Burn plus LPS injection augmented hepatic NLRP3 inflammasome activation
Burn, LPS, and burn + LPS increased NLRP3 inflammasome activation in the liver. We observed significantly increased Il-1, Emr, and Nlrp3 mRNA expression (Fig. 1, A–C) and a significant elevation of NLRP3 protein (Fig. 1, D and E) in the liver of burn and burn + LPS groups compared with the sham.
Burn and LPS injection induced hepatic ER stress
We observed significantly increased expression of Chop, Xbp1-s, and Pdi genes, markers of ER stress in liver, in sham + LPS and burn + LPS groups (Fig. 3, A, C, and D). Bip mRNA and BIP protein levels increased significantly in the LPS, burn, and burn + LPS groups (Fig. 3, B, E, and F). Therefore, burn injury, LPS, and their combination led to hepatic ER stress.
Burn induced hypermetabolism
To determine the underlying mechanism for the differences in weight and glucose, we monitored gene expression of hepatic metabolic modulators, including G6pase, which is the final-step catalyst in gluconeogenesis and glycogenolysis and therefore plays a key role in the homeostatic regulation of blood glucose level (15); Fasn, which catalyze the synthesis of palmitate from acetyl-CoA and malonyl-CoA in the presence of NADPH and thus is fundamental to energy storage and biosynthesis of hormones and other important biological molecules; Sik 1, which is a serine-threonine kinase related with steroidogenesis and metabolic regulation in adipose tissue (16). We also determined the hepatic protein level of G6Pase and SCD1, which is the rate-limiting enzyme catalyzing the biosynthesis of monounsaturated fatty acids (17). In the burn group, G6pase, Fasn, and Sik1 increased significantly compared with sham. In LPS group, G6pase mRNA level decreased compared with sham (Fig. 4, A–C). Protein level of G6Pase in liver tissue decreased significantly in LPS-only and burn + LPS groups (Fig. 4, D and F), whereas the level of SCD1decreased significantly in burn-only and burn + LPS group (Fig. 4, E and G).
We were particularly interested in the gene expression and tissue abundance of PGC-1α in the liver because it has profound impact on mitochondria energetic metabolism, glucose metabolism, and lipid metabolism (18). We observed increased gene expression of Pgc-1α in all treatment groups (Fig. 5A).
Unlike burn, which downregulates sirtuin-1, LPS inhibits PKA C/AMPKα
Lipopolysaccharide alone caused a significant decrease in the protein level of spliced PGC-1α (Fig. 5, B and D). To investigate the possible mechanisms of such PGC-1α inhibition, we measured the activation of PKA C, sirtuin-1, and AMPK, which are upstream regulators of spliced and full-length PGC-1α, respectively. Western blot analysis showed that LPS alone decreased phospho-AMPKα and phospho-PKA C, whereas there were no significant changes in phosphor-AMPKβ and PPAR-α. We found that sirtuin-1 decreased in burn and burn + LPS groups but not in the LPS-only group (data not shown) (Fig. 6).
In this study, by using a two-hit rat model of burn + LPS intraperitoneal injection, we investigated the activation of NLRP3 inflammasome and ER stress in liver, their interplay, and its impact on postburn metabolism. We found that both burn and LPS induce significant NLRP3 inflammasome activation in liver, and such NLRP3 inflammasome activation augmented liver damage and metabolic derangement in burn + LPS group. This is the first study to report NLRP3 inflammasome activation in liver tissue after burn injury.
Our previous studies have demonstrated that severe burn injury induces enormous and persisting stress response and hypermetabolism (19). The postburn stress response leads to the accumulation of unfolded and misfolded proteins in the ER and increased intracellular heat shock and chaperone proteins (20). Postburn hypermetabolism causes increased ATP production, increased oxidative phosphorylation and reactive oxygen species production in the mitochondria, and increased gene transcription (21). All of these increased intracellular biomolecules are DAMPs indicating challenging environment and have been shown to trigger NLRP3 inflammasome activation in other disease models (22, 23). Composed of NLRP, ASC, and pro–caspase 1, the NLRP3 inflammasome generally assembles in macrophages where it activates caspase 1 to ultimately produce IL-1β, which is an important mediator of the inflammatory response, and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis (24). Moreover, IL-1β interferes with insulin receptor signaling (25) and may thus directly exacerbate metabolic derangement. In the current study, we confirmed such NLRP3 inflammasome activation in the liver in burn and sepsis animals by showing increased inflammasome-related gene expression (Il-1, Nlrp3) and increased NLRP3 protein. EMR is a specific marker of macrophages (26). The very similar pattern of Emr mRNA expression with Il-1 and Nlrp3 suggests macrophage involvement of liver inflammasome activation. Moreover, our observations suggest that thermal injury with or without sepsis induces NLRP3 inflammasome activation in the liver. This is consistent with our understanding that ER stress produces DAMPs and may thus contribute to the initiation of inflammasome formation (27).
We showed in the current study that ER stress and inflammasome activation (assessed by gene expression and protein analysis) shared the same pattern in the LPS, burn, and burn + LPS groups. Further research in knockout models will be important to establish whether there is a causative relationship between these two pathological processes. We noticed that there was less robust ER stress-related genes expression in burn-only group. The possible explanation might be that the rats were recovering from initial thermal injury after 96 h. Our previous study showed that the stress response after burn peaks indeed between 24 and 48 h (28). Nevertheless, we did observe augmented responses of both ER stress and inflammasome activation in burn + LPS treatment group.
Enhanced ER stress and NLRP3 inflammasome activation affected postburn metabolism. By monitoring the metabolic modulator genes’ expression, liver function, and blood glucose level, we noticed increased metabolic demand from burn injury and lowered metabolic capacity by LPS treatment. Robust increase in G6pase, Fasn, and Sik 1 in burn group postulates an increased need for gluconeogenesis and lipid metabolism, whereas decreased protein level of G6Pase in LPS-only and burn + LPS groups indicated impaired metabolic functioning attributed to LPS treatment. Significant decrease in hepatic SCD1 might be due to insulin resistance of burn and burn + LPS groups (29).
Particularly, PGC-1α was of our research interest for its multiple, potent roles in mitochondria respiration, gluconeogenesis, lipid metabolism, and so on. PGC 1α has two isoforms: a 113-kDa full-length isoform and a 38-kDa spliced isoform. Spliced PGC-1α has the same functional domain with its full-length form but can more freely transport between the nucleus, mitochondria, ER, and cytosol and thus has a more powerful effect on metabolic modulation (30). A significant increase in Pgc-1α mRNA expression in all three treatment groups suggested increased demand for it. However, in the LPS group, we did not observe corresponding increased protein levels of full-length PGC-1α and actually observed significantly decreased levels of spliced PGC-1α. This may partly account for the metabolic impairment and liver dysfunction in the burn + LPS group.
We previously reported that post-burn ER stress induced hepatic apoptosis contributing to liver damage (31, 32). In the current study, immunohistochemical analysis showed that LPS may augment such postburn liver damage (Fig. 1, E–H).
Our results support previously reported findings of Osuka et al. (11) that macrophages actively involve NLRP3 inflammasome activation. Moreover, our observation and findings may reasonably explain the controversy of survival advantage of inflammasome activation and its detrimental effects on clinical outcome: burn- and LPS-induced inflammasome activation has more profound impact on metabolic impairment and liver dysfunction than its protective effect of cytokines balancing (12). Considering the difference of severity of injury between the two studies (60% TBSA burn with second hit of LPS vs. 25%TBSA burn), we may also postulate the different modes of inflammasome activation upon a different intensity of stimuli (i.e., being protective upon mild injury and being detrimental upon severe injury).
We did not observe significant elevation of blood glucose level in this animal model. However, considering the unwellness of the animals in the treatment groups, which included weight loss (Fig. 1I) and decreased food intake (data not shown), blood glucose level of greater than 7 mmol/L was still quite noticeable, which might still be indicative for postburn hypermetabolism.
To investigate whether LPS, ER stress, and/or inflammasome activation directly or indirectly interfere with PGC-1α expression and function, we examined the upstream modulators of PGC-1α. Three regulatory pathways were studied. The first is PKA C/PGC-1α interaction. It has been shown that activation of PKA can significantly increase the nuclear content of spliced PGC-1α, and thus, PKA modulates PGC-1α–dependent signaling (33). We found significantly decreased phospho-PKA C indicating the inhibition of PKA activity in the LPS group, which was consistent with the decreased level of spliced PGC-1α. Therefore, we infer that PKA C inhibition at least partly contributes to attenuation of spliced PGC-1α in burn + LPS treatment group.
The second regulatory pathway is AMPK. AMPK may activate PGC-1α through phosphorylation of specific serine and threonine residues (34). By Western blotting analysis of AMPK system in this two-hit model, we found that there was a significant decrease in phospho-AMPKα in the burn + LPS group. There were no significant changes in the level of phospho-AMPKβ and total AMPKα and AMPKβ. Because AMPKα is the catalyst unit, whereas AMPKβ is regulatory, our result suggests that AMPK activity is directly inhibited at its catalyst unit in burn + LPS treatment group. The consistency of attenuation of PGC-1α and AMPKα activity may also indicate the possibility of involvement of AMPKα in PGC-1α regulation.
The third upstream regulatory molecule of PGC-1α is sirtuin-1, which activates PGC-1α through NAD+-dependent deacetylation. In this way, sirtuin-1 links metabolic perturbation with cellular transcriptional output. We observed inhibition of sirtuin-1 in burn and burn + LPS treatment group but not LPS-only group. Based on this observation, we may reasonably postulate that the inhibition of PGC-1α in burn + LPS treatment group is the overlay of burn-induced sirtuin-1 inhibition and PKA C/AMPKα signaling blockage mainly induced by LPS. Further confirmative research is needed to establish such causative relationship between PGC-1a inhibition and liver dysfunction and damage.
Western blotting analysis of PPAR-α did not show significant changes among different treatment groups. It is thus unlikely that PPAR-α in liver would respond to the changes in burn or LPS-induced stress or inflammasome activation. Because PGC-1α is usually regarded as the coactivator of PPAR-α (35), our result may indicate that PGC-1α plays a regulatory role in PPAR-α transcriptional activity.
Based on this study, we summarize that there is inflammasome activation in the liver after burn and LPS administration. Second, NLRP3 inflammasome activation contributes to the post-burn ER stress response, and the two pathological processes exacerbate metabolic dysfunction in the liver. Finally, PGC-1α most likely plays an important role in the hypermetabolic response after burn and may be regulated by PKA C, AMPKα, and sirtuin-1 signaling pathways. Future studies will investigate whether PGC-1α, PKA C, AMPKα, and sirtuin-1 may represent potential therapeutic targets for the treatment of postburn ER stress, inflammasome activation, and subsequent metabolic dysfunction.
The authors thank Fangming Xiu and Mile Stanojcic for their help with the animal experiments.
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Keywords:© 2014 by the Shock Society
Thermal injury; sepsis; ER stress; inflammasome; metabolism