Interleukin-22 regulates B3GNT7 expression to induce fucosylation of glycoproteins in intestinal epithelial cells.

Interleukin (IL)-22 is a cytokine that plays a critical role in intestinal epithelial homeostasis. Its downstream functions are mediated through interaction with the heterodimeric IL-22 receptor and subsequent activation of signal transducer and activator of transcription 3 (STAT3). IL-22 signaling can induce transcription of genes necessary for intestinal epithelial cell proliferation, tissue regeneration, tight junction fortification, and antimicrobial production. Recent studies have also implicated IL-22 signaling in the regulation of intestinal epithelial fucosylation in mice. However, whether IL-22 regulates intestinal fucosylation in human intestinal epithelial cells and the molecular mechanisms that govern this process are unknown. Here, in experiments performed in human cell lines and human-derived enteroids, we show that IL-22 signaling regulates expression of the B3GNT7 transcript, which encodes a ß1-3-N-acetylglucosaminyltransferase that can participate in the synthesis of poly-N-acetyllactosamine (polyLacNAc) chains. Additionally, we find that IL-22 signaling regulates levels of the α1-3-fucosylated Lewis X (Lex) blood group antigen, and that this glycan epitope is primarily displayed on O-glycosylated intestinal epithelial glycoproteins. Moreover, we show that increased expression of B3GNT7 alone is sufficient to promote increased display of Lex-decorated carbohydrate glycan structures primarily on O-glycosylated intestinal epithelial glycoproteins. Together, these data identify B3GNT7 as an intermediary in IL-22-dependent induction of fucosylation of glycoproteins and uncover a novel role for B3GNT7 in intestinal glycosylation.

Leveraging Siglec-8 endocytic mechanisms to kill human eosinophils and malignant mast cells.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a cell-surface protein expressed selectively on human eosinophils, mast cells, and basophils, making it an ideal target for the treatment of diseases involving these cell types. However, the effective delivery of therapeutic agents to these cells requires an understanding of the dynamics of Siglec-8 surface expression. OBJECTIVES: We sought to determine whether Siglec-8 is endocytosed in human eosinophils and malignant mast cells, identify mechanisms underlying its endocytosis, and demonstrate whether a toxin can be targeted to Siglec-8-bearing cells to kill these cells. METHODS: Siglec-8 surface dynamics were examined by flow cytometry using peripheral blood eosinophils, mast cell lines, and Siglec-8-transduced cells in the presence of inhibitors targeting components of endocytic pathways. Siglec-8 intracellular trafficking was followed by confocal microscopy. The ribosome-inhibiting protein saporin was conjugated to a Siglec-8-specific antibody to examine the targeting of an agent to these cells through Siglec-8 endocytosis. RESULTS: Siglec-8 endocytosis required actin rearrangement, tyrosine kinase and protein kinase C activities, and both clathrin and lipid rafts. Internalized Siglec-8 localized to the lysosomal compartment. Maximal endocytosis in Siglec-8-transduced HEK293T cells required an intact immunoreceptor tyrosine-based inhibitory motif. Siglec-8 was also shuttled to the surface via a distinct pathway. Sialidase treatment of eosinophils revealed that Siglec-8 is partially masked by sialylated cis ligands. Targeting saporin to Siglec-8 consistently caused extensive cell death in eosinophils and the human mast cell leukemia cell line HMC-1.2. CONCLUSIONS: Therapeutic payloads can be targeted selectively to eosinophils and malignant mast cells by exploiting this Siglec-8 endocytic pathway.

Sialic acid-binding immunoglobulin-like lectin 8 (Siglec-8) is an activating receptor mediating ß2-integrin-dependent function in human eosinophils.

BACKGROUND: Siglec-8 is a CD33 subfamily cell-surface receptor selectively expressed on human eosinophils. After cytokine priming, Siglec-8 mAb or glycan ligand binding causes eosinophil apoptosis associated with reactive oxygen species (ROS) production. Most CD33-related Siglecs function as inhibitory receptors, but the ability of Siglec-8 to stimulate eosinophil ROS production and apoptosis suggests that Siglec-8 might instead function as an activating receptor. OBJECTIVE: We sought to determine the role of IL-5 priming and identify the signaling molecules involved in Siglec-8 function for human eosinophils. METHODS: We used an mAb and/or a multimeric synthetic sulfated sialoglycan ligand recognizing Siglec-8 in combination with integrin blocking antibodies, pharmacologic inhibitors, phosphoproteomics, and Western blot analysis to define the necessity of various proteins involved in Siglec-8 function for human eosinophils. RESULTS: Cytokine priming was required to elicit the unanticipated finding that Siglec-8 engagement promotes rapid ß2-integrin-dependent eosinophil adhesion. Also novel was the finding that this adhesion was necessary for subsequent ROS production and apoptosis. Siglec-8-mediated ROS was generated through reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation because pretreatment of eosinophils with catalase (an extracellular superoxide scavenger) or NSC 23766 (a Rac GTPase inhibitor) completely inhibited Siglec-8-mediated eosinophil apoptosis. Finally, engagement of Siglec-8 on IL-5-primed eosinophils resulted in increased phosphorylation of Akt, p38, and c-Jun N-terminal kinase 1 that was also ß2-integrin dependent; pharmacologic inhibition of these kinases completely prevented Siglec-8-mediated eosinophil apoptosis. CONCLUSIONS: These data demonstrate that Siglec-8 functions uniquely as an activating receptor on IL-5-primed eosinophils through a novel pathway involving regulation of ß2-integrin-dependent adhesion, NADPH oxidase, and a subset of protein kinases.

Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways.

Human siglecs are a family of 14 sialic acid-binding proteins, most of which are expressed on subsets of immune cells where they regulate immune responses. Siglec-8 is expressed selectively on human allergic inflammatory cells-primarily eosinophils and mast cells-where engagement causes eosinophil apoptosis and inhibits mast cell mediator release. Evidence supports a model in which human eosinophils and mast cells bind to Siglec-8 sialoglycan ligands on inflammatory target tissues to resolve allergic inflammation and limit tissue damage. To identify Siglec-8-binding sialoglycans from human airways, proteins extracted from postmortem human trachea were resolved by size-exclusion chromatography and composite agarose-acrylamide gel electrophoresis, blotted and probed by Siglec-8-Fc blot overlay. Three size classes of Siglec-8 ligands were identified: 250 kDa, 600 kDa and 1 MDa, each of which was purified by affinity chromatography using a recombinant pentameric form of Siglec-8. Proteomic mass spectrometry identified all size classes as the proteoglycan aggrecan, a finding validated by immunoblotting. Glycan array studies demonstrated Siglec-8 binding to synthetic glycans with a terminal Neu5Acα2-3(6-sulfo)-Gal determinant, a quantitatively minor terminus on keratan sulfate (KS) chains of aggrecan. Treating human tracheal extracts with sialidase or keratanase eliminated Siglec-8 binding, indicating sialylated KS chains as Siglec-8-binding determinants. Treating human tracheal histological sections with keratanase also completely eliminated the binding of Siglec-8-Fc. Finally, Siglec-8 ligand purified from human trachea extracts induced increased apoptosis of freshly isolated human eosinophils in vitro. We conclude that sialylated KS proteoglycans are endogenous human airway ligands that bind Siglec-8 and may regulate allergic inflammation.

Siglec-8 Signals Through a Non-Canonical Pathway to Cause Human Eosinophil Death In Vitro.

Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a glycan-binding receptor bearing immunoreceptor tyrosine-based inhibitory and switch motifs (ITIM and ITSM, respectively) that is selectively expressed on eosinophils, mast cells, and, to a lesser extent, basophils. Previous work has shown that engagement of Siglec-8 on IL-5-primed eosinophils causes cell death via CD11b/CD18 integrin-mediated adhesion and NADPH oxidase activity and identified signaling molecules linking adhesion, reactive oxygen species (ROS) production, and cell death. However, the proximal signaling cascade activated directly by Siglec-8 engagement has remained elusive. Most members of the Siglec family possess similar cytoplasmic signaling motifs and recruit the protein tyrosine phosphatases SHP-1/2, consistent with ITIM-mediated signaling, to dampen cellular activation. However, the dependence of Siglec-8 function in eosinophils on these phosphatases has not been studied. Using Siglec-8 antibody engagement and pharmacological inhibition in conjunction with assays to measure cell-surface upregulation and conformational activation of CD11b integrin, ROS production, and cell death, we sought to identify molecules involved in Siglec-8 signaling and determine the stage of the process in which each molecule plays a role. We demonstrate here that the enzymatic activities of Src family kinases (SFKs), Syk, SHIP1, PAK1, MEK1, ERK1/2, PLC, PKC, acid sphingomyelinase/ceramidase, and Btk are all necessary for Siglec-8-induced eosinophil cell death, with no apparent role for SHP-1/2, SHIP2, or c-Raf. While most of these signaling molecules are necessary for Siglec-8-induced upregulation of CD11b integrin at the eosinophil cell surface, Btk is phosphorylated and activated later in the signaling cascade and is instead necessary for CD11b activation. In contrast, SFKs and ERK1/2 are phosphorylated far earlier in the process, consistent with their role in augmenting cell-surface levels of CD11b. In addition, pretreatment of eosinophils with latrunculin B or jasplakinolide revealed that actin filament disassembly is necessary and sufficient for surface CD11b integrin upregulation and that actin polymerization is necessary for downstream ROS production. These results show that Siglec-8 signals through an unanticipated set of signaling molecules in IL-5-primed eosinophils to induce cell death and challenges the expectation that ITIM-bearing Siglecs signal through inhibitory pathways involving protein tyrosine phosphatases to achieve their downstream functions.

Mechanisms for Alternaria alternata Function in the Skin During Induction of Peanut Allergy in Neonatal Mice With Skin Barrier Mutations.

Neonatal mice with heterozygous mutations in genes encoding the skin barrier proteins filaggrin and mattrin (flaky tail mice [FT+/-]) exhibit oral peanut-induced anaphylaxis after skin sensitization. As we have previously reported, sensitization in this model is achieved via skin co- exposure to the environmental allergen Alternaria alternata (Alt), peanut extract (PNE), and detergent. However, the function of Alt in initiation of peanut allergy in this model is little understood. The purpose of this study was to investigate candidate cytokines induced by Alt in the skin and determine the role of these cytokines in the development of food allergy, namely oncostatin M (Osm), amphiregulin (Areg), and IL-33. RT-qPCR analyses demonstrated that skin of FT+/- neonates expressed Il33 and Osm following Alt or Alt/PNE but not PNE exposure. By contrast, expression of Areg was induced by either Alt, PNE, or Alt/PNE sensitization in FT+/- neonates. In scRNAseq analyses, Osm, Areg, and Il33 were expressed by several cell types, including a keratinocyte cluster that was expanded in the skin of Alt/PNE-exposed FT+/- pups as compared to Alt/PNE-exposed WT pups. Areg and OSM were required for oral PNE-induced anaphylaxis since anaphylaxis was inhibited by administration of neutralizing anti-Areg or anti-OSM antibodies prior to each skin sensitization with Alt/PNE. It was then determined if intradermal injection of recombinant IL33 (rIL33), rAreg, or rOSM in the skin could substitute for Alt during skin sensitization to PNE. PNE skin sensitization with intradermal rIL33 was sufficient for oral PNE-induced anaphylaxis, whereas skin sensitization with intradermal rAreg or rOSM during skin exposure to PNE was not sufficient for anaphylaxis to oral PNE challenge. Based on these studies a pathway for IL33, Areg and OSM in Alt/PNE sensitized FT+/- skin was defined for IgE induction and anaphylaxis. Alt stimulated two pathways, an IL33 pathway and a pathway involving OSM and Areg. These two pathways acted in concert with PNE to induce food allergy in pups with skin barrier mutations.

Single-site, five-year experience with human eosinophil isolation by density gradient centrifugation and CD16 immunomagnetic negative separation.

OBJECTIVE: Little has been reported regarding the reliability of methods for the purification of human blood eosinophils. We retrospectively reviewed our experience with 350 consecutive eosinophil isolations. RESULTS: Between January 2014 and December 2018, we conducted 350 eosinophil purifications from 83 donors. Absolute eosinophil count (AEC), calculated from hospital complete blood counts when available (n = 289), ranged from 32 to 1352 eosinophils/µL ([Formula: see text]: 179 ± 136/µL). Eosinophil yields ranged from 0.4 to 24.4 million cells per 20 mL of blood drawn ([Formula: see text]: 3.1 ± 1.9 million eosinophils) with > 98% purity. Comparing AEC to actual yield, recovery was 87% ± 29% ([Formula: see text]) and AEC strongly correlated with yield. To explore the reproducibility of yield, a subsequent analysis was limited to those donors drawn ≥ 3 times (N = 35), and there was no difference in the average coefficient of variation for yield between allergic and non-allergic donors. Viability of isolated eosinophils was consistently > 95% and after 24 h of culture did not differ between allergic and non-allergic donors. We conclude that this immunomagnetic separation method for human eosinophil isolation from whole blood is a reliable, reproducible technique for obtaining an average of 87% yield with high purity and viability.

Frontline Science: Characterization of a novel mouse strain expressing human Siglec-8 only on eosinophils.

Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a human cell surface protein expressed exclusively on eosinophils, mast cells, and basophils that, when engaged, induces eosinophil apoptosis and inhibits mast cell mediator release. This makes Siglec-8 a promising therapeutic target to treat diseases involving these cell types. However, preclinical studies of Siglec-8 targeting in vivo are lacking because this protein is only found in humans, apes, and some monkeys. Therefore, we have developed a mouse strain in which SIGLEC8 transcription is activated by Cre recombinase and have crossed this mouse with the eoCre mouse to achieve eosinophil-specific expression. We confirmed that Siglec-8 is expressed exclusively on the surface of mature eosinophils in multiple tissues at levels comparable to those on human blood eosinophils. Following ovalbumin sensitization and airway challenge, Siglec-8 knock-in mice generated a pattern of allergic lung inflammation indistinguishable from that of littermate controls, suggesting that Siglec-8 expression within the eosinophil compartment does not alter allergic eosinophilic inflammation. Using bone marrow from these mice, we demonstrated that, during maturation, Siglec-8 expression occurs well before the late eosinophil developmental marker C-C motif chemokine receptor 3, consistent with eoCre expression. Antibody ligation of the receptor induces Siglec-8 endocytosis and alters the phosphotyrosine profile of these cells, indicative of productive signaling. Finally, we demonstrated that mouse eosinophils expressing Siglec-8 undergo cell death when the receptor is engaged, further evidence that Siglec-8 is functional on these cells. These mice should prove useful to investigate Siglec-8 biology and targeting in vivo in a variety of eosinophilic disease models.

Glycobiology of Eosinophilic Inflammation: Contributions of Siglecs, Glycans, and Other Glycan-Binding Proteins.

The historical focus on protein-protein interactions in biological systems, at the expense of attention given to interactions between other classes of molecules, has overlooked important and clinically relevant processes and points of potential clinical intervention. For example, the significance of protein-carbohydrate interactions, especially in the regulation of immune responses, has recently received greater recognition and appreciation. This review discusses several ways by which cell-surface lectin-glycan interactions can modulate eosinophil function, particularly at the levels of eosinophil recruitment and survival, and how such interactions can be exploited therapeutically. A primary focus is on discoveries concerning Siglec-8, a glycan-binding protein selectively expressed on human eosinophils, and its closest functional paralog in the mouse, Siglec-F. Recent advances in the synthesis of polymeric ligands, the identification of physiological ligands for Siglec-8 and Siglec-F in the airway, and the determination of the basis of glycan ligand discrimination of Siglec-8 are discussed. Important similarities and differences between these siglecs are outlined. Eosinophil expression of additional glycan-binding proteins or their glycan ligands, including interactions involving members of the selectin, galectin, and siglec families, is summarized. The roles of these molecules in eosinophil recruitment, survival, and inflammation are described. Finally, the modulation of these interactions and potential therapeutic exploitation of glycan-binding proteins and their ligands to ameliorate eosinophil-associated diseases are considered.