Molecular approaches to understand vector-host and vector-pathogen interactions

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

The accomplishments of the section are: 1. We discovered that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrated murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We established that the structure of this 45kDa neutrophil chemotactic protein does not resemble that of known chemokines. This novel salivary chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P<0.0001) and increases parasite burden (P<0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host. 2. We demonstrated that heme oxygenase-1 (HO-1), a pleiotropic cytoprotective isoenzyme that mitigates heme-mediated tissue damage, is induced after bites of sand flies, mosquitoes, and ticks. Further, we demonstrated that erythrophagocytosis by macrophages, including a skin-residing CD163+CD91+ professional iron-recycling subpopulation, produces HO-1 after bites. Importantly, we established that global deletion or transient inhibition of HO-1 in mice increases inflammation and pathology following Leishmania-infected sand fly bites without affecting parasite number, whereas CO, an end product of the HO-1 enzymatic reaction, suppressed skin inflammation. Our results indicate that HO-1 induction by blood-feeding sand flies promotes tolerance to Leishmania infection. Collectively, our data demonstrate that HO-1 induction through erythrophagocytosis is a universal mechanism that regulates skin inflammation following blood feeding by arthropods, thus promoting early-stage disease tolerance to vector-borne pathogens. 3. We investigated the effects of Leishmania infection on sand fly midgut gene expression by performing RNA-Seq analysis of Leishmania infantum-infected Lutzomyia longipalpis midguts at different time points, each corresponding to when the insect midguts are enriched with a particular Leishmania developmental stage. These encompassed early time points when blood digestion is taking place as well as late time points when the parasites are undergoing metacyclogenesis. This approach expands our breadth of knowledge by assessing the effect of Leishmania infection on over 13,000 sand fly midgut transcripts, focusing on genes encoding secreted proteins and also on genes participating in other biological processes of midgut epithelial cells. Surprisingly only 113 sand fly transcripts, about 1%, were differentially expressed in the presence of Leishmania parasites. Two main patterns of sand fly gene expression modulation were noted. At early time points (days 14), more transcripts were down-regulated by Leishmania infection at large fold changes (>32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 2 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by Leishmania infection with small fold changes (<32 fold). The molecular functions of these genes have been associated with the metabolism of lipids and detoxification of xenobiotics. Overall, our data suggest that the presence of Leishmania produces a limited change in the midgut transcript expression profile in sand flies. Further, Leishmania modulates sand fly gene expression early on in the developmental cycle in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points where a massive number of parasites in the anterior midgut results only in modest changes in midgut gene expression. 4. We tested a dermotropic live attenuated centrin gene deleted Leishmania major (LmCen/) vaccine that was developed under Good Laboratory Practices. We demonstrated that a single intradermal injection confers robust and durable protection against lethal VL transmitted naturally via bites of L. donovani-infected sand flies and prevents mortality. Surprisingly, immunogenicity characteristics of LmCen/ parasites revealed activation of common immune pathways like L. major wild type parasites. Spleen cells from LmCen/ immunized and L. donovani challenged hamsters produced significantly higher Th1-associated cytokines including IFN-, TNF-, and reduced expression of the anti-inflammatory cytokines like IL-10, IL-21, compared to non-immunized challenged animals. PBMCs, isolated from healthy people from non-endemic region, upon LmCen/ infection also induced more IFN- compared to IL-10, consistent with our immunogenicity data in LmCen/ immunized hamsters. This study demonstrates that the LmCen/ parasites are safe and efficacious against VL and is a strong candidate vaccine to be tested in a human clinical trial. 5. We investigated in Mali, an endemic area for cutaneous leishmaniasis and filariasis the effect of Phlebotomus duboscqi salivary glands (SG) on human monocytes in Malian subjects exposed or unexposed to sand fly bites in the context of a concomitant filaria infection. Monocytes of unexposed Malian individuals selectively upregulated the production of IL-6 and CCL4 in response to SG from Pd or other vector species. In contrast, monocytes of individuals exposed to sand fly bites lost their responsiveness to SG, microfilariae antigen and LPS, irrespective of co-infection with filaria. Nevertheless, infection with filaria significantly upreguled the frequency of CCL22+monocytes, IL-10+mDCs and regulatory T cells. Together, our data suggest that repeated exposure to Phlebotomus duboscqi saliva alters human monocyte function towards a tolerized phenotype while co-infection with filaria favors a Leishmania-promoting Th2/regulatory response.