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

Altered CD4⁺ cells differentiation in NEC patients.

<p>Mucosal mRNA expression levels of <i>CD45</i> (A), <i>CD4</i> (B), <i>RORC</i> (C), <i>IL17A</i> (D), <i>FOXP3</i> (E) and <i>CTLA4</i> (F) 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

Altered relative amount of CD4⁺ cells in NEC patients.

<p>Mucosal mRNA expression levels of <i>TBX21</i> (G) and <i>GATA3</i> (H) in the ileum of patients were quantified using qPCR and normalized to the mRNA expression levels of <i>CD4</i>. Asterisks indicate statistical significant differences between indicated groups. </p

Intestinal morphology of NEC patients.

<div><p>Ileal tissues from NEC patients were stained with hematoxylin and eosin and representative sections are shown. </p> <p>(A) Ileum of an A-NEC-XBP1u patient with severe morphological damage. </p> <p>(B) Ileum of an A-NEC-XBP1u patient with mild morphological damage. </p> <p>(C) Ileum of an A-NEC-XBP1s patient with complete crypt-villus loss but remaining surface epithelium. </p> <p>(D) Ileum of an A-NEC-XBP1s patient who hardly had epithelium left. </p> <p>(E) Ileum of an R-NEC patient with mild mucosal damage. </p></div

ER stress and the UPR up-regulated the expression levels of <i>IL6</i> and <i>IL8</i>.

<p>The mRNA expression levels of <i>IL6</i> (A,C & E) and <i>IL8</i> (B, D & F) in the ileum of patients (A&B), THP-1 cells (C & D) and HIEC cells (E & F) were quantified using qPCR and normalized to the mRNA expression levels of <i>GAPDH</i>. Asterisks indicate statistical significant differences between indicated groups. </p