Group 1 Innate Lymphoid Cell Lineage Identity Is Determined by a cis-Regulatory Element Marked by a Long Non-coding RNA.

Commitment to the innate lymphoid cell (ILC) lineage is determined by Id2, a transcriptional regulator that antagonizes T and B cell-specific gene expression programs. Yet how Id2 expression is regulated in each ILC subset remains poorly understood. We identified a cis-regulatory element demarcated by a long non-coding RNA (lncRNA) that controls the function and lineage identity of group 1 ILCs, while being dispensable for early ILC development and homeostasis of ILC2s and ILC3s. The locus encoding this lncRNA, which we termed Rroid, directly interacted with the promoter of its neighboring gene, Id2, in group 1 ILCs. Moreover, the Rroid locus, but not the lncRNA itself, controlled the identity and function of ILC1s by promoting chromatin accessibility and deposition of STAT5 at the promoter of Id2 in response to interleukin (IL)-15. Thus, non-coding elements responsive to extracellular cues unique to each ILC subset represent a key regulatory layer for controlling the identity and function of ILCs.

Joint profiling of chromatin accessibility and CAR-T integration site analysis at population and single-cell levels.

Chimeric antigen receptor (CAR)-T immunotherapy has yielded impressive results in several B cell malignancies, establishing itself as a powerful means to redirect the natural properties of T lymphocytes. In this strategy, the T cell genome is modified by the integration of lentiviral vectors encoding CAR that direct tumor cell killing. However, this therapeutic approach is often limited by the extent of CAR-T cell expansion in vivo. A major outstanding question is whether or not CAR-T integration itself enhances the proliferative competence of individual T cells by rewiring their regulatory landscape. To address this question, it is critical to define the identity of an individual CAR-T cell and simultaneously chart where the CAR-T vector integrates into the genome. Here, we report the development of a method called EpiVIA (https://github.com/VahediLab/epiVIA) for the joint profiling of the chromatin accessibility and lentiviral integration site analysis at the population and single-cell levels. We validate our technique in clonal cells with previously defined integration sites and further demonstrate the ability to measure lentiviral integration sites and chromatin accessibility of host and viral genomes at the single-cell resolution in CAR-T cells. We anticipate that EpiVIA will enable the single-cell deconstruction of gene regulation during CAR-T therapy, leading to the discovery of cellular factors associated with durable treatment.

The long non-coding RNA Morrbid regulates Bim and short-lived myeloid cell lifespan.

Neutrophils, eosinophils and 'classical' monocytes collectively account for about 70% of human blood leukocytes and are among the shortest-lived cells in the body. Precise regulation of the lifespan of these myeloid cells is critical to maintain protective immune responses and minimize the deleterious consequences of prolonged inflammation. However, how the lifespan of these cells is strictly controlled remains largely unknown. Here we identify a long non-coding RNA that we termed Morrbid, which tightly controls the survival of neutrophils, eosinophils and classical monocytes in response to pro-survival cytokines in mice. To control the lifespan of these cells, Morrbid regulates the transcription of the neighbouring pro-apoptotic gene, Bcl2l11 (also known as Bim), by promoting the enrichment of the PRC2 complex at the Bcl2l11 promoter to maintain this gene in a poised state. Notably, Morrbid regulates this process in cis, enabling allele-specific control of Bcl2l11 transcription. Thus, in these highly inflammatory cells, changes in Morrbid levels provide a locus-specific regulatory mechanism that allows rapid control of apoptosis in response to extracellular pro-survival signals. As MORRBID is present in humans and dysregulated in individuals with hypereosinophilic syndrome, this long non-coding RNA may represent a potential therapeutic target for inflammatory disorders characterized by aberrant short-lived myeloid cell lifespan.

Control of Immune Cell Homeostasis and Function by lncRNAs.

The immune system is composed of diverse cell types that coordinate responses to infection and maintain tissue homeostasis. In each of these cells, extracellular cues determine highly specific epigenetic landscapes and transcriptional profiles to promote immunity while maintaining homeostasis. New evidence indicates that long non-coding RNAs (lncRNAs) play crucial roles in epigenetic and transcriptional regulation in mammals. Thus, lncRNAs have emerged as key regulatory molecules of immune cell gene expression programs in response to microbial and tissue-derived cues. We review here how lncRNAs control the function and homeostasis of cell populations during immune responses, emphasizing the diverse molecular mechanisms by which lncRNAs tune highly contextualized transcriptional programs. In addition, we discuss the new challenges faced in interrogating lncRNA mechanisms and function in the immune system.

Myeloid-derived miR-6236 potentiates adipocyte insulin signaling and prevents hyperglycemia during obesity.

Adipose tissue macrophages (ATMs) influence obesity-associated metabolic dysfunction, but the mechanisms by which they do so are not well understood. We show that miR-6236 is a bona fide miRNA that is secreted by ATMs during obesity. Global or myeloid cell-specific deletion of miR-6236 aggravates obesity-associated adipose tissue insulin resistance, hyperglycemia, hyperinsulinemia, and hyperlipidemia. miR-6236 augments adipocyte insulin sensitivity by inhibiting translation of negative regulators of insulin signaling, including PTEN. The human genome harbors a miR-6236 homolog that is highly expressed in the serum and adipose tissue of obese people. hsa-MIR-6236 expression negatively correlates with hyperglycemia and glucose intolerance, and positively correlates with insulin sensitivity. Together, our findings establish miR-6236 as an ATM-secreted miRNA that potentiates adipocyte insulin signaling and protects against metabolic dysfunction during obesity.

Hepatocytes demarcated by EphB2 contribute to the progression of nonalcoholic steatohepatitis.

Current therapeutic strategies for treating nonalcoholic steatohepatitis (NASH) have failed to alleviate liver fibrosis, which is a devastating feature leading to hepatic dysfunction. Here, we integrated single-nucleus transcriptomics and epigenomics to characterize all major liver cell types during NASH development in mice and humans. The bifurcation of hepatocyte trajectory with NASH progression was conserved between mice and humans. At the nonalcoholic fatty liver (NAFL) stage, hepatocytes exhibited metabolic adaptation, whereas at the NASH stage, a subset of hepatocytes was enriched for the signatures of cell adhesion and migration, which were mainly demarcated by receptor tyrosine kinase ephrin type B receptor 2 (EphB2). EphB2, acting as a downstream effector of Notch signaling in hepatocytes, was sufficient to induce cell-autonomous inflammation. Knockdown of Ephb2 in hepatocytes ameliorated inflammation and fibrosis in a mouse model of NASH. Thus, EphB2-expressing hepatocytes contribute to NASH progression and may serve as a potential therapeutic target.

Inflammatory adipose activates a nutritional immunity pathway leading to retinal dysfunction.

Age-related macular degeneration (AMD), the leading cause of irreversible blindness among Americans over 50, is characterized by dysfunction and death of retinal pigment epithelial (RPE) cells. The RPE accumulates iron in AMD, and iron overload triggers RPE cell death in vitro and in vivo. However, the mechanism of RPE iron accumulation in AMD is unknown. We show that high-fat-diet-induced obesity, a risk factor for AMD, drives systemic and local inflammatory circuits upregulating interleukin-1ß (IL-1ß). IL-1ß upregulates RPE iron importers and downregulates iron exporters, causing iron accumulation, oxidative stress, and dysfunction. We term this maladaptive, chronic activation of a nutritional immunity pathway the cellular iron sequestration response (CISR). RNA sequencing (RNA-seq) analysis of choroid and retina from human donors revealed that hallmarks of this pathway are present in AMD microglia and macrophages. Together, these data suggest that inflamed adipose tissue, through the CISR, can lead to RPE pathology in AMD.

The gut microbiota regulates white adipose tissue inflammation and obesity via a family of microRNAs.

The gut microbiota is a key environmental determinant of mammalian metabolism. Regulation of white adipose tissue (WAT) by the gut microbiota is a process critical to maintaining metabolic fitness, and gut dysbiosis can contribute to the development of obesity and insulin resistance (IR). However, how the gut microbiota regulates WAT function remains largely unknown. Here, we show that tryptophan-derived metabolites produced by the gut microbiota controlled the expression of the miR-181 family in white adipocytes in mice to regulate energy expenditure and insulin sensitivity. Moreover, dysregulation of the gut microbiota-miR-181 axis was required for the development of obesity, IR, and WAT inflammation in mice. Our results indicate that regulation of miR-181 in WAT by gut microbiota-derived metabolites is a central mechanism by which host metabolism is tuned in response to dietary and environmental changes. As we also found that MIR-181 expression in WAT and the plasma abundance of tryptophan-derived metabolites were dysregulated in a cohort of obese human children, the MIR-181 family may represent a potential therapeutic target to modulate WAT function in the context of obesity.