CCR7-dependent immunity during acute Toxoplasma gondii infection

Abstract

The chemokine receptor CCR7 is a well-established homing receptor for dendritic cells and T cells. Interactions with its ligands, CCL19 and CCL21, facilitate priming of immune responses in lymphoid tissue, yet CCR7-independent immune responses can be generated in the presence of sufficient antigen. In these studies, we investigated the role of CCR7 signaling in the generation of protective immune responses to the intracellular protozoan parasite Toxoplasma gondii. The results demonstrated a significant increase in the expression of CCL19, CCL21, and CCR7 in peripheral and central nervous system (CNS) tissues over the course of infection. Unexpectedly, despite the presence of abundant antigen, CCR7 was an absolute requirement for protective immunity to T. gondii, as CCR7(-/-) mice succumbed to the parasite early in the acute phase of infection. Although serum levels of interleukin 12 (IL-12), IL-6, tumor necrosis factor alpha (TNF-alpha), and IL-10 remained unchanged, there was a significant decrease in CCL2/monocyte chemoattractant protein 1 (MCP-1) and inflammatory monocyte recruitment to the site of infection. In addition, CCR7(-/-) mice failed to produce sufficient gamma interferon (IFN-gamma), a critical Th1-associated effector cytokine required to control parasite replication. As a result, there was increased parasite dissemination and a significant increase in parasite burden in the lungs, livers, and brains of infected mice. Adoptive-transfer experiments revealed that expression of CCR7 on the T-cell compartment alone is sufficient to enable T-cell priming, increase IFN-gamma production, and allow the survival of CCR7(-/-) mice. These data demonstrate an absolute requirement for T-cell expression of CCR7 for the generation of protective immune responses to Toxoplasma infection.

FIG. 1.

CCL19, CCL21, and CCR7 are upregulated during the course of T. gondii infection. Infected mice were sacrificed at days 7, 14, 21, and 42 postinfection, and total RNA was extracted from the livers and brains. Real-time RT-PCR was conducted to determine the absolute copy numbers of CCL19, CCL21, and CCR7 using a standard curve, and they were compared to those of the internal reference HPRT gene. The data are represented as mean fold changes over naïve mice, with the error bars representing standard errors of the mean (SEM) of at least 3 biological replicates.

FIG. 2.

Deficiency in proinflammatory cytokine production during acute infection in CCR7^−/− mice. (a) Serum samples were collected from C57BL/6 and CCR7^−/− mice at day 7 postinfection, and the protein levels of IFN-γ, CCL2, IL-12p70, IL-10, IL-6, and TNF-α were measured. The levels (averages ± SEM) for individual mice (n = 3) are plotted. (b) Single-cell suspensions were generated from naïve and infected spleens and restimulated with sTAg. IFN-γ production was measured after 48 h using ELISA. Averages and SEM of at least 3 biological replicates are plotted and are representative of 3 independent experiments containing a minimum of 3 biological replicates. *, P < 0.001.

FIG. 3.

CCR7^−/− mice fail to survive acute Toxoplasma infection due to uncontrolled parasite replication. (a) C57BL/6 WT (n = 5) and CCR7^−/− (n = 5) mice were infected with 10⁴ T. gondii parasites, and survival was monitored daily. (b) PECs (the arrows indicate replicating parasites) and liver and lung histology at day 7 postinfection indicating increased parasite burden and inflammation in CCR7^−/− compared to C57BL/6 mice. (c) Measurement of parasite DNA in lung, liver, and brain at days 7 and 14 postinfection using real-time RT-PCR as described in Materials and Methods. Averages and SEM of 3 biological replicates are plotted.

FIG. 4.

Analysis of cellular composition using flow cytometry at the site of infection (PECs) (a) and draining lymph nodes (b) from 7-day-infected C57BL/6 and CCR7^−/− mice. The numbers represent proportions of dendritic cells (CD11c⁺) as percentages of live cells (top) and proportions of inflammatory monocytes (CD45⁺ CD11b⁺ Ly6C^hi Ly6G⁻) and neutrophils (CD45⁺ CD11b⁺ Ly6C^med Ly6G^hi) as a percentage of CD11b⁺ cells from PECs (a, bottom). (c) Absolute numbers of neutrophils and dendritic cells in the draining lymph nodes from 7-day-infected C57BL/6 and CCR7^−/− mice. Averages and SEM are shown.

FIG. 5.

CCR7⁺ T cells are sufficient for protective immune responses in CCR7^−/− mice. (a) T cells were isolated from spleens and lymph nodes of naive C57BL/6 or CCR7^−/− mice, and 5 × 10⁶ cells were adoptively transferred into CCR7^−/− mice (n = 5). C57BL/6 (n = 5) and nontransferred CCR7^−/− mice (n = 5) were sham injected with PBS. The survival curve is representative of four independent experiments with similar results. (b) Serum IFN-γ levels at day 7 postinfection. Averages and SEM are shown.