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Infection with the obligate intracellular protozoan Leishmania is thought to be initiated by direct parasitization of macrophages, but the early events following transmission to the skin by vector sand flies have been difficult to examine directly. Using dynamic intravital microscopy and flow cytometry, we observed a rapid and sustained neutrophilic infiltrate at localized sand fly bite sites. Invading neutrophils efficiently captured Leishmania major (L.m.) parasites early after sand fly transmission or needle inoculation, but phagocytosed L.m. remained viable and infected neutrophils efficiently initiated infection. Furthermore, neutrophil depletion reduced, rather than enhanced, the ability of parasites to establish productive infections. Thus, L.m. appears to have evolved to both evade and exploit the innate host response to sand fly bite in order to establish and promote disease.
Many parasitic diseases are transmitted by the bite of an infected arthropod, yet the dynamics of the host-parasite interaction in this context remain largely uncharacterized. Transmission of Leishmania by infected sand fly bite represents an attractive experimental system to study early inflammatory responses and relate these processes to the establishment of an infectious disease. Leishmaniasis is thought to be initiated by direct parasitization of macrophages following deposition into the skin (1). However, the ability of neutrophils to rapidly respond to and efficiently phagocytose a variety of pathogens suggests they may also be an initial target of Leishmania infection (2–4). Indeed, following needle injection of L.m., infected neutrophils have been observed, and both host protective and disease promoting roles for these cells have been reported (5–10). Importantly, the role of neutrophils has never been addressed in sand fly-transmitted Leishmania infections.
Sand fly biting involves wounding of the microvasculature to create a hemorrhagic pool from which to feed, a process that initiates a strong local inflammatory response (11–13). In order to further characterize the host response at the site of sand fly bite, uninfected or L.m.-infected Phlebotomus duboscqi sand flies, a natural vector of L.m., were allowed to feed on the ears of C57BL/6 mice (14, 15) which develop self-healing cutaneous lesions similar to the human disease. Flow cytometric analysis revealed a dramatic and sustained infiltration of neutrophils into the skin accompanied by a significant recruitment of macrophages, regardless of the infectious status of the flies (Fig. 1A and B). In order to visualize the bite site in vivo, an RFP-expressing strain of L.m. (L.m.-RFP) (14) (Fig. S1, A to D) and mice expressing eGFP under the control of the endogenous lysozyme M promoter (LYS-eGFP) (16) were employed. eGFPhi cells recovered from the skin of LYS-eGFP mice following L.m. infection are CD11bhiGr-1hiF4/80−MHCII− neutrophils while eGFPlo cells represent CD11b+F4/80+MHCII+/−Gr-1− monocyte/macrophage populations (Fig. S1, E to G, see also Fig. S1, H to K and Fig. 4D). Two hours following exposure of the ventral ear pinnae of LYS-eGFP mice to either uninfected or L.m.-RFP-infected sand flies, GFPhi neutrophils accumulated at sites of proboscis penetration through the skin (Fig. 1C).
2-photon intravital microscopy (2P-IVM) revealed that as early as 30” following exposure to sand flies, neutrophils had migrated into the skin and began localizing around apparent bite sites (Fig. 1D). Over the next hour, neutrophils rapidly accumulated in, and subsequently swarmed around, the vicinity of both infected and uninfected sand fly bites (Fig. 1D, and movies S1 and S2), eventually forming an epidermal “plug” through sequential migration of neutrophils into the hole left by the sand fly proboscis (Fig. 1E and S2, and movies S3 and S4). Parasite phagocytosis by neutrophils was readily observed during this recruitment process (movie S5) leading to the presence of large numbers of parasite-containing neutrophils at later time points (Fig. 1F and movie S6). In contrast to the rapid motility reported for mosquito-transmitted Plasmodium sporozoites, which appear to actively search for blood and lymphatic vessels (17, 18), L.m. parasites appeared relatively immobile following sand fly delivery into the skin.
Due to the relatively low and variable number of parasites deposited by sand fly bite (14), intradermal (i.d.) needle inoculation of high numbers of infectious-stage L.m.-RFP metacyclic promastigotes was employed to quantitatively analyze the fate of parasites post-infection (p.i.). The pattern of neutrophil recruitment at early time points was similar to sand fly bite, although comparatively short-lived (Fig. S3). Two hours following injection of L.m-RFP into the ear, analysis of all RFP+ dermal cells revealed that the vast majority of the L.m.-RFP signal was associated with the CD11bhiGr-1hiLYS-eGFPhi neutrophil population (Fig. 2, A–D and S1N). These data are consistent with kinetic analyses of fixed tissue sections showing parasites initially interspersed between F4/80+GPlo macrophages and subsequently phagocytosed by newly arriving F4/80−eGFPhigh neutrophils (Fig. 2, E–F).
Dynamic analysis of neutrophil recruitment and parasite uptake revealed the rapid accumulation of neutrophils inside blood vessels surrounding the infection site as early as 30” p.i. and the subsequent diapedesis of these cells into the skin parenchyma (Fig. 2G and movie S7). Extravasating neutrophils were preferentially distributed to the side of the vessel facing parasite deposition and characterized by an extremely elongated uropod (Fig. 2H and movie S8). Neutrophils then moved in a highly directed manner towards the inoculation site (Fig. 2, G to I) where they rapidly and efficiently phagocytosed individual parasites (Fig. 2J). Phagocytosis occurred concurrently with migrational arrest, as demonstrated by a decrease in neutrophil mean velocity following parasite uptake (Fig. 2K). Additional data acquired following needle inoculation in the absence of parasites suggest that the initial inflammatory response to sand fly or needle-induced tissue damage drives the robust neutrophilic recruitment observed in these studies, overriding the potential contribution of any parasite-specific signals (Figure S4 and movie S9).
As macrophages are the definitive host cell for Leishmania, their relationship with neutrophils was explored. Mice expressing eGFP under the control of the endogenous MHC class II promoter (MHCII-eGFP) (19) (Fig. S1, L and M) were inoculated with L.m.-RFP. Phenotypic analysis of RFP-gated dermal cells at 18 hours p.i. revealed that the RFP signal was primarily associated with CD11bhiGr-1hiMHC-II-eGFP− neutrophils and only small numbers of monocytes/macrophages or CD11c+ DCs (Fig. 3A and S5). Strikingly, we observed an increase in the absolute number of RFP+ macrophages and a corresponding drop in the absolute number of RFP+ neutrophils over time (Fig. 3, C and D). By 6–7 days p.i the RFP signal was found primarily in the macrophage/monocyte population and only sparsely in neutrophils and CD11c+ DCs (Fig. 3, B, D, and E). Although MHC-II+CD11c+ cells represented an extremely small proportion of infected cells at 1 day p.i., their increase in numbers by day 6 suggests that dermal DCs and/or Langerhans cells participate in the infectious process (20).
To determine the fate of Leishmania promastigotes following phagocytosis by neutrophils in vivo, infected and uninfected neutrophils were isolated from the ear dermis by cell sorting (Fig. 4, A to C). GFPhiRFP+ cells retained a normal cytoplasmic and nuclear appearance and contained intracellular L.m. parasites (Fig. 4, D) (21). Limiting dilution analysis of sorted neutrophils revealed that 92% of RFP+ but only 1.2% of RFP− neutrophils contained at least one viable parasite (Fig. 4E). Furthermore, naïve mice inoculated with 103 RFP+GFP+ neutrophils or 103 cultured L.m.-RFP established equivalent infections (Fig. 4, F to H), demonstrating that L.m. phagocytosed by neutrophils are viable and can contribute to the establishment and progression of disease.
The extremely dense clusters of eGFP-expressing neutrophils and macrophages/monocytes that formed several hours following parasite inoculation (Fig. 3E) made visualization of individual cell-cell interactions difficult. To overcome this problem, sorted GFPhiRFP+ infected neutrophils were injected into the ears of transgenic animals expressing eGFP under the control of the macrophage/monocyte-specific CSF1 receptor promoter (22). Recipient animals were pre-exposed to sand flies 12 hours prior to neutrophil transfer in order to induce an inflammatory environment at the infection site. Utilizing 2P-IVM, we observed what appeared to be viable parasites (as indicated by their expression of RFP [Fig. S1, A to D]) being released from apoptotic neutrophils in the vicinity of surrounding macrophages (Fig. S6 and movies S10–S12).
We next examined the functional role of neutrophils on the establishment and progression of sand fly-transmitted Leishmaniasis. Mice treated with neutrophil-depleting antibody 16 hours prior to infected sand fly exposure had a specific and dramatic reduction of CD11b+Ly-6G+F4/80− neutrophils in the ear dermis 1 day after transmission (Fig. 4, I and J). In some, though not all experiments, reduced numbers of neutrophils were also observed in ears 6 days after transmission (Fig. 4J). Importantly, neutrophil depletion significantly reduced both the number of viable parasites detected per ear (Fig. 4L), as well as the incidence of ears with detectable parasites at 1 and 4 weeks post-transmission (Fig. 4M). Thus, the early influx and persistence of neutrophils following sand fly transmission of L. m. appears critical for the development of cutaneous disease.
The data presented here are relevant to the “Trojan Horse” model of L.m. infection (2) which postulates that uptake of infected neutrophils is a mechanism for ‘silent’ entry of parasites into macrophages. Our observations indicate that neutrophils are the initial host cell for a substantial fraction of parasites following infection and that neutrophil depletion results in reduced disease at 1 week p.i. We found no evidence, however, for uptake of intact, infected neutrophils by macrophages. In addition, macrophages were efficiently recruited to sites of infection and were able to directly phagocytose parasites in neutrophil-depleted animals (Fig. S7). Under these conditions, macrophages and DCs did not acquire more parasites compared to non-depleted animals containing competing neutrophils, suggesting that neutrophils may facilitate infection by rescuing parasites not accessible to other phagocytic cells from death in extracellular spaces. Alternatively, infected neutrophils may release transitional stage parasites better adapted for macrophage uptake and survival, or macrophages may exhibit compromised microbicidal function in a setting in which they are heavily engaged in the anti-inflammatory process of clearing apoptotic neutrophils (23, 24). This latter possibility is supported by an increase in the spontaneous release of the pro-inflammatory cytokines IL-1α and β by ear cells from neutrophil-depleted animals (Figure 4, N) (25).
The ability of phagocytic cells to rapidly localize to sites of tissue inflammation and subsequently capture and destroy pathogens is a hallmark of the innate immune response, highly conserved, and among the earliest observations in microbiology (26). We find that sand fly bites and needle inoculation induce an intense neutronphilic infiltrate into the skin, irrespective of parasite infection. These data are consistent with the finding that neutrophils are recruited to sites of sterile brain injury (27), and suggest that the predominance of L.m.-infected neutrophils at the site of parasite deposition is a byproduct of a host response aimed at wound repair and sterilization. Thus, the neutrophilic host response to the wound inflicted by arthropod vectors appears to have been a driving force in pathogen evolution aimed at counteracting and even exploiting the presence of these innate effector cells.