Neutrophil depletion causes a fatal defect in murine pulmonary Staphylococcus aureus clearance

Abstract

Background: Staphylococcus aureus is the most common cause of healthcare-associated pneumonia. Despite the significant morbidity and mortality associated with the disease, animal models of S. aureus pneumonia are rare.

Materials and methods: We examined the pathogenicity of four different strains of S. aureus (both methicillin-sensitive and -resistant as well as Panton-Valentine leukocidin-positive and -negative) in four strains of immunocompetent inbred and outbred mice (FVB/N, C57Bl/6, BALB/c, ND4; n = 148). The immunological basis for the development of murine S. aureus pneumonia was then determined by selectively depleting neutrophils, lymphocytes, or pulmonary macrophages prior to the onset of infection. An additional cohort of animals was rendered immunosuppressed by induction of abdominal sepsis via cecal ligation and puncture 2, 4, or 7 d prior to the onset of pneumonia.

Results: Nearly all immunocompetent mice survived, regardless of which strain of S. aureus was used or which strain of mouse was infected. Among animals with immune depletion or prior immunosuppression, survival was decreased only following neutrophil depletion (26% versus 90% alive at 7 d, P < 0.0001). Compared to immunocompetent animals, neutrophil-depleted mice with S. aureus pneumonia had delayed pulmonary bacterial clearance at 16 and 40 h but had no difference in levels of bacteremia. Neutrophil-depleted mice also had elevated levels of pulmonary monocyte chemotactic protein-1 (822 pg/mL versus 150 pg/mL, P < 0.05). In contrast, pulmonary histological appearance was similar in both groups as was dry/wet lung weight.

Conclusions: These results suggest that neutrophils play a critical role in the host response to S. aureus pneumonia, and the survival differences observed in neutrophil-depleted mice are associated with alterations in bacterial clearance and pulmonary cytokine response.

FIG. 1

Percentage of animals alive at 7 days by genetic background or bacterial strain of mice that received intratracheal injection of S. aureus. Regardless of whether mice (n=148) were (A) inbred or outbred or received (B) MSSA or MRSA (PVL positive or negative), essentially all immunocompetent animals survived seven days.

FIG. 2

Percentage of animals alive at 7 days in mice subjected to “two hit” model of sepsis. Animals were subjected to CLP followed by intratracheal injection of S. aureus a variable number of days later. Regardless of interval between CLP and S. aureus, survival was statistically similar to animals subjected to CLP followed by sham pneumonia.

FIG. 3

Percentage of animals alive at 7 days of immunodepleted animals that had intratracheal injection of S. aureus. Mice that had neutrophil depletion via polyclonal antibody (n=27) had decreased survival within two days while lymphocyte-deficient Rag-1^−/− mice (n=14) and animals that had pulmonary macrophage depletion (n=10) essentially all survived (p<0.0001).

FIG. 4

Bacterial clearance in neutrophil-depleted and immunocompetent (“wild type”) mice following intratracheal injection of S. aureus (n=6–7 animals/group/timepoint). Neutrophil-depleted mice had higher concentrations of bacteria in BAL fluid at both 16 and 40 hours. Data has been log transformed for presentation.

FIG. 5

Histologic severity and distribution of pneumonia in neutrophil-depleted and immunocompetent (“wild type”) mice following intratracheal injection of S. aureus (n=6–7 animals/group/timepoint). Marked interanimal variation was seen in both severity (A) and anatomic distribution (B) without significant differences seen between neutrophil-depleted or immunocompetent animals (p>0.05). Severity was graded on a 1–4 scale, with 1 representing normal lung and 4 representing severe pneumonia. Distribution was graded on a 1–3 scale with 1 representing no pneumonia and 3 representing diffuse pneumonia. Representative micrographs of immunocompetent (C) and neutrophil-depleted (D) mice with grade 3 severity pneumonia 16 hours after intratracheal injection of S. aureus demonstrate similar histologic appearance.