Siglec-9 is an inhibitory receptor on human mast cells in vitro

Background: Mast cell activation is critical for the development of allergic diseases. Ligation of sialic acid-binding immunoglobin-like lectins (Siglecs), such as Siglec-6, -7, and -8 as well as CD33, have been shown to inhibit mast cell activation. Recent studies showed that human mast cells express Siglec-9, an inhibitory receptor also expressed by neutrophils, monocytes, macrophages, and dendritic cells.Objective: We aimed to characterize Siglec-9 expression and function in human mast cells in vitro.Methods: We assessed the expression of Siglec-9 and Siglec-9 ligands on human mast cell lines and human primary mast cells by real-time quantitative PCR, flow cytometry, and confocal microscopy. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt the SIGLEC9 gene. We evaluated Siglec-9 inhibitory activity on mast cell function by using native Siglec-9 ligands, glycophorin A (GlycA), and highmolecular-weight hyaluronic acid, a monoclonal antibody against Siglec-9, and coengagement of Siglec-9 with the highaffinity receptor for IgE (Fc epsilon RI).Results: Human mast cells express Siglec-9 and Siglec-9 ligands. SIGLEC9 gene disruption resulted in increased expression of activation markers at baseline and increased responsiveness to IgE-dependent and IgE-independent stimulation. Pretreatment with GlycA or high-molecular-weight hyaluronic acid followed by IgE-dependent or -independent stimulation had an inhibitory effect on mast cell degranulation. Coengagement of Siglec-9 with Fc epsilon RI in human mast cells resulted in reduced degranulation, arachidonic acid production, and chemokine release.Conclusions: Siglec-9 and its ligands play an important role in limiting human mast cell activation in vitro.