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

Abstract

The gut virome is a complex community that exists in equilibrium with the host. Disruptions of this balance could drive the development of inflammatory diseases, such as inflammatory bowel disease (IBD). RNA editing, particularly A-to-I editing by ADAR1, prevents the excessive immune response to viral double strand (ds) RNA. Failure of RNA editing may sustain inflammation and this study explore the role of ADAR1 in IBD. ADAR1 was analyzed in IBD patients and healthy controls (CTR) using western blotting and qPCR. Colonic epithelial cells (HCEC-1CT), ex vivo organ cultures, and colonic organoids were treated poly I:C after ADAR1 silencing with an antisense oligonucleotide (AS). Inflammatory pathways and PANoptosome were measured by western blotting, flow cytometry, and ELISA. The role of ADAR1 was also studied in DSS-colitis model. ADAR1 was significantly reduced in the inflamed epithelium of ulcerative colitis (UC) gut samples. ADAR1 silencing in HCEC-1CT, ex vivo organ cultures or colonic organoids strongly increases the immune response to poly I:C and leads to activation of inflammatory pathways and PANoptosis. Inhibition of gut ADAR1 expression during DSS-colitis exacerbated gut inflammation. JAK inhibition or AhR activation mitigated the immune response that follows ADAR1 silencing. These data suggest that ADAR1 could be involved in IBD inflammation.

Keywords: adenosine deaminase acting on RNA 1; inflammatory bowel disease; innate immunity; mucosal damage; panoptosis.

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 in tissue lysates from ileal biopsies of two CTR and two patients with CD. (D) Representative western blot showing ADAR1 and β‐actin expression in tissue lysates from paired colonic biopsies of uninflamed (U) and inflamed (I) areas of two patients with UC. (E) Representative western blot showing ADAR1 and β‐actin expression in intestinal epithelial cells (IEC) and lamina propria mononuclear cells (LPMC) in colonic biopsies of one CTR and one patient with UC. Right panel shows the densitometric analysis of ADAR1/β‐actin ratio of the samples analyzed (7 CTR and 7 patients with UC). Values are expressed in arbitrary units (a.u.). Data are shown as mean ± SEM. ****p < 0.0001; two‐way ANOVA followed by Sidak's test. (F) Correlation of densitometric analysis of ADAR1/β‐actin ratio in IEC of UC patients with their relative Mayo endoscopic score (eMayo). Each dot is a single biopsy. Data analyzed by Pearson's correlation. (G) Correlation of densitometric analysis of ADAR1/β‐actin ratio in LPMC of UC patients with their relative Mayo endoscopic score (eMayo). Each dot is a single biopsy. Data analyzed by Pearson's correlation.

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 16 h and then stimulated with poly I:C for 8 h. Data are expressed as mean ± SEM of three independent experiments (*p < 0.05, **p < 0.01; Student's t test). (E) Protein concentration of TNF‐α, IFN‐α, and IFN‐β determined by ELISA in the culture supernatants harvested from colon organoids derived from colonic biopsies of healthy controls 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).

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 (cleaved caspase 3 or Cl‐Casp3), active caspase 1 (cleaved caspase 1 or Cl‐Casp1), and β‐actin expression in ex vivo organ cultures of healthy colonic mucosal samples transfected with ADAR1 sense (S) or antisense (AS) oligonucleotides for 16 h and then stimulated with poly I:C for 8 h. (E) Protein concentration of IL1‐β determined by ELISA in the culture supernatants harvested from ex vivo organ cultures treated as in (D). Data are expressed as mean ± SEM of three independent experiments (*p < 0.05, **p < 0.01; Student's t test). (F) Representative images of colon organoids derived from colonic biopsies of healthy controls transfected with ADAR1 sense (S) or antisense (AS) oligonucleotides for 24 h and then stimulated or not with poly I:C for 48 h. Three time points are shown after poly I:C challenging (0, 24, and 48 h). Scale bars: 100 µm. (G) Quantification of cell death (%) in colon organoids treated as in (F) measured by trypan blue dye. Data are expressed as mean ± SEM of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001; two‐way ANOVA followed by Sidak's test). (H) Protein concentration of IL1‐β determined by ELISA in the culture supernatants harvested from colon organoids treated as in (F). Data are expressed as mean ± SEM of three independent experiments (*p < 0.05; two‐way ANOVA followed by Sidak's test).

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 the colonic tissue lysates of mice from (A). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; one‐way ANOVA followed by Dunnett's test).

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 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 UC patients' colonic mucosal samples treated for 20 h with DMSO or Ficz (200 nM) and then stimulated with poly I:C for 1 h. Values are expressed as arbitrary units (a.u.). Right panel shows the densitometric analysis of ADAR/β‐actin, p65/ β‐actin, and pTBK1/β‐actin ratio of the samples analyzed. Values are expressed as arbitrary units (a.u.). Data are expressed as mean ± SEM of three independent experiments. (*p < 0.05, **p < 0.01, ****p < 0.0001; one‐way ANOVA followed by Dunnett's test).