PostDoc, Wayne State University
Finding non-antibiotic therapeutics for Pseudomonas aeruginosa keratits
Pseudomonas aeruginosa (PA) is an important human pathogen. It causes many infections including keratitis, a destructive blinding diseases of the cornea. Specifically in developed countries, PA is the most commonly recovered causative organism in contact lens-related keratitis. More than 140 million people wear contact lenses worldwide, making PA-induced keratitis a global cause of visual impairment and is associated with a cost, $175 million in direct healthcare expenditures in the USA alone. This imposes both a clinical and economic burden. Currently antibiotics are used to treat Pseudomonas keratitis. However with the emergence of multi-drug resistant PA , finding non-antibiotic based therapies to treat keratitis is an urgent need and is the focus of my research. In this regard I am testing the therapeutic efficacy of Glycyrrhizin (GLY), a natural, non antibiotic compound for keratitis induced PA. To this end we found that GLY is therapeutic for Keratitis induced by clinical isolates of PA and continue further research to test the impact on GLY against multi-drug resistant PA.
Abstract: Thrombomodulin (TM) is a multidomain, transmembrane protein with anti-inflammatory properties. Thrombomodulin domain (D) 1 is lectin-like, interacting with Lewis Y antigen on lipopolysaccharide, and with HMGB1, while TMD23 is associated with angiogenic and anti-inflammatory functions. Thus, we tested if TM is protective against Pseudomonas aeruginosa keratitis and whether it enhanced corneal vascularity.Eyes of C57BL/6 (B6) mice were injected with recombinant TM (rTM), rTMD1, or PBS subconjunctivally before and intraperitoneally after infection with P. aeruginosa. Clinical scores, photography with a slit lamp, RT-PCR, ELISA, myeloperoxidase (MPO) assay, viable bacterial plate counts, and India ink perfusion were used to assess the disease response and corneal vascularity (rTM only).Recombinant TM versus PBS treatment reduced clinical scores and corneal opacity. Corneal mRNA levels for HMGB1 were unchanged, but proinflammatory molecules IL-1β, CXCL2, NF-κB, TLR4, and RAGE were decreased; anti-inflammatory molecules SIGIRR and ST2 were increased. ELISA confirmed the mRNA data for HMGB1, IL-1β, and CXCL2 proteins. Both neutrophil influx and viable bacterial plate counts also were decreased after rTM treatment. Protein levels for angiogenic molecules VEGF, VEGFR-1, and VEGFR-2 were measured at 5 days post infection and were not different or reduced significantly after rTM treatment. Further, perfusion with India ink revealed similar vessel ingrowth between the two groups. Similar studies were performed with rTMD1, but disease severity, mRNA, proteins, MPO, and plate counts were not changed from controls.These data provide evidence that rTM treatment is protective against bacterial keratitis, does not reduce HMGB1, and is not angiogenic.
Pub.: 01 Jan '16, Pinned: 27 Jun '17
Abstract: High mobility group box 1 (HMGB1) contributes to poor disease outcome in Pseudomonas aeruginosa keratitis. This study tests the prophylactic effect of treatment with HMGB1 inhibitors, glycyrrhizin (GLY) and its derivative, carbenoxolone (CBX), for Pseudomonas keratitis.We treated C57BL/6 (B6) mice subconjunctivally with GLY or CBX, infected with a noncytotoxic clinical isolate (KEI 1025) or a cytotoxic strain (ATCC 19660) of P. aeruginosa, and injected intraperitoneally with either agent. Clinical score, photography with a slit lamp, real-time RT-PCR, ELISA, myeloperoxidase (MPO) assay, bacterial plate count, histopathology, and absorbance assays were used to assess treatment efficacy and bacteriostatic activity.After KEI 1025 infection, GLY treatment reduced HMGB1 (mRNA and protein levels) and improved disease outcome with significant reduction in mRNA levels of IL-1β, TLR4, CXCL2, and IL-12; protein expression (IL-1β, CXCL2); neutrophil infiltrate; and bacterial load. Treatment with GLY enhanced antimicrobial proteins, including CRAMP and mBD2, but not mBD3. Glycyrrhizin also reduced clinical scores and improved disease outcome in corneas infected with strain 19660. However, neither HMGB1 mRNA or protein levels were reduced, but rather, CXCL2 expression (mRNA and protein), neutrophil infiltrate, and bacterial load were reduced statistically. Treatment with GLY initiated 6 hours after infection reduced plate count; GLY also was bacteriostatic for KEI 1025 and ATCC 19660.Glycyrrhizin reduces HMGB1 and is protective against P. aeruginosa-induced keratitis with a clinical isolate that is noncytotoxic. It was similar, but less effective when used after infection with a cytotoxic strain, which did not reduce HMGB1.
Pub.: 30 Oct '16, Pinned: 27 Jun '17