RESUMO
Microsatellite unstable colorectal cancer (MSI-CRC) can arise through germline mutations in mismatch repair (MMR) genes in individuals with Lynch syndrome (LS), or sporadically through promoter methylation of the MMR gene MLH1. Despite the different origins of hereditary and sporadic MSI tumours, their genomic features have not been extensively compared. A prominent feature of MMR-deficient genomes is the occurrence of many indels in short repeat sequences, an understudied mutation type due to the technical challenges of variant calling in these regions. In this study, we performed whole genome sequencing and RNA-sequencing on 29 sporadic and 14 hereditary MSI-CRCs. We compared the tumour groups by analysing genome-wide mutation densities, microsatellite repeat indels, recurrent protein-coding variants, signatures of single base, doublet base, and indel mutations, and changes in gene expression. We show that the mutational landscapes of hereditary and sporadic MSI-CRCs, including mutational signatures and mutation densities genome-wide and in microsatellites, are highly similar. Only a low number of differentially expressed genes were found, enriched to interferon-γ regulated immune response pathways. Analysis of the variance in allelic fractions of somatic variants in each tumour group revealed higher clonal heterogeneity in sporadic MSI-CRCs. Our results suggest that the differing molecular origins of MMR deficiency in hereditary and sporadic MSI-CRCs do not result in substantial differences in the mutational landscapes of these tumours. The divergent patterns of clonal evolution between the tumour groups may have clinical implications, as high clonal heterogeneity has been associated with decreased tumour immunosurveillance and reduced responsiveness to immunotherapy.
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According to conventional views, colon cancer originates from stem cells. However, inflammation, a key risk factor for colon cancer, has been shown to suppress intestinal stemness. Here, we used Paneth cells as a model to assess the capacity of differentiated lineages to trigger tumorigenesis in the context of inflammation in mice. Upon inflammation, Paneth cell-specific Apc mutations led to intestinal tumors reminiscent not only of those arising in patients with inflammatory bowel disease, but also of a larger fraction of human sporadic colon cancers. The latter is possibly because of the inflammatory consequences of western-style dietary habits, a major colon cancer risk factor. Machine learning methods designed to predict the cell-of-origin of cancer from patient-derived tumor samples confirmed that, in a substantial fraction of sporadic cases, the origins of colon cancer reside in secretory lineages and not in stem cells.
Assuntos
Carcinogênese , Linhagem da Célula , Neoplasias do Colo , Inflamação , Celulas de Paneth , Animais , Camundongos , Linhagem da Célula/genética , Celulas de Paneth/patologia , Humanos , Inflamação/genética , Inflamação/patologia , Carcinogênese/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Mutação , Células-Tronco/patologia , Diferenciação Celular/genética , Transformação Celular Neoplásica/genética , Proteína da Polipose Adenomatosa do Colo/genética , Camundongos Endogâmicos C57BL , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologiaRESUMO
In monogenic autoinflammatory diseases, mutations in genes regulating innate immune responses often lead to uncontrolled activation of inflammasome pathways or the type I interferon (IFN-I) response. We describe a mechanism of autoinflammation potentially predisposing patients to life-threatening necrotizing soft tissue inflammation. Six unrelated families are identified in which affected members present with necrotizing fasciitis or severe soft tissue inflammations. Exome sequencing reveals truncating monoallelic loss-of-function variants of nuclear factor κ light-chain enhancer of activated B cells (NFKB1) in affected patients. In patients' macrophages and in NFKB1-variant-bearing THP-1 cells, activation increases both interleukin (IL)-1ß secretion and IFN-I signaling. Truncation of NF-κB1 impairs autophagy, accompanied by the accumulation of reactive oxygen species and reduced degradation of inflammasome receptor nucleotide-binding oligomerization domain, leucine-rich repeat-containing protein 3 (NLRP3), and Toll/IL-1 receptor domain-containing adaptor protein inducing IFN-ß (TRIF), thus leading to combined excessive inflammasome and IFN-I activity. Many of the patients respond to anti-inflammatory treatment, and targeting IL-1ß and/or IFN-I signaling could represent a therapeutic approach for these patients.
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Fasciite Necrosante , Interferon Tipo I , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Imunidade Inata , Inflamação/metabolismo , Subunidade p50 de NF-kappa BRESUMO
According to conventional views, colon cancer originates from stem cells. However, inflammation, a key risk factor for colon cancer, was shown to suppress intestinal stemness. Here, we employed Paneth cells (PCs) as a model to assess the capacity of differentiated lineages to trigger tumorigenesis in the context of inflammation. Upon inflammation, PC-specific Apc mutations led to intestinal tumors reminiscent not only of those arising in inflammatory bowel disease (IBD) patients but also of a larger fraction of sporadic colon cancers. The latter is likely due to the inflammatory consequences of Western-style dietary habits, the major colon cancer risk factor. Computational methods designed to predict the cell-of-origin of cancer confirmed that, in a substantial fraction of sporadic colon cancers the cells-of-origin are secretory lineages and not stem cells.
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BACKGROUND: Accurate regulation of DNA methylation is necessary for normal cells to differentiate, develop and function. TET2 catalyzes stepwise DNA demethylation in hematopoietic cells. Mutations in the TET2 gene predispose to hematological malignancies by causing DNA methylation overload and aberrant epigenomic landscape. Studies on mice and cell lines show that the function of TET2 is boosted by vitamin C. Thus, by strengthening the demethylation activity of TET2, vitamin C could play a role in the prevention of hematological malignancies in individuals with TET2 dysfunction. We recently identified a family with lymphoma predisposition where a heterozygous truncating germline mutation in TET2 segregated with nodular lymphocyte-predominant Hodgkin lymphoma. The mutation carriers displayed a hypermethylation pattern that was absent in the family members without the mutation. METHODS: In a clinical trial of 1 year, we investigated the effects of oral 1 g/day vitamin C supplementation on DNA methylation by analyzing genome-wide DNA methylation and gene expression patterns from the family members. RESULTS: We show that vitamin C reinforces the DNA demethylation cascade, reduces the proportion of hypermethylated loci and diminishes gene expression differences between TET2 mutation carriers and control individuals. CONCLUSIONS: These results suggest that vitamin C supplementation increases DNA methylation turnover and provide a basis for further work to examine the potential benefits of vitamin C supplementation in individuals with germline and somatic TET2 mutations. TRIAL REGISTRATION: This trial was registered at EudraCT with reference number of 2018-000155-41 (01.04.2019).
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Ácido Ascórbico , Proteínas de Ligação a DNA , Dioxigenases , Neoplasias Hematológicas , Ácido Ascórbico/uso terapêutico , Dioxigenases/genética , Desmetilação do DNA , Metilação de DNA , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa , Neoplasias Hematológicas/genética , Mutação , Vitaminas/uso terapêutico , HumanosRESUMO
Mechanical forces in a constrained cellular environment were recently established as a facilitator of chromosomal damage. Whether this could contribute to tumorigenesis is not known. Uterine leiomyomas are common neoplasms that display relatively few chromosomal aberrations. We hypothesized that if mechanical forces contribute to chromosomal damage, signs of this could be seen in uterine leiomyomas from parous women. We examined the karyotypes of 1946 tumors, and found a striking overrepresentation of chromosomal damage associated with parity. We then subjected myometrial cells to physiological forces similar to those encountered during pregnancy, and found this to cause DNA breaks and a DNA repair response. While mechanical forces acting in constrained cellular environments may thus contribute to neoplastic degeneration, and genesis of uterine leiomyoma, further studies are needed to prove possible causality of the observed association. No evidence for progression to malignancy was found.
Assuntos
Aberrações Cromossômicas , Reparo do DNA , Leiomioma/genética , Complexo Mediador/genética , Paridade , Neoplasias Uterinas/genética , Adulto , Fenômenos Biomecânicos , Quebras de DNA de Cadeia Dupla , Feminino , Expressão Gênica , Humanos , Histerectomia , Cariótipo , Leiomioma/etiologia , Leiomioma/patologia , Leiomioma/cirurgia , Mutação , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Miométrio/metabolismo , Miométrio/patologia , Gravidez , Cultura Primária de Células , Estudos Prospectivos , Neoplasias Uterinas/etiologia , Neoplasias Uterinas/patologia , Neoplasias Uterinas/cirurgiaRESUMO
One in four women suffers from uterine leiomyomas (ULs)-benign tumours of the uterine wall, also known as uterine fibroids-at some point in premenopausal life. ULs can cause excessive bleeding, pain and infertility1, and are a common cause of hysterectomy2. They emerge through at least three distinct genetic drivers: mutations in MED12 or FH, or genomic rearrangement of HMGA23. Here we created genome-wide datasets, using DNA, RNA, assay for transposase-accessible chromatin (ATAC), chromatin immunoprecipitation (ChIP) and HiC chromatin immunoprecipitation (HiChIP) sequencing of primary tissues to profoundly understand the genesis of UL. We identified somatic mutations in genes encoding six members of the SRCAP histone-loading complex4, and found that germline mutations in the SRCAP members YEATS4 and ZNHIT1 predispose women to UL. Tumours bearing these mutations showed defective deposition of the histone variant H2A.Z. In ULs, H2A.Z occupancy correlated positively with chromatin accessibility and gene expression, and negatively with DNA methylation, but these correlations were weak in tumours bearing SRCAP complex mutations. In these tumours, open chromatin emerged at transcription start sites where H2A.Z was lost, which was associated with upregulation of genes. Furthermore, YEATS4 defects were associated with abnormal upregulation of bivalent embryonic stem cell genes, as previously shown in mice5. Our work describes a potential mechanism of tumorigenesis-epigenetic instability caused by deficient H2A.Z deposition-and suggests that ULs arise through an aberrant differentiation program driven by deranged chromatin, emanating from a small number of mutually exclusive driver mutations.
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Montagem e Desmontagem da Cromatina , Cromatina/genética , Cromatina/metabolismo , Histonas/deficiência , Leiomioma/genética , Mutação , Neoplasias Uterinas/genética , Carcinogênese/genética , Linhagem Celular , Cromatina/química , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Leiomioma/metabolismo , Leiomioma/patologia , Ligases/genética , Complexo Repressor Polycomb 1/genética , Proteínas do Grupo Polycomb/genética , Fatores de Transcrição/genética , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologiaRESUMO
Hereditary colorectal cancer (CRC) syndromes attributable to high penetrance mutations represent 9-26% of young-onset CRC cases. The clinical significance of many of these mutations is understood well enough to be used in diagnostics and as an aid in patient care. However, despite the advances made in the field, a significant proportion of familial and early-onset cases remains molecularly uncharacterized and extensive work is still needed to fully understand the genetic nature of CRC susceptibility. With the emergence of next-generation sequencing and associated methods, several predisposition loci have been unraveled, but validation is incomplete. Individuals with cancer-predisposing mutations are currently enrolled in life-long surveillance, but with the development of new treatments, such as cancer vaccinations, this might change in the not so distant future for at least some individuals. For individuals without a known cause for their disease susceptibility, prevention and therapy options are less precise. Herein, we review the progress achieved in the last three decades with a focus on how CRC predisposition genes were discovered. Furthermore, we discuss the clinical implications of these discoveries and anticipate what to expect in the next decade.
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Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Transformação Celular Neoplásica/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Predisposição Genética para Doença , Alelos , Animais , Progressão da Doença , Estudos de Associação Genética , Ligação Genética , Genômica/métodos , Humanos , Mutação , Fenótipo , Síndrome , Sequenciamento do ExomaRESUMO
BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disorder associated with an elevated risk of colorectal cancer (CRC). IBD-associated CRC (IBD-CRC) may represent a distinct pathway of tumorigenesis compared to sporadic CRC (sCRC). Our aim was to comprehensively characterize IBD-associated tumorigenesis integrating multiple high-throughput approaches, and to compare the results with in-house data sets from sCRCs. METHODS: Whole-genome sequencing, single nucleotide polymorphism arrays, RNA sequencing, genome-wide methylation analysis, and immunohistochemistry were performed using fresh-frozen and formalin-fixed tissue samples of tumor and corresponding normal tissues from 31 patients with IBD-CRC. RESULTS: Transcriptome-based tumor subtyping revealed the complete absence of canonical epithelial tumor subtype associated with WNT signaling in IBD-CRCs, dominated instead by mesenchymal stroma-rich subtype. Negative WNT regulators AXIN2 and RNF43 were strongly down-regulated in IBD-CRCs and chromosomal gains at HNF4A, a negative regulator of WNT-induced epithelial-mesenchymal transition (EMT), were less frequent compared to sCRCs. Enrichment of hypomethylation at HNF4α binding sites was detected solely in sCRC genomes. PIGR and OSMR involved in mucosal immunity were dysregulated via epigenetic modifications in IBD-CRCs. Genome-wide analysis showed significant enrichment of noncoding mutations to 5'untranslated region of TP53 in IBD-CRCs. As reported previously, somatic mutations in APC and KRAS were less frequent in IBD-CRCs compared to sCRCs. CONCLUSIONS: Distinct mechanisms of WNT pathway dysregulation skew IBD-CRCs toward mesenchymal tumor subtype, which may affect prognosis and treatment options. Increased OSMR signaling may favor the establishment of mesenchymal tumors in patients with IBD.
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Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/genética , Neoplasias Associadas a Colite/genética , Metilação de DNA , Epigênese Genética , Doenças Inflamatórias Intestinais/genética , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/patologia , Neoplasias Associadas a Colite/imunologia , Neoplasias Associadas a Colite/patologia , Análise Mutacional de DNA , Epigenômica , Feminino , Finlândia , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Análise de Sequência de RNA , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Sequenciamento Completo do GenomaRESUMO
Microsatellite instability (MSI) is caused by defective DNA mismatch repair (MMR), and manifests as accumulation of small insertions and deletions (indels) in short tandem repeats of the genome. Another form of repeat instability, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), has been suggested to occur in 50% to 60% of colorectal cancer (CRC), of which approximately one quarter are accounted for by MSI. Unlike for MSI, the criteria for defining EMAST is not consensual. EMAST CRCs have been suggested to form a distinct subset of CRCs that has been linked to a higher tumor stage, chronic inflammation, and poor prognosis. EMAST CRCs not exhibiting MSI have been proposed to show instability of di- and trinucleotide repeats in addition to tetranucleotide repeats, but lack instability of mononucleotide repeats. However, previous studies on EMAST have been based on targeted analysis of small sets of marker repeats, often in relatively few samples. To gain insight into tetranucleotide instability on a genome-wide level, we utilized whole genome sequencing data from 227 microsatellite stable (MSS) CRCs, 18 MSI CRCs, 3 POLE-mutated CRCs, and their corresponding normal samples. As expected, we observed tetranucleotide instability in all MSI CRCs, accompanied by instability of mono-, di-, and trinucleotide repeats. Among MSS CRCs, some tumors displayed more microsatellite mutations than others as a continuum, and no distinct subset of tumors with the previously proposed molecular characters of EMAST could be observed. Our results suggest that tetranucleotide repeat mutations in non-MSI CRCs represent stochastic mutation events rather than define a distinct CRC subclass.
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Neoplasias Colorretais/genética , Testes Genéticos/métodos , Mutação INDEL , Repetições de Microssatélites , Sequenciamento Completo do Genoma/métodos , Testes Genéticos/estatística & dados numéricos , Humanos , Sequenciamento Completo do Genoma/estatística & dados numéricosRESUMO
BACKGROUND: Tumor necrosis factor α-induced protein 3 gene (TNFAIP3, also called A20) haploinsufficiency (HA20) leads to autoinflammation and autoimmunity. We have recently shown that a p.(Lys91*) mutation in A20 disrupts nuclear factor κB signaling, impairs protein-protein interactions of A20, and leads to inflammasome activation. METHODS: We now describe the clinical presentations and drug responses in a family with HA20 p.(Lys91*) mutation, consistent with our previously reported diverse immunological and functional findings. RESULTS: We report for the first time that inflammasome-mediated autoinflammatory lung reaction caused by HA20 can be treated with interleukin 1 antagonist anakinra. We also describe severe anemia related to HA20 successfully treated with mycophenolate. In addition, HA20 p.(Lys91*) was found to associate with autoimmune thyroid disease, juvenile idiopathic arthritis, psoriasis, liver disease, and immunodeficiency presenting with specific antibody deficiency and genital papillomatosis. CONCLUSIONS: We conclude that HA20 may lead to combination of inflammation, immunodeficiency, and autoimmunity. The condition may present with variable and unpredictable symptoms with atypical treatment responses.
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Artrite Juvenil , Haploinsuficiência , Autoimunidade , Humanos , Mutação , NF-kappa BRESUMO
Upon binding to pathogen or self-derived cytosolic nucleic acids cyclic GMP-AMP synthase (cGAS) triggers the production of cGAMP that further activates transmembrane protein STING. Upon activation STING translocates from ER via Golgi to vesicles. Monogenic STING gain-of-function mutations cause early-onset type I interferonopathy, with disease presentation ranging from fatal vasculopathy to mild chilblain lupus. Molecular mechanisms underlying the variable phenotype-genotype correlation are presently unclear. Here, we report a novel gain-of-function G207E STING mutation causing a distinct phenotype with alopecia, photosensitivity, thyroid dysfunction, and features of STING-associated vasculopathy with onset in infancy (SAVI), such as livedo reticularis, skin vasculitis, nasal septum perforation, facial erythema, and bacterial infections. Polymorphism in TMEM173 and IFIH1 showed variable penetrance in the affected family, implying contribution to varying phenotype spectrum. The G207E mutation constitutively activates inflammation-related pathways in vitro, and causes aberrant interferon signature and inflammasome activation in patient PBMCs. Treatment with Janus kinase 1 and 2 (JAK1/2) inhibitor baricitinib was beneficiary for a vasculitic ulcer, induced hair regrowth and improved overall well-being in one patient. Protein-protein interactions propose impaired cellular trafficking of G207E mutant. These findings reveal the molecular landscape of STING and propose common polymorphisms in TMEM173 and IFIH1 as likely modifiers of the phenotype.
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Alelos , Estudos de Associação Genética , Predisposição Genética para Doença , Helicase IFIH1 Induzida por Interferon/genética , Proteínas de Membrana/genética , Mutação , Estudos de Casos e Controles , Consanguinidade , Feminino , Perfilação da Expressão Gênica , Ligação Genética , Humanos , Masculino , Linhagem , Transcriptoma , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: CCAAT enhancer-binding protein epsilon (C/EBPε) is a transcription factor involved in late myeloid lineage differentiation and cellular function. The only previously known disorder linked to C/EBPε is autosomal recessive neutrophil-specific granule deficiency leading to severely impaired neutrophil function and early mortality. OBJECTIVE: The aim of this study was to molecularly characterize the effects of C/EBPε transcription factor Arg219His mutation identified in a Finnish family with previously genetically uncharacterized autoinflammatory and immunodeficiency syndrome. METHODS: Genetic analysis, proteomics, genome-wide transcriptional profiling by means of RNA-sequencing, chromatin immunoprecipitation (ChIP) sequencing, and assessment of the inflammasome function of primary macrophages were performed. RESULTS: Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated noncanonical inflammasome activation caused by decreased association with transcriptional repressors, leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLR family, pyrin domain-containing 3 protein (NLRP3) and constitutively expressed caspase-5 in macrophages. CONCLUSION: We describe a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBPε. Mutated C/EBPε acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency. The mechanism, including widely dysregulated transcription, is likely not unique for C/EBPε. Similar multiomics approaches should also be used in studying other transcription factor-associated diseases.
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Proteínas Estimuladoras de Ligação a CCAAT/genética , Mutação com Ganho de Função/genética , Síndromes de Imunodeficiência/genética , Inflamassomos/genética , Inflamação/genética , Macrófagos/metabolismo , Neutrófilos/fisiologia , Idoso , Caspases/genética , Caspases/metabolismo , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamassomos/metabolismo , Macrófagos/patologia , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Linhagem , Análise de Sequência de RNA , Regulação para CimaRESUMO
Clonal hematopoiesis driven by somatic heterozygous TET2 loss is linked to malignant degeneration via consequent aberrant DNA methylation, and possibly to cardiovascular disease via increased cytokine and chemokine expression as reported in mice. Here, we discover a germline TET2 mutation in a lymphoma family. We observe neither unusual predisposition to atherosclerosis nor abnormal pro-inflammatory cytokine or chemokine expression. The latter finding is confirmed in cells from three additional unrelated TET2 germline mutation carriers. The TET2 defect elevates blood DNA methylation levels, especially at active enhancers and cell-type specific regulatory regions with binding sequences of master transcription factors involved in hematopoiesis. The regions display reduced methylation relative to all open chromatin regions in four DNMT3A germline mutation carriers, potentially due to TET2-mediated oxidation. Our findings provide insight into the interplay between epigenetic modulators and transcription factor activity in hematological neoplasia, but do not confirm the putative role of TET2 in atherosclerosis.
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Aterosclerose/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Haploinsuficiência , Doença de Hodgkin/genética , Proteínas Proto-Oncogênicas/genética , Adulto , Aterosclerose/patologia , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Epigênese Genética , Feminino , Finlândia , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Hematopoese/genética , Doença de Hodgkin/sangue , Doença de Hodgkin/patologia , Humanos , Masculino , Fenótipo , Cultura Primária de Células , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/metabolismo , Sequenciamento Completo do GenomaRESUMO
OBJECTIVES: TNFAIP3 encodes A20 that negatively regulates nuclear factor kappa light chain enhancer of activated B cells (NF-κB), the major transcription factor coordinating inflammatory gene expression. TNFAIP3 polymorphisms have been linked with a spectrum of inflammatory and autoimmune diseases and, recently, loss-of-function mutations in A20 were found to cause a novel inflammatory disease 'haploinsufficiency of A20' (HA20). Here we describe a family with HA20 caused by a novel TNFAIP3 loss-of-function mutation and elucidate the upstream molecular mechanisms linking HA20 to dysregulation of NF-κB and the related inflammasome pathway. METHODS: NF-κB activation was studied in a mutation-expressing cell line using luciferase reporter assay. Physical and close-proximity protein-protein interactions of wild-type and TNFAIP3 p.(Lys91*) mutant A20 were analysed using mass spectrometry. NF-κB -dependent transcription, cytokine secretion and inflammasome activation were compared in immune cells of the HA20 patients and control subjects. RESULTS: The protein-protein interactome of p.(Lys91*) mutant A20 was severely impaired, including interactions with proteins regulating NF-κB activation, DNA repair responses and the NLR family pyrin domain containing 3 (NLRP3) inflammasome. The p.(Lys91*) mutant A20 failed to suppress NF-κB signalling, which led to increased NF-κB -dependent proinflammatory cytokine transcription. Functional experiments in the HA20 patients' immune cells uncovered a novel caspase-8-dependent mechanism of NLRP3 inflammasome hyperresponsiveness that mediated the excessive secretion of interleukin-1ß and interleukin-18. CONCLUSIONS: The current findings significantly deepen our understanding of the molecular mechanisms underlying HA20 and other diseases associated with reduced A20 expression or function, paving the way for future therapeutic targeting of the pathway.
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BACKGROUND AND AIMS: Subendothelial interaction of LDL with extracellular matrix drives atherogenesis. This interaction can be strengthened by proteolytic modification of LDL. Mast cells (MCs) are present in atherosclerotic lesions, and upon activation, they degranulate and release a variety of neutral proteases. Here we studied the ability of MC proteases to cleave apoB-100 of LDL and affect the binding of LDL to proteoglycans. METHODS: Mature human MCs were differentiated from human peripheral blood-derived CD34+ progenitors in vitro and activated with calcium ionophore to generate MC-conditioned medium. LDL was incubated in the MC-conditioned medium or with individual MC proteases, and the binding of native and modified LDL to isolated human aortic proteoglycans or to human atherosclerotic plaques ex vivo was determined. MC proteases in atherosclerotic human coronary artery lesions were detected by immunofluorescence and qPCR. RESULTS: Activated human MCs released the neutral proteases tryptase, chymase, carboxypeptidase A3, cathepsin G, and granzyme B. Of these, cathepsin G degraded most efficiently apoB-100, induced LDL fusion, and enhanced binding of LDL to isolated human aortic proteoglycans and human atherosclerotic lesions ex vivo. Double immunofluoresence staining of human atherosclerotic coronary arteries for tryptase and cathepsin G indicated that lesional MCs contain cathepsin G. In the lesions, expression of cathepsin G correlated with the expression of tryptase and chymase, but not with that of neutrophil proteinase 3. CONCLUSIONS: The present study suggests that cathepsin G in human atherosclerotic lesions is largely derived from MCs and that activated MCs may contribute to atherogenesis by enhancing LDL retention.
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Apolipoproteína B-100/metabolismo , Aterosclerose/enzimologia , Doenças das Artérias Carótidas/enzimologia , Catepsina G/metabolismo , Doença da Artéria Coronariana/enzimologia , Lipoproteínas LDL/metabolismo , Mastócitos/enzimologia , Proteoglicanas/metabolismo , Aterosclerose/patologia , Doenças das Artérias Carótidas/patologia , Degranulação Celular , Células Cultivadas , Doença da Artéria Coronariana/patologia , Ativação Enzimática , Humanos , Placa Aterosclerótica , Ligação Proteica , ProteóliseRESUMO
OBJECTIVES: To study the prevalence of asymptomatic activation of Epstein-Barr virus (EBV) in patients with rheumatoid arthritis (RA) and to analyse the correlation of serum EBV DNA with the disease activity. METHODS: The level of EBV DNA was determined by droplet digital PCR assay from the serum of 46 DMARD naive early RA (ERA) and 22 chronic RA (CRA)-patients at study onset. Follow-up samples from 31 ERA and 16 CRA patients were obtained after starting or modifying the anti-rheumatic treatment. EBV DNA was also measured from 33 healthy controls and 9 patients with adult onset Still's disease (AOSD). Disease activity was assessed by the disease activity score (DAS28). RESULTS: At baseline, EBV DNA was detected in the serum of 7 of the 46 ERA patients all of whom had moderate or high disease activity. In the follow-up samples, 11 of 31 patients were EBV DNA positive. At baseline EBV positive patients had significantly higher disease activity (p=0.036) and the concentration of EBV DNA correlated significantly with DAS28 (rs=0.333, p=0.024). EBV DNA was detected in 3 of 22 CRA patients at study onset and in 8 of 16 in the follow-up samples. At follow-up EBV positive patients had significantly higher DAS28 (p=0.027) and the concentration of EBV DNA correlated significantly with DAS28 (rs=0.724, p=0.002). Only one of the healthy controls and none of the AOSD patients were positive for EBV DNA. CONCLUSIONS: Active RA is associated with a lytic EBV infection which may have a role in the pathogenesis of RA.
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Artrite Reumatoide/diagnóstico , Artrite Reumatoide/epidemiologia , DNA Viral/genética , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/epidemiologia , Herpesvirus Humano 4/genética , Reação em Cadeia da Polimerase/métodos , Adulto , Idoso , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/virologia , Estudos de Casos e Controles , DNA Viral/sangue , Infecções por Vírus Epstein-Barr/sangue , Infecções por Vírus Epstein-Barr/virologia , Feminino , Finlândia/epidemiologia , Interações Hospedeiro-Patógeno , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prevalência , Fatores de Risco , Índice de Gravidade de Doença , Fatores de Tempo , Carga Viral , Ativação ViralRESUMO
Inflammasomes are intracellular protein platforms, which, upon activation, produce the highly proinflammatory cytokines interleukin (IL)-1ß and IL-18. Heme, hemin and their degradation products possess significant immunomodulatory functions. Here, we studied whether hemin regulates inflammasome function in macrophages. Both hemin and its derivative, cobalt protoporphyrin (CoPP), significantly reduced IL-1ß secretion by cultured human primary macrophages, the human monocytic leukemia cell line and also mouse bone marrow-derived and peritoneal macrophages. Intraperitoneal administration of CoPP to mice prior to urate crystal-induced peritonitis alleviated IL-1ß secretion to the peritoneal cavity. In cultured macrophages, hemin and CoPP inhibited NLRP3 inflammasome assembly by reducing the amount of intracellular apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). The reduction of ASC was associated with enhanced autophagosome formation and autophagic flux. Inhibition of autophagy prevented the CoPP-induced depletion of ASC, implying that the depletion was caused by increased autophagy. Our data indicate that hemin functions as an endogenous negative regulator of the NLRP3 inflammasome. The inhibition is mediated via enhanced autophagy that results in increased degradation of ASC. This regulatory mechanism may provide a novel approach for the treatment of inflammasome-related diseases.
Assuntos
Hemina/metabolismo , Inflamassomos/metabolismo , Macrófagos/fisiologia , Peritonite/imunologia , Protoporfirinas/metabolismo , Animais , Linhagem Celular , Hemina/administração & dosagem , Humanos , Imunomodulação , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Peritonite/induzido quimicamente , Protoporfirinas/administração & dosagem , Ácido ÚricoRESUMO
OBJECTIVE: Activation of the inflammasome pathway in macrophages results in the secretion of 2 potent proinflammatory and proatherogenic cytokines, interleukin (IL)-1ß, and IL-18. Atherosclerotic lesions are characterized by the presence of various endogenous activators of the NLR family pyrin domain containing 3 (NLRP3) inflammasome, including cholesterol crystals and extracellular ATP. The aim of this study was to comprehensively characterize the expression of inflammasome pathway components and regulators in human atherosclerotic lesions. APPROACH AND RESULTS: Twenty human coronary artery RNA samples from 10 explanted hearts were analyzed using an inflammasome pathway-focused quantitative polymerase chain reaction array. Advanced atherosclerotic plaques, when compared with early-to-intermediate lesions from the same coronary trees, displayed significant upregulation of 12 target genes, including the key inflammasome components apoptosis-associated speck-like protein containing a CARD domain, caspase-1, and IL-18. Immunohistochemical stainings of the advanced plaques revealed macrophage foam cells positive for NLRP3 inflammasome components around the necrotic lipid cores. The polymerase chain reaction array target p38δ mitogen-activated protein kinase was upregulated in advanced plaques and strongly expressed by lesional macrophage foam cells. In cultured human monocyte-derived macrophages, the p38δ mitogen-activated protein kinase was activated by intracellular stress signals triggered during ATP- and cholesterol crystal-induced NLRP3 inflammasome activation and was required for NLRP3-mediated IL-1ß secretion. CONCLUSIONS: Increased expression of the key inflammasome components in advanced coronary lesions implies enhanced activity of the inflammasome pathway in progression of coronary atherosclerosis. The p38δ mitogen-activated protein kinase was identified as a novel regulator of NLRP3 inflammasome activation in primary human macrophages, and thus, represents a potential target for modulation of atherosclerotic inflammation.
Assuntos
Doença da Artéria Coronariana/enzimologia , Vasos Coronários/enzimologia , Células Espumosas/enzimologia , Inflamassomos/metabolismo , Proteína Quinase 13 Ativada por Mitógeno/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Placa Aterosclerótica , Trifosfato de Adenosina/metabolismo , Células Cultivadas , Colesterol/metabolismo , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Vasos Coronários/patologia , Cristalização , Ativação Enzimática , Células Espumosas/patologia , Perfilação da Expressão Gênica/métodos , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Inflamassomos/genética , Masculino , Pessoa de Meia-Idade , Proteína Quinase 13 Ativada por Mitógeno/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Necrose , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Fatores de Tempo , Transativadores/metabolismo , Regulação para CimaRESUMO
Calcific aortic valve disease (CAVD) is a progressive pathological condition with no effective pharmacological therapy. To identify novel molecular pathways as potential targets for pharmacotherapy, we studied microRNA (miRNA) profiles of heavily stenotic aortic valves (AS). One of the most upregulated miRNAs in AS valves compared to control valves was miR-125b (1.4-fold; P < 0.05). To identify CAVD-related changes in gene expression, DNA microarray analysis was performed, including an intermediate fibro(sclero)tic stage of the disease. This revealed changes especially in genes related to inflammation and immune response, including chemokine (C-C motif) ligand 3 (CCL3) and 4 (CCL4). CCL3 mRNA level was increased 3.9-fold (P < 0.05) when AS valves were compared to control valves, and a 2.5-fold increase (P < 0.05) in CCL4 gene expression was observed when fibro(sclero)tic valves were compared to control valves. Both CCL3 and CCL4 localized to macrophages by immunofluorescence. To identify chemokine-miRNA target pairs, data from miRNA target prediction databases were combined with valvular miRNA and mRNA expression profiles. MiR-125b was computationally predicted to target CCL4, as confirmed experimentally in cultured human THP-1 macrophages. Collectively, miR-125b and CCL4 appear to be involved in the progression of CAVD and may offer novel therapeutic and diagnostic strategies related to this disease.