The inflammasome NLRP3 is activated by pathogen associated molecular patterns (PAMPs) during infection, including RNA and proteins from influenza A virus (IAV). However, chronic activation by danger associated molecular patterns (DAMPs) can be deleterious to the host. We show that blocking NLRP3 activation can be either protective or detrimental at different stages of lethal influenza A virus (IAV). Administration of the specific NLRP3 inhibitor MCC950 to mice from one day following IAV challenge resulted in hypersusceptibility to lethality. In contrast, delaying treatment with MCC950 until the height of disease (a more likely clinical scenario) significantly protected mice from severe and highly virulent IAV-induced disease. These findings identify for the first time that NLRP3 plays a detrimental role later in infection, contributing to IAV pathogenesis through increased cytokine production and lung cellular infiltrates. These studies also provide the first evidence identifying NLRP3 inhibition as a novel therapeutic target to reduce IAV disease severity.

Innate immune cells have a critical role in defense against infection and disease. Central to this is the broad specificity with which they can detect pathogen-associated patterns and danger-associated patterns via the pattern recognition receptors (PRRs) they express. Several families of PRRs have been identified including: Toll-like receptors (TLRs), C-type lectin-like receptors, retinoic acid-inducible gene-like receptors and nucleotide-binding oligomerization domain–like receptors. TLRs are one of the most largely studied families of PRRs. The binding of ligands to TLRs on antigen presenting cells (APCs), mainly dendritic cells, leads to APC maturation, induction of inflammatory cytokines and the priming of naive T cells to drive acquired immunity. Therefore, activation of TLRs promotes both innate inflammatory responses and the induction of adaptive immunity. Consequently, in the last two decades mounting evidence has inextricably linked TLR activation with the pathogenesis of immune diseases and cancer. It has become advantageous to harness these aspects of TLR signaling therapeutically to accelerate and enhance the induction of vaccine-specific responses and also target TLRs with the use of biologics and small molecule inhibitors for the treatment of disease. In these respects, TLRs may be considered a ‘Swiss Army' knife of the immune system, ready to respond in a multitude of infectious and disease states. Here we describe the latest advances in TLR-targeted therapeutics and the use of TLR ligands as vaccine adjuvants.

BACKGROUND AND PURPOSEInflammasomes are multimeric complexes that facilitate caspase-1-mediated processing of the pro-inflammatory cytokines IL-1β and IL-18. Clinical hypertension is associated with renal inflammation and elevated circulating levels of IL-1β and IL-18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces BP, markers of renal inflammation and fibrosis. EXPERIMENTAL APPROACHWild-type and inflammasome-deficient ASC −/− mice were uninephrectomized and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomized but received a placebo pellet and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real-time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry.

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4−/− and Myd88−/−, but not TRIF−/− mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.

The extensively studied cytokine IL-1β is an important mediator of the inflammatory response. However, dysregulated release of IL-1β can be detrimental and is attributed to the progression and pathogenesis of multiple inflammatory diseases including, rhuematoid arthritis (RA), atherosclerosis, type 2 diabetes (T2D), Alzheimers disease and gout. IL-1β is encoded as a pro-protein. A multi-protein molecular scaffold termed the "Inflammasome" is responsible for the tightly controlled and coordinated processing of pro-IL-1β. The activation of several NLR (nucleotide-binding oligomerization domain (NOD)-like receptor) family members and PYHIN (pyrin and HIN domain) proteins can drive the formation of inflammasomes. However, the exact biochemical mechanisms governing their activation have been the subject of much research. Different inflammasomes have been demonstrated to respond to the same pathogen inducing a cooperative immune response accountable for the clearance of infection. Here, we review current knowledge surrounding the biochemical regulation of the NLRP1, NLRP3, NLRC4, AIM2 and IFI16 inflammasomes.

The emergence of avian H7N9 influenza A virus in humans with associated high mortality has highlighted the threat of a potential pandemic. Fatal H7N9 infections are characterized by hyperinflammation and increased cellular infiltrates in the lung. Currently there are limited therapies to address the pathologies associated with H7N9 infection and the virulence factors that contribute to these pathologies. We have found that PB1-F2 derived from H7N9 activates the NLRP3 inflammasome and induces lung inflammation and cellular recruitment that is NLRP3-dependent. We have also shown that H7N9 and A/Puerto Rico/H1N1 (PR8)PB1-F2 peptide treatment induces significant mitochondrial reactive oxygen production, which contributes to NLRP3 activation. Importantly, treatment of cells or mice with the specific NLRP3 inhibitor MCC950 significantly reduces IL-1β maturation, lung cellular recruitment, and cytokine production. Together, these results suggest that PB1-F2 from H7N9 avian influenza A virus may be a major contributory factor to disease pathophysiology and excessive inflammation characteristic of clinical infections and that targeting the NLRP3 inflammasome may be an effective means to reduce the inflammatory burden associated with H7N9 infections.

Mitochondria are important regulators of macrophage polarisation. Here, we show that arginase-2 (Arg2) is a microRNA-155 (miR-155) and interleukin-10 (IL-10) regulated protein localized at the mitochondria in inflammatory macrophages, and is critical for IL-10-induced modulation of mitochondrial dynamics and oxidative respiration. Mechanistically, the catalytic activity and presence of Arg2 at the mitochondria is crucial for oxidative phosphorylation. We further show that Arg2 mediates this process by increasing the activity of complex II (succinate dehydrogenase). Moreover, Arg2 is essential for IL-10-mediated downregulation of the inflammatory mediators succinate, hypoxia inducible factor 1α (HIF-1α) and IL-1β in vitro. Accordingly, HIF-1α and IL-1β are highly expressed in an LPS-induced in vivo model of acute inflammation using Arg2−/− mice. These findings shed light on a new arm of IL-10-mediated metabolic regulation, working to resolve the inflammatory status of the cell.

Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria both in vivo and in vitro. These lipid-bound structures carry a range of immunogenic components derived from the parent cell, which are transported into host target cells and activate the innate immune system. Recent advances in the field have shed light on some of the multifaceted roles of OMVs in host-pathogen interactions. In this study, we investigated the ability of OMVs from two clinically important pathogens, Pseudomonas aeruginosa and Helicobacter pylori, to activate canonical and noncanonical inflammasomes. P. aeruginosa OMVs induced inflammasome activation in mouse macrophages, as evidenced by "speck" formation, as well as the cleavage and secretion of interleukin-1β and caspase-1. These responses were independent of AIM2 and NLRC4 canonical inflammasomes, but dependent on the noncanonical caspase-11 pathway. Moreover, P. aeruginosa OMVs alone were able to activate the inflammasome in a TLR-dependent manner, without requiring an exogenous priming signal. In contrast, H. pylori OMVs were not able to induce inflammasome activation in macrophages. Using CRISPR/Cas9 knockout THP-1 cells lacking the human caspase-11 homologs, caspase-4 and -5,we demonstrated that caspase-5 but not caspase-4 is required for inflammasome activation by P. aeruginosa OMVs in human monocytes. In contrast, free P. aeruginosa lipopolysaccharide (LPS) transfected into cells induced inflammasome responses via caspase-4. This suggests that caspase-4 and caspase-5 differentially recognize LPS depending on its physical form or route of delivery into the cell. These findings have relevance to Gram-negative infections in humans and the use of OMVs as novel vaccines.