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1.
Nat Commun ; 12(1): 836, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547321

RESUMO

Dynamic regulation of intestinal cell differentiation is crucial for both homeostasis and the response to injury or inflammation. Sprouty2, an intracellular signaling regulator, controls pathways including PI3K and MAPKs that are implicated in differentiation and are dysregulated in inflammatory bowel disease. Here, we ask whether Sprouty2 controls secretory cell differentiation and the response to colitis. We report that colonic epithelial Sprouty2 deletion leads to expanded tuft and goblet cell populations. Sprouty2 loss induces PI3K/Akt signaling, leading to GSK3ß inhibition and epithelial interleukin (IL)-33 expression. In vivo, this results in increased stromal IL-13+ cells. IL-13 in turn induces tuft and goblet cell expansion in vitro and in vivo. Sprouty2 is downregulated by acute inflammation; this appears to be a protective response, as VillinCre;Sprouty2F/F mice are resistant to DSS colitis. In contrast, Sprouty2 is elevated in chronic colitis and in colons of inflammatory bowel disease patients, suggesting that this protective epithelial-stromal signaling mechanism is lost in disease.


Assuntos
Colite/genética , Glicogênio Sintase Quinase 3 beta/genética , Homeostase/genética , Interleucina-33/genética , Proteínas de Membrana/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Criança , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Feminino , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Células HT29 , Homeostase/efeitos dos fármacos , Humanos , Interleucina-33/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Dodecilsulfato de Sódio/administração & dosagem
2.
Nat Commun ; 11(1): 5453, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33116139

RESUMO

The coronavirus SARS-CoV-2 is the causative agent of the ongoing severe acute respiratory disease pandemic COVID-19. Tissue and cellular tropism is one key to understanding the pathogenesis of SARS-CoV-2. We investigate the expression and subcellular localization of the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), within the upper (nasal) and lower (pulmonary) respiratory tracts of human donors using a diverse panel of banked tissues. Here, we report our discovery that the ACE2 receptor protein robustly localizes within the motile cilia of airway epithelial cells, which likely represents the initial or early subcellular site of SARS-CoV-2 viral entry during host respiratory transmission. We further determine whether ciliary ACE2 expression in the upper airway is influenced by patient demographics, clinical characteristics, comorbidities, or medication use, and show the first mechanistic evidence that the use of angiotensin-converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARBs) does not increase susceptibility to SARS-CoV-2 infection through enhancing the expression of ciliary ACE2 receptor. These findings are crucial to our understanding of the transmission of SARS-CoV-2 for prevention and control of this virulent pathogen.


Assuntos
Antagonistas de Receptores de Angiotensina/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Infecções por Coronavirus/patologia , Expressão Gênica/efeitos dos fármacos , Peptidil Dipeptidase A/genética , Pneumonia Viral/patologia , Sistema Respiratório/patologia , Fatores Etários , Cílios/metabolismo , Infecções por Coronavirus/virologia , Células Endoteliais , Células Caliciformes/metabolismo , Humanos , Pulmão/patologia , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Sistema Respiratório/metabolismo , Sistema Respiratório/virologia , Fatores Sexuais , Sinusite/metabolismo , Fumar
3.
Cell Rep ; 32(12): 108175, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32946807

RESUMO

To predict the tropism of human coronaviruses, we profile 28 SARS-CoV-2 and coronavirus-associated receptors and factors (SCARFs) using single-cell transcriptomics across various healthy human tissues. SCARFs include cellular factors both facilitating and restricting viral entry. Intestinal goblet cells, enterocytes, and kidney proximal tubule cells appear highly permissive to SARS-CoV-2, consistent with clinical data. Our analysis also predicts non-canonical entry paths for lung and brain infections. Spermatogonial cells and prostate endocrine cells also appear to be permissive to SARS-CoV-2 infection, suggesting male-specific vulnerabilities. Both pro- and anti-viral factors are highly expressed within the nasal epithelium, with potential age-dependent variation, predicting an important battleground for coronavirus infection. Our analysis also suggests that early embryonic and placental development are at moderate risk of infection. Lastly, SCARF expression appears broadly conserved across a subset of primate organs examined. Our study establishes a resource for investigations of coronavirus biology and pathology.


Assuntos
Infecções por Coronavirus/patologia , Mucosa Nasal/metabolismo , Pneumonia Viral/patologia , Receptores Virais/genética , Tropismo Viral/genética , Internalização do Vírus , Células A549 , Animais , Betacoronavirus/crescimento & desenvolvimento , Linhagem Celular , Chlorocebus aethiops , Enterócitos/metabolismo , Perfilação da Expressão Gênica , Células Caliciformes/metabolismo , Células HEK293 , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Mucosa Nasal/virologia , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Análise de Célula Única , Células Vero
4.
Int J Biol Sci ; 16(13): 2464-2476, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760213

RESUMO

In 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused infections worldwide. However, the correlation between the immune infiltration and coronavirus disease 2019 (COVID-19) susceptibility or severity in cancer patients remains to be fully elucidated. ACE2 expressions in normal tissues, cancers and cell lines were comprehensively assessed. Furthermore, we compared ACE2 expression between cancers and matched normal tissues through Gene Expression Profiling Interactive Analysis (GEPIA). In addition, we performed gene set enrichment analysis (GSEA) to investigate the related signaling pathways. Finally, the correlations between ACE2 expression and immune infiltration were investigated via Tumor Immune Estimation Resource (TIMER) and GEPIA. We found that ACE2 was predominantly expressed in both adult and fetal tissues from the digestive, urinary and male reproductive tracts; moreover, ACE2 expressions in corresponding cancers were generally higher than that in matched healthy tissues. GSEA showed that various metabolic and immune-related pathways were significantly associated with ACE2 expression across multiple cancer types. Intriguingly, we found that ACE2 expression correlated significantly with immune cell infiltration in both normal and cancer tissues, especially in the stomach and colon. These findings proposed a possible fecal-oral and maternal-fetal transmission of SARS-CoV-2 and suggested that cancers of the respiratory, digestive or urinary tracts would be more vulnerable to SARS-CoV-2 infection.


Assuntos
Biologia Computacional , Infecções por Coronavirus/imunologia , Neoplasias/imunologia , Pneumonia Viral/imunologia , Adulto , Betacoronavirus , Infecções por Coronavirus/complicações , Enterócitos/metabolismo , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Genótipo , Células Caliciformes/metabolismo , Hepatócitos/metabolismo , Humanos , Sistema Imunitário , Túbulos Renais/embriologia , Masculino , Neoplasias/complicações , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/complicações , Prognóstico , RNA-Seq , Transdução de Sinais
5.
Proc Natl Acad Sci U S A ; 117(35): 21519-21526, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817517

RESUMO

The intestinal epithelium is a highly dynamic structure that rejuvenates in response to acute stressors and can undergo alterations in cellular composition as animals age. The microbiota, acting via secreted factors related to indole, appear to regulate the sensitivity of the epithelium to stressors and promote epithelial repair via IL-22 and type I IFN signaling. As animals age, the cellular composition of the intestinal epithelium changes, resulting in a decreased proportion of goblet cells in the colon. We show that colonization of young or geriatric mice with bacteria that secrete indoles and various derivatives or administration of the indole derivative indole-3 aldehyde increases proliferation of epithelial cells and promotes goblet cell differentiation, reversing an effect of aging. To induce goblet cell differentiation, indole acts via the xenobiotic aryl hydrocarbon receptor to increase expression of the cytokine IL-10. However, the effects of indoles on goblet cells do not depend on type I IFN or on IL-22 signaling, pathways responsible for protection against acute stressors. Thus, indoles derived from the commensal microbiota regulate intestinal homeostasis, especially during aging, via mechanisms distinct from those used during responses to acute stressors. Indoles may have utility as an intervention to limit the decline of barrier integrity and the resulting systemic inflammation that occurs with aging.


Assuntos
Células Caliciformes/efeitos dos fármacos , Células Caliciformes/microbiologia , Indóis/farmacologia , Interleucina-10/metabolismo , Microbiota/fisiologia , Receptores de Hidrocarboneto Arílico/metabolismo , Envelhecimento/metabolismo , Animais , Bactérias/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Feminino , Células Caliciformes/citologia , Células Caliciformes/metabolismo , Interleucina-10/biossíntese , Interleucinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muco/metabolismo , Transdução de Sinais
6.
Am J Pathol ; 190(9): 1823-1832, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32561135

RESUMO

Leukotriene B4 (LTB4) is a major proinflammatory mediator important in host defense, whereas resolvins (Rvs) are produced during the resolution phase of inflammation. The authors determined the actions of both RvE1 and RvD1 on LTB4-induced responses of goblet cells cultured from rat conjunctiva. The responses measured were an increase in the intracellular [Ca2+] ([Ca2+]i) and high-molecular-weight glycoprotein secretion. Treatment with RvE1 or RvD1 for 30 minutes significantly blocked the LTB4-induced [Ca2+]i increase. The actions of RvE1 on LTB4-induced [Ca2+]i increase were reversed by siRNA for the RvE1 receptor, and the actions of RvD1 were reversed by an RvD1 receptor inhibitor. The RvE1 and RvD1 block of LTB4-stimulated increase in [Ca2+]i was also reversed by an inhibitory peptide to ß-adrenergic receptor kinase. LTB4 and block of the LTB4-stimulated increase in [Ca2+]i by RvE1 and RvD1 were partially mediated by the depletion of intracellular Ca2+ stores. RvE1, but not RvD1, counterregulated the LTB4-induced high-molecular-weight glycoprotein secretion. Thus, both RvE1 and RvD1 receptors directly inhibit LTB4 by phosphorylating the LTB4 receptor using ß adrenergic receptor kinase. RvE1 receptor counterregulates the LTB4-induced increase in [Ca2+]i and secretion, whereas RvD1 receptor only counterregulates LTB4-induced [Ca2+]i increase.


Assuntos
Cálcio/metabolismo , Túnica Conjuntiva/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Células Caliciformes/metabolismo , Leucotrieno B4/metabolismo , Mucinas/metabolismo , Animais , Ácido Eicosapentaenoico/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley
7.
Cell ; 181(5): 1016-1035.e19, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32413319

RESUMO

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.


Assuntos
Células Epiteliais Alveolares/metabolismo , Enterócitos/metabolismo , Células Caliciformes/metabolismo , Interferon Tipo I/metabolismo , Mucosa Nasal/citologia , Peptidil Dipeptidase A/genética , Adolescente , Células Epiteliais Alveolares/imunologia , Animais , Betacoronavirus/fisiologia , Linhagem Celular , Células Cultivadas , Criança , Infecções por Coronavirus/virologia , Enterócitos/imunologia , Células Caliciformes/imunologia , Infecções por HIV/imunologia , Humanos , Influenza Humana/imunologia , Interferon Tipo I/imunologia , Pulmão/citologia , Pulmão/patologia , Macaca mulatta , Camundongos , Mycobacterium tuberculosis , Mucosa Nasal/imunologia , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Receptores Virais/genética , Serina Endopeptidases/metabolismo , Análise de Célula Única , Tuberculose/imunologia , Regulação para Cima
8.
Am J Physiol Lung Cell Mol Physiol ; 319(1): L82-L90, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32401676

RESUMO

Goblet cell metaplasia (GCM) and mucin overproduction are a hallmark of chronic rhinosinusitis (CRS) and chronic obstructive pulmonary disease (COPD). In the airways, cigarette smoke (CS) induces activation of the epidermal growth factor receptor (EGFR) leading to GCM and overexpression of the gel-forming mucin MUC5AC. Although previous studies have demonstrated that a membrane-bound mucin, MUC1, modulates the activation of CS-induced EGFR, the role of MUC1 in CS-induced GCM and mucin overproduction has not been explored. In response to CS exposure, wild-type (WT) rats displayed Muc1 translocation from the apical surface of airway epithelium to the intracellular compartment of hyperplastic intermediate cells, EGFR phosphorylation, GCM, and Muc5ac overproduction. Similarly, human CRS sinonasal tissues demonstrated hyperplasia of intermediate cells enriched with MUC1 in the intracellular compartment, which was accompanied by GCM and increased MUC5AC expression. To further evaluate the role of Muc1 in vivo, a Muc1 knockout (KO) rat (MUC in humans and Muc in animals) was developed. In contrast to WT littermates, Muc1-KO rats exhibited no activation of EGFR, and were protected from GCM and Muc5ac overproduction. Genetic knockdown of MUC1 in human lung or Muc1 knockout in primary rat airway epithelial cells led to significantly diminished EGF-induced MUC5AC production. Together, these findings suggest that MUC1-dependent EGFR activation mediates CS-induced GCM and mucin overproduction. Strategies designed to suppress MUC1-dependent EGFR activation may provide a novel therapeutic approach for treating mucin hypersecretion in CRS and COPD.


Assuntos
Células Caliciformes/metabolismo , Mucina-5AC/metabolismo , Mucina-1/metabolismo , Fumar/efeitos adversos , Animais , Linhagem Celular Tumoral , Polaridade Celular , Células Epiteliais/metabolismo , Epitélio/metabolismo , Epitélio/patologia , Receptores ErbB/metabolismo , Células Caliciformes/patologia , Metaplasia , Fosforilação , Ratos Sprague-Dawley
9.
PLoS One ; 15(4): e0232023, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32352981

RESUMO

INTRODUCTION: Intestinal atresia is a rare congenital affliction that is often associated with severe bacterial infections despite adequate neonatal surgery. Previous studies have focused on enteric nervous system variations. We hypothesized that epithelial systems (ES) may also be involved in the pathophysiology of postnatal disorders. MATERIALS AND METHODS: Global gene expression was measured by transcriptomic analysis in a rat model of induced intestinal atresia. The analyses then focused on genes involved in ES (enterocytes and goblet cells). Rat fetus small intestines at various stages of development (ED15, ED17, ED19, and ED21, n = 22), were used as non-operated controls and compared to the upper and lower segments of rat fetus small intestines with an induced atresia (n = 14; ligature at ED18). The pattern of gene expression was then confirmed by histochemistry, electron microscopy, and RT-qPCR. RESULTS: From ED15 to ED21, the expression of several genes exhibited a physiological increase of ES markers, with a significant increase at the end of gestation. The operated embryos exhibited significantly higher variations of gene expression in the proximal segment than in the distal segment in terms of absorption and the epithelial barrier. An increase in goblet cells and markers was observed in the proximal segment compared to the controls. CONCLUSION: Fetal intestinal obstruction accelerates maturation in the proximal segment and disrupts the intestinal wall in the distal segment, with a decrease in the number of mucosal cells. Moreover, the epithelial cells underwent significant changes, supporting the notion that intestinal disorders involve more than the ENS.


Assuntos
Atresia Intestinal/genética , Atresia Intestinal/fisiopatologia , Mucosa Intestinal/fisiopatologia , Animais , Modelos Animais de Doenças , Sistema Nervoso Entérico , Enterócitos/metabolismo , Células Epiteliais/metabolismo , Feminino , Feto , Motilidade Gastrointestinal/fisiologia , Perfilação da Expressão Gênica/métodos , Células Caliciformes/metabolismo , Obstrução Intestinal/fisiopatologia , Intestinos/fisiopatologia , Gravidez , Ratos , Ratos Wistar , Transcriptoma/genética
10.
Am J Physiol Cell Physiol ; 318(6): C1305-C1315, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32348177

RESUMO

Dry eye is a common sight-impairing, painful disorder characterized by disruption of the preocular tear film, whose integrity is required for ~70% of the eye's refractive power. A universal feature of clinical dry eye is hyperosmolarity of the tears resulting from their accelerated evaporation due to dysfunction of tear- and oil-producing ocular glands. A key adaptive response to dryness/hyperosmolarity is release of tear-stabilizing mucin by conjunctival goblet cells. Yet the mechanisms mediating this response to hyperosmolarity remain poorly understood. In this study of freshly excised rat conjunctiva, perforated-patch recordings revealed that during sustained hyperosmolarity, the development of a nonspecific cation (NSC) conductance depolarizes the goblet cells to a near-optimal voltage for the tonic activation of their voltage-gated calcium channels (VGCCs). In turn, as demonstrated by high-resolution membrane capacitance measurements, VGCC activation boosts the exocytotic response of conjunctival goblet cells to neural input. However, over time, VGCC activation also increases the vulnerability of these cells to the lethality of hyperosmolarity. Viability assays further revealed that hyperosmotic-induced goblet cell death is critically dependent on P2X7 receptor channels. Similar to the yin-yang impact of VGCCs on goblet cell physiology and pathobiology, P2X7 activation not only compromises goblet cell viability but also enhances exocytotic activity. Thus, the NSC/VGCC and P2X7 purinoceptor pathways are components of a previously unappreciated high-gain/high-risk adaptive strategy to combat ocular dryness. These pathways boost release of tear-stabilizing mucin at the risk of jeopardizing the viability of the conjunctival goblet cells, whose loss is a histopathological hallmark of irreversible mucin-deficient dry eye.


Assuntos
Canais de Cálcio/metabolismo , Túnica Conjuntiva/metabolismo , Síndromes do Olho Seco/metabolismo , Células Caliciformes/metabolismo , Ativação do Canal Iônico , Receptores Purinérgicos P2X7/metabolismo , Lágrimas/metabolismo , Adaptação Fisiológica , Animais , Túnica Conjuntiva/patologia , Síndromes do Olho Seco/patologia , Feminino , Células Caliciformes/patologia , Masculino , Potenciais da Membrana , Concentração Osmolar , Osmorregulação , Ratos Long-Evans , Ratos Sprague-Dawley , Transdução de Sinais
11.
EMBO J ; 39(10): e105114, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32246845

RESUMO

The SARS-CoV-2 pandemic affecting the human respiratory system severely challenges public health and urgently demands for increasing our understanding of COVID-19 pathogenesis, especially host factors facilitating virus infection and replication. SARS-CoV-2 was reported to enter cells via binding to ACE2, followed by its priming by TMPRSS2. Here, we investigate ACE2 and TMPRSS2 expression levels and their distribution across cell types in lung tissue (twelve donors, 39,778 cells) and in cells derived from subsegmental bronchial branches (four donors, 17,521 cells) by single nuclei and single cell RNA sequencing, respectively. While TMPRSS2 is strongly expressed in both tissues, in the subsegmental bronchial branches ACE2 is predominantly expressed in a transient secretory cell type. Interestingly, these transiently differentiating cells show an enrichment for pathways related to RHO GTPase function and viral processes suggesting increased vulnerability for SARS-CoV-2 infection. Our data provide a rich resource for future investigations of COVID-19 infection and pathogenesis.


Assuntos
Brônquios/citologia , Expressão Gênica , Pulmão/citologia , Peptidil Dipeptidase A/genética , Serina Endopeptidases/genética , Análise de Célula Única , Adulto , Envelhecimento , Brônquios/metabolismo , Células Cultivadas , Doença Crônica/epidemiologia , Infecções por Coronavirus/genética , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Alemanha , Células Caliciformes/metabolismo , Humanos , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/genética , Padrões de Referência , Análise de Sequência de RNA , Caracteres Sexuais , Fumar , Bancos de Tecidos
12.
PLoS One ; 15(4): e0230231, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240190

RESUMO

Enteroids are cultured primary intestinal epithelial cells that recapitulate epithelial lineage development allowing for a more complex and physiologically relevant model for scientific study. The large presence of intestinal stem cells (ISC) in these enteroids allows for the study of metabolite effects on cellular processes and resulting progeny cells. Short-chain fatty acids (SCFA) such as butyrate (BUT) are bacterial metabolites produced in the gastrointestinal tract that are considered to be beneficial to host cells. Therefore, the objective was to study the effects of SCFAs on biomarkers of ISC activity, differentiation, barrier function and epithelial defense in the intestine using mouse and human enteroid models. Enteroids were treated with two concentrations of acetate (ACET), propionate (PROP), or BUT for 24 h. Enteroids treated with BUT or PROP showed a decrease in proliferation via EdU uptake relative to the controls in both mouse and human models. Gene expression of Lgr5 was shown to decrease with BUT and PROP treatments, but increased with ACET. As a result of BUT and PROP treatments, there was an increase in differentiation markers for enterocyte, Paneth, goblet, and enteroendocrine cells. Gene expression of antimicrobial proteins Reg3ß, Reg3γ, and Defb1 were stimulated by BUT and PROP, but not by ACET which had a greater effect on expression of tight junction genes Cldn3 and Ocln in 3D enteroids. Similar results were obtained with human enteroids treated with 10 mM SCFAs and grown in either 3D or Transwell™ model cultures, although tight junctions were influenced by BUT and PROP, but not ACET in monolayer format. Furthermore, BUT and PROP treatments increased transepithelial electrical resistance after 24 h compared to ACET or control. Overall, individual SCFAs are potent stimulators of cellular gene expression, however, PROP and especially BUT show great efficacy for driving cell differentiation and gene expression.


Assuntos
Ácido Acético/farmacologia , Ácido Butírico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Propionatos/farmacologia , Esferoides Celulares/efeitos dos fármacos , Animais , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Claudina-3/genética , Claudina-3/metabolismo , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Células Enteroendócrinas/citologia , Células Enteroendócrinas/efeitos dos fármacos , Células Enteroendócrinas/metabolismo , Células Caliciformes/citologia , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Humanos , Camundongos , Ocludina/genética , Ocludina/metabolismo , Proteínas Associadas a Pancreatite/genética , Proteínas Associadas a Pancreatite/metabolismo , Celulas de Paneth/citologia , Celulas de Paneth/efeitos dos fármacos , Celulas de Paneth/metabolismo , Cultura Primária de Células , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Junções Íntimas/efeitos dos fármacos , beta-Defensinas/genética , beta-Defensinas/metabolismo
13.
Am J Physiol Lung Cell Mol Physiol ; 318(6): L1270-L1279, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32348677

RESUMO

The organization of the normal airway mucus system differs in small experimental animals from that in humans and large mammals. To address normal murine airway mucociliary clearance, Alcian blue-stained mucus transport was measured ex vivo on tracheal tissues of naïve C57BL/6, Muc5b-/-, Muc5ac-/-, and EGFP-tagged Muc5b reporter mice. Close to the larynx with a few submucosal glands, the mucus appeared as thick bundles. More distally in the trachea and in large bronchi, Alcian blue-stained mucus was organized in cloud-like formations based on the Muc5b mucin. On tilted tissue, the mucus clouds moved upward toward the larynx with an average velocity of 12 µm/s compared with 20 µm/s for beads not associated with clouds. In Muc5ac-/- mice, Muc5b formed mucus strands attached to the tissue surface, while in Muc5b-/- mice, Muc5ac had a more variable appearance. The normal mouse lung mucus thus appears as discontinuous clouds, clearly different from the stagnant mucus layer in diseased lungs.


Assuntos
Mucina-5B/metabolismo , Muco/metabolismo , Sistema Respiratório/metabolismo , Animais , Transporte Biológico , Fluorescência , Células Caliciformes/metabolismo , Camundongos Endogâmicos C57BL , Mucina-5AC/metabolismo , Membrana Mucosa/metabolismo , Traqueia/metabolismo
14.
Am J Respir Cell Mol Biol ; 63(1): 46-56, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32176858

RESUMO

Goblet cell metaplasia, excessive mucus production, and inadequate mucus clearance accompany and exacerbate multiple chronic respiratory disorders, such as asthma and chronic obstructive pulmonary disease. Notch signaling plays a central role in controlling the fate of multiple cell types in the lung, including goblet cells. In the present study, we explored the therapeutic potential of modulating the Notch pathway in the adult murine lung using chemically modified antisense oligonucleotides (ASOs). To this end, we designed and characterized ASOs targeting the Notch receptors Notch1, Notch2, and Notch3 and the Notch ligands Jag1 (Jagged 1) and Jag2 (Jagged 2). Pulmonary delivery of ASOs in healthy mice or mice exposed to house dust mite, a commonly used mouse model of asthma, resulted in a significant reduction of the respective mRNAs in the lung. Furthermore, ASO-mediated knockdown of Jag1 or Notch2 in the lungs of healthy adult mice led to the downregulation of the club cell marker Scgb1a1 and the concomitant upregulation of the ciliated cell marker FoxJ1 (forkhead box J1). Similarly, ASO-mediated knockdown of Jag1 or Notch2 in the house dust mite disease model led to reduced goblet cell metaplasia and decreased mucus production. Because goblet cell metaplasia and excessive mucus secretion are a common basis for many lung pathologies, we propose that ASO-mediated inhibition of JAG1 could provide a novel therapeutic path for the treatment of multiple chronic respiratory diseases.


Assuntos
Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Proteína Jagged-1/metabolismo , Pulmão/efeitos dos fármacos , Metaplasia/tratamento farmacológico , Metaplasia/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Animais , Asma/metabolismo , Biomarcadores/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pyroglyphidae , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
15.
Parasite Immunol ; 42(6): e12709, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32145074

RESUMO

AIMS: The role of the immune response to cyathostomin infections in horses remains unknown. Intestinal goblet cell hyperplasia has previously been noted as a component in cyathostomin infection; however, the function is unclear. The goal of this study was to evaluate the local and systemic gene expression to cyathostomin infections following larvicidal treatment and explore their relation to goblet cells. METHODS AND RESULTS: Thirty-six ponies with naturally acquired cyathostomin infections were randomly allocated into three groups: fenbendazole-treated (10 mg/kg PO 5 days), moxidectin-treated (0.4 mg/kg PO once) and untreated control. Whole blood from all horses was collected weekly, and tissue samples from the large intestine collected during necropsy at 2 and 5 weeks post-treatment (WPT). Gene expression of interleukin (IL)-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-22, IFN-γ, resistin-like molecule beta (RELM-ß), Mucin 2 (MUC2) and tumour necrosis factor (TNF)-α was measured using qRT-PCR. There were statistically significant linear correlations between luminal worm burdens and MUC2 (r = -.2358) and RELM-ß (r = -.2261). CONCLUSION: This suggests an active role of immune system post-treatment in parasite expulsion, specifically in goblet cells, and that the organs respond differently to treatment and the larvae themselves. This may have implications in the disease process and treatment.


Assuntos
Anti-Helmínticos/uso terapêutico , Regulação da Expressão Gênica/imunologia , Células Caliciformes/metabolismo , Doenças dos Cavalos/imunologia , Estrongilídios/imunologia , Animais , Citocinas/metabolismo , Fenbendazol/uso terapêutico , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Doenças dos Cavalos/tratamento farmacológico , Doenças dos Cavalos/parasitologia , Cavalos , Larva/efeitos dos fármacos , Macrolídeos/uso terapêutico , Estrongilídios/efeitos dos fármacos
16.
Am J Ophthalmol ; 213: 267-282, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32006483

RESUMO

PURPOSE: The purpose of this study was to investigate an enlarged dacryoadenotic lacrimal gland and normal lacrimal glands for the presence of goblet cells (mucocytes). DESIGN: Retrospective clinicopathologic series. METHODS: An enlarged lacrimal gland (dacryoadenosis) without obvious histopathologic alterations was extensively evaluated histochemically, immunohistochemically, and ultrastructurally to detect the presence of goblet cells and to compare the findings with those in five normal lacrimal glands. RESULTS: Granular, zymogen-rich pyramidal acinar cells in normal glands predominated over a previously not reported subpopulation of nongranular, pale-staining cells in both dacryoadenotic and normal lacrimal glands. These cells histochemically stained positively with mucicarmine and Alcian blue. Immunohistochemical and electron microscopic evaluations established that there was a displacement or replacement of cytoplasmic gross cystic disease fluid protein-15 and CK 7-positive tonofilaments in the pale acinar cells by myriad mucus granules. The goblet cells constituted approximately 2% of the normal acinar cells and 5% of dacryoadenotic acinar cells. A depletion of myoepithelial cells and ectopic intra-acinar ductular cells were also observed in dacryoadenosis. CONCLUSION: Dacryoadenosis is caused by an increase in the number of acini without individual acinar cell hyperplasia. A normal cytologic feature of the lacrimal gland is the presence of acinar goblet cells that had been long overlooked; they are increased in number in dacryoadenosis. Intra-acinar ductular cells and the scattered loss of myoepithelial cells are other abnormalities in dacryoadenosis. The presence of lacrimal gland goblet cells may have physiologic implications for the precorneal tear film and its derangements as well as for the histogenesis of mucus-producing carcinomas.


Assuntos
Células Caliciformes/ultraestrutura , Doenças do Aparelho Lacrimal/patologia , Aparelho Lacrimal/ultraestrutura , Azul Alciano/metabolismo , Carmim/metabolismo , Feminino , Células Caliciformes/metabolismo , Humanos , Queratina-7/metabolismo , Doenças do Aparelho Lacrimal/diagnóstico por imagem , Doenças do Aparelho Lacrimal/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Microscopia Eletrônica , Pessoa de Meia-Idade , Estudos Retrospectivos , Coloração e Rotulagem , Tomografia Computadorizada por Raios X
17.
Sci Rep ; 10(1): 2933, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076085

RESUMO

To compare goblet cell (GC) number and area in the covered superior (SB) versus exposed temporal (TB) bulbar conjunctiva in control versus aqueous tear deficient eyes (ATD) and evaluate correlation with tear MUC5AC protein. SB and TB impression cytology performed on control eyes, Sjögren syndrome (SS) ATD, and non-SS ATD was stained with period acid Schiff. GC number and area were measured with image analysis software. Protein-normalized MUC5AC level was measured in Schirmer strip-collected tears. Compared to control conjunctiva, GC number and area were significantly lower in SS, non-SS, and combined ATD groups in exposed TB, and were also significantly lower in SS and combined ATD groups in covered SB. In all ATD, GC number and area were significantly correlated, but differences between SB and TB were non-significant. Normalized tear MUC5AC protein was lower in all ATD groups versus control eyes, and correlated only with GC area. GCs are significantly decreased in the covered and exposed conjunctiva in SS. GC area may be a better disease measure than number for ATD. Correlation between tear MUC5AC concentration and GC area suggests tear MUC5AC mucin can be used as a disease-relevant biomarker for conjunctiva GC health.


Assuntos
Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/patologia , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Mucina-5AC/metabolismo , Lágrimas/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Contagem de Células , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
18.
Am J Respir Cell Mol Biol ; 62(4): 513-523, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31922915

RESUMO

In asthma, goblet cell numbers are increased within the airway epithelium, perpetuating the production of mucus that is more difficult to clear and results in airway mucus plugging. Notch1, Notch2, or Notch3, or a combination of these has been shown to influence the differentiation of airway epithelial cells. How the expression of specific Notch isoforms differs in fully differentiated adult asthmatic epithelium and whether Notch influences mucin production after differentiation is currently unknown. We aimed to quantify different Notch isoforms in the airway epithelium of individuals with severe asthma and to examine the impact of Notch signaling on mucin MUC5AC. Human lung sections and primary bronchial epithelial cells from individuals with and without asthma were used in this study. Primary bronchial epithelial cells were differentiated at the air-liquid interface for 28 days. Notch isoform expression was analyzed by Taqman quantitative PCR. Immunohistochemistry was used to localize and quantify Notch isoforms in human airway sections. Notch signaling was inhibited in vitro using dibenzazepine or Notch3-specific siRNA, followed by analysis of MUC5AC. NOTCH3 was highly expressed in asthmatic airway epithelium compared with nonasthmatic epithelium. Dibenzazepine significantly reduced MUC5AC production in air-liquid interface cultures of primary bronchial epithelial cells concomitantly with suppression of NOTCH3 intracellular domain protein. Specific knockdown using NOTCH3 siRNA recapitulated the dibenzazepine-induced reduction in MUC5AC. We demonstrate that NOTCH3 is a regulator of MUC5AC production. Increased NOTCH3 signaling in the asthmatic airway epithelium may therefore be an underlying driver of excess MUC5AC production.


Assuntos
Asma/metabolismo , Brônquios/metabolismo , Células Epiteliais/metabolismo , Pulmão/metabolismo , Mucina-5AC/metabolismo , Receptor Notch3/metabolismo , Transdução de Sinais/fisiologia , Idoso , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Células Caliciformes/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , RNA Interferente Pequeno/metabolismo , Mucosa Respiratória/metabolismo
19.
FASEB J ; 34(2): 3289-3304, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31916636

RESUMO

The enzyme glutathione S-transferase theta 1 (GSTT1) is involved in detoxifying chemicals, including reactive oxygen species (ROS). Here, we provide a significant insight into the role of GSTT1 in inflammatory bowel disease (IBD). We identified decreased expression of GSTT1 in inflamed colons from IBD patients compared to controls. We intrarectally or intraperitoneally delivered Gstt1 gene to mice with dextran sodium sulfate (DSS)-induced colitis and noted attenuation of colitis through gene transfer of Gstt1 via an IL-22 dependent pathway. Downregulation of GSTT1 by pathogen-associated molecular patterns (PAMPs) of microbes reduced innate defense responses and goblet cell differentiation. The GSTT1 mutation in intestinal epithelial cells (IECs) and IBD patients decreased its dimerization, which was connected to insufficient phosphorylation of signal transducer and activator of transcription-3 and p38/mitogen-activated protein kinase by their common activator, IL-22. GSTT1 ameliorated colitis and contributed as a modulator of goblet cells through sensing pathogens and host immune responses. Its mutations are linked to chronic intestinal inflammation due to its insufficient dimerization. Our results provide new insights into GSTT1 mutations that are linked to chronic intestinal inflammation due to its insufficient dimerization and their functional consequences in IBDs.


Assuntos
Diferenciação Celular , Colite Ulcerativa/metabolismo , Glutationa Transferase/metabolismo , Células Caliciformes/metabolismo , Interleucinas/metabolismo , Animais , Células Cultivadas , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Enterócitos/citologia , Enterócitos/metabolismo , Feminino , Glutationa Transferase/genética , Células Caliciformes/citologia , Células HT29 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Multimerização Proteica , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Células THP-1 , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
Environ Toxicol ; 35(6): 652-664, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31925992

RESUMO

1,2-Dimethylhydrazine (DMH), an environmental toxicant specifically targets the colon. The present study was aimed to evaluate the efficacy of gallic acid (GA) against colon toxicity induced by DMH in Wistar rats. GA, a phenolic acid has numerous beneficial properties, which include antiviral, antifungal and antioxidant properties which help cells to overcome oxidative stress and balance the redox homeostasis. GA was administered orally at two doses (25 and 50 mg/kg body weight) once daily for 14 days and a single dose (40 mg/kg body weight) of DMH was administered subcutaneously on 14th day. Animals were sacrificed on the 15th day and we could find that GA at both the doses significantly ameliorates DMH-induced increased toxicity markers and also substantially increases the glutathione content level and activities of detoxifying enzymes. It also ameliorates the expression of proliferation, inflammation, apoptosis, goblet cell disintegration, and mucin depletion in the colon that was elevated upon administration of DMH. Histological alterations provide further confirmation of the protective role of GA against DMH-induced colon toxicity. The results of this study clearly indicate supplementation of GA is beneficial in ameliorating DMH-induced oxidative stress, inflammation, proliferation, apoptosis, mucin depletion, and goblet cell disintegration in colon of Wistar rats.


Assuntos
1,2-Dimetilidrazina/toxicidade , Anti-Inflamatórios/toxicidade , Proliferação de Células/efeitos dos fármacos , Ácido Gálico/farmacologia , Células Caliciformes/efeitos dos fármacos , Mucinas/metabolismo , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Glutationa/metabolismo , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Inflamação , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar
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