In vivo targeting of miR-223 in experimental eosinophilic oesophagitis

Abstract

Objectives: Eosinophilic oesophagitis (EoE) is characterised by oesophageal inflammation, fibrosis and dysfunction. Micro (mi)-RNAs interfere with pro-inflammatory and pro-fibrotic transcriptional programs, and miR-223 was upregulated in oesophageal mucosal biopsy specimens from EoE patients. The therapeutic potential of modulating miR-223 expression in vivo has not been determined. We aimed to elucidate the relevance of oesophageal miR-223 expression in an in vivo model of EoE by inhibiting miR-223 tissue expression.

Methods: The expression of miR-223 and the validated miR-223 target insulin-like growth factor receptor 1 (IGF1R) protein was determined in our paediatric cohort of EoE patients. A murine model of Aspergillus fumigatus-induced EoE was employed, and oesophagi were assessed for miR-233, IGF1R, T lymphocyte type 2 (T2) cytokine expression and eosinophil infiltration. Mice were treated with antagomirs targeting miR-223 or resveratrol targeting its upstream regulator Midline-1(MID-1).

Results: There was an inverse relationship between an increased expression of miR-223 and a decreased IGF1R protein concentration in biopsy specimens from EoE patients. TNF-related apoptosis-inducing ligand deficiency, MID-1 inhibition and resveratrol treatment suppressed miR-223 expression. Furthermore, inhibition of miR-223 and treatment with resveratrol in the oesophagus resulted in an amelioration of EoE hallmark features including eosinophilic infiltration, oesophageal circumference and a reduction in T2 cytokine expression.

Conclusion: miR-223 has a key role in the perpetuation of EoE hallmark features downstream of TNF-related apoptosis-inducing ligand and MID-1 in an experimental model. These studies highlight a potentially critical role of miRNA function in EoE aetiology. miR-223 expression in the oesophagus may be therapeutically modulated by resveratrol, providing a potential new therapeutic option to be explored in EoE patients for this increasingly prevalent condition.

Keywords: Midline‐1; eosinophilic oesophagitis; microRNA; resveratrol.

Figure 1

miR‐223 is upregulated in eosinophilic oesophagitis (EoE) patients and is inversely correlated with IGF1R protein expression. (a) Relative expression of miR‐223 in EoE patients (n = 17) normalised to U6 and healthy controls (n = 21) determined by TaqMan™ qPCR. (b) IGF1R protein as quantified by ELISA is downregulated in biopsies from EoE patients compared to control biopsies. Statistical significance was determined using Mann–Whitney analysis. (c) IGF1R protein correlated with miR‐223 expression in EoE and patient biopsies (n = 17). Statistical significance was determined using Spearman's correlations (r). Data are from a single technical replicate experiment, expressed as mean ± SEM. **P < 0.01, ***P < 0.001.

Figure 2

miR‐223 expression is upregulated in Aspergillus fumigatus (Asp F)‐exposed murine oesophagi, is associated with IGF1R downregulation and is downstream of TNF‐related apoptosis‐inducing ligand (TRAIL) and MID‐1. Relative expression of oesophageal miR‐223 in vivo in (a) wild‐type BALB/c mice treated with scrambled (SCR) antagomir or antagomir targeting miR‐223 (A.223). (b) Expression of IGF1R in BALB/c oesophagi exposed to A. fumigatus and treated with SCR or A.223. (c) Spearman's correlation of oesophageal miR‐223 and IGF1R in BALB/c mice. (d, e) miR‐223 expression of (d) A. fumigatus‐exposed wild‐type BALB/c (WT) and TRAIL‐deficient^−/− mice on a BALB/c background and (e) wild‐type BALB/c mice treated with A. fumigatus and administered either nonsense scrambled control sequence (NONc) siRNA or siRNA targeting Midline‐1 (MID‐1). Expression is normalised to Sno202 and saline (SAL) control group for miR‐223, and β‐actin and SAL control group for IGF1R (n = 5–7). Data are from a single technical replicate experiment, expressed as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 3

Inhibition of miR‐223 in the oesophagus results in reduced eosinophilic infiltration of the oesophagus and oesophageal enlargement with antagomir‐223. (a) Enumeration of eosinophils present in the oesophageal squamous‐epithelial cell layer as determined by light microscopy analysis of H&E‐stained BALB/c oesophageal transverse sections (n = 4 or 5).(b–d)Representative images of (b) SAL, (c) AspF/SCR or (d) AspF/A.223 with eosinophils circled in yellow. (e) Oesophageal fibrosis analysis with representative images of (f) SAL, (g) AspF/Scr and (h) AspF/A223‐treated BALB/c mice. (i) Oesophageal circumference analysis of oesophageal tissue determined using image analysis (n = 5–8). (j, k) Protein expression of (j) IL‐5 and (k) IL‐13 determined by ELISA and normalised to tissue BSA (n = 5–8). Data are from a single technical replicate experiment, expressed as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.001.

Figure 4

Resveratrol reduces MID‐1 and miR‐223 expression while reducing eosinophilic oesophagitis (EoE) features. (a) The E3 ubiquitin ligase Midline‐1 (MID‐1) is upregulated in Aspergillus fumigatus (Asp F)‐exposed C57Bl/6 mice and inhibited by oral resveratrol (RES; n = 3–6). (b) Resveratrol treatment decreased miR‐223 expression (n = 5 or 6). (c) Enumeration of eosinophils present in the oesophageal squamous‐epithelial cell layer (per mm²) as determined by light microscopy analysis of H&E‐stained C57Bl/6 oesophageal transverse sections (n = 6 or 7). (d) Oesophageal circumference analysis of oesophageal tissue determined using image analysis (n = 6 or 7) in mice treated with RES. (e, f) Protein expression of (e) IL‐5 and (f) IL‐13 determined by ELISA and normalised to tissue BSA (n = 5 or 6). Data are from a single technical replicate experiment, expressed as mean ± SEM. *P < 0.05, **P < 0.01.