NLRP3 and caspase-1 are involved in the control of <i>T. cruzi</i> infection.

<p>WT, MyD88^−/−, NLRP3^−/−, caspase-1^−/−, IFN-γ^−/− and iNOS^−/− mice were subcutaneously infected with 10³<i>T. cruzi</i> blood trypomastigotes. Parasitemia was quantified by counting the parasites in 5 µL of tail blood obtained on days 4 to 20 after infection (<b>A</b>). Supernatants from cultured spleen cells (3×10⁶ cells/300 µl) isolated from naive (-) or infected (10 days) mice were collected after 24 h. The levels of IL-6 (<b>B</b>), IFN-γ (<b>C</b>) and IL-1β (<b>D</b>) were assessed by ELISA, and NO secretion was assessed by the Griess reaction (<b>E</b>). Numbers represent the mean ± S.D. (n = 5). *** p>0,001, ** p>0,01 and * p>0,05 compared to the WT group. Mortality was assessed by daily inspection of the cages (<b>F</b>). Experiments were repeated two times with IFN-γ^−/− and iNOS^−/− mice (n = 6) and five times with MyD88^−/−, NLRP3^−/−, caspase-1^−/− mice (n = 6) showing similar results.</p

Caspase-1 but not IL-1R mediates <i>T. cruzi</i>-induced NO secretion.

<p>PMs (3×10⁵/well of chamber slides) from WT, MyD88^−/− and NLRP3^−/− mice were infected with <i>T. cruzi</i> Y trypomastigotes (5 parasites/cell) for 2–4 h. After the extracellular parasites were removed, AG (1 mM) was added to the cultures. After 48 h, the chambers were fixed with methanol, stained with DAPI and evaluated by fluorescence microscopy (600×). The numbers represent the increase in the number of amastigotes compared to untreated cells, and 1000 macrophages were counted from a representative experiment (<b>A</b>). PMs (3×10⁵/well of chamber slides) from WT, caspase-1^−/− and IL-1R^−/− mice (3 parasites/cell) were infected with <i>T. cruzi</i> Y trypomastigotes (5 parasites/cell) for 2–4 h. Chambers were obtained as described and evaluated by fluorescence microscopy (600×). The number of amastigotes was counted in 1000 macrophages from a representative experiment. The experiments were repeated two times with similar results (<b>B</b>). <i>T. cruzi</i>-infected PMs (3 parasites per cell) (1,5×10⁶ cells/300 µl) from WT, caspase-1^−/−, iNOS^−/− and IL-1R^−/− mice were treated or not with z-YVAD-fmk (10 µM) or AG (1 mM). After 48 h, nitrite accumulation was measured in the culture supernatants by the Griess reaction. Bars represent the mean ± S.D. of triplicate samples. *** p>0,001 compared to the untreated group (<b>C</b>). <i>T. cruzi</i>-infected PMs (3×10⁵/well of chamber slides) from WT, caspase-1^−/−, iNOS^−/− and IL-1R^−/− mice (3 parasites/cell) were treated or not with z-YVAD-fmk (10 µM) or AG (1 mM). Chambers were obtained as described above and evaluated by fluorescence microscopy (600×). The frequency of infected macrophages was counted in 1000 macrophages from a representative experiment. Bars represent the rate of increase compared to untreated cells, where *** p>0,001 compared to the untreated group. The experiments were repeated two times with similar results (<b>D</b>).</p

<i>T. cruzi</i>-induced NLRP3 activation.

<p>PMs from WT, NLRP3^−/− and caspase-1^−/− mice (1,5×10⁶ cells/300 µl) were infected with <i>T. cruzi</i> Y trypomastigotes (3 parasites per cell). The supernatants were collected after 24 h for the evaluation of IL-1β levels by ELISA. Bars represent the mean ± S.D. of triplicate samples, where ** p>0,01 compared to the WT group (<b>A</b>). <i>T. cruzi</i>-infected PMs (3 parasites per cell) (1,5×10⁶ cells/300 µl) were treated with Ca-074ME, APO or GLB in doses of 25, 50 and 100 µM, or left untreated (-). After 24 h, the supernatants were collected and IL-1β production was evaluated by ELISA. Bars represent the mean ± S.D. of triplicate samples, where ***p>0,001 compared to the untreated group. The experiments were repeated three times, and representative results are shown (<b>B</b>).</p

NLRP3-mediated NO production acts synergistically with MyD88-mediated pathways to control <i>T. cruzi</i> infection.

<p>PMs from WT mice were infected with <i>T. cruzi</i> Y (3 parasites per cell) (1,5×10⁶ cells/300 µl) in the presence of neutralizing antibodies against IL-1β, IL-18 or both cytokines in doses of 0,1, 1 and 10 µg/mL, or left untreated. After 48 h, nitrite accumulation was measured in the culture supernatants by the Griess reaction. Bars represent the mean ± S.D. of triplicate samples where ** p>0,01 and * p>0,05 when compared to untreated group (<b>A</b>). <i>T. cruzi</i>-infected PMs from WT and MyD88^−/− mice (3 parasites/cell) were treated or not with z-YVAD-fmk (10 µM). After 48 h, nitrite accumulation was measured in culture supernatants by Griess Rection. Bars represent the mean ± S.D. of triplicate samples where *** p>0,001 and * p>0,01 when compared to untreated group (<b>B</b>). PMs (3×10⁵/well of chamber slides) from MyD88^−/− mice were infected with tripomastigotes from <i>T. cruzi</i> Y (5 parasites/cell) for 2–4 h. After removal of extracellular parasites, z-YVAD-fmk (10 µM) was added to cultures. After 48 h chambers were prepared and analyzed as described above to evaluate the numbers of amastigotes. Results were obtained in 1000 counted macrophages from a representative experiment Experiments were repeated three times with the same profile of results (<b>C</b>).</p

NLRP3 Controls <i>Trypanosoma cruzi</i> Infection through a Caspase-1-Dependent IL-1R-Independent NO Production

<div><p><i>Trypanosoma cruzi</i> (<i>T. cruzi</i>) is an intracellular protozoan parasite and the etiological agent of Chagas disease, a chronic infectious illness that affects millions of people worldwide. Although the role of TLR and Nod1 in the control of <i>T. cruzi</i> infection is well-established, the involvement of inflammasomes remains to be elucidated. Herein, we demonstrate for the first time that <i>T. cruzi</i> infection induces IL-1β production in an NLRP3- and caspase-1-dependent manner. Cathepsin B appears to be required for NLRP3 activation in response to infection with <i>T. cruzi</i>, as pharmacological inhibition of cathepsin B abrogates IL-1β secretion. NLRP3^−/− and caspase1^−/− mice exhibited high numbers of <i>T. cruzi</i> parasites, with a magnitude of peak parasitemia comparable to MyD88^−/− and iNOS^−/− mice (which are susceptible models for <i>T. cruzi</i> infection), indicating the involvement of NLRP3 inflammasome in the control of the acute phase of <i>T. cruzi</i> infection. Although the inflammatory cytokines IL-6 and IFN-γ were found in spleen cells from NLRP3^−/− and caspase1^−/− mice infected with <i>T. cruzi</i>, these mice exhibited severe defects in nitric oxide (NO) production and an impairment in macrophage-mediated parasite killing. Interestingly, neutralization of IL-1β and IL-18, and IL-1R genetic deficiency demonstrate that these cytokines have a minor effect on NO secretion and the capacity of macrophages to control <i>T. cruzi</i> infection. In contrast, inhibition of caspase-1 with z-YVAD-fmk abrogated NO production by WT and MyD88^−/− macrophages and rendered them as susceptible to <i>T. cruzi</i> infection as NLRP3^−/− and caspase-1^−/− macrophages. Taken together, our results demonstrate a role for the NLRP3 inflammasome in the control of <i>T. cruzi</i> infection and identify NLRP3-mediated, caspase-1-dependent and IL-1R-independent NO production as a novel effector mechanism for these innate receptors.</p></div

The proposed model for NLRP3-mediated control of <i>T. cruzi</i>.

<p>TLR2, TLR4, TLR7 and TLR9 recognize <i>T. cruzi</i> PAMPs and control infection by MyD88 adaptor molecule-mediated effector mechanisms. <i>T. cruzi</i> infection leads to lysosomal disruption, which may result in cathepsin B release into the cytosol and NLRP3 inflammasome activation. The activation of the NLRP3 inflammasome results in caspase-1 activation, which induces NO secretion in an IL-1R-independent manner. Caspase-1-dependent NO secretion appears to cooperate with MyD88-dependent pathways to control <i>T. cruzi</i> infection.</p

Phenotypic characterization of specific CD8⁺ T cells of infected and/or AdASP-2 immunized A/Sn mice.

<p>A/Sn mice were infected or immunized as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002699#ppat-1002699-g003" target="_blank">Figure 3A</a>. Splenic cells were collected nineteen days after infection/immunization. The histograms show FACS analysis on CD8⁺ cells (Gr. 1) and H-2K^k-TEWETGQI⁺ CD8⁺ cells (Gr. 2, 3, and 4) and the indicated marker (blue). Control cells were from naive mice (red lines). Results of CD44, KLRG1 and CD183 staining are presented as MFI and frequencies of the CD44^High, KLRG1^High or CD183^High cells, respectively. On the other hand, results of CD27 and CD62L staining are presented as MFI and frequencies of the CD27^Low or CD62L^Low cells. Representative analyses are shown from pools of cells from 3 mice. Stainings were performed 2 or more times with identical results.</p

Effects of JES6-1 treatment on the early CD4⁺ T cell response to <i>P. chabaudi</i> malaria.

<p>(A) C57BL/6 mice were treated with JES6-1 mAb on days 0, 2 and 4 p.i. with 10⁶ PRBC. On day 7 p.i., splenic CD4⁺ T cells were analyzed for CD122, CD25, CD69 and CD44 expression and cell size (FSC). Data show gated CD4⁺ T cells expressing high levels of activation markers and large size (n = 3–4). (B) Non-stimulated (basal) and anti-CD3 mAb stimulated IFN-γ and IL-17 production was evaluated in CD4⁺ T cells from the same groups of mice. (C) Numbers of CD4⁺ T cells per spleen were determined in the same groups of mice. (D) On day 4 p.i., PRBC-stimulated CD4⁺ T cell proliferation was analyzed <i>in vitro</i> in the presence or absence of JES6-1 mAb. Histograms show CFSE fluorescence in gated CD4⁺ T cells. The means ± SD (n = 3–4) of the percentages of replicating (CFSE^LO) cells are shown. In A–D, significant differences compared experimental conditions *p<0.05 with cells from non-infected (NI) mice; **p<0.05 with cells from non-treated (NT) mice; and #p<0.05 with non-stimulated cells. Data are representative of three separate experiments.</p

Schematic representation of the ASP-2 antigen and its expression in intra-cellular amastigotes of <i>T. cruzi</i>.

<p><b>A</b>) The protein is a prototypical member of the <i>trans</i>-sialidase family of <i>T. cruzi</i> surface antigens containing a putative signal peptide at the N-terminal region, 2 ASP-box sequences, and a VTV-box at the C-terminal domain. The protein is attached to the membrane through a glycophosphatidylinositol anchor. <b>B</b>) ASP-2 expression in intra-cellular amastigotes was determined by immunofluorescence with the specific MAb K22. HeLa cells were infected for 48 h with trypomastigotes of the Y strain. After fixation, indirect immunofluorescence and DAPI staining were performed and imaged under fluorescence microscopy. Bar, 14 µM. Photomicrography kindly provided by Dr. Clara Claser (Singapore Immunology Network -SIgN, Singapore).</p

Effects of JES6-1 treatment on the splenic CD4⁺CD25⁺FoxP3⁺ cell population during <i>P. chabaudi</i> malaria.

<p>(A) C57BL/6 mice were infected with 10⁶ PRBC. On day 7 p.i., CD4⁺ T cells were analyzed for CD25 and FoxP3 expression and cell size (FSC). Dot plots represent gated CD4⁺ T cells. Dot plots and histograms show a representative mouse from each group. Numbers inside dot plots refer to means ± SD (n = 4) of cell percentages in each gate. Histograms show gated CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁺FoxP3⁻ cells in relation to CD4⁺CD25⁻FoxP3⁻ cells. Numbers inside histograms refer to means ± SD (n = 4) of large cell percentages. (B) On days 0, 2 and 4 p.i., C57BL/6 mice were treated with JES6-1 mAb. Data represent the means ± SD (n = 4) of CD4⁺CD25⁺FoxP3⁺ cell percentages and numbers per spleen on days 7 and 20 of infection. In A–B, significant differences compared experimental conditions *p<0.05 with cells from non-infected (NI) mice; **p<0.05 with cells from non-treated (NT) mice; and #p<0.05 with CD25⁺FoxP3⁺ cells. Data are representative of two separate experiments.</p