CD4 T lymphocytes in lung fibrosis: diverse subsets, diverse functions

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1 Review CD4 T lymphocytes in lung fibrosis: diverse subsets, diverse functions Sandra Lo Re, Dominique Lison, and François Huaux 1 Louvain Centre for Toxicology and Applied Pharmacology, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain, Brussels, Belgium RECEIVED MAY 30, 2012; REVISED SEPTEMBER 25, 2012; ACCEPTED OCTOBER 31, DOI: /jlb ABSTRACT The discovery of several subsets of CD4 Th lymphocytes has contributed to refine and to challenge our understanding of the roles of CD4 T cells in the pathogenesis of fibrotic lung diseases. Here, we review recent findings, indicating that CD4 T subpopulations possess contrasting pro- and antifibrotic activities in human and experimental lung fibrosis. Special attention is given to delineate the activity of the newly discovered CD4 T lymphocyte subsets (Tregs, Th22, and Th9) on fibroblast function and matrix deposition through the release of growth factors, cytokines, and eicosanoids. It appears that the function of a CD4 T lymphocyte subset or of a cytokine can differ with the disease stage (acute vs. chronic), pulmonary localization (bronchial vs. alveolar), cellular level (epithelial cell vs. fibroblast), or immune environment (inflammatory or immunosuppressive). Integrating our recent understanding of the contrasting functions of T lymphocyte subsets in fibrosis provides new insights and opportunities for improved treatment strategies. J. Leukoc. Biol. 93: ; Abbreviations: / deficient, -SMA -smooth muscle actin, AhR aryl hydrocarbon receptor, BALF bronchoalveolar lavage fluid, Be beryllium, CTGF connective tissue growth factor, EMT epithelial-mesenchymal transition, Foxp3 forkhead box p3, HCV hepatitis C virus, ILD interstitial lung disease, IPF idiopathic pulmonary fibrosis, SSc systemic sclerosis, Teff effector T cell, Tg transgenic, Treg regulatory T cell, TSK/ tight skin FIBROSIS AND ILDs Although initially beneficial, tissue healing processes may become pathogenic when not controlled appropriately, leading to considerable tissue remodeling and the formation of permanent scar tissue called fibrosis. In some cases, it might ultimately lead to organ failure and death [1]. Fibrosis is characterized by abnormal fibroblast proliferation and exaggerated deposition of ECM components, including collagen, altering deeply the architecture and the functions of the affected organ. According to the World Health Organization, 800 million people worldwide suffer from a fibrotic disease, including lung (pulmonary fibrosis), heart (cardiomyopathy), skin (diabetic skin, keloid formation), liver (progressive liver cirrhosis), kidney (diabetic glomerulosclerosis), and vasculature (atherosclerosis) [1]. These diseases can result from inadequate responses of the organism to diverse agents (e.g., toxics, drugs, radiations, or infections) or unknown factors. They can also accompany other chronic diseases, such as asthma, cancer, or diabetes [1]. Lung fibrosis is one the oldest recorded fibroproliferative disorders. It can be traced back to ancient Egypt, where it was recognized as a consequence of silica inhalation. Today, there are nearly 300 distinct injurious or inflammatory causes of ILD that can result in progressive lung scarring. Many other ILDs are referred to as idiopathic when arising for no obvious reason. Despite this long history, lung fibrosis remains a disease with no effective treatment [2]. Indeed, therapies currently used for pulmonary fibrosis, namely anti-inflammatory or immunosuppressive drugs, are largely ineffective, and lung transplantation remains the only viable intervention in end-stage pulmonary fibrosis [2]. Fibroblasts are mainly responsible for the increased collagen and matrix synthesis and deposition that occur in pulmonary fibrosis [3]. The origin of lung fibroblasts during pulmonary fibrosis is now better defined: the different sources, including proliferation of resident interstitial lung fibroblasts; differentiation of progenitor cells, called fibrocytes, from the bone marrow; or transition of epithelial cells to a fibroblast phenotype, a process termed EMT [3]. Resident intrapulmonary fibroblasts respond to a variety of stimuli during fibrogenesis and differentiate into myofibroblasts [3]. This myofibroblast differentiation is generally thought to represent the conversion of the quiescent nonsynthetic, noncontractile fibroblast to a hypersynthetic contractile phenotype overexpressing smooth muscle markers, such as -actin ( -SMA) [3]. Despite advances in the characterization of the fibrotic process and new understandings of the origins of fibroblasts and myofibroblasts, the exact molecular mechanisms of the disease remain poorly understood. Persistent immunological responses and inappropriate release of immune mediators have been 1. Correspondence: Louvain Centre for Toxicology and Applied Pharmacology (LTAP), Institute of Experimental and Clinical Research (IREC), Université Catholique de Louvain, Avenue Mounier 52, B , 1200 Brussels, Belgium. francois.huaux@uclouvain.be /13/ Society for Leukocyte Biology Volume 93, April 2013 Journal of Leukocyte Biology 499

2 linked to injury, damage, and scar formation into host tissues and definitely identified as crucial pathological processes. Among those, TGF-, PDGF [4], TNF- [5], and IL-1 [6] contribute to fibroproliferative reorganization of lung tissue by acting indirectly or directly on fibroblast biology. The role of macrophages during the development of fibrosis has been studied extensively, and these cells have been determined to possess deleterious functions as a key cellular source of profibrotic cytokines [7]. The presence of CD4 T lymphocytes in different organs is also invariably associated with fibrogenesis in human and animal studies, implying a potential role for these immune cells in the fibrotic process [8, 9]. The interest to explore the functions of CD4 T cells in the development of fibrosis also comes from their extraordinary capacity to produce cytokines, chemokines, and growth factors, which can stimulate fibroblast proliferation, differentiation, and collagen production [10]. CD4 Th CELLS AND EXPERIMENTAL LUNG FIBROSIS CD4 Th lymphocytes orchestrate the immune responses involved in host defense by producing cytokines. Distinct CD4 T cell lineages selectively produce distinct cytokines and express transcription factors controlling the whole immune responses. For instance, bacteria and virus are only eliminated efficiently with the help of the proinflammatory cytokine IFN-, produced by Th1 cells. In contrast, parasites are controlled by the activities of the Th2-derived cytokines, IL-4 and IL-13. Recently, this Th1 Th2 balance, established since the mid-1980s, has been completed, and new subpopulations have been discovered and implicated in autoimmune diseases (Th17), allergy (Th9), and cancer (Tregs). CD4 T lymphocyte development first occurs in the thymus before its establishment in peripheral lymphoid tissues. Upon specific TCR interaction in peripheral lymphoid tissue with cognate antigen presented by APCs, such as DCs, and with the cytokines in the microenvironment provided by the innate immune system, naive Th cells can differentiate into effector subsets [11] (Fig. 1). Lymphocyte homing is the process in which activated CD4 T cells located in peripheral lymphoid tissue return to their original challenge site to search for their cognate antigen [12]. Passage of T cells through the endothelial barrier of target tissues is a tightly controlled cascade of events depending of chemokine-mediated activation [13] (Fig. 1). The specific role of CD4 T cells during the development of fibrosis has been difficult to establish. Delineating the contribution of CD4 T cells has been complicated by their functional heterogeneity, breadth in antigen specificity, transient appearance in circulation, and sequestration in fibrotic tissue. In the following parts, we discuss recent progress in identifying the multiple roles of CD4 T subsets in orchestrating and mediating the inappropriate tissue repair during lung fibrosis. We highlight several recent reports that have used original approaches (Fig. 1) to provide new evidence for CD4 T cells as direct effectors in lung fibrosis. When appropriate, reference is also made to select examples of fibrosis in other organs (e.g., skin, liver). Figure 1. T cell homing after antigen exposure. T cell expansion is orchestrated by antigen-activated DCs in the LNs. By expressing CCR7, these presenting cells migrate from the injured tissue to LNs through a gradient of CCL21 and CCL19 chemokines. In the LNs, DCs present the antigen peptide, which is recognized by the antigen-specific TCR. Then, CD80/86 on DCs interact with their receptor, CD28, on T cells to generate activation signals. After activation, T cell homing is then directed by a gradient of chemokines (i.e., CCL22, CCL17, CCL18) and by the expression of CCR4. These chemokines are released by tissue resident cells, such as macrophages (M), epithelial cells (Ep), and fibroblasts (F) after recognition of the antigen (Ag; open circles, chemokines). General implications of T lymphocytes Mice with genetic defects leading to a total lack of T lymphocytes (SCID, nude, or Rag / ) develop alveolitis and pulmonary fibrosis similar to WT mice in response to bleomycin [14], FITC [15], or silica exposure [16], suggesting that T lymphocytes are not necessary for the development of pulmonary fibrosis, at least in these animal models. In contrast, other studies have suggested that the presence of T lymphocytes worsens pulmonary fibrosis. Nude mice exposed to silica developed reduced fibrosis and neutrophil infiltration in comparison with WT mice [17]. In addition, administration of bleomycin in athymic mice caused decreased fibroblast proliferation and less ECM accumulation compared with T lymphocyte-competent animals [18]. Finally, one animal study has suggested that T lymphocytes may be protective against fibrosis by show- 500 Journal of Leukocyte Biology Volume 93, April

3 Lo Re et al. CD4 T cells in lung fibrosis ing that exposure of nude and SCID mice to asbestos resulted in enhanced fibrosis in comparison with their WT counterparts. Reconstitution of these SCID mice with functional T lymphocytes reduced the excessive fibrotic response to asbestos [19]. The general implication of T cells in fibrogenesis was also investigated experimentally by using immunosuppressive drugs that inhibit T cell activation. Cyclosporin A-treated hamsters and rats presented less collagen accumulation than their controls in response to bleomycin [20], but the opposite was observed in BALB/c mice [21]. Treatment with rapamycin reduced skin fibrosis of TSK/ mice and skin and lung fibrosis in a bleomycin-induced SSc mouse model [22]. Rapamycin treatment inhibited proliferation and collagen production of TSK/ mouse fibroblasts in a dose-dependent manner [22]. Further, rapamycin inhibits experimentally induced urethral stricture formation in rabbits. This effect may be a result of its inhibition of fibroblast proliferation and collagen expression [22]. Altogether, these results using general inhibition of T cell activation indicate the difficulty of attributing a clear function for T cells as a whole during fibroproliferative disease of the lungs. Other studies have used an alternative approach consisting of specific T cell subset depletion by injecting blocking antibodies in WT animals. Several authors have demonstrated that the neutralization of CD4 and/or CD8 T cells after injection of anti-cd3, anti-cd4, or anti-cd8 antibodies in mice reduced the intensity of lung fibrosis in response to silica or bleomycin [23, 24], supporting the view that CD4 and CD8 T cell populations contribute to the fibrotic lesions. However, other authors have demonstrated that systemic depletion of CD4 and CD8 T lymphocytes in bleomycin or FITC pulmonary fibrosis models failed to affect the extent or severity of lung damage [15]. In T cell depletion studies, the controversial observations may be explained by variability in the degree of T cell depletion achieved [25]. The role of T cells was also investigated in the development of fibrosis by using deficient mice. In a mouse model of hypersensitivity pneumonitis that progresses to lung fibrosis upon repeated exposure to Bacillus subtilis, T cells expanded in the lung and inhibited collagen deposition through the production of IL-22 [26]. On the other hand, IL-17A-producing T cells promoted inflammation and pulmonary fibrosis in mice exposed to bleomycin [6], suggesting that depending on their cytokine production or the fibrosis model, T cells possess pro- or antifibrotic functions. These current uncertainties and conflicting results led researchers to study and develop models deficient in T cell stimulatory molecules. Mice whose T cells lack CD28 a central costimulatory cell-surface molecule necessary for full T cell activation showed markedly attenuated pulmonary fibrosis following exposure to bleomycin compared with WT mice [27]. It is important that transferring CD28-positive T cells from WT mice into CD28 / animals restored the fibrotic response to bleomycin [27]. Up-regulation of the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) mrna is clearly observed in a model of pulmonary fibrosis induced by anti-fas antibody inhalation [28]. However, other studies have suggested that activated T lymphocytes have no important role in the development of fibrosis. Indeed, B7-1 / /B7-2 / mice had only a limited reduction of the granuloma size or of the extent of fibrosis in an experimental model of liver fibrosis [29]. IL-2-Bax, an IL-2R-targeted chimeric protein, reduced the lymphocytic infiltration of the lungs in response to bleomycin, but this effect was not accompanied by a decrease in lung fibrosis [30]. The group of Luzina [31] used another approach, in which T lymphocytes were selectively attracted into healthy or injured mouse lungs. Pulmonary overexpression of CCL18, a chemokine that is highly selective for T cells, induced a prolonged perivascular and peribronchial infiltration of T lymphocytes, as well as collagen accumulation. They also showed that in this model, T lymphocytes express V 3 and V 5 integrins that are necessary for lymphocytic infiltration and T cell-associated TGF- activation [31]. A systemic depletion of T cells with antimouse lymphocyte serum prevented collagen accumulation in this model, despite the continuous expression of CCL18, suggesting that T cells were indeed the driving force of fibrosis [25]. Furthermore, Trujillo and colleagues [32] observed that mice deficient in CCR7, an essential chemokine receptor for T lymphocyte homing, presented reduced bleomycin-induced lung fibrosis compared with WT mice. Expression of CCL21 during chronic hepatitis C is implicated in the recruitment of T lymphocytes and the organization of inflammatory lymphoid tissue and may promote fibrogenesis in the inflamed areas via activation of CCR7 on hepatic stellate cells [33]. Moreover, intrapulmonary administration of bleomycin provoked lethal inflammatory and fibrotic responses in WT mice, but such responses were absent in CCR4 / mice, another chemokine receptor important for T cell homing [34]. In particular, CCR4 appears essential to drive the accumulation of T lymphocytes at the fibrotic site [35]. Finally, neutralization of CCL17, a ligand of CCR4, led to a reduction in pulmonary fibrosis induced by bleomycin [36], indicating that T lymphocytes play a role in fibrogenesis. Several mechanisms can be envisioned for T cell-driven fibrosis, as CD4 T lymphocytes produce numerous cytokines, growth factors, proteases, and other stimuli that may alter the phenotype and function of fibroblasts [37]. Luzina and colleagues [25] demonstrated that T lymphocytes cocultured with fibroblasts induced an up-regulation of TGF- 1 and stimulated collagen production. It is also likely that cytokines and growth factors produced by T cells not only directly activate resident fibroblasts but also may contribute to fibrocyte homing and fibroblastic transdifferentiation. For example, CD4 and especially CD8 T cells are capable of producing CCL3 (MIP-1 ), and this chemokine is involved in pulmonary recruitment of fibrocytes in association with development of pulmonary fibrosis in an animal model [25]. Moreover, in vitro and in vivo, the presence of TGF- -producing CD4 T cells greatly enhanced the differentiation of monocytes into fibrocytes. In vivo, in a model of renal fibrosis, the number of fibrocytes in SCID mice was significantly lower than in WT BALB/c mice, and deposition of collagen was reduced significantly in SCID mice. The differentiation of fibrocytes was also markedly reduced in mice treated with anti-cd4 antibodies [10]. EMT, in which epithelial cells acquire a fibroblast-like phenotype, is Volume 93, April 2013 Journal of Leukocyte Biology 501

4 driven by TGF-, predominantly produced by macrophages [38]. It can be also postulated that besides macrophages, TGF- -secreting CD4 T cells can also participate in this central process for fibrosis. In conclusion, numerous studies have shown that infiltrating T lymphocytes and mainly CD4 T cells are associated with the severity of fibroproliferative disorders of the lung. The discovery of several subsets of Th lymphocytes has led to a better characterization of the roles of T cells in fibrotic lung disorders as developed below. Effect of proinflammatory Th1, Th17, and Th22 cells Th1 cells and their related cytokine (IFN- ) participate in the establishment of bleomycin-induced lung fibrosis by promoting chronic lung inflammation and fibroblast proliferation [23, 39]. IFN- mrna and IFN- protein levels were increased in silica- or bleomycin-treated mice compared with their respective saline controls [39]. IFN- gene-deleted mice exposed to silica developed less extensive silicosis and reduced lung collagen accumulation than WT mice. Moreover, lung collagen content was significantly lower in IFN- / mice exposed to bleomycin compared with their WT C57BL/6J mice [6, 39]. These data suggest a profibrotic role of IFN- in two models of experimental lung fibrosis (Fig. 2). In contrast, several in vitro and in vivo studies have suggested that IFN- may control the fibrotic process. Indeed, Kiwamoto and colleagues [40] have observed that IFN- has multiple antifibrotic properties by directly suppressing fibroblast proliferation and collagen production or by indirectly attenuating the effects of IL-4 and IL-13 on fibroblasts. In addition, pretreatment of WT mice with IFN- -neutralizing antibodies enhanced fibrosis following lung injury, indicating a protective effect of IFN- against fibrosis [41] (Fig. 2). In experimental pulmonary and liver fibrosis, infiltrated NKT and NK cells control fibrosis by producing high amounts of IFN- [42]. Recently, the participation of the Th1 subset was also explored by using mice deficient in T-bet, a key transcription factor for Th1 development. After bleomycin treatment, collagen content in the lung increased twofold in T-bet / mice but was unaffected in WT BALB/c mice, indicating that preventing expression of the Th1 transcription factor T-bet renders bleomycin-resistant BALB/c mice sensitive to bleomycin [43]. These data contribute to define new, antifibrotic functions for Th1 cells. Recent experiments have strengthened the notion that inflammatory Th cells favor the establishment of fibrosis. Indeed, experimental studies have clarified the role of Th17 lymphocytes, another proinflammatory Th lymphocyte subset. IL- 17A production is markedly increased in the BAL, lung, and thoracic LNs after bleomycin instillation in mice [6]. Gasse and colleagues [44] showed that bleomycin or IL-1 -induced lung injury leads to increased expression of early IL-23p19 (inducing Th17 differentiation) and IL-17A or IL-17F expression. As shown with gene-deficient mice, IL-17A, IL-23p19, and IL- 17RA signaling but not IL-17F are required for inflammatory and fibrotic responses to bleomycin [6, 44]. Moreover, neutral- Figure 2. Beneficial and deleterious effects of CD4 T cell subsets in lung fibrosis. CD4 T cells are involved in pulmonary fibrotic disease by their cytokine production. Th1 cells induce fibroblast proliferation by producing IFN-, but other studies have reported that this subpopulation possesses antifibrotic functions. Th2 cells are the most profibrotic. They produce IL-4 and IL-13, known to induce fibroblast differentiation and ECM synthesis, leading to fibrosis. Th17 cells produce IL-17, which activates the proliferation of fibroblasts. Th22 secrete IL-22, which inhibits ECM deposition. Th9 produce IL-9 which controls the development of fibrosis through PGE 2, a well-known antifibrotic mediator, but also CTGF, which induces ECM accumulation and fibroblast differentiation. Tregs stimulate fibroblast proliferation by secreting PDGF-B and TGF- but inhibit Teff activities (open circles, chemokines; green arrows, activation; red lines segments, inhibition). Ep., Epithelial cells; Fibro., fibroblast. 502 Journal of Leukocyte Biology Volume 93, April

5 Lo Re et al. CD4 T cells in lung fibrosis ization of IL-17A in vivo promoted the resolution of bleomycin-induced acute inflammation, attenuated pulmonary fibrosis, and increased survival [44, 45]. In addition, the IL-17A antagonism inhibited silica-induced chronic inflammation and pulmonary fibrosis [46]. IL-1 -induced lung fibrosis is also dependent on IL-17A in mice and a cooperative role for IL-17A, TGF-, and IL-6 is involved in this pathological pathway [6, 45]. Blockade of IL-17 can also improve myocardial fibrosis in heart failure and liver fibrosis in cholestatic and hepatotoxic models [47, 48]. Mechanistically, a direct effect of IL-17A on tissue-resident cells has been offered recently by several authors using in vitro approaches to explain its inflammatory and fibrotic functions. First, IL-17A not only enhances cytokine (CCL2, IL-6, IL-11, and VEGF) and chemokine (IL-8, MIP-2, and keratinocyte chemottractant) production by fibroblasts [49, 50] but also induces their proliferation, differentiation, and matrix protein and metalloproteinaseproduction via a possible TGF- -dependent mechanism [48, 51]. Epithelial cells also represent an important target of IL-17A. This cytokine highly increases the synthesis and secretion of collagen, reduces the autophagic degradation of collagen, and promotes the EMT process in an apparent TGF- -dependent manner in epithelial cell cultures [46]. Finally, IL-17A stimulates fibrocyte proliferation, differentiation, and inflammatory mediator production [52]. These results strongly argue for a direct profibrotic role of IL-17A and Th17 lymphocytes (Fig. 2). Recent elements have, however, tempered these conclusions by showing that IL-17 does not represent a master regulator of fibrosis in all experimental models. IL-17R / did not strongly disrupt fibrosis, and neutralizing anti-il-17a antibodies were not able to modulate silica-induced lung fibrosis [51]. In addition, Nakashima and colleagues [53] demonstrated recently that IL-17A signaling has an antifibrogenic effect via the upregulation of mir-129-5p and the down-regulation of CTGF and 1-collagen in SSc fibroblasts. As for Th1/IFN-, itis becoming clear that Th17/IL-17 may positively or negatively affect fibrosis, probably according to the inflammatory or noninflammatory status occurring during the development of fibrosis. Th22 cells represent a new subset of T cells, producing IL- 22, clearly separate from Th17 and other known T cell subsets with distinct gene expression and function [54]. A transcriptome profile of IL-22-producing Th cells included genes encoding proteins involved in tissue remodeling, such as FGFs, and chemokines involved in angiogenesis and repair [54]. IL-22 alone efficiently induced rapid wound healing in an in vitro model of keratinocyte injury. IL-22-neutralizing antibodies reversed this effect, and ril-22 restored rapid wound healing [54]. In this context, Simonian and colleagues [26] have observed that preventing the expression of IL-22, by mutating the AhR or inhibiting AhR signaling, accelerated infectioninduced lung fibrosis. Direct blockade of IL-22 also enhanced collagen deposition in the lung, whereas administration of ril-22 inhibited lung fibrosis, demonstrating an unexpected, protective role of IL-22 against fibrosis in this case [26] (Fig. 2). Furthermore, IL-22 overexpression or treatment decreased liver -SMA expression and accelerated the resolution of liver fibrosis after CCL4 treatment [55]. Fibrosis in other tissues may be affected by the IL-22 pathway. Deletion of IL-22 exacerbated liver fibrosis, whereas administration of IL-22 or IL-17E (a negative regulator of IL-23) protected mice from bile duct ligation-induced liver fibrosis [48]. The antifibrotic effect of IL-22 is likely mediated by the release of chemokines involved in regular wound healing in the skin, the induction of fibroblast senescence in the liver, and the regeneration of the epithelial cell barrier in the lung [54 56]. Altogether, IL-22 seems a potential antifibrotic cytokine, and increasing IL-22 levels may be beneficial in pulmonary fibrosis, specifically related to inflammation. The protective effect of IL-22 during fibrosis is not observed in all mouse models of fibrosis, as IL-22 / mice developed normal silica-induced fibrotic granuloma formation [51] and hepatic fibrosis following chronic infection with the helminth parasite Schistosoma mansoni [57]. Profibrotic activities of Th2 cells Numerous studies have strengthened the concept that fibrosis is a Th2 disease and that the Th2 cytokines IL-4, IL-13, and IL-5 each have pivotal functions in the regulation of tissue remodeling and fibrosis. Receptors for IL-4 and IL-13 are found on fibroblast subtypes, and in vitro studies have shown that ECM protein synthesis [23] and myofibroblast differentiation are induced by IL-4 or IL-13 stimulation [40] (Fig. 2). Neutralizing antibodies to IL-4 or IL-13 have significantly reduced collagen deposition in diverse models of tissue fibrosis: in the lungs of Aspergillus fumigatus conidia [58] or bleomycin-treated mice [59], in the liver of S. mansoni-infected mice [60], and in the skin of mice developing scleroderma-like syndrome [61]. Overexpression of IL-13 in the lung triggered significant subepithelial airway fibrosis in mice in the absence of any additional inflammatory stimulus [62]. Liu and coworkers [63] have observed that compared with WT controls, IL-4 / and IL-13 / mice showed reduced bleomycin-induced lung expression of FIZZ1 (known to induce myofibroblast differentiation in vitro) and fibrosis, which were essentially abolished in IL-4 and IL-13 double-deficient mice. Moreover, in response to FITC, IL-13 / and IL- 4/13 / mice were significantly protected against lung fibrosis, as measured by total lung collagen levels and histology [64]. Overexpression of GATA-3, a transcription factor for Th2 development, enhanced the development of subepithelial fibrosis, whereas overexpression of T-bet, a transcription factor for Th1 differentiation, did not affect this process [40]. All of these studies have clearly identified the IL-4/IL-13 couple as a major fibrotic mediator. In animal models, IL-5 has been shown to be expressed in mice after bleomycin exposure, and treatment with anti-il-5 antibodies reduced bleomycin-induced fibrosis [65]. In mice deficient in IL-5 and/or CCL11 (eotaxin), tissue eosinophilia was abolished, and the ability of CD4 Th2 cells to produce the profibrotic cytokine IL-13 was impaired significantly [66]. IL-5-overexpressing mice developed marked lung fibrosis and highly produced IL-13 and TGF- after bleomycin treatment [23]. Thus, one of the key roles of IL-5 and eosinophils may be to facilitate the production of important profibrotic cytokines, such as IL-13 and/or TGF-, which function as key mediators of fibrosis [66]. Taken together, these results provide consistent evidence that Th2 Volume 93, April 2013 Journal of Leukocyte Biology 503

6 polarization of the immune response is distinctly associated with the development of pathological fibrosis. It is notable that the Th2/IL-13/IL-4 axis is not required in all types of pulmonary fibrosis. By evaluating the intensity of silica-induced lung fibrosis in mice deficient for IL-4 and IL- 4R, Misson and colleagues [67] showed that the establishment of pulmonary fibrosis was independent of IL-4 and IL-13. In addition bleomycin-induced fibrosis appeared to be IL-13- and IL-13R 2-independent. Indeed, C57BL/6 WT, IL-13 /, and IL-13R 2 / mice all displayed indistinguishable levels of interstitial fibrosis after intratracheal delivery of bleomycin [6]. Thus, Th2 cytokines appear to function as dominant but not obligatory profibrotic mediators. Role of Th9 lymphocytes in lung fibrosis The newly discovered Th9 subset develops in response to combined signals from TGF- and IL-4, two important profibrotic mediators. IL-9 promotes inflammation by stimulating growth of hematopoietic cells, particularly mast cells, and the secretion of proinflammatory factors, including chemokines. Several evidences support an important role for Th9 cells in allergic airway inflammation and remodeling. Anti-IL-9 antibodytreated mice were protected from OVA or house dust-mite allergen-induced pulmonary subepithelial fibrosis with a concomitant reduction in mature mast cell activation and in expression of the profibrotic mediators TGF- 1, as well as FGF-2 [68]. van den Brule and her coworkers [69] have observed that Alternaria alternata-exposed IL-9-overexpressing Tg mice (Tg5) presented a marked subepithelial fibrotic response with high collagen and fibronectin deposition in bronchial areas. This profibrotic role of IL-9 in airway fibrosis was associated with the increased accumulation of eosinophils and the growth factor CTGF. Arras et al. [70] used Tg5 mice to investigate the role of IL-9 in the alveolar fibrotic process. In contrast to the finding at the airway level, they showed that silica-treated Tg5 mice exhibited reduced alveolar collagen deposition. Moreover, bleomycin induced less lung injury and less epithelial damage in IL-9-overexpressing mice as compared with non-tg mice [71]. COX2 inhibitors, which reduced the antifibrotic PGE 2 production, suppressed the protection in Tg5 mice, supporting the idea that IL-9 controls silica- and bleomycin-induced alveolar damage through a PG-dependent mechanism [71] (Fig. 2). Altogether, these data suggest a dual role of IL-9 in lung fibrosis, as apparently anti- or profibrotic, depending on the airway or alveolar localization of the process, respectively. The fact that IL-9 possesses the remarkable ability to induce the well-characterized antifibrotic mediator PGE 2 suggests that providing IL-9 may be a beneficial molecule in alveolar fibrosis. Implication of Tregs Tregs have become important players in regulating immune responses by counterbalancing inflammation and Teff functions. Besides the number of studies investigating the contribution of Teff subsets in pulmonary fibrosis (see above Th1, Th2, and Th17), the possible role of Tregs has received relatively less attention. As Tregs highly express the master regulator of fibrosis TGF-, these immunosuppressive cells have been proposed to be involved in lung fibrosis. CD4 Foxp3 Tregs were recruited persistently in the lungs in experimental fibrosis induced by silica particles and contributed to lung fibrosis by stimulating fibroblast proliferation through the secretion of PDGF-B in noninflammatory conditions [72]. A profibrotic role of Tregs was also demonstrated by depleting them with anti-cd25 antibodies that attenuated the progression of silicainduced lung fibrosis [73]. Early depletion of Tregs has also reduced bleomycin-induced lung fibrosis and TGF- 1 expression in mice. The observation that Treg numbers in lung tissue from human TGF- 1 Tg mice with advanced lung fibrosis are increased significantly compared with their counterparts without fibrosis also supports the active role of Tregs in lung fibrosis [74]. Tregs can increase collagen production and deposition via TGF- axis and chitinase 3-like-1 activity in fibroblasts after viral infections [75]. Treg-derived growth factors are not limited to PDGF and TGF-. Indeed, Tregs could produce the ECM protein fibronectin that contributes to fibrotic responses in a cardiac graft model by stimulating fibroblast adhesion, growth, migration, and differentiation [76]. Interestingly, there is also an apparent interplay between Tregs and tissue cells. In vitro, Tregs directly increased TGF- expression by epithelial cells and proliferated, differentiated, and produced TGF- in contact with fibroblasts [10, 74, 77]. In addition, IL-10/TGF- -producing macrophages induced Treg differentiation from Teffs in vitro and in vivo [78]. By their strong capacity to dampen deleterious inflammation, Tregs have also been proposed as important regulators of inflammation-related fibrosis. In response to silica, depletion of Treg-immunosuppressive activity resulted in increased lung inflammation, accumulation of Teff lymphocytes, and sustained expression of profibrotic Teff cytokines (IL-4, IL-13, IFN-, IL-17) [72], leading to lung fibrosis. Similarly, worsened hepatic fibrosis was also observed after in vivo neutralization of Treg influx in a mouse model of bile-duct ligation, resulting in exacerbated inflammation, proinflammatory cytokine production, and Teff functions [79]. The beneficial effect of Tregs on inflammation-dependent fibrosis was confirmed in a Tregtransfer strategy in several studies. Transfer of schistosomia infection-expanded CD4 Foxp3 Tregs reduced Th2-fibrotic granulomas by containing inflammatory pathology during intestinal schistosomiasis [80]. Adoptively transferred Tregs greatly reduced ventricular fibrosis and interstitial myofibroblast accumulation by attenuating inflammatory cell numbers and activity of the TGF- 1 axis [81]. Injection of Tregs differentiated by macrophage-derived TGF/IL-10 significantly attenuated renal inflammation and structural injury in murine adriamycin nephrosis without promoting renal fibrosis [78]. Finally, retroviral transfer of the Foxp3 gene at the onset of granuloma formation enhanced Foxp3 expression in the liver granuloma CD4 CD25 T cells and strongly suppressed full granuloma development [82]. These studies indicate that the implication of Tregs differs according to the subtype of lung fibrosis (dependent or not of inflammation); they can exacerbate the fibrotic process in noninflammatory conditions by producing 504 Journal of Leukocyte Biology Volume 93, April

7 Lo Re et al. CD4 T cells in lung fibrosis growth factors for fibroblasts or limit the development of fibrosis that results from uncontrolled and persistent inflammation. CD4 Th CELLS IN HUMAN FIBROTIC DISEASE Several studies have explored the role of CD4 T lymphocytes and their polarization in the fibrotic lung disorders in humans. These studies globally strengthen the observations from animal research. CD4 T cells are consistently found in the alveolar walls and interstitial and perivascular areas in the lungs of IPF patients, and their presence is associated with a lethal deterioration of the pulmonary function and poor survival [83, 84]. CD4 T lymphocytes are also increased in the lung tissue and BAL of patients exposed to Be [9], asbestos [85], or irradiation [86], with hypersensitivity pneumonitis [87] or asymptomatic ILD [88]. Their accumulation is correlated with fibrosis in asthma [1] and an inadequate scarring process in sclerodermic patients [8]. In contrast to sarcoidosis, the CD4/CD8 ratio in BALF from patients with lung fibrosis is not increased significantly and appears as a weak indicator to discriminate fibrotic lung disorders [89]. Nevertheless, recent studies strongly suggested that particular subpopulations of CD4 T lymphocytes are accumulated in lung fibrotic disorders and play a pivotal role in fibrogenesis. Indeed, CD28 or CCR4 CD4 T lymphocytes are specifically recruited and may identify patients at high risk for clinical deterioration [90]. The Th2 cytokines IL-4 and IL-13 are found at increased levels in the BALFs of IPF patients, in the pulmonary interstitium of individuals with fibrosing alveolitis, and in PBMCs of those suffering from periportal fibrosis [1]. T cells from patients with SSc express IL-5, whereas cells from normal individuals do not [91]. In addition, imatinib treatment appears to confer its potential therapeutic effect in fibrosis by decreasing an IL-4-producing Th2 cell emergence [92]. Also, the progression of a granulomatous response in sarcoidosis toward irreversible fibrosis can be explained by a change of the alveolitis, phenotypically from a Th1 polarization toward a Th2 response, characterized by the production of IL-4, IL-13, and IL-10 [93]. Th1 cells, Th1 chemokines, and IFN- were reduced in lung of patients with lung fibrosis, comforting an imbalance of the Th1/Th2 pathway in favor of the Th2 axis [94]. However, recent human studies support for the marked presence of another inflammatory Th subtype in the fibrotic lesions. IL-17Aproducing T cells were, in particular, detected in fibrotic interstitium of IPF patients [95]. Also, IL-17A and its regulator cytokine IL-1 are increased in BALF of IPF patients [6]. Interestingly, IL-17A levels in lymphocytes and serum were also found elevated in patients developing SSc and fibrosis [96]. The contribution of IL-17-mediated mechanisms to the pathogenesis of IPF remains, however, underexplored and will require additional human studies. Cellular infiltrates in fibrotic lung, liver, lymphatic, pancreas, and renal tissue were shown to include high proportions of Tregs in situ [82, 97 99] or peripheral blood [100]. A fibrotic environment in the lung results in an increased abundance of TGF- 1 and CD4 CD25 FOXP3 Tregs and a decreased proportion of activated Teffs [101]. There is a massive tissue infiltration of T lymphocytes secreting a high level of IL-10 and TGF- in IPF and sarcoidosis, evolving into progressive fibrosis patients [98, 102]. Spatial colocalization and temporal concordance in levels of TGF- 1 Tregs and collagen deposition were also noted in lymphatic tissues [103]. Yapici et al. [104] and Taflin et al. [105] found a positive correlation between the presence of Tregs in renal granulomas and the degree of interstitial fibrosis. Furthermore, the process of fibrosis in active and diffuse forms of SSc is associated with a FOXP3 signature [106]. Other studies mentioned, however, that the presence of Tregs is correlated with a control of the development of fibrosis. Indeed, an extensive lymphocyte infiltration, abundant in CD4 FOXP3 Tregs, was present in HCV-infected livers, although absent from healthy liver, but these Tregs were more numerous in HCV-infected livers, showing only limited fibrosis [97]. Fewer FOXP3 Tregs and TGF- and IL-10 cells were found in the skin of patients with scleroderma than in comparison with healthy individuals. Similarly, there were reduced TGF- and IL-10 serum levels and fewer circulating CD4 CD25 bright FOXP3 cells in patients with SSc or morphoea (connective tissue diseases characterized by fibrosis of the skin) compared with unaffected patients [107]. In addition, Treg counts have been shown to be reduced in the lung, the BAL, or blood of patients developing IPF or silicosis [108, 109]. Treg-induced suppression of Th type 1 and 2 cytokine secretion was impaired in the BAL of patients with IPF. Moreover, the defective function of BAL Tregs correlated highly with parameters of disease severity [108]. As observed in animals, these human studies altogether reported a positive or negative correlation between the presence of Tregs and the severity of the fibrotic disease, probably in function of the amplitude of the inflammatory response. In an inflammatory microenvironment, IL-22 appears to induce, in synergy with other proinflammatory cytokines, chronic tissue inflammation and fibrotic disease progression in humans. IL-22 has been found to be pathogenic in rheumatoid arthritis via promoting neutrophil recruitment, synovial fibroblast proliferation, and CCL2 production [110, 111]. In human asthmatic lung tissue, IL-9 can drive allergic inflammation and fibrosis of the airways by increasing mast cells influx and profibrogenic mediator production [68]. Besides these roles in the disease progression, IL-22 and IL-9 are, however, assumed to possess powerful beneficial activities in other fibrotic-related diseases [112]. Indeed, their receptors are preferentially expressed on various tissue epithelial cells, and this pathway appears to be critically important at barrier surfaces where epithelial cells play an active role in the initiation, regulation, and resolution of the tissue-repair process. Interestingly, the BALF of patients with pulmonary sarcoidosis and IPF had lower levels of IL-22 than that of the normal control subjects [113]. The serum IL-9 level in patients with SSc was associated with lower frequency and severity of pulmonary fibrosis. IL-9 may play a key role in the regulation of lung fibrosis in SSc by enhancing regulatory B cell recruitment in the lungs [114]. Volume 93, April 2013 Journal of Leukocyte Biology 505

8 PARTICULARITIES OF CD4 T CELL SUBSETS IN THE DEVELOPMENT OF FIBROSIS In contrast to the dogma that a unique Th subpopulation is implicated in lung fibrosis, the new data emerging in the literature and described above strongly suggest that distinct CD4 subpopulations have diverse deleterious or beneficial activities during fibrosis, also in function of the disease context. Indeed, certain Th subsets appear to drive the fibrotic process by exacerbating inflammation, ECM production, or fibroblast and epithelial cell differentiation. Other Th cells control fibrosis by reducing inflammation or enhancing the expression of antifibrotic mediators, such as PGs. The above analysis indicates, therefore, that apparent controversies concerning the role of T lymphocytes in fibrogenesis can be resolved by integrating a higher degree of complexity. Indeed, it appears that whereas cytokines or Th cells clearly participate in the fibrotic process, the functions of a given cell subset or cytokine can differ with the disease stage (acute vs. chronic), pulmonary localization (bronchial vs. alveolar), cellular level (epithelial cell vs. fibroblast), or immune environment (inflammatory or immunosuppressive). CD4 T CELL ACTIVATION IN LUNG FIBROSIS It also remains to be determined whether specific antigen engagement by T cells is required for the development of fibrosis. Some human studies have observed an accumulation of memory T lymphocytes, which are specific for an antigen (from self or nonself) in the lung or the liver of fibrotic patients [100, 102]. Self-antigens may include neoaccessibility of a normally sequestered self-determinant for which tolerance has been lost or was never acquired and are frequently unknown [102] or identified as Annexin 1 [115]. Specific oxidation of an autoantigen can directly participate in the pathogenesis of sclerosis, for example [116]. Moreover, reactive oxygen intermediates produced during inflammation chemically modify self-proteins on APCs, thus creating neoantigenic determinants [117]. Nonself antigens associated with fibrotic pathologies are identified and can be products of a chronic microbial infection or exogenous environmental proteins [102]. However, the mechanism by which nonpeptidic fibrotic agents, such as Be, silica, or bleomycin, trigger T cell accumulation and activation remains less clear. For instance, chronic Be fibrotic disease is characterized by the accumulation of Be-specific CD4 in the lung, but it is not known whether Be-responsive T cells recognize Be alone or conformational changes in the self-peptide induced by Be [9]. In conclusion, there are different hypothetic mechanisms for T lymphocyte activation following fibrotic agent exposure in the LNs. It is possible that lymphocytes are stimulated directly by the agent presented by APCs (Fig. 3, 1) or acting as a superantigen [118, 119] (Fig. 3, 2). There is also a possible interaction between APCs and lymphocytes, by the release of broad cytokines able to unspecifically stimulate one or both cell types [118] (Fig. 3, 3). In this context, Thatcher and colleagues [120] have already noted that mice expressing a Tg TCR- gene, which prevents effective recognition of antigens other than a single epitope of hen-egg Figure 3. Hypothetic mechanisms of T cell activation during fibrosis. When antigens are present in the alveoli, they are taken up by alveolar macrophages (M). DCs interact with antigen-loaded macrophages to take over the antigen. These DCs may then migrate to the draining LNs and stimulate lymphocyte interactions. In the LNs, there are three hypothetic mechanisms for T lymphocyte activation during lung fibrosis: (1) reactive oxygen intermediates produced during inflammation chemically modify self-proteins on APCs, thus creating neoantigenic determinants; (2) lymphocytes are stimulated directly by the fibrotic agent acting as a superantigen; (3) DCs and lymphocytes interact together, possibly by the release of cytokines able to unspecifically stimulate one or both cell types. 506 Journal of Leukocyte Biology Volume 93, April

9 Lo Re et al. CD4 T cells in lung fibrosis lysozyme, instilled with bleomycin, presented no difference in the inflammatory or fibrotic response compared with control mice, suggesting that fibrogenesis involves an antigen-independent mechanism in the case of nonpeptidic molecules (as illustrated in the third hypothesis; Fig. 3, 3). POTENTIAL THERAPEUTIC IMPLICATIONS Strategies for treating lung fibrosis have been proposed, based on the assumption that the fibroproliferative disease is a result of profibrotic CD4 T lymphocytes emerging during the establishment of the disease. Unfortunately, general treatments inhibiting T lymphocytes in humans and animal models failed to convincingly control the fibrotic disease and to delineate whether T cells still represent valid and promising therapeutic targets. However, it is now evident that CD4 T cell subpopulations possess distinct roles in fibrosis and that diverse CD4 T cell pathways drive or, in contrast, control fibroproliferative disease. It is important that CD4 T cell subsets are extremely imbricated with each other, leading to the fact that inhibition of one population may be compensated by the expansion of the other. This last point is well illustrated by several experimental studies, where for instance, rapamycin or cyclosporin treatment aggravates lung fibrosis in mice by increasing expression of profibrotic Th2 cytokines and reducing expression of antifibrotic Th1 cytokines [21]. We confirmed this important point by depleting PDGF-producing Tregs in silica-induced lung fibrosis in mice [72]. Depletion of these immunosuppressive Tregs allowed a massive accumulation of Teffs possessing strong profibrotic functions by releasing IL-4, for instance. Consequently, the cytokine products of CD4 T lymphocytes appear to be more suitable biological targets than the Th subset per se. This therapeutic option has already been used in large groups of patients developing lung fibrotic diseases. Unfortunately, the efficacy of rifn- or Gleevec treatment (Fig. 4) remains controversial and disappointing, although a differential effect in specific subgroups of patients has been postulated. Interestingly, this individual susceptibility determining a clinical response to treatment could be related to the diversity of the profibrotic CD4 T subpopulations [121, 122]. Thus, the identification of the Th lymphocyte profile (inflammatory Th, Th2, or Tregs) in a fibrotic patient should certainly help the clinician to select the most appropriate therapy to inhibit key effector Th cytokines (IL-17, IL-4/ IL-13, or PDGF/TGF-, respectively; Fig. 4). It remains to be determined whether IL-9 and IL-22 might be useful in treating patients with lung fibrosis, based on the new discovery of their interesting antifibrotic activities. The direct use of these Th cytokines to control fibroblast functions and regenerate adequate lung epithelia in patients is not, however, without potential hazards. IL-22 and IL-9 are associated with autoimmune and allergic diseases or tumorigenesis, and like many cytokines, they have perplexing proinflammatory and anti-inflammatory effects. Thus, additional clinical and experimental studies are necessary to strengthen this attractive therapeutic possibility. Figure 4. Clinical perspectives: appropriate treatment for each fibrotic patient. The identification of a Th lymphocyte profile (inflammatory Th, Th2, or Tregs) in a fibrotic patient should certainly help the clinician to select the most appropriate therapy inhibiting key effector Th cytokines (IL-17, IL-4/IL-13, or PDGF/TGF-, respectively). The use of antifibrotic molecules (ril-9 or ril-22) might also be interesting to treat the fibrotic patient. AUTHORSHIP S.L.R. and F.H. were involved in the conception and design of the figures and wrote the manuscript. D.L. contributed to writing the paper. ACKNOWLEDGMENTS This work was funded by the Fonds de la Recherche Scientifique Médicale; by Actions de Recherche Concertées, Communauté Française de Belgique, Direction de la Recherche Scientifique; and by the European Commission under FP7- HEALTH-F4-2008, Contract No F.H. is a Research Associate with the Fonds de la Recherche Scientifique (FNRS), Belgium. REFERENCES 1. Wilson, M. S., Wynn, T. A. (2009) Pulmonary fibrosis: pathogenesis, etiology and regulation. Mucosal Immunol. 2, Du Bois, R. M. (2010) Strategies for treating idiopathic pulmonary fibrosis. Nat. Rev. Drug Discov. 9, Tanjore, H., Xu, X. C., Polosukhin, V. V., Degryse, A. L., Li, B., Han, W., Sherrill, T. P., Plieth, D., Neilson, E. G., Blackwell, T. S., Lawson, W. E. (2009) Contribution of epithelial-derived fibroblasts to bleomycin-induced lung fibrosis. Am. J. Respir. Crit. Care Med. 180, Bonner, J. C. (2004) Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev. 15, Distler, J. H., Schett, G., Gay, S., Distler, O. (2008) The controversial role of tumor necrosis factor in fibrotic diseases. Arthritis Rheum. 58, Wilson, M. S., Madala, S. K., Ramalingam, T. R., Gochuico, B. R., Rosas, I. O., Cheever, A. W., Wynn, T. A. (2010) Bleomycin and IL Volume 93, April 2013 Journal of Leukocyte Biology 507