Impaired expression of inflammatory cytokines and chemokines at early stages of infection with Leishmania amazonensis

Abstract

Infection of mice with Leishmania major results in disease progression or resolution, largely depending on the genetic backgrounds of the mouse strains. Infection with Leishmania amazonensis, on the other hand, causes progressive cutaneous lesions in most inbred strains of mice. We hypothesized that deficient activation of early immune responses contributes to the pathogenesis in L. amazonensis-infected mice. To distinguish early molecular events that determine the outcome of Leishmania infections, we examined cytokine gene expression in C57BL/6 mice infected with either L. amazonensis or L. major (a healing model). After 2 to 4 weeks, L. amazonensis-infected mice had significantly delayed and depressed expression of inflammatory cytokines (interleukin-12 [IL-12], gamma interferon, IL-1 alpha, IL-1 beta), CC chemokines (CC chemokine ligand 3 [CCL3]/macrophage inflammatory protein 1 alpha [MIP-1 alpha], CCL4/MIP-1 beta, CCL5/RANTES, MIP-2), and chemokine receptors (CCR1, CCR2, CCR5) in foot tissues and draining lymph nodes compared to the expression in L. major-infected controls. These findings correlated with defective T-cell responsiveness to parasite stimulation in vivo and in vitro. Adoptive transfer of L. amazonensis-specific Th1 cells prior to infection overcame the immune defects of the animals, leading to complete control of the disease. Studies with gene knockout mice suggested that IL-10, but not IL-4, contributed partially to compromised immunity in L. amazonensis-infected hosts. The data suggest that there is impairment in multiple immune functions at early stages of infection with L. amazonensis parasites and provide a compelling rationale to explore immune augmentation as an intervention in American cutaneous leishmaniasis.

FIG. 1.

Lesion development during the course of infection with L. amazonensis and L. major. C57BL/6 mice (five mice/group) were infected subcutaneously with 2 × 10⁶ stationary-phase promastigotes of L. amazonensis (La) or L. major. Lesion sizes were measured with a micrometer. The parasite load in foot tissues was assessed after 10 weeks of infection, and the numbers in parentheses are means ± standard deviations for the groups (in log scale). Two asterisks indicate that there is a statistically significant difference (P < 0.01) between the two infection groups.

FIG. 2.

Deficient expression of proinflammatory cytokines during infection with L. amazonensis. C57BL/6 mice were infected subcutaneously with 2 × 10⁶ stationary-phase promastigotes of L. amazonensis (La) or L. major. At 0, 1, 2, and 8 weeks after infection, draining LNs were harvested and pooled (three to five LNs/group) for total RNA extraction. RPA analyses were performed for the genes indicated, as described in Materials and Methods, and the results of a representative assay are shown (A). (B and C) Quantities of each mRNA species detected in LNs (B) and foot tissues (C), normalized and expressed relative to the amount of GAPDH. The data are means ± standard deviations for three separate experiments. wk, week(s).

FIG. 3.

Deficient expression of CC chemokines during infection with L. amazonensis. C57BL/6 mice (five mice/group) were infected with 2 × 10⁶ promastigotes of L. amazonensis (La) or L. major. At 0, 1, 2, and 4 weeks after infection, total RNA was extracted from foot tissues. RPA analyses were conducted for the genes indicated, and the results of a representative assay are shown (A). (B) Quantity of each mRNA species normalized and expressed relative to the amount of GAPDH. The data are means ± standard deviations for three separate experiments. wk, week(s).

FIG. 4.

Differential production of cytokines and chemokines following infection with L. amazonensis or L. major. C57BL/6 mice (six to eight mice/group) were infected with 2 × 10⁶ promastigotes of L. amazonensis (open bars) or L. major (solid bars). LN cells were cultured in vitro in the presence of parasite lysates (equivalent to 5 × 10⁶ parasites/well), and the supernatant was collected 3 days later. Foot tissue homogenates were prepared and pooled from three naive and infected mice as described in Materials and Methods. The levels of IL-4, IL-10, and IFN-γ in the supernatants of LN cell cultures and of IL-1β, MIP-1α, and MCP-1 in foot tissue homogenates were determined by ELISAs. The data are means ± standard deviations for three animals per group in three independent experiments. One asterisk (P < 0.05) and two asterisks (P < 0.01) indicate that there are statistically significant differences between the two infection groups.

FIG. 5.

Reduced expression of cytokine and chemokine receptors in L. amazonensis-infected mice. C57BL/6 mice (five to eight mice/group) were not treated or were infected with 2 × 10⁶ promastigotes of L. amazonensis (La) or L. major. At 2 weeks after infection, draining LNs were harvested and pooled for isolation of CD4⁺ T cells by positive selection. Total RNA was extracted from CD4⁺ T cells, and RPA analyses were conducted for cytokine receptors (A) and chemokine receptors (B). (C and D) Quantities of mRNA species for cytokine receptors (C) and chemokine receptors (D) normalized and expressed relative to the amount of GAPDH. The data are means ± standard deviations for three separate experiments. One asterisk (P < 0.05) and two asterisks (P < 0.01) indicate that there are statistically significant differences between the two infection groups.

FIG. 6.

Parasite-specific proliferation of draining LN cells and purified CD4⁺ LN T cells. Draining LN cells were collected from C57BL/6 mice at 2 weeks after infection with L. amazonensis (□) or L. major (▪), and CD4⁺ LN T cells were isolated by positive selection. Draining LN cells (5 × 10⁵ cells) (A) and CD4⁺ LN T cells (10⁵ cells) (B) were cultured without and with 5 × 10⁵ irradiated syngeneic splenocytes in the presence of different concentrations of promastigote lysates for 4 days in 96-well plates. One microcurie of [³H]thymidine was added 18 h before harvest, and the radioactivity was counted. The data are means ± standard deviations for two separate experiments. One asterisk (P < 0.05) and two asterisks (P < 0.01) indicate that there are statistically significant differences between the two infection groups.

FIG. 7.

Reversal of immunodeficiency and control of L. amazonensis infection following Th1-cell transfer. C57BL/6 mice (five to eight mice/group) were not treated or received different doses of Th1 cells and then were infected with 2 × 10⁶ promastigotes of L. amazonensis 1 day later. (A) Lesion sizes, as determined biweekly with a micrometer. The parasite load in foot tissues was assessed after 14 weeks of infection, and the numbers in parentheses are means ± standard deviations for the groups (in log scale). The parasite loads in mice that received 5 × 10⁶ or 1 × 10⁷ Th1 cells were lower than those in nontransferred controls (P < 0.01). Mice that received 5 × 10⁶ Th1 cells were selected for subsequent gene expression analyses. At zero time and 2 weeks after infection, draining LNs (B), foot tissues (C), and purified CD4⁺ LN T cells (D and E) were collected and used for total RNA extraction. RPA analyses were performed for different genes. The quantity of each mRNA species was normalized and expressed relative to the amount of GAPDH. The data are means ± standard deviations for three separate experiments. One asterisk (P < 0.05) and two asterisks (P < 0.01) indicate that there are statistically significant differences between the transferred and nontransferred groups.

FIG. 8.

Partial reversal of immunodeficiency in L. amazonensis-infected IL-10^−/− mice. IL-10^−/− and control (WT) C57BL/6 mice were infected with 2 × 10⁶ promastigotes of L. amazonensis. (A) Lesion sizes, as determined with a micrometer. (Inset) Parasite loads in foot tissues were assessed (log₁₀ scale) after 4 and 10 weeks of infection. One asterisk indicates that there is a statistically significant difference (P < 0.05) between the two infection groups at 4 and 10 weeks after infection. (B) Total RNAs were extracted from LNs or infected foot tissues at zero time and 2 and 4 weeks after infection and used for RPA analyses of different genes. The quantity of each mRNA species detected in LNs and foot tissues was normalized and expressed relative to the amount of GAPDH. The data are means ± standard deviations for three separate experiments.