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

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.

Macrophages Promote Oxidative Metabolism To Drive Nitric Oxide Generation in Response to Trypanosoma cruzi

“…Because ROS and NO and their by-products are strong cytotoxic agents that may directly kill the pathogen (24) or influence the cellular function by formation of adducts on DNA, proteins, and lipids (25,26), our data suggest that T. cruzi diminishes the oxidative/nitrosative response to ensure its survival in ms. Our finding of inhibition of ROS by DPI (NOX2 inhibitor) and a lack of mitochondrial ROS stimulated by LPS/IFN-␥ (Fig. 2H and I) and our prior T. cruzi infection studies in splenocytes and human THP-1 ms (27,28) suggest that NOX2 is the major source of ROS in ms. How SYL diminishes NOX2 activation to control ROS/NO levels remains to be further investigated. Others have suggested that an elaborate antioxidant network comprising peroxiredoxins that scavenge ROS and NO provides a survival advantage to T. cruzi in immune cells (6).…”

“…In the context of T. cruzi, prepared lysates are insufficient to activate an inflammatory response in ms and splenocytes. Human THP-1 ms were shown to lack induction of IL-1␤ with either live T. cruzi and T. cruzi lysate stimulation compared to LPS treatment (28). Splenocytes isolated from normal mice respond to T. cruzi lysates with low levels of production of H 2 O 2 that were enhanced only when splenic cells were primed with live T. cruzi before secondary stimulation with T. cruzi lysate (27).…”

Gene Expression Profiling and Functional Characterization of Macrophages in Response to Circulatory Microparticles Produced during <b><i>Trypanosoma cruzi</i></b> Infection and Chagas Disease

“…THP-1 human monocytes (ATCC TIB-202) were suspended in complete RPMI media and incubated at 37°C/5% CO 2 for 24 h in presence of 50 ng/ml phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich), and then for 48 h in complete RPMI media without any stimulus to generate the resting mφs [21]. RAW 264.7 murine macrophages (ATCC TIB-71) were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 2 mmol/l glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Corning, Corning NY).…”

La respuesta inmunitaria adaptativa en la infección crónica por Trypanosoma cruzi

“…El parásito puede influenciar o alterar el comportamiento de estas células a través de la infección directa ( . La activación de estas células presentadoras de antígeno, como los monocitos y macrófagos, tiene que ver con la producción de moléculas efectoras como el óxido nítrico (NO), los productos reactivos del oxígeno (reactive oxygen species, ROS) y las citocinas inflamatorias, las cuales tienen un papel fundamental en la presentación antigénica, así como en la activación subsecuente de la respuesta mediada por linfocitos T. El parásito suele disminuir dicha respuesta alterando la actividad de la enzima NADPH oxidasa, la cual regula la producción de ROS (Dey, et al, 2014). Asimismo, los antígenos derivados del parásito alteran el patrón de citocinas producidas, por ejemplo, las células dendríticas de pacientes con enfermedad de Chagas crónica producen más interleucina 10 (IL-10) que interleucina 12 (IL-12) en presencia de la proteína de choque térmico de T. cruzi.…”