Granzyme A- and B-cluster deficiency delays acute lung injury in pneumovirus-infected mice

Abstract

Lower respiratory tract infection by the human pneumovirus respiratory syncytial virus is a frequent cause of acute lung injury in children. Severe pneumovirus disease in humans is associated with activation of the granzyme pathway by effector lymphocytes, which may promote pathology by exaggerating proapoptotic caspase activity and proinflammatory activity. The main goal of this study was to determine whether granzymes contribute to the development of acute lung injury in pneumovirus-infected mice. Granzyme-expressing mice and granzyme A- and B-cluster single- and double-knockout mice were inoculated with the rodent pneumovirus pneumonia virus of mice strain J3666, and were studied for markers of lung inflammation and injury. Expression of granzyme A and B is detected in effector lymphocytes in mouse lungs in response to pneumovirus infection. Mice deficient for granzyme A and the granzyme B cluster have unchanged virus titers in the lungs but show a significantly delayed clinical response to fatal pneumovirus infection, a feature that is associated with delayed neutrophil recruitment, diminished activation of caspase-3, and reduced lung permeability. We conclude that granzyme A- and B-cluster deficiency delays the acute progression of pneumovirus disease by reducing alveolar injury.

Figure 1

PVM infection induces expression of GzmA and GzmB. A, gene expression profiles of transcripts encoding GzmA and GzmB in total lung RNA of C57Bl/6 mice (RNA from 5 mice pooled per time point) at time zero and days 1–7, 10, 14, 21 and 28 after inoculation with PVM J3666 (30 pfu). Peak of GzmA and GzmB expression at day 7 (green highlight). B, histogram plots of intracellular GzmA and GzmB expression in BALF CD8⁺CD3⁺ cells (CTLs, blue plot), NK1.1⁺CD3⁻ (NK cells, green plot) and CD8⁻CD3⁺ (CD8⁻ T lymphocytes, red plot) of PVM-infected mice at day 7 (BALF leukocytes from 3 mice pooled).

Figure 2

Lung response to PVM infection in GzmA and GzmB-cluster gene deleted mice. A, virus titer in the lung, expressed as number of PVM-sh copies per 10⁹ gapdh copies; B, lung homogenate caspase-3 activity (arbitrary fluorescence units); C, concentration of α-macroglobulin in BALF; D, BALF total neutrophil (PMN) counts; E, concentration of IL-6 (pg/ml) in BALF; and F, concentration of TNFα (pg/ml) in BALF, in the wild type (B6 wt), GzmA^−/−, GzmB^−/− and GzmA^−/−GzmB^−/− mice on day 3 (n=3 per group), 7 (n=6 per group) and 8 (n=4–6 per group) after PVM inoculation (6 × 10³ copies). * p<0.05. Data are shown as bar graphs depicting mean and standard error.

Figure 3

PVM clearance and apoptosis in GzmA and GzmB-cluster gene deleted mice. A, virus titer in the lung, expressed as number of PVM-sh copies per 10⁹ gapdh copies; B, lung homogenate caspase-3 activity (arbitrary fluorescence units), in the GzmA^+/−GzmB^+/− mice and GzmA^−/−GzmB^−/− mice on day 7 (n=6 per group) and 8 (n=6 per group) after PVM inoculation (6 × 10³ copies). * p<0.05. Data are shown as box plots depicting the median, interquartile range and full range. C, representative images of cleaved caspase-3 immunohistochemistry in lung tissues of the GzmA^+/−GzmB^+/− mice and GzmA^−/−GzmB^−/− mice on day 8 after PVM inoculation. Note the relative positive staining in alveolar wall cells (arrows) as compared to bronchial epithelial cells in the GzmA^+/−GzmB^+/− mice.

Figure 4

Lung permeability response to PVM infection in GzmA and GzmB-cluster gene deleted mice. Concentration of α-macroglobulin (A) and IgM (B) in BALF of the GzmA^+/−GzmB^+/− mice and GzmA^−/−GzmB^−/− mice on day 7 (n=6 per group) and 8 (n=6 per group) after PVM inoculation (6 × 10³ copies). * p<0.05. Data are shown as box plots depicting the median, interquartile range and full range.

Figure 5

Lung inflammatory response to PVM infection in GzmA and GzmB-cluster gene deleted mice. Total neutrophil (PMN) counts (A), and concentrations of IL-6 (B), TNFα (C), IFNγ (D), MIP-2 (E) and KC (F) in BALF of the GzmA^+/−GzmB^+/− mice and GzmA^−/−GzmB^−/− mice on day 7 (n=6 per group) and 8 (n=6 per group) after PVM inoculation (6 × 10³ copies). * p<0.05. Data are shown as box plots depicting the median, interquartile range and full range.

Figure 6

Lung tissue response to PVM infection in GzmA and GzmB-cluster gene deleted mice. Representative images of haematoxylin and eosin stained lung tissue from the GzmA^+/−GzmB^+/− mice and GzmA^−/−GzmB^−/− mice on day 8 after PVM inoculation (6 × 10³ copies). Magnification 100x.

Figure 7

Clinical response to PVM infection in GzmA and GzmB-cluster gene deleted mice. A, Kaplan-Meier curves showing the percentages of GzmA^+/−GzmB^+/− (black dots, n=9) and GzmA^−/−GzmB^−/− (white dots, n=9) mice reaching the endpoint of a clinical score of 5 (moribund) and/or weight loss > 20% after PVM inoculation (1 × 10⁴ copies). p < 0.05 by log-rank (Mantel-Cox) test. B, percentage of baseline (day 0) weight of the GzmA^+/−GzmB^+/− (black dots) and GzmA^−/−GzmB^−/− (white dots) mice after PVM inoculation (1 × 10⁴ copies). * p < 0.05. Bars show the mean.