IL-10 receptor blockade during T cell priming prevents the suppressive effects of <i>L. monocytogenes</i> within a phagosome.

<p>Mice were infected with ActA-Lm–OVA, LLO-Lm-OVA, or the combination of both strains in combination with αIL-10R antibody. (A) 30 days post infection mice were challenged with a lethal dose of wt <i>L. monocytogenes</i>-OVA. Spleens were harvested 3 days later and CFU per spleen determined. Each bar represents the mean and standard error of 5 mice per group. (B) B6.MyD88−/− mice were infected with 1×10⁵ CFU ActA-Lm-OVA, 1×10⁸ CFU LLO-Lm-OVA, or the combination of both strains. 16 weeks later, mice were challenged with 1×10³ CFU wt <i>L. monocytogenes</i>-OVA. Spleens and livers were harvested 3 days later and CFU per organ determined. Each bar represents the mean and standard error of 3–5 mice per group. Data are from one representative experiment of two.</p

Suppression of the primary T cell response is antigen-independent.

<p>(A) Mice were infected with the indicated combinations of 1×10⁵ CFU ActA-Lm, 1×10⁵ CFU ActA-Lm–OVA, 1×10⁸ CFU LLO-Lm, and 1×10⁸ CFU LLO-Lm-OVA. 7 days later, the frequency of OVA_257–264 -specific CD8 and LLO_190–201 -specific CD4 T cells was determined by IFN-γ intracellular cytokine staining. (B) ActA-Lm and LLO-Lm <i>L. monocytogenes</i> were engineered to express 4 defined epitopes from vaccinia virus. Mice were infected intravenously with the indicated combinations of ActA-Lm-QuadVacc and LLO-Lm-QuadVacc. 7 days later, spleens were harvested and the frequency of CD8 T cells specific for each epitope was determined by IFN-γ intracellular cytokine staining. Total splenocyte number and absolute CD8 T cells per spleen were consistent between all groups. Values in each plot represent the mean±SEM of IFN-γ+ cells within the CD4 or CD8 population from 5 animals per group.</p

<i>L. monocytogenes</i> within a phagosome impairs protective immunity.

<p>Mice were infected with 1×10⁵ CFU ActA-Lm–OVA alone, or in combination with increasing doses of phagosome-confined LLO-Lm-OVA. (A) Mice were challenged 60 days later with a lethal dose of wt <i>L. monocytogenes</i>-OVA. Spleens were harvested 3 days later and CFU per spleen determined. (B) Mice were infected with 1×10⁵ CFU ActA-Lm-Erm^R alone, or in combination with 1×10⁸ CFU LLO-Lm. Erythromycin-resistant colonies were enumerated from the spleen and liver over 96 hours. Each data point represents the mean and standard error of 5 mice per group. (C) Mice were infected with 1×10⁵ CFU ActA-Lm-Erm^R alone, or in combination with 1×10⁸ CFU LLO-Lm. Erythromycin-resistant colonies were enumerated from the spleen and liver at 1 and 6 hours post infection. Each data point represents the mean and standard error of 5 mice per group from one representative experiment of two. (D) Bone marrow-derived macrophages were infected with ActA-Lm-Erm^R alone (at 1∶10,000), or in combination with 1×10⁸ CFU LLO-Lm (at 1∶200). Erythromycin-resistant colonies were enumerated at the indicated timepoints. Each data point represents the mean and standard error of 3 independent coverslips per timepoint from one representative experiment of two. (E) Mice were infected with the indicated combinations of 1×10⁵ CFU ActA-Lm–OVA and 1×10⁸ heat-killed ActA-Lm–OVA. 30 days later, mice were challenged with 1×10⁵ CFU of wt <i>L. monocytogenes</i>-OVA. Spleens were harvested 3 days later and CFU per spleen determined. (F) Mice were infected with 1×10⁵ CFU ActA-Lm, 1×10⁵ CFU <i>B. subtilis</i>, or the combination of both strains. 30 days post infection, mice were challenged and CFU determined. (G) Mice were infected with the indicated combinations of 1×10³ CFU wild-type and 1×10⁶ CFU LLO-Lm. 58 days later, mice were challenged with 1×10⁵ CFU of wild-type <i>L. monocytognes</i>. Spleens were harvested 3 days later and CFU per spleen determined. In all panels, each point represents a single animal with † indicating animals that died before CFU were determined. Lines indicate the median of each group.</p

LLO activity is sufficient for autophagy activation, as measured by LC3-GFP colocalization.

<p>(<b>A</b>) Photomicrographs: liposomes containing, wild-type LLO or heat-killed LLO. Texas Red (TR) = red dye in both wild-type and heat-killed LLO containing liposomes. Images are representative of results obtained in three independent experiments. Bar = 1 µm. (<b>B</b>) Percentage of colocalization of LC3-GFP with LLO-containing liposomes over a 30 minute time course. Closed line, closed circles: liposomes containing wild-type LLO; dashed line, closed squares: liposomes containing heat-killed LLO. Data shown are representative of results obtained in 3 independent experiments.</p

<i>L. monocytogenes</i> within a phagosome suppress the host inflammatory response to bacteria within the cytosol.

<p>(A) Mice were infected with 1×10⁵ CFU ActA-Lm–OVA alone, or in combination with increasing doses of LLO-Lm-OVA. Serum was collected 24 hours later and assayed for IFN-γ, IL-12p70, IL-6, and MCP-1. (B) C57Bl/6 and B6.MyD88−/− mice were infected with 1×10⁵ CFU ActA-Lm–OVA, 1×10⁸ CFU LLO-Lm-OVA, or the combination of both strains. Serum was collected 4 hours later and assayed for IL-10 and IL-12p40. Bars represent the mean and standard error of 5 mice per group.</p

<i>L. monocytogenes</i> within a phagosome impairs the primary T cell response.

<p>Mice were infected with 1×10⁵ CFU ActA-Lm–OVA alone, or in combination with increasing doses of LLO-Lm-OVA. 7 days later, the frequency of OVA_257–264-specific CD8 T cells and LLO_190–201-specific CD4 T cells was determined by pentamer and IFN-γ intracellular cytokine staining. Total splenocyte number and absolute CD8 T cells per spleen were consistent between all groups. Values in each plot represent the mean±SEM of antigen-specific cells within the CD4 or CD8 population from 5 animals per group.</p

Autophagy is induced by wild-type, Δ<i>actA</i>, Δ<i>plcAB</i> but not by Δ<i>hly</i> or heat-killed <i>L. monocytogenes</i>.

<p>(<b>A</b>) Western blot of LC3I and LC3II in wild-type and <i>atg5^−/−</i> BMDMs infected with wild-type <i>L. monocytogenes</i>. All images are from a single gel. (<b>B</b>) Western blot of LC3I and LC3II in wild-type BMDMs at 60 minutes post-infection. BMDMs were infected with wild-type, Δ<i>hly</i>, Δ<i>actA</i>, Δ<i>plcAB</i> mutant <i>L. monocytogenes</i> as well as heat-killed <i>L. monocytogenes</i>. Images taken from a single gel, cropped and combined. Data shown are representative of results obtained in three independent experiments.</p

<i>B. subtilis</i> expressing cytolysins induced LC3I lipidation and LC3-GPF colocalization in BMDMs.

<p>(<b>A</b>) Western blot for LC3I lipidation in BMDMs infected with either wild-type <i>B. subtilis</i> or <i>B. subtilis</i> expressing LLO. (<b>B</b>) LC3 colocalization in LC3-GFP BMDMs infected with either wild-type <i>B. subtilis</i> or <i>B. subtilis</i> engineered to express LLO or PFO. Solid line, closed circles: LC3-GFP BMDMs infected with <i>B. subtilis</i> expressing PFO; dashed line, closed squares: LC3-GFP BMDMs infected with <i>B. subtilis</i> expressing LLO; solid line, open circles: LC3-GFP BMDMs infected with <i>B. subtili</i>. Graphs are a representative of three independent experiments, standard deviations have been included. (<b>C</b>) Photomicrographs: LC3-GFP BMDMs 40 minutes post-infection with either wild-type or cytolysin expressing <i>B. subtilis</i> (red). Images are representative of results obtained in 3 independent experiments. Bar = 2 µm. (<b>D</b>) Stills from time-lapse video microscopy of <i>B. subtilis</i> (red) expressing LLO in LC3-GFP BMDMs. Bar = 3.4 µm.</p

LC3-GFP co-localizes with wild-type <i>L. monocytogenes</i>, but does not co-localize with Δ<i>hly</i>.

<p>(<b>A</b>) Percent of BMDMs that contain LC3-GFP, colocalizing with wild-type and mutant (Δ<i>hly</i>) <i>L. monocytogenes</i> (red) up to three hours post-infection. (<b>B</b>) Photomicrographs of LC3-GFP BMDMs 40 minutes post-infection showing colocalization of wild-type <i>L. monocytogenes</i> (red) with LC3-GFP. Graphs are a representative of three independent experiments, standard deviations have been included. Images are representative of results obtained in three independent experiments. Bar = 2 µm.</p