Inflammation, Immune Response, and Fibrosis

J Scleroderma Relat Disord 2017; 2(Suppl 2): S1-S5 JSRD DOI: 10.5301/jsrd.5000265 ISSN 2397-1983 REVIEW Interleukin-6 as a multifunctional regulator: inflammation, immune response, and fibrosis

Ernest Choy1, Stefan Rose-John2

1 CREATE Centre, Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, Wales - UK 2 Institute of Biochemistry, University of Kiel Medical School, Kiel - Germany

Abstract Interleukin 6 (IL-6) is a 184-amino acid protein cytokine that is produced by many types of cells and is expressed during states of cellular stress, such as inflammation, infection, wound sites, and cancer. IL-6 levels may increase several thousand-fold in these states and may help to coordinate the response to dysregulation of tissue ho- meostasis. IL-6 acts through a membrane-bound IL-6 receptor (mIL-6R), which, together with a second receptor, glycoprotein 130 (gp130), leads to the initiation of intracellular signaling (classic signaling). Given that IL-6R is expressed on only a few types of cells, though all cells express gp130, direct stimulation by IL-6 is limited to cells that express mIL-6R. However, IL-6R is also produced as a soluble, secreted protein that, together with IL-6, can stimulate all gp130-expressing cells by a process termed IL-6 trans-signaling. IL-6 trans-signaling can be blocked without affecting IL-6 classic signaling through mIL-6R. IL-6 has major effects on the adaptive and innate immune system and on mesenchymal and stromal responses during inflammation. It promotes the development of pathogenic T-helper 17 T cells and the maturation of B lymphocytes. Many innate immune cells, neutrophils, and monocytes/macrophages produce and respond to IL-6, resulting in autocrine feedback loops that amplify inflammation. IL-6 has been implicated in the pathogenesis of fibrotic diseases in which IL-6 trans-signaling has been shown to stimulate the proliferation of fibroblasts and the release of procollagen and fibronectin. Keywords: Cytokine receptor gp130, Fibrosis, Inflammation, Interleukin-6, Receptors, Trans-signaling

Introduction plays a role in the body’s defense against infection, in many regenerative processes, and in the regulation of body weight Interleukin-6 (IL-6) is a proinflammatory cytokine that (8). IL-6 is produced by activated immune cells and stromal was first identified as a B-cell stimulatory factor (1). The ma- cells, including T cells, monocytes/macrophages, endothe- ture protein consists of 184 amino acids that form a four- lial cells, fibroblasts, and hepatocytes. This article provides helix bundle with the up-up-down-down topology common an overview of the pleiotropic effects of IL-6, focusing on its to most cytokines (2). On elucidation of its cDNA sequence involvement in mediating both normal and pathological pro- (1), it became apparent that IL-6 was identical to hepatocyte- cesses related to the immune system. stimulating factor (3), hybridoma/plasmacytoma growth factor (4), interferon-β2 (5), and a 26-kDa protein with unknown Interleukin-6 classic signaling and trans-signaling function (6). Although this early observation provided initial clues to the pleiotropic nature of IL-6, it is now known that Stimulation of cells by IL-6 is initiated by binding of IL-6 IL-6 has many functions in the regulation and coordination of to the IL-6 receptor (IL-6R), which belongs to the family of the immune system, metabolism, and nervous system (7, 8). type 1 cytokine receptors (2). The complex of IL-6 and IL-6R Not only is it involved in many autoimmune diseases, it also then associates with a second receptor protein, glycoprotein 130 (gp130), which dimerizes and initiates cellular signaling Accepted: October 9, 2017 (9, 10). Because IL-6 exhibits measurable affinity to IL-6R but not to gp130, only cells that express IL-6R and gp130 are ca- Published online: November 2, 2017 pable of responding directly to IL-6 (11, 12). Although gp130 Corresponding author: is expressed on all cells of the body, IL-6R is found on only a Ernest Choy few cell types, including hepatocytes, some epithelial cells, CREATE Centre, Section of Rheumatology and some leukocytes (9). However, soluble IL-6R (sIL-6R) is Division of Infection and Immunity generated by proteolytic cleavage and, to a minor extent, Cardiff University School of Medicine Tenovus Building, Health Park by alternative splicing (13-15). This sIL-6R binds IL-6 with an Cardiff CF14 4XN affinity comparable to that of membrane-bound IL-6R (mIL- Wales, United Kingdom 6R), and the complex of IL-6 and sIL-6R can also bind to mem- [email protected] brane-bound gp130, inducing dimerization and signaling

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suppressor of cytokine signaling 3, or SOCS3, which is rapidly upregulated by IL-6 and leads to the inhibition of gp130 signaling (19, 22). The four-membrane distal tyrosine residues of the cytoplasmic portion of gp130 are docking sites for signal transducer and activator of transcription 1 (STAT1) and 3 (STAT3), which become phosphorylated by JAK1, dimerize, and translocate to the nucleus, whereupon they act as transcription factors and lead to the transcription of IL-6 target genes, such as c-myc, bcl2, cyclin D1, MCP-1, Gremlin-1, several matrix metalloproteinases, and Notch (20, 23). Transcrip- tion of STAT3 is negatively regulated by protein inhibitor of activated STAT (PIAS). Intracellular IL-6 classic signaling and trans-signaling are qualitatively identical, though it has been shown that IL-6 trans-signaling leads to higher signal amplitudes because most IL-6R–expressing cells exhibit higher levels of gp130 than IL-6R (9). Therefore, stimulation of such cells leads to fewer activated gp130 molecules than stimulation with the complex of IL-6 and sIL-6R, which can stimulate virtually all Fig. 1 - IL-6 classic signaling. gp130 = glycoprotein 130; IL-6 = in- membrane-bound gp130 molecules (9). Moreover, it was terleukin-6; IL-6R = interleukin-6 receptor; JAK1/2 = Janus kinase demonstrated that stimulation of IL-6R–expressing cells with 1/2; PIAS3 = protein inhibitor of activated STAT3; sgp130 = soluble IL-6 results in rapid internalization and degradation of IL-6, glycoprotein 130; SHP2 = Src homology region 2-containing pro- whereas the complex of IL-6 and sIL-6R is internalized inef- tein tyrosine phosphatase 2; sIL-6R = soluble interleukin-6 receptor; SOCS3 = suppressor of cytokine signaling 3; STAT1 & 3 = signal fectively (24). This results in longer signaling induced by the transducer and activator of transcription 1 and 3; Tyk2 = tyrosine complex of IL-6 and sIL-6R than signaling induced by IL-6 kinase 2. alone (24). The higher signaling amplitude and the longer du- ration of signaling may result in qualitatively different IL-6 responses mediated by classic signaling and trans-signaling. For (16). Furthermore, cells that do not express IL-6R and that example, chemically induced liver cancer was recently shown are therefore not directly responsive to IL-6 can be stimulat- to be dependent on IL-6 trans-signaling even though hepato- ed by the complex of IL-6 and sIL-6R (9). This process, which cytes are one of the few cell types that express mIL-6R (25). has been termed IL-6 trans-signaling, dramatically increases the spectrum of IL-6 target cells (13). It has been shown that Interleukin-6 and the acute phase response IL-6 trans-signaling through sIL-6R is mainly proinflammatory, whereas IL-6 classic signaling through mIL-6R is associated Stimulation of hepatocytes by IL-6 leads to initiation of with protective effects against infections and regenerative the acute phase response and release of acute phase pro- activities (8). IL-6 trans-signaling can be efficiently blocked by teins, including C-reactive protein (CRP), serum amyloid A, the soluble gp130Fc protein, which consists of the entire ex- haptoglobin, ferritin, and fibrinogen (26). Elevated levels of tracellular portion of gp130 dimerized by the Fc portion of a acute phase proteins are the hallmark of inflammation and human immunoglobulin G1 (IgG1) antibody (9, 11). Blockade are routinely measured when monitoring inflammation in of IL-6 trans-signaling by the soluble gp130Fc protein leaves clinical practice (27). IL-6 is the principal driver of the acute all classic IL-6 signaling through mIL-6R intact (11) (Fig. 1). As phase response in the pathogenesis of rheumatoid arthritis a result, it has been proposed that selective inhibition of IL-6 (RA), as evidenced by neutralization of IL-6 by the anti–IL-6R trans-signaling might be a more prudent mode of IL-6 inhibi- antibody tocilizumab, which led to the normalization of CRP tion because its protective and regenerative activities are not (28). Hepcidin is an acute phase protein that regulates iron compromised by this strategy (17, 18). metabolism; it reduces intestinal iron absorption and seques- ters iron into macrophages (29), which can lead to the devel- Intracellular signaling opment of anemia of chronic diseases (29). In RA, the role of IL-6 signaling in the development of inflammatory anemia is The dimerization of gp130 leads to activation of Janus supported by evidence showing that IL-6R blockade with to- kinase (JAK) tyrosine kinases (19), which are constitutively cilizumab reduces hepcidin and haptoglobin, as well as CRP, bound to the intracellular portion of the gp130 protein (20) subsequently increasing hemoglobin levels (30). (Fig. 1). There are five tyrosine residues within the cytoplasmic tail of gp130 that are phosphorylated primarily by JAK1 Innate immunity (20). The membrane proximal tyrosine residue acts as a docking site for SHP2, which is an adapter protein and a tyrosine Many innate immune cells and stromal cells produce and phosphatase, leading to the activation of mitogen-activated respond to IL-6 (17, 31), highlighting the prominent role that protein kinase and phosphoinositide 3-kinase signaling (19, this cytokine plays in the innate immune response and in its 21). The membrane proximal tyrosine residue is also a tar- interaction with stromal cells (32). Autocrine feedback can get of the negative regulator of gp130 signaling known as amplify inflammation (33), and IL-6 is thought to be involved

© 2017 The Authors. Published by Wichtig International Choy and Rose-John S3 in the transition from acute to chronic inflammation. During Endothelial cell function acute inflammation, monocytes, macrophages, and endothelial cells produce IL-6, leading to the recruitment of neutro- IL-6 and IL-6R have been implicated in the pathogenesis phils through activation of a subset of chemokines and ad- of cardiovascular diseases. Human endothelial cells express hesion molecules by endothelial cells, smooth muscle cells, gp130 but not IL-6R (9, 37, 52), whereas sIL-6R–mediated and fibroblasts (31, 34). IL-6 has also been shown to prolong trans-signaling is known to increase the expression of ad- neutrophil survival through regulatory effects on neutrophil hesion molecules by endothelial cells and the binding of apoptosis (35). The shedding of mIL-6R from neutrophils dur- neutrophils, thereby causing vascular inflammation (37). In ing chronic inflammation allows stromal cells that do not -ex collagen-induced arthritis—an animal model of RA—inflam- press IL-6R to respond to IL-6, leading to the production of matory arthritis is associated with impaired vascular contrac- monocyte-specific chemoattractants (36, 37) and increasing tile responses when stimulated by 5-hydroxytryptophan (53). monocyte influx (36, 38). Blocking IL-6 trans-signaling with the soluble gp130Fc protein reverses vascular dysfunction to a normal state (54). Adaptive immunity Fibroblasts and fibrosis T and B cells are the main orchestrators of the adaptive immune response, which is central to the pathogenesis of IL-6 has been implicated in the pathogenesis of fibrotic many autoimmune diseases. Naive T and B cells are acti- diseases, including pulmonary (55) and hepatic (56) fibrosis vated in the germinal centers of secondary lymphoid organs and systemic sclerosis (SSc) (57). Fibrosis is a common fea- (39). These activated effector T and B cells drive cellular- and ture in chronic inflammatory diseases, whereas in fibrotic humoral-mediated immunity to amplify inflammation and diseases, such as SSc, inflammatory infiltrates and cytokines produce autoantibodies (39). IL-6 has important roles in both are often found in affected organs (58). Fibroblasts and mac- T- and B-cell activation, as evidenced by the observation that rophages, especially activated M2 macrophages, produce IL-6 IL-6–deficient animals are resistant to the development of and TGF-β, which are profibrotic cytokines (59, 60). IL-6 trans- antigen-induced arthritis (40). signaling leads to increases in collagen 1 synthesis by STAT3 and SMAD3 activation and production of Gremlin-1, a bone B cells morphogenetic protein antagonist (59). Inhibiting Gremlin-1 reduced collagen synthesis. Serum amyloid A, an acute phase On engagement with an antigen through the B-cell recep- reactant produced by the liver, can induce dermal fibroblasts tor, B cells are activated, differentiate into plasma cells, and to produce IL-6 (61). IL-6–deficient mice were resistant to produce antibodies. IL-6 was originally identified as a T-cell- fibrosis in a peritoneal model of inflammation-mediated fi- derived soluble factor that induced the maturation of B cells brosis; transfer of polarized Th1 cells reversed this effect, sug- into plasma cells (41). In addition, IL-6 causes CD4+ and CD8+ T gesting IL-6 caused fibrosis by shifting from chronic to acute cells to differentiate into subsets that produce IL-21, which in inflammation through the induction of Th1 responses (62). In turn stimulates B-cell maturation (42, 43). IL-6–deficient ani- hypertrophic scars, dermal fibroblasts express a high level of mals have diminished antibody responses and are susceptible cell surface gp130, and IL-6 trans-signaling leads to the pro- to infection (44). A recent study has demonstrated a role for liferation of fibroblasts and the excessive production of- ex IL-6 in the induction of regulatory B cells (45). IL-6 also allows tracellular matrix, including procollagen and fibronectin (63). the adaptive immune response to reciprocally impact the in- In patients with SSc, IL-6 levels are elevated in serum and nate immune response because activated mature B cells and in bronchoalveolar lavage fluid (64, 65). Serum IL-6 levels cor- regulatory B cells produce IL-6, which then triggers innate im- relate with skin scores (66). Peripheral blood monocytes from mune cells and stromal cells to produce cytokines, chemo- patients with SSc produce more IL-6, either spontaneously or kines, and other proinflammatory factors (32). when stimulated (65, 67). Elevated sIL-6R and sgp130 levels have been reported in 20 patients with localized scleroderma T cells (68). Fibroblasts from diseased skin sites of patients with SSc produce from 6- to 30-fold higher levels of IL-6 than fibro- IL-6 plays a pivotal role in T-cell–mediated immunity, in part blasts from unaffected skin and healthy controls (69). by promoting the differentiation of T-helper 17 (Th17) cells (46), which are characterized by the production of IL-17. Coun- Conclusion tering the pro-autoimmune Th17 cells are CD4+CD25+Foxp3+ regulatory T (Treg) cells. Transforming growth factor-beta IL-6 is produced by a number of cell types and exerts (TGF-β) is a critical differentiation factor for the generation of pleiotropic effects. In particular, IL-6 plays an important role Treg cells. Bettelli et al (46) showed that the presence of IL-6 in mediating innate and adaptive immune responses. Many inhibits TGF-β-induced differentiation of Treg cells. Further- innate immune cells—neutrophils and monocytes/macro- more, the presence of both IL-6 and TGF-β drives naive T cells phages—produce and respond to IL-6, which can result in the to differentiate into Th17 cells (46-48). IL-6 is also produced amplification of inflammation and a switch from an acute to a by Th17 cells, further promoting differentiation into Th17 cells chronic inflammatory state. IL-6 is also involved in mediating (49) and the development of autoimmune diseases (50). IL-6 the activation of T and B cells, which are the main drivers of and IL-17 released by Th17 cells induce fibroblasts to produce adaptive immune responses and key cells in the pathogenesis IL-6, further amplifying the immune reaction (51). of many autoimmune diseases. IL-6 has been implicated in

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