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Abstract
Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, provoking liver and spleen tissue destruction that is lethal unless treated. The parasite replicates in macrophages and modulates host microbicidal responses. We have previously reported that neutrophil elastase (NE) is required to sustain L. donovani intracellular growth in macrophages through the induction of interferon beta (IFN-β). Here, we show that the gene expression of IFN-β by infected macrophages was reduced by half when TLR4 was blocked by pre-treatment with neutralizing antibodies or in macrophages from tlr2 -/- mice, while the levels in macrophages from myd88-/- mice were comparable to those from wild-type C57BL/6 mice. The neutralization of TLR4 in tlr2 -/- macrophages completely abolished induction of IFN-β gene expression upon parasite infection, indicating an additive role for both TLRs. Induction of type I interferon (IFN-I), OASL2, SOD1, and IL10 gene expression by L. donovani was completely abolished in macrophages from NE knock-out mice (ela2 -/-) or from protein kinase R (PKR) knock-out mice (pkr -/-), and in C57BL/6 macrophages infected with transgenic L. donovani expressing the inhibitor of serine peptidase 2 (ISP2). Parasite intracellular growth was impaired in pkr -/- macrophages but was fully restored by the addition of exogenous IFN-β, and parasite burdens were reduced in the spleen of pkr -/- mice at 7 days, as compared to the 129Sv/Ev background mice. Furthermore, parasites were unable to grow in macrophages lacking TLR3, which correlated with lack of IFN-I gene expression. Thus, L. donovani engages innate responses in infected macrophages via TLR2, TLR4, and TLR3, via downstream PKR, to induce the expression of pro-survival genes in the host cell, and guarantee parasite intracellular development.
Highlights
Visceral leishmaniasis (VL) is a deadly disease caused by the parasitic protozoa Leishmania donovani or Leishmania infantum
In L. major infections, we had observed that the neutrophil elastase (NE)-Toll-like receptor 4 (TLR4) axis leading to IFN-I was conveyed by protein kinase R (PKR) [13], we asked if this pathway was employed by L. donovani
We could readily detect the gene expression of both IFN-a (Figure 1A) and IFN-b (Figure 1B) in macrophages from 129Sv/Ev mice infected with L. donovani for 2 h or 6 h, in relation to control uninfected macrophages, but not in macrophages infected with L. donovani expressing ISP2, to that which has been described for macrophages from C57BL/6 mice [15]
Summary
Visceral leishmaniasis (VL) is a deadly disease caused by the parasitic protozoa Leishmania donovani or Leishmania infantum. Infective parasites are flagellated metacyclic promastigotes that can be taken up by phagocytes at the site of infection In macrophages, they differentiate to amastigotes that multiply inside a parasitophorous vacuole generated from the phagosome through the fusion/recruitment of elements from different endosomal compartments [3]. L. amazonensis, a species associated with cutaneous disease, employs the LPG-TLR2 pathway to stimulate downstream routes typically associated to anti-viral responses, i.e., the activation of the double-stranded RNA-dependent protein kinase R (PKR), and downstream induction of type I interferon (IFN-I) expression [9, 10] PKR activation and the upregulation of its expression, leading to IFNb production, is required for the concomitant production of IL10, and sustained parasite multiplication in infected cells [10]. Lack of ISP2 impacts long-term parasite survival in infected mice, mainly due to sustained recruitment of iNOS+ monocytes to the lesion site and higher local IFN-g levels [14]
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