Toward a novel experimental model of infection to study American cutaneous leishmaniasis caused by Leishmania braziliensis

Abstract

Leishmania spp. cause a broad spectrum of diseases collectively known as leishmaniasis. Leishmania braziliensis is the main etiological agent of American cutaneous leishmaniasis (ACL) and mucocutaneous leishmaniasis. In the present study, we have developed an experimental model of infection that closely resembles ACL caused by L. braziliensis. In order to do so, BALB/c mice were infected in the ear dermis with 10(5) parasites and distinct aspects of the infection were evaluated. Following inoculation, parasite expansion in the ear dermis was accompanied by the development of an ulcerated dermal lesion which healed spontaneously, as seen by the presence of a scar. Histological analysis of infected ears showed the presence of a mixed inflammatory infiltrate consisting of both mononuclear and polymorphonuclear cells. In draining lymph nodes, parasite replication was detected throughout the infection. In vitro restimulation of draining lymph node cells followed by intracellular staining showed an up-regulation in the production of gamma interferon (IFN-gamma) and in the frequency of IFN-gamma-secreting CD4(+) and CD8(+) T cells. Reverse transcription-PCR of ears and draining lymph node cells showed the expression of CC chemokines. The dermal model of infection with L. braziliensis herein is able to reproduce aspects of the natural infection, such as the presence of an ulcerated lesion, parasite dissemination to lymphoid areas, and the development of a Th1-type immune response. These results indicate that this model shall be useful to address questions related to the concomitant immunity to reinfection and parasite persistence leading to mucocutaneous leishmaniasis.

FIG. 1.

Inoculation of L. braziliensis into the ear dermis of BALB/c mice leads to the development of an ulcerated lesion. (A) Mice were infected with 10⁵ L. braziliensis promastigotes, and the course of lesion development was monitored for 10 weeks. Lesion sizes (in millimeters) are expressed as means and standard errors of the means from three independent experiments, each performed with five mice. (B) Photomicrograph of a mouse ear at 5 weeks postinfection demonstrating lesion ulceration.

FIG. 2.

Parasite load estimate in BALB/c mice after intradermal inoculation of L. braziliensis. Mice were infected with 10⁵ L. braziliensis promastigotes. Ear (open bars) and draining lymph node (solid bars) parasite loads were determined at 2, 4, 6, 8, and 10 weeks postinfection via a limiting dilution assay. The data represent the means and standard errors of the means from three independent experiments, each performed with five mice.

FIG. 3.

Histological aspects of ear lesions in BALB/c mice after intradermal inoculation of L. braziliensis. BALB/c mice (five mice per group) were infected with 10⁵ L. braziliensis promastigotes. Ears were removed at 5 (A and B) and 9 (C and D) weeks postinfection and stained with hematoxylin and eosin stain. (A) Ulcerated lesion showingextensive fibrinoid necrosis and inflammatory infiltrate extending up to the epidermis. Magnification, ×40. (B) Parasitized macrophages are indicated by thin arrows, and polymorphonuclear cells are indicated by broad arrows. Magnification, ×1,000. (C) Epidermal reconstitution accompanies the healing process. Magnification, ×100. (D) Macrophages are indicated by thin arrows, and epithelioid cells are indicated by broad arrows. Magnification, ×400.

FIG. 4.

Intracellular cytokine production by CD4⁺ T cells from BALB/c mice after intradermal inoculation of L. braziliensis. Mice were inoculated with PBS or with 10⁵ L. braziliensis promastigotes. At 2, 4, 6, and 8 weeks postinfection, draining lymph nodes were pooled and cells were preincubated with Brefeldin A for 4 h before being stained. Data represent the percentages of cells with signals for the particular cytokine that were greater than the background signals established using isotype controls. The data represent the means and standard errors of the means from three independent experiments, each performed with five mice per group. *, P ≤ 0.05.

FIG. 5.

Intracellular cytokine production by CD8⁺ T cells from BALB/c mice after intradermal inoculation of L. braziliensis. Mice were inoculated with PBS or with 10⁵ L. braziliensis promastigotes. At 2, 4, 6, and 8 weeks postinfection, draining lymph nodes were pooled and cells were preincubated with Brefeldin A for 4 h before being stained. Data represent the percentages of cells with signals for the particular cytokine that were greater than the background signals established using isotype controls. The data represent the means and standard errors of the means from three independent experiments, each performed with five mice per group. *, P ≤ 0.05; **, P < 0.01.

FIG. 6.

Cytokine production in BALB/c mice after intradermal inoculation of L. braziliensis. Mice were infected with 10⁵ L. braziliensis promastigotes. At 2, 4, 6, and 8 weeks postinfection, draining lymph node cells were collected and stimulated (solid bars) or not (open bars) with L. braziliensis for 48 h (IL-4 and IL-10) or 72 h (IFN-γ). Cytokine levels in supernatants were determined by ELISA. The data represent the means and standard errors of the means from three independent experiments, each performed with five mice per group.

FIG. 7.

Chemokine mRNA expression in BALB/c mice after intradermal inoculation of L. braziliensis. Mice were infected with 10⁵ L. braziliensis promastigotes. At 2, 4, 6, and 8 weeks postinfection, total RNA was obtained from pooled ears (A), draining lymph nodes (B), and uninfected mice (UN). Total RNA was used in RT-PCR for the amplification of CCL2/MCP-1, CCL3/MIP1-α, CCL4-MIP1-β, CCL5/RANTES, and β-actin. Data shown are from a single experiment representative of three separate experiments, each performed with five mice.