Adult learning, memory, and social interaction partially depend on neurogenesis in two regions: the hippocampus and the subventricular zone. There is evidence that the immune system is important for these processes in pathological situations, but there is no review of its role in non-pathological or near-physiological conditions. Although further research is warranted in this area, some conclusions can be drawn. Intrusive LyC6hi monocytes and autoreactive CD4+ T cells have a positive impact on neurogenesis and behavior, but the latter are deleterious if specific to external antigens. Mildly activated microglia play a crucial role in promoting these processes, by eliminating apoptotic neuronal progenitors and producing low levels of interleukins, which increase if the cells are activated, leading to inhibition of neurogenesis. Chemokines are poorly studied, but progenitor cells and neurons express their receptors, which appear important for migration and maturation. The few works that jointly analyzed neurogenesis and behavior showed congruent effects of immune cells and cytokines. In conclusion, the immune system components -mostly local- seem of utmost importance for the control of behavior under non-pathological conditions.

The pathophysiology of a stroke is a complex process involving oxidative stress and inflammation. As a result of the actions of reactive oxygen species (ROS), not only does vascular damage occur, but the brain tissue is also damaged. It is a dynamic process, induced by a cellular-molecular immune response, focused on the development of an immediate reaction. During ischemia, inflammatory mediators are released, among which IL-6 plays a particularly important role in the acute phase of a stroke. Recently, a lot of attention has been devoted to this pleiotropic pro-inflammatory cytokine, which enhances the migration of leukocytes and is controlled by chemokines and the expression of adhesion handlers. The impact of IL-6 on the severity of neurological treatment and on patient prognosis in AIS is of interest to many researchers. More and more data indicate that it may be a reliable prognostic factor in strokes.

Cytokines are important soluble mediators that are involved in physiological and pathophysiological processes. Among them, members of the interleukin-6 (IL-6) family of cytokines have gained remarkable attention, because especially the name-giving cytokine IL-6 has been shown to be an excellent target to treat inflammatory and autoimmune diseases. The IL-6 family consists of nine members, which activate their target cells via combinations of non-signaling α- and/or signal-transducing β-receptors. While some receptor combinations are exclusively used by a single cytokine, other cytokine receptor combinations are used by multiple cytokines. Research in recent years unraveled another level of complexity: several cytokine cannot only signal via their canonical receptors, but can bind to and signal via additional α- and/or β-receptors, albeit with less affinity. While several examples of such cytokine plasticity have been reported, a systematic analysis of this phenomenon is lacking. The development of artificial intelligence programs like AlphaFold allows the computational analysis of protein complexes in a systematic manner. Here, we develop a analysis pipeline for cytokine:cytokine receptor interaction and show that AlphaFold-Multimer correctly predicts the canonical ligands of the IL-6 family. However, AlphaFold-Multimer does not provide sufficient insight to conclusively predict alternative, low-affinity ligands for receptors within the IL-6 family.

Cytokines influence cell-fate decisions of naïve lymphocytes and determine outcome of immune responses by transducing signals that regulate the initiation, intensity and duration of immune responses. However, aberrant regulation of physiological levels of cytokines contribute to the development of autoimmune and other inflammatory diseases. The Interleukin 6 (IL-6)/IL-12 superfamily of cytokines have a profound influence on all aspects of host immunity and our focus in this review is on the signaling pathways that mediate their functions, with emphasis on how this enigmatic family of cytokines promote or suppress inflammation depending on the physiological context. We also describe regulatory lymphocyte populations that suppress neuroinflammatory diseases by producing cytokines, such as IL-27 (i27-Breg) or IL-35 (i35-Breg and iTR35). We conclude with emerging immunotherapies like STAT-specific Nanobodies, Exosomes and Breg therapy that ameliorate CNS autoimmune diseases in preclinical studies.

Interleukin-11 (IL-11), a member of the IL-6 cytokine family, has potential pro-inflammatory and pro-fibrotic roles in pulmonary, hepatic, cardiovascular, renal and intestinal disease pathogenesis, including oncogenesis. The potential for therapeutic intervention in these disease spaces has therefore made the IL-11 signaling axis an attractive target in drug discovery, and antibody inhibitors of IL-11 signaling are currently under evaluation in Phase I/II clinical trials. While lower molecular weight small molecule and peptide inhibitors may offer the potential for improved tissue penetration, developability and manufacturing cost compared with a protein therapeutic, reports of such chemical matter in the literature are limited. In this work, a series of cyclic peptides derived from phage display biopanning campaigns against both IL-11 and its cognate receptor IL-11Rα are presented. The most active IL-11 binder (peptide 4, KD 140 nM) exhibited inhibition of IL-11/IL-11Rα dimerization in a biochemical AlphaLISA assay (Ki 300 nM), and alanine scanning was carried out on this sequence to identify residues important for target binding and inhibitory activity. Further structural optimization yielded lead peptide 15 (Ki 180 nM), which exhibited at least 70-fold greater activity than IL-11 inhibitors previously reported in the literature. The de novo peptide macrocycles presented serve as a robust starting point for development of therapeutic inhibitors of the IL-11/IL-11Rα interaction.

Maintenance of delicate homeostasis is very important in various diseases because it ensures appropriate immune surveillance against pathogens and prevents excessive inflammation. In a disturbed homeostatic condition, hyperactivation of immune cells takes place and interplay between these cells triggers a plethora of signaling pathways, releasing various pro-inflammatory cytokines such as Tumor necrosis factor alpha (TNFα), Interferon-gamma (IFNƴ), Interleukin-6 (IL-6), and Interleukin-1 beta (IL-1β), which marks cytokine storm formation. To be precise, dysregulated balance can impede or increase susceptibility to various pathogens. Pathogens have the ability to hijack the host immune system by interfering with the host's chromatin architecture for their survival and replication in the host cell. Cytokines, particularly IL-6, Interleukin-17 (IL-17), and Interleukin-23 (IL-23), play a key role in orchestrating innate immune responses and shaping adaptive immunity. Understanding the interplay between immune response and the role of epigenetic modification to maintain immune homeostasis and the structural aspects of IL-6, IL-17, and IL-23 can be illuminating for a novel therapeutic regimen to treat various infectious diseases. In this review, the light is shed on how the orchestration of epigenetic regulation facilitates immune homeostasis.

Interleukin-6 (IL-6) is a multifaceted cytokine essential in many immune system processes and their regulation. It also plays a key role in hematopoiesis, and in triggering the acute phase reaction. IL-6 overproduction is critical in chronic inflammation associated with autoimmune diseases like rheumatoid arthritis and contributes to cytokine storms in COVID-19 patients. Over 20 years ago, researchers proposed that IL-6, which is typically monomeric, can also form dimers via a domain-swap mechanism, with indirect evidence supporting their existence. The physiological significance of IL-6 dimers was shown in B-cell chronic lymphocytic leukemia. However, no structures have been reported so far. Here, we present the crystal structure of an IL-6 domain-swapped dimer that computational approaches could not predict. The structure explains why the IL-6 dimer is antagonistic to the IL-6 monomer in signaling complex formation and provides insights for IL-6 targeted therapies.

Breast cancer stem cells (CSCs) are resistant to most cancer therapeutics and contribute to tumor recurrence and metastasis. Two breast CSC-promoting transcription factors, truncated glioma-associated oncogene homolog 1 (tGLI1) and signal transducer and activator of transcription 3 (STAT3), have been reported to be frequently co-expressed in HER2-enriched breast cancer and triple-negative breast cancer (TNBC), undergo protein-protein interactions for gene regulation and activation, and functionally cooperate to promote breast CSCs. STAT3 can be activated by activated interleukin-6 receptor/glycoprotein-130 (IL-6R/GP130). Co-targeting of tGLI1 and IL-6R/GP130 has not been investigated in breast cancer or any tumor type. Here, we report that tGLI1 and GP130 are co-overexpressed in the majority of HER2-enriched breast cancers and TNBCs at 53.8% and 44.4%, respectively. tGLI1+IL-6/IL-6R/GP130 signaling is frequently co-enriched and co-activated in HER2-enriched breast cancer and TNBC when compared to luminal subtypes. tGLI1+GP130 co-overexpression strongly promotes CSCs of HER2-enriched breast cancer and TNBC. FDA-approved tGLI1 inhibitor Ketoconazole and GP130 inhibitor Bazedoxifene synergize against breast cancer proliferation and CSC phenotypes in vitro and reduce TNBC tumor growth and metastatic burden in vivo. Our study demonstrates, for the first time, that co-targeting tGLI1 and IL-6R/GP130/STAT3 signaling pathways is synergistic against HER2-enriched breast cancer and TNBC, warranting future clinical investigations.

Sterile inflammation has been increasingly recognized as a hallmark of non-infectious kidney diseases. Induction of pro-inflammatory cytokines in injured kidney tissue promotes infiltration of immune cells serving to clear cell debris and facilitate tissue repair. However, excessive or prolonged inflammatory response has been associated with immune-mediated tissue damage, nephron loss, and development of renal fibrosis. Interleukin 6 (IL-6) is a cytokine with pleiotropic effects including a major role in inflammation. IL-6 signals either via membrane-bound (classic signaling) or soluble receptor forms (trans-signaling) thus affecting distinct cell types and eliciting various metabolic, cytoprotective, or pro-inflammatory reactions. Antibodies neutralizing IL-6 or its receptor have been developed for therapy of autoimmune and chronic non-renal inflammatory diseases. Small molecule inhibitors of Janus kinases acting downstream of the IL-6 receptor, as well as recombinant soluble glycoprotein 130 variants suppressing the IL-6 trans-signaling add to the available therapeutic options. Animal data and accumulating clinical experience strongly suggest that suppression of IL-6 signaling pathways bears therapeutic potential in acute and chronic kidney diseases. The present work analyses the renoprotective potential of clinically relevant IL-6 signaling inhibitors in acute kidney injury, chronic kidney disease, and kidney transplantation with focus on current achievements and future prospects.

Given the increasing effectiveness of immune-based therapies, management of their associated toxicities is of utmost importance. Cytokine release syndrome (CRS), characterized by elevated levels of cytokine, poses a significant challenge following the administration of antibodies and CAR-T cell therapies. CRS also contributes to multiple organ dysfunction in severe viral infections, notably in COVID-19. Given the pivotal role of IL-6 cytokine in initiating CRS, it has been considered a most potential therapeutic target to mitigate hyperactivated immune responses. While monoclonal antibodies of IL-6 show promise in mitigating cytokine storm, concerns about immunotoxicity persist, and small molecule IL-6 antagonists remain unavailable. The present study employed sophisticated computational techniques to identify potential hit compounds as IL-6 inhibitors, with the aim of inhibiting IL-6/IL-6R protein-protein interactions. Through ligand-based pharmacophore mapping and shape similarity in combination with docking-based screening, we identified nine hit compounds with diverse chemical scaffolds as potential binders of IL-6. Further, the MD simulation of 300 ns of five virtual hits in a complex with IL-6 was employed to study the dynamic behavior. To provide a more precise prediction, binding free energy was also estimated. The identified compounds persistently interacted with the residues lining the binding site of the IL-6 protein. These compounds displayed low binding energy during MMPBSA calculations, substantiating their strong association with IL-6. This study suggests promising scaffolds as potential inhibitors of IL-6/IL-6R protein-protein interactions and provides direction for lead optimization.

Abiotic D-proteins that selectively bind to natural L-proteins have gained significant biotechnological interest. However, the underlying structural principles governing such heterochiral protein-protein interactions remain largely unknown. In this study, we present the de novo design of D-proteins consisting of 50-65 residues, aiming to target specific surface regions of L-proteins or L-peptides. Our designer D-protein binders exhibit nanomolar affinity toward an artificial L-peptide, as well as two naturally occurring proteins of therapeutic significance: the D5 domain of human tropomyosin receptor kinase A (TrkA) and human interleukin-6 (IL-6). Notably, these D-protein binders demonstrate high enantiomeric specificity and target specificity. In cell-based experiments, designer D-protein binders effectively inhibited the downstream signaling of TrkA and IL-6 with high potency. Moreover, these binders exhibited remarkable thermal stability and resistance to protease degradation. Crystal structure of the designed heterochiral D-protein-L-peptide complex, obtained at a resolution of 2.0 Å, closely resembled the design model, indicating that the computational method employed is highly accurate. Furthermore, the crystal structure provides valuable information regarding the interactions between helical L-peptides and D-proteins, particularly elucidating a novel mode of heterochiral helix-helix interactions. Leveraging the design of D-proteins specifically targeting L-peptides or L-proteins opens up avenues for systematic exploration of the mirror-image protein universe, paving the way for a diverse range of applications.

In Interleukin (IL)-6 signalling, IL-6 site I binds to the IL-6 receptor (IL-6R) first, following by IL-6 site II interaction to domain 2/3 of gp130 to form premature trimeric IL-6:IL-6R:gp130 receptor complexes. Formation of the mature hexameric receptor complex is then facilitated by the inter-trimeric interaction of IL-6 site III with domain 1 of the opposing gp130. The two gp130-associated Janus kinases (JAKs) trans-phosphorylate when their spatiotemporal pairing is correct, which causes the activation of STAT, ERK, and AKT pathways in a balanced manner. Since the intracellular domain (ICD) of IL-6R is not needed for STAT/ERK/AKT phosphorylation, we investigated the conditions under which a chimeric IL-6RECD-gp130TMD/ICD receptor protein confers biological activity. For IL-6RECD-gp130TMD/ICD, the extracellular domain (ECD) of IL-6R was fused to the transmembrane domain (TMD) and ICD of gp130. Co-expression of IL-6RECD-gp130TMD/ICD with signalling-deficient gp130 variants did not induce IL-6 signalling, suggesting that the assembly of hexameric complexes failed to dimerize the IL-6R-associated JAKs correctly. By mimicking the premature trimeric receptor complex, IL-6-mediated dimerization of IL-6RECD-gp130TMD/ICD with the single-cytokine-binding variant gp130ΔD1 induced signalling. Of note, IL-6 signalling via these synthetic gp130ΔD1:IL-6RECD-gp130TMD/ICD complexes resulted predominantly in STAT3 phosphorylation. A STAT3-dominated profile was also observed after IL-6-induced signalling mediated by a JAK-deficient IL-6RECD-gp130TMD/ICDΔJAK variant in complex with the JAK-proficient but STAT/ERK/AKT-deficient gp130JAKΔICD variant. Our data showed that effective ERK/AKT signalling could not be executed after intracellular domain swapping from gp130 to the IL-6R. Taken together, the chimeric IL-6R/gp130 receptor may be helpful in the creation of customized synthetic IL-6 signalling.

Periodontitis is a chronic inflammatory disease of the periodontium that can lead to the loss of affected teeth if left untreated. It is induced by a multifactorial process centered on microbial pathogens such as Fusobacterium nucleatum (F.n.). Non-invasive physical plasma (NIPP), a highly reactive gas, has become a focus of research, not only for its hemostatic, proliferation-enhancing and apoptotic properties, but also for its antimicrobial potential. The objective of this study was to examine the impact of NIPP on human periodontal ligament (PDL) cells that had been induced into a state of periodontal infection in vitro.

Initially, the solitary effect of NIPP was evaluated by measuring temperature and pH and analyzing reactive oxygen species (ROS). Additionally, DAPI and phalloidin staining were employed to investigate possible cytotoxic effects. The cells were pre-incubated with F.n. and treated with NIPP after 24 hours. Interleukin (IL)-6 and IL-8 were analyzed at mRNA and protein levels, respectively, by real-time PCR and ELISA.

NIPP alone had no significant effect on PDL cells. However, the F.n.-induced upregulation of IL-6 and IL-8 was counteracted by NIPP.

Thus, the utilization of NIPP may be regarded as a promising therapeutic strategy for the treatment of periodontal diseases.

Oncostatin M (OSM) is a unique Interleukin 6 (IL-6) family cytokine that plays pivotal roles in numerous biological events by signaling via two types of receptor complexes. While type I OSM receptor complex is formed by glycoprotein 130 (gp130) heterodimerization with Leukemia Inhibitory Factor receptor (LIFR), type II OSM receptor complex is composed of gp130 and OSM receptor (OSMR). OSM is an important contributor to multiple inflammatory diseases and cancers while OSM inhibition has been shown to be effective at reducing symptoms, making OSM an attractive therapeutic target. Using cryogenic electron microscopy (cryo-EM), we characterize full extracellular assemblies of human type I OSM receptor complex and mouse type II OSM receptor complex. The juxtamembrane domains of both complexes are situated in close proximity due to acute bends of the receptors. The rigid N-terminal extension of OSM contributes to gp130 binding and OSM signaling. Neither glycosylation nor pro-domain cleavage of OSM affects its activity. Mutagenesis identifies multiple OSM and OSMR residues crucial for complex formation and signaling. Our data reveal the structural basis for the assemblies of both type I and type II OSM receptor complexes and provide insights for modulation of OSM signaling in therapeutics.

gp130 functions as a shared signal-transducing subunit not only for interleukin (IL)-6 but also for eight other human cytokine receptor complexes. The IL-6 signaling pathway mediated through gp130 encompasses classical, trans, or cluster signaling, intricately regulated by a diverse array of modulators affecting IL-6, its receptor, and gp130. Currently, only a limited number of small molecule antagonists and agonists for gp130 are known. This review aims to comprehensively examine the current knowledge of these modulators and provide insights into their pharmacological properties, particularly in the context of cancer and other diseases. Notably, the prominent gp130 modulators SC144, bazedoxifene, and raloxifene are discussed in detail, with a specific focus on the discovery of SC144's iron-chelating properties. This adds a new dimension to the understanding of its pharmacological effects and therapeutic potential in conditions where iron homeostasis is significant. Our bioinformatic analysis of gp130 and genes related to iron homeostasis reveals insightful correlations, implicating the role of iron in the gp130 signaling pathway. Overall, this review contributes to the evolving understanding of gp130 modulation and its potential therapeutic applications in various disease contexts. SIGNIFICANCE STATEMENT: This perspective provides a timely and comprehensive analysis of advancements in gp130 signaling research, emphasizing the therapeutic implications of the currently available modulators. Bioinformatic analysis demonstrates potential interplay between gp130 and genes that regulate iron homeostasis, suggesting new therapeutic avenues. By combining original research findings with a broader discussion of gp130's therapeutic potential, this perspective significantly contributes to the field.

Interleukin (IL)-12 is a heterodimeric pro-inflammatory cytokine. Our cryoelectron microscopy structure determination of human IL-12 in complex with IL-12Rβ1 and IL-12Rβ2 at a resolution of 3.75 Å reveals that IL-12Rβ2 primarily interacts with the IL-12p35 subunit via its N-terminal Ig-like domain, while IL-12Rβ1 binds to the p40 subunit with its N-terminal fibronectin III domain. This binding mode of IL-12 with its receptors is similar to that of IL-23 but shows notable differences with other cytokines. Through structural information and biochemical assays, we identified Y62, Y189, and K192 as key residues in IL-12p35, which bind to IL-12Rβ2 with high affinity and mediate IL-12 signal transduction. Furthermore, structural comparisons reveal two distinctive conformational states and structural plasticity of the heterodimeric interface in IL-12. As a result, our study advances our understanding of IL-12 signal initiation and opens up new opportunities for the engineering and therapeutic targeting of IL-12.

The landscape of clinical management for metastatic melanoma (MM) and other solid tumors has been modernized by the advent of immune checkpoint inhibitors (ICI), including programmed cell death-1 (PD-1), programmed cell death-ligand 1 (PD-L1), and cytotoxic T lymphocyte antigen 4 (CTLA-4) inhibitors. While these agents demonstrate efficacy in suppressing tumor growth, they also lead to immune-related adverse events (irAEs), resulting in the exacerbation of autoimmune diseases such as rheumatoid arthritis (RA), ulcerative colitis (UC), and Crohn's disease (CD). The immune checkpoint inhibitors offer promising advancements in the treatment of melanoma and other cancers, but they also present significant challenges related to irAEs and autoimmune diseases. Ongoing research is crucial to better understand these challenges and develop strategies for mitigating adverse effects while maximizing therapeutic benefits. In this manuscript, we addressed this challenge using network-based approaches by constructing and analyzing the molecular and signaling networks associated with tumor-immune crosstalk. Our analysis revealed that IL6 is the key regulator responsible for irAEs during ICI therapies. Furthermore, we conducted an integrative network and molecular-level analysis, including virtual screening, of drug libraries, such as the Collection of Open Natural Products (COCONUT) and the Zinc15 FDA-approved library, to identify potential IL6 inhibitors. Subsequently, the compound amprenavir was identified as the best molecule that may disrupt essential interactions between IL6 and IL6R, which are responsible for initiating the signaling cascades underlying irAEs in ICI therapies.

Doxorubicin is an important anticancer drug, however, elicits dose-dependently cardiomyopathy. Given its mode of action, i.e. topoisomerase inhibition and DNA damage, we investigated genetic events associated with cardiomyopathy and searched for mechanism-based possibilities to alleviate cardiotoxicity. We treated rats at clinically relevant doses of doxorubicin. Histopathology and transmission electron microscopy (TEM) defined cardiac lesions, and transcriptomics unveiled cardiomyopathy-associated gene regulations. Genomic-footprints revealed critical components of Abl1-p53-signaling, and EMSA-assays evidenced Abl1 DNA-binding activity. Gene reporter assays confirmed Abl1 activity on p53-targets while immunohistochemistry/immunofluorescence microscopy demonstrated Abl1, p53&p73 signaling.

Doxorubicin treatment caused dose-dependently toxic cardiomyopathy, and TEM evidenced damaged mitochondria and myofibrillar disarray. Surviving cardiomyocytes repressed Parkin-1 and Bnip3-mediated mitophagy, stimulated dynamin-1-like dependent mitochondrial fission and induced anti-apoptotic Bag1 signaling. Thus, we observed induced mitochondrial biogenesis. Transcriptomics discovered heterogeneity in cellular responses with minimal overlap between treatments, and the data are highly suggestive for distinct cardiomyocyte (sub)populations which differed in their resilience and reparative capacity. Genome-wide footprints revealed Abl1 and p53 enriched binding sites in doxorubicin-regulated genes, and we confirmed Abl1 DNA-binding activity in EMSA-assays. Extraordinarily, Abl1 signaling differed in the heart with highly significant regulations of Abl1, p53 and p73 in atrial cardiomyocytes. Conversely, in ventricular cardiomyocytes, Abl1 solely-modulated p53-signaling that was BAX transcription-independent. Gene reporter assays established Abl1 cofactor activity for the p53-reporter PG13-luc, and ectopic Abl1 expression stimulated p53-mediated apoptosis.

The tyrosine kinase Abl1 is of critical importance in doxorubicin induced cardiomyopathy, and we propose its inhibition as means to diminish risk of cardiotoxicity.

Interleukin-6 (IL-6), a multifunctional cytokine, is an attractive therapeutic target for immunological and inflammatory diseases. We investigated the chemical synthesis of IL-6 and its enantiomer (d-IL-6) using a sequential N-to-C native chemical ligation strategy from six peptide segments. Solubilizing Trt-K10 tags improved the intermediate solubility and served as protecting groups during the metal-free desulfurization to facilitate the synthesis of full-length IL-6 protein. Synthetic l-IL-6 and recombinant IL-6 exhibited nearly identical structural and binding properties. The symmetrical binding property of d-IL-6 was also demonstrated by functional analysis using IL-6-binding peptides. The resulting functional d-IL-6 was employed to screen a phage-displayed antibody fragment library, leading to the identification of several d-IL-6-binding single-domain antibodies. This work will contribute to the development of novel, potent IL-6 inhibitors without the adverse effects of undesired immune activation.

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

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.

Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo-designed fibroblast growth factor receptor (FGFR)-binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.

Inflammatory bowel disease (IBD) is characterized by abnormal immune responses, including elevated proinflammatory cytokines, such as tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) in the gastrointestinal (GI) tract. This study presents the synthesis and anti-inflammatory evaluation of 2,4,5-trimethylpyridin-3-ol analogues, which exhibit dual inhibition of TNFα- and IL-6-induced inflammation. Analysis using in silico methods, including 3D shape-based target identification, modeling, and docking, identified G protein-coupled estrogen receptor 1 (GPER) as the molecular target for the most effective analogue, 6-26, which exhibits remarkable efficacy in ameliorating inflammation and restoring colonic mucosal integrity. This was further validated by surface plasmon resonance (SPR) assay results, which showed direct binding to GPER, and by the results showing that GPER knockdown abolished the inhibitory effects of 6-26 on TNFα and IL-6 actions. Notably, 6-26 displayed no cytotoxicity, unlike G1 and G15, a well-known GPER agonist and an antagonist, respectively, which induced necroptosis independently of GPER. These findings suggest that the GPER-selective compound 6-26 holds promise as a therapeutic candidate for IBD.

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.

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.

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.

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.

Ciliary neurotrophic factor (CNTF) activates cells via the non-signaling α-receptor CNTF receptor (CNTFR) and the two signaling β-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.

Interleukin-6 (IL-6)-type cytokines not only have key immunomodulatory functions that affect the pathogenesis of diseases such as autoimmune diseases, chronic inflammatory conditions, and cancer, but also fulfill important homeostatic tasks. Even though the pro-inflammatory arm has hindered the development of therapeutics based on natural-like IL-6-type cytokines to date, current synthetic trends might pave the way to overcome these limitations and eventually lead to immune-inert designer cytokines to aid type 2 diabetes and brain injuries. Those synthetic biology approaches include mutations, fusion proteins, and inter-cytokine swapping, and resulted in IL-6-type cytokines with altered receptor affinities, extended target cell profiles, and targeting of non-natural cytokine receptor complexes. Here, we survey synthetic cytokine developments within the IL-6-type cytokine family and discuss potential clinical applications.

Excessive STAT3 signalling via gp130, the shared receptor subunit for IL-6 and IL-11, contributes to disease progression and poor survival outcomes in patients with colorectal cancer. Here, we provide evidence that bazedoxifene inhibits tumour growth via direct interaction with the gp130 receptor to suppress IL-6 and IL-11-mediated STAT3 signalling. Additionally, bazedoxifene combined with chemotherapy synergistically reduced cell proliferation and induced apoptosis in patient-derived colon cancer organoids. We elucidated that the primary mechanism of anti-tumour activity conferred by bazedoxifene treatment occurs via pro-apoptotic responses in tumour cells. Co-treatment with bazedoxifene and the SMAC-mimetics, LCL161 or Birinapant, that target the IAP family of proteins, demonstrated increased apoptosis and reduced proliferation in colorectal cancer cells. Our findings provide evidence that bazedoxifene treatment could be combined with SMAC-mimetics and chemotherapy to enhance tumour cell apoptosis in colorectal cancer, where gp130 receptor signalling promotes tumour growth and progression.

Cannabidiol (CBD) is the most relevant nonpsychostimulant phytocompound found in Cannabis sativa. CBD has been extensively studied and has been proposed as a therapeutic candidate for neuroinflammation-related conditions. However, being a highly lipophilic drug, it has several drawbacks for pharmaceutical use, including low solubility and high permeability. Synthetic polymers can be used as drug delivery systems to improve CBD's stability, half-life, and biodistribution. Here, we propose using a synthetic polymer as a nanoparticulate vehicle for CBD (NPCBD) to overcome the pharmacological drawbacks of free drugs. We tested the NPCBD-engineered system in the context of ischemic events in a relevant oxygen and glucose deprivation (OGD) model in primary cortical cells (PCC). Moreover, we have characterized the inflammatory response of relevant cell types, such as THP-1 (human monocytes), HMC3 (human microglia), and PCC, to NPCBD and observed a shift in the inflammatory state of the treated cells after the ischemic event. In addition, NPCBD exhibited a promising ability to restore mitochondrial function after OGD insult in both HMC3 and PCC cells at low doses of 1 and 0.2 μM CBD. Taken together, these results suggest the potential for preclinical use.

The pancreas is a heterocrine gland that has both exocrine and endocrine parts. Most pancreatic cancer begins in the cells that line the ducts of the pancreas and is called pancreatic ductal adenocarcinoma (PDAC). PDAC is the most encountered pancreatic cancer type. One of the most important characteristic features of PDAC is neuropathy which is primarily due to perineural invasion (PNI). PNI develops tumor microenvironment which includes overexpression of fibroblasts cells, macrophages, as well as angiogenesis which can be responsible for neuropathy pain. In tumor microenvironment inactive fibroblasts are converted into an active form that is cancer-associated fibroblasts (CAFs). Neurotrophins they also increase the level of Substance P, calcitonin gene-related peptide which is also involved in pain. Matrix metalloproteases are the zinc-associated proteases enzymes which activates proinflammatory interleukin-1β into its activated form and are responsible for release and activation of Substance P which is responsible for neuropathic pain by transmitting pain signal via dorsal root ganglion. All the molecules and their role in being responsible for neuropathic pain are described below.

"Neurodegenerative disorder" is an umbrella term for a group of fatal progressive neurological illnesses characterized by neuronal loss and inflammation. Interleukin-6 (IL-6), a pleiotropic cytokine, significantly affects the activities of nerve cells and plays a pivotal role in neuroinflammation. Furthermore, as high levels of IL-6 have been frequently observed in association with several neurodegenerative disorders, it may potentially be used as a biomarker for the progression and prognosis of these diseases. This review summarizes the production and function of IL-6 as well as its downstream signaling pathways. Moreover, we make a comprehensive review on the roles of IL-6 in neurodegenerative disorders and its potential clinical application.

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.

IL-11 and IL-6 activate signalling via assembly of the cell surface receptor gp130; however, it is unclear how signals are transmitted across the membrane to instruct cellular responses. Here we solve the cryoEM structure of the IL-11 receptor recognition complex to discover how differences in gp130-binding interfaces may drive signalling outcomes. We explore how mutations in the IL6ST gene encoding for gp130, which cause severe immune deficiencies in humans, impair signalling without blocking cytokine binding. We use cryoEM to solve structures of both IL-11 and IL-6 complexes with a mutant form of gp130 associated with human disease. Together with molecular dynamics simulations, we show that the disease-associated variant led to an increase in flexibility including motion within the cytokine-binding core and increased distance between extracellular domains. However, these distances are minimized as the transmembrane helix exits the membrane, suggesting a stringency in geometry for signalling and dimmer switch mode of action.

Breast cancer is the most frequent malignancy in women. However, in the middle and late stages, some people develop distant metastases, which considerably lower the quality of life and life expectancy. The brain is one of the sites where metastasis frequently happens. According to epidemiological research, brain metastases occur at a late stage in 30-50% of patients with HER2-positive breast cancer, resulting in a poor prognosis. Additionally, few treatments are available for HER2-positive brain metastatic breast cancer, and the mortality rate is remarkable owing to the complexity of the brain's anatomical structure and physiological function. In this review, we described the stages of the brain metastasis of breast cancer, the relationship between the microenvironment and metastatic cancer cells, and the unique molecular and cellular mechanisms. It involves cancer cells migrating, invading, and adhering to the brain; penetrating the blood-brain barrier; interacting with brain cells; and activating signal pathways once inside the brain. Finally, we reviewed current clinically used treatment approaches for brain metastasis in HER2-positive breast cancer; summarized the traditional treatment, targeted treatment, immunotherapy, and other treatment modalities; compared the benefits and drawbacks of each approach; discussed treatment challenges; and emphasized the importance of identifying potential targets to improve patient survival rates and comprehend brain metastasis in breast cancer.

The spinal inflammatory signal often spreads to distant segments, accompanied by widespread pain symptom under neuropathological conditions. Multiple cytokines are released into the cerebrospinal fluid (CSF), potentially inducing the activation of an inflammatory cascade at remote segments through CSF flow. However, the detailed alteration of CSF in neuropathic pain and its specific role in widespread pain remain obscure.

A chronic constriction injury of the infraorbital nerve (CCI-ION) model was constructed, and pain-related behavior was observed on the 7th, 14th, 21st, and 28th days post surgery, in both vibrissa pads and hind paws. CSF from CCI-ION rats was transplanted to naïve rats through intracisternal injection, and thermal and mechanical allodynia were measured in hind paws. The alteration of inflammatory cytokines in CCI-ION's CSF was detected using an antibody array and bioinformatic analysis. Pharmacological intervention targeting the changed cytokine in the CSF and downstream signaling was performed to evaluate its role in widespread pain.

CCI-ION induced local pain in vibrissa pads together with widespread pain in hind paws. CCI-ION's CSF transplantation, compared with sham CSF, contributed to vibrissa pad pain and hind paw pain in recipient rats. Among the measured cytokines, interleukin-6 (IL-6) and leptin were increased in CCI-ION's CSF, while interleukin-13 (IL-13) was significantly reduced. Furthermore, the concentration of CSF IL-6 was correlated with nerve injury extent, which gated the occurrence of widespread pain. Both astrocytes and microglia were increased in remote segments of the CCI-ION model, while the inhibition of astrocytes in remote segments, but not microglia, significantly alleviated widespread pain. Mechanically, astroglial signal transducer and activator of transcription 3 (STAT3) in remote segments were activated by CSF IL-6, the inhibition of which significantly mitigated widespread pain in CCI-ION.

IL-6 was induced in the CSF of the CCI-ION model, triggering widespread pain via activating astrocyte STAT3 signal in remote segments. Therapies targeting IL-6/STAT3 signaling might serve as a promising strategy for the widespread pain symptom under neuropathological conditions.