INTRODUCTION
Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is caused by several direct insults or indirect factors to the lung. Sepsis is the main cause of indirect injury (1, 2). Sepsis and sepsis-induced multiple organ failure remain one of the leading causes of death in critically ill patients and present a major challenge for both scientists and clinicians (3). The lung is the organ most vulnerable to sepsis, which is characterized by imbalance of pro-inflammatory/anti-inflammatory cytokines, pulmonary alveolar and interstitial edema, progressive hypoxemia, and impaired gas exchange (1).
Macrophages display a remarkable ability to produce a variety of pro-inflammatory/anti-inflammatory factors to eliminate pathogenic microorganisms and ensure resolution of inflammation. Depending on the inflammatory microenvironment in the lung, alveolar macrophages can acquire specific functional phenotypes. The different phenotypes are often described as “classical activated macrophages” (M1) and “alternately activated macrophages” (M2). In the early stage of inflammation, M1 macrophages produce pro-inflammatory cytokines such as IL-1β, IL-6, IL-12, and tumor necrosis factor (TNF)-α (4), reactive nitrogen intermediates and reactive oxygen intermediates (5). Thereby, M1 macrophages inhibit cellular proliferation and even cause tissue injury. In contrast, the M2 phenotype expresses anti-inflammatory cytokines like IL-10 and other effectors including arginase 1 (Arg1), chitinase-3-like 3 (Ym1), Mrc 1 (CD206), which are involved in tissue repair and wound healing (5, 6). It is well known that both of these phenotypes play an important role in the process of pulmonary inflammation. Thus, the balance between M1/M2 macrophages is critical in controlling the excessive inflammation and triggering wound healing. Therefore, the determination of treatments that balance M1/M2 alveolar macrophage polarization would be important for treatment of septic ALI/ARDS.
Increasing evidence indicates that metabolic reprogramming is linked to the polarization state of macrophages, and the tricarboxylic acid (TCA) cycle as well as its endogenous metabolites are vital in M1/M2 macrophage activation (7). TCA cycle dysfunction was shown to be involved in the M1 polarization of macrophages, and M1 macrophages rely on glycolytic metabolism for ATP generation (5, 8), which leads to lower α-ketoglutarate (α-KG)/succinate ratio. Recent studies show that succinate acts as a pro-inflammatory metabolite and can promote the production of the pro-inflammatory cytokines and reactive oxygen species in M1 macrophages. In contrast, alternately activated M2 macrophages maintain an intact TCA cycle activity and produce ATP by oxidative metabolism to provide energy for tissue remodeling and wound healing. α-KG is a TCA cycle intermediate that serves to produce ATP. A recent study showed that α-KG extended the lifespan of adult Caenorhabditis elegans by inhibiting ATP synthase and the TOR (target of rapamycin) pathway (9). Moreover, M2 polarization of bone marrow-derived macrophages (BMDMs) rely on the α-KG-Jmjd3 pathway and α-KG/succinate ratio (10). Recently, melatonin was reported to possess anti-inflammatory properties by transportation of exosomal α-KG to macrophages and led to the increased ratio of M2 to M1 macrophages (11). Thus, these results point out that α-KG can promote macrophage polarization toward the protective M2 phenotype; however, the underlying molecular mechanisms are complex and have not been fully elucidated.
It is now clear that the nutrient and growth factor sensor-mTORC1 plays a central role in cell growth and metabolism, such as protein synthesis, glycolysis, adipogenesis, and inhibiting autophagy. p70 ribosomal protein S6 kinase (p70S6K, S6K1) is one of the key substrates of mTORC1. In addition, mTORC1 can directly phosphorylate S6K1 by inducing the phosphorylation of the hydrophobic motif site, Thr389 (12). Several studies have demonstrated that mTORC1 served as the downstream molecule of phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase (AKT) signaling pathway, which is activated by toll like receptors and plays key role in innate immunity and macrophages metabolism reprogramming (13). However, the role of mTORC1 and its key substrates-p70S6K in regulating macrophage polarization remains unclear.
Peroxisome proliferator-activated receptor γ (PPARγ) is a member of ligand-activated transcription factors superfamily PPARs and plays a significant role in lipid metabolism, energy homeostasis, and modulating the immune inflammatory response. A growing body of evidence confirmed the significance of PPARγ in regulating M2 polarization in vitro and in vivo. It has been shown that PPARγ is required for alternative macrophage polarization by generating macrophage-specific PPARγ knockout mice (14). Another study reported that PPARγ deficiency in lung macrophages induced pulmonary inflammation even increased mortality after streptococcus pneumoniae infection (15). Hence, PPARγ exhibits beneficial effects in inflammatory disorders by promoting macrophage polarization to M2 profile and repression of inflammatory response.
In the present study, we hypothesize that α-KG may orchestrate M1/M2 phenotypic balance of alveolar macrophages through the regulators of immune responses (PPARγ or mTORC1), thus has beneficial effects on septic ALI/ARDS. To test this hypothesis, we conducted our experiments with MH-S cells in vitro and a mouse model of LPS-induced ALI/ARDS in vivo.
MATERIALS AND METHODS
Cell culture and intervention
MH-S cells, the SV40 transformed mouse alveolar macrophage cell line (CRI-2019, ATCC, Baltimore, Md, USA), were grown in RPMI-1640 medium (Gibco, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Australia Origin) and 1% penicillin/streptomycin. Cells were cultured at 37°C in 5% CO2 and 95% air-humidified incubator. MH-S cells were activated as M1 or M2 macrophages with the indicated stimulants (according to previous studies): 1 μg·mL−1 LPS (L3024, Sigma, Mo) 3 h for M1 polarization (16), and 20 ng·mL−1 of recombinant mouse IL-4 (404-ML, R&D Systems) 8 h for M2 polarization (17). The Dimethyl 2-oxoglutarate (DM-αKG; 349631, Sigma, Mo) (1/2 mM) was added 24 h before LPS or IL-4 stimulation (10). To further assess the effect of DM-αKG on macrophage polarization, we used Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) (a selective inhibitor of Glutaminase, SML0601, Sigma, Mo)10 μM (10, 18) to inhibit the production of intracellular α-KG. To explore the potential molecular mechanism, we used the GW9662 (a selective PPARγ antagonist, 70785, Cayman) 3 μM (19) and Rapamycin (a prototypical allosteric mTOR inhibitor, S1039, selleckchem, Houston, Tex) 20 nM (20) respectively to inhibit the function of PPARγ and mTORC1. The cell culture and interfering conditions were detailed in Supplementary material (https://links.lww.com/SHK/A853).
Animals and drug treatment
C57BL/6 male mice were purchased from Shanghai SLAC Laboratory Animal Center Animal Center and housed in specific pathogen-free conditions. Animal care and experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH publications NO.8023, revised 1978). All studies were reviewed and approved by the Animal Experiment Administration Committee of the Shanghai Pulmonary Hospital. To acutely induce septic ALI/ARDS, C57BL/6 mice (20–23 g) were injected intraperitoneally with lipopolysaccharide (L2630, Sigma, Mo) 10 mg kg−1 or 15 mg kg−1. The Dimethyl 2-oxoglutarate (DM-αKG; 349631, Sigma, Mo) were injected intraperitoneally at a dose of 0.6 g·kg−1 (based on previous work (10)) daily for 2 consecutive days before injection with LPS. Animal experiments were detailed in Supplementary material (https://links.lww.com/SHK/A853).
RNA extraction and real-time quantitative PCR
Total RNA was purified from MH-S cells using the RNA isolation kit (RNA fast2000, Fastagen, Shanghai, China) or extracted from the lung tissues using Trizol reagent (Invitrogen, Calif), according to the manufacturer's protocols. Quantification of cytokines mRNA expression was detected by RT-qPCR as described previously (21). Complementary DNA (cDNA) was synthesized using the (RR036A, Takara, Japan) following standard protocols. RT-qPCR analysis was performed with the SYBR Premix Ex Taq II (RR820A, Takara, Japan) with a QuantStudio 6 flex Real-Time PCR System (Life Technologies, Carlsbad, Calif). The primers for RT-qPCR synthesized by Shanghai Sangon Biotech Co, Ltd (Shanghai, China) are listed in Supplementary Table 1 (https://links.lww.com/SHK/A853). All gene expression levels were calculated using the 2−ΔΔCt method and normalized with the level of β-actin gene in the same sample.
Western blot analysis
Radio-immunoprecipitation assay lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and 1 mM phenylmethanesulfonyl fluoride (PMSF)] containing phosphatase inhibitor were used for extraction of whole-cell lysates. Preparation of cytosolic and nuclear extracts was according to a commercial kit (Beyotime, P0028, Shanghai, China). Briefly, the cells were collected and washed twice with phosphate-buffered saline (1 × PBS), then lysed in cytoplasmic protein extraction reagent A supplemented with 1 mM PMSF. The mixture was vortexed for 5 s (maximum speed) and incubated on ice for 15 min. Upon addition of cytoplasmic protein extraction reagent B, the mixture was vortexed for 5 s (maximum speed) and incubated on ice for 1 min followed by centrifugation at 12,000 × g for 5 min. The supernatant was pipetted immediately and kept as cytoplasmic proteins. For precipitate: discarded the supernatant completely, lysed in nuclear protein extraction reagent (containing PMSF), and vortexed for 15 to 30 s (maximum speed) each 1 to 2 min in following 30 min. Following centrifugation at 16,000 × g for 10 min, the supernatant was collected as nuclear extract. The bicinchoninic acid assay kit (Beyotime, P0010, Shanghai, China) was used for protein concentration quantitation. For western blotting, the protein samples were denatured by boiling with 5 × SDS loading buffer and separated by 10% sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride membrane subsequently. Then the membranes were blocked with 5% bovine serum albumin in TBST for 2 h and incubated overnight at 4°C with primary antibodies. The antibodies used for western blotting were as the following: β-actin (1:1,000, 20536-1-AP, Proteintech), Lamin A/C (1:1,000, 10298-1-AP, Proteintech), PPARγ (1:900, #2443, Cell Signaling Technology), Phospho-p70S6 Kinase (Thr389) (1:800, #9234, Cell Signaling Technology), p70 S6 Kinase Antibody (1:1,000, #2708, Cell Signaling Technology) and the horse-radish peroxidase-conjugated secondary antibodies (1:8,000, #L3012-2, Signalway Antibody). After being washed, the membranes were incubated with secondary antibodies for 1 h at room temperature. Finally, the signals were detected with an ECL chemiluminescence detection kit (Beyotime, P0018A, Shanghai, China) and a Quantity One software (Bio-Rad). The density of each specific band was quantified with Image J.
Cytokine quantification by enzyme-linked immunoassay assay (ELISA)
The quantification of sera cytokines (IL-6, IL-12 p70) was assessed with Mouse ELISA MAX Standard Sets (BioLegend, San Diego, Calif) according to the protocols supplied by manufacturers using serum samples from indicated time points.
Measurement of LDH and ATP
A lactate dehydrogenase assay kit (Jiancheng Bioengineering Institute, A020-2, Nanjing, China) was used to measure the production levels of LDH in the culture medium or lung tissues according to the protocols supplied by manufacturers. Media were centrifuged at 1,500 g at 4°C for 15 min to remove cell debris and for the subsequent steps. Measurement of ATP levels was tested by a commercial ATP assay kit (Beyotime, S0026, Shanghai, China). The cells were collected and disrupted with lysis buffer, and centrifuged to collect supernatant for subsequent steps according to the manufacturer's protocols.
Clinical score
The clinical score of each animal was assessed by researcher blinded to the treatment group at indicated time points as described by previous studies (22). The clinical scoring system is detailed in Supplementary Table 2 (https://links.lww.com/SHK/A853). The four variables were summed to represent the clinical score of each mouse (total score, 0–18), the higher the score the worse the clinical situation of the animal.
Lung histopathology and injury score analysis
A portion of the mouse lung tissues were fixed in 4% paraformaldehyde for at least 24 h. Then, the lung tissues were paraffin-embedded according to the standard protocol. Subsequently, the paraffin blocks were sectioned at 5 μm thickness and stained with hematoxylin and eosin (H&E) before microscope histological exam. Finally, the lung histopathology and injury score analysis was performed by pathologists blinded to the treatment group. The severity of lung damage was scored based on the following histologic features, as has been described previously (23): alveolar congestion, hemorrhage, infiltration or aggregation of neutrophils in airspace or vessel wall, and thickness of alveolar wall/hyaline membrane formation. Each item was graded on a five-point scale from 0 to 4: 0 (minimal damage), 1 (mild damage), 2 (moderate damage), 3 (severe damage), and 4 (maximal damage), respectively. The four variables were summed to represent the lung injury score (total score, 0–16).
Lung tissues wet-to-dry weight (W/D) ratio analysis
At 3 h after lung injury, the lung tissues were harvested and the wet weight was recorded. To obtain the dry weight (constant weight), the lung tissues were placed in an oven at 80°C for 24 h and measured three times at different time points until the weight no longer changed. The ratio of wet-to-dry weight was calculated to assess pulmonary edema.
Statistical analysis
All results are presented as mean ± standard deviation (SD), n = 6/10 mice per group. The differences among the groups were determined using one-way ANOVA with Bonferroni post hoc multiple comparisons test. Statistical significance between different treatments was accepted when P < 0.05. Statistical analyses were performed with Graph Pad Prism 7 software (La Jolla, Calif). The statistical analysis details were provided in each figure legend.
RESULTS
α-KG inhibits M1 phenotype macrophage polarization in MH-S cells
To evaluate the effect of α-KG in M1/M2 phenotypic balance of alveolar macrophages in the septic ALI/ARDS, we used LPS to stimulate MH-S cells toward M1 phenotype. We found that α-KG significantly decreased the expression of IL-1β, IL-6, and TNF-α in M1-polarized MH-S cells at a concentration of 2 mM (Fig. 1A–C). In contrast, α-KG (2 mM) significantly reversed the downregulation of M2 marker gene expression (Arg1) in M1-polarized cells (Fig. 1D). To further confirm the effect of α-KG in M1-polarized MH-S cells, we pretreated MH-S cells with BPTES, a selective inhibitor of glutaminase (GLS1), which could reduce α-KG levels in the TCA cycle (24). As shown in Figure 1E to G, BPTES treatment increased the gene expression of IL-1β, IL-6, and TNF-α in LPS-treated MH-S cells, while α-KG (2 mM) significantly reversed the upregulation of M1 marker genes expression. In addition, the inhibition of α-KG on M1 macrophage polarization was also confirmed by the decreased production of LDH and enhanced ATP production in M1-polarized MH-S cells (Fig. 1, H–I). LPS stimulation significantly increased the level of LDH in media, which is associated with the expression of inflammatory cytokines (such as IL-1β, TNF-α, IL-6). However, α-KG significantly decreased the level of LDH in media as well as led to elevated intracellular ATP levels. Together, these results indicate that α-KG exerts anti-inflammatory properties by suppressing LPS-induced M1 macrophage activation in MH-S cells.
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Fig. 1: α-KG suppressed M1 macrophage activation in MH-S cells.
α-KG promoted IL-4-induced M2 phenotype macrophage polarization in MH-S cells
Next, we investigated the role of α-KG in polarization of M2 phenotype MH-S cells. We used rmIL-4 to stimulate MH-S cells toward M2 phenotype. As previously reported, α-KG (2 mM) upregulated the expression of IL-4-triggered M2-specific marker genes, including Arg1, Ym1, and Mrc1 (Fig. 2,A–C). Additionally, RT-qPCR results showed that BPTES-treated MH-S cells exhibited markedly defective M2 polarization: reduced expression of M2-specific genes such as Arg1, Ym1, and Mrc1. However, the addition of α-KG restored the M2 marker gene expression (Fig. 2, D–F). Furthermore, as shown in Figure 2G, the intracellular ATP levels were significantly higher in IL-4 and α-KG costimulation group when compared with the other groups. Interestingly, there was no significant change of intracellular ATP level in BPTES treatment group, when compared with the IL-4-stimulated group. The above-mentioned results suggested that α-KG plays an important role in promoting M2 phenotype macrophage polarization in MH-S cells.
Fig. 2: α-KG promoted IL-4 induced M2 phenotype macrophage polarization in MH-S cells.
α-KG down-regulated mTORC1/p70S6K pathway in M1-polarized MH-S cells
As a central sensor of nutrient and energy, the mTORC1 plays an important role in regulating cell metabolism, cell growth, and cell cycle progression. A previous study has found that the mTORC1 pathway can be activated by LPS, as indicated by the increasing phosphorylation of the key effector p70S6K (25). Next, we explored the effect of α-KG on the activation of mTORC1/p70S6K pathway in LPS-induced M1-polarized MH-S cells. Western blots showed that the phosphorylation of p70S6K was increased in the LPS-stimulated group and the LPS-induced phosphorylation of p70S6K was further enhanced after BPTES treatment. However, the protein levels of P-p70S6K were significantly reduced by α-KG (2 mM) (Fig. 3, A and B). To further confirm that the mTORC1/p70S6K pathway was involved in the effect of α-KG on M1 macrophage polarization, we used rapamycin, a prototypical allosteric mTOR inhibitor, to block mTORC1. As shown in Figure 3C and D, rapamycin significantly inhibited the expression of P-p70S6K in LPS treated-MH-S cells. Moreover, the inhibitory effect of rapamycin on the expression of P-p70S6K showed no obvious change after the addition of α-KG. Taken together, our results demonstrated that α-KG inhibit mTORC1/p70S6K signaling pathway in M1-polarized MH-S cells.
Fig. 3: α-KG inhibited mTORC1/p70S6K signaling pathway in LPS-induced M1-polarized MH-S cells.
PPARγ is involved in α-KG-mediated M2 macrophage polarization in MH-S cells
The transcription factor PPARγ is reported to play an essential role in regulating the M1/M2 phenotypic switch through up-regulating expression of genes involved in fatty acid oxidation. α-KG (2 mM) markedly promoted the activation of PPARγ, as indicated by increased translocation of PPARγ to the nucleus in MH-S cells while compared with IL-4 treated group. The nuclear translocation of PPARγ was significantly decreased after BPTES treatment. However, α-KG significantly reversed the inhibitory effect of BPTES on PPARγ nuclear translocation (Fig. 4, A–C). As shown in Figure 4, D to F, GW9662, a selective PPARγ antagonist, significantly inhibited the nuclear translocation of PPARγ in IL-4 stimulated MH-S cells, while α-KG effectively reversed the nuclear PPARγ protein level. In addition, GW9662 significantly inhibited the gene expression of M2 marker Arg1 in IL-4-treated MH-S cells, but α-KG effectively up-regulated the expression of Arg1 in IL-4 or IL-4 + GW9662 treatment groups (Fig. 4G). Because PPARs are key mediators of fatty acid homeostasis in many cell types (26). Examination of relevant metabolic genes expression revealed that α-KG significantly increased the mRNA levels of CPT1-α (β-oxidation), FABP4 and CD36 (fatty acid uptake), PPARγ and PGC1β (transcriptional regulators) in IL-4-treated MH-S cells (Fig. 4H). The above results indicate that α-KG promotes the alternative (M2) activation of macrophages by enhancing the nuclear translocation of PPARγ and up-regulating the expression of relevant fatty acid metabolic genes.
Fig. 4: PPARγ activation is involved in α-KG-mediated M2 macrophage polarization in MH-S cells.
α-KG attenuated LPS-induced ALI/ARDS by modulating macrophage polarization in vivo
To further validate the effects of α-KG on inflammation and macrophage polarization in vivo, we utilized an LPS-induced mouse ALI/ARDS model (16). Compared with the LPS group, α-KG pretreated mice were protected from sepsis and had better clinical scores (Fig. 5A). H&E staining revealed that LPS (10 mg·kg−1) injection could lead to inflammatory cells infiltration, interstitial edema, and interalveolar septal thickening at 3 h. It is noteworthy that α-KG (0.6 g·kg−1) treatment attenuated the pathological changes in the lung tissues (Fig. 5B). Consistently, the lung damage score was largely diminished in mice pretreated with α-KG compared with LPS group (Fig. 5C). The wet-to-dry weight ratio (W/D) of lung tissues was significantly higher at 3 h after LPS treatment when compared with normal group. Nevertheless, W/D significantly decreased in α-KG treatment group (Fig. 5D). In addition, the serum levels of IL-6 and IL-12 were significantly increased at 3 h after LPS treatment, while α-KG significantly inhibited the LPS-induced secretion of inflammatory cytokines in sera (Fig. 5, E and F). RT-qPCR analysis of lung tissues showed that pro-inflammatory M1 marker gene expression (IL-1β, IL-6, and TNF-α) was upregulated and anti-inflammatory M2 marker gene expression (Arg1 and Mrc1) was decreased following LPS injection 3 h. Conversely, α-KG pretreatment inhibited LPS-triggered M1 marker gene expression (IL-1β, IL-6, and TNF-α) and enhanced M2 marker gene expression (Arg1 and Mrc1) in lungs (Fig. 5, G–K). LDH is an enzyme involved in anaerobic glycolysis and classically activated M1 macrophages are associated with high level of LDH. Thus, we investigated the LDH production in the lung tissues. The results showed that LPS injection increased the production of LDH, while the pretreatment of α-KG significantly decreased the LDH production in lungs (Fig. 5L). Collectively, these data indicated that α-KG may ameliorate septic ALI/ARDS through regulating macrophage polarization and reducing inflammation in vivo (Fig. 6).
Fig. 5: α-KG attenuated LPS-induced ALI/ARDS by modulating macrophage polarization in vivo.
Fig. 6: The chemical structure of DM-αKG and scheme for the proposed mechanism of α-KG in modulating mouse alveolar macrophage polarization in the LPS-induced ALI mouse model.
DISCUSSION
The balance between M1/M2 alveolar macrophages is essential to control the “cytokine storm” and is a promising approach for the prevention and treatment of ALI (6). α-KG, a TCA cycle intermediate, possesses anti-inflammatory properties of promoting macrophage polarization toward a M2 phenotype (10, 11, 27). However, it is unknown whether α-KG regulates the polarization of alveolar macrophages and the underlying mechanisms are still unclear. In the present study, we found that α-KG inhibited LPS-induced MH-S cells M1 polarization and decreased the expression of pro-inflammatory genes (IL-1β, IL-6, and TNF-α). Meanwhile, α-KG enhanced alternately activated macrophages polarization by upregulating the expression of IL-4-triggered M2-specific marker genes (Arg1, Ym1, and Mrc1) in MH-S cells. Furthermore, α-KG inhibited mTORC1/p70S6K signaling pathway in LPS-triggered M1-polarized MH-S cells. On the other hand, α-KG markedly promoted PPARγ nuclear translocation and the expression of relevant fatty acid metabolic genes in IL-4-treated MH-S cells. Finally, we found that α-KG attenuated LPS-induced ALI/ARDS in a mouse model, including amelioration of the lung pathological damage and clinical score, reduction of pro-inflammatory response, and promotion of M2 alveolar macrophages polarization.
The mTOR pathway acts a pivotal part in sensing nutrients and regulating metabolism, which prompted us to explore whether mTORC1 pathway participates in these processes. However, conflicting observations have been reported. Kimura et al. (17) showed that Lamtor1-mediated activation of mTORC1 presented definite anti-inflammatory activity in BMDMs. In contrast, another study indicated that the constitutive activation of mTORC1 potently enhanced LPS-mediated pro-inflammatory response in Tsc1−/− BMDMs (25). It appears that mTORC1 presents different biological effects in the context of different stimuli. In the current study, we found that the mTORC1 pathway can be activated by LPS and α-KG inhibited M1 activation by suppressing the activation of mTORC1/p70S6K pathway in MH-S cells, which is consistent with a previous study (9). M1 and M2 macrophages have distinct metabolic phenotypes: there is an enhancement in the glycolytic flux to lactate in M1-polarized macrophages, while IL-4-induced M2 macrophages use fatty acid oxidation (FAO; also known as β-oxidation) and oxidative phosphorylation to provide ATP for tissue remodeling and repair (28). It is known that cellular LDH expression levels were regulated by myc and PI3K/Akt/mTORC1 pathways at translational and transcriptional levels (29). Consistent with previous studies (30), our results show that the LDH levels were increased under LPS stimulation while α-KG treatment significantly decreased the production of LDH in M1-polarized MH-S cells. Consequently, we speculated that α-KG orchestrated M1 phenotype macrophage polarization via mTORC1-mediated metabolic reprogramming.
It is well established that PPARγ promotes macrophage polarization into the M2 profile mainly through up-regulating the expression of a wide range of genes involved in fatty acid oxidation and inhibiting the NF-κB signal pathway. The PPARγ expressed in peritoneal macrophages after an inflammatory stimulus could inhibit a subset of LPS-induced genes (31). However, whether α-KG modulates macrophage polarization by regulating PPARγ pathway has not been fully elucidated. In the current study, we found that α-KG enhanced M2 macrophage polarization partially by augmenting PPARγ nuclear transcription. Furthermore, our data also indicated that the relevant metabolic genes, which were involved in fatty acid metabolism (FABP4, CD36, PGC1β, CPT1-α), were upregulated by α-KG. The promotion effect of α-KG on the gene expression of Arg1 was partially inhibited by GW9662, which suggested that α-KG also regulates M2-like macrophage polarization through some other mechanisms. For example, α-KG also promotes M2 macrophage polarization in a Jmjd3-dependent epigenetic reprogramming manner (10) or by attenuating STAT3/NF-κB signaling pathway (11).
However, the mechanism by which PPARγ and mTORC1/S6K1 are regulated remains incompletely understood. The master regulator of cellular metabolism, AMP-activated protein kinase (AMPK), maintains energy homeostasis by sensing the concentrations of ATP, ADP, and AMP, plays an important role in metabolic reprogramming of macrophage. Chin et al. (9) reported that α-KG might activate AMPK by inhibiting the ATP synthase (increasing AMP/ATP ratio), thus inhibited mTOR. Another study reported that AMPK directly phosphorylates TSC2 and raptor by a two-pronged mechanism to inhibit mTORC1 activity (32). AMPK also increases the expression of transcriptional regulator peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α), which might be the driving force for increased β-oxidation of fatty acids (33). Several studies have demonstrated that PPARs act as a downstream metabolic mediator of AMPK, involved in lipid metabolism (34, 35). Though the contexts in which most of these regulatory events occur are poorly defined at present, we speculate that AMPK might be involved in the mechanism of macrophage polarization modulated by α-KG. Our next challenge would be exploring the relationship between PPARγ and mTORC1/S6K1 in the context of macrophage polarization.
Alveolar macrophages play an essential role in the first line of defense against infectious pathogens, as a kind of residential cells in the lung (5). The functional and phenotypical plasticity of alveolar macrophages enables them to play different roles in the process of inflammation. Thus, the regulation of alveolar macrophages polarization phenotype would be a more favorable treatment of pulmonary inflammatory diseases. As shown in this report, α-KG attenuates LPS-induced M1 pro-inflammatory gene expression and enhanced M2 anti-inflammatory gene expression during the “cytokine storm” which ameliorated LPS-induced ALI/ARDS.
There are several limitations in the present study. First, the molecular basis of alveolar macrophage polarization by α-KG was only studied in a MH-S cell line. The protective effects and potential mechanisms of α-KG on the polarization of LPS-treated primary cultured alveolar macrophages should be conducted in the future. Second, further animal studies are warranted to confirm the regulatory effect of α-KG on alveolar macrophages in vivo.
In conclusion, our study demonstrates that α-ketoglutarate modulates macrophage polarization through regulation of PPARγ transcription and mTORC1/p70S6K pathway. Moreover, α-KG protected mice against septic ALI/ARDS by alleviating LPS-induced lung edema, inhibiting infiltration of inflammatory cells and suppressing pulmonary histological damage. All of these suggest α-KG would be beneficial for prevention of inflammatory diseases such as septic ALI/ARDS.
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