MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition.

Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and specific small-molecule inhibitor of the NLRP3 pathway, but its molecular target is not defined. Here, we show that MCC950 directly interacts with the Walker B motif within the NLRP3 NACHT domain, thereby blocking ATP hydrolysis and inhibiting NLRP3 activation and inflammasome formation.

Structural Biology of NOD-Like Receptors.

The nucleotide-binding domain (NBD) and leucine-rich repeat (LRR) containing (NLR) proteins are a large family of intracellular immune receptors conserved in both animals and plants. Mammalian NLRs function as pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) or host-derived danger associated molecular patterns (DAMPs). PAMP or DAMP perception activates NLRs which consequently recruit pro-caspase-1 directly or indirectly. These sequential events result in formation of large multimeric protein complexes termed inflammasomes that mediate caspase-1 activation for pyroptosis and cytokine secretion. Recent structural and biochemical studies provide significant insights into the acting mechanisms of NLR proteins. In this chapter, we review and discuss these studies concerning autoinhibition, ligand recognition, activation of NLRs, and assembly of NLR inflammasomes.

AIM2 Inflammasome Assembly and Signaling.

AIM2 (absent in melanoma 2) is a cytoplasmic sensor of double-stranded DNA from pathogens or damaged cellular organelles. It recruits ASC (apoptosis-associated specklike protein containing a CARD) and caspase-1 to form the AIM2 inflammasome, activate caspase-1, and elicit inflammatory responses via cytokine maturation and pyroptotic cell death. Structural studies from X-ray crystallography, NMR, and cryo-EM have revealed many details in AIM2 inflammasome activation, assembly, and regulation. Many principles learned from AIM2 inflammasome also apply to other inflammasomes. In this chapter, we discuss the interactions between dsDNA and AIM2-like receptors, between AIM2 and adaptor protein ASC, and between ASC and caspase-1 with the focus on helical filament assembly formed by PYD and CARD domains.

Altered expression of inflammasomes in Hirschsprung's disease.

AIM OF THE STUDY: The pathogenesis of Hirschsprung's disease-associated enterocolitis (HAEC) is poorly understood. Inflammasomes are a large family of multiprotein complexes that act to mediate host immune responses to microbial infection and have a regulatory or conditioning influence on the composition of the microbiota. Inflammasomes and the apoptosis-associated speck-like protein (ASC) lead to caspase-1 activation. The activated caspase-1 promotes secretion of pro-inflammatory cytokines (IL-1ß and IL-18) from their precursors (pro-IL-1ß and pro-IL-18). Inflammasomes have been implicated in a host of inflammatory disorders. Among the inflammasomes, NLRP3, NLRP12 and NLRC4 are the most widely investigated. Knock-out mice models of inflammasomes NLRP3, NLRP12, NLRC4, caspase-1 and ASC are reported to have higher susceptibility to experimental colitis. The purpose of this study was to investigate the expression of NLRP3, NLRP12, NLRC4, caspase-1, ASC, pro-IL-1ß and pro-IL-18 in the bowel specimens from patients with HSCR and controls. METHODS: Pulled-through colonic specimens were collected from HSCR patients (n = 6) and healthy controls from the proximal colostomy of children with anorectal malformations (n = 6). The gene expression of NLRP3, NLRP12, NLRC4, caspase-1, ASC, pro-IL-1ß and pro-IL-18 was assessed using qPCR. The protein distribution was assessed using immunofluorescence and confocal microscopy. MAIN RESULTS: qRT-PCR analysis revealed that NLRP3, NLRP12, NLRC4, ASC and pro-IL-1ß gene expressions was significantly downregulated in the aganglionic and ganglionic colon of patients with HSCR compared to controls. Confocal microscopy revealed a markedly decreased expression of NLRP3, NLRP12, NLRC4 and ASC protein in the colonic epithelium of aganglionic and ganglionic bowel of patients with HSCR compared to controls. CONCLUSIONS: To our knowledge, this is the first study analyzing NLRP3, NLRP12, NLRC4, ASC and pro-IL-1ß gene expressions in patients with HSCR. Decreased expression of NLRP3, NLRP12, NLRC4, ASC and pro-IL-1ß in the aganglionic and ganglionic bowel may increase susceptibility of HSCR patients to develop HAEC.

NLRC3 protein inhibits inflammation by disrupting NALP3 inflammasome assembly via competition with the adaptor protein ASC for pro-caspase-1 binding.

Inflammasomes are multiprotein complexes that sense pathogen-associated and danger-associated molecular patterns and induce inflammation in cells. The NALP3 inflammasome is tightly regulated by recently discovered control mechanisms, but other modulators still remain to be characterized. NLR family CARD-containing 3 (NLRC3) protein, a caspase recruitment domain (CARD)-containing member of the nucleotide oligomerization domain-like receptor (NLR) family, was found to down-regulate the NF-κB pathway and stimulator of interferon genes (STING)-dependent cytokine secretion. However, the effect of NLRC3 on the NALP3 inflammasome or other inflammasomes is still unknown. We hypothesized that NLRC3 might inhibit NALP3 inflammasome complex assembly. Toward this end, we tested whether NLRC3 overexpression or knockdown influences NALP3 activity in human monocyte and HEK293FT cells when the complex is ectopically reconstituted. We found that NLRC3 indeed decreases NALP3-induced IL-1ß maturation and secretion, pro-caspase-1 cleavage, and speck formation by apoptosis-associated speck-like protein containing a CARD (ASC) protein in response to NALP3 activators. We also show that endogenous NLRC3 interacts with both ASC and pro-caspase-1 but not with NALP3, disrupts ASC speck formation through its CARD, and impairs the ASC and pro-caspase-1 interaction. Moreover, the NLRC3 CARD alone could dampen IL-1ß secretion and ASC speck formation induced by NALP3 mutants associated with autoinflammatory diseases. In conclusion, we show here that, besides its role in the inhibition of the NF-κB pathway, NLRC3 interferes with the assembly and activity of the NALP3 inflammasome complex by competing with ASC for pro-caspase-1 binding.

Selective NLRP3 inflammasome inhibitor reduces neuroinflammation and improves long-term neurological outcomes in a murine model of traumatic brain injury.

The nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated inflammatory response has emerged as a prominent contributor to the pathophysiological processes of traumatic brain injury (TBI). Recently, a potent, selective, small-molecule NLRP3 inflammasome inhibitor, MCC950, was described. Here, we investigated the effect of MCC950 on inflammatory brain injury and long-term neurological outcomes in a mouse model of TBI. Male C57/BL6 mice were subjected to TBI using the controlled cortical impact injury (CCI) system. Western blotting, flow cytometry, and immunofluorescence assays were utilized to analyze post-traumatic NLRP3 inflammasome expression and determine its cellular source. We found that NLRP3 inflammasome expression was significantly increased in the peri-contusional cortex and that microglia were the primary source of this expression. The effects of MCC950 on mice with TBI were then determined using post-assessments including analyses of neurological deficits, brain water content, traumatic lesion volume, neuroinflammation, blood-brain barrier (BBB) integrity, and cell death. MCC950 treatment resulted in a better neurological outcome after TBI by alleviating brain edema, reducing lesion volume, and improving long-term motor and cognitive functions. The therapeutic window for MCC950 against TBI was as long as 6 h. Furthermore, the neuroprotective effect of MCC950 was associated with reduced microglial activation, leukocyte recruitment, and pro-inflammatory cytokine production. In addition, MCC950 preserved BBB integrity, alleviated TBI-induced loss of tight junction proteins, and attenuated cell death. Notably, the efficacy of MCC950 was abolished in microglia-depleted mice. These results indicate that microglia-derived NLRP3 inflammasome may be primarily involved in the inflammatory response to TBI, and specific NLRP3 inflammasome inhibition using MCC950 may be a promising therapeutic approach for patients with TBI.

Adipose-derived stem cells ameliorate colitis by suppression of inflammasome formation and regulation of M1-macrophage population through prostaglandin E2.

Inflammatory bowel disease (IBD) is an idiopathic disease caused by a dysregulated immune response to intestinal microbes in an individual with a genetic predisposition. Therefore, alleviation of inflammation is very important to treat IBD. Mesenchymal stem cells (MSCs) have been highlighted as new candidates for treating autoimmune disease based on their immunomodulatory properties. In this study, we investigated the anti-inflammatory mechanism and therapeutic effects of adipose tissue-derived MSCs (ASCs) using THP-1 macrophages and dextran sodium sulfate (DSS)-induced mice with chronic colitis. LPS-treated THP-1 cells expressed mRNA of CD11b, an M1 macrophage marker, at day 2. However, THP-1 co-cultured with ASCs expressed mRNA of CD206, CD68, CCL18, legumain, and IL-10, markers of M2 macrophages. In THP-1 cells co-cultured with ASCs, precursor (pro)-IL-1ß, Cox-2, and NLRP3 increased dramatically compared to LPS-treated THP-1 cells. Secretion of IL-1ß and IL-18 was significantly inhibited by ASCs, but PGE2 production was highly increased in co-culture conditions of THP-1 and ASCs. IL-18 secretion was inhibited by PGE2 treatment, and PGE2 inhibited inflammasome complex (ASC/Cas-1/NLRP3) formation in THP-1 cells. In the DSS-induced chronic colitis model, ASCs ameliorated colitis by decreasing the total number of macrophages and the M1 macrophage population. Our results suggest that ASCs can suppress the inflammatory response by controlling the macrophage population, and ASCs may be therapeutically useful for the treatment of IBD.

NLRP3 Inflammasome Is Involved in Q-VD-OPH Induced Necroptosis Following Cerebral Ischemia-Reperfusion Injury.

Necroptosis is a manner of caspase-independent cell death,which accounts for delayed ischemic cerebral injury, and can be used as a novel tool to expand the treatment time window in ischemic cerebral injury. Q-VD-OPH, a novel pan caspase inhibitor, has been identified as an inducer of necroptosis. In this study, we determined the optimal dose of Q-VD-OPH, which induces necroptosis in rats by the middle cerebral artery occlusion, followed by reperfusion. Furthermore, we report that the NLRP3 inflammasome is involved in necroptosis, with levels of NLRP3 inflammasome proteins as well as inflammatory cytokines, such as IL-1ß, being elevated. We also demonstrated that NLRP3 was not only expressed in microglia and vascular endothelial cell, but also in neurons when necroptosis is induced with Q-VD-OPH. Inhibition of NLRP3 by glyburide strongly suppressed the expression of NLRP3 inflammasome proteins and IL-1ß, and markedly reduced brain tissue damage. Our findings provide evidence that pretreatment with Q-VD-OPH suppresses apoptosis and induces necroptosis in the cerebral ischemia-reperfusion model. We also identified that the NLRP3 inflammasome plays an important role in neuronal necroptosis, and that NLRP3 inflammasome deficiency reduces brain tissue damage after cerebral ischemia-reperfusion injury in rats.

Overexpression of NLRP3, NLRC4 and AIM2 inflammasomes and their priming-associated molecules (TLR2, TLR4, Dectin-1, Dectin-2 and NFκB) in Malassezia folliculitis.

The activation of NLRP3, NLRC4 and AIM2 inflammasomes is pivotal for innate immunity against some pathogenic fungi, but their role in the pathogenesis of Malassezia folliculitis (MF) remains unclear. The objective of the study was to determine the expression of 4 canonical inflammasomes (NLRP1, NLRP3, NLRC4 and AIM2) and their priming-associated molecules (TLR2, TLR4, Dectin-1, Dectin-2 and NFκB) in MF lesion. Expression of NLRP1, NLRP3, NLRC4, AIM2, caspase-1, IL-1ß, TLR2, TLR4, Dectin-1, Dectin-2 and NFκB was detected by immunohistochemistry in skin lesion of 23 MF patients and normal skin of 12 healthy subjects. Furthermore, NLRP1, NLRP3, NLRC4, AIM2, caspase-1 and IL-1ß mRNA was measured by quantitative real-time PCR (qRT-PCR) in 12 MF cases and 10 controls. Immunohistochemical analysis revealed that NLRP3, NLRC4, AIM2, Casp-1, IL-1ß, TLR2, TLR4, Dectin-1, Dectin-2 and NFκB expression was up-regulated in the epidermis and dermal inflammatory cells of MF lesion compared with control skin (P < .01-.05), but NLRP1 expression was not different between both groups (P > .05). qRT-PCR showed that levels of NLRP3, Casp-1 and IL-1ß mRNA were significantly increased (P < .01-.05), whereas those of NLRP1, NLRC4 and AIM2 mRNA were slightly augmented compared to control skin (P > .05). Our observation suggests that simultaneous activation of NLRP3, NLRC4 and AIM2 inflammasomes may play an important role in the pathogenesis of MF.

The NLRP3 Inflammasome is Involved in the Pathogenesis of Parkinson's Disease in Rats.

The etiology and pathogenesis of Parkinson's disease (PD) are complicated and have not been fully elucidated, but an important association has been identified between inflammation and PD. In this study, we investigated the role of the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain-containing (NLRP) 3 inflammasome, consisting of NLRP3, caspase-1 and cytokines of the IL-1 family, in lipopolysaccharide (LPS)-induced and 6-hydroxydopamine (6-OHDA)-induced PD rats. Microinjection of different doses of caspase-1 inhibitor (Ac-YVAD-CMK, 300 or 1200 ng/rat) was performed for seven consecutive days. Then, rotational behavior, the number of dopamine (DA) neurons in the substantia nigra pars compacta (SNc), and the mRNA and protein expression levels of NLRP3 inflammasome components were measured 14 days after the microinjection setup was established. Results showed that high mRNA and protein expression levels of NLRP3 inflammasome components were observed in the injected side of the LPS- and 6-OHDA-induced PD rats; Ac-YVAD-CMK inhibited the mRNA and protein expression of NLRP3 inflammasome components in both LPS- and 6-OHDA-induced PD rats. Moreover, the number of rotations was significantly decreased, and the number of DA neurons in the SNc improved. Our data indicate that the NLRP3 inflammasome participates in the pathogenesis of PD and that inhibiting the downstream pathway of the NLRP3/caspase-1/IL-1ß axis can alleviate the occurrence of PD symptoms, providing a new basis for the prevention and treatment of PD.

Upregulation of NLRP3 inflammasome components in Mooren's ulcer.

PURPOSE: Mooren's ulcer (MU) is a peripheral corneal ulceration of presumed autoimmune etiology. NLRP3 inflammasome has been shown to be involved in a variety of autoimmune and auto-inflammatory diseases. However, the role of NLRP3 inflammasome in MU has not been investigated. Here, we evaluate the expression of NLRP3 inflammasome and its downstream inflammatory factors in human MU. METHODS: Conjunctival biopsy specimens were obtained from seven patients with MU and six healthy donors. The removed conjunctivas were histopathologically evaluated for NLRP3 inflammasome component expression using antibodies directed against NLRP3, Caspase-1 (CASP1), and Interleukin-1ß (IL-1ß). Quantitative real-time PCR was used to measure the mRNA expression of NLRP3 and IL-1ß, and the protein expressions of NLRP3, pro-CASP1, CASP1, and IL-1ß were detected by Western blotting. RESULTS: NLRP3 and IL-1ß mRNA expression showed higher levels in the MU group than in healthy controls. Western-blot and immunofluorescence analysis also showed that basal expression of NLRP3 inflammasome components (NLRP3, CAPS1, and IL-1ß) was elevated in patients with MU compared with healthy controls. Most importantly, we found that the cleavaged form of CASP1 and IL-1ß was significantly increased in MU patients compared with healthy donors, which indicates that the upregulation of NLRP3 inflammasome was probably responsible for the enhanced IL-1ß production in MU patients. CONCLUSIONS: This study demonstrated that the expression of the NLRP3-CASP1-IL-1ß signaling pathway was markedly increased in the conjunctival lesions of patients with MU, suggesting the involvement of NLRP3 inflammasome in the onset and development of the inflammation in MU.

Stress induces the danger-associated molecular pattern HMGB-1 in the hippocampus of male Sprague Dawley rats: a priming stimulus of microglia and the NLRP3 inflammasome.

Exposure to acute and chronic stressors sensitizes the proinflammatory response of microglia to a subsequent immune challenge. However, the proximal signal by which stressors prime microglia remains unclear. Here, high mobility group box-1 (HMGB-1) protein was explored as a potential mediator of stress-induced microglial priming and whether HMGB-1 does so via the nucleotide-binding domain, leucine-rich repeat, pyrin domain containing protein 3 (NLRP3) inflammasome. Exposure to 100 inescapable tail shocks (ISs) increased HMGB-1 and NLRP3 protein in the hippocampus and led isolated microglia to release HMGB-1 ex vivo. To determine whether HMGB-1 signaling is necessary for stress-induced sensitization of microglia, the HMGB-1 antagonist BoxA was injected into the cisterna magna before IS. Hippocampal microglia were isolated 24 h later and stimulated with LPS ex vivo to probe for stress-induced sensitization of proinflammatory responses. Previous IS potentiated gene expression of NLRP3 and proinflammatory cytokines to LPS, that is, microglia were sensitized. Treatment with BoxA abolished this effect. To determine whether HMGB-1 is sufficient to prime microglia, IS was replaced with intracerebral administration of disulfide or fully reduced HMGB-1. Intracerebral disulfide HMGB-1 mimicked the effect of the stressor, because microglia isolated from HMGB-1-treated rats expressed exaggerated NLRP3 and proinflammatory cytokine expression after LPS treatment, whereas fully reduced HMGB-1 had no effect. The present results suggest that the CNS innate immune system can respond to an acute stressor as if it were cellular damage, thereby releasing the danger signal HMGB-1 in the brain to prime microglia by acting on the NLRP3 inflammasome, in preparation for a later immune challenge.

Exosome-mediated inflammasome signaling after central nervous system injury.

Neuroinflammation is a response against harmful effects of diverse stimuli and participates in the pathogenesis of brain and spinal cord injury (SCI). The innate immune response plays a role in neuroinflammation following CNS injury via activation of multiprotein complexes termed inflammasomes that regulate the activation of caspase 1 and the processing of the pro-inflammatory cytokines IL-1ß and IL-18. We report here that the expression of components of the nucleotide-binding and oligomerization domain (NOD)-like receptor protein-1 (NLRP-1) inflammasome, apoptosis speck-like protein containing a caspase recruitment domain (ASC), and caspase 1 are significantly elevated in spinal cord motor neurons and cortical neurons after CNS trauma. Moreover, NLRP1 inflammasome proteins are present in exosomes derived from CSF of SCI and traumatic brain-injured patients following trauma. To investigate whether exosomes could be used to therapeutically block inflammasome activation in the CNS, exosomes were isolated from embryonic cortical neuronal cultures and loaded with short-interfering RNA (siRNA) against ASC and administered to spinal cord-injured animals. Neuronal-derived exosomes crossed the injured blood-spinal cord barrier, and delivered their cargo in vivo, resulting in knockdown of ASC protein levels by approximately 76% when compared to SCI rats treated with scrambled siRNA. Surprisingly, siRNA silencing of ASC also led to a significant decrease in caspase 1 activation and processing of IL-1ß after SCI. These findings indicate that exosome-mediated siRNA delivery may be a strong candidate to block inflammasome activation following CNS injury. We propose the following signaling cascade for inflammasome activation in peripheral tissues after CNS injury: CNS trauma induces inflammasome activation in the nervous system and secretion of exosomes containing inflammasome protein cargo into cerebral spinal fluid. The inflammasome containing exosomes then fuse with target cells to activate the innate immune response in peripheral tissues. We suggest that these findings may be used to develop new therapeutics to treat the devastating inflammation and cell destruction evoked by CNS injuries. IL-1ß and IL-18 = pro-inflammatory cytokines.

Pyoderma gangrenosum and its syndromic forms: evidence for a link with autoinflammation.

Pyoderma gangrenosum is a rare inflammatory neutrophilic dermatosis manifesting as painful ulcers with violaceous, undermined borders on the lower extremities. It may occur in the context of classic syndromes like PAPA (pyogenic arthritis, pyoderma gangrenosum and acne) and SAPHO (synovitis, acne, pustulosis, hyperostosis, osteitis), as well as in a recently described entity named PASH (pyoderma gangrenosum, acne and suppurative hidradenitis). Pyoderma gangrenosum has recently been included within the spectrum of autoinflammatory diseases, which are characterized by recurrent episodes of sterile inflammation, without circulating autoantibodies and autoreactive T cells. In PAPA syndrome, different mutations involving the PSTPIP1 gene, via an increased binding affinity to pyrin, induce the assembly of inflammasomes. These are molecular platforms involved in the activation of caspase 1, a protease that cleaves inactive prointerleukin (pro-IL)-1ß to its active isoform IL-1ß. The overproduction of IL-1ß triggers the release of a number of proinflammatory cytokines and chemokines, which are responsible for the recruitment and activation of neutrophils, leading to neutrophil-mediated inflammation. In SAPHO syndrome, the activation of the PSTPIP2 inflammasome has been suggested to play a role in inducing the dysfunction of the innate immune system. Patients with PASH have recently been reported to present alterations of genes involved in well-known autoinflammatory diseases, such as PSTPIP1, MEFV, NOD2 and NLRP3. Pyoderma gangrenosum and its syndromic forms can be regarded as a single clinicopathological spectrum in the context of autoinflammation.

Inhibition of the NLRP3 inflammasome provides neuroprotection in rats following amygdala kindling-induced status epilepticus.

BACKGROUND: NLRP3 inflammasome is proposed to regulate inflammation in several neurological diseases, but its role in epilepsy remains largely unknown. This study aimed to investigate the role of the NLRP3 inflammasome in neuroinflammation, spontaneous recurrent seizures (SRS) and hippocampal neuronal loss in rat brain following amygdala kindling-induced status epilepticus (SE). METHODS: We detected the protein levels of IL-1ß and NLRP3 inflammasome components by Western blot in the hippocampus of shams and SE rats at different time points following SE. To further examine whether the activation of the NLRP3 inflammasome contributes to SE-associated neuronal damage, we employed a nonviral strategy to knock down NLRP3 and caspase-1 expression in brain before undergoing SE. Proinflammatory cytokine levels and hippocampal neuronal loss were evaluated at 12 hours and at 6 weeks following SE respectively in these NLRP3 and caspase-1 deficient rats. Meanwhile, SRS occurrence was evaluated through a 4-week video recording started 2 weeks after SE in these NLRP3 and caspase-1 deficient rats. RESULTS: IL-1ß levels and NLRP3 inflammasome components levels dramatically increased at 3 hours after SE, and reached a maximum at 12 hours after SE compared with the control group. Knock down of NLRP3 or caspase-1 decreased the levels of IL-1ß and IL-18 at 12 hours after SE, which was accompanied by a significant suppression in the development and severity of SRS during the chronic epileptic phase. Meanwhile, knock down of NLRP3 or caspase-1 led to a remarkable reduction of hippocampal neuronal loss in the CA1 and CA3 area of the hippocampus at 6 weeks after SE. CONCLUSIONS: Our study provides the first evidence that the NLRP3 inflammasome was significantly up-regulated following SE. More importantly, we show that inhibition of the NLRP3 inflammasome provides neuroprotection in rats following SE. These findings suggest that NLRP3 may represent a potential target for the treatment of epileptogenesis.

Anthrax lethal factor cleavage of Nlrp1 is required for activation of the inflammasome.

NOD-like receptor (NLR) proteins (Nlrps) are cytosolic sensors responsible for detection of pathogen and danger-associated molecular patterns through unknown mechanisms. Their activation in response to a wide range of intracellular danger signals leads to formation of the inflammasome, caspase-1 activation, rapid programmed cell death (pyroptosis) and maturation of IL-1ß and IL-18. Anthrax lethal toxin (LT) induces the caspase-1-dependent pyroptosis of mouse and rat macrophages isolated from certain inbred rodent strains through activation of the NOD-like receptor (NLR) Nlrp1 inflammasome. Here we show that LT cleaves rat Nlrp1 and this cleavage is required for toxin-induced inflammasome activation, IL-1 ß release, and macrophage pyroptosis. These results identify both a previously unrecognized mechanism of activation of an NLR and a new, physiologically relevant protein substrate of LT.

Host-pathogen interaction in invasive Salmonellosis.

Salmonella enterica infections result in diverse clinical manifestations. Typhoid fever, caused by S. enterica serovar Typhi (S. Typhi) and S. Paratyphi A, is a bacteremic illness but whose clinical features differ from other Gram-negative bacteremias. Non-typhoidal Salmonella (NTS) serovars cause self-limiting diarrhea with occasional secondary bacteremia. Primary NTS bacteremia can occur in the immunocompromised host and infants in sub-Saharan Africa. Recent studies on host-pathogen interactions in Salmonellosis using genome sequencing, murine models, and patient studies have provided new insights. The full genome sequences of numerous S. enterica serovars have been determined. The S. Typhi genome, compared to that of S. Typhimurium, harbors many inactivated or disrupted genes. This can partly explain the different immune responses both serovars induce upon entering their host. Similar genome degradation is also observed in the ST313 S. Typhimurium strain implicated in invasive infection in sub-Saharan Africa. Virulence factors, most notably, type III secretion systems, Vi antigen, lipopolysaccharide and other surface polysaccharides, flagella, and various factors essential for the intracellular life cycle of S. enterica have been characterized. Genes for these factors are commonly carried on Salmonella Pathogenicity Islands (SPIs). Plasmids also carry putative virulence-associated genes as well as those responsible for antimicrobial resistance. The interaction of Salmonella pathogen-associated molecular patterns (PAMPs) with Toll-like receptors (TLRs) and NOD-like receptors (NLRs) leads to inflammasome formation, activation, and recruitment of neutrophils and macrophages and the production of pro-inflammatory cytokines, most notably interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)-α, and interferon-gamma (IFN)-γ. The gut microbiome may be an important modulator of this immune response. S. Typhimurium usually causes a local intestinal immune response, whereas S. Typhi, by preventing neutrophil attraction resulting from activation of TLRs, evades the local response and causes systemic infection. Potential new therapeutic strategies may lead from an increased understanding of infection pathogenesis.

NLRP3 inflammasome activity is negatively controlled by miR-223.

Inflammasomes are multiprotein signaling platforms that form upon sensing microbe- or damage-associated molecular patterns. Upon their formation, caspase-1 is activated, leading to the processing of certain proinflammatory cytokines and the initiation of a special type of cell death, known as pyroptosis. Among known inflammasomes, NLRP3 takes on special importance because it appears to be a general sensor of cell stress. Moreover, unlike other inflammasome sensors, NLRP3 inflammasome activity is under additional transcriptional regulation. In this study, we identify the myeloid-specific microRNA miR-223 as another critical regulator of NLRP3 inflammasome activity. miR-223 suppresses NLRP3 expression through a conserved binding site within the 3' untranslated region of NLRP3, translating to reduced NLRP3 inflammasome activity. Although miR-223 itself is not regulated by proinflammatory signals, its expression varies among different myeloid cell types. Therefore, given the tight transcriptional control of NLRP3 message itself, miR-223 functions as an important rheostat controlling NLRP3 inflammasome activity.

Cutting edge: miR-223 and EBV miR-BART15 regulate the NLRP3 inflammasome and IL-1ß production.

Although microRNA (miRNA) regulation of TLR signaling is well established, this has not yet been observed for NLR proteins or the inflammasomes they form. We have now validated a highly conserved miR-223 target site in the NLRP3 3'-untranslated region. miR-223 expression decreases as monocytes differentiate into macrophages, whereas NLRP3 protein increases during this time. However, overexpression of miR-223 prevents accumulation of NLRP3 protein and inhibits IL-1ß production from the inflammasome. Virus inhibition of the inflammasome is an emerging theme, and we have also identified an EBV miRNA that can target the miR-223 binding site in the NLRP3 3'-untranslated region. Furthermore, this virus miRNA can be secreted from infected B cells via exosomes to inhibit the NLRP3 inflammasome in noninfected cells. Therefore, we have identified both the first endogenous miRNA that limits NLRP3 inflammatory capacity during myeloid cell development and also a viral miRNA that takes advantage of this, limiting inflammation for its own purposes.

DPP-4 inhibitors repress NLRP3 inflammasome and interleukin-1beta via GLP-1 receptor in macrophages through protein kinase C pathway.

BACKGROUND: Anti-atherosclerotic effects of dipeptidyl peptidase-4 (DPP-4) inhibitors have been shown in many studies. Since inflammation and immune response play a key role in atherogenesis, we examined the effect of DPP-4 inhibitors on the expression of nod-like receptor family, pyrin domain containing 3 (NLRP3) Inflammasome and Interleukin-1beta (IL-1ß) in human macrophages. METHODS AND RESULTS: THP-1 macrophages were incubated with oxidized low density lipoprotein (ox-LDL) with or without DPP-4 inhibitors (sitagliptin and NVPDPP728). The effects of DPP-4 inhibitors on the expression of NLRP3, toll-like receptor 4 (TLR4) and pro-inflammatory cytokine IL-1ß were studied. Both DPP-4 inhibitors induced a significant reduction in NLRP3, TLR4 and IL-1ß expression; concurrently, there was an increase in glucagon like peptide 1 receptor (GLP-1R) expression. Simultaneously, DPP-4 inhibitors reduced phosphorylated-PKC, but not PKA, levels. To determine the role of PKC activation in the effects of DPP-4 inhibitors, cells were treated with PMA- which blocked the effect of DPP-4 inhibitors on NLRP3 and IL-1ß as well as TLR4 and GLP-1R. Over-expression of GLP-1R in macrophages with its agonist liraglutide also blocked the effects of PMA. CONCLUSION: DPP-4 inhibitors suppress NLRP3, TLR4 and IL-1ß in human macrophages through inhibition of PKC activity. This study provides novel insights into the mechanism of inhibition of inflammatory state and immune response in atherosclerosis by DPP-4 inhibitors.