Direct Engagement of TLR4 in Invariant NKT Cells Regulates Immune Diseases by Differential IL-4 and IFN-c Production in Mice Ji Hyung Kim1,2.¤a, Hye Sung Kim1,2., Hye Young Kim1¤b, Sae Jin Oh1,2, Doo Hyun Chung1,2* 1 Department of Pathology, Seoul National University College of Medicine, Seoul, Korea, 2 Laboratory of Immune Regulation in Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea Abstract During interaction with APCs, invariant (i) NKT cells are thought to be indirectly activated by TLR4-dependently activated APCs. However, whether TLR4 directly activates iNKT cells is unknown. Therefore, the expression and function of TLR4 in iNKT cells were investigated. Flow cytometric and confocal microscopic analysis revealed TLR4 expression on the surface and in the endosome of iNKT cells. Upon LPS stimulation, iNKT cells enhanced IFN-c production, but reduced IL-4 production, in the presence of TCR signals, depending on TLR4, MyD88, TRIF, and the endosome. However, enhanced TLR4- mediated IFN-c production by iNKT cells did not affect IL-12 production or CD1d expression by DCs. Adoptive transfer of WT, but not TLR4-deficient, iNKT cells promoted antibody-induced arthritis in CD1d2/2 mice, suggesting that endogenous TLR4 ligands modulate iNKT cell function in arthritis. Furthermore, LPS-pretreated WT, but not TLR4-deficient, iNKT cells suppressed pulmonary fibrosis, but worsened hypersensitivity pneumonitis more than untreated WT iNKT cells, indicating that exogenous TLR4 ligands regulate iNKT cell functions in pulmonary diseases. Taken together, we propose a novel direct activation pathway of iNKT cells in the presence of TCR signals via endogenous or exogenous ligand-mediated engagement of TLR4 in iNKT cells, which regulates immune diseases by altering IFN-c and IL-4 production. Citation: Kim JH, Kim HS, Kim HY, Oh SJ, Chung DH (2012) Direct Engagement of TLR4 in Invariant NKT Cells Regulates Immune Diseases by Differential IL-4 and IFN-c Production in Mice. PLoS ONE 7(9): e45348. doi:10.1371/journal.pone.0045348 Editor: Geetha P. Bansal, Tulane University, United States of America Received November 23, 2011; Accepted August 21, 2012; Published September 19, 2012 Copyright: 2012 Kim 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: This work was supported by a National Research Foundation (www.nrf.re.kr) of Korea grant funded by the Korean government (Ministry of Education, Science and Technology) (2010-0017890). 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: doohyun@snu.ac.kr ¤a Current address: Immune Disease Institute, Program in Cellular and Molecular Medicine at Children’s Hospital, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States of America ¤b Current address: Division of Immunology, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts, United States of America . These authors contributed equally to this work. Introduction Invariant (i) natural killer T (NKT) cells express intermediate levels of a semi-invariant Va24-Ja15 TCR in humans and Va14- Ja18 TCR in mice [1], which recognize the glycolipid antigens presented by CD1d [2]. NKT cells regulate various immune responses in autoimmune diseases [3,4,5], tumor rejection [6], pulmonary fibrosis [7], and infection [8,9]. Upon activation, iNKT cells rapidly produce large amounts of IL-4 and IFN-c [10], which regulate innate and adaptive immune responses [11]. Therefore, it has been suggested that iNKT cells regulate immune diseases by modulating the Th1/Th2 balance in vivo. Toll-like receptors (TLRs) are pathogen recognition molecules that activate the immune system as part of innate immunity [12]. TLRs expressed in dendritic cells (DCs) link innate and adaptive immunity by regulating the expression of chemokine receptors and adhesion molecules that allow DCs to mature and migrate into the draining lymph nodes [12]. TLR4 is complexed with MD-2 and CD14, and binds lipopolysaccharide (LPS) [13]. Upon ligand binding, TLR4 associates with myeloid differentiation factor 88 (MyD88) and Toll-interleukin-1 receptor domain-containing adaptor inducing IFN-b (TRIF), which is crucial for the recruitment of several proteins that are critical in signal trans- duction [14,15]. MyD88-deficient mice have a profound defect in the activation of antigen-specific Th1 but not Th2 immune responses, suggesting that TLR signals play a critical role in balancing Th1/Th2 responses [16]. TLR4 specifically promotes production of the Th1-inducing cytokine IL-12 [17] and the chemokine interferon-gamma inducible protein (IP)-10 [18]. In contrast, Dabbagh and colleagues demonstrated that TLR4 is required for optimal Th2 responses against nonpathogenic antigens, which are dependent on DC maturation and cytokine production [19]. These conflicting data suggest that TLR4 plays a complex role in regulating the Th1/Th2 balance, depending on target tissue microenvironment. Upon TLR4 engagement, antigen presenting cells (APCs) are activated and produce IL-12 (cytokine- driven) and/or enhance presentation of endogenous glycolipid by CD1d molecules (self-antigen-driven) to iNKT cells, triggering their activation [20,21]. The constitutive expression of high levels of IL-12 receptor endows iNKT cells with a rapid response to IL- 12 and activation [22,23]. Based on these findings, cytokine- and self-antigen-driven indirect pathways have been suggested to be PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45348