Increased Glycolytic Activity Is Part of Impeded M1(LPS) Macrophage Polarization in the Presence of Urolithin A.

Urolithin A is a gut metabolite of ellagitannins and reported to confer health benefits, e.g., by increased clearance of damaged mitochondria by macroautophagy or curbed inflammation. One targeted cell type are macrophages, which are plastic and able to adopt pro- or anti-inflammatory polarization states, usually assigned as M1 and M2 macrophages, respectively. This flexibility is tightly coupled to characteristic shifts in metabolism, such as increased glycolysis in M1 macrophages, and protein expression upon appropriate stimulation. This study aimed at investigating whether the anti-inflammatory properties of U: rolithin A may be driven by metabolic alterations in cultivated murine M1(lipopolysaccharide) macrophages. Expression and extracellular flux analyses showed that urolithin A led to reduced il1ß, il6, and nos2 expression and boosted glycolytic activity in M1(lipopolysaccharide) macrophages. The pro-glycolytic feature of UROLITHIN A: occurred in order to causally contribute to its anti-inflammatory potential, based on experiments in cells with impeded glycolysis. Mdivi, an inhibitor of mitochondrial fission, blunted increased glycolytic activity and reduced M1 marker expression in M1(lipopolysaccharide/UROLITHIN A: ), indicating that segregation of mitochondria was a prerequisite for both actions of UROLITHIN A: . Overall, we uncovered a so far unappreciated metabolic facet within the anti-inflammatory activity of UROLITHIN A: and call for caution about the simplified notion of increased aerobic glycolysis as an inevitably proinflammatory feature in macrophages upon exposure to natural products.

Corrigendum: Sulforaphane diminishes moonlighting of pyruvate kinase M2 and interleukin 1ß expression in M1 (LPS) macrophages.

[This corrects the article DOI: 10.3389/fimmu.2022.935692.].

Sulforaphane impedes mitochondrial reprogramming and histone acetylation in polarizing M1 (LPS) macrophages.

M1 (LPS) macrophages are characterized by a high expression of pro-inflammatory mediators, and distinct metabolic features that comprise increased glycolysis, a broken TCA cycle, or impaired OXPHOS with augmented mitochondrial ROS production. This study investigated whether the phytochemical sulforaphane (Sfn) influences mitochondrial reprogramming during M1 polarization, as well as to what extent this can contribute to Sfn-mediated inhibition of M1 marker expression in murine macrophages. The use of extracellular flux-, metabolite-, and immunoblot analyses as well as fluorescent dyes indicative for mitochondrial morphology, membrane potential or superoxide production, demonstrated that M1 (LPS/Sfn) macrophages maintain an unbroken TCA cycle, higher OXPHOS rate, boosted fusion dynamics, lower membrane potential, and less superoxide production in their mitochondria when compared to control M1 (LPS) cells. Sustained OXPHOS and TCA activity but not the concomitantly observed high dependency on fatty acids as fuel appeared necessary for M1 (LPS/Sfn) macrophages to reduce expression of nos2, il1ß, il6 and tnfα. M1 (LPS/Sfn) macrophages also displayed lower nucleo/cytosolic acetyl-CoA levels in association with lower global and site-specific histone acetylation at selected pro-inflammatory gene promoters than M1 (LPS), evident in colorimetric coupled enzyme assays, immunoblot and ChIP-qPCR analyses, respectively. Supplementation with acetate or citrate was able to rescue both histone acetylation and mRNA expression of the investigated M1 marker genes in Sfn-treated cells. Overall, Sfn preserves mitochondrial functionality and restricts indispensable nuclear acetyl-CoA for histone acetylation and M1 marker expression in LPS-stimulated macrophages.

Norbergenin prevents LPS-induced inflammatory responses in macrophages through inhibiting NFκB, MAPK and STAT3 activation and blocking metabolic reprogramming.

Inflammation is thought to be a key cause of many chronic diseases and cancer. However, current therapeutic agents to control inflammation have limited long-term use potential due to various side-effects. This study aimed to examine the preventive effects of norbergenin, a constituent of traditional anti-inflammatory recipes, on LPS-induced proinflammatory signaling in macrophages and elucidate the underlying mechanisms by integrative metabolomics and shotgun label-free quantitative proteomics platforms. Using high-resolution mass spectrometry, we identified and quantified nearly 3000 proteins across all samples in each dataset. To interpret these datasets, we exploited the differentially expressed proteins and conducted statistical analyses. Accordingly, we found that LPS-induced production of NO, IL1ß, TNFα, IL6 and iNOS in macrophages was alleviated by norbergenin via suppressed activation of TLR2 mediated NFκB, MAPKs and STAT3 signaling pathways. In addition, norbergenin was capable of overcoming LPS-triggered metabolic reprogramming in macrophages and restrained the facilitated glycolysis, promoted OXPHOS, and restored the aberrant metabolites within the TCA cycle. This is linked to its modulation of metabolic enzymes to support its anti-inflammatory activity. Thus, our results uncover that norbergenin regulates inflammatory signaling cascades and metabolic reprogramming in LPS stimulated macrophages to exert its anti-inflammatory potential.

Branched-chain ketoacids derived from cancer cells modulate macrophage polarization and metabolic reprogramming.

Macrophages are prominent immune cells in the tumor microenvironment that can be educated into pro-tumoral phenotype by tumor cells to favor tumor growth and metastasis. The mechanisms that mediate a mutualistic relationship between tumor cells and macrophages remain poorly characterized. Here, we have shown in vitro that different human and murine cancer cell lines release branched-chain α-ketoacids (BCKAs) into the extracellular milieu, which influence macrophage polarization in an monocarboxylate transporter 1 (MCT1)-dependent manner. We found that α-ketoisocaproate (KIC) and α-keto-ß-methylvalerate (KMV) induced a pro-tumoral macrophage state, whereas α-ketoisovalerate (KIV) exerted a pro-inflammatory effect on macrophages. This process was further investigated by a combined metabolomics/proteomics platform. Uptake of KMV and KIC fueled macrophage tricarboxylic acid (TCA) cycle intermediates and increased polyamine metabolism. Proteomic and pathway analyses revealed that the three BCKAs, especially KMV, exhibited divergent effects on the inflammatory signal pathways, phagocytosis, apoptosis and redox balance. These findings uncover cancer-derived BCKAs as novel determinants for macrophage polarization with potential to be selectively exploited for optimizing antitumor immune responses.

Sulforaphane diminishes moonlighting of pyruvate kinase M2 and interleukin 1ß expression in M1 (LPS) macrophages.

Murine macrophages activated by the Toll-like receptor 4 agonist lipopolysaccharide (LPS) polarize to the M1 type by inducing proinflammatory marker proteins and changing their energy metabolism to increased aerobic glycolysis and reduced respiration. We here show that the aliphatic isothiocyanate sulforaphane (Sfn) diminishes M1 marker expression (IL-1ß, IL-6, TNF-α, iNOS, NO, and ROS) and leads to highly energetic cells characterized by both high glycolytic and high respiratory activity as assessed by extracellular flux analysis. Focusing on a potential connection between high glycolytic activity and low IL-1ß expression in M1 (LPS/Sfn) macrophages, we reveal that Sfn impedes the moonlighting function of pyruvate kinase M2 (PKM2) in M1 macrophages. Sfn limits mono/dimerization and nuclear residence of PKM2 accompanied by reduced HIF-1α levels, Stat3 phosphorylation at tyrosine 705, and IL-1ß expression while preserving high levels of cytosolic PKM2 tetramer with high glycolytic enzyme activity. Sfn prevents glutathionylation of PKM2 in LPS-stimulated macrophages which may account for the reduced loss of PKM2 tetramer. Overall, we uncover PKM2 as a novel affected hub within the anti-inflammatory activity profile of Sfn.

Differences and similarities between mesenchymal stem cell and endothelial progenitor cell immunoregulatory properties against T cells.

Bone-marrow-derived mesenchymal stem cells and endothelial progenitor cells have some interesting biological properties that make them unique for cell therapy of degenerative and cardiovascular disorders. Although both cell populations have been already studied and used for their regenerative potentials, recently their special immunoregulatory features have brought much more attention. Mesenchymal stem cells and endothelial progenitor cells have both proangiogenic functions and have been shown to suppress the immune response, particularly T cell proliferation, activation, and cytokine production. This makes them suitable choices for allogeneic stem cell transplantation. Nevertheless, these two cells do not have equal immunoregulatory activities. Many elements including their extraction sources, age/passage, expression of different markers, secretion of bioactive mediators, and some others could change the efficiency of their immunosuppressive function. However, to our knowledge, no publication has yet compared mesenchymal stem cells and endothelial progenitor cells for their immunological interaction with T cells. This review aims to specifically compare the immunoregulatory effect of these two populations including their T cell suppression, deactivation, cytokine production, and regulatory T cells induction capacities. Moreover, it evaluates the implications of the tumor necrosis factor alpha-tumor necrosis factor receptor 2 axis as an emerging immune checkpoint signaling pathway controlling most of their immunological properties.

Corrigendum: TNFR2 Is a Crucial Hub Controlling Mesenchymal Stem Cell Biological and Functional Properties.

[This corrects the article DOI: 10.3389/fcell.2020.596831.].

TNFR2 Is a Crucial Hub Controlling Mesenchymal Stem Cell Biological and Functional Properties.

Mesenchymal stem cells (MSCs) have drawn lots of attention as gold standard stem cells in fundamental and clinical researches during the last 20 years. Due to their tissue and vascular repair capacities, MSCs have been used to treat a variety of degenerative disorders. Moreover, MSCs are able to modulate immune cells' functions, particularly T cells while inducing regulatory T cells (iTregs). MSCs are very sensitive to inflammatory signals. Their biological functions could remarkably vary after exposure to different pro-inflammatory cytokines, notably TNFα. In this article, we have explored the importance of TNFR2 expression in a series of MSCs' biological and functional properties. Thus, MSCs from wild-type (WT) and TNFR2 knockout (TNFR2 KO) mice were isolated and underwent several ex vivo experiments to investigate the biological significance of TNFR2 molecule in MSC main functions. Hampering in TNFR2 signaling resulted in reduced MSC colony-forming units and proliferation rate and diminished the expression of all MSC characteristic markers such as stem cell antigen-1 (Sca1), CD90, CD105, CD44, and CD73. TNFR2 KO-MSCs produced more pro-inflammatory cytokines like TNFα, IFNγ, and IL-6 and less anti-inflammatory mediators such as IL-10, TGFß, and NO and induced Tregs with less suppressive effect. Furthermore, the TNFR2 blockade remarkably decreased MSC regenerative functions such as wound healing, complex tube formation, and endothelial pro-angiogenic support. Therefore, our results reveal the TNFα-TNFR2 axis as a crucial regulator of MSC immunological and regenerative functions.