RESUMO
Missense mutations in PLCG2 can cause autoinflammation with phospholipase C gamma 2-associated antibody deficiency and immune dysregulation (APLAID). Here, we generated a mouse model carrying an APLAID mutation (p.Ser707Tyr) and found that inflammatory infiltrates in the skin and lungs were only partially ameliorated by removing inflammasome function via the deletion of caspase-1. Also, deleting interleukin-6 or tumor necrosis factor did not fully prevent APLAID mutant mice from autoinflammation. Overall, these findings are in accordance with the poor response individuals with APLAID have to treatments that block interleukin-1, JAK1/2 or tumor necrosis factor. Cytokine analysis revealed increased granulocyte colony-stimulating factor (G-CSF) levels as the most distinct feature in mice and individuals with APLAID. Remarkably, treatment with a G-CSF antibody completely reversed established disease in APLAID mice. Furthermore, excessive myelopoiesis was normalized and lymphocyte numbers rebounded. APLAID mice were also fully rescued by bone marrow transplantation from healthy donors, associated with reduced G-CSF production, predominantly from non-hematopoietic cells. In summary, we identify APLAID as a G-CSF-driven autoinflammatory disease, for which targeted therapy is feasible.
Assuntos
Transplante de Medula Óssea , Fator Estimulador de Colônias de Granulócitos , Animais , Camundongos , Citocinas , Interleucina-1 , Fator de Necrose Tumoral alfa/genética , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/metabolismoRESUMO
Group 3 innate lymphoid cell (ILC3)-mediated production of the cytokine interleukin-22 (IL-22) is critical for the maintenance of immune homeostasis in the gastrointestinal tract. Here, we find that the function of ILC3s is not constant across the day, but instead oscillates between active phases and resting phases. Coordinate responsiveness of ILC3s in the intestine depended on the food-induced expression of the neuropeptide vasoactive intestinal peptide (VIP). Intestinal ILC3s had high expression of the G protein-coupled receptor vasoactive intestinal peptide receptor 2 (VIPR2), and activation by VIP markedly enhanced the production of IL-22 and the barrier function of the epithelium. Conversely, deficiency in signaling through VIPR2 led to impaired production of IL-22 by ILC3s and increased susceptibility to inflammation-induced gut injury. Thus, intrinsic cellular rhythms acted in synergy with the cyclic patterns of food intake to drive the production of IL-22 and synchronize protection of the intestinal epithelium through a VIP-VIPR2 pathway in ILC3s.
Assuntos
Imunidade nas Mucosas/imunologia , Subpopulações de Linfócitos/imunologia , Linfócitos/imunologia , Periodicidade , Peptídeo Intestinal Vasoativo/imunologia , Animais , Ingestão de Alimentos/imunologia , Imunidade Inata/imunologia , Subpopulações de Linfócitos/metabolismo , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.
Assuntos
Apoptose/imunologia , Macrófagos/imunologia , Necroptose/imunologia , Piroptose/imunologia , Infecções por Salmonella/imunologia , Salmonella/imunologia , Animais , Caspase 1/deficiência , Caspase 1/genética , Caspase 12/deficiência , Caspase 12/genética , Caspase 8/genética , Caspases Iniciadoras/deficiência , Caspases Iniciadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/genéticaRESUMO
H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes in differentiation and development. Here, we investigate the molecular consequences of heterochromatin loss in cells deficient in both SUV39H1 and SUV39H2 (Suv39DKO), the major mammalian histone methyltransferase enzymes that catalyze heterochromatic H3K9me3 deposition. We reveal a paradoxical repression of protein-coding genes in Suv39DKO cells, with these differentially expressed genes principally in euchromatic (Tn5-accessible, H3K4me3- and H3K27ac-marked) rather than heterochromatic (H3K9me3-marked) or polycomb (H3K27me3-marked) regions. Examination of the three-dimensional (3D) nucleome reveals that transcriptomic dysregulation occurs in euchromatic regions close to the nuclear periphery in 3D space. Moreover, this transcriptomic dysregulation is highly correlated with altered 3D genome organization in Suv39DKO cells. Together, our results suggest that the nuclear lamina-tethering of Suv39-dependent H3K9me3 domains provides an essential scaffold to support euchromatic genome organization and the maintenance of gene transcription for healthy cellular function.
Assuntos
Eucromatina , Heterocromatina , Histona-Lisina N-Metiltransferase , Histonas , Metiltransferases , Transcrição Gênica , Animais , Camundongos , Linhagem Celular , Eucromatina/metabolismo , Eucromatina/genética , Regulação da Expressão Gênica , Heterocromatina/metabolismo , Heterocromatina/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Histonas/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genéticaRESUMO
Inhibitor of growth 4 and 5 (ING4, ING5) are structurally similar chromatin-binding proteins in the KAT6A, KAT6B and KAT7 histone acetyltransferase protein complexes. Heterozygous mutations in the KAT6A or KAT6B gene cause human disorders with cardiac defects, but the contribution of their chromatin-adaptor proteins to development is unknown. We found that Ing5-/- mice had isolated cardiac ventricular septal defects. Ing4-/-Ing5-/- embryos failed to undergo chorioallantoic fusion and arrested in development at embryonic day 8.5, displaying loss of histone H3 lysine 14 acetylation, reduction in H3 lysine 23 acetylation levels and reduced developmental gene expression. Embryonic day 12.5 Ing4+/-Ing5-/- hearts showed a paucity of epicardial cells and epicardium-derived cells, failure of myocardium compaction, and coronary vasculature defects, accompanied by reduced expression of epicardium genes. Cell adhesion gene expression and proepicardium outgrowth were defective in the ING4- and ING5-deficient state. Our findings suggest that ING4 and ING5 are essential for heart development and promote epicardium and epicardium-derived cell fates and imply mutation of the human ING5 gene as a possible cause of isolated ventricular septal defects.
Assuntos
Proteínas de Transporte , Comunicação Interventricular , Lisina , Humanos , Animais , Camundongos , Linhagem da Célula , Histonas , Acetilação , Cromatina , Fatores de Transcrição , Proteínas Supressoras de Tumor , Proteínas de Homeodomínio/genética , Proteínas de Ciclo Celular , Histona AcetiltransferasesRESUMO
Polycythemia vera (PV) is a myeloproliferative neoplasm driven by activating mutations in JAK2 that result in unrestrained erythrocyte production, increasing patients' hematocrit and hemoglobin concentrations, placing them at risk of life-threatening thrombotic events. Our genome-wide association study of 440 PV cases and 403 351 controls using UK Biobank data showed that single nucleotide polymorphisms in HFE known to cause hemochromatosis are highly associated with PV diagnosis, linking iron regulation to PV. Analysis of the FinnGen dataset independently confirmed overrepresentation of homozygous HFE variants in patients with PV. HFE influences the expression of hepcidin, the master regulator of systemic iron homeostasis. Through genetic dissection of mouse models of PV, we show that the PV erythroid phenotype is directly linked to hepcidin expression: endogenous hepcidin upregulation alleviates erythroid disease whereas hepcidin ablation worsens it. Furthermore, we demonstrate that in PV, hepcidin is not regulated by expanded erythropoiesis but is likely governed by inflammatory cytokines signaling via GP130-coupled receptors. These findings have important implications for understanding the pathophysiology of PV and offer new therapeutic strategies for this disease.
Assuntos
Policitemia Vera , Animais , Camundongos , Policitemia Vera/genética , Policitemia Vera/complicações , Hepcidinas/genética , Estudo de Associação Genômica Ampla , Ferro/metabolismo , Fenótipo , HomeostaseRESUMO
Blood vessel growth and remodelling are essential during embryonic development and disease pathogenesis. The diversity of endothelial cells (ECs) is transcriptionally evident and ECs undergo dynamic changes in gene expression during vessel growth and remodelling. Here, we investigated the role of the histone acetyltransferase HBO1 (KAT7), which is important for activating genes during development and for histone H3 lysine 14 acetylation (H3K14ac). Loss of HBO1 and H3K14ac impaired developmental sprouting angiogenesis and reduced pathological EC overgrowth in the retinal endothelium. Single-cell RNA sequencing of retinal ECs revealed an increased abundance of tip cells in Hbo1-deficient retinas, which led to EC overcrowding in the retinal sprouting front and prevented efficient tip cell migration. We found that H3K14ac was highly abundant in the endothelial genome in both intra- and intergenic regions, suggesting that HBO1 acts as a genome organiser that promotes efficient tip cell behaviour necessary for sprouting angiogenesis. This article has an associated 'The people behind the papers' interview.
Assuntos
Histona Acetiltransferases/metabolismo , Neovascularização Patológica/metabolismo , Acetilação , Animais , Movimento Celular/fisiologia , Células Cultivadas , Desenvolvimento Embrionário/fisiologia , Células Endoteliais/metabolismo , Feminino , Histonas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
The histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development, histone H3 lysine 14 acetylation (H3K14Ac), and the expression of embryonic patterning genes. In this study, we report the role of HBO1 in regulating hematopoietic stem cell function in adult hematopoiesis. We used 2 complementary cre-recombinase transgenes to conditionally delete Hbo1 (Mx1-Cre and Rosa26-CreERT2). Hbo1-null mice became moribund due to hematopoietic failure with pancytopenia in the blood and bone marrow 2 to 6 weeks after Hbo1 deletion. Hbo1-deleted bone marrow cells failed to repopulate hemoablated recipients in competitive transplantation experiments. Hbo1 deletion caused a rapid loss of hematopoietic progenitors. The numbers of lineage-restricted progenitors for the erythroid, myeloid, B-, and T-cell lineages were reduced. Loss of HBO1 resulted in an abnormally high rate of recruitment of quiescent hematopoietic stem cells (HSCs) into the cell cycle. Cycling HSCs produced progenitors at the expense of self-renewal, which led to the exhaustion of the HSC pool. Mechanistically, genes important for HSC functions were downregulated in HSC-enriched cell populations after Hbo1 deletion, including genes essential for HSC quiescence and self-renewal, such as Mpl, Tek(Tie-2), Gfi1b, Egr1, Tal1(Scl), Gata2, Erg, Pbx1, Meis1, and Hox9, as well as genes important for multipotent progenitor cells and lineage-specific progenitor cells, such as Gata1. HBO1 was required for H3K14Ac through the genome and particularly at gene loci required for HSC quiescence and self-renewal. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSC maintenance and self-renewal in adult hematopoiesis.
Assuntos
Autorrenovação Celular , Células-Tronco Hematopoéticas , Histona Acetiltransferases , Animais , Células Cultivadas , Senescência Celular , Deleção de Genes , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Venetoclax (VEN) inhibits the prosurvival protein BCL2 to induce apoptosis and is a standard therapy for chronic lymphocytic leukemia (CLL), delivering high complete remission rates and prolonged progression-free survival in relapsed CLL but with eventual loss of efficacy. A spectrum of subclonal genetic changes associated with VEN resistance has now been described. To fully understand clinical resistance to VEN, we combined single-cell short- and long-read RNA-sequencing to reveal the previously unappreciated scale of genetic and epigenetic changes underpinning acquired VEN resistance. These appear to be multilayered. One layer comprises changes in the BCL2 family of apoptosis regulators, especially the prosurvival family members. This includes previously described mutations in BCL2 and amplification of the MCL1 gene but is heterogeneous across and within individual patient leukemias. Changes in the proapoptotic genes are notably uncommon, except for single cases with subclonal losses of BAX or NOXA. Much more prominent was universal MCL1 gene upregulation. This was driven by an overlying layer of emergent NF-κB (nuclear factor kappa B) activation, which persisted in circulating cells during VEN therapy. We discovered that MCL1 could be a direct transcriptional target of NF-κB. Both the switch to alternative prosurvival factors and NF-κB activation largely dissipate following VEN discontinuation. Our studies reveal the extent of plasticity of CLL cells in their ability to evade VEN-induced apoptosis. Importantly, these findings pinpoint new approaches to circumvent VEN resistance and provide a specific biological justification for the strategy of VEN discontinuation once a maximal response is achieved rather than maintaining long-term selective pressure with the drug.
Assuntos
Antineoplásicos , Leucemia Linfocítica Crônica de Células B , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , NF-kappa B , Resistencia a Medicamentos Antineoplásicos/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Recidiva , Antineoplásicos/uso terapêuticoRESUMO
Paternal pre-conceptual environmental experiences, such as stress and diet, can affect offspring brain and behavioral phenotypes via epigenetic modifications in sperm. Furthermore, maternal immune activation due to infection during gestation can reprogram offspring behavior and brain functioning in adulthood. However, the effects of paternal pre-conceptual exposure to immune activation on the behavior and physiology of offspring (F1) and grand-offspring (F2) are not currently known. We explored effects of paternal pre-conceptual exposure to viral-like immune activation on F1 and F2 behavioral and physiological phenotypes using a C57BL/6J mouse model. Males were treated with a single injection (intraperitoneal) of the viral mimetic polyinosinic:polycytidylic acid (Poly I:C: 12 mg/kg) then bred with naïve female mice four weeks after the Poly I:C (or 0.9% saline control) injection. The F1 offspring of Poly I:C treated fathers displayed increased depression-like behavior in the Porsolt swim test, an altered stress response in the novelty-suppressed feeding test, and significant transcriptomic changes in their hippocampus. Additionally, the F1 male offspring of Poly I:C treated F0 males showed significantly increased immune responsivity after a Poly I:C immune challenge (12 mg/kg). Furthermore, the F2 male grand-offspring took longer to enter and travelled significantly shorter distances in the light zone of the light/dark box. An analysis of the small noncoding RNA profiles in sperm from Poly I:C treated males and their male offspring revealed significant effects of Poly I:C on the sperm microRNA content at the time of conception and on the sperm PIWI-interacting RNA content of the male offspring. Notably, eight miRNAs with an FDR < 0.05 (miR-141-3p, miR-126b-5p, miR-669o-5p, miR-10b-3p, miR-471-5p, miR-463-5p, miR-148b-3p, and miR-181c-5p) were found to be significantly downregulated in the sperm of Poly I:C treated males. Collectively, we demonstrate that paternal pre-conceptual exposure to a viral immune challenge results in both intergenerational and transgenerational effects on brain and behavior that may be mediated by alterations in the sperm small noncoding RNA content.
Assuntos
MicroRNAs , Pequeno RNA não Traduzido , Masculino , Feminino , Camundongos , Animais , Humanos , Camundongos Endogâmicos C57BL , Sêmen , Espermatozoides , Pai , MicroRNAs/genética , MicroRNAs/farmacologia , Pequeno RNA não Traduzido/farmacologia , Poli I/farmacologiaRESUMO
Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function1. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF)2,3. KAT6A has essential roles in normal haematopoietic stem cells4-6 and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia7,8. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers8. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus9,10, a function that requires its KAT activity10. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days11. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.
Assuntos
Benzenossulfonatos/farmacologia , Senescência Celular/efeitos dos fármacos , Histona Acetiltransferases/antagonistas & inibidores , Hidrazinas/farmacologia , Linfoma/tratamento farmacológico , Linfoma/patologia , Sulfonamidas/farmacologia , Acetilação/efeitos dos fármacos , Animais , Benzenossulfonatos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Desenvolvimento de Medicamentos , Fibroblastos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Acetiltransferases/deficiência , Histona Acetiltransferases/genética , Histonas/química , Histonas/metabolismo , Hidrazinas/uso terapêutico , Linfoma/enzimologia , Linfoma/genética , Lisina/química , Lisina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Sulfonamidas/uso terapêuticoRESUMO
Unlike clustered HOX genes, the role of nonclustered homeobox gene family members in hematopoiesis and leukemogenesis has not been extensively studied. Here we found that the hematopoietically expressed homeobox gene Hhex is overexpressed in acute myeloid leukemia (AML) and is essential for the initiation and propagation of MLL-ENL-induced AML but dispensable for normal myelopoiesis, indicating a specific requirement for Hhex for leukemic growth. Loss of Hhex leads to expression of the Cdkn2a-encoded tumor suppressors p16(INK4a) and p19(ARF), which are required for growth arrest and myeloid differentiation following Hhex deletion. Mechanistically, we show that Hhex binds to the Cdkn2a locus and directly interacts with the Polycomb-repressive complex 2 (PRC2) to enable H3K27me3-mediated epigenetic repression. Thus, Hhex is a potential therapeutic target that is specifically required for AML stem cells to repress tumor suppressor pathways and enable continued self-renewal.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Epigênese Genética , Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide Aguda/fisiopatologia , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
Clinical immunity to P. falciparum malaria is non-sterilizing, with adults often experiencing asymptomatic infection. Historically, asymptomatic malaria has been viewed as beneficial and required to help maintain clinical immunity. Emerging views suggest that these infections are detrimental and constitute a parasite reservoir that perpetuates transmission. To define the impact of asymptomatic malaria, we pursued a systems approach integrating antibody responses, mass cytometry, and transcriptional profiling of individuals experiencing symptomatic and asymptomatic P. falciparum infection. Defined populations of classical and atypical memory B cells and a TH2 cell bias were associated with reduced risk of clinical malaria. Despite these protective responses, asymptomatic malaria featured an immunosuppressive transcriptional signature with upregulation of pathways involved in the inhibition of T-cell function, and CTLA-4 as a predicted regulator in these processes. As proof of concept, we demonstrated a role for CTLA-4 in the development of asymptomatic parasitemia in infection models. The results suggest that asymptomatic malaria is not innocuous and might not support the induction of immune processes to fully control parasitemia or efficiently respond to malaria vaccines.
Assuntos
Malária Falciparum , Parasitemia , Adulto , Infecções Assintomáticas , Antígeno CTLA-4 , Humanos , Terapia de Imunossupressão , Malária Falciparum/genética , Malária Falciparum/parasitologia , Plasmodium falciparumRESUMO
BACKGROUND: Typical symptoms of uncomplicated dengue fever (DF) include headache, muscle pains, rash, cough, and vomiting. A proportion of cases progress to severe dengue hemorrhagic fever (DHF), associated with increased vascular permeability, thrombocytopenia, and hemorrhages. Progression to severe dengue is difficult to diagnose at the onset of fever, which complicates patient triage, posing a socio-economic burden on health systems. METHODS: To identify parameters associated with protection and susceptibility to DHF, we pursued a systems immunology approach integrating plasma chemokine profiling, high-dimensional mass cytometry and peripheral blood mononuclear cell (PBMC) transcriptomic analysis at the onset of fever in a prospective study conducted in Indonesia. RESULTS: After a secondary infection, progression to uncomplicated dengue featured transcriptional profiles associated with increased cell proliferation and metabolism, and an expansion of ICOS+CD4+ and CD8+ effector memory T cells. These responses were virtually absent in cases progressing to severe DHF, that instead mounted an innate-like response, characterised by inflammatory transcriptional profiles, high circulating levels of inflammatory chemokines and with high frequencies of CD4low non-classical monocytes predicting increased odds of severe disease. CONCLUSIONS: Our results suggests that effector memory T cell activation might play an important role ameliorating severe disease symptoms during a secondary dengue infection, and in the absence of that response, a strong innate inflammatory response is required to control viral replication. Our research also identified discrete cell populations predicting increased odds of severe disease, with potential diagnostic value.
Assuntos
Dengue , Dengue Grave , Humanos , Leucócitos Mononucleares , Estudos Prospectivos , Linfócitos TRESUMO
Somatically acquired mutations in PHF6 (plant homeodomain finger 6) frequently occur in hematopoietic malignancies and often coincide with ectopic expression of TLX3. However, there is no functional evidence to demonstrate whether these mutations contribute to tumorigenesis. Similarly, the role of PHF6 in hematopoiesis is unknown. We report here that Phf6 deletion in mice resulted in a reduced number of hematopoietic stem cells (HSCs), an increased number of hematopoietic progenitor cells, and an increased proportion of cycling stem and progenitor cells. Loss of PHF6 caused increased and sustained hematopoietic reconstitution in serial transplantation experiments. Interferon-stimulated gene expression was upregulated in the absence of PHF6 in hematopoietic stem and progenitor cells. The numbers of hematopoietic progenitor cells and cycling hematopoietic stem and progenitor cells were restored to normal by combined loss of PHF6 and the interferon α and ß receptor subunit 1. Ectopic expression of TLX3 alone caused partially penetrant leukemia. TLX3 expression and loss of PHF6 combined caused fully penetrant early-onset leukemia. Our data suggest that PHF6 is a hematopoietic tumor suppressor and is important for fine-tuning hematopoietic stem and progenitor cell homeostasis.
Assuntos
Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/metabolismo , Leucemia/etiologia , Proteínas Repressoras/fisiologia , Animais , Carcinogênese , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Receptores de Interferon , Proteínas Repressoras/genética , Proteínas Supressoras de TumorRESUMO
It has been proposed that interactions between mammalian chromosomes, or transchromosomal interactions (also known as kissing chromosomes), regulate gene expression and cell fate determination. Here we aimed to identify novel transchromosomal interactions in immune cells by high-resolution genome-wide chromosome conformation capture. Although we readily identified stable interactions in cis, and also between centromeres and telomeres on different chromosomes, surprisingly we identified no gene regulatory transchromosomal interactions in either mouse or human cells, including previously described interactions. We suggest that advances in the chromosome conformation capture technique and the unbiased nature of this approach allow more reliable capture of interactions between chromosomes than previous methods. Overall our findings suggest that stable transchromosomal interactions that regulate gene expression are not present in mammalian immune cells and that lineage identity is governed by cis, not trans chromosomal interactions.
Assuntos
Cromossomos de Mamíferos/genética , Regulação da Expressão Gênica , Imunidade Celular/genética , Mamíferos/fisiologia , Animais , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Cromossomos de Mamíferos/química , Cromossomos de Mamíferos/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Citometria de Fluxo , Genoma , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Conformação de Ácido Nucleico , EstereoisomerismoRESUMO
In rheumatoid arthritis (RA), extracellular vesicles (EVs) are associated with both the propagation and attenuation of joint inflammation and destruction. However, the specific EV content responsible for these processes is largely unknown. Investigations into identifying EV content are confounded by the challenges in obtaining high-quality EV preparations from synovial fluid. Implementing a size exclusion chromatography-based method of EV isolation, coupled with small RNA sequencing, we accurately characterised EV miRNAs in synovial fluid obtained from RA patients and investigated the differences between joints with high- and low-grade inflammation. Synovial fluid was obtained from the joints of 12 RA patients and, based on leukocyte counts, classified as either high (n = 7)- or low (n = 5)-grade inflammation. Using size exclusion chromatography, EVs were purified and small RNA was extracted and sequenced on a NextSeq 500. Sequencing reads were aligned to miRBase v21, and differences in miRNA profiles between RA patients with high- and low-grade joint inflammation were analysed. In total, 1972 distinct miRNAs were identified from RA synovial fluid EVs. miRNAs with less than five reads in fewer than five patients were filtered out, leaving 318 miRNAs for analysis. Analysis of the most abundant miRNAs suggested that they negatively regulate multiple genes relevant to inflammation, including signal transducer and activator of transcription 3 (STAT3), which lies downstream of IL-6 and has a pro-inflammatory role in RA. Synovial fluid from joints with high-grade inflammation contained 3.5-fold more EV miRNA per mL of synovial fluid (p = 0.0017). Seventy-eight EV miRNAs were differentially expressed between RA joints with high- and low-grade inflammation, and pathway analysis revealed that their target genes were commonly involved a variety of processes, including cellular apoptosis, proliferation and migration. Of the 49 miRNAs that were elevated in joints with high-grade inflammation, pathway analysis revealed that genes involved in cytokine-mediated signalling pathways were significantly enriched targets. In contrast, genes associated with reactive oxygen species signalling were significantly enriched as targets of the 29 miRNAs elevated in joints with low-grade inflammation. Our study identified an abundance of EV miRNAs from the synovial fluid of RA patients with the potential to modulate inflammation. In doing so, we defined potential mechanisms by which synovial fluid EVs may contribute to RA pathophysiology.
Assuntos
Artrite Reumatoide/genética , Vesículas Extracelulares/genética , Inflamação/genética , MicroRNAs/genética , Líquido Sinovial/metabolismo , Idoso , Artrite Reumatoide/complicações , Estudos de Coortes , Vesículas Extracelulares/ultraestrutura , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Fatores Imunológicos/metabolismo , Inflamação/complicações , Inflamação/patologia , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Acute rheumatic fever (ARF) and rheumatic heart disease are autoimmune consequences of group A streptococcus infection and remain major causes of cardiovascular morbidity and mortality around the world. Improved treatment has been stymied by gaps in understanding key steps in the immunopathogenesis of ARF and rheumatic heart disease. This study aimed to identify (1) effector T cell cytokine(s) that might be dysregulated in the autoimmune response of patients with ARF by group A streptococcus, and (2) an immunomodulatory agent that suppresses this response and could be clinically translatable to high-risk patients with ARF. METHODS: The immune response to group A streptococcus was analyzed in peripheral blood mononuclear cells from an Australian Aboriginal ARF cohort by a combination of multiplex cytokine array, flow cytometric analysis, and global gene expression analysis by RNA sequencing. The immunomodulatory drug hydroxychloroquine was tested for effects on this response. RESULTS: We found a dysregulated interleukin-1ß-granulocyte-macrophage colony-stimulating factor (GM-CSF) cytokine axis in ARF peripheral blood mononuclear cells exposed to group A streptococcus in vitro, whereby persistent interleukin-1ß production is coupled to overproduction of GM-CSF and selective expansion of CXCR3+CCR4-CCR6- CD4 T cells. CXCR3+CCR4-CCR6- CD4 T cells are the major source of GM-CSF in human CD4 T cells and CXCL10, a CXCR3 ligand and potent T helper 1 chemoattractant, was elevated in sera from patients with ARF. GM-CSF has recently emerged as a key T cell-derived effector cytokine in numerous autoimmune diseases, including myocarditis, and the production of CXCL10 may explain selective trafficking of these cells to the heart. We provide evidence that interleukin-1ß amplifies the expansion of GM-CSF-expressing CD4 T cells, which is effectively suppressed by hydroxychloroquine. RNA sequencing showed shifts in gene expression profiles and differentially expressed genes in peripheral blood mononuclear cells derived from patients at different clinical stages of ARF. CONCLUSIONS: Given the safety profile of hydroxychloroquine and its clinical pedigree in treating autoimmune diseases such as rheumatoid arthritis, where GM-CSF plays a pivotal role, we propose that hydroxychloroquine could be repurposed to reduce the risk of rheumatic heart disease after ARF.