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Carolina Lema, Rose Y Reins, Betty Zhang, Rachel L Redfern; High-mobility box group 1 protein: An alarmin driving dry eye inflammation?. Invest. Ophthalmol. Vis. Sci. 2017;58(8):447.
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© ARVO (1962-2015); The Authors (2016-present)
Dry eye disease (DED) is characterized by increased tear osmolarity and ocular surface damage due to chronic inflammation. Following cell injury or death high-mobility group box 1 (HMGB1) protein, an alarmin, is released and activates the innate immune response. Previously, we have reported that HMGB1 expression is increased in the tears of dry eye patients. Therefore, we sought to determine if HMGB1 expression increases with in vitro hyperosmolar stress (HOS) or in an established mouse model of experimental dry eye (EDE), thereby propagating the cycle of inflammation on the ocular surface.
Human telomerase corneal epithelial (hTCEpi) cells were treated with hyperosmolar media to induce HOS (450mOsM, 6h) or tumor necrosis factor alpha (TNF-α, 20ng/ml, 24h). Following treatment, HMGB1 expression and localization was examined by immunostaining. Alternatively, hTCEpi were stimulated with rhHMGB1 (10-1000ng/ml for 6 and 24h) and IL-6, IL-8 and TNF-α mRNA and protein were examined by qRT-PCR and ELISA, respectively. Differentiated U937 macrophages were also treated with rhHMGB1 (10ug/ml, 8h) and TNF-α secretion was measured in supernatants, to verify its biological activity. Finally, HMGB1 expression was examined in frozen tissue sections and cell lysates collected from EDE (subcutaneous scopolamine and environmental stress) and untreated (UT) C57BL/6 mice by immunostaining and qRT-PCR.
HOS and TNFα induced nucleus to cytoplasm translocation of HMGB1 in hTCEpi cells. rhHMGB1 stimulation of differentiated U937 macrophages increased TNF-α protein secretion when compared to the untreated control (298±6pg/ml vs 1056±113pg/ml; p-value≤0.05). However, rhHMGB1 treatment did not significantly increase the mRNA or protein levels of IL-6, IL-8 or TNF-α in hTCEpi cells. In the mouse corneal epithelium, EDE increased HMGB1 staining and mRNA expression (1.52±0.27 vs 3.38±0.55; p-value≤0.05) compared to UT animals.
HMGB1 levels are increased by HOS (in vitro) and desiccation stress (in vivo). However, rhHMGB1 stimulation (in vitro) does not trigger inflammatory cytokines IL-6, IL-8 and TNF-α. Whether HMGB1 is directly related to inflammation in DED remains unclear and deserves further studies.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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