Clinical and Immunological Factors That Distinguish COVID-19 From Pandemic Influenza A(H1N1).

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is a global health threat with the potential to cause severe disease manifestations in the lungs. Although COVID-19 has been extensively characterized clinically, the factors distinguishing SARS-CoV-2 from other respiratory viruses are unknown. Here, we compared the clinical, histopathological, and immunological characteristics of patients with COVID-19 and pandemic influenza A(H1N1). We observed a higher frequency of respiratory symptoms, increased tissue injury markers, and a histological pattern of alveolar pneumonia in pandemic influenza A(H1N1) patients. Conversely, dry cough, gastrointestinal symptoms and interstitial lung pathology were observed in COVID-19 cases. Pandemic influenza A(H1N1) was characterized by higher levels of IL-1RA, TNF-α, CCL3, G-CSF, APRIL, sTNF-R1, sTNF-R2, sCD30, and sCD163. Meanwhile, COVID-19 displayed an immune profile distinguished by increased Th1 (IL-12, IFN-γ) and Th2 (IL-4, IL-5, IL-10, IL-13) cytokine levels, along with IL-1ß, IL-6, CCL11, VEGF, TWEAK, TSLP, MMP-1, and MMP-3. Our data suggest that SARS-CoV-2 induces a dysbalanced polyfunctional inflammatory response that is different from the immune response against pandemic influenza A(H1N1). Furthermore, we demonstrated the diagnostic potential of some clinical and immune factors to differentiate both diseases. These findings might be relevant for the ongoing and future influenza seasons in the Northern Hemisphere, which are historically unique due to their convergence with the COVID-19 pandemic.

Characterization and Function of Tumor Necrosis Factor and Interleukin-6-Induced Osteoclasts in Rheumatoid Arthritis.

OBJECTIVE: We have previously reported that stimulation of mouse bone marrow-derived macrophages with tumor necrosis factor (TNF) and interleukin-6 (IL-6) induces differentiation of osteoclast-like cells. We undertook this study to clarify the characterization and function of human TNF and IL-6-induced osteoclasts using peripheral blood collected from patients with rheumatoid arthritis (RA) and healthy donors. METHODS: Peripheral blood monocytes were cultured with a combination of TNF and IL-6, TNF alone, IL-6 alone, or with RANKL, and their bone resorption ability was evaluated. Expression levels of NFATc1, proinflammatory cytokines, and matrix metalloproteinase 3 were analyzed. The effects of NFAT inhibitor and JAK inhibitor were examined. Furthermore, the relationship between the number of TNF and IL-6-induced osteoclasts or RANKL-induced osteoclasts differentiated from peripheral blood mononuclear cells (PBMCs) in patients with RA and the modified total Sharp score (mTSS) or whole-body bone mineral density (BMD) was examined. RESULTS: Peripheral blood monocytes stimulated with a TNF and IL-6-induced osteoclasts were shown to demonstrate the ability to absorb bone matrix. Cell differentiation was not inhibited by the addition of osteoprotegerin. Stimulation with a combination of TNF and IL-6 promoted NFATc1 expression, whereas the NFAT and JAK inhibitors prevented TNF and IL-6-induced osteoclast formation. Expression levels of IL1ß, TNF, IL12p40, and MMP3 were significantly increased in TNF and IL-6-induced osteoclasts, but not in RANKL-induced osteoclasts. The number of TNF and IL-6-induced osteoclasts differentiated from PBMCs in patients with RA positively correlated with the mTSS, whereas RANKL-induced osteoclast numbers negatively correlated with the whole-body BMD of the same patients. CONCLUSION: Our results demonstrate that TNF and IL-6-induced osteoclasts may contribute to the pathology of inflammatory arthritis associated with joint destruction, such as RA.

Iguratimod Inhibits the Aggressiveness of Rheumatoid Fibroblast-Like Synoviocytes.

OBJECTIVE: Iguratimod, a novel disease-modifying anti-rheumatic drug for the treatment of rheumatoid arthritis, has been approved in China and Japan. Here, we aimed to find whether iguratimod can inhibit the aggressive behavior and promote apoptosis of rheumatoid fibroblast-like synoviocytes (RA-FLSs). METHODS: The proliferation of RA-FLSs was assessed by 5-ethynyl-2'-deoxyuridine test and Cell Counting Kit-8. Migration and invasion were determined by the wound test and a transwell assay. Apoptosis was tested by flow cytometry. The mRNA expression of matrix metalloproteinases (MMPs) and proinflammatory cytokines in RA-FLSs were measured by quantitative PCR and ELISA. To gain insight into the molecular signaling mechanisms, we determined the effect of iguratimod on the activation of mitogen-activated protein kinases (MAPK) signaling pathways by the cellular thermal shift assay (CETSA) and western blot. RESULTS: Iguratimod treatment significantly reduced the proliferation, migration, and invasive capacities of RA-FLSs in a dose-dependent manner in vitro. MMP-1, MMP-3, MMP-9, Interleukin-6 (IL-6), and monocyte chemoattractant protein-1 mRNA and protein levels were all decreased after treatment with iguratimod. Furthermore, tumor necrosis factor-alpha- (TNF-α-) induced expression of phosphorylated c-Jun N-terminal kinases (JNK) and P38 MAPK were inhibited by iguratimod. Additionally, iguratimod promoted the apoptosis of RA-FLSs. Most importantly, iguratimod was shown to directly interact with JNK and P38 protein by CETSA assay. Moreover, activating transcription factor 2 (ATF-2), a substrate of both JNK and P38, was suppressed by iguratimod. CONCLUSIONS: Our findings suggested that the therapeutic effects of iguratimod on RA might be, in part, due to targeting the aggressive behavior and apoptosis of RA-FLSs.

Interleukin 1 beta and Matrix Metallopeptidase 3 Contribute to Development of Epidermal Growth Factor Receptor-Dependent Serrated Polyps in Mouse Cecum.

BACKGROUND & AIMS: Transgenic mice (HBUS) that express the epidermal growth factor receptor (EGFR) ligand HBEGF (heparin-binding epidermal growth factor-like growth factor) and a constitutively active G protein-coupled receptor (US28) in intestinal epithelial cells develop serrated polyps in the cecum. Development of serrated polyps depends on the composition of the gut microbiota and is associated with bacterial invasion of the lamina propria, accompanied by induction of inflammation and up-regulation of interleukin 1 beta (IL1B) and matrix metalloproteinase (MMP) 3 in the cecum. We investigated the mechanisms by which these changes contribute to development of serrated polyps. METHODS: We performed studies with C57BL/6 (control) and HBUS mice. To accelerate polyp development, we increased the exposure of the bacteria to the lamina propria by injecting HBUS mice with diphtheria toxin, which binds transgenic HBEGF expressed by the epithelial cells and causes apoptosis. Mice were given injections of IL1B-neutralizing antibody and the MMP inhibitor N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid. Intestinal tissues were collected from mice and analyzed by histology, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and flow cytometry. We examined fibroblast subsets in polyps using single-cell RNA sequencing. RESULTS: Administration of diphtheria toxin to HBUS mice accelerated development of serrated polyps (95% of treated mice developed polyps before 100 days of age, compared with 53% given vehicle). IL1B stimulated subsets of platelet-derived growth factor receptor alpha+ (PDGRFA+) fibroblasts isolated from cecum, resulting in increased expression of MMP3. Neutralizing antibodies against IL1B or administration of the MMP inhibitor reduced the number of serrated polyps that formed in the HBUS mice. Single-cell RNA sequencing analysis showed subsets of fibroblasts in serrated polyps that express genes that regulate matrix fibroblasts and inflammation. CONCLUSIONS: In studies of mice, we found that barrier breakdown and expression of inflammatory factors contribute to development of serrated polyps. Subsets of cecal PDGFRA+ fibroblasts are activated by release of IL1B from myeloid cells during the early stages of serrated polyp development. MMP3 produced by PDGFRA+ fibroblasts is important for serrated polyp development. Our findings confirm the functions of previously identified serrated polyp-associated molecules and indicate roles for immune and stromal cells in serrated polyp development.

Novel specific human and mouse stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10) antibodies for biochemical and immunohistochemical analyses.

Matrix metalloproteinases (MMP) are a family of more than 25 zinc-dependent enzymes that are centrally involved in cellular migration, tissue remodeling, cancer invasion and metastasis. Besides degrading extracellular matrix proteins, MMPs are crucial for growth factor and cytokine release and activation. At the same time, they can inactivate inflammatory mediators and enzymes themselves through protein degradation. Subclasses of MMPs include collagenases, gelatinases, stromelysins, membrane-bound MMPs, and others. With regard to the stromelysin subfamily, three members exist, e.g., stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), and stromelysin-3 (MMP-11). MMP-3, and MMP-10 share extensive similarities at the amino acid level that made it difficult to develop specific antibodies distinguishing between MMP-3 and MMP-10. Scrutinizing published data on and performing different analyses with detection of both stromelysins with commercially available or lab-made antibodies showed ambiguous results with regard to specificity of antibodies used to date. We developed new specific antibodies against the most divergent parts of the active forms of both proteins. We assessed the specificity of our novel specific anti-human and anti-mouse MMP-3 and MMP-10 antibodies in cell lysates and different human and murine skin tissues. Tests analyzing specificity of the novel antibodies included Western immunoblotting, immunofluorescence, and immunohistochemistry on paraffin sections. Analyses demonstrated specific detection of respective protein for human or mouse samples except for the anti-human MMP-3 antibody. The aim of this summary was to call attention the MMP research community to distinguish clearly between both enzymes. Our new specific anti-mouse MMP-3 and both MMP-10 antibodies allow us to address this detection problem and to enable comparative studies between both stromelysins with regard to their respective location and function in the tissue.

Expression of T follicular helper lymphocytes with different subsets and analysis of serum IL-6, IL-17, TGF-ß and MMP-3 contents in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the expression levels of T follicular helper (Tfh) with different subsets in patients with rheumatoid arthritis (RA) and their serum interleukin-6 (IL-6), interleukin-17 (IL-17), transforming growth factor-ß (TGF-ß) and matrix metalloproteinase 3 (MMP-3) contents. PATIENTS AND METHODS: The medical records of 45 RA patients in the Department of Rheumatology and Immunology in the First Affiliated Hospital of Chengdu Medical College from January 2016 to April 2018 were retrospectively analyzed. They were divided into the RA high activity group (24 cases, group A) (DAS28 score ≥ 5.0) and RA low activity group (21 cases, group B) (3.2 < DAS28 score < 5.0). At the same time, 20 healthy subjects were selected as a control group. Flow cytometry was used to detect the expression levels of Tfh1, Tfh2 and Tfh17, enzyme-linked immunosorbent assay to detect serum IL-6, IL-17, IL-21 and MMP-3 concentrations. The correlation of Tfh cells with IL-6, IL-17, IL-21 and MMP-3 was analyzed. RESULTS: Those of peripheral blood mononuclear cell (PBMC) Tfh2 and Tfh17 cells were significantly higher in group A than those in group B (p < 0.05). Compared with the control group, the concentrations of serum IL-6, IL-17 and MMP-3 significantly increased (p < 0.001), but that of serum TGF-ß markedly decreased in group A and group B (p < 0.01). The concentrations of serum IL-6, IL-17 and MMP-3 were remarkably higher in group A than those in group B (p < 0.001), but that of serum TGF-ß was significantly lower in group A than that in group B (p < 0.001). The expression level of PBMC Tfh2 cells, PBMC Tfh17 cells was positively correlated with serum IL-6, IL-17 and MMP-3. The expression levels of Tfh2 and Tfh17 cells are positively correlated with serum IL-6, IL-17 and MMP-3 concentrations, negatively correlated with serum TGF-ß concentration. CONCLUSIONS: Tfh2 and Tfh17 are expected to be new targets for immunotherapy in RA patients.

Morin Exhibits Anti-Inflammatory Effects on IL-1ß-Stimulated Human Osteoarthritis Chondrocytes by Activating the Nrf2 Signaling Pathway.

BACKGROUND/AIMS: Osteoarthritis (OA) is a multifactorial disease that is associated with inflammation in joints. The purpose of the present study was to investigate the anti-inflammatory activity and mechanism of morin on human osteoarthritis chondrocytes stimulated by IL-1ß. METHODS: The levels of NO and PGE2 were measured by the Griess method and ELISA. The levels of MMP1, MMP3, and MMP13 were also measured by ELISA. RESULTS: The results revealed that IL-1ß significantly increased the production of NO, PGE2, MMP1, MMP3, and MMP13. Additionally, the increases were significantly attenuated by treatment with morin. Furthermore, IL-1ß-induced NF-κB activation was suppressed by morin. In addition, the expression of Nrf2 and HO-1 were increased by morin and knockdown of Nrf2 could prevent the anti-inflammatory effects of morin. CONCLUSION: In conclusion, this study suggested that morin attenuated IL-1ß-induced inflammation by activating the Nrf2 signaling pathway.

IL-9 receptor: Regulatory role on FLS and pannus formation.

OBJECTIVE: Functions of the Th9 cells and its signature cytokine IL-9 in human autoimmune diseases is currently under extensive research. Here we are reporting new functions of IL-9-receptor (IL-9R); its regulatory role on (i) FLS (fibroblast like synoviocyte) biology and (ii) pannus formation in rheumatoid arthritis (RA) and psoriatic arthritis (PsA). METHODS: RA, PsA, and OA synovial tissue biopsies were obtained; FLS were derived and cultured from these tissues. T quantify protein and messenger RNA levels of IL9-receptor (IL-9R) Western blot and real-time PCR techniques were used. For Pro-growth/survival effect of IL-9 (rIL-9) Annexin-V (apoptosis assay) and MTT assays were used. RESULTS: Immunoblot and RT-PCR studies demonstrated IL9-R in FLS of RA, PsA, and OA. IL9-R was functionally active. rIL-9 induced significant proliferation of FLS (p < 0.001) and had an inhibitory effect on TNF-α induced apoptosis. Proliferation of FLS induced by rIL-9 could be significantly inhibited (p < 0.001) with an IL-9R antibody. Further we observed, rIL-9 induced increased secretion of IL-6, IL-8 and also unregulated MMP-3 expression in FLS. CONCLUSIONS: Proliferation of FLS, induction of pro-nflammatory cytokines and upregulation of metaloprotinase (MMP 3) the key pathologic events for pannus formation are regulated by IL-9 and its recptor. Thus the IL-9/IL-9R system is a new contributing factor in the cytokine network of PsA and RA.

Pannus inflammation in sacroiliitis following immune pathological injury and radiological structural damage: a study of 193 patients with spondyloarthritis.

BACKGROUND: The pathogenesis of sacroiliitis is unclear; therefore, we aimed to systematically study the immunopathology of sacroiliitis in patients with axial spondyloarthritis (axSpA), and explore the relationship between pannus formation, inflammation, and the structural damage caused by sacroiliitis. METHODS: Fine needle aspiration biopsy of the sacroiliac joint (SIJ) was performed in 193 patients with axSpA. Clinical, laboratory, and imaging data were collected at baseline and during the follow up. Immunohistochemistry analysis was performed to detect CD34+ microvessels, CD68+ osteoclasts/macrophages, vascular endothelial growth factor (VEGF), metalloproteinase-3 (MMP-3), tumor necrosis factor-α (TNF-α), and caspase-3. Autopsy subjects were used as controls. RESULTS: In early sacroiliitis (grade 0-1) all pathological features could be observed, with the most common being subchondral pannus formation. Among the 193 patients, 98 were followed up for 1-13 years (mean 3.6 years); 63.3% had radiological progression at the endpoint. Multiple regression analysis showed that cartilage pannus invasion (OR 2.99, P = 0.010) and endochondral ossification (OR 3.97, P = 0.049) at baseline were risk factors for radiological structural damage. Compared to SIJ controls, the subchondral microvessel density, number of CD68+ multinuclear osteoclasts, and the levels of VEGF, caspase-3, MMP-3, and TNF-α expressed at the interface of the bone and cartilage were significantly higher in patients with sacroiliitis. CONCLUSIONS: Subchondral fibrovascular tissue formation is the most important pathological feature in early sacroiliitis. The existence of cartilage pannus invasion or endochondral ossification at baseline can predict radiological structural damage during the follow up.

Carvacrol ameliorates inflammatory response in interleukin 1ß-stimulated human chondrocytes.

Carvacrol, a monoterpenic phenol present in Origanum vulgare (oregano) and Thymus vulgaris (thyme), possesses anti­inflammatory effects; however, little is known about the effects and underlying mechanism of carvacrol on chondrocytes in osteoarthritis (OA). The present study aimed to investigate the protective effects of carvacrol against inflammation in interleukin 1ß (IL­1ß)­stimulated human chondrocytes. The results indicated that carvacrol inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) production, and decreased the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX­2). Carvacrol also suppressed the protein expression levels of matrix metalloproteinase (MMP)­3 and MMP­13 in IL­1ß­stimulated human OA chondrocytes. Furthermore, carvacrol suppressed the activation of nuclear factor (NF)­κB signaling pathway in IL­1ß­induced human chondrocytes. In conclusion, the present results demonstrated that carvacrol was able to inhibit IL­1ß­induced NO and PGE2 production, as well as iNOS, COX­2 and MMPs expression in human chondrocytes by suppressing the activation of NF­κB signaling pathway. Thus, carvacrol may have potential therapeutic functions for the treatment of OA.

Regulatory effects of zinc on cadmium-induced cytotoxicity in chronic inflammation.

OBJECTIVES: Zinc (Zn) has major effects on immune system activation while Cadmium (Cd) has anti-inflammatory and anti-proliferative effects in several chronic inflammatory contexts. The aim of this work was to investigate by which mechanisms Zn could compete with Cd and eventually counteract its deleterious effects. Rheumatoid arthritis (RA) synoviocytes exposed to cytokines were used as a model of chronic inflammation; osteoarthritis (OA) synoviocytes were used as control. METHODS: Cell/medium fractionation constants were analyzed for different metals by inductively-coupled-plasma mass-spectrometry by comparison to the 70Zn spike. Interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-α) were used to mimic inflammation. Gene expression of ZIP-8 importer, metallothioneins-1 (MT-1s) and the ratio between metalloprotease-3 and the tissue inhibitor of metalloproteinases (MMP-3)/TIMP-1) were evaluated after pre-exposure to cytokines and Cd, with or without the addition of exogenous Zn (0.9 ppm). Cell viability was measured by neutral red assay and IL-6 production by ELISA. RESULTS: Synoviocytes selectively absorbed and retained Cd in comparison to Zn. Metal import increased with IL-17/TNF-α exposure, through the enhanced ZIP-8 expression. Zn did not modify ZIP-8 expression, while Cd reduced it (p<0.05). Zn induced a reduction of Cd-induced MT-1s expression, in particular of MT-1X (3-fold), and subsequently the final intra-cellular content of Cd. By reducing Cd accumulation in cells, Zn reversed Cd anti-proliferative and anti-inflammatory effects but preserved the low MMP-3/TIMP-1 ratio induced by Cd, which was enhanced by inflammatory conditions. CONCLUSION: Zinc counteracts the deleterious effect of Cd by reducing its import and accumulation in the cell, without the reactivation of destructive pathways such as MMPs.

Granisetron and carvedilol can protect experimental rats againstadjuvant-induced arthritis.

CONTEXT: Rheumatoid arthritis (RA), a disabling autoimmune disorder of the joints as well as other organs, affects about 1% of population. Unfortunately, all current treatments of RA cause severe gastrointestinal, renal and other complications. OBJECTIVE: We aimed to evaluate the possible antiarthritic effects of a serotonin 5-HT3 receptor blocker, granisetron, and a nonselective adrenergic receptor blocker, carvedilol, on complete Freund's adjuvant-induced RA in adult female albino rats. MATERIALS AND METHODS: Rats were allocated into a normal control group, an arthritis control group, two reference treatment groups receiving dexamethasone (1.5 mg/kg/day) and methotrexate (1 mg/kg/day), and two treatment groups receiving granisetron (2.5 mg/kg/day) and carvedilol (10 mg/kg/day). Serum-specific rheumatoid, immunological, inflammatory and oxidative stress biomarkers were assessed. A confirmatory histopathological study on joints and spleens was performed. RESULTS: Granisetron administration significantly improved all the measured biomarkers, with the values of rheumatoid factor, matrix metalloproteinase-3, cartilage oligomeric matrix protein, immunoglobulin G, antinuclear antibody and myeloperoxidase being restored back to normal levels. Carvedilol administration significantly improved all biomarkers, with serum MPO value restored back to normal levels. DISCUSSION AND CONCLUSIONS: Serotonin 5-HT3 receptor blockers and adrenergic receptor blockers, represented by granisetron and carvedilol, may represent new promising protective strategies against RA, at least owing to immune-modulator, anti-inflammatory and antioxidant potentials.

Development of recombinant matrix metalloproteinase-3 based sandwich ELISA for sero-diagnosis of canine mammary carcinomas.

Matrix metalloproteinase-3 is invariably upregulated in cancerous condition. So we aimed to determine serum level of MMP-3 in canine mammary tumors. The gene was expressed in E. coli system as ~43kDa recombinant protein, which was refolded, purified, and confirmed. Hyperimmune serum was raised against the expressed protein in rabbits and mice to standardize sandwich ELISA. ROC analysis revealed largest area under the curve of 0.998 with sensitivity (100%) and specificity (95%) for a cut-off value of 0.363 with respect to histopathological staining. The finding of the present study indicates that MMP-3 can act as a potential molecular marker for serodiagnosis of canine mammary carcinomas.

Inhibitory Effects of Viscum coloratum Extract on IgE/Antigen-Activated Mast Cells and Mast Cell-Derived Inflammatory Mediator-Activated Chondrocytes.

The accumulation and infiltration of mast cells are found in osteoarthritic lesions in humans and rodents. Nonetheless, the roles of mast cells in osteoarthritis are almost unknown. Although Viscum coloratum has various beneficial actions, its effect on allergic and osteoarthritic responses is unknown. In this study, we established an in vitro model of mast cell-mediated osteoarthritis and investigated the effect of the ethanol extract of Viscum coloratum (VEE) on IgE/antigen (IgE/Ag)-activated mast cells and mast cell-derived inflammatory mediator (MDIM)-stimulated chondrocytes. The anti-allergic effect of VEE was evaluated by degranulation, inflammatory mediators, and the FcεRI signaling cascade in IgE/Ag-activated RBL-2H3 cells. The anti-osteoarthritic action of VEE was evaluated by cell migration, and the expression, secretion, and activity of MMPs in MDIM-stimulated SW1353 cells. VEE significantly inhibited degranulation (IC50: 93.04 µg/mL), the production of IL-4 (IC50: 73.28 µg/mL), TNF-α (IC50: 50.59 µg/mL), PGD2 and LTC4, and activation of the FcεRI signaling cascade in IgE/Ag-activated RBL-2H3 cells. Moreover, VEE not only reduced cell migration but also inhibited the expression, secretion, and/or activity of MMP-1, MMP-3, or MMP-13 in MDIM-stimulated SW1353 cells. In conclusion, VEE possesses both anti-allergic and anti-osteoarthritic properties. Therefore, VEE could possibly be considered a new herbal drug for anti-allergic and anti-osteoarthritic therapy. Moreover, the in vitro model may be useful for the development of anti-osteoarthritic drugs.

Lycorine protects cartilage through suppressing the expression of matrix metalloprotenases in rat chondrocytes and in a mouse osteoarthritis model.

Extracellular matrix (ECM) degrading enzymes, including matrix metalloproteinases (MMPs), are critical for cartilage destruction in the progression of osteoarthritis (OA). Thus, identifying novel drugs, which suppress the synthesis of MMPs may facilitate the treatment of OA. The cytotoxicity of lycorine was determined using a CCK8 assay. The effects of lycorine on IL­1ß­induced upregulation of MMPs and activation of mitogen­activated protein kinase pathways were detected by western blot analysis and reverse transcription­quantitative polymerase chain reaction. Hematoxylin and eosin staining and Safranin O staining were used to evaluate the effect of lycorine in a mouse anterior cruciate ligament transection model. In the present study, it was demonstrated for the first time, to the best of our knowledge, that lycorine (LY) suppressed interleukin­1ß (IL­1ß)­induced synthesis of MMP­3 and MMP­13 in vitro. Molecular analysis revealed that LY abrogated the phosphorylation of c­Jun N­terminal kinase (JNK) and the activation of the nuclear factor (NF)­κB signaling pathway caused by IL­1ß stimulation. In addition, in vivo experiments in a mouse anterior cruciate ligament transection model confirmed the protective role of LY on cartilage. Taken together, the data obtained in the present study demonstrated that LY suppressed the IL­1ß­induced expression of MMP­3 and MMP­13 through inhibition of the JNK and NF­κB pathways, suggesting that LY may be used as a potential drug for the treatment of OA.

Matrix metalloproteinase-10 is a target of T and B cell responses that correlate with synovial pathology in patients with antibiotic-refractory Lyme arthritis.

Infection-induced autoimmunity is thought to be a contributing factor in antibiotic-refractory Lyme arthritis, but studies of autoimmunity have been hindered by difficulty in identifying relevant autoantigens. We developed a novel approach that begins with the identification of T cell epitopes in synovial tissue using tandem mass spectrometry. Herein, we identified an immunogenic HLA-DR-presented peptide (T cell epitope) derived from the source protein matrix metalloproteinase-10 (MMP-10) from the synovium of a patient with antibiotic-refractory arthritis. This finding provided a bridge for the identification of autoantibody responses to MMP-10, the "first autoimmune hit" in a subgroup of patients with erythema migrans, the initial skin lesion of the infection. Months later, after priming of the immune response to MMP-10 in early infection, a subset of patients with antibiotic-responsive or antibiotic-refractory arthritis had MMP-10 autoantibodies, but only patients with antibiotic-refractory arthritis had both T and B cell responses to the protein, providing evidence for a "second autoimmune hit". Further support for a biologically relevant autoimmune event was observed by the positive correlation of anti-MMP-10 autoantibodies with distinct synovial pathology. This experience demonstrates the power of new, discovery-based methods to identify relevant autoimmune responses in chronic inflammatory forms of arthritis.

Mice Deficient in CIZ/NMP4 Develop an Attenuated Form of K/BxN-Serum Induced Arthritis.

CIZ/NMP4 (Cas interacting zinc finger protein, Nmp4, Zfp384) is a transcription factor that is known to regulate matrix related-proteins. To explore the possible pathophysiological role of CIZ/NMP4 in arthritis, we examined CIZ/NMP4 expression in articular cartilage in arthritis model. CIZ/NMP4 was expressed in the articular chondrocytes of mice at low levels while its expression was enhanced when arthritis was induced. Arthritis induction increased clinical score in wild type mice. In contrast, CIZ/NMP4 deficiency suppressed such rise in the levels of arthritis score and swelling of soft tissue. CIZ/NMP4 deficiency also reduced invasion of inflammatory cells in joint tissue. Quantitative PCR analyses of mRNA from joints revealed that arthritis-induced increase in expressions of IL-1ß was suppressed by CIZ/NMP4 deficiency. CIZ/NMP4 bound to IL-1ß promoter and activated its transcription. The increase in CIZ/NMP4 in arthritis was also associated with enhancement in bone resorption and cartilage matrix degradation. In fact, RANKL, a signaling molecule prerequisite for osteoclastogenesis and, MMP-3, a clinical marker for arthritis were increased in joints upon arthritis induction. In contrast, CIZ/NMP4 deficiency suppressed the arthritis-induced increase in bone resorption, expression of RANKL and MMP-3 mRNA. Thus, CIZ/NMP4 plays a role in the development of arthritis at least in part through regulation of key molecules related to the arthritis.

Matrix metalloproteinases contribute to the regulation of chemokine expression and pulmonary inflammation in Cryptococcus infection.

Matrix metalloproteinases (MMPs) are a family of extracellular proteases that play roles in regulating the immune response in inflammatory processes. Previous studies indicated that different MMPs were involved in the host defence and tissue damage in response to different pathogens. However, the contributions of MMPs during Cryptococcus infection have not been addressed clearly. Here, we examined the expression and activity of MMPs during Cryptococcus infection. Among MMP family members, we found significant increases of MMP-3 and MMP-12 mRNA levels and MMP12 zymographic activities in response to C. neoformans but not C. gattii infection. The expression of MMP12 was induced in RAW cells after C. neoformans treatment and in alveolar macrophages purified from C. neoformans-infected mice. Interestingly, administration of MMP inhibitor GM6001 into C. neoformans-infected mice resulted in a significantly increased pulmonary fungal burden with attenuated inflammatory cell infiltration. Corresponding to this finding, the expression of the macrophage- and neutrophil-attracting chemokines CCL2 and CXCL1 was inhibited in the GM6001-treated group and MMP12 levels were found to be correlated strongly with CCL2 mRNA expression. Thus, our data suggest that the induction of MMPs by C. neoformans infection potentiates inflammatory cell infiltration by modulating pulmonary chemokines, thereby promoting effective host immunity to pulmonary Cryptococcus infection.

Histone Methylation and STAT-3 Differentially Regulate Interleukin-6-Induced Matrix Metalloproteinase Gene Activation in Rheumatoid Arthritis Synovial Fibroblasts.

OBJECTIVE: Synovial fibroblasts (SFs) produce matrix-degrading enzymes that cause joint destruction in rheumatoid arthritis (RA). Epigenetic mechanisms play a pivotal role in autoimmune diseases. This study was undertaken to elucidate the epigenetic mechanism that regulates the transcription of matrix metalloproteinases (MMPs) in RASFs. METHODS: MMP gene expression and histone methylation profiles in the MMP promoters were examined in RASFs. The effect of WD repeat domain 5 (WDR5) silencing on histone methylation and MMP gene expression in RASFs was analyzed. MMP gene expression, surface expression of the interleukin-6 (IL-6) receptor, phosphorylation of STAT-3, and binding of STAT-3 in the MMP promoters were investigated in RASFs stimulated with IL-6. RESULTS: The MMP-1, MMP-3, MMP-9, and MMP-13 genes were actively transcribed in RASFs. Correspondingly, the level of histone H3 trimethylated at lysine 4 (H3K4me3) was elevated, whereas that of H3K27me3 was suppressed in the MMP promoters in RASFs. The decrease in H3K4me3 via WDR5 small interfering RNA reduced the levels of messenger RNA for MMP-1, MMP-3, MMP-9, and MMP-13 in RASFs. Interestingly, IL-6 signaling significantly increased the expression of MMP-1, MMP-3, and MMP-13, but not MMP-9, in RASFs. Although the IL-6 signaling pathway was similarly active in RASFs and osteoarthritis SFs, STAT-3 bound to the MMP-1, MMP-3, and MMP-13 promoters, but not the MMP-9 promoter, after IL-6 stimulation in RASFs. CONCLUSION: Our findings indicate that histone methylation and STAT-3 regulate spontaneous and IL-6-induced MMP gene activation in RASFs. The combination of chromatin structure and transcription factors may regulate distinct arthritogenic properties of RASFs.

Inhibitory Effect of a Novel Antirheumatic Drug T-614 on the IL-6-Induced RANKL/OPG, IL-17, and MMP-3 Expression in Synovial Fibroblasts from Rheumatoid Arthritis Patients.

T-614 (also named as iguratimod), a novel antirheumatic drug, could attenuate joint inflammation and articular damage in rheumatoid arthritis (RA) patients, providing a new therapy for RA. Here, we tested the role T-614 on the IL-6-induced receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG), IL-17, and MMP-3 expression in synovial fibroblasts from rheumatoid arthritis (RASFs) patients. T-614 decreased RANKL expression and RANKL/OPG ratio in IL-6-induced RASFs. We confirmed this effect by a decrease of the mRNA and protein RANKL and mRNA RANKL/OPG in RASFs exposed in vitro to T-614 or MTX. Markedly decreased levels of IL-17, retinoid-related orphan receptor C (RORc), and MMP-3 mRNA expression were also observed in IL-6-induced RASFs in the presence of T-614 or MTX compared with those in its absence. Furthermore, T-614 blocked expression of p-ERK1/2 protein without affecting ERK1/2 expression, indicating that the way that T-614 regulated RANKL expression might be ERK1/2 pathway. Our results suggest that T-614 yields a strong improvement in arthritis via exact suppression of RANKL/OPG, IL-17, and MMP-3 expression in RASFs.