Adenoviral vectors stimulate innate immune responses in macrophages through cross-talk with epithelial cells

Although adenovirus vectors (Ads) have been widely utilized for gene delivery, their clinical application has been hampered by host immune responses. It has been shown that macrophages can induce inflammatory response against Ads in vivo, but they are not easily activated by Ads in vitro, suggesting their activation requires interaction with other cells. In this study, we investigated the interaction between macrophages and epithelial cells during Ad infection. Ad infection of the macrophage–epithelial cell co-culture resulted in rapid and drastic changes in the cell culture such as decrease in pH within 24 h, indicating macrophage activation. Ad infected co-culture showed several characteristics of inflammation including cytotoxicity, induction of pro-inflammatory cytokines, and generation of nitric oxide and reactive oxygen species. These signs of macrophage activation and inflammation were observed exclusively in the co-culture and were absent or significantly weaker in the macrophage mono-culture suggesting that there was a synergistic response by the interaction between macrophages and epithelial cells. We found that inhibition of NF-κB activation significantly reduced the inflammatory responses in the co-culture. Furthermore, we show that only the macrophages adjacent to epithelial cells were activated during Ad infection demonstrating that the interaction between macrophages and epithelial cells are crucial for Ad-induced inflammatory response.