Assessment of Glutamine as a Fuel Source for Alveolar Macrophages Exposed to Chronic Ethanol Using an Extracellular Flux Bioanalyzer

Summary

Alcohol misuse impairs alveolar macrophage (AM) immunity due to suppressed mitochondrial respiration and bioenergetics. We recently demonstrated that ethanol (EtOH) exposure increases glutamine dependency for mitochondrial respiration in AMs. Herein, methods are provided to determine the usage of glutamine for mitochondrial respiration in EtOH-treated AMs using an extracellular flux bioanalyzer.

Abstract

Alveolar macrophages (AMs) are the first line of cellular defense in the lower airway against pathogens. However, chronic and excessive alcohol use impairs the ability of AMs to phagocytize and clear pathogens from the alveolar space, in part through dysregulated fuel metabolism and bioenergetics. Our prior work has shown that chronic ethanol (EtOH) consumption impairs mitochondrial bioenergetics and increases lactate levels in AMs. Further, we recently demonstrated that EtOH increases glutamine dependency and glutamine-dependent maximal respiration while decreasing flexibility, shifting away from pyruvate-dependent respiration and towards glutamine-dependent respiration. Glutaminolysis is an important compensatory pathway for mitochondrial respiration when pyruvate is used for lactic acid production or when other fuel sources are insufficient. Using a mouse AM cell line, MH-S cells, exposed to either no EtOH or EtOH (0.08%) for 72 h, we determined the dependency of mitochondrial respiration and bioenergetics on glutamine as a fuel source using an extracellular flux bioanalyzer. Real-time measures were done in response to bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES), an inhibitor of glutaminase 1, which prevents the enzymatic conversion of glutamine to glutamate, in media vehicle or in response to vehicle alone, followed by testing mitochondrial stress. The step-by-step protocol provided herein describes our methods and calculations for analyzing average levels of glutamine-dependent basal mitochondrial respiration, mitochondrial ATP-linked respiration, maximal mitochondrial respiration, and mitochondrial spare respiratory capacity across multiple biological and experimental replicates.

Introduction

Alcohol use disorder (AUD) affects over 28 million people in the United States¹. People with AUD are 2-4 times more likely to develop respiratory infections^2,3, increasing risk for premature morbidity and mortality^3,4,5. Alcohol misuse increases susceptibility to respiratory infections, in part through suppressing lung immune function. Alveolar macrophages (AMs) are the first line of cellular defense against pathogens in the lower lung, where they phagocytize and clear inhaled microbes. However....

Protocol

1. Chronic EtOH exposure in vitro

1. Culture MH-S cells, a mouse alveolar macrophage cell line, in complete media containing RPMI-1640 with 10% fetal bovine serum, 1% penicillin/streptomycin, 11.9 mM sodium bicarbonate, 40 mg/mL gentamicin, and 50 µM 2-mercaptoethanol at 37°C with 5% CO₂ in a humidified incubator.
2. Treat cultured MH-S cells with or without EtOH (0.8 mg/mL or 0.08%) for 72 h, where EtOH should be changed every 24 h. Incubate.......

Discussion

The data presented herein are additional biological replicates for untreated and chronic EtOH-exposed MH-S cells treated with media control or BPTES, which are published in Crotty et al.¹¹. The protocol described is used to assess the dependency of EtOH-exposed MH-S cells on glutamine as a fuel source for mitochondrial respiration and bioenergetics using an extracellular flux bioanalyzer. There are several critical steps in the protocol. Firstly, MH-S cell formation and confluency are important. M.......

Materials

Name	Company	Catalog Number	Comments
15 mL conical tube	VWR International, Inc.	21008-670	Used for preparing stock concentrations of mitochondria-related reagents.
2-mercaptoethanol	Sigma Aldrich Co.	M6250	Used for culturing MH-S cells.
Cell scraper	Dot Scientific, Inc.	70-1180	Used for scraping MH-S cells from T-75 flasks.
Countess 3 FL Instrument: cell counter	Fisher Scientific Company	AMQAF2000	Used for counting the number of MH-S cells to plate for experiments.
D-glucose	Sigma Aldrich Co.	G8270	Used for the extracellular flux base medium.
Ethanol	Fisher Scientific Company	4355720	Experimental treatment for MH-S cells.
Fetal bovine serum	Sciencell Research Laboratories	500	Used for culturing MH-S cells.
Gentamicin	Sigma Aldrich Co.	G1397	Used for culturing MH-S cells.
GlutaMAX	Thermo Fisher Scientific	35050061	Used for the extracellular flux base medium.
GraphPad Prism 10.2.3	GraphPad Software	N/A	Software for statistical analysis. Downloadable after purchase at https://www.graphpad.com/features.
MH-S cells	American Type Culture Collection	CRL-2019	Mouse alveolar macrophage cell line.
Microcentrifuge tube	USA Scientific, Inc.	4036-3212	Used for preparing working concentrations of mitochondria-related reagents.
Microsoft 365 Excel: computer spreadsheet program	Microsoft	N/A	Software for data organization and mitochondrial bioenergetics calculations. Downloadable after purchase at https://www.microsoft.com/en-us/microsoft-365/excel.
Penicillin/streptomycin	Fisher Scientific Company	15140122	Used for culturing MH-S cells.
Phosphate buffered saline	VWR International, Inc.	45000-446	Used for washing MH-S cells.
RPMI-1640	VWR International, Inc.	45000-396	Used for culturing MH-S cells.
Seahorse Wave Pro Software: computer software program for assay design and analysis	Agilent Technologies, Inc.	N/A	The computer is attached to the Seahorse XF Pro Analyzer instrument. Downloadable from  https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-software/software-download-for-seahorse-wave-pro-software?productURL=https%3A%2F%2Fwww.agilent.com%2Fen%2Fproduct%2Fcell-analysis%2Freal-time-cell-metabolic-analysis%2Fxf-software%2Fseahorse-wave-pro-software-2007523.
Seahorse XF Base Medium: extracellular flux base medium, pH 7.4	Agilent Technologies, Inc.	103334-100	Used for preparing stock and working concentrations of mitochondria-related reagents and culturing MH-S cells prior to experimental runs.
Seahorse XF Glutamine Oxidation Stress Test Kit	Agilent Technologies, Inc.	103674-100	Contains stock BPTES, oligomycin, FCCP, and rotenone/antimycin A.
Seahorse XF Pro Analyzer: extracellular flux bioanalyzer	Agilent Technologies, Inc.	N/A	Extracellular flux bioanalyzer.
Seahorse XFe96 FluxPak: 96-well extracellular flux pak cartridges, 96-well extracellular flux microculture plates, and calibrant solution	Agilent Technologies, Inc.	102416-100	Used to prepare MH-S cells for assays using an extracellular flux bioanalyzer.
Serological pipet	Santa Cruz Biotechnology, Inc.	sc-550678	Used for removing media from MH-S cells.
Sodium bicarbonate	Sigma Aldrich Co.	S6014	Used for culturing MH-S cells.
Sodium pyruvate	Sigma Aldrich Co.	P4562	Used for the extracellular flux base medium.
T-75 flasks	Santa Cruz Biotechnology, Inc.	sc-200263	Used for culturing MH-S cells.