Inhibition Of IL-6 Trans-signaling Research Articles

Exclusive inhibition of IL-6 trans-signaling by soluble gp130FlyRFc

gp130 is the signal-transducing receptor for the Interleukin (IL)-6 type cytokines IL-6 and IL-11. To induce signaling, IL-6 forms a complex with IL-6 receptor (IL-6R) and IL-11 with IL-11 receptor (IL-11R). Membrane-bound IL-6R and IL-11R in complex with gp130 and the cytokine mediate classic-signaling, whereas trans-signaling needs soluble IL-6R and IL-11R variants. Interleukin (IL)-6 trans-signaling is of particular importance because it drives the development of autoimmune diseases, including rheumatoid arthritis and chronic inflammatory bowel diseases, whereas a role for IL-11 trans-signaling remains elusive. Soluble gp130 selectively inhibits trans-signaling of IL-6 whereas both, classic- and trans-signaling are abrogated by IL-6- and IL-6R-antibodies. Recently, we described an optimized sgp130 variant, which carries three amino acid substitutions T102Y/Q113F/N114L (sgp130FlyFc) resulting in reduced inhibition of IL-11 trans-signaling by increasing the affinity of sgp130 for the site I of IL-6. Moreover, we described that the patient mutation R281Q in gp130 results in reduced IL-11 signaling. Here, we show that the combination of T102Y/Q113F/N114L and R281Q in the new variant sgp130FlyRFc results in complete preservation of IL-11 mediated trans-signaling, whereas inhibition of IL-6 trans-signaling is maintained. Since sgp130Fc (olamkicept) has successfully completed a phase IIa trial in Crohn’s disease (CD) and ulcerative colitis, sgp130FlyRFc might serve as second-generation therapeutic to diminish IL-11 trans-signaling cross-reactivity.

Abstract 3213: Combination of anti-IL-6 and anti-PD-L1 antibodies synergistically reinvigorates cancer immunity by activating both CTL and Th1 cells in mice

Abstract We previously demonstrated that Th1 differentiation of tumor-specific CD4+ T cells was attenuated in tumor-bearing or aged mice in an IL-6-dependent manner (Nat. Comm. 6: e6702, 2015, Cancer Immunol. Res. 1: 64, 2013). Furthermore we found that accompanied by the systemic increase of soluble IL-6 receptor (sIL-6R) in cancer patients, inhibition of IL-6 trans-signaling mediated through the sIL-6R restored the Th1 responses, their helper activity toward CD8+ T cells, and anti-tumor activity in tumor-bearing mice (Cancer Res. 77: 2279, 2017). The systemically increased sIL-6Rs were mainly produced by myeloid cells. Moreover, cMaf-deficient CD4+ T cells were resistant to Th1 suppression and impairment of T cell-mediated anti-tumor immunity induced by IL-6/sIL-6R indicating that c-Maf activity was responsible for Th1 suppression. Myeloid cell-derived sIL-6R was also possibly associated with Th1 suppression and c-Maf expression in head and neck cancer patients. These results suggest that targeting a pro-inflammatory cytokine, IL-6 enhanced the tumor-specific Th1 responses and subsequent anti-tumor effects. However, IL-6 blockade in turn up-regulated the expression of immune-checkpoint molecule, PD-L1 on melanoma cells. This PD-L1 induction was canceled in IFN-γ-deficient mice or mice depleted of CD4+ T cells, suggesting an important role of CD4+ T cell-derived IFN-γ in the PD-L1 induction in tumor-bearing hosts. On the other hand, in some patients with melanoma, anti-PD-1 antibody, Nivolumab treatment increased the systemic level of IL-6, which were associated with their poor clinical responses. This PD-L1 blockade-evoked IL-6 induction was also observed in melanoma-bearing mice (Cancer Res. 78: 5011, 2018). Considering the mechanistic linkage between IL-6 and PD-1/PD-L1 signals, we found that PD-1/PD-L1 blockade prompted PD-1+macrophages to produce IL-6 in tumor microenvironment. Depletion of macrophages in melanoma-bearing mice revealed that macrophages functioned as a source of IL-6 during PD-L1 blockade, which was responsible for the defective Th1 response. Furthermore, combined blockade of the mutually regulated-immunosuppressive activities mediated by IL-6 and PD-1/PD-L1 signals enhanced the infiltration of IFN-γ-producing CD4+ T cells in tumor tissues, and exerted a synergistic anti-tumor effect, whereas PD-L1 blockade alone did not promote Th1 response. Collectively, these findings suggest that IL-6 is considered to be a rational immunosuppressive target to overcome a narrow therapeutic window of anti-PD-1/PD-L1 therapy. [ This research was financially supported by the JSPS KAKENHI grant nos. 26430165 and 18K07325 to HT, nos. 15H04311 and 16H06498 to YN, and the P-CREATE from the AMED, Japan to YN and HT. ] Citation Format: Yasuharu Nishimura, Koji Fujieda, Azusa Miyashita, Satoshi Fukushima, Tokunori Ikeda, Yosuke Kubo, Satoru Senju, Hironobu Ihn, Hiroyuki Oshiumi, Hirotake Tsukamoto. Combination of anti-IL-6 and anti-PD-L1 antibodies synergistically reinvigorates cancer immunity by activating both CTL and Th1 cells in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3213.

Targeting histone deacetylase 3 (HDAC3) in the bone marrow microenvironment inhibits multiple myeloma proliferation by modulating exosomes and IL-6 trans-signaling

Multiple myeloma (MM) is an incurable cancer that derives pro-survival/proliferative signals from the bone marrow (BM) niche. Novel agents targeting not only cancer cells, but also the BM-niche have shown the greatest activity in MM. Histone deacetylases (HDACs) are therapeutic targets in MM and we previously showed that HDAC3 inhibition decreases MM proliferation both alone and in co-culture with bone marrow stromal cells (BMSC). In this study, we investigate the effects of HDAC3 targeting in BMSCs. Using both BMSC lines as well as patient-derived BMSCs, we show that HDAC3 expression in BMSCs can be induced by co-culture with MM cells. Knock-out (KO), knock-down (KD), and pharmacologic inhibition of HDAC3 in BMSCs results in decreased MM cell proliferation; including in autologous cultures of patient MM cells with BMSCs. We identified both quantitative and qualitative changes in exosomes and exosomal miRNA, as well as inhibition of IL-6 trans-signaling, as molecular mechanisms mediating anti-MM activity. Furthermore, we show that HDAC3-KD in BM endothelial cells decreases neoangiogenesis, consistent with a broad effect of HDAC3 targeting in the BM-niche. Our results therefore support the clinical development of HDAC3 inhibitors based not only on their direct anti-MM effects, but also their modulation of the BM microenvironment.

Pharmacologic IL-6Rα inhibition in cholangiocarcinoma promotes cancer cell growth and survival

Biliary tract cancer (BTC) represents a malignant tumor of the biliary tract including cholangiocarcinoma (CCA) and the carcinoma of the gallbladder (GBC) with a 5-year survival rate between 5 and 18% due to late diagnosis and rapid disease progression. Chronic inflammation is one of the main risk factors for CCA and GBC in particular. IL-6, as a mediator of inflammation, can act through a membrane-bound receptor alpha-chain (mIL-6R, “IL-6 classic signaling”) or via soluble forms (sIL-6R, “IL-6 trans-signaling”). However, little is known about the impact on cellular responses of IL-6 trans-signaling on BTC. We analyzed primary tumors as whole sections and as tissue microarrays, and also searched The Cancer Genome Atlas database. Compared to non-neoplastic, non-inflamed gallbladder tissue, IL-6Rα was downregulated in GBC, and this correlated with the patients' overall survival. Furthermore, different CCA cell lines and compounds for activation (IL-6 and Hyper-IL-6) or inhibition (Tocilizumab and sgp130Fc) of IL-6 classic signaling and trans-signaling were used to determine their effects on cellular processes between the two modes of IL-6 signaling. Inhibition of IL-6 trans-signaling by sgp130Fc reduced CCA cell line viability and apoptosis, whereas migration and proliferation were increased. We conclude that IL-6Rα expression is a good prognostic marker for GBC, and that the blocking of IL-6 trans-signaling and activation of IL-6 classic signaling have tumor promoting activity. These findings warrant the exclusion of patients with GBC or other malignancies associated with bile metabolism from IL-6R inhibitor therapy.

THU-471 - Inhibition of IL6 trans-signaling leads to NAFLD, mature-onset obesity and metabolic syndrome

THU-471 - Inhibition of IL6 trans-signaling leads to NAFLD, mature-onset obesity and metabolic syndrome

P3.07-002 Blocking of ADAM17 Mitigates Kras-Induced Lung Adenocarcinoma Possibly via Inhibition of IL-6 Trans-Signaling

P3.07-002 Blocking of ADAM17 Mitigates Kras-Induced Lung Adenocarcinoma Possibly via Inhibition of IL-6 Trans-Signaling

SAT-357 - Inhibition of IL6 trans-signaling leads to gut microbiota-dependant mature-onset obesity and metabolic syndrome

SAT-357 - Inhibition of IL6 trans-signaling leads to gut microbiota-dependant mature-onset obesity and metabolic syndrome

Inhibition of IL-6 trans-signaling in the brain increases sociability in the BTBR mouse model of autism

Autism is a severe neurodevelopmental disorder with a large population prevalence, characterized by abnormal reciprocal social interactions, communication deficits, and repetitive behaviors with restricted interests. The BTBR T+Itpr3tf (BTBR) mice have emerged as strong candidates to serve as models of a range of autism-relevant behaviors. Increasing evidences suggest that interleukin (IL)-6, one of the most important neuroimmune factors, was involved in the pathophysiology of autism. It is of great importance to further investigate whether therapeutic interventions in autism can be achieved through the manipulation of IL-6. Our previous studies showed that IL-6 elevation in the brain could mediate autistic-like behaviors, possibly through the imbalances of neural circuitry and impairments of synaptic plasticity. In this study, we evaluate whether inhibiting IL-6 signaling in the brain is sufficient to modulate the autism-like behaviors on the BTBR mice. The results showed that chronic infusion of an analog of the endogenous IL-6 trans-signaling blocker sgp130Fc protein increased the sociability in BTBR mice. Furthermore, no change was observed in the number of excitatory synapse, level of synaptic proteins, density of dentitic spine and postsynaptic density in BTBR cortices after inhibiting IL-6 trans-signaling. However, inhibition of IL-6 trans-signaling increased the evoked glutamate release in synaptoneurosomes from the cerebral cortex of BTBR mice. Our findings suggest that inhibition of excessive production of IL-6 may have selective therapeutic efficacy in treating abnormal social behaviors in autism.

Different Soluble Forms of the Interleukin-6 Family Signal Transducer gp130 Fine-tune the Blockade of Interleukin-6 Trans-signaling

Soluble forms of the IL-6 receptor (sIL-6R) bind to the cytokine IL-6 with similar affinity as the membrane-bound IL-6R. IL-6·sIL-6R complexes initiate IL-6 trans-signaling via activation of the ubiquitously expressed membrane-bound β-receptor glycoprotein 130 (gp130). Inhibition of IL-6 trans-signaling has been shown to be favorable in numerous inflammatory diseases. Furthermore, different soluble forms of gp130 (sgp130) exist that, together with the sIL-6R, are thought to form a buffer for IL-6 in the blood. However, a functional role for the different sgp130 forms has not been described to date. Here we demonstrate that the metalloproteases ADAM10 and ADAM17 can produce sgp130 by ectodomain shedding of gp130, even though this mechanism only accounts for a minor proportion of sgp130 in the circulation. We further show that full-length sgp130 and the shorter forms sgp130-rheumatoid arthritis-associated peptide (RAPS) and sgp130-E10 are differentially expressed in a cell type- specific manner. Remarkably, full-length sgp130 is expressed by monocytes, but this expression is completely lost during differentiation into macrophages in vitro Using genetically engineered murine pre-B cells that secrete different forms of sgp130, we found that these secreted sgp130 proteins are able to prevent trans-signaling-driven cell proliferation of the secreting cells, whereas conditioned supernatant from these cells failed to block IL-6 trans-signaling in other cells. Thus, our data suggest that the different sgp130 forms are released from cells into their immediate surroundings and appear to form cell-associated gradients to modulate their own susceptibility for IL-6 trans-signaling.

ID: 13: The biology of Interleukin-6 and strategies of blockade

Cytokines receptors exist in membrane bound and soluble form. The IL-6/soluble IL-6R complex stimulates target cells not stimulated by IL-6 alone, since they do not express the membrane bound IL-6R. We have named this process ’trans-signaling’. The soluble IL-6R is generated via ectodomain shedding by the membrane bound metalloprotease ADAM17. Soluble gp130 is the natural inhibitor of IL-6/soluble IL-6R complex responses. The dimerized recombinant soluble gp130Fc fusion protein is a molecular tool to discriminate between gp130 responses via membrane bound and soluble IL-6R responses. Interestingly, depending on the animal model used, global blockade of IL-6 signaling by neutralizing monoclonal antibodies and selective blockade of IL-6 trans-signaling can lead to different consequences. We used neutralizing monoclonal antibodies for global blockade of IL-6 signaling and the sgp130Fc protein for selective blockade of IL-6 trans-signaling in several animal models of human diseases. Inhibition of IL-6 trans-signaling but not global IL-6 blockade was beneficial in the cecal ligation and puncture sepsis model. Defense against bacterial infections rely on the membrane bound IL-6R. Acute pancreatitis often results in subsequent acute lung injury, which is an inflammatory disease with high mortality. IL-6 is necessary for the inflammatory process to reach the lung and blockade of IL-6 trans-signaling is sufficient to block the disease. The extent of inflammation is controlled by trans-signaling via the soluble IL-6R. Using the sgp130Fc protein or sgp130Fc transgenic mice we demonstrate in animal models of inflammatory bowel disease, peritonitis, rheumatoid arthritis, atherosclerosis, pancreatitis, lupus erythematodes, colon cancer, ovarian cancer, pancreatic cancer, nephrotoxic nephritis and high fat diet induced obesity that IL-6 trans-signaling via the soluble IL-6R is the crucial step in the development and the progression of the disease. Therefore, sgp130Fc is a novel therapeutic agent for the treatment of chronic inflammatory diseases and cancer and it underwent phase I clinical trials as an anti-inflammatory in 2013/2014. Phase II clinical trials in patients with autoimmune diseases such as inflammatory bowel disease will follow in 2015/16.

Allergen-induced IL-6 trans-signaling activates γδ T cells to promote type 2 and type 17 airway inflammation

A variant in the IL-6 receptor (IL-6R) gene increases asthma risk and is predicted to decrease IL-6 classic signaling and increase IL-6 trans-signaling. This suggests that inhibition of IL-6 trans-signaling, but not classic signaling, might suppress allergic airway inflammation. We sought to determine whether IL-6 signaling contributes to (1) acute experimental asthma induced by clinically relevant allergens and (2) variation in asthma clinical phenotypes in asthmatic patients. Mice were sensitized to house dust mite (HDM) or cockroach at day 0, treated with IL-6R inhibitors at day 13, and challenged with the same allergen at days 14 to 17. End points were measured 3 hours after the final challenge. IL-6 and soluble IL-6 receptor (sIL-6R) expression in induced sputum of asthmatic patients was correlated with asthma clinical phenotypes. Both HDM and cockroach induced a type 2/type 17 cytokine profile and mixed granulocytic inflammation in the airways. Both allergens increased IL-6 expression in the airways, but only cockroach induced sIL-6R expression. Therefore HDM challenge promoted IL-6 classic signaling but not trans-signaling; in this model treatment with anti-IL-6R did not suppress airway inflammation. In contrast, cockroach-induced inflammation involved activation of IL-6 trans-signaling and production of IL-17A by γδ T cells. Anti-IL-6R, selective blockade of sIL-6R, or γδ T-cell deficiency significantly attenuated cockroach-induced inflammation. Asthmatic patients with high airway IL-6 and sIL-6R levels were enriched for the neutrophilic and mixed granulocytic subtypes. Experimental asthma associated with both high IL-6 and high sIL-6R levels in the airways is attenuated by treatment with IL-6R inhibitors.

S-21 : Blocking only the bad side of Interleukin-6

Cytokines receptors exist in membrane bound and soluble form. The IL-6/soluble IL-6R complex stimulates target cells not stimulated by IL-6 alone, since they do not express the membrane bound IL-6R. We have named this process ‘ trans-signaling ’. The soluble IL-6R is generated via ectodomain shedding by the membrane bound metalloprotease ADAM17. Soluble gp130 is the natural inhibitor of IL-6/soluble IL-6R complex responses [1] . The dimerized recombinant soluble gp130Fc fusion protein is a molecular tool to discriminate between gp130 responses via membrane bound and soluble IL-6R responses. Interestingly, depending on the animal model used, global blockade of IL-6 signaling by neutralizing monoclonal antibodies and selective blockade of IL-6 trans-signaling can lead to different consequences. We used neutralizing monoclonal antibodies for global blockade of IL-6 signaling and the sgp130Fc protein for selective blockade of IL-6 trans-signaling in several animal models of human diseases. Inhibition of IL-6 trans-signaling but not global IL-6 blockade was beneficial in the cecal ligation and puncture sepsis model [2] . Defense against bacterial infections rely on the membrane bound IL-6R [3] . Acute pancreatitis often results in subsequent acute lung injury, which is an inflammatory disease with high mortality. IL-6 is necessary for the inflammatory process to reach the lung and blockade of IL-6 trans-signaling is sufficient to block the disease [4] . The extent of inflammation is controlled by trans-signaling via the soluble IL-6R. Using the sgp130Fc protein or sgp130Fc transgenic mice we demonstrate in animal models of inflammatory bowel disease, peritonitis, rheumatoid arthritis, atherosclerosis, pancreatitis, lupus erythematodes, colon cancer, ovarian cancer and pancreatic cancer that IL-6 trans-signaling via the soluble IL-6R is the crucial step in the development and the progression of the disease [1] , [2] , [3] , [4] , [5] . Therefore, sgp130Fc is a novel therapeutic agent for treatment of chronic inflammatory diseases and cancer and it underwent phase I clinical trials as an anti-inflammatory in 2013/2014. Phase II clinical trials in patients with autoimmune diseases such as inflammatory bowel disease will follow next year.

216: The pro-inflammatory activities of interleukin-6 are mediated by the soluble interleukin-6 receptor via trans-signaling

Cytokines receptors exist in membrane bound and soluble form. The IL-6/soluble IL-6R complex stimulates target cells not stimulated by IL-6 alone, since they do not express the membrane bound IL-6R. We have named this process ‘trans-signaling’. Soluble gp130 is the natural inhibitor of IL-6/soluble IL-6R complex responses [1]. The dimerized recombinant soluble gp130Fc fusion protein is a molecular tool to discriminate between gp130 responses via membrane bound and soluble IL-6R responses. We used neutralizing monoclonal antibodies for global blockade of IL-6 signaling and the sgp130Fc protein for selective blockade of IL-6 trans-signaling in several animal models of human diseases. Inhibition of IL-6 trans-signaling but not global IL-6 blockade was beneficial in the cecal ligation and puncture sepsis model [2]. Defense against bacterial infections rely on the membrane bound IL-6R [3]. Acute pancreatitis often results in subsequent acute lung injury, which is an inflammatory disease with high mortality. IL-6 is necessary for the inflammatory process to reach the lung and blockade of IL-6 trans-signaling is sufficient to block the disease [4]. The extent of inflammation is controlled by trans-signaling via the soluble IL-6R. Using the sgp130Fc protein or sgp130Fc transgenic mice we demonstrate in animal models of inflammatory bowel disease, peritonitis, rheumatoid arthritis, atherosclerosis pancreatitis, colon cancer, ovarian cancer and pancreatic cancer, that IL-6 trans-signaling via the soluble IL-6R is the crucial step in the development and the progression of the disease [1–4]. Therefore, sgp130Fc is a novel therapeutic agent for the treatment of chronic inflammatory diseases and cancer and it will undergo phase I clinical trials as an anti-inflammatory drug in 2013. [1] Jones SA, Scheller J, Rose-John S. Therapeutic strategies for the clinical blockade of IL-6/gp130 signaling. J Clin Invest 2011;121:3375–83. [2] Barkhausen T, et al. Selective blockade of IL-6 trans-signaling improves survival in a murine polymicrobial sepsis model. Crit Care Med 2011;39:1407–13. [3] Hoge J, et al. IL-6 controls the innate immune response against Listeria monocytogenes via classical IL-6 signaling. J Immunol 2013;190:703–11. [4] Zhang H, et al. IL-6 trans-signaling promotes pancreatitis-associated lung injury and lethality. J Clin Invest 2013;123:1019–31.

I13 The pro-inflammatory activities of interleukin-6 are mediated by trans-signaling

Cytokines and cytokine receptors exist in membrane bound and soluble form. Membrane bound cytokinesneed to cleaved in order to become systemically available. While most soluble receptors are antagonists, some areagonists like the soluble IL-6 receptors. In vivo, the IL-6/soluble IL-6R complex stimulates several types of target cells notstimulated by IL-6 alone, since they do not express the membrane bound IL-6R. This process has been named transsignaling[1]. We have shown that soluble gp130 is the natural inhibitor of IL-6/soluble IL-6R complex responses.Recombinant soluble gp130 protein is a molecular tool to discriminate between gp130 responses via membrane boundand soluble IL-6R responses [1]. The soluble IL-6R is mostly generated by proteolysis of the IL-6R transmembraneprotein. Shedding of the IL-6R is mediated mainly by the metalloprotease ADAM17, which is also responsible for thecleavage of TNFa and ligands of the EGF-R. Consequently, activation of ADAM17 has different effects on the activationof the immune response as well as on induction of regenerative responses [2,3]. We used neutralizing monoclonal antibodies for global blockade of IL-6 signaling and the sgp130Fc protein forselective blockade of IL-6 trans-signaling in several animal models of human diseases. We could show that inhibition of IL-6 trans-signaling was beneficial in a sepsis model. Also bacterial infectionmodels suggest a different outcome of global blockade of IL-6 as compared to selective IL-6 trans-signaling inhibition [4].Conclusion: The extent of inflammation is controlled by trans-signaling via the soluble IL-6R. Using the sgp130Fcprotein or sgp130Fc transgenic mice we demonstrate in several chronic inflammatory diseases and cancers includinginflammatory bowel disease, peritonitis, rheumatoid arthritis, atherosclerosis, colon cancer, ovarian cancer andpancreatic cancer, that IL-6 trans-signaling via the soluble IL-6R is a crucial step in the development and the progressionof the disease. Therefore, sgp130Fc is a novel therapeutic agent for the treatment of chronic inflammatory diseases andcancer [5-7].

Cutting Edge: Inhibition of IL-6 Trans-Signaling Protects from Malaria-Induced Lethality in Mice

Circulating IL-6 levels correlate with the severity of blood-stage malaria in humans and mouse models, but the impact of IL-6 classic signaling through membrane IL-6Rα, as well as IL-6 trans-signaling through soluble IL-6Rα, on the outcome of malaria has remained unknown. In this study, we created IL-6Rα-deficient mice that exhibit a 50% survival of otherwise lethal blood-stage malaria of the genus Plasmodium chabaudi. Inducing IL-6 trans-signaling by injection of mouse recombinant soluble IL-6Rα in IL-6Rα-deficient mice restores the lethal outcome to malaria infection. In contrast, inhibition of IL-6 trans-signaling via injection of recombinant sGP130Fc protein in control mice results in a 40% survival rate. Our data demonstrate that IL-6 trans-signaling, rather than classic IL-6 signaling, contributes to malaria-induced lethality in mice, preceded by an increased inflammatory response. Therefore, inhibition of IL-6 trans-signaling may serve as a novel promising therapeutic basis to combat malaria.

Selective blockade of interleukin-6 trans-signaling improves survival in a murine polymicrobial sepsis model*

The pleiotropic cytokine interleukin (IL)-6 seems to play a pivotal role in sepsis, but contradictory findings in animal models impede a rationale for therapies directed against IL-6. IL-6 signals by two mechanisms via the ubiquitous transmembrane glycoprotein 130 (gp130): "classic" signaling using membrane-bound IL-6 receptor (IL-6R) and trans-signaling using soluble IL-6R (sIL-6R). Trans-signaling is selectively inhibited by soluble gp130 (sgp130). The aim of this study was to systematically compare complete blockade of IL-6 signaling (using a neutralizing anti-IL-6 antibody) and selective blockade of IL-6 trans-signaling (using a fusion protein of sgp130 and the crystallizable fragment of immunoglobulin G1, sgp130Fc) in a standardized cecal ligation and puncture (CLP) sepsis model. Animal study. Animal laboratory. C57BL/6J mice. We performed a 96-hr dose-response study and a 24-hr study to investigate short-term mechanisms. In the 96-hr study, CLP was performed in 120 randomized mice (20 mice received vehicle, 10 mice per dose group). Mice were treated with equimolar doses of sgp130Fc (0.01/0.1/1/10 mg/kg) or anti-IL-6 (0.008/0.08/0.8/8 mg/kg) 24 hrs before CLP. Two additional groups received 0.5 mg/kg sgp130Fc 24 hrs before or 1 mg/kg sgp130Fc 24 hrs after CLP. Survival and activity scores were obtained daily until 96 hrs after CLP. In the 24-hr study, mice were randomized into four groups with 10 animals each (sham/vehicle, CLP/vehicle, CLP/anti-IL-6 [0.8 mg/kg], and CLP/sgp130Fc [1 mg/kg]) and killed after 24 hrs. In contrast to anti-IL-6, pretreatment with sgp130Fc significantly and dose-dependently increased survival from 45% to 100%. In addition, 1 mg/kg sgp130Fc administered 24 hrs after CLP increased survival from 45% to 80%. Mechanistically, sgp130Fc efficacy was reflected by complete prevention of epithelial cell apoptosis in the jejunum after CLP, which was not achieved with anti-IL-6. Selective inhibition of IL-6 trans-signaling by sgp130Fc has considerable potential for the treatment of sepsis and related disorders.

New insights into the role and signalling processes of gp130

Cytokine receptors exist in membrane bound and soluble form. While most soluble receptors are antagonists, some soluble receptors are agonists like soluble receptors of the gp130 cytokine family. In vivo, the IL-6/soluble IL-6R complex stimulates several types of target cells not stimulated by IL-6 alone, since they do not express the membrane bound IL-6R. This process has been named trans-signaling [1]. We have shown that soluble gp130 is the natural inhibitor of IL-6/soluble IL-6R complex responses. The recombinant soluble gp130 protein is a molecular tool to discriminate between gp130 responses via membrane bound and soluble IL-6R responses. We have constructed a fusion of soluble gp130 and the Fc portion of human IgG1. This sgp130Fc protein proved to be efficient in blocking responses via the IL-6/soluble IL-6R complex without affecting IL-6 responses, which are mediated via the membrane bound IL-6R [1]. The soluble IL-6R is mostly generated by proteolysis of the IL-6R transmembrane protein. Shedding of the IL-6R is mediated by the metalloprotease ADAM17, which is also responsible for the cleavage of TNFα and ligands of the EGF-R. Consequently, activation of ADAM17 has different effects on the activation of the immune response as well as on induction of regenerative responses [2,3]. Interestingly, depending on the animal model used, global blockade of IL-6 signaling by neutralizing monoclonal antibodies and selective blockade of IL-6 trans-signaling can lead to different consequences. We could recently show that inhibition of IL-6 trans-signaling was beneficial in a caecum ligation puncture model whereas global IL-6 blockade showed no benefit in survival of the animals [4]. In contrast, in a sepsis model induced by a bolus injection of LPS, both, global blockade of IL-6 signaling by neutralizing monoclonal antibodies and selective blockade of IL-6 trans-signaling proved effective in preventing the death of the animals [5]. Also various infection models suggest a different outcome of global blockade of IL-6 as compared to selective IL-6 trans-signaling inhibition. We could show that the extent of IL-6 trans-signaling in chronic inflammatory diseases and cancer is controlled by the soluble IL-6R. Using the sgp130Fc protein or sgp130Fc transgenic mice we demonstrate that in several chronic inflammatory diseases and cancers including inflammatory bowel disease, peritonitis, rheumatoid arthritis, colon cancer, ovarian cancer and pancreatic cancer, that IL-6 trans-signaling via the soluble IL-6R is a crucial step in the development and the progression of the disease. Therefore, sgp130Fc is a novel therapeutic agent for the treatment of chronic inflammatory diseases and cancer [1,6-8].

Therapeutic Targeting of IL-6 Trans Signaling Counteracts STAT3 Control of Experimental Inflammatory Arthritis

Cytokine control of the synovial infiltrate is a central process in the development of inflammatory arthritis. In this study, we combine genetic approaches and intervention strategies to describe a fundamental requirement for IL-6-mediated STAT3 signaling in orchestrating the inflammatory infiltrate in monoarticular and systemic models of experimental arthritis. STAT3 activation via the common gp130 signal-transducing receptor for all IL-6-related cytokines led to increased retention of neutrophils and T cells within the inflamed synovium, which included STAT3-regulated IL-17A-secreting T cells. Control of leukocyte infiltration was reliant upon IL-6 signaling via its soluble receptor (termed IL-6 trans signaling), as evidenced by selective blockade of this alternative IL-6 signaling pathway using an engineered variant of soluble gp130 (sgp130Fc). This therapeutic intervention led to substantial clinical improvement in mice with emerging or established incidence of systemic arthritis. These data illustrate that IL-6 control of STAT3 is critical for regulating the synovial infiltrate in inflammatory arthritis, and suggest that selective inhibition of IL-6 trans signaling may provide a more refined intervention strategy for blocking IL-6-driven proarthritic activities.

Signal Transducer and Activator of Transcription 5b Promotes Mucosal Tolerance in Pediatric Crohn's Disease and Murine Colitis

Growth hormone (GH) regulates anabolic metabolism via activation of the STAT5b transcription factor and reduces mucosal inflammation in colitis. Peroxisome proliferator-activated receptor (PPAR) gamma suppresses mucosal inflammation and is regulated by GH through STAT5b. We hypothesized that the GH:STAT5b axis influences susceptibility to colitis via regulation of local PPARgamma abundance. Colon biopsies from children with newly diagnosed Crohn's disease (CD) and controls were exposed to GH in short-term organ culture. Trinitrobenzene sulfonic acid (TNBS) administration was used to induce colitis in STAT5b-deficient mice and wild-type controls, with and without rosiglitazone pretreatment. GH receptor, STAT5b, PPARgamma, and nuclear factor kappaB activation and expression were determined. Epithelial cell GH receptor expression and GH-dependent STAT5b activation and PPARgamma expression were reduced in CD colon. STAT5b-deficient mice exhibited reduced basal PPARgamma nuclear abundance and developed more severe proximal colitis after TNBS administration. This was associated with a significant increase in mucosal nuclear factor kappaB activation at baseline and after TNBS administration. Rosiglitazone ameliorated colitis in wild-type mice but not STAT5b-deficient mice. GH-dependent STAT5b activation is impaired in affected CD colon and contributes to chronic mucosal inflammation via down-regulation of local PPARgamma expression. Therapeutic activation of the GH:STAT5b axis therefore represents a novel target for restoring both normal anabolic metabolism and mucosal tolerance in CD.

TGF-β Suppresses Tumor Progression in Colon Cancer by Inhibition of IL-6 trans-Signaling

Alterations of TGF-β signaling have been described in colorectal cancer, although the molecular consequences are largely unknown. By using transgenic mice overexpressing TGF-β or a dominant-negative TGF-βRII, we demonstrate that TGF-β signaling in tumor infiltrating T lymphocytes controls the growth of dysplastic epithelial cells in experimental colorectal cancer, as determined by histology and a novel system for high-resolution chromoendoscopy. At the molecular level, TGF-β signaling in T cells regulated STAT-3 activation in tumor cells via IL-6. IL-6 signaling required tumor cell-derived soluble IL-6R rather than membrane bound IL-6R and suppression of such TGF-β-dependent IL-6 trans-signaling prevented tumor progression in vivo. Taken together, our data provide novel insights into TGF-β signaling in colorectal cancer and suggest novel therapeutic approaches for colorectal cancer based on inhibition of TGF-β-dependent IL-6 trans-signaling.