Ka-Kit Li

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)

Optimising risk stratification in primary biliary cirrhosis: AST/platelet ratio index predicts outcome independent of ursodeoxycholic acid response

BACKGROUND & AIMS Outcomes in primary biliary cirrhosis (PBC) can be predicted by biochemical response to ursodeoxycholic acid (UDCA). Such stratification inadequately captures cirrhosis/portal hypertension, recognised factors associated with adverse events. METHODS We evaluated a cohort of PBC patients (n=386) attending the Liver Unit in Birmingham (derivation cohort), seeking to identify risk-variables associated with transplant-free survival independent of UDCA-response. A validation cohort was provided through well-characterised patients attending the Toronto Center for Liver Diseases (n=479) and Jena University Hospital (n=150). RESULTS On multivariate analysis, factors at diagnosis associated with liver transplant (LT)/death were patient age (HR:1.06; p<0.001), elevated bilirubin (HR:1.27; p<0.001), early-onset cirrhosis (HR:2.40; p<0.001) and baseline AST/platelet ratio index (APRI) (HR:1.95; p<0.001). At 1-year, UDCA biochemical non-response predicted poorer transplant-free survival, and additional factors (multivariate) associated with adverse outcome were age (HR:1.02; p<0.05) and 1-year APRI (HR:1.15; p<0.001). Obtaining a cut-point from our derivation cohort, APRI >0.54 at baseline was predictive of LT/death (adjusted HR: 2.40; p<0.001), and retained statistical significance when applied at 1-year (APRI-r1, adjusted HR:2.75; p<0.001) despite controlling for UDCA-response. Across both cohorts, transplant-free survival was poorer for biochemical-responders with an APRI-r1 >0.54 vs. biochemical-responders with a lower APRI-r1 (p<0.01 and p<0.001, respectively); non-responders with high APRI-r1 had the poorest outcomes (p<0.001 and p<0.001). CONCLUSION In PBC, elevated APRI is associated with future risk of adverse events, independently and additively of UDCA-response. This cross-centre, robustly validated observation will contribute to ongoing efforts to refine existing risk-stratification tools, as well as direct focus for new therapies in patients with PBC.

Bidirectional transendothelial migration of monocytes across hepatic sinusoidal endothelium shapes monocyte differentiation and regulates the balance between immunity and tolerance in liver

Monocytes are versatile cells that can fulfill proinflammatory and anti‐inflammatory functions when recruited to the liver. Recruited monocytes differentiate into tissue macrophages and dendritic cells, which sample antigens and migrate to lymph nodes to elicit T‐cell responses. The signals that determine monocyte differentiation and the role of hepatic sinusoidal endothelial cells (HSECs) in this process are poorly understood. HSECs are known to modulate T‐cell activation, which led us to investigate whether transendothelial migration of monocytes across HSECs influences their phenotype and function. Subsets of blood‐derived monocytes were allowed to transmigrate across human HSECs into a collagen matrix. Most migrated cells remained in the subendothelial matrix, but ∼10% underwent spontaneous basal to apical transendothelial migration. The maturation, cytokine secretion, and T‐cell stimulatory capacity of reverse transmigrating (RT) and subendothelial (SE) monocytes were compared. SE monocytes were mainly CD16–, whereas 75%‐80% of RT monocytes were CD16+. SE monocytes derived from the CD14++CD16− subset and exhibited high phagocytic activity, whereas RT monocytes originated from CD14++CD16+ and CD14+CD16++ monocytes, displayed an immature dendritic cell–like phenotype (CD11cposHLA‐DRposCD80loCD86lo), and expressed higher levels of chemokine (C‐C motif) receptor 8. Consistent with a dendritic cell phenotype, RT monocytes secreted inflammatory cytokines and induced antigen‐specific CD4+ T‐cell activation. In contrast, SE monocytes suppressed T‐cell proliferation and activation and exhibited endotoxin tolerance. Transcriptome analysis underscored the functional differences between SE and RT monocytes. Conclusions: Migration across HSECs shapes the subsequent fate of monocytes, giving rise to anergic macrophage‐like cells in tissue and the release of immunocompetent pre–dendritic cells into the circulation. (Hepatology 2016;63:233–246)

Antitumor CD8+T cells in hepatocellular carcinoma: Present but exhausted

H epatocellular carcinoma (HCC) is the second leading cause of global cancer deaths and the incidence is rising. Advances in medical treatment have resulted in improved survival for many common malignancies, but not HCC. The lack of curative treatments and consequent dismal survival rates make elucidating HCC biology and translating this into effective treatments a priority. There is good evidence that HCC stimulates an immune response, making immunotherapy in which the host’s immune system is stimulated to attack the cancer a potential option. However, despite efficacy in animal models, most clinical trials of tumor immunotherapy have been disappointing. The article from Robert Thimme’s group in this issue highlights mechanisms that inhibit effective antitumor immunity in HCC and suggests immunotherapeutic options against a formidable foe. The host immune system has the potential to recognize a wide range of tumor-associated antigens (TAA) including alpha-fetoprotein (AFP). Moreover, there is emerging evidence that the presence of TAA-specific T cells is associated with an improved response to dendritic cell (DC) vaccination and better prognosis. However, despite mounting a detectable immune response, tumor progression frequently occurs, and understanding why the immune response fails to control the tumor is a crucial step if immunotherapy is to become clinically effective. Many mechanisms have been implicated in cancer immune evasion. These include down-regulation of major histocompatility complex (MHC) molecules and TAA on tumor cells, inhibition of effector immune cells functions, the recruitment and induction of immunosuppressive immune cells, and the disruption of effective tumor antigen presentation as a consequence of reduced costimulatory molecule expression and interference of antigen processing by DC (Fig. 1). In this month’s issue of HEPATOLOGY, Flecken et al. report new findings on the antitumor response in HCC with important therapeutic implications. The authors describe a subset of CD8T cells specific for the TAAs AFP, glypican-3 (GPC-3), melanomaassociated gene-A1 (MAGE-A1), and New Yorkesophageal squamous cell carcinoma-1 (NY-ESO-1) in the blood, liver, and tumors of patients with HCC, and quantified the magnitude and breadth of the antitumor CD8T-cell response. Previous studies have reported an association between the strength of the antitumor T-cell response and patient survival and the present work illustrates that a broad immune response early in disease is associated with a better prognosis. This has implications for the development of therapeutic vaccines. Many vaccine trials have used a specific antigen or antigenic epitope to prime T-cell responses. However, the identification of multiple immunodominant epitopes across different tumor antigens suggests that vaccines should incorporate several antigens if they are to be effective. In the context of DC-based vaccination, this study supports the use of tumor lysate to pulse DCs based on the notion that multiple tumor lysate proteins favor the development of a broader antitumor immune response. The authors go on to demonstrate that TAA-specific CD8T cells expanded from the blood of patients with HCC have markedly impaired IFN-c secretion and cytolytic activity. Interestingly, the same patients had preserved effector CD8T cells against Epstein-Barr virus (EBV) and cytomegalovirus (CMV), suggesting that this is a specific tumor effect. This is important because it shows that the TAA-specific CD8T cells are present, they have not been deleted, and thus it is potentially possible to restore their effector function. However, to do this it is important to understand the underlying mechanisms responsible for their dysfunction. These include T-cell exhaustion as a consequence Abbreviations: AFP, alpha-fetoprotein; CTLA-4, cytotoxic T-lymphocytes antigen-4; DC, dendritic cell; HCC, hepatocellular carcinoma; IFN-g, interferon gamma; MDSC, myeloid-derived suppressive cells; MHC, major histocompatility complex; PD-1, programmed death receptor-1; TAA, tumor associated antigens; TAM, tumor-associated macrophages; Tregs, regulatory T cells. Address reprint requests to: David H. Adams, FRCP, F.Med.Sci., Professor of Hepatology, National Institute for Health Research Biomedical Research Unit, University of Birmingham, Birmingham, UK. E-mail: d.h.adams@bham.ac.uk. Received September 4, 2013; accepted September 25, 2013. Copyright VC 2014 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.26779 Potential conflict of interest: Nothing to report.

CMV infection of human sinusoidal endothelium regulates hepatic T cell recruitment and activation

BACKGROUND & AIMS Human cytomegalovirus infection (HCMV) is associated with an increased morbidity after liver transplantation, by facilitating allograft rejection and accelerating underlying hepatic inflammation. We hypothesized that human hepatic sinusoidal endothelial cells infected with HCMV possess the capacity to modulate allogeneic T cell recruitment and activation, thereby providing a plausible mechanism of how HCMV infection is able to enhance hepatic immune activation. METHODS Human hepatic sinusoidal endothelial cells were isolated from explanted livers and infected with recombinant endotheliotropic HCMV. We used static and flow-based models to quantify adhesion and transendothelial migration of allogeneic T cell subsets and determine their post-migratory phenotype and function. RESULTS HCMV infection of primary human hepatic sinusoidal endothelial cells facilitated ICAM-1 and CXCL10-dependent CD4 T cell transendothelial migration under physiological levels of shear stress. Recruited T cells were primarily non-virus-specific CXCR3(hi) effector memory T cells, which demonstrated features of LFA3-dependent Th1 activation after migration, and activated regulatory T cells, which retained a suppressive phenotype following transmigration. CONCLUSIONS The ability of infected hepatic endothelium to recruit distinct functional CD4 T cell subsets shows how HCMV facilitates hepatic inflammation and immune activation and may simultaneously favor virus persistence.

Contact-Dependent Depletion of Hydrogen Peroxide by Catalase Is a Novel Mechanism of Myeloid-Derived Suppressor Cell Induction Operating in Human Hepatic Stellate Cells

Myeloid-derived suppressor cells (MDSC) represent a unique cell population with distinct immunosuppressive properties that have been demonstrated to shape the outcome of malignant diseases. Recently, human hepatic stellate cells (HSC) have been reported to induce monocytic-MDSC from mature CD14+ monocytes in a contact-dependent manner. We now report a novel and unexpected mechanism by which CD14+HLADRlow/− suppressive cells are induced by catalase-mediated depletion of hydrogen peroxide (H2O2). Incubation of CD14+ monocytes with catalase led to a significant induction of functional MDSC compared with media alone, and H2O2 levels inversely correlated with MDSC frequency (r = −0.6555, p < 0.05). Catalase was detected in primary HSC and a stromal cell line, and addition of the competitive catalase inhibitor hydroxylamine resulted in a dose-dependent impairment of MDSC induction and concomitant increase of H2O2 levels. The NADPH-oxidase subunit gp91 was significantly increased in catalase-induced MDSC as determined by quantitative PCR outlining the importance of oxidative burst for the induction of MDSC. These findings represent a so far unrecognized link between immunosuppression by MDSC and metabolism. Moreover, this mechanism potentially explains how stromal cells can induce a favorable immunological microenvironment in the context of tissue oxidative stress such as occurs during cancer therapy.

The management of patients awaiting liver transplantation.

Since it was first performed in 1963, liver transplantation has become the only effective curative treatment in patients with liver failure. During the interval between being added to the waiting list and receiving a graft, the patients condition may deteriorate as a result of disease progression or complications of the underlying liver disease. Both may result in death, removal from the waiting list because of futility of the procedure or, a worsened outcome following transplantation. The main aims during this period are to delay or prevent further deterioration in the patients condition, to optimize their general medical health, to prevent, detect and treat any complications, and to offer treatment for specific conditions to improve the patients overall outcome following liver transplantation.

Recurrent nonviral liver disease following liver transplantation

Recurrent disease after liver transplantation is well recognized and remains a potential cause of premature graft loss. The rates of recurrence are difficult to establish because of the lack of consistency in diagnostic criteria and approaches to diagnosis. Owing to the fact that recurrent parenchymal disease may occur in the presence of normal liver tests, those centers that use protocol biopsies will report greater rates of recurrence. It is important to recognize that rates of recurrence vary according to indication and show little correlation with rates of graft loss from recurrent disease. Recurrance rates are greatest for primary sclerosing cholangitis and autoimmune hepatitis, and low reccurrance rates are reported for alcoholic liver disease and recurrent primary biliary cirrhosis. The impact of recurrent nonalcoholic fatty liver disease is not yet clear. Patients and clinicians need to be aware of the possibility of recurrent disease in the differential diagnosis of abnormal liver tests, and management stategies may require alteration to reduce the impact of disease recurrence on outcome. Finally, an understanding of which diseases do recur after transplantation and identification of the risk factors may lead to a better understanding of the pathogenetic mechanisms of these conditions.

Single-gene association between GATA-2 and autoimmune hepatitis: A novel genetic insight highlighting immunologic pathways to disease.

Background & Aims Autoimmune hepatitis (AIH), an immune-mediated liver disease, originates as a consequence of interacting genetic and environmental risk factors. Treatment remains non-specific and prone to side effects. Deficiencies in regulatory T cell (Treg) function are hypothesized to contribute to the pathogenesis of AIH. Methods We describe an adult patient who presented with AIH in the context of monocytopenia. The patient was characterized by GATA2 gene sequencing, flow cytometry of peripheral blood for leucocyte subsets, ELISA for serum Flt-3 ligand, and immunohistochemistry of liver biopsy tissue. Results Sequencing confirmed a GATA2 mutation. Peripheral Treg were absent in the context of a preserved total T cell count. Immunostaining for the Treg transcription factor FOXP3 was reduced in liver tissue as compared to a control AIH specimen. There were marked deficiencies in multiple antigen-presenting cell subsets and Flt-3 ligand was elevated. These findings are consistent with previous reports of GATA2 dysfunction. Conclusions The association of a GATA2 mutation with AIH is previously unrecognized. GATA2 encodes a hematopoietic cell transcription factor, and mutations may manifest as monocytopenia, dendritic and B cell deficiencies, myelodysplasia, and immunodeficiency. Tregs may be depleted as in this case. Our findings provide support for the role of Tregs in AIH, complement reports of other deficiencies in T cell regulation causing AIH-like syndromes, and support the rationale of attempting to modulate the Treg axis for the therapeutic benefit of AIH patients.

A method for conducting suppression assays using small numbers of tissue-isolated regulatory T cells

Graphical abstract Lymphocytes were isolated from human tissue samples by enzymatic and mechanical methods. Treg and Tconv were cell sorted from the tissue cell suspension by gating the lymphocytes forward scatter–side scatter region into a CD4–Live/Dead dot plot. CD4+ live cells were then gated into a CD127–CD25 dot plot. This last dot plot was used to define Treg (Streams 1 and 2) and Tconv cells (Stream 3). Treg were gated into a CCR5 histogram to define CCR5low and CCR5high Treg. Thus, specific Treg subsets were isolated for use in downstream suppression assays. Histograms depicting percentage Foxp3 expression by colorectal cancer-isolated Treg and Tconv show representative data from 9 separate experiments. Treg isolated from colorectal cancer therefore express high amounts of Foxp3 whereas Tconv do not.