Reactive oxygen species regulate neutrophil recruitment and survival in pneumococcal pneumonia

Abstract

Rationale: The role of NADPH oxidase activation in pneumonia is complex because reactive oxygen species contribute to both microbial killing and regulation of the acute pulmonary infiltrate. The relative importance of each role remains poorly defined in community-acquired pneumonia.

Objectives: We evaluated the contribution of NADPH oxidase-derived reactive oxygen species to the pathogenesis of pneumococcal pneumonia, addressing both the contribution to microbial killing and regulation of the inflammatory response.

Methods: Mice deficient in the gp91(phox) component of the phagocyte NADPH oxidase were studied after pneumococcal challenge.

Measurements and main results: gp91(phox)(-/-) mice demonstrated no defect in microbial clearance as compared with wild-type C57BL/6 mice. A significant increase in bacterial clearance from the lungs of gp91(phox)(-/-) mice was associated with increased numbers of neutrophils in the lung, lower rates of neutrophil apoptosis, and enhanced activation. Marked alterations in pulmonary cytokine/chemokine expression were also noted in the lungs of gp91(phox)(-/-) mice, characterized by elevated levels of tumor necrosis factor-alpha, KC, macrophage inflammatory protein-2, monocyte chemotactic protein-1, and IL-6. The greater numbers of neutrophils in gp91(phox)(-/-) mice were not associated with increased lung injury. Levels of neutrophil elastase in bronchoalveolar lavage were not decreased in gp91(phox)(-/-) mice.

Conclusions: During pneumococcal pneumonia, NADPH oxidase-derived reactive oxygen species are redundant for host defense but limit neutrophil recruitment and survival. Decreased NADPH oxidase-dependent reactive oxygen species production is well tolerated and improves disease outcome during pneumococcal pneumonia by removing neutrophils from the tight constraints of reactive oxygen species-mediated regulation.

Figure 1.

Increased survival in gp91 ^{phox −/−} mice after pneumococcal infection. Mice were instilled via the trachea with 10⁷ cfu of type 2 pneumococci and monitored for 10 days. There was significantly greater survival of the gp91 ^{phox −/−} mice (n = 26) compared with the wild-type C57BL/6 control mice (n = 25); log-rank analysis.

Figure 2.

Reduced bacteria in lung and blood from gp91 ^{phox −/−} mice after high-dose pneumococcal infection. Bacteria in (A) lung homogenates and (B) blood from wild-type C57BL/6 control mice and gp91 ^{phox −/−} mice were determined 24 hours after intratracheal instillation of 10⁷ cfu of type 2 pneumococci. Means and SEM, Mann-Whitney.

Figure 3.

Increased numbers of neutrophils in lung sections from gp91 ^{phox −/−} mice after infection with pneumococci. Shown are images of lung sections obtained from (A and B) wild-type C57BL/6 control mice and (C and D) gp91 ^{phox −/−} mice 48 hours after intratracheal instillation of 10⁷ cfu of type 2 pneumococci. Original magnification: (A and C) ×20; (B and D) ×40. Histology shows changes of patchy lobar consolidation with increased cellularity and increased numbers of neutrophils in the alveolar inflammatory infiltrate of the gp91 ^{phox −/−} mice. (E and F) Semiquantitative scoring of neutrophilic infiltration in tissue sections (E) 24 hours (n = 6) and (F) 48 hours (gp91 ^{phox −/−} mice, n = 4; C57BL/6 mice, n = 6) after intratracheal instillation of 10⁷ cfu of type 2 pneumococci, showing numbers of sections with areas of confluent alveolar neutrophilic infiltration in the alveoli (area < 0.5 mm, score 0; area > 0.5 mm, score 1). (G) Myeloperoxidase (MPO) activity in lung homogenates from C57BL/6 and gp91 ^{phox −/−} mice 24 hours after intratracheal instillation of 10⁷ cfu of type 2 pneumococci; n = 7 (each group). ΔOD₄₅₀ = change in optical density at 450 nm. Means and SEM, t test.

Figure 4.

Increased numbers of neutrophils in bronchoalveolar lavage (BAL) from gp91 ^{phox −/−} mice after infection with pneumococci. (A) Representative appearances of cytospins of BAL from C57BL/6 mice (top) and gp91 ^{phox −/−} mice (bottom) 24 hours after intratracheal instillation of 10⁷ cfu of type 2 pneumococci (Streptococcus pneumoniae). (B) Absolute numbers of neutrophils (#PMN) in BAL from wild-type C57BL/6 control mice (n = 11) and gp91 ^{phox −/−} mice (n = 12) from the same experiments as in (A). (C) Absolute numbers of neutrophils in BAL 48 hours after instillation of 10⁷ cfu of type 2 S. pneumoniae; n = 5 (each group). Means and SEM, t test.

Figure 5.

gp91 ^{phox −/−} mice have reduced numbers of apoptotic cells in the lung after infection with type 2 pneumococci. (A) Representative cytospin demonstrating apoptotic cells (arrows) of bronchoalveolar lavage (BAL) from C57BL/6 mice 24 hours after instillation of 10⁷ cfu of type 2 pneumococci D39 (Streptococcus pneumoniae). (B and C) Percentage of apoptotic events (apoptotic cells and bodies) in cytospins of BAL from wild-type C57BL/6 control mice (n = 11) and gp91 ^{phox −/−} mice (n = 12) (B) 24 hours and (C) 48 hours after S. pneumoniae instillation; n = 5 (each group). (D and E) Percentage neutrophil (PMN) apoptosis (annexin-V–PE⁺/TO-PRO-3⁻, flow cytometry) in BAL from C57BL/6 and gp91 ^{phox −/−} mice (D) 24 hours and (E) 48 hours after intratracheal instillation of 10⁷ cfu of type 2 pneumococci; n = 5 (each group). Means and SEM, t test.

Figure 6.

Increase in neutrophil activation in gp91 ^{phox −/−} mice after infection with pneumococci. Mean fluorescence intensity (MFI) of (A) CD11b, (B) CD18, and (C) CD62L in bronchoalveolar lavage (BAL) from wild-type C57BL/6 control mice and gp91 ^{phox −/−} mice 24 hours after intratracheal instillation of 10⁷ cfu of type 2 pneumococci, n = 4 (each group). Means and SEM, t test.

Figure 7.

No increase in lung injury in gp91 ^{phox −/−} mice after high-dose pneumococcal infection. (A) Levels of albumin in bronchoalveolar lavage (BAL) from C57BL/6 (n = 11) and gp91 ^{phox −/−} (n = 10) mice, 48 hours after intratracheal instillation of 10⁷ cfu of type 2 pneumococci. (B) Levels of IgM in BAL from C57BL/6 (n = 23) and gp91 ^{phox −/−} (n = 16) mice 24 hours after intratracheal instillation of 10⁷ cfu of type 2 pneumococci. (C) Wet-to-dry ratio of lungs from C57BL/6 and gp91 ^{phox −/−} mice 24 hours after intratracheal instillation of 10⁷ cfu of type 2 pneumococci, n = 6 (each group). Means and SEM, Mann-Whitney.

Figure 8.

Neutrophil elastase production in gp91 ^{phox −/−} mice. (A) Levels of neutrophil elastase (NE) in bronchoalveolar lavage (BAL) from C57BL/6 and gp91 ^{phox −/−} mice 24 hours after instillation of 10⁷ cfu of type 2 pneumococci D39 or pneumococci lacking pyruvate oxidase activity (SpxB⁻); n = 7 (each group). (B) Absolute numbers of neutrophils in BAL from the same experiments as in (A). Means and SEM, Mann-Whitney.