Pathogenic Strains of Acanthamoeba Are Recognized by TLR4 and Initiated Inflammatory Responses in the Cornea

Abstract

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.

Pattern recognition receptors in microbial keratitis

“…172,173 Acanthamoeba is recognised by TLR4, which initiates the MyD88/NF-kB pathway and also the MAPK/ERK pathway, leading to release of proinflammatory and chemotactic mediators such as IL-8, CXCL2, TNF-α, and IFN-β. 174,175 Macrophage activation in the presence of antiAcanthamoeba antibody and IFN-γ has been shown to demonstrate trophozoite killing activity. 167,176 Macrophages are thought to be a crucial mediator in early infection: depletion of macrophages in a Chinese hamster model of Acanthamoeba keratitis produced a notable worsening of disease.…”

Identification of Acanthamoeba Membrane Protein That is Recognized by TLR4 on Corneal Epithelial Cells

“…Several groups have shown that TLRs recognize pathogens during ocular infection [26,27] . Recently, others and we have shown that TLR4 is expressed by HCE cells and is responsible for the recognition of Acanthamoeba trophozoites [24,40,41] . In this study, we aimed to determine the components of Acanthamoeba trophozoites that interact with TLR4 on corneal epithelial cells and trigger chemokines production.…”

“…Study of AK pathogenesis raises the hope of solving difficulties in targeting therapeutic agents to treat AK; therefore, several studies have been orchestrated on the pathogenesis of AK [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] . More recently, we observed that Acanthamoeba trophozoites activate toll-like receptor 4 (TLR4) on human and Chinese hamster corneal epithelial cells in vitro and in vivo and stimulate cytokine secretion and progression of keratitis [24] .…”

“…Chemokine IL-8 was quantified from cell supernatants using ELISA [21,23,24] . Briefly, cell culture supernatants were collected at the indicated times after treatments and centrifuged to remove cell debris.…”

“…recognize TLR4 on corneal epithelial cells in vitro and in vivo and induce chemokines production [24] . To determine the components of Acanthamoeba trophozoites that interact with TLR4 and stimulate chemokine IL-8 secretion from TLR4-expressing HEK293 and HCE cells, cells were treated with different doses of AcMP and IL-8 production was assessed by ELISA.…”