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Bile acids attenuate PKM2 pathway activation in proinflammatory microglia.

ABSTRACT: Glycolysis is the metabolic pathway that converts glucose into pyruvate. Central nervous system (CNS) pathologies, such as spinal cord injury (SCI) and ischemia, are accompanied by an increase of the glycolytic pathway in the damaged areas as part of the inflammatory response. Pyruvate kinase is a key glycolytic enzyme that converts phosphoenolpyruvate and ADP to pyruvate and ATP. The protein has two isoforms, PKM1 and PKM2, originated from the same gene. As a homodimer, PKM2 loses the pyruvate kinase activity and acts as a transcription factor that regulates the expression of target genes involved in glycolysis and inflammation. After SCI, resident microglia and hematogenous macrophages are key inducers of the inflammatory response with deleterious effects. Activation of the bile acid receptor TGR5 inhibits the pro-inflammatory NFκB pathway in microglia and macrophages. In the present study we have investigated whether bile acids affect the expression of glycolytic enzymes and their regulation by PKM2. Bacterial lipopolysaccharide (LPS) induced the expression of PKM1, PKM2 and its target genes in primary cultures of microglial and Raw264.7 macrophage cells. SCI caused an increase of PKM2 immunoreactivity in macrophages after SCI. Pretreatment with tauroursodeoxycholic acid (TUDCA) or taurolithocholic acid (TLCA) reduced the expression of PKM2 and its target genes in cell cultures. Similarly, after SCI, TUDCA treatment reduced the expression of PKM2 in the lesion center. These results confirm the importance of PKM2 in the inflammatory response in CNS pathologies and indicate a new mechanism of bile acids as regulators of PKM2 pathway.

SUBMITTER: Romero-Ramirez L

PROVIDER: S-EPMC8795255 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundNeuroinflammation is the response of the central nervous system to events that interfere with tissue homeostasis and represents a common denominator in virtually all neurological diseases. Activation of microglia, the principal immune effector cells of the brain, contributes to neuronal injury by release of neurotoxic products. Toll-like receptor 4 (TLR4), expressed on the surface of microglia, plays an important role in mediating lipopolysaccharide (LPS)-induced microglia activation and inflammatory responses. We have previously shown that curcumin and some of its analogues harboring an α,β-unsaturated 1,3-diketone moiety, able to coordinate the magnesium ion, can interfere with LPS-mediated TLR4-myeloid differentiation protein-2 (MD-2) signaling. Fluoroquinolone (FQ) antibiotics are compounds that contain a keto-carbonyl group that binds divalent ions, including magnesium. In addition to their antimicrobial activity, FQs are endowed with immunomodulatory properties, but the mechanism underlying their anti-inflammatory activity remains to be defined. The aim of the current study was to elucidate the molecular mechanism of these compounds in the TLR4/NF-κB inflammatory signaling pathway.MethodsThe putative binding mode of five FQs [ciprofloxacin (CPFX), levofloxacin (LVFX), moxifloxacin, ofloxacin, and delafloxacin] to TLR4-MD-2 was determined using molecular docking simulations. The effect of CPFX and LVFX on LPS-induced release of IL-1β and TNF-α and NF-κB activation was investigated in primary microglia by ELISA and fluorescence staining. The interaction of CPFX and LVFX with TLR4-MD-2 complex was assessed by immunoprecipitation followed by Western blotting using Ba/F3 cells.ResultsCPFX and LVFX bound to the hydrophobic region of the MD-2 pocket and inhibited LPS-induced secretion of pro-inflammatory cytokines and activation of NF-κB in primary microglia. Furthermore, these FQs diminished the binding of LPS to TLR4-MD-2 complex and decreased the resulting TLR4-MD-2 dimerization in Ba/F3 cells.ConclusionsThese results provide new insight into the mechanism of the anti-inflammatory activity of CPFX and LVFX, which involves, at least in part, the activation of TLR4/NF-κB signaling pathway. Our findings might facilitate the development of new molecules directed at the TLR4-MD-2 complex, a potential key target for controlling neuroinflammation.

Project description:Accumulating evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes various neurological symptoms in patients with coronavirus disease 2019 (COVID-19). The most dominant immune cells in the brain are microglia. Yet, the relationship between neurological manifestations, neuroinflammation, and host immune response of microglia to SARS-CoV-2 has not been well characterized. Here, we reported that SARS-CoV-2 can directly infect human microglia, eliciting M1-like proinflammatory responses, followed by cytopathic effects. Specifically, SARS-CoV-2 infected human microglial clone 3 (HMC3), leading to inflammatory activation and cell death. RNA sequencing (RNA-seq) analysis also revealed that endoplasmic reticulum (ER) stress and immune responses were induced in the early, and apoptotic processes in the late phases of viral infection. SARS-CoV-2-infected HMC3 showed the M1 phenotype and produced proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor α (TNF-α), but not the anti-inflammatory cytokine IL-10. After this proinflammatory activation, SARS-CoV-2 infection promoted both intrinsic and extrinsic death receptor-mediated apoptosis in HMC3. Using K18-hACE2 transgenic mice, murine microglia were also infected by intranasal inoculation of SARS-CoV-2. This infection induced the acute production of proinflammatory microglial IL-6 and TNF-α and provoked a chronic loss of microglia. Our findings suggest that microglia are potential mediators of SARS-CoV-2-induced neurological problems and, consequently, can be targets of therapeutic strategies against neurological diseases in patients with COVID-19. IMPORTANCE Recent studies reported neurological and cognitive sequelae in patients with COVID-19 months after the viral infection with several symptoms, including ageusia, anosmia, asthenia, headache, and brain fog. Our conclusions raise awareness of COVID-19-related microglia-mediated neurological disorders to develop treatment strategies for the affected patients. We also indicated that HMC3 was a novel human cell line susceptible to SARS-CoV-2 infection that exhibited cytopathic effects, which could be further used to investigate cellular and molecular mechanisms of neurological manifestations of patients with COVID-19.

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Bile acids attenuate PKM2 pathway activation in proinflammatory microglia.

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