Formyl-peptide Receptor Agonists and Amorphous SiO2-NPs Synergistically and Selectively Increase the Inflammatory Responses of Human Monocytes and PMNs

We tested whether amorphous SiO2-NPs and formyl-peptide receptor (FPRs) agonists synergistically activate human monocytes and neutrophil polymorphonuclear granulocytes (PMNs). Peptide ligands specifically binding to FPR1 (f-MLP) and to FPR2 (MMK-1, WKYMVM and WKYMVm) human isoforms did not modify the association of SiO2-NPs to both cell types or their cytotoxic effects. Similarly, the extent of CD80, CD86, CD83, ICAM-1 and MHCII expression in monocytes treated with SiO2-NPs was not significantly altered by any FPRs agonist. However, FPR1 stimulation with f-MLP strongly increased the secretion of IL-1β, IL-6 and IL-8 by human monocytes, and of IL-8 by PMNs in the presence of SiO2-NPs, due to the synergic stimulation of gene transcription. FPR2 agonists also up-modulated the production of IL-1β induced by monocytes treated with SiO2-NPs. In turn, SiO2-NPs increased the chemotaxis of PMNs toward FPR1-specific ligands, but not toward FPR2-specific ones. Conversely, the chemotaxis of monocytes toward FPR2-specific peptides was inhibited by SiO2-NPs. NADPH-oxidase activation triggered by FPR1- and FPR2-specific ligands in both cell types was not altered by SiO2-NPs. Microbial and tissue danger signals sensed by FPRs selectively amplified the functional responses of monocytes and PMNS to SiO2-NPs, and should be carefully considered in the assessment of the risk associated with nanoparticle exposure

Self-Assembled Biocompatible Fluorescent Nanoparticles for Bioimaging

Fluorescence is a powerful tool for mapping biological events in real-time with high spatial resolution. Ultra-bright probes are needed in order to achieve high sensitivity: these probes are typically obtained by gathering a huge number of fluorophores in a single nanoparticle (NP). Unfortunately this assembly produces quenching of the fluorescence because of short-range intermolecular interactions. Here we demonstrate that rational structural modification of a well-known molecular fluorophore N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD) produces fluorophores that self-assemble in nanoparticles in the biocompatible environment without any dramatic decrease of the fluorescence quantum yield. Most importantly, the resulting NP show, in an aqueous environment, a brightness which is more than six orders of magnitude higher than the molecular component in the organic solvent. Moreover, the NP are prepared by nanoprecipitation and they are stabilized only via non-covalent interaction, they are surprisingly stable and can be observed as individual bright spots freely diffusing in solution at a concentration as low as 1 nM. The suitability of the NP as biocompatible fluorescent probes was demonstrated in the case of HeLa cells by fluorescence confocal microscopy and MTS assays

Proinflammatory effects of bare and PEGylated ORMOSIL-, PLGA- and SUV-NPs on monocytes and PMNs and their modulation by f-MLP

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C1q-Mediated Complement Activation and C3 Opsonization Trigger Recognition of Stealth Poly(2-methyl-2-oxazoline)-Coated Silica Nanoparticles by Human Phagocytes

Poly(2-methyl-2-oxazoline) (PMOXA) is an alternative promising polymer to poly(ethylene glycol) (PEG) for design and engineering of macrophage-evading nanoparticles (NPs). Although PMOXA-engineered NPs have shown comparable pharmacokinetics and in vivo performance to PEGylated stealth NPs in the murine model, its interaction with elements of the human innate immune system has not been studied. From a translational angle, we studied the interaction of fully characterized PMOXA-coated vinyltriethoxysilane-derived organically modified silica NPs (PMOXA-coated NPs) of approximately 100 nm in diameter with human complement system, blood leukocytes, and macrophages and compared their performance with PEGylated and uncoated NP counterparts. Through detailed immunological and proteomic profiling, we show that PMOXA-coated NPs extensively trigger complement activation in human sera exclusively through the classical pathway. Complement activation is initiated by the sensing molecule C1q, where C1q binds with high affinity (Kd = 11 \ub1 1 nM) to NP surfaces independent of immunoglobulin binding. C1q-mediated complement activation accelerates PMOXA opsonization with the third complement protein (C3) through the amplification loop of the alternative pathway. This promoted NP recognition by human blood leukocytes and monocyte-derived macrophages. The macrophage capture of PMOXA-coated NPs correlates with sera donor variability in complement activation and opsonization but not with other major corona proteins, including clusterin and a wide range of apolipoproteins. In contrast to these observations, PMOXA-coated NPs poorly activated the murine complement system and were marginally recognized by mouse macrophages. These studies provide important insights into compatibility of engineered NPs with elements of the human innate immune system for translational steps

The Soluble Recombinant Neisseria meningitidis Adhesin NadAΔ351–405 Stimulates Human Monocytes by Binding to Extracellular Hsp90

The adhesin NadA favors cell adhesion/invasion by hypervirulent Neisseria meningitidis B (MenB). Its recombinant form NadAΔ351–405, devoid of the outer membrane domain, is an immunogenic candidate for an anti-MenB vaccine able to stimulate monocytes, macrophages and dendritic cells. In this study we investigated the molecular mechanism of NadAΔ351–405 cellular effects in monocytes. We show that NadAΔ351–405 (against which we obtained polyclonal antibodies in rabbits), binds to hsp90, but not to other extracellular homologous heat shock proteins grp94 and hsp70, in vitro and on the surface of monocytes, in a temperature dependent way. Pre-incubation of monocytes with the MenB soluble adhesin interfered with the binding of anti-hsp90 and anti-hsp70 antibodies to hsp90 and hsp70 at 37°C, a condition in which specific cell-binding occurs, but not at 0°C, a condition in which specific cell-binding is very diminished. Conversely, pre-incubation of monocytes with anti-hsp90 and anti-hsp70 antibodies did not affected NadAΔ351–405 cell binding in any temperature condition, indicating that it associates to another receptor on their plasma membrane and then laterally diffuses to encounter hsp90. Consistently, polymixin B interfered with NadAΔ351–405 /hsp90 association, abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due to NadAΔ351–405 and inhibited adhesin-induced cytokine/chemokine secretion without affecting monocyte-adhesin binding. Co-stimulation of monocytes with anti-hsp90 antibodies and NadAΔ351–405 determined a stronger but polymixin B insensitive cell activation. This indicated that the formation of a recombinant NadA/hsp90/hsp70 complex, although essential for full monocyte stimulation, can be replaced by anti-hsp90 antibody/hsp90 binding. Finally, the activation of monocytes by NadAΔ351–405 alone or in the presence of anti-hsp90 antibodies were both inhibited by neutralizing anti-TLR4 antibodies, but not by anti-TLR2 antibodies. We propose that hsp90-dependent recruitment into an hsp90/hsp70/TLR4 transducing signal complex is necessary for the immune-stimulating activity of NadAΔ351–405 anti-MenB vaccine candidate

The Honey-bee antimicrobial peptide Apidaecin differentially immune-modulates human macrophages, monocytes and dendritic cells.

We show that apidaecin binds to human macrophages, monocytes and dendritic cells, displaying different intracellular distributions and inducing diversified effects. An apidaecin-cell association was detectable at concentrations as low as 5 \ub5M and increased without saturation until 60 \ub5M, was receptor independent and required a physiological temperature (37\ub0C). For apidaecin, cytosolic localization was prevalent in macrophages and endosomal localization in monocytes, and associations with the plasma membrane were predominant in dendritic cells. Apidaecin upregulated T-lymphocyte co-stimulatory molecule CD80 and cytokine/chemokine production in macrophages, but not in monocytes and dendritic cells. Suboptimal stimulatory doses (5\u201310 \ub5M) of apidaecin partially inhibited the lipopolysaccharide (LPS)-induced increase in major histocompatibility complex class II (MHCII) and CD86 in macrophages, and the release of selected cytokines/chemokines by both macrophages [interleukin (IL)-6 and tumor necrosis factor (TNF)-\u3b1] and monocytes [IL-6, TNF-\u3b1, basic fibroblast growth factor (FGF) and eotaxin]. Apidaecin had a double-edged effect: at low concentrations it partially antagonized LPS-stimulatory effects on both macrophages and monocytes while it stimulated pro-inflammatory and pro-immune functions of macrophages at higher concentrations

Combined Action of Human Commensal Bacteria and Amorphous Silica Nanoparticles on the Viability and Immune Responses of Dendritic Cells

Dendritic cells (DCs) regulate the host-microbe balance in the gut and skin, tissues likely exposed to nanoparticles (NPs) present in drugs, food, and cosmetics. We analyzed the viability and the activation of DCs incubated with extracellular media (EMs) obtained from cultures of commensal bacteria (Escherichia coli, Staphylococcus epidermidis) or pathogenic bacteria (Pseudomonas aeruginosa, Staphylococcus aureus) in the presence of amorphous silica nanoparticles (SiO2 NPs). EMs and NPs synergistically increased the levels of cytotoxicity and cytokine production, with different nanoparticle dose-response characteristics being found, depending on the bacterial species. E. coli and S. epidermidis EMs plus NPs at nontoxic doses stimulated the secretion of interleukin-1 beta (IL-1 beta), IL-12, IL-10, and IL-6, while E. coli and S. epidermidis EMs plus NPs at toxic doses stimulated the secretion of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), IL-4, and IL-5. On the contrary, S. aureus and P. aeruginosa EMs induced cytokines only when they were combined with NPs at toxic concentrations. The induction of maturation markers (CD86, CD80, CD83, intercellular adhesion molecule 1, and major histocompatibility complex class II) by commensal bacteria but not by pathogenic ones was improved in the presence of noncytotoxic SiO2 NP doses. DCs consistently supported the proliferation and differentiation of CD4(+) and CD8(+) T cells secreting IFN-gamma and IL-17A. The synergistic induction of CD86 was due to nonprotein molecules present in the EMs from all bacteria tested. At variance with this finding, the synergistic induction of IL-1 beta was prevalently mediated by proteins in the case of E. coli EMs and by nonproteins in the case of S. epidermidis EMs. A bacterial costimulus did not act on DCs after adsorption on SiO2 NPs but rather acted as an independent agonist. The inflammatory and immune actions of DCs stimulated by commensal bacterial agonists might be altered by the simultaneous exposure to engineered or environmental NPs

Catastrophic inflammatory death of monocytes and macrophages by overtaking of a critical dose of endocytosed synthetic amorphous silica nanoparticles/serum protein complexes

Aims: We tested whether phagocytic monocytes/macrophages are more susceptible than nonphagocytes to nanoparticle (NP) toxicity. Materials & methods: We compared in vitro cell death and proinflammatory cytokine production in human monocytes, macrophages, lymphocytes and HeLa cells due to synthetic amorphous silica (SiO2)-NPs in different serum concentrations and correlated them with cellular uptake and distribution. Results: Phagocytes were approximately ten-times more sensitive than nonphagocytes to SiO2-NPs and more effectively endocytosed SiO2-NP\u2013serum protein nanoagglomerates, so determining their accumulation in acidic endocytic compartments well beyond a critical/cytotoxic threshold. Monocyte/macrophage death was paralleled by cytokine secretion. Conclusion: The physiological specialization of monocytes/macrophages to effectively capture NPs may expose them to the risk of catastrophic inflammatory death upon saturation of their maximal storage capacity

Water-soluble peptide-coated nanoparticles: Control of the helix structure and enhanced differential binding to immune cells

The stabilizing action of C(\u3b1)-tetrasubstituted \u3b1-amino acids inserted into a sequence of short peptides allowed for the first time the preparation of water-soluble nanoparticles of different materials coated with a helix-structured undecapeptide. This peptide coating strongly favors nanoparticle uptake by human immune system cells

The membrane expression of Neisseria meningitidis adhesin A (NadA) increases the proimmune effects of MenB OMVs on human macrophages, compared with NadA(-) OMVs, without further stimulating their proinflammatory activity on circulating monocytes

Hypervirulent MenB causing fatal human infections frequently display the oligomeric-coiled coil adhesin NadA, a 45-kDa intrinsic outer membrane protein implicated in binding to and invasion of respiratory epithelial cells. A recombinant soluble mutant lacking the 10-kDa COOH terminal membrane domain (NadA\u394351\u2013405) also activates human monocytes/macrophages/DCs. As NadA is physiologically released during sepsis as part of OMVs, in this study, we tested the hypothesis that NadA+ OMVs have an enhanced or modified proinflammatory/proimmune action compared with NadA\u2013 OMVs. To do this we investigated the activity of purified free NadA\u394351\u2013405 and of OMVs from MenB and Escherichia coli strains, expressing or not full-length NadA. NadA\u394351\u2013405 stimulated monocytes and macrophages to secrete cytokines (IL-1\u3b2, TNF-\u3b1, IL-6, IL-12p40, IL-12p70, IL-10) and chemokines (IL-8, MIP-1\u3b1, MCP-1, RANTES), and full-length NadA improved MenB OMV activity, preferentially on macrophages, and only increased cytokine release. NadA\u394351\u2013405 induced the lymphocyte costimulant CD80 in monocytes and macrophages, and NadA+ OMVs induced a wider set of molecules supporting antigen presentation (CD80, CD86, HLA-DR, and ICAM-1) more efficiently than NadA\u2013 OMVs only in macrophages. Moreover, membrane NadA effects, unlike NadA\u394351\u2013405 ones, were much less IFN-\u3b3-sensitive. The activity of NadA-positive E. coli OMVs was similar to that of control OMVs. NadA in MenB OMVs acted at adhesin concentrations 3c106 times lower than those required to stimulate cells with free NadA\u394351\u2013405