Pathophysiology of and liver immunity

Won-Mook Choi and Won‐Il Jeong* Lab of Liver Research, Graduate School of Medical Science and Engineering, KAIST, Korea

Abbreviation: BM, ; DC, ; ECM, extracellular matrix; HSC, ; IFN, in- terferon; IL, interleukin; KC, Kupffer cell; NK, natural killer; PDGF, -derived growth factor; TGF, trans- forming growth factor; Th, T helper; TLR, toll-like receptor; TNF, ; TRAIL, tumor necrosis factor-related apoptosis ligand

Acknowledgement This research was not supported by any agency in the public, commercial or not-for-profit sector.

Conflict of interest The authors declare no conflict of interest.

Abstract

Various types of chronic liver disease cause liver fibrosis. Hepatic stellate cells (HSCs) are known as the key cell type by producing a large amount of extracellular matrix, profibrotic and chemokines. Recently, emerging evi- dence suggests that the liver is not only a metabolic organ but also an immunologic organ due to enrichment of di- verse types of innate and adaptive immune cells. Moreover, the cell-to-cell interactions between HSCs and various types of immune cells are closely associated with the pathogenesis of liver fibrosis. Especially, the liver is known to play a key role in innate immune defenses against pathogens. Indeed, various innate immune cells such as Kupffer cells, /, , and dendritic cells play an important role in accelerating or ameliorat- ing liver fibrosis directly or indirectly via interactions with HSCs. Moreover, unlike other organs, innate lymphocytes such as natural killer (NK), NKT, and γδ T cell are abundant in the liver comprising up to 50% of whole liver lymphocytes. Although still controversial in their roles (i.e. profibrotic vs. antifibrotic), these innate lymphocytes are

www.kasl.org 53 제4회 임상연구방법론 워크숍 also deeply involved in the pathogenesis of liver fibrosis. Especially, NK cells seem to play a negative regulatory role in liver fibrosis via inhibiting or suppressing activated HSCs. In addition, increasing evidences have suggested that adaptive immune cells are no longer a ‘bystander’, but contribute considerably to liver fibrosis. In this review, we summarize the updated concept of pathophysiology of liver fibrosis and liver immunity.

Introduction

Liver fibrosis is the final common response to virtually all types of chronic liver injury. The mechanisms and pathophysiology of liver fibrosis have been investigated in the last several decades; however, are still far from com- plete understating.1-3 Among various liver cell types, hepatic stellate cells (HSCs) are considered to be a major type of cells responsible for liver fibrosis.4 Under normal physiologic condition, HSCs exist as a quiescent form and play a role as a retinol (vitamin A) storing cell. However, once activated by various types of liver injury, HSCs lose their lipid droplets containing retinol and become myofibroblast-like cells producing a large amount of extracellular ma- trix (ECM) proteins, which subsequently lead to liver fibrosis.5 Many other cell types including myofibroblast, bone marrow-derived progenitor cells, and , are also effector cells to the pathogenesis of liver fibrosis; how- ever, those cells play a limited role compared to HSCs.1-3 Recently, the liver has been postulated to be an immunologic organ. Especially, the liver is known to play a key role in innate immune defenses against pathogens.6-9 Since the liver plays as an organ barrier or a filter between the digestive tract and the rest of the body before entering systemic circulation, the immune system in the liver is pre- dominantly composed of innate immunity as follows.7 First, the liver synthesizes 80-90% of innate proteins includ- ing complements, secreted pattern recognition receptors, and acute phase proteins. Second, various type of liver cells also expresses various pattern recognition receptors including toll-like receptor (TLR).10-12 Third, a large num- ber of sinusoidal endothelial cells and Kupffer cells (KCs) eliminate soluble macromolecules by endocytosis and in- soluble wastes via , respectively.7 Finally, liver innate lymphocytes such as natural killer (NK), NKT, and γδ T cells are comprising up to 50% of whole liver lymphocytes.7,8 Although the adaptive immunity in the liver seems less active than the innate immunity, the immune responses of CD4+ and CD8+ T cells against liver-trophic microorganisms also occur in the liver.6,9 Intriguingly, increasing evidence suggests that various components of the immune system, as mentioned above, control the pathophysiology of liver fibrosis. Moreover, recent studies suggest that pro-inflammatory cytokines, such as transforming growth factor (TGF)-β1 and platelet-derived growth factor (PDGF), released by various liver immune cells activates HSCs and HSCs also have bidirectional immunor- egulatory roles by secreting various cytokines, chemokines, expressing TLRs, and functioning as antigen presenting cells.4,5 In the present review, we will discuss the role of immune system in the pathogenesis of liver fibrosis.

54 대한간학회 정원일 | Pathophysiology of liver fibrosis and liver immunity

Figure 1. Interactions between hepatic stellate cells and immunes cells on the pathogenesis of liver fibrosis.

Especially, we will focus on interaction between HSCs and immune cells in liver fibrogenesis (Fig. 1).

Cytokines and chemokines in liver fibrosis

Cytokines are soluble peptides secreted by various types of cells including immune cells, parenchymal and non-parenchymal cells, which mediate immune and inflammatory reactions. When hepatocytes are damaged by var- ious factors such as viral agents, alcohol, and hepatotoxins, macrophages and neutrophils are recruited and produce various cytokines and chemokines that mediate the inflammatory response, which then leads to the deposition of ECM by activated HSCs. Among various cytokines, TGF-β, tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, IL-10, and interferon (IFN)-γ are the most important cytokines produced in the liver and the main cellular source of these cytokines is KCs.13 TGF-β1, the most profibrotic , stimulates its own production by myofibroblasts, which establishes an au- tocrine cycle of myofibroblast differentiation and activation. Previous studies demonstrated that anti-TGF-β ap- proaches have been successfully tried to treat experimental models of liver fibrosis. However, these approaches (i.e. broad targeting of the TGF-β pathway) have a critical problem due to the versatile nature of TGF-β activities.14 Therefore, the fundamental mechanism of TGF-β signaling pathway on liver fibrosis should be elucidated to devel- op approaches that specifically target profibrogenic TGF-β signaling pathway. Experimental studies in murine models demonstrate that both IFN-α/β and IFN-γ are negatively associated

www.kasl.org 55 제4회 임상연구방법론 워크숍 with liver fibrosis. In the experiments with IFN-γ-deficient mice, liver fibrosis was more severely induced by ad- 15,16 ministration of CCl4 or feeding with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet. Moreover, treatment with IFN-α/β or IFN-γ improves liver fibrosis in a variety of animal models.16-19 However, direct IFN-γ treatment failed to suppress the activation of HSCs in chronically alcohol-fed mice, thus failed to prevent alcoholic liver fibrosis.20 The antifibrotic effects of IFN are mediated by inducing HSC cycle arrest and apoptosis,21,22 activating NK cell,16 and inhibiting synthesis.19 Besides, IL-8 is also involved in liver fibrogenesis by inducing a con- tractile phenotype associated with focal adhesion and upregulating α-smooth muscle actin production.23 Since the chemokine system is critical in lymphocyte recruitment, several studies have investigated the role of chemokines in the pathogenesis of liver fibrosis. Among various chemokine signaling pathways, the CC- and CXC-chemokine receptor families have consistently showed important regulatory roles. Especially, CCL2 (MCP-1) and its receptor, CCR2 have been widely studied. When the action of CCL2 was interfered by ad- enovirus-mediated expression of a mutated ligand (7ND-CCL2), and lymphocyte infiltration into in- jured was significantly decreased; thus, HSC activation was reduced and liver fibrogenesis was prevented.24 Moreover, we should note that HSCs are one of the major cellular sources of CCL2 through TLR4 ligands,25 which suggests that cell to cell interactions between HSCs and various immune cells play an important role on liver fibrogenesis. CCR5, a chemokine receptor binding with CCL5, CCL3, or CCL4, was involved in proliferation and migration of human HSCs and both recruitment of /macrophages, which causes liver fibrosis.26 In pa- tient with hepatitis C infection, expression of CXCR3 and its chemokines, particularly CXCL10, was increased in advanced and fibrosis.27 CCR7 is also involved in liver fibrogenesis. In patients with chronic hep- atitis C, expression of CCR7 was increased and its ligand CCL21 was detected by immunostaining around in- flammatory lymphoid follicles. Expression of CCR7 was also upregulated in patients with primary biliary cirrhosis. In a corresponding in vitro experiment, cultured HSCs expressed CCR7 and exposure of HSCs to CCL21 activated several signaling pathways which stimulated cell migration and accelerated wound healing by in- ducing several proinflammatory genes.28 It was also shown that HSCs express CXCR4 in vivo and in vitro. CXCR4 activation by its endogenous ligand, stromal cell-derived factor-1alpha, triggered HSC expression of α-smooth muscle actin and collagen I and increased HSC proliferation.29

Innate immune cells in liver fibrosis

Kupffer cells. KCs, which are self-renewing embryo-derived local macrophages, constitute 15% of the total liver cell population. Once activated, KCs produce various cytokines such as TGF-β1, TNF-α, MCP-1, and other in- flammatory mediators, which can activate HSCs during liver fibrogenesis.3 KC depletion by gadolinium chloride resulted in attenuation of CCl4-induced liver fibrosis with reduced histological fibrosis and prevention of the in-

56 대한간학회 정원일 | Pathophysiology of liver fibrosis and liver immunity creased expression of TGF-β in a rat model.30 KCs are located in the center of the sinusoids, which allows intimate interactions with other non-parenchymal hepatic cell populations. Especially, KCs can activate HSCs via paracrine mechanisms with TGF-β and PDGF production.31 In addition, TLR4-Myd88-NF-kB signaling pathway plays a critical role in enhancing interaction between HSCs and KCs.25 These profibrotic functions of KCs during chronic liver injury remain unaffected even after inhibition of the additional infiltration of monocyte by blocking the che- mokine CCL2.32 Monocytes/macrophages. During liver fibrosis, large amounts of monocyte-derived macrophages are infiltrated into the damaged area of liver. There are two types of macrophages including M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophage. In general, activation of M1 pro-inflammatory macrophages leads to the pro- duction of pro-inflammatory cytokines and ultimately the development of fibrosis. On the other hand, M2 mac- rophages might negatively regulate liver fibrosis via the production of anti-inflammatory cytokine such as IL-10.33 Recently, ‘restorative macrophages’ have been identified. These cells are characterized by Ly-6Clo expression and cannot be classified according to the classical M1/M2 nomenclature by transcriptome analysis. Although exact mechanisms should be further elucidated, these Ly-6Clo macrophages seem to be originated from infiltrating Ly-6Chi monocytes/macrophages, which suggests that these cells undergo a functional switch during liver fibrogenesis. These cells play a role in resolving fibrosis via production of matrix metalloproteinases (MMP-9, -12, -13) and induction of HSC apoptosis.34 It can be inferred from these versatile roles of macrophages that either dif- ferent populations of monocytes/macrophages are involved at each stage or, more likely, that macrophages are evolving during liver fibrogenesis in response to the change of local microenvironment.35 Neutrophils. In general, liver inflammation precedes liver fibrosis; thus, infiltrating leukocytes, especially neu- trophils, can potentiate the progression of liver fibrosis.36-38 In co-culturing system, collagen synthesis in human HSCs was stimulated by -derived reactive oxygen species.36 Moreover, activated rat HSCs can recruit neutrophils to the injured area of the liver by secreting neutrophil-attracting chemokines including MIP-2 and cy- tokine-induced neutrophil chemoattractant.37,38 Dendritic cells (DCs). DCs are professional antigen-presenting cells which are central to modulate both innate and adaptive immune responses.9 Recent studies indicate that liver DCs play dual roles in liver fibrosis. First, in contrast with other organs, liver DCs display a tolerogenic phenotype; thus, are believed to form the basis of im- mune tolerance to various antigens in the liver. However, during liver fibrosis, a transformation of DC function is occurred from tolerogenic to immunogenic, which subsequently enhance activities of NK cells and CD8+ cells but reduce population of regulator T cells via the production of TNF-α.39 On the other hand, liver DCs are involved in the regression of liver fibrosis by the production of MMP-9 after cessation of liver injury.40 Therefore, further studies are warranted to elucidate the exact roles of DC during liver fibrogenesis and regression. Bone marrow (BM) cells. Previous studies have suggested that BM-derived cells migrate into fibrotic liver tissue

www.kasl.org 57 제4회 임상연구방법론 워크숍 and promote liver fibrosis.41-44 In murine and human models, myofibroblasts, not HSCs, transdifferentiated from BM-derived cells contribute to the development of liver fibrosis.41-43 However, BM-derived cells have anti-fibrotic functions in different cell linages. Our recent study showed that infusion of CD11b+Gr1+BM cells ameliorated liv- er fibrosis by producing IL-10, expanding regulatory T cells, but decreasing macrophage infiltration in mice.45 Similarly, when human BM cells were co-cultured with human HSC lines (LX-2 or hTERT), the expression of IL-10 was increased. Moreover, serum IL-10 levels were significantly increased in patients with liver cirrhosis after autologous BM cell infusion.45 However, more studies should be performed to clarify the underlying mechanism and confirm the effects of autologous BM cell infusion therapy to treat advanced liver fibrosis.

Innate-like lymphocytes in liver fibrosis

As mentioned in the introduction, the liver is located between the intestine and the systemic circulation; thus, is continuously exposed to food-derived antigens, bacterial products and toxins. Therefore, liver innate lymphocytes including NK, NKT, and γδ T cells predominate in the population of whole liver lymphocytes up to 50% and play a critical role in the pathogenesis of various liver diseases.7,8 NK cells. Recently, we and other groups have illuminated NK cells as important negative regulatory cells in liver fibrogensis using rodent models.16,22,46,47 During liver fibrosis, NK cells can interact with activated HSCs via reti- noic acid early inducible gen-1/NKG2D or activating/inhibitor killer immunoglobulin receptor/MHC class I-de- pendent manner,16,46 which leads to HSC apoptosis in a TNF-related apoptosis ligand (TRAIL)-dependent man- ner and IFN-γ.16,22 Moreover, several studies showed that human NK cells can kill human HSCs via TRAIL-, Fas-ligand-, and NKG2D-dependent manners, thereby inhibiting liver fibrosis in patients.48, 49 However, our recent study demonstrate that intermediately activated HSCs in advanced liver fibrosis, in contrast to early activated HSCs, can survive against NK cell killing and IFN-γ-induced apoptosis via retinoic acid-mediated TGF-β pro- duction and suppressor of cytokine signaling 1 expression of HSCs, respectively.47 The result from our study can explain a clinical trial reporting that treatment of IFN-γ showed no beneficial effects on patients with advanced liver fibrosis.50 Based on these studies, liver NK cells seem to protect against liver fibrosis through interaction with HSCs. However, more studies should be conducted to elucidate the bidirectional interactions between HSCs and NK cells, especially the reverse inhibitory actions of HSCs against NK cells or the effects of retinol and its metabo- lites of HSCs on NK cells. NKT cells. Compared to other cell types, the role of NKT cells on the pathogenesis of liver fibrosis is still on debates. Recent studies indicate that NKT cells aggravate liver fibrosis by producing inflammatory cytokines such as IL-4 and IL-13, which leads to activate HSCs in murine models.51-53 However, NKT cells might attenuate liver fibrosis by producing anti-fibrotic cytokines or cytotoxicity against activated HSCs.53 Thus, further studies should

58 대한간학회 정원일 | Pathophysiology of liver fibrosis and liver immunity be recommended to elucidate the role of NKT cells on liver fibrogenesis. γδ T cells. While the majority of T cells express a T-cell receptor composed of αβ heterodimers, a smaller subset expresses a γδ T-cell receptor. In contrast to conventional T cells, γδ T cells show less T-cell receptor diversity; thus, are considered as innate-like lymphocytes linking innate and adaptive immunity. γδ T cells produce large amounts of immunoregulatory cytokines, especially IL-17. Although IL-17 has been considered as an important cytokine in hepatic inflammation,54 the role of γδ T cells on the pathogenesis of liver fibrosis was largely unknown. During liver injury and fibrosis, γδ T cells accumulate in the liver and contribute to IL-17 production, which ag- gravates liver inflammation and fibrosis in various experimental models of chronic live injury and liver samples of patients with chronic hepatitis.55, 56 However, γδ T cells, especially its CCR6 expressing subset, may protect against liver fibrosis via induction of apoptosis in HSC through Fas/Fas-ligand interactions.57 Since the exact role of γδ T cells on the pathogenesis of liver fibrosis is still controversial, further studies will be required.

Adaptive immune cells in liver fibrosis

Growing evidence has suggested that the adaptive immune system is important in liver fibrosis. While T helper (Th) 2 lymphocytes produce high levels of profibrotic cytokines such as IL-4, Th1 lymphocytes express high levels of antifibrotic cytokines such as IFN-γ. Thus, the Th1/Th2 ratio is associated with the balance in favor of or against liver fibrosis.58, 59 Among Th1 lymphocytes, increased numbers of CD8+ T cells and decreased CD4+/CD8+ ratio are related with the induction of liver fibrosis in mice and human.60, 61 The role of B cells in liver fibrogenesis 62 was revealed by that collagen deposition was attenuated in CCl4-induced fibrosis in B cell-deficient mice. The possible mechanisms mediating the effects of B- cells on liver fibrogenesis are as follows. First, it is possible that B cells produce the profibrotic cytokine such as IL-6, which contributes to liver fibrosis through activation of HSCs and increased collagen and TIMP synthesis. Second, alteration in T cell function in B cell-deficiency may contrib- ute to the pathogenesis of liver fibrosis. Therefore, we should not overlook B cells as a ‘bystanders’ in the patho- genesis of liver fibrosis.

Conclusion

All types of liver injury regardless of certain causes result in liver fibrosis at the end. During liver injury, HSCs are activated and transdifferentiated into myofibroblast-like cells producing a large amount of ECM proteins and immunoregulatory cytokines. Moreover, emerging evidences suggest that various types of immune cells are in- volved in the pathogenesis of liver fibrosis and interact with HSCs in favor of or against liver fibrosis. However, de- spite extensive research and advances in our understanding of the pathogenesis of liver fibrosis, the effective anti-

www.kasl.org 59 제4회 임상연구방법론 워크숍 fibrotic agents have not yet been developed. Therefore, further investigations are required to identify the main cel- lular effectors of liver fibrosis and the key cytokines regulating the fibrotic process, thereby providing novel ther- apeutic targets or developing cell-based immunotherapies for the treatment of liver fibrosis.

References

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