Intratumoral Delivery of Genetically Engineered Anti-IL-6 Trans-signaling Therapeutics.

Interleukin-6 (IL-6) is a highly pro-inflammatory cytokine involved in the etiopathology of several inflammatory diseases and cancer. As so, the inhibition of IL-6 signaling pathways has emerged as an attractive therapeutic avenue for the treatment of several chronic diseases. Since IL-6 trans-signaling was described as the pathological branch of IL-6, selective inhibitors were developed. Next-generation variants with increased trans-signaling specificity and potency emerged as great candidates for the treatment of several diseases, with reduced off-target effects. The highly time-consuming and costly processes involving recombinant protein production, however, have hampered the progress of anti-cytokine pharmaceuticals in clinic so far. Herein, we developed gene therapeutic modalities of IL-6-trans-signaling inhibitors as alternatives for sustained recombinant protein secretion. By using an IL-6-dependent lymphoma cell line and xenograft tumor model, we demonstrated the superior inhibitory potential of second-generation anti-IL-6 trans-signaling therapeutic. We compared the efficiency of distinct gene delivery modalities using a bioluminescent biomarker probe and observed consistent protein production via cell-based delivery. When delivered intratumorally, genetically engineered sgp130FlyRFc-secreting cells significantly reduced tumor burden and increased animal survival, representing a promising therapeutic avenue to be explored in clinically relevant gene delivery applications.

STAT4 Mediates IL-6 Trans-Signaling Arrhythmias in High Fat Diet Guinea Pig Heart.

Obesity is a major risk factor for the development of life-threatening malignant ventricular tachyarrhythmias (VT) and sudden cardiac death (SCD). Risks may be highest for patients with high levels of the proinflammatory cytokine interleukin (IL)-6. We used our guinea pig model of high-fat diet (HFD)-induced arrhythmias that exhibit a heightened proinflammatory-like pathology, which is also observed in human obesity arrhythmias, as well as immunofluorescence and confocal microscopy approaches to evaluate the pathological IL-6 trans-signaling function and explore the underlying mechanisms. Using blind-stick and electrocardiogram (ECG) techniques, we tested the hypothesis that heightened IL-6 trans-signaling would exhibit increased ventricular arrhythmia/SCD incidence and underlying arrhythmia substrates. Remarkably, compared to low-fat diet (LFD)-fed controls, HFD promoted phosphorylation of the IL-6 signal transducer and activator of transcription 4 (STAT4), leading to its activation and enhanced nuclear translocation of pSTAT4/STAT4 compared to LFD controls and pSTAT3/STAT3 nuclear expression. Overactivation of IL-6 trans-signaling in guinea pigs prolonged the QT interval, which resulted in greater susceptibility to arrhythmias/SCD with isoproterenol challenge, as also observed with the downstream Janus kinase (JAK) 2 activator. These findings may have potentially profound implications for more effective arrhythmia therapy in the vulnerable obese patient population.

Exploratory assessment of the effect of systemic administration of soluble glycoprotein 130 on cognitive performance and chemokine levels in a mouse model of experimental traumatic brain injury.

Uncontrolled neuroinflammation mediates traumatic brain injury (TBI) pathology and impairs recovery. Interleukin-6 (IL-6), a pleiotropic inflammatory regulator, is associated with poor clinical TBI outcomes. IL-6 operates via classical-signaling through membrane-bound IL-6 receptor (IL-6R) and trans-signaling through soluble IL-6 receptor (s)IL-6R. IL-6 trans-signaling specifically contributes to neuropathology, making it a potential precision therapeutic TBI target. Soluble glycoprotein 130 (sgp130) prevents IL-6 trans-signaling, sparing classical signaling, thus is a possible treatment. Mice received either controlled cortical impact (CCI) (6.0 ± 0.2 m/s; 2 mm; 50-60ms) or sham procedures. Vehicle (VEH) or sgp130-Fc was subcutaneously administered to sham (VEH or 1 µg) and CCI (VEH, 0.25 µg or 1 µg) mice on days 1, 4, 7, 10 and 13 post-surgery to assess effects on cognition [Morris Water Maze (MWM)] and ipsilateral hemisphere IL-6 related biomarkers (day 21 post-surgery). CCI + sgp130-Fc groups (0.25 µg and 1 µg) were combined for analysis given similar behavior/biomarker outcomes. CCI + VEH mice had longer latencies and path lengths to the platform and increased peripheral zone time versus Sham + VEH and Sham + sgp130-Fc mice, suggesting injury-induced impairments in learning and anxiety. CCI + sgp130-Fc mice had shorter platform latencies and path lengths and had decreased peripheral zone time, indicating a therapeutic benefit of sgp130-Fc after injury on learning and anxiety. Interestingly, Sham + sgp130-Fc mice had shorter platform latencies, path lengths and peripheral zone times than Sham + VEH mice, suggesting a beneficial effect of sgp130-Fc, independent of injury. CCI + VEH mice had increased brain IL-6 and decreased sgp130 levels versus Sham + VEH and Sham + sgp130-Fc mice. There was no treatment effect on IL-6, sIL6-R or sgp130 in Sham + VEH versus Sham + sgp130-Fc mice. There was also no treatment effect on IL-6 in CCI + VEH versus CCI + sgp130-Fc mice. However, CCI + sgp130-Fc mice had increased sIL-6R and sgp130 versus CCI + VEH mice, demonstrating sgp130-Fc treatment effects on brain biomarkers. Inflammatory chemokines (MIP-1ß, IP-10, MIG) were increased in CCI + VEH mice versus Sham + VEH and Sham + sgp130-Fc mice. However, CCI + sgp130-Fc mice had decreased chemokine levels versus CCI + VEH mice. IL-6 positively correlated, while sgp130 negatively correlated, with chemokine levels. Overall, we found that systemic sgp130-Fc treatment after CCI improved learning, decreased anxiety and reduced CCI-induced brain chemokines. Future studies will explore sex-specific dosing and treatment mechanisms for sgp130-Fc therapy.

Increased expression of CD38 on endothelial cells in SARS-CoV-2 infection in cynomolgus macaques.

SARS-CoV-2 infection causes activation of endothelial cells (ECs), leading to dysmorphology and dysfunction. To study the pathogenesis of endotheliopathy, the activation of ECs in lungs of cynomolgus macaques after SARS-CoV-2 infection and changes in nicotinamide adenine dinucleotide (NAD) metabolism in ECs were investigated, with a focus on the CD38 molecule, which degrades NAD in inflammatory responses after SARS-CoV-2 infection. Activation of ECs was seen from day 3 after SARS-CoV-2 infection in macaques, with increases of intravascular fibrin and NAD metabolism-associated enzymes including CD38. In vitro, upregulation of CD38 mRNA in human ECs was detected after interleukin 6 (IL-6) trans-signaling induction, which was increased in the infection. In the presence of IL-6 trans-signaling stimulation, however, CD38 mRNA silencing induced significant IL-6 mRNA upregulation in ECs and promoted EC apoptosis after stimulation. These results suggest that upregulation of CD38 in patients with COVID-19 has a protective role against IL-6 trans-signaling stimulation induced by SARS-CoV-2 infection.

MiR-449a antagonizes EMT through IL-6-mediated trans-signaling in laryngeal squamous cancer.

MicroRNAs (miRNAs) are involved in post-transcriptional gene expression regulation and in mechanisms of cancer growth and metastases. In this light, miRNAs could be promising therapeutic targets and biomarkers in clinical practice. Therefore, we investigated if specific miRNAs and their target genes contribute to laryngeal squamous cell carcinoma (LSCC) development. We found a significant decrease of miR-449a in LSCC patients with nodal metastases (63.3%) compared with patients without nodal involvement (44%). The AmpliSeq Transcriptome of HNO-210 miR-449a-transfected cell lines allowed the identification of IL6-R as a potential target. Moreover, the downregulation of IL6-R and the phosphorylation reduction of the downstream signaling effectors, suggested the inhibition of the IL-6 trans-signaling pathway. These biochemical effects were paralleled by a significant inhibition of invasion and migration in vitro and in vivo, supporting an involvement of epithelial-mesenchymal transition. These findings indicate that miR-449a contributes to suppress the metastasization of LSCC by the IL-6 trans-signaling block and affects sensitivity to external stimuli that mimic pro-inflammatory conditions.

Nonspecific Inhibition of IL6 Family Cytokine Signalling by Soluble gp130.

IL6 is a proinflammatory cytokine that binds to membrane-bound IL6 receptor (IL6R) or soluble IL6R to signal via gp130 in cis or trans, respectively. We tested the hypothesis that sgp130Fc, which is believed to be a selective IL6 trans-signalling inhibitor, is in fact a non-specific inhibitor of gp130 signalling. In human cancer and primary cells, sgp130Fc inhibited IL6, IL11, OSM and CT1 cis-signalling. The IC50 values of sgp130Fc for IL6 and OSM cis-signalling were markedly (20- to 200-fold) lower than the concentrations of sgp130Fc used in mouse studies and clinical trials. sgp130 inhibited IL6 and OSM signalling in the presence of an ADAM10/17 inhibitor and the absence of soluble IL6R or OSMR, with effects that were indistinguishable from those of a gp130 neutralising antibody. These data show that sgp130Fc does not exclusively block IL6 trans-signalling and reveal instead that broad inhibition of gp130 signalling likely underlies its therapeutic effects. This proposes global or modular inhibition of gp130 as a therapeutic approach for treating human disease.

Interleukin-6 trans-signaling regulates monocyte chemoattractant protein-1 production in immune-mediated necrotizing myopathy.

OBJECTIVE: Immune-mediated necrotizing myopathy (IMNM) is pathologically characterized by diffuse myofiber necrosis and regeneration, myophagocytosis, and a sparse inflammatory infiltrate. The monocyte chemoattractant protein-1 (MCP-1) is a key chemokine that regulates monocyte/macrophage infiltration into injured tissues. The interleukin-6 (IL-6) signalling in the induction of MCP-1 expression has not been investigated in IMNM. METHODS: MCP-1 expression in muscle specimens was assessed using immunohistochemistry and real-time quantitative polymerase chain reaction (RT-qPCR). Levels of multiple serological cytokines were evaluated using the Meso Scale Discovery electrochemiluminescence system. Flow cytometry, RT-qPCR, enzyme-linked immunosorbent assay, western blot, dual-luciferase reporter assays, and chromatin immunoprecipitation-qPCR were performed to explore the effects of IL-6 signalling on MCP-1 production in human myoblasts. RESULTS: MCP-1 was scattered and was positively expressed within myofibers and a few inflammatory cells in the muscles of patients with IMNM. Sarcoplasmic MCP-1 expression significantly correlated with myonecrosis, myoregeneration, and inflammatory infiltration. Serum MCP-1, IL-6, and the soluble form of the IL-6 receptor (sIL-6R) were elevated in patients with IMNM compared with controls. Serological MCP-1 levels were significantly associated with serum IL-6 expression and clinical disease severity in IMNM patients. The IL-6/sIL-6R complex induced MCP-1 expression via the signal transducer and activator of transcription 3 (STAT3) pathway in human myoblasts. Mechanistically, phospho-STAT3 was enriched in the MCP-1 promoter region and promoted the transcription. CONCLUSION: IL-6 trans-signalling may contribute to the immunopathogenesis of IMNM by augmenting inflammation through regulation of MCP-1 expression in IMNM.

Cytokines of the interleukin-6 family as emerging targets in inflammatory bowel disease.

INTRODUCTION: Inflammatory bowel disease (IBD) is an umbrella term that includes different chronic inflammatory diseases of the gastrointestinal tract, most commonly Crohn's disease and ulcerative colitis. IBD affects more than 6 million people worldwide and constitutes not only a debilitating disease for the patients, but also a significant factor for society due to costs for health care and reduced working capacity. Despite the introduction of biologicals for the treatment of IBD, the identification of novel targets that could lead to novel therapeutics is still needed. AREAS COVERED: In this review, we summarize current knowledge about the interleukin-6 family of cytokines as potential therapeutic targets for improving the therapy of patients with IBD. We discuss cytokines like IL-6 itself for which therapeutics such as inhibitory monoclonal antibodies have already entered the clinics, but also focus on other family members whose therapeutic potential has not been explored yet. EXPERT OPINION: The different cytokines of the IL-6 family offer multiple therapeutic targets that can potentially be used to treat patients with inflammatory bowel disease, but unwanted side effects like inhibition of epithelial regeneration have to be considered.

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.

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.

Replenishing decoy extracellular vesicles inhibits phenotype remodeling of tissue-resident cells in inflammation-driven arthritis.

The interleukin 6 (IL6) signaling pathway plays pleiotropic roles in regulating the inflammatory milieu that contributes to arthritis development. Here, we show that activation of IL6 trans-signaling induces phenotypic transitions in tissue-resident cells toward an inflammatory state. The establishment of arthritis increases the serum number of extracellular vesicles (EVs), while these EVs express more IL6 signal transducer (IL6ST, also known as gp130) on their surface. Transferring these EVs can block IL6 trans-signaling in vitro by acting as decoys that trap hyper IL6 and prevent inflammatory amplification in recipient arthritic mice. By genetically fusing EV-sorting domains with extracellular domains of receptors, we engineered EVs that harbor a higher quantity of signaling-incompetent decoy receptors. These exogenous decoy EVs exhibit significant potential in eliciting efficient anti-inflammatory effects in vivo. Our findings suggest an inherent resistance of decoy EVs against inflammation, highlighting the therapeutic potential of efficient decoy EVs in treating inflammatory diseases.

Modulation of Pro-Inflammatory IL-6 Trans-Signaling Axis by Splice Switching Oligonucleotides as a Therapeutic Modality in Inflammation.

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a crucial role in maintaining normal homeostatic processes under the pathogenesis of various inflammatory and autoimmune diseases. This context-dependent effect from a cytokine is due to two distinctive forms of signaling: cis-signaling and trans-signaling. IL-6 cis-signaling involves binding IL-6 to the membrane-bound IL-6 receptor and Glycoprotein 130 (GP130) signal-transducing subunit. By contrast, in IL-6 trans-signaling, complexes of IL-6 and the soluble form of the IL-6 receptor (sIL-6R) signal via membrane-bound GP130. Various strategies have been employed in the past decade to target the pro-inflammatory effect of IL-6 in numerous inflammatory disorders. However, their development has been hindered since these approaches generally target global IL-6 signaling, also affecting the anti-inflammatory effects of IL-6 signaling too. Therefore, novel strategies explicitly targeting the pro-inflammatory IL-6 trans-signaling without affecting the IL-6 cis-signaling are required and carry immense therapeutic potential. Here, we have developed a novel approach to specifically decoy IL-6-mediated trans-signaling by modulating alternative splicing in GP130, an IL-6 signal transducer, by employing splice switching oligonucleotides (SSO), to induce the expression of truncated soluble isoforms of the protein GP130. This isoform is devoid of signaling domains but allows for specifically sequestering the IL-6/sIL-6R receptor complex with high affinity in serum and thereby suppressing inflammation. Using the state-of-the-art Pip6a cell-penetrating peptide conjugated to PMO-based SSO targeting GP130 for efficient in vivo delivery, reduced disease phenotypes in two different inflammatory mouse models of systemic and intestinal inflammation were observed. Overall, this novel gene therapy platform holds great potential as a refined therapeutic intervention for chronic inflammatory diseases.

Selective blockade of interleukin 6 trans-signaling depresses atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) has been accepted as an inflammatory atrial myopathy. Interleukin 6 (IL-6)-dependent inflammatory signaling pathways take context-dependent effects on cardiovascular diseases. IL-6 trans-signaling is predominantly pro-inflammatory. However, its effect on AF is unclear. OBJECTIVE: The purpose of this study was to investigate the role of IL-6 trans-signaling in AF. METHODS: Circulating levels of IL-6, soluble IL-6 receptor, and soluble glycoprotein 130 (sgp130) in patients with AF and controls were measured to estimate the activation of IL-6 trans-signaling. A mouse model of AF was established by transverse aortic constriction surgery. Sgp130Fc administration was used for the selective blockade of IL-6 trans-signaling. Studies were conducted to evaluate the effects and underlying mechanisms of sgp130Fc on AF inducibility and atrial conduction abnormalities and structural remodeling. RESULTS: In patients, the elevation of IL-6 trans-signaling level was positively associated with AF occurrence. IL-6 trans-signaling activation was recapitulated in the mouse model of AF. In transverse aortic constriction-challenged mice, the selective blockade of IL-6 trans-signaling with sgp130Fc attenuated AF inducibility, which was attributable to the amelioration of slow conduction and conduction heterogeneity induced by atrial dilation, fibrosis, and reduction in connexin 40 and redistribution of connexin 43. Sgp130Fc administration also reduced immune cell infiltration and oxidative stress in the mouse atrium and abrogated IL-6 trans-signaling activation-mediated connexin dysregulation and reactive oxygen species production in atrial myocytes. CONCLUSION: IL-6 trans-signaling activation contributes to AF development, and its selective blockade may promise a novel therapeutic strategy.

Blockade of IL-11 Trans-Signaling or JAK2/STAT3 Signaling Ameliorates the Profibrotic Effect of IL-11.

OBJECTIVES: Systemic sclerosis (SSc) is a rare rheumatic disease characterized by vascular damage, dysregulated immune response, and fibrosis. Interleukin-11 (IL-11) is upregulated in SSc. This study aimed to investigate the pathological and therapeutic role of the IL-11 trans-signaling pathway in SSc. METHODS: Plasma IL-11 level was evaluated in 32 patients with SSc and 15 healthy controls, while the expression levels of ADAM10, ADAM17, IL-11, IL-11 Rα, or IL-11 co-stained with CD3 or CD163 in the skin of SSc patients and healthy controls were analyzed. Fibroblasts were treated with IL-11 and ionomycin to evaluate the profibrotic effect of IL-11 trans-signaling pathway. TJ301 (sgp130Fc) and WP1066 (a JAK2/STAT3 inhibitor) intervention groups were set up to investigate the antifibrotic effect of targeting IL-11. RESULTS: Levels of plasma IL-11 were extremely low in most SSc patients and healthy controls. In contrast, levels of IL-11, IL-11 Rα, and ADAM10, but not ADAM17, were significantly elevated in the skin of SSc patients. Moreover, the numbers of IL-11+ CD3+ cells and IL-11+ CD163+ cells were increased in the skin of SSc patients. Besides, IL-11 and ADAM10 were also elevated in the skin and pulmonary of bleomycin-induced SSc mouse. Fibroblasts co-stimulated with IL-11 and ionomycin showed increased expression of COL3 and phosphorylation of STAT3, which could be inhibited by TJ301 or WP1066. TJ301 also ameliorated skin and lung fibrosis in BLM-induced SSc mouse. CONCLUSIONS: IL-11 induces fibrosis in SSc by regulating the trans-signaling pathway. Blockage of sgp130Fc or inhibition of the JAK2/STAT3 pathway could ameliorate the profibrotic effect of IL-11.

Dissecting Interleukin-6 Classic and Trans-signaling in Inflammation and Cancer.

Interleukin-6 (IL-6) is a cytokine synthesized by many cells in the human body. IL-6 binds to a membrane-bound receptor (IL-6R), which is only present on hepatocytes, some epithelial cells, and some leukocytes. The complex of IL-6 and IL-6R binds to the ubiquitously expressed receptor subunit gp130, which forms a homodimer and thereby initiates intracellular signaling, e.g., the JAK/STAT and MAPK pathways. Proteases can cleave the membrane-bound IL-6R from the cell surface and generate a soluble IL-6R (sIL-6R), which retains its ability to bind IL-6. The IL-6/sIL-6R complex associates with gp130 and induces signaling even on cells which do not express the IL-6R. This paradigm has been called IL-6 trans-signaling, whereas signaling via the membrane-bound IL-6R is referred to as classic signaling. We have generated several molecular tools to differentiate between both pathways and to analyze the consequences of cellular IL-6 signaling in vivo. One of these tools is soluble gp130Fc, which selectively inhibits IL-6 trans-signaling. This protein under the WHO name Olamkicept has successfully undergone phase II clinical trials in patients with autoimmune diseases. Here, in this chapter, we describe several molecular tools to differentiate between IL-6 classic and trans-signaling and to analyze the consequences of cellular IL-6 signaling in vivo.

Dynamics of the Levels of Interleukin 6, Its Soluble Receptor, and Soluble Glycoprotein 130 in Patients with Chronic Heart Failure and Preserved or Reduced Ejection Fraction.

The features of IL-6 trans-signaling were studied in patients with heart failure with reduced (n=74) and preserved (n=31) ejection fraction (EF) during acute decompensation of HF (ADHF) and after 1 year. Patients with ADHF with reduced EF demonstrated higher levels of IL-6 and soluble glycoprotein 130 in comparison with those in patients with preserved EF: 10.18 (7.07; 16.14) pg/ml vs 6.35 (3.52; 11.00) pg/ml and 543.46 (455.37; 634.43) ng/ml vs 498.50 (408.16; 632.23) ng/ml, respectively. The levels of soluble IL-6 receptor little differed in these groups: 57.82 (47.55; 79.85) ng/ml vs 61.30 (44.97; 78.08) ng/ml. After 1 year, the levels of IL-6 in HF patients with reduced EF significantly decreased (5.36 (3.35; 8.35) pg/ml), while in patients with preserved EF, the decrease in this parameter was less pronounced (5.86 (4.05; 7.32) pg/ml), and the difference between groups disappeared. The levels of soluble glycoprotein 130 increased in both groups: 448.06 (357.74; 550.67) ng/ml vs 385.35 (344.29; 523.72) ng/ml. It should be noted that after 1 year (in stable patients), the levels of soluble IL-6 receptor increased in both groups: 65.75 (54.84; 75.39) ng/ml vs 70.81 (57.51; 82.25) ng/ml. Thus, despite the high levels of IL-6 in HF patients with reduced EF, the potential limiting IL-6 trans-signaling in these patients is higher than in patients with preserved EF.

Inhibition of interleukin-6 trans-signaling improves survival and prevents cognitive impairment in a mouse model of sepsis.

Sepsis-associated encephalopathy (SAE) manifests clinically as acute and chronic cognitive impairments, which is associated with increased morbidity and mortality. Interleukin-6 (IL-6), a pro-inflammatory cytokine, is consistently up-regulated in sepsis. IL-6 initiates proinflammatory effects after binding to soluble IL-6 receptor (IL-6R) through trans-signalling, which requires the transducer gp130. In this study, we investigated whether inhibition of IL-6 trans-signalling is a putative therapeutic target for sepsis and SAE. Twenty-five patients (12 septic and 13 non-septic patients) were recruited for the study. A significant increase of IL-6, IL-1ß, IL-10, and IL-8 was observed in the septic patients 24 h after ICU admission. In animal study, cecal ligation and puncture (CLP) was used to induce sepsis in male C57BL/6J mice. One hour before or after inducing sepsis, mice were treated with sgp130, a selective IL-6 trans-signaling inhibitor, respectively. Survival rate, cognition, levels of inflammatory cytokines, integrity of blood-brain barrier (BBB), and oxidative stress were assessed. In addition, immune cells activation and transmigration were evaluated in peripheral blood and brains. Sgp130 improved survival rate and cognitive functions, reduced levels of inflammatory cytokines, including IL-6, TNF-α, IL-10, and MCP-1, in plasma and hippocampus (hipp), mitigated BBB disruption, and ameliorated sepsis-induced oxidative stress. Sgp130 also affected monocytes/macrophages and lymphocytes transmigration and activation in septic mice. Our results indicate that selective inhibition of IL-6 trans-signaling by sgp130 exerts protective effects against SAE in a mouse model of sepsis, suggesting a potential therapeutic strategy.

The Alzheimer's disease-linked protease BACE1 modulates neuronal IL-6 signaling through shedding of the receptor gp130.

BACKGROUND: The protease BACE1 is a major drug target for Alzheimer's disease, but chronic BACE1 inhibition is associated with non-progressive cognitive worsening that may be caused by modulation of unknown physiological BACE1 substrates. METHODS: To identify in vivo-relevant BACE1 substrates, we applied pharmacoproteomics to non-human-primate cerebrospinal fluid (CSF) after acute treatment with BACE inhibitors. RESULTS: Besides SEZ6, the strongest, dose-dependent reduction was observed for the pro-inflammatory cytokine receptor gp130/IL6ST, which we establish as an in vivo BACE1 substrate. Gp130 was also reduced in human CSF from a clinical trial with a BACE inhibitor and in plasma of BACE1-deficient mice. Mechanistically, we demonstrate that BACE1 directly cleaves gp130, thereby attenuating membrane-bound gp130 and increasing soluble gp130 abundance and controlling gp130 function in neuronal IL-6 signaling and neuronal survival upon growth-factor withdrawal. CONCLUSION: BACE1 is a new modulator of gp130 function. The BACE1-cleaved, soluble gp130 may serve as a pharmacodynamic BACE1 activity marker to reduce the occurrence of side effects of chronic BACE1 inhibition in humans.

In vivo immunomodulation of IL6 signaling in a murine multiple trauma model.

A significant number of trauma patients die during the ICU phase of care because of a severe immune response. Interleukin-6 (IL6) plays a central role within that immune response, signaling through a membrane-bound (IL6-R) and a soluble IL6 receptor (sIL6-R). IL6 and the sIL6-R can form an agonistic IL6/sIL6-R-complex, activating numerous cells that are usually not IL6 responsive, a process called trans-signaling. We attempted to demonstrate that modulation of the IL6 signaling (classic signaling and trans-signaling) can attenuate the devastating immune response after trauma in a murine multiple trauma model. Mice were allocated to three study arms: sham, fracture or polytrauma. Half of the animals had the application of an IL6-R antibody following an intervention. After a pre-set time, blood samples were analysed for IL6 and sIL6-R serum levels, organs were analysed for neutrophil infiltration and end organ damage was evaluated. IL6 and sIL6-R showed a rapid peak after fracture, and much more markedly after polytrauma. These parameters were reduced significantly by globally blocking IL6 signaling via IL6-R antibody (Mab) application. Shock organ analysis also illustrated significant neutrophil infiltration following polytrauma, which was also abated via IL6-R Mab application. Furthermore, end organ damage was reduced by IL6-R Mab application. The study results prove the regulatory role of IL6 signaling pathways in polytrauma, with haemorrhagic shock being a major trigger of inflammatory response. Modulation of IL6 signaling shows promise in the prevention of adverse events like organ failure following major trauma and might be a target for in vivo immunomodulation to reduce mortality in severely injured patients, but further evaluation regarding classic IL6 signaling and IL6 trans-signaling is needed.

Bystander activation of microglia by Brucella abortus-infected astrocytes induces neuronal death via IL-6 trans-signaling.

Inflammation plays a key role in the pathogenesis of neurobrucellosis where glial cell interactions are at the root of this pathological condition. In this study, we present evidence indicating that soluble factors secreted by Brucella abortus-infected astrocytes activate microglia to induce neuronal death. Culture supernatants (SN) from B. abortus-infected astrocytes induce the release of pro-inflammatory mediators and the increase of the microglial phagocytic capacity, which are two key features in the execution of live neurons by primary phagocytosis, a recently described mechanism whereby B. abortus-activated microglia kills neurons by phagocytosing them. IL-6 neutralization completely abrogates neuronal loss. IL-6 is solely involved in increasing the phagocytic capacity of activated microglia as induced by SN from B. abortus-infected astrocytes and does not participate in their inflammatory activation. Both autocrine microglia-derived and paracrine astrocyte-secreted IL-6 endow microglial cells with up-regulated phagocytic capacity that allows them to phagocytose neurons. Blocking of IL-6 signaling by soluble gp130 abrogates microglial phagocytosis and concomitant neuronal death, indicating that IL-6 activates microglia via trans-signaling. Altogether, these results demonstrate that soluble factors secreted by B. abortus-infected astrocytes activate microglia to induce, via IL-6 trans-signaling, the death of neurons. IL-6 signaling inhibition may thus be considered a strategy to control inflammation and CNS damage in neurobrucellosis.