RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA

Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE-DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo

Inflammasome activation and formation of ASC specks in patients with juvenile idiopathic arthritis

Objective The formation of large intracellular protein aggregates of the inflammasome adaptor ASC is a hallmark of inflammasome activation and characteristic of autoinflammation. Inflammasome activated cells release the highly proinflammatory cytokine IL-1β in addition to ASC specks into the extracellular space. Autoinflammatory activity has been demonstrated in systemic JIA, however minimal data exist on the role of inflammasomes in other JIA subtypes. We therefore investigated, if pyroptotic cells are present in the circulation of oligo- and poly-articular JIA. Methods Peripheral blood of JIA patients (n = 46) was investigated for ASC speck formation, a key step in inflammasome activation, by flow cytometry and immunofluorescence. Free ASC and proinflammatory cytokine levels were determined by ELISA and multiplex assay. Results Oligo-articular JIA patients showed a significantly increased proportion of ASC speck+ monocytes compared to poly-articular JIA patients. In serum free ASC alone is not sufficient to assess inflammasome activity and does not correlate with ASC speck+ monocytes. Compared to control several cytokines were significantly elevated in samples of JIA patients. JIA serum containing antinuclear antibodies, incubated with ASC specks boosts a secondary inflammation by IL-1β production in macrophages. Conclusion For the first time, we detect ex vivo inflammasome activation by ASC speck formation in oligo- and poly-articular JIA patients. Most notably, inflammasome activation was significantly higher in oligo- compared to poly-articular JIA patients. This data suggests that inflammasome derived autoinflammation may have a greater influence in the previously thought autoimmune oligo-articular JIA patients

Cutting Edge: Protein Arginine Deiminase 2 and 4 Regulate NLRP3 Inflammasome-Dependent IL-1β Maturation and ASC Speck Formation in Macrophages

Protein arginine deiminase (PAD) enzymes catalyze the conversion of protein-bound arginine into citrulline, an irreversible posttranslational modification with loss of a positive charge that can influence protein-protein interactions and protein structure. Protein arginine deiminase activity depends on high intracellular calcium concentrations occurring in dying cells. In this study, we demonstrate that protein citrullination is common during pyroptotic cell death in macrophages and that inhibition of PAD enzyme activity by Cl-amidine, a pan-PAD inhibitor, blocks NLRP3 inflammasome assembly and proinflammatory IL-1β release in macrophages. Genetic deficiency of either PAD2 or PAD4 alone in murine macrophages does not impair IL-1β release; however, pharmacological inhibition or small interfering RNA knockdown of PAD2 within PAD4-/-macrophages does. Our results suggest that PAD2 and 4 activity in macrophages is required for optimal inflammasome assembly and IL-1β release, a finding of importance for autoimmune diseases and inflammation

Gadolinium-based compounds induce NLRP3-dependent IL-1beta production and peritoneal inflammation

OBJECTIVE: Nephrogenic systemic fibrosis (NSF) is a progressive fibrosing disorder that may develop in patients with chronic kidney disease after administration of gadolinium (Gd)-based contrast agents (GBCAs). In the setting of impaired renal clearance of GBCAs, Gd deposits in various tissues and fibrosis subsequently develops. However, the precise mechanism by which fibrosis occurs in NSF is incompletely understood. Because other profibrotic agents, such as silica or asbestos, activate the nucleotide-binding oligomerisation domain (NOD)-like receptor protein 3 (NLRP3) inflammasome and initiate interleukin (IL)-1beta release with the subsequent development of fibrosis, we evaluated the effects of GBCAs on inflammasome activation. METHODS: Bone marrow derived macrophages from C57BL/6, Nlrp3-/- and Asc-/- mice were incubated with three Gd-containing compounds and IL-1beta activation and secretion was detected by ELISA and western blot analysis. Inflammasome activation and regulation was investigated in IL-4- and interferon (IFN)gamma-polarised macrophages by ELISA, quantitative real time (qRT)-PCR and NanoString nCounter analysis. Furthermore, C57BL/6 and Nlrp3-/-mice were intraperitoneally injected with GBCA and recruitment of inflammatory cells to the peritoneum was analysed by fluorescence-activated cell sorting (FACS). RESULTS: Free Gd and GBCAs activate the NLRP3 inflammasome and induce IL-1beta secretion in vitro. Gd-diethylenetriaminepentaacetic acid also induces the recruitment of neutrophils and inflammatory monocytes to the peritoneum in vivo. Gd activated IL-4-polarised macrophages more effectively than IFNgamma-polarised macrophages, which preferentially expressed genes known to downregulate inflammasome activity. CONCLUSIONS: These data suggest that Gd released from GBCAs triggers a NLRP3 inflammasome-dependent inflammatory response that leads to fibrosis in an appropriate clinical setting. The preferential activation of IL-4-differentiated macrophages is consistent with the predominantly fibrotic presentation of NSF. already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions

Overexpression of membrane-bound fas ligand (CD95L) exacerbates autoimmune disease and renal pathology in pristane-induced lupus

Loss-of-function mutations in the Fas death receptor or its ligand result in a lymphoproliferative syndrome and exacerbate clinical disease in most lupus-prone strains of mice. One exception is mice injected with 2,6,10,14-tetramethylpentadecane (TMPD), a hydrocarbon oil commonly known as pristane, which induces systemic lupus erythematosus-like disease. Although Fas/Fas ligand (FasL) interactions have been strongly implicated in the activation-induced cell death of both lymphocytes and other APCs, FasL can also trigger the production of proinflammatory cytokines. FasL is a transmembrane protein with a matrix metalloproteinase cleavage site in the ectodomain. Matrix metalloproteinase cleavage inactivates membrane-bound FasL and releases a soluble form reported to have both antagonist and agonist activity. To better understand the impact of FasL cleavage on both the proapoptotic and proinflammatory activity of FasL, its cleavage site was deleted through targeted mutation to produce the deleted cleavage site (DeltaCS) mouse line. DeltaCS mice express higher levels of membrane-bound FasL than do wild-type mice and fail to release soluble FasL. To determine to what extent FasL promotes inflammation in lupus mice, TMPD-injected FasL-deficient and DeltaCS BALB/c mice were compared with control TMPD-injected BALB/c mice. We found that FasL deficiency significantly reduced the early inflammatory exudate induced by TMPD injection. In contrast, DeltaCS mice developed a markedly exacerbated disease profile associated with a higher frequency of splenic neutrophils and macrophages, a profound change in anti-nuclear Ab specificity, and markedly increased proteinuria and kidney pathology compared with controls. These results demonstrate that FasL promotes inflammation in TMPD-induced autoimmunity, and its cleavage limits FasL proinflammatory activity

Caspase-8 modulates dectin-1 and complement receptor 3-driven IL-1beta production in response to beta-glucans and the fungal pathogen, Candida albicans

Inflammasomes are central mediators of host defense to a wide range of microbial pathogens. The nucleotide-binding domain and leucine-rich repeat containing family (NLR), pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in triggering caspase-1-dependent IL-1beta maturation and resistance to fungal dissemination in Candida albicans infection. beta-Glucans are major components of fungal cell walls that trigger IL-1beta secretion in both murine and human immune cells. In this study, we sought to determine the contribution of beta-glucans to C. albicans-induced inflammasome responses in mouse dendritic cells. We show that the NLRP3-apoptosis-associated speck-like protein containing caspase recruitment domain protein-caspase-1 inflammasome is absolutely critical for IL-1beta production in response to beta-glucans. Interestingly, we also found that both complement receptor 3 (CR3) and dectin-1 play a crucial role in coordinating beta-glucan-induced IL-1beta processing as well as a cell death response. In addition to the essential role of caspase-1, we identify an important role for the proapoptotic protease caspase-8 in promoting beta-glucan-induced cell death and NLRP3 inflammasome-dependent IL-1beta maturation. A strong requirement for CR3 and caspase-8 also was found for NLRP3-dependent IL-1beta production in response to heat-killed C. albicans. Taken together, these results define the importance of dectin-1, CR3, and caspase-8, in addition to the canonical NLRP3 inflammasome, in mediating beta-glucan- and C. albicans-induced innate responses in dendritic cells. Collectively, these findings establish a novel link between beta-glucan recognition receptors and the inflammatory proteases caspase-8 and caspase-1 in coordinating cytokine secretion and cell death in response to immunostimulatory fungal components