Microglial TLR4-induced TAK1 phosphorylation and NLRP3 activation mediates neuroinflammation and contributes to chronic morphine-induced antinociceptive tolerance

Pharmacol Res. 2021 Mar:165:105482. doi: 10.1016/j.phrs.2021.105482. Epub 2021 Feb 5.

Abstract

Background and purpose: The aim of this work was to investigate the role and signal transduction of toll-like receptor 4 (TLR4), TGF-β-activated kinase 1 (TAK1) and nod-like receptor protein 3 (NLRP3) in microglial in the development of morphine-induced antinociceptive tolerance.

Methods: TLR4 and NLRP3 knockout mice and 5Z-7-oxozeaeno (a selective inhibitor against TAK1 activity) were used to observe their effect on the development of morphine tolerance. Intrathecal injections of morphine (0.75 mg/kg once daily for 7 days) were used to establish anti-nociceptive tolerance, which was measured by the tail-flick test. Spinal TLR4, TAK1, and NLRP3 expression levels and phosphorylation of TAK1 were evaluated by Western blotting and immunofluorescence.

Results: Repeated treatment with morphine increased total expression of spinal TLR4, TAK1, and NLRP3 and phosphorylation of TAK1 in wild-type mice. TLR4 knockout attenuated morphine-induced tolerance and inhibited the chronic morphine-induced increase in NLRP3 and phosphorylation of TAK1. Compared with controls, mice that received 5Z-7-oxozeaenol showed decreased development of morphine tolerance and inhibition on repeated morphine-induced increase of NLRP3 but not TLR4. NLRP3 knockout mice showed resistance to morphine-induced analgesic tolerance with no effect on chronic morphine-induced expression of TLR4 and TAK1. TLR4, TAK1, and NLRP3 were collectively co-localized together and with the microglia marker Iba1.

Conclusions: Microglial TLR4 regulates TAK1 expression and phosphorylation and results in NLRP3 activation contributes to the development of morphine tolerance through regulating neuroinflammation. Targeting TLR4-TAK1-NLRP3 signaling to regulate neuro-inflammation will be alternative therapeutics and strategies for chronic morphine-induced antinociceptive tolerance.

Keywords: 5Z-7-oxozeaeno (PubChem CID: 9863776); DAPI dihydrochloride(PubChem CID:160166); Dimethyl sulfoxide (PubChem CID: 679); Formaldehyde (PubChem CID: 712); Morphine (PubChem CID: 5288826); Morphine tolerance; Neuroinflammation; Nod-like receptor protein 3, NLRP3; Pentobarbital (PubChem CID: 4737); TAK1, TGF-β-activated kinase 1; TLR4, toll-like receptor 4.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Analgesics, Opioid / pharmacology
  • Animals
  • Drug Tolerance* / physiology
  • Inflammation Mediators* / antagonists & inhibitors
  • Inflammation Mediators* / metabolism
  • MAP Kinase Kinase Kinases / genetics
  • MAP Kinase Kinase Kinases / metabolism
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Microglia / drug effects
  • Microglia / metabolism
  • Morphine* / pharmacology
  • NLR Family, Pyrin Domain-Containing 3 Protein* / genetics
  • NLR Family, Pyrin Domain-Containing 3 Protein* / metabolism
  • Pain Measurement / drug effects
  • Pain Measurement / methods
  • Phosphorylation / drug effects
  • Phosphorylation / physiology
  • Toll-Like Receptor 4* / deficiency
  • Toll-Like Receptor 4* / genetics

Substances

  • Analgesics, Opioid
  • Inflammation Mediators
  • MAP kinase kinase kinase 7
  • MAP Kinase Kinase Kinases
  • Morphine
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • Nlrp3 protein, mouse
  • Tlr4 protein, mouse
  • Toll-Like Receptor 4