Showing papers by "John P. Iredale published in 2011"

Ly6Chi monocytes direct alternatively activated profibrotic macrophage regulation of lung fibrosis.

Abstract: Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating disease. Antiinflammatory therapies, including corticosteroids, are of no benefit. The role of monocytes and macrophages is therefore controversial.Objectives: To define the role of monocytes and macrophages during lung fibrogenesis and resolution, and explore the phenotype of the cells involved.Methods: We used multiple in vivo depletional strategies, backed up by adoptive transfer techniques. Further studies were performed on samples from patients with IPF.Measurements and Main Results: Depletion of lung macrophages during fibrogenesis reduced pulmonary fibrosis as measured by lung collagen (P = 0.0079); fibrosis score (P = 0.0051); and quantitative polymerase chain reaction for surrogate markers of fibrosis Col1 (P = 0.0083) and α-smooth muscle actin (P = 0.0349). There was an associated reduction in markers of the profibrotic alternative macrophage activation phenotype, Ym1 (P = 0.0179), and Arginase1. The alternative macrophage marker CD...

Scraping fibrosis: expressway to the core of fibrosis.

Abstract: Animal experiments using single organs as models of fibrosis spur therapeutic development toward promising targets, but testing of these therapies in human fibrosis yielded disappointing results and limited efficacy. Finding core pathways relevant in different organs that can become fibrotic will uncover molecules that will prove useful as therapeutic targets in many species, including humans. In ‘Bench to Bedside’, Scott Friedman, Wajahat Mehal and John Iredale discuss this new paradigm in fibrosis research and its potential as a new drug development approach. In ‘Bedside to Bench’, Alison Eddy peruses how the protein encoded by UMOD, a gene linked to variable risk for chronic kidney disease and hypertension in humans, may have a role in fibrosis and kidney disease. Uncovering the normal function of UMOD and its gene variants will shed light on the pathogenesis of chronic kidney disease and aid in the discovery of new targets for kidney fibrosis and hypertension.

Remodelling of extracellular matrix is a requirement for the hepatic progenitor cell response

Abstract: Background and methods In advanced liver damage, hepatic regeneration can occur through proliferation of a resident hepatic progenitor cell (HPC) population. HPCs are located within a designated niche in close association with myofibroblasts and bone marrow (BM) derived macrophages. Extra-cellular matrix (ECM) laminin invariably surrounds HPCs, but the functional requirement of this matrix-cell association is untested in vivo. Using the collagen Iα1 (r/r) mouse (r/r), which produces mutated collagen I resistant to matrix metalloproteinase degradation and has an exaggerated fibrotic response to liver injury, we test the relationship between collagen degradation, laminin deposition, and the HPC response. Results Chronic fibrotic carbon tetrachloride (CCl 4 ) injury can induce a florid HPC response associated with dense laminin deposition. In the recovery phase after chronic CCl 4 injury, r/r mice have a markedly attenuated HPC response compared to wild-types, together with persistence of collagen I and failure to deposit ECM laminin. Similar results were found in r/r mice given the choline-deficient ethionine supplemented diet, another model of the HPC response. In cross-over sex-mismatched BM transplantation (BMT) experiments between r/r mice and wild-types, the blunted HPC response of r/r mice was not rescued by wild-type BMT and likewise not conferred on to wild-type recipients by r/r BMT, demonstrating that the attenuated HPC response in r/r mice is a property intrinsic to the liver. Conclusion Failure of ECM remodelling after chronic fibrotic liver injury hinders the ability of the liver to activate HPCs. Laminin-progenitor cell interactions within the HPC niche are a critical for HPC mediated regeneration.

Persistence of functional hepatocyte-like cells in immune-compromised mice

Abstract: Background: Human embryonic stem cells (hESCs) can be efficiently differentiated to hepatocyte-like cells (HLCs) in vitro and demonstrate many of the functions and gene expression found in the adult liver. Aims: In this study, we assess the therapeutic value of HLCs in long-term cell-based therapies in vivo. Methods: hESC-derived HLCs were injected into the spleen of acutely injured NODscidIL-2Rγnull mice and analysed at various time points post-transplantation up to 3 months. Results: Large clusters of human cells engrafted in the spleen after 3 days and had expanded considerably by 31 days. At these time points, we identified human cells expressing parenchymal hepatocyte markers, exhibiting biliary duct-like structures and expressing myofibroblast markers. Three months after transplantation, we could detect human HLCs that were positive for albumin and CK18 by immunostaining and human DNA by fluorescent in situ hybridisation. Moreover, we could detect secretion of human serum albumin by enzyme-linked immunoabsorbant assay. Conclusions: We observed the persistence, engraftment and function of HLCs in vivo up to 3 months post-translation; however, all murine recipients developed large splenic and liver tumours that contained endodermal and mesodermal cell types. Although our studies demonstrate that hESC-derived HLCs have the potential to play an important role in cell-based therapies, current methodologies and transplantation strategies require substantial refinement before they can be deployed safely.

Metabolic pathways promoting intrahepatic fatty acid accumulation in methionine and choline deficiency: implications for the pathogenesis of steatohepatitis.

Abstract: The pathological mechanisms that distinguish simple steatosis from steatohepatitis (or NASH, with consequent risk of cirrhosis and hepatocellular cancer) remain incompletely defined. Whereas both a methionine- and choline-deficient diet (MCDD) and a choline-deficient diet (CDD) lead to hepatic triglyceride accumulation, MCDD alone is associated with hepatic insulin resistance and inflammation (steatohepatitis). We used metabolic tracer techniques, including stable isotope ([13C4]palmitate) dilution and mass isotopomer distribution analysis (MIDA) of [13C2]acetate, to define differences in intrahepatic fatty acid metabolism that could explain the contrasting effect of MCDD and CDD on NASH in C57Bl6 mice. Compared with control-supplemented (CS) diet, liver triglyceride pool sizes were similarly elevated in CDD and MCDD groups (24.37 ± 2.4, 45.94 ± 3.9, and 43.30 ± 3.5 μmol/liver for CS, CDD, and MCDD, respectively), but intrahepatic neutrophil infiltration and plasma alanine aminotransferase (31 ± 3, 48 ± 4, 231 ± 79 U/l, P < 0.05) were elevated only in MCDD mice. However, despite loss of peripheral fat in MCDD mice, neither the rate of appearance of palmitate (27.2 ± 3.5, 26.3 ± 2.3, and 28.3 ± 3.5 μmol·kg−1·min−1) nor the contribution of circulating fatty acids to the liver triglyceride pool differed between groups. Unlike CDD, MCDD had a defect in hepatic triglyceride export that was confirmed using intravenous tyloxapol (142 ± 21, 122 ± 15, and 80 ± 7 mg·kg−1·h−1, P < 0.05). Moreover, hepatic de novo lipogenesis was significantly elevated in the MCDD group only (1.4 ± 0.3, 2.3 ± 0.4, and 3.4 ± 0.4 μmol/day, P < 0.01). These findings suggest that important alterations in hepatic fatty acid metabolism may promote the development of steatohepatitis. Similar mechanisms may predispose to hepatocyte damage in human NASH.

Acute liver failure in Scotland between 1992 and 2009; incidence, aetiology and outcome

Abstract: Summary Aim: To describe incidence, aetiology and outcome data for Scotland since the inception of the Scottish Liver Transplant Unit (SLTU) in 1992. Background: Acute liver failure (ALF) is a rare but frequently fatal condition. Few studies have adequate patient numbers to draw convincing conclusions over demographic features, aetiology and outcome. Design: Statistical analysis of prospectively collected data on aetiology, demographic, clinical and outcome of all admissions, including those with ALF, to the SLTU. Methods: Incidence data presented for admissions and ALF. Descriptive frequencies for aetiology, clinical, demographic and outcome data presented; including split analysis for paracetamol and nonparacetamol aetiologies. Univariate and multivariate analysis of admission factors predictive of outcome is described. Results: Nine hundred and forty-nine patients were admitted to the SLTU between 1992 and 2009. Five hundred and twenty-four patients had ALF. The annual incidence of ALF in the Scottish population is 0.62 per 100 000 and paracetamol overdose (POD) was the largest causative factor; responsible for 0.43 cases of ALF per 100 000 population per year. The odds ratio (OR) of transplantation or death was 0.47 in the POD group compared to other aetiologies; yet of not being a transplant candidate having met the Kings College Hospital poor prognostic criteria OR was 4.9. Of admissions listed for transplant 76.0% were transplanted. Of those listed and not transplanted mortality was approaching 100% and 76.1% of those transplanted survived to discharge. Conclusions: This large, prospective, single centre study with a defined geographical area and well-recorded population provides accurate data regarding ALF between 1992 and 2009.

OP14 Identification and characterisation of a Novel Ly-6Cintermediate intrahepatic macrophage population which mediates the resolution of liver fibrosis, is induced by phagocytosis and can be manipulated therapeutically in vivo

Abstract: Introduction Macrophages are critical for the progression and resolution of hepatic fibrosis. Studies have identified Ly-6C hi macrophages as the pro-fibrogenic subset in mice. However, the identity of the pro-resolution hepatic macrophage population is unknown. Aim We aimed to identify and characterise the macrophage population mediating the resolution of hepatic fibrosis. Method We established a model of reversible murine hepatic fibrosis by administering 4 weeks of CCl 4 , followed by tissue harvests at serial timepoints after the final dose. Results Histological analysis identified maximal fibrosis resolution between 72 and 96 h after the final CCl 4 dose. Flow cytometry of hepatic macrophages showed that during maximal fibrosis resolution there was a loss of pro-fibrotic Ly-6C hi macrophages and large increase in a Ly-6C intermediate macrophage population, which were the most numerous macrophage subset identified at any timepoint during fibrogenesis and recovery. Using CD11B-DTR mice, macrophages were depleted during the rapid resolution phase, resulting in a failure to remodel the hepatic scar. Critically, this depletion strategy selectively ablated the Ly-6C int subset, the degree of depletion correlating significantly with the amount of persistent fibrosis. A series of bone marrow transplantation, adoptive transfer and in situ labelling experiments identified that the pro-resolution Ly-6C int macrophage population derives from recruitment of Ly-6C hi monocytes, a common origin to the pro-fibrotic Ly-6C hi macrophages, indicative of a phenotypic switch in situ. Microarray profiling of FACS sorted Ly-6C int macrophages in comparison to pro-fibrotic Ly-6C hi macrophages, demonstrated a novel phenotype outside the M1/M2 macrophage paradigm, with down regulation of pro-inflammatory and pro-fibrotic genes, upregulation of matrix degrading enzymes and enrichment for phagocytosis related pathways. Confocal microscopy indicated that the Ly-6C int population contained more intracellular apoptotic debris, confirming the post-phagocytic nature of these cells. Feeding primary murine macrophages with hepatocyte debris in vitro induced a similar phenotypic switch to that seen in vivo. Furthermore, this phagocytosis-induced switch could be modelled by feeding macrophages with liposomes in vitro. Critically, systemic administration of liposomes to mice during maximal fibrosis resolution increased the number of hepatic Ly-6C int macrophages and accelerated the resolution of fibrosis. Conclusion In summary, we have identified the specific Ly-6C int macrophage subset which mediates the resolution of hepatic fibrosis. Extensive characterisation demonstrated this macrophage phenotype is produced by the phagocytosis of dead cells and thus can be manipulated in vivo by the induction of phagocytic behaviour with a beneficial effect on fibrosis resolution.