Investigating FoxP3 in the Airway Epithelium and Developing Novel Airway Media to Enhance Culture Conditions and Immune Responses for Improved Clinical Translation

Abstract

Chronic rhinosinusitis (CRS) is a pervasive and multifaceted condition, affecting approximately 10-12% of the global population [1, 2]. Characterised by persistent inflammation of the nasal and paranasal sinus mucosa, CRS leads to a constellation of symptoms that can profoundly impact an individual's quality of life [2, 3]. The pathoaetiology of CRS is not well understood, however, a combination of microbiome dysbiosis and immune-epigenetic changes in mucosal tissue are thought to be involved [1]. Human Nasal Epithelial Cells (HNECs) are thought to play a non-professional role in immune regulation, however, the mechanism behind this is unclear [2]. Forkhead Box Protein 3 (FoxP3) is a transcription factor found in T-Regulatory Cells (T-Regs) and is responsible for many immune-regulatory processes [3]. If FoxP3 expression can be demonstrated in HNECs it may identify a mechanism for the immunoregulatory processes of the nasal epithelium. Nasal mucosa tissue samples and HNECs were collected intraoperatively from patients classified as either control or CRS without Nasal Polyps (CRSsNP). As outlined in Paper One, FoxP3 was found to be present in HNECs and the altered expression between control and CRSsNP patient samples indicates a possible immunoregulatory role for FoxP3 in the sinus epithelial layer. The controlling factors for FoxP3 expression in HNECs remain unclear. The study of FoxP3 is challenged by its heterogeneous patterns of expression and function. FoxP3 exhibits tissue-specific expression patterns and demonstrates involvement in multiple disease states, where alterations in its expression, stability, or function can contribute to immune dysregulation [4-6]. These intricacies make it difficult to clearly understand FoxP3’s role in mucosal epithelial cell immunology and immune homeostasis [6, 7]. Cell culture techniques further exacerbate these investigatory complexities and have significant effects on FoxP3 at an epigenetic level [8-10]. Advanced 3D cell culture techniques enable the study of the immunological function of respiratory epithelial cells in their differentiated ciliated phenotype. However, current methods have limited application for clinical translation due to the use of non-physiologic growth and differentiation media [9]. Subtle immunoepigenetic changes involved in chronic inflammatory diseases of the airway are altered in the presence of differentiation-inducing factors used in in vitro cell culture medium [9-11]. A novel airway mucosa stimulation medium (AMSM) was designed using Human Plasma Like Medium (HPLM) as a base. AMSM was tested on primary Human Nasal Epithelial Cells (HNEC) and grown in Air-Liquid Interface (ALI) cultures, AMSM was tested against the industry standard PneumaCult-ALI Medium. AMSM iteration 8.2 (AMSM8.2), developed in Paper Two, demonstrated equivalence or superiority to PneumaCult-ALI Medium-ALI across all metrics. AMSM8.2 represents a new fully modifiable airway epithelial cell differentiation medium using a physiologically reflective base, permitting clinically translatable in vitro research without sacrificing epithelial membrane form or function. Future work can be conducted into the role of FoxP3 in sinus immune dysregulation with the use of this novel media, with the aim of creating clinically translatable research in the effort to treat CRS.