Keratin 17- and PKCα-dependent transient amplification of neutrophil influx after repeated stress to the skin

Abstract

Neutrophils contribute to the pathogenesis of chronic inflammatory skin diseases. Little is known about the source and identity of the signals mediating their recruitment in inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin and assess whether keratinocyte-derived signals impact neutrophil recruitment. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12h and resolves within 24h. A second TPA treatment or a UVB challenge, when applied at 24h but not 48h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in ex vivo culture, and involves the K17-dependent sustainment of protein kinase Cα (PKCα) activity and release of neutrophil chemoattractants by stressed keratinocytes. We show that K17 binds RACK1, a scaffold essential for PKCα activity. Finally, analyses of RNAseq data reveal the presence of a transcriptomic signature consistent with TAR and PKCα activation in chronic inflammatory skin diseases. These findings uncover a novel, transient, and keratin-dependent mechanism that amplifies neutrophil recruitment to the skin under stress, with direct implications for inflammatory skin disorders.

Neutrophils secrete exosome-associated DNA to resolve sterile acute inflammation

“…We found that neutrophil infiltration peaks at 12 h and returns to basal levels by 48 h ( Fig. 6e-g ) - kinetics that align with previous studies 67–69 . The absence of Ly6G staining on the ventral (untreated) side of the ear indicates that neutrophil infiltration occurs in the tissue proximal to the TPA-treated area.…”

“…To assess the effect of secreted DNA on the kinetics of neutrophil infiltration in vivo , we took advantage of a well-established model of acute skin inflammation in mice, induced by topical application of a phorbol ester, tetradecanoylphorbol-13-acetate (TPA) 66,67 . The primary targets of topically applied TPA are the suprabasal differentiating keratinocytes of the epidermis 67 . DNase I or PBS control, was injected in the tail vein one hour prior to topical TPA treatment on the dorsal ear skin.…”

“…Mice were treated with 20 μl of 0.2 mg/ml of TPA (Cell Signaling Technology #4174S), topically on the dorsal side of ear skin, with the other ear treated with an equal volume of acetone as vehicle control 67 . To disintegrate secreted DNA in the inflamed tissue, mice were injected intravenously in the tail with either 100 µl of PBS or recombinant human DNase I (PROSPECT #ENZ-319; 1 mg/ml) 1 h before TPA treatment.…”