Pulmonary Fibrosis and Antioxidants: Finding the Right Target

Interstitial lung disease (ILD) describes a broad range of chronic fibrotic lung disorders with diverse etiologies (1). ILD is characterized by loss of lung parenchyma and extracellular matrix deposition, which leads to worsened lung compliance, increased work of breathing, impaired gas exchange, and eventually respiratory failure (2). Importantly, idiopathic pulmonary fibrosis, a severe form of fibrosing ILD, is fatal, having a median survival of only 3–5 years (3). Currently, therapeutic options for idiopathic pulmonary fibrosis only slow progression but do not halt or reverse the disease; thus, there is a substantial need for novel insights and treatments for this devastating disorder.

Although there is an incomplete understanding of the molecular mechanisms that lead to pulmonary fibrosis, recent data support a multicellular model. In this model, shown in Figure 1, alveolar injury is the initiating event, followed by a failure of local repair (4). Lung injury induces the recruitment of inflammatory cells from the circulation, and it has been shown that monocytes from the circulation, which differentiate into monocyte-derived alveolar macrophages (MoAMs), are key drivers of pulmonary fibrosis (5). MoAMs express profibrotic genes such as TGF-β (transforming growth factor-β), which activate fibroblasts, inducing their proliferation and secretion of extracellular matrix (e.g., collagen) (5–7). Concomitantly, fibroblasts release G-CSF (granulocyte colony–stimulating factor), whose receptor is expressed on MoAMs, which sustains a population of profibrotic MoAMs in the fibrotic niche, thus setting up a positive feedback loop (5). Proper alveolar repair requires the differentiation of alveolar type-2 (AT2) cells into AT1 cells, which disrupts the interaction between fibroblasts and MoAMs, allowing reconstitution of the alveolar–capillary space (5). However, data from mouse models and patients with lung fibrosis have revealed improper AT2–AT1 differentiation, with accumulation of an intermediate cell type expressing both AT1 and AT2 genes (6). These intermediate cells are characterized by an increase in the integrated stress response (ISR) and expression of KRT8 (mice) or KRT17 (humans) (6). Importantly, the accumulation of these intermediate cells failed to repair the alveolar epithelium, thus facilitating fibrosis (6). The ISR mediates its downstream effects in part by the phosphorylation of eIF2α (eukaryotic translation initiation factor α), which results in global and gene-specific translational changes (8). ISRIB is a potent small molecule ISR inhibitor that blocks signaling downstream of eIF2α and reverses the translational effects of the ISR, including upregulation of ATF4 and CHOP (9). Intriguingly, inhibition of the ISR with ISRIB has been shown to facilitate the differentiation of AT2 to AT1 cells, promote alveolar repair, and prevent pulmonary fibrosis (4).

Figure 1.

Diagram depicting injury of the alveolar epithelium and development of lung fibrosis. (A) The normal alveolar epithelium contains small cuboidal alveolar type-2 (AT2) cells and large, flattened AT1 cells. TR-AMs (tissue-resident alveolar macrophages) reside within the alveolar space, whereas fibroblasts and extracellular matrix (ECM) are present in the interstitium. (B) PHMG, bleomycin, etc. induce epithelial injury. Monocytes from the circulation infiltrate into the alveolar space. (C) Monocytes from the circulation differentiate into MoAMs (monocyte-derived alveolar macrophages). MoAMs secrete profibrotic molecules such as TGF-β (transforming growth factor-β), which activate fibroblasts. Activated fibroblasts proliferate, deposit ECM (e.g., collagen), and release G-CSF (granulocyte colony–stimulating factor) to maintain MoAMs in the niche. (D) Instead of AT2 cells properly differentiating into AT1 cells, they incorrectly generate an intermediate cell type with properties of both AT1 and AT2 cells, which fail to repair the epithelium. Lack of repair maintains the interaction between MoAMs and fibroblasts, reinforcing their signaling feedback loop and causing persistent fibrosis. Inhibition of the integrated stress response can promote accurate AT2–AT1 differentiation, thus improving epithelial repair and reducing fibrotic disease (4). Figure created with BioRender.com. ISRIB = integrated stress response inhibitor; PHMG = polyhexamethylene guanidine.

Lung injury and pulmonary fibrosis are characterized by an increase in reactive oxygen species (ROS) and oxidative damage (10). Although physiologic ROS concentrations are required for normal signaling, elevated concentrations can lead to aberrant signaling, DNA damage, and cell death (11, 12). Mitochondria are a major source of intracellular ROS, which are produced predominantly at complexes I and III of the electron transport chain in the form of superoxide (13). In this issue of the Journal (pp. 57–72), Jeong and colleagues describe how a mitochondrial antioxidant, NecroX, significantly reduces lung injury and fibrosis in two distinct mouse models (14). In the first model, the authors used polyhexamethylene guanidine, known to cause severe lung injury and fatal ILD in children, to induce lung injury in mice (15, 16). The second model used the more common method of intratracheal instillation of bleomycin. Both polyhexamethylene guanidine and bleomycin led to similar lung disease in mice, which was characterized by alveolar injury, immune cell infiltration, and subsequent lung fibrosis. They found that in each model there was an increase in mitochondrial ROS (mROS), endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR). In addition to oxidative stress, ER stress is also a hallmark of pulmonary fibrosis, arising because of an accumulation of misfolded proteins in the ER, which activates the UPR (8, 17, 18). This results in phosphorylation of eIF2α and induction of the ATF adaptive transcriptional pathway, which are important components of the ISR (8). The authors report that the mitochondrially targeted antioxidant, NecroX, decreased mROS, improved ER stress, and reduced UPR signaling, as evidenced by reductions in both phosphorylated eIF2α and ATF-6α. In addition, NecroX reduced inflammatory cell recruitment to the lung and ameliorated lung fibrosis. Interestingly, the authors also show that 4-phenylbuteric acid, a direct ER stress inhibitor, diminished lung fibrosis. Although it remains uncertain whether NecroX is inhibiting ER stress through decreased mROS or by modulating mitochondrial calcium influx, their whole-lung tissue transcriptomics demonstrate that NecroX reversed the aberrant expression of genes involved in both mitochondrial biology and ER stress. Finally, deconvolution of whole-lung transcriptomics revealed reduced AT2 cell gene expression, consistent with a reduced number of AT2 cells, which also improved with NecroX.

Although an in silico estimation was performed, one question that the authors leave largely unanswered is what cell type(s) NecroX is acting on to mitigate lung fibrosis. Although several cell types are involved in the development of lung injury and fibrosis, alveolar epithelial cells play a crucial role in the process. We postulate that the authors’ data are consistent with a model in which NecroX improves mitochondrial oxidative injury in alveolar epithelial cells leading to reduced ER stress and attenuation of the ISR, promoting AT2–AT1 differentiation and hastening alveolar repair. This would reduce inflammatory cell recruitment into the lung, interrupt the MoAM–fibroblast feedback loop, and ameliorate tissue fibrosis. Importantly, NecroX is almost certainly scavenging mROS from other cell types in the fibrotic niche, possibly mitigating disease through additional mechanisms that may not be mutually exclusive. For example, mitochondrial antioxidants have been shown to reduce TGF-β signaling and fibrotic gene expression in lung fibroblasts from patients with pulmonary fibrosis (19). Use of flow cytometry and single-cell RNA sequencing could facilitate identification of the cell(s) mediating the effects of NecroX.

In summary, this study provides supportive evidence for a multicellular model linking epithelial mitochondrial dysfunction to activation of the ISR, impaired alveolar repair, and inflammatory cell infiltration, setting up a positive feedback loop with fibroblasts resulting in tissue fibrosis. Although the authors show that NecroX, an mROS scavenger, mitigates fibrotic lung disease in mice, a clinical trial with the nonspecific antioxidant N-acetyl cysteine failed to reduce pulmonary fibrosis in patients (20). However, it is intriguing that NecroX was able to attenuate the ISR and that it replicates previous data demonstrating that direct inhibition of the ISR with ISRIB reduced lung fibrosis in mice (4). This suggests that targeting the ISR is promising for treating pulmonary fibrosis, and future research should test whether ISR inhibition can be a valuable treatment for patients suffering from fibrotic lung diseases.