MPC1 was inhibited in sepsis and endotoxin tolerance models
In our previous study, we discovered that the formation of the pyruvate kinase M2 (PKM2) tetramer plays a crucial role in inducing endotoxin tolerance in macrophages[9]. PKM2 primarily functions to catalyze the conversion of phosphoenolpyruvate into pyruvate, which is then transported into mitochondria by mitochondrial pyruvate carrier 1 (MPC1) to fuel the tricarboxylic acid cycle (TAC)[23]. Research has shown that MPC1 is intimately associated with the development of acute inflammation[14]. However, whether MPC1 is involved in the regulation of sepsis and endotoxin tolerance of macrophages has not been reported. To investigate the regulatory role of MPC1 in LPS-mediated sepsis in vitro, we treated RAW264.7 cells with LPS to establish a macrophage sepsis model, monitoring the protein expression of MPC1 at various times post-stimulation. As indicated in Fig. 1A, MPC1 expression decreased significantly 3 h after LPS treatment. Subsequently, we developed an endotoxin tolerance model in RAW264.7 cells and observed that MPC1 expression in the endotoxin-tolerant (ET) group was significantly higher compared to the non-endotoxin-tolerant (LPS) group (Fig. 1B).
Further, we established in vivo sepsis models in mice through either intraperitoneal injection of 4 mg/kg LPS or cecal ligation and perforation (CLP). We detected the expression of MPC1 in peritoneal macrophages (PMs), Kupffer cells (KCs) and bone marrow-derived macrophages (BMDMs) at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h and 6 h after LPS injection or after CLP operation. The in vivo results closely mirrored our in vitro findings, with significant reductions in MPC1 expression observed 3 h post-LPS injection or CLP (Fig. 1C, D). Additionally, mice that were pretreated with an intraperitoneal injection of 400 µg/kg LPS for 6 h, followed by treatment with 4 mg/kg LPS to induce endotoxin tolerance, exhibited significantly higher MPC1 expression in PMs compared to those in the LPS-only group (Fig. 1E). These findings suggest that the expression of MPC1 in macrophages is suppressed in the sepsis models in vivo and in vitro.
Overexpression of MPC1 inhibits LPS-mediated oxidative stress and inflammatory response
It is well-documented that lipopolysaccharide (LPS) triggers both oxidative stress and inflammatory responses in monocytes/macrophages[24, 25]. To further investigate the potential modulatory effects of MPC1 on LPS-mediated inflammatory response and oxidative stress, we overexpressed MPC1 in RAW264.7 cells with lentivirus. As depicted in Fig. 2A and 2B, both nucleic acid and protein levels of MPC1 were significantly elevated 72 h after lentiviral transduction. As shown in the Fig. 2C, compared with LPS group, overexpression of MPC1 inhibited the secretion of LPS-mediated pro-inflammatory factors TNF-α and IL-6, indicating that MPC1 inhibited LPS-mediated inflammatory response. Furthermore, we assessed the impact of MPC1 overexpression on oxidative stress markers within these cells. Mitochondrial reactive oxygen species (mtROS) and reactive oxygen species (ROS) serve as key indicators of oxidative stress, with ROS predominantly arising from the transfer of mtROS from mitochondria to the cytoplasm[26]. We found that overexpression of MPC1 also inhibits the production of mtROS and ROS in macrophages, that is, overexpression of MPC1 inhibits LPS-mediated oxidative stress (Fig. 2D, E). Collectively, these results demonstrate that MPC1 possesses anti-inflammatory and antioxidant properties, acting as a negative regulator of both the inflammatory and oxidative stress responses elicited by LPS.
Overexpression of MPC1 does not promote the formation of endotoxin tolerance
Endotoxin tolerance of macrophages can be induced by promoting the expression of anti-inflammatory factors or by inhibiting the expression of pro-inflammatory factors[27, 28]. In the study, we found that MPC1 is a negative regulator of LPS-mediated inflammatory response. In order to study whether MPC1 is involved in the regulation of endotoxin tolerance of macrophages, we categorized RAW264.7 cells into five groups: control (Con), sepsis (LPS), endotoxin tolerance (ET), overexpression of MPC1 with LPS treatment (OE-MPC1 + LPS), and overexpression of MPC1 in the endotoxin tolerance (OE-MPC1 + ET). As shown in Fig. 3, compared with LPS group, overexpression of MPC1 inhibited the secretion of TNF-α and IL-6 mediated by LPS (LPS vs OE-MPC1 + LPS). Surprisingly, compared with the ET group, the levels of TNF-α and IL-6 in the OE-MPC1 + ET group were significantly increased (Fig. 3). These results indicate that overexpression of MPC1 does not promote, and may in fact inhibit, the formation of endotoxin tolerance in macrophages.
Mitochondrial oxidative stress resistance promotes endotoxin tolerance
Mitohormesis describes the adaptation of mitochondria to continuous stress following a mild dose of stress stimulation, providing both mitochondrial and cellular protection. It plays a critical role in regulating a variety of physiological and pathological processes, including aging and cancer [12, 13]. Our previous research demonstrated that TEPP-46, a small molecule agonist of PKM2, promotes mitochondrial biogenesis by facilitating the formation of the PKM2 tetramer, subsequently inducing endotoxin tolerance in macrophages[9]. Similar to mitochondrial biogenesis, mitochondrial oxidative stress resistance is another biological marker of mitohormesis[14]. Whether mitochondrial oxidative stress resistance is involved in the regulation of endotoxin tolerance needs further study. Mitochondrial oxidative stress resistance refers to the adaptation of mitochondria to re-oxidative stress stimulation after mild dose of oxidative stress stimulation[14]. Mitochondrial oxidative stress resistance is an important endogenous protective way to limit the over-activation of mitochondria by oxidative stress stimulation, mainly reflected in the production of less mtROS and ROS when stimulated by re-oxidative stress. Although mtROS is considered as a pro-inflammatory mediator, it is also an important factor to induce mitochondrial oxidative stress resistance[14]. Inhibition of mtROS production will inhibit mitochondrial oxidative stress resistance, and then expand the inflammatory response when stimulated by secondary oxidative stress[14]. We found that the levels of mtROS and ROS in RAW264.7 cells were significantly increased with LPS treatment, suggesting that LPS promoted the oxidative stress response of macrophages (Fig. 2D, E). Menadione is an oxidant with obvious cytotoxicity, which can induce oxidative damage of cells[29]. As shown in Fig. 3A-C, with the concentration increased of menadione, menadione obviously induced apoptosis of RAW264.7 cells and promoted the expression of ROS. We found that compared with the RAW264.7 stimulated by menadione alone, cells stimulated by LPS for 6 h and then stimulated by menadione can effectively inhibit the production of ROS and significantly improve the cell activity (Fig. 3D, E). This shows that LPS pre-treatment may prime mitochondrial oxidative stress resistance, thus attenuating the oxidative stress response upon subsequent exposure. To further investigate the impact of mitochondrial oxidative stress resistance on endotoxin tolerance, we utilized mito-TEMPO, a selective scavenger of mtROS targeting mitochondria[30]. As shown in Fig. 3F, G, RAW264.7 cells pretreated with mito-TEMPO and then stimulated with LPS can significantly inhibit the production of mtROS mediated by LPS and inhibit mitochondrial oxidative stress resistance.Additionally, compared to untreated cells, the removal of mtROS by mito-TEMPO led to a decreased formation of endotoxin tolerance (Fig. 3H). These findings underscore the importance of mitochondrial oxidative stress resistance in promoting endotoxin tolerance in macrophages.
Overexpression of MPC1 inhibits endotoxin tolerance by inhibiting mitochondrial oxidative stress resistance
The role of MPC1 in regulating mitochondrial oxidative stress resistance in macrophages remains unclear. In this study, we observed that the expression of MPC1 decreased and the levels of mitochondrial reactive oxygen species (mtROS) increased in LPS-treated macrophages (Fig. 1A and Fig. 2D). When MPC1 was overexpressed and cells were stimulated with LPS, we found that overexpression of MPC1 significantly decreased the levels of mtROS and ROS (Fig. 2D, E). These results indicated that overexpression of MPC1 inhibited LPS-mediated oxidative stress. To further explore the impact of MPC1 on mitochondrial oxidative stress resistance, cells were categorized into four groups: control, menadione, LPS + menadione, and OE-MPC1 + LPS + menadione. We found that the ROS level in OE-MPC1 + LPS + menadione group was significantly higher than in LPS + menadione group, suggesting that MPC1 overexpression reduced the oxidative stress resistance of macrophages to menadione (Fig. 5A). This implies that MPC1 acts as a negative regulator of mitochondrial oxidative stress resistance.
As we know that overexpression of MPC1 inhibited the formation of endotoxin tolerance in macrophages (Fig. 3). In order to further study whether MPC1 regulates endotoxin tolerance of macrophages by regulating mitochondrial oxidative stress resistance. We applied Timblin's method to induce oxidative stress resistance of RAW264.7 with 4-OHE1[14]. As shown in Fig. 5B, the level of mtROS in RAW264.7 cells increased significantly after being stimulated with 4-OHE1, which inhibited menadione-mediated oxidative stress (Fig. 5C). This confirmed that 4-OHE1 successfully induces mitochondrial oxidative stress resistance.Subsequently, the experiment was expanded to include several groups: control, LPS, endotoxin tolerance (ET), overexpressed MPC1 in the ET (OE-MPC1 + ET), and 4-OHE1 treatment alongside overexpressed MPC1 in the ET (4-OHE1 + OE-MPC1 + ET). The findings showed that TNF-α and IL-6 levels in the OE-MPC1 + ET group were significantly elevated compared to the ET group, while in the 4-OHE1 + OE-MPC1 + ET group, these levels significantly decreased, suggesting that inducing mitochondrial oxidative stress resistance with 4-OHE1 could mitigate the suppressive effect of MPC1 overexpression on endotoxin tolerance (Fig. 5D). In summary, these results demonstrate that MPC1 is a negative regulator of LPS-mediated acute inflammation, and its overexpression can effectively reduce LPS-induced inflammatory responses. However, because overexpression of MPC1 also inhibits mitochondrial oxidative stress resistance, it consequently restricts the formation of endotoxin tolerance, substantially diminishing the capacity of macrophages to withstand secondary high-dose LPS stimulation.
SIRT3/SOD2 axis is involved in the regulation of mitochondrial oxidative stress resistance and endotoxin tolerance
SIRT3 and SOD2 are main located in mitochondria, the former has deacetylase activity, and the latter is mitochondrial O2− scavenger, which mainly removes mtROS[31–33]. It is reported that SIRT3 can induce the deacetylation of SOD2, and participate in the development of many diseases by reducing the level of acetylated SOD2[34]. In LPS-mediated macrophage sepsis model, we found that SIRT3 expression decreased and SOD2 acetylation level increased (Fig. 6A). It is reported that the higher the acetylation level of SOD2, the lower the activity of SOD2 and the weaker the ability to scavenge mtROS[35]. Therefore, we speculate that this may be the main pathway for LPS to promote the production of mtROS. In order to test the above hypothesis, we overexpressed RAW264.7 cells SIRT3 with lentivirus. As shown in Fig. 6B, C, the protein and nucleic acid expression of SIRT3 in RAW264.7 cells increased significantly after 72 h transfection with lentivirus. Besides, overexpression of SIRT3 inhibited the promotion of LPS on macrophage mtROS and ROS, indicating that overexpression of SIRT3 inhibited LPS-mediated oxidative stress (Fig. 6D, E). Then, we established a macrophage sepsis model, and stimulated the cells with menadione, and found that the oxidative stress resistance of macrophages to menadione was weakened (Fig. 6F). 3-TYP is a selective inhibitor targeting SIRT3[36]. After macrophages was stimulated with 3-TYP, the expression of SIRT3 decreased, the acetylation level of SOD2 increased, and mtROS increased (Fig. 6G, H). Compared with using menadione alone, pretreatment with 3-TYP and then treatment with menadione can significantly reduce the cytotoxicity of menadione (Fig. I). This suggests that SIRT3/SOD2 axis is the key way to mediate mitochondria oxidative stress resistance of macrophage.
Next, In order to further study the regulation of SIRT3/SOD2 axis on endotoxin tolerance, we constructed endotoxin tolerance and endotoxin-intolerant models of RAW264.7 cells. Compared with endotoxin-intolerant group, the expression of SIRT3 in endotoxin-tolerant group increased and the acetylation level of SOD2 decreased (Fig. 6J). Overexpression of SIRT3 inhibits the secretion of TNF-α and IL-6 (Fig. 6K). On the contrary, pretreatment of cells with 3-TYP inhibited the expression of SIRT3 and promoted the release TNF-α and IL-6 (Fig. 6K). Overexpression of macrophage SIRT3, and then established the endotoxin tolerance model of macrophage. We found that overexpression of SIRT3 inhibited the formation of endotoxin tolerance (Fig. 6L). Collectively, these results suggest that SIRT3/SOD2 axis is the key way to mediate mitochondria oxidative stress resistance and endotoxin tolerance of macrophage.
MPC1 regulates mitochondrial oxidative stress resistance and endotoxin tolerance through the SIRT3/SOD2 axis
Our study revealed that under LPS stimulation, the expression levels of MPC1 and SIRT3 decreased, while the acetylation level of SOD2 increased in RAW264.7 cells. In order to further study whether MPC1 regulates the mitochondria oxidative stress resistance and endotoxin tolerance through SIRT3/SOD2 axis, we overexpressed MPC1 with lentivirus, and found that after overexpression of MPC1, the expression of SIRT3 increased and the acetylation level of SOD2 decreased (Fig. 7A). Additionally, suppressing SIRT3 expression with 3-TYP after overexpressing MPC1 led to an increased acetylation level of SOD2 (Fig. 7B). Our experiments demonstrated that MPC1 overexpression reduced the oxidative stress resistance of macrophages to menadione (Fig. 5A). On the basis of overexpression of MPC1, inhibiting the expression of SIRT3 with 3-TYP can increase the oxidative stress resistance of macrophages to menadione and promote the formation of endotoxin tolerance of macrophages (Fig. 7C, D). This suggests that MPC1's modulation of mitochondrial oxidative stress resistance and endotoxin tolerance operates significantly through its effects on the SIRT3 pathway. In Fig. 5A we found that overexpression of MPC1 promotes the expression of SIRT3. Interestingly, overexpression of SIRT3 also promotes the expression of MPC1 (Fig. 7E). However, co-immunoprecipitation (Co-IP) experiments indicated no direct protein interaction between MPC1 and SIRT3 (Fig. 7F), suggesting that the regulatory relationship between MPC1 and SIRT3 may be mediated by other mechanisms or signaling pathways, which warrants further investigation.