High-Mobility Group Box 1: A Novel Target for Treatment of Pseudomonas aeruginosa Keratitis

Abstract

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.

Glycyrrhizin Reduces HMGB1 and Bacterial Load in Pseudomonas aeruginosa Keratitis

“…15 Treatment with siHMGB1 led to improved disease outcomes along with reduction in proinflammatory cytokines, an increase in anti-inflammatory cytokines and reduced neutrophil infiltration. For the current study, we used a higher bacterial concentration for the clinical isolate than was used in the above paper 15 where 1 × 10 6 CFU/μL was used for both the cytotoxic and the clinical isolate strains and silencing (scrambled treatment for controls siRNA for HMGB1) was our experimental approach to decrease HMGB1 levels. This proof of principle study established HMGB1 as a target for treatment.…”

“…9,10 In this regard, extracellular HMGB1, a member of a family of molecules referred to as DAMPS or alarmins, contributes to the pathogenesis of Pseudomonas keratitis, 15 as well as other infectious 13,38 and noninfectious diseases. 38,39 Extracellular HMGB1 is a late mediator of the inflammatory response, 12 in contrast to other proinflammatory molecules 12 (e.g., TNF-α, IL-1β, and IFN-α) that are released by activated immune cells early in the disease response.…”

“…Upon spectrophotometric quantification at 260 nm, 1 μg of each RNA sample was reverse transcribed using Moloney-murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen, Carlsbad, CA, USA) to produce a cDNA template for the PCR reaction. 15 Complementary (c)DNA products were diluted 1:25 with diethylpyrocarbonate (DEPC)-treated water. A 2-μL aliquot of diluted cDNA was used for the real-time RT-PCR reaction with real-time SYBR green/fluorescein PCR master mix (Bio-Rad Laboratories, Richmond, CA, USA) and primer concentrations of 10 μM (total 10 μL reaction volume).…”

“…In the study, in vivo assays using gene silencing and antibody neutralization of HMGB1 demonstrated an improvement in disease outcome. 15 …”

Macrophage Inflammatory Protein-2 in High Mobility Group Box 1 Secretion of Macrophage Cells Exposed to Lipopolysaccharide

“…When HMGB1 was silenced in mice using siRNA, fewer pro-inflammatory cytokines were observed compared with the levels in control mice; HMGB1 knockdown also decreased the mRNA and protein levels of IL-1β, MIP-2, CXCL12 and CXCR4 in mice and in mononuclear cells [51]. Moreover, neutralization of HMGB1 with an anti-HMGB1 monoclonal antibody strongly inhibited Fas-induced production of MIP-2 and pro-inflammatory cytokines [52].…”