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Caspase-1/ASC inflammasome-mediated activation of IL-1β-ROS-NF-κB pathway for control of Trypanosoma cruzi replication and survival is dispensable in NLRP3-/- macrophages.

Dey N, Sinha M, Gupta S, Gonzalez MN, Fang R, Endsley JJ, Luxon BA, Garg NJ - PLoS ONE (2014)

Bottom Line: When WT and ASC-/- macrophages were treated with inhibitors of caspase-1, IL-1β, or NADPH oxidase, we found that IL-1β production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal.Moreover, IL-1β regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages.We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1β/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch (UTMB), Galveston, Texas, United States of America.

ABSTRACT
In this study, we have utilized wild-type (WT), ASC-/-, and NLRP3-/- macrophages and inhibition approaches to investigate the mechanisms of inflammasome activation and their role in Trypanosoma cruzi infection. We also probed human macrophages and analyzed published microarray datasets from human fibroblasts, and endothelial and smooth muscle cells for T. cruzi-induced changes in the expression genes included in the RT Profiler Human Inflammasome arrays. T. cruzi infection elicited a subdued and delayed activation of inflammasome-related gene expression and IL-1β production in mφs in comparison to LPS-treated controls. When WT and ASC-/- macrophages were treated with inhibitors of caspase-1, IL-1β, or NADPH oxidase, we found that IL-1β production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal. Moreover, IL-1β regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages. NLRP3-/- macrophages, despite an inability to elicit IL-1β activation and inflammatory cytokine gene expression, exhibited a 4-fold decline in intracellular parasites in comparison to that noted in matched WT controls. NLRP3-/- macrophages were not refractory to T. cruzi, and instead exhibited a very high basal level of ROS (>100-fold higher than WT controls) that was maintained after infection in an IL-1β-independent manner and contributed to efficient parasite killing. We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1β/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages. However, NLRP3-mediated IL-1β/NFκB activation is dispensable and compensated for by ROS-mediated control of T. cruzi replication and survival in macrophages.

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IL-1β production in macrophages infected by T. cruzi.(A–D) PMA-differentiated THP-1 mφs were incubated with T. cruzi trypomastigotes (cell: parasite ratio, 1∶3), Tc lysate (10 µg protein/106 cells) or LPS (100 ng/ml) for 3 h (A&C) and 18 h (B&D). In some experiments, ATP was added during last 30 min of incubation (C&D). IL-1β release in supernatants was determined by ELISA. (E–G) IL-1β contributes to parasite control in mφs. THP-1 mφs were incubated with SYTO®11-labeled T. cruzi in the presence or absence of anti-IL-1β antibody for 18 h. (E) SYTO®11 fluorescence as an indicator of parasite uptake (shown by arrows) was determined by using an Olympus BX-15 microscope equipped with a digital camera (magnification 40X). (F) Quantitative PCR analysis of parasite burden in infected mφs by using Tc18SrDNA-specific oligonucleotides (normalized to human GAPDH). (G) Addition of anti-IL-1β antibody depletes secreted IL-1β levels in T. cruzi-infected mφs. In all figures, data are representative of three independent experiments and presented as mean ± SD. Significance is shown by *normal versus infected and #treated/infected versus infected (*,#p<0.05, **,##p<0.01, and ***,###p<0.001).
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pone-0111539-g001: IL-1β production in macrophages infected by T. cruzi.(A–D) PMA-differentiated THP-1 mφs were incubated with T. cruzi trypomastigotes (cell: parasite ratio, 1∶3), Tc lysate (10 µg protein/106 cells) or LPS (100 ng/ml) for 3 h (A&C) and 18 h (B&D). In some experiments, ATP was added during last 30 min of incubation (C&D). IL-1β release in supernatants was determined by ELISA. (E–G) IL-1β contributes to parasite control in mφs. THP-1 mφs were incubated with SYTO®11-labeled T. cruzi in the presence or absence of anti-IL-1β antibody for 18 h. (E) SYTO®11 fluorescence as an indicator of parasite uptake (shown by arrows) was determined by using an Olympus BX-15 microscope equipped with a digital camera (magnification 40X). (F) Quantitative PCR analysis of parasite burden in infected mφs by using Tc18SrDNA-specific oligonucleotides (normalized to human GAPDH). (G) Addition of anti-IL-1β antibody depletes secreted IL-1β levels in T. cruzi-infected mφs. In all figures, data are representative of three independent experiments and presented as mean ± SD. Significance is shown by *normal versus infected and #treated/infected versus infected (*,#p<0.05, **,##p<0.01, and ***,###p<0.001).

Mentions: THP-1 mφs incubated with T. cruzi trypomastigotes (1∶3, cell: parasite ratio) or Tc-lysate exhibited a ∼2-fold increase in IL-1β release at 3 h pi that was consistently increased at 6 h and 12 h pi (data not shown) and maximized to a>9.8-fold increase by 18 h pi (Fig.1A&B). Incubation with higher number of parasites (1∶4, 1∶5 or 1∶6, cell: parasite ratio) did not result in a further increase in IL-1β release at 3 h and 18 h pi (data not shown). LPS treatment (100-ng/ml) triggered a substantially higher level of IL-1β release in THP-1 mφs than was observed with T. cruzi infection, the maximal difference being noted at 3 h (Fig.1A&B). Exogenous addition of ATP, the K+ flux agent that can trigger caspase-1 cleavage and inflammasome activation in response to PAMPs, elicited a 2-fold and no increase in IL-1β release in Tc-infected and LPS-treated cells at 3 h (Fig.1A&C). No significant effect of exogenous ATP on IL-1β release was observed at 18 h post-incubation (Fig.1B&D)


Caspase-1/ASC inflammasome-mediated activation of IL-1β-ROS-NF-κB pathway for control of Trypanosoma cruzi replication and survival is dispensable in NLRP3-/- macrophages.

Dey N, Sinha M, Gupta S, Gonzalez MN, Fang R, Endsley JJ, Luxon BA, Garg NJ - PLoS ONE (2014)

IL-1β production in macrophages infected by T. cruzi.(A–D) PMA-differentiated THP-1 mφs were incubated with T. cruzi trypomastigotes (cell: parasite ratio, 1∶3), Tc lysate (10 µg protein/106 cells) or LPS (100 ng/ml) for 3 h (A&C) and 18 h (B&D). In some experiments, ATP was added during last 30 min of incubation (C&D). IL-1β release in supernatants was determined by ELISA. (E–G) IL-1β contributes to parasite control in mφs. THP-1 mφs were incubated with SYTO®11-labeled T. cruzi in the presence or absence of anti-IL-1β antibody for 18 h. (E) SYTO®11 fluorescence as an indicator of parasite uptake (shown by arrows) was determined by using an Olympus BX-15 microscope equipped with a digital camera (magnification 40X). (F) Quantitative PCR analysis of parasite burden in infected mφs by using Tc18SrDNA-specific oligonucleotides (normalized to human GAPDH). (G) Addition of anti-IL-1β antibody depletes secreted IL-1β levels in T. cruzi-infected mφs. In all figures, data are representative of three independent experiments and presented as mean ± SD. Significance is shown by *normal versus infected and #treated/infected versus infected (*,#p<0.05, **,##p<0.01, and ***,###p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4221042&req=5

pone-0111539-g001: IL-1β production in macrophages infected by T. cruzi.(A–D) PMA-differentiated THP-1 mφs were incubated with T. cruzi trypomastigotes (cell: parasite ratio, 1∶3), Tc lysate (10 µg protein/106 cells) or LPS (100 ng/ml) for 3 h (A&C) and 18 h (B&D). In some experiments, ATP was added during last 30 min of incubation (C&D). IL-1β release in supernatants was determined by ELISA. (E–G) IL-1β contributes to parasite control in mφs. THP-1 mφs were incubated with SYTO®11-labeled T. cruzi in the presence or absence of anti-IL-1β antibody for 18 h. (E) SYTO®11 fluorescence as an indicator of parasite uptake (shown by arrows) was determined by using an Olympus BX-15 microscope equipped with a digital camera (magnification 40X). (F) Quantitative PCR analysis of parasite burden in infected mφs by using Tc18SrDNA-specific oligonucleotides (normalized to human GAPDH). (G) Addition of anti-IL-1β antibody depletes secreted IL-1β levels in T. cruzi-infected mφs. In all figures, data are representative of three independent experiments and presented as mean ± SD. Significance is shown by *normal versus infected and #treated/infected versus infected (*,#p<0.05, **,##p<0.01, and ***,###p<0.001).
Mentions: THP-1 mφs incubated with T. cruzi trypomastigotes (1∶3, cell: parasite ratio) or Tc-lysate exhibited a ∼2-fold increase in IL-1β release at 3 h pi that was consistently increased at 6 h and 12 h pi (data not shown) and maximized to a>9.8-fold increase by 18 h pi (Fig.1A&B). Incubation with higher number of parasites (1∶4, 1∶5 or 1∶6, cell: parasite ratio) did not result in a further increase in IL-1β release at 3 h and 18 h pi (data not shown). LPS treatment (100-ng/ml) triggered a substantially higher level of IL-1β release in THP-1 mφs than was observed with T. cruzi infection, the maximal difference being noted at 3 h (Fig.1A&B). Exogenous addition of ATP, the K+ flux agent that can trigger caspase-1 cleavage and inflammasome activation in response to PAMPs, elicited a 2-fold and no increase in IL-1β release in Tc-infected and LPS-treated cells at 3 h (Fig.1A&C). No significant effect of exogenous ATP on IL-1β release was observed at 18 h post-incubation (Fig.1B&D)

Bottom Line: When WT and ASC-/- macrophages were treated with inhibitors of caspase-1, IL-1β, or NADPH oxidase, we found that IL-1β production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal.Moreover, IL-1β regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages.We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1β/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch (UTMB), Galveston, Texas, United States of America.

ABSTRACT
In this study, we have utilized wild-type (WT), ASC-/-, and NLRP3-/- macrophages and inhibition approaches to investigate the mechanisms of inflammasome activation and their role in Trypanosoma cruzi infection. We also probed human macrophages and analyzed published microarray datasets from human fibroblasts, and endothelial and smooth muscle cells for T. cruzi-induced changes in the expression genes included in the RT Profiler Human Inflammasome arrays. T. cruzi infection elicited a subdued and delayed activation of inflammasome-related gene expression and IL-1β production in mφs in comparison to LPS-treated controls. When WT and ASC-/- macrophages were treated with inhibitors of caspase-1, IL-1β, or NADPH oxidase, we found that IL-1β production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal. Moreover, IL-1β regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages. NLRP3-/- macrophages, despite an inability to elicit IL-1β activation and inflammatory cytokine gene expression, exhibited a 4-fold decline in intracellular parasites in comparison to that noted in matched WT controls. NLRP3-/- macrophages were not refractory to T. cruzi, and instead exhibited a very high basal level of ROS (>100-fold higher than WT controls) that was maintained after infection in an IL-1β-independent manner and contributed to efficient parasite killing. We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1β/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages. However, NLRP3-mediated IL-1β/NFκB activation is dispensable and compensated for by ROS-mediated control of T. cruzi replication and survival in macrophages.

Show MeSH
Related in: MedlinePlus