Neutrophil elastase promotes Leishmania donovani infection via interferon-ß.

Visceral leishmaniasis is a deadly illness caused by Leishmania donovani that provokes liver and spleen inflammation and tissue destruction. In cutaneous leishmaniasis, the protein of L. major, named inhibitor of serine peptidases (ISP) 2, inactivates neutrophil elastase (NE) present at the macrophage surface, resulting in blockade of TLR4 activation, prevention of TNF-α and IFN-ß production, and parasite survival. We report poor intracellular growth of L. donovani in macrophages from knockout mice for NE (ela-/-), TLR4, or TLR2. NE and TLR4 colocalized with the parasite in the parasitophorous vacuole. Parasite load in the liver and spleen of ela-/- mice were reduced and accompanied by increased NO and decreased TGF-ß production. Expression of ISP2 was not detected in L. donovani, and a transgenic line constitutively expressing ISP2, displayed poor intracellular growth in macrophages and decreased burden in mice. Infected ela-/- macrophages displayed significantly lower IFN-ß mRNA than background mice macrophages, and the intracellular growth was fully restored by exogenous IFN-ß. We propose that L. donovani utilizes the host NE-TLR machinery to induce IFN-ß necessary for parasite survival and growth during early infection. Low or absent expression of parasite ISP2 in L. donovani is necessary to preserve the activation of the NE-TLR pathway.-Dias, B. T., Dias-Teixeira, K. L., Godinho, J. P., Faria, M. S., Calegari-Silva, T., Mukhtar, M. M., Lopes, U. G., Mottram, J. C., Lima, A. P. C. A. Neutrophil elastase promotes Leishmania donovani infection via interferon-ß.

Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNFα and IFNß.

In cutaneous leishmaniasis, Leishmania amazonensis activates macrophage double-stranded, RNA-activated protein kinase R (PKR) to promote parasite growth. In our study, Leishmania major grew normally in RAW cells, RAW-expressing dominant-negative PKR (PKR-DN) cells, and macrophages of PKR-knockout mice, revealing that PKR is dispensable for L. major growth in macrophages. PKR activation in infected macrophages with poly I:C resulted in parasite death. Fifty percent of L. major-knockout lines for the ecotin-like serine peptidase inhibitor (ISP2; Δisp2/isp3), an inhibitor of neutrophil elastase (NE), died in RAW cells or macrophages from 129Sv mice, as a result of PKR activation. Inhibition of PKR or NE or neutralization of Toll-like receptor 4 or 2(TLR4 or TLR2) prevented the death of Δisp2/isp3. Δisp2/isp3 grew normally in RAW-PKR-DN cells or macrophages from 129Sv pkr(-/-), tlr2(-/-), trif(-/-), and myd88(-/-) mice, associating NE activity, PKR, and TLR responses with parasite death. Δisp2/isp3 increased the expression of mRNA for TNF-α by 2-fold and of interferon ß (IFNß) in a PKR-dependent manner. Antibodies to TNF-α reversed the 95% killing by Δisp2/isp3, whereas they grew normally in macrophages from IFN receptor-knockout mice. We propose that ISP2 prevents the activation of PKR via an NE-TLR4-TLR2 axis to control innate responses that contribute to the killing of L. major.-Faria, M. S., Calegari-Silva, T. C., de Carvalho Vivarini, A., Mottram, J. C., Lopes, U. G., Lima, A. P. C. A. Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNFα and IFNß.

Toll-like receptors in leishmania infections: guardians or promoters?

Protozoa of the genus Leishmania cause a wide variety of pathologies ranging from self-healing skin lesions to visceral damage, depending on the parasite species. The outcome of infection depends on the quality of the adaptive immune response, which is determined by parasite factors and the host genetic background. Innate responses, resulting in the generation of mediators with anti-leishmanial activity, contribute to parasite control and help the development of efficient adaptive responses. Among those, the potential contribution of members of the Toll-like receptors (TLRs) family in the control of Leishmania infections started to be investigated about a decade ago. Although most studies appoint a protective role for TLRs, there is growing evidence that in some cases, TLRs facilitate infection. This review highlights recent advances in TLR function during Leishmania infections and discusses their potential role in restraining parasite growth versus yielding disease.

Ecotin-like serine peptidase inhibitor ISP1 of Leishmania major plays a role in flagellar pocket dynamics and promastigote differentiation.

Leishmania ISPs are ecotin-like natural peptide inhibitors of trypsin-family serine peptidases, enzymes that are absent from the Leishmania genome. This led to the proposal that ISPs inhibit host serine peptidases and we have recently shown that ISP2 inhibits neutrophil elastase, thereby enhancing parasite survival in murine macrophages. In this study we show that ISP1 has less serine peptidase inhibitory activity than ISP2, and in promastigotes both are generally located in the cytosol and along the flagellum. However, in haptomonad promastigotes there is a prominent accumulation of ISP1 and ISP2 in the hemidesmosome and for ISP2 on the cell surface. An L. major mutant deficient in all three ISP genes (Δisp1/2/3) was generated and compared with Δisp2/3 mutants to elucidate the physiological role of ISP1. In in vitro cultures, the Δisp1/2/3 mutant contained more haptomonad, nectomonad and leptomonad promastigotes with elongated flagella and reduced motility compared with Δisp2/3 populations, moreover it was characterized by very high levels of release of exosome-like vesicles from the flagellar pocket. These data suggest that ISP1 has a primary role in flagellar homeostasis, disruption of which affects differentiation and flagellar pocket dynamics.

Leishmania inhibitor of serine peptidase 2 prevents TLR4 activation by neutrophil elastase promoting parasite survival in murine macrophages.

Leishmania major is a protozoan parasite that causes skin ulcerations in cutaneous leishmaniasis. In the mammalian host, the parasite resides in professional phagocytes and has evolved to avoid killing by macrophages. We identified L. major genes encoding inhibitors of serine peptidases (ISPs), which are orthologs of bacterial ecotins, and found that ISP2 inhibits trypsin-fold S1A family peptidases. In this study, we show that L. major mutants deficient in ISP2 and ISP3 (Δisp2/3) trigger higher phagocytosis by macrophages through a combined action of the complement type 3 receptor, TLR4, and unregulated activity of neutrophil elastase (NE), leading to parasite killing. Whereas all three components are required to mediate enhanced parasite uptake, only TLR4 and NE are necessary to promote parasite killing postinfection. We found that the production of superoxide by macrophages in the absence of ISP2 is the main mechanism controlling the intracellular infection. Furthermore, we show that NE modulates macrophage infection in vivo, and that the lack of ISP leads to reduced parasite burdens at later stages of the infection. Our findings support the hypothesis that ISPs function to prevent the activation of TLR4 by NE during the Leishmania-macrophage interaction to promote parasite survival and growth.

Influence of parasite encoded inhibitors of serine peptidases in early infection of macrophages with Leishmania major.

Ecotin is a potent inhibitor of family S1A serine peptidases, enzymes lacking in the protozoan parasite Leishmania major. Nevertheless, L. major has three ecotin-like genes, termed inhibitor of serine peptidase (ISP). ISP1 is expressed in vector-borne procyclic and metacyclic promastigotes, whereas ISP2 is also expressed in the mammalian amastigote stage. Recombinant ISP2 inhibited neutrophil elastase, trypsin and chymotrypsin with K(i)s between 7.7 and 83 nM. L. major ISP2-ISP3 double null mutants (Deltaisp2/3) were created. These grew normally as promastigotes, but were internalized by macrophages more efficiently than wild-type parasites due to the upregulation of phagocytosis by a mechanism dependent on serine peptidase activity. Deltaisp2/3 promastigotes transformed to amastigotes, but failed to divide for 48 h. Intracellular multiplication of Deltaisp2/3 was similar to wild-type parasites when serine peptidase inhibitors were present, suggesting that defective intracellular growth results from the lack of serine peptidase inhibition during promastigote uptake. Deltaisp2/3 mutants were more infective than wild-type parasites to BALB/c mice at the early stages of infection, but became equivalent as the infection progressed. These data support the hypothesis that ISPs of L. major target host serine peptidases and influence the early stages of infection of the mammalian host.