@article{776e1f3be53b40368b38bcf1c63dcf1f,
title = "Sex-dependent NAD(H) redox alteration in alveolar macrophages from mice expressing SP-A2 (but not from SP-A KO) in response to ozone exposure: Potential implications for COVID-19",
abstract = "Co-enzyme nicotinamide adenine dinucleotide (NAD(H)) redox plays a key role in macrophage function. Surfactant protein (SP-) A modulates the functions of alveolar macrophages (AM) and ozone (O3) exposure in the presence or absence of SP-A and reduces mouse survival in a sex-dependent manner. It is unclear whether and how NAD(H) redox status plays a role in the innate immune response in a sex-dependent manner. We investigated the NAD(H) redox status of AM from SP-A2 and SP-A knockout (KO) mice in response to O3 or filtered air (control) exposure using optical redox imaging technique. We found: (i) In SP-A2 mice, the redox alteration of AM in response to O3 showed sex-dependence with AM from males being significantly more oxidized and having a higher level of mitochondrial reactive oxygen species than females; (ii) AM from KO mice were more oxidized after O3 exposure and showed no sex differences; (iii) AM from female KO mice were more oxidized than female SP-A2 mice; and (iv) Two distinct subpopulations characterized by size and redox status were observed in a mouse AM sample. In conclusions, the NAD(H) redox balance in AM responds to O3 in a sex-dependent manner and the innate immune molecule, SP-A2, contributes to this observed sex-specific redox response.",
author = "Xu, {He N.} and Zhenwu Lin and Gandhi, {Chintan K.} and Shaili Amatya and Yunhua Wang and Li, {Lin Z.} and Joanna Floros",
note = "Funding Information: and anti‐inflammatory roles depending on the specific conditions and modulate immune reactions through cytokine release [4]. Metabolism has profound influence on macrophage polarization/activation and pathogenesis [5]. Macrophage activation is critically supported by metabolic shifts. Pro‐inflammatory macrophages have an anaerobic metabolic profile based on glycolysis whereas anti‐inflammatory macrophages generally rely on oxidative phosphorylation (OXPHOS) for energy generation [6,7]. Nicotinamide adenine dinucleotide (NAD+ and the reduced form NADH, denoted as NAD(H)) is an essential co‐enzyme for hundreds of reactions within the cell [8]. A change in the NAD+/NADH ratio (redox shift) can profoundly affect metabolism, including reactions of glycolysis and the tricarboxylic acid cycle (TCA) and OXPHOS. NAD(H) is also a key mediator in metabolic network, and an NAD+‐ coupled redox status shift has been demonstrated upon activation of macrophages [9]. Ozone (O3) exposure accelerates glycolysis in macrophages which requires constant re‐oxidation of NADH [10]. As an essential electron donor, NADH (and the reduced nicotinamide adenine dinucleotide phosphate (NADPH)) is intrinsically fluorescent and emits blue light (~450 nm) upon the excitation by UV light (~360 nm). Another intrinsically fluorescent molecule, flavin adenine dinucleotide (FAD), is an oxidized cofactor of flavoproteins that catalyze a wide range of biological redox reactions. FAD emits green light (~520 nm) when being excited by blue light (~430 nm). NADH and FADH2 (the reduced form of FAD) are fed into the electron transport chain in mitochondria to generate adenosine‐5′‐triphosphate by OXPHOS. Pioneered by Chance et al. [11–15], optical redox imaging (ORI) detects the intrinsic fluorescence signals of NADH and Fp (oxidized flavoproteins containing FAD) and provides the optical redox ratio, Fp/NADH or the normalized redox ratio, Fp/(NADH+Fp). The redox ratio provides a measure of mitochondrial redox state. It has been shown that Fp/NADH and Fp/(NADH+Fp) linearly correlate with the biochemically determined NAD+/NADH [16] and the mass‐spectrometry determined NAD+/(NADH+NAD+) [17,18], respectively. ORI is sensitive to cellular metabolic changes and reactive oxygen species (ROS) generation [19,20] and has been widely applied to study cellular metabolism and bioenergetics in both normal and diseased tissues [21–23]. ORI is also sensitive to or correlates with the genetic status and responds to genetic modulations (overexpression and knockdown) of oncogenes [17,24–29]. ORI of the fluorescence intensity and lifetime of NADH and FAD has also been shown to identify macrophage activation phenotypes [9,30] and detect metabolic heterogeneity of macrophages within the tumor microenvironment [30]. Surfactant protein A (SP‐A), an innate immune molecule in the lung, has a significant impact on the alveolar macrophages (AM) proteome [31,32] and bacterial phagocytosis [33]. Ozone (O3)‐ induced oxidative stress in mice with or without SP‐A exhibited a sex‐dependent effect on mouse survival after bacterial infection [33,34] in lung pneumonia dissemination [35,36], and on markers of inflammation and oxidative stress in the BAL [37]. However, humans (unlike rodents) have two functional genes, SFTPA1 and SFTPA2, encoding SP‐A1 and SP‐A2 proteins, respectively; and a number of genetic variants have been identified for each gene [38]. SP‐A1 and SP‐A2 have been shown to differentially affect the AM proteome [39,40], mouse survival and lung function [41,42]. Moreover, after O3‐induced oxidative stress, the AM miRNome (full spectrum of micro RNAs expressed in the genome) in mice carrying either SP‐A1 or SP‐A2 was differentially affected in a sex‐ dependent manner, with SP‐A2 having a major impact on males [43]. In the present study, we built on these previous findings in an attempt to better understand the mechanisms of O3‐induced oxidative stress in the presence or absence of SP‐A2, from the perspective of AM metabolism using optical redox imaging. Specifically, we found that ozone exposure altered the NAD(H) redox status of mouse AM in a sex‐dependent manner in the presence of SP‐A2, and SP‐A2 may play a role in females in counteracting O3‐induced oxidation by modulating the NAD(H) redox status. Funding Information: This work was supported by CHILD Research Fund Department of Pediatrics, Pennsylvania State University College of Medicine (J.F.), and by NIH R01CA191207 (L.Z.L.). We thank Annemarie Jacob for her assistance in redox heterogeneity analysis. We also thank the Cell and Developmental Biology (CDB) Microscopy Core, Perelman School of Medicine, University of Pennsylvania. Publisher Copyright: {\textcopyright} 2020 by the authors. Licensee MDPI, Basel, Switzerland.",
year = "2020",
month = oct,
doi = "10.3390/antiox9100915",
language = "English (US)",
volume = "9",
pages = "1--19",
journal = "Antioxidants",
issn = "2076-3921",
publisher = "Multidisciplinary Digital Publishing Institute (MDPI)",
number = "10",
}