Caspase-1/ASC Inflammasome-Mediated Activation of IL-1β–ROS–NF-κB Pathway for Control of <i>Trypanosoma cruzi</i> Replication and Survival Is Dispensable in NLRP3^−/− Macrophages

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.本研究利用野生型（wild-type, WT）、ASC基因敲除（ASC−/−）及NLRP3基因敲除（NLRP3−/−）巨噬细胞，并结合抑制策略，探究了炎性体（inflammasome）的激活机制及其在克氏锥虫（Trypanosoma cruzi）感染中的作用。此外，本研究还对人类巨噬细胞进行了检测，并分析了已发表的微阵列数据集（microarray datasets），这些数据集来自人类成纤维细胞、内皮细胞和平滑肌细胞，旨在探究克氏锥虫诱导的RT Profiler人类炎性体芯片（RT Profiler Human Inflammasome arrays）所涵盖基因的表达变化。与脂多糖（lipopolysaccharide, LPS）处理的对照组相比，克氏锥虫感染会使巨噬细胞中炎性体相关基因表达及白细胞介素1β（IL-1β）的产生呈现出受抑制且延迟的激活状态。当用半胱天冬酶1（caspase-1）、白细胞介素1β或烟酰胺腺嘌呤二核苷酸磷酸氧化酶（NADPH oxidase）的抑制剂处理野生型与ASC−/−巨噬细胞时，研究发现半胱天冬酶1/ASC炎性体产生白细胞介素1β需要活性氧（reactive oxygen species, ROS）作为次级信号。此外，白细胞介素1β可调控炎性细胞因子基因表达的核因子κB（NF-κB）信号通路，进而影响巨噬细胞内的寄生虫复制。尽管NLRP3−/−巨噬细胞无法激活白细胞介素1β并上调炎性细胞因子基因表达，但其胞内寄生虫数量较配对的野生型对照组下降了4倍。NLRP3−/−巨噬细胞并非对克氏锥虫不易感，反而展现出远高于野生型对照组的基础活性氧水平（较野生型高100倍以上），且该水平在感染后仍以不依赖白细胞介素1β的方式维持，这有助于高效杀伤寄生虫。综上，本研究证实：半胱天冬酶1/ASC炎性体在人类与小鼠巨噬细胞中，通过激活白细胞介素1β/活性氧及核因子κB信号通路调控细胞因子基因表达，从而参与克氏锥虫的宿主控制过程。然而，NLRP3介导的白细胞介素1β/核因子κB激活并非必需，其功能可由活性氧介导的巨噬细胞内克氏锥虫复制与存活调控所代偿。