The Role Of Mucin O-glycans In Intestinal Mucosal Barrier Of Piglets

Swine industry is the pillar industry of China’s national economy.Early weaning is one of the key technologies in modern pig production.The application improves reproductive performance of sow and the utilization of pens and facilitates disease prevention.However,early weaning stress is typically associated with intestinal mucosal barrier dysfunction,leading to reduced feed intake and sub-optimal growth,which further triggers diarrhea and high mortality.Post-weaning diarrhea(PWD)is one of the major problems of pig production.Hence,a more detailed understanding of alteration in the intestinal barrier function and potential targets to prevent PWD certainly provide insight into nutritional intervention to alleviate diarrhea in weaned piglets.Mucus layer,overlying the gut epithelium,is the first line of defense against bacterial translocation and lowers the mechanical stress on the epithelium.Mucus is a large polymeric network of mucins that are heavily O-glycosylated.Mucin-derived O-glycans play a critical role in intestinal barrier function by supplying attachment sites,serving as a source of nutrients for specific bacteria,and protecting mucus layer from bacterial protease-degradation.However,few studies focus on the characterization of the mucus layer in weaned piglets.A deeper understanding of the alteration in mucus layer in diarrheal and healthy piglets and the relationship between mucin O-glycans and microbiota is a key to develop potential targeted treatments.Hence,using diarrheal piglets and their healthy controls from large-scale pig farm,we investigated the role of mucin-derived O-glycans in intestinal barrier with reported empirical data from glycome,microbiome,targeted metabolome,and in vitro experiments.The research can provide theoretical and scientific basis for developing the targets of preventing and alleviating diarrhea in piglets.The results are shown as follows:Experiment 1 Structure and abundance of mucin O-glycans in piglets using PGCLC-MS/MSIn this study,piglets were early weaned from their sows at 21 days of age.We selected piglets with a score of 6 over 5 days on the 7 days post-weaning.Gender-matched and similar initial body weight littermates were used as the healthy controls.All piglets were euthanized and intestinal tissues were immediately separated.About 0.5 cm of colonic tissue were fixed in Carnoy’s fixative for staining of goblet cells and mucus layer.The thickness of mucus layer and the number of goblet cells were greatly reduced in diarrheal piglets as shown by UEA immunofluorescence assay and Alcian blue-periodic acid-Schiff staining.Diarrheal piglets showed reduction in mucus-related genes,including MUC2,MUC4,and RETNLB.A total of57 unique O-glycan structures were identified using PGC-LC-MS/MS.Partial least squares discriminant analysis(PLS-DA)model exhibited a significant separation of clustering pattern between the two groups.Among the mucin O-glycans from piglets,Core 1 and Core 3 glycans were lower in abundance than Core 2 and Core 4 glycans.Core 2 and Core 4 type glycans dominated the glycan spectra,with a higher amount of Core 4 glycans and a larger number of Core 2 branched glycoforms in the colon of piglets.We also observed an increased in neutral glycans and a decreased abundance of acidic glycans in the colon of diarrheal piglets.Consistently,a decrease in the relative m RNA expression of glycotransferases in the colon of diarrheal piglets were observed.The glycan chain length on mucin from both diarrheal and healthy piglets ranged from 2 to 6 monosaccharides.Relative amount of short glycan(dissaccharides)was more abundant in the colon of diarrheal piglets while longer glycan(hexasaccharides)was less abundant.These findings indicated that diarrheal piglets exhibited a breached mucus layer and aberrant mucin O-glycan profile.Experiment 2 Analyses of alteration of microbiota and its-derived metabolites in weaned piglets with diarrheaIleal mucosa,colonic mucosa and luminal contents,and feces were collected from piglets in experiment 1.There was no significant difference in the ileal mucosal α-diversity.The principal coordinates analysis(PCo A)showed a trend of separation of ileal mucosal microbiota composition between the two groups.The overall structure of mucosal and luminal microbiota in the colon investigated by PCo A showed that diarrheal piglets displayed a shift clustering of bacterial composition,which was distinct from healthy controls.Linear discriminant analysis effect size(LEf Se)analysis was employed to identify differentially abundant bacterial taxa in the diarrheal piglets and healthy controls.In the ileum,diarrheal piglets had significantly higher proportions of the genera Dorea and Holdemanella.Many of the genera,Roseburia,Propionicicella,and Atopostipes were enriched in the healthy piglets.In the colonic mucosa,diarrheal piglets had significantly higher proportions of the genera Peptococcus,Campylobacter,and Peptostreptococcus.Many of the genera,Phascolarctobacterium,Lachnospiraceae FCS020 group,and Eubacterium hallii group,from Firmicutes were also enriched in the colonic mucosa of healthy controls.In the colonic lumen,diarrheal piglets presented markedly greater proportions of the genera Escherichia Shigella,Anaerobiospirillum,Peptostreptococcus,and Alloprevotella than healthy controls did.Moreover,many taxa,Gammaproteobacteria,Enterobacteriaceae,and Enterobacterales from phyla Proteobacteria were significantly elevated as well in the diarrheal piglets.We further observed substantially lower concentrations of butyrate in both the colonic contents and feces of the diarrheal piglets.By contrast,the level of lactate was significantly increased in the diarrheal piglets.LC-MS/MS analysis revealed that the most striking difference in the bile acid profiling analysis was that two SBAs,LCA and HDCA,were significantly reduced in the feces of diarrheal piglets compared with the healthy controls.The above results indicated that diarrheal piglets exhibited colonic microbiota dysbiosis and bacterial translocation and subsequent leakage into other visceral organs.The reduced SBA levels further indicated that the disturbed microbiota could not metabolize PBA and result in abnormal metabolites.Experiment 3 Analyses of intestinal epithelial and immune barrier in piglets with diarrheaA segment of the ileum and colon measuring 1 cm was fixed in 4% paraformaldehyde for histological analysis.After gently flushing the intestine with ice-cold PBS,the ileal and colonic mucosa was collected by scraping the intestinal wall with glass microscope slides.Compared with the healthy controls,diarrheal piglets displayed a tendency towards decreased body weight.However,the liver and spleen weight were significantly increased in piglets with diarrhea.Diarrheal piglets showed a leaky gut with higher levels of serum DAO and LPS,decreased abundance of ileal and colonic ZO-1 and Occludin and colonic Claudin1 as well as expression of gene encoding for ZO-1,Occludin,Claudin2,and Claudin4.H&E staining showed that diarrheal piglets displayed decreased ileal villus height and V/C and increased colonic crypt depth.Diarrhea led to E.coli translocating into the liver and colon tissue.Moreover,diarrheal piglets showed elevated levels of cytokines including serum and colonic IL-6,serum and colonic IL-8,and colonic TNF-a.No difference in the levels of ileal cytokines was observed between the two groups.These results showed that diarrhea led to poor intestinal epithelial barrier function,as evidenced by higher concentrations of DAO and LPS in the serum and lower expression of tight junction proteins.Additionally,diarrheal piglets displayed systemic inflammatory response.More severe inflammatory response was observed in the colon compared with the ileum.Experiment 4 The mechanism study on mucin O-glycans orchestrating gut homeostasis in pigletsFresh feces from 8 piglets at 28 days of age were collected and pooled.The pooled feces were cocultured with colonic mucin glycans(CMGs)from diarrheal piglets or their healthy counterparts under anaerobic conditions.After incubation for 24 h,cultured samples were collected and used for SCFAs detection and bacterial composition analysis.Fermentation of colonic mucin glycans from diarrheal piglets by the gut microbiota resulted in a remarkable reduction of acetate,propionate,isobutyrate,butyrate,isovalerate,and valerate.16 S r RNA sequence analysis showed that CMGs from diarrheal piglet fermentation harbored a different microbiome in both a-and b-diversity within bacterial communities as compared with these from the healthy controls.Analysis of the microbiota at the phylum level revealed a significant increase in the relative abundance of Desulfobacterota and a decrease in the relative abundance of Fusobacteriota in the diarrheal piglets’ CMGs fermentation than in the healthy controls.Taxonomic classifications at the genus level revealed that CMGs from diarrheal piglets highly enriched Desulfovibrio and Lactobacillus,whereas dampened Prevotella,Ruminococcus torques group,and Faecalibacterium by 24 h of fermentation.Notably,at the genus level,the abundance of Blautia and Subdoligranulum was also significantly decreased in CMGs from diarrheal piglet fermentation,whereas the abundance of Parabacteroides,Phascolarctobacterium,and Bacteroides increased.To directly assess the potential roles of mucin O-glycan in pathogenic adhesion,benzyl-a-Gal NAc was employed to inhibit mucin Oglycosylation.The fluorescence staining indicated a significant reduction in the number of E.coli k88 adherent to the mucin-producing cell line T84 cells after 72 h treatment with the inhibitor.We also noticed that key genes related to mucins showed decreased expression after being incubated with benzyl-a-Gal NAc.The disrupted epithelial barrier function and inflammation susceptibility were further illustrated and supported by the induced cytokines,including expression of IL-18,IL-6,IL-8,and repressed ZO-1 expression.Having shown mucin O-glycan and microbiota reciprocally regulate each other in order to maintain intestinal homeostasis,we explore the potential downstream mechanisms related to the impaired mucin O-glycosylation in diarrheal piglets.We observed the elevated expression of inflammasomes,including NLRP3,ASC,and Caspase1 in diarrheal piglets.Consistent with the inflammasome activation,analysis of colonic tissues revealed increased IL-1b and IL-18 released from diarrheal piglets compared with the healthy controls.Moreover,western blotting analysis revealed that piglets presented with diarrhea led to the reduced levels of ATG7,ATG5,and LC3A/B II and accumulation of p62 in the colon.Given the fact that diarrhea affects autophagosome formation in piglets,the effects of E.coli k88 challenge on T84 cells were assessed.Compared with the CON group,a higher m RNA level of p62 was detected in the ETEC group,whereas lower m RNA levels of ATG5,ATG7,and LC3 B were detected.Moreover,pretreatment with the autophagy inhibitor 3-methyladenine(3-MA)suppressed the expression of key genes(e.g.MUC2,TFF3,MUC17,and RETNLB)related to goblet cell secretions.In conclusion,our data demonstrated that autophagy restriction and inflammasomes activation might result in aberrant mucin O-glycans,leading to an impaired gut microenvironment.To sum up,our data support that diarrhea results in inflammasomes activation and autophagy defect,which may dysregulate mucus secretion and induce aberrant mucin Oglycans,including reduction in acidic glycans and truncated glycans,thereby leading to colonic microbiota dysbiosis,epithelial barrier,and immune barrier dysfunction.We investigate the mechanisms underlying mucin O-glycans maintaining gut homeostasis.The study lays the groundwork with an alternative strategy to potentially prevent diarrheal diseases in piglets.