SPA4 peptide suppresses the lipopolysaccharide (LPS)-stimulated lactate secretion in lung epithelial cells through modulation of Toll-like receptor-4 (TLR4)

Abstract

Abstract Lung infection and inflammation can cause severe injury leading to acute respiratory distress syndrome. An increased expression and activity of the TLR4 is associated with inflammatory response. Simultaneous elevated level of lactate is used as the marker of disease severity and is associated with poor outcomes in critically ill patients. Therapeutic treatment with TLR4-interacting SPA4 peptide (amino acid sequence: GDFRYSDGTPVNYTNWYRGE) suppresses inflammation in immune cells and in mouse models of lung infection and inflammation. In this study, we evaluated the effect of SPA4 peptide on lactate production by H441 human lung epithelial cells against Pseudomonas aeruginosa-derived LPS. Briefly, the H441 cells were challenged with LPS (1 μg/ml) and treated with SPA4 peptide (100 μM) after 1 h of LPS challenge. Next day, the cell-free supernatants and cell lysates were harvested. We also transfected the cells with TLR4 siRNA to achieve suppression of TLR4. Levels of protein and lactate were quantitated in cell lysates and in filtered supernatants using biochemical assays. The lactate levels were normalized with cell protein. The unchallenged, vehicle-treated cells served as control. Our results demonstrated reduced secretion of lactate by SPA4 peptide-treated cells (187.5% of control) versus vehicle-treated cells (324.7% of control) against LPS stimuli (p<0.05). The immunostaining and confocal imaging showed knockdown of TLR4 in up to 50% cells. The LPS-stimulated lactate secretion was reduced and SPA4 peptide treatment did not further affect the lactate levels in TLR4 siRNA transfected cells. Together, our results suggest that the SPA4 peptide suppresses lactate secretion through modulation of TLR4 in lung epithelial cells. Supported by grant from NIH (R01HL136325) and 2021 American Association of Immunologists Careers in Immunology Fellowship Program.