Antiinflammatory Effects of Budesonide in Human Fetal Lung

Abstract

Lung inflammation in premature infants contributes to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease with long-term sequelae. Pilot studies administering budesonide suspended in surfactant have found reduced BPD without the apparent adverse effects that occur with systemic dexamethasone therapy. Our objective was to determine budesonide potency, stability, and antiinflammatory effects in human fetal lung. We cultured explants of second-trimester fetal lung with budesonide or dexamethasone and used microscopy, immunoassays, RNA sequencing, liquid chromatography/tandem mass spectrometry, and pulsating bubble surfactometry. Budesonide suppressed secreted chemokines IL-8 and CCL2 (MCP-1) within 4 hours, reaching a 90% decrease at 12 hours, which was fully reversed 72 hours after removal of the steroid. Half-maximal effects occurred at 0.04-0.05 nM, representing a fivefold greater potency than for dexamethasone. Budesonide significantly induced 3.6% and repressed 2.8% of 14,500 sequenced mRNAs by 1.6- to 95-fold, including 119 genes that contribute to the glucocorticoid inflammatory transcriptome; some are known targets of nuclear factor-κB. By global proteomics, 22 secreted inflammatory proteins were hormonally regulated. Two glucocorticoid-regulated genes of interest because of their association with lung disease are CHI3L1 and IL1RL1. Budesonide retained activity in the presence of surfactant and did not alter its surface properties. There was some formation of palmitate-budesonide in lung tissue but no detectable metabolism to inactive 16α-hydroxy prednisolone. We concluded that budesonide is a potent and stable antiinflammatory glucocorticoid in human fetal lung in vitro, supporting a beneficial antiinflammatory response to lung-targeted budesonide:surfactant treatment of infants for the prevention of BPD.

Keywords: RNA sequencing; chemokines; inflammatory; proteomics; surfactant.

Figure 1.

Time course of chemokine release from fetal lung explants cultured in Bud or Dex. (A) Representative CCL2 Western blot of culture media ± 30 nM Bud or Dex collected every 24 hours. At this concentration of corticosteroid, CCL2 was similarly suppressed by both Bud and Dex at all time points (7.5 and 15 kD bands) with minimal effect on the upper nonspecific band. (B) IL-8 and CCL2 levels by ELISA in culture media collected and changed every 4 hours, maintaining the original treatment conditions. Bud reduced levels by 4 hours with ∼90% suppression within 12–16 hours and 8–12 hours for CCL2 and IL-8, respectively (n = 4, 30 nM Bud). (C and D) Effect of removing corticosteroid after 4 hours of exposure. Explants were exposed to 3 nM Bud or Dex continuously (solid bars) or for 4 hours only (gray bars) followed by a saline rinse and replacement with steroid-free medium. For CCL2, the differences between washout levels and continuous exposure were statistically significant at all time points for both steroids, whereas IL-8 levels were significantly different at 48 and 72 hours. Dashed line indicates control level. Data are presented as mean ± SE (n = 4). Bud, budesonide; Dex, dexamethasone; wo, steroid washout. CCL2, C-C motif chemokine ligand 2.

Figure 2.

Cytokine-related genes regulated by budesonide. Twenty-eight budesonide-regulated genes for cytokines, their receptors, or interacting proteins from Tables 2 and 3 are shown. Ovals designate cytokines (n = 13), boxes indicate membrane receptors (11), green designates up-regulation of mRNA by budesonide (5), and red represents suppression (23). Related proteins include CARD16 and BCL2L1, which both inhibit CASP1 and its activation of IL1, and CHI3L1, which mediates some effects of IL6 and IL13. Some of the known inflammatory-related functions of each cytokine are listed to the right; with two possible exceptions (down-regulation of IFNAR2 [receptor for IFNW1] and inhibition of CARD16), budesonide effects are antiinflammatory.