Potential conflict of interest: Nothing to report.
Supported by the intramural program of the NIAAA, NIH.
Prednisolone is a corticosteroid that has been used to treat inflammatory liver diseases such as autoimmune hepatitis and alcoholic hepatitis. However, the results have been controversial, and how prednisolone affects liver disease progression remains unknown. In the current study we examined the effect of prednisolone treatment on several models of liver injury, including T/NKT cell hepatitis induced by concanavalin A (ConA) and α-galactosylceramide (α-GalCer), and hepatotoxin-mediated hepatitis induced by carbon tetrachloride (CCl4) and/or ethanol. Prednisolone administration attenuated ConA- and α-GalCer-induced hepatitis and systemic inflammatory responses. Treating mice with prednisolone also suppressed inflammatory responses in a model of hepatotoxin (CCl4)-induced hepatitis, but surprisingly exacerbated liver injury and delayed liver repair. In addition, administration of prednisolone also enhanced acetaminophen-, ethanol-, or ethanol plus CCl4-induced liver injury. Immunohistochemical and flow cytometric analyses demonstrated that prednisolone treatment inhibited hepatic macrophage and neutrophil infiltration in CCl4-induced hepatitis and suppressed their phagocytic activities in vivo and in vitro. Macrophage and/or neutrophil depletion aggravated CCl4-induced liver injury and impeded liver regeneration. Finally, conditional disruption of glucocorticoid receptor in macrophages and neutrophils abolished prednisolone-mediated exacerbation of hepatotoxin-induced liver injury. Conclusion: Prednisolone treatment prevents T/NKT cell hepatitis but exacerbates hepatotoxin-induced liver injury by inhibiting macrophage- and neutrophil-mediated phagocytic and hepatic regenerative functions. These findings may not only increase our understanding of the steroid treatment mechanism but also help us to better manage steroid therapy in liver diseases. (Hepatology 2014;59:1094–1106)
signal transducer and activator of transcription 3
tumor necrosis factor-α
Liver inflammation is associated with acute and chronic liver diseases, including alcoholic liver disease, nonalcoholic fatty liver disease, viral hepatitis, and autoimmune hepatitis, which are characterized by the activation and infiltration of inflammatory cells in the liver.[1-7] It is generally believed that liver inflammation is triggered by pathogen infections (e.g., hepatitis viruses) and/or damage-associated molecular patterns (DAMPs) released by damaged hepatocytes.[6-8] However, the mechanisms through which DAMPs induce liver inflammation and how such inflammation contributes to the progression of liver disease remain largely unknown.[6-8] For example, ∼95% of heavy drinkers develop fatty liver, but only 20%-35% of them progress to alcoholic hepatitis and fibrosis, in which inflammation is thought to play a critical role.[1, 2, 9] Currently, the mechanisms underlying inflammation development in alcoholic liver injury are not fully understood, and many mechanisms have been proposed,[1, 2, 9] including the alcohol consumption-associated elevation of hepatic endotoxin levels, hepatocellular damage, elevation of reactive oxygen species (ROS), activation of innate immunity, and activation of Kupffer cells. All of these factors up-regulate the expression of proinflammatory cytokines and chemokines in the liver, followed by the recruitment of inflammatory cells, such as neutrophils and macrophages.[1, 2, 9] The general purpose of early neutrophil infiltration is to remove dead or dying cells as a prerequisite for wound repair and regeneration, but neutrophils also cause cell injury, primarily through the formation of ROS and release of proteases.[10-13] Despite its beneficial effect on liver repair and regeneration, inflammation remains an actively investigated therapeutic target and immunosuppressive drugs, such as steroids, have been used for the treatment of several types of inflammatory liver diseases. However, the results of steroid therapy for inflammatory liver diseases, especially its results on alcoholic hepatitis, have been controversial.[14-20]
Glucocorticoids (GCs) are steroid hormones that are synthesized in the adrenal cortex and play a wide variety of important functions in the body, including the modulation of metabolism, regulation of stress responses, and suppression of inflammation. The actions of GCs are mediated by binding to the GC receptor (GR) and subsequently inhibiting the activity of transcription factors, which control the expression of proinflammatory cytokines, chemokines, and adhesion molecules. Because of its broad antiinflammatory effects, synthetic GCs, such as prednisolone, have been widely used to treat inflammatory liver diseases that are caused by an overactive immune system. Prednisolone treatment has been shown to ameliorate symptoms and improve biochemical and histologic abnormalities in many types of liver diseases, including autoimmune hepatitis, cirrhosis patients with septic shock, and liver transplantation. Steroids have also been used for the treatment of alcoholic hepatitis for many years, but the results have been mixed.[15-20] Surprisingly, although steroids have been used to treat inflammatory liver diseases for more than five decades, the mechanism through which prednisolone affects liver disease pathogenesis has not been explored. The aim of the present study was to examine the effect of prednisolone on liver injury in different experimental mouse models of inflammatory liver injury, including concanavalin A (ConA)-induced T-cell hepatitis, α-galactosylceramide (α-GalCer)-induced natural killer T (NKT) cell hepatitis, hepatotoxin carbon tetrachloride (CCl4)- or acetaminophen (APAP)-induced hepatitis,[27, 28] chronic plus binge ethanol feeding model,[29, 30] and ethanol plus CCl4-induced liver fibrosis. Our results demonstrate that prednisolone treatment prevented ConA- and α-GalCer-induced acute T/NKT cell hepatitis but exacerbated CCl4-, APAP-, ethanol-, or ethanol plus CCl4-induced liver injury. Subsequent studies suggested that prednisolone treatment worsened CCl4-induced hepatocellular damage by inhibiting macrophage- and neutrophil-mediated phagocytic and regenerative functions.
Materials and Methods
C57BL/6N mice were purchased from the NCI (Frederick, MD). Myeloid-specific glucocorticoid receptor knockout (LysCre+GRflox/flox) (mGR KO) mice were kindly provided by Dr. Louis J. Muglia (University of Cincinnati). LysCre−/− GRflox/flox mice were used as littermate wild-type (WT) controls. All animal experiments were approved by the National Institute on Alcohol Abuse and Alcoholism Animal Care and Use Committee.
For the acute liver injury model, 8- to 10-week old male mice were injected with ConA (20 mg/kg body weight, intravenously) (Sigma-Aldrich, St. Louis, MO), α-GalCer (100 mg/kg body weight, intraperitoneally) (Enzo Life Science, Farmingdale, NY), or CCl4 (0.2 mL/kg body weight, intraperitoneally) (Sigma-Aldrich). Subsequently, prednisolone (10 mg/kg body weight in 5% ethanol, intraperitoneally; Sigma-Aldrich) or vehicle (5% ethanol) was administered to mice, as described in Supporting Fig. 1. In some experiments, mice were injected intraperitoneally with 50 mg/kg body weight 5-bromo-2′-deoxy-uridine (BrdU; Sigma-Aldrich) 2 hours before sacrifice for the examination of liver regeneration.
The chronic plus binge ethanol model was described previously. For ethanol plus CCl4-induced liver fibrosis model, mice were fed a Liber-DeCarli liquid diet for 8 weeks, and CCl4 (0.1 mg/kg) was administrated twice a week during the 8-week feeding. Prednisolone was injected intraperitoneally daily for the last 2 weeks. For APAP-induced liver injury, mice were fasting for 24 hours and subsequently injected with APAP (200 mg/kg, intraperitoneally), and prednisolone or vehicle was administrated as described in the figure legends.
Other materials and methods are included in the Supporting document.
Cotreatment With Prednisolone Ameliorates Acute T/NKT Cell Hepatitis Induced by ConA or α-GalCer
To investigate the role of prednisolone in experimental T-cell hepatitis, mice were cotreated with ConA plus prednisolone or vehicle (Supporting Fig. 1A). Treatment of mice with prednisolone alone did not induce elevation of serum alanine aminotransferase (ALT) and no obvious liver necrosis was observed in mice after prednisolone treatment alone (Supporting Fig. 2). ConA injection induced an elevation of serum ALT levels and massive necrosis in the liver, which were markedly decreased in mice cotreated with prednisolone (Supporting Fig. 3A,B). Prednisolone treatment also markedly prevented ConA-induced elevation of serum levels of various cytokines (Supporting Fig. 3C). Similarly, prednisolone treatment prevented the NKT activator α-GalCer-induced systemic inflammatory responses, liver inflammation, and injury (Supporting Fig. 4).
Cotreatment With Prednisolone Delays Liver Repair in CCl4-Induced Acute Hepatitis
CCl4 is a hepatotoxic agent that has been widely used to induce liver injury in a hepatotoxin-mediated hepatitis model that allows for the evaluation of both necrosis and subsequent inflammation. To determine the role of prednisolone in toxin-induced acute liver injury, mice were injected with CCl4 and cotreated with prednisolone or vehicle (Supporting Fig. 1C). Liver histology analyses in Fig. 1A,B show that in the CCl4+vehicle group, injection of a single dose of CCl4 induced massive liver necrosis 24, 48, and 72 hours postinjection, but such necrosis was markedly recovered 96 hours postinjection; surprisingly, in the CCl4+prednisolone group, massive liver necrosis was not improved 96 hours postinjection and still remained until 120 hours. In agreement with liver histology data, prednisolone cotreatment induced higher levels of serum ALT levels 48 hours post-CCl4 injection (Fig. 1C). Treatment with prednisolone alone did not induce obvious liver necrosis (Fig. 1B) nor affected serum ALT levels (Fig. 1C). Moreover, the serum levels of several proinflammatory cytokines (e.g., tumor necrosis factor alpha [TNF-α] and interleukin [IL]-6), which are important for liver regeneration,[33, 34] were elevated after CCl4 injection with vehicle cotreatment. These effects were completely attenuated in prednisolone-cotreated mice (Fig. 1D). Finally, treatment of mice with prednisolone did not affect hepatic expression of p450CYP2E1, a key enzyme to metabolize CCl4 (Supporting Fig. 5). This suggests that prednisolone exacerbation of CCl4-induced liver injury is not mediated by way of the regulation of CCl4 metabolism.
In addition, we also examined the effects of prednisolone treatment on APAP-induced liver injury (Supporting Fig. 6). Liver histology analyses show a single injection of APAP induced massive liver necrosis 48 hours postinjection, but such necrosis still remained until 72 hours in the APAP+prednisolone group (Supporting Fig. 6C). In addition, prednisolone cotreatment markedly attenuated BrdU incorporation in hepatocytes 48 hours after APAP injection (Supporting Fig. 6C,D).
Cotreatment With Prednisolone Exacerbates Ethanol or Ethanol Plus CCl4-Induced Chronic Liver Injury and Fibrosis
To investigate the effect of prednisolone on alcohol-induced liver injury, mice were fed an ethanol diet for 10 days, followed by a single gavage of ethanol plus prednisolone or vehicle treatment (Supporting Fig. 7A). Chronic-binge ethanol feeding elevated serum ALT levels, which were markedly increased in mice cotreated with prednisolone (Supporting Fig. 7B). In addition, hematoxylin and eosin (H&E) and Oil Red O staining revealed higher degree of steatosis in mice cotreated with prednisolone than in mice cotreated with vehicle (Supporting Fig. 7C).
To further investigate the effect of prednisolone in chronic liver injury and fibrosis, mice were treated with ethanol plus CCl4 and coinjected with prednisolone or vehicle (Supporting Fig. 8A). As illustrated in Supporting Fig. 8B, prednisolone cotreatment induced higher levels of serum ALT levels, greater levels of Sirius red and α-SMA staining, and higher hepatic expression levels of α-SMA compared with vehicle-cotreated groups (Supporting Fig. 8E,F).
Prednisolone Administration Inhibits Acute CCl4-Induced Macrophage and Neutrophil Recruitment in the Liver
CCl4-induced liver injury is associated with activation of Kupffer cells and triggers migration of macrophages into hepatic cords. As shown in Fig. 2A, administration of CCl4 plus vehicle cotreatment induced accumulation of mononuclear cells (MNCs) and Gr-1intCD11b+ macrophages in the liver 24 hours posttreatment, but such accumulation was attenuated in prednisolone-cotreated mice. This reduction of macrophages infiltration in prednisolone-cotreated mice was not due to the increased macrophage apoptosis because there were no differences in macrophage apoptosis between prednisolone-cotreated and vehicle-cotreated groups (Supporting Fig. 9).
Neutrophil infiltration was determined by immunohistochemical analyses of myeloperoxidase (MPO, a marker for neutrophils). As illustrated in Fig. 2B and Supporting Fig. 10, CCl4 injection resulted in neutrophil infiltration in the liver. The number of MPO+ cells was significantly increased 24 and 48 hours post-CCl4 injection and declined thereafter in the CCl4+vehicle group. Prednisolone cotreatment delayed hepatic neutrophil recruitment, with a lower number of MPO+ cells at 24 and 48 hours but a higher number at 72 hours post-CCl4 administration compared with the vehicle-cotreated group. Furthermore, fluorescence-activated cell sorting (FACS) analyses also confirmed that prednisolone cotreatment attenuated hepatic neutrophil infiltration post-CCl4 injection. As illustrated in Fig. 2C, in the CCl4+vehicle group, the total number of Gr-1hiCD11b+ neutrophils was elevated, with a peak effect occurring 24 hours and then declining 72 hours postinjection. Prednisolone cotreatment resulted in a lower number of neutrophils 24 hours but a slightly higher number 72 hours post-CCl4 injection compared with the vehicle-cotreated mice. Such prednisolone-mediated inhibition of hepatic neutrophil number was not due to promotion of neutrophil apoptosis because FACS analyses showed that prednisolone cotreatment delayed liver neutrophil apoptosis (Supporting Fig. 11).
Finally, the hepatic expression of several cytokines and chemokines, which are associated with macrophage and neutrophil recruitment, was suppressed in the prednisolone-cotreated mice compared with those in the vehicle-cotreated groups (Fig. 2D).
Coadministration With Prednisolone Attenuates Macrophage and Neutrophil Functions
Macrophages and neutrophils are known to be professional phagocytes that play a significant role in the clearance of apoptotic cells and in the resolution of inflammation. To analyze the effect of prednisolone on macrophage and neutrophil phagocytic activity, mice were injected with latex beads post-CCl4 plus prednisolone or vehicle cotreatment. As illustrated in Fig. 3A,B, CCl4 injection increased the percentage of macrophage or neutrophil engulfing latex beads, which was significantly reduced in the prednisolone-cotreated group. Moreover, an in vitro phagocytosis assay (Fig. 3C,D) revealed that prednisolone exposure decreased the fluorescence intensity of latex beads in peritoneal macrophages and neutrophils.
Kupffer cells, liver resident macrophages, stimulate tissue damage and repair by secreting cytokines. Therefore, we examined whether prednisolone treatment altered cytokine production by Kupffer cells as well as hepatic stellate cells (HSCs) and hepatocytes. As illustrated in Supporting Fig. 12, Kupffer cells produced the highest levels of TNF-α and IL-6 after CCl4 treatment, followed by HSCs and hepatocytes. Such expression in Kupffer cells from CCl4 plus prednisolone-cotreated mice was much lower than those from CCl4 plus vehicle-cotreated mice. Expression of TNF-α but not IL-6 was also attenuated in HSCs from the CCl4 plus prednisolone group compared with the CCl4 plus vehicle.
Finally, the effects of prednisolone on neutrophil-mediated ROS production were also examined. As illustrated in Fig. 3E, without in vitro treatment with phorbol myristate acetate (PMA), neutrophils from CCl4-treated mice produced a slightly higher ROS burst compared with those from mice without CCl4 treatment. This ROS burst production was suppressed in prednisolone-cotreated mice compared with vehicle-cotreated mice 24 hours but not 72 hours post-CCl4 injection. In vitro incubation with PMA, which induces cells to undergo an NOX-dependent respiratory burst, markedly increased the ROS levels of neutrophils (Fig. 3E). This PMA-mediated elevation of ROS production was lower in neutrophils from prednisolone-cotreated mice compared with those from vehicle-cotreated mice 24 hours post-CCl4 injection (Fig. 3E).
Prednisolone Treatment Delays Liver Regeneration by Inhibiting Hepatic Signal Transducer and Activator of Transcription 3 (STAT3) and Nuclear Factor Kappa B (NF-κB) Activation in CCl4-Induced Acute Hepatitis
The effect of prednisolone on liver regeneration was examined to further understand why prednisolone treatment delayed liver repair post-CCl4 injection as observed in Fig. 1. As illustrated in Fig. 4A, CCl4 challenge markedly increased BrdU incorporation in hepatocytes, with a peak effect 48 hours postchallenge. However, prednisolone cotreatment delayed this peak to 72 hours.
We next investigated the mechanisms underlying the prednisolone-mediated interruption of liver regeneration in CCl4-induced acute hepatitis by examining the hepatic expression of pSTAT3, pNF-κB, and proliferative genes. As shown in Fig. 4B, hepatic STAT3 was activated, with a peak effect occurring at 3-12 hours after CCl4 injection in the CCl4+vehicle group. The hepatic expression levels of pSTAT3 were lower at 6 and 12 hours but higher at 24 to 72 hours after CCl4 injection in the CCl4+prednisolone group, which suggests that prednisolone treatment caused a delay in hepatic STAT3 activation. The hepatic expression of pNF-κB was also decreased at 3 and 6 hours in CCl4+prednisolone group compared with the CCl4+vehicle group (Fig. 4C). In addition, prednisolone treatment slightly reduced NF-κB acetylation at 6 hours post-CCl4 injection, but it did not affect STAT3 acetylation (Supporting Fig. 13). The induction of PCNA and cyclin D1 expression was delayed in the prednisolone-cotreated mice compared with the vehicle-cotreated mice (Fig. 4B).
The above findings suggest that prednisolone inhibits neutrophil and macrophage recruitment and functions; however, the roles of these inflammatory cells in CCl4-induced liver injury and regeneration remain largely unclear. To understand the functions of macrophages and neutrophils, we used anti-Gr1Ab antibody to deplete these cells prior to CCl4 challenge. As illustrated in Supporting Fig. 14A, pretreatment with anti-Gr1Ab markedly reduced both macrophages (Gr1intCD11b+) and neutrophils (Gr1hiCD11b+) in the liver. The depletion of both macrophages and neutrophils with anti-Gr-1 Ab induced greater elevation of serum ALT and liver injury (Supporting Fig. 14B), increased the area of necrosis (Supporting Fig. 14C), and delayed liver regeneration as determined by BrdU incorporation (Supporting Fig. 14D).
To further dissect the roles of macrophages and neutrophils, we used anti-Ly6G and clodronate to deplete specifically neutrophils and macrophages, respectively. As illustrated in Fig. 5A, treatment with anti-Ly6G depleted neutrophils (Gr1hiCD11b+) without affecting macrophages (Gr1intCD11b+) in the liver. Such treatment increased CCl4-induced elevation of serum ALT and liver necrosis, and delayed liver regeneration (Fig. 5B-D). Additionally, treatment with clodronate depleted F4/80 positive macrophages in the liver (Fig. 6A) and also exacerbated CCl4-induced elevation of serum ALT and liver necrosis but attenuated liver regeneration (Fig. 6B-D).
Treatment With Prednisolone Does Not Affect CCl4-Induced Liver Injury and Regeneration in mGR KO Mice
To further explore whether the detrimental effects of prednisolone on CCl4-induced hepatitis are mediated by targeting macrophages and/or neutrophils, we used myeloid-specific GR knockout (mGR KO) mice, in which the GR gene was deleted in macrophages and neutrophils. Western blot analyses confirmed a loss of GR protein expression in both macrophages and neutrophils from mGR KO mice (Fig. 7A). Moreover, the effects of prednisolone on CCl4-induced acute hepatitis were compared in WT and mGR KO mice. As illustrated in Fig. 7B, CCl4 treatment resulted in an elevation of serum ALT levels in WT mice, and such elevation was higher in prednisolone-cotreated WT mice 48 hours post-CCl4 injection. However, prednisolone cotreatment did not affect CCl4-induced elevation of serum ALT levels in mGR KO mice. Liver histology analyses in Fig. 7C show that prednisolone cotreatment increased CCl4-induced necrotic area in WT mice but did not affect this in mGR KO mice. Immunohistochemical analyses in Fig. 7D show that prednisolone cotreatment delayed neutrophil infiltration in CCl4-treated WT mice (the peak of neutrophil infiltration occurred 48 hours post-CCl4 injection alone but occurred 72 hours post-CCl4 plus prednisolone cotreatment). In contrast, prednisolone cotreatment did not have an effect on neutrophil infiltration in CCl4-treated mGR KO mice. Similarly, prednisolone cotreatment delayed liver regeneration in CCl4-treated WT mice but not in mGR KO mice (Fig. 7E).
Prednisolone has been used in the treatment of various types of liver diseases, especially autoimmune/inflammatory conditions in which the immune system is overactive. However, these treatments have varying degrees of responsiveness among individuals and in different types of liver diseases. This is partly because the effects of prednisolone treatment on liver disease pathogenesis remain unknown. In the current study, several novel findings were demonstrated. First, prednisolone treatment is very effective in preventing T/NKT cell hepatitis but exacerbates hepatotoxin-induced hepatitis and fibrosis. Second, prednisolone treatment inhibits macrophage- and neutrophil-mediated phagocytosis. Third, depletion of macrophages or neutrophils exacerbates liver injury and attenuates liver regeneration in CCl4-induced liver injury. Fourth, prednisolone treatment delays liver regeneration in CCl4-induced liver injury. We have integrated all of these findings into a model in Fig. 8.
ConA- and α-GalCer-induced hepatitis are mouse models of T/NKT cell-mediated liver injury that resemble autoimmune hepatitis in humans.[36, 37] Both models are characterized by the rapid activation of NKT and T cells and subsequent production of various proinflammatory cells and infiltration of neutrophils and macrophages, followed by hepatocyte necrosis and apoptosis.[36, 37] ConA activates a wide array of T cells regardless of their antigen specificity, and α-GalCer is not an endogenous lipid antigen for NKT cells; thus, neither ConA- nor α-GalCer-induced hepatitis represent autoimmune liver disease models in a strict sense. However, many features of autoimmune hepatitis are observed in these models. Thus, both of these models are often considered to represent autoimmune hepatitis models and are used to study the mechanisms underlying T/NKT cell-mediated liver injury and test the effectiveness of immunosuppressive drugs for the treatment of autoimmune hepatitis.[36, 37] Here we demonstrated that cotreating mice with prednisolone markedly prevented ConA- and α-GalCer-induced liver inflammation and injury (Supporting Figs. 1, 2. This finding is consistent with the experimental and clinical data that steroid therapy is effective in a mouse autoimmune hepatitis model of adenoviral infection and in patients with autoimmune hepatitis.[39, 40] The protective effects of prednisolone treatment on T/NKT cell hepatitis are likely mediated by its immunosuppressive function on T/NKT cell activation and cytokine production and the subsequent inhibition of T/NKT cell hepatitis.
In contrast to ConA- or α-GalCer-induced hepatitis, which is initiated by activated immune cells, hepatotoxin ethanol and/or CCl4-induced liver injury is instigated by the direct induction of hepatocyte damage, which subsequently activates and recruits macrophages/Kupffer cells and neutrophils. Activated macrophages/Kupffer cells produce free radicals and proinflammatory cytokines that further trigger hepatocellular damage and induce neutrophil accumulation and activation. The resulting neutrophil influx may promote additional tissue damage by way of the release of oxygen-reactive species and proteases.[10, 41] However, macrophages and neutrophils also play an important role in removing necrotic cell debris and activating regenerative pathways, ultimately facilitating tissue repair and the resolution of the inflammatory response.[13, 41-43] In the current study, we demonstrated that the depletion of macrophages and/or neutrophils markedly increased serum levels of ALT and hepatic necrotic areas but delayed liver regeneration in CCl4-induced acute hepatitis (Figs. 5 and 6; Supporting Fig. 14). These findings suggest that macrophage and neutrophil infiltration contribute to the removal of necrotic hepatocytes and the subsequent promotion of liver repair in this hepatotoxin-induced liver injury model.
Although prednisolone treatment markedly prevented T/NKT cell hepatitis, the same treatment delayed liver repair and exacerbated liver injury in response to CCl4, APAP, ethanol, or ethanol plus CCl4. Given our above-mentioned findings that macrophages and neutrophils play important roles in promoting liver repair (Figs. 5 and 6; Supporting Fig. 14), the inhibition of macrophage and neutrophil infiltration and their functions is the major mechanism contributing to the detrimental effect of prednisolone in hepatotoxin-induced hepatitis. In addition, prednisolone treatment markedly reduced serum levels of TNF-α and IL-6 (Fig. 1) and inhibited the hepatic activation of the major TNF-α and IL-6 downstream signaling pathway STAT3 and NF-κB, respectively (Fig. 4), which likely contributed to the inhibitory effect of prednisolone on liver regeneration because these factors promote liver regeneration.[33, 34]
Although GR is expressed ubiquitously in many cell types, we provided evidence suggesting that the detrimental effect of prednisolone on CCl4-induced hepatitis is mediated, at least in part, by targeting GR in macrophages and neutrophils. First, prednisolone cotreatment exacerbated CCl4-induced liver injury in WT mice but not in mGR KO mice, in which the GR gene is deleted in macrophages and neutrophils (Fig. 7A). Second, the inhibitory effects of prednisolone on neutrophil infiltration and liver regeneration in CCl4-treated WT mice were not observed in mGR KO mice (Fig. 7B-E). Third, in vitro treatment with prednisolone directly inhibited macrophage and neutrophil phagocytic functions (Fig. 3). Collectively, these findings suggest that prednisolone targets GR in macrophages and neutrophils, and subsequently inhibits their phagocytic and regenerative functions, thereby exacerbating hepatotoxin-induced liver injury.
To our knowledge, this is the first study to extensively examine the effects of the glucocorticoid drug prednisolone on liver injury and inflammation in several mouse models. Our findings may help us to not only understand the effects of steroid therapy on liver disease pathogenesis and progression but also select steroid therapy for liver disease treatments. For example, prednisolone treatment effectively prevented ConA- and α-GalCer-induced hepatitis, which are two models that resemble human autoimmune hepatitis.[36, 37] These findings are in agreement with other clinical results that demonstrate that steroids are effective in many patients with this disease when they are included in the standard treatment regimens of autoimmune hepatitis. In contrast, reports regarding steroid therapy for alcoholic hepatitis have been controversial.[15-20] Alcoholic hepatitis is a syndrome characterized by inflammatory cell infiltration in the liver (mainly neutrophils) and hepatocellular injury; however, the mechanisms underlying the development of alcoholic hepatitis remain obscure, and there are no animal models of the severe form of this disease.[1, 2, 9] Alcohol is a hepatotoxin that is primarily metabolized by hepatocytes. This metabolism produces oxidative stress and inflammation, followed by hepatocellular damage and neutrophil infiltration, mechanisms that are similar to those involved in hepatotoxin CCl4-induced liver damage. Therefore, inflammation is likely not the primary source of hepatocellular damage in alcoholic hepatitis, and it may occur as a result of hepatocyte necrosis and contribute to tissue repair and the healing process. If this is the case, steroid treatment may exacerbate liver injury and delay liver regeneration in alcoholic hepatitis. However, steroid treatment may retain some beneficial effects in attenuating the systemic inflammatory responses and improving short-term survival in patients with severe alcoholic hepatitis.[15, 16] Interestingly, patients with alcoholic hepatitis often have increased levels of circulating antibodies against lipid peroxidation adducts and an increased number of T cells in the liver, suggesting that autoimmune responses may contribute to the pathogenesis of alcoholic hepatitis.[44, 45] Steroid therapy may be effective if autoimmune responses are the primary source of liver injury in alcoholic hepatitis. Future studies are urgently needed to identify the major mechanisms that contribute to hepatocellular damage in alcoholic hepatitis, which may help us to create a more effective strategy using steroids to treat these patients.
We thank Dr. Philippe Mathurin (Université Lille 2, CHRU Lille, Lille, France) and Dr. Michael Lucey (University of Wisconsin, Madison) for suggestions during this study, and Dr. Louis J. Muglia (University of Cincinnati) for providing myeloid-specific GR KO mice.