Although nonalcoholic fatty liver disease (NAFLD) is conventionally assessed histologically for lobular features of inflammation, development of portal fibrosis appears to be associated with disease progression. We investigated the composition of the portal inflammatory infiltrate and its relationship to the ductular reaction (DR), a second portal phenomenon implicated in fibrogenesis. The portal inflammatory infiltrate may contribute directly to fibrogenesis as well as influence the fate of the DR hepatic progenitor cells (HPCs), regulating the balance between liver repair and fibrosis. The presence of portal inflammation in NAFLD was strongly correlated with disease severity (fibrosis stage) and the DR. The portal infiltrate was characterized by immunostaining NAFLD liver biopsy sections (n = 33) for broad leukocyte subset markers (CD68, CD3, CD8, CD4, CD20, and neutrophil elastase) and selected inflammatory markers (matrix metalloproteinase 9 and interleukin [IL]-17). Cells expressing all markers examined were identified throughout the liver lobules and in portal tracts, although portal tracts were more densely populated (P < 0.01), and dominated by CD68+ macrophages and CD8+ lymphocytes, at all stages of disease. An increase in portal macrophages in NAFLD patients with steatosis alone (P < 0.01) was the earliest change detected, even before elevated expression of the proinflammatory cytokines, IL1B and TNF, in patients with early NASH (P < 0.05). Portal and periductal accumulation of all other cell types examined occurred in progressed NASH (all P < 0.05). Conclusion: Knowledge of the complex cellular composition of the portal inflammatory infiltrate and HPC/DR niche in NAFLD will shape future functional studies to elucidate the contribution of portal inflammation to HPC differentiation and NAFLD pathogenesis. (Hepatology 2014;59:1393-1405)
Nonalcoholic fatty liver disease (NAFLD) has become the most common cause of chronic liver injury, yet only a proportion of patients progress to cirrhosis, liver failure, and hepatocellular carcinoma. NAFLD is conventionally assessed histologically for the presence of lobular features of injury, including hepatocyte ballooning, Mallory bodies, zone 3 inflammation, and perisinusoidal fibrosis. However, recent studies have identified the importance of portal fibrosis in predicting the subgroup of NAFLD patients that develop progressive liver disease and liver-related mortality.[1, 2] Clearly, identifying the mechanisms responsible for development and progression of portal fibrosis in fatty liver disease is critically important. In this regard, portal inflammation is observed in the majority of subjects with nonalcoholic steatohepatitis (NASH) and was acknowledged as a component of the inflammatory score in the original NAFLD grading and staging system of Brunt et al. Mild portal inflammation correlates with more severe liver disease[4, 5] as well as with clinical features associated with risk of progressive disease, such as increased age, body mass index (BMI), and markers of insulin resistance.[4, 5]
Few data are available regarding phenotype of the portal inflammatory infiltrate or the stimuli for its presence. Previous studies describe a mixed infiltrate of lymphocytes, neutrophils, monocytes, and occasional eosinophils,[4, 6] but the relative numbers of different cells and their relationship to disease progression are unknown. The degree of portal inflammation does not correlate with grade of lobular inflammation, but is associated instead with portal-based changes, such as a ductular reaction (DR).[7, 8] The DR, a reactive lesion at the portal tract interface comprising small biliary ductules with an accompanying complex of stroma and inflammatory cells, develops when hepatocyte regeneration is impaired and hepatic progenitor cell (HPC) proliferation takes over. HPCs are bipotential and capable of proliferation and differentiation into hepatocytes, to replace injured cells, or into cholangiocytes. HPC activation and a DR are common responses to chronic liver injury, including NAFLD, and are thought to precede progressive, portal fibrosis. We previously demonstrated a correlation between the DR and portal inflammation in NAFLD, and both factors were independently associated with stage of fibrosis.
Within portal tracts, inflammatory cells and their mediators influence the differentiation and fate choice of HPC, which, in turn, may determine the balance between liver repair and fibrogenesis. The portal inflammatory infiltrate may contribute directly to fibrogenesis through release of profibrogenic cytokines or play a key role in determining HPC fate toward a fibrogenic DR.[11-13] To identify the potential cellular sources of local fibrogenic mediators, we characterized the cellular profile of the portal and periductal inflammatory infiltrate in patients with NAFLD and analyzed the data with respect to histological and clinical features as well as whole-liver cytokine and chemokine expression. A cohort of patients with alcohol-related liver disease (ALD) was also assessed for comparison.
Although portal inflammation is neither a diagnostic criterion for NAFLD nor a component of the NAS, it is reported to be associated with clinical and histological features of advanced disease. The current study was undertaken to profile the cells comprising the portal inflammatory infiltrate and their association with the DR and portal fibrosis. The findings demonstrate that a mix of inflammatory cells, including T cells, B cells, macrophages, and neutrophils, accumulate within portal tracts of NASH patients with portal fibrosis, supporting a role for portal inflammation in progressive disease. Despite differences in the underlying etiology of disease, the portal inflammatory infiltrate was similar in NASH and ASH patients, suggesting that these diseases may share inflammatory and fibrogenic mechanisms.
An important new finding of this study is the significant increase in portal macrophages in subjects with steatosis alone. In contrast, other inflammatory cell numbers did not show a significant increase over control subjects until portal fibrosis was present. The early accumulation of macrophages, and elevated expression of the proinflammatory cytokine mRNAs, TNF and IL1B, supports an initiating role for the innate immune system in NAFLD, but the specific stimulus for its activation is unclear. Portal macrophage accumulation was further amplified in steatohepatitis and was positively associated with hepatocyte ballooning and lobular inflammation, but not with the extent of steatosis or senescent hepatocyte burden. So, whereas lobular injury could plausibly trigger macrophage activation, the factors that recruit or retain macrophages, and subsequently other immune cells, specifically in portal tracts are unknown. Candidate chemoattractants include CCL2/MCP-1, chemokine (C-X3-C motif) ligand 1, and chemokine (C-X-C motif) ligand 12, which have been shown to be produced by inflamed biliary epithelia or proliferating ductules in inflammatory liver diseases.[27-29] We observed elevated CCL2 expression in early-stage NASH, consistent with the early accumulation of portal macrophages. The CCL2/CCR2 (C-C chemokine receptor 2) axis has been implicated in the pathogenesis of CLD in some mouse models, but seems to be irrelevant in others.[30, 31] Elevated hepatic and systemic CCL2 have been reported in human CLD, including NASH. However, as we previously demonstrated in chronic HCV infection, lobular, but not portal, macrophages expressed CCR2 in NASH (data not shown). This may suggest that CCR2 is not important for portal macrophage homing in human CLD or simply that the receptor is dynamically regulated in the inflammatory environment.
Although the most significant portal accumulation of inflammatory cells occurred in progressed NASH, the activated hepatic environment that arises early in disease—as evidenced by proinflammatory cytokine expression—likely influences the phenotype and function of resident cells. Having identified MMP-9 as a marker of a subpopulation of portal macrophages associated with fibrosis in chronic HCV infection, we investigated its expression in NASH. MMP-9+ macrophages were present in portal tracts of NASH patients, but, in contrast to HCV, also formed crown-like structures around large steatotic hepatocytes in centrilobular regions. MMP-9 functions in multiple pathways have been implicated in fibrogenesis and its resolution, and may play different roles at different stages of disease.[33, 34] The role of MMP-9 in NASH progression is not known. However, the different localization of MMP-9+ macrophages in NASH and HCV may correspond to different patterns of fibrosis in the two diseases, supporting the conclusion that MMP-9 acts locally to regulate tissue remodeling.
Portal inflammation can persist after surgical or drug treatment for NAFLD, even as steatohepatitis and fibrosis improve.[35-37] After rosiglitazone therapy, the ratio of portal to lobular inflammation increased, but actual portal inflammation severity was not specified. Others found that semiquantitative portal inflammation scores did not change significantly.[36, 37] Currently, it remains unclear how treatment of NASH affects the extent and composition of portal inflammation or whether inflammation associated with the DR changes. This warrants further study. Additionally, although the current study suggests that portal, rather than lobular, inflammatory populations correlated more closely with fibrosis, until changes in portal inflammation post-therapy are better understood, it cannot yet be used as an endpoint in clinical trials of NAFLD treatment.
Portal, but not lobular, inflammatory cell populations were significantly associated with extent of periportal DR. Inflammatory cells form an important component of the DR—a complex of matrix and cells, including cholangiocytes, stem cells, and myofibroblasts. Inflammatory cells were not only present in the periductular niche, but also made direct contact with strings of K7-positive, immature HPCs. Cellular cross-talk between leukocytes, matrix, and other cells in the ductular niche may have a pivotal role in regulating both fibrogenesis and liver repair. Although HPCs are at least bipotential, capable of differentiating into hepato- and cholangiocytes, their capacity for epithelial to mesenchymal transition is controversial. The context of the inflammatory niche may determine the outcome of their activation. In mouse liver injury models, for example, the capacity of activated HPCs to replace injured hepatocytes and restore liver function was dependent upon the type of liver injury and resulting DR. The factors underpinning this heterogeneity are not known, but several candidates have been identified, some of which are produced by macrophages, including TWEAK (TNF-like weak inducer of apoptosis)[13, 40] and WNT proteins. Other inflammatory cell populations colocalizing with expanding and migrating HPCs are also likely to contribute regulatory and effector cytokines, including IL-17+ cells, NK cells, and T lymphocytes.[22, 25, 26, 41]
Whether HPC activation and proliferation directly drives portal fibrosis as well as liver regeneration is controversial. In a recent study of liver regeneration in mice with carbon-tetrachloride–induced fibrosis, HPC expansion was accompanied by increased profibrogenic gene expression and de novo collagen deposition. Blocking the HPC mitogen, TWEAK, inhibited HPC proliferation, prevented fibrogenic response, and increased hepatocyte replication. However, it is unclear whether HPC and stellate cell activation and proliferation are interdependent or whether they occur in parallel as a result of a common stimulus (e.g., TWEAK) or to secretion of profibrogenic cytokines and growth factors from the DR or surrounding inflammatory cells.
In summary, we show that early macrophage infiltration and subsequent portal inflammation are key features of NAFLD progression. Portal, but not lobular, inflammatory cell populations were significantly associated with stage of fibrosis, suggesting that recruitment and consequences of portal and lobular inflammation should be considered separately, because different immunopathogenic processes may be involved. Understanding the cellular composition of the periductular niche and the cross-talk occurring within this niche will help shape functional studies to elucidate the cellular and molecular mediators of HPC differentiation, as well as the role of HPCs in liver fibrosis and repair.