Unveiling novel insights into human IL-6 - IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis.

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.

IL-6 Blockade in Cytokine Storm Syndromes.

Interleukin-6 (IL-6) is a pro-inflammatory cytokine elevated in cytokine storm syndromes, including hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS). It is also elevated in cytokine release syndrome (CRS) after immune activating cancer therapies such as chimeric antigen receptor (CAR) T-cells or bispecific T-cell engagers (BITEs) and in some patients after infection with SARS-CoV-2. The interaction of IL-6 with its receptor complex can happen in several forms, making effectively blocking this cytokine's effects clinically challenging. Fortunately, effective clinical agents targeting the IL-6 receptor (tocilizumab) and IL-6 directly (siltuximab) have been developed and are approved for use in humans. IL-6 blockade has now been used to safely and effectively treat several cytokine storm syndromes (CSS). Other methods of investigation in effective IL-6 blockade are underway.

Acovenosigenin A ß-glucoside mediates JAK2-STAT3 signaling pathway by targeting GP130 in A549 and H460 cells based on integrative analysis of transcriptome and proteome and biological verification.

Acovenosigenin A ß-glucoside (AAG) is a cardiac glycoside derived from Streptocaulon juventas (Lour.) Merr, which exhibited the potential in treating lung cancer in our previous research. However, the action mechanism remains unclear. In this research, JAK2-STAT3 signaling pathway was predicted to be the critical regulation pathway based on the integrative analysis of transcriptome and proteome. Western blotting and qPCR assays were performed to identify that AAG can regulate JAK2-STAT3 signaling pathway and its downstream genes, such as c-Myc, Survivin, Cyclin B1, CDK1, Bcl-2. And this action of AAG depended on the suppression of STAT3 phosphorylation and its nuclear translocation through the experiments of Immunofluorescence, transient transfection and cryptotanshinone treatment. Additionally, AAG was discovered to mediate the JAK2-STAT3 pathway in IL-6-driven A549 and H460 cells, which in turn inhibited cell proliferation, promoted mitochondria-related apoptosis, and arrested the cell cycle progression. By molecular docking analysis, CETSA and SIP experiments, the protein of GP130 was identified as the specific target of AAG in A549 and H460 cells. Further studies suggested that AAG inhibited JAK2-STAT3 pathway and its downstream genes by targeting GP130 in nude mice xenograft model in vivo. This research presented that AAG exhibits the promising potential in the treatment of NSCLC.

Myeloid cells coordinately induce glioma cell-intrinsic and cell-extrinsic pathways for chemoresistance via GP130 signaling.

The DNA damage response (DDR) and the blood-tumor barrier (BTB) restrict chemotherapeutic success for primary brain tumors like glioblastomas (GBMs). Coherently, GBMs almost invariably relapse with fatal outcomes. Here, we show that the interaction of GBM and myeloid cells simultaneously induces chemoresistance on the genetic and vascular levels by activating GP130 receptor signaling, which can be addressed therapeutically. We provide data from transcriptomic and immunohistochemical screens with human brain material and pharmacological experiments with a humanized organotypic GBM model, proteomics, transcriptomics, and cell-based assays and report that nanomolar concentrations of the signaling peptide humanin promote temozolomide (TMZ) resistance through DDR activation. GBM mouse models recapitulating intratumoral humanin release show accelerated BTB formation. GP130 blockade attenuates both DDR activity and BTB formation, resulting in improved preclinical chemotherapeutic efficacy. Altogether, we describe an overarching mechanism for TMZ resistance and outline a translatable strategy with predictive markers to improve chemotherapy for GBMs.

Mg-Cross-Linked Alginate Hydrogel Induces BMSC/Macrophage Crosstalk to Enhance Bone Tissue Regeneration via Dual Promotion of the Ligand-Receptor Pairing of the OSM/miR-370-3p-gp130 Signaling Pathway.

Macrophages play a pivotal role in the crosstalk between the immune and skeletal systems, while Mg-based biomaterials demonstrate immunomodulatory capabilities in this procedure. However, the mechanism of how Mg2+ promotes osteogenesis through the interplay of bone marrow-derived mesenchymal stem cells (BMSCs) and macrophages remains undescribed. Here, we demonstrated that a Mg-cross-linked alginate hydrogel exerted a dual enhancement of BMSCs osteogenic differentiation through the ligand-receptor pairing of the OSM/miR-370-3p-gp130 axis. On the one hand, Mg2+, released from the Mg-cross-linked hydrogel, stimulates bone marrow-derived macrophages to produce and secrete more OSM. On the other hand, Mg2+ lowers the miR-370-3p level in BMSCs and in turn, reverses its suppression on gp130. Then, the OSM binds to the gp130 heterodimer receptor and activates intracellular osteogenic programs in BMSCs. Taken together, this study reveals a novel cross-talk pattern between the skeletal and immune systems under Mg2+ stimulation. This study not only brings new insights into the immunomodulatory properties of Mg-based biomaterials for orthopedic applications but also enriches the miRNA regulatory network and provides a promising target to facilitate bone regeneration in large bone defects.

Pharmacological modulation of gp130 signalling enhances Achilles tendon repair by regulating tenocyte migration and collagen synthesis via SHP2-mediated crosstalk of the ERK/AKT pathway.

Tendon injuries typically display limited reparative capacity, often resulting in suboptimal outcomes and an elevated risk of recurrence or rupture. While cytokines of the IL-6 family are primarily recognised for their inflammatory properties, they also have multifaceted roles in tissue regeneration and repair. Despite this, studies examining the association between IL-6 family cytokines and tendon repair remained scarce. gp130, a type of glycoprotein, functions as a co-receptor for all cytokines in the IL-6 family. Its role is to assist in the transmission of signals following the binding of ligands to receptors. RCGD423 is a gp130 modulator. Phosphorylation of residue Y759 of gp130 recruits SHP2 and SOCS3 and inhibits activation of the STAT3 pathway. In our study, RCGD423 stimulated the formation of homologous dimers of gp130 and the phosphorylation of Y759 residues without the involvement of IL-6 and IL-6R. Subsequently, the phosphorylated residues recruited SHP2, activating the downstream ERK and AKT pathways. These mechanisms ultimately promoted the migration ability of tenocytes and matrix synthesis, especially collagen I. Moreover, RCGD423 also demonstrated significant improvements in collagen content, alignment of collagen fibres, and biological and biomechanical function in a rat Achilles tendon injury model. In summary, we demonstrated a promising gp130 modulator (RCGD423) that could potentially enhance tendon injury repair by redirecting downstream signalling of IL-6, suggesting its potential therapeutic application for tendon injuries.

A derivative of 3-(1,3-diarylallylidene)oxindoles inhibits dextran sulfate sodium-induced colitis in mice.

BACKGROUND: IA-0130 is a derivative of 3-(1,3-diarylallylidene)oxindoles, which is a selective estrogen receptor modulator (SERM). A previous study demonstrated that SERM exhibits anti-inflammatory effects on colitis by promoting the anti-inflammatory phenotype of monocytes in murine colitis. However, the therapeutic effects of oxindole on colitis remain unknown. Therefore, we evaluated the efficacy of IA-0130 on dextran sulfate sodium (DSS)-induced mouse colitis. METHODS: The DSS-induced colitis mouse model was established by administration of 2.5% DSS for 5 days. Mice were orally administered with IA-0130 (0.01 mg/kg or 0.1 mg/kg) or cyclosporin A (CsA; 30 mg/kg). Body weight, disease activity index score and colon length of mice were calculated and histological features of mouse colonic tissues were analyzed using hematoxylin and eosin staining. The expression of inflammatory cytokines and tight junction (TJ) proteins were analyzed using quantitative real-time PCR and enzyme-linked immunosorbent assay. The expression of interleukin-6 (IL-6) signaling molecules in colonic tissues were investigated using Western blotting and immunohistochemistry (IHC). RESULTS: IA-0130 (0.1 mg/kg) and CsA (30 mg/kg) prevented colitis symptom, including weight loss, bleeding, colon shortening, and expression of pro-inflammatory cytokines in colon tissues. IA-0130 treatment regulated the mouse intestinal barrier permeability and inhibited abnormal TJ protein expression. IA-0130 down-regulated IL-6 expression and prevented the phosphorylation of signaling molecules in colonic tissues. CONCLUSIONS: This study demonstrated that IA-0130 suppressed colitis progression by inhibiting the gp130 signaling pathway and expression of pro-inflammatory cytokines, and maintaining TJ integrity.

Assessment of serum inflammatory parameters in RRMS and SPMS patients.

OBJECTIVES: Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease. Patients with relapsing-remitting MS (RRMS) and secondary progressive MS (SPMS) differ in their responses to treatment; therefore, the correct diagnosis of the particular type of MS is crucial, and biomarkers that can differentiate between the forms of MS need to be identified. The aim of this study was to compare the levels of inflammatory parameters in serum samples from patients with RRMS and SPMS. METHODS: The study group consisted of 60 patients with diagnosed MS. The patients were divided into RRMS and SPMS groups. In the RRMS patients, the usage of disease-modifying treatment was included in our analysis. The serum levels of inflammatory parameters were evaluated. RESULTS: The serum levels of BAFF, gp130 and osteopontin were significantly higher in SPMS patients than in RRMS patients. The serum levels of BAFF correlated with age in both RRMS and SPMS patients. The serum levels of MMP-2 were significantly higher in RRMS patients than in SPMS patients and correlated with the number of past relapses. The serum levels of IL-32 were significantly higher in RRMS treatment-naïve patients than in RRMS patients treated with disease-modifying therapy. DISCUSSION: Significant differences were found in BAFF, gp130, MMP-2 and osteopontin levels between RRMS and SPMS patients. Serum IL-32 levels were statistically lower in RRMS patients treated with disease-modifying therapy than in treatment-naïve patients.

Plasma IL-1 and IL-6 Family Cytokines with Soluble Receptor Levels at Diagnosis in Head and Neck Squamous Cell Carcinoma: High Levels Predict Decreased Five-Year Disease-Specific and Overall Survival.

Activation of the acute-phase cascade (APC) has been correlated with outcomes in various cancers, including head and neck squamous cell carcinoma (HNSCC). Primary drivers of the APC are the cytokines within the interleukin-6 (IL-6) and IL-1 families. Plasma levels of IL-6 family cytokines/soluble receptors (IL-6, IL-27, IL-31, OSM, CNTF, soluble (s-)gp130, s-IL-6Rα) and IL-1 family members (IL-1RA, s-IL-33Rα) were determined at diagnosis for 87 human papillomavirus (HPV)-negative (-) HNSCC patients. We then studied the 5-year Disease-Specific Survival (DSS) and Overall Survival (OS). Increased plasma levels of IL-6 (p < 0.001/p < 0.001) (DSS/OS), IL-31 (p = 0.044/p = 0.07), IL-1RA (p = 0.004/p = 0.035), soluble (s)-IL-6Rα p = 0.022/p = 0.035), and s-gp130 (p = 0.007/p = 0.003) at diagnosis were predictors of both OS and DSS from HPV(-) HNSCC patients. The cytokine DSS/OS predictions were associated with TNM stage and smoking history, whereas the soluble receptors IL-6Rα, gp130, and IL33Rα more uniquely predicted DSS/OS. Clinically, IL-6 levels above 2.5 pg/mL yielded 75% specificity and 70% sensitivity for DSS. In conclusion, high plasma levels of IL-6, IL-31, and IL-1RA, as well as the soluble receptors IL-6Rα, gp130, and IL33Rα, predicted clinical outcome. This shows their potential as candidates for both general therapy and immune therapy stratification, as well as being future platforms for the development of new immunotherapy.

E3 Ubiquitin Ligase NEDD4L Negatively Regulates Skin Tumorigenesis by Inhibiting IL-6/GP130 Signaling Pathway.

IL-6 signaling plays a crucial role in the survival and metastasis of skin cancer. NEDD4L acts as a suppressor of IL-6 signaling by targeting GP130 degradation. However, the effects of the NEDD4L-regulated IL-6/GP130 signaling pathway on skin cancer remain unclear. In this study, protein expression levels of NEDD4L and GP130 were measured in tumor tissues from patients with cutaneous squamous cell carcinoma. Skin tumors were induced in wild-type and Nedd4l-knockout mice, and activation of the IL-6/GP130/signal transducer and activator of transcription 3 signaling pathway was detected. The results indicated a negative correlation between the protein expression levels of NEDD4L and GP130 in cutaneous squamous cell carcinoma tissues from patients. Nedd4l deficiency significantly promoted 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate-induced skin tumorigenesis and benign-to-malignant conversion by activating the IL-6/GP130/signal transducer and activator of transcription 3 signaling pathway, which was abrogated by supplementation with the GP130 inhibitor SC144. Furthermore, our findings suggested that NEDD4L can interact with GP130 and promote its ubiquitination in skin tumors. In conclusion, our results indicate that NEDD4L could act as a tumor suppressor in skin cancer, and inhibition of GP130 could be a potential therapeutic method for treating this disease.

Engineered interleukin-6-derived cytokines recruit artificial receptor complexes and disclose CNTF signaling via the OSMR.

Ciliary neurotrophic factor (CNTF) activates cells via the non-signaling α-receptor CNTF receptor (CNTFR) and the two signaling ß-receptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). The CNTF derivate, Axokine, was protective against obesity and insulin resistance, but clinical development was halted by the emergence of CNTF antibodies. The chimeric cytokine IC7 used the framework of interleukin (IL-)6 with the LIFR-binding site from CNTF to activate cells via IL-6R:gp130:LIFR complexes. Similar to CNTF/Axokine, IC7 protected mice from obesity and insulin resistance. Here, we developed CNTF-independent chimeras that specifically target the IL-6R:gp130:LIFR complex. In GIL-6 and GIO-6, we transferred the LIFR binding site from LIF or OSM to IL-6, respectively. While GIO-6 signals via gp130:IL-6R:LIFR and gp130:IL-6R:OSMR complexes, GIL-6 selectively activates the IL-6R:gp130:LIFR receptor complex. By re-evaluation of IC7 and CNTF, we discovered the Oncostatin M receptor (OSMR) as an alternative non-canonical high-affinity receptor leading to IL-6R:OSMR:gp130 and CNTFR:OSMR:gp130 receptor complexes, respectively. The discovery of OSMR as an alternative high-affinity receptor for IC7 and CNTF designates GIL-6 as the first truly selective IL-6R:gp130:LIFR cytokine, whereas GIO-6 is a CNTF-free alternative for IC7.

Langat virus inhibits the gp130/JAK/STAT signaling by reducing the gp130 protein level.

The tick-borne encephalitis virus (TBEV) serocomplex includes several medically important flavivirus members endemic to Europe, Asia, and North America, which can induce severe neuroinvasive or viscerotropic diseases with unclear mechanisms of pathogenesis. Langat virus (LGTV) shares a high sequence identity with TBEV but exhibits lower pathogenic potential in humans and serves as a model for virus-host interactions. In this study, we demonstrated that LGTV infection inhibits the activation of gp130/JAK/STAT (Janus kinases (JAK) and signal transducer and activator of transcription (STAT)) signaling, which plays a pivotal role in numerous biological processes. Our data show that the LGTV-infected cells had significantly lower phosphorylated STAT3 (pSTAT3) protein upon oncostatin M (OSM) stimulation than the mock-infected control. LGTV infection blocked the nuclear translocation of STAT3 without a significant effect on total STAT3 protein level. LGTV inhibited JAK1 activation and reduced gp130 protein expression in infected cells, with the viral NS5 protein mediating this effect. TBEV infection also reduces gp130 level. On the other hand, pretreatment of Vero cells with OSM significantly reduces LGTV replication, and STAT1/STAT2 knockdown had little effect on OSM-mediated antiviral effect, which suggests it is independent of STAT1/STAT2 and, instead, it is potentially mediated by STAT3 signlaing. These findings shed light on the LGTV and TBEV-cell interactions, offering insights for the future development of antiviral therapeutics and improved vaccines.

ART1 knockdown decreases the IL-6-induced proliferation of colorectal cancer cells.

Colorectal cancer (CRC) is a worldwide health concern. Chronic inflammation is a risk factor for CRC, and interleukin-6 (IL-6) plays a pivotal role in this process. Arginine-specific mono-ADP-ribosyltransferase-1 (ART1) positively regulates inflammatory cytokines. ART1 knockdown reduces the level of glycoprotein 130 (gp130), a key transducer in the IL-6 signalling pathway. However, the relationship between ART1 and IL-6 and the resulting effects on IL-6-induced proliferation in CRC cells remain unclear. The aims of this study were to investigate the effects of ART1 knockdown on IL-6-induced cell proliferation in vitro and use an in vivo murine model to observe the growth of transplanted tumours. The results showed that compared with the control, ART1-sh cancer cells induced by IL-6 exhibited reduced viability, a lower rate of colony formation, less DNA synthesis, decreased protein levels of gp130, c-Myc, cyclin D1, Bcl-xL, and a reduced p-STAT3/STAT3 ratio (P < 0.05). Moreover, mice transplanted with ART1-sh CT26 cells that had high levels of IL-6 displayed tumours with smaller volumes (P < 0.05). ART1 and gp130 were colocalized in CT26, LoVo and HCT116 cells, and their expression was positively correlated in human CRC tissues. Overall, ART1 may serve as a promising regulatory factor for IL-6 signalling and a potential therapeutic target for human CRC.

Craniosynostosis-associated variants in the IL-11R complex: new insights and questions.

Skull growth involves the expansion of both the flat calvarial bones of the skull and the fibrous marginal zones, termed sutures, between them. This process depends on co-ordinated proliferation of mesenchymal-derived progenitor cells within the sutures, and their differentiation to osteoblasts which produce the bone matrix required to expand the size of the bony plates. Defects lead to premature closure of these sutures, termed craniosynostosis, resulting in heterogeneous head shape differences due to restricted growth of one or more sutures. The impact on the individual depends on how many and which sutures are affected and the severity of the effect. Several genetic loci are responsible, including a wide range of variants in the gene for the interleukin 11 receptor (IL11RA, OMIM#600939). Recent work from Kespohl and colleagues provides new insights into how some of these variants influence IL-11R function; we discuss their influences on IL-11R structure and IL-11 function as a stimulus of osteoblast differentiation.

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 Family of Cytokines in Cancers.

Nine soluble ligands [interleukin-6 (IL-6), interleukin-11 (IL-11), leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine, interleukin-27 (IL-27), and interleukin-31] share the ubiquitously expressed transmembrane protein-glycoprotein-130 beta-subunit (gp130) and thus form IL-6 family cytokines. Proteins that may be important for cancerogenesis, CT-1, IL-11, IL-27, LIF, OSM, and CNTF, belong to the superfamily of IL-6. Cytokines such as IL-6, IL-11, and IL-27 are better investigated in comparison with other members of the same family of cytokines, eg, CT-1. Gp130 is one of the main receptors through which these cytokines exert their effects. The clinical implication of understanding the pathways of these cytokines in oncology is that targeted therapy to inhibit or potentiate cytokine activity may lead to remission in some cases.

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.

Molecular characterization of the craniosynostosis-associated interleukin-11 receptor variants p.T306_S308dup and p.E364_V368del.

Interleukin-11 (IL-11) is a member of the IL-6 family of cytokines and is an important factor for bone homeostasis. IL-11 binds to and signals via the membrane-bound IL-11 receptor (IL-11R, classic signaling) or soluble forms of the IL-11R (sIL-11R, trans-signaling). Mutations in the IL11RA gene, which encodes the IL-11R, are associated with craniosynostosis, a human condition in which one or several of the sutures close prematurely, resulting in malformation of the skull. The biological mechanisms of how mutations within the IL-11R are linked to craniosynostosis are mostly unexplored. In this study, we analyze two variants of the IL-11R described in craniosynostosis patients: p.T306_S308dup, which results in a duplication of three amino-acid residues within the membrane-proximal fibronectin type III domain, and p.E364_V368del, which results in a deletion of five amino-acid residues in the so-called stalk region adjacent to the plasma membrane. The stalk region connects the three extracellular domains to the transmembrane and intracellular region of the IL-11R and contains cleavage sites for different proteases that generate sIL-11R variants. Using a combination of bioinformatics and different biochemical, molecular, and cell biology methods, we show that the IL-11R-T306_S308dup variant does not mature correctly, is intracellularly retained, and does not reach the cell surface. In contrast, the IL-11R-E364_V368del variant is fully biologically active and processed normally by proteases, thus allowing classic and trans-signaling of IL-11. Our results provide evidence that mutations within the IL11RA gene may not be causative for craniosynostosis and suggest that other regulatory mechanism(s) are involved but remain to be identified.

The Human GP130 Cytokine Receptor and Its Expression-an Atlas and Functional Taxonomy of Genetic Variants.

Genetic variants in IL6ST encoding the shared cytokine receptor for the IL-6 cytokine family GP130 have been associated with a diverse number of clinical phenotypes and disorders. We provide a molecular classification for 59 reported rare IL6ST pathogenic or likely pathogenic variants and additional polymorphisms. Based on loss- or gain-of-function, cytokine selectivity, mono- and biallelic associations, and variable cellular mosaicism, we grade six classes of IL6ST variants and explore the potential for additional variants. We classify variants according to the American College of Medical Genetics and Genomics criteria. Loss-of-function variants with (i) biallelic complete loss of GP130 function that presents with extended Stüve-Wiedemann Syndrome; (ii) autosomal recessive hyper-IgE syndrome (HIES) caused by biallelic; and (iii) autosomal dominant HIES caused by monoallelic IL6ST variants both causing selective IL-6 and IL-11 cytokine loss-of-function defects; (iv) a biallelic cytokine-specific variant that exclusively impairs IL-11 signaling, associated with craniosynostosis and tooth abnormalities; (v) somatic monoallelic mosaic constitutively active gain-of-function variants in hepatocytes that present with inflammatory hepatocellular adenoma; and (vi) mosaic constitutively active gain-of-function variants in hematopoietic and non-hematopoietic cells that are associated with an immune dysregulation syndrome. In addition to Mendelian IL6ST coding variants, there are common non-coding cis-acting variants that modify gene expression, which are associated with an increased risk of complex immune-mediated disorders and trans-acting variants that affect GP130 protein function. Our taxonomy highlights IL6ST as a gene with particularly strong functional and phenotypic diversity due to the combinatorial biology of the IL-6 cytokine family and predicts additional genotype-phenotype associations.