RESUMO
During the 2015-2016 Zika virus (ZIKV) epidemic, ZIKV-associated neurological diseases were reported in adults, including microcephaly, Guillain-Barre syndrome, myelitis, meningoencephalitis, and fatal encephalitis. However, the mechanisms underlying the neuropathogenesis of ZIKV infection are not yet fully understood. In this study, we used an adult ZIKV infection mouse model (Ifnar1-/-) to investigate the mechanisms underlying neuroinflammation and neuropathogenesis. ZIKV infection induced the expression of proinflammatory cytokines, including interleukin-1ß (IL-1ß), IL-6, gamma interferon, and tumor necrosis factor alpha, in the brains of Ifnar1-/- mice. RNA-seq analysis of the infected mouse brain also revealed that genes involved in innate immune responses and cytokine-mediated signaling pathways were significantly upregulated at 6 days postinfection. Furthermore, ZIKV infection induced macrophage infiltration and activation and augmented IL-1ß expression, whereas microgliosis was not observed in the brain. Using human monocyte THP-1 cells, we confirmed that ZIKV infection promotes inflammatory cell death and increases IL-1ß secretion. In addition, expression of the complement component C3, which is associated with neurodegenerative diseases and known to be upregulated by proinflammatory cytokines, was induced by ZIKV infection through the IL-1ß-mediated pathway. An increase in C5a produced by complement activation in the brains of ZIKV-infected mice was also verified. Taken together, our results suggest that ZIKV infection in the brain of this animal model augments IL-1ß expression in infiltrating macrophages and elicits IL-1ß-mediated inflammation, which can lead to the destructive consequences of neuroinflammation. IMPORTANCE Zika virus (ZIKV) associated neurological impairments are an important global health problem. Our results suggest that ZIKV infection in the mouse brain can induce IL-1ß-mediated inflammation and complement activation, thereby contributing to the development of neurological disorders. Thus, our findings reveal a mechanism by which ZIKV induces neuroinflammation in the mouse brain. Although we used adult type I interferon receptor IFNAR knockout (Ifnar1-/-) mice owing to the limited mouse models of ZIKV pathogenesis, our conclusions contributed to the understanding ZIKV-associated neurological diseases to develop treatment strategies for patients with ZIKV infection based on these findings.
Assuntos
Encéfalo , Interleucina-1beta , Macrófagos , Infecção por Zika virus , Animais , Humanos , Camundongos , Encéfalo/imunologia , Citocinas/imunologia , Inflamação/imunologia , Interleucina-1beta/imunologia , Macrófagos/imunologia , Doenças Neuroinflamatórias/imunologia , Doenças Neuroinflamatórias/virologia , Zika virus , Infecção por Zika virus/imunologia , Transcriptoma/imunologia , Modelos Animais de Doenças , Neurônios/imunologia , Neurônios/virologiaRESUMO
Accumulating evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes various neurological symptoms in patients with coronavirus disease 2019 (COVID-19). The most dominant immune cells in the brain are microglia. Yet, the relationship between neurological manifestations, neuroinflammation, and host immune response of microglia to SARS-CoV-2 has not been well characterized. Here, we reported that SARS-CoV-2 can directly infect human microglia, eliciting M1-like proinflammatory responses, followed by cytopathic effects. Specifically, SARS-CoV-2 infected human microglial clone 3 (HMC3), leading to inflammatory activation and cell death. RNA sequencing (RNA-seq) analysis also revealed that endoplasmic reticulum (ER) stress and immune responses were induced in the early, and apoptotic processes in the late phases of viral infection. SARS-CoV-2-infected HMC3 showed the M1 phenotype and produced proinflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor α (TNF-α), but not the anti-inflammatory cytokine IL-10. After this proinflammatory activation, SARS-CoV-2 infection promoted both intrinsic and extrinsic death receptor-mediated apoptosis in HMC3. Using K18-hACE2 transgenic mice, murine microglia were also infected by intranasal inoculation of SARS-CoV-2. This infection induced the acute production of proinflammatory microglial IL-6 and TNF-α and provoked a chronic loss of microglia. Our findings suggest that microglia are potential mediators of SARS-CoV-2-induced neurological problems and, consequently, can be targets of therapeutic strategies against neurological diseases in patients with COVID-19. IMPORTANCE Recent studies reported neurological and cognitive sequelae in patients with COVID-19 months after the viral infection with several symptoms, including ageusia, anosmia, asthenia, headache, and brain fog. Our conclusions raise awareness of COVID-19-related microglia-mediated neurological disorders to develop treatment strategies for the affected patients. We also indicated that HMC3 was a novel human cell line susceptible to SARS-CoV-2 infection that exhibited cytopathic effects, which could be further used to investigate cellular and molecular mechanisms of neurological manifestations of patients with COVID-19.
Assuntos
Apoptose , COVID-19 , Microglia , Animais , Linhagem Celular , Citocinas/metabolismo , Humanos , Interleucina-6 , Camundongos , Camundongos Transgênicos , Microglia/virologia , SARS-CoV-2 , Fator de Necrose Tumoral alfaRESUMO
BACKGROUND: Although the prognostic biomarkers associated with colorectal cancer (CRC) survival are well known, there are limited data on the association between the molecular characteristics and survival after recurrence (SAR). The purpose of this study was to assess the association between pathway mutations and SAR. METHODS: Of the 516 patients with stage III or high risk stage II CRC patients treated with surgery and adjuvant chemotherapy, 87 who had distant recurrence were included in the present study. We analyzed the association between SAR and mutations of 40 genes included in the five critical pathways of CRC (WNT, P53, RTK-RAS, TGF-ß, and PI3K). RESULTS: Mutation of genes within the WNT, P53, RTK-RAS, TGF-ß, and PI3K pathways were shown in 69(79.3%), 60(69.0%), 57(65.5%), 21(24.1%), and 19(21.8%) patients, respectively. Patients with TGF-ß pathway mutation were younger and had higher incidence of mucinous adenocarcinoma (MAC) histology and microsatellite instability-high. TGF-ß pathway mutation (median SAR of 21.6 vs. 44.4 months, p = 0.021) and MAC (20.0 vs. 44.4 months, p = 0.003) were associated with poor SAR, and receiving curative resection after recurrence was associated with favorable SAR (Not reached vs. 23.6 months, p < 0.001). Mutations in genes within other critical pathways were not associated with SAR. When MAC was excluded as a covariate, multivariate analysis revealed TGF-ß pathway mutation and curative resection after distant recurrence as an independent prognostic factor for SAR. The impact of TGF-ß pathway mutations were predicted using the PROVEAN, SIFT, and PolyPhen-2. Among 25 mutations, 23(92.0%)-24(96.0%) mutations were predicted to be damaging mutation. CONCLUSIONS: Mutation in genes within TGF-ß pathway may have negative prognostic role for SAR in CRC. Other pathway mutations were not associated with SAR.
Assuntos
Adenocarcinoma Mucinoso/genética , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Recidiva Local de Neoplasia/genética , Transdução de Sinais/genética , Adenocarcinoma Mucinoso/mortalidade , Adenocarcinoma Mucinoso/patologia , Adenocarcinoma Mucinoso/terapia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/metabolismo , Capecitabina , Quimioterapia Adjuvante/métodos , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Feminino , Fluoruracila/análogos & derivados , Fluoruracila/uso terapêutico , Seguimentos , Humanos , Leucovorina/uso terapêutico , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/terapia , Compostos Organoplatínicos/uso terapêutico , Oxaloacetatos , Cuidados Paliativos/métodos , Prognóstico , Reto/patologia , Reto/cirurgia , Análise de Sobrevida , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: Colorectal cancer (CRC) develops through the alteration of several critical pathways. This study was aimed at evaluating the influence of critical pathways on survival outcomes for patients with CRC. METHODS: Targeted next-generation sequencing of 40 genes included in the 5 critical pathways of CRC (WNT, P53, RTK-RAS, phosphatidylinositol-4,5-bisphosphate 3-kinase [PI3K], and transforming growth factor ß [TGF-ß]) was performed for 516 patients with stage III or high-risk stage II CRC treated with surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy. The associations between critical pathway mutations and relapse-free survival (RFS) and overall survival were analyzed. The associations were further analyzed according to the tumor location. RESULTS: The mutation rates for the WNT, P53, RTK-RAS, PI3K, and TGF-ß pathways were 84.5%, 69.0%, 60.7%, 30.0%, and 28.9%, respectively. A mutation in the PI3K pathway was associated with longer RFS (adjusted hazard ratio [HR], 0.59; 95% confidence interval [CI], 0.36-0.99), whereas a mutation in the RTK-RAS pathway was associated with shorter RFS (adjusted HR, 1.60; 95% CI, 1.01-2.52). Proximal tumors showed a higher mutation rate than distal tumors, and the mutation profile was different according to the tumor location. The mutation rates of Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), and B-Raf proto-oncogene serine/threonine kinase (BRAF) were higher in proximal tumors, and the mutation rates of adenomatous polyposis coli (APC), tumor protein 53 (TP53), and neuroblastoma RAS viral oncogene homolog (NRAS) were higher in distal tumors. The better RFS with the PI3K pathway mutation was significant only for proximal tumors, and the worse RFS with the RTK-RAS pathway mutation was significant only for distal tumors. CONCLUSIONS: A PI3K pathway mutation was associated with better RFS for CRC patients treated with adjuvant chemotherapy, and an RTK-RAS pathway mutation was associated with worse RFS. The significance of the prognostic impact differed according to the tumor location. Cancer 2017;123:3513-23. © 2017 American Cancer Society.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Regulação Neoplásica da Expressão Gênica , Mutação , Proto-Oncogenes/genética , Adulto , Idoso , Quimioterapia Adjuvante , Estudos de Coortes , Colectomia/métodos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Terapia Combinada , Intervalos de Confiança , Procedimentos Clínicos , Receptores ErbB/genética , Feminino , Genes ras , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Valor Preditivo dos Testes , Prognóstico , Proto-Oncogene Mas , República da Coreia , Estudos Retrospectivos , Análise de Sobrevida , Resultado do TratamentoRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease characterized by the formation of multiple fluid-filled cysts in bilateral kidneys. Although mutations in polycystic kidney disease 1 (PKD1) are predominantly responsible for ADPKD, the focal and sporadic property of individual cystogenesis suggests another molecular mechanism such as epigenetic alterations. To determine the epigenomic alterations in ADPKD and their functional relevance, ADPKD and non-ADPKD individuals were analyzed by unbiased methylation profiling genome-wide and compared with their expression data. Intriguingly, PKD1 and other genes related to ion transport and cell adhesion were hypermethylated in gene-body regions, and their expressions were downregulated in ADPKD, implicating epigenetic silencing as the key mechanism underlying cystogenesis. Especially, in patients with ADPKD, PKD1 was hypermethylated in gene-body region and it was associated with recruitment of methyl-CpG-binding domain 2 proteins. Moreover, treatment with DNA methylation inhibitors retarded cyst formation of Madin-Darby Canine Kidney cells, accompanied with the upregulation of Pkd1 expression. These results are consistent with previous studies that knock-down of PKD1 was sufficient for cystogenesis. Therefore, our results reveal a critical role for hypermethylation of PKD1 and cystogenesis-related regulatory genes in cyst development, suggesting epigenetic therapy as a potential treatment for ADPKD.
Assuntos
Cistos/genética , Metilação de DNA , Epigênese Genética , Estudo de Associação Genômica Ampla , Rim/patologia , Rim Policístico Autossômico Dominante/genética , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Hibridização Genômica Comparativa , Biologia Computacional , Cistos/patologia , Cães , Regulação para Baixo , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Células Madin Darby de Rim Canino , Mutação , Rim Policístico Autossômico Dominante/patologia , RNA/genética , RNA/isolamento & purificação , Análise de Sequência de DNA , Transdução de Sinais , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Lipopolysaccharide (LPS), the major causative agent of bacterial sepsis, has been used by many laboratories in genome-wide expression profiling of the LPS response. However, these studies have predominantly used in vitro cultured macrophages (Macs), which may not accurately reflect the LPS response of these innate immune cells in vivo. To overcome this limitation and to identify inflammatory genes in vivo, we have profiled genome-wide expression patterns in non-lymphoid, splenic myeloid cells extracted directly from LPS-treated mice. Genes encoding factors known to be involved in mediating or regulating inflammatory processes, such as cytokines and chemokines, as well as many genes whose immunological functions are not well known, were strongly induced by LPS after 3 h or 8 h of treatment. Most of the highly LPS-responsive genes that we randomly selected from the microarray data were independently confirmed by quantitative RT-PCR, implying that our microarray data are quite reliable. When our in vivo data were compared to previously reported microarray data for in vitro LPS-treated Macs, a significant proportion (â¼20%) of the in vivo LPS-responsive genes defined in this study were specific to cells exposed to LPS in vivo, but a larger proportion of them (â¼60%) were influenced by LPS in both in vitro and in vivo settings. This result indicates that our in vivo LPS-responsive gene set includes not only previously identified in vitro LPS-responsive genes but also novel LPS-responsive genes. Both types of genes would be a valuable resource in the future for understanding inflammatory responses in vivo.
Assuntos
Macrófagos/imunologia , Células Mieloides/imunologia , Sepse/genética , Baço/imunologia , Animais , Células Cultivadas , Quimiocinas/genética , Citocinas/genética , Perfilação da Expressão Gênica , Imunidade Inata/genética , Inflamação/genética , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise em MicrossériesRESUMO
Psoriasis and atopic dermatitis (AD) are skin diseases that are characterized by polarized CD4+ T cell responses. During the polarization of naïve CD4+ T cells, DNA methylation plays an important role in the regulation of gene transcription. In this study, we profiled the genome-wide DNA methylation status of naïve CD4+ T cells in patients with psoriasis or AD and healthy controls using a ChIP-seq method. Only psoriasis patient T cells, not those of AD patients, showed distinct hypomethylation (>4-fold) compared to healthy control T cells in twenty-six regions of the genome ranging in size from 10 to 70 kb. These regions were mostly pericentromeric on 10 different chromosomes and incidentally coincided with various strong epigenomic signals, such as histone modifications and transcription factor binding sites, that had been observed in the ENCODE project implying the potential epigenetic regulation in psoriasis development. The gene-centric analysis indicated that the promoter regions of 121 genes on the X chromosome had dramatically elevated methylation levels in psoriasis patient T-cells compared to those from healthy controls (>4-fold). Moreover, immune-related genes on the X chromosome had higher hypermethylation than other genes (P=0.046). No such patterns were observed with AD patient T cells. These findings imply that methylation changes in naïve CD4+ T cells may affect CD4+ T cell polarization, especially in the pathogenesis of psoriasis.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Metilação de DNA , Dermatite Atópica/genética , Epigênese Genética , Imunidade/genética , Psoríase/genética , Cromossomos Humanos X/genética , Genoma Humano , Humanos , Regiões Promotoras GenéticasRESUMO
BACKGROUND: Epigenetic alteration of gene expression is a common event in human cancer. DNA methylation is a well-known epigenetic process, but verifying the exact nature of epigenetic changes associated with cancer remains difficult. METHODS: We profiled the methylome of human gastric cancer tissue at 50-bp resolution using a methylated DNA enrichment technique (methylated CpG island recovery assay) in combination with a genome analyzer and a new normalization algorithm. RESULTS: We were able to gain a comprehensive view of promoters with various CpG densities, including CpG Islands (CGIs), transcript bodies, and various repeat classes. We found that gastric cancer was associated with hypermethylation of 5' CGIs and the 5'-end of coding exons as well as hypomethylation of repeat elements, such as short interspersed nuclear elements and the composite element SVA. Hypermethylation of 5' CGIs was significantly correlated with downregulation of associated genes, such as those in the HOX and histone gene families. We also discovered long-range epigenetic silencing (LRES) regions in gastric cancer tissue and identified several hypermethylated genes (MDM2, DYRK2, and LYZ) within these regions. The methylation status of CGIs and gene annotation elements in metastatic lymph nodes was intermediate between normal and cancerous tissue, indicating that methylation of specific genes is gradually increased in cancerous tissue. CONCLUSIONS: Our findings will provide valuable data for future analysis of CpG methylation patterns, useful markers for the diagnosis of stomach cancer, as well as a new analysis method for clinical epigenomics investigations.
Assuntos
Metilação de DNA/genética , Neoplasias Gástricas/genética , Bioensaio , Cromossomos Humanos/genética , Análise por Conglomerados , Ilhas de CpG/genética , Mucosa Gástrica/metabolismo , Inativação Gênica , Genoma Humano/genética , Humanos , Metástase Linfática/genética , Anotação de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNA , Estômago/patologiaRESUMO
BACKGROUND: Nucleosome deposition downstream of transcription initiation and DNA methylation in the gene body suggest that control of transcription elongation is a key aspect of epigenetic regulation. RESULTS: Here we report a genome-wide observation of distinct peaks of nucleosomes and methylation at both ends of a protein coding unit. Elongating polymerases tend to pause near both coding ends immediately upstream of the epigenetic peaks, causing a significant reduction in elongation efficiency. Conserved features in underlying protein coding sequences seem to dictate their evolutionary conservation across multiple species. The nucleosomal and methylation marks are commonly associated with high sequence-encoded DNA-bending propensity but differentially with CpG density. As the gene grows longer, the epigenetic codes seem to be shifted from variable inner sequences toward boundary regions, rendering the peaks more prominent in higher organisms. CONCLUSIONS: Recent studies suggest that epigenetic inhibition of transcription elongation facilitates the inclusion of constitutive exons during RNA splicing. The epigenetic marks we identified here seem to secure the first and last coding exons from exon skipping as they are indispensable for accurate translation.