Figure 1.
p47phox−/− mice are susceptible to T. cruzi infection and exhibit increased mortality and parasite burden.
(A) Western blotting of splenic macrophages and heart tissue, probed with anti-p47phox antibody. The data presented are from mice infected with 10,000 parasites and harvested at day 7 pi. (B–E) C57BL/6 (WT and p47phox−/−) mice were infected with T. cruzi (2,000 or 10,000 parasites/mouse). (B.a) Percent survival from infection was monitored daily. (B.b) Tissue parasite foci in skeletal muscle tissue sections of infected mice were monitored by light microscopy at weekly intervals. (C) Histologic examination of heart tissue-sections for parasite foci (data shown is at day 30 post-infection, original magnification: 200×; arrows mark the parasite nests). (D&E) Semi-quantitative PCR (D) and real time quantitative PCR (E) analysis of parasite burden in the heart tissue of infected mice at day 30 pi. Data in all figures are presented as mean ± SD and significance presented as *normal-versus-infected, #WT/infected-versus-p47phox−/−/infected (*, #p<0.05, **,##p<0.01, n = 8/group).
Table 1.
Tissue burden of parasite foci and inflammation in wild type and p47phox−/− mice infected by T. cruzi.
Figure 2.
T. cruzi uptake, intracellular replication and release by p47phox−/− phagocytes.
(A) Bone marrow (BM) and splenic cells were isolated from WT and p47phox−/− mice and in vitro incubated for 24 h with CFSE-labeled T. cruzi followed by 30 min with fluorescence conjugated anti-CD11b (macrophage marker) and anti-Ly6G (neutrophil marker) antibodies. The antibody-labeled cells were fixed, and analyzed by flow cytometry. Representative flow cytometry analyses of BM cells infected with CFSE-labeled T. cruzi are shown. (B) Bar graphs of the percentage of BM (Ba) and splenic (Bb) cell populations (CFSE+CD11b+: infected macrophages, CFSE+Ly6+: infected neutrophils). Data are derived from 3 independent experiments. (C) Supernatants of infected splenic monocytes were monitored for parasite release at 24 h and 48 h post-incubation by light microscopy. Data are presented as mean ± SD and significance presented as *normal-versus-infected, #WT/infected-versus-p47phox−/−/infected (*, #p<0.05, **,##p<0.01.
Figure 3.
In vitro functional activation of p47phox−/− macrophages in response to T. cruzi infection.
(A&B) Isolated splenocytes from WT and p47phox−/− mice were in vitro infected with live or heat-inactivated (HIA) T. cruzi for 24 h (± IFNγ). Cells incubated with phorbol myristate acetate (PMA) were used as positive controls. (A.a) H2DCFDA oxidation by intracellular ROS was monitored by fluorimetry. (A.b) Nitroblue tetrazolium (NBT) reduction to formazan blue crystals by cellular superoxide was visualized by light microscopy. (B) RT-PCR quantitation of iNOS mRNA level. (C) BM (panels a&c) and splenic (panels c&d) cells were stimulated with IFN-γ, washed, and then exposed to T. cruzi (live or heat-inactivated) for 24 h. The release of IFN-γ (panels a&b) and TNF-α (panels c&d) in supernatants was monitored by an ELISA. ND: not detectable.
Figure 4.
Histological analysis of inflammatory infiltrate in T. cruzi-infected p47phox−/− mice.
Shown are representative images of H&E staining (blue: nuclear, pink: muscle/cytoplasm/keratin) of skeletal muscle sections from WT (panels a–d) and p47phox−/− (panels e–h) mice infected with T. cruzi (2000 parasites/mouse), and harvested at day 7 (a&e), 14 (b&f), 21 (c&g), and 30 (d&h) post-infection. H&E stained images from normal mice are shown as insets in panels a and e (magnification: 40×).
Figure 5.
In vivo ROS, nitric oxide, and cytokine profile in p47phox−/− mice infected with T. cruzi.
Mice (WT and p47phox−/−) were infected with T. cruzi as described in Materials and Methods. (A) Splenic single cell population from infected mice (30 dpi) were cyto-spinned on glass slides. Shown is fluorescence staining for dihydroethidium (measures intracellular ROS, binds DNA). Cells stained with DAPI (binds nuclei, blue) are shown as controls. (B) Splenocytes were harvested from T. cruzi-infected mice at day 30 pi, and in vitro incubated for 24 h (± TcL). Shown is intracellular nitric oxide (panel a) and nitrite levels in the supernatant (panel b), determined by using DAF-FM staining/fluorimetry and Griess reagent assay, respectively. (C) Splenocytes were harvested from infected mice at day 7, 14, 21, and 30 post-infection, and incubated in vitro (± TcL) for 48 h. Shown are the TNF-α (a&b), IFN-γ (c&d) and IL-10 (e&f) levels in cell-free supernatants, determined by an ELISA. ND: not detectable.
Figure 6.
The p47phox−/− mice lacked the ability to develop CD8+T cell response to T. cruzi infection.
Mice were infected with T. cruzi, and harvested at day 30 pi. Splenocytes were in vitro incubated for 48 h in presence or absence of TcL as 2nd antigenic stimulus (controls: concanavalin A). (A) Cell proliferation determined by an MTT assay. (B&C) After in vitro stimulation (±TcL), splenocytes from infected mice were labeled for 30-min with fluorescent- conjugated specific antibodies as described in Materials and Methods, and analyzed by flow cytometry. Splenic frequency of CD4+ and CD8+ T cells (B.a) are shown. Shown are also the mean percentage of CD8+ T cells that were Ki67+ (PerCPCy 5.5, B.b), IFN-γ+ (e-Fluor, B.c), and CD107a+ (PerCPA, B.d). Representative quandrant images of flow cytometry analysis of T cell subsets are shown in (C).