Potentiation of IL-4 and Vitamin D3 Receptor Signaling by Retinoic Acid in Intestinal Epithelial Cells and Macrophages - Mechanisms and Targets

Previously we demonstrated all-trans retinoic acid (ATRA) increased mRNA expression of several IL-4 induced chemokines associated with alternative activation (M2a) in porcine macrophages.  Herein, we describe several mechanisms whereby ATRA affects IL-4 signaling and extend these findings to epithelial cells. Treatment of primary explanted pig alveolar macrophages, blood-derived monocytes, and immortalized pig cell lines with ATRA alone induced expression of additional markers of M2a activation as shown by next generation sequencing (NGS) and real-time RT-PCR analysis. These markers included the adenosine A2b receptor ADORA2B, interleukin 1 receptor antagonist (IL1RN), Tissue inhibitor of metalloproteinase 1 (TIMP1) and the vitamin D3 (1,25- dihydroxyvitamin D3) receptor (VDR). Next generation sequencing also showed that an abundance of genes in the VDR activation pathway are also regulated by ATRA.  Treatment with both ATRA and vitamin D₃ co-regulated the activities of each other and differentially affected the markers of M2a macrophages. In two intestinal epithelial cell lines, we observed that ATRA increased RNA and protein for the IL-4 receptor alpha chain and the VDR.  ATRA increased IL-4 induced phosphorylation of signal transducer and activator of transcription 6 (STAT6) and mRNA expression of the chloride channel, calcium activated, family member 1 (CLCA1), important for mucus formation, and chemokine (C-C motif) ligand 26 (CCL26) a potent eosinophil chemoattractant. ATRA also increased vitamin D₃ induced expression of CYP24A1.  Thus, in intestinal epithelial cells and macrophages, ATRA increased signaling in response to IL-4 and VD3. These findings have important implications for the regulation of inflammation at mucosal surfaces.