Effect of the Antimicrobial Peptide LL-37 on Gene Expression of Chemokines and 29 Toll-like Receptor-Associated Proteins in Human Gingival Fibroblasts Under Stimulation with Porphyromonas gingivalis Lipopolysaccharide

The antimicrobial peptide LL-37 neutralizes the biological activity of lipopolysaccharide (LPS), while it upregulates the expression of several immune-related genes. We investigated the effect of LL-37 on gene regulation of human gingival fibroblasts (HGFs), stimulated with or without Porphyromonas gingivalis-derived LPS, a ligand for Toll-like receptor (TLR). LL-37 was non-toxic to HGFs up to a concentration of 10 μg/ml. P. gingivalis LPS upregulated the expression of IL8, CXCL10, and CCL2, whereas LL-37 reduced this upregulation. In absence of LPS, LL-37 itself upregulated the expression of IL8 and CCL2. LL-37 increased the expression of P2X7, which was constitutively expressed in HGFs. The P2X7 antagonist A-438079 suppressed the cytotoxicity and upregulatory effect of LL-37 on chemokine response, but not its downregulatory effect on P. gingivalis LPS–induced chemokine response. Whether LL-37 alters the expression of 29 genes that encode TLR-associated proteins, including TLRs, co-receptors, signaling molecules, and negative regulators, in HGFs, under stimulation with LPS, was examined. Among TLRs, P. gingivalis LPS upregulated the level of TLR4, whereas LL-37 reduced it. In co-receptors, LL-37 downregulated the level of CD14. Among signaling molecules, LL-37 augmented the LPS-upregulated expression of IRAK1. Similar effects were observed in the specific negative regulators TNFAIP3, RNF216, TOLLIP, and SIGIRR. Our results suggest that LL-37 exerts cytotoxicity and upregulation of chemokine response via the P2X7 receptor, while it induces downregulation of P. gingivalis LPS–induced chemokine response through alteration in the expression of 7 specific TLR-associated genes: downregulation of TLR4 and CD14 and upregulation of IRAK1, TNFAIP3, RNF216, TOLLIP, and SIGIRR.