Repeated stress to the skin amplifies neutrophil infiltration in a keratin 17- and PKCα-dependent manner.

Neutrophils are the first immune cells to reach inflamed sites and contribute to the pathogenesis of chronic inflammatory skin diseases. Yet, little is known about the pattern of neutrophil infiltration in inflamed skin in vivo and the mechanisms mediating their recruitment. Here, we provide insight into the dynamics of neutrophil infiltration in skin in response to acute or repeated inflammatory stress, highlighting a novel keratinocyte- and keratin 17 (K17)-dependent mechanism that regulates neutrophil recruitment to inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12 h and resolves within 24 h. A subsequent TPA treatment or a UVB challenge, when applied 24 h but not 48 h 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 chemoattractants by stressed keratinocytes. K17 binds RACK1, a scaffold protein essential for PKCα activity. The N-terminal head domain of K17 is crucial for its association with RACK1 and regulation of PKCα activity. Analysis of RNAseq data reveals a signature consistent with TAR and PKCα activation in inflammatory skin diseases. These findings uncover a novel, keratin-dependent mechanism that amplifies neutrophil recruitment in skin under stress, with direct implications for inflammatory skin disorders.

Neutrophils secrete exosome-associated DNA to resolve sterile acute inflammation.

Acute inflammation, characterized by a rapid influx of neutrophils, is a protective response that can lead to chronic inflammatory diseases when left unresolved. Secretion of LTB 4 -containing exosomes is required for effective neutrophil infiltration during inflammation. In this study, we show that neutrophils release nuclear DNA in a non-lytic, rapid, and repetitive manner, via a mechanism distinct from suicidal NET release and cell death. The packaging of nuclear DNA occurs in the lumen of nuclear envelope (NE)-derived multivesicular bodies (MVBs) that harbor the LTB 4 synthesizing machinery and is mediated by the lamin B receptor (LBR) and chromatin decondensation. Disruption of secreted exosome-associated DNA (SEAD) in a model of sterile inflammation in mouse skin amplifies and prolongs the presence of neutrophils, impeding the onset of resolution. Together, these findings advance our understanding of neutrophil functions during inflammation and the physiological significance of NETs, with implications for novel treatments for inflammatory disorders.