American Journal of Respiratory Cell and Molecular Biology

Transforming growth factor-β (TGFB) regulates cell proliferation, differentiation, apoptosis, and matrix homeostasis and is intimately involved in fibrosis. TGFB expression is increased in fibrotic lung diseases, such as idiopathic pulmonary fibrosis, and in chronic inflammatory conditions, such as chronic obstructive pulmonary disease and asthma. In addition to exhibiting profibrotic activities, the protein exhibits profound immune-suppressive actions involving both innate and adaptive responses, but often this aspect of TGFB biology is overlooked. Recent investigations have demonstrated that TGFB causes wide-ranging immune suppression, including blunting of pivotal early innate IFN responses. These activities permit severe virus infections, often followed by secondary bacterial infections, which may last longer, with augmented inflammation, scarring, fibrosis, and loss of lung function. Strategies to oppose TGFB actions or to enhance IFN responses may help ameliorate the detrimental consequences of infection in patients with diseases characterized by TGFB overexpression, inflammation, and fibrosis.

The profibrotic role of transforming growth factor-β (TGFB) in chronic lung diseases is well established; however, the immune-suppressive activities of this cytokine are often overlooked. Recent investigations have demonstrated that TGFB suppresses innate immune responses, enabling severe virus infections. This review highlights the immune-suppressive actions of TGFB, focusing on the clinical relevance of excess TGFB in the context of intercurrent virus infections in chronic lung diseases. Strategies to oppose TGFB actions are discussed.

Transforming growth factor-β (TGFB) is a member of a superfamily of dimeric polypeptide growth factors that includes bone morphogenetic proteins and activins. The TGFB family comprises pleiotropic regulatory molecules with effects on cell proliferation, differentiation, migration, and survival and impacts on biological processes that include development, carcinogenesis, fibrosis, wound healing, and immune responses (1, 2). Three TGFB isoforms (TGFB1, TGFB2, and TGFB3) are encoded by separate, independently regulated genes. The expression of TGFB is widespread, and the molecules are produced by inflammatory cells such as neutrophils and eosinophils, structural cells including epithelial, fibroblast, and smooth muscle cells (3, 4), and macrophages and regulatory T cells (5, 6). TGFB1 mRNA is present in endothelial, hematopoietic, and connective-tissue cells, and TGFB1 knockout mice develop extensive multiorgan inflammation and die shortly after birth (7).

TGFB peptides are synthesized as a biologically latent pre–pro-TGFB precursor that forms a dimer and then is proteolytically cleaved into the C-terminal noncovalently linked mature TGFB and the N-terminal latency-associated peptide. On secretion, the latency-associated peptide–TGFB complex is bound by latent TGFB-binding protein (8, 9) that is activated by several mechanisms including proteases, thrombospodin-1, reactive oxygen species, and low pH (1012). The integrin αvβ6 is a key activator in experimental lung fibrosis and is overexpressed by human lung tissue in idiopathic pulmonary fibrosis (IPF) (13). After activation, TGFB signals by binding to two distinct TGFB receptor subunits, type I and type II (TGFR1 and TGFR2). TGFR2 exists as a homodimer (14, 15), and after TGFB binding, TGFR1 is recruited and activated, leading to phosphorylation of SMAD2 and SMAD3, which complex with SMAD4, translocate to the nucleus, and activate target gene transcription (15). The inhibitory SMAD7 forms a negative feedback mechanism by competing with SMAD2/3 for receptor binding (16). TGFB also activates SMAD-independent signaling pathways, including mitogen-activated protein kinase, RhoA, and phosphatidylinositol-3 kinase (15). Key aspects of TGFB biology most relevant to pulmonary diseases have been reviewed recently by Aschner and Downey (17).

TGFB also has profound immune-suppressive actions involving both innate and adaptive responses (9, 18), an aspect of TGFB biology that is frequently overlooked. This review highlights the early innate immune suppressive actions of TGFB, chiefly focusing on the clinical relevance of excess TGFB in the context of intercurrent infections in chronic lung disease.

TGFB is recognized increasingly as a key lung cytokine, and associations with lung diseases, characterized by various degrees of fibrosis, have been demonstrated (1922). These include important conditions such as IPF (florid fibrosis), chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) (moderate fibrosis), and asthma (mild fibrosis). Studies in lung diseases have used a combination of techniques to measure TGFB using peripheral blood, bronchoalveolar lavage (BAL), endobronchial biopsy tissues, lung resection specimens, and lung cells with in vitro culture.

IPF is the prototypical fibrotic lung disease, and the role of TGFB in its pathogenesis is widely recognized. Studies have reported elevated TGFB mRNA and protein expression levels in lung tissue from patients with IPF compared with that of healthy control subjects (19), and levels of TGFB in BAL were more than fivefold higher in those with IPF than in nondiseased individuals (23). Studies in human lung tissue obtained from patients with IPF have shown that epithelial cells, endothelial cells, macrophages, and connective tissue cells all may produce excess TGFB (19). However, the molecule has also been implicated in the pathogenesis of other chronic lung diseases. Joseph and coworkers measured peripheral blood bioactive TGFB in those with atopic asthma, those with nonatopic asthma, and healthy control subjects. The researchers found higher levels in patients with asthma, mainly in those who had nonatopic asthma (24). Similar observations were made in a study that reported detection of higher levels of TGFB in patients with asthma compared with healthy individuals (21). Studies of BAL have tallied with observations in peripheral blood. Redington and coworkers measured immunoreactive TGFB1 in BAL obtained from patients with relatively severe asthma and from healthy control subjects, both at baseline and after segmental allergen challenge (25). TGFB1 was significantly increased at baseline and increased further after allergen exposure (approximately fivefold increases 24 h after allergen challenge) in subjects with asthma. Studies in milder asthma have confirmed these findings in the segmental allergen challenge model, but differences compared with healthy control subjects at baseline were not observed (26). Recently, Brown and colleagues (27) investigated TGFB1 expression in BAL obtained from children with mild/moderate and severe asthma and compared it with that of atopic healthy adults. TGFB1 was increased predominantly in severe asthma and correlated with the degree of airway obstruction. Finally, in patients with CF, TGFB1 was raised in BAL and was directly correlated with airway neutrophil counts, diminished lung function, and hospitalization (20). In mouse models of CF, TGFB released by airway epithelial cells after influenza A virus infection induced IL-6 in BAL (28).

TGFB has also been noted in lung tissue specimens and TGFB1–3 was detectable in airway epithelium in healthy individuals (29). Minshall and coworkers obtained endobronchial biopsy specimens from individuals with mild, moderate, and severe asthma (30). They demonstrated greater expression of TGFB1 mRNA by polymerase chain reaction and TGFB protein on immunohistochemistry. Increases in TGFB1 levels were related to asthma severity, with double-staining immunohistochemistry techniques revealing that eosinophils (EG2-positive cells) produced up to 50% of the detectable TGFB1. Immunohistochemical examination of endobronchial biopsy specimens taken from patients with asthma and COPD (mainly a chronic bronchitis group) also demonstrated increased TGFB1 immunoreactivity in both conditions; this was particularly evident in COPD and was associated with evidence of airway remodeling (22). TGFB1 was shown to be produced by eosinophils and fibroblasts in these sections. Finally, in vitro investigations demonstrated that airway macrophages obtained from the BAL of patients with asthma and COPD released more TGFB1 compared with those from healthy individuals (31). One early study failed to detect increases of TGFB in asthma and COPD (32). However, archival tissue obtained from lung resection specimens or postmortem lung tissues were used in this study, and TGFB mRNA and protein epitopes may have been degraded. Studies in CF tissue have not been reported.

Studies in human lung disease indicate that TGFB, chiefly TGFB1, is increased in a spectrum of airway diseases characterized by severe to mild lung fibrosis (1921, 23, 25, 27). Importantly, these relatively diverse clinical lung conditions are unified by their susceptibility to exacerbations often caused by infections, chiefly virus infections. Infections are more frequent and severe if the condition itself is severe and long standing (3335). For example, asthma exacerbations have been attributed to intercurrent virus infections in up to 80% of children (36) and >50% of adults (37). Virus infections are strongly linked to COPD exacerbations (38, 39), and IPF/lung fibrosis exacerbations requiring hospitalization are believed to result from bacterial infections, often after initial virus infections (40). Several factors such as reduced immunity, excess secretions, and airway obstruction and airway remodeling with anatomical distortion and structural damage can partly explain a propensity to exacerbations caused by infection. However, given the ubiquitous presence of TGFB overproduction in all these lung conditions, it has to be considered that the molecule may act as an additional aggravating factor predisposing the individual to immune suppression and enhancing susceptibility to damaging infections.

It is clear that TGFB participates in airway fibrosis, but a key role of the molecule during infections has been underappreciated. There are as yet no clinical studies that have examined the influence and role of TGFB as an immune-suppressant factor influencing susceptibility to lung infections.

An immunoregulatory role for TGFB was first acknowledged in 1986 when it was shown to be produced by T cells and to regulate their responses (41). Subsequent reports have described the effects of TGFB on virtually all leukocyte lineages and have identified a pivotal role for the molecule in regulating innate and adaptive immune responses (2). Studies in Tgfb1 knockout mice further emphasized the importance of this molecule in the regulation of inflammation and immunity (7).

The regulatory actions of TGFB on innate immune responses are well documented. For example, invading pathogens are recognized by pattern recognition receptors, and Toll-like receptor (TLR) 4 is a pattern recognition receptor that, in conjunction with CD14, recognizes LPS, a key component of the bacterial cell wall. TGFB can inhibit CD14 expression, thus attenuating TLR4 signaling and the innate immune response (42). TGFB has also been shown to promote the degradation of MyD88 in macrophages and is capable of inhibiting MyD88-dependent TLR signaling pathways (43). Other studies have demonstrated that TGFB negatively regulates natural killer (NK) cell development and maturation during infancy (44) but that it can also act as a potent chemoattractant for mast cells, neutrophils, and eosinophils (4547). Finally, TGFB can suppress the antigen-presentation properties of differentiated dendritic cells and macrophages (48, 49).

TGFB has pleiotropic effects on adaptive immunity and can act in either a pro- or an antiinflammatory fashion. This occurs by several mechanisms. It can regulate T-cell responses by inhibiting T-cell proliferation and inducing T-cell death, thus limiting T-cell expansion after activation. Conversely, it can promote survival of activated T cells (50). TGFB also controls both antiinflammatory (Treg, Th1, Th2) and proinflammatory (Th17, Th9) T-cell responses. Th1 and Th2 T-cell differentiation is inhibited via TGFB-mediated down-regulation of the transcription factors T-bet and GATA3, whereas TGFB works in combination with IL-2 to regulate Foxp3+ regulatory T cells (reviewed in Reference 50). TGFB acts in combination with IL-1, IL-6, and IL-21 to induce Th17 cells that are central to autoimmunity (51) and with IL-4 to induce Th9 cells (52). TGFB also contributes to the generation of B cells from precursor cells and can have both stimulatory and inhibitory effects on mature B cells by promoting IgA class switching and resting B-cell apoptosis, or by inhibiting B-cell proliferation (53). Finally, TGFB controls priming and contraction of CD8+ T cells in response to viral or bacterial infections, thereby preventing prolonged activation of innate responses. However, reduction of TGFB control on reinfection permits early proliferation of NK1.1 CD8+ T cells and rapid production of IFN-γ and granzyme B (54). It is not known if this process is impaired in conditions characterized by increased TGFB activities.

In summary, most cell types can produce and secrete TGFB, and it is now recognized that this molecule acts as a central key regulator of innate and adaptive immune responses. Excessive production of TGFB could result in the suppression of appropriate immune responses and could amplify exacerbations caused by intercurrent infections.

There has been limited evaluation of the role of virus infections in IPF exacerbations. In animal models of IPF, virus infection has been shown to amplify established fibrosis (55). A study by Wootton and colleagues evaluated virus infection during exacerbation in 43 patients with IPF, with viruses detected in only four cases (56). However, the onset of IPF exacerbations is often insidious and it is possible that by the time of presentation, viruses may no longer be detectable and secondary bacterial infection may have ensued (57). In asthma and COPD exacerbations, symptoms develop more rapidly, and viruses, particularly rhinovirus (RV), but also respiratory syncytial virus (RSV), human metapneumovirus, parainfluenza virus, and influenza A and B viruses, are detected frequently (37, 5860).

Substantial evidence has emerged recently from laboratory investigations, indicating that TGFB blunts innate immune responses, thereby enhancing virus infections. Investigators have utilized in vitro respiratory cell lines and primary cell culture models to examine the effects of TGFB on virus infection. Overexpression of TGFB observed in the lungs of patients with IPF, asthma, COPD, and other lung diseases has been modeled by pretreating cells in culture with recombinant TGFB1.

In studies using primary culture human airway fibroblasts to investigate the effects of TGFB on RV infection (61), pretreatment of fibroblasts with TGFB significantly increased RV replication, an outcome elicited by suppression of innate immune responses (Figure 1). Similar observations were made in fibroblasts obtained from patients with asthma and in myofibroblasts transdifferentiated from naive fibroblasts by TGFB. Importantly, innate immune responses could be restored through the addition of recombinant IFNβ, a strategy that reversed enhanced RV replication. Similar findings were made in primary human bronchial epithelial cells in which TGFB pretreatment increased RV replication and decreased type I and III IFN protein secretion ([62] and unpublished observations). The suppression of innate immune responses was attributed to the induction of SOCS1 and SOCS3, molecules that interfere with interferon signaling and that are up-regulated by TGFB. The addition of anti-TGFB antibodies to asthmatic epithelial cells before RV infection significantly reduced the levels of SOCS1 and SOCS3 (62).

Other studies have investigated the effects of TGFB on RSV, a respiratory virus that commonly infects young children. Using primary human bronchial epithelial cells and the alveolar epithelial A549 cell line, McCann and Imani (63) established that TGFB enhanced RSV replication and RSV-induced TNF-α production, the latter through the activation of p38 MAPK, a signaling molecule pivotal in inflammatory cytokine production. They concluded that the presence of TGFB in the lungs of patients with asthma exaggerates inflammatory responses to respiratory pathogens, supporting enhanced viral replication (63). Further studies used RSV to examine the autocrine effects of TGFB on infection and the influence of cell cycle arrest, which TGFB has been reported to regulate (64). These studies found that RSV infection induced TGFB, which stimulated cell cycle arrest in the G0/G1 and G2/M phases and enhancement of RSV replication (65), but it is not known if this mechanism pertains to other types of infection.

The interactions between TGFB and virus infections are complex and are likely to be contingent on disease and pathogen settings. Carlson and coworkers demonstrated activation of TGFB by influenza A virus neuraminidase and found that administration of exogenous TGFB after infection resulted in delayed mortality and reduced virus titers (66). However, if TGFB expression in tissue was increased before influenza A virus infection (as in chronic lung diseases), these benefits were lost, and detrimental effects were observed (66). The actions of TGFB may also be influenced by virus characteristics. For example, herpes simplex virus (HSV) ocular infection establishes a permanent latent infection in the neurons of the trigeminal ganglia, and overexpression of TGFB in a mouse model of HSV infection reduced T cell–mediated immune responses and increased systemic infection (67). However, if genetic approaches are used to reduce TGFB production in response to infection (using a dominant negative TGFB receptor type II transgenic mouse model), HSV latency is enhanced and HSV replication is reduced (68). It is not clear whether these findings apply to conditions in which TGFB is overly abundant before infection.

Immune responses may also influence TGFB activities. Recent evidence indicates that signaling by RIG-I-like receptors that drives innate immune responses can repress TGFB activities (69). Activated IFN-regulatory factor 3 was shown to interact with TGFB pathways, thereby disrupting transcription complexes by competing with coregulators. Silencing IFN-regulatory factor 3 expression enhanced TGFB-induced epithelial-mesenchymal transition and Treg cell responses to virus infection. These findings suggest that interactions between TGFB and immune responses to infection may be bidirectional and that intercurrent infections could exacerbate some of the detrimental actions of TGFB, or oppose some of its beneficial regulatory functions. Earlier studies have shown that infection with a murine herpesvirus increased TGFB and fibrogenesis in a virus-induced model of lung fibrosis (70). TGFB expression and fibrogenesis were reduced by antiviral therapy.

Bacterial infections are a key problem in COPD and CF. In COPD, nontypeable Haemophilus influenzae (NTHi) is the most common pathogen (71) and in CF, Pseudomonas aeruginosa is the most persistent and serious infection (72). To date, there have been few studies of interactions between TGFB overexpression and bacterial infections. TGFB activation by NTHi infection may act synergistically to amplify inflammation, but it is unknown if this is the result of greater NTHi replication. This bacterium may also cause perturbation of TGFB receptors and nuclear factor-κB activation to enhance inflammatory responses (73). Studies in a bleomycin mouse model of TGFB overexpression and lung fibrosis failed to show increased P. aeruginosa replication or exacerbated fibrosis (74). In a mouse model of influenza A virus infection, TGFB activation was increased, followed by enhanced expression of fibronectin that increased group A Streptococcus adherence and infection (75).

Taken together, these studies demonstrate that the interactions between TGFB, respiratory virus, and bacterial infections may be exceptionally detrimental in lung diseases characterized by elevated TGFB. The multiple actions of TGFB on early immune responses as evidenced by suppression of type I and III interferons, down-regulation of interferon-regulated genes, and up-regulation of the negative regulators SOCS1 and SOCS3 hint at a highly complex system. To date, detailed investigations of the immune suppressive actions of TGFB on intercurrent infections in a fibrotic setting have not been reported, and further studies are needed to assess these interactions. Novel therapeutic approaches targeting TGFB itself or strengthening innate immune responses may potentially reduce the clinical deterioration and morbidity caused by infections in diseases such as IPF, COPD, and asthma.

The TGFB signaling pathway is being targeted extensively for the development of therapeutic agents effective for treatment of lung diseases characterized by fibrosis. Several strategies have been used to design compounds that have progressed through various stages of preclinical and clinical investigation (76). In addition, older compounds with anti-TGFB activities have been shown recently to display considerable promise for the treatment of lung fibrosis, chiefly in patients with IPF. However, responses to TGFB inhibition are likely to be individualized and to be determined by innate genetic variations in patients, as well by individual disease characteristics and stage at time of clinical presentation.

Pirfenidone is a compound that has dual antiinflammatory and antifibrotic effects (77). It has been demonstrated to inhibit fibroblast activity, collagen synthesis, and matrix deposition via TGFB activity blockade (reviewed in Reference 77). Open-label studies and subsequent prospective randomized studies found clinical benefit in early IPF, and, on the basis of these findings, it was first approved for therapeutic use in 2008 in Japan. After the Assessment of Pirfenidone to Confirm Efficacy and Safety in Idiopathic Pulmonary Fibrosis (ASCEND) study published in 2014 (78), pirfenidone was approved by the Food and Drug Administration for the treatment of IPF in the United States. Several lines of evidence indicate that the compound can attenuate inflammation and modulate airway remodeling. In a chronic allergen challenge model, Hirano and coworkers demonstrated reductions in TGFB1, IL-4, IL-5, and IL-13 in BAL after treatment with pirfenidone (79). Pirfenidone also inhibited type I collagen, fibronectin and heat-shock protein-47 production in response to TGFB in vitro (80) and in vivo in a bleomycin model of murine lung fibrosis (81). The effect of pirfenidone on TGFB-mediated immune suppression has not been evaluated, but it is feasible that some of the clinical benefits may derive from normalized immune responses and fewer intercurrent infections. A recent metaanalysis found that pirfenidone reduced episodes of acute worsening in IPF (many of which may be triggered by infections) by 46% (82).

The specificity and stability of monoclonal antibodies make them a promising therapeutic strategy for neutralizing extracellular ligands for diseases in which TGFB is overexpressed. STX-100 (Stromedix), a humanized antiintegrin β6 monoclonal antibody designed to prevent TGFB activation, and LY2382770, a pan-TGFB ligand-specific blocking antibody, are currently in phase II clinical trials for the treatment of IPF (NCT01371305) and kidney fibrosis (NCT01113801), respectively (reviewed in Reference 76).

Other novel therapeutic strategies have shown encouraging outcomes, albeit in separate conditions characterized by overexpression of TGFB. LY2157299 is a small molecule inhibitor of the TGFB receptor type I kinase that inhibits TGFB signaling by preventing SMAD2 phosphorylation (83). It is currently in phase Ib/II clinical trials for pancreatic cancer (NCT01373164) and may be suitable for studies in IPF. However, early toxicological studies have raised concerns that type I receptor kinase inhibitors may have detrimental cardiac (84) and other organ toxicities (85), potentially limiting their use for human purposes. Further studies are clearly indicated to establish safety in humans. Pyrrole-imidazole polyamides are molecules that bind specifically in the DNA minor groove of the TGFB1 gene promoter to prevent transcription factor binding and to attenuate gene expression (86). The large size of these molecules and the high concentrations required to achieve the requisite bioactivity have precluded progression to clinical testing to date.

Studies to date indicate that inhibitors of TGFB activities and signaling are generally well tolerated, and it is conceivable that in the future, surrogate markers will be useful to select patient populations likely to respond to anti-TGFB strategies or to identify those with predictable adverse effects. This concept has been demonstrated in studies measuring P-SMAD2 levels in peripheral blood cells to predict responses to treatment (87).

Overall, the outlook for compounds opposing TGFB activities appears bright, and several companies have progressed drug development to the stage of early clinical application. On-going studies are needed to determine the lung diseases likely to respond, optimize patient selection, determine dosing schedules, and identify subjects likely to develop serious adverse effects.

TGFB is ubiquitous in chronic inflammatory lung disease and may contribute to an immune-suppressed state. Consequently, individuals with IPF, asthma, COPD, CF, and other chronic conditions may be prone to more troublesome and longer-lasting virus and bacterial infections, or to severe virus infections followed by secondary bacterial infections (Figure 2). This, in turn, may cause amplified inflammation with amplified scarring and fibrosis accompanied by increased TGFB production, which leads to further immune suppression.

A vicious circle may result, with recurrent infection, lung scarring, and remodeling, leading to loss of lung function and clinical decline. Treatments opposing TGFB itself and compounds that enhance innate immunity are under development, and these are likely to limit infection-related exacerbations. These novel approaches may help arrest or retard progressive clinical deterioration in chronic inflammatory lung diseases.

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Correspondence and requests for reprints should be addressed to Belinda J. Thomas, Ph.D., Hudson Institute of Medical Research, 27-31 Wright Street, Clayton, Melbourne, Victoria, Australia, 3168. E-mail:

This work was supported by funding from the Victorian Government’s Operational Infrastructure Support Program, a Banyu Life Science Foundation International Fellowship (K.K.), and a Senior Research Fellowship from the Australian NHMRC (K.L.L., ID1079646).

Author Contributions: All authors contributed to the development and writing of the manuscript, and have approved the final version.

Originally Published in Press as DOI: 10.1165/rcmb.2016-0248PS on September 7, 2016

Author disclosures are available with the text of this article at


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