Malaria-induced NLRP12/NLRP3-dependent caspase-1 activation mediates IL-1β and hypersensitivity to bacterial superinfection.

<p>Step 1 –Phagocytes internalize <i>Plasmodium</i> DNA bound to hemozoin that activates TLR9 and the adaptor molecule named MyD88. <b>Step 2 –</b> MyD88 signaling triggers the expression of IL-12, which will initiate the production of IFN-γ by T lymphocytes and NK cells. <b>Step 3 –</b> Low levels of caspase-1-independent IL-1β induced by malaria infection. <b>Step 4 –</b> IFN-γ priming and MyD88 signaling in phagocytes will lead to enhanced expression of pro-caspase-1. K⁺ efflux as well as rupture (by hemozoin crystals) and release of lysosome contents will induce the assembly of ASC, NLRP3 and NLR12 inflammasomes and promote cleavage of pro-caspase-1. <b>Step 5 -</b> Bacterial superinfection triggers expression of high pro-IL-1β levels, in a TNF-α-dependent manner. Pro-IL-1β will be cleaved by active caspase-1 generated on <b>step 4</b>. Upon secondary bacterial infection, the malaria-primed macrophages will release deleterious amounts of IL-1β.</p

NLRP3/NLRP12 containing inflammasomes and caspase-1 activation in PBMCs from <i>P. vivax</i> malaria patients.

<p>(<b>A</b>) PBMCs derived from <i>P. vivax</i> malaria patients and healthy donors were lysed, cross-linked by treatment with disuccinimidyl suberate <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003885#ppat.1003885-Moulds1" target="_blank">[70]</a>, and ASC oligomerization assessed by Western blot analysis. PBMCs from a healthy donor stimulated with LPS and nigericin were used as positive control. . (<b>B</b>) NLRP3, NLRP12 and AIM2 containing inflammasomes (specks) in monocytes from <i>P. vivax</i> malaria patients were visualized in a confocal microscope. (<b>C</b>) The bar graphs show the frequency of specks in monocytes derived from <i>P. vivax</i> malaria patients. We saw no specks on cells from healthy donors or cells from malaria patients incubated with the secondary antibody only. See also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003885#ppat.1003885.s006" target="_blank">Figure S6</a></b>.</p

NLRP3/NLRP12-dependent activation of caspase-1 and pyroptosis in mice infected with <i>P. chabaudi</i>.

<p>(<b>A</b>) At 9 days post-infection, splenocytes from C57BL/6, and P2X7R^−/− mice were lysed and analyzed by Western blot employing caspase-1-specific antibody. (<b>B</b>) At 7 days post-infection, active caspase-1 by FLICA reagent, membrane integrity by propidium iodide, and cell size change by shift on FSC axis were assessed in splenic macrophages (CD11b⁺F4/80⁺) and DCs (CD11c⁺MHC-II⁺). (<b>C</b>) Splenocytes lysates from mice at 7 days post-infection were used to detected active caspase-1 by Western blot. A faint band of similar molecular weight of active caspase-1 that corresponds to IgG light chain is seen in the uninfected controls or in various infected knockout mice. The results presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003885#ppat-1003885-g003" target="_blank">figures 3A, 3B and 3C</a> are representative of 2 experiments that yield similar results. (<b>D</b>) A LPS dose of 10 µg/mouse was given intravenously at 7 days post-infection with <i>P.chabaudi</i> and sera collected 9 hours later, for measuring IL-1β and TNF-α levels. The numbers within parenthesis indicate the percentage of lethality 48 hours after LPS challenge (10 µg/mouse). The levels of IL-1β measured in the sera of infected C57BL/6, NLRP3^−/−, NLRP12, ASC^−/− and Casp-1^−/− were not different. The results are means + SEM of 10 mice from 2 independent experiments. Significant differences are indicated by **<i>p<0.001</i> obtained in the Mann-Whitney test.</p

Treatment with IL-1RA prevents lethality in mice infected with <i>P. chabaudi</i> and challenged with a secondary bacterial infection.

<p>(<b>A</b>) At 7 days post-infection, mice were challenged with 10 µg of LPS and serum samples collected 9 hours later for cytokine measurements. The numbers within parenthesis indicate the percentage of lethality 24 hours after low dose (10 µg/mouse) LPS challenge. (<b>B</b>) Splenic macrophages (CD11b⁺F4/80⁺) and DCs (CD11c⁺MHC-II⁺) from mice at 7 days post-infection were stained with FLICA reagent in order to detect active caspase-1. (<b>C</b>) At day 7 post-infection the mice were treated with IL-1RA (anakinra) immediately prior to LPS challenge. Lethality was assessed from 12 to 48 hours post-LPS challenge. (<b>D</b>) At 7 days post-infection with <i>P. chabaudi</i>, sub-lethal sepsis was induced by CLP. A group of mice received treatment with IL-1RA (100 mg/kg/day) beginning 24 hours before the CLP procedure. Levels of circulating IL-1β were measured 24 hs after CLP. (<b>E</b>) Mice received peroral challenge with 10⁸ of <i>Salmonella typhimurium</i> at 5 days post-infection with <i>P. chabaudi.</i> A group of mice received treatment with IL-1RA (100 mg/kg/day) beginning 48 hours after bacterial challenge. The levels of circulating IL-1β were measured at 3 days post-<i>Salmonella</i> challenge. (<b>F</b>) Translocation of aerobic bacteria was quantified 24 hours after the CLP procedure. (<b>G</b>) Translocation of <i>S. typhimurium</i> was quantified 3 days after peroral challenge. We used 5 to 8 mice per group and results shown are representative of 2 independent experiments. Significant differences are *<i>p<0.01</i>, <i>**p<0.005 ***p<0.001</i> obtained in a Chi-square test.</p

Association of circulating IL-1β levels and high susceptibility to endotoxic shock in mice primed by infection with <i>P.chabaudi</i>.

<p>IL-1β release and high susceptibility to endotoxic shock. Mice were infected with 10⁵ parasitized red blood cells (i.p). At 7 days post-infection, a low dose LPS (10 µg/mouse) was inoculated and lethality evaluated 24 hours later. Parasitemia was defined by smears giemsa- stained. FLICA reagent was used to assess active caspase-1 in total splenocytes from mice at 7 days post-infection. The data were collected by flow cytometry and median fluorescence intensity (MFI) analyses performed using Flowjo software. The range of detection of circulating IL-1β was 15.6–1000 pg/ml and determined using and ELISA Duoset kit from R&D Systems.</p

Monocytes are the major source of active caspase-1 during malaria.

<p>PBMCs were obtained from either <i>P. vivax</i> or <i>P. falciparum</i> malaria patients as well as from healthy donors. (<b>A</b>) The gate was set based on monocytes profile in PBMCs from <i>P. vivax</i> infected patients. PBMCs were stained with anti-CD14 and anti-CD16 antibodies to determine the presence of different monocyte subsets. These cells were gated based on FSC and SSC to avoid neutrophil contamination. When the CD14^dimCD16⁺ gate was moved down in PBMCs from healthy donors or from malaria patients after treatment, we did not detect any active caspase-1. The bar graphs show the flow cytometry analysis of PBMCs from five <i>P. vivax</i> infected subjects before and after malaria treatment primaquine and chloroquine, as well as eight healthy donors. To determine the median fluorescence intensity (MFI) and frequency of (CD14⁺CD16⁻) and (CD14^dimCD16⁺) that are active caspase-1, we used the FLICA assay. (<b>B – top panel</b>) Active caspase-1 (p10) was detected in lysates of PBMCs from <i>P. vivax</i> or (<b>C – top panel</b>) <i>P. falciparum</i> infected individuals by Western blot. (<b>B and C – bottom panel</b>) PBMCs were stimulated with LPS (100 ng/ml) for 24 hours, and levels of IL-1β assessed in the culture supernatants by ELISA. Significant differences are *<i>p</i><<i>0.05</i> and **<i>p<0.005</i> as indicated by the unpaired <i>t</i> test with Welch correction or Mann-Whitney test when a normality test failed.</p

Caspase-1 activation, IL-1β production and pyroptosis in splenic macrophages and DCs from <i>P. chabaudi</i> infected mice.

<p>(<b>A</b>) Gene expression was determined in splenocytes of 3 C57BL/6 or MyD88^−/− mice at 6 days post-infection over 3 non-infected controls by Microarray analysis. (<b>B and F</b>) Splenocytes from C57BL/6, ASC^−/−, Casp-1^−/− or MyD88^−/− mice were stained and analyzed by FACs to gate macrophages (CD11b⁺F4/80⁺) and DCs (CD11c⁺MHC-II⁺). Active caspase-1 was evaluated by FLICA reagent, membrane integrity by nuclei staining with 7AAD, and cell size change by shift on FSC axis. The results are representative from 3 experiments that yield similar results. (<b>C</b>) On day 7 post-infection splenocytes from C57BL/6, ASC^−/−, Casp-1^−/− and MyD88^−/− mice were lysed by RIPA buffer and analyzed by Western blot employing an anti-caspase-1 antibody. A faint band of similar molecular weight of active caspase-1 corresponds to IgG light chain is seen in the infected ASC^−/− and Casp-1^−/− mice. (<b>D and G</b>) At 7 days post-infection mice were inoculated intravenously with 10 µg of LPS per mouse, and 9 hours later, sera was collected for measuring the levels of circulating IL-1β. The average levels of IL-1β in control and infected mice, before LPS challenge, were 64.2 and 434.2 pg/ml in figure D, and 82.2 and 602.4 pg/ml in figure G. These results are the means + SEM of 10–15 animals from 3 independent experiments that yield similar results. (<b>E</b>) CD11c⁺ and CD11b⁺ cells highly purified from spleens of C57BL/6 mice at day post-infection were cultured with LPS (1 µg/ml) and supernatants harvested 18 h later to measure the levels of IL-1β. As positive control we used the purified cells stimulated with LPS at same concentration followed by nigericin at 5 µM. Significant differences are indicated by *<i>p</i><0.01, **<i>p<0.001</i> and ***<i>p<0.0005</i> obtained in the Mann-Whitney test.</p