TRIF Is a Critical Negative Regulator of TLR Agonist Mediated Activation of Dendritic Cells In Vivo Sergey S. Seregin., Yasser A. Aldhamen . , Daniel M. Appledorn, Charles F. Aylsworth, Sarah Godbehere, Chyong-Jy Joyce Liu, Dionisia Quiroga, Andrea Amalfitano* Department of Microbiology and Molecular Genetics, Department of Pediatrics, College of Osteopathic Medicine, Michigan State University, East Lansing, Michigan, United States of America Abstract Despite recent advances in developing and licensing adjuvants, there is a great need for more potent formulations to enhance immunogenicity of vaccines. An Eimeria tenella derived antigen (rEA) augments immune responses against several pathogens in animal models and recently was confirmed to be safe for human use. In this study, we have analyzed the molecular mechanisms underlying rEA activity in mice, and confirmed that rEA activates multiple immune cell types, including DCs, macrophages, NK, B, and T cells. The rEA adjuvant also elicits the induction of pleiotropic pro-inflammatory cytokines, responses that completely depend upon the presence of the TLR adaptor protein MyD88. Surprisingly, we also found that the TRIF adaptor protein acts as a potent negative regulator of TLR agonist-triggered immune responses. For example, IL12 production and the induction of co-stimulatory molecule expression by DCs and IFNc production by NK cells in vivo were significantly increased in rEA-treated TRIF-KO mice. Importantly, however, TRIF suppressive effects were not restricted to rEA-mediated responses, but were apparent in LPS- or ODN2006-activated DCs as well. Taken together, our findings confirm that rEA is a potent adjuvant, triggering robust activation of the innate immune system, in a manner that is augmented by MyD88 and inhibited by TRIF; thereby unveiling the potential complexities of modulating TLR activity to augment vaccine efficacy. Citation: Seregin SS, Aldhamen YA, Appledorn DM, Aylsworth CF, Godbehere S, et al. (2011) TRIF Is a Critical Negative Regulator of TLR Agonist Mediated Activation of Dendritic Cells In Vivo. PLoS ONE 6(7): e22064. doi:10.1371/journal.pone.0022064 Editor: Karin E. Peterson, National Institute of Allergy and Infectious Diseases - Rocky Mountain Laboratories, United States of America Received March 8, 2011; Accepted June 14, 2011; Published July 8, 2011 Copyright: 2011 Seregin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: National Institutes of Health grants RO1DK-069884, P01 CA078673, the MSU Foundation (http://www.msufoundation.msu.edu/) as well the Osteopathic Heritage Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: amalfit1@msu.edu . These authors contributed equally to this work Introduction There is a great need to develop more efficient vaccines to combat or prevent infections by a number of detrimental pathogens that continue to plague mankind [1,2,3]. The use of novel adjuvants capable of beneficially stimulating the immune system to maximize efficacy of various vaccination strategies is a rapidly developing field. Most adjuvants augment the induction of innate immune responses by triggering robust activation of dendritic cells (DCs) and macrophages, actions that can result in improved induction of antigen specific adaptive immune respons- es. Upon migration to the draining lymph nodes, these highly active antigen presenting cells (APCs) are capable of presenting specific antigens to responsive T cells, thereby generating significant pools of antigen-specific T cells [2,4]. Incorporation of Toll-like receptor (TLR) ligands into vaccine formulations represent a class of adjuvants proposed for usage in next generation vaccines. This is primarily due to TLRs being expressed at high levels on important immune cell types (DCs, macrophages, NK cells) and their ability to potently activate the innate immune system [4,5,6]. In light of these facts, the recombinant, Eimeria tenella derived antigen (rEA) has been proven to be capable of inducing IL-12p70 production, enhancing Th1 cellular responses, and yielding protection against Toxoplasma gondii infection in mice [7]. rEA has also been shown to be an efficient immunomodulator, having both antiviral and anti-cancer properties [8,9,10]. Previous studies have also shown that HIV-gag-specific T cell responses are significantly increased when rEA formulations are administered together with the antigen [11,12]. Moreover, rEA showed no evidence of toxicity in pre-clinical [8] and clinical trials [13]. Specifically, no severe adverse reactions were reported in human clinical trials despite detection of increased IL-12 responses in 30% of the treated cancer patients [13]. The rEA protein has a relatively high amino acid sequence homology (67%) and shares very similar biological activities in vitro and in vivo with T. gondii-derived profilin-like protein, both of which trigger potent IL-12 responses in DCs. The profilin induced responses were completely dependent upon the adaptor protein MyD88 and at least partially mediated via TLR11 [14]. Moreover, it has been shown in vitro that human TLRs (TLR2, TLR3, TLR4, TLR5, TLR7, TLR8 and TLR9) do not transduce rEA signaling [9]. Therefore, TLR11 has been suggested as the rEA receptor mediating rEA signaling, but this notion remains to be confirmed. Since no functional human TLR11 homolog has been discovered, these facts leave unidentified the mechanism underlying rEA action in humans, and opens a discussion regarding other pattern recognition receptors (PRRs) that may PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e22064