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1.
Biomedicines ; 9(9)2021 Sep 07.
Article in English | MEDLINE | ID: mdl-34572362

ABSTRACT

Human respiratory syncytial virus (HRSV) is the most common cause of severe respiratory infections in infants and young children, often leading to hospitalization. In addition, this virus poses a serious health risk in immunocompromised individuals and the elderly. HRSV is also a major nosocomial hazard in healthcare service units for patients of all ages. Therefore, the development of antiviral treatments against HRSV is a global health priority. In this study, mitoxantrone, a synthetic anthraquinone with previously reported in vitro antiprotozoal and antiviral activities, inhibits HRSV replication in vitro, but not in vivo in a mice model. These results have implications for preclinical studies of some drug candidates.

2.
Front Genet ; 11: 552949, 2020.
Article in English | MEDLINE | ID: mdl-33193627

ABSTRACT

The CD69 gene encodes a C-type lectin glycoprotein with immune regulatory properties which is expressed on the cell surfaces of all activated hematopoietic cells. CD69 activation kinetics differ by developmental stage, cell linage and activating conditions, and these differences have been attributed to the participation of complex gene regulatory networks. An evolutionarily conserved regulatory element, CNS2, located 4kb upstream of the CD69 gene transcriptional start site, has been proposed as the major candidate governing the gene transcriptional activation program. To investigate the function of human CNS2, we studied the effect of its endogenous elimination via CRISPR-Cas9 on CD69 protein and mRNA expression levels in various immune cell lines. Even when the entire promoter region was maintained, CNS2-/- cells did not express CD69, thus indicating that CNS2 has promoter-like characteristics. However, like enhancers, inverted CNS2 sustained transcription, although at a diminished levels, thereby suggesting that it has dual promoter and enhancer functions. Episomal luciferase assays further suggested that both functions are combined within the CNS2 regulatory element. In addition, CNS2 directs its own bidirectional transcription into two different enhancer-derived RNAs molecules (eRNAs) which are transcribed from two independent transcriptional start sites in opposite directions. This eRNA transcription is dependent on only the enhancer sequence itself, because in the absence of the CD69 promoter, sufficient RNA polymerase II levels are maintained at CNS2 to drive eRNA expression. Here, we describe a regulatory element with overlapping promoter and enhancer functions, which is essential for CD69 gene transcriptional regulation.

3.
Genes (Basel) ; 10(9)2019 08 28.
Article in English | MEDLINE | ID: mdl-31466317

ABSTRACT

The immune regulatory receptor CD69 is expressed upon activation in all types of leukocytes and is strongly regulated at the transcriptional level. We previously described that, in addition to the CD69 promoter, there are four conserved noncoding regions (CNS1-4) upstream of the CD69 promoter. Furthermore, we proposed that CNS2 is the main enhancer of CD69 transcription. In the present study, we mapped the transcription factor (TF) binding sites (TFBS) from ChIP-seq databases within CNS2. Through luciferase reporter assays, we defined a ~60 bp sequence that acts as the minimum enhancer core of mouse CNS2, which includes the Oct1 TFBS. This enhancer core establishes cooperative interactions with the 3' and 5' flanking regions, which contain RUNX1 BS. In agreement with the luciferase reporter data, the inhibition of RUNX1 and Oct1 TF expression by siRNA suggests that they synergistically enhance endogenous CD69 gene transcription. In summary, we describe an enhancer core containing RUNX1 and Oct1 BS that is important for the activity of the most potent CD69 gene transcription enhancer.


Subject(s)
Antigens, CD/genetics , Antigens, Differentiation, T-Lymphocyte/genetics , Enhancer Elements, Genetic , Lectins, C-Type/genetics , Animals , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Conserved Sequence , Core Binding Factor Alpha 2 Subunit/metabolism , Humans , Jurkat Cells , Lectins, C-Type/metabolism , Mice , Octamer Transcription Factor-1/metabolism , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism
4.
J Virol ; 93(19)2019 10 01.
Article in English | MEDLINE | ID: mdl-31315995

ABSTRACT

CD69 is highly expressed on the leukocyte surface upon viral infection, and its regulatory role in the vaccinia virus (VACV) immune response has been recently demonstrated using CD69-/- mice. Here, we show augmented control of VACV infection using the anti-human CD69 monoclonal antibody (MAb) 2.8 as both preventive and therapeutic treatment for mice expressing human CD69. This control was related to increased natural killer (NK) cell reactivity and increased numbers of cytokine-producing T and NK cells in the periphery. Moreover, similarly increased immunity and protection against VACV were reproduced over both long and short periods in anti-mouse CD69 MAb 2.2-treated immunocompetent wild-type (WT) mice and immunodeficient Rag2-/- CD69+/+ mice. This result was not due to synergy between infection and anti-CD69 treatment since, in the absence of infection, anti-human CD69 targeting induced immune activation, which was characterized by mobilization, proliferation, and enhanced survival of immune cells as well as marked production of several innate proinflammatory cytokines by immune cells. Additionally, we showed that the rapid leukocyte effect induced by anti-CD69 MAb treatment was dependent on mTOR signaling. These properties suggest the potential of CD69-targeted therapy as an antiviral adjuvant to prevent derived infections.IMPORTANCE In this study, we demonstrate the influence of human and mouse anti-CD69 therapies on the immune response to VACV infection. We report that targeting CD69 increases the leukocyte numbers in the secondary lymphoid organs during infection and improves the capacity to clear the viral infection. Targeting CD69 increases the numbers of gamma interferon (IFN-γ)- and tumor necrosis factor alpha (TNF-α)-producing NK and T cells. In mice expressing human CD69, treatment with an anti-CD69 MAb produces increases in cytokine production, survival, and proliferation mediated in part by mTOR signaling. These results, together with the fact that we have mainly worked with a human-CD69 transgenic model, reveal CD69 as a treatment target to enhance vaccine protectiveness.


Subject(s)
Immunologic Factors/antagonists & inhibitors , Killer Cells, Natural/immunology , Lectins, C-Type/antagonists & inhibitors , T-Lymphocytes/immunology , Vaccinia virus/immunology , Vaccinia/prevention & control , Animals , Antibodies, Monoclonal/administration & dosage , Antigens, CD/administration & dosage , Antigens, CD/genetics , Antigens, Differentiation, T-Lymphocyte/administration & dosage , Antigens, Differentiation, T-Lymphocyte/genetics , Disease Models, Animal , Humans , Immunologic Factors/administration & dosage , Immunologic Factors/genetics , Lectins, C-Type/administration & dosage , Lectins, C-Type/genetics , Mice , Mice, Transgenic , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Vaccinia/immunology , Vaccinia/therapy
5.
Circ Res ; 124(2): 279-291, 2019 01 18.
Article in English | MEDLINE | ID: mdl-30582456

ABSTRACT

RATIONALE: CD69 is an immunomodulatory molecule induced during lymphocyte activation. Following stroke, T-lymphocytes upregulate CD69 but its function is unknown. OBJECTIVE: We investigated whether CD69 was involved in brain damage following an ischemic stroke. METHODS AND RESULTS: We used adult male mice on the C57BL/6 or BALB/c backgrounds, including wild-type mice and CD69-/- mice, and CD69+/+ and CD69-/- lymphocyte-deficient Rag2-/- mice, and generated chimeric mice. We induced ischemia by transient or permanent middle cerebral artery occlusion. We measured infarct volume, assessed neurological function, and studied CD69 expression, as well as platelet function, fibrin(ogen) deposition, and VWF (von Willebrand factor) expression in brain vessels and VWF content and activity in plasma, and performed the tail-vein bleeding test and the carotid artery ferric chloride-induced thrombosis model. We also performed primary glial cell cultures and sorted brain CD45-CD11b-CD31+ endothelial cells for mRNA expression studies. We blocked VWF by intravenous administration of anti-VWF antibodies. CD69-/- mice showed larger infarct volumes and worse neurological deficits than the wild-type mice after ischemia. This worsening effect was not attributable to lymphocytes or other hematopoietic cells. CD69 deficiency lowered the time to thrombosis in the carotid artery despite platelet function not being affected. Ischemia upregulated Cd69 mRNA expression in brain endothelial cells. CD69-deficiency increased fibrin(ogen) accumulation in the ischemic tissue, and plasma VWF content and activity, and VWF expression in brain vessels. Blocking VWF reduced infarct volume and reverted the detrimental effect of CD69-/- deficiency. CONCLUSIONS: CD69 deficiency promotes a prothrombotic phenotype characterized by increased VWF and worse brain damage after ischemic stroke. The results suggest that CD69 acts as a downregulator of endothelial activation.


Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Brain/blood supply , Brain/metabolism , Endothelial Cells/metabolism , Infarction, Middle Cerebral Artery/metabolism , Lectins, C-Type/metabolism , Lymphocyte Activation , T-Lymphocytes/metabolism , Animals , Antigens, CD/genetics , Antigens, Differentiation, T-Lymphocyte/genetics , Blood Coagulation , Blood Platelets/metabolism , Brain/pathology , Cells, Cultured , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Disease Models, Animal , Endothelial Cells/pathology , Infarction, Middle Cerebral Artery/genetics , Infarction, Middle Cerebral Artery/pathology , Lectins, C-Type/deficiency , Lectins, C-Type/genetics , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Signal Transduction , T-Lymphocytes/pathology , von Willebrand Factor/metabolism
6.
Leukemia ; 32(6): 1445-1457, 2018 06.
Article in English | MEDLINE | ID: mdl-29483712

ABSTRACT

CD69 regulates lymphocyte egress from the thymus and lymph nodes through cis-interactions and the downregulation of surface sphingosine-1-phosphate (S1P) receptor-1 (S1P1). However, its role in the regulation of cell egress from bone marrow has not been extensively studied. We show here that CD69 targeting induced rapid and massive mobilization of BM leukocytes, which was inhibited by desensitization to S1P with FTY720. This mobilization was reproduced with anti-human CD69 mAb treatment of mice expressing human CD69. In this strain, the mobilization occurred to the same extent as that induced by AMD3100. The anti-human CD69 treatment highly increased LSK and CLP cell proliferation and numbers, both in the periphery and in the BM, and also augmented S1P1 and CXCR4 expression. Additionally, increased mTOR, p70S6K, S6, and 4E-BP1 phosphorylation was detected after in vivo anti-CD69 treatment in the bone marrow. Importantly, mTOR inhibition with rapamycin inhibited anti-huCD69-induced mobilization of hematopoietic stem and progenitor cells (HSPCs). Together, our results indicated that CD69 targeting induces not only mobilization but also high proliferation of HSPCs, and thus is crucial for precursor cell replenishment over time. These results suggest that anti-CD69 mAbs are putative novel candidates for mobilization strategies.


Subject(s)
Hematopoietic Stem Cell Mobilization/methods , Hematopoietic Stem Cells/physiology , Lectins, C-Type/antagonists & inhibitors , Membrane Proteins/physiology , Phosphoric Monoester Hydrolases/physiology , TOR Serine-Threonine Kinases/physiology , Animals , Antibodies, Monoclonal/pharmacology , Antigens, CD , Antigens, Differentiation, T-Lymphocyte , Benzylamines , Cell Movement/drug effects , Cell Proliferation/drug effects , Chemokine CXCL12/biosynthesis , Cyclams , Female , Fingolimod Hydrochloride/pharmacology , Heterocyclic Compounds/pharmacology , Male , Mice
7.
J Virol ; 90(14): 6464-6474, 2016 07 15.
Article in English | MEDLINE | ID: mdl-27147744

ABSTRACT

UNLABELLED: During the host response to viral infection, the transmembrane CD69 protein is highly upregulated in all immune cells. We have studied the role of CD69 in the murine immune response to vaccinia virus (VACV) infection, and we report that the absence of CD69 enhances protection against VACV at both short and long times postinfection in immunocompetent and immunodeficient mice. Natural killer (NK) cells were implicated in the increased infection control, since the differences were greatly diminished when NK cells were depleted. This role of NK cells was not based on an altered NK cell reactivity, since CD69 did not affect the NK cell activation threshold in response to major histocompatibility complex class I NK cell targets or protein kinase C activation. Instead, NK cell numbers were increased in the spleen and peritoneum of CD69-deficient infected mice. That was not just secondary to better infection control in CD69-deficient mice, since NK cell numbers in the spleens and the blood of uninfected CD69(-/-) mice were already augmented. CD69-deficient NK cells from infected mice did not have an altered proliferation capacity. However, a lower spontaneous cell death rate was observed for CD69(-/-) lymphocytes. Thus, our results suggest that CD69 limits the innate immune response to VACV infection at least in part through cell homeostatic survival. IMPORTANCE: We show that increased natural killer (NK) cell numbers augment the host response and survival after infection with vaccinia virus. This phenotype is found in the absence of CD69 in immunocompetent and immunodeficient hosts. As part of the innate immune system, NK lymphocytes are activated and participate in the defense against infection. Several studies have focused on the contribution of NK cells to protection against infection with vaccinia virus. In this study, it was demonstrated that the augmented early NK cell response in the absence of CD69 is responsible for the increased protection seen during infection with vaccinia virus even at late times of infection. This work indicates that the CD69 molecule may be a target of therapy to augment the response to poxvirus infection.


Subject(s)
Antigens, CD/physiology , Antigens, Differentiation, T-Lymphocyte/physiology , Immunity, Innate/immunology , Killer Cells, Natural/immunology , Lectins, C-Type/physiology , Peritoneum/immunology , Spleen/immunology , Vaccinia virus/immunology , Vaccinia/virology , Animals , Female , Homeostasis , Killer Cells, Natural/metabolism , Killer Cells, Natural/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Vaccinia/immunology
8.
Mol Immunol ; 66(2): 171-9, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25801305

ABSTRACT

The CD69 type II C-type lectin is one of the earliest indicators of leukocyte activation acting in lymphocyte migration and cytokine secretion. CD69 expression in hematopoietic lineage undergoes rapid changes depending on the cell-lineage, the activation state or the localization of the cell where it is expressed, suggesting a complex and tightly controlled regulation. Here we provide new insights on the transcriptional regulation of CD69 gene in mammal species. Through in silico studies, we analyzed several regulatory features of the 4 upstream conserved non-coding sequences (CNS 1-4) previously described, confirming a major function of CNS2 in the transcriptional regulation of CD69. In addition, multiple transcription binding sites are identified in the CNS2 region by DNA cross-species conservation analysis. By functional approaches we defined a core region of 226bp located within CNS2 as the main enhancer element of CD69 transcription in the hematopoietic cells analyzed. By chromatin immunoprecipitation, binding of RUNX1 to the core-CNS2 was shown in a T cell line. In addition, we found an activating but not essential role of RUNX1 in CD69 gene transcription by site-directed mutagenesis and RNA silencing, probably through the interaction with this potent enhancer specifically in the hematopoietic lineage. In summary, in this study we contribute with new evidences to the landscape of the transcriptional regulation of the CD69 gene.


Subject(s)
5' Flanking Region , Antigens, CD/genetics , Antigens, Differentiation, T-Lymphocyte/genetics , Core Binding Factor Alpha 2 Subunit/genetics , Enhancer Elements, Genetic , Gene Expression Regulation , Lectins, C-Type/genetics , Transcription, Genetic , Amino Acid Sequence , Animals , Antigens, CD/chemistry , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/chemistry , Antigens, Differentiation, T-Lymphocyte/metabolism , Binding Sites , Cell Line, Tumor , Conserved Sequence , Core Binding Factor Alpha 2 Subunit/chemistry , Core Binding Factor Alpha 2 Subunit/metabolism , Genes, Reporter , Humans , Jurkat Cells , K562 Cells , Lectins, C-Type/chemistry , Lectins, C-Type/metabolism , Luciferases/genetics , Luciferases/metabolism , Molecular Sequence Data , Protein Binding , Transfection , Transgenes
9.
PLoS One ; 7(10): e48593, 2012.
Article in English | MEDLINE | ID: mdl-23119065

ABSTRACT

CD69 is rapidly upregulated on T cells upon activation. In this work we show that this is also the case for CD69 expression on dendritic cells (DC). Thus, the expression kinetics of CD69 on both cell types is reminiscent of the one of costimulatory molecules. Using mouse models of transgenic T cells, we aimed at evaluating the effect of monoclonal antibody (MAb)-based targeting and gene deficiency of CD69 expressed by either DC or T cells on the extent of antigen (Ag)-specific T cell priming, which could be the result of a putative role in costimulation as well as on DC maturation and Ag-processing and presentation. CD69 targeting or deficiency of DC did not affect their expression of costimulatory molecules nor their capacity to induce Ag-specific T cell proliferation in in vitro assays. Also, CD69 targeting or deficiency of transgenic T cells did not affect the minimal proliferative dose for different peptide agonists in vitro. In in vivo models of transgenic T cell transfer and local Ag injection, CD69 deficiency of transferred T cells did not affect the extent of the proliferative response in Ag-draining lymph nodes (LN). In agreement with these results, CD69 MAb targeting or gene deficiency of Vaccinia-virus (VACV) infected mice did not affect the endogenous formation of virus-specific CD8(+) T cell populations at the peak of the primary immune response. Altogether our results argue against a possible role in costimulation or an effect on Ag processing and presentation for CD69.


Subject(s)
Antigen Presentation/immunology , Antigens, CD/immunology , Antigens, Differentiation, T-Lymphocyte/immunology , Dendritic Cells/immunology , Lectins, C-Type/immunology , T-Lymphocytes/immunology , Adoptive Transfer , Animals , Antigen Presentation/genetics , Antigens/immunology , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/genetics , Antigens, Differentiation, T-Lymphocyte/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Dendritic Cells/metabolism , Female , Flow Cytometry , Lectins, C-Type/genetics , Lectins, C-Type/metabolism , Lipopolysaccharides/immunology , Lipopolysaccharides/pharmacology , Lymph Nodes/immunology , Lymph Nodes/metabolism , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Oligodeoxyribonucleotides/immunology , Oligodeoxyribonucleotides/pharmacology , T-Lymphocytes/metabolism , Toll-Like Receptor 9/agonists , Up-Regulation/drug effects , Up-Regulation/immunology , Vaccinia/immunology , Vaccinia/virology , Vaccinia virus/immunology
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