Simultaneous induction of autophagy and toll-like receptor signaling pathways by graphene oxide

Abstract

Graphene oxide (GO) nanosheets have sparked growing interests in biological and medical applications. This study examined how macrophage, the primary immune cell type engaging microbes, responded to GO treatment. We uncovered that incubation of macrophage cell RAW264.7 with GO elicited autophagy in a concentration-dependent manner, as evidenced by the appearance of autophagic vacuoles and activation of autophagic marker proteins. Such GO-induced autophagy was observed in various cell lines and in macrophage treated with GO of different sizes. Strikingly, GO treatment of macrophage provoked the toll-like receptor (TLR) signaling cascades and triggered ensuing cytokine responses. Molecular analysis identified that TLR4 and TLR9 and their downstream signaling mediators MyD88, TRAF6 and NF-κB played pivotal roles in the GO-induced inflammatory responses. By silencing individual genes in the signaling pathway, we further unveiled that the GO-induced autophagy was modulated by TLR4, TLR9 and was dependent on downstream adaptor proteins MyD88, TRIF and TRAF6. Altogether, we demonstrated that GO treatment of cells simultaneously triggers autophagy and TLR4/TLR9-regulated inflammatory responses, and the autophagy was at least partly regulated by the TLRs pathway. This study thus suggests a mechanism by which cells respond to nanomaterials and underscores the importance of future safety evaluation of nanomaterials.

Introduction

Graphene and its oxidized form, graphene oxide (GO), have drawn intense attention in recent years for biological and medical applications [1]. The surface of GO contains hydrophilic oxygen-containing functional groups (i.e. hydroxyl, epoxyl and carboxyl tails) on the basal plane and edges, rendering GO amenable to stable dispersion in water and functionalization. These attributes have prompted the use of GO for bioimaging [2], cellular probing [3], cellular growth and differentiation [4], gene and drug delivery [5], [6] and photothermal therapy [7]. These burgeoning applications in biomedicine entail the need to evaluate the in vitro and in vivo safety of GO.

Autophagy is a process that degrades intracellular components in response to stressful conditions (e.g. starvation and infection) and is linked to cellular processes as diverse as cell survival, cell death, pathogen clearance and antigen presentation [8], [9], [10]. Autophagy involves the formation of double-membraned vesicles termed autophagosomes, which sequester cytoplasm and organelles and then fuse with lysosomes to form autolysosomes, thus degrading the contents of the vacuole [10]. Autophagy is negatively controlled by mTOR (mammalian target of rapamycin) complex 1 (mTORC1) and inhibition of mTORC1 kinase activity initiates the formation of autophagosome that comprises a complex consisting of Beclin 1 and other factors. The autophagosome formation also involves the conversion of microtubule-associated protein light chain 3 (LC3-I) to the lipidated form LC3-II, consequently conversion from LC3-I to LC3-II is a common indicator of autophagy.

Toll-like receptors (TLRs) are important receptors for the detection of microbial antigens and subsequent induction of innate immune responses [11]. Among the TLRs, TLR2 recognizes bacterial lipoproteins while TLR3 detects virus-derived dsRNA. TLR4 recognizes lipopolysaccharides (LPS) and TLR5 recognizes bacterial flagellin. TLR7 mediates recognition of viral ssRNA while TLR9 senses unmethylated DNA with CpG motifs derived from bacterial and viruses [12]. Upon engagement with cognate ligands, the TLRs transduce signals by first recruiting adaptor proteins including myeloid differentiating factor 88 (MyD88) and TIR domain-containing adaptor inducing IFN-beta (TRIF), followed by activation of downstream signaling proteins such as TRAF6 and NF-κB, eventually resulting in various cellular responses including secretion of cytokines and interferons (IFNs).

The connection between autophagy and TLRs was discovered in 2007 as it was found that TLRs signaling in macrophages links the autophagy pathway to phagocytosis [13] and TLR4 stimulation enhances the autophagic elimination of phagocytosed mycobacteria in macrophages [14]. Ensuing studies further reported that TLR2, TLR3 and TLR7 play roles in autophagy induction [15], [16]. The TLR-activated autophagy is regulated by the interaction of MyD88 or TRIF with Beclin 1 [17] and TLR engagement induces the interaction, thereby reducing the binding of Beclin 1 to inhibitory molecules. Beclin 1 also interacts with TRAF6 so as to facilitate its activation and subsequent formation of autophagosomes [18], thus TRAF6 seems to be crucial for TLR-activated autophagy [18]. The regulatory interaction of heat shock protein HSP90 with Beclin 1 is also required for autophagy induction downstream of TLR3 and TLR4 [19].

Since GO holds promise for diverse in vivo applications and macrophage is the primary immune cell type that engages foreign substances and modulates the immune responses in vivo, the overriding objective of this study was to explore how macrophage responded to GO treatment in order to evaluate the safety of GO.