RESUMO
BACKGROUND: Allergic inflammation affects the epithelial cell populations resulting in goblet cell hyperplasia and decreased ciliated cells. Recent advances in single-cell RNA sequencing (scRNAseq) have enabled the identification of new cell subtypes and genomic features of single cells. In this study, we aimed to investigate the effect of allergic inflammation in nasal epithelial cell transcriptomes at the single-cell level. METHODS: We performed scRNAseq in cultured primary human nasal epithelial (HNE) cells and in vivo nasal epithelium. The transcriptomic features and epithelial cell subtypes were determined under IL-4 stimulation, and cell-specific marker genes and proteins were identified. RESULTS: We confirmed that cultured HNE cells were similar to in vivo epithelial cells through scRNAseq. Cell-specific marker genes were utilized to cluster the cell subtypes, and FOXJ1+ -ciliated cells were sub-classified into multiciliated and deuterosomal cells. PLK4 and CDC20B were specific for deuterosomal cells, and SNTN, CPASL, and GSTA2 were specific for multiciliated cells. IL-4 altered the proportions of cell subtypes, resulting in a decrease in multiciliated cells and loss of deuterosomal cells. The trajectory analysis revealed deuterosomal cells as precursor cells of multiciliated cells and deuterosomal cells function as a bridge between club and multiciliated cells. A decrease in deuterosomal cell marker genes was observed in nasal tissue samples with type 2 inflammation. CONCLUSION: The effects of IL-4 appear to be mediated through the loss of the deuterosomal population, resulting in the reduction in multiciliated cells. This study also newly suggests cell-specific markers that might be pivotal for investigating respiratory inflammatory diseases.
Assuntos
Células Epiteliais , Interleucina-4 , Humanos , Diferenciação Celular/genética , Células Cultivadas , Células Epiteliais/metabolismo , Inflamação/metabolismo , Interleucina-4/metabolismo , Mucosa Nasal , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Allergic rhinitis (AR) is a multifactorial airway disease characterized by basal and goblet cell hyperplasia. Hyaluronic acid (HA) is a major component of extracellular matrix and a critical contributor to tissue repair and remodeling after injury. We previously demonstrated that the intermediate progenitor cell (IPC) surface marker CD44v3 is upregulated in the basal and suprabasal layers of well-differentiated primary human nasal epithelial (HNE) cells after stimulation with the Th2 (T-helper cell type 2) cytokine IL-4, and an antibody blocking the CD44v3-HA interaction suppressed IL-4-induced goblet cell hyperplasia. We now show that the expression of HA and two HA synthases, HAS2 and HAS3, was upregulated in both the nasal surface epithelium of subjects with AR and IL-4-stimulated HNE cells. Inhibition of HA synthesis by 4-methylumbelliferone suppressed IL-4-induced goblet cell hyperplasia. Moreover, HAS2 and HAS3 were expressed in IPCs depending on the differentiation events, as follows: the rapid, transient upregulation of HAS2 induced basal IPC proliferation and basal-to-suprabasal transition, whereas the delayed upregulation of HAS3 promoted the transition of suprabasal IPCs to a goblet cell fate. 4-methylumbelliferone treatment in a house dust mite-induced murine AR model attenuated goblet cell metaplasia. Last, HA concentrations in nasal epithelial lining fluids from patients with AR positively correlated with the concentrations of mediators causing allergic inflammation. These data suggest that HA produced after the sequential upregulation of HAS2 and HAS3 contributes to goblet cell hyperplasia in allergic airway inflammation and modulates disease progression.
Assuntos
Células Caliciformes , Hialuronan Sintases , Rinite Alérgica , Animais , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/enzimologia , Células Caliciformes/patologia , Humanos , Hialuronan Sintases/metabolismo , Ácido Hialurônico/metabolismo , Himecromona/farmacologia , Himecromona/uso terapêutico , Hiperplasia/genética , Hiperplasia/patologia , Interleucina-4/metabolismo , Camundongos , Rinite Alérgica/tratamento farmacológico , Rinite Alérgica/enzimologia , Rinite Alérgica/patologiaRESUMO
Microfluidic-based biosensors have been developed for their precise automatic reaction control. However, these biosensors require external devices that are difficult to transport and use. To overcome this disadvantage, our group made an easy-to-use, cheap, and light pumpless three-dimensional photo paper-based microfluidic analytical device (3D-µPAD; weight: 1.5 g). Unlike conventional paper-based microfluidic analytical devices, the 3D-µPAD can be used to control fluid flow in a 3D manner, thus allowing sophisticated multi-step reaction control. This device can control fluid flow speed and direction accurately using only the capillary-driven flow without an external device like a pump. The flow speed is controlled by the width of the microfluidic channel and its surface property. In addition, fluid speed control and 3D-bridge structure enable the control of fluid flow direction. Using these methods, multi-step enzyme-linked immunosorbent assay (ELISA) can be done automatically in sequence by injecting solutions (sample, washing, and enzyme's substrate) at the same time in the 3D-µPAD. All the steps can be performed in 14 min, and data can be analyzed immediately. To test this device, thioredoxin-1 (Trx-1), a biomarker of breast cancer, is used as the target. In the 3D-µPAD, it can detect 0-200 ng/mL of Trx-1, and the prepared 3D-µPAD Trx-1 sensor displays excellent selectivity. Moreover, by analyzing the concentration of Trx-1 in real patients and healthy individuals' blood serum samples using the 3D-µPAD, and comparing results to ELISA, it can be confirmed that the 3D-µPAD is a good tool for cancer diagnosis.
Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Ensaio de Imunoadsorção Enzimática , Humanos , Papel , TiorredoxinasRESUMO
BACKGROUND: Arginine methylation is a posttranslational modification mediated by protein arginine methyltransferases (PRMTs). Although previous studies have shown that PRMT1 contributes to the severity of allergic airway inflammation or asthma, the underlying mechanism is poorly understood. OBJECTIVE: This study aimed to explore the role of PRMT1 and its relevant mechanism in the development of allergic rhinitis (AR). METHODS: The expression levels of PRMTs and cytokines were determined by RT-PCR, and the localization of PRMT1 was determined by immunohistochemistry and confocal microscopy. The levels of house dust mite (HDM)-specific immunoglobulins in serum and of cytokines in nasal lavage fluids were determined by ELISA. PRMT1 inhibition was achieved by siRNA and treatment with the pan PRMT inhibitor arginine N-methyltransferase inhibitor-1. RESULTS: PRMT1 expression was significantly increased in the nasal mucosa of patients and mice with AR. The degree of eosinophilic infiltration in the nasal mucosa was reduced in PRMT1+/- AR mice compared with wild-type mice. PRMT1 haploinsufficiency reduced the levels of HDM-specific immunoglobulins in serum and those of TH2 (IL-4, IL-5, and IL-13) and epithelial (thymic stromal lymphopoietin [TSLP], IL-25, and IL-33) cytokines in the nasal lavage fluids of AR mice. In nasal epithelial cells, HDM and IL-4 cooperate to enhance PRMT1 expression through a mitogen-activated protein kinase-dependent pathway. In addition, PRMT1 was essential for the production of TSLP, IL-25, and IL-33 in response to HDM and IL-4. Arginine N-methyltransferase inhibitor-1 treatment alleviated AR in the mouse model. CONCLUSIONS: PRMT1 plays an important role in AR development by regulating epithelial-derived cytokine production and might be a new therapeutic target for AR.
Assuntos
Citocinas/imunologia , Células Epiteliais/imunologia , Proteína-Arginina N-Metiltransferases/imunologia , Proteínas Repressoras/imunologia , Rinite Alérgica/imunologia , Alérgenos/imunologia , Animais , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Líquido da Lavagem Nasal/imunologia , Mucosa Nasal/imunologia , Proteína-Arginina N-Metiltransferases/genética , Pyroglyphidae/imunologiaRESUMO
In allergic airway diseases, intermediate progenitor cells (IPCs) increase in number in the surface epithelium. IPCs arise from basal cells, the origin of hallmark pathological changes, including goblet cell hyperplasia and mucus hypersecretion. Thus, targeting IPCs will benefit future treatment of allergic airway diseases. However, the lack of adequate cell surface markers for IPCs limits their identification and characterization. We now show that CD44 containing exon v3 (CD44v3) is a surface marker for IPCs that are capable of both proliferating and generating differentiated goblet cells in allergic human nasal epithelium. In primary human nasal epithelial cells that had differentiated at an air-liquid interface, IL-4 upregulated mRNA expression of three CD44v variants that include exon v3 (CD44v3-v6, CD44v3,v8-v10, and CD44v3-v10), and it induced expression of CD44v3 protein in the basal and suprabasal layers of the culture. FACS analysis revealed two subpopulations differing in CD44v3 concentrations, as follows: CD44v3low cells expressed high amounts of proliferative and basal cell markers (Ki-67 and TP63), whereas CD44v3high cells strongly expressed progenitor and immature and mature goblet cell markers (SOX2, CA2, and SPDEF). Importantly, a blocking anti-CD44 antibody suppressed IL-4-induced mucin production by human nasal epithelial cells. Furthermore, CD44v3 was coexpressed with TP63, KRT5, or SOX2 and was upregulated in the basal and suprabasal layers of the nasal surface epithelium of subjects with allergic rhinitis. Taken together, these data demonstrate that high CD44v3 expression contributes to goblet cell hyperplasia in inflammation of the allergic airway.
Assuntos
Células Caliciformes/metabolismo , Receptores de Hialuronatos/metabolismo , Hiperplasia/metabolismo , Sistema Respiratório/metabolismo , Células-Tronco/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Éxons/genética , Células Caliciformes/patologia , Humanos , Hiperplasia/patologia , Hipersensibilidade/metabolismo , Hipersensibilidade/patologia , Inflamação/metabolismo , Inflamação/patologia , Mucinas/metabolismo , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , RNA Mensageiro/genética , Sistema Respiratório/patologia , Células-Tronco/patologia , Regulação para Cima/fisiologiaRESUMO
Recent developments in the fields of biomedical chemistry and immune bioengineering have enabled innovative therapeutic approaches that can enhance the efficacy, accuracy, and safety of cancer immunotherapy. Among the numerous strategies utilized in cancer immunotherapy, Toll-like receptor (TLR) agonist-based approaches have been studied for a long time since they trigger the innate immune system and generate antigen-specific T cell responses to fight against tumors. In addition to these immunostimulatory functions, TLR agonists also contribute to the reprogramming of immune suppressive tumor microenvironments. Although TLR agonists are now being intensively studied in clinical trials due to their substantial immunomodulatory properties, they still show a low therapeutic index. Nonspecific and random stimulation of various immune cells produces excess levels of proinflammatory cytokines, resulting in cytokine storms and chronic diseases. Therefore, the development of chemical strategies to enhance the therapeutic efficacy as well as the safety of TLR agonist-based immunotherapy is essential and in high demand.In this Account, we summarize and discuss recent developments in biomedical chemistry and bioengineering techniques for the immunomodulation of TLR agonists that have addressed the limitations in current cancer immunotherapy. Immunomodulation of TLR agonists can be classified into two different approaches: (1) molecular modulation via chemical structure modification and (2) macroscopic modulation via an engineered drug delivery system. In molecular modulation, based on prodrug and antedrug principles, activity is modulated (active or inactive) through immolative chemical linkers that can respond to extrinsic or intrinsic biological stimulation and the plasmatic environment, respectively. To increase the effectiveness of TLR agonists as immunostimulatory agents, researchers have conjugated TLR agonists with other immunotherapeutic moieties (antigen, antibody, other TLR agonist, etc.). For macroscopic modulation, bioengineering of delivery carriers differing in size or with albumin hitchhiking moieties has been utilized to increase the efficiency of the targeting of these carriers to secondary lymphoid organs (lymph nodes (LNs) and spleen). The conjugation of specific targeting ligands and incorporation of stimulus-triggering moieties can promote the delivery of TLR agonists into specific cells or intracellular compartments. Implantable porous scaffolds for specific immune cell recruitment and in situ depot-forming gel systems for controlled release of immunomodulatory drugs can increase the therapeutic efficacy of TLR agonists while reducing systemic toxicity. Taken together, these findings show that well-designed and precisely controlled chemical strategies for the immunomodulation of TLR agonists at both the molecular and macroscopic levels are expected to play key roles in improving the therapeutic efficacy of cancer immunotherapy while minimizing immune-related toxicity.
Assuntos
Imunoterapia , Neoplasias/terapia , Receptores Toll-Like/agonistas , Portadores de Fármacos/química , Endossomos/imunologia , Endossomos/metabolismo , Humanos , Linfonodos/imunologia , Linfonodos/metabolismo , Nanopartículas/química , Neoplasias/imunologia , Pró-Fármacos/química , Pró-Fármacos/uso terapêutico , Linfócitos T/imunologia , Linfócitos T/metabolismo , Receptores Toll-Like/metabolismoRESUMO
Although immune checkpoint inhibitors have significantly improved clinical outcomes in various malignant cancers, only a small proportion of patients reap benefits, likely due to the low number of T cells and high number of immunosuppressive cells in the tumor microenvironment (TME) of patients with advanced disease. We developed a cancer vaccine adjuvanted with nanoemulsion (NE) loaded with TLR7/8 agonist (R848) and analyzed its therapeutic effect alone or in combination with immune checkpoint inhibitors, on antitumor immune responses and the reprogramming of suppressive immune cells in the TME. NE (R848) demonstrated robust local and systemic antitumor immune responses in both subcutaneous and orthotopic mouse lung cancer models, inducing tumor-specific T cell activation and mitigating T cell exhaustion. Combination with anti-PD-1 antibodies showed synergistic effects with respect to therapeutic efficacy and survival rate. Thus, NE (R848)-based cancer vaccines could prevent tumor recurrence and prolong survival by activating antitumor immunity and reprogramming immunosuppression.
Assuntos
Vacinas Anticâncer/farmacologia , Neoplasias Pulmonares/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor 7 Toll-Like/genética , Receptor 8 Toll-Like/genética , Adjuvantes Imunológicos/farmacologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Emulsões/química , Emulsões/farmacologia , Humanos , Imidazóis/farmacologia , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Receptor de Morte Celular Programada 1/imunologia , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Microambiente Tumoral/efeitos dos fármacosRESUMO
A positive link between persistent cellular motion and a defective tight junction barrier allows increased antigenic penetration and contact between ligand-receptor pairs, leading to exacerbated allergic airway inflammation and remodeling. Given that collective cell migration involves cell-cell and cell-extracellular matrix adhesions, and given that IL-4 induces epithelial barrier dysfunction and decreases cell-extracellular matrix adhesions, we hypothesized that IL-4 may induce collective migration in the well-differentiated primary human nasal epithelial cells (HNECs). Well-differentiated HNECs were treated with IL-4, and the effects of IL-4 on cell migration were investigated using genetic and pharmacological approaches, live-cell imaging, a vertex model, and immunostaining. IL-4 disrupted the expression and localization of the tight junction proteins zonula occludens 1 and occludin, and it induced the cleavage and asymmetric distribution of E-cadherin in the HNEC layers. It also induced collective epithelial migration and cell shape changes driven by actin cytoskeleton reorganization. In addition, the effect of IL-4 on collective HNEC migration was reversed by pharmacologic and genetic inhibition of the αv-integrin-activating enzyme furin, and function-blocking antibodies for αvß5 or αvß6. In IL-4-stimulated cells, both anti-αvß5 and anti-αvß6 inhibited the phosphorylation of focal adhesion kinase. Furthermore, both ß5- and ß6-integrins were enriched in basal cells in the injured airway epithelium with allergic rhinitis. These findings suggest that αvß5 and αvß6 serve as critical mechanoreceptors in IL-4-induced collective HNEC migration through the focal adhesion kinase signaling pathway. These results have implications for targeting treatment of exacerbation of respiratory allergic diseases.
Assuntos
Antígenos de Neoplasias/metabolismo , Movimento Celular/fisiologia , Células Epiteliais/metabolismo , Integrinas/metabolismo , Interleucina-4/metabolismo , Receptores de Vitronectina/metabolismo , Hipersensibilidade Respiratória/patologia , Caderinas/metabolismo , Adesão Celular , Forma Celular/fisiologia , Matriz Extracelular/patologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Furina/genética , Humanos , Ocludina/metabolismo , Hipersensibilidade Respiratória/imunologia , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Rinite Alérgica/patologia , Junções Íntimas/patologia , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Histamine is an important mediator of allergic reactions, and mucus hypersecretion is a major allergic symptom. However, the direct effect of histamine on mucus secretion from airway mucosal epithelia has not been clearly demonstrated. TMEM16A is a Ca2+-activated chloride channel, and it is closely related to fluid secretion in airway mucosal epithelia. We investigated whether histamine directly induces fluid secretion from epithelial cells or submucosal glands (SMG) and mechanisms related, therewith, in allergic airway diseases. In pig airway tissues from the nose or trachea, histamine was a potent secretagogue that directly induced strong responses. However, gland secretion from human nasal tissue was not induced by histamine, even in allergic rhinitis patients. Histamine type 1 receptor (H1R) and histamine type 2 receptor (H2R) were not noted in SMG by in situ hybridization. Cultured primary human nasal epithelial (NHE) cells were used for the measurement of short-circuit current changes with the Ussing chamber. Histamine-induced slight responses of anion secretions under normal conditions. The response was enhanced by IL-4 stimulation through TMEM16A, which might be related to fluid hypersecretion in allergic rhinitis. Pretreatment with IL-4 augmented the histamine response that was suppressed by a TMEM16A inhibitor. TMEM16A expression was enhanced by 24-h treatment of IL-4 in human nasal epithelial cells. The expression of TMEM16A was significantly elevated in an allergic rhinitis group, compared with a control group. We elucidated histamine-induced fluid secretions in synergy with IL-4 through TMEM16A in the human airway epithelium. In addition, we observed species differences between pigs and humans in terms of gland secretion of histamine.
Assuntos
Canais de Cloreto/metabolismo , Histamina/farmacologia , Interleucina-4/farmacologia , Muco/metabolismo , Proteínas de Neoplasias/metabolismo , Mucosa Respiratória/metabolismo , Adulto , Animais , Anoctamina-1 , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Modelos Biológicos , Mucosa Nasal/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Histamínicos/genética , Receptores Histamínicos/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Rinite Alérgica/metabolismo , Rinite Alérgica/patologia , Sus scrofa , Traqueia/patologia , Conchas Nasais/metabolismoRESUMO
The aim of this study was to observe whether Polycal has inhibitory activity on ligation-induced experimental periodontitis and related alveolar bone loss in rats following topical application to the gingival regions. One day after the ligation placements, Polycal (50, 25, and 12.5 mg/mL solutions at 200 µL/rat) was topically applied to the ligated gingival regions daily for 10 days. Changes in bodyweight, alveolar bone loss index, and total number of buccal gingival aerobic bacterial cells were monitored, and the anti-inflammatory effects were investigated via myeloperoxidase activity and levels of the pro-inflammatory cytokines IL-1ß and TNF-α. The activities of inducible nitric oxide synthase (iNOS) and lipid peroxidation (MDA) were also evaluated. Bacterial proliferation, periodontitis, and alveolar bone loss induced by ligature placements were significantly inhibited after 10 days of continuous topical application of Polycal. These results indicate that topical application of Polycal has a significant inhibitory effect on periodontitis and related alveolar bone loss in rats mediated by antibacterial, anti-inflammatory, and anti-oxidative activities.
Assuntos
Perda do Osso Alveolar/prevenção & controle , Anti-Infecciosos/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Antioxidantes/administração & dosagem , Gluconato de Cálcio/administração & dosagem , Periodontite/tratamento farmacológico , beta-Glucanas/administração & dosagem , Administração Tópica , Perda do Osso Alveolar/metabolismo , Animais , Anti-Infecciosos/farmacologia , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Bactérias/efeitos dos fármacos , Gluconato de Cálcio/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Combinação de Medicamentos , Humanos , Interleucina-1beta/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Periodontite/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo , beta-Glucanas/farmacologiaRESUMO
Squamous metaplasia in airway epithelium is a pathological process arising from abnormal remodeling/repair responses to injury. Proteolytic maturation of many growth and differentiation factors involved in tissue remodeling is controlled by proprotein convertases (PCs). However, the role of these convertases in airway remodeling remains poorly understood. Using a retinoic acid deficiency-induced squamous metaplasia model of cultured human nasal epithelial cells (HNECs), we observed a significant increase in the expression of PC5/6A, a PC member, and bone morphogenetic protein-2 (BMP-2), a candidate substrate for PC5/6A. Specific lentiviral short hairpin RNA-mediated PC5/6A knockdown decreased BMP-2 expression and maturation, decreased expression of squamous cell markers, and increased expression of ciliated cell markers. Decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK), a PC inhibitor, and LDN-193189, a BMP receptor inhibitor, suppressed squamous differentiation, promoted mucociliary differentiation, and down-regulated the BMP-2/Smad1/5/8/p38 signaling pathways. Dec-RVKR-CMK also decreased expression of PC5/6A, but not furin, another PC member, suggesting the involvement of PC5/6A in squamous differentiation of HNECs. Overexpression of PC5/6A and BMP-2 in the human nasal epithelial cell line RPMI-2650 demonstrated that PC5/6A can activate BMP-2. Under retinoic acid-sufficient culture conditions for mucociliary differentiation of HNECs, short-term expression of PC5/6A by the adenovirus system and addition of exogenous BMP-2 induced squamous differentiation. Furthermore, PC5/6A and BMP-2 were highly expressed in metaplastic squamous epithelium of human nasal polyps. Taken together, PC5/6A is involved in squamous differentiation of HNECs, possibly through up-regulation of the BMP-2/pSmad1/5/8/p38 signaling pathway, pointing to a potential therapeutic target for the prevention of chronic airway diseases that exhibit squamous metaplasia.
Assuntos
Proteína Morfogenética Óssea 2/fisiologia , Diferenciação Celular , Células Epiteliais/fisiologia , Pró-Proteína Convertase 5/fisiologia , Células Cultivadas , Humanos , Mucosa Nasal/citologia , Proteínas Smad/metabolismo , Tretinoína/metabolismoRESUMO
Neurodegenerative diseases such as Alzheimer (AD) and Parkinson (PD) are characterized by abnormal aggregation of misfolded ß-sheet-rich proteins, including amyloid-ß (Aß)-derived peptides and tau in AD and α-synuclein in PD. Correct folding and assembly of these proteins are controlled by ubiquitously expressed molecular chaperones; however, our understanding of neuron-specific chaperones and their involvement in the pathogenesis of neurodegenerative diseases is limited. We here describe novel chaperone-like functions for the secretory protein 7B2, which is widely expressed in neuronal and endocrine tissues. In in vitro experiments, 7B2 efficiently prevented fibrillation and formation of Aß(1-42), Aß(1-40), and α-synuclein aggregates at a molar ratio of 1:10. In cell culture experiments, inclusion of recombinant 7B2, either in the medium of Neuro-2A cells or intracellularly via adenoviral 7B2 overexpression, blocked the neurocytotoxic effect of Aß(1-42) and significantly increased cell viability. Conversely, knockdown of 7B2 by RNAi increased Aß(1-42)-induced cytotoxicity. In the brains of APP/PSEN1 mice, a model of AD amyloidosis, immunoreactive 7B2 co-localized with aggregation-prone proteins and their respective aggregates. Furthermore, in the hippocampus and substantia nigra of human AD- and PD-affected brains, 7B2 was highly co-localized with Aß plaques and α-synuclein deposits, strongly suggesting physiological association. Our data provide insight into novel functions of 7B2 and establish this neural protein as an anti-aggregation chaperone associated with neurodegenerative disease.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Doenças Neurodegenerativas/metabolismo , Proteína Secretora Neuroendócrina 7B2/fisiologia , alfa-Sinucleína/antagonistas & inibidores , Idoso , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Animais , Morte Celular/fisiologia , Feminino , Hipocampo/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Proteína Secretora Neuroendócrina 7B2/química , Proteína Secretora Neuroendócrina 7B2/metabolismo , Substância Negra/metabolismo , alfa-Sinucleína/metabolismoRESUMO
BACKGROUND: Allergic rhinitis (AR) and asthma are 2 entities of allergic airway diseases that frequently occur together, which is referred to as united airways. In contrast to this general concept, we hypothesized that innate immunity of the upper and lower airways is respectively distinctive, because the immunologic conditions of the nasal and lung mucosa as well as the functions of the immune cells within their epithelia are different. OBJECTIVE: We wanted to identify distinctive mechanisms of innate immunity in the nose and lung mucosa, which are responsible for house dust mite (HDM)-induced AR and allergic asthma (AA), respectively. METHODS: We constructed a mouse model of AR or AA induced by sensitization and consequent provocation with HDM extracts. RESULTS: HDM-derived ß-glucans, rather than LPS, were proven to be essential to activating innate immunity in the nasal mucosa and triggering AR, which depended on Toll-like receptor 2 (TLR2), but not on TLR4; however, the LPS/TLR4 signaling axis, rather than ß-glucans/TLR2, was critical to HDM-induced AA. These differences were attributed to the specific role of ß-glucans and LPS in inducing the surface expression of TLR2 and TLR4 and their translocation to lipid rafts in nasal and bronchial epithelial cells, respectively. We also showed that dual oxidase 2-generated reactive oxygen species mediate both ß-glucan-induced TLR2 activation and LPS-induced TLR4 activation. CONCLUSIONS: We describe a novel finding of distinctive innate immunity of the nose and lungs, respectively, which trigger AR and AA, by showing the critical role of HDM-induced TLR activation via dual oxidase 2-mediated reactive oxygen species.
Assuntos
Hipersensibilidade/imunologia , Pulmão/imunologia , Mucosa Nasal/imunologia , Pyroglyphidae/imunologia , Sistema Respiratório/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Asma/imunologia , Asma/metabolismo , Oxidases Duais , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Hipersensibilidade/metabolismo , Imunidade Inata/imunologia , Lipopolissacarídeos/imunologia , Pulmão/metabolismo , Camundongos , NADPH Oxidases/imunologia , NADPH Oxidases/metabolismo , Mucosa Nasal/metabolismo , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Sistema Respiratório/metabolismo , Rinite Alérgica , Rinite Alérgica Perene/imunologia , Rinite Alérgica Perene/metabolismo , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , beta-Glucanas/imunologia , beta-Glucanas/metabolismoRESUMO
Allergic asthma (AA) is a common inflammatory airway disease characterized by increased airway hyper-responsiveness (AHR), inflammation, and remodeling. Akkermansia muciniphila is a strictly anaerobic bacterium residing in the gut and is a promising next-generation probiotic to improve metabolic inflammatory syndrome. A recent study suggested the beneficial effect of live A. muciniphila on allergic airway inflammation (AAI) in mice. However, whether the heat-killed form can improve AAI requires further investigation. Mice sensitized and challenged with house dust mites (HDM) develop AA hallmarks including inflammatory cell infiltration, goblet cell hyperplasia, and subepithelial collagen deposition in the lungs. These phenomena were reversed by oral administration of the heat-killed A. muciniphila strain EB-AMDK19 (AMDK19-HK) isolated from the feces of healthy Koreans. Furthermore, AMDK19-HK diminished the HDM-induced AHR to inhaled methacholine, lung mast cell accumulation, and serum HDM-specific IgE levels. It also led to the overall suppression of IL-4, IL-13, and eotaxin production in bronchoalveolar lavage fluids, and Il4, Il5, Il13, and Ccl17 gene expression in lung tissues. Moreover, AMDK19-HK suppressed Th2-associated cytokine production in the splenocytes of HDM-sensitized mice in vitro. Additionally, a combination of 16S rRNA gene sequencing and short-chain fatty acid (SCFA) analysis in cecal samples revealed that AMDK19-HK modulated the relative abundance of circulating SCFA-associated gut genera, including a positive correlation with Lachnospiraceae_ NK4A136_group and a negative correlation with Lachnoclostridium and significantly increased cecal SCFA concentrations. Finally, AMDK19-HK improved intestinal mucosal barrier function. These results suggest that the oral administration of AMDK19-HK ameliorates HDM-induced AAI in mice by suppressing Th2-mediated immune responses and could have a protective effect against AA development.
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The proprotein convertases (PCs) are serine proteases responsible for the proteolytic maturation of many precursor proteins involved in upper airway remodelling during nasal polyposis. We have previously found that PC1/3 is expressed in human nasal mucosa. However, whether PC1/3 is related to nasal polyp formation has not been investigated. To gain insight into the functional role of PC1/3 in nasal polyps, we determined PC1/3 expression in nasal polyps by immunostaining, Western blotting and enzyme assays and generated stable cells expressing PC1/3 using airway epithelial cell line NCI-H292. Nasal polyps exhibit increased PC1/3 expression compared to normal nasal mucosa. PC1/3 was expressed in neuroendocrine cells in normal nasal mucosa and it was also expressed in goblet and ciliated cells in nasal polyps. NCI-H292 cells stably expressing PC1/3 displayed morphological changes, enhanced cell proliferation and migration, downregulation of E-cadherin and cytokeratins and upregulation of N-cadherin, vimentin, matrix metalloproteinase-2, collagen-I, snail and twist. Importantly, PC1/3 expression was positively correlated with epithelial-mesenchymal transition in cultured human nasal epithelial cells and in nasal polyps. Taken together, our data suggest that PC1/3 overexpression induces morphological and phenotypic epithelial-mesenchymal transition changes of airway epithelial cells and these changes may contribute to the pathogenesis of nasal polyps.
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Células Epiteliais/citologia , Transição Epitelial-Mesenquimal , Epitélio/metabolismo , Regulação da Expressão Gênica , Pólipos Nasais/metabolismo , Pró-Proteína Convertase 1/fisiologia , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Células Caliciformes/metabolismo , Humanos , Inflamação , Camundongos , Mucosa Nasal/metabolismo , Células Neuroendócrinas/citologia , Fenótipo , Sistema Respiratório/citologia , Sinusite/metabolismo , CicatrizaçãoRESUMO
OBJECTIVE: For several decades, radiotherapy has been widely used to treat metastatic vertebral tumors. This study was designed to assess the feasibility and early clinical outcomes of high-dose radiotherapy to treat such tumors, using helical tomotherapy. METHODS: Between June 2009 and December 2011, 51 sites in 36 patients were treated with high-dose radiotherapy using helical tomotherapy for vertebral metastasis. Treatment outcomes and dosimetric analyses of spinal cord were retrospectively evaluated. RESULTS: Median follow-up was 11.5 months (range, 6-34.6) for surviving patients. The median total dose and the number of fractions in the primary helical tomotherapy arm were 2700 cGy and 3 fractions, respectively. Actuarial 6-month local control rates were 85.7%, and symptomatic vertebral compression fractures developed in five patients after a median of 4.2 (range, 2.9-5.7) months. Among 13 patients with 19 metastatic sites who showed pre-treatment impairment in neurologic function, five patients (with seven sites) in whom symptoms were mild showed improvement in neuronal function. The median pre-treatment pain visual analog scale score of 7 decreased to a median of 3 after helical tomotherapy (P < 0.001) at a median of 1 month (range, 0.5-3.2) of follow-up. No significant morbidity developed during follow-up except for one grade 3 esophagitis. CONCLUSIONS: The use of helical tomotherapy to treat metastatic vertebral tumors appears to be both safe and reliable in terms of local tumor control and early pain relief. Local progression and the risk of compression fracture in patients with pre-existing spinal instability remain the principal factors of limiting improved clinical and functional outcomes. Optimal dose-fractionation schemes and appropriate patient selection are required to achieve better outcomes with high-dose radiotherapy using helical tomotherapy.
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Radioterapia de Intensidade Modulada/métodos , Neoplasias da Coluna Vertebral/radioterapia , Neoplasias da Coluna Vertebral/secundário , Adulto , Idoso , Idoso de 80 Anos ou mais , Fracionamento da Dose de Radiação , Feminino , Seguimentos , Fraturas por Compressão/etiologia , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Dosagem Radioterapêutica , Planejamento da Radioterapia Assistida por Computador , Radioterapia de Intensidade Modulada/efeitos adversos , Neoplasias da Coluna Vertebral/tratamento farmacológico , Neoplasias da Coluna Vertebral/mortalidade , Resultado do TratamentoRESUMO
The beneficial effects of Akkermansia muciniphila (Akk) on gut health and inflammation reduction have been demonstrated; however, scientific evidence of hair growth enhancement by Akk has not been reported. Therefore, this study was undertaken to investigate the effect of Akk on improving testosterone-mediated hair growth inhibition. Hair growth inhibition was induced through subcutaneous injection of testosterone into the shaved dorsal skin of C57BL/6 male mice. Live and pasteurized Akk were orally administered at a concentration of 1 × 108 colony-forming unit. After 5 weeks, hair length and skin tissues were analyzed. The live and pasteurized Akk significantly stimulated hair growth, countering the inhibitory effect of testosterone compared to the testosterone-alone group. Hematoxylin and eosin staining revealed a significant increase in hair follicle size in the Akk-treated group. An increase in ß-catenin levels, which are associated with hair growth and cell cycle progression, was also observed. Moreover, the Akk-treated group exhibited increased levels of fibroblast growth factors, including Fgf7, Igf1, Fgf7, Fgf10, and Fgf21. However, no significant difference was observed between the live and pasteurized Akk groups. These results underscore the potential of live and pasteurized Akk in improving testosterone-mediated hair growth inhibition.
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Although conventional innate immune stimuli contribute to immune activation, they induce exhausted immune cells, resulting in suboptimal cancer immunotherapy. Here we suggest a kinetically activating nanoadjuvant (K-nanoadjuvant) that can dynamically integrate two waves of innate immune stimuli, resulting in effective antitumour immunity without immune cell exhaustion. The combinatorial code of K-nanoadjuvant is optimized in terms of the order, duration and time window between spatiotemporally activating Toll-like receptor 7/8 agonist and other Toll-like receptor agonists. K-nanoadjuvant induces effector/non-exhausted dendritic cells that programme the magnitude and persistence of interleukin-12 secretion, generate effector/non-exhausted CD8+ T cells, and activate natural killer cells. Treatment with K-nanoadjuvant as a monotherapy or in combination therapy with anti-PD-L1 or liposomes (doxorubicin) results in strong antitumour immunity in murine models, with minimal systemic toxicity, providing a strategy for synchronous and dynamic tailoring of innate immunity for enhanced cancer immunotherapy.
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Linfócitos T CD8-Positivos , Neoplasias , Animais , Camundongos , Imunoterapia/métodos , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/uso terapêutico , Imunidade Inata , Neoplasias/terapiaRESUMO
Introduction: Nonalcoholic steatohepatitis (NASH) is an advanced nonalcoholic fatty liver disease characterized by chronic inflammation and fibrosis. A dysbiosis of the gut microbiota has been associated with the pathophysiology of NASH, and probiotics have proven helpful in its treatment and prevention. Although both traditional and next-generation probiotics have the potential to alleviate various diseases, studies that observe the therapeutic effect of next-generation probiotics on NASH are lacking. Therefore, we investigated whether a next-generation probiotic candidate, Faecalibacterium prausnitzii, contributed to the mitigation of NASH. Methods: In this study, we conducted 16S rRNA sequencing analyses in patients with NASH and healthy controls. To test F. prausnitzii could alleviate NASH symptoms, we isolated four F. prausnitzii strains (EB-FPDK3, EB-FPDK9, EB-FPDK11, and EB-FPYYK1) from fecal samples collected from four healthy individuals. Mice were maintained on a high-fructose high-fat diet for 16 weeks to induce a NASH model and received oral administration of the bacterial strains. Changes in characteristic NASH phenotypes were assessed via oral glucose tolerance tests, biochemical assays, and histological analyses. Results: 16S rRNA sequencing analyses confirmed that the relative abundance of F. prausnitzii reduced significantly in patients with NASH compared to healthy controls (pâ<â0.05). In the NASH mice, F. prausnitzii supplementation improved glucose homeostasis, prevented hepatic lipid accumulation, curbed liver damage and fibrosis, restored damaged gut barrier functions, and alleviated hepatic steatosis and liver inflammation. Furthermore, real-time PCR assays documented that the four F. prausnitzii strains regulated the expression of genes related to hepatic steatosis in these mice. Discussion: Our study, therefore, confirms that the administration of F. prausnitzii bacteria can alleviate NASH symptoms. We propose that F. prausnitzii has the potential to contribute to the next-generation probiotic treatment of NASH.
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Introduction: Obesity and related metabolic issues are a growing global health concern. Recently, the discovery of new probiotics with anti-obesity properties has gained interest. Methods: In this study, four Faecalibacte-rium prausnitzii strains were isolated from healthy human feces and evaluated on a high-fat diet-induced mouse model for 12 weeks. Results: The F. prausnitzii strains reduced body weight gain, liver and fat weights, and calorie intake while improving lipid and glucose metabolism in the liver and adipose tissue, as evidenced by regulating lipid metabolism-associated gene expression, including ACC1, FAS, SREBP1c, leptin, and adiponectin. Moreover, the F. prausnitzii strains inhibited low-grade inflammation, restored gut integrity, and ameliorated hepatic function and insulin resistance. Interestingly, the F. prausnitzii strains modulated gut and neural hormone secretion and reduced appetite by affecting the gut-brain axis. Supplementation with F. prausnitzii strains noticeably changed the gut microbiota composition. Discussion: In summary, the novel isolated F. prausnitzii strains have therapeutic effects on obesity and associated metabolic disorders through modulation of the gut-brain axis. Additionally, the effectiveness of different strains might not be achieved through identical mechanisms. Therefore, the present findings provide a reliable clue for developing novel therapeutic probiotics against obesity and associated metabolic disorders.