The Intersection of Epigenetics and Metabolism in Trained Immunity.

The last few years have witnessed an increasing body of evidence that challenges the traditional view that immunological memory is an exclusive trait of the adaptive immune system. Myeloid cells can show increased responsiveness upon subsequent stimulation with the same or a different stimulus, well after the initial challenge. This de facto innate immune memory has been termed "trained immunity" and is involved in infections, vaccination and inflammatory diseases. Trained immunity is based on two main pillars: the epigenetic and metabolic reprogramming of cells. In this review we discuss the latest insights into the epigenetic mechanisms behind the induction of trained immunity, as well as the role of different cellular metabolites and metabolic networks in the induction, regulation and maintenance of trained immunity.

Chromosomal contact permits transcription between coregulated genes.

Transcription of coregulated genes occurs in the context of long-range chromosomal contacts that form multigene complexes. Such contacts and transcription are lost in knockout studies of transcription factors and structural chromatin proteins. To ask whether chromosomal contacts are required for cotranscription in multigene complexes, we devised a strategy using TALENs to cleave and disrupt gene loops in a well-characterized multigene complex. Monitoring this disruption using RNA FISH and immunofluorescence microscopy revealed that perturbing the site of contact had a direct effect on transcription of other interacting genes. Unexpectedly, this effect on cotranscription was hierarchical, with dominant and subordinate members of the multigene complex engaged in both intra- and interchromosomal contact. This observation reveals the profound influence of these chromosomal contacts on the transcription of coregulated genes in a multigene complex.

Long noncoding RNA CHROMR regulates antiviral immunity in humans.

Long noncoding RNAs (lncRNAs) have emerged as critical regulators of gene expression, yet their contribution to immune regulation in humans remains poorly understood. Here, we report that the primate-specific lncRNA CHROMR is induced by influenza A virus and SARS-CoV-2 infection and coordinates the expression of interferon-stimulated genes (ISGs) that execute antiviral responses. CHROMR depletion in human macrophages reduces histone acetylation at regulatory regions of ISG loci and attenuates ISG expression in response to microbial stimuli. Mechanistically, we show that CHROMR sequesters the interferon regulatory factor (IRF)-2-dependent transcriptional corepressor IRF2BP2, thereby licensing IRF-dependent signaling and transcription of the ISG network. Consequently, CHROMR expression is essential to restrict viral infection of macrophages. Our findings identify CHROMR as a key arbitrator of antiviral innate immune signaling in humans.

Structural Variants Create New Topological-Associated Domains and Ectopic Retinal Enhancer-Gene Contact in Dominant Retinitis Pigmentosa.

The cause of autosomal-dominant retinitis pigmentosa (adRP), which leads to loss of vision and blindness, was investigated in families lacking a molecular diagnosis. A refined locus for adRP on Chr17q22 (RP17) was delineated through genotyping and genome sequencing, leading to the identification of structural variants (SVs) that segregate with disease. Eight different complex SVs were characterized in 22 adRP-affected families with >300 affected individuals. All RP17 SVs had breakpoints within a genomic region spanning YPEL2 to LINC01476. To investigate the mechanism of disease, we reprogrammed fibroblasts from affected individuals and controls into induced pluripotent stem cells (iPSCs) and differentiated them into photoreceptor precursor cells (PPCs) or retinal organoids (ROs). Hi-C was performed on ROs, and differential expression of regional genes and a retinal enhancer RNA at this locus was assessed by qPCR. The epigenetic landscape of the region, and Hi-C RO data, showed that YPEL2 sits within its own topologically associating domain (TAD), rich in enhancers with binding sites for retinal transcription factors. The Hi-C map of RP17 ROs revealed creation of a neo-TAD with ectopic contacts between GDPD1 and retinal enhancers, and modeling of all RP17 SVs was consistent with neo-TADs leading to ectopic retinal-specific enhancer-GDPD1 accessibility. qPCR confirmed increased expression of GDPD1 and increased expression of the retinal enhancer that enters the neo-TAD. Altered TAD structure resulting in increased retinal expression of GDPD1 is the likely convergent mechanism of disease, consistent with a dominant gain of function. Our study highlights the importance of SVs as a genomic mechanism in unsolved Mendelian diseases.

Enhancer-Derived lncRNAs Regulate Genome Architecture: Fact or Fiction?

How does the non-coding portion of the genome contribute to the regulation of genome architecture? A recent paper by Tan et al. focuses on the relationship between cis-acting complex-trait-associated lincRNAs and the formation of chromosomal contacts in topologically associating domains (TADs).

A chromatin-regulated biphasic circuit coordinates IL-1ß-mediated inflammation.

Inflammation is characterized by a biphasic cycle consisting initially of a proinflammatory phase that is subsequently resolved by anti-inflammatory processes. Interleukin-1ß (IL-1ß) is a master regulator of proinflammation and is encoded within the same topologically associating domain (TAD) as IL-37, which is an anti-inflammatory cytokine that opposes the function of IL-1ß. Within this TAD, we identified a long noncoding RNA called AMANZI, which negatively regulates IL-1ß expression and trained immunity through the induction of IL37 transcription. We found that the activation of IL37 occurs through the formation of a dynamic long-range chromatin contact that leads to the temporal delay of anti-inflammatory responses. The common variant rs16944 present in AMANZI augments this regulatory circuit, predisposing individuals to enhanced proinflammation or immunosuppression. Our work illuminates a chromatin-mediated biphasic circuit coordinating expression of IL-1ß and IL-37, thereby regulating two functionally opposed states of inflammation from within a single TAD.

Genetic regulators of cytokine responses upon BCG vaccination in children from West Africa.

Genetic variation is a key factor influencing cytokine production capacity, but which genetic loci regulate cytokine production before and after vaccination, particularly in African population is unknown. Here, we aimed to identify single-nucleotide polymorphisms (SNPs) controlling cytokine responses after microbial stimulation in infants of West-African ancestry, comprising of low-birth-weight neonates randomized to bacillus Calmette-Guérin (BCG) vaccine-at-birth or to the usual delayed BCG. Genome-wide cytokine cytokine quantitative trait loci (cQTL) mapping revealed 12 independent loci, of which the LINC01082-LINC00917 locus influenced more than half of the cytokine-stimulation pairs assessed. Furthermore, nine distinct cQTLs were found among infants randomized to BCG. Functional validation confirmed that several complement genes affect cytokine response after BCG vaccination. We observed a limited overlap of common cQTLs between the West-African infants and cohorts of Western European individuals. These data reveal strong population-specific genetic effects on cytokine production and may indicate new opportunities for therapeutic intervention and vaccine development in African populations.

Delayed caspase-8 activation and enhanced integrin ß1-activated FAK underpins anoikis in oesophageal carcinoma cells harbouring mt p53-R175H.

FAK (focal adhesion kinase)-mediated signalling reportedly suppresses caspase-8 activation and, as a consequence, rescues epithelial cells from Fas-mediated anoikis. Critical was the use of a HOSCC (human oesophageal squamous carcinoma) cell line harbouring mt (mutant) p53-R175H and displaying resistance to detachment and Tyr397 dephosphorylation of FAK. Here we show, although caspase-8 activation is delayed in the mt p53-R175H cell line, comparable apoptotic events evidenced in the wt (wild type) p53 HOSCC cell lines could be induced in the mt p53-R175H cell line by strengthening the apoptotic stimulus. Significant to anoikis-related regulation, the delay in caspase-8 activation was accompanied by the maintenance of FAK Tyr397 phosphorylation, integrin ß1-associated FAK and a FAK/caspase-8 complex. Thus, mt p53-R175H may desensitize tumours to Fas-mediated anchorage-independent death via a FAK-dependent mechanism.

Visualization of Chromatin Dynamics by Live Cell Microscopy Using CRISPR/Cas9 Gene Editing and ANCHOR Labeling.

The organization of the eukaryotic nucleus facilitates functional chromatin contacts which regulate gene transcription. Despite this being extensively studied through population-based chromatin contact mapping and microscopic observations in single cells, the spatiotemporal dynamics of chromatin behavior have largely remained elusive. The current methods to label and observe specific endogenous genomic loci in living cells have been challenging to implement and too invasive to biological processes. In this protocol, we describe the use of a recently developed DNA labelling strategy (ANCHOR) with CRISPR/Cas9 gene editing, to discreetly label genes for live cell imaging to study chromatin dynamics. Our approach improves on some of the fundamental shortfalls associated with current labelling strategies and has the potential for multiplexed observations.

Advances in understanding molecular regulation of innate immune memory.

The epigenetic and functional reprogramming of immune genes during induction of trained immunity is accompanied by the metabolic rewiring of cellular state. This memory is induced in the hematopoietic niche and propagated to daughter cells, generating epigenetically and metabolically reprogrammed innate immune cells that are greatly enhanced in their capacity to resolve inflammation. In particular, these cells show accumulation of H3K4me3 and H3K27Ac epigenetic marks on multiple immune gene promoters and associated enhancers. However, the mechanism governing how these epigenetic marks accumulate at discrete immune gene loci has been poorly understood, until now. Here, we discuss some recent advances in the regulation of trained immunity, with a particular focus on the mechanistic role of a novel class of long non-coding RNAs in the establishment of epigenetic marks on trained immune gene promoters.

BCG Vaccination Induces Long-Term Functional Reprogramming of Human Neutrophils.

The tuberculosis vaccine bacillus Calmette-Guérin (BCG) protects against some heterologous infections, probably via induction of non-specific innate immune memory in monocytes and natural killer (NK) cells, a process known as trained immunity. Recent studies have revealed that the induction of trained immunity is associated with a bias toward granulopoiesis in bone marrow hematopoietic progenitor cells, but it is unknown whether BCG vaccination also leads to functional reprogramming of mature neutrophils. Here, we show that BCG vaccination of healthy humans induces long-lasting changes in neutrophil phenotype, characterized by increased expression of activation markers and antimicrobial function. The enhanced function of human neutrophils persists for at least 3 months after vaccination and is associated with genome-wide epigenetic modifications in trimethylation at histone 3 lysine 4. Functional reprogramming of neutrophils by the induction of trained immunity might offer novel therapeutic strategies in clinical conditions that could benefit from modulation of neutrophil effector function.

The Set7 Lysine Methyltransferase Regulates Plasticity in Oxidative Phosphorylation Necessary for Trained Immunity Induced by ß-Glucan.

Trained immunity confers a sustained augmented response of innate immune cells to a secondary challenge, via a process dependent on metabolic and transcriptional reprogramming. Because of its previous associations with metabolic and transcriptional memory, as well as the importance of H3 histone lysine 4 monomethylation (H3K4me1) to innate immune memory, we hypothesize that the Set7 methyltransferase has an important role in trained immunity induced by ß-glucan. Using pharmacological studies of human primary monocytes, we identify trained immunity-specific immunometabolic pathways regulated by Set7, including a previously unreported H3K4me1-dependent plasticity in the induction of oxidative phosphorylation. Recapitulation of ß-glucan training in vivo additionally identifies Set7-dependent changes in gene expression previously associated with the modulation of myelopoiesis progenitors in trained immunity. By revealing Set7 as a key regulator of trained immunity, these findings provide mechanistic insight into sustained metabolic changes and underscore the importance of characterizing regulatory circuits of innate immune memory.

Lnc-ing Trained Immunity to Chromatin Architecture.

Human innate immune cells exposed to certain infections or stimuli develop enhanced immune responses upon re-infection with a different second stimulus, a process termed trained immunity. Recent studies have revealed that hematopoietic stem cells (HSCs) are integral to trained immune responses as they are able to "remember" transcriptional responses and transmit this state to their progeny to educate them how to respond to future infections. The macrophages that arise from trained HSCs are epigenetically reprogrammed and as a result robustly express immune genes, enhancing their capability to resolve infection. Accumulation of H3K4me3 epigenetic marks on multiple immune gene promoters underlie robust transcriptional responses during trained immune responses. However, the mechanism underpinning how these epigenetic marks accumulate at discrete immune gene loci has been poorly understood. In this review, we discuss the previously unexplored contributions of nuclear architecture and long non-coding RNAs on H3K4me3 promoter priming in trained immunity. Altering the activity of these lncRNAs presents a promising therapeutic approach to achieve immunomodulation in inflammatory disease states.

Publisher Correction: Immune genes are primed for robust transcription by proximal long noncoding RNAs located in nuclear compartments.

In the version of this article initially published, '+' and '-' labels were missing from the graph keys at the bottom of Fig. 8d. The error has been corrected in the HTML and PDF versions of the article.

Immune genes are primed for robust transcription by proximal long noncoding RNAs located in nuclear compartments.

Accumulation of trimethylation of histone H3 at lysine 4 (H3K4me3) on immune-related gene promoters underlies robust transcription during trained immunity. However, the molecular basis for this remains unknown. Here we show three-dimensional chromatin topology enables immune genes to engage in chromosomal contacts with a subset of long noncoding RNAs (lncRNAs) we have defined as immune gene-priming lncRNAs (IPLs). We show that the prototypical IPL, UMLILO, acts in cis to direct the WD repeat-containing protein 5 (WDR5)-mixed lineage leukemia protein 1 (MLL1) complex across the chemokine promoters, facilitating their H3K4me3 epigenetic priming. This mechanism is shared amongst several trained immune genes. Training mediated by ß-glucan epigenetically reprograms immune genes by upregulating IPLs in manner dependent on nuclear factor of activated T cells. The murine chemokine topologically associating domain lacks an IPL, and the Cxcl genes are not trained. Strikingly, the insertion of UMLILO into the chemokine topologically associating domain in mouse macrophages resulted in training of Cxcl genes. This provides strong evidence that lncRNA-mediated regulation is central to the establishment of trained immunity.

The lncRNA Connection Between Cellular Metabolism and Epigenetics in Trained Immunity.

Trained immunity describes the ability of innate immune cells to form immunological memories of prior encounters with pathogens. Recollection of these memories during a secondary encounter manifests a broadly enhanced inflammatory response characterized by the increased transcription of innate immune genes. Despite this phenomenon having been described over a decade ago, our understanding of the molecular mechanisms responsible for this phenotype is still incomplete. Here we present an overview of the molecular events that lead to training. For the first time, we highlight the mechanistic role of a novel class of long non-coding RNAs (lncRNAs) in the establishment and maintenance of discrete, long lasting epigenetic modifications that are causal to the trained immune response. This recent insight fills in significant gaps in our understanding of trained immunity and reveals novel ways to exploit trained immunity for therapeutic purposes.

Visualization of Enhancer-Derived Noncoding RNA.

Enhancers are principal regulators that allow spatiotemporal tissue-specific control of gene expression. While mounting evidence suggests that enhancer-derived long noncoding RNAs (long ncRNAs), including enhancer RNAs (eRNAs), are an important component of enhancer function, their expression has not been broadly analyzed at a single cell level via imaging techniques. This protocol describes a method to image eRNA in single cells by in situ hybridization followed by tyramide signal amplification (TSA). The procedure can be multiplexed to simultaneously visualize both eRNA and protein-coding transcript at the site of transcriptional elongation, thereby permitting analysis of dynamics between the two transcript species in single cells. Our approach is not limited to eRNAs, but can be implemented on other transcripts.

The emerging molecular biology toolbox for the study of long noncoding RNA biology.

Long noncoding RNAs (lncRNAs) have been implicated in many biological processes. However, due to the unique nature of lncRNAs and the consequential difficulties associated with their characterization, there is a growing disparity between the rate at which lncRNAs are being discovered and the assignment of biological function to these transcripts. Here we present a molecular biology toolbox equipped to help dissect aspects of lncRNA biology and reveal functionality. We outline an approach that begins with a broad survey of genome-wide, high-throughput datasets to identify potential lncRNA candidates and then narrow the focus on specific methods that are well suited to interrogate the transcripts of interest more closely. This involves the use of imaging-based strategies to validate these candidates and observe the behaviors of these transcripts at single molecule resolution in individual cells. We also describe the use of gene editing tools and interactome capture techniques to interrogate functionality and infer mechanism, respectively. With the emergence of lncRNAs as important molecules in healthy and diseased cellular function, it remains crucial to deepen our understanding of their biology.