Neutrophil-derived lactoferrin induces the inflammatory responses of rheumatoid arthritis synovial fibroblasts via Toll-like receptor 4.

Damage-associated molecular patterns (DAMPs) are proposed to drive aberrant stimulation of Toll-like receptors (TLRs) in rheumatoid arthritis (RA) inflamed joints. In the current study we investigated the role of the neutrophil-derived lactoferrin (LTF), as an endogenous ligand for TLR4 in the inflammatory response of RA synovial fibroblasts (RASFs). RASFs were stimulated with LTF, and the expressions of inflammatory cytokines in RASFs were measured. To clarify the TLR4 signalling pathway associated with LTF stimulation, a small molecular inhibitor of TLR4 (TAK242) and NF-κB inhibitor were used. The role of nuclear factor of activated T cells 5 (NFAT5) was identified using small interfering RNA. To reveal the interaction between NF-κB and NFAT5, cerulenin, which disrupts their interaction, was used. Stimulation of RASFs with LTF significantly increased the expressions of inflammatory cytokines and chemokines, such as IL-6, CCL20 and IL-8, in RASFs. LTF enhanced the mRNA expressions of these cytokines in RASFs stimulated by TNF-α. TAK242 almost completely inhibited the expressions of inflammatory cytokines and chemokines in RASFs stimulated by LTF. The NF-κB inhibitor partially repressed the expressions of IL-6 and IL-8 mRNAs induced by LTF, but not CCL20 mRNA expression. On the other hand, NFAT5 silencing decreased the expressions of CCL20 and IL-8 mRNAs induced by LTF, but not IL-6 mRNA expression. Cerulenin repressed the expressions of IL-6, CCL20 and IL-8 in RASFs stimulated by LTF. Neutrophil-derived LTF may play a role as an endogenous ligand for TLR4 expressed on RASFs. NFAT5-NF-κB enhanceosome might regulate the expressions of LTF-TLR4-responsive genes in RASFs.