Neutrophil reduction attenuates the severity of lung injury in the early phase of pneumococcal pneumonia in mice

Abstract

Neutrophils are the first leukocytes to be recruited to sites of inflammation in response to chemotactic factors released by activated macrophages and pulmonary epithelial and endothelial cells in bacterial pneumonia, a common cause of acute respiratory distress syndrome (ARDS). Although neutrophilic inflammation facilitates the elimination of pathogens, neutrophils also may cause bystander tissue injury. Even though the presence of neutrophils in alveolar spaces is a key feature of acute lung injury and ARDS especially from pneumonia, their contribution to the pathogenesis of lung injury is uncertain. The goal of this study was to elucidate the role of neutrophils in a clinically relevant model of bacterial pneumonia. We investigated the effect of reducing neutrophils in a mouse model of pneumococcal pneumonia treated with antibiotics. Neutrophils were reduced with anti-lymphocyte antigen 6 complex locus G6D (Ly6G) monoclonal antibody 24 h before and immediately preceding infection. Mice were inoculated intranasally with Streptococcus pneumoniae and received ceftriaxone 12 h after bacterial inoculation. Neutrophil reduction in mice treated with ceftriaxone attenuated hypoxemia, alveolar permeability, epithelial injury, pulmonary edema, and inflammatory biomarker release induced by bacterial pneumonia, even though bacterial loads in the distal air spaces of the lung were modestly increased as compared with antibiotic treatment alone. Thus, when appropriate antibiotics are administered, lung injury in the early phase of bacterial pneumonia is mediated in part by neutrophils. In the early phase of bacterial pneumonia, neutrophils contribute to the severity of lung injury, although they also participate in host defense.NEW & NOTEWORTHY Neutrophil accumulation is a key feature of ARDS, but their contribution to the pathogenesis is still uncertain. We investigated the effect of reducing neutrophils in a clinically relevant mouse model of pneumococcal pneumonia treated with antibiotics. When appropriate antibiotics were administered, neutrophil reduction with Ly6G antibody markedly attenuated lung injury and improved oxygenation. In the early phase of bacterial pneumonia, neutrophils contribute to the severity of lung injury, although they also participate in host defense.

Keywords: Streptococcus pneumoniae; acute lung injury; acute respiratory distress syndrome; bacterial pneumonia; neutrophils.

Graphical abstract

Figure 1.

Ly6G antibody reduces neutrophils in BAL and blood at 24 h after infection with Streptococcus pneumoniae. A: 10- to 12-wk-old female C57BL/6 mice received 25 mg/kg of anti-Ly6G monoclonal antibody (mAb) or isotype control (ISO) intraperitoneally (IP) at 24 h and immediately before bacterial inoculation, then inoculated intranasally (IN) with 10⁸ colony-forming units of Streptococcus pneumoniae (SP), followed by 150 mg/kg of ceftriaxone or vehicle control IP at 12 h after bacterial inoculation. All mice were euthanized 24 h after inoculation. B: neutrophils in bronchoalveolar lavage (BAL) fluid were quantified by flow cytometry. Representative panels from normal control (NC), infected untreated mice (SP+ISO), ceftriaxone treatment (SP+CTRX+ISO), or anti-Ly6G mAb and ceftriaxone treatment (SP+CTRX+Ly6G) are shown. Inoculation of Streptococcus pneumoniae caused a marked neutrophil infiltration into the airspaces in the lungs at 24 h after infection. Neutrophils accounted for approximately 80% of all live leukocytes in the BAL. Administration of anti-Ly6G mAb reduced neutrophils in the BAL to <1%. C and D: ceftriaxone did not alter the number of neutrophils, while administration of anti-Ly6G mAb reduced neutrophils in the BAL and blood. E: the number of alveolar macrophages in BAL was not altered by SP infection, ceftriaxone, nor anti-Ly6G mAb. F: inflammatory monocytes in BAL were increased by pneumococcal infection, but not altered by ceftriaxone nor anti-Ly6G mAb. G: neutrophil elastase, a measure inflammatory protease released by neutrophils, in the BAL was reduced by ceftriaxone compared with untreated mice, and further reduced by anti-Ly6G mAb, compared with ceftriaxone alone. All data are shown as means ± SD. Statistical differences between groups were calculated with one-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. †P < 0.05 compared with other groups. CTRX, ceftriaxone; IN, intranasal inoculation; IP, intraperitoneal injection; ISO, isotype control; NC, normal control; SP, Streptococcus pneumoniae.

Figure 2.

Neutrophil reduction attenuates indices of acute lung injury in mice with pneumonia treated with antibiotics. A: arterial oxygen saturation ($\mathrm{S}\mathrm{p}{\mathrm{O}}_2$) was measured 12, 18, and 24 h after bacterial inoculation. $\mathrm{S}\mathrm{p}{\mathrm{O}}_2$ in the infected untreated mice (SP) declined 18 h after infection. Ceftriaxone treatment improved $\mathrm{S}\mathrm{p}{\mathrm{O}}_2$ at 18 and 24 h after infection (SP+CTRX). Combination of neutrophil reduction by anti-Ly6G monoclonal antibody (mAb) with ceftriaxone treatment (SP+CTRX+Ly6G) had higher oxygenation than mice with ceftriaxone treatment alone. n = 5/group. B–D: excess lung water, a measure of edema in the interstitial and alveolar spaces (B), total protein concentration in bronchoalveolar lavage (BAL) fluid, a measure of alveolar-capillary barrier disruption (C), and RAGE in BAL fluid, a biomarker of epithelial cell injury (D) were attenuated by ceftriaxone treatment compared with untreated infected mice, and further attenuated by neutrophil reduction when compared with untreated or ceftriaxone treatment alone. n = 9–10/group. Mice were administered either isotype control IgG or anti-Ly6G mAb at 24 h and immediately before bacterial inoculation. All data are shown as means ± SD. Statistical differences between groups were calculated with two-way repeated measures ANOVA followed by Tukey’s multiple comparison test (A) or one-way ANOVA followed by Tukey’s multiple comparison test (B–D). *P < 0.05, **P < 0.01, ****P < 0.0001. †P < 0.05 compared with other groups. BAL, bronchoalveolar lavage; CTRX, ceftriaxone; NC, normal control; SP, Streptococcus pneumoniae; SpO₂, arterial oxygen saturation.

Figure 3.

Neutrophil reduction increases bacterial load in BAL but not systemically. A: ceftriaxone treatment (SP+CTRX) significantly reduced the bacterial loads in BAL at 24 h after infection compared with untreated infected mice (SP). Neutrophil reduction with ceftriaxone treatment (SP+CTRX+Ly6G) increased the load of bacteria in BAL compared with the ceftriaxone-treated mice but reduced the load of bacteria compared with the untreated mice (n = 9 or 10/group). B: ceftriaxone significantly reduced the bacterial loads in spleen at 24 h after infection. Neutrophil reduction did not significantly increase the bacterial loads compared with the ceftriaxone-treated mice (n = 9 or 10/group). Mice were administered either isotype control IgG or anti-Ly6G mAb at 24 h and immediately before bacterial inoculation. All data are shown as means ± SD. Statistical differences between groups were calculated with (A) one-way ANOVA followed by Tukey’s multiple comparison test. B: Kruskal–Wallis followed by Dunn’s multiple comparison. *P < 0.05, ***P < 0.001, ****P < 0.0001. BAL, bronchoalveolar lavage; CTRX, ceftriaxone; SP, Streptococcus pneumoniae.

Figure 4.

Neutrophil reduction reduces inflammatory biomarkers in BAL. Inflammatory biomarkers IL-1β, IL-6, KC, MIP-2, IL-10, and MCP-1 in BAL were significantly higher in untreated infected mice (SP) compared with normal healthy control (NC). Ceftriaxone treatment (SP+CTRX) reduced IL-1β, IL-6, KC, and MIP-2, but did not impact the levels of IL-10 and MCP-1. Neutrophil reduction (SP+CTRX+Ly6G) significantly attenuated the release of all these inflammatory cytokines and chemokines in the air spaces except for KC compared with untreated mice and ceftriaxone treatment alone (n = 9–10/group). Mice were administered either isotype control IgG or anti-Ly6G mAb at 24 h and immediately before bacterial inoculation. All data are shown as means ± SD. Statistical differences between groups were calculated with one-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. †P < 0.05 compared with other groups. CTRX, ceftriaxone; KC, keratinocyte chemoattractant; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; NC, normal control; SP, Streptococcus pneumoniae.

Figure 5.

Correlation between excess lung water and oxygenation. Arterial oxygen saturation ($\mathrm{S}\mathrm{p}{\mathrm{O}}_2$) and excess lung water were measured in the same mice. $\mathrm{S}\mathrm{p}{\mathrm{O}}_2$ was measured just before euthanization. Black, blue, red, and white circles represent values obtained from pneumococcus-infected mice with neutrophil reduction with anti-Ly6G monoclonal antibody (mAb) and ceftriaxone treatment (SP+CTRX+Ly6G), antibiotics treatment alone (SP+CTRX), untreated infected mice (SP), and neutrophil reduction alone without antibiotics (SP+Ly6G), respectively. All groups were combined to analyze the correlation. The black line represents the linear regression. Excess lung water and $\mathrm{S}\mathrm{p}{\mathrm{O}}_2$ had a strong negative correlation (n = 5/group). Mice were administered either isotype control IgG or anti-Ly6G mAb at 24 h and immediately before bacterial inoculation. Spearman’s correlation was used to test the relationship. CTRX, ceftriaxone; $\mathrm{S}\mathrm{p}{\mathrm{O}}_2$, arterial oxygen saturation; SP, Streptococcus pneumoniae.