Imaging NAD(H) Redox Alterations in Cryopreserved Alveolar Macrophages from Ozone-Exposed Mice and the Impact of Nutrient Starvation during Long Lag Times.

He N Xu,Shaili Amatya,Lin Z Li,Joanna Floros

doi:10.3390/antiox10050767

He N Xu, Shaili Amatya + Show 2 more

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https://doi.org/10.3390/antiox10050767

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Abstract

Employing the optical redox imaging technique, we previously identified a significant redox shift of nicotinamide adenine dinucleotide (NAD and the reduced form NADH) in freshly isolated alveolar macrophages (AM) from ozone-exposed mice. The goal here was twofold: (a) to determine the NAD(H) redox shift in cryopreserved AM isolated from ozone-exposed mice and (b) to investigate whether there is a difference in the redox status between cryopreserved and freshly isolated AM. We found: (i) AM from ozone-exposed mice were in a more oxidized redox state compared to that from filtered air (FA)-exposed mice, consistent with the results obtained from freshly isolated mouse AM; (ii) under FA exposure, there was no significant NAD(H) redox difference between fresh AM that had been placed on ice for 2.5 h and cryopreserved AM; however, under ozone exposure, fresh AM were more oxidized than cryopreserved AM; (iii) via the use of nutrient starvation and replenishment and H2O2-induced oxidative stress of an AM cell line, we showed that this redox difference between cryopreserved and freshly isolated AM is likely the result of the double “hit”, i.e., the ozone-induced oxidative stress plus nutrient starvation that prevented freshly isolated AM from a full recovery after being on ice for a prolonged time period. The cryopreservation technique we developed eliminates/minimizes the effects of oxidative stress and nutrient starvation on cells. This method can be adopted to preserve lung macrophages from animal models or clinical patients for further investigations.

Highlights

Alveolar macrophages (AM) are the sentinel phagocytic cells of the innate immune system in the lungs [1] and are essential in mediating lung innate immunity and maintaining tissue homeostasis
Using a mouse alveolar macrophage cell line, we further investigated why fresh AM had larger redox shifts than the cryopreserved ones by studying the hypothesis that the ~2.5 h lag time of fresh cells on ice prior to experimentation resulted in nutrient starvation
AM had a similar density of attached cells and morphology to fresh AM from SP-A KO

Summary

Introduction

Alveolar macrophages (AM) are the sentinel phagocytic cells of the innate immune system in the lungs [1] and are essential in mediating lung innate immunity and maintaining tissue homeostasis. Lung macrophages are commonly collected through bronchoalveolar lavage (BAL) or tracheal aspiration for subsequent analysis. To minimize the potential impact lag time may have on AM metabolism from the time of macrophage removal from the lung microenvironment to subsequent experimentation, cell ex vivo time should be minimized. As an alternative, collected lung macrophages can be cryopreserved and revived upon future studies. Cryopreservation is a common technique for cell culture studies, cryopreserving primary cells can be context-specific and may require fine-tuning of the protocol for better cell revival for subsequent analysis

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