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Cytokine release syndrome (CRS), commonly known as cytokine storm, is an acute systemic inflammatory response that is a significant global health threat. Interleukin-6 (IL-6) and interleukin-1 (IL-1) are key pro-inflammatory cytokines involved in CRS and are hence critical therapeutic targets. Current antagonists, such as tocilizumab and anakinra, target IL-6R/IL-1R but have limitations due to their long half-life and systemic anti-inflammatory effects, making them less suitable for acute or localized treatments. Here we present the de novo design of small protein antagonists that prevent IL-1 and IL-6 from interacting with their receptors to activate signaling. The designed proteins bind to the IL-6R, GP130 (an IL-6 co-receptor), and IL-1R1 receptor subunits with binding affinities in the picomolar to low-nanomolar range. X-ray crystallography studies reveal that the structures of these antagonists closely match their computational design models. In a human cardiac organoid disease model, the IL-1R antagonists demonstrated protective effects against inflammation and cardiac damage induced by IL-1β. These minibinders show promise for administration via subcutaneous injection or intranasal/inhaled routes to mitigate acute cytokine storm effects. Here, the authors computationally designed and produced small protein antagonists to target IL-6 and IL-1β signaling to develop modulators of CRS.

Crohn’s disease is a chronic inflammatory intestinal disease that is frequently accompanied by aberrant healing and stricturing complications. Crosstalk between activated myeloid and stromal cells is critical in the pathogenicity of Crohn’s disease 1 , 2 , and increases in intravasating monocytes are correlated with a lack of response to anti-TNF treatment 3 . The risk alleles with the highest effect on Crohn’s disease are loss-of-function mutations in NOD2 4 , 5 , which increase the risk of stricturing 6 . However, the mechanisms that underlie pathogenicity driven by NOD2 mutations and the pathways that might rescue a lack of response to anti-TNF treatment remain largely uncharacterized. Here we use direct ex vivo analyses of patients who carry risk alleles of NOD2 to show that loss of NOD2 leads to dysregulated homeostasis of activated fibroblasts and macrophages. CD14 + peripheral blood mononuclear cells from carriers of NOD2 risk alleles produce cells that express high levels of collagen, and elevation of conserved signatures is observed in nod2- deficient zebrafish models of intestinal injury. The enrichment of STAT3 regulation and gp130 ligands in activated fibroblasts and macrophages suggested that gp130 blockade might rescue the activated program in NOD2-deficient cells. We show that post-treatment induction of the STAT3 pathway is correlated with a lack of response to anti-TNF treatment in patients, and demonstrate in vivo in zebrafish the amelioration of the activated myeloid–stromal niche using the specific gp130 inhibitor bazedoxifene. Our results provide insights into NOD2-driven fibrosis in Crohn’s disease, and suggest that gp130 blockade may benefit some patients with Crohn’s disease—potentially as a complement to anti-TNF therapy. NOD2 deficiency drives fibrosis and stricturing complications in Crohn’s disease through dysregulated homeostasis of activated fibroblasts and macrophages, which is ameliorated by gp130 blockade in human cell and zebrafish models.

Background Sepsis and inflammation can cause intensive care unit‐acquired weakness (ICUAW). Increased interleukin‐6 (IL‐6) plasma levels are a risk factor for ICUAW. IL‐6 signalling involves the glycoprotein 130 (gp130) receptor and the JAK/STAT‐pathway, but its role in sepsis‐induced muscle wasting is uncertain. In a clinical observational study, we found that the IL‐6 target gene, SOCS3, was increased in skeletal muscle of ICUAW patients indicative for JAK/STAT‐pathway activation. We tested the hypothesis that the IL‐6/gp130‐pathway mediates ICUAW muscle atrophy. Methods We sequenced RNA (RNAseq) from tibialis anterior (TA) muscle of cecal ligation and puncture‐operated (CLP) and sham‐operated wildtype (WT) mice. The effects of the IL‐6/gp130/JAK2/STAT3‐pathway were investigated by analysing the atrophy phenotype, gene expression, and protein contents of C2C12 myotubes. Mice lacking Il6st, encoding gp130, in myocytes (cKO) and WT controls, as well as mice treated with the JAK2 inhibitor AG490 or vehicle were exposed to CLP or sham surgery for 24 or 96 h. Results Analyses of differentially expressed genes in RNAseq (≥2‐log2‐fold change, P < 0.01) revealed an activation of IL‐6‐signalling and JAK/STAT‐signalling pathways in muscle of septic mice, which occurred after 24 h and lasted at least for 96 h during sepsis. IL‐6 treatment of C2C12 myotubes induced STAT3 phosphorylation (three‐fold, P < 0.01) and Socs3 mRNA expression (3.1‐fold, P < 0.01) and caused myotube atrophy. Knockdown of Il6st diminished IL‐6‐induced STAT3 phosphorylation (−30.0%; P < 0.01), Socs3 mRNA expression, and myotube atrophy. JAK2 (− 29.0%; P < 0.01) or STAT3 inhibition (−38.7%; P < 0.05) decreased IL‐6‐induced Socs3 mRNA expression. Treatment with either inhibitor attenuated myotube atrophy in response to IL‐6. CLP‐operated septic mice showed an increased STAT3 phosphorylation and Socs3 mRNA expression in TA muscle, which was reduced in septic Il6st‐cKO mice by 67.8% (P < 0.05) and 85.6% (P < 0.001), respectively. CLP caused a loss of TA muscle weight, which was attenuated in Il6st‐cKO mice (WT: −22.3%, P < 0.001, cKO: −13.5%, P < 0.001; WT vs. cKO P < 0.001). While loss of Il6st resulted in a reduction of MuRF1 protein contents, Atrogin‐1 remained unchanged between septic WT and cKO mice. mRNA expression of Trim63/MuRF1 and Fbxo32/Atrogin‐1 were unaltered between CLP‐treated WT and cKO mice. AG490 treatment reduced STAT3 phosphorylation (−22.2%, P < 0.05) and attenuated TA muscle atrophy in septic mice (29.6% relative reduction of muscle weight loss, P < 0.05). The reduction in muscle atrophy was accompanied by a reduction in Fbxo32/Atrogin‐1‐mRNA (−81.3%, P < 0.05) and Trim63/MuRF1‐mRNA expression (−77.6%, P < 0.05) and protein content. Conclusions IL‐6 via the gp130/JAK2/STAT3‐pathway mediates sepsis‐induced muscle atrophy possibly contributing to ICUAW.

Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer. Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3. Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion. gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation. This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function. This study demonstrates the activation of a STAT3-independent healing pathway in response to mucosal injury which involves the co-receptor for IL-6 cytokines gp130 and downstream effectors Src, Yes, YAP and Notch. Beneficial effects of inflammation As well as activating innate and adaptive immunity, inflammation triggers tissue repair and regeneration through mechanisms that are largely unknown. This study demonstrates the activation of a STAT3-independent healing pathway in a mouse model of experimental colitis through a process involving gp130 (a co-receptor for interleukin-6 family cytokines) and the downstream effectors Src, Yes, YAP and Notch.

The activation of STAT3 by tyrosine phosphorylation, essential for normal development and for a normal inflammatory response to invading pathogens, is kept in check by negative regulators. Abnormal constitutive activation of STAT3, which contributes to the pathology of cancer and to chronic inflammatory diseases such as rheumatoid arthritis, occurs when negative regulation is not fully effective. SOCS3, the major negative regulator of STAT3, is induced by tyrosine-phosphorylated STAT3 and terminates STAT3 phosphorylation about 2 h after initial exposure of cells to members of the IL-6 family of cytokines by binding cooperatively to the common receptor subunit gp130 and JAKs 1 and 2. We show here that when the epidermal growth factor receptor (EGFR) is present and active, STAT3 is rephosphorylated about 4 h after exposure of cells to IL-6 or oncostatin M and remains active for many hours. Newly synthesized IL-6 drives association of the IL-6 receptor and gp130 with EGFR, leading to EGFR-dependent rephosphorylation of STAT3, which is not inhibited by the continued presence of SOCS3. This second wave of STAT3 activation supports sustained expression of a subset of IL-6-induced proteins, several of which play important roles in inflammation and cancer, in which both IL-6 secretion and EGFR levels are often elevated.

The IL-6 family of cytokines consists of IL-6, IL-11, IL-27, IL-31, oncostatin M (OSM), leukaemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophin 1 (CT-1) and cardiotrophin-like cytokine factor 1 (CLCF1). Membership of this cytokine family is defined by usage of common β-receptor signalling subunits, which activate various intracellular signalling pathways. Each IL-6 family member elicits responses essential to the physiological control of immune homeostasis, haematopoiesis, inflammation, development and metabolism. Accordingly, distortion of these cytokine activities often promotes chronic disease and cancer; the pathological importance of this is exemplified by the successful treatment of certain autoimmune conditions with drugs that target the IL-6 pathway. Here, we discuss the emerging roles for IL-6 family members in infection, chronic inflammation, autoimmunity and cancer and review therapeutic strategies designed to manipulate these cytokines in disease.

Activating Janus kinase (JAK) and signal transducer and activator of transcription (STAT) mutations have been discovered in many T-cell malignancies, including anaplastic lymphoma kinase (ALK)⁻ anaplastic large cell lymphomas (ALCLs). However, such mutations occur in a minority of patients. To investigate the clinical application of targeting JAK for ALK− ALCL, we treated ALK− cell lines of various histological origins with JAK inhibitors. Interestingly, most exogenous cytokine-independent cell lines responded to JAK inhibition regardless of JAK mutation status. JAK inhibitor sensitivity correlated with the STAT3 phosphorylation status of tumor cells. Using retroviral shRNA knockdown, we have demonstrated that these JAK inhibitor-sensitive cells are dependent on both JAK1 and STAT3 for survival. JAK1 and STAT3 gain-of-function mutations were found in some, but not all, JAK inhibitor-sensitive cells. Moreover, the mutations alone cannot explain the JAK1/STAT3 dependency, given that wild-type JAK1 or STAT3 was sufficient to promote cell survival in the cells that had either JAK1or STAT3 mutations. To investigate whether other mechanisms were involved, we knocked down upstream receptors GP130 or IL-2Rγ. Knockdown of GP130 or IL-2Rγ induced cell death in selected JAK inhibitor-sensitive cells. High expression levels of cytokines, including IL-6, were demonstrated in cell lines as well as in primary ALK− ALCL tumors. Finally, ruxolitinib, a JAK1/2 inhibitor, was effective in vivo in a xenograft ALK− ALCL model. Our data suggest that cytokine receptor signaling is required for tumor cell survival in diverse forms of ALK− ALCL, even in the presence of JAK1/STAT3 mutations. Therefore, JAK inhibitor therapy might benefit patients with ALK− ALCL who are phosphorylated STAT3⁺.

IL-6 family cytokines are defined by the common use of the signal-transducing receptor chain glycoprotein 130 (gp130). Increasing evidence indicates that these cytokines are essential in the regulation of metabolic homeostasis as well as in the pathophysiology of multiple gastrointestinal and liver disorders, thus making them attractive therapeutic targets. Over the past few years, therapies modulating gp130 signalling have grown exponentially in several clinical settings including obesity, cancer and inflammatory bowel disease. A newly engineered gp130 cytokine, IC7Fc, has shown promising preclinical results for the treatment of type 2 diabetes, obesity and liver steatosis. Moreover, drugs that modulate gp130 signalling have shown promise in refractory inflammatory bowel disease in clinical trials. A deeper understanding of the main roles of the IL-6 family of cytokines during homeostatic and pathological conditions, their signalling pathways, sources of production and target cells will be crucial to the development of improved treatments. Here, we review the current state of the role of these cytokines in hepatology and gastroenterology and discuss the progress achieved in translating therapeutics targeting gp130 signalling into clinical practice.In this Review, the authors describe the biology of IL-6 family cytokines relevant to metabolism, liver disease and gastrointestinal disease, and discuss the potential of IL-6 family members as therapeutic agents in these areas.

The cytokine interleukin-6 (IL-6) plays a crucial role in autoimmune and inflammatory diseases. Understanding the precise mechanism of IL-6 interaction at the amino acid level is essential to develop IL-6-inhibiting compounds. In this study, we employed computer-guided drug design tools to predict the key residues that are involved in the interaction between IL-6 and its receptor IL-6R. Subsequently, we generated IL-6 mutants and evaluated their binding affinity to IL-6R and the IL-6R − gp130 complex, as well as monitoring their biological activities. Our findings revealed that the R167A mutant exhibited increased affinity for IL-6R, leading to enhanced binding to IL-6R − gp130 complex and subsequently elevated intracellular phosphorylation of STAT3 in effector cells. On the other hand, although E171A reduced its affinity for IL-6R, it displayed stronger binding to the IL-6R − gp130 complex, thereby enhancing its biological activity. Furthermore, we identified the importance of R178 and R181 for the precise recognition of IL-6 by IL-6R. Mutants R181A/V failed to bind to IL-6R, while maintaining an affinity for the IL-6 − gp130 complex. Additionally, deletion of the D helix resulted in complete loss of IL-6 binding affinity for IL-6R. Overall, this study provides valuable insights into the binding mechanism of IL-6 and establishes a solid foundation for future design of novel IL-6 inhibitors.

ObjectiveTo discover and confirm blood-based colon cancer early-detection markers.DesignWe created a high-density antibody microarray to detect differences in protein levels in plasma from individuals diagnosed with colon cancer <3 years after blood was drawn (ie, prediagnostic) and cancer-free, matched controls. Potential markers were tested on plasma samples from people diagnosed with adenoma or cancer, compared with controls. Components of an optimal 5-marker panel were tested via immunoblotting using a third sample set, Luminex assay in a large fourth sample set and immunohistochemistry (IHC) on tissue microarrays.ResultsIn the prediagnostic samples, we found 78 significantly (t-test) increased proteins, 32 of which were confirmed in the diagnostic samples. From these 32, optimal 4-marker panels of BAG family molecular chaperone regulator 4 (BAG4), interleukin-6 receptor subunit beta (IL6ST), von Willebrand factor (VWF) and CD44 or epidermal growth factor receptor (EGFR) were established. Each panel member and the panels also showed increases in the diagnostic adenoma and cancer samples in independent third and fourth sample sets via immunoblot and Luminex, respectively. IHC results showed increased levels of BAG4, IL6ST and CD44 in adenoma and cancer tissues. Inclusion of EGFR and CD44 sialyl Lewis-A and Lewis-X content increased the panel performance. The protein/glycoprotein panel was statistically significantly higher in colon cancer samples, characterised by a range of area under the curves from 0.90 (95% CI 0.82 to 0.98) to 0.86 (95% CI 0.83 to 0.88), for the larger second and fourth sets, respectively.ConclusionsA panel including BAG4, IL6ST, VWF, EGFR and CD44 protein/glycomics performed well for detection of early stages of colon cancer and should be further examined in larger studies.