Heterogeneity in Macrophage Phagocytosis of Staphylococcus aureus Strains: High-Throughput Scanning Cytometry-Based Analysis

Alveolar macrophages (AMs) can phagocytose unopsonized pathogens such as S. aureus via innate immune receptors, such as scavenger receptors (SRs). Cytoskeletal events and signaling pathways involved in phagocytosis of unopsonized bacteria likely govern the fate of ingested pathogens, but are poorly characterized. We have developed a high-throughput scanning cytometry-based assay to quantify phagocytosis of S. aureus by adherent human blood-derived AM-like macrophages in a 96-well microplate format. Differential fluorescent labeling of internalized vs. bound bacteria or beads allowed automated image analysis of collapsed confocal stack images acquired by scanning cytometry, and quantification of total particles bound and percent of particles internalized. We compared the effects of the classic SR blocker polyinosinic acid, the cytoskeletal inhibitors cytochalasin D and nocodazole, and the signaling inhibitors staurosporine, Gö 6976, JNK Inhibitor I and KN-93, on phagocytosis of a panel of live unopsonized S. aureus strains, (Wood, Seattle 1945 (ATCC 25923), and RN6390), as well as a commercial killed Wood strain, heat-killed Wood strain and latex beads. Our results revealed failure of the SR inhibitor polyinosinic acid to block binding of any live S. aureus strains, suggesting that SR-mediated uptake of a commercial killed fluorescent bacterial particle does not accurately model interaction with viable bacteria. We also observed heterogeneity in the effects of cytoskeletal and signaling inhibitors on internalization of different S. aureus strains. The data suggest that uptake of unopsonized live S. aureus by human macrophages is not mediated by SRs, and that the cellular mechanical and signaling processes that mediate S. aureus phagocytosis vary. The findings also demonstrate the potential utility of high-throughput scanning cytometry techniques to study phagocytosis of S. aureus and other organisms in greater detail

Signaling pathways required for macrophage scavenger receptor-mediated phagocytosis: analysis by scanning cytometry

Background: Scavenger receptors are important components of the innate immune system in the lung, allowing alveolar macrophages to bind and phagocytose numerous unopsonized targets. Mice with genetic deletions of scavenger receptors, such as SR-A and MARCO, are susceptible to infection or inflammation from inhaled pathogens or dusts. However, the signaling pathways required for scavenger receptor-mediated phagocytosis of unopsonized particles have not been characterized. Methods: We developed a scanning cytometry-based high-throughput assay of macrophage phagocytosis that quantitates bound and internalized unopsonized latex beads. This assay allowed the testing of a panel of signaling inhibitors which have previously been shown to target opsonin-dependent phagocytosis for their effect on unopsonized bead uptake by human in vitro-derived alveolar macrophage-like cells. The non-selective scavenger receptor inhibitor poly(I) and the actin destabilizer cytochalasin D were used to validate the assay and caused near complete abrogation of bead binding and internalization, respectively. Results: Microtubule destabilization using nocodazole dramatically inhibited bead internalization. Internalization was also significantly reduced by inhibitors of tyrosine kinases (genistein and herbimycin A), protein kinase C (staurosporine, chelerythrine chloride and Gö 6976), phosphoinositide-3 kinase (LY294002 and wortmannin), and the JNK and ERK pathways. In contrast, inhibition of phospholipase C by U-73122 had no effect. Conclusion: These data indicate the utility of scanning cytometry for the analysis of phagocytosis and that phagocytosis of unopsonized particles has both shared and distinct features when compared to opsonin-mediated phagocytosis

Estimating the effective density of engineered nanomaterials for in vitro dosimetry

The need for accurate in vitro dosimetry remains a major obstacle to the development of cost-effective toxicological screening methods for engineered nanomaterials. An important key to accurate in vitro dosimetry is the characterization of sedimentation and diffusion rates of nanoparticles suspended in culture media, which largely depend upon the effective density and diameter of formed agglomerates in suspension. Here we present a rapid and inexpensive method for accurately measuring the effective density of nano-agglomerates in suspension. This novel method is based on the volume of the pellet obtained by bench-top centrifugation of nanomaterial suspensions in a packed cell volume tube, and is validated against gold-standard analytical ultracentrifugation data. This simple and cost-effective method allows nanotoxicologists to correctly model nanoparticle transport, and thus attain accurate dosimetry in cell culture systems, which will greatly advance the development of reliable and efficient methods for toxicological testing and investigation of nano-bio interactions in vitro

Advanced computational modeling for in vitro nanomaterial dosimetry

Background: Accurate and meaningful dose metrics are a basic requirement for in vitro screening to assess potential health risks of engineered nanomaterials (ENMs). Correctly and consistently quantifying what cells “see,” during an in vitro exposure requires standardized preparation of stable ENM suspensions, accurate characterizatoin of agglomerate sizes and effective densities, and predictive modeling of mass transport. Earlier transport models provided a marked improvement over administered concentration or total mass, but included assumptions that could produce sizable inaccuracies, most notably that all particles at the bottom of the well are adsorbed or taken up by cells, which would drive transport downward, resulting in overestimation of deposition. Methods: Here we present development, validation and results of two robust computational transport models. Both three-dimensional computational fluid dynamics (CFD) and a newly-developed one-dimensional Distorted Grid (DG) model were used to estimate delivered dose metrics for industry-relevant metal oxide ENMs suspended in culture media. Both models allow simultaneous modeling of full size distributions for polydisperse ENM suspensions, and provide deposition metrics as well as concentration metrics over the extent of the well. The DG model also emulates the biokinetics at the particle-cell interface using a Langmuir isotherm, governed by a user-defined dissociation constant, KD, and allows modeling of ENM dissolution over time. Results: Dose metrics predicted by the two models were in remarkably close agreement. The DG model was also validated by quantitative analysis of flash-frozen, cryosectioned columns of ENM suspensions. Results of simulations based on agglomerate size distributions differed substantially from those obtained using mean sizes. The effect of cellular adsorption on delivered dose was negligible for KD values consistent with non-specific binding (> 1 nM), whereas smaller values (≤ 1 nM) typical of specific high-affinity binding resulted in faster and eventual complete deposition of material. Conclusions: The advanced models presented provide practical and robust tools for obtaining accurate dose metrics and concentration profiles across the well, for high-throughput screening of ENMs. The DG model allows rapid modeling that accommodates polydispersity, dissolution, and adsorption. Result of adsorption studies suggest that a reflective lower boundary condition is appropriate for modeling most in vitro ENM exposures. Electronic supplementary material The online version of this article (doi:10.1186/s12989-015-0109-1) contains supplementary material, which is available to authorized users

Genome-Wide RNAi Screen in IFN-γ-Treated Human Macrophages Identifies Genes Mediating Resistance to the Intracellular Pathogen Francisella tularensis

Interferon-gamma (IFN-γ) inhibits intracellular replication of Francisella tularensis in human monocyte-derived macrophages (HMDM) and in mice, but the mechanisms of this protective effect are poorly characterized. We used genome-wide RNA interference (RNAi) screening in the human macrophage cell line THP-1 to identify genes that mediate the beneficial effects of IFN-γ on F. tularensis infection. A primary screen identified ∼200 replicated candidate genes. These were prioritized according to mRNA expression in IFN-γ-primed and F. tularensis-challenged macrophages. A panel of 20 top hits was further assessed by re-testing using individual shRNAs or siRNAs in THP-1 cells, HMDMs and primary human lung macrophages. Six of eight validated genes tested were also found to confer resistance to Listeria monocytogenes infection, suggesting a broadly shared host gene program for intracellular pathogens. The F. tularensis-validated hits included ‘druggable’ targets such as TNFRSF9, which encodes CD137. Treating HMDM with a blocking antibody to CD137 confirmed a beneficial role of CD137 in macrophage clearance of F. tularensis. These studies reveal a number of important mediators of IFN-γ activated host defense against intracellular pathogens, and implicate CD137 as a potential therapeutic target and regulator of macrophage interactions with Francisella tularensis

An integrated methodology for assessing the impact of food matrix and gastrointestinal effects on the biokinetics and cellular toxicity of ingested engineered nanomaterials

Background Engineered nanomaterials (ENMs) are increasingly added to foods to improve their quality, sensory appeal, safety and shelf-life. Human exposure to these ingested ENMs (iENMS) is inevitable, yet little is known of their hazards. To assess potential hazards, efficient in vitro methodologies are needed to evaluate particle biokinetics and toxicity. These methodologies must account for interactions and transformations of iENMs in foods (food matrix effect) and in the gastrointestinal tract (GIT) that are likely to determine nano-biointeractions. Here we report the development and application of an integrated methodology consisting of three interconnected stages: 1) assessment of iENM-food interactions (food matrix effect) using model foods; 2) assessment of gastrointestinal transformations of the nano-enabled model foods using a three-stage GIT simulator; 3) assessment of iENMs biokinetics and cellular toxicity after exposure to simulated GIT conditions using a triculture cell model. As a case study, a model food (corn oil-in-water emulsion) was infused with Fe2O3 (Iron(III) oxide or ferric oxide) ENMs and processed using this three-stage integrated platform to study the impact of food matrix and GIT effects on nanoparticle biokinetics and cytotoxicity . Methods A corn oil in phosphate buffer emulsion was prepared using a high speed blender and high pressure homogenizer. Iron oxide ENM was dispersed in water by sonication and combined with the food model. The resulting nano-enabled food was passed through a three stage (mouth, stomach and small intestine) GIT simulator. Size distributions of nano-enabled food model and digestae at each stage were analyzed by DLS and laser diffraction. TEM and confocal imaging were used to assess morphology of digestae at each phase. Dissolution of Fe2O3 ENM along the GIT was assessed by ICP-MS analysis of supernatants and pellets following centrifugation of digestae. An in vitro transwell triculture epithelial model was used to assess biokinetics and toxicity of ingested Fe2O3 ENM. Translocation of Fe2O3 ENM was determined by ICP-MS analysis of cell lysates and basolateral compartment fluid over time. Results It was demonstrated that the interactions of iENMs with food and GIT components influenced nanoparticle fate and transport, biokinetics and toxicological profile. Large differences in particle size, charge, and morphology were observed in the model food with and without Fe2O3 and among digestae from different stages of the simulated GIT (mouth, stomach, and small intestine). Immunoflorescence and TEM imaging of the cell culture model revealed markers and morphology of small intestinal epithelium including enterocytes, goblet cells and M cells. Fe2O3 was not toxic at concentrations tested in the digesta. In biokinetics studies, translocation of Fe2O3 after 4 h was \u3c1% and ~2% for digesta with and without serum, respectively, suggesting that use of serum proteins alters iENMs biokinetics and raises concerns about commonly-used approaches that neglect iENM – food-GIT interactions or dilute digestae in serum-containing media. Conclusions We present a simple integrated methodology for studying the biokinetics and toxicology of iENMs, which takes into consideration nanoparticle-food-GIT interactions. The importance of food matrix and GIT effects on biointeractions was demonstrated, as well as the incorporation of these critical factors into a cellular toxicity screening model. Standardized food models still need to be developed and used to assess the effect of the food matrix effects on the fate and bioactivity of iENMs since commercial foods vary considerably in their compositions and structures

Lipid and protein corona of food-grade TiO2 nanoparticles in simulated gastrointestinal digestion

In the presence of biological matrices, engineered nanomaterials, such as TiO2, develop a biomolecular corona composed of lipids, proteins, etc. In this study, we analyzed the biocorona formed on the food grade TiO2 (E171) going through an in vitro simulated gastrointestinal digestion system in either a fasting food model (FFM), a standardized food model (SFM), or a high fat food model (HFFM). Lipids and proteins were extracted from the biocorona and underwent untargeted lipidomic and label-free shotgun proteomic analyses. Our results showed that the biocorona composition was different before and after food digestion. After digestion, more diverse lipids were adsorbed compared to proteins, most of which were the enzymes added to the simulated digestion system. The corona lipid profile was distinct from the digested food model they presented in, although similarity in the lipid profiles between the corona and the food matrix increased with the fat content in the food model. The corona formed in the two low-fat environments of FFM and SFM shared a higher degree of similarity while very different from their corresponding matrix, with some lipid species adsorbed with high enrichment factors, indicating specific interaction with the TiO2 surface outperforming lipid matrix concentration in determination of corona formation. Formation of the biocorona may have contributed to the reduced oxidative stress as well as toxicological impacts observed in cellular studies. The present work is the first to confirm persistent adsorption of biomolecules could occur on ingested nanomaterials in food digestae. More future studies are needed to study the in vivo impacts of the biocorona, and shed lights on how the biocorona affects the biotransformations and fate of the ingested nanomaterials, which may impose impacts on human health

Scanning cytometry fluorescence imaging with GFP-S. aureus RN6390.

<p>Adherent GM-MФs were incubated with unopsonized GFP-<i>S. aureus</i> RN6390. External <i>S. aureus</i> were labeled with an IgG₃ monoclonal mouse anti-<i>S. aureus</i> primary antibody and Texas-RedX-conjugated goat anti-mouse secondary antibody. Collapsed confocal stack images were acquired by scanning cytometry. (A) CellTracker Blue and Hoechst channel (cells). (B) GFP channel (all bacteria). (C) Texas Red channel (external bacteria). (D) Composite image. (Original magnification, 200X).</p

Summary of binding inhibition results.

<p>Beads: biotinlyated carboxylated 1 micron green fluorescent latex beads. CK Wood: commercial killed AlexaFluor 488-conjugated <i>S. aureus</i> Wood strain.</p><p>Wood: live S. aureus Wood strain. Seattle 1945: Live S. aureus Seattle 1945 strain. RN 6390: live GFP+S. aureus RN6390 strain. IgG ops. RN6390: IgG α-S. aureus opsonized RN6390.</p><p>+ <25% inhibition. ++25–50% inhibition. +++50–75% inhibition. ++++ >75% inhibition.</p><p>- not significantly changed. ̂̂ 25–50% increased. nd: not determined.</p