L1 glycosylation [Figure 5B]. Overexpression of CHI3L1mutant plasmid N68P, which lacks Nglycosylation, in SW480 cells with subsequent infection with AIEC LF82WT strain resulted in much less bacterial association, as in comparison with cells overexpressing WT or CHI3L1 mutant N211P, which have conserved Nglycosylation [Figure 5C]. We additional investigated how CHI3L1 N68P mutantoverexpressing cells responded to different chiA mutants by overexpressing N68P or N211Pmutant CHI3L1 or WT CHI3L1 in IECs and then infecting the cells with LF82WT or the 4 LF82 mutants. There was significantly enhanced bacterial adhesion with LF82WT and chiA/chiALF82 in CHI3L1WToverexpressing cells, also as the N211P mutant CHI3L1overexpressing cells [Figure 5D, Supplementary Figure 5B]. Bacterial counts within the groups infected together with the other mutant LF82 strains (LF82chiA, chiA/chiAK12 and chiA/chiALF825MU) remained substantially decrease. Even so, there was no apparent distinction in bacterial association across all groups of infected cells that overexpressed CHI3L1 mutant N68P. This indicates that Nglycosylation at the single 68th asparagine residue in mouse CHI3L1, which corresponds to human CHI3L1 60th asparagine residue, is critical for ChiAmediated host/ microbial interactions.941-43-5 Data Sheet NIHPA Author Manuscript NIHPA Author Manuscript NIHPA Author ManuscriptGastroenterology.549531-11-5 manufacturer Author manuscript; readily available in PMC 2014 September 01.PMID:23522542 Low et al.PageLF82 ChiA plays a essential part in effective infection with the host and in exacerbating infectious colitis in vivo To further confirm our in vitro findings and investigate the in vivo relevance with the observed virulence of LF82WT and its 4 chiA mutants, 80weekold C57Bl/6 mice were provided 1.five DSS in their drinking water to induce mild intestinal epithelial harm, and orally gavaged with 108 LF82WT or its 4 chiA mutants for 15 consecutive days. The body weight of every mouse was monitored day-to-day. Mice infected with LF82WT or chiA/ chiALF82 strains didn’t show any indicators of weight recovery till the endpoint and had greater clinical scores [Figure 6A]. Conversely, LF82chiA, chiA/chiAk12 or chiA/ chiALF825MUinfected mice too as uninfected mice showed recovery right after DSS day ten, with milder clinical scores [Figure 6A]. On remedy day 7, LF82WTinfected mouse stools contained the highest number of bacteria as in comparison with each of the other groups of mice [Figure 6B]. On day 14, the stool bacterial count was highest in mice infected with either LF82WT or chiA/chiALF82. Bacteria translocation assays revealed that only LF82WT and chiA/chiALF82infected mice showed appreciable bacterial counts in the liver, spleen, mesenteric lymph nodes (MLNs) and colon [Figure 6C], in association with considerably lowered colonic length as in comparison with the other groups [Supplementary Figure 6A]. Colonic production of CHI3L1 was upregulated just after DSS therapy with or with out AIEC infection [Supplementary Figure 6B]. Moreover, colonic histological sections clearly showed serious colitis improvement in LF82WT and chiA/chiALF82infected mice, with massive number of infiltrating inflammatory cells in colonic LP. Conversely, mice infected using the remaining LF82chiA mutants had milder colitis, as determined by histologic scores, and significantly less LP cellular infiltration [Figures 6D and 6E; Supplementary Figures 7A and 7B]. Upregulation of IL6, TNF and IL1 in LF82WT and chiA/chiALF82infected mice additional supports the colitis severity and proinflammatory environment, as in comparison to chiA, c.