Mucin Muc2 Deficiency and Weaning Influences the Expression of the Innate Defense Genes Reg3β, Reg3γ and Angiogenin-4

Background Mucin Muc2 is the structural component of the intestinal mucus layer. Absence of Muc2 leads to loss of this layer allowing direct bacterial-epithelial interactions. We hypothesized that absence of the mucus layer leads to increased expression of innate defense peptides. Specifically, we aimed to study the consequence of Muc2 deficiency (Muc2-/-) on the expression of regenerating islet-derived protein 3 beta (Reg3ß), regenerating islet-derived protein 3 gamma (Reg3¿), and angiogenin-4 (Ang4) in the intestine shortly before and after weaning. Methods Intestinal tissues of Muc2-/- and wild-type (WT) mice were collected at postnatal day 14 (P14, i.e. pre-weaning) and P28 (i.e. post-weaning). Reg3ß, Reg3¿, and Ang4 expression was studied by quantitative real-time PCR, Western-blot, in situ hybridization, and immunohistochemistry. Results Reg3ß and Reg3¿ were expressed by diverging epithelial cell types; namely enterocytes, Paneth cells, and goblet cells. Additionally, Ang4 expression was confined to Paneth cells and goblet cells. Expression of Reg3ß, Reg3¿, and Ang4 differed between WT and Muc2-/- mice before and after weaning. Interestingly, absence of Muc2 strongly increased Reg3ß and Reg3¿ expression in the small intestine and colon. Finally, morphological signs of colitis were only observed in the distal colon of Muc2-/- mice at P28, where and when expression levels of Reg3ß, Reg3¿, and Ang4 were the lowest. Conclusions Expression of Reg3 proteins and Ang4 by goblet cells point to an important role for goblet cells in innate defense. Absence of Muc2 results in up-regulation of Reg3ß and Reg3¿ expression, suggesting altered bacterial-epithelial signaling and an innate defense response in Muc2-/- mice. The inverse correlation between colitis development and Reg3ß, Reg3¿, and Ang4 expression levels might point toward a role for these innate defense peptides in regulating intestinal inflammatio

Colitis development during the suckling-weaning transition in mucin Muc2-deficient mice

The mucin Muc2 is the structural component of the colonic mucus layer. Adult Muc2 knockout (Muc2(-/-)) mice suffer from severe colitis. We hypothesized that Muc2 deficiency induces inflammation before weaning of mother's milk [postnatal day (P) 14] with aggravation of colitis after weaning (P28). Muc2(-/-) and wild-type mice were killed at embryonic day 18.5 and P1.5, P7.5, P14, P21, and P28. Colonic morphology, influx of T cells, and goblet cell-specific protein expression was investigated by (immuno)histochemistry. Cytokine and Toll-like receptor (TLR) profiles in the colon were analyzed by quantitative RT-PCR. Muc2(-/-) mice showed an increased and persistent influx of Cd3ε-positive T cells in the colonic mucosa as of P1.5. This was accompanied by mucosal damage at P28 in the distal colon but not in the proximal colon. At P14, the proinflammatory immune response [i.e., increased interleukin (IL)-12 p35, IL-12 p40, and tumor necrosis factor-α, expression] in the distal colon of Muc2(-/-) mice presented with an immune suppressive response [i.e., increased Foxp3, transforming growth factor (TGF)-β1, IL-10, and Ebi3 expression]. In contrast, at P28, a proinflammatory response remained in the distal colon, whereas the immune suppressive response (i.e., Foxp3 and TGF-β1 expression) declined. The proximal colon of Muc2(-/-) mice did not show morphological damage and was dominated by an immune suppressive response at P14 and P28. Interestingly, changes in expression of TLRs and TLR-related molecules were observed in the distal colon at P14 and P28 and in the proximal colon only at P28. Colitis in Muc2(-/-) mice is limited before weaning by immune suppressive responses and exacerbates in the distal colon after weaning because of the decline in the immune suppressive respons

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

<div><p>Background</p><p>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.</p> <p>Methods</p><p>Ileal tissues from NEC and control patients were obtained during surgical resection and/or at stoma closure. Splicing of <i>XBP1</i> was detected using PCR, and gene expression was quantified using qPCR and Western blot.</p> <p>Results</p><p>Splicing of <i>XBP1</i> 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 <i>XBP1</i> splicing (A-NEC-XBP1s) had increased mucosal expression of <i>GRP78</i>, <i>CHOP</i>, <i>IL6</i> and <i>IL8</i>. Similar results were obtained by inducing ER stress and the UPR <i>in</i><i>vitro</i>. A-NEC-XBP1s patients showed altered T cell differentiation indicated by decreased mucosal expression of <i>RORC, IL17A</i> and <i>FOXP3</i>. 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 <i>IL6</i> and <i>IL8</i> expression and higher mucosal <i>FOXP3</i> expression.</p> <p>Conclusions</p><p>XBP1 splicing, ER stress and the UPR in NEC are associated with increased <i>IL6</i> and <i>IL8</i> expression levels, altered T cell differentiation and severe epithelial injury.</p> </div

Occurrence of ER stress and the UPR in a subset of A-NEC patients.

<div><p>(A) Splicing of <i>XBP1</i> mRNA was checked in all patients, and representative PCR products of <i>XBP1u</i> and <i>XBP1s</i> are shown using DNA electrophoresis. Dimethyl sulfoxide (DMSO)-treated HIEC cells were used as negative control for <i>XBP1s</i>, and TM-treated HIEC cells were used as positive control for <i>XBP1s</i>. </p> <p>Mucosal mRNA expression levels of <i>GRP78</i> (B) and <i>CHOP</i> (C) in the ileum of patients were quantified using qPCR and normalized to <i>GAPDH</i> mRNA levels. Asterisks indicate statistical significant differences between indicated groups. </p> <p>(D) The Western blot result shows the representative protein expression of GRP78 and CHOP in A-NEC-XBP1u and A-NEC-XBP1s patients, and beta ACTIN was used as loading control. </p></div