Effects of inflammatory and anti-inflammatory environments on the macrophage mitochondrial function

Mitochondrial response to inflammation is crucial in the metabolic adaptation to infection. This study aimed to explore the mitochondrial response under inflammatory and anti-inflammatory environments, with a focus on the tricarboxylic acid (TCA) cycle. Expression levels of key TCA cycle enzymes and the autophagy-related protein light chain 3b (LC3b) were determined in raw 264.7 cells treated with lipopolysaccharide (LPS) and metformin (Met). Additionally, reactive oxygen species (ROS) levels and mitochondrial membrane potential were assessed using flow cytometry. Moreover, 8-week-old C57BL/6J mice were intraperitoneally injected with LPS and Met to assess the mitochondrial response in vivo. Upon LPS stimulation, the expression of key TCA enzymes, including citrate synthase, α-ketoglutarate dehydrogenase, and isocitrate dehydrogenase 2, and the mitochondrial membrane potential decreased, whereas the levels of LC3b and ROS increased. However, treatment with Met inhibited the reduction of LPS-induced enzyme levels as well as the elevation of LC3b and ROS levels. In conclusion, the mitochondrial TCA cycle is affected by the inflammatory environment, and the LPS-induced effects can be reversed by Met treatment.

Scientific Reports | (2020) 10:20324 | https://doi.org/10.1038/s41598-020-77370-x www.nature.com/scientificreports/ mitochondrial function is poorly understood. Previous studies suggested that abnormal mitochondrial function is associated with diseases such as Parkinson's disease and diabetes mellitus 18,19 . Therefore, studying the correlation between inflammation and macrophage mitochondrial function could lead to a better understanding of the link between inflammation and diseases. This study aims to explore the energy metabolism of macrophages under either inflammatory or anti-inflammatory conditions by adding LPS and Met, respectively, using an in vitro and an in vivo model.

Methods
Ethics statement. The procedures for care and use of mice model was approved by the Shanghai Jiao Tong University Affiliated Sixth People's Hospital of Medicine Ethics Committee (Shanghai, China). All animalrelated methods were carried out in accordance with relevant guidelines and regulations.
Flow cytometer analyses were performed using a BD FACS Celesta analytical flow cytometer, quantitative PCR using a Roche Light Cycler 480II.
Macrophage culture. RAW264.7 cells were cultured in DMEM low glycemic medium supplemented with 100 IU/mL of penicillin, 100 µg/mL of streptomycin, and 10% heat-inactivated fetal bovine serum. Cells were cultured at 37 °C with 5% CO 2 . Cells were used for the experiments when they reached the logarithmic growth phase. RAW264.7 cells were inoculated on 6-well plates with a density of 5 × 10 5 cells/mL per well and 2 mL of medium were added to each well for 24 h. Then, cells were treated as follows: LPS group, 1 μg/mL of LPS for 8 h; Met plus LPS group, 5 mmol/L 20 of Met for 1 h, and then 1 μg/mL of LPS for 8 h. LPS was dissolved in pure dd water and Met was dissolved in pure PBS.

Real-time PCR (qPCR).
Total RNA was extracted from RAW264.7 cells using the RNA isolater Total RNA Extraction Reagent (Vazyme Co., Ltd., Nanjing, China). RNA concentration and purity were determined by ultraviolet spectrophotometer. Then RNA samples were reverse transcribed using the Fast Quant RT kit (Tiangen Co., Ltd., Shanghai, China), following the manufacturer's protocol. qPCR was performed using the Super Real Pre Mix Plus (Tiangen Co., Ltd., Shanghai, China) with the following PCR profile: 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s. Relative expression levels were measured using the 2 −ΔΔCt method. Primer sequences for qPCR in this study is seen Table 1.
Western blotting. Cells and lung tissues were washed twice with PBS and lysed in RIPA Lysis buffer (Beyotime Co., Ltd., Shanghai, China). Total protein concentrations were measured using a BCA Protein Assay Kit (Beyotime Co., Ltd., Shanghai, China), and an equal amount of protein (30 µg/lane) was loaded into an SDSpolyacrylamide gel for electrophoresis and then transferred onto a polyvinylidene difluoride membrane. Membranes were then blocked by incubating them in TBS containing 5% non-fat milk and 0.1% Tween-20 for 2 h at room temperature. Membranes were then incubated sequentially with primary antibodies overnight at 4 °C. The following primary antibody dilutions were used: anti-IDH2 (1:1000), anti-CS (1:1000), anti-OGDH (1:1000), anti-MDH2 (1:10,000), anti-LC3b (1:1000), and anti-GAPDH (1:1000). Membranes were then washed 3 times in 0.1% TBST and incubated with a goat anti-mouse secondary antibody and a goat anti-rabbit secondary anti- Reactive oxygen species detection. Intracellular total ROS were detected using the Reactive Oxygen Species Assay Kit (50101ES01, Yisheng Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer's protocol. Briefly, following the treatment with LPS and Met, the cell culture medium was removed and dichloro-dihydro-fluorescein diacetate (DCFH-DA) was added at a final concentration of 10 µM. Then, cells were incubated in a CO 2 incubator for 20 min at 37 °C and washed 3 times with PBS. The relative FITC fluorescence intensity was measured by flow cytometry as a measure of the ROS levels. The FITC excitation wavelength was 488 nm and the emission wavelength was 525 nm.
Mitochondrial membrane potential detection. Mitochondrial membrane potential was detected using the JC-1 Mitochondrial Membrane Potential Assay Kit (40706ES60, Yisheng Biotechnology Co., Ltd., Shanghai, China), according to the manufacturer's protocol. Briefly, following treatment, the cell culture medium was removed and cells were resuspended in 0.5 mL of JC-1 staining solution and then incubated at 37 °C for 20 min in a light-proof cell incubator. Then, cells were centrifuged at 4 °C for 3 min at 600 × g, the supernatant was discarded, cells were washed twice with the JC-1 dye buffer, and cells were resuspended. FITC and PE fluorescence reading were used as a measure of mitochondrial membrane potential. FITC fluorescence was detected at 488 nm and 530 nm by flow cytometry, while the PE fluorescence signal was excited at 530 nm and detected at 630 nm.

Scientific Reports
Statistical analysis. Median between two groups was compared using the Mann-Whitney test, while the medians of multiple groups were compared using the Kruskal-Wallis test. All statistical analyses were performed using the GraphPad Prism 5.0 (USA) statistical software and a P < 0.05 was considered statistically significant.

ROS are increased following inflammation.
After treating RAW264.7 macrophages with LPS, we observed an increase of ROS levels, compared to the control cells (P < 0.05) (Fig. 1). Interestingly, this increase was inhibited when cells were pre-treated with Met (P < 0.05). Conversely, when cells where pre-treated with Met, we observed a lack of IL-6 induction (P < 0.05) and the concomitant increase of the anti-inflammatory marker IL-10 (P < 0.05) (Fig. 2).
Expression levels of key TCA cycle enzymes decrease following inflammation. We then measured the mRNA levels of 4 key enzymes of the TCA cycle: OGDH, IDH2, CS, and MDH2. The expression levels of all 4 genes were decreased upon LPS stimulation (P < 0.05), but this effect was reversed when pretreating RAW264.7 cells with Met (P < 0.05) (Fig. 3).
We also detected the protein levels by Western blotting and observed a significant decrease of OGDH, IDH2, and CS following LPS stimulation (P < 0.05), which was reversed by Met pre-treatment (P < 0.05). However, differently from the mRNA levels, the protein levels of MDH2 did not significantly change (Fig. 4). Mitochondrial membrane potential decreases upon inflammation. Finally, we measured the mitochondrial membrane potential in RAW264.7 macrophages treated with LPS and observed a significant decrease of the potential (P < 0.05), which was reversed upon pre-treatment with Met (P < 0.05), (Fig. 6).

Immunohistochemical examination of lung tissues following LPS and Met/LPS treatment.
We then investigated the effect of LPS and Met treatment in vivo. We observed that the injection of mice with 20 mg/kg of LPS induced the following pathological changes in the lung tissue 24 h later: significant enlargement of the alveolar septum, injury and rupture of the alveolar wall and capillary blockage, accumulation of erythrocyte in the interstitial fluid, secretion of a large number of inflammatory factors in the alveolar cavity, and infiltration of inflammatory neutrophils in the interstitium. All these parameters improved when mice where pre-treated for 1 h with 250 mg/kg Met (Fig. 7).
Expression levels of key TCA cycle enzymes decreases following inflammation in the lung tissue. We observed that, upon LPS treatment, the expression of OGDH, CS, and MDH2 in the lung tissue significantly decreased (P < 0.05), while the expression of IDH2 did not significantly change. However, upon Met pre-treatment, the expression levels of OGDH and MDH2 were reverted (P < 0.05), while the expression of IDH2 and CS was not impacted (Fig. 8). However, this increase was reversed upon Met pre-treatment (P < 0.05) (Fig. 9).

Discussion
In this study, we analyzed the expression of four enzymes involved in the TCA cycle upon LPS stimulation alone (inflammatory stimulation) and in the presence of Met (anti-inflammatory stimulation). Results showed that, in vitro, LPS induced the decrease of CS, OGDH, and IDH2, both at the mRNA and protein levels, which reverted in presence of Met. Similar results were obtained in vivo in a mouse model: upon LPS, the expression of OGDH, CS, and MDH2 in lung tissue decreased and the expression of OGDH and MDH2 was reverted upon Met pre-treatment. A recent study described the role of IDH2 in cancer development 22 , while another suggested that the dysregulation of IDH2 in various cancers is correlated with inflammatory response disorders, such as the excessive production of pro-inflammatory factors 23 . In septic patients, the maximum level of CS activity in monocytes is significantly lower than that observed in normal people 24 . Thus, it has been suggested that a decrease in the expression of key enzymes of the TCA cycle could lead to an increase of reactive oxygen species, resulting in mitochondrial dysfunction, which may be correlated to septicemia. In the liver, the inflammatory reaction intensifies the acetylation of mitochondrial proteins such as MDH2. When MDH2 is acetylated, it activates the malate aspartate shuttle activity to maintain glycolysis 25 . OGDH is a key pro-inflammatory metabolic intermediate: through the NF-κB pathway, OGDH can increase the production of LPS-induced pro-inflammatory factors. The expression of OGDH is IDH2-dependent. When IDH2 is lost, the expression of OGDH decreases, reducing the LPS-induced pulmonary inflammatory response 26 .
Metabolites such as OGDH may be used as specific biomarkers of the human inflammatory response in the context of systemic infections, sepsis, and autoimmune diseases. The accurate determination of citric acid cycle intermediates is commonly performed using mass spectrometry, which is a fast, sensitive, and reproducible method, easy to standardize for clinical application 27 . Itaconic acid is a TCA cycle metabolite, which is believed to be released by macrophages during inflammation 28 . Succinic acid and citric acid have also been shown to be significantly reduced in the urine of patients affected with inflammatory bowel disease, compared to those observed in healthy people 29 . These limited clinical studies suggest that TCA cycle intermediates such as succinic acid, citric acid, itaconic acid, and fumaric acid may be easily measured in the body fluids of patients with different inflammatory conditions as biomarkers of the inflammation status 30 .
Our results also showed that, upon LPS treatment, ROS and autophagy increased, while the mitochondrial membrane potential decreased. These results suggest that inflammation would trigger autophagy to clear organelles damaged by ROS. Moreover, the decrease in the mitochondrial membrane potential suggests that mitochondrial function is negatively affected. We speculate that the expression of key enzymes and the efficiency of the TCA cycle decrease during the inflammatory state, leading to the accumulation of TCA cycle metabolites such as citric acid and succinic acid. The accumulation of these metabolites increases ROS levels. Increased ROS levels may inhibit the oxidative phosphorylation process and ATP formation, resulting in a damaged TCA cycle imbalanced mitochondrial energy metabolism, and a drop of membrane potential. In these conditions, autophagy can maintain cell homeostasis and protect the body from the inflammatory response triggered by ROS. Therefore, we hypothesize that inflammation in macrophages disrupts the TCA cycle, affecting the mitochondrial and cellular functions.
In this study, we used Met as an anti-inflammatory agent. Met is the first-line drug for the treatment of type 2 diabetes mellitus, which has been known for its anti-inflammatory effects 9,31 . Met can reduce the production of different pro-inflammatory factors and induce anti-inflammatory factors, regulating the differentiation of   32 . Previous studies have shown that Met could be a promising therapeutic agent for treating heat damage, as it significantly improved the mitochondrial energy in elderly mice 33 . Our results suggest that Met can protect cells from the inflammation-induced dysregulation of the TCA cycle. Therefore, we speculate that the mechanism underlying the use of Met to treat heat injury may be correlated to its ability to protect mitochondrial activity. Considering the great potential of Met for the treatment of different diseases [34][35][36][37][38][39][40][41][42][43] , further investigation of its anti-inflammatory potential and its role in energy metabolism and mitochondria regulation are needed. Our study has some limitations as following: (1) the absence of the experimental group with alone metformin affects the interpretability of the results; (2) GAPDH as an internal reference in this study may increase the uncertainty of the results; (3) metformin administered to the animal model might induce an acute hypoglycemia episode that may affect the results; however, Met plus LPS group in this study did not occur an hypoglycemia episode, and its mechanism may be related to insulin resistance and hyperglycemia induced by LPS injection 44 ; and (4) unfortunately, we have not been able to test the activities of the TCA cycle enzymes from other markers of oxidative stress and autophagy. Therefore, a validation study that investigates the molecular mechanisms regulating the TCA cycle upon inflammation stimulation should continue in the future. Figure 9. The expression levels of autophagy-related protein LC3b in macrophages in mice treated with lipopolysaccharide and metformin. GAPDH: glyceraldehyde-3-phosphate dehydrogenase, LPS: lipopolysaccharide, Met: Metformin, LC3b: microtubule associated protein 1 light chain 3 beta. LPS group: compared with blank control group. Met + LPS group: compared with LPS group. *P < 0.05, **P < 0.01, ***P < 0.0001.