RESUMO
The differentiation of B cells into plasmablasts (PBs) and then plasma cells (PCs) is associated with extensive cell reprogramming and new cell functions. By using specific inhibition strategies (including a novel morpholino RNA antisense approach), we found that early, sustained upregulation of the proviral integrations of Moloney virus 2 (PIM2) kinase is a pivotal event during human B-cell in vitro differentiation and then continues in mature normal and malignant PCs in the bone marrow. In particular, PIM2 sustained the G1/S transition by acting on CDC25A and p27Kip1 and hindering caspase 3-driven apoptosis through BAD phosphorylation and cytoplasmic stabilization of p21Cip1. In PCs, interleukin-6 triggered PIM2 expression, resulting in antiapoptotic effects on which malignant PCs were particularly dependent. In multiple myeloma, pan-PIM and myeloid cell leukemia-1 (MCL1) inhibitors displayed synergistic activity. Our results highlight a cell-autonomous function that links kinase activity to the newly acquired secretion ability of the PBs and the adaptability observed in both normal and malignant PCs. These findings should finally prompt the reconsideration of PIM2 as a therapeutic target in multiple myeloma.
Assuntos
Mieloma Múltiplo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Plasmócitos/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genéticaRESUMO
BACKGROUND: Allergy regroups numerous complex and various diseases classified as IgE-dependent or non-IgE-dependent hypersensitivities. IgEs are expressed as membrane and secreted forms by B cells and plasma cells, respectively. In IgE-mediated hypersensitivity, IgE secretion and binding to the high-affinity IgE receptor FcεRI on effector cells are responsible for the onset of allergic symptoms; in contrast, surface IgE expression as a B-cell receptor is barely detectable. OBJECTIVE: Our aim was to test an innovative antisense approach to reducing IgE secretion. METHODS: We designed an antisense oligonucleotide (ASO) targeting the polyadenylation signal of human secreted IgE to redirect IgE transcript polyadenylation from the secreted form to the membrane form. ASO treatments were performed on B cells from transgenic mice expressing humanized IgE (InEps mice), as well as on human primary B cells and myeloma cells. In vivo ASO delivery was tested by using an InEps mouse model. RESULTS: We demonstrated that treatment with a morpholino ASO targeting the secreted IgE polyadenylation signal drastically decreased IgE secretion and inversely increased membrane IgE mRNA expression. In addition, ASO treatment induced apoptosis of IgE-expressing U266 myeloma cells, and RNA sequencing revealed attenuation of their plasma cell phenotype. Remarkably, systemic administration of an ASO coupled with Pip6a as an arginine-rich cell-penetrating peptide decreased IgE secretion in vivo. CONCLUSION: Altogether, this ASO strategy could be an effective way to decrease IgE secretion and allergic symptoms in patients with IgE-dependent allergies, and it could also promote allergen tolerance through apoptosis of IgE+ antibody-secreting cells.
Assuntos
Hipersensibilidade , Mieloma Múltiplo , Animais , Sobrevivência Celular , Humanos , Imunoglobulina E/metabolismo , Camundongos , Oligonucleotídeos Antissenso/farmacologia , Plasmócitos/metabolismo , Poliadenilação , Receptores de IgE/metabolismoRESUMO
Currently, several biologics are used for the treatment of cutaneous pathologies such as atopic dermatitis (AD), psoriasis or skin cancers. The main administration routes are subcutaneous and intravenous injections. However, little is known about antibody penetration through the skin. The aim was to study the transcutaneous penetration of a reduced-size antibody as a single-chain variable fragment (scFv) compared to a whole antibody (Ab) and to determine its capacity to neutralize an inflammatory cytokine involved in AD such as human interleukin-4 (hIL-4). Transcutaneous penetration was evaluated by ex vivo studies on tape-stripped pig ear skin. ScFv and Ab visualization through the skin was measured by Raman microspectroscopy. In addition, hIL-4 neutralization was studied in vitro using HEK-Blue™ IL-4/IL-13 cells and normal human keratinocytes (NHKs). After 24 h of application, analysis by Raman microspectroscopy showed that scFv penetrated into the upper dermis while Ab remained on the stratum corneum. In addition, the anti-hIL4 scFv showed very efficient and dose-dependent hIL-4 neutralization. Thus, scFv penetrates through to the upper papillary dermis while Ab mostly remains on the surface, the anti-hIL4 scFv also neutralizes its target effectively suggesting its potential use as topical therapy for AD.
RESUMO
Skin is a vital protective organ, the main role of which is to provide a physical barrier and to prevent the entry of pathogens. Various pathologies, such as atopic dermatitis (AD), psoriasis (PSO), or skin cancers, can affect the skin, and all show a high and increasing prevalence. Many antibodies are currently used in the treatment of these diseases. However, various studies are underway for the development of new biologics directed against specific targets. In this review, we describe current biologics used in skin pathologies as well as antibodies in development. We also discuss various immunotherapy examples that use new delivery technologies, such as microneedle patch, nanoparticles (NPs), liposomes, or gel formulation.
Assuntos
Produtos Biológicos/farmacologia , Imunoterapia , Terapia de Alvo Molecular , Dermatopatias , Fármacos Dermatológicos/farmacologia , Composição de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/tendências , Humanos , Imunoterapia/métodos , Imunoterapia/tendências , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Dermatopatias/classificação , Dermatopatias/tratamento farmacológico , Dermatopatias/imunologiaRESUMO
Light chain (LC) deposition disease (LCDD) is a rare disorder characterized by glomerular and peritubular amorphous deposits of a monoclonal immunoglobulin LC, leading to nodular glomerulosclerosis and nephrotic syndrome. We developed a transgenic model using site-directed insertion of the variable domain of a pathogenic human LC gene into the mouse immunoglobulin κ locus, ensuring its production by all plasma cells (PCs). High free LC levels were achieved after backcrossing with mice presenting increased PC differentiation and no immunoglobulin heavy chain production. Our mouse model recapitulates the characteristic features of LCDD, including progressive glomerulosclerosis, nephrotic-range proteinuria, and finally kidney failure. The variable domain of the LC bears alone the structural properties involved in its pathogenicity. RNA sequencing conducted on PCs demonstrated that LCDD LC induces endoplasmic reticulum stress, likely accounting for the high efficiency of proteasome inhibitor-based therapy. Accordingly, reduction of circulating pathogenic LC was efficiently achieved and not only preserved renal function but also partially reversed kidney lesions. Finally, transcriptome analysis of presclerotic glomeruli revealed that proliferation and extracellular matrix remodeling represented the first steps of glomerulosclerosis, paving the way for future therapeutic strategies in LCDD and other kidney diseases featuring diffuse glomerulosclerosis, particularly diabetic nephropathy.
Assuntos
Cadeias Leves de Imunoglobulina/metabolismo , Paraproteinemias/diagnóstico , Paraproteinemias/etiologia , Animais , Biomarcadores , Ciclo Celular/genética , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Matriz Extracelular , Citometria de Fluxo , Perfilação da Expressão Gênica , Ordem dos Genes , Marcação de Genes , Vetores Genéticos/genética , Cadeias Leves de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/metabolismo , Imuno-Histoquímica , Rim/metabolismo , Rim/patologia , Testes de Função Renal , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Camundongos , Camundongos Transgênicos , Paraproteinemias/complicações , Paraproteinemias/mortalidade , Agregados Proteicos , Agregação Patológica de Proteínas , Insuficiência Renal/diagnóstico , Insuficiência Renal/etiologia , Insuficiência Renal/metabolismo , Insuficiência Renal/mortalidadeRESUMO
Class switch recombination (CSR) changes antibody isotype by replacing Cµ constant exons with different constant exons located downstream on the immunoglobulin heavy (IgH) locus. During CSR, transcription through specific switch (S) regions and processing of non-coding germline transcripts (GLTs) are essential for the targeting of activation-induced cytidine deaminase (AID). While CSR to IgG1 is abolished in mice lacking an Iγ1 exon donor splice site (dss), many questions remain regarding the importance of I exon dss recognition in CSR. To further clarify the role of I exon dss in CSR, we first evaluated RNA polymerase II (RNA pol II) loading and chromatin accessibility in S regions after activation of mouse B cells lacking Iγ1 dss. We found that deletion of Iγ1 dss markedly reduced RNA pol II pausing and active chromatin marks in the Sγ1 region. We then challenged the post-transcriptional function of I exon dss in CSR by using antisense oligonucleotides (ASOs) masking I exon dss on GLTs. Treatment of stimulated B cells with an ASO targeting Iγ1 dss, in the acceptor Sγ1 region, or Iµ dss, in the donor Sµ region, did not decrease germline transcription but strongly inhibited constitutive splicing and CSR to IgG1. Supporting a global effect on CSR, we also observed that the targeting of Iµ dss reduced CSR to IgG3 and, to a lesser extent, IgG2b isotypes. Altogether, this study reveals that the recognition of I exon dss first supports RNA pol II pausing and the opening of chromatin in targeted S regions and that GLT splicing events using constitutive I exon dss appear mandatory for the later steps of CSR, most likely by guiding AID to S regions.
Assuntos
Éxons , Switching de Imunoglobulina , Oligonucleotídeos Antissenso/genética , Sítios de Splice de RNA , Animais , Citidina Desaminase/genética , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Polimerase II/genética , Transcrição GênicaRESUMO
The presence of premature termination codons (PTCs) in transcripts is dangerous for the cell as they encode potentially deleterious truncated proteins that can act with dominant-negative or gain-of-function effects. To avoid the synthesis of these shortened polypeptides, several RNA surveillance systems can be activated to decrease the level of PTC-containing mRNAs. Nonsense-mediated mRNA decay (NMD) ensures an accelerated degradation of mRNAs harboring PTCs by using several key NMD factors such as up-frameshift (UPF) proteins. Another pathway called nonsense-associated altered splicing (NAS) upregulates transcripts that have skipped disturbing PTCs by alternative splicing. Thus, these RNA quality control processes eliminate abnormal PTC-containing mRNAs from the cells by using positive and negative responses. In this review, we describe the general mechanisms of NMD and NAS and their respective involvement in the decay of aberrant immunoglobulin and TCR transcripts in lymphocytes.
Assuntos
Processamento Alternativo , Linfócitos B/metabolismo , Códon sem Sentido/genética , Degradação do RNAm Mediada por Códon sem Sentido , Diferenciação Celular , Códon sem Sentido/metabolismo , Mutação da Fase de Leitura , Mutação com Ganho de Função , Plasmócitos/metabolismo , Estabilidade de RNA , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Regulação para CimaRESUMO
The random V(D)J recombination process ensures the diversity of the primary immunoglobulin (Ig) repertoire. In two thirds of cases, imprecise recombination between variable (V), diversity (D), and joining (J) segments induces a frameshift in the open reading frame that leads to the appearance of premature termination codons (PTCs). Thus, many B lineage cells harbour biallelic V(D)J-rearrangements of Ig heavy or light chain genes, with a productively-recombined allele encoding the functional Ig chain and a nonproductive allele potentially encoding truncated Ig polypeptides. Since the pattern of Ig gene expression is mostly biallelic, transcription initiated from nonproductive Ig alleles generates considerable amounts of primary transcripts with out-of-frame V(D)J junctions. How RNA surveillance pathways cooperate to control the noise from nonproductive Ig genes will be discussed in this review, focusing on the benefits of nonsense- mediated mRNA decay (NMD) activation during B-cell development and detrimental effects of nonsense-associated altered splicing (NAS) in terminally differentiated plasma cells. [BMB Reports 2019; 52(12): 671-678].
Assuntos
Linfócitos B/imunologia , Genes de Imunoglobulinas , Imunoglobulinas/genética , Degradação do RNAm Mediada por Códon sem Sentido , Plasmócitos/imunologia , Recombinação V(D)J/genética , Alelos , Animais , Formação de Anticorpos/genética , Linfócitos B/metabolismo , Códon sem Sentido/metabolismo , Humanos , Imunoglobulinas/imunologia , Plasmócitos/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Recombinação V(D)J/imunologiaRESUMO
The error-prone V(D)J recombination process generates considerable amounts of nonproductive immunoglobulin (Ig) pre-mRNAs. We recently demonstrated that aberrant Ig chains lacking variable (V) domains can be produced after nonsense-associated altered splicing (NAS) events. Remarkably, the expression of these truncated Ig polypeptides heightens endoplasmic reticulum stress and shortens plasma cell (PC) lifespan. Many questions remain regarding the molecular mechanisms underlying this new truncated Ig exclusion (TIE-) checkpoint and its restriction to the ultimate stage of B-cell differentiation. To address these issues, we evaluated the extent of NAS of Ig pre-mRNAs using an Ig heavy chain (IgH) knock-in model that allows for uncoupling of V exon skipping from TIE-induced apoptosis. We found high levels of V exon skipping in PCs compared with B cells, and this skipping was correlated with a biallelic boost in IgH transcription during PC differentiation. Chromatin analysis further revealed that the skipped V exon turned into a pseudo-intron. Finally, we showed that hypertranscription of Ig genes facilitated V exon skipping upon passive administration of splice-switching antisense oligonucleotides (ASOs). Thus, V exon skipping is coupled to transcription and increases as PC differentiation proceeds, likely explaining the late occurrence of the TIE-checkpoint and opening new avenues for ASO-mediated strategies in PC disorders.
Assuntos
Linfócitos B/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Plasmócitos/metabolismo , Alelos , Animais , Diferenciação Celular , Estresse do Retículo Endoplasmático , Éxons/genética , Variação Genética , Íntrons/genética , Camundongos , Camundongos Endogâmicos C57BL , Degradação do RNAm Mediada por Códon sem Sentido , Splicing de RNA , Recombinação V(D)JRESUMO
Pax5 is the guardian of the B cell identity since it primes or enhances the expression of B cell specific genes and concomitantly represses the expression of B cell inappropriate genes. The tight regulation of Pax5 is therefore required for an efficient B cell differentiation. A defect in its dosage can translate into immunodeficiency or malignant disorders such as leukemia or lymphoma. Pax5 is expressed from two different promoters encoding two isoforms that only differ in the sequence of their first alternative exon. Very little is known regarding the role of the two isoforms during B cell differentiation and the regulation of their expression. Our work aims to characterize the mechanisms of regulation of the expression balance of these two isoforms and their implication in the B cell differentiation process using murine ex vivo analyses. We show that these two isoforms are differentially regulated but have equivalent function during early B cell differentiation and may have functional differences after B cell activation. The tight control of their expression may thus reflect a way to finely tune Pax5 dosage during B cell differentiation process.
RESUMO
Aberrantly rearranged immunoglobulin (Ig) alleles are frequent. They are usually considered sterile and innocuous as a result of nonsense-mediated mRNA decay. However, alternative splicing can yield internally deleted proteins from such nonproductively V(D)J-rearranged loci. We show that nonsense codons from variable (V) Igκ exons promote exon-skipping and synthesis of V domain-less κ light chains (ΔV-κLCs). Unexpectedly, such ΔV-κLCs inhibit plasma cell (PC) differentiation. Accordingly, in wild-type mice, rearrangements encoding ΔV-κLCs are rare in PCs, but frequent in B cells. Likewise, enforcing expression of ΔV-κLCs impaired PC differentiation and antibody responses without disturbing germinal center reactions. In addition, PCs expressing ΔV-κLCs synthesize low levels of Ig and are mostly found among short-lived plasmablasts. ΔV-κLCs have intrinsic toxic effects in PCs unrelated to Ig assembly, but mediated by ER stress-associated apoptosis, making PCs producing ΔV-κLCs highly sensitive to proteasome inhibitors. Altogether, these findings demonstrate a quality control checkpoint blunting terminal PC differentiation by eliminating those cells expressing nonfunctionally rearranged Igκ alleles. This truncated Ig exclusion (TIE) checkpoint ablates PC clones with ΔV-κLCs production and exacerbated ER stress response. The TIE checkpoint thus mediates selection of long-lived PCs with limited ER stress supporting high Ig secretion, but with a cost in terms of antigen-independent narrowing of the repertoire.
Assuntos
Alelos , Formação de Anticorpos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Rearranjo Gênico do Linfócito B , Imunoglobulinas/genética , Plasmócitos/imunologia , Plasmócitos/metabolismo , Processamento Alternativo , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Códon sem Sentido , Estresse do Retículo Endoplasmático , Éxons , Região Variável de Imunoglobulina/genética , Camundongos , Plasmócitos/citologia , Transcrição GênicaRESUMO
Randall-type heavy chain deposition disease (HCDD) is a rare disorder characterized by glomerular and peritubular amorphous deposits of a truncated monoclonal immunoglobulin heavy chain (HC) bearing a deletion of the first constant domain (CH1). We created a transgenic mouse model of HCDD using targeted insertion in the immunoglobulin κ locus of a human HC extracted from a HCDD patient. Our strategy allows the efficient expression of the human HC in mouse B and plasma cells, and conditional deletion of the CH1 domain reproduces the major event underlying HCDD. We show that the deletion of the CH1 domain dramatically reduced serum HC levels. Strikingly, even with very low serum level of truncated monoclonal HC, histologic studies revealed typical Randall-type renal lesions that were absent in mice expressing the complete human HC. Bortezomib-based treatment resulted in a strong decrease of renal deposits. We further demonstrated that this efficient response to proteasome inhibitors mostly relies on the presence of the isolated truncated HC that sensitizes plasma cells to bortezomib through an elevated unfolded protein response (UPR). This new transgenic model of HCDD efficiently recapitulates the pathophysiologic features of the disease and demonstrates that the renal damage in HCDD relies on the production of an isolated truncated HC, which, in the absence of a LC partner, displays a high propensity to aggregate even at very low concentration. It also brings new insights into the efficacy of proteasome inhibitor-based therapy in this pathology.
Assuntos
Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Doença das Cadeias Pesadas/tratamento farmacológico , Cadeias Pesadas de Imunoglobulinas/química , Nefropatias/tratamento farmacológico , Inibidores de Proteassoma/farmacologia , Agregação Patológica de Proteínas/tratamento farmacológico , Pirazinas/farmacologia , Sequência de Aminoácidos , Animais , Bortezomib , Modelos Animais de Doenças , Expressão Gênica , Loci Gênicos , Doença das Cadeias Pesadas/genética , Doença das Cadeias Pesadas/imunologia , Doença das Cadeias Pesadas/patologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Nefropatias/genética , Nefropatias/imunologia , Nefropatias/patologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/imunologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologia , Plasmócitos/metabolismo , Plasmócitos/patologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/imunologia , Agregação Patológica de Proteínas/patologia , Estrutura Terciária de Proteína , Deleção de Sequência , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Resposta a Proteínas não Dobradas/imunologiaRESUMO
B-cell terminal differentiation into antibody secreting plasma cells (PCs) features a transcriptional shift driven by the activation of plasma cell lineage determinants such as Blimp-1 and Xbp-1, together with the extinction of Pax5. Little is known about the signals inducing this change in transcriptional networks and the role of the B-cell receptor (BCR) in terminal differentiation remains especially controversial. Here, we show that tonic BCR signal strength influences PC commitment in vivo. Using immuno-globulin light chain transgenic mice expressing suboptimal surface BCR levels and latent membrane protein 2A knock-in animals with defined BCR-like signal strengths, we show that weak, antigen-independent constitutive BCR signaling facilitates spontaneous PC differentiation in vivo and in vitro in response to TLR agonists or CD40/IL-4. Conversely, increasing tonic signaling completely prevents this process that is rescued by lowering surface BCR expression or through the inhibition of Syk phosphorylation. These findings provide new insights into the role of basal BCR signaling in PC differentiation and point to the need to resolve a strong BCR signal in order to guarantee terminal differentiation.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Diferenciação Celular , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular/imunologia , Camundongos , Camundongos Knockout , Plasmócitos/citologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , Sindecana-1/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Receptores Toll-Like/metabolismo , Proteínas da Matriz Viral/metabolismoRESUMO
Neurotensin, a neuropeptide growth factor, and its two specific neurotensin receptors, NTSR1 and NTSR2, were shown to be expressed by human B cell lines. Another NTSR, sortilin, which is common to neurotensin and neurotrophins, was also detected as we have previously described. Neurotensin was functional in B cell lines; it induced their proliferation and inhibited apoptosis induced by serum deprivation or Fas activation. Quantitative study of gene expression in two malignant B cell diseases showed that NTSR2 was overexpressed, NTSR1 decreased, and neurotensin was unexpressed in B cell leukemia patient's cells, as compared with healthy B cells. However, these expressions did not significantly change in large diffuse B cell lymphoma lymph nodes compared with benign ones. This study points out that neurotensin and its two specific receptors are expressed in human B lymphocytes. Such expressions were not described, and their relationship in B cell diseases, especially in chronic B cell leukemia, needs to be considered further in regard to these findings.
Assuntos
Linfócitos B/metabolismo , Regulação Neoplásica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Linfoma Difuso de Grandes Células B/genética , Neurotensina/genética , Receptores de Neurotensina/genética , Apoptose , Linfócitos B/patologia , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Neurotensina/metabolismo , Especificidade de Órgãos , Cultura Primária de Células , Receptores de Neurotensina/metabolismo , Transdução de Sinais , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Remodeling of immunoglobulin genes by activation-induced deaminase (AID) is required for affinity maturation and class-switch recombination in mature B lymphocytes. In the immunoglobulin heavy chain locus, these processes are predominantly controlled by the 3' cis-regulatory region. We now show that this region is transcribed and undergoes AID-mediated mutation and recombination around phylogenetically conserved switchlike DNA repeats. Such recombination, which we term locus suicide recombination, deletes the whole constant region gene cluster and thus stops expression of the immunoglobulin of the B cell surface, which is critical for B cell survival. The frequency of this event is approaching that of class switching and makes it a potential regulator of B cell homeostasis.
Assuntos
Linfócitos B/fisiologia , Citidina Desaminase/metabolismo , Deleção de Genes , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Genes de Cadeia Pesada de Imunoglobulina , Recombinação Genética , Animais , Linfócitos B/imunologia , Sequência de Bases , Linhagem Celular , Sobrevivência Celular , Homeostase , Humanos , Switching de Imunoglobulina , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Transcrição GênicaRESUMO
Immunoglobulin (Ig) genes naturally acquire frequent premature termination codons during the error-prone V(D)J recombination process. Although B cell differentiation is linked to the expression of productive Ig alleles, the transcriptional status of nonfunctionally recombined alleles remains unclear. Here, we tracked transcription and posttranscriptional regulation for both Ig heavy-chain (IgH) alleles in mice carrying a nonfunctional knock-in allele. We show that productively and nonproductively VDJ-rearranged alleles are transcribed throughout B cell development, carry similar active chromatin marks, and even display equivalent RNA polymerase II (RNAPII) loading after B cell stimulation. Hence, these results challenge the idea that the repositioning of one allele to heterochromatin could promote the silencing of nonproductive alleles. Interestingly, the efficiency of downstream RNA surveillance mechanisms fluctuates according to B cell activation and terminal differentiation: unspliced nonfunctional transcripts accumulate in primary B cells, while B cell activation promotes IgH transcription, RNA splicing, and nonsense-mediated mRNA decay (NMD). Altogether, IgH transcription and RNA splicing rates determine by which RNA surveillance mechanisms a B cell can get rid of nonproductive IgH mRNAs.
Assuntos
Linfócitos B/citologia , Cadeias Pesadas de Imunoglobulinas/genética , Splicing de RNA , RNA/genética , Transcrição Gênica , Recombinação V(D)J , Alelos , Animais , Linfócitos B/metabolismo , Diferenciação Celular , Células Cultivadas , Técnicas de Introdução de Genes , Camundongos , Camundongos Endogâmicos C57BL , RNA/análise , Precursores de RNA/análise , Precursores de RNA/genéticaRESUMO
Estrogen treatment exerts a protective effect on experimental autoimmune encephalomyelitis (EAE) and is under clinical trial for multiple sclerosis therapy. Estrogens have been suspected to protect from CNS autoimmunity through their capacity to exert anti-inflammatory as well as neuroprotective effects. Despite the obvious impacts of estrogens on the pathophysiology of multiple sclerosis and EAE, the dominant cellular target that orchestrates the anti-inflammatory effect of 17ß-estradiol (E2) in EAE is still ill defined. Using conditional estrogen receptor (ER) α-deficient mice and bone marrow chimera experiments, we show that expression of ERα is critical in hematopoietic cells but not in endothelial ones to mediate the E2 inhibitory effect on Th1 and Th17 cell priming, resulting in EAE protection. Furthermore, using newly created cell type-specific ERα-deficient mice, we demonstrate that ERα is required in T lymphocytes, but neither in macrophages nor dendritic cells, for E2-mediated inhibition of Th1/Th17 cell differentiation and protection from EAE. Lastly, in absence of ERα in host nonhematopoietic tissues, we further show that ERα signaling in T cells is necessary and sufficient to mediate the inhibitory effect of E2 on EAE development. These data uncover T lymphocytes as a major and nonredundant cellular target responsible for the anti-inflammatory effects of E2 in Th17 cell-driven CNS autoimmunity.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Células Th1/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Animais , Southern Blotting , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Separação Celular , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Receptor alfa de Estrogênio/imunologia , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Células Th1/citologia , Células Th1/imunologia , Células Th17/citologia , Células Th17/imunologiaRESUMO
DREAM/KChIP-3 is a calcium-dependent transcriptional repressor highly expressed in immune cells. Transgenic mice expressing a dominant active DREAM mutant show reduced serum Ig levels. In vitro assays show that reduced Ig secretion is an intrinsic defect of transgenic B cells that occurs without impairment in plasma cell differentiation, class switch recombination, or Ig transcription. Surprisingly, transgenic B cells show an accelerated entry in cell division. Transcriptomic analysis of transgenic B cells revealed that hyperproliferative B cell response could be correlated with a reduced expression of Klf9, a cell-cycle regulator. Pulse-chase experiments demonstrated that the defect in Ig production is associated with reduced translation rather than with increased protein degradation. Importantly, transgenic B cells showed reduced expression of the Eif4g3 gene, which encodes a protein related to protein translation. Our results disclose, to our knowledge, a novel function of DREAM in proliferation and Ig synthesis in B lymphocytes.
Assuntos
Formação de Anticorpos/imunologia , Diferenciação Celular/imunologia , Rearranjo Gênico do Linfócito B/imunologia , Imunoglobulinas/imunologia , Proteínas Interatuantes com Canais de Kv/imunologia , Plasmócitos/imunologia , Proteínas Repressoras/imunologia , Animais , Formação de Anticorpos/genética , Diferenciação Celular/genética , Proliferação de Células , Fator de Iniciação Eucariótico 4G/biossíntese , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Rearranjo Gênico do Linfócito B/genética , Imunoglobulinas/biossíntese , Imunoglobulinas/genética , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/imunologia , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Plasmócitos/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Sex hormones influence immune responses and the development of autoimmune diseases including MS and its animal model, EAE. Although it has been previously reported that ovariectomy could worsen EAE, the mechanisms implicated in the protective action of endogenous ovarian hormones have not been addressed. In this report, we now show that endogenous estrogens limit EAE development and CNS inflammation in adult female mice through estrogen receptor α expression in the host non-hematopoietic tissues. We provide evidence that the enhancing effect of gonadectomy on EAE development was due to quantitative rather than qualitative changes in effector Th1 or Th17 cell recruitment into the CNS. Consistent with this observation, adoptive transfer of myelin oligodendrocyte glycoprotein-specific encephalitogenic CD4(+) T lymphocytes induced more severe EAE in ovariectomized mice as compared to normal female mice. Finally, we show that gonadectomy accelerated the early recruitment of inflammatory cells into the CNS upon adoptive transfer of encephalitogenic CD4(+) T cells. Altogether, these data show that endogenous estrogens, through estrogen receptor α, exert a protective effect on EAE by limiting the recruitment of blood-derived inflammatory cells into the CNS.
Assuntos
Encéfalo/patologia , Linfócitos T CD4-Positivos/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Receptor alfa de Estrogênio/metabolismo , Estrogênios/imunologia , Transferência Adotiva , Animais , Encéfalo/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/transplante , Movimento Celular/genética , Células Cultivadas , Citoproteção/genética , Citoproteção/imunologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/cirurgia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/imunologia , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esclerose Múltipla , Proteínas da Mielina , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito , OvariectomiaRESUMO
17Beta-estradiol (E2) has been shown to promote the expression of inflammatory mediators by LPS-activated tissue resident macrophages through estrogen receptor alpha (ERalpha) signaling. However, it remained to be determined whether E2 similarly influences macrophages effector functions under inflammatory conditions in vivo, and whether this action of E2 resulted from a direct effect on macrophages. We show in this study that chronic E2 administration to ovariectomized mice significantly increased both cytokine (IL-1beta, IL-6, and TNF-alpha) and inducible NO synthase mRNA abundance in thioglycolate (TGC)-elicited macrophages. The proinflammatory action of E2 was also evidenced at the level of released IL-1beta and IL-6 by ex vivo LPS-activated macrophages. E2 concomitantly inhibited PI3K activity as well as Akt phosphorylation in TGC-elicited macrophages, suggesting that E2 promoted TLR-dependent macrophage activation by alleviating this suppressive signaling pathway. Indeed, this effect was abolished in the presence of the inhibitor wortmannin, demonstrating a key functional link between inhibition of PI3K activity and the E2 action on macrophage functions. Endogenous estrogens levels circulating in ovary-intact mice were sufficient to promote the above described actions. Finally, thanks to a CreLox strategy, targeted disruption of ERalpha gene in macrophages totally abolished the effect of E2 on the expression of inflammatory mediators by both resident and TGC-elicited peritoneal macrophages. In conclusion, we demonstrate that estrogens, through the activation of ERalpha in macrophages in vivo, enhance their ability to produce inflammatory mediators and cytokines upon subsequent TLR activation.