BMDC maturation in response to infection with rough <i>Brucella</i> strain RB51 or its parent smooth strain S2308.

<p>WT or Casp2KO BMDCs were infected with S2308, RB51, or <i>S. typhimurium</i> strain SL1344 (MOI: 5). At 24 h post infection, the expression of three costimulatory cell surface markers CD40 (A), CD80 (B), and CD86 (C) was measured by flow cytometry. For each surface molecule studied, a cytometry analysis histogram from one representative experiment is presented on the left, and the complication histogram on the right includes summarized results (means ± standard deviations of the means) from four independent experiments. The asterisk sign (*) denotes statistically significant difference (P-value <0.05) between an infection group and the medium control group. The sign (**) represents statistically significant difference (P-value <0.05) between WT and Casp2KO BMDCs infected with the same bacteria.</p

The effects of caspase-2 on production of cytokines in <i>Brucella</i>-infected BMDCs.

<p> To assess the effect of caspase-2 on DC function, the protein levels of TNF-α (A), IL-6 (B), IL12/IL23p40 (C), and IFN-γ (D) from the culture supernatants of S2308- or RB51-infected WT or Casp2KO BMDCs were collected and analyzed using ELISA. Supernatants were obtained at 24 h post infection. The results represent cytokine secretion (means ± standard deviations of the means of three experiments). The asterisk sign (*) denotes statistically significant difference (P-value <0.05) between an infection group and the medium control group. The sign (**) represents statistically significant difference (P-value <0.05) between WT and Casp2KO BMDCs infected with the same bacteria.</p

Cell death of macrophages infected with <i>B. abortus</i> strains RB51,RA1 and their parent wild type S2308.

<p>(A) RB51 and RA1 induced 61.5%±8.7% and 74.7%±5.6% macrophage cells death, respectively, at a MOI of 200 at 24 h post infection. <i>B. abortus</i> strain 2308 induced limited macrophage cell death even at the MOI of 2,000. (B) LDH release from RB51, RA1 and S2308 infected macrophages. The data represent the means±standard deviations for three independent experiments. The asterisk (*) represents the significant differences (<i>P</i><0.05) of the LDH release level from macrophages infected by smooth S2308 compared to that from macrophages infected with rough strain RB51 or RA1. (C) Growth kinetics of RB51, RA1 and S2308 in macrophages. The number of internalized cells of S2308 at MOI 2,000 found was similar to the number of cells of internalized RB51 and RA1 at a MOI 200 at 1 h post infection. The asterisk (*) represents the significant differences (<i>P</i><0.05) in survival level of smooth S2308 compared to that of rough strain RB51 or RA1 inside infected macrophages.</p

Apoptotic and necrotic macrophage cell death induced by RB51.

<p>(A) Annexin V/PI staining shows both apoptosis (solid arrow) and necrosis (hollow arrow) of macrophages at as early as 2 h post infection. (B) Staining with Hoechst 33342 shows cell shrinkage and nuclear condensation (diamond head arrow) of infected macrophages at 6 h post infection at a MOI of 200. The results are a representative of three independent experiments.</p

Kinetic analysis of <i>Brucella</i>-induced dendritic cell death.

a<p>Hours Post Infection.</p>b<p>Results of simultaneous Annexin V and PI staining of dendritic cells infected with indicated bacteria: ++++, 75–100% positive; +++, 50–75% positive; ++, 25–50% positive; +, 5–20% positive; ±, <5% positive; −, no positive staining. A staining positive cell means it is positive in Annexin V staining, PI staining, or staining with both dyes.</p

Decreased cell death of infected macrophages and increased RB51 survival inside macrophages with the usage of the caspase-2 inhibitor.

<p>Macrophages pretreated with Z-VDVAD-FMK were infected with RB51 for 24 h at a MOI of 200. (A) Macrophages pretreated with the caspase-2 inhibitor and infected with RB51 released less LDH than infected cells lacking inhibitor. (B) Growth kinetics of RB51 in macrophages. The number of RB51 cells surviving inside macrophages pretreated with the caspase-2 inhibitor was approximately 120 times greater than the number of cells found in untreated macrophages (<i>P</i><0.05). The asterisk (*) represents significant difference (<i>P</i><0.05) in the LDH release or bacterial CFUs from RB51-infected macrophages with caspase-2 inhibitor treatment compared to that without caspase-2 inhibitor. Data represent the means±standard deviations from three independent experiments.</p

Apoptotic and necrotic cell death in peritoneal primary macrophages isolated from RB51-vaccinated mice.

<p>Annexin V/PI staining shows both apoptosis and necrosis of peritoneal macrophages at 24 h after infection with RB51. No obvious cell death was observed in peritoneal macrophages from mice injected (i.p.) with S2308 or heat-killed RB51 (HK-RB51). The results are a representative of three independent experiments.</p

Decreased mitochondrial membrane potential (MMP) and increased cytochrome <i>c</i> release from mitochondria in RB51-infected macrophages (MOI: 200).

<p>Infection with S2308 at a MOI of 2,000 did not cause decrease of MMP or cytochrome <i>c</i> release. (A) The results of flow cytometry results were summarized in graphic form, and represent the means±standard deviations of three independent experiments. The asterisk (*) represents significant differences (<i>P</i><0.05) compared to the medium control group. (B) Cytochrome <i>c</i> release was analyzed by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006830#s4f" target="_blank">Western blot analysis</a>. β-actin was used as a reference. Only cytoplasmic portion of cell lysates was assayed. The results are a representative of three independent experiments.</p

Caspase-2 enzyme activity in infected macrophages induced by RB51 but not by S2308.

<p>(A) Analysis of caspase-2 activities by a colorimetric assay. The asterisk (*) represents significant differences (<i>P</i><0.05) compared to the medium control group. (B) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006830#s4f" target="_blank">Western blot analysis</a> of precaspase-2 in infected macrophages. The numbers shown in the figure represent the amounts of procaspase-2 quantified by densitometry and normalized to the β-actin content. The data represent the means±standard deviations from three independent experiments.</p

Apoptotic and necrotic cell death of bone marrow derived macrophages infected with viable rough <i>B. abortus</i> strains RB51 or RA1 (MOI: 200) at 24 h post infection.

<p>Smooth strain S2308 (MOI: 2000) and heat-killed RB51 and RA1 (equivalent MOI: 200) did not induce obvious macrophage cell death. The results are a representative of three independent experiments.</p