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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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IL-1α and IL-1β control bacterial burden and neutrophil recruitment in vivo.(A) 8–12 week old B6 or Il1r1−/− mice were infected with 1×106 ΔflaA L. pneumophila 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 and C) B6 or Il1r1−/− mice were infected with 1×106 ΔflaA 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. (D, E, and F) 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 ΔflaA 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.
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ppat-1003400-g007: IL-1α and IL-1β control bacterial burden and neutrophil recruitment in vivo.(A) 8–12 week old B6 or Il1r1−/− mice were infected with 1×106 ΔflaA L. pneumophila 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 and C) B6 or Il1r1−/− mice were infected with 1×106 ΔflaA 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. (D, E, and F) 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 ΔflaA 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.

Mentions: As caspase-11 contributes to flagellin-independent IL-1α and IL-1β release from infected macrophages in vitro and IL-1α and IL-1β secretion is flagellin-independent in vivo, we wanted to determine the contribution of IL-1α and IL-1β to host defense against L. pneumophila in vivo. IL-1α and IL-1β both bind the IL-1 receptor (IL-1R), which signals through the MyD88 adaptor protein [59]–[61]. As MyD88 is critical for control of L. pneumophila replication during in vivo infection but deletion of an individual MyD88-dependent TLR or a combination of TLRs does not recapitulate MyD88 deficiency, it is likely that other MyD88-dependent receptors, including the IL-1R, may play a role [62]–[66]. IL-1R signaling contributes to chemokine production by non-hematopoietic cells during infection with wild-type, flagellin-expressing L. pneumophila[67]. However, the role of IL-1R signaling during infection with flagellin-deficient L. pneumophila, which do not activate the NAIP5/NLRC4 inflammasome, has not been investigated. We therefore infected B6 and IL-1R-deficient (Il1r1−/−) mice intranasally with ΔflaA Lp and measured bacterial burden in the lung over the course of seven days. Though both B6 and Il1r1−/− mice received similar initial bacterial burdens, Il1r1−/− mice show a defect in bacterial clearance as early as 24 hours post-infection (Figure 7A). Bacterial burden remains elevated in the absence of IL-1R signaling, with the Il1r1−/− mice still exhibiting a log-increase in bacterial load at 120 hours post-infection. Since IL-1R signaling is important for neutrophil recruitment [68], we examined whether Il1r1−/− mice have a defect in neutrophil recruitment to the pulmonary airway during L. pneumophila infection. Indeed, Il1r1−/− mice exhibit a significant decrease in neutrophil recruitment to the airway 24 hours post-infection, possibly contributing to their inability to efficiently clear the pathogen (Figure 7B–C).


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)

IL-1α and IL-1β control bacterial burden and neutrophil recruitment in vivo.(A) 8–12 week old B6 or Il1r1−/− mice were infected with 1×106 ΔflaA L. pneumophila 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 and C) B6 or Il1r1−/− mice were infected with 1×106 ΔflaA 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. (D, E, and F) 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 ΔflaA 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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675167&req=5

ppat-1003400-g007: IL-1α and IL-1β control bacterial burden and neutrophil recruitment in vivo.(A) 8–12 week old B6 or Il1r1−/− mice were infected with 1×106 ΔflaA L. pneumophila 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 and C) B6 or Il1r1−/− mice were infected with 1×106 ΔflaA 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. (D, E, and F) 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 ΔflaA 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.
Mentions: As caspase-11 contributes to flagellin-independent IL-1α and IL-1β release from infected macrophages in vitro and IL-1α and IL-1β secretion is flagellin-independent in vivo, we wanted to determine the contribution of IL-1α and IL-1β to host defense against L. pneumophila in vivo. IL-1α and IL-1β both bind the IL-1 receptor (IL-1R), which signals through the MyD88 adaptor protein [59]–[61]. As MyD88 is critical for control of L. pneumophila replication during in vivo infection but deletion of an individual MyD88-dependent TLR or a combination of TLRs does not recapitulate MyD88 deficiency, it is likely that other MyD88-dependent receptors, including the IL-1R, may play a role [62]–[66]. IL-1R signaling contributes to chemokine production by non-hematopoietic cells during infection with wild-type, flagellin-expressing L. pneumophila[67]. However, the role of IL-1R signaling during infection with flagellin-deficient L. pneumophila, which do not activate the NAIP5/NLRC4 inflammasome, has not been investigated. We therefore infected B6 and IL-1R-deficient (Il1r1−/−) mice intranasally with ΔflaA Lp and measured bacterial burden in the lung over the course of seven days. Though both B6 and Il1r1−/− mice received similar initial bacterial burdens, Il1r1−/− mice show a defect in bacterial clearance as early as 24 hours post-infection (Figure 7A). Bacterial burden remains elevated in the absence of IL-1R signaling, with the Il1r1−/− mice still exhibiting a log-increase in bacterial load at 120 hours post-infection. Since IL-1R signaling is important for neutrophil recruitment [68], we examined whether Il1r1−/− mice have a defect in neutrophil recruitment to the pulmonary airway during L. pneumophila infection. Indeed, Il1r1−/− mice exhibit a significant decrease in neutrophil recruitment to the airway 24 hours post-infection, possibly contributing to their inability to efficiently clear the pathogen (Figure 7B–C).

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