Exposure to plant-oriented microbiome altered jejunal and colonic innate immune response and barrier function more strongly in suckling than in weaned piglets

Abstract

Weaning often leaves the piglet vulnerable to gut dysfunction. Little is known about the acute response of a gut mucosa primed by a milk-oriented microbiome before weaning to a plant-oriented microbiome (POM) after weaning. We evaluated the epithelial structure, secretory response and permeability in the small and large intestines of piglets receiving a milk-based (i.e. preweaning) or plant-based diet (i.e. postweaning) to POM inocula using intestinal loop perfusion assays (ILPA). The POM were prepared from jejunal and colonic digesta of four 7 week-old weaned (day 28 of life) piglets, having gut-site specific microbial and metabolite composition. Two consecutive ILPA were performed in 16 piglets pre- (day 24-27) and 16 piglets postweaning (day 38-41) in two replicate batches. Two jejunal and colonic loops per piglet were perfused with Krebs-Henseleit buffer (control) or the respective POM. The outflow fluid was analyzed for antimicrobial secretions. Jejunal and colonic loop tissue was collected after each ILPA for histomorphology and electrophysiology using Ussing chambers. ANOVA was performed using the MIXED procedure in SAS. The POM stimulated the secretory response by increasing mucin in the jejunal and colonic outflow by 99.7 and 54.1%, respectively, and jejunal IgA by 19.2%, whereas colonic lysozyme decreased 25.6% compared to the control (P < 0.05). Fittingly, the POM raised the number of goblet cells by 96.7% in jejunal and 56.9% in colonic loops compared to control loops (P < 0.05). The POM further flattened jejunal villi by 18.3% and reduced crypt depth in jejunal and colonic loops by 53.8 and 9.0% compared to the control (P < 0.05); observations typically made postweaning and indicative for mucosal recognition of ‘foreign’ compounds. The POM altered the jejunal and colonic net ion flux as indicated by 22.7 and 59.2% greater short-circuit current compared to control loops, respectively; the effect being stronger postweaning (P < 0.05). Colonic barrier function improved with age (P < 0.05), whereas POM perfusion compromised the mucosal barrier as suggested by 17.7 and 54.1% greater GT and mucosal-to-serosal flux of fluorescein-isothiocyanate dextran, respectively, compared to the control (P < 0.05). In conclusion, results demonstrated that the preweaning gut epithelium acutely responds to novel compounds in postweaning digesta by upregulating the first line of defense (i.e. mucin and lysozyme secretion) and impairment of the structural integrity.

An insight into the temporal dynamics in the gut microbiome, metabolite signaling, immune response, and barrier function in suckling and weaned piglets under production conditions

“…Likewise, expression levels of the four investigated TLRs did not show a weaning-associated upregulation on DoL 31, which may indicate that either the sampling timepoint was too late, other pattern-recognition receptors were activated, or the activation was only visible at translational and hence functional protein levels. The first assumption would be supported by recent observations from our group using the intestinal loop perfusion assay that the milk-primed gut mucosa (jejunum and colon) reacts to postweaning digesta with an upregulation of the innate immune response within 2 h of exposure ( 25 ). Deeper crypts, recruitment of goblet cells, expression of MUC4 (only cecum), and intraepithelial lymphocytes post-weaning at both gut sites indicated increased secretory processes, supporting the upregulation of an innate immune response ( 15 ).…”

“…Preparation, imaging, and evaluation of histo-morphological parameters of the jejunal and cecal tissue were performed as previously described ( 25 ). Briefly, per gut site, piglet, and DoL, one discontinuous 3 to 4 μm-thick section was cut, deparaffinized, and stained with hematoxylin and eosin, and digital images were taken (Pannoramic Scan II slidescanner, 3DHISTECH Ltd., Budapest, Hungary) and analyzed (FIJI software) ( 26 ).…”

“…To assess changes in mucosal gene expression, total RNA isolates from 24 mg frozen jejunal and cecal tissue were mechanically homogenized and analyzed using the RNeasy Mini Kit (RNeasy Mini Qiacube Kit, Qiagen, Hilden, Germany) ( 25 ). Each RNA isolate was treated with DNase I (Invitrogen TURBO DNA-free Kit, Thermo Fisher Scientific Inc.) to remove genomic DNA and quantified with the Qubit RNA HS Assay Kit on the Quibt 4 Fluorometer (Thermo Fisher Scientific Inc.).…”