Targeted proteomics and spectral flow cytometry analysis of cutaneous lupus erythematosus

Cutaneous Lupus Erythematosus (CLE) is an inflammatory skin disease characterized by perivascular and periadnexal lymphohistiocytic infiltrate and interface dermatitis. Using suction blister biopsies of lesional and nonlesional skin from four CLE patients and three healthy donors, we did spectral flow cytometry to study the immune cells infiltrating the skin and inflammatory cytokines in interstitial skin fluid. We identified a significant increase in HLA-DR+antigen-presenting cells in lesional (P<0.0001) and nonlesional (P<0.0001) skin compared to healthy controls. We detected the presence of T-cells, B-cells, natural killer cells, and plasmacytoid dendritic cells, key cell populations thought to drive disease immunopathogenesis. We found different clusters of cells expressing CXCR3 in the skin, while the total frequency and MFI of CXCR3+cells were not significantly different in healthy skin compared to CLE. Further, we measured 184 protein analytes in interstitial skin fluid using targeted proteomics. Our data confirmed the elevation of chemokine ligands previously reported to increase in highly inflamed CLE skin, CXCL9 (P<0.0001) and CXCL11 (P=0.0007), validating the blister biopsy as a minimally invasive method to monitor disease activity. Further, we identified potential novel biomarkers, including CASP8 (P=0.002), CTF1 (P=0.01), HGF (P=0.0005), Flt3L (P<0.0001), IFNL1 (P=0.02), CXCL6 (P<0.0001), CCL25 (P=0.005), and CCL28 (P=0.03) that are increased in the lesional skin compared to healthy. We uncovered proteins with increased concentration in clinically normal skin of CLE patients, which may define the preclinical stage of the disease. These differentially expressed proteins (DEPs) between nonlesional skin and healthy controls included increased CXCL6 (P=0.002), HGF (0.01), Flt3L (P<0.0001), and CCL25 (P=0.003), and decreased CEACAM3 (P=0.0002). We hypothesize that these proteins may serve as early disease activity biomarkers or predictors of disease progression.