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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 ESX-1 secretion system of Mtb does not affect secretion of proinflammatory cytokines in dendritic cells but is important for complete inflammasome activation.BMDCs were left uninfected (UI), infected with wild-type Mtb (Mtb) or the esxA deletion mutant (ΔesxA) for 4 h at MOI of 10, washed and incubated for an additional 24 h (A+B) or the indicated timepoints (C+D). (A) The cytokine profile in the supernatants was analyzed using a bead-based immunoassay (black =  uninfected, white =  Mtb, gray =  ΔesxA). (B) and (C) IL-1β secretion was analyzed by ELISA. (D) The percent of cells with activated caspase-1 was determined via flow cytometry using fluorescent caspase-1 substrates (FLICA). (E) Pro-IL-1β protein levels in BMDCs. (F) GFP-labeled bacteria were used to infect BMDCs and rate of infection was determined via flow cytometry. Shown are means and standard deviation of triplicate measurements of one representative experiment out of three. In all figures, the asterisks denote range of p values (* = p<0.05, ** = 0.01>p>0.001,***p<0.001, ns =  not significant ) as determined by one way ANOVA with Tukey's post test.
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pone-0040722-g001: The ESX-1 secretion system of Mtb does not affect secretion of proinflammatory cytokines in dendritic cells but is important for complete inflammasome activation.BMDCs were left uninfected (UI), infected with wild-type Mtb (Mtb) or the esxA deletion mutant (ΔesxA) for 4 h at MOI of 10, washed and incubated for an additional 24 h (A+B) or the indicated timepoints (C+D). (A) The cytokine profile in the supernatants was analyzed using a bead-based immunoassay (black =  uninfected, white =  Mtb, gray =  ΔesxA). (B) and (C) IL-1β secretion was analyzed by ELISA. (D) The percent of cells with activated caspase-1 was determined via flow cytometry using fluorescent caspase-1 substrates (FLICA). (E) Pro-IL-1β protein levels in BMDCs. (F) GFP-labeled bacteria were used to infect BMDCs and rate of infection was determined via flow cytometry. Shown are means and standard deviation of triplicate measurements of one representative experiment out of three. In all figures, the asterisks denote range of p values (* = p<0.05, ** = 0.01>p>0.001,***p<0.001, ns =  not significant ) as determined by one way ANOVA with Tukey's post test.

Mentions: First, the inflammatory cytokine profile of dendritic cells upon infection with Mtb and the esxA deletion mutant of Mtb (MtbΔesxA ) were investigated using a bead-based immunoassay. Both strains induced a significant secretion of the pro-inflammatory cytokines IL-6 and TNF from negligible amounts (<0.1ng/ml) in the supernatants of uninfected cells to 4-6ng/ml in infected cells. There was no induction of IL-10, MCP-1 or IFN-γ secretion by DCs after infection by either strain (Fig. 1A). Next the activation of the inflammasome was investigated via the detection of secreted IL-1β. DCs infected with wild-type Mtb secreted about 3ng/ml of IL-1β which was very similar to the positive control (LPS plus ATP treatment). Interestingly, Mtb deficient in functional ESX-1 secretion system induces less IL-1β secretion of approximately 1.5ng/ml in wild-type BMDCs (Fig. 1B). The secreted IL-1β was the mature form because these supernatants were devoid of pro-IL-1β as tested by a pro-IL-1βspecific ELISA (data not shown). Next we demonstrated if these differences in IL-1β secretion can be detected already right after the 4h infection period and continued to persist during the 4 h and 8 h post infection timepoints(Fig. 1C). To test more directly if the ESX-1 complex was involved in inflammasome activation, we analyzed the activation of caspase-1 via fluorescent substrates and flow cytometry. We could show that only Mtb induced significantly more caspase-1 activation (∼30% positive cells) when compared to uninfected and Mtb esxA mutant infected cells (∼8–12%) at 0h and 4h post infection (Fig. 1D). The induction of pro-IL-1β production is very similar right after the infection period as determined by western-blotting (Fig. 1E). At 4 hpi and 8 hpi the pro-IL-1β levels were consistently lower in esxA mutant infected cells (Fig. 1E). The rate of infection of BMDCs after 4h is around 80% for both wild-type and mutant Mtb as measured by flow cytometry after infection with GFP-expressing bacteria (Fig. 1F). These results suggest that the inflammasome of DCs is activated by Mtb infection in a manner that is partially dependent upon ESX-1 secreted proteins.


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 ESX-1 secretion system of Mtb does not affect secretion of proinflammatory cytokines in dendritic cells but is important for complete inflammasome activation.BMDCs were left uninfected (UI), infected with wild-type Mtb (Mtb) or the esxA deletion mutant (ΔesxA) for 4 h at MOI of 10, washed and incubated for an additional 24 h (A+B) or the indicated timepoints (C+D). (A) The cytokine profile in the supernatants was analyzed using a bead-based immunoassay (black =  uninfected, white =  Mtb, gray =  ΔesxA). (B) and (C) IL-1β secretion was analyzed by ELISA. (D) The percent of cells with activated caspase-1 was determined via flow cytometry using fluorescent caspase-1 substrates (FLICA). (E) Pro-IL-1β protein levels in BMDCs. (F) GFP-labeled bacteria were used to infect BMDCs and rate of infection was determined via flow cytometry. Shown are means and standard deviation of triplicate measurements of one representative experiment out of three. In all figures, the asterisks denote range of p values (* = p<0.05, ** = 0.01>p>0.001,***p<0.001, ns =  not significant ) as determined by one way ANOVA with Tukey's post test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3404059&req=5

pone-0040722-g001: The ESX-1 secretion system of Mtb does not affect secretion of proinflammatory cytokines in dendritic cells but is important for complete inflammasome activation.BMDCs were left uninfected (UI), infected with wild-type Mtb (Mtb) or the esxA deletion mutant (ΔesxA) for 4 h at MOI of 10, washed and incubated for an additional 24 h (A+B) or the indicated timepoints (C+D). (A) The cytokine profile in the supernatants was analyzed using a bead-based immunoassay (black =  uninfected, white =  Mtb, gray =  ΔesxA). (B) and (C) IL-1β secretion was analyzed by ELISA. (D) The percent of cells with activated caspase-1 was determined via flow cytometry using fluorescent caspase-1 substrates (FLICA). (E) Pro-IL-1β protein levels in BMDCs. (F) GFP-labeled bacteria were used to infect BMDCs and rate of infection was determined via flow cytometry. Shown are means and standard deviation of triplicate measurements of one representative experiment out of three. In all figures, the asterisks denote range of p values (* = p<0.05, ** = 0.01>p>0.001,***p<0.001, ns =  not significant ) as determined by one way ANOVA with Tukey's post test.
Mentions: First, the inflammatory cytokine profile of dendritic cells upon infection with Mtb and the esxA deletion mutant of Mtb (MtbΔesxA ) were investigated using a bead-based immunoassay. Both strains induced a significant secretion of the pro-inflammatory cytokines IL-6 and TNF from negligible amounts (<0.1ng/ml) in the supernatants of uninfected cells to 4-6ng/ml in infected cells. There was no induction of IL-10, MCP-1 or IFN-γ secretion by DCs after infection by either strain (Fig. 1A). Next the activation of the inflammasome was investigated via the detection of secreted IL-1β. DCs infected with wild-type Mtb secreted about 3ng/ml of IL-1β which was very similar to the positive control (LPS plus ATP treatment). Interestingly, Mtb deficient in functional ESX-1 secretion system induces less IL-1β secretion of approximately 1.5ng/ml in wild-type BMDCs (Fig. 1B). The secreted IL-1β was the mature form because these supernatants were devoid of pro-IL-1β as tested by a pro-IL-1βspecific ELISA (data not shown). Next we demonstrated if these differences in IL-1β secretion can be detected already right after the 4h infection period and continued to persist during the 4 h and 8 h post infection timepoints(Fig. 1C). To test more directly if the ESX-1 complex was involved in inflammasome activation, we analyzed the activation of caspase-1 via fluorescent substrates and flow cytometry. We could show that only Mtb induced significantly more caspase-1 activation (∼30% positive cells) when compared to uninfected and Mtb esxA mutant infected cells (∼8–12%) at 0h and 4h post infection (Fig. 1D). The induction of pro-IL-1β production is very similar right after the infection period as determined by western-blotting (Fig. 1E). At 4 hpi and 8 hpi the pro-IL-1β levels were consistently lower in esxA mutant infected cells (Fig. 1E). The rate of infection of BMDCs after 4h is around 80% for both wild-type and mutant Mtb as measured by flow cytometry after infection with GFP-expressing bacteria (Fig. 1F). These results suggest that the inflammasome of DCs is activated by Mtb infection in a manner that is partially dependent upon ESX-1 secreted proteins.

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