Effects of SPA4 peptide on lipopolysaccharide‐disrupted lung epithelial barrier, injury, and function in a human cell system and mouse model of lung injury

Abstract

Disrupted epithelial barrier, fluid accumulation, inflammation, and compromised physiology are hallmarks of lung injury. Here we investigated the structural stability of the Toll‐like receptor‐4 (TLR4)‐interacting SPA4 peptide, its effect on Pseudomonas aeruginosa lipopolysaccharide (LPS)‐disrupted epithelial barrier in a human cell system, and lung injury markers in a mouse model of LPS‐induced lung inflammation. The structural properties of SPA4 peptide were investigated using circular dichroism and UV–VIS spectroscopy. The transepithelial electrical resistance (TEER), an indicator of barrier function, was measured after the cells were challenged with 1 μg/ml LPS and treated with 10 or 100 μM SPA4 peptide. The expression and localization of tight junction proteins were studied by immunoblotting and immunocytochemistry, respectively. Mice were intratracheally challenged with 5 μg LPS per g body weight and treated with 50 μg SPA4 peptide. The lung wet/dry weight ratios or edema, surfactant protein‐D (SP‐D) levels in serum, lung function, tissue injury, body weights, and temperature, and survival were determined as study parameters. The spectroscopy results demonstrated that the structure was maintained among different batches of SPA4 peptide throughout the study. Treatment with 100 μM SPA4 peptide restored the LPS‐disrupted epithelial barrier, which correlated with the localization pattern of Zonula Occludens (ZO)‐1 and occludin proteins. Correspondingly, SPA4 peptide treatment helped suppress the lung edema and levels of serum SP‐D, improved some of the lung function parameters, and reduced the mortality risk against LPS challenge. Our results suggest that the anti‐inflammatory activity of the SPA4 peptide facilitates the resolution of lung pathology.

Stability and structure–activity relationship of the SPA4 peptide under ambient and stressed conditions of lung injury

“…3) as described previously. 5,27 Lower absorbance readings were noted between 200–250 nm wavelengths for the SPA4 peptide maintained in respective buffer solutions at pH 3.0 and 10.0. Similarly, the shifts in peaks were noted in the fourth-derivative spectra.…”

“…10C) compared to that in water as published earlier. 5,27,33 However, low absorbance readings between 200-220 nm wavelengths suggest an effect on peptide backbone of SPA4 peptide in the presence of 500 mM sodium lactate (Fig. 10B).…”

“…The UV-VIS spectroscopy was used to determine any changes in primary structure of the SPA4 peptide as described earlier. 5,27 Briey, an aliquot of 2 ml was loaded onto a well of microvolume glass slide plate (Take3 plate, Agilent, Santa Clara, CA). The UV-VIS absorbance readings or optical density (OD) values were recorded at 200-750 nm wavelengths with an interval of 5 nm and optical pathlength of 0.5 mm.…”

“…2,3 Intratracheal administration of the SPA4 peptide suppresses inammation and injury and improves the pulmonary function parameters and survival in mouse models of lung infection and inammation. [3][4][5] Although therapeutic administration of SPA4 peptide has been effective in lung inammation models, it is likely to get exposed to varying stressed conditions in respiratory tract. The lung microenvironment undergoes signicant changes during inammation.…”

“…2,3 Intratracheal administration of the SPA4 peptide suppresses inflammation and injury and improves the pulmonary function parameters and survival in mouse models of lung infection and inflammation. 3–5…”