Expression and function of resolvin RvD1n-3 DPA receptors in oral epithelial cells

Abstract

Chronic inflammatory responses can inflict permanent damage to host tissues. Specialized pro-resolving mediators downregulate inflammation but also can have other functions. The aim of this study was to examine whether oral epithelial cells express the receptors FPR2/ALX and DRV1/GPR32, which bind RvD1_{n-3 DPA} , a recently described pro-resolving mediator derived from omega-3 docosapentaenoic acid (DPA), and whether RvD1_{n-3 DPA} exposure induced significant responses in these cells. Gingival biopsies were stained using antibodies to FPR2/ALX and DRV1/GPR32. Expression of FPR2/ALX and DRV1/GPR32 was examined in primary oral epithelial cells by qRT-PCR, flow cytometry, and immunofluorescence. The effect of RvD1_{n-3 DPA} on intracellular calcium mobilization and transcription of beta-defensins 1 and 2, and cathelicidin was evaluated by qRT-PCR. FPR2/ALX and DRV1/GPR32 were expressed by gingival keratinocytes in situ. In cultured oral epithelial cells, FPR2/ALX was detected on the cell surface, whereas FPR2/ALX and DRV1/GPR32 were detected intracellularly. Exposure to RvD1_{n-3 DPA} induced intracellular calcium mobilization, FPR2/ALX internalization, DRV1/GPR32 translocation to the nucleus, and significantly increased expression of genes coding for beta-defensin 1, beta-defensin 2, and cathelicidin. This shows that the signal constituted by RvD1_{n-3 DPA} is recognized by oral keratinocytes and that this can strengthen the antimicrobial and regulatory potential of the oral epithelium.

Keywords: DRV1/GPR32; FPR2/ALX; beta defensin; cathelicidin; oral epithelium.

FIGURE 1

Expression of FPR2/ALX and DRV1/GPR32 in oral gingival epithelium. Immunohistochemical staining of sections of healthy human gingival epithelium. Staining was performed on formalin‐fixed and paraffin‐embedded tissues with antibodies reactive for the receptors FPR2/ALX (A–D) and DRV1/GPR32 (E–H) or the isotype‐ and concentration‐matched controls (I). Figures A, E, and I present 10× original magnification. Inserts show higher magnification of the marked areas. Results are representative of six separate human donors

FIGURE 2

Expression of FPR2/ALX and DRV1/GPR32 in cultured primary oral epithelial cells. (A) Box (25‐75 percentiles) and whiskers (10‐90 percentiles) plot with median (horizontal line) of ΔCt values for FPR2/ALX and DRV1/GPR32 mRNA expression, n = 7 donors. (B) Representative histograms from flow cytometric analyses of FPR2/ALX and DRV1/GPR32 staining on non‐permeabilized (left) and permeabilized (right) oral keratinocytes. Pink dotted lines represent primary oral epithelial cells stained for FPR2/ALX and DRV1/GPR32. The corresponding isotypes are shown in black solid lines. The Y‐axis shows percent normalized to mode. (C) Representative immunofluorescence microscopy images of FPR2/ALX (green) and DRV1/GPR32 (red) expression in quiescent primary oral epithelial cells. Nuclei were stained with DAPI (blue). Results are representative of three separate human donors

FIGURE 3

FPR2/ALX internalization, nuclear translocation of DRV1/GPR32 and intracellular Ca²⁺ mobilization after addition of RvD1_{n‐3 DPA} to primary oral epithelial cells. (A) Primary oral epithelial cells were incubated with vehicle (<0.1% ethanol) or RvD1_{n‐3 DPA} (10⁻¹¹–10⁻⁹ M) for 15 min at 37°C. Percent internalization was calculated (see Material and Methods) and shown as mean ± SD (n = 3; *p < 0.05; paired t‐tests comparing RvD1_{n‐3 DPA} vs vehicle after Bonferroni adjustment for multiple comparisons). (B) Representative confocal microscopy images of expression of FPR2/ALX (green) and DRV1/GPR32 (red) in vehicle‐treated primary oral epithelial cells (left column) and after 20 min incubation with RvD1_{n‐3 DPA} (0.1 nM; right column). Nuclei were stained with DAPI (blue). Results are representative of three separate human donors. (C) Primary oral epithelial cells were loaded with Fluo‐3 AM (3 µM in keratinocyte serum‐free medium) for 45 min, washed twice with Ca²⁺‐free PBS, incubated for 30 min to allow complete de‐esterification of intracellular Fluo‐3 AM ester and then stimulated with RvD1_{n‐3 DPA} (10⁻¹²–10⁻⁸ M). Increase in intracellular Ca²⁺ mobilization was quantified by flow cytometry. Results are expressed as mean% ± SD, n = 3 donors

FIGURE 4

Effect of RvD1_{n‐3 DPA} addition on mRNA levels of antimicrobial peptides in oral epithelial cells. DEFB1, DEFB4A, and CAMP mRNA expression patterns after incubation with RvD1_n‐3DPA (0.1 nM) in primary oral epithelial cells were evaluated by qRT‐PCR and compared with vehicle‐treated (<0.1% ethanol) control oral epithelial cells (Ctr). Box (25–75 percentiles) and whiskers (10‐90 percentiles) plot with median (horizontal line) of fold change after adjustment to the house keeping gene (n = 4–5 donors). * p < 0.05, two‐sided paired t‐test