<i>Y</i>. <i>enterocolitica</i> internalization is reduced in hypoxic incubated cells.

<p><i>Y</i>. <i>enterocolitica</i> serotype O:8 8081v was used to infect Caco-2 cells (MOI 10) pre-incubated at normoxia or hypoxia for 24 hr. The infection was also performed at normoxia or hypoxia. (A) The percentage of internalized bacteria was significantly reduced in hypoxia pre-incubated cells. There was no significant difference in the number of associated bacteria (B) or in bacterial growth (C) in the cells grown under either condition. (D) Twenty-four hr incubation under hypoxia did not result in significant differences in cytotoxicity as compared to 24 hr under normoxia. * p ≤ 0.05 using one-way ANOVA. Plotted values represent mean ±SEM.</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

<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

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

Treatment with DMOG mimics hypoxic results.

<p><i>Y</i>. <i>enterocolitica</i> serotype O:8 8081v was used to infect Caco-2 cells (MOI 10) incubated at normoxia and treated with DMOG for 7 hr. The infection was performed at normoxia. (A) The percentage of internalized bacteria was significantly reduced in DMOG treated cells. There was no significant difference in the number of associated bacteria (B) or in bacterial growth (C) in the cells between treated and untreated. (D) Treatment with DMOG did not result in significant differences in cytotoxicity. (E) Western blots of β1 integrin and HIF-1α and their (F) quantification in Caco-2 cells treated with DMOG for 7 hr as compared to untreated controls. * p ≤ 0.05; ** p ≤ 0.01 using two-tailed Student’s <i>t</i>-test.</p

Quantification of Western blots of β1 integrin and HIF-1α.

<p>(A) Caco-2 cells pre-incubated under hypoxia as compared to the normoxic controls for 24 hr. Quantification of Western blot values are presented as a ratio over the respective normoxic value. (B) Representative Western blots showing β₁ integrin at 130 kDa and HIF-1α at 120kDa. ** p ≤ 0.01, *** p ≤ 0.001 using two-tailed Student’s <i>t</i>-test.</p

Analysis of total and phosphorylated FAK.

<p>Levels of total FAK and p-FAK Y397 from Caco-2 cells pre-incubated under hypoxia or normoxia for 24 hr and then infected with <i>Y</i>. <i>enterocolitica</i>. (A) Western blots and (B) their quantification. Values are presented as a ratio of infected over uninfected in case of normoxia or hypoxia, respectively. ** p ≤ 0.01 using two-tailed Student’s <i>t</i>-test.</p

Decreased β1 integrin under hypoxia.

<p>Representative fluorescent micrographs of β₁ integrin abundance under normoxia (A) or hypoxia (C) with the mouse IgG1 isotype control (B and D). Green: β1 integrin, Blue: DAPI.</p

Integrin blocking decreases internalization.

<p>Cells were treated with 45 μg/ml of 6S6 integrin blocking antibody, 45 μg/ml of IgG1 isotype control or left untreated for one hour before infection. (A) The percentage of internalized bacteria in cells blocked with anti-integrin antibody was significantly decreased. There was no significant difference between untreated or antibody blocked cells under hypoxia. (B) There was no significant difference in the number of associated bacteria under any condition. **** p<0.0001 using One way ANOVA test, and ** p ≤ 0.01 and ns = non-significant using Tukey's multiple comparisons test.</p