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Cited by 123Inflammasome and immune disordersTrypanosoma species research and implicationsImmune Response and Inflammation

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

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Gonçalves, Virginia M., Matteucci, Kely C., Buzzo, Carina L., Miollo, Bruna H., Ferrante, Danny, Torrecilhas, Ana C., Rodrigues, Mauricio M., Alvarez, Jose M., Bortoluci, Karina R.·2013·PLoS Neglected Tropical Diseases
DOI 10.1371/journal.pntd.0002469PMC PMC3789781
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Abstract

Trypanosoma cruzi (T. cruzi) 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 T. cruzi infection is well-established, the involvement of inflammasomes remains to be elucidated. Herein, we demonstrate for the first time that T. cruzi 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 T. cruzi, as pharmacological inhibition of cathepsin B abrogates IL-1β secretion. NLRP3(-/-) and caspase1(-/-) mice exhibited high numbers of T. cruzi parasites, with a magnitude of peak parasitemia comparable to MyD88(-/-) and iNOS(-/-) mice (which are susceptible models for T. cruzi infection), indicating the involvement of NLRP3 inflammasome in the control of the acute phase of T. cruzi infection. Although the inflammatory cytokines IL-6 and IFN-γ were found in spleen cells from NLRP3(-/-) and caspase1(-/-) mice infected with T. cruzi, 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 T. cruzi 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 T. cruzi infection as NLRP3(-/-) and caspase-1(-/-) macrophages. Taken together, our results demonstrate a role for the NLRP3 inflammasome in the control of T. cruzi infection and identify NLRP3-mediated, caspase-1-dependent and IL-1R-independent NO production as a novel effector mechanism for these innate receptors.

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Figure 1 T. cruzi-induced NLRP3 activation. PMs from WT, NLRP3−/− and caspase-1−/− mice (1,5×106 cells/300 µl) were infected with T. cruzi 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 (A). T. cruzi-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).
Figure 1 T. cruzi-induced NLRP3 activation. PMs from WT, NLRP3−/− and caspase-1−/− mice (1,5×106 cells/300 µl) were infected with T. cruzi 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 (A). T. cruzi-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).CC BY · PubMed Central
Figure 2 NLRP3 and caspase-1 are involved in the control of T. cruzi infection. WT, MyD88−/−, NLRP3−/−, caspase-1−/−, IFN-γ−/− and iNOS−/− mice were subcutaneously infected with 103 T. cruzi blood trypomastigotes. Parasitemia was quantified by counting the parasites in 5 µL of tail blood obtained on days 4 to 20 after infection (A). 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), IFN-γ (C) and IL-1β (D) were assessed by ELISA, and NO secretion was assessed by the Griess reaction (E). 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 (F). 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.
Figure 2 NLRP3 and caspase-1 are involved in the control of T. cruzi infection. WT, MyD88−/−, NLRP3−/−, caspase-1−/−, IFN-γ−/− and iNOS−/− mice were subcutaneously infected with 103 T. cruzi blood trypomastigotes. Parasitemia was quantified by counting the parasites in 5 µL of tail blood obtained on days 4 to 20 after infection (A). 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), IFN-γ (C) and IL-1β (D) were assessed by ELISA, and NO secretion was assessed by the Griess reaction (E). 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 (F). 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.CC BY · PubMed Central
Figure 3 Macrophages isolated from NLRP3−/− and caspase-1−/− mice are impaired in NO secretion and the control of T. cruzi infection. PMs (3×105/well of chamber slides) from WT, MyD88−/−, NLRP3−/− and caspase-1−/− mice were infected with T. cruzi Y trypomastigotes (1∶5) for 2–4 h. Extracellular parasites were removed by washing. After 48 hours, the chambers were fixed with methanol, stained with DAPI and evaluated by fluorescence microscopy (600×). The number of amastigotes (A) and the frequency of the infected cells (B) were counted in 1000 macrophages from a representative experiment. Experiments were repeated four times with similar results. Supernatants from non-infected (-) and T. cruzi-infected PMs (3 parasites per cell) (1,5×106 cells/300 µl) were collected after 24 h to evaluate the IL-6 levels by ELISA (C), and after 48 h to measure NO secretion by the Griess reaction (D). Numbers represent the mean ± S.D. (n = 5). *** p>0,001 and ** p>0,01 compared to the WT group. T. cruzi-infected PMs (3 parasites per cell) (1,5×106 cells/300 µl) from WT mice were treated with Ca-074ME, GLB or APO in doses of 25, 50 and 100 µM, or left untreated. After 48 h, supernatants were collected to evaluate the level of NO production by the Griess reaction. Bars represent the mean ± S.D. of triplicate samples. *** p>0,001 compared to the untreated group. (E). PMs (3×105/well of chamber slides) from WT mice were infected with T. cruzi Y trypomastigotes (5 parasites/cell) for 2-4 h. After removing extracellular parasites, Ca-074ME (50 µM) was added to the cultures. After 48 h, the chambers were fixed with methanol, stained with DAPI and evaluated by fluorescence microscopy (600×) to determine the frequency of infected cells. Bars represent the mean ± S.D. of triplicate samples, and ** p>0,001 and * p>0,01 compared to the untreated group (F).
Figure 3 Macrophages isolated from NLRP3−/− and caspase-1−/− mice are impaired in NO secretion and the control of T. cruzi infection. PMs (3×105/well of chamber slides) from WT, MyD88−/−, NLRP3−/− and caspase-1−/− mice were infected with T. cruzi Y trypomastigotes (1∶5) for 2–4 h. Extracellular parasites were removed by washing. After 48 hours, the chambers were fixed with methanol, stained with DAPI and evaluated by fluorescence microscopy (600×). The number of amastigotes (A) and the frequency of the infected cells (B) were counted in 1000 macrophages from a representative experiment. Experiments were repeated four times with similar results. Supernatants from non-infected (-) and T. cruzi-infected PMs (3 parasites per cell) (1,5×106 cells/300 µl) were collected after 24 h to evaluate the IL-6 levels by ELISA (C), and after 48 h to measure NO secretion by the Griess reaction (D). Numbers represent the mean ± S.D. (n = 5). *** p>0,001 and ** p>0,01 compared to the WT group. T. cruzi-infected PMs (3 parasites per cell) (1,5×106 cells/300 µl) from WT mice were treated with Ca-074ME, GLB or APO in doses of 25, 50 and 100 µM, or left untreated. After 48 h, supernatants were collected to evaluate the level of NO production by the Griess reaction. Bars represent the mean ± S.D. of triplicate samples. *** p>0,001 compared to the untreated group. (E). PMs (3×105/well of chamber slides) from WT mice were infected with T. cruzi Y trypomastigotes (5 parasites/cell) for 2-4 h. After removing extracellular parasites, Ca-074ME (50 µM) was added to the cultures. After 48 h, the chambers were fixed with methanol, stained with DAPI and evaluated by fluorescence microscopy (600×) to determine the frequency of infected cells. Bars represent the mean ± S.D. of triplicate samples, and ** p>0,001 and * p>0,01 compared to the untreated group (F).CC BY · PubMed Central
Figure 4 Caspase-1 but not IL-1R mediates T. cruzi-induced NO secretion. PMs (3×105/well of chamber slides) from WT, MyD88−/− and NLRP3−/− mice were infected with T. cruzi 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 (A). PMs (3×105/well of chamber slides) from WT, caspase-1−/− and IL-1R−/− mice (3 parasites/cell) were infected with T. cruzi 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). T. cruzi-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 (C). T. cruzi-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 (D).
Figure 4 Caspase-1 but not IL-1R mediates T. cruzi-induced NO secretion. PMs (3×105/well of chamber slides) from WT, MyD88−/− and NLRP3−/− mice were infected with T. cruzi 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 (A). PMs (3×105/well of chamber slides) from WT, caspase-1−/− and IL-1R−/− mice (3 parasites/cell) were infected with T. cruzi 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). T. cruzi-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 (C). T. cruzi-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 (D).CC BY · PubMed Central
Figure 5 NLRP3-mediated NO production acts synergistically with MyD88-mediated pathways to control T. cruzi infection. PMs from WT mice were infected with T. cruzi 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 (A). T. cruzi-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). PMs (3×105/well of chamber slides) from MyD88−/− mice were infected with tripomastigotes from T. cruzi 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 (C).
Figure 5 NLRP3-mediated NO production acts synergistically with MyD88-mediated pathways to control T. cruzi infection. PMs from WT mice were infected with T. cruzi 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 (A). T. cruzi-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). PMs (3×105/well of chamber slides) from MyD88−/− mice were infected with tripomastigotes from T. cruzi 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 (C).CC BY · PubMed Central
Figure 6 The proposed model for NLRP3-mediated control of T. cruzi. TLR2, TLR4, TLR7 and TLR9 recognize T. cruzi PAMPs and control infection by MyD88 adaptor molecule-mediated effector mechanisms. T. cruzi 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 T. cruzi infection.
Figure 6 The proposed model for NLRP3-mediated control of T. cruzi. TLR2, TLR4, TLR7 and TLR9 recognize T. cruzi PAMPs and control infection by MyD88 adaptor molecule-mediated effector mechanisms. T. cruzi 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 T. cruzi infection.CC BY · PubMed Central

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Authors
Gonçalves, V. M., Matteucci, K. C., Buzzo, C. L., Miollo, B. H., Ferrante, D., Torrecilhas, A. C., Rodrigues, M. M., Alvarez, J. M., & Bortoluci, K. R.
Affiliations
  • Universidade Federal de São Paulo (BR)
  • Instituto de Ciências Ambientais, Químicas e Farmacêuticas (BR)
Corresponding author
Bortoluci, Karina R.
ORCID
Venue
PLoS Neglected Tropical Diseases
Publisher
Public Library of Science (PLoS)
Published
2013-10-03
Volume
7
Issue
10
Pages
e2469
ISSN
  • 1935-2727 (ISSN-L)
  • 1935-2735
DOI
10.1371/journal.pntd.0002469
PMC
PMC3789781
OpenAlex
W2140577495
Cited by
123
References
68
Citations by year
2026: 4 · 2025: 7 · 2024: 4 · 2023: 4 · 2022: 7 · 2021: 5 · 2020: 15 · 2019: 14 · 2018: 17 · 2017: 12 · 2016: 14 · 2015: 6 · 2014: 14
FWCI
5.08
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS neglected tropical diseases (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • LA Referencia (Red Federada de Repositorios Institucionales de Publicaciones Científicas) (publishedVersion, other-oa)
  • Americanae (AECID Library) (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • Fundação de Amparo à Pesquisa do Estado de São Paulo (08/50958-8)
  • Conselho Nacional de Desenvolvimento Científico e Tecnológico
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Inflammasome and immune disorders
  • Health Sciences › Medicine › Epidemiology › Trypanosoma species research and implications
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
Keywords
Trypanosoma cruzi, Chagas disease, NLRP3 inflammasome, caspase-1, nitric oxide production, IL-1β, cathepsin B, innate immunity, MyD88, IL-18, parasitemia control
MeSH terms
NLR Family, Pyrin Domain-Containing 3 Protein, Animals, Carrier Proteins, Disease Models, Animal, Female, Gene Expression Regulation, Nitric Oxide, Trypanosoma cruzi, Chagas Disease, Mice, Knockout, Caspase 1, Mice, Interleukin-1beta, Host-Pathogen Interactions

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