Faculty Bibliography

The regression, or resolution, of inflammation in atherosclerotic plaques is impaired in diabetes. However, the factors mediating this effect remain incomplete. We identified protein arginine methyltransferase 2 (PRMT2) as a protein whose expression in macrophages is reduced in hyperglycemia and diabetes. PRMT2 catalyzes arginine methylation to target proteins to modulate gene expression. Because PRMT2 expression is reduced in cells in hyperglycemia, we wanted to determine whether PRMT2 plays a causal role in the impairment of atherosclerosis regression in diabetes. We, therefore, examined the consequence of deleting PRMT2 in myeloid cells during the regression of atherosclerosis in normal and diabetic mice. Remarkably, we found significant impairment of atherosclerosis regression under normoglycemic conditions in mice lacking PRMT2 (Prmt2^-/-) in myeloid cells that mimic the decrease in regression of atherosclerosis in WT mice under diabetic conditions. This was associated with increased plaque macrophage retention, as well as increased apoptosis and necrosis. PRMT2-deficient plaque CD68+ cells under normoglycemic conditions showed increased expression of genes involved in cytokine signaling and inflammation compared to WT cells. Consistently, Prmt2^-/- bone marrow-derived macrophages (BMDMs) showed an increased response of proinflammatory genes to LPS and a decreased response of inflammation resolving genes to IL-4. This increased response to LPS in Prmt2^-/- BMDMs occurs via enhanced NF-kappa B activity. Thus, the loss of PRMT2 is causally linked to impaired atherosclerosis regression via a heightened inflammatory response in macrophages. That PRMT2 expression was lower in myeloid cells in plaques from human subjects with diabetes supports the relevance of our findings to human atherosclerosis.

Aerobic exercise is associated with decreased cancer incidence and cancer-associated mortality. However, little is known about the effects of exercise on pancreatic ductal adenocarcinoma (PDA), a disease for which current therapeutic options are limited. Herein, we show that aerobic exercise reduces PDA tumor growth, by modulating systemic and intra-tumoral immunity. Mechanistically, exercise promotes immune mobilization and accumulation of tumor-infiltrating IL15Rα⁺ CD8 T cells, which are responsible for the tumor-protective effects. In clinical samples, an exercise-dependent increase of intra-tumoral CD8 T cells is also observed. Underscoring the translational potential of the interleukin (IL)-15/IL-15Rα axis, IL-15 super-agonist (NIZ985) treatment attenuates tumor growth, prolongs survival, and enhances sensitivity to chemotherapy. Finally, exercise or NIZ985 both sensitize pancreatic tumors to αPD-1, with improved anti-tumor and survival benefits. Collectively, our findings highlight the therapeutic potential of an exercise-oncology axis and identify IL-15 activation as a promising treatment strategy for this deadly disease.

Mammalian cells autonomously activate hypoxia-inducible transcription factors (HIFs) to ensure survival in low-oxygen environments. We report here that injury-induced hypoxia is insufficient to trigger HIF1α in damaged epithelium. Instead, multimodal single-cell and spatial transcriptomics analyses and functional studies reveal that retinoic acid-related orphan receptor γt⁺ (RORγt⁺) γδ T cell-derived interleukin-17A (IL-17A) is necessary and sufficient to activate HIF1α. Protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling proximal of IL-17 receptor C (IL-17RC) activates mammalian target of rapamycin (mTOR) and consequently HIF1α. The IL-17A-HIF1α axis drives glycolysis in wound front epithelia. Epithelial-specific loss of IL-17RC, HIF1α, or blockade of glycolysis derails repair. Our findings underscore the coupling of inflammatory, metabolic, and migratory programs to expedite epithelial healing and illuminate the immune cell-derived inputs in cellular adaptation to hypoxic stress during repair.

The Kv1.3 potassium channel is expressed abundantly on activated T cells and mediates the cellular immune response. This role has made the channel a target for therapeutic immunomodulation to block its activity and suppress T cell activation. Here, we report structures of human Kv1.3 alone, with a nanobody inhibitor, and with an antibody-toxin fusion blocker. Rather than block the channel directly, four copies of the nanobody bind the tetramer's voltage sensing domains and the pore domain to induce an inactive pore conformation. In contrast, the antibody-toxin fusion docks its toxin domain at the extracellular mouth of the channel to insert a critical lysine into the pore. The lysine stabilizes an active conformation of the pore yet blocks ion permeation. This study visualizes Kv1.3 pore dynamics, defines two distinct mechanisms to suppress Kv1.3 channel activity with exogenous inhibitors, and provides a framework to aid development of emerging T cell immunotherapies.

Dysregulation of cholesterol homeostasis is associated with many diseases such as cardiovascular disease and cancer. Liver X receptors (LXRs) are major upstream regulators of cholesterol homeostasis and are activated by endogenous cholesterol metabolites such as 27-hydroxycholesterol (27HC). LXRs and various LXR ligands such as 27HC have been described to influence several extra-hepatic biological systems. However, disparate reports of LXR function have emerged, especially with respect to immunology and cancer biology. This would suggest that, similar to steroid nuclear receptors, the LXRs can be selectively modulated by different ligands. Here, we use RNA-sequencing of macrophages and single-cell RNA-sequencing of immune cells from metastasis-bearing murine lungs to provide evidence that LXR satisfies the 2 principles of selective nuclear receptor modulation: (1) different LXR ligands result in overlapping but distinct gene expression profiles within the same cell type, and (2) the same LXR ligands differentially regulate gene expression in a highly context-specific manner, depending on the cell or tissue type. The concept that the LXRs can be selectively modulated provides the foundation for developing precision pharmacology LXR ligands that are tailored to promote those activities that are desirable (proimmune), but at the same time minimizing harmful side effects (such as elevated triglyceride levels).

Computational methods and tools are a powerful complementary approach to experimental work for studying regulatory interactions in living cells and systems. We demonstrate the use of formal reasoning methods as applied to the Caenorhabditis elegans germ line, which is an accessible system for stem cell research. The dynamics of the underlying genetic networks and their potential regulatory interactions are key for understanding mechanisms that control cellular decision-making between stem cells and differentiation. We model the "stem cell fate" versus entry into the "meiotic development" pathway decision circuit in the young adult germ line based on an extensive study of published experimental data and known/hypothesized genetic interactions. We apply a formal reasoning framework to derive predictive networks for control of differentiation. Using this approach we simultaneously specify many possible scenarios and experiments together with potential genetic interactions, and synthesize genetic networks consistent with all encoded experimental observations. In silico analysis of knock-down and overexpression experiments within our model recapitulate published phenotypes of mutant animals and can be applied to make predictions on cellular decision-making. A methodological contribution of this work is demonstrating how to effectively model within a formal reasoning framework a complex genetic network with a wealth of known experimental data and constraints. We provide a summary of the steps we have found useful for the development and analysis of this model and can potentially be applicable to other genetic networks. This work also lays a foundation for developing realistic whole tissue models of the C. elegans germ line where each cell in the model will execute a synthesized genetic network.

Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA-Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate-binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with submicromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.

BACKGROUND:miRNA therapeutics have gained attention during the past decade. These oligonucleotide treatments can modulate the expression of miRNAs in vivo and could be used to correct the imbalance of gene expression found in human diseases such as obesity, metabolic syndrome, and atherosclerosis. The in vivo efficacy of current anti-miRNA technologies hindered by physiological and cellular barriers to delivery into targeted cells and the nature of miRNAs that allows one to target an entire pathway that may lead to deleterious off-target effects. For these reasons, novel targeted delivery systems to inhibit miRNAs in specific tissues will be important for developing effective therapeutic strategies for numerous diseases including atherosclerosis. METHODS:We used pH low-insertion peptide (pHLIP) constructs as vehicles to deliver microRNA-33-5p (miR-33) antisense oligonucleotides to atherosclerotic plaques. Immunohistochemistry and histology analysis was performed to assess the efficacy of miR-33 silencing in atherosclerotic lesions. We also assessed how miR-33 inhibition affects gene expression in monocytes/macrophages by single-cell RNA transcriptomics. RESULTS:in macrophages from atherosclerotic lesions targeted by pHLIP-anti-miR-33. CONCLUSIONS:This study provides proof of principle for the application of pHLIP for treating advanced atherosclerosis via pharmacological inhibition of miR-33 in macrophages that avoid the deleterious effects in other metabolic tissues. This may open new therapeutic opportunities for atherosclerosis-associated cardiovascular diseases via selective delivery of other protective miRNAs.