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1.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-33799879

RESUMEN

The development of nanocarriers (NC) for biomedical applications has gained large interest due to their potential to co-deliver drugs in a cell-type-targeting manner. However, depending on their surface characteristics, NC accumulate serum factors, termed protein corona, which may affect their cellular binding. We have previously shown that NC coated with carbohydrates to enable biocompatibility triggered the lectin-dependent complement pathway, resulting in enhanced binding to B cells via complement receptor (CR)1/2. Here we show that such NC also engaged all types of splenic leukocytes known to express CR3 at a high rate when NC were pre-incubated with native mouse serum resulting in complement opsonization. By focusing on dendritic cells (DC) as an important antigen-presenting cell type, we show that CR3 was essential for binding/uptake of complement-opsonized NC, whereas CR4, which in mouse is specifically expressed by DC, played no role. Further, a minor B cell subpopulation (B-1), which is important for first-line pathogen responses, and co-expressed CR1/2 and CR3, in general, engaged NC to a much higher extent than normal B cells. Here, we identified CR-1/2 as necessary for binding of complement-opsonized NC, whereas CR3 was dispensable. Interestingly, the binding of complement-opsonized NC to both DC and B-1 cells affected the expression of activation markers. Our findings may have important implications for the design of nano-vaccines against infectious diseases, which codeliver pathogen-specific protein antigen and adjuvant, aimed to induce a broad adaptive cellular and humoral immune response by inducing cytotoxic T lymphocytes that kill infected cells and pathogen-neutralizing antibodies, respectively. Decoration of nano-vaccines either with carbohydrates to trigger complement activation in vivo or with active complement may result in concomitant targeting of DC and B cells and thereby may strongly enhance the extent of dual cellular/humoral immune responses.


Asunto(s)
Subgrupos de Linfocitos B/inmunología , Linfocitos B/inmunología , Antígeno CD11b/inmunología , Proteínas del Sistema Complemento/inmunología , Células Dendríticas/inmunología , Receptores de Complemento/inmunología , Animales , Subgrupos de Linfocitos B/metabolismo , Linfocitos B/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Células Cultivadas , Activación de Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Células Dendríticas/metabolismo , Dextranos/química , Portadores de Fármacos/química , Humanos , Activación de Linfocitos/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Nanopartículas/química , Proteínas Opsoninas/inmunología , Proteínas Opsoninas/metabolismo , Fagocitosis/inmunología , Receptores de Complemento/metabolismo
2.
BMC Geriatr ; 21(1): 225, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33794800

RESUMEN

BACKGROUND: Sepsis is a critical challenge for the older adults as the immune function is less responsive by aging. Although cell numbers seem preserved in the older adults, macrophages present age-related function decline, which including reduced chemokines, phagocytosis, and autophagy. ABT-263, an inhibitor of the anti-apoptotic protein Bcl-2, is reported had a senolytic effect which can selectively clear the senescent cells in vivo and rejuvenate the aged tissues. METHODS: We treated the aged (12-16 months) and young (4-6 months) C57BL/6 mouse with ABT-263, then gave the animals cecal slurry injection to induce sepsis to observe the effect of senolytic compound ABT-263 on the survival rate of sepsis. Additionally, we isolated peritoneal macrophages from the aged mouse to investigate the cell function and molecular mechanism. 3-methyladenine (3-MA), a phosphatidylinositol 3-kinases (PI3K) inhibitor, and rapamycin, an autophagy-enhancer, were used to block or mimic the autophagy, respectively. RT-PCR and Western Blot were used to detect autophagy related gene and protein changes in sepsis. EGFP-expressing E. coli was used as a marker to evaluate the phagocytic ability of macrophages. RESULTS: The results showed ABT-263 treatment improved the survival rate of sepsis in the aged mouse which related to autophagy, while blocking the autophagy can eliminate this effect. It is revealed that ABT-263 enhanced the phagocytic ability of the peritoneal macrophages by increasing the Trem-2 receptor. Additionally, ABT-263 blocked the binding of Bcl-2 to Beclin-1, thus induced Beclin-1-dependent autophagy. CONCLUSION: ABT-263 enhanced the macrophage function in aged mouse by increasing the Trem-2 receptors and inducing a beclin-1-dependent autophagy, consequently, protected the aged mouse from sepsis.


Asunto(s)
Escherichia coli , Fosfatidilinositol 3-Quinasas , Anciano , Compuestos de Anilina , Animales , Autofagia , Beclina-1 , Humanos , Macrófagos , Ratones , Ratones Endogámicos C57BL , Fagocitosis , Sulfonamidas
3.
J Appl Oral Sci ; 29: e20200770, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33825754

RESUMEN

OBJECTIVE: Neutrophils are key effector cells of the innate immune system. They recognize antigens through membrane receptors, which are expressed during their maturation and activation. Neutrophils express FcγRII (CD32), FcγRIII (CD16), and FcγRI (CD64) after being activated by different factors such as cytokines and bacterial products. These receptors are involved with phagocytosis of IgG-opsonized microbes and enhance defense mechanisms. Based on that, our study seeks to compare the expression of FcγRII, FcγRIII, FcγRI, and CD11b on neutrophils from elderly and young subjects and their expression after in vitro activation with cytokines and LPS. METHODOLOGY: Neutrophils were isolated from human peripheral blood and from mice bone marrow by density gradient. After isolation, FCγRs expression was immediately analyzed by flow cytometry or after in vitro stimulation. RESULTS: In freshly isolated cells, the percentage of FcγRIIIb+ and CD11b+ neutrophils were higher in samples from young individuals; FcγRIIIa expression was more prominent on aged neutrophils; FcγRIA expression was similar in all samples analyzed. Exposure to CXCL8 and LPS resulted in a higher percentage of FcγRIa+ neutrophils on elderly individuals' samples but lower when compared with neutrophils from young donors. We observed that LPS caused an increase in FcγRIIa expression on aging human neutrophils. In contrast, FcγRIIIb expression in response to CXCL8 and LPS stimulation was not altered in the four groups. CD11b expression was lower in neutrophils from elderly individuals even in response to LPS and CXCL8. In mice, we observed differences only regarding CD11b expression, which was increased on aged neutrophils. LPS exposure caused an increase in all FcγRs. CONCLUSIONS: Our results suggest that, in humans, the overall pattern of FcγR expression and integrin CD11b are altered during aging and immunosenescence might contribute to age-related infection.


Asunto(s)
Neutrófilos , Receptores de IgG , Animales , Recuento de Células , Citometría de Flujo , Ratones , Fagocitosis
4.
mBio ; 12(2)2021 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-33879585

RESUMEN

Convalescent plasma is a promising therapy for coronavirus disease 2019 (COVID-19), but the antibody characteristics that contribute to efficacy remain poorly understood. This study analyzed plasma samples from 126 eligible convalescent blood donors in addition to 15 naive individuals, as well as an additional 20 convalescent individuals as a validation cohort. Multiplexed Fc Array binding assays and functional antibody response assays were utilized to evaluate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody composition and activity. Donor convalescent plasma samples contained a range of antibody cell- and complement-mediated effector functions, indicating the diverse antiviral activity of humoral responses observed among recovered individuals. In addition to viral neutralization, convalescent plasma samples contained antibodies capable of mediating such Fc-dependent functions as complement activation, phagocytosis, and antibody-dependent cellular cytotoxicity against SARS-CoV-2. Plasma samples from a fraction of eligible donors exhibited high activity across all activities evaluated. These polyfunctional plasma samples could be identified with high accuracy with even single Fc Array features, whose correlation with polyfunctional activity was confirmed in the validation cohort. Collectively, these results expand understanding of the diversity of antibody-mediated antiviral functions associated with convalescent plasma, and the polyfunctional antiviral functions suggest that it could retain activity even when its neutralizing capacity is reduced by mutations in variant SARS-CoV-2.IMPORTANCE Convalescent plasma has been deployed globally as a treatment for COVID-19, but efficacy has been mixed. Better understanding of the antibody characteristics that may contribute to its antiviral effects is important for this intervention as well as offer insights into correlates of vaccine-mediated protection. Here, a survey of convalescent plasma activities, including antibody neutralization and diverse effector functions, was used to define plasma samples with broad activity profiles. These polyfunctional plasma samples could be reliably identified in multiple cohorts by multiplex assay, presenting a widely deployable screening test for plasma selection and investigation of vaccine-elicited responses.


Asunto(s)
Anticuerpos Antivirales/sangre , /terapia , /inmunología , Adulto , Anciano , Anticuerpos Neutralizantes/sangre , Especificidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos Virales/inmunología , Fenómenos Biofísicos , Estudios de Cohortes , Activación de Complemento , Convalecencia , Femenino , Humanos , Inmunización Pasiva , Masculino , Persona de Mediana Edad , Fagocitosis , Adulto Joven
5.
mBio ; 12(2)2021 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-33879594

RESUMEN

Beyond neutralization, antibodies binding to their Fc receptors elicit several innate immune functions including antibody-dependent complement deposition (ADCD), antibody-dependent cell-mediated phagocytosis (ADCP), and antibody-dependent cell-mediated cytotoxicity (ADCC). These functions are beneficial, as they contribute to pathogen clearance; however, they also can induce inflammation. We tested the possibility that qualitative differences in SARS-CoV-2-specific antibody-mediated innate immune functions contribute to coronavirus disease 2019 (COVID-19) severity. We found that anti-S1 and anti-RBD antibodies from hospitalized COVID-19 patients elicited higher ADCD but lower ADCP compared to antibodies from nonhospitalized COVID-19 patients. Consistently, higher ADCD was associated with higher systemic inflammation, whereas higher ADCP was associated with lower systemic inflammation during COVID-19. Our study points to qualitative, differential features of anti-SARS-CoV-2 specific antibodies as potential contributors to COVID-19 severity. Understanding these qualitative features of natural and vaccine-induced antibodies will be important in achieving optimal efficacy and safety of SARS-CoV-2 vaccines and/or COVID-19 therapeutics.IMPORTANCE A state of hyperinflammation and increased complement activation has been associated with coronavirus disease 2019 (COVID-19) severity. However, the pathophysiological mechanisms that contribute to this phenomenon remain mostly unknown. Our data point to a qualitative, rather than quantitative, difference in SARS-CoV-2-specific antibodies' ability to elicit Fc-mediated innate immune functions as a potential contributor to COVID-19 severity and associated inflammation. These data highlight the need for further studies to understand these qualitative features and their potential contribution to COVID-19 severity. This understanding could be essential to develop antibody-based COVID-19 therapeutics and SARS-CoV-2 vaccines with an optimal balance between efficacy and safety.


Asunto(s)
Anticuerpos Antivirales , Inmunidad Innata , /inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Biomarcadores/sangre , /virología , Estudios de Casos y Controles , Estudios de Cohortes , Activación de Complemento , Femenino , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Inflamación/sangre , Inflamación/etiología , Inflamación/inmunología , Masculino , Persona de Mediana Edad , Pandemias , Fagocitosis , Receptores Fc/inmunología , Índice de Severidad de la Enfermedad , Glicoproteína de la Espiga del Coronavirus/inmunología
6.
Nat Commun ; 12(1): 1742, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741975

RESUMEN

A highly protective vaccine will greatly facilitate achieving and sustaining malaria elimination. Understanding mechanisms of antibody-mediated immunity is crucial for developing vaccines with high efficacy. Here, we identify key roles in humoral immunity for Fcγ-receptor (FcγR) interactions and opsonic phagocytosis of sporozoites. We identify a major role for neutrophils in mediating phagocytic clearance of sporozoites in peripheral blood, whereas monocytes contribute a minor role. Antibodies also promote natural killer cell activity. Mechanistically, antibody interactions with FcγRIII appear essential, with FcγRIIa also required for maximum activity. All regions of the circumsporozoite protein are targets of functional antibodies against sporozoites, and N-terminal antibodies have more activity in some assays. Functional antibodies are slowly acquired following natural exposure to malaria, being present among some exposed adults, but uncommon among children. Our findings reveal targets and mechanisms of immunity that could be exploited in vaccine design to maximize efficacy.


Asunto(s)
Inmunidad Humoral , Malaria/inmunología , Malaria/prevención & control , Receptores de IgG/inmunología , Esporozoítos/inmunología , Adulto , Anciano , Anticuerpos Antiprotozoarios/inmunología , Niño , Femenino , Humanos , Kenia , Vacunas contra la Malaria/inmunología , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Neutrófilos/inmunología , Fagocitosis/inmunología , Plasmodium falciparum/inmunología , Receptores de IgG/metabolismo , Células THP-1 , Adulto Joven
7.
Cell Prolif ; 54(4): e13022, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33686740

RESUMEN

OBJECTIVES: This study aimed to investigate the protective effect of SCARF1 on acute rejection (AR), phagocytic clearance of Kupffer cells (KCs), M2 polarization and the exact mechanism underlying these processes. METHODS: AAV was transfected into the portal vein of rats, and AR and immune tolerance (IT) models of liver transplantation were established. Liver tissue and blood samples were collected. The level of SCARF1 was detected via WB and immunohistochemical staining. Pathological changes in liver tissue were detected using HE staining. Apoptotic cells were detected using TUNEL staining. KC polarization was assessed via immunohistochemical staining. Primary KCs were isolated and co-cultured with apoptotic T lymphocytes. Phagocytosis of apoptotic cells and polarization of KCs were both detected using immunofluorescence. Calcium concentration was determined using immunofluorescence and a fluorescence microplate reader. The levels of PI3K, p-AKT and P-STAT3 were assessed via WB and immunofluorescence. RESULTS: Compared to the IT group, the level of SCARF1 was significantly decreased in the AR group. Overexpression of SCARF1 in KCs improved AR and liver function markers. Enhanced phagocytosis mediated by SCARF1 is beneficial for improving the apoptotic clearance of AR and promoting M2 polarization of KCs. SCARF1-mediated enhancement of phagocytosis promotes increased calcium concentration in KCs, thus further activating the PI3K-AKT-STAT3 signalling pathway. CONCLUSIONS: SCARF1 promotes the M2 polarization of KCs by promoting phagocytosis through the calcium-dependent PI3K-AKT-STAT3 signalling pathway.


Asunto(s)
Calcio/metabolismo , Trasplante de Hígado , Receptores Depuradores de Clase F/metabolismo , Transducción de Señal , Animales , Apoptosis , Polaridad Celular , Células Cultivadas , Técnicas de Cocultivo , Macrófagos del Hígado/citología , Macrófagos del Hígado/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Fagocitosis , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Endogámicas Lew , Factor de Transcripción STAT3/metabolismo , Receptores Depuradores de Clase F/genética , Linfocitos T/citología , Linfocitos T/metabolismo
8.
Int J Mol Sci ; 22(4)2021 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-33672445

RESUMEN

Hereditary retinal dystrophies (HRD) represent a significant cause of blindness, affecting mostly retinal pigment epithelium (RPE) and photoreceptors (PRs), and currently suffer from a lack of effective treatments. Highly specialized RPE and PR cells interact mutually in the functional retina, therefore primary HRD affecting one cell type leading to a secondary HRD in the other cells. Phagocytosis is one of the primary functions of the RPE and studies have discovered that mutations in the phagocytosis-associated gene Mer tyrosine kinase receptor (MERTK) lead to primary RPE dystrophy. Treatment strategies for this rare disease include the replacement of diseased RPE with healthy autologous RPE to prevent PR degeneration. The generation and directed differentiation of patient-derived human-induced pluripotent stem cells (hiPSCs) may provide a means to generate autologous therapeutically-relevant adult cells, including RPE and PR. However, the continued presence of the MERTK gene mutation in patient-derived hiPSCs represents a significant drawback. Recently, we reported the generation of a hiPSC model of MERTK-associated Retinitis Pigmentosa (RP) that recapitulates disease phenotype and the subsequent creation of gene-corrected RP-hiPSCs using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9. In this study, we differentiated gene-corrected RP-hiPSCs into RPE and found that these cells had recovered both wild-type MERTK protein expression and the lost phagocytosis of fluorescently-labeled photoreceptor outer segments observed in uncorrected RP-hiPSC-RPE. These findings provide proof-of-principle for the utility of gene-corrected hiPSCs as an unlimited cell source for personalized cell therapy of rare vision disorders.


Asunto(s)
Edición Génica , Células Madre Pluripotentes Inducidas/patología , Fagocitosis , Epitelio Pigmentado de la Retina/patología , Retinitis Pigmentosa/patología , Diferenciación Celular/genética , Línea Celular , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/ultraestructura , Mutación/genética , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Segmento Externo de las Células Fotorreceptoras Retinianas/patología , Segmento Externo de las Células Fotorreceptoras Retinianas/ultraestructura , Epitelio Pigmentado de la Retina/ultraestructura , Retinitis Pigmentosa/genética , Tirosina Quinasa c-Mer/genética , Tirosina Quinasa c-Mer/metabolismo
9.
J Vis Exp ; (168)2021 02 10.
Artículo en Inglés | MEDLINE | ID: mdl-33645569

RESUMEN

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.


Asunto(s)
Disección , Cultivo Primario de Células/métodos , Epitelio Pigmentado de la Retina/citología , Animales , Bioensayo , Diferenciación Celular , Polaridad Celular , Separación Celular , Impedancia Eléctrica , Células Epiteliales/citología , Humanos , Ratones Endogámicos C57BL , Fagocitosis , Factores de Tiempo
10.
J Vis Exp ; (168)2021 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-33645588

RESUMEN

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an "eat-me" signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.


Asunto(s)
Bioensayo/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Macrófagos/metabolismo , Neuroblastoma/patología , Fagocitosis , Animales , Muerte Celular , Línea Celular Tumoral , Análisis de Datos , Colorantes Fluorescentes/química , Células Madre Embrionarias Humanas/citología , Humanos , Concentración de Iones de Hidrógeno , Células Madre Pluripotentes Inducidas/citología , Control de Calidad , Reproducibilidad de los Resultados , Imagen de Lapso de Tiempo
11.
Carbohydr Polym ; 260: 117796, 2021 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-33712144

RESUMEN

The polysaccharide (DRP) was gained from dandelion roots by ultrasonic-assisted enzymatic extraction (UAEE) followed by two-step column purification. Then selenylation of DRP has been accomplished by HNO3-Na2SeO3 method. sDRP-1 and sDRP-2 with the selenium content of 170 ± 1.13 and 710 ± 4.00 µg/g were prepared for further structural characterization and bioactivity determination. DRP, sDRP-1, and sDRP-2 were composed of the same monosaccharides in different molar ratios, and the molecular weights of DRP, sDRP-1 and sDRP-2 were 8700, 7900, and 5600 Da, respectively. Fourier transform infrared (FT-IR) spectra confirmed that DRP, sDRP-1, and sDRP-2 possessed similar functional groups. The results of Congo red test, X-ray diffraction (XRD) and scanning electron microscopy (SEM) showed that DRP, sDRP-1, and sDRP-2 had no three helix structure, did not form single crystal, and all belonged to amorphous morphology. sDRP-1 and sDRP-2 possessed greater antioxidant activities in vitro than the native polysaccharide DRP. At the same time, the selenized polysaccharides showed better immunomodulatory ability and could be used as new-type immunoenhancer. The present conclusions provided theoretical basis for the new application of dandelion polysaccharides and the development of dandelion resources.


Asunto(s)
Antioxidantes/química , Polisacáridos/química , Selenio/química , Taraxacum/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Factores Inmunológicos/química , Factores Inmunológicos/metabolismo , Factores Inmunológicos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Peso Molecular , Fagocitosis/efectos de los fármacos , Raíces de Plantas/metabolismo , Polisacáridos/metabolismo , Polisacáridos/farmacología , Células RAW 264.7
12.
Nat Commun ; 12(1): 1508, 2021 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-33686057

RESUMEN

LC3-associated phagocytosis (LAP) contributes to a wide range of cellular processes and notably to immunity. The stabilization of phagosomes by the macroautophagy machinery in human macrophages can maintain antigen presentation on MHC class II molecules. However, the molecular mechanisms involved in the formation and maturation of the resulting LAPosomes are not completely understood. Here, we show that reactive oxygen species (ROS) produced by NADPH oxidase 2 (NOX2) stabilize LAPosomes by inhibiting LC3 deconjugation from the LAPosome cytosolic surface. NOX2 residing in the LAPosome membrane generates ROS to cause oxidative inactivation of the protease ATG4B, which otherwise releases LC3B from LAPosomes. An oxidation-insensitive ATG4B mutant compromises LAP and thereby impedes sustained MHC class II presentation of exogenous Candida albicans antigens. Redox regulation of ATG4B is thereby an important mechanism for maintaining LC3 decoration of LAPosomes to support antigen processing for MHC class II presentation.


Asunto(s)
Presentación de Antígeno/fisiología , Autofagia/fisiología , Antígenos de Histocompatibilidad Clase II/metabolismo , Fagosomas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Antígenos Fúngicos , Proteínas Relacionadas con la Autofagia , Candida albicans , Fosfatidilinositol 3-Quinasas Clase III , Cisteína Endopeptidasas/metabolismo , Células HEK293 , Humanos , Macroautofagia , Macrófagos/metabolismo , NADPH Oxidasa 2/metabolismo , Oxidación-Reducción , Fagocitosis/fisiología , Especies Reactivas de Oxígeno/metabolismo
13.
Int J Mol Sci ; 22(5)2021 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-33668084

RESUMEN

The interaction of macrophages with apoptotic cells is required for efficient resolution of inflammation. While apoptotic cell removal prevents inflammation due to secondary necrosis, it also alters the macrophage phenotype to hinder further inflammatory reactions. The interaction between apoptotic cells and macrophages is often studied by chemical or biological induction of apoptosis, which may introduce artifacts by affecting the macrophages as well and/or triggering unrelated signaling pathways. Here, we set up a pure cell death system in which NIH 3T3 cells expressing dimerizable Caspase-8 were co-cultured with peritoneal macrophages in a transwell system. Phenotype changes in macrophages induced by apoptotic cells were evaluated by RNA sequencing, which revealed an unexpectedly dominant impact on macrophage proliferation. This was confirmed in functional assays with primary peritoneal macrophages and IC-21 macrophages. Moreover, inhibition of apoptosis during Zymosan-induced peritonitis in mice decreased mRNA levels of cell cycle mediators in peritoneal macrophages. Proliferation of macrophages in response to apoptotic cells may be important to increase macrophage numbers in order to allow efficient clearance and resolution of inflammation.


Asunto(s)
Apoptosis , Proliferación Celular , Macrófagos Peritoneales/citología , Peritonitis/patología , Animales , Células Cultivadas , Técnicas de Cocultivo , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Peritonitis/inducido químicamente , Peritonitis/metabolismo , Fagocitosis , Zimosan/toxicidad
14.
Int J Mol Sci ; 22(4)2021 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-33673336

RESUMEN

The mammalian immune system senses foreign antigens by mechanisms that involve the interplay of various kinds of immune cells, culminating in inflammation resolution and tissue clearance. The ability of the immune cells to communicate (via chemokines) and to shift shape for migration, phagocytosis or antigen uptake is mainly supported by critical proteins such as aquaporins (AQPs) that regulate water fluid homeostasis and volume changes. AQPs are protein channels that facilitate water and small uncharged molecules' (such as glycerol or hydrogen peroxide) diffusion through membranes. A number of AQP isoforms were found upregulated in inflammatory conditions and are considered essential for the migration and survival of immune cells. The present review updates information on AQPs' involvement in immunity and inflammatory processes, highlighting their role as crucial players and promising targets for drug discovery.


Asunto(s)
Acuaporinas/inmunología , Movimiento Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Desarrollo de Medicamentos , Fagocitosis/efectos de los fármacos , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/inmunología , Movimiento Celular/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/patología
15.
Life Sci ; 273: 119150, 2021 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-33662426

RESUMEN

As a transmembrane protein, CD47 plays an important role in mediating cell proliferation, migration, phagocytosis, apoptosis, immune homeostasis, inhibition of NO signal transduction and other related reactions. Upon the interaction of innate immune checkpoint CD47-SIRPα occurrence, they send a "don't eat me" signal to the macrophages. This signal ultimately helps tumors achieve immune escape by inhibiting macrophage contraction to prevent tumor cells from phagocytosis. Therefore, the importance of CD47-SIRPα immune checkpoint inhibitors in tumor immunotherapy has attracted more attention in recent years. Based on the cognitive improvement of the effect with CD47 in tumor microenvironment and tumor characteristics, the pace of tumor treatment strategies for CD47-SIRPα immune checkpoint inhibitors has gradually accelerated. In this review, we introduced the high expression of CD47 in cancer cells to avoid phagocytosis by immune cells and the importance of CD47 in the structure of cancer microenvironment and the maintenance of cancer cell characteristics. Given the role of the innate immune system in tumorigenesis and development, an improved understanding of the anti-tumor process of innate immune checkpoint inhibitors can lay the foundation for more effective combinations with other anti-tumor treatment strategies.


Asunto(s)
Antígenos de Diferenciación/inmunología , Antígeno CD47/inmunología , Inmunidad Innata/inmunología , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Receptores Inmunológicos/inmunología , Animales , Humanos , Inmunoterapia/métodos , Macrófagos , Neoplasias/inmunología , Fagocitosis , Escape del Tumor , Microambiente Tumoral
16.
Nat Commun ; 12(1): 1704, 2021 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-33731716

RESUMEN

GPR37 was discovered more than two decades ago, but its biological functions remain poorly understood. Here we report a protective role of GPR37 in multiple models of infection and sepsis. Mice lacking Gpr37 exhibited increased death and/or hypothermia following challenge by lipopolysaccharide (LPS), Listeria bacteria, and the mouse malaria parasite Plasmodium berghei. Sepsis induced by LPS and Listeria in wild-type mice is protected by artesunate (ARU) and neuroprotectin D1 (NPD1), but the protective actions of these agents are lost in Gpr37-/- mice. Notably, we found that ARU binds to GPR37 in macrophages and promotes phagocytosis and clearance of pathogens. Moreover, ablation of macrophages potentiated infection, sepsis, and their sequelae, whereas adoptive transfer of NPD1- or ARU-primed macrophages reduced infection, sepsis, and pain-like behaviors. Our findings reveal physiological actions of ARU in host cells by activating macrophages and suggest that GPR37 agonists may help to treat sepsis, bacterial infections, and malaria.


Asunto(s)
Macrófagos/metabolismo , Dolor/prevención & control , Receptores Acoplados a Proteínas G/metabolismo , Sepsis/prevención & control , Traslado Adoptivo , Animales , Artesunato/metabolismo , Artesunato/farmacología , Artesunato/uso terapéutico , Modelos Animales de Enfermedad , Ácidos Docosahexaenoicos/metabolismo , Ácidos Docosahexaenoicos/farmacología , Ácidos Docosahexaenoicos/uso terapéutico , Lipopolisacáridos/toxicidad , Listeria monocytogenes/patogenicidad , Macrófagos/efectos de los fármacos , Macrófagos/patología , Macrófagos/trasplante , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Simulación del Acoplamiento Molecular , Dolor/inmunología , Dolor/mortalidad , Fagocitosis/efectos de los fármacos , Plasmodium berghei/patogenicidad , Receptores Acoplados a Proteínas G/deficiencia , Sepsis/inmunología , Sepsis/mortalidad , Sepsis/terapia
17.
Nat Commun ; 12(1): 1792, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741926

RESUMEN

In both sickle cell disease and malaria, red blood cells (RBCs) are phagocytosed in the spleen, but receptor-ligand pairs mediating uptake have not been identified. Here, we report that patches of high mannose N-glycans (Man5-9GlcNAc2), expressed on diseased or oxidized RBC surfaces, bind the mannose receptor (CD206) on phagocytes to mediate clearance. We find that extravascular hemolysis in sickle cell disease correlates with high mannose glycan levels on RBCs. Furthermore, Plasmodium falciparum-infected RBCs expose surface mannose N-glycans, which occur at significantly higher levels on infected RBCs from sickle cell trait subjects compared to those lacking hemoglobin S. The glycans are associated with high molecular weight complexes and protease-resistant, lower molecular weight fragments containing spectrin. Recognition of surface N-linked high mannose glycans as a response to cellular stress is a molecular mechanism common to both the pathogenesis of sickle cell disease and resistance to severe malaria in sickle cell trait.


Asunto(s)
Anemia de Células Falciformes/metabolismo , Eritrocitos/metabolismo , Manosa/metabolismo , Fagocitos/metabolismo , Polisacáridos/metabolismo , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/parasitología , Eritrocitos/parasitología , Citometría de Flujo/métodos , Hemólisis , Humanos , Ligandos , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Glicoproteínas de Membrana/metabolismo , Fagocitosis , Plasmodium falciparum/fisiología , Unión Proteica , Receptores Inmunológicos/metabolismo
18.
Nat Rev Mol Cell Biol ; 22(4): 242-243, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33594279
19.
Nature ; 590(7847): 618-623, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33568811

RESUMEN

Errors in early embryogenesis are a cause of sporadic cell death and developmental failure1,2. Phagocytic activity has a central role in scavenging apoptotic cells in differentiated tissues3-6. However, how apoptotic cells are cleared in the blastula embryo in the absence of specialized immune cells remains unknown. Here we show that the surface epithelium of zebrafish and mouse embryos, which is the first tissue formed during vertebrate development, performs efficient phagocytic clearance of apoptotic cells through phosphatidylserine-mediated target recognition. Quantitative four-dimensional in vivo imaging analyses reveal a collective epithelial clearance mechanism that is based on mechanical cooperation by two types of Rac1-dependent basal epithelial protrusions. The first type of protrusion, phagocytic cups, mediates apoptotic target uptake. The second, a previously undescribed type of fast and extended actin-based protrusion that we call 'epithelial arms', promotes the rapid dispersal of apoptotic targets through Arp2/3-dependent mechanical pushing. On the basis of experimental data and modelling, we show that mechanical load-sharing enables the long-range cooperative uptake of apoptotic cells by multiple epithelial cells. This optimizes the efficiency of tissue clearance by extending the limited spatial exploration range and local uptake capacity of non-motile epithelial cells. Our findings show that epithelial tissue clearance facilitates error correction that is relevant to the developmental robustness and survival of the embryo, revealing the presence of an innate immune function in the earliest stages of embryonic development.


Asunto(s)
Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Desarrollo Embrionario , Células Epiteliales/citología , Fagocitos/citología , Fagocitosis , Pez Cebra/embriología , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Animales , Apoptosis , Movimiento Celular , Forma de la Célula , Extensiones de la Superficie Celular , Inmunidad Innata , Ratones , Fosfatidilserinas/metabolismo , Proteína de Unión al GTP rac1/metabolismo
20.
Nat Commun ; 12(1): 1158, 2021 02 24.
Artículo en Inglés | MEDLINE | ID: mdl-33627648

RESUMEN

Niemann-Pick type C disease is a rare neurodegenerative disorder mainly caused by mutations in NPC1, resulting in abnormal late endosomal/lysosomal lipid storage. Although microgliosis is a prominent pathological feature, direct consequences of NPC1 loss on microglial function remain not fully characterized. We discovered pathological proteomic signatures and phenotypes in NPC1-deficient murine models and demonstrate a cell autonomous function of NPC1 in microglia. Loss of NPC1 triggers enhanced phagocytic uptake and impaired myelin turnover in microglia that precede neuronal death. Npc1-/- microglia feature a striking accumulation of multivesicular bodies and impaired trafficking of lipids to lysosomes while lysosomal degradation function remains preserved. Molecular and functional defects were also detected in blood-derived macrophages of NPC patients that provide a potential tool for monitoring disease. Our study underscores an essential cell autonomous role for NPC1 in immune cells and implies microglial therapeutic potential.


Asunto(s)
Colesterol/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microglía/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismo , Animales , Western Blotting , Células Cultivadas , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Vaina de Mielina/metabolismo , Enfermedad de Niemann-Pick Tipo C/genética , Fagocitosis/genética , Fagocitosis/fisiología , Proteómica/métodos
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