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-γ 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-γ 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 CD1d−/− 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-γ and IL-4 production.
Highlights
Invariant (i) natural killer T (NKT) cells express intermediate levels of a semi-invariant Va24-Ja15 TCR in humans and Va14Ja18 TCR in mice [1], which recognize the glycolipid antigens presented by CD1d [2]
Results iNKT Cells Constitutively Express TLR4 on the Surface and in the Early Endosome To explore whether iNKT cells express TLR4, liver mononuclear cells were obtained from wild type (WT) B6 or TLR42/2 mice and TLR4 expression was examined both on the cell surface and in the cytoplasm of iNKT cells, macrophages, and conventional T cells
TLR4 was co-localized with the early endosome maker EEA-1 in the cytoplasm of a-GalCer/CD1d tetramer+ iNKT cells from WT B6 mice, indicating that TLR4 is localized in the endosomal compartment of the cytoplasm in iNKT cells
Summary
Invariant (i) natural killer T (NKT) cells express intermediate levels of a semi-invariant Va24-Ja15 TCR in humans and Va14Ja18 TCR in mice [1], which recognize the glycolipid antigens presented by CD1d [2]. 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]. 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. The constitutive expression of high levels of IL-12 receptor endows iNKT cells with a rapid response to IL12 and activation [22,23] Based on these findings, cytokine- and self-antigen-driven indirect pathways have been suggested to be the mechanisms by which TLR4-mediated activation of APCs regulates iNKT cell functions [21]. It is generally accepted that iNKT cells are activated indirectly by TLR4dependently activated DCs rather than directly by self-expressed TLR4 expressed
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