Staphylococcus aureus protein A induced inflammatory response in human corneal epithelial cells

Abstract

In the present study, we examined the role of Staphylococcus aureus protein A (SpA) in inducing inflammatory response in human corneal epithelial cells (HCECs). Exposure of HCECs to SpA induces rapid NF-kappaB activation and secretion of proinflammatory cytokine/chemokines (TNF-alpha and IL-8) in both concentration and time-dependent manner. Challenge of HCECs with live SpA(-/-) mutant S. aureus strains resulted in significantly reduced production of the cytokines when compared to the wild-type S. aureus strain. SpA also elicited the activation of MAP Kinases P38, ERK, but not JNK, in HCECs. SpA-induced production of proinflammatory cytokine were completely blocked by the NF-kappaB and p38 inhibitors and partially inhibited by the Jnk inhibitor. Pretreatment with anti-TLR2 neutralizing antibody had no effect on SpA-induced inflammatory response in HCECs, suggesting that this response is independent of TLR2 signaling. Moreover, unlike TLR2 ligands, SpA failed to induce the expression of antimicrobial peptides (hBD2 and LL-37) in HCECs. These studies indicate that SpA is a S. aureus virulence factor that stimulates HCEC inflammatory response through a pathway distinct from TLR2 in HCECs.

Fig 1. Effect of SpA on NF-κB activation in HUCL and primary HCECs: dose and time-dependent course studies

HUCL (A, B & D) and Primary (C) cells were incubated with various concentrations of SpA for 1 h (A), or 100 μg/ml for different time points (B & C) with unstimulated cells as control. A, B, and C, Western blotting of total cell lysate for phospho-IκB-α (IκB-α) and total IκB-α. D. NF-κB p65 DNA binding activity in HUCL nuclear extracts measured by ELISA using a Colorimetric Transfactor p65 kit.. Results shown are representative of three independent experiments.

Fig 2. Effect of SpA stimulation on TNF-α and IL-8 production in HCECs

HUCL (A&B) and Primary HCEC (C) were cultured with various concentration of SpA for 4 h (A) or with 100 μg/ml SpA for different time periods up to 24 h (B). Secretion of IL-8 (open bars) or TNF-α (gray bars) into culture media was assayed by ELISA and presented as pg/ml for 1 × 10⁶ cells. Data represent means ± SD of n = 3 independent experiments; *P < 0.05.

Fig 3. Effect of MAPKs inhibitors and TLR2 neutralizing antibody on SpA-induced IL-8 secretion

HUCL cell were incubated with inhibitors of MAPKs-mediated signal pathways for 30 min prior to the addition of SpA and the induced secretion of IL-8 after 4 h was assessed by IL-8 cytokine ELISA. The inhibitors used include the NF-κB inhibitor isohelenin (IS, 25 μM) and three MAP kinase pathways, SB (p38 MAP kinase inhibitor, 5 μM); SP (JNK inhibitor, 25 μM) and U0126 (ERK inhibitor, 10 μM). Anti-TLR2 antibody (20 μg/ml) was used to block the TLR2 pathway. The data is representative of three individual experiments.

Fig 4. SpA and TLR2 agonist Pam3Cys mediated gene expression in HCECs

HUCL cells cultured in KBM were challenged with SpA (100 μg/ml) or Pam3Cys (10μg/ml). At the indicated times, cells were processed for semiquantitative RT-PCR to assess mRNA expression of hBD2, LL-37, IL-8 and TNF-α with GAPH as internal control. Results shown are representative of two independent experiments.