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.

The human MHC class I-related neonatal Fc receptor, hFcRn, mediates bidirectional transport of IgG across mucosal barriers. Here, we find that at steady state hFcRn distributes predominantly to an apical intracellular compartment and almost exclusively to the basolateral cell surface of polarized epithelial cells. It moves only transiently to the apical membrane. Ligand binding does not redistribute the steady state location of the receptor. Removal of the cytoplasmic tail that contains di-leucine and tryptophan-based endocytosis motifs or incubation at low temperature (18°C) redistributes the receptor apically. The rates of endocytosis of the full-length hFcRn from the apical or basolateral membrane domains, however, are equal. Thus, the strong cell surface polarity displayed by hFcRn results from dominant basolateral sorting by motifs in the cytoplasmic tail that nonetheless allows for a cycle of bidirectional transcytosis. INTRODUCTIONThe numerous studies on the cell biology of the polymeric immunoglobulin receptor (pIgR) and transferrin receptor (TfnR) as expressed in the Madin-Darby canine kidney (MDCK) cell line have provided a detailed characterization of the itineraries of these proteins undergoing such processes as basolateral recycling, basolateral to apical transcytosis, and apical recycling in MDCK cells (Mostov and Deitcher, 1986;Leung et al., 1999;Brown et al., 2000;. Comparatively little is known about trafficking in the apical to basolateral direction because of the lack of a model protein that physiologically harnesses this pathway, and whether the trafficking of pIgR and TfnR can be generalized to the trafficking of other proteins in polarized cells remains to be tested.The MHC class I-related neonatal Fc receptor, FcRn, is responsible for the absorption of maternal IgG across the rabbit and rodent yolk sac, the human placenta, and the proximal small intestine of the neonatal rodent (Rodewald, 1970;Simister and Mostov, 1989;Roberts et al., 1990;Medesan et al., 1996;Firan et al., 2001). Absorption of IgG depends on the ability of FcRn to bind IgG and traffic bidirectionally across the epithelial cells that line the lumen of these tissues (Jones and Waldmann, 1972;Abrahamson and Rodewald, 1981;Dickinson et al., 1999;McCarthy et al., 2000;Kobayashi et al., 2002). Almost nothing is known about the cellular mechanisms that explain how any membrane receptor can move bidirectionally across polarized epithelial cells.Like most other MHC class I-related molecules, FcRn is an obligate heterodimer consisting of a glycosylated heavy (␣) chain (40 -44 kDa in humans, 48 -50 kDa in rodents) that associates noncovalently with ␤2-microglobulin (␤ 2 m; Simister and Mostov, 1989). The association with ␤ 2 m is species dependent (Claypool et al., 2002), and the functional receptor is likely a dimer of heterodimers that binds one IgG molecule (Burmeister et al., 1994;Kim et al., 1994). The Fc fragment of IgG binds to FcRn at acidic pH (pH Յ6.5) and releases from the receptor at neutral pH (Rodewald, 1976). In cells...

Secretory IgA (SIgA) is widely held to be responsible for the defense of the mucosae against pathogenics and other potentially harmful agents. In this study, polymeric Ig receptor (pIgR) knockout mice, which lack secretory antibodies (SAb), were used to investigate the role of vaccine‐elicited SAb in protection against gastrointestinal bacterial infections. An essential role for specific SAb in protection against Vibrio cholerae was evident from experiments showing that vaccinated pIgR–/– mice, but not vaccinated C57BL/6 mice, were susceptible to cholera toxin challenge. Vaccination of C57BL/6 mice with Salmonella typhimurium elicited strong antigen‐specific, mucosal responses, which blocked in vitro invasion of epithelia. However, vaccinated C57BL/6 and pIgR–/– mice were equally resistant to challenge infection with virulent S. typhimurium. Finally, we investigated the importance of SIgA in protection against recurrent infections with Citrobacter rodentium. Although higher numbers of bacteria were detected early after challenge infection in feces of vaccinated pIgR–/– mice compared with vaccinated C57BL/6 mice, both mouse strains showed complete clearance after 9 days. These results suggested that, in immune animals, SIgA is crucial for the protection of gastrointestinal surfaces against secreted bacterial toxins, may inhibit early colonization by C. rodentium, but is not essential for protection against re‐infection with S. typhimurium or C. rodentium.

The importance of IgA for protection at mucosal surfaces remains unclear, and in fact, it has been reported that IgA-deficient mice have fully functional vaccine-induced immunity against several bacterial and viral pathogens. The role of respiratory Ab in preventing colonization by Streptococcus pneumoniae has now been examined using polymeric IgR knockout (pIgR−/−) mice, which lack the ability to actively secrete IgA into the mucosal lumen. Intranasal vaccination with a protein conjugate vaccine elicited serotype-specific anti-capsular polysaccharide Ab locally and systemically, and pIgR−/− mice produced levels of total serum Ab after vaccination that were similar to wild-type mice. However, pIgR−/− mice had ∼5-fold more systemic IgA and 6-fold less nasal IgA Ab than wild-type mice due to defective transport into mucosal tissues. Wild-type, but not pIgR−/− mice were protected against infection with serotype 14 S. pneumoniae, which causes mucosal colonization but does not induce systemic inflammatory responses in mice. The relative importance of secretory IgA in host defense was further shown by the finding that intranasally vaccinated IgA gene-deficient mice were not protected from colonization. Although secretory IgA was found to be important for protection against nasal carriage, it does not appear to have a crucial role in immunity to systemic pneumococcus infection, because both vaccinated wild-type and pIgR−/− mice were fully protected from lethal systemic infection by serotype 3 pneumococci. The results demonstrate the critical role of secretory IgA in protection against pneumococcal nasal colonization and suggest that directed targeting to mucosal tissues will be needed for effective vaccination in humans.