Regulation of mast cells by overlapping but distinct protein interactions of Siglec-6 and Siglec-8.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 and Siglec-8 are closely related mast cell (MC) receptors with broad inhibitory activity, but whose functional differences are incompletely understood. METHODS: Proteomic profiling using quantitative mass spectrometry was performed on primary mouse MCs to identify proteins associated with Siglec-6 and Siglec-8. For functional characterization, each receptor was evaluated biochemically and in ex vivo and in vivo inhibition models of IgE and non-IgE-mediated MC activation in Siglec-6- or Siglec-8-expressing transgenic mice. RESULTS: Siglec-6 and Siglec-8 were found in MCs within large complexes, interacting with 66 and 86 proteins, respectively. Strikingly, Siglec-6 and Siglec-8 interacted with a large cluster of proteins involved in IgE and non-IgE-mediated MC activation, including the high affinity IgE receptor, stem cell factor (SCF) receptor KIT/CD117, IL-4 and IL-33 receptors, and intracellular kinases LYN and JAK1. Protein interaction networks revealed Siglec-6 and Siglec-8 had overlapping yet distinct MC functions, with a potentially broader regulatory role for Siglec-6. Indeed, Siglec-6 preferentially interacted with the mature form of KIT at the cell surface, and treatment with an anti-Siglec-6 antibody significantly inhibited SCF-mediated MC activation more in comparison to targeting Siglec-8. CONCLUSION: These data demonstrate a central role for Siglec-6 and Siglec-8 in controlling MC activation through interactions with multiple activating receptors and key signaling molecules. Our findings suggest that Siglec-6 has a role distinct from that of Siglec-8 in regulating MC function and represents a distinct potential therapeutic target in mast cell-driven diseases.

A DLG1-ARHGAP31-CDC42 axis is essential for the intestinal stem cell response to fluctuating niche Wnt signaling.

A central factor in the maintenance of tissue integrity is the response of stem cells to variations in the levels of niche signals. In the gut, intestinal stem cells (ISCs) depend on Wnt ligands for self-renewal and proliferation. Transient increases in Wnt signaling promote regeneration after injury or in inflammatory bowel diseases, whereas constitutive activation of this pathway leads to colorectal cancer. Here, we report that Discs large 1 (Dlg1), although dispensable for polarity and cellular turnover during intestinal homeostasis, is required for ISC survival in the context of increased Wnt signaling. RNA sequencing (RNA-seq) and genetic mouse models demonstrated that DLG1 regulates the cellular response to increased canonical Wnt ligands. This occurs via the transcriptional regulation of Arhgap31, a GTPase-activating protein that deactivates CDC42, an effector of the non-canonical Wnt pathway. These findings reveal a DLG1-ARHGAP31-CDC42 axis that is essential for the ISC response to increased niche Wnt signaling.

Discovery of an agonistic Siglec-6 antibody that inhibits and reduces human mast cells.

Mast cells (MC) are key drivers of allergic and inflammatory diseases. Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 is an immunoregulatory receptor found on MCs. While it is recognized that engaging Siglecs with antibodies mediates inhibition across immune cells, the mechanisms that govern this agonism are not understood. Here we generated Siglec-6 mAb clones (AK01 to AK18) to better understand Siglec-6-mediated agonism. Siglec-6 mAbs displayed epitope-dependent receptor internalization and inhibitory activity. We identified a Siglec-6 mAb (AK04) that required Fc-mediated interaction for receptor internalization and induced inhibition and antibody-dependent cellular phagocytosis against MCs. AK04-mediated MC inhibition required Siglec-6 immunoreceptor tyrosine-based inhibitory motif (ITIM) and ITIM-like domains and was associated with receptor cluster formation containing inhibitory phosphatases. Treatment of humanized mice with AK04 inhibited systemic anaphylaxis with a single dose and reduced MCs with chronic dosing. Our findings suggest Siglec-6 activity is epitope dependent and highlight an agonistic Siglec-6 mAb as a potential therapeutic approach in allergic disease.

Functional and Phenotypic Characterization of Siglec-6 on Human Mast Cells.

Mast cells are tissue-resident cells that contribute to allergic diseases, among others, due to excessive or inappropriate cellular activation and degranulation. Therapeutic approaches to modulate mast cell activation are urgently needed. Siglec-6 is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptor selectively expressed by mast cells, making it a promising target for therapeutic intervention. However, the effects of its engagement on mast cells are poorly defined. Siglec-6 expression and endocytosis on primary human mast cells and mast cell lines were assessed by flow cytometry. SIGLEC6 mRNA expression was examined by single-cell RNAseq in esophageal tissue biopsy samples. The ability of Siglec-6 engagement or co-engagement to prevent primary mast cell activation was determined based on assessments of mediator and cytokine secretion and degranulation markers. Siglec-6 was highly expressed by all mast cells examined, and the SIGLEC6 transcript was restricted to mast cells in esophageal biopsy samples. Siglec-6 endocytosis occurred with delayed kinetics relative to the related receptor Siglec-8. Co-crosslinking of Siglec-6 with FcεRIα enhanced the inhibition of mast cell activation and diminished downstream ERK1/2 and p38 phosphorylation. The selective, stable expression and potent inhibitory capacity of Siglec-6 on human mast cells are favorable for its use as a therapeutic target in mast cell-driven diseases.

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

Immunomodulation of mast cell (MC) activity is warranted in allergic and inflammatory diseases where MCs have a central role in pathogenesis. Targeting Siglec-8, an inhibitory receptor on MCs and eosinophils, has shown promising activity in preclinical and clinical studies. While the intracellular pathways that regulate Siglec-8 activity in eosinophils have been well studied, the signaling mechanisms that lead to MC inhibition have not been fully elucidated. Here, we evaluate the intracellular signaling pathways of Siglec-8-mediated inhibition in primary MCs using an anti-Siglec-8 monoclonal antibody (mAb). Phospho-proteomic profiling of FcεRI-activated MCs revealed Siglec-8 mAb-treatment globally inhibited proximal and downstream kinases, leading to attenuated MC activation and degranulation. In fact, Siglec-8 was found to directly interact with FcεRI signaling molecules. Siglec-8 inhibition was dependent on both cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that interact with the SH2 containing protein phosphatase Shp-2 upon Siglec-8 phosphorylation. Taken together, these data support a model in which Siglec-8 regulates proximal FcεRI-induced phosphorylation events through phosphatase recruitment and interaction with FcεRIγ, resulting in global inhibition of MCs upon Siglec-8 mAb engagement.

Mast Cell and Eosinophil Activation Are Associated With COVID-19 and TLR-Mediated Viral Inflammation: Implications for an Anti-Siglec-8 Antibody.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection represents a global health crisis. Immune cell activation via pattern recognition receptors has been implicated as a driver of the hyperinflammatory response seen in COVID-19. However, our understanding of the specific immune responses to SARS-CoV-2 remains limited. Mast cells (MCs) and eosinophils are innate immune cells that play pathogenic roles in many inflammatory responses. Here we report MC-derived proteases and eosinophil-associated mediators are elevated in COVID-19 patient sera and lung tissues. Stimulation of viral-sensing toll-like receptors in vitro and administration of synthetic viral RNA in vivo induced features of hyperinflammation, including cytokine elevation, immune cell airway infiltration, and MC-protease production-effects suppressed by an anti-Siglec-8 monoclonal antibody which selectively inhibits MCs and depletes eosinophils. Similarly, anti-Siglec-8 treatment reduced disease severity and airway inflammation in a respiratory viral infection model. These results suggest that MC and eosinophil activation are associated with COVID-19 inflammation and anti-Siglec-8 antibodies are a potential therapeutic approach for attenuating excessive inflammation during viral infections.

A monoclonal antibody to Siglec-8 suppresses non-allergic airway inflammation and inhibits IgE-independent mast cell activation.

In addition to their well characterized role in mediating IgE-dependent allergic diseases, aberrant accumulation and activation of mast cells (MCs) is associated with many non-allergic inflammatory diseases, whereby their activation is likely triggered by non-IgE stimuli (e.g., IL-33). Siglec-8 is an inhibitory receptor expressed on MCs and eosinophils that has been shown to inhibit IgE-mediated MC responses and reduce allergic inflammation upon ligation with a monoclonal antibody (mAb). Herein, we evaluated the effects of an anti-Siglec-8 mAb (anti-S8) in non-allergic disease models of experimental cigarette-smoke-induced chronic obstructive pulmonary disease and bleomycin-induced lung injury in Siglec-8 transgenic mice. Therapeutic treatment with anti-S8 inhibited MC activation and reduced recruitment of immune cells, airway inflammation, and lung fibrosis. Similarly, using a model of MC-dependent, IL-33-induced inflammation, anti-S8 treatment suppressed neutrophil influx, and cytokine production through MC inhibition. Transcriptomic profiling of MCs further demonstrated anti-S8-mediated downregulation of MC signaling pathways induced by IL-33, including TNF signaling via NF-κB. Collectively, these findings demonstrate that ligating Siglec-8 with an antibody reduces non-allergic inflammation and inhibits IgE-independent MC activation, supporting the evaluation of an anti-Siglec-8 mAb as a therapeutic approach in both allergic and non-allergic inflammatory diseases in which MCs play a role.

Discovery, Function, and Therapeutic Targeting of Siglec-8.

Siglecs (sialic acid-binding immunoglobulin-like lectins) are single-pass cell surface receptors that have inhibitory activities on immune cells. Among these, Siglec-8 is a CD33-related family member selectively expressed on human mast cells and eosinophils, and at low levels on basophils. These cells can participate in inflammatory responses by releasing mediators that attract or activate other cells, contributing to the pathogenesis of allergic and non-allergic diseases. Since its discovery in 2000, initial in vitro studies have found that the engagement of Siglec-8 with a monoclonal antibody or with selective polyvalent sialoglycan ligands induced the cell death of eosinophils and inhibited mast cell degranulation. Anti-Siglec-8 antibody administration in vivo to humanized and transgenic mice selectively expressing Siglec-8 on mouse eosinophils and mast cells confirmed the in vitro findings, and identified additional anti-inflammatory effects. AK002 (lirentelimab) is a humanized non-fucosylated IgG1 antibody against Siglec-8 in clinical development for mast cell- and eosinophil-mediated diseases. AK002 administration has safely demonstrated the inhibition of mast cell activity and the depletion of eosinophils in several phase 1 and phase 2 trials. This article reviews the discovery and functions of Siglec-8, and strategies for its therapeutic targeting for the treatment of eosinophil- and mast cell-associated diseases.

An anti-siglec-8 antibody depletes sputum eosinophils from asthmatic subjects and inhibits lung mast cells.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is expressed on mast cells and eosinophils, but information about Siglec-8 expression and function in the lung is limited. A humanized antibody, AK002, targeting Siglec-8 is undergoing development for treatment of diseases associated with mast cell and eosinophil-driven inflammation. OBJECTIVE: To characterize Siglec-8 expression in the airway in asthma and determine whether antibodies that target Siglec-8 (S8mAbs) can decrease airway eosinophils in asthma or inhibit lung mast cell activation. METHODS: Gene expression profiling and flow cytometry were used to characterize Siglec-8 expression in sputum cells from stable asthma. An antibody-dependent cellular cytotoxicity (ADCC) assay was used to determine whether an S8mAb can decrease eosinophils in sputum from asthma patients ex vivo. A mast cell activation assay was used to determine whether an S8mAb can inhibit mast cell activation in human lung tissue ex vivo. RESULTS: Gene expression for Siglec-8 is increased in sputum cells in asthma and correlates with gene expression for eosinophils and mast cells. Gene expression for Siglec-8 is inversely and significantly correlated with measures of airflow obstruction in asthma patients. Siglec-8 is prominently expressed on the surface of eosinophils and mast cells in sputum. S8mAbs decrease eosinophils in sputum from patients with asthma and inhibit FcεR1-activated mast cells in lung tissues. CONCLUSIONS AND CLINICAL RELEVANCE: Siglec-8 is highly expressed on eosinophils and mast cells in asthmatic sputum and targeting Siglec-8 with an antibody is a plausible strategy to decrease sputum eosinophils and inhibit lung mast cells in asthma.