Combined NADPH Oxidase 1 and Interleukin 10 Deficiency Induces Chronic Endoplasmic Reticulum Stress and Causes Ulcerative Colitis-Like Disease in Mice

<div><p>Ulcerative colitis (UC) is a chronic inflammatory bowel disease affecting the rectum which progressively extents. Its etiology remains unknown and the number of treatments available is limited. Studies of UC patients have identified an unbalanced endoplasmic reticulum (ER) stress in the non-inflamed colonic mucosa. Animal models with impaired ER stress are sensitive to intestinal inflammation, suggesting that an unbalanced ER stress could cause inflammation. However, there are no ER stress-regulating strategies proposed in the management of UC partly because of the lack of relevant preclinical model mimicking the disease. Here we generated the IL10/Nox1^dKO mouse model which combines immune dysfunction (IL-10 deficiency) and abnormal epithelium (NADPH oxidase 1 (Nox1) deficiency) and spontaneously develops a UC-like phenotype with similar complications (colorectal cancer) than UC. Our data identified an unanticipated combined role of IL10 and Nox1 in the fine-tuning of ER stress responses in goblet cells. As in humans, the ER stress was unbalanced in mice with decreased eIF2α phosphorylation preceding inflammation. In IL10/Nox1^dKO mice, salubrinal preserved eIF2α phosphorylation through inhibition of the regulatory subunit of the protein phosphatase 1 PP1R15A/GADD34 and prevented colitis. Thus, this new experimental model highlighted the central role of epithelial ER stress abnormalities in the development of colitis and defined the defective eIF2α pathway as a key pathophysiological target for UC. Therefore, specific regulators able to restore the defective eIF2α pathway could lead to the molecular remission needed to treat UC.</p></div

Altered goblet cells and mucin expression in IL10/Nox1^dKO mice.

<p>(<b>A</b>) Representative sections of the distal colon of 7-week old WT (n = 10), Nox1^KO (n = 10), IL10^KO (n = 10), and IL10/Nox1^dKO (n = 20) mice stained with AB/PAS. The distal colon of 7-week old WT (n = 5), Nox1^KO (n = 5), IL10^KO (n = 5), and IL10/Nox1^dKO (n = 5) mice is shown both in transverse and longitudinal semi-thin sections. (<b>B</b>) Immunohistological analysis of Muc2 and Muc4 in distal colonic sections of 7-weeks old WT (n = 5), Nox1^KO (n = 5), IL10^KO (n = 5), and IL10/Nox1^dKO (n = 5) mice. (<b>C</b>) Representative sections of the distal colon of 3-week old WT (n = 5) and IL10/Nox1^dKO (n = 5) mice stained with AB/PAS (upper panels) or with anti-Muc2 antibody (lower panels).</p

Spontaneous colitis in IL10/Nox1^dKO mice.

<p>(<b>A</b>) The DAI is measured daily in WT (n = 10), Nox1^KO (n = 10), IL10^KO (n = 10), and IL10/Nox1^dKO (n = 25) mice. Statistics: <i>p</i>-values for Kruskal-Wallis non-parametric analysis are shown, Dunn’s multiple comparison test versus WT; *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. (<b>B</b>) Clinical symptoms of IL10/Nox1^dKO mice. Body weight changes, rectal bleeding and stool scores were assessed daily. The weight-to-length ratio was determined for each individual colon of WT (n = 10), Nox1^KO (n = 10), IL10^KO (n = 10), and IL10/Nox1^dKO (n = 25) mice aged 7 and 12 weeks. Statistics: box plots show median, quartiles, and range; <i>p</i>-values for Kruskal-Wallis non-parametric analysis are shown, Dunn's multiple comparison test <i>vs</i>. WT, NS, not significant. (<b>C</b>) Percentage of WT (n = 10), Nox1^KO (n = 10), IL10^KO (n = 20), and IL10/Nox1^dKO (n = 30) mice with prolapse at 7 and 12 weeks of age. Statistics are as in (B).</p

Similar ultrastructural abnormalities in the colonic epithelium of IL10/Nox1^dKO mice and patients with UC.

<p>(<b>A</b>) Representative transmission electron micrographs of the unaffected colon of 4-week old WT (n = 5), Nox1^KO (n = 5), IL10^KO (n = 10), and IL10/Nox1^dKO (n = 10) mice. (<b>B</b>) Representative transmission electron micrographs of the unaffected colon of 10 healthy subjects and 10 patients with UC. Note that IL10/Nox1^dKO mice display morphological goblet cell abnormalities similar to those found in patients with UC including reduced size of thecae containing stored mucins, immature mucus granules, and swollen mitochondria. (<b>C</b>) Representative scanning electron micrographs (SEM) of distal colonic crypts of 6-week old WT (n = 5), Nox1^KO (n = 5), IL10^KO (n = 10), and IL10/Nox1^dKO (n = 10) mice. Note that the Lieberkhün’s crypts are longer in IL10/Nox1^dKO mice. (<b>D</b>) Representative scanning electron micrographs of the distal colonic epithelium of 6-week old WT (n = 5), Nox1^KO (n = 5), IL10^KO (n = 8), and IL10/Nox1^dKO (n = 10) mice. Note the inappropriate pattern of crypt openings (arrowheads) on SEM with enlarged extrusive zones and dilated gland lumen in the distal colon of IL10/Nox1^dKO mice. (<b>E</b>) Representative SEM of the unaffected colonic mucosa of 10 healthy subjects and 10 patients with UC. Note the regular pattern of crypt openings with diffuse edema, enlarged extrusive zones, and dilated gland lumen similar to those found in IL10/Nox1^dKO mice. Abbreviations: GC: goblet cell, C: colonocyte, T: thecae, LM: lamina propria, M: mitochondria.</p

Nox1 and IL10 negatively regulate the UPR in cultured intestinal goblet cells.

<p>(<b>A</b>) HT-29Cl16E cells carrying siRNA scrambled or Nox1 siRNA were treated in triplicate with vehicle (Ctrl), IL10 (50 ng/ml), TM (5 µg/ml), or IL10+TM. <i>NOX1</i> mRNA levels were determined by qPCR and normalized to β-actin with the mean ratio of the control group corrected to 1. Statistics: <i>p</i>-values for Kruskal-Wallis non-parametric analysis are shown, Dunn’s multiple comparison test versus Ctrl, **<i>p</i><0.01, ***<i>p</i><0.001. (<b>B</b>) eIF2α phosphorylation was measured using an Alphascreen <i>SureFire</i> P-Ser⁵¹-eIF2α assay in three independent experiments (mean +/− SD). (<b>C</b>) Upper panel – HT-29Cl16E cells carrying siRNA scrambled or Nox1 siRNA were treated with vehicle (Ctrl), IL10 (50 ng/ml), TM (5 µg/ml), or IL10+TM. Proximity between PP1c and GADD34 was detected by PLA. Nuclei were stained with TO-PRO-3 iodide. Confocal photomicrographs are representative of four independent experiments (original magnification x40). Lower panel – Quantification of PLA signals for PP1c and GADD34 proximity (n = 8 per condition). Fluorescent signals were counted using Imaris software and the average number of spots per cell is represented (mean ± SD). (<b>D</b>) Upper panel - HT-29Cl16E cells were treated in triplicate with vehicle (Ctrl), thapsigargin (Tg, 5 µM), IL10 (50 ng/ml), or IL10+Tg. Lower panel - HT-29Cl16E cells carrying scrambled (si) or Nox1 siRNA (si NOX1) were treated in triplicate with vehicle (Ctrl) or Tg (5 µM). <i>XBP1s</i> and <i>GADD34</i> mRNA levels were determined by qPCR and normalized to β-actin Statistics: <i>p</i>-values for Kruskal-Wallis non-parametric analysis are shown, Dunn’s multiple comparison test versus Ctrl, *<i>p</i><0.05, ***<i>p</i><0.001. (<b>E</b>) Concentration of IL8 in supernatants from HT-29Cl16E cells carrying scrambled (si) or Nox1 siRNA (si NOX1) treated in triplicate with vehicle (Ctrl),), thapsigargin (Tg, 5 µM), IL10 (50 ng/ml), or IL10+Tg (mean S.D).</p

The IL10 protein and <i>NOX1</i> mRNA are detected in the non-inflamed colonic mucosa of controls (Ctrl, n = 12) and UC patients (n = 12) by ELISA and qPCR, respectively.

<p>Statistics: Mann-Whitney U-test (<i>p-</i>values shown).</p

Salubrinal rebalances the altered ER stress and prevents colitis.

<p>Three-4-week old IL10/Nox1^dKO mice received 1 mg/kg salubrinal (Sal) intraperitonally or vehicle (Vh) 3 times per week for 3 weeks. (<b>A</b>) Histological colitis scores were determined at 6–7 weeks of age from H&E-stained colonic sections. Statistics: box plots show median, quartiles, and range; Mann-Whitney U-test, <i>p</i>-values are shown. (<b>B</b>) Representative H&E-stained sections of the distal colon of Vh- (n = 10) or Sal (n = 15)-treated IL10/Nox1^dKO mice. (<b>C</b>) Left panels - Goblet cell staining with blue alcian/periodic acid Schiff stain on distal colonic sections of Vh- or Sal-treated IL10/Nox1^dKO mice (n = 15/group). Right panels - Representative transmission electron micrographs of the distal colon of Vh- (n = 6) or Sal- (n = 8) treated IL10/Nox1^dKO mice. (<b>D</b>). Representative immunoblot analysis of indicated protein expression in the distal colon of Vh- (n = 10) or Sal- (n = 15) treated IL10/Nox1^dKO mice aged 6–7 weeks. β-actin is used as loading control. The P-eIF2α/eIF2α ratio was measured and densitometric analyses are shown. <i>P</i>-values for Mann-Whitney U-test analysis are shown. (<b>E</b>) Representative immunohistological analysis of P-eIF2αβ (Ser⁵¹) in WT (n = 8), Vh- (n = 10) or Sal- (n = 15) treated IL10/Nox1^dKO mice aged 6–7 weeks. Note that the P-eIF2α epithelial staining in salubrinal-treated IL10/Nox1^dKO mice is similar to that of WT mice.</p