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

Inhibitor of serine peptidase 2 enhances Leishmania major survival in the skin through control of monocytes and monocyte-derived cells

Leishmania major is the causative agent of the neglected tropical disease, cutaneous leishmaniasis. In the mouse, protective immunity to Leishmania is associated with inflammatory responses. Here, we assess the dynamics of the inflammatory responses at the lesion site during experimental long-term, low-dose intradermal infection of the ear, employing noninvasive imaging and genetically modified L. major Significant infiltrates of neutrophils and monocytes occurred at 1-4 d and 2-4 wk, whereas dermal macrophage and dendritic cell (DC) numbers were only slightly elevated in the first days. Quantitative whole-body bioluminescence imaging of myeloperoxidase activity and the quantification of parasite loads indicated that the Leishmania virulence factor, inhibitor of serine peptidase 2 (ISP2), is required to modulate phagocyte activation and is important for parasite survival at the infection site. ISP2 played a role in the control of monocyte, monocyte-derived macrophage, and monocyte-derived DC (moDC) influx, and was required to reduce iNOS expression in monocytes, monocyte-derived cells, and dermal DCs; the expression of CD80 in moDCs; and levels of IFN-γ in situ. Our findings indicate that the increased survival of L. major in the dermis during acute infection is associated with the down-regulation of inflammatory monocytes and monocyte-derived cells via ISP2.-Goundry, A., Romano, A., Lima, A. P. C. A., Mottram, J. C., Myburgh, E. Inhibitor of serine peptidase 2 enhances Leishmania major survival in the skin through control of monocytes and monocyte-derived cells

TLR2, TLR4 and Protein kinase R (PKR) 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-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

Attempts to Image the Early Inflammatory Response during Infection with the Lymphatic Filarial Nematode Brugia pahangi in a Mouse Model

Helminth parasites remain a major constraint upon human health and well-being in many parts of the world. Treatment of these infections relies upon a very small number of therapeutics, most of which were originally developed for use in animal health. A lack of high throughput screening systems, together with limitations of available animal models, has restricted the development of novel chemotherapeutics. This is particularly so for filarial nematodes, which are long-lived parasites with a complex cycle of development. In this paper, we describe attempts to visualise the immune response elicited by filarial parasites in infected mice using a non-invasive bioluminescence imaging reagent, luminol, our aim being to determine whether such a model could be developed to discriminate between live and dead worms for in vivo compound screening. We show that while imaging can detect the immune response elicited by early stages of infection with L3, it was unable to detect the presence of adult worms or, indeed, later stages of infection with L3, despite the presence of worms within the lymphatic system of infected animals. In the future, more specific reagents that detect secreted products of adult worms may be required for developing screens based upon live imaging of infected animals

<i>In vivo</i> imaging of MPO-specific bioluminescence in mice implanted with adult <i>B</i>. <i>pahangi</i>.

<p>BALB/c mice were infected intraperitoneally by transplantation with 10 female adult <i>B</i>. <i>pahangi (B</i>.<i>p)</i>. Sham-operated mice (Sham) received no worms and control mice (Ctl) received an intraperitoneal injection of HBSS but underwent no surgery. On day 17 post-infection mice were imaged using an IVIS spectrum 20 minutes after subcutaneous injection of 200 mg/kg luminol. Representative images of 2 mice per group are shown. The colour scale indicates bioluminescence radiance in photons/second/cm²/steradian. Graphs show the bioluminescence total flux (in photons/second) within the same abdominal region of interest. Each symbol shows the total flux for a single mouse, lines indicate the means (n = 4–6 mice) and error bars show SD (*<i>p</i> < 0.05 using a one-way ANOVA with Dunn’s post-test).</p

<i>Trypanosoma brucei rhodesiense</i> Inhibitor of Cysteine Peptidase (ICP) Is Required for Virulence in Mice and to Attenuate the Inflammatory Response

The protozoan Trypanosoma brucei rhodesiense causes Human African Trypanosomiasis, also known as sleeping sickness, and penetrates the central nervous system, leading to meningoencephalitis. The Cathepsin L-like cysteine peptidase of T. b. rhodesiense has been implicated in parasite penetration of the blood–brain barrier and its activity is modulated by the chagasin-family endogenous inhibitor of cysteine peptidases (ICP). To investigate the role of ICP in T. b. rhodesiense bloodstream form, ICP-null (Δicp) mutants were generated, and lines re-expressing ICP (Δicp:ICP). Lysates of Δicp displayed increased E-64-sensitive cysteine peptidase activity and the mutant parasites traversed human brain microvascular endothelial cell (HBMEC) monolayers in vitro more efficiently. Δicp induced E-selectin in HBMECs, leading to the adherence of higher numbers of human neutrophils. In C57BL/6 mice, no Δicp parasites could be detected in the blood after 6 days, while mice infected with wild-type (WT) or Δicp:ICP displayed high parasitemia, peaking at day 12. In mice infected with Δicp, there was increased recruitment of monocytes to the site of inoculation and higher levels of IFN-γ in the spleen. At day 14, mice infected with Δicp exhibited higher preservation of the CD4+, CD8+, and CD19+ populations in the spleen, accompanied by sustained high IFN-γ, while NK1.1+ populations receded nearly to the levels of uninfected controls. We propose that ICP helps to downregulate inflammatory responses that contribute to the control of infection

<i>In vivo</i> imaging of MPO-specific bioluminescence in mice infected with <i>B</i>. <i>pahangi</i> L3 larvae.

<p>BALB/c mice were injected with 50 L3 of <i>B</i>. <i>pahangi</i> into the RHS footpad (<i>B</i>.<i>p</i>) and with HBSS into the LHS footpad. On day 4 (<b>A</b>) and day 11 (<b>B</b>) post-infection mice were imaged using an IVIS spectrum 20 minutes after subcutaneous injection of 200 mg/kg luminol. Representative images of 3 mice per group are shown. The colour scale indicates bioluminescence radiance in photons/second/cm²/steradian. Graphs show the bioluminescence total flux (in photons/second) within the same footpad region of interest (ROI, red ovals in images). Each symbol shows the total flux for a single mouse, lines indicate the means (n = 7 mice) and error bars show SD (**<i>p</i> < 0.01 using a Mann-Whitney test).</p

Histopathology of <i>B</i>. <i>pahangi</i> infected and uninfected limbs in a mouse model.

<p>BALB/c mice were injected with 50 L3 of <i>B</i>. <i>pahangi</i> into the RHS footpad and with HBSS into the LHS footpad and analysed on the days p.i. as indicated. <b>(A)</b> uninfected mouse, Day 21, HBSS-injected limb: there is no inflammation in the fascial plane <b>(B)</b> infected mouse, Day 7: a filarial nematode is present within the lumen of a dilated lymphatic vessel in the fascial plane. <b>(C)</b> infected mouse, Day 14 prominent lymphangiectasis and lymphangitis in the fascial plane. <b>(D)</b> infected mouse, Day 21: marked inflammatory infiltration obliterating the lumen of a lymphatic vessel. Inset: remnants of degenerate nematodes within the lumen surrounded by inflammatory cells. <b>(E)</b> infected mouse (same animal as section C), Day 14: the inflammatory infiltrate affecting the lymphatic vessel in the fascial plane is characterised by the presence of many eosinophils highlighted by the modified haematoxylin eosin method. <b>(F)</b> infected mouse, Day 21: nematodes surrounded by intense inflammatory infiltration comprising large numbers of eosinophils highlighted by the modified Congo Red protocol.</p

Role of the inhibitor of serine peptidase 2 (ISP2) of Trypanosoma brucei rhodesiense in parasite virulence and modulation of the inflammatory responses of the host

Trypanosoma brucei rhodesiense is one of the causative agents of Human African Trypanosomiasis (HAT), known as sleeping sickness. The parasite invades the central nervous system and causes severe encephalitis that is fatal if left untreated. We have previously identified ecotin-like inhibitors of serine peptidases, named ISPs, in trypanosomatid parasitic protozoa. Here, we investigated the role of ISP2 in bloodstream form T. b. rhodesiense. We generated gene-deficient mutants lacking ISP2 (Δisp2), which displayed a growth profile in vitro similar to that of wild-type (WT) parasites. C57BL/6 mice infected with Δisp2 displayed lower blood parasitemia, a delayed hind leg pathological phenotype and survived longer. The immune response was examined at two time-points that corresponded with two peaks of parasitemia. At 4 days, the spleens of Δisp2-infected mice had a greater percentage of NOS2+ myeloid cells, IFN-γ+-NK cells and increased TNF-α compared to those infected with WT and parasites re-expressing ISP2 (Δisp2:ISP2). By 13 days the increased NOS2+ population was sustained in Δisp2-infected mice, along with increased percentages of monocyte-derived dendritic cells, as well as CD19+ B lymphocytes, and CD8+ and CD4+ T lymphocytes. Taken together, these findings indicate that ISP2 contributes to T. b. rhodesiense virulence in mice and attenuates the inflammatory response during early infection