Suneil Koliwad, MD, PhD
My laboratory investigates the role of chronic inflammation in the development of Type 2 Diabetes and how dietary lipids modulate the activation of innate immune cells that trigger and maintain inflammation in metabolic tissues.
Beyond comprising our bodily energy stores, dietary lipids are necessary for several critical processes, including the synthesis of cellular membranes, vitamins, skin lipids and waxes, blood clotting, gene expression, and cell signaling cascades. However, when chronically in excess, dietary fat intake is associated with several diseases, including diabetes, neurodegenerative diseases, certain cancers, and atherosclerosis, which together exact a huge toll on human health. Given the high prevalence of high-fat diets in our world, determining the links between dietary lipids and these diseases is of biomedical and socioeconomic importance.
One emerging possibility involves chronic inflammation. Excess dietary fat intake often produces tissue inflammation that manifests prior to disease onset. For type 2 diabetes, this process is characterized by the inflammatory (M1) activation of innate immune cells, including macrophages and dendritic cells, in metabolic tissues such as white adipose, muscle, and liver. In exploring how dietary lipids may act as proximal triggers of macrophage activation, we showed that genetically-altered murine macrophages with an increased capacity to store dietary fats as intracellular triacylglycerol are less vulnerable to M1 inflammatory activation both in culture and in living mice. Remarkably, by transplanting these protective macrophages into mice protected them against developing diabetes driven by diet-induced obesity.
We are now deeply focused on probing the mechanisms by which intracellular fatty acids modulate inflammatory activation in macrophages, dendritic cells, and CNS microglia and determine how these impact type 2 diabetes, neurodegeneration, and other diseases in which chronic inflammation precedes overt disease.
Scientific Overview of our Research Program
Hypothesis 1: Fatty acids and their metabolites act intracellularly to modulate key pathways that regulate the inflammatory function of macrophages, dendritic cells, microglia, and other innate immune cells.
We are using tools, including mice with selected mutations in metabolic and inflammatory cells and mice that express specific reporter molecules to probe potential mechanisms by which intracellular fatty acids modulate inflammation in myeloid cells and in tissues.
- We have identified that altering the intracellular storage of fatty acids can modulate two signaling pathways that impinge on NF-kB activation, a direct transcriptional regulator of several classical secreted inflammatory factors. These include Toll-like receptor 4 (TLR4) signaling and the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress. We are currently using several approaches to determine how this modulation takes place.
- Palmitate and oleate represent the two most highly abundant FAs in the diet, in the blood of individuals, and in the WAT. Along with prenylation and myristoylation, protein palmitoylation has been described in diverse mammalian cell types, but not in macrophages. More intriguingly, modification of proteins by acylation with oleate (oleoylation) is very poorly described. Additionally, it is not known whether palmitate and oleate have differential effects on gene transcription. For example, oleate is normally stored in cells as triacylglycerol, but what happens when the capacity for such storage is exceeded? We are using proteomic and genomic screening approaches in macrophages with varying capacities for fat storage to answer this question.
- We are using metabolomics to measure the effects of different classes of lipids and their storage on the flux through energy pathways in macrophages.
Hypothesis 2: The ratio of free intracellular lipids to those neutralized and stored in intracellular compartments, such as triacylglycerol, is a determinant of inflammatory activation in myeloid cells.
The two predominant dietary fatty acids, oleate (monounsaturated) and palmitate (saturated), are taken up equally well by cultured macrophages. However, intracellular oleate routs primarily into the triacylglycerol fraction while only ~12% of palmitate does so. Rather, we have observed palmitate to route into several different phospholipid fractions, while oleate did not do this. We are actively studying how monounsaturated fatty acids such as oleic acid, which are known to have health-promoting effects, influence the intracellular routing of pro-inflammatory saturated fatty acids. We are able to use specific genetic modifications to alter the fatty acid routing within macrophages and are asking how these alterations affect inflammatory function.
Hypothesis 3: Reducing diet-induced inflammatory activation of innate immune cells in metabolic tissues and in the brain can preserve normal tissue function and stave off disease.
- White Adipose Tissue Inflammation: We aim to more clearly discern how two cell types—macrophages and adipocytes—interact during the process by which high-fat diets induce chronic inflammation within the white adipose tissue (WAT) and ultimately metabolic disease (1-4). Specifically we are able to use genetic tools to explore how fat storage capacity by macrophages and adipocytes modulates inflammatory and metabolic processes in vitro and in vivo.
- Neuroinflammation: We are beginning to study how dietary lipids regulate the inflammatory function of microglia, the resident innate immune cells of the brain, and to correlate this regulation with the function and lifespan of neurons which are lost in neurodegenerative diseases.
- Metabolic Tissues: We are studying how altering lipid-induced macrophage activation can modulate the progression of other metabolic diseases, such as atherosclerosis, fatty liver disease, and potentially cancers.
New Experimental Tools
- We have developed a culture system to study inflammatory responsiveness in macrophages with triacylglycerol storage capacity over a range starting at 10-fold above normal all the way down to zero.
- We have crossed murine lines expressing conditional (“floxed”) alleles with those expressing Cre recombinase in myeloid cells (Cd11b-Cre) in order to generate mice lacking TLR4 (TLR4-MacKO), the ER stress sensor IRE1α (IRE-MacKO), or DGAT1 (DGAT-MacKO) only in macrophages and microglia.
- We have obtained mice expressing fluorescent proteins or luciferase coupled to the activation IRE1α (ERAI-venus, ERAI-luc; from Dr. T. Iwawaki, Riken, Japan) or NF-κB (NF-κB-luc, NF-κB-GFP, the latter from Dr. C. Jobin, UNC, Chapel Hill). These reporters can be used in high-throughput screens to identify lipids that modulate the UPR or M1 response in cells and in mice.
- By controlling the expression of an oncoprotein, Hoxb8, we have immortalized murine myeloid progenitors harvested from mouse bone marrow or embryonic liver cells and can differentiated these into macrophages or dendritic cells on demand. These cells mirror macrophages harvested from mice. Immortalized progenitors will allow for high-throughput screens without the associated mouse costs.
- We have cultured primary microglia from mouse brains and observed that the response of these cells to treatment with proinflammatory fatty acids mirrors that of peripheral macrophages.
- We have applied a methodology called click chemistry to the macrophage proteome to detect the reversible posttranslational covalent modification of proteins by acyl-thioester linkages of cysteine residues to fatty acyl-CoA derivates of nutritional fatty acids. In brief, cells are metabolically labeled with a fatty acid analog amenable to copper-catalyzed azide-alkyne cycloaddition (click chemistry). Labeled cells can be lysed, and the membrane fractions treated with a rhodamine or biotin-azide reporter group in the presence of Copper. Proteins with linkages to click analogs can be detected by avidin-HRP blotting and gel- or LC-MS-based characterization. We are synthesizing analogs of several nutritional lipids and expect this methodology to provide robust results in primary murine macrophages.
Significance: Long-Term Goals of our Research
- Defining lipid-modification of proteins as a mechanism regulating macrophage function.
- Identifying metabolic targets beyond DGAT1 that modulate inflammation in macrophages.
- Exploring the role of DGAT1 (an ACAT homolog) in cholesterol storage and atherosclerosis.
- Determining if altering lipid metabolism in microglia can modulate neuroinflammation in neurodegenerative conditions
- Discover rational nutraceuticals that counteract the effects of high-fat diets to improve lifespan.