RESUMO
A Disintegrin And Metalloproteinase domain-containing protein 10 (ADAM10), is involved in several metabolic and inflammatory pathways. We speculated that ADAM10 plays a modulatory role in adipose tissue inflammation and metabolism. To this end, we studied adipose tissue-specific ADAM10 knock-out mice (aKO). While young, regular chow diet-fed aKO mice showed increased insulin sensitivity, following prolonged (33 weeks) high-fat diet (HFD) exposure, aKO mice developed obesity and insulin resistance. Compared to controls, aKO mice showed less inflammatory adipokine profile despite the significant increase in adiposity. In brown adipose tissue, aKO mice on HFD had changes in CD8+ T cell populations indicating a lesser inflammatory pattern. Following HFD, both aKO and control littermates demonstrated decreased adipose tissue pro-inflammatory macrophages, and increased anti-inflammatory accumulation, without differences between the genotypes. Collectively, our observations indicate that selective deletion of ADAM10 in adipocytes results in a mitigated inflammatory response, leading to increased insulin sensitivity in young mice fed with regular diet. This state of insulin sensitivity, following prolonged HFD, facilitates energy storage resulting in increased fat accumulation which ultimately leads to the development of a phenotype of obesity and insulin resistance. In conclusion, the data indicate that ADAM10 has a modulatory effect of inflammation and whole-body energy metabolism.
Assuntos
Proteína ADAM10 , Tecido Adiposo , Dieta Hiperlipídica , Camundongos Knockout , Animais , Masculino , Camundongos , Proteína ADAM10/metabolismo , Proteína ADAM10/genética , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Dieta Hiperlipídica/efeitos adversos , Inflamação/metabolismo , Resistência à Insulina , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Obesidade/metabolismo , Obesidade/etiologia , FenótipoRESUMO
There is a clinical need for new treatment options addressing allergic disease. Selective serotonin reuptake inhibitors (SSRIs) are a class of antidepressants that have anti-inflammatory properties. We tested the effects of the SSRI fluoxetine on IgE-induced function of mast cells, which are critical effectors of allergic inflammation. We showed that fluoxetine treatment of murine or human mast cells reduced IgE-mediated degranulation, cytokine production, and inflammatory lipid secretion, as well as signaling mediated by the mast cell activator ATP. In a mouse model of systemic anaphylaxis, fluoxetine reduced hypothermia and cytokine production. Fluoxetine was also effective in a model of allergic airway inflammation, where it reduced bronchial responsiveness and inflammation. These data show that fluoxetine suppresses mast cell activation by impeding an FcÉRI-ATP positive feedback loop and support the potential repurposing of this SSRI for use in allergic disease.
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Fluoxetina , Mastócitos , Humanos , Animais , Camundongos , Fluoxetina/farmacologia , Retroalimentação , Inflamação/tratamento farmacológico , Citocinas , Trifosfato de Adenosina , Imunoglobulina ERESUMO
Physiochemical cues like topography and wettability can impact the inflammatory response and tissue integration after biomaterial implantation. T cells are essential for immunomodulation of innate immune cells and play an important role in the host response to biomaterial implantation. This study aimed to understand how CD4+ and CD8+ T cell subsets, members of the αß T cell family, polarize in response to smooth, rough, or rough-hydrophilic titanium (Ti) implants and whether their presence modulates immune cell crosstalk and mesenchymal stem cell (MSC) recruitment following biomaterial implantation. Post-implantation in mice, we found that CD4+ and CD8+ T cell subsets polarized differentially in response to modified Ti surfaces. Additionally, mice lacking αß T cells had significantly more pro-inflammatory macrophages, fewer anti-inflammatory macrophages, and reduced MSC recruitment in response to modified Ti post-implantation than αß T cell -competent mice. Our results demonstrate that T cell activation plays a significant role during the inflammatory response to implanted biomaterials, contributing to macrophage polarization and MSC recruitment and proliferation, and the absence of αß T cells compromises new bone formation at the implantation site. STATEMENT OF SIGNIFICANCE: T cells are essential for immunomodulation and play an important role in the host response to biomaterial implantation. Our results demonstrate that T cells actively participate during the inflammatory response to implanted biomaterials, controlling macrophage phenotype and recruitment of MSCs to the implantation site.
Assuntos
Células-Tronco Mesenquimais , Titânio , Camundongos , Animais , Titânio/farmacologia , Materiais Biocompatíveis/metabolismo , Macrófagos/metabolismo , Linfócitos T , Proliferação de CélulasRESUMO
IgE-mediated mast cell activation is a driving force in allergic disease in need of novel interventions. Statins, long used to lower serum cholesterol, have been shown in multiple large-cohort studies to reduce asthma severity. We previously found that statins inhibit IgE-induced mast cell function, but these effects varied widely among mouse strains and human donors, likely due to the upregulation of the statin target, 3-hydroxy-3-methylgutaryl-CoA reductase. Statin inhibition of mast cell function appeared to be mediated not by cholesterol reduction but by suppressing protein isoprenylation events that use cholesterol pathway intermediates. Therefore, we sought to circumvent statin resistance by targeting isoprenylation. Using genetic depletion of the isoprenylation enzymes farnesyltransferase and geranylgeranyl transferase 1 or their substrate K-Ras, we show a significant reduction in FcεRI-mediated degranulation and cytokine production. Furthermore, similar effects were observed with pharmacological inhibition with the dual farnesyltransferase and geranylgeranyl transferase 1 inhibitor FGTI-2734. Our data indicate that both transferases must be inhibited to reduce mast cell function and that K-Ras is a critical isoprenylation target. Importantly, FGTI-2734 was effective in vivo, suppressing mast cell-dependent anaphylaxis, allergic pulmonary inflammation, and airway hyperresponsiveness. Collectively, these findings suggest that K-Ras is among the isoprenylation substrates critical for FcεRI-induced mast cell function and reveal isoprenylation as a new means of targeting allergic disease.
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Anafilaxia , Inibidores de Hidroximetilglutaril-CoA Redutases , Camundongos , Humanos , Animais , Receptores de IgE/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Farnesiltranstransferase/metabolismo , Mastócitos/metabolismo , Anafilaxia/metabolismo , Transdução de Sinais , Degranulação Celular , Imunoglobulina E/metabolismo , Inflamação/metabolismo , Colesterol/metabolismo , PrenilaçãoAssuntos
Asma , Humanos , Animais , Camundongos , Modelos Animais de Doenças , Glutaratos , AlérgenosRESUMO
Patients with cystic fibrosis (CF) have decreased severity of severe acute respiratory syndrome-like coronavirus-2 (SARS-CoV-2) infections, but the underlying cause is unknown. Patients with CF have high levels of neutrophil elastase (NE) in the airway. We examined whether respiratory epithelial angiotensin-converting enzyme 2 (ACE-2), the receptor for the SARS-CoV-2 spike protein, is a proteolytic target of NE. Soluble ACE-2 levels were quantified by ELISA in airway secretions and serum from patients with and without CF, the association between soluble ACE-2 and NE activity levels was evaluated in CF sputum. We determined that NE activity was directly correlated with increased ACE-2 in CF sputum. Additionally, primary human bronchial epithelial (HBE) cells, exposed to NE or control vehicle, were evaluated by Western analysis for the release of cleaved ACE-2 ectodomain fragment into conditioned media, flow cytometry for the loss of cell surface ACE-2, its impact on SARS-CoV-2 spike protein binding. We found that NE treatment released ACE-2 ectodomain fragment from HBE and decreased spike protein binding to HBE. Furthermore, we performed NE treatment of recombinant ACE-2-Fc-tagged protein in vitro to assess whether NE was sufficient to cleave recombinant ACE-2-Fc protein. Proteomic analysis identified specific NE cleavage sites in the ACE-2 ectodomain that would result in loss of the putative N-terminal spike-binding domain. Collectively, data support that NE plays a disruptive role in SARS-CoV-2 infection by catalyzing ACE-2 ectodomain shedding from the airway epithelia. This mechanism may reduce SARS-CoV-2 virus binding to respiratory epithelial cells and decrease the severity of COVID19 infection.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Fibrose Cística , Elastase de Leucócito , Humanos , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , Fibrose Cística/metabolismo , Elastase de Leucócito/metabolismo , Ligação Proteica , Proteômica , Mucosa Respiratória/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Neutrophils are the most abundant immune cells in the blood and the first cells to be recruited to the biomaterial implantation site. Neutrophils are fundamental in recruiting mononuclear leukocytes to mount an immune response at the injury site. Neutrophils exert significant pro-inflammatory effects through the release of cytokines and chemokines, degranulation and release of myeloperoxidase (MPO) and neutrophil elastase (NE), and the production of large DNA-based networks called neutrophil extracellular traps (NETs). Neutrophils are initially recruited and activated by cytokines and pathogen- and damage-associated molecular patterns, but little is known about how the physicochemical composition of the biomaterial affects their activation. This study aimed to understand how ablating neutrophil mediators (MPO, NE, NETs) affected macrophage phenotype in vitro and osseointegration in vivo. We discovered that NET formation is a crucial mediator of pro-inflammatory macrophage activation, and inhibition of NET formation significantly suppresses macrophage pro-inflammatory phenotype. Furthermore, reducing NET formation accelerated the inflammatory phase of healing and produced greater bone formation around the implanted biomaterial, suggesting that NETs are essential regulators of biomaterial integration. Our findings emphasize the importance of the neutrophil response to implanted biomaterials and highlight innate immune cells' regulation and amplification signaling during the initiation and resolution of the inflammatory phase of biomaterial integration. STATEMENT OF SIGNIFICANCE: Neutrophils are the most abundant immune cells in blood and are the first to be recruited to the injury/implantation site where they exert significant pro-inflammatory effects. This study aimed to understand how ablating neutrophil mediators affected macrophage phenotype in vitro and bone apposition in vivo. We found that NET formation is a crucial mediator of pro-inflammatory macrophage activation. Reducing NET formation accelerated the inflammatory phase of healing and produced greater appositional bone formation around the implanted biomaterial, suggesting that NETs are essential regulators of biomaterial integration.
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Armadilhas Extracelulares , Titânio/farmacologia , Osteogênese , Neutrófilos , Citocinas/farmacologia , Materiais Biocompatíveis/farmacologiaRESUMO
Materials for craniofacial and orthopedic implants are commonly selected based on mechanical properties and corrosion resistance. The biocompatibility of these materials is typically assessed in vitro using cell lines, but little is known about the response of immune cells to these materials. This study aimed to evaluate the inflammatory and immune cell response to four common orthopedic materials [pure titanium (Ti), titanium alloy (TiAlV), 316L stainless steel (SS), polyetheretherketone (PEEK)]. Following implantation into mice, we found high recruitment of neutrophils, pro-inflammatory macrophages, and CD4+ T cells in response to PEEK and SS implants. Neutrophils produced higher levels of neutrophil elastase, myeloperoxidase, and neutrophil extracellular traps in vitro in response to PEEK and SS than neutrophils on Ti or TiAlV. Macrophages co-cultured on PEEK, SS, or TiAlV increased polarization of T cells towards Th1/Th17 subsets and decreased Th2/Treg polarization compared to Ti substrates. Although SS and PEEK are considered biocompatible materials, both induce a more robust inflammatory response than Ti or Ti alloy characterized by high infiltration of neutrophils and T cells, which may cause fibrous encapsulation of these materials. STATEMENT OF SIGNIFICANCE: Materials for craniofacial and orthopedic implants are commonly selected based on their mechanical properties and corrosion resistance. This study aimed to evaluate the immune cell response to four common orthopedic and craniofacial biomaterials: pure titanium, titanium-aluminum-vanadium alloy, 316L stainless steel, and PEEK. Our results demonstrate that while the biomaterials tested have been shown to be biocompatible and clinically successful, the inflammatory response is largely driven by chemical composition of the biomaterials.
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Materiais Biocompatíveis , Titânio , Animais , Camundongos , Materiais Biocompatíveis/farmacologia , Materiais Biocompatíveis/química , Titânio/farmacologia , Titânio/química , Aço Inoxidável/química , Polímeros/farmacologia , Polietilenoglicóis/farmacologia , Polietilenoglicóis/química , Cetonas/farmacologia , Cetonas/química , Ligas/farmacologia , Teste de Materiais , Propriedades de SuperfícieRESUMO
Genome-wide association studies have linked the ORM (yeast)-like protein isoform 3 (ORMDL3) to asthma severity. Although ORMDL3 is a member of a family that negatively regulates serine palmitoyltransferase (SPT) and thus biosynthesis of sphingolipids, it is still unclear whether ORMDL3 and altered sphingolipid synthesis are causally related to non-Th2 severe asthma associated with a predominant neutrophil inflammation and high interleukin-17 (IL-17) levels. Here, we examined the effects of ORMDL3 overexpression in a preclinical mouse model of allergic lung inflammation that is predominantly neutrophilic and recapitulates many of the clinical features of severe human asthma. ORMDL3 overexpression reduced lung and circulating levels of dihydrosphingosine, the product of SPT. However, the most prominent effect on sphingolipid levels was reduction of circulating S1P. The LPS/OVA challenge increased markers of Th17 inflammation with a predominant infiltration of neutrophils into the lung. A significant decrease of neutrophil infiltration was observed in the Ormdl3 transgenic mice challenged with LPS/OVA compared to the wild type and concomitant decrease in IL-17, that plays a key role in the pathogenesis of neutrophilic asthma. LPS decreased survival of murine neutrophils, which was prevented by co-treatment with S1P. Moreover, S1P potentiated LPS-induced chemotaxis of neutrophil, suggesting that S1P can regulate neutrophil survival and recruitment following LPS airway inflammation. Our findings reveal a novel connection between ORMDL3 overexpression, circulating levels of S1P, IL-17 and neutrophil recruitment into the lung, and questions the potential involvement of ORMDL3 in the pathology, leading to development of severe neutrophilic asthma.
Assuntos
Asma , Interleucina-17 , Proteínas de Membrana , Animais , Humanos , Camundongos , Asma/metabolismo , Estudo de Associação Genômica Ampla , Inflamação/metabolismo , Interleucina-17/genética , Interleucina-17/uso terapêutico , Lipopolissacarídeos , Proteínas de Membrana/metabolismo , Camundongos Transgênicos , Esfingolipídeos/metabolismoRESUMO
BACKGROUND: Immune activation, neuroinflammation, and cell death are the hallmarks of multiple sclerosis (MS), which is an autoimmune demyelinating disease of the central nervous system (CNS). It is well-documented that the cellular inhibitor of apoptosis 2 (cIAP2) is induced by inflammatory stimuli and regulates adaptive and innate immune responses, cell death, and the production of inflammatory mediators. However, the impact of cIAP2 on neuroinflammation associated with MS and disease severity remains unknown. METHODS: We used experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of MS, to assess the effect of cIAP2 deletion on disease outcomes. We performed a detailed analysis on the histological, cellular, and molecular levels. We generated and examined bone-marrow chimeras to identify the cIAP2-deficient cells that are critical to the disease outcomes. RESULTS: cIAP2-/- mice exhibited increased EAE severity, increased CD4+ T cell infiltration, enhanced proinflammatory cytokine/chemokine expression, and augmented demyelination. This phenotype was driven by cIAP2-deficient non-hematopoietic cells. cIAP2 protected oligodendrocytes from cell death during EAE by limiting proliferation and activation of brain microglia. This protective role was likely exerted by cIAP2-mediated inhibition of the non-canonical NLRP3/caspase-8-dependent myeloid cell activation during EAE. CONCLUSIONS: Our findings suggest that cIAP2 is needed to modulate neuroinflammation, cell death, and survival during EAE. Significantly, our data demonstrate the critical role of cIAP2 in limiting the activation of microglia during EAE, which could be explored for developing MS therapeutics in the future.
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Encefalomielite Autoimune Experimental , Esclerose Múltipla , Animais , Proteína 3 com Repetições IAP de Baculovírus/genética , Proteína 3 com Repetições IAP de Baculovírus/metabolismo , Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Esclerose Múltipla/patologia , Doenças NeuroinflamatóriasRESUMO
Non-alcoholic steatohepatitis (NASH) is linked to adipose tissue dysfunction, with weight loss being the only treatment shown to reverse it. Due to this correlation with obesity, the study of adipose tissue and adipocytes is an important step in understanding the pathogenesis of this disease. Here, we describe the isolation process of the stromal vascular fraction (SVF) of adipose tissue. The SVF contains the foundational cells that will differentiate into adipocytes. These cells can be isolated and subsequently differentiated in vitro into white and beige adipocytes. We outline the in vitro differentiation of pre-adipocytes into cultured white and beige adipocytes using both human and mouse adipose tissue.
Assuntos
Adipócitos Bege , Adipócitos , Adipócitos Brancos , Tecido Adiposo , Animais , Diferenciação Celular , Camundongos , Fração Vascular EstromalRESUMO
Obesity is an enormous global health problem, and obesity-induced nonalcoholic steatohepatitis (NASH) is contributing to a rising incidence and mortality for hepatocellular carcinoma (HCC). Increase in de novo lipogenesis and decrease in fatty acid ß-oxidation (FAO) underlie hepatic lipid accumulation in NASH. Astrocyte-elevated gene-1/metadherin (AEG-1) overexpression contributes to both NASH and HCC. AEG-1 harbors an LXXLL motif through which it blocks activation of peroxisome proliferator activated receptor α (PPARα), a key regulator of FAO. To better understand the role of LXXLL motif in mediating AEG-1 function, using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, we generated a mouse model (AEG-1-L24K/L25H) in which the LXXLL motif in AEG-1 was mutated to LXXKH. We observed increased activation of PPARα in AEG-1-L24K/L25H livers providing partial protection from high-fat diet-induced steatosis. Interestingly, even with equal gene dosage levels, compared with AEG-1-wild-type livers, AEG-1-L24K/L25H livers exhibited increase in levels of lipogenic enzymes, mitogenic activity and inflammation, which are attributes observed when AEG-1 is overexpressed. These findings indicate that while LXXLL motif favors steatotic activity of AEG-1, it keeps in check inflammatory and oncogenic functions, thus maintaining a homeostasis in AEG-1 function. AEG-1 is being increasingly appreciated as a viable target for ameliorating NASH and NASH-HCC, and as such, in-depth understanding of the functions and molecular attributes of this molecule is essential. Conclusion: The present study unravels the unique role of the LXXLL motif in mediating the balance between the metabolic and oncogenic functions of AEG-1.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas de Membrana , Hepatopatia Gordurosa não Alcoólica , Proteínas de Ligação a RNA , Animais , Astrócitos/metabolismo , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas de Membrana/genética , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Obesidade/genética , PPAR alfa/genética , Proteínas de Ligação a RNA/genética , Fatores de TranscriçãoRESUMO
SARS-CoV-2, the virus responsible for COVID-19, emerged in late 2019 and has since spread throughout the world, infecting over 200 million people. The fast spread of SARS-CoV-2 showcased the need for rapid and sensitive testing methodologies to help track the disease. Over the past 18 months, numerous SARS-CoV-2 variants have emerged. Many of these variants are suggested to be more transmissible as well as less responsive to neutralization by vaccine-induced antibodies. Viral whole-genome sequencing is the current standard for tracking these variants. However, whole-genome sequencing is costly and the technology and expertise are limited to larger reference laboratories. Here, we present the feasibility of a fast, inexpensive methodology using snapback primer-based high-resolution melting to test for >20 high-consequence SARS-CoV-2 spike mutations. This assay can distinguish between multiple variant lineages and be completed in roughly 2 h for less than $10 per sample.
RESUMO
Since the start of the coronavirus disease 2019 (COVID-19) pandemic, molecular diagnostic testing for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has faced substantial supply chain shortages and noteworthy delays in result reporting after sample collection. Supply chain shortages have been most evident in reagents for RNA extraction and rapid diagnostic testing. This study explored the kinetic limitations of extraction-free rapid cycle quantitative real-time RT-PCR for SARS-CoV-2 virus detection using the commercially available capillary-based LightCycler. After optimizing for time and reaction conditions, a protocol for sensitive and specific quantitative RT-PCR of SARS-CoV-2 RNA from nasopharyngeal swabs in <20 minutes was developed, with minimal hands-on time requirements. This protocol improves detection speed while maintaining the sensitivity and specificity of hydrolysis probe-based detection. Percentage agreement between the developed assay and previously tested positive patient samples was 97.6% (n = 40/41), and negative patient samples was 100% (40/40). The study further demonstrates that using purified RNA, SARS-CoV-2 testing using extreme RT-PCR, and product verification by melting can be completed in <3 minutes. Overall, these studies provide a framework for increasing the speed of SARS-CoV-2 and other infectious disease testing.
Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , Humanos , Técnicas de Diagnóstico Molecular/métodos , Pandemias/prevenção & controle , RNA Viral/genética , Sensibilidade e Especificidade , Manejo de Espécimes/métodosRESUMO
Mast cells are found primarily at interfaces with the external environment, where they provide protection from pathogens but also elicit allergic inflammation. Mast cell activation by antigen-induced aggregation of IgE bound to the high affinity receptor, FcεRI, is a critical factor leading to inflammation and bronchoconstriction. We previously found that Stat5 is activated by FcεRI and that Stat5B suppression decreased IgE-induced cytokine production in vitro, but in vivo responses have not been assessed. We now show that Stat5B-deficient (KO) mice have reduced responses to IgE-mediated anaphylaxis, despite normal mast cell tissue distribution. Similarly, Stat5B KO mast cells have diminished IgE-induced degranulation and cytokine secretion in vitro. These mice have elevated IgE production that is not correlated with an intrinsic B cell defect. The current work demonstrates that the Stat5B isoform is required for normal mast cell function and suggests it limits IgE production in vivo.
Assuntos
Anafilaxia/imunologia , Linfócitos B/imunologia , Hipersensibilidade/imunologia , Imunoglobulina E/metabolismo , Mastócitos/imunologia , Receptores de IgE/metabolismo , Fator de Transcrição STAT5/metabolismo , Animais , Degranulação Celular , Células Cultivadas , Citocinas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fator de Transcrição STAT5/genéticaRESUMO
The Adipoq-Cre transgenic mouse is widely used in the development of adipocyte-specific genetic manipulations for the study of obesity and type 2 diabetes. In the process of developing a new mouse model utilizing the adipocyte selective Adipoq-Cre transgenic mouse, strong genetic linkage between a gene of interest, Adam10, and the Adipoq-Cre transgene was discovered. Whole-genome sequencing of the Adipoq-Cre transgenic mouse model identified the genomic insertion site within the Tbx18 gene locus on chromosome 9 and this insertion causes a significant decrease in Tbx18 gene expression in adipose tissue. Insertion of genes Kng2, Kng1, Eif4a2 and Rfc4 also occurred in the Adipoq-Cre transgenic mouse, and these passenger genes may have functional consequences in various tissues.
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Adiponectina/genética , Transgenes/genética , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Expressão Gênica/genética , Integrases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Obesidade/metabolismo , Especificidade de Órgãos/genética , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismoRESUMO
BACKGROUND: Nothing is known about the mechanisms by which increased ceramide levels in the lung contribute to allergic responses and asthma severity. OBJECTIVE: We sought to investigate the functional role of ceramide in mouse models of allergic airway disease that recapitulate the cardinal clinical features of human allergic asthma. METHODS: Allergic airway disease was induced in mice by repeated intranasal administration of house dust mite or the fungal allergen Alternaria alternata. Processes that can be regulated by ceramide and are important for severity of allergic asthma were correlated with ceramide levels measured by mass spectrometry. RESULTS: Both allergens induced massive pulmonary apoptosis and also significantly increased reactive oxygen species in the lung. Prevention of increases in lung ceramide levels mitigated allergen-induced apoptosis, reactive oxygen species, and neutrophil infiltration. In contrast, dietary supplementation of the antioxidant α-tocopherol decreased reactive oxygen species but had no significant effects on elevation of ceramide level or apoptosis, indicating that the increases in lung ceramide levels in allergen-challenged mice are not mediated by oxidative stress. Moreover, specific ceramide species were altered in bronchoalveolar lavage fluid from patients with severe asthma compared with in bronchoalveolar lavage fluid from individuals without asthma. CONCLUSION: Our data suggest that elevation of ceramide level after allergen challenge contributes to the apoptosis, reactive oxygen species generation, and neutrophilic infiltrate that characterize the severe asthmatic phenotype. Ceramide might be the trigger of formation of Creola bodies found in the sputum of patients with severe asthma and could be a biomarker to optimize diagnosis and to monitor and improve clinical outcomes in this disease.
Assuntos
Asma/imunologia , Ceramidas/imunologia , Pulmão/imunologia , Estresse Oxidativo , Adulto , Alérgenos/imunologia , Alternaria/imunologia , Animais , Apoptose , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/imunologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Pyroglyphidae/imunologia , Espécies Reativas de Oxigênio/imunologia , Adulto JovemRESUMO
The role of the inducible costimulatory of T cells (ICOS) has been shown to be important for many different T cell outcomes and is indispensable for follicular helper T cell (TFH) responses. Since its discovery, there have been several studies on the regulation of ICOS at a transcriptional level. However, the post-translational regulation of ICOS has not been well characterized. Here, we demonstrate that ICOS is internalized following ligation. We show that costimulation with CD3 results in differential internalization and fate than stimulation of ICOS alone. Additionally, we show that ICOS internalization is PI3K and clathrin mediated. The studies presented here not only increase the mechanistic understanding of ICOS post-translational regulation but also give insight into the potential mechanisms by which T cells expressing high affinity receptors may be preferentially chosen to become TFH cells with increased ICOS levels.
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The role of ADAM17, its substrates, and its natural inhibitor has been well studied in the context of inflammation, including metabolic inflammation, with mixed results. Previous studies examining global Adam17 knockdown models and ADAM17 inhibition using overexpression of endogenous ADAM17 inhibitors have shown improved metabolic health and decreased metabolic inflammation. However, there have been no studies examining the role of adipocyte ADAM17 using in vivo models. In this study, we developed an adipocyte-specific Adam17 knockout model using Adipoq-Cre-expressing mice crossed with Adam17-floxed mice. Using this model, we show that loss of adipocyte ADAM17 plays no evident role in baseline metabolic responses. Surprisingly, in a state of metabolic stress using high-fat diet (HFD), we observed that adipocyte ADAM17 had little effect overall on the metabolic phenotype as well as inflammatory cell populations. Using whole-body metabolic phenotyping, we show that loss of ADAM17 has no effect on energy utilization both at a baseline state as well as following HFD. However, lastly, using high-parameter flow cytometry, we show that loss of adipocyte ADAM17 alters macrophage and eosinophil populations following HFD. Overall, the studies presented here give more insight into the role of ADAM17 in metabolic responses and metabolic inflammation, specifically in adipocytes.