Epithelial cell continuity is preserved during DC extension. Three sequential images of a villus in an ileal explant from an MHC CII-EGFP mouse given Salmonella orally and immersed in buffer containing TRITC-dextran showing the exclusion of the tracer from the basolateral surfaces of the epithelial layer (also see Video S5).

Invasive and noninvasive Salmonella induce a similar number of DC extensions. MHC CII-EGFP mice were treated orally with noninvasive (NIS; open bars) or invasive (IS; filled bars) Salmonella for 1, 3, or 5 h. The small intestine peri-cecal region was explanted and imaged, and the average number of DC extensions was calculated as described in Fig. 2. Data represent the average number of extensions ± SE/villus from >50 villi.

Interaction of inert particles and bacteria with DC extensions in the bowel lumen. (A) A time sequence of images from the same region of a villus in the terminal ileum of an MHC CII-EGFP mouse. The mouse was given noninvasive Salmonella (red) orally. A segment of the terminal ileum was isolated 5 h later, the epithelium was stained using Cell Tracker Blue, and the preparation was imaged by two-photon microscopy. A BB can be seen interacting with a fluorescent bacterium before internalizing it. (B) A three-dimensional section of a villus in the terminal ileum from an MHC CII-EGFP mouse. Noninvasive Salmonella (red) was given orally 5 h before the terminal ileum was explanted and stained with Cell Tracker Blue. A Salmonella bacterium just internalized by a DC extension with the typical BB shape is shown by the colocalization of green and red in the three-dimensional section (also see Videos S6 and S7).

Role of epithelial TLRs and MyD88-dependent signaling in the induction of DC extensions. (A) Extensions of DCs across the small bowel epithelium were measured in MHC CII-EGFP animals as described in Fig. 2 either without treatment or after PG, poly(I:C), LPS, flagellin, or CpGs administration. (B) TLR4-, TLR2-, or MyD88-deficient recipient mice were irradiated and reconstituted with bone marrow cells from CD11c-EGFP (for TLR4-deficient recipients) or MHC CII-EGFP (for TLR2- and MyD88-deficient recipients) mice, respectively, to create CD11c-EGFP/TLR4^−/−, MHC CII-EGFP/TLR2^−/−, and MHC CII-EGFP/MyD88^−/− bone marrow chimeras. The extensions of DCs across the small bowel epithelium were measured in these chimeric animals as described in Fig. 2 either without treatment or after noninvasive Salmonella gavage, as indicated. Data are represented as the average number of extensions ± SE/villus from two independent experiments for each chimera type.

Characteristics of DC trans-epithelial extensions. (A) MHC CII-EGFP mice were deprived of food for 4 h before oral Salmonella administration. The terminal ileum was then imaged by intravital two-photon microscopy. The nuclei of all the cells were labeled with Hoechst 33342 (blue), and the epithelium was stained with SNARF (red). Two different DCs can be seen extending dendritic processes into the intestinal lumen. In this example, both BB (#) and finger-like (*) dendritic extensions can be seen in the same villus (also see Video S3). (B) MHC CII-EGFP mice were deprived of food for 4 h before oral noninvasive Salmonella administration. The terminal ileal epithelium was then labeled with SNARF-1 (red) and imaged by two-photon microscopy. Sequential images from a time series are shown at high magnifications and reveal a dendrite reaching the luminal side and acquiring the BB shape. The protrusion is almost completely retracted after 22 min (see Video S4).

Inducible DC extensions in the terminal ileum. Explanted small intestinal segments from untreated (open bars) or noninvasive Salmonella-treated (filled bars) mice were analyzed by two-photon microscopy for the presence of trans-epithelial DC extensions. (A) The number of DC extensions was determined by inspection of each z-slice in image stacks collected in multiple sites within the indicated subregions of the small bowel. The data are presented as the average number of extensions ± SE/villus. The data show representative results from one of three independent experiments, each involving five random acquisitions per intestinal region with three to eight villi visualized per acquisition. (B) Average numbers of DC extensions in the proximal ileum of untreated (open bar) or antibiotic-treated (striped bar) mice were analyzed as in A. Results are representative of three separate experiments.

DCs extend through the apical epithelium of villi. (A) Static two-photon microscopy image of the villi in the peri-cecal region of a CD11c-EGFP mouse given noninvasive Salmonella orally 3 h before imaging (top). The image is a three-dimensional reconstruction of 65 serial optical sections (1.5 μm/z-section) comprising a total depth of 97.5 μm. Extensions of DCs (green) into the intestinal lumen can be observed. The surface of the villi was outlined by SNARF-1 staining of the epithelial cells (red). Bar, 25 μm. Boxed region in the top panel is shown in a higher magnification in the bottom panel to better illustrate how DC extensions (green) protrude through the intestinal epithelium (red) into the luminal cavity. The arrows indicate examples of spherical DC extensions into the intestinal lumen (also see Video S1). (B) A representative static image of an ileal villus from an MHC CII-EGFP mouse stained with SNARF-1 to highlight the epithelial cells (red) shows the preferential location of DC extensions (green) near the apical region of the villus (white arrows). The villus was arbitrarily subdivided in 10 equal compartments from the bottom to the apical region (z = 0 to z = 100), and the average number of DC extensions in each compartment is displayed in the histogram. Values are expressed as the number of DC extensions per villus in the indicated section (mean ± SE). The top regions (z = 70–100; open bars) show a significant difference in the number of DC extensions as compared with the middle and lower parts (z = 40–70; striped bars) of the villi. Also see Video S 2. (C) A single optical z-slice of a villus from the ileum of an MHC CII-EGFP mouse. Epithelial cells are stained with SNARF-1 (red), and the cell nuclei are stained with Hoechst 33342 (blue). The higher magnification in the bottom panel demonstrates a DC extending an MHC CII⁺ (green) dendrite through the epithelium. Once reaching the intestinal lumen, the DC dendrite assumes the characteristic shape of a sphere (white arrow).