RESUMO
The recent development of single cell sequencing technologies has revolutionized the state-of-art of cell biology, allowing the simultaneous measurement of thousands of genes in single cells. This technology has been applied to study the transcriptome of single cells in homeostasis and also in response to pathogenic exposure, greatly increasing our knowledge of the immune response to infectious agents. Yet the number of these studies performed in aquacultured fish species is still very limited. Thus, in the current study, we have used the 10x Genomics single cell RNA sequencing technology to study the response of rainbow trout (Oncorhynchus mykiss) peripheral blood leukocytes (PBLs) to infectious pancreatic necrosis virus (IPNV), an important trout pathogen. The study allowed us to obtain a transcriptomic profile of 12 transcriptionally distinct leukocyte cell subpopulations that included four different subsets of B cells, T cells, monocytes, two populations of dendritic-like cells (DCs), hematopoietic progenitor cells, non-specific cytotoxic cells (NCC), neutrophils and thrombocytes. The transcriptional pattern of these leukocyte subpopulations was compared in PBL cultures that had been exposed in vitro to IPNV for 24 h and mock-infected cultures. Our results revealed that monocytes and neutrophils showed the highest number of upregulated protein-coding genes in response to IPNV. Interestingly, IgM+IgD+ and IgT+ B cells also upregulated an important number of genes to the virus, but a much fainter response was observed in ccl4 + or plasma-like cells (irf4 + cells). A substantial number of protein-coding genes and genes coding for ribosomal proteins were also transcriptionally upregulated in response to IPNV in T cells and thrombocytes. Interestingly, although genes coding for ribosomal proteins were regulated in all affected PBL subpopulations, the number of such genes transcriptionally regulated was higher in IgM+IgD+ and IgT+ B cells. A further analysis dissected which of the regulated genes were common and which were specific to the different cell clusters, identifying eight genes that were transcriptionally upregulated in all the affected groups. The data provided constitutes a comprehensive transcriptional perspective of how the different leukocyte populations present in blood respond to an early viral encounter in fish.
Assuntos
Infecções por Birnaviridae , Doenças dos Peixes , Vírus da Necrose Pancreática Infecciosa , Leucócitos , Oncorhynchus mykiss , Análise de Célula Única , Animais , Oncorhynchus mykiss/imunologia , Oncorhynchus mykiss/virologia , Vírus da Necrose Pancreática Infecciosa/imunologia , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/veterinária , Infecções por Birnaviridae/virologia , Análise de Célula Única/métodos , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Leucócitos/imunologia , Leucócitos/virologia , Transcriptoma , Perfilação da Expressão Gênica/métodosRESUMO
Enterovirus A71 (EV-A71) is associated with neurological conditions such as acute meningitis and encephalitis. The virus is detected in the bloodstream, and high blood viral loads are associated with central nervous system (CNS) manifestations. We used an in vitro blood-brain barrier (BBB) model made up of human brain-like endothelial cells (hBLECs) and brain pericytes grown in transwell systems to investigate whether three genetically distinct EV-A71 strains (subgenogroups C1, C1-like, and C4) can cross the human BBB. EV-A71 poorly replicated in hBLECs, which released moderate amounts of infectious viruses from their luminal side and trace amounts of infectious viruses from their basolateral side. The barrier properties of hBLECs were not impaired by EV-A71 infection. We investigated the passage through hBLECs of EV-A71-infected white blood cells. EV-A71 strains efficiently replicated in immune cells, including monocytes, neutrophils, and NK/T cells. Attachment to hBLECs of immune cells infected with the C1-like virus was higher than attachment of cells infected with C1-06. EV-A71 infection did not impair the transmigration of immune cells through hBLECs. Overall, EV-A71 targets different white blood cell populations that have the potential to be used as a Trojan horse to cross hBLECs more efficiently than cell-free EV-A71 particles.IMPORTANCEEnterovirus A71 (EV-A71) was first reported in the USA, and numerous outbreaks have since occurred in Asia and Europe. EV-A71 re-emerged as a new multirecombinant strain in 2015 in Europe and is now widespread. The virus causes hand-foot-and-mouth disease in young children and is involved in nervous system infections. How the virus spreads to the nervous system is unclear. We investigated whether white blood cells could be infected by EV-A71 and transmit it across human endothelial cells mimicking the blood-brain barrier protecting the brain from adverse effects. We found that endothelial cells provide a strong roadblock to prevent the passage of free virus particles but allow the migration of infected immune cells, including monocytes, neutrophils, and NK/T cells. Our data are consistent with the potential role of immune cells in the pathogenesis of EV-A71 infections by spreading the virus in the blood and across the human blood-brain barrier.
Assuntos
Barreira Hematoencefálica , Células Endoteliais , Enterovirus Humano A , Infecções por Enterovirus , Barreira Hematoencefálica/virologia , Humanos , Enterovirus Humano A/genética , Enterovirus Humano A/fisiologia , Infecções por Enterovirus/virologia , Infecções por Enterovirus/imunologia , Células Endoteliais/virologia , Replicação Viral , Monócitos/virologia , Monócitos/imunologia , Pericitos/virologia , Leucócitos/virologia , Leucócitos/imunologia , Encéfalo/virologia , Células Matadoras Naturais/imunologia , Neutrófilos/imunologia , Neutrófilos/virologiaRESUMO
BACKGROUND: Chronic/latent viral infections may accelerate immunological aging, particularly among people living with HIV (PLWH). We characterized chronic/latent virus infections across their lifespan and investigated their associations with leukocyte telomere length (LTL). METHODS: Participants enrolled in the CARMA cohort study were randomly selected to include n = 15 for each decade of age between 0 and >60 y, for each sex, and each HIV status. Cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 8 (HHV-8), herpes simplex virus 1 (HSV-1), and HSV-2 infection were determined serologically; HIV, hepatitis C (HCV), and hepatitis B (HBV) were self-reported. LTLs were measured using monochrome multiplex qPCR. Associations between the number of viruses, LTL, and sociodemographic factors were assessed using ordinal logistic and linear regression modeling. RESULTS: The study included 187 PLWH (105 female/82 male) and 190 HIV-negative participants (105 female/84 male), ranging in age from 0.7 to 76.1 years. Living with HIV, being older, and being female were associated with harbouring a greater number of chronic/latent non-HIV viruses. Having more infections was in turn bivariately associated with a shorter LTL. In multivariable analyses, older age, living with HIV, and the female sex remained independently associated with having more infections, while having 3-4 viruses (vs. 0-2) was associated with a shorter LTL. CONCLUSIONS: Our results suggest that persistent viral infections are more prevalent in PLWH and females, and that these may contribute to immunological aging. Whether this is associated with comorbidities later in life remains an important question.
Assuntos
Infecções por HIV , Leucócitos , Humanos , Feminino , Infecções por HIV/virologia , Infecções por HIV/imunologia , Masculino , Leucócitos/virologia , Pessoa de Meia-Idade , Adulto , Idoso , Adulto Jovem , Adolescente , Criança , Telômero/genética , Lactente , Pré-Escolar , Infecção Latente/virologia , Viroses/virologia , Viroses/imunologia , Doença Crônica , Estudos de Coortes , Recém-NascidoRESUMO
Bovine viral diarrhea virus (BVDV) infections cause USD 1.5-2 billion in losses annually. Maternal BVDV after 150 days of gestation causes transient fetal infection (TI) in which the fetal immune response clears the virus. The impact of fetal TI BVDV infections on postnatal growth and white blood cell (WBC) methylome as an index of epigenetic modifications was examined by inoculating pregnant heifers with noncytopathic type 2 BVDV or media (sham-inoculated controls) on Day 175 of gestation to generate TI (n = 11) and control heifer calves (n = 12). Fetal infection in TI calves was confirmed by virus-neutralizing antibody titers at birth and control calves were seronegative. Both control and TI calves were negative for BVDV RNA in WBCs by RT-PCR. The mean weight of the TI calves was less than that of the controls (p < 0.05). DNA methyl seq analysis of WBC DNA demonstrated 2349 differentially methylated cytosines (p ≤ 0.05) including 1277 hypomethylated cytosines, 1072 hypermethylated cytosines, 84 differentially methylated regions based on CpGs in promoters, and 89 DMRs in islands of TI WBC DNA compared to controls. Fetal BVDV infection during late gestation resulted in epigenomic modifications predicted to affect fetal development and immune pathways, suggesting potential consequences for postnatal growth and health of TI cattle.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina , Metilação de DNA , Vírus da Diarreia Viral Bovina , Epigênese Genética , Leucócitos , Animais , Bovinos , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/genética , Feminino , Gravidez , Leucócitos/virologia , Vírus da Diarreia Viral Bovina/genética , Anticorpos Antivirais/sangue , Doenças Fetais/virologia , Doenças Fetais/veterinária , Doenças Fetais/genética , Vírus da Diarreia Viral Bovina Tipo 2/genética , Feto/virologiaRESUMO
Introduction: The causative agent of COVID-19 (Coronavirus Disease 2019) is an enveloped RNA (ribonucleic acid) virus of the SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) family. The effects of SARS-CoV-2 on the differentiation and maturation of blood cells have been the subject of several studies, we report our experience of an investigation of the morphologic abnormalities of leukocytes observed during COVID-19. Patients and methods: This is a prospective study of 5 months, from February 2021 to June 2021. Forty COVID-19 patients and 20 healthy controls were included in this study. We performed complete blood count and peripheral blood smear of all patients and control samples. Leukocytes abnormalities were quantified as a percentage of 100 leukocytes of the same lineage. Results: The morphological abnormalities of the leukocytes found in percentage of patients have been mainly neutrophils bilobed 72,5%, hypogranulation 45%, acquired pseudo Pelger-Huet 35%, vacuolated neutrophils 42,5%, Apoptotic neutrophils 35,5 %, neutrophils with toxic granulations 30%, myelemia 45%, atypical lymphocytes 52,5%, lymphoplasmocytes 60% and vacuolated monocytes 27, 5%. Conclusion: Our study revealed several morphological abnormalities of the different cells of the leukocyte lineage. The presence of toxic granulations in the cytoplasm of the myelocytes was specific to this study.
Assuntos
COVID-19 , Leucócitos , SARS-CoV-2 , Humanos , COVID-19/patologia , Leucócitos/patologia , Leucócitos/virologia , Estudos Prospectivos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Neutrófilos/patologia , Estudos de Casos e Controles , Idoso , Contagem de LeucócitosRESUMO
Patients with COVID-19 generally raise antibodies against SARS-CoV-2 following infection, and the antibody level is positively correlated to the severity of disease. Whether the viral antibodies exacerbate COVID-19 through antibody-dependent enhancement (ADE) is still not fully understood. Here, we conducted in vitro assessment of whether convalescent serum enhanced SARS-CoV-2 infection or induced excessive immune responses in immune cells. Our data revealed that SARS-CoV-2 infection of primary B cells, macrophages and monocytes, which express variable levels of FcγR, could be enhanced by convalescent serum from COVID-19 patients. We also determined the factors associated with ADE, and found which showed a time-dependent but not viral-dose dependent manner. Furthermore, the ADE effect is not associated with the neutralizing titer or RBD antibody level when testing serum samples collected from different patients. However, it is higher in a medium level than low or high dilutions in a given sample that showed ADE effect, which is similar to dengue. Finally, we demonstrated more viral genes or dysregulated host immune gene expression under ADE conditions compared to the no-serum infection group. Collectively, our study provides insight into the understanding of an association of high viral antibody titer and severe lung pathology in severe patients with COVID-19.
Assuntos
Anticorpos Facilitadores/imunologia , Leucócitos/virologia , SARS-CoV-2/patogenicidade , COVID-19/imunologia , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Soros Imunes/imunologia , Leucócitos/metabolismo , Receptores de IgG/metabolismo , Replicação Viral/imunologiaRESUMO
Small ruminant lentiviruses (SRLVs) exist as populations of closely related genetic variants, known as quasispecies, within an individual host. The privileged way of SRLVs transmission in goats is through the ingestion of colostrum and milk of infected does. Thus, characterization of SRLV variants transmitted through the milk, including milk epithelial cells (MEC), may provide useful information about the transmission and evolution of SRLVs. Therefore, the aim of this study was to detect SRLVs in peripheral blood leukocytes (PBLs) and milk epithelial cells of goats naturally infected with SRLVs and perform single nucleotide variations analysis to characterize the extent of genetic heterogeneity of detected SRLVs through comparison of their gag gene sequences. Blood and milk samples from 24 seropositive goats were tested in this study. The double immunolabeling against p28 and cytokeratin demonstrated that milk epithelial cells originated from naturally infected goats were infected by SRLVs. Moreover, PCR confirmed the presence of the integrated SRLVs proviral genome indicating that MECs may have a role as a reservoir of SRLVs and can transmit the virus through milk. The blood and MEC derived sequences from 7 goats were successfully sequenced using NGS and revealed that these sequences were genetically similar. The MEC and blood-derived sequences contained from 3 to 30 (mean, 10.8) and from 1 to 10 (mean, 5.4) unique SNVs, respectively. In five out of seven goats, SNVs occurred more frequent in MEC derived sequences. Non-synonymous SNVs were found in both, PBLs and MEC-derived sequences of analyzed goats and their total number differed between animals. The results of this study add to our understanding of SRLVs genomic variability. Our data provides evidence for the existence of SRLVs quasispecies and to our knowledge, this is the first study that showed quasispecies composition and minority variants of SRLVs present milk epithelial cells.
Assuntos
Cabras/virologia , Lentivirus/isolamento & purificação , Leucócitos/virologia , Leite/virologia , Animais , Células Cultivadas , Células Epiteliais/virologia , Lentivirus/genética , Polimorfismo de Nucleotídeo ÚnicoAssuntos
COVID-19/diagnóstico , Síndrome da Liberação de Citocina/diagnóstico , Coagulação Intravascular Disseminada/diagnóstico , SARS-CoV-2/patogenicidade , Aspartato Aminotransferases/sangue , Doenças Assintomáticas , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , COVID-19/mortalidade , COVID-19/patologia , Creatina Quinase/sangue , Síndrome da Liberação de Citocina/tratamento farmacológico , Síndrome da Liberação de Citocina/mortalidade , Síndrome da Liberação de Citocina/patologia , Coagulação Intravascular Disseminada/tratamento farmacológico , Coagulação Intravascular Disseminada/mortalidade , Coagulação Intravascular Disseminada/patologia , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Humanos , Interleucina-6/sangue , L-Lactato Desidrogenase/sangue , Leucócitos/imunologia , Leucócitos/virologia , Linfopenia/diagnóstico , Linfopenia/patologia , Linfopenia/virologia , Prognóstico , Índice de Gravidade de Doença , Análise de Sobrevida , Trombocitopenia/diagnóstico , Trombocitopenia/patologia , Trombocitopenia/virologia , Tratamento Farmacológico da COVID-19RESUMO
Severe coronavirus disease 2019 (COVID-19) is characterized by overproduction of immune mediators, but the role of interferons (IFNs) of the type I (IFN-I) or type III (IFN-III) families remains debated. We scrutinized the production of IFNs along the respiratory tract of COVID-19 patients and found that high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity. Production of specific IFN-III, but not IFN-I, members denotes patients with a mild pathology and efficiently drives the transcription of genes that protect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In contrast, compared to subjects with other infectious or noninfectious lung pathologies, IFNs are overrepresented in the lower airways of patients with severe COVID-19 that exhibit gene pathways associated with increased apoptosis and decreased proliferation. Our data demonstrate a dynamic production of IFNs in SARS-CoV-2-infected patients and show IFNs play opposing roles at distinct anatomical sites.
Assuntos
COVID-19/patologia , Interferons/metabolismo , Sistema Respiratório/virologia , Índice de Gravidade de Doença , Fatores Etários , Envelhecimento/patologia , COVID-19/genética , COVID-19/imunologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Regulação da Expressão Gênica , Humanos , Interferons/genética , Leucócitos/patologia , Leucócitos/virologia , Pulmão/patologia , Pulmão/virologia , Síndrome do Desconforto Respiratório/patologia , Síndrome do Desconforto Respiratório/virologia , Carga ViralRESUMO
Hantaan virus (HTNV) infects humans and causes hemorrhagic fever with renal syndrome (HFRS). The development of well-characterized animal models of HFRS could accelerate the testing of vaccine candidates and therapeutic agents and provide a useful tool for studying the pathogenesis of HFRS. Because NLRC3 has multiple immunoregulatory roles, we investigated the susceptibility of Nlrc3-/- mice to HTNV infection in order to establish a new model of HFRS. Nlrc3-/- mice developed weight loss, renal hemorrhage, and tubule dilation after HTNV infection, recapitulating many clinical symptoms of human HFRS. Moreover, infected Nlrc3-/- mice showed higher viral loads in serum, spleen, and kidney than wild type C57BL/6 (WT) mice, and some of them manifested more hematological disorders and significant pathological changes within multiple organs than WT mice. Our results identify that HTNV infected Nlrc3-/- mice can develop clinical symptoms and pathological changes resembling patients with HFRS, suggesting a new model for studying the pathogenesis and testing of candidate vaccines and therapeutics.
Assuntos
Vírus Hantaan/patogenicidade , Febre Hemorrágica com Síndrome Renal/virologia , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Rim/virologia , Animais , Citocinas/sangue , Modelos Animais de Doenças , Predisposição Genética para Doença , Vírus Hantaan/imunologia , Febre Hemorrágica com Síndrome Renal/imunologia , Febre Hemorrágica com Síndrome Renal/metabolismo , Febre Hemorrágica com Síndrome Renal/patologia , Interações Hospedeiro-Patógeno , Peptídeos e Proteínas de Sinalização Intercelular/genética , Rim/imunologia , Rim/metabolismo , Rim/patologia , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/virologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Carga ViralRESUMO
Blood cell analysis is a major pillar of biomedical research and healthcare. These analyses are performed in central laboratories. Rapid shipment from collection site to the central laboratories is currently needed because cells and biomarkers degrade rapidly. The dried blood spot from a fingerstick allows the preservation of cellular molecules for months but entire cells are never recovered. Here leucocyte elution is optimized from dried blood spots. Flow cytometry and mRNA expression profiling are used to analyze the recovered cells. 50-70% of the leucocytes that are dried on a polyester solid support via elution after shaking the support with buffer are recovered. While red blood cells lyse upon drying, it is found that the majority of leucocytes are preserved. Leucocytes have an altered structure that is improved by adding fixative in the elution buffer. Leucocytes are permeabilized, allowing an easy staining of all cellular compartments. Common immunophenotyping and mRNAs are preserved. The ability of a new biomarker (CD169) to discriminate between patients with and without Severe Acute Respiratory Syndrome induced by Coronavirus 2 (SARS-CoV-2) infections is also preserved. Leucocytes from blood can be dried, shipped, and/or stored for at least 1 month, then recovered for a wide variety of analyses, potentially facilitating biomedical applications worldwide.
Assuntos
Doenças Transmissíveis/diagnóstico , Testes Diagnósticos de Rotina/métodos , Teste em Amostras de Sangue Seco/métodos , Hematologia/métodos , Imunofenotipagem/métodos , Anticorpos Antivirais/sangue , Biomarcadores/sangue , Coleta de Amostras Sanguíneas/métodos , COVID-19/diagnóstico , Separação Celular/métodos , Doenças Transmissíveis/virologia , Eritrócitos/virologia , Citometria de Fluxo/métodos , Humanos , Leucócitos/virologia , RNA Mensageiro/sangue , SARS-CoV-2/genéticaRESUMO
Mucosal exposure to infected semen accounts for the majority of HIV-1 transmission events, with rectal intercourse being the route with the highest estimated risk of transmission. Yet, the impact of semen inflammation on colorectal HIV-1 transmission has never been addressed. Here we use cynomolgus macaques colorectal tissue explants to explore the effect of leukocytospermia, indicative of male genital tract inflammation, on SIVmac251 infection. We show that leukocytospermic seminal plasma (LSP) has significantly higher concentration of a number of pro-inflammatory molecules compared to normal seminal plasma (NSP). In virus-exposed explants, LSP enhance SIV infection more efficiently than NSP, being the increased viral replication linked to the level of inflammatory and immunomodulatory cytokines. Moreover, LSP induce leukocyte accumulation on the apical side of the colorectal lamina propria and the recruitment of a higher number of intraepithelial dendritic cells than with NSP. These results suggest that the outcome of mucosal HIV-1 infection is influenced by the inflammatory state of the semen donor, and provide further insights into mucosal SIV/HIV-1 pathogenesis.
Assuntos
Colo/virologia , Células Dendríticas/virologia , Reto/virologia , Sêmen/virologia , Vírus da Imunodeficiência Símia/fisiologia , Replicação Viral/fisiologia , Animais , Colo/metabolismo , Citocinas/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/fisiologia , Leucócitos/metabolismo , Leucócitos/patologia , Leucócitos/virologia , Macaca mulatta , Masculino , Reto/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/transmissão , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Técnicas de Cultura de TecidosRESUMO
Increased mortality in COVID-19 cases is often associated with microvascular complications. We have recently shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein promotes an inflammatory cytokine interleukin 6 (IL-6)/IL-6R-induced trans signaling response and alarmin secretion. Virus-infected or spike-transfected human epithelial cells exhibited an increase in senescence, with a release of senescence-associated secretory phenotype (SASP)-related inflammatory molecules. Introduction of the bromodomain-containing protein 4 (BRD4) inhibitor AZD5153 to senescent epithelial cells reversed this effect and reduced SASP-related inflammatory molecule release in TMNK-1 or EAhy926 (representative human endothelial cell lines), when cells were exposed to cell culture medium (CM) derived from A549 cells expressing SARS-CoV-2 spike protein. Cells also exhibited a senescence phenotype with enhanced p16, p21, and senescence-associated ß-galactosidase (SA-ß-Gal) expression and triggered SASP pathways. Inhibition of IL-6 trans signaling by tocilizumab and inhibition of inflammatory receptor signaling by the Bruton's tyrosine kinase (BTK) inhibitor zanubrutinib, prior to exposure of CM to endothelial cells, inhibited p21 and p16 induction. We also observed an increase in reactive oxygen species (ROS) in A549 spike-transfected and endothelial cells exposed to spike-transfected CM. ROS generation in endothelial cell lines was reduced after treatment with tocilizumab and zanubrutinib. Cellular senescence was associated with an increased level of the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), which have in vitro leukocyte attachment potential. Inhibition of senescence or SASP function prevented VCAM-1/ICAM-1 expression and leukocyte attachment. Taken together, we identified that human endothelial cells exposed to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers, leading to enhanced leukocyte adhesion. IMPORTANCE The present study was aimed at examining the underlying mechanism of extrapulmonary manifestations of SARS-CoV-2 spike protein-associated pathogenesis, with the notion that infection of the pulmonary epithelium can lead to mediators that drive endothelial dysfunction. We utilized SARS-CoV-2 spike protein expression in cultured human hepatocytes (Huh7.5) and pneumocytes (A549) to generate conditioned culture medium (CM). Endothelial cell lines (TMNK-1 or EAhy926) treated with CM exhibited an increase in cellular senescence markers by a paracrine mode and led to leukocyte adhesion. Overall, the link between these responses in endothelial cell senescence and a potential contribution to microvascular complication in productively SARS-CoV-2-infected humans is implicated. Furthermore, the use of inhibitors (BTK, IL-6, and BRD4) showed a reverse effect in the senescent cells. These results may support the selection of potential adjunct therapeutic modalities to impede SARS-CoV-2-associated pathogenesis.
Assuntos
Senescência Celular , Células Endoteliais/metabolismo , Leucócitos/metabolismo , Comunicação Parácrina , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células A549 , Adesão Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Células Endoteliais/patologia , Células Endoteliais/virologia , Compostos Heterocíclicos com 2 Anéis/farmacologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Leucócitos/patologia , Leucócitos/virologia , Piperazinas/farmacologia , Pirazóis , Piridazinas , Espécies Reativas de Oxigênio/metabolismo , Receptores de Interleucina-6/metabolismo , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Grass carp reovirus (GCRV) causes serious losses to the grass carp industry. At present, infectious tissues of GCRV have been studied, but target cells remain unclear. In this study, peripheral blood cells were isolated, cultured, and infected with GCRV. Using quantitative real-time polymerase chain reaction (qRT-PCR), Western Blot, indirect immunofluorescence, flow cytometry, and transmission electron microscopy observation, a model of GCRV infected blood cells in vitro was established. The experimental results showed GCRV could be detectable in leukocytes only, while erythrocytes and thrombocytes could not. The virus particles in leukocytes are wrapped by empty membrane vesicles that resemble phagocytic vesicles. The empty membrane vesicles of leukocytes are different from virus inclusion bodies in C. idella kidney (CIK) cells. Meanwhile, the expression levels of IFN1, IL-1ß, Mx2, TNFα were significantly up-regulated in leukocytes, indicating that GCRV could cause the production of the related immune responses. Therefore, GCRV can infect leukocytes in vitro, but not infect erythrocytes and thrombocytes. Leukocytes are target cells in blood cells of GCRV infections. This study lays a theoretical foundation for the study of the GCRV infection mechanism and anti-GCRV immunity.
Assuntos
Carpas , Doenças dos Peixes/virologia , Leucócitos/virologia , Infecções por Reoviridae/veterinária , Reoviridae/fisiologia , Animais , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Plaquetas/virologia , Células Cultivadas , Eritrócitos/metabolismo , Eritrócitos/ultraestrutura , Eritrócitos/virologia , Citometria de Fluxo , Leucócitos/metabolismo , Leucócitos/ultraestrutura , Reoviridae/ultraestrutura , Carga ViralRESUMO
Coronavirus disease 2019 (COVID-19) is a global epidemic disease caused by a novel virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing serious adverse effects on human health. In this study, we obtained a blood leukocytes sequencing data set of COVID-19 patients from the GEO database and obtained differentially expressed genes (DEGs). We further analyzed these DEGs by protein-protein interaction analysis and Gene Ontology enrichment analysis and identified the DEGs closely related to SARS-CoV-2 infection. Then, we constructed a six-gene model (comprising IFIT3, OASL, USP18, XAF1, IFI27, and EPSTI1) by logistic regression analysis and calculated the area under the ROC curve (AUC) for the diagnosis of COVID-19. The AUC values of the training group, testing group, and entire group were 0.930, 0.914, and 0.921, respectively. The six genes were highly expressed in patients with COVID-19 and positively correlated with the expression of SARS-CoV-2 invasion-related genes (ACE2, TMPRSS2, CTSB, and CTSL). The risk score calculated by this model was also positively correlated with the expression of TMPRSS2, CTSB, and CTSL, indicating that the six genes were closely related to SARS-CoV-2 infection. In conclusion, we comprehensively analyzed the functions of DEGs in the blood leukocytes of patients with COVID-19 and constructed a six-gene model that may contribute to the development of new diagnostic and therapeutic ideas for COVID-19. Moreover, these six genes may be therapeutic targets for COVID-19.
Assuntos
COVID-19/metabolismo , Regulação Viral da Expressão Gênica , Leucócitos/metabolismo , Leucócitos/virologia , SARS-CoV-2/metabolismo , 2',5'-Oligoadenilato Sintetase , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , COVID-19/genética , Feminino , Estudo de Associação Genômica Ampla , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Logísticos , Masculino , Proteínas de Membrana , Pessoa de Meia-Idade , Proteínas de Neoplasias , SARS-CoV-2/genética , Ubiquitina TiolesteraseRESUMO
Mammalian three-dimensional (3D) enteroids mirror in vivo intestinal organisation and are powerful tools to investigate intestinal cell biology and host-pathogen interactions. We have developed complex multilobulated 3D chicken enteroids from intestinal embryonic villi and adult crypts. These avian enteroids develop optimally in suspension without the structural support required to produce mammalian enteroids, resulting in an inside-out enteroid conformation with media-facing apical brush borders. Histological and transcriptional analyses show these enteroids comprise of differentiated intestinal epithelial cells bound by cell-cell junctions, and notably, include intraepithelial leukocytes and an inner core of lamina propria leukocytes. The advantageous polarisation of these enteroids has enabled infection of the epithelial apical surface with Salmonella Typhimurium, influenza A virus and Eimeria tenella without the need for micro-injection. We have created a comprehensive model of the chicken intestine which has the potential to explore epithelial and leukocyte interactions and responses in host-pathogen, food science and pharmaceutical research.
Assuntos
Eimeria tenella/patogenicidade , Células Epiteliais , Vírus da Influenza A/patogenicidade , Mucosa Intestinal , Leucócitos , Salmonella typhimurium/patogenicidade , Animais , Células Cultivadas , Microambiente Celular , Galinhas , Eimeria tenella/imunologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Células Epiteliais/parasitologia , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno , Vírus da Influenza A/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/parasitologia , Mucosa Intestinal/virologia , Leucócitos/imunologia , Leucócitos/microbiologia , Leucócitos/parasitologia , Leucócitos/virologia , Camundongos Endogâmicos C57BL , Organoides , Permeabilidade , Fagocitose , Fenótipo , Codorniz , Salmonella typhimurium/imunologiaRESUMO
Mass cytometry (CyTOF) represents one of the most powerful tools in immune phenotyping, allowing high throughput quantification of over 40 parameters at single-cell resolution. However, wide deployment of CyTOF-based immune phenotyping studies are limited by complex experimental workflows and the need for specialized CyTOF equipment and technical expertise. Furthermore, differences in cell isolation and enrichment protocols, antibody reagent preparation, sample staining, and data acquisition protocols can all introduce technical variation that can confound integrative analyses of large data-sets of samples processed across multiple labs. Here, we present a streamlined whole blood CyTOF workflow which addresses many of these sources of experimental variation and facilitates wider adoption of CyTOF immune monitoring across sites with limited technical expertise or sample-processing resources or equipment. Our workflow utilizes commercially available reagents including the Fluidigm MaxPar Direct Immune Profiling Assay (MDIPA), a dry tube 30-marker immunophenotyping panel, and SmartTube Proteomic Stabilizer, which allows for simple and reliable fixation and cryopreservation of whole blood samples. We validate a workflow that allows for streamlined staining of whole blood samples with minimal processing requirements or expertise at the site of sample collection, followed by shipment to a central CyTOF core facility for batched downstream processing and data acquisition. We apply this workflow to characterize 184 whole blood samples collected longitudinally from a cohort of 72 hospitalized COVID-19 patients and healthy controls, highlighting dynamic disease-associated changes in circulating immune cell frequency and phenotype.
Assuntos
COVID-19/diagnóstico , Separação Celular , Citometria de Fluxo , Imunofenotipagem , Leucócitos/imunologia , SARS-CoV-2/imunologia , Fluxo de Trabalho , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , COVID-19/sangue , COVID-19/imunologia , COVID-19/virologia , Estudos de Casos e Controles , Feminino , Ensaios de Triagem em Larga Escala , Interações Hospedeiro-Patógeno , Humanos , Leucócitos/metabolismo , Leucócitos/virologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , SARS-CoV-2/patogenicidade , Índice de Gravidade de Doença , Adulto JovemRESUMO
Novel coronavirus disease 2019 (COVID-19) is associated with a hypercoagulable state, characterized by abnormal coagulation parameters and by increased incidence of cardiovascular complications. With this study, we aimed to investigate the activation state and the expression of transmembrane proteins in platelets of hospitalized COVID-19 patients. We investigated transmembrane proteins expression with a customized mass cytometry panel of 21 antibodies. Platelets of 8 hospitalized COVID-19 patients not requiring intensive care support and without pre-existing conditions were compared to platelets of healthy controls (11 donors) with and without in vitro stimulation with thrombin receptor-activating peptide (TRAP). Mass cytometry of non-stimulated platelets detected an increased surface expression of activation markers P-Selectin (0.67 vs. 1.87 median signal intensity for controls vs. patients, p = 0.0015) and LAMP-3 (CD63, 0.37 vs. 0.81, p = 0.0004), the GPIIb/IIIa complex (4.58 vs. 5.03, p < 0.0001) and other adhesion molecules involved in platelet activation and platelet-leukocyte interactions. Upon TRAP stimulation, mass cytometry detected a higher expression of P-selectin in COVID-19 samples compared to controls (p < 0.0001). However, we observed a significantly reduced capacity of COVID-19 platelets to increase the expression of activation markers LAMP-3 and P-Selectin upon stimulation with TRAP. We detected a hyperactivated phenotype in platelets during SARS-CoV-2 infection, consisting of highly expressed platelet activation markers, which might contribute to the hypercoagulopathy observed in COVID-19. In addition, several transmembrane proteins were more highly expressed compared to healthy controls. These findings support research projects investigating antithrombotic and antiplatelet treatment regimes in COVID-19 patients, and provide new insights on the phenotypical platelet expression during SARS-CoV-2 infection.
Assuntos
Plaquetas/patologia , COVID-19/complicações , Leucócitos/patologia , SARS-CoV-2/isolamento & purificação , Trombose/epidemiologia , Adulto , Plaquetas/metabolismo , Plaquetas/virologia , COVID-19/transmissão , COVID-19/virologia , Estudos de Casos e Controles , Feminino , Alemanha/epidemiologia , Humanos , Leucócitos/metabolismo , Leucócitos/virologia , Masculino , Pessoa de Meia-Idade , Selectina-P/metabolismo , Fragmentos de Peptídeos/metabolismo , Fenótipo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Trombose/virologiaRESUMO
In dairy cattle infected with bovine leukemia virus (BLV), the proviral load (PVL) level is directly related to the viral transmission from infected animals to their healthy herdmates. Two contrasting phenotypic groups can be identified when assessing PVL in peripheral blood of infected cows. A large number of reports point to bovine genetic variants (single nucleotide polymorphisms) as one of the key determinants underlying PVL level. However, biological mechanisms driving BLV PVL profiles and infection progression in cattle have not yet been elucidated. In this study, we evaluated whether a set of candidate genes affecting BLV PVL level according to whole genome association studies are differentially expressed in peripheral blood mononuclear cells derived from phenotypically contrasting groups of BLV-infected cows. During a 10-mo-long sampling scheme, 129 Holstein cows were phenotyped measuring anti-BLV antibody levels, PVL quantification, and white blood cell subpopulation counts. Finally, the expression of 8 genes (BOLA-DRB3, PRRC2A, ABT1, TNF, BAG6, BOLA-A, LY6G5B, and IER3) located within the bovine major histocompatibility complex region harboring whole genome association SNP hits was evaluated in 2 phenotypic groups: high PVL (n = 7) and low PVL (n = 8). The log2 initial fluorescence value (N0) transformed mean expression values for the ABT1 transcription factor were statistically different in high- and low-PVL groups, showing a higher expression of the ABT1 gene in low-PVL cows. The PRRC2A and IER3 genes had a significant positive (correlation coefficient = 0.61) and negative (correlation coefficient = -0.45) correlation with the lymphocyte counts, respectively. Additionally, the relationships between gene expression values and lymphocyte counts were modeled using linear regressions. Lymphocyte levels in infected cows were better explained (coefficient of determination = 0.56) when fitted a multiple linear regression model using both PRRC2A and IER3 expression values as independent variables. The present study showed evidence of differential gene expression between contrasting BLV infection phenotypes. These genes have not been previously related to BLV pathobiology. This valuable information represents a step forward in understanding the BLV biology and the immune response of naturally infected cows under a commercial milk production system. Efforts to elucidate biological mechanisms leading to BLV infection progression in cows are valuable for BLV control programs. Further studies integrating genotypic data, global transcriptome analysis, and BLV progression phenotypes are needed to better understand the BLV-host interaction.