Caspase-11 activation is independent of ASC and NLRC4.

<p>(<b>A</b>) Unprimed B6, <i>Casp1^−/−Casp11^−/−</i>, or <i>Asc^−/−Nlrc4^−/−</i> BMDMs were infected with WT <i>L. pneumophila</i> (WT Lp), Δ<i>dotA</i> Lp, Δ<i>flaA</i> Lp, or PBS (mock infection) for 20 hours, and levels of IL-1α and IL-1β in the supernatants were measured by ELISA. Graphs show the mean ± SEM of triplicate wells. (<b>B</b>) Unprimed B6, <i>Casp1^−/−Casp11^−/−</i>, or <i>Asc^−/−Nlrc4^−/−</i> BMDMs were infected with WT Lp, Δ<i>dotA</i> Lp, Δ<i>flaA</i> Lp, or PBS (mock infection) for 20 hours or treated with LPS+ATP for 1 hour. Levels of processed caspase-1 (casp-1 p10) and caspase-11 (casp-11 p26) in the supernatants, and pro-caspase-1, pro-caspase-11, and β-actin (loading control) in the cell lysates were determined by immunoblot analysis. (<b>C</b>) 8–12 week old B6 and <i>Asc^−/−</i> mice were infected intranasally with either 1×10⁶ Δ<i>flaA</i> Lp or PBS. Bronchoalveolar lavage fluid (BALF) was collected 24 hours post-infection, and levels of IL-1α and IL-1β were measured by ELISA. Graphs show the mean ± SEM of 9 mice per group. Dashed line represents the limit of detection. Data are representative of three independent experiments (A,B) or are displayed as the pooled results of two independent experiments (C). *** is p<0.001 by two-way ANOVA with Bonferroni post-test. ** is p<0.01 by unpaired t-test. NS is not significant.</p

Caspase-11 mediates inflammasome activation in response to a functional <i>Yersinia</i> type III secretion system.

<p>BMDMs from B6, <i>Casp1^−/−Casp11^−/−</i>, <i>Casp1^−/−</i>, or <i>Casp11^−/−</i> mice were primed with 0.05 µg/mL LPS for 4 hours and infected with type III secretion system-deficient <i>Y. pseudotuberculosis</i> (Δ<i>yopB</i> Yp), effectorless <i>Y. pseudotuberculosis</i> ΔHOJMEK (Δ6 Yp), or PBS (mock infection) or treated with 2.5 mm ATP for 4 hours. (<b>A</b>) Levels of IL-1α and IL-1β in the supernatants were measured by ELISA. (<b>B</b>) Cell death (% cytotoxicity) was measured by lactate dehydrogenase (LDH) release relative to Triton X-100-lysed cells. Graphs show the mean ± SEM of triplicate wells. Data are representative of two independent experiments. *** is p<0.001 and ** is p<0.01 by two-way ANOVA with Bonferroni post-test. NS is not significant.</p

Caspase-11 controls multiple pathways of inflammasome activation in response to bacterial secretion systems that access the host cytosol.

<p>Three distinct inflammasome pathways are induced upon interaction of virulent bacteria with host cells. Translocation of flagellin into the host cytosol by specialized secretion systems triggers a NAIP5/NLRC4/caspase-1 inflammasome that leads to cell death, IL-1α, and IL-1β release. Virulent bacteria induce two separate pathways of caspase-11-dependent inflammasome activation through a two-signal model. First, TLR stimulation by PAMPs (signal one) leads to upregulation of pro-IL-1α, pro-IL-1β, NLRP3, and pro-caspase-11. Next, cytosolic detection of virulence activity, namely type III or type IV secretion (signal two), leads to caspase-11 processing and activation. Active caspase-11 contributes to NLRP3-mediated inflammasome activation and caspase-1-dependent IL-1β secretion. Caspase-11 also mediates caspase-1-independent cell death and IL-1α release through a pathway that is independent of the NLRP3/ASC and NAIP5/NLRC4 inflammasomes and involves an unknown host sensor.</p

Caspase-11 mediates both NLRP3-dependent and NLRP3-independent immune responses.

<p>(<b>A</b>) B6, <i>Casp1^−/−Casp11^−/−</i>, <i>Asc</i>^−/−, <i>Nlrc4</i>^−/−, or <i>Asc^−/−Nlrc4^−/−</i> BMDMs were primed with 0.5 µg/mL LPS for 2.5 hours and infected with WT <i>L. pneumophila</i> (WT Lp), Δ<i>dotA</i> Lp, Δ<i>flaA</i> Lp, or PBS (mock infection) or treated with 2.5 mm ATP for 4 hours. Levels of IL-1α and IL-1β in the supernatants were measured by ELISA and cell death (% cytotoxicity) was measured by LDH release into the supernatants relative to Triton X-100-lysed cells. Graphs show the mean ± SEM of triplicate wells. (<b>B</b> and <b>C</b>) B6 or <i>Nlrp3^−/−</i> BMDMs were primed with 0.5 µg/mL LPS for 2.5 hours and infected with WT Lp, Δ<i>dotA</i> Lp, Δ<i>flaA</i> Lp, or PBS (mock infected) or treated with 2.5 mm ATP for 1 hour (C) or 4 hours (B). (B) Levels of IL-1α and IL-1β in the supernatants were measured by ELISA and cell death (% cytotoxicity) was measured by LDH release into the supernatants relative to Triton X-100-lysed cells. Graphs show the mean ± SEM of triplicate wells. (C) Levels of processed caspase-1 (casp-1 p10) in the supernatants and pro-caspase-1 in the cell lysates were determined by immunoblot analysis. Data are representative of two (A,C) or three (B) independent experiments. *** is p<0.001 by one-way ANOVA with Tukey post-test. NS is not significant.</p

Non-canonical inflammasome responses to <i>L.</i><i>pneumophila</i> occur independently of TRIF and IFNAR.

<p>(<b>A</b>) Unprimed B6, <i>Ifnar^−/−</i>, or <i>Trif^−/−</i> BMDMs were infected with WT <i>L. pneumophila</i> (WT Lp), Δ<i>dotA</i> Lp, Δ<i>flaA</i> Lp, <i>E. coli</i>, or PBS (mock infection) for 16 hours. Levels of IL-1α and IL-1β in the supernatants were measured by ELISA. (<b>B</b>) Unprimed B6, <i>Ifnar^−/−</i>, or <i>Trif^−/−</i> BMDMs were infected with WT Lp, Δ<i>dotA</i> Lp, Δ<i>flaA</i> Lp, or PBS (mock infection) for 16 hours. Cell death (% cytotoxicity) was measured by LDH release into the supernatants relative to Triton X-100-lysed cells. Graphs show the mean ± SEM of triplicate wells. (<b>C</b>) B6, <i>Ifnar^−/−</i>, or <i>Trif^−/−</i> BMDMs were primed with 0.4 µg/mL Pam3CSK4 for 4 hours and infected with WT Lp, Δ<i>dotA</i> Lp, Δ<i>flaA</i> Lp, or PBS for 16 hours. Levels of full-length caspase-11 (pro-casp-11) and processed caspase-11 (casp11 p26) in the supernatants and pro-casp-11 and β-actin (loading control) in the cell lysates were determined by immunoblot analysis. Data are representative of two independent experiments.</p

Caspase-11 controls the release of IL-1α and IL-1β and pyroptosis in response to flagellin-deficient <i>L.</i><i>pneumophila</i>.

<p>B6, <i>Casp1^−/−Casp11^−/−</i>, <i>Casp1^−/−</i>, or <i>Casp11^−/−</i> BMDMs were primed with 0.5 µg/mL LPS for 2.5 hours and infected with WT <i>L. pneumophila</i> (WT Lp), Δ<i>dotA</i> Lp, Δ<i>flaA</i> Lp, or PBS (mock infection) or treated with 2.5 mm ATP for 1(C) or 4(A,B) hours. (<b>A</b>) Levels of IL-1α and IL-1β in the supernatants were measured by ELISA. Graphs show the mean ± SEM of triplicate wells. (<b>B</b>) Cell death (% cytotoxicity) was measured by LDH release into the supernatants relative to Triton X-100-lysed cells. Graphs show the mean ± SEM of triplicate wells. (<b>C</b>) Levels of processed caspase-1 (casp-1 p10) in the supernatants and full-length caspase-1 (pro-casp-1) and β-actin in the cell lysates were determined by immunoblot analysis. Data are representative of three independent experiments. *** is p<0.001 by two-way ANOVA with Bonferroni post-test, ** is p<0.01 by two-way ANOVA with Bonferroni post-test, and * is p<0.05 by unpaired t-test. NS is not significant.</p

IL-1α and IL-1β control bacterial burden and neutrophil recruitment <i>in vivo</i>.

<p>(<b>A</b>) 8–12 week old B6 or <i>Il1r1^−/−</i> mice were infected with 1×10⁶ Δ<i>flaA L. pneumophila</i> intranasally (IN). Lungs were plated to quantify CFU per gram. Graph shows the mean ± SEM of three or four infected mice per group. Dashed line represents the limit of detection. (<b>B</b> and <b>C</b>) B6 or <i>Il1r1^−/−</i> mice were infected with 1×10⁶ Δ<i>flaA</i> Lp IN. 24 hours post-infection, bronchoalveolar lavage fluid (BALF) was collected and the percentage of neutrophils in the BALF was quantified by flow cytometry. Percentages are reported as the frequency of live cells in the BALF. (B) Representative flow cytometry plots showing the percentage of Gr-1⁺Ly6G⁺ neutrophils. (C) Graph showing the percentage of neutrophils. Each point represents an individual mouse and lines indicate the mean of 4 mice per group. (<b>D, E</b>, and <b>F</b>) B6 mice were injected intraperitoneally (IP) with either PBS, 100 µg isotype control antibody (iso), 100 µg anti-IL-1α antibody, 100 µg anti-IL-1β antibody, or 100 µg each of anti-IL-1α and anti-IL-1β (anti-IL-1α/β) 16 hours before infection. The mice were then intranasally infected with either 1×10⁶ Δ<i>flaA</i> Lp or mock infected with PBS. (D and E) 24 hours post-infection, BALF was collected and flow cytometry was performed to quantify the percentage of neutrophils. (D) Representative flow cytometry plots showing the percentage of Gr-1⁺Ly6G⁺ neutrophils. (E) Graph showing the percentage of neutrophils. Each point represents an individual mouse, lines indicate the mean of 8 mice per group, and error bars represent SEM. Shown are the pooled results of two independent experiments. (F) 72 hours post-infection, the lungs were plated to quantify CFU per gram. Each point represents an individual mouse. Line indicates the mean of 4 infected mice per group with error bars representing SEM. *** is p<0.001 by one-way ANOVA with Tukey post-test or unpaired t-test (C). **is p<0.01 and *is p<0.05 by unpaired t-test. NS is not significant.</p