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1.
Ann Rheum Dis ; 2024 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-38609158

RESUMEN

INTRODUCTION: Systemic lupus erythematosus with antiphospholipid syndrome (SLE-APS) represents a challenging SLE endotype whose molecular basis remains unknown. METHODS: We analysed whole-blood RNA-sequencing data from 299 patients with SLE (108 SLE-antiphospholipid antibodies (aPL)-positive, including 67 SLE-APS; 191 SLE-aPL-negative) and 72 matched healthy controls (HC). Pathway enrichment analysis, unsupervised weighted gene coexpression network analysis and machine learning were applied to distinguish disease endotypes. RESULTS: Patients with SLE-APS demonstrated upregulated type I and II interferon (IFN) pathways compared with HC. Using a 100-gene random forests model, we achieved a cross-validated accuracy of 75.6% in distinguishing these two states. Additionally, the comparison between SLE-APS and SLE-aPL-negative revealed 227 differentially expressed genes, indicating downregulation of IFN-α and IFN-γ signatures, coupled with dysregulation of the complement cascade, B-cell activation and neutrophil degranulation. Unsupervised analysis of SLE transcriptome identified 21 gene modules, with SLE-APS strongly linked to upregulation of the 'neutrophilic/myeloid' module. Within SLE-APS, venous thromboses positively correlated with 'neutrophilic/myeloid' and 'B cell' modules, while arterial thromboses were associated with dysregulation of 'DNA damage response (DDR)' and 'metabolism' modules. Anticardiolipin and anti-ß2GPI positivity-irrespective of APS status-were associated with the 'neutrophilic/myeloid' and 'protein-binding' module, respectively. CONCLUSIONS: There is a hierarchical upregulation and-likely-dependence on IFN in SLE with the highest IFN signature observed in SLE-aPL-negative patients. Venous thrombotic events are associated with neutrophils and B cells while arterial events with DDR and impaired metabolism. This may account for their differential requirements for anticoagulation and provide rationale for the potential use of mTOR inhibitors such as sirolimus and the direct fIIa inhibitor dabigatran in SLE-APS.

2.
Ann Rheum Dis ; 82(5): 646-657, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36898766

RESUMEN

INTRODUCTION: Inflammatory mediators are detected in the cerebrospinal fluid of systemic lupus erythematosus patients with central nervous system involvement (NPSLE), yet the underlying cellular and molecular mechanisms leading to neuropsychiatric disease remain elusive. METHODS: We performed a comprehensive phenotyping of NZB/W-F1 lupus-prone mice including tests for depression, anxiety and cognition. Immunofluorescence, flow cytometry, RNA-sequencing, qPCR, cytokine quantification and blood-brain barrier (BBB) permeability assays were applied in hippocampal tissue obtained in both prenephritic (3-month-old) and nephritic (6-month-old) lupus mice and matched control strains. Healthy adult hippocampal neural stem cells (hiNSCs) were exposed ex vivo to exogenous inflammatory cytokines to assess their effects on proliferation and apoptosis. RESULTS: At the prenephritic stage, BBB is intact yet mice exhibit hippocampus-related behavioural deficits recapitulating the human diffuse neuropsychiatric disease. This phenotype is accounted by disrupted hippocampal neurogenesis with hiNSCs exhibiting increased proliferation combined with decreased differentiation and increased apoptosis in combination with microglia activation and increased secretion of proinflammatory cytokines and chemokines. Among these cytokines, IL-6 and IL-18 directly induce apoptosis of adult hiNSCs ex vivo. During the nephritic stage, BBB becomes disrupted which facilitates immune components of peripheral blood, particularly B-cells, to penetrate into the hippocampus further augmenting inflammation with locally increased levels of IL-6, IL-12, IL-18 and IL-23. Of note, an interferon gene signature was observed only at nephritic-stage. CONCLUSION: An intact BBB with microglial activation disrupting the formation of new neurons within the hippocampus represent early events in NPSLE. Disturbances of the BBB and interferon signature are evident later in the course of the disease.


Asunto(s)
Lupus Eritematoso Sistémico , Vasculitis por Lupus del Sistema Nervioso Central , Adulto , Humanos , Ratones , Animales , Lactante , Barrera Hematoencefálica , Interleucina-6 , Interleucina-18 , Microglía , Citocinas , Neurogénesis , Interferones , Hipocampo
3.
Ann Rheum Dis ; 81(10): 1409-1419, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35906002

RESUMEN

OBJECTIVES: Patients with lupus nephritis (LN) are in urgent need for early diagnosis and therapeutic interventions targeting aberrant molecular pathways enriched in affected kidneys. METHODS: We used mRNA-sequencing in effector (spleen) and target (kidneys, brain) tissues from lupus and control mice at sequential time points, and in the blood from 367 individuals (261 systemic lupus erythematosus (SLE) patients and 106 healthy individuals). Comparative cross-tissue and cross-species analyses were performed. The human dataset was split into training and validation sets and machine learning was applied to build LN predictive models. RESULTS: In murine SLE, we defined a kidney-specific molecular signature, as well as a molecular signature that underlies transition from preclinical to overt disease and encompasses pathways linked to metabolism, innate immune system and neutrophil degranulation. The murine kidney transcriptome partially mirrors the blood transcriptome of patients with LN with 11 key transcription factors regulating the cross-species active LN molecular signature. Integrated protein-to-protein interaction and drug prediction analyses identified the kinases TRRAP, AKT2, CDK16 and SCYL1 as putative targets of these factors and capable of reversing the LN signature. Using murine kidney-specific genes as disease predictors and machine-learning training of the human RNA-sequencing dataset, we developed and validated a peripheral blood-based algorithm that discriminates LN patients from normal individuals (based on 18 genes) and non-LN SLE patients (based on 20 genes) with excellent sensitivity and specificity (area under the curve range from 0.80 to 0.99). CONCLUSIONS: Machine-learning analysis of a large whole blood RNA-sequencing dataset of SLE patients using human orthologs of mouse kidney-specific genes can be used for early, non-invasive diagnosis and therapeutic targeting of LN. The kidney-specific gene predictors may facilitate prevention and early intervention trials.


Asunto(s)
Lupus Eritematoso Sistémico , Nefritis Lúpica , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Proteínas de Unión al ADN/genética , Diagnóstico Precoz , Perfilación de la Expresión Génica , Humanos , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/genética , Nefritis Lúpica/diagnóstico , Nefritis Lúpica/tratamiento farmacológico , Nefritis Lúpica/genética , Ratones , ARN
4.
Ann Rheum Dis ; 79(2): 242-253, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31780527

RESUMEN

OBJECTIVES: Haematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to both myeloid and lymphoid cell lineages. We reasoned that the aberrancies of immune cells in systemic lupus erythematosus (SLE) could be traced back to HSPCs. METHODS: A global gene expression map of bone marrow (BM)-derived HSPCs was completed by RNA sequencing followed by pathway and enrichment analysis. The cell cycle status and apoptosis status of HSPCs were assessed by flow cytometry, while DNA damage was assessed via immunofluorescence. RESULTS: Transcriptomic analysis of Lin-Sca-1+c-Kit+ haematopoietic progenitors from diseased lupus mice demonstrated a strong myeloid signature with expanded frequencies of common myeloid progenitors (CMPs)-but not of common lymphoid progenitors-reminiscent of a 'trained immunity' signature. CMP profiling revealed an intense transcriptome reprogramming with suppression of granulocytic regulators indicative of a differentiation arrest with downregulation trend of major regulators such as Cebpe, Cebpd and Csf3r, and disturbed myelopoiesis. Despite the differentiation arrest, frequencies of BM neutrophils were markedly increased in diseased mice, suggesting an alternative granulopoiesis pathway. In patients with SLE with severe disease, haematopoietic progenitor cells (CD34+) demonstrated enhanced proliferation, cell differentiation and transcriptional activation of cytokines and chemokines that drive differentiation towards myelopoiesis, thus mirroring the murine data. CONCLUSIONS: Aberrancies of immune cells in SLE can be traced back to the BM HSPCs. Priming of HSPCs and aberrant regulation of myelopoiesis may contribute to inflammation and risk of flare. TRIAL REGISTRATION NUMBER: 4948/19-07-2016.


Asunto(s)
Reprogramación Celular/inmunología , Células Madre Hematopoyéticas/inmunología , Lupus Eritematoso Sistémico/inmunología , Células Mieloides/inmunología , Transcriptoma/inmunología , Animales , Apoptosis/inmunología , Proteína delta de Unión al Potenciador CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Ciclo Celular/inmunología , Mapeo Cromosómico , Daño del ADN , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Factor Estimulante de Colonias de Granulocitos/metabolismo , Granulocitos/inmunología , Linfocitos/inmunología , Ratones
5.
Front Immunol ; 15: 1383358, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38779657

RESUMEN

Introduction: Immune cells that contribute to the pathogenesis of systemic lupus erythematosus (SLE) derive from adult hematopoietic stem and progenitor cells (HSPCs) within the bone marrow (BM). For this reason, we reasoned that fundamental abnormalities in SLE can be traced to a BM-derived HSPC inflammatory signature. Methods: BM samples from four SLE patients, six healthy controls, and two umbilical cord blood (CB) samples were used. CD34+ cells were isolated from BM and CB samples, and single-cell RNA-sequencing was performed. Results: A total of 426 cells and 24,473 genes were used in the analysis. Clustering analysis resulted in seven distinct clusters of cell types. Mutually exclusive markers, which were characteristic of each cell type, were identified. We identified three HSPC subpopulations, one of which consisted of proliferating cells (MKI67 expressing cells), one T-like, one B-like, and two myeloid-like progenitor subpopulations. Differential expression analysis revealed i) cell cycle-associated signatures, in healthy BM of HSPC clusters 3 and 4 when compared with CB, and ii) interferon (IFN) signatures in SLE BM of HSPC clusters 3 and 4 and myeloid-like progenitor cluster 5 when compared with healthy controls. The IFN signature in SLE appeared to be deregulated following TF regulatory network analysis and differential alternative splicing analysis between SLE and healthy controls in HSPC subpopulations. Discussion: This study revealed both quantitative-as evidenced by decreased numbers of non-proliferating early progenitors-and qualitative differences-characterized by an IFN signature in SLE, which is known to drive loss of function and depletion of HSPCs. Chronic IFN exposure affects early hematopoietic progenitors in SLE, which may account for the immune aberrancies and the cytopenias in SLE.


Asunto(s)
Perfilación de la Expresión Génica , Células Madre Hematopoyéticas , Interferones , Lupus Eritematoso Sistémico , Análisis de la Célula Individual , Transcriptoma , Humanos , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Células Madre Hematopoyéticas/metabolismo , Interferones/metabolismo , Interferones/genética , Femenino , Adulto , Reprogramación Celular/genética , Masculino
6.
Lupus Sci Med ; 11(1)2024 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-38471723

RESUMEN

OBJECTIVES: In SLE, deregulation of haematopoiesis is characterised by inflammatory priming and myeloid skewing of haematopoietic stem and progenitor cells (HSPCs). We sought to investigate the role of extramedullary haematopoiesis (EMH) as a key player for tissue injury in systemic autoimmune disorders. METHODS: Transcriptomic analysis of bone marrow (BM)-derived HSPCs from patients with SLE and NZBW/F1 lupus-prone mice was performed in combination with DNA methylation profile. Trained immunity (TI) was induced through ß-glucan administration to the NZBW/F1 lupus-prone model. Disease activity was assessed through lupus nephritis (LN) histological grading. Colony-forming unit assay and adoptive cell transfer were used to assess HSPCs functionalities. RESULTS: Transcriptomic analysis shows that splenic HSPCs carry a higher inflammatory potential compared with their BM counterparts. Further induction of TI, through ß-glucan administration, exacerbates splenic EMH, accentuates myeloid skewing and worsens LN. Methylomic analysis of BM-derived HSPCs demonstrates myeloid skewing which is in part driven by epigenetic tinkering. Importantly, transcriptomic analysis of human SLE BM-derived HSPCs demonstrates similar findings to those observed in diseased mice. CONCLUSIONS: These data support a key role of granulocytes derived from primed HSPCs both at medullary and extramedullary sites in the pathogenesis of LN. EMH and TI contribute to SLE by sustaining the systemic inflammatory response and increasing the risk for flare.


Asunto(s)
Lupus Eritematoso Sistémico , Nefritis Lúpica , beta-Glucanos , Humanos , Animales , Ratones , Hematopoyesis , Células Madre Hematopoyéticas
7.
Front Immunol ; 14: 1203848, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37475860

RESUMEN

Objective: A blood-based biomarker is needed to assess lupus nephritis (LN) disease activity, minimizing the need for invasive kidney biopsies. Long non-coding RNAs (lncRNAs) are known to regulate gene expression, appear to be stable in human plasma, and can serve as non-invasive biomarkers. Methods: Transcriptomic data of whole blood samples from 74 LN patients and 20 healthy subjects (HC) were analyzed to identify differentially expressed (DE) lncRNAs associated with quiescent disease and flares. Weighted gene co-expression network analysis (WGCNA) was performed to uncover lncRNAs with a central role (hub lncRNAs) in regulating key biological processes that drive LN disease activity. The association of hub lncRNAs with disease activity was validated using RT-qPCR on an independent cohort of 15 LN patients and 9 HC. cis- and trans-targets of validated lncRNAs were explored in silico to examine potential mechanisms of their action. Results: There were 444 DE lncRNAs associated with quiescent disease and 6 DE lncRNAs associated with flares (FDR <0.05). WGCNA highlighted IFN signaling and B-cell activity/adaptive immunity as the most significant processes contributing to nephritis activity. Four disease-activity-associated lncRNAs, namely, NRIR, KLHDC7B-DT, MIR600HG, and FAM30A, were detected as hub genes and validated in an independent cohort. NRIR and KLHDC7B-DT emerged as potential key regulators of IFN-mediated processes. Network analysis suggests that FAM30A and MIR600HG are likely to play a central role in the regulation of B-cells in LN through cis-regulation effects and a competing endogenous RNA mechanism affecting immunoglobulin gene expression and the IFN-λ pathway. Conclusions: The expression of lncRNAs NRIR, KLHDC7B-DT, FAM30A, and MIR600HG were associated with disease activity and could be further explored as blood-based biomarkers and potential liquid biopsy on LN.


Asunto(s)
Nefritis Lúpica , ARN Largo no Codificante , Humanos , Nefritis Lúpica/diagnóstico , Nefritis Lúpica/genética , Biomarcadores , Perfilación de la Expresión Génica , Biopsia Líquida
8.
Front Immunol ; 14: 1072598, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37051253

RESUMEN

Introduction: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) present with a complex phenotype and are associated with high mortality and multi-organ involvement. We sought to define the transcriptional landscape and molecular endotypes of AAVs and compare it to systemic lupus erythematosus (SLE). Methods: We performed whole blood mRNA sequencing from 30 patients with AAV (granulomatosis with polyangiitis/GPA and microscopic polyangiitis/MPA) combined with functional enrichment and network analysis for aberrant pathways. Key genes and pathways were validated in an independent cohort of 18 AAV patients. Co-expression network and hierarchical clustering analysis, identified molecular endotypes. Multi-level transcriptional overlap analysis to SLE was based on our published data from 142 patients. Results: We report here that "Pan-vasculitis" signature contained 1,982 differentially expressed genes, enriched in leukocyte differentiation, cytokine signaling, type I and type II IFN signaling and aberrant B-T cell immunity. Active disease was characterized by signatures linked to cell cycle checkpoints and metabolism pathways, whereas ANCA-positive patients exhibited a humoral immunity transcriptional fingerprint. Differential expression analysis of GPA and MPA yielded an IFN-g pathway (in addition to a type I IFN) in the former and aberrant expression of genes related to autophagy and mRNA splicing in the latter. Unsupervised molecular taxonomy analysis revealed four endotypes with neutrophil degranulation, aberrant metabolism and B-cell responses as potential mechanistic drivers. Transcriptional perturbations and molecular heterogeneity were more pronounced in SLE. Molecular analysis and data-driven clustering of AAV uncovered distinct transcriptional pathways that could be exploited for targeted therapy. Discussion: We conclude that transcriptomic analysis of AAV reveals distinct endotypes and molecular pathways that could be targeted for therapy. The AAV transcriptome is more homogenous and less fragmented compared to the SLE which may account for its superior rates of response to therapy.


Asunto(s)
Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos , Lupus Eritematoso Sistémico , Humanos , Anticuerpos Anticitoplasma de Neutrófilos , Lupus Eritematoso Sistémico/genética , Genómica , ARN Mensajero
9.
Front Immunol ; 14: 1257321, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38022524

RESUMEN

Chronic inflammatory diseases (CIDs), including inflammatory bowel disease (IBD), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) are thought to emerge from an impaired complex network of inter- and intracellular biochemical interactions among several proteins and small chemical compounds under strong influence of genetic and environmental factors. CIDs are characterised by shared and disease-specific processes, which is reflected by partially overlapping genetic risk maps and pathogenic cells (e.g., T cells). Their pathogenesis involves a plethora of intracellular pathways. The translation of the research findings on CIDs molecular mechanisms into effective treatments is challenging and may explain the low remission rates despite modern targeted therapies. Modelling CID-related causal interactions as networks allows us to tackle the complexity at a systems level and improve our understanding of the interplay of key pathways. Here we report the construction, description, and initial applications of the SYSCID map (https://syscid.elixir-luxembourg.org/), a mechanistic causal interaction network covering the molecular crosstalk between IBD, RA and SLE. We demonstrate that the map serves as an interactive, graphical review of IBD, RA and SLE molecular mechanisms, and helps to understand the complexity of omics data. Examples of such application are illustrated using transcriptome data from time-series gene expression profiles following anti-TNF treatment and data from genome-wide associations studies that enable us to suggest potential effects to altered pathways and propose possible mechanistic biomarkers of treatment response.


Asunto(s)
Artritis Reumatoide , Enfermedades Inflamatorias del Intestino , Lupus Eritematoso Sistémico , Humanos , Inhibidores del Factor de Necrosis Tumoral , Artritis Reumatoide/etiología , Artritis Reumatoide/genética , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/genética , Resultado del Tratamiento , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/genética
10.
Front Immunol ; 13: 860726, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35615355

RESUMEN

Objectives: Treatment of Systemic Lupus Erythematosus (SLE) is characterized by a largely empirical approach and relative paucity of novel compound development. We sought to stratify SLE patients based on their molecular phenotype and identify putative therapeutic compounds for each molecular fingerprint. Methods: By the use of whole blood RNA-seq data from 120 SLE patients, and in a data-driven, clinically unbiased manner, we established modules of commonly regulated genes (molecular endotypes) and re-stratified patients through hierarchical clustering. Disease activity and severity were assessed using SLEDAI-2K and Lupus Severity Index, respectively. Through an in silico drug prediction pipeline, we investigated drugs currently in use, tested in lupus clinical trials, and listed in the iLINCS prediction databases, for their ability to reverse the gene expression signatures in each molecular endotype. Drug repurposing analysis was also performed to identify perturbagens that counteract group-specific SLE signatures. Results: Molecular taxonomy identified five lupus endotypes, each characterized by a unique gene module enrichment pattern. Neutrophilic signature group consisted primarily of patients with active lupus nephritis, while the B-cell expression group included patients with constitutional features. Patients with moderate severity and serologic activity exhibited a signature enriched for metabolic processes. Mild disease was distributed in two groups, exhibiting enhanced basic cellular functions, myelopoiesis, and autophagy. Bortezomib was predicted to reverse disturbances in the "neutrophilic" cluster, azathioprine and ixazomib in the "B-cell" cluster, and fostamatinib in the "metabolic" patient subgroup. Conclusion: The clinical spectrum of SLE encompasses distinct molecular endotypes, each defined by unique pathophysiologic aberrancies potentially reversible by distinct compounds.


Asunto(s)
Lupus Eritematoso Sistémico , Nefritis Lúpica , Linfocitos B/metabolismo , Análisis por Conglomerados , Humanos , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/genética , Índice de Severidad de la Enfermedad
11.
Front Immunol ; 13: 964274, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36159832

RESUMEN

Background: An interplay between immune cells and resident skin and joint stromal cells is implicated in psoriatic arthritis (PsA), yet the mechanisms remain elusive with a paucity of molecular biomarkers for activity and response. Combined transcriptomic and immunophenotypic analysis of whole blood and skin fibroblasts could provide further insights. Methods: Whole blood RNA-seq was performed longitudinally in 30 subjects with PsA at the beginning, one and six months after treatment, with response defined at six months. As control groups, 10 healthy individuals and 10 subjects with rheumatoid arthritis (RA) were recruited combined with public datasets from patients with psoriasis (PsO) and systemic lupus erythematous (SLE). Differential expression analysis and weighted gene co-expression network analysis were performed to identify gene expression signatures, while deconvolution and flow cytometry to characterize the peripheral blood immune cell profile. In a subset of affected and healthy individuals, RNA-seq of skin fibroblasts was performed and subjected to CellChat analysis to identify the blood-skin fibroblast interaction network. Results: PsA demonstrated a distinct "activity" gene signature in the peripheral blood dominated by TNF- and IFN-driven inflammation, deregulated cholesterol and fatty acid metabolism and expansion of pro-inflammatory non-classical monocytes. Comparison with the blood transcriptome of RA, PsO, and SLE revealed a "PsA-specific signature" enriched in extracellular matrix remodeling. This was further supported by the skin fibroblast gene expression profile, displaying an activated, proliferating phenotype, and by skin-blood interactome analysis revealing interactions with circulating immune cells through WNT, PDGF and immune-related semaphorins. Of note, resistance to treatment was associated with upregulation of genes involved in TGFß signaling and angiogenesis and persistent increase of non-classical monocytes. Differentially expressed genes related to platelet activation and hippo signaling discriminated responders and non-responders as early as one month after treatment initiation. Conclusion: Transcriptome analysis of peripheral blood and skin fibroblasts in PsA reveals a distinct disease activity signature and supports the involvement of skin fibroblasts through their activation and interaction with circulating immune cells. Aberrant TGFß signaling and persistently increased non-classical monocytes characterize treatment-resistant PsA, with pro-inflammatory pathways related to platelet activation and Hippo signaling predicting early response to treatment.


Asunto(s)
Artritis Psoriásica , Artritis Reumatoide , Lupus Eritematoso Sistémico , Psoriasis , Semaforinas , Artritis Psoriásica/tratamiento farmacológico , Artritis Psoriásica/genética , Biomarcadores/metabolismo , Ácidos Grasos/metabolismo , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Humanos , Lupus Eritematoso Sistémico/metabolismo , Psoriasis/metabolismo , Semaforinas/metabolismo , Transcriptoma , Factor de Crecimiento Transformador beta/metabolismo
12.
Sci Adv ; 8(43): eabo5840, 2022 10 28.
Artículo en Inglés | MEDLINE | ID: mdl-36306362

RESUMEN

B cells orchestrate autoimmune responses in patients with systemic lupus erythematosus (SLE), but broad-based B cell-directed therapies show only modest efficacy while blunting humoral immune responses to vaccines and inducing immunosuppression. Development of more effective therapies targeting pathogenic clones is a currently unmet need. Here, we demonstrate enhanced activation of the ATR/Chk1 pathway of the DNA damage response (DDR) in B cells of patients with active SLE disease. Treatment of B cells with type I IFN, a key driver of immunity in SLE, induced expression of ATR via binding of interferon regulatory factor 1 to its gene promoter. Pharmacologic targeting of ATR in B cells, via a specific inhibitor (VE-822), attenuated their immunogenic profile, including proinflammatory cytokine secretion, plasmablast formation, and antibody production. Together, these findings identify the ATR-mediated DDR axis as the orchestrator of the type I IFN-mediated B cell responses in SLE and as a potential novel therapeutic target.


Asunto(s)
Lupus Eritematoso Sistémico , Humanos , Lupus Eritematoso Sistémico/metabolismo , Linfocitos B , Daño del ADN , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo
13.
Sci Rep ; 11(1): 15759, 2021 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-34344937

RESUMEN

Systemic lupus erythematosus (SLE) is an autoimmune disease where bone-marrow-derived haematopoietic cells have a key role in its pathogenesis with accumulating evidence suggesting an aberrant function of haematopoietic stem/progenitor cells (HSPCs). We examined whether patrolling HSPCs differ from bone-marrow HSPCs both in SLE and healthy individuals, and how they participate in peripheral tissue injury. By employing next-generation RNA sequencing, the transcriptomes of CD34+ HSPCs deriving from the bone marrow and those patrolling the bloodstream of both healthy and individuals with SLE were compared. Patrolling SLE and Healthy human HSPC kinetics were examined through their inoculation into humanised mice. Patrolling and bone-marrow HSPCs have distinct molecular signatures, while patrolling SLE HSPCs showed an enhanced extramedullary gene expression profile. Non-mobilised, SLE-derived circulating HSPCs demonstrated altered homing capacities. Xenotransplantation of circulating HSPCs in humanised mice showed that human peripheral blood HSPCs possess the ability for extramedullary organ colonisation to the kidneys. Circulating and bone marrow-derived HSPCs are distinct in steady and diseased states. Patrolling SLE CD34+ HSPCs are able to home at extramedullary sites such as the spleen and kidneys, potentially participating in peripheral tissue injury.


Asunto(s)
Médula Ósea/patología , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/patología , Riñón/patología , Lupus Eritematoso Sistémico/patología , Bazo/patología , Transcriptoma , Adulto , Anciano , Animales , Médula Ósea/metabolismo , Estudios de Casos y Controles , Femenino , Células Madre Hematopoyéticas/metabolismo , Humanos , Riñón/lesiones , Riñón/metabolismo , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Persona de Mediana Edad , Bazo/lesiones , Bazo/metabolismo
14.
Blood Adv ; 5(1): 129-142, 2021 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-33570632

RESUMEN

CD4+ T cells orchestrate immune responses and are actively engaged in shaping tumor immunity. Signal transducer and activator of transcription (STAT) signaling controls the epigenetic tuning of CD4+ T-cell differentiation and polarization, and perturbed STAT signaling networks in CD4+ T cells subvert antitumor immunity in malignancies. Azacitidine (AZA), the mainstay therapy for high-risk myelodysplastic syndromes (HR-MDS), affects CD4+ T-cell polarization and function, but whether this contributes to AZA efficacy is currently unknown. By using functional proteomic, transcriptomic, and mutational analyses in 73 HR-MDS patients undergoing AZA therapy, we demonstrate that responding patients exhibited a coordinated CD4+ T-cell immune response and downregulated the inflammatory cytokine signaling pathways in CD4+ T cells after AZA, in contrast to nonresponders who upregulated the same pathways. We further observed an AZA-mediated downregulation of intereukin-6 (IL-6)-induced STAT3 phosphorylation in CD4+FOXP3- conventional T cells (Tcons) that correlated independently with better response and survival, whereas it was also not associated with the mutation number and profile of the patients. The AZA-induced downregulation of IL-6/STAT3 axis in Tcons restored the STAT signaling architecture in CD4+ T-cell subsets, whereas STAT signaling networks remained disorganized in patients who upregulated IL-6/STAT3 activity in Tcons. Given the pivotal role of CD4+ T cells in adaptive immunity, our findings suggest that the downregulation of the IL-6/STAT3 pathway in Tcons potentially constitutes a previously unrecognized immune-mediated mechanism of action of AZA and sets the scene for developing rational strategies of AZA combinations with IL-6/STAT3 axis inhibitors.


Asunto(s)
Azacitidina , Interleucina-6 , Azacitidina/farmacología , Linfocitos T CD4-Positivos , Factores de Transcripción Forkhead , Humanos , Interleucina-6/genética , Proteómica , Factor de Transcripción STAT3 , Transducción de Señal
15.
Cell Metab ; 32(4): 591-604.e7, 2020 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-32738205

RESUMEN

Regulatory T cells (Tregs) are vital for the maintenance of immune homeostasis, while their dysfunction constitutes a cardinal feature of autoimmunity. Under steady-state conditions, mitochondrial metabolism is critical for Treg function; however, the metabolic adaptations of Tregs during autoimmunity are ill-defined. Herein, we report that elevated mitochondrial oxidative stress and a robust DNA damage response (DDR) associated with cell death occur in Tregs in individuals with autoimmunity. In an experimental autoimmune encephalitis (EAE) mouse model of autoimmunity, we found a Treg dysfunction recapitulating the features of autoimmune Tregs with a prominent mtROS signature. Scavenging of mtROS in Tregs of EAE mice reversed the DDR and prevented Treg death, while attenuating the Th1 and Th17 autoimmune responses. These findings highlight an unrecognized role of mitochondrial oxidative stress in defining Treg fate during autoimmunity, which may facilitate the design of novel immunotherapies for diseases with disturbed immune tolerance.


Asunto(s)
Autoinmunidad/inmunología , Mitocondrias/inmunología , Linfocitos T Reguladores/inmunología , Animales , Línea Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción
16.
Mediterr J Rheumatol ; 31(3): 366-368, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-33163873

RESUMEN

ANCA-associated vasculitides (AAVs) are characterised by heterogeneous molecular and pathophysiological traits, causing ambiguous differential diagnosis and taxonomy. Response to therapy has proven far from successful, contributing to high mortality. Transcriptome analysis of different vasculitis subtypes adds new leads in elucidating mechanisms of disease and the role of specific cell subsets to them. Recent findings have shown that mitophagy is a procedure whose imbalance could lead to immune dysregulation with certain involvement to autoimmunity. Inflammatory response related mitophagy is yet to be described in AAVs. We here describe a research protocol to investigate mitophagy in monocytes, neutrophils, and T cells in AAV patients, and the relationship of disturbed mitophagy with ANCA seropositivity.

17.
Sci Rep ; 10(1): 7431, 2020 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-32366993

RESUMEN

Age-related hearing loss (ARHL) is a threat to future human wellbeing. Multiple factors contributing to the terminal auditory decline have been identified; but a unified understanding of ARHL - or the homeostatic maintenance of hearing before its breakdown - is missing. We here present an in-depth analysis of homeostasis and ageing in the antennal ears of the fruit fly Drosophila melanogaster. We show that Drosophila, just like humans, display ARHL. By focusing on the phase of dynamic stability prior to the eventual hearing loss we discovered a set of evolutionarily conserved homeostasis genes. The transcription factors Onecut (closest human orthologues: ONECUT2, ONECUT3), Optix (SIX3, SIX6), Worniu (SNAI2) and Amos (ATOH1, ATOH7, ATOH8, NEUROD1) emerged as key regulators, acting upstream of core components of the fly's molecular machinery for auditory transduction and amplification. Adult-specific manipulation of homeostatic regulators in the fly's auditory neurons accelerated - or protected against - ARHL.


Asunto(s)
Envejecimiento , Antenas de Artrópodos/fisiología , Drosophila melanogaster/fisiología , Pérdida Auditiva/genética , Audición/genética , Homeostasis , Neuronas/fisiología , Animales , Proteínas de Drosophila/genética , Femenino , Genotipo , Proteínas de Homeodominio/genética , Humanos , Masculino , Ratones , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Interferencia de ARN , Análisis de Secuencia de ARN , Sonido , Factores de Tiempo , Transactivadores/genética , Factores de Transcripción/genética , Transcriptoma
18.
Cancer Res ; 79(23): 5986-5998, 2019 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-31690667

RESUMEN

1α,25-Dihydroxyvitamin D3 signals via the vitamin D receptor (VDR). Higher serum vitamin D is associated with thinner primary melanoma and better outcome, although a causal mechanism has not been established. As patients with melanoma commonly avoid sun exposure, and consequent vitamin D deficiency might worsen outcomes, we interrogated 703 primary melanoma transcriptomes to understand the role of vitamin D-VDR signaling and replicated the findings in The Cancer Genome Atlas metastases. VDR expression was independently protective for melanoma-related death in both primary and metastatic disease. High tumor VDR expression was associated with upregulation of pathways mediating antitumor immunity and corresponding with higher imputed immune cell scores and histologically detected tumor-infiltrating lymphocytes. High VDR-expressing tumors had downregulation of proliferative pathways, notably Wnt/ß-catenin signaling. Deleterious low VDR levels resulted from promoter methylation and gene deletion in metastases. Vitamin D deficiency (<25 nmol/L ∼ 10 ng/mL) shortened survival in primary melanoma in a VDR-dependent manner. In vitro functional validation studies showed that elevated vitamin D-VDR signaling inhibited Wnt/ß-catenin signaling genes. Murine melanoma cells overexpressing VDR produced fewer pulmonary metastases than controls in tail-vein metastasis assays. In summary, vitamin D-VDR signaling contributes to controlling pro-proliferative/immunosuppressive Wnt/ß-catenin signaling in melanoma and this is associated with less metastatic disease and stronger host immune responses. This is evidence of a causal relationship between vitamin D-VDR signaling and melanoma survival, which should be explored as a therapeutic target in primary resistance to checkpoint blockade. SIGNIFICANCE: VDR expression could potentially be used as a biomarker to stratify patients with melanoma that may respond better to immunotherapy.


Asunto(s)
Calcitriol/deficiencia , Melanoma/inmunología , Receptores de Calcitriol/metabolismo , Neoplasias Cutáneas/inmunología , Deficiencia de Vitamina D/inmunología , Animales , Calcitriol/sangre , Línea Celular Tumoral , Conjuntos de Datos como Asunto , Progresión de la Enfermedad , Femenino , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Melanoma/sangre , Melanoma/mortalidad , Melanoma/patología , Ratones , Piel/metabolismo , Neoplasias Cutáneas/sangre , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Análisis de Supervivencia , Factores de Tiempo , Deficiencia de Vitamina D/sangre , Deficiencia de Vitamina D/patología , Vía de Señalización Wnt , beta Catenina/metabolismo
19.
Sci Rep ; 9(1): 8908, 2019 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-31222134

RESUMEN

Systematic tumour profiling is essential for biomarker research and clinically for assessing response to therapy. Solving the challenge of delivering informative copy number (CN) profiles from formalin-fixed paraffin embedded (FFPE) material, the only likely readily available biospecimen for most cancers, involves successful processing of small quantities of degraded DNA. To investigate the potential for analysis of such lesions, whole-genome CNVseq was applied to 300 FFPE primary tumour samples, obtained from a large-scale epidemiological study of melanoma. The quality and the discriminatory power of CNVseq was assessed. Libraries were successfully generated for 93% of blocks, with input DNA quantity being the only predictor of success (success rate dropped to 65% if <20 ng available); 3% of libraries were dropped because of low sequence alignment rates. Technical replicates showed high reproducibility. Comparison with targeted CN assessment showed consistency with the Next Generation Sequencing (NGS) analysis. We were able to detect and distinguish CN changes with a resolution of ≤10 kb. To demonstrate performance, we report the spectrum of genomic CN alterations (CNAs) detected at 9p21, the major site of CN change in melanoma. This successful analysis of CN in FFPE material using NGS provides proof of principle for intensive examination of population-based samples.


Asunto(s)
Variaciones en el Número de Copia de ADN , Neoplasias/genética , Adhesión en Parafina , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias/patología , Reproducibilidad de los Resultados , Fijación del Tejido
20.
Clin Cancer Res ; 25(24): 7424-7435, 2019 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-31515461

RESUMEN

PURPOSE: Previously identified transcriptomic signatures have been based on primary and metastatic melanomas with relatively few American Joint Committee on Cancer (AJCC) stage I tumors, given difficulties in sampling small tumors. The advent of adjuvant therapies has highlighted the need for better prognostic and predictive biomarkers, especially for AJCC stage I and stage II disease. EXPERIMENTAL DESIGN: A total of 687 primary melanoma transcriptomes were generated from the Leeds Melanoma Cohort (LMC). The prognostic value of existing signatures across all the AJCC stages was tested. Unsupervised clustering was performed, and the prognostic value of the resultant signature was compared with that of sentinel node biopsy (SNB) and tested as a biomarker in three published immunotherapy datasets. RESULTS: Previous Lund and The Cancer Genome Atlas signatures predicted outcome in the LMC dataset (P = 10-8 to 10-4) but showed a significant interaction with AJCC stage (P = 0.04) and did not predict outcome in stage I tumors (P = 0.3-0.7). Consensus-based classification of the LMC dataset identified six classes that predicted outcome, notably in stage I disease. LMC class was a similar indicator of prognosis when compared with SNB, and it added prognostic value to the genes reported by Gerami and colleagues. One particular LMC class consistently predicted poor outcome in patients receiving immunotherapy in two of three tested datasets. Biological characterization of this class revealed high JUN and AXL expression and evidence of epithelial-to-mesenchymal transition. CONCLUSIONS: A transcriptomic signature of primary melanoma was identified with prognostic value, including in stage I melanoma and in patients undergoing immunotherapy.


Asunto(s)
Biomarcadores de Tumor/genética , Biología Computacional/métodos , Regulación Neoplásica de la Expresión Génica , Inmunoterapia/mortalidad , Melanoma/patología , Neoplasias Cutáneas/patología , Transcriptoma , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Femenino , Humanos , Masculino , Melanoma/genética , Melanoma/terapia , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/terapia , Tasa de Supervivencia , Resultado del Tratamiento , Adulto Joven , Melanoma Cutáneo Maligno
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