Innate lymphoid cells and fibrotic regulation

Accepted Manuscript Title: INNATE LYMPHOID CELLS AND FIBROTIC REGULATION Authors: Steven Horsburgh, Stephen Todryk, Andreas Ramming, J¨org H.W. Distler, Steven O’Reilly PII: DOI: Reference:

S0165-2478(17)30286-9 http://dx.doi.org/10.1016/j.imlet.2017.08.022 IMLET 6100

To appear in:

Immunology Letters

Received date: Revised date: Accepted date:

19-6-2017 15-8-2017 18-8-2017

Please cite this article as: Horsburgh Steven, Todryk Stephen, Ramming Andreas, Distler J¨org HW, O’Reilly Steven.INNATE LYMPHOID CELLS AND FIBROTIC REGULATION.Immunology Letters http://dx.doi.org/10.1016/j.imlet.2017.08.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

INNATE LYMPHOID CELLS AND FIBROTIC REGULATION *Steven Horsburgh1 Stephen Todryk1 Andreas Ramming2 Jörg H. W. Distler2 Steven O’Reilly1

1

Department of Applied Sciences, Faculty of Health and Life Sciences, Northumbria University,

Newcastle upon Tyne, UK 2

Department of Internal Medicine 3 and Institute for Clinical Immunology, University of

ErlangenNuremberg and University Hospital Erlangen, Erlangen, Germany

*corresponding author.

Highlights •

Innate lymphoid cells (ILCs) are immune cells that do not possess B or T cell receptors.

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They produce a variety of cytokines which regulate fibrosis.

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These cytokines activate TGF-β signalling and other immune cells.

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Dysregulation of ILC numbers has been observed in a number of pathologies.

Abstract Innate lymphoid cells (ILCs) are innate immune cells that do not possess B or T cell receptors but belong to the lymphoid lineage. While these cells have not yet been extensively investigated since their classification as a homogenous group, emerging evidence suggests that they exert

significant regulatory roles in both tissue remodelling and inflammation, and are therefore, also involved in fibrotic regulation. The following review will serve to outline the transcription factors, surface markers, and cytokines that define each subgroup, and the process by which these cells differentiate. Furthermore, the diverse functions of these cells in non-pathogenic states will be discussed, in addition to the interactions between ILCs and other cells of the immune system, both innate and adaptive, and how these pathways can elicit both pro- and anti-inflammatory and –fibrotic effects in varying tissues.

Keywords: lymphocytes, cytokines, inflammation, fibrosis, scleroderma.

1.0 - Introduction Innate lymphoid cells (ILCs) are a relatively recent reclassification of a collection of cells that do not share expression of cell surface markers with other immune cell lineages (i.e. Lin-), nor do they respond in an antigen-specific manner given that they also lack T cell receptor (TCR) expression. However, they are CD45+ and produce a variety of cytokines with similar functionality to other lymphocytes. Furthermore, despite evidence which suggests that ILCs may not be essential for survival in humans [1] in contrast to adaptive lymphocytes, these cells exert a number of protective functions in a variety of tissues, in addition to contributing to deleterious responses such as fibrosis. It has been suggested that the overlapping functions of innate and adaptive lymphocytes and potential ILC redundancy could be an evolutionary mechanism in order to protect against immunopathology and reduce susceptibility to autoimmunity [2].

2.0 - Classification of ILCs – Subgroups and Functions Overlapping and unique gene-expression signatures have been reported between the subgroups [3], highlighting that whilst they all exhibit a vast number of genetic and phenotypic similarities, nuanced differences in how they develop and function results in the distinct subgroup classification. Although once controversial, it is now recognised that group 1 ILCs comprises ‘ILC1’ and conventional natural killer cell (‘cNK’) subgroups, both of which express the transcription factor T-bet, with only the latter expressing Eomes [4, 5]. Various other transcriptional differences have been reported which supports the distinction of the two subgroups [3]. Whilst the majority of ILCs are CD127+ (i.e. IL-7Rα+)

and thus rely on IL-7 signalling as a master regulator of development [6], each ILC subgroup is also tightly regulated by other cytokines. ILC1s express high levels of IL12rb1 (i.e. IL-12 receptor) and are the key producers of IFNγ and TNFα in response to IL-12 stimulation [5]. Co-stimulation with IL-18 was also shown to significantly augment IFNγ protein levels [7]. CD127- ILC1s are able to produce both TNFα and IFNγ, whereas CD127+ ILCs only produce IFNγ [8]. As such, ILC1s are largely noncytotoxic, and are involved in immunity to intracellular bacteria and chronic inflammation due, in part, to expression of CXCR3 which mediates Th1 cell chemoattraction and maturation [7]. In addition, IFNγ is involved in a positive feedback loop as it is not only produced by T h1 cells but also elicits the differentiation of naive CD4+ cells into more Th1 cells. Interestingly, stimulation of NKp44+ ILC3s with IL-12 and IL-2 results in ILC1s, demonstrating the plasticity of ILC phenotypes depending on the environment and cytokines that are present [7]. In their role as the main cytotoxic lymphocytes of the immune system, and the only ILC subgroup which possess considerable cytotoxic activity, cNKs are activated by IL-15 [9] and secrete the cytolytic enzymes granzyme and perforin, as well as TNFα and IFNγ in small amounts. Functionally, these cells possess a key role in tumour surveillance, viral immunity, and the inflammatory response. With no currently defined subgroups, ILC2s are presently the most homogenous ILC population. The transcription factor GATA3, alongside IL-25 and IL-33 signalling [10], appears to be essential in ILC2 differentiation; high levels of expression was reported in these cells, with other Lin- CD127+ ILCs expressing GATA3 at lower levels [11]. Furthermore, GATA3-/- mice exhibited an absence of ILC2s, with a concomitant reduction in IL-13 and IL-5 mRNA. Other transcription factors such as RORα [12], TCF-1 [13], and Notch [14] are also required for ILC2 differentiation. In contrast to ILC1s, ILC2s produce Th2-associated cytokines such as IL-4, IL-5, IL-9, and IL-13. Since the latter cytokine is essential in the ‘type-2’ response to Helminth infection, as it induces eosinophilia and smooth muscle contraction [15], one of the main roles of ILC2s is to act as an innate effector cell; IL-25 and IL-33 receptor KO mice exhibited a significant attenuation in ILC2 cell numbers, concomitantly with an impaired ability to expel worms (N.brasiliensis) [10]. Again, due to the cytokines that are produced, these cells also contribute to allergic responses in the airways. Specifically, infiltrating ILC2s have been shown to be the predominant source of IL-13 in the lung, in addition to CD4+ T cells [16]. Clearly, a considerable overlap exists between ILC2 and Th2 cells. For example, IL-2 produced by CD4+ T cells promotes ILC2 proliferation [17] and stimulates Th2 cytokine release from ILC2s, whilst differentiation of naïve T cells is enhanced by ILC2s, thereby heightening the Th2 cytokine response [18]. Similarly, IL-13 release from ILC2s stimulates the production of CCL17, known for Th2 cell chemoattractant ability [19]. The reported strong correlation between ILC2 numbers and skin infiltrating Th2 cells seems logical therefore, especially given that ILC2 depletion prior to an allergen challenge significantly attenuated Th2 numbers. Finally, another function of ILC2s is tissue homeostasis; the expression of a number of genes associated with tissue repair are expressed at high levels, at least within lung ILC2s

[20]. This will be discussed further later in this review as these mechanisms overlap with those involved in fibrosis. Both group 3 ILC subgroups, ‘ILC3’ and Lymphoid Tissue inducers (‘LTi’), rely upon the Th17associated transcription factor RORγt and IL-7 for their development. ILC3s are also T-bet+ and can be further divided based upon the presence of Natural Cytotoxicity Receptors (NCRs) such as NKp46 [21]. Further support for the division of this group based on NCRs was provided by Rankin et al [22], who demonstrated that NCR+ and NCR- ILC3s exhibited distinct gene expression profiles, particularly with regard to the expression of a number of transcriptional regulators. IL-23 and IL-1β elicit the production of IL-22, IL-17 (NCR- cells only) and GM-CSF from ILC3s [8, 23]. These cytokines are also produced by LTi cells [24], in addition to TNFα and Lymphotoxin [25]. In terms of function, both ILC3s and LTi’s are involved in immunity to extracellular bacteria [26, 27]; due to the production of the aforementioned cytokines, particularly IL-17 and IL-22, group 3 ILCs can act as innate effector cells during host defence [24]. Additionally, NCR+ ILC3s are required for epithelial homeostasis [28], whereas LTi’s contribute to homeostasis of the intestinal tract [29]. Furthermore, LTi’s possess a key role in secondary lymphoid organ formation during embryogenesis [25, 30]. The role of ILC3s is made more complex when their interaction with T cells is considered. NCR- ILC3s have been found to express MHCII, and can, therefore, process and present antigens but are unable to stimulate naïve T cell proliferation due to the lack of the co-stimulatory markers CD40, CD80, and CD86. It had previously been purported that due to the absence of these markers, antigen presentation serves to impair T cell responses. Indeed, Hepworth et al [31] discovered this to be the case in vivo. Furthermore, memory CD4+ T cell survival is impaired in RORγ-/- mice; the researchers went on to demonstrate that LTi’s aid in the survival of memory CD4+ T cells, but not CD8+ T cells [32]. Table 1 provides a summary of the transcription factors, surface markers, and cytokines associated with each ILC subgroup.

3.0 - ILC Development As with all lymphocytes, ILCs differentiate from a common lymphoid progenitor; however, unlike T and B cell lineages, ILC precursors appear to express Integrin-α4β7 and have been termed α-lymphoid progenitor cells (αLPs). CXCR6+ αLPs are able to differentiate into ILC3s and cNKs [33], whilst all other sub groups (ILC1, ILC2, ILC3, LTi) develop from Id2+ αLPs [5]. The importance of Id2 is demonstrated by Id2-depleted mice, which are deficient in ILCs [20]. Furthermore, Id2, Notch, and

RORγt are all essential in LTi production from common lymphoid progenitors [34]. Early innate lymphoid cell progenitors (EILP) are Integrin-α4β7+ and CXCR3-, and have been shown to develop into both cytotoxic and helper-like ILC lineages in vitro [35]. Epigenetic mechanisms also likely contribute to the differentiation and regulation of these cells, although this is currently an under-investigated area. For example, expression of the human KIR and murine Ly49a genes, which encode a transmembrane NK cell receptor for MHC class I molecules, are regulated by DNA methylation [36], as well as histone acetylation in the case of Ly49a [37]. The histone deacetylase inhibitor trichostatin A was able to significantly reduce the number of IL-5 and IL-13 expressing ILC2s in the lungs of Alternaria-challenged mice [38]. Non-coding RNA also appear to possess regulatory roles; the long non-coding RNA lncKdm2b has been implicated in the proliferation of ILC3s via remodelling complex recruitment and activation of the transcription factor Zfp292 [39]. Moreover, of particular interest within the context of this review is the regulation of ILC2s by microRNA-155 (miR-155). Elevated expression of miR-155 in both peripheral blood mononuclear cells and skin of Systemic Sclerosis (SSc) patients has been reported previously [40]. Resistance to bleomycin-induced fibrosis of the skin and lungs was evident in miR-155-/- mice [41], and similarly, silencing of miR-155 in dermal fibroblasts elicited a significant attenuation of not only Wnt/β-catenin signalling, but functionally, collagen synthesis [40]. ILC2 numbers were also impaired in allergenchallenged miR-155-/- mice [42], suggesting that this microRNA regulates ILC2 expansion and therefore IL-33 signalling which are inherently linked to downstream inflammatory and fibrotic processes. It must be noted that ILCs development is still an emerging area of investigation and is only included briefly in the current review to highlight both the similarities and differences between ILC and regular lymphocyte development pathways. Gronke et al [43] have recently published a concise overview of this area. Furthermore, while there is agreement on the definitions and nomenclature of the ILC subgroups, there are also some reported inconsistencies in terms of the exact expression profiles of these newly defined cell types.

4.0 - ILCs in Normal Skin The skin is the largest and one of the most diverse organs of the body as it is populated by a large number of different cell types, and consequently, possesses a vast number of functions. The epidermis and dermis are the two predominant layers; the former is comprised primarily of keratinocytes, but also includes melanocytes, Langerhans and Merkel cells, and a small number of CD8+ memory T cells. Conversely, the dermis is populated by dendritic cells, mast cells, macrophages, B cells, CD4+ and CD8+ T cells, and NK cells, which, as previously alluded to, were the first ILC subgroup to be identified [44].

More recently, all ILC subgroups have been detected, primarily within the dermis. Roediger et al [45] reported that while ILCs were found in both the epidermis and dermis, the latter was the predominant source, with ILC2s comprising up to 10% of all CD45+ cells. One analysis concluded that approximately 40% of quantified ILCs in healthy skin were ILC2s, with NKs, NCR+ ILC3 and LTi cells contributing approximately 10%, 20%, and 30%, respectively [46]. ILC1 and ILC3 cells have also been found in healthy and diseased skin [46, 47]. Importantly, keratinocytes, as well as the less abundant dendritic and Langerhans cells, are able to release all of the cytokines that are essential for ILC subgroup activation [48-52].

5.0 - ILCs and Fibrosis Accumulating evidence highlights that ILCs play a central regulatory role in healthy skin and wound healing, with disruption to the tissue repair mechanisms resulting in aberrant fibrosis. Cells of both the innate and adaptive immune systems are involved in this process, with ILCs considered to act as mediators between the two. Release of a number of cytokines from a variety of cell types coordinates this pathway and will be discussed, both generally, and in the context of fibrotic diseases. Fibrosis involves inflammatory cell infiltration, fibroblast activation and myofibroblast differentiation, followed by excessive and regular deposition of extracellular matrix components (i.e. collagen, amongst others), and is involved in a large number of debilitating chronic diseases. Under normal physiological conditions, the steps of inflammation, proliferation and remodelling coordinate appropriate wound repair, however, pathological aberrations in these processes can result in persistent activation of these pathways, which leads to fibrosis. Chronic inflammation, therefore, significantly contributes to the pathogenesis of fibrosis, and as such, it is logical that numerous cells of both the innate and adaptive immune systems would play a key role. Indeed, evidence is mounting that ILCs are involved in driving fibrosis in a number of tissues.

5.1 - Mechanisms Table 1 outlines the cytokines produced by each ILC subgroup, of which regulation of inflammation and fibrosis are key functions. IL-25 and IL-33 begin the fibrotic cascade; the former can be secreted by innate cells such as eosinophils and macrophages, as well as Th2 cells, whereas the latter is released upon cell damage [53]. In combination with TSLP, these cytokines induce ILC2 expansion [54]. IL-2 secretion from Th2 cells occurs following antigen-specific interaction with ILC2s [17], which elicits the release of IL-13, in addition to IL-4 and IL-5, from ILC2s. In addition, intragastric antigen challenge-induced elevations in

Th2 numbers was shown to augment IL-25-induced IL-13 secretion by ILC2s [55]. The pro-fibrotic consequences of the ‘Type 2’ response are primarily mediated through the functions of these cytokines. IL-13 signalling induces TGF-β1, concomitantly with reduced matrix metalloproteinase (MMP) expression [56, 57]. Both IL-4 and IL-13 have been shown to cause excess collagen deposition in dermal fibroblasts, hepatic stellate cells (HSCs), and in the lung [58-62], as well as increase α-SMA expression [63]. Furthermore, both cytokines are involved in the polarisation of fibrotic alternatively activated (i.e. M2) macrophages [64], and have been shown to elicit myofibroblast differentiation [63]. Dendritic cells contribute to this pathway through IL-13 mediated suppression of the anti-fibrotic IFNγ, while IL-4 stimulates maturation of these cells [65]. Although not purported to possess as large a role as IL-4 and IL-13, IL-5, which upon secretion by Th2, cells causes eosinophils differentiation [66]; cells known to produce TGF-β. Additionally, in response to IL-33, eosinophils have been shown to secrete IL-4 [67], which would serve to exacerbate the fibrotic response. The ILC3 secreted cytokine IL-17 exerts both pro- and anti-fibrotic functions. For example, it contributes to pulmonary inflammation and fibrosis, as demonstrated by IL-17ra-/- mice that exhibited significant attenuation of collagen deposition, as well as reduced macrophage, CD4+ and CD8+ T cell counts in the lungs [68]. IL-17A-induced pulmonary fibrosis is dependent upon TGF-β signalling in the lung [69]. Similar results have been reported with regard to liver fibrosis; expression of α-SMA, Col1α1, TIMP1, and TGF-β1 were all significantly lower in HSCs from IL-17ra-/- mice [70]. Similarly, IL-17 induced activation of HSCs, in addition to augmentation of TGF-β1, IL-6, TNF-α, and α-SMA expression concurrently with increased production of collagen [70, 71]. IL-17 expression and concentrations have been reported as elevated in skin and serum of SSc patients, which was postulated to be due to a negative feedback mechanism given that IL-17RA was suppressed, thereby inhibiting this signalling pathway [72]. This initially appears counterintuitive; however, antifibrotic effects of IL-17A were also reported in this study. IL-17A reduced CTGF and Col1α1 protein levels in normal fibroblasts, mediated by miR-129-5p, which is likely an inhibitor of TGF-β1 signalling [73]. Moreover, pulmonary fibrosis can occur in the absence of IL-17, demonstrating that it may not be a potent fibrogenic factor [74]. As already alluded to briefly, IFNγ also exerts anti-fibrotic effects via apoptosis and attenuated activation of HSCs [75-78], in addition to inhibition of TGF-β1 [79], and the accumulation of collagen, fibronectin, and laminin [75].

ILCs have been described in the pathogenesis of a number of inflammatory skin disorders such as Atopic Dermatitis and Psoriasis [46, 47, 80, 81]. With a specific emphasis on fibrosis, there is a considerable paucity of literature detailing the role that ILCs play in fibrotic disorders of the skin, such as SSc, as well as fibrosis of the liver, lungs, and gut. Discussion of the involvement of ILCs in other forms of fibrosis is worthwhile, therefore, given the considerable overlap in mechanisms.

5.2 - Skin IL-33 expressing cells are present in normal skin [82]; however, this expression is augmented in SSc patient serum samples, which correlates with the extent of fibrosis [83]. Administration of recombinant IL-33 elicits a multitude of effects, including an increase in IL-13, IL-4, IL-5, TIMP-1, Col3α1, and Col6α1 mRNA [84]. Not only this, but local accumulation of eosinophils and CD3+ cells also occurred. The importance of IL-33, secreted by eosinophils in response to IL-13, was also demonstrated by the lack of inflammation and fibrosis in ST2-/- mice. While these studies did not specifically identify ILCs in this pathway linking IL-13/IL-33 signalling to fibrosis, these particular cytokines are well established as being intrinsically linked with ILC2s. Focusing specifically on SSc, an autoimmune disorder characterised by fibrosis of the skin, significant elevations in ILC1 (primarily CD4+) and NKp44+ ILC3 numbers were observed in peripheral blood samples in comparison to age and gender matched controls [85]. In contrast, NKp44- ILC3s frequencies were attenuated, while ILC2s remained unchanged; a surprising finding given the involvement of Th2/ILC2 cytokines such as IL-4, IL-5, and IL-13. Furthermore, it was also reported that co-expression of IL-6rα and gp130 on CD4+ ILC1s was attenuated, which may appear counterintuitive given that IL6 concentrations are significantly elevated in the sera of SSc patients [86], and elevated IL-6 expression in early SSc has been shown to be correlated with severity of sclerosis of the skin and long term survival [87]. The authors do postulate, however, that CD4+ ILC1s and ILC2s are sources of soluble IL-6Rα, which can activate the ‘trans-signalling’ pathway, independently from membrane bound IL-6 receptor. The lack of observable alterations in ILC2 frequency described above contrasts with data previously reported by Wohlfahrt et al [88]. Using two different panels of surface markers, they discovered that ILC2 numbers in the skin were elevated 10-fold and 3.8-fold compared with healthy controls (panel 1: ICOS+ ST2+ CD3- CD11b-; panel 2: ST2+ IL-17RB+ KLRG1+ Lin-). This elevation in ILC2 frequency was corroborated in blood samples. Of importance was that in both skin and blood, higher counts were observed in patients with diffuse cutaneous SSc compared with limited cutaneous SSc, and also correlated with the extent of skin fibrosis, suggesting that these cells do indeed contribution to the manifestation of fibrosis.

Although tissue-specific differences in ILC counts likely occur in samples taken from differing sources (i.e. skin vs blood), this does not account for the conflicting findings between these two studies given that Wohlfahrt et al [88] corroborated their skin-specific data in the blood. Rather, these discrepancies point to the specific panel of markers used to identify ILC subsets, and shows that this clearly plays a significant role in accurate quantification, especially considering that there is no current consensus. As alluded to above, the release of a number of cytokines from ILCs results in aberrant wound healing mechanisms which cause fibrosis. In vitro experiments revealed that co-culture of ILC2s and fibroblasts lead to activation of fibroblasts and trans-differentiation into myofibroblasts with excessive release of ECM (Ramming et al, unpublished data). However, ILCs are also involved in normal, non-pathogenic wound healing, both within the skin and other tissues. In an excisional wound model, IL-33 mRNA was found to be elevated concomitantly with elevated numbers of ILC2s. Upon deletion of IL-33, the ILC2 response was attenuated, in addition to a delayed healing response, whereas administration of IL-33 elicited the opposite response [89]. Furthermore, it has been shown that Notch1 upregulates TNFα which directs RORyt+ ILC3s to the wound site [90]. Whilst specific to lung ILCs and not the skin, ST2 and IL-25 receptors, IL-2, and IL-5 were reported to be the top transcripts in naïve mouse samples [20]. In addition to this, amphiregulin and the extracellular matrix (ECM) proteins dermatopontin, asporin, and decorin i.e. genes associated with tissue remodelling, were also elevated. Given that wound healing and immune defence response were the primary enriched pathways, combined with ILC-induced epithelial proliferation following influenza infection, the data suggest that ILCs promote pulmonary tissue remodelling.

5.3 - Lungs A number of studies have reported that IL-33 expression is elevated in the lungs of idiopathic pulmonary fibrosis (IPF) patients [91, 92]. A series of experiments was able to demonstrate that IL-33/ST2 signalling causes the polarisation of macrophages to a pro-fibrotic ‘M2’ phenotype in the lungs, thereby eliciting the production of IL-13 and TGF-β1. Additionally, IL-13 release was also elevated by IL33induced ILC2 expansion. Functionally, ST2 or macrophage deficiency resulted in attenuated lung inflammation and fibrosis, whereas exogenous IL-33 administration or ILC2 transfer exacerbated bleomycin-induced lung inflammation and fibrosis. This study focussed on the importance of IL-33, however, another group reported that only IL-25 expression, and not IL-33 or TSLP, was elevated in the mouse lung, albeit following the S.mansoni egginduced pulmonary granuloma model [58], rather than bleomycin as in the previous study [91]. Nevertheless, IL-25 is also known to be involved in ILC2 differentiation, with IL-25-/- mice producing less IL-4, IL-13, and TGF-β, resulting in a significant reduction in collagen deposition in both untreated

and egg-treated mice [58]. The importance of IL-13 in this instance was emphasised by the lack of collagen induction by IL-13 deficient ILC2s. Lung tissue of IPF patients also exhibited elevated IL-25 levels and ILC2 counts, which combined with the above experimental evidence, strongly supports the involvement of this cytokine-cell interaction in aberrant fibrotic progression.

5.4 - Gut When combined, these data substantiate the theoretical pathway linking IL-33, IL-25, and TSLP, and the subsequent signalling cascade involving ILC2s/Th2 cells, macrophages, and fibroblasts. In addition to this pathway, IL-13 may also be pro-fibrotic due to modifications to MMPs, which degrade ECM proteins; IL-13 production by ILCs (postulated to be ILC2s due to phenotypic similarities) in Crohn’s disease intestinal muscle inhibited MMP synthesis by fibroblasts, thereby abrogating ECM degradation [93]. Similar results were reported in conjunctival fibroblasts, specifically, that IL-13 inhibited IL1βinduced MMP1 synthesis [94]. The significance of ILC2s in the gut has been emphasised by the lack of collagen deposition in RORα, an ILC2 transcription factor, deficient mice [95]. Of interest is that IL22 and IL-17A were reduced in this model, suggesting that these effects are ILC3-dependent. These data give further support to ILC involvement in fibrosis, but also serve to highlight the considerable complexity of the subgroups and their development.

5.5 - Liver The aforementioned augmentation of IL-33 expression reported in other tissues has also been found in mouse and human fibrotic livers, alongside an associated elevation in ST2 mRNA [96]. These data have since been corroborated [97]. Furthermore, not only did overexpression of IL-33 result in excessive collagen deposition at four weeks, but also elevated RORα mRNA and ILC2 numbers. S.mansoni treated mice also exhibited an increase in ILC2 frequency in the liver. The authors were able to demonstrate that these cells were regulators of hepatic fibrosis via depletion of ILC2s prior to IL-33 administration, resulting in attenuation of collagen deposition.

6.0 - Conclusion It is clear from the available data that ILCs, in particular ILC2s, are central in the pro-fibrotic ‘Type 2’ pathway, and as such, are able to elicit a downstream cascade of events by which TGF-β signalling is upregulated, thereby causing aberrant fibrosis due to excessive deposition of collagen. During chronic inflammation such as rheumatoid arthritis, however, ILC2s foster the resolution of inflammation and restore immune homeostasis in chronic inflammatory diseases [98]. Given that is now known that

epigenetic mechanisms are involved in the pathogenesis of fibrotic diseases such as SSc [99], it would be of great interest to further elucidate how aberrations in these mechanisms elicit downstream alterations in ILCs, and subsequently, fibrotic signalling pathways. This could pave the way for therapeutic intervention.

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Table 1 – Currently accepted transcription factors, human and murine surface markers, and cytokines required for differentiation, in addition to proteins produced by each ILC subgroup.

Group

1

Subgroup

ILC1

Transcription Factors

T-bet+ Eomes-

2

3

cNK

ILC2

ILC3

LTi

T-bet+ Eomes+

Gata-3+ RORα+

RORγt+ T-bet+/-

RORγt+ T-bet-

TCF-1+ Notch+ Human Surface Markers*

CD45+ CD2+

CD45+

CD27+ CD28+

CD94+

CD56+

CD45+

CD127+ CD45+ CD127+

Nkp46+ CRTH2+ KLRG1+ +/-

CD117+ CD90+

CD117+

CD56+/-

CD4+ CD56+/-

NKp46+ TRAIL+

CD127

CD69+

CD11bint

CD25+ CD90+

NKp44+/-

CD127+/-

NKp44+/-

ST2+/- IL-17RB+/-

CD4-

ICOS+

Sca-1+

CD45+

CD127+

CRTH2- CD117Murine Surface Markers

Cytokines Required

CD49b+

CD45+

CD127+ CD45+ CD127+

CD45+

CD127+

CD45+ CD2+

CD45+

CD27+ CD28+

NKp46+ NK1.1+§

KLRG1+ ICOS+

CD117+

CD117+ CD4+

CD49a+ CD69+

CD127+/-

Sca-1+ CD25+

NKp46+/-

NKp46-

NK1.1+§ NKp46+

CD11bint

CD90+

NK1.1-§

NK1.1-§

TRAIL+

ST2+/- IL-17RB+/-

KLRG- CD49b-

CD127+/- ICOSCD117-

CD49b- NK1.1-§

IL-7, IL-12, IL-15, IL-18

IL-2, IL-7, IL-9, IL-

CD117IL-1, IL-7, IL-23

25, IL-33, TSLP Lacking human markers

CD3, CD11c, CD14, CD19, CD34, Fc RI

Lacking murine markers

CD3, B220, CD11c, GR-1, Ly6B, Fc RI

Produce

IFNγ, TNFα

Granzyme,

IL-4, IL-5, IL-9,

IL-7,

IL-23,

RANKL

IL-17A, IL-22

IL-17,

IL-22,

perforin, TNFα

IFNγ,

IL-13,

TNFα, RANKL,

Amphiregulin

Lymphotoxin

*By no means a fully comprehensive list of surface markers, which may also vary depending on the source of ILC and the particular function within that tissue. Rather, these are surface markers that have been shown to generally characterise ILC subpopulations (Zhang et al, 2015; Roan et al, 2016). § strain specific (e.g. Balb/c)