Dysregulation of Macrophage Activation Profiles by Engineered Nanoparticles

Although the potential human health impacts from exposure to engineered nanoparticles (ENPs) are uncertain, past epidemiological studies have established correlations between exposure to ambient air pollution particulates and the incidence of pneumonia and lung infections. Using amorphous silica and superparamagnetic iron oxide (SPIO) as model high production volume ENPs, we examined how macrophage activation by bacterial lipopolysaccharide (LPS) or the lung pathogen <i>Streptococcus pneumoniae</i> is altered by ENP pretreatment. Neither silica nor SPIO treatment elicited direct cytotoxic or pro-inflammatory effects in bone marrow-derived macrophages. However, pretreatment of macrophages with SPIO caused extensive reprogramming of nearly 500 genes regulated in response to LPS challenge, hallmarked by exaggerated activation of oxidative stress response pathways and suppressed activation of both pro- and anti-inflammatory pathways. Silica pretreatment altered regulation of only 67 genes, but there was strong correlation with gene sets affected by SPIO. Macrophages exposed to SPIO displayed a phenotype suggesting an impaired ability to transition from an M1 to M2-like activation state, characterized by suppressed IL-10 induction, enhanced TNFα production, and diminished phagocytic activity toward <i>S. pneumoniae</i>. Studies in macrophages deficient in scavenger receptor A (SR-A) showed SR-A participates in cell uptake of both the ENPs and <i>S. pneumonia</i> and co-regulates the anti-inflammatory IL-10 pathway. Thus, mechanisms for dysregulation of innate immunity exist by virtue that common receptor recognition pathways are used by some ENPs and pathogenic bacteria, although the extent of transcriptional reprogramming of macrophage function depends on the physicochemical properties of the ENP after internalization. Our results also illustrate that biological effects of ENPs may be indirectly manifested only after challenging normal cell function. Nanotoxicology screening strategies should therefore consider how exposure to these materials alters susceptibility to other environmental exposures

Quantitative Profiling of Protein S‑Glutathionylation Reveals Redox-Dependent Regulation of Macrophage Function during Nanoparticle-Induced Oxidative Stress

Engineered nanoparticles (ENPs) are increasingly utilized for commercial and medical applications; thus, understanding their potential adverse effects is an important societal issue. Herein, we investigated protein S-glutathionylation (SSG) as an underlying regulatory mechanism by which ENPs may alter macrophage innate immune functions, using a quantitative redox proteomics approach for site-specific measurement of SSG modifications. Three high-volume production ENPs (SiO₂, Fe₃O₄, and CoO) were selected as representatives which induce low, moderate, and high propensity, respectively, to stimulate cellular reactive oxygen species (ROS) and disrupt macrophage function. The SSG modifications identified highlighted a broad set of redox sensitive proteins and specific Cys residues which correlated well with the overall level of cellular redox stress and impairment of macrophage phagocytic function (CoO > Fe₃O₄ ≫ SiO₂). Moreover, our data revealed pathway-specific differences in susceptibility to SSG between ENPs which induce moderate <i>versus</i> high levels of ROS. Pathways regulating protein translation and protein stability indicative of ER stress responses and proteins involved in phagocytosis were among the most sensitive to SSG in response to ENPs that induce subcytoxic levels of redox stress. At higher levels of redox stress, the pattern of SSG modifications displayed reduced specificity and a broader set pathways involving classical stress responses and mitochondrial energetics (<i>e.g.,</i> glycolysis) associated with apoptotic mechanisms. An important role for SSG in regulation of macrophage innate immune function was also confirmed by RNA silencing of glutaredoxin, a major enzyme which reverses SSG modifications. Our results provide unique insights into the protein signatures and pathways that serve as ROS sensors and may facilitate cellular adaption to ENPs, <i>versus</i> intracellular targets of ENP-induced oxidative stress that are linked to irreversible cell outcomes

Dysregulation of Macrophage Activation Profiles by Engineered Nanoparticles

Although the potential human health impacts from exposure to engineered nanoparticles (ENPs) are uncertain, past epidemiological studies have established correlations between exposure to ambient air pollution particulates and the incidence of pneumonia and lung infections. Using amorphous silica and superparamagnetic iron oxide (SPIO) as model high production volume ENPs, we examined how macrophage activation by bacterial lipopolysaccharide (LPS) or the lung pathogen <i>Streptococcus pneumoniae</i> is altered by ENP pretreatment. Neither silica nor SPIO treatment elicited direct cytotoxic or pro-inflammatory effects in bone marrow-derived macrophages. However, pretreatment of macrophages with SPIO caused extensive reprogramming of nearly 500 genes regulated in response to LPS challenge, hallmarked by exaggerated activation of oxidative stress response pathways and suppressed activation of both pro- and anti-inflammatory pathways. Silica pretreatment altered regulation of only 67 genes, but there was strong correlation with gene sets affected by SPIO. Macrophages exposed to SPIO displayed a phenotype suggesting an impaired ability to transition from an M1 to M2-like activation state, characterized by suppressed IL-10 induction, enhanced TNFα production, and diminished phagocytic activity toward <i>S. pneumoniae</i>. Studies in macrophages deficient in scavenger receptor A (SR-A) showed SR-A participates in cell uptake of both the ENPs and <i>S. pneumonia</i> and co-regulates the anti-inflammatory IL-10 pathway. Thus, mechanisms for dysregulation of innate immunity exist by virtue that common receptor recognition pathways are used by some ENPs and pathogenic bacteria, although the extent of transcriptional reprogramming of macrophage function depends on the physicochemical properties of the ENP after internalization. Our results also illustrate that biological effects of ENPs may be indirectly manifested only after challenging normal cell function. Nanotoxicology screening strategies should therefore consider how exposure to these materials alters susceptibility to other environmental exposures

Dysregulation of Macrophage Activation Profiles by Engineered Nanoparticles

Although the potential human health impacts from exposure to engineered nanoparticles (ENPs) are uncertain, past epidemiological studies have established correlations between exposure to ambient air pollution particulates and the incidence of pneumonia and lung infections. Using amorphous silica and superparamagnetic iron oxide (SPIO) as model high production volume ENPs, we examined how macrophage activation by bacterial lipopolysaccharide (LPS) or the lung pathogen <i>Streptococcus pneumoniae</i> is altered by ENP pretreatment. Neither silica nor SPIO treatment elicited direct cytotoxic or pro-inflammatory effects in bone marrow-derived macrophages. However, pretreatment of macrophages with SPIO caused extensive reprogramming of nearly 500 genes regulated in response to LPS challenge, hallmarked by exaggerated activation of oxidative stress response pathways and suppressed activation of both pro- and anti-inflammatory pathways. Silica pretreatment altered regulation of only 67 genes, but there was strong correlation with gene sets affected by SPIO. Macrophages exposed to SPIO displayed a phenotype suggesting an impaired ability to transition from an M1 to M2-like activation state, characterized by suppressed IL-10 induction, enhanced TNFα production, and diminished phagocytic activity toward <i>S. pneumoniae</i>. Studies in macrophages deficient in scavenger receptor A (SR-A) showed SR-A participates in cell uptake of both the ENPs and <i>S. pneumonia</i> and co-regulates the anti-inflammatory IL-10 pathway. Thus, mechanisms for dysregulation of innate immunity exist by virtue that common receptor recognition pathways are used by some ENPs and pathogenic bacteria, although the extent of transcriptional reprogramming of macrophage function depends on the physicochemical properties of the ENP after internalization. Our results also illustrate that biological effects of ENPs may be indirectly manifested only after challenging normal cell function. Nanotoxicology screening strategies should therefore consider how exposure to these materials alters susceptibility to other environmental exposures

Hierarchical cluster analyses showing temporal changes in expression ratios for significant RNA and protein changes.

<p>The scale bar indicates the log₁₀ expression ratio compared to 0 hr controls. Values in gray indicate the protein/phosphorylated protein was not detected at that time point.</p

Summary of network statistics derived from individual and integrated datasets.

<p>Summary of network statistics derived from individual and integrated datasets.</p

Experimental design and characterization of cell cycle transition with EGF treatment.

<p>A) Experiments were scaled to provide sufficient sample for parallel analyses by gene microarray, global proteomics and Western blot technologies. B) Flow cytometry results showing the time course for transitions between G₁/S and G₂/M phases during EGF-induced mitosis.</p

Example network inferred from the integrated datasets.

<p>Shown is the overall structure of one of the largest network clusters identified from the combined microarray, LC-FTICR and Powerblot datasets from the 0–4 hr early time domain. A) The source of the data contributing to each element (node) in this network cluster is coded by color, and connections between elements (edges) were inferred from the literature using the MetaCore database. B) The cellular processes represented by each node in the network are coded by color, using the process categories from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034515#pone-0034515-g005" target="_blank">Figure 5</a>.</p

Major cell processes represented by each high-dimensional dataset.

<p>The biological processes represented by each data type across all time points (Panel A) were determined by gene set enrichment and significance values are p-values calculated within the MetaCore software. Only the cell processes showing the highest significance values are shown. The results in panel B show the major cell processes for all combined data, separated based on early (0–4 hr), intermediate (8–13 hr) or late (18–24 hr) time points after EGFR activation.</p

K-means cluster analysis comparison of microarray and LC-FTICR expression ratio data.

<p>The left panel shows the overall results for 446 RNA/protein pairs expressed as the log₁₀ ratio over 0 hr control samples. Panels A–C highlight 3 different clusters that show different overall temporal patterns between the RNA and protein data.</p