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.

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.

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.

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.

TNF regulates transcription of NLRP3 inflammasome components and inflammatory molecules in cryopyrinopathies.

The NLRP3 inflammasome is a protein complex responsible for caspase-1-dependent maturation of the proinflammatory cytokines IL-1ß and IL-18. Gain-of-function missense mutations in NLRP3 cause the disease spectrum known as the cryopyrin-associated periodic syndromes (CAPS). In this study, we generated Nlrp3-knockin mice on various KO backgrounds including Il1b/Il18-, caspase-1-, caspase-11- (Casp1/11-), and Tnf-deficient strains. The Nlrp3L351P Il1b-/- Il18-/- mutant mice survived and grew normally until adulthood and, at 6 months of age, exhibited marked splenomegaly and leukophilia. Injection of these mice with low-dose LPS resulted in elevated serum TNF levels compared with Nlrp3L351P Casp1/11-/- mice and Il1b-/- Il18-/- littermates. Treatment of Nlrp3A350V mice with the TNF inhibitor etanercept resulted in all pups surviving to adulthood, with normal body and spleen/body weight ratios. Nlrp3A350V Tnf-/- mice showed a similar phenotypic rescue, with marked reductions in serum IL-1ß and IL-18, reduced myeloid inflammatory infiltrate in the skin and spleen, and substantial decreases in splenic mRNA expression of both inflammasome components (Nlrp3, Pycard, pro-Casp1) and pro-cytokines (Il1b, Il18). Likewise, we observed a reduction in the expression of both pro-Casp1 and pro-Il1b in cultured Nlrp3A350V Tnf-/- BM-derived DCs. Our data show that TNF is an important transcriptional regulator of NLRP3 inflammasome components in murine inflammasomopathies. Moreover, these results may have therapeutic implications for CAPS patients with partial responses to IL-1-targeted therapies.

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.

Expression of Inflammasome-Associated Proteins in Human Oropharyngeal Squamous Cell Carcinoma.

Inflammasomes, large protein complexes typically consisting of a Nod-like receptor (NLR), adapter protein apoptosis-associated speck-like protein containing CARD (ASC) and caspase-1, are postulated to be activated in response to danger signals arising from tumors. Inflammasomes are thought to have critical but contrasting roles through facilitating antitumor immunity and inducing oncogenic factors. However, the role and function of inflammasomes in oropharyngeal carcinoma remain unclear. We analyzed nine specimens of oropharyngeal squamous cell carcinoma (SCC) and determined the expression of NLRP3, ASC, interleukin (IL)-1ß, IL-18 and caspase-1 in the specimens with and without human papilloma virus (HPV) infection using immunohistochemistry, and analyzed the correlations between the altered expression of these proteins and clinicopathological factors of oropharyngeal SCC. We found strong expression of NLRP3, ASC, IL-1ß, IL-18 and caspase-1 in human oropharyngeal SCC and weak or no expression of these proteins in normal tonsils. Furthermore, the distribution of mindbomb E3 ubiquitin protein ligase 1 and inflammasome-associated proteins in oropharyngeal SCC was not significantly different; there was no correlation between the expression of inflammasome-associated proteins and HPV infection. These findings suggest that inflammasomes in oropharyngeal SCC play a key role through facilitating antitumor immunity and the possibility of new roles for inflammasomes in the oropharynx.

Dysregulation of the NLRP3 inflammasome complex and related cytokines in patients with multiple myeloma.

OBJECTIVE: The NLRP3 inflammasome complex, an important regulatory factor of inflammation and cell apoptosis, has attracted considerable attention in the development of tumor. Here, we analyzed the expression and clinical significance of NLRP3 inflammasome complex and related cytokines in patients with multiple myeloma (MM). METHODS: Peripheral blood and bone marrow of 38 newly diagnosed myeloma patients and 25 age- and gender-matched healthy people were studied. NLRP3 and caspase-1 were analyzed using quantitative real-time polymerase chain reaction and Western blot and IL-1beta, IL-18, RANKL, and OPG were evaluated by enzyme-linked immunosorbent assay. RESULTS: We showed that aberrant NLRP3 and caspase-1 expression were observed in MM and down-regulated compared with the healthy people. We further demonstrated that NLRP3 mRNA was negatively correlated with beta2-microglobulin and plasma cell percentage in MM. The downstream cytokines IL-18 and sRANKL/OPG in MM patients were higher than that in control group. Moreover, the lower mRNA levels of NLRP3 and caspase-1 were shown to be positively correlated with IL-1beta in newly diagnosed MM patients. CONCLUSIONS: This study demonstrated that dysregulated expression of NLRP3-caspase-1-IL-1beta axis was observed in patients with MM, suggesting they might be involved in the pathogenesis of MM.

Defective mitochondrial fission augments NLRP3 inflammasome activation.

Despite the fact that deregulated NLRP3 inflammasome activation contributes to the pathogenesis of chronic inflammatory or metabolic disorders, the underlying mechanism by which NLRP3 inflammasome signaling is initiated or potentiated remains poorly understood. Much attention is being paid to mitochondria as a regulator of NLRP3 inflammasome activation, but little is known about the role of mitochondrial dynamics for the inflammasome pathway. Here, we present evidence that aberrant mitochondrial elongation caused by the knockdown of dynamin-related protein 1 (Drp1) lead to a marked increase in NLRP3-dependent caspase-1 activation and interleukin-1-beta secretion in mouse bone marrow-derived macrophages. Conversely, carbonyl cyanide m-chlorophenyl hydrazone, a chemical inducer of mitochondrial fission, clearly attenuated NLRP3 inflammasome assembly and activation. Augmented activation of NLRP3 inflammasome by mitochondrial elongation is not resulted from the increased mitochondrial damages of Drp1-knockdown cells. Notably, enhanced extracellular signal-regulated kinase (ERK) signaling in Drp1-knockdown macrophages is implicated in the potentiation of NLRP3 inflammasome activation, possibly via mediating mitochondrial localization of NLRP3 to facilitate the assembly of NLRP3 inflammasome. Taken together, our results provide a molecular insight into the importance of mitochondrial dynamics in potentiating NLRP3 inflammasome activation, leading to aberrant inflammation.

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.

Pyroptotic death storms and cytopenia.

For over two decades, we have embraced the cytokine storm theory to explain sepsis, severe sepsis and septic shock. The failure of numerous large-scale clinical trials, which aimed to treat sepsis by neutralizing inflammatory cytokines and LPS, indicates that alternative pathophysiological mechanisms are likely to account for sepsis and the associated immune suppression in patients with severe infection. Recent insights that extricate pyroptotic death from inflammatory cytokine production in vivo have highlighted a need to investigate the consequences of apoptotic and non-apoptotic death in contributing to cytopenia and immune suppression. In this review, we will focus on the biochemical and cellular mechanisms controlling pyroptosis, a Caspase-1/11 dependent form of cell death during infection.

Inhibition of P2X4 suppresses joint inflammation and damage in collagen-induced arthritis.

Recent data have shown that the purinergic receptor P2X4 plays key roles in inflammatory responses. We evaluated whether P2X4 inhibition could affect the development of arthritis and autoimmunity in collagen-induced arthritis (CIA) model. P2X4 antisense oligonucleotide (asODN) was injected intravenously via tail vein into the CIA mice to selectively inhibit P2X4 expression daily for 14 days. P2X4 asODN treatment reduced the clinical score of CIA in mice. P2X4 asODN also decreased the levels of serum IL-1ß, TNF-α, IL-6, and IL-17. P2X4 asODN treatment significantly inhibited synovial inflammation and joint destruction. P2X4 asODN treatment also suppressed the NLR family, pyrin domain containing 1 (NLRP1) inflammasome activation in CIA mice and synovial cells of human rheumatoid arthritis. These data show that P2X4 asODN confers a therapeutic benefit on CIA. Inhibition of the NLRP1 inflammasome signaling pathway is the underlying mechanism of action.

Involvement of the spinal NALP1 inflammasome in neuropathic pain and aspirin-triggered-15-epi-lipoxin A4 induced analgesia.

Neuroinflammation plays an important role in nerve-injury-induced neuropathic pain, but the explicit molecular mechanisms of neuroinflammation in neuropathic pain remain unclear. As one of the most critical inflammatory cytokines, interleukin-1ß (IL-1ß) has been regarded as broadly involved in the pathology of neuropathic pain. The inflammasome caspase-1 platform is one primary mechanism responsible for the maturation of IL-1ß. Lipoxins, a type of endogenous anti-inflammatory lipid, have proved to be effective in relieving neuropathic pain behaviors. The present study was designed to examine whether the inflammasome caspase-1 IL-1ß platform is involved in chronic constriction injury (CCI)-induced neuropathic pain and in lipoxin-induced analgesia. After rats were subjected to the CCI surgery, mature IL-1ß was significantly increased in the ipsilateral spinal cord, and the inflammasome platform consisting of NALP1 (NAcht leucine-rich-repeat protein 1), caspase-1 and ASC (apoptosis-associated speck-like protein containing a caspase-activating recruitment domain) was also activated in spinal astrocytes and neurons, especially at the superficial laminae of the spinal dorsal horn; The aspirin-triggered-15-epi-lipoxin A4 (ATL), which shares the potent actions of the endogenous lipoxins, was administered to the CCI rats. Repeated intrathecal injection with ATL markedly attenuated the CCI-induced thermal hyperalgesia and significantly inhibited NALP1 inflammasome activation, caspase-1 cleavage, and IL-1ß maturation. These results suggested that spinal NALP1 inflammasome was involved in the CCI-induced neuropathic pain and that the analgesic effect of ATL was associated with suppressing NALP1 inflammasome activation.

LRRFIP2 negatively regulates NLRP3 inflammasome activation in macrophages by promoting Flightless-I-mediated caspase-1 inhibition.

The NLRP3 inflammasome is the most characterized inflammasome activated by cellular infection or stress, which is responsible for the maturation of proinflammatory cytokines IL-1ß and IL-18. The precise molecular mechanism for negative regulation of NLRP3 inflammasome activation needs to be further defined. Here we identify leucine-rich repeat Fli-I-interacting protein 2 (LRRFIP2) as an NLRP3-associated protein and an inhibitor for NLRP3 inflammasome activation. LRRFIP2 binds to NLRP3 via its N terminus upon NLRP3 inflammasome activation, and also interacts with Flightless-I, a pseudosubstrate of caspase-1, via its Coil motif. Knockdown of Flightless-I significantly promotes NLRP3 inflammasome activation. LRRFIP2 enhances the interaction between Flightless-I and caspase-1, facilitating the inhibitory effect of Flightless-I on caspase-1 activation. Furthermore, silencing of Flightless-I abrogates the inhibitory effect of LRRFIP2 on NLRP3 inflammasome. These data demonstrate that LRRFIP2 inhibits NLRP3 inflammasome activation by recruiting the caspase-1 inhibitor Flightless-I, thus outlining a new mechanism for negative regulation of NLRP3 inflammasome.

Expression of the NLRP3 inflammasome in cerebral cortex after traumatic brain injury in a rat model.

Inflammatory response plays an important role in the pathogenesis of secondary damage after traumatic brain injury (TBI). The inflammasome is a multiprotein complex involved in innate immunity and a number of studies have suggested that the inflammasome plays a critical role in a host inflammatory signaling. Nucleotide-binding domain, leucine-rich repeat, pyrin domain containing 3 (NLRP3) is a key component of the NLRP3-inflammasome, which also includes apoptotic speck-containing protein (ASC) with a cysteine protease (caspase)-activating recruitment domain and pro-caspase1. Activation of the NLRP3-inflammasome causes the processing and release of the interleukin 1 beta (IL-1ß) and interleukin 18 (IL-18). Based on this, we hypothesized that the NLRP3-inflammasome could participate in the inflammatory response following TBI. However, the expression of NLRP3-inflammasome in cerebral cortex after TBI is not well known. Rats were randomly divided into control, sham and TBI groups (including 6 h, 1 day, 3 day and 7 day sub-group). TBI model was induced, and animals were sacrificed at each time point respectively. The expression of NLRP3-inflammasome was measured by quantitative real-time polymerase chain reaction, western blot and immunohistochemistry respectively. Immunofluorescent double labeling was performed to identify the cell types of NLRP3-inflammasome's expression. Moreover, enzyme linked immunosorbent assay was used to detect the alterations of IL-1ß and IL-18 at each time point post-injury. The results showed that, TBI could induce assembly of NLRP3-inflammasome complex, increased expression of ASC, activation of caspase1, and processing of IL-1ß and IL-18. These results suggested that NLRP3-inflammasome might play an important role in the inflammation induced by TBI and could be a target for TBI therapy.

NLRP3 inflammasomes show high expression in aorta of patients with atherosclerosis.

OBJECTIVES: The aim of the present study was to investigate the expression of NLRP3 in aorta of patients with coronary atherosclerosis and to explore the association between aortic expression levels of NLRP3 and atherosclerotic risk factors. METHODS: We collected small pieces of ascending aorta from 36 patients undergoing coronary artery bypass graft (CABG) surgery, and the arterial tissues from 10 subjects without atherosclerosis through the kidney donation program were taken as control. The expression of NLRP3 of the research and control group was determined by immunohistochemistry. Gensini score was used to evaluate the severity of coronary atherosclerosis. RESULTS: NLRP3 was strongly expressed in aorta of CABG patients. The aortic NLRP3 expression was elevated in patients with hypertension or diabetes, and smokers. The NLRP3 expression in aorta was positively correlated with total cholesterol, low density lipoprotein cholesterol, and lipoprotein(a) (P<0.05); but negatively correlated with high density lipoprotein cholesterol (P<0.05). Spearman correlation revealed that aortic NLRP3 expression had significant correlation with Gensini coronary severity scores (P<0.05). CONCLUSIONS: NLRP3 was overexpressed in aorta of patients with coronary atherosclerosis and the aortic NLRP3 expression is correlated with the severity of coronary artery disease and the atherosclerotic risk factors.

Silica induces NLRP3 inflammasome activation in human lung epithelial cells.

BACKGROUND: In myeloid cells the inflammasome plays a crucial role in innate immune defenses against pathogen- and danger-associated patterns such as crystalline silica. Respirable mineral particles impinge upon the lung epithelium causing irreversible damage, sustained inflammation and silicosis. In this study we investigated lung epithelial cells as a target for silica-induced inflammasome activation. METHODS: A human bronchial epithelial cell line (BEAS-2B) and primary normal human bronchial epithelial cells (NHBE) were exposed to toxic but nonlethal doses of crystalline silica over time to perform functional characterization of NLRP3, caspase-1, IL-1ß, bFGF and HMGB1. Quantitative RT-PCR, caspase-1 enzyme activity assay, Western blot techniques, cytokine-specific ELISA and fibroblast (MRC-5 cells) proliferation assays were performed. RESULTS: We were able to show transcriptional and translational upregulation of the components of the NLRP3 intracellular platform, as well as activation of caspase-1. NLRP3 activation led to maturation of pro-IL-1ß to secreted IL-1ß, and a significant increase in the unconventional release of the alarmins bFGF and HMGB1. Moreover, release of bFGF and HMGB1 was shown to be dependent on particle uptake. Small interfering RNA experiments using siNLRP3 revealed the pivotal role of the inflammasome in diminished release of pro-inflammatory cytokines, danger molecules and growth factors, and fibroblast proliferation. CONCLUSION: Our novel data indicate the presence and functional activation of the NLRP3 inflammasome by crystalline silica in human lung epithelial cells, which prolongs an inflammatory signal and affects fibroblast proliferation, mediating a cadre of lung diseases.

Dental infection of Porphyromonas gingivalis exacerbates high fat diet-induced steatohepatitis in mice.

BACKGROUND: We investigated the effects of dental infection with Porphyromonas gingivalis (P.g.), an important periodontal pathogen, on NASH progression, by feeding mice a high fat diet (HFD)and examining P.g. infection in the liver of NASH patients. METHODS: C57BL/6J mice were fed either chow-diet (CD) or HFD for 12 weeks, and then half of the mice in each group were infected with P.g. from the pulp chamber (HFD-P.g.(-), HFD-P.g.(+), CD-P.g.(-) and CD-P.g.(+)). Histological and immunohistochemical examinations, measurement of serum lipopolysaccharide (LPS) levels and ELISA for cytokines in the liver were performed. We then studied the effects of LPS from P.g. (P.g.-LPS) on palmitate-induced steatotic hepatocytes in vitro, and performed immunohistochemical detection of P.g. in liver biopsy specimens of NASH patients. RESULTS: Serum levels of LPS are upregulated in P.g.(+) groups. Steatosis of the liver developed in HFD groups, and foci of Mac2-positive macrophages were prominent in HFD-P.g.(+). P.g. was detected in Kupffer cells and hepatocytes. Interestingly, areas of fibrosis with proliferation of hepatic stellate cells and collagen formation were only observed in HFD-P.g.(+). In steatotic hepatocytes, expression of TLR2, one of the P.g.-LPS receptors, was upregulated. P.g.-LPS further increased mRNA levels of palmitate-induced inflammasome and proinflammatory cytokines in steatotic hepatocytes. We demonstrated for the first time that P.g. existed in the liver of NASH patients with advanced fibrosis. CONCLUSIONS: Dental infection of P.g. may play an important role in NASH progression through upregulation of the P.g.-LPS-TLR2 pathway and activation of inflammasomes. Therefore, preventing and/or eliminating P.g. infection by dental therapy may have a beneficial impact on management of NASH.

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.

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.