BackgroundInappropriate cross talk between mammals and their gut microbiota may trigger intestinal inflammation and drive extra-intestinal immune-mediated diseases. Epithelial cells constitute the interface between gut microbiota and host tissue, and may regulate host responses to commensal enteric bacteria. Gnotobiotic animals represent a powerful approach to study bacterial-host interaction but are not readily accessible to the wide scientific community. We aimed at refining a protocol that in a robust manner would deplete the cultivable intestinal microbiota of conventionally raised mice and that would prove to have significant biologic validity.Methodology/Principal FindingsPreviously published protocols for depleting mice of their intestinal microbiota by administering broad-spectrum antibiotics in drinking water were difficult to reproduce. We show that twice daily delivery of antibiotics by gavage depleted mice of their cultivable fecal microbiota and reduced the fecal bacterial DNA load by 400 fold while ensuring the animals' health. Mice subjected to the protocol for 17 days displayed enlarged ceca, reduced Peyer's patches and small spleens. Antibiotic treatment significantly reduced the expression of antimicrobial factors to a level similar to that of germ-free mice and altered the expression of 517 genes in total in the colonic epithelium. Genes involved in cell cycle were significantly altered concomitant with reduced epithelial proliferative activity in situ assessed by Ki-67 expression, suggesting that commensal microbiota drives cellular proliferation in colonic epithelium.ConclusionWe present a robust protocol for depleting conventionally raised mice of their cultivatable intestinal microbiota with antibiotics by gavage and show that the biological effect of this depletion phenocopies physiological characteristics of germ-free mice.

Innate and adaptive mucosal defense mechanisms ensure a homeostatic relationship with the large and complex mutualistic gut microbiota. Dimeric IgA and pentameric IgM are transported across the intestinal epithelium via the epithelial polymeric Ig receptor (pIgR) and provide a significant portion of the first line of natural or adaptive antibodymediated immune defense of the intestinal mucosa. We found that colonic epithelial cells from pIgR KO mice differentially expressed (more than twofold change) more than 200 genes compared with cells from WT mice, and upregulated the expression of antimicrobial peptides in a commensal-dependent manner. Detailed profiling of microbial communities based on 16S rRNA genes revealed differences in the commensal microbiota between pIgR KO and WT mice. Furthermore, we found that pIgR KO mice showed increased susceptibility to dextran sulfate sodium-induced colitis, and that this was driven by their conventional intestinal microbiota. Thus, in the absence of pIgR, the stability of the commensal microbiota is disturbed, gut homeostasis is compromised, and the outcome of colitis is significantly worsened. Keywords: Antimicrobial peptide r Colitis r Polymeric Ig receptor Supporting Information available online IntroductionMucus membranes lining the gastrointestinal tract are constantly bombarded by an enormous number of foreign antigens derived Correspondence: Prof. Finn-Eirik Johansen e-mail: f.e.johansen@imbv.uio.no from dietary products and the commensal microbiota. The microbial load of the human colon (about 10 14 bacteria) is estimated to be more than ten times the number of eukaryotic cells in the body [1,2]. The commensal microbiota lives in a mutualistic relationship with their host and provides several benefits. These include * These authors contributed equally to this work. Eur. J. Immunol. 2012Immunol. . 42: 2959Immunol. -2970 the digestion of insoluble fibers and increased energy usage of foods, synthesis of vitamin K [3,4], and niche occupation that could otherwise be exploited by pathogens [5]. The aggregate gene pool of the microbiota, a.k.a. the metagenome, contains 150 times more genes than the human genome [6,7]. Although the human microbiome varies considerably between hosts, our core microbiome has been classified into only three types of communities termed enterotypes [8].A first line of immune defense mediated by nonspecific innate immune effector components has evolved to protect the epithelial barrier without causing inflammatory immune responses [9]. The primary effector component of the adaptive immune system at mucosal sites is secretory IgA (SIgA) [10]. These antibodies are generated by cooperation between dimeric IgA (dIgA)-producing plasma cells and mucosal epithelial cells (ECs), which actively transport dIgA antibodies to the lumen by polymeric Ig receptor (pIgR)-mediated transfer. During transcytosis, the extracellular domain of the pIgR, known as secretory component, becomes covalently coupled to the IgA molecule and final release of receptor-carg...

Summaryb-Glucans are glucose polymers with a variety of stimulatory effects on the immune system. The objective of this study was to determine the effect of prophylactic oral administration of soluble Saccharomyces cerevisiae-derived b-1,3/1,6-glucan (SBG) on the outcome of experimental endotoxaemia and shock-associated organ injury. Male Wistar rats were pretreated with SBG orally (SBGpo, 20 mg/kg/day) for 14 days, subcutaneously (SBGsc, 2 mg/kg/ day) for 3 days, or vehicle (placebo). Rats were anaesthetized and subjected to endotoxaemia by intravenous infusion of Escherichia coli lipopolysaccharide (LPS) (6 mg/kg) or saline infusion (sham). We observed significant levels of plasma b-glucan in the SBGpo group (P < 0·5), although the SBGsc group had levels approximately 40-fold higher despite a 10-fold lower dose. SBG prophylaxis caused enhanced blood pressure recovery following LPS-induced blood pressure collapse. Oral treatment with SBG attenuated the LPS-induced rise in plasma creatinine levels (P < 0·05), indicating protection against renal injury. SBG also attenuated the plasma levels of aspartate aminotransferase and alanine aminotransferase (SBGpo, P < 0·01; SBGsc, P < 0·01), indicating protection against LPS-induced hepatic injury. A moderate increase in baseline interleukin (IL)-1b levels was observed in the SBGsc group (P < 0·05). In the LPS-challenged rats, plasma levels of proinflammatory cytokines was moderately reduced in both SBG-treated groups compared to placebo. SBG treatment, particularly oral administration, had a striking effect on the haemodynamics of LPS-treated rats, although only a minute fraction of the orally administered b-glucan translocated to the circulation. Enhanced organ perfusion may thus be responsible for the attenuated levels of indicators of kidney and liver injury seen in SBG-treated rats.

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