Effect of IL-10 in macrophages during <i>B</i>. <i>pseudomallei</i> infection.

<p>(A) Quantitative real-time PCR analysis of IL-10 expression in macrophages from C57BL/6 WT and <i>caspase6</i>^<i>-/-</i> mice 6 h after infection with <i>B</i>. <i>pseudomallei</i> strain E8 (MOI ~ 50). The data were logarithmized to achieve normal distribution and compared using Student’s <i>t</i>-test. Values are means ± standard deviations from three independent experiments. (B) Intracellular bacterial burden of IL-10-treated and non-treated C57BL/6 macrophages after infection with <i>B</i>. <i>pseudomallei</i> strain E8 (MOI ~ 25). Data were analysed using Student’s <i>t</i>-test. Values are means ± standard deviations from triplicate determinations. The experiment was repeated three times.</p

Cytokine expression in caspase-6 deficient mice 24 hours after infection with <i>B</i>. <i>pseudomallei</i>.

<p>Quantitative real-time PCR analysis of inflammatory parameters in the spleens of C57BL/6 WT and <i>caspase6</i>^<i>-/-</i> mice. Mice were infected with 5 × 10⁴ CFU of <i>B</i>. <i>pseudomallei</i> strain E8 i.v. for 24 h. Uninfected control animals received PBS. Each of the 4 groups contained 9 mice from 3 replicates (n = 36). Data were analysed using Student’s <i>t</i> test. Values are means ± standard deviations from three independent experiments.</p

Cytokine expression in caspase-6 deficient mice 6 hours after infection with <i>B</i>. <i>pseudomallei</i>.

<p>Quantitative real-time PCR analysis of inflammatory parameters in the spleens of C57BL/6 WT (n = 8) and <i>caspase6</i>^<i>-/-</i> mice (n = 8) 6 hours after infection with 5 × 10⁴ CFU <i>B</i>. <i>pseudomallei</i> strain E8. Data were analysed using Student’s <i>t</i> test. Values are means ± standard deviations from two independent experiments.</p

Invasion and replication of <i>B</i>. <i>pseudomallei</i> in caspase-6 deficient macrophages.

<p>Intracellular bacterial burden of C57BL/6-WT and C57BL/6-<i>caspase6</i>^<i>-/-</i> macrophages after infection with <i>B</i>. <i>pseudomallei</i> strain E8 at an MOI of ~ 25. The CFU data were logarithmized to achieve normal distribution and compared using Student’s t-test. Values are means ± standard deviations from triplicate determinations. The experiment was repeated three times.</p

Cell death induction in caspase-6 deficient macrophages after infection with <i>B</i>. <i>pseudomallei</i>.

<p>Course of cell damage in C57BL/6 WT and <i>caspase6</i>^<i>-/-</i> macrophages after infection with <i>B</i>. <i>pseudomallei</i> strain E8. (A) For the LDH release assay cells were infected with an MOI of ~10. Data were analysed using Student’s <i>t</i> test. Values are means ± standard deviations from triplicate determinations. (B) Real-time cell status analysis of C57BL/6 WT (blue, triangles) and <i>caspase6</i>^<i>-/-</i> macrophages (red, circles). Infected cells are represented by brighter lines (top-down triangle, small circle) and received an MOI of ~5. Uninfected controls are represented by darker lines (upright triangle, big circle). The y-axis is a relative scale for the cell status measured by the XCelligence system. The experiments were repeated twice.</p

Caspase-6 mediates resistance against <i>Burkholderia pseudomallei</i> infection and influences the expression of detrimental cytokines

<div><p>Caspase-6 is a member of the executioner caspases and known to play a role in innate and adaptive immune processes. However, its role in infectious diseases has rarely been addressed yet. We here examined the impact of caspase-6 in an <i>in vivo</i> infection model using the Gram-negative rod <i>Burkholderia pseudomallei</i>, causing the infectious disease melioidosis that is endemic in tropical and subtropical areas around the world. <i>Caspase-6</i>^-/- and C57BL/6 wild type mice were challenged with <i>B</i>. <i>pseudomallei</i> for comparing mortality, bacterial burden and inflammatory cytokine expression. Bone-marrow derived macrophages were used to analyse the bactericidal activity in absence of caspase-6. Caspase-6 deficiency was associated with higher mortality and bacterial burden <i>in vivo</i> after <i>B</i>. <i>pseudomallei</i> infection. The bactericidal activity of <i>caspase-6</i>^-/- macrophages was impaired compared to wild type cells. <i>Caspase-6</i>^-/- mice showed higher expression of the IL-1β gene, known to be detrimental in murine melioidosis. Expression of the IL-10 gene was also increased in <i>caspase-6</i>^-/- mice as early as 6 hours after infection. Treatment with exogenous IL-10 rendered mice more susceptible against <i>B</i>. <i>pseudomallei</i> challenge. Thus, caspase-6 seems to play a crucial role for determining resistance against the causative agent of melioidosis. To our knowledge this is the first report showing that caspase-6 is crucial for mediating resistance in an <i>in vivo</i> infection model. Caspase-6 influences the expression of detrimental cytokines and therefore seems to be important for achieving a well-balanced immune response that contributes for an efficient elimination of the pathogen.</p></div

<i>B. pseudomallei</i> BsaK is involved in caspase-1-dependent pyroptosis and IL-1β production in macrophages in the early phase of infection.

<p>(<b>A</b>) Processing of caspases-1, -9, -7 and PARP was detected by immunoblot in cell lysates of C57BL/6 BMM infected with <i>B. pseudomallei</i> E8 wild-type or mutants ΔBsaK, ΔFliC and ΔFliCΔBsaK at MOI of 50∶1 at 1.5, 6 and 24 hours post infection. One experiment of at least three performed is shown. non-infected (n.i.). (<b>B</b>) Invasion and intracellular replication of respective <i>B. pseudomallei</i> strains was examined in BMM infected at MOI of 2∶1 at the indicated time points. (<b>C</b>) Induction of pyroptosis was measured as lactate dehydrogenase (LDH) release in cell culture supernatants of <i>B. pseudomallei</i> infected BMM (MOI 200∶1). (<b>D</b>) Secretion of mature IL-1β was measured in supernatants of <i>B. pseudomallei</i> infected BMM (MOI 50∶1) at 6 and 24 hours after infection. (B, C) Data are presented as mean with standard error of the mean (SEM) of triplicate determinations. One representative experiment out of three independent experiments is shown. (D) Data are presented as mean with SEM of four independent experiments (n = 4). Statistical analyses were performed using one-way ANOVA (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001 compared to non-infected macrophages or as indicated). below detection (bd), non-infected (n.i.).</p

<i>Burkholderia</i>-mediated activation of caspase-1 in macrophages is strain- dependent.

<p>Cleavage of caspases-1, -9, -7, and PARP was detected by immunoblot in cell lysates of C57BL/6 wild-type BMM infected with <i>B. pseudomallei</i> strains E8, K96243 (K9), E212, 1026b, and <i>B. thailandensis</i> strain E264 at MOI of (<b>A</b>) 50∶1 and (<b>B</b>) 200∶1 at 1.5 hours post infection. One experiment of at least three performed is shown. non-infected (n.i.).</p

<i>B. pseudomallei</i> ΔBsaK infected mice show reduced cytokine levels and neutrophil influx.

<p>BALB/c mice were intranasally infected with <i>B. pseudomallei</i> E8 wild-type and ΔBsaK at 40 CFU. (<b>A</b>) Cytokine (TNF-α, IL-6, MCP-1, IFN-γ, IL-1β) and myeloperoxidase (MPO) levels were measured in BALF obtained 48 hours post infection. (<b>B</b>) Flow cytometric analyses for neutrophils in BALF, lung and spleen was performed 48 hours after challenge with <i>B. pseudomallei</i> E8 wild-type, ΔBsaK or PBS. (A, B) Pooled data from two independent experiments are presented as mean with standard error of the mean (n = 10). Statistical analyses were performed using (A) a Student's <i>t</i> test or (B) one-way ANOVA (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001 compared to PBS infected mice or as indicated).</p

Caspase-1-Dependent and -Independent Cell Death Pathways in <i>Burkholderia pseudomallei</i> Infection of Macrophages

<div><p>The cytosolic pathogen <i>Burkholderia pseudomallei</i> and causative agent of melioidosis has been shown to regulate IL-1β and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during <i>B. pseudomallei</i> infection have not been addressed so far in detail. Furthermore, the role of <i>B. pseudomallei</i> factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a <i>B. pseudomallei</i> flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1β secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1β, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1-dependent and -independent cell death mechanisms in the pathogenesis of <i>B. pseudomallei</i> infection.</p></div