Nanoparticles Displaying Allergen and Siglec-8 Ligands Suppress IgE-FcεRI–Mediated Anaphylaxis and Desensitize Mast Cells to Subsequent Antigen Challenge

Abstract

Siglec-8 is an inhibitory receptor expressed on eosinophils and mast cells. In this study, we took advantage of a novel Siglec-8 transgenic mouse model to assess the impact of modulating IgE-dependent mast cell degranulation and anaphylaxis using a liposomal platform to display an allergen with or without a synthetic glycan ligand for Siglec-8 (Sig8L). The hypothesis is that recruitment of Siglec-8 to the IgE–FcεRI receptor complex will inhibit allergen-induced mast cell degranulation. Codisplay of both allergen and Sig8L on liposomes profoundly suppresses IgE-mediated degranulation of mouse bone marrow–derived mast cells or rat basophilic leukemia cells expressing Siglec-8. In contrast, liposomes displaying only Sig8L have no significant suppression of antigenic liposome-induced degranulation, demonstrating that the inhibitory activity by Siglec-8 occurs only when Ag and Sig8L are on the same particle. In mouse models of anaphylaxis, display of Sig8L on antigenic liposomes completely suppresses IgE-mediated anaphylaxis in transgenic mice with mast cells expressing Siglec-8 but has no protection in mice that do not express Siglec-8. Furthermore, mice protected from anaphylaxis remain desensitized to subsequent allergen challenge because of loss of Ag-specific IgE from the cell surface and accelerated clearance of IgE from the blood. Thus, although expression of human Siglec-8 on murine mast cells does not by itself modulate IgE-FcεRI–mediated cell activation, the enforced recruitment of Siglec-8 to the FcεRI receptor by Sig8L-decorated antigenic liposomes results in inhibition of degranulation and desensitization to subsequent Ag exposure.

The Inhibitory Receptor Siglec-8 Interacts With FcεRI and Globally Inhibits Intracellular Signaling in Primary Mast Cells Upon Activation

“…In contrast to the cell death-inducing activity mediated by Siglec-8 mAb antibodies in eosinophils (12,32), our in vitro assessments of Siglec-8 mediated MC activity revealed little evidence of inhibition by a Siglec-8 mAb in the absence of FceRI-induced activation. These data suggest that a close physical association of activating and inhibitory receptors is required for inhibition and are consistent with recent work studying CD33 (Siglec-3) and Siglec-8 (15,33). The authors demonstrate that FceRI and Siglec-8/CD33 are not entirely colocalized in the same membrane microdomain and active recruitment of the inhibitory receptor to the lipid rafts containing the IgE-FceRI complex is required.…”

“…The inhibitory receptor Siglec-8 has emerged as a promising drug target in MC-driven diseases and the work described herein was carried out to study in detail the effects of Siglec-8 engagement on intracellular signaling upon MC activation through FceRI. While inhibition of FceRI-mediated activation of MC through Siglec-8 engagement has been demonstrated previously (9,15,16), the consequences of this inhibition on intracellular signaling have not been thoroughly investigated.…”

“…Indeed, Siglec-8 mAbs have been shown to inhibit both IgE-dependent and -independent MC activation in vivo, including systemic anaphylaxis in humanized mice and IL-33and Substance P-mediated MC activation in Siglec-8 transgenic mice (12)(13)(14). Similarly, nanoparticles displaying allergen and Siglec-8 ligands have been shown to suppress phosphorylation of kinases activated by high affinity IgE receptor (FceRI) signaling and reduce IgE-mediated murine anaphylaxis (15). Consistent with Siglec-8 functioning as an inhibitory receptor, the membrane proximal ITIM was shown to be required for Siglec-8 mAbmediated inhibition of FceRI-dependent calcium flux and secretory responses in MCs (9).…”

Mouse Models of Food Allergy in the Pursuit of Novel Treatment Modalities

“…Targeting Siglec 8 directly with antibodies shows some promise for inhibiting mast cell degranulation ( 54 ). However, liposomes co-displaying Siglec 8 and antigen, which recruits Siglec 8 to the IgE-FcεRI complex, suppresses and desensitizes mast cells ( 55 ).…”

Siglec Signaling in the Tumor Microenvironment

“…To study cis-bindingmediated Siglec activation, studies have installed lipidconjugated glycopolypeptides into cell membranes (187,193,194). Similarly, to study trans-binding-mediated Siglec activation, Siglec-ligand containing liposomes have been used (195)(196)(197). However, addition of Siglec ligands in such manners requires careful interpretation, as this can lead to overexpression of unnatural sialic acids that can be recognized by Siglecs either in cis or trans and does not represent a natural situation.…”