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Caspase-11 activation in response to bacterial secretion systems that access the host cytosol.

Casson CN, Copenhaver AM, Zwack EE, Nguyen HT, Strowig T, Javdan B, Bradley WP, Fung TC, Flavell RA, Brodsky IE, Shin S - PLoS Pathog. (2013)

Bottom Line: Many bacterial pathogens use specialized secretion systems to translocate effector proteins into the cytosol of host cells.Unlike IL-1β, IL-1α secretion does not require caspase-1.Furthermore, we find both overlapping and non-redundant roles for IL-1α and IL-1β in mediating neutrophil recruitment and bacterial clearance in response to pulmonary infection by L. pneumophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Inflammasome activation is important for antimicrobial defense because it induces cell death and regulates the secretion of IL-1 family cytokines, which play a critical role in inflammatory responses. The inflammasome activates caspase-1 to process and secrete IL-1β. However, the mechanisms governing IL-1α release are less clear. Recently, a non-canonical inflammasome was described that activates caspase-11 and mediates pyroptosis and release of IL-1α and IL-1β. Caspase-11 activation in response to Gram-negative bacteria requires Toll-like receptor 4 (TLR4) and TIR-domain-containing adaptor-inducing interferon-β (TRIF)-dependent interferon production. Whether additional bacterial signals trigger caspase-11 activation is unknown. Many bacterial pathogens use specialized secretion systems to translocate effector proteins into the cytosol of host cells. These secretion systems can also deliver flagellin into the cytosol, which triggers caspase-1 activation and pyroptosis. However, even in the absence of flagellin, these secretion systems induce inflammasome activation and the release of IL-1α and IL-1β, but the inflammasome pathways that mediate this response are unclear. We observe rapid IL-1α and IL-1β release and cell death in response to the type IV or type III secretion systems of Legionella pneumophila and Yersinia pseudotuberculosis. Unlike IL-1β, IL-1α secretion does not require caspase-1. Instead, caspase-11 activation is required for both IL-1α secretion and cell death in response to the activity of these secretion systems. Interestingly, whereas caspase-11 promotes IL-1β release in response to the type IV secretion system through the NLRP3/ASC inflammasome, caspase-11-dependent release of IL-1α is independent of both the NAIP5/NLRC4 and NLRP3/ASC inflammasomes as well as TRIF and type I interferon signaling. Furthermore, we find both overlapping and non-redundant roles for IL-1α and IL-1β in mediating neutrophil recruitment and bacterial clearance in response to pulmonary infection by L. pneumophila. Our findings demonstrate that virulent, but not avirulent, bacteria trigger a rapid caspase-11-dependent innate immune response important for host defense.

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Caspase-11 activation is independent of ASC and NLRC4.(A) Unprimed B6, Casp1−/−Casp11−/−, or Asc−/−Nlrc4−/− BMDMs were infected with WT L. pneumophila (WT Lp), ΔdotA Lp, ΔflaA 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) Unprimed B6, Casp1−/−Casp11−/−, or Asc−/−Nlrc4−/− BMDMs were infected with WT Lp, ΔdotA Lp, ΔflaA 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. (C) 8–12 week old B6 and Asc−/− mice were infected intranasally with either 1×106 ΔflaA 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.
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ppat-1003400-g003: Caspase-11 activation is independent of ASC and NLRC4.(A) Unprimed B6, Casp1−/−Casp11−/−, or Asc−/−Nlrc4−/− BMDMs were infected with WT L. pneumophila (WT Lp), ΔdotA Lp, ΔflaA 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) Unprimed B6, Casp1−/−Casp11−/−, or Asc−/−Nlrc4−/− BMDMs were infected with WT Lp, ΔdotA Lp, ΔflaA 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. (C) 8–12 week old B6 and Asc−/− mice were infected intranasally with either 1×106 ΔflaA 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.

Mentions: The ASC and NAIP5/NLRC4 inflammasomes are required for caspase-1 activation and IL-1β secretion in response to L. pneumophila[47]. To determine if these inflammasomes are also required for caspase-11 activation and IL-1α release, we infected ASC/NLRC4-deficient (Asc−/−Nlrc4−/−) BMDMs with L. pneumophila. Asc−/−Nlrc4−/− BMDMs do not secrete IL-1β in response to either WT Lp, ΔflaA Lp, or LPS+ATP. However, Asc−/−Nlrc4−/− BMDMs still release IL-1α in response to ΔflaA Lp in primed and unprimed macrophages (Figures 3A, S4). Thus, unlike IL-1β, IL-1α is released independently of flagellin, ASC, and NLRC4. Accordingly, despite an absence of processed caspase-1 p10, robust levels of processed caspase-11 p26 are detected in the supernatants of Asc−/−Nlrc4−/− cells infected with either WT or ΔflaA Lp but not in response to LPS+ATP (Figure 3B).


Caspase-11 activation in response to bacterial secretion systems that access the host cytosol.

Casson CN, Copenhaver AM, Zwack EE, Nguyen HT, Strowig T, Javdan B, Bradley WP, Fung TC, Flavell RA, Brodsky IE, Shin S - PLoS Pathog. (2013)

Caspase-11 activation is independent of ASC and NLRC4.(A) Unprimed B6, Casp1−/−Casp11−/−, or Asc−/−Nlrc4−/− BMDMs were infected with WT L. pneumophila (WT Lp), ΔdotA Lp, ΔflaA 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) Unprimed B6, Casp1−/−Casp11−/−, or Asc−/−Nlrc4−/− BMDMs were infected with WT Lp, ΔdotA Lp, ΔflaA 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. (C) 8–12 week old B6 and Asc−/− mice were infected intranasally with either 1×106 ΔflaA 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.
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Related In: Results  -  Collection

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ppat-1003400-g003: Caspase-11 activation is independent of ASC and NLRC4.(A) Unprimed B6, Casp1−/−Casp11−/−, or Asc−/−Nlrc4−/− BMDMs were infected with WT L. pneumophila (WT Lp), ΔdotA Lp, ΔflaA 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) Unprimed B6, Casp1−/−Casp11−/−, or Asc−/−Nlrc4−/− BMDMs were infected with WT Lp, ΔdotA Lp, ΔflaA 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. (C) 8–12 week old B6 and Asc−/− mice were infected intranasally with either 1×106 ΔflaA 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.
Mentions: The ASC and NAIP5/NLRC4 inflammasomes are required for caspase-1 activation and IL-1β secretion in response to L. pneumophila[47]. To determine if these inflammasomes are also required for caspase-11 activation and IL-1α release, we infected ASC/NLRC4-deficient (Asc−/−Nlrc4−/−) BMDMs with L. pneumophila. Asc−/−Nlrc4−/− BMDMs do not secrete IL-1β in response to either WT Lp, ΔflaA Lp, or LPS+ATP. However, Asc−/−Nlrc4−/− BMDMs still release IL-1α in response to ΔflaA Lp in primed and unprimed macrophages (Figures 3A, S4). Thus, unlike IL-1β, IL-1α is released independently of flagellin, ASC, and NLRC4. Accordingly, despite an absence of processed caspase-1 p10, robust levels of processed caspase-11 p26 are detected in the supernatants of Asc−/−Nlrc4−/− cells infected with either WT or ΔflaA Lp but not in response to LPS+ATP (Figure 3B).

Bottom Line: Many bacterial pathogens use specialized secretion systems to translocate effector proteins into the cytosol of host cells.Unlike IL-1β, IL-1α secretion does not require caspase-1.Furthermore, we find both overlapping and non-redundant roles for IL-1α and IL-1β in mediating neutrophil recruitment and bacterial clearance in response to pulmonary infection by L. pneumophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Inflammasome activation is important for antimicrobial defense because it induces cell death and regulates the secretion of IL-1 family cytokines, which play a critical role in inflammatory responses. The inflammasome activates caspase-1 to process and secrete IL-1β. However, the mechanisms governing IL-1α release are less clear. Recently, a non-canonical inflammasome was described that activates caspase-11 and mediates pyroptosis and release of IL-1α and IL-1β. Caspase-11 activation in response to Gram-negative bacteria requires Toll-like receptor 4 (TLR4) and TIR-domain-containing adaptor-inducing interferon-β (TRIF)-dependent interferon production. Whether additional bacterial signals trigger caspase-11 activation is unknown. Many bacterial pathogens use specialized secretion systems to translocate effector proteins into the cytosol of host cells. These secretion systems can also deliver flagellin into the cytosol, which triggers caspase-1 activation and pyroptosis. However, even in the absence of flagellin, these secretion systems induce inflammasome activation and the release of IL-1α and IL-1β, but the inflammasome pathways that mediate this response are unclear. We observe rapid IL-1α and IL-1β release and cell death in response to the type IV or type III secretion systems of Legionella pneumophila and Yersinia pseudotuberculosis. Unlike IL-1β, IL-1α secretion does not require caspase-1. Instead, caspase-11 activation is required for both IL-1α secretion and cell death in response to the activity of these secretion systems. Interestingly, whereas caspase-11 promotes IL-1β release in response to the type IV secretion system through the NLRP3/ASC inflammasome, caspase-11-dependent release of IL-1α is independent of both the NAIP5/NLRC4 and NLRP3/ASC inflammasomes as well as TRIF and type I interferon signaling. Furthermore, we find both overlapping and non-redundant roles for IL-1α and IL-1β in mediating neutrophil recruitment and bacterial clearance in response to pulmonary infection by L. pneumophila. Our findings demonstrate that virulent, but not avirulent, bacteria trigger a rapid caspase-11-dependent innate immune response important for host defense.

Show MeSH
Related in: MedlinePlus