Temporal Expression of Bacterial Proteins Instructs Host CD4 T Cell Expansion and Th17 Development

Pathogens can substantially alter gene expression within an infected host depending on metabolic or virulence requirements in different tissues, however, the effect of these alterations on host immunity are unclear. Here we visualized multiple CD4 T cell responses to temporally expressed proteins in Salmonella-infected mice. Flagellin-specific CD4 T cells expanded and contracted early, differentiated into Th1 and Th17 lineages, and were enriched in mucosal tissues after oral infection. In contrast, CD4 T cells responding to Salmonella Type-III Secretion System (TTSS) effectors steadily accumulated until bacterial clearance was achieved, primarily differentiated into Th1 cells, and were predominantly detected in systemic tissues. Thus, pathogen regulation of antigen expression plays a major role in orchestrating the expansion, differentiation, and location of antigen-specific CD4 T cells in vivo

Salmonella Transiently Reside in Luminal Neutrophils in the Inflamed Gut

Enteric pathogens need to grow efficiently in the gut lumen in order to cause disease and ensure transmission. The interior of the gut forms a complex environment comprising the mucosal surface area and the inner gut lumen with epithelial cell debris and food particles. Recruitment of neutrophils to the intestinal lumen is a hallmark of non-typhoidal Salmonella enterica infections in humans. Here, we analyzed the interaction of gut luminal neutrophils with S. enterica serovar Typhimurium (S. Tm) in a mouse colitis model.Upon S. Tm(wt) infection, neutrophils transmigrate across the mucosa into the intestinal lumen. We detected a majority of pathogens associated with luminal neutrophils 20 hours after infection. Neutrophils are viable and actively engulf S. Tm, as demonstrated by live microscopy. Using S. Tm mutant strains defective in tissue invasion we show that pathogens are mostly taken up in the gut lumen at the epithelial barrier by luminal neutrophils. In these luminal neutrophils, S. Tm induces expression of genes typically required for its intracellular lifestyle such as siderophore production iroBCDE and the Salmonella pathogenicity island 2 encoded type three secretion system (TTSS-2). This shows that S. Tm at least transiently survives and responds to engulfment by gut luminal neutrophils. Gentamicin protection experiments suggest that the life-span of luminal neutrophils is limited and that S. Tm is subsequently released into the gut lumen. This "fast cycling" through the intracellular compartment of gut luminal neutrophils would explain the high fraction of TTSS-2 and iroBCDE expressing intra- and extracellular bacteria in the lumen of the infected gut. In conclusion, live neutrophils recruited during acute S. Tm colitis engulf pathogens in the gut lumen and may thus actively engage in shaping the environment of pathogens and commensals in the inflamed gut

Energy Taxis toward Host-Derived Nitrate Supports a Salmonella Pathogenicity Island 1-Independent Mechanism of Invasion

Salmonella enterica serovar Typhimurium can cross the epithelial barrier using either the invasion-associated type III secretion system (T3SS-1) or a T3SS-1-independent mechanism that remains poorly characterized. Here we show that flagellum-mediated motility supported a T3SS-1-independent pathway for entering ileal Peyer’s patches in the mouse model. Flagellum-dependent invasion of Peyer’s patches required energy taxis toward nitrate, which was mediated by the methyl-accepting chemotaxis protein (MCP) Tsr. Generation of nitrate in the intestinal lumen required inducible nitric oxide synthase (iNOS), which was synthesized constitutively in the mucosa of the terminal ileum but not in the jejunum, duodenum, or cecum. Tsr-mediated invasion of ileal Peyer’s patches was abrogated in mice deficient for Nos2, the gene encoding iNOS. We conclude that Tsr-mediated energy taxis enables S. Typhimurium to migrate toward the intestinal epithelium by sensing host-derived nitrate, thereby contributing to invasion of Peyer’s patches

Mucosal flagellin-specific CD4 T cells produce IL-22 upon restimulation.

<p>C57BL/6 mice were infected three times orally with 5×10⁹ Salmonella (BRD509) at one-month intervals. On day 7 after the third infection, CD4 T cells from spleen and MLN, including Peyer's patches, were isolated. Purified CD4 T cells were restimulated with 10 µM peptide (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002499#ppat-1002499-t003" target="_blank">Table 3</a>) for 16 h in the presence of irradiated splenocytes. Culture supernatants were collected and IL-22 production measured by ELISA. Data show IL-22 production in culture supernatants as scatter plots representing individual mice. Data are pooled from three separate experiments. Numbers above each group indicate statistical significance and show the <i>p</i> value of a comparison between media alone (no stimulation) and peptide stimulation group.</p

Natural <i>Salmonella</i> class-II epitopes used in this study.

<p>Natural <i>Salmonella</i> class-II epitopes used in this study.</p

Differential regulation of bacterial Flagellin (FliC) and SseJ in vitro and in vivo.

<p>For in vitro detection of bacterial mRNA, <i>Salmonella</i> (BRD509) were cultured under SPI1-inducing conditions (LB broth, for 3hours) or SPI2-inducing conditions (modified N minimal medium, for 6hours) (<i>Top)</i>. For in vivo detection, C57BL/6 mice were infected intravenously with 5X10⁵<i>Salmonella</i> (BRD509), and spleens harvested 30 min or 5 hours later (<i>Bottom</i>). Bacterial RNA was isolated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002499#s4" target="_blank">Materials and Methods</a>. Expression of Flagellin (FliC) or SseJ mRNA was quantified by real-time qPCR. Bar graphs show the mean number ± SEM of Flagellin (FliC) or SseJ mRNA transcripts normalized to the respective amount of 16S rRNA in each sample. Data are pooled from two separate experiments (in vitro) or three separate experiments using a total of nine mice (in vivo). Numbers above indicate statistical significance and show the <i>p</i> value of a comparison between the groups.</p

Expansion of endogenous flagellin_427–441- or SseJ_329–341-specific CD4 T cells after peptide immunization or <i>Salmonella</i> infection of mice.

<p>C57BL/6 mice were immunized sub-cutaneously with either 100 µg of flagellin_427–441 or SseJ_329–341 peptide in the presence of CFA or infected with 5x10⁵<i>Salmonella</i> (BRD509). On day 7, inguinal, axillary, and brachial lymph nodes were isolated from peptide-immunized mice (Immunized). On day 35, spleens were harvested from C57BL/6 mice infected intravenously with <i>Salmonella</i> BRD509 (Infected). Endogenous flagellin_427–441- or SseJ_329–341-specific CD4 T cells were detected using Flagellin:I-A^b or SseJ:I-A^b tetramers, enriched with anti-fluorochrome microbeads, stained with antibodies to several surface markers, and examined by flow cytometry. CD4 T cells from naive, immunized, and <i>Salmonella</i>-infected mice, were detected among CD11c⁻CD11b⁻F4/80⁻B220⁻CD3⁺ cells, and further analyzed for expression of CD44 and Flagellin:I-A^b or SseJ:I-A^b tetramer positive cells. CD4 T cells from the unbound column fraction and CD8 T cells within the bound fraction are also shown as controls. FACS plots are representative of three mice per group and three replicate experiments.</p

Detection of flagellin-specific CD4 Th17 cells in intestinal tissues.

<p>C57BL/6 mice were infected three times orally with 5×10⁹<i>Salmonella</i> (BRD509) at one-month intervals. On day 7 after the third infection, CD4 T cells from the spleens, MLN, including Peyer's patches, livers (LV), and laminar propria (LP) were purified. Isolated CD4 T cells (1×10⁵) were restimulated with 10 µM of various peptides (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002499#ppat-1002499-t003" target="_blank">Table 3</a>) for 16 h in the presence of irradiated splenocytes. (A) IFN-γ or, (B) IL-17A production was measured by ELISPOT. Scatter graphs show the number of (A) IFN-γ or, (B) IL-17A-producing CD4 T cells in infected tissues from individual mice. (C) Bar graphs show the mean ratio of IFN-γ to IL-17 production after in vitro stimulation. Numbers above indicate statistical significance and show <i>p</i> value of a comparison between media alone (no stimulation) and peptide stimulation group.</p

Salmonella peptides screened for immunogenicity in C57BL/6 mice.

<p>Salmonella peptides screened for immunogenicity in C57BL/6 mice.</p

Discovery of novel <i>Salmonella</i> I-A^b epitopes.

<p>(A) C57BL/6 mice were immunized with a mixture of 4 groups of 11 peptides from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002499#ppat-1002499-t001" target="_blank">Table 1</a> at 5 µg/peptide in CFA. Eight days later, draining lymph nodes were isolated and purified CD4 T cells restimulated with individual peptides (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002499#ppat-1002499-t001" target="_blank">Table 1</a>) in the presence of irradiated splenocytes. IFN-γ production was measured 24–36 hours later by ELISPOT. (B) 129Sv mice were orally infected with virulent 5x10⁷<i>Salmonella</i> (SL1344) and spleens harvested 11 weeks later. Purified CD4 T cells were restimulated with peptides from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002499#ppat-1002499-t002" target="_blank">Table 2</a> for 24–36 h in the presence of irradiated splenocytes and IFN-γ production measured by ELISPOT. ConA and no peptide wells constitute the positive and negative controls respectively. Each plate is representative of 2–3 individual experiments.</p