Linling Cheng

Identification and characterization of infiltrating macrophages in acetaminophen-induced liver injury

The role of macrophages in the pathogenesis of acetaminophen (APAP)‐induced liver injury remains controversial, as it has been demonstrated that these cells display pro‐toxicant and hepato‐protective functions. This controversy may stem from the heterogeneity and/or plasticity of macrophages and the difficulty in distinguishing and differentially studying subpopulations of macrophages in the liver. In the present study, using flow cytometric analysis and fluorescence‐labeled antibodies against specific cell surface macrophage markers, we were able to, for the first time, identify an APAP‐induced macrophage (IM) population distinct from resident Kupffer cells. The data demonstrated that the IMs were derived from circulating monocytes that infiltrated the liver following APAP‐induced liver injury. The IMs exhibited a phenotype consistent with that of alternatively activated macrophages and demonstrated the ability to phagocytize apoptotic cells and induce apoptosis of neutrophils. Furthermore, in the absence of the IMs, the resolution of hepatic damage following APAP‐induced hepatotoxicity was delayed in CCR2−/− mice compared with wild‐type mice. These findings likely contribute to the role of the IMs in the processes of tissue repair, including counteracting inflammation and promoting angiogenesis. The present study also demonstrated the ability of separating populations of macrophages and delineating distinct functions of each group in future studies of inflammatory disease in the liver and other tissues.

Mechanism of T cell tolerance induction by murine hepatic Kupffer cells

The liver is known to favor the induction of immunological tolerance rather than immunity. Although Kupffer cells (KC) have been indicated to play a role in liver tolerance to allografts and soluble antigens, the mechanisms involved remain unclear. We hypothesized that KCs could promote immune tolerance by acting as incompetent antigen‐presenting cells (APC), as well as actively suppressing T cell activation induced by other potent APCs. The expression of antigen presentation‐related molecules by KCs was phenotyped by flow cytometry. The abilities of KCs to act as APCs and to suppress T cell activation induced by splenic dendritic cells (DC) were examined by in vitro proliferation assays using CD4+ OVA‐TCR (ovalbumin T cell receptor) transgenic T cells. We found that, compared with DCs, KCs expressed significantly lower levels of major histocompatibility complex (MHC) II, B7‐1, B7‐2, and CD40. This result is consistent with our observation that KCs were not as potent as DCs in eliciting OVA‐specific T cell proliferation. However, KCs isolated from polyinosinic:polycytidylic acid–treated mice expressed significantly higher levels of MHC II and costimulatory molecules than did naïve KCs and could stimulate stronger T cell responses. More importantly, we found that KCs could inhibit DC‐induced OVA‐specific T cell activation. Further investigation of the underlying mechanism revealed that prostaglandins produced by KCs played an important role. The results ruled out the possible involvement of interleukin‐10, nitric oxide, 2,3‐dioxygenase, and transforming growth factor β in KC‐mediated T cell suppression. Conclusion: Our data indicate that KCs are a tolerogenic APC population within the liver. These findings suggest that KCs may play a critical role in regulating immune reactions within the liver and contributing to liver‐mediated systemic immune tolerance. (HEPATOLOGY 2008.)

Increased natural killer cell cytotoxicity and NKp30 expression protects against hepatitis C virus infection in high-risk individuals and inhibits replication in vitro.

CD56pos natural killer (NK)/natural T (NT) cells are important innate effectors providing the first line of defense against viral infection. Enhanced NK activity has been shown to protect from human immunodeficiency virus‐1 infection. However, the role played by these innate effectors in protection against or development of hepatitis C virus (HCV) infection is unknown. We characterized CD56pos populations in 11 injection drug users (IDUs) who remained uninfected despite being repeatedly exposed to HCV. NK profiles in exposed but uninfected (EU) individuals were compared with preinfection samples (median 90 days prior to HCV seroconversion) collected from 14 IDUs who were exposed and subsequently became infected (EI) and unexposed normal control subjects (n = 8). Flow cytometric analysis of CD56pos populations demonstrated that EUs had a higher proportion of CD56low mature (P = 0.0011) NK cells compared with EI subjects. Bead‐isolated NKs (>90% purity) from EUs had significantly higher interleukin‐2 (IL‐2)–induced cytolytic activity against the NK‐sensitive cell line K562 at an effector‐to‐target ratio of 10:1 (P < 0.0001). NKp30, a natural cytotoxicity receptor involved in NK activation, is highest on NK/NT cells in EUs relative to infected subjects. Using the JFH‐1 infection system, we demonstrated that NKp30high cells in the absence of exogenous stimulation significantly reduce infection of hepatocytes. Conclusion: CD56pos populations in EUs are enriched for effector NKs displaying enhanced IL‐2–induced cytolytic activity and higher levels of the natural cytotoxicity receptor NKp30‐activating receptor. In addition, NKp30high cells are more effective in preventing infection of Huh‐7.5 cells than their NKp30low/neg counterparts. These data support the hypothesis that NK cells contribute to anti‐HCV defense in vivo in the earliest stages of infection, providing innate protection from HCV acquisition. (HEPATOLOGY 2010)

Role of neutrophils in a mouse model of halothane-induced liver injury

Drug‐induced liver injury (DILI) is a major safety concern in drug development. Its prediction and prevention have been hindered by limited knowledge of the underlying mechanisms, in part the result of a lack of animal models. We developed a mouse model of halothane‐induced liver injury and characterized the mechanisms accounting for tissue damage. Female and male Balb/c, DBA/1, and C57BL/6J mice were injected intraperitoneally with halothane. Serum levels of alanine aminotransferase and histology were evaluated to determine liver injury. Balb/c mice were found to be the most susceptible strain, followed by DBA/1, with no significant hepatotoxicity observed in C57BL/6J mice. Female Balb/c and DBA/1 mice developed more severe liver damage compared with their male counterparts. Bioactivation of halothane occurred similarly in all three strains based on detection of liver proteins adducted by the reactive metabolite. Mechanistic investigations revealed that hepatic message levels of tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β); IL‐6, and IL‐8 were significantly higher in halothane‐treated Balb/c mice compared to DBA/1 and C57BL/6J mice. Moreover, a higher number of neutrophils were recruited into the liver of Balb/c mice upon halothane treatment compared with DBA/1, with no obvious neutrophil infiltration detected in C57BL/6J mice. Neutrophil depletion experiments demonstrated a crucial role for these cells in the development of halothane‐induced liver injury. The halothane‐initiated hepatotoxicity and innate immune response‐mediated escalation of tissue damage are consistent with events that occur in many cases of DILI. In conclusion, our model provides a platform for elucidating strain‐based and gender‐based susceptibility factors in DILI development. (HEPATOLOGY 2006;44:1421–1431.)

Natural killer inhibitory receptor expression associated with treatment failure and interleukin‐28B genotype in patients with chronic hepatitis C

Natural killer (NK) cells constitute a first line of defense against viral infections; their function is governed by the integration of signals from multiple activating and inhibitory surface receptors. We hypothesized that because NKs become rapidly activated by cytokines, response to anti‐hepatitis C virus (HCV) therapy would be predicted by the phenotype and function of NKs. We used a cohort of 101 patients (55 African, 46 Caucasian‐American) who received pegylated‐interferon (IFN) and ribavirin for 48 weeks. Multiparameter FACS analysis was used to examine relative expression of 14 different inhibitory/activating receptors. Interleukin (IL)‐28B genotyping (rs12979860) was also performed. Pretreatment levels of inhibitory receptors CD158a, CD158b, and CD158e were higher in patients who demonstrated poor viral decline within the first 28 days of therapy. Higher expression levels of inhibitory receptors NKG2A, CD158b, and CD158e were demonstrable in patients who failed to achieve sustained virologic response (SVR). Patients carrying the IL‐28B T allele had higher NKG2A expression on effector NKs. We created a mathematical regression model incorporating race, viral level, and two inhibitory receptors. The area‐under‐the curve was 0.88, which is highly predictive of SVR. Moreover, the model performed complementarily with IL‐28B across the CC, CT, and TT genotypes. Purified NKG2Aneg NKs treated with pegylated‐IFN‐α for 4 hours demonstrated higher levels of IFN‐γ‐inducible protein‐10 (IP‐10) and tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) compared with their NKG2Apos counterparts. Conclusions: These results provide novel insights into the associations of NK phenotype with IL‐28B genotype and gene expression patterns, as well as the role of NKs in mediating IFN‐induced viral clearance of chronic HCV infection. (HEPATOLOGY 2011;)

Hepatitis C virus pathogen associated molecular pattern (PAMP) triggers production of lambda-interferons by human plasmacytoid dendritic cells.

Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell in the defense against viruses. Through pattern recognition receptors (PRRs), these cells detect viral pathogen associated molecular patterns (PAMPs) and initiate an Interferon (IFN) response. pDCs produce the antiviral IFNs including the well-studied Type I and the more recently described Type III. Recent genome wide association studies (GWAS) have implicated Type III IFNs in HCV clearance. We examined the IFN response induced in a pDC cell line and ex vivo human pDCs by a region of the HCV genome referred to as the HCV PAMP. This RNA has been shown previously to be immunogenic in hepatocytes, whereas the conserved X-region RNA is not. We show that in response to the HCV PAMP, pDC-GEN2.2 cells upregulate and secrete Type III (in addition to Type I) IFNs and upregulate PRR genes and proteins. We also demonstrate that the recognition of this RNA is dependent on RIG-I-like Receptors (RLRs) and Toll-like Receptors (TLRs), challenging the dogma that RLRs are dispensable in pDCs. The IFNs produced by these cells in response to the HCV PAMP also control HCV replication in vitro. These data are recapitulated in ex vivo pDCs isolated from healthy donors. Together, our data shows that pDCs respond robustly to HCV RNA to make Type III Interferons that control viral replication. This may represent a novel therapeutic strategy for the treatment of HCV.

Race‐ and gender‐related variation in natural killer p46 expression associated with differential anti‐hepatitis c virus immunity

Major racial and gender differences have been documented in the natural history and treatment responses of chronic hepatitis C virus (HCV) infection; however, distinct mechanisms have remained enigmatic. We hypothesized that racial‐ and gender‐related differences in natural killer (NK) cell populations may explain altered natural history and treatment responses. Our study cohort consisted of 29 African‐American (AA; 55% male) and 29 Caucasian‐American (CA; 48% male) healthy uninfected control subjects. Multiparameter flow cytometric analysis was used to characterize levels, phenotype with respect to 14 NK receptors, and lymphokine‐activated killing (LAK) function. Gene expression was assessed by real‐time reverse‐transcriptase polymerase chain reaction after 6‐hour in vitro stimulation with Toll‐like receptor (TLR) ligands. The ability to control HCV infection was assessed in the Huh‐7.5/JFH‐1 coculture system. NK expression of natural cytotoxicity receptor NKp46 was strongly associated with CA race and female gender and correlated positively with LAK activity (P = 0.0054). NKp46high NKs were more efficient at controlling HCV than their NKp46low counterparts (P < 0.001). Similarly, ligation of NKp46 on isolated NK cells resulted in a significant reduction in the HCV copy number detected in Huh‐7.5/JFH‐1 coculture (multiplicity of infection: 0.01) at an effector:target ratio of 5:1 (P < 0.005). After TLR stimulation, genes involved in cytotoxicity, but not cytokine genes, were significantly up‐regulated in NKp46high NKs. Cytokine stimulation (interleukin [IL]‐12 and IL‐15) demonstrated that NKp46high NK cells have significantly higher interferon‐gamma production than NKp46low cells. TLR stimulation significantly induced degranulation as well as tumor necrosis factor alpha (TNF‐α)‐related apoptosis‐inducing ligand, Fas, and TNF‐α protein expression in NKp46high NKs. NKp46 ligand was induced on HCV‐infected hepatocytes. Conclusions: NKp46 expression may contribute to differential HCV responses. NKp46 expression correlates with anti‐HCV activity in vitro and thus may prove to be a useful therapeutic target. (HEPATOLOGY 2012)

Prostaglandin I2 and E2 mediate the protective effects of cyclooxygenase‐2 in a mouse model of immune‐mediated liver injury

Studies of the molecular and cellular mechanisms of concanavalin A (ConA)‐induced liver injury have provided important knowledge on the pathogenesis of many liver diseases involving hepatic inflammation. However, studies identifying hepato‐protective factors based on the mechanistic understanding of this model are lacking. Evidence suggests that certain prostaglandin (PG) products of cyclooxygenase (COX)‐1 and COX‐2 provide important anti‐inflammatory and cytoprotective functions in some pathophysiological states. In the present study, we demonstrate a protective role of COX‐2 derived PGs in ConA‐induced liver injury. COX‐2−/− mice developed much more severe liver damage upon ConA treatment compared with wild‐type and COX‐1−/− mice. Treatment of COX‐2−/− mice with misoprostol (a PGE1/2 analog) or beraprost (a PGI2 analog) significantly decreased ConA‐induced liver injury. Data from both in vivo and in vitro experiments demonstrated that misoprostol and beraprost acted directly on hepatic leukocytes, including natural killer (NK)T and T cells, and down‐regulated their production of interferon (IFN)‐γ, which are critical in mediating ConA‐induced tissue damage. Collectively, the results provide strong evidence that the protective effects of COX‐2 within the liver are mediated through the production of PGE2 and PGI2, which exert anti‐inflammatory functions. These findings suggest that COX‐2‐derived PGs may have great therapeutic potentials in treating patients with inflammatory liver diseases. (HEPATOLOGY 2007;45:159–169.)

Galectin-9 Functionally Impairs Natural Killer Cells in Humans and Mice

ABSTRACT Galectin-9 is a pleiotropic immune modulator affecting numerous cell types of innate and adaptive immunity. Patients with chronic infection with either hepatitis C virus (HCV) or HIV have elevated circulating levels. Limited data exist on the regulation of natural killer (NK) cell function through interaction with galectin-9. We found that galectin-9 ligation downregulates multiple immune-activating genes, including eight involved in the NK cell-mediated cytotoxicity pathway, impairs lymphokine-activated killing, and decreases the proportion of gamma interferon (IFN-γ)-producing NK cells that had been stimulated with interleukin-12 (IL-12)/IL-15. We demonstrate that the transcriptional and functional changes induced by galectin-9 are independent of Tim-3. Consistent with these results for humans, we find that the genetic absence of galectin-9 in mice is associated with greater IFN-γ production by NK cells and enhanced degranulation. We also show that in the setting of a short-term (4-day) murine cytomegalovirus infection, terminally differentiated NKs accumulate in the livers of galectin-9 knockout mice, and that hepatic NKs spontaneously produce significantly more IFN-γ in this setting. Taken together, our results indicate that galectin-9 engagement impairs the function of NK cells, including cytotoxicity and cytokine production.

Effect of polyI:C cotreatment on halothane‐induced liver injury in mice

Drug‐induced liver injury (DILI) is a challenging problem in drug development and clinical practice. Patient susceptibility to DILI is multifactorial, making these reactions difficult to predict and prevent. Clinical observations have suggested that concurrent bacterial and viral infections represent an important risk factor in determining patient susceptibility to developing adverse drug reactions, although the underlying mechanism is not clear. In the present study, we employed the viral RNA mimetic (polyinosinic‐polycytidylic acid [polyI:C]) to emulate viral infection and examined its effect on halothane‐induced liver injury. Although pretreatment of mice with polyI:C attenuated halothane hepatotoxicity due to its inhibitory effect on halothane metabolism, posttreatment significantly exacerbated liver injury with hepatocellular apoptosis being significantly higher than that in mice treated with polyI:C alone or halothane alone. The pan‐caspase inhibitor z‐VAD‐fmk suppressed liver injury induced by polyI:C/posthalothane cotreatment, suggesting that the increased hepatocyte apoptosis contributes to the exacerbation of liver injury. Posttreatment with polyI:C also caused activation of hepatic Kupffer cells (KCs) and natural killer (NK) cells and upregulated multiple proapoptotic factors, including tumor necrosis factor‐α (TNF‐α), NK receptor group 2, member D (NKG2D), and Fas ligand (FasL). These factors may play important roles in mediating polyI:C‐induced hepatocyte apoptosis. Conclusion: This is the first study to provide evidence that concurrent viral infection can inhibit cytochrome (CYP)450 activities and activate the hepatic innate immune system to proapoptotic factors. DILI may be attenuated or exacerbated by pathogens depending on the time of infection. (HEPATOLOGY 2009;49:215–226.)