RESUMO
Cytomegalovirus (CMV) infection poses risks to newborns, necessitating effective therapies. Given that the damage includes both viral infection of brain cells and immune system-related damage, here we investigate the involvement of cellular prion protein (PrP), which plays vital roles in neuroprotection and immune regulation. Using a murine model, we show the role of PrP in tempering neonatal T cell immunity during CMV infection. PrP-null mice exhibit enhanced viral control through elevated virus-specific CD8 T cell responses, leading to reduced viral titers and pathology. We further unravel the molecular mechanisms by showing CMV-induced upregulation followed by release of PrP via the metalloproteinase ADAM10, impairing CD8 T cell response specifically in neonates. Additionally, we confirm PrP downregulation in human CMV (HCMV)-infected fibroblasts, underscoring the broader relevance of our observations beyond the murine model. Furthermore, our study highlights how PrP, under the stress of viral pathogenesis, reveals its impact on neonatal immune modulation.
Assuntos
Animais Recém-Nascidos , Linfócitos T CD8-Positivos , Infecções por Citomegalovirus , Citomegalovirus , Camundongos Knockout , Animais , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/imunologia , Humanos , Camundongos , Linfócitos T CD8-Positivos/imunologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/virologia , Proteínas Priônicas/metabolismo , Proteínas Priônicas/genética , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Proteína ADAM10/metabolismo , Proteína ADAM10/genéticaRESUMO
Congenital human cytomegalovirus (HCMV) infection may cause life-threatening disease and permanent damage to the central nervous system. The mouse model of CMV infection is most commonly used to study mechanisms of infection and pathogenesis. While essential to limit mouse CMV (MCMV) replication, the inflammatory responses, particularly IFNγ and TNFα, cause neurodevelopmental abnormalities. Other soluble mediators of the immune response in most tissues remain largely unexplored. To address this gap, we quantified 48 soluble mediators of the immune response, including 32 cytokines, 10 chemokines, 3 growth factors/regulators, and 3 soluble receptors in the spleen, liver, lungs, and brain at 9 and 14 days postinfection (dpi). Our analysis found 25 induced molecules in the brain at 9 dpi, with an additional 8 showing statistically elevated responses at 14 dpi. Specifically, all analyzed CCL group cytokines (CCL2, CCL3, CCL4, CCL5, CCL7, and CCL11) were upregulated at 14 dpi in the brain. Furthermore, data revealed differentially regulated analytes across tissues, such as CCL11, CXCL5, and IL-10 in the brain, IL-33/IL-33R in the liver, and VEGF-a and IL-5 in the lungs. Overall, this study provides an overview of the immune dynamics of soluble mediators in congenital CMV.
Assuntos
Infecções por Citomegalovirus , Muromegalovirus , Animais , Humanos , Camundongos , Citocinas , Encéfalo , Fator de Necrose Tumoral alfaRESUMO
Metabolic-associated fatty liver disease (MAFLD) is a spectrum of clinical manifestations ranging from benign steatosis to cirrhosis. A key event in the pathophysiology of MAFLD is the development of nonalcoholic steatohepatitis (NASH), which can potentially lead to fibrosis and hepatocellular carcinoma, but the triggers of MAFLD-associated inflammation are not well understood. We have observed that lipid accumulation in hepatocytes induces expression of ligands specific to the activating immune receptor NKG2D. Tissue-resident innate-like T cells, most notably γδ T cells, are activated through NKG2D and secrete IL-17A. IL-17A licenses hepatocytes to produce chemokines that recruit proinflammatory cells into the liver, which causes NASH and fibrosis. NKG2D-deficient mice did not develop fibrosis in dietary models of NASH and had a decreased incidence of hepatic tumors. The frequency of IL-17A+ γδ T cells in the blood of patients with MAFLD correlated directly with liver pathology. Our findings identify a key molecular mechanism through which stressed hepatocytes trigger inflammation in the context of MAFLD.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Humanos , Camundongos , Hepatócitos/metabolismo , Hepatócitos/patologia , Inflamação/patologia , Interleucina-17/metabolismo , Cirrose Hepática/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Linfócitos T/metabolismoRESUMO
Photodynamic therapy (PDT) is broadly used to treat different tumors, and it is a rapidly developing approach to inactivating or inhibiting the replication of fungi, bacteria, and viruses. Herpes simplex virus 1 (HSV-1) is an important human pathogen and a frequently used model to study the effects of PDT on enveloped viruses. Although many photosensitizers (PSs) have been tested for their antiviral properties, analyses are usually limited to assessing the reduction in viral yield, and thus the molecular mechanisms of photodynamic inactivation (PDI) remain poorly understood. In this study, we investigated the antiviral properties of TMPyP3-C17H35, a tricationic amphiphilic porphyrin-based PS with a long alkyl chain. We show that light-activated TMPyP3-C17H35 can efficiently block virus replication at certain nM concentrations without exerting obvious cytotoxicity. Moreover, we show that the levels of viral proteins (immediate-early, early, and late genes) were greatly reduced in cells treated with subtoxic concentrations of TMPyP3-C17H35, resulting in markedly decreased viral replication. Interestingly, we observed a strong inhibitory effect of TMPyP3-C17H35 on the virus yield only when cells were treated before or shortly after infection. In addition to the antiviral activity of the internalized compound, we show that the compound dramatically reduces the infectivity of free virus in the supernatant. Overall, our results demonstrate that activated TMPyP3-C17H35 effectively inhibits HSV-1 replication and that it can be further developed as a potential novel treatment and used as a model to study photodynamic antimicrobial chemotherapy.
RESUMO
COVID-19 vaccines prevent severe forms of the disease, but do not warrant complete protection against breakthrough infections. This could be due to suboptimal mucosal immunity at the site of virus entry, given that all currently approved vaccines are administered via the intramuscular route. In this study, we assessed humoral and cellular immune responses in BALB/c mice after intranasal and intramuscular immunization with adenoviral vector ChAdOx1-S expressing full-length Spike protein of SARS-CoV-2. We showed that both routes of vaccination induced a potent IgG antibody response, as well as robust neutralizing capacity, but intranasal vaccination elicited a superior IgA antibody titer in the sera and in the respiratory mucosa. Bronchoalveolar lavage from intranasally immunized mice efficiently neutralized SARS-CoV-2, which has not been the case in intramuscularly immunized group. Moreover, substantially higher percentages of epitope-specific CD8 T cells exhibiting a tissue resident phenotype were found in the lungs of intranasally immunized animals. Finally, both intranasal and intramuscular vaccination with ChAdOx1-S efficiently protected the mice after the challenge with recombinant herpesvirus expressing the Spike protein. Our results demonstrate that intranasal application of adenoviral vector ChAdOx1-S induces superior mucosal immunity and therefore could be a promising strategy for putting the COVID-19 pandemic under control.
Assuntos
COVID-19 , Vacinas Virais , Adenoviridae/genética , Administração Intranasal , Animais , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Imunidade Celular , Imunidade nas Mucosas , Camundongos , Camundongos Endogâmicos BALB C , Pandemias/prevenção & controle , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Vacinação/métodosRESUMO
Broad tissue tropism of cytomegaloviruses (CMVs) is facilitated by different glycoprotein entry complexes, which are conserved between human CMV (HCMV) and murine CMV (MCMV). Among the wide array of cell types susceptible to the infection, mononuclear phagocytes (MNPs) play a unique role in the pathogenesis of the infection as they contribute both to the virus spread and immune control. CMVs have dedicated numerous genes for the efficient infection and evasion of macrophages and dendritic cells. In this study, we have characterized the properties and function of M116, a previously poorly described but highly transcribed MCMV gene region that encodes M116.1p, a novel protein necessary for the efficient infection of MNPs and viral spread in vivo. Our study further revealed that M116.1p shares similarities with its positional homologs in HCMV and RCMV, UL116 and R116, respectively, such as late kinetics of expression, N-glycosylation, localization to the virion assembly compartment, and interaction with gH-a member of the CMVs fusion complex. This study, therefore, expands our knowledge about virally encoded glycoproteins that play important roles in viral infectivity and tropism. IMPORTANCE Human cytomegalovirus (HCMV) is a species-specific herpesvirus that causes severe disease in immunocompromised individuals and immunologically immature neonates. Murine cytomegalovirus (MCMV) is biologically similar to HCMV, and it serves as a widely used model for studying the infection, pathogenesis, and immune responses to HCMV. In our previous work, we have identified the M116 ORF as one of the most extensively transcribed regions of the MCMV genome without an assigned function. This study shows that the M116 locus codes for a novel protein, M116.1p, which shares similarities with UL116 and R116 in HCMV and RCMV, respectively, and is required for the efficient infection of mononuclear phagocytes and virus spread in vivo. Furthermore, this study establishes the α-M116 monoclonal antibody and MCMV mutants lacking M116, generated in this work, as valuable tools for studying the role of macrophages and dendritic cells in limiting CMV infection following different MCMV administration routes.
Assuntos
Sistema Fagocitário Mononuclear/virologia , Muromegalovirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Animais , Fibroblastos/metabolismo , Fibroblastos/virologia , Glicosilação , Infecções por Herpesviridae/virologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Sistema Fagocitário Mononuclear/metabolismo , Transcrição Gênica , Proteínas do Envelope Viral/genética , Vírion/metabolismo , Montagem de Vírus , Internalização do Vírus , Replicação ViralRESUMO
The twentieth century witnessed a huge expansion in the number of vaccines used with great success in combating diseases, especially the ones caused by viral and bacterial pathogens. Despite this, several major public health threats, such as HIV, tuberculosis, malaria, and cancer, still pose an enormous humanitarian and economic burden. As vaccines based on the induction of protective, neutralizing antibodies have not managed to effectively combat these diseases, in recent decades, the focus has increasingly shifted towards the cellular immune response. There is substantial evidence demonstrating CD8 T cells as key players in the protection not only against many viral and bacterial pathogens, but also in the fight against neoplastic cells. Here, we present arguments for CD8 T cells to be considered as promising candidates for vaccine targeting. We discuss the heterogeneity of CD8 T cell populations and their contribution in the protection of the host. We also outline several strategies of using a common human pathogen, cytomegalovirus, as a vaccine vector since accumulated data strongly suggest it represents a promising approach to the development of novel vaccines against both pathogens and tumors.
RESUMO
Viral vectors have emerged as a promising alternative to classical vaccines due to their great potential for induction of a potent cellular and humoral immunity. Cytomegalovirus (CMV) is an attractive vaccine vector due to its large genome with many non-essential immunoregulatory genes that can be easily manipulated to modify the immune response. CMV generates a strong antigen-specific CD8 T cell response with a gradual accumulation of these cells in the process called memory inflation. In our previous work, we have constructed a mouse CMV vector expressing NKG2D ligand RAE-1γ in place of its viral inhibitor m152 (RAE-1γMCMV), which proved to be highly attenuated in vivo. Despite attenuation, RAE-1γMCMV induced a substantially stronger CD8 T cell response to vectored antigen than the control vector and provided superior protection against bacterial and tumor challenge. In the present study, we confirmed the enhanced protective capacity of RAE-1γMCMV as a tumor vaccine vector and determined the phenotypical and functional characteristics of memory CD8 T cells induced by the RAE-1γ expressing MCMV. RNAseq data revealed higher transcription of numerous genes associated with effector-like CD8 T cell phenotype in RAE-1γMCMV immunized mice. CD8 T cells primed with RAE-1γMCMV were enriched in TCF1 negative population, with higher expression of KLRG1 and lower expression of CD127, CD27, and Eomes. These phenotypical differences were associated with distinct functional features as cells primed with RAE-1γMCMV showed inferior cytokine-producing abilities but comparable cytotoxic potential. After adoptive transfer into naive hosts, OT-1 cells induced with both RAE-1γMCMV and the control vector were equally efficient in rejecting established tumors, suggesting the context of latent infection and cell numbers as important determinants of enhanced anti-tumor response following RAE-1γMCMV vaccination. Overall, our results shed new light on the phenotypical and functional distinctness of memory CD8 T cells induced with CMV vector expressing cellular ligand for the NKG2D receptor.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas contra Citomegalovirus/imunologia , Memória Imunológica , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Fenótipo , Animais , Vacinas Anticâncer/imunologia , Biologia Computacional/métodos , Citomegalovirus/imunologia , Citotoxicidade Imunológica , Perfilação da Expressão Gênica , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/prevenção & controle , Imunofenotipagem , Ativação Linfocitária/imunologia , Camundongos , Muromegalovirus/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , TranscriptomaRESUMO
Congenital human cytomegalovirus (cHCMV) infection of the brain is associated with a wide range of neurocognitive sequelae. Using infection of newborn mice with mouse cytomegalovirus (MCMV) as a reliable model that recapitulates many aspects of cHCMV infection, including disseminated infection, CNS infection, altered neurodevelopment, and sensorineural hearing loss, we have previously shown that mitigation of inflammation prevented alterations in cerebellar development, suggesting that host inflammatory factors are key drivers of neurodevelopmental defects. Here, we show that MCMV infection causes a dramatic increase in the expression of the microglia-derived chemokines CXCL9/CXCL10, which recruit NK and ILC1 cells into the brain in a CXCR3-dependent manner. Surprisingly, brain-infiltrating innate immune cells not only were unable to control virus infection in the brain but also orchestrated pathological inflammatory responses, which lead to delays in cerebellar morphogenesis. Our results identify NK and ILC1 cells as the major mediators of immunopathology in response to virus infection in the developing CNS, which can be prevented by anti-IFN-γ antibodies.
Assuntos
Encéfalo/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Inflamação/imunologia , Células Matadoras Naturais/imunologia , Linfócitos/imunologia , Animais , Animais Recém-Nascidos , Encéfalo/patologia , Encéfalo/virologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Quimiocina CXCL10/metabolismo , Quimiocina CXCL9/genética , Quimiocina CXCL9/imunologia , Quimiocina CXCL9/metabolismo , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/virologia , Regulação da Expressão Gênica/imunologia , Humanos , Imunidade Inata/imunologia , Inflamação/genética , Inflamação/virologia , Células Matadoras Naturais/metabolismo , Linfócitos/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/imunologia , Microglia/metabolismo , Microglia/virologia , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Receptores CXCR3/metabolismoRESUMO
Viruses utilize microRNAs (miRNAs) in a vast variety of possible interactions and mechanisms, apparently far beyond the classical understanding of gene repression in humans. Likewise, herpes simplex virus 1 (HSV-1) expresses numerous miRNAs and deregulates the expression of host miRNAs. Several HSV-1 miRNAs are abundantly expressed in latency, some of which are encoded antisense to transcripts of important productive infection genes, indicating their roles in repressing the productive cycle and/or in maintenance/reactivation from latency. In addition, HSV-1 also exploits host miRNAs to advance its replication or repress its genes to facilitate latency. Here, we discuss what is known about the functional interplay between HSV-1 and the host miRNA machinery, potential targets, and the molecular mechanisms leading to an efficient virus replication and spread.
RESUMO
Human herpes simplex virus 1 (HSV-1) expresses numerous miRNAs, the function of which is not well understood. Several qualitative and quantitative analyses of HSV-1 miRNAs have been performed on infected cells in culture and animal models, however, there is very limited knowledge of their expression in human samples. We sequenced small-RNA libraries of RNA derived from human trigeminal ganglia latently infected with HSV-1 and Varicella zoster virus (VZV) and detected only a small subset of HSV-1 miRNA. The most abundantly expressed miRNAs are miR-H2, miRNA that regulates the expression of immediate early gene ICP0, and miR-H3 and -H4, both miRNAs expressed antisense to the transcript encoding the major neurovirulence factor ICP34.5. The sequence of many HSV-1 miRNAs detected in human samples was different from the sequences deposited in miRBase, which might significantly affect targeted functional analyses.
Assuntos
Variação Genética , Herpes Simples/virologia , Herpesvirus Humano 1/genética , MicroRNAs/genética , RNA Viral/genética , Gânglio Trigeminal/virologia , Latência Viral , Herpesvirus Humano 3/genética , Humanos , Análise de Sequência de DNARESUMO
The DNA damage response (DDR) pathway and ARF function as barriers to cancer development. Although commonly regarded as operating independently of each other, some studies proposed that ARF is positively regulated by the DDR. Contrary to either scenario, we found that in human oncogene-transformed and cancer cells, ATM suppressed ARF protein levels and activity in a transcription-independent manner. Mechanistically, ATM activated protein phosphatase 1, which antagonized Nek2-dependent phosphorylation of nucleophosmin (NPM), thereby liberating ARF from NPM and rendering it susceptible to degradation by the ULF E3-ubiquitin ligase. In human clinical samples, loss of ATM expression correlated with increased ARF levels and in xenograft and tissue culture models, inhibition of ATM stimulated the tumour-suppressive effects of ARF. These results provide insights into the functional interplay between the DDR and ARF anti-cancer barriers, with implications for tumorigenesis and treatment of advanced tumours.