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. 2012 Apr 30;44(4):260-7.
doi: 10.3858/emm.2012.44.4.021.

Toll-like receptor 4 on islet β cells senses expression changes in high-mobility group box 1 and contributes to the initiation of type 1 diabetes

Min Li  1 , Lujun Song, Xiaodong Gao, Wenju Chang, Xinyu Qin
Affiliations

Toll-like receptor 4 on islet β cells senses expression changes in high-mobility group box 1 and contributes to the initiation of type 1 diabetes

Min Li et al. Exp Mol Med. .

Abstract

Type 1 diabetes mellitus is caused by the autoimmune destruction of β cells within the islets. In recent years, innate immunity has been proposed to play a key role in this process. High-mobility group box 1 (HMGB1), an inflammatory trigger in a number of autoimmune diseases, activates proinflammatory responses following its release from necrotic cells. Our aim was to determine the significance of HMGB1 in the natural history of diabetes in non-obese diabetic (NOD) mice. We observed that the rate of HMGB1 expression in the cytoplasm of islets was much greater in diabetic mice compared with non-diabetic mice. The majority of cells positively stained for toll-like receptor 4 (TLR4) were β cells; few α cells were stained for TLR4. Thus, we examined the effects of anti-TLR4 antibodies on HMGB1 cell surface binding, which confirmed that HMGB1 interacts with TLR4 in isolated islets. Expression changes in HMGB1 and TLR4 were detected throughout the course of diabetes. Our findings indicate that TLR4 is the main receptor on β cells and that HMGB1 may signal via TLR4 to selectively damage β cells rather than α cells during the development of type 1 diabetes mellitus.

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Figures

Figure 1
Figure 1
Hematoxylin and eosin staining of pancreatic sections demonstrates extensive islet destruction in diabetic NOD mice (B) compared with 4-week-old, non-diabetic NOD mice (A). Immunohistochemical staining shows preferential localization of HMGB1 in the nuclei of islet cells in 4-week-old non-diabetic mice (C, E). HMGB1 expression in the cytoplasm of the islets is much higher with the development of diabetes (D, F). The rate of HMGB1 expression in the cytoplasm of islets was much greater in diabetic mice compared with non-diabetic mice (E) **P < 0.01 (n = 9 per group).
Figure 2
Figure 2
Expression of HMGB1 receptors (TLR2, TLR4, TLR9 and RAGE) and insulin in pancreatic islets of 4-week-old non-diabetic NOD mice. (A, E, I, M) Insulin immunostaining (red) of β cells. (B, F, J, N) TLR2, TLR4, TLR9 and RAGE immunostaining (green). (C, G, K, O) DAPI nuclear staining (blue). Pancreatic islets from 4-week-old non-diabetic NOD mice show little or no expression of TLR2 (B), TLR9 (J) or RAGE (N). In contrast, TLR4 is highly expressed on the islets (green) in 4-week-old non-diabetic NOD mice (F).Co-localization (yellow) of TLR4 and insulin (H) suggested that TLR4 is mainly expressed in β cells.
Figure 3
Figure 3
TLR4 is not expressed in α cells. Islets from 4-week-old non-diabetic NOD mice were double-labeled with TLR4 and glucagon. (A) Glucagon immunostaining (red). (B) TLR4 receptor immunostaining (green). (C) DAPI nuclear staining (blue). (D) Co-localization of TLR4 and glucagon indicate sparse expression of TLR4 in α cells.
Figure 4
Figure 4
Effects of TLR antibodies on cell surface binding of HMGB1. Islets were isolated from 4-week-old non-diabetic NOD mice, plated in six-well plates and used at 70% confluence. (A) Incubation of islets with NHS-fluorescein-HMGB1 for 6 h at 4℃ resulted in an annular staining pattern. (B-G) Cells were also preincubated with 25 µg/mL of anti-TLR2 (B), TLR9 (C), RAGE (D), IgG (E), TLR4 (F) or unlabeled HMGB1 (G) for 30 min at 4℃ in Opti-MEM I medium, followed by 5 µg/mL NHS-fluorescein-labeled HMGB1 for 6 h at 4℃. Cells were then washed three times with PBS (pH 7.2) and fixed for 20 min at room temperature in 2% paraformaldehyde-PBS. After fixing, fluorescence was recorded with a confocal microscope.
Figure 5
Figure 5
Western blotting analysis of HMGB1 and TLR-4 protein expression. Pancreatic expression of HMGB1 and TLR4 was low in non-diabetic NOD mice (4-6 weeks of age). In the early stage of diabetes (10-14 weeks of age), the expression of HMGB1 and TLR4 protein was significantly upregulated. In contrast, the expression of HMGB1 and TLR4 was decreased in mice with established diabetes (28-32 weeks of age). The data are from three independent experiments. **P < 0.01 versus control and late-stage normal mice.
Figure 6
Figure 6
Pancreatic mRNA expression of HMGB1 and TLR-4 mRNA. Pancreatic tissue was obtained from NOD mice (8 mice per time) every 3-5 weeks from 4 to 32 weeks of age and subjected to qRT-PCR. Values are means ± standard error of the mean. The data are from three independent experiments. *P < 0.05 (HMGB1) for 8 versus 10 weeks of age, 10 versus 12 weeks of age, 12 versus 16 weeks of age and 20 versus 26 weeks of age (Wilcoxon's signed rank test). **P < 0.05 (TLR4) for 4 versus 6 weeks of age, 6 versus 8 weeks of age, 8 versus 10 weeks of age, 12 versus 16 weeks of age, 16 versus 20 weeks of age and 26 versus 32 weeks of age (Wilcoxon's signed rank test).
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