Diagnostic-and therapeutic release-aimed nanoparticles require the highest degree of biocompatibility. Some physical and chemical characteristics of such nanomaterials are often at odds with this requirement. For instance, metals with specific features used as contrast agents in magnetic resonance imaging need particular coatings to improve their blood solubility and increase their biocompatibility. Other examples come from the development of nanocarriers exploiting the different characteristics of two or more materials, i.e., the ability to encapsulate a certain drug by one core-material and the targeting capability of a different coating surface. Furthermore, all these "human-non-self" modifications necessitate proofs of compatibility with the immune system to avoid inflammatory reactions and resultant adverse effects for the patient. In the present review we discuss the molecular interactions and responses of the immune system to the principal nanoparticle surface modifications used in nanomedicine.

BackgroundObesity is a chronic low inflammatory state. In the obesity condition the white adipose tissue (WAT) is massively infiltrated with monocytes/macrophages, and the nature of the signals recruiting these inflammatory cells has yet to be fully elucidated. Haptoglobin (Hp) is an inflammatory marker and its expression is induced in the WAT of obese subjects. In an effort to elucidate the biological significance of Hp presence in the WAT and of its upregulation in obesity we formulated the hypothesis that Hp may serve as a macrophage chemoattractant.ResultsWe demonstrated by chemotaxis assay that Hp is able to attract chemokine (C-C motif) receptor 2 (CCR2)-transfected pre-B lymphocytes and monocytes in a dose-dependent manner. Moreover, Hp-mediated migration of monocytes is impaired by CCR2-specific inhibition or previous cell exposure to monocyte chemoattractant protein 1 (MCP1) (also known as CCR2 ligand or chemokine (C-C motif) ligand 2 (CCL2)). Downstream effects of Hp/CCR2 interaction were also investigated: flow cytometry proved that monocytes treated with Hp show reduced CCR2 expression on their surface; Hp interaction induces calcium release that is reduced upon pretreatment with CCR2 antagonist; extracellular signal-regulated kinase (ERK)1/2, a signal transducer activated by CCR2, is phosphorylated following Hp treatment and this phosphorylation is reduced when cells are pretreated with a specific CCR2 inhibitor. Consistently, blocking the ERK1/2 pathway with U0126, the selective inhibitor of the ERK upstream mitogen-activated protein (MAP)-ERK kinase (MEK), results in a dramatic reduction (by almost 100%) of the capability of Hp to induce monocyte migration.ConclusionsOur data show that Hp is a novel monocyte chemoattractant and that its chemotactic potential is mediated, at least in part. by its interaction with CCR2.

OBJECTIVEHaptoglobin (Hp) is upregulated in both inflammation and obesity. The low chronic inflammatory state, caused by massive adipose tissue macrophage (ATM) infiltration found in obesity, and low adiponectin have been implicated in the development of insulin resistance and hepatosteatosis. The aim of this work was to investigate whether and how Hp interferes with the onset of obesity-associated complications.RESEARCH DESIGN AND METHODSHp-null (Hp−/−) and wild-type (WT) mice were metabolically profiled under chow-food diet (CFD) and high-fat diet (HFD) feeding by assessing physical parameters, glucose tolerance, insulin sensitivity, insulin response to glucose load, liver triglyceride content, plasma levels of leptin, insulin, glucose, and adiponectin. ATM content was evaluated by using immunohistochemistry (anti-F4/80 antibody). Adiponectin expression was measured in Hp-treated, cultured 3T3-L1 and human adipocytes.RESULTSNo genotype-related difference was found in CFD animals. HFD-Hp−/− mice revealed significantly higher glucose tolerance, insulin sensitivity, glucose-stimulated insulin secretion, and adiponectin expression and reduced hepatomegaly/steatosis compared with HFD-WT mice. White adipose tissue (WAT) of HFD-Hp−/− mice showed higher activation of insulin signaling cascade, lower ATM, and higher adiponectin expression. Hp was able to inhibit adiponectin expression in cultured adipocytes.CONCLUSIONSWe demonstrated that in the absence of Hp, obesity-associated insulin resistance and hepatosteatosis are attenuated, which is associated with reduced ATM content, increased plasma adiponectin, and higher WAT insulin sensitivity.

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Haptoglobin (Hp) is an inflammatory and adiposity marker, its expression during obesity being specifically induced in the white adipose tissue (WAT). We previously reported that when challenged with a high fat diet (HFD) Hp−/− mice are partially protected from the onset of insulin resistance and hepatosteatosis. The aim of the present study was to get further insights into Hp function in WAT. To this end, we performed histological and gene expression analysis of the Hp−/− WAT, both in standard and obesity conditions, and investigated how Hp deficiency impacts adipogenesis and WAT development. The average size and percentage of very large adipocytes were respectively smaller and reduced in HFD Hp−/− mice as compared with HFD WT. The expression of perilipin, HSL and angiogenesis related markers were increased in HFD Hp−/− mice. Lean adult Hp−/− showed significantly larger adipocytes and lower subcutaneous WAT expression of aP2 and LPL with respect to WT. Hp−/− young mice (P30) were characterized by larger adipocyte size and lower expression of adipocyte and adipogenesis markers. Comparison of adipocyte size distribution between young and adult mice revealed attenuated changes in Hp−/− mice compared with WT. Mouse embryonic fibroblasts from Hp−/− mice were less capable of accumulating triglycerides and exhibited lower expression of PPARγ, aP2, FAS, LPL and Leptin. In conclusion, Hp deficiency tends to blunt the effect of age and diet on the size of adipocytes, which show less susceptibility to develop hypertrophy during obesity and a reduced adipogenic/hyperplastic potential during youth. In addition, Hp deficiency impacts negatively on adipogenesis.

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO 2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO 2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Cerium dioxide nanoparticles (CeO2 NPs) are known as scavengers of reactive oxygen species for the coexistence of Ce 3+ / Ce 4+ oxidation states. Cell treatments with CeO2 NPs often lead to controversial proinflammatory and anti-inflammatory results. The aim of the study was to investigate the immune events following the administration of ceria nanoparticles to THP-1 monocytes. To address this issue, we performed flow cytometry, chemotaxis and ELISA experiments on THP-1 monocytes treated with different concentrations of CeO2 NPs. CeO2 nanoparticle treatments induced a significant pro-inflammatory C-C chemokine receptor 2 (CCR2) up-regulation within the first 6 hours lasting over-expressed for 24 hours. Differently, CCR5 showed no response at any concentration tested. Enhanced chemotaxis towards the CCR2 specific ligand MCP-1 reinforced the observation demonstrating a functional immune outcome. The pro-inflammatory profile of the treated monocytes was also supported by CD16 upregulation but no differences in CX3CR1 or other monocyte receptors, like CD11b and CD14, were detectable. Moreover, CeO2 NPs exposure did not promote any release of inflammatory cytokines suggesting a specific and direct effect of the nanoparticles on CCR2 and CD16.Our in vitro results reveal a specific role of CeO2 NPs in the upregulation of CCR2, which might contribute to increase the proinflammatory monocyte/macrophage migration toward the sites of CCL2 expression.

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