Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium

Nadzeya Marozkina,James F Chmiel,Benjamin Gaston,Rebekah S Cunningham,Laura Smith,Yi Zhao,Jeeho Kim,Janna Kiselar,Michael D Davis,Scott H Randell,Joe Zein

doi:10.1038/s41598-021-94782-5

Abstract

Endothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase (eNOS) biochemistry. We hypothesized that Hb could also be expressed and biochemically active in the ciliated human airway epithelium. Primary human airway epithelial cells, cultured at air–liquid interface (ALI), were obtained by clinical airway brushings or from explanted lungs. Human airway Hb mRNA data were from publically available databases; or from RT-PCR. Hb proteins were identified by immunoprecipitation, immunoblot, immunohistochemistry, immunofluorescence and liquid chromatography- mass spectrometry. Viral vectors were used to alter Hbβ expression. Heme and nitrogen oxides were measured colorimetrically. Hb mRNA was expressed in human ciliated epithelial cells. Heme proteins (Hbα, β, and δ) were detected in ALI cultures by several methods. Higher levels of airway epithelial Hbβ gene expression were associated with lower FEV1 in asthma. Both Hbβ knockdown and overexpression affected cell morphology. Hbβ and eNOS were apically colocalized. Binding heme with CO decreased extracellular accumulation of nitrogen oxides. Human airway epithelial cells express Hb. Higher levels of Hbβ gene expression were associated with airflow obstruction. Hbβ and eNOS were colocalized in ciliated cells, and heme affected oxidation of the NOS product. Epithelial Hb expression may be relevant to human airways diseases.

Highlights

Endothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase biochemistry
We were interested in the possibility that Hb could be expressed in ciliated epithelium because it could modify chemistry downstream of NOS activity, as it does in the vasculature, nitrogen oxide signaling has been difficult to understand in a number of airways diseases including a sthma[6], cystic fibrosis (CF)[3], sickle cell a nemia[7], and primary ciliary dyskinesia (PCD)[8]
Hemoglobin β (Hbβ) RNA, hemoglobin α1 (Hbα1) RNA and hemoglobin α2 (Hbα2) RNA expression, shown as box-and-whisker plots, were expressed in human airway epithelium collected in 48 samples of the healthy donors and obtained from the GEO database, Series GSE97036

Summary

Introduction

Endothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase (eNOS) biochemistry. Higher levels of airway epithelial Hbβ gene expression were associated with lower FEV1 in asthma. Both Hbβ knockdown and overexpression affected cell morphology. Human airway epithelial cells express Hb. Higher levels of Hbβ gene expression were associated with airflow obstruction. Hbβ and eNOS were colocalized in ciliated cells, and heme affected oxidation of the NOS product. We were interested in the possibility that Hb could be expressed in ciliated epithelium because it could modify chemistry downstream of NOS activity, as it does in the vasculature, nitrogen oxide signaling has been difficult to understand in a number of airways diseases including a sthma[6], cystic fibrosis (CF)[3], sickle cell a nemia[7], and primary ciliary dyskinesia (PCD)[8]

Methods

Results

Conclusion