The Role of γδ T Cells in Fibrotic Diseases

Inflammation induced by toxins, micro-organisms, or autoimmunity may result in pathogenic fibrosis, leading to long-term tissue dysfunction, morbidity, and mortality. Immune cells play a role in both induction and resolution of fibrosis. γδ T cells are an important group of unconventional T cells characterized by their expression of non-major histocompatibility complex restricted clonotypic T cell receptors for non-peptide antigens. Accumulating evidence suggests that subsets of γδ T cells in experimentally induced fibrosis following bleomycin treatment, or infection with Bacillus subtilis, play pro-inflammatory roles that instigate fibrosis, whereas the same cells may also play a role in resolving fibrosis. These processes appear to be linked at least in part to the cytokines produced by the cells at various stages, with interleukin (IL)-17 playing a central role in the inflammatory phase driving fibrosis, but later secretion of IL-22, interferon γ, and CXCL10 preventing pathologic fibrosis. Moreover, γδ T cells appear to be involved, in an antigen-driven manner, in the prototypic human fibrotic disease, systemic sclerosis (SSc). In this paper we review in brief the scientific publications that have implicated γδ T cells in fibrotic diseases and their pro- and anti-fibrotic effects.


INT RODUCTION
Ex tensiv e tissue deposition of ex tracellular matrix proteins by activated fibroblasts may lead to structural and functional tissue damage. Uncontrolled fibrosis may be a consequence of inflammation triggered by pathogens, autoimmunity or malignancies, and is related to dysregulation of multiple types of immune cells including subsets of T cells. 1 γ δ T cells, a "non-conventional" T cell population, were discovered in 1 986 and, in contrast to "conventional" T cells ex pressing the αβ T cell receptor (TCR), recognize non-peptidic antigens independent of major histocompatibility (MHC) molecules. [2][3][4][5] In humans there are two major subsets; the first ex presses TCR γδ That use variable (V) region genes V γ 9 and V δ2 in the γ and δ TCR poly peptides, respectiv ely . V γ 9δ2 TCR sense low-molecularweight phosphoantigens of microbes, and host cellproduced phosphoantigens in the mevalonate pathway . 6 These phosphoantigens bind to the extra-or intracellular domains of the cell surface membrane molecule buty rophilin 3A1 (CD27 7 ), inducing a nov el structure or conformation that is detected by cells ex pressing the V γ9δ2 TCR, triggering their cy tokine production and/or cyto tox icity . 7 ,8 Thus, V γ 9δ2 γδ T cells are poised to detect and respond to infections or altered intracellular metabolism induced, for example, by intracellular infections, or a malignant transformation. The second human γδ T cell subset is characterized by the Vδ1 genes in the δ TCR poly peptide. V δ1 + γδ T cells are distributed along epithelial barriers. Their TCR detects lipid antigens presented by CD1 molecules, similar to natural killer T (NKT) cells. 9,10 Although the murine immune sy stem lacks phosphoantigen-reactive γδ T cells, the role of buty ro philins in γ δ T cell dev elopment is retained in mice, at least for some subsets, as ex emplified by the dependence of entire subsets of murine γδ T cells on specific buty rophilins for their development and homing to the skin and gut. [11][12][13] Readers are referred to comprehensiv e rev iews of murine γδ T cells by V antourout and Hay day . 4,5 Despite obvious distinctions between the murine and human γδ T cells, there is ample ev idence to indicate that the functional repertoire of γδ T cells in both humans and mice includes cytokine production, cy totoxicity, and help for B cells. 4 Uniquely , moreover, subsets of these cells acquire their full functional potential during maturation in the thymus, contrasting with αβ T cells that fully mature functionally only after encountering antigens in the peripheral lymphatic system. In this regard, in both humans and mice, γ δ T cells are similar to innate ly mphocytes, which positions them at the forefront of the response to foreign inv aders and internal "stress" conditions, including, for ex ample, metabolic aberrations induced by malignancy, infections or other stressogens. 4 Indeed, inflammatory, malignant, and infectious conditions are associated with numerical alterations of γ δ T cells in humans. 14 Giv en their unique abilities to detect non-peptide antigens, that may evade adaptiv e αβ T cells, and their rapid, non-MHC-dependent responsiveness, these cells may thus play a critical and unique role in diseases. Here, we review the involvement of the γδ T cell subset in pathological fibrotic responses. Specifically, we concentrate on systemic sclerosis (SSc), the prototypic systemic fibrosing disease in humans, and on animal models in ex perimental settings mimicking SSc, as well as in organ-localized pulmonary and liv er fibrosis.

γ δ T CELLS IN HUMAN FIBROSIS
Most of the ev idence linking human γ δ T cells to fibrosis comes from studies of the systemic sclerosis (SSc). Thus, in SSc , V δ1 + γ δ T cells were identified in the skin during v ery early stages of SSc. 15 Furthermore, the div ersity of V δ1 junctional regions (composed of the v ariable [V ], diversity [D], and joining [J] gene segments) in peripheral blood (PB) mononuclear cells (PBMC), lung, esophagus, stomach, or skin of patients was limited in SSc patients, and the same V δ1-Jδ junctional sequences could be isolated from multiple tissues suggesting an antigen-driven ex pansion of V δ1 + γ δ T cells in SSc. 16 In a large group of patients, percentages of PB γ δ T cells were significantly lower in SSc patients with diffuse and late-stage disease with pulmonary inv olv ement , muscle involvement, and the presence of anti-Scl-7 0 antibodies, mimicking the University of California at Dav is line (UCD)-200 chicken model described below. 17 In addition, V γ9 + γ δ T cells persist in SSc patients' PB, respond by ex pression of CD25 and CD69 to a phosphoantigen, isopentenyl pyrophosphate (IPP), and induce contact-dependent, tumor necrosis factor (TNF) α-independent apoptosis of cultured sy nov ial fibroblasts. 18 Howev er, higher concentrations of zoledronate, an aminobisphosphonate that increases IPP by inhibiting intracellular farnesy l py rophosphate (FPP) sy nthase, were required for maximal proliferation of V γ9 + T cells in SSc patients than in healthy controls, suggesting their dy sfunc tion in SSc; y et these cells still secreted factors that inhibited collagen production. 19 Furthermore, less anti-fibrotic cytokines TNF-α and IFN-γ were secreted in response to IPP in SSc. Indeed, reduction of procollagen secretion by fibroblasts cultured with supernatants of IPP-stimulated PBMC was observed only in some SSc patients. 19 On the other hand, γδ T cell supernatants from patients induced more proliferation of fibroblasts than αβ T cell supernatants, and doubling of collagen synthesis in human skin fibroblasts maintained in supernatants of SSc -derived γδ T cells was observed, which was inhibited by anti-transforming growth factor-beta (TGFβ) antibody and anti-basicfibroblast growth factor antibodies. 20 Furthermore, PB γδ T cells of SSc patients expressed higher levels of CD1 6 and CD69 compared to healthy controls, and collagen gene 1 (COL1 ) A2 mRNA ex pression was significantly higher in fibroblasts co -cultured with γδ T cells from SSc patients. 21

Sy stemic Sclerosis
The first indication that γδ T cells participate in the pathogenesis of fibrotic conditions arose from research in UCD-200 chickens. These animals dev elop a hereditary connectiv e tissue disease characterized by severe lymphocytic infiltration and fibrosis of skin and internal organs, a model of human progressiv e SSc. The skin infiltrating mononuclear cells in the deeper dermis were mainly TCR αβ cells, whereas the perivascular area of the papillary dermis was enriched for TCR γ δ + ly mphocy tes. 22

Bleom ycin model: evidence for involvement of γδ T cells
In the bleomy cin (BLM) model of lung fibrosis induced by a single intratracheal instillation of BLM, >80% of the γδ T cells in bronchoalv eolar lav age (BAL) fluid ex pressed the E-cadherin binding αEβ7 integrin, at lev els that were 2-3 times higher than on CD4 + or CD8 + T cells, suggesting a critical role for γδ T cells in the pathogenesis of BLM-induced lung fibrosis. 23 After ex posure to BLM, but not to Schistosoma mansoni eggs, the interleukin (IL)-17A that was produced by CD4 + and γδ T cells induced significant neutrophilia and pulmonary fibrosis. In parallel, IL-17A and IL-1β were increased in the BAL fluid of patients with idiopathic pulmonary fibrosis (IPF). 24 Bleomycin or IL-1β-induced lung injury also led to increased expression of early IL-23p19 and IL-1 7 A or IL-1 7 F. A v ery early IL-1 7 A and IL-1 7 F ex pression by ROR γ t(+) γ δ T cells could be demonstrated 24 h after BLM administration. In addition, IL-23p19 and IL-17A ex pressions or IL-1 7 RA signaling were necessary for pulmonary TGFβ1 production, collagen deposition, and ev olution to fibrosis. 25 Likewise, in the surfactant protein C/TNFα (SP-C/TNF) transgenic mouse, where the TNFα transgene is ov erex pressed in ty pe II pneumocytes, the absolute number of ly mphocytes recovered were approx imately four times that in littermates, and included γ δ T cells and B1 cells. In these mice the pulmonary lymphocytic infiltration is followed by fibrotic changes including accumulation of fibroblasts and deposition of ex tracellular matrix . 26 Moreov er, when ex perimental animals were injected intravenously with saline or collagen (Col)V 1 0 day s before intratracheal instillation of BLM, ColV -pretreated animals showed a significant reduction in lung inflammation compared with nontreated animals which associated with a lower proportion of γδ and CD4 + T cells. 27 After lung injury by BLM, γ δ T cells localized to the lung lesions and were the predominant source of IL-1 7 by flow cy tometry and real-time polymerase chain reaction (PCR). γ δ T cell knockout (KO) mice showed a significant reduction in cellular infiltration into the airway s, reduced ex pression of IL-6 in the lung, a significant delay in epithelial repair, and increased inflammation and fibrosis. 28 In another study , although γ δ T cell populations increased after BLM administration, pulmonary fibrosis was more severe in γ δ KO mice, as measured by collagen deposition (hy droxyproline) and histopathological features. Furthermore, there was no evidence of resolution of the fibrotic response up to 45 day s after BLM therapy. γ δ KO mice had decreased concentrations of IL-6, granulocy te colony -stimulating factor, chemokine C-X-C ligand (CXCL) 1 , and interferoninducible protein 1 0/CXCL1 0. Importantly , γ δ T cells produced all four of these cytokines, and γ δ T cells sorted from BLM-treated lung were sufficient to resolve fibrosis in γ δ KO mice. Ov erexpression of CXCL1 0 in the lung decreased the severity of fibrosis seen in the γ δ KO mice, and adoptive transfer of γδ T cells from CXCL1 0(-/-) mice failed to rev erse the sev ere fibrosis in γ δ KO mice. Thus, γ δ T cells promote resolution of fibrosis through production of CXCL1 0. 29 In addition, BLM-treated mice showed decreased lev els of IL-22 in the lung, and IL-22producing γ δ T cells were also decreased significant-ly in the lungs and spleens. Blockade of IL-22 deteriorated pulmonary fibrosis, and was associated with elev ated α-smooth muscle actin and ov eractivated Smad2. Thus, IL-22 produced by γδ T cells may play a protectiv e role in BLM-induced pulmonary fibrosis. 30 Furthermore, BLM-induced lung inflammation and subsequent fibrosis was ameliorated in osteopontin (OPN)-deficient mice, whereas OPN was ex pressed ubiquitously in the lung parenchymal and bone marrow-derived components. The TH1 7 differentiation of CD4 + αβ T cells and IL-17producing γδ T cells was reduced in OPN-deficient mice compared to wild-type mice, whereas TH1 differentiation and the percentage of IFN-γ-producing γδ T cells increased. Thus, OPN ex pressed in both parenchymal and bone marrow cell components contributed to BLM-induced lung inflammation and fibrosis by affecting the ratio of pathogenic IL-1 7 /protectiv e IFN-γ T cells. 31

Silicosis m odel
Silicosis ev olv ed ov er months after ex posure of inbred mice to cristobalite silica with accumulation of ly mphocytes in alv eolar spaces, in lung parenchy mal lesions and nodules, and in enlar ged bronchial-associated lymphoid tissues and thoracic ly mph nodes. The lung ly mphocytes were predominantly CD4 + T cells, with numerous CD8 + T cells, natural killer cells, and γδ T cells. 32 In another study upregulation of IL-1 7 A was associated with the dev elopment of ex perimental silicosis, but was markedly reduced in athy mic, γδ T cell-deficient or CD4 + T cell-depleted mice. γδ T ly mphocy tes and CD4 + T cells, but not macrophages, neutrophils, NK cells, or CD8 T cells, purified from the lungs of silicotic mice, markedly expressed IL-1 7 A. Acute alv eolitis induced by silica was IL-17A-dependent, but was dispensable for the late inflammatory and fibrotic lung responses. 33

Melphalan m odel
Ex posure to melphalan, a nitrogen mustard, induced an early burst of the pro-inflammatory cy tokines IL-1β, IL-6, and IL-23 in airways, followed by ex tensive infiltration of neutrophils in the lung tissue and airways. The acute phase was followed by a sustained ly mphocytic response that persisted for at least 1 4 days with resulting lung fibrosis. Engagement of T ly mphocytes, particularly the γ δ T cell subset, was crucial both for the acute cytokine and neutrophil response and for the late -phase lung fibrosis as indicated by the lack of response in γδ T cell-deficient mice. 34

Bacillus subtilis
C57 BL/6 mice repeatedly ex posed to Bacillus subtilis dev elop mononuclear infiltrates containing Vγ6 + /Vδ1 + γ δ T cells in the lung. In the absence of these, mice treated with B. subtilis had significantly increased collagen deposition in the lung, consistent with a regulatory role for V γ 6 + /Vδ1 + γ δ T cells. Ex posing transgenic Vγ6 + /Vδ1 + mice to B. subtilis decreased collagen content in the lung compared with wild-ty pe C57 BL/6 mice. Cy tokine analysis of lungs from wild-ty pe mice repeatedly exposed to B. subtilis demonstrated increased IL-17A concentrations. In the absence of IL-17 receptor signaling, IL-1 7 ra(-/-) mice had delayed clearance of B. subtilis, with increased lung inflammation and fibrosis. Although IL-17A was predominantly expressed by V γ 6 + /Vδ1 + γδ T cells, a compensatory increase in IL-1 7 A ex pression by CD4 + T cells was seen in the absence of γ δ T cells that resulted in similar levels of IL-1 7 A in the lungs of TCRδ(-/-) and wild-ty pe C57 BL/6 mice, suggesting an important role for IL-1 7 A-expressing γδ or αβ T ly mphocy tes in eliminating the micro-organism and preventing excessive inflammation and eventual lung fibrosis. 35 Likewise, in another study of this mouse model, γ δ T cells ex panded in the lung and inhibited collagen deposition. A subset of these γ δ cells represents the predominant source of the TH1 7 cytokine IL-22 in this model. Prev enting ex pression of IL-22 by mutating the ary l hydrocarbon receptor (AhR)-or inhibiting AhR signaling-accelerated lung fibrosis. Moreover, the presence of protective γ δ T cells and IL-22 diminished recruitment of CD4 + T cells to lung. 36 Finally , repeatedly exposing C57 BL/6 mice to B. subtilis resulted in a 33-fold increase in the number of CD4 + T cells and a 354-fold increase in γδ T cells in the lung. The γ δ T cells consisted almost entirely of V γ 6 + /Vδ1 + γ δ T cells. Treatment of C57 BL/6 mice with heat-killed versus live B. subtilis resulted in a 2-fold increase in the number of CD4 + T cells in the lung but no ex pansion of γ δ T cells. In addition, mice treated with heat-killed B. subtilis dev eloped significantly increased pulmonary fibrosis compared with mice treated with the liv e microorganism. Mice deficient in V γ 6 + /V δ1 + γ δ T cells, when treated with B. subtilis, had a 231 -fold increase in lung CD4+ T cells and significantly increased collagen deposition compared with wildty pe C57 BL/6 mice, again consistent with an im -munoregulatory role for the V γ 6 + /Vδ1 + γ δ T cell subset. 35

Tuberculosis
The acute phase of pulmonary tuberculosis induced in BALB/c mice by the intratracheal instillation of the liv e virulent strain H-37 Rv was characterized by an inflammatory infiltrate in the alv eolar capillary interstitium, blood v essel, and bronchial wall with formation of granulomas from 1 to 28 day s after infection and a predominance of TH1 cells. The chronic phase was characterized by pneumonia, focal necrosis, and fibrosis. γδ T ly mphocytes were inv olved both at the beginning (3 days) and the later stages of the infection. 37 In bov ine tuberculosis, there was an increase in the expression of TGFβ, and of ty pe I procollagen in advanced stage granulomas. As the granulomas adv anced, there was a steady increase in the number of CD68 + cells and γ δ T cells. 38

Carbon tetrachloride model
Increased IL-17A production was mainly detected in hepatic γδ T cells in wild-ty pe mice. Liv er fibrosis and IL-1 7 A production by γδ T cells were both significantly attenuated in toll-like receptor (TLR)-3 KO mice compared with wild-ty pe mice. Interleukin-17A-producing γδ T cells were in close contact with activated hepatic stellate cells (HSCs), suggesting a role for HSCs in IL-1 7 A production by γδ T cells. Interleukin-17A production by γ δ T cells was substantially increased upon co-culturing with ex osome-treated wild-ty pe HSCs or conditioned medium from TLR3-activated wild-type HSCs. Tolllike receptor-3 deficiency in HSCs contributed to decreased IL-17A production by γδ T cells, as well as liv er fibrosis. Thus, in liv er injury , the ex osomemediated activation of TLR3 in HSCs ex acerbates liv er fibrosis by enhancing IL-17A production by γδ T cells, which might be associated with HSC stimulation by unknown self-TLR3 ligands from damaged hepatocytes. 39 Chemokine receptor 6 (CCR6) and chemokine ligand (CCL) 20 ex pression were intrahepatically upregulated in patients with chronic liv er diseases compared to control liv er , with periportal accumulation of CCR6(+) mononuclear cells and CCL20 induction by hepatic parenchymal cells. In murine liv ers CCR6 was ex pressed by macrophages, CD4 + , and γδ T cells and upregulated in fibrosis, whereas CCL20 was induced by injury in primary hepatocytes. In the carbon tetrachloride (CCl4) and methionine-choline-deficient dietinduced murine models of chronic liv er injury , Ccr6(-/-) mice developed more severe fibrosis with enhanced immune cell infiltration than wild-ty pe mice, and CCR6 was required by IL-17-and IL-22ex pressing γ δ T cells for accumulation in injured liv er. Adoptiv e transfer of wild-ty pe γ δ, but not CD4 + T cells, into Ccr6(-/-) mice reduced hepatic inflammation and fibrosis in chronic injury to wildty pe lev el. The anti-inflammatory function of hepatic γδ T cells was independent of IL-17, whereas γ δ T cells co-localized with HSCs in vivo and promoted apoptosis of primary murine HSCs in a cell-cell contact-dependent manner, involving Fasligand (CD95L). 40

Fasciola hepatica (fluke)
Ten day s after primary infection with Fasciola hepatica (fluke), portal tract areas surrounding migratory tunnels were infiltrated with T cells and B cells. Micro-abscesses were distributed sporadically in the liv er parenchy ma, and y oung flukes were observed in the liv er tissue free from inflammatory cells. Chronic primary infections were characterized by perilobular fibrosis and a predominance of CD8 + and γ δ T cells. 41

Cryptosporidium parvum
Inoculation of mice deficient in αβ and γ δ T cells with Cryptosporidium parvum resulted in persistent infection and severe inflammatory bowel disease-like lesions contrasting with neonatal immunocompetent strains of mice which results in a transient, noninflammatory enteric infection. Glandular hy perplasia, abscess formation, and extensive fibrosis of the lamina propria and extensive hepatic periportal fibrosis were noted in persistently infected mice, which were not observed in mice deficient only in αβ T cells. 42

Rotavirus
Liv ers from rhesus rotav irus-infected mice that dev elop biliary atresia (BA) had 7 -fold more IL-1 7 messenger RNA than control mice (P=0.02). γ δ T cells were the ex clusive source of IL-1 7 . Mice that were developing BA and given antibodies against IL-1 7 had lower levels of liv er inflammation. Likewise, liv er tissues from patients with BA had 4.6 -fold higher lev els of IL-17 messenger RNA than control liv er tissues (P=0.02). 43

Schistosoma japonicum
In C57 BL/6 mice infected with S. japonicum expression and release of IL-17 was significantly higher in hepatic ly mphocy tes from infected mice. Interleukin-17 was induced in all CD4 + and NK cells by PMA and ionomy cin, but γδ T ly mphocy tes ex hibited the largest increase. Reducing IL-1 7 activ ity using anti-IL-1 7 A antibodies decreased infiltration of inflammatory cells and collagen deposition in the liv ers of infected C57 BL/6 mice. 44

CONCLUSION
In summary , the data clearly indicate the inv olv ement of γ δ T cells in major human fibrotic diseases, as well as in models of post-inflammatory fibrosis in animals. The ex perimental models, however, suggest BLM , Bleomycin; CXCL10, C-X-C motif chemokine 10; IL, interleukin; TH, T helper; TL,toll-like.
dual inv olv ement: a role in induction of inflammation that can lead to fibrosis by IL-17-secreting γδ T cells, contrasting with a role in prev ention of fibrosis related to γδ T cells that mediate either killing of cells responsible for secreting the ex tracellular matrix , or by subsets of these cells that secrete either matrix -degrading enzy mes, IL-22, CXCL1 0, or IFNγ (Table 1 , Figure 1 , and Workalemahu et al. 45 ). Much further study is required to elucidate the mechanisms that control pro-and antifibrotic effects of γδ T cells in human disease, since manipulation of these responses might enable prev ention or alleviation of sev ere human fibrotic diseases.

Figure 1. Hypothetical Model of γδ T Cell Involvement in Fibrosis.
A hypothetical model is depicted of how two types of γδ T cells, a T helper (TH) cell antigen-presenting cell (APC) and a myofibroblast, are involved in induction collagen secretion. The APCs are depicted presenting a peptidic antigen in M HC to the TH17 αβ T cell receptor, or a lipid antigen to a γδ T cell via a CD1 molecule, eliciting release of IL-17 that activates the myofibroblast to secrete collagen. Other γδ T cells, of the phosphoantigen -recognizing variety in humans, or, in the murine system, a subset secreting IL-22 and CXCL10, may become activated by other antigens presented by butyrophilins, to exert anti-fibrotic activity by inducing apoptosis of the myofibroblast or hepatic stellate cells, or by suppressing TH17 cells. Red depicts pro -and blue anti-fibrotic functions.