Tri-culture system for pro-hapten sensitizer identification and potency classification

Abstract

Allergic contact dermatitis (ACD) is an inflammatory disease that impacts 15-20% of the general population and accurate screening methods for chemical risk assessment are needed. However, most approaches poorly predict pre- and pro-hapten sensitizers, which require abiotic or metabolic conversion prior to inducing sensitization. We developed a tri-culture system comprised of MUTZ-3-derived Langerhans cells, HaCaT keratinocytes, and primary dermal fibroblasts to mimic the cellular and metabolic environments of skin sensitization. A panel of non-sensitizers and sensitizers was tested and the secretome was evaluated. A support vector machine (SVM) was used to identify the most predictive sensitization signature and classification trees identified statistical thresholds to predict sensitizer potency. The SVM computed 91% tri-culture prediction accuracy using the top 3 ranking biomarkers (IL-8, MIP-1β, and GM-CSF) and improved the detection of pre- and pro-haptens. This in vitro assay combined with in silico data analysis presents a promising approach and offers the possibility of multi-metric analysis for enhanced ACD sensitizer screening.

A new cell line based coculture system for skin sensitisation testing in one single assay using T cells, aryl hydrocarbon receptor knockout, and co-inhibitory blockage

“…In a further study with THP-1 cells, all sensitising chemicals tested, including DNCB, induced release of MIP-1β and non-sensitizers with one exception were truly negative (Lim et al 2008 ). In a sensitisation assay of MUTZ-3-derived Langerhans cells, HaCaT and primary dermal fibroblasts, MIP-1 β was used in combination with further biomarkers GM-CSF and IL-8 to improve the detection of pre- and pro-haptens (Lee et al 2018 ). However, until now application of blocking for immunoregulatory molecules and its effect on MIP-1β were not reported.…”

Impact of mono-culture vs. Co-culture of keratinocytes and monocytes on cytokine responses induced by important skin sensitizers

“…One may indeed also consider a model with more than two cell types. A tri-culture model with MUTZ-3-derived Langerhans cells, HaCaT keratinocytes, and primary dermal fibroblasts has been proposed to improve the detection of pre-and pro-haptens when some cytokines (IL-8, MIP-1b, GM-CSF) were assessed as potential biomarkers for skin sensitization (Lee et al 2018). Together, the data here and from the cited studies indicate that co-culture models seem promising for predicting toxicity.…”

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

“…Moreover, there is already an improved correlation reported between the EC3 from LLNA and the concentration of chemicals to give a positive result, namely ECΔ10 (CD86) and ECΔ50 (CD54), from COCAT set-up compared to THP-1 monoculture suggesting a more fine-scale prediction of skin sensitisation potency (Hennen and Blomeke, 2016). Using another co-culture model comprising MUTZ-LC (Lee et al, 2018), skin sensitisation potency prediction is also experienced and shows that the co-culture of MUTZ-LC with KCs and fibroblasts outperforms the monoculture concerning the overall accuracy.…”

“…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…”