Role of TGF-β3 in the regulation of immune responses T. Okamura1,2, K. Morita1, Y. Iwasaki1, M. Inoue1, T. Komai1, K. Fujio1, K. Yamamoto1

1Department of Allergy and ABSTRACT ences, indicating their non-redundancy Rheumatology, Graduate School of Transforming -betas (12-16). The TGF-βs have opposite ef- Medicine, The University of Tokyo, (TGF-βs) are multifunctional fects on tissue fibrosis. Wound- Tokyo, Japan; that have been implicated in the regu- experiments revealed that TGF-β1 2Max Planck-The University of Tokyo Center for Integrative Inflammology, lation of a broad range of biological and TGF-β2 cause fibrotic scarring The University of Tokyo, Tokyo, Japan. processes, including , responses and that TGF-β3 induces a Tomohisa Okamura, MD, PhD cell survival, and cell differentiation. scar-free response (17). Both TGF-β1 Kaoru Morita, MD The three isoforms identified in mam- and -β2 activate the collagen α2 (I) Yukiko Iwasaki, MD, PhD mals, TGF-β1, TGF-β2, and TGF-β3, promoter, resulting in increased Mariko Inoue, MD have high , bind to collagen synthesis (18). It was also re- Toshihiko Komai, MD the same receptors, and show similar ported that TGF-β1, but not TGF-β3, is Keishi Fujio, MD, PhD biological functions in many in vitro a crucial factor in the development of Kazuhiko Yamamoto, MD, PhD studies. However, analysis of the in vivo pulmonary fibrosis (19). Most of the in- Please address correspondence to: functions of the three isoforms and mice formation on the immunological activ- Keishi Fujio, MD, PhD, deficient for each reveals strik- Department of Allergy and ity of TGF-βs derives from studies of Rheumatology, ing differences, illustrating their unique TGF-β1 and, in part, TGF-β2, whereas Graduate School of Medicine, biological importance and functional recent investigations have begun to il- The University of Tokyo, non-redundancy. Although increasing luminate the importance of TGF-β3 in 7-3-1 Hongo, Bunkyo-ku, evidence suggests that TGF-β1 and, to the . In this review, we Tokyo 113-8655, Japan. a lesser extent, TGF-β2 play an integral focus on recent novel insights into the E-mail: [email protected] role in maintaining immune tolerance, immunological role of TGF-β3, with Received and accepted on August 28, 2015. the immunological role of TGF-β3 has comparison to TGF-β1 and -β2. Clin Exp Rheumatol 2015; 33 (Suppl. 92): not been carefully evaluated. Recent S63-S69. studies have focused on the multifunc- TGF-β family synthesis © Copyright Clinical and tional role of TGF-β3. In this review, The three isoforms of TGF-β each uti- Experimental Rheumatology 2015. we provide an overview of the role of lise a unique activation mechanism TGF-β3 in immunity, with comparison from their latent forms (20). TGF-βs are Key words: TGF-β3, TGF-β1, to TGF-β1 and -β2. initially synthesised as larger precursor TGF-β2, humoral immunity, polypeptides containing a hydropho- autoimmune disease, CD4+CD25- + Introduction bic signal sequence, latency-associated LAG3 regulatory T cells, Egr-2 Three structurally similar isoforms of peptide (LAP), and mature peptide (pre- TGF-β (TGF-β1, TGF-β2, and TGF-β3) pro-TGF-β). Pre-pro-TGF-β monomers have been identified in humans (1). dimerise through disulfide bridges - be TGF-β1 was first cloned from a human tween cysteine residues in LAP and cDNA library (2) and human TGF-β2 cysteine residues in the mature peptide and TGF-β3 cDNA were subsequently (pro-TGF-β), and are cleaved by cloned (3, 4). TGF-β1 is highly homol- convertase but remain non-covalently ogous to the TGF-β2 and -β3 isoforms associated with the mature peptide. (5). TGF-β2 and -β3 share 71% (β2 vs. LAP confers latency to the mature pep- Competing interests: β1) and 76% (β3 vs. β1) sequence iden- tide by shielding the receptor-binding K. Yamamoto has received financial support or fees from AbbVie, Astellas, tity to TGF-β1 (6). Although TGF-β4 region of active TGF-β (21). Similar BMS, Daiichi-Sankyo, MitsubishiTanabe, and -β5 were identified from cDNA li- latent complexes have not yet been re- Pfizer, Sanofi, Santen, Takeda, Teijin, braries of chicken chondrocyte and frog ported for other members of the TGF-β Boehringer Ingelheim, Chugai, Eisai, oocyte, respectively (6, 7), mammalian superfamily. Ono, Taisho Toyama, UCB, Immuno- TGF-β4 and -β5 have not yet been de- The activity of secreted TGF-β is pri- Future, Asahi Kasei and Janssen; scribed. In vitro experiments indicated marily regulated by the conversion K. Fujio has received financial support or that TGF-β1, -β2, and -β3 often exhibit of its latent form to its active form. fees from Astellas, BMS, Daiichi-Sankyo, MitsubishiTanabe, Pfizer, Santen, Takeda, similar biological activities (8-11). Although tissues contain significant Chugai, Eisai, Taisho Toyama, UCB, and Despite this high sequence homology, quantities of latent TGF-β, the activa- Janssen; the other co-authors have analysis of the in vivo functions of the tion of only a small fraction of this la- declared no competing interests. three isoforms reveals marked differ- tent TGF-β exerts maximal effects on

S-63 Role of TGF-β3 in the regulation of immune responses / T. Okamura et al. target cells. In lymphoid tissues, LAP whereas αvβ8 integrin is expressed on endothelial cells (42). TGF-β3 induces is thought to be removed by throm- most leukocytes. Leucocyte-specific the activation of TGF-β receptor II ki- bospondin, plasmin, and acidification conditional deletion of Itgb8 (which nase and phosphorylates ALK5, which during the inflammatory process (22). encodes integrin β8) causes severe recruits ALK1 into a TGF-β receptor The TGF-β-LAP complex, called the inflammatory bowel disease and the complex. ALK5 kinase activity is re- small latent complex (SLC), is further development of high levels of auto- quired for optimal ALK1 activation. bound to latent TGF-β-binding protein against double-stranded Activated ALK1 and ALK5 induce (LTBP). There are four isoforms of DNA (32). These findings indicate the the phosphorylation of Smad1/5 and

LTBP in humans (LTBP-1, -2, -3, and importance of αvβ8 integrin-mediated Smad2/3, respectively, whereas ALK1 -4). LTBP-2 is unique in the LTBP fam- TGF-β activation by lymphoid cells directly inhibits ALK5/Smad3 signal- ily as it is the only isoform that does for preventing the immune dysfunction ling (41). Intriguingly, , a co- not bind to latent TGFβ (23). TGF-β- that results in inflammatory bowel dis- receptor for TGF-β1 and TGF-β3, func- LAP-LTBP is termed the large latent ease and autoimmunity. The expression tions as a modulator of the balance be- complex (LLC). The LTBP is not nec- of αvβ8 integrin on dendritic cells and tween TGF-β/ALK1 and TGF-β/ALK5 essary for latency (24), but it does play activated CD4+CD25+Foxp3+ regulato- signalling. Endoglin, which stimulates a critical role in the assembly and secre- ry T cells (Tregs) (32, 33) might play a TGF-β/ALK1 signalling and indirectly tion of TGF-β (25), as well as target- key role in promoting immune regula- inhibits TGF-β/ALK5 signalling, regu- ing the LLC for storage through inter- tion by activating TGF-β1. Consistent lates endothelial cell proliferation (43). actions with fibronectin and fibrillin in with these reports, Itgb8-deficient mice These findings suggest that the ratio be- the extracellular matrix (ECM) through show perinatal lethality with profound tween ALK1 and ALK5 signalling may non-covalent interactions (26). LTBPs defects in vascular development, par- provide one explanation for the bifunc- covalently bind to the LAP portion of ticularly in the brain (34), and a simi- tionality of the TGF-βs. Smad-inde- SLC through the third eight-cysteine lar phenotype is also seen in mice with pendent non-canonical pathways medi- type domain of LTBP. The third eight- mutation in Tgfb1 or deletion in the ated by TGF-β have also been reported. cysteine type domain of LTBP-1 and Tgfb3 gene (35). TGF-β1 induces profibrotic signalling LTBP-3 can associate efficiently with by activating tyrosine-protein kinase pro-TGF-β1, -β2, and -β3. Conversely, TGF-β family receptors and signal c-Abl, which is a mediator for fibrotic LTBP-4 binds to TGF-β1-LAP more transduction responses (44). Several studies have weakly than the other two LTBPs, indi- TGF-βs are secreted as latent proteins. suggested other possible non-canonical cating that LTBP-1 and LTBP-3 might After activation, TGF-βs interact with pathways, such as mitogen-activated be the primary proteins responsible for the same receptor heterodimers, TGF-β protein kinases (MAPK), phosphati- binding to SLC (27). The C-termini of receptor I (TGFBR1/ALK-5) and dylinositol-4,5-biphosphate 3-kinase LTBP-1, -2, and -4 bind to a similar TGF-β receptor II (TGFBR2), to acti- (PI3K), AKT, and the NF-κB pathway region in the N-terminus of fibrillin-1 vate the canonical Smad2/3 signalling (45). More extensive studies of TGF-β and -2 through non-covalent interac- pathway. However, TGF-β2 requires in immune cells are tions, whereas LTBP-3 reportedly does binding of TGF-β receptor III (TGF- needed to elucidate the specific role of not (28). However, LTBP-3 co-localises BR3) prior to biding TGFBR2 (36). each TGF-β isoform in the immune with fibrillin fibres in tissues, and loss TGF-β3 transmits signals biologically system. of fibrillin-1 prevents the incorporation similar to and more potently than those of LTBP-3 into a matrix, indicating that transmitted by TGF-β1 and -β2, de- TGF-β-deficient animals LTBP-3 association with the matrix de- pending on the cell type and assay used TGF-β1-deficient mice die by 4 weeks pends on fibrillin-1 microfibrils (29). (10, 37-39). Phosphorylated Smad2 and of age with massive infiltrations of lym- As for secretion of LTBPs from cells, Smad3 form heteromeric complexes phocytes and resulting in LTBP-1 and LTBP-4 do not require with Smad4 and translocate to the nu- multi-organ inflammation, including the binding to SLC, whereas LTBP-3 is se- cleus where they activate target gene lungs (vasculitis, perivascular cuffing, creted only as LLC (30, 31). transcription. The function of TGF-β3 and interstitial pneumonia) and heart TGF-β1-LAP and TGF-β3-LAP, but in palate fusion is mediated by Smad2, (endocarditis and myocarditis) (46, 47). not TGF-β2-LAP, contain an Arg-Gly- since the overexpression of a Smad2 The progressive inflammatory process Asp (RGD) integrin-binding motif, and transgene in TGF-β3 mutant palate epi- is accompanied by several autoim- several RGD-binding integrins, such as thelial cells rescues the cleft palate phe- mune manifestations, such as produc-

αvβ6 and αvβ8, are able to activate latent notype in TGF-β3-deficient mice (40). tion of antibodies to nuclear antigens TGF-β. Thus, the expression of αvβ6 Intriguingly, TGF-β3 can also activate (single-stranded DNA, double-stranded and αvβ8 integrins is thought to be cru- ALK-1, but only in the presence of DNA, Sm, and RNP), immune complex cial for the activation of TGF-β1 and ALK5 (41). ALK-1 was first identified deposition, and increased expression of

-β3. The expression of αvβ6 integrin is as a receptor for BMP-9 and BMP-10, both major histocompatibility complex restricted to a subset of epithelial cells and was shown to mediate downstream (MHC) class I and class II antigens (48, and is not expressed on immune cells, signals associated with Smad1/5/8 in 49). Mice with TGF-β2 deletion exhibit

S-64 Role of TGF-β3 in the regulation of immune responses / T. Okamura et al. perinatal mortality and show cardiac, 17-producing CD4+ T cells, known as Th17 cells, TGF-β3-induced Th17 cells lung, craniofacial, limb, spinal column, Th17 cells. The differentiation of Th17 induced higher expression of Smad1/5 eye, inner ear, and urogenital defects. cells in mice is initiated synergistical- and lower expression of Smad2/3. T-bet Around 23% of TGF-β2 mutant embry- ly by TGF-β1 and IL-6 (57-59). Th17 might have a crucial role for the induc- os on the 129 background, but not C57, cells induced by TGF-β1 plus IL-6 do tion of TGF-β3-induced Th17 cells, displayed cleft palate (50). TGF-β3- not readily induce autoimmune disease because Th17 cells from T-bet-deficient deficient mice survive throughout the unless they are further exposed to IL-23 mice failed to induce severe EAE due prenatal period, only to die within 20 (60). TGF-β1 is able to stimulate im- to lower TGF-β3 expression, and ex- hr of birth due to defective palatogen- munoglobulin A (IgA) production by ogenous administration of TGF-β3 esis and delayed pulmonary develop- lipopolysaccharide (LPS)-stimulated overcomes the T-bet requirement for ment (51, 52). Although TGF-β2- and (61). In humans, TGF-β1 and the induction of pathogenic Th17 cells. TGF-β3-deficient mice both develop IL-21, produced by follicular helper T However, the mechanism for inducing cleft palates, the structural features of (TFH) cells, synergistically promote TGF-β3 by T-bet remains to be eluci- cleft palate are quite different between naïve B cell proliferation and differen- dated. Chikuma et al. also reported that the mutants (50). Thus, there is little tiation to IgA-producing plasmablasts mice with -specific deficiency in phenotypic overlap between TGF-β1-, (62). The combination of IL-21 and TRIM28 show derepression of TGF-β, -β2– and -β3-deficient mice, indicating TGF-β1 up-regulates the expression especially the TGF-β3 isoform, which non-compensated functions between of CCR10, a common mucosal hom- contributed to the development and ac- the three isoforms of TGF-β. However, ing receptor, which mediates migration cumulation of autoreactive Th17 cells vascular pathology is a common fea- to and retention in the germinal cent- (69). These findings indicate a pro- ture of TGF-β1, -β2 and -β3 deficiency. ers, on B cells. Although a high serum inflammatory role for TGF-β3 in some Around 50% of TGF-β1-deficient mice concentration of TGF-β1 was found in inflammatory conditions. show prenatal lethality due to impaired patients with SLE, it did not correlate development of the yolk sac vascula- with Systemic lupus erythematosus Anti-inflammatory role of TGF-β3 ture (53). TGFB2 and TGFB3 muta- (SLE) disease activity (63), and the role Deletion of Tgfr2 in various leukocyte tions are both associated with aortic of TGF-β1 in lupus pathogenesis has subsets clarified the immunoregulatory aneurysms and dissections in humans yet to be elucidated. roles of the TGF-βs (70), and many (54, 55). Intriguingly, a knock-in mouse In mammals, TGF-β1 is predominant- studies have revealed immunoregulato- in which the coding region of exon1 of ly expressed by haematopoietic cells, ry roles for TGF-β1. Naïve CD4+ T cells the Tgfb3 gene replaced the full-length such as CD4+CD25+ Tregs, TFH cells, can differentiate into Th17 cells when Tgfb1 cDNA display a significant, but dendritic cells, and B cells, whereas TGF-β1 synergises with IL-6, while not complete, improvement in the cleft TGF-β2 and TGF-β3 are present in TGF-β1 induces CD4+CD25+Foxp3+ palate phenotype when compared to negligible amounts and have been Treg, which is an anti-inflammatory TGF-β3-deficient mice, indicating that thought to play an insignificant role in subset of CD4+ T cells in humans and an isoform-specific role for TGF-β3 the immune system (64-66). In humans, animals (71). It should be noted that in the palatal epithelium during pal- TGF-β1 is the most abundant circulat- TGF-β1 negatively regulates the dif- ate formation can be at least partially ing isoform in plasma (67). However, ferentiation of Th1 and Th2 cells by compensated by TGF-β1 (56). One can several recent lines of evidence suggest blocking the induction of T-bet and argue that mice with TGF-β2 and -β3 a role for TGF-β3 in the pathogenesis GATA3, respectively (72, 73). Sys- deletion do not survive long enough of autoimmune diseases. In lymphoid temic administration of TGF-β1 sup- to develop autoimmune disease due to tissue, TGF-β3 is expressed in CD4+ pressed experimental autoimmune en- their perinatal mortality. The neonatal T cells, CD8+ T cells, γδ T cells and B cephalitis (EAE) in mice (74) and the mortality in TGF-β2- and -β3-deficient cells, but not in myeloid cells (CD11b+ acute and chronic phases of streptococ- mice make it difficult to investigate the and/or CD11c+ cells). Kuchroo and col- cal cell wall (SCW)-induced arthritis significance of these two TGF-β iso- leagues reported that TGF-β3 is a key in rats (75). TGF-β1 potently inhibits forms in the immune system. Hence, molecule expressed by pathogenic Th17 both proliferation and Ig production by more detailed studies with conditional cells (68). Acute ablation of TGF-β3- Staphylococcus aureus-activated hu- TGF-β2 and -β3 deletion in immune expressing cells in MOG-immunised man B cells (76). TGF-β1 suppresses cells are needed. mice led to selective loss of IL-17+ cells B cells by inhibiting both the synthesis with no obvious change in IFN-γ+ cells. of Ig mRNA and the switch from the Pro-inflammatory role of TGF-β3 TGF-β3-induced Th17 cells highly ex- membrane form to the secreted forms It is recognised that TGF-β is a multi- press Il22 and Il23r mRNA compared to of μ and γ mRNA (77). Consistent with functional cytokine. Of particular inter- TGF-β1-induced Th17 cells. The adop- these studies, the addition of TGF-β1 est, TGF-β1 is known to play a crucial tive transfer of TGF-β3-induced Th17 neutralising partially abro- role in immune responses. TGF-β1 has cells, but not TGF-β1-induced Th17 gated the CD4+CD25+CD69- Treg-me- been shown to induce a distinct lineage cells, induced severe EAE. Intrigu- diated suppression of B cell antibody of pro-inflammatory (IL)- ingly, in contrast to TGF-β1-induced production in vitro (78).

S-65 Role of TGF-β3 in the regulation of immune responses / T. Okamura et al.

Unlike TGF-β1, the studies referred to in which autoimmune inflammation is ing (87). Membrane-bound TGF-β1 is above did not investigate the suppres- responsible for multiple organ dam- important for the suppressive activity sive activity of TGF-β3. There are few age, and autoantibodies play a pivotal of CD4+CD25+ Tregs in spite of the studies examining the distinct expres- role in triggering the inflammation. It fact that Tgfb1 mRNA is not elevated sion of TGF-β3 in immune cells at the was also reported that lymphocyte- in CD4+CD25+ Tregs (88, 89). These protein level. However, indirect evi- specific Egr2 conditional knockout findings highlight the importance of dence suggested that TGF-β3 plays an mice develop a lupus-like autoimmune evaluating TGF-βs at the protein lev- important role in immune regulation. disease (85). We have recently re- el. However, most of previous studies In the mouse experimental autoim- vealed that Egr2-expressing wild type have examined TGF-β3 expression mune encephalomyelitis (EAE) model, (WT) LAG3+ Tregs repress excessive only at the mRNA level. the induction of EAE was associated humoral immunity in T cell-specific Some studies have illustrated the with high expression of Tgfb1 mRNA Egr2 conditional knockout mice (11). importance of the concentration of and low expression of Tgfb3 mRNA Intriguingly, LAG3+ Tregs, but not TGF-β1 in regulating regulatory and in spinal cord tissue. The treatment of CD4+CD25+ Tregs, produce enormous pro-inflammatory T cells. TGF-β1 can EAE with 17β-estradiol elevated the amounts of TGF-β3 in an Egr2- and induce both Treg and Th17 cell devel- expression of TGF-β3, but lowered the Fas-dependent manner. TGF-β3 ef- opment, depending on whether pro-in- expression of TGF-β1 (79). TGF-β2 fectively suppresses both B cell pro- flammatory cytokines such as IL-6 and expression was not altered by EAE in- liferation and antibody production by IL-23 are present (58, 90). Further, the duction or treatment. The inverse rela- inhibiting several important pathways effect of the TGF-β family is affected tionship between TGF-β1 and TGF-β3 for B cell function, such as STAT6, by their concentration. Lower con- indicates that the two TGF-β isoforms Syk, and NF-κB p65. In lupus-prone centrations of TGF-β1 synergise with play opposing roles in the control of MRL-Faslpr/lpr mice with a Fas muta- IL-6 and IL-21 to promote the initial EAE. tion, adoptive transfer of LAG3+ Treg differentiation of Th17 cells. Higher We recently reported that CD4+CD25- from Fas-sufficient MRL-Fas+/+ mice concentrations of TGF-β1 suppress LAG3+ Tregs (LAG3+ Treg) regulate effectively suppressed the progres- Th17 cell differentiation by inducing humoral immunity and lupus disease sion of lupus in a TGF-β3-dependent Foxp3-mediated ROR-γt antagonism in MRL-Faslpr/lpr mice via TGF-β3 pro- manner. TGF-β3-mediated B cell sup- and repressing IL-23R expression (91, duction (11). LAG3+ Treg characteristi- pression requires programmed death-1 92). The concentration of TGF-β1 also cally express the transcriptional factor (PD-1) expression on B cells. PD-1 is affects its ability to directly suppress early growth response gene 2 (Egr2), an immunoinhibitory receptor of the B cells. Although TGF-β1 (1.0 ng/ml) which is required for T cell anergy in- CD28/B7 family, and polymorphism of selectively stimulates IgG2b secretion duction (80), and were first identified the PDCD1 gene is associated with a from LPS-stimulated B cells, higher as Foxp3-negative induced Treg that higher incidence of SLE (86). Intrigu- concentrations of TGF-β1 typically produced high amounts of IL-10 (81). ingly, PD-L1, the ligand for PD-1, is suppresses all Ig isotypes (93). These Egr2 mediates IL-27-induced IL-10 highly expressed on the cell surface of physiological complexities make it production in CD4+ T cells via signal LAG3+ Treg in the steady state (11). difficult to assess the isotype-specific transducer and activator of transcrip- These findings revealed the previously roles of each TGF-β isoform in the im- tion 3 (STAT3) (82). Ectopic expression unknown cellular and molecular basis mune system. of Egr2 on naïve CD4+ T cells induces of TGF-β3 for the physiological con- Of special interest are the possible not only LAG3 and B-lymphocyte-in- trol of humoral immunity. side effects of TGF-β treatment. High duced maturation protein-1 (Blimp-1) concentrations of TGF-β1 may induce expression, but also IL-10 production, Summary and perspective undesirable side effects. Systemic ad- and confers in vivo suppressive activ- TGF-βs have bi-functional roles in the ministration of TGF-β1 at >20 μg/day, ity (81), indicating that Egr2 plays a immune system, whereby they regu- but not 5 μg/day (75), increases the major role in the suppressive activity late both pro-inflammatory and anti- incidence of anaemia and thrombo- of LAG3+ Tregs. Subsequently, Gagli- inflammatory activities. The biological cytopenia in mice (94). These effects ani et al. reported that co-expression of activity of the three isoforms of TGF-β are transient and completely reversed LAG3 and CD49b is specific for IL- is similar in most in vitro experiments two weeks after cessation of treatment. 10-producing type 1 T regulatory (Tr1) (10), whereas accumulating evidence Other possible side effects of TGF-β1 cells (83). Thus, LAG3 is considered demonstrates differences in their in treatment are fibrosis (17, 19), - obe to be one of the phenotypic markers of vivo capabilities. However, we should sity, and impaired glucose tolerance IL-10-producing Foxp3-independent be cautious when evaluating the roles (95). Intriguingly, TGF-β3 not only CD4+ Tregs. We and our collabora- of TGF-βs in the immune system. It has induces scar-free healing (17), but also tors have shown that polymorphisms been reported that the levels of Tgfb1 improves glucose tolerance and pheno- in EGR2 influence SLE susceptibil- mRNA do not correlate with the dis- typic changes in adipocyte morphology ity in humans (84). SLE is regarded tribution of TGF-β1 protein as meas- (96), suggesting the potential thera- as a prototypic autoimmune disease, ured by immunohistochemical stain- peutic advantages of TGF-β3. Further

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