Quantum dots induced interferon beta expression via TRIF-dependent signaling pathways by promoting endocytosis of TLR4

Abstract

Quantum dots (QDs) are nano-sized semiconductors. Previously, intratracheal instillation of QD705s induces persistent inflammation and remodeling in the mouse lung. Expression of interferon beta (IFN-β), involved in tissue remodeling, was induced in the mouse lung. The objective of this study was to understand the mechanism of QD705 induced interferon beta (IFN-β) expression. QD705-COOH and QD705-PEG increased IFN-β and IP-10 mRNA levels during day1 to 90 post-exposure in mouse lungs. QD705-COOH increased IFN-β expression via Toll/interleukin-1 receptor domain-containing adapter protein (TRIF) dependent Toll-like receptor (TLR) signaling pathways in macrophages RAW264.7. Silencing TRIF expression with siRNA or co-treatment with a TRIF inhibitor tremendously abolished QD705s-induced IFN-β expression. Co-treatment with a TLR4 inhibitor completely prevented IFN-β induction by QD705-COOH. QD705-COOH readily entered cells, and co-treatment with either inhibitors of endocytosis or intracellular TLRs prevented IFN-β induction. Thus, activation of the TRIF dependent TLRs pathway by promoting endocytosis of TLR4 is one of the mechanisms for immunomodulatory effects of nanoparticles.

Introduction

Newly engineered quantum dots (QDs) are semiconductor nanocrystals with a unique autofluorescent property with great potential for diagnosis and drug delivery, and imaging agents in biomedicine (Xing et al., 2006, Gao and Dave, 2007, Zhang et al., 2008, Mulder et al., 2010, Obonyo et al., 2010). QD705 emits stable near-infrared fluorescence and is approximately 12 ∼ 20 nm in diameter with a metalloid crystalline core (Cd/Se/Te) surrounded by a thin shell of ZnS (Dabbousi et al., 1997). The surface of QD705 can be applied for specific applications, such as fluorescence efficiency, solubility in biological media or bio-conjugation to antibodies for specific diagnostic and therapeutic purposes (Gao et al., 2004, Mulder et al., 2010). QD705-COOH has carboxyl groups conjugated onto the surface of QD705. QD705-PEG has been surface modified with methoxy-polyethylene glycol, which is an inert biologically compatible polymer.

QDs have great potential in terms of clinical applications, but they have been shown to cause immune responses in vitro and in vivo. Fischer et al. (2010) showed that QD and QD-PEG exposure increased Interleukin 6 (IL-6) expression in Kupffer cells. Organic, COOH and NH₂(PEG) QDs induced tumor necrosis factor alpha (TNF-α) expression at 24 h in macrophage cells (J774A.1 cell) (Clift et al., 2010). Previously, we demonstrated that intravenous administration of QD705-PEG increased TNF-α and IL-6 expression in mouse liver (Lin et al., 2011). In addition, QD705-COOH and QD705-PEG simultaneously induced inflammatory cytokines (TNF-α, IL-6, chemokine (C-X-C motif) ligand 1, CXCL1 and monocyte chemotactic protein 1, MCP-1) and anti-inflammatory cytokines (Interleukin 10, IL-10 and Interleukin 13, IL-13) in mouse lung (Ho et al., 2013a). Romoser et al. (2011) reported that CdSe/ZnS-COOH quantum dots exposure unregulated of inflammatory and immunoregulatory proteins in human skin cells. However, the mechanisms by which QDs induce immunoregulatory responses remain unclear.

The Toll-like receptors (TLRs) in mammals is the essential effectors to activation of innate immunity, inflammation and adaptive immunity by recognizing wide variety of pathogen-associated molecular patterns (Iwasaki and Medzhitov, 2004). TLRs include 10 (TLRs1-10) and 13 (TLRs1-13) family in human and mice. The TLR family includes cell surface receptor and intracellular receptor (Blasius and Beutler, 2010). The intracellular TLRs, including TLR3, TLR7, TLR8, and TLR9, are originally found to detect nucleic acid. On the other hand, TLR1, TLR2, TLR4, TLR5, and TLR6 are expressed largely on the cell surface where they recognize microbial components such as lipopeptides, Lipopolysaccharides (LPS) and flagellin (Kawai and Akira, 2009, Kumar et al., 2009). TLRs induce immune responses through their coupling with intracellular adaptor molecules, MyD88 and TRIF (Cook et al., 2004, Michelsen et al., 2004). While TLR4 mediates immune responses through both the MyD88 and TRIF pathways, TLR3 signals through the TRIF and all the other TLRs mediate through the MyD88 pathway (Pandey and Agrawal, 2006, Foster et al., 2007). In TRIF-dependent pathway, activation of the TLR triggers formation of a TRAM/TRIF/TRAF3/TBK1/IKKi complex, which leads to the activation of various transcription factors including interferon (IFN)-regulatory factor 3 (IRF3) and IRF7, and hence to the production of type I IFN genes, such as interferon beta (IFN-β), IFN-inducible protein 10 (IP-10) and regulated on activation, normal T cell expressed and secreted (RANTES) (Takeda and Akira, 2004, Premkumar et al., 2010).

Type I IFN mainly activate intracellular antimicrobial programmes and play roles in the development of innate and adaptive immune responses (Ivashkiv and Donlin, 2014). IFN-β is secreted by many cell types including macrophages, lymphocytes and others. IFN-β modulates innate immunity which promote antigen presentation and NK cell functions. Also, IFN-β promote the development of antigen-specific T cell and B cell responses and immunological memory (Ivashkiv and Donlin, 2014). IFN-β also functions as a deactivator of mononuclear phagocytes, and hence as an inhibitor of inflammation (Billiau, 1995). IFN-β-induced anti-tumor activities can also abrogate tumorigenicity and eradicate established tumors (Dong et al., 1999) and regulates antivirus immunological activity through the Th1/Th2 balance (Biron, 1998, Opal and DePalo, 2000). We previously demonstrated that intratracheal instillation of QD705-PEG and QD-COOH induced severe inflammation, but accompanied immunoregulatory responses (Ho et al., 2011). However, the mechanisms by which nanoparticles induce immunoregulatory responses remain unclear. Thus, we hypothesized that TRIF and TLRs might be involved in the immunoregulatory responses that are induced by nanoparticles such as QD705 in mouse lungs. Macrophages are pioneers of recruited innate immune cells that come into contact with foreign agents in the lungs. In our present study, we utilize the production of IFN-β in RAW264.7 cells as a model to investigate the role of TRIF and TLRs in QD705 induced immunoregulatory responses. Our findings indicate that endocytosis of QD705, accompanied with endocytosis of TLR4, triggered TRIF dependent TLR signaling for subsequent IFN-β induction, which provides important new insights regarding the mechanisms by which these nanoparticles induced immunoregulatory responses.