Trypanosoma cruzi Induces the PARP1/AP-1 Pathway for Upregulation of Metalloproteinases and Transforming Growth Factor β in Macrophages: Role in Cardiac Fibroblast Differentiation and Fibrosis in Chagas Disease

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  FIG 1. Macrophages express and release metalloproteinases in response to T. cruzi infection. RAW 264.7 Mϕ were incubated with medium or T. cruzi (Tc) trypomastigotes (cell-to-parasite ratio, 1:3) in the presence and absence of 20 ng/ml IFN-γ and 10 ng/ml CCL2 for 0 to 24 h. (A to C) Real-time RT-qPCR evaluation of mRNA levels for Mmp2, Mmp9, and Mmp12 (normalized to Gapdh). (D) Western blot images for MMP2 and MMP9 in Mϕ cell homogenates. Cells were incubated with T. cruzi trypomastigotes (cell-to-parasite ratio, 1:3) in the presence and absence of 20 ng/ml IFN-γ and 10 ng/ml CCL2 for 24 h. (E and F) Cell supernatants were collected after 24 h of incubation, concentrated 10-fold, and used for MMP2 (pro- form, 72 kDa; cleaved form, 66 kDa) and MMP9 (pro- form, 92 kDa) zymography. Bar graphs (F) show relative gelatin degradation scores. (G and H) Western blot images for MMP12 (pro- form, 54 kDa; cleaved form, 45 kDa [G]) and relative densitometry scores for MMP12 (normalized to GAPDH [H]) in cell homogenates and supernatants (obtained after 24 h of incubation). Data in bar graphs are means and SD and are representative of two independent experiments with two biological replicates per…

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  FIG 2. Profibrotic response of splenic and cardiac macrophages in CD-infected mice. Mice were infected with T. cruzi (10,000 parasites per mouse) and euthanized at 150 days postinfection (controls had no infection). Spleen (SMϕ) and heart (HMϕ) macrophages were isolated using CD11b+ magnetic beads and incubated in vitro for 24 h. (A to C) Real-time RT-qPCR evaluation of mRNA levels. (D to F) TGF-β, TNF-α, and MCP3 release in supernatants, determined by an ELISA. (G to J) Gelatin zymography for MMP2 and MMP9 in culture supernatants of splenic and heart Mϕ (J) and relative gelatin degradation scores for pro-MMP2 (G), cleaved MMP2 (H), and pro-MMP9 (I). Data (means and standard deviation [SD]) are representative of two independent experiments with two biological replicates per treatment (triplicate and duplicate observations per sample for RT-qPCR and zymography, respectively). Significance was calculated by Student’s two-tailed t test or a nonparametric Mann-Whitney U test. +++, P ≤ 0.001.

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  FIG 3. MMP2 and MMP9 are required for TGF-β production by Mϕ in response to T. cruzi infection. RAW 264.7 Mϕ were incubated with T. cruzi trypomastigotes in the presence and absence of 10 μM ARP-100 (which inhibits MMP2), 10 μM MMP9 inhibitor-1 (which inhibits MMP9), 10 μM MMP408 (which inhibits MMP12), or 10 μM uk122 (which inhibits urokinase-type plasminogen activator [uPA]). Cell culture supernatants were used for the detection of TGF-β (A), TNF-α (B), and MCP3/CCL7 (C) by an ELISA. Data are representative of two independent experiments (three biological replicates per treatment and duplicate observations per sample) and are means and SD. ^, P ≤ 0.05; +++ and ^̂̂, P ≤ 0.001 (+, noninfected versus infected by Student’s two-tailed t test or nonparametric Mann-Whitney U test; ^, infected versus infected/treated by one-way ANOVA and Tukey’s post hoc test).

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  FIG 4. Inhibition of Mϕ MMP9 arrests TGF-β-dependent cardiac fibroblast transition to myofibroblasts in response to T. cruzi. Human THP-1 Mϕ were incubated for 24 h with T. cruzi in the presence or absence of 10 μM concentrations of inhibitors of MMP2 or MMP9. Next, human cardiac fibroblast (HCF) cells were incubated for 48 to 96 h with spent medium from THP-1 Mϕ that were noninfected (A to D), infected (E to H), or infected and treated with inhibitors of MMP2 (I to L) or MMP9 (M to P). Positive controls were HCF cells incubated with recombinant TGF-β (Q to T). Transition of HCF to myofibroblasts was determined by immunofluorescence staining for S1000A4 (red) and α-SMA (green), respectively. DAPI (4′,6-diamidino-2-phenylindole; blue) staining marks nuclei. Representative images are shown at a magnification of ×40. Bar graphs show percentages of S100A4+ and α-SMA+ HCF at 48 to 96 h post-incubation. Data (means and SD) are derived from two independent experiments, with three biological replicates per treatment, and each sample was analyzed in 9 microscopic fields. Significance comparing the expression of S100A4 (+) or α-SMA (*) at different time points was calculated by repeated-measures…

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  FIG 5. MMP and TGF-β expression in response to T. cruzi infection is PARP1 dependent. (A to E) RAW 264.7 Mϕ were incubated with T. cruzi for 72 h and extracellular vesicles (EV) released in supernatants were isolated. Next, a new batch of Mϕ was incubated for 18 h with T. cruzi or T. cruzi-induced EV (TEV) in the presence or absence of 10 μM iniparib (which inhibits PARP1). The mRNA levels for Mmp2, Mmp9, Mmp12, Mmp13, and Tgfb1, normalized to Gapdh, were measured by real-time RT-qPCR. (F to J) RT-qPCR evaluation of mRNAs in heart tissue of WT and Parp1−/− mice euthanized at 150 days postinfection (controls had no infection). Data are representative of two independent experiments with 3 biological replicates per treatment (triplicate observations per sample). Significance is annotated as follows: +, noninfected versus infected; ^, infected versus infected with chemical or genetic deletion of PARP1 (Student’s two-tailed t test or nonparametric Mann-Whitney U test). ^, P ≤ 0.05; ++ and ^̂, P ≤ 0.01; +++ and ^̂̂, P ≤ 0.001. iParp1, inhibitor of PARP1.

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  FIG 6. Profibrotic macrophages and tissue fibrosis in myocardium of CD-infected mice (with and without PARP1). Mice (WT and Parp1−/−) were challenged with T. cruzi and euthanized at 150 days postinfection. Paraffin-embedded left ventricular tissue sections were subjected to immunohistochemical staining. Representative images show colocalization of CD68 (magenta, Mϕ marker) with MMP9 (A to D), TGF-β (G to J), galectin (M to P), and vimentin (S to V) (magnification, ×60). Semiquantitative immunohistochemistry scores for tissue levels of MMP9, TGF-β, galectin, and vimentin (E, K, Q, and W, respectively) and CD68+ Mϕ colocalized with MMP9, TGF-β, galectin, and vimentin (F, L, R, and X, respectively) are shown. Bar graphs show means and SD (3 mice per group, two tissue sections per mouse, 9 microscopic fields per tissue section; magnification, ×20). Significance was calculated by Student’s two-tailed t test or nonparametric Mann-Whitney U t test and is annotated as follows: +, WT versus WT T. cruzi-infected or Parp1−/− versus Parp1−/− T. cruzi-infected; ^, WT T. cruzi-infected versus Parp1−/− T. cruzi-infected. ++, P ≤ 0.01; +++ and ^̂̂, P < 0.001).

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  FIG 7. PARP1 induces T. cruzi-mediated fibrotic response through transcriptional activation of AP-1. (A to C) RAW 264.7 Mϕ were incubated with T. cruzi in the presence and absence of 10 μM concentrations of specific inhibitors of NF-κB, AP-1, RUNX-2, and STAT3. Results of real-time RT-qPCR for Mmp2, Mmp9, and Tgfb1 (normalized to Gapdh) are shown. (D to I) BM-derived primary Mϕ were infected with T. cruzi for 24 h, and nuclear fractions were used to evaluate c-Fos (D), Fos B (E), Fra-1 (F), JunB (G), JunD (H), and c-Jun (I) by using AP-1 transcription factor family ELISA. Data (means and SD) are representative of two independent experiments, 2 or 3 biological replicates per treatment, and 2 or 3 observations per sample. Significance is annotated as follows: +, noninfected versus infected (Student’s two-tailed t test or nonparametric Mann-Whitney U test); ^, infected versus infected and treated (one-way ANOVA followed by Tukey’s post hoc correction test). ++, P ≤ 0.01; +++ and ^̂̂, P ≤ 0.001).