Role of ASM/Cer/TXNIP signaling module in the NLRP3 inflammasome activation

Lipids Health Dis. 2021 Feb 21;20(1):19. doi: 10.1186/s12944-021-01446-4.

Abstract

Background: This study aimed to explore the effects of ceramide (Cer) on NLRP3 inflammasome activation and their underlying mechanisms.

Methods: Lipopolysaccharide (LPS)/adenosine triphosphate (ATP)-induced NLRP3 inflammasome activation in J774A.1 cells and THP-1 macrophages was used as an in vitro model of inflammation. Western blotting and real-time PCR (RT-PCR) were used to detect the protein and mRNA levels, respectively. IL-1β and IL-18 levels were measured by ELISA. ASM assay kit and immunofluorescence were used to detect ASM activity and Cer content.

Results: Imipramine, a well-known inhibitor of ASM, significantly inhibited LPS/ATP-induced activity of ASM and the consequent accumulation of Cer. Additionally, imipramine suppressed the LPS/ATP-induced expression of thioredoxin interacting protein (TXNIP), NLRP3, caspase-1, IL-1β, and IL-18 at the protein and mRNA level. Interestingly verapamil, a TXNIP inhibitor, suppressed LPS/ATP-induced activation of TXNIP/NLRP3 inflammasome but did not affect LPS/ATP-induced ASM activation and Cer formation. TXNIP siRNA and verapamil inhibited C2-Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome. In addition, the pretreatment of cells with sulfo-N-succinimidyl oleate (SSO), an irreversible inhibitor of the scavenger receptor CD36, blocked Cer-induced upregulation of nuclear factor-κB (NF-κB) activity, TXNIP expression, and NLRP3 inflammasome activation. Inhibition of NF-κB activation by SN50 prevented Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome but did not affect CD36 expression.

Conclusion: This study demonstrated that the ASM/Cer/TXNIP signaling pathway is involved in NLRP3 inflammasome activation. The results documented that the CD36-dependent NF-κB-TXNIP signaling pathway plays an essential role in the Cer-induced activation of NLRP3 inflammasomes in macrophages.

Keywords: Acid sphingomyelinase; Ceramide; J774A.1 cells; Lipopolysaccharide; NOD-like receptor Protein3; THP-1 macrophages; Thioredoxin interacting protein.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Carrier Proteins / genetics*
  • Caspase 1 / genetics
  • Ceramides / genetics*
  • Gene Expression Regulation / genetics
  • Humans
  • Inflammasomes / genetics
  • Inflammation / genetics*
  • Inflammation / metabolism
  • Inflammation / pathology
  • Interleukin-18 / genetics
  • Interleukin-1beta / genetics
  • Lipopolysaccharides / pharmacology
  • Macrophages / drug effects
  • NF-kappa B / antagonists & inhibitors
  • NF-kappa B / genetics
  • NLR Family, Pyrin Domain-Containing 3 Protein / genetics*
  • Oleic Acids / pharmacology
  • Peptides / pharmacology
  • Signal Transduction / drug effects
  • Sphingomyelin Phosphodiesterase / genetics
  • Succinimides / pharmacology
  • Verapamil / pharmacology

Substances

  • Carrier Proteins
  • Ceramides
  • IL1B protein, human
  • Inflammasomes
  • Interleukin-18
  • Interleukin-1beta
  • Lipopolysaccharides
  • NF-kappa B
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • Oleic Acids
  • Peptides
  • SN50 peptide
  • Succinimides
  • TXNIP protein, human
  • sulfo-N-succinimidyl oleate
  • Adenosine Triphosphate
  • Verapamil
  • Sphingomyelin Phosphodiesterase
  • Caspase 1