Unsaturated compounds induce up-regulation of CD86 on dendritic cells in the in vitro sensitization assay LCSA

Abstract

Unsaturated compounds are known to cause false-positive reactions in the local lymph node assay (LLNA) but not in the guinea pig maximization test. We have tested a panel of substances (succinic acid, undecylenic acid, 1-octyn-3-ol, fumaric acid, maleic acid, linoleic acid, oleic acid, alpha-linolenic acid, squalene, and arachidonic acid) in the loose-fit coculture-based sensitization assay (LCSA) to evaluate whether unspecific activation of dendritic cells is a confounder for sensitization testing in vitro. Eight out of 10 tested substances caused significant up-regulation of CD86 on dendritic cells cocultured with keratinocytes and would have been classified as sensitizers; only succinic acid was tested negative, and squalene had to be excluded from data analysis due to poor solubility in cell culture medium. Based on human data, only undecylenic acid can be considered a true sensitizer. The true sensitizing potential of 1-octyn-3-ol is uncertain. Fumaric acid and its isomer maleic acid are not known as sensitizers, but their esters are contact allergens. A group of 18- to 20-carbon chain unsaturated fatty acids (linoleic acid, oleic acid, alpha-linolenic acid, and arachidonic acid) elicited the strongest reaction in vitro. This is possibly due to the formation of pro-inflammatory lipid mediators in the cell culture causing nonspecific activation of dendritic cells. In conclusion, both the LLNA and the LCSA seem to provide false-positive results for unsaturated fatty acids. The inclusion of T cells in dendritic cell-based in vitro sensitization assays may help to eliminate false-positive results due to nonspecific dendritic cell activation. This would lead to more accurate prediction of sensitizers, which is paramount for consumer health protection and occupational safety.

Assessing the causal relationship between circulating immune cells and abdominal aortic aneurysm by bi-directional Mendelian randomization analysis

“…CD86 is an immune co-stimulatory molecule predominantly expressed on antigen-presenting cells like dendritic cells and macrophages. It serves as a crucial facilitator for both innate and adaptive immune responses 30 , 31 . Elevated levels of circulating CD86 + myeloid DCs may potentiate the risk of AAA by amplifying proinflammatory T-cell activities.…”

“…This interaction effectively lowers the threshold for T-cell receptor-mediated responses. Once activated, T cells may contribute to the inflammatory cascade by releasing metalloproteinases (MMPs) which degrade the aortic wall's extracellular matrix 30 , 33 .…”

Immune-competent in vitro co-culture models as an approach for skin sensitisation assessment

“…Loose-fit Co-culture-based Sensitization Assay (LCSA): 48h treatment CD86 DCrc expression and cell viability by flow cytometry (Schreiner et al, 2007); (Schreiner et al, 2008); (Wanner and Schreiner, 2008); (Wanner et al, 2010); (Sonnenburg et al, 2012); (Sonnenburg et al, 2015); (Frohwein et al, 2016); (Frombach et al, 2017) Primary KC + PBMC with addition of cytokines cocktail after co-culture for 48h in serum free condition LCSA-ly: 48h treatment CD44, CD119, CD124 and IL-23R lymphocyte cell expression and cell viability by flow cytometry, extracellular release of IL-4, IFN-ɣ and IL-17 by ELISA (Frombach et al, 2018) NCTC2544 KC cell line + THP-1 cell line 24h treatment CD86, CD54, CCR7 and IL-8 co-culture expression by RT-qPCR (Meloni et al, 2010) HaCaT KC cell line + moDC 2 treatment protocols: either 1h treatment of HaCaT cell line, removal of treatment and co-culture with DC for 20h or 1h treatment of HaCaT cell line, removal of treatment, post-incubation for 20h and conditioned supernatant in contact with DC for 20h CD83 and CD86 moDC cell expression by flow cytometry (Matjeka et al, 2012) HaCaT KC cell line + primary dermal fibroblasts + MUTZ-LC 48h treatment Cell viability (Alamar Blue test), extracellular release of 27 cytokines using bio-plex assay and of IL-8 by ELISA (Lee et al, 2018) ELISA: enzyme-linked immunosorbent assay; moDC: monocytes-derived dendritic cell; MUTZ-LC: MUTZ-3 acute myeloid leukemia cell line differentiated into LC; PBMC: peripheral blood mononuclear cell; PI: propidium iodide…”