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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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LPS priming induces rapid IL-1α and IL-1β secretion in response to L.pneumophila T4SS activity.(A) Unprimed B6 or Casp1−/−Casp11−/− BMDMs were infected with WT L. pneumophila (WT Lp), Δdot Lp, ΔflaA Lp, or PBS (mock infection) for 20 hours. (B) B6 or Casp1−/−Casp11−/− BMDMs were either unprimed or primed with 0.5 µg/mL LPS for 2.5 hours and infected with WT Lp, ΔdotA Lp, ΔflaA Lp, or PBS for 4 hours. (C) B6 BMDMs were pretreated with either 20 µM or 40 µM of the caspase-1 inhibitor YVAD-cmk or DMSO vehicle control for 0.5 hours and infected with WT Lp, ΔdotA Lp, ΔflaA Lp, or PBS for 20 hours. Levels of IL-1α and IL-1β in the supernatants were measured by ELISA. Graphs show the mean ± SEM of triplicate wells. Data are representative of three or four independent experiments. *** is p<0.001 by 2-way ANOVA with Bonferroni post-test. NS is not significant.
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ppat-1003400-g001: LPS priming induces rapid IL-1α and IL-1β secretion in response to L.pneumophila T4SS activity.(A) Unprimed B6 or Casp1−/−Casp11−/− BMDMs were infected with WT L. pneumophila (WT Lp), Δdot Lp, ΔflaA Lp, or PBS (mock infection) for 20 hours. (B) B6 or Casp1−/−Casp11−/− BMDMs were either unprimed or primed with 0.5 µg/mL LPS for 2.5 hours and infected with WT Lp, ΔdotA Lp, ΔflaA Lp, or PBS for 4 hours. (C) B6 BMDMs were pretreated with either 20 µM or 40 µM of the caspase-1 inhibitor YVAD-cmk or DMSO vehicle control for 0.5 hours and infected with WT Lp, ΔdotA Lp, ΔflaA Lp, or PBS for 20 hours. Levels of IL-1α and IL-1β in the supernatants were measured by ELISA. Graphs show the mean ± SEM of triplicate wells. Data are representative of three or four independent experiments. *** is p<0.001 by 2-way ANOVA with Bonferroni post-test. NS is not significant.

Mentions: L. pneumophila infection induces IL-1α and IL-1β secretion that requires T4SS activity [47], [52]. IL-1β secretion is regulated by a flagellin-dependent NAIP5/NLRC4 inflammasome and a poorly defined ASC inflammasome that both activate caspase-1 [47], [51]. The mechanisms underlying IL-1α secretion are less clear, but IL-1α secretion is still robustly induced by flagellin-deficient L. pneumophila, which do not activate the NAIP5/NLRC4 inflammasome [52]. Recent studies have described a non-canonical inflammasome triggered in response to Gram-negative bacteria. This non-canonical inflammasome requires lipopolysaccharide (LPS) for the upregulation and activation of caspase-11 and subsequent IL-1α and IL-1β release [26]–[29]. Whether caspase-11 is also activated in response to bacteria that use specialized secretion systems to translocate bacterial molecules into the host cytosol is unknown. We thus hypothesized that LPS priming would upregulate caspase-11, pro-IL-1α, and pro-IL-1β and allow for more robust and rapid IL-1α and IL-1β secretion in response to T4SS activity. To test this, we first compared IL-1α and IL-1β release in unprimed and LPS-primed bone marrow-derived macrophages (BMDMs). As shown previously [48], [52], unprimed BMDMs secrete robust levels of IL-1α and IL-1β by 20 hours post-infection with wild-type L. pneumophila (WT Lp) (Figure 1A). Slightly attenuated levels of secreted IL-1α and IL-1β are observed with flagellin-deficient L. pneumophila (ΔflaA Lp), which do not activate the NAIP5/NLRC4 inflammasome [17], [18]. Secretion of both cytokines is significantly diminished during infection with L. pneumophila lacking DotA, an essential component of the T4SS (ΔdotA Lp), and is significantly diminished in caspase-1/caspase-11-deficient (Casp1−/−Casp11−/−) macrophages as well (Figure 1A). The diminished IL-1 secretion induced by ΔdotA Lp is not due to a lack of pro-IL-1 production, as ΔdotA Lp and WT Lp induce robust levels of pro-IL-1β (Figure S1A). At 4 hours post-infection, unprimed macrophages do not secrete IL-1 (Figure 1B). However, LPS-primed cells rapidly secrete IL-1α and IL-1β, and this secretion is abrogated in Casp1−/−Casp11−/− macrophages (Figure 1B). Secretion of IL-18, another IL-1 family cytokine, also requires T4SS activity and is eliminated in Casp1−/−Casp11−/− cells (Figure S1B). Comparable levels of the caspase-1/caspase-11-independent cytokines IL-12 and TNF-α are secreted in the absence and presence of LPS priming (Figure S1C–D). These data suggest that LPS priming upregulates a factor required for rapid IL-1α and IL-1β release in response to L. pneumophila T4SS activity.


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)

LPS priming induces rapid IL-1α and IL-1β secretion in response to L.pneumophila T4SS activity.(A) Unprimed B6 or Casp1−/−Casp11−/− BMDMs were infected with WT L. pneumophila (WT Lp), Δdot Lp, ΔflaA Lp, or PBS (mock infection) for 20 hours. (B) B6 or Casp1−/−Casp11−/− BMDMs were either unprimed or primed with 0.5 µg/mL LPS for 2.5 hours and infected with WT Lp, ΔdotA Lp, ΔflaA Lp, or PBS for 4 hours. (C) B6 BMDMs were pretreated with either 20 µM or 40 µM of the caspase-1 inhibitor YVAD-cmk or DMSO vehicle control for 0.5 hours and infected with WT Lp, ΔdotA Lp, ΔflaA Lp, or PBS for 20 hours. Levels of IL-1α and IL-1β in the supernatants were measured by ELISA. Graphs show the mean ± SEM of triplicate wells. Data are representative of three or four independent experiments. *** is p<0.001 by 2-way ANOVA with Bonferroni post-test. NS is not significant.
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Related In: Results  -  Collection

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ppat-1003400-g001: LPS priming induces rapid IL-1α and IL-1β secretion in response to L.pneumophila T4SS activity.(A) Unprimed B6 or Casp1−/−Casp11−/− BMDMs were infected with WT L. pneumophila (WT Lp), Δdot Lp, ΔflaA Lp, or PBS (mock infection) for 20 hours. (B) B6 or Casp1−/−Casp11−/− BMDMs were either unprimed or primed with 0.5 µg/mL LPS for 2.5 hours and infected with WT Lp, ΔdotA Lp, ΔflaA Lp, or PBS for 4 hours. (C) B6 BMDMs were pretreated with either 20 µM or 40 µM of the caspase-1 inhibitor YVAD-cmk or DMSO vehicle control for 0.5 hours and infected with WT Lp, ΔdotA Lp, ΔflaA Lp, or PBS for 20 hours. Levels of IL-1α and IL-1β in the supernatants were measured by ELISA. Graphs show the mean ± SEM of triplicate wells. Data are representative of three or four independent experiments. *** is p<0.001 by 2-way ANOVA with Bonferroni post-test. NS is not significant.
Mentions: L. pneumophila infection induces IL-1α and IL-1β secretion that requires T4SS activity [47], [52]. IL-1β secretion is regulated by a flagellin-dependent NAIP5/NLRC4 inflammasome and a poorly defined ASC inflammasome that both activate caspase-1 [47], [51]. The mechanisms underlying IL-1α secretion are less clear, but IL-1α secretion is still robustly induced by flagellin-deficient L. pneumophila, which do not activate the NAIP5/NLRC4 inflammasome [52]. Recent studies have described a non-canonical inflammasome triggered in response to Gram-negative bacteria. This non-canonical inflammasome requires lipopolysaccharide (LPS) for the upregulation and activation of caspase-11 and subsequent IL-1α and IL-1β release [26]–[29]. Whether caspase-11 is also activated in response to bacteria that use specialized secretion systems to translocate bacterial molecules into the host cytosol is unknown. We thus hypothesized that LPS priming would upregulate caspase-11, pro-IL-1α, and pro-IL-1β and allow for more robust and rapid IL-1α and IL-1β secretion in response to T4SS activity. To test this, we first compared IL-1α and IL-1β release in unprimed and LPS-primed bone marrow-derived macrophages (BMDMs). As shown previously [48], [52], unprimed BMDMs secrete robust levels of IL-1α and IL-1β by 20 hours post-infection with wild-type L. pneumophila (WT Lp) (Figure 1A). Slightly attenuated levels of secreted IL-1α and IL-1β are observed with flagellin-deficient L. pneumophila (ΔflaA Lp), which do not activate the NAIP5/NLRC4 inflammasome [17], [18]. Secretion of both cytokines is significantly diminished during infection with L. pneumophila lacking DotA, an essential component of the T4SS (ΔdotA Lp), and is significantly diminished in caspase-1/caspase-11-deficient (Casp1−/−Casp11−/−) macrophages as well (Figure 1A). The diminished IL-1 secretion induced by ΔdotA Lp is not due to a lack of pro-IL-1 production, as ΔdotA Lp and WT Lp induce robust levels of pro-IL-1β (Figure S1A). At 4 hours post-infection, unprimed macrophages do not secrete IL-1 (Figure 1B). However, LPS-primed cells rapidly secrete IL-1α and IL-1β, and this secretion is abrogated in Casp1−/−Casp11−/− macrophages (Figure 1B). Secretion of IL-18, another IL-1 family cytokine, also requires T4SS activity and is eliminated in Casp1−/−Casp11−/− cells (Figure S1B). Comparable levels of the caspase-1/caspase-11-independent cytokines IL-12 and TNF-α are secreted in the absence and presence of LPS priming (Figure S1C–D). These data suggest that LPS priming upregulates a factor required for rapid IL-1α and IL-1β release in response to L. pneumophila T4SS activity.

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