Human Fibroblasts from Patients with Airway Stenosis Have Unique, Targetable Signatures and Retain Transcriptional Memory

Introduction:: Subglottic stenosis (SGS) is the narrowing of the upper airway because of a fibrotic response to prolonged endotracheal tube intubation.¹ In SGS scar tissue builds up in the airway as resident fibroblasts in the lamina propria – the layer between tracheal cartilage and epithelium – differentiate into myofibroblasts and secrete copious amounts of extracellular matrix. Recently, transcriptomic and functional assays have stratified distinct fibroblast populations in human fibrotic skin diseases based on their capacity to secrete extracellular matrix, remodel scar tissue, or induce inflammation.² In SGS, however, this has not been examined, offering a unique opportunity to further characterize the disease to better create treatments. Therefore, the objective of this study was to compare fibroblasts derived from diseased and internal control regions of pediatric SGS airways and delineate their distinct subpopulations based on characteristic myofibrotic markers. We also sought to correlate the response of different fibroblast subpopulations to the pro-fibrotic cytokine TGF-β1 with surface markers, intracellular protein abundance, and gene expression. Overall, we demonstrate that fibroblasts from the diseased region are phenotypically matrix-depositing and pro-inflammatory fibroblasts that respond strongly to TGF-β1, while those from the internal control region are largely matrix-remodeling and resist fibrosis, indicating that targeted treatment approaches accounting for the distinct fibroblast populations are necessary to achieve superior treatment.


Materials and Methods:: Two laryngeal biopsies were obtained from each consented pediatric patient with SGS at the Children’s Hospital of Philadelphia from the diseased (stenosed) airway region and from a distal, healthy region in the trachea. Biopsies were cut with a scalpel into small pieces and cultured in growth media for 2 days to allow fibroblasts to grow out and adhere to the tissue culture flask. Then, cells were subcultured following established protocols. Successful isolation was validated with known surface markers via flow cytometry and immunofluorescence. Fibroblasts from both regions were stimulated by 10ng/mL TGF-β1 for 5 days and assessed for their fibrotic and inflammatory response by quantifying levels of the surface markers Thy-1, platelet-derived growth factor receptor-α (PDGFR-α) via flow cytometry. Additionally, RT-qPCR quantification of α-SMA, IL-6, MMP-1, MMP-3, and MMP-13, along with ELISA staining of IL-1β, IL-6, and active MMPs were performed to further characterize inflammatory responses and fibroblast phenotypes.


Results, Conclusions, and Discussions:: Results

Subcultured fibroblasts exhibited surface negativity for leukocyte markers CD45, CD31, and CD326, and positivity for PDGFR-α, indicating correct isolation of airway fibroblasts. Flow cytometry revealed increases in α-smooth muscle actin (α-SMA)— the hallmark of myofibrotic differentiation— and decreases in platelet-derived growth factor receptor-α (PDGFR-α) between fibroblasts in the stenosed and non-stenosed regions, which has been shown to be characteristic of myofibrotic differentiation.³ Additionally, flow cytometry indicated a decrease in Thy-1 in SGS fibroblasts. Notably, TGF-β1 stimulation increased expression of α-SMA in fibrotic myofibroblasts more strongly than in healthy fibroblasts. Lastly, diseased fibroblasts exhibited increased expression and secretion of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α while healthy fibroblasts showed increases in MMP-1, MMP-3, and MMP-13.



Discussions

Diseased fibroblasts exhibited a baseline higher α-SMA expression compared to controls indicating an initial, more pronounced myofibrotic state. Furthermore, the differential response of diseased and internal control fibroblasts when exposed to a pro-fibrotic stimulus (TGF-β1) suggests that each population retains a certain degree of transcriptional memory dependent on the physiological niche from which they were biopsied. Specifically, the SGS fibroblasts retained pro-fibrotic predisposition, expressing lower levels of PDGFR-α and higher levels of α-SMA, while normal fibroblasts expressed more matrix-remodeling genes. These findings possess significant potential for informing future pharmaceutical and gene-editing therapies that target specific fibroblast subpopulations to reduce or prevent scar formation and airway occlusion.



Conclusions

We demonstrated that fibroblasts from stenosed and non-stenosed regions of the airway are phenotypically different and respond differentially to TGF-β1 stimulation. These phenotypic differences offer a way of identifying fibrotic and non-fibrotic airway cells, suggesting a method to screen for and target stenosis-inducing cells and potentially prevent stenosis or re-stenosis.


Acknowledgements (Optional): :

References (Optional): : 1) Children’s Hospital of Philadelphia https://www.chop.edu/conditions-diseases/subglottic-stenosis

2)Deng, CC., Hu, YF., Zhu, DH. et al. Single-cell RNA-seq reveals fibroblast heterogeneity and increased mesenchymal fibroblasts in human fibrotic skin diseases. Nat Commun 12, 3709 (2021). https://doi.org/10.1038/s41467-021-24110-y

3)Yao et al. Temporal control of PDGFRα regulates the fibroblast-to-myofibroblast transition in wound healing Cell Reports 40, 111192 (2022). https://doi.org/10.1016/j.celrep.2022.111192