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.

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.

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.

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.

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.

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.

Hypoxia-inducible factor-1α perpetuates synovial fibroblast interactions with T cells and B cells in rheumatoid arthritis.

Synovial fibroblast hyperplasia, T-cell hyperactivity, B-cell overactivation, and the self-perpetuating interactions among these cell types are major characteristics of rheumatoid arthritis (RA). The inflamed joints of RA patients are hypoxic, with upregulated expression of hypoxia-inducible factor-1α (HIF-1α) in RA synovial fibroblasts (RASFs). It remains unknown whether HIF-1α regulates interactions between RASFs and T cells and B cells. We report here that HIF-1α promotes the expression of inflammatory cytokines IL-6, IL-8, TNF-α, and IL-1ß, and cell-cell contact mediators IL-15, vascular cell adhesion molecule (VCAM)-1, thrombospondin (TSP)-1, and stromal cell-derived factor (SDF)-1 in RASFs. Furthermore, HIF-1α perpetuates RASF-mediated inflammatory Th1- and Th17-cell expansion while differentially inhibiting regulatory B10 and innate-like B cells, leading to increased IFN-γ, IL-17, and IgG production and decreased protective natural IgM secretion. Our findings suggest that HIF-1α perpetuates the interactions between RASFs and T cells and B cells to induce inflammatory cytokine and autoantibody production, thus exacerbating the severity of RA. Targeting HIF-1α may provide new therapeutic strategies for overcoming this persistent disease.

Pathogenic conversion of regulatory B10 cells into osteoclast-priming cells in rheumatoid arthritis.

Regulatory B10 cells were functionally impaired in rheumatoid arthritis (RA), yet the mechanisms were unclear. B cells are recently recognized as important participants in osteoclastogenesis by producing RANKL. In this study, we investigated whether regulatory B10 cells could convert into RANKL-producing cells, thus impairing their immunosuppressive functions in RA and exacerbating the disease progression. Our results showed that human regulatory B10 cells could ectopically express RANKL. Under RA circumstance, RANKL-producing B10 cells expanded dramatically, partially induced by TNF-α. The frequencies of these cells were positively correlated with RA patient disease activities and tender joint counts, but negatively correlated with the frequencies of regulatory B10 cells. Strikingly, RANKL-producing B10 cells from RA patients, but not healthy individuals significantly promoted osteoclast differentiation and bone erosion in a paracrine and cell-cell contact-dependent manner. Moreover, these pathogenic RANKL-producing B10 cells declined while regulatory IL-10-producing B10 cells increased in RA patients with disease remission after therapy. Collectively, these results showed that in RA, regulatory B10 cells demonstrated the potential of converting into RANKL-producing cells, thus exacerbating osteoclast formation, bone destruction and disease progression. Modulating the status of B10 cells might provide novel therapeutic strategies for RA.

Myeloid-derived suppressor cells have a proinflammatory role in the pathogenesis of autoimmune arthritis.

OBJECTIVES: Although myeloid-derived suppressor cells (MDSCs) have been linked to T cell tolerance, their role in autoimmune rheumatoid arthritis (RA) remains elusive. Here we investigate the potential association of MDSCs with the disease pathogenesis using a preclinical model of RA and specimen collected from patients with RA. METHODS: The frequency of MDSCs in blood, lymphoid tissues, inflamed paws or synovial fluid and their association with disease severity, tissue inflammation and the levels of pathogenic T helper (Th) 17 cells were examined in arthritic mice or in patients with RA (n=35) and osteoarthritis (n=15). The MDSCs in arthritic mice were also characterised for their phenotype, inflammation status, T cell suppressive activity and their capacity of pro-Th17 cell differentiation. The involvement of MDSCs in the disease pathology and a Th17 response was examined by adoptive transfer or antibody depletion of MDSCs in arthritic mice or by coculturing mouse or human MDSCs with naïve CD4+ T cells under Th17-polarising conditions. RESULTS: MDSCs significantly expanded in arthritic mice and in patients with RA, which correlated positively with disease severity and an inflammatory Th17 response. While displaying T cell suppressive activity, MDSCs from arthritic mice produced high levels of inflammatory cytokines (eg, interleukin (IL)-1ß, TNF-α). Mouse and human MDSCs promoted Th17 cell polarisation ex vivo. Transfer of MDSCs facilitated disease progression, whereas their elimination in arthritic mice ameliorated disease symptoms concomitant with reduction of IL-17A/Th17 cells. CONCLUSIONS: Our studies suggest that proinflammatory MDSCs with their capacity to drive Th17 cell differentiation may be a critical pathogenic factor in autoimmune arthritis.

Hypoxia and hypoxia-inducible factor-1α provoke toll-like receptor signalling-induced inflammation in rheumatoid arthritis.

OBJECTIVES: Hyperplasia of synovial fibroblasts, infiltration with lymphocytes and tissue hypoxia are major characteristics of rheumatoid arthritis (RA). Extensive data support a key role for toll-like receptors (TLRs) in RA. Little is known regarding the impact of hypoxia on TLR-induced inflammation in RA. The aim of this study was to reveal the effects of hypoxia and its regulator, hypoxia-inducible factor-1α (HIF-1α), on the inflammatory response of RA synovial fibroblasts (RASF) to TLR ligands. METHODS: Hypoxia was induced in RASF by incubation with Na2S2O4. TLR3 ligand polyIC, TLR2 ligand peptidoglycan, TLR4 ligand LPS and TLR9 ligand CpG were used to stimulate the cells. Effects of hypoxia on TLR-induced inflammatory mediators were determined by RT-PCR, qPCR and ELISA. Overexpression of HIF-1α as well as knocking-down its expression was used to reveal its fundamental role. RASF-induced inflammatory T cell expansion was determined by flow cytometry analysis of T helper (Th)1/Th17 cells, and IFN-γ/IL-17 production by ELISA after RASF/T cell coculture. RESULTS: Hypoxia potentiated the expression of inflammatory cytokines, metalloproteinases and VEGF in RASF stimulated by different TLR ligands, especially polyIC, a synthetic mimic of dsRNA from viruses or apoptotic cells. HIF-1α played a fundamental role in this synergy. Moreover, HIF-1α overexpression enhanced RASF-mediated expansion of inflammatory Th1 and Th17 cells, leading to proinflammatory IFN-γ and IL-17 production. CONCLUSIONS: Our findings suggest that hypoxia and HIF-1α may function in conjunction with TLR-stimulated innate immune responses to drive inflammation in RA. This pathway may serve as a therapeutic target for the disease.

[Effects of Dickkopf-1 on synovitis of rheumatoid arthritis].

OBJECTIVE: As an important feature of rheumatoid arthritis (RA), an excessive amount of pro-inflammatory cytokines in RA synovial lesions induces the proliferation of synovial fibroblasts, cartilage erosion and systemic inflammatory immune response. Wnt inhibitor, Dkk-1, contributes to joint remodeling. This study was conducted to explore the role of Dkk-1 in the regulation of pro-inflammatory cytokine secretion. METHODS: Rheumatoid arthritis synovial fibroblasts (RASF) were cultured from surgical synovial specimens and utilized at passages 4-8. For Dkk-1 silencing assay, Dkk-1-specific siRNA and control scrambled siRNA were transfected into RASF respectively. For Dkk-1 over-expression assay, Dkk-1 plasmid and control vector were transfected into RASFs respectively. Production of pro-inflammatory cytokines, such as interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8), were measured by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR) at pre- and post-transfection respectively. Differences between various groups were evaluated by Wilcoxon signed-rank test. RESULTS: After a treatment of Dkk-1-specific siRNA, RASF exhibited decreased production of pro-inflammatory cytokines in association with down-regulated Dkk-1 levels while Dkk-1 over-expression could up-regulate the production of pro-inflammatory cytokines. The effects were marked when RAFS was activated by TNF-α and IL-1ß after transfection. CONCLUSION: Dkk-1 promotes the production of pro-inflammatory cytokines in RASF so as to exacerbate synovitis of RA. And targeting Dkk-1 may provide a novel therapeutic strategy for RA.

Superior molecularly altered influenza virus hemagglutinin peptide 308-317 inhibits collagen-induced arthritis by inducing CD4+ Treg cell expansion.

OBJECTIVE: To investigate the inhibitory effect and possible mechanism of a novel influenza virus hemagglutinin 308-317 peptide (altered HA308-317 peptide) in collagen-induced arthritis (CIA). METHODS: CIA was induced in DBA/1 mice by immunization with type II collagen (CII). Altered HA308-317 peptide, wild HA308-317 peptide, wild CII263-272 peptide, and irrelevant peptide were administered intranasally beginning at arthritis onset. Clinical and histologic scores were assessed, and cytokine levels were determined in the serum or in supernatants from splenocytes. Characteristics of T cell subsets in response to different peptides were analyzed both in vivo and in vitro. RESULTS: Intranasal administration of wild CII263-272 peptide, wild HA308-317 peptide, or altered HA308-317 peptide could significantly ameliorate CIA, but altered HA308-317 peptide showed greater therapeutic effects than wild CII263-272 peptide and wild HA308-317 peptide. The effect of altered HA308-317 peptide was associated with a substantial decrease in production of interleukin-17 (IL-17) and interferon-γ (IFNγ) and with a marked increase in production of IL-10 and transforming growth factor ß, both in serum and in supernatants from splenocytes treated with altered HA308-317 peptide. Both the number and function of CD4+ Treg cells were significantly up-regulated by altered HA308-317 peptide, with a decreased induction of Th1 cells (CD4+IFNγ+) and Th17 cells (CD4+IL-17+). Adoptive transfer of CD4+CD25+ T cells from altered HA308-317 peptide-treated mice resulted in greater suppressive capacity in ameliorating CIA severity than did adoptive transfer of CD4+CD25+ T cells from wild HA308-317 peptide-treated, wild CII263-272 peptide-treated, or irrelevant peptide-treated mice. CONCLUSION: Intranasal administration of altered HA308-317 peptide potently suppressed the severity of CIA by increasing the number and function of CD4+ Treg cells, suggesting that altered HA308-317 peptide might be a promising candidate for treatment of rheumatoid arthritis.

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.

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.

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.

Diagnostic significance of measuring antibodies to cyclic type 3 muscarinic acetylcholine receptor peptides in primary Sjogren's syndrome.

OBJECTIVE: SS is an autoimmune disease characterized by salivary and lacrimal gland dysfunction leading to dry mouth (xerostomia) and dry eyes (xerophthalmia). Anti-muscarinic acetylcholine type-3 receptor (anti-M3R) autoantibodies have been shown to be a good serum marker in primary SS (pSS). The aim of this study was to assess the clinical correlations of anti-M3R-derived peptide antibodies in patients with pSS. METHODS: Sequences of the first to fourth cycle-M3R (c1M3R-c4M3R)-derived peptide was synthesized by a solid-phase technique on an Applied Biosytems Peptide Synthesizer. Synthesized cM3R peptide (cM3RP) was used as substrate in an ELISA to detect IgG anti-cM3RP antibodies in serum samples of patients and controls. The clinical and biological parameters of the diseases were also evaluated. The EULAR SS disease activity index (ESSDAI) score was used to measure disease activity in patients with primary SS. RESULTS: (i) Anti-c2M3RP antibodies were highly prevalent in pSS patients, and the titre is much higher than anti-c1,3,4M3RP antibodies. (ii) The prevalence of anti-c2M3RP antibodies in pSS, SLE, RA and healthy controls was 62.2, 7.1, 5.3 and 1.6%, respectively. The prevalence of anti-linear-2-M3RP antibodies in pSS, SLE and RA patients and healthy controls were 56.1, 20.0, 14.7 and 9.4%. (iii) The specificity of anti-c2M3RP antibodies was 95.1%, much higher than that of linear polypeptide (84.7%) for pSS diagnosis. (iv) In pSS patients, anti-c2M3RP positivity had significantly increased frequency in patients who were RF or ANA positive, and had several haematological abnormalities, such as leucopenia, anaemia and thrombocytopenia. Furthermore, the ESSDAI score was significantly higher in anti-c2M3RP-positive pSS patients (P < 0.05). CONCLUSION: Anti-c2M3RP antibody was highly specific for patients with pSS. The presence of anti-c2M3RP antibody in pSS indicates that c2M3RP may act as an autoantigen that may play a role in the pathogenesis of pSS.