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.

Low dose methotrexate (LD-MTX) remains the gold standard in rheumatoid arthritis (RA) therapy. Multiple mechanisms on a variety of immune cells contribute to the anti-inflammatory effects of LD-MTX. Inflammatory signaling is deeply implicated in hematopoiesis by regulating hematopoietic stem and progenitor cell (HSPC) fate decisions; raising the question of whether HSPC are also modulated by LD-MTX. This is the first study to characterize the effects of LD-MTX on HSPC. CD34+ HSPC were isolated from healthy donors' non-mobilized peripheral blood. Resting and/or cycling HSPCs were treated with LD-MTX [dose equivalent to that used in RA patients]. Flow cytometry was performed to assess HSPC viability, cell cycle, surface abundance of reduced folate carrier 1 (RFC1), proliferation, reactive oxygen species (ROS) levels, DNA double-strand breaks, p38 activation, and CD34+ subpopulations. HSPC clonogenicity was tested in colony-forming cell assays. Our results indicate that in cycling HSPC, membrane RFC1 is upregulated and, following LD-MTX treatment, they accumulate more intracellular MTX than resting HSPC. In cycling HSPC, LD-MTX inhibits HSPC expansion by promoting S-phase cell-cycle arrest, increases intracellular HSPC ROS levels and DNA damage, and reduces HSPC viability. Those effects involve the activation of the p38 MAPK pathway and are rescued by folinic acid. The effects of LD-MTX are more evident in CD34+ CD38High progenitors. In non-cycling HSPC, LD-MTX also reduces the proliferative response while preserving their clonogenicity. In summary, HSPC uptake LD-MTX differentially according to their cycling state. In turn, LD-MTX results in reduced proliferation and the preservation of HSPC clonogenicity.

Cytokine storm (CS) is a severe systemic inflammatory syndrome characterized by the excessive activation of immune cells and a significant increase in circulating levels of cytokines. This pathological process is implicated in the development of life-threatening conditions such as fulminant myocarditis (FM), acute respiratory distress syndrome (ARDS), primary or secondary hemophagocytic lymphohistiocytosis (HLH), cytokine release syndrome (CRS) associated with chimeric antigen receptor-modified T (CAR-T) therapy, and grade III to IV acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation. The significant involvement of the JAK-STAT pathway, Toll-like receptors, neutrophil extracellular traps, NLRP3 inflammasome, and other signaling pathways has been recognized in the pathogenesis of CS. Therapies targeting these pathways have been developed or are currently being investigated. While novel drugs have demonstrated promising therapeutic efficacy in mitigating CS, the overall mortality rate of CS resulting from underlying diseases remains high. In the clinical setting, the management of CS typically necessitates a multidisciplinary team strategy encompassing the removal of abnormal inflammatory or immune system activation, the preservation of vital organ function, the treatment of the underlying disease, and the provision of life supportive therapy. This review provides a comprehensive overview of the key signaling pathways and associated cytokines implicated in CS, elucidates the impact of dysregulated immune cell activation, and delineates the resultant organ injury associated with CS. In addition, we offer insights and current literature on the management of CS in cases of FM, ARDS, systemic inflammatory response syndrome, treatment-induced CRS, HLH, and other related conditions.

Hantavirus causes hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Strong inflammatory responses and vascular leakage are important hallmarks of these often fatal diseases. The mechanism behind pathogenesis is unknown and no specific treatment is available. IL-6 was recently highlighted as a biomarker for HPS/HFRS severity. IL-6 signaling is complex and context dependent: while classical signaling generally provide protective responses, trans-signaling can cause severe pathogenic responses. Here, we investigated a potential role for IL-6 trans-signaling in hantavirus pathogenesis.

Effects of IL-6 trans-signaling during in vitro hantavirus infection were assessed using primary human endothelial cells treated with recombinant soluble IL-6 receptor (sIL-6R). Plasma from Puumala orthohantavirus-infected HFRS patients (n=28) were analyzed for IL-6 trans-signaling potential and its associations to severity.

In vitro, sIL-6R treatment of infected cells enhanced IL-6 and CCL2 secretion, upregulated ICAM-1, and affected VE-cadherin leading to a disrupted cell barrier integrity. HFRS patients showed altered plasma levels of sIL-6R and soluble gp130 (sgp130) resulting in an increased sIL-6R/sgp130 ratio suggesting enhanced IL-6 trans-signaling potential. Plasma sgp130 levels negatively correlated with number of interventions and positively with albumin levels. Patients receiving oxygen treatment displayed a higher sIL-6R/sgp130 ratio compared to patients that did not.

IL-6 trans-signaling is linked to hantavirus pathogenesis. Targeting IL-6 trans-signaling might provide a therapeutic strategy for treatment of severe HFRS and perhaps also HPS.

A novel neutral water-soluble polysaccharide (HCBP1-1) was isolated and purified from Hemerocallis citrina Borani by column chromatography. The fine structure of HCBP1-1 was determined by a series of physical and chemical means. HCBP1-1 was a homogeneous low molecular weight polysaccharide of 7.25 kDa. Methylation and NMR analysis revealed that HCBP1-1 consisted of α-D-Glcp-(1→, →4)-α-D-Glcp-(1→, →4)-β-D-Galp-(1→, →4,6)-α-D-Glcp-(1→, →3,4)-α-D-Glcp-(1→. In particular, the main backbone of HCBP1-1 consisted of unsubstituted (4-α-D-Glcp and 4-β-D-Galp) and monosubstituted (4,6-α-D-Glcp and 3,4-α-D-Glcp) glucose and galactose units linked by α-(1 → 4) and β-(1 → 4) glycosidic linkages. The side chain consisted of α-D-Glcp-(1 → and →4)-α-D-Glcp-(1→. Furthermore, bioactivity assays show that HCBP1-1 significantly promoted the secretion of NO, TNF-α, IL-1β, and IL-6 and the expression of NF-κB p65 in RAW264.7 cells, which had good immune-enhancing activity. These results suggest that HCBP1-1 had beneficial immunomodulatory effects and could be developed into functional foods or nutraceuticals with immune-enhancing properties.

The study objective was to determine if a common single nucleotide polymorphism in the interleukin 6 (IL-6) receptor (rs2228145, p.Asp358Ala) predicted treatment response to tocilizumab in giant cell arteritis (GCA).

Genetic sequencing of the rs2228145 locus was performed in 2 independent cohorts of patients with GCA. Peripheral blood mononuclear cells (PBMCs) from patients and controls were evaluated for expression of the interleukin 6 receptor (IL-6R) and its coreceptor, gp130, using flow cytometry. The same PBMCs were stimulated with IL-6 and evaluated for downstream targets of IL-6: STAT3 phosphorylation (pSTAT3) and IL-17A expression.

In total, 100 patients with GCA were included (derivation cohort n = 58; validation cohort n = 42). The rs2228145 variant predicted tocilizumab response in each cohort. In the derivation cohort, a gene dose-dependent response was observed with a 36% response rate in the homozygous patients and 95% response rate in patients without the variant (P = .003). In the validation cohort, tocilizumab response rates were 50% for homozygotes and 85% for patients without the variant (P = .04). pSTAT3 levels were significantly increased in response to IL-6 stimulation in a gene dose-dependent manner in CD4 T cells from patients with GCA but not controls. CD4 T cells from patients with GCA had significantly higher membrane expression of gp130 than healthy controls, and response to IL-6 correlated with gp130 expression. IL-17 producing CD4 T cells were increased in a gene dose-dependent response to IL-6 (P < .01).

The rs2228145 variant is associated with decreased treatment response to tocilizumab and worse outcomes in GCA by enhancing CD4 T cell response to IL-6.

There have been remarkable advancements in understanding the complex and dynamic immune biological processes engaged during all stages of pregnancy. Exquisite control of immune processes is critical to successful outcome in all stages of pregnancy from ovulation to birth. There are many immunomodulatory therapeutics that may offer beneficial treatment options for a variety of diseases (e.g., inflammation/autoimmunity, cancer) to patients that are or desire to become pregnant. It is important to understand the potential for these immunomodulatory therapeutics to alter the critical immune processes in pregnancy to inform clinical risk relative to successful pregnancy. The Health and Environmental Sciences Institute-Developmental and Reproductive Toxicology/Immuno-safety Technical Committee (HESI DART/ITC) conducted a survey on approaches to assess adverse pregnancy outcomes with immunomodulators. HESI DART/ITC also organized a workshop for an extended discussion on immune mechanisms during pregnancy, the adequacy of current tools/methodologies to identify concerns for potential pregnancy hazards from immunomodulatory therapies, ways to identify and address scientific gaps, and global regulatory considerations across various immunomodulatory modalities and indications. In this manuscript we summarize learnings from these efforts to characterize risk within this patient population, promote more informed treatment decisions, and enable safer pharmacological interventions during pregnancy.

Surface nano-modification has emerged as an effective strategy to enhance osseointegration of titanium (Ti) implants. Despite its promise, rational optimization of surface nanomorphology for ameliorated osseointegration remains a significant challenge. Our research pioneering developed a one-step alkali etching technique to produce a gradient nanowire coating with continuously varied dimensions on Ti surfaces, which was subsequently served as a versatile platform for high-throughput screening of optimal dimensions to enhance osseointegration. The results showed that macrophages (MФs) that mainly governed the initial inflammatory reaction exhibited a polarization tendency towards pro-healing M2 phenotype with decreased nanowire dimension due to nanomorphology-mediated focal adhesion formation and activation of its downstream signaling pathways (typically PI3K-Akt). Simultaneously, small-sized nanowires with diameter of 5.63-14.25 nm and inter-spacing of 29.42-57.97 nm were conductive to angiogenesis of endothelial cells (ECs) and osteogenesis of bone marrow mesenchymal stem cells (BMSCs), which may share similar mechanisms of MФs. The in vivo results well corroborated these in vitro observations. The knowledge gained from the present work not only advance our understanding of the interaction between surface morphology and cells, but also potentially pave the way for efficient and cost-effective design of advanced biomaterial surfaces for better osseointegration.

The interactions between viruses and the host immune response are nuanced and intricate. The cytokine response arguably plays a central role in dictating the outcome of virus infection, balancing inflammation, and healing, which is crucial to resolving infection without destructive immunopathologies.

Early innate immune responses are key to the generation of a beneficial or detrimental immune response. These initial responses are regulated by a plethora of surface bound, endosomal, and cytoplasmic innate immune receptors known as pattern recognition receptors. Of these, the Toll-like receptors (TLRs) play an important role in the induction of cytokines during virus infection. Recognizing pathogen-associated molecular patterns (PAMPs) such as viral proteins and/or nucleotide sequences, the TLRs act as sentinels for the initiation and propagation of immune responses.

TLRs are important receptors for initiating the innate response to single-stranded RNA (ssRNA) viruses like influenza A virus (IAV), severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), SARS-CoV-2, Middle East respiratory syndrome coronavirus, dengue virus, and Ebola virus. Infection with these viruses is also associated with aberrant expression of proinflammatory cytokines that contribute to a harmful cytokine storm response. Herein we discuss the connections between these ssRNA viruses, cytokine storm, and the roles of TLRs.

The interactions between viruses and the host immune response are nuanced and intricate. The cytokine response arguably plays a central role in dictating the outcome of virus infection, balancing inflammation, and healing, which is crucial to resolving infection without destructive immunopathologies.

Early innate immune responses are key to the generation of a beneficial or detrimental immune response. These initial responses are regulated by a plethora of surface bound, endosomal, and cytoplasmic innate immune receptors known as pattern recognition receptors. Of these, the Toll-like receptors (TLRs) play an important role in the induction of cytokines during virus infection. Recognizing pathogen-associated molecular patterns (PAMPs) such as viral proteins and/or nucleotide sequences, the TLRs act as sentinels for the initiation and propagation of immune responses.

TLRs are important receptors for initiating the innate response to single-stranded RNA (ssRNA) viruses like influenza A virus (IAV), severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), SARS-CoV-2, Middle East respiratory syndrome coronavirus, dengue virus, and Ebola virus. Infection with these viruses is also associated with aberrant expression of proinflammatory cytokines that contribute to a harmful cytokine storm response. Herein we discuss the connections between these ssRNA viruses, cytokine storm, and the roles of TLRs.

Follicular dendritic cell-secreted protein (FDC-SP) is produced by follicular dendritic cells, periodontal ligament and junctional epithelium (JE). JE exists immediately apical to the bottom of the pocket and binds enamel with hemidesmosomes to protect the periodontium from bacterial infection. To analyze the transcriptional regulation of the FDC-SP gene by interleukin-6 (IL-6), we performed real-time PCR, Western blotting, immunofluorescence, luciferase (LUC) assays, gel mobility shift and chromatin immunoprecipitation (ChIP) assays using Ca9-22 and Sa3 gingival epithelial cells. IL-6 increased FDC-SP mRNA and protein levels at 3-24 h. IL-6 increased LUC activities of the LUC constructs containing FDC-SP gene promoter sequences from -116 to -717 bp upstream from the transcriptional start site. IL-6 induced LUC activities of -345FDCSP were inhibited by protein kinase A, tyrosine kinase, mitogen-activated protein kinase kinase, phosphoinositide 3-kinase, signal transducer, activator of transcription 3 (STAT3) and glycoprotein 130 inhibitors. Gel shift and ChIP assays showed that IL-6 induced Yin Yang1 (YY1), GATA binding protein (GATA), CCAAT/enhancer-binding protein (C/EBP) β, phosphorylated STAT3 (p-STAT3) binding to YY1, GATA, C/EBP2, C/EBP3 and GAS2-3 elements. These results indicate that IL-6 induces FDC-SP gene transcription YY1, GATA, C/EBP2, GAS2-3 and C/EBP3 elements in the human FDC-SP gene promoter, and suggesting that FDC-SP may be involved in the defense against JE in periodontium during the progression of periodontitis.

Structural, functional, and molecular-level changes in the aging heart are influenced by a dynamic interplay between immune signaling and cellular metabolism that is referred to as immunometabolism. This review explores the crosstalk between cellular metabolic pathways including glycolysis, oxidative phosphorylation, fatty acid metabolism, and the immune processes that govern cardiac aging. With a rapidly aging population that coincides with increased cardiovascular risk and cancer incidence rates, understanding the immunometabolic underpinnings of cardiac aging provides a foundation for identifying therapeutic targets to mitigate cardiac dysfunction. Aging alters the immune environment of the heart by concomitantly driving the changes in immune cell metabolism, mitochondrial dysfunction, and redox signaling. Shifts in these metabolic pathways exacerbate inflammation and impair tissue repair, creating a vicious cycle that accelerates cardiac functional decline. Treatment with cancer therapy further complicates this landscape, as aging-associated immunometabolic disruptions augment the susceptibility to cardiotoxicity. The current review highlights therapeutic strategies that target the immunometabolic axis to alleviate cardiac aging pathologies. Interventions include modulating metabolic intermediates, improving mitochondrial function, and leveraging immune signaling pathways to restore cardiac health. Advances in immunometabolism thus hold significant potential for translating preclinical findings into therapies that improve the quality of life for the aging population and underscore the need for approaches that address the immunometabolic mechanisms of cardiac aging, providing a framework for future research.

Relapsing-remitting multiple sclerosis is associated with changes in Jak/STAT pathways in immune cells, but the influence of disease-modifying drugs on these pathways is poorly understood. The aim of this study was to evaluate the impact of first-line disease-modifying drugs used in treatment of RRMS on expression of the STAT pathway and T-cell-related genes in the blood and on serum concentrations of sgp130 and TGF-β1 in women, as well as on the level of phosphorylated STAT3 and STAT5 proteins in T cells of untreated patients and heathy controls. Expression of STAT1, STAT3, STAT5A, STAT5B, SOCS1, SOCS3, FOXP3, IKZF2, RORC and ICOS genes in the blood of untreated RRMS patients, in the blood of patients treated with interferon-β, glatiramer acetate, dimethyl fumarate or teriflunomide and in the blood of healthy controls was evaluated using droplet digital PCR. Serum concentrations of sgp130 and TGF-β1 were evaluated by ELISA. Phosphorylated STAT3 and STAT5 protein levels in T cells were evaluated by flow cytometry. STAT3 gene expression was significantly higher in untreated patients than in healthy control, but the level of phosphorylated STAT3 in T cells was significantly lower. Patients treated with interferon-β or dimethyl fumarate had significantly lower STAT3 gene expression. Patients treated with teriflunomide had higher STAT1 gene expression, than untreated patients. Patients treated with dimethyl fumarate also had significantly lower RORC gene expression than untreated patients. The study shows the impact of drugs used in first-line treatment of relapsing-remitting multiple sclerosis on expression of STAT and T-cell-related genes.

Cardiovascular disease (CVD) remains a major global health concern and a leading cause of morbidity and mortality worldwide. Early-diagnosis and prompt medical attention are crucial in managing and reducing overall impact on health-and-wellbeing, necessitating the development of innovative diagnostics, which transcend traditional methodologies. Raman spectroscopy uniquely provides molecular fingerprinting and structural information, offering insights into biochemical composition. Integration of Raman spectroscopy with advanced machine learning is established as a powerful clinical adjunct for point-of-care detection of CVDs. A non-invasive, label-free spectroscopic platform coupled with neural network algorithm, 'SKiNET' has been developed to accurately detect the biomolecular changes within plasma of CVD versus healthy cohorts, enabling rapid diagnosis and longer-term monitoring, where the real-time capabilities provide dynamic assessment of progression, aligning treatment strategies with evolving states. CVD has been detected and classified via SKiNET with 88.6 %-accuracy, 92.9 %-specificity and 85.1 %-sensitivity and with 83.8 %-accuracy. The hybrid RS-SKiNET bio-molecularly specific detection signposted a comprehensive panel of CVD-indicative biomarkers, including SIL-6, IL-9, LpA, ApoB, PCSK9 and NT-ProBNP, offering important insights into disease mechanisms and risk-stratification. This multidimensional technique holds potential for improved patient-and-healthcare management for CVDs, laying the platform toward high-throughput biomolecular profiling of CVD-indicative macromolecular biomarkers, particularly vital for widespread point-of-care diagnostics and monitoring.

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.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA gamma herpesvirus. Like other herpesviruses, KSHV establishes a latent infection with limited gene expression, while KSHV occasionally undergoes the lytic replication phase, which produces KSHV progenies and infects neighboring cells. KSHV genome encodes 80+ open reading frames. One of the KSHV genes, K2, encodes viral interleukin 6 (vIL-6), a homolog of human IL-6 (hIL-6), mainly expressed in the lytic phase of the virus. vIL-6 plays a crucial role in regulating the expression of other viral genes and is also associated with inducing angiogenesis, cell survival, and immune evasion, which is suggested to promote the development of KSHV-associated diseases. This review summarizes the current knowledge on vIL-6. We focus on the vIL-6 regarding its protein structure, transcriptional regulation, cell signaling pathways, and contribution to the KSHV-associated diseases.

Protein-protein interactions (PPIs) are fundamental to cellular signaling and transduction which marks them as attractive therapeutic drug development targets. What were once considered to be undruggable targets have become increasingly feasible due to the progress that has been made over the last two decades and the rapid technological advances. This work explores the influence of technological innovations on PPI research and development. Additionally, the diverse strategies for discovering, modulating, and characterizing PPIs and their corresponding modulators are examined with the aim of presenting a streamlined pipeline for advancing PPI-targeted therapeutics. By showcasing carefully selected case studies in PPI modulator discovery and development, we aim to illustrate the efficacy of various strategies for identifying, optimizing, and overcoming challenges associated with PPI modulator design. The valuable lessons and insights gained from the identification, optimization, and approval of PPI modulators are discussed with the aim of demonstrating that PPI modulators have transitioned beyond early-stage drug discovery and now represent a prime opportunity with significant potential. The selected examples of PPI modulators encompass those developed for cancer, inflammation and immunomodulation, as well as antiviral applications. This perspective aims to establish a foundation for the effective targeting and modulation of PPIs using PPI modulators and pave the way for future drug development.

Addressing global challenges in wound management has greatly encouraged the emergence of home diagnosis and monitoring devices. This technological shift has accelerated the development of new skin patch sensors for continuous health monitoring. A key requirement is the creation of flexible platforms capable of mimicking human skin features. Here, for the first time, an innovative, highly adaptable electrochemical biosensor with molecularly imprinted polymers (MIPs) is customized for the detection of the inflammatory biomarker interleukin-6 (IL-6). The 3-electrode gold pattern is geometrically standardized onto a 6 µm thick polyimide flexible membrane, an optically transparent, and biocompatible polymeric substrate. Subsequently, a biomimetic sensing layer specifically designed for the detection of IL-6 target is produced on these transducers. The obtained MIP biosensor shows a good linear response within the concentration range 50 pg mL-1-50 ng mL-1, with a low limit of detection (8 pg mL-1). X-ray photoelectron spectroscopy, scanning electron microscopy, and Fourier transform infrared spectroscopy characterizations confirm the modifications of the flexible gold transducer. After optimization, the biosensing device shows remarkable potential in terms of sensitivity, selectivity, and reproducibility. Overall, the integration of a low-cost electrochemical sensor on biocompatible flexible polymers opens the way for a new generation of monitoring tools with higher accuracy, less invasiveness, and greater patient comfort.

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.

Osteoarthritis (OA) is a prevalent joint disorder characterized by joint pain, functional impairment, and disability. The current study investigated the therapeutic effects of intra-articular injection of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) in rats with knee OA.

Fourty five male Wistar rats were randomly divided into three groups (A-C) and received either an intra-articular injection of normal saline (NS) or rBM-MSCs. The normal control group (A, n = 15) received NS, the OA control group (B, n = 15) received NS, and the OA treated group (C, n = 15) received rBM-MSCs (0.5 × 106 cells in 25 μL NS). Knee OA was induced using monosodium iodoacetate (MIA). rBM-MSCs were sourced from female Wistar rats and their stem cells were characterized using flow cytometry. Histomorphometric analyses were performed on knee sections from both normal and OA knee. Serum biomarkers, including hyaluronic acid (HA), cross-linked N-telopeptide of type I collagen-1 (NTX-1), NGF, calcitonin gene-related peptide (CGRP), matrix metalloproteinase-3 (MMP-3), oligomeric cartilage matrix protein COMP, interleukin-6 (IL-6), and soluble IL-6 receptor (sIL-6R), were analyzed using ELISA kits. Ingenuity Pathway Analysis (IPA) was used to determine the genes regulated by MSCs in OA, and the protective mechanisms were determined using the Molecular Activity Predictor (MAP).

rBM-MSCs were positive for CD29 and CD90 and negative for CD45 surface markers. OA biomarkers were significantly elevated in the untreated OA group but decreased after treatment with intra-articular MSCs. The OA group treated with MSCs showed significant repair of the damaged cartilage compared to the control group.

Cartilage damage leads to an increase in inflammatory cytokine levels and is associated with an increase in serum biomarkers related to cartilage degradation. Intra-articular administration of MSCs showed beneficial effects, including regeneration of damaged cartilage and a reduction in inflammation-related serum biomarker levels.

Paget's disease of bone (PDB) is characterised by increased and disorganised bone remodelling leading to various complications, such as bone deformity, deafness, secondary osteoarthritis, and pathological fracture. Pain is the most common presenting symptom of PDB, but it is unclear to what extent this is due to increased metabolic activity of the disease, complications, or unrelated causes. We conducted a cross-sectional study of 168 people with PDB attending secondary care referral centres in the UK. We documented the presence of musculoskeletal pain and sought to determine its underlying causes. Musculoskeletal pain was reported by 122/168 (72.6%) individuals. The most common cause was osteoarthritis of joints distant from an affected PDB site in 54 (44.3%), followed by metabolically active PDB in 18 (14.7%); bone deformity in 14 (11.4%); osteoarthritis of a joint neighbouring an affected site in 11 (9.0%), neuropathic pain in 10 (8.2%), and various other causes in the remainder. Pain was more common in women (p<0.019) and in older individuals (p<0.001). Circulating concentrations of macrophage colony-stimulating factor (M-CSF) were significantly higher in those with pain (p = 0.008), but there was no difference between groups of patients with and without pain in concentrations of interleukin-6 (IL-6) or biochemical markers of bone turnover. Pain is a common symptom in PDB but is most often due to osteoarthritis at an unaffected site. The study illustrates the importance of fully evaluating people with PDB to determine the underlying cause of pain so that management can be tailored appropriately.

In previous study, lower levels of serum GP130 were reported in children with MPP. GP130 is an important signal transducer, the down regulation of which may influence host immune responses. In this study, we aimed to analyze the regulatory mechanism of GP130 during MP infection. Firstly, the mRNA and protein levels of GP130 both decrease and then increase with increasing multiplicity of infection (MOI: 1 to 40) of MP. The lowest levels of GP130 were detected at MOI of 5. Then, heat treated MP but not trypsin treated MP or MP extracted proteins show regulatory effect to the expression of GP130. These indicate that the down regulation of GP130 is related to protein mediate adhesion process of MP. Gene expression analysis revealed that MP affected apoptosis and the TLR4 pathway in infected cells, and the mRNA level of IL-6 was correlated with that of GP130. Further, Z-VAD-FMK (pan-caspase inhibitor) can suppress the apoptosis induced by MP infection and restore GP130 at protein level. Further studies revealed that MP infection promoted TLR4 internalization but did not activate the NF-κB pathway. The levels of surface TLR4 showed correlation with the transcription of IL-6 and GP130. TAK242 (TLR4 inhibitor) and PS341 (proteasome inhibitor) can restore the decreased transcription of GP130, both of which were able to promote NF-κB pathway activation in MP-infected cells. These suggested that the regulation of TLR4/NF-κB pathway and induced apoptosis post MP infection are involved in the down-regulation of GP130 at transcription and protein levels, respectively.

An ineffective immune response resulting from dysregulation of cytokine production might encourage viral persistence and cause chronic viral hepatitis to worsen. This study examined the relationship between alterations in interleukin-6 (IL-6) levels and the IL-6 - 174 G > C (rs1800795) polymorphism, as well as how this polymorphism affects the development and progression of chronic hepatitis brought on by hepatitis B (HBV) and hepatitis C (HCV) into hepatocellular carcinoma (HCC).

Whole blood samples from 126 Egyptian patients with HCC (111 with HCV and 15 with HBV), as well as 126 age- and sex-matched healthy individuals, were used to extract DNA. Using PCR-based allele-specific amplification (ASA), the existence of the IL-6 G-174C polymorphism was investigated. Additionally, each participant's serum IL-6 levels were determined using an enzyme-linked immunosorbent assay (ELISA).

The primary observations revealed that HCC patients had greater serum levels of IL-6 compared to the control groups (p < 0.001). Patients with the variant (CG and GG) genotype in the HCC group were found to have more disease severity indicated by higher levels of alpha-fetoprotein (AFP) and a higher ascites grade, as well as increased inflammatory activity as defined by higher levels of IL-6 and C-reactive protein (CRP) (p < 0.001 for both) in comparison to patients with the wild-type (CC) genotype (p < 0.001 and p = 0.002, respectively).

The rs1800795 SNP in the IL-6 gene was associated with increased inflammatory activity and high levels of IL-6, indicating that this SNP may play a role in the development of HCC in Egyptian patients with chronic viral hepatitis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by abdominal pain, diarrhea, rectal bleeding, and weight loss. The pathogenesis and treatment of UC remain key areas of research interest. Various factors, including genetic predisposition, immune dysregulation, and alterations in the gut microbiota, are believed to contribute to the pathogenesis of UC. Current treatments for UC include 5-aminosalicylic acids, corticosteroids, immunosuppressants, and biologics. However, study reported that the one-year clinical remission rate is only around 40%. It is necessary to prompt the exploration of new treatment modalities. Biologic therapies, such as anti-TNF-α monoclonal antibody and JAK inhibitor, primarily consist of small molecules targeting specific pathways, effectively inducing and maintaining remission. Given the significant role of the gut microbiota, research into intestinal microecologics, such as probiotics and prebiotics, and fecal microbiota transplantation (FMT) shows promising potential in UC treatment. Additionally, medicinal herbs, such as chili pepper and turmeric, used in complementary therapy have shown promising results in UC management. This article reviews recent findings on the mechanisms of UC, including genetic susceptibility, immune cell dynamics and cytokine regulation, and gut microbiota alterations. It also discusses current applications of biologic therapy, herbal therapy, microecologics, and FMT, along with their prospects and challenges.

Understanding the pathogenesis and mechanisms of prion diseases can significantly expand our knowledge in the field of neurodegenerative diseases. Prion biology is increasingly recognized as being relevant to the pathophysiology of Alzheimer's disease and Parkinson's disease, both of which affect millions of people each year. This bioinformatics study used a theoretical protein-RNA recognition code (1-L transcription) to reveal the post-transcriptional regulation of the prion protein (PrPC). The principle for this method is directly elucidated on PrPC, in which an octa-repeat can be 1-L transcribed into a GGA triplet repeat RNA aptamer known to reduce the misfolding of normal PrPC into abnormal PrPSc. The identified genes/proteins are associated with mitochondria, cancer, COVID-19 and ER-stress, and approximately half are directly or indirectly associated with prion diseases. For example, the octa-repeat supports CD44, and regions of the brain with astrocytic prion accumulation also display high levels of CD44.

Multiple sclerosis (MS) is a chronic autoimmune disorder characterized by inflammation, demyelination, and neurodegeneration, resulting in significant disability and reduced quality of life. Current therapeutic strategies primarily target immune dysregulation, but limitations in efficacy and tolerability highlight the need for alternative treatments. Plant-derived compounds, including alkaloids, phenylpropanoids, and terpenoids, have demonstrated anti-inflammatory effects in both preclinical and clinical studies. By modulating immune responses and promoting neuroregeneration, these compounds offer potential as novel adjunctive therapies for MS. This review provides insights into the molecular and cellular basis of MS pathogenesis, emphasizing the role of inflammation in disease progression. It critically evaluates emerging evidence supporting the use of plant-derived compounds to attenuate inflammation and MS symptomology. In addition, we provide a comprehensive source of information detailing the known mechanisms of action and assessing the clinical potential of plant-derived compounds in the context of MS pathogenesis, with a focus on their anti-inflammatory and neuroprotective properties.

The Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway serves as a paradigm for signal transduction from the extracellular environment to the nucleus. It plays a pivotal role in physiological functions, such as hematopoiesis, immune balance, tissue homeostasis, and surveillance against tumors. Dysregulation of this pathway may lead to various disease conditions such as immune deficiencies, autoimmune diseases, hematologic disorders, and cancer. Due to its critical role in maintaining human health and involvement in disease, extensive studies have been conducted on this pathway, ranging from basic research to medical applications. Advances in the structural biology of this pathway have enabled us to gain insights into how the signaling cascade operates at the molecular level, laying the groundwork for therapeutic development targeting this pathway. Various strategies have been developed to restore its normal function, with promising therapeutic potential. Enhanced comprehension of these molecular mechanisms, combined with advances in protein engineering methodologies, has allowed us to engineer cytokines with tailored properties for targeted therapeutic applications, thereby enhancing their efficiency and safety. In this review, we outline the structural basis that governs key nodes in this pathway, offering a comprehensive overview of the signal transduction process. Furthermore, we explore recent advances in cytokine engineering for therapeutic development in this pathway.

The interplay between inflammation, immune dysregulation, and the onset of neurological disorders, including epilepsy, has become increasingly recognized. Interleukin (IL)-6, a pro-inflammatory cytokine, is suspected to not only mediate traditional inflammatory pathways but also contribute to neuroinflammatory responses that could underpin neuropsychiatric symptoms and broader psychiatric disorders in epilepsy patients. The role of IL-6 receptor (IL6R) blockade presents an intriguing target for therapeutic intervention due to its potential to attenuate these processes.

To explore the potential of IL6R blockade in reducing the risk of epilepsy and investigate whether this pathway might also influence associated psychiatric and neuropsychiatric conditions due to neuroinflammation.

Mendelian randomization (MR) analysis employing single nucleotide polymorphisms (SNPs) in the vicinity of the IL6R gene (total individuals = 408225) was used to evaluate the putative causal relationship between IL6R blockade and epilepsy (total cases/controls = 12891/312803), focal epilepsy (cases/controls = 7526/399290), and generalized epilepsy (cases/controls = 1413/399287). SNP weights were determined by their effect on C-reactive protein (CRP) levels and integrated using inverse variance-weighted meta-analysis as surrogates for IL6R effects. To address potential outlier and pleiotropic influences, sensitivity analyses were conducted employing a variety of MR methods under different modeling assumptions.

The genetic simulation targeting IL6R blockade revealed a modest but significant reduction in overall epilepsy risk [inverse variance weighting: Odds ratio (OR): 0.827; 95% confidence interval (CI): 0.685-1.000; P = 0.05]. Subtype analysis showed variability, with no significant effect observed in generalized, focal, or specific childhood and juvenile epilepsy forms. Beyond the primary inflammatory marker CRP, the findings also suggested potential non-inflammatory pathways mediated by IL-6 signaling contributing to the neurobiological landscape of epilepsy, hinting at possible links to neuroinflammation, psychiatric symptoms, and associated mental disorders.

The investigation underscored a tentative causal relationship between IL6R blockade and decreased epilepsy incidence, likely mediated via complex neuroinflammatory pathways. These results encouraged further in-depth studies involving larger cohorts and multifaceted psychiatric assessments to corroborate these findings and more thoroughly delineate the neuro-psychiatric implications of IL-6 signaling in epilepsy. The exploration of IL6R blockade could herald a novel therapeutic avenue not just for seizure management but also for addressing the broader psychiatric and cognitive disturbances often associated with epilepsy.

This study aimed to comprehensively investigate hypervirulent carbapenem-resistant Klebsiella pneumoniae (CR-hvKP) in the Ningbo region. Importantly, we sought to elucidate its molecular characteristics and pathogenic mechanisms. This information will provide evidence-based insights for preventing and controlling nosocomial infections and facilitate improved clinical diagnosis and treatment in this region.

96 carbapenem-resistant Klebsiella pneumoniae strains were collected from the Ningbo region between January 2021 and December 2022. Whole genome sequencing and bioinformatic methods were employed to identify and characterize CR-hvKP strains at the molecular level. The minimum inhibitory concentrations (MICs) of common clinical antibiotics were determined using the VITEK-2 Compact automatic microbiological analyzer. Plasmid conjugation experiments evaluated the transferability of resistance plasmids. Finally, mouse virulence assays were conducted to explore the pathogenic mechanisms.

Among the 96 strains, a single CR-hvKP strain, designated CR-hvKP57, was identified, with an isolation frequency of 1.04%. Whole-genome sequencing revealed the strain to be ST23 serotype with a K1 capsule. This strain harbored three plasmids. Plasmid 1, a pLVPK-like virulence plasmid, carried multiple virulence genes, including rmpA, rmpA2, iroB, iucA, and terB. Plasmid 2 contained transposable element sequences such as IS15 and IS26. Plasmid 3, classified as a resistance plasmid, harbored the bla KPC-3 carbapenem resistance gene. Mouse virulence assays demonstrated a high mortality rate associated with CR-hvKP57 infection. Additionally, there was a significant increase in IL-1β, IL-6, and TNF-α levels in response to CR-hvKP57 infection, indicating varying degrees of inflammatory response. Western blot experiments further suggested that the pathogenic mechanism involves activation of the NF-κB signaling pathway.

This study confirms the emergence of hypervirulent CR-hvKP in the Ningbo region, which likely resulted from the acquisition of a pLVPK-like virulence plasmid and a bla KPC-3 resistance plasmid by the ST23-K1 type Klebsiella pneumoniae. Our findings highlight the urgent need for more judicious use of antibiotics to limit the emergence of resistance. Additionally, strengthening infection prevention and control measures is crucial to minimize the spread of virulence and resistance plasmids.

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.

Introduction: IL-6 and IL-10 may affect the activity of cytochrome P450 (CYP) 3A enzymes involved in tacrolimus (Tac) metabolism. Moreover, the effect of IL-6 and IL-10 on Tac pharmacokinetics may differ with respect to the genetic variations in their genes.Aim: To examine the influence of IL-6 and IL-10 gene polymorphisms on Tac dose requirements and exposure over a 5-year period following kidney transplantation. Univariate and standard multivariate linear regression and Monte Carlo analysis were performed to investigate potential covariates influencing Tac dose-adjusted trough concentration (C0/D) in various post-transplantation periods.Materials & methods: IL-6 (-174G > C), IL-10 (-1082G > A, -819C > T and -592C > A) genotype, Tac daily dose, C0, C0/D and intrapatient variability data were collected from 113 patients.Results: Multivariate regression analysis and accompanied Monte Carlo simulation underscore the importance of considering IL-6 -174G > C and IL-10 -1082G > A gene polymorphisms, alongside Tac metabolic phenotype and post-transplantation period, when tailoring Tac dosage regimen.Conclusion: This study provides valuable insights regarding the individualized adjustment of Tac treatment in various post-transplantation periods.

Our purpose is to unveil Andrographolide's potential multi-target and multi-mechanism therapeutic effects in treating OA via systematic network pharmacological analysis and cell experimental validation.

Initially, we gathered data from Andrographolide and OA-related databases to obtain information on Andrographolide's biological properties and the targets linked with OA. We developed a bioinformatic network about Andrographolide and OA, whereby we analyzed the network to identify potential therapeutic targets and mechanisms of action of Andrographolide. Subsequently, we used molecular docking to analyze the binding sites of Andrographolide to the target proteins. At the same time, SDF-1 was used to construct an OA cell model to verify the therapeutic effect of Andrographolide on OA and its effect on target proteins.

Our experimental results show that Andrographolide has excellent pharmaceutical properties, by Lipinski's rules for drugs, suggesting that this compound can be considered to have a high therapeutic potential in drug development. 233 targets were preliminarily investigated, the mechanisms through which Andrographolide targets OA primarily involve the TNF signaling pathway, PI3K-AKT signaling pathway, IL-17 signaling pathway, and TLR signaling pathway. These mechanisms target OA by influencing immune and inflammatory responses in the joints, regulating apoptosis to prevent chondrocyte death. Finally, TNF-α, STAT3, TP53, IL-6, JUN, IL-1β, HIF-1α, TGF-β1, and AKT1 were identified as 9 key targets of Andrographolide anti-OA. In addition, our molecular docking analyzes with cell experimental validation further confirm the network pharmacology results. According to our molecular docking results, Andrographolide can bind to all the hub target proteins and has a good binding ability (binding energy < -5 kcal/mol), with the strongest binding affinity to AKT1 of -9.2 kcal/ mol. The results of cell experiments showed that Andrographolide treatment significantly increased the cell viability and the expression of COL2A1 and ACAN proteins. Moreover, 30 μM Andrographolide significantly reversed SDF-1-induced increases in the protein expression of TNF-α, STAT3, TP53, IL-6, JUN, IL-1β, HIF-1α, and TGF-β1, and decreases in the protein expression of AKT1.

This study provides a comprehensive understanding of the potential therapeutic targets and mechanisms of action of Andrographolide in OA treatment. Our findings suggest that Andrographolide is a promising candidate for drug development in the management of OA.

IL6 signaling plays an important role in triggering labor and IL6 is an established biomarker of intrauterine infection/inflammation (IUI) driven preterm labor (PTL). The biology of IL6 during IUI at the maternal-fetal interface was investigated in samples from human subjects and non-human primates (NHP).

Pregnant women with histologic chorioamnionitis diagnosed by placenta histology were recruited (n=28 term, n=43 for preterm pregnancies from 26-36 completed weeks of gestation). IUI was induced in Rhesus macaque by intraamniotic injection of lipopolysachharide (LPS, n=23). IL1 signaling was blocked using Anakinra (human IL-1 receptor antagonist, n=13), and Tumor necrosis factor (TNF) signaling was blocked by anti TNF-antibody (Adalimumab n=14). The blockers were given before LPS. All animals including controls (intraamniotic injection of saline n=27), were delivered 16h after LPS/saline exposure at about 80% gestation.

IUI induced a robust expression of IL6 mRNAs in the fetal membranes (chorion-amnion-decidua tissue) both in humans (term and preterm) and NHP. The major sources of IL6 mRNA expression were the amnion mesenchymal cells (AMC) and decidua stroma cells. Additionally, during IUI in the NHP, ADAM17 (a protease that cleaves membrane bound IL6 receptor (IL6R) to release a soluble form) and IL6R mRNA increased in the fetal membranes, and the ratio of IL6 and soluble forms of IL6R, gp130 increased in the amniotic fluid signifying upregulation of IL6 trans-signaling. Both IL1 and TNF blockade suppressed LPS-induced IL6 mRNAs in the AMC and variably decreased elements of IL6 trans-signaling.

These data suggest that IL1 and TNF blockers may be useful anti-inflammatory agents via suppression of IL6 signaling at the maternal-fetal interface.

Interleukin (IL)-6 has anti- and pro-inflammatory functions, controlled by IL-6 classic and trans-signaling, respectively. Differences in the downstream signaling mechanism between IL-6 classic and trans-signaling have not been identified. Here, we report that IL-6 activates glycolysis to regulate the inflammatory response. IL-6 regulates glucose metabolism by forming a complex containing signal-transducing activators of transcription 3 (STAT3), hexokinase 2 (HK2), and voltage-dependent anion channel 1 (VDAC1). The IL-6 classic signaling directs glucose flux to oxidative phosphorylation (OxPhos), while IL-6 trans-signaling directs glucose flux to anaerobic glycolysis. Classic IL-6 signaling promotes STAT3 translocation into mitochondria to interact with pyruvate dehydrogenase kinase-1 (PDK1), leading to pyruvate dehydrogenase α (PDHA) dissociation from PDK1. As a result, PDHA is dephosphorylated, and STAT3 is phosphorylated at Ser727. By contrast, IL-6 trans-signaling promotes the interaction of sirtuin 2 (SIRT2) and lactate dehydrogenase A (LDHA), leading to the dissociation of STAT3 from SIRT2. As a result, LDHA is deacetylated, and STAT3 is acetylated and phosphorylated at Tyr705. IL-6 classic signaling promotes the differentiation of regulatory T cells via the PDK1/STAT3/PDHA axis, whereas IL-6 trans-signaling promotes the differentiation of Th17 cells via the SIRT2/STAT3/LDHA axis. Conclusion: IL-6 classic signaling generates anti-inflammatory functions by shifting energy metabolism to OxPhos, while IL-6 trans-signaling generates pro-inflammatory functions by shifting energy metabolism to anaerobic glycolysis.

Several autoimmune disorders have been linked to polymorphisms in IL10 and IL6R genes. This research aimed to study whether single nucleotide polymorphisms (SNPs) in the genes of IL10 and IL6R were associated with acute anterior uveitis (AAU) in Han Chinese.

Genotyping was carried out by the iPLEX Gold Genotyping Assay. Our study comprised 420 patients with AAU and 918 healthy subjects from Han Chinese. Using the chi-square (χ2) test, alleles and genotypes were analyzed between AAU subjects and healthy controls.

All ten SNPs were successfully genotyped and four SNPs (IL10/rs1800871, IL10/rs3021094, IL10/rs2222202, IL6R/rs4845618) exhibited weak associations with AAU, as indicated by their Puncorr values. However, upon applying the Bonferroni correction, there was no significant association between AAU and the control subjects. Additionally, the haplotype analysis of the ten SNPs revealed no association with AAU.

Our findings suggested that polymorphisms of the tested ten SNPs on the IL10 and IL6R genes did not show any association with the risk of developing AAU among the Han Chinese population.

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.