A Differentiated Epithelial Layer Graft Improves Fibrosis and Survival in Airway Stenosis via <scp>IL</scp>‐37
Authors: Roza Khalmuratova, Yi Sook Kim, Dae Woo Kim, Hyun‐Woo Shin
Journal: Allergy (2024)

The airway epithelium serves as the first line of defense, protecting against airborne pathogens and pollutants while regulating immune responses. This layer comprises various cell types, including goblet cells, ciliated columnar cells, and basal cells. Airway basal stem cells have the ability to self-renew and differentiate into all pseudostratified epithelial cell types under both steady-state conditions and following acute injury [1-4]. Extensive and recurrent airway damage can induce fibrosis and stenosis, making normal function restoration challenging [5, 6]. Tissue engineering has shown promise in replacing irreversibly damaged airway epithelium, but no definitive methods have been established [7]. This study aimed to investigate the potential of a differentiated human airway epithelial layer graft to improve animal survival and reduce fibrosis in airway stenosis, offering a novel approach compared to conventional methods that focus on stem or mesenchymal stem cells. In our study, we investigated the efficacy of transplanting a fully differentiated human airway epithelial layer graft onto de-epithelialized trachea to enhance survival and maintain tracheal patency. First, we evaluated the protective effect of a fully differentiated human airway epithelial layer graft on de-epithelialized trachea (Figures S1 and S2). C57BL/6 mice were randomly divided into three groups: trachea with native intact epithelium (intact trachea group), de-epithelialized trachea (sham control group), or de-epithelialized trachea with transplanted human airway epithelial layer graft (graft group) (Figure 1A). The tracheal grafts were harvested from recipient mice on Day 14 post-transplantation for histological and immunohistochemical analyses. We observed the connection between the donor and host trachea under a microscope with hematoxylin–eosin (H&E) staining and found that they were anastomosed (Figure 1B, Figure S3). All animals in the intact trachea group survived, whereas four out of six mice died in the sham control group (Figure 1C). The tracheal lumen in the intact trachea group was not narrowed, and the mucosa of the trachea was similar to that of the host trachea. The lumen of the de-epithelialized trachea was narrowed, and granulation tissue proliferation was clearly observed (Figure 1D). The epithelial layer graft of fully differentiated human nasal cells to de-epithelialized trachea provided a better survival outcome, with only one animal out of six dying after surgery. An inflammatory response, characterized by an increased presence of neutrophils and macrophages, was observed in the de-epithelialized sham control trachea. In contrast, reduced inflammation and less fibrous proliferation was observed in the de-epithelialized trachea of the group that received an epithelial layer graft (Figure 1E,F). An epithelial surface was observed in all animals within the group that received an intact trachea (Figure 1B). STEM101-positive cells (a human nuclear marker) were found exclusively in the transplanted human airway epithelial layer graft (Figure 1G, Figure S3). Immunofluorescence staining demonstrated positive expression of ciliated (acetylated-tubulin positive) and basal (p63 positive) cells in both the intact trachea and graft groups (Figure 1H,I). This indicates that the transplanted human epithelial layer graft was successfully integrated into the de-epithelialized trachea. Next, we investigated any potential epithelial-derived cytokines that could suppress both inflammation and fibrosis. Recent studies have demonstrated that IL-37 plays a protective role against airway inflammation and fibrosis [8, 9]. IL-37 is a member of the IL-1 cytokine family that exhibits anti-inflammatory and immunosuppressive properties [10]. Treatment with recombinant IL-37 (50 ng/mL) inhibited the upregulated expression of α-smooth muscle actin (α-SMA) and collagen type I in NIH/3T3 cells induced by TGF-β1 (Figure S4). Furthermore, human airway epithelial cells co-cultured with NIH/3T3 cells suppressed TGF-β1-induced α-SMA and collagen type I expression. Notably, blocking IL-37 with a neutralizing antibody (20 μg/mL) restored α-SMA and collagen type I expression. These results demonstrate that IL-37 can inhibit fibroblast activation and reduce α-SMA and collagen type I production, in accordance with previous findings. To investigate the role of IL-37, we introduced lentiviral vectors into the airway epithelium (Figure S5). We initially assessed the transduction efficiency of small hairpin RNA targeting IL-37 in an ALI and discovered that fully differentiated airway epithelium transduced with lentiviral-mediated IL-37-specific small hairpin RNAs effectively inhibited IL-37 expression. To further confirm the function of epithelial-derived IL-37, C57BL/6 mice were subsequently randomly assigned to receive either de-epithelialized trachea or de-epithelialized trachea with a shControl or shIL37-treated epithelial layer graft (Figure 2A). Figure 2B,C display the results of in vivo experiments, revealing a significant impact of IL-37 knockdown on survival rates. Moreover, a noticeable increase in the number of neutrophils and macrophages was observed (Figure 2D,E). The human airway epithelial layer graft showed the presence of STEM101-positive cells in both the shControl and shIL-37-treated groups (Figure 2F). There was no significant difference in the number of ciliated cells between the two groups (Figure 2G). Immunofluorescence analysis revealed the presence of p63 with positive staining (Figure 2H). In the shCont-treated group, the human airway epithelial layer graft exhibited IL37-positive cells, while a decreased level of IL37-positive cells was seen in the shIL-37-treated group (Figure 2I and Figures S6 and S7). Epithelial cells play a crucial role in regeneration, primarily due to the differentiation potential of basal cells. Recent in vivo studies have specifically utilized epithelial basal cells, demonstrating their potential to seed artificial tracheas, leading to the formation of functional tracheal linings [11, 12]. These studies highlight the potential of epithelial cells in tissue engineering and regenerative medicine. Our study demonstrated that a fully differentiated human airway epithelial layer graft on a de-epithelialized trachea provides survival benefits and restores airway function in an animal model. In conclusion, a differentiated human airway epithelial layer graft has the potential to suppress fibrosis and improve animal survival in airway stenosis. Moreover, the beneficial effect of the epithelial layer graft is dependent on the expression of IL-37, suggesting that IL-37 could be a potential therapeutic target for airway stenosis. These findings suggest that this method could effectively treat airway diseases and injuries that result in tracheal epithelial damage. Conceptualization: H.-W. S. Methodology: R.K. Resources: D.W.K. Investigation: R.K. Animal experiments: R.K. and Y.S.K. Formal analysis and interpretation of data: R.K. and Y.S.K. Writing – original draft: R.K. Writing – review and editing: R.K. and H.-W.S. Supervision: H.-W.S. Funding acquisition: H.-W.S. This work was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI17C1669 and RS-2024-00439991) and a grant from the National Research Foundation of Korea (2022R1A4A3034038). R.K. and H.-W. S. have an intellectual property on the epithelial graft technique. Other author has declared that no conflicts of interest exist. The data that support the findings of this study are available from the corresponding author upon reasonable request. Figure S1. Figure S2. Figure S3. Figure S4. Figure S5. Figure S6. Figure S7. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.