Integrated lncRNA and mRNA analysis reveals the immune modulatory mechanisms of antimicrobial peptide BSN-37 in mouse peritoneal macrophages

Abstract

Antimicrobial peptides (AMPs) possess vaccine adjuvant activity; however, their specific targets and molecular mechanisms remain incompletely understood, which hinders their clinical application. This study aimed to elucidate the key targets and pathways through which the antimicrobial peptide BSN-37 modulates immune responses in macrophages, providing evidence for its potential clinical translation. In this investigation, Balb/c mice were administered BSN-37 for 12 h, after which total RNA was extracted from peritoneal macrophages to assess the mRNA expression levels of cytokines and key molecules on the cell surface, followed by transcriptomic sequencing. The results demonstrated that BSN-37 significantly upregulated the mRNA expression of these molecules and cytokines. A total of 228 differentially expressed long non-coding RNAs (lncRNAs) (121 upregulated, 107 downregulated) and 149 differentially expressed mRNAs (104 upregulated, 45 downregulated) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed significant enrichment of differentially expressed mRNAs in immune response pathways, PI3K-Akt signaling, and NOD-like receptor signaling. Differentially expressed lncRNA target genes were associated with T cell receptor signaling, PD-1 checkpoint regulation, and other immune regulatory pathways. Protein-protein interaction network analysis identified core genes such as CCchemokine receptor 1 (CCR1) and Toll Like Receptor 8 (TLR8). Molecular docking studies confirmed that BSN-37 exhibited strong binding affinity to TLR8 and CCR1, with binding energies less than - 5 kcal/mol. RT-qPCR validation confirmed the reliability of the sequencing data. These findings indicate that BSN-37 activates multiple immune response pathways in macrophages by targeting immune-related genes such as TLR8 and CCR1, offering theoretical support for the development of novel immune adjuvants.

Keywords: Adjuvants; Antimicrobial peptide; Long non-coding RNA; Macrophages.

Fig. 1

A flow chart for the procedures done within the current study.

Fig. 2

Detection of the expression of co-stimulatory molecules (A) and cytokines (B) by RT-qPCR.

Fig. 3

Analysis of DE lncRNA and mRNA in peritoneal macrophages stimulated by BSN-37. (A) Venn diagram of lncRNA; (B) Type of lncRNA; (C) Cluster heatmap of DE lncRNA; (D) Cluster heatmap of DE mRNA; (E) Volcano map of DE lncRNA; (F) Volcano map of DE mRNA.

Fig. 4

Characteristic analysis of mRNA and lncRNA. (A) Length analysis of lncRNA. (B) Length analysis of mRNA. (C) Exon number analysis of lncRNA. (D) Exon number analysis of mRNA. (E) ORF length analysis of lncRNA. (F) ORF length analysis of mRNA. (G) Variable shear isomer comparison diagram of lncRNA and mRNA. (H) Chromosome distribution ring diagram of DE lncRNA and DE mRNA.

Fig. 5

GO enrichment analysis of the target genes of the DE lncRNAs (A–C) and the DE mRNAs (D–I).

Fig. 6

KEGG enrichment analysis of the target genes of the DE mRNAs (A) and the DE lncRNAs (B,C).

Fig. 7

Validation of the expression of DE mRNA (A,B) and lncRNA (C,D) by RT-qPCR. The calculated data were processed and analyzed by GraphPad Prism software, P < 0.05*, P < 0.01**, P < 0.001***, P < 0.0001****, statistically significant.

Fig. 8

Protein interaction network of differentially expressed mRNA (A) and core gene screening (B). (A) DE mRNA interaction network diagram. (B) PPI network diagram.

Fig. 9

Molecular docking pattern between BSN-37 and core targets. (A) to (B) represented the binding of BSN-37 with CCR1, TLR8, respectively. The receptor protein is represented in green, the polypeptide ligand in purple, the rod-like structure of the receptor protein docking site in orange, and the rod-like structure of the BSN-37 docking site in blue.

Fig. 10

Proposed immune modulatory mechanisms of antimicrobial peptide BSN-37 on peritoneal macrophages in mice.