Leishmania virulence factors: inhibitors of serine peptidases

Leishmania spp. are protozoan parasites that cause a spectrum of pathologies in humans and other vertebrates, ranging symptomatically from cutaneous ulceration to visceral dissemination. In order to survive within the host, Leishmania are able to evade and modulate the host immune responses through the actions of their virulence factors; however, few putative virulence factors have been characterised during in vivo infection with Leishmania. The Leishmania major genome has revealed the presence of three peptide inhibitors of S1A family serine peptidases (ISPs), which are orthologues of a bacterial protease inhibitor, ecotin. Serine peptidases of the S1A family are absent in Leishmania; therefore, the ISPs have been proposed to inhibit the activity of host serine peptidases, such as those expressed by cells of the innate immune response. ISP2, which is expressed in the mammalian-infective metacyclic promastigote and amastigote stages, has previously been shown to inhibit neutrophil elastase (NE), a serine peptidase expressed by neutrophils, monocytes, and macrophages. This inhibition prevents the activation of a Toll-like receptor 4 (TLR4)-NE pathway during Leishmania-macrophage interaction promoting Leishmania survival and growth in macrophages in vitro. The aims of this project were to assess whether the presence or absence of ISP2 in L. major affects parasite survival in vivo, and to investigate the effects of ISP2 on immune cell dynamics in vivo, specifically with regards to cell recruitment and activation, using the C57BL/6 mouse model. Parasite burdens were performed in mice infected with L. major wild-type (WT) parasites, a cell line deficient in ISP2/3 (Δisp2/3), and a cell line re-expressing ISP2/3 (Δisp2/3:ISP2/3). L. major Δisp2/3 parasites could not be detected at the site of inoculation by 5 wk post-infection compared with WT and Δisp2/3:ISP2/3 parasites, but parasites of all three cell lines were detected in the draining lymph nodes (dLNs) throughout the course of infection. These data were corroborated using in vivo bioluminescence imaging (BLI) of luciferase-expressing (LUC2) versions of these cell lines, in which only a low bioluminescent signal was observed at the site of inoculation with the LUC2-expressing Δisp2/3 cell line over the course of infection, compared with the LUC2-expressing L. major and Δisp2/3:ISP2/3 cell lines. These results suggest that ISP2 may be important in the establishment and persistence of Leishmania infection by conferring parasite survival, particularly at the site of infection. Serine peptidases of innate immune cells, such as NE, function in the proteolytic cleavage of cytokines, chemokines, and cell receptors, to regulate immune cell recruitment and activation. Flow cytometric analysis of innate cell populations at the site of inoculation, in response to L. major WT and Δisp2/3 parasites over 5 wk of infection, was conducted. This line of investigation revealed significantly higher numbers of monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells (moDCs) at 2 wk in Δisp2/3 infection. MoDCs have crucial functions in the induction of antigen-specific T helper 1 responses, which are considered to be important for parasite clearance. MoDCs at the site of Δisp2/3 infection showed an upregulation of the DC co-stimulatory molecule CD80 compared with those from WT infection suggesting an upregulation of DC maturation. MoDCs have also been shown to be the major producers of inducible nitric oxide synthase (iNOS) during L. major infection, which catalyses the production of nitric oxide that is responsible for the killing of Leishmania. Intracellular staining of iNOS through flow cytometric techniques showed that iNOS expression in moDCs was not affected by the presence or absence of ISP2; there was, however, an increase in iNOS expression in other innate cell types, the resident macrophages and DCs, monocytes, and monocyte-derived macrophages, at the site of Δisp2/3 infection compared with those from WT and Δisp2/3:ISP2/3 infections. At the 2 wk time-point, there was also a significant increase in the concentration of IFN-γ, a cytokine that induces iNOS expression, in response to Δisp2/3 infection compared with WT and Δisp2/3:ISP2/3 infections, as determined by ELISA. Quantitative in vivo BLI of myeloperoxidase (MPO) activity of activated phagocytes was determined over a period of 7 wk, which, also, indicated differences in phagocyte activation at the site of inoculation in L. major WT and Δisp2/3 infections. Taken together, these results indicate that the immune response is more primed towards Leishmania killing in Δisp2/3 infection compared with WT infection, which suggests that ISP2 modulates these immune responses to facilitate Leishmania survival. Infections in transgenic mice deficient in NE, Ela-/-, showed similar monocyte recruitment and moDC activation responses in Δisp2/3 infection compared with WT and Δisp2/3:ISP2/3 infections, as those observed in the C57BL/6 mice. This indicates that NE may not be the major target for ISP2 in vivo, or that there may be compensations for the loss of NE by other serine peptidases in this model. Although the exact mechanism by which ISP2 modulates the recruitment and activation of the innate immune cells in vivo remains to be determined, this study has, for the first time, shown numerous differences in the innate immune responses induced following infection with either L. major WT or a mutant deficient in a putative virulence factor using in vivo techniques, such as in vivo imaging (IVIS) and flow cytometry, to compare the infections