Specific depletion reveals a novel role for neutrophil-mediated protection in the liver during Listeria monocytogenes infection

Abstract

Previous studies have suggested that neutrophils are required for resistance during infection with multiple pathogenic microorganisms. However, the depleting antibody used in those studies binds to both Ly6G and Ly6C (anti-Gr-1; clone RB6-8C5). This antibody has been shown to deplete not only neutrophils but also monocytes and a subset of CD8(+) T cells. Recently, an antibody against Ly6G, which specifically depletes neutrophils, was characterized. In the present study, neutrophils are depleted using the antibody against Ly6G during infection with the intracellular bacterium Listeria monocytogenes (LM). Our data show that neutrophil-depleted mice are much less susceptible to infection than mice depleted with anti-Gr-1. Although neutrophils are required for clearance of LM, their importance is more pronounced in the liver and during a high-dose bacterial challenge. Furthermore, we demonstrate that the protection mediated by neutrophils is due to the production of TNF-α, but not IFN-γ. Additionally, neutrophils are not required for the recruitment of monocytes or the generation of adaptive T-cell responses during LM infection. This study highlights the importance of neutrophils during LM infection, and indicate that depletion of neutrophils is less detrimental to the host than depletion of all Gr-1-expressing cell populations.

Figure 1

Anti-Ly6G depletes neutrophils, while anti-Gr-1 depletes neutrophils and monocytes during LM infection. B6 mice received PBS, anti-Gr-1, or anti-Ly6G one day prior to infection with ~10⁵ LM for 1 day (A, B, C, D, & E) or ~10³ LM for 3 days (B). Flow cytometric analysis of size (forward scatter) and granularity (side scatter) on peripheral blood leukocytes, splenocytes, and liver leukocytes is displayed (A). The number above the gated sub-region indicates the percentage of the granulocyte population out of the whole live cell gate after treatment with PBS (top), anti-Gr-1 (middle), and anti-Ly6G (bottom). The percentage of neutrophils in the peripheral blood was determined by cytospins performed at days 1 and 3 pi in mice treated with PBS, anti-Gr-1, or anti-Ly6G (B). Splenocytes and liver leukocytes were analyzed based on expression of Ly6C and CD11b after treatment with PBS, anti-Gr-1 or anti-Ly6G (C). Percentages of Ly6C^hiCD11b^int monocytes (red population) and Ly6C^intCD11b^hi neutrophils (blue population) are illustrated in the live cell gates (A) based on their respective colors. Graphical depictions of neutrophil (D) and monocyte (E) populations are expressed as a percentage of live cells. Two-way ANOVAs detected significant effects of antibody treatment (p ≤ 0.05). An * indicates a significant difference from B6 mice treated with PBS (p ≤ 0.05). These data are representative of two independent experiments. Data are expressed as the mean ± SEM (n = 5/group).

Figure 2

Neutrophils are important for resistance against a high-dose LM challenge. B6 mice received PBS, anti-Gr-1, or anti-Ly6G one day prior to infection with ~10⁴ LM (A) or ~3.5×10⁴ LM (B), or at days -1 and 3 pi with ~10⁴ LM (C) or ~3.5×10⁴ LM (D). Survival data are presented on a Kaplan-Meier plot and log rank tests were used to compare the susceptibility between groups. A significant effect of anti-Gr-1 is observed in A (p ≤ 0.05). A significant effect of anti-Ly6G is observed in D (p ≤ 0.05). In A, n=9–10/group, and in B, C, and D, n=10/group.

Figure 3

Neutrophils are required for clearance of LM from the liver during low dose infection and are required for clearance of LM from the spleen and liver during high dose infection. B6 mice received PBS, anti-Gr-1, or anti-Ly6G one day prior to infection. After infection, spleen and liver CFUs were determined at one day (A–C), three days (DE), and five days (F) pi. The infectious doses used were: ~10³ LM (A, D, F), ~10⁴ LM (E), ~10⁵ LM (B), or ~10⁶ LM (C). Two-way ANOVAs detected significant effects of antibody treatment on bacterial CFUs (p ≤ 0.05). An * indicates a significant difference from B6 mice treated with PBS (p ≤ 0.05). An ** indicates a significant difference from both B6 mice treated with PBS or treated with anti-Ly6G. These data are representative of two independent experiments. All data are expressed as the mean ± SEM (n = 5/group).

Figure 4

Neutrophils are not required for the production of IFN-γ during LM infection. B6 mice received either PBS, anti-Gr-1, or anti-Ly6G one day prior to infection with ~10⁵ LM. At day 1 pi, serum was harvested, and splenocytes and liver leukocytes were cultured overnight and the supernatants were utilized for an IFN-γ ELISA (A). Splenocytes were also cultured for 4 hours with BFA and used for intracellular cytokine staining (B). An * indicates a significant difference from B6 mice treated with PBS (p ≤ 0.05). These data are representative of two independent experiments. All data are expressed as the mean ± SEM (n = 5/group).

Figure 5

Neutrophils do not regulate the generation of T cell responses against LM. B6 mice received either PBS or anti-Ly6G one day prior to infection with ~10⁴ LM/OVA ΔactA. Splenocytes were harvested from uninfected mice, PBS treated, or anti-Ly6G treated mice at day 7 pi with LM/OVA ΔactA. Splenocytes were stimulated overnight with 10 μM SIINFEKL peptide (A&B) or 50:1 HKLM/OVA ΔactA (C&D), and were utilized for IFN-γ intracellular cytokine staining. Percentages (A&C) or total cell numbers (B&D) of IFN-γ+CD8+ T cells (A&B) and IFN-γ+CD4+ T cells (C&D) are depicted. An * indicates a significant difference between uninfected mice and both PBS and anti-Ly6G treated infected mice (p ≤ 0.05). No other differences were significant (p ≥ 0.05). These data are representative of two independent experiments. All data are expressed as the mean ± SEM (n = 3–5/group).

Figure 6

Neutrophils are necessary for the production of TNF-α during LM infection. B6 mice received either PBS, anti-Gr-1, or anti-Ly6G one day prior to infection with ~10⁵ LM (A, C, & D) or ~10⁶ LM (B). At day 1 pi, splenocyte and liver leukocyte cultures were stimulated overnight with 50:1 HKLM and utilized for a TNF-α ELISA (A & B). Splenocytes and liver leukocytes were cultured for 4 hrs with BFA and prepared for TNF-α intracellular cytokine staining, and percentages (C) or total cell numbers (D) of TNF-α+ monocytes and neutrophils are depicted. For A & B an * indicates a significant difference mice treated with PBS (p ≤ 0.05). An ** indicates a significant difference from both B6 mice treated with PBS or treated with anti-Ly6G. For C and D, an * indicates a significant difference from monocytes (p ≤ 0.05). These data are representative of two independent experiments. All data are expressed as the mean ± SEM (n = 5/group). N.D. = not detectable.