Nonalcoholic steatohepatitis (NASH) is the most common etiology of chronic liver dysfunction in the United States and can progress to cirrhosis and liver failure. Inflammatory insult resulting from fatty infiltration of the liver is central to disease pathogenesis. Dendritic cells (DCs) are antigen-presenting cells with an emerging role in hepatic inflammation. We postulated that DCs are important in the progression of NASH. We found that intrahepatic DCs expand and mature in NASH liver and assume an activated immune phenotype. However, rather than mitigating the severity of NASH, DC depletion markedly exacerbated intrahepatic fibroinflammation. Our mechanistic studies support a regulatory role for DCs in NASH by limiting sterile inflammation through their role in the clearance of apoptotic cells and necrotic debris. We found that DCs limit CD8+ T-cell expansion and restrict Toll-like receptor expression and cytokine production in innate immune effector cells in NASH, including Kupffer cells, neutrophils, and inflammatory monocytes. Consistent with their regulatory role in NASH, during the recovery phase of disease, ablation of DC populations results in delayed resolution of intrahepatic inflammation and fibroplasia. Conclusion: Our findings support a role for DCs in modulating NASH. Targeting DC functional properties may hold promise for therapeutic intervention in NASH. (HEPATOLOGY 2013;58:589–602)
Nonalcoholic fatty liver disease (NAFLD) is the hepatic consequence of metabolic syndrome, which includes insulin resistance, hypertension, hyperlipidemia, and visceral adiposity. Obesity itself is an independent risk factor for NAFLD, which is currently recognized as the most common cause of liver dysfunction in the United States, representing 75% of all cases of chronic liver disease (CLD). Moreover, future projections estimate that 50% of all Americans will have elements characteristic of NAFLD by 2030. In most cases of NAFLD, liver steatosis is mild and reversible; however, 10%-20% of cases progress to nonalcoholic steatohepatitis (NASH), characterized by intense intrahepatic inflammation, exacerbated steatosis, hepatocellular injury, and incipient fibrosis. Furthermore, NASH can progress to cirrhosis, liver failure, and hepatocellular carcinoma. Between 2000 and 2010, the percentage of orthotopic liver transplants performed for NASH in the United States increased from 1.2% to 7.4%.
The precise cellular and biochemical pathogeneses of NASH are incompletely understood. However, a “two-hit” hypothesis has been gaining experimental traction. In general terms, hepatic lipid accumulation, the “first hit,” is thought to induce oxidative stress and hepatocyte damage, which subjects the liver to inflammatory cell infiltration—the “second hit”—leading to the cyclical development of further inflammatory injury and eventual fibrosis. A number of inflammatory mediators have been implicated. Kupffer cells (KCs) reside in liver sinusoids and contribute to hepatocyte cell death by Toll-like receptor (TLR)9-mediated production of interleukin (IL)-1β. TNF-α production by activated KCs is essential for fibrosis development in NASH. Moreover, NASH is mitigated in mice fed a methionine-choline–deficient (MCD) diet in the absence of KCs. Neutrophils are also important mediators of hepatocellular damage in NASH. Neutrophils are activated by necrotic hepatocytes and perpetuate hepatitis through the release of proinflammatory cytokines and the secretion of myeloperoxidase (MPO), an abundant source of free radicals that contributes to disease progression by increasing oxidative hepatocyte damage. An increased liver neutrophil/lymphocyte ratio has been shown to increase the likelihood of progression of steatosis to steatohepatitis and, ultimately, fibrosis in patients with NASH.
Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that initiate potent adaptive immune responses. DCs have also recently emerged as important mediators in noninfectious chronic fibroinflammatory conditions. For example, DCs modulate the severity of inflammation during exacerbations of asthma and are necessary for bleomycin-mediated pulmonary fibrosis. Mucosal DCs in the small and large intestine are thought to be responsible for triggering deleterious T-cell responses to the endogenous microflora in inflammatory bowel disease. We recently showed that, despite their activated phenotype, DCs can have a protective role in acute pancreatitis by limiting sterile inflammation. The role of DCs in CLD is incompletely defined. We reported that DCs become highly proinflammatory in thioacetamide-induced chronic liver fibrosis. However, the resolution of murine liver fibrosis was recently found to be accelerated by the recruitment of DCs. In NASH liver, our initial investigations uncovered a robust recruitment of phenotypically activated DCs early in disease. Based on these data, we postulated that DCs augment the cycle of inflammation in NASH. However, our investigations, utilizing continuous in vivo depletion of DC populations, revealed a more-complex relationship, because DCs limit fibroinflammation in NASH by curtailing the destructive effects of KCs and neutrophils. Furthermore, during the recovery phase of disease, DC depletion delays the resolution of intrahepatic inflammation and fibroplasia. This work offers novel insight to the pathogenesis and resolution of NASH and has potential implications for targeting DCs in experimental therapeutics.
This is the first investigation to report a significant role for hepatic DCs in NASH. We demonstrated that DCs are recruited to the liver soon after MCD diet initiation, plateau at 3-4 times normal levels by 2 weeks, and remain at an elevated level, unless there is disease resolution. NASH DCs exhibit an activated surface phenotype and increase their production of proinflammatory cytokines. Consistent with their mature phenotype, our in vitro experimentation shows that NASH DCs potently induce proliferation of both allogeneic T cells and antigen-restricted CD4+ T cells while reducing CD4+ T-cell expression of the CD25+FoxP3+ Treg phenotype. The finding of intrahepatic DC activation after hepatic insult is consistent with our previous reports showing immunogenic transformation of liver DCs in thioacetamide-induced liver fibrosis and acute hepatic injury induced by acetaminophen overdose.[12, 21] However, despite their phenotypic and functional activation, ablation of DC in NASH results in increased hepatic inflammation, diminished numbers of Tregs, expansion of CD8+ T cells, enhanced viability and production of proinflammatory cytokines by immune effector cells, increased hepatocyte apoptosis, and, ultimately, accelerated liver fibrosis. These ostensibly paradoxical findings are not entirely unprecedented. Recent studies have shown that, despite adopting a proinflammatory phenotype, hepatic DCs can accelerate the regression of hepatic fibrosis and ameliorate hepatic ischemia-reperfusion (I/R) injury.[13, 28] For example, exogenous expansion of hepatic DC populations by Flt3 ligand administration accelerates the regression of CCl4-induced liver fibrosis, despite phenotypic activation of DCs.
Our findings show that effects of DC contrast sharply with the role of KCs, whose expansion have been strongly linked to worsening intrahepatic fibroinflammation in NASH. Our investigations suggest multiple parallel mechanisms by which DC may regulate hepatitis. Importantly, we found that DCs in NASH liver are differentially capable of activating CD4+ T cells, in comparison with CD8+ T cells. Furthermore, upon DC depletion, the CD8/CD4 T-cell ratio is skewed markedly upward with associated diminution of Tregs. The protective role of Tregs in CLD is well established.[29, 30] Furthermore, relative suppression of CD8+ T-cell expansion may be protective, because CD8+ T cells have recently been shown to drive adipose tissue inflammation and have an emerging role in NASH pathogenesis.[31, 32]
Additionally, the exacerbated hepatic insult associated with ablation of DC populations may be mechanistically related to the DC's role in limiting sterile inflammation through clearance of apoptotic bodies and necrotic debris. Sterile inflammation in the liver increases recruitment, viability, and activation of innate immune cells. We show that liver DCs express high CLEC9A, which recognizes and binds death signals on necrotic cells and is primary in DC capacity to clear necrotic products.[26, 27] Accordingly, we found that NASH liver DCs have remarkable capacity to capture necrotic cellular debris and apoptotic targets, when compared to other hepatic APC subsets and DCs from control liver. Furthermore, we found that DC depletion leads to an accentuation of sterile inflammation within the liver, because NASH(-DC) liver contains modestly higher HMGB1 and elevated markers of apoptosis, including p53, which has been demonstrated to play a pivotal role as a mediator of apoptosis in experimental NASH. This also results in augmented production of proinflammatory cytokines—including IL-1β, TNF-α, and IL-6—and enhanced viability and expression of TLR4 and TLR9 in innate effector cells. Miura et al. demonstrated that signaling through TLR9 leads to progression of NASH by KC production of IL-1β. TLR4 signaling in KCs has also been linked to severity of steatohepatitis.
DC production of IL-10 may also have an important role in limiting hepatic damage in NASH. Bamboat et al. recently showed that DNA released from apoptotic hepatocytes stimulates liver DC to secrete IL-10 in a TLR9-dependent manner. Furthermore, IL-10 derived from hepatic DCs can ameliorate liver injury through suppression of inflammatory monocyte function. Additional studies in contexts such as allergen-induced asthma and cisplatin-induced nephrotoxicity have shown that DCs attenuate sterile inflammation through release of IL-10.[34, 35] We found that NASH DCs exhibited markedly elevated IL-10 production, compared to normal liver DCs. Moreover, IL-10 production by NASH NPC was reduced by 40%-60% in the absence of DCs, suggesting that DC production of IL-10 may have an important regulatory role in NASH.
Interestingly, despite showing varied evidence of increased inflammation, fibrosis, hepatocyte apoptosis, and delayed recovery from NASH upon DC depletion, we did not find significant elevations in serum ALT in NASH(-DC), compared with NASH mice with intact DC populations. However, this is consistent with previous reports showing that the severity of NASH may not correlate with serum ALT levels.[36, 37] Furthermore, clinically severe NASH can exist without overt elevations in serum ALT. These studies suggest that ALT alone cannot be used as a “hard endpoint” in NASH.
Numerous studies have used the CD11c.DTR model to investigate the role of DCs in diverse inflammatory conditions within the liver, including I/R injury and acute acetaminophen hepatotoxicity.[21, 28] Similarly, the CD11c.DTR model has been useful in determining the role of DCs in many extrahepatic diseases, including allergic asthma, acute lung injury, pancreatitis, and renal I/R injury.[11, 28, 39] However, a sobering report by Tittel et al. recently showed that DC depletion in CD11c.DTR mice is associated with an early nonspecific neutrophilia in multiple organs, including a modest neutrophilia within the liver, implying that conclusions drawn using the CD11c.DTR model may be confounded by nonspecific effects. The mechanism for the reported neutrophilia in CD11c.DTR mice depleted of DCs remains uncertain. However, we did not observe unintended changes in leukocyte composition in BM chimeric CD11c.DTR mice upon DC depletion that were independent of NASH. Possible explanations for our disparate results may be that the BM chimeric CD11c.DTR model is not affected by the neutrophilia associated with the endogenous model. Endogenous CD11c.DTR mice are distinct from the chimeric model in that repeated administration of diphtheria toxin is lethal. Furthermore, chronic DC depletion as in NASH may not cause the neutrophilia associated with acute single-dose depletion. Nevertheless, the CD11c.DTR model, though it is the best available tool to study the role of DCs in vivo in mice, is not the perfect model because the effects of DC depletion may not necessarily faithfully mimic the role of DC in situ. Thus, additional insight on the role of DCs in NASH and other inflammatory diseases may be forthcoming pending the advent of additional experimental tools to study DC effects in vivo.
In summary, our data suggest that DCs have complex influences on both the pathogenesis and resolution of steatohepatitis, which may have implications to human disease. However, a limitation of our study is that there is no perfect murine model of NASH mimicking human disease. Additionally, direct comparison of the current data, even to other murine studies of NASH employing an MCD diet, may be confounded by alternate durations of treatment between studies. That is, because the development of NASH, as well as its resolution, is a dynamic process, examining the intrahepatic phenotype and immune milieu after varied durations of feeding mice an MCD diet may yield inconsistent findings. Furthermore, interrupting the pathogenesis of NASH by targeting DCs in experimental therapeutics may prove challenging, given the technical limitations in modulating human DC function in vivo. Thus, additional investigations are needed to evaluate the clinical utility of these findings in treating patients with NASH or preventing disease onset.