NLRP1-variant dependent macrophage death depends on parasite invasion and controls parasite proliferation.

<p>(A) Viability of LEW BMDMs infected with Mycalolide-treated (3 µM, 15 min) RH tachyzoites (MOI 1∶1) after 24 h as measured by MTS assay (P-value comparing Mycalolide group to untreated = 0.0002). (B, C) Radiance emission analyses of metabolically active, viable Type II <i>Toxoplasma</i> 76K parasites (B, graph MOI 3∶1, 6 h; inset shows representative plate from one experiment) or Type I RH parasites (C, MOI 1∶1 over 48 h) in BMDMs from different rat strains. P-value comparing NLRP1^{variant 1,2} expressing strains to NLRP1^{variant 5} expressing strains are <0.01 in I by t-test and <0.0001 in J by two-way ANOVA. (D) Number of parasites/vacuole in infected BMDMS (24 h, 3∶1 MOI) as assessed by microscopy is shown. CDF, BN infections were with 76K, and SD, LEW infections were with RH. Between 50–100 vacuoles counted per experiment. Average values from 3 experiments are shown for all strains, except SD (n = 2). P-values are <0.01 (two-way ANOVA) when comparing NLRP1^{variant 1, 2} expressing strains to NLRP1^{variant 5} expressing strains. (E) Left panels show light microscopy images of CDF and LEW monolayers infected with 76K (MOI 6∶1, 6 h). Right panels show fluorescence microscopy image of single SD and LEW BMDMs infected with RH (MOI 1∶1, 2 h). Blue is Hoechst stained nucleus, green are GFP-expressing parasites. Dividing parasites in SD cells (upper right) or a single parasite in LEW cells (lower right) are shown. (F) LEW BMDMs were infected with PRU (MOI 3;1) and at 5 h post infection culture supernatants from dying cells was spun, filtered and transferred to similarly infected (PRU, MOI 3∶1) CDF BMDMs. Viability of CDF BMDMs was assessed at 10 h post-infection by MTT staining. All values were calculated relative to uninfected control BMDMs (G) SD BMDMs were infected with RH parasites (2 h, MOI 1∶1), washed with PBS and medium replaced with fresh media, media from RH-infected (24 h, MOI 1∶1) or uninfected LEW BMDMs. Parasites/vacuole counted at 24 h. P-values >0.1 (ns) for comparison of any of three groups for 1, 2, 4 and 8 parasites/vacuole counts (by two-way ANOVA).