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1.
PLoS Genet ; 15(6): e1007721, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31199803

RESUMO

B-cell activation yields abundant cell death in parallel to clonal amplification and remodeling of immunoglobulin (Ig) genes by activation-induced deaminase (AID). AID promotes affinity maturation of Ig variable regions and class switch recombination (CSR) in mature B lymphocytes. In the IgH locus, these processes are under control of the 3' regulatory region (3'RR) super-enhancer, a region demonstrated in the mouse to be both transcribed and itself targeted by AID-mediated recombination. Alternatively to CSR, IgH deletions joining Sµ to "like-switch" DNA repeats that flank the 3' super-enhancer can thus accomplish so-called "locus suicide recombination" (LSR) in mouse B-cells. Using an optimized LSR-seq high throughput method, we now show that AID-mediated LSR is evolutionarily conserved and also actively occurs in humans, providing an activation-induced cell death pathway in multiple conditions of B-cell activation. LSR either focuses on the functional IgH allele or is bi-allelic, and its signature is mainly detected when LSR is ongoing while it vanishes from fully differentiated plasma cells or from "resting" blood memory B-cells. Highly diversified breakpoints are distributed either within the upstream (3'RR1) or downstream (3'RR2) copies of the IgH 3' super-enhancer and all conditions activating CSR in vitro also seem to trigger LSR although TLR ligation appeared the most efficient. Molecular analysis of breakpoints and junctions confirms that LSR is AID-dependent and reveals junctional sequences somehow similar to CSR junctions but with increased usage of microhomologies.


Assuntos
Linfócitos B/imunologia , Citidina Desaminase/genética , Região de Troca de Imunoglobulinas/genética , Imunoglobulinas/imunologia , Alelos , Animais , Diferenciação Celular/genética , Citidina Desaminase/imunologia , Marcação de Genes , Humanos , Região de Troca de Imunoglobulinas/imunologia , Tecido Linfoide/imunologia , Camundongos , Tonsila Palatina/imunologia , Tonsila Palatina/metabolismo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Sequências Reguladoras de Ácido Nucleico
2.
J Immunol ; 198(10): 4148-4155, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28416601

RESUMO

B cells ensure humoral immune responses due to the production of Ag-specific memory B cells and Ab-secreting plasma cells. In secondary lymphoid organs, Ag-driven B cell activation induces terminal maturation and Ig isotype class switch (class switch recombination [CSR]). CSR creates a virtually unique IgH locus in every B cell clone by intrachromosomal recombination between two switch (S) regions upstream of each C region gene. Amount and structural features of CSR junctions reveal valuable information about the CSR mechanism, and analysis of CSR junctions is useful in basic and clinical research studies of B cell functions. To provide an automated tool able to analyze large data sets of CSR junction sequences produced by high-throughput sequencing (HTS), we designed CSReport, a software program dedicated to support analysis of CSR recombination junctions sequenced with a HTS-based protocol (Ion Torrent technology). CSReport was assessed using simulated data sets of CSR junctions and then used for analysis of Sµ-Sα and Sµ-Sγ1 junctions from CH12F3 cells and primary murine B cells, respectively. CSReport identifies junction segment breakpoints on reference sequences and junction structure (blunt-ended junctions or junctions with insertions or microhomology). Besides the ability to analyze unprecedentedly large libraries of junction sequences, CSReport will provide a unified framework for CSR junction studies. Our results show that CSReport is an accurate tool for analysis of sequences from our HTS-based protocol for CSR junctions, thereby facilitating and accelerating their study.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Switching de Imunoglobulina/genética , Recombinação Genética , Software , Linfócitos B/imunologia , Quebras de DNA de Cadeia Dupla , Isotipos de Imunoglobulinas/genética , Região de Troca de Imunoglobulinas/genética
4.
Gene ; 861: 147232, 2023 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-36736508

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection which is commonly known as COVID-19 (COronaVIrus Disease 2019) has creeped into the human population taking tolls of life and causing tremendous economic crisis. It is indeed crucial to gain knowledge about their characteristics and interactions with human host cells. It has been shown that the majority of our genome consists of non-coding RNAs. Non-coding RNAs including micro RNAs (miRNAs) and long non-coding RNAs (lncRNAs) display significant roles in regulating gene expression in almost all cancers and viral diseases. It is intriguing that miRNAs and lncRNAs remarkably regulate the function and expression of major immune components of SARS-CoV-2. MiRNAs act via RNA interference mechanism in which they bind to the complementary sequences of the viral RNA strand, inducing the formation of silencing complex that eventually degrades or inhibits the viral RNA and viral protein expression. LncRNAs have been extensively shown to regulate gene expression in cytokine storm and thus emerges as a critical target for COVID-19 treatment. These lncRNAs also act as competing endogenous RNAs (ceRNAs) by sponging miRNAs and thus affecting the expression of downstream targets during SARS-CoV-2 infection. In this review, we extensively discuss the role of miRNAs and lncRNAs, describe their mechanism of action and their different interacting human targets cells during SARS-CoV-2 infection. Finally, we discuss possible ways how an interference with their molecular function could be exploited for new therapies against SARS-CoV-2.


Assuntos
COVID-19 , MicroRNAs , RNA Longo não Codificante , Humanos , MicroRNAs/genética , RNA Longo não Codificante/genética , SARS-CoV-2/genética , Tratamento Farmacológico da COVID-19 , RNA Viral
5.
Front Immunol ; 14: 1155906, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37359540

RESUMO

Introduction: In mature B cells, activation-induced deaminase reshapes Ig genes through somatic hypermutation and class switch recombination of the Ig heavy chain (IgH) locus under control of its 3' cis-regulatory region (3'RR). The 3'RR is itself transcribed and can undergo "locus suicide recombination" (LSR), then deleting the constant gene cluster and terminating IgH expression. The relative contribution of LSR to B cell negative selection remains to be determined. Methods: Here, we set up a knock-in mouse reporter model for LSR events with the aim to get clearer insights into the circumstances triggering LSR. In order to explore the consequences of LSR defects, we reciprocally explored the presence of autoantibodies in various mutant mouse lines in which LSR was perturbed by the lack of Sµ or of the 3'RR. Results: Evaluation of LSR events in a dedicated reporter mouse model showed their occurrence in various conditions of B cell activation, notably in antigen-experienced B cells Studies of mice with LSR defects evidenced increased amounts of self-reactive antibodies. Discussion: While the activation pathways associated with LSR are diverse, in vivo as well as in vitro, this study suggests that LSR may contribute to the elimination of self-reactive B cells.


Assuntos
Linfócitos B , Suicídio , Camundongos , Animais , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Switching de Imunoglobulina/genética , Antígenos/metabolismo
6.
Neurochem Int ; 154: 105301, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35121011

RESUMO

Traumatic Brain Injury (TBI) is one of the leading causes of death and disability worldwide. Aspirin (ASA) and clopidogrel (CLOP) are antiplatelet agents that inhibit platelet aggregation. They are implicated in worsening the intracerebral haemorrhage (ICH) risk post-TBI. However, antiplatelet drugs may also exert a neuroprotective effect post-injury. We determined the impact of ASA and CLOP treatment, alone or in combination, on ICH and brain damage in an experimental rat TBI model. We assessed changes in platelet aggregation and measured serum thromboxane by enzyme immune assay. We also explored a panel of brain damage and apoptosis biomarkers by immunoblotting. Rats were treated with ASA and/or CLOP for 48 h prior to TBI and sacrificed 48 h post-injury. In rats treated with antiplatelet agents prior to TBI, platelet aggregation was completely inhibited, and serum thromboxane was significantly decreased, compared to the TBI group without treatment. TBI increases UCHL-1 and GFAP, but decreases hexokinase expression compared to the non-injured controls. All groups treated with antiplatelet drugs prior to TBI had decreased UCH-L1 and GFAP serum levels compared to the TBI untreated group. Furthermore, the ASA and CLOP single treatments increased the hexokinase serum levels. We confirmed that αII-spectrin cleavage increased post-TBI, with the highest cleavage detected in CLOP-treated rats. Aspirin and/or CLOP treatment prior to TBI is a double-edged sword that exerts a dual effect post-injury. On one hand, ASA and CLOP single treatments increase the post-TBI ICH risk, with a further detrimental effect from the ASA + CLOP treatment. On the other hand, ASA and/or CLOP treatments are neuroprotective and result in a favourable profile of TBI injury markers. The ICH risk and the neuroprotection benefits from antiplatelet therapy should be weighed against each other to ameliorate the management of TBI patients.


Assuntos
Lesões Encefálicas Traumáticas , Lesões Encefálicas , Animais , Aspirina/farmacologia , Aspirina/uso terapêutico , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas Traumáticas/tratamento farmacológico , Clopidogrel/farmacologia , Humanos , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Ratos
7.
Front Immunol ; 12: 737427, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34777346

RESUMO

Activation-induced deaminase (AID) is the major actor of immunoglobulin (Ig) gene diversification in germinal center B-cells. From its first description, it was considered as mandatory for class switch recombination (CSR), and this discovery initiated a long quest for all of the AID-interacting factors controlling its activity. The mechanisms focusing AID-mediated DNA lesions to given target sequences remain incompletely understood with regards the detailed characterization of optimal substrates in which cytidine deamination will lead to double strand breaks (DSBs) and chromosomal cleavage. In an effort to reconsider whether such CSR breaks absolutely require AID, we herein provide evidence, based on deep-sequencing approaches, showing that this dogma is not absolute in both human and mouse B lymphocytes. In activated B-cells from either AID-deficient mice or human AID-deficient patients, we report an intrinsic ability of the IgH locus to undergo "on-target" cleavage and subsequent synapsis of broken regions in conditions able to yield low-level CSR. DNA breaks occur in such conditions within the same repetitive S regions usually targeted by AID, but their repair follows a specific pathway with increased usage of microhomology-mediated repair. These data further demonstrate the role of AID machinery as not initiating de novo chromosomal cleavage but rather catalyzing a process which spontaneously initiates at low levels in an appropriately conformed IgH locus.


Assuntos
Linfócitos B/enzimologia , Citidina Desaminase/deficiência , Switching de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas/genética , Síndromes de Imunodeficiência/genética , Ativação Linfocitária , Animais , Linfócitos B/imunologia , Citidina Desaminase/genética , Quebras de DNA , Reparo do DNA por Junção de Extremidades , Modelos Animais de Doenças , Loci Gênicos , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Síndromes de Imunodeficiência/enzimologia , Síndromes de Imunodeficiência/imunologia , Camundongos Knockout
8.
Clin Transl Immunology ; 10(6): e1280, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34136216

RESUMO

OBJECTIVES: Inhibitors of bromodomain and extra terminal domain (BET) proteins are a new and growing class of anti-cancer drugs, which decrease oncogene expression by targeting superenhancers. Antibody production is another physiological process relying on superenhancers, and it remains to be clarified whether potential immunomodulatory properties of BET inhibitors might impact humoral immunity and allergy. METHODS: We thus evaluated humoral immune responses and their Th2 context in vitro and in vivo in mice following treatment with the classical BET-inhibitor JQ1. We quantified immunoglobulin (Ig) and antibody production by B cells either stimulated in vitro or obtained from immunised mice. JQ1 effects on class switching and activation-induced deaminase loading were determined, together with modifications of B, T follicular helper (Tfh) and T helper 2 (Th2) populations. JQ1 was finally tested in B-cell-dependent models of immune disorders. RESULTS: Bromodomain and extra terminal domain inhibition reduced class switching, Ig expression on B cells and antibody secretion and was correlated with decreased numbers of Tfh cells. However, JQ1 strongly increased the proportion of GATA3+ Th2 cells and the secretion of corresponding cytokines. In a mouse allergic model of lung inflammation, JQ1 did not affect eosinophil infiltration or mucus production but enhanced Th2 cytokine production and aggravated clinical manifestations. CONCLUSION: Altogether, BET inhibition thus interweaves intrinsic negative effects on B cells with a parallel complex reshaping of T-cell polarisation which can increase type 2 cytokines and eventually promote B-cell-dependent immunopathology. These opposite and potentially hazardous immunomodulatory effects raise concerns for clinical use of BET inhibitors in patients with immune disorders.

9.
Front Immunol ; 8: 1277, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29180995

RESUMO

Memory B cells and long-lived plasma cells are key elements of adaptive humoral immunity. Regardless of the immunoglobulin class produced, these cells can ensure long-lasting protection but also long-lasting immunopathology, thus requiring tight regulation of their generation and survival. Among all antibody classes, this is especially true for IgE, which stands as the most potent, and can trigger dramatic inflammatory reactions even when present in minute amounts. IgE responses and memory crucially protect against parasites and toxic components of venoms, conferring selective advantages and explaining their conservation in all mammalian species despite a parallel broad spectrum of IgE-mediated immunopathology. Long-term memory of sensitization and anaphylactic responses to allergens constitute the dark side of IgE responses, which can trigger multiple acute or chronic pathologic manifestations, some punctuated with life-threatening events. This Janus face of the IgE response and memory, both necessary and potentially dangerous, thus obviously deserves the most elaborated self-control schemes.

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