Won-Il Jeong, Lab of Liver Research, GSMSE/KAIST, 335 Gwahangno, Building E7, Room 8107, Daejeon 305-701, Korea. Email: email@example.com
Activated hepatic stellate cells (HSCs) have been considered as a major type of cells in liver fibrosis by producing a huge amount of extracellular matrix, especially collagen fibers, and profibrotic mediators such as transforming growth factor-beta, interleukin-6 and monocyte chemoattractant protein-1. Recently, accumulated evidence suggests that the liver is an immunologic organ because of enrichment of diverse types of immune cells and that their interactions with HSCs are closely related with the progression of liver fibrosis. However, the underlying mechanisms of interaction between HSCs and immune cells remain largely unknown. Recently, several studies have demonstrated that natural killer cells, M2 macrophages, regulatory T cells, and bone marrow derived CD11b+Gr1+ immature cells ameliorate liver fibrosis, whereas neutrophils, M1 macrophages, CD8 T cells, natural killer T cells and interleukin-17-producing cells accelerate liver fibrosis. However, there are still controversial issues about their functions during liver fibrogenesis. In this review, we summarize the diversity roles of immune cells (e.g. profibrotic/antifibrotic or both) in regulating the activation of HSCs during hepatic fibrogenesis, in which several producible mediators by HSCs play important roles in the interaction with them. Moreover, the current cell-based therapies using immune cells against liver fibrosis are discussed.
Liver fibrosis is well characterized by abnormal accumulation of extracellular matrix (ECM) and HSCs are considered as a major type of cells responsible for liver fibrosis. Generally, HSCs are located in the space between hepatocytes and sinusoidal endothelial cells. Under normal condition, quiescent HSCs store retinol (vitamin A) lipid droplets in their cytoplasm, whereas activated HSCs during liver injury lose their droplets and become myofibroblast-like cells producing a huge amount of ECM, especially collagen fibers, and expressing alpha-smooth muscle actin, subsequently leading to liver fibrosis. After liver injuries, inflammatory cytokines released by several cell types including HSCs play a crucial role in liver fibrosis. Among those cytokines, platelet-derived growth factor and transforming growth factor (TGF)-β1 are the most powerful mitogen and fibrogenic effector to HSCs, respectively. In addition, many recent studies suggest that HSCs have immunoregulatory roles by secreting chemokines such as monocyte chemoattractant protein-1 (MCP-1), regulated and normal T cell expressed and secreted (RANTES), and macrophage inflammatory proteins (MIPs), expressing toll-like receptors (TLRs) and chemokine receptors including CCR5, CCR7, CXCR3, and CXCR7, and functioning as antigen presenting cells.[1, 2] Moreover, phagocytosis of apoptotic lymphocytes by HSCs contributes to the enhanced activation of HSCs, whereas the fusion of T cell microparticles with cell membrane of HSCs induces up-regulation of fibrolytic genes in HSCs leading to down-regulation of procollagen α1 messenger RNA and blunting of activities of transforming growth factor-beta 1 (TGF-β1).[3, 4] Furthermore, activation of TLR4 signaling pathway in HSCs promotes liver fibrosis by enhanced TGF-β signaling. Therefore, these studies implicate that not only interaction between HSCs and immune cells but also immune signaling pathways within HSCs play important roles in liver fibrogenesis.
Immunologic organ of the liver
Recent emerging reports have suggested that the liver is an immunologic organ in humans and rodents because of its structure, location, and function.[6-9] Generally, the liver consists of parenchymal cells (hepatocytes) and non-parenchymal cells enriched with innate and adaptive immune cells. For example, approximately 60–80% of the hepatic cell number is composed of hepatocytes, and the remaining 20–40% is non-parenchymal cells including endothelial cells, Kupffer cells, lymphocytes, biliary cells, and HSCs. Among non-parenchymal cells, endothelial cells and Kupffer cells play important roles in the elimination of wastes and antigen presenting by engulfing wastes and expressing major histocompatibility complex (MHC) and co-stimulated molecules, respectively.[6, 7] Endothelial cells usually remove soluble macromolecules via endocytosis, whereas Kupffer cells are responsible for the elimination of insoluble wastes via phagocytosis. Especially, Kupffer cells, consisting of about 20% of non-parenchymal cells, are activated by circulating diverse stimuli of blood through various receptor systems (e.g. pattern recognition receptors), subsequently inducing inflammation.[7, 9] In addition, liver innate lymphocytes such as natural killer (NK), NKT, and γδ T cells are abundant in the liver compared with those of peripheral blood, and they are comprising up to 50% of whole liver lymphocytes, implicating that the liver is an another special site of recognizing invading antigens.[7, 8] The immune responses and priming of CD4+ and CD8+ T cells against liver-trophic microorganisms also occurred in the liver.[6, 9] Intriguingly, these immune cells in the liver are also involved in the pathogenesis of liver fibrosis, which are discussed in this review.
Pro-fibrotic immune cells
Hepatic Kupffer cells/resident macrophages have been implicated as key mediators of liver fibrosis through production of various cytokines such as tumor necrosis factor-alpha (TNF-α), TGF-β1, monocyte chemotactic protein-1 (MCP-1), and other inflammatory mediators, which can activate HSCs during liver fibrogenesis. In addition, TLR4-Myd88-NF-kB signaling plays a key role in enhancing interaction between HSCs and Kupffer cells, in which MCP-1 and its receptor C-C chemokine receptor 2 (CCR2) play critical roles not only in the infiltration of macrophages and but also in the activation of HSCs in injured liver.[11, 12] Mutated MCP-1 significantly reduced dimethylnitrosamine-induced liver fibrosis by inhibiting infiltration of macrophages and by reducing TGF-β1 production, leading to suppressed activation of HSCs. The pro-fibrotic roles of MCP-1 are also supported by findings from experiments using mice deficiency in its receptor CCR2 in murine liver fibrosis models induced by bile duct ligation or carbon tetrachloride (CCl4) injection.
Liver inflammation coincides with liver fibrosis, and immigrating leukocytes, especially neutrophils, can potentiate the progression of liver fibrosis.[13-15] In co-culturing system, neutrophil-derived reactive oxygen species stimulates collagen synthesis in human HSCs, whereas treatment with various antioxidants attenuates it. In addition, activating rat HSCs can recruit neutrophils by producing neutrophil-attracting chemokines such as MIP-2 and cytokine-induced neutrophil chemoattractant.[14, 15] Furthermore, recent studies suggest that interleukin-17 (IL-17) produced by several type cells including neutrophils has potent roles to recruit and activate neutrophils, which is closely related with liver fibrosis of both human and mice.[16-18]
Activation of HSCs and liver fibrosis are negatively or positively regulated by lymphocyte population or its inflammatory cytokines such as IL-10 and IL-17, respectively. For example, increased numbers of CD8+ T cells and decreased CD4+/CD8+ ratio are associated with induction of liver fibrosis in mice and human.[19, 20] Adoptive transfer of CD8+ T cells to SCID mice showed more liver injury and fibrosis induced by CCl4 than those of mice transferred with CD3+ or CD4+ T lymphocytes, whereas CD8+ T cell-mediated liver fibrosis was attenuated by IL-10. In terms of the effects of CD4+ T cells on liver fibrosis, the role of IL-17-producing CD4+ T cells (Th17 cells) has been extensively investigated in the pathogenesis of liver fibrosis. IL-17 cytokines including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (IL-25), and IL-17F are central players not only in various adaptive immune responses to certain pathogens but also in autoimmune diseases. Except IL-17E, IL-17 family cytokines can be produced by Th17 cells (dominant cell type), CD8+ T cells, γδ T cells, NK cells, and neutrophils. Interestingly, recent several studies have suggested that IL-17 plays important roles in exacerbating liver fibrosis in both human and mice.[17, 18] These studies demonstrated that IL-17-stimulated human HSCs recruited neutrophils via chemokine production such as IL-8 and GROα, and it directly stimulated collagen production in primary murine HSCs and human HSC cell line LX-2 via STAT3 activation, leading to accelerated liver fibrosis. In summary, IL-17 and its producing CD4+ T cells are involved in promoting the liver fibrogenesis via several mechanisms.
NKT cells are a subtype of lymphocytes that shares cell surface receptors of both NK and T cells. Mouse liver lymphocytes contain approximately 30% NKT cells, while human liver lymphocytes contain up to 10%.[7, 22] Recent studies demonstrate that NKT cells promote liver fibrosis by producing inflammatory cytokines such as IL-4 and IL-13, leading to activation of HSCs in several murine models including HBV transgenic mice and xenobiotics-induced liver injury.[23-25] However, there are still several debates in the role of NKT cells on liver fibrosis because of anti-fibrotic cytokine production and cytotoxicity of NKT cells against activated HSCs. Thus, further studies will be required to determine the effects of NKT cells.
Bone marrow (BM) cells
Over the past decade, many studies have suggested that BM-derived cells migrating into fibrotic liver tissue promote liver fibrogenesis.[26-29] In mice and humans, BM-derived cells may transdifferentiate into collagen-producing myofibroblasts in hepatotoxin-induced mouse liver fibrosis model and in patients with hepatitis virus-derived fibrosis.[26, 27] In addition, BM-derived fibrocytes also contribute to bile duct ligation-induced liver fibrosis in mice, while HSCs are not originated from BM cells. Furthermore, adoptive transfer of Gr1+ monocyte subset isolated from BM cells promoted CCl4-treated liver fibrosis of mice via direct activation of HSCs in a TGF-β-dependent manner. In contrast, other types of BM cells have shown to ameliorate liver fibrosis, which is discussed later.
Anti-fibrotic immune cells
Recently, we and other groups have suggested that hepatic NK cells play a negative regulatory role in liver fibrosis in mice.[30-33] During liver fibrogenesis, NK cells can interact with activated HSCs via retinoic acid early inducible gene-1/NKG2D- or activating/inhibitory killer immunoglobulin receptor/MHC class I-dependent manners,[30, 31] leading to kill or suppress activated HSCs by modulating the production of NK cell-mediated tumor necrosis factor-related ligand (TRAIL) and interferon-γ.[30, 32] Although NK cells inhibit liver fibrosis by producing IFN-γ, which induces HSC apoptosis and cell cycle arrest, a clinical trial reported that treatment of IFN-γ showed no beneficial effects on patients with advanced liver fibrosis. This discrepancy was elucidated by our recent study that in contrast to early activated HSCs, intermediately activated HSCs in advanced liver fibrosis were resistant to NK cell killing and interferon-γ treatment because of retinoic acid-mediated TGF-β production and suppressor of cytokine signaling (SOCS) 1 expression of HSCs, respectively. In addition, several papers show human NK cells kill human HSCs, thereby inhibiting liver fibrosis in patients.[35, 36] Isolated NK cells from HCV-infected patients efficiently induce apoptosis of activated HSCs in TRAIL-, FasL-, and NKG2D-dependent manners. NKp46high NK cell subset potentially suppresses HCV replication and HCV-associated liver damage, leading to amelioration of liver fibrosis. However, chronic alcohol consumption accelerates liver fibrosis by suppressing the anti-fibrotic effects of NK cell/interferon-γ. Based on these studies, hepatic NK cells seem to have an anti-fibrotic role through interaction with HSCs. Nevertheless, the bidirectional interactions between HSCs and NK cells are still not fully understood, especially the reverse suppressive effects of HSCs against NK cells or the effects of retinol and its metabolites of HSCs on NK cells.
For the past decade, several studies and clinical trials have suggested that autologous BM cells and BM-derived progenitor cells improved liver function and ameliorated fibrosis in patients with cirrhosis and in mice with advanced liver fibrosis.[38-40] Recently, we demonstrated that CD11b+Gr1+ BM cells ameliorated liver fibrosis by IL-10 production in mice. In this study, CD11b+Gr1+ BM cells were specifically contacted with activated HSCs in the fibrous septa, which was associated with attenuated liver fibrosis, enhanced hepatic expression of IL-10 and expanded regulatory T cells (Tregs) but decreased macrophage infiltration within 24 h after infusion of BM cells. Similarly, human BM cells expressed more IL-10 after interaction with human HSC lines (LX-2 or hTERT) in vitro, and serum levels of IL-10 were significantly increased in patients with liver cirrhosis after autologous BM cell infusion. Furthermore, in vitro studies revealed that IL-10 production of CD11b+Gr1+ BM cells was dependent on HSC-derived retinoic acid and IL-6 production. In addition, increased IL-10 levels were also detected in CCl4-induced liver fibrosis of mice after colony stimulatory factor-1-derived BM macrophage delivery. However, further extensive studies will be required to elucidate the underlying mechanism of autologous BM cell infusion therapy to patients with cirrhosis even though several underlying mechanisms of BM cells for anti-fibrotic effects were reported.
Regulatory T cells (Tregs) and Th17 cells
Tregs differentiated from CD4+ T cells are characterized by expression of Foxp3, and the naturally occurring CD4+CD25+Foxp3+ Tregs are present in normal naïve mice and healthy individuals from birth. Under certain conditions, Tregs might acquire the ability to produce TGF-β and/or IL-10. In contrast to healthy liver, Tregs are more abundant in the livers of patients with chronic viral hepatitis, autoimmune liver disease, and primary biliary cirrhosis.[44, 45] Recent studies suggest that HSCs are directly or indirectly involved in the expansion or generation of Tregs, subsequently driving liver-induced tolerance.[46, 47] In addition, Tregs were found closely associated with HSCs in lipopolysaccharide-treated and cold ischemic preserved liver. Thus, these studies suggest that Tregs might play a negative role by IL-10 production in suppressing liver inflammation, which is related with HSC interaction. Moreover, Th17 cells can produce IL-22, which can induce senescence of activated HSCs via a STAT3/SOCS3-dependent manner, thereby leading to amelioration of liver fibrosis in mice.
During liver fibrogenesis, huge amounts of macrophages are infiltrated in the damaged area of liver, where they play a key role in driving activation of HSCs by producing pro-inflammatory cytokines such as TNF-α, leading to liver fibrosis.[1, 10] However, recent immunologic studies have suggested that there are two classified populations of macrophages including M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages polarized by cytokines from Th1 cells and Th2 cells, respectively. Especially, M2 macrophages might negatively regulate liver fibrosis via the production of anti-inflammatory cytokine IL-10. However, under certain conditions, M2 macrophages may also promote liver fibrosis via TGF-β- and MCP-1/CCR2-dependent manners. Although, macrophages can be classified into M1 and M2, there are no significant differences in their morphologic characteristics. Other macrophages such as scar-associated macrophages and BM-derived macrophages have shown to suppress liver fibrosis via matrix metalloproteinase (MMP) productions.[42, 52]
Dendritic cells (DCs)
Generally, DCs play important roles in both innate and adaptive immune responses as professional antigen-presenting cells. However, the roles of DCs in liver fibrosis are not clearly demonstrated yet. Recent studies show dual roles of liver DCs in liver fibrosis. In thioacetamide-induced liver fibrosis, the characteristics of liver DCs are transformed from tolerogenic to immunogenic, which subsequently enhance inflammatory changes (enhanced activities of NK cells and CD8+ T cells but reduced population of Tregs) in liver fibrosis via the production of TNF-α. In contrast, after cessation of liver injury, liver DCs are implicated in the regression of CCl4-induced liver fibrosis via the production of MMP-9. Therefore, further detailed studies are required to clarify the roles of DC during liver fibrogenesis and regression.
Interaction of HSCs and immune cells in alcoholic liver disease
HSCs are involved in the pathogenesis of all stages of alcoholic liver disease such as alcoholic steatosis (fatty liver), steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma by producing endocannabinoids, proinflammatory cytokines and chemokines, collagen fibers, and retinol metabolites.[55-57] Besides alcohol-mediated activation of HSCs, diverse liver immune cells such as NK cells, Kupffer cells/macrophages, and IL-17-producing cells are under the influence of alcohol, leading to various interactions with HSCs compared with those in normal circumstances. Chronic alcohol consumption suppresses the cytotoxicity of NK cells against activated HSCs, while alcohol-mediated TLR4 activation in Kupffer cells/macrophages induces enhanced activation of HSCs by producing proinflammatory cytokines such as TNF-α, subsequently accelerating liver fibrosis. In addition, alcohol consumption accumulates IL-17-producing cells including neutrophils in the liver, which subsequently enhance activation of HSCs.[17, 18] However, the interactions between HSCs and other types of liver immune cells, especially adaptive immune cells, in alcoholic liver disease are still unclear. Thus, further studies are strongly required to address those matters.
During liver injury, activated HSCs participate in various liver diseases via abnormal ECM accumulation and cytokine productions. However, recent studies suggest novel immunomodulatory roles of HSCs and bidirectional interactions between HSCs and immune cells, which may regulate positively or negatively to the pathogenesis of liver disease. In Fig. 1, we depicted possible interactions between HSCs and immune cells during liver diseases, especially liver fibrosis. Several types of immune cells have protective roles, but the others have opposite effects on liver fibrosis. Certain types of immune cells such as NKT, macrophages, and Th17 cells have dual roles in liver fibrogenesis. Therefore, further investigations are needed to identify more specific functions of each cell type, thereby providing therapeutic targets or developing cell-based therapies for the treatment of liver fibrosis.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (no. 2011–008306) and the KAIST High Risk High Return Project (HRHRP).