Role of gamma-delta T cells in liver inflammation and fibrosis

Abstract

Conventional adaptive T cell responses contribute to liver inflammation and fibrogenesis, especially in chronic viral infections and autoimmune hepatitis. However, the role of unconventional gamma-delta (γδ) T cells in liver diseases is less clear. In the past two decades, accumulating evidence revealed that γδ T cell numbers remarkably increase in the liver upon various inflammatory conditions in mice and humans. More recent studies demonstrated that the functional effect of γδ T cells on liver disease progression depends on the subsets involved, which can be identified by the expression of distinct T cell receptor chains and of specific cytokines. Fascinatingly, γδ T cells may have protective as well as pathogenic functions in liver diseases. Interferon γ-producing γδ T cells, for example, induce apoptosis in hepatocytes but also in hepatic tumor cells; while interleukin-17-expressing γδ T cells can downregulate pathogenic effector functions of other immune cells and can promote apoptosis of fibrogenic stellate cells. However, the results obtained in human liver disease as well as murine models are not fully conclusive at present, and the effects of γδ T cells on the outcome of liver disease might vary dependent on etiology and stage of disease. Further definitions of the γδ T cell subsets involved in acute and chronic liver inflammation, as well as their effector cytokines might uncover whether interference with γδ T cells could be a useful target for the treatment of liver disease.

Key Words: Liver fibrosis, Liver cirrhosis, Interleukin-17, Gamma/delta T cells, Cytokines

Core tip: The liver is particularly enriched in unconventional T cells expressing the gamma-delta T cell receptor and the functional role of these gamma-delta (γδ) T cells in liver diseases is being intensively investigated at present. γδ T cells accumulate in inflamed liver and their function appears highly dependent on the distinct subsets. In principle, γδ T cells can be protective as well as pathogenic in the context of liver inflammation. This review summarizes the current knowledge of γδ T cell effector functions and the cytokines produced by these cells in human liver diseases and murine experimental models of acute and chronic liver injury.

INTRODUCTION

Despite its various metabolic functions, the liver is also an important immunological organ. The blood coming from the gastrointestinal tract via the portal vein carries manifold potential antigens, derived from the commensal microflora of the gut, food or invading pathogens[1]. Hepatic leukocytes are able to either mount immune responses against pathogenic antigens or to induce tolerance against harmless substances[2]. Innate immune cells are important triggers of hepatic inflammation and it is well known that the liver is selectively enriched in macrophages (Kupffer cells), natural killer (NK) and natural killer T (NKT) cells, and also one of the richest sources for gamma/delta T cells (γδ T cells) in the body[3,4]. About 15%-25% of the hepatic T cells express the gamma/delta T cell receptor (TCR), indicating that this specific lymphocyte population might exert important functions in liver homeostasis and diseases. Moreover, the liver is also a site of extrathymic generation of γδ T cells during human fetal development, where the first transcripts of γδ TCR genes appear before a functional thymus is developed[5]. γδ T cells are a specific subpopulation of non-conventional T cells that are identified by expression of the γδ TCR instead of the αβ TCR[6,7]. In secondary lymphoid organs they account for only 2%-3% of all CD3⁺ cells, while the highest abundance of γδ T cells is seen in the gut mucosa[8].

γδ T cells are often described to link innate and adaptive immunity as they share features with innate immune cells as well as with conventional T cells of the adaptive immune system[9,10]. In contrast to αβ T cells, γδ T cells leave the thymus after their maturation as mature T cells with a defined functional potential in a so-called pre-activated status[11]. Although γδ T cells are able to recognize antigens presented on MHC molecules, they express only a restricted TCR repertoire and also recognize a lot of non-peptide ligands without the need for TCR engagement[12,13]. In the periphery, γδ T cells can also be sufficiently activated through cytokines without TCR engagement, allowing them to respond much faster than αβ T cells. Similar to conventional T cells, γδ T cells can kill target cells via death receptor mediated apoptosis or release of cytolytic granules[14,15]. They also produce large amounts of immunomodulatory cytokines, including interferon (IFN)γ, interleukin (IL)-17, IL-4, IL-5, IL-10, IL-13, TGFβ and GM-CSF[16].

According to their functional potential, γδ T cells can be subdivided into different effector populations. γδ T cells expressing a specific cytokine or with particular tissue localization often show a bias towards use of the same TCR V gene segments. IFNγ secreting γδ T cells, for example, often express Vδ1 or Vγ9Vδ2 chains[17-19], while γδ T cells expressing Vγ4 are frequently associated with production of IL-17[20,21] and/or IL-10[19]. In mice, these subtypes can also be distinguished by expression of surface markers, with the IFNγ secreting subpopulation expressing NK1.1 and CD27[11,22], while the IL-17⁺ subpopulation expresses CCR6 and CD25[22]. Interestingly, γδ T cells have been shown to be the major source of IL-17 in different immune-mediated diseases, often producing much higher amounts of this cytokine than (conventional) CD4⁺ Th17 cells, even if responding in similar or lower numbers than Th17 cells[23,24].

The functional role of γδ T cells during the pathogenesis of inflammatory disorders seems to be very diverse as they have been associated with pathogenic as well as protective functions, depending on the inflamed organ and disease model studied. In experimental glomerulonephritis, collagen-induced arthritis or experimental silicosis, for example, γδ T cells promote disease progression through production of IL-17[25-27]. In contrast, during adriamycin-induced nephropathy or concanavalin A-induced hepatitis, γδ T cells play a protective role through downregulation of the pathogenic functions of CD4⁺ or NKT cells, respectively[20,28].

In recent years, a number of studies using material from patients with liver diseases as well as experimental models of liver injury revealed that γδ T cell subsets are altered during the progression of liver diseases, indicating that this unconventional lymphocyte population might be of utmost importance for determining the fate of inflammatory processes in the liver. In this review article, we aim to present and discuss the current knowledge about the functional role of γδ T cells and their subsets in the pathogenesis of liver disease in mice and humans, as well as possible mechanisms of their pro- or anti-inflammatory activities in the context of liver diseases (Table 1).

Table 1 Role of gamma-delta T cells in human and experimental liver disease.

Species	Liver disease	TCR usage	Cytokine production	Other markers	Effector function(s)	Ref.
Protective functions of γδ T cells
Mouse	Concanavalin A-induced hepatitis	Vγ4	IL-17		γδ T cells inhibit NKT cell function	[20]
Mouse	Experimental fibrosis	Vγ4?	IL-17, IL-22	CCR6, CD95L	γδ T cells induce stellate cell apoptosis and limit collagen production	[47]
Mouse	Listeria monocytogenes infection	Vγ4	IL-10		γδ T cells downregulate CD8+ T cell effector function	[39]
		Vγ4/Vγ6	IL-17		γδ T cells are protective during early infection	[24]
Human	Liver metastasis of colon cancer	Vδ1	IFNγ, TNFα, IL-2	CD56, CD161	hepatic γδ T cells are cytotoxic against tumor cell lines in culture	[17]
Human	Pediatric tumor cell culture	Vγ9Vδ2	?		γδ T cells are cytotoxic against hepatoma cells in culture	[18]
Mouse	Adenoviral infection	Vγ4	IL-17		γδ T cells are critical for establishment of functional adaptive immune responses	[21]
Pathogenic functions of γδ T cells
Mouse	Schistosoma japonicum infection	?	IL-17		γδ T cells contribute to immune-mediated pathology	[40]
Mouse	Adenoviral infection	?	IFN-γ	CXCR3	γδ T cells contribute to hepatocyte apoptosis via FasL engagement and recruitment of cytotoxic T cells	[37]
Mouse	MHV infection	?	TNF-α, IFN-γ, IL-17, IL-2	CD69, CD44	γδ T cells induce hepatocyte apoptosis via TNF-α-signaling	[38]
Human	HCV infection	Vδ1	IFN-γ	HLA-DR, CD95, CD45-RO	Activated γδ T cells contribute to HCV-mediated immunopathology	[19]

γδT cells: Gamma-delta T cells; IFN: Interferon; IL: Interleukin; TNF: Tumor necrosis factor; MHV: Mouse hepatitis virus; HCV: Hepatitis C virus.

AUTOIMMUNE LIVER DISEASE

γδ T cells were already implicated in human autoimmune liver diseases two decades ago. Patients with primary sclerosing cholangitis or autoimmune hepatitis have been shown to display elevated numbers of γδ T cells in blood and liver when compared to healthy controls[29]. In the liver, γδ T cells were predominantly found in portal infiltrates and areas of bile duct proliferation or fibrogenesis, but the exact contribution of these cells to liver immunopathology remained elusive. Further insight into the functional role of γδ T cells in autoimmune hepatitis was provided more recently in a study of Zhao et al[20] by using the mouse model of concanavalin A (ConA)-induced fulminant hepatitis. This disease model of rapid liver inflammation and necrosis is dependent on the activation of CD4⁺ T cells[30] and the role of IL-17 in this condition is controversially discussed (reviewed in[31]). In this study, the authors suggest a protective role of IL-17 produced by Vγ4⁺γδ T cells through downregulation of the pathogenic function of NKT cells. NKT cells accumulate early after injury in the liver and promote the initiation of inflammatory responses and subsequent tissue damage by releasing pro-inflammatory cytokines[32]. Vγ4⁺γδ T cells were the primary source of IL-17 in ConA-induced hepatitis and adoptive transfer of wild type (wt) γδ T cells was able to reduce the aggravated disease phenotype in γδ T cell deficient mice, associated with higher liver damage and IFNγ levels, to the level of wt mice. This function was critically dependent on IL-17 as this effect could not be observed when TCRδ^-/- mice were reconstituted with IL-17^-/-γδ T cells[20]. These data indicate possible protective functions of IL-17⁺γδ T cells via NKT cell inhibition in immune-mediated liver diseases such as autoimmune hepatitis (Table 1).

VIRAL INFECTION

The essential role of T cell mediated immune responses in either clearing viral hepatitis or allowing persistent chronic infections is well established[33]. However, less data exist on γδ T cells in hepatitis B or C. In patients with chronic hepatitis B virus (HBV) infection, intrahepatic as well as peripheral γδ T cell numbers inversely correlate with disease severity[34]. Wu et al[34] showed that mainly Vδ2⁺γδ T cells are reduced and that these cells display an effector-memory phenotype with expression of CD45RA, MHC class II molecule human leukocyte antigens (HLA)-DR and CD38. Furthermore, these cells produce high levels of IFNγ but not IL-17 and are able to inhibit cytokine production of pathogenic CD4⁺ Th17 cells through cell contact- as well as IFNγ-dependent mechanisms. Therefore, the authors concluded that reduced numbers of γδ T cells account for decreased inhibition of Th17 cells, resulting in higher liver damage and pathology.

In contrast, several studies have shown that γδ T cells are enriched in the livers of patients with chronic hepatitis C virus (HCV) infection when compared to healthy controls or peripheral blood[19,35,36]. Agrati and colleagues demonstrated that these γδ T cells are predominantly Vδ1⁺ and display an effector-memory phenotype as they express HLA-DR and CD95[19]. These cells also produce increased levels of IFNγ during HCV infection and therefore very likely contribute to HCV-induced immunopathology in the liver. Furthermore, an additional study by Tseng et al[36] showed that γδ T cells isolated from livers of HCV patients are cytotoxic against primary human hepatocytes in culture, suggesting that γδ T cells might contribute to HCV-triggered liver injury.

A similar effect is seen in mice with adenoviral infection. IFN-γ-producing γδ T cells accumulate around infected hepatocytes and contribute to hepatocyte death through Fas-mediated apoptosis[37]. Furthermore, IFNγ production induces the release of chemokines like CXCL9 by hepatocytes, which further recruits γδ T cells and CD8⁺ cytotoxic T cells. The importance of γδ T cells for these pathogenic processes is underlined by the fact that γδ T cell deficient mice are protected from adenovirus-induced liver injury. However, these mice show no difference in viral clearance. Another study by Hou et al[21] shows that IL-17 producing γδ T cells also increase in adenovirus-infected murine liver. Consistent with the results obtained in ConA-induced hepatitis, Vγ4⁺γδ T cells are the major IL-17 producers and IL-17 secretion by these cells is critical for the development of a functional antiviral immune response and subsequent clearance of the virus.

In mouse hepatitis virus (MHV) infection, γδ T cells play a clearly pathogenic role but via a different mechanism[38]. Although IFNγ- and IL-17- producing γδ T cells accumulate in the liver also in this model, their function seems to be rather dependent on tumor necrosis factor (TNF) α-production. Activated hepatic γδ T cells are cytotoxic against MHV infected hepatocytes but this effect does not require cell-cell contact or IFNγ-/IL-17-signaling, while blockade of TNFα leads to markedly reduced hepatocytotoxicity[38].

Taken together, the functional role of γδ T cells during viral infection of the liver seems to be highly dependent on the subset involved. While Vδ1⁺ and Vδ2⁺ T cells are associated with production of IFNγ and progression of liver immunopathology, the Vγ4⁺ IL-17 producing subset of γδ T cells seems to be rather important for viral clearance. The fact that liver injury during MHV infection is dependent on TNF-α production by γδ T cells might suggest that a third subset of γδ T cells is functionally involved in viral-induced liver diseases.

BACTERIAL AND PARASITIC LIVER INFECTIONS

γδ T cells have been shown to exert protective functions in bacterial infections of the liver. γδ T cell deficient mice infected with Listeria monocytogenes develop increased liver pathology which is caused by infiltrating CD8⁺ T cells producing high levels of TNF-α[39]. This pathogenic effect can be prevented through adoptive transfer of Vγ4⁺γδ T cells. These cells produce high levels of IL-10, which in turn downregulates TNF-α production in CD8⁺ T cells (Figure 1). Furthermore, Vγ4⁺ T cells are also the major IL-17 producing cell type during Listeria infection and γδ T cell-derived IL-17 is critically needed for protective immunity during early infection[24]. IL-17 deficient mice reconstituted with γδ T cell-deficient bone marrow, meaning that γδ T cells are able to produce IL-17 but γδ T cells are not, show a much higher bacterial burden in the liver than mice reconstituted with wt bone marrow[24]. In contrast, during Schistosoma japonicum infection IL-17 production by γδ T cells seems to have a more pathogenic role[40]. Although γδ T cells are the major IL-17 producing cell type also in this model, neutralization of IL-17 reduced liver inflammation and pathology in this case.

Figure 1 Role of gamma-delta T cells in liver disease. Upon liver damage several subsets of gamma-delta (γδ) T cells are recruited to the liver, where they can exert different functions on numerous cell types, ultimately resulting in protective or pathogenic effects on the outcome of liver disease. Pathogenic effects include induction of hepatocyte apoptosis by interferon (IFN)γ - and/or tumor necrosis factor (TNF) α-producing γδ T cells, mediated via death receptor signaling (TNF receptors or Fas/CD95). However, the Vδ1⁺ IFNγ-producing subset can also have beneficial functions as they drive tumor cells apoptosis. Other protective functions can be attributed to Vγ4⁺ T cells, which produce interleukin (IL)-17 and IL-10, and can downregulate pathogenic effector functions of other lymphocytes like natural killer T (NKT) cells or cytotoxic T cells, respectively. IL-17⁺γδ T cells have also been shown to induce Fas-mediated apoptosis of hepatic stellate cells (the main producer of collagen during hepatofibrogenesis), thereby limiting liver fibrosis.

During malaria infection, however, γδ T cells play only a minor role as long as conventional adaptive T cell responses are intact, demonstrated by the fact that γδ T cell deficient mice survive plasmodium infection without extensive organ failure[41]. γδ T cells are needed for protective immunity against the parasite only in mice deficient for γδ T cells. In this case, depletion of γδ T cells leads to severe immunopathology because development of the parasite is not inhibited, an effect that can be reversed through adoptive transfer of γδ T cells[41].

As described above, γδ T cells can have opposing effects in different infection models. This further underlines the functional heterogeneity of the different γδ T cell subsets distinguished by cytokine production or usage of specific receptor chains. The impact that γδ T cells have on the outcome of different infectious diseases might also be influenced by the nature of the adaptive immune response induced by the microorganism itself as this could change the local cytokine milieu dramatically.

LIVER FIBROSIS

Independent from the underlying etiology of liver disease, such as viral hepatitis, alcoholic and non-alcoholic steatohepatitis or other origins, chronic liver diseases characteristically progress from tissue injury to chronic hepatitis and fibrosis to liver cirrhosis as the end-stage of chronic liver diseases[42]. Persistent inflammation in the liver is considered the driving force for disease progression. Over recent years, several studies have emphasized the crucial role of various immune cell subsets for controlling inflammation and fibrogenesis in the liver and the interplay between the different leukocyte populations, including monocytes, Kupffer cells, NK/NKT or T lymphocytes, appears to be tightly regulated by cytokines and chemokines[43,44]. Although IL-17 has been recognized as an important regulatory cytokine in hepatic inflammation[31], relatively few data exist on the contribution of γδ T cells to the pathogenesis of liver fibrosis. γδ T cells accumulate in fibrotic liver and contribute to IL-17 production in different experimental models of chronic liver injury, as well as liver samples of patients with chronic hepatitis[45,46]. Interestingly, IL-17 itself, produced mainly by αβ T cells and neutrophils, was found to promote fibrosis progression through activation of hepatic stellate cells (HSC) and Kupffer cells.

In contrast, hepatic γδ T cells can be associated with protective functions in murine chronic liver injury but these functions appear to be independent from the signature cytokine IL-17. We recently showed that specifically the CCR6 expressing subtype of γδ T cells, producing IL-17 and IL-22, accumulates in fibrotic livers of mice subjected to experimental liver injury models[47]. These cells are capable of limiting fibrosis progression through induction of apoptosis in HSC, the major collagen producing cell type in the liver. Nevertheless, this effect does not depend on their IL-17 or IL-22 production but is rather mediated through Fas/Fas-ligand (FasL) interactions. IL-17 deficient γδ T cells are able to limit liver fibrogenesis to the same extent as wt γδ T cells and blockade of IL-22 could not reduce HSC apoptosis, while use of a FasL-blocking antibody significantly inhibited HSC apoptosis (Figure 1). Thus, these data indicate that γδ T cells, at least its CCR6 expressing subset, represent an important anti-fibrotic pathway in hepatic inflammation by ameliorating the inflammatory reaction and the activation of collagen-producing stellate cells in chronically injured liver.

LIVER CANCER

More than two decades ago the first studies showed that γδ T cells accumulate in tumor bearing liver. Patients with hepatic malignancies as well as tumor bearing mice show elevated levels of γδ T cells in the liver when compared to healthy controls[17,48]. Usually these cells display an activated phenotype with expression of CD56, CD161 and LFA-1 and are cytotoxic against hepatoma cells and Daudi targets in culture[17,18]. Furthermore, murine Vδ1⁺γδ T cells induced in response to cytomegalovirus (CMV) infection have been shown to inhibit development of liver metastases in a colon cancer model[49]. These findings suggest that γδ T cells might contribute to anti-tumoral immune responses, likely by promoting direct cytotoxic responses to malignant parenchymal cells (Figure 1). However, tumor cells can escape γδ T cell responses through downregulation of the respective ligands[18].

Although detailed mechanistical studies on anti-tumoral responses of γδ T cells in the liver are still lacking, further insight into these mechanism might be provided by a recent study on recruitment of γδ T cells in the B16 melanoma model[50]. In this model, γδ T cells inhibit tumor growth as γδ T cell-deficient mice develop larger tumors than their wild type counterparts. A similar effect is seen in CCR2- as well as CCL2-deficient mice, which display reduced γδ T cell infiltrates in B16 lesion and a higher tumor growth rate. Moreover, this study also shows that murine as well as human peripheral γδ T cells migrate toward CCL2 in vitro[50]. Since this effect could only be observed with Vδ1⁺ but not Vδ2⁺γδ T cells, this mechanism might very well also play a role in hepatic malignancies.

CONCLUSION

γδ T cells have been shown to accumulate in the liver upon various inflammatory conditions which lead to hepatic fibrosis and other types of immunopathology when becoming chronic. The exact contribution of these lymphocytes to liver inflammation seems to be highly dependent on the subsets involved, which can be identified by the specific cytokines they produce and their expression of different T cell receptor chains. γδ T cells producing IFNγ often co-express TNFα and the Vδ1 chain but usually do not produce IL-17, which is often co-expressed with Vγ4 chains. The effect of these subsets on the outcome of liver disease also depends in part on the underlying liver disease etiology. Accordingly, the IFNγ⁺ subset is able to induce apoptosis in different cell types, which might have pathogenic or beneficial effects on liver immunopathology depending on whether hepatocytes or tumor cells are affected. In contrast, IL-17 producing γδ T cells are often associated with protective functions in liver inflammation as they can inhibit pathogenic effector functions of cytotoxic T cells or NKT cells, as well as limit hepatofibrogenesis through inhibition of hepatic stellate cells. Nevertheless, the results obtained in human liver disease as well as murine models are not fully conclusive at present as many studies lack detailed analysis on the correlation of cytokine production with specific surface markers such as TCR chains. Therefore, it is not clear whether the diverse functions that γδ T cells have during different liver diseases are executed by very few subsets according to the cytokines they produce or by a huge variety of γδ T cells with redundant cytokine profiles. Thus, it is of utmost importance to further define γδ T cell subsets in acute and chronic liver inflammation as well as the cytokines they produce in order to assess whether interference with γδ T cells might be useful as a therapeutic target for the treatment of liver disease.