Anti-dsDNA antibodies bind to TLR4 and activate NLRP3 inflammasome in lupus monocytes/macrophages

Journal of Translational Medicine, Jun 2016

Background NLRP3 inflammasome has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The activation of NLRP3 inflammasome results in the production of IL-1β and the subsequent inflammation. Anti-dsDNA antibodies (anti-dsDNA Abs) play critical roles in the development and progression of SLE. However, the mechanism of NLRP3 inflammasome activation in SLE is still not known. This study investigated the activation of NLRP3 inflammasome stimulated by anti-dsDNA Abs in monocytes/macrophages from SLE patients. Methods Monocytes/macrophages from SLE patients or healthy controls were stimulated with anti-dsDNA Ab-positive serum or purified anti-dsDNA Abs. Activation of inflammasome was measured by flow cytometry or Western blot. Anti-dsDNA Abs isolated from active SLE patients were injected into female (NZB × NZW) F1 mice and the activation of NLRP3 inflammasome and the frequencies of Th17 and Treg were examined. Results The activity of caspase-1 was significantly increased in active SLE patients and was correlated with serum levels of anti-dsDNA Abs and disease activities. The concentrations of IL-1β and IL-17A were also significantly higher in SLE patients compared to healthy controls. Anti-dsDNA Ab-positive serum rather than healthy serum or RF (rheumatoid factor)-positive serum stimulated the activation of caspase-1 in monocytes. Anti-dsDNA Abs bound to TLR4 on macrophages and induced the production of ROS. Mitochondria-targeting antioxidant Mito-TEMPO, IκB kinase inhibitor peptide or TLR4 siRNA inhibited the activation of NLRP3 inflammasome and the secretion of IL-1β induced by anti-dsDNA Abs. Injection of anti-dsDNA Abs into (NZB × NZW) F1 mice resulted in increased caspase-1 activation and production of IL-1β and IL-17A. The Th17/Treg cell ratio also significantly increased following anti-dsDNA Ab injection. Conclusions Anti-dsDNA Abs activated NLRP3 inflammasome in monocytes/macrophages from SLE patients by binding to TLR4 and inducing the production of mitochondrial ROS.

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Anti-dsDNA antibodies bind to TLR4 and activate NLRP3 inflammasome in lupus monocytes/macrophages

Zhang et al. J Transl Med Anti-dsDNA antibodies bind to TLR4 and activate NLRP3 inflammasome in lupus monocytes/macrophages Hui Zhang 0 Rong Fu 0 Chaohuan Guo 0 Yuefang Huang 1 Hongyue Wang 0 Shuang Wang 0 Jijun Zhao 0 Niansheng Yang 0 0 Department of Rheumatology, First Affiliated Hospital, Sun Yat-sen University , 58 Zhongshan Road II, Guangzhou 510080 , China 1 Department of Pediatrics, First Affiliated Hospital, Sun Yat-sen University , Zhongshan Road II, Guang- zhou 510080 , China Background: NLRP3 inflammasome has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The activation of NLRP3 inflammasome results in the production of IL-1β and the subsequent inflammation. Anti-dsDNA antibodies (anti-dsDNA Abs) play critical roles in the development and progression of SLE. However, the mechanism of NLRP3 inflammasome activation in SLE is still not known. This study investigated the activation of NLRP3 inflammasome stimulated by anti-dsDNA Abs in monocytes/macrophages from SLE patients. Methods: Monocytes/macrophages from SLE patients or healthy controls were stimulated with anti-dsDNA Ab-positive serum or purified anti-dsDNA Abs. Activation of inflammasome was measured by flow cytometry or Western blot. Anti-dsDNA Abs isolated from active SLE patients were injected into female (NZB × NZW) F1 mice and the activation of NLRP3 inflammasome and the frequencies of Th17 and Treg were examined. Results: The activity of caspase-1 was significantly increased in active SLE patients and was correlated with serum levels of anti-dsDNA Abs and disease activities. The concentrations of IL-1β and IL-17A were also significantly higher in SLE patients compared to healthy controls. Anti-dsDNA Ab-positive serum rather than healthy serum or RF (rheumatoid factor)-positive serum stimulated the activation of caspase-1 in monocytes. Anti-dsDNA Abs bound to TLR4 on macrophages and induced the production of ROS. Mitochondria-targeting antioxidant Mito-TEMPO, IκB kinase inhibitor peptide or TLR4 siRNA inhibited the activation of NLRP3 inflammasome and the secretion of IL-1β induced by antidsDNA Abs. Injection of anti-dsDNA Abs into (NZB × NZW) F1 mice resulted in increased caspase-1 activation and production of IL-1β and IL-17A. The Th17/Treg cell ratio also significantly increased following anti-dsDNA Ab injection. Conclusions: Anti-dsDNA Abs activated NLRP3 inflammasome in monocytes/macrophages from SLE patients by binding to TLR4 and inducing the production of mitochondrial ROS. SLE; Anti-dsDNA antibodies; NLRP3 inflammasome; TLR4; Mitochondrial ROS Background Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with multiple organs involvement [ 1 ]. Despite the advances in the treatment of SLE, the morbidity and mortality of the disease remain high [ 2 ]. Dysregulation of immune system plays a critical role in the initiation and progression of SLE. Overproduction of cytokines and the subsequent inflammation are involved in the pathogenesis of SLE [ 3 ]. IL-1β is a proinflammatory cytokine that has been linked to SLE. It has been demonstrated that IL-1β is elevated in the serum of SLE patients [ 3, 4 ] and IL-1 receptor antagonist led to clinical and serological improvement [5]. In addition, IL-1β−/− and IL-1α/β−/− mice are resistant to the induction of experimental lupus [ 6 ]. Inflammasomes are a family of molecular platforms mostly expressed in the cytoplasm of monocytes/ macrophages. Inflammasomes are activated by infection or danger signals and trigger the maturation of proinflammatory cytokines such as IL-1β to engage in innate immune response [ 7 ]. NLRP3 inflammasome is one of the most studied members. Activation of NLRP3 inflammasome involves the recruitment of adapter protein apoptosis-associated speck like protein (ASC) through homotypic PYD-PYD interaction. ASC subsequently recruits pro-caspase-1 via CARD-CARD contact in turn. The oligomerization of NLRP3 inflammasome leads to autocatalytic activation of caspase-1, which converts inactive pro-IL-1 into bioactive form [ 8 ]. NLRP3 inflammasome can be activated by either exogenous or endogenous stimuli [ 9–11 ]. Anti-double stranded DNA (anti-dsDNA) antibodies are the hallmark antibodies of SLE [ 12 ]. Anti-dsDNA antibodies are correlated with disease activity [ 13 ] and involved in the pathogenesis of SLE [ 14 ]. Anti-dsDNA antibodies presented in the blood before disease onset [ 15 ] and administration of anti-dsDNA antibodies from SLE patients into NZBWF1/J mice resulted in accelerated lupus [ 16 ]. Data showed that anti-dsDNA antibodies stimulated the expression and secretion of IL-1β from mononuclear cells and monocytes [ 17, 18 ]. It implies that anti-dsDNA antibodies might initiate and promote the disease by stimulating the production of IL-1β. Recently, we have showed that NLRP3 inflammasome was activated in a mouse model of SLE, and inhibition of NLRP3 inflammsome activation resulted in (...truncated)


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Hui Zhang, Rong Fu, Chaohuan Guo, Yuefang Huang, Hongyue Wang, Shuang Wang, Jijun Zhao, Niansheng Yang. Anti-dsDNA antibodies bind to TLR4 and activate NLRP3 inflammasome in lupus monocytes/macrophages, Journal of Translational Medicine, 2016, pp. 156, 14, DOI: 10.1186/s12967-016-0911-z