Neutrophils contribute to development of a protective immune response during onset of infection with Leishmania donovani

Abstract

Neutrophils are key components of the inflammatory response and as such contribute to the killing of microorganisms. In addition, recent evidence suggests their involvement in the development of the immune response. The role of neutrophils during the first weeks post-infection with Leishmania donovani was investigated in this study. When L. donovani-infected mice were selectively depleted of neutrophils with the NIMP-R14 monoclonal antibody, a significant increase in parasite numbers was observed in the spleen and bone marrow and to a lesser extent in the liver. Increased susceptibility was associated with enhanced splenomegally, a delay in the maturation of hepatic granulomas, and a decrease in inducible nitric oxide synthase expression within granulomas. In the spleen, neutrophil depletion was associated with a significant increase in interleukin 4 (IL-4) and IL-10 levels and reduced gamma interferon secretion by CD4(+) and CD8(+) T cells. Increased production of serum IL-4 and IL-10 and higher levels of Leishmania-specific immunoglobulin G1 (IgG1) versus IgG2a revealed the preferential induction of Th2 responses in neutrophil-depleted mice. Altogether, these data suggest a critical role for neutrophils in the early protective response against L. donovani, both as effector cells involved in the killing of the parasites and as significant players influencing the development of a protective Th1 immune response.

FIG. 1.

Role of neutrophils in the early control of L. donovani growth. (A) Parasite burdens following infection with L. donovani were measured in the spleen, liver, and bone marrow at day 14 postinfection. Mice were treated with 250 μg of either a control MAb or an antineutrophil MAb (NIMP-R14) on days 0, 3, 6, 9, and 12 postinfection. A significant increase in parasite burdens in the spleens and bone marrow of neutrophil-depleted mice compared to controls was observed (P < 0.0006 and P < 0.0001, respectively). (B) Spleens and livers were removed and weighed 14 days postinfection. A significant increase (P < 0.0001) in spleen weight was observed in the neutrophil-depleted group compared to control MAb-treated mice.

FIG. 2.

Liver granuloma formation in L. donovani-infected BALB/c mice 14 days postinfection. Granuloma maturation was assessed at day 14 postinfection on paraffin-embedded liver sections from control MAb-treated or antineutrophil MAb (NIMP-R14)-treated mice. Sections were stained with hematoxylin and eosin, and granuloma formation was determined at magnification ×1,000. A significant decrease in mature (P < 0.012) and sterile (P < 0.006) granulomas was observed for neutrophil-depleted mice compared to results for mice treated with the control MAb, while levels of infected Kupffer cells were significantly increased (P < 0.016).

FIG. 3.

Decreased iNOS in hepatic granulomas of neutrophil-depleted mice 14 days postinfection. iNOS-stained liver sections from 14-day-infected BALB/c mice treated with a control MAb (A) or the neutrophil-depleting MAb (B). Sections were counterstained with hematoxylin and eosin. Original magnification, ×100; insert magnification, ×400.

FIG. 4.

Cytokine mRNA expression in spleen and liver. Quantitative real-time PCR was performed on spleen (A) or liver (B) of L. donovani-infected mice treated with PBS, control MAb, or the NIMP-R14 neutrophil-depleting MAb (α-neutrophil) 14 days postinfection. Basal levels of mRNA were analyzed with spleen cells from uninfected mice similarly treated (naive). Values of IFN-γ, TNF, IL-4, and IL-10 mRNA were normalized to endogenous levels of HPRT mRNA and are represented as arbitrary units. Each bar is the mean of triplicate measurement for three or four spleens or livers. Data are representative of three distinct experiments. n.d., not detectable.

FIG. 5.

Analysis of IFN-γ production by splenic T cells of neutrophil-depleted mice. (A) The number of IFN-gamma-producing CD4⁺ and CD8⁺ spleen cells was measured in control MAb-treated and antineutrophil MAb-treated mice at day 14 postinfection. Cells were restimulated with phorbol myristate acetate-ionomycin and gated on CD4⁺ and CD8⁺ T cells, and the FACSDiva software was used to analyze the results. The results are representative of three experiments performed. A significant reduction in CD4⁺ IFN-γ⁺ cells (P < 0.001) and CD8⁺ IFN-γ⁺ cells (P < 0.0005) for antineutrophil-treated mice compared to results for control mice was observed. (B) Cytokine levels in supernatants of splenocytes stimulated or not with ConA for L. donovani-infected mice depleted of neutrophils or not depleted. Data are the means ± standard errors for splenocyte cultures from four mice, representative of three experiments.

FIG. 6.

Increased IL-4 and IL-10 cytokine levels and increased IgG1 levels in serum of neutrophil-depleted mice 14 days postinfection. (A to C) L. donovani-infected mice were treated with neutrophil-depleting MAb or a control MAb and serum cytokine levels analyzed 14 days postinfection. Serum levels of IL-4 (A), IL-10 (B), or IL-12 (C) were analyzed by ELISA. Results are the means ± standard errors of the cytokine levels for four to six mice per group from one representative experiment of two performed. n.d., not detectable. (D) Parasite-specific IgG1 and IgG2a levels were measured by ELISA in the serum of mice treated with control MAb or antineutrophil MAb at day 14 postinfection. A significant increase in IgG1 levels (P < 0.0001) in the NIMP-R14-treated group over levels for control MAb-treated mice was observed.