Lymphocytic Choriomeningitis Virus Expands a Population of NKG2D+CD8+ T Cells That Exacerbates Disease in Mice Coinfected with Leishmania major

Abstract

Leishmaniasis is a significant neglected tropical disease that is associated with a wide range of clinical presentations and a lifelong persistent infection. Because of the chronic nature of the disease, there is a high risk for coinfection occurring in patients, and how coinfections influence the outcome of leishmaniasis is poorly understood. To address this issue, we infected mice with Leishmania major and 2 wk later with lymphocytic choriomeningitis virus (LCMV) and then monitored the course of infection. Leishmania parasites are controlled by production of IFN-γ, which leads to macrophage-mediated parasite killing. Thus, one might predict that coinfection with LCMV, which induces a strong systemic type 1 response, would accelerate disease resolution. However, we found that infection with LCMV led to significantly enhanced disease in L. major-infected animals. This increased disease correlated with an infiltration into the leishmanial lesions of NKG2D(+) CD8(+) T cells producing granzyme B, but surprisingly little IFN-γ. We found that depletion of CD8 T cells after viral clearance, as well as blockade of NKG2D, reversed the increased pathology seen in coinfected mice. Thus, this work highlights the impact a secondary infection can have on leishmaniasis and demonstrates that even pathogens known to promote a type 1 response may exacerbate leishmanial infections.

Figure 1

Co-infection of L. major infected mice with LCMV exacerbates lesion formation. (A) Mice infected with L. major in the ear were challenged 2 weeks later with LCMV and ear thickness was measured weekly. (B) Parasite burden in the lesions was determined at 3, 5, 7 and 8 weeks post L. major infection. (C) Correlation of the lesion size and parasite burden of co-infected mice. Data are representative of at least 2 independent experiments (n=4–5 mice per group). Error bars represent SEM. NS = not significant

Figure 2

Co-infection with L. major does not alter the immune response to LCMV. (A) Mice infected with L. major in the ear were challenged 2 weeks later with LCMV and 7 days after LCMV infection spleens were harvested. Splenocytes were incubated with a pool of LCMV peptides for 5 hours with BFA and monensin. Cells were pregated on live, CD45+, CD8+ before IFNγ staining was assessed. Representative plots are shown with the mean percentage of total CD45+ cells ± SEM. (B) Number of IFNγ+ CD8 T cells is shown. (C) Spleen samples were taken to assess viral titers by plaque assay 3, 5 and 10 days following LCMV infection. Data are representative of a single experiment (A and B; n=5 mice per group) or 2 independent experiments (C; n=4–5 mice per group). Percentages are shown as mean ± SEM. Error bars represent SEM. NS = not significant

Figure 3

Co-infected mice exhibit increased inflammation. (A) Lesion size of co-infected mice at 5 weeks. (B) Lesion of mice infected for 5 weeks with L. major. (C) Lesion of mice infected for 5 weeks with L. major, which were co-infected at 2 weeks with LCMV. (D) H&E section of a lesion of mice infected for 5 weeks with L. major. (E) H&E section of a lesion of mice infected for 5 weeks with L. major, that were co-infected at 2 weeks with LCMV. Error bars represent 100 μM. (F) At 5 weeks after L. major infection infected skin was harvested, digested, and stained with antibodies for myeloid cells. Myeloid cells were pregated on live, CD45+, CD11b+ before the gates shown for monocytes and neutrophils. Representative plots are shown with the mean percentage of total CD45+ cells ± SEM. (G) The number of neutrophils and monocytes is shown. (H) Correlation between lesion size and neutrophils. Data are representative of 4 independent experiments (n=4–5 mice per group). Percentages are shown as mean ± SEM. Error bars represent SEM.

Figure 4

Co-infection with L. major leads to a transient immunosuppression. (A) Lymph nodes draining the site of L. major infection were harvested 3 weeks post-infection with LCMV and single cell suspensions stimulated with leishmanial antigen. Supernatants were collected at 72 hr and IFN-γ levels assessed by ELISA. (B) Lymph nodes draining the site of L. major infection were harvested 7 (B) post-infection with LCMV, and single cell suspensions stimulated with leishmanial antigen. Supernatants were collected at 72 hr and IFN-γ, IL-4, IL-10 and IL-17 levels assessed by ELISA. (C and D) Bead purified CD4+ T cells from the spleens of mice 3 weeks after infection with either L. major alone or L. major and LCMV were cultured with infected or uninfected DCs overnight. BFA and monensin were added to cultures for the final 4 hours prior to staining and analysis by flow cytometry. Cells were gated on live, CD45+,CD4+,CD44hi prior to analysis of expression of CD69 (C) and IFNγ (D). Data are representative of 3 independent experiments (A and B; n=3–5 mice per group) or a single experiment (C and D; n=3 mice per group). Error bars represent SEM. NS = not significant

Figure 5

Co-infection with LCMV results in a significant increase in T cells and gzmB in the infected skin. (A) Mice were infected with L. major or left uninfected for 2 weeks. Some mice from each group were then infected with LCMV. At the peak of lesion formation, 5 weeks after the initial infection with L. major, infected skin was harvested, digested, and stained with antibodies for T cells. T cells were pregated on live, CD45+, CD11b- cells before the gates shown for CD4 and CD8 T cells. Representative plots are shown with the mean percentage of total CD45+ cells ± SEM. (B) The number of CD4 and CD8 T cells is shown. (C) In addition to surface staining, cells from the skin were also incubated with BFA alone for 5 hours prior to intracellular staining for GzmB, IFN-γ, and IL-17 in CD8 T cells. Cells were pregated on live, CD45+, CD8+ and representative plots are shown. (D) Number of CD8 T cells expressing GzmB, IFN-γ, and IL-17 in lesions is shown. (E) RNA was isolated from whole ear tissue at 3 weeks post L. major infection and message levels for GzmB, IFN-γ, and IL-17 were determined. Whole ear tissue was homogenized and supernatants were analyzed for GzmB, IFN-γ, and IL-17 by ELISA at 3 weeks (F) and 5 weeks (G) post L. major infection. Data are representative of two independent experiments (A–E; n=4–5 mice per group) and ear supernatant data is representative of a single experiment at each time point (F and G; n=4 mice per group). Percentages are shown as mean ± SEM. NS = not significant

Figure 6

CD8 T cells induce immunopathology in co-infected mice. (A) Mice were infected with L. major or left uninfected for 2 weeks. Some mice from each group were then infected with LCMV. Beginning on day 8 post infection with LCMV, some mice in each group were treated with anti-CD8 depleting antibody biweekly for the remainder of the experiment. (B) Ear thickness was measured weekly. (C) Photographs of lesions 5 weeks after the initial infection with L. major. (D) Parasite burden was assessed by limiting dilution at 5 weeks. (E) Cells were isolated from lesions at 5 weeks post L. major infection and stained. The cells were pregated on live, CD45+, CD11b+ cells before the gates shown for monocytes and neutrophils. Representative plots are shown with the mean percentage of total CD45+ cells ± SEM. (F) Number of monocytes and neutrophils is shown. Data is representative of two independent experiments (n=5 mice per group). Percentages are shown as mean ± SEM. NS = not significant

Figure 7

The induction of immunopathology in co-infected mice is dependent upon NKG2D. (A) CD8 T cells from the blood, DLN and lesion (ear) 5 weeks post L. major infection were pregated on live, CD45+, CD8+, CD44hi cells and analyzed for NKG2D expression. Representative histograms are shown. (B) Beginning on day 3 post infection with LCMV, mice were treated with NKG2D blocking antibody biweekly for the remainder of the experiment. (C) Ear thickness was measured weekly. (D) Photographs of lesions 5 weeks after the initial infection with L. major. (E) Parasite burden was assessed by limiting dilution at 5 weeks. (F) Cells were isolated from lesions 5 weeks after L. major infection and stained for antibodies to myeloid cells. The flow plots were pregated on live, CD45+, CD11b+ cells before the gates shown for monocytes and neutrophils. Representative plots are shown with the mean percentage of total CD45+ cells ± SEM. (G) Number of monocytes and neutrophils is shown. (I) Cells were isolated from lesions 5 weeks after L. major infection and stained for CD107a as described. The data shown is representative of 2 or more experiments (n=4 or 5). Percentages are shown as mean ± SEM. NS = not significant