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Mycobacterium tuberculosis infection of dendritic cells leads to partially caspase-1/11-independent IL-1β and IL-18 secretion but not to pyroptosis.

Abdalla H, Srinivasan L, Shah S, Mayer-Barber KD, Sher A, Sutterwala FS, Briken V - PLoS ONE (2012)

Bottom Line: Furthermore, NLRP3 inflammasome activation and apoptosis induction were both reduced in BMDCs infected with the esxA deletion mutant of Mtb demonstrating the importance of a functional ESX-1 secretion system.Altogether we demonstrate the partially caspase-1/11-independent, but NLRP3- and ASC-dependent IL-1β secretion in Mtb-infected BMDCs.These findings point towards a potential role of DCs in the host innate immune response to mycobacterial infections via their capacity to induce IL-1β and IL-18 secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT

Background: Interleukin-1β (IL-1β) is important for host resistance against Mycobacterium tuberculosis (Mtb) infections. The response of the dendritic cell inflammasome during Mtb infections has not been investigated in detail.

Methodology/principal findings: Here we show that Mtb infection of bone marrow-derived dendritic cells (BMDCs) induces IL-1β secretion and that this induction is dependent upon the presence of functional ASC and NLRP3 but not NLRC4 or NOD2. The analysis of cell death induction in BMDCs derived from these knock-out mice revealed the important induction of host cell apoptosis but not necrosis, pyroptosis or pyronecrosis. Furthermore, NLRP3 inflammasome activation and apoptosis induction were both reduced in BMDCs infected with the esxA deletion mutant of Mtb demonstrating the importance of a functional ESX-1 secretion system. Surprisingly, caspase-1/11-deficient BMDCs still secreted residual levels of IL-1βand IL-18 upon Mtb infection which was abolished in cells infected with the esxA Mtb mutant.

Conclusion: Altogether we demonstrate the partially caspase-1/11-independent, but NLRP3- and ASC-dependent IL-1β secretion in Mtb-infected BMDCs. These findings point towards a potential role of DCs in the host innate immune response to mycobacterial infections via their capacity to induce IL-1β and IL-18 secretion.

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The inflammasome is not involved in BMDC host cell death induction after Mtb infection.BMDCs from indicated wild-type or knockout mouse strains were infected with Mtb (white bars) or left uninfected (black bars). After 24 h the amount of TUNEL positive cells was determined in (A) and in (B). In (C) the amount of necrosis was determined via analysis of the release of adenylate kinase into the supernatant of infected cells relative to detergent lysed cells (PC). Shown are means and standard deviation of triplicate measurements of one representative experiment out of three.
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pone-0040722-g004: The inflammasome is not involved in BMDC host cell death induction after Mtb infection.BMDCs from indicated wild-type or knockout mouse strains were infected with Mtb (white bars) or left uninfected (black bars). After 24 h the amount of TUNEL positive cells was determined in (A) and in (B). In (C) the amount of necrosis was determined via analysis of the release of adenylate kinase into the supernatant of infected cells relative to detergent lysed cells (PC). Shown are means and standard deviation of triplicate measurements of one representative experiment out of three.

Mentions: Inflammasome activation is a defining feature of pyroptosis (caspase-1-dependent) and pyronecrosis (ASC-dependent) [4]. We investigated if the Mtb-infected BMDCs undergo one of this forms of cell death. Pyroptosis also leads to disruption of the cell membrane and release of cytosolic proteins into the extracellular environment, whereas apoptotic cell death maintains the membrane integrity [4], [5]. The permeabilization of the cell membrane of infected or uninfected BMDCs was analyzed by the detection of the cytosolic enzyme, adenylate kinase (AK), in the cell supernatant. First, the percentage of TUNEL positive cells in BMDCs derived from Nlrc4−/− and Nod2−/− was measured and found to be not significantly different from wild-type BMDCs which was as expected since these cells did not show any difference in inflammasome activation (Fig. 4A.). As shown in Figure 3, the BMDCs of the Caspase-1/11, ASC- and NLRP3- deficient mice all had a clear defect in inflammasome activation after Mtb infection and thus they were now compared for their propensity to undergo cell death by measuring DNA fragmentation (TUNEL staining) and cell membrane disruption (AK release assay) (Fig. 4B and 4C, respectively ). The induction of TUNEL positive cells by Mtb was similar, about 40%, in BMDCs of all the different mouse strains, except for the Nlrp3−/− cells in which the level of TUNEL positive cells increased to about 50% after 24h of infection (Fig. 4B). These results demonstrate that none of the inflammasome components is important for the cell death induction as measured by DNA degradation. Furthermore, Mtb infection did not lead to an increase in cell membrane permeability after infection of wild-type and Casp1/11−/− BMDCs. Infected and uninfected BMDCs from these mice had about 20% cell lysis relative to the detergent lysis control, PC, which was set to 100% lysis (Fig. 4C). Surprisingly, we did detect a significant increase of cell lysis in BMDCs of Asc−/− and Nlrp3−/− BMDCs which increased in both cases from about 20% in uninfected cells to about 40% in Mtb-infected cells. These results would suggest that these inflammasome components may help to prevent necrosis induction under some circumstances. In conclusion, these results underscored the lack of pyroptosis and pyronecrosis induction in BMDCs after Mtb infection. Furthermore, Il1r1−/− BMDCs did not demonstrate any defect in IL-1β secretion after 24 h of infection with Mtb, since in wild-type and knock-out BMDCs about 4ng/ml of IL-1β were secreted (Fig. 5A). The IL-1RI-deficient cells showed a statistically significant reduction in TUNEL staining over the same time period with 50.25% in Mtb infected wild-type cells and 37.00% in Mtb-infected Il1r1−/− BMDCs (Fig. 5B), suggesting a minor involvement of IL-1RI signaling in BMDC apoptosis induction.


Mycobacterium tuberculosis infection of dendritic cells leads to partially caspase-1/11-independent IL-1β and IL-18 secretion but not to pyroptosis.

Abdalla H, Srinivasan L, Shah S, Mayer-Barber KD, Sher A, Sutterwala FS, Briken V - PLoS ONE (2012)

The inflammasome is not involved in BMDC host cell death induction after Mtb infection.BMDCs from indicated wild-type or knockout mouse strains were infected with Mtb (white bars) or left uninfected (black bars). After 24 h the amount of TUNEL positive cells was determined in (A) and in (B). In (C) the amount of necrosis was determined via analysis of the release of adenylate kinase into the supernatant of infected cells relative to detergent lysed cells (PC). Shown are means and standard deviation of triplicate measurements of one representative experiment out of three.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3404059&req=5

pone-0040722-g004: The inflammasome is not involved in BMDC host cell death induction after Mtb infection.BMDCs from indicated wild-type or knockout mouse strains were infected with Mtb (white bars) or left uninfected (black bars). After 24 h the amount of TUNEL positive cells was determined in (A) and in (B). In (C) the amount of necrosis was determined via analysis of the release of adenylate kinase into the supernatant of infected cells relative to detergent lysed cells (PC). Shown are means and standard deviation of triplicate measurements of one representative experiment out of three.
Mentions: Inflammasome activation is a defining feature of pyroptosis (caspase-1-dependent) and pyronecrosis (ASC-dependent) [4]. We investigated if the Mtb-infected BMDCs undergo one of this forms of cell death. Pyroptosis also leads to disruption of the cell membrane and release of cytosolic proteins into the extracellular environment, whereas apoptotic cell death maintains the membrane integrity [4], [5]. The permeabilization of the cell membrane of infected or uninfected BMDCs was analyzed by the detection of the cytosolic enzyme, adenylate kinase (AK), in the cell supernatant. First, the percentage of TUNEL positive cells in BMDCs derived from Nlrc4−/− and Nod2−/− was measured and found to be not significantly different from wild-type BMDCs which was as expected since these cells did not show any difference in inflammasome activation (Fig. 4A.). As shown in Figure 3, the BMDCs of the Caspase-1/11, ASC- and NLRP3- deficient mice all had a clear defect in inflammasome activation after Mtb infection and thus they were now compared for their propensity to undergo cell death by measuring DNA fragmentation (TUNEL staining) and cell membrane disruption (AK release assay) (Fig. 4B and 4C, respectively ). The induction of TUNEL positive cells by Mtb was similar, about 40%, in BMDCs of all the different mouse strains, except for the Nlrp3−/− cells in which the level of TUNEL positive cells increased to about 50% after 24h of infection (Fig. 4B). These results demonstrate that none of the inflammasome components is important for the cell death induction as measured by DNA degradation. Furthermore, Mtb infection did not lead to an increase in cell membrane permeability after infection of wild-type and Casp1/11−/− BMDCs. Infected and uninfected BMDCs from these mice had about 20% cell lysis relative to the detergent lysis control, PC, which was set to 100% lysis (Fig. 4C). Surprisingly, we did detect a significant increase of cell lysis in BMDCs of Asc−/− and Nlrp3−/− BMDCs which increased in both cases from about 20% in uninfected cells to about 40% in Mtb-infected cells. These results would suggest that these inflammasome components may help to prevent necrosis induction under some circumstances. In conclusion, these results underscored the lack of pyroptosis and pyronecrosis induction in BMDCs after Mtb infection. Furthermore, Il1r1−/− BMDCs did not demonstrate any defect in IL-1β secretion after 24 h of infection with Mtb, since in wild-type and knock-out BMDCs about 4ng/ml of IL-1β were secreted (Fig. 5A). The IL-1RI-deficient cells showed a statistically significant reduction in TUNEL staining over the same time period with 50.25% in Mtb infected wild-type cells and 37.00% in Mtb-infected Il1r1−/− BMDCs (Fig. 5B), suggesting a minor involvement of IL-1RI signaling in BMDC apoptosis induction.

Bottom Line: Furthermore, NLRP3 inflammasome activation and apoptosis induction were both reduced in BMDCs infected with the esxA deletion mutant of Mtb demonstrating the importance of a functional ESX-1 secretion system.Altogether we demonstrate the partially caspase-1/11-independent, but NLRP3- and ASC-dependent IL-1β secretion in Mtb-infected BMDCs.These findings point towards a potential role of DCs in the host innate immune response to mycobacterial infections via their capacity to induce IL-1β and IL-18 secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT

Background: Interleukin-1β (IL-1β) is important for host resistance against Mycobacterium tuberculosis (Mtb) infections. The response of the dendritic cell inflammasome during Mtb infections has not been investigated in detail.

Methodology/principal findings: Here we show that Mtb infection of bone marrow-derived dendritic cells (BMDCs) induces IL-1β secretion and that this induction is dependent upon the presence of functional ASC and NLRP3 but not NLRC4 or NOD2. The analysis of cell death induction in BMDCs derived from these knock-out mice revealed the important induction of host cell apoptosis but not necrosis, pyroptosis or pyronecrosis. Furthermore, NLRP3 inflammasome activation and apoptosis induction were both reduced in BMDCs infected with the esxA deletion mutant of Mtb demonstrating the importance of a functional ESX-1 secretion system. Surprisingly, caspase-1/11-deficient BMDCs still secreted residual levels of IL-1βand IL-18 upon Mtb infection which was abolished in cells infected with the esxA Mtb mutant.

Conclusion: Altogether we demonstrate the partially caspase-1/11-independent, but NLRP3- and ASC-dependent IL-1β secretion in Mtb-infected BMDCs. These findings point towards a potential role of DCs in the host innate immune response to mycobacterial infections via their capacity to induce IL-1β and IL-18 secretion.

Show MeSH
Related in: MedlinePlus