A low dose of LPS induces ET in vitro and in vivo
LPS has the ability to incite a vigorous inflammatory response and result in all the physiological manifestations of septic shock (Su 2002
). However, after exposure to low concentrations of LPS, cells or organisms can enter into a transient unresponsive state and are unable to respond to further challenges with LPS (Seeley and Ghosh 2017
). This phenomenon is ET, one of the important mechanisms for host protection against endotoxin shock.
To explore the mechanism of ET, we stimulated C57BL/6 mice with low-dose LPS and established ET models in vivo and KCs in vitro. KCs isolated from C57BL/6 mice were randomly divided into three groups: the control (CON) group, the nonendotoxin tolerance (NET) group and the endotoxin tolerance (ET) group (NET: 1000 ng/ml; ET: 10 ng/ml+1000 ng/ml). The ET and NET groups were cultured with complete medium containing 1000 ng/ml LPS for 12 h, but before that, the ET group was pretreated with 10 ng/mL LPS for 24 h. The ET group had lower expression of proinflammatory cytokines including TNF-α and iNOS (Fig. 1A). Additionally, the level of TNF-α in the cell supernatants of the NET group was significantly increased compared to that in the ET group, while the level of IL-10 in the NET group was lower than that in the ET group (Fig. 1B).
To study the effect of ET in vivo, mice in the ET group were pretreated with LPS (intraperitoneal injections of 50 µg/kg, 250 µg/kg, 500 µg/kg LPS for three consecutive days) and then the two groups (NET and ET) were restimulated with LPS (20 mg/kg, intraperitoneally) for 24 h. All of the mice were observed for 6 days, and we found that compared to NET group, mice in ET group had significantly improved survival (Fig. 1
C). No mice died during the observation period in the saline-treated group (CON), which was not reflected in the survival curve. The liver sections from each group showed less tissue damage and inflammatory cell infiltration in the ET group (Fig. 1
D), and the ET group had reduced levels of proinflammatory cytokines, such as TNF-ɑ and iNOS (Fig. 1
E). Consistent with the in vitro results, the serum levels of TNF-α in the NET group were higher than that in the ET group, and the levels of IL-10 in the NET group were decreased compared to that in the ET group (Fig. 1
F). In addition, the serum AST and ALT levels showed that the ET group experienced lower inflammatory activation and tissue damage (Fig. 1
G). The above results demonstrated that the ET model was successfully established in vitro and in vivo.
TBK1 can mediate inflammatory regulation in ET
It was reported that promoting TBK1 phosphorylation can regulate the host innate immune response during infection (Chen 2021). To determine whether TBK1 was involved in ET, mice were treated as previously described to establish ET and NET models in vivo. In both the NET and ET groups, the phosphorylation of TBK1 increased; however, the phosphorylation of TBK1 was higher in the ET group than in the NET group. In addition, the ET group had fewer proinflammatory cytokines, such as TNF-ɑ and iNOS (Fig. 2A). A higher level of TBK1 phosphorylation was also found in the ET group according to immunofluorescence staining (Fig. 2B). To clarify the relationship between TBK1 and ET in vitro, KCs isolated from normal C57BL/6 mice were treated as previously described. While total TBK1 was not affected in either the NET or ET groups (Fig. 2C), the phosphorylation of TBK1 in the ET group was increased to a greater extent (Fig. 2C-D), which suggested that TBK1 might be related to ET in vivo and in vitro.
To explore the role of TBK1 in ET, TBK1 expression was knocked down in RAW264.7 cells with siRNA (TBK1-siRNA). The TBK1-siRNA+ET group exhibited downregulated phosphorylation of TBK1 compared with the ET group, and the expression of proinflammatory cytokines, including TNF-ɑ and iNOS, was increased, which was the reverse of the previous results (Fig. 2E). As TBK1 was knocked down by siRNA, the phosphorylation level of TBK1 was decreased, and the protective effect of ET was also weakened. Correspondingly, the TBK1 gene was overexpressed in RAW264.7 cells by shRNA-TBK1 lentiviral transduction (LV-TBK1-OE). As predicted, the protein level of TBK1 increased significantly after lentiviral transduction for 72 h. The LV-TBK1-OE+ET group had the lower expression of proinflammatory cytokines (including TNF-ɑ and iNOS) and higher phosphorylation of TBK1 (Fig. 2F). The serum level of TNF-α in the TBK1-siRNA+ET group was significantly increased compared to the LV-TBK1-OE+ET group, while the serum level of IL-10 in the TBK1-siRNA+ET group was lower than that in the LV-TBK1-OE+ET group (Fig. 2G). These results revealed that the ET was partly dependent upon TBK1 in mediating the reduction of inflammation, and the phosphorylation level of TBK1 was closely related to the level of inflammation during ET: when TBK1 was knocked down and the phosphorylation level decreased, inflammation was aggravated and the secretion of inflammatory factors increased, while when TBK1 was overexpressed and the phosphorylation level increased, the inflammation was alleviated. The above results indicated that TBK1 could mediate inflammatory regulation in ET through protein phosphorylation.
TBK1 can regulate the phosphorylation of RIPK3
Recent studies focusing on macrophages indicated that RIPK3 can regulate the production of proinflammatory cytokines in particular settings (Kang et al. 2013; Wong et al. 2014; Newton 2015). RIPK3 may be involved in regulating cell death and inflammatory signaling pathways (Sun et al. 1999; Zhang et al. 2009), therefore, we tried to explore whether RIPK3 played a role in ET. After stimulation with LPS, KCs in the ET group showed high expression of phosphorylated RIPK3 compared to KCs in the NET group (Fig. 3A). Immunofluorescence staining also confirmed that the phosphorylation level of RIPK3 was increased in the ET group (Fig. 3B). These results revealed that RIPK3 might be involved in ET. In addition, RIPK3 was found to interact with TBK1 by coimmunoprecipitation (co-IP) experiments established in KCs with coexpressed proteins in the ET group (Fig. 3C), which indicated that TBK1 and RIPK3 can interact during macrophage tolerance to LPS.
To further explore the relationship between TBK1 and RIPK3 in ET in macrophages, we first knocked down the expression of TBK1 in RAW264.7 cells to observe whether the change in TBK1 expression would affect the expression of RIPK3. As the phosphorylation level of TBK1 decreased, the level of phosphorylated RIPK3 was decreased (Fig. 3D). As predicted, the phosphorylation levels of TBK1 and RIPK3 were both upregulated after increasing the expression of TBK1 by shRNA-TBK1 lentiviral transduction in the LV-TBK1-OE+ET group (Fig. 3E). These results revealed that TBK1 could affect the phosphorylation level of RIPK3 in ET, but the functional significance of the RIPK3 phosphorylation event and downstream substrates have not been fully clarified (Sun et al. 2012; Declercq et al. 2009).
Increasing the enzymatic activity of GLUD1 can induce ET in macrophages
GLUD1 is known as a mitochondrial matrix enzyme that converts glutamine to α-KG (Zhang et al. 2009; He et al. 2020; Goossens et al. 1996), and growing evidence has shown that glutamine can transfer into mitochondria to function as an energy substrate (Zhang et al. 2009). The protein expression of GLUD1 and phosphorylated RIPK3 was higher in the ET group than in the NET group (Fig. 4A). Immunofluorescence staining showed that the expression of GLUD1 increased in the ET group (Fig. 4B). Furthermore, the enzyme activities of GLUD1, both in the liver and in BMDMs (the models were established as previously described), were significantly increased in the ET group compared with the NET group (Fig. 4C), while immunohistochemistry was also confirmed that the ET group had a higher expression of GLUD1 than the NET group (Fig. 4D). These results suggested that GLUD1 expression was indeed increased in ET, but how GLUD1 promotes LPS tolerance remains unclarified.
Macrophages represent with distinct phenotypes with different functions according to the tissue microenvironment and different stimuli (Wynn et al. 2013; Murray and Wynn 2011). Liu reported that α-KG generated from glutaminolysis could promote M2 activation via Jmjd3-dependent metabolic and epigenetic reprogramming, while glutaminolysis restricted M1 polarization (Liu et al. 2017). Therefore, we carried out ELISAs to detect the contents of α-KG in the different groups. The levels of α-KG in serum and cell supernatants in the ET group were than those in the NET group (Fig. 4E). We then checked the ratios of M1-type and M2-type macrophages among BMDMs. As predicted, the ratio of M1-type macrophages in the ET group was significantly decreased (Fig. 4F). The expression of CD86 was increased in the NET group (Fig. 4G). As the enzyme activity of GLUD1 increased, the content of α-KG also increased restricting M1 polarization and promoting macrophage tolerance to LPS. Increasing the enzyme activity of GLUD1 could be an effective measure to induce ET in macrophages.
The regulatory effect of GLUD1 on ET was reversed after eliminating macrophages in mice
Macrophages are critical to maintain the tissue homeostasis in innate and adaptive immune responses (Wynn et al. 2013). To determine whether the elimination of macrophages would have an effect on the induction of ET by GLUD1, we depleted macrophages in mice with clodronate (neutral) liposomes. Male C56BL/6J mice (8-10 weeks old, 22±2 g) were randomly divided into two groups: the control liposome group (injected peritoneally with 200 µl control liposomes) and the neutral liposome group (injected peritoneally with 200 µl neutral liposomes). The mice in the two groups were treated as previously described to establish ET in vivo. The control liposome group was divided into three groups: the control liposome control (C-CON) group, the control liposome NET (C-NET) group and the control liposome ET (C-ET) group, while the neutral liposome group was also divided into three groups: the neutral liposome CON (N-CON) group, the neutral liposome NET (N-NET) group and the neutral liposome ET (N-ET) group (Fig. 5A).
The KCs in the livers of mice in the control liposome group remained essentially unchanged, while the KCs in the neutral liposome group were basically eliminated (Fig. 5B). All of the mice were observed for 6 days, and we found that compared to the C-NET group, mice in the C-ET group had significantly improved survival. However, compared to the N-NET group, the survival of mice in the N-ET group was significantly decreased which was the reverse of the above results. Compared with the C-ET group, the percent survival of mice in the N-ET group was reduced by approximately 93%. Moreover, all mice in the N-ET group died before the sixth day of observation (Fig. 5C). H&E staining of the liver sections from each group showed that there was less tissue damage and inflammatory cell infiltration in the C-ET group than in the C-NET group, while there were no significant differences in tissue injury and inflammatory cell infiltration between the N-NET group and the N-ET group (Fig. 5D). Consistent with the H&E results: the serum levels of TNF-α and IL-10 were not significantly different between the N-NET and N-ET groups (Fig. 5E). Furthermore, the expression of GLUD1 was lower in the N-ET group than that in the C-ET group (Fig. 5F). The enzyme activity of GLUD1 was decreased in the N-ET group compared with the C-ET group, resulting in a decrease in the content of α-KG in the liver (Fig. 5G-H). The inflammatory response in the neutral liposome group was still severe because the protective mechanism of ET was not induced, even if mice were stimulated with low dose LPS in advance. Taken together, these results indirectly proved that GLUD1 participated in ET by regulating macrophages and that the regulatory effect of GLUD1 on ET was reversed after eliminating the macrophages in mice.
RIPK3 induces ET by promoting the enzyme activity of GLUD1
The interaction of endogenous RIPK3 with GLUD1 increases after TNF-α stimulation, and in cells that have sufficient RIPK3 expression, the gateway to glutamine use is readily opened (Zhang et al. 2009). In addition, it could directly reduce the catalytic activity of GLUD1 after inhibiting the activation of RIPK3 induced by LPS (Nelson et al. 2018). Based on the facts of previous studies, we tried to explore the roles of RIPK3 and GLUD1 in ET. According to our co-IP results, the GLUD1 and RIPK3 proteins interact in ET models in vivo and in vitro (Fig. 6A-B).
To further determine the regulatory effect of RIPK3 on GLUD1, we first knocked down RIPK3 in RAW264.7 cells using siRNA and established ET and NET models (RIPK3-siRNA). The protein levels of RIPK3 and phosphorylated RIPK3 decreased significantly in the RIPK3-siRNA+ET group. As shown in Fig. 6C, with the decrease in RIPK3, the expression of GLUD1 in the RIPK3-siRNA+ET group was significantly lower than that in the ET group, while the expression of TNF-ɑ, and iNOS was increased. In addition, the enzyme activity of GLUD1 in the RIPK3-siRNA+ET group was much lower than that in the ET group (Fig. 6D). As the level of α-KG was decreased in the RIPK3-siRNA+ET group compared with the ET group (Fig. 6E), the ratio of M1 macrophages in the RIPK3-siRNA+ET group was increased (Fig. 6F). After knocking down the expression of RIPK3, the enzyme activity of GLUD1 decreased, and the production of α-KG decreased, resulting in a reduction in macrophage tolerance to LPS.
Correspondingly, when the RIPK3 gene was overexpressed in RAW264.7 cells by shRNA-RIPK3 lentiviral transduction (LV-RIPK3-OE), GLUD1 expression increased, while the enzyme activity of GLUD1 also increased (Fig. 6G-H), which led to a higher level of α-KG in the LV-RIPK3-OE+ET group (Fig. 6I). These results indicated that increasing RIPK3 can promote the enzyme activity of GLUD1 to increase the level of α-KG. The expression of iNOS and TNF-ɑ in the LV-RIPK3-OE+ET group was significantly decreased after increasing the expression of RIPK3, which showed that overexpression of RIPK3 can promote the enzyme activity of GLUD1, thus strengthening the effect of α-KG on promoting ET and alleviating the inflammatory reaction (Fig. 6G). Taken together, RIPK3 can mediate the inflammatory response induced by LPS and enhance ET through glutaminolysis.
TBK1 participates in glutaminolysis by mediating the phosphorylation of RIPK3 to promote ET
TBK1 is required in ET both in vivo and in vitro; however, the specific mechanism of how TBK1 participate in ET is still unclear. As TBK1 promoted the phosphorylation of RIPK3, we next sought to determine whether TBK1 participated in the glutamine metabolic pathway to promote ET. We knocked down the expression of TBK1 by using siRNA and found that the expression of GLUD1 in the TBK1-siRNA+ET group was lower than that in the ET group (Fig. 7A), as well as the enzyme activity of GLUD1 (Fig. 7B). In addition, the level of α-KG was also decreased in the TBK1-siRNA+ET group compared with the ET group (Fig. 7C). Flow cytometry showed that the ratio of M1 macrophages was lower in the TBK1-siRNA+ET group than in the ET group (Fig. 7D). Taken together, these findings demonstrated that the decrease in TBK1 expression could reduce the expression of phosphorylated RIPK3 and then affect the enzyme activity of GLUD1, resulting in a decrease in the production of α-KG. Finally, shRNA-TBK1-OE lentiviral particles were used to increase TBK1 expression in RAW264.7 cells. Compared with the ET group, overexpression of TBK1 could successfully increased the expression of GLUD1 (Fig. 7E). Additionally, the enzyme activity of GLUD1 increased, and the level of α-KG increased in the LV-TBK1-OE+ET group compared with the ET group (Fig. 7F-G). All of these findings indicated that the expression of TBK1 can mediate the phosphorylation of RIPK3, thereby inducing GLUD1 to decompose glutamine to produce α-KG. These results suggested that the modulation of glutamine metabolism, which plays a vital role in macrophage ET, would be an attractive strategy for harnessing macrophage-mediated immune responses.