Down-regulation of human enteric antimicrobial peptides by NOD2 during differentiation of the paneth cell lineage.
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ABSTRACT:
SUBMITTER: Tan G
PROVIDER: S-EPMC4323657 | biostudies-other | 2015
REPOSITORIES: biostudies-other
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Project description:ObjectiveAlthough polymorphisms of the NOD2 gene predispose to the development of ileal Crohn's disease, the precise mechanisms of this increased susceptibility remain unclear. Previous work has shown that transcript expression of the Paneth cell (PC) antimicrobial peptides (AMPs) α-defensin 4 and α-defensin-related sequence 10 are selectively decreased in Nod2(-/-) mice. However, the specific mouse background used in this previous study is unclear. In light of recent evidence suggesting that mouse strain strongly influences PC antimicrobial activity, we sought to characterise PC AMP function in commercially available Nod2(-/-) mice on a C57BL/6 (B6) background. Specifically, we hypothesised that Nod2(-/-) B6 mice would display reduced AMP expression and activity.DesignWild-type (WT) and Nod2(-/-) B6 ileal AMP expression was assessed via real-time PCR, acid urea polyacrylamide gel electrophoresis and mass spectrometry. PCs were enumerated using flow cytometry. Functionally, α-defensin bactericidal activity was evaluated using a gel-overlay antimicrobial assay. Faecal microbial composition was determined using 454-sequencing of the bacterial 16S gene in cohoused WT and Nod2(-/-) littermates.ResultsWT and Nod2(-/-) B6 mice displayed similar PC AMP expression patterns, equivalent α-defensin profiles, and identical antimicrobial activity against commensal and pathogenic bacterial strains. Furthermore, minimal differences in gut microbial composition were detected between the two cohoused, littermate mouse groups.ConclusionsOur data reveal that Nod2 does not directly regulate PC antimicrobial activity in B6 mice. Moreover, we demonstrate that previously reported Nod2-dependent influences on gut microbial composition may be overcome by environmental factors, such as cohousing with WT littermates.
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Project description:Transcription factor Neurod1 is required for enteroendocrine progenitor differentiation and maturation. Several earlier studies indicated that ectopic expression of Neurod1 converted non- neuronal cells into neurons. However, the functional consequence of ectopic Neurod1 expression has not been examined in the GI tract, and it is not known whether Neurod1 can similarly switch cell fates in the intestine. We generated a mouse line that would enable us to conditionally express Neurod1 in intestinal epithelial cells at different stages of differentiation. Forced expression of Neurod1 throughout intestinal epithelium increased the number of EECs as well as the expression of EE specific transcription factors and hormones. Furthermore, we observed a substantial reduction of Paneth cell marker expression, although the expressions of enterocyte-, tuft- and goblet-cell specific markers are largely not affected. Our earlier study indicated that Neurog3+ progenitor cells give rise to not only EECs but also Goblet and Paneth cells. Here we show that the conditional expression of Neurod1 restricts Neurog3+ progenitors to adopt Paneth cell fate, and promotes more pronounced EE cell differentiation, while such effects are not seen in more differentiated Neurod1+ cells. Together, our data suggest that forced expression of Neurod1 programs intestinal epithelial cells more towards an EE cell fate at the expense of the Paneth cell lineage and the effect ceases as cells mature to EE cells.
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Project description:Canonical Wnt signaling plays a rate-limiting role in regulating self-renewal and differentiation in mouse embryonic stem cells (ESCs). We have previously shown that mutation in the Apc (adenomatous polyposis coli) tumor suppressor gene constitutively activates Wnt signaling in ESCs and inhibits their capacity to differentiate towards ecto-, meso-, and endodermal lineages. However, the underlying molecular and cellular mechanisms through which Wnt regulates lineage differentiation in mouse ESCs remain to date largely unknown. To this aim, we have derived and studied the gene expression profiles of several Apc-mutant ESC lines encoding for different levels of Wnt signaling activation. We found that down-regulation of Tcf3, a member of the Tcf/Lef family and a key player in the control of self-renewal and pluripotency, represents a specific and primary response to Wnt activation in ESCs. Accordingly, rescuing Tcf3 expression partially restored the neural defects observed in Apc-mutant ESCs, suggesting that Tcf3 down-regulation is a necessary step towards Wnt-mediated suppression of neural differentiation. We found that Tcf3 down-regulation in the context of constitutively active Wnt signaling does not result from promoter DNA methylation but is likely to be caused by a plethora of mechanisms at both the RNA and protein level as shown by the observed decrease in activating histone marks (H3K4me3 and H3-acetylation) and the upregulation of miR-211, a novel Wnt-regulated microRNA that targets Tcf3 and attenuates early neural differentiation in mouse ESCs. Our data show for the first time that Wnt signaling down-regulates Tcf3 expression, possibly at both the transcriptional and post-transcriptional levels, and thus highlight a novel mechanism through which Wnt signaling inhibits neuro-ectodermal lineage differentiation in mouse embryonic stem cells.
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Project description:Paneth cells at the base of small intestinal crypts of Lieberkühn secrete high levels of α-defensins in response to cholinergic and microbial stimuli. Paneth cell α-defensins are broad spectrum microbicides that function in the extracellular environment of the intestinal lumen, and they are responsible for the majority of secreted bactericidal peptide activity. Paneth cell α-defensins confer immunity to oral infection by Salmonella enterica serovar Typhimurium, and they are major determinants of the composition of the small intestinal microbiome. In addition to host defense molecules such as α-defensins, lysozyme, and Pla2g2a, Paneth cells also produce and release proinflammatory mediators as components of secretory granules. Disruption of Paneth cell homeostasis, with subsequent induction of endoplasmic reticulum stress, autophagy, or apoptosis, contributes to inflammation in diverse genetic and experimental mouse models.
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Project description:The intestinal epithelial barrier plays critical role in the intestinal homeostasis and dysfunction of the epithelial barrier causes microbial invasion that would lead to inflammation in the gut. Antimicrobial peptides (AMPs) are essential components of the epithelial barrier to inhibit bacterial invasion. The regulatory mechanisms of the expression of AMPs are not fully characterized. Here, we report a cell-type-specific role of ovarian tumor family deubiquitinase 4 (OTUD4) in experimental colitis and bacterial infection. OTUD4 is upregulated in the inflamed mucosa of IBD patients and in the colon of mice treated with dextran sulfate sodium salt (DSS). Knockout of OTUD4 in intestinal organoids promotes the expression of AMPs after the stimulation with lipopolysaccharide (LPS), peptidoglycan (PGN), or Salmonella typhimurium (S.t.). In addition, the expression of AMPs was upregulated in intestinal epithelial cells (IECs) after DSS treatment or S.t. infection. Consistently, Vil-Cre;Otud4fl/fl mice and Def-Cre;Otud4fl/fl mice exhibit hyper-resistance to DSS-induced colitis and S.t. infection compared to Otud4fl/fl mice. Mechanistically, Knockout of OTUD4 results in hyper K63-linked ubiquitination of MyD88 and increases the activation of the NF-κB signaling pathway and MAPK signaling pathway to promote the expression of AMPs. These finds collectively highlight an indispensable role of OTUD4 in paneth cells to modulate AMPs production, indicating OTUD4 as a promoter and potential druggable target for gastrointestinal inflammation and bacterial infection.
2023-03-01 | GSE222836 | GEO
Project description:The biological effects of 1α,25-dihydroxyvitamin D3 (1,25 (OH)2D3) on osteoblast differentiation and function differ significantly depending upon the cellular state of maturation. To explore this phenomenon mechanistically, we examined the impact of 1,25(OH)2D3 on the transcriptomes of both pre-osteoblastic (POBs) and differentiated osteoblastic (OBs) MC3T3-E1 cells, and assessed localization of the vitamin D receptor (VDR) at sites of action on a genome-scale using ChIP sequence analysis. We observed that the 1,25(OH)2D3-induced transcriptomes of POBs and OBs were quantitatively and qualitatively different, supporting not only the altered biology observed but the potential for a change in VDR interaction at the genome as well. This idea was confirmed through discovery that VDR cistromes in POBs and OBs were also strikingly different. Depletion of VDR-binding sites in OBs, due in part to reduced VDR expression, was the likely cause of the loss of VDR-target gene interaction. Continued novel regulation by 1,25(OH)2D3, however, suggested that factors in addition to the VDR might also be involved. Accordingly, we show that transcriptomic modifications are also accompanied by changes in genome binding of the master osteoblast regulator RUNX2 and the chromatin remodeler CCAAT/enhancer-binding protein β. Importantly, genome occupancy was also highlighted by the presence of epigenetic enhancer signatures that were selectively changed in response to both differentiation and 1,25(OH)2D3. The impact of VDR, RUNX2, and C/EBPβ on osteoblast differentiation is exemplified by their actions at the Runx2 and Sp7 gene loci. We conclude that each of these mechanisms may contribute to the diverse actions of 1,25(OH)2D3 on differentiating osteoblasts.
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Project description:This SuperSeries is composed of the SubSeries listed below.
2018-09-15 | GSE113347 | GEO
Project description:The mammalian intestine is one of the most rapidly self-renewing tissues, driven by stem cells residing at the crypt bottom. Paneth cells form a major element of the niche microenvironment providing various growth factors to orchestrate intestinal stem cell homeostasis, such as Wnt3. Different Wnt ligands can selectively activate β-catenin-dependent (canonical) or -independent (noncanonical) signaling. Here, we report that the Dishevelled-associated activator of morphogenesis 1 (Daam1) and its paralogue Daam2 asymmetrically regulate canonical and noncanonical Wnt (Wnt/PCP) signaling. Daam1/2 interacts with the Wnt inhibitor RNF43, and Daam1/2 double knockout stimulates canonical Wnt signaling by preventing RNF43-dependent degradation of the Wnt receptor, Frizzled (Fzd). Single-cell RNA sequencing analysis revealed that Paneth cell differentiation is impaired by Daam1/2 depletion because of defective Wnt/PCP signaling. Together, we identified Daam1/2 as an unexpected hub molecule coordinating both canonical and noncanonical Wnt, which is fundamental for specifying an adequate number of Paneth cells.
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Project description:CXCL13 is a constitutively expressed chemokine that controls migration of immune cells to lymphoid follicles. Previously, we found CXCL13 mRNA levels increased in rhesus macaque spleen tissues during AIDS. This led us to examine the levels and locations of CXCL13 by detailed in situ methods in cynomolgus macaque lymphoid and intestinal tissues. Our results revealed that there were distinct localization patterns of CXCL13 mRNA compared to protein in germinal centers. These patterns shifted during the course of simian immunodeficiency virus (SIV) infection, with increased mRNA expression within and around follicles during AIDS compared to uninfected or acutely infected animals. Unexpectedly, CXCL13 expression was also found in abundance in Paneth cells in crypts throughout the small intestine. Therefore, we expanded our analyses to include chemokines and antimicrobial peptides (AMPs) not previously demonstrated to be expressed by Paneth cells in intestinal tissues. We examined the expression patterns of multiple chemokines, including CCL25, as well as ?-defensin 6 (DEFA6), ?-defensin 2 (BDEF2), rhesus ?-defensin 1 (RTD-1), and Reg3? in situ in intestinal tissues. Of the 10 chemokines examined, CXCL13 was unique in its expression by Paneth cells. BDEF2, RTD-1, and Reg3? were also expressed by Paneth cells. BDEF2 and RTD-1 previously have not been shown to be expressed by Paneth cells. These findings expand our understanding of mucosal immunology, innate antimicrobial defenses, homeostatic chemokine function, and host protective mechanisms against microbial translocation.
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