Hypoxia-altered signaling pathways of toll-like receptor 4 (TLR4) in human corneal epithelial cells

Abstract

Purpose: Toll-like receptor 4 (TLR4), a member of the TLR family, is an important pattern recognition molecule that plays a role in the host's innate immune responses to lipopolysaccharide (LPS), a component of gram-negative bacteria. Contact lens wear is one of the risk factors for bacterial keratitis. The purpose of this study was to determine whether hypoxia or contact lens wear alters the TLR4 signaling pathways in human corneal epithelial cells (HCECs).

Method: A simian virus 40-immortalized human corneal epithelial cell (SV40-HCEC) line was cultured under 20% O2 or 2% O2 and exposed to LPS. The expression of TLR4, interleukin-6 (IL-6), and IL-8 was determined using a real-time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunoblotting. Immunoblotting was also used to determine whether the nuclear factor kappa B (NFkappaB) was activated in the SV40-HCEs. HCECs were obtained from 17 healthy volunteers and 18 hydrogel soft contact lens (SCL) wearers using impression cytology (IC), and the expression of the mRNA of TLR4 was determined using real-time RT-PCR.

Results: A reduction in the expression of the mRNA and protein of TLR4 was detected in SV40-HCECs cultured under hypoxic conditions. Hypoxia also attenuated both the LPS-induced expression of IL-6 and IL-8, and the activation of NFkappaB in SV40-HCECs. The expression of the mRNA of TLR4 was down-regulated in the HCECs of soft contact lens wearers.

Conclusions: These results indicate that hypoxia attenuates the TLR4 signaling pathway in HCECs, suggesting that the increase in the susceptibility to bacterial infections under hypoxic conditions may be related to the TLR4 signaling pathways.

Figure 1

Expression of TLR4 mRNA in SV40-HCEs cultured under 20% O₂ (control group) or 2% O₂ (hypoxia group). The expression of the mRNA of TLR4 decreased to about 10% of the control group under hypoxic conditions. The p-values were calculated using two-tailed paired t tests. The asterisk indicates a p<0.05.

Figure 2

Immunoblotting for TLR4 protein expression in SV40-HCECs cultured under 20% O₂ (control group) or 2% O₂ (hypoxia group). TLR4 protein expression of the hypoxia group decreased to about 30% of the control group.

Figure 3

Effects of hypoxia on the expression of cytokines in SV40-HCECs exposed to LPS. Total RNA was isolated from SV40-HCECs cultured under 20% O₂ (control group) or 2% O₂ (Hypoxia group) for 72 h and stimulated with LPS for 24 h. The expression of the mRNA of IL-6 and IL-8 was determined using real-time PCR. The relative level of mRNA expression for each cytokine is normalized to G3PDH mRNA expression. The p-values were calculated using a two-tailed paired t test. The asterisk indicates a p<0.05 and the double asterisk indicates a p<0.01).

Figure 4

Cytokine secretion by SV40-HCEs cultured under 20% O₂ (control group) or 2% O₂ (hypoxia group) stimulated with LPS. The culture medium was collected 24 h after LPS exposure and analyzed for the presence of IL-6 and IL-8 proteins using ELISA. The p-values were calculated using a two-tailed paired t tests. The triple asterisk indicates a p<0.001).

Figure 5

Immunoblotting for NFκB 24 h after exposure to LPS. Immunoblotting showed that NFκB protein expression in the hypoxia group was reduced compared to that in the control group.

Figure 6

Expression of TLR4 mRNA in the CECs of normal volunteers and SCL wearers. TLR4 mRNA was detected from all samples, but the expression of TLR4 from SCL wearers was one-half the amount from normal volunteers. The p-values were calculated using a two-tailed paired t test. The asterisk indicates a p<0.05.