Effect of (R)‐salbutamol on the switch of phenotype and metabolic pattern in LPS‐induced macrophage cells

Abstract Evidence demonstrates that M1 macrophage polarization promotes inflammatory disease. Here, we discovered that (R)‐salbutamol, a β2 receptor agonist, inhibits and reprograms the cellular metabolism of RAW264.7 macrophages. (R)‐salbutamol significantly inhibited LPS‐induced M1 macrophage polarization and downregulated expressions of typical M1 macrophage cytokines, including monocyte chemotactic protein‐1 (MCP‐1), interleukin‐1β (IL‐1β) and tumour necrosis factor α (TNF‐α). Also, (R)‐salbutamol significantly decreased the production of inducible nitric oxide synthase (iNOS), nitric oxide (NO) and reactive oxygen species (ROS), while increasing the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio. In contrast, (S)‐salbutamol increased the production of NO and ROS. Bioenergetic profiles showed that (R)‐salbutamol significantly reduced aerobic glycolysis and enhanced mitochondrial respiration. Untargeted metabolomics analysis demonstrated that (R)‐salbutamol modulated metabolic pathways, of which three metabolic pathways, namely, (a) phenylalanine metabolism, (b) the pentose phosphate pathway and (c) glycerophospholipid metabolism were the most noticeably impacted pathways. The effects of (R)‐salbutamol on M1 polarization were inhibited by a specific β2 receptor antagonist, ICI‐118551. These findings demonstrated that (R)‐salbutamol inhibits the M1 phenotype by downregulating aerobic glycolysis and glycerophospholipid metabolism, which may propose (R)‐salbutamol as the major pharmacologically active component of racemic salbutamol for the treatment of inflammatory diseases and highlight the medicinal value of (R)‐salbutamol.

activities. Recent studies have shown that the relative proportion of macrophage subsets, rather than the absolute number of macrophages, significantly affects disease progression. 4,5 By combining gene expression profiles and surface marker quantification, a series of different macrophage populations, namely M1 pro-inflammatory and M2 anti-inflammatory macrophages, have been characterized. M1 macrophages mediate inflammation by upregulating monocyte chemotactic protein (MCP)-1, interleukin (IL)-1β and tumour necrosis factor-alpha (TNF-α), while M2 macrophages mediate the resolution of inflammation by inducing the expression of CD206, arginase-1 (arg-1) and IL-10. 6 Failure to regulate these mediators can lead to tissue damage and cell destruction due to production of reactive oxygen species (ROS) and nitric oxide (NO). Pro-inflammatory cytokines associated with M1 macrophages are increasingly recognized as central mediators in chronic inflammatory diseases and some cardiovascular diseases (CVDs). [7][8][9][10] The persistent polarization of M1 macrophages leads to an inflammatory milieu that prevents the transition to inflammation regression. 11 It is well known that redox imbalance and oxidative stress contribute to the inflammatory development. Glutathione (GSH), low-molecular weight thiol compound, can protect cells from oxidative stress by scavenging excess radicals. 12 Therefore, inhibiting the polarization of M1 macrophages is key to reducing the level of inflammation in the progression of various diseases.
Metabolic changes in macrophages are associated with different inflammatory responses. Pathogen infection, for example can lead to the "reprogramming" of immune cell metabolism. There is an increase in intracellular glucose metabolism (increased aerobic glycolysis (ie Warburg effect) and accelerated glucose uptake) upon pathogen infection, which mobilizes immune cells to destroy foreign bodies. 13 Other studies have reported that modifications of aerobic glycolysis result in altered immune cell activities. 14 Taken together, these data suggest that immune cell activity may be moderated via intracellular glucose metabolism regulation. β 2 adrenergic receptor agonists are known treatments of obstructive lung diseases. 15 The activation of adenylate cyclase increases cyclic AMP synthesis and relaxes bronchial smooth muscle.
Moreover, β 2 adrenergic receptor agonists possess a number of antiinflammatory effects. 16 Racemic salbutamol is a 50:50 mixture of the (S)-and (R)-isomers of salbutamol, of which the latter acts as the active enantiomer. 17 (S)-salbutamol was found to induce exaggerated airway reactivity and exacerbate asthmatic conditions. 18,19 It increases intracellular calcium, causing airway hypersensitivity and leading to bronchoconstriction, 18 and thus may contribute to the cumulative adverse effects. In addition, salbutamol has been shown to modulate the macrophage immune response. 16  However, the anti-inflammatory mechanisms of (R)-salbutamol are not fully understood, and the association of the anti-inflammatory potential of (R)-salbutamol with macrophage metabolism and polarization has yet to be investigated. The RAW264.7 cell line is an established model for the study of macrophage function and was used to investigate the in vitro effects of (R)-salbutamol on macrophage polarization and metabolism.
In this study, we evaluated the effect of (R)-salbutamol on the inhibition of the lipopolysaccharide (LPS)-induced activation of RAW264.7 macrophages. We examined the effect of (R)-salbutamol on the typical cytokines of M1 macrophages at the messenger RNA (mRNA) and protein levels. In addition, we investigated the suppressive effects of (R)-salbutamol on M1 macrophage polarization and cellular metabolism reprogramming. These findings may provide evidence for (R)-salbutamol to be a candidate drug in treating inflammatory diseases.

RAW264.7 cell lines were gifted from the Southern Medical
University. DMEM supplemented with 0.1% (v/v) penicillin/streptomycin, 10% (v/v) heat-inactivated FBS and 4.5 g/L glucose was used to nurture the cells in a humidified incubator (5% (v/v) CO 2 at 37°C), and cells at passages 5-10 were used for all experiments. It was reported that treatment with 100 ng/mL LPS for 12 hours was found enough to induce the largest mRNA expressions of IL-12, IL-1, TNF-α, IL-1Ra, IL-6 and IFN-γ, 20 This is consistent with another study indicating that treatment with 100 ng/mL LPS for 12 hours upregulated M1 macrophage cytokines. 21 Based on these studies, a concentration of 100 ng/mL LPS and 12-hour treatment period were selected to induce M1 polarization in RAW264.7 cells for subsequent experiments.

| Cell viability assay
A CCK-8 assay (Dojindo) was used to determine the cell viability following the manufacturer's instructions. RAW264.7 cells were treated with various concentrations of (R)-salbutamol for one hour prior to LPS induction (100 ng/mL). Cells were then incubated for a further 2 hours with the addition of 10 μL of CCK-8. Cells were visualized at 450 nm with an Enspire-2300 Multimode Reader (PerkinElmer).

| Cell phenotype identification
Six-well plates were used for seeding RAW264.7 cells (1 × 10 5 cells/ well) overnight. LPS (100 ng/mL) was used to treat the cells after the addition of (R)-salbutamol. Upon completion of treatment, all cells were extracted and rinsed twice with PBS, before being blocked on ice for 30 minutes with magnetic-activated cell sorting (MACS) buffer. Then, the cells were labelled with the following antibodies: PE-conjugated anti-mouse F4/80, APC-conjugated anti-mouse CD11c and FITC-conjugated anti-mouse CD206. FITC-, APC-and PE-conjugated rat anti-mouse IgG antibodies served as an isotype control for nonspecific background signals. Labelled cells were analysed using a BD FACSAriaIII cell sorter (BDIS). FlowJo software (Tree Star, Inc) was used to analyse data.

| ROS and NO detection
The intracellular ROS levels were examined using DCFH-DA (Life Technologies-Thermo Fisher Scientific) before visualization with a LSM710 Laser Scanning Confocal Microscope (Carl Zeiss) to quantify the fluorescence signals of the oxidized product (2′,7′-dichlorofluorescein, DCF).
The Griess assay (Beyotime) was used to evaluate the amount of NO in the culture supernatant by measuring the concentration of nitrite (a stable NO breakdown product). An NO − sensitive fluorescence probe DAF-FM DA (Sigma) was used to detect intracellular NO. 22 DAF-FM DA (10 μmol/L) was used to label the cells at 37°C for 30 minutes before they were washed thrice with PBS. Fluorescence was detected using a LSM710 Laser Scanning Confocal Microscope (scale bars, 100 μm) (Carl Zeiss).

| Intracellular GSH/GSSG ratio determination
The total levels of intracellular total GSH and oxidized glutathione (GSSG) in the cells were measured using a total GSH and GSSG assay kit (Beyotime), respectively.

| Evaluation of cytokine levels by enzyme-linked immunosorbent assay
Mouse enzyme-linked immunosorbent assay (ELISA) kits were used to determine the concentrations of MCP-1, IL-1β and TNF-α in the cell supernatants (Neobioscience).

| RNA isolation and real-time PCR analysis
TRIzol reagent (Life Technologies Inc, Gibco) was used to isolate total RNA from RAW264.7 cells prior to cDNA synthesis using the M-MLV 1st Strand Kit from Invitrogen. Quantitative real-time PCR was performed with the SYBR Green Mix (Life Technologies Inc, Gibco). The relative expression level of each mRNA (MCP-1, IL-1β and TNF-α) was compared against the levels of the endogenous protein β-actin with the 2 −∆∆Ct cycle threshold method. Table S1 lists all gene sequences related to this experiment.

| Western blotting analysis
Western blotting was used to assess the relative expression levels of iNOS in RAW264.7 cells. Briefly, total protein was extracted, and protein concentrations were then determined with the BCA kit (Thermo Scientific). Proteins were denatured and then subjected to 8% (v/v) sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Bio-Rad, CA, USA) before being exposed to 5% (w/v) skim milk for 1 hour. The membranes were then incubated with primary antibodies against iNOS (1:1000; Cell Signaling Technology) and β-actin (1:1000; Affinity Biosciences) overnight at 4°C. The membranes were subsequently incubated with the appropriate secondary antibodies for 1 hour at room temperature. The membranes were rinsed with 0.1% (v/v) Tween-20 in Tris-buffered saline between each step. Finally, the signals were detected by Image Lab software (Bio-Rad Laboratories) after incubation with an enhanced luminescence kit (Thermo Scientific).

| Seahorse analysis
The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of the RAW264.7 cells were measured in real-time using a Seahorse XF96 extracellular flux analyser (Agilent). The cells were seeded in an XF96 cell culture plate 2 days before the experiment and cultivated in a humidified atmosphere (5% (v/v) CO 2 at 37°C).
The next day, 200 µL of XF calibrator was inserted into all XF cartridge wells before being subjected to an overnight incubation at 37°C in a humidified atmosphere without CO 2 . One hour prior to the experiment, cells were rinsed with PBS, and then, XF assay medium was added to each well and then incubation at 37°C for 1 hour in a humidified atmosphere without CO 2 . For OCR analysis, 1 mmol/L sodium pyruvate, 2 mmol/L l-glutamine and 10 mmol/L glucose were added into the XF assay medium. After measuring basal respiration, rotenone/antimycin A (1 μmol/L), carbonyl cyanide m-chlorophenyl hydrazone (0.5 μmol/L) and oligomycin (1 μmol/L) were injected into each sequence to evaluate respiratory chain coupling and the maximal and nonmitochondrial oxygen consumption. In the ECAR assay, 2 mmol/L l-glutamine was added to the XF assay medium. Glycolytic flux (glycolytic reserve, glycolytic capacity and glycolytic reserve) was assessed by sequentially adding 10 mmol/L glucose, 1 μmol/L oligomycin and 50 mmol/L 2-deoxyglucose. The OCR and ECAR values were automatically calculated by the Seahorse XF-96 software.

| Metabolite extraction of cells
After incubation, cells were harvested and disrupted using a tissue grinder (SCIENTZ-48). A three-solvent biphasic system with a methyl-T-butyl-ether:methanol:water (MTBE solution, v/v/v) at a volume ratio of 6:3:1 was used to extract metabolites in this study. [23][24][25] A total of 40 µL of cell lysate was added to 160 µL of MTBE solution, and the sample was vigorously vortexed at 4°C for  All samples were kept at 4°C, and 5 µL of each sample was used for analysis.

| Sample injection description
Two extract fractions from biphasic extractions were generated from each sample, 28 an organic layer and an aqueous layer. The first injection of 5 µL of the organic layer was followed by a second injection of 5 µL of the aqueous phase onto the same column for the gradient described above. However, the gradient had not yet begun, and the method lasted only 1 minutes without increasing the concentration of the mobile phase of acetonitrile (B solvent), ensuring that the organic phase of hydrophobic lipids remained at the head of the column. Immediately afterwards (via the next line in the sequence table), 5 µL of the aqueous phase was injected into the same column, and the complete gradient was executed.

| Statistical analysis
The UHPLC/ESI-TIMS TOF-MS/MS data were analysed using software (Waters) for peak alignment, selection and normalization to determine the peak intensities for retention time (RT) and m/z data pairs. The potential biomarkers responsible for the discrimination between these groups were identified based on variable importance in projection (VIP) values > 1.0, P values < .05 and max fold change > 2. The resultant data matrices were exported to the

| (R)-salbutamol inhibits the polarization of M1 macrophages in LPS-induced RAW264.7 cells via the β 2 adrenergic receptor
(R)-salbutamol is a well-known asthma bronchodilator ( Figure   S1A). (R)-salbutamol cytotoxicity on RAW2647 cells was exam- The results showed that treatment with LPS led to a significant NO and ROS elevation in macrophage cells as evidenced by increased fluorescence intensity, while treatment with various concentrations of (R)-salbutamol prior to LPS exposure significantly reduced the amount of intracellular NO and ROS induced by LPS in a dose-dependent manner (data not shown). The average peak plasma concentration after a single 4 mg tablet of salbutamol is 30-60 nmol/L. 29,30 The peak plasma concentrations of salbutamol post-inhalation are 7.5-23 nmol/L. 31 The concentration of salbutamol in the lungs is inevitably higher than that in the blood.
(R)-salbutamol is a eutomer of salbutamol. Therefore in view of the effect of (R)-salbutamol in various concentration on NO and ROS, the concentration of (R)-salbutamol (10 µmol/L) used in this study is acceptable and mirrors dosages administered in other studies on human airway epithelial cells. 32 Next, to investigate the effect of β 2 adrenergic receptor acti-  (Figures 1 and S2B). In the LPS-induced macrophage cell model, only a few M2 macrophages can be observed.
In addition, the MFI of M2 macrophages upon stimulation with LPS and pretreatment with (R)-salbutamol were not significantly different. To investigate whether (R)-salbutamol mediates its effects on M1 macrophage polarization via the β2 adrenergic receptor, a specific β2 adrenergic receptor antagonist, ICI-118551, was employed in this study. These findings indicate that the MFI and counts of M1 macrophages were raised to 77.5% and 97.3%, respectively, when cells were pretreated with (R)-salbutamol following incubation with ICI-118551 (Figures 1 and S2B). Taken together, these findings suggested that (R)-salbutamol exerted its inhibitory effects on M1 macrophage polarization through the β 2 adrenergic receptor.

| (R)-salbutamol decreases the production of MCP-1, IL-1β and TNF-α in LPS-induced RAW264.7 cells
To confirm that M1 polarization was more predominant than M2 polarization after stimulation with LPS, the levels of typical M1 macrophage cytokines (ie MCP-1, IL-1β and TNF-α) were determined using ELISA; these cytokines are mainly synthesized by macrophages. 34,35 These pro-inflammatory cytokines are mediators of many human chronic inflammatory diseases and have been associated with acute phase reactions. 34,36 As shown in Figure 2A

| (R)-salbutamol decreases NO and ROS production in LPS-induced RAW264.7 cells
Lipopolysaccharide can cause chronic inflammation, which is usually linked with higher NO levels. 37 We hypothesized that (R)-salbutamol exhibited anti-inflammatory properties. To determine the anti-inflammatory impact of (R)-salbutamol on M1 macrophage polarization, the intracellular NO levels were determined using DAF-

| (R)-salbutamol increases the ratio of GSH/ GSSG in LPS-induced RAW264.7 cells
Since glutathione (GSH) is a direct scavenger for excessive ROS. 38 The effect of (R)-salbutamol on intracellular GSH was also evaluated in this study. Intracellular GSH/GSSG ratios decreased to 70.60% of control levels when cells were induced with LPS, and the ratio F I G U R E 3 Effects of (R)-salbutamol on LPS-induced NO production and the expression of iNOS in RAW264.7

| (R)-salbutamol rescues mitochondrial respiration and inhibits aerobic glycolysis in the LPSinduced RAW264.7 cells
Macrophage activation elicits changes in metabolic profiles according to activation state. It has been shown that LPS-induced macrophages adopt glycolytic metabolic profiles. 39 , 100 μm). C, The GSH/GSSG ratio. Data are presented as the mean ± SD. ** P < .01, * P < .05; ns, not significant metabolic plasticity to maintain intracellular ATP content ( Figure 5D).
Collectively, these data suggested that (R)-salbutamol likely mediated the metabolism shift in LPS-induced cells and that it protected against LPS-induced inflammation. An in-depth investigation is needed to clarify the intrinsic mechanism as well as to identify the molecular target of (R)-salbutamol in the process of LPS-induced inflammation.
To obtain an improved overall understanding of the bioenergetic profiles of (R)-salbutamol in LPS-induced cells, basal ECAR was plotted against mitochondrial OCR ( Figure 5E). Two distinct groups of cellular bioenergetic profiles were identified. The control cells and LPS-induced cells pretreated with (R)-salbutamol had a more aerobic phenotype than LPS-induced cells and ICI-118551-treated cells, which were more glycolytic ( Figure 5E). The ratio of OCR/ECAR was reduced in LPS-induced cells compared with control cells, whereas the ratio of OCR/ECAR was raised when LPS-induced cells were pretreated with (R)-salbutamol ( Figure 5F). Similarly, ICI-118551 treatment did not change the ratio of OCR/ECAR in LPS-induced cells.
Collectively, cells pretreated with (R)-salbutamol displayed lower glycolytic capacities than those with LPS-induced cells, indicating that (R)-salbutamol can restore the maximal glycolytic and respiratory capacities to near-normal levels.

| Method validation
To investigate the metabolic mechanisms of (R)-salbutamol, an un-  respectively, while the RSDs of peak area were within the ranges of 2.45%-13.95%, 1.51%-7.92% and 1.69%-8.79% for repeatability, injection precision and system stability, respectively (Table S2). These findings demonstrated that the present analytical method is appropriate for metabolomics analysis, as the data showed great stability and reproducibility.

| Multivariate analysis and identification of potential biomarkers
Powerful To compare metabolite changes among these samples, volcano plots were constructed, and the data revealed metabolites that were significantly upregulated (red plots) and downregulated (green plots) ( Figure 6E-F). In addition, Venn diagrams were constructed to compare the characteristics of each metabolite in the LPS-induced group, (R)-salbutamol-treated group and ICI-118551-treated group ( Figure 6G-H). Based on VIP values > 1, P < .05 and fold change > 2, a total of 11 potential biomarkers were ascertained from the peak profile of metabolomics by HMDB, EZinfo software and LipidMAPS (Table S3). These identified metabolites were deoxyribose-5-phosphate, lysophosphatidylcholine (LysoPC) (14:0), LysoPC

| Metabolic pathways
To further elucidate the metabolic pathways that were regulated by (R)-salbutamol in LPS-induced cells, the above-mentioned biomarkers were further analysed using MetaboAnalyst 4.0. Several pathways, including glycerophospholipid metabolism, phenylalanine metabolism and the pentose phosphate pathway, were highly impacted, suggesting that these pathways are involved in the (R)-salbutamol-mediated M1 polarization of LPS-induced cells ( Figure 6J).
In particular, glycerophospholipid metabolism was the most highly impacted pathway, suggesting that glycerophospholipid metabolism, but not phenylalanine metabolism and the pentose phosphate pathway, is likely involved in (R)-salbutamol-mediated M1 polarization.
Taken together, our findings suggested that the gradual variation in effects was due to perturbations of endogenous metabolites in macrophages under different conditions.

| D ISCUSS I ON
This study investigates the effects of (R)-salbutamol, a β 2 receptor agonist, on M1 macrophage polarization and metabolic alterations in LPS-induced RAW264.7 cells. β 2 receptor agonists are the cornerstone bronchodilating agents used to treat obstructive lung diseases. 15 These agents have also been demonstrated to possess anti-inflammatory properties on airways and may reduce pro-inflammatory mediators as well as prevent tissue oedema and exudate. 40,41 A commonly used β 2 receptor agonist is racemic salbutamol which contains both (R)-salbutamol and (S)-salbutamol. Racemic salbutamol reduces carrageenan-induced paw oedema in rodents 42 via a β 2 receptor-dependent mechanism. 43 On the other hand, studies showed that (S)-salbutamol likely exacerbates asthma 18 and results in pro-inflammatory influences. 44 In this study, we demonstrated that the (S)-enantiomer of salbutamol differs from the (R)-enanti-

omer. (S)-salbutamol increases NO and ROS levels in macrophages
instead of inhibiting these molecules like its counterpart. This finding suggests that the (S)-enantiomer of salbutamol may play different roles in macrophage polarization in the inflammatory response.
The mechanism of the differences between the salbutamol (S)-and (R)-enantiomers needs further investigation. Collectively, we found that (R)-salbutamol inhibited the LPS-induced M1 phenotype of macrophages, which may be associated with the anti-inflammatory mechanism of (R)-salbutamol.
Macrophages are crucial in host defence against infections.
Inflammatory diseases and cancer have been documented to possess an excess of pro-inflammatory molecules such as IL-1β, TNF-α, NO and ROS. Inflammation is a double-edged sword. On one hand, it is responsible for stimulating tissue regrowth and halting worsening cellular injury. However, prolonged and uncontrolled inflammatory responses lead to severe tissue damage culminating with multi-organ failure with high mortality rates. The present study showed that 10 µmol/L (R)-salbutamol could reduce the expression of typical cytokines found in M1 macrophages (ie MCP-1, IL-1β and TNF-α) more effectively than 100 nmol/L salbutamol. 43 These results mirror those of Tanaka et al, who found that salbutamol exhibited protective anti-inflammatory effects on LPS-treated rat peritoneal macrophages. 16 Additionally, we found that (R)-salbutamol reduced the production of NO, iNOS and ROS ( Figure 3). NO works at almost all stages of inflammation by regulating of inflammatory cell transmission. 45 In LPS-induced inflammation, NO is produced by iNOS. Racemic salbutamol reportedly inhibited the mRNA and protein levels of iNOS via the ERK pathway in rat peritoneal macrophages. 46 Similarly, both exogenous and endogenous ROS cause oxidative DNA damage that alters cell signal transduction, a deleterious process that is observed in several stages of tumorigenesis such as tumour development and progression. ROS production is amplified in cells exposed to LPS. Our data showed that (R)-salbutamol can prevent excessive ROS through the LPS-mediated macrophage pro-inflammatory response. Furthermore, the inhibitory properties of (R)-salbutamol could be blocked by specific β 2 receptor antagonists, ICI-118551.
In addition, the ratio of GSH/GSSG was increased in LPS-induced cells pretreated with (R)-salbutamol. GSH is a cytosol sulfhydryl antioxidant and can scavenge excessive ROS, which results in the reduction of intracellular ROS in LPS-induced macrophages. This reduction in oxidative stress inhibits manufacturing of pro-inflammatory cytokines, such as MCP-1, IL-1β and TNF-α. 47 In summary, we discovered that (R)-salbutamol, the (R)-enantiomer of a widely prescribed β 2 receptor agonist, may be critical in the LPS-induced switch of RAW264.7 cells to the M1 phenotype via the β 2 adrenergic receptor. Nevertheless, its effects on M2 macrophage polarization is less clear, requiring further investigation.
Cell metabolism reprogramming is essential for the inflammatory process, especially during macrophage polarization. 48 Immune cell activation depends heavily on intracellular glucose metabolism. 49 In this study, we investigated the bioenergetic profiles of mitochondrial respiration and aerobic glycolysis in LPS-induced cells and compared them with cells pretreated with (R)-salbutamol and ICI-118551. We found that (R)-salbutamol significantly inhibited intracellular aerobic glycolysis. Thus, these data revealed that (R)-salbutamol rescued basal respiration and reduced OCR, respiratory reserves, ATP production and maximal respiration in LPS-induced cells, suggesting that LPS markedly altered cellular metabolism. In the presence of oxygen, the metabolic phenotype was characterized by the production of glycolytic energy, which is highly similar to the Warburg effect seen in tumour cells. 50 Our data suggested that LPS stimulation leads to metabolic reprogramming via switching OXPHOS to aerobic glycolysis. These results are similar to reports demonstrating a decrease in bone marrow-derived macrophage glycolysis after pretreatment with racemic salbutamol. 51 LPS could disrupt mitochondrial homeostasis by enhancing aerobic glycolysis accompanied, and this was accompanied by a decrease in mitochondrial respiration. In this study, we found that cells pretreated with (R)-salbutamol could reverse this phenotype by normalizing its metabolic manner. However, we should consider the underlying mechanism of how (R)-salbutamol inhibits the aerobic effect facilitated by LPS.
The Warburg effect suggests that cells under severe oxidative stress benefit from transitioning from oxidative to reductive metabolism.
Taken together, further studies are needed to investigate how (R)salbutamol downregulates aerobic glycolysis and how this enhances mitochondrial respiration.
Previous studies showed that metabolic reprogramming is critical for the maturation and polarization of immune cells. 52  Recent studies revealed that LysoPCs promote and stabilize a strong M1 phenotype during macrophage polarization 56 and thereby increase ROS and NO production. [57][58][59] The levels of PE and LysoPE increased in activated human macrophages. 60 The present study showed that

CO N FLI C T O F I NTE R E S T S
The authors have declared no conflict of interest. F I G U R E 7 Potential inhibitory effects of (R)-salbutamol on M1 macrophage polarization and metabolism. Red and blue boxes represent the activation and inhibition of some indices that were evaluated in this study. Words in black are metabolites that were not detected in this experiment. Italic words in the blue boxes represent the pathways that were most affected in this study the manuscript. All authors drafted or critically revised the manuscript for important intellectual content and approved the final version of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed in this study are available from the corresponding author on reasonable request.