The ecotin-like peptidase inhibitor of Trypanosoma cruzi prevents TMPRSS2-PAR2-TLR4 crosstalk downmodulating infection and inflammation.

Trypanosoma cruzi is the causative agent of Chagas disease, a chronic pathology that affects the heart and/or digestive system. This parasite invades and multiplies in virtually all nucleated cells, using a variety of host cell receptors for infection. T. cruzi has a gene that encodes an ecotin-like inhibitor of serine peptidases, ISP2. We generated ISP2-null mutants (Δisp2) in T. cruzi Dm28c using CRISPR/Cas9. Epimastigotes of Δisp2 grew normally in vitro but were more susceptible to lysis by human serum compared to parental and ISP2 add-back lines. Tissue culture trypomastigotes of Δisp2 were more infective to human muscle cells in vitro, which was reverted by the serine peptidase inhibitors aprotinin and camostat, suggesting that host cell epitheliasin/TMPRSS2 is the target of ISP2. Pretreatment of host cells with an antagonist to the protease-activated receptor 2 (PAR2) or an inhibitor of Toll-like receptor 4 (TLR4) selectively counteracted the increased cell invasion by Δisp2, but did not affect invasion by parental and add-back lines. The same was observed following targeted gene silencing of PAR2, TLR4 or TMPRSS2 in host cells by siRNA. Furthermore, Δisp2 caused increased tissue edema in a BALB/c mouse footpad infection model after 3 h differently to that observed following infection with parental and add-back lines. We propose that ISP2 contributes to protect T. cruzi from the anti-microbial effects of human serum and to prevent triggering of PAR2 and TLR4 in host cells, resulting in the modulation of host cell invasion and contributing to decrease inflammation during acute infection.

Toll-Like Receptor 3 (TLR3) Is Engaged in the Intracellular Survival of the Protozoan Parasite Leishmania (Leishmania) amazonensis.

The protozoan parasite Leishmania (L.) amazonensis infects and replicates inside host macrophages due to subversion of the innate host cell response. In the present study, we demonstrate that TLR3 is required for the intracellular growth of L. (L.) amazonensis. We observed restricted intracellular infection of TLR3-/- mouse macrophages, reduced levels of IFN1ß and IL-10, and increased levels of IL-12 upon L. (L.) amazonensis infection, compared with their wild-type counterparts. Accordingly, in vivo infection of TLR3-/- mice with L. (L.) amazonensis displayed a significant reduction in lesion size. Leishmania (L.) amazonensis infection induced TLR3 proteolytic cleavage, which is a process required for TLR3 signaling. The chemical inhibition of TLR3 cleavage or infection by CPB-deficient mutant L. (L.) mexicana resulted in reduced parasite load and restricted the expression of IFN1ß and IL-10. Furthermore, we show that the dsRNA sensor molecule PKR (dsRNA-activated protein kinase) cooperates with TLR3 signaling to potentiate the expression of IL-10 and IFN1ß and parasite survival. Altogether, our results show that TLR3 signaling is engaged during L. (L.) amazonensis infection and this component of innate immunity modulates the host cell response.

Toll-Like Receptor- and Protein Kinase R-Induced Type I Interferon Sustains Infection of Leishmania donovani in Macrophages.

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-ß). Here, we show that the gene expression of IFN-ß 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-ß 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-ß, 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.

Leishmania amazonensis downregulates macrophage iNOS expression via Histone Deacetylase 1 (HDAC1): a novel parasite evasion mechanism.

The induced expression of nitric oxide synthase (iNOS) controls the intracellular growth of Leishmania in infected macrophages. Histones deacetylases (HDACs) negatively regulate gene expression through the formation of complexes containing transcription factors such as NF-κB p50/50. Herein, we demonstrated the occupancy of p50/p50_HDAC1 to iNOS promoter associated with reduced levels of H3K9Ac. Remarkably, we found increased levels of HDAC1 in L. amazonensis-infected macrophages. HDAC1 upregulation was not found in L. major-infected macrophages. The parasite intracellular load was reduced in HDAC1 knocked-down macrophages, which presented increased nitric oxide levels. HDAC1 silencing led to the occupancy of CBP/p300 to iNOS promoter and the rise of H3K9Ac modification. Importantly, the immunostaining of skin samples from hiporeactive cutaneous leishmaniasis patients infected with L. amazonensis, revealed high levels of HDAC1. In brief, L. amazonensis induces HDAC1 in infected macrophages, which contribute to parasite survival and is associated to hiporeactive stage found in L. amazonensis infected patients.

Emerging Role for the PERK/eIF2α/ATF4 in Human Cutaneous Leishmaniasis.

Leishmania parasites utilize adaptive evasion mechanisms in infected macrophages to overcome host defenses and proliferate. We report here that the PERK/eIF2α/ATF4 signaling branch of the integrated endoplasmic reticulum stress response (IERSR) is activated by Leishmania and this pathway is important for Leishmania amazonensis infection. Knocking down PERK or ATF4 expression or inhibiting PERK kinase activity diminished L. amazonensis infection. Knocking down ATF4 decreased NRF2 expression and its nuclear translocation, reduced HO-1 expression and increased nitric oxide production. Meanwhile, the increased expression of ATF4 and HO-1 mRNAs were observed in lesions derived from patients infected with the prevalent related species L.(V.) braziliensis. Our data demonstrates that Leishmania parasites activate the PERK/eIF2α/ATF-4 pathway in cultured macrophages and infected human tissue and that this pathway is important for parasite survival and progression of the infection.

The human parasite Leishmania amazonensis downregulates iNOS expression via NF-κB p50/p50 homodimer: role of the PI3K/Akt pathway.

Leishmania amazonensis activates the NF-κB transcriptional repressor homodimer (p50/p50) and promotes nitric oxide synthase (iNOS) downregulation. We investigated the role of PI3K/Akt in p50/p50 NF-κB activation and the effect on iNOS expression in L. amazonensis infection. The increased occupancy of p50/p50 on the iNOS promoter of infected macrophages was observed and we demonstrated that both p50/p50 NF-κB induction and iNOS downregulation in infected macrophages depended on PI3K/Akt activation. Importantly, the intracellular growth of the parasite was also impaired during PI3K/Akt signalling inhibition and in macrophages knocked-down for Akt 1 expression. It was also observed that the increased nuclear levels of p50/p50 in L. amazonensis-infected macrophages were associated with reduced phosphorylation of 907 Ser p105, the precursor of p50. Corroborating these data, we demonstrated the increased levels of phospho-9 Ser GSK3ß in infected macrophages, which is associated with GSK3ß inhibition and, consequently, its inability to phosphorylate p105. Remarkably, we found that the levels of pPTEN 370 Ser, a negative regulator of PI3K, increased due to L. amazonensis infection. Our data support the notion that PI3K/Akt activity is sustained during the parasite infection, leading to NF-κB 105 phosphorylation and further processing to originate p50/p50 homodimers and the consequent downregulation of iNOS expression.