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Caspase-3 mediates the pathogenic effect of Yersinia pestis YopM in liver of C57BL/6 mice and contributes to YopM's function in spleen.

Ye Z, Gorman AA, Uittenbogaard AM, Myers-Morales T, Kaplan AM, Cohen DA, Straley SC - PLoS ONE (2014)

Bottom Line: The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids.By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells.This result identified caspase-3 as a co-factor or effector in YopM's action and supports the hypothesis that in liver YopM's main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, United States of America.

ABSTRACT
The virulence protein YopM of the plague bacterium Yersinia pestis has different dominant effects in liver and spleen. Previous studies focused on spleen, where YopM inhibits accumulation of inflammatory dendritic cells. In the present study we focused on liver, where PMN function may be directly undermined by YopM without changes in inflammatory cell numbers in the initial days of infection, and foci of inflammation are easily identified. Mice were infected with parent and ΔyopM-1 Y. pestis KIM5, and effects of YopM were assessed by immunohistochemistry and determinations of bacterial viable numbers in organs. The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids. A new in-vivo phenotype of YopM was revealed: death of inflammatory cells, evidenced by TUNEL staining beginning at d 1 of infection. Based on distributions of Ly6G(+), F4/80(+), and iNOS(+) cells within foci, the cells that were killed could have included both PMNs and macrophages. By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells. The destruction depended on the presence of both PMNs in the mice and YopM in the bacteria. In mice that lacked the apoptosis mediator caspase-3 the infection dynamics were novel: the parent Y. pestis was limited in growth comparably to the ΔyopM-1 strain in liver, and in spleen a partial growth limitation for parent Y. pestis was seen. This result identified caspase-3 as a co-factor or effector in YopM's action and supports the hypothesis that in liver YopM's main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs.

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Early in infection many Y. pestis KIM5 bacteria were associated with inflammatory cells including PMNs.Mice were infected IV for 17 h with 107 28/37°C-grown Y. pestis KIM5. Sections of formalin-fixed paraffin-embedded livers were stained with cell- or Y. pestis-specific antibody, developed with DAB (black or brown), and counterstained with hematoxylin. Antibodies used were: Panel A, anti-Ly6G (for PMNs); Panel B, anti-F4/80 (for KCs and MOs); Panels C and D, anti-Y. pestis antibodies; Panels E and F, the YPF19 monoclonal antibody against the F1 fibril. Solid arrows in panels A and B indicate foci of inflammation; open arrows indicate either small foci or “polar cap” (tangential) sections of foci. The arrow in panel C points to a bacterium on a mononuclear cell adjacent to a focus where numerous bacteria were contained in the plane of the section. The arrow in panel D indicates several bacteria associated with PMNs in a hepatic sinus. Arrows in panels E and F point to bacteria associated with mononuclear cells. The bars in panels A and B represent 100 µm; in panels C-F, the bars represent 10 µm.
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pone-0110956-g003: Early in infection many Y. pestis KIM5 bacteria were associated with inflammatory cells including PMNs.Mice were infected IV for 17 h with 107 28/37°C-grown Y. pestis KIM5. Sections of formalin-fixed paraffin-embedded livers were stained with cell- or Y. pestis-specific antibody, developed with DAB (black or brown), and counterstained with hematoxylin. Antibodies used were: Panel A, anti-Ly6G (for PMNs); Panel B, anti-F4/80 (for KCs and MOs); Panels C and D, anti-Y. pestis antibodies; Panels E and F, the YPF19 monoclonal antibody against the F1 fibril. Solid arrows in panels A and B indicate foci of inflammation; open arrows indicate either small foci or “polar cap” (tangential) sections of foci. The arrow in panel C points to a bacterium on a mononuclear cell adjacent to a focus where numerous bacteria were contained in the plane of the section. The arrow in panel D indicates several bacteria associated with PMNs in a hepatic sinus. Arrows in panels E and F point to bacteria associated with mononuclear cells. The bars in panels A and B represent 100 µm; in panels C-F, the bars represent 10 µm.

Mentions: Our previous studies had indicated that in liver YopM undermines PMN antibacterial function without a diffusible mediator [19]. This could occur through direct binding and delivery of YopM to PMNs or through delivery of YopM to a cell such as a KC that PMNs must directly interact with to have their antibacterial effect. To establish the context in which YopM functions early, we used IHC to identify inflammatory cells and locate Y. pestis in livers of B6 mice infected for 17 h with thermally pre-induced Y. pestis KIM5 at a dose high enough (106 or 107) that bacteria would be found in thin sections. Figure 3A shows that these early inflammatory foci were populated by PMNs, which tended to be evenly distributed as illustrated (based on nuclear morphology in H&E-stained sections from 4 experiments and Ly6G staining in the experiment of Figure 3: see Table S1). F4/80+ cells (Kupffer cells [KCs] and MOs) were scattered in sinusoids and clustered around foci (Fig. 3B). If the focus was small, they tended to be present over the entire focus (∼70% of such foci), but in larger foci they were restricted to the periphery (∼84% of such foci) (quantification is given in Table 1 and Table S1). This observation indicates that as foci enlarge, KC/MOs are lost from the centers.


Caspase-3 mediates the pathogenic effect of Yersinia pestis YopM in liver of C57BL/6 mice and contributes to YopM's function in spleen.

Ye Z, Gorman AA, Uittenbogaard AM, Myers-Morales T, Kaplan AM, Cohen DA, Straley SC - PLoS ONE (2014)

Early in infection many Y. pestis KIM5 bacteria were associated with inflammatory cells including PMNs.Mice were infected IV for 17 h with 107 28/37°C-grown Y. pestis KIM5. Sections of formalin-fixed paraffin-embedded livers were stained with cell- or Y. pestis-specific antibody, developed with DAB (black or brown), and counterstained with hematoxylin. Antibodies used were: Panel A, anti-Ly6G (for PMNs); Panel B, anti-F4/80 (for KCs and MOs); Panels C and D, anti-Y. pestis antibodies; Panels E and F, the YPF19 monoclonal antibody against the F1 fibril. Solid arrows in panels A and B indicate foci of inflammation; open arrows indicate either small foci or “polar cap” (tangential) sections of foci. The arrow in panel C points to a bacterium on a mononuclear cell adjacent to a focus where numerous bacteria were contained in the plane of the section. The arrow in panel D indicates several bacteria associated with PMNs in a hepatic sinus. Arrows in panels E and F point to bacteria associated with mononuclear cells. The bars in panels A and B represent 100 µm; in panels C-F, the bars represent 10 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4220956&req=5

pone-0110956-g003: Early in infection many Y. pestis KIM5 bacteria were associated with inflammatory cells including PMNs.Mice were infected IV for 17 h with 107 28/37°C-grown Y. pestis KIM5. Sections of formalin-fixed paraffin-embedded livers were stained with cell- or Y. pestis-specific antibody, developed with DAB (black or brown), and counterstained with hematoxylin. Antibodies used were: Panel A, anti-Ly6G (for PMNs); Panel B, anti-F4/80 (for KCs and MOs); Panels C and D, anti-Y. pestis antibodies; Panels E and F, the YPF19 monoclonal antibody against the F1 fibril. Solid arrows in panels A and B indicate foci of inflammation; open arrows indicate either small foci or “polar cap” (tangential) sections of foci. The arrow in panel C points to a bacterium on a mononuclear cell adjacent to a focus where numerous bacteria were contained in the plane of the section. The arrow in panel D indicates several bacteria associated with PMNs in a hepatic sinus. Arrows in panels E and F point to bacteria associated with mononuclear cells. The bars in panels A and B represent 100 µm; in panels C-F, the bars represent 10 µm.
Mentions: Our previous studies had indicated that in liver YopM undermines PMN antibacterial function without a diffusible mediator [19]. This could occur through direct binding and delivery of YopM to PMNs or through delivery of YopM to a cell such as a KC that PMNs must directly interact with to have their antibacterial effect. To establish the context in which YopM functions early, we used IHC to identify inflammatory cells and locate Y. pestis in livers of B6 mice infected for 17 h with thermally pre-induced Y. pestis KIM5 at a dose high enough (106 or 107) that bacteria would be found in thin sections. Figure 3A shows that these early inflammatory foci were populated by PMNs, which tended to be evenly distributed as illustrated (based on nuclear morphology in H&E-stained sections from 4 experiments and Ly6G staining in the experiment of Figure 3: see Table S1). F4/80+ cells (Kupffer cells [KCs] and MOs) were scattered in sinusoids and clustered around foci (Fig. 3B). If the focus was small, they tended to be present over the entire focus (∼70% of such foci), but in larger foci they were restricted to the periphery (∼84% of such foci) (quantification is given in Table 1 and Table S1). This observation indicates that as foci enlarge, KC/MOs are lost from the centers.

Bottom Line: The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids.By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells.This result identified caspase-3 as a co-factor or effector in YopM's action and supports the hypothesis that in liver YopM's main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, United States of America.

ABSTRACT
The virulence protein YopM of the plague bacterium Yersinia pestis has different dominant effects in liver and spleen. Previous studies focused on spleen, where YopM inhibits accumulation of inflammatory dendritic cells. In the present study we focused on liver, where PMN function may be directly undermined by YopM without changes in inflammatory cell numbers in the initial days of infection, and foci of inflammation are easily identified. Mice were infected with parent and ΔyopM-1 Y. pestis KIM5, and effects of YopM were assessed by immunohistochemistry and determinations of bacterial viable numbers in organs. The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids. A new in-vivo phenotype of YopM was revealed: death of inflammatory cells, evidenced by TUNEL staining beginning at d 1 of infection. Based on distributions of Ly6G(+), F4/80(+), and iNOS(+) cells within foci, the cells that were killed could have included both PMNs and macrophages. By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells. The destruction depended on the presence of both PMNs in the mice and YopM in the bacteria. In mice that lacked the apoptosis mediator caspase-3 the infection dynamics were novel: the parent Y. pestis was limited in growth comparably to the ΔyopM-1 strain in liver, and in spleen a partial growth limitation for parent Y. pestis was seen. This result identified caspase-3 as a co-factor or effector in YopM's action and supports the hypothesis that in liver YopM's main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs.

Show MeSH
Related in: MedlinePlus