Canonical PI3Kγ signaling in myeloid cells restricts Trypanosoma cruzi infection and dampens chagasic myocarditis

Figures

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  Fig. 1. The PI3Kγ signaling is enhanced in the heart tissue of mice after experimental infection with T. cruzi. a Transcriptome analysis for the expression of Pik3ca, Pik3cd, and Pik3cg genes in non-infected C57BL/6J mice or 18 days post infection with T. cruzi. b RT-PCR analysis of the mRNA expression of Pik3ca, Pik3cb, Pik3cd, and Pik3cg genes in the heart tissue of C57BL/6 non-infected mice (n = 7) or 18 days post infection with T. cruzi Y strain (n = 11). Gapdh was used as a housekeeping gene. c Representative western blots and analysis of phosphorylated (p) and total (t) AKT1 expression in the heart tissue of non-infected C57BL/6 mice or 18 days post infection with T. cruzi (n = 6). GAPDH was used as a loading control. d Representative western blots and analysis of phosphorylated (p) and total (t) AKT2 expression in the heart tissue of non-infected C57BL/6 mice or 18 days post infection with T. cruzi (n = 6). GAPDH was used as a loading control. AU refers to arbitrary units (c, d). *P < 0.05, **P < 0.01, ***P < 0.001, and ns = no statistical significance (unpaired Student’s t-test in b–d). Data are representative of two (b–d) independent experiments (mean ± s.e.m in b–d)

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  Fig. 2. PI3Kγ signaling in hematopoietic cells is essential for mouse resistance to T. cruzi infection. a–c Body weight (a) and survival rate of C57BL/6 WT (n = 10) and Pik3cg−/− (n = 10) (b) or Pik3cgKD/KD (n = 10) (c) mice infected with 103 trypomastigote forms of T. cruzi Y strain. d, e Blood parasitemia of WT and Pik3cg−/− (d) or Pik3cgKD/KD (e) mice infected with 103 trypomastigote forms of T. cruzi Y strain. f Survival rate of chimera mice infected with 500 trypomastigote forms of T. cruzi Y strain. *P < 0.05, ***P < 0.001, and ns = no statistical significance (unpaired Student’s t-test in a, d, e and Mantel–Cox log-rank test in b, c, f). Data are representative of two (f) of three (a–e) independent experiments (mean ± s.e.m in a, d, e)

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  Fig. 3. PI3Kγ is important for the heart function of T. cruzi-infected mice. a, b Serum CK-MB level of WT (n = 4), Pik3cg−/− (n = 4) (a) and Pik3cgKD/KD (n = 7) (b) non-infected mice or 18 days post infection with 103 trypomastigote forms of T. cruzi Y strain. c Representative images of echocardiography in WT (n = 6) and Pik3cg−/− (n = 6) non-infected mice or 18 days after infection with T. cruzi. Red arrows indicate diastolic ventricular diameter and yellow arrow indicates diastolic ventricular wall thickness. d–g Diastolic left ventricle diameter (d), diastolic left ventricular posterior wall thickness (e), ejection fraction (f), and cardiac output (g) of WT (n = 6) and Pik3cg−/− (n = 6) non-infected mice or 18 days after infection with 103 trypomastigote forms of T. cruzi Y strain. *P < 0.05 and **P < 0.01 relative to WT-infected mice and #P < 0.05 and ##P < 0.01 relative to non-infected group (one-way ANOVA with Tukey’s post hoc test in a, b, d–g). Data are representative of two independent experiments (mean ± s.e.m in a, b, d–g)

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  Fig. 4. PI3Kγ controls the heart inflammation after infection with T. cruzi. a Representative images from H&E staining of heart tissue from non-infected WT (n = 6) and Pik3cg−/− (n = 6) mice or 18 days after infection with 103 trypomastigote forms of T. cruzi Y strain. Scale bar = 50 μm. b Heat map demonstrating the profile of cytokines and chemokines produced in the heart tissue of non-infected WT and Pik3cg−/− mice or 18 days post infection with 103 trypomastigote forms of T. cruzi Y strain. c Representative flow cytometry plots for the analysis of CD4 staining and quantification of the number of positive cells in the heart tissue of WT (n = 5) and Pik3cg−/− (n = 4) naive mice or infected with T. cruzi Y strain. d PCR analysis of mRNA expression of the transcription factors Tbet, Gata-3, and Rorγt in the heart tissue of WT (n = 4) and Pik3cg−/− (n = 4) mice. Gapdh was used as a housekeeping gene. e Survival rate of Pik3cg−/− mice infected with 103 trypomastigote forms of T. cruzi Y strain and treated at day 9 post infection with 2 mg Kg− and at days 12 and 15 post infection with 1 mg Kg− of dexamethasone (n = 5) or vehicle (n = 5). *P < 0.05 and ***P < 0.001 (one-way ANOVA with Tukey’s…

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  Fig. 5. The absence of PI3Kγ does not affect the number and suppressive function of regulatory T-cells in the heart tissue of infected mice. a Representative flow cytometry plots for the analysis of Foxp3 staining and quantification of the number of positive cells in the heart tissue of WT (n = 4) and Pik3cg−/− (n = 3) naive mice or infected with T. cruzi Y strain. b PCR analysis of mRNA expression of Foxp3 gene in the heart tissue of WT (n = 4) and Pik3cg−/− (n = 4) mice. Gapdh was used as a housekeeping gene. c–e Flow cytometry analysis measured by the mean fluorescence intensity (MFI) of the cell surface markers Foxp3 (c), CTLA-4 (d), and CD39 (e) expressed in CD4+Foxp3+ cells from the heart tissue of infected of WT (n = 4) and Pik3cg−/− (n = 4) mice 18 days after infection with 103 trypomastigote forms of T. cruzi Y strain. f In vitro T-cell suppression by Treg cells isolated from the lymph nodes of WT and Pik3cg−/− naive mice. ns = no statistical significance (unpaired Student’s t-test in a–f). Data are from one experiment (f) or representative of two (a–e) independent experiments (mean ± s.e.m in a–f)

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  Fig. 6. PI3Kγ controls the heart parasitism after infection with T. cruzi. a Representative images from H&E staining of heart tissue from WT (n = 6) and Pik3cg−/− (n = 6) mice 18 days post infection with T. cruzi Y strain. Black arrows indicate amastigote nests of the parasite. Scale bars = 20 µm. b, c Quantitative PCR analysis of T. cruzi DNA isolated from Pik3cg−/− (b) and Pik3cgKD/KD (c) mice 18 days post infection with T. cruzi Y strain. d Survival rate of Pik3cg−/− mice infected with 103 trypomastigote forms of T. cruzi Y strain and treated with 100 mg Kg− of benznidazole (BNZ) (n = 8) or vehicle (n = 6). *P < 0.05, by unpaired Student’s t-test, relative to WT mice (a–c). *P < 0.05 and ***P < 0.001 (unpaired Student’s t-test in a–c and Mantel–Cox log-rank test in d). Data are from one experiment (d) or representative of two (c) or three (a, b) independent experiments (mean ± s.e.m in a–c)

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  Fig. 7. Disruption of PI3Kγ signaling in human or murine macrophages harms the killing of intracellular T. cruzi. a Representative flow cytometry dot plots for the analysis of CD11b staining and quantification of the absolute number of positive cells in the heart tissue of non-infected WT (n = 5) and Pik3cg−/− (n = 5) mice or 18 days post infection with 103 trypomastigote forms of T. cruzi Y strain. b Quantitative PCR analysis of nanogram of T. cruzi DNA presents in 1 ng of DNA from CD11b− and CD11b+ cells isolated from the heart tissue of WT and Pik3cg−/− or Pik3cgKD/KD-infected mice. c Representative images of BMDMs stimulated with 0.1 or 1 ng of IFN-γ and infected for 48 h with trypomastigote forms of T. cruzi Y strain at MOI 3:1. Scale bars = 100 µm. d Trypomastigote forms of T. cruzi Y strain released from culture of WT and Pik3cg−/− BMDM stimulated with 0.1 ng ml− of IFN-γ. Experiments were performed in triplicate. e Nitrite quantification by Griess reaction in the supernatant of WT and Pik3cg−/− BMDMs stimulated with 1 ng of IFN-γ and infected with T. cruzi Y strain at MOI 3:1. f Nitrate quantification in the heart tissue of WT (n = 7) and Pik3cg−/− (n = 7) naive mice or 18 days…

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  Fig. 8. AKT1 signaling in myeloid cells confers resistance to T. cruzi infection. a Survival rate of littermate control (n = 7) and Akt1−/−Lysmcre (n = 7) mice after infection with 103 trypomastigote forms of T. cruzi Y strain. b Quantitative PCR analysis of picogram of T. cruzi DNA presents in 1 ng of heart tissue DNA isolated from WT (n = 5) and Akt1−/−Lysmcre (n = 5) mice. c Survival rate of WT (n = 7) and AKT2−/− (n = 7) mice infected with 103 trypomastigote forms of T. cruzi Y strain. *P < 0.05 and ns = no statistical significance (Mantel–Cox log-rank test in a, c and unpaired Student’s t-test in b). Data are representative of two independent experiments (mean ± s.e.m in b)

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  Fig. 9. The PI3Kγ signaling is enhanced in the human heart tissue with Chagas disease. a Transcriptome analysis of family I PIK3 genes expression (PIK3CA, PIK3CB, PIK3CD, and PIK3CG) in healthy control (Ctl; n = 7) and patients with idiopathic dilated cardiomyopathy (DCM; n = 14) or chronic chagasic cardiomyopathy (CCC; n = 10). b RT-PCR analysis of the mRNA expression of PIK3CA, PIK3CB, PIK3CD, and PIK3CG genes in the heart tissue of Ctl (n = 5); DCM (n = 10); and CCC (n = 10) patients. GAPDH was used as a housekeeping gene. c Representative western blots and analysis of phosphorylated (p) and total (t) AKT1 expression in the heart tissue of Ctl (n = 5), DCM (n = 10), and CCC (n = 10) patients. GAPDH was used as a loading control. d Representative western blots and analysis of phosphorylated (p) and total (t) AKT2 expression in the heart tissue of Ctl (n = 5), DCM (n = 10), and CCC (n = 10) patients. GAPDH was used as a loading control. AU refers to arbitrary units. ns = no statistical significance (one-way ANOVA with Tukey’s post hoc test in b–d). *P < 0.05 and **P < 0.01 relative to Ctl and #P < 0.05, ##P < 0.01, and ###P < 0.001 relative to DCM (one-way ANOVA with Tukey’s post hoc…

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  Fig. 10. Relationship between parasitism and PI3Kγ expression in the heart tissue of CCC patients. a Quantitative real-time PCR analysis of T. cruzi DNA levels (pg/50 ng of human DNA) in heart tissue from CCC patients. The black line indicates the lowest amount of parasite detected by reaction. b qRT-PCR analysis of the mRNA expression of PIK3CG in the heart tissue of CCC patients segregated by high (n = 5) or low (n = 6) heart parasitism. Unpaired Student’s t-test in b. Data are from one experiment (mean ± s.e.m in b)