Modulation of proinflammatory activity by the engineered cationic antimicrobial peptide WLBU-2

Background: Host-derived (LL-37) and synthetic (WLBU-2) cationic antimicrobial peptides (CAPs) are known for their membrane-active bactericidal properties. LL-37 is an important mediator for immunomodulation, while the mechanism of action of WLBU-2 remains unclear. Objective: To determine if WLBU-2 induces an early proinflammatory response that facilitates bacterial clearance in cystic fibrosis (CF). Methods: C57BL6 mice were given intranasal or intraperitoneal 1×10 (6) cfu/mL Pseudomonas aeruginosa (PA) and observed for 2h, followed by instillation of LL-37 or WLBU-2 (2-4mg/kg) with subsequent tissue collection at 24h for determination of bacterial colony counts and quantitative RT-PCR measurement of cytokine transcripts. CF airway epithelial cells (IB3-1, ΔF508/W1282X) were cultured in appropriate media with supplements. WLBU-2 (25μM) was added to the media with RT-PCR measurement of TNF-α and IL-1β transcripts after 20, 30, and 60min. Flow cytometry was used to determine if WLBU-2 assists in cellular uptake of Alexa 488-labeled LPS. Results: In murine lung exposed to intranasal or intraperitoneal WLBU-2, there was a reduction in the number of surviving PA colonies compared to controls. Murine lung exposed to intraperitoneal WLBU-2 showed fewer PA colonies compared to LL-37. After 24h WLBU-2 exposure, PA-induced IL-1β transcripts from lungs showed a twofold decrease (p<0.05), while TNF-α levels were unchanged. LL-37 did not significantly change transcript levels. In IB3-1 cells, WLBU-2 exposure resulted in increased TNF-α and IL-1β transcripts that decreased by 60min. WLBU-2 treatment of IB3-1 cells displayed increased LPS uptake, suggesting a potential role for CAPs in inducing protective proinflammatory responses. Taken together, the cytokine response, LPS uptake, and established antimicrobial activity of WLBU-2 demonstrate its ability to modulate proinflammatory signaling as a protective mechanism to clear infection. Conclusions: The immunomodulatory properties of WLBU-2 reveal a potential mechanism of its broad-spectrum antibacterial activity and warrant further preclinical evaluation to study bacterial clearance and rescue of chronic inflammation.