C-Jun NH2 terminal kinase (JNK) is an essential mediator of Toll-like receptor 2-induced corneal inflammation

Abstract

TLRs play an important role in the host inflammatory response to bacteria and bacterial products by activating a cascade of intracellular events leading to production of proinflammatory and chemotactic cytokines. To determine the role of MAPKs in TLR- induced corneal inflammation, we stimulated human corneal epithelial (HCE) cells with TLR2 ligands, tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3Cys) or inactivated Staphylococcus aureus, and examined the time course of expression of MAPKs and the effect of MAPK inhibition on IkBalpha degradation and CXC chemokine production. We found that S. aureus and Pam3Cys stimulate phosphorylation of JNK, p38 MAPK, and ERK within 4 h and that blockade of JNK, but not p38 or ERK phosphorylation, had an inhibitory effect on IkBalpha degradation and CXC chemokine production. To determine if JNK is also important in TLR2-induced corneal inflammation in vivo, we examined JNK1(-/-) mice and pharmacological inhibitors in a murine model of TLR2-induced corneal inflammation which is characterized by neutrophil recruitment to the corneal stroma and development of corneal haze. We found that corneal inflammation was significantly impaired in JNK1(-/-) mice compared with control mice, and in mice treated with the JNK inhibitor compared with vehicle control. Taken together with results from HCE cells, these findings demonstrate that JNK has an essential role in TLR2-induced corneal inflammation.

Fig. 1

S. aureus- and Pam₃Cys-induced activation of MAPKs and NF-κB in HCE cells. (Left) Human primary corneal epithelial cells were isolated from donor eyes obtained from the Cleveland Eye Bank and incubated with 1 × 10⁸ UV-killed S. aureus strain 8325. At indicate times, cells were harvested, lysed, and processed for SDS-PAGE and Western blot analysis using antibody specific for total or phosphorylated (P) JNK, p38, ERK, and IκBα. Actin was used as a loading control. The SV40-immortalized HCE cell line (HCE-T) was stimulated with S. aureus (Middle) or the synthetic TLR2 ligand Pam3Cys (Right). At indicated times, cells were examined as described for the left panel. The experiment is representative of four repeat experiments.

Fig. 2

IkBα phosphorylation is selectively blocked by the JNK inhibitor SP600125. HCE-T cells were stimulated with S. aureus or Pam₃Cys, together with inhibitors for ERK, p38, or JNK. After 6 h, cells were harvested and processed for SDS-PAGE/Western blot analysis, as described in the legend to Figure 1. Densitometry shows inhibition of IkBα phosphorylation by the JNK inhibitor SP600125 but not by other MAPK inhibitors. This experiment was repeated three times with similar results.

Fig. 3

CXC chemokine production in HCE-T cells is blocked by SP600125. (A) HCE-T cells were incubated with S. aureus for 6 h or 24 h, and supernatants were examined for production of CXCL1, CXCL5, and CXCL8 by ELISA. (B) HCE cells were incubated 24 h with S. aureus in the presence of the JNK inhibitor SP600125, and supernatants were examined as before. Note that cells treated with SP600125 had significantly impaired CXC chemokine production compared with cells incubated with vehicle alone (*, P<0.05). This experiment was repeated twice with similar results. Cont, Control.

Fig. 4

Phosphorylation of MAPKs and IkBα in TLR2-induced corneal inflammation. Corneas of C57BL/6 mice were gently scarified by three parallel scratches and treated with H₂O (trauma control) or with the TLR2 ligand Pam₃Cys. At indicated times, corneas were dissected and processed for SDS-PAGE and Western blot analysis. Each sample represents a pool of two corneas and is representative of two repeat experiments.

Fig. 5

SP600125 inhibits TLR2-induced corneal inflammation. Corneal epithelium of C57BL/6 mice was scarified using three parallel scratches and treated topically with SP600125 or with vehicle alone 1 h prior to and at the same time as and 6 h after corneas were treated topically with Pam₃Cys. After 24 h, neutrophils in the corneal stroma were detected by immunohistochemistry (A), and corneal thickness and haze were measured by in vivo confocal microscopy (B, C). Data are mean ± SEM for five corneas per group, and the experiment was repeated twice.

Fig. 6

TLR2-induced corneal inflammation in JNK1^−/− mice. Corneas of JNK-1^−/− and C57BL/6 mice were scarified as described above and stimulated by topical application of Pam₃Cys (P3C). After 24 h, corneal inflammation was measured as described above. Data are mean ± SEM for five corneas per group and are representative of two repeat studies.