Synthetic peptides of IL-1Ra and HSP70 have anti-inflammatory activity on human primary monocytes and macrophages: Potential treatments for inflammatory diseases.

Chronic inflammatory diseases are increasing in developed societies, thus new anti-inflammatory approaches are needed in the clinic. Synthetic peptides complexes can be designed to mimic the activity of anti-inflammatory mediators, in order to alleviate inflammation. Here, we evaluated the anti-inflammatory efficacy of tethered peptides mimicking the interleukin-1 receptor antagonist (IL-1Ra) and the heat-shock protein 70 (HSP70). We tested their biocompatibility and anti-inflammatory activity in vitro in primary human monocytes and differentiated macrophages activated with two different stimuli: the TLR agonists (LPS + IFN-γ) or Pam3CSK4. Our results demonstrate that IL-1Ra and HSP70 synthetic peptides present a satisfactory biocompatible profile and significantly inhibit the secretion of several pro-inflammatory cytokines (IL-6, IL-8, IL-1ß and TNFα). We further confirmed their anti-inflammatory activity when peptides were coated on a biocompatible material commonly employed in surgical implants. Overall, our findings support the potential use of IL-1Ra and HSP70 synthetic peptides for the treatment of inflammatory conditions.

An in vitro model for osteoarthritis using long-cultured inflammatory human macrophages repeatedly stimulated with TLR agonists.

Osteoarthritis (OA) is characterized by an abundance of inflammatory M1-like macrophages damaging local tissues. The search for new potential drugs for OA suffers from the lack of appropriate methods of long-lasting inflammation. Here we developed and characterized an in vitro protocol of long-lasting culture of primary human monocyte-derived macrophages differentiated with a combination of M-CSF+GM-CSF that optimally supported long-cultured macrophages (LC-Mϕs) for up to 15 days, unlike their single use. Macrophages repeatedly stimulated for 15 days with the TLR2 ligand Pam3CSK4 (LCS-Mϕs), showed sustained levels over time of IL-6, CCL2, and CXCL8, inflammatory mediators that were also detected in the synovial fluids of OA patients. Furthermore, macrophages isolated from the synovia of two OA patients showed an expression profile of inflammation-related genes similar to that of LCS-Mϕs, validating our protocol as a model of chronically activated inflammatory macrophages. Next, to confirm that these LCS-Mϕs could be modulated by anti-inflammatory compounds, we employed dexamethasone and/or celecoxib, two drugs widely used in OA treatment, that significantly inhibited the production of inflammatory mediators. This easy-to-use in vitro protocol of long-lasting inflammation with primary human macrophages could be useful for the screening of new compounds to improve the therapy of inflammatory disorders.

In Vivo Analysis of Tumor-Associated Macrophages in the Tumor Microenvironment.

In most solid cancers, tumor-associated macrophages (TAMs) infiltrating the tumor microenvironment (TME) represent a major population of immunosuppressive cells. This correlates with poor prognosis and resistance to antitumoral therapies, including immune checkpoint inhibitors. Although initial preclinical studies were primarily meant to deplete macrophages in the TME or prevent their recruitment at tumor sites, recent evidence has indicated that the reprogramming of macrophages into cytotoxic effectors might be more beneficial in eliciting an effective antitumor immune response. Taking this into consideration, the comprehensive analysis of the phenotype and function of macrophages in the TME, and their interaction with cancer cells or other immune cells, has become of paramount importance in oncological research. Accordingly, here we explain the experimental procedures for the in vivo evaluation of tumor progression and response to therapy, with a particular focus on the detailed analysis of TAMs and related immune cells in the TME by flow cytometry, RNA analysis, and multiplex immunophenotyping. The output generated through these experiments allow researchers to test the efficacy of new therapeutic strategies on targeting.

Recent Discoveries in Nanoparticle-Macrophage Interactions: In Vitro Models for Nanosafety Testing and Novel Nanomedical Approaches for Immunotherapy.

Nanoparticles (NPs) offer unique properties for biomedical applications, leading to new nanomedicines [...].

Drug Delivery Systems for the Treatment of Knee Osteoarthritis: A Systematic Review of In Vivo Studies.

Many efforts have been made in the field of nanotechnology to improve the local and sustained release of drugs, which may be helpful to overcome the present limitations in the treatment of knee OA. Nano-/microparticles and/or hydrogels can be now engineered to improve the administration and intra-articular delivery of specific drugs, targeting molecular pathways and pathogenic mechanisms involved in OA progression and remission. In order to summarize the current state of this field, a systematic review of the literature was performed and 45 relevant studies were identified involving both animal models and humans. We found that polymeric nanoparticles loaded with anti-inflammatory drugs (i.e., dexamethasone or celecoxib) are the most frequently investigated drug delivery systems, followed by microparticles and hydrogels. In particular, the nanosystem most frequently used in preclinical research consists of PLGA-nanoparticles loaded with corticosteroids and non-steroidal anti-inflammatory drugs. Overall, improvement in histological features, reduction in joint inflammation, and improvement in clinical scores in patients were observed. The last advances in the field of nanotechnology could offer new opportunities to treat patients affected by knee OA, including those with previous meniscectomy. New smart drug delivery approaches, based on nanoparticles, microparticles, and hydrogels, may enhance the therapeutic potential of intra-articular agents by increasing the permanence of selected drugs inside the joint and better targeting specific receptors and tissues.

Zebrafish Models for the Safety and Therapeutic Testing of Nanoparticles with a Focus on Macrophages.

New nanoparticles and biomaterials are increasingly being used in biomedical research for drug delivery, diagnostic applications, or vaccines, and they are also present in numerous commercial products, in the environment and workplaces. Thus, the evaluation of the safety and possible therapeutic application of these nanomaterials has become of foremost importance for the proper progress of nanotechnology. Due to economical and ethical issues, in vitro and in vivo methods are encouraged for the testing of new compounds and/or nanoparticles, however in vivo models are still needed. In this scenario, zebrafish (Danio rerio) has demonstrated potential for toxicological and pharmacological screenings. Zebrafish presents an innate immune system, from early developmental stages, with conserved macrophage phenotypes and functions with respect to humans. This fact, combined with the transparency of zebrafish, the availability of models with fluorescently labelled macrophages, as well as a broad variety of disease models offers great possibilities for the testing of new nanoparticles. Thus, with a particular focus on macrophage-nanoparticle interaction in vivo, here, we review the studies using zebrafish for toxicological and biodistribution testing of nanoparticles, and also the possibilities for their preclinical evaluation in various diseases, including cancer and autoimmune, neuroinflammatory, and infectious diseases.

Cellular and Molecular Mechanisms Underlying Glioblastoma and Zebrafish Models for the Discovery of New Treatments.

Glioblastoma (GBM) is the most common of all brain malignant tumors; it displays a median survival of 14.6 months with current complete standard treatment. High heterogeneity, aggressive and invasive behavior, the impossibility of completing tumor resection, limitations for drug administration and therapeutic resistance to current treatments are the main problems presented by this pathology. In recent years, our knowledge of GBM physiopathology has advanced significantly, generating relevant information on the cellular heterogeneity of GBM tumors, including cancer and immune cells such as macrophages/microglia, genetic, epigenetic and metabolic alterations, comprising changes in miRNA expression. In this scenario, the zebrafish has arisen as a promising animal model to progress further due to its unique characteristics, such as transparency, ease of genetic manipulation, ethical and economic advantages and also conservation of the major brain regions and blood-brain-barrier (BBB) which are similar to a human structure. A few papers described in this review, using genetic and xenotransplantation zebrafish models have been used to study GBM as well as to test the anti-tumoral efficacy of new drugs, their ability to interact with target cells, modulate the tumor microenvironment, cross the BBB and/or their toxicity. Prospective studies following these lines of research may lead to a better diagnosis, prognosis and treatment of patients with GBM.

Targeting Tumor-Associated Macrophages in Anti-Cancer Therapies: Convincing the Traitors to Do the Right Thing.

In the last decade, it has been well-established that tumor-infiltrating myeloid cells fuel not only the process of carcinogenesis through cancer-related inflammation mechanisms, but also tumor progression, invasion, and metastasis. In particular, tumor-associated macrophages (TAMs) are the most abundant leucocyte subset in many cancers and play a major role in the creation of a protective niche for tumor cells. Their ability to generate an immune-suppressive environment is crucial to escape the immune system and to allow the tumor to proliferate and metastasize to distant sites. Conventional therapies, including chemotherapy and radiotherapy, are often not able to limit cancer growth due to the presence of pro-tumoral TAMs; these are also responsible for the failure of novel immunotherapies based on immune-checkpoint inhibition. Several novel therapeutic strategies have been implemented to deplete TAMs; however, more recent approaches aim to use TAMs themselves as weapons to fight cancer. Exploiting their functional plasticity, the reprogramming of TAMs aims to convert immunosuppressive and pro-tumoral macrophages into immunostimulatory and anti-tumor cytotoxic effector cells. This shift eventually leads to the reconstitution of a reactive immune landscape able to destroy the tumor. In this review, we summarize the current knowledge on strategies able to reprogram TAMs with single as well as combination therapies.

Therapeutic Manipulation of Macrophages Using Nanotechnological Approaches for the Treatment of Osteoarthritis.

Osteoarthritis (OA) is the most common joint pathology causing severe pain and disability. Macrophages play a central role in the pathogenesis of OA. In the joint microenvironment, macrophages with an M1-like pro-inflammatory phenotype induce chronic inflammation and joint destruction, and they have been correlated with the development and progression of the disease, while the M2-like anti-inflammatory macrophages support the recovery of the disease, promoting tissue repair and the resolution of inflammation. Nowadays, the treatment of OA in the clinic relies on systemic and/or intra-articular administration of anti-inflammatory and pain relief drugs, as well as surgical interventions for the severe cases (i.e., meniscectomy). The disadvantages of the pharmacological therapy are related to the chronic nature of the disease, requiring prolonged treatments, and to the particular location of the pathology in joint tissues, which are separated anatomical compartments with difficult access for the drugs. To overcome these challenges, nanotechnological approaches have been investigated to improve the delivery of drugs toward macrophages into the diseased joint. This strategy may offer advantages by reducing off-target toxicities and improving long-term therapeutic efficacy. In this review, we describe the nanomaterial-based approaches designed so far to directly or indirectly manipulate macrophages for the treatment of osteoarthritis.

Arginine-Based Poly(I:C)-Loaded Nanocomplexes for the Polarization of Macrophages Toward M1-Antitumoral Effectors.

Background: Tumor-associated macrophages (TAMs), with M2-like immunosuppressive profiles, are key players in the development and dissemination of tumors. Hence, the induction of M1 pro-inflammatory and anti-tumoral states is critical to fight against cancer cells. The activation of the endosomal toll-like receptor 3 by its agonist poly(I:C) has shown to efficiently drive this polarization process. Unfortunately, poly(I:C) presents significant systemic toxicity, and its clinical use is restricted to a local administration. Therefore, the objective of this work has been to facilitate the delivery of poly(I:C) to macrophages through the use of nanotechnology, that will ultimately drive their phenotype toward pro-inflammatory states. Methods: Poly(I:C) was complexed to arginine-rich polypeptides, and then further enveloped with an anionic polymeric layer either by film hydration or incubation. Physicochemical characterization of the nanocomplexes was conducted by dynamic light scattering and transmission electron microscopy, and poly(I:C) association efficiency by gel electrophoresis. Primary human-derived macrophages were used as relevant in vitro cell model. Alamar Blue assay, ELISA, PCR and flow cytometry were used to determine macrophage viability, polarization, chemokine secretion and uptake of nanocomplexes. The cytotoxic activity of pre-treated macrophages against PANC-1 cancer cells was assessed by flow cytometry. Results: The final poly(I:C) nanocomplexes presented sizes lower than 200 nm, with surface charges ranging from +40 to -20 mV, depending on the envelopment. They all presented high poly(I:C) loading values, from 12 to 50%, and great stability in cell culture media. In vitro, poly(I:C) nanocomplexes were highly taken up by macrophages, in comparison to the free molecule. Macrophage treatment with these nanocomplexes did not reduce their viability and efficiently stimulated the secretion of the T-cell recruiter chemokines CXCL10 and CCL5, of great importance for an effective anti-tumor immune response. Finally, poly(I:C) nanocomplexes significantly increased the ability of treated macrophages to directly kill cancer cells. Conclusion: Overall, these enveloped poly(I:C) nanocomplexes might represent a therapeutic option to fight cancer through the induction of cytotoxic M1-polarized macrophages.

Introducing WikiPathways as a Data-Source to Support Adverse Outcome Pathways for Regulatory Risk Assessment of Chemicals and Nanomaterials.

A paradigm shift is taking place in risk assessment to replace animal models, reduce the number of economic resources, and refine the methodologies to test the growing number of chemicals and nanomaterials. Therefore, approaches such as transcriptomics, proteomics, and metabolomics have become valuable tools in toxicological research, and are finding their way into regulatory toxicity. One promising framework to bridge the gap between the molecular-level measurements and risk assessment is the concept of adverse outcome pathways (AOPs). These pathways comprise mechanistic knowledge and connect biological events from a molecular level toward an adverse effect outcome after exposure to a chemical. However, the implementation of omics-based approaches in the AOPs and their acceptance by the risk assessment community is still a challenge. Because the existing modules in the main repository for AOPs, the AOP Knowledge Base (AOP-KB), do not currently allow the integration of omics technologies, additional tools are required for omics-based data analysis and visualization. Here we show how WikiPathways can serve as a supportive tool to make omics data interoperable with the AOP-Wiki, part of the AOP-KB. Manual matching of key events (KEs) indicated that 67% could be linked with molecular pathways. Automatic connection through linkage of identifiers between the databases showed that only 30% of AOP-Wiki chemicals were found on WikiPathways. More loose linkage through gene names in KE and Key Event Relationships descriptions gave an overlap of 70 and 71%, respectively. This shows many opportunities to create more direct connections, for example with extended ontology annotations, improving its interoperability. This interoperability allows the needed integration of omics data linked to the molecular pathways with AOPs. A new AOP Portal on WikiPathways is presented to allow the community of AOP developers to collaborate and populate the molecular pathways that underlie the KEs of AOP-Wiki. We conclude that the integration of WikiPathways and AOP-Wiki will improve risk assessment because omics data will be linked directly to KEs and therefore allow the comprehensive understanding and description of AOPs. To make this assessment reproducible and valid, major changes are needed in both WikiPathways and AOP-Wiki.

Carbon Nanotubes as Optical Sensors in Biomedicine.

Single-walled carbon nanotubes (SWCNTs) have become potential candidates for a wide range of medical applications including sensing, imaging, and drug delivery. Their photophysical properties (i.e., the capacity to emit in the near-infrared), excellent photostability, and fluorescence, which is highly sensitive to the local environment, make SWCNTs promising optical probes in biomedicine. In this Perspective, we discuss the existing strategies for and challenges of using carbon nanotubes for medical diagnosis based on intracellular sensing as well as discuss also their biocompatibility and degradability. Finally, we highlight the potential improvements of this nanotechnology and future directions in the field of carbon nanotubes for biomedical applications.

Nanomedicine and cancer immunotherapy - targeting immunosuppressive cells.

The search for pharmacological strategies to reach and impact on immunosuppressive cells is, currently, one of the most exciting areas in cancer immunology and clinical oncology. In this context, it is increasingly accepted that the success of these therapies will largely depend on the availability of appropriate drug delivery strategies. Considering the critical role that nanotechnology plays in the development of these novel therapies, the main goal of this article is to provide an overview of the potential of nanomedicines targeted to immunosuppressive cells for the treatment of cancer. We present, first, a brief description of classical cancer immunotherapies based on therapeutic vaccination and monoclonal antibodies, with a special focus on the use of nanotechnologies and the targeting of immunological checkpoints. Second, we provide a thoughtful analysis of the possibilities to target the immunosuppressive cells, namely tumour-associated macrophages, myeloid-derived suppressor cells, tumour-associated neutrophils and regulatory T cells, at the tissue level (i.e. tumour, spleen, blood, lymph) and, also, at the cellular level. Finally, we wrap the article with a disclosure of strategies used to impair the generation, kill or re-educate these immunosuppressive cells, thus providing an up-to-date picture of the choices available for therapeutic intervention.

Comprehensive In Vitro Toxicity Testing of a Panel of Representative Oxide Nanomaterials: First Steps towards an Intelligent Testing Strategy.

Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO2 NMs with different surface chemistry - hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO - uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques - precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO2>TiO2). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially 'weak-embryotoxic' and ZnO and SiO2 NMs as 'non-embryotoxic'. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103). This ranking was different in the case of embryonic tissues, for which TiO2 displayed higher toxicity compared with ZnO and SiO2. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.