Flagellin/NLRC4 Pathway Rescues NLRP3-Inflammasome Defect in Dendritic Cells From HIV-Infected Patients: Perspective for New Adjuvant in Immunocompromised Individuals.

Introduction: NLRP3 inflammasome plays a key role in dendritic cells (DC) activation in response to vaccine adjuvants, however we previously showed that it is not properly activated in DC from HIV-infected patients (HIV-DC), explaining, at least in part, the poor response to immunization of these patients. Taking in account that several cytoplasmic receptors are able to activate inflammasome, and that bacterial components are considered as a novel and efficient adjuvant, we postulated that bacterial flagellin (FLG), a natural ligand of NAIP/NLRC4 inflammasome, could rescue the activation of the complex in HIV-DC. Objective: Demonstrate that FLG is able to activate monocyte-derived dendritic cells from HIV-infected individuals better than LPS, and to what extent the entity of inflammasome activation differs between DC from HIV-infected patients and healthy donors. Methods: Monocyte-derived dendritic cells from HIV-infected patients (HIV-DC) and healthy donors (HD-DC) were stimulated with FLG, and inflammasome as well as DC activation (phenotypic profile, cytokine production, autologous lymphocytes activation) were compared. Chemical and genetic inhibitors were used to depict the relative contribution of NLRC4 and NLRP3 in HIV/HD-DC response to FLG. Results: FLG properly activates HD-DC and HIV-DC. FLG induces higher inflammasome activation than LPS in HIV-DC. FLG acts through NLRC4 and NLRP3 in HD-DC, but at a lesser extent in HIV-DC due to intrinsic NLRP3 defect. Conclusions: FLG by-passes NLRP3 defect in HIV-DC, through the activation of NAIP/NLRC4 inflammasome, indicating possible future use of the bacterial component as an efficient adjuvant in immunocompromised individuals.

Interleukin-18 diagnostically distinguishes and pathogenically promotes human and murine macrophage activation syndrome.

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperferritinemic systemic inflammatory disorders. Although profound cytotoxic impairment causes familial HLH (fHLH), the mechanisms driving non-fHLH and MAS are largely unknown. MAS occurs in patients with suspected rheumatic disease, but the mechanistic basis for its distinction is unclear. Recently, a syndrome of recurrent MAS with infantile enterocolitis caused by NLRC4 inflammasome hyperactivity highlighted the potential importance of interleukin-18 (IL-18). We tested this association in hyperferritinemic and autoinflammatory patients and found a dramatic correlation of MAS risk with chronic (sometimes lifelong) elevation of mature IL-18, particularly with IL-18 unbound by IL-18 binding protein, or free IL-18. In a mouse engineered to carry a disease-causing germ line NLRC4T337S mutation, we observed inflammasome-dependent, chronic IL-18 elevation. Surprisingly, this NLRC4T337S-induced systemic IL-18 elevation derived entirely from intestinal epithelia. NLRC4T337S intestines were histologically normal but showed increased epithelial turnover and upregulation of interferon-γ-induced genes. Assessing cellular and tissue expression, classical inflammasome components such as Il1b, Nlrp3, and Mefv predominated in neutrophils, whereas Nlrc4 and Il18 were distinctly epithelial. Demonstrating the importance of free IL-18, Il18 transgenic mice exhibited free IL-18 elevation and more severe experimental MAS. NLRC4T337S mice, whose free IL-18 levels were normal, did not. Thus, we describe a unique connection between MAS risk and chronic IL-18, identify epithelial inflammasome hyperactivity as a potential source, and demonstrate the pathogenicity of free IL-18. These data suggest an IL-18-driven pathway, complementary to the cytotoxic impairment of fHLH, with potential as a distinguishing biomarker and therapeutic target in MAS.

Fungal-derived cues promote ocular autoimmunity through a Dectin-2/Card9-mediated mechanism.

Uveitis (intraocular inflammation) is a leading cause of loss of vision. Although its aetiology is largely speculative, it is thought to arise from complex genetic-environmental interactions that break immune tolerance to generate eye-specific autoreactive T cells. Experimental autoimmune uveitis (EAU), induced by immunization with the ocular antigen, interphotoreceptor retinoid binding protein (IRBP), in combination with mycobacteria-containing complete Freund's adjuvant (CFA), has many clinical and histopathological features of human posterior uveitis. Studies in EAU have focused on defining pathogenic CD4+ T cell effector responses, such as those of T helper type 17 (Th17) cells, but the innate receptor pathways precipitating development of autoreactive, eye-specific T cells remain poorly defined. In this study, we found that fungal-derived antigens possess autoimmune uveitis-promoting function akin to CFA in conventional EAU. The capacity of commensal fungi such as Candida albicans or Saccharomyces cerevisae to promote IRBP-triggered EAU was mediated by Card9. Because Card9 is an essential signalling molecule of a subgroup of C-type lectin receptors (CLRs) important in host defence, we evaluated further the proximal Card9-activating CLRs. Using single receptor-deficient mice we identified Dectin-2, but not Mincle or Dectin-1, as a predominant mediator of fungal-promoted uveitis. Conversely, Dectin-2 activation by α-mannan reproduced the uveitic phenotype of EAU sufficiently, in a process mediated by the Card9-coupled signalling axis and interleukin (IL)-17 production. Taken together, this report relates the potential of the Dectin-2/Card9-coupled pathway in ocular autoimmunity. Not only does it contribute to understanding of how innate immune receptors orchestrate T cell-mediated autoimmunity, it also reveals a previously unappreciated ability of fungal-derived signals to promote autoimmunity.

Cholera toxin, and the related nontoxic adjuvants mmCT and dmLT, promote human Th17 responses via cyclic AMP-protein kinase A and inflammasome-dependent IL-1 signaling.

We have examined the molecular pathways involved in the adjuvant action of cholera toxin (CT) and two novel nontoxic molecules, multiple-mutated CT (mmCT) and double-mutant heat-labile toxin (dmLT) on human T cell responses. Human PBMCs or isolated monocytes were stimulated in vitro with CT, mmCT, or dmLT plus a polyclonal stimulus (staphylococcal enterotoxin B) or specific bacterial Ags, and effects on expression of cytokines and signaling molecules were determined. CT, mmCT, and dmLT strongly enhanced IL-17A and to a lesser extent IL-13 responses, but had little effect on IFN-γ production or cell proliferation. Intracellular cytokine staining revealed that the enhanced IL-17A production was largely confined to CD4(+) T cells and coculture experiments showed that the IL-17A promotion was effectively induced by adjuvant-treated monocytes. Relative to CT, mmCT and dmLT induced at least 100-fold lower levels of cAMP, yet this cAMP was enough and essential for the promotion of Th17 responses. Thus, inhibition of cAMP-dependent protein kinase A was abolished, and stimulation with a cAMP analog mimicked the adjuvant effect. Furthermore, CT, mmCT, and dmLT induced IL-1ß production and caspase-1 activation in monocytes, which was associated with increased expression of key proinflammatory and inflammasome-related genes, including NLRP1, NLRP3, and NLRC4. Inflammasome inhibition with a specific caspase-1 inhibitor, or blocking of IL-1 signaling by IL-1 receptor antagonist, abrogated the Th17-promoting effect. We conclude that CT, mmCT, and dmLT promote human Th17 responses via cAMP-dependent protein kinase A and caspase-1/inflammasome-dependent IL-1 signaling.

Inflammasomes.

Inflammasomes are large cytosolic multiprotein complexes that assemble in response to detection of infection- or stress-associated stimuli and lead to the activation of caspase-1-mediated inflammatory responses, including cleavage and unconventional secretion of the leaderless proinflammatory cytokines IL-1ß and IL-18, and initiation of an inflammatory form of cell death referred to as pyroptosis. Inflammasome activation can be induced by a wide variety of microbial pathogens and generally mediates host defense through activation of rapid inflammatory responses and restriction of pathogen replication. In addition to its role in defense against pathogens, recent studies have suggested that the inflammasome is also a critical regulator of the commensal microbiota in the intestine. Finally, inflammasomes have been widely implicated in the development and progression of various chronic diseases, such as gout, atherosclerosis, and metabolic syndrome. In this perspective, we discuss the role of inflammasomes in infectious and noninfectious inflammation and highlight areas of interest for future studies of inflammasomes in host defense and chronic disease.