the-murine-choline-deficient-ethionine-supplemented-cde-diet-model

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

Curtin University

Human monocyte-derived macrophages (HMM) internalise proatherogenic chylomicron remnants via several high affinity receptor pathways. However, the endocytic ultrastructures responsible for the uptake of chylomicron remnants by macrophages have not previously been described. In this study, we have utilised transmission electron microscopy together with colloidal gold-labelling of chylomicron remnants to investigate the pathways involved in macrophage uptake of chylomicron remnants. We found that macrophages internalise chylomicron remnants via surface-connected compartments of up to 600 nm as well as non-clathrin coated pits. Chylomicron remnants were found to be distributed internally in a number of endocytic vesicles including early cysternal endosomes, spherical late endosomes and tubular vesicular compartments. Uptake of chylomicron remnants by HMM via phagocytosis or macropinocytosis was excluded based on the observations that lipoproteins were not found in phagolysosomes nor modified by inhibitors of these two processes, respectively. The latter observation contrasts with previous reports of chylomicron remnant internalisation by macrophages of other species.

An investigation by electron microscopy of chylomicron remnant uptake by human monocyte-derived macrophages.

The macrophage colony stimulating factor receptor (cFms) and alpha(V)beta(3) integrin are both abundantly expressed and play critical roles in the differentiation, survival and migration of osteoclasts. We have previously demonstrated that cross-talk between cFms- and alpha(V)beta(3)-mediated signaling pathways regulated the cytoskeletal organization required for osteoclast migration. To investigate the nature of interaction between the two receptors, we sequentially used anion-exchange chromatography and immunoprecipitation to purify alpha(V)beta(3)-associated protein complexes. We have demonstrated that cFms stably associated with alpha(V)beta(3) in osteoclasts during adhesion, and that the association was induced by macrophage colony stimulating factor (M-CSF) stimulation. However, the kinetics of association of alpha(V)beta(3) and cFms did not correlate with the kinetics of tyrosine phosphorylation of cFms. Instead, maximally observed alpha(V)beta(3)/cFms association was after the peak of cFms tyrosine phosphorylation and correlated inversely with the total amount of cFms remaining. Furthermore, the complex containing cFms and alpha(V)beta(3) also contained a number of other signaling molecules including Pyk2, p130(Cas) and c-Cbl, known downstream regulators of the integrin-mediated signaling pathways in osteoclasts. In the presence of M-CSF, co-localization of alpha(V)beta(3) integrin and cFms was identified in the podosomal actin ring of the osteoclast during adhesion on glass. Interestingly, co-localization of both receptors was not found in the sealing zone, but in punctate structures associated with adhesion- or transcytosis-like structures in osteoclasts on bone. Taken together, we suggest that the association of alpha(V)beta(3) and cFms could be the result of signaling following tyrosine phosphorylation of cFms. The recruitment of cFms to alpha(V)beta(3) integrin may be an integral part of a larger signaling complex via which both of adhesion- and growth factor receptors coordinately regulate osteoclast adhesion, motility and membrane trafficking.

M-CSF induces the stable interaction of cFms with alphaVbeta3 integrin in osteoclasts.

Uptake by macrophages forms an important part of the mode of action of particulate adjuvants such as oil-in-water emulsions and alum. We have found previously that such adjuvants promote macrophage survival and suggested that this response may contribute to their efficacy. To explore this adjuvant activity further, we have investigated whether oil-in-water emulsion stimulates glucose uptake in macrophages and whether such uptake is relevant to the promotion of survival. We found that oil-in-water emulsion stimulated glucose uptake in a biphasic manner. The first acute phase was independent of mRNA and protein synthesis but appeared to require PI3K activity. In contrast, the second chronic phase was dependent on mRNA and protein synthesis. Importantly, the second phase of glucose uptake required MEK1/2 as well as PI3K activity, indicating that the MEK1/2 pathway can also contribute to cellular glucose uptake. The increased glucose transporter 1 expression during the second phase and long-term survival also appeared to be dependent on PI3K and MEK1/2 signaling pathways. Metabolism of the glucose was required for the emulsion-stimulated survival as well as the increase of prosurvival Bcl-2 transcript levels and maintenance of Bcl-2 protein expression. As transgenic overexpression of Bcl-2 enhances the survival of macrophages in the absence of growth factor, the glycolytic control of Bcl-2 levels may play a central role in emulsion-stimulated macrophage survival. Enhanced glucose uptake by macrophages may therefore be critical to the action of particulate adjuvants.

Glycolytic control of adjuvant-induced macrophage survival: role of PI3K, MEK1/2, and Bcl-2.

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.

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

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.

Hypoxia prolongs monocyte/macrophage survival and enhanced glycolysis is associated with their maturation under aerobic conditions.

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.

Glucose metabolism is required for oxidized LDL-induced macrophage survival: role of PI3K and Bcl-2 family proteins.

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.

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

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.

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

Complications of end-stage chronic liver disease signify a major cause of mortality worldwide. Irrespective of the underlying cause, most chronic liver diseases are characterized by hepatocellular necrosis, inflammation, fibrosis, and proliferation of liver progenitor cells or ductular reactions. Vast differences exist between experimental models that mimic these processes, and their identification is fundamental for translational research. We compared two common murine models of chronic liver disease: the choline-deficient, ethionine-supplemented (CDE) diet versus thioacetamide (TAA) supplementation. Markers of liver injury, including serum alanine transaminase levels, apoptosis, hepatic fat loading, and oxidative stress, as well as inflammatory, fibrogenic and liver progenitor cell responses, were assessed at days 3, 7, 14, 21, and 42. This study revealed remarkable differences between the models. It identified periportal injury and fibrosis with an early peak and slow normalization of all parameters in the CDE regimen, whereas TAA-treated mice had pericentral patterns of progressive injury and fibrosis, resulting in a more severe hepatic injury phenotype. This study is the first to resolve two different patterns of injury and fibrosis in the CDE and TAA model and to indisputably identify the fibrosis pattern in the TAA model as driven from the pericentral vein region. Our data provide a valuable foundation for future work using the CDE and TAA regimens to model a variety of human chronic liver diseases.

Divergent Inflammatory, Fibrogenic, and Liver Progenitor Cell Dynamics in Two Common Mouse Models of Chronic Liver Injury.

The availability of non-tumorigenic and tumorigenic liver progenitor cell (LPC) lines affords a method to screen putative anti-liver cancer agents to identify those that are selectively effective. To prove this principle we tested thalidomide and a range of its derivatives and compared them to lenalidomide and sorafenib, to assess their growth-inhibitory effects.

Identification of a thalidomide derivative that selectively targets tumorigenic liver progenitor cells and comparing its effects with lenalidomide and sorafenib.

Article	Year
An investigation by electron microscopy of chylomicron remnant uptake by human monocyte-derived macrophages. Atherosclerosis\| PubMed ID: 16310792	2006
M-CSF induces the stable interaction of cFms with alphaVbeta3 integrin in osteoclasts. The international journal of biochemistry & cell biology\| PubMed ID: 16600665	2006
Glycolytic control of adjuvant-induced macrophage survival: role of PI3K, MEK1/2, and Bcl-2. Journal of leukocyte biology\| PubMed ID: 19270084	2009
Phosphatidylinostitol-3 kinase and phospholipase C enhance CSF-1-dependent macrophage survival by controlling glucose uptake. Cellular signalling\| PubMed ID: 19376223	2009
Hypoxia prolongs monocyte/macrophage survival and enhanced glycolysis is associated with their maturation under aerobic conditions. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 19494322	2009
Glucose metabolism is required for oxidized LDL-induced macrophage survival: role of PI3K and Bcl-2 family proteins. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 19667115	2009
Invading macrophages play a major role in the liver progenitor cell response to chronic liver injury. Journal of hepatology\| PubMed ID: 20561705	2010
Kupffer cell-monocyte communication is essential for initiating murine liver progenitor cell-mediated liver regeneration. Hepatology (Baltimore, Md.)\| PubMed ID: 26173184	2015
Divergent Inflammatory, Fibrogenic, and Liver Progenitor Cell Dynamics in Two Common Mouse Models of Chronic Liver Injury. The American journal of pathology\| PubMed ID: 27181403	2016
Identification of a thalidomide derivative that selectively targets tumorigenic liver progenitor cells and comparing its effects with lenalidomide and sorafenib. European journal of medicinal chemistry\| PubMed ID: 27208658	2016

Caryn L. Elsegood

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