Capsid proteins of foot-and-mouth disease virus interact with TLR2 and CD14 to induce cytokine production
Introduction
Foot-and-mouth disease (FMD) occurs worldwide and frequently causes considerable economic loss in the animal husbandry industry. The impact of an FMD outbreak includes disruptions in the animal feed, veterinary, pharmaceutical and tourism associated industries. The etiological agent of FMD is the FMD virus (FMDV) that is highly infectious to cloven-hoofed animals, including all commonly held livestock such as cattle, sheep, goats and pigs. As FMD inflicts heavy losses in livestock productivity, it is considered to be the biggest disease threat to the trade of animals and animal products in the world [1].
FMDV belongs to the Aphthovirus genus of the Picornaviridae family and is a very contagious RNA virus [2]. Its viral particle contains a RNA genome within an icosahedral capsid. The FMDV capsid is composed of 4 structural proteins, VP1, VP2, VP3 and VP4, in which VP4 is located internally, whereas VP1, VP2 and VP3 are surface oriented [3]. The immune responses of the host during FMDV infection and vaccination have been studied and found to involve IFNs [4]. Other cytokines such as IL-6, IL-8 and IL-12 have also been shown to increase in the plasma of FMDV vaccinated pigs [5]. It is thus generally believed that monocytes/macrophages are activated by FMDV antigens during FMDV infection and vaccination. However, the binding epitope(s) of FMDV and the receptors on host immune cells responsible for immune responses remain unclear.
The host innate immune system recognizes specific patterns of microbes by pattern recognition receptors (PRRs) expressed by antigen presenting cells such as monocytes/macrophages and dendritic cells. There are a variety of PRRs that include TLRs, nod-like receptors (NLRs), and RIG-I-like receptors (RLRs) [6,7]. Among these PRRs, TLRs are the most well-characterized so far. When binding to specific ligands, TLRs recruit signaling adaptor protein MyD88 or TRIF to initiate responses and activate the NF-κB and MAPK pathways resulting in the production of pro-inflammatory cytokines and type I interferons [8,9]. There is selective interaction between various TLRs and their respective ligands. For instance, TLR3, TLR7 and TLR8 recognize microbial RNA; TLR9 recognizes microbial DNA; TLR5 recognizes bacterial flagellin; TLR4 recognizes lipopolysaccharides (LPS) from bacteria; TLR2 alone and heterodimers with either TLR1 or TLR6 recognize a broad range of cell surface components of microbes including bacteria, fungi, virus and parasites [10,11]. The responses of TLRs to their ligands can be regulated by accessory molecules such as monocyte differentiation antigen CD14 that has been found to bind bacterial LPS and viruses on the cell surface to induce cytokines after viral or bacterial infection [12].
Expression of TLR4, 5, 7, 8, and 9 has been reported in swine immune cells [7]. Zhang [13] reported increased mRNA expression levels of TLR4 and IFNs in cattle infected with FMDV. Other than TLRs, RLRs such as helicase MDA-5 in porcine kidney cell line PK-15 cells have been shown to recognize FMDV RNA [14]. However, which specific component(s) of FMDV is recognized by which specific immune cell PRRs to activate which signal pathway(s) are unknown.
In this study, we found that BEI-FMDV activated TLR2 via its capsid proteins VP1 and VP3 by using the NF-κB reporter assay. Our results demonstrated that rVP1 and rVP3 but not rVP0 bind to TLR2 and CD14 to activate NF-κB and induce IL-6 in a MyD88 dependent manner. Although TLR1 and TLR6 did not bind directly to rVP1 and rVP3, they enhanced rVP1- and rVP3-mediated activation of TLR2 signaling most likely via the formation of the TLR1/TLR2 and TLR2/TLR6 heterodimer. Further studies were also conducted to understand the recognition of BEI-FMDV, VP1 and VP3 by swine macrophages.
Section snippets
Cell lines and cell culture
The 293 T human embryonic kidney cells and mouse RAW264.7 macrophages were obtained from American Type Culture Collection (VA, USA). HEK 293 cells stably transfected with the HA-tagged human TLR2 gene (293-hTLR2-HA), human TLR6 gene (293-hTLR6-HA), and human CD14 gene (293-hCD14-HA) were purchased from InvivoGen (CA, USA). 293 T and RAW264.7 cells were maintained in complete DMEM that contained 10% heat-inactivated FBS, 50 μg/ml penicillin, 50 μg/ml streptomycin sulfate and 100 μg/ml neomycin
FMDV capsid proteins activate TLR2 signaling pathway in immune cells
Although BEI-FMDV with structurally intact capsids has been shown to induce cytokines such as IL-6, IL-8, IL-12, IFN-α, IFN-β, and IFN-γ in pigs [5], its underlying molecular mechanism of action is not well delineated. To investigate whether PRRs, particularly TLRs are required for elicitation of immune responses to FMDV, human embryonic kidney (HEK) 293 T cells that lack constitutive TLRs were transiently transfected with each kind of specific TLR-expressing plasmid and co-transfected with
Discussion
Infection of cattle with FMDV has been shown to increase the TLR4 mRNA expression level in nasal-associated lymphoid tissue [13]. However, it has not been shown whether or not TLR4 is linked to the FMDV-induced immune responses. By screening nine TLRs, we found that BEI-FMDV expressing structurally intact capsid of FMDV was recognized by TLR2, but not TLR4 or other TLRs, to activate NF- кB in an MyD 88-dependent manner (Fig. 1) and produce IL-6 in macrophages (Fig. 2). Energy transfer analysis
Acknowledgments
We thank Dr. Ming-Hwa Jong from the National Institute for Animal Health for providing the BEI-FMDV (O/Taiwan/97). We also thank Dr. Chi-Min Cheng from the Agricultural Technology Research Institute for providing swine macrophages and Ms. Miranda Loney of the Agricultural Biotechnology Research Center English Editors’ Office, Academia Sinica, for English editorial assistance. This work was supported partly by the National Science Council (NSC96-2313-B-001-002) and partly by funding from
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These authors contributed equally to this work.