title: 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.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Vijay
authors.initials: V
authors.lastname: Ramani
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA Shanjana-Awasthi@ouhsc.edu.
authors.forename: Shanjana
authors.initials: S
authors.lastname: Awasthi
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002352:Carrier Proteins / Q000276:immunology*; D002460:Cell Line; D005260:Female; D058847:Inflammasomes / Q000276:immunology*; D053583:Interleukin-1beta / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D010446:Peptide Fragments / Q000494:pharmacology*; D012333:RNA, Messenger / Q000276:immunology; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000276:immunology*
chemical_list: D002352:Carrier Proteins; C587672:IL1B protein, mouse; D058847:Inflammasomes; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C492716:Nlrp3 protein, mouse; D010446:Peptide Fragments; D012333:RNA, Messenger; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D000255:Adenosine Triphosphate
keywords: anti-inflammatory activity; immunomodulation; surfactant protein A