Anti-Cholestatic Therapy with Obeticholic Acid Improves Short-Term Memory in Bile Duct-Ligated Mice.

Patients with cholestatic liver disease, including those with primary biliary cholangitis, can experience symptoms of impaired cognition or brain fog. This phenomenon remains unexplained and is currently untreatable. Bile duct ligation (BDL) is an established rodent model of cholestasis. In addition to liver changes, BDL animals develop cognitive symptoms early in the disease process (before development of cirrhosis and/or liver failure). The cellular mechanisms underpinning these cognitive symptoms are poorly understood. Herein, the study explored the neurocognitive symptom manifestations, and tested potential therapies, in BDL mice, and used human neuronal cell cultures to explore translatability to humans. BDL animals exhibited short-term memory loss and showed reduced astrocyte coverage of the blood-brain barrier, destabilized hippocampal network activity, and neuronal senescence. Ursodeoxycholic acid (first-line therapy for most human cholestatic diseases) did not reverse symptomatic or mechanistic aspects. In contrast, obeticholic acid (OCA), a farnesoid X receptor agonist and second-line anti-cholestatic agent, normalized memory function, suppressed blood-brain barrier changes, prevented hippocampal network deficits, and reversed neuronal senescence. Co-culture of human neuronal cells with either BDL or human cholestatic patient serum induced cellular senescence and increased mitochondrial respiration, changes that were limited again by OCA. These findings provide new insights into the mechanism of cognitive symptoms in BDL animals, suggesting that OCA therapy or farnesoid X receptor agonism could be used to limit cholestasis-induced neuronal senescence.

TGF-ß2 silencing to target biliary-derived liver diseases.

OBJECTIVE: TGF-ß2 (TGF-ß, transforming growth factor beta), the less-investigated sibling of TGF-ß1, is deregulated in rodent and human liver diseases. Former data from bile duct ligated and MDR2 knockout (KO) mouse models for human cholestatic liver disease suggested an involvement of TGF-ß2 in biliary-derived liver diseases. DESIGN: As we also found upregulated TGFB2 in liver tissue of patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), we now fathomed the positive prospects of targeting TGF-ß2 in early stage biliary liver disease using the MDR2-KO mice. Specifically, the influence of TgfB2 silencing on the fibrotic and inflammatory niche was analysed on molecular, cellular and tissue levels. RESULTS: TgfB2-induced expression of fibrotic genes in cholangiocytes and hepatic stellate cellswas detected. TgfB2 expression in MDR2-KO mice was blunted using TgfB2-directed antisense oligonucleotides (AON). Upon AON treatment, reduced collagen deposition, hydroxyproline content and αSMA expression as well as induced PparG expression reflected a significant reduction of fibrogenesis without adverse effects on healthy livers. Expression analyses of fibrotic and inflammatory genes revealed AON-specific regulatory effects on Ccl3, Ccl4, Ccl5, Mki67 and Notch3 expression. Further, AON treatment of MDR2-KO mice increased tissue infiltration by F4/80-positive cells including eosinophils, whereas the number of CD45-positive inflammatory cells decreased. In line, TGFB2 and CD45 expression correlated positively in PSC/PBC patients and localised in similar areas of the diseased liver tissue. CONCLUSIONS: Taken together, our data suggest a new mechanistic explanation for amelioration of fibrogenesis by TGF-ß2 silencing and provide a direct rationale for TGF-ß2-directed drug development.

Role of Histopathology in Autoimmune Hepatitis.

The diagnosis of autoimmune hepatitis (AIH) is based on a combination of biochemical, immunological and histological features and exclusion of other causes of liver disease. Typical histological features include a chronic hepatitis pattern of injury with portal inflammation and interface activity, predominance of plasma cells in the portal infiltrate, emperipolesis, and hepatocellular rosette formation. Centrilobular injury with prominent hepatocellular necrosis and mononuclear inflammation is now recognised in the histological spectrum of AIH and may represent an early stage of the disease. Liver histology plays a major role in clinical diagnostic scoring systems and is important to confirm or support the clinical diagnosis of AIH. This review focuses on the role of histopathology in AIH and highlights the contribution of histological interpretation to the diagnosis of AIH, differential diagnosis from other entities, recognition of concurrent liver disease, and identification of the so-called overlap or variant syndromes, and addresses the importance of liver biopsy in the management and prognosis of patients with AIH.

Breast cancer metastasis: demonstration that FOXP3 regulates CXCR4 expression and the response to CXCL12.

The X-linked transcription factor FOXP3 is expressed by epithelial cells of organs including the breast, where it is considered a tumour suppressor. The chemokine receptor CXCR4 also regulates the development of breast cancer by stimulating cell migration towards CXCL12-expressing sites of metastatic spread. During activation, human T cells show reciprocal regulation of FOXP3 and CXCR4. This study was designed to examine the role FOXP3 plays in metastatic breast cancer, with a particular focus on its potential to regulate CXCR4. Human breast cancer samples showed significantly decreased FOXP3 protein expression but an increased number of CXCR4 transcripts. In comparison with normal primary breast epithelial cells, FOXP3 was down-regulated at both transcript and protein levels in the breast cancer cell lines MCF-7 and MDA-MB-231. In the invasive MDA-MB-231 cells, the remaining FOXP3 was located predominately within the cytoplasm. Following stable FOXP3 overexpression in MDA-MB-231 cells, significant decreases were observed in the expression of ErbB2/HER2, SKP2, c-MYC, and CXCR4. In contrast, an increase in p21 expression led to inhibition of cell proliferation, with a greater proportion in the G1 phase of the cell cycle suggesting the induction of senescence. Specific knockdown of FOXP3 in normal human breast epithelial cells with siRNA significantly increased ErbB2/HER2, SKP2, c-MYC, and CXCR4, and decreased p21 expression. These cells also showed a significantly increased chemotactic response towards CXCL12, consistent with a role for FOXP3 in the regulation of cell migration. Results from this study are consistent with FOXP3 functioning as an important tumour suppressor in breast cancer. Indeed, the potential functions of FOXP3 in breast epithelium can now be extended to include regulation of CXCR4 expression and response to the pro-metastatic chemokine CXCL12.

T cell-mediated biliary epithelial-to-mesenchymal transition in liver allograft rejection.

Loss of bile duct epithelium is characteristic of early chronic rejection following liver transplantation. Recent studies have suggested that intrahepatic biliary epithelial cells can transform into myofibroblasts. This study examines the induction and molecular regulation of this transition during allograft rejection. Immortalized human cholangiocytes were stimulated with either transforming growth factor beta1 (TGFbeta1) or a T cell line, and they were examined for morphological, proteomic, and functional features. Posttransplant liver biopsy sections were also examined. Treatment of cholangiocytes with TGFbeta1 or TGFbeta-presenting T cells induced a bipolar morphology, reduced expression of E-cadherin and zona occludens 1 (ZO-1), and increased vimentin, fibronectin, matrix metalloproteinase 2 (MMP-2), MMP-9, and S100 calcium binding protein A4 (S100A4); treated cells invaded a model basement membrane. Chemokines induced T cell penetration of 3-dimensional, cultured bile duct-like structures and bile ducts in liver biopsy sections. A spatial association was observed between duct-infiltrating T cells and cholangiocyte expression of mesenchymal markers, including S100A4. Inhibition of S100A4 expression in vitro blocked TGFbeta1-mediated loss of E-cadherin and ZO-1 but did not reduce induction of fibronectin, MMP-2, or MMP-9. This study demonstrates the potential for T cells to induce an intrahepatic biliary epithelial-to-mesenchymal cell transition during chronic rejection. Furthermore, S100A4 expression by cholangiocytes was identified as a crucial regulator of this transition.

Bile acid receptor agonists in primary biliary cholangitis: Regulation of the cholangiocyte secretome and downstream T cell differentiation.

Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease. Approximately 30% of patients do not respond to therapy with ursodeoxycholic acid (UDCA). Previous studies have implicated increased senescence of cholangiocytes in patients who do not respond to UDCA. This may increase the release of cytokines which drive pathogenic T cell polarization. As FXR agonists are beneficial in treating UDCA non-responsive patients, the current study was designed to model the interactions between cholangiocytes and CD4+ T cells to investigate potential immunomodulatory mechanisms of bile acid receptor agonists. Human cholangiocytes were co-cultured with CD4+ T cells to model the biliary stress response. Senescent cholangiocytes were able to polarize T cells toward a Th17 phenotype and suppressed expression of FoxP3 (P = 0.0043). Whilst FXR and TGR5 receptor agonists were unable directly to alter cholangiocyte cytokine expression, FGF19 was capable of significantly reducing IL-6 release (P = 0.044). Bile acid receptor expression was assessed in PBC patients with well-characterized responsiveness to UDCA therapy. A reduction in FXR staining was observed in both cholangiocytes and hepatocytes in PBC patients without adequate response to UDCA. Increased IL-6 expression by senescent cholangiocytes represents a potential mechanism by which biliary damage in PBC could contribute to excessive inflammation.

Activation and modulation of cardiac poly-adenosine diphosphate ribose polymerase activity in a rat model of brain death.

DNA damage during transplantation can activate poly-adenosine diphosphate ribose polymerase (PARP) resulting in the generation of polymers of adenosine diphosphate-ribose (PAR). Excessive linkage of PAR to nuclear proteins can induce cell death, thereby limiting the function of transplanted organs. This study uses a rat model of brain death to determine the profile of PARP activation and whether mechanisms that lead to cell death can be ameliorated by appropriate donor resuscitation. The expression of PAR-linked nuclear proteins within cardiac myocytes was greatly increased after the induction of donor brain death. Importantly, infusion of noradrenaline or vasopressin to normalize the chronic hypotension produced by brain death reduced the expression of PAR to a level below baseline. These data suggest that chronic hypotension after donor brain death has the potential to limit cardiac function through the activation of PARP; however, this early cause of graft damage can be mitigated by appropriate donor resuscitation.

Chemokine-mediated inflammation: Identification of a possible regulatory role for CCR2.

The chemokine receptor CCR2 binds four pro-inflammatory monocyte chemoattractant proteins, designated MCP1/CCL2, MCP2/CCL8, MCP3/CCL7 and MCP4/CCL13. This study demonstrates the important biology of this receptor during the response to the chemokine milieu. Competitive chemotaxis and calcium flux assays were performed utilising mixtures of chemokines to assess a hierarchal arrangement of chemokine prepotency; these demonstrated that the MCP2-CCR2 interaction is able to supersede signals generated by RANTES, another pro-inflammatory chemokine, or the homeostatic chemokine SDF1. These observations were validated using three physiologically relevant monocytic cell lines. Having identified the importance of CCR2, experiments were then performed to examine the signal transduction processes coupled to this receptor. G protein coupling was initially examined; Cholera toxin reduced the chemotactic response to MCP2 (p<0.001), whilst the response to the other MCP chemokines remained normal. The response to MCP2 was uniquely inhibited by elevated concentrations of cAMP and, unlike MCP1, 3 and 4 (p<0.05), MCP2 failed to inhibit adenylate cyclase. Expression of dominant negative H-ras demonstrated that each MCP chemokine required active ras in order to elicit ERK activation and a chemotactic response. Unlike MCP1, MCP2 failed to induce nuclear translocation of activated ERK1 or subsequent induction of c-Myc expression. Akt activation also showed ligand-specific differences, with MCP2 producing a delayed response compared to the other MCP chemokines. Together these data highlight the importance of CCR2 and suggest that it is a powerful tool for fine tuning the immune response.

Development of a robust protocol for gene expression analysis using formalin-fixed, paraffin-embedded liver transplant biopsy specimens.

Liver transplant biopsies are routinely archived following formalin fixation and paraffin embedding and may provide an additional source of diagnostic information following transcriptomic biomarker analysis. This study was designed to compare gene transcription between resting and stressed biliary cells in culture, these cells after fixation and embedding and archival liver transplant biopsy tissue. The transcription of p21/WAF1 and transforming growth factor (TGF)-ß1 showed similar changes in the fresh and embedded liver cells. However, the expression of TGF-ß2 was markedly different between the fresh and embedded samples, suggesting that fixation can produce sequence-specific artefacts. Sufficient quantities of pure RNA were recovered from all the liver transplant biopsies to allow complementary DNA production. Measurement of the transcription of all three genes showed variability between the cases. Although the results for individual transcripts should be interpreted with care, these data do suggest the feasibility of performing a larger biomarker discovery studies using archival tissue.

Kidney transplantation: analysis of the expression and T cell-mediated activation of latent TGF-ß.

Activated T cells infiltrate a renal allograft during rejection and can respond to TGF-ß within the tubules, causing local differentiation and expression of the αE(CD103)ß7 integrin. This study was performed to examine the expression of latent TGF-ß within renal allograft tissues and to define a mechanism by which T cells can activate and respond to this latent factor. Rejecting renal allograft biopsy tissues showed increased expression of the latent TGF-ß complex, which was localized around the tubules by a mechanism that might involve interaction with heparan sulfate in the basement membrane. A cultured renal TEC line also expressed the latent complex, but these cells did not respond to this form of TGF-ß by pSmad 3. However, coculture of these cells with activated T cells induced the expression of CD103, suggesting that T cells can activate and respond to the latent TGF-ß associated with TEC. Although activated T cells expressed little cell-surface TSP-1, this was increased by culture with fibronectin or fibronectin-expressing renal TEC. Blockade of TSP-1 using LSKL peptides reduced the potential of activated T cells to differentiate in response to latent TGF-ß. This study suggests that penetration of renal tubules by activated T cells leads to increased expression of T cell-surface TSP-1, allowing activation of latent TGF-ß sequestered on heparan sulfate within the microenvironment. This mechanism may be important for localized phenotypic maturation of T cells that have infiltrated the kidney during allograft rejection.