The role of interleukin (IL)-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti-IL-10 receptor antibody for sterile cure

Abstract

Some pathogens (e.g., Mycobacterium tuberculosis, Toxoplasma gondii, Leishmania spp) have been shown to persist in their host after clinical cure, establishing the risk of disease reactivation. We analyzed the conditions necessary for the long term maintenance of Leishmania major in genetically resistant C57BL/6 mice after spontaneous healing of their dermal lesions. Interleukin (IL)-10 was found to play an essential role in parasite persistence as sterile cure was achieved in IL-10-deficient and IL-4/IL-10 double-deficient mice. The requirement for IL-10 in establishing latency associated with natural infection was confirmed in IL-10-deficient mice challenged by bite of infected sand flies. The host-parasite equilibrium was maintained by CD4+ and CD8+ T cells which were each able to release IL-10 or interferon (IFN)-gamma, and were found to accumulate in chronic sites of infection, including the skin and draining lymph node. A high frequency of the dermal CD4+ T cells released both IL-10 and IFN-gamma. Wild-type mice treated transiently during the chronic phase with anti-IL-10 receptor antibodies achieved sterile cure, suggesting a novel therapeutic approach to eliminate latency, infection reservoirs, and the risk of reactivation disease.

Figure 1.

Chronic phase L. major infection in C57BL/6 mice. (A) Number of parasites per ear (○) and diameter of induration (•) after intradermal inoculation of 100 L. major metacyclics in the ear of C57BL/6 mice. Values represent mean induration in mm ± SD, 3–5 mice per group, and geometric mean parasite number per ear ± SD, 5 mice and 10 ears per group. (B) Number of parasites in the ear 5 mo after intradermal inoculation of 100 L.major metacyclics and treated for 2.5 (•) or 4.5 (▪) wk with anti-CD4, anti-CD8, or isotype control before titration. Bars represent geometric mean parasite number per ear, four mice and eight ears per group. The experiment is representative of three separate experiments. (C) Dot plots of TCRβ¹CD4⁺ and TCRβ¹CD8⁺ cells present in the ear dermis 6 mo after challenge. The dermal sheets of four mice were pooled. The data shown are from a single experiment, representative of three separate experiments.

Figure 2.

IL-10 and IL-10/4 KO mice achieve sterile immunity. (A) Diameter of induration after intradermal inoculation of 100 L.major metacyclics in C57BL/6 (•), C57BL10 (▿), IL-10^−/− (▴), IL-4^−/− (□), and IL-10/4^−/− (○) mice. Values represent mean induration in mm ± SD, 12–9 mice and 24–18 ears per group. (B) Number of parasites 6 wk, and (C) and 9.5 wk after challenge in the dermal site (▪) or in the LN (♦), three mice and six ears per group. The experiment is representative of four separate experiments. WT, wild-type. (D) Number of parasites in the dermis or (E) in the draining LN 6 mo after challenge and treated with anti–IFN-γ or isotype control for 2.5 wk before parasite titration; three mice, six ears or LNs per group. The experiment is representative of three separate experiments.

Figure 3.

Natural infection of IL-10 KO mice. (A) The course of infection after transmission of L. major by bite of P. papatasi in C57BL/10 (•) and C57BL/10 IL-10^−/− mice (○); 6 to 8 mice, 12 to 16 ears per time point. The value shown at each time point are the sum of the lesion's diameter ± 1 SD. (B) Parasite number in the ear (▪) and draining LN (♦), 9 wk after bite. The two draining LNs from individual mice were pooled. The bar represents geometric mean parasite number; 6–8 LNs and 12–16 ears per group.

Figure 4.

Anti–IL-10 receptor treatment of chronically infected mice. Number of parasites in the dermis (•) or LN (♦) of C57BL/6 mice treated beginning at 7 mo after infection with anti–IL-10R mAb or isotype control for 2 wk before titration. Bars represent the geometric mean of the parasite number per ear, 13 mice and 26 ears or draining LNs per group. The data represent a pool of three separate experiments.

Figure 5.

Antigen-specific cytokine release by LN cells. (A) LN cells from C57BL/6 mice were pooled at each time point from four mice and eight LNs, stimulated in vitro with SLA, and the supernatants were collected at 48 h for determination of IL-10 (○) and IFN-γ (•). Values represent the mean cytokine concentration of four separate experiments ± 1 SD.

Figure 6.

Single cell analysis of cytokine production by dermal and LN T cells. (A) 7 mo after infection, dermal leukocytes were pooled from four mice (eight ears), restimulated with L. major infected FSDDCs, and stained for detection of surface markers and intracellular cytokines. Cells were gated on TCRβ¹ cells. The numbers represent the percentage of CD4⁺ or CD8⁺ cells also positive for IL-10 or IFN-γ. (B) Dermal or LN cells from mice 7 mo after infection were gated on CD4⁺TCRβ¹ or CD8⁺TCRβ¹ cells. The numbers represent the percentage of gated cells positive for IL-10, IFN-γ, or both cytokines. The data shown in A and B are from a single experiment, representative of five separate experiment. (C) 6 mo after infection, C57Bl/6 mice were treated for 1 wk with anti–IL-10R mAb or isotype control and dermal cells from four mice (eight ears) per group were pooled, restimulated with anti-CD28, IL-2, and SLA, and stained. Cells were gated on TCRβ¹ cells. Numbers shown are the absolute number of CD4⁺ or CD8⁺ cells per ear positive or negative for IL-10 or IFN-γ. The data are from a single experiment that is representative of two separate experiments.