Combination of scavenger receptor-A with anti-cyclic citrullinated peptide antibody for the diagnosis of rheumatoid arthritis.

OBJECTIVES: The routine biomarkers for rheumatoid arthritis (RA), including anticyclic citrullinated peptide antibody (anti-CCP), rheumatoid factor (RF), immunoglobulin M (IgM), erythrocyte sedimentation rate (ESR), and C-reaction protein (CRP) have limited sensitivity and specificity. Scavenger receptor-A (SR-A) is a novel RA biomarker identified by our group recently, especially for seronegative RA. Here, we performed a large-scale multicentre study to further assess the diagnostic value of SR-A in combination with other biomarkers for RA. METHODS: The performance of SR-A in combination with other biomarkers for RA diagnosis was first revealed by a pilot study, and was further elucidated by a large-scale multicentre study. A total of 1129 individuals from 3 cohorts were recruited in the study, including RA patients, healthy controls, and patients with other common rheumatic diseases. Diagnostic properties were evaluated by the covariate-adjusted receiver-operating characteristic (AROC) curve, sensitivity, specificity and clinical association, respectively. RESULTS: Large-scale multicentre analysis showed that SR-A and anti-CCP dual combination was the optimal method for RA diagnosis, increasing the sensitivity of anti-CCP by 13% (87% vs 74%) while maintaining a specificity of 90%. In early RA patients, SR-A and anti-CCP dual combination also showed promising diagnostic value, increasing the sensitivity of anti-CCP by 7% (79% vs 72%) while maintaining a specificity of 94%. Moreover, SR-A and anti-CCP dual combination was correlated with ESR, IgM, and autoantibodies of RA patients, further revealing its clinical significance. CONCLUSION: SR-A and anti-CCP dual combination could potentially improve early diagnosis of RA, thus improving the prognosis and reducing mortality.

Proinflammatory phenotype of B10 and B10pro cells elicited by TNF-α in rheumatoid arthritis.

OBJECTIVES: B10 and B10pro cells suppress immune responses via secreting interleukin (IL)-10. However, their regulators and underlying mechanisms, especially in human autoimmune diseases, are elusive. This study aimed to address these questions in rheumatoid arthritis (RA), one of the most common highly disabling autoimmune diseases. METHODS: The frequencies and functions of B10 and B10pro cells in healthy individuals and patients with RA were first analysed. The effects of proinflammatory cytokines, particularly tumour necrosis factor (TNF)-α on the quantity, stability and pathogenic phenotype of these cells, were then assessed in patients with RA before and after anti-TNF therapy. The underlying mechanisms were further investigated by scRNA-seq database reanalysis, transcriptome sequencing, TNF-α-/- and B cell-specific SHIP-1-/- mouse disease model studies. RESULTS: TNF-α was a key determinant for B10 cells. TNF-α elicited the proinflammatory feature of B10 and B10pro cells by downregulating IL-10, and upregulating interferon-γ and IL-17A. In patients with RA, B10 and B10pro cells were impaired with exacerbated proinflammatory phenotype, while anti-TNF therapy potently restored their frequencies and immunosuppressive functions, consistent with the increased B10 cells in TNF-α-/- mice. Mechanistically, TNF-α diminished B10 and B10pro cells by inhibiting their glycolysis and proliferation. TNF-α also regulated the phosphatidylinositol phosphate signalling of B10 and B10pro cells and dampened the expression of SHIP-1, a dominant phosphatidylinositol phosphatase regulator of these cells. CONCLUSIONS: TNF-α provoked the proinflammatory phenotype of B10 and B10pro cells by disturbing SHIP-1 in RA, contributing to the disease development. Reinstating the immunosuppressive property of B10 and B10pro cells might represent novel therapeutic approaches for RA.

Leukocyte Ig-like receptor A3 facilitates inflammation, migration and invasion of synovial tissue-derived fibroblasts via ERK/JNK activation.

OBJECTIVE: Leukocyte Ig-like receptor A3 (LILRA3) is a soluble receptor belongs to the immunoglobulin superfamily. Our previous studies demonstrated that LILRA3 is a common genetic risk for multiple autoimmune diseases, including RA. Functional LILRA3 conferred increased risk of joint destruction in patients with early RA. We undertook this study to further investigate the pathological role of LILRA3 in joint inflammation of RA. METHODS: Soluble LILRA3 was measured by ELISA. LILRA3 plasmids were transfected into human fibroblast-like synoviocytes (FLSs) using electroporation. Activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was determined by western blots. Cytokine transcripts were quantified by real-time PCR. Migratory and invasive capacities of FLSs were evaluated using transwell migration and Matrigel invasion assays. FLS apoptosis was analysed using flow cytometry. Colocalization of LILRA3, LILRB1 and HLA-G in RA-FLSs was visualized by immunofluorescence staining. RESULTS: Soluble LILRA3 was specifically expressed in synovial fluid and serum LILRA3 was significantly increased and positively correlated with disease activity/severity in RA patients. LILRA3 induced an increased expression of IL-6, IL-8 and MMP3 in RA-FLSs. In vitro LILRA3 stimulation or overexpression promoted RA-FLS migration and invasion, and enhanced phosphorylation of ERK/JNK. Inhibition of ERK/JNK resulted in suppression of IL-6/IL-8 expression in LILRA3-stimulated RA-FLSs. LILRA3 was co-localized with its homologue LILRB1 and shared ligand HLA-G in RA-FLSs. CONCLUSION: The present study provides the first evidence that soluble LILRA3 is a novel proinflammatory mediator involved in synovial inflammation by promoting RA-FLS activation, migration and invasion, probably through the ERK/JNK signalling pathways.

Serum sCD25 is an indicator for rheumatoid arthritis-associated interstitial lung disease.

OBJECTIVES: CD25 (IL-2Rα) is one of IL-2 receptor's polypeptide subunits, and its soluble form is increased in patients with various inflammatory or autoimmune diseases. This study aimed to evaluate the clinical correlation of serum soluble CD25 (sCD25) with interstitial lung disease (ILD) in rheumatoid arthritis (RA) patients. METHODS: 294 RA patients, including 72 in the discovery cohort (15 patients with ILD, 57 patients without ILD), 222 in the validation cohort (41 patients with ILD and 181 patients without ILD), and 58 healthy controls (HCs) were recruited. High-resolution computed tomography (HRCT) scan provided evidence and patterns of RA-ILD. Serum sCD25 concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Clinical and laboratory data were recorded and the association with sCD25 was also analysed. RESULTS: In the discovery cohort, 16 RA-related molecules including cytokines, chemokines and functional soluble cell surface proteins were investigated. The results showed that sCD25 was significantly higher in RA-ILD than in RA-no-ILD group (p=0.004). ROC analysis also showed RA-ILD was discriminated with RA-no-ILD by sCD25 (AUC=0.695, 95% CI=0.541-0.849). Logistics regression demonstrated that sCD25 was one of the risk factors of RA-ILD. This result was further confirmed in validation cohort (p<0.001). According to the cut-off value in the discovery cohort, the sensitivity and specificity of sCD25 in RA-ILD were 51.2%, 77.3%, respectively. Compared with RA-no-ILD, serum level of sCD25 was also higher in different HRCT patterns including UIP, NSIP and RA-ILA. The ROC curves revealed sCD25 as diagnostic marker in UIP, NSIP and RA-ILA (with AUCs of 0.730, 0.761, and 0. 694, respectively, p<0.05). The result indicated that sCD25 was a biomarker for RA-ILD subtypes. Although sCD25 was not correlated with HRCT scores, it was significantly higher in consolidation pattern by HRCT. CONCLUSIONS: sCD25 was significantly elevated in RA-ILD (including UIP, NSIP and RA-ILA) compared to RA-no-ILD and HCs, which supports their value as a potential biomarker in RA-ILD screening and assessment.

Increased human neutrophil lipocalin and its clinical relevance in adult-onset Still's disease.

BACKGROUND: Human neutrophil lipocalin (HNL) has been used extensively to differentiate acute bacterial infection from febrile diseases as a biomarker to reflect the activation of the neutrophil. The serum HNL levels in the adult-onset Still's disease (AOSD) patients with and without infection, as well as the healthy controls (HCs), were analyzed statistically in this study to evaluate the value of HNL for the diagnosis of AOSD. METHODS: A total of 129 AOSD patients were enrolled, from whom blood samples were drawn and the AOSD diagnosis was confirmed through the review of the medical records, where the systemic score, demographic characteristics, clinical manifestations, and laboratory parameters were also collected for the patients; in addition, a total of 40 HCs were recruited among the blood donors from the healthcare center with the relevant information collected. The HNL test was done for the blood samples with the enzyme-linked immunosorbent assay and the analyses were done for the correlations of HNL with clinical manifestations and diagnostic effectiveness. RESULTS: The serum HNL increased significantly in the patients with only AOSD as compared with that in the HCs (139.76 ±â€Š8.99 ng/mL vs . 55.92 ±â€Š6.12 ng/mL; P  < 0.001). The serum HNL level was correlated with the white blood cell (WBC) count ( r  = 0.335, P  < 0.001), neutrophil count ( r  = 0.334, P  < 0.001), erythrocyte sedimentation rate ( r  = 0.241, P  = 0.022), C-reactive protein ( r  = 0.442, P  < 0.0001), and systemic score ( r  = 0.343, P  < 0.0001) in the AOSD patients significantly. Patients with fever, leukocytosis ≥15,000/mm 3 , and myalgia in the HNL-positive group were observed relatively more than those in the HNL-negative group ( P  = 0.009, P  = 0.023, and P  = 0.007, respectively). HNL was a more sensitive indicator than ferritin and C-reactive protein (CRP) to differentiate the AOSD patients with bacterial infection from AOSD-only patients, and the Youden index was 0.6 for HNL and 0.29 for CRP. CONCLUSION: Serum HNL can be used as a biomarker for the diagnosis of the AOSD, and HNL is also observed to be associated with the disease activity.

Decreased natural killer T-like cells correlated to disease activity in systemic lupus erythematosus.

OBJECTIVES: To evaluate the absolute numbers and frequencies of natural killer T-like (NKT-like) cells in systemic lupus erythematosus (SLE) and to characterize the possible role of the cells. METHODS: Seventy-nine patients with SLE together with 30 age- and sex-matched healthy controls were enrolled. Flow cytometric determination of peripheral NKT-like cells was carried out for all participants by detecting the absolute counts (Abs) and percentage (%) of CD3 + CD16 + CD56 + cells. Disease activity index, laboratory parameters, and clinical manifestations were collected. The correlation between the cells and these parameters was analyzed. RESULTS: SLE patients had, with respect to controls, considerably decreased values of NKT-like cells (P < 0.001 in both absolute number and percentage). The absolute number of NKT-like cells was found to have positive correlations with WBC, RBC, PLT, C3, C4, IgM and negative correlations with the disease duration, SLEDAI-2 K, anti-dsDNA, anti-nucleosome, anti-ribosomal protein, CRP, ESR. Meanwhile, it was found that the percentage values of NKT-like cells decreased in SLE patients with nephritis which was correlated with anti-ribosomal protein and CRP in comparison to SLE patients without nephritis. Moreover, an increase in the NKT-like cell counts was also observed in the patients with a clinical response to the treatment. CONCLUSIONS: The absolute counts and frequencies of NKT-like cells decreased in SLE patients significantly, which correlated to disease activities and could recover to normal after the treatment. The NKT-like cells may play an important role in the pathogenesis of SLE and could be a useful marker in the disease assessment. Key Points • The absolute counts and frequencies of NKT-like cells decreased in SLE patients significantly. • NKT-like cells were related to the disease activities and could restore after the treatment. • NKT-like cells may be a useful marker in the disease assessment.

Immune phenotype changes in IgG4-related disease: CD161 + Treg and Foxp3 + Treg.

OBJECTIVE: We aimed to characterize the alterations in the immune phenotypes and explore the potential relevance to pathogenesis in IgG4-RD. METHODS: Forty-two IgG4-RD patients and thirty-eight healthy controls were recruited in this study. Peripheral immunocompetent cells including T cells, CD4 + T cells, CD8 + T cells, B cells, NK cells CD4 + CD45RA + T cells (naïve T cells), CD4 + CD25 - / + Foxp3 - T cells (Teff), CD4 + CD25hiCD127lowCD161 + T cells (CD161 + Treg), CD4 + CD25hiFoxp3 + T cells (Foxp3 + Treg), CD4 + CD4RA-CXCR5 + PD1 + CCR7low T cells (pTfh), T helper (Th) 1, Th2, and Th17 before and after treatment were immunophenotyped by flow cytometry. RESULTS: Compared with healthy controls, IgG4-RD patients showed higher proportions of NK (20.1% vs 13.6%, p < 0.01), Th1 (CD4 + IFN-γ + : 17.9% vs 14.2%, p = 0.061; TNF-α: 43.7% vs 36.7%, p < 0.05), Th2 (CD4 + IL-4 + : 2.4% vs 1.3%, p < 0.0001), CD161 + Treg (14.9% vs 11.6%, p < 0.01), pTfh (3.2% vs 2.4%, p < 0.05), and Foxp3 + Treg (8.3% vs 7.0%, p < 0.01) and lower proportions of B lymphocytes (8.4% vs 13.1%, p < 0.001), Teff (91.6% vs 92.6%, p < 0.01), and naïve Th cells (19.9% vs 32.1%, p < 0.01) before treatment. Foxp3 + Treg percentage decreased significantly after treatment (8.6% vs 6.9%, p < 0.05). Both serum C3 (r = - 0.6374, p < 0.01) and C4 (r = - 0.6174, p < 0.01) levels were in negative correlation with CD161 + Treg. The eosinophil percentage was positively correlated with Foxp3 + Treg (r = 0.5435, p < 0.05). Serum IgE level was positively correlated with Th2 (r = 0.5545, p < 0.05). There was a positive correlation between CD161 + Treg and pTfh (r = 0.4974, p < 0.05) while a negative correlation between Th2 and B cells (r = - 0.4925, p < 0.05). CONCLUSION: Immune phenotypes were altered in IgG4-RD. Treg/Teff balance was shifted toward Treg in IgG4-RD. CD161 + Treg was likely to be involved in the pathogenesis of IgG4-RD. Key Points •Immune phenotypes were altered in B cells, T cells, and NK cells in IgG4-RD. •Treg/Teff balance was shifted toward Treg in IgG4-RD. •CD161+ Treg maybe play a proinflammatory role in IgG4-RD.

Diminished natural killer T-like cells correlates with aggravated primary Sjögren's syndrome.

OBJECTIVE: To reveal the characteristics and potential role of natural killer T-like cells (NKT-like cells) in the pathogenesis of primary Sjögren's syndrome (pSS). METHODS: Forty-six patients with pSS and 30 healthy subjects were enrolled in the study. The frequencies and cell count of NKT-like cells as well as other lymphocyte subsets were analyzed by flow cytometry. The clinical and laboratory indicators of pSS patients were also collected. Then, the correlation between NKT-like cells and pSS patient manifestations was analyzed by Spearman's rank test. In addition, NKT-like cells before and after therapy were also compared. RESULTS: Both the number and the frequencies of NKT-like cells were significantly decreased in pSS patients. The counts of NKT-like cells were positively correlated with CD4+ T cells (r = 0.464, P = 0.001), CD8+ T cells (r = 0.363, P = 0.013), NK cells (r = 0.488, P = 0.001), and IgM levels (r = 0.443, P = 0.002), while negatively correlated with the disease duration (r = - 0.33, P = 0.027). Moreover, after effective therapy, NKT-like cells were recovered both in the cell counts and frequencies. CONCLUSION: In pSS, NKT-like cells were fundamentally decreased, potentially contributing to the disease pathogenesis. Modulating the status of NKT-like cells might provide a novel strategy for treating the disease. KEY POINTS: • NKT-like cells were significantly decreased in pSS patients. • NKT-like cells were correlated with pSS patient manifestations. • NKT-like cells might be serverd as a new marker for assessing the status of pSS.

Dickkopf-1 perpetuated synovial fibroblast activation and synovial angiogenesis in rheumatoid arthritis.

OBJECTIVE: Dickkopf-1 (Dkk-1), a regulatory molecule of the Wnt pathway, is elevated and leads to bone resorption in patients with RA. This study is aimed to investigate the contribution of Dkk-1 to synovial inflammation and synovial fibroblast-mediated angiogenesis in RA. METHODS: The expression of Dkk-1 in RA synovial fibroblasts (RASF) and osteoarthritis synovial fibroblasts (OASF) was detected by real-time PCR and ELISA, respectively. RASF were stimulated with different pro-inflammatory factors. The expression of angiogenic factors, pro-inflammatory cytokines, and MMPs in RASF was analyzed by real-time PCR when Dkk-1 was inhibited or overexpressed. Meanwhile, the concentrations of MCP-1, IL-6, IL-8, and MMP-3 in the cell culture supernatant were assessed by ELISA. The effects of Dkk-1 on the MAPK signaling pathway were evaluated by western blot. Matrigel tube formation assay was employed to reveal the direct and indirect effects of Dkk-1 on synovial angiogenesis. RESULTS: Dkk-1 expression was elevated in synovial fluids and synovial fibroblasts of RA patients. Treatment with various pro-inflammatory cytokines significantly promoted DKK-1 expression in RASF. The production of potent angiogenic factors, pro-inflammatory cytokines, and MMPs in RASF was elevated, whereas the reverse results were found in the inhibitor groups. Silenced Dkk-1expression in RASF dampened capillary tube organization in both direct and indirect manners, resulting in restrained ERK, JNK, and p38 signaling pathway activation. CONCLUSION: We concluded that Dkk-1 exacerbated the inflammation, cartilage erosion, and angiogenesis mediated by synovial fibroblasts in RA. Modulation of DKK-1 expression may facilitate development of novel strategies to control RA. Key points • Dkk-1 expression was elevated in synovial fluids and synovial fibroblasts of RA patients. • Treatment with various pro-inflammatory cytokines significantly promoted DKK-1 expression. • Silenced Dkk-1expression in RASF dampened capillary tube organization.

Nanocage-Based Capture-Detection System for the Clinical Diagnosis of Autoimmune Disease.

The detection of autoantibodies is critical for diagnosis of autoimmune diseases. However, the sensitivity is often limited by the properties of the antigens and the detection systems such as enzyme-linked immunosorbent assay (ELISA). Here, employing the multidisplay ability of ferritin, a highly sensitive nanocage-based capture-detection system is designed, of which the sensitivity is 100-1000-fold higher than that of conventional ELISA methods. The capture nanocages are constructed by displaying the primary Sjögren's syndrome (pSS)-related antigenic peptides on ferritin nanocage, which present epitopes effectively and high affinity, leading to tenfold higher capture capability for autoantibodies. Human IgG Fc-binding peptides are also engineered on ferritin nanocage, which enable high binding affinity and efficient horseradish peroxidase (HRP)-labeling. Compared with commercial HRP-conjugated anti-human IgG antibody, the nanocage-based detecting probe exhibited more than tenfold increased sensitivity. Autoantibodies are then examined in 91 sera from patients with pSS, 51 from rheumatoid arthritis, 54 from systemic lupus erythematosus, and 55 from healthy individuals by using the nanocage-based ELISA. The results indicate that the nanocage-based capture-detection system is an effective detection platform and provide a novel and more sensitive method for the diagnosis of autoimmune diseases.

Antibody to peptidoglycan recognition protein (PGLYRP)-2 as a novel biomarker in rheumatoid arthritis.

OBJECTIVES: To identify novel autoantigens from circulating immune complexes (CICs) in rheumatoid arthritis (RA) patients and further explore their clinical significance. METHODS: From serum samples of 10 early RA (ERA) patients and 10 healthy donors, CICs were isolated and subjected to orbitrap mass spectrometry for autoantigen identification. Antibodies against the peptidoglycan recognition protein-2 (PGLYRP-2) derived from CICs were further detected by indirect enzyme-linked immunosorbent assay (ELISA) in 178 patients with RA, compared with 59 osteoarthritis (OA), 59 systemic lupus erythematosus (SLE), 55 ankylosing spondylitis (AS), 95 primary Sjögren's syndrome (pSS) and 50 healthy controls (HC). RESULTS: Thirty-three potential antigens out of 323 proteins were identified from CICs of RA patients. The autoantibodies to PGLYRP-2 were significantly increased in RA patients with 42.70% sensitivity and 85.20% specificity in comparison to other rheumatic diseases and healthy controls. The prevalence of anti-PGLYRP-2 was also elevated in subgroups of RA, with 34.72% in ERA, 35.29% in RF negative and 42.86% in anti-CCP negative patients. Further analysis suggested that anti-PGLYRP-2 was potentially accompanied with production of other autoantibodies in RA. In addition, we found by homology analysis that an epitope of PGLYRP-2442-447 mimics amino acid residues 431-436 of N-acetylmuramoyl-L-alanine amidase (NAMLAA) in actinomyces naeslundii. CONCLUSIONS: Autoantibody against PGLYRP-2 was identified as a promising biomarker in RA, especially in early and seronegative patients.

Scavenger receptor-A is a biomarker and effector of rheumatoid arthritis: A large-scale multicenter study.

Early diagnosis is critical to improve outcomes in rheumatoid arthritis (RA), but current diagnostic tools have limited sensitivity. Here we report a large-scale multicenter study involving training and validation cohorts of 3,262 participants. We show that serum levels of soluble scavenger receptor-A (sSR-A) are increased in patients with RA and correlate positively with clinical and immunological features of the disease. This discriminatory capacity of sSR-A is clinically valuable and complements the diagnosis for early stage and seronegative RA. sSR-A also has 15.97% prevalence in undifferentiated arthritis patients. Furthermore, administration of SR-A accelerates the onset of experimental arthritis in mice, whereas inhibition of SR-A ameliorates the disease pathogenesis. Together, these data identify sSR-A as a potential biomarker in diagnosis of RA, and targeting SR-A might be a therapeutic strategy.

Activity of fibroblast-like synoviocytes in rheumatoid arthritis was impaired by dickkopf-1 targeting siRNA.

BACKGROUND: Fibroblast-like synoviocytes (FLSs), resident mesenchymal cells of synovial joints, play an important role in the pathogenesis of rheumatoid arthritis (RA). Dickkopf-1 (DKK-1) has been proposed to be a master regulator of bone remodeling in inflammatory arthritis. Here, potential impairation on the activity of FLSs derived from RA to small interfering RNAs (siRNAs) targeting DKK-1 was investigated. METHODS: siRNAs targeting DKK-1 were transfected into FLSs of patients with RA. Interleukin (IL)-1ß, IL-6, IL-8, matrix metalloproteinase (MMP) 2, MMP3, MMP9, transforming growth factor (TGF)-ß1, TGF-ß2 and monocyte chemoattractant protein (MCP)-1 levels in the cell culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Invasion assay and H incorporation assay were utilized to investigate the effects of siRNAs targeting DKK-1 on FLSs invasion and cell proliferation, respectively. Western blotting was performed to analyze the expression of nuclear factor (NF)-κB, interleukin-1 receptor-associated kinase (IRAK)1, extracellular regulated protein kinases (ERK)1, Jun N-terminal kinase (JNK) and ß-catenin in FLSs. RESULTS: DKK-1 targeting siRNAs inhibited the expression of DKK-1 in FLSs (P < 0.01). siRNAs induced a significant reduction of the levels of IL-6, IL-8, MMP2, MMP3 and MMP9 in FLSs compared to the control group (P < 0.05). DKK-1 targeting siRNAs inhibited the proliferation and invasion of FLSs (P < 0.05). Important molecules of pro-inflammatory signaling in FLSs, including IRAK1 and ERK1, were decreased by the inhibition of DKK-1 in FLSs. In contrast, ß-catenin, a pivotal downstream molecule of the Wnt signaling pathway was increased. CONCLUSIONS: By inhibiting DKK-1, we were able to inhibit the proliferation, invasion and pro-inflammatory cytokine secretion of FLSs derived from RA, which was mediated by the ERK or the IRAK-1 signaling pathway. These data indicate the application of DKK-1 silencing could be a potential therapeutic approach to RA.

CD70-mediated CD27 expression downregulation contributed to the regulatory B10 cell impairment in rheumatoid arthritis.

Regulatory B10 cells have been shown to exhibit impaired functions in autoimmune diseases. However, the underlying mechanism is still obscure. In the present study, we aimed to understand the regulatory characteristics of regulatory B10 cells and how these cells are involved in the development of rheumatoid arthritis (RA). Here, we chose CD19+CD24hiCD27+ as the phenotype of regulatory B10 cells. We found that the frequencies of CD19+CD24hiCD27+ regulatory B10 cells were decreased and that their IL-10-producing function was impaired in patients with RA compared with healthy controls (HCs). The impairment in CD19+CD24hiCD27+ B10 cells was partially attributed to the decreased expression of CD27 induced by the upregulated CD70 expression on CD19 + B cells and CD4 + T cells. The proportion of CD19+CD24hiCD27+ regulatory B10 cells could be restored by blocking the CD70-CD27 interaction with an anti-CD70 antibody. Furthermore, the CD70-CD27 interaction significantly elevated IL-10 expression and might compensate for the decreased number of CD19+CD24hiCD27+ B cells. Hence, the CD70-CD27 interaction might play a critical role in the numerical and functional impairments of regulatory B10 cells, thus contributing to RA pathogenesis. In conclusion, the change in CD19+CD24hiCD27+ regulatory B10 cells in RA was only a consequence, not the cause, of RA development, but the increased expression of CD70 might be the culprit.

Efficacy and safety of iguratimod on patients with relapsed or refractory IgG4-related disease.

OBJECTIVE: To evaluate the therapeutic efficacy and safety of iguratimod on patients with relapsed or refractory IgG4-related disease (IgG4-RD). METHODS: We conducted a retrospective single-center study in 17 IgG4-RD patients admitted to Peking University People's Hospital. Patients were given iguratimod, 25 mg, twice daily and clinical data were collected at 0, 12, and 24 weeks. The baseline treatments include prednisone, cyclophosphamide, leflunomide, mycophenolate mofetil, and methotrexate. Clinical manifestation, IgG4-RD responder index (IgG-RD RI), serological indexes, gland ultrasound findings, and adverse drug effect were recorded. IgG4-RD RI scores < 3 and declining ≥ 2 were recognized as complete response (CR); IgG4-RD RI scores declining ≥ 2 but remaining ≥ 3 were recognized as partial response (PR). If a patient's IgG4-RD RI score was 3 at the beginning, PR was considered as a 1-point decrease after the therapy. RESULTS: Serum IgG4 decreased significantly from 708 (321-902) mg/dl at baseline to 446 (138-396) mg/dl at 24 weeks (P = 0.0016). IgG4-RD RI decreased significantly from 9.79 ± 3.07 at baseline to 3.57 ± 1.09 at 24 weeks (P < 0.0001). Overall, 2 (14.3%) patients achieved CR, 11 (78.6%) patients achieved PR, and 1 (7.14%) patient had no response to treatment at week 24. Serum IgG level and salivary glands major diameter also decreased significantly at week 12 and 24 after treatment. CONCLUSION: Iguratimod can be a therapeutic strategy to achieve remission in relapsed or refractory IgG4-RD patients inadequately responding to corticosteroid treatment with or without other immunosuppressant treatment. Key messages • Iguratimod was effective for relapsed or refractory IgG4-RD patients. • Iguratimod can improve the clinical symptoms of patients, reduce the serum IgG and IgG4 levels, and can also reduce the volume of involved glands.

Upregulated microRNA-15b alleviates ovarian cancer through inhitbition of the PI3K/Akt pathway by targeting LPAR3.

Ovarian cancer characterizes as the fourth leading consequence of death associated with cancer for women. Accumulating evidence underscores the vital roles of microRNAs (miRNAs) in preventing ovarian cancer development. Besides, induction of the phosphatidylinositol-3 kinase/serine/threonine kinase (PI3K/Akt) pathway associated with the ovarian cancer cell migration and invasion. The study aims to examine the effects of miR-15b on the proliferation, apoptosis, and senescence of human ovarian cancer cells by binding to lysophosphatidic acid receptor 3 (LPAR3) with the involvement of the PI3K/Akt pathway. The positive expression of LPAR3 protein was detected by immunohistochemistry. Then the interaction between miR-15b and LPAR3 was examined. The possible role of miR-15b in ovarian cancer was explored using gain- and loss-of-function experiments. Subsequently, the functions of miR-15b on PI3K/Akt pathway, proliferation, migration, invasion, senescence and apoptosis of ovarian cancer cells were assessed. Furthermore, in vivo tumorigenicity assay in nude mice was performed. LPAR3 was overexpressed, whereas miR-15b was poorly expressed in ovarian cancer tissues. LPAR3 is a direct target of miR-15b. Restored miR-15b promoted Bax expression, apoptosis, and senescence, inhibited expression of LPAR3 and Bcl-2, the extent of PI3K and Akt phosphorylation, as well as ovarian cancer cell proliferation, migration, and invasion. Further, tumor growth was observed to be prevented by miR-15b overexpression. Collectively, our study demonstrates that miR-15b represses the proliferation and drives the senescence and apoptosis of ovarian cancer cells through the suppression of LPAR3 and the PI3K/Akt pathway, highlighting an antitumorigenic role of miR-15b.

Reversal of cisplatin resistance by microRNA-139-5p-independent RNF2 downregulation and MAPK inhibition in ovarian cancer.

Some microRNAs (miRs) are dysregulated in cancers, and aberrant miR expression has been reported to correlate with chemoresistance of cancer cells. Therefore, the present study aims at investigating the effects of microRNA-139-5p (miR-139-5p) on cisplatin resistance of ovarian cancer (OC) with involvement of ring finger protein 2 (RNF2) and the mitogen-activated protein kinase (MAPK) signaling pathway. OC tissues were obtained from 66 primary OC patients. The cisplatin-sensitive A2780 and cisplatin-resistant A2780/DDP cell lines were collected for construction of RNF2 silencing and overexpressed plasmids. Cell vitality and apoptosis were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and annexin V-FITC/propidium iodide double-staining, respectively. Next, expression of RNF2, extracellular signal-related kinase, and p38 was determined by quantitative reverse transcription-quantitative polymerase chain reaction and Western blot analysis. Finally, the volume of xenograft tumors in BALB/c nude mice was detected. RNF2 and miR-139-5p were identified to be involved in OC. In addition, MAPK activation and RNF2 were related to cisplatin resistance of OC. miR-139-5p was downregulated in cisplatin-resistant OC tissues, and miR-139-5p overexpression could inhibit cell vitality, reduce cisplatin resistance, and promote apoptosis of OC cells. Furthermore, miR-139-5p combined with MAPK inhibitors more obviously reduced cisplatin resistance of OC. Taken together, this study demonstrated that miR-139-5p overexpression combined with inactivation of the MAPK signaling pathway can reverse the cisplatin resistance of OC by suppressing RNF2. Thus, miR-139-5p overexpression might be a future therapeutic strategy for OC.

Impaired CD27+IgD+ B Cells With Altered Gene Signature in Rheumatoid Arthritis.

Natural antibodies, particularly natural IgM, are proved to play indispensable roles in the immune defenses against common infections. More recently, the protective roles of these natural IgM were also recognized in autoimmune diseases. They are mainly produced by B-1 and innate-like B cells (ILBs). Human CD19+CD27+IgD+ B cells, also termed as un-switched memory B cells, were proposed to be a kind of ILBs. However, functional features and characteristics of these cells in rheumatoid arthritis (RA) remained poorly understood. In this study, we found that human CD27+IgD+ B cells could produce natural antibody-like IgM. Under RA circumstance, the frequencies of these cells were significantly decreased. Moreover, the IgM-producing capacities of these cells were also dampened. Interestingly, the BCR repertoire of these cells was altered in RA, demonstrating decreased diversity with preferential usage alteration from VH3-23D to VH1-8. Single cell sequencing further revealed the proinflammatory biased features of these cells in RA. These CD27+IgD+ B cells were negatively correlated with RA patient disease activities and clinical manifestations. After effective therapy with disease remission in RA, these cells could be recovered. Taken together, these results have revealed that CD27+IgD+ B cells were impaired in RA with dysfunctional features, which might contribute to the disease perpetuation.

MicroRNA-374b inhibits cervical cancer cell proliferation and induces apoptosis through the p38/ERK signaling pathway by binding to JAM-2.

Cervical cancer (CC) remains a highly prevalent cancer and mortality globally among women globally. The aim of the present study was to assess the ability of miR-374b to regulate CC cells through JAM-2, whilst exploring whether the underlying mechanism and its relation to the p38/ERK signaling pathway. During the study, microRNA-374b (miR-374b) was observed to have been expressed at a low level among CC tissues. Hence, a series of miR-374b mimics, miR-374b inhibitors, siRNA against JAM-2, SB202190 (an inhibitor for p38), and PD98059 (an inhibitor for ERK) were introduced to treat CC Siha cells and normal cervical Ect1/E6E7 cells. MTT, flow cytometry, scratch test, and transwell assays were applied to determine cell viability, apoptosis, migration, and invasion. The inhibitory role of the p38/ERK signaling pathway was observed in the CC cells treated with miR-374b mimics or siRNA against JAM-2. miR-374b mimic exposure was found to reduce cell viability, migration, and invasion, but induce apoptosis. MiR-374b inhibitor exposure was observed to have induced effects on the CC cells in a contrary manner to those induced by that of the miR-374b mimics. The key findings of the study demonstrated that miR-374b significantly inhibits cell proliferation, migration, and invasion through the blockade of the p38/ERK signaling pathway activation, as well as negatively binding to JAM-2, highlighting its potential as a therapeutic target for CC.

Elevated Levels of Soluble ST2 were Associated with Rheumatoid Arthritis Disease Activity and Ameliorated Inflammation in Synovial Fibroblasts.

BACKGROUND: Much evidence has demonstrated that interleukin (IL)-33 plays an important role in rheumatoid arthritis (RA). However, there have been limited studies about soluble ST2, a receptor for IL-33, in RA. The aims of this study were to detect the levels of ST2 in the serum and synovial fluid of RA patients and to reveal the association of these levels with disease activity and the function of ST2 in RA. METHODS: A total of 56 RA patients and 38 age-matched healthy controls were enrolled in this study. Synovial fluid samples were collected from another 30 RA patients and 20 osteoarthritis patients. Serum and synovial fluid levels of ST2 were measured by ELISA. In addition, the levels of ST2 in the serum of RA patients before and after therapy were detected. The function of ST2 in RA was revealed by the results of an in vitro cell assay, where recombinant ST2 proteins were used to treat peripheral blood mononuclear cells (PBMCs) and RA synovial fibroblasts (RASFs). RESULTS: Serum-soluble ST2 levels were significantly higher in RA patients (127.14 ± 61.43 pg/ml) than those in healthy controls (78.37 ± 41.93 pg/ml, P < 0.01). Synovial fluid-soluble ST2 levels (41.90 ± 33.58 pg/ml) were much higher in RA patients than those in osteoarthritis patients (19.71 ± 16.72 pg/ml, P < 0.05). RA patients who received effective therapy for 6 months showed decreased serum-soluble ST2 levels (113.01 ± 53.90 pg/ml) compared to baseline (139.59 ± 68.36 pg/ml) (P = 0.01). RA patients with high disease activity had higher serum-soluble ST2 levels (162.02 ± 56.78 pg/ml) than those with low disease activity (94.67 ± 40.27 pg/ml, P = 0.001). Soluble ST2 did not affect IL-1ß, IL-6, IL-8, or tumor necrosis factor-α (TNF-α) expression in PBMCs from RA patients. However, soluble ST2 ameliorated the expressions of IL-33 and IL-1ß but not that of IL-6, IL-8, or TNF-α in resting RASFs. Interestingly, in the RASFs stimulated by TNF-α plus IL-1ß, soluble ST2 showed extensive suppressive effects on the expression of IL-6, IL-8, and TNF-α. CONCLUSION: Elevated levels of ST2 in the serum and synovial fluid were associated with disease activity and ameliorated IL-33 expression and IL-33-induced inflammation in RASFs, suggesting that soluble ST2 might be a potential therapeutic candidate for RA.