Lianjie Shi

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.

Toll-Like Receptors Expressed by Synovial Fibroblasts Perpetuate Th1 and Th17 Cell Responses in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial fibroblast hyperplasia and bone and cartilage erosion. Synovial fibroblast- and T cell-mediated inflammation plays crucial roles in the pathogenesis of RA. However how this inflammation is initiated, propagated, and maintained remains controversial. Here, we systemically examined the contribution of toll-like receptors (TLRs) to the inflammatory mediator production as well as Th1 and Th17 cell hyperactivity in RA. Our results show that rheumatoid arthritis synovial fibroblasts (RASF) express a series of TLRs, including TLR2, TLR3, TLR4, and TLR9, with the predominant expression of TLR3. Moreover, the expression levels of these TLRs were higher than those in osteoarthritis synovial fibroblasts (OASF). Ligation of TLR3, as well as TLR2 and TLR4, resulted in vigorous production of inflammatory cytokines, matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF) in RASF, with activation of the NF-κB, MAPK, and IRF3 pathways. More important, activation of these TLRs expressed by RASF exacerbated inflammatory Th1 and Th17 cell expansion both in cell-cell contact-dependent and inflammatory cytokine-dependent manners, which induced more IFN-γ and IL-17 accumulation. Targeting TLRs may modulate the inflammation in RA and provide new therapeutic strategies for overcoming this persistent disease.

Hypoxia-Inducible Factor-1α and Interleukin 33 Form a Regulatory Circuit to Perpetuate the Inflammation in Rheumatoid Arthritis

Hyperplasia of synovial fibroblasts, infiltration with inflammatory cytokines, and tissue hypoxia are the major characteristics of rheumatoid arthritis (RA). Interleukin 33 (IL-33) is a newly identified inflammatory cytokine exacerbating the disease severity of RA. Hypoxia-inducible factor-1α (HIF-1α) showed increased expression in RA synovium and could regulate a number of inflammatory cytokine productions. Nevertheless, its correlation with IL-33 remains largely unknown. Here, we showed that elevated levels of IL-33 were demonstrated in RA patient synovial fluids, with upregulated expression of HIF-1α and IL-33 in the synovial fibroblasts. Knocking down HIF-1α compromised IL-33 expression in rheumatoid arthritis synovial fibroblasts (RASF), while enforcing HIF-1α expression in RASF substantially upregulated IL-33 levels. HIF-1α promoted the activation of the signalling pathways controlling IL-33 production, particularly the p38 and ERK pathways. Moreover, we showed for the first time that IL-33 in turn could induce more HIF-1α expression in RASF, thus forming a HIF-1α/IL-33 regulatory circuit that would perpetuate the inflammatory process in RA. Targeting this pathological pathway and HIF-1α may provide new therapeutic strategies for overcoming the persistent and chronic inflammatory disease.

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.

Different expression patterns and clinical significance of mAxl and sAxl in systemic lupus erythematosus

Axl is one of the TAM family members that downregulates activated immune responses to maintain immune homeostasis. We analyzed the expression and clinical relevance of Axl on the surface of CD14+ monocytes/macrophages (mAxl, membrane Axl) and in the plasma (sAxl, soluble Axl) from patients with systemic lupus erythematosus (SLE). Compared to healthy subjects, the concentrations of sAxl were significantly elevated in plasma from SLE patients, while the mAxl expression on CD14+ monocytes/macrophages from SLE patients was significantly downregulated. A series of severe disease clinical manifestations and laboratory features such as presence of autoantibodies, 24-hour proteinuria excretion or SLEDAI ≥10 were associated with decreased mAxl expression on monocytes/macrophages but elevated sAxl levels in plasma. The plasma level of Gas6, the main ligand of Axl, was slightly decreased in SLE patients, and was negatively correlated with anti-dsDNA antibodies and C-reactive protein. SLE patients with SLEDAI ≥10 showed significantly lower Gas6 levels. Our study suggests that abnormal mAxl and sAxl expression may be involved in the imbalance of immune regulation in SLE.

The Expression and Clinical Significance of Different Forms of Mer Receptor Tyrosine Kinase in Systemic Lupus Erythematosus

Objective. To investigate the expression and clinical significance of trans-membrane MerTK (mMer) on circulating CD14+ monocytes/macrophages and soluble MerTK (sMer) levels in plasma in systemic lupus erythematosus (SLE). Method. 108 SLE patients and 42 healthy controls were recruited in this study. The expression of mMer on the surfaces of CD14+ monocytes/macrophages was evaluated by flow cytometry (FCM). The sMer levels were measured by ELISA. Real-time quantitative PCR was applied to evaluate the mRNA levels of MerTK and ADAM17. Results. Both mMer expression on CD14+ monocytes/macrophages and sMer levels in plasma significantly increased in SLE patients compared to healthy subjects. The frequency of anti-inflammatory MerTK expressing CD14+CD16+ monocytes decreased in SLE. mMer expression was positively correlated with CD163 expression on CD14+ cells. Both the mMer expression on CD14+ monocytes/macrophages and sMer levels in plasma were positively correlated with SLEDAI. Furthermore, more elevated mMer and sMer levels were found in patients with higher SLEDAI, presence of anti-SSA, anti-Sm autoantibodies, and lupus nephritis. Conclusion. Both mMer and sMer levels significantly increased in SLE and positively correlated with disease activity and severity. The upregulation of MerTK expression may serve as a biomarker of the disease activity and severity of SLE.

CD16+ Monocyte Subset Was Enriched and Functionally Exacerbated in Driving T-Cell Activation and B-Cell Response in Systemic Lupus Erythematosus

Background The roles that CD16+ monocyte subset plays in T-cell activation and B-cell response have not been well studied in systemic lupus erythematosus (SLE). Objective The present study aimed to investigate the distribution of CD16+ monocyte subsets in SLE and explore their possible roles in T-cell activation and B-cell differentiation. Methods The frequencies of monocyte subsets in the peripheral blood of healthy controls (HCs) and patients with SLE were determined by flow cytometry. Monocyte subsets were sorted and cocultured with CD4+ T cells and CD19+ B cells. Then, T and B cells were collected for different subset detection, while the supernatants were collected for immunoglobulin G, IgA, and IgM or interferon-γ and interleukin-17A detection by enzyme-linked immunosorbent assay. Results Our results showed that CD16+ monocytes exhibited a proinflammatory phenotype with elevated CD80, CD86, HLA-DR, and CX3CR1 expression on the cell surface. It’s further demonstrated that CD16+ monocytes from patients and HCs shared different cell-surface marker profiles. The CD16+ subset was enriched in SLE and had an exacerbated capacity to promote CD4+ T cell polarization into a Th17 phenotype. Also, CD16+ monocytes had enhanced impacts on CD19+ B cells to differentiate into plasma B cells and regulatory B cells with more Ig production. Conclusion This study demonstrated that CD16+ monocytes, characterized by different cell-surface marker profiles, were enriched and played a critical role in driving the pathogenic T- and B-cell responses in patients with SLE.

Soluble TAM receptor tyrosine kinases in rheumatoid arthritis: correlation with disease activity and bone destruction

The TAM receptor tyrosine kinases (TAM RTK) are a subfamily of receptor tyrosine kinases, the role of which in autoimmune diseases such as systemic lupus erythematosus has been well explored, while their functions in rheumatoid arthritis (RA) remain largely unknown. In this study, we investigated the role of soluble TAM receptor tyrosine kinases (sAxl/sMer/sTyro3) in patients with RA. A total of 306 RA patients, 100 osteoarthritis (OA) patients and 120 healthy controls (HCs) were enrolled into this study. The serum concentrations of sAxl/sMer/sTyro3 were measured by enzyme‐linked immunosorbent assay (ELISA), then the associations between sAxl/sMer/sTyro3 levels and clinical features of RA patients were analysed. We also investigated whether sTyro3 could promote osteoclast differentiation in vitro in RA patients. The results showed that compared with healthy controls (HCs), sTyro3 levels in the serum of RA patients were elevated remarkably and sMer levels were decreased significantly, whereas there was no difference between HCs and RA patients on sAxl levels. The sTyro3 levels were correlated weakly but positively with white blood cells (WBC), immunoglobulin (Ig)M, rheumatoid factor (RF), swollen joint counts, tender joint counts, total sharp scores and joint erosion scores. Conversely, there were no significant correlations between sMer levels and the above indices. Moreover, RA patients with high disease activity also showed higher sTyro3 levels. In‐vitro osteoclast differentiation assay showed further that tartrate‐resistant acid phosphatase (TRAP)+ osteoclasts were increased significantly in the presence of sTyro3. Collectively, our study indicated that serum sTyro3 levels were elevated in RA patients and correlated positively with disease activity and bone destruction, which may serve as an important participant in RA pathogenesis.

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.