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1.
Medicine (Baltimore) ; 100(35): e27058, 2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34477137

RESUMO

ABSTRACT: The treatment for squamous cell lung cancer (SqCLC) is limited, and the prognosis of SqCLC is poor. In this article, we aimed to analyze and identify immune-related cells and competition endogenous RNA (ceRNA) that influence the prognosis of SqCLC. SqCLC and lung adenocarcinoma data were downloaded from TCGA-GDC. A total of 22 types of immune cell fractions were estimated using CIBERSORT. R software was used to identify any significantly different transcriptome data, including mRNA, LncRNA, and miRNA. The univariate cox regression method was applied to screen for prognosis-related lncRNA, miRNA, mRNA and tumor-infiltrating immune cells. There were 504 patients included in this study. There was a higher proportion of memory activated CD4+ T cells and CD8+ T cells in younger women. Follicular helper T (Tfh) cells were predictive of a good prognosis and reflected immune activation in SqCLC. The SFTA1P/NKX2-1-AS1, hsa-mir-503, GREM2 ceRNA axes and NKX2-1-AS1, hsa-mir-96, PROK2 ceRNA axes were found to be important for the immune function, pathogenesis, and prognosis of SqCLC. Collectively, the immune-related ceRNA and tumor-infiltrating immune cells in SqCLC are likely important determinants of SqCLC pathogenesis, prognosis, and immune status.


Assuntos
Neoplasias Pulmonares/imunologia , Neoplasias de Células Escamosas/imunologia , Adolescente , Idoso , Criança , Células Epiteliais/patologia , Feminino , Ontologia Genética , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias de Células Escamosas/patologia , Estudos Retrospectivos , Tomografia Computadorizada por Raios X/métodos
2.
Int J Mol Sci ; 22(16)2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34445100

RESUMO

Endometriosis is a common gynecological disorder characterized by ectopic growth of endometrium outside the uterus and is associated with chronic pain and infertility. We investigated the role of the long intergenic noncoding RNA 01133 (LINC01133) in endometriosis, an lncRNA that has been implicated in several types of cancer. We found that LINC01133 is upregulated in ectopic endometriotic lesions. As expression appeared higher in the epithelial endometrial layer, we performed a siRNA knockdown of LINC01133 in an endometriosis epithelial cell line. Phenotypic assays indicated that LINC01133 may promote proliferation and suppress cellular migration, and affect the cytoskeleton and morphology of the cells. Gene ontology analysis of differentially expressed genes indicated that cell proliferation and migration pathways were affected in line with the observed phenotype. We validated upregulation of p21 and downregulation of Cyclin A at the protein level, which together with the quantification of the DNA content using fluorescence-activated cell sorting (FACS) analysis indicated that the observed effects on cellular proliferation may be due to changes in cell cycle. Further, we found testis-specific protein kinase 1 (TESK1) kinase upregulation corresponding with phosphorylation and inactivation of actin severing protein Cofilin, which could explain changes in the cytoskeleton and cellular migration. These results indicate that endometriosis is associated with LINC01133 upregulation, which may affect pathogenesis via the cellular proliferation and migration pathways.


Assuntos
Endometriose/genética , Endométrio/patologia , Células Epiteliais/patologia , RNA Longo não Codificante/genética , Adulto , Linhagem Celular , Proliferação de Células , Endometriose/patologia , Endométrio/citologia , Endométrio/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Regulação para Cima , Adulto Jovem
3.
Biomed Res Int ; 2021: 5522964, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34337019

RESUMO

Ziziphora (Cacotti in Persian) belongs to the Lamiaceae family (mint group) and is vastly found in Iran and Asia. This traditional medicinal plant is normally used as analgesic and for treatment of particular gastrointestinal diseases. Since colorectal cancer is one of the most common causes of death in the world and the second leading cause of cancer death among adults, there is a pressing need to inhibit this malignancy by using methods with minimal side effects. One of these methods is the use of natural resources such as medical plants. This study is aimed at investigating the expression of apoptosis-related genes in the adjacent culture of colorectal cancer epithelial cells (HT-29) with Ziziphora essential oil (ZEO). The essential oil was extracted from Ziziphora leaves, and its compounds were determined and then added to the HT-29 culture medium at different concentrations. After 24 hours, the HT-29 cells were harvested from the medium and cytotoxicity was analyzed by MTT assay. After MTT assay and determination of the percentage of apoptosis by flow cytometry, RNA extraction was performed and the expression levels of Bax, Bcl-2, caspase 3 (C3), and caspase 9 (C9) were analyzed using newly designed primers by reverse transcription (RT) qPCR method and GeniX6 software. Also, specific antibodies were used for western blot analyses of those molecules. GC analysis revealed 42 different compounds in the ZEO, including pulegone (26.65%), menthone (5.74%), thymol (5.51%), and menthol (1.02%). MTT assay showed that the concentration of 200 µg/ml of ZEO had the highest HT-29 cell death during 24 hours. After incubation with the concentration of 50 µg/ml of ZEO for 24 and 48 hours, caspase 3 and 9 gene expressions in the treated group increased compared to those in the control group (P < 0.001), while the Bcl-2 expression decreased. The results showed that having anticancer compounds, ZEO can increase C3 and C9 and decrease Bcl-2 expressions, causing apoptosis in HT-29 cells in vitro. This can lead to the use of ZEO as a factor for colorectal cancer treatment.


Assuntos
Apoptose , Neoplasias Colorretais/patologia , Lamiaceae/química , Óleos Voláteis/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Monoterpenos Cicloexânicos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Cromatografia Gasosa-Espectrometria de Massas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Modelos Biológicos , Proteína X Associada a bcl-2/metabolismo
4.
Nature ; 596(7871): 262-267, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34349263

RESUMO

Regulated cell death is an integral part of life, and has broad effects on organism development and homeostasis1. Malfunctions within the regulated cell death process, including the clearance of dying cells, can manifest in diverse pathologies throughout various tissues including the gastrointestinal tract2. A long appreciated, yet elusively defined relationship exists between cell death and gastrointestinal pathologies with an underlying microbial component3-6, but the direct effect of dying mammalian cells on bacterial growth is unclear. Here we advance a concept that several Enterobacteriaceae, including patient-derived clinical isolates, have an efficient growth strategy to exploit soluble factors that are released from dying gut epithelial cells. Mammalian nutrients released after caspase-3/7-dependent apoptosis boosts the growth of multiple Enterobacteriaceae and is observed using primary mouse colonic tissue, mouse and human cell lines, several apoptotic triggers, and in conventional as well as germ-free mice in vivo. The mammalian cell death nutrients induce a core transcriptional response in pathogenic Salmonella, and we identify the pyruvate formate-lyase-encoding pflB gene as a key driver of bacterial colonization in three contexts: a foodborne infection model, a TNF- and A20-dependent cell death model, and a chemotherapy-induced mucositis model. These findings introduce a new layer to the complex host-pathogen interaction, in which death-induced nutrient release acts as a source of fuel for intestinal bacteria, with implications for gut inflammation and cytotoxic chemotherapy treatment.


Assuntos
Apoptose , Enterobacteriaceae/crescimento & desenvolvimento , Enterobacteriaceae/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Intestinos/citologia , Intestinos/microbiologia , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/patologia , Feminino , Doenças Transmitidas por Alimentos/microbiologia , Vida Livre de Germes , Interações Hospedeiro-Patógeno , Inflamação/metabolismo , Inflamação/microbiologia , Inflamação/patologia , Masculino , Camundongos , Mucosite/induzido quimicamente , Salmonella/enzimologia , Salmonella/genética , Salmonella/crescimento & desenvolvimento , Salmonella/metabolismo , Transcriptoma , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
5.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445556

RESUMO

Neutrophil extracellular traps (NETs), built from mitochondrial or nuclear DNA, proteinases, and histones, entrap and eliminate pathogens in the course of bacterial or viral infections. Neutrophils' activation and the formation of NETs have been described as major risk factors for acute lung injury, multi-organ damage, and mortality in COVID-19 disease. NETs-related lung injury involves both epithelial and endothelial cells, as well as the alveolar-capillary barrier. The markers for NETs formation, such as circulating DNA, neutrophil elastase (NE) activity, or myeloperoxidase-DNA complexes, were found in lung specimens of COVID-19 victims, as well as in sera and tracheal aspirates obtained from COVID-19 patients. DNA threads form large conglomerates causing local obstruction of the small bronchi and together with NE are responsible for overproduction of mucin by epithelial cells. Various components of NETs are involved in the pathogenesis of cytokine storm in SARS-CoV-2 pulmonary disease. NETs are responsible for the interplay between inflammation and thrombosis in the affected lungs. The immunothrombosis, stimulated by NETs, has a poor prognostic significance. Better understanding of the role of NETs in the course of COVID-19 can help to develop novel approaches to the therapeutic interventions in this condition.


Assuntos
COVID-19/imunologia , Armadilhas Extracelulares/virologia , Pulmão/imunologia , Neutrófilos/imunologia , SARS-CoV-2/imunologia , COVID-19/patologia , COVID-19/virologia , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/virologia , Células Endoteliais/patologia , Células Epiteliais/patologia , Armadilhas Extracelulares/imunologia , Histonas/imunologia , Humanos , Elastase de Leucócito/deficiência , Elastase de Leucócito/imunologia , Pulmão/patologia , Pulmão/virologia , Ativação de Neutrófilo , Neutrófilos/virologia , Peroxidase/imunologia
6.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445577

RESUMO

Infections by the zoonotic foodborne bacterium Campylobacter jejuni (C. jejuni) are among the most frequent causes of bacterial gastroenteritis worldwide. The aim was to evaluate the relationship between epithelial barrier disruption, mucosal immune activation, and vitamin D (VD) treatment during C. jejuni infection, using intestinal epithelial cells and mouse models focused on the interaction of C. jejuni with the VD signaling pathway and VD treatment to improve C. jejuni-induced barrier dysfunction. Our RNA-Seq data from campylobacteriosis patients demonstrate inhibition of VD receptor (VDR) downstream targets, consistent with suppression of immune function. Barrier-preserving effects of VD addition were identified in C. jejuni-infected epithelial cells and IL-10-/- mice. Furthermore, interference of C. jejuni with the VDR pathway was shown via VDR/retinoid X receptor (RXR) interaction. Paracellular leakiness of infected epithelia correlated with tight junction (TJ) protein redistribution off the TJ domain and apoptosis induction. Supplementation with VD reversed barrier impairment and prevented inhibition of the VDR pathway, as shown by restoration of transepithelial electrical resistance and fluorescein (332 Da) permeability. We conclude that VD treatment restores gut epithelial barrier functionality and decreases bacterial transmigration and might, therefore, be a promising compound for C. jejuni treatment in humans and animals.


Assuntos
Infecções por Campylobacter/complicações , Permeabilidade da Membrana Celular , Células Epiteliais/efeitos dos fármacos , Interleucina-10/fisiologia , Mucosa Intestinal/efeitos dos fármacos , Vitamina D/farmacologia , Animais , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/isolamento & purificação , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Junções Íntimas/metabolismo , Vitaminas/farmacologia
7.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445350

RESUMO

Following an intraventricular hemorrhage (IVH), red blood cell lysis and hemoglobin (Hb) oxidation with the release of heme can cause sterile neuroinflammation. In this study, we measured Hb derivates and cellular adhesion molecules ICAM-1 and VCAM-1 with cell-free miRNAs in cerebrospinal fluid (CSF) samples obtained from Grade-III and Grade-IV preterm IVH infants (IVH-III and IVH-IV, respectively) at multiple time points between days 0-60 after the onset of IVH. Furthermore, human choroid plexus epithelial cells (HCPEpiCs) were incubated with IVH and non-IVH CSF (10 v/v %) for 24 h in vitro to investigate the IVH-induced inflammatory response that was investigated via: (i) HMOX1, IL8, VCAM1, and ICAM1 mRNAs as well as miR-155, miR-223, and miR-181b levels by RT-qPCR; (ii) nuclear translocation of the NF-κB p65 subunit by fluorescence microscopy; and (iii) reactive oxygen species (ROS) measurement. We found a time-dependent alteration of heme, IL-8, and adhesion molecules which revealed a prolonged elevation in IVH-IV vs. IVH-III with higher miR-155 and miR-181b expression at days 41-60. Exposure of HCPEpiCs to IVH CSF samples induced HMOX1, IL8, and ICAM1 mRNA levels along with increased ROS production via the NF-κB pathway activation but without cell death, as confirmed by the cell viability assay. Additionally, the enhanced intracellular miR-155 level was accompanied by lower miR-223 and miR-181b expression in HCPEpiCs after CSF treatment. Overall, choroid plexus epithelial cells exhibit an abnormal cell phenotype after interaction with pro-inflammatory CSF of IVH origin which may contribute to the development of later clinical complications in preterm IVH.


Assuntos
Hemorragia Cerebral/patologia , Plexo Corióideo/metabolismo , Síndrome de Resposta Inflamatória Sistêmica/patologia , Proteína C-Reativa/líquido cefalorraquidiano , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Hemorragia Cerebral/complicações , Hemorragia Cerebral/congênito , Hemorragia Cerebral/metabolismo , Plexo Corióideo/patologia , Estudos de Coortes , Citocinas/líquido cefalorraquidiano , Citocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Heme/metabolismo , Hemoglobinas/metabolismo , Humanos , Hungria , Recém-Nascido , Recém-Nascido Prematuro , Molécula 1 de Adesão Intercelular/líquido cefalorraquidiano , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Síndrome de Resposta Inflamatória Sistêmica/congênito , Síndrome de Resposta Inflamatória Sistêmica/etiologia , Síndrome de Resposta Inflamatória Sistêmica/metabolismo , Molécula 1 de Adesão de Célula Vascular/líquido cefalorraquidiano , Molécula 1 de Adesão de Célula Vascular/metabolismo
8.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361111

RESUMO

Maternal smoking is a risk factor of preterm prelabor rupture of the fetal membranes (pPROM), which is responsible for 30% of preterm births worldwide. Cigarettes induce oxidative stress and inflammation, mechanisms both implicated in fetal membranes (FM) weakening. We hypothesized that the receptor for advanced glycation end-products (RAGE) and its ligands can result in cigarette-dependent inflammation. FM explants and amniotic epithelial cells (AECs) were treated with cigarette smoke condensate (CSC), combined or not with RAGE antagonist peptide (RAP), an inhibitor of RAGE. Cell suffering was evaluated by measuring lactate dehydrogenase (LDH) medium-release. Extracellular HMGB1 (a RAGE ligand) release by amnion and choriodecidua explants were checked by western blot. NF-κB pathway induction was determined by a luciferase gene reporter assay, and inflammation was evaluated by cytokine RT-qPCR and protein quantification. Gelatinase activity was assessed using a specific assay. CSC induced cell suffering and HMGB1 secretion only in the amnion, which is directly associated with a RAGE-dependent response. CSC also affected AECs by inducing inflammation (cytokine release and NFκB activation) and gelatinase activity through RAGE engagement, which was linked to an increase in extracellular matrix degradation. This RAGE dependent CSC-induced inflammation associated with an increase of gelatinase activity could explain a pathological FM weakening directly linked to pPROM.


Assuntos
Âmnio/patologia , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/patologia , Efeitos Tardios da Exposição Pré-Natal/patologia , Fumaça/efeitos adversos , Adulto , Âmnio/efeitos dos fármacos , Âmnio/imunologia , Âmnio/metabolismo , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/imunologia , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Receptor para Produtos Finais de Glicação Avançada
9.
Front Immunol ; 12: 663303, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34194429

RESUMO

The release of neutrophil extracellular traps (NETs), a process termed NETosis, avoids pathogen spread but may cause tissue injury. NETs have been found in severe COVID-19 patients, but their role in disease development is still unknown. The aim of this study is to assess the capacity of NETs to drive epithelial-mesenchymal transition (EMT) of lung epithelial cells and to analyze the involvement of NETs in COVID-19. Bronchoalveolar lavage fluid of severe COVID-19 patients showed high concentration of NETs that correlates with neutrophils count; moreover, the analysis of lung tissues of COVID-19 deceased patients showed a subset of alveolar reactive pneumocytes with a co-expression of epithelial marker and a mesenchymal marker, confirming the induction of EMT mechanism after severe SARS-CoV2 infection. By airway in vitro models, cultivating A549 or 16HBE at air-liquid interface, adding alveolar macrophages (AM), neutrophils and SARS-CoV2, we demonstrated that to trigger a complete EMT expression pattern are necessary the induction of NETosis by SARS-CoV2 and the secretion of AM factors (TGF-ß, IL8 and IL1ß). All our results highlight the possible mechanism that can induce lung fibrosis after SARS-CoV2 infection.


Assuntos
COVID-19/fisiopatologia , Transição Epitelial-Mesenquimal , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Adulto , Biópsia , Líquido da Lavagem Broncoalveolar/citologia , COVID-19/complicações , COVID-19/imunologia , Linhagem Celular , Células Epiteliais/patologia , Humanos , Pulmão/patologia , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/metabolismo
10.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199463

RESUMO

Little is known about the ability for epithelial regeneration and wound healing in patients with inflammatory bowel diseases. We evaluated the epithelial proliferation and wound healing ability of patients with Crohn's disease (CD) using patient-derived intestinal organoids. Human intestinal organoids were constructed in a three-dimensional intestinal crypt culture of enteroscopic biopsy samples from controls and CD patients. The organoid-forming efficiency of ileal crypts derived from CD patients was reduced compared with those from control subjects (p < 0.001). Long-term cultured organoids (≥6 passages) derived from controls and CD patients showed an indistinguishable microscopic appearance and culturing behavior. Under TNFα-enriched conditions (30 ng/mL), the organoid reconstitution rate and cell viability of CD patient-derived organoids were significantly lower than those of the control organoids (p < 0.05 for each). The number of EdU+ cells was significantly lower in TNFα-treated organoids derived from CD patients than in TNFα-treated control organoids (p < 0.05). In a wound healing assay, the unhealed area in TNFα-treated CD patient-derived organoids was significantly larger than that of TNFα-treated control organoids (p < 0.001). The wound healing ability of CD patient-derived organoids is reduced in TNFα-enriched conditions, due to reduced cell proliferation. Epithelial regeneration ability may be impaired in patients with CD.


Assuntos
Proliferação de Células/genética , Doença de Crohn/terapia , Células Epiteliais/metabolismo , Organoides/crescimento & desenvolvimento , Adulto , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Células Epiteliais/patologia , Feminino , Humanos , Íleo/crescimento & desenvolvimento , Íleo/lesões , Íleo/patologia , Mucosa Intestinal/crescimento & desenvolvimento , Mucosa Intestinal/patologia , Intestinos/diagnóstico por imagem , Intestinos/lesões , Masculino , Pessoa de Meia-Idade , Organoides/metabolismo , Regeneração/genética , Transdução de Sinais/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Fator de Necrose Tumoral alfa/genética , Cicatrização/genética
11.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209135

RESUMO

Radiation-induced damage to normal lung parenchyma remains a dose-limiting factor in thorax-associated radiotherapy (RT). Severe early and late complications with lungs can increase the risk of morbidity in cancer patients after RT. Herein, senescence of lung epithelial cells following RT-induced cellular stress, or more precisely the respective altered secretory profile, the senescence-associated secretory phenotype (SASP), was suggested as a central process for the initiation and progression of pneumonitis and pulmonary fibrosis. We previously reported that abrogation of certain aspects of the secretome of senescent lung cells, in particular, signaling inhibition of the SASP-factor Ccl2/Mcp1 mediated radioprotection especially by limiting endothelial dysfunction. Here, we investigated the therapeutic potential of a combined metformin treatment to protect normal lung tissue from RT-induced senescence and associated lung injury using a preclinical mouse model of radiation-induced pneumopathy. Metformin treatment efficiently limited RT-induced senescence and SASP expression levels, thereby limiting vascular dysfunctions, namely increased vascular permeability associated with increased extravasation of circulating immune and tumor cells early after irradiation (acute effects). Complementary in vitro studies using normal lung epithelial cell lines confirmed the senescence-limiting effect of metformin following RT finally resulting in radioprotection, while fostering RT-induced cellular stress of cultured malignant epithelial cells accounting for radiosensitization. The radioprotective action of metformin for normal lung tissue without simultaneous protection or preferable radiosensitization of tumor tissue might increase tumor control probabilities and survival because higher radiation doses could be used.


Assuntos
Brônquios , Células Epiteliais , Metformina/farmacologia , Lesões Experimentais por Radiação , Protetores contra Radiação/farmacologia , Animais , Brônquios/metabolismo , Brônquios/patologia , Senescência Celular/efeitos dos fármacos , Senescência Celular/efeitos da radiação , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Camundongos , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Lesões Experimentais por Radiação/prevenção & controle
12.
Life Sci ; 282: 119827, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34273373

RESUMO

AIMS: We aimed to investigate the effect of PBDEs (47, 99, 209) on cellular events involved in epigenetic modification, inflammation, and epithelial mesenchymal transition (EMT). MATERIALS AND METHODS: We studied: 1) ERK1/2 phosphorylation; 2) Enhancer of Zester Homolog 2 (EZH2); 3) Histone H3 tri-methylated in lysine 27 (H3K27me3); 4) K-RAS; 5) silencing disabled homolog 2-interacting protein gene (DAB2IP), 6) let-7a; 7) Muc5AC/Muc5B, and 8) IL-8 in a 3D in vitro model of epithelium obtained with primary Normal Human Bronchial Epithelial cells (pNHBEs) or A549 cell line, chronically exposed to PBDEs (47, 99, 209). KEY FINDINGS: PBDEs (10 nM, 100 nM and 1 µM) increased ERK1/2 phosphorylation, and EZH2, H3K27me3, and K-RAS protein expression, while decreased DAB2IP and Let-7a transcripts in pNHBEs ALI culture. Furthermore PBDEs (47, 99) (100 nM) increased Muc5AC and Muc5B mRNA, and PBDE 47 (100 nM) IL-8 mRNA via EZH2 in pNHBEs. Finally, PBDEs (100 nM) affected EZH2, H3K27me3, K-RAS protein expression, and DAB2IP, Let-7a transcripts and cell invasion in A549 cells. Gsk343 (methyltransferase EZH2 inhibitor) (1 mM) and U0126 (inhibitor of MEK1/2) (10 µM) were used to show the specific effect of PBDEs. SIGNIFICANCE: PBDE inhalation might promote inflammation/cancer via EZH2 methyltransferase activity and H3K27me3, k-RAS and ERk1/2 involvement, generating adverse health outcomes of the human lung.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Células Epiteliais , Retardadores de Chama/administração & dosagem , Éteres Difenil Halogenados/efeitos adversos , Neoplasias Pulmonares , Proteínas de Neoplasias/metabolismo , Mucosa Respiratória , Células A549 , Idoso , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Feminino , Retardadores de Chama/farmacologia , Éteres Difenil Halogenados/farmacologia , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mucosa Respiratória/enzimologia , Mucosa Respiratória/patologia
13.
NPJ Biofilms Microbiomes ; 7(1): 57, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230496

RESUMO

Bacterial vaginosis (BV) is a gynecologic disorder characterized by a shift in cervicovaginal microbiota from Lactobacillus spp. dominance to a polymicrobial biofilm composed of diverse anaerobes. We utilized a well-characterized human three-dimensional cervical epithelial cell model in conjunction with untargeted metabolomics and immunoproteomics analyses to determine the immunometabolic contribution of three members of the Veillonellaceae family: Veillonella atypica, Veillonella montpellierensis and Megasphaera micronuciformis at this site. We found that Veillonella spp. infections induced significant elevation of polyamines. M. micronuciformis infections significantly increased soluble inflammatory mediators, induced moderate levels of cell cytotoxicity, and accumulation of cell membrane lipids relative to Veillonella spp. Notably, both V. atypica and V. montpellierensis infections resulted in consumption of lactate, a key metabolite linked to gynecologic and reproductive health. Collectively our approach and data provide unique insights into the specific contributions of Veillonellaceae members to the pathogenesis of BV and women's health.


Assuntos
Metabolismo Energético , Membrana Mucosa/metabolismo , Membrana Mucosa/microbiologia , Vagina/metabolismo , Vagina/microbiologia , Veillonellaceae/fisiologia , Aminoácidos/metabolismo , Técnicas de Cultura de Células , Biologia Computacional/métodos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Feminino , Interações Hospedeiro-Patógeno/imunologia , Humanos , Metabolismo dos Lipídeos , Metaboloma , Metabolômica/métodos , Vaginose Bacteriana/metabolismo , Vaginose Bacteriana/microbiologia
14.
Molecules ; 26(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299636

RESUMO

Age-related macular degeneration (AMD) occurs due to an abnormality of retinal pigment epithelium (RPE) cells that leads to gradual degeneration of the macula. Currently, AMD drug pipelines are endowed with limited options, and anti-VEGF agents stand as the dominantly employed therapy. Despite the proven efficacy of such agents, the evidenced side effects associated with their use underscore the need to elucidate other mechanisms involved and identify additional molecular targets for the sake of therapy improvement. The previous literature provided us with a solid rationale to preliminarily explore the potential of selective HDAC6 and HSP90 inhibitors to treat wet AMD. Rather than furnishing single-target agents (either HDAC6 or HSP90 inhibitor), this study recruited scaffolds endowed with the ability to concomitantly modulate both targets (HDAC6 and HSP90) for exploration. This plan was anticipated to accomplish the important goal of extracting amplified benefits via dual inhibition (HDAC6/HSP90) in wet AMD. As a result, G570 (indoline-based hydroxamate), a dual selective HDAC6-HSP90 inhibitor exerting its effects at micromolar concentrations, was pinpointed in the present endeavor to attenuate blue light-induced cell migration and retinal neovascularization by inhibiting VEGF production. In addition to the identification of a potential chemical tool (G570), the outcome of this study validates the candidate HDAC6-HSP90 as a compelling target for the development of futuristic therapeutics for wet AMD.


Assuntos
Movimento Celular , Células Epiteliais/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Luz , Neovascularização Retiniana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Células Epiteliais/patologia , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/química , Humanos , Masculino , Camundongos , Neovascularização Retiniana/induzido quimicamente , Neovascularização Retiniana/patologia , Epitélio Pigmentado da Retina/irrigação sanguínea , Epitélio Pigmentado da Retina/patologia
15.
Am J Physiol Cell Physiol ; 321(3): C415-C428, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34260299

RESUMO

Leucine-rich α-2-glycoprotein-1 (LRG1) is a novel profibrotic factor that modulates transforming growth factor-ß (TGF-ß) signaling. However, its role in the corneal fibrotic response remains unknown. In the present study, we found that the LRG1 level increased in alkali-burned mouse corneas. In the LRG1-treated alkali-burned corneas, there were higher fibrogenic protein expression and neutrophil infiltration. LRG1 promoted neutrophil chemotaxis and CXCL-1 secretion. Conversely, LRG1-specific siRNA reduced fibrogenic protein expression and neutrophil infiltration in the alkali-burned corneas. The clearance of neutrophils effectively attenuated the LRG1-enhanced corneal fibrotic response, whereas the presence of neutrophils enhanced the effect of LRG1 on the fibrotic response in cultured TKE2 cells. In addition, the topical application of LRG1 elevated interleukin-6 (IL-6) and p-Stat3 levels in the corneal epithelium and in isolated neutrophils. The clearance of neutrophils inhibited the expression of p-Stat3 and IL-6 promoted by LRG1 in alkali-burned corneas. Moreover, neutrophils significantly increased the production of IL-6 and p-Stat3 promoted by LRG1 in TKE2 cells. Furthermore, the inhibition of Stat3 signaling by S3I-201 decreased neutrophil infiltration and alleviated the LRG1-enhanced corneal fibrotic response in the alkali-burned corneas. S3I-201 also reduced LRG1 or neutrophil-induced fibrotic response in TKE2 cells. In conclusion, LRG1 promotes the corneal fibrotic response by stimulating neutrophil infiltration via the modulation of the IL-6/Stat3 signaling pathway. Therefore, LRG1 could be targeted as a promising therapeutic strategy for patients with corneal fibrosis.


Assuntos
Queimaduras Químicas/genética , Quimiotaxia/efeitos dos fármacos , Queimaduras Oculares/genética , Glicoproteínas/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética , Álcalis , Ácidos Aminossalicílicos/farmacologia , Animais , Benzenossulfonatos/farmacologia , Queimaduras Químicas/tratamento farmacológico , Queimaduras Químicas/metabolismo , Queimaduras Químicas/patologia , Linhagem Celular , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/tratamento farmacológico , Queimaduras Oculares/patologia , Fibrose/prevenção & controle , Regulação da Expressão Gênica , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
16.
Cell Death Dis ; 12(7): 708, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267179

RESUMO

The dysregulated microRNAs (miRNAs) are involved in diabetic retinopathy progression. Epithelial mesenchymal transition (EMT) and cell permeability are important events in diabetic retinopathy. However, the function and mechanism of miR-195 in EMT and cell permeability in diabetic retinopathy remain largely unclear. Diabetic retinopathy models were established using streptozotocin (STZ)-induced diabetic mice and high glucose (HG)-stimulated ARPE-19 cells. Retina injury was investigated by hematoxylin-eosin (HE) staining. EMT and cell permeability were analyzed by western blotting, immunofluorescence, wound healing, and FITC-dextran assays. MiR-195 expression was detected via qRT-PCR. YY1, VEGFA, Snail1, and Smurf2 levels were detected via western blotting. The interaction relationship was analyzed via ChIP, Co-IP, or dual-luciferase reporter assay. The retina injury, EMT, and cell permeability were induced in STZ-induced diabetic mice. HG induced EMT and cell permeability in ARPE-19 cells. MiR-195, YY1, VEGFA, and Snail1 levels were enhanced, but Smurf2 abundance was reduced in STZ-induced diabetic mice and HG-stimulated ARPE-19 cells. VEGFA knockdown decreased Snail1 expression and attenuated HG-induced EMT and cell permeability. YY1 silence reduced VEGFA and Snail1 expression, and mitigated HG-induced EMT and cell permeability. YY1 could bind with VEGFA and Snail1, and it was degraded via Smurf2-mediated ubiquitination. MiR-195 knockdown upregulated Smurf2 to decrease YY1 expression and inhibited HG-induced EMT and cell permeability. MiR-195 targeted Smurf2, increased expression of YY1, VEGFA, and Snail1, and promoted HG-induced EMT and cell permeability. MiR-195 promotes EMT and cell permeability of HG-stimulated ARPE-19 cells by increasing VEGFA/Snail1 via inhibiting the Smurf2-mediated ubiquitination of YY1.


Assuntos
Retinopatia Diabética/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , MicroRNAs/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Fator de Transcrição YY1/metabolismo , Animais , Linhagem Celular , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Células Epiteliais/patologia , Glucose/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Permeabilidade , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais , Fatores de Transcrição da Família Snail/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator de Transcrição YY1/genética
17.
Am J Physiol Lung Cell Mol Physiol ; 321(3): L576-L594, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34318710

RESUMO

The paramyxoviridae, respiratory syncytial virus (RSV), and murine respirovirus are enveloped, negative-sense RNA viruses that are the etiological agents of vertebrate lower respiratory tract infections (LRTIs). We observed that RSV infection in human small airway epithelial cells induced accumulation of glycosylated proteins within the endoplasmic reticulum (ER), increased glutamine-fructose-6-phosphate transaminases (GFPT1/2) and accumulation of uridine diphosphate (UDP)-N-acetylglucosamine, indicating activation of the hexosamine biosynthetic pathway (HBP). RSV infection induces rapid formation of spliced X-box binding protein 1 (XBP1s) and processing of activating transcription factor 6 (ATF6). Using pathway selective inhibitors and shRNA silencing, we find that the inositol-requiring enzyme (IRE1α)-XBP1 arm of the unfolded protein response (UPR) is required not only for activation of the HBP, but also for expression of mesenchymal transition (EMT) through the Snail family transcriptional repressor 1 (SNAI1), extracellular matrix (ECM)-remodeling proteins fibronectin (FN1), and matrix metalloproteinase 9 (MMP9). Probing RSV-induced open chromatin domains by ChIP, we find XBP1 binds and recruits RNA polymerase II to the IL6, SNAI1, and MMP9 promoters and the intragenic superenhancer of glutamine-fructose-6-phosphate transaminase 2 (GFPT2). The UPR is sustained through RSV by an autoregulatory loop where XBP1 enhances Pol II binding to its own promoter. Similarly, we investigated the effects of murine respirovirus infection on its natural host (mouse). Murine respirovirus induces mucosal growth factor response, EMT, and the indicators of ECM remodeling in an IRE1α-dependent manner, which persists after viral clearance. These data suggest that IRE1α-XBP1s arm of the UPR pathway is responsible for paramyxovirus-induced metabolic adaptation and mucosal remodeling via EMT and ECM secretion.


Assuntos
Endorribonucleases/metabolismo , Células Epiteliais/metabolismo , Hexosaminas/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Mucosa Respiratória/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sincicial Respiratório Humano/fisiologia , Resposta a Proteínas não Dobradas , Replicação Viral , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Linhagem Celular Transformada , Endorribonucleases/genética , Células Epiteliais/patologia , Células Epiteliais/virologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Hexosaminas/genética , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/genética , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/patologia , Proteína 1 de Ligação a X-Box/genética
18.
Nat Cell Biol ; 23(7): 796-807, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34239062

RESUMO

Inflammatory bowel diseases present with elevated levels of intestinal epithelial cell (IEC) death, which compromises the gut barrier, activating immune cells and triggering more IEC death. The endogenous signals that prevent IEC death and break this vicious cycle, allowing resolution of intestinal inflammation, remain largely unknown. Here we show that prostaglandin E2 signalling via the E-type prostanoid receptor 4 (EP4) on IECs represses epithelial necroptosis and induces resolution of colitis. We found that EP4 expression correlates with an improved IBD outcome and that EP4 activation induces a transcriptional signature consistent with resolution of intestinal inflammation. We further show that dysregulated necroptosis prevents resolution, and EP4 agonism suppresses necroptosis in human and mouse IECs. Mechanistically, EP4 signalling on IECs converges on receptor-interacting protein kinase 1 to suppress tumour necrosis factor-induced activation and membrane translocation of the necroptosis effector mixed-lineage kinase domain-like pseudokinase. In summary, our study indicates that EP4 promotes the resolution of colitis by suppressing IEC necroptosis.


Assuntos
Colite/metabolismo , Colo/metabolismo , Dinoprostona/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Necroptose , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Colite/induzido quimicamente , Colite/patologia , Colite/prevenção & controle , Colo/efeitos dos fármacos , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células HT29 , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necroptose/efeitos dos fármacos , Organoides , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/genética , Transdução de Sinais
19.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200296

RESUMO

Recent studies found that expression of NEDD4-2 is reduced in lung tissue from patients with idiopathic pulmonary fibrosis (IPF) and that the conditional deletion of Nedd4-2 in lung epithelial cells causes IPF-like disease in adult mice via multiple defects, including dysregulation of the epithelial Na+ channel (ENaC), TGFß signaling and the biosynthesis of surfactant protein-C proprotein (proSP-C). However, knowledge of the impact of congenital deletion of Nedd4-2 on the lung phenotype remains limited. In this study, we therefore determined the effects of congenital deletion of Nedd4-2 in the lung epithelial cells of neonatal doxycycline-induced triple transgenic Nedd4-2fl/fl/CCSP-rtTA2S-M2/LC1 mice, with a focus on clinical phenotype, survival, lung morphology, inflammation markers in BAL, mucin expression, ENaC function and proSP-C trafficking. We found that the congenital deletion of Nedd4-2 caused a rapidly progressive lung disease in neonatal mice that shares key features with interstitial lung diseases in children (chILD), including hypoxemia, growth failure, sterile pneumonitis, fibrotic lung remodeling and high mortality. The congenital deletion of Nedd4-2 in lung epithelial cells caused increased expression of Muc5b and mucus plugging of distal airways, increased ENaC activity and proSP-C mistrafficking. This model of congenital deletion of Nedd4-2 may support studies of the pathogenesis and preclinical development of therapies for chILD.


Assuntos
Células Epiteliais/patologia , Pulmão/patologia , Ubiquitina-Proteína Ligases Nedd4/fisiologia , Alvéolos Pulmonares/patologia , Fibrose Pulmonar/patologia , Animais , Animais Recém-Nascidos , Células Epiteliais/metabolismo , Feminino , Mediadores da Inflamação/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/metabolismo , Fibrose Pulmonar/etiologia
20.
J Virol ; 95(18): e0085321, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34232065

RESUMO

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus causing acute intestinal infection in pigs, with high mortality often seen in neonatal pigs. The newborns rely on innate immune responses against invading pathogens because of lacking adaptive immunity. However, how PEDV disables the innate immunity of newborns toward severe infection remains unknown. We found that PEDV infection led to reduced expression of histone deacetylases (HDACs), especially HDAC1, in porcine IPEC-J2 cells. HDACs are considered important regulators of innate immunity. We hypothesized that PEDV interacts with certain host factors to regulate HDAC1 expression in favor of its replication. We show that HDAC1 acted as a negative regulator of PEDV replication in IPEC-J2 cells, as shown by chemical inhibition, gene knockout, and overexpression. A GC-box (GCCCCACCCCC) within the HDAC1 promoter region was identified for Sp1 binding in IPEC-J2 cells. Treatment of the cells with Sp1 inhibitor mithramycin A inhibited HDAC1 expression, indicating direct regulation of HDAC1 expression by Sp1. Of the viral proteins that were overexpressed in IPEC-J2 cells, the N protein was found to be present in the nuclei and more inhibitory to HDAC1 transcription. The putative nuclear localization sequence 261PKKNKSR267 contributed to its nuclear localization. The N protein interacted with Sp1 and interfered with its binding to the promoter region, thereby inhibiting its transcriptional activity for HDAC1 expression. Our findings reveal a novel mechanism of PEDV evasion of the host responses, offering implications for studying the infection processes of other coronaviruses. IMPORTANCE The enteric coronavirus porcine epidemic diarrhea virus (PEDV) causes fatal acute intestinal infection in neonatal pigs that rely on innate immune responses. Histone deacetylases (HDACs) play important roles in innate immune regulation. Our study found PEDV suppresses HDAC1 expression via the interaction of its N protein and porcine Sp1, which identified a novel mechanism of PEDV evasion of the host responses to benefit its replication. This study suggests that other coronaviruses, including SARS-CoV and SARS-CoV-2, also make use of their N proteins to intercept the host immune responses in favor of their infection.


Assuntos
Infecções por Coronavirus/veterinária , Células Epiteliais/virologia , Histona Desacetilase 1/antagonistas & inibidores , Mucosa Intestinal/virologia , Fator de Transcrição Sp1/metabolismo , Doenças dos Suínos/virologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Células Cultivadas , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Fator de Transcrição Sp1/genética , Suínos , Doenças dos Suínos/metabolismo , Doenças dos Suínos/patologia , Proteínas não Estruturais Virais/genética
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