Siglec-8 on murine eosinophils: A new model for an old target

Abstract

Dysregulated type 2 immune responses are characterized by increased accumulation and activation of eosinophils, mast cells, and basophils in inflamed tissues. Eosinophils, basophils, and mast cells are granulocytes that contain many preformed potent mediators that can be released rapidly into the local environment. These effector cells are critical to the pathophysiology of many allergic disorders, including asthma, allergic rhinitis, chronic urticaria, and eosinophil gastrointestinal disorders; thus, therapeutics that impact the survival of these effector cells in affected tissues remain a priority for development.

Significant advances have been made in targeting eosinophil survival in patients via the biologic agents that inhibit the eosinophilia-promoting cytokine IL-5, and these biologics are now FDA-approved for asthma and eosinophilic granulomatosis with polyangiitis. These biologics that target IL-5 aim to reduce eosinophil survival in tissues; though they are effective in reducing blood eosinophilia, they are less efficacious in eliminating eosinophils in tissues. Several mechanisms for the persistent elevation in tissue eosinophil counts have been proposed, including the decreased dependence for eosinophils on IL-5 for survival in the inflamed tissue due to the presence of other cytokines (e.g., GM-CSF) that promote viability. Benralizumab is an FDA-approved biologic that binds IL-5Rα, a component of the IL-5 receptor, which is expressed by human eosinophils and basophils. Benralizumab inhibits IL-5 from binding to its receptor and, in addition, results in eosinophil and basophil apoptosis via Ab-dependent cell-mediated cytotoxicity (ADCC). Importantly, airway eosinophil level in clinical trial participants that received benralizumab was markedly reduced compared with that of those who received placebo,1 highlighting the effectiveness of cell death-inducing therapies in reducing tissue eosinophil counts. Decreased surface expression of IL-5Rα has been noted on eosinophils following allergen challenge or exposure to type 2 cytokines2,3; thus, the ADCC function of benralizumab could theoretically be impacted by an active inflammatory response.

The protein sialic acid-binding immunoglobulin-like lectin 8 (Siglec-8) is a potential therapeutic target for allergic disorders as it is expressed on the surface of human eosinophils, basophils, and mast cells.4 Siglec-8 is a single-pass transmembrane surface receptor that contains an intracellular ITIM and a membrane-distal immunoreceptor tyrosine-based switch motif. Importantly, engaging Siglec-8 on cytokine-primed eosinophils results in apoptosis.5,6 The functional paralog in mice, Siglec-F, has a differential expression pattern and binding properties and has comparably weaker proapoptotic effects than Siglec-8, creating challenges for preclinical in vivo studies.7

In this issue, O’Sullivan et al. describe a new mouse strain in which the human SIGLEC8 gene is knocked into the murine Rosa26 genomic locus (SIGLEC8^LSL). The upstream loxP-flanked stop cassette limits SIGLEC8 expression to cells that express the Cre recombinase. In the model described in this issue, the new SIGLEC8 strain is crossed with an established strain with eosinophil-specific expression of Cre recombinase,8 and consequently, Siglec-8 expression is detected exclusively on the surface of all developmental stages of the eosinophil lineage (SIGLEC8^Eo), from the eosinophil progenitor (EoP) to mature eosinophils. Notably, engaging Siglec-8 ex vivo on the transgenic eosinophils isolated from the SIGLEC8^Eo mice induced cell death at rates comparable to cytokine-primed human eosinophils, indicating that the human Siglec-8 protein is functional in murine eosinophils. Critical signaling pathways that are responsible for Siglec-8-mediated cell death have been identified utilizing ex vivo studies.7 It remains unclear whether additional and/or alternative pathways are utilized in vivo in inflamed tissues for Siglec-8-mediated cell death. Investigators will be able to identify required molecules and specific mechanisms by crossing this novel eosinophil-restricted Siglec-8-expressing strain (SIGLEC8^Eo) with other strains lacking signaling and effector molecules.

The important potential of the knock-in Siglec-8 strain (SIGLEC8^LSL) is the specificity of expression in relevant cell types, permitting Siglec-8 expression not only on the surface of eosinophils (SIGLEC8^Eo), but also on the surface of mast cells (Mcpt5-Cre9) and/or basophils (Mcpt8-Cre, 2011) via lineage-restricted expression of the Cre recombinase. No publications to date have reported downstream effects of Siglec-8 engagement on basophil viability or functional responses, likely due to the rarity of basophils in the peripheral blood and the low level of Siglec-8 expression on basophils. In addition, studies on the effect of Siglec-8 engagement on mast cells have been limited to cell lines, as mast cells mature from progenitors in tissues and mature mast cells are normally not detected in the peripheral circulation. The effect of Siglec-8 targeting on mast cells in vivo and under conditions in which mast cell numbers are increased in inflamed tissue is an exciting opportunity for this knock-in SIGLEC8^LSL strain.

Little is known about the regulation of SIGLEC8 gene expression, and a limitation of this new knock-in strain (SIGLEC8^LSL) is that SIGLEC8 mRNA expression is not controlled by its endogenous regulatory elements. SIGLEC8 expression is induced in later stages of eosinophil maturation,7 but in the model in this issue (SIGLEC8^Eo), Siglec-8 is detected on the surface of EoPs in addition to mature eosinophils; thus targeting Siglec-8 in vivo may have a greater impact as multiple developmental stages will be affected. Eosinophils express two splice variants of SIGLEC8, comprising a long variant that results in the full-length Siglec-8 protein and a truncated form lacking cytoplasmic signaling motifs.11 The function of the shorter form remains unknown, and this novel strain does not take into account any possible interaction between the long and short forms in vivo. In addition, environmental or developmental signals released by inflamed tissues could potentially regulate endogenous SIGLEC8 gene expression, which would not be taken into consideration in this mouse model system.

Yet, Siglec-8 surface expression on eosinophils in the peripheral blood does not seem to vary much between individuals with eosinophilic asthma (unpublished data from author). It is unclear how much variability in Siglec-8 surface expression is observed in inflamed tissues. In addition, Siglec-8 appears to be partially masked by sialylated cis ligands on the surface of human eosinophils,12 which is likely a protective mechanism to prevent engagement except by high-affinity ligands. It remains to be determined whether murine eosinophils are able to similarly mask human Siglec-8 on the surface. Siglec-8 does undergo endocytosis following engagement by ligands, which presumably occurs in tissues where its endogenous ligands are expressed.13 Intriguingly, ligand-induced endocytosis can be leveraged to deliver therapeutic payloads to target Siglec-8-expressing cells.12 This is especially interesting as Siglec-8 engagement does not induce mast cell death but instead inhibits mediator release following activation; thus, exploiting the endocytosis of Siglec-8 to deliver a toxin to induce mast cell death is an exciting possibility. This novel murine knock-in strain (SIGLEC8^LSL) will allow for testing the therapeutic potential and consequences in vivo of inducing cell death via endocytosis of a therapeutic agent, such as ligand-coated nanoparticles.

This newly described mouse strain that permits eosinophil-specific expression (SIGLEC8^Eo) of a targetable human surface protein will be ideal to study the mechanism, efficacy and consequences of engaging Siglec-8 on eosinophils in a variety of relevant inflammatory models (Fig. 1).

Figure 1

The new mouse strain described in this issue (O’Sullivan et al.) expresses functional human Siglec-8 protein (hSiglec-8) on the surface of murine eosinophils. This new strain can be utilized to test the efficacy and consequences in vivo of Siglec-8 engagement by endogenous ligands, ligand-coated beads or antibodies. The effect of an inflammatory milieu on Siglec-mediated cell death in vivo can be tested in experimental models of allergic disorders, including asthma and eosinophil gastrointestinal disorders. Mice deficient in specific signaling or effector molecules, or with defects in endocytic pathways, can be crossed with the Siglec-8-expressing strain to delineate downstream mechanisms. Finally, the human SIGLEC8 knock-in strain could potentially be crossed with already established strains with Cre recombinase expression limited to mast cells and basophils to permit selective expression of human Siglec-8 to these relevant cell types

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