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An Experimental Approach to Rigorously Assess Paneth Cell ?-Defensin (Defa) mRNA Expression in C57BL/6 Mice.

ABSTRACT: Abundant evidence from many laboratories supports the premise that ?-defensin peptides secreted from Paneth cells are key mediators of host-microbe interactions in the small intestine that contribute to host defense and homeostasis. ?-defensins are among the most highly expressed antimicrobial peptides at this mucosal surface in many mammals, including humans and mice; however, there is striking variation among species in the number and primary structure of ?-defensin paralogs. Studies of these biomolecules in vivo are further complicated by striking variations between laboratory mouse strains. Herein, we report an experimental approach to determine with precision and specificity expression levels of ?-defensin (Defa) mRNA in the small intestine of C57BL/6 mice through an optimized set of oligonucleotide primers for qRT-PCR assays and cloned cDNA plasmids corresponding to the Defa paralogs. This approach demonstrated marked differences in ?-defensin expression in C57BL/6 mice with respect to proximal/distal anatomical location and developmental stage, which have not been described previously. These data underscore the importance of careful attention to method (primer choice, proximal vs. distal location, and developmental stage) in analysis of antimicrobial peptide expression and their impact.

SUBMITTER: Castillo PA

PROVIDER: S-EPMC6739474 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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An Experimental Approach to Rigorously Assess Paneth Cell α-Defensin (Defa) mRNA Expression in C57BL/6 Mice.

Castillo Patricia A PA Nonnecke Eric B EB Ossorio Daniel T DT Tran Michelle T N MTN Goley Stephanie M SM Lönnerdal Bo B Underwood Mark A MA Bevins Charles L CL

Scientific reports 20190911 1

Abundant evidence from many laboratories supports the premise that α-defensin peptides secreted from Paneth cells are key mediators of host-microbe interactions in the small intestine that contribute to host defense and homeostasis. α-defensins are among the most highly expressed antimicrobial peptides at this mucosal surface in many mammals, including humans and mice; however, there is striking variation among species in the number and primary structure of α-defensin paralogs. Studies of these ...[more]

PMID: 31511628

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Project description:IntroductionThe mechanism underlying radiation-induced gut microbiota dysbiosis is undefined. This study examined the effect of radiation on the intestinal Paneth cell α-defensin expression and its impact on microbiota composition and mucosal tissue injury and evaluated the radio-mitigative effect of human α-defensin 5 (HD5).MethodsAdult mice were subjected to total body irradiation, and Paneth cell α-defensin expression was evaluated by measuring α-defensin mRNA by RT-PCR and α-defensin peptide levels by mass spectrometry. Vascular-to-luminal flux of FITC-inulin was measured to evaluate intestinal mucosal permeability and endotoxemia by measuring plasma lipopolysaccharide. HD5 was administered in a liquid diet 24 hours before or after irradiation. Gut microbiota was analyzed by 16S rRNA sequencing. Intestinal epithelial junctions were analyzed by immunofluorescence confocal microscopy and mucosal inflammatory response by cytokine expression. Systemic inflammation was evaluated by measuring plasma cytokine levels.ResultsIonizing radiation reduced the Paneth cell α-defensin expression and depleted α-defensin peptides in the intestinal lumen. α-Defensin down-regulation was associated with the time-dependent alteration of gut microbiota composition, increased gut permeability, and endotoxemia. Administration of human α-defensin 5 (HD5) in the diet 24 hours before irradiation (prophylactic) significantly blocked radiation-induced gut microbiota dysbiosis, disruption of intestinal epithelial tight junction and adherens junction, mucosal barrier dysfunction, and mucosal inflammatory response. HD5, administered 24 hours after irradiation (treatment), reversed radiation-induced microbiota dysbiosis, tight junction and adherens junction disruption, and barrier dysfunction. Furthermore, HD5 treatment also prevents and reverses radiation-induced endotoxemia and systemic inflammation.ConclusionThese data demonstrate that radiation induces Paneth cell dysfunction in the intestine, and HD5 feeding prevents and mitigates radiation-induced intestinal mucosal injury, endotoxemia, and systemic inflammation.

Dataset Information

An Experimental Approach to Rigorously Assess Paneth Cell ?-Defensin (Defa) mRNA Expression in C57BL/6 Mice.

Publications

An Experimental Approach to Rigorously Assess Paneth Cell α-Defensin (Defa) mRNA Expression in C57BL/6 Mice.

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