Production of IL-1β and Inflammasome with Up-Regulated Expressions of NOD-Like Receptor Related Genes in Toxoplasma gondii-Infected THP-1 Macrophages

Abstract

Toxoplasma gondii is an obligate intracellular parasite that stimulates production of high levels of proinflammatory cytokines, which are important for innate immunity. NLRs, i.e., nucleotide-binding oligomerization domain (NOD)-like receptors, play a crucial role as innate immune sensors and form multiprotein complexes called inflammasomes, which mediate caspase-1-dependent processing of pro-IL-1β. To elucidate the role of inflammasome components in T. gondii-infected THP-1 macrophages, we examined inflammasome-related gene expression and mechanisms of inflammasome-regulated cytokine IL-1β secretion. The results revealed a significant upregulation of IL-1β after T. gondii infection. T. gondii infection also upregulated the expression of inflammasome sensors, including NLRP1, NLRP3, NLRC4, NLRP6, NLRP8, NLRP13, AIM2, and NAIP, in a time-dependent manner. The infection also upregulated inflammasome adaptor protein ASC and caspase-1 mRNA levels. From this study, we newly found that T. gondii infection regulates NLRC4, NLRP6, NLRP8, NLRP13, AIM2, and neuronal apoptosis inhibitor protein (NAIP) gene expressions in THP-1 macrophages and that the role of the inflammasome-related genes may be critical for mediating the innate immune responses to T. gondii infection.

Keywords: THP-1 macrophage; Toxoplasma gondii; inflammasome; nucleotide-binding oligomerization domain (NOD)-like receptor (NLR); qRT-PCR.

Fig. 1

T. gondii triggers the expression and secretion of mature IL-1β. (A, B) THP-1 macrophages were mock-infected or infected with RH strain of T. gondii at an MOI of 10 for 4 hr and 8 hr. IL-1β expression levels were quantified by western blotting (A) and qRT-PCR (B). (C) Culture supernatants were collected, and IL-1β levels were measured in the supernatants by ELISA. Results represent the means ± SD from a representative of 3 independent experiments (*P<0.05 compared with the control).

Fig. 2

T. gondii infection induces caspase-1 activation and elevates NLRP1, NLRP3, NLRC4, and AIM2. (A) THP-1 macrophages were mock-infected or infected with RH strain of T. gondii using different MOIs for 8 hr. Cells were collected, and caspase-1 expression levels were quantified by western blotting. The α-tubulin was used as a loading control. (B) THP-1 macrophages were mock-infected or infected with RH strain of T. gondii at an MOI of 10. Cells were harvested at 4 hr or 8 hr post infection, RNA was extracted, and NLRP1, NLRP3, NLRC4, and AIM2 expressions were determined by qRT-PCR. HPRT1 was used as an internal control. (C) THP-1 macrophages were mock-infected or infected with RH strain of T. gondii at an MOI of 10 for 4 hr or 8 hr. NLRP3 and NLRC4 protein levels in the cells were quantified by western blotting. The α-tubulin was used as a loading control. A representative of 3 independent replicates with similar results is shown (*P<0.05, compared with the control).

Fig. 3

T. gondii upregulates ASC mRNA and protein levels and caspase-1 mRNA levels. (A) THP-1 macrophages were mock-infected or infected with RH strain of T. gondii at an MOI of 10 for 4 hr or 8 hr. ASC protein levels in the cells were quantified by western blotting. The α-tubulin was used as a loading control. (B) THP-1 macrophages were mock-infected or infected with RH strain of T. gondii at an MOI of 10. Cells were harvested at 4 hr or 8 hr post infection, RNA was extracted, and ASC and caspase-1 gene expression was determined by qRT-PCR. HPRT1 was used as an internal control. A representative of 3 independent replicates with similar results is shown (*P<0.05 compared with the control).

Fig. 4

T. gondii infection regulates a subset of NLRP genes and NAIP. THP-1 macrophages were mock-infected or infected with RH strain of T. gondii at an MOI of 10. Cells were harvested at 4 hr or 8 hr post infection, RNA was extracted, and the expression of various genes was determined by qRT-PCR. HPRT1 was used as an internal control. A representative of 3 independent replicates with similar results is shown (*P<0.05, compared with the control).