Toll-like receptor 4-interacting SPA4 peptide suppresses the NLRP3 inflammasome in response to LPS and ATP stimuli

Abstract

Inflammation is induced because of interplay among multiple signaling pathways and molecules during infectious and noninfectious tissue injuries. Crosstalk between Toll-like receptor-4 signaling and the neuronal apoptosis inhibitor protein, major histocompatibility class 2 transcription activator, incompatibility locus protein from Podospora anserina, and telomerase-associated protein (NACHT), leucine-rich repeat (LRR), and pyrin domain-containing protein 3 (NLRP3) inflammasome against pathogen- or damage-associated molecular patterns can cause exaggerated inflammation. We previously established that the Toll-like receptor-4-interacting SPA4 peptide suppresses gram-negative bacterial lipopolysaccharide (Toll-like receptor-4 ligand)-induced nuclear factor-κB and inflammatory response. In the present study, we hypothesized that the SPA4 peptide exerts its anti-inflammatory effects by suppressing the crosstalk between Toll-like receptor-4 signaling and the NLRP3 inflammasome. We evaluated binding of the lipopolysaccharide-ligand to cell-surface Toll-like receptor-4 in the presence or absence of adenosine triphosphate (an NLRP3 inflammasome inducer) by flow cytometry. The expression and activity of NLRP3 inflammasome-related parameters were studied in cells challenged with lipopolysaccharide and adenosine triphosphate using molecular and immunologic methods. The cells were challenged with lipopolysaccharide and treated with SPA4 peptide before (pre-adenosine triphosphate) or after (post-adenosine triphosphate) secondary challenge with adenosine triphosphate. Our data demonstrate that the Toll-like receptor-4-interacting SPA4 peptide does not affect the binding of lipopolysaccharide to Toll-like receptor-4 in the presence or absence of adenosine triphosphate. We also found that the SPA4 peptide inhibits mRNA and cellular protein levels of pro-interleukin-1β and NLRP3, formation of the NLRP3 inflammasome, caspase activity, and release of interleukin-1β. Furthermore, the SPA4 peptide treatment reduced the secreted levels of interleukin-1β from cells overexpressing Toll-like receptor-4 compared with cells expressing the dominant-negative form of Toll-like receptor-4. Together our results suggest that the SPA4 peptide exerts its anti-inflammatory activity by suppressing Toll-like receptor-4-priming of the NLRP3 inflammasome