GM-CSF induces proinflammatory macrophages, but the underlying mechanisms have not been studied thus far. In this study, we investigated the mechanisms of how GM-CSF induces inflammatory macrophages. First, we observed that GM-CSF increased the extent of LPS-induced acute glycolysis in murine bone marrow-derived macrophages. This directly correlates with an inflammatory phenotype because glycolysis inhibition by 2-deoxyglucose abolished GM-CSF-mediated increase of TNF-α, IL-1β, IL-6, and IL-12p70 synthesis upon LPS stimulation. Increased glycolytic capacity is due to de novo synthesis of glucose transporter (GLUT)-1, -3, and -4, as well as c-myc. Meanwhile, GM-CSF increased 3-hydroxy-3-methyl-glutaryl-CoA reductase, which is the rate-limiting enzyme of the mevalonate pathway. Inhibition of acute glycolysis or 3-hydroxy-3-methyl-glutaryl-CoA reductase abrogated the inflammatory effects of GM-CSF priming in macrophages. Finally, mice with inflamed colons exposed to dextran sodium sulfate containing GLUT-1 macrophages led to massive uptake of [F]-fluorodeoxyglucose, but GM-CSF neutralization reduced the positron-emission tomography signal in the intestine and also decreased GLUT-1 expression in colonic macrophages. Collectively, our results reveal glycolysis and lipid metabolism created by GM-CSF as the underlying metabolic constructs for the function of inflammatory macrophages.
Granulocyte-macrophage colony stimulating factor (GM-CSF) has a role in inducing emergency hematopoiesis upon exposure to inflammatory stimuli. Although GM-CSF generated murine bone marrow derived cells have been widely used as macrophages or dendritic cells in research, the exact characteristics of each cell population have not yet been defined. Here we discriminated GM-CSF grown bone marrow derived macrophages (GM-BMMs) from dendritic cells (GM-BMDCs) in several criteria. After C57BL/6J mice bone marrow cell culture for 7 days with GM-CSF supplementation, two main populations were observed in the attached cells based on MHCII and F4/80 marker expressions. GM-BMMs had MHCIIlowF4/80high as well as CD11c+CD11bhighCD80−CD64+MerTK+ phenotypes. In contrast, GM-BMDCs had MHCIIhighF4/80low and CD11chighCD8α− CD11b+CD80+CD64−MerTKlow phenotypes. Interestingly, the GM-BMM population increased but GM-BMDCs decreased in a GM-CSF dose-dependent manner. Functionally, GM-BMMs showed extremely high phagocytic abilities and produced higher IL-10 upon LPS stimulation. GM-BMDCs, however, could not phagocytose as well, but were efficient at producing TNFα, IL-1β, IL-12p70 and IL-6 as well as inducing T cell proliferation. Finally, whole transcriptome analysis revealed that GM-BMMs and GM-BMDCs are overlap with in vivo resident macrophages and dendritic cells, respectively. Taken together, our study shows the heterogeneicity of GM-CSF derived cell populations, and specifically characterizes GM-CSF derived macrophages compared to dendritic cells.
Interleukin-10 (IL-10) is an important anti-inflammatory cytokine that is produced primarily by macrophages. We investigated mechanisms by which the timing of IL-10 production was controlled in macrophages and found that cyclin-dependent kinase 5 (CDK5) activity was markedly increased in lipopolysaccharide (LPS)-stimulated macrophages through the synthesis of the CDK5-binding partner and activator p35. Degradation of p35 released the inhibition on anti-inflammatory signaling mediated by CDK5-p35 complexes. The transiently active CDK5-p35 complexes limited the LPS-stimulated phosphorylation and activation of various mitogen-activated protein kinases (MAPKs), thereby preventing the premature production of SOCS3 (suppressor of cytokine signaling 3), an inhibitor of inflammatory responses in macrophages, and IL-10. Furthermore, we showed that dextran sodium sulfate failed to induce colitis in p35-deficient mice, which was associated with the enhanced production of IL-10 by macrophages. Together, our results suggest that CDK5 enhances the inflammatory function of macrophages by inhibiting the MAPK-dependent production of IL-10.
Macrophages play important roles in defense against infection, as well as in homeostasis maintenance. Thus alterations of macrophage function can have unexpected pathological results. Cyclooxygenase (COX) inhibitors are widely used to relieve pain, but the effects of long-term usage on macrophage function remain to be elucidated. Using bone marrow-derived macrophage culture and long-term COX inhibitor treatments in BALB/c mice and zebrafish, we showed that chronic COX inhibition drives macrophages into an inflammatory state. Macrophages differentiated in the presence of SC-560 (COX-1 inhibitor), NS-398 (COX-2 inhibitor) or indomethacin (COX-1/2 inhibitor) for 7 days produced more TNFα or IL-12p70 with enhanced p65/IκB phosphoylation. YmI and IRF4 expression was reduced significantly, indicative of a more inflammatory phenotype. We further observed that indomethacin or NS-398 delivery accelerated zebrafish death rates during LPS induced sepsis. When COX inhibitors were released over 30 days from an osmotic pump implant in mice, macrophages from peritoneal cavities and adipose tissue produced more TNFα in both the basal state and under LPS stimulation. Consequently, indomethacin-exposed mice showed accelerated systemic inflammation after LPS injection. Our findings suggest that macrophages exhibit a more inflammatory phenotype when COX activities are chronically inhibited.
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