Faculty Bibliography

Non-coding RNAs, once considered "genomic junk", are now known to play central roles in the dynamic control of transcriptional and post-transcriptional gene expression. Long non-coding RNAs (lncRNAs) are an expansive class of transcripts broadly described as greater than 200 nucleotides in length. While most lncRNAs are species-specific, their lack of conservation does not imbue a lack of function. LncRNAs have been found to regulate numerous diverse biological functions, including those central to macrophage differentiation and activation. Through their ability to form RNA-DNA, RNA-protein and RNA-RNA interactions, lncRNAs have been implicated in the regulation of myeloid lineage determination, and innate and adaptive immune functions, among others. In this review, we discuss recent advances, current challenges and future opportunities in understanding the roles of lncRNAs in macrophage functions in homeostasis and disease.

AIMS/OBJECTIVE:Acute myocardial infarction rapidly increases blood neutrophils (<2 hours). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 hours after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 minutes) release of extracellular vesicles (EVs), which have emerged as an important means of cell-cell signalling and are thus a potential mechanism for communicating with remote tissues. METHODS AND RESULTS/RESULTS:Here, we show that injury to the myocardium rapidly mobilises neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilised splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilisation of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. CONCLUSIONS:Our findings show a novel EV-dependent mechanism for the rapid mobilisation of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells. TRANSLATIONAL PERSPECTIVE/UNASSIGNED:Peripheral blood neutrophils are rapidly elevated following acute myocardial infarction (AMI) and prior to alterations in systemic cytokines. Extracellular vesicles (EVs) are membrane enclosed particles that carry protein and miRNAs and are rapidly liberated from endothelial cells (EC). Here, we show that following AMI EC-derived-EVs (EC-EVs) mediate neutrophil mobilisation from the spleen via EC-EV-VCAM-1 and induce transcriptional activation of neutrophils in the blood to favour miRNA-126-mRNA targets; miRNA-126 antagomir treatment lowers infarct size. EC-EV-VCAM-1 and EC-EV-miRNA-126 are novel mechanisms that mobilise splenic reserve of neutrophils, a previously unidentified source of neutrophils in sterile ischaemic injury.

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.

Psychological stress (PS) is associated with systemic inflammation and accelerates inflammatory disease progression (e.g., atherosclerosis). The mechanisms underlying stress-mediated inflammation and future health risk are poorly understood. Monocytes are key in sustaining systemic inflammation, and recent studies demonstrate that they maintain the memory of inflammatory insults, leading to a heightened inflammatory response upon rechallenge. We show that PS induces remodeling of the chromatin landscape and transcriptomic reprogramming of monocytes, skewing them to a primed hyperinflammatory phenotype. Monocytes from stressed mice and humans exhibit a characteristic inflammatory transcriptomic signature and are hyperresponsive upon stimulation with Toll-like receptor ligands. RNA and ATAC sequencing reveal that monocytes from stressed mice and humans exhibit activation of metabolic pathways (mTOR and PI3K) and reduced chromatin accessibility at mitochondrial respiration-associated loci. Collectively, our findings suggest that PS primes the reprogramming of myeloid cells to a hyperresponsive inflammatory state, which may explain how PS confers inflammatory disease risk.

Rationale: MicroRNA-33 post-transcriptionally represses genes involved in lipid metabolism and energy homeostasis. Targeted inhibition of miR-33 increases plasma HDL cholesterol and promotes atherosclerosis regression, in part, by enhancing reverse cholesterol transport and dampening plaque inflammation. However, how miR-33 reshapes the immune microenvironment of plaques remains poorly understood. Objective: To define how miR-33 inhibition alters the dynamic balance and transcriptional landscape of immune cells in atherosclerotic plaques. Methods and Results: We used single cell RNA-sequencing of aortic CD45⁺ cells, combined with immunohistologic, morphometric and flow cytometric analyses to define the changes in plaque immune cell composition, gene expression and function following miR-33 inhibition. We report that anti-miR-33 treatment of Ldlr^-/- mice with advanced atherosclerosis reduced plaque burden and altered the plaque immune cell landscape by shifting the balance of pro- and anti-atherosclerotic macrophage and T cell subsets. By quantifying the kinetic processes that determine plaque macrophage burden, we found that anti-miR-33 reduced levels of circulating monocytes and splenic myeloid progenitors, decreased macrophage proliferation and retention, and promoted macrophage attrition by apoptosis and efferocytotic clearance. scRNA-sequencing of aortic arch plaques showed that anti-miR-33 reduced the frequency of MHCIIhi "inflammatory" and Trem2hi "metabolic" macrophages, but not tissue resident macrophages. Furthermore, anti-miR-33 led to derepression of distinct miR-33 target genes in the different macrophage subsets: in resident and Trem2hi macrophages, anti-miR-33 relieved repression of miR-33 target genes involved in lipid metabolism (e.g., Abca1, Ncoa1, Ncoa2, Crot), whereas in MHCIIhi macrophages, anti-miR-33 upregulated target genes involved in chromatin remodeling and transcriptional regulation. Anti-miR-33 also reduced the accumulation of aortic CD8+ T cells and CD4+ Th1 cells, and increased levels of FoxP3+ regulatory T cells in plaques, consistent with an immune-dampening effect on plaque inflammation. Conclusions: Our results provide insight into the immune mechanisms and cellular players that execute anti-miR-33's atheroprotective actions in the plaque.

OBJECTIVE:in vitro and in vivo. Treatment of mice with inhibitors of miR-33 increased expression of these miR-33 target genes in brown and subcutaneous white adipose tissue, upregulating expression of UCP1, and rendering mice resistant to cold challenge. CONCLUSIONS:Collectively, our findings demonstrate that miR-33 targets key genes involved in BAT activation and white adipose beiging and expand our understanding of how miR-33 coordinately regulates pathways involved in metabolic homeostasis.