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The skin has a role of a physical barrier between the host and the environment, but it has also a major role in detecting invading pathogens. Keratinocytes, as the main component of the skin, act as first-line responders against pathogenic microorganisms and initiate the cutaneous innate immune response.

Toll-like receptors (TLRs), are pattern recognition receptors which are expressed by many cell types in human skin, including keratinocytes.

They are involved in cutaneous host defense mechanisms against a variety of microorganisms including bacteria, fungi, and viruses.

The innate immune response is directed against conserved pathogen-associated molecular patterns (PAMPs) of microorganisms, and each TLR recognizes a different microbial component [1].

The interleukin-1 family members (IL-1fm) and TLR families share similar functions. More than any other cytokine family, the IL-1fm is primarily associated with innate immunity.

Whereas TLRs trigger inflammation via PAMPs recognition, IL-1fm cytokines trigger innate inflammation via receptors of IL-1fm.

The primary IL-1fm member IL-1α, which functions as a DAMP (danger associated molecular pattern), is considered as the main alarmin of the skin through its release by damaged keratinocytes in lesional sites.

In this study, our goal was first to functionally profile and characterize the response of normal human keratinocytes (NHEK) to microbial patterns (TLR agonists) and IL-1α in parallel.

To do so, the effects of multiple TLR agonists and IL-1α were evaluated on the gene expression profile, on signal transduction (NF-ƘB translocation) and also on IL-8 and IL-6 release.

Secondly, we also tried to link these responses to those of pathogens of interest in skin microbiota (namely, C. acnes, S. aureus and P. aeruginosa), by evaluating the effect on IL-8 release by NHEK infected with living bacteria from these 3 strains.

NHEK were grown in defined SFM medium. To profile the effects of selected TLR agonists: Pam3CSK4 (TLR1/2); Zymosan (TLR2); Poly (I:C) (TLR3); LPS ultrapure (TLR4); Flagellin (TLR5); FSL-1 (TLR6); Imiquimod (TLR7/8); ODN-2006 (TLR9), and the effect of IL-1α, cells were incubated with the different molecules for 4 hours (gene expression analysis), 20 minutes or 1 hour (signal transduction assays) or 48 hours (cytokine release analysis).

Gene expression analysis was achieved using Affymetrix human genome strip array technology, the effects on NF-kB translocation was evidenced by immunofluorescence and image analysis and the release of IL-8 and IL-6 by cells was measured using ELISA.

For bacterial infections, keratinocytes were treated with the bacterial strains at the following ratios (C. acnes MOI 100, S. aureus MOI 10 and P. aeruginosa MOI 1) and the cells were incubated for 24 hours.

Alternatively, NHEK were also infected in parallel either with a wildtype strain of P. aeruginosa (PAK), alone or in presence of an anti-TLR5 antibody, or with a flagellin-deleted mutant of P. aeruginosa (PAK-∆fliC (Fla-)).