Effect of Dermatophagoides farinae extract and calcitriol on canine primary sublingual epithelial cells. Effect of Dermatophagoides farinae extract and calcitriol on canine 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 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 24h incubation with Dermatophagoides farinae extract, TLR2 ligands (FSL-1, heat-killed Listeria monocytogenes, Pam3CSK4), a TLR3 ligand (poly I:C), a TLR4 ligand (LPS) and calcitriol (1,25-dihydroxyvitamin D3), viability of the cells was analyzed using an MTT test and their secretion of IL-6, IL-10, CXCL8 and TGF-β1 was measured by ELISA. Additionally, to evaluate its potential effect as an adjuvant, sublingual epithelial cells were incubated with calcitriol in combination with 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 co-administration 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. IL-6 and IL-10 could not be detected at the protein nor at the mRNA level. It can be concluded that D. farinae extract and TLR ligands augment the secretion of the pro-inflammatory chemokine CXCL8, which might interfere with sublingual desensitization. On the other hand, CXCL8 secretion was reduced by co-application of calcitriol and D. farinae extract. Calcitriol therefore seems to be a suitable candidate to be used as adjuvant during sublingual immunotherapy. Overall design: 5 biological replicates were analysed which were all subjected to 4 different conditions

将变应原涂布于舌下上皮以对过敏个体实施脱敏治疗，该疗法称为舌下免疫疗法（sublingual immunotherapy）。然而，目前尚未有研究探讨舌下上皮细胞对屋尘螨变应原，以及Toll样受体配体、骨化三醇这类潜在促耐受佐剂的应答反应。为探究该问题，本研究从犬体内分离原代舌下上皮细胞并开展体外培养。将细胞分别与屋尘螨（Dermatophagoides farinae）提取物、TLR2配体（FSL-1、热灭活单核细胞增生李斯特菌、Pam3CSK4）、TLR3配体（聚肌胞苷酸poly I:C）、TLR4配体脂多糖（LPS）以及骨化三醇（1,25-二羟基维生素D3）孵育24小时后，采用MTT法检测细胞活力，通过酶联免疫吸附试验（ELISA）检测细胞分泌的IL-6、IL-10、CXCL8及TGF-β1水平。此外，为评估骨化三醇作为佐剂的潜在效能，研究人员将舌下上皮细胞与骨化三醇联合屋尘螨提取物共同孵育，随后检测CXCL8的分泌量。另外，通过RNA测序（RNA-sequencing）评估屋尘螨提取物与骨化三醇对细胞转录组的影响。实验结果显示，聚肌胞苷酸可显著降低舌下上皮细胞的活力，其余刺激物无此效应；屋尘螨提取物与除脂多糖外的所有TLR配体，均可显著上调CXCL8的分泌水平；骨化三醇可显著抑制CXCL8的分泌，且与屋尘螨提取物联合给药时，可将CXCL8的浓度降至未受刺激的舌下上皮细胞的基础水平。尽管可检测到TGF-β1的分泌，但所有刺激物均无法对其分泌水平产生调控作用；IL-6与IL-10无论在蛋白水平还是mRNA水平均未检测到。综上，屋尘螨提取物与TLR配体可促进促炎性趋化因子CXCL8的分泌，这可能会干扰舌下脱敏治疗的效果；而骨化三醇与屋尘螨提取物联合应用时可降低CXCL8的分泌，因此骨化三醇有望成为舌下免疫疗法中的合格佐剂。整体实验设计：共纳入5个生物学重复样本，所有样本均接受4种不同的处理条件。