Exploring the Molecular Mechanisms Behind Inflammatory Disease
November 17, 2020
Alison B. Kohan, PhD
Associate Professor of Medicine
Endocrinology and Metabolism
Critical Importance of Plasma Triglycerides in Disease
Cardiovascular disease (CVD) is the leading cause of mortality in the United States1. In the past 40+ years, research and epidemiology has largely focused on the role of cholesterol in the blood as a major modifiable risk. As a result, we now have a variety of clinical approaches to lowering plasma cholesterol (most notably statin therapies)2. Despite the widespread and successful use of statins in patients to reduce blood cholesterol (by lowering concentrations of low-density lipoprotein, LDL), patients who present at the emergency room with myocardial infarction are almost all already prescribed statins3,4. This highlights the fact that lowering blood cholesterol level, and successful statin therapy, does not fully reduce the risk of cardiovascular disease. There are additional residual risk factors that we must find and treat in order to reduce CVD mortality.
In recent years, there has been significant progress in identifying and defining these residual risk factors. Elevated plasma triglycerides, especially after a meal, has long been identified as an independent CVD risk factor5–7. In addition, post-meal plasma triglyceride concentrations are strongly predictive of ischemic events in both sexes, even considering differences in protective high-density lipoprotein (HDL) concentration6. Despite this known relationship between plasma triglycerides and CVD risk, approximately 30% of the US population still have moderate-to-high plasma triglyceride levels (greater than 150 mg/dL)8,9. Pharmaceutical and lifestyle interventions that reduce plasma triglycerides are clearly critical. The anti-PCSK-9 drugs (Repatha®) and anti-apoC-III drugs (by both Staten Biotechnology and Ionis Pharmaceuticals10,11) are an example of pharmaceutical targets of plasma triglycerides.
Chylomicrons are the Intestinal Lipoprotein Responsible for Dietary Fat Absorption and Metabolism
Physiologically, chylomicrons and very-low-density lipoprotein (VLDL), two types of triglyceride-rich lipoproteins, make up the largest pool of extracellular lipid substrates in vivo. Immediately following a meal containing fat, there is a transient rise in circulating plasma triglyceride. The triglyceride comes largely from the intestinal enterocyte, where lipids from the diet are taken up and packaged into chylomicrons. This process occurs predominantly in the duodenum and jejunum12,13. Two hours after a fatty meal, the small intestine reaches its peak chylomicron synthesis and secretion rate, though chylomicrons are secreted as early as 13 minutes from fat absorption and for as long as ~6 hours14.
Chylomicrons deliver dietary-derived lipids from the intestine while hepatic-derived lipids are packaged into VLDL and secreted into circulation primarily during fasting. Triglycerides from these lipoproteins are delivered to tissues for energy through uptake via the low-density lipoprotein receptor (LDLr). In addition, these lipoproteins are hydrolyzed by lipoprotein lipase (LPL) and release free fatty acids which can be taken up by cells through fatty acid transporters or passive diffusion. Because of this delivery, chylomicrons can provide a source of lipid fuel for almost all cells in the body. Chylomicrons are unique from VLDL because they also carry antigens from diet, intestinal microbiota, and intestine cells to the gut immune system, and thus interact extensively with mucosal immune cells. Since chylomicrons have a dual role in immune modulation, factors that can regulate the digestion and absorption of dietary lipid, as well as the secretion of chylomicrons, can also regulate the exposure of the immune system to potential antigen and lipid fuel.
Chylomicrons contain triglyceride and cholesterol in their core, surrounded by phospholipids, and contain apolipoproteins B-48, A-I, A-IV, and C-III. Apolipoproteins are biologically active in metabolic processes (including lipid clearance and glucose homeostasis). ApoB-48 is essential to the structure of the chylomicron, but interestingly, the other apolipoprotein components serve as signaling molecules and enzymatic modulators that are essential for chylomicron metabolism and clearance from the blood. Moderating post-prandial lipids and the apolipoproteins associated with them is an important part of moderating cardiovascular disease risk.
Apolipoprotein C-III (apoC-III) is another apolipoprotein that is a potent cardiovascular risk factor. It is an exchangeable apolipoprotein produced by both the intestine and liver, found on both chylomicrons and very low-density lipoproteins15. In humans, plasma apoC-III levels are elevated during both hyperlipidemia and diabetes16–18. In plasma, apoC-III delays chylomicron and VLDL clearance, and in the liver it stimulates VLDL secretion. Through both of these actions, apoC-III stimulates plasma hyperlipidemia19,20. In addition to its role in the maintenance of the hyperlipidemic state, apoC-III levels are themselves an independent predictor of cardiovascular disease risk21,22. Recent large-scale epidemiological studies have revealed that mutations in apoC-III result in a striking decrease in ischemic cardiovascular disease and coronary heart disease risk in humans23,24. This finding has generated significant new interest in apoC-III.
ApoC-III has also recently been shown to act in the intestine as an inhibitor of dietary lipid absorption25. This new intestinal role for apoC-III is likely important in understanding the mechanism by which apoC-III mediates cardiovascular disease risk given that fat absorption and intestinal lipoprotein secretion contribute to cardiovascular disease progression6,26,27. How intestinal apoC-III is regulated is unknown and may be quite different from hepatic apoC-III regulation since the hepatic and intestinal lipoprotein synthesis and pathways are unique and regulated at different steps28–30. There may be a valuable difference in the regulation and function of apoC-III in these tissues and it is likely that dietary nutrients moderate this effect.
Hurdles to Studying Chylomicron Secretion and Small Intestinal Physiology
Despite the importance of studying the intestine and its lipoprotein secretion in metabolism and disease, it has been notoriously difficult to study because the tissue rapidly degrades during isolation, as well as due to the lack of cell culture models31,32. Primary enterocytes are short-lived (~24h); everted gut sacs cannot be transfected; and Caco-2 cells are a monolayer colon cancer cell line that lacks essential biology of the small intestine. Overall, the lack of a culture model has been a significant roadblock to gaining mechanistic insights into the function of the small intestine as a metabolic organ.
Intact intestine is a complex tissue comprised of multiple cell types, including enterocytes (the absorptive cells of the intestine), enteroendocrine cells (which secrete incretin hormones), goblet cells (which secret mucus), and mast cells (which secrete immune modulators). There are two primary structures in the intestinal epithelium: the villus and crypt. The crypt is found at the base of the villus, and these structures contain the multi-potent intestinal stem cells (ISCs), which express the transcription factor LGR5 and give rise to all of the cells lining the intestinal epithelium33,34. In vivo, the intestinal epithelium is in a constant state of differentiation, renewal, and replacement driven by these ISCs within the crypt niche.
The isolation and propagation of these stem cells was first established by Sato and Clevers in 2009 and has had a major impact on the field34. They showed that the crypt, when plated into 3D Matrigel and treated with growth factors, will differentiate into a 3D enteroid. In the enteroid culture, the LGR5+ stem cells within those crypts grow and differentiate into all the cell types normally found in the intestinal epithelium, including new stem cells.
As the primary stem cell within an isolated crypt differentiates in response to growth factors, it forms a three-dimensional enteroid. Mature enteroids (by convention “enteroid” refers to mouse-derived cultures, whereas “organoids” refer to human-derived cultures) form at approximately day 10 in growth media and retain intestinal barrier function, express amino acid transporters, and intestine-specific stem cell markers31,35,36. These cells maintain their physiological orientation around a central lumen (the apical surface) and a basolateral surface facing media.
The stem cells differentiate in culture by sloughing off cells into the luminal compartment followed by regeneration of crypt epithelium. Enteroids are therefore not only powerful models of intestinal function, but also represent a significant advance in our ability to determine intestinal mechanisms for dietary fat absorption and lipoprotein synthesis and secretion.
Beyond Cardiovascular Disease: The Role of Intestinal Lipoproteins in Other Diseases
Short-bowel syndrome is an extreme example of what happens without small intestinal lipid absorption processes. Patient energy needs cannot be met through carbohydrate feeding, nor through parenteral nutrition. In addition, the liver cannot handle the increased burden of clearing portal nutrients while also secreting VLDL to keep up with energy demands. Ultimately, patients die of liver failure. This brutal disease illustrates key physiological processes of the small intestine: ability to absorb dietary triglycerides and present these triglycerides to the rest of the body in easily metabolizable form (chylomicrons), the importance of the lymphatic route of lipid absorption (because otherwise all nutrients are shunted to the liver via portal circulation), and finally the inability to sustain energy homeostasis without dietary lipids.
Diseases where these lipases are absent or reduced also cause a significant defect in dietary fat absorption, including cystic fibrosis (CF), where stricture of the pancreas seriously reduces the secretion of lipases and bicarbonate during fat ingestion. Thus, deficiency in pancreatic enzyme secretion leads to an inability to hydrolyze dietary lipids in the intestinal lumen. Cystic fibrosis transmembrane conductance regulator (CFTR) is also critical in bile acid secretion from the liver to the intestinal lumen. Therefore, luminal conditions in the CF intestine are antagonistic to lipid absorption, which requires hydrolysis of dietary lipid with pancreatic lipase and emulsification with bile salts. These are relatively well-understood physio-chemical defects in the CF intestine, and CF patients are prescribed Pancreatic Enzyme Replacement Therapy (PERT) to bring total fat absorption to normal levels. Despite PERT, when human intestinal explants are cultured, they secrete reduced numbers of intestinal triglyceride-rich lipoproteins, as well as exhibit reduced apoB synthesis37. With the advent of CFTR tri-modulator Trikafta, it will be interesting to see whether the small intestinal manifestations of CF become a larger focus.
Conclusion and Future Directions
Lipid absorption by the small intestine is absolutely critical for whole-body metabolism and overall triglyceride concentrations, both of which are risk factors for cardiovascular disease. Chylomicron metabolism also plays a critical role in determining plasma levels of triglyceride and dietary antigens. The rates of chylomicron secretion and remnant clearance are controlled by intracellular and extracellular factors including apolipoprotein CIII (apoC-III). Functionally, therefore, humans are almost always in the post-prandial state. Understanding how chylomicron synthesis and secretion, metabolism, and interaction with immune cells is a critical frontier for understanding inflammatory disease.
The Kohan Lab has been using primary intestinal organoids to dissect these chylomicron-driven effects on human disease. Dr. Kohan's team was the first to show that organoids recapitulate intestinal fat absorption by taking up 3H-FFA and secreting 3H-TAG along with apoB-48 in a chylomicron particle31,36.
Chylomicrons are difficult to isolate, and the intestine is notoriously finicky to study. The Kohan Lab uses primary intestinal organoids to get around these issues and discover new metabolic processes that are chylomicron driven. Researchers in the Kohan lab have recently discovered a mechanism of regulatory T cell (Treg) regulation by intestinal chylomicrons. Specifically, they discovered that mice overexpressing human apolipoprotein C-III are protected from dextran-sulfate sodium (DSS)-induced colitis and its associated symptoms. Conversely, apoC-III knockout mice are susceptible to severe colitis and have fewer colonic Tregs than their wild-type counterparts.
The canonical role of apoC-III is to inhibit lipid uptake from triglyceride-rich lipoproteins like chylomicrons, by inhibiting lipoprotein lipase (LPL) and low-density lipoprotein receptor (LDLr). Building upon our team’s expertise in intestinal lipid metabolism and lipoprotein clearance, we found that intestinal Tregs and T cells express high levels of LDLr and, in response to apoC-III, T cells take up less triglyceride. Our data suggest that inhibiting lipid uptake from chylomicrons into Tregs stimulates intestinal Tregs and protects against colitis. We are now dissecting the molecular mechanisms involved, in the hopes that this pathway might be a pharmaceutical target for treating inflammatory bowel diseases.
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