CXCL10/gamma interferon-inducible protein 10-mediated protection against Leishmania amazonensis infection in mice

Abstract

Leishmania amazonensis can cause progressive disease in most inbred strains of mice. We have previously shown that L. amazonensis-infected C57BL/6 mice have profound impairments in expression of proinflammatory cytokines and chemokines and in activation of antigen-specific CD4(+) T cells. These impairments are independent of interleukin-4 (IL-4) but partially due to IL-10 production. The precise mechanism of pathogenesis associated with L. amazonensis infection remains largely unresolved. Since chemokines are essential mediators of leukocyte recruitment and effector cell function, we hypothesized that these molecules are important for the initiation of early responses locally and for the eventual control of the infection. In this study, we examined the roles of CXCL10/gamma interferon-inducible protein 10 (IP-10) and CCL2/monocyte chemoattractant protein 1 (MCP-1) in the activation of the macrophage effector function in vitro and their efficacy in ameliorating infection in vivo. Bone marrow-derived macrophages of both BALB/c and C57BL/6 mice were treated with increasing concentrations of recombinant chemokines prior to infection with either stationary-phase promastigotes or tissue-derived amastigotes. We found that treatment with IP-10 or MCP-1 significantly reduced parasite burdens, in a dose-dependent manner, and triggered nitric oxide production. When susceptible C57BL/6 mice were injected locally with IP-10 following L. amazonensis infection, there was a significant delay in lesion development and a reduction in parasite burdens, accompanied by 7- and 3.5-fold increases in gamma interferon and IL-12 secretion, respectively, in restimulated lymph node cells. This study confirms that IP-10 plays a protective role in promoting the reduction of intracellular parasites and thereby opens new avenues for therapeutic control of nonhealing cutaneous leishmaniasis in the New World.

FIG. 1.

CXCR3 is expressed on murine BM-Mφs. BM-Mφs of BALB/c mice were seeded into six-well plates (2.5 × 10⁶ cells/well) and allowed to rest for 24 h. (A) Total RNA (100 ng) was extracted for RT-PCR analyses of CXCR3 and β-actin transcripts. Splenocytes from BALB/c mice were treated with LPS/IFN-γ and used as positive controls. (B) Mφs were collected and stained for surface expression of F4/80 and CXCR3 molecules. For flow cytometry, the single gate was set up according to the isotype control, and the number represents the percentage of cells within the gate. Staining profiles of the isotype control (dashed line) and the anti-CXCR3 group (solid line) were included in the histogram overlay representing the shift in fluorescence.

FIG. 2.

Pretreatment with IP-10 and MCP-1 reduces parasite burdens in Mφs. BM-Mφs of BALB/c, C57BL/6, or C3H/HeJ mice were seeded into 24-well plates (3 × 10⁵ cells/coverslip/well). Cells were left untreated or treated with either IP-10 (100 ng/ml), MCP-1 (100 ng/ml), or LPS (20 ng/ml) plus IFN-γ (20 ng/ml) for 4 h prior to infection with 2.4 × 10⁶ stationary-phase promastigotes (at an 8:1 parasite-to-cell ratio). All groups of cells were subsequently stained for parasites, using pooled sera from infected mice and FITC-conjugated goat anti-mouse IgG. The nuclei of the cells were stained with DAPI. Images of 10 random fields per condition were taken for counting of parasite loads. Data are presented as numbers of parasites per 100 Mφs and are shown as means ± standard deviations (SD) for three independent experiments (representing 30 fields per condition). Each group was compared to untreated controls (*, P < 0.5; **, P < 0.01; ***, P < 0.001).

FIG. 3.

Pretreatment with IP-10 and MCP-1 promotes reduction of L. amazonensis amastigotes. BM-Mφs of BALB/c mice in 24-well plates (3 × 10⁵/well) were left untreated (white bar) or treated (black bars) with 100 ng/ml of IP-10 or MCP-1 for 4 h prior to infection with 6 × 10⁵ tissue-derived amastigotes (at a 2:1 parasite-to-cell ratio). At 48 h postinfection, cells were lysed with 0.01% SDS, and the number of parasites per well was counted with a hemocytometer (triplicate wells per condition). Data are presented as numbers of parasites per well and shown as the means ± SD for three independent experiments (representing nine wells per condition). Each group was compared to untreated controls (*, P < 0.05; ***, P < 0.001).

FIG. 4.

Nitric oxide and proinflammatory chemokines contribute to IP-10- and MCP-1-mediated parasite killing. (A) BM-Mφs of B6 mice (3 × 10⁵ cells/well) in 24-well plates were left untreated or treated with the indicated stimuli, as described in the legend to Fig. 2, for 4 h prior to infection with 2.4 × 10⁶ stationary-phase promastigotes (at an 8:1 parasite-to-cell ratio). At 48 h postinfection, supernatants from uninfected (black bars) and infected (dotted bars) groups were collected for measurement of nitrite via the Griess reagent. (B) BM-Mφs were generated from wild-type B6 mice (black bars) or iNOS^−/− B6 mice (white bars) and treated with 100 ng/ml of IP-10, MCP-1, or IFN-γ for 4 h prior to infection with L. amazonensis lesion-derived amastigotes as described in the legend to Fig. 2. Cells treated with LPS (20 ng/ml) plus IFN-γ (20 ng/ml) served as positive controls. At 48 h postinfection, cells were treated with 0.01% SDS to release intracellular parasites, and the parasite number per well was counted. Data are presented as numbers of parasites per well and expressed as means ± SD for triplicate wells per condition. The iNOS^−/− groups were compared with their wild-type counterparts (, P < 0.05; , P < 0.01); , P < 0.001). The data shown are representative of three independent repeats. (C) BM-Mφs of BALB/c mice were seeded into six-well plates (1 × 10⁶ cells/well) and infected with 8 × 10⁶ stationary-phase promastigotes (8:1 parasite-to-cell ratio). At 48 h postinfection, cell-free supernatants were collected for the measurement of cytokine profiles via protein cytokine arrays. The intensities of protein spots for the IP-10-treated group were compared with those of the corresponding spots for the untreated controls, and data are presented as x-fold increases above the infection control levels. The data shown are the results for those molecules that displayed ≥2-fold increases over the infection control levels and are representative of three independent repeats.

FIG. 5.

Local administration of IP-10 delays lesion development in susceptible B6 mice. (A) B6 mice (five per group) were infected s.c. with 2 × 10⁵ L. amazonensis metacyclic promastigotes and treated s.c. with IP-10 (100 ng in 5 μl) or PBS on days 1, 3, and 7 of infection. Lesion size (in mm; size of the infected foot minus size of control foot for each mouse) was monitored weekly, and the data shown are the means ± SD for each group. Results are representative of three independent experiments. The parasite burden per foot was determined at 10 weeks postinfection and shown as the mean for each group. , P < 0.05 (treated groups in comparison to the PBS controls). (B) At 10 weeks postinfection, PBS- and IP-10-treated mice were sacrificed, and total RNAs were extracted from infected foot tissues. RT-PCR analysis was performed to determine expression changes in IFN-γ. Band intensities were analyzed via spot densitometry, and expression changes were determined after normalization with β-actin.

FIG. 6.

Local administration of IP-10 triggers the production of multiple Th1-favored cytokines and chemokines. B6 mice (five per group) were infected and treated as described in the legend to Fig. 5. At 3 weeks (A) and 6 weeks (B) postinfection, LN cells (5 × 10⁶/ml/well) were collected from IP-10-treated groups or PBS controls, pooled from five mice, and stimulated with parasite antigen (1 × 10⁷ parasite equivalents) for 48 h. Cell-free supernatants were collected for the measurement of cytokine profiles via protein cytokine arrays. The intensities of protein spots for the IP-10-treated group were compared with those of the corresponding spots for the infection controls, and data are presented as x-fold increases above the infection control levels. Data shown in panels A and B represent those molecules with ≥2-fold increases compared to the infection control levels at 3 and 6 weeks postinfection, respectively. Example membranes for infection control and IP-10 groups at 6 weeks postinfection are shown in panel C, and all tested cytokines/chemokines in the membranes are illustrated to the right. Membranes for medium controls using cells from infected mice detected no or minimal expression of cytokines and chemokines (data not shown).