Maraviroc Prevents HCC Development by Suppressing Macrophages and the Liver Progenitor Cell Response in a Murine Chronic Liver Disease Model.

Maraviroc (MVC), a CCR5 antagonist, reduces liver fibrosis, injury and tumour burden in mice fed a hepatocarcinogenic diet, suggesting it has potential as a cancer therapeutic. We investigated the effect of MVC on liver progenitor cells (LPCs) and macrophages as both have a role in hepatocarcinogenesis. Mice were fed the hepatocarcinogenic choline-deficient, ethionine-supplemented diet (CDE) ± MVC, and immunohistochemistry, RNA and protein expression were used to determine LPC and macrophage abundance, migration and related molecular mechanisms. MVC reduced LPC numbers in CDE mice by 54%, with a smaller reduction seen in macrophages. Transcript and protein abundance of LPC-associated markers correlated with this reduction. The CDE diet activated phosphorylation of AKT and STAT3 and was inhibited by MVC. LPCs did not express Ccr5 in our model; in contrast, macrophages expressed high levels of this receptor, suggesting the effect of MVC is mediated by targeting macrophages. MVC reduced CD45+ cells and macrophage migration in liver and blocked the CDE-induced transition of liver macrophages from an M1- to M2-tumour-associated macrophage (TAM) phenotype. These findings suggest MVC has potential as a re-purposed therapeutic agent for treating chronic liver diseases where M2-TAM and LPC numbers are increased, and the incidence of HCC is enhanced.

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 inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

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.

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

BACKGROUND & AIMS: 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. METHODS: Cell growth, the mitotic and apoptotic index of cell cultures were measured using the Cellavista instrument (SynenTec) using commercially available reagents. RESULTS: Neither lenalidomide nor thalidomide (100 µM) affected tumorigenic LPCs but killed their non-tumorigenic counterparts. Sorafenib arrested growth in both cell types. All but two derivatives of thalidomide were ineffective; of the two effective derivatives, one (thalidomide C1) specifically affected the tumorigenic cell line (10 µM). Mitotic and apoptotic analyses revealed that thalidomide C1 induced apoptotic cell death and not mitotic arrest. CONCLUSIONS: This study shows that screens incorporating non-tumorigenic and tumorigenic liver cell lines are a sound approach to identify agents that are effective and selective. A high throughput instrument such as the Cellavista affords robust and reproducible objective measurements with a large number of replicates that are reliable. These experiments show that neither lenalidomide nor thalidomide are potentially useful for anti-liver cancer therapy as they kill non-tumorigenic liver cells and not their tumorigenic counterparts. Sorafenib in contrast, is highly effective, but not selective. One tested thalidomide derivative has potential as an anti-tumor drug since it induced growth arrest; and importantly, it selectively induced apoptotic cell death only in tumorigenic liver progenitor cells.

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

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.

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

UNLABELLED: 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.

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.

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-x(L), 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-x(L) protein levels. MEK1/2 and PI3K activities were also required for oxLDL-induced Bcl-2 and Bcl-x(L) 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-x(L) protein levels.

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.

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.

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

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.

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

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.

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

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.