A secondary wave of neutrophil infiltration causes necrosis and ulceration in lesions of experimental American cutaneous leishmaniasis

<div><p>We evaluated the importance of neutrophils in the development of chronic lesions caused by <i>L</i>. <i>Viannia spp</i>. using the hamster as experimental model of American Cutaneous Leishmaniasis (ACL). Neutrophils infiltrated the lesion within the first six hours post-infection. Inhibition of this early infiltration using a polyclonal antibody or cyclophosphamide was associated with transient parasite control but the protective effect vanished when lesions became clinically apparent. At lesion onset (approximately 10 days p.i.), there was an increased proportion of both uninfected and infected macrophages, and subsequently a second wave of neutrophils infiltrated the lesion (after 19 days p.i.) This second neutrophil infiltration was associated with lesion necrosis and ulceration (R² = 0.75) and maximum parasite burden. Intradermal delivery of N-formylmethionyl-leucyl-phenylalanine (fMLP), aimed to increase neutrophil infiltration, resulted in larger lesions with marked necrosis and higher parasite burden than in mock treated groups (p<0.001 each). In contrast, reduced neutrophil infiltration via cyclophosphamide-mediated depletion led to more benign lesions and lower parasite loads compared to controls (p<0.001 each). Neutrophils of the second wave expressed significantly lower GM-CSF, reactive oxygen species and nitric oxide than those of the first wave, suggesting that they had less efficient anti-leishmania activity. However, there was increased inflammatory cytokines and expression of neutrophil proteases (myeloperoxidase, cathepsin G and elastase) in lesions during the second wave of neutrophil infiltration compared with the levels reached during the first wave (6h p.i.). This suggests that augmented neutrophil proteases and inflammatory cytokines during the secondary wave of neutrophils could contribute to skin inflammation, ulceration and necrosis in ACL. The overall results indicate that neutrophils were unable to clear the infection in this model, and that the second wave of neutrophils played an important role in the severity of ACL.</p></div

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

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

Transcriptional Profiling in Experimental Visceral Leishmaniasis Reveals a Broad Splenic Inflammatory Environment that Conditions Macrophages toward a Disease-Promoting Phenotype

<div><p>Visceral Leishmaniasis (VL), caused by the intracellular protozoan <i>Leishmania donovani</i>, is characterized by relentlessly increasing visceral parasite replication, cachexia, massive splenomegaly, pancytopenia and ultimately death. Progressive disease is considered to be due to impaired effector T cell function and/or failure of macrophages to be activated to kill the intracellular parasite. In previous studies, we used the Syrian hamster (<i>Mesocricetus auratus</i>) as a model because it mimics the progressive nature of active human VL. We demonstrated previously that mixed expression of macrophage-activating (IFN-γ) and regulatory (IL-4, IL-10, IL-21) cytokines, parasite-induced expression of macrophage arginase 1 (Arg1), and decreased production of nitric oxide are key immunopathologic factors. Here we examined global changes in gene expression to define the splenic environment and phenotype of splenic macrophages during progressive VL. We used RNA sequencing coupled with <i>de novo</i> transcriptome assembly, because the Syrian hamster does not have a fully sequenced and annotated reference genome. Differentially expressed transcripts identified a highly inflammatory spleen environment with abundant expression of type I and type II interferon response genes. However, high IFN-γ expression was ineffective in directing exclusive M1 macrophage polarization, suppressing M2-associated gene expression, and restraining parasite replication and disease. While many IFN-inducible transcripts were upregulated in the infected spleen, fewer were induced in splenic macrophages in VL. Paradoxically, IFN-γ enhanced parasite growth and induced the counter-regulatory molecules Arg1, Ido1 and Irg1 in splenic macrophages. This was mediated, at least in part, through IFN-γ-induced activation of STAT3 and expression of IL-10, which suggests that splenic macrophages in VL are conditioned to respond to macrophage activation signals with a counter-regulatory response that is ineffective and even disease-promoting. Accordingly, inhibition of STAT3 activation led to a reduced parasite load in infected macrophages. Thus, the STAT3 pathway offers a rational target for adjunctive host-directed therapy to interrupt the pathogenesis of VL.</p></div

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

Protease expression was enhanced with the infection and during the second wave of neutrophil infiltration.

<p>(A-C), Expression of myeloperoxidase, cathepsin G, matrix metallopeptidase 8 (MMP8), proteinase-3, neutrophil elastase and matrix metallopeptidase 9 (MMP9) determined in skin from uninfected hamsters (Un), or in the infection site of hamsters infected with <i>L</i>. <i>V</i>. <i>panamensis</i>. Lesions obtained during the first wave of neutrophil infiltration (1^st wave, 6h p.i.) or during the second wave of neutrophil infiltration (2^nd wave, 28d p.i.). Determined by qPCR.*p<0.01; **p<0.001; ***p<0.001, n = 5–7 lesions per group. One-way Anova, Kruskal-Wallis statistic.</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

IFN-γ signaling leads to altered gene expression and increased parasite load in infected splenic macrophages.

<p>Differentially expressed transcripts from spleen tissue <b>(A)</b> and splenic macrophages <b>(B)</b> were loaded into IPA and the canonical IFN-γ signaling pathway generated. Transcripts upregulated in infection are shaded in red. <b>(C)</b> Expression of Cxcl9 and Cxcl10 in bone marrow derived macrophages (BMDMs) that were uninfected (Un) or infected <i>in vitro</i> with <i>L</i>. <i>donovani</i> (Ld), and left unstimulated (C) or stimulated with IFN-α, IFN-γ, or a combination of both (IFNα/γ) for 24 hrs. Data are shown as the mean and SEM of the fold-change relative to the uninfected, unstimulated group. <b>(D)</b> Parasite burden in bone marrow derived macrophages infected <i>in vitro</i> with <i>L</i>. <i>donovani</i> and left unstimulated (Con) or stimulated by IFN-α, IFN-γ, or a combination of both (IFNα/γ) for 24 hrs. <b>(E)</b> Relative parasite burden in splenic macrophages isolated from hamsters at 7, 14, 21, and 42 days after <i>L</i>. <i>donovani</i> infection, cultured and stimulated <i>ex vivo</i> for 24 hrs with hamster IFNγ (+) or mock supernatant (-). Parasite load was determined by expression of Leishmania 18S gene and fold-increase calculated against uninfected cells. <b>(F)</b> Expression of Arg1, Ido1, and Irg1 in uninfected (Un) and infected (Ld) BMDMs treated for 24 hrs with recombinant mouse IFN-α, recombinant hamster IFN-γ, or a combination of both (IFNα/γ). <b>(G)</b> Expression of Arg1 in uninfected BMDMs treated for 24 hrs with recombinant hamster IL-4, IFN-γ, or a combination of both (IL4-IFNγ). *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p