Ye Htun Oo

Analysis of CD161 expression on human CD8+ T cells defines a distinct functional subset with tissue-homing properties.

CD8+ T lymphocytes play a key role in host defense, in particular against important persistent viruses, although the critical functional properties of such cells in tissue are not fully defined. We have previously observed that CD8+ T cells specific for tissue-localized viruses such as hepatitis C virus express high levels of the C-type lectin CD161. To explore the significance of this, we examined CD8+CD161+ T cells in healthy donors and those with hepatitis C virus and defined a population of CD8+ T cells with distinct homing and functional properties. These cells express high levels of CD161 and a pattern of molecules consistent with type 17 differentiation, including cytokines (e.g., IL-17, IL-22), transcription factors (e.g., retinoic acid-related orphan receptor γ-t, P = 6 × 10−9; RUNX2, P = 0.004), cytokine receptors (e.g., IL-23R, P = 2 × 10−7; IL-18 receptor, P = 4 × 10−6), and chemokine receptors (e.g., CCR6, P = 3 × 10−8; CXCR6, P = 3 × 10−7; CCR2, P = 4 × 10−7). CD161+CD8+ T cells were markedly enriched in tissue samples and coexpressed IL-17 with high levels of IFN-γ and/or IL-22. The levels of polyfunctional cells in tissue was most marked in those with mild disease (P = 0.0006). These data define a T cell lineage that is present already in cord blood and represents as many as one in six circulating CD8+ T cells in normal humans and a substantial fraction of tissue-infiltrating CD8+ T cells in chronic inflammation. Such cells play a role in the pathogenesis of chronic hepatitis and arthritis and potentially in other infectious and inflammatory diseases of man.

Accumulation of natural killer T cells in progressive nonalcoholic fatty liver disease

Liver inflammation is greater in nonalcoholic steatohepatitis (NASH) than steatosis, suggesting that immune responses contribute to nonalcoholic fatty liver disease (NAFLD) progression. Livers normally contain many natural killer T (NKT) cells that produce factors that modulate inflammatory and fibrogenic responses. Such cells are relatively depleted in steatosis, but their status in more advanced NAFLD is uncertain. We hypothesized that NKT cells accumulate and promote fibrosis progression in NASH. We aimed to determine if livers become enriched with NKT cells during NASH‐related fibrosis; identify responsible mechanisms; and assess if NKT cells stimulate fibrogenesis. NKT cells were analyzed in wildtype mice and Patched‐deficient (Ptc+/−) mice with an overly active Hedgehog (Hh) pathway, before and after feeding methionine choline‐deficient (MCD) diets to induce NASH‐related fibrosis. Effects of NKT cell‐derived factors on hepatic stellate cells (HSC) were examined and fibrogenesis was evaluated in CD1d‐deficient mice that lack NKT cells. NKT cells were quantified in human cirrhotic and nondiseased livers. During NASH‐related fibrogenesis in wildtype mice, Hh pathway activation occurred, leading to induction of factors that promoted NKT cell recruitment, retention, and viability, plus liver enrichment with NKT cells. Ptc+/− mice accumulated more NKT cells and developed worse liver fibrosis; CD1d‐deficient mice that lack NKT cells were protected from fibrosis. NKT cell‐conditioned medium stimulated HSC to become myofibroblastic. Liver explants were 2‐fold enriched with NKT cells in patients with non‐NASH cirrhosis, and 4‐fold enriched in patients with NASH cirrhosis. Conclusion: Hh pathway activation leads to hepatic enrichment with NKT cells that contribute to fibrosis progression in NASH. (HEPATOLOGY 2010;)

Distinct Roles for CCR4 and CXCR3 in the Recruitment and Positioning of Regulatory T Cells in the Inflamed Human Liver

Regulatory T cells (Tregs) are found at sites of chronic inflammation where they mediate bystander and Ag-specific suppression of local immune responses. However, little is known about the molecular control of Treg recruitment into inflamed human tissues. We report that up to 18% of T cells in areas of inflammation in human liver disease are forkhead family transcriptional regulator box P3 (FoxP3)+ Tregs. We isolated CD4+CD25+CD127lowFoxP3+ Tregs from chronically inflamed human liver removed at transplantation; compared with blood-derived Tregs, liver-derived Tregs express high levels of the chemokine receptors CXCR3 and CCR4. In flow-based adhesion assays using human hepatic sinusoidal endothelium, Tregs used CXCR3 and α4β1 to bind and transmigrate, whereas CCR4 played no role. The CCR4 ligands CCL17 and CCL22 were absent from healthy liver, but they were detected in chronically inflamed liver where their expression was restricted to dendritic cells (DCs) within inflammatory infiltrates. These DCs were closely associated with CD8 T cells and CCR4+ Tregs in the parenchyma and septal areas. Ex vivo, liver-derived Tregs migrated to CCR4 ligands secreted by intrahepatic DCs. We propose that CXCR3 mediates the recruitment of Tregs via hepatic sinusoidal endothelium and that CCR4 ligands secreted by DCs recruit Tregs to sites of inflammation in patients with chronic hepatitis. Thus, different chemokine receptors play distinct roles in the recruitment and positioning of Tregs at sites of hepatitis in chronic liver disease.

Osteopontin is induced by hedgehog pathway activation and promotes fibrosis progression in nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a leading cause of cirrhosis. Recently, we showed that NASH‐related cirrhosis is associated with Hedgehog (Hh) pathway activation. The gene encoding osteopontin (OPN), a profibrogenic extracellular matrix protein and cytokine, is a direct transcriptional target of the Hh pathway. Thus, we hypothesize that Hh signaling induces OPN to promote liver fibrosis in NASH. Hepatic OPN expression and liver fibrosis were analyzed in wild‐type (WT) mice, Patched‐deficient (Ptc+/−) (overly active Hh signaling) mice, and OPN‐deficient mice before and after feeding methionine and choline–deficient (MCD) diets to induce NASH‐related fibrosis. Hepatic OPN was also quantified in human NASH and nondiseased livers. Hh signaling was manipulated in cultured liver cells to assess direct effects on OPN expression, and hepatic stellate cells (HSCs) were cultured in medium with different OPN activities to determine effects on HSC phenotype. When fed MCD diets, Ptc+/− mice expressed more OPN and developed worse liver fibrosis (P < 0.05) than WT mice, whereas OPN‐deficient mice exhibited reduced fibrosis (P < 0.05). In NASH patients, OPN was significantly up‐regulated and correlated with Hh pathway activity and fibrosis stage. During NASH, ductular cells strongly expressed OPN. In cultured HSCs, SAG (an Hh agonist) up‐regulated, whereas cyclopamine (an Hh antagonist) repressed OPN expression (P < 0.005). Cholangiocyte‐derived OPN and recombinant OPN promoted fibrogenic responses in HSCs (P < 0.05); neutralizing OPN with RNA aptamers attenuated this (P < 0.05). Conclusion: OPN is Hh‐regulated and directly promotes profibrogenic responses. OPN induction correlates with Hh pathway activity and fibrosis stage. Therefore, OPN inhibition may be beneficial in NASH (HEPATOLOGY 2011)

Mycophenolate mofetil as second line therapy in autoimmune hepatitis

INTRODUCTION:In patients with autoimmune hepatitis, efficient immunosuppressive therapy is essential to avoid progression to cirrhosis. There is no established second line therapy for patients failing standard therapy with steroids and azathioprine. The aim of this study was to examine the possible role of mycophenolate mofetil (MMF) as second line treatment of autoimmune hepatitis (AIH).PATIENTS AND ETHODS:We were able to identify 37 patients (29 women, 8 men) with AIH proven according to International AIH Group criteria who failed standard therapy. One patient on MMF was excluded due to non-compliance. A total of 28 of 36 patients had experienced side effects necessitating stop of treatment. One patient stopped azathioprine due to pregnancy. A total of nine patients did not respond sufficiently to azathioprine. A total of four patients with a treatment duration of 3 months or less because of severe side effects were considered as intolerant to MMF. Remission was defined as aspartate transaminase (ASP) < twice upper normal limit (UNL).RESULTS:Of 36 patients on MMF included in the analysis, 14 patients (39%) experienced remission. A total of 22 patients (61%) did not respond sufficiently to MMF. The response rate to MMF was dependent on the cause of treatment cessation of azathioprine. Of eight patients with prior nonresponse to azathioprine, six (75%) did not respond to MMF and only two (25%) reached biochemical remission. Of 28 patients with azathioprine intolerance in 16 (57%) patients, the response to MMF was insufficient and in 12 patients (43%) remission was reached. The difference did not reach statistical significance due to the relatively small numbers included.CONCLUSION:In the light of its good tolerability, MMF seems to be an alternative for patients who could not tolerate azathioprine previously. However, our data suggest that a majority of patients fail MMF particularly if they are switched because of an insufficient response to azathioprine.

The role of chemokines in the recruitment of lymphocytes to the liver

Chemokines direct leukocyte trafficking and positioning within tissues. They thus play critical roles in regulating immune responses and inflammation. The chemokine system is complex involving interactions between multiple chemokines and their receptors that operate in combinatorial cascades with adhesion molecules. The involvement of multiple chemokines and chemokine receptors in these processes brings flexibility and specificity to recruitment. The hepatic vascular bed is a unique low flow environment through which leukocyte are recruited to the liver during homeostatic immune surveillance and in response to infection or injury. The rate of leukocyte recruitment and the nature of cells recruited through the sinusoids in response to inflammatory signals will shape the severity of disease. At one end of the spectrum fulminant liver failure results from a rapid recruitment of leukocytes that leads to hepatocyte destruction and liver failure at the other diseases such as chronic hepatitis C infection may progress over many years from hepatitis to fibrosis and cirrhosis. Chronic hepatitis is charactezised by a T lymphocyte rich infiltrate and the nature and outcome of hepatitis will depend on the T cell subsets recruited, their activation and function within the liver. Different subsets of effector T cells have been described based on their secretion of cytokines and specific functions. These include Th1 and Th2 cells and more recently Th17 and Th9 cells which are associated with different types of immune response and which express distinct patterns of chemokine receptors that promote their recruitment under particular conditions. The effector function of these cells is balanced by the recruitment of regulatory T cells that are able to suppress antigen-specific effectors to allow resolution of immune responses and restoration of immune homeostasis. Understanding the signals that are responsible for recruiting different lymphocyte subsets to the liver will elucidate disease pathogenesis and open up new therapeutic approaches to modulate recruitment in favour of resolution rather than injury.

Monocyte subsets in human liver disease show distinct phenotypic and functional characteristics

Liver fibrosis is a wound healing response to chronic liver injury and inflammation in which macrophages and infiltrating monocytes participate in both the development and resolution phase. In humans, three monocyte subsets have been identified: the classical CD14++CD16−, intermediate CD14++CD16+, and nonclassical CD14+CD16++ monocytes. We studied the phenotype and function of these monocyte subsets in peripheral blood and liver tissue from patients with chronic inflammatory and fibrotic liver diseases. The frequency of intrahepatic monocytes increased in disease compared with control liver tissue, and in both nondiseased and diseased livers there was a higher frequency of CD14++CD16+ cells with blood. Our data suggest two nonexclusive mechanisms of CD14++CD16+ accumulation in the inflamed liver: (1) recruitment from blood, because more than twice as many CD14++CD16+ monocytes underwent transendothelial migration through hepatic endothelial cells compared with CD14++CD16− cells; and (2) local differentiation from CD14++CD16− classical monocytes in response to transforming growth factor β and interleukin (IL)‐10. Intrahepatic CD14++CD16+ cells expressed both macrophage and dendritic cell markers but showed high levels of phagocytic activity, antigen presentation, and T cell proliferation and secreted proinflammatory (tumor necrosis factor α, IL‐6, IL‐8, IL‐1β) and profibrogenic cytokines (IL‐13), chemokines (CCL1, CCL2, CCL3, CCL5), and growth factors (granulocyte colony‐stimulating factor and granulocyte‐macrophage colony‐stimulating factor), consistent with a role in the wound healing response. Conclusion: Intermediate CD14++CD16+ monocytes preferentially accumulate in chronically inflamed human liver as a consequence of enhanced recruitment from blood and local differentiation from classical CD14++CD16− monocytes. Their phagocytic potential and ability to secrete inflammatory and profibrogenic cytokines suggests they play an important role in hepatic fibrogenesis. (HEPATOLOGY 2013)

Innate immune cells in liver inflammation.

Innate immune system is the first line of defence against invading pathogens that is critical for the overall survival of the host. Human liver is characterised by a dual blood supply, with 80% of blood entering through the portal vein carrying nutrients and bacterial endotoxin from the gastrointestinal tract. The liver is thus constantly exposed to antigenic loads. Therefore, pathogenic microorganism must be efficiently eliminated whilst harmless antigens derived from the gastrointestinal tract need to be tolerized in the liver. In order to achieve this, the liver innate immune system is equipped with multiple cellular components; monocytes, macrophages, granulocytes, natural killer cells, and dendritic cells which coordinate to exert tolerogenic environment at the same time detect, respond, and eliminate invading pathogens, infected or transformed self to mount immunity. This paper will discuss the innate immune cells that take part in human liver inflammation, and their roles in both resolution of inflammation and tissue repair.

CXCR3-dependent recruitment and CCR6-mediated positioning of Th-17 cells in the inflamed liver

Background & Aims IL-17 secreting CD4 (Th17) and CD8 (Tc17) T cells have been implicated in immune-mediated liver diseases, but the molecular basis for their recruitment and positioning within the liver is unknown. Methods The phenotype and migratory behaviour of human liver-derived Th17 and Tc17 cells were investigated by flow cytometry and chemotaxis and flow-based adhesion assays. The recruitment of murine Th17 cells to the liver was studied in vivo using intra-vital microscopy. Results IL-17+ T cells comprised 1–3% of the T cell infiltrate in inflammatory liver diseases and included both CD4 (Th17) and CD8 (Tc17) cells. They expressed RORC and the IL-23 receptor and included subsets that secreted IL-22 and interferon-γ. Th17 and Tc17 cells expressed high levels of CXCR3 and CCR6, Tc17 cells also expressed CXCR6. Binding to human sinusoidal endothelium from flow was dependent on β1 and β2 integrins, CXCR3, and, in the case of Th17 cells, VAP-1. Th17 recruitment via sinusoids in mice with liver inflammation was reduced by treatment with antibodies against CXCR3 ligands, confirming the role of CXCR3 in Th17 recruitment in vivo. In human liver, IL-17+ cells were detected in portal infiltrates close to inflamed bile ducts expressing the CCR6 ligand CCL20. Cytokine-treated human cholangiocytes secreted CCL20 and induced CCR6-dependent migration of Th17 cells suggesting that local cholangiocyte chemokine secretion localises Th17 cells to bile ducts. Conclusions CXCR3 promotes recruitment of Th17 cells from the blood into the liver in both human and murine liver injury. Their subsequent positioning near bile ducts is dependent on cholangiocyte-secreted CCL20.

MAIT cells are activated during human viral infections.

Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation—driving cytokine release and Granzyme B upregulation—is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology.