieren. Die Aktivierung von ILC2s durch IL-9 fördert im Gegensatz dazu pro-aktiv die Entzundungskontrolle¨ und stellt somit ein neues Therapiekonzept dar.

2 2 English abstract

Inflammation is the tightly controlled physiological process of activation of the body’s self-defence mechanisms. Dysbalanced inflammation leads to a misdirected self-defence and manifests as autoimmune disease, marked by a chronic inflammation and the destruction of the normal tissue physiology. This ultimately leads to the loss of the tissue’s functionality. Rheumatoid arthritis is one of the most chronic forms of inflammatory disease in humans. It affects roughly 1 % of the population world wide. Understanding the underlying mechanisms of chronic inflammation in rheumatoid arthritis is of pivotal clinical and socio-economic interest. Novel therapies and diagnostic biomarkers therefore are desirable. In this study, it was shown that interleukin-9 (IL-9) induced the resolution of inflammation by the activation and expansion of type 2 innate lymphoid cells (ILC2s). IL-9 was found to be an autocrine factor produced by ILC2s. The induction and activation of ILC2s by IL-9 positively affected the capacity of regulatory T cells. IL-9 up-regulated the expression of inducible T cell co-stimulator ligand (ICOS-L) and glucocorticoid-induced tumour necrosis factor-related ligand (GITR-L) on ILC2s. Engagement of the respective receptors on regulatory T cells promoted their activity and efficiently broke down the persistent inflammation. The data presented in this study highlight IL-9 as a checkpoint of inflammation control. In the murine system, the deficiency of IL-9 led to a persisting inflammatory arthritis, which was marked by the failure to resolve. Conversely, gene transfer therapy with Il9 enhanced the resolution of arthritis. In humans, ILC2s expressing IL-9 were specifically enriched in the synovial joint tissue of patients in a stable condition of disease inactivity, the so-called remission. The numbers of circulating ILC2s in the peripheral blood were predictive for the disease activity, as patients in remission displayed a particularly higher number of circulating ILC2s as compared to patients with a high disease activity. This highlighted the potential of ILC2s to serve as a novel biomarker in rheumatoid arthritis.

3 In conclusion, gaining the control to induce the resolution of inflammation is not only relevant for numerous autoimmune disorders, but also for conditions of chronic infection. The current anti-rheumatic therapies exclusively suppress the activation pathways of inflammation. IL-9 driven activation of ILC2s and the subsequent boost of regulatory T cells hence describes a novel therapeutic concept, which fosters the activation of the resolution inducing pathways, rather than suppressing the pro-inflammatory cascades.

4 3 Introduction

Research on innate lymphoid cells (ILCs) is an emerging field in immunology. An inquiry on PubMed, the web search engine of the MEDLINE medical literature database, revealed a continuously increasing number of classical articles with one of the following terms, innate lymphoid cell(s), ILC, ILC1, ILC2 or ILC3, in the title or abstract, starting from 2010 (Figure 1). Of note, ILCs have been discovered much earlier, but it was only in 2013, when the research community came to an agreement for a uniformed nomenclature (Spits, Artis et al. 2013). Taking into account the constantly growing knowledge base, the nomenclature was updated recently (Vivier, Artis et al. 2018). According to the current classification, ILCs are defined as lymphoid cells lacking adaptive recombination-activating gene (RAG)-rearranged receptors. They are distinct from other innate immune cells such as myeloid or dendritic cells, not only because they follow a different developmental trajectory, but also because they rely on the interleukin 2 receptor gamma (IL2Rγ) family members interleukin (IL)-7 or -15 for their development and activation (Cao et al. 1995; DiSanto et al. 1995; Mebius, Rennert et al. 1997; Moro et al. 2010; Neill et al. 2010; Satoh-Takayama, Lesjean-Pottier et al. 2010; Robinette et al. 2017). They are grouped into five subtypes that are cytotoxic natural killer cells (NK cells), three types of helper-like ILCs and lymphoid tissue inducer cells (LTis). The latter are a very unique cell type triggering the development of lymph nodes (LNs) and Peyer’s patches in the embryo. In contrast to LTis, the four other ILC populations, that are NK cells and helper-like ILCs, rather functionally and phenotypically mirror the adaptive cytotoxic T cells (TCs) and helper T cells (THs), respectively. The field of ILC research is still quite novel and a lot of effort was put into understanding the role of ILCs in tissue homoeostasis and in immune reactions against infectious agents. Barrier sites are at the first line of defence, and many ILCs accumulate at these sites. They provide an immediate cytokine response, while adaptive T cell responses set on after several days (Vivier, Artis et al. 2018). Thus, while ILCs have been well studied under homoeostatic and infectious or allergic conditions,

5 3.1 Innate lymphoid cells and the bridge between primary and adaptive immune response

250

200

150

100

matching classical articles matching

0 2010 2015 year Figure 1: Publications on innate lymphoid cells over time. This bar graph shows the number of publications containing one the following terms in the title or the abstract: innate lymphoid cell(s), ILC, ILC1, ILC2 or ILC3. The articles shown were published between 2010 and 2018. Before 2010, no article matching the research criteria was published referring the abbreviation ILC to innate lymphoid cells.

autoimmune diseases are still a likewise blind spot. The early studies focused on inﬂammatory bowl disease and ﬁbrosis, while other autoimmune diseases such as multiple sclerosis and rheumatic diseases have only been studied within the last four years (Walker et al. 2013; Sonnenberg et al. 2015; Xiong et al. 2018). Therefore, the aim of the present study was to elucidate the activity of type 2 innate lymphoid cells (ILC2s) in rheumatoid arthritis (RA). Unexpectedly, ILC2s were found to be protective (Rauber et al. 2017), which has been corroborated by further studies (Omata et al. 2018; Soare et al. 2018).

3.1 Innate lymphoid cells and the bridge between primary and

adaptive immune response

To understand the importance of ILCs within the immune system, one key question that needs to be answered is, where do ILCs integrate in the classic understanding of host defence and autoimmunity. Therefore, it is necessary to understand in which historic context ILCs have been discovered and how this revolutionized the understanding of immunity.

6 3.1 Innate lymphoid cells and the bridge between primary and adaptive immune response

3.1.1 The discovery

The discovery of ILCs started already 40 years ago, which is about 80 years after the kick-off of modern immunological research (Figure 2). The first ILC population discovered, were NK cells in 1975 at the Karolinska Institute (Kiessling et al. 1975) and at the National Cancer Institute (Herberman, Nunn et al. 1975). They were found to be ”small lymphocytes” with an ”in vitro cytotoxic behavior” (Kiessling et al. 1975) against various tumour cell lines present in athymic nude mice, which are devoid of T cells and thymus-dependant B cells, but they also were found in normal spleen preparations after the depletion of T and B cells. Thus, they exerted a natural cytotoxic effect against tumour cells without the prior exposure to the target, which is required for cytolytic effector TC cells. The first reliable isolations of human NK cells (large granular lymphocytes, LGLs) from peripheral blood mononuclear cells (PBMCs) succeeded five years later in Helsinki (Timonen et al. 1980). With the first cytometers being on the market in the nineteen seventies, it furthermore was possible to identify more and more surface markers such as the cluster of differentiation (CD) molecules. The finding that among PBMCs, T cells and monocytes share some markers with NK cells, fueled the discussion about their lineage belonging (Ortaldo et al. 1981). The characteristics of NK cells, i.e. the natural cytotoxicity against some tumour cells as well as virus infected cells, stupefied researchers and fundamentally opposed the prevalent understanding of the immune system (Herberman and Ortaldo 1981). Indeed, the state of understanding of the immune system was limited in 1975. It was just the previous decade that the developmental and functional differences between T and B lymphocytes have been deciphered by Jacques Miller (Miller 1961; Miller and Mitchell 1968; Mitchell et al. 1968). Hence, the state of the art concepts of the immune system stricktely divided into a humoral and a cellular branch mediated by B and T cells, respectively (Miller 1975; Jucker 1976, pp. 574-592). Consequently, all other known immune cells, i.e. monocytes, macrophages and polymorphonucelar cells (PMNs), were considered to be accessory. Splitting the immune system in a cellular and a humoral branch is a concept that dates back to the beginnings of immunological research. The research field ‘immunology’ emerged in the ending 19th century, when Louis Pastuer (1822-1895), Robert Koch (1843-1910), Ilya Mechnikov (1845–1916) and Paul Ehrlich (1854-1915) started to systematically test and descibe host defence mechanisms and immunological reactions (reviewed in Kaufmann (2008)). The idea of a cell-mediated immunity came up from Mechnikov’s work, who

7 3.1 Innate lymphoid cells and the bridge between primary and adaptive immune response described macrophages and neutrophils (microphages) as phagocyting migratory immune cells. He was contrasted by Ehrlich, who published his observation that the host defence is mediated by antibodies and antibody-producing cells. Ehrlich thus represented a concept of humoral immunity. It is noteworthy that in 1975, macrophages were considered to be the only presenters of antigen. In fact, it also was at that time that splenic dendritic cells (DCs) have been discovered by Ralph Steinman (Steinman et al. 1973). It then took a while until the second population of ILCs was discovered. In 1992, it was reported that the first cells found in peripheral LNs in mice after birth were CD4+ and CD3- (Kelly et al. 1992). Reina Mebius and her colleagues then refined these findings in 1996 and 1997, deciphering that these cells indeed arose in the fetal liver and that they seeded the LN anlagen already from embryonic day E14.5 onwards (Mebius, Streeter et al. 1996; Mebius, Rennert et al. 1997). Subsequent studies underpinned the essential role of the LTi derived cytokines lymphotoxin and receptor of nuclear factor κ B ligand (RANKL) for the embryonic LN organogenesis and showed that LTis depend on the transcription factor retinoic acid receptor related orphan receptor γ (RORγ)t (Futterer¨ et al. 1998; Ansel et al. 2000; D. Kim et al. 2000; Sun et al. 2000). In the first trimester of human embryogenesis, a CD4+ CD3- cell population was found in the lamina propria (Spencer et al. 1986). Despite the fact that CD4 is widely expressed on different leukocyte populations in humans and not exclusively restricted to lymphoid cells as in mice, the findings on murine LTis created a room to speculate about human LTis. However, in 2009, it was clearly shown by Hergen Spits and colleagues that these cells did not correspond to human LTis (Cupedo et al. 2009). Instead, human embryonic CD4+ CD3- cells emerged to be monocytic precursors, while cells with true LTi capacities emerged to express CD127 (IL-7 receptor alpha) but lacked CD4. Up until then, the two lines of ILCs discovered were committed to execute very unique tasks. They enriched the immunological concept, but the confusion only started, when helper-like ILC were discovered. These ILCs are functionally that close to TH cells, that researchers struggeled with their evolutionary anchorage and speculated about their redundancy (Spits and Di Santo 2011; Walker et al. 2013; Tanriver et al. 2014; Vivier, Van De Pavert et al. 2016; Ebbo et al. 2017). In contrast to NK cells and LTis, the discovery of helper-like ILCs was supported by the advances in the immunological research making new genetic knock out (KO) models available. In particular, there was the discovery of the two RAG genes in David Baltimore’s laboratory in 1989/90 (Schatz et al. 1989; Oettinger

8 3.1 Innate lymphoid cells and the bridge between primary and adaptive immune response et al. 1990) and the discovery of the IL2Rγ in 1992 by the team of Kazuo Sugamura (Takeshita et al. 1992). The essential contribution of the IL2Rγ-family cytokines in lymphocyte development was corroborated by genetically deﬁcient mice described in 1995 (Cao et al. 1995; DiSanto et al. 1995).

When then the TH2 associated cytokine IL-25 was cloned for the first time (Fort et al. 2001), the third ILC population discovered emerged as ILC2s. It was found that an innate cell population responded to IL-25 in a similar manner to TH2 cells (Fort et al. 2001). They were innate, since they were still present in RAG1 deficient mice lacking both B and T cells. The authors claimed acocording to the state of science that these cells were ”nonlymphoid accessory cell[s]” (Fort et al. 2001). However, since they also were absent in mice deficient for RAG2 and IL2Rγ, it was more likely that they belonged to the lymphoid lineage (Hurst et al. 2002). Later publications from Richard Locksley’s and Andrew McKenzie’s laboratories studying infections with the parasitic helminth Nippostrongylus brasiliensis, also implied the presence of a dedicated ILC2 population (Fallon et al. 2006; Voehringer et al. 2006). However it took until 2010, when a series of publications by the two afore mentioned groups as well as the team arround Shigeo Koyasu provided definitive descriptions of ILC2s. As a consequence, these cells were named natural helper cells (Moro et al. 2010), nuocytes (Neill et al. 2010) and innate helper cells (Price et al. 2010). The excitement of having found a new cell type was only partially limited by David Artis and collegues, who showed that the ILC2s population, as identified by the markers used in the aforementioned publications, might be intermingled with some type 2 committed multipotent progenior cells (MMPtype2), which have a myelogenic potential (Saenz, Siracusa, Perrigoue et al. 2010; Saenz, Siracusa, Monticelli et al. 2013). Despite their responsiveness to IL-25, ILC2s showed a marked response to IL-2 and -33. The IL2Rγ-family member IL-7 was essential for their development (Moro et al. 2010). Based on the expression of surface markers that were similar to mouse ILC2s, human ILC2s were initially described in the mesentery (Moro et al. 2010) and in the lung (Monticelli, Sonnenberg et al. 2011). However, it was then again Hergen Spits and colleagues, who first succeeded with their isolation and thus could provide the functional proofs of human ILC2s (J. M. Mjösberg et al. 2011; J. Mjösberg et al. 2012). It appeared that the human ILC2 population is characterized by the very uniform expression of the prostaglandin receptor CRTH2 (chemoattractant receptor-homologous molecule expressed on T helper type 2 cells), which was a unique feature among all human ILC subsets (J. M. Mjösberg et al. 2011). Hence, the isolation

9 3.1 Innate lymphoid cells and the bridge between primary and adaptive immune response and characterisation of human ILC2s appeared to be more straightforwardly as compared to murine ILC2s, which express a variety of markers that are either not representing the whole population or are shared with other ILC populations. Between 2008 and 2009, several reports described an uncommon phenotype of NK cells in the intestinal lamina propria of humans and mice (Satoh-Takayama, Vosshenrich et al. 2008; Cella et al. 2009; Hughes et al. 2009; Luci et al. 2009; Sanos et al. 2009; Takatori et al. 2009). A first report by James DiSanto and colleagues showed a unique NK cell diversity in the murine gut lamina propria (Satoh-Takayama, Vosshenrich et al. 2008). These putative NK cells, despite expressing the natural cytotoxicity receptors (NCRs) NKp46 and NK1.1, were distinct from conventional NK cells as they did not express interferon gamma (IFN-γ) or perforin, but RORγ and IL-22. In a similar approach, Eric Vivier and colleagues studied bona fide NK cells in various murine tissues by their expression of NKp46 and the lack of CD3 (Luci et al. 2009). It was found that there were ”true” NK cells in tissues like the bone marrow, spleen and skin, but in the gut, the NKp46+CD3- cells rather resembled LTis. Both reports were corroborated by a report from Andreas Diefenbach’s team, who described NKp46+ cells that expressed RORγt in the murine gut as a distinct population from conventional NK cells (Sanos et al. 2009). In line with Satoh-Takayama, Vosshenrich et al. (2008), the expression of IL-22 in these cells was triggered by the commensal flora and was abrogated in germ-free mice. Takatori et al. (2009) complemented the story describing an IL-17-producing LTi-like population in the spleen of RAG2 deficient mice. Complementary to mice, Marco Colonna and colleagues studied NKp44+ and NKp44- CD3-CD56+ NK cells at human epithelial barrier sites and found that tonsillar NKp44+ NK cells were distinct from cytolytic conventional NKp44- NK cells (Cella et al. 2009). Based on the significant production of IL-22 in these cells, they called them NK22 cells. Since in all these descriptions, cells were similar to TH17 cells mediating type 3 immunity, later on, RORγ expressing NK cells were classified as type 3 innate lymphoid cells (ILC3s) (Spits, Artis et al. 2013; Vivier, Artis et al. 2018). Finally, it needed to be shown that also type 1 immunity disposes of an innate counterpart. Finding a discrete type 1 innate lymphoid cell (ILC1) population, however was hampered by the fact that NK cells, ILC3s and LTis express a similar set of markers. Hence, when Andreas Diefenbach and colleagues were fate mapping the development of ILC3s, first of all they were able to developmentally delineate ILC3s as a distinct lineage from conventional NK cells. However, using a fate mapping mouse, they

10 3.1 Innate lymphoid cells and the bridge between primary and adaptive immune response

1980/81 2009 2010 2011 Timonen/Herberman: Spits/Cupedo: Diefenbach/Powrie Spits/Mjösberg: Human Human Murine Human LGL Cells LTi Cells ILC1 ILC2 1975 2008/09 Di Santo/Colonna/Vivier/ Kiessling/Herberman: 1996 2010 2013 Diefenbach/Littman: Murine Mebius: Koyasu/McKezie/ Spits: Human and Murine Natural Killer Murine Locksley/Artis: Human ILC3 Cells LTi Cells Murine ILC1 ILC2

1883-87 Mechnikov: cellular Immunity 1961-68 Miller: 1992 T cells Sugamura: and B cells common gamma Chain 1899-1901 1989 Baltimore: Ehrlich: RAG humoral 1973 Genes Immunity Steinman: Dendritic Cells Figure 2: The discovery of innate lymphoid cells - A timeline. Given the space limitations, not all authors and reports have been credited. The author is apologiz- ing to the uncredited researchers. The graphic indicates the names of the main investigators and the years, when deﬁnitive publications on the respective cell population were published. Distances on the ribbon are not true to scale. Black dots indicate events of general importance to immunological and especially ILC research. Red dots indicate the discovery of NK cells, magenta dots that of LTis, ochre dots that of ILC3s, blue dots that of ILC2s and green dots that of ILC1s.

found to their surprise that RORγt- NKp46+ ILCs that arose from a RORγt+ precorsor were poor producers of IL-22, but produced vast amounts of IFN-γ exceeding the levels of conventional NK cells (Vonarbourg et al. 2010). Importantly, like ILC3s, IFN-γ-expressing ILCs were dependant on IL-7 signalling. They also could conﬁrm the results from Fiona Powrie’s team, who showed that the IFN-γ production by ILCs in the small intestine was IL-12 (a type 1 associated cytokine) triggered, while in the large intestine, it exclusively was induced through the type 3 associated cytokine IL-23 (Buonocore et al. 2010). Existence of a human ILC1 population was again validated by Hergen Spits’ team (Bernink et al. 2013). In contrast to the murine ILC1 subset, human ILC1s were found to be devoid of NCRs and they fundamentally diﬀered from NK cells by the expression of the IL-7 receptor.

3.1.2 The classiﬁcation of innate lymphoid cells and their integration into the immune system

Unlike TH cells, whose classiﬁcation refers to their key cytokine, e.g. TH9 cells secrete IL-9, ILCs are classiﬁed by the type of immunity that they are mediating. There are three types of cell-mediated

+ + immunity mediated by CD4 TH, CD8 TC and ILCs (Annunziato et al. 2015). Type 1 immune responses target intracellular pathogens such as viruses and intracellular bacteria and protozoa. The key eﬀector cytokine is IFN-γ produced by TH1 cells, TC1 cells, ILC1s and NK

11 3.1 Innate lymphoid cells and the bridge between primary and adaptive immune response cells.

Type 2 immunity is mediated by TH2 cells, TC2 cells and ILC2s responsible for the secretion of the key cytokines IL-5 and -13. Inﬁltration of eosinophils and class switching of B cells towards immunoglobulin (Ig)E are hallmarks of type 2 immunity. It is mostly directed against parasitic helminth infections. Therefore, IL-9-secreting cells such as ILC2s, mast cells and TH9 cells also fall in the type 2 category. Last but not least, type 3 responses raise immunity against extracellular bacteria and fungi. Key cytokines are IL-17A and -17F as well as IL-21 and -22 produced by TH17 and TH22 cells, TC17 cells, ILC3s and LTis. NK cells and LTis ﬁrtstly have been attributed to the type 1 and type 3 ILCs, respectively, because they rely on the same transcription factors and produce the same cytokines. i.e. T-box transcription factor TBX21 (T-bet) as well as IFN-γ and RORγt as well as IL-17A and -22, respectively (Spits, Artis et al. 2013). The nomenclature recently was updated and now separates helper-like ILCs that develop from the hierarchical lower ILC precursor, and NK cells as well as LTis that develop from the common innate lymphoid progenitor and the common helper innate lymphoid progenitor, respectively (Figure 3).

3.1.3 Are innate lymphoid cells only innate mirrors or fully-ﬂedged immune cells

To pick up the threat, it seems appealing to start from the evolution of immunity. Host defence is litterally intrinsic to life itself. Thus, simple procaryotic organisms already dispose of an anti-viral defence mechanism (Barrangou et al. 2007). This defence mechanism relies on small heritable but convertible deoxyribonucleic acid (DNA) sequences, so called clustered regularly interspaced short palindromic repeatss (CRISPRs). In the past years, researchers took advantage of this mechanism to generate the genetic engeneering system CRISPR/Cas-9 (Jinek et al. 2012; Doudna et al. 2014). Unorthodoxly, CRISPR/Cas-9 uniﬁes the dichotomy of innate (heritable) and adaptive (convertible) immunity. Another ancient immune mechanism that has found wide application to laboratories, is ribonucleic acid (RNA) intereference mediated gene knock-down. RNA interference was ﬁrstly described in detail in Caenorhabditis elegans, a nematode (Fire et al. 1998). It is conserved in most eucaryotes and it is most likely to be an ancient defence mechanism against genomic parasites (Cerutti et al. 2006). Other ancient immune mechanisms comprise soluble factors such as the complement

12 3.1 Innate lymphoid cells and the bridge between primary and adaptive immune response

NKP T-BET NK EOMES IL-12

TOX NFIL3 ILC1 ID2 IL-12 ETS1 IL-23 T-bet NFIL3 RUNX3 RORγT NFIL3 RORα ID2 IL-25 Bcl11B TOX GATA3 PLZF CLP CILP CHILP ILCP GATA3 ILC2 TCF-1 GFI1 IL-33 ETS1

RORγT AHR ID2

IL-23

ILC3

RORγT TOX RA ID2 LTiP LTi

Figure 3: Developmental trajectories of innate lymphoid cells and their classification. Transcription factors and inducing cytokines required for the development of the respective ILC population. The origin is the CLP (common lymphoid progenitor), that differentiates to a CILP (common innate lymphoid progenitor) or to adaptive lymphocytes. The CILP can either develop into the NKP (NK cell precursor) and then into NK cells, or into the CHILP (common helper innate lymphoid progenitor). The CHILP in turn develops into either the LTiP (lymphoid tissue inducer progenitor) and subsequently into LTis, or into the ILCP (innate lymphoid cell precursors) giving rise to helper-like ILC populations. Each stage is characterized by the requirement of specific transcription factors. The expression of the transcription factors is controlled by cytokines. Key inducing cytokines are shown. The transcription factors are: NFIL3 (Nuclear factor IL-3 induced); ID2 (Inhibitor of DNA binding 2); TOX (Thymocyte selection-associated high mobility group box protein); TCF-1 (T cell factor 1), ETS1 (avian erythroblastosis virus E26 homolog-1); GATA3 (GATA binding protein 3), PLZF (promyelocytic leukemia zinc finger); T-BET (T-box transcription factor); EOMES (Eomesodermin); RUNX3 (Runt-related transcription factor 3); RORγt (RAR- related orphan receptor gamma t); RORα (RAR-related orphan receptor alpha); Bcl11b (B cell lymphoma/leukemia 11B); GFI1 (Growth factor independent 1); AhR (Aryl hydro-carbon receptor). Figure modified from Vivier, Artis et al. (2018).

13 3.1 Innate lymphoid cells and the bridge between primary and adaptive immune response system. Soluble factors are of pivotal importance in sessile organisms that lack motile cellular defence mechanisms such as plants (Jones et al. 2006). The general public understanding of the term immune system nonetheless comprises ”the complex system of organs, cells, and molecules responsible for producing the body’s protective response to a foreign organism or substance or its own abnormal cells” as it is described in the Oxford English Dictionary (Oxford English Dictionary 2008). As such, the immune system only can be found in the subphylum Vertebrata, in which specialized lymphogenic tissues and the branch of adaptive immunity emerged (Boehm, Iwanami et al. 2012; Boehm, Hess et al. 2012). The complexity of the immune system increased with its evolution. Early vertebrates, jawless fishes (Agnatha), are devoid of B and T cells, but developed a distinct type of adaptive immune cells. These cells carry variable lymphocyte receptors (VLRs) that structurally differ from B cell receptor (BCR) and T cell receptor (TCR). The organization into primary and secondary lymphoid tissues also is a characteristic shared among all vertebrates. The most ancient lymphoid organs are the thymus and gut associated lymphoid tissues. More recent, in jawed fishes (Gnathostomata), B and T cells arose, but also an additional lymphoid organ, the spleen. Fully organized LNs are a likewise recent development in mammals and birds. Both, LNs and Peyer’s patches, require the activity of LTis for their organogenesis (Mebius, Rennert et al.

1997; Futterer¨ et al. 1998; Ansel et al. 2000; D. Kim et al. 2000; Sun et al. 2000). Lymphotoxin, which is one of the essential cytokines for the organ development of LNs, can only be found in mammals, which are evolutionary late vertebrates (Vivier, Van De Pavert et al. 2016). In conclusion, this suggests that LTis emerged far after B and T cells in the lymphoid lineage. A similar evoltionary pattern can be found among NK cells. Cells displaying an NK cell behaviour can be found accross virtually all vertebrate classes (Yoder et al. 2011). However, mammalian killer cell lectin-like receptors are not conserved over all other vertebrate classes (Yoder et al. 2011; Vivier, Van De Pavert et al. 2016). This suggest a certain degree of co-evolution between ILCs and adaptive lymphocytes. In this regard it is noteworthy that several reports have established interactions between ILCs and adaptive lymphocytes, especially CD4+ T lymphocytes (Hepworth et al. 2013; Oliphant et al. 2014; Burg et al. 2014; Molofsky et al. 2015). It also is worth a short comment that in humans, ILCs might be generally redundant, at least in countries with western hygenic standard. Severe combined immunodeﬁciency (SCID) patients

14 3.2 The interleukin-9 with mutations of the IL2Rγ or its signal transducer Janus kinase (JAK)3 are devoid of ILCs and adaptive lymphocytes (Vély et al. 2016). In mice, ILCs were found to be mostly tissue resident and the pool of peripheral tissue resident ILCs was not replenished by circulating ILCs or their precursors (Gasteiger et al. 2015). Hence, SCID patients reconstituted with healthy donor stem cells, displayed a dramatically reduced frequency of ILCs. Nonetheless, the investigators were not able to deduce specifically ILC-linked diseases in a follow-up time of almost 40 years (Vély et al. 2016). In conclusion, although the term innate lymphoid cells is suggestive of describing an ancestral cell population, it is unlikely that ILCs are either the precursors of T cells nor evolutionary more ancient. It is more likely that both populations have co-evolved despite some redundancy.

3.2 The interleukin-9

As aforementioned (subsection 3.1.2), IL-9 is a type 2 immunity cytokine. Among others, ILC2s produce this cytokine in an autocrine fashion (Wilhelm, Hirota et al. 2011; Turner et al. 2013).

Interestingly, the presence of transcripts of the receptor of IL-9 has been found to discriminate TH2 cells from ILC2s as well as ILC2s from ILC3s and LTis (Price et al. 2010; Hoyler et al. 2012). The cytokine was independently discovered in 1988/89 in Brussels, Munich and Mainz as P40, mast cell growth-enhancing activity (MEA) and T cell growth factor 3 (TCGFIII), respectively (Uyttenhove et al. 1988; Moeller et al. 1989; Schmitt, Brandwijk et al. 1989). Since then, IL-9 was extensively studied and several times reviewed (Noelle et al. 2010; Goswami et al. 2011; Perumal et al. 2011; Wilhelm, Turner et al. 2012; Stassen et al. 2012; Kaplan 2013; Pan et al. 2013; Zhao et al. 2013; Schmitt, Klein et al. 2014; Kaplan et al. 2015; Ciccia, Guggino, Ferrante et al. 2016; Gong et al. 2017; Rivera Vargas et al. 2017).

3.2.1 The cellular source of interleukin-9

Since initially, activated and long term cultures of CD4+ T cells were identified as the main source of the cytokine (Schmitt, Brandwijk et al. 1989), most research was focussed on T cells. However, there was a certain degree of confusion, which T cell subset would be the responsible producer of IL-9. At the beginnings, research on IL-9 was hindered by the lack of a commercialized monoclonal antibody and the lack of genetic reporters that would allow the identification of a specific population

15 3.2 The interleukin-9 ex vivo (Wilhelm, Turner et al. 2012). Another obstacle to identify IL-9-producing cells emerged from the expression kinetics. Hence, in most cases IL-9 only appears transiently during the ignition of inﬂammation and after this initial peak, it rapidly vanishes (Gessner et al. 1993; Tan et al. 2010;

Wilhelm, Hirota et al. 2011; Licona-Limón et al. 2013; Monteiro et al. 2015; Peng et al. 2015). IL-9 therefore often precedes other readily detectable cytokines. Nevertheless, very soon after its discovery, a team of researchers from Erlangen described IL-9 to be a TH2 cytokine (Gessner et al. 1993). They found relevant in vivo expression in T cells, since athymic mice had no detectable Il9 messenger RNA and the blockade of TH2 differentiation with an anti-IL-4 antibody also abrogated the IL-9 expression. In contrast, later reports showed regulatory T cells (Tregs) and TH17 cells to be the prominent source of IL-9 (Lu et al. 2006; Elyaman et al. 2009; Nowak et al. 2009). In 2008, two independent definite reports from the laboratories of Brigitta Stockinger and Vijay Kuchroo showed that indeed a distinct subset of T cells expresses the IL-9 and hence thereafter they were called TH9 cells (Dardalhon et al. 2008; Veldhoen et al.

2008). Interestingly, the IL-9 production by highly pro-inﬂammatory TH17 cells could be reproduced redundandly (Elyaman et al. 2009; Nowak et al. 2009; Stephens et al. 2011; Wilhelm, Hirota et al.

2011; Licona-Lim´on et al. 2013), but its production by suppressive Tregs is less clear (Lu et al. 2006; Nowak et al. 2009; Elyaman et al. 2009; Wilhelm, Hirota et al. 2011). These partially contradicting

findings might be explained by the fact that like Tregs, also TH9 as well as TH17 cells require transforming growth factor beta (TGF-β) for their differentiation from na¨ıve T cells. Hence, their fate is only determined by the differential presence of the cytokines IL-4 (TH9) and IL-6 (TH17) or their absence (Tregs) (Dardalhon et al. 2008; Veldhoen et al. 2008). Furthermore, it was observed in vivo using fate mapping reporters, that at least Tregs and TH17 are very versatile and naturally undergo conversion of their identity (Gagliani et al. 2015; Komatsu et al. 2014). Accordingly, under steady state in the small intestine approximately 1 % of the T cells switched from a TH17 to a Treg phenotype (Gagliani et al. 2015).

Besides T cells, also activated mast cells and esoinophiles were claimed to produce IL-9 (Hultner,¨ K¨olsch et al. 2000; Gounni, Nutku et al. 2000). In 2011, it was again the team of Stockinger, who showed that ILC2s not only signiﬁcantly produced IL-9, but also required the cytokine for their survival in vivo (Wilhelm, Hirota et al. 2011; Turner et al. 2013). As mentioned previously, the receptor of IL-9 served to discriminate ILC2 from other lymphoid populations (Price et al. 2010; Hoyler et al.

16 3.2 The interleukin-9

2012). Stockinger and colleagues demonstrated that signalling through the IL-9 receptor is essential for the survival of ILC2s, but not TH2 cells (Turner et al. 2013). Accordingly, ILC2s expressed in response to IL-9 the anti-apoptotic transcriptional co-activator BCL-3. This autocrine feedback loop governed cytokine release and tissue repair in various inflammatory lung models. Of note, only IL-33, but not IL-25 was able to induce the IL-9 expression in ILC2s (Wilhelm, Hirota et al. 2011). These findings were confirmed by Richard Locksley’s team (Mohapatra et al. 2016). They furthermore showed that even under steady state, ILC2s contribute to 80% of the basal IL-9 production. The control of IL-9 expression has been very well studied in T cells, and only very recently in ILC2s

(Mohapatra et al. 2016). The production of IL-9 in TH9 cells stricktly relies on the transcription factors PU.1 (encoded by Spi1) and IRF4 (Chang et al. 2010; Staudt et al. 2010). While the expression of PU.1 among T cell seemingly is a unique feature of TH9 cells, IRF4 acts on a hirarchical higher level and also is involved in TH2 and TH17 generation. During the polarisation of TH9 cells, the promotor regions of both, Il9 and Spi1, undergo epigenetic remoddeling to enable IL-9 expression in murine and human T cells (Chang et al. 2010; Ramming et al. 2012; Perumal et al. 2011). Like in T cells, the production of IL-9 in ILC2s is dependant on the transcription factor IRF4. The role of PU.1 in ILC2s, if any, remains to be determined.

3.2.2 The immune-modulatory capacities of interleukin-9

IL-9 emerged as a pleiotropic immuneregulator from the beginning. Two early reports described IL-9 as a T cell growth factor (Uyttenhove et al. 1988; Schmitt, Brandwijk et al. 1989). Another article reported a mast cell growth-enhancing activity of IL-9 (Moeller et al. 1989). IL-9 belongs to the superfamiliy of IL-2, whose members IL-2, -4, -7, -9, -15, and -21 have receptors that share the common-γ chain (IL2Rγ) (Rochman et al. 2009). Curiously, the IL-9 receptor α-chain is encoded on the chromosome 11 in mouse, but on the chromosomes X and Y in humans (Kermouni et al. 1995). The heterodimeric IL-9 receptor signals through JAK1 (α-chain) and JAK3 (γ-chain) (Miyazaki et al. 1994; Russell et al. 1994; Demoulin et al. 1996), which subsequently activates the signal transducer and activator of transcription (STAT)1, 3 and 5 (Demoulin et al. 1996). Notewithstanding the employment of IL-9 receptor transcripts as a discriminator in transcriptomic analyses of T cells and ILC2s, the expression of the IL-9 receptor protein has been reported on a variety of cells thereof bonna ﬁde TH2, TH9, TH17, Tregs, but not na¨ıve T cells, mast cells,

17 3.2 The interleukin-9 eosinophils, ILC2s and also epithelial cells (Druez et al. 1990; Gounni, Gregory et al. 2000; Nowak et al. 2009; Elyaman et al. 2009; Turner et al. 2013; Gerlach et al. 2014).

3.2.2.1 Interleukin-9 in infection, tumour and allergy

The pro-inflammatory activity of IL-9 was shown to be beneficial in acute reactions to infections with nematodes (Faulkner et al. 1998; Townsend et al. 2000; Turner et al. 2013), but also against tumours (reviewed in Rivera Vargas et al. (2017)). In contrast, the pro-inflammatory activity is undesired in inappropriate immune responses like allergic reactions. The depletion of IL-9-expressing T cells and ILC2s ameliorated asthmatic models (Staudt et al. 2010; Chang et al. 2010; Wilhelm, Hirota et al. 2011) and IL-9-producing mast cells were shown to promote food allergy (C.-Y. Chen et al. 2015). Also inadequate immune responses in cystic fibrosis were shown to be maintained by

IL-9 as a self-enhancing loop of ILC2s, TH9 cells and mast cells (Moretti et al. 2017). In this regard it is worth mentionning MEDI-528, a humanized IgG1 that binds IL-9, and designed to reduce the activity of IL-9 dependant cells in asthma. It turned out that not only bench side scientists struggled to capture IL-9, but also bed side clinical scientists. A phase 2b clinical trial concluded that the blockade of IL-9 in patients under basal therpapy ”was not associated with an improvement [...] or any major safety concern” (Oh et al. 2013).

3.2.2.2 Interleukin-9 in autoimmune diseases

Since chronic inflammation is the underlying reason for autoimmmune diseases, therapeutical targeting of pro-inflammotory cytokine pathways is the standard therapy (see subsection 3.3.2). However, the role of IL-9 in autoimmune disease is less clear and it does not solely enhance the pathologies. One of the first autoimmune models in which IL-9 was studied, was experimental auto-immune encephalitis (EAE), a preclinical model of multiple sclerosis. Two contradicting reports were published in 2009 using the same IL-9 receptor deficient mouse strain to study EAE, but slightly different immunization protocols against myelin oligodendrocyte glycoprotein (MOG induced EAE). The first report found that the deficiency of IL-9 signalling ameliorated the model. The KO mice displayed a reduced number of TH17 cells, IL-6-producing macrophages and mast cells in the central nervous system (Nowak et al. 2009). IL-9 was produced by TH17 cells and served as an intrinsic amplifier. In sharp contrast, the second report showed that the KO mice had an aggravated EAE with an earlier

18 3.3 Rheumatoid arthritis onset (Elyaman et al. 2009). Even though the authors could corroborate that IL-9 was produced by

TH17 cells, the self-sustaining effect of IL-9 on TH17 cells was overwritten by a significant stronger effect on Tregs enhancing their activity. Thus, TH17 cell driven inflammation was self-limited via the

IL-9-Treg axis. IL-9-producing TH17 also were found in another report studying autoimmune gastritis

(Stephens et al. 2011). In this model, in vitro polarized TH17 cells are adoptively transferred into athymic nude mice to induce an experimental autoimmune gastritis. Unexpectedly, symptoms were aggravated, when T cells were isolated from IL-9 KO mice. The authors speculated that IL-9 would activate mast cells, thereby dampening the extend of the TH17 response. Indeed, Treg derived IL-9 was shown to prime mast cells torwards a tolerogenic phenotype, which created an environment allowing for the enhanced survival and acceptance of allogenic grafts (Lu et al. 2006).

In contrast to TH17 derived IL-9, classical TH9 were shown to aggravate inﬂammatory bowel disease, another autoimmune pathology. In this model, the accumulation of TH9 cells in the intestine, detected by the expression of PU.1 in T cells, was found to be the patho-physiologically instructive event in humans and mice (Gerlach et al. 2014). IL-9 receptor signalling on epithelial cells induced intestinal barrier loss and thus prevented healing. In other rheumatic diseases, mostly smaller clinical reports and descriptive studies stressed the point that IL-9 would mainly be TH9 derived, and that higher IL-9 levels would be associated with a more sever pathology in patients with e.g. psoriasis arthritis, RA, atopic dermatitis and others (Ciccia, Guggino, Ferrante et al. 2016). However, an in depth analysis for most of the rheumatic diseases is lacking.

3.3 Rheumatoid arthritis

RA is a chronic inflammatory autoimmune disease, which affects up to 1 % of the population world- wide (recent reviews: Smolen, Aletaha et al. (2016), McInnes et al. (2017) and Z. Chen, Bozec et al. (2018)). Patients suffer from pain and stiffness of the affected joints, which is caused by leukocyte infiltration and the subsequent destruction of the tissue. Erosions of the catrilage and the underlying bone are hallmarks of late disease stages. It is the characteristic of chronic autoimmune diseases that they hardly resolve and usually accompany the patients during their entire life. In this context, arthritis can be particularly challenging, when it already arises during childhood as

19 3.3 Rheumatoid arthritis juvenile idiopathic arthritis (Prakken et al. 2011). Much progress has been made within the recent years understanding the etiology. Risk factors comprise similar to other autoimmune diseases genetic predisposition of the human leukocyte antigen (HLA) locus, but also other loci including co-receptor stimulation and cytokine responsiveness (Stahl et al. 2010). The socio-economic status and thus heritable and acquired epigenetic variations as well as the commensal flora have been linked to a predisposition. Smoking is for instance one of the best established environmental factors (Spagnolo et al. 2018). Smoking furthermore increases the risk of RA accompanied complications in the lung (interstitial lung disease). Despite the lung, the extra-articular manifestation that accompany RA can virtually affect any organ thereby decreasing the patient’s health condition while increasing the morbidity (Scott et al. 2010; McInnes et al. 2011). Hence, understanding how inflammation is maintained in RA and how a decline could be induced is a compelling question with outstanding clinical relevance.

3.3.1 Molecular and cellular pathways in rheumatoid arthritis

In the preclinical phase of RA, a breakdown of self-tolerance kicks-off a cascade of events ultimately leading to the pathological manifestations of RA. To explain the breakdown of self-tolerance, the Knudson two hit theory was adopted from cancer to autoimmune disease (Smolen, Aletaha et al. 2016; McInnes et al. 2017). Knudson postulated that cancer is induced by the accumulation of at least two mutations in the genome (Knudson 1971). Non of the single mutations would be able to induce cancer by itself. In the context of auto-immune dieseases, in an organ distinct from the organ, where the disease will manifest, an immune reaction is initiated (first hit). In RA, the lung might be a candidate, since smoking increases the disease-risk (Smolen, Aletaha et al. 2016). Upon a second event, e.g. mechanical stress in the joint, which would be accompanied by a mild but resolving inflammatory reaction in unbiased individuals, the reactive immune cells gain the ability to locate to the joint, inducing there a destructive inflammation.

3.3.1.1 Auto-antibodies

The breakdown of self-tolerance preclinically becomes apparent in some patients as seropositivity for auto-antibodies. The two classical types of auto-antibodies in RA are antibodies against IgG known as rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA). Both types have been

20 3.3 Rheumatoid arthritis detected more than 10 years before clinical appearance af RA in seemingly healthy individuals (Nielen et al. 2004). Approximately half of the later RA patients were seropositive before onset of clinical symptoms, while in established RA the prevalence is about two-third. Hence, auto-antibodies may not be a pathophysiological key trigger for disease induction, however seropositivity is associated with a more severe disease (Smolen, Aletaha et al. 2016; Veale, Orr et al. 2017). In several mouse models of RA, immune complex deposition on the synovial cartilage is observed and thought to be the trigger for joint inﬂammation in these models (Brackertz et al. 1977; Matsumoto et al. 2002; Cope 2007). In humans, ACPA were shown to directly activate osteoclasts and osteoclastogenesis, and thus increased the degree of bone errosion (Harre et al. 2012). RFs were shown to activate macrophages and the complement and beyond that, they could bind to IgG-type ACPAs, which further boosted their pro-inﬂammatory potential (Anquetil et al. 2015). Accordingly, the anti-CD20 antibody rituximab is approved for therapy of RA, when other disease modifying anti-rheumatic drugs

(DMARDs) failed (Smolen, Landew´e et al. 2017). It is especially eﬃcient in seropositive patients (Cohen et al. 2015).

3.3.1.2 Alterations of the synovial joint micro-architecture in early and in clinically manifested rheumatoid arthritis

A synovial joint is a joint with a fibrous capsule (Figure 4). The capsule encloses two adjacent bones, which are covered by cartilage. The synovium is a thin adventitious layer of cells that is covering the inside of the joint capsule in regions that are not directly involved in the mechanical hinging of the joint (Asif Amin et al. 2017; Orr et al. 2017). The synovium of healthy individuals displays a rather poor cellular density (Smith 2003). The majority of cells forming the synovial lining are either haematopoietic macrophages or mesenchymal fibroblast like synoviocytes (FLSs). The lining cells seal the underlining connective tissue matrix, containing the sub-lining fibroblasts, the vascular network and a few tissue resident immune cells (Asif Amin et al. 2017). The onset of synovitis in RA is accompanied by a severe remodelling of the synovial joint. Synovitis is characterized by the massive influx of immune cells, proliferation of FLSs, and neo-vascularisation. Activation of FLSs leads to a destructive phenotype that is partially independent of immune cell activation and therefore already could occur as an early event in RA. It was observed that FLSs from RA patients showed an intrinsic invasive and matrix-degrading phenotype when transplanted into

21 3.3 Rheumatoid arthritis

Healthy Femur Rheumatoid

Hyperplastic Synovial membrane Capsule

Ligaments

Meniscus Infiltrates

Synovial membrane Pannus Synovial fluid

Tibia Bone marrow

Figure 4: Schematic illustration of a synovial joint. Schematic view of a healthy (left) and a rheumatic (right) knee joint highlighting the degenerative processes in RA.

untreated immunodeficient SCID mice (Lefèvre et al. 2009). There also is evidence that different subsets of FLS develop in the RA synovium. These were characterized by their different capacities of invasion and matrix degradation, support of osteoclastogenesis and sustaining the recruitment and activation of immune cells (Croft et al. 2016; Mizoguchi et al. 2018). Activation of FLSs furthermore is linked to angiogenesis (MacDonald et al. 2018). Spreading of vessels is a similar invasive mechanism to FLS activation including the induction of matrix degrading enzymes, cell proliferation and recruitment of accessory cells. An invasive proliferative accumulation of cells in RA is called pannus. Pannus formation destroying the underlying cartilage and bone is the major reason for the loss of functionality of joints in RA (Figure 4).

22 3.3 Rheumatoid arthritis

3.3.1.3 T cells in rheumatoid arthritis

T cells are important actors in RA. The synovium of highly active RA patients is characterized by lymphoid clusters. T cells are present as either diffuse aggregates or together with B cells forming ectopic lymphoid structures with germinal centres (Young et al. 1984; Humby et al. 2009). Between 70 and 90 % of the infiltrating CD4+ T cells were found to be CD45RO+ antigen experienced or memory cells (Kohem et al. 1996; Nanki et al. 2000; Rao et al. 2017). In a recent study on RF and ACPA seropostive patients, the CD4+ T cell compartment was studied by high dimensional mass cytometry. About one quarter of the cells consisted of peripheral helper cells, a unique cell type, which differs from classical TH cells and Tregs, but resembles follicular helper cells (Rao et al. 2017). The follicular helper subset is the predominant T cell type in B cell follicles of secondary lymphoid organs (SLOs) (Fazilleau et al. 2009). They express the same chemotactic receptor C- X-C motif chemokine receptor (CXCR)5 as B cells do, but not the T zone homing receptor C-C motif chemokine receptor (CCR)7. They are antigen experienced and by their expression of CD40 ligand and inducible T cell co-stimulator (ICOS), they are able to induce the formation of germinal centres. Similarly, peripheral helpers were able to promote plasma cell differentiation in vitro. They fundamentally differed from follicular helpers by the lack of CXCR5. Instead, they expressed the B cell recruiting chemoattractant chemokine C-X-C motif ligand (CXCL)13 and thus might help B cell differentiation in the periphery underlining the importance of T cells in rheumatoid arthritis. The complexity of synovial T cell biology is still not fully captured.

3.3.1.4 The pro-inﬂammatory cyotkine cascades in rheumatoid arthritis

The pro-inflammatory network that ultimately leads to joint destruction in RA is composed of two major cytokines (Schett et al. 2013). These are tumor necrosis factor alpha (TNF-α) and IL-6. TNF-α is thought to be upstream of IL-6 (Charles et al. 1999). TNF-α has been among the first targeted therapies and several TNF-α transgenic mice have been established as models of RA (P. Li et al. 2003). The vast TNF-α dependant cytokine network in RA pathogenesis has been extensively reviewed (McInnes et al. 2007). Hence, in the following a putative TNF-α induced T cell-ostecolast axis is outlined, considering most recent reports. ACPA immune complexes are a possible trigger for TNF-α release from monocytes (Sokolove et al. 2011). TNF-α was shown to prevent the decline of inflammation by its ability to inactivate the transcription factor FoxP3 in Tregs

23 3.3 Rheumatoid arthritis

(Nie et al. 2013). This was associated with an increase of IFN-γ and IL-17-producing TH cells in the RA synovium. In addition, TNF-α signlaling to FLSs induced the very robust and sustained expression of IL-6 (Loupasakis et al. 2017). Both, loss of FoxP3 and FLS derived IL-6 also was required for the generation of TH17 cells from inactivated Tregs (Komatsu et al. 2014). Now, these ex-Treg-TH17 cells were a prominent source of the cytokine RANKL. Ultimatly, TNF-α and RANKL directly synergized in the osteoclastogenesis (Y. H. Zhang et al. 2001). In conclusion, TNF-α might lead via the T cell axis to bone destruction.

3.3.2 Current therapies

Compulsory by their nature, autoimmune diseases dispose of an inexhaustible source of immunogen and therefore are characterized by persistent inflammations (Cho et al. 2015). Persistent inflammation leads to profound tissue remodelling and destruction by activation of fibroblasts, vascular alterations and/or to the formation of ectopic lymphoid structures (Muller-Ladner¨ et al. 2007; Pitza- lis et al. 2014; Asif Amin et al. 2017; Orr et al. 2017; MacDonald et al. 2018). Reconditioning of the tissue can only be archived by the terminal resolution of inflammation. The tissue integrity is maintained through a thoroughly balanced network of different cell populations. Stromal cells and fibroblasts structure any tissue by the creation of specific micro-environments. Those are then popu- lated and maintained by a tissue specific set of immune cells. Acute inflammation rapidly declines and does not profoundly alter the micro-environments and thus integrity. Chronic inflammation however will lead to the dysregulation of microenvironements and to in-depth structural changes. In systemic sclerosis, activation of fibroblasts results in an abnormally increased deposition of the extracellular matrix and a subsequent loss of function of the tissue affected (Distler et al. 2017). In contrast to fibrosis, in a highly inflammatory disease like RA, activation of fibroblasts leads to an abnormally erosive phenotype with degradation of extracellular matrix (Neumann et al. 2010). Ectopic lymphoid structures form as a result of chronic inflammatory autoimmune disorders and in RA, they serve as a long term reservoir of antigen and centers of immune cell maturation (Pitzalis et al. 2014). Therapeutics for rheumatic diseases and other autoimmune diseases are called disease modifying anti-rheumatic drugs. This is due to the fact that the concept of disease modifying drugs firstly was developed in the field of rheumatology. Although a more generalized term remission inducing drug (RID) was proposed, it could not be established (Buer 2015). DMARDs either can be small

24 3.3 Rheumatoid arthritis molecules or recombinant proteins (”biologicals”). Small molecules are called sDMARDs and comprise conventional csDMARDs therapeutics such as methotrexate (MTX) and specifically pathway targeting tsDMARDs. tsDMARDs can act as tyrosine kinase inhibitors, so called ”-nibs”, e.g. for JAK pathways tofacitinib and baricitinib, or as inhibitors of second messenger producing enzymes, e.g. apremilast, which is approved for psoriasis and psoriatic arthritis and inhibits PDE4. The second group of DMARDs, the biologicals, contains genetically engeneered humanized and human recombinant proteins, which are mostly antibodies or decoy receptors. These are called bDMARDs and their generic-like products are called biosimilar bsDMARDs (Coates et al. 2016; Smolen, Landewé et al. 2017). The acute treatment of highly inflammatory arthritis aimes to dampen the immune system in a generalized fashion to dispel the symptomes and reduce pain. To treat pain and stiffness, non- opioid analgetics, especially non-steroidal anti-inflammatory drugs (NSAIDs) are accepted (Smolen,

Landewé et al. 2017). To convey the patient in a relatively stable state of low disease activity or remission, a glucocorticoid and a conventional DMARD are applied. The anchor DMARD therapy is the low dose application of MTX (Smolen, Landewé et al. 2017). MTX is an antagonist of folic acid (Vitamin B9) inhibiting DNA synthesis. Therefore, in a higher dose it is also used as chemotherapeutic for neoplastic diseases (Groff et al. 1983). If this first therapy fails, it is recommended to change to another conventional DMARD, given the prognosis would be favourable. In the other cases, it is most common to use a biological DMARD, i.e a blocking antibody. Most commonly used are anti-TNF-α therapies. It also remains the possibility to use a targeted DMARD. In case of RA, this would be a JAK inhibitor like tofacitinib, but clinical experience is lower and thus TNF-α blockade is generally preferred (Smolen, Landewé et al. 2017). This scheme iterates until the patient reaches remission.

3.3.2.1 Biological disease modifying anti-rheumatic drugs that block cytokine pathways

Commonly the biological treatments target the pro-inﬂammatory cytokines or their receptors and therefore aim to reduce the pool of potentially self-reactive immune cells by interfering with their activation. These therapies comprise drugs that target TNF-α (adalimumab, certolizumab, golim- umab, inﬂiximab and etanercept), IL-6 (tocilizumab, sarilumab, clazakizumab, sirukumab), IL-17

(secukinumab) or IL-23 (guselkumab, ustekinumab) (Coates et al. 2016; Smolen, Landew´e et al.

25 3.3 Rheumatoid arthritis

2017). Since their developement, TNF-α-targeting biologicals turned out to be most efficient block- busters. Advantageously, they can be used in many different kinds of autoimmune diseases. Besides RA, they are used in Crohn’s Disease, psoriatic disorders and ankylosing spondylitis and others (Choo- Kang et al. 2005). By contrast, targeting the IL-17/23 axis only is efficient in psoriatic arthritis, but not in RA. Thus, in case an RA patient would not respond to an anti-TNF-α treatment, targeting the IL-6 pathway would be another option. Biological DMARDs can either block the receptor (tocilizumab) or the cytokine itself (clazakizumab).

3.3.2.2 Other pathways targeted by disease modifying anti-rheumatic drugs

Programmed cell death (PD)-1 is an important immune checkpoint that under normal circumstances mediates peripheral tolerance. Many tumours create immunosuppressive micro-environments increasing the availability of the ligands PD-L1 and PD-L2 in their milieu (Bardhan et al. 2016). Furthermore, there is evidence that impaired PD-1 signalling is involved in RA and other autoimmune diseases (Francisco et al. 2010). Therefore, the PD-1 pathway is likewise a promissing anchor point in autoimmune diseases and cancer. The latter ﬁeld already disposes of FDA approved therapies (nivolumab, pembrolizumab) (Bardhan et al. 2016). Other therapies include the blockage of co-stimulation via CD80 and CD86 with decoy receptors (abatacept, belatacept) to minimize T cell activity. Both decoy receptors are cytotoxic T-lymphocyte- associated protein (CTLA)4-IgG1-Fc fusion proteins that bind CD80 and CD86 thereby preventing the co-stimulatory through CD28 (Blair et al. 2017). Targeting the survival signals of B cells by blocking B cell activating factor (BAFF) (belimumab), however has turned out to be ineﬃcient in RA (Stohl et al. 2012).

3.3.2.3 Antibody dependant cell mediated cytotoxicity induced by disease modifying anti-rheumatic drugs

Now ”the more-the better”, the survival of B cells also can be tackled by antibody dependant cell mediated cytotoxicity (ADCC). Antibody binding to CD20 (rituximab) inducing ADCC turned out to be an eﬀective treatment of RA under circumstances where TNF-α blockade is unfavourable (e.g. after cancer therapy) (Edwards et al. 2004). Other CD20-blocking therapies (ocrelizumab,

26 3.4 Aim and rationale of the study ofatumumab and obinutuzumab) are in clinical trials with mixed outcome and not yet licenced for the treatment of RA (Du et al. 2017). Thus rituximab in a combined therapy with MTX is the only approved ADCC therapy in Europe and the United States (Smolen, Landew´e et al. 2017).

3.3.2.4 Are disease modifying anti-rheumatic drugs pro-resolving?

The currently approved DMARDs turn out to act mostly anti-inflammatory (Bluestone et al. 2012). The term anti-inflammatory reflects that the therapies antagonise the pro-inflammatory machinery (Serhan, Brain et al. 2007). Controlling inflammation is important to prevent tissue destruction, yet it does not per se finally terminate the inflammation and resolve it. Resolution of inflammation is an active process that requires the participation of immune cells as well as of surrounding stromal cells (Serhan, Brain et al. 2007; Serhan, Chiang et al. 2008; Tabas et al. 2013; Buckley et al. 2013;

Ortega-Gómez et al. 2013; Sonnenberg et al. 2015; Fullerton et al. 2016). It not only implies the termination of the pro-inflammatory cascade, but also the active clearance of inflammatory cells from the tissue, the induction of tissue regeneration and wound healing to finally re-establish the tissue functionality. Resolution normally would start by the clearance of the causing agent and the pro-inflammatory milieu from the site of inflammation to prevent further recruitment of leukocytes. Since in autoimmune diseases the clearance of the causing agent does not harmonize with the host survival, resolution promoting therapies inevitable need to induce or restore tolerance to the causing agent. Yet, none of the above mentioned therapies aims to resolve inflammation by fostering the intrinsic property of the immune system to limit an immune response.

3.4 Aim and rationale of the study

Discovering the pathways that impair the resolution processes in RA may not only be of clinical relevance for this disease entity, but beyond that it might be critical for uncovering general pathophysiological aspects of chronic inflammatory and autoimmune diseases. Likewise, there is no resolving or tolerance-inducing therapy approved. The advantage of a pro- resolving therapy over conventional DMARD therapies is that these therapies would specifically modify the immune response preventing tissue destruction but importantly maintaining a virtually immunity against pathogens. Accordingly, reported side effects of DMARDs are infections with

27 3.4 Aim and rationale of the study opportunists, or malignancies and patients with an antibody therapy (biological DMARDs) tend to have a slightly higher risk of infections than those treated with synthetic DMARDs only (Ramiro et al. 2017). Moreover, another field that to date is almost completely unaddressed in rheumatology are the therapeutic interventions on ILCs. Certainly, helper-like ILC2s just have been discovered ten years ago and medical research on them is still in the starting phase. However, while their activity in infectious diseases already has been studied with much enthusiasm, knowledge about helper-like ILC2s in autoimmune disorders is still limited. It is also noteworthy, that laboratory inbreed mice that are housed under strict hygienic conditions hardly bear any helper-like ILCs in peripheral blood. In contrast, in humans they are readily detectable. So far, peripheral blood ILC counts are not routinely examined in clinic despite their high potential as biomarkers of the health status. In this study, the pivotal aim was to understand how chronic autoimmune diseases such as RA persist. Using a genetically IL-9 deficient mouse and over-expression approaches, the study was designed to elucidate the participation of this cytokine in rheumatoid inflammatory disease. It was the ultimate aim of the study to translate the findings from the murine system to humans and thus to make them available for diagnostic and therapeutic strategies.

28 4 Material and methods

4.1 Instruments, equipment, reagents, kits, buﬀers and media

The instruments used in this study are listed in Table 1. These instruments were critical for the data generation and acquisition.

Table 1: List of instruments. This list contains the instruments that essentially were required for the generation of the data. ELISA, enzyme-linked immunosorbent assay.

Name Type Supplier Specification Gallios flow cytometer Beckman Coulter 3-laser/10-channel flow cytometer Navios CE-licensed version of Gallios for human blood analysis Astrios MoFlow cell sorter Beckman Coulter high-speed jet-in-air sorter Step One Plus light cycler Applied Biosystems 96-well plate real time PCR device Eclipse Ni-U microscope Nikon 4-channel epifluorescence microscope Eclipse Ci-E microscope Nikon upright transmitted light microscope Axioskop 2 microscope Zeiss upright transmitted light microscope connected to the Osteomeasure system Epoch spectrometer BioTek microplate photometer for ELISA µCT35 µCT Scanco Medical X-ray microtomography instrument

29 4.1 Instruments, equipment, reagents, kits, buﬀers and media

4.1.1 Auxiliary equipments

Other auxiliary equipments and standard molecular biology laboratory devices that were used are listed in Table 2.

Table 2: List of auxiliary equipment. This list contains additional devices and the auxiliary standard molecular biology laboratory equipments and auxiliary materials. Name Description Supplier Cell culture AE2000 Trinocular inverted cell culture microscope Motic ENVAIR eco safe Basic Plus biological safety cabinet Envair WNB 45 water bath Memmert CO2 Incubator ICOmed cell culture incubator Memmert Heraeus Megafuge 16R tempered centrifuge Thermo Fisher Millivac vacuum pump EMD Millipore Neubauer haemocytometer cell counting aid Paul Marienfeld Flow cytometry and cell sorting ThermoMixer C heated thermoshaker Eppendorf Heraeus Megafuge 16 centrifuge with plate rotor Thermo Fisher Centrifuge 5424 R desktop centrifuge Eppendorf Vortex-Genie 2 sample mixer Scientific Industries DynaMag 15 Magnet Dynabead separator Thermo Fisher Histology Citadel 1000 tissue processor system Thermo Fisher TES 99 tissue embedding system Medite COP 30 tissue cool plate Medite Meditome A550 microtome Medite TFB 55 tissue flotation bath Medite Universal oven U oven Memmert Genius microwave Panasonic StarFrost adhesive microscope slides Knittel Cover slips 24 x 500 mm Menzel Whatman prepleated filter paper, grade 1V Sigma Plastics Tissue culture plates 48-well, 96-well U and V shapes Greiner 96-well HTS transwell plates Corning Petri dishes, 60 mm diameter Sarstedt MicroAmp 96-well optical PCR plates with adhesive film Thermo Fisher 8-well PCR stips Nerbe 1.4 ml conic polypropylene tubes micronic Haemolysis tubes, 5 ml Greiner Conical tubes, 15 ml and 50 ml, sterile, polystyrene Nerbe Conical tubes, 2 ml, 1 ml and 0.5 ml sterile, polystyrene Eppendorf

30 4.1 Instruments, equipment, reagents, kits, buﬀers and media

Serological pipettes, 2 ml, 5 ml, 10 ml, 25 ml, 50 ml Nerbe Low-density polyethylene Pasteur pipettes Brand SafeSeal filter tips, 1,000, 200, 100, 10 µl Biozym Pipette tips, 1,000, 200, 100, 10 µl Nerbe Monovette, EDTA vacuum tubes Sarstedt Inject-F, 1 ml single use syringe B. Braun Inject Solo, 2 ml single use syringe, luer lock B. Braun BD Microlance 27G 25G, 21G BD 300 µl insulin syringe, 27G Therumo Precellys Ceramic Beads kit 2.8 mm ceramic beads in 1.5 ml Bertin Instruments reaction tubes with screw cap SteriCup Vacuum filtration system 0.2 µm EMD Millipore Nunc MaxiSorp adsorbent 96-well ELISA plates Thermo Fisher Cell strainer 40 and 70 µm cell filters BD Falcon Various G 5216 -20 ◦C freezer Liebherr TSE400A -80 ◦C freezer Thermo Fisher Provit 2200 autoclave ASS Aigner RF0244A ice machine Manitowoc Barnstead GenPure water purification system Thermo Fisher PeqStar 96 universal 96-well thermal cycler Peqlab EasyCast D2 gel electrophoresis systems Thermo Fisher Power Pack P25T power supply for electrophoresis Core Gel Doc XRS gel documentation system Bio Rad Precellys 24 tissue homogenizer Bertin Instruments CS series CS200 balance Ohaus PH-100 ATC pH meter Voltcraft RCT basic magnetic heated stirrer IKA C110T electronic calliper Kröplin Tail vein restrainer restrainer for mouse Braintree Scientific IL11 infrared lamp Beurer Research Plus pipettes for 1,000, 200, 100, 10, Eppendorf 2.5 µl single channel and 300 µl 8x multichannel Pipetteboy pipette aid for serological Integra Biosciences pipettes RNase ZAP RNase decontamination solution Ambion

4.1.2 Inorganic and organic chemicals

All chemicals used in this study are listed in Table 3.

31 4.1 Instruments, equipment, reagents, kits, buﬀers and media

Table 3: List of chemicals, organic and inorganic compounds. This list contains all chemicals used in the study. The column ”Name” contains either the IUPAC nomenclature, the trivial name or the molecular formula. CAS identiﬁers are included. EDTA, ethylenediaminetetra-acetic acid. EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetra-acetic acid. PMSF, phenylmethylsulfonylﬂuorid. SDS, sodium dodecyl sulfate. Tris,2-Amino-2-(hydroxymethyl)propane-1,3-diol.

Name CAS Catalogue Supplier 0.4 % trypan blue 72-57-1 15250-061 Thermo Fisher solution 4 % formaldehyde, 50-00-0 P087 Carl Roth phosphate buﬀered β-mercaptoethanol 60-24-2 4227 Carl Roth Acetic acid 64-19-7 6755 Carl Roth Agarose 9012-36-6 3810 Carl Roth Citric acid 77-92-9 7624 Carl Roth Chloral hydrate 302-17-0 K318 Carl Roth Eosin B 548-24-3 0306 Carl Roth Ethanol 64-17-5 9065 Carl Roth Ethanol, denatured 64-17-5 K928 Carl Roth EDTA 60-00-4 8040 Carl Roth EGTA 67-42-5 3054 Carl Roth Fast Green FCF 2353-45-9 0301 Carl Roth Hydrochloric acid 7647-01-0 4625 Carl Roth Isopropanol 67-63-0 0733 Carl Roth KAl(SO42) 7784-24-9 CN78 Carl Roth KHCO3 298-14-6 P748 Carl Roth NaCl 7647-14-5 9265 Carl Roth NaIO3 7681-55-2 HN17 Carl Roth NaOH 1310-73-2 9356 Carl Roth NH4Cl 12125-02-9 K298 Carl Roth Percoll 65455-52-9 17-0891-02 GE Healthcare PMSF 329-98-6 6367 Carl Roth Safranin O 477-73-6 T129 Carl Roth SDS 151-21-3 0183 Carl Roth Sodium hydroxide 1310-73-2 9356 Carl Roth Tris 77-86-1 A411 Carl Roth Tris-HCl 1185-53-1 9090 Carl Roth Triton-X 100 9002-93-1 3051 Carl Roth Tween-20 9005-64-5 9127 Carl Roth Uric acid 69-93-2 4999 Carl Roth Xylol 1330-20-7 9713 Carl Roth

32 4.1 Instruments, equipment, reagents, kits, buﬀers and media

Table 4: List of kits and other material. This list contains multi-component kits that were used in the study and also lists additional reagents such as enzymes. bp, base pair. BSA, bovine serum albumin. DNA, deoxyribonucleic acid. TRAP, tartrate-resistant acetic phosphatase.

Name Catalogue Supplier Kits FoxP3 Transcription factor buffer set 00-5523-00 eBioscience IC fixation kit 88-8824-00 eBioscience Ledgendplex T helper cytokine panel 740740 Biolegend mouse IL-9 ELISA 442704 Biolegend mouse IL-22 ELISA 88-7002-88 eBioscience mouse IL-33 ELISA 436407 Biolegend RNeasy RNA isolation kit 740406.50 Macherey-Nagel SuperScript IV first-strand synthesis kit 18091050 Invitrogen TRAP Detection Kit 387A Sigma Other reagents 100 bp DNA Ladder N3231 New England Biolabs BSA, fraction V 8076 Carl Roth Collagenase D 11 088 882 001 Roche DNase I, grade II 10 104 159 001 Roche Faramount aqueous mounting medium S3025 Dako Fluorescent mounting medium S3023 Dako Gelstain Red 0984 Carl Roth Horse serum 16050130 gibco Protease Inhibitor cocktail I3786 Sigma Aldrich RedMastermix (2x) M3029.0000 Genaxxon Proteinase K, PCR grade 03 115 879 001 Roche RBC lysis/fixation solution 422401 Biolegend Roti-Histokitt non aqueous mounting medium 6638 Carl Roth SYBR select master mix 4472903 Applied Biosystems

4.1.3 Kits and other reagents

Kits and other materials used in the study are listed in Table 4.

4.1.4 Buﬀers and media for cell culture and lymphocyte isolations

The buﬀers and media used for the sample preparation and cell culture are listed in Table 5. Foetal bovine serum (FBS) was inactivated at 60 ◦C for 40 min prior to ﬁltration through a 0.22 µm sterile membrane.

33 4.2 Software

Table 5: List of buffers and cell culture media. This table lists the media, buffers and cell culture supplements used in the study. Hank’s Balanced Salt Solution (HBSS), Iscove’s modified Dulbecco’s medium (IMDM), phosphate buffered saline (PBS), Roswell Park Memorial Institute medium (RPMI 1640). w/ with, w/o without.

Buﬀer/Media Supplier Catalogue Amphotericin B solution gibco 15240-062 Dubelco’s PBS gibco 14190-144 Dubelco’s PBS, 10x gibco 14200-059 FBS gibco 10500-064 HBSS, w/ phenol red gibco 14180-046 HBSS, w/o phenol red gibco 14175-095 IMDM gibco 12440-053 L-glutamin gibco 250300-81 Penicillin-streptomycin gibco 15140-122 solution Ringer B. Braun 3570010 RPMI 1640 gibco 31870-025 sterile water B. Braun 387873 β-mercaptoethanol Carl Roth 4227

4.2 Software

The ﬁnal graphic output was generated with Adobe Illustrator CS6, Microsoft PowerPoint 2010, and the image J distribution Fiji (daily built). Statistical analysis were performed with Microsoft Excel 2010 and GraphPad Prism Version 5. Flow cytometry data were analysed using Beckman Coulter Kaluza Version 1.5. Microscopic images were analysed in Fiji (Schindelin et al. 2012; Schneider et al. 2012). Representative panorama images were reconstructed using the TurboReg and MosaicJ plugins (Thevenaz et al. 1998; Th´evenaz et al. 2007). Cell numbers were determined using the Cell Counter plugin and the Count Particles algorithm. For photo-histo-morphometry of antigen induced arthrits (AIA) bones, the OsteoMeasure (OsteoMetrics) software was used. This tool is useful for the annotation of bone and joint histologic images according to the ASBMR standard nomenclature.

This thesis was written in LATEX.

4.3 Patients

All patients included for the blood analysis were in care at the hospital of the University of Erlangen- Nuremberg in Erlangen. Synovial biopsies were obtained from rheumatology centres in Erlangen,

34 4.3 Patients

Barcelona, London, Zurich and Dublin. All RA patients fulfilled the 2010 American College of Rheumatology (ACR) classification criteria (Neogi et al. 2010). Patients were defined to be in remission, when they displayed a diseases activity score 28 (DAS28) of less than 2.6 (Prevoo et al. 1995; Sheehy et al. 2014).

4.3.1 Ethical statement

The study was approved by the local ethical committee of the University of Erlangen-Nuremberg. Written informed consent was obtained from all subjects.

4.3.2 Blood donors

Venous blood was collected as part of the routine diagnostic at the outpatient clinic of the Department of Internal Medicine 3 at the hospital of the University of Erlangen-Nuremberg in Erlangen. Small aliquots were used for research purpose (subsubsection 4.5.1.3). In total, 111 patients with RA were included. The cohort was characterized by a sex distribution of 73.9 % females, a mean age of 59.9 ± 14.3 years, a mean DAS28 of 3.2 ± 1.3. The seropositivity for RF was 69.4 % and 59.5 % for ACPA in the cohort. 56.8 % of the patients in the cohort were treated with MTX and 28.8 % of them were treated with biological DMARDs, mostly an anti-TNF-α therapy. 63 of the 111 patients (59.5 %) were included for the follow-up longitudinal assessment of circulating ILC2s 6 to 12 month after the baseline measurement.

4.3.3 Donors of synovial biopsies

Synovial biopsies were obtained by arthroscopic and ultrasound-guided biopsy procedures or after joint replacement surgeries. In total, 38 RA patients were included, 19 of them had an active RA and were biopsied before treatment, and 19 patients were in remission and under DMARD treatment (15 with MTX, 5 with TNF-α inhibitors). The active RA patients displayed a mean DAS28 of 5.8 ± 1.4. The patients in remission displayed a mean DAS28 of 1.9 ± 0.5. No signiﬁcant diﬀerence was observed between patients with active RA or patients in remission in the sex distribution (65.0 % versus 73.7 %), age distribution (52.3 ± 19.8

35 4.4 Animals years versus 52.9 ± 18.2 years), RF seropositivity (67.4 % versus 63.1 %) and ACPA seropositivity (58.5 % versus 68.4 %). Patients with an acute joint trauma were included as controls (n = 8). These patients were characterized by an acute synovitis not related to RA. Normal control synovium was obtained from patients with no articular disease process (n = 11). In the longitudinal biopsy cohort obtained at the centre in London, 10 RA patients with an early disease (duration of less than 12 months) were included that underwent a 6 months follow-up biopsy after initiation of an anti-rheumatic therapy with conventional synthetic DMARDs. The anti-rheumatic therapies included MTX monotherapy or a combination of methotrexate and/or sulfasalazine and/or hydroxychloroquine. The baseline DAS28 was 5.1 ± 1.2 and 2.0 ± 0.6 at the follow-up. The cohort was characterized by a sex distribution of 70 % females and a mean age of 49.8 ± 22.6 years. The seropositivity for RF or ACPA was 70 % in the cohort.

4.4 Animals

All genetically modified animals were bred in an in-house facility. Wildtype (WT) control mice were purchased from the Janvier Labs and imported into the in-house facility. This facility was classified as an specific pathogen (SPF)-free housing and breeding facility. Table 6 lists the mouse strains, their background and their origin. The Il9 -/- animals were kindly provided by Andrew McKenzie from the MRC Laboratory of Molecular Biology in Cambridge, UK. The Il9 Citrine reporter mice were a kind gift from Benno Weigmann from the department of Internal Medicine 1 at the hospital of the University of Erlangen-Nuremberg in Erlangen. David Vöhringer from the department of Infection Biology at the hospital of the University of Erlangen-Nuremberg in Erlangen, provided the Balb/cLy5.1 mice. Spi1 fl/fl x Lck-cre+ mice were a kind gift from Mark Kaplan from the department of Microbiology and Immunology at the Indiana University, USA.

4.4.1 Genotypting

Mice were genotyped by polymerase chain reaction (PCR) to identify the mutations. Tail biopsies were obtained from mice at the age of weaning and digested with proteinase K. The digest buﬀer was composed of 100 mM Tris-HCl, 3 mM EDTA, 0.3 % mass fraction (w/v) SDS, 200 mM NaCl and

36 4.4 Animals

Table 6: List of the mouse strains. The table speciﬁes their trivial name, the oﬃcial MGI nomenclature with the MGI ID and their genetic background. NA, not applicable.

Strain Nomenclature MGI Background wild type Balb/c BALB/cJRj NA BALB/c reporter Il9 Citrine IL9Cit/Balb NA BALB/c mixed Foxp3 GFP C.Cg-Foxp3tm2Tch 3699400 BALB/c Ly5.1 CByJ.SJL(B6)-Ptprca 4819849 BALB/c knock-out Il9 -/- Il9tm1Anjm 2656470 BALB/c Spi1 ﬂ/ﬂ Spi1tm1.2Nutt 3578011 C57BL/6 Lck-cre B6.Cg-Tg(Lck-cre)548Jxm 2176199 C57BL/6

2 µg/ml proteinase K (added freshly). Tail biopsies were digested for 4 ha at 56 ◦C. Proteinase K was denatured for 15 min at 95 ◦C. The digest was brieﬂy centrifuged and diluted 1:5 in water to be used as input in the PCR. PCR primers are listed in Table 7. The PCR program was as follows:

• Initial denaturation: 95 ◦C 5 min • Cylce: 35x

– Denaturation 95 ◦C 10 sec – Annealing Yb ◦C 30 sec – Elongation 72 ◦C 45 sec • Final elongation 72 ◦C 5 min • Final cooling 4 ◦C

The PCR-products were purified using a gel-electrophoresis system. The gels usually contained 1.5 % (w/v) agarose, and 0.01 % volume fraction (v/v) of the DNA stain in TAE buffer (40 mM Tris, pH 8.5, 20 mM acetic acid, 1 mM EDTA). The running time was usually 20 min and the voltage fixed at 100 V. The Ly5 allele Ptprc was identified by flow cytometry of a blood sample with isoform specific antibodies (Table 9).

aTime units: sec, second(s). min, minute(s). h, hour(s). d, day(s). bAnnealing temperature Y: Il9 -/- 64 ◦C, Il9 Citrine 64 ◦C, Foxp3 GFP 59 ◦C, Spi1 ﬂ/ﬂ 65 ◦C, Lck-cre 58 ◦C.

37 4.4 Animals

Table 7: List of genotyping primers. bp, base pairs. F, forward primer. R, reverse primer. WT, wildtype. KO, knock out. TG, transgene. ctrl, internal control.

Strain Name Sequence Products Il9 -/--F 5’-TGATTGTACCACACCCTGCTACAGGG-3’ WT: 100 bp Il9 -/- Il9 -/--R 5’-CGGACACGTTATGTTCTTTAGGACTTC-3’ KO: 1,000 bp Il9 Citrine-5419 5’-AAGAGCATCTTTTCTGAGGAA-3’ WT: 320 bp Il9 Citrine Il9 Citrine-5420 5’-AGGTAATTGGTGTCTTGATGC-3’ TG: 650 bp Il9 Citrine-5422 5’-CTGTTGTAGTTGTACTCCAGC-3’ Foxp3 GFP-R 5’-GCGTAAGCAGGGCAATAGAGG-3’ WT: 275 bp Foxp3 GFP Foxp3 GFP-R 5’-GCATGAGGTCAAGGGTGATG-3’ TG: 325 bp Spi1 fl/fl-F 5’-CTGTCTGCCACCACCTGCCTACATT-3’ WT: 680 bp Spi1 fl/fl Spi1 fl/fl-R 5’-GTGCTTCCTTGGGAGTCTGGCGCT-3’ flox: 738 bp Cre-F 5’-GCGGTCTGGCAGTAAAAGTATC-3’ TG: 102 bp Cre-R 5’-GTGAAACAGCATTGCTGTCACTT-3’ ctrl: 74 bp Lck-cre ctrl-F 5’-CACGTGGGCTCCAGCATT-3’ ctrl-R 5’-TCACCAGTCATTTCTGCCTTTG-3’

4.4.2 Ethical statement

All experiments carried out on mice were approved by the animal ethical committee of the government of Unterfranken, Wurzburg.¨

4.4.3 Animal models

The animal models of arthritis used in the present study have been described in detail in Cope (2007). The gouty arthritis was induced as described in Schauer et al. (2014). The principles of hydrodynamic gene transfer (HDGT) were described in G. Zhang et al. (1999) and Liu et al. (1999).

4.4.3.1 Antigen induced arthritis

Protocol AIA was induced in mice by an immunization against mehtylated bovine serum albumin (mBSA) followed by the intra-articular challenge with mBSA inducing a remitting and locally-limited joint inflammation. The protocol is schematically described in Figure 5. Briefly, preferentially female mice at the age of 8 - 10 weeks were immunized with 100 µg mBSA emulsified in 100 µl complete Freund’s adjuvant (CFA) by a s.c.c injection into the right flank. Additionally, as an adjuvant, 5 x 108 heat-inactivated Bordetella pertussis were administrated i.p. in 100 µl PBS. The immunization was

cAdministration routes: i.p., intra-peritoneally. i.v., intra-venously. s.c. sub-cutaneously.

38 4.4 Animals boosted after 7 d by a s.c. injection of 100 µg mBSA emulsiﬁed in 100 µl CFA above the tail root and an i.p. injection of 5 x 108 heat-inactivated B. pertussis in 100 µl PBS. After 14 d (day 21 after the ﬁrst immunization), mice were challenged by an intra-articular injection of 100 µg mBSA in 25 µl PBS into the right knee joint with a insulin syringe through the skin. The injection of an equal volume of PBS into the left knee joint served as control. Additional controls did only receive PBS after immunization.

Monitoring The weight of the mice was monitored twice weekly. The joint swelling was measured with an electronic calliper every second day. The baseline knee joint diameter was measured immediately prior to challenge.

Adoptive transfer of regulatory T cells and type 2 innate lymphoid cells ILC2s and Tregs were isolated as described below (section 4.7). The anti-CD25 clone 3C7 was used for in vivo applications given the reported in vivo depletion activity of the otherwise preferred PC61 (Table 9 and Takahashi

6 et al. 2000). 0.5 x 10 Tregs were either directly transferred i.v. in 100 µl of Ringer solution or pre-stimulated over night. For pre-stimulation, cells were concentrated to 1 x 106 cells/ml and pre- incubated with recombinant mouse IL-9 at 50 ng/ml or with a combination of recombinant mouse ICOS ligand (ICOS-L) at 1 µg/ml and agonistic antibody to glucocorticoid induced TNFR related (GITR) at 10 µg/ml in complete IMDM medium supplemented as described in section 4.6. For the adoptive transfer of ILC2s, 5 x 103 cells were co-administrated by intra-articular injection together with the 100 µg of mBSA at day 21 of AIA.

4.4.3.2 Serum transfer induced arthritis

Protocol Serum transfer induced arthritis (SIA) was induced in mice by a single i.p. injection of 150 µl serum generated from the K/BxN mouse line. Serum was harvested every other week from the retro-orbital plexus of K/BxN mice starting at the age of 5 weeks, when mice start to display symptoms of arthritis. Serum from litters was collected, pooled, and frozen at -20 ◦C up until use. The arthritis inducing capacity of the serum was routinely tested for each batch. All experiments were carried out with the same batch of serum.

39 4.4 Animals

Monitoring All clinical parameters, including weight, grip strength and paw swelling, were monitored every second day. Grip strength was assessed on a scale ranging from 3 (normal grip) to 0 (no grip). For the assessment, mice were placed on the cage top grid and cautiously pulled at the tail root, which induces a gripping reﬂex. The measurement was repeated three times for each mouse and time point. Swelling of the knee joints, paws and ankle joints was measured with an electronic calliper. The baseline measurements were taken immediately prior to the serum injection.

4.4.3.3 Gouty arthritis

Protocol Monosodium urate (MSU) crystals were prepared as described in Schauer et al. (2014). The crystals were grown in a solution of 10 mM uric acid and 154 mM NaCl at pH 7.2 under constant agitation for 3 d. The crystals were washed in ethanol and dried under sterile conditions prior to their sterilized at 180 ◦C for 2 h. The sterilized crystals were then solved in neural pH PBS at a concentration of 300 mg/ml. For induction of the gouty arthritis model, 50 µl of the crystal solution was injected s.c. into the right paw of mice. The contralateral paw was challenged with 50 µl of PBS only as control.

Monitoring Mice were monitored daily, including weight and paw swelling. Swelling of the paws was measured with an electronic calliper. The baseline measurements were taken immediately prior to the injection of the crystals.

4.4.3.4 Hydrodynamic gene transfer

Protocol HDGT is a method to induce non-viral ectopic gene expression. In mice, the targeted tissue is the liver. By the fast administration of naked plasmid DNA solved in a high volume of a physiological solution, the plasmid DNA is taken up by hepatocytes initiating the ectopic gene expression. In this study, the plasmid DNA was solved in a volume of ringer solution corresponding to 10.5% of the body weight. The plasmid solution was applied through the tail vein within approximately 3 sec. To dilatate the vein, the tails of mice were spotlighted for approximately 1 min with an infrared lamp. To induce the expression of IL-9, 10 µg of an Il9 minicircle (MC) was applied. To induce the expression of IL-25 and -33, 10 µg of each plasmid, encoding Il25 and Il33, was applied. The IL9 MC and the Il25 and Il33 plasmids were a kind gift from Stefan Wirtz at the department

40 4.5 Flow cytometry and cell sorting onf Internal Medicine 1 at the hospital of the University of Erlangen-Nuremberg in Erlangen (Mched- lidze et al. 2013, and unpublished). All vectors were under the control of an hepatocyte speciﬁc albumin promoter and contained a secretion sequence to ensure the release of the product into the bloodstream.

Plasmid and minicircle production MC were produced from a parental vector by removing the backbone and the resistance genes resulting in a vector that only contains the promoter and the multiple cloning site (Kay et al. 2010). All material required is lited in Table 8. MCs were produced in ZYCY10P3S2T, which is a Escherichia coli strain derived from the strain BW27783, but stabely expresses a set of inducible MC-assembly enzymes, including the PhiC31 integrase and the I-SceI homing endonuclease (Kay et al. 2010). Competent ZYCY10P3S2T E. coli were transformed with a parental pMC.EF1α-MCS-SV40polyA vector containing Il9 in its multiple cloning site. A heat-shock transformation protocol was used with 45 sec heat shock at 37 ◦C and 1 µg input vector. Transformed bacteria were recovered for 1 h in super optimal broth (SOC) medium and then clones were selected on lysogeny broth (LB) agar plates containing 50 µg/ml kanamycin. ZYCY10P3S2T clones were grown in terrific broth (TB) medium containing 50 µg/ml kanamycin and 0.2% (v/v) glycerol at 30 ◦C and agitation until they reached an optical density at 600 nm of 4-6. Recombination was induced adding an equal volume of LB medium containing 0.02% (w/v) L-arabinose for 4 h. IL-25 and -33 expression plasmids were produced in DH5α E. coli and grown in LB medium containing 50 µg/ml kanamycin as over night culture at 37 ◦C. Vectors were isolated using the PureLink HiPure Plasmid Maxiprep-kit according to the manufacturer’s instructions. Residual parental plasmid was removed with a parental vector specific restriction enzyme (PvuII) and a subsequent exo-nuclease digest. Enzymes were removed by ammonium acetate precipitation and free nucleotides were removed by washing the plasmids through a centrifugal filter with a 30 kDa molecular weight cut-off. Plasmids were stored in Tris-EDTA buffer at -20 ◦C.

4.5 Flow cytometry and cell sorting

The applications ”cell sorting” and ”flow cytometry” are highly similar. Both require the dissociation of tissues into single-cell suspensions that are subsequently stained with fluorochrome labelled antibodies. Both application rely on the detection of a fluorescence signal and subsequent grouping of

41 4.5 Flow cytometry and cell sorting

Table 8: List of reagents and material required for the minicircle and plasmid production. LB, lysogeny broth. SOC, super optimal broth. TB, terriﬁc broth. TE, Tris-EDTA.

Type Supplier Order No E. coli strains DH5α New England Biolabs C2987 ZYCY10P3S2T System Biosciences MN900A-1 outgrowth media Agar-agar Carl Roth 5210 Glycerol Carl Roth 6967 L-arabinose Carl Roth 5118 LB medium powder Carl Roth X968 SOC medium New England Biolabs B9020 TB medium powder Carl Roth X972 Kanamycin Carl Roth T832 Plasmid purification 30 kDa Amicon Ultra centrifugal Filter Merck UFC203024 Ammonium acetate 7.5 M Affymetrix 75908 Plasmid Safe DNase Lucigen E3110K PureLink HiPure Plasmid Maxiprep Thermo Fisher K210007 PvuII high fidelity New England Biolabs R3151 TE Buffer Lonza 51235

cells with similar patterns into diﬀerent populations.

4.5.1 Sample preparation

In the present study, knee joints, spleens and LNs were dissociated into single-cell suspensions by enzymatic digestion. Joints and SLOs were dissected out from mice after they were sacriﬁced by cervical dislocation. Fat, tendons and muscle tissue was thoroughly removed from the dissected tissue. The digestion protocol was as follows:

4.5.1.1 Secondary lymphoid organs

LNs were cut in quarters, spleen was cut longitudinally and transversally into nine to twelve pieces. Cutting ensured a good drainage of the digestive medium, which consisted of RPMI 1640 medium supplemented with 0.4 mg/ml collagenase D and 0.2 mg/ml DNase I. A single LN was digested in 1 ml of digestion medium at 37 ◦C for 1 h on a thermoshaker at 1,000 rounds per minute. To ensure a good dissociation of the tissue, pipetting the organ pieces with a 1,000 µl pipette was performed every 20 min. For spleens, the volume of the digest was scaled up. The resulting single-

42 4.5 Flow cytometry and cell sorting cell suspensions were washed in 30 ml of ice-cold RPMI 1640 supplemented with 10 % (v/v) FBS and 10 mM EDTA. Erythrocytes in spleen preparations were lysed using a hypo-tonic ACK lysis buﬀer

(150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA). Finally, single-cell suspensions were ﬁltered through a 70 µm nylon ﬁlter. The viability and cell number was determined with trypan blue and a Neubauer haemocytometer prior to the antibody staining.

For sorting of ILC2s and Tregs from spleen and mesenteric LNs, the dissected SLOs of one mouse were pooled and digested in 3 ml of digestive medium for in total 1 h without shaking. The medium was collected and replaced after every 15 min for four times. Samples were carefully pipetted up and down before every exchange of the digestion medium. The resulting single-cell suspensions were washed in 30 ml of ice-cold RPMI 1640 supplemented with 10 % (v/v) FBS and 10 mM EDTA. Erythrocytes were lysed using a hypo-tonic ACK lysis buffer. The single-cell suspensions were filtered through a 70 µm nylon filter. Cells were washed in 50 ml PBS and centrifuged at low g (100 g instead of 300 g) to remove small debris influencing the sorting purity. The viability and cell number was determined with trypan blue and a Neubauer haemocytometer prior to the antibody staining.

4.5.1.2 Joints

After removing all the surrounding tissue from the legs, especially poplietal LNs, the femur and tibia were cut at the proximal sites to the knee joint leaving fragments that were not longer than a few millimetres. The joint capsule was then opened and digested in total for 2 h in 2 ml of RPMI 1640 medium supplemented with 1 mg/ml collagenase D and 0.2 mg/ml DNase I at 37 ◦C. The medium was collected and replaced after 1 h. Samples were carefully vortexed every 10 min. The resulting single-cell suspensions after 1 h and 2 h were washed in 30 ml of ice-cold RPMI 1640 supplemented with 10 % (v/v) FBS and 10 mM EDTA. Single-cell suspensions were filtered through a 70 µm nylon filter. The single cell suspensions were then further purified using a percoll gradient centrifugation. The bottom layer consisted of 70 % percoll diluted in clear HBSS, the middle layer consisted of 35 % percoll diluted in HBSS with phenol red, the top layer contained the cell suspension in PBS. The percoll was layered in 15 ml reaction tubes and centrifuged at 800 g for 30 min without break. The 70-to-35% interface was collected and washed in PBS. The viability and cell number was determined with trypan blue and a Neubauer haemocytometer prior to the antibody staining.

43 4.5 Flow cytometry and cell sorting

4.5.1.3 Blood

In EDTA collected venous human blood from patients was processed for routine automated complete blood count. In addition, 100 µl of whole blood was directly incubated with antibodies. Erythrocytes were lysed using RBC Lysis and Fixation solution according to the instructions of use. Prior to analysis, samples were ﬁltered through a 40 µm nylon ﬁlter.

4.5.2 Antibody staining

Commonly antibodies for flow cytometry and cell sorting are monoclonal IgGs and originate mostly from rat. In contrast to microscopy of paraffin embedded sections, the antibodies mostly recognize natural surface antigens and are directly labelled. Table 9 and Table 10 list the antibodies used in this study and specifies their supplier, the name of the clone and the isotype as well as their fluorochrome label. Before any staining, low affinity Fcγ receptors were blocked with an anti-CD16/32 antibody. For flow cytometry, surface antibody staining was performed in PBS supplemented with 2 % (v/v) FBS and 5 mM EDTA for 20 min on ice in 96-well V-shaped micro-titre plates. For sorting, staining was performed in larger volumes and hence in 50 ml reaction tubes. For intracellular staining, the IC Fixation kit was used according to the manufacturer’s instructions. For staining of transcription factors, a FoxP3-staining kit was used according to the instructions of use.

44 4.5 Flow cytometry and cell sorting

Table 9: List of flow cytometry and cell sorting antibodies mouse. NA, not applicable. Conjugated dyes: APC, allophycocyanin; CY, cyanine; FITC, fluorescin; PB, pacific blue; PE, phycoerythrin. All other dyes are proprietary names by the respective supplier. For the viability dyes, ”clone” indicates the catalogue number. Dilutions indicate the final concentrations or the dilution used, when no stock concentration was indicated by the manufacturer. Target Supplier Clone Label Dilution Viability/DNA Sigma D9542 NA 0.1 µg / ml Fixable viability Dye eBioscience 65-0865 APC/e780 1 / 4,000 CD3 Biolegend 145-2C11 FITC 5.0 µg / ml APC 0.2 µg / ml CD4 Biolegend RM4.5 FITC 0.5 µg / ml PE/CY7 0.2 µg / ml CD11b Biolegend M1/70 APC 0.2 µg / ml APC/CY7 1.0 µg / ml CD11c Biolegend N418 PB 2.5 µg / ml CD16/CD32 Biolegend 93 no label 10 µg / ml CD25 (in vivo) Biolegend 3C7 PE 2.0 µg / ml PE 0.4 µg / ml CD25 (in vitro) Biolegend PC61 PE/CY7 0.4 µg / ml CD44 Biolegend IM7 PE 0.1 µg / ml CD45 Biolegend 30F11 PE/Dazzel 0.1 µg / ml CD45.1 Biolegend A20 PE/Dazzel 0.2 µg / ml CD45.2 Biolegend 104 APC/Alexa700 0.5 µg / ml CD45R/B220 Biolegend RA3-6B2 FITC 5.0 µg / ml CD49b PB 2.5 µg / ml Biolegend DX5 (pan NK) PE/CY 1.0 µg / ml CD62L eBioscience MEL14 APC/e780 0.1 µg / ml CD90.2 Biolegend 30-H12 PE/CY7 0.2 µg / ml CD127 (IL-7 Rα) Miltenyi A7R34 vioFITC 7.5 µg / ml CD278 (ICOS) Miltenyi 7E.17G9 PercP/Vio700 6.0 µg / ml PB 5.0 µg / ml FcRIa Biolegend MAR1 PE/CY7 2.0 µg / ml FoxP3 eBioscience FJK-16s APC 1.0 µg / ml IFN-γ Biolegend XMG1.2 APC 1.0 µg / ml IL-4 Biolegend 11B11 Alexa488 2.0 µg / ml IL-9 Biolegend RM9A4 PE 1.0 µg / ml IL-17A eBioscience eBio17B7 PE/CY7 0.4 µg / ml 17A2 premixed RB6-8C5 Lineage Biolegend RA3-6B2 PB 1 / 10 Cocktail Ter-119 M1/70

45 4.6 T cell polarisation

PE 2.0 µg / ml KLRG1 Biolegend 2F1 PE/CY7 0.7 µg / ml Ki67 Biolegend 16A8 APC 0.2 µg / m Sca-1 Biolegend D7 PE/CY7 0.2 µg / ml ST2 (IL-33 R) eBioscience RMST2-2 PE 5.0 µg / ml TCR-β Biolegend H57-597 APC 0.2 µg / ml

Table 10: List of flow cytometry antibodies human. Conjugated dyes: APC, allophycocyanin; CY, cyanine; FITC, fluorescin; PE, phycoerythrin. All other dyes are artificial names from the respective supplier. Dilutions are indicated since anti-human flow cytometry antibodies usually lack the indication of a concentration by the manufacturer.

Target Supplier Clone Label Dilution CD3 Biolegend UCHT1 FITC 1 / 50 CD11c Biolegend 3.9 Alexa488 1 / 50 CD14 Biolegend HCD14 FITC 1 / 100 CD19 Biolegend HIB19 FITC 1 / 50 CD34 Biolegend 561 FITC 1 / 50 CD45 Mitenyi 5B1 VioGreen 1 / 500 CD94 Biolegend DX22 FITC 1 / 50 CD117 (c-Kit) eBioscience 104D2 PE/CY5 1 / 20 CD127 (IL-7 Rα) Biolegend A019D5 APC 1 / 20 CD294 (CRTH2) Biolegend BM16 APC/CY7 1 / 20 FcRIa Biolegend AER-37 FITC 1 / 50

4.6 T cell polarisation

For the in vitro polarisation of TH9 and TH17 cells, single-cell suspension from spleens were generated as described in subsubsection 4.5.1.1. The resulting suspensions were magnetically enriched for CD4+ T cells with a negative selection. From those CD4 enriched T cells, 1 x106 cells were cultured in 48-well plates. The wells were pre-coated at 37 ◦C for 1 h with 200 µl of an antibody to CD3 diluted to 5 µg/ml in PBS (Table 11). CD4 enriched T cells were cultured in IMDM supplemented with 100 µg/ml streptomycin, 100 U/ml penicillin, 0.25 µg/ml amphotericin B, 2 mM L-glutamine, 0.5 mM β-mercaptoethanol and 10 % FBS for 72 h at 37 ◦C in a humidiﬁed atmosphere containing

5 % CO2 (Table 5). Following 72 h of culture under polarising conditions, cells were restiumlated with a cell activation

46 4.7 Restimulation of sorted ILC2s and T cells cocktail consisting of 80 nM phorbol 12-myristate 13-acetate (PMA) and 1.3 µM ionomycin for 5 h in the presence of 5 ng/ml brefeldin A and 2 nM monensin prior to their analysis by ﬂow cytometry.

T helper 0 As control served the undirected T cell activation (TH0) with only CD28 co-stimulation at 3 µg/ml.

T helper 9 For the polarisation to TH9 cells, the medium was supplemented as for TH0, but further included 10 µg/ml antibody to IFN-γ, 5 ng/ml recombinant murine TGF-β and 10 ng/ml recombinant murine IL-4.

Conventional T helper 17 For the conventional TH17 diﬀerentiation, the medium was supplemented as for TH0, but further included 10 µg/ml antibody to IFN-γ, 10 µg/ml antibody to IL-4, 5 ng/ml recombinant murine TGF-β and 20 ng/ml recombinant murine IL-6.

Inflammatory T helper 17 For the inflammatory TH17 differentiation, the medium was supplemented as for TH0, but further included 10 µg/ml antibody to IFN-γ, 10 µg/ml antibody to IL-4, 5 ng/ml recombinant murine TGF-β, 100 ng/ml recombinant murine IL-6, 20 ng/ml recombinant murine IL-1β, 20 ng/ml recombinant murine IL-21 and 30 ng/ml recombinant murine IL-23.

4.7 Restimulation of sorted ILC2s and T cells

4.7.1 Culture of type 2 innate lymphoid cells

ILC2s were expanded in vivo as described in subsubsection 4.4.3.4 with HDGT of Il25 and Il33 for 3 d prior to sorting. ILC2s were sorted as KLRG1+ ICOS+ cells lacking CD3, B220, CD1b, CD11c, CD49b and FcRIa from pooled spleens and mesenteric LNs. Post-sort, 0.25 x 106 ILC2s were cultured in U-shape 96-well plates for 72 h in in complete IMDM medium supplemented as for T cell diﬀerentiation (section 4.6). Occasionally, 50 ng/ml recombinant murine IL-9 was added. RNA was isolated as described below (section 4.11).

4.7.2 Restimulation of sorted regulatory T cells

+ Tregs were sorted from na¨ıve spleens as the CD25 high expressing population (hi) of CD4 cells. The

47 4.7 Restimulation of sorted ILC2s and T cells

Table 11: List of antibodies and cytokines required for T cell and ILC assays. CFSE, carboxyﬂuorescein succinimidyl ester. rh, recombinant human. rm, recombinant murine.

Type Clone Supplier Order No anti-CD3 145-2C11 Biolegend 100313 anti-CD28 37.51 Biolegend 102111 anti-GITR-L 5F1 Biolegend 147404 anti-acgitr DTA-1 Biolegend 126303 anti-ICOS-L HK5.3 Biolegend 107407 anti-IFN-γ AN-18 Biolegend 517903 anti-IL-4 11B11 Biolegend 504121 Brefeldin A - Biolegend 420601 CFSE - Thermo Fisher C34554 Cell activation cocktail - Biolegend 423301 CD4 enrichment Kit - Biolegend 480005 Dynabeads T-Activator CD3/CD28 - Thermo Fisher 11452D Monensin - Biolegend 420701 rat IgG2a, κ isotype RTK2758 Biolegend 400515 rm-ICOS-L - abcam ab214990 rm-IL-1β - Biolegend 575102 rh-IL-2 - Biolegend 589102 rm-IL-4 - Biolegend 574302 rm-IL-6 - Biolegend 575702 rm-IL-9 - Biolegend 556002 rm-IL-21 - Biolegend 574502 rm-IL-23 - Biolegend 589002 rm-TGF-β - Biolegend 763102

48 4.7 Restimulation of sorted ILC2s and T cells

6 expression of Foxp3 in Tregs was ensured by ﬂow cytometry post-sort reanalysis. 0.125 x 10 Tregs were cultured in U-shape 96-well plates for 48 h in the presence of 200 ng/ml recombinant human IL-2 in complete RPMI 1640 medium supplemented with 100 µg/ml streptomycin, 100 U/ml penicillin, 0.25 µg/ml amphotericin B, 2 mM L-glutamine and 10 % FBS. Occasionally, 50ng/ml recombinant murine IL-9 was added. To activate Tregs, anti-CD3/28 beads were added at a cell-to-bead ratio of 1:2. RNA was isolated as described below (section 4.11).

4.7.3 Suppression assay

+ + + - GFP CD4 FoxP3 Tregs and CD4 FoxP3 eﬀector T cells (Teﬀs) were sorted from Foxp3 and

GFP -/- 6 Foxp3 x Il9 spleens. Teﬀs were concentrated to 10 x 10 cells/ml and labelled with 5 mM CFSE solved in PBS for 8 min at 37 ◦C. CFSE was quenched by adding 2 volumes of FBS. Cells were washed twice in PBS supplemented with 2 % (v/v) FBS and 5 mM EDTA.

6 GFP 0.25 x 10 CFSE-labelled Teﬀs from Foxp3 spleens were co-cultured in U-shape 96-well plates

GFP GFP -/- for 72 h with Tregs from Foxp3 or Foxp3 x Il9 mice at indicated ratios of Teﬀ :Treg. To activate Tregs, anti-CD3/28 beads were added at a cell-to-bead ratio of 1:2 and 200 ng/ml recombinant human IL-2. Culture medium was the same as for TH polarisation (section 4.6). Occasionally, 0.25 x 106 sorted ILC2s were added together with blocking anti-ICOS-L and anti-GITR-L antibodies or isotypes (Table 11). In other instances, ILC2s were added in the transwell-chambers of a 96-transwell system. In an another set of experiments, suppression assays were performed in the presence of either 50 ng/ml recombinant murine IL-9 or of a combination of recombinant murine ICOS-L at 1 µg/ml and agonistic antibody to GITR at 10 µg/ml.

After 72 h, the suppression of Teff cell proliferation was determined by flow cytometry as the dilution of CFSE and supernatants were collected. Briefly, cells were isolated and dissociated from the beads by pipetting, beads were removed with a magnet, cells were washed, stained for the TCR β-chain to distinguish T cells from ILC2s and analysed by flow cytometry. The fluorescence signals

GFP of the CFSE reporter and the endogenous GFP expression of Foxp3 Tregs being detected in the same channel could be distinguished by their diﬀerent qualities of spillover to the adjacent channel (CFSE high, GFP low). The cell proliferation was assessed by dilution of CFSE among generations.

The suppression was calculated using the division index (DI) with unsuppressed Teﬀ as reference: suppression (index) = 100 – DIprobe/DIreference x 100.

49 4.8 Enzyme-linked immunosorbent assays

4.8 Enzyme-linked immunosorbent assays

The cytokine expression was assessed by ELISA. IL-9, -25 and -33 serum levels were measured with a sandwich ELISA according to the manufacturer’s instructions of use. Cytokine profiling was performed with a multiplex flow cytometry bead assay. Data analysis was done using Kaluza software. For the protein extraction from knees, joints were dissected and snap frozen in liquid nitrogen. Frozen joints were homogenized in a non-denaturing protein extraction buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 % (v/v) Triton X-100, 0.5 % (w/v) SDS, 1 mM PMSF, 1 % (v/v) protease inhibitor cocktail) using ceramic beads and a tissue homogenizer. Homogenized samples were centrifuged (18,000 g, 20 min, 4 ◦C) to remove debris. The supernatant was frozen at -80 ◦C until analysis by ELISA.

4.9 Histology and microscopy

The tissues analysed in the present study comprised human arthroscopic and surgical biopsies of synovial tissue as well as knee joints and paws from mice.

4.9.1 Sample preparation

All tissue was fixed for 16 - 24 h in PBS containing 4 % (w/v) formaldehyde. Cutting bony tissue requires either harsh embedding and cutting methods or the fixation and decalcification of tissue as performed in this study. Murine tissue containing bony parts was decalcified in 0.5 M EDTA prior to embedding and cutting. For the decalcification, the tissue was kept at 4 ◦C. The EDTA solution was replaced twice per week. After 14 d the tissue was washed in deionized water. All tissues were kept in 70 % (v/v) ethanol until dehydration and paraffin embedding. Dehydration was achieved with an automated system by a series of ethanol bathes with decreasing water content, ethanol/xylol clearing and finally infiltration with paraffin.

4.9.2 Thin section cutting

Paraﬃn embedded tissue blocks were cooled down to -15 ◦C on a tissue cooling plate prior to cutting. Human tissue was cut to thin sections of 5 µm thickness, murine bone tissue to 1 µm thickness.

50 4.9 Histology and microscopy

Sections were unfolded in a water bath, mounted on glass slides and dried for at least 24 h at ambient temperature.

4.9.3 Deparaﬃnization and rehydration

Paraffin embedded thin sections were deparaffinized by heating the slides for 30 min at 65 ◦C. The slides were then washed twice with xylol for 10 min to remove residual wax. For subsequent rehydration, the slides were sunk ethanol baths with increasing water content for 5 min each (2x 100 % (v/v) ethanol, 1x 95 % (v/v) ethanol, 1x 80 % (v/v) ethanol). Finally, the slides were briefly washed in deionized water.

4.9.4 Conventional histological stainings

Deparaﬃnized and rehydrated thin sections were stained with haematoxylin and eosin or safranin O. TRAP activity was revealed with an in in situ precipitation assay.

4.9.4.1 Haematoxylin and eosin staining

The haematoxylin solution (0.1 % (w/v) haematoxylin, 0.02 % (w/v) NaIO3, 105 mM KAl(SO4)2, 300 mM Chloral hydrate, 0.1 % (w/v) citric acid) was filtered through Whatman filter paper and sections were incubated for 10 min in the filtered solution. Excessive stain was removed by rinsing the slides briefly in deionized water, followed by a decolonization solution (70 % (v/v) isopropanol containing 1 % (v/v) hydrochloric acid) and finally washed for 10 min in a bath fuelled with running tap water. Eosin counter stain (0.3 % (w/v) (w/v) Eosin, 0.01 % (v/v) acetic acid) was incubated for 3 min and washed in deionized water. For mounting, the tissue was dehydrated by a series of baths with increasing isopropanol content and xylol (1x 70 % (v/v) isopropanol, 1x 80 % (v/v) isopropanol, 1x 90 % (v/v) isopropanol, 2x 100 % isopropanol, 1x xylol). Slides were mounted with a water free mounting medium.

4.9.4.2 Safranin O staining

Safranin O was diluted in deionized water to 0.1 % (w/v) and deparaﬃnized and rehydrated tissue thins sections were bathed in this solution for 45 min. Excessive stain was removed by bathing the slides in deionized water for a short time. Slides were counter stained with Fast Green (0.1 % (w/v)

51 4.9 Histology and microscopy diluted in deionized water) for 10 min and excessive dye was removed bathing the slides in deionized water. Mounting was performed as described above (subsubsection 4.9.4.2).

4.9.4.3 Detection of tartrate-resistant acetic phosphatase

TRAP activity was revealed using a commercial kit according to the manufacturer’s instructions (Table 4). Brieﬂy, other endogenous phosphatases were blocked with a tartrate solution (Naphthol AS-BI phosphoric acid, acetate solution and tartrate solution) at 37 ◦C for 1 h. Enzymatic activity was revealed by adding Fast Green and sodium nitrite solutions for 2 to 5 min. Slides were mounted with aqueous mounting media.

4.9.5 Immunoﬂuorescence staining

Dehydration and paraﬃn embedding leads to antigen denaturation. To stain tissue sections with antibodies, the epitopes must be recovered.

4.9.5.1 Epitope retrieval

The epitopes for immunofluorescence staining were retrieved from deparaffinized and rehydrated sections using a heat-induced method. The slides were 5-times alternately bathed for 2 min each in a boiling sodium citrate solution (10 mM sodium citrate, pH 6.0) and a boiling Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA, 0.05 % (v/v) Tween-20, pH 9.0). Finally, the slides were briefly washed in deionized water.

4.9.5.2 Antibody staining

Despite of the epitope retrieval, antibodies are specifically raised to recognize paraffin embedded tissue epitopes. For signal amplification, immunoflorescence microscopy mostly bases on antigen- specific polyclonal primary antibodies from different species and species-restricted polyclonal secondary antibodies coupled to fluorochromes for signal detection. Table 12 lists the primary and secondary antibodies used in this study and specifies their supplier, the species as well as their fluorochrome label. To avoid unspecific binding of the antibodies, the thin sections were blocked for 1 h in PBS supplemented with 5 % (w/v) BSA and 2 % (v/v) horse serum. The primary antibodies were incubated overnight at 4 ◦C. The sections were then intensely washed in PBS and then incubated

52 4.10 X-ray microtomography

Table 12: List of antibodies used for immunoﬂuorescence staining. The column ”clone” denominates monoclonal antibodies or catalogue entries for polyclonal antibodies. NA, not applicable. SN, cell culture supernatant.

Target Reactivity Host Supplier Clone Label Dilution CD3 hu mu abcam Ps1 (SN) no lable 1 / 50 CD3e mu ah Biolegend 145-2C11 no lable 10 µg / ml IL-9 hu, mu rb abcam ab203386 no lable 10 µg / ml ICOS hu, mu gt abcam ab111247 no lable 5.0 µg / ml FoxP3 mu rt eBioscience FJK-16s no lable 2.5 µg / ml Tryptase hu, mu mu Thermo AA1 no lable 0.025 µg / ml CD11b hu, mu rt Biolegend M1/70 no lable 5.0 µg / ml CD16 hu ms Santa Cruz 2Q1240 no lable 2.0 µg / ml IgG rb dk abcam ab150062 Alexa555 4.0 µg / ml IgG rb gt invitrogen A21244 Alexa647 4.0 µg / ml IgG gt dk abcam ab150129 Alexa488 4.0 µg / ml IgG gt dk invitrogen A11058 Alexa594 4.0 µg / ml IgG rt dk invitrogen A21208 Alexa488 4.0 µg / ml IgG rt dk invitrogen A21209 Alexa594 4.0 µg / ml IgG mu dk abcam ab150105 Alexa488 4.0 µg / ml IgG ah gt abcam ab175680 Alexa405 4.0 µg / ml IgG ah gt abcam ab173004 Alexa647 4.0 µg / ml DNA NA NA Sigma D9542 DAPI 0.1 µg / ml

with the secondary antibodies and occasionally in combination 1 µg/ml 4’,6-diamidino-2-phenylindole (DAPI) for 2 h at ambient temperature. The antibodies and their dilutions are listed in Table 12 and the staining are described in chapter 5. For imaging, the slides were mounted in aqueous ﬂuorescence mounting medium and sealed with commercial nail polish.

4.10 X-ray microtomography

µCT analysis was performed on tibia (AIA) and an area of the paw including calcaneus, the tarsal bones and the distal part of the tibia (SIA). The acquisition parameters were as follows: voltage 40 kV, x-ray current 250 µA, exposure time 5000 ms/projection, matrix 1024 x 1024 pixel and voxel size in reconstructed image 9 µm. The number of projections was dependant on the bone analysed. For tibial bone, mineral density and degree of erosion was calculated as the ratio of bone volume to total volume and the mean thickness of single trabecel, respectively.

53 4.11 Semi-quantitative real-time expression analysis

4.11 Semi-quantitative real-time expression analysis

Total RNA was isolated with silica membrane columns according to the instructions of the manufacturer (Table 4). Reverse transcription into cDNA was performed using random hexamers primers. The instrument used for reverse transcription and PCR as well as the light cycler are listed in Table 1. Specific PCR products were detected using SYBR green and custom primers (Table 13). Specificity of the products and primers was controlled using untranscribed sample and water control. Refer- ence gene was beta microglobulin (B2m) for T cells and ex vivo analysis of tissue extracts. Gene expression in ILC2 cultures was normalized to (Hprt1) (Wilhelm, Hirota et al. 2011). All samples were measured in triplicates. Differences in gene expression were calculated with the comparative Ct method (Schmittgen et al. 2008).

4.11.1 Primers

All primers were synthesized by Metabion, Planegg, Germany. The primers used for real time PCR are listed in Table 13.

4.12 Statistical analysis

Data are always represented as mean and mean of the standard errors (SEM). When two groups were compared, two-tailed Student’s t-test was used. In cases were patient follow-ups were tested, the paired test was used, in all other cases unpaired tests were performed. When more than two groups were compared, variance was analysed by ordinary analysis of variance (ANOVA) test and signiﬁcance was tested with Tukey’s post hoc test. Alpha-levels were encoded by symbols as follows: ns, p ≥0.05 *, p < 0.05; **, p < 0.01; ***, p < 0.001.

54 4.12 Statistical analysis

Table 13: List of real time primers.

Gene Accession Sequence Product reference genes 5’-GGTGCTTGTCTCACTGACCG-3’ B2m NM 009735.3 50 bp 5’-TTTGAGGGGTTTTCTGGATAGCAT-3’ 5’-TCAGTCAACGGGGGACATAAAAG-3’ Hprt NM 013556.2 142 bp 5’-GGGGCTGTACTGCTTAACCAG-3’ target genes 5’-TCACTGTGCGGGAAGGTCTAT-3’ Cma1 NM 010780.3 78 bp 5’-AGCTTCTGCCACGTGTCTTC-3’ 5’-TGGCTACACATTCAAACTGCCT-3’ Cpa3 NM 007753.2 55 bp 5’-CCTTGCAACTTTCAATAGGTCCTG-3’ 5’-CACCCAGGAAAGACAGCAACCT-3’ Foxp3 NM 054039.2 93 bp 5’-CCTTCTCACAACCAGGCCACTT-3’ 5’-GTCACTGGAAGGTCTGCCCA-3’ Fcer1a NM 010184.2 59 bp 5’-ATGACATCAAGAGACATGAACAGCA-3’ 5’-GAAGCCGTACTTCTGCCGTG-3’ Icos NM 017480.2 74 bp 5’-CCGAGCCATTGATTTCTCCTGTT-3’ 5’-AGTCCTTGTCCCCGTCCTTG-3’ Icosl NM 015790.3 134 bp 5’-GTCAGGCGTGGTCTGTAAGTTC-3’ 5’-TGGGCAGTCCCAGAAAGAAAAG-3’ Mcpt4 NM 010779.2 50 bp 5’-TCCTCCAGAGTCTCCCTTGTATG-3’ 5’-CAGACTTTGGACCAACTGTTCTCAG-3’ Tnfrsf18 NM 009400.3 50 bp 5’-CAGGGAACATGGTGAGAAATCCAA-3’ 5’-ACTGCCATCGAGTCCTGCAT-3’ Tnfsf18 NM 183391.3 194 bp 5’-ACTACGAAGGGGGCATTGTCT-3’ 5’-GACTCCTGCCAGGGCGATTC-3’ Tpsb2 NM 010781.3 63 bp 5’-CTGCAGCCAGGTACCCTTCA-3’

55 5 Results

5.1 Interleukin-9 deﬁciency leads to chronic inﬂammation in antigen

induced arthritis

A murine standard model of RA is AIA (Brackertz et al. 1977). It is very well reflecting T lymphocyte- driven aspects of inflammation during RA and also is characterized by clinical features such as immune complex depositions on the cartilage, progressive cartilage and bone erosions as well as synovial infiltrations. Being based on an immunization-challenge protocol, it disposes of tightly controlled kinetics, yet it undeniably lacks other aspects of human RA such as the auto-immune nature or genetic predispositions (Cope 2007, pp. 243-254). Briefly described, mice are immunized against mBSA by two consecutive s.c. injections (Figure 5). 21 days after starting the immunization (day 21), mBSA or PBS serving as control is injected into the knee joint. A rapid onset of inflammation is observed within 24 h accompanied by oedema which in turn serves as a measure of the severity of AIA. Swelling usually declines within the following 14 d (until day 42) in WT animals. In contrast, it could be observed that in mice deficient of the cytokine IL-9 (Il9 -/-), symptoms of AIA remained constantly present beyond day 42 (Figure 6). Histological analysis of knee joints collected at day 42 confirmed the persistence of inflammation in Il9 -/- mice in contrast to a mostly resolved inflammation in WT mice (Figure 7 A and B). In line with the chronicity of the inflammation, an extended cartilage damage was observed in Il9 -/- mice, as determined by the decrease of the Safranin O staining and a reduction of the number of chondrocytes (Figure 7 C and D). Enzymatic revelation of TRAP revealed an increased number of TRAP-positive osteoclasts in the trabecular part of the tibiae in Il9 -/- mice (Figure 7 E and F). Accordingly, the high resolution µCT analysis demonstrated a substantial destruction of the trabecular meshwork in Il9 -/- mice challenged with mBSA (Figure 8).

56 5.1 Interleukin-9 deﬁciency leads to chronic inﬂammation in antigen induced arthritis

Time [d]* 0 7 21 23 42

Knee joint swelling

1st Immunization Boost Induction 21 42

* Days with red background: blood serum collected Figure 5: The antigen induced arthrits model of rheumatoid arthritis. Schematic representation of the AIA model as used in this study.

A 2.0 B 30 WT *** WT 1.5 Il9 -/- -/- 20 Il9 1.0

AUC 10 0.5 [jointswelling]

Jointswelling [mm] 0.0 0 21 26 31 36 41 Time [d] Figure 6: Antigen induced arthrits in wildtype and Il9 -/- mice. AIA was induced in WT and Il9 -/- mice according to Figure 5. Knee joint swelling was measured from the time point of inoculation of mBSA into the knee joint at day 21 till the expected resolution of AIA at day 42 in WT mice. (A) Joint swelling [mm] over time [d]. (B) Integral quantiﬁcation of the swelling shown as area under the curve (AUC). Data are represented as mean, error bars indicate the SEM. ***, p < 0.001 determined by Student’s t-test. (A+B) WT n = 9. Il9-/- n = 7.

57 5.1 Interleukin-9 deﬁciency leads to chronic inﬂammation in antigen induced arthritis

A Ctrl AIA

B WT 200 ** WT ] 2 150 Il9 -/- mm

3 100

[x10 50 Il9-/-

Inflamedsynovium 0

C D WT 4 80 80 * *** * 3 60 60

2 40 40 [score] 1 20 20 Il9-/-

0 Chondrocytenumber 0 0 Cartilagethickness [µm] Loss of safraninstaining Lossof O

F WT 150 15 *** *** 100 10

50 5 N.Oc / B.Pm / N.Oc Il9-/- [%] BS / Oc.S 0 0

Figure 7: Histological analysis of knee joints from the antigen induced arthrits model of rheumatoid arthritis at day 42. (A) Representative images of haematoxylin and eosin staining for the quantification of inflammation. (B) Quantification of the inflamed synovium [mm]. (C) Representative images of safranin O staining with methylene blue counter stain to detect chondrocytes and to determine the amount of cartilage degradation. (D) Quantification of the loss of safranin O staining in joints [score]. Quantification of chondrocytes [number] and the cartilage thickness [µm] of the articular cartilage. (E) Representative images of the TRAP staining detecting osteoclasts. (F) Quantification of osteoclasts [number] per bone perimeter (N.Oc/B.Pm). Quantification of the cumulative osteoclast surface in relation to the bone surface [%] (Oc.S/BS). Scale bars indicate 100 µm. Data are represented as mean, error bars indicate the SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001 determined by Student’s t-test. (B) WT n = 6. Il9-/- n = 6. (D) WT n = 7. Il9-/- n = 6. (F) WT n = 5. Il9-/- n = 5.

58 5.1 Interleukin-9 deﬁciency leads to chronic inﬂammation in antigen induced arthritis

A Ctrl AIA B 5 50 WT ** ** Il9 -/- 4 45 WT 3 40 2 BV/TV [%] BV/TV 1 [µm] Tb.Th 35 0 30

Il9-/-

Figure 8: X-ray microtomography analysis of tibiae from the antigen induced arthrits model of rheumatoid arthritis at day 42. (A) Representative µCT images showing the destruction of the trabecular meshwork in the tibia. (B) Quantitation of the bone volume (over total, BV/TV) and the trabecular thickness (Tb.th) in WT and Il9 -/- mice. Data are represented as mean, error bars indicate the SEM. **, p < 0.01 determined by Student’s t-test. (B) WT n = 6. Il9-/- n = 6.

Since AIA is strongly T cell dependant and IL-9 is a cytokine with pleiotropic effects on T cells (reviewed in section 3.2), the observations from the Il9 -/- mice arouse the suspicion that the phenotype was related to alterations of either the T cell development or the immunization efficiency in these mice. To rule out such an effect in this model, WT and Il9 -/- mice were subjected to AIA and additionally received IL-9 by HDGT only at day 22, that is after inoculation with mBSA. The HDGT of an Il9 MC led to a fast increase of the IL-9 serum levels (Figure 9 A) and thus to a systemic availability of the cytokine. There was no significant alteration of the clinical features in WT mice treated with the HDGT of Il9 MC as compared to normal WT mice. Importantly, the clinical feature of Il9 -/- mice were rescued by the systemic availability of IL-9 (Figure 9 B and C). These results indicated that the phenotype of Il9 -/- did not originate from an altered response of T cells to the immunization in AIA. Next, the effects of IL-9 deficiency were studied in an acute inflammatory setting. Gouty arthritis is an inflammatory arhritis that is mainly triggered by neutrophile activation (Schauer et al. 2014). Intra articular inoculation of MSU crystals induces the rapid recruitment and activation of neutrophiles and consequently a local inflammation. There was no detectable difference between WT and Il9 -/- mice in terms of severity of the acute inflammatory arthritis model (Figure 10), explaining why IL-9 did not interfere with the very acute onset of AIA within the very first days. Collectively these experiments showed that the deficiency of IL-9 is responsible for a chronic phenotype in a RA-like model in mice. The chronicity is supposedly not related to an intrinsic

59 5.1 Interleukin-9 deﬁciency leads to chronic inﬂammation in antigen induced arthritis

A B C *** 1.5 2.0 WT 30 ns *** WT 1.5 Il9 -/- Il9 -/- 1.0 20 Il9 Il9 1.0 WT + MC WT + MC -/- 0.5 Il9 + Il9 MC AUC 10 Il9 -/- + Il9 MC 0.5 [jointswelling]

0 Jointswelling [mm] 0.0 0 21 23 28 35 21 26 31 36 41

IL-9 serumIL-9level [µg/ml] Time [d] Time [d] Figure 9: A re-established spontaneous resolution of antigen induced arthrits in Il9 -/- mice after the hydrodynamic gene transfer of Il9. AIA was induced in WT and Il9 -/- mice as described before (Figure 5). Some mice additionally received an IL-9 over-expressing MC by HDGT (Il9 MC) at day 22. (A) IL-9 levels in the serum of Il9 -/- treated with Il9 MC. (B) Joint swelling [mm] was monitored over time [d] starting with the inoculation of mBSA into the knee joint (day 21). (C) Quantiﬁcation of (B) shown as AUC. Data are represented as mean, error bars indicate the SEM. ns, p ≥ 0.05; ***, p < 0.001 determined by one-way ANOVA with Tukey’s post hoc test. (A) n = 5. (B+C) WT n = 6. Il9-/- n = 8. WT + Il9 MC = 7. Il9-/- + Il9 MC = 8.

A B 1.0 8 WT + MSU crystals ns WT + MSU crystals Il9 -/- + MSU crystals 6 Il9 -/- + MSU crystals WT + PBS WT + PBS 0.5 Il9 -/- + PBS 4 Il9 -/- + PBS AUC 2 [pawswelling]

Pawswelling [mm] 0.0 0 0 1 3 5 7 9 Time [d] Figure 10: The gout-like inflammatory arthritis induced by monosodium urate crystals in wildtype and Il9 -/- animals. A gout-like inflammation was induced in WT and Il9 -/- mice by the injection of MSU crystals into the paw. Saline injections with PBS into the contralateral paw served as controls. (A) Paw swelling [mm] was measured over time [d]. (B) Quantification of (A) shown as AUC. Data are represented as mean, error bars indicate the SEM. ns, p ≥ 0.05 determined by one-way ANOVA with Tukey’s post hoc test. (A + B) WT n = 9. Il9-/- n = 9.

60 5.2 Interleukin-9 limits chronic inﬂammation in the serum transfer induced arthritis developmental defect of T cells.

5.2 Interleukin-9 limits chronic inﬂammation in the serum transfer

induced arthritis

When mice, which carry the major histocompability complex (MHC) class II allel H-2g7 are in- tercrossed with KRN mice carrying a transgenic TCR, the F1 generation (K/BxN) spontaneously develops an inflammatory and chronic arthritis (Kouskoff et al. 1996). The serum generated from the F1 generation induces after transfer a similar phenotype in other mice strains, which are devoid of this specific MHCII-TCR constellation. The phenotyp is thought to be induced by the abundant anti-glucose-6-phosphate isomerase (GPI) auto-antibodies in the serum (Cope 2007, pp. 269-282). In contrast to AIA, the serum transfer induced arthritis (SIA) model is relatively devoid of T cell activation in its initiation. It thus reflects the effector phase of arthritis with clinical manifestations that are mostly induced by the immune complex deposition on the cartilage surface, similar to AIA (Matsumoto et al. 2002). In this regard, the SIA model was used to investigate the therapeutic potential of IL-9 in arthritis. Wildtypic Balb/c mice received either the Il9 MC by HDGT, serum from the K/BxN line (SIA), or a combination of both. An empty parental MC vector served as control for the HDGT. The paw swelling, which usually lasts for more than 14-21 d in SIA, was monitored over time. While the paw swelling was constantly high in mice receiving the empty vector together with the K/BxN serum, mice that received instead the Il9 MC showed only a very moderate swelling that rapidly declined till day 10 (Figure 11). Histological analysis revealed the protective activity of IL-9. Infiltrations of inflammatory cells into the paw as well as the cartilage destruction were significantly reduced in mice treated with Il9 HDGT (Figure 12 A-D). Likewise, the presence of TRAP+ osteoclasts was reduced and the calcaneus bone was protected from erosive remodelling (Figure 12 E-G). Taken together, while the deficiency of IL-9 was responsible for the chronicity of arthritis, its over-expression allowed a therapeutic intervention and led to the faster resolution of arthritis.

61 5.3 Alterations of the cytokine signature in chronic arthritis

A 1.5 B 10 *** ctrl + ctrl MC 8 *** ctrl + ctrl MC *** Il9 1.0 *** ctrl + MC 6 ctrl + Il9 MC

SIA + ctrl MC AUC 4 SIA + ctrl MC 0.5 Il9 [pawswelling] Il9 SIA + MC 2 SIA + MC

Pawswelling [mm] 0.0 0 2 4 6 8 10 Time [d] C Ctrl + ctrl MC Ctrl + Il9 MC SIA + ctrl MC SIA + Il9 MC

Figure 11: Resolution of serum transfer induced arthritis by the hydrodynamic gene transfer of Il9. Arthritis was induced by the transfer of serum generated from K/BxN mice. Arthritic and non- arthritic mice received Il9 by HDGT (Il9 MC) leading to a systemic availability of IL-9. (A) Paw swelling [mm] was monitored over time [d]. (B) Data shown in (A) represented as AUC. (C) Representative photographs showing the paw swelling at day 8. Data are represented as mean, error bars indicate the SEM. ***, p< 0.001 determined by one-way ANOVA with Tukey’s post hoc test. (A + B) ctrl + ctrl MC n = 10. ctrl + Il9 MC n = 10. SIA + ctrl MC n = 16. SIA + Il9 MC n = 16.

5.3 Alterations of the cytokine signature in chronic arthritis

To get a deeper insight into how IL-9 promotes the resolution of arthritis, WT and Il9 -/- mice were subjected to AIA again and their serum was collected at days 0, 22 and 42. The kinetics of type 1, 2 and 3 immunity signature cytokines were determined by multiplex ELISA assays (Figure 13 A). As expected, there was a strong increase of the type 1 cytokines IL-6, IFN-γ and TNF-α directly after the inoculation of mBSA into the knee joint. The type 1 response was only transient and declined thereafter. Strikingly, the type 3 cytokine IL-17A displayed constantly elevated serum levels in Il9 -/-, but not in WT mice. Next, the cytokine release was directly measured in the joint tissue (Figure 13 B). In concordance with the levels in the serum, the levels of IL-17A in the joint were signiﬁcantly higher and remained on a high level throughout the whole measuring period in Il9 -/- as compared to WT mice. The elevated IL-17A levels appeared to be a result of the enduring presence

+ of CD4 TH17 cells in the draining LNs and the aﬀected joints (Figure 13 C-E).

-/- These results raised the question whether Il9 mice would be intrinsically biased towards a TH17 polarisation. Indeed, it was reported that IL-9 signalling would stabilise the TH17 response in the EAE model (Nowak et al. 2009). Therefore, CD4 enriched splenocytes from WT and Il9 -/- mice

62 5.3 Alterations of the cytokine signature in chronic arthritis

Ctrl + ctrl MC Ctrl + Il9 MC SIA + ctrl MC SIA + Il9 MC B 1.5 ctrl + ctrl MC A Il9 * ctrl + MC 1.0 SIA + ctrl MC 0.5 SIA + Il9 MC

Infiltrates [AU] Infiltrates ns C 0.0

D 4 ***

3 E 2 Loss of 1 ns

Safranin[score] O 0 F 2.0 G * 1.5 area + 1.0

0.5 ns TRAP

[% of paw of perimeter] [% 0.0 Figure 12: Histological analysis of paws from the serum transfer induced arthritis model of rheumatoid arthritis. (A) Representative images of the haematoxylin and eosin staining for the quantification of inflammation. (B) Quantification of the inflammaroty area in arbitrary units (AU). (C) Representative images of the safranin O staining with methylene blue counter stain to detect chondrocytes and to determine the cartilage degradation. (D) Quantification of the loss of safranin O staining in paws [score]. (E) Representative dark field images of the TRAP reaction (orange) staining osteoclasts. (F) Quantification of the TRAP+ area over paw perimeter on histological sections [%]. (G) Representative µCT images showing the erosions of the calcaneus (arrow). Data are represented as mean, error bars indicate the SEM. ns, p ≥ 0.05; *, p < 0.05; ***, p < 0.001 determined by one-way ANOVA with Tukey’s post hoc test. (B+D) ctrl + ctrl MC n = 4. ctrl + Il9 MC n = 4. SIA + ctrl MC n = 4. SIA + Il9 MC n = 4. (F) ctrl + ctrl MC n = 4. ctrl + Il9 MC n = 4. SIA + ctrl MC n = 10. SIA + Il9 MC n = 10.

63 5.3 Alterations of the cytokine signature in chronic arthritis

A Time [d] 0 22 42 B 150 WT ns ns ns

-/- -/- -/- Il9-/- 0-1,99 WT Il9 WT Il9 WT Il9 100 2-3,99 IL-2 4-7,99 IL-4 50 8-15,99

IL-6 [pg/ml] TNF-a 16-31,99 IL-9 0 32-63,99 IL-17A ns

Conc. [pg/ml] Conc. 64-127,99 IFN-γ 1500 ns ≥128 TNF-α ns 1000

C WT Il9-/- 500 CD4+ cells / knee CD4+ cells / knee [pg/ml] IL-6 10 3 10 3 0 10 2 10 2

1 1 400 10 10 ns 300 10 0 10 0

0 0 200 ns

IL-17A ns 0 10 0 10 1 10 2 10 3 0 10 0 10 1 10 2 10 3 IFN-g [pg/ml] IFN-g 100 IL-5 0 D 10 WT 40 ns 8 Il9-/- ns 30 6 ns ns

17/ 20 ns H cells[%] ** + 4 T

2 [pg/ml] IL-4 10 Lymph node Lymph CD4 0 0

E 10 150 ns ** 8 * ns 100 * 6 * 17/ H 4 T 50

Kneejoint 2 IL-17A [pg/ml] IL-17A synovialcells[%] 0 0 ctrl AIA ctrl AIA ctrl AIA ctrl AIA ctrl AIA ctrl AIA 24 27 42 24 27 42 Time [d] Time [d] Figure 13: Analysis of the cytokine patterns in antigen induced arthrits of wildtype and Il9 -/- animals. (A) Heat-map of the multiplex ELISA measuring the serum cytokines. Serum was collected from WT and Il9 -/- mice at days 0, 22 and 42 after the first immunization in AIA. (B) Multiplex ELISA of protein extracts generated from the knee joints of WT and Il9 -/- mice at days 24, 27 and 42 of AIA. (C) Representative flow cytometry plots of CD4+ synovial cells stained for IL-17A and -5 at -/- + day 42 of AIA in WT and Il9 mice. (D) Percentage of TH17 cells among CD4 cells in control and draining LNs of WT and Il9 -/- mice assessed by flow cytometry at days 24, 27 and 42 of AIA. -/- (E) Percentage of TH17 cells among synovial cells in control and arthritic joints of WT and Il9 mice assessed by flow cytometry at days 24, 27 and 42 of AIA. Data are represented as mean, error bars indicate the SEM. ns, p ≥ 0.05; *, p < 0.05; **, p < 0.01 determined by Student’s t-test. (A) n = 3-6 per cytokine/ time point/ genotype. (B) n = 4-7 per cytokine/ time point/ genotype. (D) n = 3-7 per cytokine/ time point/ genotype. (E) n = 3-7 per cytokine/ time point/ genotype.

64 5.3 Alterations of the cytokine signature in chronic arthritis

ns 10 ns WT 8 Il9-/- 6 4 2 ns Th17cells[%] 0 conventional inflammatory

TH0 TH17 -/- Figure 14: In vitro TH17 polarization capacity in wildtype and Il9 mice. + -/- Magnetically separated CD4 splenocytes from WT and Il9 mice were cultured under TH0, conventional TH17 (TGF-β + IL-6) and inﬂammatory TH17 (TGF-β + IL-6 + IL-1β + IL-21 + IL-23) polarizing conditions for 3 d. Data are represented as mean, error bars indicate the SEM. ns, p ≥ 0.05 determined by Student’s t-test. WT n = 4. Il9-/- n = 4.

were cultured under TH17-polarising conditions (Figure 14). There was a slight trend of a lowered induction eﬃciency under conventional conditions in Il9 -/- mice, but not under inﬂammatory conditions. Hence it was unlikely that an intrinsic variance of the TH17 cells would be the key trigger of chronic arthritis in the Il9 -/- mice.

-/- Though there was an exacerbated TH17 response in Il9 mice, the TH17 cells initially did not account for the alterations among the total CD4+ T cell compartment (Figure 15 A). In mice, inexperienced na¨ıve T cells are characterised by the expression of L-selectin (CD62L) and experienced T cells acquire CD44 while loosing CD62L (Swain et al. 1996). Memory T cells are a CD44 high expressing population (hi) that has completely lost the L-selectin expression. Effector cells at the transition are a CD44 low expressing population (lo). From the composition of the T cell subsets in draining LNs of arthritic joints, no definitive deduction was possible as there were only slight but no significant changes in the ratios of na¨ıve (CD44lo CD62L+) vs. effector (CD44lo CD62L-) vs. memory (CD44hi CD62L-) T cells (Figure 15 B and C). The increase of the CD44 high expressing population (hi) in Il9 -/- may not necessarily be interpreted as an increase of memory T cells, but rather might reflect a higher percentage of activated T cells in AIA, which in turn substantiates the higher levels of IL-17A.

-/- In summary, the chronic arthritic phenotype of AIA in Il9 mice was linked to a persistent TH17 response. Since this observation could not be linked to an intrinsic defect of TH17 cells, it seemed more likely that it was linked to an intrinsic failure to stop the lymphocyte response.

65 5.4 Regulatory T cells are impaired in interleukin-9 deﬁcient mice

A ns WT B C WT Il9-/- 6.0 10 3 6.0 Il9-/- Memory Memory 10 2 Naïve 4.0 4.0 Effector 1 10 cells + Naïve 2.0 ns 10 0 2.0 / lymph node] / / lymph node] / CD4 6 6 CD4+cells 0 Effector 0 CD44 0 [x10 [x10 ctrl draining 0 10 0 10 1 10 2 10 3 ctrl draining CD62L Figure 15: Composition of the CD4+ T cell compartment in wildtype and Il9 -/- mice during antigen induced arthrits. (A) Number of CD4+ cells per poplietal LN in AIA at day 27. (B) Representative flow cytometry plot of the CD4+ T cells in the draining lymph node of AIA showing the distribution of CD44 and hi CD62L expressing cells. Three different TH subsets can be identified: memory T cells (CD44 CD62L-), effector T cells (CD44lo CD62L-) and na¨ıve T cells (CD44lo CD62L+). (C) Quantitative analysis of the CD4+ T cell compartment as shown in (B). Data are represented as mean, error bars indicate the SEM. ns, p ≥ 0.05 determined by Student’s t-test. (A) WT n = 5. Il9-/- n = 5. (C) WT n = 5. Il9-/- n = 5.

5.4 Regulatory T cells are impaired in interleukin-9 deﬁcient mice

Indeed, it was suggested that Tregs would display a higher capacity to suppress the activity of responder cells when stimulated by IL-9 in vitro (Elyaman et al. 2009). Therefore, Treg numbers were assessed in the draining lymph nodes of healthy and arthritic joints of mice subjected to AIA (Figure 16 A). There was no signiﬁcant diﬀerence in terms of numbers between WT and Il9 -/- mice. Instead and in accordance with the aforementioned report, there was a reduced capacity to

-/- suppress the proliferation of responder Teﬀ cells by Tregs isolated from Il9 mice as compared to those isolated from WT mice (Figure 16 B and C). Yet and in contrast, the addition of recombinant murine IL-9 to the assay failed to unambiguously deduce a direct eﬀect of the interleukin on Tregs

(Figure 17 A). When Tregs were sorted and stimulated for 48 h, there was a down-regulation by half of the co-stimulatory receptors GITR (Tnfrsf18) and ICOS (Icos) in Il9 -/- mice compared to WT. In the presence of recombinant IL-9 this down-regulation could not be rescued. By contrast, the expression of Foxp3 remained invariant under all circumstances (Figure 17 B).

-/- At this point, the chronic arthritis still could have been related to resistant Teﬀ cells in Il9 and not to the deﬁciency of Treg to suppress. To reject this hypothesis, the reciprocal suppression assay

-/- was performed with Teﬀs isolated from Il9 and WT animals, while Tregs were exclusively isolated from the non-mutants (Figure 18 A). In this setting, there was no impaired suppression observed.

-/- Furthermore, WT and Il9 Teﬀs produced similar amounts of the cytokines IFN-γ, TNF-α as well

66 5.4 Regulatory T cells are impaired in interleukin-9 deﬁcient mice

A B C WT T + WT T ns 40 eff reg 8.0 WT T 40 eff * Il9 -/- WT T eff + T reg Il9 -/- Il9 -/- cells 6.0 30 T + T 30

+ eff reg * T + WT T * 4.0 20 eff reg 20

CD25 ns +

/ lymph / node] 10 10 5 2.0 CD4 [10 Counts 0 0 Suppression[Index] 0 ctrl draining 10 0 10 1 10 2 1:4 1:8 1:16

CFSE Ratio T reg : T eff Figure 16: An impaired suppressive capacity of regulatory T cells from Il9 -/- mice. + + (A) Numbers of CD4 CD25 Tregs per poplietal LN in AIA at day 27. (B) Representative flow cytometry chart showing the dilution of the CFSE dye, which is indicating the proliferation of Teff -/- cells. Suppression mediated by WT and Il9 Tregs reduces the dilution. (C) Quantitation of the -/- suppression mediated by WT and Il9 Tregs on WT Teffs at different ratios of Teff and Treg cells. Data are represented as mean, error bars indicate the SEM. ns, p ≥ 0.05; *, p < 0.05 determined by Student’s t-test. (A) WT n = 5. Il9-/- n = 5. (C) WT n = 17. Il9-/- n = 17.

A B Foxp3 Tnfrsf18 Icos WT T + WT T 20 eff reg 1.5 1.5 1.5 WT T * ** * reg WT T + Il9 -/- T ns eff reg * WT T + rmIL-9 15 ** reg -/- WT T + Il9 T 1.0 1.0 1.0 -/- ns eff reg Il9 T 10 + rmIL-9 reg Il9 -/- T + rmIL-9 0.5 0.5 0.5 reg 5 relativeexpression Suppression[Index] 0 0.0 0.0 0.0 1:16

Ratio Treg : Teff Figure 17: The impaired suppression is not a direct result of IL-9 shortage. -/- -/- (A) Quantitation of the suppression mediated by WT and Il9 Tregs on WT Teﬀs. Il9 Tregs mediated suppression was additionally performed in presence of 50 ng/ml recombinant murine IL-9. (B) Expression levels of Foxp3, Tnfrsf18 and Icos in sorted and stimulated (anti-CD3/CD28) Tregs in the presence and absence of recombinant murine IL-9 for 48 h. Expression was normalized to expression of B2m. Data are represented as mean, error bars indicate the SEM. ns, p ≥ 0.05; *, p < 0.05; **, p < 0.01 determined by one-way ANOVA with Tukey’s post hoc test. (A) WT n = X. Il9-/- n = Y. Il9-/-+ rmIL-9 n = Z. (B) WT n = 4. Il9-/- n = 4. WT + rmIL-9 n = 4. Il9-/- + rmIL-9 n = 4.

67 5.4 Regulatory T cells are impaired in interleukin-9 deﬁcient mice

A B ns WT T + WT T 40 ns WT Treg + WT Teff 100 reg eff ns ns WT T + Il9 -/- T WT T + Il9 -/- T 30 reg eff 80 reg eff ns ns ns 60 20 40 10 20 Suppression of Suppression[Index] 0 0 1:4 1:8 1:16 cytokineproduction [%] IFN-γ TNF-α IL-2 IL-4

Ratio T reg : T eff Figure 18: The impaired suppression is not linked to a different responses of effector T cells. -/- (A) Quantitation of the suppression mediated by WT Tregs on WT or Il9 Teffs at different ratios of Teff and Treg cells. (B) Suppression of the cytokine release into the culture supernatant [%] measured by multiplex ELISA. The Treg to Teff ratio was 1:16. Data are normalized to the cytokine release of Teff cells cultured under same conditions in the absence of Tregs. Data are represented as mean, error bars indicate the SEM. ns, p ≥ 0.05 determined by Student’s t-test. (A) WT n = 5. Il9-/- n = 5. (B) WT n = 6. Il9-/- n = 6.

A B WT ** WT 3.0 40 ns Il9 -/- ns Il9 -/- 30 * Il9-/- Il9-/- 2.0 + WT T reg + WT T reg Il9 -/- + Il9 -/- T 20 Il9 -/- + Il9 -/- T

reg AUC reg 1.0 Il9 -/- Il9 -/- Il9 -/- Il9 -/- + rmIL-9ps T reg 10 + rmIL-9ps T reg [jointsweling]

Jointswelling [mm] 0.0 0 21 26 31 36 41 Time [d] Figure 19: The adoptive transfer of regulatory T cells in antigen induced arthrits. -/- AIA was induced in WT and Il9 mice as before. Sorted Tregs were adoptively transferred by an i.v. -/- injection. Occasionally, Tregs from Il9 mice were pre-stimulated (ps) with recombinant murine IL-9. (A) Joint swelling [mm] was monitored over time [d]. (B) Data shown in (A) represented as AUC. Data are shown as mean, error bars indicate the SEM. ns, p ≥ 0.05; *, p < 0.05; **, p < 0.01 determined by one-way ANOVA with Tukey’s post hoc test. (A+B) WT n = 6. Il9-/- -/- -/- -/- -/- -/- n = 6. Il9 + WT Treg n = 6. Il9 + Il9 Treg n = 6. Il9 + rmIL-9ps Il9 Treg n = 6.

as IL-2 and -4 in vitro (not shown) and the suppression of the cytokine release by WT Tregs was

-/- equal in WT and Il9 Teﬀs (Figure 18 B).

To conﬁrm these ﬁndings, Tregs were adoptively transferred into the AIA model (Figure 19). While

-/- the transfer of WT Tregs prevented the chroniﬁcation of the model in Il9 animals, the transfer of

-/- -/- either Il9 Tregs or Il9 Tregs pre-exposed to recombinant IL-9, both failed to rescue the model from chronicity.

Taken together, the lack of IL-9 primarily led to an insuﬃcient activation of Tregs which in turn led to a failure to stop and to resolve the inﬂammation.

68 5.5 Type 2 innate lymphoid cells are the main producers of interleukin-9 in the arthritic joint

A 3 B 10 Spi1fl/fl x Lck-cre+ 2.0 Spi1fl/fl x Lck-cre+ 10 2 Spi1 fl/fl x Lck-cre - 1.5 Spi1 fl/fl x Lck-cre -

1 -/- 10 1.0 Il9

0 10 0.5 IL-17A

10 0 10 1 10 2 10 3 Jointswelling [mm] 0.0 21 26 31 36 41 IL-9 Time [d] C 20 *** Spi1fl/fl x Lck-cre+ *** 15 Spi1 fl/fl x Lck-cre - ns 10 Il9 -/- AUC 5 [jointswelling] 0 Figure 20: Antigen induced arthrits in Spi1 fl/fl x Lck-cre mice. (A) Representative flow cytometry plot of magnetically separated CD4+ splenocytes from fl/fl + fl/fl - Spi1 x Lck-cre and Spi1 x Lck-cre mice cultured under TH9 polarizing conditions for 3 d. (B) Joint swelling [mm] over time [d] during AIA in Spi1 fl/fl x Lck-cre+ and Spi1 fl/fl x Lck-cre- and Il9 -/- animals. Inoculation of mBSA into the knee joint at day 21 after the first immunization. (C) Integral quantification of the swelling shown in (B) as AUC. Data are sown as mean, error bars indicate the SEM. ns, p ≥ 0.05; ***, p < 0.001 determined by one-way ANOVA with Tukey’s post hoc test. (A) Spi1fl/fl x Lck-cre+ n = 3. Spi1fl/fl x Lck-cre- n = 3. (B+C) Spi1fl/fl x Lck-cre+ n = 5. Spi1fl/fl x Lck-cre- n = 6. Il9-/- n = 4.

5.5 Type 2 innate lymphoid cells are the main producers of

interleukin-9 in the arthritic joint

At this stage, the source of IL-9 remained unassigned and its way of action was still obscure. Current reports pointed out that IL-9 derived from TH9 cells in patients with a highly active RA (Ciccia, Gug- gino, Rizzo et al. 2015; Kundu-Raychaudhuri et al. 2016). Therefore, it seemed quite straightforward to test the possibility that TH9 cells would be directly involved in the chronic arthritis phenotype observed in the Il9 -/- animals. To this end, the transcription factor Pu.1 (encoded by the gene Spi1), which is critically involved in the generation of TH9 cells (Chang et al. 2010), was knocked out in T cells using the Lck-cre driver line crossed on the Spi1 fl/fl responders (Orban et al. 1992; Dakic et al. 2005). It was confirmed that CD4 enriched splenocytes isolated from the Spi1 fl/fl x Lck-cre+

fl/fl + mice were inefficient to generate TH9 cells in vitro (Figure 20 A). Then, Spi1 x Lck-cre mice and Cre- littermates were subjected to AIA (Figure 20 B and C). There was no difference observed between Spi1 fl/fl x Lck-cre+ mice and their Spi1 fl/fl litter mates lacking the cre recombinase, which ruled out the involvement of TH9 cells in this model. From its discovery on, IL-9 was described as a mast cell promoting growth factor (a first name was

69 5.5 Type 2 innate lymphoid cells are the main producers of interleukin-9 in the arthritic joint

A Fcer1a Cpa3 Mcpt4 Cma1 Tpsb2 1.5 1.5 1.5 1.5 1.5 ns ns WT ns ns * Il9 -/- 1.0 1.0 1.0 1.0 1.0

0.5 0.5 0.5 0.5 0.5

relativeexpression 0.0 0.0 0.0 0.0 0.0

B Fcer1a Cpa3 Mcpt4 Cma1 Tpsb2 3 3 3 3 3 ctrl SIA + ctrl MC ns ns 2 2 ns 2 ns 2 2 ns SIA + Il9 MC

1 1 1 1 1

relativeexpression 0 0 0 0 0 Figure 21: Mast cells in antigen induced arthrits or serum transfer induced arthritis in wildtype and Il9 -/- animals. Analysis of the expression of common mast cell associated enzymes in AIA and SIA. Analysed were Fcer1a, Cpa3 (mast cell carboxypeptidase A), Mcpt4 (mast cell protease (MCPT) 4), Cma1 (Chymase, MCPT5) and Tpbs2 (tryptase beta-2, MCPT6). The expression of mast cell genes was normalized to the expression of B2m.(A) Comparison of the expression levels in WT and Il9 -/- arthritic joints of AIA at day 27. (B) Comparison of the expression levels at day 11 in WT control paws and WT arthritic paws of SIA with HDGT of either control (ctrl) or Il9 MC. Data are sown as mean, error bars indicate the SEM. (A) WT n = 6. Il9-/- n = 6. ns, p ≥ 0.05; *, p < 0.05 determined by Student’s t-test. (B) ctrl n = 4. SIA + ctrl MC n = 6. SIA + Il9 MC n = 6. ns, p ≥ 0.05 determined by one-way ANOVA with Tukey’s post hoc test.

mast cell growth-enhancing activity) (Hultner,¨ Druez et al. 1990). Therefore, the activation of these cells in arthritis was explored, considering that first, SIA fails in lines defiecent of mast cells (Lee et al. 2002) but second, mast cells get supported from Tregs via IL-9 (Lu et al. 2006). There was no significant difference detected in the expression of common mast cell activation genes in the joints isolated from either WT or Il9 -/- mice subjected to AIA (Figure 21 A) and WT mice subjected to SIA with or without the HDGT of Il9 (Figure 21 B). Therefore, it seemed unlikely that mast cells would be involved in mediating chronicity. To definitively pinpoint the source of IL-9, AIA was induced in Il9 Citrine mice, in which Citrine replaces the first exon of Il9 (Gerlach et al. 2014). The expression of Citrine was inspected at day 27, which is during the resolution interval. The resolution phase was defined as the interval between the peak of the inflammation and its 50 % regression (Bannenberg et al. 2005). The

+ majority of IL-9 was produced neither by T cells (bonna ﬁde TH9) nor by FcR1a mast cells or basophils, but to about 80 % by leukocytes devoid of speciﬁc lineage markers (Figure 22 A and C). These cells could be further characterized as ILC2s based on their expression of ST2 and ICOS. In

70 5.6 Type 2 innate lymphoid cells signiﬁcantly rely on interleukin-9 addition, Citrine+ ILC2s expressed the common markers CD25 (IL2Rα), CD90.2 (Thy1.2), stem cell antigen 1 (sca-1) and were negative for the high aﬃnity IgE receptor FcR1a (Figure 22 B and D). Collectively, these experiments indicated that IL-9 was mainly produced by ILC2s during the resolution interval of AIA, while other potential IL-9 producers such as mast cells or TH9 cells were bare sources.

5.6 Type 2 innate lymphoid cells signiﬁcantly rely on interleukin-9

One of the initial reports on ILC2s described that the receptor for IL-9 served as discriminator between

TH2 cells and ILC2s in micro array data (Price et al. 2010). However, it was also stated in other reports that IL-9 itself was not a proliferative stimulus to ILC2s in vitro (Moro et al. 2010; Price et al. 2010). Nevertheless, later reports using IL-9 fate mapping reporter mice clearly identified IL-9 receptor signalling as a survival factor of ILC2s, but not of TH2 cells in inflammatory models of the lung (Wilhelm, Hirota et al. 2011; Turner et al. 2013). To investigate the autocrine IL-9 signalling among ILC2s in arthritis, WT and Il9 -/- mice were challenged by the HDGT of IL-25 and -33 in combination with or without IL-9. The mesenteric and inguinal LNs as well as the spleen were analysed five days after the challenge. ILC2s were identified as CD45+ CD127 (IL7Ra)+ ICOS+ KLRG1+ cells lacking the expression of lineage markers (CD3, B220, CD11b, CD11c, GR-1, FcR1a). The sub-population of ILC2s expressing the IL-33 receptor ST2 was strongly dependant on IL-9 under theses conditions (Figure 23). Thus, ST2+ ILC2s were strongly reduced in Il9 -/- mice compared to the WT, and the rescue by the HDGT of Il9 resulted in an exceeded recompensation for ST2+ ILC2s. In congruence with the findings in the Il9 Citrine mice, there was a significant increase of ILC2s in the arthritic joints of WT mice in the AIA model at day 27 (Figure 24 A). In contrast, there was no increase observed in the Il9 -/- animals. Furthermore, ILC2s in the WT strain expressed the proliferation marker Ki67, whereas ILC2s in the KO strain did not (Figure 24 B and C). In view of the findings described in section 5.5, it emerged that the autocrine feedback loop of IL-9 and ILC2s was responsible for the resolution of inflammation in the joints.

71 5.6 Type 2 innate lymphoid cells signiﬁcantly rely on interleukin-9

A 1000 10 3 10 3 20 22.6 800 10 2 2 15 10 600 10 1 10 89.1 10 1 400 10 0 10 0 200 5 0 77.4 Lineage IL-9 Counts SSC-I 0 0 0 200 400 600 800 1000 10 0 10 1 10 2 10 3 10 0 10 1 10 2 10 3 0 200 400 600 800 1000 FSC-I Viability CD45 FSC-I

3 B 10 100 ILC2 87.8 80 *** 10 2 TH9 60 10 1 Basophils 40 / Mast cells 0 10 [%] leukocytes Other + 20 ST2

IL-9 0 10 0 10 1 10 2 10 3 ICOS

C CD25 (IL-2Ra) CD90.2 (Thy1.2) FMO stained

D 10 2

10 1 0 10 0 10 1 10 2 0 10 0 10 1 10 2

sca-1 FcER1A gMFI 10 0

10 -1 FMO CD25 Thy1.2 Sca-1 FcER1A count 0 10 0 10 1 10 2 0 10 0 10 1 10 2 marker Figure 22: Interleukin-9-producing cells in antigen induced arthrits. (A) Flow cytometry gating strategy to identify Citrine+ ILC2s. Citrine reflects the IL-9 production in Il9 Citrine mice. (B) Composition of all Citrine+ cells during AIA at day 27. (C) Representative flow charts of CD25, CD90, sca-1 and FcR1a with fluorescence minus one (FMO) control gated on ILC2s. (D) Expression levels of CD25, CD90, sca-1 and FcR1a with FMO control on ILC2s represented as as mean fluorescence indices (MFIs). Data are represented as mean, error bars indicate SEM. ***, p < 0.01 determined by one-way ANOVA with Tukey’s post hoc test. (A+B) n = 6. (C+D) n = 8.

72 5.6 Type 2 innate lymphoid cells signiﬁcantly rely on interleukin-9

A cells doublets exclusion viabilty 10 3 1000 2 800 10

1 600 10 SSC-A FSC-H FSC-TOF 0 400 10 FSC-A FSC-A SSC-H 200 10 -1 Viability SSC-I 0 10 -2 0 200 400 600 800 1000 0 200 400 600 800 1000 FSC-I FSC-I

ILCs WT Il9-/- Il9-/- + Il9 MC 10 3 10 3 10 3 10 3 10 3

10 2 10 2 10 2 10 2 10 2

10 1 10 1 10 1 10 1 10 1

10 0 10 0 10 0 10 0 10 0 ST2 ICOS Lineage 0 10 0 10 1 10 2 10 3 10 0 10 1 10 2 10 3 10 0 10 1 10 2 10 3 10 0 10 1 10 2 10 3 10 0 10 1 10 2 10 3 CD45 IL7Ra ICOS B

*** WT 2.0 [%]

+ Il9 -/- WT + Il9 MC * 1.0 *** Il9 -/- + Il9 MC ** *** * ILC2s of CD45 ILC2s of 0.0 spleen mesenteric inguinal Figure 23: Type 2 innate lymphoid cells require interleukin-9 signalling. ILC2s were induced in WT and Il9 -/- mice by the HDGT of Il25 and Il33. Some mice received additionally Il9.(A) Flow cytometry gating strategy to identify ILC2s. (B) Frequency of ILC2s in spleen, mesenteric and inguinal LNs among all CD45+ cells. Data are shown as mean, error bars indicate the SEM. ns, p ≥ 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.01 determined by one-way ANOVA with Tukey’s post hoc test. (A+B) WT n = 6. Il9-/- n = 5. WT + Il9 MC n = 3. Il9-/- + Il9 MC n = 4.

A B *** C ** 10 * 30 * WT WT 210 WT 8 Il9-/- Il9-/- 28 Il9-/-

/ knee] / 20 3 6 6

ILC2s 2 + 4 10 24 Ki67

ILC2s [n / knee] / ILC2s [n 2 2 +

[% of ILC2 / knee] ILC2 of / [% 2 ILC2s [x10 ILC2s 0

0 0 Ki67 2 ctrl arthritic Figure 24: Impaired proliferation of type 2 innate lymphoid cells in antigen induced arthrits in Il9 -/- mice. (A) Number of ILC2s per joint in AIA at day 27. (B) Frequency of Ki67+ ILC2s per arthritic joint in AIA at day 27. (C) Data represented in (B) as absolute numbers. Data are shown as mean, error bars indicate the SEM. (A) WT n = 5. Il9-/- n = 5. *, p <0.05 determined by one-way ANOVA with Tukey’s post hoc test. (B+C) WT n = 5. Il9-/- n = 5. **, p < 0.01; ***, p < 0.001 determined by Student’s t-test.

73 5.7 Type 2 innate lymphoid cells reinforce regulatory T cells

A FoxP3 IL-9 ICOS CD3ε MERGE Haematoxylin & Eosin

10 µm

B *** 200 µm 8 *** IL-9neg T cells *** IL-9neg ILC2s

] 6

reg pos 4 IL-9 T cells pos [n / T / [n IL-9 ILC2s 2 surrounding cells reg

T 0 IL-9neg IL-9pos Figure 25: Co-localization of regulatory T cells with type 2 innate lymphoid cells in the inflamed synovium. (A) Representative immunofluorescence micrographs of FoxP3, IL-9, ICOS and CD3 allowing to + - + + detect the co-localization of IL-9 expressing ILC2s (ICOS CD3 ) and Tregs (FoxP3 CD3 ). (B) Quantitative analysis of IL-9-producing T cells and ILC2s being surrounded by Tregs in the inflamed synovium. Sections were obtained from WT AIA at day 27. Data are shown as mean, error bars indicate the SEM. ***, p <0.001 determined by one-way ANOVA with Tukey’s post hoc test. n = 24 cells per group on sections of 4 individual animals.

5.7 Type 2 innate lymphoid cells reinforce regulatory T cells

Numerous reports have shown the direct interaction of ILC2s and CD4+ T cells (Mirchandani et al. 2014; Oliphant et al. 2014; Molofsky et al. 2015). Among those, a direct interaction between

Tregs and ILC2s was described in visceral fat pads, where IL-33 dependant ILC2s were required for the activation and accumulation of tissue Tregs (Molofsky et al. 2015). To test weather IL-9 would play a similar role in the inﬂamed synovium, arthritic joints were analysed at day 27 of

AIA. Immunoﬂuorescence images stained for the Treg markers FoxP3 and CD3 as well as the ILC2 markers ICOS and IL-9 revealed a close spacial proximity of both cell types in the inﬂamed synovium (Figure 25).

Stimulation via the co-stimulatory receptors ICOS and GITR is substantially enhancing Treg activity in vitro and in vivo (Burmeister et al. 2008; Nishioka et al. 2008; Y. H. Kim et al. 2015). Tregs from Il9 -/- mice were characterized by a signiﬁcant down-regulation of theses co-activators (Figure 17).

This raised the question whether ILC2s would modulate Treg activity by these receptors. Indeed, when ILC2s were sorted from SLOs and stimulated for 72 h with IL-9, they showed a marked up-regulation

74 5.7 Type 2 innate lymphoid cells reinforce regulatory T cells of the receptor ligands Icosl and Tnfsf18 encoding ICOS-L and GITR-L, respectively (Figure 26 A). To tackle the functional implication of this receptor-ligand couples, the suppression assays were performed in the presence of ILC2s. ILC2s eﬀectively restored the suppressive activity of previously

-/- impaired Il9 Tregs (Figure 26 B and C). Blocking the ILC2-Treg interaction by the interception of the ligands with speciﬁc antibodies led to an exacerbated proliferation of the Teﬀ responder cells.

Conversely, the combined engagement of both receptors, GITR and ICOS, on Tregs by an agonistic anti-GITR antibody and recombinant ICOS-L, respectively, potentiated the suppressive capacity of

-/- Il9 Tregs in the absence of ILC2s (Figure 26 D and E). Nevertheless, the ILC2-Treg interaction appeared to be cell-cell contact dependant, since the restoration of the suppressive activity of Il9 -/-

Tregs by ILC2s failed in trans-well assays (Figure 26 F and G).

Legend to Figure 26 continued from the next page: (A) In vivo expanded ILC2 (Il25 + Il33 HDGT) were sorted and stimulated in vitro with 50 ng/ml recombinant murine IL-9 for 72 h. The expression levels of GITR-L and ICOS-L were compared to freshly sorted ILC2s. The expression was normalized to the expression of Hprt1.(B + D + F) Representative flow cytometry charts showing the dilution of the CFSE dye in responder cells. The dilution indicates the proliferation of the Teff cells. The suppression of proliferation is mediated by Tregs. The suppression assay was performed in the presence or absence of ILC2s and anti-GITR-L or anti-ICOS-L capturing antibody or the isotype control (B); in the presence or absence of an agonistic anti-GITR antibody and recombinant ICOS-L (D); in the presence or absence of ILC2s separated from Tregs and Teffs by a trans-well insert (G). (C + E + F) Quantitation of the suppression mediated by WT -/- or Il9 Tregs on WT Teffs in the presence or absence of ILC2s and anti-GITR-L or anti-ICOS-L capturing antibody or the isotype contro (B); in the presence or absence of an agonistic anti-GITR antibody and recombinant ICOS-L (D); in the presence or absence of ILC2s separated from Tregs and Teffs by a trans-well insert (G). Data are shown as mean, error bars indicate the SEM. (A) freh n = 5-7. 72 h IL-9 n = 3-6. *, p < 0.05; **, p < 0.01 determined by Student’s t-test. (C) iso ctrl: WT n = 17. Il9-/- n = 17. WT + ILC2s + antibody n = 15. Il9-/- + ILC2s + antibody n = 10. anti-GITR-L: WT n = 8. Il9-/- n = 8. WT + ILC2s + antibody n = 12. Il9-/- + ILC2s + antibody n = 7. anti-ICOS-L: WT n = 10. Il9-/- n = 10. WT + ILC2s + antibody n = 11. Il9-/- + ILC2s + antibody n = 10. (E) WT n = 4. Il9-/- n = 4. WT + stimulation n = 4. Il9-/- + stimulation n = 4. (G) WT n = 4. Il9-/- n = 4. WT + ILC2s n = 4. Il9-/- + ILC2s n = 4. (C, E, G) ns, p ≥0.05, *, p < 0.05; **, p < 0.01; ***, p <0.001 determined by one-way ANOVA with Tukey’s post hoc test.

75 5.7 Type 2 innate lymphoid cells reinforce regulatory T cells

A Tnfsf18 Icosl 80 4 Fresh * *** 60 3 72h IL-9 40 2

20 1

relative expression relative 0 expression relative 0 B control anti-GITR-L anti-ICOS-L 40 30 Il9 -/- T 30 30 reg Il9 -/- T + ILC2s + iso ctrl 20 reg 20 20 Il9 -/- T reg + ILC2s + blocking antibody 10 10 10

Counts 0 0 0 10 0 10 1 10 2 10 0 10 1 10 2 10 0 10 1 10 2 CFSE *** *** C 30 * 30 *** 30 *** ** * ** WT T 20 20 20 reg Il9 -/- T 10 10 10 reg WT T + ILC2s + antibody 0 0 0 reg Il9 -/- T + ILC2s + antibody -10 -10 -10 reg -20 -20 -20 Suppression[index] -30 -30 -30

D 30 F T Teff eff 10 -/- 20 Il9 -/- T + Il9 T (ctrl) Teff + T reg (ctrl) eff reg

T + WT T (ctrl) Teff + WT Treg (ctrl) eff reg 5 10 Il9 -/- T + Il9 -/- T Teff + T reg (GITR/ICOS) eff reg + ILC2s (transwell) Counts 0 Counts 0 10 0 10 1 10 2 10 3 -10 1 0 10 1 10 2 10 3 CFSE CFSE ns E 5 0 G 40 *** WT T * ns WT T ** reg 30 reg 4 0 Il9 -/- T Il9 -/- T reg 20 reg 3 0 WT T ( GITR/ICOS ) WT T + ILC2s (transewll) reg 10 reg Il9 -/- T ( GITR/ICOS ) Il9 -/- T + ILC2s (transewll) 2 0 reg 0 reg 1 0 -10 Suppression[index] Suppression[index] 0 -20 Figure 26: Mechanism of ILC2-mediated enhancement of the suppressive activity of regulatory T cells. Continued on the previous page.

-/- In order to validate these ﬁndings in vivo,Tregs were sorted from Il9 animals and prior to their adoptive transfer into the AIA model, they were stimulated with the agonistic GITR antibody and

-/- recombinant ICOS-L for 24 h. The transfer of stimulated but not of control Il9 Tregs resulted in a limited course of the AIA model in Il9 -/- mice with a full resolution of inflammation comparable to that of AIA in WT animals (Figure 27). Finally, AIA was induced in Il9 -/- mice and purified ILC2s from congenic CD45.1 Balb/c mice were adoptively transferred directly into the arthritic joint. Adoptive transfer of ILC2s efficiently induced

76 5.8 Innate lymphoid cells as a potential diagnostic biomarker in human rheumatoid arthritis

A B 2.0 40 ns WT ** WT * 1.5 30 *** Il9 -/- Il9 -/- 1.0 20 Il9 -/- Il9 -/- + AUC + Il9 -/- T (ctrl) Il9 -/- T (ctrl) 0.5 reg 10 reg -/- Il9 -/- Il9 + [jointswelling] + Il9-/- T ( αGITR/ICOS-L) Il9 -/- T ( αGITR/ICOS-L) Jointswelling [mm] 0.0 reg 0 reg 21 26 32 37 41 Time [d] Figure 27: Adoptive transfer of stimulated regulatory T cells in antigen induced arthrits. -/- -/- AIA was induced in WT and Il9 mice as before (Figure 5). Sorted Il9 Tregs were adoptively -/- transferred by i.v. injection at day 22. Occasionally, Tregs from Il9 mice were pre-stimulated with an agonistic anti-GITR antibody and recombinant ICOS-L. (A) Joint swelling [mm] was monitored over time [d]. (B) Data shown in (A) represented as AUC. Data are shown as mean, error bars indicate the SEM. ns, p ≥ 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001 determined by one-way -/- -/- -/- ANOVA with Tukey’s post hoc test. (A+B) WT n = 6. Il9 n = 10. Il9 + Il9 Treg n = 4. -/- -/- Il9 + Il9 Treg (αGITR/ICOS-L) n = 10.

the resolution of AIA in Il9 -/- animals (Figure 28 A and B). Transferred ILC2s were found to be viable and present in comparable numbers in the knee joints until the complete resolution of AIA in both WT and Il9 -/- animals indicating the self sustained survival via IL-9 (Figure 28 C and D). Importantly, the adoptive transfer of ILC2s also abrogated the excessive release of IL-17 in Il9 -/- mice (Figure 28 E). In summary, these data explained the link between IL-9 and the resolution of inﬂammation in the inﬂamed synovium. It could be shown that IL-9 instructed ILC2s to express the Treg co-activators

ICOS-L and GITR-L. ILC2s thereby guided Tregs to promote the resolution of inﬂammation and to shrink the TH17 response in the joints.

5.8 Innate lymphoid cells as a potential diagnostic biomarker in

human rheumatoid arthritis

To assess whether a similar ILC2-Treg axis is involved in the remission of human RA, biopsies from the human synovium were analysed by an immunofluorescence staining. RA patients were stratified according to the standardized DAS28 criteria into two goups. The first group contained the patients with a high disease activity displaying a DAS28 of ≥3.2 and the second group contained patients in remission with a DAS28 of <2.6 (Prevoo et al. 1995; Sheehy et al. 2014). The biopsies from the rheumatoid synovium were compared to biopsies taken from acute orthopaedic trauma and

77 5.8 Innate lymphoid cells as a potential diagnostic biomarker in human rheumatoid arthritis

A B 2.0 40 *** WT * WT 1.5 Il9 -/- 30 Il9 -/- Il9 -/- 1.0 + ILC2 20 Il9 -/- + ILC2 AUC 0.5 10 [jointswelling]

Jointswelling [mm] 0.0 0 21 26 31 36 41 46 Time [d] C cells doublets exclusion lineage exclusion ILC2s 1000 10 3 10 3

800 10 2 10 2

600 1 1 SSC-A FSC-H FSC-TOF 10 10 400 FSC-A FSC-A SSC-H 2 10 0 200 0 -2 0 SSC-I Lineage 0 ICOS 0 200 400 600 800 1000 -20 2 10 1 10 2 10 3 0 10 0 10 1 10 2 10 3 FSC-I KLRG1 IL7Ra * E ** CD45.2 vs CD45.1 D ns WT 10 3 40 WT 80 Il9 -/- Il9 -/- 10 2 60 30 Il9 -/- + ILC2 10 1 20 40 2 10 20 0 IL-17A [pg/ml] IL-17A

CD45.1 -2 NDND CD45.1+ILC2s[%] 0 1 2 3 0 -5 0 10 10 10 ctrl arthritic 0 22 44 CD45.2 Time [d] Figure 28: Adoptive transfer of type 2 innate lymphoid cells in antigen induced arthrits. AIA was induced in WT and Il9 -/- mice as before (Figure 5). ILC2s were sorted from CD45.1+ mice and adoptively transferred by intra-articular injection at day 21 into Il9 -/- animals. (A) Joint swelling [mm] was monitored over time [d]. (B) Data shown in (A) represented as AUC. (C) The presence of ILC2s in the knee joint at the end of the model was analysed by ﬂow cytometry. Representative plots are shown highlighting the adoptively transferred CD45.1+ in red. CD45.2+ ILC2s are host derived. (D) Frequency of adoptively transferred CD45.1+ ILC2s among all ILC2s in the joint at at the end of the model. (E) Serum levels of IL-17 at day 0, 22 and 44 of AIA were determined by multiplex ELISA in WT, Il9 -/- and Il9 -/- mice having received adoptively transferred ILC2s. Data are shown as mean, error bars indicate the SEM. ns, p ≥ 0.05; *, p < 0.05; **, p < 0.01 determined by one-way ANOVA with Tukey’s post hoc test. WT n = 5. Il9-/- n = 4. Il9-/- + ILC2s n = 8.

78 5.8 Innate lymphoid cells as a potential diagnostic biomarker in human rheumatoid arthritis healthy synovium. The tissue was stained for IL-9, ICOS and lineage (CD3, CD11b, CD16, mast

+ + - + + cell tryptase) to detect IL-9-producing ILC2s (IL-9 ICOS lineage ) and TH9 cells (IL-9 lineage , Figure 29 A). Low levels of IL-9 were found in healthy and traumatic injured synovium. In contrast, a high abundancy of IL-9+ cells was found in the rheumatic synovium (Figure 29 B). Here, IL-9 was mainly produced by lineage+ cells in patients with an active RA corroborating published observations (Ciccia, Guggino, Rizzo et al. 2015; Kundu-Raychaudhuri et al. 2016). In sharp contrast, patients in a phase of stable disease remission displayed an IL-9 production by mainly lineage- ILC2s. To rule out the possibility that the predominating IL-9 production by ILC2s during remission was a characteristic of individual patients rather than being directly linked to disease activity, longitudinal analysis were performed. Patients with an early active RA (DAS28 = 5.1 ± 1.2, disease onset < 12 month, n = 10) were biopsied before treatment with conventional synthetic DMARDs and six month later again, when the anti-rheumatic therapy had led to remission (DAS28 = 2.0 ± 0.6, Figure 29 C). The remission of RA was confirmed by a significant reduction of synovial leukocytes. Importantly, the production of IL-9 shifted from lineage+ cells in the active disease to lineage- ILC2s in the phase of remission, confirming that the shift of the IL-9 producing cell type was disease related and not a contribution of the individual patients. These observations authenticated the previous results on the importance of IL-9+ ILC2s for the resolution of inflammation in RA obtained in the murine system. The logical next step therefore was to determine the diagnostic potential of peripheral blood ILC2s. To this end, the patients were stratified according to their disease activity and counts of circulating ILC2s were determined by flow cytometry (Figure 30 A). Indeed, the patients with a high disease activity displayed particularly lower counts of peripheral blood ILC2s than the patients in remission Figure 30 B). The numbers of circulating ILC2s significantly correlated with the disease activity measured by the DAS28 (Figure 30 C). Finally, the diagnostic potential of circulating ILC2s was substantiated by the longitudinal observation of RA patients (Figure 30 D). Patients that remained in remission during the entire observation period, also kept high levels of ILC2, whereas patients that constantly retained an active RA, did not increase their low levels of circulating ILC2s. Those patients that switched from an active disease to remission increased their peripheral blood ILC2 counts and vice versa. In conclusion, the murine and human data corroborated the outstanding role of ILC2s in the resolution of inflammation and the prevention of chronic inflammatory conditions.

79 5.8 Innate lymphoid cells as a potential diagnostic biomarker in human rheumatoid arthritis

A Haematoxylin Lineage ICOS IL-9 DAPI Merge & Eosin

normal control

acute trauma

active RA

RA remission

25 µm 100 µm B *** C *** *** *** ns Lin+ IL-9+ Baseline 8 *** 300 ** 6 * 8 *** IL-9 + ILC2s 6 month 6 *** *** 6 200 4 treatment 4 4 100 2 cellsHPF / 2 HPF cells / 2

0 0 0 0 normal acute active RA leukocytes Lin + IL-9 + IL-9 + ILC2s control trauma RA remission Figure 29: Interleukin-9 expressing cells in different phases of human arthritis. (A) Human synovial biopsies were stained for the lineage (Lin) markers (CD3, CD11b, CD16 and mast cell tryptase) as well as ICOS, IL-9 and DAPI. Representative micrographs of the immunofluorescence and haematoxylin and eosin staining. (B) Quantification of lineage+ IL-9+ and lineage- ICOS+ IL-9+ cells (ILC2s) per 0.3 mm2 (high power field (HPF)) of synovial tissue. (C) Lon- gitudinal monitoring of IL-9+ cells in biopsies of patients with active RA and after six months of treatment with anti-rheumatic drugs inducing disease remission. Data are shown as mean, error bars indicate the SEM. (B) ns, p ≥ 0.05; ***, p < 0.001 determined by one-way ANOVA with Tukey’s post hoc test. Normal control n = 11. Acute trauma n = 8. Active RA n = 19 (DAS28 ≥3.2). RA remission n = 19 (DAS28 ≤2.6). (C) *, p < 0.05; **, p < 0.01; ***, p < 0.001 determined by Student’s t-test. n = 10.

80 5.8 Innate lymphoid cells as a potential diagnostic biomarker in human rheumatoid arthritis

A cells doublets exclusion B

1000 RA remission 800 active RA 600 SSC-A 400 FSC-H FSC-TOF *** FSC-A FSC-A SSC-H 80 200 40 0 0 200 400 600 800 1000 40 SSC-I FSC-I 30

lymphocytes ILCs ILC2s ILC2s 20 3 3 1000 10 10 [cells / µl blood] µl [cells/ 10 800 10 2 10 2

600 1 0 10 10 1 400 1 10 0 200 0 -1 0 0 1 2 3 0 1 2 3 1 2 3

10 10 10 10 0 10 10 10 10 c-KIT 0 10 10 10 SSC-I Lineage CD45 IL7Ra CRTH2

persistent induction inflammatory disease flare stable remission of remission C D activity 80 80 80 80 80 R = -0.6651 40 p < 0.0001 40 40 40 40 40 40 40 40 40 30 30 30 30 30 ILC2s ILC2s 20 20 20 20 20 [cells / µlblood] [cells / [cells / µlblood] [cells/ 10 10 10 10 10

0 0 0 0 0 0 2 4 6 8 baseline follow-up baseline follow-up baseline follow-up baseline follow-up Disease Activity Score inactive active inactive inactive active inactive active active (DAS28) Figure 30: Circulating type 2 innate lymphoid cells in patients with rheumatoid arthritis. (A) Gating strategy to identify ILC2s in human peripheral blood by flow cytometry. (B) Quantifica- tion of circulating ILC2s in patients in remission and with active RA. (C) Correlation of ILC2 counts in peripheral blood with DAS28. (D) Longitudinal monitoring of ILC2 counts in peripheral blood of RA patients at baseline and at a 6–12 month later follow-up. Patients were stratified into four groups according to baseline and follow-up disease activity according to the DAS28 criteria. Data are shown as mean, error bars indicate the SEM. (B) ***, p < 0.001 determined by Student’s t-test. RA remission n = 50 (DAS28 ≤2.6). active RA n = 61 (DAS28 = ≥3.2). (C) non-parametric Spearman’s correlation. n = 110. (D) n = 63.

81 6 Discussion

RA is a highly chronic and inflammatory autoimmune disease. In the past, several cytokine pathways have been investigated. Among those, TNF-α and IL-6 emerged to be the major enhancers, which predestined them as therapeutic targets. This study was designed to reveal the intersection of IL-9 within the cytokine network of chronic inflammatory arthritides. The data obtained suggest a novel mechanism, in which IL-9 acts as a pro-resolving cytokine (Figure 31). The main effector function of IL-9 in inflammatory arthritis was the activation of ILC2s. The activation of ILC2s subsequently provided a favourable environment for Tregs, as they provided co-stimulation through GITR and ICOS.

Tregs were required for the termination of inﬂammation and the subsequent decline of synovitis. In both, the human and the murine synovium, ILC2s produced IL-9. Of note, the accumulation of IL-9-producing synovial ILC2s was associated with the regression of the disease. Not only synovial, but also peripheral blood ILC2s counts inversely correlated with the disease activity, suggesting that ILC2s may serve as a diagnostic biomarker in the future.

6.1 Skewing the innate compartment by interleukin-9 to a

pro-resolving milieu in arthritis

Rheumatoid arthritis is unambiguously a T cell dependant disease, but also T-independent mechanisms are involved in inducing and regulating the inflammation. IL-9 is not a classical pro-resolving cytokine and has a pro- and anti-inflammatory activity (subsection 3.2.2). The classical regulatory cytokines are TGF-β and IL-10. This is because the disruption of TGF-β signalling on T cells leads to a spontaneous multi-organ auto-immunity, which fatally ends at the age of weaning (Gorelik et al. 2000; Marie et al. 2006; M. O. Li et al. 2006). The regulatory activity of IL-10 is mainly restricted to the gut. Hence, IL-10 deficient mice develop a chronic enterocolitits (Kuhn¨ et al. 1993). The

82 6.1 Skewing the innate compartment by interleukin-9 to a pro-resolving milieu in arthritis

GITR ligand

ILC2 IL-9 GITR T IL-9R reg ILC2 ILC2 ICOS ILC2 ILC2 ILC2 Activation ICOS ligand

IL-9

Figure 31: Proposed mechanism for the type 2 innate lymphoid cell induced licensing of regulatory T cells in rheumatoid arthritis. In congruence with published reports, IL-9 was found to be an autocrine cytokine secreted by ILC2s. It was found that ILC2s were the major source of IL-9 in the arthritic joint of mice. Furthermore, IL-9 provided a proliferative stimulus to the ILC2 population in vivo. Activated ILC2s produced the co-stimulatory molecules ICOS-L and GITR-L. The blockage of this pathways resulted in impaired Treg suppression and chronic arthritis. Murine data were corroborated by the analysis of human synoial joint biopsies. In particular, patients with a low disease activity had elevated numbers of joint resident IL-9-producing ILC2s. Counts of peripheral blood ILC2s inversely correlated with the disease activity.

83 6.1 Skewing the innate compartment by interleukin-9 to a pro-resolving milieu in arthritis absence of either of the two cytokines TGF-β or IL-10 results in an exacerbated type 1/3 immune response characterized by the upregulation of IFN-γ, IL-12, TNF-α and IL-6 (Fiorentino, Zlotnik, Mosmann et al. 1991; Fiorentino, Zlotnik, Vieira et al. 1991; Gorelik et al. 2000; Marie et al. 2006; M. O. Li et al. 2006). Since these signal mediators are the anchors of the cytokine network in RA, skewing the immune response from a type 1/3 immunity towards a type 2 immunity is thought to be beneficial in this disease entity. Accordingly, an IL-4 therapy resulted in decreased clinical manifestations of collagen or proteoglycan induced arthritis (Horsfall et al. 1997; Finnegan et al. 1999). Double deficient mice for IL-4 and -13 displayed a more sever phenotype of SIA (Z. Chen, Andreev et al. 2016). Interestingly, in the latter study, infection with the nematode N. brasiliensis protected WT mice subjected to SIA and mice of the TNF-α-transgenic line. It was proposed that skewing the inflammation torwards a TH2 phenotype induced a robust eosinophilia and subsequently repolarised joint resident macrophages. Indeed, infection models with N. brasiliensis robustly induced ILC2s that not only secreted IL-4,

-5 and -13, but also secreted IL-9 (Turner et al. 2013; Licona-Limón et al. 2013; Mohapatra et al. 2016). An induction of IL-5-producing ILC2s following serum challange in the SIA model was observed recently (Omata et al. 2018). In the present study, ILC2s were activated and amplified by the HDGT of Il9 and the over-expression of IL-9 led to elevated serum levels of IL-5 in the SIA model (Figure 23 and data not shown). It might be speculated that the ILC2-eosinophil axis might synergize with the ILC2-Treg axis in the resolution of inflammatory arthritis, however there is multiple evidence that Tregs are the major cell type that is responsible for the control of the resolution of inflammation. First, eosinophils were reported to produce IL-9 and thus are able to foster the ILC2 response (Gounni, Nutku et al. 2000). Second, indeed both models, the SIA and the TNF-α transgenic model, are controlled by innate immune cells as both models were applicable in RAG deficient mice (Korganow et al. 1999; Kontoyiannis et al. 1999). However, SIA was ameliorated in TCR-WT mice, when exclusively B cells were lacking, and worsened, when the remaining T cells were exclusively TCR-KRN transgenic (Korganow et al. 1999).

It was shown that TCR-KRN transgenic T cells preferentially acquire a TH17 phenotype in the SIA model, which explains the aggravation of the model on the B cell deficient KRN background (Jacobs et al. 2009). The amelioration of SIA in the B cell deficient TCR-WT mice implies the control of inflammation by unspecific T cells including Tregs.

84 6.2 Mast cell - regulatory T cell cooperation during inﬂammation control

Actually, Tregs not only restrict inflammation as they suppress other T cells, but also they can directly suppress the innate immune system. In the lung, Tregs were shown to alter the phenotype of alveolar macrophages to a pro-resolving M2 type and to inhibit pro-inflammatory alveolar ILC2s by the sectretion of TGF-β and the sequestration of ICOS-L (D’Alessio et al. 2009; Krishnamoorthy et al. 2015; Rigas et al. 2017). The alternative activation of human macrophages by Tregs was shown to require cell-cell contact as well as IL-10, but not TGF-β (Tiemessen et al. 2007). In a model of T cell independent colonic inflammation, the adoptive transfer of Tregs reduced the intestinal inflammation and neutrophil splenomegaly in a IL-10 and TGF-β dependant manner (Maloy et al. 2003). Efferocytosis, that is the clearance of apoptotic and nectrotic cell debris by phagocytes, is an important step in the resolution of inflammation (Ortega-Gómez et al. 2013). Tregs were shown to enhance the efferocytosis of alveolar and peritoneal macrophages (D’Alessio et al. 2009; Proto et al.

2018). It also was shown that IL-13 produced by Tregs is involved in the alternative M2 polarisation of macrophages in human and mice (Tiemessen et al. 2007; Proto et al. 2018). To induce the resolution of inflammation, IL-10 only was required as an autocrine amplifier for macrophage efferocytosis, but not as an inducing factor secreted by Tregs (D’Alessio et al. 2009; Proto et al. 2018). Moreover, Tregs reduced the amount of TNF-α and IL-6 secreted by macrophages (Tiemessen et al. 2007; D’Alessio et al. 2009; Proto et al. 2018).

In conclusion, these reports highlight how the activation of Tregs not only stops the lymphocyte reaction, but also that Tregs are important inducers of resolution by macrophages. In the present study, Tregs were activated by ILC2s that themselves were dependant on IL-9. Hence, the IL-9-ILC2-

Treg axis appears to be a multifocal mechanism to control disease ﬂares in RA as this mechanism reduces the abundance of TNF-α and IL-6 from macrophages and IL-17 form pro-inﬂammatory TH17 cells.

6.2 Mast cell - regulatory T cell cooperation during inﬂammation

control

Mast cells are also likely to be involved in IL-9 driven immuntiy. IL-9 was discovered as a growth factor for mast cells (Moeller et al. 1989). Furthermore, it has been suggested that they are critically involved in the induction of arthritis in the SIA model and human arthritis (Lee et al. 2002; Rivellese

85 6.2 Mast cell - regulatory T cell cooperation during inflammation control et al. 2018). In the skin, it was found that patroling ILC2s interacted with mast cells for prolonged periods (Roediger et al. 2013). The authors also claimed that IL-9 positively impacted the release of IL-6 and TNF-α from bone marrow derived mast cells. In the lamina propria of the gut, the degranulation of mast cells releasing proteases was impaired in mice deficient of IL-9 or its receptor (C.-Y. Chen et al. 2015). Considering these reports, it could be speculated that the application of IL-9 would indeed enhance SIA by activating the pro-inflammatory repertoire of mast cells. However, in the present study, the over-expression of IL-9 did not result in an aggravated arthritis, but led to its enhanced resolution (Figure 9 and 11). In addition, pro-inflammatory mast cells are mostly associated with IL-9 synthesis

(Hultner,¨ Kölsch et al. 2000; C.-Y. Chen et al. 2015; Moretti et al. 2017). In this study, analysis of Il9 Citrine mice subjected to AIA, revealed that only a marginal number of Citrine+ cells was positive for the mast cell and basophilic marker FcRIa (Figure 22). Intriguingly and similar to ILC2s, mast cells are Janus-faced immune cells acting pro- and anti- inflammatory. Two reports showed that mast cells stimulated by IL-9 derived from Tregs created an immunosuppressive environment. In the first study, skin allografts were studied in a system, in which the recipients were pre-exposed to allogenic cells combined with the blockade of CD40 ligand creating a tolerogenic environment (Honey et al. 1999). The authors found that Tregs as well as mast cells were recruited to the graft (Lu et al. 2006). The model failed in mast cell deficient mice as well as in mice treated with an anti-IL-9 antibody. The second report analysed a model of acute renal inflammation that was aggravated in mast cell deficient mice (Eller et al. 2011). The adoptive transfer of Tregs failed to rescue the phenotype in mast cell deficient mice, but ameliorated it in WT

-/- mice. The authors could link the positive effect of Tregs on mast cells to IL-9, since Il9 Tregs also failed to improve the model. The exact mechanism of mast cell mediated control of inflammation is still elusive, but a tight crosstalk with Tregs in both directions is likely. Interestingly, in a smaller in vitro study it was conceptualized that TNF-α blocks degranulation of mast cells and in turn up-regulates the expression of surface ICOS-L (Gao et al. 2017). Hence, mast cells gained the ability to reinforce Tregs. Papain induced airway inflammation is characterized by IL-33 triggered expression of IL-9 in ILC2s (Wilhelm, Hirota et al. 2011). In mast cell deficient mice, papain induced a much more severe inflammation (H. Morita et al. 2015). The authors described that IL-33 instructed mast cells to produce IL-2 and co-stimulators such as ICOS-L, which resulted

86 6.3 The balance of type 2 and type 3 innate lymphoid cells in arthritis

in the increased generation of Tregs. In view of these reports, it emerges that mast cells can act as downstream effectors of resolution induced by IL-9 and Tregs. In the present study, resolution of inflammation was induced in T cell dependant and T cell independent models by IL-9. In contrast to Lu et al. (2006), there was no significant up-regulation of the messenger RNA of those genes highly sensitive for mast cell activity neither in AIA between Il9 -/- and WT mice nor in SIA in response to K/BxN or IL-9 (Figure 21). It therefore is unlikely, that mast cells are involved in the resolution processes of inflammatory arthritis.

6.3 The balance of type 2 and type 3 innate lymphoid cells in

arthritis

In the present study, ILC2s emerged to be a critical parameter for disease activity. Not only in the synovium, but also in the peripheral blood the frequency of ILC2s correlated with the disease activity of RA (Figure 29 and Figure 30). The homogeneity of the longitudinal analysis prompts to establish peripheral blood ILC2 counts as a diagnostic tool. Another study on a different RA cohort showed a similar correlation between DAS28 and circulating ILC2s (Omata et al. 2018). Psoriatic arthritis is a different class of rheumatic disease. While RA is thought to be mainly mediated by the TNF-α-IL-6 network, psoriasis (only cutaneous involvement) and especially psoriatic arthritis (involvement of skin, joints and entheses) is thought to be anchored in the IL-23/17-TNF-α network (Veale and Fearon 2018). According to this cytokine profile, ILC3s are likely to be critically involved. It was described that mainly NCR+ ILC3s were enriched in the synovial fluid of psoriatic arthritis patients (Leijten et al. 2015). In the peripheral blood, ILCs with a ILC3-like phenotype (cKit+CRTH2-) were proposed to be mainly circulating precursors in healthy individuals (Lim et al. 2017). Nonetheless, in psoriatic arthritis patients, cKit+CRTH2- ILCs were found to be primed to secrete IL-17A and to express RORγt (Soare et al. 2018). Importantly, the number of circulating ILC2s inversely correlated with the disease activity stressing ILC2s as a generalized diagnostic marker in rheumatic diseases.

87 6.4 Regulatory T cells in rheumatoid arthritis

6.4 Regulatory T cells in rheumatoid arthritis

T cells are involved in the initiation and the maintenance of chronic inflammation in RA. Among the various T cell subsets that can be found in the RA synovium are also Tregs. Their disease-limiting nature has been proven in collagen induced arthritis and AIA, but their involvement in the human disease is not fully understood (Morgan, Sutmuller et al. 2003; Morgan, Flierman et al. 2005; Frey et al. 2005; Wright et al. 2009; Komatsu et al. 2014). It was suggested, that TNF-α down-regulates the expression of FOXP3 in human Tregs thereby limiting their activity (Valencia et al. 2006). A meta study of case reports on Tregs in RA concluded a significant reduction of Treg numbers in the peripheral blood of active RA patients in comparison to healthy individuals and also a lower number in active disease patients in comparison to patients in remission (T. Morita et al. 2016). The authors selected 31 publications and in a first attempt, they did not find a conclusive result. This

+ hi + was because Tregs were heterogeneously deﬁned among studies as either CD25 , CD25 , FoxP3

+ + hi or CD25 FoxP3 . Conclusive results were only obtained, when Tregs were deﬁned as either CD25 or CD25+ FoxP3+. Indeed, the IL-2 receptor α-chain CD25 is generally up-regulated on activated

T cells and therefore only the high expressing population might contain a suﬃcient purity of Tregs. In this context it is worth to recall the study from Komatsu et al. (2014), who described that the transfer

+ lo of FoxP3 CD25 T cells into the collagen induced arthritis model resulted in the conversion of Tregs

hi + to TH17 cells. Accordingly, only CD25 FoxP3 T cells were able to induce disease amelioration.

In this study, several adoptive transfer experiments and in vitro experiments with Tregs were conducted. Despite the fact that it previously was reported that Foxp3 GFP mice might be a save tool for the isolation of a stable Treg population for adoptive transfer experiments in arthritis models (Rubtsov et al. 2010), in the case of the present study, for in vivo studies only the CD25hi population was isolated. And indeed, the adoptive transfer of WT Tregs could rescue chronic non-resolving AIA in Il9 -/- animals validating this strategy (Figure 19). The insuﬃcient suppressive activity of Il9 -/-

Tregs was not a result of either altered CD25 expression or altered FoxP3 expression. Neither the

Treg population size nor its mean ﬂuorescence intensity of CD25 was altered (Figure 16 and data not shown). Expression levels of Foxp3 were similar between WT and Il9 -/- mice (Figure 17). Sorting of

-/- Tregs resulted in equal yields between Il9 and WT mice across experiments and post-sort analysis revealed consistent expression of FoxP3 in sorted cells (not shown).

88 6.5 Co-stimulation of regulatory T cells and the interaction with type 2 innate lymphoid cells

For the in vitro experiments, IL-9 deficient and sufficient Foxp3 GFP lines were used confirming

-/- the in vivo experiments. Il9 Tregs had a reduced capacity to suppress proliferating Teﬀ responders (Figure 16), which was linked to the down-regulation of co-stimulatory receptors. However, in this

-/- study it remained unaddressed at which developmental stage Il9 Tregs failed to fully mature. It was reported that ILC2s can be found in the human thymus (Gentek et al. 2013), which would put them in the position to interfere early in the Treg development. Both, Tregs and ILC2s require a similar signal repertoire for their development and their activation including the stimulation by Notch signalling, IL-2 and -33 (Hoyler et al. 2012; Gentek et al. 2013; Weist et al. 2015). Hence, both cell types might occupy the same thymic and peripheral niches. In the absence of IL-9, which promotes the survival of ILC2s, the competition for these other factors is likely to be increased, which then results in an insuﬃcient development of both cell types.

6.5 Co-stimulation of regulatory T cells and the interaction with

type 2 innate lymphoid cells

For the thymic development of Tregs, besides the stimulation by IL-2, the stimulation by GITR-L, TNF-α and OX40L was found to be essential (Mahmud et al. 2014). GITR deﬁciency resulted in an impaired generation of Tregs. Furthermore, it can be speculated that GITR is a more reliable marker for Tregs than CD25. Transfer experiments with either CD25 or GITR depleted thymocytes into athymic nude mice showed that only the transfer of the GITR deprived population resulted in a high incidence of inﬂammatory myocarditis and rapid death of the recipients (Ono et al. 2006).

By contrast, many other studies found that GITR stimulation impaired Tregs. This might be due to an epigenetically controlled down-regulation of FoxP3 expression upon GITR stimulation (I.-K. Kim et al. 2015; Xiao et al. 2015). Interestingly, GITR stimulation opened the Il9 locus and hence this mechanism would explain the previously reported high production of IL-9 in Tregs upon GITR stimulation (Lu et al. 2006). GITR mediates signals through the acceptor TRAF6 and then through both, the canonical and the non-canonical pathway of NF-κB (I.-K. Kim et al. 2015; Xiao et al. 2015). Detailed analysis showed that only the alternative pathway was responsible for the instability of the Foxp3 locus (Xiao et al. 2015).

Balancing canonical and alternate signalling thus appears to be essential for Treg stability. Activ-

89 6.5 Co-stimulation of regulatory T cells and the interaction with type 2 innate lymphoid cells ation of the canonical pathway was shown to inhibit the non-canonical pathway and thus canonical

NF-κB signalling might reduce the instability of Tregs (Gray et al. 2014). Modulation of the NF-κB pathway also is an important component of co-stimulation by the receptors of the B7 family CD28, ICOS, CTLA4 and PD-1 (Schmitz et al. 2006). Stimulation of TCR and CD28 activated exclusively the canonical NF-κB pathway in na¨ıve T cells, but non-canonical signalling was required for the activation of memory T cells (Ishimaru et al. 2006). Furthermore, it has been shown that the in vitro and in vivo stimulation of Tregs by either the agonistic anti-GITR antibody g3c or pentameric

GITR-L induces the proliferation of Tregs (Y. H. Kim et al. 2015; Nishioka et al. 2008). The inhibi- tion of tumour growth by the agonistic anti-GITR antibody DTA-1 was shown to be dependant on ADCC (Bulliard et al. 2013). Thus anti-GITR therapies might not per se depend on the functional inactivation of Tregs.

In conclusion, the eﬀect of GITR stimulation on Tregs might be only transient. Furthermore, the eﬀect appears to be dependant on additional co-stimulation that modulates the NF-κB pathway.

Notably, GITR-L transgenic mice displayed increased Treg counts (Olﬀen et al. 2009). In this study, the in vitro suppression assays revealed that the sequestration of GITR-L induced the loss of Treg suppression (Figure 26). Given that GITR was found to be almost exclusively expressed on Tregs but not responders (Ono et al. 2006), this ﬁnding is in line with the reports showing an enhanced activity of Treg after GITR stimulation (Y. H. Kim et al. 2015; Nishioka et al. 2008).

+ Besides GITR, ICOS is established as one of the key co-receptor of Tregs. FoxP3 Tregs can be

+ subdivided in two populations on basis of the expression of ICOS. ICOS Tregs have been shown to be more suppressive, more proliferative and less prone to cell death (Y. Chen et al. 2012). Accordingly,

TH17 converted from Tregs express lower amounts of ICOS highlighting the essential role of ICOS stimulation for Treg stability (Komatsu et al. 2014). In line with these reports, in this study the sequestration of ICOS-L impaired the suppression of

Teﬀ proliferation by Tregs (Figure 26). Furthermore, a combined stimulation of ICOS and GITR led to a particularly strong suppression of Teﬀ proliferation, arguing for a synergistic co-stimulation

-/- by both pathways. In vivo, adoptively transferred Il9 Tregs were pre-stimulated with DTA-1 and recombinant ICOS-L resulting in a restored resolution of AIA in Il9 -/- mice (Figure 27). The pre- stimulation might have elicited a highly stable and proliferative Treg population thus having led to a dilution of the antibody to cell ratio, which obviously was protective against ADCC.

90 6.5 Co-stimulation of regulatory T cells and the interaction with type 2 innate lymphoid cells

In this study it was found that Tregs received co-stimulation from activated ILC2s via ICOS-L and GITR-L. The interaction of both cell types has been described in diﬀerent physiological settings.

Based on the observation that both, ILC2s and Tregs can be activated by IL-33, Molofsky et al. (2015) studied visceral adipose tissue, which disposes of particularly high endogenous IL-33 levels.

Visceral Tregs, but not ILC2s, failed to accumulate in mice with deﬁcient IL-33 signalling. In addition, the depletion of ILC2s resulted in an ablated Treg accumulation. It was found, that ILC2s expressed high levels of ICOS-L and in vitro as in vivo ICOS signalling expanded the Treg population. It was found by another study that the ICOS-ICOS-L interaction is an autocrine requirement for the homeostatic survival and the activation of ILC2s in the lung (Maazi et al. 2015). Accordingly,

Tregs can reversely signal to ILC2s sequestering ICOS-L in the lung and limiting allergic inflammation and eosinophilia (Rigas et al. 2017). However, the requirement of ICOS signalling for the survival of ILC2s remains controversial. While IL-33 failed to elicit ILC2 accumulation in the lung of ICOS deficient mice, ICOS-L deficient mice displayed a normal ILC2 response to IL-33 (Molofsky et al. 2015; Maazi et al. 2015).

TGF-β was suggested as an additional mediator of the Treg-ILC2 crosstalk, since the Treg mediated restriction of ILC2 derived IL-13 was abolished in presence of an inhibitor of SMAD3, the signal transducer of the TGF-β receptor (Krishnamoorthy et al. 2015). A very attractive candidate however is amphiregulin, which was shown to enhance Treg activity (Zaiss et al. 2013). The reason is that ILC2s were identified as the major source of amphiregulin during influenza airway infection and IL-33 robustly induced amphiregulin expression in ILC2s (Monticelli, Sonnenberg et al. 2011). Indeed, an amphiregulin mediated ILC2-Treg mechanism of inflammation control recently was suggested to take place in liver inflammation (Ochel et al. 2018). Of note, the receptor for amphiregulin is also expressed on epithelial cells and in a colonic inflammation model, ILC2s directly controlled the epithelial barrier integrity by amphiregulin (Monticelli, Osborne et al. 2015). In this study, ILC2s were shown to express ICOS-L and GITR-L upon stimulation with IL-9 (Fig- ure 26). The expression of both ligands was sufficient to rescue the impaired suppressive activity

-/- of Il9 Tregs in vitro and in vivo. Despite the multiple evidence of soluble mediators, trans-well experiments impaired the pro-resolution eﬀect of ILC2s.

91 7 Conclusion and outlook

The findings in this study unveiled a novel mechanism of inflammation control and induction of resolution. Resolution mainly has been attributed to myeloid-derived lipid metabolites, so-called specialized pro-resolving lipid mediators and lipoxins. Actually, these molecules have been shown to be involved in the regulation of ILC2 activity (Konya et al. 2016). By contrast, Tregs are established regulators of peripheral tolerance and tissue homoeostasis and as such they control the initiation of autoimmune diseases. Hence, therapeutic modulation of Tregs is a compelling approach. Finally, IL-9 is described as a cyotkine with a pro- and anti-inflammatory activity. All together, the following conclusions can be drawn. First, IL-9 is a pro-resolving cytokine. However its pro-resolving activity is restricted to the right environment. This became apparent by the comparison of IL-9-producing cells in the synovium of active and inactive RA patients. Second, ILC2s exert immune regulatory functions. Besides their pro-inflammatory function, many reports have shown that ILC2s indirectly and directly contribute to the tissue integrity and inflammation control by altering the cytokine milieu. Hence, the activation of ILC2s emerges as a potential therapeutic anchor point to induce resolution of inflammation. Third, ILC2s interact with the adaptive immune system, namely Tregs, in a cell-cell contact dependant and potentially bi-directional manner. Fourth, ILC2s are a potential bio-marker for the independent diagnosis of the disease activity in rheumatic diseases. In conclusion, ILC2s emerge as a cell population orchestrating inflammation with the ability to promote its resolution. Mono-therapies with anti-inflammatory DMARDs are limited, as they fail to restore immune tolerance. Hence, combined therapies limiting the pro-inflammatory cascades together with an ILC2 induction to restore the tolerance might be the future of anti-rheumatic therapy. Further studies also will be required to establish ILC2s in routine diagnostic.

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121 9 Appendix

122 9.1 List of acronyms

9.1 List of acronyms

ACPA anti-citrullinated protein antibodies ADCC antibody dependant cell mediated cytotoxicity AIA antigen induced arthrits ANOVA analysis of variance AUC area under the curve BAFF B cell activating factor BCR B cell receptor BSA bovine serum albumin CCR C-C motif chemokine receptor CD cluster of diﬀerentiation CFA complete Freund’s adjuvant CFSE carboxyﬂuorescein succinimidyl ester CRISPR clustered regularly interspaced short palindromic repeats CRTH2 chemoattractant receptor-homologous molecule expressed on T helper type 2 cells CTLA cytotoxic T-lymphocyte-associated protein CXCL chemokine C-X-C motif ligand CXCR C-X-C motif chemokine receptor DAPI 4’,6-diamidino-2-phenylindole DAS28 diseases activity score 28 DC dendritic cell DMARD disease modifying anti-rheumatic drug DNA deoxyribonucleic acid EAE experimental auto-immune encephalitis EDTA ethylenediaminetetra-acetic acid EGTA ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetra-acetic acid ELISA enzyme-linked immunosorbent assay FBS foetal bovine serum

I 9.1 List of acronyms

FLS fibroblast like synoviocyte FMO fluorescence minus one GITR glucocorticoid induced TNFR related GPI glucose-6-phosphate isomerase HBSS Hank’s Balanced Salt Solution HDGT hydrodynamic gene transfer hi high expressing population HLA human leukocyte antigen HPF high power field ICOS inducible T cell co-stimulator ICOS-L ICOS ligand IFN-γ interferon gamma Ig immunoglobulin IL interleukin IL2Rγ interleukin 2 receptor gamma ILC innate lymphoid cell ILC1 type 1 innate lymphoid cell ILC2 type 2 innate lymphoid cell ILC3 type 3 innate lymphoid cell IMDM Iscove’s modified Dulbecco’s medium JAK Janus kinase KLRG1 killer cell lectin-like receptor subfamily G member 1 KO knock out LB lysogeny broth LN lymph node lo low expressing population LTi lymphoid tissue inducer cell mBSA mehtylated bovine serum albumin MC minicircle MCPT mast cell protease

II 9.1 List of acronyms

MFI mean fluorescence index MHC major histocompability complex MSU monosodium urate MTX methotrexate NCR natural cytotoxicity receptor NK cell natural killer cell NSAID non-steroidal anti-inflammatory drug PBMC peripheral blood mononuclear cell PBS phosphate buffered saline PCR polymerase chain reaction PD programmed cell death PMA phorbol 12-myristate 13-acetate PMN polymorphonucelar cell PMSF phenylmethylsulfonylfluorid RA rheumatoid arthritis RAG recombination-activating gene RANKL receptor of nuclear factor κ B ligand RF rheumatoid factor RID remission inducing drug RNA ribonucleic acid RORγ retinoic acid receptor related orphan receptor γ RPMI 1640 Roswell Park Memorial Institute medium sca-1 stem cell antigen 1 SCID severe combined immunodeficiency SDS sodium dodecyl sulfate SEM mean of the standard errors SIA serum transfer induced arthritis SLO secondary lymphoid organ SOC super optimal broth STAT signal transducer and activator of transcription

III 9.1 List of acronyms

TB terriﬁc broth T-bet T-box transcription factor TBX21

TC cytotoxic T cell TCR T cell receptor

Teﬀ eﬀector T cell TGF-β transforming growth factor beta

TH helper T cell TNF-α tumor necrosis factor alpha TRAP tartrate-resistant acetic phosphatase

Treg regulatory T cell Tris 2-Amino-2-(hydroxymethyl)propane-1,3-diol VLR variable lymphocyte receptor (v/v) volume fraction WT wildtype (w/v) mass fraction

IV 9.2 List of ﬁgures

9.2 List of ﬁgures

1 Publications on ILCs over time ...... 6 2 The discovery of ILCs - A timeline ...... 11 3 Developmental trajectories of ILCs and their classiﬁcation ...... 13 4 Schematic illustration of a synovial joint ...... 22

5 The AIA model of RA ...... 57 6 AIA in WT and Il9 -/- mice ...... 57 7 Histological analysis of knee joints from the AIA model of RA at day 42 ...... 58 8 µCT analysis of tibiae from the AIA model of RA at day 42 ...... 59 9 A re-established spontaneous resolution of AIA in Il9 -/- mice after the HDGT of Il9 . 60 10 The gout-like inﬂammatory arthritis induced by MSU crystals in WT and Il9 -/- animals 60 11 Resolution of SIA by the HDGT of Il9 ...... 62 12 Histological analysis of paws from the SIA model of RA ...... 63 13 Analysis of the cytokine patterns in AIA of WT and Il9 -/- animals ...... 64

-/- 14 In vitro TH17 polarization capacity in WT and Il9 mice ...... 65 15 Composition of the CD4+ T cell compartment in WT and Il9 -/- mice during AIA . . 66

-/- 16 An impaired suppressive capacity of Tregs from Il9 mice ...... 67 17 The impaired suppression is not a direct result of IL-9 shortage ...... 67

18 The impaired suppression is not linked to a diﬀerent responses of Teﬀs ...... 68

19 The adoptive transfer of Tregs in AIA ...... 68 20 AIA in Spi1 ﬂ/ﬂ x Lck-cre mice ...... 69 21 Mast cells in AIA or SIA in WT and Il9 -/- animals ...... 70 22 IL-9-producing cells in AIA ...... 72 23 ILC2s require IL-9 signalling ...... 73 24 Impaired proliferation of ILC2s in AIA in Il9 -/- mice ...... 73

25 Co-localization of Tregs with ILC2s in the inﬂamed synovium ...... 74

26 Mechanism of ILC2-mediated enhancement of the suppressive activity of Tregs .... 76

27 Adoptive transfer of stimulated Tregs in AIA ...... 77

V LIST OF FIGURES

28 Adoptive transfer of ILC2s in AIA ...... 78 29 IL-9 expressing cells in diﬀerent phases of human arthritis ...... 80 30 Circulating ILC2s in patients with RA ...... 81

31 Proposed mechanism for the ILC2 licensing of Tregs in RA ...... 83

VI 9.3 List of tables

9.3 List of tables

1 List of instruments ...... 29 2 List of auxiliary equipment ...... 30 3 List of chemicals, organic and inorganic compounds ...... 32 4 List of kits and other material ...... 33 5 List of buffers and cell culture media ...... 34 6 List of mouse strains ...... 37 7 List of genotyping primers ...... 38 8 List of reagents and material required for the MC and plasmid production ...... 42 9 List of flow cytometry and cell sorting antibodies mouse ...... 45 10 List of flow cytometry antibodies human ...... 46 11 List of antibodies and cytokines required for T cell and ILC assays ...... 48 12 List of antibodies used for immunofluorescence staining ...... 53 13 List of real time primers ...... 55

VII 9.4 Acknowledgments

9.4 Acknowledgments

There are numerous colleagues to who I have to express my gratitude. They made this study possible and paved my way till here. First of all, I would like to express my gratitude to Dr. Andreas Ramming for welcoming me in his team. He gave me the great opportunity to work on a really fascinating story. I thank you, Andreas, for your trust and confidence, your support and your encouragement. A special thank you to my supervisor, Prof. Steffen Backert, for your willingness to supervise and examine my thesis and hence to accompany me in this important moment. I also want to express my gratitude to Prof. Georg Schett and Prof. Jörg Distler, not only for your willingness to join the thesis supervisory committee, but also and especially for your stimulating input as well as the help and support preparing and submitting the manuscript. Last but not least, my thanks belongs to Prof. Lars Nitschke for completing my committee and heading the doctoral examination. A really big thank you to all the team members of AG Ramming and Distler for your helping hands in several experiments and your critical and honest comments in all the data discussions. Thanks to Markus Luber for helping with the T cell polarisation assays. Thanks to Katja Dreissigacker, Regina Kleinlein, Rita Weinkam and Monika Pascual Mate for their assistance. Thanks to Dana Weidner for the µCT acquisitions. Thanks to Uwe Appelt and Markus Mroz for the hours and hours of sorting. I would like to thank all our co-authors and cooperation partners of the paper for their contribution. Thanks to the patients for their precious donations and thanks to the animal keepers in Med3 and PETZ. You all made this study possible.

VIII