Combined action of influenza virus and Staphylococcus aureus panton-valentine leukocidin provokes severe lung epithelium damage

Abstract

Staphylococcus aureus necrotizing pneumonia is a life-threatening disease that is frequently preceded by influenza infection. The S. aureus toxin Panton-Valentine leukocidin (PVL) is most likely causative for necrotizing diseases, but the precise pathogenic mechanisms of PVL and a possible contribution of influenza virus remain to be elucidated. In this study, we present a model that explains how influenza virus and PVL act together to cause necrotizing pneumonia: an influenza infection activates the lung epithelium to produce chemoattractants for neutrophils. Upon superinfection with PVL-expressing S. aureus, the recruited neutrophils are rapidly killed by PVL, resulting in uncontrolled release of neutrophil proteases that damage the airway epithelium. The host counteracts this pathogen strategy by generating PVL-neutralizing antibodies and by neutralizing the released proteases via protease inhibitors present in the serum. These findings explain why necrotizing infections mainly develop in serum-free spaces (eg, pulmonary alveoli) and open options for new therapeutic approaches.

Figure 1.

The proinflammatory and cytotoxic effects of Panton–Valentine leukocidin (PVL) and/or influenza virus on polymorphonuclear neutrophils (PMNs). Human neutrophils were freshly isolated and infected with influenza virus (multiplicity of infection [MOI]: 0, 1, 5). Virus titers were determined in standard plaque assay 4, 8, and 16 hours postinfection (A). For analyzing the proinflammatory and cytotoxic effects of PVL and/or influenza virus, human neutrophils were freshly isolated, and 1 × 10⁶ cells were infected with influenza virus (MOI: 0, 1, 5) for 3 hours and subsequently incubated with increasing concentrations of PVL (0, 1, 5 nM) for 1 hour. Cells were then washed and stained with allophycocyanin (APC)-labeled anti-CD11b antibodies to measure cell activation (B) or stained with propidium iodide (PI) to determine cell death (C) by flow cytometry. The values represent the mean ± standard deviation of at least 3 independent experiments. *P ≤ .05, **P ≤ .01, ***P ≤ .001 (independent t test). Abbreviations: NS, not significant, AU, arbitrary unit.

Figure 2.

The effect of Panton–Valentine leukocidin (PVL) and/or influenza virus on lung epithelial cells. Lung epithelial A549 cells were infected with influenza virus (multiplicity of infection [MOI]: 0, 1, 5) 2, 4, 6, and 8 hours postinfection. Viral PB1-protein accumulation was detected by Western blot analysis, equal protein loads were verified using anti-ERK2 antibodies, and virus titers were determined in standard plaque assay 8 and 24 hours postinfection (A). For analyzing the effect of PVL and/or influenza virus on the A549 cells, the cells were infected with influenza virus (MOI: 0, 1, 5) for 18 hours and subsequently incubated with different concentrations of PVL (0, 1, 5 nM) or phenol soluble modulins (PSMs) (35 µg/mL) for 2 hours. Cells were then washed, and whole RNA was extracted to measure the expression of the chemokine CCL5 (RANTES) and CXCL10 (IP-10) (B). Additionally, cells were stained with propidium iodide (PI) to determine cell death by flow cytometry (C) and examined by light microscopy to determine the integrity of the cell monolayer (D). The values represent the mean ± standard deviation of at least 3 independent experiments. **P ≤ .01, *** P ≤ .001 (independent t test).

Figure 3.

Effect of supernatants from Panton–Valentine leukocidin (PVL) and/or influenza-treated neutrophils on epithelial A549 cells. In a 2-step cell culture model, human neutrophils were stimulated with influenza virus (multiplicity of infection [MOI]: 0, 1, 5) and PVL (0, 1, 5 nM) (step 1) and the obtained supernatants were used to incubate epithelial cell monolayers for 18 hours (step 2). Epithelial cells were then washed and whole RNA was extracted to measure the expression of the chemokine CCL5 (RANTES) (A). Additionally, all cells (attached and detached) were stained with propidium iodide (PI) to determine cell death by flow cytometry (B). Furthermore, the remaining attached cells were detached by trypsinisation and counted by flow cytometry (C). Attached epithelial cells were examined by light microscopy to determine the integrity of the cell monolayer (D). The values represent the mean ± standard deviation of at least 3 independent experiments. Abbreviations: NS, not significant, PSM, phenol soluble modulin.

Figure 4.

Bacterial supernatants from Panton–Valentine leukocidin (PVL)–producing strains kill neutrophils and have a destructive effect on epithelial cell monolayers. First, human polymorphonuclear neutrophils (PMNs) were infected with influenza virus (multiplicity of infection [MOI]: 0, 1, 5) for 3 hours and were subsequently incubated for 30 minutes with bacterial culture medium (2%, 5%, 10%) obtained from different bacterial strains including TM300, TM300 + PVL, USA300, and USA300ΔPVL (step 1). Cells were then stained with propidium iodide (PI) to determine cell death by flow cytometry (A); data only shown for strain USA300. The supernatants from treated neutrophils were collected and used to incubate epithelial cell monolayers for 18 hours (step 2). The numbers of remaining attached epithelial cells were counted by flow cytometry (B). Attached epithelial cells were examined by light microscopy to determine the integrity of the cell monolayer (C). The values represent the mean ± standard deviation of at least 3 independent experiments. *P ≤ .05, ** P ≤ .01, *** P ≤ .001 (independent t test). Abbreviations: S. aureus, Staphylococcus aureus; S. carnosus, Staphylococcus carnosus.

Figure 5.

Human serum and protease inhibitors can prevent the destructive effect of Panton–Valentine leukocidin (PVL). First, human neutrophils (PMN) were stimulated with PVL (5 nM) in the presence or absence of serum or a protease inhibitor cocktail. Cells were then stained with propidium iodide (PI) to determine cell death by flow cytometry (A). In a second experiment, neutrophils were killed with 5 nM PVL, and different human serum preparations and fetal bovine serum were tested for their ability to inhibit the cytotoxic effect (B) (step 1). Supernatants from PVL-treated neutrophils were used to incubate epithelial cell monolayers for 18 hours with or without human serum or a protease inhibitor cocktail as indicated (step 2). The numbers of remaining attached epithelial cells were counted by flow cytometry (C) and examined by light microscopy to determine the integrity of the cell monolayer (D). The values represent the mean ± standard deviation of at least 3 independent experiments. *P ≤ .05, *** P ≤ .001 (independent t test). Abbreviation: IgG, immunoglobulin G.

Figure 6.

In vivo model of necrotizing pneumonia. Freshly isolated human neutrophils (PMNs) (1 × 10⁶ cells) were incubated for 30 minutes with Panton–Valentine leukocidin (PVL) (5 nM) or with 2% of bacterial culture medium obtained from strains USA300 or USA300ΔPVL in the presence or absence of human serum (1%). Subsequently, 40 µL of the supernatants of treated neutrophils were instilled intranasally in the lung of mice. After 24 hours, mice were killed, and the lungs were macroscopically evaluated (A) and were subjected to histopathological examination (B and C).

Figure 7.

Schematic model of the proposed pathogenic mechanisms leading to the development of necrotizing pneumonia. Abbreviations: PVL, Panton–Valentine leukocidin; S. aureus, Staphylococcus aureus.