ABSTRACT
Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation and swelling. Several studies have demonstrated that RA fibroblast-like synovial cells (RA-FLS) play an important role in RA pathogenesis. Activated RA-FLS contribute to synovial inflammation by secreting inflammatory cytokines including interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α. LMT-28 is derivative of oxazolidone and exerts anti-inflammatory effects on RA via IL-6 signaling pathway regulation. LMT-28 also regulates T cell differentiation in RA condition. However, the effect of LMT-28 on the migration and invasion of RA-FLS remains unknown. Kaempferol has been reported to have pharmacological effects on various diseases, such as inflammatory diseases, autoimmune diseases, and cancer. Additionally, kaempferol has been reported to inhibit RA-FLS migration and invasion, but it is not known about the therapeutic mechanism including molecular mechanism such as receptor. The present study aimed to investigate the synergistic effects of the combined treatment of LMT-28 and kaempferol on RA-FLS activation and RA pathogenesis in mouse model. LMT-28 and kaempferol co-administration inhibited RA disease severity and histological collapse in the joint tissues of CIA mice, as well as downregulated the levels of pro-inflammatory cytokines in mouse serum. Additionally, the combined treatment inhibited excessive differentiation of T helper 17 cells and osteoclasts. Furthermore, compared with single treatments, combined treatment showed enhanced inhibitory effects on the hyperactivation of IL-6-induced signaling pathway in RA-FLS. Combined treatment also inhibited RA-FLS cell proliferation, migration, and invasion and suppressed the expression of matrix metalloproteinase in RA-FLS. Furthermore, we confirmed that the combined treatment inhibited chondrocyte proliferation, migration, and invasion. In conclusion, our results suggest that the combined treatment of LMT-28 and kaempferol exerts a synergistic effect on the RA development via the regulation of IL-6-induced hyperactivation of RA-FLS. Furthermore, this study suggests that combination therapies can be an effective therapeutic option for arthritis.
Subject(s)
Anti-Inflammatory Agents , Arthritis, Experimental , Kaempferols , Animals , Kaempferols/pharmacology , Kaempferols/therapeutic use , Kaempferols/administration & dosage , Mice , Arthritis, Experimental/drug therapy , Arthritis, Experimental/pathology , Arthritis, Experimental/metabolism , Anti-Inflammatory Agents/pharmacology , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/pathology , Arthritis, Rheumatoid/metabolism , Mice, Inbred DBA , Disease Models, Animal , Male , Cell Movement/drug effects , Interleukin-6/metabolism , Cell Proliferation/drug effects , Cytokines/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Cell Differentiation/drug effects , Drug Therapy, Combination , Synoviocytes/drug effects , Synoviocytes/metabolismABSTRACT
Human Natural Killer (NK) cells are heterogeneous lymphocytes regulated by variegated arrays of germline-encoded activating and inhibitory receptors. They acquire the ability to detect polymorphic self-antigen via NKG2A/HLA-E or KIR/HLA-I ligand interactions through an education process. Correlations among HLA/KIR genes, kidney transplantation pathology and outcomes suggest that NK cells participate in allograft injury, but mechanisms linking NK HLA/KIR education to antibody-independent pathological functions remain unclear. We used CyTOF to characterize pre- and post-transplant peripheral blood NK cell phenotypes/functions before and after stimulation with allogeneic donor cells. Unsupervised clustering identified unique NK cell subpopulations present in varying proportions across patients, each of which responded heterogeneously to donor cells based on donor ligand expression patterns. Analyses of pre-transplant blood showed that educated, NKG2A/KIR-expressing NK cells responded greater than non-educated subsets to donor stimulators, and this heightened alloreactivity persisted > 6 months post-transplant despite immunosuppression. In distinct test and validation sets of patients participating in two clinical trials, pre-transplant donor-induced release of NK cell Ksp37, a cytotoxicity mediator, correlated with 2-year and 5-year eGFR. The findings explain previously reported associations between NK cell genotypes and transplant outcomes and suggest that pre-transplant NK cell analysis could function as a risk-assessment biomarker for transplant outcomes.
ABSTRACT
BACKGROUND: Long-term persistence of Ebola virus (EBOV) in immunologically privileged sites has been implicated in recent outbreaks of Ebola virus disease (EVD) in Guinea and the Democratic Republic of Congo. This study was designed to understand how the acute course of EVD, convalescence, and host immune and genetic factors may play a role in prolonged viral persistence in semen. METHODS: A cohort of 131 male EVD survivors in Liberia were enrolled in a case-case study. "Early clearers" were defined as those with 2 consecutive negative EBOV semen test results by real-time reverse-transcription polymerase chain reaction (rRT-PCR) ≥2 weeks apart within 1 year after discharge from the Ebola treatment unit or acute EVD. "Late clearers" had detectable EBOV RNA by rRT-PCR >1 year after discharge from the Ebola treatment unit or acute EVD. Retrospective histories of their EVD clinical course were collected by questionnaire, followed by complete physical examinations and blood work. RESULTS: Compared with early clearers, late clearers were older (median, 42.5 years; P < .001) and experienced fewer severe clinical symptoms (median 2, P = .006). Late clearers had more lens opacifications (odds ratio, 3.9 [95% confidence interval, 1.1-13.3]; P = .03), after accounting for age, higher total serum immunoglobulin G3 (IgG3) titers (P = .005), and increased expression of the HLA-C*03:04 allele (0.14 [.02-.70]; P = .007). CONCLUSIONS: Older age, decreased illness severity, elevated total serum IgG3 and HLA-C*03:04 allele expression may be risk factors for the persistence of EBOV in the semen of EVD survivors. EBOV persistence in semen may also be associated with its persistence in other immunologically protected sites, such as the eye.
Subject(s)
Ebolavirus , Hemorrhagic Fever, Ebola , Humans , Male , Ebolavirus/genetics , Hemorrhagic Fever, Ebola/epidemiology , Semen , Liberia/epidemiology , Retrospective Studies , HLA-C Antigens , Survivors , Risk FactorsABSTRACT
OBJECTIVES: The interleukin-6 (IL-6)-mediated signaling pathway plays an essential role in the development of rheumatoid arthritis. LMT-28 suppresses the activation of the IL-6-mediated signaling by direct targeting of gp130. Although LMT-28 and metformin both possess anti-inflammatory activity, the beneficial effect of LMT-28 and metformin combination on a collagen-induced arthritis (CIA) model has not yet been investigated. This study aimed to investigate the anti-inflammatory effect and mechanism of a combination of LMT-28 and metformin in a CIA model. METHODS: In MH7A cells, cell proliferation and the IL-6-mediated signaling pathway following administration of LMT-28 and metformin combination was analyzed through MTT assay and Western blotting. The level of T helper 17 (Th17) cell differentiation from CD4+ T cells was analyzed in mouse splenocytes and human peripheral blood mononuclear cells. Arthritis score, incidence rate, inflammatory cytokine, and T-cell subsets were measured in CIA mice following administration of LMT-28 and metformin combination. RESULTS: Combination treatment with LMT-28 and metformin diminished proliferation of MH7A cells and IL-6-mediated gp130, STAT3, and ERK signaling more than in individual treatments. Furthermore, the differentiation of CD4+ T cells into Th17 cells was attenuated more by combination treatment with LMT-28 and metformin than individual treatments. The combination of LMT-28 and metformin ameliorated the arthritic score better than individual treatments. The combination significantly reduced tumor necrosis factor and IL-6 levels in the sera and had an anti-inflammatory effect on the distribution of Treg/Th17 cells in the lymph nodes. CONCLUSION: Combination treatment with LMT-28 and metformin significantly ameliorates arthritic symptoms in CIA by suppressing Th17 differentiation and IL-6 signaling.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Arthritis, Experimental/drug therapy , Metformin/pharmacology , Oxazolidinones/pharmacology , Animals , Anti-Inflammatory Agents/therapeutic use , Arthritis, Experimental/chemically induced , Cell Differentiation/drug effects , Cell Line , Collagen/toxicity , Drug Therapy, Combination , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Interleukin-17/metabolism , Interleukin-6/antagonists & inhibitors , Interleukin-6/metabolism , Male , Metformin/therapeutic use , Mice, Inbred C57BL , Mice, Inbred DBA , Oxazolidinones/therapeutic use , Synoviocytes/drug effects , Synoviocytes/metabolism , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/metabolism , Th17 Cells/drug effects , Th17 Cells/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Rheumatoid arthritis is a chronic inflammatory disease associated with joint inflammation and destruction driven by T helper 17 (Th17) cells. Interleukin-6 (IL-6) is secreted by many cell types, including macrophages and synovial fibroblasts. It induces the differentiation and function of Th17 cells that can increase lymphocytic infiltration in the joint. LMT-28 can suppress IL-6 signalling through direct binding to glycoprotein-130 and alleviate inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. The purpose of this study was to assess whether LMT-28 could potently inhibit Th17 differentiation and to determine the mechanism involved in the attenuating effect of LMT-28 on rheumatoid arthritis through the IL-6 signalling pathway. LMT-28 reduced the arthritis score and showed protective effects against bone and cartilage destruction in collagen-induced arthritis (CIA) mice. In mice with CIA, LMT-28 markedly decreased serum levels of IL-6, TNF and IL-1ß compared to vehicle control. Moreover, LMT-28 attenuated Th17 cell activation in lymph nodes of CIA mice. We demonstrated that LMT-28 suppressed differentiation of Th17 in mouse splenocytes and human peripheral blood mononuclear cells (PBMCs). Additionally, LMT-28 inhibited phosphorylation of GP130, STAT3 and ERK induced by Hyper-IL-6 in human fibroblast-like synoviocytes (FLS). Collectively, these results suggest that LMT-28 can inhibit differentiated/activated-Th17 cells in rheumatoid arthritis by blocking activation of the STAT3 pathway. LMT-28 can attenuate rheumatoid arthritis by inhibiting differentiation/activation of Th17 cells and suppressing the proliferation and signalling activation of the IL-6/solubleIL-6 receptor complex stimulated FLS.
Subject(s)
Arthritis, Experimental/drug therapy , Arthritis, Rheumatoid/drug therapy , Cytokine Receptor gp130/metabolism , Interleukin-6/metabolism , Molecular Targeted Therapy , Small Molecule Libraries/pharmacology , Th17 Cells/drug effects , Animals , Arthritis, Experimental/immunology , Arthritis, Experimental/metabolism , Arthritis, Experimental/pathology , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Cell Differentiation/drug effects , Mice , Signal Transduction/drug effects , Small Molecule Libraries/therapeutic use , Synoviocytes/drug effects , Synoviocytes/pathology , Th17 Cells/cytologyABSTRACT
Rheumatoid arthritis (RA) is a systemic, chronic inflammatory disease that is characterized by T helper 17 (Th17) cell- and osteoclast-induced joint destruction and inflammation. In RA, several cytokines (interleukin (IL)-1, 6,17, and tumor necrosis factor (TNF)) are involved in almost all aspects of articular inflammation and destruction. This study aimed to evaluate the combinatorial effect of TNF and IL-6 inhibitors on the differentiation and activation of Th17 cells and osteoclasts in the context of RA, and to identify the RA-related mechanisms through IL-6 signaling. Tetrahydropapaverine (THP) showed direct binding to TNF in screening-ELISA, and SPR and TNF-neutralization assays. In a previous study, the therapeutic effect of gp130-targeting LMT-28 was confirmed in RA. Combinatorial treatment with LMT-28 and THP reduced the arthritis index and showed protective effects against bone and cartilage destruction in CIA mice. The secretion levels of TNF, IL-6, and IL-1ß significantly decreased upon combinatorial treatment with LMT-28 and THP. Further, the LMT-28 and THP combination suppressed the differentiation and activation of Th17 cells in mouse splenocytes and human PBMCs. In human RA-FLS, the LMT-28 and THP combination inhibited cell proliferation and downregulated IL-6 and/or TNF-mediated signaling relative to that observed upon independent treatment with LMT-28 or THP. Furthermore, the combination of LMT-28 and THP significantly inhibited the differentiation of mouse bone marrow monocytes (BMMs) into osteoclasts. In conclusion, the LMT-28 and THP combination can attenuate RA through the inhibition of Th17 differentiation and osteoclastogenesis, and suppression of IL-6 or TNF-induced signaling pathways. This combinatorial therapy could be used as a new strategy for the treatment of RA.
Subject(s)
Arthritis, Rheumatoid/drug therapy , Cell Differentiation , Cytokine Receptor gp130/antagonists & inhibitors , Down-Regulation , Osteogenesis , Small Molecule Libraries/therapeutic use , Th17 Cells/cytology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Animals , Arthritis, Experimental/pathology , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/pathology , Cell Differentiation/drug effects , Cell Line , Cytokine Receptor gp130/metabolism , Cytokines/metabolism , Down-Regulation/drug effects , Drug Therapy, Combination , Fibroblasts/drug effects , Fibroblasts/pathology , Humans , Inflammation Mediators/metabolism , Isoquinolines/pharmacology , Isoquinolines/therapeutic use , Male , Mice , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteoclasts/pathology , Osteogenesis/drug effects , Oxazolidinones/pharmacology , Oxazolidinones/therapeutic use , Protein Binding/drug effects , RANK Ligand/pharmacology , Receptors, Tumor Necrosis Factor/metabolism , Small Molecule Libraries/pharmacology , Synoviocytes/drug effects , Synoviocytes/pathology , Th17 Cells/drug effects , Tumor Necrosis Factor-alpha/metabolismABSTRACT
A series of oxazolidinone and indole derivatives were synthesized and evaluated as IL-6 signaling blockers by measuring the effects of these compounds on IL-6-induced luciferase expression in human hepatocarcinoma HepG2 cells transfected with p-STAT3-Luc. Among different compounds screened, compound 4d was emerged as the most potent IL-6 signaling blockers with IC50 value of 5.9 µM which was much better than (+)-Madindoline A (IC50=21 µM), a known inhibitor of IL-6.
Subject(s)
Interleukin-6/metabolism , Oxazolidinones/chemistry , Signal Transduction/drug effects , Binding Sites , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Crystallography, X-Ray , Hep G2 Cells , Humans , Indoles/chemistry , Inhibitory Concentration 50 , Interleukin-6/antagonists & inhibitors , Molecular Docking Simulation , Oxazolidinones/chemical synthesis , Oxazolidinones/pharmacology , Protein Structure, Tertiary , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Structure-Activity RelationshipABSTRACT
IL-6 is a major causative factor of inflammatory disease. Although IL-6 and its signaling pathways are promising targets, orally available small-molecule drugs specific for IL-6 have not been developed. To discover IL-6 antagonists, we screened our in-house chemical library and identified LMT-28, a novel synthetic compound, as a candidate IL-6 blocker. The activity, mechanism of action, and direct molecular target of LMT-28 were investigated. A reporter gene assay showed that LMT-28 suppressed activation of STAT3 induced by IL-6, but not activation induced by leukemia inhibitory factor. In addition, LMT-28 downregulated IL-6-stimulated phosphorylation of STAT3, gp130, and JAK2 protein and substantially inhibited IL-6-dependent TF-1 cell proliferation. LMT-28 antagonized IL-6-induced TNF-α production in vivo. In pathologic models, oral administration of LMT-28 alleviated collagen-induced arthritis and acute pancreatitis in mice. Based on the observation of upstream IL-6 signal inhibition by LMT-28, we hypothesized IL-6, IL-6Rα, or gp130 to be putative molecular targets. We subsequently demonstrated direct interaction of LMT-28 with gp130 and specific reduction of IL-6/IL-6Rα complex binding to gp130 in the presence of LMT-28, which was measured by surface plasmon resonance analysis. Taken together, our data suggest that LMT-28 is a novel synthetic IL-6 inhibitor that functions through direct binding to gp130.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Arthritis, Experimental/drug therapy , Cytokine Receptor gp130/antagonists & inhibitors , Interleukin-6/antagonists & inhibitors , Oxazolidinones/pharmacology , Pancreatitis/drug therapy , Small Molecule Libraries/pharmacology , Administration, Oral , Animals , Arthritis, Experimental/genetics , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Cell Line, Tumor , Cytokine Receptor gp130/genetics , Cytokine Receptor gp130/immunology , Gene Expression Regulation , Hep G2 Cells , Humans , Interleukin-6/genetics , Interleukin-6/immunology , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/genetics , Janus Kinase 2/immunology , Leukocytes/drug effects , Leukocytes/immunology , Leukocytes/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred DBA , Pancreatitis/genetics , Pancreatitis/immunology , Pancreatitis/pathology , Phosphorylation , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/immunology , Signal Transduction , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunologyABSTRACT
AIM: To evaluate protective effects of Chunggan extract (CGX), a traditional herbal formula, under 4 wk of alcohol consumption-induced liver injury. METHODS: Male Sprague-Dawley Rats were orally administered 30% ethanol daily for 4 wk with or without CGX. The pharmaceutical properties were assessed through liver enzymes, histopathology, fibrogenic cytokines, and alcohol metabolism in hepatic tissues as well as by in vitro experiment using HSC-T6 cells. RESULTS: Four weeks of alcohol consumption notably increased liver enzymes and malondialdehyde levels in serum and hepatic tissue. CGX not only prevented the collagen deposition determined by histopathology and hydroxyproline content, but also normalized transforming growth factor-beta, platelet-derived growth factor-beta and connective tissue growth factor at the gene expression and protein levels in liver tissue. Moreover, CGX treatment also significantly normalized the abnormal changes in gene expression profiles of extracellular matrix proteins, matrix metalloproteinase and their inhibitors, alcohol metabolism, and inflammatory reactions. In the acetaldehyde-stimulated HSC-T6 cells, CGX considerably inhibited collagen production and normalized fibrogenic cytokines in both gene expression and protein levels. CONCLUSION: The present study evidenced that CGX has hepatoprotective properties via modulation of fibrogenic cytokines and alcohol metabolism in alcoholic liver injury.
Subject(s)
Drugs, Chinese Herbal/pharmacology , Hepatocytes/drug effects , Liver Diseases, Alcoholic/prevention & control , Liver/drug effects , Protective Agents/pharmacology , Animals , Cell Line , Collagen/metabolism , Connective Tissue Growth Factor/genetics , Connective Tissue Growth Factor/metabolism , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Gene Expression Regulation , Hepatocytes/metabolism , Hepatocytes/pathology , Hydroxyproline/metabolism , Liver/metabolism , Liver/pathology , Liver Diseases, Alcoholic/blood , Liver Diseases, Alcoholic/genetics , Liver Diseases, Alcoholic/pathology , Male , Malondialdehyde/blood , Phytotherapy , Plants, Medicinal , Proto-Oncogene Proteins c-sis/genetics , Proto-Oncogene Proteins c-sis/metabolism , RNA, Messenger/metabolism , Rats, Sprague-Dawley , Time Factors , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolismABSTRACT
The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, are thought to play a role in the regulation of cell adhesion and migration during development by mediating cell-to-cell signalling events. The transmembrane ephrinB protein is a bidirectional signalling molecule that sends a forward signal through the activation of its cognate receptor tyrosine kinase residing on another cell. The reverse signal is transduced into the ephrinB-expressing cell via tyrosine phosphorylation of its conserved C-terminal cytoplasmic domain. Previous work from our laboratory has implicated the activated FGFR1 (fibroblast growth factor receptor 1) as a regulator of a de-adhesion signal that results from overexpression of ephrinB1. In the present study, we report the isolation of Xenopus Grb4 (growth-factor-receptor-bound protein 4), an ephrinB1-interacting protein, and we show that when expressed in Xenopus oocytes, ephrinB1 interacts with Grb4 in the presence of an activated FGFR1. Amino acid substitutions were generated in Grb4, and the resulting mutants were expressed along with ephrinB1 and an activated FGFR in Xenopus oocytes. Co-immunoprecipitation analysis shows that the FLVR motif within the Src homology 2 domain of Xenopus Grb4 is vital for this phosphorylation-dependent interaction with ephrinB1. More importantly, using deletion and substitution analysis we identify the tyrosine residue at position 298 of ephrinB1 as being required for the physical interaction with Grb4, whereas Tyr-305 and Tyr-310 are dispensable. Moreover, we show that the region between amino acids 301 and 304 of ephrinB1 is also required for this critical tyrosine-phosphorylation-dependent event.