Cell Type-specific Mechanisms Research Articles

Cell type specific roles of FOXP1 during early neocortical murine development.

Cortical development is a tightly controlled process and any deviation during development may increase the susceptibility to neurodevelopmental disorders, such as autism spectrum disorders (ASD). Numerous studies identified mutations in FOXP1, a transcription factor enriched in the neocortex, as causal for ASD and FOXP1 syndrome. Our group has shown that Foxp1 deletion in the mouse cortex leads to overall reduced cortex thickness, alterations in cortical lamination, and changes in the relative thickness of cortical layers. However, the developmental and cell type-specific mechanisms underlying these changes remained unclear. This work characterizes the developmental requirement of neocortical Foxp1 at key embryonic and perinatal ages using a conditional knock-out of Foxp1. We find that Foxp1 deletion results in accelerated pseudo-age during early neurogenesis, increased cell cycle exit during late neurogenesis, altered gene expression and chromatin accessibility, and selective migration deficits in a subset of upper-layer neurons. These data explain the postnatal differences observed in cortical layers and relative cortical thickness. We also highlight genes regulated by FOXP1 and their enrichment with high-confidence ASD or synaptic genes. Together, these results underscore a network of neurodevelopmental disorder-related genes that may serve as potential modulatory targets for postnatal modification relevant to ASD and FOXP1 syndrome.

Single-Cell Transcriptomic Profiling Unveils Dynamic Immune Cell Responses during Haemonchus contortus Infection.

Haemonchus contortus is a parasite widely distributed in tropical, subtropical, and warm temperate regions, causing significant economic losses in the livestock industry worldwide. However, little is known about the genetics of H. contortus resistance in livestock. In this study, we monitor the dynamic immune cell responses in diverse peripheral blood mononuclear cells (PBMCs) during H. contortus infection in goats through single-cell RNA sequencing (scRNA-Seq) analysis. A total of four Boer goats, two goats with oral infection with the L3 larvae of H. contortus and two healthy goats as controls, were used in the animal test. The infection model in goats was established and validated by the fecal egg count (FEC) test and qPCR analysis of the gene expression of IL-5 and IL-6. Using scRNA-Seq, we identified seven cell types, including T cells, monocytes, natural killer cells, B cells, and dendritic cells with distinct gene expression signatures. After identifying cell subpopulations of differentially expressed genes (DEGs) in the case and control groups, we observed the upregulation of multiple inflammation-associated genes, including NFKBIA and NFKBID. Kyoto Encyclopedia of the Genome (KEGG) enrichment analysis revealed significant enrichment of NOD-like receptor pathways and Th1/Th2 cell differentiation signaling pathways in CD4 T cells DEGs. Furthermore, the analysis of ligand-receptor interaction networks showed a more active state of cellular communication in the PBMCs from the case group, and the inflammatory response associated MIF-(CD74 + CXCR4) ligand receptor complex was significantly more activated in the case group, suggesting a potential inflammatory response. Our study preliminarily revealed transcriptomic profiling characterizing the cell type specific mechanisms in host PBMCs at the single-cell level during H. contortus infection.

A single-cell profile reveals the transcriptional regulation responded for Abelmoschus manihot (L.) treatment in diabetic kidney disease

BackgroundHuangkui capsule (HKC), as an ethanol extract of Abelmoschus manihot (L.), has a significant efficacy in treatment of the patients with diabetic kidney disease (DKD). The bioactive ingredients of HKC mainly include the flavonoids such as rutin, hyperoside, hibifolin, isoquercetin, myricetin, quercetin and quercetin-3-O-robinobioside. PurposeTo explore the molecular mechanisms of A. manihot in treatment of DKD. Study designA single-cell RNA sequencing analysis of kidneys in db/db mice with and without HKC administration. MethodsUrinary biochemical and histopathological examination in C57BL/6 and db/db mice of DKD and HKC groups was done. Single-cell RNA sequencing pipeline was then performed. The regulatory mechanisms of seven flavonoids in HKC were revealed by cell communication, prediction of transcription factor regulatory network, and molecular docking. ResultsBy constructing ligand-receptor regulatory network and performing molecular docking between 75 receptors with different activities and seven flavonoids. 11 key receptors in 4 cell types (segment 3 proximal convoluted tubular cell, ascending limbs of the loop of Henle, distal convoluted tubule, and T cell) in kidneys were found to be directly interacted with HKC. The interactions regulated 8 downstream regulons. The docking receptors in T cell led to transcriptional event differences in the regulons such as Cebpb, Rel, Tbx21 and Klf2 and consequently affected the activation, differentiation, and infiltration of T cell, while the receptors Tgfbr1 and Ldlr in stromal cells of kidneys were closely associated with the downstream transcriptional events of renal injury and proteinuria in DKD. ConclusionThe current study provides novel information of the key receptors and regulons in renal cells for a better understanding of the cell type specific molecular mechanisms of A. manihot in treatment of DKD.

Cell-Type Specific Connectivity of Whisker-Related Sensory and Motor Cortical Input to Dorsal Striatum.

The anterior dorsolateral striatum (DLS) is heavily innervated by convergent excitatory projections from the primary motor (M1) and sensory cortex (S1) and considered an important site of sensorimotor integration. M1 and S1 corticostriatal synapses have functional differences in their connection strength with striatal spiny projection neurons (SPNs) and fast-spiking interneurons (FSIs) in the DLS and, as a result, exert distinct influences on sensory-guided behaviors. In the present study, we tested whether M1 and S1 inputs exhibit differences in the subcellular anatomical distribution of striatal neurons. We injected adeno-associated viral vectors encoding spaghetti monster fluorescent proteins (sm.FPs) into M1 and S1 in male and female mice and used confocal microscopy to generate 3D reconstructions of corticostriatal inputs to single identified SPNs and FSIs obtained through ex vivo patch clamp electrophysiology. We found that M1 and S1 dually innervate SPNs and FSIs; however, there is a consistent bias towards the M1 input in SPNs that is not found in FSIs. In addition, M1 and S1 inputs were distributed similarly across the proximal, medial, and distal regions of SPN and FSI dendrites. Notably, closely localized M1 and S1 clusters of inputs were more prevalent in SPNs than FSIs, suggesting that cortical inputs are integrated through cell-type specific mechanisms. Our results suggest that the stronger functional connectivity from M1 to SPNs compared to S1, as previously observed, is due to a higher quantity of synaptic inputs. Our results have implications for how sensorimotor integration is performed in the striatum through cell-specific differences in corticostriatal connections.

Single-cell transcriptomic atlas of goat ovarian aging

BackgroundThe ovaries are one of the first organs that undergo degenerative changes earlier in the aging process, and ovarian aging is shown by a decrease in the number and quality of oocytes. However, little is known about the molecular mechanisms of female age-related fertility decline in different types of ovarian cells during aging, especially in goats. Therefore, the aim of this study was to reveal the mechanisms driving ovarian aging in goats at single-cell resolution.ResultsFor the first time, we surveyed the single-cell transcriptomic landscape of over 27,000 ovarian cells from newborn, young and aging goats, and identified nine ovarian cell types with distinct gene-expression signatures. Functional enrichment analysis showed that ovarian cell types were involved in their own unique biological processes, such as Wnt beta-catenin signalling was enriched in germ cells, whereas ovarian steroidogenesis was enriched in granulosa cells (GCs). Further analysis showed that ovarian aging was linked to GCs-specific changes in the antioxidant system, oxidative phosphorylation, and apoptosis. Subsequently, we identified a series of dynamic genes, such as AMH, CRABP2, THBS1 and TIMP1, which determined the fate of GCs. Additionally, FOXO1, SOX4, and HIF1A were identified as significant regulons that instructed the differentiation of GCs in a distinct manner during ovarian aging.ConclusionsThis study revealed a comprehensive aging-associated transcriptomic atlas characterizing the cell type-specific mechanisms during ovarian aging at the single-cell level and offers new diagnostic biomarkers and potential therapeutic targets for age-related goat ovarian diseases.

Mapping individual cardiac cell types and their contribution in the development of heart failure in a multiple comorbidity porcine model

Abstract Background Heart failure with preserved ejection fraction (HFpEF) is characterized by impaired left ventricular (LV) relaxation and filling due to increased myocardial stiffness and hypertrophy. The development of HFpEF is associated with multiple, interrelated risk factors including obesity, chronic kidney disease (CKD), diabetes mellitus (DM), hypertension. Mechanistic studies have implied that comorbidity-driven chronic inflammation, oxidative stress, and endothelial dysfunction act as contributors to the onset and development of HFpEF. Multiple studies that support this HFpEF paradigm highlight the complexity of the disease with different pathways active in and across different cell types. A recently developed multiple comorbidity swine (MCS) model with DM, CKD hypercholesterolemia, demonstrate inflammation, microvascular endothelial dysfunction and LV diastolic dysfunction. This model mimics the slow progression of disease in patients and is therefore ideal to study the earliest mechanisms underlying HFpEF. Purpose Here we hypothesize that single nuclei RNA (snRNA) sequencing aid in mapping the contribution of each individual cardiac cell cluster to disease development, to improve our mechanistic understanding of HFpEF. Methods We performed snRNA sequencing to analyze the transcriptional landscape of the cardiac environment in the well validated MCS model. Frozen left ventricle myocardial samples of MCS (n=4) and healthy control (n=3) swine were used. Results The analysis included ∼100.000 individual nuclei and offers new insights in cell type specific mechanisms during HFpEF onset and development against the background of common comorbidities. We identified 34 different cell clusters, which together represent 6 major cell types. Besides cardiomyocytes (CMs), endothelial cells (ECs) and fibroblasts, a large population of immune cells and smaller populations of mural cells and neurons were present in both MCS and control swine. IPA pathway analysis predicts transcriptomic changes within the cell clusters in response to comorbidities including activation of semaphoring and estrogen signaling in the largest CM cluster (CM2) and activation of EIF2 signaling in ECs (Fig. 1). Gene set enrichment analysis confirmed the activation of HFpEF paradigm pathways in the MCS dataset. Overlap of human HFpEF heart bulk RNA sequencing data and the MCS dataset showed overlapping disease processes including interferon gamma (IFNG) signaling. Moreover, IFNG signaling and IFNG associated transcription factor STAT1 were activated in most of the CM and EC clusters. Conclusion These findings suggest an essential role of multiple pathways during the onset and development of HFpEF. Further improvement of our understanding of underlying molecular mechanisms and crosstalk between cell populations, could pinpoint drug targets and aid the development of new treatments for HFpEF.Top 5 IPA predicted canonical pathways

Integrative Transcriptome and Proteome Profiling of Insulin-Resistant Kidney Cell Models and Patient Biopsies Reveals Common and Cell Type-Specific Mechanisms Underpinning Diabetic Kidney Disease

Integrative Transcriptome and Proteome Profiling of Insulin-Resistant Kidney Cell Models and Patient Biopsies Reveals Common and Cell Type-Specific Mechanisms Underpinning Diabetic Kidney Disease

Cell type-specific molecular mechanisms and implications of necroptosis in inflammatory respiratory diseases.

Necroptosis is generally considered as an inflammatory cell death form. The core regulators of necroptotic signaling are receptor-interacting serine-threonine protein kinases 1 (RIPK1) and RIPK3, and the executioner, mixed lineage kinase domain-like pseudokinase (MLKL). Evidence demonstrates that necroptosis contributes profoundly to inflammatory respiratory diseases that are common public health problem. Necroptosis occurs in nearly all pulmonary cell types in the settings of inflammatory respiratory diseases. The influence of necroptosis on cells varies depending upon the type of cells, tissues, organs, etc., which is an important factor to consider. Thus, in this review, we briefly summarize the current state of knowledge regarding the biology of necroptosis, and focus on the key molecular mechanisms that define the necroptosis status of specific cell types in inflammatory respiratory diseases. We also discuss the clinical potential of small molecular inhibitors of necroptosis in treating inflammatory respiratory diseases, and describe the pathological processes that engage cross talk between necroptosis and other cell death pathways in the context of respiratory inflammation. The rapid advancement of single-cell technologies will help understand the key mechanisms underlying cell type-specific necroptosis that are critical to effectively treat pathogenic lung infections and inflammatory respiratory diseases.

Impaired autophagy in the lower airways and lung parenchyma in stable COPD.

There is increasing evidence of autophagy activation in COPD, but its role is complex and probably regulated through cell type-specific mechanisms. This study aims to investigate the autophagic process at multiple levels within the respiratory system, using different methods to clarify conflicting results reported so far. This cross-sectional study was performed on bronchial biopsies and peripheral lung samples obtained from COPD patients (30 and 12 per sample type, respectively) and healthy controls (25 and 22 per sample type, respectively), divided by smoking history. Subjects were matched for age and smoking history. We analysed some of the most important proteins involved in autophagosome formation, such as LC3 and p62, as well as some molecules essential for lysosome function, such as lysosome-associated membrane protein 1 (LAMP1). Immunohistochemistry was used to assess the autophagic process in both sample types. ELISA and transcriptomic analysis were performed on lung samples. We found increased autophagic stimulus in smoking subjects, regardless of respiratory function. This was revealed by immunohistochemistry through a significant increase in LC3 (p<0.01) and LAMP1 (p<0.01) in small airway bronchiolar epithelium, alveolar septa and alveolar macrophages. Similar results were obtained in bronchial biopsy epithelium by evaluating LC3B (p<0.05), also increased in homogenate lung tissue using ELISA (p<0.05). Patients with COPD, unlike the others, showed an increase in p62 by ELISA (p<0.05). No differences were found in transcriptomics analysis. Different techniques, applied at post-transcriptional level, confirm that cigarette smoke stimulates autophagy at multiple levels inside the respiratory system, and that autophagy failure may characterise COPD.

A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation.

Communication and contact between neurons and astrocytes is important for proper brain physiology. How neuron/astrocyte crosstalk is affected by intraneuronal tau aggregation in neurodegenerative tauopathies is largely elusive. Human induced pluripotent stem cell (iPSC)-derived neurons provide the opportunity to model tau pathology in a translationally relevant in vitro context. However, current iPSC models inefficiently develop tau aggregates, and co-culture models of tau pathology have thus far utilized rodent astrocytes. In this article, we describe the co-culture of human iPSC-derived neurons with primary human astrocytes in a 96-well format compatible with high-content microscopy. By lentiviral overexpression of different mutated tau variants, this protocol can be flexibly adapted for the efficient induction of seeded or spontaneous tau aggregation. We used this novel co-culture model to identify cell type-specific disease mechanisms and to provide proof of concept for intervention by antisense therapy. These results show that this human co-culture model provides a highly translational tool for target discovery and drug development for human tauopathies. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Human neuron/astrocyte co-culture for seeded and spontaneous intraneuronal tau aggregation Support Protocol 1: Human induced pluripotent stem cell culture Support Protocol 2: Human primary astrocyte culture.

Uncovering specific mechanisms across cell types in dynamical models.

Ordinary differential equations are frequently employed for mathematical modeling of biological systems. The identification of mechanisms that are specific to certain cell types is crucial for building useful models and to gain insights into the underlying biological processes. Regularization techniques have been proposed and applied to identify mechanisms specific to two cell types, e.g., healthy and cancer cells, including the LASSO (least absolute shrinkage and selection operator). However, when analyzing more than two cell types, these approaches are not consistent, and require the selection of a reference cell type, which can affect the results. To make the regularization approach applicable to identifying cell-type specific mechanisms in any number of cell types, we propose to incorporate the clustered LASSO into the framework of ordinary differential equation modeling by penalizing the pairwise differences of the logarithmized fold-change parameters encoding a specific mechanism in different cell types. The symmetry introduced by this approach renders the results independent of the reference cell type. We discuss the necessary adaptations of state-of-the-art numerical optimization techniques and the process of model selection for this method. We assess the performance with realistic biological models and synthetic data, and demonstrate that it outperforms existing approaches. Finally, we also exemplify its application to published biological models including experimental data, and link the results to independent biological measurements.

Whole genome deconvolution unveils Alzheimer’s resilient epigenetic signature

Assay for Transposase Accessible Chromatin by sequencing (ATAC-seq) accurately depicts the chromatin regulatory state and altered mechanisms guiding gene expression in disease. However, bulk sequencing entangles information from different cell types and obscures cellular heterogeneity. To address this, we developed Cellformer, a deep learning method that deconvolutes bulk ATAC-seq into cell type-specific expression across the whole genome. Cellformer enables cost-effective cell type-specific open chromatin profiling in large cohorts. Applied to 191 bulk samples from 3 brain regions, Cellformer identifies cell type-specific gene regulatory mechanisms involved in resilience to Alzheimer’s disease, an uncommon group of cognitively healthy individuals that harbor a high pathological load of Alzheimer’s disease. Cell type-resolved chromatin profiling unveils cell type-specific pathways and nominates potential epigenetic mediators underlying resilience that may illuminate therapeutic opportunities to limit the cognitive impact of the disease. Cellformer is freely available to facilitate future investigations using high-throughput bulk ATAC-seq data.

Abstract P1140: Acute Mechanosensitive Adaptation To Phenylephrine In Cardiomyocytes And Fibroblasts

Mechanosensitive pathways are fine tuned to the physiological environment of the cell. Adrenergic stimulation induces cell type specific responses in the heart, including cardiomyocyte hypertrophy and fibroblast proliferation. We hypothesized acute induction of hypertrophy by phenylephrine (PHE) would activate mechanosensitive pathways in fibroblasts and myocytes, revealing novel molecular features of early hypertrophic adaptation. Male mice were subcutaneously injected with 20 mg/kg of PHE or PBS every other day for one week. Echocardiography revealed significant cardiac hypertrophy with increased ejection fraction (72.45% + 6.15 PHE v 56.32 + 5.32 PBS, p&lt;0.0001) and LV wall thickness (2.25 mm + 0.29 PHE v 1.62 + 0.13 PBS, p&lt;0.0001), along with diastolic impairment, as measured by elevated E/e’ ratio (37.25 + 7.29 PHE v 23.67 + 3.62 PBS, p&lt;0.0001). Interestingly, histological analysis showed increased fibrotic deposition and cardiomyocyte cross-sectional area was preferential increased in fibrotic areas (265.5 um 2 + 67.5 PHE fibrotic v 195.1 + 47.46 PHE non-fibrotic v 165.6 + 46.96 PBS). While cardiac fibroblasts from PHE-treated animals did not show alpha smooth muscle actin staining, a traditional marker for activated myofibroblasts, fibroblasts from PHE-treated mice showed increased f-actin stress fibers and nuclear localization of Mrtfa, a mechanosensitive transcriptional coactivator. We performed RNA-seq on isolated cardiomyocytes, demonstrating activation of myocyte-specific responses to increased pressure ( Des, Nppa ), as well as activation of fibrotic genes ( Tgfbr1, Tgfbr2, Tgfb2 ) in myocytes . We sought to investigate mechanosensitive pathways initiated by PHE treatment in cardiomyocytes and identified transcriptional changes in Ankrd1, a Titin binding protein which translocates to the nucleus upon stress. We demonstrate a PHE induced increase in Ankrd1 protein expression and nuclear localization (nuclear area occupancy, as measured by microscopy, 17.04% + 8.76 PHE v 11.91% + 9.134 PBS, p=0.002) in cardiomyocytes. Together these findings demonstrate distinct acute phenotyptic adaptation to adrenergic stress in myocytes and fibroblasts, revealing cell type-specific mechanosensitive signaling mechanisms.

Motor neurons within a network use cell-type specific feedback mechanisms to constrain relationships among ion channel mRNAs.

Recently, activity has been proposed as a primary feedback mechanism used by continuously bursting neurons to coordinate ion channel mRNA relationships that underlie stable output. However, some neuron types only have intermittent periods of activity and so may require alternative mechanisms that induce and constrain the appropriate ion channel profile in different states of activity. To address this, we used the pyloric dilator (PD; constitutively active) and the lateral gastric (LG; periodically active) neurons of the stomatogastric ganglion (STG) of the crustacean Cancer borealis. We experimentally stimulated descending inputs to the STG to cause release of neuromodulators known to elicit the active state of LG neurons and quantified the mRNA abundances and pairwise relationships of 11 voltage-gated ion channels in active and silent LG neurons. The same stimulus does not significantly alter PD activity. Activation of LG upregulated ion channel mRNAs and lead to a greater number of positively correlated pairwise channel mRNA relationships. Conversely, this stimulus did not induce major changes in ion channel mRNA abundances and relationships of PD cells, suggesting their ongoing activity is sufficient to maintain channel mRNA relationships even under changing modulatory conditions. In addition, we found that ion channel mRNA correlations induced by the active state of LG are influenced by a combination of activity- and neuromodulator-dependent feedback mechanisms. Interestingly, some of these same correlations are maintained by distinct mechanisms in PD, suggesting that these motor networks use distinct feedback mechanisms to coordinate the same mRNA relationships across neuron types.NEW & NOTEWORTHY Neurons use various feedback mechanisms to adjust and maintain their output. Here, we demonstrate that different neurons within the same network can use distinct signaling mechanisms to regulate the same ion channel mRNA relationships.

Emotionally clocked out: cell-type specific regulation of mood and anxiety by the circadian clock system in the brain.

Circadian rhythms are self-sustained oscillations of biological systems that allow an organism to anticipate periodic changes in the environment and optimally align feeding, sleep, wakefulness, and the physiological and biochemical processes that support them within the 24 h cycle. These rhythms are generated at a cellular level by a set of genes, known as clock genes, which code for proteins that inhibit their own transcription in a negative feedback loop and can be perturbed by stress, a risk factor for the development of mood and anxiety disorders. A role for circadian clocks in mood and anxiety has been suggested for decades on the basis of clinical observations, and the dysregulation of circadian rhythms is a prominent clinical feature of stress-related disorders. Despite our understanding of central clock structure and function, the effect of circadian dysregulation in different neuronal subtypes in the suprachiasmatic nucleus (SCN), the master pacemaker region, as well as other brain systems regulating mood, including mesolimbic and limbic circuits, is just beginning to be elucidated. In the brain, circadian clocks regulate neuronal physiological functions, including neuronal activity, synaptic plasticity, protein expression, and neurotransmitter release which in turn affect mood-related behaviors via cell-type specific mechanisms. Both animal and human studies have revealed an association between circadian misalignment and mood disorders and suggest that internal temporal desynchrony might be part of the etiology of psychiatric disorders. To date, little work has been conducted associating mood-related phenotypes to cell-specific effects of the circadian clock disruptions. In this review, we discuss existing literature on how clock-driven changes in specific neuronal cell types might disrupt phase relationships among cellular communication, leading to neuronal circuit dysfunction and changes in mood-related behavior. In addition, we examine cell-type specific circuitry underlying mood dysfunction and discuss how this circuitry could affect circadian clock. We provide a focus for future research in this area and a perspective on chronotherapies for mood and anxiety disorders.

Cell-Specific Mechanisms in the Heart of COVID-19 Patients.

From the onset of the pandemic, evidence of cardiac involvement in acute COVID-19 abounded. Cardiac presentations ranged from arrhythmias to ischemia, myopericarditis/myocarditis, ventricular dysfunction to acute heart failure, and even cardiogenic shock. Elevated serum cardiac troponin levels were prevalent among hospitalized patients with COVID-19; the higher the magnitude of troponin elevation, the greater the COVID-19 illness severity and in-hospital death risk. Whether these consequences were due to direct SARS-CoV-2 infection of cardiac cells or secondary to inflammatory responses steered early cardiac autopsy studies. SARS-CoV-2 was reportedly detected in endothelial cells, cardiac myocytes, and within the extracellular space. However, findings were inconsistent and different methodologies had their limitations. Initial autopsy reports suggested that SARS-CoV-2 myocarditis was common, setting off studies to find and phenotype inflammatory infiltrates in the heart. Nonetheless, subsequent studies rarely detected myocarditis. Microthrombi, cardiomyocyte necrosis, and inflammatory infiltrates without cardiomyocyte damage were much more common. In vitro and ex vivo experimental platforms have assessed the cellular tropism of SARS-CoV-2 and elucidated mechanisms of viral entry into and replication within cardiac cells. Data point to pericytes as the primary target of SARS-CoV-2 in the heart. Infection of pericytes can account for the observed pericyte and endothelial cell death, innate immune response, and immunothrombosis commonly observed in COVID-19 hearts. These processes are bidirectional and synergistic, rendering a definitive order of events elusive. Single-cell/nucleus analyses of COVID-19 myocardial tissue and isolated cardiac cells have provided granular data about the cellular composition and cell type-specific transcriptomic signatures of COVID-19 and microthrombi-positive COVID-19 hearts. Still, much remains unknown and more in vivo studies are needed. This review seeks to provide an overview of the current understanding of COVID-19 cardiac pathophysiology. Cell type-specific mechanisms and the studies that provided such insights will be highlighted. Given the unprecedented pace of COVID-19 research, more mechanistic details are sure to emerge since the writing of this review. Importantly, our current knowledge offers significant clues about the cardiac pathophysiology of long COVID-19, the increased postrecovery risk of cardiac events, and thus, the future landscape of cardiovascular disease.

Human ESC-derived vascular cells promote vascular regeneration in a HIF-1α dependent manner.

Hypoxia-inducible factor (HIF-1α), a core transcription factor responding to changes in cellular oxygen levels, is closely associated with a wide range of physiological and pathological conditions. However, its differential impacts on vascular cell types and molecular programs modulating human vascular homeostasis and regeneration remain largely elusive. Here, we applied CRISPR/Cas9-mediated gene editing of human embryonic stem cells and directed differentiation to generate HIF-1α-deficient human vascular cells including vascular endothelial cells, vascular smooth muscle cells, and mesenchymal stem cells (MSCs), as a platform for discovering cell type-specific hypoxia-induced response mechanisms. Through comparative molecular profiling across cell types under normoxic and hypoxic conditions, we provide insight into the indispensable role of HIF-1α in the promotion of ischemic vascular regeneration. We found human MSCs to be the vascular cell type most susceptible to HIF-1α deficiency, and that transcriptional inactivation of ANKZF1, an effector of HIF-1α, impaired pro-angiogenic processes. Altogether, our findings deepen the understanding of HIF-1α in human angiogenesis and support further explorations of novel therapeutic strategies of vascular regeneration against ischemic damage.

Abstract LB176: Interrogating the single cell immune checkpoint inhibition atlas to decipher ICI response mechanisms

Abstract Introduction: Immune Checkpoint Inhibition (ICI) therapies have demonstrated significant clinical success in multiple cancer types. However, many patients do not develop durable responses to ICI treatments and the underlying mechanisms of action are not fully resolved. Recent single cell RNA-seq (scRNA-seq) studies provide a window into the complex interplay between cancer and heterogenous immune cell populations underlying ICI response. However, individual studies are typically focused on specific therapies in specific cancer types making it challenging to identify universal mechanisms and response markers. Approach: To facilitate a more comprehensive study of cell-type specific ICI mechanisms and response determinants, we developed the Single Cell Immune Checkpoint Inhibition Atlas (ICI Atlas) which integrates 27 scRNA-seq datasets (including 17 human and 10 mice datasets) spanning close to 1.9 million cells. Of the 27 datasets, 25 contain both pre- and post-treatment samples, one contains only pre-treatment samples and one contains only post-treatment samples. The Atlas spans over 30 cell types and 16 different cancer types as well as 20 treatment strategies spanning 11 ICI therapies and 28 other drugs. Results: To begin exploring the cell-type specific responses to ICI, we conducted comparative analyses of the ICI Atlas T cells in 4 groups: 1) responders vs. non-responders pre-treatment; 2) responders vs. non-responders post-treatment; 3) responders post-treatment vs. untreated; and 4) non-responders post-treatment vs. untreated leading to 62 pairwise comparisons across ICI Atlas datasets and treatment strategies. First, we found that T cells were either more abundant or more differentiated after ICI treatment and were more differentiated or more abundant in responders (post-treatment) compared to non-responders. We also found that genes previously identified in a meta-analysis of bulk mRNA datasets (Litchfield et al. 2021) as being effective predictors of ICI response are also more highly expressed in T cells of responders post-treatment compared to non-responders or compared to untreated subjects. Using an IFN-related gene signature, we found that post treatment as compared to baseline, IFN genes increased more substantially in responders than in non-responders. Across a large set of studies, we found that the IFN signature clusters T cells into IFN-effector/producers (effector/activated T cells) and IFN-insensitive cells (naive/memory T cells). Finally, using Atlas-derived ICI response signatures in T cells and data from a recently published study of mismatch repair deficient colorectal cancers (MMRd CRC, Pelka et al. 2021), we showed that T cells from MMRd CRC patients recapitulate signatures of ICI responders. Our results suggest that large-scale scRNA-seq atlases can serve as valuable tools for corroborating existing hypotheses, as well as providing key new insights into cell-type specific mechanisms underlying ICI therapy response. Citation Format: Piotr Topolewski, Aleksandra Możwiłło, Marlena Osipowicz, Roy Ronen, Janusz Dutkowski. Interrogating the single cell immune checkpoint inhibition atlas to decipher ICI response mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB176.

Genetic variation in cis-regulatory domains suggests cell type-specific regulatory mechanisms in immunity

Studying the interplay between genetic variation, epigenetic changes, and regulation of gene expression is crucial to understand the modification of cellular states in various conditions, including immune diseases. In this study, we characterize the cell-specificity in three key cells of the human immune system by building cis maps of regulatory regions with coordinated activity (CRDs) from ChIP-seq peaks and methylation data. We find that only 33% of CRD-gene associations are shared between cell types, revealing how similarly located regulatory regions provide cell-specific modulation of gene activity. We emphasize important biological mechanisms, as most of our associations are enriched in cell-specific transcription factor binding sites, blood-traits, and immune disease-associated loci. Notably, we show that CRD-QTLs aid in interpreting GWAS findings and help prioritize variants for testing functional hypotheses within human complex diseases. Additionally, we map trans CRD regulatory associations, and among 207 trans-eQTLs discovered, 46 overlap with the QTLGen Consortium meta-analysis in whole blood, showing that mapping functional regulatory units using population genomics allows discovering important mechanisms in the regulation of gene expression in immune cells. Finally, we constitute a comprehensive resource describing multi-omics changes to gain a greater understanding of cell-type specific regulatory mechanisms of immunity.

The biotoxin BMAA promotes dysfunction via distinct mechanisms in neuroblastoma and glioblastoma cells.

Chronic exposure to the Cyanobacteria biotoxin Beta-methylamino-L-alanine (BMAA) has been associated with development of a sporadic form of ALS called Amyotrophic Lateral Sclerosis/Parkinsonism-Dementia Complex (ALS/PDC), as observed within certain Indigenous populations of Guam and Japan. Studies in primate models and cell culture have supported the association of BMAA with ALS/PDC, yet the pathological mechanisms at play remain incompletely characterized, effectively stalling the development of rationally-designed therapeutics or application of preventative measures for this disease. In this study we demonstrate for the first time that sub-excitotoxic doses of BMAA modulate the canonical Wnt signaling pathway to drive cellular defects in human neuroblastoma cells, suggesting a potential mechanism by which BMAA may promote neurological disease. Further, we demonstrate here that the effects of BMAA can be reversed in cell culture by use of pharmacological modulators of the Wnt pathway, revealing the potential value of targeting this pathway therapeutically. Interestingly, our results suggest the existence of a distinct Wnt-independent mechanism activated by BMAA in glioblastoma cells, highlighting the likelihood that neurological disease may result from the cumulative effects of distinct cell-type specific mechanisms of BMAA toxicity.