P2008 Lipopolysaccharide-induced gene expression of CD14 in TRIF pathway is epigenetically regulated by sulforaphane in porcine pulmonary alveolar macrophages

Abstract

Cluster of differentiation 14 (CD14) is the pattern recognition receptor (PRR) involved in the recognition of bacterial component lipopolysaccharide (LPS) through the MyD88-dependent and TRIF pathway of innate immunity. It may be a modulator to prevent and mitigate the LPS-induced lung inflammation in pigs. However, reports on CD14 activation induced by LPS in TRIF pathway are controversial. Furthermore, the gene expression regulation of CD14 by the epigenetic factor sulforaphane (SFN) is still poorly understood. To identify the epigenetic changes of CD14 mediated with SFN in LPS-induced TRIF pathway, the PAMs model in vitro was investigated. For this, the mRNA expression of CD14 and downstream genes of TRIF pathway were quantified using qPCR. The cytokine levels of tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) were measured by enzyme-linked immunosorbent assay (ELISA). The gene expression of the epigenetic enzymes DNA methyltransferase-1 (DNMT1) and DNMT3a were quantified. The protein level of NF-κB was analyzed by western blot. Furthermore, the DNA methylation alterations of CD14 at promotor and gene-body (CDS region) were analyzed using bisulfite sequencing in SFN- and LPS-treated PAMs. It was shown that CD14 gene expression was induced by 5 µg/ml LPS in time-dependent manner. At time point 12 h, the gene expression of CD14 and downstream genes in TRIF pathway, including TRIF, TRAF6, NFγB, TRAF3, IRF7, and cytokines, such as TNF-α, IL-1β, IL-6, and IFN-β, were significantly induced by LPS. The LPS-induced gene expression was suppressed by SFN in a dose-dependent manner. The LPS-induced cytokine levels, including TNFα, IL-1β, and NF-κB levels, were also inhibited by SFN. Similarly, the DNMT3a mRNA expression was increased by LPS and downregulated by SFN at a dose of 5 µM. Furthermore, the bisulfite sequencing results showed that gene body methylation of CD14 was positively associated with gene expression of LPS-treated PAMs and this methylation status was inhibited by SFN in a dose-dependent manner. This in vitro study suggests that CD14 is involved in TRIF pathway, including TRIF-TRAF6 and TRIF-TRAF3 pathway, by LPS induction. Furthermore, this LPS-CD14 activation was suppressed by SFN via the epigenetic regulation of CD14 gene body methylation associated with DNMT3a. This study provided novel insights into SFN-mediated epigenetic downregulation of CD14 gene in LPS-induced TRIF pathway inflammation and may open new avenues for approaches to prevent and mitigate LPS-induced inflammation in pigs.