RESUMEN
RATIONALE: Type 2 (T2) asthma is characterized by airflow limitations and elevated levels of blood and sputum eosinophils, fractional exhaled nitric oxide, IgE, and periostin. While eosinophils are associated with exacerbations, the contribution of eosinophils to lung inflammation, remodeling and function remains largely hypothetical. OBJECTIVES: To determine the effect of T2 cytokines IL-4, IL-13 and IL-5 on eosinophil biology and compare the impact of depleting just eosinophils versus inhibiting all aspects of T2 inflammation on airway inflammation. METHODS: Human eosinophils or endothelial cells stimulated with IL-4, IL-13 or IL-5 were assessed for gene changes or chemokine release.Mice exposed to house dust mite extract received anti-IL-4Rα (dupilumab), anti-IL-5 or control antibodies and were assessed for changes in lung histological and inflammatory endpoints. MEASUREMENTS AND MAIN RESULTS: IL-4 or IL-13 stimulation of human eosinophils and endothelial cells induced gene expression changes related to granulocyte migration; whereas, IL-5 induced changes reflecting granulocyte differentiation.In a mouse model, blocking IL-4Rα improved lung function by impacting multiple effectors of inflammation and remodeling, except peripheral eosinophil counts, thereby disconnecting blood eosinophils from airway inflammation, remodeling and function. Blocking IL-5 globally reduced eosinophil counts but did not impact inflammatory or functional measures of lung pathology. Whole lung transcriptome analysis revealed that IL-5 or IL-4Rα blockade impacted eosinophil associated genes, whereas IL-4Rα blockade also impacted genes associated with multiple cells, cytokines and chemokines, mucus production, cell:cell adhesion and vascular permeability. CONCLUSIONS: Eosinophils are not the sole contributor to asthma pathophysiology or lung function decline and emphasizes the need to block additional mediators to modify lung inflammation and impact lung function.
Asunto(s)
Asma , Neumonía , Animales , Humanos , Ratones , Asma/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Endoteliales/metabolismo , Inflamación/metabolismo , Interleucina-13/metabolismo , Pulmón/metabolismo , Neumonía/metabolismo , Interleucina-4/farmacologíaRESUMEN
Plasmacytoid dendritic cells (pDCs) are a dendritic cell subset that secrete type I IFNs in response to microbial stimuli. The scaffold protein, CD2-associated protein (CD2AP), is a marker of human pDCs as it is highly expressed in this cell type. Recently, in human pDCs, decreased CD2AP expression appeared to enhance the production of type I IFNs via an inhibitory receptor-induced signaling cascade. In this study, we sought to determine the role of CD2AP in murine pDCs using CD2AP knockout (KO) mice. CD2AP was dispensable for the development of pDCs and for the upregulation of activation markers following stimulation. Loss of CD2AP expression did not affect the production of type I IFNs stimulated by TLR ligation, and only slightly impaired type I IFN production when inhibitory pathways were engaged in vitro. This was also confirmed by showing that CD2AP deficiency did not influence type I IFN production by pDCs in vivo. Because CD2AP plays a role in regulating actin dynamics, we examined the actin cytoskeleton in pDCs and found that activated CD2AP KO pDCs had significantly higher levels of actin polymerization than wild-type pDCs. Using two different inflammation models, we found that CD2AP KO pDCs have a defect in lymph node migration, correlating with the defects in actin dynamics. Our work excludes a role for CD2AP in the regulation of type I IFNs in pDCs, and suggests that the major function of CD2AP is on the actin cytoskeleton, affecting migration to local lymph nodes under conditions of inflammation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Citocinas/biosíntesis , Proteínas del Citoesqueleto/fisiología , Células Dendríticas/citología , Inflamación/patología , Ganglios Linfáticos/patología , Actinas/fisiología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Movimiento Celular/fisiología , Citocinas/genética , Proteínas del Citoesqueleto/deficiencia , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Células Dendríticas/metabolismo , Inflamación/inmunología , Interferón-alfa/biosíntesis , Interferón-alfa/genética , Ganglios Linfáticos/inmunología , Tejido Linfoide/citología , Ratones , Ratones Congénicos , Ratones Noqueados , Mycobacterium tuberculosis/inmunología , Quimera por Radiación , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Vesiculovirus/inmunologíaRESUMEN
Monocytes are highly plastic immune cells that modulate antitumor immunity. Therefore, identifying factors that regulate tumor monocyte functions is critical for developing effective immunotherapies. Here, we determine that endogenous cancer cell-derived type I interferons (IFNs) control monocyte functional polarization. Guided by single-cell transcriptomic profiling of human and mouse tumors, we devise a strategy to distinguish and separate immunostimulatory from immunosuppressive tumor monocytes by surface CD88 and Sca-1 expression. Leveraging this approach, we show that cGAS-STING-regulated cancer cell-derived IFNs polarize immunostimulatory monocytes associated with anti-PD-1 immunotherapy response in mice. We also demonstrate that immunosuppressive monocytes convert into immunostimulatory monocytes upon cancer cell-intrinsic cGAS-STING activation. Consistently, we find that human cancer cells can produce type I IFNs that polarize monocytes, and our immunostimulatory monocyte gene signature is enriched in patient tumors that respond to anti-PD-1 immunotherapy. Our work exposes a role for cancer cell-derived IFNs in licensing monocyte functions that influence immunotherapy outcomes.
Asunto(s)
Interferón Tipo I , Neoplasias , Humanos , Ratones , Animales , MonocitosRESUMEN
In the mammalian gastrointestinal tract, the cell fate decisions that specify the development of multiple, diverse lineages are governed in large part by interactions of stem and early lineage progenitor cells with their microenvironment, or niche. Here, we show that the gastric parietal cell (PC) is a key cellular component of the previously undescribed niche for the gastric epithelial neck cell, the progenitor of the digestive enzyme secreting zymogenic (chief) cell (ZC). Genetic ablation of PCs led to failed patterning of the entire zymogenic lineage: progenitors showed premature expression of differentiated cell markers, and fully differentiated ZCs failed to develop. We developed a separate mouse model in which PCs localized not only to the progenitor niche, but also ectopically to the gastric unit base, which is normally occupied by terminally differentiated ZCs. Surprisingly, these mislocalized PCs did not maintain adjacent zymogenic lineage cells in the progenitor state, demonstrating that PCs, though necessary, are not sufficient to define the progenitor niche. We induced this PC mislocalization by knocking out the cytoskeleton-regulating gene Cd2ap in Mist1(-/-) mice, which led to aberrant E-cadherin localization in ZCs, irregular ZC-ZC junctions, and disruption of the ZC monolayer by PCs. Thus, the characteristic histology of the gastric unit, with PCs in the middle and ZCs in the base, may depend on establishment of an ordered adherens junction network in ZCs as they migrate into the base.
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Diferenciación Celular , Linaje de la Célula , Células Principales Gástricas/citología , Células Epiteliales/citología , Nicho de Células Madre/citología , Células Madre/citología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cadherinas/metabolismo , Polaridad Celular , Proliferación Celular , Células Principales Gástricas/metabolismo , Células Principales Gástricas/ultraestructura , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Uniones Intercelulares/metabolismo , Uniones Intercelulares/ultraestructura , Ratones , Modelos Biológicos , Células Parietales Gástricas/citología , Células Parietales Gástricas/metabolismo , Células Parietales Gástricas/ultraestructura , Unión Proteica , Transporte de Proteínas , Nicho de Células Madre/metabolismo , Nicho de Células Madre/ultraestructura , Células Madre/metabolismo , Células Madre/ultraestructuraRESUMEN
BACKGROUND: CD2-associated protein (CD2AP) is an SH3-containing scaffold adaptor protein which regulates the actin cytoskeleton. Recently, CD2AP was identified as a genetic risk factor for Alzheimer's disease (AD) by several genome-wide association studies. One of the hallmarks of AD is the accumulation of aggregated forms of Amyloid-ß (Aß) in the brain. In humans, CD2AP AD susceptibility locus (rs9349407) is associated with an increased plaque burden. Aß production is highly regulated by endocytosis and is influenced by lysosomal function. Lysosomal trafficking is influenced by CD2AP. In this study, we decreased CD2AP levels in N2a neuroblastoma cultures and PS1APP mice and analyzed Aß levels and plaque burden. RESULTS: Our data show that suppressing CD2AP expression using shRNA in N2a-APP695 cells results in decreased cell membrane amyloid precursor protein, decreased Aß release and a lower Aß42/Aß40 ratio. CD2AP protein is expressed in the brain as detected by western blot, and the expression level is dependent on gene dosage. In 1-month old PS1APP mice, complete loss of CD2AP in brain resulted in a decreased Aß42/Aß40 ratio in brain tissue lysates while there was no effect on Aß deposition or accumulation in PS1APP mice expressing one copy of CD2AP. CONCLUSION: CD2-Associated Protein affects Aß levels and Aß42/Aß40 ratio in vitro. The effect of CD2-Associated Protein on Aß metabolism is subtle in vivo.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Precursor de Proteína beta-Amiloide/genética , Encéfalo/metabolismo , Proteínas del Citoesqueleto/metabolismo , Neuroblastoma/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Proteínas del Citoesqueleto/deficiencia , Modelos Animales de Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Ratones Transgénicos , Neuroblastoma/genéticaRESUMEN
Here we address the assumption that the massive intact albuminuria accompanying mutations of structural components of the slit diaphragm is due to changes in glomerular permeability. The increase in intact albumin excretion rate in Cd2ap knockout mice by >100-fold was not accompanied by equivalent changes in urine flow rate, glomerular filtration rate or increases in dextran plasma clearance rate, which demonstrates that changes in glomerular permeability alone could not account for the increase in intact albumin excretion. The albuminuria could be accounted for by inhibition of the tubule degradation pathway associated with degrading filtered albumin. There are remarkable similarities between these results and all types of podocytopathies in acquired and toxin-induced renal disease, and nephrotic states seen in mice with podocyte mutations.
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Proteínas Adaptadoras Transductoras de Señales/genética , Albuminuria/metabolismo , Proteínas del Citoesqueleto/genética , Enfermedades Renales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Albuminuria/fisiopatología , Animales , Proteínas del Citoesqueleto/deficiencia , Femenino , Tasa de Filtración Glomerular , Enfermedades Renales/fisiopatología , Glomérulos Renales/metabolismo , Glomérulos Renales/fisiopatología , Masculino , Ratones , Ratones Noqueados , Permeabilidad , ProteolisisRESUMEN
Ubiquitination of the epidermal growth factor receptor (EGFR) by cbl and its cognate adaptor cbl-interacting protein of 85 kDa (CIN85) is known to play an essential role in directing this receptor to the lysosome for degradation. The mechanisms by which this ubiquitin modification is regulated are not fully defined, nor is it clear where this process occurs. In this study we show that EGFR activation leads to a pronounced src-mediated tyrosine phosphorylation of CIN85 that subsequently influences EGFR ubiquitination. Of importance, phospho-CIN85 interacts with the Rab5-positive endosome, where it mediates the sequestration of the ubiquitinated receptor into multivesicular bodies (MVBs) for subsequent degradation. These findings provide novel insights into how src- kinase-based regulation of a cbl adaptor regulates the fate of the EGFR.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Receptores ErbB/metabolismo , Cuerpos Multivesiculares/metabolismo , Familia-src Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Endocitosis , Endosomas/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Modelos Biológicos , Mutación , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-cbl/genética , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Interferencia de ARN , Ubiquitinación , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismo , Familia-src Quinasas/genéticaRESUMEN
Previous studies suggest that the forkhead transcription factor Foxj1 inhibits spontaneous autoimmunity in part by antagonizing NF-kappaB activation. To test this hypothesis, we ectopically expressed Foxj1 in the T cells of lupus-prone MRL/lpr mice by backcrossing a CD2-Foxj1 transgene against the MRL/lpr background. Strikingly, CD2-Foxj1-MRL/lpr animals showed a significant reduction in lymphadenopathy, pathogenic autoantibodies, and end-organ disease-but surprisingly, reversion of autoimmunity was not attributable to modulation of NF-kappaB. Instead, CD2-Foxj1 transgenic mice exhibited a peripheral T cell lymphopenia, associated with an accumulation of mature single-positive thymocytes. Transgenic thymocytes demonstrated unimpaired lymphoid organ entry in adoptive transfer studies but demonstrated impaired thymic exodus in response to CCL19, apparently independent of CCR7, S1P1, and NF-kappaB. These findings confirm the importance of Foxj1 in the regulation of T cell tolerance but furthermore suggest a novel and specific role for Foxj1 in regulating thymic egress.
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Quimiotaxis de Leucocito , Factores de Transcripción Forkhead/fisiología , Tolerancia Inmunológica , Timo/citología , Traslado Adoptivo , Animales , Autoinmunidad , Antígenos CD2/genética , Quimiocina CCL19 , Quimiocinas CC , Factores de Transcripción Forkhead/genética , Humanos , Linfopenia/etiología , Ratones , Ratones Transgénicos , Linfocitos T/metabolismo , Linfocitos T/fisiologíaRESUMEN
TRPM4 has recently been described as a calcium-activated nonselective (CAN) cation channel that mediates membrane depolarization. However, the functional importance of TRPM4 in the context of calcium (Ca2+) signaling and its effect on cellular responses are not known. Here, the molecular inhibition of endogenous TRPM4 in T cells was shown to suppress TRPM4 currents, with a profound influence on receptor-mediated Ca2+ mobilization. Agonist-mediated oscillations in intracellular Ca2+ concentration ([Ca2+]i), which are driven by store-operated Ca2+ influx, were transformed into a sustained elevation in [Ca2+]i. This increase in Ca2+ influx enhanced interleukin-2 production. Thus, TRPM4-mediated depolarization modulates Ca2+ oscillations, with downstream effects on cytokine production in T lymphocytes.