Hydroxychloroquine inhibits proteolytic processing of endogenous TLR7 protein in human primary plasmacytoid dendritic cells

Toll-like receptor 7 (TLR7) triggers antiviral immune responses through its capacity to recognize single-stranded RNA. Proteolytic cleavage of TLR7 protein is required for its functional maturation in the endosomal compartment. Structural studies demonstrated that the N- and C-terminal domains of TLR7 are connected and involved in ligand binding after cleavage. Hydroxychloroquine (HCQ), an antimalarial drug, has been studied for its antiviral effects. HCQ increases pH in acidic organelles and has been reported to potently inhibit endosomal TLR activation. Whether HCQ can prevent endogenous TLR7 cleavage in primary immune cells, such as plasmacytoid dendritic cells (pDCs), had never been examined. Here, using a validated anti-TLR7 antibody suitable for biochemical detection of native TLR7 protein, we show that HCQ-treatment of fresh PBMCs, CAL-1 leukemic and primary human pDCs inhibits TLR7 cleavage and results in accumulation of full-length protein. As a consequence, we observe an inhibition of pDC activation in response to TLR7 stimulation with synthetic ligands and viruses including inactivated SARS-CoV2, which we show herein activates pDCs through TLR7-signaling. Together, our finding suggests that the major pathway by which HCQ inhibits ssRNA-sensing by pDCs may rely on its capacity to inhibit endosomal acidification and the functional maturation of TLR7 protein.