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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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Non-canonical inflammasome responses to L.pneumophila occur independently of TRIF and IFNAR.(A) Unprimed B6, Ifnar−/−, or Trif−/− BMDMs were infected with WT L. pneumophila (WT Lp), ΔdotA Lp, ΔflaA Lp, E. coli, or PBS (mock infection) for 16 hours. Levels of IL-1α and IL-1β in the supernatants were measured by ELISA. (B) Unprimed B6, Ifnar−/−, or Trif−/− BMDMs were infected with WT Lp, ΔdotA Lp, ΔflaA 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. (C) B6, Ifnar−/−, or Trif−/− BMDMs were primed with 0.4 µg/mL Pam3CSK4 for 4 hours and infected with WT Lp, ΔdotA Lp, ΔflaA 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.
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ppat-1003400-g005: Non-canonical inflammasome responses to L.pneumophila occur independently of TRIF and IFNAR.(A) Unprimed B6, Ifnar−/−, or Trif−/− BMDMs were infected with WT L. pneumophila (WT Lp), ΔdotA Lp, ΔflaA Lp, E. coli, or PBS (mock infection) for 16 hours. Levels of IL-1α and IL-1β in the supernatants were measured by ELISA. (B) Unprimed B6, Ifnar−/−, or Trif−/− BMDMs were infected with WT Lp, ΔdotA Lp, ΔflaA 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. (C) B6, Ifnar−/−, or Trif−/− BMDMs were primed with 0.4 µg/mL Pam3CSK4 for 4 hours and infected with WT Lp, ΔdotA Lp, ΔflaA 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.

Mentions: Recent data demonstrate that caspase-11 activation in response to a wide variety of Gram-negative bacteria requires TLR4 signaling through its adaptor TRIF and subsequent type I IFN production [27]–[29]. To determine if L. pneumophila engages a similar TRIF and type I IFN receptor (IFNAR)-dependent pathway for caspase-11 activation, we infected TRIF-deficient (Trif−/−) and IFNAR-deficient (Ifnar−/−) BMDMs. Unlike the response to E. coli, L. pneumophila infection of unprimed macrophages triggered robust cell death and secretion of IL-1α and IL-1β that was independent of IFNAR and TRIF (Figure 5A–B). Consistently, priming with the TLR1/2 agonist Pam3CSK4, which results in TRIF- and IFNAR-dependent cytokine secretion and cell death in response to E. coli[27], still induced cell death and cytokine secretion in TRIF- and IFNAR-deficient cells in response to L. pneumophila (Figure S8A–B). These data suggest that during L. pneumophila infection, caspase-11 is upregulated and activated independently of TRIF and IFNAR signaling. Indeed, caspase-11 is still robustly processed and secreted independently of IFNAR and TRIF (Figures 5C, S9). Notably, substantially upregulated levels of pro-caspase-11 are not observed in the lysates of cells infected with WT or ΔflaA Lp because both the pro and cleaved forms of caspase-11 are rapidly secreted into the cell supernatant upon infection (Figures 5C, S9). Accordingly, lysates from IFNAR- and TRIF-deficient macrophages infected with L. pneumophila express comparable levels of pro-caspase-11 to wild-type macrophages, whereas TRIF and IFNAR do contribute to upregulation of pro-caspase-11 in response to E. coli (Figure S10A–C). When the macrophages are primed with LPS prior to infection, there is a moderate contribution of TRIF and IFNAR signaling to inflammasome activation, consistent with the observation that LPS stimulates the TLR4-TRIF-IFNAR axis involved in caspase-11 upregulation (Figure S8C–D). Because the caspase-11-dependent response to L. pneumophila is TRIF-independent, we investigated whether the TLR signaling adaptor MyD88 contributes to caspase-11 upregulation. When immortalized macrophages deficient for both MyD88 and Trif (iMyd88−/−Trif−/−) were infected, caspase-11 upregulation was abrogated in response to both WT and ΔflaA Lp (Figure S11A–B), and we were unable to detect caspase-11 activation (data not shown). Thus, although TRIF is not required for caspase-11 activation, a TLR-dependent signal is likely required as the loss of both MyD88 and TRIF eliminates caspase-11 upregulation and activation.


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)

Non-canonical inflammasome responses to L.pneumophila occur independently of TRIF and IFNAR.(A) Unprimed B6, Ifnar−/−, or Trif−/− BMDMs were infected with WT L. pneumophila (WT Lp), ΔdotA Lp, ΔflaA Lp, E. coli, or PBS (mock infection) for 16 hours. Levels of IL-1α and IL-1β in the supernatants were measured by ELISA. (B) Unprimed B6, Ifnar−/−, or Trif−/− BMDMs were infected with WT Lp, ΔdotA Lp, ΔflaA 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. (C) B6, Ifnar−/−, or Trif−/− BMDMs were primed with 0.4 µg/mL Pam3CSK4 for 4 hours and infected with WT Lp, ΔdotA Lp, ΔflaA 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675167&req=5

ppat-1003400-g005: Non-canonical inflammasome responses to L.pneumophila occur independently of TRIF and IFNAR.(A) Unprimed B6, Ifnar−/−, or Trif−/− BMDMs were infected with WT L. pneumophila (WT Lp), ΔdotA Lp, ΔflaA Lp, E. coli, or PBS (mock infection) for 16 hours. Levels of IL-1α and IL-1β in the supernatants were measured by ELISA. (B) Unprimed B6, Ifnar−/−, or Trif−/− BMDMs were infected with WT Lp, ΔdotA Lp, ΔflaA 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. (C) B6, Ifnar−/−, or Trif−/− BMDMs were primed with 0.4 µg/mL Pam3CSK4 for 4 hours and infected with WT Lp, ΔdotA Lp, ΔflaA 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.
Mentions: Recent data demonstrate that caspase-11 activation in response to a wide variety of Gram-negative bacteria requires TLR4 signaling through its adaptor TRIF and subsequent type I IFN production [27]–[29]. To determine if L. pneumophila engages a similar TRIF and type I IFN receptor (IFNAR)-dependent pathway for caspase-11 activation, we infected TRIF-deficient (Trif−/−) and IFNAR-deficient (Ifnar−/−) BMDMs. Unlike the response to E. coli, L. pneumophila infection of unprimed macrophages triggered robust cell death and secretion of IL-1α and IL-1β that was independent of IFNAR and TRIF (Figure 5A–B). Consistently, priming with the TLR1/2 agonist Pam3CSK4, which results in TRIF- and IFNAR-dependent cytokine secretion and cell death in response to E. coli[27], still induced cell death and cytokine secretion in TRIF- and IFNAR-deficient cells in response to L. pneumophila (Figure S8A–B). These data suggest that during L. pneumophila infection, caspase-11 is upregulated and activated independently of TRIF and IFNAR signaling. Indeed, caspase-11 is still robustly processed and secreted independently of IFNAR and TRIF (Figures 5C, S9). Notably, substantially upregulated levels of pro-caspase-11 are not observed in the lysates of cells infected with WT or ΔflaA Lp because both the pro and cleaved forms of caspase-11 are rapidly secreted into the cell supernatant upon infection (Figures 5C, S9). Accordingly, lysates from IFNAR- and TRIF-deficient macrophages infected with L. pneumophila express comparable levels of pro-caspase-11 to wild-type macrophages, whereas TRIF and IFNAR do contribute to upregulation of pro-caspase-11 in response to E. coli (Figure S10A–C). When the macrophages are primed with LPS prior to infection, there is a moderate contribution of TRIF and IFNAR signaling to inflammasome activation, consistent with the observation that LPS stimulates the TLR4-TRIF-IFNAR axis involved in caspase-11 upregulation (Figure S8C–D). Because the caspase-11-dependent response to L. pneumophila is TRIF-independent, we investigated whether the TLR signaling adaptor MyD88 contributes to caspase-11 upregulation. When immortalized macrophages deficient for both MyD88 and Trif (iMyd88−/−Trif−/−) were infected, caspase-11 upregulation was abrogated in response to both WT and ΔflaA Lp (Figure S11A–B), and we were unable to detect caspase-11 activation (data not shown). Thus, although TRIF is not required for caspase-11 activation, a TLR-dependent signal is likely required as the loss of both MyD88 and TRIF eliminates caspase-11 upregulation and activation.

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