Faculty Bibliography

OBJECTIVE:mice revealed an increase in the stress granule-specific markers Ras-G3BP (GTPase-activating protein SH3 domain-binding protein) and PABP (poly-A-binding protein) in intimal macrophages and smooth muscle cells that correlated with disease progression. In vitro, PABP+ and G3BP+ SGs were rapidly induced in VSMC and bone marrow-derived macrophages in response to atherosclerotic stimuli, including oxidized low-density lipoprotein and mediators of mitochondrial or oxidative stress. We observed an increase in eIF2α phosphorylation, a requisite for stress granule formation, in cells exposed to these stimuli. Interestingly, SG formation, PABP expression, and eIF2α phosphorylation in VSMCs is reversed by treatment with the anti-inflammatory cytokine interleukin-19. Microtubule inhibitors reduced stress granule accumulation in VSMC, suggesting cytoskeletal regulation of stress granule formation. SG formation in VSMCs was also observed in other vascular disease pathologies, including vascular restenosis. Reduction of SG component G3BP1 by siRNA significantly altered expression profiles of inflammatory, apoptotic, and proliferative genes. CONCLUSIONS:These results indicate that SG formation is a common feature of the vascular response to injury and disease, and that modification of inflammation reduces stress granule formation in VSMC.

Macrophages accumulate prominently in the visceral adipose tissue (VAT) of obese humans and high fat diet (HFD) fed mice, and this is linked to insulin resistance and type II diabetes. While the mechanisms regulating macrophage recruitment in obesity have been delineated, the signals directing macrophage persistence in VAT are poorly understood. We previously showed that the neuroimmune guidance cue netrin-1 is expressed in the VAT of obese mice and humans, where it promotes macrophage accumulation. To better understand the source of netrin-1 and its effects on adipose tissue macrophage (ATM) fate and function in obesity, we generated mice with myeloid-specific deletion of netrin-1 (Ntn1^fl/flLysMCre^+/-; Ntn1^Δmac). Interestingly, Ntn1^Δmac mice showed a modest decrease in HFD-induced adiposity and adipocyte size, in the absence of changes in food intake or leptin, that was accompanied by an increase in markers of adipocyte beiging (Prdm16, UCP-1). Using single cell RNA-seq, combined with conventional histological and flow cytometry techniques, we show that myeloid-specific deletion of netrin-1 caused a 50% attrition of ATMs in HFD-fed mice, particularly of the resident macrophage subset, and altered the phenotype of residual ATMs to enhance lipid handling. Pseudotime analysis of single cell transcriptomes showed that in the absence of netrin-1, macrophages in the obese VAT underwent a phenotypic switch with the majority of ATMs activating a program of genes specialized in lipid handling, including fatty acid uptake and intracellular transport, lipid droplet formation and lipolysis, and regulation of lipid localization. Furthermore, Ntn1^Δmac macrophages had reduced expression of genes involved in arachidonic acid metabolism, and targeted LCMS/MS metabololipidomics analysis revealed decreases in proinflammatory eicosanoids (5-HETE, 6-trans LTB₄, TXB₂, PGD₂) in the obese VAT. Collectively, our data show that targeted deletion of netrin-1 in macrophages reprograms the ATM phenotype in obesity, leading to reduced adipose inflammation, and improved lipid handling and metabolic function.

Obesity can lead to type 2 diabetes and is an epidemic. A major contributor to its adverse effects is inflammation of the visceral adipose tissue (VAT). Life-long caloric restriction (CR), in contrast, results in extended lifespan, enhanced glucose tolerance/insulin sensitivity, and other favorable phenotypes. The effects of CR following obesity are incompletely established, but studies show multiple benefits. Many leukocyte types, macrophages predominantly, reside in VAT in homeostatic and pathological states. CR following obesity transiently increases VAT macrophage content prior to resolution of inflammation and obesity, suggesting that macrophage content and phenotype play critical roles. Here, we examined the heterogeneity of VAT leukocytes and the effects of obesity and CR. In general, our single-cell RNA-sequencing data demonstrate that macrophages are the most abundant and diverse subpopulation of leukocytes in VAT. Obesity induced significant transcriptional changes in all 15 leukocyte subpopulations, with many genes showing coordinated changes in expression across the leukocyte subpopulations. Additionally, obese VAT displayed expansion of one major macrophage subpopulation, which, in silico, was enriched in lipid binding and metabolic processes. This subpopulation returned from dominance in obesity to lean proportions after only 2 weeks of CR, although the pattern of gene expression overall remained similar. Surprisingly, CR VAT is dominated by a different macrophage subpopulation, which is absent in lean conditions. This subpopulation is enriched in genes related to phagocytosis and we postulate that its function includes clearance of dead cells, as well as excess lipids, contributing to limiting VAT inflammation and restoration of the homeostatic state.

Macrophages in the heart have dual roles in injury and repair after myocardial infarction, and understanding the two sides of this coin using traditional 'bulk cell' technologies has been challenging. By combining genetic fate-mapping and single-cell transcriptomics, a new study (Nat. Immunol. 2019;20:29-39) reveals how distinct macrophage populations expand and diverge across the healthy heart and after infarction.

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.

Long non-coding RNAs (lncRNAs), once considered 'genomic junk', have been found to regulate diverse biological processes and their study continues to reveal novel insights into lncRNA functions. Recent studies revealed that some lncRNAs may harbor small open reading frames (ORFs) that code for functional micropeptides. While investigating an unannotated primate-specific lncRNA, lncVLDLR, that is altered in patients with type II diabetes and cardiovascular disease, we discovered a previously unrecognized ORF encoding a 44 amino acid micropeptide. In vitro transcription and translation of the IMP coding sequence in the presence of ³⁵S-methionine produced a single 8 kDa peptide, which we have named Inflammation-modulating MicroPeptide (IMP). To dissect IMP function, we focused on its amino acid sequence and putative structure. These analyses revealed high sequence homology between IMP and transcription factors such as NFKB, c-myb and zinc finger proteins, and the presence of a hydrophobic region with an LxxLL motif often found in transcriptional regulators. Circular dichroism spectroscopy of synthesized IMP predicted an intrinsically disordered peptide, which is a common characteristic of transcriptional coactivators. To investigate a potential role of IMP in regulating gene transcription, we cloned a MYC-epitope tag in-frame with IMP within the full-length transcript of lncVLDLR and expressed it in HEK293 cells. Immunofluorescence staining, and cell fractionation combined with western blotting, confirmed nuclear localization of IMP RNA-seq analysis of THP1 macrophages overexpressing IMP revealed an increase in inflammatory genes, including cytokines and chemokines. Moreover, analysis of upstream regulators of these genes suggests that IMP may interact with KIX domaincontaining transcriptional coactivators to regulate inflammatory gene expression. Together our data identify a novel human micropeptide, encoded within a putative lncRNA that is dysregulated in diabetes and cardiovascular disease, that regulates inflammatory gene transcription. Further characterization of IMP and its regulatory network may uncover novel opportunities for therapeutic intervention in cardiovascular and other inflammatory diseases

Abdominal aortic aneurysms (AAA) are characterized by extensive extracellular matrix (ECM) fragmentation and inflammation. However, the mechanisms by which these events are coupled thereby fueling focal vascular damage are undefined. Here we report through single-cell RNA-sequencing of diseased aorta that the neuronal guidance cue netrin-1 can act at the interface of macrophage-driven injury and ECM degradation. Netrin-1 expression peaks in human and murine aneurysmal macrophages. Targeted deletion of netrin-1 in macrophages protects mice from developing AAA. Through its receptor neogenin-1, netrin-1 induces a robust intracellular calcium flux necessary for the transcriptional regulation and persistent catalytic activation of matrix metalloproteinase-3 (MMP3) by vascular smooth muscle cells. Deficiency in MMP3 reduces ECM damage and the susceptibility of mice to develop AAA. Here, we establish netrin-1 as a major signal that mediates the dynamic crosstalk between inflammation and chronic erosion of the ECM in AAA.

Atherosclerosis is characterized by the retention of modified lipoproteins in the arterial wall. These modified lipoproteins activate resident macrophages and the recruitment of monocyte-derived cells, which differentiate into mononuclear phagocytes that ingest the deposited lipoproteins to become "foam cells": a hallmark of this disease. In this Part 2 of a 4-part review series covering the macrophage in cardiovascular disease, we critically review the contributions and relevant pathobiology of monocytes, macrophages, and foam cells as relevant to atherosclerosis. We also review evidence that via various pathways, a failure of the resolution of inflammation is an additional key aspect of this disease process. Finally, we consider the likely role played by genomics and biological networks in controlling the macrophage phenotype in atherosclerosis. Collectively, these data provide substantial insights on the atherosclerotic process, while concurrently offering numerous molecular and genomic candidates that appear to hold great promise for selective targeting as clinical therapies.