Background/Purpose: Ultraviolet B irradiation (UVB) contributes to skin inflammation. As UVB mostly affects the epidermis, with just 10% getting into the dermis, the crosstalk between epidermis and dermis in the response to UVB warrants investigation. Extracellular vesicles (EVs), lipid bilayer membrane vesicles secreted by many cells, can carry lipids, proteins and nucleic acids to mediate signal transduction. As a critical sensor and adaptor for the host immune response to cytosolic DNA and cyclic dinucleotides, stimulator of interferon genes (STING) plays critical roles in immunity and inflammation. Initiated by cytosolic pattern recognition receptors, inflammasome activation-mediated pyroptosis is a highly inflammatory form of programmed cell death. Thus, we hypothesized that EVs derived from UVB-irradiated keratinocytes might trigger STING and inflammasome-mediated proinflammatory responses in dermal cells; The goal here was to evaluate the crosstalk between STING and the inflammasome during EVs-mediated proinflammatory responses in dermal cells.

Methods: Human keratinocytes (HaCaT) cells were irradiated with UVB light. After irradiation, cells were cultured for 24 hours and the supernatant was harvested for EV collection. EVs were isolated by ultracentrifugation and used to stimulate fibroblasts or macrophages with/without STING or inflammasome signaling inhibitors. The supernatant was harvested for ELISA and the lysed cells were collected for Western blot. C57BL/6J (Stock No.: 000664) and C57BL/6J-Sting1gt/J (Stock No.: 017537) mice purchased from Jackson Laboratory were treated with or 100 mJ/cm² UVB for five consecutive days. Dorsal skin samples were collected for histological analysis.

Results: UVB irradiated HaCaT cells released more extracellular vesicles than unirradiated cells (1.78×10⁹/mL vs. 3.31×10⁸/mL), with a similar mean size (74.7nm vs 73.5nm). UVB-irradiated-keratinocyte-derived EVs (KEV-UVB) expressed more small extracellular vesicle surface markers (CD81, CD9, CD63) than non-irradiated-KEVs. KEV-UVB triggered more interferonb (IFNb) release from macrophages than fibroblasts (111.1±21.45 vs. 4.85±0.72 pg/mL P< 0.05 n=3). STING antagonist H-151 attenuated KEV-UVB triggered IFNb production in macrophages (13.18±6.38 vs. 111.1±21.45 pg/mL P< 0.05). TBK1 inhibitor MRT67307 also showed a similar effect (12.6±0.71 vs. 304.6±94.4 pg/mL P< 0.05). Inhibition of the STING signaling pathway also suppressed KEV-UVB triggered interleukin 1b (IL1b) production in macrophages (60.52±11.41 vs. 325.2±62.68 pg/mL P< 0.05), while suppression of the inflammasome pathway by VX765 and Ac-YVAD-cmk only attenuated KEV-UVB triggered IL1b (22.00±5.01 vs. 34.67±5.85 pg/mL P< 0.05) but not IFNb production in macrophages (636.9±165.1 vs. 680.8±147.1 pg/mL P>0.05). Furthermore, UVB irradiation triggered more skin inflammation in STING knockout mice than WT mice in vivo.

Conclusion: KEV-UVB were mediators of inflammation, and triggered both STING and inflammasome-mediated cytokine release. Targeting the STING signaling pathway may provide insight into a potential therapeutic approach for UVB-induced skin inflammation.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/uvb-irradiated-keratinocyte-derived-extracellular-vesicles-induced-proinflammatory-responses-in-macrophages/