The crucial role of alveolar fibroblasts in pulmonary fibrosis

INTRODUCTION

Fibroblasts support each organ by maintaining the structure of the extracellular matrix (ECM)^[1]. Following tissue injury, new subsets of fibroblasts, including those that produce large amounts of ECM, emerge at injury sites. Studies have shown that in chronic diseases, these fibroblasts may contribute to pathological fibrosis^[2,3]. However, the cellular origin of fibrotic pathology remains controversial. Some reports suggest that these cells may transdifferentiate from other lineages, such as hematopoietic cells, epithelial cells, pericytes, or endothelial cells^[4,5]. In contrast, a previous computational cell atlas analysis from this group indicated that pro-fibrotic fibroblasts responding to alveolar injury originate from alveolar fibroblasts^[6]. This concept-that a specific cellular lineage can be activated by microenvironmental cues to drive fibrosis-likely extends beyond the lung. For instance, a high-fat diet has been shown to create a pro-tumorigenic microenvironment that synergizes with occult hepatitis B virus infection to accelerate hepatocellular carcinoma development, underscoring the importance of metabolic and inflammatory cross-talk in disease progression^[7]. Furthermore, dietary stressors can induce profound epigenetic reprogramming, as demonstrated in the intestine, where a high-fat diet promotes tumorigenesis through such mechanisms^[8]. The article “Alveolar fibroblast lineage orchestrates lung inflammation and fibrosis” clarifies the developmental process of pro-fibrotic fibroblasts in lung injury and may provide insights into therapeutic strategies for pulmonary fibrosis^[9].

TREATMENT OF HUMAN IDIOPATHIC PULMONARY FIBROSIS

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease characterized by persistent fibrotic remodeling. Following diagnosis, the median survival is approximately 2.8 years, and mortality exceeds that of many cancers, earning IPF the designation of a “cancer-like” disorder. Currently, there is no cure for IPF. Nevertheless, comprehensive treatment strategies can help slow disease progression and alleviate symptoms. The current first-line therapies are pirfenidone and nintedanib. Long-term use of corticosteroids should be avoided - unless clear autoimmune features are present - as they provide no proven benefit and increase the risk of infection^[10]. Nintedanib, a tyrosine kinase inhibitor, exerts therapeutic effects by blocking pro-fibrotic signaling pathways such as Platelet-Derived Growth Factor Receptor (PDGFR), Fibroblast Growth Factor Receptor (FGFR), and Vascular Endothelial Growth Factor Receptor (VEGFR)^[11]. Its common side effects include diarrhea, elevated liver enzymes, and nausea. Pirfenidone is thought to exert anti-fibrotic effects by inhibiting Transforming Growth Factor Beta (TGFβ)^[12]. It also has anti-inflammatory properties, which are consistent with the findings of this study. However, its exact mechanism of action still needs further verification. Reported adverse effects include gastrointestinal disturbances (e.g., nausea and diarrhea), photosensitivity-related rashes, and generalized fatigue. Combination therapy may be considered for certain patients, but careful evaluation of potential risks is essential. Findings from this study suggest that pirfenidone used in combination with anti-inflammatory agents may have synergistic effects in attenuating both pulmonary inflammation and fibrosis. Moreover, this study identified a panel of candidate biomarkers-including SAA3 for inflammatory lung fibroblasts and CTHRC1 for fibrotic fibroblasts-that may facilitate the development of targeted therapies for IPF. Nevertheless, clinical validation remains necessary before these biomarkers can be translated into therapeutic applications.

MARKING TOOLS USED IN THE STUDY

Under normal conditions, the lungs contain multiple fibroblast subpopulations, each defined by distinct anatomical locations within the alveolar interstitium and peribronchial regions^[6]. Commonly used fibroblast markers, such as Pdgfra and Collagen, type I, alpha 1-Green Fluorescent Protein (Col1a1-GFP), cannot fully distinguish these subpopulations. Pdgfra is widely expressed in lung fibroblasts and can enrich the overall fibroblast population^[6,13]; however, it labels all fibroblast subtypes indiscriminately and thus lacks the specificity needed to separate alveolar, interstitial, or perivascular fibroblasts. Similarly, the Col1a1-GFP reporter marks all collagen I-producing fibroblasts, offering broad coverage, but its signal is predominantly concentrated around bronchovascular bundles-regions of high cellular density. As a result, this system lacks the resolution required to clearly visualize alveolar fibroblasts or distinguish between functionally distinct fibroblast subpopulations. In short, although Pdgfra and Col1a1-GFP are widely used, their lack of subpopulation specificity limits investigations into lung fibroblast heterogeneity and function.

To overcome this limitation and enable precise labeling of alveolar fibroblasts, the experimental group generated and utilized Scube2-creER transgenic mice. In these mice, Scube2 is highly expressed in alveolar fibroblasts but not in other subpopulations (e.g., perivascular or peribronchial fibroblasts)^[6]. Moreover, unlike conventional surface markers that reflect only transient cell states, Scube2-creER allows for long-term, specific lineage tracing of alveolar fibroblasts^[9]. To validate the specificity of Scube2-creER, the researchers performed hybridization assays, whole-lung imaging, and thick-section imaging, all of which confirmed that labeling was restricted to alveolar fibroblasts, with no signal observed in other fibroblast subtypes. This validation was essential for subsequent experimental phases and represented a significant highlight of the investigation.

THE RESPONSE OF ALVEOLAR FIBROBLASTS TO INJURY

Having confirmed the specificity of Scube2-creER, the research team next investigated how alveolar fibroblasts respond to injury. To this end, they performed single-cell RNA sequencing using Scube2-creER; Rosa26-tdTomato mice. Lung tissues were harvested at days 0, 7, 14, and 21 post-injury, with three biologically independent replicate groups included in the experimental design. In addition to previously described mesenchymal subtypes, the researchers identified four distinct fibroblast clusters at the injury site, which they termed fibrotic fibroblasts, inflammatory fibroblasts, stress-activated fibroblasts, and proliferative fibroblasts^[9].

Inflammatory fibroblasts were characterized by elevated expression of chemokines-such as CXCL1, CXCL2, and SAA3-as well as interferon-response genes, and played a key role in mediating early inflammatory cell recruitment. Fibrotic fibroblasts expressed high levels of collagen (COL1A1) and fibrosis markers (CTHRC1) and drove extracellular matrix deposition. Stress-activated fibroblasts expressed cell cycle arrest genes (e.g., CDKN1A/p21) and may represent a senescent state. Proliferative fibroblasts emerged transiently during the early stages of injury and contributed to tissue repair. The study mainly focused on inflammatory, fibrotic, and stress-activated fibroblasts^[14]. Pseudo-temporal analysis indicated that stress-activated fibroblasts represent the terminal state of inflammatory fibroblasts, while fibrotic fibroblasts appear to differentiate through an alternative trajectory and may also represent a terminal state. Importantly, the TGFβ signaling pathway was found to play a key role in the transition of inflammatory fibroblasts into fibrotic fibroblasts [Figure 1]. Blocking TGFβ signaling significantly reduced fibrosis (with a 50% decrease in collagen deposition)^[9] but concurrently exacerbated inflammation. This finding suggests that TGFβ indirectly alleviates excessive inflammatory responses by inhibiting the differentiation of inflammatory fibroblasts, while also functioning as a central pro-fibrotic factor. These results highlight how complex interactions between molecular signaling and the immune microenvironment shape disease progression-a concept with implications beyond fibrosis, extending to cancer biology. For instance, in lung squamous cell carcinoma, m6A RNA modification has been identified as a key regulator of the tumor immune landscape and a predictor of immunotherapy response^[15]. Thus, targeting upstream regulatory mechanisms-such as TGFβ in fibrosis or m6A-related pathways in cancer-to remodel the pathological microenvironment represents a promising cross-disease therapeutic strategy. In IPF, researchers identified highly conserved fibroblast subpopulations-including SFRP2⁺ inflammatory fibroblasts and CTHRC1⁺ fibrotic fibroblasts-through a combination of in situ hybridization and single-cell RNA sequencing in murine models. These findings confirm the cross-species conservation of inflammatory and pro-fibrotic fibroblast subtypes. Moreover, the spatial distribution of these cells in fibrotic lesions paralleled that observed in mice, verifying the translational relevance of animal models for human diseases. Finally, based on experimental and analytical data from tgfbr2 knockout models, the researchers demonstrated that following lung injury, alveolar fibroblasts transition from an inflammatory phenotype to a pro-fibrotic state, and further confirmed that these activated fibrotic fibroblasts are the primary drivers of pulmonary fibrosis.

Figure 1. Transformation pathways of fibroblast subpopulations during pulmonary fibrosis. In homeostasis, SCUBE2⁺ alveolar fibroblasts support alveolar type II (AT2) cells. Under fibrotic stimulation, mediated by TGF-β signaling, these fibroblasts shift into an SAA3⁺ inflammatory state that recruits neutrophils and monocytes, and subsequently differentiate into CTHRC1⁺ fibrotic fibroblasts, which deposit collagen within the alveolar lumen.

EXPLORATION OF CTHRC1⁺ FIBROTIC FIBROBLASTS

To elucidate the functional role of CTHRC1⁺ fibrotic fibroblasts in pulmonary fibrosis-particularly their potential to directly drive collagen deposition-the experimental group generated a Cthrc1-creER knock-in mouse model by inserting a P2A-creERT2-T2A-GFP cassette into the terminal exon of the endogenous Cthrc1 locus. This model specifically labels CTHRC1-expressing fibrotic fibroblasts after injury upon tamoxifen induction. However, because the GFP signal generated by this construct was too weak to be detected by flow cytometry or microscopy, the tdTomato reporter gene was used for cell tracking instead. To achieve this, the researchers crossed Cthrc1-creER mice with Rosa26-tdTomato reporter mice, replacing GFP with tdTomato (red fluorescence) for visualization and lineage tracing. Using this system, they monitored the spatiotemporal dynamics of CTHRC1⁺ fibroblasts during the progression of lung fibrosis. Similar to the Scube2-creER system, both models rely on tamoxifen induction; however, they target distinct fibroblast populations: Scube2-creER specifically labels alveolar fibroblasts, whereas Cthrc1-creER labels fibrotic fibroblasts [Figure 2]. Following tamoxifen induction, approximately 50% of tdTomato-labeled cells were ablated, accompanied by a significant reduction in pulmonary fibrosis, as evidenced by decreased hydroxyproline content and reduced collagen I deposition. These findings establish CTHRC1⁺ fibroblasts as key effector cells in driving fibrotic progression. Accordingly, therapeutic approaches could be developed by targeting Cthrc1 with antibodies or small-molecule inhibitors. Moreover, since CTHRC1⁺ fibroblasts are modulated by TGFβ signaling, inhibition of the TGFβ pathway also represents a promising therapeutic strategy.

Figure 2. Spatial distribution of Scube2-creER- and Cthrc1-creER-labeled fibroblasts. In homeostasis, Scube2-creER-labeled fibroblasts are located within the alveolar septa, where they support AT2 cells. During fibrosis, Cthrc1-creER‑labeled fibroblasts localize to the alveolar lumen, where they secrete collagen I and drive intra-alveolar fibrosis.

DISADVANTAGES

Although this study investigated the role of alveolar fibroblasts in pulmonary fibrosis, it did not fully rule out potential contributions from other fibroblast subpopulations to disease pathogenesis. Thus, the findings cannot be generalized to all fibroblasts under all pathological conditions. Moreover, mouse models do not replicate the chronicity, microenvironment complexity (e.g., aging, comorbidities), or heterogeneous disease progression observed in human IPF. In addition, the number of IPF samples used for comparison was limited (n = 3), which may have led to biased conclusions. Although the study partially addressed this limitation by integrating two large databases containing over 16 IPF samples^[16,17], further expansion of the dataset is required to verify cross-species consistency. A larger sample size would improve both the statistical power and experimental robustness of the study findings. Furthermore, TGFβ-targeted therapy should be evaluated in large animal models to enhance clinical relevance. While the study focused on the TGFβ signaling pathway, it did not identify specific downstream effectors or clarify its synergistic or antagonistic interactions with other signaling pathways (such as IL-1β). Notably, simultaneous knockout of the tgfbr2 gene to inhibit TGFβ signaling significantly reduced pulmonary fibrosis, lowering collagen deposition by approximately 50%. However, this intervention also appeared to exacerbate inflammatory responses, indicating the need for concurrent administration of anti-inflammatory drugs.

CONCLUSION

This study employed an ingeniously designed Scube2-creER system to achieve precise labeling of alveolar fibroblasts. Through a cross-species, multi-omics systematic investigation, it revealed the critical functions of alveolar fibroblasts and the TGFβ signaling pathway in pulmonary fibrosis pathogenesis, thereby providing an important foundation for understanding IPF mechanisms and facilitating the development of targeted therapies. These findings identify TGFβ or CTHRC1 as potential therapeutic targets, although validation in large animal IPF models remains necessary before clinical translation. Moreover, the study may also offer valuable insights into fibrotic mechanisms in other organs.