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STAT3 signaling axis: recent advances towards specific inhibition
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ScienceDirect The IL-6/gp130/STAT3 signaling axis: recent advances towards specific inhibition Christoph Garbers, Samadhi Aparicio-Siegmund and Stefan Rose-John Interleukin-6 has long been recognized as a prototypic proinflammatory cytokine that is involved in the pathogenesis of all inflammatory diseases. Activation of the gp130 homodimer by IL-6 leads to the initiation of Jak/STAT signaling, a pathway that is often constitutively switched on in inflammatory malignancies. However, a plethora of studies in the last decade has convincingly shown that only signaling via the soluble IL-6R (trans-signaling) accounts for the deleterious effects of IL-6, whereas classic signaling via the membrane-bound receptor is essential for the regenerative and anti-bacterial effects of IL-6 (classic signaling). In this review, we highlight recent developments in the field of IL-6 research, and specifically focus on advances towards a safe and specific inhibition of IL-6 trans-signaling. Address Institute of Biochemistry, Kiel University, Olshausenstrasse 40, Kiel, Germany Corresponding author: Rose-John, Stefan ([email protected])
Current Opinion in Immunology 2015, 34:75–82 This review comes from a themed issue on Cytokines Edited by Christopher A Hunter and Steven F Ziegler
http://dx.doi.org/10.1016/j.coi.2015.02.008 0952-7915/# 2015 Elsevier Ltd. All rights reserved.
Introduction The pleiotropic cytokine interleukin-6 (IL-6) has multiple beneficial functions in hematopoiesis and regeneration. Overshooting reactions of IL-6, however, play a central role in the progression and development of cancer. The expression of IL-6 is up-regulated in many malignancies, and the enhanced IL-6 signaling leads to a constant activation of the transcription factor STAT3 which in turn increases IL-6 expression. This positive feedback loop confers a tumor microenvironment that promotes tumor growth [1]. Furthermore, IL-6 has been recognized as a potent driving force in many chronic inflammatory diseases. Consequently, tocilizumab, a human IL-6R specific antibody that blocks binding of IL-6 to its receptor, has been developed and is clinically www.sciencedirect.com
approved for the treatment of rheumatoid arthritis (RA) in more than 100 countries [2]. Besides this, tocilizumab has been approved for the treatment of polyarticular juvenile idiopathic arthritis (PJIA) and systemic juvenile idiopathic arthritis (SJIA) by both the FDA and the EMEA. In Japan, it has further been approved to medicate people suffering from Castleman’s disease [3]. The signal transduction of IL-6 is induced by binding of IL-6 to its specific alpha receptor. In complex with the IL6R, IL-6 activates a homodimer of the signal-transducing b-receptor gp130 with high affinity. The b-receptor is shared by all IL-6 family members [4]. For signal transduction the IL-6R can either be membrane-bound or soluble and the signaling process is accordingly called classic or trans-signaling (Figure 1). The two different signaling events have divergent functions. Trans-signaling is believed to mediate chronic inflammation and cancer development and is therefore thought to be a target for therapeutic intervention [5]. When IL-6 binds to its receptor complex it induces the activation of several intracellular signaling cascades. The most prominent among them appears to be the Jak/STAT pathway, where gp130-associated Janus Kinases (Jaks), particularly Jak1, are activated after ligand binding. Jak1 phosphorylates distinct intracellular tyrosine residues of the receptor and thereby recruits STAT molecules, which also become phosphorylated by the Jaks. These translocate afterwards into the nucleus to activate gene transcription. The main negative feedback regulator of the IL-6 signaling axis is SOCS3, which is a STAT3 target gene and thus transcriptionally up-regulated after cellular gp130 activation by IL-6. SOCS3 binds to the region around pY-759 within the cytoplasmic portion of gp130 as well as to the interacting Jak kinases. This leads to the recruitment of an E3 ubiquitin ligase complex and subsequently to degradation of gp130 as well as the Jak kinase [6]. In addition, SOCS3 can directly inhibit Jak kinases through binding to their kinase domains, which abrogates further phosphorylation of for example STAT proteins. Other negative regulators of IL-6 signaling are the phosphatase SHP2 and the E3 SUMO-protein ligase protein inhibitor of activated STAT (PIAS). In the present review, we highlight recent advances and developments within the still flourishing field of IL-6 science with a special focus on concepts of specific therapeutic intervention. Current Opinion in Immunology 2015, 34:75–82
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Figure 1
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Interleukin-6 classic and trans-signaling. Interleukin-6 can either signal via the membrane-bound receptor (classic signaling, left) or in complex with the soluble IL-6 receptor (trans-signaling, right). In both cases, homodimerization of gp130 leads to the activation of intracellular signaling pathways, in particular phosphorylation of STAT3 via the tyrosine kinase Jak1. Negative feedback inhibition is achieved through induced expression of SOCS3. Whereas the monoclonal antibody tocilizumab blocks both classic and trans-signaling, sgp130Fc has been engineered to selectively target the IL-6/sIL-6R complex.
IL-6 classic versus trans-signaling The soluble IL-6R is a rare example of an agonistic soluble cytokine receptor. After binding its ligand IL-6, the IL-6/sIL-6R can in principle activate all cells of the human body through homodimerization of the ubiquitously expressed b-receptor gp130. We have termed this process trans-signaling, which accounts predominantly for the pro-inflammatory and thus potentially deleterious actions of IL-6 (Figure 1). The critical role of IL-6 trans-signaling was established by the use of genetically engineered mice, which overexpress the IL-6 trans-signaling inhibitor sgp130Fc, which is a fusion protein of the gp130 ectodomain with the Fc portion of a human IgG antibody [7]. It turned out in several animal models that IL-6 acting via the membrane bound IL-6R acted in a protective fashion and that selective blockade of IL-6 trans-signaling was superior to global blockade of IL-6 activity by neutralizing antibodies. In the caecum puncture ligation model, treatment of mice with sgp130Fc led to up to 100% survival of mice whereas global blockade of IL-6 showed no beneficial effect [8]. In a recent model of Current Opinion in Immunology 2015, 34:75–82
nephrotoxic nephritis we could demonstrate, that IL-6 suppressed the activity of inflammatory macrophages [9], which was in line with the recently discovered activity of IL-6 to induce the differentiation of regenerative M2 macrophages [10]. The soluble IL-6R is generated by alternative splicing of the IL-6R mRNA and by limited proteolysis of the membrane-bound precursor, the latter seems to be mediated predominantly by ADAM17 [11]. Besides its proinflammatory effects, IL-6 trans-signaling is also required and sufficient for the rapid development of cytotoxic T cells in the liver [12]. In a mouse model of pancreatic intraepithelial neoplasia, IL-6 trans-signaling dependent activation of STAT3 was shown to be required for disease progression and the full development of pancreatic ductal adenocarcinoma [13]. Several soluble forms of gp130 occur naturally in the human blood, which are generated by differential mRNA splicing and alternative polyadenylation [14]. These www.sciencedirect.com
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sgp130 variants contain different numbers of the six extracellular domains of gp130 and differ in their efficiency to inhibit IL-6 trans-signaling as well as their biological half-life. It is tempting to speculate that the different forms of sgp130 are used in vivo to ‘fine-tune’ the blockade of IL-6/sIL-6R signaling. Elevated serum levels of IL-6 are a disease severity marker for the inflammatory acute lung injury, a common complication in severe acute pancreatitis. Recently, it was shown that IL-6 mediates its effects via trans-signaling in this disease, as sgp130Fc blocked the pancreatitis-induced acute lung injury [15]. Finally, the authors could show that IL-6 and sIL-6R are present in samples from human acute lung injury patients, and that these two proteins can be used as a disease predictor [15]. In a mouse model of atherosclerosis, sgp130Fc did not only reduce disease, but led to a significant regression of advanced atherosclerosis, most probably through reduced monocyte recruitment and the progression of atherosclerotic plaques [16]. Inhibition of IL-6 trans-signaling was further shown to be protective in an otherwise lethal infection with Plasmodium chabaudi, which causes malaria [17]. The damaging effects of IL-6 in the central nervous system also appear to depend solely upon trans-signaling, as mice which express sgp130 under the GFAP promoter are fully protected [18]. In contrast to trans-signaling, signal transduction of IL-6 via the membrane-bound receptor has been named classic signaling. Classic signaling on hepatocytes, which is of crucial importance for the hepatic acute phase response, is essential for the protective properties of IL-6, as it helps the immune system to cope with for example bacterial infections. Accordingly, IL-6 classic signaling was found to be essential to eliminate the intracellular pathogen Listeria monocytogenes, and sgp130Fc did not compromise this important immune reaction [19]. In line with this, sgp130Fc did not impede with protective immune responses after infection with Mycobacterium tuberculosis, whereas global blockade of IL-6 with an anti-IL-6 antibody (blocking classic and trans-signaling) did so [20]. Recently it was shown that IL-6 plays an important role in the defense of the body against viral infections [21]. Deletion of the gp130 gene in T-cells compromised Tcell survival and IL-27 induced IL-21 production upon lymphocytic choriomeningitis virus infection [22].
The soluble IL-6R: a buffer in the blood In healthy individuals levels of IL-6 in the plasma are extremely low, close to the detection limit of 1 pg/ml [23]. Plasma levels of sIL-6R range between 50 and 75 ng/ml and levels of sgp130 are around 400 ng/ml [24] (Figure 2). During inflammatory states, the levels of sIL-6R and sgp130 do not largely change but levels of IL-6 can increase dramatically. In patients with rheumatoid www.sciencedirect.com
arthritis, IL-6 levels of 150 ng/ml have been reported [25]. Under septic conditions levels of IL-6 in the low microgram range have been reported [26]. Therefore, IL6 secreted by cells such as neutrophils, monocytes/macrophages, endothelial cells, fibroblasts and T-cells [27] will bind to the sIL-6R with an affinity of around 1 nM. Subsequently, the complex of IL-6 and sIL-6R will bind to sgp130 with an affinity of 10 pM where its activity is neutralized [28]. Thus, sIL-6R and sgp130 form a buffer for IL-6 in the blood with a capacity equal to the molar concentration of the sIL-6R, which corresponds to a concentration of IL-6 of 100-150 ng/ml (Figure 2). Interestingly, a single nucleotide polymorphism in the IL-6R gene (rs2228145) has been identified, which changes aspartate 358 in the juxtamembrane region of the receptor protein to alanine and which facilitates shedding of the IL-6R by the metalloprotease ADAM17 [29]. Individuals with alanine 358 show significantly higher sIL-6R levels in the blood and are less susceptible to autoimmune diseases such as rheumatoid arthritis [30]. Two meta-analyses, which incorporated clinical data from more than 100 000 individuals could show that for every copy of the Asp358Ala SNP inherited, the concentrations of C-reactive protein and other acute-phase proteins were reduced, as well as the overall risk to suffer from coronary heart disease [31,32]. In this context, increased levels of sIL-6R appear to have an anti-inflammatory impact, which might be taken as an argument for the validity of the concept of the IL-6 buffer in the blood formed by sIL-6R and sgp130.
Plasticity of gp130 and IL-6R Gp130 is the common b-receptor for all cytokines of the IL-6 family, including IL-6, IL-11, CLC, CNTF, CT-1, OSM, LIF and IL-27 [33]. Signal transduction is initiated after dimer formation with LIFR, OSMR, WSX-1 or gp130 itself. This fact illustrates a remarkable kind of plasticity, as gp130 is able to engage signaling with a variety of ligands, non-signaling alpha-receptors and at least three other breceptors [33]. Deregulated cytokine signaling via gp130 is associated with chronic inflammation and cancer development. Accordingly, ectopic expression of a constitutively active gp130 variant (L-gp130) in mice induces the formation of multiple myeloma in concert with myc [34]. Besides IL-6, CNTF has been described as a second ligand for the IL-6R. The signal is transduced via a gp130/ LIFR heterodimer, the same signaling complex that CNTF uses when it binds to the CNTFR. Recently, the IL-27 subunit p28 (also known as IL-30) has been identified as another ligand for the IL-6R [35–37]. In contrast to CNTF, IL-30 signals via a gp130 homodimer like IL-6, whereas IL-27 (p28/EBI3) engages a heterodimer of gp130/WSX1. Thus, IL-30 is able to activate two different b-receptor complexes, depending to which non-signaling alpha-receptor (EBI3 or IL-6R) it initially Current Opinion in Immunology 2015, 34:75–82
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Figure 2
(a)
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Soluble IL-6R and soluble gp130 constitute a buffer for IL-6 in the blood. (a) In healthy humans IL-6 (1–5 pg/ml), soluble IL-6R (30–70 ng/ml) and soluble gp130 (250–400 ng/ml) are constitutively present. IL-6 binds with an affinity of 1 nM to the sIL-6R. The complex of IL-6/sIL-6R binds to sgp130 with 100 times higher affinity (10 pM). The molar ratios indicate that the concentration of the soluble IL-6R controls the capacity of the IL-6 buffer in the blood. (b) The IL-6R SNP rs2228145 leads to higher concentrations of sIL-6R in the blood and thus to a higher capacity of the IL-6 buffer leading to protection of certain inflammatory diseases [30].
binds [37] (Figure 3a). Interestingly, p28 has also been described as an antagonist of IL-6 family cytokines, as transgenic overexpression of p28 resulted in impaired B cell responses in a gp130-dependent manner [38]. In a mouse model of inflammation-induced liver injury, p28 acted as an anti-inflammatory cytokine [39]. Furthermore, p28 either alone or in combination with p40 was able to suppress disease severity in a model of experimental autoimmune uveitis (EAU) [40]. How these different Current Opinion in Immunology 2015, 34:75–82
findings can be incorporated into one functional model is not clear at the present time. The most recent addition to the IL-6 family is IL-35 (EBI3/p35), which can be considered as a cytokine that bridges IL-6 and IL-12 cytokine families [41]. Until now, four different b-receptor pairs for IL-35 have been described (IL-12Rb2/IL-12Rb2, gp130/gp130, IL-12Rb2/ gp130 and IL-12Rb2/WSX-1) [42,43]. This suggests a www.sciencedirect.com
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Figure 3
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Plasticity within the IL-6 family of cytokines. (a) IL-6 binds with high affinity to the IL-6R, which induces recruitment of a gp130 homodimer. CNTF, which usually binds to CNTFR/LIFR/gp130 complexes with high affinity, can also bind IL-6R/LIFR/gp130 complexes, although with much lower affinity. IL-30 binds efficiently to EBI3 (to form the heterodimeric cytokine IL-27, which signals via the gp130/WSX-1 complex), but can also bind to a gp130 homodimer via the IL-6R with low affinity. (b) IL-35 (p35/EBI3) shares its subunits with IL-27 (p28/EBI3) and IL-12 (p35/p40).
remarkable plasticity on both the involved receptors and the heterodimeric cytokine itself, especially if one considers that both subunits are further shared with other cytokines (EBI3 with IL-27 and p35 with IL-12, respectively, Figure 3b). Binding of IL-6 family cytokines to their respective receptors has been biochemically studied for more than 20 years. The term chemical plasticity has been introduced for a receptor interacting with many www.sciencedirect.com
ligands in a specific manner [44]. However, the ability of IL-35 to bind to such a wide variety of receptor combinations has not been reported for any other cytokine. This is also true for the finding that different receptor combinations induce the phosphorylation of different combinations of STAT proteins, which appears unlikely given the knowledge about other cytokines and receptors of the same family [42]. Therefore, additional Current Opinion in Immunology 2015, 34:75–82
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and more rigorous studies are needed to fully understand the different receptor complexes and the signal transduction of the cytokine IL-35. Biochemical analysis are further complicated by the fact that p35 protein produced in bacteria can be used to assemble IL-12, but not IL-35 [45].
Therapeutic inhibition of IL-6 activity IL-6 together with IL-1 and TNFa play a pivotal role in the maintenance of chronic inflammatory diseases [46,47]. Blockade of TNFa has been very successful in the treatment of rheumatoid arthritis and inflammatory bowel disease [47]. For the blockade of IL-6, a small molecule inhibitor of JAK1, JAK2 and JAK3 (tofacitinib) and a humanized monoclonal antibody targeting the human IL-6R (tocilizumab) have been approved by the FDA for the treatment of rheumatoid arthritis [24]. A number of monoclonal antibodies against human IL-6 and the human IL-6R are in phase I, phase II or phase III of clinical development [24]. While inhibitors of JAK1, JAK2 and JAK3 have the potential to block the activity of most cytokines and interferons [48] the monoclonal antibody tocilizumab is specific for the human IL-6R and is therefore expected only to block the activity of IL-6 and to some unknown extent the activity of CNTF [49] and p28 [37], since these cytokines can also act via the IL-6R [37,49]. Other small molecules that interfere with the Jak/STAT signaling cascade, for example inhibitors of protein kinase II [50], are just entering clinical trials.
[28,54]. Interestingly, sgp130Fc could theoretically block signaling of IL-11 via a soluble form of the IL11R [55]. However, to date no naturally occurring sIL11R has been described. Such a notion might have important implications for the treatment of inflammatory disease in which both processes, inflammation and regeneration, play a role. The intestine is the organ with the highest regenerative activity. The fact that IL-6 / mice upon treatment with the irritant dextran sodium sulfate show more inflammation than wt animals was explained by the compromised STAT3 activation in the intestinal epithelial cells [56]. Interestingly, treatment by recombinant IL-6 can be beneficial in a mouse model of human inflammatory bowel disease [57]. Consequently, in patients upon global IL-6 blockade, gastrointestinal perforations have been observed. This might be the main reason why the antibody tocilizumab is not approved for the treatment of inflammatory bowel disease. Selective blockade of IL-6 trans-signaling by the sgp130Fc protein was demonstrated to be beneficial in different animal models of human inflammatory bowel disease [58,59] and colon cancer [60,61]. The sgp130Fc protein was produced under GMP conditions and phase I clinical trials were conducted in 2013/14. It is planned to move to phase II clinical trials in inflammatory bowel disease patients in the year 2015 [24].
Conclusions In the past years it has been noted that IL-6 not only is involved in pro-inflammatory activities but also in tissue regeneration after wounding and in the defense of the body against bacterial infection. The discovery that the natural sgp130 protein found in the blood at high concentrations (see above) was a specific inhibitor of IL-6 trans-signaling via the sIL-6R [28] paved the way to systematically analyze the contribution of IL-6 classic and trans-signaling in many animal models of human inflammatory diseases and cancer [51]. Using a sgp130Fc protein [28] it turned out that in most cases the blockade of IL-6 trans-signaling was as efficient as the global blockade of IL-6 classic and trans-signaling with an IL-6 or IL-6R specific monoclonal antibody [52]. In many cases, however, the regenerative activity of IL6 was preserved upon selective blockade of IL-6 transsignaling. From such experiments it was deduced that the pro-inflammatory activities of IL-6 are mediated via trans-signaling whereas the anti-inflammatory and regenerative activities are mediated via the membrane bound IL-6R [53]. Sgp130Fc does not efficiently interfere with signaling of other IL-6 family cytokines including IL-27 because all these cytokines (with the exception of IL-11) bind to a heterodimer of gp130 and the receptor proteins LIF-R, OSM-R or WSX-1 Current Opinion in Immunology 2015, 34:75–82
Since its first description as an interferon b-like protein in 1980 [4], our understanding of the biology of IL-6 and its physiologic and pathophysiologic implications have dramatically increased. The development of the monoclonal antibody tocilizumab and its worldwide approval for the treatment of several inflammatory diseases have proven the importance of IL-6 in the pathology of these diseases and the therapeutic value of its inhibition. The identification of soluble forms of the IL-6R have revealed a second mode of IL-6 signaling, which has been termed trans-signaling, and which is mainly controlled by the metalloprotease ADAM17. Proteolysis of the membrane-bound IL-6R leads to the generation of agonistic IL-6/sIL-6R complexes that can be specifically blocked by the sgp130Fc protein. The effectiveness of specific inhibition of IL-6 trans-signaling has been proven in numerous mouse models of inflammatory diseases and cancer, and sgp130Fc has just recently successfully completed phase I clinical trials and will enter phase II in 2015. It will be interesting to see if the promising results from animal models hold true and if sgp130Fc can be used in the future to treat patients with inflammatory diseases with less side effects. www.sciencedirect.com
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Acknowledgements The work of CG and SR-J is supported by the Deutsche Forschungsgemeinschaft (SFB877 Project Grants A1 and A10), the Bundesministerium fu¨r Bildung und Forschung (BMBF grant ‘InTraSig’, project B) and the Cluster of Excellence ‘Inflammation at Interfaces’.
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31. Interleukin-6 receptor pathways in coronary heart disease: a collaborative meta-analysis of 82 studies. Lancet 2012, 379:1205-1213. 32. The interleukin-6 receptor as a target for prevention of coronary heart disease: a Mendelian randomisation analysis. Lancet 2012, 379:1214-1224. 33. Garbers C, Hermanns H, Schaper F, Mu¨ller-Newen G, Gro¨tzinger J, Rose-John S, Scheller J: Plasticity and cross-talk of interleukin 6type cytokines. Cytokine Growth Factor Rev 2012, 23:85-182. 34. Dechow T, Steidle S, Go¨tze KS, Rudelius M, Behnke K, Pechloff K, Kratzat S, Bullinger L, Fend F, Soberon V et al.: GP130 activation induces myeloma and collaborates with MYC. J Clin Invest 2014, 124:5263-5274. 35. Crabe´ S, Guay-Giroux A, Tormo AJ, Duluc D, Lissilaa R, Guilhot F, Mavoungou-Bigouagou U, Lefouili F, Cognet I, Ferlin WG et al.: The IL-27 p28 subunit binds cytokine-like factor 1 to form a cytokine regulating NK and T cell activities requiring IL-6R for signaling. J Immunol 2009, 183:7692-7702. 36. Tormo A, Meliani Y, Beaupre´ L, Sharma M, Fritz J, Elson G, Crabe´ S, Gauchat J-F: The composite cytokine p28/cytokinelike factor 1 sustains B cell proliferation and promotes plasma cell differentiation. J Immunol 2013, 191:1657-1665. 37. Garbers C, Spudy B, Aparicio-Siegmund S, Waetzig GH, Sommer J, Ho¨lscher C, Rose-John S, Gro¨tzinger J, Lorenzen I, Scheller J: An interleukin-6 receptor-dependent molecular switch mediates signal transduction of the IL-27 cytokine subunit p28 (IL-30) via a gp130 protein receptor homodimer. J Biol Chem 2013, 288:4346-4354. 38. Stumhofer JS, Tait ED, Quinn WJ, Hosken N, Spudy B, Goenka R, Fielding CA, O’Hara AC, Chen Y, Jones ML et al.: A role for IL27p28 as an antagonist of gp130-mediated signaling. Nat Immunol 2010, 11:1119-1126. 39. Dibra D, Cutrera J, Xia X, Kallakury B, Mishra L, Li S: Interleukin30: a novel antiinflammatory cytokine candidate for prevention and treatment of inflammatory cytokine-induced liver injury. Hepatology (Baltim, MD) 2012, 55:1204-1214. 40. Wang R-XX, Yu C-RR, Mahdi RM, Egwuagu CE: Novel IL27p28/ IL12p40 cytokine suppressed experimental autoimmune uveitis by inhibiting autoreactive Th1/Th17 cells and promoting expansion of regulatory T cells. J Biol Chem 2012, 287:36012-36021.
48. Leonard WJ, O’Shea JJ: Jaks and STATs: biological implications. Annu Rev Immunol 1998, 16:293-322. 49. Schuster B, Kovaleva M, Sun Y, Regenhard P, Matthews V, Grotzinger J, Rose-John S, Kallen KJ: Signaling of human ciliary neurotrophic factor (CNTF) revisited. The interleukin-6 receptor can serve as an alpha-receptor for CTNF. J Biol Chem 2003, 278:9528-9535. 50. Aparicio-Siegmund S, Sommer J, Monhasery N, Schwanbeck R, Keil E, Finkensta¨dt D, Pfeffer K, Rose-John S, Scheller J, Garbers C: Inhibition of protein kinase II (CK2) prevents induced signal transducer and activator of transcription (STAT) 1/3 and constitutive STAT3 activation. Oncotarget 2014, 5:2131-2148. 51. Waetzig GH, Rose-John S: Hitting a complex target: an update on interleukin-6 trans-signalling. Expert Opin Ther Targets 2012, 16:225-236. 52. Jones SA, Scheller J, Rose-John S: Therapeutic strategies for the clinical blockade of IL-6/gp130 signaling. J Clin Invest 2011, 121:3375-3383. 53. Scheller J, Chalaris A, Schmidt-Arras D, Rose-John S: The proand anti-inflammatory properties of the cytokine interleukin-6. Biochim Biophys Acta 2011, 1813:878-888. 54. Scheller J, Schuster B, Ho¨lscher C, Yoshimoto T, Rose-John S: No inhibition of IL-27 signaling by soluble gp130. Biochem Biophys Res Commun 2005, 326:724-728. 55. Pflanz S, Tacken I, Grotzinger J, Jacques Y, Minvielle S, Dahmen H, Heinrich PC, Muller-Newen G: A fusion protein of interleukin-11 and soluble interleukin-11 receptor acts as a superagonist on cells expressing gp130. FEBS Lett 1999, 450:117-122. 56. Grivennikov S, Karin E, Terzic J, Mucida D, Yu GY, Vallabhapurapu S, Scheller J, Rose-John S, Cheroutre H, Eckmann L et al.: IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitisassociated cancer. Cancer Cell 2009, 15:103-113. In this study the role of IL-6 in colon cancer is analyzed. The authors show that IL-6 is a critical tumor promoter during early tumorigenesis. Importantly, a protective and regenerative role of IL-6 on intestinal epithelial cells is demonstrated. 57. Spehlmann ME, Manthey CF, Dann SM, Hanson E, Sandhu SS, Liu LY, Abdelmalak FK, Diamanti MA, Retzlaff K, Scheller J et al.: Trp53 deficiency protects against acute intestinal inflammation. J Immunol 2013, 191:837-847.
42. Collison L, Delgoffe G, Guy C, Vignali K, Chaturvedi V, Fairweather D, Satoskar A, Garcia K, Hunter C, Drake C et al.: The composition and signaling of the IL-35 receptor are unconventional. Nat Immunol 2012, 13:290-299.
58. Atreya R, Mudter J, Finotto S, Mu¨llberg J, Jostock T, Wirtz S, Schu¨tz M, Bartsch B, Holtmann M, Becker C et al.: Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in crohn disease and experimental colitis in vivo. Nat Med 2000, 6:583-588. In this study it is shown for the first time that the specifc blockade of IL-6 trans-signaling with the soluble gp130Fc protein can protect mice from colitis and induce apoptosis of mucosal T-cells.
43. Wang R-XX, Yu C-RR, Dambuza IM, Mahdi RM, Dolinska M, Sergeey YV, Wingfield PT, Kim S-HH, Egwuagu CE: Interleukin35 induces regulatory B cells that suppress autoimmune disease. Nat Med 2014, 20:633-641.
59. Mitsuyama K, Matsumoto S, Rose-John S, Suzuki A, Hara T, Tomiyasu N, Handa K, Tsuruta O, Funabashi H, Scheller J et al.: STAT3 activation via interleukin 6 trans-signalling contributes to ileitis in SAMP1/Yit mice. Gut 2006, 55:1263-1269.
44. Chow D, He X, Snow AL, Rose-John S, Garcia KC: Structure of an extracellular gp130 cytokine receptor signaling complex. Science 2001, 291:2150-2155.
60. Becker C, Fantini MC, Schramm C, Lehr HA, Wirtz S, Nikolaev A, Burg J, Strand S, Kiesslich R, Huber S et al.: TGF-beta suppresses tumor progression in colon cancer by inhibition of IL-6 trans-signaling. Immunity 2004, 21:491-501.
41. Collison L, Workman C, Kuo T, Boyd K, Wang Y, Vignali K, Cross R, Sehy D, Blumberg R, Vignali DA: The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature 2007, 450:566-569.
45. Aparicio-Siegmund S, Moll JM, Lokau J, Grusdat M, Schro¨der J, Plo¨hn S, Rose-John S, Gro¨tzinger J, Lang PA, Scheller J et al.: Recombinant p35 from bacteria can form interleukin (IL-)12, but not IL-35. PLOS ONE 2014, 9:e107990. 46. Ferraccioli G, Bracci-Laudiero L, Alivernini S, Gremese E, Tolusso B, De Benedetti F: Interleukin-1b and interleukin-6 in arthritis animal models: roles in the early phase of transition Current Opinion in Immunology 2015, 34:75–82
61. Matsumoto S, Hara T, Mitsuyama K, Yamamoto M, Tsuruta O, Sata M, Scheller J, Rose-John S, Kado S, Takada T: Essential roles of IL-6 trans-signaling in colonic epithelial cells, induced by the IL-6/soluble-IL-6 receptor derived from lamina propria macrophages, on the development of colitis-associated premalignant cancer in a murine model. J Immunol 2010, 184:1543-1551. www.sciencedirect.com
ScienceDirect The IL-6/gp130/STAT3 signaling axis: recent advances towards specific inhibition Christoph Garbers, Samadhi Aparicio-Siegmund and Stefan Rose-John Interleukin-6 has long been recognized as a prototypic proinflammatory cytokine that is involved in the pathogenesis of all inflammatory diseases. Activation of the gp130 homodimer by IL-6 leads to the initiation of Jak/STAT signaling, a pathway that is often constitutively switched on in inflammatory malignancies. However, a plethora of studies in the last decade has convincingly shown that only signaling via the soluble IL-6R (trans-signaling) accounts for the deleterious effects of IL-6, whereas classic signaling via the membrane-bound receptor is essential for the regenerative and anti-bacterial effects of IL-6 (classic signaling). In this review, we highlight recent developments in the field of IL-6 research, and specifically focus on advances towards a safe and specific inhibition of IL-6 trans-signaling. Address Institute of Biochemistry, Kiel University, Olshausenstrasse 40, Kiel, Germany Corresponding author: Rose-John, Stefan ([email protected])
Current Opinion in Immunology 2015, 34:75–82 This review comes from a themed issue on Cytokines Edited by Christopher A Hunter and Steven F Ziegler
http://dx.doi.org/10.1016/j.coi.2015.02.008 0952-7915/# 2015 Elsevier Ltd. All rights reserved.
Introduction The pleiotropic cytokine interleukin-6 (IL-6) has multiple beneficial functions in hematopoiesis and regeneration. Overshooting reactions of IL-6, however, play a central role in the progression and development of cancer. The expression of IL-6 is up-regulated in many malignancies, and the enhanced IL-6 signaling leads to a constant activation of the transcription factor STAT3 which in turn increases IL-6 expression. This positive feedback loop confers a tumor microenvironment that promotes tumor growth [1]. Furthermore, IL-6 has been recognized as a potent driving force in many chronic inflammatory diseases. Consequently, tocilizumab, a human IL-6R specific antibody that blocks binding of IL-6 to its receptor, has been developed and is clinically www.sciencedirect.com
approved for the treatment of rheumatoid arthritis (RA) in more than 100 countries [2]. Besides this, tocilizumab has been approved for the treatment of polyarticular juvenile idiopathic arthritis (PJIA) and systemic juvenile idiopathic arthritis (SJIA) by both the FDA and the EMEA. In Japan, it has further been approved to medicate people suffering from Castleman’s disease [3]. The signal transduction of IL-6 is induced by binding of IL-6 to its specific alpha receptor. In complex with the IL6R, IL-6 activates a homodimer of the signal-transducing b-receptor gp130 with high affinity. The b-receptor is shared by all IL-6 family members [4]. For signal transduction the IL-6R can either be membrane-bound or soluble and the signaling process is accordingly called classic or trans-signaling (Figure 1). The two different signaling events have divergent functions. Trans-signaling is believed to mediate chronic inflammation and cancer development and is therefore thought to be a target for therapeutic intervention [5]. When IL-6 binds to its receptor complex it induces the activation of several intracellular signaling cascades. The most prominent among them appears to be the Jak/STAT pathway, where gp130-associated Janus Kinases (Jaks), particularly Jak1, are activated after ligand binding. Jak1 phosphorylates distinct intracellular tyrosine residues of the receptor and thereby recruits STAT molecules, which also become phosphorylated by the Jaks. These translocate afterwards into the nucleus to activate gene transcription. The main negative feedback regulator of the IL-6 signaling axis is SOCS3, which is a STAT3 target gene and thus transcriptionally up-regulated after cellular gp130 activation by IL-6. SOCS3 binds to the region around pY-759 within the cytoplasmic portion of gp130 as well as to the interacting Jak kinases. This leads to the recruitment of an E3 ubiquitin ligase complex and subsequently to degradation of gp130 as well as the Jak kinase [6]. In addition, SOCS3 can directly inhibit Jak kinases through binding to their kinase domains, which abrogates further phosphorylation of for example STAT proteins. Other negative regulators of IL-6 signaling are the phosphatase SHP2 and the E3 SUMO-protein ligase protein inhibitor of activated STAT (PIAS). In the present review, we highlight recent advances and developments within the still flourishing field of IL-6 science with a special focus on concepts of specific therapeutic intervention. Current Opinion in Immunology 2015, 34:75–82
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Figure 1
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Interleukin-6 classic and trans-signaling. Interleukin-6 can either signal via the membrane-bound receptor (classic signaling, left) or in complex with the soluble IL-6 receptor (trans-signaling, right). In both cases, homodimerization of gp130 leads to the activation of intracellular signaling pathways, in particular phosphorylation of STAT3 via the tyrosine kinase Jak1. Negative feedback inhibition is achieved through induced expression of SOCS3. Whereas the monoclonal antibody tocilizumab blocks both classic and trans-signaling, sgp130Fc has been engineered to selectively target the IL-6/sIL-6R complex.
IL-6 classic versus trans-signaling The soluble IL-6R is a rare example of an agonistic soluble cytokine receptor. After binding its ligand IL-6, the IL-6/sIL-6R can in principle activate all cells of the human body through homodimerization of the ubiquitously expressed b-receptor gp130. We have termed this process trans-signaling, which accounts predominantly for the pro-inflammatory and thus potentially deleterious actions of IL-6 (Figure 1). The critical role of IL-6 trans-signaling was established by the use of genetically engineered mice, which overexpress the IL-6 trans-signaling inhibitor sgp130Fc, which is a fusion protein of the gp130 ectodomain with the Fc portion of a human IgG antibody [7]. It turned out in several animal models that IL-6 acting via the membrane bound IL-6R acted in a protective fashion and that selective blockade of IL-6 trans-signaling was superior to global blockade of IL-6 activity by neutralizing antibodies. In the caecum puncture ligation model, treatment of mice with sgp130Fc led to up to 100% survival of mice whereas global blockade of IL-6 showed no beneficial effect [8]. In a recent model of Current Opinion in Immunology 2015, 34:75–82
nephrotoxic nephritis we could demonstrate, that IL-6 suppressed the activity of inflammatory macrophages [9], which was in line with the recently discovered activity of IL-6 to induce the differentiation of regenerative M2 macrophages [10]. The soluble IL-6R is generated by alternative splicing of the IL-6R mRNA and by limited proteolysis of the membrane-bound precursor, the latter seems to be mediated predominantly by ADAM17 [11]. Besides its proinflammatory effects, IL-6 trans-signaling is also required and sufficient for the rapid development of cytotoxic T cells in the liver [12]. In a mouse model of pancreatic intraepithelial neoplasia, IL-6 trans-signaling dependent activation of STAT3 was shown to be required for disease progression and the full development of pancreatic ductal adenocarcinoma [13]. Several soluble forms of gp130 occur naturally in the human blood, which are generated by differential mRNA splicing and alternative polyadenylation [14]. These www.sciencedirect.com
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sgp130 variants contain different numbers of the six extracellular domains of gp130 and differ in their efficiency to inhibit IL-6 trans-signaling as well as their biological half-life. It is tempting to speculate that the different forms of sgp130 are used in vivo to ‘fine-tune’ the blockade of IL-6/sIL-6R signaling. Elevated serum levels of IL-6 are a disease severity marker for the inflammatory acute lung injury, a common complication in severe acute pancreatitis. Recently, it was shown that IL-6 mediates its effects via trans-signaling in this disease, as sgp130Fc blocked the pancreatitis-induced acute lung injury [15]. Finally, the authors could show that IL-6 and sIL-6R are present in samples from human acute lung injury patients, and that these two proteins can be used as a disease predictor [15]. In a mouse model of atherosclerosis, sgp130Fc did not only reduce disease, but led to a significant regression of advanced atherosclerosis, most probably through reduced monocyte recruitment and the progression of atherosclerotic plaques [16]. Inhibition of IL-6 trans-signaling was further shown to be protective in an otherwise lethal infection with Plasmodium chabaudi, which causes malaria [17]. The damaging effects of IL-6 in the central nervous system also appear to depend solely upon trans-signaling, as mice which express sgp130 under the GFAP promoter are fully protected [18]. In contrast to trans-signaling, signal transduction of IL-6 via the membrane-bound receptor has been named classic signaling. Classic signaling on hepatocytes, which is of crucial importance for the hepatic acute phase response, is essential for the protective properties of IL-6, as it helps the immune system to cope with for example bacterial infections. Accordingly, IL-6 classic signaling was found to be essential to eliminate the intracellular pathogen Listeria monocytogenes, and sgp130Fc did not compromise this important immune reaction [19]. In line with this, sgp130Fc did not impede with protective immune responses after infection with Mycobacterium tuberculosis, whereas global blockade of IL-6 with an anti-IL-6 antibody (blocking classic and trans-signaling) did so [20]. Recently it was shown that IL-6 plays an important role in the defense of the body against viral infections [21]. Deletion of the gp130 gene in T-cells compromised Tcell survival and IL-27 induced IL-21 production upon lymphocytic choriomeningitis virus infection [22].
The soluble IL-6R: a buffer in the blood In healthy individuals levels of IL-6 in the plasma are extremely low, close to the detection limit of 1 pg/ml [23]. Plasma levels of sIL-6R range between 50 and 75 ng/ml and levels of sgp130 are around 400 ng/ml [24] (Figure 2). During inflammatory states, the levels of sIL-6R and sgp130 do not largely change but levels of IL-6 can increase dramatically. In patients with rheumatoid www.sciencedirect.com
arthritis, IL-6 levels of 150 ng/ml have been reported [25]. Under septic conditions levels of IL-6 in the low microgram range have been reported [26]. Therefore, IL6 secreted by cells such as neutrophils, monocytes/macrophages, endothelial cells, fibroblasts and T-cells [27] will bind to the sIL-6R with an affinity of around 1 nM. Subsequently, the complex of IL-6 and sIL-6R will bind to sgp130 with an affinity of 10 pM where its activity is neutralized [28]. Thus, sIL-6R and sgp130 form a buffer for IL-6 in the blood with a capacity equal to the molar concentration of the sIL-6R, which corresponds to a concentration of IL-6 of 100-150 ng/ml (Figure 2). Interestingly, a single nucleotide polymorphism in the IL-6R gene (rs2228145) has been identified, which changes aspartate 358 in the juxtamembrane region of the receptor protein to alanine and which facilitates shedding of the IL-6R by the metalloprotease ADAM17 [29]. Individuals with alanine 358 show significantly higher sIL-6R levels in the blood and are less susceptible to autoimmune diseases such as rheumatoid arthritis [30]. Two meta-analyses, which incorporated clinical data from more than 100 000 individuals could show that for every copy of the Asp358Ala SNP inherited, the concentrations of C-reactive protein and other acute-phase proteins were reduced, as well as the overall risk to suffer from coronary heart disease [31,32]. In this context, increased levels of sIL-6R appear to have an anti-inflammatory impact, which might be taken as an argument for the validity of the concept of the IL-6 buffer in the blood formed by sIL-6R and sgp130.
Plasticity of gp130 and IL-6R Gp130 is the common b-receptor for all cytokines of the IL-6 family, including IL-6, IL-11, CLC, CNTF, CT-1, OSM, LIF and IL-27 [33]. Signal transduction is initiated after dimer formation with LIFR, OSMR, WSX-1 or gp130 itself. This fact illustrates a remarkable kind of plasticity, as gp130 is able to engage signaling with a variety of ligands, non-signaling alpha-receptors and at least three other breceptors [33]. Deregulated cytokine signaling via gp130 is associated with chronic inflammation and cancer development. Accordingly, ectopic expression of a constitutively active gp130 variant (L-gp130) in mice induces the formation of multiple myeloma in concert with myc [34]. Besides IL-6, CNTF has been described as a second ligand for the IL-6R. The signal is transduced via a gp130/ LIFR heterodimer, the same signaling complex that CNTF uses when it binds to the CNTFR. Recently, the IL-27 subunit p28 (also known as IL-30) has been identified as another ligand for the IL-6R [35–37]. In contrast to CNTF, IL-30 signals via a gp130 homodimer like IL-6, whereas IL-27 (p28/EBI3) engages a heterodimer of gp130/WSX1. Thus, IL-30 is able to activate two different b-receptor complexes, depending to which non-signaling alpha-receptor (EBI3 or IL-6R) it initially Current Opinion in Immunology 2015, 34:75–82
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Figure 2
(a)
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Soluble IL-6R and soluble gp130 constitute a buffer for IL-6 in the blood. (a) In healthy humans IL-6 (1–5 pg/ml), soluble IL-6R (30–70 ng/ml) and soluble gp130 (250–400 ng/ml) are constitutively present. IL-6 binds with an affinity of 1 nM to the sIL-6R. The complex of IL-6/sIL-6R binds to sgp130 with 100 times higher affinity (10 pM). The molar ratios indicate that the concentration of the soluble IL-6R controls the capacity of the IL-6 buffer in the blood. (b) The IL-6R SNP rs2228145 leads to higher concentrations of sIL-6R in the blood and thus to a higher capacity of the IL-6 buffer leading to protection of certain inflammatory diseases [30].
binds [37] (Figure 3a). Interestingly, p28 has also been described as an antagonist of IL-6 family cytokines, as transgenic overexpression of p28 resulted in impaired B cell responses in a gp130-dependent manner [38]. In a mouse model of inflammation-induced liver injury, p28 acted as an anti-inflammatory cytokine [39]. Furthermore, p28 either alone or in combination with p40 was able to suppress disease severity in a model of experimental autoimmune uveitis (EAU) [40]. How these different Current Opinion in Immunology 2015, 34:75–82
findings can be incorporated into one functional model is not clear at the present time. The most recent addition to the IL-6 family is IL-35 (EBI3/p35), which can be considered as a cytokine that bridges IL-6 and IL-12 cytokine families [41]. Until now, four different b-receptor pairs for IL-35 have been described (IL-12Rb2/IL-12Rb2, gp130/gp130, IL-12Rb2/ gp130 and IL-12Rb2/WSX-1) [42,43]. This suggests a www.sciencedirect.com
Therapeutic blockade of IL-6 activity Garbers, Aparicio-Siegmund and Rose-John 79
Figure 3
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Plasticity within the IL-6 family of cytokines. (a) IL-6 binds with high affinity to the IL-6R, which induces recruitment of a gp130 homodimer. CNTF, which usually binds to CNTFR/LIFR/gp130 complexes with high affinity, can also bind IL-6R/LIFR/gp130 complexes, although with much lower affinity. IL-30 binds efficiently to EBI3 (to form the heterodimeric cytokine IL-27, which signals via the gp130/WSX-1 complex), but can also bind to a gp130 homodimer via the IL-6R with low affinity. (b) IL-35 (p35/EBI3) shares its subunits with IL-27 (p28/EBI3) and IL-12 (p35/p40).
remarkable plasticity on both the involved receptors and the heterodimeric cytokine itself, especially if one considers that both subunits are further shared with other cytokines (EBI3 with IL-27 and p35 with IL-12, respectively, Figure 3b). Binding of IL-6 family cytokines to their respective receptors has been biochemically studied for more than 20 years. The term chemical plasticity has been introduced for a receptor interacting with many www.sciencedirect.com
ligands in a specific manner [44]. However, the ability of IL-35 to bind to such a wide variety of receptor combinations has not been reported for any other cytokine. This is also true for the finding that different receptor combinations induce the phosphorylation of different combinations of STAT proteins, which appears unlikely given the knowledge about other cytokines and receptors of the same family [42]. Therefore, additional Current Opinion in Immunology 2015, 34:75–82
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and more rigorous studies are needed to fully understand the different receptor complexes and the signal transduction of the cytokine IL-35. Biochemical analysis are further complicated by the fact that p35 protein produced in bacteria can be used to assemble IL-12, but not IL-35 [45].
Therapeutic inhibition of IL-6 activity IL-6 together with IL-1 and TNFa play a pivotal role in the maintenance of chronic inflammatory diseases [46,47]. Blockade of TNFa has been very successful in the treatment of rheumatoid arthritis and inflammatory bowel disease [47]. For the blockade of IL-6, a small molecule inhibitor of JAK1, JAK2 and JAK3 (tofacitinib) and a humanized monoclonal antibody targeting the human IL-6R (tocilizumab) have been approved by the FDA for the treatment of rheumatoid arthritis [24]. A number of monoclonal antibodies against human IL-6 and the human IL-6R are in phase I, phase II or phase III of clinical development [24]. While inhibitors of JAK1, JAK2 and JAK3 have the potential to block the activity of most cytokines and interferons [48] the monoclonal antibody tocilizumab is specific for the human IL-6R and is therefore expected only to block the activity of IL-6 and to some unknown extent the activity of CNTF [49] and p28 [37], since these cytokines can also act via the IL-6R [37,49]. Other small molecules that interfere with the Jak/STAT signaling cascade, for example inhibitors of protein kinase II [50], are just entering clinical trials.
[28,54]. Interestingly, sgp130Fc could theoretically block signaling of IL-11 via a soluble form of the IL11R [55]. However, to date no naturally occurring sIL11R has been described. Such a notion might have important implications for the treatment of inflammatory disease in which both processes, inflammation and regeneration, play a role. The intestine is the organ with the highest regenerative activity. The fact that IL-6 / mice upon treatment with the irritant dextran sodium sulfate show more inflammation than wt animals was explained by the compromised STAT3 activation in the intestinal epithelial cells [56]. Interestingly, treatment by recombinant IL-6 can be beneficial in a mouse model of human inflammatory bowel disease [57]. Consequently, in patients upon global IL-6 blockade, gastrointestinal perforations have been observed. This might be the main reason why the antibody tocilizumab is not approved for the treatment of inflammatory bowel disease. Selective blockade of IL-6 trans-signaling by the sgp130Fc protein was demonstrated to be beneficial in different animal models of human inflammatory bowel disease [58,59] and colon cancer [60,61]. The sgp130Fc protein was produced under GMP conditions and phase I clinical trials were conducted in 2013/14. It is planned to move to phase II clinical trials in inflammatory bowel disease patients in the year 2015 [24].
Conclusions In the past years it has been noted that IL-6 not only is involved in pro-inflammatory activities but also in tissue regeneration after wounding and in the defense of the body against bacterial infection. The discovery that the natural sgp130 protein found in the blood at high concentrations (see above) was a specific inhibitor of IL-6 trans-signaling via the sIL-6R [28] paved the way to systematically analyze the contribution of IL-6 classic and trans-signaling in many animal models of human inflammatory diseases and cancer [51]. Using a sgp130Fc protein [28] it turned out that in most cases the blockade of IL-6 trans-signaling was as efficient as the global blockade of IL-6 classic and trans-signaling with an IL-6 or IL-6R specific monoclonal antibody [52]. In many cases, however, the regenerative activity of IL6 was preserved upon selective blockade of IL-6 transsignaling. From such experiments it was deduced that the pro-inflammatory activities of IL-6 are mediated via trans-signaling whereas the anti-inflammatory and regenerative activities are mediated via the membrane bound IL-6R [53]. Sgp130Fc does not efficiently interfere with signaling of other IL-6 family cytokines including IL-27 because all these cytokines (with the exception of IL-11) bind to a heterodimer of gp130 and the receptor proteins LIF-R, OSM-R or WSX-1 Current Opinion in Immunology 2015, 34:75–82
Since its first description as an interferon b-like protein in 1980 [4], our understanding of the biology of IL-6 and its physiologic and pathophysiologic implications have dramatically increased. The development of the monoclonal antibody tocilizumab and its worldwide approval for the treatment of several inflammatory diseases have proven the importance of IL-6 in the pathology of these diseases and the therapeutic value of its inhibition. The identification of soluble forms of the IL-6R have revealed a second mode of IL-6 signaling, which has been termed trans-signaling, and which is mainly controlled by the metalloprotease ADAM17. Proteolysis of the membrane-bound IL-6R leads to the generation of agonistic IL-6/sIL-6R complexes that can be specifically blocked by the sgp130Fc protein. The effectiveness of specific inhibition of IL-6 trans-signaling has been proven in numerous mouse models of inflammatory diseases and cancer, and sgp130Fc has just recently successfully completed phase I clinical trials and will enter phase II in 2015. It will be interesting to see if the promising results from animal models hold true and if sgp130Fc can be used in the future to treat patients with inflammatory diseases with less side effects. www.sciencedirect.com
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Acknowledgements The work of CG and SR-J is supported by the Deutsche Forschungsgemeinschaft (SFB877 Project Grants A1 and A10), the Bundesministerium fu¨r Bildung und Forschung (BMBF grant ‘InTraSig’, project B) and the Cluster of Excellence ‘Inflammation at Interfaces’.
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