High-mobility group box 1: A novel target for treatment of Pseudomonas aeruginosa keratitis

摘要

High mobility group box 1 (HMGB1), a prototypic alarmin, mediates the systemic inflammatory response syndrome. Treatment with vasoactive intestinal peptide (VIP), an anti-inflammatory neuropeptide, down-regulates pro-inflammatory cytokines and promotes healing in a susceptible (cornea perforates) model of Pseudomonas (P.) aeruginosa keratitis, also significantly down-regulates HMGB1 expression. Therefore, we examined targeting HMGB1 for treatment of P. aeruginosa keratitis to avoid delivery and other issues associated with VIP. For this, HMGB1 was silenced using siRNA, while controls were treated with a non-specific scrambled sequence siRNA. Less disease was seen after infection in siHMGB1 over control mice and was documented by clinical score and photographs with a slit lamp. Real time RT-PCR and ELISA confirmed HMGB1 knockdown. RT-PCR analysis also revealed reduced mRNA levels of IL-1β, MIP-2, TNF-α, TLR4, and receptor for advanced glycation end products (RAGE), while mRNA levels of anti-inflammatory TLRs SIGIRR and ST2 were significantly increased. HMGB1 knockdown also decreased IL-1β and MIP-2 proteins, reducing PMN number in the infected cornea. mRNA and protein levels of CXCL12 and CXCR4, as well as mononuclear cells, were significantly reduced after HMGB1 knockdown. Antibody neutralization of HMGB1, infection with a clinical isolate and rHMGB1 treatment of resistant mice, supported the silencing studies. These data provide evidence that silencing HMGB1 promotes better resolution of P. aeruginosa keratitis by decreasing levels of pro-inflammatory mediators, (decreasing PMN infiltration), increasing anti-inflammatory TLRs, reducing CXCL12 (preventing HMGB1/CXCL12 heterodimer formation), and signaling through CXCR4, reducing monocyte/macrophage infiltration.