Innate immune response of corneal epithelial cells to Staphylococcus aureus infection: role of peptidoglycan in stimulating proinflammatory cytokine secretion

Abstract

Purpose: This study sought to elucidate the innate immune responses of cultured human corneal epithelial cells (HCECs) to infection by the Gram-positive bacterium Staphylococcus aureus and to determine the underlying mechanisms.

Methods: HUCL, a telomerase-immortalized HCEC line, and primary cultures of HCECs were challenged with live or heat-killed S. aureus, its exoproducts, or cell wall components lipoteichoic acid (LTA) and peptidoglycan (PGN). IkappaB-alpha phosphorylation and degradation as well as phosphorylation of MAPKs, p38, and JNK-1/2, were assessed by Western blot analysis. The expression of interleukin (IL)-6, IL-8, TNF-alpha, and beta-defensin-2 were determined using RT-PCR and secretion of IL-6, IL-8, TNF-alpha, and beta-defensin were measured using enzyme-linked immunosorbent assay and immunoblot analysis of culture medium.

Results: Exposure of HUCL cells to live, but not heat-killed, S. aureus resulted in NF-kappaB activation in a time-dependent manner, as assessed by the increase in IkappaB-alpha phosphorylation and degradation. Live bacteria also activated the p38 and JNK pathways. The effects of live bacteria on HUCL cells may be attributable to bacterial exoproducts, since the conditioned medium of S. aureus also effectively stimulated these signaling pathways. PGN, but not LTA, activated the NF-kappaB and MAPK pathways in a dose- and time-dependent manner. Concomitant with activation of NF-kappaB and MAPKs, transcriptional expression of IL-6, IL-8, TNF-alpha, and beta-defensin-2 were induced in cells challenged with bacterial exoproducts and PGN. Secretion of IL-6, IL-8, TNF-alpha, and beta-defensin-2 were also significantly increased in HCECs in response to bacterial exoproducts and PGN challenge.

Conclusions: Corneal epithelial cells possess the ability to recognize the presence of Gram-positive bacteria and to initiate the innate immune responses by the expression and/or release of proinflammatory cytokines and beta-defensin-2 in the cornea.

Figure 1

Expression of TLR-1, -2, -6 and -9 in primary HCECs (HCE), HUCL, and THP-1 was analyzed by PCR after reverse transcription of mRNA. GAPDH was analyzed to verify similar cDNA loading. THP-1 was used as a positive control. Results shown are representative of three experiments with similar results.

Figure 2

HCECs were responsive to S. aureus exposure. HUCL cells were exposed to live S. aureus (A) or heat-killed bacteria (B) for the indicated times, and cells were lysed for Western blot analysis with antibodies against phospho-IκB-α (P-IκB-α), phospho-p38 (P-p38), phospho-JNK (P-JNK), and IκB-α (IκB). Antibodies against non-phosphorylated p38 and JNK were used to show that there were no changes in the protein levels and to normalize sample loading. HCECs challenged with live bacteria showed significant activation of NF-κB, p-38, and JNK signaling pathways in a time-dependent manner, whereas no such change was observed in cells treated with heat-inactivated bacteria. Results shown are representative of three independent experiments.

Figure 3

S. aureus–conditioned medium induced activation of NF-κB and MAPKs. HUCL cells (A) or primary HCECs (B) isolated from human corneas were incubated with 1:10 dilution of conditioned medium in KBM for the indicated times (in minutes) with KBM alone (C) and KBM + synthetic medium (C1) as controls. Cells were lysed and results displayed as described in Figure 2. S. aureus–conditioned medium induced rapid activation of NF-κB, p-38, and JNK in HCECs. Results shown are representative of three independent experiments.

Figure 4

Dose-dependent effect of PGN and LTA on NF-κB activation. HUCL cells (A) or THP-1 (B) cells were stimulated with increasing concentrations of PGN or LTA for 1 hour and analyzed for IκB-α phosphorylation (P-IκB) and degradation (IκB) by Western blot analysis. PGN, but not LTA, induced NF-κB activation in HUCL cells in a concentration-dependent manner. Both TLA and PGN induced rapid and strong responses in the control THP-1 cells. Results shown are representative of four independent experiments.

Figure 5

PGN mediated activation of NF-κB and MAPKs. HUCL cells (A) and primary HCECs (B) were challenged for various time intervals with PGN (20 μg/mL). Cells were lysed for Western blot analysis using antibodies against phospho-IκB-α (P-IκB-α), phospho-p38 (P-p38), phospho-JNK (P-JNK), and IκB-α (IκB). Antibodies against p38 and JNK were used to show that there are no changes in protein levels and to normalize sample loading. PGN triggered NF-κB and MAPK activation in HUCL cells as well as in primary HCECs in a time-dependent manner. Results shown are representative of two independent experiments.

Figure 6

LTA did not induce activation of NF-κB and MAPKs. HUCL cells (A) and primary HCECs (B) were stimulated with LTA (25 μg/mL) for various time intervals. Cells were lysed for Western blot analysis using antibodies against phospho-IκB-α (P-IκB-α), phospho-p38 (P-p38), phospho-JNK (P-JNK), and IκB-α (IκB). Antibodies against p38 and JNK were used to show that there are no changes in protein levels and to normalize sample loading. LTA did not trigger NF-κB and MAPK activation in HUCL cells or primary HCECs. Results shown are representative of three independent experiments.

Figure 7

PGN and S. aureus– conditioned medium induced IL-6, IL-8, hBD2, and TNF-α mRNA expression in HUCL cells. HUCL cells grown overnight in KBM were stimulated with 20 μg/mL PGN or 1:10 dilution of conditioned medium in KBM for the indicated times. Total RNA was extracted, reverse transcribed, and amplified using specific primers (Table 1), with GAPDH as the control. PCR products were separated by electrophoresis and stained. HUCL cells expressed the proinflammatory cytokines IL-6, IL-8, and TNF-α, as well as β-defensin-2, in response to PGN and S. aureus– conditioned medium challenge. Results shown are representative of three independent experiments.

Figure 8

IL-6, -8, and TNF-α secretion in HCECs in response to PGN and S. aureus–conditioned medium challenge. HUCL cells (A) and primary HCECs (B) grown overnight in KBM were treated with 20 μg/mL PGN or 1:10 dilution of S. aureus–conditioned medium for 6 hours, and the released IL-6, IL-8, and TNF-α in cell culture supernatants were measured by ELISA. The amount of cytokines was normalized with protein concentration of cell lysate (nanograms per milligram cell lysate). The data shown are representative of triplicate experiments.

Figure 9

PGN and S. aureus–conditioned medium induced hBD-2 secretion in HCECs. HUCL cells (B) or primary HCECs (C) were incubated with serum-free medium alone (Cont), media containing 20 μg/mL PGN (PGN), or a 1:10 dilution of S. aureus–conditioned medium (CM) for 6 hours. The secretion of hBD-2 peptide into the culture medium was detected by immunoblot analysis. To each slot-blot well, 100 μL culture medium was added and probed with anti-hBD-2 antibody. Recombinant human (r)hBD-2 peptide (10 ng/mL and 50 ng/mL) was included as positive control (A). Results are representative of triplicate experiments.