Role of the lipoxygenase pathway in RSV-induced alternatively activated macrophages leading to resolution of lung pathology

Abstract

Resolution of severe Respiratory Syncytial Virus (RSV)-induced bronchiolitis is mediated by alternatively activated macrophages (AA-Mφ) that counteract cyclooxygenase (COX)-2-induced lung pathology. Herein, we report that RSV infection of 5-lipoxygenase (LO)(-/-) and 15-LO(-/-) macrophages or mice failed to elicit AA-Mφ differentiation and concomitantly exhibited increased COX-2 expression. Further, RSV infection of 5-LO(-/-) mice resulted in enhanced lung pathology. Pharmacologic inhibition of 5-LO or 15-LO also blocked differentiation of RSV-induced AA-Mφ in vitro and, conversely, treatment of 5-LO(-/-) macrophages with downstream products, lipoxin A4 and resolvin E1, but not leukotriene B4 or leukotriene D4, partially restored expression of AA-Mφ markers. Indomethacin blockade of COX activity in RSV-infected macrophages increased 5-LO and 15-LO, as well as arginase-1 mRNA expression. Treatment of RSV-infected mice with indomethacin also resulted not only in enhanced lung arginase-1 mRNA expression and decreased COX-2, but also decreased lung pathology in RSV-infected 5-LO(-/-) mice. Treatment of RSV-infected cotton rats with a COX-2-specific inhibitor resulted in enhanced lung 5-LO mRNA and AA-Mφ marker expression. Together, these data suggest a novel therapeutic approach for RSV that promotes AA-Mφ differentiation by activating the 5-LO pathway.

Figure 1

RSV infection induces 5-LO and 15-LO mRNA and lipoxygenase activity in vitro. (A) Highly purified WT C57BL/6J alveolar and peritoneal macrophage cultures were treated with medium only, rIL-4 (40 ng/ml), or infected with RSV (MOI = 2). WT C57BL/6J mice were mock infected with PBS or infected with RSV. Lungs were collected on the days indicated. Gene expression was analyzed by qRT-PCR. Data represent means +/− s.e.m from three (macrophages) and 2 (whole lungs) separate experiments. (B) WT C57BL/6J and cotton rat macrophages were treated as in (A). Lipoxygenase enzymatic activity was determined by the FOX assay. Data represent means +/− s.e.m. from 3 separate assays. (C) WT C57BL/6J mice and cotton rats were infected with RSV and lipoxygenase enzymatic activity was determined as in (B). Data represent means +/− s.e.m. from two separate assays with 3–6 animals per treatment group in 2 experiments (mouse) and 1 experiment (cotton rat; representative of 3 separate experiments with similar results).

Figure 2

The 5-LO pathway is required for RSV-induced AA-Mϕ differentiation and lack of 5-LO results in increased pathology in response to RSV infection in vivo. (A) WT C57BL/6J, 5-LO^−/−, and 15-LO^−/− peritoneal macrophages were treated as in Figure 1 and mRNA expression measured. Data are means +/− s.e.m. from 2 separate experiments. (B) WT and 5-LO−/− mice were mock- or RSV-infected. Mice were killed 6 days post-infection, and arginase-1 and COX-2 mRNA measured in lungs by qRT-PCR. (C) WT and 5-LO^−/− mice were treated as in panel (B). Lung pathology was scored as described in “Methods.” Results are compiled from 2 independent experiments. (D) Lung sections derived from WT and 5LO^−/− mice that were RSV-infected. Lungs were harvested 6 days post infection. Images were taken at 400X magnification and the bars shown are 200 µM.

Figure 3

Pharmacological blockade of the LO pathway inhibits AA-Mϕ differentiation. (A) WT C57BL/6J peritoneal macrophages were pre-treated with AA-861 (5-LO inhibitor; 1 µg/ml), PD-146176 (15-LO inhibitor; 1 µg/ml), or both AA-861 and PD-146176 for 2 h. Macrophages were then treated with medium alone, rIL-4, or infected with RSV for 48 h. Gene expression was measured by qRT-PCR. (B) 5-LO^−/− peritoneal macrophages were pre-treated with LXA4 (1 µg/ml) overnight, RvE1 (1 µg/ml), LTB4 (1 µg/ml), or LTD4 (1 µg/ml) for 1 h. Macrophages were treated with medium alone or infected with RSV for 48 h. Gene expression was measured by qRT- PCR. (C and D) WT C57BL/6J peritoneal macrophages were pre-treated with LY25583 (100 µg/ml) or zileuton (1 µM) for 1 h. Macrophages were then treated with medium alone, LTB₄ (1 µg/ml) or LTD₄ (1 µg/ml), rIL-4 (40 ng/ml) or infected with RSV for 48 h. Gene expression was measured by qRT-PCR. All data represent means +/− s.e.m. from 2 experiments.

Figure 4

Blocking COX activity enhances AA-Mϕ and 5-LO mRNA. (A) WT C57BL/6J macrophages were treated with medium alone or infected with RSV (MOI = 2) in the absence or presence of indomethacin (10 µM/ml) for 48 h. Gene expression for mRNA was measured by quantitative real-time PCR. Data represents means +/− s.e.m. from 2 experiments (* p < 0.05). (B-D) WT and 5-LO^−/− mice were mock- or RSV-infected. Starting at day 2 post-infection, mice were either treated with saline or indomethacin (0.3 mg/kg) for 5 consecutive days. Mice were killed 6 days post-infection, and (B) arginase-1 and COX-2 mRNA measured in lungs by qRT-PCR. (C) Lung pathology was scored as described in “Methods.” (B-C: * is WT vs. WT comparison, p < 0.05; # is 5-LO^−/− vs. 5-LO^−/− comparison, p < 0.05; ** WT vs. 5-LO^−/− comparison, p < 0.05). (D) Lung sections derived from 5-LO^−/− mice that were RSV-infected and treated with either saline or indomethacin. Lungs were harvested 6 days post infection. Images were taken at 100X magnification and the bars shown are 500 µM. (E) Cotton rats were treated daily with vehicle (saline) or parecoxib (50 mg/kg) in 200 µl volume i.p. starting two days prior to infection for prophylactic group and starting on the day of infection (day 0) for therapeutic group. Animals were infected i.n. with RSV A/Long (1×10⁶ pfu/100µl/animal) i.n. on day 0. Treatment on day 0 was done ~4 h post-infection. Animals were sacrificed on day 6 p.i. and 5-LO mRNA expression was analyzed by qRT-PCR. Uninfected and non-treated animals were used as controls.