TLR4: the receptor bridging Acanthamoeba challenge and intracellular inflammatory responses in human corneal cell lines

Abstract

Acanthamoeba keratitis (AK) is a painful, vision-threatening infection caused by pathogenic strains of the protozoan, Acanthamoeba. Toll-like receptors (TLRs), which are important components of innate immunity, have an important role in the detection of foreign pathogens and the signaling cascades in host cells. However, no report on the interaction between Acanthamoeba and TLR has been found. In this study we analyzed the role of the TLR and its signaling pathway in human telomerase-immortalized corneal epithelial cells (HUCLs) and stromal fibroblasts (THSFs) challenged by Acanthamoeba. We show that the expressions of TLR4, myeloid differentiation protein 88 (MyD88), nuclear factor (NF)-jB, phospho-IjB, phospho-extracellular signal-regulated kinases 1/2 (p-Erk1/2) and the inflammatory cytokines interleukin (IL)-8, tumor necrosis factor (TNF)-a and interferon (IFN)-b were significantly increased in Acanthamoeba-treated cells. Pretreatment with anti-TLR antibodies or the specific inhibitors pyrrolidine dithiocarbamate (PDTC) (for the NF-jB pathway) and U0126 (for the ERK pathway) was conducted. It was found that anti-TLR4 antibody attenuated the production of cytokines induced by Acanthamoeba infection. PDTC inhibited the production of IL-8 and TNF-a whereas U0126 inhibited the synthesis of IFN-b. Thus, TLR4 is a receptor for Acanthamoeba and exerts an effect through TLR4-MyD88-NF-jB and TLR4-ERK1/2 pathways to induce the secretion of cytokines in human corneal cell lines challenged by Acanthamoeba.

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

“…These findings are in agreement with those of Ren et al [40] who found TLR4 was the main receptor that upregulated in HCE cells and induced upregulation of IL-8 and other cytokines after Acanthamoeba challenge. It is also possible that AcMP-P2 induced proinflammatory chemokines production by HCE cells through other mechanisms.…”

“…Previous findings showed that while several TLRs, including TLR1, TLR2, TLR3, TLR4, TLR5, and TLR9, are expressed constitutively, only TLR4 is involved in Acanthamoeba recognition on corneal epithelial cells [24,40,41] . Acanthamoeba spp.…”

“…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.…”

“…Previous studies showed that neutralizing TLR4 antibody inhibits the interaction of Acanthamoeba trophozoites in corneal epithelial cells and diminishes IL-8 secretion [24,40] . In the present study, we determined whether the neutralizing TLR4 antibody has the capacity to inhibit AcMP-P2-stimulated IL-8 secretion from HCE and TLR4-expressing HEK293 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.…”