NLRP12 attenuates colon inflammation by maintaining colonic microbial diversity and promoting protective commensal bacterial growth

Inflammatory bowel diseases involve the dynamic interplay of host genetics, microbiome and inflammatory response. Here, we report that NLRP12, a negative regulator of innate immunity, is reduced in human ulcerative colitis by comparing monozygotic twins and other patient cohorts. In parallel, Nlrp12-deficiency in mice caused increased colonic basal inflammation, leading to a less-diverse microbiome, loss of protective gut commensal strains (Lachnospiraceae) and increased colitogenic strains (Erysipelotrichaceae). Dysbiosis and colitis susceptibility associated with Nlrp12-deficency were reversed equally by treatment with antibodies targeting inflammatory cytokines or by administration of beneficial commensal Lachnospiraceae isolates. Fecal transplants from specific pathogen free reared mice into germ-free Nlrp12-deficient mice showed that NLRP12 and the microbiome each contribute to immune signaling that culminates in colon inflammation. These findings reveal a feed-forward loop where NLRP12 promotes specific commensals that can reverse gut inflammation, while cytokine blockade during NLRP12-deficiency can reverse dysbiosis

A Cell Permeable Peptide Inhibitor of NFAT Inhibits Macrophage Cytokine Expression and Ameliorates Experimental Colitis

Nuclear factor of activated T cells (NFAT) plays a critical role in the development and function of immune and non-immune cells. Although NFAT is a central transcriptional regulator of T cell cytokines, its role in macrophage specific gene expression is less defined. Previous work from our group demonstrated that NFAT regulates Il12b gene expression in macrophages. Here, we further investigate NFAT function in murine macrophages and determined the effects of a cell permeable NFAT inhibitor peptide 11R-VIVIT on experimental colitis in mice. Treatment of bone marrow derived macrophages (BMDMs) with tacrolimus or 11R-VIVIT significantly inhibited LPS and LPS plus IFN-γ induced IL-12 p40 mRNA and protein expression. IL-12 p70 and IL-23 secretion were also decreased. NFAT nuclear translocation and binding to the IL-12 p40 promoter was reduced by NFAT inhibition. Experiments in BMDMs from IL-10 deficient (Il10−/−) mice demonstrate that inhibition of IL-12 expression by 11R-VIVIT was independent of IL-10 expression. To test its therapeutic potential, 11R-VIVIT was administered systemically to Il10−/− mice with piroxicam-induced colitis. 11R-VIVIT treated mice demonstrated significant improvement in colitis compared to mice treated with an inactive peptide. Moreover, decreased spontaneous secretion of IL-12 p40 and TNF in supernatants from colon explant cultures was demonstrated. In summary, NFAT, widely recognized for its role in T cell biology, also regulates important innate inflammatory pathways in macrophages. Selective blocking of NFAT via a cell permeable inhibitory peptide is a promising therapeutic strategy for the treatment of inflammatory bowel diseases

MicroRNAs:New players in IBD

MicroRNAs (miRNAs) are small non-coding RNAs, 18–23 nucleotides long, which act as post-transcriptional regulators of gene expression. miRNAs are strongly implicated in the pathogenesis of many common diseases, including IBDs. This review aims to outline the history, biogenesis and regulation of miRNAs. The role of miRNAs in the development and regulation of the innate and adaptive immune system is discussed, with a particular focus on mechanisms pertinent to IBD and the potential translational applications

Tacrolimus inhibits IL-12 p40 protein and mRNA expression.

<p>(<b>A</b>) Murine bone marrow-derived macrophages (BMDMs) were pretreated with the indicated concentrations of tacrolimus for 1 h. Cells were then either left untreated (NS) or stimulated with LPS (100 ng/ml) or LPS and IFN-γ (10 ng/ml) for 24 h. IL-12 p40 protein secretion was assayed from supernatants by ELISA. (<b>B</b>) BMDMs were incubated with the indicated concentrations of tacrolimus for 1 h followed by 1 h treatment with IFN-γ (10 ng/ml, where indicated) prior to LPS (100 ng/ml) stimulation for 4 h or left untreated (NS). Cells were harvested and total RNA was assayed for <i>Il12b</i> mRNA levels by real-time RT-PCR. Each result represents the mean ± standard error (SD) for duplicate assays from three independent experiments. * p<0.05.</p

A 5-day course of oral antibiotics followed by faecal transplantation to eradicate carriage of multidrug-resistant Enterobacteriaceae: a randomized clinical trial

International audienceOBJECTIVES: Intestinal carriage with extended spectrum β-lactamase Enterobacteriaceae (ESBL-E) and carbapenemase-producing Enterobacteriaceae (CPE) can persist for months. We aimed to evaluate whether oral antibiotics followed by faecal microbiota transplantation (FMT) can eradicate intestinal carriage with ESBL-E/CPE.METHODS: Randomized, open-label, superiority trial in four tertiary-care centres (Geneva (G), Paris (P), Utrecht (U), Tel Aviv (T)). Non-immunocompromised adult patients were randomized 1: 1 to either no intervention (control) or a 5-day course of oral antibiotics (colistin sulphate 2 × 106 IU 4×/day; neomycin sulphate 500 mg 4×/day) followed by frozen FMT obtained from unrelated healthy donors. The primary outcome was detectable intestinal carriage of ESBL-E/CPE by stool culture 35-48 days after randomization (V4). ClinicalTrials.govNCT02472600. The trial was funded by the European Commission (FP7).RESULTS: Thirty-nine patients (G = 14; P = 16; U = 7; T = 2) colonized by ESBL-E (n = 36) and/or CPE (n = 11) were enrolled between February 2016 and June 2017. In the intention-to-treat analysis 9/22 (41%) patients assigned to the intervention arm were negative for ESBL-E/CPE at V4 (1/22 not receiving the intervention imputed as positive) whereas in the control arm 5/17 (29%) patients were negative (one lost to follow up imputed as negative) resulting in an OR for decolonization success of 1.7 (95% CI 0.4-6.4). Study drugs were well tolerated overall but three patients in the intervention group prematurely stopped the study antibiotics because of diarrhoea (all received FMT).CONCLUSIONS: Non-absorbable antibiotics followed by FMT slightly decreased ESBL-E/CPE carriage compared with controls; this difference was not statistically significant, potentially due to early trial termination. Further clinical investigations seem warranted

VIVIT treatment reduces spontaneous secretion of colonic inflammatory cytokines.

<p>Explants from colons of VIVIT (n = 12 for <b>A</b>, <b>B</b>; n = 7 for <b>C</b>) and VEET (n = 9 for <b>A</b>, <b>B</b>; n = 6 for <b>C</b>) treated mice were incubated in RPMI medium for 24 h, released cytokines were then determined by ELISA. We noted a marked difference in the gut spontaneous secretion of TNF-α (<b>B</b>) in gut supernatants from VIVIT treated mice, with a statistically significant change in IL-12 p40 (<b>A</b>) and in interferon-γ (<b>C</b>) production compared to VEET treated mice. Values are normalized to weight of intestinal explants, and data are presented as mean ± SEM.</p

11R-VIVIT reduces DNA binding to the NFAT/IRF8 site in the murine IL-12 p40 promoter.

<p>BMDMs were either untreated (lane 1) or pretreated with the indicated concentrations of 11R-VEET (lanes 2–4) or 11R-VIVIT (lanes 5–7) for 1 h. Cells were then treated for 1 h with IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) stimulation for 4 h. Cells were harvested and nuclear extracts were prepared for EMSA. ³²P labeled oligonucleotide probe spanning the NFAT/IRF8 site of the IL-12 p40 promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034172#pone.0034172-Zhu1" target="_blank">[10]</a> was incubated with 10 mg of nuclear extracts on ice for 30 min prior to electrophoresis. For supershift assays (lanes 3,4 and 6,7), 10 mg nuclear extract were incubated with 3 mg of anti-NFAT c1 (lanes 3 and 6) or anti-IRF8 (lanes 4 and 7) antibodies for 30 min on ice prior to addition of the ³²P labeled probe and 30 min incubation on ice followed by electrophoresis. The arrows represent DNA-protein complexes formed before and after incubation with the indicated antibodies. The above result is representative of five independent experiments.</p

11R-VIVIT inhibits pro-inflammatory cytokine expression in murine macrophages.

<p>BMDMs from WT mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or 11R-VEET for 1 h followed by LPS (100 ng/ml) and IFN-γ (10 ng/ml) for 24 h. IL-12 p70 (<b>A</b>), IL-23 (<b>B</b>) and TNF (<b>C</b>) protein secretion were assayed from supernatants by ELISA. Each result represents mean ± SE of duplicate assays from three independent experiments. * p<0.05.</p

NFAT binds to <i>Nos2</i> promoter and its selective inhibition abrogates nitric oxide secretion in macrophages.

<p>(<b>A</b>) Nuclear extracts were prepared from BMDMs stimulated with LPS (lane 1) or LPS and IFN-γ (lanes 2–7). Extracts were either incubated with a labeled probe NFAT/ISRE (element from region II of the promoter scheme), a competitor ISRE oligonucleotide, or with the indicated antibodies. DNA-protein complexes (indicated by arrow) were separated by electrophoresis. EMSA revealed binding of both IRF8 and NFATc1 to the same <i>Nos2</i> promoter element. (<b>B</b>) BMDMs from WT mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or the control peptide for 1 h followed by LPS (100 ng/ml) and IFN-γ (10 ng/ml) for 24 h. Nitric oxide secretion was assayed from supernatants by Greiss reaction. Experiments were performed in duplicate and repeated three times (mean ± SEM). (<b>C</b>) BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT for 1 h followed by 1 h treatment with LPS alone or IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) treatment for 4 h. Cells were harvested and total RNA was assayed for <i>Nos2</i> mRNA levels by real-time RT PCR. Data is representative of three independent experiments. * p<0.05.</p