NOD-like Receptor Family Pyrin Domain Containing 6 Research Articles

A mouse model to distinguish NLRP6-mediated inflammasome-dependent and -independent functions

The NOD-like receptor (NLR) family pyrin domain containing 6 (NLRP6) serves as a sensor for microbial dsRNA or lipoteichoic acid (LTA) in intestinal epithelial cells (IECs), and initiating multiple pathways including inflammasome pathway and type I interferon (IFN) pathway, or regulating nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. NLRP6 can exert its function in both inflammasome-dependent and inflammasome-independent manners. However, there is no tool to distinguish the contribution of individual NLRP6-mediated pathway to the physiology and pathology in vivo. Here, we validated that Arg39 and Trp50 residues in the pyrin domain (PYD) of murine NLRP6 are required for ASC recruitment and inflammasome activation, but are not important for the RNA binding and PYD-independent NLRP6 oligomerization. We further generated the Nlrp6R39E&W50E mutant mice, which showed reduced inflammasome activation in either steady state intestine or during viral infection. However, the type I IFN production in cells or intestine tissue from Nlrp6R39E&W50E mutant mice remain normal. Interestingly, NLRP6-mediated inflammasome activation or the IFN-I production seems to play distinct roles in the defense responses against different types of RNA viruses. Our work generated a useful tool to study the inflammasome-dependent role of NLRP6 in vivo, which might help to understand the complexity of multiple pathways mediated by NLRP6 in response to the complicated and dynamic environmental cues in the intestine.

Spotlight on NLRP6 and Tumor Research Situation: A Potential Cancer Participant.

NOD-like receptor family pyrin domain containing 6 (NLRP6) is a new pattern recognition receptor in the mammalian innate immune system. Both the liver and the gut exhibit substantial levels of cytoplasmic expression. It can speed up cell response to endogenous danger signals or exogenous pathogen infection. NLRP6 can function in various ways as an inflammasome or a noninflammasome. The understanding of NLRP6 is steadily increasing thanks to ongoing investigations, but due to discrepancies in how those studies have described their link with tumors, the significance of NLRP6 in the emergence of cancer is still debatable as of this writing. This article will use the structure and function of NLRP6 as the pivotal point and thoroughly explain the present interactions between NLRP6 and tumors and any possible clinical benefits.

The Milk Active Ingredient, 2'-Fucosyllactose, Inhibits Inflammation and Promotes MUC2 Secretion in LS174T Goblet Cells In Vitro.

In several mice inflammatory models, human milk oligosaccharides (HMOs) were shown to protect the intestinal barrier by promoting mucin secretion and suppressing inflammation. However, the functions of the individual HMOs in enhancing mucin expression in vivo have not been compared, and the related mechanisms are not yet to be clarified. In this study, we investigated the modulatory effects of 2′-fucosyllactose (2′-FL), 3′-sialyllactose (3′-SL), galacto-oligosaccharide (GOS) and lactose (Lac) on goblet cells’ functions in vitro. The appropriate dosage of the four chemicals was assessed in LS174T cells using the CCK-8 method. Then they were supplemented into a homeostasis and inflammatory environment to further investigate their effects under different conditions. Mucin secretion-related genes, including mucin 2 (MUC2), trefoil factor family 3 (TFF3), resistin-like β (RETNLB), carbohydrate sulfotransferase 5 (CHST5) and galactose-3-O-sulfotransferase 2 (GAL3ST2), in LS174T cells were detected using quantitative RT-qPCR. The results showed that 2′-FL (2.5 mg/mL, 72 h) was unable to increase MUC2 secretion in a steady-state condition. Comparatively, it exhibited a greater ability to improve mucin secretion under an inflammatory condition compared with GOS, demonstrated by a significant increase in TFF3 and CHST5 mRNA expression levels (p > 0.05). However, 3′-SL and Lac exhibited no effects on mucin secretion. To further investigate the underlying mechanism via which 2′-FL enhanced goblet cells’ secretion function, the NOD-like receptor family pyrin domain containing 6 (NLRP6) gene, which is closely related to MUC2 secretion, was silenced using the siRNA method. After silencing the NLRP6 gene, the mRNA expression levels of MUC2, TFF3 and CHST5 in the (2′-FL + tumor necrosis factor α (TNF-α) + NLRP6 siRNA) group were significantly decreased compared with the (2′-FL + TNF-α) group (p > 0.05), indicating that NLRP6 was essential for MUC2 expression in goblet cells. We further found that 2′-FL could significantly decrease toll-like receptor 4 (TLR4, p < 0.05), myeloid differential protein-88 (MyD88, p < 0.05) and nuclear factor kappa-B (NF-κB, p < 0.05) levels in LS174T inflammatory cells, even when the NLRP6 was silenced. Altogether, these results indicated that in goblet cells, 2′-FL exerts its function via multiple processes, i.e., by promoting mucin secretion through NLRP6 and suppressing inflammation by inhibiting the TLR4/MyD88/NF-κB pathway.

Estrogen receptor β activation inhibits colitis by promoting NLRP6-mediated autophagy.

Estrogen receptor β (ERβ) and NOD-like receptor family pyrin domain containing 6 (NLRP6) are highly expressed in intestinal tissues. Loss of ERβ and NLRP6 exacerbate colitis in mouse models; however, the underlying mechanisms are incompletely understood. Here, we report that ERβ directly activates the NLRP6 gene expression via binding to estrogen responsive element of Nlrp6 gene promoter. ERβ also physically interacts with the NLRP6 nucleotide-binding domain and promotes NLRP6 inflammasome assembly. The ERβ-NLRP6 axis then interacts with multiple autophagy-related proteins, including ULK1, BECN1, ATG16L1, LC3B, and p62, and affects the autophagosome biogenesis and autophagic flux. Finally, NLRP6-mediated autophagy suppresses the inflammatory response by promoting the K48-linked polyubiquitination of ASC, Casp-1 p20, IL-1β, TNF-α, and prohibitin-2. Thus, ERβ-NLRP6 direct an anti-inflammatory response by promoting autophagy. Our work uncovers an ERβ-NLRP6-autophagy pathway as a regulatory mechanism that maintains intestinal epithelial cell homeostasis and facilitates tissue repair in colitis.

Qing-Chang-Hua-Shi granule ameliorates DSS-induced colitis by activating NLRP6 signaling and regulating Th17/Treg balance

BackgroundChinese herbal medicine Qing-Chang-Hua-Shi granule (QCHS) is widely used to treat ulcerative colitis in China. However, the molecular mechanisms of QCHS remains largely unknown. PurposeTo assess the therapeutic effects of QCHS on colitis and to reveal its mechanisms of action. MethodsThe main components of QCHS were identified using a UHPLC-QTOF-MS method and the efficacy of QCHS was evaluated using an DSS-induced mice model. The inflammatory responses and mucosal integrity in colon were comprehensively assessed. Flow cytometry was used to analysis the proportion of Th17 and Treg cells. Detect the signal transduction of the NOD-like receptor family pyrin domain containing 6 (NLRP6) both in vitro and in vivo. Furthermore, siNLRP6 transfection was used to validate the functional targets of QCHS. ResultsQCHS treatment significantly alleviated colitis in mice by improving symptoms and pathological damage. Moreover, QCHS treatment suppressed the inflammatory response and preserved the integrity of colon tissue. Most importantly, QCHS balanced the Th17/Treg response of UC mice. Mechanistically, by activating NLRP6 inflammasome pathway, QCHS regulated the maturation of interleukin (IL)-1β and IL-18 to affect inflammation and drive Th17 cell differentiation. ConclusionsThe effect of QCHS on UC mice is dose-dependent, with high-dose QCHS being superior to 5-Aminosalicylic acid (200 mg/kg/day). QCHS acts through the NLRP6 signaling pathway to modulate Th17/Treg balance, resulting in the protective effects against colitis. This study investigated the relevant pharmacological mechanisms of QCHS, providing further evidence for the application of QCHS in UC treatment.

NLRP6 negatively regulates type 2 immune responses in mice.

Inflammasomes are large protein complexes that assemble in the cytosol in response to danger such as tissue damage or infection. Following activation, inflammasomes trigger cell death and the release of biologically active forms of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. NOD-like receptor family pyrin domain containing 6 (NLRP6) inflammasome is required for IL-18 secretion by intestinal epithelial cells, macrophages, and T cells, contributing to homeostasis and self-defense against pathogenic microbes. However, the involvement of NLRP6 in type 2 lung inflammation remains elusive. Wild-type (WT) and Nlrp6-/- mice were used. Birch pollen extract (BPE)-induced allergic lung inflammation, eosinophil recruitment, Th2-related cytokine and chemokine production, airway hyperresponsiveness, and lung histopathology, Th2 cell differentiation, GATA3, and Th2 cytokines expression, were determined. Nippostrongylus brasiliensis (Nb) infection, worm count in intestine, type 2 innate lymphoid cell (ILC2), and Th2 cells in lungs were evaluated. We demonstrate in Nlrp6-/- mice that a mixed Th2/Th17 immune responses prevailed following birch pollen challenge with increased eosinophils, ILC2, Th2, and Th17 cell induction and reduced IL-18 production. Nippostrongylus brasiliensis infected Nlrp6-/- mice featured enhanced early expulsion of the parasite due to enhanced type 2 immune responses compared to WT hosts. In vitro, NLRP6 repressed Th2 polarization, as shown by increased Th2 cytokines and higher expression of the transcription factor GATA3 in the absence of NLRP6. Exogenous IL-18 administration partially reduced the enhanced airways inflammation in Nlrp6-/- mice. In summary, our data identify NLRP6 as a negative regulator of type 2 immune responses.

NLRP6 Serves as a Negative Regulator of Neutrophil Recruitment and Function During Streptococcus pneumoniae Infection.

Streptococcus pneumoniae is an invasive pathogen with high morbidity and mortality in the immunocompromised children and elderly. NOD-like receptor family pyrin domain containing 6 (NLRP6) plays an important role in the host innate immune response against pathogen infections. Our previous studies have shown that NLRP6 plays a negative regulatory role in host defense against S. pneumoniae, but the underlying mechanism is still unclear. The further negative regulatory role of NLRP6 in the host was investigated in this study. Our results showed that NLRP6−/− mice in the lung had lower bacterial burdens after S. pneumoniae infection and expressed higher level of tight junction (TJ) protein occludin compared to WT mice, indicating the detrimental role of NLRP6 in the host defense against S. pneumoniae infection. Transcriptome analysis showed that genes related to leukocytes migration and recruitment were differentially expressed between wild-type (WT) and NLRP6 knockout (NLRP6−/−) mice during S. pneumoniae infection. Also, NLRP6−/− mice showed higher expression of chemokines including C-X-C motif chemokine ligand 1 (CXCL1) and 2 (CXCL2) and lower gene expression of complement C3a receptor 1 (C3aR1) and P-selectin glycoprotein ligand-1 (PSGL-1) which are the factors that inhibit the recruitment of neutrophils. Furthermore, NLRP6−/− neutrophils showed increased intracellular bactericidal ability and the formation of neutrophil extracellular traps (NETs) during S. pneumoniae infection. Taken together, our study suggests that NLRP6 is a negative regulator of neutrophil recruitment and function during S. pneumoniae infection. Our study provides a new insight to develop novel strategies to treat invasive pneumococcal infection.

2'-Fucosyllactose Ameliorates Inflammatory Bowel Disease by Modulating Gut Microbiota and Promoting MUC2 Expression.

Gut microbiota dysbiosis, together with goblet cells dysfunction has been observed in ulcerative colitis cases. This study aims to evaluate the potential of 2′-fucosyllactose (2′-FL) supplementation in inhibiting intestinal inflammation through regulating gut microbiota, protecting goblet cells, and stimulating mucin secretion. 2′-FL was orally administered to C57BL/6J mice daily (400 mg/kg bw) for 21 days and 5% dextran sulfate sodium (DSS) was used to induce the colitis in the last 7 days. Meanwhile, fecal microbiota transplantation (FMT) was conducted to test the roles of gut microbiota in the remission of colitis by 2′-FL. Gut microbiota alteration was analyzed through 16S ribosomal RNA (16S rRNA) sequencing. Periodic acid-Schiff (PAS), immunofluorescence staining, as well as mucin 2 (MUC2) and NOD-like receptor family pyrin domain containing 6 (NLRP6) messenger RNA (mRNA) expression in colon fragments was performed and detected. The results showed that the DSS + 2′-FL mice were found to have a slower rate of weight loss, lower disease activity index (DAI) scores, and longer colon lengths than the DSS group (p < 0.05), so in the FMT recipient mice which received fecal microbiota from the DSS + 2′-FL group. In addition, the data revealed that 2′-FL relieved the disorder of DSS-induced gut microbiota, including decreasing the high abundance of mucin-utilizing bacteria in the DSS group, such as Bacteroides, Lachnospiraceae NK4A136, Lachnospiraceae, and Bacteroides vulgatus. PAS and immunofluorescence staining showed that 2′-FL treatment promoted the recovery of goblet cells and enhanced MUC2 and NLRP6 expression, which was also observed in the FM (DSS + 2′-FL) group. Moreover, NLRP6, which has been proved to be a negative regulator for Toll-like receptor 4/myeloid differential protein-8/nuclear factor-kappa B (TLR4/MyD88/NF-κB) pathway, was upregulated by 2′-FL in colon tissue. In conclusion, this study suggests that 2′-FL ameliorates colitis in a gut microbiota-dependent manner. The underlying protective mechanism associates with the recovery of goblet cells number and improves MUC2 secretion through TLR4-related pathway.

BRCC3 promotes activation of the NLRP6 inflammasome following cerebral ischemia/reperfusion (I/R) injury in rats

NOD-like receptor family pyrin domain containing 6 (NLRP6), a novel member of the NLR family, has been confirmed to have an inflammasome-dependent proinflammatory effect in cerebral ischemia/reperfusion injury. NLRP6 assembles a multimeric inflammasome complex comprising the adaptor ASC and the effector pro-caspase-1 to mediate the activation of caspase-1. The molecular mechanism regulating activation of the NLRP6 inflammasome remains unclear. Previous studies have shown that BRCA1-BRCA2-containing complex subunit 3 (BRCC3), a JAMM domain-containing Zn2+ metalloprotease deubiquitinating enzyme, participates in a variety of cellular activities. In this study, we found that BRCC3 expression was increased in the middle cerebral artery occlusion (MCAO) model. BRCC3 siRNA could reduce nerve damage and inflammation. Interestingly, the result of co-immunoprecipitation showed that the interaction between BRCC3 and NLRP6 was enhanced after model, and the result of immunofluorescence showed that the co-localization of BRCC3 and NLRP6 was increased. At the same time, the expression of NLRP6, cleavated-caspase-1 and IL-1β was decreased after BRCC3 interference. These results illustrate a regulatory mechanism involving the BRCC3-NLRP6 pathway and highlight NLRP6 as a potential therapeutic target for inflammatory diseases.

The intestinal parasite Cryptosporidium is controlled by an enterocyte intrinsic inflammasome that depends on NLRP6.

The apicomplexan parasite Cryptosporidium infects the intestinal epithelium. While infection is widespread around the world, children in resource-poor settings suffer a disproportionate disease burden. Cryptosporidiosis is a leading cause of diarrheal disease, responsible for mortality and stunted growth in children. CD4 T cells are required to resolve this infection, but powerful innate mechanisms control the parasite prior to the onset of adaptive immunity. Here, we use the natural mouse pathogen Cryptosporidium tyzzeri to demonstrate that the inflammasome plays a critical role in initiating this early response. Mice lacking core inflammasome components, including caspase-1 and apoptosis-associated speck-like protein, show increased parasite burden and caspase 1 deletion solely in enterocytes phenocopies whole-body knockout (KO). This response was fully functional in germfree mice and sufficient to control Cryptosporidium infection. Inflammasome activation leads to the release of IL-18, and mice that lack IL-18 are more susceptible to infection. Treatment of infected caspase 1 KO mice with recombinant IL-18 is remarkably efficient in rescuing parasite control. Notably, NOD-like receptor family pyrin domain containing 6 (NLRP6) was the only NLR required for innate parasite control. Taken together, these data support a model of innate recognition of Cryptosporidium infection through an NLRP6-dependent and enterocyte-intrinsic inflammasome that leads to the release of IL-18 required for parasite control.

The NLRP6 inflammasome.

The NOD-like receptor family pyrin domain containing 6 (NLRP6), a member of the NOD-like receptor (NLR) family, acts as a cytosolic innate immune sensor that recognizes microbe-associated molecular patterns. In some circumstances upon activation, NLRP6 recruits the adaptor apoptosis-associated speck-like protein (ASC) and the inflammatory caspase-1 or caspase-11 to form an inflammasome, which mediates the maturation and secretion of the pro-inflammatory cytokines IL-18 and IL-1β. In other contexts, NLRP6 can exert its function in an inflammasome-independent manner. Tight regulation of the NLRP6 inflammasome is critical in maintaining tissue homeostasis, while improper inflammasome activation may contribute to the development of multiple diseases. In intestinal epithelial cells, the NLRP6 inflammasome is suggested to play a role in regulating gut microbiome composition, goblet cell function and related susceptibility to gastrointestinal inflammatory, infectious and neoplastic diseases. Additionally, NLRP6 may regulate extra-intestinal diseases. In this review, we summarize current knowledge on the NLRP6 inflammasome and its activation and regulation patterns, as well as its effector functions contributing to disease modulation. We discuss current challenges in NLRP6 research and future prospects in harnessing its function into potential human interventions.

NLRP6 Regulates CD4 Mediated Graft-Versus-Host Disease in Experimental Murine BMT

NLRP6 (NOD-like receptor family pyrin domain containing 6) is an important inflammasome component and is highly expressed in intestinal epithelial and immune cells. NLRP6 mediated inflammasome activation plays a critical role in response to intestinal infection and preventing dysbiosis of gut microbiota. However, we recently found that NLRP6 plays a pathogenic role in GVHD. Interestingly, we also found that activated T cells increased NLRP6 expression, but the T cell autonomous role of NLRP6 in regulating T cell responses is unknown. Because NLRP6 is an important regulator of GVH responses, we tested the hypothesis that NLRP6 deficiency in donor T cells would ameliorate GVHD. To test our hypothesis, we first performed a detailed phenotypic analysis of various T cell subsets and activation markers in naive NLRP6−/− and wild-type (WT) B6 animals and found a similar distribution of naive, memory, effector and regulatory T cells. In order to examine whether the absence of NLRP6 in donors affects GVHD, WT-BALB/c animals were lethally irradiated and transplanted on day 0 with 5 × 106 bone marrow and 1.0 × 106 splenic CD90+T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or NLRP6−/− animals. Contrary to our hypothesis, the recipients receiving donor T cells from NLRP6−/− animals showed a significantly worse survival compared to allogeneic WT-B6 animals (p We hypothesized that GVHD severity and mortality was similar in the B6 into C3H.sw model because NLRP6 regulates CD4+ and CD8+ T cell responses, differently. In order to test this, we transplanted C3H.sw recipients as above except we infused either 1 × 106 CD4+ or CD8+ T cells from B6-WT or NLRP6−/− animals. GVHD severity and mortality (P To explore how NLRP6 effects T cell responses independent of inflammasome activation, we tested naive T cell proliferation in vitro after allogeneic or non-specific TCR stimulation by anti-CD3 and CD28 antibody and found that NLRP6−/− CD4+ but not CD8+ T cells proliferated more than WT-B6 CD4+ or CD8+ T cells, respectively, following either stimulus. In addition, activated NLRP6−/− CD4+ T cells showed less apoptosis than WT. Absence of NLRP6 in T cells promotes naive CD4 T cells into Th1 cell differentiation. These data suggest that NLRP6 regulates CD4+ T cell mediated immune responses and that NLRP6 in donor T cells is critical for controlling CD4+ T cell mediated GVHD.

NLRP6 in Donor T Cells Separately Regulates CD4 and CD8 Mediated Graft-Versus-Host Disease in Experimental Murine BMT

NLRP6 (NOD-like receptor family pyrin domain containing 6) is an important inflammasome component and is highly expressed in intestinal epithelial and in immune cells. NLRP6 mediated inflammasome activation plays a critical role in response to intestinal infection and preventing dysbiosis of gut microbiota through the secretion of IL-18 and mucus. However, we recently found that NLRP6 plays a pathogenic role in GVHD that is independent of microbial dysbiosis, which is in contrast to its well-appreciated microbiome-dependent protective role in intestinal colitis and tumorigenesis. Interestingly, we also found that activated T cells increased NLRP6 expression, but the T cell autonomousrole of NLRP6 in regulating T cell responses is unknown. Because NLRP6 is an important regulator of GVH responses, we tested the hypothesis that NLRP6 deficiency in donor T cells would ameliorate GVHD. To test our hypothesis, we first performed adetailed phenotypic analysis of various T cell subsets and activation markers in naïve NLRP6-/-and wild-type (WT) B6 animals and found a similar distribution of naïve, memory, effector and regulatory T cells. In order to examine whether the absence of NLRP6 in donors affects GVHD, WT-BALB/canimals were lethally irradiated (700cGy) and transplanted on day 0 with 5x106bone marrow and 1.0x106 splenic CD90+T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or NLRP6-/-animals. Contrary to our hypothesis, the recipients receiving donor T cells from NLRP6-/-animals showed a significantly worse survival compared to allogeneic WT-B6 animals (p&lt;0.05). GVHD mortality and severity were also increased in an MHC mismatched B6 into B10.BR model, and in an MHC mismatched haploidentical B6 into F1model (p&lt;0.05). In contrast, GVHD severity and mortality were similar in an MHC matched multiple minor antigen mismatched B6 into C3H.sw model. We hypothesized that GVHD severity and mortality was similar in the B6 into C3H.sw model because NLRP6 regulates CD4+ and CD8+ T cell responses, differently. In order to test this, we transplanted C3H.sw recipients as above except we infused either 1x106CD4+ or CD8+ T cells from B6-WT or NLRP6-/-animals. GVHD severity and mortality (P&lt;0.05) were enhanced only when NLRP6-/-CD4+ T cells transplanted. We confirmed enhanced GVHD mortality and severity mediated by donor NLRP6-/-CD4+ T cells in a second MHC-mismatched GVHD model, B6 into BALB/c (p&lt;0.05). To explore how NLRP6 effects T cell responses independent ofinflammasome activation, we tested naïve T cell proliferation in vitro after allogeneic or non-specific TCR stimulation by anti-CD3 and CD28 antibody and found that NLRP6-/-CD4+ but not CD8+T cells proliferated more than WT-B6 CD4+ or CD8+ T cells, respectively, following either stimulus. Furthermore, allogeneicNLRP6-/-T cells also caused greater mortality compared to WT allogenic T cells in a non-irradiated B6 into F1 model, which lacks inflammasome activation associated with conditioning induced DAMPs and PAMPs. Microarray analysis of activated T cells from NLRP6-/-animals showed higher expression of IL-2 and IFN-γ than WT B6 T cells, and we observed no effect of NLRP6 in a Treg suppression assay. These data suggest that NLRP6 regulates CD4+ T cell- mediated immune responses and that NLRP6 in donor T cells is critical for controlling CD4+ T cell mediated GVHD. The effect of NLRP6 on T cell mediated GVL is currently under investigation. Disclosures Tawara: Kyowa Hakko Kirin: Honoraria, Research Funding; Ono Pharmaceutical: Research Funding; Astellas Pharma: Research Funding. Ishizawa:Otsuka Pharmaceutical: Research Funding; Pfizer: Research Funding; Novartis: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau.

Effect of mild moxibustion on intestinal microbiota and NLRP6 inflammasome signaling in rats with post-inflammatory irritable bowel syndrome.

BACKGROUNDAbout one-third of refractory irritable bowel syndrome (IBS) cases are caused by gastrointestinal (GI) infection/inflammation, known as post-infectious/post-inflammatory IBS (PI-IBS). Although it is known that intestinal microbiota and host NOD-like receptor family pyrin domain containing 6 (NLRP6) inflammsome signaling are closely related to PI-IBS and moxibustion has a therapeutic effect on PI-IBS, whether moxibustion regulates the intestinal flora and host NLRP6 events in PI-IBS remains unclear.AIMTo examine the regulatory effect of moxibustion on intestinal microbiota and host NLRP6 inflammatory signaling in PI-IBS.METHODSSprague-Dawley rats were divided into a normal control group, a model control group, a mild moxibustion group, and a sham mild moxibustion group. PI-IBS rats in the mild moxibustion group were treated with moxibusiton at bilateral Tianshu (ST 25) and Zusanli (ST36) for 7 consecutive days for 10 min each time. The sham group rats were given the same treatment as the mild moxibustion group except the moxa stick was not ignited. Abdominal withdrawal reflex (AWR) score was measured to assess the visceral sensitivity, and colon histopathology and ultrastructure, colonic myeloperoxidase (MPO) activity, and serum C-reactive protein (CRP) level were measured to evaluate low-grade colonic inflammation in rats. The relative abundance of selected intestinal bacteria in rat feces was detected by 16S rDNA PCR and the NLRP6 inflammsome signaling in the colon was detected by immunofluorescence, qRT-PCR, and Western blot.RESULTSThe AWR score was significantly decreased and the low-grade intestinal inflammation reflected by serum CRP and colonic MPO levels was inhibited in the mild moxibustion group compared with the sham group. Mild moxibustion remarkably increased the relative DNA abundances of Lactobacillus, Bifidobacterium, and Faecalibacterium prausnitzii but decreased that of Escherichia coli in the gut of PI-IBS rats. Additionally, mild moxibustion induced mRNA and protein expression of intestine lectin 1 but inhibited the expression of IL-1β, IL-18, and resistance-like molecule β by promoting the NLRP6 and reducing the mRNA and protein expression of apoptosis-associated speck-like protein containing CARD (ASC) and cysteinyl-aspartate-specific proteinase 1 (Caspase-1). The relative DNA abundances of Lactobacillus, Bifidobacteria, Faecalibacterium prausnitzii, and Escherichia coli in each group were correlated with the mRNA and protein expression of NLRP6, ASC, and Caspase-1 in the colon.CONCLUSIONThese findings indicated that mild moxibustion can relieve low-grade GI inflammation and alleviate visceral hypersensitivity in PI-IBS by regulating intestinal microbes and controlling NLRP6 inflammasome signaling.

Effect of mild moxibustion on intestinal microbiota and NLRP6 inflammasome signaling in rats with post-inflammatory irritable bowel syndrome

BACKGROUND About one-third of refractory irritable bowel syndrome (IBS) cases are caused by gastrointestinal (GI) infection/inflammation, known as post-infectious/post-inflammatory IBS (PI-IBS). Although it is known that intestinal microbiota and host NOD-like receptor family pyrin domain containing 6 (NLRP6) inflammsome signaling are closely related to PI-IBS and moxibustion has a therapeutic effect on PI-IBS, whether moxibustion regulates the intestinal flora and host NLRP6 events in PI-IBS remains unclear.

Dietary fructose-induced gut dysbiosis promotes mouse hippocampal neuroinflammation: a benefit of short-chain fatty acids

BackgroundWestern-style diets arouse neuroinflammation and impair emotional and cognitive behavior in humans and animals. Our previous study showed that a high-fructose diet caused the hippocampal neuroinflammatory response and neuronal loss in animals, but the underlying mechanisms remained elusive. Here, alterations in the gut microbiota and intestinal epithelial barrier were investigated as the causes of hippocampal neuroinflammation induced by high-fructose diet.ResultsA high-fructose diet caused the hippocampal neuroinflammatory response, reactive gliosis, and neuronal loss in C57BL/6N mice. Depletion of the gut microbiota using broad-spectrum antibiotics suppressed the hippocampal neuroinflammatory response in fructose-fed mice, but these animals still exhibited neuronal loss. Gut microbiota compositional alteration, short-chain fatty acids (SCFAs) reduction, intestinal epithelial barrier impairment, NOD-like receptor family pyrin domain-containing 6 (NLRP6) inflammasome dysfunction, high levels of serum endotoxin, and FITC-dextran were observed in fructose-fed mice. Of note, SCFAs, as well as pioglitazone (a selective peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist), shaped the gut microbiota and ameliorated intestinal epithelial barrier impairment and NLRP6 inflammasome dysfunction in fructose-fed mice. Moreover, SCFAs-mediated NLRP6 inflammasome activation was inhibited by histamine (a bacterial metabolite) in ex vivo colonic explants and suppressed in murine CT26 colon carcinoma cells transfected with NLRP6 siRNA. However, pioglitazone and GW9662 (a PPAR-γ antagonist) exerted no impact on SCFAs-mediated NLRP6 inflammasome activation in ex vivo colonic explants, suggesting that SCFAs may stimulate NLRP6 inflammasome independently of PPAR-γ activation. SCFAs and pioglitazone prevented fructose-induced hippocampal neuroinflammatory response and neuronal loss in mice. Additionally, SCFAs activated colonic NLRP6 inflammasome and increased DCX+ newborn neurons in the hippocampal DG of control mice.ConclusionsOur findings reveal that gut dysbiosis is a critical factor for a high-fructose diet-induced hippocampal neuroinflammation in C57BL/6N mice possibly mediated by impairing intestinal epithelial barrier. Mechanistically, the defective colonic NLRP6 inflammasome is responsible for intestinal epithelial barrier impairment. SCFAs can stimulate NLRP6 inflammasome and ameliorate the impairment of intestinal epithelial barrier, resulting in the protection against a high-fructose diet-induced hippocampal neuroinflammation and neuronal loss. This study addresses a gap in the understanding of neuronal injury associated with Western-style diets. A new intervention strategy for reducing the risk of neurodegenerative diseases through SCFAs supplementation or dietary fiber consumption is emphasized.

Host NLRP6 exacerbates graft-versus-host disease independent of gut microbial composition.

Host NOD-like receptor family pyrin domain-containing 6 (NLRP6) regulates innate immune responses and gastrointestinal homeostasis. Its protective role in intestinal colitis and tumorigenesis is dependent on the host microbiome. Host innate immunity and microbial diversity also play a role in the severity of allogeneic immune-mediated gastrointestinal graft-versus-host disease (GVHD), the principal toxicity after allogeneic haematopoietic cell transplantation. Here, we examined the role of host NLRP6 in multiple murine models of allogeneic bone marrow transplantation. In contrast to its role in intestinal colitis, host NLRP6 aggravated gastrointestinal GVHD. The impact of host NLRP6 deficiency in mitigating GVHD was observed regardless of co-housing, antibiotic treatment or colonizing littermate germ-free wild-type and NLRP6-deficient hosts with faecal microbial transplantation from specific pathogen-free wild-type and Nlrp6-/- animals. Chimaera studies were performed to assess the role of NLRP6 expression on host haematopoietic and non-haematopoietic cells. The allogeneic [B6Ly5.2 → Nlrp6-/-] animals demonstrated significantly improved survival compared to the allogeneic [B6Ly5.2 → B6] animals, but did not alter the therapeutic graft-versus-tumour effects after haematopoietic cell transplantation. Our results unveil an unexpected, pathogenic role for host NLRP6 in gastrointestinal GVHD that is independent of variations in the intestinal microbiome and in contrast to its well-appreciated microbiome-dependent protective role in intestinal colitis and tumorigenesis.

NLRP6 in Host Target Tissues Exacerbates Graft-Versus-Host Disease

Intestinal epithelial cells play an important role in the maintenance of host microbiota as well as intestinal homeostasis by expression of pattern recognition receptors (PRRs). NLRP6 (NOD-like receptor family pyrin domain containing 6) is an important inflammasome component and is highly expressed on the intestinal epithelium and in immune cells. NLRP6 mediated inflammasome activation plays a critical role in intestinal infection and in preventing dysbiosis of gut normal microbiota thorough the secretion of IL-18 and mucus. In addition, NLRP6 knock out (NLRP6-/-) mice show greater severity and mortality of inflammatory bowel diseases (IBD). Given its unique functions in GI tract and in immune cells, we determined its role in the severity of GI GVHD by testing the hypothesis that the absence of the NLRP6 in hosts would enhance GVHD.We utilized the well-characterized MHC disparate BALB/c→B6 model of GVHD. B6 wild type (WT) and B6 NLRP6-/- animals were lethally irradiated (10Gy) and transplanted with 5x106 BM and 3x106 splenic T-cells from either syngeneic WT-B6 or allogeneic BALB/c donors. Contrary to the hypothesis and in contrast to IBD model, allogeneic B6 NLRP6-/- animals showed significantly improved survival compared to the allo-B6 WT animals (p<0.01). The decrease in mortality was associated with reduced GVHD specific clinical severity and histopathology of the GI tract (p<0.05). Similar results were obtained in a MHC matched, minor antigen mismatched C3H.sw into B6 model for eliminating strain dependent artifacts. Despite the reduction in GVHD mortality and GI damage, donor T-cells demonstrated equivalent expansion and cytokine secretion (IFNγ, IL-17 and TNFα) in both allogeneic WT and NLRP6-/- animals (days 7, 14, and 21) both in the spleen and in the GI tract. Similar recruitment of donor derived CD11b+ cells in the gut (day21) was observed in both the allo-groups. The macrophages and the dendritic cells from the WT and NLRP6-/- B6 animals showed similar allogeneic T cell proliferation in an MLR. Next, to dissect the cause for reducing GVHD mortality and to determine whether NLRP6 expression in host hematopoietic derived cells is critical, we generated [B6→B6Ly5.2] and [NLRP6-/- B6→B6Ly5.2] chimeras and utilized them as hosts in 2nd allo-BMT and found that both the chimeras demonstrated similar severity and mortality. These data collectively demonstrate that the reduction in GI GVHD, observed in the absence of NLRP6, is independent of NLRP6 expression in host hematopoietic derived cells.Because the absence of NLRP6 could alter endogenous microbiota colonization, we co-housed the NLRP6-/- and WT B6 for 3 weeks and then utilized them as hosts in allo-BMT. Co-housed B6 NLRP6-/- animals still showed significantly improved survival and decreased GVHD severity when compared to WT B6 animals (p<0.05), suggesting that alteration of microbiota is dispensable for protection from GVHD.Because NLRP6 is highly expressed on the intestinal epithelium we next evaluated whether the reduction of GVHD is due to the absence of NLRP6 on the host non-hematopoietic cells such as the intestinal epithelium. To this end, we generated [B6→B6Ly5.2] and [B6Ly5.2→NLRP6-/-] chimeras and utilized them as hosts. The allogeneic [B6Ly5.2→NLRP6-/-] animals demonstrated significantly improved survival compared to the allogeneic [B6→B6Ly5.2] recipient animals (p<0.05). Furthermore, allo- NLRP6-/- animals showed significantly less barrier permeability and increased level of Claudin 3 and EphB3 which are critical in the repair process in intestinal epithelium (p<0.05). Interestingly, serum level of IL-18, an inflammasome-dependent cytokine, and the expression of anti (bcl-2 or bcl-xl)- or pro- apoptotic genes (Bax or Bak) in epithelium of NLRP6-/- animals after allo-BMT was equivalent to WT-B6 animals. These data suggest that the absence of NLRP6 in host non-hematopoietic cells, likely the intestinal epithelial cells, is crucial for protection from GVHD.Our results indicate that NLRP6 has various functions and has differential effects in GVHD pathogenesis in contrast to IBD where NLRP6 has protective effect. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Microbiota-induced activation of epithelial IL-6 signaling links inflammasome-driven inflammation with transmissible cancer

The microbiota is pivotal in the pathogenesis of inflammatory bowel disease (IBD)-associated inflammation-induced colorectal cancer (CRC), yet mechanisms for these effects remain poorly characterized. Here, we demonstrate that aberrant inflammasome-induced microbiota plays a critical role in CRC development, where mice deficient in the NOD-like receptor family pyrin domain containing 6 (NLRP6) inflammasome feature enhanced inflammation-induced CRC formation. Intriguingly, WT mice cohoused either with inflammasome-deficient mice or with mice lacking IL-18 feature exacerbated inflammation-induced CRC compared with singly housed WT mice. Enhanced tumorigenesis is dependent on microbiota-induced chemokine (C-C motif) ligand 5 (CCL5)-driven inflammation, which in turn promotes epithelial cell proliferation through local activation of the IL-6 pathway, leading to cancer formation. Altogether, our results mechanistically link the altered microbiota with the pathogenesis of inflammation-induced CRC and suggest that in some conditions, microbiota components may transfer CRC susceptibility between individuals.