Effect of Gram Negative Anaerobic Bacteria on Normal Human Skin Keratinocytes (HEKa cells)

Objectives: To define the inflammatory signature of healthy keratinocytes induced by gram-negative anaerobe bacteria commonly found in HS and investigate pathways of activation. Methods: Type strains of P. nigrescens, P. melanogenica, P. intermedia, P. asaccharolytica, F. nucleatum, as well as S. aureus and the normal skin commensal S. epidermidis were heat-killed and co-incubated with normal human keratinocytes. RNA was analyzed using RNAseq and RT-qPCR. TLR4 and JAK inhibitors were used to investigate mRNA and protein inhibition of inflammatory cytokines. Results: All GNAs tested induced significantly higher levels of CXCL8 than S. epidermidis or negative control. P. nigrescens, P. melanogenica, and F. nucleatum were the most stimulatory and produced CXCL8 levels much higher than S. aureus. RNASeq revealed a broad inflammatory profile most strongly induced by F. nucleatum. All three bacteria strongly activated the IL-17 pathway and correlated with HS skin transcriptomes. Both TLR4 and JAK inhibition significantly reduced keratinocyte inflammation. Results: All GNAs tested induced significantly higher levels of CXCL8 than S. epidermidis or negative control. P. nigrescens, P. melanogenica, and F. nucleatum were the most stimulatory and produced CXCL8 levels much higher than S. aureus. RNASeq revealed a broad inflammatory profile most strongly induced by F. nucleatum. All three bacteria strongly activated the IL-17 pathway and correlated with HS skin transcriptomes. To compare gene signatures induced by anaerobes we stimulated keratinocytes for 3 hours with 2*10^9 bacteria and then harvested RNA and subjected the samples to RNASeq