Balancing anti-inflammatory and anti-oxidant responses in murine bone marrow derived macrophages

Abstract

Rationale: The underlying pathophysiology of bronchopulmonary dysplasia includes a macrophage-mediated host response orchestrated by anti-inflammatory peroxisome proliferator-activated receptor gamma (PPARγ) and anti-oxidant nuclear factor (erythroid-derived 2)-like 2 (Nrf2). These have not yet been studied in combination. This study tested the hypothesis that combined inflammatory and oxidative stressors would interact and change PPARγ- and Nrf2-regulated gene expression and antioxidant capacity. Therefore, we investigated the effect of dual stimulation with lipopolysaccharide and hyperoxia in murine bone marrow-derived macrophages (BMDM).

Methods: Sub-confluent BMDM from wild-type C57BL/6J mice were treated with lipopolysaccharide (LPS) 1ug/mL for 2 hours followed by room air (21% oxygen) or hyperoxia (95% oxygen) for 24 hours. Taqman real time-polymerase chain reaction gene expression assays, total antioxidant capacity assays, and Luminex assays were performed.

Results: Supernatants of cultured BMDM contained significant antioxidant capacity. In room air, LPS treatment decreased expression of PPARγ and Nrf2, and increased expression of tumor necrosis factor-alpha and heme oxygenase-1; similar findings were observed under hyperoxic conditions. LPS treatment decreased cellular total antioxidant capacity in room air but not in hyperoxia. Increased expression of sulfiredoxin-1 in response to hyperoxia was not observed in LPS-treated cells. Dual stimulation with LPS treatment and exposure to hyperoxia did not have synergistic effects on gene expression. Cellular total antioxidant capacity was not changed by hyperoxia exposure.

Conclusions: Our hypothesis was supported and we demonstrate an interaction between inflammatory and oxidative stressors in a model system of bronchopulmonary dysplasia pathogenesis. The protective anti-oxidant effect of cell culture media may have protected the cells from the most deleterious effects of hyperoxia.

Fig 1. Experimental design.

BMDM were isolated from wild-type C57BL/6J mice and grown to subconfluence, then were treated with LPS or saline, followed 2 hours later by hyperoxia or room air, and were harvested 24 hours later.

Fig 2. Total antioxidant capacity of BMDM supernatant.

Supernatant from BMDM treated with LPS or saline and hyperoxia (HOx) or room air (RA) was assayed for total antioxidant capacity. Values are presented as mM Trolox equivalents, an antioxidant standard. Values are mean ± SD.

Fig 3. ΔCt values for genes of interest in BMDM.

Cells treated with LPS or saline control, and placed in room air (RA) or hyperoxia (HOx). ΔCt values represent difference in threshold cycle between gene of interest and GAPDH. Values are mean ± SD. * p<0.05, ** p<0.01.

Fig 4. TNFα values in BMDM supernatant.

Cells were treated with LPS or saline control, and placed in room air (RA) or hyperoxia (HOx). TNFα levels of the cell culture supernatant were measured via Luminex ELISA. Values are mean ± SD. ** p<0.01.

Fig 5. Total antioxidant capacity of BMDM homogenate.

Relative total antioxidant capacity per protein content, normalized to room air (RA) samples. Values are mean ± SD. ** p<0.01.