High glucose may decrease the innate immune through TLRs in cornea epithelium

Abstract

Purpose: The purpose of this study was to investigate the high potential of glucose in inhibiting the innate immune in cultured human cornea epithelial cells (HCEC) and try to determine whether the role of high glucose on the HCEC relate to toll-like receptor (TLR)2 and TLR4.

Methods: Cells were cultured for 3 days in 5 mmol/l (normal glucose). Then high glucose (25 mmol/l) was added along with normal glucose with daily changes in media for 24 h. The cells were also treated with mannitol as an osmotic control. The cellular abundance of the mRNAs for TLR2 and TLR4 was determined by real-time polymerase chain reaction analysis. The proteins of TLR2 and TLR4 were also compared by immunofluorescent staining and western blot. The release of interleukin 6 (IL-6) and IL-8 from cultured HCEC was measured using enzyme-linked immunosorbent assays (ELISA) in the presence and absence of specific blocking antibodies to TLR2 and TLR4.

Results: Incubation of HCEC with high glucose showed that the mRNA expression of TLR2 and TLR4 was markedly inhibited. Immunofluorescent staining and western blot analysis confirmed that the protein expression of TLR2 and TLR4 was downregulated in response to high glucose. The result of ELISA also showed that the release of IL-6 and IL-8 can be inhibited by high glucose, but these inhibitions were partly counteracted after pretreatment with anti-TLR2 and/or anti-TLR4 monoclonal antibody. The results also showed that the osmotic control did not affect the expression of TLR2, TLR4, and IL-6, 8.

Conclusions: High glucose may decrease the innate immune through TLRs in cornea epithelium.

Figure 1

Real time PCR shows the relative expression of TLR2 and TLR4 mRNA in high glucose and high mannitol treated HCEC compared with untreated HCEC. The untreated HCEC is regarded as standard control (RQ=1), treated cells are expressed as the multiple of the untreated HCEC. Bars represent means±SEM of 3 independent experiments. *represent a p<0.05 versus control. RQ represent relative quantity.

Figure 2

Immunofluorescent staining detect the protein expression of TLR2 and TLR4. The high glucose and high mannitol stimulated cells treated with anti-TLR2 (A) and anti-TLR4 (B) antibodies and stained by Cy3 and DAPI dihydrochloride. There was no immunoreactivity in the negative control (isotype IgG). Merge means overlap the DAPI and Cy3.

Figure 3

Western blot detect the protein expression of TLR2 and TLR4. A: western blot analyses detect the expression of TLR2 and TLR4 at the protein level in HCEC under stimulation of high glucose and high mannitol. Equal amounts of proteins were loaded. B: Column diagrams and Bars represent the ratio of the scanned immunoblots of TLR2 and TLR4 to that of GAPDH. Untreated HCEC was regarded as control. Data are the mean±SEM of triplicates from an experiment that was repeated three times with similar results. *represent a p<0.05 versus control.

Figure 4

The results of ELISA showed the release of IL-6 and IL-8 from HCEC under high glucose and high mannitol stimulation with and without TLR2 and TLR4 monoclonal antibody (1:200). The untreated HCEC is regarded as control. Data are the mean±SEM of triplicates from an experiment that was repeated three times with similar results. *represent a p<0.05 versus control.