Granzyme B as a regulator of IFNα production in cutaneous lupus erythematosus

Summary: Abstract Body: Recent studies have shown that monocytes, macrophages, and conventional DCs, but not plasmacytoid dendritic cells (pDC), are major source of IFNα in cutaneous lupus erythematosus(CLE). However, novel medications targeting pDCs reduce IFNα levels and improve CLE. Granzyme B is a proteolytic enzyme that induces apoptosis, and large concentrations are produced by pDCs in CLE, yet its role in modulating signaling pathways, including IFNα release, has not been elucidated. Peripheral blood mononuclear cells (PBMCs) were obtained from 18 CLE patients. PBMCs were co-incubated for 24 hours with 500U recombinant human granzyme B alone or with either 10ug/mL TL8-506 (TLR8 stimulator), 1.5uM CpG (TLR9 stimulator) or 60ug/mL cGAMP (STING stimulator). Positive controls were PBMCs stimulated separately with only TL8-506, CpG or cGAMP. Supernatants were then analyzed for IFNα via ELISA. PBMCs incubated with granzyme B alone did not produce any IFNα (n=5). PBMCs co-incubated with granzyme B and CpG (n=5) significantly reduced IFNα production compared to PBMCs incubated with CpG alone (mean IFNα 20.6±9.1 vs. 95.2±21.5 pg/mL respectively, p<0.01). However, PBMCs co-incubated with granzyme B with either TL8-506 (n=3) or cGAMP (n=5) produced significantly increased IFNα compared to PBMCs incubated with either TL8-506 or cGAMP alone (for TL8-506, mean IFNα 21.0±3.4 vs. 3.0±1.0 pg/mL respectively, p<0.05; for cGAMP, mean IFNα 14.1±2.2 vs 6.1±2.0 pg/mL respectively, p<0.01). CpG stimulates the TLR9 pathway primarily on pDCs and granzyme B may have a negative feedback effect in suppressing TLR9-induced IFNα release from pDCs, as suggested by recent studies. However, monocytes and macrophages primarily use the TLR8 and STING pathways for IFNα production. Granzyme B which is produced in large numbers by pDCs may potentiate IFNα release by enhancing the TLR8 or STING pathways on macrophages and monocytes in CLE. Outside of its role in inducing apoptosis, granzyme B may be a bridge between pDCs and other immune cells by modulating signaling pathways, in particular that of type-1 IFN in CLE. Touraj Khosravi-Hafshejani^{1, 2}, Ahmed Eldaboush^{1, 2}, Victoria Werth^{1, 2} 1. CMCVAMC, Philadelphia, PA, United States. 2. Dermatology, Penn Medicine, Philadelphia, PA, United States. Adaptive and Auto-Immunity