RvD1n-3 DPA Downregulates the Transcription of Pro-Inflammatory Genes in Oral Epithelial Cells and Reverses Nuclear Translocation of Transcription Factor p65 after TNF-α Stimulation

Abstract

Specialized pro-resolving mediators (SPMs) are multifunctional lipid mediators that participate in the resolution of inflammation. We have recently described that oral epithelial cells (OECs) express receptors of the SPM resolvin RvD1_{n-3 DPA} and that cultured OECs respond to RvD1_{n-3 DPA} addition by intracellular calcium release, nuclear receptor translocation and transcription of genes coding for antimicrobial peptides. The aim of the present study was to assess the functional outcome of RvD1_{n-3 DPA}-signaling in OECs under inflammatory conditions. To this end, we performed transcriptomic analyses of TNF-α-stimulated cells that were subsequently treated with RvD1_{n-3 DPA} and found significant downregulation of pro-inflammatory nuclear factor kappa B (NF-κB) target genes. Further bioinformatics analyses showed that RvD1_{n-3 DPA} inhibited the expression of several genes involved in the NF-κB activation pathway. Confocal microscopy revealed that addition of RvD1_{n-3 DPA} to OECs reversed TNF-α-induced nuclear translocation of NF-κB p65. Co-treatment of the cells with the exportin 1 inhibitor leptomycin B indicated that RvD1_{n-3 DPA} increases nuclear export of p65. Taken together, our observations suggest that SPMs also have the potential to be used as a therapeutic aid when inflammation is established.

Keywords: NF-κB; gingival; oral epithelium; oral inflammation; p65; periodontitis; resolvin; specialized pro-resolving mediators.

Figure 1

Experimental setup used for mRNA sequencing. Primary oral epithelial cells were incubated for 30 min with TNF-α or vehicle. After washing, RvD1_{n-3 DPA} or vehicle was added for 5 h. The cells were then harvested and processed for mRNA sequencing. Part of the figure was created with Biorender.com.

Figure 2

Differentially expressed genes in TNF-α-activated oral epithelial cells treated with RvD1_{n-3 DPA}. (A) Based on FDR, 28 genes were differentially expressed between oral epithelial cells treated with TNF-α + RvD1_{n-3 DPA} versus TNF-α alone (blue). All genes were down-regulated. In red, the expression of the same genes is shown when TNF-α addition is compared with control (vehicle). (B) Volcano plot of the differentially expressed genes when TNF-α + RvD1_{n-3 DPA} and TNF-α addition were compared.

Figure 3

Gene ontology (GO) biological process and KEGG pathway enrichment analysis. (A) GO and (B) KEGG pathway enrichment analysis of biological processes on the 28 differentially expressed genes in oral epithelial cells when TNF-α + RvD1_{n-3 DPA} and TNF-α addition were compared.

Figure 4

RvD1_{n-3 DPA} downregulates the NF-κB canonical pathway in TNF-α-activated oral epithelial cells. (A) The canonical pathway of NF-κB activation. Red arrows indicate transcriptional changes after TNF-α addition. Blue arrows show changes after addition of TNF-α + RvD1_{n-3 DPA}. (B) Causal network analysis centered on RELA, showing that RELA regulates a variety of downstream pro-inflammatory targets for which RvD1_{n-3 DPA} appears to partly or fully reverse TNF-α-induced expression changes (based on TNF-α + RvD1_{n-3 DPA} and TNF-α comparison).

Figure 5

RvD1_{n-3 DPA} inhibits p65 translocation to the cell nucleus. (A) Experimental setup. Primary oral epithelial cells were treated for 1 h with leptomycin B or left untreated. Then, the cells were washed and incubated for 15 min with TNF-α or left untreated. After a final washing, RvD1_{n-3 DPA} or vehicle was added for 15 min after which the cover slips with the cells were processed for immunocytofluorescence and analysis with CellProfiler (see Material and Methods). (B) Microscopic pictures of p65 immunocytofluorescence (green) of cells as described in (A). Stainings combined with DAPI are displayed in Figure S1. Original magnification ×20. (C) Distribution of p65 in oral epithelial cells treated as indicated on the x-axis. The y-axis shows the ratio of nuclear/cytoplasmic p65 staining. Part of Figure 5A was created with BioRender.com. ** Statistically significant differences (p < 0.05) as assayed by repeated measures ANOVA with adjustment for multiple comparisons based on False Discovery Rate (FDR).