The tyrosine kinase inhibitor imatinib prevents lung injury and death after intravenous LPS in mice

Abstract

Severe sepsis and septic shock are frequent causes of the acute respiratory distress syndrome, and important sources of human mortality. Lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls, plays a major role in the pathogenesis of severe sepsis and septic shock. LPS exposure induces the production of harmful reactive oxygen species, and the resultant oxidant injury has been implicated in the pathogenesis of both severe sepsis and ARDS. We previously showed that the tyrosine kinase inhibitor imatinib increases lung endothelial antioxidant enzymes and protects against pulmonary endothelial antioxidant injury. In the present study, we tested the hypothesis that imatinib would protect against lung injury and systemic inflammation caused by intravenous LPS in an intact mouse model of endotoxemia mimicking early sepsis. We found that intravenous LPS induced a significant increase in the activity of lung xanthine oxidoreductase (XOR), an enzyme which is a major source of reactive oxygen species and implicated in the pathogenesis of acute lung injury. Imatinib had no effect of LPS-induced XOR activity. However, pretreatment of mice with imatinib increased lung catalase activity and decreased intravenous LPS-induced lung oxidant injury as measured by γ-H2AX, a marker of oxidant-induced DNA damage, lung apoptosis, and pulmonary edema. Imatinib also attenuated systemic cytokine expression after intravenous LPS exposure. Finally, imatinib completely prevented mortality in an in vivo, intravenous LPS mouse model of endotoxemia and lung injury. These results support the testing of imatinib as a novel pharmacologic agent in the treatment of Gram-negative sepsis and sepsis-induced ARDS.

Keywords: Endotoxin; imatinib; lung injury; sepsis.

© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Figures

Figure 1

Effect of imatinib on LPS-induced lung XOR and catalase activity. C57BL/6J mice were exposed to IV LPS or PBS (control) for 24 h and lung tissue was harvested for enzymatic assessment. (A) Mice exposed to IV LPS demonstrate a significant (3.38-fold) increase in XOR activity compared to control. A subset of mice pretreated with imatinib and exposed to LPS also shows a significant (4.08-fold) increase in XOR activation. There was no statistical difference in lung XOR activity between mice exposed to LPS alone compared to those pretreated with imatinib and exposed to LPS. (B) Lung catalase activity was reduced in mice exposed to LPS compared to control mice (0.055 ± 0.03 vs. 0.18 ± 0.05). A subset of mice pretreated with imatinib and exposed to LPS showed a marked increase in catalase activity when compared to control mice or those exposed to LPS, 0.36 ± 0.05, 0.18 ± 0.05, and 0.055 ± 0.03, respectively. N = 5 per group. One-way ANOVA, P = 0.001. *P < 0.05 versus all others.

Figure 2

Imatinib prevents LPS-induced pulmonary oxidative damage. C57BL/6J mice were exposed to IV LPS or PBS (control) for 24 h and lung tissue was harvested for markers of oxidative damage. Top panel: Lung tissue homogenates were immunoblotted with antibodies recognizing γ-H2AX, a marker of oxidant-induced DNA damage. As shown, there is a marked increase in γ-H2AX in mice exposed to LPS. Mice pretreated with imatinib were significantly protected against LPS-induced γ-H2AX expression. Bottom panel: Quantification of densitometry confirms a significant increase in γ-H2AX expression in response to LPS, which is markedly reduced with imatinib pretreatment. Representative immunoblot of five mice per condition. One-way ANOVA, P < 0.0001. *P < 0.05 versus control and LPS + Imatinib. #P < 0.05 versus control and LPS.

Figure 3

Imatinib prevents LPS-induced lung damage. C57BL/6J mice were exposed to IV LPS or PBS (control) for 24 h and lung tissue was harvested for markers of lung damage. (A) Mice exposed to IV LPS demonstrate a significant (2.2-fold) increase in caspase 3/7 activity compared to control. Mice pretreated with imatinib were significantly protected against LPS-induced caspase 3/7 activation. There was no statistical difference in lung caspase 3/7 activity between control mice and those pretreated with imatinib and exposed to LPS. (B) Mice exposed to IV LPS demonstrated a significant increase in lung edema formation, as assessed by lung wet weight to body weight ratios, compared to control, 4.6 ± 0.23 and 3.8 ± 0.07, respectively. Mice pretreated with imatinib were significantly protected against LPS-induced lung edema formation. There was no statistical difference in pulmonary edema formation between control mice and those pretreated with imatinib and exposed to LPS. N = 5–7 per group. One-way ANOVA, P < 0.0001. *P < 0.05 versus all others.

Figure 4

Imatinib prevents LPS-induced systemic inflammation. C57BL/6J mice were exposed to IV LPS or PBS (control) for 24 h and serum was collected for evaluation of cytokines as a marker of systemic inflammation. (A) Mice exposed to IV LPS demonstrate a ∼ninefold increase in serum TNFα levels compared to control, 125.6, and 14.25 pg/mL, respectively. Mice pretreated with imatinib were significantly protected against LPS-induced TNFα induction. (B) Mice exposed to IV LPS demonstrate a substantial increase in serum IL-6 levels compared to control, 4666 pg/mL, and 0 pg/mL, respectively. Mice pretreated with imatinib were significantly protected against LPS-induced IL-6 induction. (C) Percent weight change from baseline was recorded at 6 h and 24 h after exposure to LPS. As shown, there is a similar amount of weight loss in both LPS-exposed mice and those pretreated with imatinib at 6 h, but by 24 h, mice pretreated with imatinib demonstrate a borderline trend toward reduced weight loss (P = 0.05). N = 5–8 per group. One-way ANOVA, P < 0.0001. *P < 0.05 versus all others. #P < 0.05 versus LPS.

Figure 5

Imatinib prevents LPS-induced mortality. C57BL/6J mice were exposed to IV LPS and followed for mortality. Survival curves for IV LPS and imatinib-treated mice after IV LPS exposure show a significant reduction in mortality with imatinib. N = 15 for LPS alone and 5 for imatinib + LPS. Log-rank test with P < 0.05.