Co-option of Neutrophil Fates by Tissue Environments.

Classically considered short-lived and purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional, and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion of neutrophils compromised angiogenesis during early age, genotoxic injury, and viral infection, and impaired hematopoietic recovery after irradiation. Neutrophils acquired these properties in target tissues, a process that, in the lungs, occurred in CXCL12-rich areas and relied on CXCR4. Our results reveal that tissues co-opt neutrophils en route for elimination to induce programs that support their physiological demands.

γ-Linolenic acid in maternal milk drives cardiac metabolic maturation.

Birth presents a metabolic challenge to cardiomyocytes as they reshape fuel preference from glucose to fatty acids for postnatal energy production1,2. This adaptation is triggered in part by post-partum environmental changes3, but the molecules orchestrating cardiomyocyte maturation remain unknown. Here we show that this transition is coordinated by maternally supplied γ-linolenic acid (GLA), an 18:3 omega-6 fatty acid enriched in the maternal milk. GLA binds and activates retinoid X receptors4 (RXRs), ligand-regulated transcription factors that are expressed in cardiomyocytes from embryonic stages. Multifaceted genome-wide analysis revealed that the lack of RXR in embryonic cardiomyocytes caused an aberrant chromatin landscape that prevented the induction of an RXR-dependent gene expression signature controlling mitochondrial fatty acid homeostasis. The ensuing defective metabolic transition featured blunted mitochondrial lipid-derived energy production and enhanced glucose consumption, leading to perinatal cardiac dysfunction and death. Finally, GLA supplementation induced RXR-dependent expression of the mitochondrial fatty acid homeostasis signature in cardiomyocytes, both in vitro and in vivo. Thus, our study identifies the GLA-RXR axis as a key transcriptional regulatory mechanism underlying the maternal control of perinatal cardiac metabolism.

Behavioural immune landscapes of inflammation.

Transcriptional and proteomic profiling of individual cells have revolutionized interpretation of biological phenomena by providing cellular landscapes of healthy and diseased tissues1,2. These approaches, however, do not describe dynamic scenarios in which cells continuously change their biochemical properties and downstream 'behavioural' outputs3-5. Here we used 4D live imaging to record tens to hundreds of morpho-kinetic parameters describing the dynamics of individual leukocytes at sites of active inflammation. By analysing more than 100,000 reconstructions of cell shapes and tracks over time, we obtained behavioural descriptors of individual cells and used these high-dimensional datasets to build behavioural landscapes. These landscapes recognized leukocyte identities in the inflamed skin and trachea, and uncovered a continuum of neutrophil states inside blood vessels, including a large, sessile state that was embraced by the underlying endothelium and associated with pathogenic inflammation. Behavioural screening in 24 mouse mutants identified the kinase Fgr as a driver of this pathogenic state, and interference with Fgr protected mice from inflammatory injury. Thus, behavioural landscapes report distinct properties of dynamic environments at high cellular resolution.

Apolipoprotein E-ε2 and Resistance to Atherosclerosis in Midlife: The PESA Observational Study.

BACKGROUND: APOE is a known genetic contributor to cardiovascular disease, but the differential role APOE alleles play in subclinical atherosclerosis remains unclear. METHODS: The PESA (Progression of Early Subclinical Atherosclerosis) is an observational cohort study that recruited 4184 middle-aged asymptomatic individuals to be screened for cardiovascular risk and multiterritorial subclinical atherosclerosis. Participants were APOE-genotyped, and omics data were additionally evaluated. RESULTS: In the PESA study, the frequencies for APOE -ε2, -ε3, and -ε4 alleles were 0.060, 0.844, and 0.096, respectively. This study included a subcohort of 3887 participants (45.8±4.3 years of age; 62% males). As expected, APOE-ε4 carriers were at the highest risk for cardiovascular disease and had significantly greater odds of having subclinical atherosclerosis compared with ε3/ε3 carriers, which was mainly explained by their higher levels of low-density lipoprotein (LDL)-cholesterol. In turn, APOE-ε2 carriers were at the lowest risk for cardiovascular disease and had significantly lower odds of having subclinical atherosclerosis in several vascular territories (carotids: 0.62 [95% CI, 0.47-0.81]; P=0.00043; femorals: 0.60 [0.47-0.78]; P=9.96×10-5; coronaries: 0.53 [0.39-0.74]; P=0.00013; and increased PESA score: 0.58 [0.48-0.71]; P=3.16×10-8). This APOE-ε2 atheroprotective effect was mostly independent of the associated lower LDL-cholesterol levels and other cardiovascular risk factors. The protection conferred by the ε2 allele was greater with age (50-54 years: 0.49 [95% CI, 0.32-0.73]; P=0.00045), and normal (<150 mg/dL) levels of triglycerides (0.54 [0.44-0.66]; P=4.70×10-9 versus 0.90 [0.57-1.43]; P=0.67 if ≥150 mg/dL). Omics analysis revealed an enrichment of several canonical pathways associated with anti-inflammatory mechanisms together with the modulation of erythrocyte homeostasis, coagulation, and complement activation in ε2 carriers that might play a relevant role in the ε2's atheroprotective effect. CONCLUSIONS: This work sheds light on the role of APOE in cardiovascular disease development with important therapeutic and prevention implications on cardiovascular health, especially in early midlife. REGISTRATION: URL: https://www.clinicaltrials.gov: NCT01410318.

SpatialDDLS: an R package to deconvolute spatial transcriptomics data using neural networks.

SUMMARY: Spatial transcriptomics has changed our way to study tissue structure and cellular organization. However, there are still limitations in its resolution, and most available platforms do not reach a single cell resolution. To address this issue, we introduce SpatialDDLS, a fast neural network-based algorithm for cell type deconvolution of spatial transcriptomics data. SpatialDDLS leverages single-cell RNA sequencing data to simulate mixed transcriptional profiles with predefined cellular composition, which are subsequently used to train a fully connected neural network to uncover cell type diversity within each spot. By comparing it with two state-of-the-art spatial deconvolution methods, we demonstrate that SpatialDDLS is an accurate and fast alternative to the available state-of-the art tools. AVAILABILITY AND IMPLEMENTATION: The R package SpatialDDLS is available via CRAN-The Comprehensive R Archive Network: https://CRAN.R-project.org/package=SpatialDDLS. A detailed manual of the main functionalities implemented in the package can be found at https://diegommcc.github.io/SpatialDDLS.

Retinoid X receptor promotes hematopoietic stem cell fitness and quiescence and preserves hematopoietic homeostasis.

Hematopoietic stem cells (HSCs) balance self-renewal and differentiation to maintain hematopoietic fitness throughout life. In steady-state conditions, HSC exhaustion is prevented by the maintenance of most HSCs in a quiescent state, with cells entering the cell cycle only occasionally. HSC quiescence is regulated by retinoid and fatty-acid ligands of transcriptional factors of the nuclear retinoid X receptor (RXR) family. Herein, we show that dual deficiency for hematopoietic RXRα and RXRß induces HSC exhaustion, myeloid cell/megakaryocyte differentiation, and myeloproliferative-like disease. RXRα and RXRß maintain HSC quiescence, survival, and chromatin compaction; moreover, transcriptome changes in RXRα;RXRß-deficient HSCs include premature acquisition of an aging-like HSC signature, MYC pathway upregulation, and RNA intron retention. Fitness loss and associated RNA transcriptome and splicing alterations in RXRα;RXRß-deficient HSCs are prevented by Myc haploinsufficiency. Our study reveals the critical importance of RXRs for the maintenance of HSC fitness and their protection from premature aging.

A Human Hereditary Cardiomyopathy Shares a Genetic Substrate With Bicuspid Aortic Valve.

BACKGROUND: The complex genetics underlying human cardiac disease is evidenced by its heterogenous manifestation, multigenic basis, and sporadic occurrence. These features have hampered disease modeling and mechanistic understanding. Here, we show that 2 structural cardiac diseases, left ventricular noncompaction (LVNC) and bicuspid aortic valve, can be caused by a set of inherited heterozygous gene mutations affecting the NOTCH ligand regulator MIB1 (MINDBOMB1) and cosegregating genes. METHODS: We used CRISPR-Cas9 gene editing to generate mice harboring a nonsense or a missense MIB1 mutation that are both found in LVNC families. We also generated mice separately carrying these MIB1 mutations plus 5 additional cosegregating variants in the ASXL3, APCDD1, TMX3, CEP192, and BCL7A genes identified in these LVNC families by whole exome sequencing. Histological, developmental, and functional analyses of these mouse models were carried out by echocardiography and cardiac magnetic resonance imaging, together with gene expression profiling by RNA sequencing of both selected engineered mouse models and human induced pluripotent stem cell-derived cardiomyocytes. Potential biochemical interactions were assayed in vitro by coimmunoprecipitation and Western blot. RESULTS: Mice homozygous for the MIB1 nonsense mutation did not survive, and the mutation caused LVNC only in heteroallelic combination with a conditional allele inactivated in the myocardium. The heterozygous MIB1 missense allele leads to bicuspid aortic valve in a NOTCH-sensitized genetic background. These data suggest that development of LVNC is influenced by genetic modifiers present in affected families, whereas valve defects are highly sensitive to NOTCH haploinsufficiency. Whole exome sequencing of LVNC families revealed single-nucleotide gene variants of ASXL3, APCDD1, TMX3, CEP192, and BCL7A cosegregating with the MIB1 mutations and LVNC. In experiments with mice harboring the orthologous variants on the corresponding Mib1 backgrounds, triple heterozygous Mib1 Apcdd1 Asxl3 mice showed LVNC, whereas quadruple heterozygous Mib1 Cep192 Tmx3;Bcl7a mice developed bicuspid aortic valve and other valve-associated defects. Biochemical analysis suggested interactions between CEP192, BCL7A, and NOTCH. Gene expression profiling of mutant mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes revealed increased cardiomyocyte proliferation and defective morphological and metabolic maturation. CONCLUSIONS: These findings reveal a shared genetic substrate underlying LVNC and bicuspid aortic valve in which MIB1-NOTCH variants plays a crucial role in heterozygous combination with cosegregating genetic modifiers.

Axial skeleton anterior-posterior patterning is regulated through feedback regulation between Meis transcription factors and retinoic acid.

Vertebrate axial skeletal patterning is controlled by co-linear expression of Hox genes and axial level-dependent activity of HOX protein combinations. MEIS transcription factors act as co-factors of HOX proteins and profusely bind to Hox complex DNA; however, their roles in mammalian axial patterning remain unknown. Retinoic acid (RA) is known to regulate axial skeletal element identity through the transcriptional activity of its receptors; however, whether this role is related to MEIS/HOX activity remains unknown. Here, we study the role of Meis in axial skeleton formation and its relationship to the RA pathway in mice. Meis elimination in the paraxial mesoderm produces anterior homeotic transformations and rib mis-patterning associated to alterations of the hypaxial myotome. Although Raldh2 and Meis positively regulate each other, Raldh2 elimination largely recapitulates the defects associated with Meis deficiency, and Meis overexpression rescues the axial skeletal defects in Raldh2 mutants. We propose a Meis-RA-positive feedback loop, the output of which is Meis levels, that is essential to establish anterior-posterior identities and patterning of the vertebrate axial skeleton.

A specific natural killer cells phenotypic signature associated to long term elite control of HIV infection.

Elite controllers (ECs) are an exceptional group of people living with HIV (PLWH) that control HIV replication without therapy. Among the mechanisms involved in this ability, natural killer (NK)-cells have recently gained much attention. We performed an in-deep phenotypic analysis of NK-cells to search for surrogate markers associated with the long term spontaneous control of HIV. Forty-seven PLWH (22 long-term EC [PLWH-long-term elite controllers (LTECs)], 15 noncontrollers receiving antiretroviral treatment [ART] [PLWH-onART], and 10 noncontrollers cART-naïve [PLWH-offART]), and 20 uninfected controls were included. NK-cells homeostasis was analyzed by spectral flow cytometry using a panel of 15 different markers. Data were analyzed using FCSExpress and R software for unsupervised multidimensional analysis. Six different subsets of NK-cells were defined on the basis of CD16 and CD56 expression, and the multidimensional analysis revealed the existence of 68 different NK-cells clusters based on the expression levels of the 15 different markers. PLWH-offART presented the highest disturbance of NK-cells homeostasis and this was not completely restored by long-term ART. Interestingly, long term spontaneous control of HIV (PLWH-LTEC group) was associated with a specific profile of NK-cells homeostasis disturbance, characterized by an increase of CD16dimCD56dim subset when compared to uninfected controls (UC) group and also to offART and onART groups (p < 0.0001 for the global comparison), an increase of clusters C16 and C26 when compared to UC and onART groups (adjusted p-value < 0.05 for both comparisons), and a decrease of clusters C10 and C20 when compared to all the other groups (adjusted p-value < 0.05 for all comparisons). These findings may provide clues to elucidate markers of innate immunity with a relevant role in the long-term control of HIV.

Single cell clonal analysis identifies an AID-dependent pathway of plasma cell differentiation.

Germinal centers (GC) are microstructures where B cells that have been activated by antigen can improve the affinity of their B cell receptors and differentiate into memory B cells (MBCs) or antibody-secreting plasma cells. Here, we have addressed the role of activation-induced deaminase (AID), which initiates somatic hypermutation and class switch recombination, in the terminal differentiation of GC B cells. By combining single cell transcriptome and immunoglobulin clonal analysis in a mouse model that traces AID-experienced cells, we have identified a novel subset of late-prePB cells (L-prePB), which shares the strongest clonal relationships with plasmablasts (PBs). Mice lacking AID have various alterations in the size and expression profiles of transcriptional clusters. We find that AID deficiency leads to a reduced proportion of L-prePB cells and severely impairs transitions between the L-prePB and the PB subsets. Thus, AID shapes the differentiation fate of GC B cells by enabling PB generation from a prePB state.

ISG20L2: an RNA nuclease regulating T cell activation.

ISG20L2, a 3' to 5' exoribonuclease previously associated with ribosome biogenesis, is identified here in activated T cells as an enzyme with a preferential affinity for uridylated miRNA substrates. This enzyme is upregulated in T lymphocytes upon TCR and IFN type I stimulation and appears to be involved in regulating T cell function. ISG20L2 silencing leads to an increased basal expression of CD69 and induces greater IL2 secretion. However, ISG20L2 absence impairs CD25 upregulation, CD3 synaptic accumulation and MTOC translocation towards the antigen-presenting cell during immune synapsis. Remarkably, ISG20L2 controls the expression of immunoregulatory molecules, such as AHR, NKG2D, CTLA-4, CD137, TIM-3, PD-L1 or PD-1, which show increased levels in ISG20L2 knockout T cells. The dysregulation observed in these key molecules for T cell responses support a role for this exonuclease as a novel RNA-based regulator of T cell function.

Technologies to Study Genetics and Molecular Pathways.

Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

Subclinical atherosclerosis and accelerated epigenetic age mediated by inflammation: a multi-omics study.

AIMS: Epigenetic age is emerging as a personalized and accurate predictor of biological age. The aim of this article is to assess the association of subclinical atherosclerosis with accelerated epigenetic age and to investigate the underlying mechanisms mediating this association. METHODS AND RESULTS: Whole blood methylomics, transcriptomics, and plasma proteomics were obtained for 391 participants of the Progression of Early Subclinical Atherosclerosis study. Epigenetic age was calculated from methylomics data for each participant. Its divergence from chronological age is termed epigenetic age acceleration. Subclinical atherosclerosis burden was estimated by multi-territory 2D/3D vascular ultrasound and by coronary artery calcification. In healthy individuals, the presence, extension, and progression of subclinical atherosclerosis were associated with a significant acceleration of the Grim epigenetic age, a predictor of health and lifespan, regardless of traditional cardiovascular risk factors. Individuals with an accelerated Grim epigenetic age were characterized by an increased systemic inflammation and associated with a score of low-grade, chronic inflammation. Mediation analysis using transcriptomics and proteomics data revealed key pro-inflammatory pathways (IL6, Inflammasome, and IL10) and genes (IL1B, OSM, TLR5, and CD14) mediating the association between subclinical atherosclerosis and epigenetic age acceleration. CONCLUSION: The presence, extension, and progression of subclinical atherosclerosis in middle-aged asymptomatic individuals are associated with an acceleration in the Grim epigenetic age. Mediation analysis using transcriptomics and proteomics data suggests a key role of systemic inflammation in this association, reinforcing the relevance of interventions on inflammation to prevent cardiovascular disease.

T Cell Homeostasis Disturbances in a Cohort of Long-Term Elite Controllers of HIV Infection.

Elite controllers (ECs) are people living with HIV (PLWH) able to control HIV replication without antiretroviral therapy and have been proposed as a model of a functional HIV cure. Much evidence suggests that this spontaneous control of HIV has a cost in terms of T cell homeostasis alterations. We performed a deep phenotypic study to obtain insight into T cell homeostasis disturbances in ECs maintaining long-term virologic and immunologic control of HIV (long-term elite controllers; LTECs). Forty-seven PLWH were included: 22 LTECs, 15 non-controllers under successful antiretroviral therapy (onART), and 10 non-controllers not receiving ART (offART). Twenty uninfected participants (UCs) were included as a reference. T cell homeostasis was analyzed by spectral flow cytometry and data were analyzed using dimensionality reduction and clustering using R software v3.3.2. Dimensionality reduction and clustering yielded 57 and 54 different CD4 and CD8 T cell clusters, respectively. The offART group showed the highest perturbation of T cell homeostasis, with 18 CD4 clusters and 15 CD8 clusters significantly different from those of UCs. Most of these alterations were reverted in the onART group. Interestingly, LTECs presented several disturbances of T cell homeostasis with 15 CD4 clusters and 13 CD8 clusters different from UC. Moreover, there was a specific profile of T cell homeostasis alterations associated with LTECs, characterized by increases in clusters of naïve T cells, increases in clusters of non-senescent effector CD8 cells, and increases in clusters of central memory CD4 cells. These results demonstrate that, compared to ART-mediated control of HIV, the spontaneous control of HIV is associated with several disturbances in CD4 and CD8 T cell homeostasis. These alterations could be related to the existence of a potent and efficient virus-specific T cell response, and to the ability to halt disease progression by maintaining an adequate pool of CD4 T cells.

Heteroplasmy of Wild-Type Mitochondrial DNA Variants in Mice Causes Metabolic Heart Disease With Pulmonary Hypertension and Frailty.

BACKGROUND: In most eukaryotic cells, the mitochondrial DNA (mtDNA) is transmitted uniparentally and present in multiple copies derived from the clonal expansion of maternally inherited mtDNA. All copies are therefore near-identical, or homoplasmic. The presence of >1 mtDNA variant in the same cytoplasm can arise naturally or result from new medical technologies aimed at preventing mitochondrial genetic diseases and improving fertility. The latter is called divergent nonpathologic mtDNA heteroplasmy (DNPH). We hypothesized that DNPH is maladaptive and usually prevented by the cell. METHODS: We engineered and characterized DNPH mice throughout their lifespan using transcriptomic, metabolomic, biochemical, physiologic, and phenotyping techniques. We focused on in vivo imaging techniques for noninvasive assessment of cardiac and pulmonary energy metabolism. RESULTS: We show that DNPH impairs mitochondrial function, with profound consequences in critical tissues that cannot resolve heteroplasmy, particularly cardiac and skeletal muscle. Progressive metabolic stress in these tissues leads to severe pathology in adulthood, including pulmonary hypertension and heart failure, skeletal muscle wasting, frailty, and premature death. Symptom severity is strongly modulated by the nuclear context. CONCLUSIONS: Medical interventions that may generate DNPH should address potential incompatibilities between donor and recipient mtDNA.

The SRSF4-GAS5-Glucocorticoid Receptor Axis Regulates Ventricular Hypertrophy.

[Figure: see text].

Interplay between UNG and AID governs intratumoral heterogeneity in mature B cell lymphoma.

Most B cell lymphomas originate from B cells that have germinal center (GC) experience and bear chromosome translocations and numerous point mutations. GC B cells remodel their immunoglobulin (Ig) genes by somatic hypermutation (SHM) and class switch recombination (CSR) in their Ig genes. Activation Induced Deaminase (AID) initiates CSR and SHM by generating U:G mismatches on Ig DNA that can then be processed by Uracyl-N-glycosylase (UNG). AID promotes collateral damage in the form of chromosome translocations and off-target SHM, however, the exact contribution of AID activity to lymphoma generation and progression is not completely understood. Here we show using a conditional knock-in strategy that AID supra-activity alone is not sufficient to generate B cell transformation. In contrast, in the absence of UNG, AID supra-expression increases SHM and promotes lymphoma. Whole exome sequencing revealed that AID heavily contributes to lymphoma SHM, promoting subclonal variability and a wider range of oncogenic variants. Thus, our data provide direct evidence that UNG is a brake to AID-induced intratumoral heterogeneity and evolution of B cell lymphoma.

JNK-mediated disruption of bile acid homeostasis promotes intrahepatic cholangiocarcinoma.

Metabolic stress causes activation of the cJun NH2-terminal kinase (JNK) signal transduction pathway. It is established that one consequence of JNK activation is the development of insulin resistance and hepatic steatosis through inhibition of the transcription factor PPARα. Indeed, JNK1/2 deficiency in hepatocytes protects against the development of steatosis, suggesting that JNK inhibition represents a possible treatment for this disease. However, the long-term consequences of JNK inhibition have not been evaluated. Here we demonstrate that hepatic JNK controls bile acid production. We found that hepatic JNK deficiency alters cholesterol metabolism and bile acid synthesis, conjugation, and transport, resulting in cholestasis, increased cholangiocyte proliferation, and intrahepatic cholangiocarcinoma. Gene ablation studies confirmed that PPARα mediated these effects of JNK in hepatocytes. This analysis highlights potential consequences of long-term use of JNK inhibitors for the treatment of metabolic syndrome.

Sox17 Controls Emergence and Remodeling of Nestin-Expressing Coronary Vessels.

RATIONALE: The molecular mechanisms underlying the formation of coronary arteries during development and during cardiac neovascularization after injury are poorly understood. However, a detailed description of the relevant signaling pathways and functional TFs (transcription factors) regulating these processes is still incomplete. OBJECTIVE: The goal of this study is to identify novel cardiac transcriptional mechanisms of coronary angiogenesis and vessel remodeling by defining the molecular signatures of coronary vascular endothelial cells during these complex processes. METHODS AND RESULTS: We demonstrate that Nes-gfp and Nes-CreERT2 transgenic mouse lines are novel tools for studying the emergence of coronary endothelium and targeting sprouting coronary vessels (but not ventricular endocardium) during development. Furthermore, we identify Sox17 as a critical TF upregulated during the sprouting and remodeling of coronary vessels, visualized by a specific neural enhancer from the Nestin gene that is strongly induced in developing arterioles. Functionally, genetic-inducible endothelial deletion of Sox17 causes deficient cardiac remodeling of coronary vessels, resulting in improper coronary artery formation. CONCLUSIONS: We demonstrated that Sox17 TF regulates the transcriptional activation of Nestin's enhancer in developing coronary vessels while its genetic deletion leads to inadequate coronary artery formation. These findings identify Sox17 as a critical regulator for the remodeling of coronary vessels in the developing heart.