SLX4IP Antagonizes Promiscuous BLM Activity during ALT Maintenance.

Cancer cells acquire unlimited proliferative capacity by either re-expressing telomerase or inducing alternative lengthening of telomeres (ALT), which relies on telomere recombination. Here, we show that ALT recombination requires coordinate regulation of the SMX and BTR complexes to ensure the appropriate balance of resolution and dissolution activities at recombining telomeres. Critical to this control is SLX4IP, which accumulates at ALT telomeres and interacts with SLX4, XPF, and BLM. Loss of SLX4IP increases ALT-related phenotypes, which is incompatible with cell growth following concomitant loss of SLX4. Inactivation of BLM is sufficient to rescue telomere aggregation and the synthetic growth defect in this context, suggesting that SLX4IP favors SMX-dependent resolution by antagonizing promiscuous BLM activity during ALT recombination. Finally, we show that SLX4IP is inactivated in a subset of ALT-positive osteosarcomas. Collectively, our findings uncover an SLX4IP-dependent regulatory mechanism critical for telomere maintenance in ALT cancer cells.

SDE2 is an essential gene required for ribosome biogenesis and the regulation of alternative splicing.

RNA provides the framework for the assembly of some of the most intricate macromolecular complexes within the cell, including the spliceosome and the mature ribosome. The assembly of these complexes relies on the coordinated association of RNA with hundreds of trans-acting protein factors. While some of these trans-acting factors are RNA-binding proteins (RBPs), others are adaptor proteins, and others still, function as both. Defects in the assembly of these complexes results in a number of human pathologies including neurodegeneration and cancer. Here, we demonstrate that Silencing Defective 2 (SDE2) is both an RNA binding protein and also a trans-acting adaptor protein that functions to regulate RNA splicing and ribosome biogenesis. SDE2 depletion leads to widespread changes in alternative splicing, defects in ribosome biogenesis and ultimately complete loss of cell viability. Our data highlight SDE2 as a previously uncharacterized essential gene required for the assembly and maturation of the complexes that carry out two of the most fundamental processes in mammalian cells.

Single molecule, long-read Apoer2 sequencing identifies conserved and species-specific splicing patterns.

Apolipoprotein E receptor 2 (Apoer2) is a synaptic receptor in the brain that binds disease-relevant ligand Apolipoprotein E (Apoe) and is highly alternatively spliced. We examined alternative splicing (AS) of conserved Apoer2 exons across vertebrate species and identified gain of exons in mammals encoding functional domains such as the cytoplasmic and furin inserts, and loss of an exon in primates encoding the eighth LDLa repeat, likely altering receptor surface levels and ligand-binding specificity. We utilized single molecule, long-read RNA sequencing to profile full-length Apoer2 isoforms and identified 68 and 48 unique full-length Apoer2 transcripts in the mouse and human cerebral cortex, respectively. Furthermore, we identified two exons encoding protein functional domains, the third EGF-precursor like repeat and glycosylation domain, that are tandemly skipped specifically in mouse. Our study provides new insight into Apoer2 isoform complexity in the vertebrate brain and highlights species-specific differences in splicing decisions that support functional diversity.

Direct recruitment of polycomb repressive complex 1 to chromatin by core binding transcription factors.

Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFß transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFß and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFß deficiencies generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via PRC2-independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.

Evaluation of logistic regression models and effect of covariates for case-control study in RNA-Seq analysis.

BACKGROUND: Next generation sequencing provides a count of RNA molecules in the form of short reads, yielding discrete, often highly non-normally distributed gene expression measurements. Although Negative Binomial (NB) regression has been generally accepted in the analysis of RNA sequencing (RNA-Seq) data, its appropriateness has not been exhaustively evaluated. We explore logistic regression as an alternative method for RNA-Seq studies designed to compare cases and controls, where disease status is modeled as a function of RNA-Seq reads using simulated and Huntington disease data. We evaluate the effect of adjusting for covariates that have an unknown relationship with gene expression. Finally, we incorporate the data adaptive method in order to compare false positive rates. RESULTS: When the sample size is small or the expression levels of a gene are highly dispersed, the NB regression shows inflated Type-I error rates but the Classical logistic and Bayes logistic (BL) regressions are conservative. Firth's logistic (FL) regression performs well or is slightly conservative. Large sample size and low dispersion generally make Type-I error rates of all methods close to nominal alpha levels of 0.05 and 0.01. However, Type-I error rates are controlled after applying the data adaptive method. The NB, BL, and FL regressions gain increased power with large sample size, large log2 fold-change, and low dispersion. The FL regression has comparable power to NB regression. CONCLUSIONS: We conclude that implementing the data adaptive method appropriately controls Type-I error rates in RNA-Seq analysis. Firth's logistic regression provides a concise statistical inference process and reduces spurious associations from inaccurately estimated dispersion parameters in the negative binomial framework.

Novel microRNA discovery using small RNA sequencing in post-mortem human brain.

BACKGROUND: MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression mainly through translational repression of target mRNA molecules. More than 2700 human miRNAs have been identified and some are known to be associated with disease phenotypes and to display tissue-specific patterns of expression. METHODS: We used high-throughput small RNA sequencing to discover novel miRNAs in 93 human post-mortem prefrontal cortex samples from individuals with Huntington's disease (n = 28) or Parkinson's disease (n = 29) and controls without neurological impairment (n = 36). A custom miRNA identification analysis pipeline was built, which utilizes miRDeep* miRNA identification and result filtering based on false positive rate estimates. RESULTS: Ninety-nine novel miRNA candidates with a false positive rate of less than 5 % were identified. Thirty-four of the candidate miRNAs show sequence similarity with known mature miRNA sequences and may be novel members of known miRNA families, while the remaining 65 may constitute previously undiscovered families of miRNAs. Nineteen of the 99 candidate miRNAs were replicated using independent, publicly-available human brain RNA-sequencing samples, and seven were experimentally validated using qPCR. CONCLUSIONS: We have used small RNA sequencing to identify 99 putative novel miRNAs that are present in human brain samples.

Extensive changes in DNA methylation are associated with expression of mutant huntingtin.

The earliest stages of Huntington disease are marked by changes in gene expression that are caused in an indirect and poorly understood manner by polyglutamine expansions in the huntingtin (HTT) protein. To explore the hypothesis that DNA methylation may be altered in cells expressing mutated HTT, we use reduced representation bisulfite sequencing (RRBS) to map sites of DNA methylation in cells carrying either wild-type or mutant HTT. We find that a large fraction of the genes that change in expression in the presence of mutant huntingtin demonstrate significant changes in DNA methylation. Regions with low CpG content, which have previously been shown to undergo methylation changes in response to neuronal activity, are disproportionately affected. On the basis of the sequence of regions that change in methylation, we identify AP-1 and SOX2 as transcriptional regulators associated with DNA methylation changes, and we confirm these hypotheses using genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq). Our findings suggest new mechanisms for the effects of polyglutamine-expanded HTT. These results also raise important questions about the potential effects of changes in DNA methylation on neurogenesis and cognitive decline in patients with Huntington disease.

Targeting H3K4 trimethylation in Huntington disease.

Transcriptional dysregulation is an early feature of Huntington disease (HD). We observed gene-specific changes in histone H3 lysine 4 trimethylation (H3K4me3) at transcriptionally repressed promoters in R6/2 mouse and human HD brain. Genome-wide analysis showed a chromatin signature for this mark. Reducing the levels of the H3K4 demethylase SMCX/Jarid1c in primary neurons reversed down-regulation of key neuronal genes caused by mutant Huntingtin expression. Finally, reduction of SMCX/Jarid1c in primary neurons from BACHD mice or the single Jarid1 in a Drosophila HD model was protective. Therefore, targeting this epigenetic signature may be an effective strategy to ameliorate the consequences of HD.

Linking proteomic and transcriptional data through the interactome and epigenome reveals a map of oncogene-induced signaling.

Cellular signal transduction generally involves cascades of post-translational protein modifications that rapidly catalyze changes in protein-DNA interactions and gene expression. High-throughput measurements are improving our ability to study each of these stages individually, but do not capture the connections between them. Here we present an approach for building a network of physical links among these data that can be used to prioritize targets for pharmacological intervention. Our method recovers the critical missing links between proteomic and transcriptional data by relating changes in chromatin accessibility to changes in expression and then uses these links to connect proteomic and transcriptome data. We applied our approach to integrate epigenomic, phosphoproteomic and transcriptome changes induced by the variant III mutation of the epidermal growth factor receptor (EGFRvIII) in a cell line model of glioblastoma multiforme (GBM). To test the relevance of the network, we used small molecules to target highly connected nodes implicated by the network model that were not detected by the experimental data in isolation and we found that a large fraction of these agents alter cell viability. Among these are two compounds, ICG-001, targeting CREB binding protein (CREBBP), and PKF118-310, targeting ß-catenin (CTNNB1), which have not been tested previously for effectiveness against GBM. At the level of transcriptional regulation, we used chromatin immunoprecipitation sequencing (ChIP-Seq) to experimentally determine the genome-wide binding locations of p300, a transcriptional co-regulator highly connected in the network. Analysis of p300 target genes suggested its role in tumorigenesis. We propose that this general method, in which experimental measurements are used as constraints for building regulatory networks from the interactome while taking into account noise and missing data, should be applicable to a wide range of high-throughput datasets.

The CoREST repressor complex mediates phenotype switching and therapy resistance in melanoma.

Virtually all patients with BRAF-mutant melanoma develop resistance to MAPK inhibitors largely through nonmutational events. Although the epigenetic landscape is shown to be altered in therapy-resistant melanomas and other cancers, a specific targetable epigenetic mechanism has not been validated. Here, we evaluated the corepressor for element 1-silencing transcription factor (CoREST) epigenetic repressor complex and the recently developed bivalent inhibitor corin within the context of melanoma phenotype plasticity and therapeutic resistance. We found that CoREST was a critical mediator of the major distinct melanoma phenotypes and that corin treatment of melanoma cells led to phenotype reprogramming. Global assessment of transcript and chromatin changes conferred by corin revealed specific effects on histone marks connected to epithelial-mesenchymal transition-associated (EMT-associated) transcription factors and the dual-specificity phosphatases (DUSPs). Remarkably, treatment of BRAF inhibitor-resistant (BRAFi-R) melanomas with corin promoted resensitization to BRAFi therapy. DUSP1 was consistently downregulated in BRAFi-R melanomas, which was reversed by corin treatment and associated with inhibition of p38 MAPK activity and resensitization to BRAFi therapies. Moreover, this activity was recapitulated by the p38 MAPK inhibitor BIRB 796. These findings identify the CoREST repressor complex as a central mediator of melanoma phenotype plasticity and resistance to targeted therapy and suggest that CoREST inhibitors may prove beneficial for patients with BRAFi-resistant melanoma.

Inflammation and neuronal gene expression changes differ in early versus late chronic traumatic encephalopathy brain.

BACKGROUND: Our understanding of the molecular underpinnings of chronic traumatic encephalopathy (CTE) and its associated pathology in post-mortem brain is incomplete. Factors including years of play and genetic risk variants influence the extent of tau pathology associated with disease expression, but how these factors affect gene expression, and whether those effects are consistent across the development of disease, is unknown. METHODS: To address these questions, we conducted an analysis of the largest post-mortem brain CTE mRNASeq whole-transcriptome dataset available to date. We examined the genes and biological processes associated with disease by comparing individuals with CTE with control individuals with a history of repetitive head impacts that lack CTE pathology. We then identified genes and biological processes associated with total years of play as a measure of exposure, amount of tau pathology present at time of death, and the presence of APOE and TMEM106B risk variants. Samples were stratified into low and high pathology groups based on McKee CTE staging criteria to model early versus late changes in response to exposure, and the relative effects associated with these factors were compared between these groups. RESULTS: Substantial gene expression changes were associated with severe disease for most of these factors, primarily implicating diverse, strongly involved neuroinflammatory and neuroimmune processes. In contrast, low pathology groups had many fewer genes and processes implicated and show striking differences for some factors when compared with severe disease. Specifically, gene expression associated with amount of tau pathology showed a nearly perfect inverse relationship when compared between these two groups. CONCLUSIONS: Together, these results suggest the early CTE disease process may be mechanistically different than what occurs in late stages, that total years of play and tau pathology influence disease expression differently, and that related pathology-modifying risk variants may do so via distinct biological pathways.

Widespread perturbation of ETS factor binding sites in cancer.

Although >90% of somatic mutations reside in non-coding regions, few have been reported as cancer drivers. To predict driver non-coding variants (NCVs), we present a transcription factor (TF)-aware burden test based on a model of coherent TF function in promoters. We apply this test to NCVs from the Pan-Cancer Analysis of Whole Genomes cohort and predict 2555 driver NCVs in the promoters of 813 genes across 20 cancer types. These genes are enriched in cancer-related gene ontologies, essential genes, and genes associated with cancer prognosis. We find that 765 candidate driver NCVs alter transcriptional activity, 510 lead to differential binding of TF-cofactor regulatory complexes, and that they primarily impact the binding of ETS factors. Finally, we show that different NCVs within a promoter often affect transcriptional activity through shared mechanisms. Our integrated computational and experimental approach shows that cancer NCVs are widespread and that ETS factors are commonly disrupted.

Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia.

Activated microglia release extracellular vesicles (EVs) as modulators of brain homeostasis and innate immunity. However, the molecules critical for regulating EV production from microglia are poorly understood. Here we establish a murine microglial cell model to monitor EV secretion by measuring the fluorescence signal of tdTomato, which is linked to tetraspanin CD63. Stimulation of tdTomato+ cells with ATP induces rapid secretion of EVs and a reduction in cellular tdTomato intensity, reflecting EV secretion. We generate a GFP+ tdTomato+ cell library expressing TurboGFP and barcoded short hairpin RNAs for genome-wide screening using next-generation sequencing. We identify Mcfd2, Sepp1, and Sdc1 as critical regulators of ATP-induced EV secretion from murine microglia. Small interfering RNA (siRNA-based) silencing of each of these genes suppresses lipopolysaccharide- and ATP-induced inflammasome activation, as determined by interleukin-1ß release from primary cultured murine microglia. These molecules are critical for microglial EV secretion and are potential therapeutic targets for neuroinflammatory disorders.

MicroRNA Alterations in Chronic Traumatic Encephalopathy and Amyotrophic Lateral Sclerosis.

Repetitive head impacts (RHI) and traumatic brain injuries are risk factors for the neurodegenerative diseases chronic traumatic encephalopathy (CTE) and amyotrophic lateral sclerosis (ALS). ALS and CTE are distinct disorders, yet in some instances, share pathology, affect similar brain regions, and occur together. The pathways involved and biomarkers for diagnosis of both diseases are largely unknown. MicroRNAs (miRNAs) involved in gene regulation may be altered in neurodegeneration and be useful as stable biomarkers. Thus, we set out to determine associations between miRNA levels and disease state within the prefrontal cortex in a group of brain donors with CTE, ALS, CTE + ALS and controls. Of 47 miRNAs previously implicated in neurological disease and tested here, 28 (60%) were significantly different between pathology groups. Of these, 21 (75%) were upregulated in both ALS and CTE, including miRNAs involved in inflammatory, apoptotic, and cell growth/differentiation pathways. The most significant change occurred in miR-10b, which was significantly increased in ALS, but not CTE or CTE + ALS. Overall, we found patterns of miRNA expression that are common and unique to CTE and ALS and that suggest shared and distinct mechanisms of pathogenesis.

Differential gene expression in the cortical sulcus compared to the gyral crest within the early stages of chronic traumatic encephalopathy.

Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative tauopathy found in individuals with a history of repetitive head impacts (RHI). Previous work has demonstrated that neuroinflammation is involved in CTE pathogenesis, however, the specific inflammatory mechanisms are still unclear. Here, using RNA-sequencing and gene set enrichment analysis (GSEA), we investigated the genetic changes found in tissue taken from the region CTE pathology is first found, the cortical sulcus, and compared it to neighboring gryal crest tissue to identify what pathways were directly related to initial hyperphosphorylated tau (p-tau) deposition. 21 cases were chosen for analysis: 6 cases had no exposure to RHI or presence of neurodegenerative disease (Control), 5 cases had exposure to RHI but no presence of neurodegenerative disease (RHI), and 10 cases had exposure to RHI and low stage CTE (CTE). Two sets of genes were identified: genes that changed in both the sulcus and crest and genes that changed specifically in the sulcus relative to the crest. When examining genes that changed in both the sulcus and crest, GSEA demonstrated an increase in immune related processes and a decrease in neuronal processes in RHI and CTE groups. Sulcal specific alterations were observed to be driven by three mechanisms: anatomy, RHI, or p-tau. First, we observed consistent sulcal specific alterations in immune, extracellular matrix, vascular, neuronal, and endocytosis/exocytosis categories across all groups, suggesting the sulcus has a unique molecular signature compared to the neighboring crest independent of pathology. Second, individuals with a history of RHI demonstrated impairment in metabolic and mitochondrial related processes. Finally, in individuals with CTE, we observed impairment of immune and phagocytic related processes. Overall, this work provides the first observation of biological processes specifically altered in the sulcus that could be directly implicated in CTE pathogenesis and provide novel targets for biomarkers and therapies.

Gene expression in the dorsolateral and ventromedial prefrontal cortices implicates immune-related gene networks in PTSD.

Studies evaluating neuroimaging, genetically predicted gene expression, and pre-clinical genetic models of PTSD, have identified PTSD-related abnormalities in the prefrontal cortex (PFC) of the brain, particularly in dorsolateral and ventromedial PFC (dlPFC and vmPFC). In this study, RNA sequencing was used to examine gene expression in the dlPFC and vmPFC using tissue from the VA National PTSD Brain Bank in donors with histories of PTSD with or without depression (dlPFC n = 38, vmPFC n = 35), depression cases without PTSD (n = 32), and psychopathology-free controls (dlPFC n = 24, vmPFC n = 20). Analyses compared PTSD cases to controls. Follow-up analyses contrasted depression cases to controls. Twenty-one genes were differentially expressed in PTSD after strict multiple testing correction. PTSD-associated genes with roles in learning and memory (FOS, NR4A1), immune regulation (CFH, KPNA1) and myelination (MBP, MOBP, ERMN) were identified. PTSD-associated genes partially overlapped depression-associated genes. Co-expression network analyses identified PTSD-associated networks enriched for immune-related genes across the two brain regions. However, the immune-related genes and association patterns were distinct. The immune gene IL1B was significantly associated with PTSD in candidate-gene analysis and was an upstream regulator of PTSD-associated genes in both regions. There was evidence of replication of dlPFC associations in an independent cohort from a recent study, and a strong correlation between the dlPFC PTSD effect sizes for significant genes in the two studies (r = 0.66, p < 2.2 × 10-16). In conclusion, this study identified several novel PTSD-associated genes and brain region specific PTSD-associated immune-related networks.

Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii.

BACKGROUND: Genome-wide computational analysis of alternative splicing (AS) in several flowering plants has revealed that pre-mRNAs from about 30% of genes undergo AS. Chlamydomonas, a simple unicellular green alga, is part of the lineage that includes land plants. However, it diverged from land plants about one billion years ago. Hence, it serves as a good model system to study alternative splicing in early photosynthetic eukaryotes, to obtain insights into the evolution of this process in plants, and to compare splicing in simple unicellular photosynthetic and non-photosynthetic eukaryotes. We performed a global analysis of alternative splicing in Chlamydomonas reinhardtii using its recently completed genome sequence and all available ESTs and cDNAs. RESULTS: Our analysis of AS using BLAT and a modified version of the Sircah tool revealed AS of 498 transcriptional units with 611 events, representing about 3% of the total number of genes. As in land plants, intron retention is the most prevalent form of AS. Retained introns and skipped exons tend to be shorter than their counterparts in constitutively spliced genes. The splice site signals in all types of AS events are weaker than those in constitutively spliced genes. Furthermore, in alternatively spliced genes, the prevalent splice form has a stronger splice site signal than the non-prevalent form. Analysis of constitutively spliced introns revealed an over-abundance of motifs with simple repetitive elements in comparison to introns involved in intron retention. In almost all cases, AS results in a truncated ORF, leading to a coding sequence that is around 50% shorter than the prevalent splice form. Using RT-PCR we verified AS of two genes and show that they produce more isoforms than indicated by EST data. All cDNA/EST alignments and splice graphs are provided in a website at http://combi.cs.colostate.edu/as/chlamy. CONCLUSIONS: The extent of AS in Chlamydomonas that we observed is much smaller than observed in land plants, but is much higher than in simple unicellular heterotrophic eukaryotes. The percentage of different alternative splicing events is similar to flowering plants. Prevalence of constitutive and alternative splicing in Chlamydomonas, together with its simplicity, many available public resources, and well developed genetic and molecular tools for this organism make it an excellent model system to elucidate the mechanisms involved in regulated splicing in photosynthetic eukaryotes.

Prediction of genome-wide effects of single nucleotide variants on transcription factor binding.

Single nucleotide variants (SNVs) located in transcriptional regulatory regions can result in gene expression changes that lead to adaptive or detrimental phenotypic outcomes. Here, we predict gain or loss of binding sites for 741 transcription factors (TFs) across the human genome. We calculated 'gainability' and 'disruptability' scores for each TF that represent the likelihood of binding sites being created or disrupted, respectively. We found that functional cis-eQTL SNVs are more likely to alter TF binding sites than rare SNVs in the human population. In addition, we show that cancer somatic mutations have different effects on TF binding sites from different TF families on a cancer-type basis. Finally, we discuss the relationship between these results and cancer mutational signatures. Altogether, we provide a blueprint to study the impact of SNVs derived from genetic variation or disease association on TF binding to gene regulatory regions.

A glycomics and proteomics study of aging and Parkinson's disease in human brain.

Previous studies on Parkinson's disease mechanisms have shown dysregulated extracellular transport of α-synuclein and growth factors in the extracellular space. In the human brain these consist of perineuronal nets, interstitial matrices, and basement membranes, each composed of a set of collagens, non-collagenous glycoproteins, proteoglycans, and hyaluronan. The manner by which amyloidogenic proteins spread extracellularly, become seeded, oligomerize, and are taken up by cells, depends on intricate interactions with extracellular matrix molecules. We sought to assess the alterations to structure of glycosaminoglycans and proteins that occur in PD brain relative to controls of similar age. We found that PD differs markedly from normal brain in upregulation of extracellular matrix structural components including collagens, proteoglycans and glycosaminoglycan binding molecules. We also observed that levels of hemoglobin chains, possibly related to defects in iron metabolism, were enriched in PD brains. These findings shed important new light on disease processes that occur in association with PD.

Pneumonia recovery reprograms the alveolar macrophage pool.

Community-acquired pneumonia is a widespread disease with significant morbidity and mortality. Alveolar macrophages are tissue-resident lung cells that play a crucial role in innate immunity against bacteria that cause pneumonia. We hypothesized that alveolar macrophages display adaptive characteristics after resolution of bacterial pneumonia. We studied mice 1 to 6 months after self-limiting lung infections with Streptococcus pneumoniae, the most common cause of bacterial pneumonia. Alveolar macrophages, but not other myeloid cells, recovered from the lung showed long-term modifications of their surface marker phenotype. The remodeling of alveolar macrophages was (a) long-lasting (still observed 6 months after infection), (b) regionally localized (observed only in the affected lobe after lobar pneumonia), and (c) associated with macrophage-dependent enhanced protection against another pneumococcal serotype. Metabolomic and transcriptomic profiling revealed that alveolar macrophages of mice that recovered from pneumonia had new baseline activities and altered responses to infection that better resembled those of adult humans. The enhanced lung protection after mild and self-limiting bacterial respiratory infections includes a profound remodeling of the alveolar macrophage pool that is long-lasting; compartmentalized; and manifest across surface receptors, metabolites, and both resting and stimulated transcriptomes.