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1.
Clin Exp Allergy ; 50(8): 904-914, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32542913

RESUMO

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.


Assuntos
Antiasmáticos/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Asma/tratamento farmacológico , Eosinófilos/efeitos dos fármacos , Lectinas/antagonistas & inibidores , Pulmão/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Adulto , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Diferenciação de Linfócitos B/metabolismo , Apoptose/efeitos dos fármacos , Asma/imunologia , Asma/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Eosinófilos/imunologia , Eosinófilos/metabolismo , Feminino , Humanos , Lectinas/genética , Lectinas/imunologia , Lectinas/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Pessoa de Meia-Idade , Receptores de IgE/genética , Receptores de IgE/metabolismo , Escarro/citologia , Adulto Jovem
2.
J Allergy Clin Immunol ; 143(6): 2227-2237.e10, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30543818

RESUMO

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec) 8 is selectively expressed on eosinophils, mast cells, and basophils and, when engaged on eosinophils, can cause cell death. OBJECTIVE: We sought to characterize surface and soluble Siglec-8 (sSiglec-8) levels in normal donors (NDs) and eosinophilic donors (EOs) and assess the efficacy of anti-Siglec-8 antibodies in inducing eosinophil cell death in vitro. METHODS: Eosinophil expression of Siglec-8 was assessed by using flow cytometry and quantitative PCR. Serum sSiglec-8 levels were measured by means of ELISA. Induction of eosinophil death by IgG4 (chimeric 2E2 IgG4) and afucosylated IgG1 (chimeric 2E2 IgG1 [c2E2 IgG1]) anti-Siglec-8 antibodies was evaluated in vitro by using flow cytometry and in vivo in humanized mice. RESULTS: Siglec-8 was consistently expressed on eosinophils from NDs and EOs and did not correlate with absolute eosinophil count or disease activity. sSiglec-8 levels were measurable in sera from most donors unrelated to absolute eosinophil counts or Siglec-8 surface expression. c2E2 IgG1 and chimeric 2E2 IgG4 were equally effective at inducing cell death (Annexin-V positivity) of purified eosinophils from NDs and EOs after overnight IL-5 priming. In contrast, killing of purified eosinophils without IL-5 was only seen in EOs, and natural killer cell-mediated eosinophil killing was seen only with c2E2 IgG1. Finally, treatment of humanized mice with anti-Siglec antibody led to robust depletion of IL-5-induced eosinophilia in vivo. CONCLUSIONS: Siglec-8 is highly expressed on blood eosinophils from EOs and NDs and represents a potential therapeutic target for eosinophilic disorders. Enhanced killing of eosinophils in the presence of IL-5 might lead to increased efficacy in patients with IL-5-driven eosinophilia.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Eosinofilia/imunologia , Eosinófilos/imunologia , Células Matadoras Naturais/imunologia , Lectinas/metabolismo , Animais , Anticorpos Bloqueadores/genética , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/imunologia , Morte Celular , Células Cultivadas , Citotoxicidade Imunológica , Eosinofilia/terapia , Humanos , Imunoglobulina G/genética , Interleucina-5/metabolismo , Lectinas/genética , Lectinas/imunologia , Contagem de Leucócitos , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , Proteínas Recombinantes de Fusão/genética , Transcriptoma
3.
Int Arch Allergy Immunol ; 180(2): 91-102, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31401630

RESUMO

INTRODUCTION: Pathologic accumulation and activation of mast cells and eosinophils are implicated in allergic and inflammatory diseases. Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is an inhibitory receptor selectively expressed on mast cells, eosinophils and, at a lower extent, basophils. When engaged with an antibody, Siglec-8 can induce apoptosis of activated eosinophils and inhibit mast cell activation. AK002 is a humanized, non-fucosylated IgG1 anti-Siglec-8 antibody undergoing clinical investigation for treatment of allergic, inflammatory, and proliferative diseases. Here we examine the human tissue selectivity of AK002 and evaluate the in vitro, ex vivo, and in vivo activity of AK002 on eosinophils and mast cells. METHODS: The affinity of AK002 for Siglec-8 and CD16 was determined by biolayer interferometry. Ex vivo activity of AK002 on human eosinophils from blood and dissociated human tissue was tested in apoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. The in vivo activity of a murine precursor of AK002 (mAK002) was tested in a passive systemic anaphylaxis (PSA) humanized mouse model. RESULTS: AK002 bound selectively to mast cells, eosinophils and, at a lower level, to basophils in human blood and tissue and not to other cell types examined. AK002 induced apoptosis of interleukin-5-activated blood eosinophils and demonstrated potent ADCC activity against blood eosinophils in the presence of natural killer cells. AK002 also significantly reduced eosinophils in dissociated human lung tissue. Furthermore, mAK002 prevented PSA in humanized mice through mast cell inhibition. CONCLUSION: AK002 selectively evokes potent apoptotic and ADCC activity against eosinophils and prevents systemic anaphylaxis through mast cell inhibition.


Assuntos
Anafilaxia/prevenção & controle , Anticorpos Monoclonais Humanizados/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Eosinófilos/imunologia , Lectinas/imunologia , Mastócitos/imunologia , Anafilaxia/imunologia , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Basófilos/imunologia , Humanos , Camundongos , Ácido N-Acetilneuramínico/imunologia , Receptores de IgG/imunologia
4.
Commun Biol ; 5(1): 1226, 2022 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-36369358

RESUMO

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.


Assuntos
Antígenos CD , Mastócitos , Humanos , Camundongos , Animais , Antígenos CD/química , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Anticorpos Monoclonais/farmacologia , Epitopos
5.
Front Immunol ; 13: 833728, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35154156

RESUMO

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.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Lectinas/metabolismo , Mastócitos/imunologia , Receptores de IgE/metabolismo , Animais , Degranulação Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteômica , Transdução de Sinais
6.
Front Immunol ; 12: 650331, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33777047

RESUMO

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.


Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , COVID-19/imunologia , Eosinófilos/imunologia , Lectinas/imunologia , Mastócitos/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , SARS-CoV-2/imunologia , Receptores Toll-Like/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/metabolismo , COVID-19/metabolismo , COVID-19/prevenção & controle , COVID-19/virologia , Estudos de Casos e Controles , Citocinas/metabolismo , Modelos Animais de Doenças , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Eosinófilos/virologia , Interações Hospedeiro-Patógeno , Humanos , Lectinas/antagonistas & inibidores , Lectinas/genética , Lectinas/metabolismo , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Mastócitos/virologia , Camundongos Transgênicos , Peptídeo Hidrolases/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/virologia , Receptores Toll-Like/metabolismo
7.
Mucosal Immunol ; 14(2): 366-376, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32814824

RESUMO

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.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Lectinas/metabolismo , Mastócitos/imunologia , Pneumonia/imunologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Sistema Respiratório/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos B/genética , Degranulação Celular , Fumar Cigarros , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Imunoglobulina E/metabolismo , Interleucina-33/metabolismo , Lectinas/genética , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Ativação de Neutrófilo , Transdução de Sinais
8.
Cells ; 10(1)2020 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-33374255

RESUMO

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.


Assuntos
Anticorpos Monoclonais Humanizados , Antígenos CD , Antígenos de Diferenciação de Linfócitos B , Eosinófilos/imunologia , Hipersensibilidade , Inflamação , Lectinas , Mastócitos/imunologia , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/farmacologia , Antígenos CD/imunologia , Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Diferenciação de Linfócitos B/fisiologia , Ensaios Clínicos como Assunto , Eosinófilos/patologia , Humanos , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/imunologia , Inflamação/tratamento farmacológico , Inflamação/imunologia , Lectinas/imunologia , Lectinas/fisiologia , Mastócitos/patologia , Camundongos , Camundongos Transgênicos
9.
JCI Insight ; 4(19)2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31465299

RESUMO

Aberrant accumulation and activation of eosinophils and potentially mast cells (MCs) contribute to the pathogenesis of eosinophilic gastrointestinal diseases (EGIDs), including eosinophilic esophagitis (EoE), gastritis (EG), and gastroenteritis (EGE). Current treatment options, such as diet restriction and corticosteroids, have limited efficacy and are often inappropriate for chronic use. One promising new approach is to deplete eosinophils and inhibit MCs with a monoclonal antibody (mAb) against sialic acid-binding immunoglobulin-like lectin 8 (Siglec-8), an inhibitory receptor selectively expressed on MCs and eosinophils. Here, we characterize MCs and eosinophils from human EG and EoE biopsies using flow cytometry and evaluate the effects of an anti-Siglec-8 mAb using a potentially novel Siglec-8-transgenic mouse model in which EG/EGE was induced by ovalbumin sensitization and intragastric challenge. MCs and eosinophils were significantly increased and activated in human EG and EoE biopsies compared with healthy controls. Similar observations were made in EG/EGE mice. In Siglec-8-transgenic mice, anti-Siglec-8 mAb administration significantly reduced eosinophils and MCs in the stomach, small intestine, and mesenteric lymph nodes and decreased levels of inflammatory mediators. In summary, these findings suggest a role for both MCs and eosinophils in EGID pathogenesis and support the evaluation of anti-Siglec-8 as a therapeutic approach that targets both eosinophils and MCs.


Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Enterite/tratamento farmacológico , Eosinofilia/tratamento farmacológico , Eosinófilos/efeitos dos fármacos , Gastrite/tratamento farmacológico , Lectinas/efeitos dos fármacos , Mastócitos/imunologia , Animais , Modelos Animais de Doenças , Enterite/imunologia , Eosinofilia/imunologia , Esofagite Eosinofílica/tratamento farmacológico , Eosinófilos/imunologia , Feminino , Gastrite/imunologia , Gastroenterite , Humanos , Lectinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina
10.
Elife ; 82019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31631836

RESUMO

Chronic itch remains a highly prevalent disorder with limited treatment options. Most chronic itch diseases are thought to be driven by both the nervous and immune systems, but the fundamental molecular and cellular interactions that trigger the development of itch and the acute-to-chronic itch transition remain unknown. Here, we show that skin-infiltrating neutrophils are key initiators of itch in atopic dermatitis, the most prevalent chronic itch disorder. Neutrophil depletion significantly attenuated itch-evoked scratching in a mouse model of atopic dermatitis. Neutrophils were also required for several key hallmarks of chronic itch, including skin hyperinnervation, enhanced expression of itch signaling molecules, and upregulation of inflammatory cytokines, activity-induced genes, and markers of neuropathic itch. Finally, we demonstrate that neutrophils are required for induction of CXCL10, a ligand of the CXCR3 receptor that promotes itch via activation of sensory neurons, and we find that that CXCR3 antagonism attenuates chronic itch.


Assuntos
Dermatite Atópica/imunologia , Neutrófilos/imunologia , Prurido/imunologia , Receptores CXCR3/imunologia , Pele/imunologia , Animais , Calcitriol/administração & dosagem , Calcitriol/análogos & derivados , Linhagem Celular , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Quimiocina CXCL10/metabolismo , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Queratinócitos/imunologia , Queratinócitos/metabolismo , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Prurido/induzido quimicamente , Prurido/genética , Receptores CXCR3/genética , Receptores CXCR3/metabolismo , Células Receptoras Sensoriais/imunologia , Células Receptoras Sensoriais/metabolismo , Pele/inervação , Pele/metabolismo
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