Recent Insights into the Involvement of Novel Transcription Factors, The Microbiome, and Dysregulated Cellular Metabolism in Pulmonary Fibrosis Pathogenesis

Recommended Reading from Cedars-Sinai Medical Center Fellows

Prof. Peter Chen, M.D., Director, Division of Pulmonary and Critical Care, and Ting Xie, M.D., Ph.D., Project Scientist

Wohlfhart T, et al. PU.1 Controls Fibroblast Polarization and Tissue Fibrosis. Nature (1)

Reviewed by Simon Coyle Rowan and Ting Xie

In their recent Nature publication, Wohlfhart and colleagues identified PU.1 (PU box binding-1), an ETS (E26 transformation-specific) family transcription factor, as an essential regulator of the genetic switch between extracellular matrix–producing profibrotic fibroblasts, as seen in idiopathic pulmonary fibrosis (IPF), and catabolic proinflammatory fibroblasts, as seen in rheumatoid arthritis (1). PU.1 has previously been implicated in diseases such as acute myeloid leukemia, Hodgkin lymphoma, and Alzheimer’s.

However, a link between PU.1, fibroblasts, and fibrosis has only been inferred by a resistance to thioacetamide-induced fibrosis, and apparent scar-free wound healing, in PU.1-deficient mouse models (2, 3).

The authors report that PU.1, long known for its fundamental role in hematopoiesis and immune system development (4), is highly expressed in matrix-producing fibrotic fibroblasts (isolated from patients with pulmonary fibrosis), but is silenced by epigenetic mechanisms in resting (isolated from healthy control subjects) and inflammatory (isolated from patients with asthma) fibroblasts by repressive histone methylation and elevated expression of miR-155 (microRNA-155), respectively. This finding is consistent with the emerging role of microRNAs and/or epigenetic regulation in the pathogenesis of IPF. Manipulations of SPI1 (Spi-1 proto-oncogene) (the gene encoding PU.1 protein in humans) in the phenotypically different fibroblasts examined in this work polarized fibroblasts toward a profibrotic gene signature by binding to the promotors of profibrotic genes (e.g., COL1A1 and ACTA2) and induced a profibrotic phenotype, supporting the link among PU.1, fibroblasts, and fibrosis suggested in previous publications.

Wohlfhart and colleagues report that PU.1 is associated with a network of profibrotic factors, including members of the TEAD-HIPPO (Transcriptional enhanced associate domain- Salvador/Warts/Hippo pathway), canonical transforming growth factor β (TGF-β)–SMAD (suppressor of Mothers against decapentaplegic), and AP1 (activator protein 1) signaling pathways. Other transcription factors linked to fibrosis, such as SNAI2 (Snail family transcriptional repressor 2), were found to bind in close proximity to PU.1 binding sites within the genome, and may contribute to the recruitment of transcription factors and the switch toward a fibrotic phenotype. Of note, many of the transcription factors identified differ from those previously reported to interact with PU.1 in shaping cell phenotypes in macrophages and immune cells.

Interestingly, a recent study found that overexpression of Spi1 in fibroblasts induced senescence (5). The question of what is driving abnormally high numbers of fibroblasts into senescence in IPF remains unanswered. Transcription factors that are known to cooperate and interact with PU.1, such as STAT3 (signal transducer and activator of transcription 3) and NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells), have been implicated in fibroblast senescence in IPF (6). Potentially, PU.1, acting as a genetic switch, driving fibroblast polarization toward a fibrotic phenotype, may also lead or contribute to the accumulation of senescent fibroblasts in the fibrotic lung.

The authors used the heterocyclic dication DB1976 to show that inhibition of PU.1 was efficacious in preventing uncontrolled fibrosis in murine models without a perceptible inhibition of hematopoietic cell differentiation. Using DB1976, the authors found that it is possible to safely interfere with a transcription factor, such as PU.1, and enable the restoration of tissue homeostasis. This study supports the work of Antony-Debré and colleagues, who also recently demonstrated in experimental models of acute myeloid leukemia that inhibition of PU.1 was therapeutically beneficial in decreasing tumor burden and increasing survival (7). In combination, these studies provide proof of concept that PU.1 inhibition has potential as a therapeutic strategy for a number of conditions that are currently poorly served by available therapeutic modalities.

Yang D, et al. Dysregulated Lung Commensal Bacteria Drive Interleukin-17B Production to Promote Pulmonary Fibrosis through Their Outer Membrane Vesicles. Immunity (8)

Reviewed by Stephanie Bora and Peter Chen

IPF is a severe disease with no cure and a median survival rate of <5 years (9). Research indicates that the cytokine IL-17 is part of the profibrotic inflammatory response (10). The critical influence of the gut microbiome on IL-17 immune responses has been established (11), yet the role of the lung microbiota in lung inflammation, and specifically IL-17 responses that promote IPF, has been overlooked. In a study published in Immunity, Yang and colleagues used the bleomycin model of fibrosis to determine if IL-17B, one of six IL-17 cytokines, caused lung fibrosis, and if the lung microbiome regulated profibrotic IL-17 production (8). They report that depletion of the microbiome with broad-spectrum antibiotics alleviated fibrosis and rescued mortality. The authors sequenced 16s DNA from lungs of control and bleomycin-treated mice, and found that bleomycin caused dysbiosis. The researchers narrowed their results to three species that were increased in the bleomycin-treated group: Bacteroides ovatus, B. stercoris, and Prevotella melaninogenica. Inoculation of antibiotics+bleomycin-treated mice with any of these bacteria restored the severity of fibrosis. The researchers further determined that bacterial outer-membrane vesicles (OMVs) isolated from B. stercoris, P. melaninogenica, or the lungs of bleomycin-treated mice caused more severe fibrosis when transferred into antibiotics+bleomycin-treated recipients, and LPS packaged in the OMVs mediated the fibrotic pathogenesis. This is a novel finding, and it would be interesting to know if these bacteria (or something equivalent) are found in IPF, and what role bacterial OMVs play in IPF, as OMVs have been shown to be potent inducers of proinflammatory responses and cell death elsewhere (12).

All of the microbiome-associated effects were found to be partially mediated by IL-17. Knocking out either IL-17A or IL-17B resulted in a 25% mortality rate in bleomycin-treated mice, as opposed to 50% in wild-type mice, which suggests that both IL-17A and IL-17B are important drivers of fibrosis, but neither is solely responsible. The authors determined the signaling mechanism used by IL-17B and described downstream targets that are similar to IL-17A, which raises the question: why produce redundant IL-17 cytokines? One answer is that although IL-17 is strongly associated with T cells, the authors find, surprisingly, that IL-17B is produced by innate immune cells. This suggests a complementary function in major branches of the immune system.

Yang and colleagues have produced a wide scope of data that open further avenues for investigation. More than one new finding was described in their work. However, there were some limitations. Microbiome depletion was achieved by means of oral and intranasal antibiotics, and oral antibiotics alone had no effect on bleomycin pathogenesis. It was a curious omission not to include a group that was only treated with intranasal antibiotics and left with an intact gut microbiome. The authors also claim that the IL-17B–producing innate immune cells were alveolar macrophages, but the markers used are not sufficient to confidently label that population (13). Further studies of these IL-17B–producing cells in vitro and better characterizations in vivo are needed. Even with these limitations, however, their work shows a new role for lung bacteria that promote fibrosis, describes mechanistic details of IL-17B signaling and OMVs, and provides evidence of innate immune cells producing IL-17. There is a correlation between fibrosis in humans and dysbiosis in the lung, and preclinical studies such as this provide insight into the potential mechanisms of a poorly understood disease (14).

Yu G, et al. Thyroid Hormone Inhibits Lung Fibrosis in Mice by Improving Epithelial Mitochondrial Function. Nat Med (15)

Reviewed by Ankita Burman and Ting Xie

Although metabolomic studies have implicated dysregulated cellular metabolism in the pathogenesis of IPF (16, 17), the molecular mechanisms by which aberrant metabolic signaling pathways contribute to lung fibrosis are not well understood. In their recent work published in Nature Medicine, Yu and colleagues found a marked increase in expression and activity of the thyroid hormone (TH)-activating enzyme iodothyronine deiodinase 2 (DIO2) in lungs of patients with IPF, potentially reflecting either TH deficiency or increased cellular metabolic requirements (15). DIO2 gene expression was negatively correlated with disease severity, suggesting an adaptive role of DIO2 in IPF. Consistent with this observation in patients, in mouse studies it was found that 1) genetic deletion of DIO2 exacerbated bleomycin-induced lung fibrosis, 2) aerosolized delivery of TH led to resolution of fibrosis in bleomycin and inducible TGF-β1 (transforming growth factor-β1) models, and 3) oral delivery of sobetirome, a thyromimetic drug, decreased established lung fibrosis in the bleomycin model. Mechanistic studies in Ppargc1a (peroxisome proliferator-activated receptor γ coactivator 1a)- or Pink1 (PTEN-induced putative kinase 1)-knockout mice and type II alveolar epithelial cells (AECs) revealed that TH acts through the PGC-1α and PINK1 pathways to attenuate mitochondria-regulated cell death pathways in type II AECs. A limitation here is the lack of evaluation of the effect of TH on cell types other than type II AECs; experiments using mice with knockdown of the TH receptor in different fibrosis-relevant cell types would be beneficial in this regard. Nevertheless, taken together, their data make a strong case for an antifibrotic role of TH in type II AECs in IPF.

In 2015, Bueno and colleagues found a prominent accumulation of dysmorphic and dysfunctional mitochondria in type II AECs in lungs of patients with IPF, which was associated with low expression of PINK1 (18). The current study by Yu and colleagues shows upstream regulation of PINK1 by TH-mediated signaling and supports the idea that dysfunctional mitochondria in type II AECs play an important role in the pathogenesis of IPF. More recently, Bueno and colleagues reported that the endoplasmic reticulum (ER) stress effector ATF3 (activating transcription factor 3) can repress PINK1 expression in type II AECs and lead to dysregulation of mitochondria homeostasis (19). Given that ER stress is a critical pathogenic mechanism in lung fibrosis (20), further studies aimed at determining the relationship among TH signaling, ER stress, and mitochondrial function could be enlightening.

The only two U.S. Food and Drug Administration–approved drugs, nintedanib and pirfenidone (and saracatinib, which was recently granted orphan drug designation for IPF by the Food and Drug Administration), target fibroblasts that are the effector cells in IPF. Because Yu and colleagues provide evidence for a beneficial effect of the thyromimetic sobetirome on the function of epithelial cells, which are important in the initiation of fibrosis, it is tempting to speculate about a potential role of thyromimetics in early stages of the disease or in combination therapy. Also, given that mitochondrial dysfunction is a hallmark of cellular aging, an open question is whether thyromimetics could prove beneficial in other aging-related conditions. However, it is important to keep in mind that animal models often fail to predict human responses to drugs (21), and it will be critical to conduct extensive studies, including assessments of dosing, delivery, safety, and efficacy, to fully elucidate the clinical potential of thyromimetics in IPF.