Mechanical ventilation enhances lung inflammation and caspase activity in a model of mouse pneumovirus infection

Abstract

Severe infection with respiratory syncytial virus (RSV) in children can progress to respiratory distress and acute lung injury (ALI). Accumulating evidence suggests that mechanical ventilation (MV) is an important cofactor in the development of ALI by modulating the host immune responses to bacteria. This study investigates whether MV enhances the host response to pneumonia virus of mice (PVM), a mouse pneumovirus that has been used as a model for RSV infection in humans. BALB/c mice were inoculated intranasally with diluted clarified lung homogenates from mice infected with PVM strain J3666 or uninfected controls. Four days after inoculation, the mice were subjected to 4 h of MV (tidal volume, 10 ml/kg) or allowed to breathe spontaneously. When compared with that of mice inoculated with PVM only, the administration of MV to PVM-infected mice resulted in increased bronchoalveolar lavage fluid concentrations of the cytokines macrophage inflammatory protein (MIP)-2, MIP-1alpha (CCL3), and IL-6; increased alveolar-capillary permeability to high molecular weight proteins; and increased caspase-3 activity in lung homogenates. We conclude that MV enhances the activation of inflammatory and caspase cell death pathways in response to pneumovirus infection. We speculate that MV potentially contributes to the development of lung injury in patients with RSV infection.

Fig. 1.

A: mean clinical score in mice treated with medium only (no virus) or 3 different doses of pneumonia virus of mice (PVM; low, 1 × 10⁷; mid, 3 × 10⁷; and high, 9 × 10⁷ copies of PVM), evaluated on days 1–4 after inoculation. Total bronchoalveolar lavage fluid (BALF) alveolar macrophages (AM), polymorphonuclear leukocytes (PMN), and lymphocytes (Ly) (B); BALF cytokine concentrations (C); BALF permeability markers (IgM and α-macroglobulin; D); and lung homogenate caspase-3 activity (E) in mice treated with medium only (no virus) or 3 different doses of PVM (low, 1 × 10⁷; mid, 3 × 10⁷; and high, 9 × 10⁷ copies of PVM) on day 4 after inoculation are shown. *P < 0.05 compared with the uninfected mice; n = 5/group. MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein.

Fig. 2.

Virus titer in the lungs of uninfected mice allowed spontaneous breathing (SB; n = 6) or subjected to mechanical ventilation (MV; n = 5) and in PVM-infected mice allowed spontaneous breathing (PVM + SB; n = 6) or subjected to mechanical ventilation (PVM + MV; n = 6). Data are expressed as numbers of PVM-sh copies (×10⁵) per 10⁹ gapdh copies. Data are shown as individual data points and box plots depicting the median, interquartile range, and range.

Fig. 3.

Total AM (A), PMN (B), and Ly (C) detected in uninfected mice allowed SB (n = 6) or subjected to MV (n = 5) and in PVM + SB (n = 6) or PVM + MV (n = 6). *P < 0.05. Data are shown as individual data points and box plots depicting the median, interquartile range, and range.

Fig. 4.

Concentrations of MIP-2 (A), MIP-1α (B), KC (C), IL-6 (D), and VEGF (E) in the BALF of uninfected mice allowed SB (n = 6) or subjected to MV (n = 5) and in PVM + SB (n = 6) or PVM + MV (n = 6). *P < 0.05. Data are shown as individual data points and box plots depicting the median, interquartile range, and range.

Fig. 5.

Concentrations of IgM (A), α-macroglobulin (B), and total protein (C) in the BALF of uninfected mice allowed SB (n = 6) or subjected to MV (n = 5) and in PVM + SB (n = 6) or PVM + MV (n = 6). *P < 0.05. Data are shown as individual data points and box plots depicting the median, interquartile range, and range.

Fig. 6.

Caspase-3 activity in lung homogenates (A), number of terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL)-positive cells per 12 high-power fields (hpf) in lung tissue sections (B), BALF soluble Fas ligand (FasL) concentrations (C), and BALF granzyme B (GrB) concentrations (D) in uninfected mice allowed SB (n = 6) or subjected to MV (n = 5) and in PVM + SB (n = 6) or PVM + MV (n = 6). *P < 0.05. The cellular distribution of caspase-3 activation is shown in merged differential interference contrast and fluorescence images (cleaved caspase-3 in pink, and nuclei in blue) from a mouse in the SB (E), MV (F), PVM (G), and PVM + MV (H) group. The figures show the presence of signal in cells of the alveolar walls (white arrows) or macrophages (black arrow). NS, not significant.

Fig. 7.

Hematoxylin-eosin-stained lung tissue sections of lung tissue from uninfected mice allowed SB (A) or subjected to MV (B) and in PVM + SB (C) or PVM + MV (D). Magnification, ×400. Note the peribronchiolar cellular infiltration in both the PVM + SB and PVM + MV group (arrows). E: mean peak inspiratory pressures in uninfected mice subjected to MV (n = 5) and PVM + MV (n = 6). Means are ± SE.