Danielle T. Reid

Combined effects of oligofructose and Bifidobacterium animalis on gut microbiota and glycemia in obese rats

Prebiotics and probiotics may be able to modify an obesity‐associated gut microbiota. The aim of this study was to examine the individual and combined effects of the prebiotic oligofructose (OFS) and the probiotic Bifidobacterium animalis subsp. lactis BB‐12 (BB‐12) on gut microbiota and host metabolism in obese rats.

Osteopontin neutralisation abrogates the liver progenitor cell response and fibrogenesis in mice

Background Chronic liver injury triggers a progenitor cell repair response, and liver fibrosis occurs when repair becomes deregulated. Previously, we reported that reactivation of the hedgehog pathway promotes fibrogenic liver repair. Osteopontin (OPN) is a hedgehog-target, and a cytokine that is highly upregulated in fibrotic tissues, and regulates stem-cell fate. Thus, we hypothesised that OPN may modulate liver progenitor cell response, and thereby, modulate fibrotic outcomes. We further evaluated the impact of OPN-neutralisation on murine liver fibrosis. Methods Liver progenitors (603B and bipotential mouse oval liver) were treated with OPN-neutralising aptamers in the presence or absence of transforming growth factor (TGF)-β, to determine if (and how) OPN modulates liver progenitor function. Effects of OPN-neutralisation (using OPN-aptamers or OPN-neutralising antibodies) on liver progenitor cell response and fibrogenesis were assessed in three models of liver fibrosis (carbon tetrachloride, methionine-choline deficient diet, 3,5,-diethoxycarbonyl-1,4-dihydrocollidine diet) by quantitative real time (qRT) PCR, Sirius-Red staining, hydroxyproline assay, and semiquantitative double-immunohistochemistry. Finally, OPN expression and liver progenitor response were corroborated in liver tissues obtained from patients with chronic liver disease. Results OPN is overexpressed by liver progenitors in humans and mice. In cultured progenitors, OPN enhances viability and wound healing by modulating TGF-β signalling. In vivo, OPN-neutralisation attenuates the liver progenitor cell response, reverses epithelial-mesenchymal-transition in Sox9+ cells, and abrogates liver fibrogenesis. Conclusions OPN upregulation during liver injury is a conserved repair response, and influences liver progenitor cell function. OPN-neutralisation abrogates the liver progenitor cell response and fibrogenesis in mouse models of liver fibrosis.

Role of IL-17A and neutrophils in fibrosis in experimental hypersensitivity pneumonitis

BACKGROUND Chronic hypersensitivity pneumonitis is characterized by pulmonary inflammation and fibrosis in response to repeated inhalation of mainly organic antigens. It is recognized that IL-17A is crucial for the development of pulmonary inflammation in murine models of experimental hypersensitivity pneumonitis, but its role in the development of pulmonary fibrosis has not been determined. Furthermore, the main cell type(s) that produce IL-17A in experimental hypersensitivity pneumonitis have not yet been identified. OBJECTIVE Our objectives were to test the hypothesis that IL-17A plays a central role in the development of pulmonary fibrosis in experimental hypersensitivity pneumonitis and to determine the main inflammatory cell type(s) responsible for IL-17A production. METHODS We used a mouse model of experimental hypersensitivity pneumonitis in which IL-17A was inhibited or neutrophils were depleted. We also used IL-17RA-deficient and RAG-2-deficient mice. Lung IL-17A-producing cells were identified by fluorescence-activated cell sorting of myeloid versus lymphoid cell populations, intracellular IL-17A staining, flow cytometry, and quantitative reverse transcription PCR for IL-17A mRNA. RESULTS We found that the development of pulmonary fibrosis depended on IL-17A and was significantly attenuated by neutrophil depletion. Neutrophils and monocytes/macrophages were the main cell types that expressed IL-17A in our model. CONCLUSIONS We have identified the central roles of IL-17A and neutrophils in the pathogenesis of fibrosis in experimental hypersensitivity pneumonitis. We have also established that nonlymphocytic innate immune cells, specifically neutrophils and monocytes/macrophages, rather than TH17 lymphocytes, are the predominant source of IL-17A in experimental hypersensitivity pneumonitis.

Kupffer Cells Undergo Fundamental Changes during the Development of Experimental NASH and Are Critical in Initiating Liver Damage and Inflammation.

Non-alcoholic fatty liver disease has become the leading liver disease in North America and is associated with the progressive inflammatory liver disease non-alcoholic steatohepatitis (NASH). Considerable effort has been made to understand the role of resident and recruited macrophage populations in NASH however numerous questions remain. Our goal was to characterize the dynamic changes in liver macrophages during the initiation of NASH in a murine model. Using the methionine-choline deficient diet we found that liver-resident macrophages, Kupffer cells were lost early in disease onset followed by a robust infiltration of Ly-6C+ monocyte-derived macrophages that retained a dynamic phenotype. Genetic profiling revealed distinct patterns of inflammatory gene expression between macrophage subsets. Only early depletion of liver macrophages using liposomal clodronate prevented the development of NASH in mice suggesting that Kupffer cells are critical for the orchestration of inflammation during experimental NASH. Increased understanding of these dynamics may allow us to target potentially harmful populations whilst promoting anti-inflammatory or restorative populations to ultimately guide the development of effective treatment strategies.

Vascular cell adhesion molecule 1 expression by biliary epithelium promotes persistence of inflammation by inhibiting effector T-cell apoptosis

Chronic hepatitis occurs when effector lymphocytes are recruited to the liver from blood and retained in tissue to interact with target cells, such as hepatocytes or bile ducts (BDs). Vascular cell adhesion molecule 1 (VCAM‐1; CD106), a member of the immunoglobulin superfamily, supports leukocyte adhesion by binding α4β1 integrins and is critical for the recruitment of monocytes and lymphocytes during inflammation. We detected VCAM‐1 on cholangiocytes in chronic liver disease (CLD) and hypothesized that biliary expression of VCAM‐1 contributes to the persistence of liver inflammation. Hence, in this study, we examined whether cholangiocyte expression of VCAM‐1 promotes the survival of intrahepatic α4β1 expressing effector T cells. We examined interactions between primary human cholangiocytes and isolated intrahepatic T cells ex vivo and in vivo using the Ova‐bil antigen‐driven murine model of biliary inflammation. VCAM‐1 was detected on BDs in CLDs (primary biliary cirrhosis, primary sclerosing cholangitis, alcoholic liver disease, and chronic hepatitis C), and human cholangiocytes expressed VCAM‐1 in response to tumor necrosis factor alpha alone or in combination with CD40L or interleukin‐17. Liver‐derived T cells adhered to cholangiocytes in vitro by α4β1, which resulted in signaling through nuclear factor kappa B p65, protein kinase B1, and p38 mitogen‐activated protein kinase phosphorylation. This led to increased mitochondrial B‐cell lymphoma 2 accumulation and decreased activation of caspase 3, causing increased cell survival. We confirmed our findings in a murine model of hepatobiliary inflammation where inhibition of VCAM‐1 decreased liver inflammation by reducing lymphocyte recruitment and increasing CD8 and T helper 17 CD4 T‐cell survival. Conclusions: VCAM‐1 expression by cholangiocytes contributes to persistent inflammation by conferring a survival signal to α4β1 expressing proinflammatory T lymphocytes in CLD. (Hepatology 2014;59:1932–1943)

Unique microbial-derived volatile organic compounds in portal venous circulation in murine non-alcoholic fatty liver disease

BACKGROUND AND AIMS Non-alcoholic fatty liver disease is now the leading liver disease in North America. The progression of non-alcoholic fatty liver disease to the inflammatory condition, non-alcoholic steatohepatitis is complex and currently not well understood. Intestinal microbial dysbiosis has been implicated in the development of non-alcoholic fatty liver disease and progression of non-alcoholic steatohepatitis. Volatile organic compounds are byproducts of microbial metabolism in the gut that may enter portal circulation and have hepatotoxic effects contributing to the pathogenesis of non-alcoholic steatohepatitis. To test this hypothesis, we measured volatile organic compounds in cecal luminal contents and portal venous blood in a mouse model of non-alcoholic steatohepatitis. METHODS Gas chromatography-mass spectrometry analysis was conducted on cecal content and portal vein blood for volatile organic compound detection from mice fed a methionine and choline deficient diet, which induces non-alcoholic steatohepatitis. The colonic microbiome was studied by 16S rRNA gene amplification using the Illumina MiSeq platform. RESULTS Sixty-eight volatile organic compounds were detected in cecal luminal content, a subset of which was also present in portal venous blood. Importantly, differences in portal venous volatile organic compounds were associated with diet-induced steatohepatitis establishing a biochemical link between gut microbiota-derived volatile organic compounds and increased susceptibility to non-alcoholic steatohepatitis. CONCLUSION Our model creates a novel tool to further study the role of gut-derived volatile organic compounds in the pathogenesis of non-alcoholic steatohepatitis.

Osteopontin is a proximal effector of leptin-mediated non-alcoholic steatohepatitis (NASH) fibrosis

INTRODUCTION Liver fibrosis develops when hepatic stellate cells (HSC) are activated into collagen-producing myofibroblasts. In non-alcoholic steatohepatitis (NASH), the adipokine leptin is upregulated, and promotes liver fibrosis by directly activating HSC via the hedgehog pathway. We reported that hedgehog-regulated osteopontin (OPN) plays a key role in promoting liver fibrosis. Herein, we evaluated if OPN mediates leptin-profibrogenic effects in NASH. METHODS Leptin-deficient (ob/ob) and wild-type (WT) mice were fed control or methionine-choline deficient (MCD) diet. Liver tissues were assessed by Sirius-red, OPN and αSMA IHC, and qRT-PCR for fibrogenic genes. In vitro, HSC with stable OPN (or control) knockdown were treated with recombinant (r)leptin and OPN-neutralizing or sham-aptamers. HSC response to OPN loss was assessed by wound healing assay. OPN-aptamers were also added to precision-cut liver slices (PCLS), and administered to MCD-fed WT (leptin-intact) mice to determine if OPN neutralization abrogated fibrogenesis. RESULTS MCD-fed WT mice developed NASH-fibrosis, upregulated OPN, and accumulated αSMA+ cells. Conversely, MCD-fed ob/ob mice developed less fibrosis and accumulated fewer αSMA+ and OPN+ cells. In vitro, leptin-treated HSC upregulated OPN, αSMA, collagen 1α1 and TGFβ mRNA by nearly 3-fold, but this effect was blunted by OPN loss. Inhibition of PI3K and transduction of dominant negative-Akt abrogated leptin-mediated OPN induction, while constitutive active-Akt upregulated OPN. Finally, OPN neutralization reduced leptin-mediated fibrogenesis in both PCLS and MCD-fed mice. CONCLUSION OPN overexpression in NASH enhances leptin-mediated fibrogenesis via PI3K/Akt. OPN neutralization significantly reduces NASH fibrosis, reinforcing the potential utility of targeting OPN in the treatment of patients with advanced NASH.

OC-054 Hepatic Expression of CCl25 Mediates Recruitment of Plasmacytoid Dendritic Cells to Limit Liver Injury

Introduction Primary Sclerosing Cholangitis (PSC) is an incurable chronic immune mediated biliary disease that occurs in individuals with IBD. We have previously reported aberrant expression of the gut chemokine, CCL25 in the human PSC liver and the recruitment of CCR9 expressing gut derived T cells. In order to gain further insights into the consequences of aberrant CCL25 expression in the liver in PSC, we induced CCL25 in the murine liver and assessed biliary inflammation in-vivo. Methods To clarify the functional role of CCL25 expression in the liver, we generated a murine liver specific knock-in of CCL25 expression and tested the effects on immune mediated cholangitis using the Ova-Bil model of antigen driven biliary injury. Immune cell phenotyping and isolation were performed using flow cytometry. Liver injury was assessed by ALT measurements and histopathology. pDC function was assessed ex-vivo in co-culture with naive transgenic TCR T cells Results Ova-Bil x CCL25KI mice developed significantly less liver injury than wt Ova-Bil controls. Flow cytometry revealed increased numbers of CCR9+ pDCA-1+ plasmacytoid dendritic cells (pDC) in the Ova-Bil x CCL25KI livers. CCR9-/- x Ova-Bil mice developed significantly worse liver injury compared to wt Ova-Bil controls and severely lacked pDCs in the liver. Adoptive transfer of wt pDCs to CCR9-/- x Ova-Bil mice rescued the phenotype and reduced the degree of liver injury comparable to wt Ova-Bil controls. In vitro studies demonstrated the ability of liver-derived pDCs to induce regulatory T cells in a retinoic acid dependent manner as a possible mechanism by which CCR9+ pDCs are able to control liver injury. Conclusion Aberrant expression of CCL25 in the liver enhances recruitment of CCR9+ pDCs and appears to be an attempt to limit the extent of hepatic inflammation in PSC. Regulatory effects of CCR9+ pDCs appears to be at least in part mediated through the expansion of hepatic regulatory T cells. Disclosure of Interest None Declared Reference Eksteen B, Grant AJ, Miles A, et al. Hepatic endothelial CCL25 mediates the recruitment of CCR9+ gut-homing lymphocytes to the liver in primary sclerosing cholangitis. J.Exp.Med. 2004; 200:1511–1517.

OC-008 Kupffer Cells Play a Dual Pro-Inflammatory and Protective Role in Non-Alcoholic Steatohepatitis

Introduction Non-alcoholic fatty liver disease (NAFLD) is the leading liver disease in Europe and North America. 30% of patients with NAFLD are estimated to develop inflammatory non-alcoholic steatohepatitis (NASH) with the potential to lead to cirrhosis and hepatocellular carcinoma. Current evidence suggests that gut bacterial products can drive hepatic inflammation by activating specific innate pattern recognition receptors (PRRs) such as TLR4 and the NALP3 inflammasome. Both receptors are expressed by liver resident macrophages, Kupffer cells (KCs). Methods To determine the role of KCs and interactions with PRRs in NASH, mice were fed a methionine choline deficient (MCD) diet for three weeks to induce NASH. Liposomal clodronate was used to deplete KCs. Serum ALT levels were measured and hepatic inflammatory infiltrates characterised by flow cytometry. Real-time qPCR was used to assess changes in gene expression. Murine findings were correlated with human liver tissue from NASH patients. Groups were compared by one-way ANOVA and significance set at P < 0.05. Results NALP3 KO (knock-out), TLR4 KO or KC deficient WT mice on a MCD diet developed reduced liver damage and decreased T lymphocyte recruitment compared to WT MCD controls. Combined KC depletion and NLRP3 KO however lead to significantly worse liver injury and progressive fibrosis as measured by collagen expression. Further investigation revealed an as yet unrecognised role for KC expressed NALP6 activation which mediated anti-inflammatory responses and modulation of hepatic IL-22 responsiveness to reduce liver injury. Conclusion KC activation through NALP3 and TLR4 increases hepatic inflammation in the MCD model of NASH. KCs have a dual role in NASH as they also express NALP6 with anti-inflammatory properties and are able to reduce hepatic injury through modulation of IL-22 responsiveness modulation. Disclosure of Interest None Declared