Role of high-temperature requirement serine protease A 2 in rheumatoid inflammation.

BACKGROUND: High-temperature requirement serine protease A 2 (HtrA2) is known to be involved in growth, unfolded protein response to stress, apoptosis, and autophagy. However, whether HtrA2 controls inflammation and immune response remains elusive. METHODS: Expression of HtrA2 in the synovial tissue of patients was examined using immunohistochemistry and immunofluorescence staining. Enzyme-linked immunosorbent assay was used to determine the concentrations of HtrA2, interleukin-6 (IL-6), interleukin-8 (IL-8), chemokine (C-C motif) ligand 2 (CCL2), and tumor necrosis factor α (TNFα). Synoviocyte survival was assessed by MTT assay. For the downregulation of HtrA2 transcripts, cells were transfected with HtrA2 siRNA. RESULTS: We found that the concentration of HtrA2 was elevated in rheumatoid arthritis (RA) synovial fluid (SF) than in osteoarthritis (OA) SF, and its concentrations were correlated with the number of immune cells in the RA SF. Interestingly, HtrA2 levels in the SF of RA patients were elevated in proportion to synovitis severity and correlated with the expression of proinflammation cytokines and chemokines, such as IL-6, IL-8, and CCL2. In addition, HtrA2 was highly expressed in RA synovium and primary synoviocytes. RA synoviocytes released HtrA2 when stimulated with ER stress inducers. Knockdown of HtrA2 inhibited the IL1ß-, TNFα-, and LPS-induced release of proinflammatory cytokines and chemokines by RA synoviocytes. CONCLUSION: HtrA2 is a novel inflammatory mediator and a potential target for the development of an anti-inflammation therapy for RA.

Identification of MYH9 as a key regulator for synoviocyte migration and invasion through secretome profiling.

OBJECTIVES: 'Invasive pannus' is a pathological hallmark of rheumatoid arthritis (RA). This study aimed to investigate secretome profile of synovial fibroblasts of patients with RA (RA-FLSs), a major cell type comprising the invasive pannus. METHODS: Secreted proteins from RA-FLSs were first identified using liquid chromatography-tandem mass spectrometry analysis. Ultrasonography was performed for affected joints to define synovitis severity at the time of arthrocentesis. Expression levels of myosin heavy chain 9 (MYH9) in RA-FLSs and synovial tissues were determined by ELISA, western blot analysis and immunostaining. A humanised synovitis model was induced in immuno-deficient mice. RESULTS: We first identified 843 proteins secreted from RA-FLSs; 48.5% of the secretome was associated with pannus-driven pathologies. Parallel reaction monitoring analysis of the secretome facilitated discovery of 16 key proteins related to 'invasive pannus', including MYH9, in the synovial fluids, which represented synovial pathology based on ultrasonography and inflammatory activity in the joints. Particularly, MYH9, a key protein in actin-based cell motility, showed a strong correlation with fibroblastic activity in the transcriptome profile of RA synovia. Moreover, MYH9 expression was elevated in cultured RA-FLSs and RA synovium, and its secretion was induced by interleukin-1ß, tumour necrosis factor α, toll-like receptor ligation and endoplasmic reticulum stimuli. Functional experiments demonstrated that MYH9 promoted migration and invasion of RA-FLSs in vitro and in a humanised synovitis model, which was substantially inhibited by blebbistatin, a specific MYH9 inhibitor. CONCLUSIONS: This study provides a comprehensive resource of the RA-FLS-derived secretome and suggests that MYH9 represents a promising target for retarding abnormal migration and invasion of RA-FLSs.

Regulation of tumor growth by leukocyte-specific protein 1 in T cells.

BACKGROUND: Clinical efficacy of T cell-based cancer immunotherapy is limited by the lack of T cell infiltration in the tumor mass, especially in solid tumors. Our group demonstrated previously that leukocyte-specific protein 1 (LSP1), an intracellular signal regulator, negatively regulates T cell infiltration in inflamed tissues. METHODS: To determine the immuno-regulatory effects of LSP1 in T cells on tumor progression, we investigated the growth of B16 melanoma in Lsp1 knockout (KO) mice and T cell-specific Lsp1 transgenic (Tg) mice. The immune cell subpopulation infiltrated into the tumor mass as well as the expression of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in T cells was assessed by flow cytometry and/or immunohistochemistry. Chemotactic migration was assayed with Lsp1 KO and Lsp1 Tg T cells. Adoptive transfer of Lsp1 KO or Lsp1 Tg T cells was performed in B16 melanoma-challenged Rag1 KO mice. RESULTS: Lsp1 KO mice showed decreased growth of B16 melanoma and increased infiltration of T cells in the tumor mass, which were completely reversed in T cell-specific Lsp1 Tg mice. Lsp1 KO CD8+ T cells also exhibited elevated migratory capacity in response to CXCL9 and CXCL10, whereas Lsp1 Tg CD8+ T cells did the opposite. LSP1 expression was increased in tumor-infiltrating T cells and could be induced by T cell receptor activation. Intriguingly, gene expression profiling of Lsp1 KO T cells suggested enhanced cytotoxicity. Indeed, expression of IFN-γ and TNF-α was increased in tumor-infiltrating CD4+ and CD8+ T cells of Lsp1 KO mice, while it was markedly reduced in those of Lsp1 Tg mice. Adoptive transfer of Lsp1 KO T cells to Rag1 KO mice was more effective in suppressing melanoma growth than transfer of Lsp1 Tg T cells. Of note, when treated with antiprogrammed cell death protein 1 (PD-1) antibody, inhibition of melanoma growth was more pronounced in Lsp1 KO mice than in Lsp1-sufficient mice, suggesting that Lsp1 depletion additively increases the antitumor effects of anti-PD-1 antibody. CONCLUSIONS: LSP1 in T cells regulates the growth of B16 melanoma in mice, possibly by affecting migration and infiltration of T cells into the tumor and by modulating production of antitumor effector cytokines by CD8+ T cells. These findings provide evidence that LSP1 can be a target to improve the efficacy of T cell-based immunotherapy.