PARP1-cGAS-NF-κB pathway of proinflammatory macrophage activation by extracellular vesicles released during Trypanosoma cruzi infection and Chagas disease

Fig 1. T. cruzi induced Ev elicit proinflammatory cytokines release in macrophages. Raw 264.7 Mφ and C2C12 muscle cells were incubated with media only or T. cruzi (cell: parasite ratio, 1: 3) for 72 h. Extracellular vesicles (Ev) released in supernatants were isolated as described in Materials and Methods. Next, cultured Mφ were incubated with Ev isolated from supernatants of Raw Mφ (A-C) and C2C12 cells (D-F) for 0–48 h (± 20 ng/mL IFN-γ) and an ELISA was performed to measure the release of TNF-α (A&D), IL-6 (B&E), and IL-1β (C&F) cytokines. Raw Mφ infected with T. cruzi or incubated with media alone were used as positive and negative controls, respectively. NEv: Ev isolated from non-infected cells; TEv: Ev isolated from supernatants of T. cruzi-infected cells. Data are representative of ≥ 2 independent experiments (three biological replicates per treatment, duplicate observations per sample) and plotted as mean value ± SD. Significance is annotated as + TEv vs. NEv and * TEv+IFN-γ vs TEv, and p values of ≤ 0.05, ≤ 0.01, and ≤ 0.001 are marked with one, two, and three symbol characters, respectively.

Fig 2. (A-F) Parp1-/- primary Mφ exhibit decreased cytokines’ expression in response to TEv stimulation. Supernatants of Raw 264.7 Mφ incubated with media only or T. cruzi were used to purify NEvRAW and TEvRAW, respectively. Primary bone-marrow Mφ (WT or Parp1-/-) were incubated with Ev (± 20 ng/mL IFN-γ) for 3 h and 48 h. (A-C) Real time RT-qPCR analysis of mRNA levels for cytokine encoding genes at 3 h post-incubation. (D-F) ELISA determination of cytokines release at 48 h post-incubation. Primary Mφ incubated with T. cruzi (cell: parasite ratio, 1:3) and IFN-γ were used as controls. (G-I) Immune characteristics of Ev produced during the development of CD (± PARP1 inhibitor). B6129S/J WT mice (n = 10 per group) were infected with T. cruzi (10,000 parasites per mouse). Plasma of normal and chronically infected (150 days post-infection) mice were used to isolate NEvmice and TEvmicech, respectively. Next, Mφ were incubated with murine plasma Ev (± 20 ng/ml IFN-γ and 5 μM iniparib) for 48 h, and an ELISA was performed to determine the TNF-α (G), IL-6 (H), and IL-1β (I) release in supernatants. Data are representative of ≥ 2 independent experiments (two biological replicates per treatment…

Fig 3. (A-E) Physical characterization of Tc-induced extracellular vesicles. Shown in A-C are representative peaks for the absolute number (panels a) and volume per nm3 (panels b) in relation to size distribution (diameter/nm) for TcEv purified from spent media of T. cruzi culture, and Ev purified from supernatants of infected RAW 264.7 Mφ (TEvRaw) and plasma of chronically infected mice (TEvmicech). Scatter dot plots for size and concentration measurements of TcEv, TEvRaw, and TEvmicech were obtained from two cycles of observations per sample, scanning 11 cell positions and capturing 60 frames. The mean and the standard deviation values for each measurement is presented below the scattered dot plots (D&E). (F&G) Protein markers by western blot analysis. Purified TEvRaw, TEvmicech and controls (NEvRaw, and Mφ, splenic, and T. cruzi lysates, 10 μg) were resolved by SDS-PAGE. Representative western blot images show CD11b, CD68 and, GAPDH levels (F) and reactivity to anti-T.cruzi polyclonal sera from infected mice (G). (H&I) PCR analysis of DNA in Ev. Total TEvDNA was purified from TEv isolated from supernatants of infected Raw Mφ and plasma of acutely (ac) and chronically (ch) infected WT…

Fig 4. Molecular contents of TEv that induce proinflammatory Mφ activation in PARP1- dependent manner. Supernatants of T. cruzi-infected Raw 264.7 Mφ were used to isolate TEv, and the latter were processed to purify TEvDNA and TEvprotein fractions, as described in Materials and Methods. (A) Representative 1.5% agarose gels show the EvDNA (± DNase I) of TEv of infected Raw Mφ, and acutely and chronically infected WT and Parp1-/- mice (controls: Matched NEv). (B) Representative 10% PAGE gels stained with Coomassie blue show TEvprotein fractions (± DNase I or protease). (C-H) Cultured Mφ were incubated with TEv, and DNase-treated TEvprotein and protease-treated TEvDNA fractions (± 5 μM iniparib) for 3 h or 48 h. The mRNA levels (3 h) and protein levels (48 h) of TNF-α, IL-6 and IL-1β cytokines were determined by real time RT-qPCR (C-E) and ELISA (F-H), respectively. Data in bar graphs are representative of ≥ 2 independent experiments (two biological replicates per treatment and triplicate observations per sample for RT-qPCR analysis; and three biological replicates per treatment and duplicate observations per sample for ELISA), and presented as mean ± SD. Statistical significance is…

Fig 5. Innate immune receptors engaged in signaling of TEv-induced macrophage activation. NEv and TEv were purified from supernatants of Raw 264.7 Mφ incubated for 72 h with media only or T. cruzi, respectively. Next, Raw Mφ were incubated with NEv or TEv for 3 h or 18 h in presence or absence of 5 μM chloroquine (inhibits endosomal TLR3/7/9), 5 μM quinacrine (inhibits TLR3/9), 5 μM ODN-2088 (ODN, inhibits TLR9), 10 μM PF-06928215 (inhibits cGAS) or 5 μM JSH-23 (inhibits NF-κB activation). The mRNA levels for genes encoding TNF-α (A), IL-6 (B), and IL-1β (C) were evaluated by RT-qPCR. Data are representative of ≥ 2 independent experiments (two biological replicates per treatment and three observations per sample) and presented as mean ± SD. Unless indicated with horizontal bar, statistical significance comparing TEv vs. TEv + inhibitor is annotated as + p ≤ 0.05, ++ p ≤ 0.01, and +++ p ≤ 0.001.

Fig 6. (A) T. cruzi-induced EvDNA is oxidized. NEv and TEv were isolated from normal and T. cruzi-infected Raw Mφ (triplicate samples per group), and from normal and chronically infected WT and Parp1-/- mice (n = 10 mice per group). Total DNA was purified from all Ev preparations, and an OxiSelect DNA Damage ELISA was performed to evaluate the 8-hydroxy-2’- deoxy guanosine (8-OHdG) levels. (B-D) Cross talk between cGAS and PARP1 regulates the TEv-induced NF-κB activity in Mφ. Raw 264.7 Mφ were transfected with NF-κB luciferase reporter and control renilla luciferase. Transfected cells were incubated for 3 h with TEvRAW purified from supernatants of T. cruzi-infected Mφ (B), or TEv purified from plasma of chronically infected WT (C) and Parp1-/- (D) mice (± 5 μM or 10 μM concentrations of PF-06928215 (inhibits cGAS) and iniparib (inhibits PARP1)). Cells were used in a Dual Luciferase (Firefly-Renilla) Assay, and the ratio of relative luminescence for firefly to renilla luciferase (control) was calculated as a measure of NF-κB activity. Matched NEv were used as controls. Data are representative of ≥ 2 independent experiments (six biological replicates per treatment) and presented as mean…

Fig 7. Histological evaluation of myocardial inflammation in chronically infected mice (± PARP1). Mice (WT, Parp1+/-, and Parp1-/-) were challenged with T. cruzi (10,000 parasites per mouse) and euthanized at 150 days post-infection. Paraffin-embedded left ventricular heart tissue sections (5 μM) were examined by hematoxylin/eosin staining (blue: nuclear; pink: muscle/cytoplasm). Shown are representative H&E stained images of tissue sections from (A-C) non-infected and (D-F) chronically infected WT (A&B), Parp1+/- (B&E), and Parp1-/- (C&F) mice. Average histological score ± SD values were derived from analysis of n = 3 mice per group, 2–3 tissue sections per mouse, 10 microscopic fields per tissue section, and discussed in Results section.

Fig 8. Myocardial macrophage profile in Chagas disease (± PARP1). Mice (WT and Parp1-/-) were euthanized at 150 days post-infection and myocardial tissue sections of non-infected and infected mice were subjected to immunohistochemistry staining. Shown are representative cardiac tissue images of WT and Parp1-/- mice (± T. cruzi infection, 60X magnification). Bar graphs show semi-quantitative, immunohistochemistry quick score ± SD (n = 3 mice per group, two tissue sections per mouse, 9 microscopic fields per tissue section, 20X magnification), and significance is annotated as + infected vs. non-infected and *WT.T. cruzi vs. Parp1-/-.T. cruzi. The p values of ≤ 0.05, ≤ 0.01, and ≤ 0.001 are presented by one, two, and three symbol characters, respectively. A-E: CD11b+CD68+ (Mφ markers), F-J: CD11b+CD206+ (detects Mφ of anti-inflammatory phenotype), K-O: CD11b+IL-10+ (indicates immuno-regulatory activation of Mφ), and P-T: CD11b+TNF-α+ (indicates proinflammatory activation of Mφ).