Caryn L. Elsegood

Hypoxia Prolongs Monocyte/Macrophage Survival and Enhanced Glycolysis Is Associated with Their Maturation under Aerobic Conditions

In chronic inflammatory lesions macrophages are abundant and adapt to the low oxygen concentrations often present there. In low oxygen some cell types die by apoptosis, as reported for macrophage cell lines, while others survive better as they shift their metabolism to anaerobic glycolysis. It was found here that hypoxia prolongs the survival of murine bone marrow-derived macrophages, either in the absence or presence of low CSF-1 (M-CSF) concentrations. Although Akt activity increased in bone marrow-derived macrophages in the low oxygen conditions, the levels of both anti- and proapoptotic Bcl-2 family members decreased. Glycolysis was enhanced as judged by increased glucose uptake, glucose transporter expression, lactate dehydrogenase mRNA expression, and lactate secretion. Human monocytes responded similarly to low oxygen, and a number of genes associated with glycolysis were shown by microarray analysis and quantitative PCR to be up-regulated. Interestingly, human monocyte-derived macrophages showed evidence of enhanced glycolysis even under aerobic conditions. It is proposed that certain monocyte/macrophage populations survive better under conditions of low oxygen, thereby contributing to their increased numbers at sites of chronic inflammation and tumors; it is also proposed that as macrophages differentiate from monocytes they begin to adopt a glycolytic metabolism allowing them to adapt readily when exposed to low oxygen conditions.

Invading macrophages play a major role in the liver progenitor cell response to chronic liver injury

BACKGROUND & AIMS Although a strong association between liver progenitor cells (LPCs) and inflammation exists in many chronic liver diseases, the exact role of the immune system in LPC-mediated hepatic regeneration remains unclear. A number of pro-inflammatory factors were identified in cytokine knockout mice in which the LPC response was attenuated but neither the mechanism nor the producing cells are known. METHODS To identify the critical immune cells and cytokines required in the LPC response, we compared two diet-induced models of liver injury with two recently established transgenic models of immune-mediated hepatitis. RESULTS Despite severe inflammation being observed in all models, the generation of LPCs was highly dependent on the cause and kinetics of liver damage. The LPC response was associated with an increase of macrophages and CD8(+) T cells but not natural killer cells. T cell-deficient mice were able to mount a LPC response, albeit delayed, suggesting that T cells are not essential. Mice mounting an LPC response showed elevated numbers of Kupffer cells and invading CX(3)CR1(high)CCR2(high) macrophages secreting persistent high levels of tumour necrosis factor alpha (TNFalpha), a major cytokine involved in the LPC response. CONCLUSIONS Liver macrophages are an important determinant of LPC expansion during liver regeneration in models of diet- and immune-mediated liver injury. Invading macrophages in particular provide pro-mitogenic cytokines such as TNFalpha that underpin the process. LPC themselves are a source of chemokines (CCL2, CX(3)CL1) that attract infiltrating macrophages.

Kupffer cell–monocyte communication is essential for initiating murine liver progenitor cell–mediated liver regeneration

Liver progenitor cells (LPCs) are necessary for repair in chronic liver disease because the remaining hepatocytes cannot replicate. However, LPC numbers also correlate with disease severity and hepatocellular carcinoma risk. Thus, the progenitor cell response in diseased liver may be regulated to optimize liver regeneration and minimize the likelihood of tumorigenesis. How this is achieved is currently unknown. Human and mouse diseased liver contain two subpopulations of macrophages with different ontogenetic origins: prenatal yolk sac–derived Kupffer cells and peripheral blood monocyte–derived macrophages. We examined the individual role(s) of Kupffer cells and monocyte‐derived macrophages in the induction of LPC proliferation using clodronate liposome deletion of Kupffer cells and adoptive transfer of monocytes, respectively, in the choline‐deficient, ethionine‐supplemented diet model of liver injury and regeneration. Clodronate liposome treatment reduced initial liver monocyte numbers together with the induction of injury and LPC proliferation. Adoptive transfer of monocytes increased the induction of liver injury, LPC proliferation, and tumor necrosis factor‐α production. Conclusion: Kupffer cells control the initial accumulation of monocyte‐derived macrophages. These infiltrating monocytes are in turn responsible for the induction of liver injury, the increase in tumor necrosis factor‐α, and the subsequent proliferation of LPCs. (Hepatology 2015;62:1272‐1284)

Effect of probucol on plasma clearance and organ uptake of chylomicrons and VLDLs in normal and diabetic rats.

Probucol was given to rats made diabetic by streptozotocin. Compared with diabetic rats not receiving probucol or with nondiabetic rats, probucol lowered the plasma concentrations of triglycerides, phospholipids, cholesterol, and apolipoprotein B. The concentrations of serum chylomicrons and very low density lipoprotein (VLDL) were also reduced. In control and diabetic rats, probucol enhanced the clearance of endogenously radiolabeled VLDL from the plasma. Clearances from the plasma of rat lymph chylomicrons or chylomicron-like lipid emulsions were slow in diabetic rats. Probucol normalized chylomicron clearance in diabetic rats primarily by restoring hepatic uptake of remnants, which was decreased in diabetes. In diabetic rats, uptake of chylomicron remnants was increased in a number of extrahepatic tissues, including the heart and kidney. Probucol significantly decreased uptake in some extrahepatic tissues. Increased plasma clearance of VLDL and chylomicrons was associated with an increase in the apolipoprotein CII/CIII and apolipoprotein E/C ratios. Orally administered probucol was specifically incorporated into lymph chylomicrons, and clearance of probucol from the plasma exactly paralleled the clearance of chylomicron remnants, as traced with radiolabeled cholesteryl esters. Chylomicron-like emulsions incorporating probucol were exclusively cleared from the plasma by the liver in normal rats. We conclude that in streptozotocin diabetic rats, probucol is an effective hypolipidemic agent because it promotes the clearance of the triglyceride-rich lipoproteins.

IS ATHEROSCLEROSIS EXCLUSIVELY A POSTPRANDIAL PHENOMENON

1. Atherosclerosis begins with the deposition of cholesterol in arterial tissue that is thought to be derived from circulating lipoproteins. There is considerable evidence implicating low density lipoprotein (LDL) as a primary source of plaque cholesterol and, consequently, there are many articles that deal with various aspects of LDL metabolism.

Defective plasma clearance of chylomicron-like lipid emulsions in Watanabe heritable hyperlipidemic rabbits

Human patients with familial hypercholesterolemia (FH) and Watanabe heritable hyperlipidemic rabbits (WHHL), while lacking normal receptors recognizing low-density lipoproteins (LDL), are said to have normal clearance of chylomicrons. In the present study, emulsions with a similar lipid composition to chylomicrons were injected intravenously in homozygous WHHL rabbits and normal control rabbits fed diet with low or high cholesterol. Radioactive labels tracing emulsion triolein and cholesteryl oleate were both removed rapidly from the bloodstream, with the removal rate of triolein always faster than that of cholesteryl oleate. This pattern was similar to the clearance of normal chylomicrons in rabbits or rats, and was consistent with the formation of remnant lipoproteins after hydrolysis of emulsion triolein by lipoprotein lipase, followed by hepatic uptake of the remnants. The removal of cholesteryl oleate was significantly slower in WHHL rabbits than in normal controls, suggesting that the absence of LDL receptor function led to impaired remnant clearance. Measured in post-heparin plasma, the activity of lipoprotein lipase was decreased in WHHL rabbits, but this was not associated with clear evidence of defective lipolysis of emulsion triolein. Apolipoprotein E did not appear to be deficient in WHHL rabbits. Plasma devoid of lipoproteins less than 1.006 g/ml from WHHL and normal control rabbits transferred similar amounts of apolipoprotein E to chylomicron-like emulsions after incubation. Impaired clearance of chylomicron remnants possibly contributes to the hypertriglyceridemia of WHHL rabbits and to accelerated atherogenesis when the function of LDL receptors is defective.

Phosphatidylinostitol-3 kinase and phospholipase C enhance CSF-1-dependent macrophage survival by controlling glucose uptake.

Colony stimulating factor-1 (CSF-1)-dependent macrophages play crucial roles in the development and progression of several pathological conditions including atherosclerosis and breast cancer metastasis. Macrophages in both of these pathologies take up increased amounts of glucose. Since we had previously shown that CSF-1 stimulates glucose uptake by macrophages, we have now investigated whether glucose metabolism is required for the survival of CSF-1-dependent macrophages as well as examined the mechanism by which CSF-1 stimulates glucose uptake. Importantly, we found that CSF-1-induced macrophage survival required metabolism of the glucose taken up in response to CSF-1 stimulation. Kinetic studies showed that CSF-1 stimulated an increase in the number of glucose transporters at the plasma membrane, including Glut1. The uptake of glucose induced by CSF-1 required intact PI3K and PLC signalling pathways, as well as the downstream effectors Akt and PKC, together with a dynamic actin cytoskeleton. Expression of constitutively active Akt partially restored glucose uptake and macrophage survival in the absence of CSF-1, suggesting that Akt is necessary but not sufficient for optimal glucose uptake and macrophage survival. Taken together, these results suggest that CSF-1 regulates macrophage survival, in part, by stimulating glucose uptake via Glut1, and PI3K and PLC signalling pathways.

Glucose Metabolism is Required for Oxidized LDL-Induced Macrophage Survival: Role of PI3K and Bcl-2 Family Proteins

Objective—Oxidized low-density lipoprotein (oxLDL) induces survival of colony stimulating factor-1 (CSF-1)-dependent macrophages in vitro. Because atherosclerotic lesion–associated macrophages take up large amounts of glucose, we investigated whether, and how, oxLDL promotes glucose uptake and how glucose metabolism regulates oxLDL-induced macrophage survival. Methods and Results—OxLDL-induced macrophage survival required glucose metabolism. OxLDL stimulated 2 phases of glucose uptake, namely acute and chronic, which required PI3K but not MEK1/2 activity. PI3K appeared to regulate glucose transport via glucose transporter affinity and/or mobilization. OxLDL also maintained levels of the prosurvival proteins, Bcl-2 and Bcl-xL, after CSF-1 had been removed through a combination of mechanisms including transcription, translation, and protein stabilization. Significantly, inhibition of glucose metabolism reduced Bcl-2 and Bcl-xL protein levels. MEK1/2 and PI3K activities were also required for oxLDL-induced Bcl-2 and Bcl-xL mRNA upregulation. Conclusions—These results suggest that oxLDL enhances macrophage survival in the absence of CSF-1 by inducing PI3K-dependent glucose uptake, which is metabolized to maintain Bcl-2 and Bcl-xL protein levels.

Effects of adrenoreceptor antagonists and agonists on clearance of emulsion models of triacylglycerol-rich lipoproteins from plasma in rats.

1. We previously found that adrenaline and noradrenaline exert essentially opposite effects on clearance from plasma of chylomicron‐like emulsions injected intravenously in rats, suggesting mechanisms that may be implicated in the atherogenic effects of chronic stress and hypertension and conversely in the protective effect of regular exercise.

Immune checkpoint inhibition: prospects for prevention and therapy of hepatocellular carcinoma

The global prevalence of liver cancer is rapidly rising, mostly as a result of the amplified incidence rates of viral hepatitis, alcohol abuse and obesity in recent decades. Treatment options for liver cancer are remarkably limited with sorafenib being the gold standard for advanced, unresectable hepatocellular carcinoma but offering extremely limited improvement of survival time. The immune system is now recognised as a key regulator of cancer development through its ability to protect against infection and chronic inflammation, which promote cancer development, and eliminate tumour cells when present. However, the tolerogenic nature of the liver means that the immune response to infection, chronic inflammation and tumour cells within the hepatic environment is usually ineffective. Here we review the roles that immune cells and cytokines have in the development of the most common primary liver cancer, hepatocellular carcinoma (HCC). We then examine how the immune system may be subverted throughout the stages of HCC development, particularly with respect to immune inhibitory molecules, also known as immune checkpoints, such as programmed cell death protein‐1, programmed cell death 1 ligand 1 and cytotoxic T lymphocyte antigen 4, which have become therapeutic targets. Finally, we assess preclinical and clinical studies where immune checkpoint inhibitors have been used to modify disease during the carcinogenic process. In conclusion, inhibitory molecule‐based immunotherapy for HCC is in its infancy and further detailed research in relevant in vivo models is required before its full potential can be realised.