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Linda D Hazlett, Xiaoyu Jiang, Sharon A McClellan, Ronald P Barrett; Hmgb1: A target for treatment of Pseudomonas aeruginosa keratitis. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1688.
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The purpose of this study is to examine high mobility group box 1 (Hmgb1), a prototypic alarmin and member of a family of danger associated molecular patterns (DAMPS), that mediates the systemic inflammatory response syndrome, and is elevated late in bacterial infection/sepsis, as an attractive target for disease treatment in P. aeruginosa infection.
C57BL/6 (B6) mice were treated with vasoactive intestinal peptide (VIP) or PBS, infected and Hmgb1 expression assessed by real-time RT-PCR, ELISA and immunostaining. Mice were then treated with Hmgb1 siRNA or scrambled control and effects also assessed by real-time RT-PCR, immunostaining and ELISA. Neutrophils (PMNs) were quantitated using a myeloperoxidase assay.
VIP versus PBS treatment decreased Hmgb1 mRNA and protein expression levels. Immunohistochemistry showed less Hmgb1 positive staining in corneal epithelium and stroma after VIP treatment. To test the effects of Hmgb1 in disease, siRNA versus scrambled control treatment was used. siRNA treated B6 mice had improved disease outcome and also demonstrated downregulation of corneal mRNA and/or protein levels for Hmgb1, IL-1β, MIP-2, TNF-α, TLR4 and RAGE; antiinflammatory SIGIRR and ST2 were upregulated. Immunohistochemistry showed that silencing Hmgb1 decreased Hmgb1 and IL-1β positive staining in cornea. Hmgb1 siRNA treatment also reduced PMN levels and CXCL12 and CXCR4 expression.
These data provide evidence that silencing Hmgb1 leads to better resolution of P. aeruginosa keratitis, as its reduction results in decreased proinflammatory mediator levels, increased antiinflammatory TLR production, decreased, but sufficient, PMN infiltration and reduced Hmgb1/CXCL12 heterodimer formation required for signaling through CXCR4.
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