Pathogenic strains of Acanthamoeba are recognized by TLR4 and initiated inflammatory responses in the cornea.

Free-living amoebae of the Acanthamoeba species are the causative agent of Acanthamoeba keratitis (AK), a sight-threatening corneal infection that causes severe pain and a characteristic ring-shaped corneal infiltrate. Innate immune responses play an important role in resistance against AK. The aim of this study is to determine if Toll-like receptors (TLRs) on corneal epithelial cells are activated by Acanthamoeba, leading to initiation of inflammatory responses in the cornea. Human corneal epithelial (HCE) cells constitutively expressed TLR1, TLR2, TLR3, TLR4, and TLR9 mRNA, and A. castellanii upregulated TLR4 transcription. Expression of TLR1, TLR2, TLR3, and TLR9 was unchanged when HCE cells were exposed to A. castellanii. IL-8 mRNA expression was upregulated in HCE cells exposed to A. castellanii. A. castellanii and lipopolysaccharide (LPS) induced significant IL-8 production by HCE cells as measured by ELISA. The percentage of total cells positive for TLR4 was higher in A. castellanii stimulated HCE cells compared to unstimulated HCE cells. A. castellanii induced upregulation of IL-8 in TLR4 expressing human embryonic kidney (HEK)-293 cells, but not TLR3 expressing HEK-293 cells. TLR4 neutralizing antibody inhibited A. castellanii-induced IL-8 by HCE and HEK-293 cells. Clinical strains but not soil strains of Acanthamoeba activated TLR4 expression in Chinese hamster corneas in vivo and in vitro. Clinical isolates but not soil isolates of Acanthamoeba induced significant (P< 0.05) CXCL2 production in Chinese hamster corneas 3 and 7 days after infection, which coincided with increased inflammatory cells in the corneas. Results suggest that pathogenic species of Acanthamoeba activate TLR4 and induce production of CXCL2 in the Chinese hamster model of AK. TLR4 may be a potential target in the development of novel treatment strategies in Acanthamoeba and other microbial infections that activate TLR4 in corneal cells.

Acanthamoeba-cytopathic protein induces apoptosis and proinflammatory cytokines in human corneal epithelial cells by cPLA2α activation.

PURPOSE: We have shown that Acanthamoeba interacts with a mannosylated protein on corneal epithelial cells and stimulates trophozoites to secrete a mannose-induced 133 kDa protease (MIP-133), which facilitates corneal invasion and induces apoptosis. The mechanism of MIP-133-induced apoptosis is unknown. The aim of this study was to determine if MIP-133 induces apoptosis and proinflammatory cytokines/chemokines in human corneal epithelial (HCE) cells via the cytosolic phospholipase A(2α) (cPLA(2α)) pathway. METHODS: HCE cells were incubated with or without MIP-133 at doses of 7.5, 15, and 50 µg/mL for 6, 12, and 24 hours. The effects of cPLA(2α) inhibitors on cPLA(2α), arachidonic acid (AA) release, and apoptosis were tested in vitro. Inhibition of cPLA(2α) involved preincubating HCE cells for 1 hour with cPLA(2α) inhibitors (10 µM methyl-arachidonyl fluorophosphonate [MAFP] or 20 µM arachidonyl trifluoromethyl ketone [AACOCF3]) with or without MIP-133 for 24 hours. Expression of cPLA(2α) mRNA and enzyme was examined by RT-PCR and cPLA(2) activity assays, respectively. Apoptosis of corneal epithelial cells was determined by caspase-3 and DNA fragmentation assays. Expression of IL-8, IL-6, IL-1ß, and IFN-γ was examined by RT-PCR and ELISA. RESULTS: MIP-133 induced significant cPLA(2α) (approximately two to four times) and AA release (approximately six times) from corneal cells while cPLA(2α) inhibitors significantly reduced cPLA(2α) (approximately two to four times) and AA release (approximately three times) (P < 0.05). cPLA(2α) inhibitors significantly inhibited MIP-133-induced DNA fragmentation approximately 7 to 12 times in HCE cells (P < 0.05). MIP-133 specifically activates cPLA(2α) enzyme activity in HCE cells, which is blocked by preincubation with anti-MIP-133 antibody. In addition, MIP-133 induced significant IL-8, IL-6, IL-1ß, and IFN-γ production, approximately two to three times (P < 0.05). CONCLUSIONS: MIP-133 interacts with phospholipids on plasma membrane of HCE cells and activates cPLA(2α). cPLA(2α) is involved in apoptosis, AA release, and activation of proinflammatory cytokines/chemokines from HCE cells. cPLA(2α) inhibitors may be a therapeutic target in Acanthamoeba keratitis.