Gold salts have long been used to treat various diseases such as rheumatoid arthritis and tuberculosis[17]. Today, AuNPs are widely used in targeted drug delivery, cell imaging, cancer diagnostics, and treatments[1]. The effectiveness of these particles depends on their ability to target specific cells followed by their internalisation. The nanometric size of AuNPs plays a critical role in their internalization; 50 nm AuNPs have been shown to be the most efficiently internalized by cells[18]. As these nanoparticles are internalised and accumulated inside cells, they may interfere with the cellular fate and functions. This issue is especially important concerning the immune system, because of the capacity of some cells of this system to actively capture extra cellular materials and control immune responses by the delivery of inflammatory signals for stimulation and orientation of antigen presentation, leading to specific immune responses. A precise knowledge of the effects of AuNPs on APCs, including macrophages (M) and dendritic cells (DCs), provides a valuable insight into the biological consequences of AuNPs exposures and to define putative adverse effects. It is yet well established that NPs immunotoxicity should be tested on APCs [19, 20], because these cells are involved in nonspecific innate defences as well as in specific immune responses. Furthermore, DCs play a critical role as a bridge between innate and adaptive immune systems by initiating the T cell-mediated response[21].Therefore, perturbation of the functions of these cells may result in altered immune response in NP-exposed individuals. In consequence, M and DCs appear to be appropriate tools for discriminating between NPs that interfere with the immune system or not. The current work provides a comparative study on the influence of AuNPs at subtoxic concentration (10 and 50 µg/ml) on two primary professional phagocytic cells derived from the bone marrow of mice, BMDCs and BMDMs. Their activities and characteristics: phagocytosis, cell activation, cytokine secretions, redox status together with their ability to present antigens was analysed in details. In addition, we provide a study into the metabolic activity, pointing at different influences of AuNPs on mitochondrial metabolism and glycolysis of BMDCs and BMDMs.
The phagocytic capacity of APCs leads to the accumulation of NP inside cells therefore it is important to investigate the sub cellular localization of AuNPs in APCs. Accumulation of AuNPs inside intracellular vesicles of J774.A1 M cell line [22] and more precisely in lysosome of Raw 264.7 M cell line [23] has been shown by TEM and confocal microscopy. In the present study, we demonstrate the presence of AuNPs in early (EEA1+) and late (LAMP1+) endosomes of APCs by confocal microscopy. In addition to AuNPs identified in EEA1 and LAMP1 vesicles aggregated clusters of AuNPs were observed in the cytosol of APCs using confocal microscopy and more precisely with TEM.
One of the key point of APCs is their ability to engulf foreign material by phagocytosis. The exposure to 10 nm AuNPs was shown to significantly reduce the phagocytic capacity of Raw 264.7 M cell line and BMDMs[24]. In the present study, we show that the phagocytic capacity of J774.1A M cell line is not impaired after 24 h exposure with AuNPs. A possible explanation for these different effects could be that different cell lines of M were used and /or to the nature of the phagocytosis assay that is based on the use of E coli [24] and of polystyrene beads in our study.
Accumulation of nanoparticle inside the APCs could be an important perturbation for the activation state of the cells. Cell interaction with nanoparticle may hinder their activation state which could be evaluated by the cell surface expression of CD86 and MHC-II. We found that AuNPs did not activate the BMDCs by themselves but increase the response of BMDCs to LPS. This finding is consistent with previous studies showing the increased expression of MHC-II [14]and CD86[25].Interestingly, BMDMs display a different behaviour: treatment with AuNPs mildly reduced the expression of activation markers in a dose dependent manner. However, AuNPs treated BMDMs did not show any alteration of the expression of activation markers upon LPS stimulation, which may be due to saturation of CD86 and MHC-II expression. Although in case of BMDMs increase of CD86 expression upon LPS stimulation is more evident than both the CD86 and MHC-II expression level.
Upon activation, APCs secrete several immune-regulatory molecules. We showed that AuNPs by themselves did not fuel any cytokine secretion. These data are in the direct line of previous studies on DCs [14] and on M [24], indicating that AuNPs do not promote production of TNF-α, IL-6 and IL-10. These findings were also validated by other previous publications in various models [23, 26]. However, we see a mild increase in TNF-α and MCP-1 production in AuNPs-treated BMDMs upon LPS stimulation at a high concentration (50 µg/ml).
We observed that AuNPs neither induce NO and ROS production in unstimulated BMDMs nor alter their production after LPS stimulation. This is consistent with previous finding [26]. Concerning BMDCs, we have seen a significant reduction of ROS production after LPS activation but only at high concentration of AuNPs while NO production remains unchanged.
To evaluate the effect of AuNPs on the metabolism of BMDMs and BMDCs, we stimulated them either with LPS or IL-4 as representatives of different microenvironments which may affect cellular functions. For example, conventional activation of pro-inflammatory cells by LPS facilitate inflammation and participate to the host defence against various kinds of microbial threats. On the other hand, alternative activation by IL4 induced anti-inflammatory cells, a potent suppressors and controllers of ongoing immune responses. Such stimulated cells exhibit a distinguishable regulation of their metabolism: LPS-activated proinflammatory cells undergoing a metabolic switch to enhance glycolysis[27, 28]. Alternatively, IL-4 stimulated cells relay on both fatty acid oxidation (FAO) and mitochondrial oxidative phosphorylation (OXPHOS) for sustained energy [10]. Thus, altered metabolism is not only a key feature of stimulated cell function but also a prerequisite for a proper response to immune stimuli. To analyse the effect of AuNPs on mitochondrial metabolism and glycolysis, mitostress and glycostress assays were performed using BMDCs and BMDMs. We showed that though AuNPs were found not to alter the basal mitochondrial respiration of BMDCs they increase it for BMDMs. The possible explanation for this phenomenon is the different phagocytic capacity of these cells. Indeed it is established that the phagocytic index of M is higher than that of DCs [29], thereby enabling accumulation of a large quantities of AuNPs in BMDMs, and increasing the basal respiration to meet the endogenous ATP demand of the cell. Unaltered proton leakage in BMDCs and BMDMs suggests that AuNPs did not induce any mitochondrial damage in these cells. Measurement of ATP production shows that pre-treatment with AuNPs did not alter the ATP production of BMDCs but increased it in BMDMs, which is consistent with basal respiration.
The basal respiration rate does not accurately reflect the ability of cellular respiration to respond to increased energy demand. As such, estimating the maximum capacity of substrate oxidation can be extremely valuable for the discovery of mechanisms by which AuNPs could affect cell metabolism.
Conversely, spare respiratory capacity indicates the reserve capacity of a cell to respond to an increased ATP demand and withstand periods of stress. Here, we show that pre-treatment with AuNPs significantly decreases the maximal and spare respiratory capacities of unstimulated and IL-4-stimulated BMDCs (cells, largely dependent on mitochondrial metabolism) but increases them significantly in unstimulated and IL-4-stimulated BMDMs. The increase in these two parameters in BMDMs can be explained by the increase in basal respiration as well as ATP production due to the increase in cellular energy demand. However, a significant decrease in both these parameters in BMDCs shows the impact of AuNPs on BMDCs. The possible explanation for this could be either alteration of the mitochondrial content and cristae density or alteration of the respiratory substrate transport system or alteration of the respiratory chain complex activity or a combination of these parameters [30].
To be activated, T cells have to recognize the antigen presented by MHC-II molecules and to be stimulated by CD86 accessory molecules, which are both expressed at the surface of DCs, in the context of inflammatory signal. We observed that BMDCs challenged by LPS as inflammatory signal showed an increased expression of CD86 and MHC-II molecules when they have been exposed to AuNPs. We investigated the impact of BMDCs on T cell antigen responses by the analysis of the secreted cytokines. A significant increase of IFN-γ, IL-13, and IL-17 productions, reflecting Th1, Th2 and Th17 cell polarisation, could be correlated with the activation of the BMDCs seen by high CD86 and MHC-II expression levels. A study with 50 nm AuNPs reported the induction of a Th17 response in humans, but no significant change in Th1 and Th2 responses[31]. This difference can be explained by the cellular model, the surface coating of AuNPs, the maturation, and activation states of DCs and the routes of antigen uptake. Interestingly, the fact that increase T cell responses to antigen affects Th1, Th2, and Th17 subsets of T cells may preserve the balance between pro-inflammatory and anti-inflammatory responses.