Leukocytes Infiltrate the Skin and Draining Lymph Nodes in Response to the ProtozoanLeishmania infantum chagasi

Abstract

The vector-borne protozoan Leishmania infantum chagasi causes minimal inflammation after inoculation into skin but disseminates to cause fatal visceral leishmaniasis. To define the inflammatory response at the parasite inoculation site, we introduced metacyclic L. infantum chagasi promastigotes intradermally into BALB/c mouse ears and studied inflammatory cells over 7 days. Ly6G؉ neutrophils rapidly infiltrated the dermis, peaking after 6 to 24 h. Macrophages and NK cells next infiltrated the dermis, and NK followed by B cells expanded in draining lymph nodes. Parasite-containing phagocytes were tracked with fluorescent mCherry-labeled L. infantum chagasi. Ly6G؉ neutrophils contained the greatest proportion of intracellular parasites 6 to 24 h after inoculation, whereas dermal macrophages harbored the majority of intracellular parasites after 2 to 7 days. These observations were validated microscopically. Low doses of antibody transiently depleted mice of neutrophils, leaving other cells intact. Combined results of in vivo imaging, flow cytometry, and quantitative PCR showed that neutrophil depletion slowed the clearance of extracellular (luciferase-positive) promastigotes during the first 24 h after inoculation yet decreased the numbers of leukocytes containing intracellular (mCherry-positive) parasites. From 3 days onward, total L. infantum chagasi-containing dermal leukocytes and total L. infantum chagasi parasites in draining lymph nodes were similar in both groups. Nonetheless, a second wave of L. infantum chagasi-containing neutrophils occurred 7 days after parasite inoculation into neutrophildepleted mice, corresponding to the time of neutrophil recovery. Thus, neutrophils were recruited to the dermis even late after inoculation, and L. infantum chagasi trafficked through neutrophils in both neutrophil-depleted and control mice, albeit with different kinetics. Recruitment of neutrophils and transient parasite residence in neutrophils may play a role in nonulcerative forms of leishmaniasis.Parasites belonging to the genus Leishmania cause a spectrum of human diseases, the most deadly of which is visceral leishmaniasis. Leishmania infantum chagasi is one of the two most common etiologic agents of visceral leishmaniasis in humans. During natural infection, a bolus of metacyclic promastigotes is delivered into a hemorrhagic dermal lesion formed by a feeding female phlebotomine sand fly (5). Parasites quickly encounter soluble and cellular microbicidal immune elements. Rather than succumb, many are taken up by phagocytic host cells, where they transform to intracellular amastigotes, a form that can multiply and survive in phagolysosomes (9, 48).Although the majority of host cells harboring Leishmania sp. amastigotes are macrophages, intracellular amastigotes have been observed in other mammalian cell types as well, including dendritic cells (DCs), fibroblasts, and neutrophils (6,21,28,33). Recent studies suggest that neutrophils can promote the early establishment of intradermal infection with Leishmania major...

Infection and Activation of Human Neutrophils with Fluorescent Leishmania infantum

“…survive inside macrophages throughout chronic infection of their mammalian hosts [3]. Reports over the past 5 years have made it clear that neutrophils are among first cells to arrive at the site of a sand fly bite and to take up the parasite [5,6]. An essential step in our understanding of the role of PMNs in leishmaniasis is the bidirectional effect of parasites on neutrophils and vice versa.…”

“…One thing that is clear is that neutrophils are abundant and interact extensively with the parasite during the first hours of infection [5,6,19]. Many other diseases such as those causing lung inflammation [43] or autoimmune disorders [44] document a role for highly activated neutrophils contributing to off-target cellular pathology.…”

“…It is clear that PMNs play a role in Leishmania infections, but their effect on disease outcome remains unclear [5,6,8,13]. Neutrophils can internalize and kill at least some Leishmania promastigotes in vitro [14–18].…”

“…Neutrophils can internalize and kill at least some Leishmania promastigotes in vitro [14–18]. They are the first cell to migrate to, and take up parasites at the site of in vivo infection with either L. major [5,18–20] or with L. infantum [6] species. Indeed, there appears to be distinct differences in the way various species of Leishmania result in activation and otherwise affect neutrophils [10,21–23].…”

In Vitro Screening Test Using Leishmania Promastigotes Stably Expressing mCherry Protein

“…On the basis of these considerations, there is a strong need to develop appropriate screening technologies. The use of fluorescent reporter genes such as those coding for green fluorescent protein (GFP) (17)(18)(19) and the red versions red fluorescent protein (RFP) (20,21) and mCherry (21,22) have considerably facilitated the screening and testing of antimicrobial agents against axenic amastigotes and intracellular amastigotes, allowing both in vitro and real-time visualization in vivo. Although animal models are well established for large-scale primary drug screening, its practical use is limited due to high costs.…”