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The evolution of T cell molecular signatures in the distal lung of patients with severe pneumonia is understudied. Here, we analyzed T cell subsets in longitudinal bronchoalveolar lavage fluid samples from 273 patients with severe pneumonia, including unvaccinated patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or with respiratory failure not linked to pneumonia. In patients with SARS-CoV-2 pneumonia, activation of interferon signaling pathways, low activation of the NF-κB pathway and preferential targeting of spike and nucleocapsid proteins early after intubation were associated with favorable outcomes, whereas loss of interferon signaling, activation of NF-κB-driven programs and specificity for the ORF1ab complex late in disease were associated with mortality. These results suggest that in patients with severe SARS-CoV-2 pneumonia, alveolar T cell interferon responses targeting structural SARS-CoV-2 proteins characterize individuals who recover, whereas responses against nonstructural proteins and activation of NF-κB are associated with poor outcomes., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

FOXP3+ regulatory T (Treg) cells are necessary to coordinate resolution of lung inflammation and a return to homeostasis after respiratory viral infections, but the specific molecular requirements for these functions and the cell types governed by Treg cells remain unclear. This question holds significance as clinical trials of Treg cell transfer therapy for respiratory viral infection are being planned and executed. Here, we report causal experiments in mice determining that Treg cells are necessary to control the numbers of activated CD8+ T cells during recovery from influenza infection. Using a genetic strategy paired with adoptive transfer techniques, we determined that Treg cells require the transcription factor TBET to regulate these potentially pro-inflammatory CD8+ T cells. Surprisingly, we found that Treg cells are dispensable for the generation of CD8+ lung tissue resident-memory T (Trm) cells yet similarly influence the transcriptional programming of CD8+ Trm and activated T cells. Our study highlights the role of Treg cells in regulating the CD8+ T cell response during recovery from influenza infection., Competing Interests: Competing Interest Statement: NM is currently an employee and owns stock in Vertex Pharmaceuticals. BDS holds United States Patent No. US 10,905,706 B2, Compositions and Methods to Accelerate Resolution of Acute Lung Inflammation, and serves on the Scientific Advisory Board of Zoe Biosciences. The other authors have no competing interests to declare.

Newborn mammalian cardiomyocytes quickly transition from a fetal to an adult phenotype that utilizes mitochondrial oxidative phosphorylation but loses mitotic capacity. We tested whether forced reversal of adult cardiomyocytes back to a fetal glycolytic phenotype would restore proliferative capacity. We deleted Uqcrfs1 (mitochondrial Rieske iron-sulfur protein, RISP) in hearts of adult mice. As RISP protein decreased, heart mitochondrial function declined, and glucose utilization increased. Simultaneously, the hearts underwent hyperplastic remodeling during which cardiomyocyte number doubled without cellular hypertrophy. Cellular energy supply was preserved, AMPK activation was absent, and mTOR activation was evident. In ischemic hearts with RISP deletion, new cardiomyocytes migrated into the infarcted region, suggesting the potential for therapeutic cardiac regeneration. RNA sequencing revealed upregulation of genes associated with cardiac development and proliferation. Metabolomic analysis revealed a decrease in α-ketoglutarate (required for TET-mediated demethylation) and an increase in S-adenosylmethionine (required for methyltransferase activity). Analysis revealed an increase in methylated CpGs near gene transcriptional start sites. Genes that were both differentially expressed and differentially methylated were linked to upregulated cardiac developmental pathways. We conclude that decreased mitochondrial function and increased glucose utilization can restore mitotic capacity in adult cardiomyocytes, resulting in the generation of new heart cells, potentially through the modification of substrates that regulate epigenetic modification of genes required for proliferation.

Introduction: Ataxia telangiectasia (A-T) is an autosomal recessive neurodegenerative disease with widespread systemic manifestations and marked variability in clinical phenotypes. In this study, we sought to determine whether transcriptomic profiling of peripheral blood mononuclear cells (PBMCs) defines subsets of individuals with A-T beyond mild and classic phenotypes, enabling identification of novel features for disease classification and treatment response to therapy., Methods: Participants with classic A-T (n = 77), mild A-T (n = 13), and unaffected controls (n = 15) were recruited from two outpatient clinics. PBMCs were isolated and bulk RNAseq was performed. Plasma was also isolated in a subset of individuals. Affected individuals were designated mild or classic based on ATM mutations and clinical and laboratory features., Results: People with classic A-T were more likely to be younger and IgA deficient and to have higher alpha-fetoprotein levels and lower % forced vital capacity compared to individuals with mild A-T. In classic A-T, the expression of genes required for V(D)J recombination was lower, and the expression of genes required for inflammatory activity was higher. We assigned inflammatory scores to study participants and found that inflammatory scores were highly variable among people with classic A-T and that higher scores were associated with lower ATM mRNA levels. Using a cell type deconvolution approach, we inferred that CD4 + T cells and CD8 + T cells were lower in number in people with classic A-T. Finally, we showed that individuals with classic A-T exhibit higher SERPINE1 (PAI-1) mRNA and plasma protein levels, irrespective of age, and higher FLT4 (VEGFR3) and IL6ST (GP130) plasma protein levels compared with mild A-T and controls., Conclusion: Using a transcriptomic approach, we identified novel features and developed an inflammatory score to identify subsets of individuals with different inflammatory phenotypes in A-T. Findings from this study could be used to help direct treatment and to track treatment response to therapy., (© 2024. The Author(s).)

The biological role of the repetitive DNA sequences in the human genome remains an outstanding question. Recent long-read human genome assemblies have allowed us to identify a function for one of these repetitive regions. We have uncovered a tandem array of conserved primate-specific retrogenes encoding the protein Elongin A3 (ELOA3), a homolog of the RNA polymerase II (RNAPII) elongation factor Elongin A (ELOA). Our genomic analysis shows that the ELOA3 gene cluster is conserved among primates and the number of ELOA3 gene repeats is variable in the human population and across primate species. Moreover, the gene cluster has undergone concerted evolution and homogenization within primates. Our biochemical studies show that ELOA3 functions as a promoter-associated RNAPII pause-release elongation factor with distinct biochemical and functional features from its ancestral homolog, ELOA. We propose that the ELOA3 gene cluster has evolved to fulfil a transcriptional regulatory function unique to the primate lineage that can be targeted to regulate cellular hyperproliferation.

Bacterial pulmonary infections are a major cause of morbidity and mortality in neonates, with less severity in older children. Previous studies demonstrated that the DNA of CD4 + T cells in the mouse lung, whose primary responsibility is to coordinate the immune response to foreign pathogens, is differentially methylated in neonates compared with juveniles. Nevertheless, the effect of this differential DNA methylation on CD4 + T cell gene expression and response to infection remains unclear. Here we treated E. coli-infected neonatal (4-day-old) and juvenile (13-day-old) mice with decitabine (DAC), a DNA methyltransferase inhibitor with broad-spectrum DNA demethylating activity, and performed simultaneous genome-wide DNA methylation and transcriptional profiling on lung CD4 + T cells. We show that juvenile and neonatal mice experienced differential demethylation in response to DAC treatment, with larger methylation differences observed in neonates. By cross-filtering differentially expressed genes between juveniles and neonates with those sites that were demethylated in neonates, we find that interferon-responsive genes such as Ifit1 are the most down-regulated methylation-sensitive genes in neonatal mice. DAC treatment shifted neonatal lung CD4 + T cells toward a gene expression program similar to that of juveniles. Following lung infection with E. coli, lung CD4 + T cells in neonatal mice exhibit epigenetic repression of important host defense pathways, which are activated by inhibition of DNA methyltransferase activity to resemble a more mature profile., (© 2023. The Author(s).)

Alveolar epithelial cell (AEC) mitochondrial (mt) DNA damage and fibrotic monocyte-derived alveolar macrophages (Mo-AMs) are implicated in the pathobiology of pulmonary fibrosis. We showed that sirtuin 3 (SIRT3), a mitochondrial protein regulating cell fate and aging, is deficient in the AECs of idiopathic pulmonary fibrosis (IPF) patients and that asbestos- and bleomycin-induced lung fibrosis is augmented in Sirt3 knockout ( Sirt3 -/- ) mice associated with AEC mtDNA damage and intrinsic apoptosis. We determined whether whole body transgenic SIRT3 overexpression ( Sirt3 Tg ) protects mice from asbestos-induced pulmonary fibrosis by mitigating lung mtDNA damage and Mo-AM recruitment. Crocidolite asbestos (100 µg/50 µL) or control was instilled intratracheally in C57Bl6 (Wild-Type) mice or Sirt3 Tg mice, and at 21 d lung fibrosis (histology, fibrosis score, Sircol assay) and lung Mo-AMs (flow cytometry) were assessed. Compared to controls, Sirt3 Tg mice were protected from asbestos-induced pulmonary fibrosis and had diminished lung mtDNA damage and Mo-AM recruitment. Further, pharmacologic SIRT3 inducers (i.e., resveratrol, viniferin, and honokiol) each diminish oxidant-induced AEC mtDNA damage in vitro and, in the case of honokiol, protection occurs in a SIRT3-dependent manner. We reason that SIRT3 preservation of AEC mtDNA is a novel therapeutic focus for managing patients with IPF and other types of pulmonary fibrosis.

The mechanistic target of rapamycin complex 1 (mTORC1) regulates metabolism and cell growth in response to nutrient, growth, and oncogenic signals. We found that mTORC1 stimulates the synthesis of the major methyl donor, S-adenosylmethionine (SAM), through the control of methionine adenosyltransferase 2 alpha (MAT2A) expression. The transcription factor c-MYC, downstream of mTORC1, directly binds to intron 1 of MAT2A and promotes its expression. Furthermore, mTORC1 increases the protein abundance of Wilms' tumor 1-associating protein (WTAP), the positive regulatory subunit of the human N 6 -methyladenosine (m 6 A) RNA methyltransferase complex. Through the control of MAT2A and WTAP levels, mTORC1 signaling stimulates m 6 A RNA modification to promote protein synthesis and cell growth. A decline in intracellular SAM levels upon MAT2A inhibition decreases m 6 A RNA modification, protein synthesis rate, and tumor growth. Thus, mTORC1 adjusts m 6 A RNA modification through the control of SAM and WTAP levels to prime the translation machinery for anabolic cell growth., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Regulatory T (Treg) cells orchestrate resolution and repair of acute lung inflammation and injury after viral pneumonia. Compared with younger patients, older individuals experience impaired recovery and worse clinical outcomes after severe viral infections, including influenza and SARS coronavirus 2 (SARS-CoV-2). Whether age is a key determinant of Treg cell prorepair function after lung injury remains unknown. Here, we showed that aging results in a cell-autonomous impairment of reparative Treg cell function after experimental influenza pneumonia. Transcriptional and DNA methylation profiling of sorted Treg cells provided insight into the mechanisms underlying their age-related dysfunction, with Treg cells from aged mice demonstrating both loss of reparative programs and gain of maladaptive programs. Strategies to restore youthful Treg cell functional programs could be leveraged as therapies to improve outcomes among older individuals with severe viral pneumonia.

Alveolar macrophages orchestrate the response to viral infections. Age-related changes in these cells may underlie the differential severity of pneumonia in older patients. We performed an integrated analysis of single-cell RNA-Seq data that revealed homogenous age-related changes in the alveolar macrophage transcriptome in humans and mice. Using genetic lineage tracing with sequential injury, heterochronic adoptive transfer, and parabiosis, we found that the lung microenvironment drove an age-related resistance of alveolar macrophages to proliferation that persisted during influenza A viral infection. Ligand-receptor pair analysis localized these changes to the extracellular matrix, where hyaluronan was increased in aged animals and altered the proliferative response of bone marrow-derived macrophages to granulocyte macrophage colony-stimulating factor (GM-CSF). Our findings suggest that strategies targeting the aging lung microenvironment will be necessary to restore alveolar macrophage function in aging.

Tregs require Foxp3 expression and induction of a specific DNA hypomethylation signature during development, after which Tregs persist as a self-renewing population that regulates immune system activation. Whether maintenance DNA methylation is required for Treg lineage development and stability and how methylation patterns are maintained during lineage self-renewal remain unclear. Here, we demonstrate that the epigenetic regulator ubiquitin-like with plant homeodomain and RING finger domains 1 (Uhrf1) is essential for maintenance of methyl-DNA marks that stabilize Treg cellular identity by repressing effector T cell transcriptional programs. Constitutive and induced deficiency of Uhrf1 within Foxp3+ cells resulted in global yet nonuniform loss of DNA methylation, derepression of inflammatory transcriptional programs, destabilization of the Treg lineage, and spontaneous inflammation. These findings support a paradigm in which maintenance DNA methylation is required in distinct regions of the Treg genome for both lineage establishment and stability of identity and suppressive function.

Background: The three-dimensional genome organization is critical for gene regulation and can malfunction in diseases like cancer. As a key regulator of genome organization, CCCTC-binding factor (CTCF) has been characterized as a DNA-binding protein with important functions in maintaining the topological structure of chromatin and inducing DNA looping. Among the prolific binding sites in the genome, several events with altered CTCF occupancy have been reported as associated with effects in physiology or disease. However, hitherto there is no comprehensive survey of genome-wide CTCF binding patterns across different human cancers., Results: To dissect functions of CTCF binding, we systematically analyze over 700 CTCF ChIP-seq profiles across human tissues and cancers and identify cancer-specific CTCF binding patterns in six cancer types. We show that cancer-specific lost and gained CTCF binding events are associated with altered chromatin interactions, partially with DNA methylation changes, and rarely with sequence mutations. While lost bindings primarily occur near gene promoters, most gained CTCF binding events exhibit enhancer activities and are induced by oncogenic transcription factors. We validate these findings in T cell acute lymphoblastic leukemia cell lines and patient samples and show that oncogenic NOTCH1 induces specific CTCF binding and they cooperatively activate expression of target genes, indicating transcriptional condensation phenomena., Conclusions: Specific CTCF binding events occur in human cancers. Cancer-specific CTCF binding can be induced by other transcription factors to regulate oncogenic gene expression. Our results substantiate CTCF binding alteration as a functional epigenomic signature of cancer.

The COMPASS protein family catalyzes histone H3 Lys 4 (H3K4) methylation and its members are essential for regulating gene expression. MLL2/COMPASS methylates H3K4 on many developmental genes and bivalent clusters. To understand MLL2-dependent transcriptional regulation, we performed a CRISPR-based screen with an MLL2-dependent gene as a reporter in mouse embryonic stem cells. We found that MLL2 functions in gene expression by protecting developmental genes from repression via repelling PRC2 and DNA methylation machineries. Accordingly, repression in the absence of MLL2 is relieved by inhibition of PRC2 and DNA methyltransferases. Furthermore, DNA demethylation on such loci leads to reactivation of MLL2-dependent genes not only by removing DNA methylation but also by opening up previously CpG methylated regions for PRC2 recruitment, diluting PRC2 at Polycomb-repressed genes. These findings reveal how the context and function of these three epigenetic modifiers of chromatin can orchestrate transcriptional decisions and demonstrate that prevention of active repression by the context of the enzyme and not H3K4 trimethylation underlies transcriptional regulation on MLL2/COMPASS targets.

People with ataxia-telangiectasia (A-T) display phenotypic variability with regard to progression of immunodeficiency, sino-pulmonary disease, and neurologic decline. To determine the association between differential gene expression, epigenetic state, and phenotypic variation among people with A-T, we performed transcriptional and genome-wide DNA methylation profiling in patients with mild and classic A-T progression as well as healthy controls. RNA and genomic DNA were isolated from peripheral blood mononuclear cells for transcriptional and DNA methylation profiling with RNA-sequencing and modified reduced representation bisulfite sequencing, respectively. We identified 555 genes that were differentially expressed among the control, mild A-T, and classic A-T groups. Genome-wide DNA methylation profiling revealed differential promoter methylation in cis with 146 of these differentially expressed genes. Functional enrichment analysis identified significant enrichment in immune, growth, and apoptotic pathways among the methylation-regulated genes. Regardless of clinical phenotype, all A-T participants exhibited downregulation of critical genes involved in B cell function (PAX5, CD79A, CD22, and FCRL1) and upregulation of several genes associated with senescence and malignancy, including SERPINE1. These findings indicate that gene expression differences may be associated with phenotypic variability and suggest that DNA methylation regulates expression of critical immune response genes in people with A-T.

Tregs require specific epigenetic signatures to induce and maintain their suppressive function in the context of inflammation and cancer surveillance. In this issue of the JCI, Xiong and colleagues identify a critical role for the epigenetic repressor REST corepressor 1 (CoREST) in promoting Treg suppressive transcriptional and functional programs. Pharmacologic inhibition and genetic loss of CoREST in Tregs impaired organ allograft tolerance and unleashed antitumor immunity via epigenetic activation of effector T cell programs. We propose that exploiting epigenetic control mechanisms will further the translation of Treg-based therapeutics to target inflammatory and malignant disorders.

Using biochemical characterization of fusion proteins associated with endometrial stromal sarcoma, we identified JAZF1 as a new subunit of the NuA4 acetyltransferase complex and CXORF67 as a subunit of the Polycomb Repressive Complex 2 (PRC2). Since CXORF67's interaction with PRC2 leads to decreased PRC2-dependent H3K27me2/3 deposition, we propose a new name for this gene: CATACOMB (catalytic antagonist of Polycomb; official gene name: EZHIP ). We map CATACOMB's inhibitory function to a short highly conserved region and identify a single methionine residue essential for diminution of H3K27me2/3 levels. Remarkably, the amino acid sequence surrounding this critical methionine resembles the oncogenic histone H3 Lys 27 -to-methionine (H3K27M) mutation found in high-grade pediatric gliomas. As CATACOMB expression is regulated through DNA methylation/demethylation, we propose CATACOMB as the potential interlocutor between DNA methylation and PRC2 activity. We raise the possibility that similar regulatory mechanisms could exist for other methyltransferase complexes such as Trithorax/COMPASS.

Regulatory T cells (T reg cells), a distinct subset of CD4 + T cells, are necessary for the maintenance of immune self-tolerance and homeostasis 1,2 . Recent studies have demonstrated that T reg cells exhibit a unique metabolic profile, characterized by an increase in mitochondrial metabolism relative to other CD4 + effector subsets 3,4 . Furthermore, the T reg cell lineage-defining transcription factor, Foxp3, has been shown to promote respiration 5,6 ; however, it remains unknown whether the mitochondrial respiratory chain is required for the T cell-suppression capacity, stability and survival of T reg cells. Here we report that T reg cell-specific ablation of mitochondrial respiratory chain complex III in mice results in the development of fatal inflammatory disease early in life, without affecting T reg cell number. Mice that lack mitochondrial complex III specifically in T reg cells displayed a loss of T cell-suppression capacity without altering T reg cell proliferation and survival. T reg cells deficient in complex III showed decreased expression of genes associated with T reg function, whereas Foxp3 expression remained stable. Loss of complex III in T reg cells increased DNA methylation as well as the metabolites 2-hydroxyglutarate (2-HG) and succinate that inhibit the ten-eleven translocation (TET) family of DNA demethylases 7 . Thus, T reg cells require mitochondrial complex III to maintain immune regulatory gene expression and suppressive function.

The tet methylcytosine dioxygenase 2 (TET2) enzyme catalyzes the conversion of the modified DNA base 5-methylcytosine to 5-hydroxymethylcytosine. TET2 is frequently mutated or dysregulated in multiple human cancers, and loss of TET2 is associated with changes in DNA methylation patterns. Here, using newly developed TET2-specific antibodies and the estrogen response as a model system for studying the regulation of gene expression, we demonstrate that endogenous TET2 occupies active enhancers and facilitates the proper recruitment of estrogen receptor α (ERα). Knockout of TET2 by CRISPR-CAS9 leads to a global increase of DNA methylation at enhancers, resulting in attenuation of the estrogen response. We further identified a positive feedback loop between TET2 and ERα, which further requires MLL3 COMPASS at these enhancers. Together, this study reveals an epigenetic axis coordinating a transcriptional program through enhancer activation via DNA demethylation.

Pneumonia represents the leading infectious cause of death in the United States. Foxp3+ regulatory T cells promote recovery from severe pneumonia in mice, but T cell responses in patients with pneumonia remain incompletely characterized because of the limited ability to serially sample the distal airspaces and perform multidimensional molecular assessments on the small numbers of recovered cells. As T cell function is governed by their transcriptional and epigenetic landscape, we developed a method to safely perform high-resolution transcriptional and DNA methylation profiling of T cell subsets from the alveoli of critically ill patients. Our method involves nonbronchoscopic bronchoalveolar lavage combined with multiparameter fluorescence-activated cell sorting, unsupervised low-input RNA-sequencing, and a modified reduced-representation bisulfite sequencing protocol. Here, we demonstrate the safety and feasibility of our method and use it to validate functional genomic elements that were predicted by mouse models. Because of its potential for widespread application, our techniques allow unprecedented insights into the biology of human pneumonia.

Pediatric acute lung injury, usually because of pneumonia, has a mortality rate of more than 20% and an incidence that rivals that of all childhood cancers combined. CD4 + T-cells coordinate the immune response to pneumonia but fail to function robustly among the very young, who have poor outcomes from lung infection. We hypothesized that DNA methylation represses a mature CD4 + T-cell transcriptional program in neonates with pneumonia. Here, we found that neonatal mice (3-4 days old) aspirated with Escherichia coli bacteria had a higher mortality rate than juvenile mice (11-14 days old). Transcriptional profiling with an unsupervised RNA-Seq approach revealed that neonates displayed an attenuated lung CD4 + T-cell transcriptional response to pneumonia compared with juveniles. Unlike neonates, juveniles up-regulated a robust set of canonical T-cell immune response genes. DNA methylation profiling with modified reduced representation bisulfite sequencing revealed 44,119 differentially methylated CpGs, which preferentially clustered around transcriptional start sites and CpG islands. A methylation difference-filtering algorithm detected genes with a high likelihood of differential promoter methylation regulating their expression; these 731 loci encoded important immune response and tissue-protective T-cell pathway components. Disruption of DNA methylation with the hypomethylating agent decitabine induced plasticity in the lung CD4 + T-cell marker phenotype. Altogether, multidimensional profiling suggested that DNA methylation within the promoters of a core set of CD4 + T-cell pathway genes contributes to the hyporesponsive neonatal immune response to pneumonia. These findings also suggest that DNA methylation could serve as a mechanistic target for disease-modifying therapies in pediatric lung infection and injury., (© 2018 McGrath-Morrow et al.)