Imbalance in the immune response in CCR5^-/- mice infected with <i>T. gondii</i>.

<p>Expression of mRNA for the cytokines IL-12p40, IFN-γ, TNF, IL-1β, IL-17, IL-6 and IL-4 (A), transcription factors Foxp3, ROR-γt, GATA-3 and T-bet (B), chemokines CCL3, CCL4, CCL5 (C) and the enzyme iNOS (D) were determined by quantitative real time PCR (qPCR) of liver and ileum tissues collected on day 8 pi from WT and CCR5^-/- mice orally infected with 5 cysts of <i>T. gondii</i>. Data were normalized to GAPDH and Ct values were analyzed by 2^-ΔΔCt method, as shown in Materials and Methods. The data represent the mean ± SEM of results from three to five mice per group and are representative of two independent experiments. NI: non-infected. * p<0.05.</p

Chemokine and chemokine receptors expression after infection and CCR5^-/- mice susceptibility to <i>Toxoplasma gondii</i>.

<p>C57BL/6 (WT) mice were infected with 5 cysts of ME-49 strain of <i>T. gondii</i>. At day 8 pi, ileum and liver RNA were extracted and levels of CCR1, CCR2, CCR3, CCR4, CCR5, CXCR3, CCL3, CCL4 and CCL5 mRNA (A) were determined by real-time quantitative PCR (qPCR). WT and CCR5^-/- mice were infected and evaluated for survival until day 30 pi (B). At day 8 pi, small intestine and liver (C) were harvested, total DNA extracted and the tissue parasitism determined by qPCR based on curve with <i>T. gondii</i> DNA. * p<0.05 compared to WT naïve mice. Data are presented as mean ± SEM of three to seven mice per group and are a representative of at least two independent experiments.</p

<i>Toxoplasma gondii</i> leads to inflammation, cellular infiltrate and ileum necrosis in the absence of CCR5.

<p>WT and CCR5^-/- mice were infected with 5 cysts of <i>T. gondii</i> and at day 8 pi, small intestine was harvested, formalin-fixed, paraffin-embedded, stained with Hematoxylin and Eosin (H&E) and analyzed by light microscopy (A). The leukocytes were isolated from lamina propria of small intestine of WT and CCR5^-/- mice at day 8 after <i>T. gondii</i> infection and characterized by flow cytometry. The percentage of CD3⁺CD4⁺ T (B), CD3⁺CD8⁺ T (C), CD3⁺NK1.1⁺ (D) and CD3^-NK1.1⁺ (E) cells was evaluated by FlowJo software. Data represent the mean ± SEM of results from three to five mice per group and are representative of two independent experiments. NI: non-infected. * p<0.05 compared to WT mice.</p

Lipid alterations and liver damage in CCR5^-/- infected mice.

<p>WT and CCR5^-/- mice were infected with 5 cysts of <i>T. gondii</i> and at day 8 pi the liver was harvested, photographed (A), weighed (B) and formalin-fixed for Hematoxylin and Eosin (H&E) staining (D, upper pannel) or frozen in OCT medium for Oil Red O staining (D lower pannel) and analysis by light microscopy. Lipid quantification was performed using specific software and expressed as percentage of area (E). Blood was collected and processed to serum triglycerides quantification (C), alanine aminotransferase-ALT (F) and aspartate aminotransferase-AST (G). The liver was also harvested in Trizol reagent, homogenized and total RNA extracted. Expression of PPARα (H) and PPARγ (I) was determined by quantitative real time PCR (qPCR). The data represent the mean ± SEM of results from three to five mice per group and are representative of two independent experiments. * p<0.05 compared to WT mice. NI: non-infected. PS: portal space, CV: centrolobular veins. Arrows: hepatocyte vacuolization. Scale bar: 100 µm.</p

Primer sequences used in qPCR.

<p>Primer sequences used in qPCR.</p

PPARα agonist partially restores liver damage but does not rescue CCR5^-/- infected mice from mortality.

<p>WT and CCR5^-/- mice were infected with 5 cysts of <i>T. gondii</i> and treated with Gemfibrozil, 100 mg/Kg/day, during 7 days. At day 8 pi, livers were harvested, formalin-fixed, paraffin-embedded, stained with Hematoxylin and Eosin (H&E) and analyzed by light microscopy (A). Blood was collected and processed to serum analysis of triglycerides (B). Survival was accompanied until day 40 pi and treatment. The results were obtained with three to five animals per group and are representative of two independent experiments. GEM: Gemfibrozil; VEH: vehicle. * p<0.05 compared to WT mice. Scale bar: 100 µm.</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

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

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

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