Small protein blockers of human IL-6 receptor alpha inhibit proliferation and migration of cancer cells.

BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that controls the immune response, and its role has been described in the development of autoimmune diseases. Signaling via its cognate IL-6 receptor (IL-6R) complex is critical in tumor progression and, therefore, IL-6R represents an important therapeutic target. METHODS: An albumin-binding domain-derived highly complex combinatorial library was used to select IL-6R alpha (IL-6Rα)-targeted small protein binders using ribosome display. Large-scale screening of bacterial lysates of individual clones was performed using ELISA, and their IL-6Rα blocking potential was verified by competition ELISA. The binding of proteins to cells was monitored by flow cytometry and confocal microscopy on HEK293T-transfected cells, and inhibition of signaling function was examined using HEK-Blue IL-6 reporter cells. Protein binding kinetics to living cells was measured by LigandTracer, cell proliferation and toxicity by iCELLigence and Incucyte, cell migration by the scratch wound healing assay, and prediction of binding poses using molecular modeling by docking. RESULTS: We demonstrated a collection of protein variants called NEF ligands, selected from an albumin-binding domain scaffold-derived combinatorial library, and showed their binding specificity to human IL-6Rα and antagonistic effect in HEK-Blue IL-6 reporter cells. The three most promising NEF108, NEF163, and NEF172 variants inhibited cell proliferation of malignant melanoma (G361 and A2058) and pancreatic (PaTu and MiaPaCa) cancer cells, and suppressed migration of malignant melanoma (A2058), pancreatic carcinoma (PaTu), and glioblastoma (GAMG) cells in vitro. The NEF binders also recognized maturation-induced IL-6Rα expression and interfered with IL-6-induced differentiation in primary human B cells. CONCLUSION: We report on the generation of small protein blockers of human IL-6Rα using directed evolution. NEF proteins represent a promising class of non-toxic anti-tumor agents with migrastatic potential.

The fibroid phenotype of biological naïve patients with rheumatoid arthritis are less likely to respond to anti-IL-6R treatment.

Type III collagen gene expression is upregulated in the synovium of patients with rheumatoid arthritis (RA) presenting the fibroid phenotype. The soluble type III collagen formation biomarker, PRO-C3, is known to measure fibrogenesis in fibrotic diseases. In this exploratory study, we aimed to investigate the association between fibrogenesis (PRO-C3) and the disease- and treatment response in patients with RA. We measured PRO-C3 in subsets of two clinical trials assessing the effect of the anti-interleukin-6 (IL-6) receptor treatment tocilizumab (TCZ) as monotherapy or polytherapy with methotrexate. PRO-C3 levels had weak or very weak correlations with the clinical parameters (Spearman's). However, when the patients were divided into Disease Activity Score-28 groups characterized by the erythrocyte sedimentation rate (DAS28-ESR), there was a statistical difference between the PRO-C3 levels of the different groups (p < 0.05). To determine the response in relation to PRO-C3, a cut-off based on PRO-C3 levels and patients in remission (DAS28-ESR ≤ 2.6) was identified. This showed that a reduction in PRO-C3 after treatment initiation was associated with decreased DAS28-ESR and a higher response rate in patients with low PRO-C3 levels than in those with high PRO-C3 levels. This indicates that a fibrotic component affects the responsiveness of patients.

[Immune Regulation by TNF Receptor-associated Factor 5].

The tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family of molecules are intracellular adaptors that regulate cellular signaling through members of the TNFR and Toll-like receptor superfamily. Mammals have seven TRAF molecules numbered sequentially from TRAF1 to TRAF7. Although TRAF5 was identified as a potential regulator of TNFR superfamily members, the in vivo function of TRAF5 has not yet been fully elucidated. We identified an unconventional role of TRAF5 in interleukin-6 (IL-6) receptor signaling involving CD4+ T cells. Moreover, TRAF5 binds to the signal-transducing glycoprotein 130 (gp130) receptor for IL-6 and inhibits the activity of the janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. In addition, Traf5-deficient CD4+ T cells exhibit significantly enhanced IL-6-driven differentiation of T helper 17 (Th17) cells, which exacerbates neuroinflammation in experimental autoimmune encephalomyelitis. Furthermore, TRAF5 demonstrates a similar activity to gp130 for IL-27, another cytokine of the IL-6 family. Additionally, Traf5-deficient CD4+ T cells display significantly increased IL-27-mediated differentiation of Th1 cells, which increases footpad swelling in delayed-type hypersensitivity response. Thus, TRAF5 functions as a negative regulator of gp130 in CD4+ T cells. This review aimed to explain how TRAF5 controls the differentiation of CD4+ T cells and discuss how the expression of TRAF5 in T cells and other cell types can influence the development and progression of autoimmune and inflammatory diseases.

Targeting IL-6 trans-signalling by sgp130Fc attenuates severity in SARS-CoV-2 -infected mice and reduces endotheliopathy.

BACKGROUND: SARS-CoV-2 infection is considered as a relapsing inflammatory process with a dysregulation of IL-6 signalling. Classic IL-6 signalling is thought to represent a defence mechanism against pathogens. In contrast, IL-6 trans-signalling has pro-inflammatory effects. In severe COVID-19, therapeutic strategies have focused on global inhibition of IL-6, with controversial results. We hypothesized that specific blockade of IL-6 trans-signalling could inhibit inflammatory response preserving the host defence activity inherent to IL-6 classic signalling. METHODS: To test the role of the specific IL-6 trans-signalling inhibition by sgp130Fc in short- and long-term consequences of COVID-19, we used the established K18-hACE2 transgenic mouse model. Histological as well as immunohistochemical analysis, and pro-inflammatory marker profiling were performed. To investigate IL-6 trans-signalling in human cells we used primary lung microvascular endothelial cells and fibroblasts in the presence/absence of sgp130Fc. FINDINGS: We report that targeting IL-6 trans-signalling by sgp130Fc attenuated SARS-CoV-2-related clinical symptoms and mortality. In surviving mice, the treatment caused a significant decrease in lung damage. In vitro, IL-6 trans-signalling induced strong and persisting JAK1/STAT3 activation in endothelial cells and lung fibroblasts with proinflammatory effects, which were attenuated by sgp130Fc. Our data also suggest that in those cells with scant amounts of IL-6R, the induction of gp130 and IL-6 by IL-6:sIL-6R complex sustains IL-6 trans-signalling. INTERPRETATION: IL-6 trans-signalling fosters progression of COVID-19, and suggests that specific blockade of this signalling mode could offer a promising alternative to mitigate both short- and long-term consequences without affecting the beneficial effects of IL-6 classic signalling. These results have implications for the development of new therapies of lung injury and endotheliopathy in COVID-19. FUNDING: The project was supported by ISCIII, Spain (COV-20/00792 to MB, PI23/01351 to MARH) and the European Commission-Next generation EU (European Union) (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global, SGL2103029 to MB). PID2019-110587RB-I00 (MB) supported by MICIN/AEI/10.13039/501100011033/and PID2022-143034OB-I00 (MB) by MICIN/AEI/10.13039/501100011033/FEDER. MAR-H acknowledges support from ISCIII, Spain and the European Commission-Next generation EU (European Union), through CSIC's Global Health PTI.

Extracellular CIRP co-stimulated T cells through IL6R/STAT3 in pediatric IgA vasculitis.

Immunoglobulin A vasculitis (IgAV) is the most common vasculitis of childhood. Disordered immune responses play important roles in its pathogenesis, but the comprehensive immune profile of the disease and the underlying mechanisms are still largely unknown. Here we found a potential disease biomarker cold inducible RNA binding protein (CIRP) in our pediatric IgAV cohort. Serum CIRP level in these patients were elevated and positively correlated with the increased early memory (CD45RA+CD62L+CD95+) T cells revealed using multicolor flow cytometry. Immune phenotyping of the patients showed they had more activated T cells with higher IL6Ra expression. T cell culture experiment showed CIRP further activated both human CD4+ and CD8+ T cells as indicated by increased perforin secretion and phosphorylation of STAT3. Blockade of IL6Rα attenuated CIRP-induced T cell toxicity in vitro. RNA-sequencing data further supported CIRP stimulation promoted human T cell activation and migration, fueled inflammation through the JAK-STAT signaling pathway. Therefore, IL6Ra-mediated T cell activation by extracellular CIRP may contribute to pathogenesis of IgAV in children, both CIRP and IL6Ra could be new therapeutic targets for IgAV.

Mitochondrial fission enhances IL-6-induced metastatic potential in ovarian cancer via ERK1/2 activation.

Ovarian cancer is a lethal gynecologic cancer mostly diagnosed in an advanced stage with an accumulation of ascites. Interleukin-6 (IL-6), a pro-inflammatory cytokine is highly elevated in malignant ascites and plays a pleiotropic role in cancer progression. Mitochondria are dynamic organelles that undergo fission and fusion in response to external stimuli and dysregulation in their dynamics has been implicated in cancer progression and metastasis. Here, we investigate the effect of IL-6 on mitochondrial dynamics in ovarian cancer cells (OVCs) and its impact on metastatic potential. Treatment with IL-6 on ovarian cancer cell lines (SKOV3 and PA-1) led to an elevation in the metastatic potential of OVCs. Interestingly, a positive association was observed between dynamin-related protein 1 (Drp1), a regulator of mitochondrial fission, and IL-6R in metastatic ovarian cancer tissues. Additionally, IL-6 treatment on OVCs was linked to the activation of Drp1, with a notable increase in the ratio of the inhibitory form p-Drp1(S637) to the active form p-Drp1(S616), indicating enhanced mitochondrial fission. Moreover, IL-6 treatment triggered the activation of ERK1/2, and inhibiting ERK1/2 mitigated IL-6-induced mitochondrial fission. Suppressing mitochondrial fission through siRNA transfection and a pharmacological inhibitor reduced the IL-6-induced migration and invasion of OVCs. This was further supported by 3D invasion assays using patient-derived spheroids. Altogether, our study suggests the role of mitochondrial fission in the metastatic potential of OVCs induced by IL-6. The inhibition of mitochondrial fission could be a potential therapeutic approach to suppress the metastasis of ovarian cancer.

Structural insights into IL-6 signaling inhibition by therapeutic antibodies.

Antibody inhibitors of the interleukin-6 (IL-6) signaling pathway, such as tocilizumab and sarilumab, have been used to treat rheumatoid arthritis, chimeric antigen receptor T cell-induced cytokine storm, and severe COVID-19 pneumonia. Here, we solve the cryogenic electron microscopy structures of sarilumab and tocilizumab in complex with IL-6R to resolutions of 3.2 and 3.3 Å, respectively. These structures reveal that both tocilizumab and sarilumab bind to the D3 domain of IL-6R. The binding surfaces of the two antibodies largely overlap, but the detailed interactions are different. Functional studies of various mutants show results consistent with our structural analysis of the antibodies and IL-6R interactions. Structural comparisons with the IL-6/IL-6R/gp130 complex indicate that sarilumab and tocilizumab probably inhibit IL-6/IL-6R signaling by competing for the IL-6 binding site. In summary, this work reveals the antibody-blocking mechanism of the IL-6 signaling pathway and paves the way for future antibody discovery.

Importance of IL-6 trans-signaling and high autocrine IL-6 production in human osteoarthritic chondrocyte metabolism.

OBJECTIVE: Neutralization of Interleukin (IL)-6-signaling by antibodies is considered a promising tool for the treatment of osteoarthritis (OA). To gain further insight into this potential treatment, this study investigated the effects of IL-6-signaling and IL-6 neutralization on chondrocyte metabolism and the release of IL-6-signaling-related mediators by human chondrocytes. DESIGN: Chondrocytes were collected from 49 patients with advanced knee/hip OA or femoral neck fracture. Isolated chondrocytes were stimulated with different mediators to analyze the release of IL-6, soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130). The effect of IL-6 and IL-6/sIL-6R complex as well as neutralization of IL-6-signaling on the metabolism was analyzed. RESULTS: OA chondrocytes showed high basal IL-6 production and release, which was strongly negatively correlated with the production of cartilage-matrix-proteins. Chondrocytes produced and released sIL-6R and sgp130. The IL-6/sIL-6R complex significantly increased nitric oxide, prostaglandin E2 and matrix metalloproteinase 1 production, decreased Pro-Collagen Type II and mitochondrial ATP production, and increased glycolysis in OA chondrocytes. Neutralization of IL-6-signaling by antibodies did not significantly affect the metabolism of OA chondrocytes, but blocking of glycoprotein 130 (gp130)-signaling by SC144 significantly reduced the basal IL-6 release. CONCLUSION: Although IL-6 trans-signaling induced by IL-6/sIL-6R complex negatively affects OA chondrocytes, antibodies against IL-6 or IL-6R did not affect chondrocyte metabolism. Since inhibition of gp130-signaling reduced the enhanced basal release of IL-6, interfering with gp130-signaling may ameliorate OA progression because high cellular release of IL-6 correlates with reduced production of cartilage-matrix-proteins.

[Research Progress on Effect of Bone Marrow Microenvironment Regulated by IL-6/IL-6R/JAK2/STAT3 Pathway on Biological Behavior of Multiple Myeloma--Review].

Multiple myeloma (MM) is a malignant disease with abnormal proliferation of clonal plasma cells. The development of the disease shows a vast heterogeneity, which is closely related to the interaction between MM cells and bone marrow microenvironment (BMM). The interleukin-6 (IL-6)/interleukin-6 receptor (IL-6R)/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway can regulate the transcription of related soluble factors in BMM, promote the proliferation, anti-apoptosis, drug resistance and guide related bone destruction of MM cells. This article reviews the research progress on the effect of BMM regulated by IL-6/IL-6R/JAK2/STAT3 pathway on the biological behavior of MM, in order to provide new research ideas for targeted therapy and precise therapy of MM.

Autocrine Regulation of Interleukin-6 via the Activation of STAT3 and Akt in Cardiac Myxoma Cells.

Plasma concentrations of a pleiotropic cytokine, interleukin (IL)-6, are increased in patients with cardiac myxoma. We investigated the regulation of IL-6 in cardiac myxoma. Immunohistochemical staining and reverse transcription-polymerase chain reaction (RT-PCR) revealed that IL-6 and its receptors, IL-6 receptor (IL-6R) and gp130, co-existed in the myxoma cells. Myxoma cells were cultured, and an antibody array assay showed that a conditioned medium derived from the cultured myxoma cells contained increased amounts of IL-6. Signal transducer and activator of transcription (STAT) 3 and Akt were constitutively phosphorylated in the myxoma cells. An enzyme-linked immunosorbent assay (ELISA) showed that the myxoma cells spontaneously secreted IL-6 into the culture medium. Real-time PCR revealed that stimulation with IL-6 + soluble IL-6R (sIL6R) significantly increased IL-6 mRNA in the myxoma cells. Pharmacological inhibitors of STAT3 and Akt inhibited the IL-6 + sIL-6R-induced gene expression of IL-6 and the spontaneous secretion of IL-6. In addition, IL-6 + sIL-6R-induced translocation of phosphorylated STAT3 to the nucleus was also blocked by STAT3 inhibitors. This study has demonstrated that IL-6 increases its own production via STAT3 and Akt pathways in cardiac myxoma cells. Autocrine regulation of IL-6 may play an important role in the pathophysiology of patients with cardiac myxoma.

Upregulated microRNA-125b-5p in patients with asthma-COPD overlap mediates oxidative stress and late apoptosis via targeting IL6R/TRIAP1 signaling.

BACKGROUND: Among patients with chronic obstructive pulmonary disease (COPD), some have features of both asthma and COPD-a condition categorized as asthma-COPD overlap (ACO). Our aim was to determine whether asthma- or COPD-related microRNAs (miRNAs) play a role in the pathogenesis of ACO. METHODS: A total of 22 healthy subjects and 27 patients with ACO were enrolled. We selected 6 miRNAs that were found to correlate with COPD and asthma. The expression of miRNAs and target genes was analyzed using quantitative reverse-transcriptase polymerase chain reaction. Cell apoptosis and intracellular reactive oxygen species production were evaluated using flow cytometry. In vitro human monocytic THP-1 cells and primary normal human bronchial epithelial (NHBE) cells under stimuli with cigarette smoke extract (CSE) or ovalbumin (OVA) allergen or both were used to verify the clinical findings. RESULTS: We identified the upregulation of miR-125b-5p in patients with ACO and in THP-1 cells stimulated with CSE plus OVA allergen. We selected 16 genes related to the miR-125b-5p pathway and found that IL6R and TRIAP1 were both downregulated in patients with ACO and in THP-1 cells stimulated with CSE plus OVA. The percentage of late apoptotic cells increased in the THP-1 cell culture model when stimulated with CSE plus OVA, and the effect was reversed by transfection with miR-125b-5p small interfering RNA (siRNA). The percentage of reactive oxygen species-producing cells increased in the NHBE cell culture model when stimulated with CSE plus OVA, and the effect was reversed by transfection with miR-125b-5p siRNA. In NHBE cells, siRNA transfection reversed the upregulation of STAT3 under CSE+OVA stimulation. CONCLUSIONS: Our study revealed that upregulation of miR-125b-5p in patients with ACO mediated late apoptosis in THP-1 cells and oxidative stress in NHBE cells via targeting IL6R and TRIAP1. STAT3 expression was also regulated by miR-125b-5p.

Role of interleukin-6 and endothelin-1 receptors in enhanced melanocyte dendricity of facial spots and suppression of their ligands by niacinamide and tranexamic acid.

BACKGROUND: Hyperpigmented spots are common issues in all ethnicities with a hallmark characteristic of increased melanocyte dendricity. OBJECTIVES: To determine (1) potential receptors and/or cytokines that are involved in increased melanocyte dendricity in multiple facial spot types; (2) treatment effects of skin-lightening compounds on identified cytokine release from keratinocytes and on dendricity in melanocytes. METHODS: Facial spots (melasma, solar lentigo, acne-induced post-inflammatory hyperpigmentation) and adjacent non-spot skin biopsies were collected from Chinese women (age 20-70). The epidermal supra and basal layers were laser dissected to enrich keratinocyte or melanocyte biology respectively for transcriptome analysis. Melanocyte dendricity was assessed histologically by immunofluorescent staining. Effect of interleukin-6 (IL-6) and endothelin-1 (ET-1) on melanocyte dendricity and melanosome transfer were assessed in human melanocytes or melanocyte-keratinocyte co-culture models. Treatment effects of skin-lightening compounds (niacinamide, tranexamic acid [TxA], sucrose laurate/dilaurate mixture [SDL]) were assessed on IL-6 or ET-1 release from keratinocytes and on dendricity in melanocytes. RESULTS: Transcriptome analysis revealed IL-6 receptor and ET-1 receptor were significantly upregulated compared to the adjacent normal skin, visually confirmed at the protein level through immunostaining. Melanocytes in spot areas are more dendritic than melanocytes in adjacent non-spot skin. The addition of IL-6 and ET-1 to cell culture models increased melanocyte dendricity and melanosome transfer. IL-6 release was significantly suppressed by niacinamide and its combination, while ET-1 release was significantly reduced by both niacinamide and TxA. In contrast, SDL acted directly upon melanocytes to reduce dendricity. CONCLUSION: Interleukin-6 and ET-1 receptors are significantly upregulated in multiple facial spot types. The in vitro testing demonstrated their respective ligands increased melanocyte dendricity. Tested skin-lightening compounds showed reduction in release of IL-6/ET-1 from epidermal keratinocytes and/or inhibition of melanocyte dendricity. This work sheds light on pathophysiological mechanism of facial spots and potential new mechanisms of these skin-lightening compounds which warrant further human clinical validation.

Dynamic IL-6R/STAT3 signaling leads to heterogeneity of metabolic phenotype in pancreatic ductal adenocarcinoma cells.

Malignancy is enabled by pro-growth mutations and adequate energy provision. However, global metabolic activation would be self-terminating if it depleted tumor resources. Cancer cells could avoid this by rationing resources, e.g., dynamically switching between "baseline" and "activated" metabolic states. Using single-cell metabolic phenotyping of pancreatic ductal adenocarcinoma cells, we identify MIA-PaCa-2 as having broad heterogeneity of fermentative metabolism. Sorting by a readout of lactic acid permeability separates cells by fermentative and respiratory rates. Contrasting phenotypes persist for 4 days and are unrelated to cell cycling or glycolytic/respiratory gene expression; however, transcriptomics links metabolically active cells with interleukin-6 receptor (IL-6R)-STAT3 signaling. We verify this by IL-6R/STAT3 knockdowns and sorting by IL-6R status. IL-6R/STAT3 activates fermentation and transcription of its inhibitor, SOCS3, resulting in delayed negative feedback that underpins transitions between metabolic states. Among cells manifesting wide metabolic heterogeneity, dynamic IL-6R/STAT3 signaling may allow cell cohorts to take turns in progressing energy-intense processes without depleting shared resources.

An epithelial gene signature of trans-IL-6 signaling defines a subgroup of type 2-low asthma.

BACKGROUND: Asthma is stratified into type 2-high and type 2-low inflammatory phenotypes. Limited success has been achieved in developing drugs that target type 2-low inflammation. Previous studies have linked IL-6 signaling to severe asthma. IL-6 cooperates with soluble-IL-6Rα to activate cell signaling in airway epithelium. OBJECTIVE: We sought to study the role of sIL-6Rα amplified IL-6 signaling in airway epithelium and to develop an IL-6+ sIL-6Rα gene signature that may be used to select asthma patients who potentially respond to anti-IL-6 therapy. METHODS: Human airway epithelial cells were stimulated with combinations of IL-6, sIL-6Rα, and inhibitors, sgp130 (Olamkicept), and anti-IL-6R (Tocilizumab), to assess effects on pathway activation, epithelial barrier integrity, and gene expression. A gene signature was generated to identify IL-6 high patients using bronchial biopsies and nasal brushes. RESULTS: Soluble-IL-6Rα amplified the activation of the IL-6 pathway, shown by the increase of STAT3 phosphorylation and stronger gene induction in airway epithelial cells compared to IL-6 alone. Olamkicept and Tocilizumab inhibited the effect of IL-6 + sIL-6Rα on gene expression. We developed an IL-6 + sIL-6Rα gene signature and observed enrichment of this signature in bronchial biopsies but not nasal brushes from asthma patients compared to healthy controls. An IL-6 + sIL-6Rα gene signature score was associated with lower levels of sputum eosinophils in asthma. CONCLUSION: sIL-6Rα amplifies IL-6 signaling in bronchial epithelial cells. Higher local airway IL-6 + sIL-6Rα signaling is observed in asthma patients with low sputum eosinophils.

CTRP4/interleukin-6 receptor signaling ameliorates autoimmune encephalomyelitis by suppressing Th17 cell differentiation.

C1q/TNF-related protein 4 (CTRP4) is generally thought to be released extracellularly and plays a critical role in energy metabolism and protecting against sepsis. However, its physiological functions in autoimmune diseases have not been thoroughly explored. In this study, we demonstrate that Th17 cell-associated experimental autoimmune encephalomyelitis was greatly exacerbated in Ctrp4-/- mice compared with WT mice due to increased Th17 cell infiltration. The absence of Ctrp4 promoted the differentiation of naive CD4+ T cells into Th17 cells in vitro. Mechanistically, CTRP4 interfered with the interaction between IL-6 and the IL-6 receptor (IL-6R) by directly competing to bind with IL-6R, leading to suppression of IL-6-induced activation of the STAT3 pathway. Furthermore, the administration of recombinant CTRP4 protein ameliorated disease symptoms. In conclusion, our results indicate that CTRP4, as an endogenous regulator of the IL-6 receptor-signaling pathway, may be a potential therapeutic intervention for Th17-driven autoimmune diseases.

Blockade of interleukin-6 trans-signaling prevents mitochondrial dysfunction and cellular senescence in retinal endothelial cells.

Interleukin-6 (IL-6) is a multifaceted cytokine implicated in the pathogenesis of diabetic retinopathy (DR). Its activity extends through cis- and trans-signaling (TS) pathways, with cis-signaling limited to specific cell types possessing the membrane-bound IL-6 receptor, while trans-signaling broadly activates various cells without the membrane bound IL-6 receptor, including retinal endothelial cells. In this study, we determined the effects of interleukin-6 trans-signaling on mitochondrial dysfunction and cellular senescence in human retinal endothelial cells (HRECs). HRECs were cultured and treated with IL-6 + soluble IL-6R or Hyper IL-6 to activate trans-signaling, along with sgp130Fc for inhibition. RT-PCR was used to analyze gene expression changes associated with inflammation and senescence. Cellular senescence was assessed using SA ß-gal staining. Mitochondrial function was evaluated using Seahorse XFe24 Bioanalyzer. IL-6 trans-signaling induced inflammatory gene expression as indicated by the upregulation of ICAM1, MCP1, and SERPINA3 levels. Additionally, it reduced mitochondrial respiration and oxidative phosphorylation, and these effects were counteracted by sgp130Fc. Moreover, IL-6 trans-signaling led to altered expression of apoptosis-associated genes, including downregulation of FIS1, BCL2, and MCL1, while promoting cellular senescence, a phenomenon mitigated by sgp130Fc. These results not only deepen our understanding of IL-6 in DR but also carry broader implications for age-related diseases and the aging process itself. This study underscores the potential therapeutic value of targeting IL-6 trans-signaling with sgp130Fc as a promising anti-inflammatory approach for DR and potentially other inflammatory conditions. Further in-vivo investigations are warranted to elucidate the function of IL-6 trans-signaling in aging-related pathologies and overall organismal health.

Structures of the interleukin 11 signalling complex reveal gp130 dynamics and the inhibitory mechanism of a cytokine variant.

Interleukin (IL-)11, an IL-6 family cytokine, has pivotal roles in autoimmune diseases, fibrotic complications, and solid cancers. Despite intense therapeutic targeting efforts, structural understanding of IL-11 signalling and mechanistic insights into current inhibitors are lacking. Here we present cryo-EM and crystal structures of the human IL-11 signalling complex, including the complex containing the complete extracellular domains of the shared IL-6 family ß-receptor, gp130. We show that complex formation requires conformational reorganisation of IL-11 and that the membrane-proximal domains of gp130 are dynamic. We demonstrate that the cytokine mutant, IL-11 Mutein, competitively inhibits signalling in human cell lines. Structural shifts in IL-11 Mutein underlie inhibition by altering cytokine binding interactions at all three receptor-engaging sites and abrogating the final gp130 binding step. Our results reveal the structural basis of IL-11 signalling, define the molecular mechanisms of an inhibitor, and advance understanding of gp130-containing receptor complexes, with potential applications in therapeutic development.

Bispecific soluble cytokine receptor-nanobody fusions inhibit Interleukin (IL-)6 trans-signaling and IL-12/23 or tumor necrosis factor (TNF) signaling.

At least 0.5% of people in the Western world develop inflammatory bowel disease (IBD). While antibodies that block tumor necrosis factor (TNF) α and Interleukin (IL-)23 have been approved for the treatment of IBD, IL-6 antibodies failed in the phase II clinical trial due to non-tolerable side effects. However, two clinical phase II studies suggest that inhibiting IL-6/soluble IL-6R (sIL-6R)-induced trans-signaling via the cytokine receptor gp130 benefit IBD patients with fewer adverse events. Here we develop inhibitors targeting a combination of IL-6/sIL-6R and TNF or IL-12/IL-23 signaling, named cs130-TNFVHHFc and cs130-IL-12/23VHHFc. Surface plasmon resonance experiments showed that recombinant cs130-TNFVHHFc and cs130-IL-12/23VHHFc bind with high affinity to IL-6/sIL-6R complexes and human TNFα (hTNFα) or IL-12/IL-23, respectively. Immunoprecipitation experiments have verified the higher ordered complex formation of the inhibitors with IL-6/sIL-6R and IL-12. We demonstrated that cs130-TNFVHHFc and cs130-IL-12/23VHHFc block IL-6/sIL-6R trans-signaling-induced proliferation and STAT3 phosphorylation of Ba/F3-gp130 cells, as well as hTNFα- or IL-23-induced signaling, respectively. In conclusion, cs130-TNFVHHFc and cs130-IL-12/23VHHFc represent a class of dimeric and bispecific chimeric cytokine inhibitors that consist of a soluble cytokine receptor fused to anti-cytokine nanobodies.

Biophysical insight into protein-protein interactions in the Interleukin-11/Interleukin-11Rα/glycoprotein 130 signaling complex.

Interleukin-11 (IL-11) is a member of the interleukin-6 (IL-6) family of cytokines. IL-11 is a regulator of multiple events in hematopoiesis, and IL-11-mediated signaling is implicated in inflammatory disease, cancer, and fibrosis. All IL-6 family cytokines signal through the signal-transducing receptor, glycoprotein 130 (gp130), but these cytokines have distinct as well as overlapping biological functions. To understand IL-11 signaling at the molecular level, we performed a comprehensive interaction analysis of the IL-11 signaling complex, comparing it with the IL-6 complex, one of the best-characterized cytokine complexes. Our thermodynamic analysis revealed a clear difference between IL-11 and IL-6. Surface plasmon resonance analysis showed that the interaction between IL-11 and IL-11 receptor α (IL-11Rα) is entropy driven, whereas that between IL-6 and IL-6 receptor α (IL-6Rα) is enthalpy driven. Our analysis using isothermal titration calorimetry revealed that the binding of gp130 to the IL-11/IL-11Rα complex results in entropy loss, but that the interaction of gp130 with the IL-6/IL-6Rα complex results in entropy gain. Our hydrogen-deuterium exchange mass spectrometry experiments suggested that the D2 domain of gp130 was not involved in IL-6-like interactions in the IL-11/IL-11Rα complex. It has been reported that IL-6 interaction with gp130 in the signaling complex was characterized through the hydrophobic interface located in its D2 domain of gp130. Our findings suggest that unique interactions of the IL-11 signaling complex with gp130 are responsible for the distinct biological activities of IL-11 compared to IL-6.

IL-6 Trans-Signaling Is Increased in Diabetes, Impacted by Glucolipotoxicity, and Associated With Liver Stiffness and Fibrosis in Fatty Liver Disease.

Many people living with diabetes also have nonalcoholic fatty liver disease (NAFLD). Interleukin-6 (IL-6) is involved in both diseases, interacting with both membrane-bound (classical) and circulating (trans-signaling) soluble receptors. We investigated whether secretion of IL-6 trans-signaling coreceptors are altered in NAFLD by diabetes and whether this might associate with the severity of fatty liver disease. Secretion patterns were investigated with use of human hepatocyte, stellate, and monocyte cell lines. Associations with liver pathology were investigated in two patient cohorts: 1) biopsy-confirmed steatohepatitis and 2) class 3 obesity. We found that exposure of stellate cells to high glucose and palmitate increased IL-6 and soluble gp130 (sgp130) secretion. In line with this, plasma sgp130 in both patient cohorts positively correlated with HbA1c, and subjects with diabetes had higher circulating levels of IL-6 and trans-signaling coreceptors. Plasma sgp130 strongly correlated with liver stiffness and was significantly increased in subjects with F4 fibrosis stage. Monocyte activation was associated with reduced sIL-6R secretion. These data suggest that hyperglycemia and hyperlipidemia can directly impact IL-6 trans-signaling and that this may be linked to enhanced severity of NAFLD in patients with concomitant diabetes. ARTICLE HIGHLIGHTS: IL-6 and its circulating coreceptor sgp130 are increased in people with fatty liver disease and steatohepatitis. High glucose and lipids stimulated IL-6 and sgp130 secretion from hepatic stellate cells. sgp130 levels correlated with HbA1c, and diabetes concurrent with steatohepatitis further increased circulating levels of all IL-6 trans-signaling mediators. Circulating sgp130 positively correlated with liver stiffness and hepatic fibrosis. Metabolic stress to liver associated with fatty liver disease might shift the balance of IL-6 classical versus trans-signaling, promoting liver fibrosis that is accelerated by diabetes.