S100A alarmins and thymic stromal lymphopoietin (TSLP) regulation in severe asthma following bronchial thermoplasty

Abstract

Rationale: Severe asthma affects a small proportion of asthmatics but represents a significant healthcare challenge. Bronchial thermoplasty (BT) is an interventional treatment approach preconized for uncontrolled severe asthma after considering biologics therapy. It was showed that BT long-lastingly improves asthma control. These improvements seem to be related to the ability of BT to reduce airway smooth muscle remodeling, reduce the number of nerve fibers and to modulate bronchial epithelium integrity and behavior. Current evidence suggest that BT downregulates epithelial mucins expression, cytokine production and metabolic profile. Despite these observations, biological mechanisms explaining asthma control improvement post-BT are still not well understood.

Objectives: To assess whether BT affects gene signatures in bronchial epithelial cells (BECs).

Methods: In this study we evaluated the transcriptome of cultured bronchial epithelial cells (BECs) of severe asthmatics obtained pre- and post-BT treatment using microarrays. We further validated gene and protein expressions in BECs and in bronchial biopsies with immunohistochemistry pre- and post-BT treatment.

Measurements and main results: Transcriptomics analysis revealed that a large portion of differentially expressed genes (DEG) was involved in anti-viral response, anti-microbial response and pathogen induced cytokine storm signaling pathway. S100A gene family stood out as five members of this family where consistently downregulated post-BT. Further validation revealed that S100A7, S100A8, S100A9 and their receptor (RAGE, TLR4, CD36) expressions were highly enriched in severe asthmatic BECs. Further, these S100A family members were downregulated at the gene and protein levels in BECs and in bronchial biopsies of severe asthmatics post-BT. TLR4 and CD36 protein expression were also reduced in BECs post-BT. Thymic stromal lymphopoietin (TSLP) and human β-defensin 2 (hBD2) were significantly decreased while no significant change was observed in IL-25 and IL-33.

Conclusions: These data suggest that BT might improve asthma control by downregulating epithelial derived S100A family expression and related downstream signaling pathways.

Keywords: Alarmin; Bronchial thermoplasty; Severe asthma.

Fig. 1

Transcriptomic analysis of BECs of severe asthmatics pre- and post-BT measured using microarray. A Volcano plot showing the distribution of transcript expression P values and fold changes. All transcripts were tested using the transcriptome analysis console (TAC) software. B Heat map. The molecular signatures pre-BT and post-BT were visualized by hierarchical clustering based on the differential transcripts’ expression. C Interaction network constructed using the ingenuity pathway analysis software. Nodes shaded in pink or red represent genes that are upregulated, and blue nodes are downregulated post-BT. A solid line represents a direct interaction and a dotted line an indirect interaction. D Several S100A family genes were interconnected in the same network in the post-BT signature. The color intensity indicates the degrees of up-regulation or down-regulation

Fig. 2

Validation of S100A family gene and protein expressions in BECs of non-asthmatic-controls and severe asthmatics pre- and post-BT. A Bar plots representing qPCR data. B Bar plot representing Western Blot data on top with representative blots below

Fig. 3

S100A family protein expressions in tissues of severe asthmatics pre- and post-BT. A Representative immunostainings for S100A family proteins. B Graphical representations of immunostaining quantification. S100A family protein expression is represented as % of epithelial area

Fig. 4

Expression of S100A family receptors in BECs. Western Blots of selected S100A family receptors (RAGE, TLR4, CD36) on top with associated graphical representations below