Proenkephalin deletion in hematopoietic cells induces intestinal barrier failure resulting in clinical feature similarities with irritable bowel syndrome in mice

Opioid-dependent immune-mediated analgesic effects have been broadly reported upon inflammation. In preclinical mouse models of intestinal inflammatory diseases, the local release of enkephalins (endogenous opioids) by colitogenic T lymphocytes alleviate inflammationinduced pain by down-modulating gut-innervating nociceptor activation in periphery. In this study, we wondered whether this immune cell-derived enkephalin-mediated regulation of the nociceptor activity also operates under steady state conditions. Here, we show that chimeric mice engrafted with enkephalin-deficient bone marrow cells exhibit not only visceral hypersensitivity but also an increase in both epithelial paracellular and transcellular permeability, an alteration of the microbial topography resulting in increased bacteriaepithelium interactions and a higher frequency of IgA-producing plasma cells in Peyer's patches. All these alterations of the intestinal homeostasis are associated with an anxiety-like behavior despite the absence of an overt inflammation as observed in patients with irritable bowel syndrome. Thus, our results show that immune cell-derived enkephalins play a pivotal role in maintaining gut homeostasis and normal behavior in mice. Because a defect in the mucosal opioid system remarkably mimics some major clinical symptoms of the irritable bowel syndrome, its identification might help to stratify subgroups of patients. Overall design: Transcriptome profiling was performed on total RNAs (extracted as described below) of colonic biopsies originating from 4 animals in each group of penk+/+ and penk-/- chimeric mice. The corresponding RNA-seq paired-end libraries were prepared according to Illumina's 18 protocol with some adjustments, using the TruSeq Stranded mRNA library prep Kit (Illumina, San Diego, USA). Briefly, mRNAs were first selected from 10 µg total RNA using poly-T beads. Then, RNAs were fragmented during 2 minutes and retrotranscribed to generate double stranded cDNA. Compatible adaptors were ligated, allowing the barcoding of the samples with unique dual indices. Twelve cycles of PCR were applied to amplify libraries, and a final purification step allowed to obtain 280-1000 pb fragments. Libraries quality was assessed using the HS NGS kit on the Fragment Analyzer (Agilent Technologies, Santa Clara, USA). Libraries quantification and sequencing were performed at the GeT-PlaGe core facility (INRAe, Toulouse, France). Libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche, Basel, Switzerland) to obtain an accurate quantification.