Loss of Adenosine Deaminase Acting on RNA 1 Induces Panoptosis and Immune Response in Ulcerative Colitis Gut Mucosa

Figures

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  FIGURE 1. ADAR1 expression is reduced in inflamed gut of ulcerative colitis patients. (A) RNA expression of ADAR was evaluated by qRT‐PCR in colonic biopsies taken from 10 healthy controls (CTR), 15 patients with ulcerative colitis (UC), and 11 patients with Crohn's disease (CD). Values were normalized to β‐actin mRNA. Results are shown as box plots (25th to 75th percentile; horizontal lines represent median, maximum, and minimum values). *p < 0.05; one‐way ANOVA followed by Dunnett's test. (B) Representative western blot showing ADAR1 p150, ADAR1 p110, and β‐actin expression in tissue lysates from colonic biopsies of two CTR, three patients with UC, and three patients with CD. Lower panel shows the densitometric analysis of ADAR1/β‐actin ratio of the samples analyzed (left panel p150 isoform, right panel p110 isoform; 10 CTR, 15 patients with UC, and 11 patients with CD). Values are expressed as arbitrary units (a.u.). Results are shown as box plots (25th to 75th percentile; horizontal lines represent median, maximum, and minimum values). **p < 0.01, ***p < 0.001, ****p < 0.0001; one‐way ANOVA followed by Dunnett's test. (C) Representative western blot showing ADAR1 and β‐actin expression…

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  FIGURE 2. Knock‐down of ADAR1 increases gut epithelial inflammation. (A) Representative western blot showing ADAR1, active p65 (phosphorylated p65 or p‐p65), active TBK1 (phosphorylated TBK1 or p‐TBK1), and β‐actin expression in HCEC‐1CT transfected with ADAR1 sense (S) or antisense (AS) oligonucleotides for 24 h and then stimulated or not with poly I:C for 1 h. (B) Protein concentration of TNF‐α and IFN‐β determined by ELISA in the culture supernatants harvested from HCEC‐1CT transfected with ADAR1 S or AS for 24 h and then stimulated or not with poly I:C for 16 h. Data are expressed as mean ± SEM of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; two‐way ANOVA followed by Sidak's test). (C) Representative western blot showing ADAR1, active p65 (phosphorylated p65 or p‐p65), active TBK1 (phosphorylated TBK1 or p‐TBK1), and β‐actin expression in ex vivo organ cultures of healthy colonic mucosal samples transfected with ADAR1 S or AS for 20 h and then stimulated with poly I:C for 1 h. (D) Protein concentration of TNF‐α, IFN‐α, and IFN‐β determined by ELISA in the culture supernatants harvested from ex vivo organ cultures transfected with ADAR1 S or AS for…

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  FIGURE 3. Knock‐down of ADAR1 induces PANoptosis. (A) Representative western blot showing ADAR1, active RIPK3 (phosphorylated RIPK3 or p‐RIPK3), active caspase 3 (cleaved caspase 3 or Cl‐Casp3), active caspase 1 (cleaved caspase 1 or Cl‐Casp1), and β‐actin expression in HCEC‐1CT transfected with ADAR1 sense (S) or antisense (AS) oligonucleotides for 24 h and then stimulated or not with poly I:C for 48 h. (B) Representative dot plot of annexin V (AnnV)‐ and propidium iodide (PI)‐positive HCEC‐1CT treated as indicated in (A). Right panel shows quantification of the percentage of AnnV and/or PI‐positive cells. Data are expressed as mean ± SEM of three independent experiments (the p value refers to comparison of AnnV+/PI+ cells among the groups; *p < 0.05, ***p < 0.001, ****p < 0.0001; two‐way ANOVA followed by Sidak's test). (C) Protein concentration of IL1‐β determined by ELISA in the culture supernatants harvested from HCEC‐1CT treated as in (A). Data are expressed as mean ± SEM of three independent experiments (**p < 0.01, ****p < 0.0001; two‐way ANOVA followed by Sidak's test). (D) Representative western blot showing ADAR1, active RIPK3 (phosphorylated RIPK3 or p‐RIPK3), active caspase 3…

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  FIGURE 4. Mice with reduced expression of ADAR1 develop a more severe DSS‐induced colitis. (A) Scheme depicting DSS‐mediated colitis in which mice were given orally ADAR1 S or AS oligonucleotides 24 and 6 h before starting DSS administration (Day 0) and then every other day until Day 8. Control mice received regular drinking water (CTR). Two independent experiments were performed and at least 5 mice per group were used in each experiment. (B) Body weights of mice, from (A), were monitored daily and each point on the graph indicates the cumulative mean ± SEM of the body weights. (*p < 0.05, ***p < 0.001; two‐way ANOVA followed by Sidak's test). (C) Representative H&E‐stained colonic sections of mice from (A). Original magnification 20×. Right inset shows the histologic score of the colonic sections. Data are expressed as mean ± SEM. (**p < 0.01, ****p < 0.0001; one‐way ANOVA followed by Dunnett's test). (D) Representative western blot showing ADAR1, active p65, active TBK1, active RIPK3, active caspase 3, active caspase 1, and β‐actin expression of colonic tissue lysates of mice from (A). (E) Protein concentration of TNF‐α, IL‐6, IFN‐γ, IL‐17A, IL1‐β, IFN‐α, and IFN‐β determined by ELISA in…

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  FIGURE 5. JAK inhibition or AhR activation prevent ADAR1 knock‐down mediated excessive immune responses to dsRNA. (A) Representative western blot showing ADAR1 and β‐actin expression in tissue lysates from colon biopsies of three patients with UC before JAKi treatment (R = responder, NR = non responder). (B) Representative western blot showing ADAR1, active STAT1 (phosphorylated STAT1 or p‐STAT1), active STAT3 (phosphorylated STAT3 or p‐STAT3), and β‐actin expression in ex vivo organ cultures of healthy colonic mucosal samples transfected with ADAR1 sense (S) or antisense (AS) oligonucleotides together or not with 1 µM of JAKis (upadacitinib or UPA, tofacitinib or TOFA, filgotinib or FILGO) for 20 h and then stimulated with poly I:C for 1 h. Right panels show the densitometric analysis of pSTAT1/β‐actin (upper panel) and pSTAT3/β‐actin (lower panel) ratio of the samples analyzed. Values are expressed as arbitrary units (a.u.). Data are representative of one experiment. (C, D) Representative western blot showing ADAR1 and β‐actin expression in HCEC‐1CT (C) or ex vivo organ cultures of UC patients' colonic mucosal samples (D) stimulated for 24 h with DMSO or Ficz (200 nM). (E) Representative…