Directional Secretomes Reflect Polarity-Specific Functions in an In Vitro Model of Human Bronchial Epithelium

Abstract

The polarity of the conducting airway epithelium is responsible for its directional secretion. This is an essential characteristic of lung integrity and function that dictates interactions between the external environment (apical) and subepithelial structures (basolateral). Defining the directional secretomes in the in vitro human bronchial epithelial (HBE) differentiated model could bring valuable insights into lung biology and pulmonary diseases. Normal primary HBE cells (n = 3) were differentiated into respiratory tract epithelium. Apical and basolateral secretions (24 h) were processed for proteome profiling and pathway analysis. A total of 243 proteins were identified in secretions from all HBE cultures combined. Of these, 51% were classified as secreted proteins, including true secreted proteins (36%) and exosomal proteins (15%). Close examination revealed consistent secretion of 69 apical proteins and 13 basolateral proteins and differential secretion of 25 proteins across all donors. Expression of Annexin A4 in apical secretions and Desmoglein-2 in basolateral secretions was validated using Western blot or ELISA in triplicate independent experiments. To the best of our knowledge, this is the first study defining apical and basolateral secretomes in the in vitro differentiated HBE model. The data demonstrate that epithelial polarity directs protein secretion with different patterns of biological processes to the apical and basolateral surfaces that are consistent with normal bronchial epithelium homeostatic functions. Applying this in vitro directional secretome model to lung diseases may elucidate their molecular pathophysiology and help define potential therapeutic targets.

Quantitative Proteomics Reveals an Altered Cystic FibrosisIn VitroBronchial Epithelial Secretome

“…Immunochemical analysis of MUC5AC and MUC5B mucins secreted by primary and life-extended cells validated the markedly increased levels in CF secretions identified by SILAC/MS/MS analysis, which quantified multiple MUC5AC and MUC5B peptides in bands that spanned agarose gels. This further supports the concept that the protein backbones of MUC5AC and MUC5B are spontaneously cleaved in a consistent manner that represents overall abundance of the mature mucins and can be reliably quantified by our MS/MS approach (35,49). Although it is generally accepted that MUC5AC and MUC5B mucins are increased in CF secretions (48,50), it has been thought that this primarily reflects the inflammatory milieu of CF airways (5,51).…”

“…Many of the most abundant proteins in UNC life-extended CF and non-CF HBE ALI apical secretions (Table E1) were also among the most abundant proteins in normal primary HBE ALI secretions (27,35). A scatter plot of the fold change of CF (labeled peptides)/non-CF (N; unlabeled peptides) (log 2 CF/N) showed that the relative expression of most proteins was unchanged (ratio of < 61.5 or log 2 < 60.58) (Figure 1).…”

“…To assess the validity of using life-extended HBE cell lines to identify differentially secreted proteins, we compared secretions from primary normal differentiated HBE cells (35) with secretions from life-extended non-CF differentiated HBE cell lines (both cohorts analyzed in the Children's National Proteomic Core Facility) regarding categorization of proteins contributing to biological processes ( Figure E1). Percentages are based on the number of proteins/total proteins identified that contribute to specific categories (out of 100%) for each cohort; many proteins appear in more than one category.…”

An in vitro model of murine middle ear epithelium

“…, 2003) and also in the apical secretions from human bronchioalveolar epithelial (HBE) cultures (Kesimer et al . , 2009; Pillai et al . , 2014).…”

Rhinovirus-Induced Airway Disease: A Model to Understand the Antiviral and Th2 Epithelial Immune Dysregulation in Childhood Asthma

“…HNEC and HBEC can be grown submerged as a cellular monolayer, or can be differentiated at air-liquid interface (ALI) to generate a pseudostratified polarized structure that closely resembles the in vivo human respiratory epithelium (18, 19). Ex vivo dsRNA exposure activates TLR-3, retinoic acid inducible gene (RIG-I), and melanoma differentiation-associated gene 5 (MDA5) signaling pathways in HNEC or HBEC, which downstream initiate transcription of IFN genes and other NFκβ-related pro-inflammatory genes (20–22).…”