The Endoplasmic Reticulum Stress Sensor IRE1α in Intestinal Epithelial Cells Is Essential for Protecting against Colitis

Intestinal epithelial cells (IECs) have critical roles in maintaining homeostasis of intestinal epithelium. Endoplasmic reticulum (ER) stress is implicated in intestinal epithelium homeostasis and inflammatory bowel disease; however, it remains elusive whether IRE1α, a major sensor of ER stress, is directly involved in these processes. We demonstrate here that genetic ablation of Ire1α in IECs leads to spontaneous colitis in mice. Deletion of IRE1α in IECs results in loss of goblet cells and failure of intestinal epithelial barrier function. IRE1α deficiency induces cell apoptosis through induction of CHOP, the pro-apoptotic protein, and sensitizes cells to lipopolysaccharide, an endotoxin from bacteria. IRE1α deficiency confers upon mice higher susceptibility to chemical-induced colitis. These results suggest that IRE1α functions to maintain the intestinal epithelial homeostasis and plays an important role in defending against inflammation bowel diseases.     

Matricellular Protein Periostin Mediates Intestinal Inflammation through the Activation of Nuclear Factor κB Signaling

Periostin is a matricellular protein that interacts with various integrin molecules on the cell surface. Although periostin is expressed in inflamed colonic mucosa, its role in the regulation of intestinal inflammation remains unclear. We investigated the role of periostin in intestinal inflammation using Postn-deficient (Postn^-/-) mice. Intestinal epithelial cells (IECs) were transfected by Postn small interfering RNAs. Periostin expression was determined in colon tissue samples from ulcerative colitis (UC) patients. Oral administration of dextran sulfate sodium (DSS) or rectal administration of trinitrobenzene sulfonic acid, induced severe colitis in wild-type mice, but not in Postn^-/- mice. Administration of recombinant periostin induced colitis in Postn^-/- mice. The periostin neutralizing-antibody ameliorated the severity of colitis in DSS-treated wild-type mice. Silencing of Postn inhibited inteleukin (IL)-8 mRNA expression and NF-κB DNA-binding activity in IECs. Tumor necrosis factor (TNF)-α upregulated mRNA expression of Postn in IECs, and recombinant periostin strongly enhanced IL-8 expression in combination with TNF-α, which was suppressed by an antibody against integrin α_v (CD51). Periostin and CD51 were expressed at significantly higher levels in UC patients than in controls. Periostin mediates intestinal inflammation through the activation of NF-κB signaling, which suggests that periostin is a potential therapeutic target for inflammatory bowel disease.     

Differential expression of tumor-associated genes and altered gut microbiome with decreased Akkermansia muciniphila confer a tumor-preventive microenvironment in intestinal epithelial Pten-deficient mice

Phosphatase and tensin homolog (Pten) antagonizes PI3K-Akt signaling; therefore, Pten impairment causes tumorigenesis. However, the correlation between Pten deficiency and colon cancer has remained elusive due to numerous opposite observations. To study this correlation, we examined whether Pten deficiency in intestinal epithelial cells (IECs) induces tumorigenesis.With mucosal biopsies of human colon cancer and normal colon, Pten mRNA was evaluated by quantitative PCR. Using IEC-specific Pten knockout mice (Pten^ΔIEC/ΔIEC), we examined the mitotic activity of IECs; and Pten^ΔIEC/ΔIEC; Apc^min/+ mice were generated by combining Pten^ΔIEC/ΔIEC with Apc^min/+ mice. Tumor-associated gene was evaluated by micro-array analysis. Fecal microbiome was analyzed through 16S rRNA gene sequencing.We found that Pten mRNA level was reduced in human colon cancer relative to normal tissues. Augmented chromatids, increased Ki-67 and PCNA expression, and enhanced Akt activation were identified in IECs of Pten^ΔIEC/ΔIEC mice compared to Pten^+/+ littermate. Combining Pten^ΔIEC/ΔIEC with Apc^min/+ condition caused rapid and aggressive intestinal tumorigenesis. However, Pten^ΔIEC/ΔIEC mice did not develop any tumors. While maintaining the tumor-driving potential, these data indicated that IEC-Pten deficiency alone did not induce tumorigenesis in mice. Furthermore, the expression of tumor-promoting and tumor-suppressing genes was decreased and increased, respectively, in the intestine of Pten^ΔIEC/ΔIEC mice compared to controls. The abundance of Akkermansia muciniphila, capable of inducing chronic intestinal inflammation, was diminished in Pten^ΔIEC/ΔIEC mice compared to controls.These findings suggested that altered tumor-associated gene expression and changed gut microbiota shape a tumor-preventive microenvironment to counteract the tumor-driving potential, leading to the tumor prevention in Pten^ΔIEC/ΔIEC mice.     

Endophilin A2-mediated alleviation of endoplasmic reticulum stress-induced cardiac injury involves the suppression of ERO1α/IP3R signaling pathway

Cardiac injury upon myocardial infarction (MI) is the leading cause of heart failure. The present study aims to investigate the role of EndoA2 in ischemia-induced cardiomyocyte apoptosis and cardiac injury. In vivo, we established an MI mouse model by ligating the left anterior descending (LAD) coronary artery, and intramyocardial injection of adenoviral EndoA2 (Ad-EndoA2) was used to overexpress EndoA2. In vitro, we used the siRNA and Ad-EndoA2 transfection strategies. Here, we reported that EndoA2 expression was remarkably elevated in the infarct border zone of MI mouse hearts and neonatal rat cardiomyocytes (NRCMs) stimulated with oxygen and glucose deprivation (OGD) which mimicked ischemia. We showed that intramyocardial injection of Ad-EndoA2 attenuated cardiomyocyte apoptosis and reduced endoplasmic reticulum (ER) stress in response to MI injury. Using siRNA for knockdown and Ad-EndoA2 for overexpression, we validated that knockdown of EndoA2 in NRCMs exacerbated OGD-induced NRCM apoptosis, whereas overexpression of EndoA2 attenuates OGD-induced cardiomyocyte apoptosis. Mechanistically, knockdown of EndoA2 activated ER stress response, which increases ER oxidoreductase 1α (ERO1α) and inositol 1, 4, 5-trisphosphate receptor (IP₃R) activity, thus led to increased intracellular Ca²⁺ accumulation, followed by elevated calcineurin activity and nuclear factor of activated T-cells (NFAT) dephosphorylation. Pretreatment with the IP₃R inhibitor 2-Aminoethoxydiphenylborate (2-APB) attenuated intracellular Ca²⁺ accumulation, and pretreatment with the Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) or the calcineurin inhibitor Cyclosporin A (CsA) inhibited EndoA2-knockdown-induced NRCM apoptosis. Overexpression of EndoA2 led to the opposite effects by suppressing ER-stress-mediated ERO1α/IP₃R signaling pathway. This study demonstrated that EndoA2 protected cardiac function in response to MI via attenuating ER-stress-mediated ERO1α/IP₃R signaling pathway. Targeting EndoA2 is a potential therapeutic strategy for the prevention of postinfarction-induced cardiac injury and heart failure.     

The Role of Estrogen Signaling in a Mouse Model of Inflammatory Bowel Disease: A Helicobacter Hepaticus Model

The pathogenesis of inflammatory bowel diseases (IBD), Crohn''s disease and ulcerative colitis, is due in part to interactions between the immune system, genetics, the environment, and endogenous microbiota. Gonadal sex hormones (GSH), such as estrogen, are thought to be involved in the development of IBD as variations in disease severity occur during pregnancy, menopause, or oral contraceptives use. In certain strains of mice, infection with Helicobacter hepaticus triggers IBD-like mucosal inflammation that is more severe in female mice than in males, suggesting a role for GSH in this model. To determine the role of estrogen signaling in microbiota-induced intestinal inflammation, estrogen receptor (ER) α and β knock-out (KO) mice, ER agonists, and adoptive transfers were utilized. We demonstrate that, when signaling is limited to ERβ on a non-CD4⁺ cell subset, disease is less severe and this correlates with decreased expression of pro-inflammatory mediators.     

Epithelial p38α Controls Immune Cell Recruitment in the Colonic Mucosa

Intestinal epithelial cells (IECs) compose the first barrier against microorganisms in the gastrointestinal tract. Although the NF-κB pathway in IECs was recently shown to be essential for epithelial integrity and intestinal immune homeostasis, the roles of other inflammatory signaling pathways in immune responses in IECs are still largely unknown. Here we show that p38α in IECs is critical for chemokine expression, subsequent immune cell recruitment into the intestinal mucosa, and clearance of the infected pathogen. Mice with p38α deletion in IECs suffer from a sustained bacterial burden after inoculation with Citrobacter rodentium. These animals are normal in epithelial integrity and immune cell function, but fail to recruit CD4⁺ T cells into colonic mucosal lesions. The expression of chemokines in IECs is impaired, which appears to be responsible for the impaired T cell recruitment. Thus, p38α in IECs contributes to the host immune responses against enteric bacteria by the recruitment of immune cells.     

Endoplasmic Reticulum Stress,Unfolded Protein Response and Altered T Cell Differentiation in Necrotizing Enterocolitis

Background

Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) play important roles in chronic intestinal inflammation. Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants and is characterized by acute intestinal inflammation and necrosis. The objective of the study is to investigate the role of ER stress and the UPR in NEC patients.

Methods

Ileal tissues from NEC and control patients were obtained during surgical resection and/or at stoma closure. Splicing of XBP1 was detected using PCR, and gene expression was quantified using qPCR and Western blot.

Results

Splicing of XBP1 was only detected in a subset of acute NEC (A-NEC) patients, and not in NEC patients who had undergone reanastomosis (R-NEC). The other ER stress and the UPR pathways, PERK and ATF6, were not activated in NEC patients. A-NEC patients showing XBP1 splicing (A-NEC-XBP1s) had increased mucosal expression of GRP78, CHOP, IL6 and IL8. Similar results were obtained by inducing ER stress and the UPR in vitro. A-NEC-XBP1s patients showed altered T cell differentiation indicated by decreased mucosal expression of RORC, IL17A and FOXP3. A-NEC-XBP1s patients additionally showed more severe morphological damage and a worse surgical outcome. Compared with A-NEC patients, R-NEC patients showed lower mucosal IL6 and IL8 expression and higher mucosal FOXP3 expression.

Conclusions

XBP1 splicing, ER stress and the UPR in NEC are associated with increased IL6 and IL8 expression levels, altered T cell differentiation and severe epithelial injury.     

β-Arrestin-1 protects against endoplasmic reticulum stress/p53-upregulated modulator of apoptosis-mediated apoptosis via repressing p-p65/inducible nitric oxide synthase in portal hypertensive gastropathy

Portal hypertensive gastropathy (PHG) is a serious cause of bleeding in patients, and is associated with portal hypertension. β-Arrestins (β-arrestin-1 and β-arrestin-2) are well-established mediators of endocytosis of G-protein-coupled receptors (GPCRs), ubiquitination, and G-protein-independent signaling. The role of β-arrestin-1 (β-arr1) in mucosal apoptosis in PHG remains unclear. The aim of this study was to investigate the involvement of β-arr1 in PHG via its regulation of endoplasmic reticulum (ER) stress/p53-upregulated modulator of apoptosis (PUMA) apoptotic signaling. Gastric mucosal injury and apoptosis were studied in PHG patients and in PHG mouse models. The induction of β-arr1 and the ER stress/PUMA signaling pathway were investigated, and the mechanisms of β-arr1-regulated gastric mucosal apoptosis were analyzed in vivo and in vitro experiments. β-arr1 and ER stress/PUMA signaling elements were markedly induced in the gastric mucosa of PHG patients and mouse models. Blockage of ER stress demonstrably attenuated the mucosal apoptosis of PHG, while targeted deletion of β-arr1 significantly aggravated the injury and ER stress/PUMA-mediated apoptosis. β-arr1 limited the activation of p65 to repress TNF-α-induced inducible nitric oxide synthase (iNOS) expression and NO release, which could regulate ER stress/PUMA-mediated mucosal apoptosis in PHG. In vivo and in vitro experiments further demonstrated that β-arr1 protected against mucosal apoptosis by repressing TNF-α-induced iNOS expression via inhibiting the activation of p65. These results indicated that β-arr1 regulated ER stress/PUMA-induced mucosal epithelial apoptosis through suppression of the TNF-α/p65/iNOS signaling pathway activation and that β-arr1 is a potential therapeutic target for PHG.     

Fibroblast Growth Factor Receptor 2c Signaling Is Required for Intestinal Cell Differentiation in Zebrafish

Background

There are four cell lineages derived from intestinal stem cells that are located at the crypt and villus in the mammalian intestine the non-secretory absorptive enterocytes, and the secretory cells, which include mucous-secreting goblet cells, regulatory peptide-secreting enteroendocrine cells and antimicrobial peptide-secreting Paneth cells. Although fibroblast growth factor (Fgf) signaling is important for cell proliferation and differentiation in various tissues, its role in intestinal differentiation is less well understood.

Methodology/Principal Findings

We used a loss of function approach to investigate the importance of Fgf signaling in intestinal cell differentiation in zebrafish; abnormal differentiation of goblet cells was observed when Fgf signaling was inhibited using SU5402 or in the Tg(hsp70ldnfgfr1-EGFP) transgenic line. We identified Fgfr2c as an important receptor for cell differentiation. The number of goblet cells and enteroendocrine cells was reduced in fgfr2c morphants. In addition to secretory cells, enterocyte differentiation was also disrupted in fgfr2c morphants. Furthermore, proliferating cells were increased in the morphants. Interestingly, the loss of fgfr2c expression repressed secretory cell differentiation and increased cell proliferation in the mib^ta52b mutant that had defective Notch signaling.

Conclusions/Significance

In conclusion, we found that Fgfr2c signaling derived from mesenchymal cells is important for regulating the differentiation of zebrafish intestine epithelial cells by promoting cell cycle exit. The results of Fgfr2c knockdown in mib^ta52b mutants indicated that Fgfr2c signaling is required for intestinal cell differentiation. These findings provide new evidences that Fgf signaling is required for the differentiation of intestinal cells in the zebrafish developing gut.     

Pathogenic Intestinal Bacteria Enhance Prostate Cancer Development via Systemic Activation of Immune Cells in Mice

A role for microbes has been suspected in prostate cancer but difficult to confirm in human patients. We show here that a gastrointestinal (GI) tract bacterial infection is sufficient to enhance prostate intraepithelial neoplasia (PIN) and microinvasive carcinoma in a mouse model. We found that animals with a genetic predilection for dysregulation of wnt signaling, Apc ^Min/+ mutant mice, were significantly susceptible to prostate cancer in an inflammation-dependent manner following infection with Helicobacter hepaticus. Further, early neoplasia observed in infected Apc ^Min/+ mice was transmissible to uninfected mice by intraperitoneal injection of mesenteric lymph node (MLN) cells alone from H. hepaticus-infected mutant mice. Transmissibility of neoplasia was preventable by prior neutralization of inflammation using anti-TNF-α antibody in infected MLN donor mice. Taken together, these data confirm that systemic inflammation triggered by GI tract bacteria plays a pivotal role in tumorigenesis of the prostate gland.     

PERK Limits Drosophila Lifespan by Promoting Intestinal Stem Cell Proliferation in Response to ER Stress

Intestinal homeostasis requires precise control of intestinal stem cell (ISC) proliferation. In Drosophila, this control declines with age largely due to chronic activation of stress signaling and associated chronic inflammatory conditions. An important contributor to this condition is the age-associated increase in endoplasmic reticulum (ER) stress. Here we show that the PKR-like ER kinase (PERK) integrates both cell-autonomous and non-autonomous ER stress stimuli to induce ISC proliferation. In addition to responding to cell-intrinsic ER stress, PERK is also specifically activated in ISCs by JAK/Stat signaling in response to ER stress in neighboring cells. The activation of PERK is required for homeostatic regeneration, as well as for acute regenerative responses, yet the chronic engagement of this response becomes deleterious in aging flies. Accordingly, knocking down PERK in ISCs is sufficient to promote intestinal homeostasis and extend lifespan. Our studies highlight the significance of the PERK branch of the unfolded protein response of the ER (UPR^ER) in intestinal homeostasis and provide a viable strategy to improve organismal health- and lifespan.     

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.     

Rosiglitazone Reverses Inflammation in Epididymal White Adipose Tissue in Hormone-Sensitive Lipase-Knockout Mice

Hormone-sensitive lipase (HSL) plays a crucial role in intracellular lipolysis, and loss of HSL leads to diacylglycerol (DAG) accumulation, reduced FA mobilization, and impaired PPARγ signaling. Hsl knockout mice exhibit adipose tissue inflammation, but the underlying mechanisms are still not clear. Here, we investigated if and to what extent HSL loss contributes to endoplasmic reticulum (ER) stress and adipose tissue inflammation in Hsl knockout mice. Furthermore, we were interested in how impaired PPARγ signaling affects the development of inflammation in epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) of Hsl knockout mice and if DAG and ceramide accumulation contribute to adipose tissue inflammation and ER stress. Ultrastructural analysis showed a markedly dilated ER in both eWAT and iWAT upon loss of HSL. In addition, Hsl knockout mice exhibited macrophage infiltration and increased F4/80 mRNA expression, a marker of macrophage activation, in eWAT, but not in iWAT. We show that treatment with rosiglitazone, a PPARγ agonist, attenuated macrophage infiltration and ameliorated inflammation of eWAT, but expression of ER stress markers remained unchanged, as did DAG and ceramide levels in eWAT. Taken together, we show that HSL loss promoted ER stress in both eWAT and iWAT of Hsl knockout mice, but inflammation and macrophage infiltration occurred mainly in eWAT. Also, PPARγ activation reversed inflammation but not ER stress and DAG accumulation. These data indicate that neither reduction of DAG levels nor ER stress contribute to the reversal of eWAT inflammation in Hsl knockout mice.     

Intestinal epithelial cell-derived semaphorin 7A negatively regulates development of colitis via αvβ1 integrin

The intestinal immune system is constantly challenged by commensal bacteria; therefore, it must maintain quiescence via several regulatory mechanisms. Although intestinal macrophages (Ms) have been implicated in repression of excessive inflammation, it remains unclear how their functions are regulated during inflammation. In this study, we report that semaphorin 7A (Sema7A), a GPI-anchored semaphorin expressed in intestinal epithelial cells (IECs), induces IL-10 production by intestinal M?s to regulate intestinal inflammation. Sema7A-deficient mice showed severe signs of dextran sodium sulfate-induced colitis due to reduced intestinal IL-10 levels. We further identified CX3CR1(+)MHC class II(int)F4/80(hi)CD11b(hi) M?s as the main producers of IL-10 via αvβ1 integrin in response to Sema7A. Notably, Sema7A was predominantly expressed on the basolateral side of IECs, and its expression pattern was responsible for protective effects against dextran sodium sulfate-induced colitis and IL-10 production by M?s during interactions between IECs and M?s. Furthermore, we determined that the administration of recombinant Sema7A proteins ameliorated the severity of colitis, and these effects were diminished by IL-10-blocking Abs. Therefore, our findings not only indicate that Sema7A plays crucial roles in suppressing intestinal inflammation through αvβ1 integrin, but also provide a novel mode of IL-10 induction via interactions between IECs and M?s.