A TLR4-interacting SPA4 peptide inhibits LPS-induced lung inflammation

Abstract

The interaction between surfactant protein-A (SP-A) and TLR4 is important for host defense. We have recently identified an SPA4 peptide region from the interface of SP-A-TLR4 complex. Here, we studied the involvement of the SPA4 peptide region in SP-A-TLR4 interaction using a two-hybrid system, and biological effects of SPA4 peptide in cell systems and a mouse model. HEK293 cells were transfected with plasmid DNAs encoding SP-A or a SP-A-mutant lacking SPA4 peptide region and TLR4. Luciferase activity was measured as the end-point of SP-A-TLR4 interaction. NF-κB activity was also assessed simultaneously. Next, the dendritic cells or mice were challenged with Escherichia coli-derived LPS and treated with SPA4 peptide. Endotoxic shock-like symptoms and inflammatory parameters (TNF-α, NF-κB, leukocyte influx) were assessed. Our results reveal that the SPA4 peptide region contributes to the SP-A-TLR4 interaction and inhibits the LPS-induced NF-κB activity and TNF-α. We also observed that the SPA4 peptide inhibits LPS-induced expression of TNF-α, nuclear localization of NF-κB-p65 and cell influx, and alleviates the endotoxic shock-like symptoms in a mouse model. Our results suggest that the anti-inflammatory activity of the SPA4 peptide through its binding to TLR4 can be of therapeutic benefit.

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

“…Mouse bone marrow-derived JAWSII dendritic cells (1 3 10 6 cells) were transfected with 2 mg of each plasmid construct encoding WTTLR4 or TLR4DN (Pro at position 712 substituted with His), using TransIT-TKO transfection reagent (Mirus Bio, Madison, WI, USA) per the previously published method [16,19]. The plasmid DNA constructs were obtained from Dr. Lynn Hajjar (University of Washington, Seattle, WA, USA).…”

“…Furthermore, upon treatment of LPS-and ATP-challenged cells with SPA4 peptide, the mRNA and cellular protein levels of NLRP3 and pro-IL-1b, NLRP3 inflammasome formation, caspase activity, and secreted levels of IL-1b are significantly reduced. Together, our results indicate that the suppression of LPS-TLR4 signaling by SPA4 peptide [14][15][16] further reduces the NLRP3 inflammasome and alleviates inflammatory response.…”

“…Our results demonstrated that the SPA4 peptide binds to TLR4 and suppresses NF-kB and the inflammatory response against gramnegative bacterial LPS (a TLR4 ligand) in cell systems [14][15][16] and in a mouse model of endotoxic shock-induced lung inflammation [16]. Results from cells transfected with the dominant-negative construct of MYD88 demonstrated that the SPA4 peptide suppresses TLR4-MYD88-dependent NF-kB activity and TNF-a cytokine [16]. In this study, we hypothesized that the SPA4 peptide would interfere with the crosstalk between TLR4 signaling and the NLRP3 inflammasome to suppress an exaggerated inflammatory response in cells challenged with LPS and ATP.…”

Eosinophil-Associated Lung Diseases. A Cry for Surfactant Proteins A and D Help?

“…In both cases, exogenous given SP-A led to "rescue" of the innate and adaptive immune phenotypes observed in SP-A-deficient mice (134)(135)(136)(137). More recently, Awasthi and colleagues has found that a specific fragment of SP-A (20-mer) has the ability to inhibit LPS-induced cytokine production and lung inflammation by interaction with TLR-4 (138). This finding suggests the possibility of other SP-A fragments that may be active against a specific receptor target for which SP-A is known to associate, such as TLR-2, SIRP-a, and CD91.…”

Anticoagulant modulation of inflammation in severe sepsis

“…TLR4 on platelets recognizes PAMPs of pathogens specifically LPS of gram negative bacteria. These receptors also recognize endogenous DAMPs which include heat shock proteins and fibronectin [21] . Platelets are further able to present microorganisms to neutrophils other immune cells, activating a portion of the innate immune system.…”