Innate immune-induced depletion of bone marrow neutrophils aggravates systemic bacterial infections

Abstract

Neutrophils are the most abundant leukocytes in circulation and provide a primary innate immune defense function against bacterial pathogens before development of a specific immune response. These specialized phagocytes are short lived (12-24 hours) and continuously replenished from bone marrow. We found that if the host is overwhelmed by a high inoculum of Listeria monocytogenes, neutrophils are depleted despite high granulocyte-colony stimulating factor induction. In contrast to a low-dose innocuous L. monocytogenes infection, high-dose Listeria challenge blocks neutrophil recruitment to infectious abscesses and bacterial proliferation is not controlled, resulting in lethal outcomes. Administering synthetic TLR2-ligand or heat-killed bacteria during the innocuous L. monocytogenes infection reproduced these effects, once again leading to overwhelming bacterial propagation. The same stimuli also severely aggravated Salmonella typhimurium, Staphylococcus aureus, and Streptococcus pyogenes systemic infection. These data implicate systemic innate immune stimulation as a mechanism of bone marrow neutrophil exhaustion which negatively influences the outcome of bacterial infections.

Fig. 1.

Transient versus terminal depletion of BM neutrophils by low- or high-dose Listeria monocytogenes (L.m.) infection. (A) L.m. counts in spleen, liver, BM (cfu/femur), and blood (cfu/μl) at the respective timepoints (n = 4 per timepoint, mean ± SEM, one of two experiments with similar outcomes). (B) Ly6G⁺CD11b⁺ (neutrophils) and CD11b⁺Ly6G⁻ (macrophages) cells in BM and Ly6G⁺CD11b⁺ (neutrophils) in blood at the respective days after low-dose L.m. infection (10³ cfu) (n = 3–4 per time point, mean ± SEM, one of two experiments with similar outcomes) (C) Survival of WT mice after infection with low-dose (10³ cfu) or high-dose (10⁵ cfu) L.m. i.v. (n = 5–10/group) (D) Serum concentration of G-CSF after infection low-dose (10³ cfu) or high-dose (10⁵ cfu) L.m. i.v. (n = 3–4 per time point, mean ± SEM) (E) Ly6G⁺CD11b⁺ (neutrophils) and CD11b⁺Ly6G⁻ (macrophages) cells in BM and Ly6G⁺CD11b⁺ (neutrophils) in blood at the respective days after infection with 10⁵ cfu L.m. (n = 3–4 per time point, mean ± SEM, one of two experiments with similar outcomes) (F) Bacterial counts in spleen, liver, BM (cfu/femur), and blood (cfu/μl) at the respective timepoints (n = 4 per timepoint, mean ± SEM, one of two experiments with similar outcome)

Fig. 2.

Histological time course of neutrophils in livers and spleens during infection with low- or high-dose L.m. Livers and spleens taken at the indicated time points were stained immunohistochemically for presence of GR-1⁺ cells, in (A) and (B) during infection with 10³ cfu L.m. and in (C) and (D) with 10⁵ cfu L.m.

Fig. 3.

Activation and death of neutrophils during infection with low- and high-dose L.m. (A and B) Mean fluorescence intensity of CD11b on the surface of Ly6G⁺CD11b⁺ (neutrophils) and CD11b⁺Ly6G⁻ (macrophages) cells in BM and blood after infection with 10³ cfu (A) or 10⁵ cfu L.m. (B) (n = 3–4, mean ± SEM, one of two experiments with similar outcomes) (C and D) Percentage of Annexin-V⁺ neutrophils, macrophages, and Ly6G⁻CD11b⁻ cells (other cells) in BM after infection with 10³ cfu (C) or 10⁵ cfu (D) L.m. (n = 3–4, mean ± SEM, one of two experiments with similar outcomes)

Fig. 4.

BM neutrophil depletion 1 day after systemic TLR2 stimulation. (A and B) BM neutrophils (Ly6G⁺CD11b⁺) numbers (A) and percentages (B) measured by FACS 24 hours after administration of 100 μg Pam2Cys i.v. (mean ± SEM, n = 3–5 animals/group, one of two experiments with similar outcomes). (C) Percentage of dead (Annexin-V⁺) neutrophils in BM 24 hours after 100 μg Pam2Cys i.v. (mean ± SEM, n = 3–5 animals/group, one of two experiments with similar outcomes). (D) Number of neutrophils in spleens 24 hours after administration of 100 μg Pam2Cys. Lower bars show viable (7AAD⁻Annexin-V⁻) neutrophils; upper bars show Annexin-V⁺7AAD⁺/⁻ neutrophils (mean ± SEM, n = 3–5 animals/group, one of two experiments with similar outcomes). (E) Mean fluorescence intensity of CD11b surface staining on BM neutrophils 24 hours after administration of 100 μg Pam2Cys i.v. (mean ± SEM, n = 3–5 animals/group, one of two experiments with similar outcomes). (F) Mean fluorescence intensity of DHR123 staining in BM neutrophils 24 hours after administration of 100 μg Pam2Cys i.v. (mean ± SEM, n = 3–5 animals/group, one of two experiments with similar outcomes).

Fig. 5.

Systemic TLR2 ligation leads to impaired neutrophil infiltration, uncontrolled bacterial propagation, and host death. (A) Experimental protocol. (B) Survival curve of tlr2^−/− or WT mice infected with 10³ cfu L.m. with or without administration of 100 μg Pam2Cys 24 hours after infection. (n = 5–11/group, two experiments) (C) Bacterial counts at day 3 of infection with 10³ cfu L.m. with or without administration of 100 μg Pam2Cys 24 hours after infection. Grey bars show WT controls that had received 100 μg α-GR1 antibody (clone NimpR14) at 24 hours after infection (mean ± SEM, n = 3 per group, one of two experiments shown with similar outcomes). (D) BM neutrophils at day 3 of the L.m. infection (mean ± SEM, n = 3, one of two experiments with similar outcomes). (E) Immunohistochemistry of livers at day 3 of Listeria infection stained with anti-L.m. serum (left row) or α-GR1 (neutrophils, right row). Conditions shown are WT PBS (1), WT Pam2Cys (2), tlr2^−/− PBS (3), tlr2^−/− Pam2Cys (4), WT α-GR1 with hematoxylin and eosin–stained inlet (5) to confirm specific staining and absence of neutrophils. (F) Bacterial counts at day 3 of infection with 4 × 10⁴ cfu Salmonella typhimurium with or without administration of 100 μg Pam2Cys 24 hours after infection (mean ± SEM, n = 5/group). (G) Bacterial counts at day 3 of infection with 5 × 10⁶ cfu Streptococcus pyogenes with or without administration of 100 μg Pam2Cys 24 hours after infection (mean ± SEM, n = 10/group, two experiments pooled). (H) Bacterial counts at day 3 of infection with 5 × 10⁶ cfu Staphylococcus aureus with or without administration of 100 μg Pam2Cys 24 hours after infection (mean ± SEM, n = 10 per group, two experiments pooled).