UNASSIGNED: Interleukin 6 (IL6) is both a pleiotropic cytokine and an immune-related gene. Interleukin 6 receptor (IL6R) is the receptor for IL6. It may be closely connected to the development of lung cancer. This research aims to explore the prognostic value of IL6R and prevent overtreatment of patients with lung adenocarcinoma (LUAD).
UNASSIGNED: In this study, the expression of IL6R in tumor tissues and surrounding tissues was first analyzed by immunohistochemistry in the Affiliated Hospital of Nantong University (NTU) cohort. Secondly, we downloaded information from The Cancer Genome Atlas (TCGA) for the TCGA cohort and used this information to explore the messenger RNA (mRNA) level of IL6R. We then used Kaplan-Meier survival analyses, univariate and multivariate Cox analyses, nomogram models, and decision curve analyses to assess the prognostic value of IL6R. In addition, we also analyzed immune cell infiltration and the signaling pathways related to IL6R through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA).
UNASSIGNED: Through the data analysis of the NTU cohort and the TCGA cohort, it was found that the expression of IL6R in normal tissues around the tumor was higher than that in tumor tissue, and was positively correlated with the overall survival (OS) of LUAD patients. Additionally, low expression of IL6R was found to be an independent predictor of poor prognosis among the patients in these two research cohorts. Next, using GO, KEGG, and GSEA analyses, we found that partially infiltrated tumor immune cells might be related to earlier staging and better prognosis of patients with LUAD. Finally, the study of the 3-5-year survival rate of LUAD patients through the nomogram showed that the expression of IL6R could improve the accuracy of prediction to prevent the overtreatment of some LUAD patients.
UNASSIGNED: In summary, our study indicated that the low expression of IL6R was associated with poor prognosis among LUAD patients and that low expression of IL6R is a potential independent risk factor that could provide a basis for strengthening postoperative classification management of such patients.

BACKGROUND: ALOX5, IL6R and SFTPD are all immune related genes that may be involved in the development of lung cancer. We sought to explore the effect of polymorphisms of these genes on the risk of lung cancer.
METHODS: Six single nucleotide polymorphisms (SNPs) were genotyped using a MassARRAY platform in a case-control cohort including 550 patients with lung cancer and 550 healthy controls.
RESULTS: The rs4845626-T and rs4329505-C alleles were associated with a decreased risk of lung cancer (p < 0.001), while the rs745986-G and rs2245121-A alleles were correlated with an increased risk of lung cancer (p < 0.01). The rs4845626-GT/GG and rs4329505-TC genotypes were protective against lung cancer (p < 0.001). However, the rs745986-AG and rs2245121-AG/AA genotypes were associated with an increased risk of lung cancer (p < 0.01). Stratification analysis showed that the rs4845626 and rs4329505 polymorphisms of IL6R were associated with a reduced risk of lung cancer in both smokers and nonsmokers (p < 0.05). However, rs892690, rs745986 and rs2115819 of ALOX5 were associated with an increased risk of disease in nonsmokers, while rs2245121 of SFTPD was correlated with a higher risk of disease in smokers (p < 0.05).
CONCLUSIONS: Our results provide candidate SNPs for early screening for lung cancer and new clues for further study of the pathogenesis of the disease.

UNASSIGNED: Mesenchymal stem cells (MSCs) have the multipotent capacity to differentiate into multiple tissue lineages as well as to self-renew, which is the main origin of adipocytes. IL6/IL6R pathway exerts a significant role in tissue regeneration and cell differentiation. Whereas, the underlying mechanism between IL6/IL6R pathway and MSCs adipogenesis differentiation remains elusive.
UNASSIGNED: MSCs from healthy donors were cultured in adipogenesis differentiation medium for 0∼14 days, during which their adipogenesis differentiation degree was evaluated by Oil Red O staining. The expression of IL6R was detected in MSCs during adipogenesis differentiation. Knockdown and overexpression of IL6R were respectively performed using siRNA and lentivirus to investigate its effect on MSCs adipogenesis differentiation. The adipogenesis marker genes expression and MAPK pathway activation were detected by Western blotting. The role of P38 pathway in the adipogenesis differentiation of MSCs was determined using the specific inhibitor SB203580.
UNASSIGNED: The expression of IL6 and IL6R increased during adipogenesis differentiation in MSCs, which were positively correlated with Oil Red O quantification result. Knockdown and overexpression experiments demonstrated a positive correlation between the expressions of IL6R and MSCs adipogenesis differentiation, accompanied by same trend of P38 phosphorylation. Besides, the specific P38 inhibitor SB203580 markedly inhibited the adipogenesis differentiation potential of MSCs.
UNASSIGNED: This study reveals IL6R facilitates the adiogenesis differentiation of MSCs via activating P38 pathway.

Glaucoma is one of the major causes of blindness, and transforming growth factor-β2 (TGF-β2) has been found to be elevated in the aqueous humor of eyes with primary open-angle glaucoma (POAG). TGF-β2 in aqueous humor causes the glaucoma-related fibrosis of human trabecular meshwork (HTM), suggesting an important role of TGF-β in POAG pathogenesis. Here, we sought to elucidate the effects of IL-6 trans-signaling on TGF-β signaling in HTM cells. Using a multiplex immunoassay, POAG patients decreased IL-6 levels and increased soluble IL-6 receptor (sIL-6R) levels compared with the controls. In in vitro experiments, we observed that the IL-6 level was increased in the conditioned medium of HTM cells after TGF-β2 stimulation. To elucidate the relationship between TGF-β2 and IL-6 in HTM cells, we conducted Western blotting and immunohistochemical analyses, and we noted that the combination of IL-6 and sIL-6R (IL6/sIL-6R) suppressed TGF-β-induced up-regulation of α-smooth muscle actin in HTM cells, whereas IL-6 alone did not. This suggests that trans-signaling, not classic signaling, of IL-6 suppresses TGF-β-induced fibrosis of HTM. IL6/sIL-6R also suppressed TGF-β-mediated activation of myosin light chain 2 (MLC2), Smad2, and p38. Of note, these inhibitory effects of IL6/sIL-6R on TGF-β were partly reduced by siRNA-mediated knockdown of STAT3. Moreover, IL-6/sIL-6R partly inhibited TGF-β-induced activation of the Smad-sensitive promoter detected with luciferase reporter gene assays and up-regulation of TGFRI and TGFRII, evaluated by quantitative real-time RT-PCR. Strikingly, overexpression of TGFRI and TGFRII diminished these inhibitory effects of IL-6/sIL-6R. We conclude that of IL-6-mediated trans-signaling potently represses TGF-β signaling in HTM cells.

Interleukin (IL-)6 is the major pro-inflammatory cytokine within the IL-6 family. IL-6 signals via glycoprotein 130 (gp130) and the membrane-bound or soluble IL-6 receptor (IL-6R), referred to as classic or trans-signaling, respectively. Whereas inflammation triggers IL-6 expression, eventually rising to nanogram/ml serum levels, soluble IL-6R (sIL-6R) and soluble gp130 (sgp130) are constitutively present in the upper nanogram/ml range. Calculations based on intermolecular affinities have suggested that systemic IL-6 is immediately trapped in IL-6·sIL-6R and IL-6·sIL-6R·sgp130 complexes, indicating that sIL-6R and sgp130 constitute a buffer system that increases the serum half-life of IL-6 or restricts systemic IL-6 signaling. However, this scenario has not been experimentally validated. Here, we quantified IL-6·sIL-6R and IL-6·sIL-6R·sgp130 complexes over a wide concentration range. The amounts of IL-6 used in this study reflect concentrations found during active inflammatory events. Our results indicated that most IL-6 is free and not complexed with sIL-6R or sgp130, indicating that the level of endogenous sgp130 in the bloodstream is not sufficient to block IL-6 trans-signaling via sIL-6R. Importantly, addition of the single-domain antibody VHH6, which specifically stabilizes IL-6·sIL-6R complexes but did not bind to IL-6 or sIL-6R alone, drove free IL-6 into IL-6·sIL-6R complexes and boosted trans-signaling but not classic signaling, demonstrating that endogenous sIL-6R has at least the potential to form complexes with IL-6. Our findings indicate that even though high concentrations of sIL-6R and sgp130 are present in human serum, the relative ratio of free IL-6 to IL-6·sIL-6R allows for simultaneous classic and trans-signaling.

Epstein-Barr virus-induced gene 3 (EBI3) is a subunit of the composite cytokines IL-27 and IL-35. Both have beneficial functions or effects in models of infectious and autoimmune diseases. This suggests that administration of EBI3 could be therapeutically useful by binding free p28 and p35 to generate IL-27 and IL-35. IL-27- and IL-35-independent functions of EBI3 could compromise its therapeutic uses. We therefore assessed the effects of EBI3 on cytokine receptor-expressing cells. We observed that EBI3 activates STAT3 and induces the proliferation of the IL-6-dependent B9 mouse plasmacytoma cell line. Analyses using blocking mAbs and Ba/F3 transfectants expressing gp130 indicate that EBI3 activity was linked to its capacity to mediate IL-6 trans-signaling, albeit less efficiently than soluble IL-6Rα. In line with this interpretation, co-immunoprecipitation and SPR experiments indicated that EBI3 binds IL-6. An important pro-inflammatory function of IL-6 trans-signaling is to activate blood vessel endothelial cells. We observed that EBI3 in combination with IL-6 could induce the expression of chemokines by human venal endothelial cells. Our results indicate that EBI3 can promote pro-inflammatory IL-6 functions by mediating trans-signaling. These unexpected observations suggest that use of EBI3 as a therapeutic biologic for autoimmune diseases will likely require co-administration of soluble gp130 to prevent the side effects associated with IL-6 trans-signaling. Together with previous studies that demonstrated activation of IL-6R by p28 (IL-30), new findings further suggest a complex interrelation between IL-27 and IL-6.

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

Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.