Adjuvanting allergen extracts for sublingual immunotherapy : calcitriol downregulates CXCL8 production in primary sublingual epithelial cells

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 culturedin vitro. After 24-h incubation with aDermatophagoides farinaeextract, aDermatophagoides pteronyssinusextract, 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 D-3), 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 beta 1 (TGF-beta 1) 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 aD. farinaeextract followed by measurement of CXCL8 secretion. Furthermore, the effect ofD. farinaeand 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 byD. farinaeextract and all TLR ligands apart from LPS. Calcitriol significantly decreased CXCL8 secretion, and coadministration withD. farinaeextract reduced CXCL8 concentrations to levels seen in unstimulated sublingual epithelial cells. Although detectable, TGF-beta 1 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 aD. farinaeextract 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 aD. farinaeextract. Calcitriol therefore seems to be a suitable candidate to be used as adjuvant during sublingual immunotherapy.

Publication year:2020

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