Adjuvanting Allergen Extracts for Sublingual Immunotherapy: Calcitriol Downregulates CXCL8 Production in Primary Sublingual Epithelial Cells

Abstract

Application of allergens onto the sublingual epithelium is used to desensitize allergic individuals, a treatment known as sublingual immunotherapy. However, the response of sublingual epithelial cells to house dust mite allergen and potential tolerance-promoting adjuvants such as Toll-like receptor (TLR) ligands and calcitriol has not been investigated. In order to study this, primary sublingual epithelial cells were isolated from dogs and cultured in vitro. After 24-h incubation with a Dermatophagoides farinae extract, a Dermatophagoides pteronyssinus extract, TLR2 ligands (FSL-1, heat-killed Listeria monocytogenes, Pam3CSK4), a TLR3 ligand (poly I:C), a TLR4 ligand [lipopolysaccharide (LPS)], and calcitriol (1,25-dihydroxyvitamin D₃), viability of the cells was analyzed using an MTT test, and their secretion of interleukin 6 (IL-6), IL-10, CXCL8, and transforming growth factor β1 (TGF-β1) was measured by enzyme-linked immunosorbent assay. Additionally, to evaluate its potential effect as an adjuvant, sublingual epithelial cells were incubated with calcitriol in combination with a D. farinae extract followed by measurement of CXCL8 secretion. Furthermore, the effect of D. farinae and calcitriol on the transcriptome was assessed by RNA sequencing. The viability of the sublingual epithelial cells was significantly decreased by poly I:C, but not by the other stimuli. CXCL8 secretion was significantly increased by D. farinae extract and all TLR ligands apart from LPS. Calcitriol significantly decreased CXCL8 secretion, and coadministration with D. farinae extract reduced CXCL8 concentrations to levels seen in unstimulated sublingual epithelial cells. Although detectable, TGF-β1 secretion could not be modulated by any of the stimuli. Interleukin 6 and IL-10 could not be detected at the protein or at the mRNA level. It can be concluded that a D. farinae extract and TLR ligands augment the secretion of the proinflammatory chemokine CXCL8, which might interfere with sublingual desensitization. On the other hand, CXCL8 secretion was reduced by coapplication of calcitriol and a D. farinae extract. Calcitriol therefore seems to be a suitable candidate to be used as adjuvant during sublingual immunotherapy.

Keywords: CXCL8; Dermatophagoides farinae; Toll-like receptor; calcitriol; dog; epithelium; sublingual; sublingual immunotherapy.

Figure 1

CD49f is expressed by canine sublingual epithelial, but not by sublingual subepithelial cells. (A) Immunofluorescence of canine sublingual tissue in which cryosections were stained with CD49f (FITC) and counterstained with Hoechst. E = epithelium, S = subepithelial tissue. (B) Histograms generated by flow cytometric analysis that are representative for all donors. Isotype controls are given in red; stained cells are shown in green. (C) Flow cytometric analysis: mean fluorescence intensity (MFI) of all samples stained with CD49f for sublingual epithelial cells (n = 6) and subepithelial cells (n = 4). Mean fluorescence intensity of the isotype control was subtracted from the MFI of the stained cells. Horizontal bars indicate the mean.

Figure 2

A Dermatophagoides farinae extract significantly increases CXCL8 secretion by canine sublingual epithelial cells (n = 6). The cells were cultured for 24 h with 20 μg/mL D. farinae extract or 20 μg/mL D. pteronyssinus extract and CXCL8 secretion was measured. *p < 0.05; **p < 0.01 to control; paired Student t-test.

Figure 3

Ligands for TLR2 and TLR3 significantly increase CXCL8 secretion by canine sublingual epithelial cells (n = 6). The cells were cultured for 24 h with Pam3CSK4 at 1 μg/mL, FSL-1 at 1 μg/mL, HKLM at 10⁸ cells/mL, poly I:C high (HMW) and low molecular weight (LMW) at 10 μg/mL, and LPS at 10 μg/mL, and CXCL8 secretion was measured by ELISA. *p < 0.05; **p < 0.01 to control; paired Student t-test.

Figure 4

Calcitriol significantly suppresses CXCL8 secretion by canine sublingual epithelial cells (n = 6). The cells were stimulated with 1, 0.1, and 0.01 μM calcitriol for 24 h, and CXCL8 secretion was measured by ELISA. **p < 0.01 to control; paired Student t-test.

Figure 5

The TLR3 ligand poly I:C significantly decreases the viability of canine sublingual epithelial cells (n = 6). The cells were cultured for 24 h with 0.1 μM calcitriol, D. farinae extract at 20 μg/mL, Pam3CSK4 at 1 μg/mL, FSL-1 at 1 μg/mL, HKLM at 10⁸ cells/mL, poly I:C high (HMW) and low molecular weight (LMW) at 10 μg/mL, and LPS at 10 μg/mL, and the effect on cell viability was assessed using an MTT test. ***p < 0.001 to control; paired Student t-test. Error bars: standard deviation.

Figure 6

Coadministration of a D. farinae extract with calcitriol suppresses CXCL8 secretion by canine sublingual epithelial cells (n = 6). The cells were cultured with 20 μg/mL D. farinae extract or a combination of 20 μg/mL D. farinae extract with 0.1 μM calcitriol for 24 h, and CXCL8 secretion was measured by ELISA. **p < 0.01; ***p < 0.001 to control; paired Student t-test.