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bioRxiv · 10.1101/2020.06.15.151621

Lack of mucosal cholinergic innervation is associated with increased IL-23-induced T-helper-17 response in Hirschsprung disease

Keck, S.·Galati-Fournier, V.·Kym, U.·Moesch, M.·Usemann, J.·Subotic, U.·Tharakan, S.·Krebs, T.·Stathopoulos, E.·Schmittenbecher, P.·Cholewa, D.·Romero, P.·Reingruber, B.·Bruder, E.·NIG Study Group,·Holland-Cunz, S.
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Abstract

Background & AimsHirschsprung’s disease (HSCR) is a congenital intestinal motility disorder defined by the absence of enteric nervous cells (ganglia). The development of HSCR-associated enterocolitis remains a life-threatening complication. Absence of enteric ganglia implicates extramural innervation of acetylcholine-secreting (cholinergic) nerve fibers. Cholinergic signals have been reported to control excessive inflammation, but the impact on HSCR-associated enterocolitis is unknown.Methods We enrolled 44 HSCR patients in a prospective multicenter study and grouped them according to their degree of colonic mucosal cholinergic innervation using immunohistochemistry. The fiber phenotype was correlated with the tissue cytokine profile as well as immune cell frequencies using quantitative reverse-transcribed real-time polymerase chain reaction (qRT-PCR) of whole colonic tissue and fluorescence-activated cell sorting (FACS) analysis of isolated colonic immune cells. Fiber-associated immune cells were identified using confocal immunofluorescence microscopy and characterized by RNA-seq analysis. Microbial dysbiosis was analyzed in colonic patient tissue using 16S rDNA gene sequencing. Finally, the fiber phenotype was correlated with postoperative enterocolitis manifestation.Results We provided evidence that extrinsic mucosal innervation correlated with reduced interleukin (IL)-17 cytokine levels and T-helper-17 (Th17) cell frequencies. Bipolar CD14high macrophages colocalized with neurons and expressed significantly less interleukin-23, a Th17-promoting cytokine. HSCR patients lacking mucosal cholinergic nerve fibers showed microbial dysbiosis and had a higher incidence of postoperative enterocolitis.Conclusion The mucosal fiber phenotype might serve as a new prognostic marker for enterocolitis development in HSCR patients and may offer an approach to personalized patient care and new future therapeutic options. (www.clinicaltrials.gov accessing number NCT03617640)Competing Interest StatementThe authors have declared no competing interest.Abbrevations used in this paperHSCRHirschsprung’s diseaseFACSFluorescence-activated cell sortingqRT-PCRQuantitative reverse-transcribed real-time polymerase chain reactionILInterleukinTh17T-helper-17ENSEnteric nervous systemAChAcetylcholineα7nAChRα7-nicotinic ACh receptorCAIPCholinergic-anti-inflammatory-pathwayVNVagus nerveMΦMacrophagesMMΦMuscle macrophagesTNFtumor necrosis factorPOIPostoperative ileusCDColon descendensRECRectumSCSigmoid colonVIPVasoactive intestinal peptideTHTyrosine hydroxylaseNOSNitric oxide synthaseNKNatural killerNCRNK cell receptorsAPCAntigen-presenting cellPCAPrincipal component analysisHLAHuman leucocyte antigenGDFGrowth/differentiation factorNRP1Neuropilin-1UNC5BUNC-5 homology BEPHB2Ephrin type B-receptor 2SEMASemaphorinLPLamina propriaFISHFluorescence in situ hybridizationOTUsOperational taxonomic unitsBMPBone morphogenetic proteinTLRToll-like receptorView Full Text

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Keck, S., Galati-Fournier, V., Kym, U., Moesch, M., Usemann, J., Subotic, U., Tharakan, S., Krebs, T., Stathopoulos, E., Schmittenbecher, P., Cholewa, D., Romero, P., Reingruber, B., Bruder, E., NIG Study Group,, Holland-Cunz, S.. 2020-06-15. Lack of mucosal cholinergic innervation is associated with increased IL-23-induced T-helper-17 response in Hirschsprung disease. https://doi.org/10.1101/2020.06.15.151621

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