J., van der Cammen M. Preassociation of bacteria with nonspecific SIgA increased their dynamics of access and restored the reduced transport observed in germ-free mice known to have a marked reduction in intestinal SIgA production. Selective SIgA-mediated targeting of bacteria is restricted to the tolerogenic CD11c+CD11b+CD8? dendritic cell subset located in the subepithelial dome region of PPs, confirming that this GSK1278863 (Daprodustat) host is not ignorant of its resident commensals. In conclusion, our work supports the concept that SIgA-mediated monitoring of commensal bacteria targeting dendritic cells in the subepithelial dome region of PPs represents a mechanism whereby the host mucosal immune system controls the continuous dialogue between the host and commensal bacteria. (24). The combination of these mechanistic data led us to speculate that SIgA may govern the mucosal sensing of commensals by facilitating communication with partners of the underlying immune system. To tackle this open issue ((serotype 5a LPS, produced at 37 C in RPMI 1640 medium complemented with 10% fetal calf serum (FCS) were used as a source of IgA (24); to reconstitute SIgA, equimolar purified polymeric IgAC5 mAb and recombinant mouse SC were combined as explained (26). Bacteria The commensal strains (LPR, CGMCC 1.3724 and DSM 2079 (Bt) were cultured as published (24). Fluorescent labeling was performed by Erg incubating 1 109 bacteria in 1 ml of PBS made up of 50 g/ml FITC (Sigma-Aldrich) for 30 min at room temperature in the dark. Washed labeled bacteria were resuspended at a concentration of 4 108/100 l and used freshly as such or after association with 2 g of reconstituted SIgA for immediate administration into mouse ligated ileal loops. Injection into a Mouse Ligated Ileal Loop The procedure of Rey was used (12) To comply with Swiss law governing animal experimentation, the investigation protocol had to be limited to 6 h. 100 l of the solution made up of 4 108 bacteria associated or not with SIgA was delivered into the lumen of a ligated ileal loop made up of a PP. Mice were sacrificed 1, 2, 4, or 6 h later, and ligated ileal loop tissue samples were collected. Preparation of Tissue Sections and Immunolabeling Intestinal segments made up of a PP were immediately fixed in 1 ml of GSK1278863 (Daprodustat) PBS/4% paraformaldehyde (Fluka) for 2 h at room temperature and further processed as explained (14). Frozen sections (average thickness of 7 m) were generated on a Leica cryostat, washed in PBS, and blocked for 20 min with first 5% FCS and then with purified anti-CD16/32 mAbs (1/100; BD Biosciences) at room temperature. Numerous cell types in PPs were detected upon incubation in PBS-5% FCS with biotinylated anti-CD11c mAb (1/10; BD Biosciences), purified anti-CD4 rat IgG (1/50; BD Biosciences), or purified anti-CD45R rat IgG (1/50; Invitrogen), followed by Cy3- or Cy5-labeled streptavidin (1/500; both from Amersham Biosciences) or anti-rat IgG conjugated to Alexa Fluor 647 (1/100; Invitrogen). Endogenous SIgA was detected with rabbit anti-mouse SC antiserum at 1/200 dilution (26) and goat anti-rabbit IgG coupled to Alexa Fluor 647 (1/200; Invitrogen). M cells were stained with rhodamine-coupled UEA-1 (Vector Laboratories, Burlingame, CA) used at a concentration of 20 g/ml. The DNA stain 4,6-diamidino-2-phenylindole (DAPI) was used at a concentration of 100 ng/ml. Sections were finally washed with PBS and mounted with anti-fading Vectashield reagent (Vector Laboratories). Laser Scanning Confocal Microscopy (LSCM) LSCM images were obtained using a Zeiss LSM 710 Quasar confocal microscope (Zeiss, G?ttingen, Germany) in multi-track mode. Natural images were analyzed and processed around the Zen 2009 software. All of the images offered in the paper are representative of 5C10 sections prepared from at least three individual experiments. Bacterial counts in the FAE and the SED region were obtained from three-dimensional acquisitions generated from GSK1278863 (Daprodustat) six individual sections prepared from three mice. Numbering of fluorescent bacteria on sections was blindly carried out by three trained individuals. Flow Cytometry Indie mice were administered 4 108 FITC-LPR (= 3) or PBS as control (= 3) into a ligated ileal loop made up of a PP. Mice were sacrificed 6 h later, and the three individual PPs per condition were pooled and digested with Liberase (Roche Applied Science) in RPMI 1640 medium complemented with 2 mm CaCl2 for 30 min at 37 C. Cells were resuspended.