Jijun Zhao

Nucleotide-binding, oligomerization domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) gene polymorphism was reported to be associated with susceptibility, disease activity or anti-tumour necrosis factor (TNF) treatment response in rheumatoid arthritis (RA). However, the roles of NLRP3 inflammasome in the development of RA have not yet been elucidated fully. The present study aimed to study the role of NLRP3 inflammasome in RA. NLRP3 inflammasome activation in synovial tissues from RA and osteoarthritis (OA) patients were assessed by Western blot. Active caspase-1 in synovia was stained by a FAM-FLICA caspase-1 probe. Mice with collagen-induced arthritis (CIA) were treated with MCC950, a selective NLRP3 inhibitor, or vehicle for 2 weeks. The clinical score of arthritis, synovial inflammation and cartilage erosion were assessed. Proinflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that NLRP3 inflammasome was highly activated in both synovia from RA patients and CIA mice. Activation of NLRP3 inflammasome occurred mainly in the infiltrating monocyte/macrophages in synovia, but not in fibroblast-like synoviocytes. Treatment with MCC950 resulted in significantly less severe joints inflammation and bone destruction. NLRP3 inflammasome activation in the synovia was inhibited significantly by MCC950 with reduced production of interleukin (IL)-1β. The inhibition of NLRP3 inflammasome activation by MCC950 was confirmed further in a human monocytic cell line, THP-1. In conclusion, NLRP3 inflammasome is involved in the pathogenesis of RA. Targeting NLRP3 inflammasome with a small molecule inhibitor might be a novel therapeutic strategy for RA.

Toll-like receptor 9 (TLR9) recognizes self-DNA and plays intricate roles in systemic lupus erythematosus (SLE). However, the molecular mechanism regulating the endosomal TLR9 response is incompletely understood. Here, we report that palmitoyl-protein thioesterase 1 (PPT1) regulates systemic autoimmunity by removing S-palmitoylation from TLR9 in lysosomes. PPT1 promotes the secretion of IFNα by plasmacytoid dendritic cells (pDCs) and TNF by macrophages. Genetic deficiency in or chemical inhibition of PPT1 reduces anti-nuclear antibody levels and attenuates nephritis in B6.Sle1yaa mice. In healthy volunteers and patients with SLE, the PPT1 inhibitor, HDSF, reduces IFNα production ex vivo. Mechanistically, biochemical and mass spectrometry analyses demonstrated that TLR9 is S-palmitoylated at C258 and C265. Moreover, the protein acyltransferase, DHHC3, palmitoylates TLR9 in the Golgi, and regulates TLR9 trafficking to endosomes. Subsequent depalmitoylation by PPT1 facilitates the release of TLR9 from UNC93B1. Our results reveal a posttranslational modification cycle that controls TLR9 response and autoimmunity.

OBJECTIVE: Development of proteinuria in lupus nephritis (LN) is associated with podocyte dysfunction. The NLRP3 inflammasome has been implicated in the pathogenesis of LN. The purpose of this study was to investigate whether NLRP3 inflammasome activation is involved in the development of podocyte injury in LN. METHODS: A fluorescence-labeled caspase 1 inhibitor probe was used to detect the activation of NLRP3 inflammasomes in podocytes derived from lupus-prone NZM2328 mice and from renal biopsy tissues obtained from patients with LN. MCC950, a selective inhibitor of NLRP3, was used to treat NZM2328 mice. Proteinuria, podocyte ultrastructure, and renal pathology were evaluated. In vitro, sera from diseased NZM2328 mice were used to stimulate a podocyte cell line, and the cells were analyzed by flow cytometry. RESULTS: NLRP3 inflammasomes were activated in podocytes from lupus-prone mice and from patients with LN. Inhibition of NLRP3 with MCC950 ameliorated proteinuria, renal histologic lesions, and podocyte foot process effacement in lupus-prone mice. In vitro, sera from diseased NZM2328 mice activated NLRP3 inflammasomes in the podocyte cell line through the production of reactive oxygen species. CONCLUSION: NLRP3 inflammasomes were activated in podocytes from lupus-prone mice and from LN patients. Activation of NLRP3 is involved in the pathogenesis of podocyte injuries and the development of proteinuria in LN.

OBJECTIVE: The NLRP3 inflammasome plays key roles in inflammation and autoimmunity, and purinergic receptor P2X7 has been proposed to be upstream of NLRP3 activation. The aim of the present study, using murine models, was to investigate whether the P2X7 /NLRP3 inflammasome pathway contributes to the pathogenesis of lupus nephritis (LN). METHODS: MRL/lpr mice were treated with the selective P2X7 antagonist brilliant blue G (BBG) for 8 weeks. Following treatment, the severity of renal lesions, production of anti-double-stranded DNA (anti-dsDNA) antibodies, rate of survival, activation of the NLRP3/ASC/caspase 1 inflammasome pathway, and ratio of Th17 cells to Treg cells were evaluated. P2X7 -targeted small interfering RNA (siRNA) was also used for in vivo intervention. Similar evaluations were carried out in NZM2328 mice, a model of LN in which the disease was accelerated by administration of adenovirus-expressing interferon-α (AdIFNα). RESULTS: Significant up-regulation of P2X7 /NLRP3 inflammasome signaling molecules was detected in the kidneys of MLR/lpr mice as compared with normal control mice. Blockade of P2X7 activation by BBG suppressed NLRP3/ASC/caspase 1 assembly and the subsequent release of interleukin-1β (IL-1β), resulting in a significant reduction in the severity of nephritis and circulating anti-dsDNA antibodies. The lifespan of the treated mice was significantly prolonged. BBG treatment reduced the serum levels of IL-1β and IL-17 and the Th17:Treg cell ratio. Similar results were obtained by specific siRNA silencing of P2X7 in vivo. The effectiveness of BBG treatment in modulating LN was confirmed in NZM2328 mice with AdIFNα-accelerated disease. CONCLUSION: Activation of the P2X7 signaling pathway accelerates murine LN by activating the NLRP3/ASC/caspase 1 inflammasome, resulting in increased IL-1β production and enhanced Th17 cell polarization. Thus, targeting of the P2X7 /NLRP3 pathway should be considered as a novel therapeutic strategy in patients with lupus.