Background/Purpose: Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by overproduction of type I interferons (IFNs) and sensitivity to ultraviolet (UV) light. Though UV is a known trigger of SLE and SLE-associated skin lesions, mechanisms of UV-induced flare remain elusive. Previous work from our lab suggests the importance of myeloid cells in cutaneous inflammatory responses in SLE. However, mechanisms of recruitment, differentiation, and function of these cells are unknown and represent the goal of this study.

Methods:
Mice: 8-12 week old female wild-type Balb/c, lupus-prone New Zealand Mixed (NZM) 2328, and lupus-prone iNZM (NZM2328 background with type I IFN receptor alpha chain knockout) mice were used per our University of Michigan IACUC-approved protocol.

UV irradiation: Dorsal fur was removed with Veet cream. Mice were placed in a restrainer with facial protection and treated with 100mJ/cm2 UVB daily for 5 days (=chronic exposure) or a single dose of 300mJ/cm2 UVB (=acute exposure) using the UV-2 ultraviolet irradiation system (Tyler Research) at a wavelength of 310 nm.

Flow Cytometry: UV-exposed full-thickness dorsal skin was harvested, mechanically dissociated, and incubated with digestion enzymes (0.1g/ml deoxyribonuclease type I, 0.1g/ml hyaluronidase type V, 0.5g/ml collagenase) for 30 minutes at 37ºC. Cells were stained for: viability, CD11b, CD11c, Ly6C, MHC-II, Ly6G, CD3. Data was collected on a Cytek Aurora flow cytometer and analyzed using FlowJo flow cytometry analysis software.

Results: RNA sequencing of UV-exposed skin of NZM, iNZM, and Balb/c mice demonstrated upregulation of monocyte chemoattractants, including CCL2, in NZM >iNZM and Balb/c. Using cell type enrichment analysis through xCell webtool, we further identified enrichment of myeloid populations in UV-exposed NZM skin >iNZM and Balb/c, suggesting that type I IFNs are driving myeloid gene signatures in lupus-prone skin. We verified myeloid enrichment by flow cytometry, wherein we identified type I IFN-enhanced monocyte recruitment in NZM skin vs. Balb/c following acute UV exposure. Exposing mice to chronic UV dosing led to increased monocyte-derived dendritic cells (moDCs) in irradiated NZM skin. Furthermore, we identified increased MHC class II expression in skin-recruited moDCs of chronically UV-exposed NZM compared to iNZM and Balb/c, suggesting type I IFNs are aberrantly activating antigen presenting cells after UV exposure. Blockade of CCL2 with neutralizing antibodies blocked the interferon-enhanced recruitment of monocytes to UV-irradiated NZM skin, with little effect on iNZM and Balb/c monocyte recruitment. Applying the same CCL2 blockade to chronically UV exposed mice resulted in diminished moDC numbers in UV-exposed NZM, but not iNZM or Balb/c, skin. Sources of CCL2 production in human SLE skin were examined and SLE compared to healthy control keratinocytes produce CCL2 following UVB exposure.

Conclusion: We have identified that CCL2 drives the observed type I IFN-enhanced increase in monocyte and moDC numbers in UV-irradiated lupus-prone skin. Further studies will elucidate mechanisms of differentiation and function of IFN-educated moDCs in UV-driven immune activation in SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ccl2-drives-interferon-enhanced-monocytosis-in-uv-mediated-skin-injury-in-murine-lupus/