The IRG1-itaconate axis protects from cholesterol-induced inflammation and atherosclerosis.

Atherosclerosis is fueled by a failure to resolve lipid-driven inflammation within the vasculature that drives plaque formation. Therapeutic approaches to reverse atherosclerotic inflammation are needed to address the rising global burden of cardiovascular disease (CVD). Recently, metabolites have gained attention for their immunomodulatory properties, including itaconate, which is generated from the tricarboxylic acid-intermediate cis-aconitate by the enzyme Immune Responsive Gene 1 (IRG1/ACOD1). Here, we tested the therapeutic potential of the IRG1-itaconate axis for human atherosclerosis. Using single-cell RNA sequencing (scRNA-seq), we found that IRG1 is up-regulated in human coronary atherosclerotic lesions compared to patient-matched healthy vasculature, and in mouse models of atherosclerosis, where it is primarily expressed by plaque monocytes, macrophages, and neutrophils. Global or hematopoietic Irg1-deficiency in mice increases atherosclerosis burden, plaque macrophage and lipid content, and expression of the proatherosclerotic cytokine interleukin (IL)-1ß. Mechanistically, absence of Irg1 increased macrophage lipid accumulation, and accelerated inflammation via increased neutrophil extracellular trap (NET) formation and NET-priming of the NLRP3-inflammasome in macrophages, resulting in increased IL-1ß release. Conversely, supplementation of the Irg1-itaconate axis using 4-octyl itaconate (4-OI) beneficially remodeled advanced plaques and reduced lesional IL-1ß levels in mice. To investigate the effects of 4-OI in humans, we leveraged an ex vivo systems-immunology approach for CVD drug discovery. Using CyTOF and scRNA-seq of peripheral blood mononuclear cells treated with plasma from CVD patients, we showed that 4-OI attenuates proinflammatory phospho-signaling and mediates anti-inflammatory rewiring of macrophage populations. Our data highlight the relevance of pursuing IRG1-itaconate axis supplementation as a therapeutic approach for atherosclerosis in humans.

Molecular basis for substrate recruitment to the PRMT5 methylosome.

PRMT5 is an essential arginine methyltransferase and a therapeutic target in MTAP-null cancers. PRMT5 uses adaptor proteins for substrate recruitment through a previously undefined mechanism. Here, we identify an evolutionarily conserved peptide sequence shared among the three known substrate adaptors (CLNS1A, RIOK1, and COPR5) and show that it is necessary and sufficient for interaction with PRMT5. We demonstrate that PRMT5 uses modular adaptor proteins containing a common binding motif for substrate recruitment, comparable with other enzyme classes such as kinases and E3 ligases. We structurally resolve the interface with PRMT5 and show via genetic perturbation that it is required for methylation of adaptor-recruited substrates including the spliceosome, histones, and ribosomal complexes. Furthermore, disruption of this site affects Sm spliceosome activity, leading to intron retention. Genetic disruption of the PRMT5-substrate adaptor interface impairs growth of MTAP-null tumor cells and is thus a site for development of therapeutic inhibitors of PRMT5.

Accelerated adipogenic differentiation of hMSCs in a microfluidic shear stimulation platform.

The use of transplanted adipose tissue to repair crucial defects is clinically interesting for surgical reconstruction. Terminally differentiated adipocytes are utilized to promote the healthy regeneration of defective tissue. Use of differentiated mesenchymal stem cells, capable of differentiation into adipocytes, is advantageous because of their regenerative properties. Conventionally, the differentiation of hMSCs toward adipocytes occurs through chemical stimulation. We designed a microfluidic system, consisting of plastic tubing and a syringe pump, to create an environment of shear to accelerate this differentiation process. This system employed a flow rate equivalent to the accelerated flow rates found within the arterial system in order to promote and activate intracellular and extracellular proteins associated with the adipogenic lineage. Confirmation of sustained viability following shear exposure was obtained using a fluorescent live-dead assay. Visualization of intracellular lipid accumulation was achieved via Oil Red O staining. When placed into culture, shear stimulated hMSCs were further induced toward brown adipose tissue, as evidenced by a greater quantity of lipid triglycerides, relative to unstimulated hMSCs. qRT-PCR analysis validated the phenotypic changes observed when the hMSCs were later cultured in adipogenic differentiation media. Additionally, increased fold change for adipogenic markers such as LPL1, CFL1, and SSP1 were observed as a result of shear stimulation. The significance of this work lies in the demonstration that transient fluid shear exposure of hMSCs in suspension can influence differentiation into adipocytes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:440-446, 2016.

Suspension-based differentiation of adult mesenchymal stem cells toward chondrogenic lineage.

Human mesenchymal stem cells (hMSCs) are derived from bone marrow and have the ability to differentiate into cartilage and other mesenchymal cell types found throughout the body. Traditionally, the differentiation of hMSCs toward chondrocytes occurs through a combination of pelleted static cell culture and chemical stimuli. As an alternative to these protocols, we developed an in vitro flow through microfluidic method to induce the differentiation of hMSCs into chondrocytes. Suspensions of unattached hMSCs were exposed to a constant shear flow over a period of 20 minutes, which promoted phenotypic and gene expression changes toward the chondrogenic lineage. These internal and external changes of chondrogenic differentiation were then observed over 3 weeks later in culture, as confirmed through fluorescent immunocytochemical staining and real-time quantitative reverse transcriptase polymerase chain reaction. The increased concentration of Type II collagen on the surface of shear stimulated hMSCs with the upregulation of MAPK1 and SOX9 demonstrated the capabilities of our approach to induce sustained differentiation. In conclusion, our shear stimulation method, in combination with chemical stimuli, illustrates enhanced differentiation of hMSCs toward the chondrogenic lineage.