Mechanisms underlying divergent responses of genetically distinct macrophages to IL-4

Mechanisms by which noncoding genetic variation influences gene expression remain only partially understood but are considered to be major determinants of phenotypic diversity and disease risk. Here, we evaluated effects of >50 million single-nucleotide polymorphisms and short insertions/deletions provided by five inbred strains of mice on the responses of macrophages to interleukin-4 (IL-4), a cytokine that plays pleiotropic roles in immunity and tissue homeostasis. Of >600 genes induced >2-fold by IL-4 across the five strains, only 26 genes reached this threshold in all strains. By applying deep learning and motif mutation analyses to epigenetic data for macrophages from each strain, we identified the dominant combinations of lineage-determining and signal-dependent transcription factors driving IL-4 enhancer activation. These studies further revealed mechanisms by which noncoding genetic variation influences absolute levels of enhancer activity and their dynamic responses to IL-4, thereby contributing to strain-differential patterns of gene expression and phenotypic diversity

SALL1 enforces microglia-specific DNA binding and function of SMADs to establish microglia identity

Spalt-like transcription factor 1 (SALL1) is a critical regulator of organogenesis and microglia identity. Here we demonstrate that disruption of a conserved microglia-specific super-enhancer interacting with the Sall1 promoter results in complete and specific loss of Sall1 expression in microglia. By determining the genomic binding sites of SALL1 and leveraging Sall1 enhancer knockout mice, we provide evidence for functional interactions between SALL1 and SMAD4 required for microglia-specific gene expression. SMAD4 binds directly to the Sall1 super-enhancer and is required for Sall1 expression, consistent with an evolutionarily conserved requirement of the TGFβ and SMAD homologs Dpp and Mad for cell-specific expression of Spalt in the Drosophila wing. Unexpectedly, SALL1 in turn promotes binding and function of SMAD4 at microglia-specific enhancers while simultaneously suppressing binding of SMAD4 to enhancers of genes that become inappropriately activated in enhancer knockout microglia, thereby enforcing microglia-specific functions of the TGFβ–SMAD signaling axis.</p

Tumor microenvironment gene expression profiles associated to complete pathological response and disease progression in resectable NSCLC patients treated with neoadjuvant chemoimmunotherapy

Background Neoadjuvant chemoimmunotherapy for non-small cell lung cancer (NSCLC) has improved pathological responses and survival rates compared with chemotherapy alone, leading to Food and Drug Administration (FDA) approval of nivolumab plus chemotherapy for resectable stage IB-IIIA NSCLC (AJCC 7th edition) without ALK or EGFR alterations. Unfortunately, a considerable percentage of tumors do not completely respond to therapy, which has been associated with early disease progression. So far, it is impossible to predict these events due to lack of knowledge. In this study, we characterized the gene expression profile of tumor samples to identify new biomarkers and mechanisms behind tumor responses to neoadjuvant chemoimmunotherapy and disease recurrence after surgery. Methods Tumor bulk RNA sequencing was performed in 16 pretreatment and 36 post-treatment tissue samples from 41 patients with resectable stage IIIA NSCLC treated with neoadjuvant chemoimmunotherapy from NADIM trial. A panel targeting 395 genes related to immunological processes was used. Tumors were classified as complete pathological response (CPR) and non-CPR, based on the total absence of viable tumor cells in tumor bed and lymph nodes tested at surgery. Differential-expressed genes between groups and pathway enrichment analysis were assessed using DESeq2 and gene set enrichment analysis. CIBERSORTx was used to estimate the proportions of immune cell subtypes. Results CPR tumors had a stronger pre-established immune infiltrate at baseline than non-CPR, characterized by higher levels of IFNG, GZMB, NKG7, and M1 macrophages, all with a significant area under the receiver operating characteristic curve (ROC) >0.9 for CPR prediction. A greater effect of neoadjuvant therapy was also seen in CPR tumors with a reduction of tumor markers and IFN gamma signaling after treatment. Additionally, the higher expression of several genes, including AKT1, BST2, OAS3, or CD8B; or higher dendritic cells and neutrophils proportions in post-treatment non-CPR samples, were associated with relapse after surgery. Also, high pretreatment PD-L1 and tumor mutational burden levels influenced the post-treatment immune landscape with the downregulation of proliferation markers and type I interferon signaling molecules in surgery samples. Conclusions Our results reinforce the differences between CPR and non-CPR responses, describing possible response and relapse immune mechanisms, opening the possibility of therapy personalization of immunotherapy-based regimens in the neoadjuvant setting of NSCLC

Customized Treatment in Non-Small-Cell Lung Cancer Based on EGFR Mutations and BRCA1 mRNA Expression

BACKGROUND: Median survival is 10 months and 2-year survival is 20% in metastatic non-small-cell lung cancer (NSCLC) treated with platinum-based chemotherapy. A small fraction of non-squamous cell lung cancers harbor EGFR mutations, with improved outcome to gefitinib and erlotinib. Experimental evidence suggests that BRCA1 overexpression enhances sensitivity to docetaxel and resistance to cisplatin. RAP80 and Abraxas are interacting proteins that form complexes with BRCA1 and could modulate the effect of BRCA1. In order to further examine the effect of EGFR mutations and BRCA1 mRNA levels on outcome in advanced NSCLC, we performed a prospective non-randomized phase II clinical trial, testing the hypothesis that customized therapy would confer improved outcome over non-customized therapy. In an exploratory analysis, we also examined the effect of RAP80 and Abraxas mRNA levels. METHODOLOGY/PRINCIPAL FINDINGS: We treated 123 metastatic non-squamous cell lung carcinoma patients using a customized approach. RNA and DNA were isolated from microdissected specimens from paraffin-embedded tumor tissue. Patients with EGFR mutations received erlotinib, and those without EGFR mutations received chemotherapy with or without cisplatin based on their BRCA1 mRNA levels: low, cisplatin plus gemcitabine; intermediate, cisplatin plus docetaxel; high, docetaxel alone. An exploratory analysis examined RAP80 and Abraxas expression. Median survival exceeded 28 months for 12 patients with EGFR mutations, and was 11 months for 38 patients with low BRCA1, 9 months for 40 patients with intermediate BRCA1, and 11 months for 33 patients with high BRCA1. Two-year survival was 73.3%, 41.2%, 15.6% and 0%, respectively. Median survival was influenced by RAP80 expression in the three BRCA1 groups. For example, for patients with both low BRCA1 and low RAP80, median survival exceeded 26 months. RAP80 was a significant factor for survival in patients treated according to BRCA1 levels (hazard ratio, 1.3 [95% CI, 1-1.7]; P = 0.05). CONCLUSIONS/SIGNIFICANCE: Chemotherapy customized according to BRCA1 expression levels is associated with excellent median and 2-year survival for some subsets of NSCLC patients , and RAP80 could play a crucial modulating effect on this model of customized chemotherapy. TRIAL REGISTRATION: (ClinicalTrials.gov) NCT00883480

Role of NR5A2 in pancreatic homeostasis and disease

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 28-05-2018Esta tesis tiene embargado el acceso al texto completo hasta el 28-11-2019Chronic inflammation is a major risk factor for the development of several tumour types Inflammation acts as a double-edged sword: on one hand, it is a precise mechanism that allows proper response upon damage. However, when de-regulated or persistent, inflammation can promote tumour development. NR5A2 is indispensable for complete pancreatic acinar cell differentiation and homeostasis. SNPs in the vicinity of the NR5A2 locus have been associated with higher risk of developing pancreatic cancer. In this work, I have demonstrated that, in addition to its role in pancreas homeostasis, NR5A2 restrains an inflammatory program in the pancreas. In this regard, reduced NR5A2 expression both in normal mouse and human pancreas, leads to a basal pre-inflammatory state that resembles that one observed in the early stages of pancreatitis. During the pre-inflammatory state, NR5A2 is genomically relocated from the promoter of acinar differentiation genes to the promoter of inflammatory genes in a process that is dependent on AP-1 proteins. Our work opens the possibility of identifying scenarios of pre-inflammation in other tissues even when histology and functionality seems normal. In addition, I have drilled down into the tumour supressor role of NR5A2. I have found that the combination of mutant KRas activation and Nr5a2 heterozigosity in pancreatic epithelial cells leads to the formation of aggressive cystic lesions, in the absence of exogenous pancreatitis induction. ! Through the analysis of the pancreatic NR5A2 interactome , we ha identified NFIC as a strong NR5A2 interactor. Our data show that NFIC participates with NR5A2 in the transcriptional regulation of the acinar differentiation program and regulates mTOR signalling pathway in the adult pancreas. ! ! Finally, harnessing the pharmacological manipulation of NR5A2, our data suggest that pretreatment of wild type y Nr5a2+/- mice with an NR5A2 agonist reduces the damage associated with pancreatitis both in wild type and Nr5a2+/- mice. Similarly, treatment with the agonist during pancreatitis reduces the pancreatitis damage. Our data open the possibility of modulating NR5A2 activity as mechanism to prevent and treat acute pancreatitis.La inflamación crónica es un factor de riesgo para el desarrollo de muchos tipos de tumores. La inflamación actúa como una espada de doble filo. Por un lado, es un mecanismo que permite al tejido responder frente a un insulto externo. Sin embargo, una inflamación pobremente regulada y/o persistente, puede favorecer el desarrollo de tumores. NR5A2 es un gen cuyas variaciones polimórficas (SNP) están asociadas a un mayor riesgo de padecer cáncer de páncreas y es necesario para la formación del páncreas. En este trabajo hemos demostrado que NR5A2 no solamente participa en la función normal del páncreas, sino que reprime un programa inflamatorio basal. De esta manera, la expresión reducida de NR5A2, tanto en ratones como en humanos, lleva a una estado pre-inflamatorio basal que se asemeja al escenario de los primeros estadíos de la pancreatitis. En este estado pre-inflamatorio basal, NR5A2 sufre una translocación genómica, de los promotores de genes de diferenciación acinar a los de los genes inflamatorios, en un proceso que depende de la sobre-expresión de las proteínas de la familia AP-1. ! Además, hemos profundizado en el papel de NR5A2 como supresor tumoural. En el presente trabajo hemos descubierto que la heterozigosidad de Nr5a2 en células epiteliales del páncreas, en combinación con la expresión de KRas mutado y en ausencia de la inducción exógena de pancreatitis, lleva a la formación de lesiones quísticas agresivas. ! A través del análisis del interactoma de NR5A2 en el páncreas, he identificado NFIC como un interactor estable de NR5A2, que participa con NR5A2 en el programa de diferenciación acinar y regula la vía de mTOR en el páncreas adulto. ! Finalmente, aprovechando la posibilidad de manipular farmacológicamente la actividad transcriptional de NR5A2, muestro que el pre-tratamiento de ratones wild type y Nr5a2+/- con un agonista de NR5A2 previene el daño pancreático durante la pancreatitis. El tratamiento con el agonista en el transcurso de la pancreatitis reduce también el daño asociado. Estos datos abren la posibilidad de incrementar la actividad transcripcional de NR5A2 como un mecanismo de prevención y tratamiento de la pancreatitis

Protocol for preparing monosodium urate crystals for in vitro and in vivo studies in mouse and human cells

Summary: Gout is caused by the deposition of monosodium urate crystals (MSUc) in the joints, triggering a unique inflammatory and metabolic response in macrophages. Here, we present a protocol to generate MSUc for in vitro and in vivo studies in mouse and human cells. We describe steps for dissolving uric acid followed by crystallizing, purifying, evaluating, and analyzing MSUc. We then detail procedures for stimulating human/mouse-derived macrophages and determining endotoxin levels in MSUc preparation. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Clonal hematopoiesis driven by DNMT3A and TET2 mutations: role in monocyte and macrophage biology and atherosclerotic cardiovascular disease.

PURPOSE OF REVIEW: Clonal hematopoiesis of indeterminate potential (CHIP), defined by the presence of somatic mutations in hematopoietic cells, is associated with advanced age and increased mortality due to cardiovascular disease. Gene mutations in DNMT3A and TET2 are the most frequently identified variants among patients with CHIP and provide selective advantage that spurs clonal expansion and myeloid skewing. Although DNMT3A and TET2 appear to have opposing enzymatic influence on DNA methylation, mounting data has characterized convergent inflammatory pathways, providing insights to how CHIP may mediate atherosclerotic cardiovascular disease (ASCVD). RECENT FINDINGS: We review a multitude of studies that characterize aberrant inflammatory signaling as result of DNMT3A and TET2 deficiency in monocytes and macrophages, immune cells with prominent roles in atherosclerosis. Although specific DNA methylation signatures associated with these known epigenetic regulators have been identified, many studies have also characterized diverse modulatory functions of DNTM3A and TET2 that urge cell and context-specific experimental studies to further define how DNMT3A and TET2 may nonenzymatically activate inflammatory pathways with clinically meaningful consequences. SUMMARY: CHIP, common in elderly individuals, provides an opportunity understand and potentially modify age-related chronic inflammatory ASCVD risk