Background/Purpose:

Neutrophil NETosis is a form of cell death characterized by nuclear envelope rupture, nuclear chromatin release, and neutrophil extracellular trap formation. NETosis is important in various human diseases, including autoimmune diseases. We recently found involvement of neutrophil NETosis in UVB-induced murine skin inflammation. UVB is known to induce multiple agents including the lipid mediator Platelet activating factor (PAF). We also found that PKCα may mediate neutrophil NET release through regulation of nuclear envelope rupture. The goal of our study is to explore the causal role of PAF Receptor (PAFR) signaling and PKCα in neutrophil NETosis and its effect on skin inflammation.

Methods:

Peritoneal- and bone marrow-derived neutrophils were isolated from wildtype, PAFR KO mice, or PKCα deficient mice and purified by anti-Ly-6G micro bead kit. To study NETosis, neutrophils were stimulated with or without PAF for 3 hours, then fixed with 2% PFA, stained by Sytox Green, and assayed for NET formation, finally confirmed by fluorescent microscopy. For UVB-irradiation, PKCα deficiency mice and their litter wildtype mice were exposed with/without 150 mJ/cm²/day under anesthesia for consecutive 5 days. Dorsal skin tissues were harvested. H&E and IHC staining were conducted.

Results:

We found that PAF can induce NETosis in neutrophils from human and WT mice, while depletion of PAF receptor (PAFR) attenuated NETosis in neutrophils from PAFR KO mice, suggesting the role of PAF in neutrophil NETosis was mediated by PAFR. To study the role of PKCα, we found PAF increased PKCα phosphorylation in a time-dependent manner in parallel to the induction of neutrophil NETosis. Moreover, pharmacologic inhibition of conventional PKC with Go6976 decreased PKCα phosphorylation, nuclear envelope rupture and neutrophil NETosis. Furthermore, PKCα deficiency significantly impaired PAF-induced NETosis in neutrophils from PKCα deficient mice. Importantly, PKCα deficiency attenuated UVB-induced skin inflammation with decreased neutrophil NETosis and decreased proinflammatory cytokines (TNFα and IL-17A) in the inflamed skin of PKCα deficient mice as compared to those of the wildtype mice under UVB exposure.

Conclusion:

These findings suggested that PAF might be a potential mediator of neutrophil NETosis in the skin of the UVB irradiated mice. PKCα appears to play vital roles in neutrophil NETosis in UVB-induced skin inflammation. The PAFR and PKCα might be potential therapeutic targets in human diseases related to UVB-induced skin inflammation.

« Back to 2018 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/pkc%ce%b1-deficiency-protected-mice-from-uvb-induced-skin-inflammation-through-attenuation-of-neutrophil-netosis/