Loss of the adhesion G-protein coupled receptor ADGRF5 in mice induces airway inflammation and the expression of CCL2 in lung endothelial cells

Fumimasa Kubo,Nobuhiro Nakamura,Shigehisa Hirose,Souta Oki,Tomoya Sato,Ryotaro Demizu,Donna Maretta Ariestanti,Rahmaningsih Mara Sabirin,Taku Fukuzawa

doi:10.1186/s12931-019-0973-6

Fumimasa Kubo, Nobuhiro Nakamura + Show 7 more

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https://doi.org/10.1186/s12931-019-0973-6

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Abstract

BackgroundAdhesion G-protein coupled receptor F5 (ADGRF5) was recently identified as an essential regulator of pulmonary surfactant homeostasis in alveolar type II cells. We previously showed that in addition to abnormal surfactant accumulation, Adgrf5-deficient (Adgrf5−/−) mice exhibit emphysema-like signs, suggesting a possible role for ADGRF5 in immune regulation. Here, we extended the phenotypic analysis of Adgrf5−/− mice to help understand its biological role in the lung, and especially in immune regulation.MethodsHistological features of lungs were evaluated by Alcian blue and Masson’s trichrome staining. Quantitative real-time PCR (qPCR) and western blot analyses were performed to analyze the differential expression of genes/proteins related to airway inflammation in lungs between wildtype and Adgrf5−/− mice. Acid–base status was assessed by performing blood gas tests and urine pH measurements. Inflammatory cell counting was performed using Giemsa-stained bronchoalveolar lavage cells. Serum IgE concentrations were determined by enzyme-linked immunosorbent assay. The expression of Ccl2, S100a8, S100a9, and Saa3 in primary lung endothelial cells (ECs) was determined by qPCR and/or western blotting. Finally, the effect of administrating RS504393 to 2-week-old Adgrf5−/− mice on gene expression in the lungs was analyzed by qPCR.ResultsAdgrf5−/− mice exhibited several features of chronic airway inflammation (mucous cell metaplasia, mucus hyperproduction, subepithelial fibrosis, respiratory acidosis, high serum IgE, mast cell accumulation, and neutrophilia) in parallel with elevated expression of genes involved in mucous cell metaplasia (Muc5ac, Muc5b, Slc26a4, and Clca1), fibrosis (Tgfb1, Col1a1, Fn1, and Tnc), and type 2 immune response (Il4, Il5, Il13, IL-25, and IL-33) at 12 and/or 30 weeks of age. In contrast, mRNA expression of Ccl2, S100a8, and S100a9 was upregulated in embryonic or neonatal Adgrf5−/− lungs as well as in lung ECs of Adgrf5−/− mice at 1 week of age. RS504393 treatment suppressed the upregulation of S100a8, S100a9, Slc26a4, and Il5 in Adgrf5−/− lungs.ConclusionsTargeted disruption of ADGRF5 results in the development of airway inflammation, which is likely mediated by the type 2 immune response and possibly CCL2-mediated inflammation. ADGRF5 also has a potential role in the regulation of genes encoding CCL2 in lung ECs, thereby maintaining immune homeostasis.

Highlights

Adhesion G-protein coupled receptor F5 (ADGRF5) was recently identified as an essential regulator of pulmonary surfactant homeostasis in alveolar type II cells
Increased expression of airway mucin genes and related mediators of mucus production in Adgrf5−/− lungs We determined the mRNA expression levels of lung parenchymal Muc5ac and Muc5b, which encode the major gel-forming mucins found in airway mucus [29]. Quantitative real-time PCR (qPCR) was performed using total RNA isolated from whole lungs of 1-week-old mice or post-lavage lungs of 3, 6, 12, and 30-week-old mice
SLC26A4 and CLCA1 are known to be involved in mucus production and allergic inflammation in this disease [31,32,33]. mRNA expression of these markers was upregulated in Adgrf5−/− lungs compared to that in WT lungs at 3 weeks of age (33.6-fold, p < 0.05 for Slc26a4 and 4.3-fold, p < 0.05 for Clca1), whereas no such significant differences were observed at 1 week of age (Fig. 1i, j)

Summary

Introduction

Adhesion G-protein coupled receptor F5 (ADGRF5) was recently identified as an essential regulator of pulmonary surfactant homeostasis in alveolar type II cells. We previously showed that in addition to abnormal surfactant accumulation, Adgrf5-deficient (Adgrf5−/−) mice exhibit emphysema-like signs, suggesting a possible role for ADGRF5 in immune regulation. Adhesion G protein-coupled receptors (GPCRs) comprise the second largest group of seven transmembrane receptors (33 members in humans), and are defined by exceptionally long N-terminal extracellular domains. These proteins are expressed in a cell typeand tissue-specific manner and are associated with diverse cellular and physiological functions such as cell adhesion and migration, immune responses, and tumorigenesis [1]. The function of ADGRF5 in lung ECs is not fully understood

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