Loss of dendritic cell migration and impaired resistance to Leishmania donovani infection in mice deficient in CCL19 and CCL21

Abstract

The encounter between APC and T cells is crucial for initiating immune responses to infectious microorganisms. In the spleen, interaction between dendritic cells (DC) and T cells occurs in the periarteriolar lymphoid sheath (PALS) into which DC and T cells migrate from the marginal zone (MZ) along chemokine gradients. However, the importance of DC migration from the MZ into the PALS for immune responses and host resistance to microbial infection has not yet been elucidated. In this study, we report that following Leishmania donovani infection of mice, the migration of splenic DC is regulated by the CCR7 ligands CCL19/CCL21. DC in plt/plt mutant mice that lack these chemokines are less activated and produce less IL-12, compared with those in wild-type mice. Similar findings are seen when mice are treated with pertussis toxin, which blocks chemokine signaling in vivo. plt/plt mice had increased susceptibility to L. donovani infection compared with wild-type mice, as determined by spleen and liver parasite burden. Analysis of splenic cytokine profiles at day 14 postinfection demonstrated that IFN-gamma and IL-4 mRNA accumulation was comparable in wild-type and plt/plt mice. In contrast, accumulation of mRNA for IL-10 was elevated in plt/plt mice. In addition, plt/plt mice mounted a delayed hepatic granulomatous response and fewer effector T cells migrated into the liver. Taken together, we conclude that DC migration from the MZ to the PALS is necessary for full activation of DC and the optimal induction of protective immunity against L. donovani.

Figure 1. The course of L. donovani infection in B6 and plt/plt mice

B6 (.) and plt/plt (.) mice were injected 2 × 10⁷ L. donovani amastigotes and parasite burden was measured in the liver (A) or the spleen (B) at the live parts indicated. The data are expressed as mean ± SEM for 4 to 8 mice pooled from two experiments. * p<0.05.

Figure 2. Distribution of parasite-infected macrophages, IL-12p40 production, and DC following L. donovani infection of B6 and plt/plt mice

The distribution of L. donovani amastigotes (green: arrowheads) and CD68⁺ macrophages (red) in the spleens of B6 and plt/plt mice (A) at 1h p.i. Original magnification is x800. B. The distribution and number of parasites in B6 (closed bar) or plt/plt (open bar) mice was determined from multiple spleen sections of individual mice. Data are expressed as mean ± SEM for 3-5 mice per strain. The distribution of IL-12p40 producing cells in the spleens of B6 (C) and plt/plt (D) mice at 5h p.i. IL-12p40 (arrowhead) was visualized with immunohistochemistry (brown), and MZ macrophages were labeled with India ink (black). Original magnification is x200. E. The number of IL-12p40 producing cells in B6 (closed bar) or plt/plt (open bar) mice was determined. Data are expressed as mean ± SEM for 3-5 mice per strain* p<0.05. F. IL-12p40 mRNA was detected by real time RT-PCR in the spleen of naïve and 5 h-infected. B6 (closed bar) or plt/plt (open bar) mice.. The values are expressed with mean ± SEM for 3 to 5 mice * p<0.05. G-J. DC were stained using CD11c in naïve B6 (G) and plt/plt (H) and in B6 (I) and plt/plt (J) mice at 5h.p.i. Dotted lines indicated the border of the MZ. Original magnification is x200. Wp; white pulp, Rp; red pulp. Data are representative of one of three experiments.

Figure 3. IL-12p40 expression in L. donovani infected mice treated with PTX

IL-12p40 mRNA accumulation was determined by real time RT-PCR in PBS-treated mice (open bar) or mice treated with 500 ng PTX at 1 day and 3days before infection (closed bar). The values are expressed with mean ± SEM for 3 to 5 mice. Data are representative of one of three experiments. * p<0.05.

Figure 4. DC activation during early L. donovani infection of B6 and plt/plt mice

(A) Spleen DC from B6 (left panels) and plt/plt (right panels) were identified as CD11c⁺ MHC Class II^hi and then analysed for expression of CD80 (top), CD86 (middle) and CD40 (bottom). Histograms show representative staining of naïve (dotted line) and 5hr-infected (solid line) mice. Data are representative of 3 mice from 3 independent experiments. (B) Mean fluorescence intensities (MFI) of MHC-II, CD80, CD86 and CD40 on splenic DC from infected B6 (solid bar) and plt/plt (open bar) mice. Data are shown as fold-increase over DC in naïve mice. The values are expressed with mean ± SEM for 3 independent experiments. * p<0.05.

Figure 5. Cytokine responses in L. donovani-infected B6 and plt/plt mice

(A) Spleen cells from naïve and d7 infected B6 and plt/plt mice were cultured for 72hr in the absence (black bars) or presence (open bars) of L. donovani amastigote antigen. Proliferation was determined by scintillation counting. (B) IFN was determined in culture dependents from restructured B6 and plt/plt mice in the absence (black bars) or presence (open bars) of L. donovani amastigote antigen. (C-E) mRNA was extracted from the spleens of naïve or day 14 p.i. B6 (dark hatch) or plt/plt (light hatch) mice and accumulation of IFNγ (C), IL-4 (D), and IL-10 (E) mRNA was detected by real time RT-PCR. * p<0.05.

Figure 6. Granuloma formation in the L. donovani infected livers of B6 and plt/plt mice

Liver sections at day 14 p.i. (A, D), day 28 p.i. (B, E), and day 56 p.i. (C, F), in B6 (A, B, C) or plt/plt (D, E, F) mice were stained with anti-L. donovani sera. Original magnification is x400. (G.) The number of hepatic granulomas in infected B6 (closed bar) or plt/plt (open bar) mice at each time point. (H) Granuloma maturation during L. donovani infection of B6 or plt/plt mice. Data represent the frequency of infected Kupffer cells (iKC), immature granulomas (IG), mature granulomas (MG), and empty granulomas (EG) per mouse at each time point.

Figure 7. Hepatic T cell and macrophage recruitment during L. donovani infection of B6 and plt/plt mice

Liver sections from B6 (A, B, C) or plt/plt (D, E, F) mice at day 14 p.i were stained for CD4 (A, D), CD8α (B, E), and F4/80 (C, F). Original magnification is x200. Data are representative of one of three experiments.