The gut epithelium exhibits several pathways that protect the integrity of this organ.

<p>Intestinal epithelial cells (ECs) produce stem cell factor (SCF), which induces proliferation and resistance to bacterial invasion. In addition, neighboring γδ intraepithelial lymphocytes (IELs) produce keratinocyte growth factor (KGF), which also stabilizes ECs. IL-22 produced by Th17, Th22, and γδ T cells as well as natural killer (NK) and lymphoid tissue inducer (LTi) cells plays a key role in both early and late phases of innate immunity in order to maintain the EC barrier. In addition, monocyte chemotactic protein (MCP-1) produced by Paneth cells and goblet cells down-regulates migration of plasmacytoid DCs (pDCs) into the intestinal lamina propria in order to decrease TNF-α-induced EC apoptosis.</p

The mucosal immune system (MIS) is interconnected, enabling it to protect vast surface areas.

<p>This is accomplished by inductive sites of organized lymphoid tissues, e.g., in the gut the Peyer's patches (PPs) and mesenteric lymph nodes (MLNs) comprise the GALT. Lumenal Ags can be easily sampled via M cells or by epithelial DCs since this surface is not covered by mucus due to an absence of goblet cells. Engested Ags in DCs trigger specific T and B cell responses in Peyer's patches and MLNs. Homing of lymphocytes expressing specific receptors helps guide their eventual entry into major effector tissues, e.g., the lamina propria of the gut, the upper respiratory (UR) tract, the female reproductive tract, or acinar regions of exocrine glands. Terminal differentiation of plasma cells producing polymeric (mainly dimeric) IgA is then transported across ECs via the pIgR for subsequent release as S-IgA Abs.</p

APRIL and BAFF expression by CD11c⁺ DCs.

<p>Mice were nasally immunized with TNP-LPS with or without nCT, nCT alone or PBS. (A) Five days after immunization, mononuclear cells were isolated from SMGs, NPs and NALT and stained for CD11c, APRIL and BAFF using the respective fluorescence-conjugated mAbs described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025396#s2" target="_blank">Materials and Methods</a> section. Samples were subjected to flow cytometric analysis by FACSCalibur®. *<i>p</i><0.05 when compared with mice given TNP-LPS or nCT alone. The values are presented as the mean ± SEM of 10 mice for each group and represent a total of five separate experiments. (B) CD11c⁺ DCs were isolated from SMGs, NPs and NALT of mice given nasal TNP-LPS with or without nCT by AutoMACS two days after the immunization. The expression of BAFF- and APRIL-specific mRNA by mucosal DCs was determined by quantitative real-time PCR. Relative expression of BAFF- and APRIL- specific mRNA were displayed as the fold change of respective transcript expression by experimental group (TNP-LPS plus nCT) over the expression by controls (TNP-LPS alone). The values are presented as the mean ± SEM of 10 mice for each group and represent a total of five separate experiments.</p

TACI, BCMA and BAFF-R expression by mucosal B-1 (CD5⁺ B220⁺) B cells.

<p>Wild-type mice were nasally immunized with TNP-LPS with or without nCT, nCT alone or PBS. (A) Five days after immunization, mononuclear cells were isolated and stained for CD5, B220, TACI, BCMA and BAFF using the respective fluorescence-conjugated mAbs as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025396#s2" target="_blank">Materials and Methods</a> section. Samples were then subjected to flow cytometric analysis by FACSCalibur®. *<i>p</i><0.05 when compared with mice given TNP-LPS or nCT alone. The values are presented as the mean ± SEM of 10 mice for each group and represent a total of five separate experiments. (B) B-1 B cells were purified by FACSAria from SMGs, NPs and NALT of mice given nasal TNP-LPS with or without nCT two days after the immunization. The expression of TACI, BCMA and BAFF-R mRNA by B1-B cells was assessed by quantitative real-time PCR. Relative expression of TACI, BCMA and BAFF-R mRNA are displayed as the fold change of respective transcript expression by the experimental group (TNP-LPS plus nCT) over the expression by controls (TNP-LPS alone). The values are presented as the mean ± SEM of 10 mice for each group and represent a total of five separate experiments.</p

The frequencies of CD11c⁺ DCs and co-stimulatory molecule expression in wild-type mice given nasal TNP-LPS with or without nCT^a.

a<p>Mononuclear cells from SMGs, NPs and NALT were isolated five days after immunization with TNP-LPS plus nCT or TNP-LPS alone, and were stained with FITC-conjugated anti-CD11c mAb.</p>b<p>Mononuclear cells were stained with FITC-conjugated anti-CD11c and PE-labeled anti-CD40, -CD80, -CD-86 or -MHC class II mAbs and were then subjected to flow cytometry analysis by FACSCalibur®.</p><p>*<i>p</i><0.05 compared with immunized mice given TNP-LPS alone.</p

Expression of AID, αCT, Iμ-Cα transcripts in B-1 B cells from the oral-nasopharyngeal effector and inductive tissues of mice nasally immunized with TNP-LPS with/without nCT or nCT alone.^a^,^b^,^c

a<p>Five days after nasal immunization, B-1 B (CD5⁺ B220⁺) cells from the SMGs, NPs and NALT (as a positive control) were purified by FACS and were then subjected to semi-quantitative RT-PCR.</p>b<p>The numbers are mean the expression percentages of the respective CSR-associated molecules when the density of β-actin expression on respective lymphoid tissues was calculated as 100 with ChemiDoc XRS Quantity one Analysis software (Bio-Rad).</p>c<p>The values are presented as the mean ± SEM of 10 mice for each group and represent a total of five separate experiments.</p><p>*<i>p</i><0.05 when compared with mice given TNP-LPS alone.</p>#<p><i>p</i><0.05 when compared with mice given nCT alone.</p

Induction of IgA CSR by B-1 B cells in the presence of mucosal DCs.

<p>IgA negative B cells were purified from the peritoneal cavity of wild-type mice by magnetic sorting. Purified cells (8×10⁶ cells/ml) were then cultured with DCs (4×10⁵ cells/ml) from SMGs, NPs and CLNs of mice given nasal TNP-LPS with/without nCT or nCT alone in the presence of 1 ng/ml TGF-β₁, and 100 ng/ml IL-5. After 5 days of incubation, non-adherent cells were harvested and stained with FITC-conjugated anti-IgA, and PE-labeled anti-CD5 mAbs. The data represent a typical profile of 10 mice for each group.</p

Rectal administration of a chlamydial subunit vaccine protects against genital infection and upper reproductive tract pathology in mice

<div><p>In this study, we tested the hypothesis that rectal immunization with a VCG-based chlamydial vaccine would cross-protect mice against heterologous genital <i>Chlamydia trachomatis</i> infection and <i>Chlamydia</i>-induced upper genital tract pathologies in mice. Female mice were immunized with a <i>C</i>. <i>trachomatis</i> serovar D-derived subunit vaccine or control or live serovar D elementary bodies (EBs) and the antigen-specific mucosal and systemic immune responses were characterized. Vaccine efficacy was determined by evaluating the intensity and duration of genital chlamydial shedding following intravaginal challenge with live serovar E chlamydiae. Protection against upper genital tract pathology was determined by assessing infertility and tubal inflammation. Rectal immunization elicited high levels of chlamydial-specific IFN-gamma-producing CD4 T cells and humoral immune responses in mucosal and systemic tissues. The elicited immune effectors cross-reacted with the serovar E chlamydial antigen and reduced the length and intensity of genital chlamydial shedding. Furthermore, immunization with the VCG-vaccine but not the rVCG-gD2 control reduced the incidence of tubal inflammation and protected mice against <i>Chlamydia</i>-induced infertility. These results highlight the potential of rectal immunization as a viable mucosal route for inducing protective immunity in the female genital tract.</p></div

Antigen-specific T cell-mediated immune responses.

<p>Pooled CD4+ T cells purified from the spleens of immunized mice and controls were restimulated <i>in vitro</i> with <i>C</i>. <i>trachomatis</i> serovar E antigen (UV-irradiated EBs; 10 microgram/ml). The amount of cross-reactive chlamydial-specific Th1 (IFN-gamma, TNF-alpha, IL-2) and Th2 (IL-5, IL-10) as well as IL-17 cytokines contained in supernatants of culture-stimulated CD4+ T cells was measured using Bio-Plex cytokine assay kit. The concentration of the cytokines in each sample was obtained by extrapolation from a standard calibration curve generated simultaneously. Data were calculated as the mean values (± S.D.) for triplicate cultures for each experiment. The controls (cultures without antigen) did not contain detectable levels of cytokine and so the data were excluded from the results. The results are from two independent experiments and are shown as mean cytokine concentrations (pg/ml) ± SD (A). Significant differences between Th1 and Th2 cytokines (IFN-gamma and IL-5) are indicated by asterisk (<i>P</i> <0.05). Antigen-specific CD4+T cell proliferative responses were assessed for their ability to proliferate in response to <i>in vitro</i> restimulation in culture with chlamydial serovar E antigen. Results are expressed as stimulation index (SI) values (B), the ratio between absorbance values of stimulated and non-stimulated cells and the bars represent the mean and S.D. of three independent experiments. *<i>p</i><0.05 (rVCG-PmpD/PorB vaccine versus rVCG-gD2 control and rVCG-PmpD/PorB vaccine versus live EBs).</p

Histopathology of oviduct tissues from vaccinated and control mice reinfected with live serovar E chlamydiae.

<p>The H&E-stained sections of excised oviduct tissues were evaluated microscopically for distribution of inflammation and pathologic lesions. Representative images of oviducts from (a) Uninfected control mice with normal oviducts (arrows); insert shows enlarged section of villi with abundant cilia (thin arrows), (b) rVCG-PmpD/PorB vaccine-immunized mice showing intact normal villi with abundant cilia (arrows) but with numerous vacuoles (arrowhead), and (c) rVCG-gD2-immunized mice showing diffusely attenuated villi (arrow); there is villus atrophy and most of the cilia from the apical surfaces of the lining epithelia are absent (arrowhead). Scale bar, 50 microns.</p