RESUMEN
The oral cavity is the major site for transmission of Kaposi's sarcoma-associated herpesvirus (KSHV), but how KSHV establishes infection and replication in the oral epithelia remains unclear. Here, we report a KSHV spontaneous lytic replication model using fully differentiated, three-dimensional (3D) oral epithelial organoids at an air-liquid interface (ALI). This model revealed that KSHV infected the oral epithelia when the basal epithelial cells were exposed by damage. Unlike two-dimensional (2D) cell culture, 3D oral epithelial organoid ALI culture allowed high levels of spontaneous KSHV lytic replication, where lytically replicating cells were enriched at the superficial layer of epithelial organoid. Single cell RNA sequencing (scRNAseq) showed that KSHV infection induced drastic changes of host gene expression in infected as well as uninfected cells at the different epithelial layers, resulting in altered keratinocyte differentiation and cell death. Moreover, we identified a unique population of infected cells containing lytic gene expression at the KSHV K2-K5 gene locus and distinct host gene expression compared to latent or lytic infected cells. This study demonstrates an in vitro 3D epithelial organoid ALI culture model that recapitulates KSHV infection in the oral cavity, where KSHV undergoes the epithelial differentiation-dependent spontaneous lytic replication with a unique cell population carrying distinct viral gene expression.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida , Infecciones por Herpesviridae , Herpesvirus Humano 8 , Regulación Viral de la Expresión Génica , Herpesvirus Humano 8/fisiología , Humanos , Análisis de la Célula Individual , Latencia del Virus , Replicación ViralRESUMEN
Enhancers play indispensable roles in cell proliferation and survival through spatiotemporally regulating gene transcription. Active enhancers and superenhancers often produce noncoding enhancer RNAs (eRNAs) that precisely control RNA polymerase II activity. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic gamma-2 herpesvirus that causes Kaposi's sarcoma and primary effusion lymphoma (PEL). It is well characterized that KSHV utilizes host epigenetic machineries to control the switch between two lifecycles, latency and lytic replication. However, how KSHV impacts host epigenome at different stages of viral lifecycle is not well understood. Using global run-on sequencing (GRO-seq) and chromatin-immunoprecipitation sequencing (ChIP-seq), we profiled the dynamics of host transcriptional regulatory elements during latency and lytic replication of KSHV-infected PEL cells. This revealed that a number of critical host genes for KSHV latency, including MYC proto-oncogene, were under the control of superenhancers whose activities were globally repressed upon viral reactivation. The eRNA-expressing MYC superenhancers were located downstream of the MYC gene in KSHV-infected PELs and played a key role in MYC expression. RNAi-mediated depletion or dCas9-KRAB CRISPR inhibition of eRNA expression significantly reduced MYC mRNA level in PELs, as did the treatment of an epigenomic drug that globally blocks superenhancer function. Finally, while cellular IRF4 acted upon eRNA expression and superenhancer function for MYC expression during latency, KSHV viral IRF4 repressed cellular IRF4 expression, decreasing MYC expression and thereby, facilitating lytic replication. These results indicate that KSHV acts as an epigenomic driver that modifies host epigenomic status upon reactivation by effectively regulating host enhancer function.
Asunto(s)
Regulación Viral de la Expresión Génica/genética , Herpesvirus Humano 8/genética , Linfoma de Efusión Primaria/genética , Línea Celular , Epigenómica/métodos , Genes myc/genética , Herpesvirus Humano 8/patogenicidad , Humanos , Proteínas Inmediatas-Precoces/genética , Linfoma de Efusión Primaria/metabolismo , Linfoma de Efusión Primaria/virología , Proteínas Nucleares/metabolismo , Proto-Oncogenes Mas , ARN/metabolismo , Sarcoma de Kaposi/virología , Transactivadores/metabolismo , Transcripción Genética/genética , Proteínas Virales/metabolismo , Activación Viral/genética , Latencia del Virus/genética , Replicación Viral/genéticaRESUMEN
RATIONALE: Quantification of type 2 inflammation provided a molecular basis for heterogeneity in asthma. Non-type 2 pathways that contribute to asthma pathogenesis are not well understood. OBJECTIVES: To identify dysregulated pathways beyond type 2 inflammation. METHODS: We applied RNA sequencing to airway epithelial brushings obtained from subjects with stable mild asthma not on corticosteroids (n = 19) and healthy control subjects (n = 16). Sequencing reads were mapped to human and viral genomes. In the same cohort, and in a separate group with severe asthma (n = 301), we profiled blood gene expression with microarrays. MEASUREMENTS AND MAIN RESULTS: In airway brushings from mild asthma on inhaled corticosteroids, RNA sequencing yielded 1,379 differentially expressed genes (false discovery rate < 0.01). Pathway analysis revealed increased expression of type 2 markers, IFN-stimulated genes (ISGs), and endoplasmic reticulum (ER) stress-related genes. Airway epithelial ISG expression was not associated with type 2 inflammation in asthma or with viral transcripts but was associated with reduced lung function by FEV1 (ρ = -0.72; P = 0.0004). ER stress was confirmed by an increase in XBP1 (X-box binding protein 1) splicing in mild asthma and was associated with both type 2 inflammation and ISG expression. ISGs were also the most activated genes in blood cells in asthma and were correlated with airway ISG expression (ρ = 0.55; P = 0.030). High blood ISG expression in severe asthma was similarly unrelated to type 2 inflammation. CONCLUSIONS: ISG activation is prominent in asthma, independent of viral transcripts, orthogonal to type 2 inflammation, and associated with distinct clinical features. ER stress is associated with both type 2 inflammation and ISG expression.
Asunto(s)
Asma/genética , Asma/fisiopatología , Retículo Endoplásmico/genética , Regulación de la Expresión Génica , Factor 3 Regulador del Interferón/genética , Adulto , Estudios de Casos y Controles , Eosinófilos/inmunología , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Persona de Mediana Edad , Estrés Oxidativo/genética , ARN/genética , Valores de Referencia , Sensibilidad y Especificidad , Transducción de SeñalRESUMEN
BACKGROUND: Congenital Zika virus (ZIKV) infection leads to severe newborn abnormalities, but its long-term impact on childhood immunity is not well understood. This study aims to investigate the serum proteomics in children exposed to ZIKV during pregnancy to understand potential immunological consequences during early childhood. METHODS: The study included ZIKV-exposed infants (ZEI) at birth (n = 42) and children exposed to ZIKV (ZEC) at two years of age (n = 20) exposed to ZIKV during pregnancy, as well as healthy controls. Serum proteomic analysis was performed on these groups to assess inflammation and immune profiles. Additionally, antibody titres against two common childhood vaccines, DTaP and MMR, were measured in healthy controls (n = 50) and ZEC (n = 92) to evaluate vaccine-induced immunity. FINDINGS: Results showed elevated inflammation in ZEI with birth abnormalities. Among ZEC, despite most having normal clinical outcomes at two years, their serum proteomics indicated a bias towards Th1-mediated immune responses. Notably, ZEC displayed reduced anti-Diphtheria toxin and anti-Clostridium tetani IgG levels against DTaP and MMR vaccines. They also exhibited lower antibody titres particularly against Th2-biased DTaP vaccines, but not Th1-biased MMR vaccines. INTERPRETATION: In conclusion, the study highlights the long-term immunological consequences of congenital ZIKV exposure. Heightened inflammation was observed in ZEI with abnormalities at birth, while ZEC maintained a chronic Th1-biased immune profile. The impaired response to Th2-biased vaccines raises concerns about lasting effects of ZIKV exposure on immune responses. Consequently, there is a need for continued longitudinal clinical monitoring to identify potential immune-related complications arising from prenatal exposure to ZIKV. FUNDING: This work was partially funded by the National Institute of Allergy and Infectious Diseases (NIAID) and National Institute of Dental and Craniofacial Research (NIDCR).
Asunto(s)
Inflamación , Infección por el Virus Zika , Virus Zika , Humanos , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/sangre , Femenino , Virus Zika/inmunología , Embarazo , Masculino , Inflamación/inmunología , Preescolar , Lactante , Proteómica/métodos , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Recién Nacido , Complicaciones Infecciosas del Embarazo/inmunología , Complicaciones Infecciosas del Embarazo/virologíaRESUMEN
Almost 20% of patients with COVID-19 experience long-term effects, known as post-COVID condition or long COVID. Among many lingering neurologic symptoms, chronic headache is the most common. Despite this health concern, the etiology of long COVID headache is still not well characterized. Here, we present a longitudinal multi-omics analysis of blood leukocyte transcriptomics, plasma proteomics and metabolomics of long COVID patients with chronic headache. Long COVID patients experienced a state of hyper-inflammation prior to chronic headache onset and maintained persistent inflammatory activation throughout the progression of chronic headache. Metabolomic analysis also revealed augmented arginine and lipid metabolisms, skewing towards a nitric oxide-based pro-inflammation. Furthermore, metabolisms of neurotransmitters including serotonin, dopamine, glutamate, and GABA were markedly dysregulated during the progression of long COVID headache. Overall, these findings illustrate the immuno-metabolomics landscape of long COVID patients with chronic headache, which may provide insights to potential therapeutic interventions.
RESUMEN
Spermidine is essential for cellular growth and acts as a prerequisite of hypusination, a post-translational modification of eukaryotic initiation factor 5A (eIF5A), allowing the translation of polyproline-containing proteins. Here, we show that oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) increases spermidine synthesis and eIF5A hypusination to enhance expression of polyproline-containing latency-associated nuclear antigen (LANA) for viral episomal maintenance. KSHV upregulates intracellular spermidine levels by dysregulating polyamine metabolic pathways in three-dimensional (3D) culture and 2D de novo infection conditions. Increased intracellular spermidine leads to increased eIF5A hypusination, ultimately enhancing LANA expression. In contrast, inhibition of spermidine synthesis or eIF5A hypusination alleviates LANA expression, decreasing viral episomal maintenance and KSHV-infected cell proliferation in vitro and in vivo, which is reversed by spermidine supplement. This demonstrates that KSHV hijacks spermidine synthesis and eIF5A hypusination pathways to enhance LANA expression for viral episomal maintenance, suggesting polyamine metabolism and eIF5A hypusination as therapeutic targets for KSHV-induced tumorigenesis.
Asunto(s)
Herpesvirus Humano 8 , Espermidina , Antígenos Virales/metabolismo , Línea Celular , Herpesvirus Humano 8/fisiología , Factores de Iniciación de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Espermidina/metabolismo , Espermidina/farmacologíaRESUMEN
Dabie bandavirus (severe fever with thrombocytopenia syndrome virus [SFTSV]) induces an immunopathogenic disease with a high fatality rate; however, the mechanisms underlying its clinical manifestations are largely unknown. In this study, we applied targeted proteomics and single-cell transcriptomics to examine the differential immune landscape in SFTS patient blood. Serum immunoprofiling identified low-risk and high-risk clusters of SFTS patients based on inflammatory cytokine levels, which corresponded to disease severity. Single-cell transcriptomic analysis of SFTS patient peripheral blood mononuclear cells (PBMCs) at different infection stages showed pronounced expansion of B cells with alterations in B-cell subsets in fatal cases. Furthermore, plasma cells in which the interferon (IFN) pathway is downregulated were identified as the primary reservoir of SFTSV replication. This study identified not only the molecular signatures of serum inflammatory cytokines and B-cell lineage populations in SFTSV-induced fatalities but also plasma cells as the viral reservoir. Thus, this suggests that altered B-cell function is linked to lethality in SFTSV infections.IMPORTANCE SFTSV is an emerging virus discovered in China in 2009; it has since spread to other countries in East Asia. Although the fatality rates of SFTSV infection range from 5.3% to as high as 27%, the mechanisms underlying clinical manifestations are largely unknown. In this study, we demonstrated that SFTSV infection in fatal cases caused an excessive inflammatory response through high induction of proinflammatory cytokines and chemokines and the aberrant inactivation of adaptive immune responses. Furthermore, single-cell transcriptome sequencing (RNA-seq) analysis of SFTS patient PBMCs revealed that SFTSV targets the B-cell lineage population, especially plasma cells, as the potential viral reservoir in patients for whom the infection is fatal. Thus, SFTSV infection may inhibit high-affinity antibody maturation and secretion of plasma B cells, suppressing neutralizing antibody production and thereby allowing significant virus replication and subsequent fatality.
Asunto(s)
Linfocitos B/inmunología , Citocinas/genética , Inflamación/genética , Phlebovirus/inmunología , Síndrome de Trombocitopenia Febril Grave/inmunología , Transcriptoma , Anciano , Anticuerpos Antivirales/sangre , Citocinas/inmunología , Reservorios de Enfermedades/virología , Femenino , Humanos , Inflamación/inmunología , Masculino , Persona de Mediana Edad , Células Plasmáticas/virología , Proteómica , Síndrome de Trombocitopenia Febril Grave/sangre , Síndrome de Trombocitopenia Febril Grave/genética , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
While pregnancy increases the risk for severe COVID-19, the clinical and immunological implications of COVID-19 on maternal-fetal health remain unknown. Here, we present the clinical and immunological landscapes of 93 COVID-19 mothers and 45 of their SARS-CoV-2-exposed infants through comprehensive serum proteomics profiling for >1,400 cytokines of their peripheral and cord blood specimens. Prenatal SARS-CoV-2 infection triggers NF-κB-dependent proinflammatory immune activation. Pregnant women with severe COVID-19 show increased inflammation and unique IFN-λ antiviral signaling, with elevated levels of IFNL1 and IFNLR1. Furthermore, SARS-CoV-2 infection re-shapes maternal immunity at delivery, altering the expression of pregnancy complication-associated cytokines, inducing MMP7, MDK, and ESM1 and reducing BGN and CD209. Finally, COVID-19-exposed infants exhibit induction of T cell-associated cytokines (IL33, NFATC3, and CCL21), while some undergo IL-1ß/IL-18/CASP1 axis-driven neonatal respiratory distress despite birth at term. Our findings demonstrate COVID-19-induced immune rewiring in both mothers and neonates, warranting long-term clinical follow-up to mitigate potential health risks.