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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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Feedback cycle of NOX2/ROS and IL-1β activation in mφs infected by T. cruzi.THP-1 mφs were infected with T. cruzi as in Fig. 1, and incubated for 3 h (A,C,E) or 18 h (B,D,F,G) in presence of NOX2 inhibitors (diphenylene iodinium (DPI) or apoCynin), ROS scavenger (N-acetylcysteine (NAC)) or IL-1β antibody. (A–D) NOX2 inhibitors decreased ROS and IL-1β levels in infected mφs. Shown are (A&B) H2DCFDA oxidation by intracellular ROS, resulting in formation of fluorescent DCF by fluorimetry and (C&D) IL-1β release in supernatants determined by an ELISA. (E&F) Treatment with anti-IL-1β antibody decreased the ROS levels in infected mφs. (G) Effect of ROS inhibitors on intracellular parasite burden, as determined by qPCR, in infected mφs.
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pone-0111539-g004: Feedback cycle of NOX2/ROS and IL-1β activation in mφs infected by T. cruzi.THP-1 mφs were infected with T. cruzi as in Fig. 1, and incubated for 3 h (A,C,E) or 18 h (B,D,F,G) in presence of NOX2 inhibitors (diphenylene iodinium (DPI) or apoCynin), ROS scavenger (N-acetylcysteine (NAC)) or IL-1β antibody. (A–D) NOX2 inhibitors decreased ROS and IL-1β levels in infected mφs. Shown are (A&B) H2DCFDA oxidation by intracellular ROS, resulting in formation of fluorescent DCF by fluorimetry and (C&D) IL-1β release in supernatants determined by an ELISA. (E&F) Treatment with anti-IL-1β antibody decreased the ROS levels in infected mφs. (G) Effect of ROS inhibitors on intracellular parasite burden, as determined by qPCR, in infected mφs.

Mentions: Ingenuity iReport analysis of differentially expressed genes in this study (Table 2) and our previously published reports [22] have led us to suggest that infection by T. cruzi would elicit ROS by changes in mitoChondrial MPT or NOX2 activation in mφs, and that ROS may serve as a 2nd signal for inflammasome activation [11], [23]. We, therefore, determined whether ROS is induced and plays a direct role in IL-1β production in infected mφs. THP-1 mφs exhibited 3.4-fold and 2.7-fold increases in DCF fluorescence (detects intracellular ROS) at 3 h and 18 h pi, respectively (Fig.4A). When NOX2 inhibitors (DPI or apoCynin) were added during incubation with T. cruzi, we noted a 21–44% and 68–80% decline in ROS (Fig.4A&B) and 37% and 45% decline in IL-β release (Fig.4C&D) at 3 h and 18 h pi, respectively. These data suggested that NOX2, at least partially, regulates ROS-dependent IL-1β activation in infected mφs. The observation of a moderate, but a significant (35–45%, p<0.05) decline in DCF fluorescence in infected mφs treated with anti-IL-1β antibody (Fig.4E&F) implied that IL-1β also contributes to activation of ROS production. However, we found no increase in intracellular parasite burden in THP-1 mφs treated with NOX2 inhibitors (Fig.4G). Together, the data presented in Figs.3&4 suggest that a feed-back cycle of caspase-1/IL-1β and ROS activation oCcurs in response to T. cruzi infection in THP-1 mφs and is required for control of intracellular parasite replication. While direct inhibition of IL-1β (Fig.1F) or caspase-1 (Fig.3C) affected the WT mφs ability to control T. cruzi, inhibition of ROS-dependent IL-1β was compensated for, likely by activation of other immune defenses capable of controlling the intracellular parasite replication and survival (Fig.4G).


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)

Feedback cycle of NOX2/ROS and IL-1β activation in mφs infected by T. cruzi.THP-1 mφs were infected with T. cruzi as in Fig. 1, and incubated for 3 h (A,C,E) or 18 h (B,D,F,G) in presence of NOX2 inhibitors (diphenylene iodinium (DPI) or apoCynin), ROS scavenger (N-acetylcysteine (NAC)) or IL-1β antibody. (A–D) NOX2 inhibitors decreased ROS and IL-1β levels in infected mφs. Shown are (A&B) H2DCFDA oxidation by intracellular ROS, resulting in formation of fluorescent DCF by fluorimetry and (C&D) IL-1β release in supernatants determined by an ELISA. (E&F) Treatment with anti-IL-1β antibody decreased the ROS levels in infected mφs. (G) Effect of ROS inhibitors on intracellular parasite burden, as determined by qPCR, in infected mφs.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111539-g004: Feedback cycle of NOX2/ROS and IL-1β activation in mφs infected by T. cruzi.THP-1 mφs were infected with T. cruzi as in Fig. 1, and incubated for 3 h (A,C,E) or 18 h (B,D,F,G) in presence of NOX2 inhibitors (diphenylene iodinium (DPI) or apoCynin), ROS scavenger (N-acetylcysteine (NAC)) or IL-1β antibody. (A–D) NOX2 inhibitors decreased ROS and IL-1β levels in infected mφs. Shown are (A&B) H2DCFDA oxidation by intracellular ROS, resulting in formation of fluorescent DCF by fluorimetry and (C&D) IL-1β release in supernatants determined by an ELISA. (E&F) Treatment with anti-IL-1β antibody decreased the ROS levels in infected mφs. (G) Effect of ROS inhibitors on intracellular parasite burden, as determined by qPCR, in infected mφs.
Mentions: Ingenuity iReport analysis of differentially expressed genes in this study (Table 2) and our previously published reports [22] have led us to suggest that infection by T. cruzi would elicit ROS by changes in mitoChondrial MPT or NOX2 activation in mφs, and that ROS may serve as a 2nd signal for inflammasome activation [11], [23]. We, therefore, determined whether ROS is induced and plays a direct role in IL-1β production in infected mφs. THP-1 mφs exhibited 3.4-fold and 2.7-fold increases in DCF fluorescence (detects intracellular ROS) at 3 h and 18 h pi, respectively (Fig.4A). When NOX2 inhibitors (DPI or apoCynin) were added during incubation with T. cruzi, we noted a 21–44% and 68–80% decline in ROS (Fig.4A&B) and 37% and 45% decline in IL-β release (Fig.4C&D) at 3 h and 18 h pi, respectively. These data suggested that NOX2, at least partially, regulates ROS-dependent IL-1β activation in infected mφs. The observation of a moderate, but a significant (35–45%, p<0.05) decline in DCF fluorescence in infected mφs treated with anti-IL-1β antibody (Fig.4E&F) implied that IL-1β also contributes to activation of ROS production. However, we found no increase in intracellular parasite burden in THP-1 mφs treated with NOX2 inhibitors (Fig.4G). Together, the data presented in Figs.3&4 suggest that a feed-back cycle of caspase-1/IL-1β and ROS activation oCcurs in response to T. cruzi infection in THP-1 mφs and is required for control of intracellular parasite replication. While direct inhibition of IL-1β (Fig.1F) or caspase-1 (Fig.3C) affected the WT mφs ability to control T. cruzi, inhibition of ROS-dependent IL-1β was compensated for, likely by activation of other immune defenses capable of controlling the intracellular parasite replication and survival (Fig.4G).

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