A novel method to generate monocyte-derived dendritic cells during coculture with HaCaT facilitates detection of weak contact allergens in cosmetics

Abstract

The in vitro sensitization assay LCSA (Loose-fit Coculture-based Sensitization Assay) has proved reliable for the detection of contact sensitizers in the past. However, the coculture of human monocyte-derived dendritic cells (DCs) with primary human keratinocytes (KCs) in serum-free medium is relatively complex compared to other sensitization assays which use continuous cell lines. To facilitate high-throughput screening of chemicals, we replaced KCs with the HaCaT cell line under various culture conditions. Coculture of HaCaT with peripheral blood mononuclear cells in serum-supplemented medium leads to generation of CD1a/CD1c DCs after addition of GM-CSF, IL-4, and TGF-β1 (as opposed to CD1a/CD1c DCs which arise in the "classic" LCSA coculture). These cells resemble monocyte-derived DCs generated in monoculture, but, unlike those, they show a marked upregulation CD86 after treatment with contact allergens. All of the nine sensitizers in this study were correctly identified by CD1a/CD1c DCs in coculture with HaCaT. Among the substances were weak contact allergens such as propylparaben (which is false negative in the local lymph node assay in mice) and resorcinol (which was not detected by CD1a/CD1c DCs in the "classic" LCSA). The level of CD86 upregulation on CD1a/CD1c DCs was higher for most allergens compared to CD1a/CD1c DCs, thus improving the assay's discriminatory power. Three out of four non-sensitizers were also correctly assessed by the coculture assay. A false-positive reaction to caprylic (octanoic) acid confirms earlier results that some fatty acids are able to induce CD86 on DC in vitro. In conclusion, change of the LCSA protocol led to reduction of time and cost while even increasing the assay's sensitivity and discriminatory power.

Skin sensitization to fragrance hydroperoxides: interplay between dendritic cells, keratinocytes and free radicals

“…Keratinocytes and DCs conduct dynamic communication and exchange material with each other 28 . Also, different responsiveness of DCs and THP‐1 cells in coculture with keratinocytes was reported for contact allergens 29,30 . Here, we investigated the potential of important hydroperoxides to activate DCs in the presence of keratinocytes without adding a radical initiator (Figure 1b and Figure S2; see Supporting Information).…”

“…28 Also, different responsiveness of DCs and THP-1 cells in coculture with keratinocytes was reported for contact allergens. 29,30 Here, we investigated the potential of important hydroperoxides to activate DCs in the presence of keratinocytes without adding a radical initiator (Figure 1b and Figure S2; see Supporting Information). We also included cumene-OOH, as it is known to generate radicals in addition to oxidative stress in 2D cultured human keratinocytes.…”

Chemical-induced contact allergy: from mechanistic understanding to risk prevention

“…The recently improved test identified all sensitizers including weak contact allergens (propylparaben) and resorcinol (that is not detected by the "classic" LCSA). Three out of four non-sensitizers were correctly identified; thus the improved LCSA has an increased sensitivity and discriminatory power (Frombach et al 2017).…”

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

“…Primary cell cultures are physiologically relevant to human biology, phenotypically similar to normal cells and retain the normal regulation of their growth pathways (Berg et al, 2014). Nevertheless, due to potential inter-donor variability, the procedure, selection of suitable donors, consideration of the number of donors required, pooling cells, characterization, can be cumbersome and expensive if pooled cells from multiple donors are not proposed by the cell supplier (Frombach et al, 2017). Besides decreasing the variability, pooling cells enables to overcome the relative limited cells numbers due to low availability or poor isolation and short life span set to avoid phenotypic drift (Abbott and Kaplan, 2015;Berg et al, 2014).…”

“…The use of KCs or DC surrogates cell lines enables the construction of reporter cell lines to monitor specific signaling pathway activation including cytokine/inflammation pathways or Keap1-Nrf2-ARE pathway (Natsch and Emter, 2015). However, cell lines are associated with difficulties such as genomic and phenotypic drift especially at high passages, metabolic deficiencies and dysregulated signaling mechanisms (dos Santos et al, 2009;Frombach et al, 2017).…”