IL15.3, a synthetic antimicrobial peptide, attenuates LPS-induced inflammatory responses in RAW 264.7 macrophages via transcriptional and proteomic modulation

Inflammation underlies numerous pathological conditions, driving the need for safe and effective anti-inflammatory agents. Anti-inflammatory peptides (AIPs) are promising therapeutics due to their specificity, efficacy, and safety. This study evaluated the cationic antimicrobial peptide IL15.3 for anti-inflammatory activity and cytocompatibility in vitro. IL15.3 demonstrated low cytotoxicity in MTT assays using RAW 264.7 macrophages, normal human dermal fibroblasts (NHDF), and HaCaT keratinocytes. In LPS-stimulated RAW 264.7 cells, IL15.3 significantly reduced nitric oxide (NO) production and reactive oxygen species (ROS) in a dose-dependent manner without affecting cell viability. qRT-PCR analysis revealed significant suppression of iNOS, COX-2, TNF-α, and IL-6 mRNA expression, alongside downregulation of NF-κB, JNK, STAT1, and STAT3, and a reciprocal shift in Keap1/Nrf2 expression suggestive of possible antioxidant pathway activation. Label-free proteomic profiling identified 5,191 proteins, revealing a multi-modal mechanism of action. IL15.3 treatment exclusively induced 4 anti-inflammatory proteins (ISG15, PEX14, TNIP3, PR) and upregulated negative regulators NRG1, DAB2IP, IL-18BP and SOCS5. Critically, IL15.3 suppressed key pro-inflammatory mediators including IKKβ, JAK1, JAK3, JNK, and IL-1RAcP, demonstrating comprehensive modulation of NF-κB, JAK/STAT, and MAPK pathways. In silico molecular docking further predicted potential interactions of IL15.3 with iNOS and the Keap1 Kelch domain, offering preliminary structural insight consistent with its putative dual anti-inflammatory and antioxidant activity. Together, these findings indicate that IL15.3 exhibits potent anti-inflammatory and antioxidant activity through multiple mechanisms in LPS-stimulated macrophages at transcriptional and proteomic levels. The coordinated upregulation of negative regulators and suppression of central inflammatory mediators collectively suggest that IL15.3 may represent a promising therapeutic candidate for inflammatory disorders.