Changes in metabolic parameters and serum YKL-40 levels in Chinese rheumatoid arthritis patients during tocilizumab therapy.

OBJECTIVE: To investigate the metabolic changes during therapy of tocilizumab (TCZ) and methotrexate (MTX) in non-diabetic rheumatoid arthritis (RA) patients and for the first time explore the associations between metabolic parameters and serum YKL-40 (sYKL-40) levels. METHODS: We enrolled active non-diabetic RA patients who were refractory to MTX. Patients received intravenous TCZ (8 mg/kg) once every 4 weeks combined with MTX for 24 weeks. Metabolic parameters and sYKL-40 levels were measured before TCZ infusion at baseline, week 4, week 12, and week 24. Correlations were assessed by the Spearman's rank correlation analysis. RESULTS: A total of 91 non-diabetic RA patients were enrolled in this study. At week 24, we observed a significant elevation in body mass index (BMI), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) levels. In contrast, there was a significant decrease in TC/HDL­C ratio. No apparent changes in insulin resistance were found. Additionally, we detected a significant reduction in sYKL-40 levels during the study. At week 24, changes in sYKL-40 levels showed a significant negative correlation (r = -0.334, p = 0.002) with changes in TC levels. CONCLUSION: The combined therapy of TCZ and MTX resulted in a significant increase in BMI and lipid levels, while an evident decrease in the TC/HDL­C ratio and sYKL-40 levels in RA patients. Additionally, there was a significant correlation between the decrease in sYKL-40 levels and the increase in TC levels during treatment with TCZ and MTX. Key Points • Lipid levels elevated significantly and sYKL-40 levels decreased obviously after therapy of TCZ combined with MTX in Chinese RA patients. • There was a significant correlation between the increase in TC levels and the decrease in sYKL-40 levels during treatment with TCZ and MTX in RA patients.

Plasma proteomic profiling reveals KRT19 could be a potential biomarker in patients with anti-MDA5+ dermatomyositis.

OBJECTIVE: To investigate the immune response-related protein profiling in plasma of patients with idiopathic inflammatory myopathies (IIMs), especially in anti-MDA5+ dermatomyositis (DM). METHODS: A total of 166 IIM patients and 107 healthy controls (HCs) were enrolled in our study. Ninety-two plasma immune response-related proteins were detected by Olink proteomics in 36 IIM patients and 25 HCs. The expression of plasma KRT19 was validated in another 130 IIM patients, 82 HCs, and 55 other rheumatic diseases. RESULTS: A total of 46 differentially expressed proteins were detected, including 12 upregulated proteins and 34 downregulated proteins in IIM patients compared with HCs. Pathway analysis revealed lactoferrin danger signal response pathway, TLR4 signaling and tolerance, infection, and IL-10 signaling pathway were activated. The immune response-related protein profiling significantly altered in anti-MDA5+ DM patients, with LAMP3, HSD11B1, and KRT19 significantly increased, while SH2D1A, ITGA11, TRIM21, CD28, ITGB6, and HEXIM1 tremendously decreased. In addition, KRT19 was significantly increased in IIM patients, especially in anti-MDA5+ DM patients with the diagnostic value of a significant area under the ROC curve of 0.881. CONCLUSION: Immune response-related proteins are significantly altered in patients with anti-MDA5+ DM patients. KRT19 could be a potential biomarker for anti-MDA5+ DM patients. Key Points • What is already known on this topic? Anti-MDA5+ DM is a distinctive subtype of IIM. Plasma immune response-related proteins panel needs to be investigated. • What this study adds? Plasma protein profiling of immune response-related proteins significantly altered in patients with idiopathic inflammatory myopathies (IIM), especially in anti-MDA5+ DM patients. • How this study might affect research, practice, or policy? KRT19 could be a potential biomarker in patients with anti-MDA5+ dermatomyositis.

Methylprednisolone pulse therapy promotes the differentiation of regulatory T cells by inducing the apoptosis of CD4+ T cells in patients with systemic lupus erythematosus.

OBJECTIVES: To investigate the differentiation of regulatory T cells (Tregs) induced by methylprednisolone (MP) pulse therapy in patients with Systemic Lupus Erythematosus (SLE). METHODS: We enrolled 30 patients with SLE and analyzed peripheral blood mononuclear cells (PBMCs) before and after MP pulse therapy. Peripheral Tregs, apoptosis of PBMCs subsets, and TGFß production by monocytes was quantified by flow cytometry. Proliferation and IFN-γ production of CD4+ T cells were measured. Furthermore, TGFß1 production by human monocyte-derived macrophages (HMDM) stimulated with MP-treated CD4+ T cells were quantified by ELISA. RESULTS: Peripheral Tregs was significantly increased after MP pulse therapy (6.76 ± 1.46% vs. 3.82 ± 1.02%, p < 0.01), with an expansion of Nrp1- induced Tregs (4.54 ± 0.46% vs. 1.75 ± 0.38%, p < 0.01). Proliferation and IFN-γ production of CD4+ T cells were significantly decreased after MP pulse therapy. MP pulse therapy induced CD4+ T cell apoptosis (early apoptosis, 26.34 ± 3.54% vs. 14.81 ± 2.89%, p < 0.01) and TGFß expression on monocytes (6.02% vs. 2.45%, p < 0.01). Furthermore, MP induced CD4+ T cell apoptosis in vitro, which stimulated HMDM to produce TGFß. Moreover, elevated TGFß level in supernatant from HMDM stimulated with MP-treated CD4+ T cells promoted Tregs differentiation. CONCLUSIONS: MP pulse therapy induces CD4+ T cell apoptosis, which promotes monocytes to produce TGFß and further facilitates Tregs differentiation. Newly-differentiated Tregs suppress proliferation and IFN-γ production of CD4+ T cells and contribute to immunoregulatory milieu after MP pulse therapy.

Circular RNA circHIPK3 Activates Macrophage NLRP3 Inflammasome and TLR4 Pathway in Gouty Arthritis via Sponging miR-561 and miR-192.

Increasing evidences indicate that circular RNAs (circRNAs) play important roles in regulating gene expressions in various diseases. However, the role of circRNAs in inflammatory response of gouty arthritis remains unknown. This study aims to investigate the role and underlying mechanism of circHIPK3 in inflammatory response of gouty arthritis. Quantitative real-time PCR was used to detect the expressions of circHIPK3, miR-192 and miR-561. Western blot was used to detect the protein levels of TLR4, NLRP3, nuclear factor-κB (NF-κB) related proteins, and Caspase-1. Dual luciferase reporter assay, RNA pull-down assay, and FISH assay were used to confirm the interaction between circHIPK3 and miR-192/miR-561. ELISA was used to detect interleukin (IL)-1ß and tumor necrosis factor (TNF)-α levels. circHIPK3 was elevated in synovial fluid mononuclear cells (SFMCs) from patients with gouty arthritis and monosodium urate (MSU)-stimulated THP-1 cells. circHIPK3 overexpression promoted the inflammatory cytokines levels in MSU-stimulated THP-1 cells, and circHIPK3 silencing obtained the opposite effect. Mechanistically, circHIPK3 sponged miR-192 and miR-561, and subsequently promoted the expressions of miR-192 and miR-561 target gene TLR4 and NLRP3. In vivo experiments confirmed circHIPK3 knockdown suppressed gouty arthritis. circHIPK3 sponges miR-192 and miR-561 to promote TLR4 and NLRP3 expressions, thereby promoting inflammatory response in gouty arthritis.

Aberrant distribution of CD3+CD56+ NKT-like cells in patients with primary Sjögren's syndrome.

OBJECTIVES: To elucidate the potential role of CD3+CD56+ NKT-like cells in the pathogenesis of primary Sjögren's syndrome (pSS). METHODS: We enrolled pSS patients and healthy controls and examined the peripheral population, the surface chemokine receptors and the proinflammatory cytokine production of NKT-like cells by flow cytometry. The infiltration of NKT-like cells in the labial salivary gland (LSG) was examined by immunofluorescence. Serum and tissue levels of CX3CL1 were detected by Cytometric Bead Array and immunohistochemistry, respectively. The chemotaxis of NKT-like cells was examined by transwell migration assay. RESULTS: Peripheral NKT-like cells from pSS patients were significantly lower than those from HC (3.09±2.35% vs. 5.37±4.06%, p=0.0002), which was negatively correlated with European League Against Rheumatism Sjögren's Syndrome Disease Activity index. NKT-like cells infiltrated into the LSG of pSS patients. Serum and LSG epithelial CX3CL1 levels were higher in pSS patients than those in HC, which promoted the chemotaxis of the NKT-like cells. NKT-like cells from pSS patients expressed a higher level of CD69, and secreted high level of TNF-α and IFN-γ, which was promoted by CX3CL1 in vitro. CONCLUSIONS: NKT-like cells decreased in peripheral and infiltrated into the LSG of the pSS patients, which could be driven by CX3CL1-CX3CR1 axis. NKT-like cells might be implicated in the pathogenesis of pSS.

Role of cell autophagy in the generation of IgM and hepatic fibrosis in primary biliary cholangitis.

OBJECTIVE: Autophagy in the immune and autoimmune diseases has been concerned more and more. We investigated the potential role of cell autophagy of B cells generating IgM in primary biliary cholangitis (PBC), and explored the relationship between the cell autophagy of hepatic stellate cells (HSCs) and hepatic fibrosis in PBC. METHODS: We examined the aggregation degree of microtubule-associated protein light chain 3II (LC3II) in B cells of PBC (n = 21), health control (HC, n = 15) and disease control (DC, n = 8, active hepatitis B). The expression level of p62/SQSTM1 (p62) in B cells was detected by flow cytometry. ELISA was adopted to detect the level of IgM in the B cell culture supernatant under the basal condition, activated condition(stimulated by pokeweed mitogen, PWM) and inhibited condition(inhibited by autophagy inhibitor, 3-methyladenine, 3-MA) respectively. We detected the expression of α-smooth muscle actin (α-SMA), the infiltration level of LC3II, transforming growth factor-ß (TGF-ß), platelet-derived growth factor-bb (PDGF-bb), and collagen I in the hepatic tissues of five PBC patients and two HC individuals. RESULTS: When B cells were stimulated and activated by PWM, the expression of p62 increased, and the mean fluorescence intensity (MFI, 2404.8 ± 689.0) of p62 in PBC was lower than that in HC (2966.8 ± 488.9,P = 0.0227). Under 3-MA treatment, the MFI expressed of p62 in B cells weakened. The reduction difference in PBC (466.4 ± 214.9) was smaller than that in HC (1166.6 ± 231.2, P = 0.0000) and DC (1184.8 ± 197.7, P = 0.0001). The level of generating IgM in the PBC group was obviously higher than that in the HC group (65.7 ± 15.4 vs. 49.5 ± 20.4 ng/ml, P = 0.0357). Before and after B cells were treated with 3-MA, the peak level of IgM did not significantly diminish in PBC and DC (65.7 ± 15.4 vs. 59.6 ± 18.7 ng/ml, P = 0.2965; 50.1 ± 17.4 vs. 48.4 ± 12.3 ng/ml, P = 0.8336). The immunohistochemical result revealed that the expression level of collagen I, α-SMA, TGF-ß and PDGF-bb in PBC was higher than that in HC, but the expression of LC3II was lower. Similarly, immunofluorescence assay revealed that the fluorescence intensity of collagen I was higher but LC3II was lower in PBC than that in HC. CONCLUSION: The high level autophagy of B cells from PBC patients is one important factor to synthesize and secrete IgM. Hepatic fibrosis of PBC is probably associated with the weakened autophagy of HSCs. Key Points • High-level autophagy of B cells in PBC patients is an important factor to synthesize and secrete IgM. • Pro-fibrogenic cytokines in PBC influence the increase of collagen-I through HSC autophagy, and promote the occurrence and development of hepatic fibrosis.

Elevated EPSTI1 promote B cell hyperactivation through NF-κB signalling in patients with primary Sjögren's syndrome.

BACKGROUND: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterised by aberrant B cell hyperactivation, whose mechanism is partially understood. METHODS: We performed whole transcriptome sequencing of B cells from three pSS patients and three matched healthy controls (HC). Differentially expression genes (DEGs) were confirmed with B cells from 40 pSS patients and 40 HC by quantitative PCR and western blot. We measured the proliferation potential and immunoglobulins production of siRNA-transfected or plasmid-transfected B cells stimulated with cytosine-phosphate-guanine (CpG) or anti-IgM. We also explored Toll-like receptor 9 (TLR9) signalling to reveal the potential mechanism of B cell hyperactivation in pSS. RESULTS: We identified 77 upregulated and 32 downregulated DEGs in pSS B cells. We confirmed that epithelial stromal interaction (EPST1) expression in pSS B cells was significantly higher than that from HCs. EPSTI1-silencing B cells stimulated with CpG were less proliferated and produced lower level of IgG and IgM comparing with control B cells. EPSTI1-silencing B cells expressed lower level of p-p65 and higher level of IκBα, and B cells with overexpressed EPSTI1 showed higher level of p-p65 and lower level of IκBα. Finally, IκBα degradation inhibitor Dehydrocostus Lactone treatment attenuated p65 phosphorylation promoted by EPSTI1. CONCLUSION: Elevated EPSTI1 expression in pSS B cells promoted TLR9 signalling activation and contributed to the abnormal B cell activation, which was promoted by facilitating p65 phosphorylation and activation of NF-κB signalling via promoting IκBα degradation. EPSTI1 might be implicated in pSS pathogenesis and was a potential therapeutic target of pSS.