Focus
Authors: Scott L. Friedman
Journal: Journal of Hepatology (2011)

This Focus article highlights three unrelated studies, the first exploring the role of gene polymorphism in risk of spontaneous bacterial peritonitis (SBP) ("Nature"), the inhibitory activity of a grapefruit flavonoid on hepatitis C virus (HCV) replication ("Natural"), and the role of multipotent stromal cells in correcting defects in a mouse fatty liver disease model ("NAFLD"). In the first study by Nischalke and colleagues, sequence variants in the gene encoding for the membrane receptor Toll-like receptor 2 were linked to an increased prevalence of SBP among cirrhotics who had ascites. TLR2 is a well characterized cell membrane pattern recognition receptor that is a vital component of the innate immune system, whose primary role is to recognize gram positive bacterial patterns and elicit protective signaling cascades [1Mele T. Madrenas J. TLR2 signalling: at the crossroads of commensalism, invasive infections and toxic shock syndrome by Staphylococcus aureus.Int J Biochem Cell Biol. 2010; 42: 1066-1071Crossref PubMed Scopus (39) Google Scholar, 2Testro A.G. Visvanathan K. Toll-like receptors and their role in gastrointestinal disease.J Gastroenterol Hepatol. 2009; 24: 943-954Crossref PubMed Scopus (94) Google Scholar]. Thus, the genetic variants that reduce the activity of TLR2 could amplify the risk of infection from gut bacteria among cirrhotics with ascites, who are already at heightened risk for infections. The two polymorphism conferring increased susceptibility, the −16934 TT and TLR2 GT microsatellite variants may reduce the activity of the receptor such that there is decreased release of inflammatory cytokines in these susceptible patients in response to gut bacteria. The findings are reminiscent of a recent study highlighting how variants of the TLR4 gene, which signals in response to gram negative bacterial patterns, affects the progression rate of fibrosis in patients with HCV [[3]Guo J. Loke J. Zheng F. Hong F. Yea S. Fukata M. et al.Functional linkage of cirrhosis-predictive single nucleotide polymorphisms of Toll-like receptor 4 to hepatic stellate cell responses.Hepatology. 2009; 49: 960-968Crossref PubMed Scopus (187) Google Scholar]. The TLR2 findings represent another example of the progressive assimilation of host genetic variants into our classification of disease risk that will subtly, but inevitably, change the way we practice medicine. And while the findings make sense based on our knowledge of TLR2 function, this study lacks a specific demonstration that the susceptibility variants negatively affect the function of cells directly responsible for preventing SBP. Specifically, in which cells are the TLR2 variants failing to respond adequately to gram positive bacteria – gut, peritoneal or hepatic macrophages, other resident cells of liver, or circulating immune cells? Are peritoneal macrophages from ascites in patients with the variants less responsive to bacterial antigens? Also, what is the mechanism by which these variants affect signaling in the responsible cells? Increasingly, experts demand that disease susceptibility variants be linked to a clear underlying mechanism, and while one is suggested by studies of TLR2 in other tissues [[4]Veltkamp M. Wijnen P.A. van Moorsel C.H. Rijkers G.T. Ruven H.J. Heron M. et al.Linkage between Toll-like receptor (TLR) 2 promotor and intron polymorphisms: functional effects and relevance to sarcoidosis.Clin Exp Immunol. 2007; 149: 453-462Crossref PubMed Scopus (74) Google Scholar], there remains a gap that future studies in SBP patients will hopefully fill. Still, if the findings were replicated in multiple cohorts, the identification of susceptibility variants in patients with ascites might prompt the testing of primary prophylactic antibiotics in high-risk patients in a prospective randomized trial. Moreover, since additional variants in the nucleotide binding oligomerisation domain (NOD2) gene conferred an independent, additive risk of SBP in this study, one could envision an aggregate score that could predict the risk of this infection, and define those who would benefit from prophylactic therapies. In the article by Goldwasser et al., the activity of a grapefruit flavonoid, naringenin, has been shown to interfere with HCV assembly in the widely used viral replication system of Huh 7.5.1 cells. 'Flavonoids' refer to a class of plant-derived polyphenolic yellow pigments containing ketones, which are commonly found in the human diet. Their remarkable breadth of biologic activity includes anti-cancer, anti-oxidant, and anti-bacterial properties, among many others. Flavonoids' appeal as a natural therapy for which disease is well known, but less appreciated is that approximately 25% of all FDA-approved drugs have originated from botanicals, and the global market for botanical and plant derived drugs is more than $20 billion and growing. In the case of naringenin, the compound has already been shown to modestly lower LDL cholesterol in human subjects [[5]Jung U.J. Kim H.J. Lee J.S. Lee M.K. Kim H.O. Park E.J. et al.Naringin supplementation lowers plasma lipids and enhances erythrocyte antioxidant enzyme activities in hypercholesterolemic subjects.Clin Nutr. 2003; 22: 561-568Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar] through a number of effects on lipid homeostasis, and has anti-cancer, anti-oxidant, and anti-diabetogenic activities in a range of animal models. The Goldwasser study in HCV was prompted in part by the recognition that viral replication is dependent on the lipid machinery of susceptible cells [[6]Syed G.H. Amako Y. Siddiqui A. Hepatitis C virus hijacks host lipid metabolism.Trends Endocrinol Metab. 2010; 21: 33-40Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar]. Their experiments carefully and systematically link an antiviral effect of naringenin to stimulation of PPARα, a key nuclear receptor that regulates lipid homeostasis, blocking VLDL production and significantly reducing ApoB secretion without affecting cell viability or leading to lipid accumulation. The result is a blockade to the assembly of infectious particles, but without an effect on viral replication per se. While the activity of this compound in reducing HCV RNA secretion was relatively modest (1.4 log) compared to a recently approved protease inhibitor, VX-950 (telaprevir) (4.7 log) [[7]Lin K. Perni R.B. Kwong A.D. Lin C. VX-950, a novel hepatitis C virus (HCV) NS3-4A protease inhibitor, exhibits potent antiviral activities in HCv replicon cells.Antimicrob Agents Chemother. 2006; 50: 1813-1822Crossref PubMed Scopus (191) Google Scholar], the safety of naringenin and its potential synergy with existing therapies make this compound especially appealing. Moreover, because the drug has already been safely tested in humans, the path toward its inclusion into multi-drug regimens for HCV seems straightforward, provided there is a willing commercial sponsor that can support its development. Whether naringenin can find such a suitor in the already crowded landscape of HCV agents that may partly determine its market viability. Regardless, the findings underscore the vast potential of natural compounds to emerge as tomorrow's therapies. The third study by Ezquer et al., adds additional evidence, albeit preliminary, of the potential power of stem cells to treat liver disease. A growing number of reports have used a range of bone-marrow derived and 'stem' cells as potential therapies for liver disease in both animal models and human trials (reviewed in [[8]Stutchfield B.M. Forbes S.J. Wigmore S.J. Prospects for stem cell transplantation in the treatment of hepatic disease.Liver Transpl. 2010; 16: 827-836Crossref PubMed Scopus (62) Google Scholar]). A wide number of tissue sources, cell characterization, mode of administration, and endpoints of efficacy have been utilized, which make direct comparisons difficult. However, some general principles help define whether a study is meaningful, including the requirements to: (1) meticulously characterize the phenotype and purity of administered cells; this is especially critical to avoid the risk of sowing a malignancy if undifferentiated stem cells are administered; (2) track the longevity and sites of administered cells; (3) clarify whether therapeutic effects are a direct result of the administrated cells or due to indirect effects on host effector cells; (4) define specific mechanism and mediators underlying a therapeutic effect; (5) establish the safety and durability of a response; and (6) define the types and stages of disease(s) that will benefit from potential therapies. In the context of these requirements, the Ezquer study has some strengths, but also some notable deficiencies. Perhaps most appealing is their use of cell therapy to correct histologic abnormalities associated with a high fat diet in mice, rather than to either rescue liver function or reverse fibrosis as in prior studies. The authors treated mice that had been on a high fat diet for 33 weeks with two doses, 4 weeks apart, of 5 × 105 'multipotent stromal cells' (MSCs) that had been harvested and expanded ex vivo (defined by flow cytometry using a panel of cell surface markers), followed by sacrifice of the mice at 50 weeks. MSCs were also labeled with green fluorescent protein to track their fate in situ. The results of MSC administration were striking but largely unexplained. MSC-treated mice had reduced transaminases, hepatomegaly, and fibrosis, and had attenuated steatosis compared to high fat diet-fed mice that were untreated. At the time of sacrifice 17 weeks after their administration, donor cells were only detectable in obese but not in control mice, distributed amongst liver, bone marrow, heart, and kidney, and visible as single cells, primarily in the portal areas. There was no definitive evidence of cell fusion with hepatocytes since the GFP positive cells did not express albumin. The findings are intriguing but not wholly satisfying, as no underlying mechanism was offered and it is unclear if the result is direct or indirect via recruitment of host cells. In contrast, a recent study carefully distinguished these two possibilities in a murine model of CCl4-induced injury and fibrosis, demonstrating that only differentiated macrophages had a salutary effect, whereas whole bone marrow exacerbated the disease [[9]Thomas J.A. Pope C. Wojtacha D. Robson A.J. Gordon-Walker T.T. Hartland S. et al.Macrophage therapy for murine liver fibrosis recruits host effector cells improving fibrosis, regeneration, and function.Hepatology. 2011; 53: 2003-2015Crossref PubMed Scopus (254) Google Scholar]. Additionally, in the Ezquer study, the preferential engraftment of cells in obese but not normal livers is also quite interesting but not explored. In summary, this study using a partially characterized stromal cell type raises the possibility that defects associated with fatty liver may be correctable, at least transiently, by cell based therapy. Continued refinement of the cell type(s) that confer benefit, use of a larger number of controls, application to other fatty liver disease models, and elucidation of specific mechanism(s) that account for these remarkable findings could bring real value to this approach, but much work remains to be done. The author declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.