Background/Purpose: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by increased sensitivity to ultraviolet radiation (UVR). UVR can trigger cutaneous and systemic disease fares, yet mechanisms driving these responses are not well characterized. Type I interferons (IFNs), which have been previously implicated in driving apoptosis of cancer cells, are expressed at higher levels at baseline in non-lesional skin of SLE patients and drive hyperinflammatory responses after UVR. In addition, these type I IFNs promote increased death of SLE keratinocytes after UVR; however, the manner in which they regulate cell death after UVR is currently unknown. This study explores the cell death pathways activated in keratinocytes by type I IFNs and UVR.

Methods: Immortalized human keratinocytes (N/TERTs) were treated overnight with IFNα (0-1000 U/ml) prior to UVB exposure (0-50 mJ/cm²). Reactive oxygen species (ROS) production was measured using the cell-permeable oxidative stress indicator CM-H₂DCFDA and fluorescence was measured 5 minutes post UVB exposure. Annexin V (AV) and propidium iodide (PI) staining was performed 4 hours post UVB exposure and cells were analyzed by flow cytometry. Cells were pretreated with apoptosis, necroptosis, and pyroptosis inhibitors to examine activated death pathways. Caspase-3 cleavage was measured by flow cytometry.

Results: Intracellular ROS were significantly increased by 5 minutes after UVB exposure. Surprisingly, priming with IFNα resulted in a dose-dependent decrease in ROS production. IFNα priming prior to UVB exposure also resulted in a significant increase in the percentage of AV⁺PI^– cells, indicative of early apoptosis. In addition, IFNα treatment promoted apoptosis at a lower dose of UVB (20 mJ/cm²). IFNα treatment prior to UVB exposure resulted in a significantly higher percentage of both cleaved-caspase-3⁺ and AV⁺PI^–, but not AV⁺PI⁺, cells compared to treatment with UVB alone. Pre-treatment with Z-VAD-FMK (pan-caspase inhibitor) or Z-IETD-FMK (caspase-8 inhibitor) significantly reduced AV⁺PI^– cells following IFNα and UVB treatment while treatment with Z-LEHD-FMK (caspase-9 inhibitor) did not, suggesting enhanced activation of the extrinsic apoptosis pathway. Further, use of Necrostatin-1 (RIPK1 inhibitor) or AC-YVAD-CMK (caspase-1 inhibitor) showed no effect on death at 4 hours post UVB exposure in the presence of IFNα suggesting that enhanced death of type I IFN treated cells after UVB likely does not involve necroptosis or pyroptosis.

Conclusion: Treatment of keratinocytes with IFNα results in increased sensitivity to UVB-mediated cell death, which takes the primary form of caspase-8 driven apoptosis, independently of ROS. It is well documented that SLE skin is rich in type I IFNs and that high circulating IFNs are a feature of the disease. Together, these data suggest that photosensitive responses exhibited by lupus patients are likely due to type I IFN priming of keratinocytes that sensitizes the cells to undergo increased apoptosis after exposure to minimal amounts of UVB. Continued investigation into the mechanism by which IFN drives UVR-mediated cell death will be crucial for developing novel preventative strategies for photosensitivity.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/interferon-alpha-promotes-caspase-dependent-apoptosis-independently-of-reactive-oxygen-species-in-ultraviolet-b-exposed-keratinocytes/