Novel role for protein kinase D3 (PKD3) in airway epithelial activation and lung inflammation in response to double stranded RNA

Abstract

Abstract Airway epithelial cells (AEC) are among the first to encounter inhaled particles and viruses and initiate subsequent immune responses. We recently reported that protein kinase D (PKD) regulates the barrier function of AEC in vitro after stimulation with the double stranded RNA polyI:C (PMID 21996340). Exposure to polyI:C mimics many features of respiratory viral infection in AEC, including induction of pro-inflammatory cytokines. It remains unknown whether PKD plays a role in AEC cytokine secretion, which we explored both in vitro and in vivo. First, we found that polyI:C-induced IL-8 production in 16HBE cells (a human AEC line) was markedly attenuated by pretreatment with PKD antagonist (CRT0066101 from Tocris (from 13000 ± 3752 to 3676 ± 2224 pg/ml at 5 μM CRT, n=4, p<0.05). Next, we challenged mice with inhaled polyI:C (10 μg, 3 consecutive days) and observed a significant increase in bronchoalveolar lavage (BAL) neutrophils 24 hours post challenge (from 0.4 ± 0.8 to 31.2 ± 9.4%; n=10, p<0.01). Strikingly, BAL neutrophil influx was reduced to near baseline values in mice pre-treated with CRT in a dose-dependent manner (e.g. 3.7 ± 4.7% with 100 μg CRT, n=10, p<0.01). The PKD antagonist also markedly reduced BAL levels of the neutrophil chemoattractant KC (p<0.01). PKD3 is the most abundant isoform in the lung by Western blot and RT-PCR. Finally, we found that polyI:C challenge of PKD3+/− mice (Prkd3Gt(OST191038)Lex/MMUCD from MMRRC) caused roughly 50% less BAL neutrophilia and KC release compared to age- and sex-matched wild type littermates (n=5). We conclude that PKD3 plays a previously unsuspected role in regulating AEC activation in response to double stranded RNA, suggesting a novel role for PKD3 in anti-viral immunity.