Parasitic infection with <i>T. cruzi</i> induces a sustained and cell type-independent activation of STAT1.

<p>(A) Immunoblots demonstrating tyrosine phosphorylation and expression levels of STAT1 in differentially treated HFF (left) and A549 cells (right) using antibodies against tyrosine-phosphorylated STAT1 (αpY-STAT1) and, after stripping off of bound immunoreactivity from the membranes, pan-STAT1 antibody (αSTAT1). Cells were either left untreated (lane 2), stimulated for 18 h with 5 ng/ml IFNγ (lane 3) or challenged for 18 h with <i>T. cruzi</i> trypomastigotes at an MOI of 10 (lane 4). Cellular extracts from IFNγ-treated U3A cells (45 min) expressing recombinant STAT1 were used as control (lane 1) (n = 3). (B) Infectious dose-dependent increase in STAT1 tyrosine and serine phosphorylation in cytokine-unstimulated HFF (left) and A549 cells (right). Equal numbers of cells were exposed to parasites for 18 h at different MOIs. Representative immunoblotting experiments using antibodies specifically reacting with phospho-Y701- (αpY-STAT1), phospho-S727- (αpS-STAT1) and pan-STAT1 antibody (αSTAT1) are shown (n = 4). (C) Quantification of Western blot results for expression of phospho-Y701-, phospho-S727- and total STAT1 in extracts from cells infected with increasing doses of <i>T. cruzi</i>, as depicted in Figure 2B. Phosphorylation and expression levels were compared to the signal intensity in uninfected cells stimulated for 3 h with IFNγ set as 100. (D-G) <i>T. cruzi</i> infection elicits GAS-binding activity. Similar extracts as used for Western blotting (B) were incubated for 30 min with [³³P]-labeled DNA containing a single STAT binding site (M67) and then loaded onto a polyacrylamide gel for detection by EMSA. (D,F) The band corresponding to M67-bound STAT1 dimers was identified by supershift with a STAT1-, but not STAT3 antibody (Ab) and, in addition, by competition with a 750-fold molar excess of unlabeled M67-DNA (Comp.). STAT1-M67 complexes are marked with an arrowhead, asterisks indicate an unspecific band. (E,G) The histograms demonstrate the M67-binding activity in cytokine-untreated HFF cells (E) and A549 cells (G) plotted against the infection dose (MOI). (H) Immunocytochemical staining of STAT1 in <i>T. cruzi</i>-infected HFF cells using anti-STAT1 antibody C-24 and Cy3-labeled secondary antibody. The fluorescence micrographs show the intracellular distribution of endogenous STAT1 in methanol-fixed, Hoechst-stained HFF cells and, in contrast, the lack of STAT1 expression in uninfected (w/o <i>T. cruzi</i>) and infected (+<i>T. cruzi</i>) U3A cells (scale bar 20 µm). Note that there was no co-localization of cytoplasmic STAT1 and Hoechst-stained amastigotes and that some parasite-containing cells showed nuclear accumulation of STAT1.</p

Induction of STAT1-regulated genes in <i>T. cruzi</i>-infected cells.

<p>(A) HFF cells were either left untreated, stimulated for 6 h with 5 ng/ml of IFNγ or infected for 18 h with <i>T. cruzi</i> parasites at an MOI of 10 in the absence of cytokine exposure, as determined by real-time PCR assays. Histograms depict expression levels of the <i>mig1</i>, <i>gbp1</i>, <i>irf1</i>, <i>ido</i>, <i>stat1</i>, <i>inos</i>, and <i>nf-kb</i> gene before (white columns) and after 6 h stimulation of cells with IFNγ (grey columns) as well as after parasite infection (black columns). Specific gene induction was normalized to the expression level of the housekeeping gene <i>gapdh</i>. The data are presented as means and standard deviations from at least three independent experiments. (B) U3A cells lacking STAT1 expression showed no induction of these genes under the same experimental conditions as used in (A) (n.d. =  not detectable). (C) Infectious dose-dependent increase in STAT1-regulated gene expression in <i>T. cruzi</i>-infected HFF cells. The <i>adam19</i> gene was used as a negative control (n = 3).</p

STAT1 serine phosphorylation is impaired in <i>T. cruzi</i>-infected cells.

<p><i>In vitro</i> dephosphorylation assays were performed by incubating equal amounts of cell extracts from STAT1-GFP-expressing U3A cells (20 µl) with cell extracts from uninfected or parasite-infected STAT1-negative U3A cells. Co-incubation of the lysates lasted for 0 min and 45 min at room temperature, respectively. Before cell lysis, the U3A cells expressing the marker protein STAT1-GFP had either been treated with IFNγ (A,B) or left untreated (C,D). The reactions were then loaded on SDS polyacrylamide gels and tested for the amount of phosphorylated and total STAT1 by means of Western blotting using the antibodies indicated. Representative Western blots (A,C) and a quantification of these results (B,D) are shown. Arrowheads at the right-hand margin of the membranes mark bands corresponding to STAT1. Note that the faster-migrating band labeled with the anti-phospho-S727 antibody is an unknown non-STAT1 protein, whose expression is typically reduced in extracts from parasite-infected cells. The experiment was repeated at least four times with similar results. (E) Detection of intracellular parasites in STAT1-reconstituted U3A cells. Cells expressing a GFP fusion of wild-type STAT1 or the tyrosine-phosphorylation-deficient point mutant Y701F were treated for 6 h with 5 ng/ml IFNγ or left untreated, as indicated. Subsequently, cells were infected for 18 h with parasites at an MOI of 2, and parasitic and human nuclei were stained in fixed cells with Hoechst dye. Adjacent non-transfected cells were used as control. Arrowheads mark localization of parasites in the cytoplasm of human cells. The experiment was performed twice for each condition with similar results. (F) Quantification of results from (E) in at least n = 14 cells per sample showing that STAT1-GFP expression did not prevent replication of amastigotes.</p

STAT1-regulated suppression of <i>T. cruzi</i> replication and its inhibition by amastigotes.

<p>The model depicts two phases in host-parasite interactions: (A) Induction of STAT1 expression by phosphorylated STAT1 via a positive feedback loop which resulted from the stimulation of cells with IFNγ. (B) Inhibition of STAT1 transcriptional activity by <i>T. cruzi</i>-mediated dephosphorylation of serine 727. Serine dephosphorylation of STAT1 leads to blunted indoleamine 2,3-dioxygenase (IDO) expression as part of a parasitic defense mechanism. The established autocrine production of interferons through a TLR-dependent pathway in the parasite-infected cells is not depicted in the figure.</p

STAT1-regulated expression of <i>ido</i> is involved in the control of <i>T. cruzi</i> infection.

<p>(A,B) Exposure of cells to the JAK inhibitor AG-490 resulted in decreased STAT1 phosphorylation both at tyrosine residue 701 and serine residue 727 and is associated with a reduced intracellular STAT1 expression. Equal numbers of HFF and A549 cells were either pre-treated with 5 ng/ml of IFNγ or challenged for 18 h with parasites at an MOI of 15, in the absence or presence of AG-490 (50 µM). Representative Western blot results (A) and the corresponding quantification of STAT1 expression (B) are shown (n = 4). (C,D) Stimulation of HFF cells with IFNγ leads to increased enzymatic activity of indoleamine 2,3-dioxygenase (IDO, C) and elevated NO production (D), as measured with Ehrlich and Griess reagent, respectively (n = 3 in triplicate). (E) Inhibition of IDO by 1-methyltryptophan (1-MT, 1.5 mM) or iNOS by S-methylisothiourea sulfate (SMT, 1 mM) in IFNγ-pre-treated HFF cells resulted in significantly elevated numbers of <i>T. cruzi</i>-replicating cells (n = 4 in triplicate).</p

STAT1 deficiency in U3A cells is associated with high susceptibility to <i>T. cruzi</i> infection and impaired cellular responses to IFNγ.

<p>(A) Stimulation of HFF cells with IFNγ (5 ng/ml for 6 h) resulted in reduced numbers of intracellular <i>T. cruzi</i> amastigotes as determined by conventional crystal violet and May-Grünwald staining. As indicated, cells were either left untreated (w/o IFNγ) or treated with IFNγ (+IFNγ) before being infected with trypomastigotes of the Brazilian Y strain of <i>T. cruzi</i> at MOIs of 0, 2 and 10, respectively (n = 2 in duplicate). (B-E) The histograms depict the average number of infected cells per microscopic field (B,D) and the average number of intracellular amastigotes per cell (C,E) in untreated HFF cells (black columns) and cells pre-treated with IFNγ (grey columns) for 6 h (B,C) and 24 h (D,E), respectively. Fibroblasts were infected with parasites at 2 or 10 MOI before parasite burden was quantified microscopically. (F,G) No inhibitory effects of IFNγ pre-treatment on <i>T. cruzi</i>-infected U3A cells lacking STAT1 expression. Similar experiment with respect to stimulation of cells with IFNγ, incubation with parasites and measurement of parasite invasion as described in (D,E), except that U3A cells were used.</p

Protein-free Glc-GalNAc-substituted GPIs are clustered on extracellular parasites.

<p>Staining after permeabilization of HFF cells infected (72 h p.i.) with RH (upper panel) and PTG (lower panel) strains was performed using mAb T54 E10, recognizing both the EtN-PO₄ and the Glc-GalNAc side-branch epitopes of protein-free GPIs. Filled arrowheads point to intracellular parasites residing inside parasitophorous vacuoles. Unfilled arrowheads point to extracellular parasites. DIC, differential interference contrast.</p

GPIs of the PTG strain activate TLR4/NF-κB signaling.

<p>(<b>A</b>) CHO cells expressing TLR2 (TLR2⁺), TLR4 (TLR4⁺), or neither (TLR2⁻/TLR4⁻) were either untreated (black line) or exposed to the four different GPIs (II, III, V and VI) extracted from 1×10⁹ parasites (gray line). CD25 expression was measured by FACS analysis 18 h after stimulation. The figure is representative of three independent experiments. Percentage = [percentage of CD25 expression (M2) on GPI-stimulated cells] minus [percentage of CD25 expression (M2) on medium stimulated cells]. (<b>B</b>) Macrophages were incubated for 15, 30, or 60 min with GPI VI extracted from 4×10⁸ PTG strain parasites. Total nuclear protein content was tested using a NF-κB assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085386#s2" target="_blank">Materials and Methods</a>. The figure is a representative experiment with PTG GPI VI and similar results were obtained with GPIs II, III and V of the PTG strain. ^**P<0.005 compared to medium control.</p

Comparison of AHM and inositol quantifications of protein-free GPIs and GPI-anchored proteins of RH and PTG strains.

a<p>Copies per cell. In parentheses are percentages of PTG values compared to RH values. Values are means ± SD of three replicates.</p

GPIs of RH and PTG strains induce TNF-α and IL-12p40 secretion by macrophages.

<p>(<b>A</b>) Macrophages were incubated for 24 h with medium alone, or with individual GPIs of the PTG strain extracted from 1×10⁸ parasites and assayed for TNF-α cytokine production. (<b>B</b>) Macrophages were incubated for 24 h with medium alone, or with individual GPIs of the RH (left panel) and the PTG strain (right panel) extracted from 2×10⁸ parasites, respectively, and assayed for IL-12p40 cytokine production. (<b>C</b>) Macrophages were incubated for 24 h with medium alone, or with GPIa (3 mM), a chemically synthesized structure of RH strain GPI III core glycan and assayed for IL-12p40 cytokine production. ^***P<0.0005 PTG GPI II compared to medium control. ND, not determined.</p