Researchers first tested the peptide for safety using MTT assays across three cell types: RAW 264.7 macrophages, normal human dermal fibroblasts, and HaCaT keratinocytes. IL15.3 demonstrated low cytotoxicity in all of them, an important early signal that the molecule could be compatible with living tissue.

The team then challenged RAW 264.7 macrophages with lipopolysaccharide (LPS), a bacterial molecule that triggers strong inflammatory responses. Treating these cells with IL15.3 significantly reduced the production of nitric oxide and reactive oxygen species in a dose-dependent manner, and it did so without compromising cell viability.

Gene expression analysis by quantitative RT-PCR revealed that the peptide significantly suppressed the mRNA of iNOS, COX-2, TNF-α, and IL-6, all central drivers of inflammation. It also downregulated NF-κB, JNK, STAT1, and STAT3, key signaling components, while shifting the balance of Keap1 and Nrf2 expression in a way suggestive of possible antioxidant pathway activation.

To probe the mechanism more deeply, the researchers performed label-free proteomic profiling, identifying 5,191 proteins in the treated cells. The results pointed to a multi-modal mechanism of action rather than a single molecular target.

Among the proteomic changes, IL15.3 treatment exclusively induced four anti-inflammatory proteins: ISG15, PEX14, TNIP3, and PR. It also upregulated negative regulators of inflammation, including NRG1, DAB2IP, IL-18BP, and SOCS5, effectively strengthening the cell’s own braking systems.

At the same time, the peptide suppressed key pro-inflammatory mediators such as IKKβ, JAK1, JAK3, JNK, and IL-1RAcP. Taken together, these changes demonstrate comprehensive modulation of the NF-κB, JAK/STAT, and MAPK pathways, three of the most important signaling networks controlling inflammatory responses.

The researchers also ran in silico molecular docking simulations, which predicted potential interactions between IL15.3 and both iNOS and the Kelch domain of Keap1. The authors emphasize that these computational results offer only preliminary structural insight, but they are consistent with the peptide’s putative dual anti-inflammatory and antioxidant activity.

Overall, the study indicates that IL15.3 exhibits anti-inflammatory and antioxidant activity through multiple mechanisms in LPS-stimulated macrophages, acting at both the transcriptional and proteomic levels. The coordinated upregulation of negative regulators alongside suppression of central inflammatory mediators suggests the peptide may represent a promising candidate for treating inflammatory disorders, though the evidence so far comes entirely from cell culture experiments and will need validation in more complex biological systems.

Subject of Research: Anti-inflammatory activity of the synthetic peptide IL15.3 in LPS-stimulated macrophages

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

Article References: Boonmark, C., Roytrakul, S., Saengyotha, F., Tankrathok, A., Wanthong, A., Daduang, J., Daduang, S., Chaveerach, A., & Jangpromma, N. (2026). IL15.3, a synthetic antimicrobial peptide, attenuates LPS-induced inflammatory responses in RAW 264.7 macrophages via transcriptional and proteomic modulation. PLOS One, 21(10), e0361031. https://doi.org/10.1371/journal.pone.0361031

Image Credits: AI Generated

DOI: 10.1371/journal.pone.0361031

Keywords: IL15.3, antimicrobial peptide, anti-inflammatory, macrophages, NF-κB, JAK/STAT, MAPK, nitric oxide, proteomics, Nrf2, LPS, in vitro