Neurochemical Research

, Volume 44, Issue 12, pp 2755–2764 | Cite as

PKC Mediates LPS-Induced IL-1β Expression and Participates in the Pro-inflammatory Effect of A2AR Under High Glutamate Concentrations in Mouse Microglia

  • Sheng-Yu Fu
  • Ren-Ping Xiong
  • Yan Peng
  • Zhuo-Hang Zhang
  • Xing Chen
  • Yan Zhao
  • Ya-Lei Ning
  • Nan Yang
  • Yuan-Guo Zhou
  • Ping LiEmail author
Original Paper


Pathogens such as bacterial lipopolysaccharide (LPS) play an important role in promoting the production of the inflammatory cytokines interleukin-1 beta (IL-1β) and tumour necrosis factor-α (TNF-α) in response to infection or damage in microglia. However, whether different signalling pathways regulate these two inflammatory factors remains unclear. The protein kinase C (PKC) family is involved in the regulation of inflammation, and our previous research showed that the activation of the PKC pathway played a key role in the LPS-induced transformation of the adenosine A2A receptor (A2AR) from anti-inflammatory activity to pro-inflammatory activity under high glutamate concentrations. Therefore, in the current study, we investigated the role of PKC in the LPS-induced production of these inflammatory cytokines in mouse primary microglia. GF109203X, a specific PKC inhibitor, inhibited the LPS-induced expression of IL-1β messenger ribonucleic acid and intracellular protein in a dose-dependent manner. Moreover, 5 µM GF109203X prevented LPS-induced IL-1β expression but did not significantly affect LPS-induced TNF-α expression. PKC promoted IL-1β expression by regulating the activity of NF-κB but did not significantly impact the activity of ERK1/2. A2AR activation by CGS21680, an A2AR agonist, facilitated LPS-induced IL-1β expression through the PKC pathway at high glutamate concentrations but did not significantly affect LPS-induced TNF-α expression. Taken together, these results suggest a new direction for specific intervention with LPS-induced inflammatory factors in response to specific signalling pathways and provide a mechanism for A2AR targeting, especially after brain injury, to influence inflammation by interfering with A2AR.


Inflammation Microglial LPS IL-1β expression TNF-α expression Protein kinase C 





Interleukin-1 beta


Tumour necrosis factor-α


Traumatic brain injury


Nuclear factor- kappa-B


Adenosine A2A receptor


Extracellular regulated protein kinases 1/2


Mitogen-activated protein kinase


Toll-like receptor 4


Myeloid differentiation primary response 88


Ionized calcium-binding adaptor molecule 1





This work was supported by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

11064_2019_2895_MOESM1_ESM.tif (7.4 mb)
Supplementary Fig. 1Inhibition of PKC abrogates changes in microglial morphology after LPS stimulation. Morphological changes with LPS stimulation in microglia after PKC inhibition (a). Nuclei are labelled with DAPI (blue), and Iba-1 is indicated by red fluorescence; scale bar = 50 µm. Evaluation of microglial cell sphericity (an index of cell activation) in four different groups.*P < 0.05 and **P < 0.01 compared with the LPS stimulation group; #P < 0.05 and ##P < 0.01 compared with the untreated control group. Supplementary material 1 (TIF 7591.1 kb)


  1. 1.
    Bachiller S et al (2018) Microglia in neurological diseases: a road map to brain-disease dependent-inflammatory response. Front Cell Neurosci 12:488PubMedPubMedCentralGoogle Scholar
  2. 2.
    Kettenmann H et al (2011) Physiology of microglia. Physiol Rev 91(2):461–553PubMedGoogle Scholar
  3. 3.
    Norris GT, Kipnis J (2019) Immune cells and CNS physiology: microglia and beyond. J Exp Med 216(1):60–70PubMedPubMedCentralGoogle Scholar
  4. 4.
    Ransohoff RM (2016) How neuroinflammation contributes to neurodegeneration. Science 353(6301):777–783PubMedGoogle Scholar
  5. 5.
    Moller B, Villiger PM (2006) Inhibition of IL-1, IL-6, and TNF-alpha in immune-mediated inflammatory diseases. Springer Semin Immunopathol 27(4):391–408PubMedGoogle Scholar
  6. 6.
    Li T et al (2019) Synergistic anti-inflammatory effects of quercetin and catechin via inhibiting activation of TLR4-MyD88-mediated NF-kB and MAPK signaling pathways. Phytother Res 33(3):756–767PubMedGoogle Scholar
  7. 7.
    Huang X et al (2009) An atypical protein kinase C (PKC zeta) plays a critical role in lipopolysaccharide-activated NF-kB in human peripheral blood monocytes and macrophages. J Immunol 182(9):5810–5815PubMedGoogle Scholar
  8. 8.
    Taupin V et al (1993) Increase in IL-6, IL-1 and TNF levels in rat brain following traumatic lesion. Influence of pre- and post-traumatic treatment with Ro5 4864, a peripheral-type (p site) benzodiazepine ligand. J Neuroimmunol 42(2):177–185PubMedGoogle Scholar
  9. 9.
    Dalgard CL et al (2012) The cytokine temporal profile in rat cortex after controlled cortical impact. Front Mol Neurosci 5:6PubMedPubMedCentralGoogle Scholar
  10. 10.
    Newton AC (2018) Protein kinase C: perfectly balanced. Crit Rev Biochem Mol Biol 53(2):208–230PubMedPubMedCentralGoogle Scholar
  11. 11.
    Garcia-Bernal F et al (2018) Protein kinase C inhibition mediates neuroblast enrichment in mechanical brain injuries. Front Cell Neurosci 12:462PubMedPubMedCentralGoogle Scholar
  12. 12.
    Zhao EY et al (2016) The role of Akt (protein kinase B) and protein kinase C in ischemia–reperfusion injury. Neurol Res 38(4):301–308PubMedGoogle Scholar
  13. 13.
    Ma Y et al (2015) Protein kinase cα regulates the expression of complement receptor Ig in human monocyte-derived macrophages. J Immunol 194(6):2855–2861PubMedGoogle Scholar
  14. 14.
    Yu W et al (2017) Fumigaclavine C exhibits anti-inflammatory effects by suppressing high mobility group box protein 1 relocation and release. Eur J Pharmacol 812:234–242PubMedGoogle Scholar
  15. 15.
    Gordon R et al (2016) Protein kinase Cδ upregulation in microglia drives neuroinflammatory responses and dopaminergic neurodegeneration in experimental models of Parkinson's disease. Neurobiol Dis 93:96–114PubMedPubMedCentralGoogle Scholar
  16. 16.
    Sejimo S, Hossain MS, Akashi K (2018) Scallop-derived plasmalogens attenuate the activation of PKCδ associated with the brain inflammation. Biochem Biophys Res Commun 503(2):837–842PubMedGoogle Scholar
  17. 17.
    Yang J et al (2015) Perfluorooctane sulfonate mediates microglial activation and secretion of TNF-α through Ca(2)(+)-dependent PKC-NF-small ka, CyrillicB signaling. Int Immunopharmacol 28(1):52–60PubMedGoogle Scholar
  18. 18.
    Kim DC et al (2005) Effect of rottlerin, a PKC-δ inhibitor, on TLR-4-dependent activation of murine microglia. Biochem Biophys Res Commun 337(1):110–115PubMedGoogle Scholar
  19. 19.
    Santiago AR et al (2014) Role of microglia adenosine A(2A) receptors in retinal and brain neurodegenerative diseases. Mediators Inflamm 2014:465694PubMedPubMedCentralGoogle Scholar
  20. 20.
    Dai SS et al (2010) Local glutamate level dictates adenosine A2A receptor regulation of neuroinflammation and traumatic brain injury. J Neurosci 30(16):5802–5810PubMedPubMedCentralGoogle Scholar
  21. 21.
    Dai SS et al (2013) Plasma glutamate-modulated interaction of A2AR and mGluR5 on BMDCs aggravates traumatic brain injury-induced acute lung injury. J Exp Med 210(4):839–851PubMedPubMedCentralGoogle Scholar
  22. 22.
    Wardas J, Konieczny J, Pietraszek M (2003) Influence of CGS 21680, a selective adenosine A(2A) agonist, on the phencyclidine-induced sensorimotor gating deficit and motor behaviour in rats. Psychopharmacology 168(3):299–306PubMedGoogle Scholar
  23. 23.
    Saura J et al (2005) Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia. J Neurochem 95(4):919–929PubMedGoogle Scholar
  24. 24.
    Papa S et al (2013) Selective nanovector mediated treatment of activated proinflammatory microglia/macrophages in spinal cord injury. ACS Nano 7(11):9881–9895PubMedGoogle Scholar
  25. 25.
    Nakajima K et al (2003) Activation of microglia with lipopolysaccharide leads to the prolonged decrease of conventional protein kinase C activity. Brain Res Mol Brain Res 110(1):92–99PubMedGoogle Scholar
  26. 26.
    Sharma N, Sharma S, Nehru B (2017) Curcumin protects dopaminergic neurons against inflammation-mediated damage and improves motor dysfunction induced by single intranigral lipopolysaccharide injection. Inflammopharmacology 25(3):351–368PubMedGoogle Scholar
  27. 27.
    Jayaprakash K et al (2017) PKC, ERK/p38 MAP kinases and NF-kB targeted signalling play a role in the expression and release of IL-1β and CXCL8 in Porphyromonas gingivalis-infected THP1 cells. APMIS 125(7):623–633PubMedGoogle Scholar
  28. 28.
    Hua KF et al (2012) High glucose increases nitric oxide generation in lipopolysaccharide-activated macrophages by enhancing activity of protein kinase C-α/δ and NF-kB. Inflamm Res 61(10):1107–1116PubMedGoogle Scholar
  29. 29.
    Song XM et al (2017) Aldose reductase inhibitors attenuate β-amyloid-induced TNF-α production in microlgia via ROS-PKC-mediated NF-kB and MAPK pathways. Int Immunopharmacol 50:30–37PubMedGoogle Scholar
  30. 30.
    Shin EJ et al (2016) PKCδ knockout mice are protected from para-methoxymethamphetamine-induced mitochondrial stress and associated neurotoxicity in the striatum of mice. Neurochem Int 100:146–158PubMedGoogle Scholar
  31. 31.
    Hoogland IC et al (2015) Systemic inflammation and microglial activation: systematic review of animal experiments. J Neuroinflammation 12:114PubMedPubMedCentralGoogle Scholar
  32. 32.
    Norden DM et al (2016) Sequential activation of microglia and astrocyte cytokine expression precedes increased Iba-1 or GFAP immunoreactivity following systemic immune challenge. Glia 64(2):300–316PubMedGoogle Scholar
  33. 33.
    Sun X et al (2018) Glycyrrhizin ameliorates inflammatory pain by inhibiting microglial activation-mediated inflammatory response via blockage of the HMGB1-TLR4-NF-kB pathway. Exp Cell Res 369(1):112–119PubMedGoogle Scholar
  34. 34.
    Markoutsa E, Xu P (2017) Redox potential-sensitive N-acetyl cysteine-prodrug nanoparticles inhibit the activation of microglia and improve neuronal survival. Mol Pharm 14(5):1591–1600PubMedPubMedCentralGoogle Scholar
  35. 35.
    Meotti FC et al (2017) The transient receptor potential ankyrin-1 mediates mechanical hyperalgesia induced by the activation of B1 receptor in mice. Biochem Pharmacol 125:75–83PubMedGoogle Scholar
  36. 36.
    Hsia CH et al (2018) Mechanisms of TQ-6, a novel ruthenium-derivative compound, against lipopolysaccharide-induced in vitro macrophage activation and liver injury in experimental mice: the crucial role of p38 MAPK and NF-kB signaling. Cells 7(11):217PubMedCentralGoogle Scholar
  37. 37.
    Muili KA et al (2013) Pancreatic acinar cell nuclear factor kB activation because of bile acid exposure is dependent on calcineurin. J Biol Chem 288(29):21065–21073PubMedPubMedCentralGoogle Scholar
  38. 38.
    Chai W et al (2013) Pyocyanin from Pseudomonas induces IL-8 production through the PKC and NF-kappaB pathways in U937 cells. Mol Med Rep 8(5):1404–1410PubMedGoogle Scholar
  39. 39.
    Shi Y et al (2017) Activated niacin receptor HCA2 inhibits chemoattractant-mediated macrophage migration via Gβγ/PKC/ERK1/2 pathway and heterologous receptor desensitization. Sci Rep 7:42279PubMedPubMedCentralGoogle Scholar
  40. 40.
    Mohanraj M et al (2018) The mycobacterial adjuvant analogue TDB attenuates neuroinflammation via mincle-independent PLC-gamma1/PKC/ERK signaling and microglial polarization. Mol Neurobiol 56(2):1167–1187PubMedGoogle Scholar
  41. 41.
    Kontny E et al (2000) Rottlerin, a PKC isozyme-selective inhibitor, affects signaling events and cytokine production in human monocytes. J Leukoc Biol 67(2):249–258PubMedGoogle Scholar
  42. 42.
    Pham TH et al (2017) Fargesin exerts anti-inflammatory effects in THP-1 monocytes by suppressing PKC-dependent AP-1 and NF-kB signaling. Phytomedicine 24:96–103PubMedGoogle Scholar
  43. 43.
    He X et al (2012) Inhibitory effect of Astragalus polysaccharides on lipopolysaccharide-induced TNF-a and IL-1β production in THP-1 cells. Molecules 17(3):3155–3164PubMedPubMedCentralGoogle Scholar
  44. 44.
    Talwar H et al (2017) MKP-1 negatively regulates LPS-mediated IL-1β production through p38 activation and HIF-1α expression. Cell Signal 34:1–10PubMedPubMedCentralGoogle Scholar
  45. 45.
    Gomes CV et al (2011) Adenosine receptors and brain diseases: neuroprotection and neurodegeneration. Biochim Biophys Acta 1808(5):1380–1399PubMedGoogle Scholar
  46. 46.
    Madeira MH et al (2015) Adenosine A2AR blockade prevents neuroinflammation-induced death of retinal ganglion cells caused by elevated pressure. J Neuroinflammation 12:115PubMedPubMedCentralGoogle Scholar
  47. 47.
    Borroto-Escuela DO et al (2018) Understanding the role of adenosine A2AR heteroreceptor complexes in neurodegeneration and neuroinflammation. Front Neurosci 12:43PubMedPubMedCentralGoogle Scholar
  48. 48.
    Chiu GS et al (2014) Adenosine through the A2A adenosine receptor increases IL-1β in the brain contributing to anxiety. Brain Behav Immun 41:218–231PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.The Molecular Biology Center, State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping HospitalThird Military Medical UniversityChongqingChina

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