Primary human dermal fibroblasts selectively sense LPS, poly I:C and ADP heptose and initiate immune response mostly through chemokine secretion.

Fibroblasts are considered structural cells that maintain tissue homeostasis and facilitate repair. They also participate in innate immunity, although their pattern recognition capabilities remain incompletely characterized. Here, we systematically assessed the innate immune responses of commercially available primary human dermal fibroblasts from a male and a female donor. Fibroblasts were stimulated with a panel of microbe-associated molecular patterns (MAMPs) targeting various pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NODs), Alpha kinase 1 (ALPK1) and STING. We quantified innate immune activation by measuring the nuclear translocation of NF-κB via high-content microscopy, and cytokines and chemokines secretion by ELISA. Baseline PRRs expression was determined using quantitative PCR. Only a restricted subset of agonists induced robust NF-κB activation with 30-70% positive cells, specifically E. coli LPS (TLR4), Poly I:C (TLR3/RIG-I), and unexpectedly ADP heptose (ALPK1). Dose-response analysis revealed surprisingly high sensitivity to LPS, as low as 1 ng/mL. Apart from IL-6 and RANTES, which were produced exclusively following Poly I:C stimulation, the secretion of pro-inflammatory cytokines IL-1β, and TNF, as well as the anti-inflammatory cytokines IL-10 and IFN-β remained undetectable compared to that of the chemokines IL-8 and MCP-1. Consistent with this limited reactivity, qPCR of PRRs revealed the basal gene expression of TLR4 co-receptors MD2, CD14, and ALPK1, while most other receptors were expressed at very low or undetectable levels. Notably, NOD1 was highly expressed despite the absence of cell activation with several NOD1 agonists. In conclusion, primary human dermal fibroblasts exhibit a highly selective yet sensitive innate immune response, that is largely restricted to chemokine production. This unexpected dissociation between chemokine and cytokine responses suggests that fibroblasts function as sentinel cells in early skin defence, capable of detecting key microbial patterns at low concentrations, and orchestrating local immune surveillance. Further investigation into interindividual variability and context-dependent activation is required.