Volume 105, Issue 2 p. 211-213
EDITORIAL COMMENTARY
Free Access

Metabolism drives monocytes during inflammation: What we do and do not know

Naeem K. Patil

Naeem K. Patil

Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN, USA

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Julia K. Bohannon

Julia K. Bohannon

Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN, USA

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Edward R. Sherwood

Corresponding Author

Edward R. Sherwood

Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN, USA

Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA

Correspondence

Edward R. Sherwood, Department of Anesthesiology, Vanderbilt University Medical Center, 1161 Medical Center Drive, T-4202 MCN, Nashville, TN 37232-2520, USA.

Email: [email protected]

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First published: 07 January 2019
Citations: 2

See corresponding article on Page 215

Abstract

Discussion on leukocyte metabolism and the subsequent changes in intracellular metabolite concentrations.

The quest for discovering novel therapeutics to treat acute inflammatory conditions such as sepsis has been a significant challenge for scientists and physicians. Despite promising discoveries using animal models, innumerable clinical trials have failed to yield any definitive treatment to reduce morbidity and mortality associated with human sepsis. There is a critical need to better understand the cellular and molecular pathogenesis of sepsis and other acute inflammatory conditions and explore novel therapies to improve outcomes. As such, the field of immunometabolism is being widely explored to better understand the metabolic basis for activation and resolution of inflammation in a quest to discover novel therapeutic targets. Alterations in leukocyte metabolism and subsequent changes in the concentrations of intracellular metabolites has recently emerged as one of the major mechanisms that guide immune cell responses during exposure to an inflammatory insult such a LPS.1, 2 Activation of myeloid cells leads to significant intracellular accumulation of metabolic intermediates such as succinate, itaconate, citrate, and many others.2 Exploration of the signaling mechanisms mediated by these metabolic intermediates has created a paradigm shift regarding how we view the role of metabolic intermediates as drivers of leukocyte responses. These findings have uncovered a novel area of investigation that may improve our understanding of leukocyte functions during sepsis for discovering novel therapeutic targets.

In this issue of the Journal of Leukocyte Biology, Zhu and colleagues report new data that provide insights into LPS-induced temporal changes in concentrations of major intracellular metabolites in THP-1 monocytic cells during periods of activation, transition, and resolution. The authors categorized post-LPS exposure periods into three sequential phases—anabolic activation (0–8 h), catabolic deactivation (24–48 h), and transition to early resolution (48–96 h). As summarized in Figure 1, the findings presented in this manuscript represents a characterization of the carbohydrate, protein, lipid, and nucleic acid metabolites induced by LPS exposure during the aforementioned sequential phases.

Details are in the caption following the image
Temporal metabolic reprogramming of THP1 cells after LPS stimulation. Shown is a listing of key carbohydrate (glycogenolysis, glycolysis, lactate, pyruvate, acetyl CoA, succinate, itaconate, 6-phosphogluconate), lipid (glycerophosphocholine, choline, polyunsaturated fatty acids, acyl-carnitine, sphingosine), protein (amino acids, methionine, tryptophan, kynurenine, valine), nucleotide (adenosine, uracil, xanthine), and cellular redox (glutathione, glutathione disulfide, S-nitrosoglutathione) related metabolic intermediates. The heat map indicates direction and degree of level changes of each over time after LPS stimulation in THP1 cells, during activation (0–8 h post-LPS), deactivation (24–48 h post-LPS) and resolution (48–96 h post-LPS) phases as reported by Zhu and colleagues. Red indicates up-regulation from baseline, blue indicates down-regulation from baseline, and white indicates no change from baseline

The authors report that during the initial activation phase the concentrations of glucose-1-phosphate and fructose-1,6-bisphosphate are significantly increased implying an increase in glycogenolysis and glycolysis, respectively.3 Increased glycogenolysis was associated with increased UDP-glucose levels during the initial phase. This is an interesting finding as UDP-glucose can glycosylate proteins and plasma protein glycosylation patterns have been shown to be distinctly regulated in sepsis survivors versus nonsurvivors.4 Glucose-1-phosphate and fructose-1,6-bisphosphate levels declined over the deactivation and resolution phases indicating a reduced rate of glycolysis and glycogenolysis over time. Despite the observed increase in glycolysis intermediates, the levels of glycolysis end products, pyruvate and lactate, remained at low levels throughout all phases. This is a perplexing finding given that high cellular lactate production and lactate accumulation are hallmarks of severe sepsis. Tracking of carbon flux using stable isotope radiolabeling to determine the exact fate of glucose as it travels via glycolysis into Krebs cycle would provide more definitive information.

Zhu et al. report that the initial activation phase was not associated with an increase in pentose phosphate pathway (PPP) metabolites. This finding is in contrast to some of the previous findings that show an acute increase in flux through the PPP pathway in LPS-activated macrophages.5 The observed difference might be due to differences in cell types employed in the respective studies. Sedoheptulose-7-phosphate, another metabolite of the PPP pathway, was transiently increased at the 24 h time point. Increased sedoheptulose-7-phosphate, derived through the action of carbohydrate response kinase-like protein (CARKL), has previously been shown to be increased in anti-inflammatory M2 like macrophages,5 thereby supporting the authors hypothesis in the current study, implicating the role of CARKL in the induction of THP-1 cell deactivation phase.

The alterations in Krebs cycle metabolic intermediates presented in this paper are in line with previous studies showing increased intracellular succinate and itaconate accumulation during the initial acute activation phase of myeloid cells.1-3 The authors state that fumarate levels were also decreased during activation, but the data were not shown in the paper. In accordance with the previous reports,1 these findings suggest potential breaks in the Krebs cycle at succinate dehydrogenase and isocitrate dehydrogenase. Previous studies had not reported the temporal changes in these metabolites after LPS exposure over a prolonged period. The current paper assumes importance in that respect and shows that the concentration of succinate continues to remain high up to 96 h after LPS exposure, whereas the concentration of itaconate peaks at 8 h and then continues to decline, reaching baseline levels by 72 h. Succinate has been shown to play a critical role in mediating hypoxia inducible factor-1α stabilization and increased mitochondrial reactive oxygen species production and increased IL-1β secretion, thereby promoting a proinflammatory phenotype in macrophages.2 On the contrary, itaconate has been shown to sustain an anti-inflammatory phenotype via inhibition of succinate dehydrogenase activity and activation of nuclear respiratory factor 2 (Nrf2), along with possessing major antimicrobial properties.2 Acetyl CoA levels tended to decline during the activation and deactivation phases and reached a minimum at 48 to 72 h challenge and recovered at 72 to 96 h. The authors do not provide a rationale for this finding, but it correlates with decreased pyruvate levels, which is a major glycolytic precursor of acetyl CoA. Concentration of other major metabolic intermediates of the Krebs cycle such as citrate, isocitrate, malate, and oxaloacetate are not reported in this paper. Previous studies have shown that glutamine anapleurosis is the major source of succinate during rewiring of the Krebs cycle during LPS activation of macrophages.2 However, the paper by Zhu et al. also demonstrates a role of succinate derived from succinylcarnitine, which is a major degradation product of valine catabolism. The authors report that levels of valine and succinylcarnitine continued to decline after LPS exposure, whereas succinate levels correspondingly increased during the same time frame.

With respect to lipid metabolism, authors report the activation phase was also associated with more than double the levels of glycerophosphocholine, choline, glycerol-3-phosphate, and acyl-carnitine derivatives, the plasma levels of which have been previously shown to correlate with septic patient outcomes.6 The levels of acyl carnitine tended to decrease at the 96 h time point, whereas a previous clinical study by Langley et al. showed that acyl carnitine derivatives were significantly increased among sepsis nonsurvivors at 28 days,6 a time point well beyond the early resolution phase. The detailed correlation among intracellular metabolite concentrations, plasma metabolite concentrations, and sepsis patient outcomes in a single study remains to be elucidated. Interestingly, the authors report increased sphingosine concentrations at the peak activation phase of 8 h. Sphingosine has previously been shown to possess direct antimicrobial effects and improve host resistance to infection, although the exact mechanisms for its antimicrobial activity are not completely understood.7 Therefore, increased sphingosine concentrations upon activation of myeloid cells might serve as an endogenous defense mechanism to protect against infecting microbes, and this finding needs to be further investigated for its therapeutic potential. Overall, these findings reveal a sequential increase in fatty acid uptake and lipolysis in THP-1 cells upon LPS stimulation.

Zhu et al. report an overall degradation of proteins up to 96 h after LPS stimulation, as demonstrated by significant decreases in dipeptides, increased lysine degradation products, and decreased concentration of majority of the amino acids, except for cysteine.3 One of the important findings reported is significant reduction of the tryptophan levels over time and associated increases in its metabolites. Specifically, the tryptophan metabolite kynurenine was significantly increased during the activation and deactivation phases, and quinolate and NAD+ levels peaked during the resolution phase. Based on their previous studies, the authors hypothesize that tryptophan catabolism supports deactivation and an immune repressive phenotype, through enhanced availability of de novo produced NAD+, which supports SIRT1 activity.8 Studies from the same group have shown that SIRT1 inhibition restores metabolic homeostasis and reverses sepsis-induced alterations in mitochondrial bioenergetics.8 They presumptively ascribe increased tryptophan metabolism to a possible increase in indoleamine 2,3-dioxyenase activity (IDO), although this was not directly demonstrated. IDO enzyme activity has been suggested to play a dual role during microbial infections and sepsis. On one hand, it is byproduct kynurenine exerts antimicrobial effects and on the other hand it might induce an immune-suppressive phenotype through dampening of T-cell-mediated immune responses, increased anti-inflammatory cytokine secretion (IL-10), and increased recruitment of regulatory T cells.9 Nevertheless, the findings by Zhu and colleagues bring to light another potential therapeutic target involving tryptophan metabolism, which merits further investigation in inflammatory conditions.

It is well established that LPS and other infectious stimuli cause a rapid increase in ROS production, known as oxidative burst, within myeloid cells such as neutrophils and macrophages. ROS production aids in clearing invading microbes. However, excess ROS can damage the cells and intracellular antioxidant defenses must be balanced with the pro-oxidant environment to counteract the ill effects of oxidative stress. Zhu et al. report increased levels of reduced glutathione (GSH), oxidized glutathione (GSSG), and S-nitrosoglutathione (GSNO) during the activation and deactivation phases, followed by significant decrease during the resolution phase, as a result of methionine catabolism.3 They also show that nucleic acid catabolism is increased at early time points after LPS exposure leading to increased xanthine levels which remain elevated up to 96 h (resolution phase). Xanthine is known to be a significant endogenous source of superoxide radical contributing to cellular stress. GSH is a major intracellular antioxidant and, as shown by Zhu et al., increased GSH levels at 8 h after LPS exposure might be beneficial in keeping oxidative stress under check. However, further detailed studies need to be undertaken to examine alterations in other endogenous antioxidant defense pathways. For example, mitochondrial superoxide dismutase (MnSOD) has been shown to be inactivated via a post-translational mechanism known as glutathionylation, which can be induced by increased intracellular GSNO and GSH levels.10 MnSOD is a major mitochondrial antioxidant enzyme which keeps in check the excess superoxide radicals generated from the mitochondrial electron transport chain. As discussed earlier, Zhu and colleagues report a significant increase in itaconate levels up to 48 h after LPS exposure, which later decline between 72 and 96 h. Itaconate has recently been shown to activate Nrf2, which is a master regulator of intracellular antioxidant responses including stimulation of increased MnSOD expression. Future studies correlating the levels of the endogenous metabolites reported in this paper with the cellular antioxidant defense mechanisms will be critical to uncover novel therapeutic targets.

The findings presented by Zhu and colleagues are important and lay the foundation for future studies evaluating the temporal changes in intracellular metabolites as monocytes and macrophages transition from inflammatory activation to resolution of inflammation. Some caution should be exercised because these were generated in monocyte like THP-1 cells and further studies need to be conducted in primary human myeloid cells to confirm similar processes. Additionally, a relatively higher concentration of LPS (1 μg/mL) was used to stimulate the cells, as compared to majority of the other in vitro studies. The concertation of LPS employed is a very critical determinant while interpreting and comparing results across such mechanistic studies. Nonetheless, this paper highlights an important fact that myeloid cells sequentially rewire their metabolism after exposure to inflammatory stimuli and suggests opportunities for better understanding monocyte and macrophage functions during sepsis and identifying novel therapeutic targets.