Sampling of GP and PLGA microspheres by SED DCs.

<p>PLGA microspheres (blue) were either (<b>Panel A</b>) co-gavaged with FITC-GPs (PLGA+GP) or (<b>Panel B</b>) administered to mice 1 hr after FITC-GPs (PLGA>GP). Twenty-four hours later PP were collected, cryosectioned, immunostained for CD11c (arrowheads; red) and viewed by confocal laser scanning microscopy. Selected GPs are noted with an arrow. The top pair of panels is a merge of the FITC, APC and PE channels, the middle panels are red and green, and the bottom red and blue, as indicated by the vertical annotation on right. Scale bar corresponds to 100 μm.</p

A Population of Langerin-Positive Dendritic Cells in Murine Peyer's Patches Involved in Sampling β-Glucan Microparticles

<div><p>Glucan particles (GPs) are 2–4 μm hollow, porous shells composed of 1,3-β-D-glucan that have been effectively used for oral targeted–delivery of a wide range of payloads, including small molecules, siRNA, DNA, and protein antigens. While it has been demonstrated that the transepithelial transport of GPs is mediated by Peyer's patch M cells, the fate of the GPs once within gut-associated lymphoid tissue (GALT) is not known. Here we report that fluorescently labeled GPs administered to mice by gavage accumulate in CD11c⁺ DCs situated in Peyer's patch sub-epithelial dome (SED) regions. GPs appeared in DCs within minutes after gavage and remained within the SED for days afterwards. The co-administration or sequential administration of GPs with differentially labeled GPs or poly(lactic-co-glycolic acid) nanoparticles demonstrated that the SED DC subpopulation in question was capable of internalizing particles of different sizes and material compositions. Phenotypic analysis identified the GP-containing DCs as being CD8α^- and CD11b^lo/-, suggesting they are the so-called myeloid and/or double negative (DN) subset(s) of PP DCs. A survey of C-type lectin receptors (CLRs) known to be expressed by leukocytes within the intestinal mucosa revealed that GP-containing SED DCs were positive for Langerin (CD207), a CLR with specificity for β-D-glucan and that has been shown to mediate the internalization of a wide range of microbial pathogens, including bacteria, viruses and fungi. The presence of Langerin⁺ DCs in the SED as determined by immunofluorescence was confirmed using Langerin E-GFP transgenic mice. In summary, our results demonstrate that following M cell-mediated transepithelial transport, GPs (and other micro/nanoparticles) are sampled by a population of SED DCs distinguished from other Peyer's patch DC subsets by their expression of Langerin. Future studies will be aimed at defining the role of Langerin in antigen sampling and antigen presentation within the context of the GALT.</p></div

Accumulation of GPs within CD11c⁺ DCs in the SED after oral gavage.

<p>GPs were administered to BALB/c mice by gavage, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091002#s2" target="_blank">Materials and Methods</a>. PPs were collected 2.5 hr later, cryosectioned, immunostained and viewed by confocal laser scanning microscopy. (<b>Panel A</b>) PBS control PP follicles stained in blue with B-cell marker CD45R/B220, CD11c⁺ DCs (magenta;arrowheads). Scale bar is 100 μm. (<b>Panels B-C</b>) GPs (green; arrows) were detected within CD11c⁺ DCs (magenta;arrowheads) located within the SED. Scale bar is 50 μm. Abbreviations: FAE, follicle-associated epithelium; SED, sub-epithelial dome; IFR, interfollicular region.</p

GPs persist within SED DCs.

<p>FITC-GPs were administered to BALB/c mice by gavage, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091002#s2" target="_blank">Materials and Methods</a>. PPs were collected at indicated time points thereafter and then cryosectioned, immunostained with anti-CD11c antibodies and viewed by confocal laser scanning microscopy. FITC-GPs are shown in green (arrows) and CD11c⁺ DCs in magenta (arrow heads). The panels correspond to the following time points (in hours) (<b>A</b>) 2.5; (<b>B</b>) 6; (<b>C</b>) 24; (<b>D</b>) 48; (<b>E</b>) 72 hrs. (F) Close up of SED CD11c⁺ DCs from tissues taken at 24 hr. In Panels A-E the scale bar corresponds to 50 μm. In Panel F the scale bar is 20 μm.</p

CLR expression in BALB/c PPs.

a<p>Positive staining in PPs (PP) or lamina propria (LP) detected by immunofluorescent microscopy (IFM);</p>b<p>Percent of total PP cells positive for indicated markers, as determined by flow cytometry (FC).</p>c<p>Detection of indicated marker in isolated PP DCs.</p

Sampling of differentially labeled GP microparticles by CD11c⁺ DCs.

<p>FITC-labeled GPs (green) or APC-labeled GPs (blue) particles were administered to mice by gavage, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091002#s2" target="_blank">Materials and Methods</a>. APC-GPs were either (<b>Panel A</b>) co-gavaged with FITC-GPs (GP+GP) or (<b>Panel B</b>) administered to mice 1 hr after FITC-GPs (GP>GP). Twenty-four hours later PP were collected, cryosectioned, immunostained with PE-labeled CD11c (arrowheads; red) and viewed by confocal laser scanning microscopy. The top pair of panels is a merge of the FITC, APC and PE channels, the middle panels are red and green, and the bottom red and blue, as indicated by the vertical annotation on right. In the left panels, the scale bar corresponds to 100 μm, while the scale bar in the right panel corresponds to 20 μm.</p

GPs localize within Langerin⁺ SED DCs.

<p>APC-labeled GPs (blue) were administered to (<b>Panel A</b>) BALB/c or (<b>Panel B</b>) Langerin E-GFP-DTR transgenic mice by gavage, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091002#s2" target="_blank">Materials and Methods</a>. PP were collected 24 h later, cryosectioned, immunostained and viewed by three color, confocal laser scanning microscopy. (<b>Panel A</b>) In panel A, BALB/c PP cryosections were immunostained with anti-Langerin mAb RMUL.2 (green; arrows) and anti-CD11c (red). In Panel B, PP cryosections from Langerin-EGFP-DTR transgenic mice were immunostained with anti-CD11c (red) only. In both panels, the <u>left</u> box shows Langerin (green) and CD11c (red) signals only, the <u>middle</u> box shows Langerin (green), CD11c (red), and the APC-GPs (blue) signals, while the <u>right</u> boxes represent a magnification of the dashed square in the middle box. Scale bars correspond to 100 μm (right and middle) or 50 μm.</p

Accumulation of GPs within MLN after oral gavage.

<p>MLNs were collected from mice 15(Panels A,B) or 24 hrs (Panel C) after there had been gavaged with GPs. Tissues were cryosectioned, immunostained, and viewed by confocal laser scanning microscopy. (<b>Panel A</b>) Cryosections of MLN from vehicle-treated (i.e., PBS) animals. Tissue sections were stained for CD45R/B220 (blue) to detect B cells and CD11c⁺ (red; arrowheads) to detect DCs. (<b>Panels B-C</b>) Cryosections of MLN demonstrating that GPs (green; arrows) were regularly associated with CD11c⁺ DCs (red; arrowheads) In panel B the scale bar corresponds to 100 μm, while Panel C it is 50 μm.</p

Experimental protocols.

<p>Grey shaded boxes indicate periods of oral peanut exposure. Mothers were exposed to ground peanut for 4 weeks during either in the pre-conception period only (PC), during preconception, pregnancy and lactation (PC+PG+LC); or were not fed peanut, serving as controls (None). All the offspring underwent oral sensitization to peanut by being gavage-fed crude peanut extract (CPE) with cholera toxin or were tolerized by being gavage-fed CPE without adjuvant. In some experiments, young mice born to naïve mothers were exposed to the milk of naïve (naïve milk) or peanut-exposed (immune milk) mice concurrently with CPE and cholera toxin to induce peanut sensitization or CPE alone to induce oral tolerance.</p

Offspring’s response to peanut sensitization based on maternal feeding of peanut.

<p>(A) Experimental protocol. Serum antibodies (B-E) and an anaphylactic response to the peanut challenge as indicated by the elevated score during the oral (PO) challenge (F) (baseline symptom score 0 in all the mice), the lowered body temperature during the intraperitoneal (IP) challenge (G), and after the peanut sensitization were assessed in offspring born to mothers exposed to peanut preconceptionally, who either continued to feed peanut during pregnancy and lactation (PC+PG+LC) and those who did not (PC). Offspring born to mothers never exposed to peanut served as controls (None). Naïve, non-sensitized mice served as controls. Shown is a mean with SEM. >15 mice were used per group.</p