哺乳动物肠上皮屏障中非编码RNAs的调控作用

哺乳动物肠上皮是一种拥有快速自我更新能力的组织,在维持机体免疫稳态与肠道应激后的损伤修复中发挥重要作用。源于隐窝底部的多能肠干细胞不断进行增殖、迁移与分化,并沿隐窝 绒毛轴向上移动,从而维持肠上皮完整性。该过程受严格而复杂的基因调控网络参与。越来越多的数据表明,肠上皮完整性受到广泛的非编码RNA的调控,主要包括肠黏膜再生、保护与上皮屏障功能等方面。本文重点讨论了两类非编码RNA(包括microRNAs和lncRNAs)转录后调控肠上皮屏障功能的研究进展。其中,miR-503、miR-146和lnc-uc.173、lnc-SPRY4-IT1、lnc-plncRNA1、lnc-Gata6等,能够促进肠黏膜的更新,增强上皮屏障功能;相反,miR-222、miR-29b、miR-195和lnc-H19与lnc-BC012900等,抑制肠上皮再生并破坏肠上皮屏障功能。miRNAs、mRNAs与lncRNAs间构成复杂的分子网络,共同调控肠上皮稳态。深入研究与肠上皮相关的miRNAs和IncRNAs分子及其作用机制,探寻引起肠黏膜炎症的关键分子靶标,为肠道炎症临床诊治提供新方向与新方法。     

哺乳动物肠上皮屏障中非编码RNAs的调控作用

哺乳动物肠上皮是一种拥有快速自我更新能力的组织,在维持机体免疫稳态与肠道应激后的损伤修复中发挥重要作用。源于隐窝底部的多能肠干细胞不断进行增殖、迁移与分化,并沿隐窝 绒毛轴向上移动,从而维持肠上皮完整性。该过程受严格而复杂的基因调控网络参与。越来越多的数据表明,肠上皮完整性受到广泛的非编码RNA的调控,主要包括肠黏膜再生、保护与上皮屏障功能等方面。本文重点讨论了两类非编码RNA(包括microRNAs和lncRNAs)转录后调控肠上皮屏障功能的研究进展。其中,miR-503、miR-146和lnc-uc.173、lnc-SPRY4-IT1、lnc-plncRNA1、lnc-Gata6等,能够促进肠黏膜的更新,增强上皮屏障功能;相反,miR-222、miR-29b、miR-195和lnc-H19与lnc-BC012900等,抑制肠上皮再生并破坏肠上皮屏障功能。miRNAs、mRNAs与lncRNAs间构成复杂的分子网络,共同调控肠上皮稳态。深入研究与肠上皮相关的miRNAs和IncRNAs分子及其作用机制,探寻引起肠黏膜炎症的关键分子靶标,为肠道炎症临床诊治提供新方向与新方法。     

Probiotic Escherichia coli Nissle 1917 inhibits leaky gut by enhancing mucosal integrity

Background

Probiotics are proposed to positively modulate the intestinal epithelial barrier formed by intestinal epithelial cells (IECs) and intercellular junctions. Disruption of this border alters paracellular permeability and is a key mechanism for the development of enteric infections and inflammatory bowel diseases (IBDs).

Methodology and Principal Findings

To study the in vivo effect of probiotic Escherichia coli Nissle 1917 (EcN) on the stabilization of the intestinal barrier under healthy conditions, germfree mice were colonized with EcN or K12 E. coli strain MG1655. IECs were isolated and analyzed for gene and protein expression of the tight junction molecules ZO-1 and ZO-2. Then, in order to analyze beneficial effects of EcN under inflammatory conditions, the probiotic was orally administered to BALB/c mice with acute dextran sodium sulfate (DSS) induced colitis. Colonization of gnotobiotic mice with EcN resulted in an up-regulation of ZO-1 in IECs at both mRNA and protein levels. EcN administration to DSS-treated mice reduced the loss of body weight and colon shortening. In addition, infiltration of the colon with leukocytes was ameliorated in EcN inoculated mice. Acute DSS colitis did not result in an anion secretory defect, but abrogated the sodium absorptive function of the mucosa. Additionally, intestinal barrier function was severely affected as evidenced by a strong increase in the mucosal uptake of Evans blue in vivo. Concomitant administration of EcN to DSS treated animals resulted in a significant protection against intestinal barrier dysfunction and IECs isolated from these mice exhibited a more pronounced expression of ZO-1.

Conclusion and Significance

This study convincingly demonstrates that probiotic EcN is able to mediate up-regulation of ZO-1 expression in murine IECs and confer protection from the DSS colitis-associated increase in mucosal permeability to luminal substances.     

The role of autophagy in maintaining intestinal mucosal barrier

The intestinal mucosal barrier is the first line to defense against luminal content penetration and performs numerous biological functions. The intestinal epithelium contains a huge surface that is lined by a monolayer of intestinal epithelial cells (IECs). IECs are dominant mediators in maintaining intestinal homeostasis that drive diverse functions including nutrient absorption, physical segregation, secretion of antibacterial peptides, and modulation of immune responses. Autophagy is a cellular self-protection mechanism in response to various stresses, and accumulating studies have revealed its importance in participating physiological processes of IECs. The regulatory effects of autophagy depend on the specific IEC types. This review aims to elucidate the myriad roles of autophagy in regulating the functions of different IECs (stem cells, enterocytes, goblet cells, and Paneth cells), and present the progress of autophagy-targeting therapy in intestinal diseases. Understanding the involved mechanisms can provide new preventive and therapeutic strategies for gastrointestinal dysfunction and diseases.     

Epithelial autophagy controls chronic colitis by reducing TNF-induced apoptosis

Genome-wide association studies (GWAS) linking polymorphisms in ATG16L1 with susceptibility to inflammatory bowel disease (IBD) have prompted mucosal immunologists to investigate the functional roles of macroautophagy/autophagy in different cell types in the gut. Here we present a recent study that addressed 2 key questions: in which cell type is autophagy deficiency most detrimental during chronic colitis and what is the functional role of autophagy in those cells? We report that autophagy in intestinal epithelial cells (IECs) acts to limit intestinal inflammation by protecting them from TNF-induced apoptosis and we discuss the potential implications for IBD treatment.     

Adrenomedullin treatment reduces intestinal inflammation and maintains epithelial barrier function in mice administered dextran sulphate sodium

Hyperactivation and hyperpermeability of the intestinal epithelium is a hallmark of IBD. AM has been shown to reduce the severity of colitis in the acetic acid and TNBS-induced colitis model, however the mechanism of the therapeutic effect of AM against the colitis has not been clarified. Here, we show that the protective capability of AM is associated with suppression of inflammation and maintenance of the intestinal epithelial barrier function. In the DSS-induced colitis model, intra-rectal AM-treated mice showed a reduction in loss of body weight and severity of colitis. AM-treatment suppressed phosphorylation of STAT1 and STAT3 in the colonic epithelium, and altered the cytokine balance in the intestinal T cells, with lower levels of IFN-γ and TNF-α but higher levels of TGF-β. Expression of the epithelial intercellular junctions such as tight and adherence junctions were sustained in the AM-treated mice. In contrast, the epithelial junctions were down-regulated in the control mice, leading to loss of epithelial barrier integrity and enhanced permeability. Collectively, these data indicate a broad spectrum of AM-induced effects with respect to protection against DSS-induced colitis, and suggest a potential therapeutic value of this treatment for IBD.     

Long noncoding RNA SPRY4-IT1 regulates intestinal epithelial barrier function by modulating the expression levels of tight junction proteins

Epithelial cells line the intestinal mucosa and form an important barrier to a wide array of noxious substances in the lumen. Disruption of the barrier integrity occurs commonly in various pathologies. Long noncoding RNAs (lncRNAs) control diverse biological processes, but little is known about the role of lncRNAs in regulation of the gut permeability. Here we show that the lncRNA SPRY4-IT1 regulates the intestinal epithelial barrier function by altering expression of tight junction (TJ) proteins. SPRY4-IT1 silencing led to dysfunction of the epithelial barrier in cultured cells by decreasing the stability of mRNAs encoding TJ proteins claudin-1, claudin-3, occludin, and JAM-1 and repressing their translation. In contrast, increasing the levels of SPRY4-IT1 in the intestinal mucosa protected the gut barrier in mice exposed to septic stress by increasing the abundance of TJ proteins. SPRY4-IT1 directly interacted with TJ mRNAs, and this process was enhanced through the association with the RNA-binding protein HuR. Of interest, the intestinal mucosa from patients with increased gut permeability exhibited a decrease in the levels of SPRY4-IT1. These findings highlight a novel role for SPRY4-IT1 in controlling the intestinal epithelial barrier and define a mechanism by which SPRY4-IT1 modulates TJ expression by altering the stability and translation of TJ mRNAs.     

Intestinal Anti-Inflammatory Activity of Lentinan: Influence on IL-8 and TNFR1 Expression in Intestinal Epithelial Cells

Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract. It is unknown whether β-1,3;1,6-glucan can induce immune suppressive effects. Here, we study intestinal anti-inflammatory activity of Lentinula edodes-derived β-1,3;1,6-glucan, which is known as lentinan. Dextran sulfate sodium (DSS)-induced colitis mice were used to elucidate effects of lentinan in vivo. In the cellular level assessment, lentinan was added into a co-culture model consisting of intestinal epithelial Caco-2 cells and LPS-stimulated macrophage RAW264.7 cells. Ligated intestinal loop assay was performed for assessing effects of lentinan on intestinal epithelial cells (IECs) in vivo. Oral administration of lentinan (100 µg/mouse) significantly ameliorated DSS-induced colitis in body weight loss, shortening of colon lengths, histological score, and inflammatory cytokine mRNA expression in inflamed tissues. Lentinan reduced interleukin (IL)-8 mRNA expression and nuclear factor (NF)-κB activation in Caco-2 cells without decreasing of tumor necrosis factor (TNF)-α production from RAW264.7 cells. Flow cytometric analysis revealed that surface levels of TNF receptor (TNFR) 1 were decreased by lentinan treatment. A clathrin-mediated endocytosis inhibitor, monodansylcadaverine, canceled lentinan inhibition of IL-8 mRNA expression. Moreover, lentinan inhibited TNFR1 expression in Caco-2 cells in both protein and mRNA level. Lentinan also inhibited TNFR1 mRNA expression in mouse IECs. These results suggest that lentinan exhibits intestinal anti-inflammatory activity through inhibition of IL-8 mRNA expression associated with the inhibition of NF-κB activation which is triggered by TNFR1 endocytosis and lowering of their expression in IECs. Lentinan may be effective for the treatment of gut inflammation including IBD.     

Autotaxin (ATX) inhibits autophagy leading to exaggerated disruption of intestinal epithelial barrier in colitis

Inflammatory bowel disease (IBD) is an immune-mediated disease. Autotaxin (ATX) is associated with increased inflammatory molecules, however, its effect on IBD is not well understood. Autophagy plays an important role in IBD, whether ATX and autophagy act in concert in IBD remains unknown. This study is to explore the possible mechanisms of ATX affecting autophagy leading to the disruption of intestinal epithelial barrier, thereby exacerbating colitis. The expression of ATX was upregulated in UC patients and dextran sulfate sodium (DSS)-induced colitis mice. Here, we described that providing an ATX inhibitor during DSS colitis increased autophagy and ameliorated colonic inflammation. Conversely, intrarectal administration with recombinant (r)ATX increased colitis and decreased autophagy. This pro-colitic effect was attenuated in mice treated with rapamycin, resulting in increased autophagy activity and mild colitis. Moreover, the inhibitory effect of rATX on autophagy was confirmed in vitro and was reversed by the addition of rapamycin. The damaging effects of ATX on epithelial barrier function were reversed by ATX inhibitor or rapamycin treatment. In sum, our results show that ATX can inhibit autophagy through the mTOR pathway, resulting in exaggerated damage to the intestinal epithelial barrier during colitis. These findings suggest that ATX may be a key pro-colitic factor, and represent a potential therapeutic target for treating IBD in the future.     

Polyamines are required for expression of Toll-like receptor 2 modulating intestinal epithelial barrier integrity

The Toll-like receptors (TLRs) allow mammalian intestinal epithelium to detect various microbes and activate innate immunity after infection. TLR2 and TLR4 have been identified in intestinal epithelial cells (IECs) as fundamental components of the innate immune response to bacterial pathogens, but the exact mechanism involved in control of TLR expression remains unclear. Polyamines are implicated in a wide variety of biological functions, and regulation of cellular polyamines is a central convergence point for the multiple signaling pathways driving different epithelial cell functions. The current study determined whether polyamines regulate TLR expression, thereby modulating intestinal epithelial barrier function. Depletion of cellular polyamines by inhibiting ornithine decarboxylase (ODC) with alpha-difluoromethylornithine decreased levels of TLR2 mRNA and protein, whereas increased polyamines by ectopic overexpression of the ODC gene enhanced TLR2 expression. Neither intervention changed basal levels of TLR4. Exposure of normal IECs to low-dose (5 microg/ml) LPS increased ODC enzyme activity and stimulated expression of TLR2 but not TLR4, while polyamine depletion prevented this LPS-induced TLR2 expression. Decreased TLR2 in polyamine-deficient cells was associated with epithelial barrier dysfunction. In contrast, increased TLR2 by the low dose of LPS enhanced epithelial barrier function, which was abolished by inhibition of TLR2 expression with specific, small interfering RNA. These results indicate that polyamines are necessary for TLR2 expression and that polyamine-induced TLR2 activation plays an important role in regulating epithelial barrier function.     

Repression of TXNIP–NLRP3 axis restores intestinal barrier function via inhibition of myeloperoxidase activity and oxidative stress in nonalcoholic steatohepatitis

Dysfunction of the intestinal barrier function occurs in hepatic injury, but the specific mechanisms responsible are largely unknown. Recently, NOD-like receptor 3 (NLRP3) inflammasome functions in impairing endothelial barrier function. In this study, we test the hypothesis that TXNIP–NLRP3 axis repression prevents against intestinal barrier function disruption in nonalcoholic steatohepatitis (NASH). First, lipopolysaccharide (LPS)-induced alterations in expression of ZO-1 and occludin, myeloperoxidase (MPO) activity, reactive oxygen species (ROS) level, and transepithelial electric resistance (TEER) in intestinal epithelial cells (IECs) isolated from C57BL/6 wild-type (WT) and TXNIP^−/− mice were evaluated. The underlying regulatory mechanisms of TXNIP knockout in vivo were investigated with the detection of expressions of TXNIP, NLRP3 and ZO-1, and occludin, the interaction of TXNIP–NLRP3, MPO activity, ROS level, permeability of intestinal mucosa, levels of inflammatory factors in serum, and LPS concentration. We identified that TXNIP knockout promoted ZO-1 and occludin expression, yet reduced MPO activity, ROS level, and cell permeability in IECs, indicating restored the intestinal barrier function. However, LPS upregulated TXNIP and NLRP3 expression, as well as contributed to the interaction between TXNIP and NLRP3 in vitro. Furthermore, TXNIP was significantly upregulated in the intestinal mucosa of NASH mice and its knockout repaired the intestinal barrier disrupt, inhibited expression of inflammatory factors, and reduced LPS concentration as well as hepatic injury in vivo. Taken together, our findings demonstrated that inhibited the activation of the TXNIP–NLRP3 axis reduced MPO activity and oxidative stress and thus restoring the intestinal barrier function in NASH. TXNIP–NLRP3 axis may be a promising therapeutic strategy for the NASH treatment.     

Regulation of intestinal epithelial intercellular adhesion and barrier function by desmosomal cadherin desmocollin-2

The role of desmosomal cadherin desmocollin-2 (Dsc2) in regulating barrier function in intestinal epithelial cells (IECs) is not well understood. Here, we report the consequences of silencing Dsc2 on IEC barrier function in vivo using mice with inducible intestinal–epithelial-specific Dsc2 knockdown (KD) (Dsc2^ERΔIEC). While the small intestinal gross architecture was maintained, loss of epithelial Dsc2 influenced desmosomal plaque structure, which was smaller in size and had increased intermembrane space between adjacent epithelial cells. Functional analysis revealed that loss of Dsc2 increased intestinal permeability in vivo, supporting a role for Dsc2 in the regulation of intestinal epithelial barrier function. These results were corroborated in model human IECs in which Dsc2 KD resulted in decreased cell–cell adhesion and impaired barrier function. It is noteworthy that Dsc2 KD cells exhibited delayed recruitment of desmoglein-2 (Dsg2) to the plasma membrane after calcium switch-induced intercellular junction reassembly, while E-cadherin accumulation was unaffected. Mechanistically, loss of Dsc2 increased desmoplakin (DP I/II) protein expression and promoted intermediate filament interaction with DP I/II and was associated with enhanced tension on desmosomes as measured by a Dsg2-tension sensor. In conclusion, we provide new insights on Dsc2 regulation of mechanical tension, adhesion, and barrier function in IECs.     

Metformin protects against intestinal barrier dysfunction via AMPKα1‐dependent inhibition of JNK signalling activation

Disruption of the intestinal epithelial barrier, that involves the activation of C‐Jun N‐terminal kinase (JNK), contributes to initiate and accelerate inflammation in inflammatory bowel disease. Metformin has unexpected beneficial effects other than glucose‐lowering effects. Here, we provided evidence that metformin can protect against intestinal barrier dysfunction in colitis. We showed that metformin alleviated dextran sodium sulphate (DSS)‐induced decreases in transepithelial electrical resistance, FITC‐dextran hyperpermeability, loss of the tight junction (TJ) proteins occludin and ZO‐1 and bacterial translocation in Caco‐2 cell monolayers or in colitis mice models. Metformin also improved TJ proteins expression in ulcerative colitis patients with type 2 diabetes mellitus. We found that metformin ameliorated the induction of colitis and reduced the levels of pro‐inflammatory cytokines IL‐6, TNF‐a and IL‐1β. In addition, metformin suppressed DSS‐induced JNK activation, an effect dependent on AMP‐activated protein kinase α1 (AMPKα1) activation. Consistent with this finding, metformin could not maintain the barrier function of AMPKα1‐silenced cell monolayers after DSS administration. These findings highlight metformin protects against intestinal barrier dysfunction. The potential mechanism may involve in the inhibition of JNK activation via an AMPKα1‐dependent signalling pathway.     

MiR-155 promotes colitis-associated intestinal fibrosis by targeting HBP1/Wnt/β-catenin signalling pathway

Intestinal fibrosis is the most common complication of Crohn's disease (CD) that is one major disorder of inflammatory bowel disease (IBD), but the precise mechanism remains unclear. MiR-155 has been involved in fibrotic diseases. Here, we determined the role of miR-155 in regulating intestinal fibrosis. MiR-155 levels were significantly up-regulated in CD patients with intestinal stricture CD. The overexpression of miR-155 significantly aggravated TNBS-induced CD-associated intestinal fibrosis. Mechanistically, we identified that HBP1, a negative regulator of the Wnt/β-catenin signalling pathway, is a direct target of miR-155. Moreover, in vitro and in vivo experiments suggested that the miR-155/HBP1 axis activates Wnt/β-catenin signalling pathway to induce intestinal fibrosis. Taken together, we demonstrated that miR-155 directly targets HBP1 to induce CD-associated intestinal fibrosis via Wnt/β-catenin signalling pathway.     

PHD3 Stabilizes the Tight Junction Protein Occludin and Protects Intestinal Epithelial Barrier Function

Prolyl hydroxylase domain proteins (PHDs) control cellular adaptation to hypoxia. PHDs are found involved in inflammatory bowel disease (IBD); however, the exact role of PHD3, a member of the PHD family, in IBD remains unknown. We show here that PHD3 plays a critical role in maintaining intestinal epithelial barrier function. We found that genetic ablation of Phd3 in intestinal epithelial cells led to spontaneous colitis in mice. Deletion of PHD3 decreases the level of tight junction protein occludin, leading to a failure of intestinal epithelial barrier function. Further studies indicate that PHD3 stabilizes occludin by preventing the interaction between the E3 ligase Itch and occludin, in a hydroxylase-independent manner. Examination of biopsy of human ulcerative colitis patients indicates that PHD3 is decreased with disease severity, indicating that PHD3 down-regulation is associated with progression of this disease. We show that PHD3 protects intestinal epithelial barrier function and reveal a hydroxylase-independent function of PHD3 in stabilizing occludin. These findings may help open avenues for developing a therapeutic strategy for IBD.