Growth Factor and Th2 Cytokine Signaling Pathways Converge at STAT6 to Promote Arginase Expression in Progressive Experimental Visceral Leishmaniasis

<div><p>Host arginase 1 (arg1) expression is a significant contributor to the pathogenesis of progressive visceral leishmaniasis (VL), a neglected tropical disease caused by the intracellular protozoan <i>Leishmania donovani</i>. Previously we found that parasite-induced arg1 expression in macrophages was dependent on STAT6 activation. Arg1 expression was amplified by, but did not require, IL-4, and required <i>de novo</i> synthesis of unknown protein(s). To further explore the mechanisms involved in arg1 regulation in VL, we screened a panel of kinase inhibitors and found that inhibitors of growth factor signaling reduced arg1 expression in splenic macrophages from hamsters with VL. Analysis of growth factors and their signaling pathways revealed that the Fibroblast Growth Factor Receptor 1 (FGFR-1) and Insulin-like Growth Factor 1 Receptor (IGF-1R) and a number of downstream signaling proteins were activated in splenic macrophages isolated from hamsters infected with <i>L. donovani</i>. Recombinant FGF-2 and IGF-1 increased the expression of arg1 in <i>L. donovani</i> infected hamster macrophages, and this induction was augmented by IL-4. Inhibition of FGFR-1 and IGF-1R decreased arg1 expression and restricted <i>L. donovani</i> replication in both <i>in vitro</i> and <i>ex vivo</i> models of infection. Inhibition of the downstream signaling molecules JAK and AKT also reduced the expression of arg1 in infected macrophages. STAT6 was activated in infected macrophages exposed to either FGF-2 or IGF-1, and STAT6 was critical to the FGFR-1- and IGF-1R-mediated expression of arg1. The converse was also true as inhibition of FGFR-1 and IGF-1R reduced the activation of STAT6 in infected macrophages. Collectively, these data indicate that the FGFR/IGF-1R and IL-4 signaling pathways converge at STAT6 to promote pathologic arg1 expression and intracellular parasite survival in VL. Targeted interruption of these pathological processes offers an approach to restrain this relentlessly progressive disease.</p></div

Activation of signaling proteins in the IGF-1R canonical pathway in splenic macrophages from hamsters with VL.

<p>(<b>A–B, D–J</b>) Immunoblot analysis of expression of proteins in the IGF-1R canonical signaling pathway in splenic macrophages was performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004165#ppat-1004165-g002" target="_blank">Fig. 2</a>. <b>C</b>) Detection of phospho-IGFR by immunoblot in BMDMs uninfected (Un) or infected <i>in vitro</i> with <i>L. donovani</i> for 20 min to 24 hrs. Shown is an immunoblot from a single experiment. <b>K</b>) Network analysis showing the activation of the FGF and IGF-1 canonical signaling pathways generated by comparing the fold change of 32 signaling proteins in splenic macrophages from uninfected and infected (7, 14, and 28 days) hamsters using Ingenuity Pathway Analysis software (Ingenuity Systems). The –log of the <i>p</i> value (vertical axis) represents the probability that the association of the data set in that pathway is due to chance. <b>L</b>) Simplified schematic of the canonical IGF-1 signaling pathway for reference.</p

IL-4 and growth factors amplify STAT6 activation.

<p><b>A, B</b>) STAT6 activation measured in a luciferase reporter assay in BHK cells stimulated for 24 h with growth factors and a sub-maximal concentration of IL-4. <b>A</b>) IL-4 (3 IU/mL) and/or FGF-2 (20 ng/mL) and <b>B</b>) IL-4 (6 IU/mL) and/or IGF-1 (100 ng/mL). Shown is the mean and SEM of STAT6 activity determined by luminometry from a single experiment that was representative of 3 independent experiments. <b>C, D</b>) Immunoblots of cell lysates (not immunoprecipitation as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004165#ppat-1004165-g008" target="_blank">Fig. 8B</a>) showing phospho-STAT6 in BMDMs stimulated for 20 min with <i>L. donovani</i> promastigotes and <b>C</b>) IL-4 (8 IU/mL) and/or FGF-2 (20 ng/mL) or <b>D</b>) IL-4 (20 IU/mL) and/or IGF-1 (100 ng/mL). Bars represent the mean and SEM of fold change with reference to the uninfected, unstimulated (Uns) controls calculated by densitometry analysis of immunoblot bands from 3 independent experiments. Also shown is a representative individual immunoblot. <b>E</b>) IL-4-mediated activation of STAT6 in infected BHK fibroblasts is reduced by inhibition of FGFR and IGF-1R but not ERK. The cells were exposed to the control (DMSO) or FGFR inhibitor (FGFRi; PD166866; 10 µM), IGFR inhibitor (IGFRi; PPP; 5 µM), or ERK inhibitor (ERKi; PD98059; 5 µM) for 1 hr and then stimulated for another 24 hrs with IL-4 (25 IU/mL) in the presence or absence of inhibitor. Shown is the mean and SEM of STAT6 activity determined by luminometry from a single experiment that was representative of 2 independent experiments.</p

Inhibitors of Receptor Tyrosine Kinases reduce Arg1 transcription.

1<p>Inhibitors (dissolved in DMSO) were used at twice the concentration reported to cause 50% inhibition of kinase activity.</p><p>No attempt was made to maximize concentration for complete kinase inhibition.</p>2<p>Arg1 mRNA expression was determined in ex vivo cultured spleen cells from hamsters infected with <i>L. donovani</i> exposed to the RTK inhibitor for 24 hrs.</p><p>The percent inhibition of arg1 transcription was calculated with reference to untreated control cells exposed to DMSO and determined by qPCR.</p

Activation of signaling proteins in the FGFR canonical pathway in splenic macrophages from hamsters with VL.

<p>(<b>A–L</b>) Splenic macrophages were isolated by adherence from the spleens of uninfected hamsters (time 0) or hamsters infected for 7, 14, and 28 days and whole cell lysates probed with antibodies directed against arg1 (panel A, representative blot A) or members of the FGF signaling pathway (panels and representative blots B–L). Bars represent the fold change with reference to control cells of uninfected hamsters calculated by densitometry analysis of immunoblot bands from samples pooled from 1–4 hamsters per determination from 2–3 independent experiments. <b>M</b>) Simplified schematic of the canonical FGF signaling pathway for reference.</p

Activation of Receptor Tyrosine Kinases and downstream signaling proteins in splenic macrophages from hamsters infected with <i>L. donovani</i>.¹

1<p>Determined using a PathScan Antibody Array (Chemiluniscent readout, Cell Signaling; 4 arrays per group) and analyzed with the IPA Software.</p>2<p>Fold-change (mean ± SEM) of the phosphoprotein expressed in splenic macrophages isolated from 28-day infected hamsters compared to splenic macrophages from uninfected hamsters.</p><p>RTKs and signaling proteins with a fold change >1.5 are shown.</p

Growth factors upregulate arginase 1 in macrophages.

<p><b>A</b>) Induction of arg1 mRNA expression in macrophages exposed to recombinant growth factors. Uninfected and <i>L. donovani</i> infected hamster BMDMs were stimulated with EGF (100 ng/mL), FGF-2 (20 ng/mL), IGF-1 (100 ng/mL), PDGF (100 ng/mL), or IL-4 (25 IU/mL) for 24 hrs and the expression of arg1 mRNA determined by qRT-PCR. Shown is the mean and standard error of the mean (SEM; error bars) of 4 replicates from a single experiment that is representative of 2 independent experiments. <b>B</b>) Dose-dependent induction of arginase activity (urea production) in hamster BMDMs infected with <i>L. donovani</i> and exposed to 2-fold increasing concentrations of growth factors for 48 h. The concentration of the growth factors was: EGF: 12.5–100 ng/mL; FGF-2: 6.25–50 ng/mL; IGF-1: 50–400 ng/mL; and PDGF: 25–100 ng/mL. Shown is the mean and SEM of 2 replicates per dose that is representative of 4 independent experiments. *p<0.05; **p<0.01; ***p<0.001.</p

IL-4 enhances growth factor-induced arg1 in <i>L. donovani</i> infected macrophages.

<p>Infected hamster BMDM were exposed or not to hamster IL-4 (25 IU/mL), recombinant human IL-10 (100 ng/mL), recombinant human FGF-2 (20 ng/mL) and/or recombinant human IGF-1 (100 ng/mL) for 24 or 48 hrs. <b>A, C, E</b>) Arg1 mRNA expression determined by qRT-PCR at 24 hrs post-treatment. Shown is the mean and SEM of 6 replicates from a single experiment that was representative of 2 independent experiments. <b>B, D, F</b>) Arg1 protein expression determined in <i>L. donovani</i> infected BMDM exposed to IL-4, IL-10, and growth factors, alone or in combination, for 48 hrs. The membranes were stripped and stained with antibody against GAPDH to confirm equivalent protein loading. Bars represent the fold change with reference to control cells of uninfected hamsters calculated by densitometry analysis of immunoblot bands from 3 independent experiments. Also shown is a representative individual immunoblot. <b>G</b>) Arg1 protein expression determined in splenic macrophages from uninfected and <i>L. donovani</i> infected hamsters exposed <i>ex vivo</i> to IL-4 and growth factors, alone or in combination, for 48 hrs. The membranes were stripped and stained with antibody against GAPDH to confirm equivalent protein loading. Bars represent the fold change with reference to control cells of uninfected hamsters calculated by densitometry analysis of immunoblot bands from 3 independent experiments. Also shown is a representative individual immunoblot. <b>H</b>) Expression of IL-13Rα1 and IL-4Rα mRNA in splenic macrophages from uninfected (0) or 18-day infected hamsters determined by qRT-PCR. <b>I</b>) Expression of IL-13Rα1 and IL-4Rα mRNA in BMDM from uninfected (Un) and <i>L. donovani</i> infected (Inf) BMDMs (24 hrs p.i.) stimulated or not with IGF-1 or FGF-2. Shown is mean and SEM of the fold increase of receptor expression over uninfected, unstimulated controls from a single experiment representative of 2 independent experiments. *p<0.05; **p<0.01; ***p<0.001.</p

Parasite-induced arg1 expression in macrophages is dependent on STAT6.

<p>Expression of <b>A</b>) STAT6 mRNA and <b>B</b>) arg1 mRNA in BMDMs that were uninfected (Un) or infected <i>in vitro</i> with <i>L. donovani</i> (Inf) for 24 h after transfection with STAT6-specific knockdown siRNA (STAT6 KD) or scrambled siRNA (Control). Shown is the mean and SEM of the fold-change in mRNA compared to unstimulated controls as determined by qRT-PCR in 6 replicates from 2 independent experiments. <b>C</b>) Parasite burden at 24 h post-infection of STAT6 KD BMDMs or control. Shown is the mean and SEM of the parasite burden with reference to control (uninfected) cells in 4 replicates determined by qRT-PCR. <b>D</b>) STAT6 and arg1 mRNA expression in splenic macrophages from <i>L. donovani</i> infected hamsters 48 hrs after <i>ex vivo</i> transfection with STAT6-specific siRNA (STAT6 KD) or scrambled siRNA (Control). Data are shown as the mean and SEM of the percent of maximal mRNA expression in the control samples. *p<0.05; ***p<0.001.</p