Neutrophil myeloperoxidase diminishes the toxic effects and mortality induced by lipopolysaccharide

Abstract

Neutrophils have crucial antimicrobial functions but are also thought to contribute to tissue injury upon exposure to bacterial products, such as lipopolysaccharide (LPS). To study the role of neutrophils in LPS-induced endotoxemia, we developed a new mouse model, PMN^DTR mice, in which injection of diphtheria toxin induces selective neutrophil ablation. Using this model, we found, surprisingly, that neutrophils serve to protect the host from LPS-induced lethal inflammation. This protective role was observed in conventional and germ-free animal facilities, indicating that it does not depend on a particular microbiological environment. Blockade or genetic deletion of myeloperoxidase (MPO), a key neutrophil enzyme, significantly increased mortality after LPS challenge, and adoptive transfer experiments confirmed that neutrophil-derived MPO contributes importantly to protection from endotoxemia. Our findings imply that, in addition to their well-established antimicrobial properties, neutrophils can contribute to optimal host protection by limiting the extent of endotoxin-induced inflammation in an MPO-dependent manner.

Figure 1.

Antibody-mediated neutrophil depletion results in increased mortality after LPS injection. (A–C) Representative flow cytometry profile (A) and quantification of CD62L (B) and CD11b (C) by geometric mean fluorescence intensity (GeoMean) on Ly6G⁺ CD11b⁺ blood neutrophils 6 h after LPS injection at the indicated concentrations. Red areas outlined in A provide a visual indication of CD62L and CD11b on the neutrophil population from the control 0 group. B and C show values from individual mice; bars indicate means ± SEM pooled from two independent experiments. **, P < 0.01; ***, P < 0.001 versus control 0 group by two-tailed Mann–Whitney U test. (D–I) Changes in body temperature (Δ°C [mean ± SEM]) and survival (percentage of live animals) after LPS injection in C57BL/6J mice treated i.p. with anti-Ly6G (D and E), anti–Gr-1 (F–I) neutrophil-depleting antibodies, or respective isotype control antibodies. Data in D–G are pooled from three independent experiments performed at Stanford University (total n = 10–12/group). Data in H are pooled from three independent experiments performed in the Institut Pasteur conventional SPF animal facility (total n = 21–26/group). Data in I are pooled from two independent experiments performed in the Institut Pasteur GF animal facility (total n = 18/group). **, P < 0.01; ***, P < 0.001 versus the corresponding isotype control group by Mantel–Cox log-rank test. Arrows in D–I indicate days of i.p. injection of the neutrophil-depleting or isotype control antibodies.

Figure 2.

Injection of DT in PMN^DTR mice induces marked depletion of neutrophils and enhances susceptibility to LPS-induced endotoxemia. (A) GFP expression (mean fluorescence intensity [MFI]) among leukocytes in the blood, BM, spleen, and peritoneal lavage fluid of MRP8-Cre/iresGFP⁺ mice and MRP8-Cre/iresGFP⁻ littermate controls. Results in A are pooled from three independent experiments, each column representing data from one mouse. (B–D) Numbers of blood (B), spleen (C), and BM (D) neutrophils 24 h after i.p. injection of 500 ng DT into PMN^DTR mice and PMN^WT littermate control mice. Results in B–D show values from individual mice; bars indicate means ± SEM pooled from three (C and D; total n = 7–8/group) or four (B; total n = 15–16/group) independent experiments. (E and F) Changes in body temperature (Δ°C [mean ± SEM]; E) and survival (percentage of live animals; F) after LPS injection in DT-treated PMN^DTR and PMN^WT mice. Data in E and F are pooled from three independent experiments (total n = 11/group); arrows indicate days of i.p. injection of DT. (G) Percentage of blood neutrophils before (time 0) and 3, 6, and 20 h after LPS injection in DT-treated PMN^DTR mice and PMN^WT controls. Data show values from individual mice; bars indicate means ± SEM pooled from three independent experiments (n = 6–10/group). (H–L) Levels of TNF-α (H), IL-6 (I), IL-10 (J), IFN-γ (K), and MCP-1 (L) in the plasma of DT-treated PMN^DTR mice and PMN^WT littermate controls before (time 0) and 6 h after LPS injection (n = 6–16/group). Results in H–L are means ± SEM pooled from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus PMN^WT group and ^#, P < 0.05; ^##, P < 0.01; ^###, P < 0.001 versus same group at time 0 by Mann–Whitney U test (B–D and G–L) or Mantel–Cox log-rank test (E and F).

Figure 3.

Neutrophil-derived MPO diminishes LPS-induced hypothermia and mortality. (A) Representative flow cytometry profile of Ly6G⁺ CD11b⁺ blood neutrophils 0 or 6 h after LPS injection in WT or Mpo^−/− mice. Areas outlined in red indicate values for neutrophils from the control (time 0) group; data are representative of results obtained in two independent experiments. (B and C) Changes in body temperature (Δ°C [mean ± SEM]; B) and survival (percentage of live animals; C) after LPS injection in WT and Mpo^−/− mice. (D) Survival after LPS injection in WT mice treated with the MPO inhibitor 4-ABAH or vehicle. Data in B–D are pooled from three independent experiments (total n = 11–13/group). **, P < 0.01; ***, P < 0.001 versus respective WT group (C) or vehicle-treated group (D) by Mantel–Cox log-rank test. (E) MPO in the peritoneal lavage fluid 6 h after injection with PBS (n = 4) or LPS (n = 7/group). Values from individual mice are shown; bars indicate means ± SEM pooled from two independent experiments. **, P < 0.01; ***, P < 0.001 versus PBS group and ^###, P < 0.001 versus LPS-treated control group by unpaired Student’s t test. (F–I) Bioluminescent visualization of MPO activity in various organs 6 h after injection of PBS or LPS in the indicated group. (F–H) Quantification of bioluminescence in spleen (F), liver (G), and lung (H). Data in F–H are means + SEM from three independent experiments (total n = 6–14/group) except for Mpo^−/− mice (two independent experiments with a total of three to four mice). *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus PBS-treated WT group and ^#, P < 0.05; ^##, P < 0.01; ^###, P < 0.001 versus corresponding LPS-treated control group by unpaired Student’s t test. NS, not significant (P > 0.05). (I) Representative images of different organs (Sp, spleen; L, liver; St, stomach; C, cecum; Du, duodenum; Ki, kidney; H, heart; and L, lung). (J and K) Changes in body temperature (J) and survival (K) after LPS injection in DT-treated PMN^DTR mice (n = 9), PMN^WT littermate controls (n = 24), and PMN^DTR mice engrafted i.v. with 10⁷ purified BM neutrophils from WT mice (WT PMNs → PMN^DTR; n = 16) or from Mpo^−/− mice (Mpo^−/− PMNs → PMN^DTR; n = 10); arrows indicate days of i.p. injection of DT. Data are pooled from two (Mpo^−/− PMNs → PMN^DTR group), three (PMN^DTR group), or five (PMN^WT and WT PMNs → PMN^DTR groups) independent experiments. ***, P < 0.001 versus PMN^WT group and ^#, P < 0.05; ^##, P < 0.01 versus WT PMNs → PMN^DTR group by Mantel–Cox log-rank test.