Revistas
Revista:
GLIA
ISSN:
0894-1491
Año:
2023
Vol.:
71
N°:
3
Págs.:
571 - 587
Inflammation is a common feature in neurodegenerative diseases that contributes to neuronal loss. Previously, we demonstrated that the basal inflammatory tone differed between brain regions and, consequently, the reaction generated to a pro-inflammatory stimulus was different. In this study, we assessed the innate immune reaction in the midbrain and in the striatum using an experimental model of Parkinson's disease. An adeno-associated virus serotype 9 expressing the alpha-synuclein and mCherry genes or the mCherry gene was administered into the substantia nigra. Myeloid cells (CD11b(+)) and astrocytes (ACSA2(+)) were purified from the midbrain and striatum for bulk RNA sequencing. In the parkinsonian midbrain, CD11b(+) cells presented a unique anti-inflammatory transcriptomic profile that differed from degenerative microglia signatures described in experimental models for other neurodegenerative conditions. By contrast, striatal CD11b(+) cells showed a pro-inflammatory state and were similar to disease-associated microglia. In the midbrain, a prominent increase of infiltrated monocytes/macrophages was observed and, together with microglia, participated actively in the phagocytosis of dopaminergic neuronal bodies. Although striatal microglia presented a phagocytic transcriptomic profile, morphology and cell density was preserved and no active phagocytosis was detected. Interestingly, astrocytes presented a pro-inflammatory fingerprint in the midbrain and a low number of differentially displayed transcripts in the striatum. During alpha-synuclein-dependent degeneration, microglia and astrocytes experience context-dependent activation states with a different contribution to the inflammatory reaction. Our results point towards the relevance of selecting appropriate cell targets to design neuroprotective strategies aimed to modulate the innate immune system during the active phase of dopaminergic degeneration.
Revista:
FRONTIERS IN IMMUNOLOGY
ISSN:
1664-3224
Año:
2023
Vol.:
14
Págs.:
1270843
Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-IKO/TCRKO CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allows the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.
Revista:
ELIFE
ISSN:
2050-084X
Año:
2023
Vol.:
12
Págs.:
e79363
Early hematopoiesis is a continuous process in which hematopoietic stem and progenitor cells (HSPCs) gradually differentiate toward specific lineages. Aging and myeloid malignant transformation are characterized by changes in the composition and regulation of HSPCs. In this study, we used single-cell RNA sequencing (scRNA-seq) to characterize an enriched population of human HSPCs obtained from young and elderly healthy individuals.
Based on their transcriptional profile, we identified changes in the proportions of progenitor compartments during aging, and differences in their functionality, as evidenced by gene set enrichment analysis. Trajectory inference revealed that altered gene expression dynamics accompanied cell differentiation, which could explain aging-associated changes in hematopoiesis. Next, we focused on key regulators of transcription by constructing gene regulatory networks (GRNs) and detected regulons that were specifically active in elderly individuals. Using previous findings in healthy cells as a reference, we analyzed scRNA-seq data obtained from patients with myelodysplastic syndrome (MDS) and detected specific alterations of the expression dynamics of genes involved in erythroid differentiation in all patients with MDS such as TRIB2. In addition, the comparison between transcriptional programs and GRNs regulating normal HSPCs and MDS HSPCs allowed identification of regulons that were specifically active in MDS cases such as SMAD1, HOXA6, POU2F2, and RUNX1 suggesting a role of these transcription factors (TFs) in the pathogenesis of the disease.
In summary, we demonstrate that the combination of single-cell technologies with computational analysis tools enable the study of a variety of cellular mechanisms involved in complex biological systems such as early hematopoiesis and can be used to dissect perturbed differentiation trajectories associated with perturbations such as aging and malignant transformation. Furthermore, the identification of abnormal regulatory mechanisms associated with myeloid malignancies could be exploited for personalized therapeutic approaches in individual patients.
Revista:
SCIENCE ADVANCES
ISSN:
2375-2548
Año:
2022
Vol.:
8
N°:
39
Págs.:
eabo0514
Identification of new markers associated with long-term efficacy in patients treated with CAR T cells is a current medical need, particularly in diseases such as multiple myeloma. In this study, we address the impact of CAR density on the functionality of BCMA CAR T cells. Functional and transcriptional studies demonstrate that CAR T cells with high expression of the CAR construct show an increased tonic signaling with up-regulation of exhaustion markers and increased in vitro cytotoxicity but a decrease in in vivo BM infiltration. Characterization of gene regulatory networks using scRNA-seq identified regulons associated to activation and exhaustion up-regulated in CARHigh T cells, providing mechanistic insights behind differential functionality of these cells. Last, we demonstrate that patients treated with CAR T cell products enriched in CARHigh T cells show a significantly worse clinical response in several hematological malignancies. In summary, our work demonstrates that CAR density plays an important role in CAR T activity with notable impact on clinical response.
Revista:
PLANT CELL AND ENVIRONMENT
ISSN:
0140-7791
Año:
2022
Vol.:
45
N°:
5
Págs.:
1428 - 1441
Epigenetic regulation is necessary for optimal organism development and preservation of gene expression profiles in the cell. In plants, the trimethylation of histone H3 lysine 27 (H3K27me3) is a silencing epigenetic mark relevant for developmental transitions like flowering. The floral transition is a key agronomic trait; however, the epigenetic mechanisms of flowering time regulation in crops remain poorly understood. Here we study the Jumonji H3K27me3 demethylases BraA.REF6 and BraA.ELF6 in Brassica rapa. Phenotypic characterization of novel mutant lines and genome-wide H3K27me3 chromatin immunoprecipitation and transcriptomic analyses indicated that BraA.REF6 plays a greater role than BraA.ELF6 in fine-tuning H3K27me3 levels. In addition, we found that braA.elf6 mutants were early flowering due to high H3K27me3 levels at B. rapa homologs of the floral repressor FLC. Unlike mutations in Arabidopsis thaliana, braA.ref6 mutants were late flowering without altering the expression of B. rapa FLC genes. Remarkably, we found that BraA.REF6 regulated a number of gibberellic acid (GA) biosynthetic genes, including a homolog of GA1, and that GA-treatment complemented the late flowering mutant phenotype. This study increases our understanding of the epigenetic regulation of flowering time in B. rapa, highlighting conserved and distinct regulatory mechanisms between model and crop species.
Revista:
ISCIENCE
ISSN:
2589-0042
Año:
2022
Vol.:
25
N°:
5
Págs.:
104225
Understanding the regulation of normal and malignant human hematopoiesis requires comprehensive cell atlas of the hematopoietic stem cell (HSC) regulatory microenvironment. Here, we develop a tailored bioinformatic pipeline to integrate public and proprietary single-cell RNA sequencing (scRNA-seq) datasets. As a result, we robustly identify for the first time 14 intermediate cell states and 11 stages of differentiation in the endothelial and mesenchymal BM compartments, respectively. Our data provide the most comprehensive description to date of the murine HSC-regulatory microenvironment and suggest a higher level of specialization of the cellular circuits than previously anticipated. Furthermore, this deep characterization allows inferring conserved features in human, suggesting that the layers of microenvironmental regulation of hematopoiesis may also be shared between species. Our resource and methodology is a stepping-stone toward a comprehensive cell atlas of the BM microenvironment.
Revista:
MOLECULAR THERAPY. METHODS & CLINICAL DEVELOPMENT
ISSN:
2329-0501
Año:
2022
Vol.:
25
Págs.:
137 - 146
Genome-editing strategies, especially CRISPR-Cas9 systems, have substantially increased the efficiency of innovative therapeutic approaches for monogenic diseases such as primary hyperoxalurias (PHs). We have previously demonstrated that inhibition of glycolate oxidase using CRISPR-Cas9 systems represents a promising therapeutic option for PH type I (PH1). Here, we extended our work evaluating the efficacy of liver-specific inhibition of lactate dehydrogenase (LDH), a key enzyme responsible for converting glyoxylate to oxalate; this strategy would not be limited to PH1, being applicable to other PH subtypes. In this work, we demonstrate a liver-specific inhibition of LDH that resulted in a drastic reduction of LDH levels in the liver of PH1 and PH3 mice after a single-dose delivery of AAV8 vectors expressing the CRISPR-Cas9 system, resulting in reduced urine oxalate levels and kidney damage without signs of toxicity. Deep sequencing analysis revealed that this approach was safe and specific, with no off-targets detected in the liver of treated animals and no on-target/off-tissue events. Altogether, our data provide evidence that in vivo genome editing using CRISPR-Cas9 systems would represent a valuable tool for improved therapeutic approaches for PH.
Revista:
LEUKEMIA
ISSN:
0887-6924
Año:
2022
Vol.:
36
N°:
8
Págs.:
1969 - 1979
Eradicating leukemia requires a deep understanding of the interaction between leukemic cells and their protective microenvironment. The CXCL12/CXCR4 axis has been postulated as a critical pathway dictating leukemia stem cell (LSC) chemoresistance in AML due to its role in controlling cellular egress from the marrow. Nevertheless, the cellular source of CXCL12 in the acute myeloid leukemia (AML) microenvironment and the mechanism by which CXCL12 exerts its protective role in vivo remain unresolved. Here, we show that CXCL12 produced by Prx1+ mesenchymal cells but not by mature osteolineage cells provide the necessary cues for the maintenance of LSCs in the marrow of an MLL::AF9-induced AML model. Prx1+ cells promote survival of LSCs by modulating energy metabolism and the REDOX balance in LSCs. Deletion of Cxcl12 leads to the accumulation of reactive oxygen species and DNA damage in LSCs, impairing their ability to perpetuate leukemia in transplantation experiments, a defect that can be attenuated by antioxidant therapy. Importantly, our data suggest that this phenomenon appears to be conserved in human patients. Hence, we have identified Prx1+ mesenchymal cells as an integral part of the complex niche-AML metabolic intertwining, pointing towards CXCL12/CXCR4 as a target to eradicate parenchymal LSCs in AML.
Revista:
NATURE COMMUNICATIONS
ISSN:
2041-1723
Año:
2022
Vol.:
13
N°:
1
Págs.:
7619
Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis, with increased incidence in older individuals. Here we analyze the transcriptome of human HSCs purified from young and older healthy adults, as well as MDS patients, identifying transcriptional alterations following different patterns of expression. While aging-associated lesions seem to predispose HSCs to myeloid transformation, disease-specific alterations may trigger MDS development. Among MDS-specific lesions, we detect the upregulation of the transcription factor DNA Damage Inducible Transcript 3 (DDIT3). Overexpression of DDIT3 in human healthy HSCs induces an MDS-like transcriptional state, and dyserythropoiesis, an effect associated with a failure in the activation of transcriptional programs required for normal erythroid differentiation. Moreover, DDIT3 knockdown in CD34+ cells from MDS patients with anemia is able to restore erythropoiesis. These results identify DDIT3 as a driver of dyserythropoiesis, and a potential therapeutic target to restore the inefficient erythroid differentiation characterizing MDS patients. © 2022, The Author(s).
Revista:
LEUKEMIA
ISSN:
0887-6924
Año:
2021
Vol.:
35
N°:
5
Págs.:
1438 - 1450
Multiple myeloma (MM) is an incurable disease, whose clinical heterogeneity makes its management challenging, highlighting the need for biological features to guide improved therapies. Deregulation of specific long non-coding RNAs (lncRNAs) has been shown in MM, nevertheless, the complete lncRNA transcriptome has not yet been elucidated. In this work, we identified 40,511 novel lncRNAs in MM samples. lncRNAs accounted for 82% of the MM transcriptome and were more heterogeneously expressed than coding genes. A total of 10,351 overexpressed and 9,535 downregulated lncRNAs were identified in MM patients when compared with normal bone-marrow plasma cells. Transcriptional dynamics study of lncRNAs in the context of normal B-cell maturation revealed 989 lncRNAs with exclusive expression in MM, among which 89 showed de novo epigenomic activation. Knockdown studies on one of these lncRNAs, SMILO (specific myeloma intergenic long non-coding RNA), resulted in reduced proliferation and induction of apoptosis of MM cells, and activation of the interferon pathway. We also showed that the expression of lncRNAs, together with clinical and genetic risk alterations, stratified MM patients into several progression-free survival and overall survival groups. In summary, our global analysis of the lncRNAs transcriptome reveals the presence of specific lncRNAs associated with the biological and clinical behavior of the disease.
Revista:
JOURNAL OF EXTRACELLULAR VESICLES
ISSN:
2001-3078
Año:
2020
Vol.:
9
N°:
1
Págs.:
1729646
Peripheral arterial disease (PAD) is associated with a high risk of cardiovascular events and death and is postulated to be a critical socioeconomic cost in the future. Extracellular vesicles (EVs) have emerged as potential candidates for new biomarker discovery related to their protein and nucleic acid cargo. In search of new prognostic and therapeutic targets in PAD, we determined the prothrombotic activity, the cellular origin and the transcriptomic profile of circulating EVs. This prospective study included control and PAD patients. Coagulation time (Procoag-PPL kit), EVs cellular origin and phosphatidylserine exposure were determined by flow cytometry in platelet-free plasma (n = 45 PAD). Transcriptomic profiles of medium/large EVs were generated using the MARS-Seq RNA-Seq protocol (n = 12/group). The serum concentration of the differentially expressed gene S100A9, in serum calprotectin (S100A8/A9), was validated by ELISA in control (n = 100) and PAD patients (n = 317). S100A9 was also determined in EVs and tissues of human atherosclerotic plaques (n = 3). Circulating EVs of PAD patients were mainly of platelet origin, predominantly Annexin V positive and were associated with the procoagulant activity of platelet-free plasma. Transcriptomic analysis of EVs identified 15 differentially expressed genes. Among them, serum calprotectin was elevated in PAD patients (p < 0.05) and associated with increased amputation risk before and after covariate adjustment (mean follow-up 3.6 years, p < 0.01). The combination of calprotectin with hs-CRP in the multivariate analysis further improved risk stratification (p < 0.01). Furthermore, S100A9 was also expressed in femoral plaque derived EVs and tissues. In summary, we found that PAD patients release EVs, mainly of platelet origin, highly positive for AnnexinV and rich in transcripts related to platelet biology and immune responses. Amputation risk prediction improved with calprotectin and was significantly higher when combined with hs-CRP. Our results suggest that EVs can be a promising component of liquid biopsy to identify the molecular signature of PAD patients.
Revista:
CIRCULATION
ISSN:
0009-7322
Año:
2020
Vol.:
142
N°:
19
Págs.:
1831 - 1847
BACKGROUND: Cardiac fibroblasts (CFs) have a central role in the ventricular remodeling process associated with different types of fibrosis. Recent studies have shown that fibroblasts do not respond homogeneously to heart injury. Because of the limited set of bona fide fibroblast markers, a proper characterization of fibroblast population heterogeneity in response to cardiac damage is lacking. The purpose of this study was to define CF heterogeneity during ventricular remodeling and the underlying mechanisms that regulate CF function. METHODS: Collagen1 alpha 1-GFP (green fluorescent protein)-positive CFs were characterized after myocardial infarction (MI) by single-cell and bulk RNA sequencing, assay for transposase-accessible chromatin sequencing, and functional assays. Swine and patient samples were studied using bulk RNA sequencing. RESULTS: We identified and characterized a unique CF subpopulation that emerges after MI in mice. These activated fibroblasts exhibit a clear profibrotic signature, express high levels of Cthrc1 (collagen triple helix repeat containing 1), and localize into the scar. Noncanonical transforming growth factor-beta signaling and different transcription factors including SOX9 are important regulators mediating their response to cardiac injury. Absence of CTHRC1 results in pronounced lethality attributable to ventricular rupture. A population of CFs with a similar transcriptome was identified in a swine model of MI and in heart tissue from patients with MI and dilated cardiomyopathy. CONCLUSIONS: We report CF heterogeneity and their dynamics during the course of MI and redefine the CFs that respond to cardiac injury and participate in myocardial remodeling. Our study identifies CTHRC1 as a novel regulator of the healing scar process and a target for future translational studies.
Revista:
GIGASCIENCE
ISSN:
2047-217X
Año:
2019
Vol.:
8
N°:
12
Págs.:
giz147
BACKGROUND:
Genome-wide maps of histone modifications have been obtained for several plant species. However, most studies focus on model systems and do not enforce FAIR data management principles. Here we study the H3K27me3 epigenome and associated transcriptome of Brassica rapa, an important vegetable cultivated worldwide.
FINDINGS:
We performed H3K27me3 chromatin immunoprecipitation followed by high-throughput sequencing and transcriptomic analysis by 3'-end RNA sequencing from B. rapa leaves and inflorescences. To analyze these data we developed a Reproducible Epigenomic Analysis pipeline using Galaxy and Jupyter, packaged into Docker images to facilitate transparency and reuse. We found that H3K27me3 covers roughly one-third of all B. rapa protein-coding genes and its presence correlates with low transcript levels. The comparative analysis between leaves and inflorescences suggested that the expression of various floral regulatory genes during development depends on H3K27me3. To demonstrate the importance of H3K27me3 for B. rapa development, we characterized a mutant line deficient in the H3K27 methyltransferase activity. We found that braA.clf mutant plants presented pleiotropic alterations, e.g., curly leaves due to increased expression and reduced H3K27me3 levels at AGAMOUS-like loci.
CONCLUSIONS:
We characterized the epigenetic mark H3K27me3 at genome-wide levels and provide genetic evidence for its relevance in B. rapa development. Our work reveals the epigenomic landscape of H3K27me3 in B. rapa and provides novel genomics datasets and bioinformatics analytical resources. We anticipate that this work will lead the way to further epigenomic studies in the complex genome of Brassica crops.
Revista:
CANCER RESEARCH
ISSN:
0008-5472
Año:
2019
Vol.:
79
N°:
3
Págs.:
625 - 638
Because of the refractory nature of mutant KRAS lung adenocarcinoma (LUAD) to current therapies, identification of new molecular targets is essential. Genes with a prognostic role in mutant KRAS LUAD have proven to be potential molecular targets for therapeutic development. Here we determine the clinical, functional, and mechanistic role of inhibitor of differentiation-1 (Id1) in mutant KRAS LUAD. Analysis of LUAD cohorts from TCGA and SPORE showed that high expression of Id1 was a marker of poor survival in patients harboring mutant, but not wild-type KRAS. Abrogation of Id1 induced G(2)-M arrest and apoptosis in mutant KRAS LUAD cells. In vivo, loss of Id1 strongly impaired tumor growth and maintenance as well as liver metastasis, resulting in improved survival. Mechanistically, Id1 was regulated by the KRAS oncogene through JNK, and loss of Id1 resulted in down-regulation of elements of the mitotic machinery via inhibition of the transcription factor FOSL1 and of several kinases within the KRAS signaling network. Our study provides clinical, functional, and mechanistic evidence underscoring Id1 as a critical gene in mutant KRAS LUAD and warrants further studies of Id1 as a therapeutic target in patients with LUAD.
Significance: These findings highlight the prognostic significance of the transcriptional regulator Id1 in KRAS-mutant lung adenocarcinoma and provide mechanistic insight into how it controls tumor growth and metastasis.
Revista:
JOURNAL OF NEUROINFLAMMATION
ISSN:
1742-2094
Año:
2019
Vol.:
16
N°:
1
Págs.:
233
Background Inflammation is a critical process for the progression of neuronal death in neurodegenerative disorders. Microglia play a central role in neuroinflammation and may affect neuron vulnerability. Next generation sequencing has shown the molecular heterogeneity of microglial cells; however, the variability in their response to pathological inputs remains unknown. Methods To determine the effect of an inflammatory stimulus on microglial cells, lipopolysaccharide (LPS) was administered peripherally to mice and the inflammatory status of the cortex, hippocampus, midbrain, and striatum was assessed. Microglial activation and interaction with the immune system were analyzed in single cell suspensions obtained from the different brain regions by fluorescence-activated cell sorting, next generation RNA sequencing, real-time PCR, and immunohistochemical techniques. Antigen-presenting properties of microglia were evaluated by the ability of isolated cells to induce a clonal expansion of CD4(+) T cells purified from OT-II transgenic mice. Results Under steady-state conditions, the midbrain presented a high immune-alert state characterized by the presence of two unique microglial subpopulations, one expressing the major histocompatibility complex class II (MHC-II) and acting as antigen-presenting cells and another expressing the toll-like receptor 4 (TLR4), and by the presence of a higher proportion of infiltrating CD4(+) T cells. This state was not detected in the cortex, hippocampus, or striatum. Systemic LPS administration induced a general increase in classic pro-inflammatory cytokines, in co-inhibitory programmed death ligand 1 (PD-L1), and in cytotoxic T lymphocyte antigen 4 (CTLA-4) receptors, as well as a decrease in infiltrating effector T cells in all brain regions. Interestingly, a specific immune-suppressive response was observed in the midbrain which was characterized by the downregulation of MHC-II microglial expression, the upregulation of the anti-inflammatory cytokines IL10 and TGF beta, and the increase in infiltrating regulatory T cells. Conclusions These data show that the midbrain presents a high immune-alert state under steady-state conditions that elicits a specific immune-suppressive response when exposed to an inflammatory stimulus. This specific inflammatory tone and response may have an impact in neuronal viability.