Qiang You

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.)

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.)

Role of hepatic resident and infiltrating macrophages in liver repair after acute injury

Treatment of liver disease, caused by hepatotoxins, viral infections, alcohol ingestion, or autoimmune conditions, remains challenging and costly. The liver has a powerful capacity to repair and regenerate, thus a thorough understanding of this tightly orchestrated process will undoubtedly improve clinical means of restoring liver function after injury. Using a murine model of acute liver injury caused by overdose of acetaminophen (APAP), our studies demonstrated that the combined absence of liver resident macrophages (Kupffer cells, KCs), and infiltrating macrophages (IMs) resulted in a marked delay in liver repair, even though the initiation and extent of peak liver injury was not impacted. This delay was not due to impaired hepatocyte proliferation but rather prolonged vascular leakage, which is caused by APAP-induced liver sinusoidal endothelial cell (LSEC) injury. We also found that KCs and IMs express an array of angiogenic factors and induce LSEC proliferation and migration. Our mechanistic studies suggest that hypoxia-inducible factor (HIF) may be involved in regulating the angiogenic effect of hepatic macrophages (Macs), as we found that APAP challenge resulted in hypoxia and stabilization of HIF in the liver and hepatic Macs. Together, these data indicate an important role for hepatic Macs in liver blood vessel repair, thereby contributing to tissue recovery from acute injury.

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.)

Involvement of natural killer T cells in halothane-induced liver injury in mice

Drug-induced liver injury (DILI) causes significant patient morbidity and mortality, and is the most common reason for drug withdrawals. It is imperative to gain a thorough understanding of the underlying mechanisms of DILI to effectively predict and prevent these reactions. We have recently developed a murine model of halothane-induced liver injury (HILI). The aim of the present study was to investigate the role of hepatic natural killer T (NKT) cells in the pathogenesis of HILI. The degrees of HILI were compared between WT and CD1d(-/-) mice, which are deficient in NKT cells. The data revealed that CD1d(-/-) mice were resistant in developing HILI. This resistance appeared to be a direct result of NKT cell depletion rather than an indirect one due to the absence of cross-talk between NKT cells and other hepatic innate immune cells. Compared with WT mice, CD1d(-/-) mice exhibited a significantly lower number of hepatic infiltrating neutrophils upon halothane challenge (470,000+/-100,000/liver in WT vs. 120,000+/-31,500/liver in CD1d(-/-) mice). This result in conjunction with our previous finding of an indispensable role of neutrophils in HILI strongly suggests that NKT cells play a critical role in regulating neutrophil recruitment, thereby contributing to the development of HILI. Collectively, the current study and published reports indicate that this murine model of HILI provides an experimental system for the investigation of the underlying mechanisms of DILI. In addition, this model may yield the discovery of susceptibility factors that may control the development of liver injury in patients treated with halothane and potentially other drugs.

Mice Lacking Natural Killer T Cells Are More Susceptible to Metabolic Alterations following High Fat Diet Feeding

Current estimates suggest that over one-third of the adult population has metabolic syndrome and three-fourths of the obese population has non-alcoholic fatty liver disease (NAFLD). Inflammation in metabolic tissues has emerged as a universal feature of obesity and its co-morbidities, including NAFLD. Natural Killer T (NKT) cells are a subset of innate immune cells that abundantly reside within the liver and are readily activated by lipid antigens. There is general consensus that NKT cells are pivotal regulators of inflammation; however, disagreement exists as to whether NKT cells exert pathogenic or suppressive functions in obesity. Here we demonstrate that CD1d−/− mice, which lack NKT cells, were more susceptible to weight gain and fatty liver following high fat diet (HFD) feeding. Compared with their WT counterparts, CD1d−/− mice displayed increased adiposity and greater induction of inflammatory genes in the liver suggestive of the precursors of NAFLD. Calorimetry studies revealed a significant increase in food intake and trends toward decreased metabolic rate and activity in CD1d−/− mice compared with WT mice. Based on these findings, our results suggest that NKT cells play a regulatory role that helps to prevent diet-induced obesity and metabolic dysfunction and may play an important role in mechanisms governing cross-talk between metabolism and the immune system to regulate energy balance and liver health.

Covalent binding of the nitroso metabolite of sulfamethoxazole is important in induction of drug-specific T-cell responses in vivo.

Immune-mediated drug hypersensitivity reactions (IDHRs) represent a significant problem due to their unpredictable and severe nature, as well as the lack of understanding of the pathogenesis. Sulfamethoxazole (SMX), a widely used antibiotic, has been used as a model compound to investigate the underlying mechanism of IDHRs because it has been associated with a relatively high incidence of hypersensitivity. Previous studies by others showed that administration of 4-(nitroso)-N-(5-methyl-1,2-oxazol-3-yl)benzenesulfonamide (SMX-NO), the reactive metabolite of SMX, to rats resulted in the generation of SMX-specific antibodies and ex vivo splenocyte proliferative responses, as well as haptenation of skin keratinocytes, circulating peripheral blood mononuclear cells, and splenocytes. The objective of the present study was to further investigate SMX-NO-protein binding in relationship to its immunogenicity. In female DBA/1 mice treated with SMX-NO, varying degrees of SMX-NO-dependent T-cell responses and SMX-NO-protein adduct formation were observed in the spleen and in inguinal, brachial, and axillary lymph nodes. The data suggested a tissue-specific threshold of SMX-NO dosage that triggers the detection of adducts and immune response. Furthermore, serum albumin and immunoglobulin were identified as protein targets for SMX-NO modification. It seemed that these adducts were formed in the blood, circulated to lymphoid tissues, and initiated SMX-NO-dependent immune responses. Collectively, these data revealed a causal link between the deposition of SMX-NO-protein adducts in a lymphoid tissue and the induction of immune response in that tissue. Our findings also suggest that the immunogenicity of SMX-NO is determined by the immunogenic nature of the hapten, rather than special characteristics of the adducted protein.

19-substituted benzoquinone ansamycin heat shock protein-90 inhibitors: biological activity and decreased off-target toxicity.

The benzoquinone ansamycins (BQAs) are a valuable class of antitumor agents that serve as inhibitors of heat shock protein (Hsp)-90. However, clinical use of BQAs has resulted in off-target toxicities, including concerns of hepatotoxicity. Mechanisms underlying the toxicity of quinones include their ability to redox cycle and/or arylate cellular nucleophiles. We have therefore designed 19-substituted BQAs to prevent glutathione conjugation and nonspecific interactions with protein thiols to minimize off-target effects and reduce hepatotoxicity. 19-Phenyl– and 19-methyl–substituted versions of geldanamycin and its derivatives, 17-allylamino-17-demethoxygeldanamycin and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), did not react with glutathione, whereas marked reactivity was observed using parent BQAs. Importantly, although 17-DMAG induced cell death in primary and cultured mouse hepatocytes, 19-phenyl and 19-methyl DMAG showed reduced toxicity, validating the overall approach. Furthermore, our data suggest that arylation reactions, rather than redox cycling, are a major mechanism contributing to BQA hepatotoxicity. 19-Phenyl BQAs inhibited purified Hsp90 in a NAD(P)H:quinone oxidoreductase 1 (NQO1)–dependent manner, demonstrating increased efficacy of the hydroquinone ansamycin relative to its parent quinone. Molecular modeling supported increased stability of the hydroquinone form of 19-phenyl-DMAG in the active site of human Hsp90. In human breast cancer cells, 19-phenyl BQAs induced growth inhibition also dependent upon metabolism via NQO1 with decreased expression of client proteins and compensatory induction of Hsp70. These data demonstrate that 19-substituted BQAs are unreactive with thiols, display reduced hepatotoxicity, and retain Hsp90 and growth-inhibitory activity in human breast cancer cells, although with diminished potency relative to parent BQAs.

Development of a screening assay to evaluate the potential of drugs to cause immune-mediated hypersensitivity reactions.

Abstract Evidence suggests that bio-activation of drugs to generate chemically reactive metabolites (RM) that act as haptens to form immunogenic protein conjugates may be an important cause of immune-mediated drug hypersensitivity reactions (IDHR). Although many drugs that form RMs raise concerns about producing IDHR, standard non-clinical testing methods are rarely able to identify compounds with the potential to produce IDHR in humans. The objective of this study was to develop a predictive assay for IDHR that involves: (1) the use of an in vitro drug-metabolizing system to generate the RM that is captured by GSH, (2) conjugating the RM-GSH conjugate to mouse serum albumin (MSA) by using a chemical cross-linker, (3) immunization of mice with RM-GSH-MSA adducts, and (4) ex vivo challenge with RM-GSH-MSA adduct and measurement of lymphocyte proliferation to determine if the RM is immunogenic. The predictivity of the assay was evaluated by using drugs that produce RM and have been strongly, weakly, or not associated with IDHRs in the clinic. While this method requires additional validation with more drugs, the results demonstrate the feasibility of identifying drugs strongly associated with IDHR and the utility of the assay for rank ordering drugs with respect to their potential to cause IDHR.

Interaction of AIM with insulin-like growth factor-binding protein-4.

Apoptosis inhibitor of macrophages (AIM/cluster of differentiation 5 antigen-like/soluble protein α) has been shown to inhibit cellular apoptosis; however, the underlying molecular mechanism has not been elucidated. Using yeast two-hybrid screening, the present study uncovered that AIM binds to insulin-like growth factor binding protein-4 (IGFBP-4). AIM interaction with IGFBP-4, as well as IGFBP-2 and -3, but not with IGFBP-1, -5 and -6, was further confirmed by co-immunoprecipitation (co-IP) using 293 cells. The binding activity and affinity between AIM and IGFBP-4 in vitro were analyzed by co-IP and biolayer interferometry. Serum depletion-induced cellular apoptosis was attenuated by insulin-like growth factor-I (IGF-I), and this effect was abrogated by IGFBP-4. Of note, in the presence of AIM, the inhibitory effect of IGFBP-4 on the anti-apoptosis function of IGF-I was attenuated, possibly through binding of AIM with IGFBP-4. In conclusion, to the best of our knowledge, the present study provides the first evidence that AIM binds to IGFBP-2, -3 and -4. The data suggest that this interaction may contribute to the mechanism of AIM-mediated anti-apoptosis function.