High-Mobility Group Box 1 in Dry Eye Inflammation

Abstract

Purpose: To determine high-mobility group box 1 (HMGB1) expression during experimental dry eye (EDE) and dry eye-like culture conditions and elucidate its role in corneal dry eye-related inflammation.

Methods: EDE was induced in 8- to 12-week-old C57BL/6 mice. Corneal tissue sections and lysates from EDE and untreated mice were evaluated for HMGB1 expression by immunostaining and quantitative real-time PCR (qPCR). For in vitro studies, human corneal epithelial cells (HCEC) were treated with hyperosmolar media, toll-like receptor (TLR) agonists, or proinflammatory cytokines to determine HMGB1 expression. HCEC were also treated with human recombinant HMGB1 (hrHMGB1) alone or in combination with inflammatory stimuli, and TNFα, IL-6, and IL-8 expression evaluated by qPCR and ELISA. Nuclear factor-κB (NF-κB) p65 nuclear translocation was determined by immunostaining.

Results: EDE mice had higher corneal HMGB1 RNA and protein expression compared to untreated animals. In HCEC, hyperosmolar stress and TNFα treatment stimulated HMGB1 production and secretion into culture supernatants. However, in vitro stimulation with hrHMGB1 did not induce secretion of TNFα, IL-6, or IL-8 or NF-κB p65 nuclear translocation. In addition, the inflammatory response elicited by TLR agonists fibroblast-stimulating lipopeptide-1 and lipopolysaccharide was not enhanced by hrHMGB1 treatment.

Conclusions: HMGB1 expression was enhanced by dry eye conditions in vivo as well as in vitro, during hyperosmolar stress and cytokine exposure, suggesting an important role for HMGB1 in dry eye disease. However, no direct inflammatory effect was observed with HMGB1 treatment. Therefore, under these conditions, HMGB1 does not contribute directly to dry eye-induced inflammation and its function at the ocular surface needs to be explored further.

Figure 1

HMGB1 is increased in the corneas of EDE mice. (A) Corneal epithelial cells were removed from EDE and UT control mice for evaluation of HMGB1 mRNA expression by qPCR. Graph represents mean ± SEM (n = 3), where tissue from three mice was pooled for each sample (nine mice per condition). (B) Frozen corneal tissue sections were stained for HMGB1 expression by immunohistochemistry. Images are representative of three mice per condition; scale bar: 50 μm. (C) Dry eye was assessed by ocular surface fluorescein staining using OCT imaging. Graph represents mean ± SEM pixel intensity quantitated from OCT images (right panel) using ImageJ analysis (n = 6 for UT and n = 5 for EDE). Statistical comparison between UT controls and EDE was performed by unpaired t-test. *P ≤ 0.05.

Figure 2

Hyperosmolar stress and TNFα increase HMGB1 cellular expression and secretion in HCEC. (A) hTCEpi were cultured with 450 mOsM media or in the presence of TNFα (10 ng/mL). After 6 hours, both hyperosmolar stress and TNFα induced increase of nuclear and cytoplasmic HMGB1 expression when compared to the UT control. Images are representative of n = 2 independent experiments; scale bar: 50 μm. (B) SV40 HCEC were cultured for 6, 12, or 24 hours in hyperosmolar media (400, 450, or 500 mOsM) (left graph). hTCEpi were stimulated with TNFα (10 ng/mL) for 1 or 6 hours (right graph). HMGB1 was measured in cell culture supernatants by ELISA. Graphs represent mean ± SEM of n = 2 independent experiments. ANOVA was used to test for statistical significance with Bonferroni's test for multiple comparisons. ***P ≤ 0.0001; **P ≤ 0.001; *P ≤ 0.05.

Figure 3

HMGB1 does not induce secretion of inflammatory cytokines in HCEC. hTCEpi (A, B) and macrophage-differentiated U937 (Mϕ-U937) cells (C, D) were stimulated with HMGB1 (10 μg/mL) for 4 or 8 hours. mRNA expression (left graphs) and secreted IL-6, IL-8, and TNFα (right graphs) were measured by qPCR and ELISA, respectively. Graphs represent mean ± SEM of n = 2 (Mϕ-U937) and n = 4 (HTCEpi) independent experiments. ANOVA was used to test for statistical significance with Bonferroni's test for multiple comparisons. ***P ≤ 0.0001; **P ≤ 0.001; *P ≤ 0.01.

Figure 4

HMGB1 does not synergize with FSL-1 to increase inflammatory cytokines in HCEC. hTCEpi (A) and primary HCEC (B) were cultured in the presence of HMGB1 (10 μg/mL) or FSL-1 (1 μg/mL) or both for 24 hours and levels of IL-6 and IL-8 were measured in culture supernatants. Graphs represent mean ± SEM of n = 3 independent experiments. ANOVA was used to test for statistical significance with Bonferroni's test for multiple comparisons. ***P ≤ 0.0001.

Figure 5

HMGB1 does not synergize with LPS to increase inflammatory cytokines in HCEC. hTCEpi (A, B) and macrophage-differentiated U937 (Mϕ-U937) cells (C, D) were stimulated with HMGB1 (10 μg/mL) or LPS (1 μg/mL) or both for 8 hours. mRNA expression (left graphs) and secreted cytokines (right graphs) were measured by qPCR and ELISA, respectively. Graphs represent mean ± SEM of n = 2 (Mϕ-U937) and n = 4 (hTCEpi) independent experiments. ANOVA was used to test for statistical significance with Bonferroni's test for multiple comparisons. ***P ≤ 0.0001; **P ≤ 0.001; *P ≤ 0.01.

Figure 6

IFNγ does not improve HCEC responsiveness to LPS or HMGB1. Primary HCEC were stimulated with IFNγ (200 U/mL) prior to treatment with HMGB1, LPS, or both for 8 hours. Cell lysates were collected for RNA extraction and qPCR determination of relative IL-6, IL-8, and TNFα mRNA expression. Graphs represent mean ± SEM of n = 2 independent experiments.

Figure 7

HMGB1 does not induce NF-κB translocation in HCEC. hTCEpi were cultured in the presence of HMGB1 (50 ng/mL) for 2 hours and immunostained for NF-κB p65 expression. TNFα (10 ng/mL) was used as a positive control for NF-κB p65 cytoplasm-nucleus translocation. No translocation of NF-κB p65 was observed with HMGB1 treatment. Images are representative of n = 2 independent experiments; scale bar: 50 μm.