An essential role of adenosine deaminase acting on RNA 1 in coeliac disease mucosa

Figures

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  Figure 1. ADAR1 expression is down-regulated in duodenal mucosa of patients with active CD. (A) Total RNA was obtained by duodenal biopsies of 15 normal controls (CTR), 8 inactive CD patients (ICD) and 10 active CD patients (ACD) and ADAR1 expression analyzed by Real-time PCR. *p<0.05. (B) Representative expression of ADAR1 (upper blot) and β-actin (lower blot) protein in duodenal mucosal samples taken from normal control, inactive and active CD patients. The blot is representative of four separate experiments analyzing total mucosal samples from 15 normal controls, 8 inactive CD patients and 10 active CD patients. Quantitative data are shown in the right panel as measured by densitometry scanning of all Western blots. Values are expressed in arbitrary units (a.u.). Each point represents the value of ADAR1/β-actin ratio in mucosal samples taken from a single subject. Horizontal bars indicate the median value. *p<0.01. (C) Immunohistochemical images, representative of 3 separate experiments in which similar results were obtained, showing ADAR1-positive cells in duodenal sections of normal controls, inactive CD and active CD patients. Staining with isotype control IgG is also shown. (D)…

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  Figure 2. Incubation of inactive CD biopsy with a peptic-tryptic digest of gliadin (PT) results in reduced expression of ADAR1. (A) Representative expression of ADAR1 (upper blot) and β-actin (lower blot) protein in duodenal mucosal samples taken from duodenal biopsies of one inactive CD patient left untreated (Unst), stimulated with BSA or with PT for 36 hours. Quantitative data are shown in the right panel as measured by densitometry scanning of all Western blots. Values are expressed in arbitrary units (a.u.) as mean ± SEM of 4 separate experiments. *p<0.05. (B) Representative expression of ADAR1 (upper blot) and β-actin (lower blot) in duodenal mucosal samples taken from duodenal biopsies of inactive CD patient cultured without (Unst, unstimulated) or with recombinant human IL-15 (50 ng/ml), IFN-γ (100 ng/ml) and IL-21 (50 ng/ml) for 24h. Quantitative data are shown in the right panel as measured by densitometry scanning of all Western blots. Values are expressed in arbitrary units (a.u.) as mean ± SEM of 3 separate experiments. *p<0.05.

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  Figure 3. Knock-down of ADAR1 in inactive CD LPMCs enhances inflammatory pathway. (A) Active IRF3 (phosphorylated, p-IRF3, upper panel), active IRF7 (phosphorylated, p-IRF7, middle panel) and β-actin (lower panel) protein expression in LPMCs isolated from one inactive CD patient and stimulated with poly I:C for 1 hour and pre-incubated in presence of a specific ADAR1 antisense oligonucleotide (AS) or a control oligonucleotide (S) for 24h. Quantitative data (right panel) are presented as mean ± SEM of 4 separate experiments. *p<0.05. (B) LMPCs were isolated from one inactive CD patient and stimulated with poly I:C for 12h and pre-incubated with the specific ADAR1 antisense oligonucleotide (AS) or a control oligonucleotide (S) for 24h. Representative histoplots showing IFN-α/β, IFN-γ and TNF-α expression in CD45+ cells analyzed by flow-cytometry. Data are shown as mean ± SEM of 4 separate experiments. *p<0.05, **p<0.01.

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  Figure 4. Mice treated with ADAR1 antisense develop a severe poly I:C-induced villous atrophy. (A) Representative H&E-stained small intestinal sections of mice left untreated (CTR), receiving poly I:C with a control oligonucleotide (poly I:C+S) or poly I:C with a specific ADAR1 antisense oligonucleotide (poly I:C+AS). Right inset shows the histological score of the small intestine sections taken from all groups of mice. *p<0.01. (B) ADAR1, TNF-α and IFN-γ mRNAs were analyzed in the small intestine of control mice (CTR), mice treated with poly I:C+S and mice receiving poly I:C+AS by real-time PCR and normalized to β-actin. *p<0.05. (C) Colonic samples taken from all mice treated as above, were analyzed for TNF-α, IFN-γ and IFN-α by enzyme-linked immunosorbent assay. *p=0.05. All data indicate mean ± SEM of 3 separated experiments in which at least 5 mice/group were considered.