Neutrophils have a protective role during early stages of Leishmania amazonensis infection in BALB/c mice

Abstract

Neutrophils are involved in the early stages of immune responses to pathogens. Here, we investigated the role of neutrophils during the establishment of Leishmania amazonensis infection in BALB/c and C57BL/6 mice. First, we showed an accumulation of neutrophils between 6 and 24 h post-infection, followed by a reduction in neutrophil numbers after 72 h. Next, we depleted neutrophils prior to infection using RB6-8C5 or 1A8 mAb. Neutrophil depletion led to faster lesion development, increased parasite numbers and higher arginase activity during the first week of infection in BALB/c mice, but not in C57BL/6 mice. Increased susceptibility was accompanied by augmented levels of anti-L. amazonensis IgG and increased production of IL-10 and IL-17. Because IL-10 is a mediator of susceptibility to Leishmania infection, we blocked IL-10 signalling in neutrophil-depleted mice using anti-IL-10R. Interestingly, inhibition of IL-10 signalling abrogated the increase in parasite loads observed in neutrophil-depleted mice, suggesting that parasite proliferation is at least partially mediated by IL-10. Additionally, we tested the effect of IL-17 in inflammatory macrophages and observed that IL-17 increased arginase activity and favoured parasite growth. Taken together, our data indicate that neutrophils control parasite numbers and limit lesion development during the first week of infection in BALB/c mice.

Keywords: IL-10; Leishmania amazonensis; immune response; neutrophils.

Figure 1

Kinetics of neutrophil recruitment to the site of Leishmania amazonensis infection. (a) Left Panel BALB/c and C57BL/6 mice were injected intradermically with 1 × 10⁶ L. amazonensis metacyclic promastigotes. At the indicated time points, the ears were collected and myeloperoxidase (MPO) activity was assessed. Data represent mean ± SE from at least three independent experiments. Statistical differences were observed between groups (BALB/c and C57BL/6) and times (two-way anova). *P < 0·001 (Bonferroni post-tests). (a) Right Panel MPO activity in ears isolated from BALB/c and C57BL/6 mice that received the following treatments: (1) noninfected animals; (2) PBS inoculated intradermically into the ears; (3) infection with 1 × 10⁶ L. amazonensis metacyclic promastigotes; (4) treatment with 0·5 mg of anti-Ly6G/Ly6C antibody clone RB6-8C5 (RB6) 16 h before infection with 1 × 10⁶ metacyclic promastigote forms of L. amazonensis. Twenty-four hours post-infected animals were euthanized, ears were collected and MPO activity was assessed. Data represent mean ± SE from 4 to 6 mice/group. Results are representative of at least three independent experiments. #P < 0·001, *P < 0·01 (one-way anova followed by Tukeys multiple comparison test). (b, c) BALB/c mice were treated with RB6 (0·5 mg) 16 h before infection with 1 × 10⁶ L. amazonensis metacyclic promastigotes. At the indicated time points, animals were euthanized and ears were collected and processed for immunophenotyping by flow cytometry. The kinetics of neutrophil accumulation at the site of infection is represented by the percentage of Ly6G⁺ cells in the live cell population at the indicated time points (b). The absolute number of neutrophils at the site of infection in BALB/c mice is shown in (c). In (d), BALB/c mice were either left untreated or they were treated with 0·5 mg of RB6 or rat IgG 16 h before infection with 1 × 10⁶ L. amazonensis. After 24 h, MPO activity was assessed. Data represent mean ± SE from 5 to 6 mice/group. Results are representative of two independent experiments. *P < 0·05 (Mann–Whitney test).

Figure 2

Course of infection after the depletion of neutrophils in BALB/c and C57BL/6 mice. BALB/c and C57BL/6 mice were injected intradermically with 1 × 10⁶ Leishmania amazonensis metacyclic promastigotes. The groups treated with RB6 received one i.p. injection of RB6 (0·5 mg) 16 h before infection (a, b). Ear thickness was monitored weekly (a). In (b), lesion development was monitored daily during the first week post-infection. The insert in (b) shows that treatment with 0·5 mg of rat IgG 16 h prior to infection does not affect the course of infection in BALB/c mice. In (c), BALB/c and C57BL/6 mice were infected as described above and the groups treated with RB6 received i.p. injection of RB6 (0·5 mg) on days −1, 1, 3, 5 and 7, and ear thickness was monitored biweekly. Data represent mean ± SE from 5 to 10 mice/group. Results are representative of at least three independent experiments. *P < 0·05 (Mann–Whitney test).

Figure 3

Neutrophil depletion increases parasite burden and arginase activity in BALB/c mice. BALB/c (a, c, e, g, h) and C57BL/6 (b, d, f) mice were treated with 0 5 mg of RB6, and 16 h later, they were infected intradermically into the ears with 1 × 10⁶ Leishmania amazonensis metacyclic promastigotes. One week after infection, mice were euthanized and ears were collected for the assessment of parasite burdens by limiting dilution analysis (a, b) and arginase activity (e, f). Lymph nodes were also collected for the assessment of parasite burdens (c, d). The dotted line in (e) and (f) represents the arginase activity in ears of noninfected mice. Parasite burdens are represented as the negative log of the titre. Data represent mean ± SE from 5 to 6 mice/group. Results are representative of at least three independent experiments. Parasite burdens in the ears (g) and in the draining lymph nodes (h) were also assessed by limiting dilution analysis 1 day post-infection (p.i.). Data represent mean ± SE from 5 to 6 mice/group. Results are the mean of two independent experiments. *P < 0·05 (Mann–Whitney test).

Figure 4

Effect of neutrophil depletion on the secretion of IL-10 and IL-17 by draining lymph node cells during recall responses. BALB/c mice were treated with 0 5 mg of RB6, and 16 h later, they were infected intradermically into the ears with 1 × 10⁶ Leishmania amazonensis metacyclic promastigotes. Three and 7 days post-infection draining lymph node cells were harvested and stimulated with L. amazonensis antigen (Ag). After 72 h, supernatants were collected for the measurement of IL-17 (a) and IL-10 (b) levels by enzyme-linked immunosorbent assay. Data represent mean ± SE from 5 to 6 mice/group. Results are representative of at least two independent experiments. *P < 0·05 (Mann–Whitney test).

Figure 5

Treatment with 1A8 mAb yields similar results as treatment with RB6-8C5 mAb. BALB/c mice were treated with 1 mg of 1A8 mAb, and 16 h later, they were infected intradermically into the ears with 1 × 10⁶ Leishmania amazonensis metacyclic promastigotes. Seven days after infection, ear thickness was measured (a); then, mice were euthanized and ears were collected for the assessment of parasite burdens by limiting dilution analysis (b) and arginase activity (c); draining lymph node cells were harvested and stimulated with L. amazonensis antigen (Ag). After 72 h, supernatants were collected for the measurement of IL-10 levels by enzyme-linked immunosorbent assay (d). Data represent mean ± SE from 5 to 6 mice/group. *P < 0·05 (Mann–Whitney test).

Figure 6

IgG1 and IgG2a in the serum of neutrophil-depleted infected BALB/c mice. BALB/c mice were treated with 0·5 mg of RB6, and 16 h later, they were infected intradermically into the ears with 1 × 10⁶ Leishmania amazonensis metacyclic promastigotes. Seven days after infection, animals were anesthetized and blood was collected from the femoral vein. After centrifugation, serum was collected and used for the detection of anti-L. amazonensis IgG1 and IgG2a by enzyme-linked immunosorbent assay. Data represent mean ± SE from 5 to 6 mice/group. Results are representative of two independent experiments. *P < 0·05 (Mann–Whitney test).

Figure 7

Neutrophil depletion alters the percentage of Tregs at the site of infection during the first week post-infection. BALB/c mice were treated with 0 5 mg of RB6, and 16 h later, they were infected intradermically into the ears with 1 × 10⁶ Leishmania amazonensis metacyclic promastigotes. After 7 days of infection, animals were euthanized and ears were collected and processed for immunophenotyping by flow cytometry. Accumulation of Treg cells in the infected ears 7 days post-infection is represented by the percentage of CD3⁺ CD4⁺ Foxp3⁺ cells gated in the population of CD3⁺ CD4⁺ cells in the ear, which represents 1–2% of the cells at the site of infection. The dotted line represents the percentage of Treg cells in ears of noninfected mice. Data represent mean ± SE from 5 to 6 mice/group. Results are representative of two independent experiments. *P < 0·05 (Mann–Whitney test).

Figure 8

The increase in IL-10 production is in part dependent on the increase in IgG levels and drives parasite growth in neutrophil-depleted mice. (a) Bone marrow-derived macrophages were infected with stationary-phase promastigotes of Leishmania amazonensis at 5 : 1 (parasite/macrophage) ratio in the presence or in the absence of LPS (10 ng/mL) or LPS + immune complexes. After 24 h, supernatants were harvested and IL-10 levels were determined by enzyme-linked immunosorbent assay (ELISA). Values are expressed as the means SD. Results represent the mean of two independent experiments performed in triplicates. *P < 0·01 (Mann–Whitney test). (b) For blockage of Fcγ receptor, BALB/c mice received i.p. injections of 2.4G2 (0·2 mg) or rat IgG on days 2, 4 and 6 after infection with 1 × 10⁶ L. amazonensis metacyclic promastigotes. The groups treated with RB6 received one i.p. injection of RB6-8C5 (0·5 mg) 16 h before infection. Seven days post-infection, draining lymph node cells were harvested and stimulated with L. amazonensis antigen (Ag). After 72 h, supernatants were collected for the measurement of IL-10 levels by ELISA. Data represent mean ± SE from 6 mice/group. *P < 0·05 (Mann–Whitney test). (c–g) For the inhibition of IL-10, BALB/c mice were infected with 1 × 10⁶ L. amazonensis metacyclic promastigotes and were inoculated i.p. every 3 days with 0·5 mg of anti-IL-10 receptor monoclonal antibody (c, d, e, f). As a control, mice were treated i.p. every 3 days with 0·5 mg of rat IgG (d, e, g). Ear thickness was monitored daily (c). *P < 0·001 between control and RB6-treated group; #P < 0·001 between control and RB6 + anti-IL-10R-treated group; $P < 0·001 between RB6 and RB6 + anti-IL-10R-treated groups (one-way anova followed by Tukeys multiple comparison test). (d) Ear thickness after 1 week of infection. ns indicates no statistic difference was observed between RB6-treated and RB6+ rat IgG-treated groups. *P < 0·05 between the indicated group and all the other groups (one-way anova followed by Tukeys multiple comparison test). One week after infection, mice were euthanized and ears were collected for the assessment of arginase activity (e) and parasite burdens by limiting dilution analysis (f, g). Data represent mean ± SE from 5 to 6 mice/group. Results are representative of two independent experiments. *P < 0·05 (Mann–Whitney test).

Figure 9

IL-17 increases arginase activity and favours parasite growth in inflammatory macrophages. Peritoneal macrophages were collected from BALB/c mice 5 days after i.p. injection of thioglycolate and cultured in the presence of 10 ng/mL of IL-17, 100 ng/mL of LPS and 5 : 1 parasites/macrophage. Phagocytosis was investigated 24 h after addition of parasites, and intracellular parasite growth was analysed after 72 h (a). After 48 h, cells were assayed for arginase activity (b). *P < 0·05 (one-way anova followed by Tukeys multiple comparison test).