NALP1 influences susceptibility to human congenital toxoplasmosis, proinflammatory cytokine response, and fate of Toxoplasma gondii-infected monocytic cells

Abstract

NALP1 is a member of the NOD-like receptor (NLR) family of proteins that form inflammasomes. Upon cellular infection or stress, inflammasomes are activated, triggering maturation of proinflammatory cytokines and downstream cellular signaling mediated through the MyD88 adaptor. Toxoplasma gondii is an obligate intracellular parasite that stimulates production of high levels of proinflammatory cytokines that are important in innate immunity. In this study, susceptibility alleles for human congenital toxoplasmosis were identified in the NALP1 gene. To investigate the role of the NALP1 inflammasome during infection with T. gondii, we genetically engineered a human monocytic cell line for NALP1 gene knockdown by RNA interference. NALP1 silencing attenuated progression of T. gondii infection, with accelerated host cell death and eventual cell disintegration. In line with this observation, upregulation of the proinflammatory cytokines interleukin-1β (IL-1β), IL-18, and IL-12 upon T. gondii infection was not observed in monocytic cells with NALP1 knockdown. These findings suggest that the NALP1 inflammasome is critical for mediating innate immune responses to T. gondii infection and pathogenesis. Although there have been recent advances in understanding the potent activity of inflammasomes in directing innate immune responses to disease, this is the first report, to our knowledge, on the crucial role of the NALP1 inflammasome in the pathogenesis of T. gondii infections in humans.

FIG. 1.

Analysis of NALP1 SNPs. The upper diagram shows the positions of genotyped SNPs relative to the intron-exon structure of the gene. The lower diagram shows the LD plots generated in Haploview, using NALP1 gene data from our North American patient cohort. LD values (D′ × 100) between markers are indicated at the intercept of the 2 markers on the matrix. Where there is no value, D′ = 1 (i.e., 100). In outlining gene SNP associations with susceptibility to congenital toxoplasmosis, D′ values between loci were calculated and displayed using Haploview. Where a high confidence for the value of D′ existed (LOD score [decimal logarithm of the odds of linkage between two loci] of ≥2), shades of pink and bright red were used. If the confidence was low (LOD score of <2), blue shading (D′ = 1) or no shading (D′ < 1) was used.

FIG. 2.

Analysis of the effect of NALP1 and TetR shRNAs on expression of NALP1. (A) Illustration of the promoter features of the pLenti4/Block-iT-DEST shRNA expression vector. In the absence of a Tet repressor protein, there is constitutive expression of the cloned NALP1 or TetR shRNA fragment. (B) Quantitative real-time PCR analysis of NALP1 transcript levels in wild-type MonoMac6 cells (MC) and MonoMac6 cells engineered to express either NALP1 gene shRNA (N-KO) or tetracycline repressor gene shRNA (TetRep) after 72 h of culture. The relative transcript levels of NALP1 were derived by dividing the obtained NALP1 value by the GAPDH value for each respective sample. The data shown are means for three independent experiments with standard error bars. The reduction in NALP1 transcript levels in N-KO cells was found to be significant (**, P < 0.001) compared to both the wild-type and TetR shRNA-expressing cells. (C) Western blotting of NALP1 protein expression in N-KO, TetRep, and MC cells was performed on cell lysates after 72 h of culture, using anti-NALP1 antibody. A similar blot was hybridized with anti-GAPDH primary antibody to check for loading. The NALP1 protein was detectable as an ∼155-kDa band.

FIG. 3.

Effect of NALP1 knockdown on viability of human monocytic cells during infection with T. gondii (A), rate of host cell infection (B), and rate of parasite multiplication within a parasitophorous vacuole in an infected cell (C). Wild-type MonoMac6 cells (solid lines with circles) and those genetically modified for stable knockdown of NALP1 (dotted lines with squares) or tetracycline repressor (dashed lines with rhombuses) gene transcription were cultured with or without parasites. The relative cell viability values were derived by dividing the absorbance obtained from the infected cells by that of the uninfected cells for each respective cell line, while the percent infected cells per field was derived as an average percentage of infected cells for 50 microscopic fields. The mean number of parasites per vacuole was determined as the average number of parasites in one parasitophorous vacuole for 200 infected cells. The data shown represent means for three independent experiments with standard error bars, and levels of statistical significance are depicted by asterisks (*, P < 0.05; **, P < 0.001).

FIG. 4.

Giemsa-stained preparations of monocytic cells examined by light microscopy to determine the effect of NALP1 gene knockdown on monocytic cell viability when cells were cultured with or without parasites. Preparations of monocytic cells genetically modified for stable knockdown of NALP1 (NALP1 KO) or tetracycline repressor (TetRep KO) gene transcription and of wild-type cells are shown at different days postinfection (DPI). Images are representative of three independent experiments, with red arrowheads depicting parasite-laden cells.

FIG. 5.

Analysis of effects of NALP1 gene knockdown on expression of proinflammatory cytokines (IL-1β, IL-18, TNF-α, and IL-12) in a human monocytic cell line (MonoMac6). Quantitative real-time PCR was performed on cDNAs synthesized using equal amounts of total RNA from wild-type MonoMac6 cells (MC) and MonoMac6 cells engineered to express either NALP1 gene shRNA (NALP1-KO) or tetracycline repressor gene shRNA (TetR) that had been cultured with (gray columns) or without (white columns) parasites for 36 h. The transcript values for IL-1β (A), IL-18 (B), TNF-α (C), and IL-12 (D) were normalized by dividing the observed value by the value for GAPDH for each respective sample to derive the relative gene transcript level. The significance of augmentation of cytokine expression attributed to infection is shown by asterisks (*, P < 0.05; **, P < 0.001). The data are shown as means for three independent experiments with standard error bars.

FIG. 6.

Analysis of effects of NALP1 gene knockdown on expression of caspase-1 and BCL2A1 in MonoMac6 cells infected with T. gondii. Quantitative real-time PCR was performed on cDNAs synthesized from equal amounts of total RNA from wild-type MonoMac6 cells (MC) and MonoMac6 cells engineered to express either NALP1 gene shRNA (NALP1-KO) or tetracycline repressor gene shRNA (TetR) that had been cultured with (gray columns) or without (white columns) parasites for 36 h. The obtained transcript values for caspase-1 (A) and BCL2A1 (B) were normalized by dividing the observed value by the value for GAPDH for each respective sample to derive the relative gene transcript level. The significance of the levels of augmentation of caspase-1 and BCL2A1 expression attributed to infection is shown by asterisks (**, P < 0.001). The data are shown as means for three independent experiments with standard error bars.

FIG. 7.

Western blot of the effect of NALP1 knockdown on the expression of IL-1β in MonoMac6 cells with or without T. gondii infection for 36 h. Wt, wild-type MonoMac6 cells; Tet, MonoMac6 cells engineered to express tetracycline repressor gene shRNA; KO, MonoMac6 cells expressing NALP1 gene shRNA. The pro-IL-1β protein was detectable as an ∼35-kDa band, while mature IL-1β was detectable as an ∼17-kDa band. The GAPDH protein band is shown as a loading check.