Fanlei Hu

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.

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)-1β, 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.

Lipopolysaccharide Increases the Incidence of Collagen‐Induced Arthritis in Mice Through Induction of Protease HTRA‐1 Expression

OBJECTIVE The protease HTRA-1 is closely associated with rheumatoid arthritis (RA). The molecular mechanisms that control HTRA-1 expression are currently unknown. This study was undertaken to determine the regulatory role of Toll-like receptors (TLRs) on HTRA-1 expression in mice with collagen-induced arthritis (CIA) and in synovial cells from RA patients. METHODS HTRA-1 messenger RNA and protein production in mouse fibroblasts, mouse macrophages, and freshly isolated RA patient synovial cells treated with TLR ligands were detected by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Arthritis incidence and severity were determined using clinical scores and histopathologic analysis. Involvement of HTRA-1 in lipopolysaccharide (LPS)-increased arthritis incidence and severity in mice was determined using anti-HTRA-1 monoclonal antibody. The signal pathways involved in HTRA-1 expression were accessed by specific inhibitors, RNA interference, dual-luciferase reporter, and chromatin immunoprecipitation methods. RESULTS LPS and tenascin-C, but not the other TLR ligands tested, strongly induced HTRA-1 expression. LPS significantly increased HTRA-1 expression in the joint tissue as well as arthritis incidence and severity in mice with CIA. Blocking HTRA-1 by antibody significantly decreased LPS-promoted CIA severity. Inhibiting NF-κB significantly decreased LPS-induced HTRA-1 expression in mouse and human cells. Dual-luciferase reporter assay and ChIP analysis showed that p65 directly binds to HTRA-1 promoter (amino acid 347). CONCLUSION Our findings indicate that TLR-4 activation increases HTRA-1 expression through the NF-κB pathway in fibroblasts and macrophages. HTRA-1 expression is involved in the enhancing effects of LPS on CIA. This study offers new insights into the regulation of HTRA-1 expression via LPS/TLR-4 and the role of HTRA-1 in RA pathogenesis.

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‐1β, 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.

The Inhibitory Effect of IFN-γ on Protease HTRA1 Expression in Rheumatoid Arthritis

The high temperature requirement A1 (HTRA1) is a potent protease involved in many diseases, including rheumatoid arthritis (RA). However, the regulatory mechanisms that control HTRA1 expression need to be determined. In this study, we demonstrated that IFN-γ significantly inhibited the basal and LPS-induced HTRA1 expression in fibroblasts and macrophages, which are two major cells for HTRA1 production in RA. Importantly, the inhibitory effect of IFN-γ on HTRA1 expression was evidenced in collagen-induced arthritis (CIA) mouse models and in human RA synovial cells. In parallel with the enhanced CIA incidence and pathological changes in IFN-γ–deficient mice, HTRA1 expression in the joint tissues was also increased as determined by real-time PCR and Western blots. IFN-γ deficiency increased the incidence of CIA and the pathological severity in mice. Neutralization of HTRA1 by Ab significantly reversed the enhanced CIA frequency and severity in IFN-γ–deficient mice. Mechanistically, IFN-γ negatively controls HTRA1 expression through activation of p38 MAPK/STAT1 pathway. Dual luciferase reporter assay and chromatin immunoprecipitation analysis showed that STAT1 could directly bind to HTRA1 promoter after IFN-γ stimulation. This study offers new insights into the molecular regulation of HTRA1 expression and its role in RA pathogenesis, which may have significant impact on clinical therapy for RA and possibly other HTRA1-related diseases, including osteoarthritis, age-related macular degeneration, and cancer.

Contribution of functional LILRA3, but not nonfunctional LILRA3, to sex bias in susceptibility and severity of anti-citrullinated protein antibody-positive rheumatoid arthritis.

Leukocyte immunoglobulin‐like receptor A3 belongs to a family of receptors with inhibitory or activating functions. Since Caucasian individuals lacking LILRA3 have been found to be susceptible to multiple sclerosis and Sjögrens syndrome, we undertook this study to examine whether LILRA3 deletion is a novel genetic risk factor for rheumatoid arthritis (RA) (another autoimmune disease), whether there are sex‐specific effects, and whether LILRA3 influences the subtype and severity of RA.

Therapeutic Effects of NK-HDAC-1, a Novel Histone Deacetylase Inhibitor, on Collagen-Induced Arthritis Through the Induction of Apoptosis of Fibroblast-Like Synoviocytes

The purpose of this study is to investigate the therapeutic effects of a novel histone deacetylase inhibitor (HDACi), NK-HDAC-1, on collagen-induced arthritis (CIA) and pathogenic fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA). The proliferation and apoptosis of FLSs treated with NK-HDAC-1 were evaluated by flow cytometry and fluorescence staining. The effect of NK-HDAC-1 treatment on pro-inflammatory cytokine production was determined by ELISA. CIA was established in DBA/1 mice, and NK-HDAC-1 or vehicle was administered daily after the onset of arthritis. Clinical and histological scores were calculated to assess the therapeutic efficacy of NK-HDAC-1. NK-HDAC-1 significantly inhibited the proliferation of FLSs through cell cycle arrest at the G2/M checkpoint and enhanced apoptosis of FLSs. The activity of caspases was increased during NK-HDAC-1 treatment. IL-6 production by FLSs was also suppressed by NK-HDAC-1. Furthermore, the oral administration of NK-HDAC-1 significantly enhanced synoviocyte apoptosis in vivo and inhibited CIA progression. Compared with subcroylanilide hydroxamic acid which exhibited moderate prophylactic efficacy, NK-HDAC-1 demonstrated therapeutic efficacy in CIA. NK-HDAC-1 is a novel HDACi that may ameliorate inflammatory arthritis by regulating the activation, apoptosis, and inflammatory responses of FLSs. This is the first study to support that NK-HDAC-1 may be a potential therapeutic agent for RA.

Ultrasonographic evaluation of major salivary glands in primary Sjögren’s syndrome: comparison of two scoring systems

OBJECTIVE To assess the diagnostic value of salivary gland ultrasonography (SGUS) for primary SS (pSS) and to compare the usefulness of two existing SGUS scoring systems. METHODS Ultrasonography examination of major salivary glands was conducted for 105 pSS patients and 41 disease control subjects without SS and 16 healthy control subjects. The imaging features were graded using two different scoring systems (0-16 and 0-48, respectively) obtained from the grades of bilateral parotid and submandibular glands. Receiver operating characteristic curves were used to describe and compare the diagnostic accuracy of the two ultrasonography echostructure scoring systems for pSS. The agreement of diagnosis for pSS between the two scoring systems was determined by κ-statistics. RESULTS SGUS scores for the pSS group were significantly higher than those for the non-pSS group (P < 0.001). The best score cut-off was 7 in the 0-16 system (80% sensitivity and 93% specificity, respectively), and it was 15 in the 0-48 system (88.6% sensitivity and 84.2% specificity, respectively). Compared with the 0-16 system, combined evaluation of all four glands when using the 0-48 system improved the diagnostic accuracy. Association analysis of both scoring systems showed a positive correlation of SGUS scores with RF and γ-globulin% (P < 0.05, overall). CONCLUSION SGUS is a feasible method for pSS diagnosis with higher sensitivity using the 0-48 system and better specificity using the 0-16 system. SGUS scores are related to RF and γ-globulin%.

Impact of the leucocyte immunoglobulin-like receptor A3 (LILRA3) on susceptibility and subphenotypes of systemic lupus erythematosus and Sjögren's syndrome

Background Recently, our research group identified the non-deleted (functional) leucocyte immunoglobulin-like receptor A3 (LILRA3) as a new genetic risk for rheumatoid arthritis. Objectives To further investigate whether the functional LILRA3 is a new susceptibility factor for other autoimmune diseases—for example, systemic lupus erythematosus (SLE) and primary Sjögrens syndrome (pSS). Methods The LILRA3 deletion polymorphism and its tagging single nucleotide polymorphism rs103294 were genotyped for 1099 patients with SLE, 403 patients with pSS and 2169 healthy controls. Association analyses were performed in whole dataset or clinical/serological subsets. The impact of LILRA3 on SLE activity and LILRA3 expression was evaluated. Results The functional LILRA3 conferred high susceptibility to both SLE (p=3.51×10−7, OR=2.03) and pSS (p=1.40×10−3, OR=2.32). It was associated with almost all the clinical/serological features in SLE, especially with leucopenia (p=4.09×10−7, OR=2.19) and thrombocytopenia (p=1.68×10−5, OR=1.70). In pSS, functional LILRA3 was specifically associated with leucopenia (p=4.39×10−4, OR=3.25), anti-Ro/SSA-positive subphenotypes (p=4.54×10−3, OR=2.34) and anti-La/SSB-positive subphenotypes (p=0.012, OR=2.49). Functional LILRA3 conferred higher disease activity in patients with SLE (p=0.044) and higher LILRA3 expression in both SLE (p=5.57×10−8) and pSS (p=1.49×10−7) than in controls. Conclusions Functional LILRA3 is a new susceptibility factor for SLE and pSS. It highly predisposes to certain phenotypes such as leucopenia and thrombocytopenia in SLE, and may confer increased disease activity in SLE and a higher risk of leucopenia and autoantibody-positive subphenotypes in pSS.

Serum anti-lipocalin 2 IgG is a novel biomarker in the diagnosis of systemic lupus erythematosus

Background Previous work suggests that lipocalin 2 is involved in the pathogenesis of systemic lupus erythematosus (SLE) and that this novel antigen could serve as a high-quality renal biomarker of acute kidney injury in SLE. However, serum lipocalin 2 antibody levels remain unclear. We have therefore undertaken this study to assess the level of serum IgG antibody against lipocalin 2 in different disease states and to evaluate the diagnostic value of this potential biomarker in SLE. Methods Serum levels of anti-lipocalin IgG antibodies were measured by ELISA in 103 SLE patients, 93 rheumatoid arthritis (RA) patients, 29 primary Sjögren’s syndrome (pSS) patients, 13 systemic sclerosis (SSc) patients, and 91 healthy controls. Diagnostic properties of anti-lipocalin IgG were determined by receiver-operating characteristic (ROC) curve analysis. Results The level of serum anti-lipocalin IgG in patients with SLE was significantly higher than in patients with RA, pSS, SSc, or healthy controls (p < 0.05), effectively distinguishing SLE from other conditions with high sensitivity and specificity (49.5% and 90.7%, respectively). In ROC curve analysis, the area under the curve (AUC) is 0.783, with a 95% confidence interval (CI) extending from 0.729 to 0.839. Anti-lipocalin antibodies were present in 48.1% of anti-Sm-negative SLE patients, and also occurred in SLE patients lacking anti-dsDNA (52%) or anti-nucleosome antibodies (46.3%) antibodies. Finally, SLE patients with positive anti-lipocalin IgG possessed higher levels of IgA and CRP than the negative group (p < 0.05), clearly demonstrating a positive correlation between anti-lipocalin IgG and these laboratory parameters. Conclusions Anti-lipocalin 2 IgG is a promising biomarker for the diagnosis of SLE, particularly when obtained in conjunction with anti-Sm, anti-dsDNA, and anti-nucleosome antibody levels.