Interleukin-6 Trans-signaling Research Articles

ADAM17 Activity and IL-6 Trans-Signaling in Inflammation and Cancer.

All ligands of the epidermal growth factor receptor (EGF-R) are transmembrane proteins, which need to be proteolytically cleaved in order to be systemically active. The major protease responsible for this cleavage is the membrane metalloprotease ADAM17, which also has been implicated in cleavage of TNFα and interleukin-6 (IL-6) receptor. It has been recently shown that in the absence of ADAM17, the main protease for EGF-R ligand processing, colon cancer formation is largely abrogated. Intriguingly, colon cancer formation depends on EGF-R activity on myeloid cells rather than on intestinal epithelial cells. A major activity of EGF-R on myeloid cells is the stimulation of IL-6 synthesis. Subsequently, IL-6 together with the ADAM17 shed soluble IL-6 receptor acts on intestinal epithelial cells via IL-6 trans-signaling to induce colon cancer formation, which can be blocked by the inhibitor of IL-6 trans-signaling, sgp130Fc. Blockade of IL-6 trans-signaling therefore offers a new therapeutic window downstream of the EGF-R for the treatment of colon cancer and possibly of other EGF-R related neoplastic diseases.

1438MicroRNA editing is integral for interleukin-6 trans-signalling and leukocyte trafficking to ischemic tissues

Abstract Background/Aim Adenosine to inosine RNA editing is an essential post-transcriptional RNA modification catalysed by adenosine deaminase acting on RNA-1 and -2 (ADAR1; ADAR2). Endothelial cells (ECs) attract and guide leukocytes to sites of ischemic tissue injury. Here we studied the role of RNA editing in ischemic disease. Methods Primary human and murine vascular endothelial cell cultures were used to assess the EC responses to interleukin-6 (IL-6) or ischemia. For the animal studies, the effect of ADAR2 in acute and chronic ischemic disease was evaluated in cremaster muscle microcirculation by intravital microscopy, in peritoneal cavity after sterile peritonitis and in gastrocnemius muscle after hind-limb ischemia by 8-colour flow cytometry and immunohistochemistry (IHC) studies of Adar2−/−/tg as well as of i(nducible)EC-ADAR2 knockout (KO) mice. For the mechanistic studies, deep RNA sequencing, qRT-PCR, western blot, confocal microscopy, target-specific microRNA (miRNA) editing studies, RNA-immunoprecipitation, miRNA/plasmid silencing/overexpression and luciferase reporter assays were used among others. For human studies, ischemic tissues derived from patients with acute or chronic ischemic heart disease were processed. Results ADAR2, but not ADAR1, expression is induced by >2-fold in hypoxic ECs and in ischemic vascular ECs in mice and humans. Unbiased gene ontology analysis of the EC transcriptome indicated that ADAR2 controls inflammatory responses and predominantly the expression of interleukin-6-signal transducer (IL6ST), the co-receptor of IL-6. Subsequently, ADAR2 controls IL-6 trans-signalling in ECs as documented by the STAT3 phosphorylation and expression of the downstream leukocyte adhesion molecules, E-selectin and VCAM-1. IL-6-inflamed cremaster muscles showed that rolling and adhesion of leukocyte subsets to vascular wall were severely impaired in Adar2−/−/tg mice. Leukocyte transmigration was also diminished by >2-fold in Adar2−/−/tg and in iEC-ADAR2 KO mice in response to IL-6 or ischemia. Similar results were obtained for leukocyte rolling, adhesion and infiltration after acute (4h) and chronic (3d; 21d) ischemia from iEC-ADAR2 KO mice and human ischemic muscle tissues. Next we studied how ADAR2 controls IL6ST expression. ADAR2-deficient vascular EC miRNAome revealed the upregulation of a conserved group of miRNAs targeting the IL6ST mRNA including miR-199a-5p and miR-335-3p. At a single-nucleotide level, ADAR2-induced RNA editing of the stem loops of the primary miR-199a1/2 and miR-335 directly disrupted Drosha recruitment to both and thus inhibited their maturation process. Accordingly, rescue experiments using miRNA-inhibitors restored IL6ST levels after ADAR2 deficiency. Conclusion Taking together, inhibition of the microRNA maturation process by ADAR2-mediated RNA editing is integral for IL-6 trans-signalling in vascular endothelium and subsequent leukocyte trafficking to ischemic tissues in mice and humans.

IL-6 trans-signaling in the brain influences the behavioral and physio-pathological phenotype of the Tg2576 and 3xTgAD mouse models of Alzheimer’s disease

Alzheimer’s disease (AD) is the most commonly diagnosed dementia but its underlying pathological mechanisms still unclear. Neuroinflammation and secretion of cytokines such as interleukin-6 (IL-6) accompany the main hallmarks of the disease: amyloid plaques and neurofibrillary tangles. In this study, we analyzed the role of IL-6 trans-signaling in two mouse models of AD, Tg2576 and 3xTg-AD mice. The inhibition of IL-6 trans-signaling partially rescued the AD-induced mortality in females of both models. Before amyloid plaques deposition, it reversed AD-induced changes in exploration and anxiety (but did not affect locomotion) in Tg2576 female mice. However, after plaque deposition the only behavioral trait affected by the inhibition of IL-6 trans-signaling was locomotion. Results in the Morris water maze suggest that cognitive flexibility was reduced by the blocking of the IL-6 trans-signaling in young and old Tg2576 female mice. The inhibition of IL-6 trans-signaling also decreased amyloid plaque burden in cortex and hippocampus, and Aβ40 and Aβ42 levels in the cortex, of Tg2576 female mice. The aforementioned changes might be correlated with changes in blood vessels and matrix structure and organization rather than changes in neuroinflammation. 3xTgAD mice showed a very mild phenotype regarding amyloid cascade, but results were in accordance with those of Tg2576 mice. These results strongly suggest that the inhibition of the IL-6 trans-signaling could represent a powerful therapeutic target in AD.

Novel Treatment Targets Based on Insights in the Etiology of Depression: Role of IL-6 Trans-Signaling and Stress-Induced Elevation of Glutamate and ATP.

Inflammation and psychological stress are risk factors for major depression and suicide. Both increase central glutamate levels and activate the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system. Both factors also affect the function of the chloride transporters, Na-K-Cl-cotransporter-1 (NKCC1) and K-Cl-cotransporter-2 (KCC2), and provoke interleukin-6 (IL-6) trans-signaling. This leads to measurable increases in circulating corticosteroids, catecholamines, anxiety, somatic and psychological symptoms, and a decline in cognitive functions. Recognition of the sequence of pathological events allows the prediction of novel targets for therapeutic intervention. Amongst others, these include blockade of the big-K potassium channel, blockade of the P2X4 channel, TYK2-kinase inhibition, noradrenaline α2B-receptor antagonism, nicotinic α7-receptor stimulation, and the Sgp130Fc antibody. A better understanding of downstream processes evoked by inflammation and stress also allows suggestions for tentatively better biomarkers (e.g., SERPINA3N, MARCKS, or 13C-tryptophan metabolism).

Interleukin-6 (IL-6) mediates protection against glucose toxicity in human Müller cells via activation of VEGF-A signaling

Interleukin-6 (IL-6) has become a target of interest for drug development aiming to treat diabetic retinopathy. Since IL-6 signaling can promote beneficial as well as detrimental effects via two different signaling pathways, the objective of the present study was to investigate the effects of classical IL-6 and IL-6 trans-signaling on human Müller cells (HMC), which are important for the development of diabetic retinopathy. HMCs were cultured in normal (5 mmol/L) and high (25 mmol/L) glucose plus or minus IL-6 or IL-6/sIL-6R. IL-6 receptor expression using immunohistochemistry and flow cytometry and cytokine release using magnetic bead assays were determined. HMCs express the membrane bound form of the IL-6 receptor (mIL-6R), gp130, and can release the soluble forms sIL-6R and sgp130 demonstrating that HMCs are capable of responding to classical IL-6 and IL-6 trans-signaling. IL-6 protected HMCs from glucose toxicity via VEGF-A signaling. IL-6/sIL-6R caused only modest protection, which was not mediated by VEGF-A. Our data show for the first time that classical IL-6 signaling exerts its beneficial effects through VEGF-A action contrary to IL-6 trans-signaling, which was VEGF-A independent. These results have clinical implications for drug development targeting IL-6 since strict anti-IL-6 therapies might further decrease neuroretinal functions in the diabetic retina.

Interleukin 6 Dependent Synaptic Plasticity in a Social Defeat-Susceptible Prefrontal Cortex Circuit

The role of the pro-inflammatory cytokine interleukin-6 (IL-6) in the etiology of stress-induced synaptic plasticity is yet unknown. We took advantage of a genetically modified mouse (TG) in which IL-6 trans-signaling via the soluble IL-6 receptor was blocked, to determine the role of IL-6 trans-signaling in the effects of a Social Defeat protocol (SD) on synaptic function of the medial prefrontal cortex (mPFC). Synaptic function in stress-sensitive (S) and stress-resilient (R) animals was studied in a mPFC slice preparation with whole-cell patch-clamp recording. SD altered numerous synaptic properties of the mPFC: R WT (but not TG) displayed a decreased ratio between N methyl-D-aspartate receptor (NMDAR-) dependent and amino propionic acid receptor (AMPAR-) dependent-current (INMDA/IAMPA), while S WT animals (but not TG) showed a reduced ratio between AMPA and γ-amino-butyric acid receptor type A (GABAAR)-dependent currents (IAMPA/IGABA). Also, SD induced an increase in the frequency but a decrease in the amplitude of excitatory action-potential dependent PSCs (sEPSCs), both in an IL-6 dependent manner, as well as a generalized (S/R-independent) decrease in the frequency of action potential independent (miniature) excitatory (IL-6 dependent) as well as inhibitory (IL-6 independent) postsynaptic current frequency. Interestingly, corner preference (measuring the intensity of social defeat) correlated positively with INMDA/IAMPA and eEPSC frequency and negatively with IAMPA/IGABA. Our results suggest that SD induces behaviorally-relevant synaptic rearrangement in mPFC circuits, part of which is IL-6 dependent. In particular, IL-6 is necessary to produce synaptic plasticity leading to stress resilience in some individuals, but to stress sensitivity in others.

IL-6 Trans-signaling Controls Liver Regeneration After Partial Hepatectomy.

Interleukin-6 (IL-6) is critically involved in liver regeneration after partial hepatectomy (PHX). Previous reports suggest that IL-6 trans-signaling through the soluble IL-6/IL-6R complex is involved in this process. However, the long-term contribution of IL-6 trans-signaling for liver regeneration after PHX is unknown. PHX-induced generation of the soluble IL-6R by ADAM (a disintegrin and metallo) proteases enables IL-6 trans-signaling, in which IL-6 forms an agonistic complex with the soluble IL-6 receptor (sIL-6R) to activate all cells expressing the signal-transducing receptor chain glycoprotein 130 (gp130). In contrast, without activation of ADAM proteases, IL-6 in complex with membrane-bound IL-6R and gp130 activates classic signaling. Here, we describe the generation of IL-6 trans-signaling mice, which exhibit boosted IL-6 trans-signaling and abrogated classic signaling by genetic conversion of all membrane-bound IL-6R into sIL-6R proteins phenocopying hyperactivation of ADAM-mediated shedding of IL-6R as single substrate. Importantly, although IL-6R deficient mice were strongly affected by PHX, survival and regeneration of IL-6 trans-signaling mice was indistinguishable from control mice, demonstrating that IL-6 trans-signaling fully compensates for disabled classic signaling in liver regeneration after PHX. Moreover, we monitored the long-term consequences of global IL-6 signaling inhibition versus IL-6 trans-signaling selective blockade after PHX by IL-6 monoclonal antibodies and soluble glycoprotein 130 as fragment crystallizable fusion, respectively. Both global IL-6 blockade and selective inhibition of IL-6 trans-signaling results in a strong decrease of overall survival after PHX, accompanied by decreased signal transducer and activator of transcription 3 phosphorylation and proliferation of hepatocytes. Mechanistically, IL-6 trans-signaling induces hepatocyte growth factor production by hepatic stellate cells. Conclusion: IL-6 trans-signaling, but not classic signaling, controls liver regeneration following PHX.

Relationship of Interleukin-6 Trans-Signaling and Coronary Heart Disease in Young Adults: a Case-Control Study in Indonesia

Interleukin-6 (IL-6) has been reported to be related to coronary heart disease (CHD). It is proposed that the IL-6 trans-signaling pathway is responsible for the inflammatory effect in diseases, including CHD. In Asian countries, CHD tends to occur in younger age. However, no study has yet been done to assess the relationship between IL-6 trans-signaling pathway and young CHD patients in Indonesia. To assess whether there is a relationship between the levels of some components in the IL-6 trans-signaling pathway, including soluble interleukin-6 receptor (sIL-6R), soluble glycoprotein 130 (sgp130), and intercellular adhesion molecule 1 (ICAM-1) and CHD in young adults. A case-control study was conducted including 33 young CHD patients and 33 non-CHD patients as the control group (age and sex matched with CHD group) at Eka Hospital Pekanbaru, Indonesia, from July to November 2018. CHD was confirmed by coronary angiography, while non-CHD patients were subjects with normal ECG, without history of chest pain and family history of CHD. All participants were checked for sIL-6R, sgp130, and ICAM-1 serum levels using ELISA assays tests. The results were evaluated statistically using Student’s t test. The sIL-6R level tended to be higher in the CHD group compared to the control group (70.19+49.38 ng/ml vs 49.42+38.79 ng/ml) but did not reach statistical significance (p=0.062). The sgp130 level was 428.38+358.79 ng/ml and 474.08+389.43 ng/ml in CHD and control group, respectively (p=0.622). While the ICAM-1 level was 1829.53+1882.37 pg/ml and 2078.16+1595.25 pg/ml in CHD and control group, respectively (p=0.565). The IL-6 trans-signaling pathway, reflected by sIL-6R, sgp130, and ICAM-1 serum levels, was not significantly related with CHD in young adults.

IL-6 exhibits both cis- and trans-signaling in osteocytes and osteoblasts, but only trans-signaling promotes bone formation and osteoclastogenesis

Interleukin 6 (IL-6) supports development of bone-resorbing osteoclasts by acting early in the osteoblast lineage via membrane-bound (cis) or soluble (trans) receptors. Here, we investigated how IL-6 signals and modifies gene expression in differentiated osteoblasts and osteocytes and determined whether these activities can promote bone formation or support osteoclastogenesis. Moreover, we used a genetically altered mouse with circulating levels of the pharmacological IL-6 trans-signaling inhibitor sgp130-Fc to determine whether IL-6 trans-signaling is required for normal bone growth and remodeling. We found that IL-6 increases suppressor of cytokine signaling 3 (Socs3) and CCAAT enhancer-binding protein δ (Cebpd) mRNA levels and promotes signal transducer and activator of transcription 3 (STAT3) phosphorylation by both cis- and trans-signaling in cultured osteocytes. In contrast, RANKL (Tnfsf11) mRNA levels were elevated only by trans-signaling. Furthermore, we observed soluble IL-6 receptor release and ADAM metallopeptidase domain 17 (ADAM17) sheddase expression by osteocytes. Despite the observation that IL-6 cis-signaling occurs, IL-6 stimulated bone formation in vivo only via trans-signaling. Although IL-6 stimulated RANKL (Tnfsf11) mRNA in osteocytes, these cells did not support osteoclast formation in response to IL-6 alone; binucleated TRAP+ cells formed, and only in response to trans-signaling. Finally, pharmacological, sgp130-Fc-mediated inhibition of IL-6 trans-signaling did not impair bone growth or remodeling unless mice had circulating sgp130-Fc levels > 10 μg/ml. At those levels, osteopenia and impaired bone growth occurred, reducing bone strength. We conclude that high sgp130-Fc levels may have detrimental off-target effects on the skeleton.

Upregulation of interleukin-6 on Cav3.2 T-type calcium channels in dorsal root ganglion neurons contributes to neuropathic pain in rats with spinal nerve ligation

The T-type calcium channels Cav3.2, one of the low voltage-activated (LVA) calcium channels, have been found to play important roles in the neuronal excitability. Recently, we and others have demonstrated that accumulation of Cav3.2 channels in the dorsal root ganglion (DRG) neurons and sensory nerves contributes to neuropathic pain after peripheral nerve injury. In the present study, we aimed to further investigate the regulation of Cav3.2 channels by interleukin-6 (IL-6) in DRG neurons in neuropathic pain rats after spinal nerve ligation (SNL). The results showed that Cav3.2 channel protein expression in L5 DRG neurons was upregulated and blockade of this channel decreased the hyperexcitability of DRG neurons and mechanical allodynia in SNL neuropathic pain rats. Furthermore, inhibition of IL-6 trans-signaling reduced the upregulation of Cav3.2 T-type channel induced by FIL-6 (a fusion protein of IL-6 and sIL-6R) in primary cultured DRG neurons in vitro. In vivo, inhibition of IL-6 trans-signaling reversed the upregulation of Cav3.2, reduced the hyperexcitability of L5 DRG neurons and alleviated mechanical allodynia in SNL rats. Our results suggest that IL-6 upregulates Cav3.2 T-type channels expression and function through the IL-6/sIL-6R trans-signaling pathway in DRG neurons, thus contributes to the development of neuropathic pain in SNL rats.

Blocking interleukin-6 trans-signaling protects against renal fibrosis by suppressing STAT3 activation.

Rationale: Renal fibrosis is the terminal manifestation of chronic and irreversible renal disease. Effective therapies other than dialysis are extremely limited. In this study, we investigated the potential effects of targeting elevated interleukin-6 (IL-6) levels in the treatment of renal fibrosis.Methods: Fc-gp130 was used to specifically block IL-6 trans-signaling. Unilateral ureteral occlusion (UUO) and ischemia reperfusion (IR) mouse models were constructed to investigate the therapeutic effect of Fc-gp130 on renal fibrosis. The role of IL-6 trans-signaling and phosphorylation of signal transducer and activator of transcription (STAT) 3 in regulating fibroblast accumulation and extracellular matrix protein deposition were evaluated in cell experiments and mouse models.Results: The kidneys of mice with UUO were found to have elevated soluble IL-6 receptor (sIL-6R) levels in the progression of fibrosis. Fc-gp130 attenuated renal fibrosis in mice, as evidenced by reductions in tubular atrophy and the production of extracellular matrix protein. Blockade of IL-6 trans-signaling with Fc-gp130 also reduced inflammation levels, immune cell infiltration, and profibrotic cytokines expression in renal tissue, with decreased STAT3 phosphorylation and reduced fibroblast accumulation in the renal tissue. In vitro, Fc-gp130 also reduced the phosphorylation of STAT3 induced by transforming growth factor (TGF)-β1 in fibroblasts. Furthermore, the therapeutic effect of Fc-gp130 was confirmed in a model of acute kidney injury-chronic kidney disease.Conclusion: Overall, IL-6 trans-signaling may contribute to crucial events in the development of renal fibrosis, and the targeting of IL-6 trans-signaling by Fc-gp130 may provide a novel therapeutic strategy for the treatment of renal fibrosis.

Effects of Interleukin-6 trans-signalling in a mouse model of Alzheimer's disease and high fat diet-induced obesity

Effects of Interleukin-6 trans-signalling in a mouse model of Alzheimer's disease and high fat diet-induced obesity

РОЛЬ ИНТЕРЛЕЙКИН-6 СИГНАЛИНГА В РАЗВИТИИ СИСТЕМНОГО ВОСПАЛЕНИЯ ПРИ ХРОНИЧЕСКОЙ ОБСТРУКТИВНОЙ БОЛЕЗНИ ЛЕГКИХ

The review focuses on the role of interleukin-6 (IL-6) signaling in the development of a systemic inflammatory process in chronic obstructive pulmonary disease (COPD). In most researches the attention is paid to local inflammation in COPD. However, it is known that the pathology is characterized by a systemic inflammatory process, which is manifested in the increased levels of proinflammatory mediators in blood flow, and the study of the molecular mechanisms of its development is very important for the therapy of the disease. One of the key mediators of systemic inflammation is cytokine IL-6 which has pro- and antiinflammatory properties. Its effect on the cells is determined by the type of signaling. Nowadays three types of IL-6 signaling are identified: transsignaling, classical and cluster signaling. The review presents the known pathophysiological mechanisms of the development of systemic inflammation in COPD involving IL-6. As a proinflammatory cytokine, IL-6 performs the following functions: transmission of a signal on lung tissue damage, initiation of leukocyte migration into the inflammation site, inhibition of T-cell apoptosis into the inflammation site, influence on T helper differentiation, participation in pathophysiological reactions of development of emphysema and fibrosis. The significance of IL-6 transsignaling for the development of inflammation in COPD has been confirmed by many studies, while there are practically no works devoted to the study of classical IL-6 signaling in COPD. The data presented in the review indicate the need for further study of the role of different types of IL-6 signaling, especially classical signaling, in the regulation of systemic inflammation in COPD.

Inhibition of interleukin-6 trans-signaling prevents inflammation and endothelial barrier disruption in retinal endothelial cells

Vascular inflammation plays a critical role in the pathogenesis of diabetic retinopathy. Recently, Interleukin-6 (IL-6) trans-signaling via soluble IL-6 receptor (sIL-6R) has emerged as a prominent regulator of inflammation in endothelial cells. This study was designed to test the hypothesis that selective inhibition of the IL-6 trans-signaling pathway will attenuate inflammation and subsequent barrier disruption in retinal endothelial cells. Human retinal endothelial cells (HRECs) were exposed to IL-6 and sIL-6R to induce IL-6 trans-signaling and the commercially available compound sgp130Fc (soluble gp-130 fused chimera) was used to selectively inhibit IL-6 trans-signaling. IL-6 trans-signaling activation caused a significant increase in STAT3 phosphorylation, expression of adhesion molecules, ROS production and apoptosis in HRECs whereas a significant decrease in mitochondrial membrane potential and NO production was observed in IL-6 trans-signaling activated cells. These changes were not observed in cells pre-treated with sgp130Fc. IL-6 trans-signaling activation was sufficient to cause barrier disruption in endothelial monolayers and pre-treatment of HRECs with sgp130Fc, maintained endothelial barrier function similar to that of untreated cells. Thus, in conclusion, these results indicate that IL-6 trans-signaling is an important mediator of inflammation, apoptosis and barrier disruptive effects in the retinal endothelial cells and inhibition of the IL-6 trans-signaling pathway using sgp130-Fc attenuates vascular inflammation and endothelial barrier disruption.

Abstract 549: Interleukin-6 Trans-Signaling in Acute Myocardial Infarction in Male BALB/c Mice

Interleukin-6 (IL-6) is a pleiotropic cytokine historically associated with development and/or maintenance of cardiovascular disease (CVD). Despite this long-standing association, the discrete roles of IL-6 on the pathogenesis or severity of CVD and notably acute myocardial infarction (AMI) remain unclear. Dogma contends that IL-6 mediates effects largely through direct interaction with a membrane-bound IL-6 receptor (IL-6R) on leukocytes and hepatocytes. Intriguingly, identification of a soluble receptor for IL-6 (sIL-6R) has invigorated study into a regulatory role of IL-6 trans-signaling (IL-6trans) in CVD pathogenesis. IL-6trans provokes signal transduction in cells lacking the membrane-bound receptor, including notably cardiomyocytes, and ultimately activates STAT3 gene transcription and stabilizes mitochondrial function, thereby promoting cardioprotection. In this study we hypothesized that IL-6trans plays a crucial role in the injury response to AMI by increasing circulating IL-6 and sIL-6R and inducing cardioprotection via STAT3. To test this hypothesis, male BALB/c mice were subjected to acute ischemia (30 min) of the LAD coronary artery (to mimic AMI) with/without reperfusion and with/without inclusion of a pre- or post-ischemic conditioning event in order to determine regulatory involvement of IL-6trans in acute cardioprotection. Following surgery, ischemic regions of left ventricle (LV) and sera were analyzed for IL-6, sIL-6R, and activated (phosphorylated) STAT3. Results show that AMI led to increases in serum IL-6 (p=0.0053) and sIL-6R (p=0.0023) as well as in LV phosphorylated (Tyr705) STAT3 (p=0.0059) compared to naïve LV tissues. Preliminary data from ischemic pre- and post-conditioning regimens were independently associated with increases in IL-6trans demonstrated by trending increases in LV STAT3 phosphorylation (Ser727, Tyr705) compared to naïve controls. These inclusive data on IL-6trans suggest it serves a biologically significant intrinsic role in mediating acute protection following AMI. We anticipate that continued investigation will firmly establish IL-6trans as a unique protective pathway that could be targeted in order to improve morbidity and mortality of AMI patients.

Interleukin 6 trans-signalling and risk of future cardiovascular events.

The pro-inflammatory response to interleukin 6 (IL6) trans-signalling in atherosclerosis is driven by the IL6 and soluble IL6 receptor (sIL6R) binary complex. The binary IL6:sIL6R complex is inactivated by sgp130 through the formation of the ternary IL6:sIL6R:sgp130 complex. The aim of this study was to investigate if IL6 trans-signalling, estimated by a ratio between the binary and ternary complexes, associates with the risk of future cardiovascular events (CVE) in a Swedish cohort of 60-year-old men and women (n = 4232). Binary and ternary complex levels expressed in nanomol/Litre were derived from serum concentrations of IL6, sIL6R, and sgp130. Cox regression models were used to assess the risk of CVE (myocardial infarction, angina pectoris, and ischaemic stroke, n = 525), expressed as hazard ratio (HR) with 95% confidence interval (CI), associated with increasing circulating levels of the three molecules and with the binary/ternary complex ratio. Estimates were adjusted for the common cardiovascular (CV) risk factors. To assess the level of IL6-trans-signalling, we estimated the binary/ternary complex ratio and then analysed the association with CVE risk. A ratio higher than the median, representing a relative excess of the active binary complex was associated with increased CVE risk (adjusted HR 1.44, 95% CI 1.21-1.72). The ratio between the functional moieties of IL6 trans-signalling, IL6:sIL6R, and IL6:sIL6R:sgp130, was associated with CVE risk indicating that it could be a promising marker of CV risk and possibly be used in selecting patients for anti-inflammatory therapy.

Abstract 5611: Inhibition of IL-6 trans-signaling in HNSCC

Abstract Various types of cancers including head and neck squamous cell carcinomas (HNSCCs) are highly inflammatory, and the cytokine interleukin-6 (IL-6) is believed to be associated with this inflammatory phenotype. Unfortunately, global inhibition of IL-6 signaling has demonstrated little to no clinical efficacy in various types of cancers. Previous studies in the field have reported major differences between IL-6 classical signaling through the membrane bound IL-6R (mIL-6R) and IL-6 trans-signaling through the soluble IL-6R (sIL-6R), and the idea that these 2 pathways may have vastly different functions. Recent studies in models of inflammation have proposed that IL-6 classical signaling serves an anti-inflammatory/homeostatic role while IL-6 trans-signaling stimulates proinflammatory events. Therefore, IL-6 trans-signaling (and not classical signaling) may be associated with the proinflammatory phenotype observed in HNSCCs and may also be responsible for the increased tumor progression and treatment resistance frequently encountered with this disease. The goal of this work is to investigate if selective inhibition of IL-6 trans-signaling will demonstrate superior antitumor efficacy compared to global inhibition of IL-6 signaling in HNSCC cells and if selective inhibition of IL-6 trans-signaling will enhance the antitumor efficacy of chemo/radiotherapy in HNSCC cells. We observed that the HNSCC cell lines SQ20B, SCC-25, FaDu and Cal-27 all express mIL-6R and secrete IL-6, sIL-6R, and the natural inhibitor of sIL-6R signaling–soluble glycoprotein 130 (sgp130)–at varying levels. Simultaneous inhibition of IL-6 classical and trans-signaling using the IL-6R antagonist tocilizumab, and selective inhibition of IL-6 trans-signaling using the extracellular portion of gp130 fused to the Fc portion of human IgG1 (sgp130-Fc), showed no significant effects on cell survival in vitro. However, sgp130-Fc significantly suppressed SQ20B tumor growth in athymic nude mice while tocilizumab had no effect. Significant antitumor effects of sgp130-Fc were also observed in a murine SCCVII/C3H syngeneic mouse model. Cell lines treated with cisplatin and/or X-ray radiation (0-4 Gy) increased IL-6, sIL-6R and sgp130 in a dose-dependent manner and sgp130-Fc significantly increased SQ20B tumor response to chemo/radiotherapy in vivo. Altogether, this work suggests that selective inhibition of IL-6 trans-signaling using sgp130-Fc may be more effective than global IL-6 inhibition with respect to suppressing HNSCC tumor growth and warrants further study as an adjuvant to standard HNSCC therapy. Citation Format: Andrean L. Simons, Rachel A. Dahl, Madelyn Espinosa-Cotton, Samuel N. Rodman, Stefan Rose-John. Inhibition of IL-6 trans-signaling in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5611.

Paradoxical Relationship Between Glycated Hemoglobin and Longitudinal Change in Physical Functioning in Older Adults: A Prospective Cohort Study.

Hyperglycemia with high hemoglobin A1c (HbA1c) levels is associated with significant health risks. However, the relationship between HbA1c levels and the physical functioning status in later life remains uncertain and so is the possible underlying mechanism. We conducted a prospective study of 2,565 initially well-functioning community-dwelling older adult aged 55 years and older from the Healthy Aging Longitudinal Study in Taiwan. Each participant received baseline measurements of blood HbA1c and inflammatory markers levels and repeated assessments of physical functioning over a mean follow-up period of 5.3 years. We used generalized linear mixed-effects regression to estimate the adjusted changes in the odds ratio for self-reported physical functioning impairment and Short Physical Performance Battery (SPPB) score according to baseline HbA1c levels (categorized into 0.5% increments from <5.5% to ≥7.0%). HbA1c levels showed a U-shaped relationship with changes in the odds ratio for physical functioning impairment and SPPB score (p for quadratic term < .001). Compared with participants with an HbA1c of 5.5% to <6.0%, those with an HbA1c of <5.5% or ≥7.0% had a higher annual increase in the odds ratio for physical functioning impairment (odds ratio [95% confidence interval] per year, 1.25 [1.04-1.50] and 1.21 [1.04-1.41]) and a higher annualized decrease in SPPB score (coefficient [95% confidence interval], -0.05 [-0.10 to 0.00] and -0.04 [-0.08 to 0.00]). These relationships were nonlinear only in participants with high soluble interleukin-6 receptor levels (>48,124 pg/mL; p for interaction < .05). High and low HbA1c levels at baseline are associated with faster physical functioning decline, particularly among individuals with elevated circulating soluble interleukin-6 receptor, a sign of enhanced interleukin-6 trans-signaling.

Interleukin-6: designing specific therapeutics for a complex cytokine.

Interleukin-6 (IL-6) is a pivotal cytokine with a diverse repertoire of physiological functions that include regulation of immune cell proliferation and differentiation. Dysregulation of IL-6 signalling is associated with inflammatory and lymphoproliferative disorders such as rheumatoid arthritis and Castleman disease, and several classes of therapeutics have been developed that target components of the IL-6 signalling pathway. So far, monoclonal antibodies against IL-6 or IL-6 receptor (IL-6R) and Janus kinases (JAK) inhibitors have been successfully developed for the treatment of autoimmune diseases such as rheumatoid arthritis. However, clinical trials of agents targeting IL-6 signalling have also raised questions about the diseases and patient populations for which such agents have an appropriate benefit-risk profile. Knowledge from clinical trials and advances in our understanding of the complexities of IL-6 signalling, including the potential to target an IL-6 trans-signalling pathway, are now indicating novel opportunities for therapeutic intervention. In this Review, we overview the roles of IL-6 in health and disease and analyse progress with several approaches of inhibiting IL-6-signalling, with the aim of illuminating when and how to apply IL-6 blockade.

DISTINCT IL-6 SIGNALING PATHWAYS DRIVE ALTERNATE PATHOGENIC T CELL DIFFERENTIATION AND GVHD AFTER TRANSPLANT IN VIVO

Abstract Introduction Dysregulation of interleukin 6 (IL-6) is pivotal for the development of graft-versus-host-disease (GVHD) after allogeneic stem cell transplantation. IL-6 drives many protective as well as inflammatory effects, the latter involving differentiation of T cells into pathogenic Th22, Th17, and Tc17 subsets. IL-6 signals through three principal pathways; classical, trans, and cluster, but their contribution to T cell differentiation and GVHD remains unclear. Importantly, these pathways are differentially targeted by clinical IL-6 inhibitors. Aims To dissect the contribution of the IL-6 signaling pathways to T cell differentiation and GVHD in order to guide therapy. Methods Donor bone marrow and T cells were transplanted from lineage-specific Cre × IL-6fl/flor IL-6Rfl/fl mice into irradiated, MHC disparate, wild-type or sgp130:Fc transgenic recipient mice with or without IL-6R mAb. Donor T cell differentiation and GVHD was assessed after transplant. Results Surprisingly, IL-6 trans-signaling was not required for T cell differentiation and GVHD, but instead regulated Th22 cell development. Indeed, inhibition of IL-6 trans-signaling in sgp130:Fc recipients promoted the expansion of Th22 cells and generated severe cutaneous GVHD. In contrast, IL-6 classical-signaling was critical for the development of Th22 and Th17 cells and its inhibition significantly attenuated GVHD broadly. Interestingly, unlike Th17 differentiation, Tc17 differentiation was highly IL-6-dependent but independent of classical and trans-signaling pathways, consistent with induction by cluster-signaling. Conclusion These data highlight cluster, in addition to classical IL-6-signalling as key therapeutic targets.