Natural Killer Cells Mediate Protection against Yersinia pseudotuberculosis in the Mesenteric Lymph Nodes.

Natural killer cells play a crucial role in the initial defense against bacterial pathogens. The crosstalk between host cells infected with intracellular pathogens and NK cells has been studied intensively, but not much attention has been given to characterize the role of NK cells in the response to extracellular bacterial pathogens such as yersiniae. In this study we used antibody-mediated NK cell depletion to address the importance of this immune cell type in controlling a Y. pseudotuberculosis infection. Analysis of the bacterial counts was used to follow the infection and flow cytometry was performed to characterize the composition and dynamic of immune cells. Depletion of NK cells led to higher bacterial loads within the mesenteric lymph nodes. We further show that in particular CD11b+ CD27+ NK cells which express higher levels of the activation marker CD69 increase within the mesenteric lymph nodes during a Y. pseudotuberculosis infection. Moreover, in response to the activation NK cells secrete higher levels of IFNy, which in turn triggers the production of the proinflammatory cytokine TNFα. These results suggest, that NK cells aid in the clearance of Y. pseudotuberculosis infections mainly by triggering the expression of proinflammatory cytokines manipulating the host immune response

Influence of PhoP and intra-species variations on virulence of Yersinia pseudotuberculosis during the natural oral infection route.

The two-component regulatory system PhoP/PhoQ has been shown to (i) control expression of virulence-associated traits, (ii) confer survival and growth within macrophages and (iii) play a role in Yersinia infections. However, the influence of PhoP on virulence varied greatly between different murine models of infection and its role in natural oral infections with frequently used representative isolates of Y. pseudotuberculosis was unknown. To address this issue, we constructed an isogenic set of phoP+ and phoP- variants of strain IP32953 and YPIII and analyzed the impact of PhoP using in vitro functionality experiments and a murine oral infection model, whereby we tested for bacterial dissemination and influence on the host immune response. Our results revealed that PhoP has a low impact on virulence, lymphatic and systemic organ colonization, and on immune response modulation by IP32953 and YPIII, indicating that PhoP is not absolutely essential for oral infections but may be involved in fine-tuning the outcome. Our work further revealed certain strain-specific differences in virulence properties, which do not strongly rely on the function of PhoP, but affect tissue colonization, dissemination and/or persistence of the bacteria. Highlighted intra-species variations may provide a potential means to rapidly adjust to environmental changes inside and outside of the host

<i>Y</i>. <i>pseudotuberculosis</i> YPIII killing by macrophages is increased after IFNγ prestimulation.

<p>1 x 10⁶ macrophages were infected with MOI 10 of YPIII, 1 hour post infection gentamycin treatment killed extracellular bacteria and after additional 60 minutes of incubation cells were lysed and bacteria were plated onto selective plates. The mean of the survival of untreated macrophage was used as baseline. Data from two independent experiments were pooled and analyzed with a Student’s t-test (*, p<0.05).</p

Infection with <i>Y</i>. <i>pseudotuberculosis</i> results in an increased frequency and numbers of IFNγ producing NK cells.

<p>Mice were infected with 2 x 10⁷ CFUs of <i>Y</i>. <i>pseudotuberculosis</i> strain YPIII. Three days post infection mLN were excised, single cell suspensions were prepared and used for extracellular (CD3, NK1.1) and intracellular staining (IFNγ). Data from three independent experiments were pooled and analyzed with a Student’s t-test, and the frequency (A) and total number (B) are given (**, p < 0.01; ***, p < 0.001).</p

CD11b⁺, CD27⁺ NK cell frequency increases in the mLN during infection with <i>Y</i>. <i>pseudotuberculosis</i> YPIII.

<p>(A) Mice were infected with 2 x 10⁷ CFU of <i>Y</i>. <i>pseudotuberculosis</i> strain YPIII. Three days post infection mLN were excised and NK cell population distribution was analyzed by flow cytometry. To do so, cells were stained with Live/Dead, CD3, NK1.1, CD11b, CD27, CD69 and CD107a. Grey filled squares represent uninfected mice, black filled circles illustrate <i>Y</i>. <i>pseudotuberculosis</i> infected mice. CD11b⁺, CD27⁺ NK cells from the mLN were further analyzed for their expression profiles of the surface markers CD107a (B) and CD69 (C). Grey filled squares represent uninfected mice, while black filled circles represent YPIII infected mice. Data from three independent experiments were pooled and analyzed with a Student’s t-test (*, p < 0.05; ***, p<0.001).</p

Global depletion of NK cells lead to increased bacterial titres of <i>Y</i>. <i>pseudotuberculosis</i> in the mLNs.

<p>7-week old female C57BL/6 mice were injected with 100 μg of anti NK1.1 antibody i. p. 24 hours prior to infection. Mice were challenged with 2 x 10⁷ CFU of <i>Y</i>. <i>pseudotuberculosis</i> strain YPIII. (A) Three days post infection mLNs were excised and homogenates were plated onto selective plates. Data from three independent experiments were pooled. Bacterial loads were compared using a Mann-Whitney U test (**, p < 0.01). (B) Three days post infection mLNs were excised and single cell suspensions were stained with Live/Dead, CD3, CD4, CD8, CD19, NK1.1, CD11b, CD11c, CD49b, F4/80, Ly6C, Ly6G. Living cell numbers of macrophages, neutrophils, inflammatory monocytes, B cells, DCs, monocytes, pDCs, TH cells, CTL, NKT-, and NK cells were assessed. Cross striped bars represent undepleted mice, black bars represent NK depleted mice. Data from three independent experiments were pooled and analyzed with a Student’s t-test (**p < 0.01; ***, p < 0.001). DCs: dendritic cells, TH: T helper cells, CTL: cytotoxic lymphocytes, NKT: natural killer T-cells, NK: natural killer cells.</p

TNFα production by macrophages in the mLN is dependent on the presence of NK cells.

<p>24 hours prior to infection mice were injected with 100 μg of anti NK1.1 antibody i.p. mice were infected with 2 x 10⁷ CFUs of <i>Y</i>. <i>pseudotuberculosis</i> YPIII. Three days post infection mLNs were isolated, single cell suspension were prepared and used for extracellular (CD3, CD11b, CD19, CD86, CD49b, MHCII, F4/80) and intracellular staining (TNFα). (A) Depicted are frequencies of CD86⁺/MHCII⁺ macrophages among living cells. CD11b⁺ F4/80⁺ cells were defined as macrophages. Subset analysis with CD86 / MHCII surface markers was performed. (B) Single cells were analyzed for their expression of TNFα and subsequently the expression of either CD3/CD19/CD49b or F4/80 to determine their cell type. Depicted are F4/80⁺ macrophages producing TNFα. Data from two independent experiments were pooled and analyzed with a Student’s t-test (*, p<0.05; ***, p<0.001).</p

Immune response analysis in the PPs of BALB/c mice induced by <i>phoP</i>⁺ and <i>phoP</i>⁻ derivatives of <i>Y. pseudotuberculosis</i> YPIII or IP32953.

<p>Mice were challenged with 2×10⁸ CFU of <i>Y. pseudotuberculosis</i> strains YPIII (<i>phoP</i>⁻) (n = 20), YP149 (YPIII <i>phoP⁺</i>) (n = 20), IP32953 (<i>phoP</i>⁺) (n = 20) and YPIP06 (IP32953 <i>phoP</i>⁻) (n = 20). A control group of uninfected mice (n = 20) was included. At day three postinfection, living cell numbers of B cells and T cells (A), plasmacytoid and conventional DCs (B), NK cells (C), neutrophils (D), monocytes (E) and macrophages (F) in the PPs were analyzed. The data represents the total cell number per organ in a logarithmic scale. Data from four independent experiments were pooled. Statistical analysis was performed using One-way ANOVA with Tukey’s post hoc test (*, p<0.05; **, p<0.01; ***, p<0.001).</p

PhoP increases expression of <i>cnfY</i> in <i>Y. pseudotuberculosis</i> YPIII.

<p><i>Y. pseudotuberculosis</i> strain YPIII and the isogenic YPIII <i>phoP⁺</i> variant carrying a <i>luxCDABE</i>-reporter plasmid fused to the promoter of <i>cnfY</i> (pJNS02) or the empty vector (pFU54) were grown in LB medium. The <i>luxCDABE</i>-reporter activity and OD₆₀₀ was measured over time. The figure represents three independent experiments done in triplicate. Statistical analysis of the RLU/OD₆₀₀ was performed with the student’s t-test (**, p<0.01; ***, p<0.001).</p

Colonization of <i>Y. pseudotuberculosis</i> YPIII (<i>phoP</i>⁻), YP149 (YPIII <i>phoP⁺</i>), IP32953 (<i>phoP</i>⁺) and YPIP06 (IP32953 <i>phoP</i>⁻) in host tissues.

<p>BALB/c mice were challenged with 2×10⁸ CFU of <i>Y. pseudotuberculosis</i> strains YPIII (<i>phoP</i>⁻) (n = 15), YP149 (YPIII <i>phoP⁺</i>) (n = 15), IP32953 (<i>phoP</i>⁺) (n = 15) and YPIP06 (IP32953 <i>phoP</i>⁻) (n = 15). At day 3 postinfection mice were sacrificed. Numbers of bacteria cells from the different strains were determined in the lymphatic organs PPs (A), MLNs (B) and the systemic organs liver (C) and spleen (D). Data from four independent experiments were pooled. Bacterial loads were compared using One-way ANOVA with Tukey’s post hoc test (*, p<0.05; **, p<0.01; ***, p<0.001).</p