Leukocytes infiltrate the skin and draining lymph nodes in response to the protozoan Leishmania infantum chagasi.

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 neutrophil-depleted 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.

In vivo imaging of transgenic Leishmania parasites in a live host.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.