Inhibition Of Epstein-Barr Virus Binding To Host Cells And Virus-induced Inflammation By Lactoferrin

Lactoferrin (LF) is an iron-binding glycoprotein of the transferrin protein family, which is expressed by mucosal epithelial cells and polymorphonuclear leukocytes and is secreted into most biological fluids including human milk, saliva, nasal secretions, tears, intestinal mucus and genital secretions. A wide range of functions have now been described for LF, including iron homeostasis, anti-microbials, anti-inflammatory a ctivity and cancer protection. As a key element of innate immunity, LF plays vital roles in host defense against infection and excessive inflammation.Epstein-Barr virus (EBV) is widespread in human populations, which causes infectious mononucleosis and has a strong association with various malignancies such as Hodgkin's disease (FDD), Burkitt's lymphoma (BL), and nasopharyngeal carcinoma (NPC). EBV exhibits a marked tropism for B lymphocytes. Its selective binding to B cells is initiated by interaction of the major viral envelope glycoprotein with its receptor CD21on the B cell surface and after entering into cells, it will immortalize B cells. EBV can also be detected in epithelial cells and is potentially oncogenic for epithelial cells. Recent studies demonstrated that EBV can infect monocytes/marcophages and dendritic cells as well as T cells in vitro and trigger the cells of the immune system to produce inflammatory mediators, which will be beneficial for preventing against infection at early stage, but cause damages to the organisms due to excessive inflammation.Through the linkage analysis of eighteen pedigrees from the Hunan province of South China, our previous study revealed that potential susceptibility loci related to NPC are located on chromosome3p21. Further isolation and identification of a list of new tumor suppressor genes for NPC from this region was pursued and LF gene is one of the candidates. At the same time, by means of NPC TMA and ELISA assay, we found that LF proteins are absent or significantly down-regulated in most NPC tissues and that its expression levels are reversely correlated with EBV VCA-IgA titers. Therefore, we speculated whether absent or low LF protein levels raise more risks for EBV infection and subsequent damages to organisms following EBV infection.LF has been reported to have a notable activity against a wide variety of viruses such as HCMV, HSV-1,2, HIV, HBV and HCV. The mechanism of LF in preventing from virus infection is considered to be the result of LF preventing virus adsorption to host cells through its binding to the virus particle directly or to the viral receptor of the targeting cells. Meanwhile, LF has also been showed to have the inhibitory activity of inflammatory response induced by a variety of viruses. Until now, the protection activities of LF against EBV infection and EBV induced inflammatory response have not been reported.In this study, we have identified LF as a potential inhibitor against EBV infection and EBV induced inflammation, and also provided the mechanisms of its functions.1. LF blocks EBV binding to B cells via interacting with EBV receptor CD21, and inhibits EBV entering into B cellsIn initial virus binding experiment, we observed that LF pre-treatment could inhibit EBV binding to human primary B lymphocyte cells surface at a certain extent by both flow cytometry and immunofluorescence assay. We next demonstrated that LF has the ability of preventing EBV entering into B cells by Q-PCR and FISH assay. To clarify the possible mechanism of LF preventing EBV binding to B cells, we used confocal microscopy experiments and coimmunoprecipitation assay to demonstrate the interaction of LF and EBV receptor CD21. These findings suggested that LF is likely to inhibit EBV entering into B cells through interfering with the virus binding to its receptor CD21.2. LF has no effects on the replication of EBV in B lymphoblastoid cellsLF has a notable inhibitory activity against the replication of some viruses. We next investigated the effect of LF on the replication of EBV in transformed B cells and human lymphoma cells P3HR1. The data showed that LF has no effect on the EBV replication.3. LF inhibits EBV transmission from B cells to epithelial cellsEBV targets not only primary B cells but also some epithelial cells including nasopharyngeal epithelial cells. Now it has been considered that the pathway that EBV enters into epithelial cells mainly depends on EBV positive B cells as a transfer vehicle. A cell and cell co-culture system was employed to determine whether LF has inhibitory effects on the infection of nasopharyngeal epithelial cells by EBV. It was showed that LF inhibits EBV transmission from B cells to epithelial cells by Q-PCR and FISH assay.4. LF reduces synthesis of IL-8and MCP-1induced by EBV via its suppression of NF-κB activityAlthough EBV is considered to be a B-lymphotrophic virus, it can also infect epithelial cells, and growing evidence shows that many other cell types are permissive to EBV infection. Stimulation of immune cells with EBV leads to the synthesis of several inflammatory factors including IL-8, MCP-1, TNF-α and IL-6. Inflammation will protect host against EBV infection and also can bring about damages owing to nonresolving inflammation. It is well known that LF has anti-inflammatory activity. We showed that LF inhibits the synthesis of IL-8and MCP-1induced by EBV infection. Since NF-κB is a key link in the course of inflammatory response, we verified that LF inhibits EBV-indcued NF-κB activation, which indicated that LF negatively regulated the inflammatory response by interfering with the upstreams of NF-κB.5. LF suppresses NF-κB activation by binding to TLR2EBV gp350binds to surface protein TLR2and thus can activate NF-κB signaling in monocytes. We showed that LF inhibits NF-κB activation mediated by TLR2by means of luciferase assay. Our previous study showed that LF binds to EBV receptor CD21. Since CD21also can bind to EBV gp350, it implied that LF is likely to have a similar structure or component with gp350, and it may also interact with TLR2. We demonstrated that LF can bind to TLR2through confocal microscopy experiments and coimmunoprecipitation assay.6. LF inhibits EBV DNA induced TLR9activation, decreasing the synthesis of IL-8In the above results, it was showed that LF inhibits the synthesis of IL-8and MCP-1induced by EBV, and the inhibitory effects of LF on IL-8is more significant than MCP-1. EBV particles, EBV DNA and some EBV encoded proteins can stimulate IL-8secretion, among which EBV DNA can be recognized by TLR9and then induce IL-8expression in the monocytes/macrophages. We showed that LF inhibits EBV DNA-induced IL-8expression through modulating TLR9's location in endosome and thus inhibiting TLR9activation.7. CD14enhances EBV DNA induced TLR9activation, leading to up-regulation of IL-8Through recognizing EBV DNA, TLR9can activate MYD88-dependent NF-κB signaling and induce IL-8synthesis and release in macrophages. However, the specific process that TLR9recognizes and responses to EBV DNA is not known. A recent study showed that CD14could promote CpG DNA-induced inflammatory response by means of being a partner of CpG DNA and the co-receptor of TLR9. In the current study, CD14is confirmed to promote the both EBV and EBV DNA-induced IL-8up-regulation.8. LF negatively regulates TLR9pathway through binding and inhibiting CD14LF can bind to CD14and thus inhibit LPS-induced inflammation. To explore the role of LF in the process that EBV DNA induced TLR9activation, we showed that LF partially inhibits the effects of CD14on promoting EBV or EBV DNA induced inflammation as well as IL-8up-regulation, whereas deletion mutant of LF, which cannot bind to CD14, does not have this ability. Then it was further confirmed that LF inhibits the TLR9activation by means of interfering CD14binding to TLR9, which will inhibit the ability of TLR9recognizing EBV DNA.In conclusion, LF protects both B cells and epithelial cells from EBV infection. LF prevents EBV infection of B cells through its interacting with EBV receptors CD21. LF shows no effects on the replication of EBV in B cells. LF inhibits EBV-induced inflammation through two mechanisms. One is LF suppresses EBV gp350-induced TLR2pathway, leading to down-regulation of NF-κB signaling. The another is LF inhibits TLR9activation by binding to CD14, which blocks the interaction between CD14and TLR9.