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. 2022 Jun 3;25(7):104520. doi: 10.1016/j.isci.2022.104520

Figure 3.

Figure 3

Infection with L. mexicana is worsened in Cd82−/− mice

(A–D) WT and Cd82−/− mice were infected intradermally with the obligate intracellular parasite Leishmania (L) mexicana. (A) Scheme for the intradermal (ear) infection of WT and Cd82−/− mice with L. mexicana. Cutaneous lesion development was measured for a total of 12 weeks post infection. At 6 weeks, mice were sacrificed and the immune composition of lesions and draining lymph nodes (DLN) was measured by flow cytometry. (B) Cutaneous lesion sizes post infection with L. mexicana. Data from N = 10–13 mice pooled from two independent experiments. Shading indicates ±SEM (C) Cutaneous lesions sizes from B only at 6 weeks. Note that several WT mice had no lesions at this time point, indicated as 0 mm. Each data point represents a single mouse. (D) Cutaneous lesions and DLNs were enzymatically digested and analyzed by flow cytometry. The proportion of macrophages, monocytes, dendritic cells (DCs), and T cells was determined by staining with fluorescently conjugated antibodies. Y axes indicate how populations were determined. Cutaneous lesions are shown as proportion of CD11b+ cells while DLNs are shown as absolute numbers. Full gating strategy is shown in Figure S3.

(E and F) Macrophages were differentiated in vitro from the bone marrow of WT and Cd82−/− mice. After 7 days differentiation, macrophages were infected with L. mexicana-TurboRFP, parasite line expressing the fluorescent protein TurboRFP. The proportion of macrophages infected with L. mexicana-TurboRFP was measured 4 h (E) and 3 days (F) later by flow cytometry. Gating strategy to determine the proportion of viable, L. mexicana-TurboRFP-infected macrophages is shown in Figure S3. Parasitic burden is measured as the TurboRFP geometric mean fluorescence intensity (MFI) of infected macrophages. Parasitic index is calculated by the multiplication of the proportion infected and parasitic burden. Each dot represents the relative mean from four independent experiments, each with separate mice. All data are presented as mean ± SEM. Significance was measured by t-test (C–F) or 2-way ANOVA (B) with the overall significance indicated in the top left corner of the graph. For 2-way ANOVA, Sidak post-hoc multiple comparison analysis was performed of individual time points. In all graphs, significance is indicated by ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S3.