Jared M. Brown

Bone marrow stromal cells attenuate sepsis via prostaglandin E 2 –dependent reprogramming of host macrophages to increase their interleukin-10 production

Sepsis causes over 200,000 deaths yearly in the US; better treatments are urgently needed. Administering bone marrow stromal cells (BMSCs—also known as mesenchymal stem cells) to mice before or shortly after inducing sepsis by cecal ligation and puncture reduced mortality and improved organ function. The beneficial effect of BMSCs was eliminated by macrophage depletion or pretreatment with antibodies specific for interleukin-10 (IL-10) or IL-10 receptor. Monocytes and/or macrophages from septic lungs made more IL-10 when prepared from mice treated with BMSCs versus untreated mice. Lipopolysaccharide (LPS)-stimulated macrophages produced more IL-10 when cultured with BMSCs, but this effect was eliminated if the BMSCs lacked the genes encoding Toll-like receptor 4, myeloid differentiation primary response gene-88, tumor necrosis factor (TNF) receptor-1a or cyclooxygenase-2. Our results suggest that BMSCs (activated by LPS or TNF-α) reprogram macrophages by releasing prostaglandin E2 that acts on the macrophages through the prostaglandin EP2 and EP4 receptors. Because BMSCs have been successfully given to humans and can easily be cultured and might be used without human leukocyte antigen matching, we suggest that cultured, banked human BMSCs may be effective in treating sepsis in high-risk patient groups.

Bone marrow stromal cells use TGF-β to suppress allergic responses in a mouse model of ragweed-induced asthma

Bone marrow stromal cells [BMSCs; also known as mesenchymal stem cells (MSCs)] effectively suppress inflammatory responses in acute graft-versus-host disease in humans and in a number of disease models in mice. Many of the studies concluded that BMSC-driven immunomodulation is mediated by the suppression of proinflammatory Th1 responses while rebalancing the Th1/Th2 ratio toward Th2. In this study, using a ragweed induced mouse asthma model, we studied if BMSCs could be beneficial in an allergic, Th2-dominant environment. When BMSCs were injected i.v. at the time of the antigen challenge, they protected the animals from the majority of asthma-specific pathological changes, including inhibition of eosinophil infiltration and excess mucus production in the lung, decreased levels of Th2 cytokines (IL-4, IL-5, and IL-13) in bronchial lavage, and lowered serum levels of Th2 immunoglobulins (IgG1 and IgE). To explore the mechanism of the effect we used BMSCs isolated from a variety of knockout mice, performed in vivo blocking of cytokines and studied the effect of asthmatic serum and bronchoalveolar lavage from ragweed challenged animals on the BMSCs in vitro. Our results suggest that IL-4 and/or IL-13 activate the STAT6 pathway in the BMSCs resulting in an increase of their TGF-β production, which seems to mediate the beneficial effect, either alone, or together with regulatory T cells, some of which might be recruited by the BMSCs. These data suggest that, in addition to focusing on graft-versus-host disease and autoimmune diseases, allergic conditions—specifically therapy resistant asthma—might also be a likely target of the recently discovered cellular therapy approach using BMSCs.

Silver Nanoparticle Protein Corona Composition in Cell Culture Media

The potential applications of nanomaterials as drug delivery systems and in other products continue to expand. Upon introduction into physiological environments and driven by energetics, nanomaterials readily associate proteins forming a protein corona (PC) on their surface. This PC influences the nanomaterial’s surface characteristics and may impact their interaction with cells. To determine the biological impact of nanomaterial exposure as well as nanotherapeutic applications, it is necessary to understand PC formation. Utilizing a label-free mass spectrometry-based proteomics approach, we examined the composition of the PC for a set of four silver nanoparticles (AgNPs) including citrate-stabilized and polyvinlypyrrolidone-stabilized (PVP) colloidal silver (20 or 110 nm diameter). To simulate cell culture conditions, AgNPs were incubated for 1 h in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, washed, coronal proteins solubilized, and proteins identified and quantified by label-free LC-MS/MS. To determine which attributes influence PC formation, the AgNPs were characterized in both water and cell culture media with 10% FBS. All AgNPs associated a common subset of 11 proteins including albumin, apolipoproteins, keratins, and other serum proteins. 110 nm citrate- and PVP-stabilized AgNPs were found to bind the greatest number of proteins (79 and 85 respectively) compared to 20 nm citrate- and PVP-stabilized AgNPs (45 and 48 respectively), suggesting a difference in PC formation based on surface curvature. While no relationships were found for other protein parameters (isoelectric point or aliphatic index), the PC on 20 nm AgNPs (PVP and citrate) consisted of more hydrophobic proteins compared to 110 nm AgNPs implying that this class of proteins are more receptive to curvature-induced folding and crowding in exchange for an increased hydration in the aqueous environment. These observations demonstrate the significance of electrostatic and hydrophobic interactions in the formation of the PC which may have broad biological and toxicological implications.

Interlaboratory Evaluation of Rodent Pulmonary Responses to Engineered Nanomaterials: The NIEHS Nano GO Consortium

Background: Engineered nanomaterials (ENMs) have potential benefits, but they also present safety concerns for human health. Interlaboratory studies in rodents using standardized protocols are needed to assess ENM toxicity. Methods: Four laboratories evaluated lung responses in C57BL/6 mice to ENMs delivered by oropharyngeal aspiration (OPA), and three labs evaluated Sprague-Dawley (SD) or Fisher 344 (F344) rats following intratracheal instillation (IT). ENMs tested included three forms of titanium dioxide (TiO2) [anatase/rutile spheres (TiO2-P25), anatase spheres (TiO2-A), and anatase nanobelts (TiO2-NBs)] and three forms of multiwalled carbon nanotubes (MWCNTs) [original (O), purified (P), and carboxylic acid “functionalized” (F)]. One day after treatment, bronchoalveolar lavage fluid was collected to determine differential cell counts, lactate dehydrogenase (LDH), and protein. Lungs were fixed for histopathology. Responses were also examined at 7 days (TiO2 forms) and 21 days (MWCNTs) after treatment. Results: TiO2-A, TiO2-P25, and TiO2-NB caused significant neutrophilia in mice at 1 day in three of four labs. TiO2-NB caused neutrophilia in rats at 1 day in two of three labs, and TiO2-P25 and TiO2-A had no significant effect in any of the labs. Inflammation induced by TiO2 in mice and rats resolved by day 7. All MWCNT types caused neutrophilia at 1 day in three of four mouse labs and in all rat labs. Three of four labs observed similar histopathology to O-MWCNTs and TiO2-NBs in mice. Conclusions: ENMs produced similar patterns of neutrophilia and pathology in rats and mice. Although interlaboratory variability was found in the degree of neutrophilia caused by the three types of TiO2 nanoparticles, similar findings of relative potency for the three types of MWCNTs were found across all laboratories, thus providing greater confidence in these interlaboratory comparisons.

Multi-walled carbon nanotube instillation impairs pulmonary function in C57BL/6 mice

BackgroundMulti-walled carbon nanotubes (MWCNTs) are widely used in many disciplines due to their unique physical and chemical properties. Therefore, some concerns about the possible human health and environmental impacts of manufactured MWCNTs are rising. We hypothesized that instillation of MWCNTs impairs pulmonary function in C57BL/6 mice due to development of lung inflammation and fibrosis.MethodsMWCNTs were administered to C57BL/6 mice by oropharyngeal aspiration (1, 2, and 4 mg/kg) and we assessed lung inflammation and fibrosis by inflammatory cell infiltration, collagen content, and histological assessment. Pulmonary function was assessed using a FlexiVent system and levels of Ccl3, Ccl11, Mmp13 and IL-33 were measured by RT-PCR and ELISA.ResultsMice administered MWCNTs exhibited increased inflammatory cell infiltration, collagen deposition and granuloma formation in lung tissue, which correlated with impaired pulmonary function as assessed by increased resistance, tissue damping, and decreased lung compliance. Pulmonary exposure to MWCNTs induced an inflammatory signature marked by cytokine (IL-33), chemokine (Ccl3 and Ccl11), and protease production (Mmp13) that promoted the inflammatory and fibrotic changes observed within the lung.ConclusionsThese results further highlight the potential adverse health effects that may occur following MWCNT exposure and therefore we suggest these materials may pose a significant risk leading to impaired lung function following environmental and occupational exposures.

Comparison of nanotube-protein corona composition in cell culture media.

In biological environments, nanomaterials associate with proteins forming a protein corona (PC). The PC may alter the nanomaterials pharmacokinetics and pharmacodynamics, thereby influencing toxicity. Using a label-free mass spectrometry-based proteomics approach, the composition of the PC is examined for a set of nanotubes (NTs) including unmodified and carboxylated single- (SWCNT) and multi-walled carbon nanotubes (MWCNT), polyvinylpyrrolidone (PVP)-coated MWCNT (MWCNT-PVP), and nanoclay. NTs are incubated for 1 h in simulated cell culture conditions, then washed, resuspended in PBS, and assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for their associated PC. To determine those attributes that influence PC formation, the NTs are extensively characterized. NTs had negative zeta potentials in water (SWCNT-COOH < MWCNT-COOH < unmodified NTs) while carboxylation increases their hydrodynamic sizes. All NTs are also found to associate a common subset of proteins including albumin, titin, and apolipoproteins. SWCNT-COOH and MWCNT-COOH are found to bind the greatest number of proteins (181 and 133 respectively) compared to unmodified NTs (<100), suggesting covalent binding to protein amines. Modified NTs bind a number of unique proteins compared to unmodified NTs, implying hydrogen bonding and electrostatic interactions are involved in PC formation. PVP-coating of MWCNT did not influence PC composition, further reinforcing the possibility of hydrogen bonding and electrostatic interactions. No relationships are found between PC composition and corresponding isoelectric point, hydropathy, or aliphatic index, implying minimal roles of hydrophobic interaction and pi-stacking.

Toxicity of Engineered Nanomaterials: A Physicochemical Perspective

The global market for nanomaterial‐based products is forecasted to reach 100 billion dollars per annum for 2011–2015. Extensive manufacturing and the use of engineered nanomaterials have raised concerns regarding their impact on biological response in living organisms and the environment at large. The fundamental properties of nanomaterials exhibit a complex dependence upon several factors such as their morphology, size, defects, and chemical stability. Therefore, it is exceedingly difficult to correlate their biological response with their intricate physicochemical properties. For example, varying toxic response may ensue due to different methods of nanomaterial preparation, dissimilar impurities, and defects. In this review, we surveyed the existing literature on the dependence of cytotoxicity on physicochemical properties. We found that ENM size, shape, defect density, physicochemical stability, and surface modification to be the main causes that elicit altered physiological response or cytotoxicity.

Effects of surface functional groups on the formation of nanoparticle-protein corona.

Herein, we examined the dependence of protein adsorption on the nanoparticle surface in the presence of functional groups. Our UV-visible spectrophotometry, transmission electron microscopy, infrared spectroscopy, and dynamic light scattering measurements evidently suggested that the functional groups play an important role in the formation of nanoparticle-protein corona. We found that uncoated and surfactant-free silver nanoparticles derived from a laser ablation process promoted a maximum protein (bovine serum albumin) coating due to increased changes in entropy. On the other hand, bovine serum albumin displayed a relatively lower affinity for electrostatically stabilized nanoparticles due to the constrained entropy changes.

Formation of a Protein Corona on Silver Nanoparticles Mediates Cellular Toxicity via Scavenger Receptors

Addition of a protein corona (PC) or protein adsorption layer on the surface of nanomaterials following their introduction into physiological environments may modify their activity, bio-distribution, cellular uptake, clearance, and toxicity. We hypothesize that silver nanoparticles (AgNPs) will associate with proteins common to human serum and cell culture media forming a PC that will impact cell activation and cytotoxicity. Furthermore, the role of scavenger receptor BI (SR-BI) in mediating this toxicity was evaluated. Citrate-suspended 20 nm AgNPs were incubated with human serum albumin (HSA), bovine serum albumin (BSA), high-density lipoprotein (HDL), or water (control) to form a PC. AgNPs associated with each protein (HSA, BSA, and HDL) forming PCs as assessed by electron microscopy, hyperspectral analysis, ζ-potential, and hydrodynamic size. Addition of the PC decreased uptake of AgNPs by rat lung epithelial and rat aortic endothelial cells. Hyperspectral analysis demonstrated a loss of the AgNP PC following internalization. Cells demonstrated concentration-dependent cytotoxicity following exposure to AgNPs with or without PCs (0, 6.25, 12.5, 25 or 50 μg/ml). All PC-coated AgNPs were found to activate cells by inducing IL-6 mRNA expression. A small molecule SR-BI inhibitor was utilized to determine the role of SR-BI in the observed effects. Pretreatment with the SR-BI inhibitor decreased internalization of AgNPs with or without PCs, and reduced both cytotoxicity and IL-6 mRNA expression. This study characterizes the formation of a PC on AgNPs and demonstrates its influence on cytotoxicity and cell activation through a cell surface receptor.

Direct observation of a single nanoparticle-ubiquitin corona formation†

The advancement of nanomedicine and the increasing applications of nanoparticles in consumer products have led to administered biological exposure and unintentional environmental accumulation of nanoparticles, causing concerns over the biocompatibility and sustainability of nanotechnology. Upon entering physiological environments, nanoparticles readily assume the form of a nanoparticle-protein corona that dictates their biological identity. Consequently, understanding the structure and dynamics of a nanoparticle-protein corona is essential for predicting the fate, transport, and toxicity of nanomaterials in living systems and for enabling the vast applications of nanomedicine. Here we combined multiscale molecular dynamics simulations and complementary experiments to characterize the silver nanoparticle-ubiquitin corona formation. Notably, ubiquitins competed with citrates for the nanoparticle surface, governed by specific electrostatic interactions. Under a high protein/nanoparticle stoichiometry, ubiquitins formed a multi-layer corona on the particle surface. The binding exhibited an unusual stretched-exponential behavior, suggesting a rich binding kinetics. Furthermore, the binding destabilized the α-helices while increasing the β-sheet content of the proteins. This study revealed the atomic and molecular details of the structural and dynamic characteristics of nanoparticle-protein corona formation.