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. 2014 Feb 18;5(1):e01117-13.
doi: 10.1128/mBio.01117-13.

Dual Role for Inflammasome Sensors NLRP1 and NLRP3 in Murine Resistance to Toxoplasma Gondii

Free PMC article

Dual Role for Inflammasome Sensors NLRP1 and NLRP3 in Murine Resistance to Toxoplasma Gondii

Gezahegn Gorfu et al. mBio. .
Free PMC article

Abstract

Induction of immunity that limits Toxoplasma gondii infection in mice is critically dependent on the activation of the innate immune response. In this study, we investigated the role of cytoplasmic nucleotide-binding domain and leucine-rich repeat containing a pyrin domain (NLRP) inflammasome sensors during acute toxoplasmosis in mice. We show that in vitro Toxoplasma infection of murine bone marrow-derived macrophages activates the NLRP3 inflammasome, resulting in the rapid production and cleavage of interleukin-1β (IL-1β), with no measurable cleavage of IL-18 and no pyroptosis. Paradoxically, Toxoplasma-infected mice produced large quantities of IL-18 but had no measurable IL-1β in their serum. Infection of mice deficient in NLRP3, caspase-1/11, IL-1R, or the inflammasome adaptor protein ASC led to decreased levels of circulating IL-18, increased parasite replication, and death. Interestingly, mice deficient in NLRP1 also displayed increased parasite loads and acute mortality. Using mice deficient in IL-18 and IL-18R, we show that this cytokine plays an important role in limiting parasite replication to promote murine survival. Our findings reveal T. gondii as a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis.

Importance: Inflammasomes are multiprotein complexes that are a major component of the innate immune system. They contain "sensor" proteins that are responsible for detecting various microbial and environmental danger signals and function by activating caspase-1, an enzyme that mediates cleavage and release of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Toxoplasma gondii is a highly successful protozoan parasite capable of infecting a wide range of host species that have variable levels of resistance. We report here that T. gondii is a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis. Using mice deficient in IL-18 and IL-18R, we show that the IL-18 cytokine plays a pivotal role by limiting parasite replication to promote murine survival.

Figures

FIG 1
FIG 1
Toxoplasma activates the inflammasome in C57BL/6 and 129S BMDMs. BMDMs were primed with 100 ng/ml LPS or left unstimulated for 2 h and subsequently infected with type II parasites (Pru; average MOI, 1) for 24 h. (A) Quantification of IL-1β in supernatants was performed using ELISA. (B) IL-1β cleavage was monitored by Western blotting of cell lysates from C57BL/6NTac or caspase-1/11−/− BMDMs that were infected with type II parasites (Pru; MOI, 0.8) for 24 h. The positions of both pro-IL-1β (37 kDa) and cleaved IL-1β (17 kDa) are indicated. (C) Cell viability of infected cells in panel A was determined at different time points using an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Panels A and C are averages of three experiments. Error bars, ±standard deviations. (D) BMDMs were primed with LPS for 2 h and infected for 24 h with type I parasites (RH) that were pretreated with dimethyl sulfoxide vehicle or mycalolide B (3 µM) for 20 min. IL-1β was measured using ELISA. Data are averages of 2 experiments. Error bars, +standard deviations. (E) C57BL/6 BMDMs were infected with the indicated strains for 24 h. IL-1β was measured using ELISA. Data are the averages of at least 3 experiments per strain. The haplogroup to which the strain belongs is indicated above. Error bars, +standard deviations. (F) C57BL/6 BMDMs were infected for 18 h with Pru (type II) or PruΔGRA15 (MOI, 4) for 18 h, and microarrays were used to determine the fold change in IL-1β mRNA expression levels compared to uninfected macrophages. (G) BMDMs were infected with Pru (type II) or PruΔGRA15 for 24 h. IL-1β was measured using ELISA. Data are representative of 3 experiments. Error bars, +standard deviations. (H) BMDMs were primed for 2 h with LPS and then infected with indicated strains for 24 h (MOI, 4). The haplogroup to which the strain belongs is indicated above. Data are the averages of 3 experiments. Error bars, +standard deviations. (I) IL-1β secretion from primed immortalized murine WT and caspase-1/11−/− macrophages, infected for 24 h with RH. Data shown are from an experiment representative of three. Error bars, +standard deviations. (J) IL-1β secretion from BMDMs prepared from wild-type C57BL/6 mice (blue) or C57BL/6 mice lacking Nlrp1b (red), Nlrp3 (green), Nlrp1b and Nlrp3 (orange), or Asc (yellow), primed for 4 h, and then infected with type I (RH; average MOI, 1) for 24 h. Cytokine secretion below the detection level is indicated on the graph with arrowheads and labeled not detected (n.d.). Data are averages of four experiments. Error bars, +standard deviations. (K) BMDMs described for panel J were primed for 3 h with LPS and then infected with type II parasites (MOI, 1.5) for 24 h. Western blot analysis on concentrated supernatants (25-fold), probing for cleaved IL-1β (17 kDa). (L) Host cell viability in panel L was measured using the MTS assay. Error bars, +standard deviations. (M) BMDMs from C57BL/6 and 129S mice were primed with 100 ng/ml LPS for 2 h and subsequently infected with type II parasites (Pru; average MOI, 0.7) for 24 h. Quantification of IL-1β in supernatants was performed using ELISA. Panels L and M are the averages of 3 experiments. (N) 129S BMDMs were primed for 2 h and infected with type II parasites (MOI, 1.6) for 24 h. Western blot analysis on concentrated supernatants (25-fold), probing for cleaved IL-1β (17 kDa). Nig, nigericin; Tg, T. gondii.
FIG 2
FIG 2
Parasite load, survival, and systemic IL-18 levels in caspase-1/11-deficient mice. (A) Bioluminescence imaging (BLI) of infected caspase-1/11 knockouts and controls on days 5 to 7 following infection with 76K GFP-LUC (10,000 tachyzoites i.p.). Images shown are for 3 mice. (B) Quantifications are from 8 mice imaged/group. (C) Aggregate survival of caspase-1/11-knockout mice (n = 13/group) compared with WT C57BL/6NTac control mice (n = 10/group). (D) IL-18 measurements in serum of caspase-1/11-deficient mice on day 5 after infection were significantly different from WT (P < 0.001) when infected with 76K GFP-LUC. No detectable levels of IL-1β were detected in circulation.
FIG 3
FIG 3
Parasite load, survival, and systemic IL-18 levels in ASC- and NLRP3-knockout mice following Toxoplasma challenge. (A) Bioluminescence imaging of 76K GFP-LUC-infected mice (various strains, 10,000 tachyzoites, i.p. route) on days 5 to 7 following infection is shown. Images shown are from two or three representative mice from 2 to 6 mice/group from one representative experiment. The experiment shown is one of 3 (WT) or 2 (ASC and NLRP3) independent experiments. (B) P values (t test) comparing luciferase activity for each knockout strain to C57BL/6J are <0.05. (C) Aggregate survival curve of ASC (n = 8)- and NLRP3 (n = 5)-knockout mice compared with WT C57BL/6J control mice (n = 13). (D) IL-18 measurements in serum of deficient mice on day 5 after infection. No detectable levels of IL-1β were detected in circulation.
FIG 4
FIG 4
Parasite load, survival, and systemic IL-18 levels in NLRP1-knockout mice following Toxoplasma challenge. (A) Bioluminescence imaging of 76K GFP-LUC-infected mice (various strains, 10,000 tachyzoites, i.p. route) on days 5 to 7 following infection is shown. Images shown are three representative mice from 4 to 6 mice/group from one representative experiment. The experiment shown is one of 2 WT or 2 NLRP1 independent experiments. (B) P values (t test) comparing luciferase activity for each knockout strain to C57BL/6NTac are <0.05. (C) Aggregate survival curve of NLRP1 (n = 10)-knockout mice compared with WT C57BL/6NTac control mice (n = 10). (D) IL-18 measurements in serum of deficient mice on day 5 after infection. No detectable levels of IL-1β were detected in circulation.
FIG 5
FIG 5
Parasite load, survival, and systemic IL-18 levels in IL-18- and IL-18R-knockout mice. (A) Bioluminescence imaging of 76K GFP-LUC-infected mice (various strains, 10,000 tachyzoites, i.p. route) on days 4 to 7 following infection is shown. Images shown are 2 to 3 representative mice from 3 to 6 mice/group. The experiment shown is one of 2 IL-1β, 4 IL-18, or 4 IL-18R independent experiments. (B) P values (t test) comparing luciferase activity for each knockout strain to C57BL/6J are <0.05. (C) Aggregate survival curve of IL-1R (n = 9)-, IL-18 (n = 19)-, and IL-18R (n = 13)-knockout mice compared with WT C57BL/6J control mice (n = 13). (D) IL-18 measurements in serum of deficient mice on day 5 after infection. No detectable levels of IL-1β were detected in circulation.

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