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1.
Viruses ; 13(6)2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205098

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a 'cytokine storm.' In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.


Assuntos
COVID-19/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL11/genética , Quimiocina CXCL9/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação para Cima , Enzima de Conversão de Angiotensina 2/genética , Animais , Linhagem Celular , Quimiocina CXCL10/imunologia , Quimiocina CXCL11/imunologia , Quimiocina CXCL9/imunologia , Síndrome da Liberação de Citocina/genética , Síndrome da Liberação de Citocina/imunologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Feminino , Humanos , Inflamação , Pulmão/citologia , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia
2.
Viruses ; 13(7)2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202029

RESUMO

The current COVID-19 pandemic has highlighted the urgent need to develop effective therapeutic strategies. We evaluated the in vitro antiviral effect against SARS-CoV-2 of a hepatitis B virus (HBV) hexamer peptide, Poly6, which is capable of eliciting an antiviral effect against human immunodeficiency virus -1 (HIV-1), as a novel HIV-1 integrase inhibitor, and a strong anticancer immune response in an IFN-I-dependent manner, as a novel potential adjuvant in anticancer immunotherapy. Here, we report that Poly6 exerts an anti-SARS-CoV-2 effect, with an estimated 50% inhibitory concentration of 2.617 µM, in the human bronchial epithelial cell line, Calu-3 but not in Vero-E6 cells, which are deficient in type 1 interferon (IFN-I) signaling. We proved via assays based on mRNA profiles, inhibitors, or blocking antibodies that Poly6 can exert an anti-SARS-CoV-2 effect in an IFN-I-dependent manner. We also found that Poly6 inhibits IL-6 production enhanced by SARS-CoV-2 in infected Calu-3 cells at both the transcription and the translation levels, mediated via IL-10 induction in an IFN-I-dependent manner. These results indicate the feasibility of Poly6 as an IFN-I-inducing COVID-19 drug with potent antiviral and anti-inflammatory activities.


Assuntos
Antivirais/farmacologia , Células Epiteliais/efeitos dos fármacos , Vírus da Hepatite B/química , Interferon Tipo I/imunologia , Peptídeos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Brônquios/citologia , Brônquios/virologia , Chlorocebus aethiops , Células Epiteliais/imunologia , Células Epiteliais/virologia , Vírus da Hepatite B/genética , Humanos , Pulmão/citologia , Pulmão/virologia , Peptídeos/imunologia , SARS-CoV-2/imunologia , Células Vero
3.
Cells ; 10(6)2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208037

RESUMO

Neutrophils are the most abundant circulating innate immune cells and comprise the first immune defense line, as they are the most rapidly recruited cells at sites of infection or inflammation. Their main microbicidal mechanisms are degranulation, phagocytosis, cytokine secretion and the formation of extracellular traps. Neutrophil extracellular traps (NETs) are a microbicidal mechanism that involves neutrophil death. Since their discovery, in vitro and in vivo neutrophils have been challenged with a range of stimuli capable of inducing or inhibiting NET formation, with the objective to understand its function and regulation in health and disease. These networks composed of DNA and granular components are capable of immobilizing and killing pathogens. They comprise enzymes such as myeloperoxidase, elastase, cathepsin G, acid hydrolases and cationic peptides, all with antimicrobial and antifungal activity. Therefore, the excessive formation of NETs can also lead to tissue damage and promote local and systemic inflammation. Based on this concept, in this review, we focus on the role of NETs in different infectious and inflammatory diseases of the mucosal epithelia and skin.


Assuntos
Armadilhas Extracelulares/fisiologia , Membrana Mucosa/imunologia , Dermatopatias/imunologia , Células Epiteliais/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/fisiologia , Neutrófilos/imunologia , Neutrófilos/fisiologia , Dermatopatias/patologia
4.
Int J Mol Sci ; 22(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200743

RESUMO

Mastitis is a common disease in dairy cows that is mostly caused by E. coli, and it brings massive losses to the dairy industry. N6-Methyladenosine (m6A), a methylation at the N6 position of RNA adenine, is a type of modification strongly associated with many diseases. However, the role of m6A in mastitis has not been investigated. In this study, we used MeRIP-seq to sequence the RNA of bovine mammary epithelial cells treated with inactivated E. coli for 24 h. In this in vitro infection model, there were 16,691 m6A peaks within 7066 mRNA transcripts in the Con group and 10,029 peaks within 4891 transcripts in the E. coli group. Compared with the Con group, 474 mRNAs were hypermethylated and 2101 mRNAs were hypomethylated in the E. coli group. Biological function analyses revealed differential m6A-modified genes mainly enriched in the MAPK, NF-κB, and TGF-ß signaling pathways. In order to explore the relationship between m6A and mRNA expression, combined MeRIP-seq and mRNA-seq analyses revealed 212 genes with concomitant changes in the mRNA expression and m6A modification. This study is the first to present a map of RNA m6A modification in mastitis treated with E. coli, providing a basis for future research.


Assuntos
Adenosina/análogos & derivados , Metilação de DNA , Células Epiteliais/metabolismo , Infecções por Escherichia coli/veterinária , Regulação da Expressão Gênica , Glândulas Mamárias Animais/metabolismo , Mastite Bovina/genética , Adenosina/química , Animais , Bovinos , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Feminino , Perfilação da Expressão Gênica , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/imunologia , Mastite Bovina/microbiologia
6.
Front Immunol ; 12: 600017, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34025636

RESUMO

Epidermal growth factor (EGF) acts as a paracrine and autocrine mediator of cell proliferation and differentiation in various types of epithelial cells, such as sebocytes, which produce the lipid-rich sebum to moisturize the skin. However, sebum lipids via direct contact and by penetrating through the epidermis may have regulatory roles on epidermal and dermal cells as well. As EGF receptor (EGFR) is expressed throughout the proliferating and the lipid-producing layers of sebaceous glands (SGs) in healthy and acne-involved skin, we investigated the effect of EGF on SZ95 sebocytes and how it may alter the changes induced by palmitic acid (PA), a major sebum component with bioactive roles. We found that EGF is not only a potent stimulator of sebocyte proliferation, but also induces the secretion of interleukin (IL)6 and down-regulates the expression of genes involved in steroid and retinoid metabolism. Importantly, when applied in combination with PA, the PA-induced lipid accumulation was decreased and the cells secreted increased IL6 levels. Functional clustering of the differentially regulated genes in SZ95 sebocytes treated with EGF, PA or co-treated with EGF+PA further confirmed that EGF may be a potent inducer of hyperproliferative/inflammatory pathways (IL1 signaling), an effect being more pronounced in the presence of PA. However, while a group of inflammatory genes was up-regulated significantly in EGF+PA co-treated sebocytes, PA treatment in the absence of EGF, regulated genes only related to cell homeostasis. Meta-analysis of the gene expression profiles of whole acne tissue samples and EGF- and EGF+PA -treated SZ95 sebocytes showed that the EGF+PA co-activation of sebocytes may also have implications in disease. Altogether, our results reveal that PA-induced lipid accumulation and inflammation can be modulated by EGF in sebocytes, which also highlights the need for system biological approaches to better understand sebaceous (immuno)biology.


Assuntos
Fator de Crescimento Epidérmico/imunologia , Células Epiteliais/imunologia , Ácido Palmítico/farmacologia , Glândulas Sebáceas/imunologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/patologia , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Interleucina-6/imunologia , Ácido Palmítico/efeitos adversos , Glândulas Sebáceas/patologia
7.
Am J Pathol ; 191(7): 1292-1302, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33964217

RESUMO

Hyperactivation of the CXCL12-CXCR4 axis occurs in endometriosis; the therapeutic potential of treatments aimed at global inhibition of the axis was recently reported. Because CXCR4 is predominantly expressed on epithelial cells in the uterus, this study explored the effects of targeted disruption of CXCR4 in endometriosis lesions. Uteri derived from adult female mice homozygous for a floxed allele of CXCR4 and co-expressing Cre recombinase under control of progesterone receptor promoter were sutured onto the peritoneum of cycling host mice expressing the green fluorescent protein. Four weeks after endometriosis induction, significantly lower number of lesions developed in Cxcr4-conditional knockout lesions relative to those in controls (37.5% vs. 68.8%, respectively). In lesions that developed in Cxcr4-knockout, reduced epithelial proliferation was associated with a lower ratio of epithelial to total lesion area compared with controls. Furthermore, while CD3+ lymphocytes were largely excluded from the epithelial compartment in control lesions, in Cxcr4-knockout lesions, CD3+ lymphocytes infiltrated the Cxcr4-deficient epithelium in the diestrus and proestrus stages. Current data demonstrate that local CXCR4 expression is necessary for proliferation of the epithelial compartment of endometriosis lesions, that its downregulation compromises lesion numbers, and suggest a role for epithelial CXCR4 in lesion immune evasion.


Assuntos
Endometriose/imunologia , Endometriose/metabolismo , Linfócitos Intraepiteliais/imunologia , Receptores CXCR4/metabolismo , Animais , Proliferação de Células , Células Epiteliais/imunologia , Feminino , Camundongos
8.
Virulence ; 12(1): 1111-1121, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34034617

RESUMO

Coronaviruses and influenza viruses are circulating in humans and animals all over the world. Co-infection with these two viruses may aggravate clinical signs. However, the molecular mechanisms of co-infections by these two viruses are incompletely understood. In this study, we applied air-liquid interface (ALI) cultures of well-differentiated porcine tracheal epithelial cells (PTECs) to analyze the co-infection by a swine influenza virus (SIV, H3N2 subtype) and porcine respiratory coronavirus (PRCoV) at different time intervals. Our results revealed that in short-term intervals, prior infection by influenza virus caused complete inhibition of coronavirus infection, while in long-term intervals, some coronavirus replication was detectable. The influenza virus infection resulted in (i) an upregulation of porcine aminopeptidase N, the cellular receptor for PRCoV and (ii) in the induction of an innate immune response which was responsible for the inhibition of PRCoV replication. By contrast, prior infection by coronavirus only caused a slight inhibition of influenza virus replication. Taken together, the timing and the order of virus infection are important determinants in co-infections. This study is the first to show the impact of SIV and PRCoV co- and super-infection on the cellular level. Our results have implications also for human viruses, including potential co-infections by SARS-CoV-2 and seasonal influenza viruses.


Assuntos
Células Epiteliais/virologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Coronavirus Respiratório Porcino/fisiologia , Interferência Viral , Animais , Antígenos CD13/metabolismo , Células Cultivadas , Coinfecção/virologia , Infecções por Coronavirus/virologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Imunidade Inata , Infecções por Orthomyxoviridae/virologia , Suínos , Traqueia/citologia , Replicação Viral
9.
Nat Commun ; 12(1): 2405, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893305

RESUMO

Kidney intercalated cells are involved in acid-base homeostasis via vacuolar ATPase expression. Here we report six human intercalated cell subtypes, including hybrid principal-intercalated cells identified from single cell transcriptomics. Phagosome maturation is a biological process that increases in biological pathway analysis rank following exposure to uropathogenic Escherichia coli in two of the intercalated cell subtypes. Real time confocal microscopy visualization of murine renal tubules perfused with green fluorescent protein expressing Escherichia coli or pHrodo Green E. coli BioParticles demonstrates that intercalated cells actively phagocytose bacteria then acidify phagolysosomes. Additionally, intercalated cells have increased vacuolar ATPase expression following in vivo experimental UTI. Taken together, intercalated cells exhibit a transcriptional response conducive to the kidney's defense, engulf bacteria and acidify the internalized bacteria. Intercalated cells represent an epithelial cell with characteristics of professional phagocytes like macrophages.


Assuntos
Células Epiteliais/imunologia , Rim/imunologia , Fagócitos/imunologia , Escherichia coli Uropatogênica/imunologia , Ácidos/química , Animais , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Rim/citologia , Rim/metabolismo , Túbulos Renais Coletores/citologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Fagócitos/citologia , Análise de Célula Única/métodos , ATPases Vacuolares Próton-Translocadoras/metabolismo
10.
Proc Natl Acad Sci U S A ; 118(16)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33811184

RESUMO

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection; induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung; and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, whereas PKR activation is evident in iAT2 and iCM. In SARS-CoV-2-infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however, activation of OAS-RNase L and PKR is observed. This is in contrast to Middle East respiratory syndrome (MERS)-CoV, which effectively inhibits IFN signaling and OAS-RNase L and PKR pathways, but is similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.


Assuntos
Células Epiteliais/imunologia , Células Epiteliais/virologia , Imunidade Inata , Pulmão/patologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/virologia , RNA de Cadeia Dupla/metabolismo , SARS-CoV-2/imunologia , Células A549 , Endorribonucleases/metabolismo , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Nariz/virologia , Replicação Viral , eIF-2 Quinase
11.
FASEB J ; 35(5): e21535, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33817835

RESUMO

Thymic epithelial cells (TECs) are indispensable for T cell development, T cell receptor (TCR) repertoire selection, and specific lineage differentiation. Medullary thymic epithelial cells (mTECs), which account for the majority of TECs in adults, are critical for thymocyte selection and self-tolerance. CD74 is a nonpolymorphic transmembrane glycoprotein of major histocompatibility complex class II (MHCII) that is expressed in TECs. However, the exact role of CD74 in regulating the development of mTEC is poorly defined. In this research, we found that loss of CD74 resulted in a significant diminution in the medulla, a selective reduction in the cell number of mature mTECs expressing CD80 molecules, which eventually led to impaired thymic CD4+ T cell development. Moreover, RNA-sequence analysis showed that CD74 deficiency obviously downregulated the canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway in mTECs. Our results suggest that CD74 positively controls mTEC cellularity and maturation partially by activating the canonical NF-κB signaling pathway.


Assuntos
Antígenos de Diferenciação de Linfócitos B/fisiologia , Diferenciação Celular , Células Epiteliais/patologia , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe II/fisiologia , Ativação Linfocitária/imunologia , NF-kappa B/metabolismo , Timo/patologia , Animais , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , Transdução de Sinais , Timo/imunologia , Timo/metabolismo
12.
FASEB J ; 35(5): e21525, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33817836

RESUMO

Glycolysis is a well-known process by which metabolically active cells, such as tumor or immune cells meet their high metabolic demands. Previously, our laboratory has demonstrated that in airway epithelial cells, the pleiotropic cytokine, interleukin-1 beta (IL1B) induces glycolysis and that this contributes to allergic airway inflammation and remodeling. Activation of glycolysis is known to increase NADPH reducing equivalents generated from the pentose phosphate pathway, linking metabolic reprogramming with redox homeostasis. In addition, numerous glycolytic enzymes are known to be redox regulated. However, whether and how redox chemistry regulates metabolic reprogramming more generally remains unclear. In this study, we employed a multi-omics approach in primary mouse airway basal cells to evaluate the role of protein redox biochemistry, specifically protein glutathionylation, in mediating metabolic reprogramming. Our findings demonstrate that IL1B induces glutathionylation of multiple proteins involved in metabolic regulation, notably in the glycolysis pathway. Cells lacking Glutaredoxin-1 (Glrx), the enzyme responsible for reversing glutathionylation, show modulation of multiple metabolic pathways including an enhanced IL1B-induced glycolytic response. This was accompanied by increased secretion of thymic stromal lymphopoietin (TSLP), a cytokine important in asthma pathogenesis. Targeted inhibition of glycolysis prevented TSLP release, confirming the functional relevance of enhanced glycolysis in cells stimulated with IL1B. Collectively, data herein point to an intriguing link between glutathionylation chemistry and glycolytic reprogramming in epithelial cells and suggest that glutathionylation chemistry may represent a therapeutic target in pulmonary pathologies with perturbations in the glycolysis pathway.


Assuntos
Reprogramação Celular , Glutarredoxinas/fisiologia , Glutationa/metabolismo , Glicólise , Inflamação/imunologia , Interleucina-1beta/farmacologia , Pulmão/imunologia , Animais , Citocinas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução
13.
Life Sci ; 276: 119432, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33794253

RESUMO

BACKGROUND: Adenoid hypertrophy (AH) can cause harmful effects on untreated children, which include mouth breathing, chronic intermittent hypoxia, sleep disordered breathing (SDB), and even some behavioral problems. However, the molecular mechanisms underlying this pathophysiological process have remained poorly understood. METHODS: In this study, SUMO was induced silencing and overexpression using RNAi and lentiviral-mediated vector. FITC-Dextran and TEER were performed to examine the role of SUMO in cell permeability. Co-immunoprecipitation (Co-IP) assay was performed to examine the interaction between SUMO1 and HIF-1α. Immunohistochemistry staining was used to examine the expressions of ZO-1, Claudin-1 and occluding respectively. RESULTS: We found that a hypoxic condition caused a dramatic upregulation of SUMO-1 expression in a time-dependent manner, a member of the ubiquitin-like protein family. Knockdown of SUMO-1 deeply suppressed the secretions of pro-inflammation cytokines including IL-6, IL-8, and TNF-α, and decreased the permeability of HTECs. Moreover, the HIF-1α inhibitor 2-MeOE2 abolished the function of SUMO-1 in HTECs. Furthermore, results obtained from CO-IP had suggested that SUMO-1 interacted with HIF-1α, and prevented its ubiquitination and degradation in HTECs by sumoylating. Importantly, our data showed that hypoxia-induced inflammation was markedly inhibited by M2 macrophages that possess potent anti-inflammatory function. CONCLUSION: Our results suggest that selectively inhibiting the SUMO-1-HIF-1α signaling pathway leads to anti-inflammatory responses in human tonsil epithelial cells, which might be a novel therapeutic approach for managing hypoxia-induced SDB resulting from AH.


Assuntos
Citocinas/metabolismo , Células Epiteliais/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/fisiopatologia , Mediadores da Inflamação/metabolismo , Tonsila Palatina/patologia , Proteína SUMO-1/metabolismo , Permeabilidade da Membrana Celular , Células Cultivadas , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Tonsila Palatina/imunologia , Tonsila Palatina/metabolismo , Proteína SUMO-1/genética
14.
Int J Mol Sci ; 22(7)2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33806254

RESUMO

The global COVID-19 pandemic caused by SARS-CoV-2 has resulted in over 2.2 million deaths. Disease outcomes range from asymptomatic to severe with, so far, minimal genotypic change to the virus so understanding the host response is paramount. Transcriptomics has become incredibly important in understanding host-pathogen interactions; however, post-transcriptional regulation plays an important role in infection and immunity through translation and mRNA stability, allowing tight control over potent host responses by both the host and the invading virus. Here, we apply ribosome profiling to assess post-transcriptional regulation of host genes during SARS-CoV-2 infection of a human lung epithelial cell line (Calu-3). We have identified numerous transcription factors (JUN, ZBTB20, ATF3, HIVEP2 and EGR1) as well as select antiviral cytokine genes, namely IFNB1, IFNL1,2 and 3, IL-6 and CCL5, that are restricted at the post-transcriptional level by SARS-CoV-2 infection and discuss the impact this would have on the host response to infection. This early phase restriction of antiviral transcripts in the lungs may allow high viral load and consequent immune dysregulation typically seen in SARS-CoV-2 infection.


Assuntos
Citocinas/genética , Processamento Pós-Transcricional do RNA , Ribossomos/metabolismo , Ribossomos/virologia , SARS-CoV-2/imunologia , Fatores de Transcrição/genética , Animais , Antivirais/antagonistas & inibidores , Linhagem Celular Tumoral , Chlorocebus aethiops , Biologia Computacional , Citocinas/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Interações entre Hospedeiro e Microrganismos , Humanos , Imunidade Inata/genética , Pulmão/imunologia , Pulmão/virologia , RNA Mensageiro/metabolismo , RNA-Seq , Ribossomos/genética , SARS-CoV-2/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma , Células Vero
15.
J Allergy Clin Immunol ; 147(6): 2083-2097.e6, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33852936

RESUMO

BACKGROUND: Excessive inflammation triggered by a hitherto undescribed mechanism is a hallmark of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and is associated with enhanced pathogenicity and mortality. OBJECTIVE: Complement hyperactivation promotes lung injury and was observed in patients suffering from Middle East respiratory syndrome-related coronavirus, SARS-CoV-1, and SARS-CoV-2 infections. Therefore, we investigated the very first interactions of primary human airway epithelial cells on exposure to SARS-CoV-2 in terms of complement component 3 (C3)-mediated effects. METHODS: For this, we used highly differentiated primary human 3-dimensional tissue models infected with SARS-CoV-2 patient isolates. On infection, viral load, viral infectivity, intracellular complement activation, inflammatory mechanisms, and tissue destruction were analyzed by real-time RT-PCR, high content screening, plaque assays, luminex analyses, and transepithelial electrical resistance measurements. RESULTS: Here, we show that primary normal human bronchial and small airway epithelial cells respond to SARS-CoV-2 infection by an inflated local C3 mobilization. SARS-CoV-2 infection resulted in exaggerated intracellular complement activation and destruction of the epithelial integrity in monolayer cultures of primary human airway cells and highly differentiated, pseudostratified, mucus-producing, ciliated respiratory tissue models. SARS-CoV-2-infected 3-dimensional cultures secreted significantly higher levels of C3a and the proinflammatory cytokines IL-6, monocyte chemoattractant protein 1, IL-1α, and RANTES. CONCLUSIONS: Crucially, we illustrate here for the first time that targeting the anaphylotoxin receptors C3a receptor and C5a receptor in nonimmune respiratory cells can prevent intrinsic lung inflammation and tissue damage. This opens up the exciting possibility in the treatment of COVID-19.


Assuntos
Brônquios/imunologia , COVID-19/imunologia , Ativação do Complemento , Células Epiteliais/imunologia , Receptor da Anafilatoxina C5a/imunologia , Mucosa Respiratória/imunologia , SARS-CoV-2/imunologia , Brônquios/patologia , Brônquios/virologia , COVID-19/patologia , COVID-19/virologia , Linhagem Celular , Complemento C3/imunologia , Citocinas/imunologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia
16.
Mol Med Rep ; 23(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33760139

RESUMO

Proximal tubular epithelial cells (PTECs) have innate immune characteristics, and produce proinflammatory factors, chemokines and complement components that drive epithelial­mesenchymal transition (EMT). Our previous studies revealed that human mesangial cells and podocytes were able to synthesize and secrete immunoglobulin (Ig)A and IgG, respectively. The aim of the present study was to evaluate the expression of Igs in PTECs. Firstly, IgG was detected in the cytoplasm, the cell membrane and the lumen of PTECs in the normal renal cortex by immunohistochemistry. Secondly, Igγ gene transcription and V(D)J recombination were detected in single PTECs by nested PCR and Sanger sequencing. Thirdly, Igγ, Igκ and Igλ were clearly detected in an immortalized PTEC line (HK­2) by immunostaining and western blotting, in which RP215 (an antibody that predominantly binds to non­B cell­derived IgG) was used. In addition, Igγ, Igκ and Igλ gene transcripts, conservative V(D)J recombination in the Igγ variable region, recombination activating gene 1/2 and activation­induced cytidine deaminase were all detected in HK­2 cells. These data suggested that PTECs may express IgG in a similar manner to B cells. Furthermore, IgG expression was upregulated by TGF­ß1 and may be involved in EMT.


Assuntos
Fibrose/genética , Imunoglobulina G/genética , Túbulos Renais Proximais/imunologia , Fator de Crescimento Transformador beta1/genética , Linhagem Celular , Células Epiteliais/imunologia , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal , Fibrose/patologia , Regulação da Expressão Gênica , Humanos , Imunoglobulina G/imunologia , Túbulos Renais Proximais/patologia , Podócitos/imunologia , Podócitos/metabolismo , RNA Mensageiro/genética , Análise de Célula Única
17.
Front Immunol ; 12: 580454, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33679733

RESUMO

Numerous types of viruses have been found in human semen, which raises concerns about the sexual transmission of these viruses. The overall effect of semen on viral infection and transmission have yet to be fully investigated. In the present study, we aimed at the effect of seminal plasma (SP) on viral infection by focusing on the mumps viral (MuV) infection of HeLa cells. MuV efficiently infected HeLa cells in vitro. MuV infection was strongly inhibited by the pre-treatment of viruses with SP. SP inhibited MuV infection through the impairment of the virus's attachment to cells. The antiviral activity of SP was resistant to the treatment of SP with boiling water, Proteinase K, RNase A, and DNase I, suggesting that the antiviral factor would not be proteins and nucleic acids. PNGase or PLA2 treatments did not abrogate the antiviral effect of SP against MuV. Further, we showed that the prostatic fluid (PF) showed similar inhibition as SP, whereas the epididymal fluid and seminal vesicle extract did not inhibit MuV infection. Both SP and PF also inhibited MuV infection of other cell types, including another human cervical carcinoma cell line C33a, mouse primary epididymal epithelial cells, and Sertoli cell line 15P1. Moreover, this inhibitory effect was not specific to MuV, as the herpes simplex virus 1, dengue virus 2, and adenovirus 5 infections were also inhibited by SP and PF. Our findings suggest that SP contains a prostate-derived pan-antiviral factor that may limit the sexual transmission of various viruses.


Assuntos
Antivirais/imunologia , Células Epiteliais/imunologia , Vírus da Caxumba/imunologia , Sêmen/imunologia , Vírus/imunologia , Animais , Antivirais/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Células HeLa , Interações Hospedeiro-Patógeno/imunologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Vírus da Caxumba/fisiologia , Sêmen/metabolismo , Sêmen/virologia , Células Vero
18.
Int J Radiat Oncol Biol Phys ; 110(5): 1283-1294, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-33722770

RESUMO

PURPOSE: Severe pneumonia and acute respiratory distress syndrome (ARDS) have been described in patients with severe coronavirus disease 2019 (COVID-19). Recently, early clinical data reported the feasibility of low doses of radiation therapy (RT) in the treatment of ARDS in patients with severe COVID-19. However, the involved mechanisms remained unknown. METHODS AND MATERIALS: Here, we used airways-instilled lipopolysaccharide (LPS) and influenza virus (H1N1) as murine models of pneumonia, and toll-like receptor (TLR)-3 stimulation in human lung macrophages. RESULTS: Low doses of RT (0.5-1 Gray) decreased LPS-induced pneumonia, and increased the percentage of nerve- and airway-associated macrophages producing interleukin (IL) 10. During H1N1 viral infection, we observed decreased lung tissue damage and immune cell infiltration in irradiated animals. Low doses of RT increased IL-10 production by infiltrating immune cells into the lung. Irradiation of TLR-3 ligand-stimulated human lung macrophages ex vivo increased IL-10 secretion and decreased interferon γ production in the culture supernatant. The percentage of human lung macrophages producing IL-6 was also decreased. CONCLUSIONS: Our data highlight a mechanism by which low doses of RT regulate lung inflammation and skew lung macrophages toward an anti-inflammatory profile. These data provide a preclinical mechanistic support to clinical trials evaluating low doses of RT, such as COVID-19-induced ARDS.


Assuntos
Células Epiteliais/efeitos da radiação , Vírus da Influenza A Subtipo H1N1 , Interleucina-10/biossíntese , Macrófagos/efeitos da radiação , Pneumonia Viral/radioterapia , Síndrome do Desconforto Respiratório/radioterapia , Animais , Anti-Inflamatórios/farmacologia , COVID-19/complicações , Dexametasona/farmacologia , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Citometria de Fluxo , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos da radiação , Interferon gama/biossíntese , Interleucina-6/biossíntese , Lipopolissacarídeos , Pulmão/citologia , Pulmão/patologia , Pulmão/efeitos da radiação , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia Viral/etiologia , Pneumonia Viral/prevenção & controle , Poli I-C , Dosagem Radioterapêutica , Síndrome do Desconforto Respiratório/etiologia , Receptor 3 Toll-Like , Carga Viral/efeitos da radiação
19.
Methods Mol Biol ; 2291: 253-272, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704757

RESUMO

Therapeutic antibodies (Abs) inhibiting bacterial adhesion to host epithelia are an attractive option to reduce the load of Shiga toxin-producing E. coli (STEC) in the intestine of the patient and also in the bovine reservoir, thereby minimizing the risk of STEC contamination in the food chain. Of particular interest are recombinant single-domain Ab fragments called nanobodies (Nbs) derived from the variable domain of camelid heavy chain-only antibodies (VHH). The outer membrane adhesin intimin and the translocated intimin receptor (Tir) are essential for the attachment of STEC to host epithelia. In addition, EspA filaments of the bacterial type III protein secretion system are needed for Tir translocation into the host cell. Given their importance for bacterial adhesion and colonization, we developed Nbs against intimin, Tir and EspA proteins of STEC serotype O157:H7. Here, we report the screening methods used to isolate inhibitory Nbs blocking intimin-Tir protein-protein interaction, actin-pedestal formation, and intimate adhesion of STEC to epithelial cells in vitro. First, we describe how VHH gene repertoires can be produced as Nbs secreted by E. coli using the α-hemolysin (HlyA) protein secretion system. Next, we report the methods for identification of inhibitors of intimin-Tir protein-protein interaction and of STEC intimate adhesion to HeLa cells in culture. These methods can be adapted for the screening of Nbs against different adhesin-receptor complexes to block the adhesion of other pathogens to host cells.


Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/imunologia , Aderência Bacteriana/imunologia , Células Epiteliais , Escherichia coli O157/imunologia , Proteínas de Escherichia coli/imunologia , Receptores de Superfície Celular/imunologia , Anticorpos de Domínio Único/imunologia , Animais , Bovinos , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Escherichia coli O157/patogenicidade , Humanos
20.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669494

RESUMO

Campylobacter concisus is a human-pathogenic bacterium of the gastrointestinal tract. This study aimed at the contribution of the mucosal immune system in the context of intestinal epithelial barrier dysfunction induced by C. concisus. As an experimental leaky gut model, we used in vitro co-cultures of colonic epithelial cell monolayers (HT-29/B6-GR/MR) with M1-macrophage-like THP-1 cells on the basal side. Forty-eight hours after C. concisus infection, the decrease in the transepithelial electrical resistance in cell monolayers was more pronounced in co-culture condition and 22 ± 2% (p < 0.001) higher than the monoculture condition without THP-1 cells. Concomitantly, we observed a reduction in the expression of the tight junction proteins occludin and tricellulin. We also detected a profound increase in 4 kDa FITC-dextran permeability in C. concisus-infected cell monolayers only in co-culture conditions. This is explained by loss of tricellulin from tricellular tight junctions (tTJs) after C. concisus infection. As an underlying mechanism, we observed an inflammatory response after C. concisus infection through pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) released from THP-1 cells in the co-culture condition. In conclusion, the activation of subepithelial immune cells exacerbates colonic epithelial barrier dysfunction by C. concisus through tricellulin disruption in tTJs, leading to increased antigen permeability (leaky gut concept).


Assuntos
Campylobacter/imunologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Apoptose , Linhagem Celular , Sobrevivência Celular , Técnicas de Cocultura , Impedância Elétrica , Células Epiteliais/patologia , Humanos , Inflamação/patologia , Intestinos/microbiologia , Intestinos/patologia , Macrófagos/metabolismo , Modelos Biológicos , Ocludina/metabolismo , Frações Subcelulares/metabolismo , Junções Íntimas/metabolismo
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