RESUMO
The DNA binding protein AT-rich interacting domain 3a (ARID3a)2 is expressed in healthy human hematopoietic cord blood progenitors where its modulation influences myeloid versus B lineage development. ARID3a is also variably expressed in subsets of adult peripheral blood hematopoietic progenitors where the consequences of ARID3a expression are unknown. In B lymphocytes, Toll-like receptor (TLR)3 signaling induces ARID3a expression in association with Type I interferon inflammatory cytokines. We hypothesized that TLR ligand stimulation of peripheral blood hematopoietic progenitors would induce ARID3a expression resulting in interferon production, and potentially influencing lineage decisions. Our data revealed that the TLR9 agonist CpG induces ARID3a expression with interferon alpha synthesis in human hematopoietic progenitors. However, ARID3a expression was not associated with increased B lineage development. These results demonstrate the need for further experiments to better define how pathogen-associated responses influence hematopoiesis.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Células-Tronco Hematopoéticas/metabolismo , Interferon-alfa/biossíntese , Receptores Toll-Like/sangue , Fatores de Transcrição/biossíntese , Adulto , Linfócitos B/citologia , Linfócitos B/metabolismo , Ilhas de CpG , Citocinas/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/sangue , Feminino , Citometria de Fluxo/métodos , Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Interferon-alfa/genética , Interferon-alfa/imunologia , Ligantes , Pessoa de Meia-Idade , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/sangueRESUMO
BACKGROUND: Immunologic aging leads to immune dysfunction, significantly reducing the quality of life of the elderly. Aged-related defects in early hematopoiesis result in reduced lymphoid cell development, functionally defective mature immune cells, and poor protective responses to vaccines and pathogens. Despite considerable progress understanding the underlying causes of decreased immunity in the elderly, the mechanisms by which these occur are still poorly understood. The DNA-binding protein ARID3a is expressed in a subset of human hematopoietic progenitors. Inhibition of ARID3a in bulk human cord blood CD34+ hematopoietic progenitors led to developmental skewing toward myeloid lineage at the expense of lymphoid lineage cells in vitro. Effects of ARID3a expression in adult-derived hematopoietic stem cells (HSCs) have not been analyzed, nor has ARID3a expression been assessed in relationship to age. We hypothesized that decreases in ARID3a could explain some of the defects observed in aging. RESULTS: Our data reveal decreased frequencies of ARID3a-expressing peripheral blood HSCs from aged healthy individuals compared with young donor HSCs. Inhibition of ARID3a in young donor-derived HSCs limits B lineage potential, suggesting a role for ARID3a in B lymphopoiesis in bone marrow-derived HSCs. Increasing ARID3a levels of HSCs from aged donors in vitro alters B lineage development and maturation. Finally, single cell analyses of ARID3a-expressing HSCs from young versus aged donors identify a number of differentially expressed genes in aged ARID3A-expressing cells versus young ARID3A-expressing HSCs, as well as between ARID3A-expressing and non-expressing cells in both young and aged donor HSCs. CONCLUSIONS: These data suggest that ARID3a-expressing HSCs from aged individuals differ at both molecular and functional levels compared to ARID3a-expressing HSCs from young individuals.
RESUMO
Type I interferons (IFN) causes inflammatory responses to pathogens, and can be elevated in autoimmune diseases such as systemic lupus erythematosus (SLE). We previously reported unexpected associations of increased numbers of B lymphocytes expressing the DNA-binding protein ARID3a with both IFN alpha (IFNα) expression and increased disease activity in SLE. Here, we determined that IFNα producing low density neutrophils (LDNs) and plasmacytoid dendritic cells (pDCs) from SLE patients exhibit strong associations between ARID3a protein expression and IFNα production. Moreover, SLE disease activity indices correlate most strongly with percentages of ARID3a+ LDNs, but were also associated, less significantly, with IFNα expression in LDNs and pDCs. Hierarchical clustering and transcriptome analyses of LDNs and pDCs revealed SLE patients with low ARID3a expression cluster with healthy controls and identified gene profiles associated with increased proportions of ARID3a- and IFNα-expressing cells of each type. These data identify ARID3a as a potential transcription regulator of IFNα-related inflammatory responses and other pathways important for SLE disease activity.
Assuntos
Linfócitos B/fisiologia , Proteínas de Ligação a DNA/genética , Células Dendríticas/fisiologia , Interferon-alfa/metabolismo , Lúpus Eritematoso Sistêmico/genética , Neutrófilos/fisiologia , Fatores de Transcrição/genética , Adulto , Idoso , Progressão da Doença , Feminino , Regulação da Expressão Gênica , Estudos de Associação Genética , Humanos , Imunidade Inata , Interferon-alfa/genética , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , TranscriptomaRESUMO
We recently reported that the transcription factor ARID3a is expressed in a subset of human hematopoietic progenitor stem cells in both healthy individuals and in patients with systemic lupus erythematosus. Numbers of ARID3a(+) lupus hematopoietic stem progenitor cells were associated with increased production of autoreactive Abs when those cells were introduced into humanized mouse models. Although ARID3a/Bright knockout mice died in utero, they exhibited decreased numbers of hematopoietic stem cells and erythrocytes, indicating that ARID3a is functionally important for hematopoiesis in mice. To explore the requirement for ARID3a for normal human hematopoiesis, hematopoietic stem cell progenitors from human cord blood were subjected to both inhibition and overexpression of ARID3a in vitro. Inhibition of ARID3a resulted in decreased B lineage cell production accompanied by increases in cells with myeloid lineage markers. Overexpression of ARID3a inhibited both myeloid and erythroid differentiation. Additionally, inhibition of ARID3a in hematopoietic stem cells resulted in altered expression of transcription factors associated with hematopoietic lineage decisions. These results suggest that appropriate regulation of ARID3a is critical for normal development of both myeloid and B lineage pathways.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Fatores de Transcrição/metabolismo , Linhagem da Célula , Sangue Fetal , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Humanos , Técnicas In Vitro , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Transdução GenéticaRESUMO
Although hematopoietic stem/progenitor cells (HSPCs) are used for transplantation, characterization of the multiple subsets within this population in humans has lagged behind similar studies in mice. We found that expression of the DNA-binding protein, ARID3a, in mouse stem cells was important for normal development of hematopoietic lineages; however, progenitors expressing ARID3a in humans have not been defined. We previously showed increased numbers of ARID3a(+) B cells in nearly half of systemic lupus erythematosus (SLE) patients, and total numbers of ARID3a(+) B cells were associated with increased disease severity. Because expression of ARID3a in those SLE patients occurred throughout all B cell subsets, we hypothesized that ARID3a expression in patient HSPCs might also be increased relative to expression in healthy controls. Our data now show that ARID3a expression is not limited to any defined subset of HSPCs in either healthy controls or SLE patients. Numbers of ARID3a(+) HSPCs in SLE patients were increased over numbers of ARID3a(+) cells in healthy controls. Although all SLE-derived HSPCs exhibited poor colony formation in vitro compared with controls, SLE HSPCs with high numbers of ARID3a(+) cells yielded increased numbers of cells expressing the early progenitor marker, CD34. SLE HSPCs with high numbers of ARID3a(+) cells also more readily generated autoantibody-producing cells than HSPCs with lower levels of ARID3a in a humanized mouse model. These data reveal new functions for ARID3a in early hematopoiesis and suggest that knowledge regarding ARID3a levels in HSPCs could be informative for applications requiring transplantation of those cells.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Fatores de Transcrição/genética , Adulto , Idoso , Animais , Anticorpos Antinucleares/imunologia , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Antígenos de Superfície/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Estudos de Casos e Controles , Expressão Gênica , Humanos , Imunofenotipagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Pessoa de Meia-Idade , Adulto JovemRESUMO
Previously, we determined that enhanced disease activity in patients with systemic lupus erythematosus (SLE) was associated with dramatic increases in numbers of B lymphocytes expressing the transcription factor ARID3a. Our data now indicate ARID3a is important for interferon alpha (IFNa) expression and show a strong association between ARID3a expression and transcription of genes associated with lupus IFN signatures. Furthermore, both ARID3a and IFNa production were elicited in healthy control B cells upon stimulation with the TLR 9 agonist, CpG. Importantly, secretion of IFNa from ARID3a+ healthy B lymphocytes stimulated increased IFNa production in plasmacytoid dendritic cells. These data identify ARID3a+ B cells as a novel type of effector B cell, and link ARID3a expression in B lymphocytes to IFN-associated inflammatory responses in SLE.
Assuntos
Subpopulações de Linfócitos B/imunologia , Proteínas de Ligação a DNA/imunologia , Expressão Gênica/imunologia , Interferon-alfa/imunologia , Fatores de Transcrição/imunologia , Subpopulações de Linfócitos B/efeitos dos fármacos , Subpopulações de Linfócitos B/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Humanos , Interferon-alfa/sangue , Interferon-alfa/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a-/- kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development.
Assuntos
Rim/anatomia & histologia , Animais , Divisão Celular , Linhagem Celular , Proteínas de Ligação a DNA/genética , Rim/citologia , Camundongos , Camundongos Knockout , Oryzias , Células-Tronco/citologia , Fatores de Transcrição/genéticaRESUMO
Compensation or unmixing is essential in analyzing multiparameter flow cytometry data. Errors in data correction, either by compensation or unmixing, can completely change the outcome or mislead the researchers. Owing to limited cell numbers, researchers often use synthetic beads to generate the required single stains for the necessary calculation. In this study, the capacity of synthetic beads to influence data correction is evaluated. Corrected data for human peripheral blood cells were generated using cell-based compensation from the same cells or bead-based compensation to identify differences between the methods. These data suggest that correction with beads on full-spectrum and conventional cytometers does not always follow the basic flow compensation/unmixing expectations and alters the data. Overall, the best approach for bead-based correction for an experiment is to evaluate which beads and fluorochromes are most accurately compensated/unmixed.
Assuntos
Corantes Fluorescentes , Humanos , Citometria de Fluxo/métodos , Contagem de Células , Coloração e RotulagemRESUMO
In conventional flowcytometry one detector (primary) is dedicated for one fluorochrome. However, photons usually end up in other detectors too (fluorescence spillover). 'Compensation' is a process that corrects the spillover signal from all detectors except the primary detector. Post 'compensation', the photon counting error of spillover signals become evident as spreading of the data. The spreading induced by spillover impairs the ability to resolve stained cell population from the unstained one, potentially reducing or completely losing cell populations. For successful multi-color panel design, it is important to know the expected spillover to maximize the data resolution. The Spillover Spreading Matrix (SSM) can be used to estimate the spread, but the outcome is dependent on detector sensitivity. Simply, the same single stained sample produces different spillover spread values when detector(s) sensitivity is altered. Many researchers mistakenly use this artifact to "reduce" the spread by decreasing detector sensitivity. This can result in diminished capacity to resolve dimly expressing cell populations. Here, we introduce SQI (Spread Quantification Index), that can quantify the spillover spread independent of detector sensitivity and independent of dynamic range. This allows users to compare spillover spread between instruments having different types of detectors, which is not possible using SSM.
Assuntos
Citometria de Fluxo/normas , Artefatos , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodosRESUMO
Systemic lupus erythematosus (SLE) is a devastating and heterogeneous autoimmune disease that affects multiple organs, and for which the underlying causes are unknown. The majority of SLE patients produce autoantibodies, have increased levels of type-I inflammatory cytokines, and can develop glomerulonephritis. Recent studies indicate an unexpected but strong association between increased disease activity in SLE patients and the expression of the DNA-binding protein ARID3a (A + T rich interaction domain protein 3a) in a number of peripheral blood cell types. ARID3a expression was first associated with autoantibody production in B cells; however, more recent findings also indicate associations with expression of the inflammatory cytokine interferon alpha in SLE plasmacytoid dendritic cells and low-density neutrophils. In addition, ARID3a is expressed in hematopoietic stem cells and some adult kidney progenitor cells. SLE cells expressing enhanced ARID3a levels show differential gene expression patterns compared with homologous healthy control cells, identifying new pathways potentially regulated by ARID3a. The associations of ARID3a expression with increased disease severity in SLE, suggest that it, or its downstream targets, may provide new therapeutic targets for SLE.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/terapia , Terapia de Alvo Molecular/tendências , Fatores de Transcrição/fisiologia , Adulto , Células-Tronco Adultas/fisiologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proteínas de Ligação a DNA/genética , Células Dendríticas/fisiologia , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Terapia de Alvo Molecular/métodos , Fatores de Transcrição/genéticaRESUMO
Previously published studies revealed that variation in expression of the DNA-binding protein ARID3a in B lymphocytes from patients with systemic lupus erythematosus (SLE) correlated with levels of disease activity ("Disease activity in systemic lupus erythematosus correlates with expression of the transcription factor AT-rich-interactive domain 3A" (J.M. Ward, K. Rose, C. Montgomery, I. Adrianto, J.A. James, J.T. Merrill et al., 2014) [1]). The data presented here compare DNA methylation patterns from SLE peripheral blood mononuclear cells obtained from samples with high numbers of ARID3a expressing B cells (ARID3a(H)) versus SLE samples with normal numbers of ARID3a(+) B cells (ARID3a(N)). The methylation data is available at the gene expression omnibus (GEO) repository, "Gene Expression Omnibus: NCBI gene expression and hybridization array data repository" (R. Edgar, M. Domrachev, A.E. Lash, 2002) [2]. Isolated B cells from SLE ARID3a(H) and ARID3a(N) B samples were also evaluated via qRT-PCR for Type I interferon (IFN) signature and pathway gene expression levels by qRT-PCR. Similarly, healthy control B cells and B cells stimulated to express ARID3a with the TLR agonist, CpG, were also compared via qRT-PCR. Primers designed to detect 6 IFNa subtype mRNAs were tested in 4 IFNa, Epstein-Barr Virus-transformed B cell lines ("Reduced interferon-alpha production by Epstein-Barr virus transformed B-lymphoblastoid cell lines and lectin-stimulated lymphocytes in congenital dyserythropoietic anemia type I" (S.H. Wickramasinghe, R. Hasan, J. Smythe, 1997) [3]). The data in this article support the publication, "Human effector B lymphocytes express ARID3a and secrete interferon alpha" (J.M. Ward, M.L. Ratliff, M.G. Dozmorov, G. Wiley, J.M. Guthridge, P.M. Gaffney, J.A. James, C.F. Webb, 2016) [4].
RESUMO
ARID3a/Bright is a DNA-binding protein that was originally discovered for its ability to increase immunoglobulin transcription in antigen-activated B cells. It interacts with DNA as a dimer through its ARID, or A/T-rich interacting domain. In association with other proteins, ARID3a increased transcription of the immunoglobulin heavy chain and led to improved chromatin accessibility of the heavy chain enhancer. Constitutive expression of ARID3a in B lineage cells resulted in autoantibody production, suggesting its regulation is important. Abnormal ARID3a expression has also been associated with increased proliferative capacity and malignancy. Roles for ARID3a in addition to interactions with the immunoglobulin locus were suggested by transgenic and knockout mouse models. Over-expression of ARID3a resulted in skewing of mature B cell subsets and altered gene expression patterns of follicular B cells, whereas loss of function resulted in loss of B1 lineage B cells and defects in hematopoiesis. More recent studies showed that loss of ARID3a in adult somatic cells promoted developmental plasticity, alterations in gene expression patterns, and lineage fate decisions. Together, these data suggest new regulatory roles for ARID3a. The genes influenced by ARID3a are likely to play pivotal roles in lineage decisions, highlighting the importance of this understudied transcription factor.