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.

Important functional role of the protein osteopontin in the progression of malignant pleural mesothelioma.

Background: Malignant Pleural Mesothelioma (MPM) is an aggressive cancer of the mesothelial lining associated with exposure to airborne non-degradable asbestos fibers. Its poor response to currently available treatments prompted us to explore the biological mechanisms involved in its progression. MPM is characterized by chronic non-resolving inflammation; in this study we investigated which inflammatory mediators are mostly expressed in biological tumor samples from MPM patients, with a focus on inflammatory cytokines, chemokines and matrix components. Methods: Expression and quantification of Osteopontin (OPN) was detected in tumor and plasma samples of MPM patients by mRNA, immunohistochemistry and ELISA. The functional role of OPN was investigated in mouse MPM cell lines in vivo using an orthotopic syngeneic mouse model. Results: In patients with MPM, the protein OPN was significantly more expressed in tumors than in normal pleural tissues and predominantly produced by mesothelioma cells; plasma levels were elevated in patients and associated with poor prognosis. However, modulation of OPN levels was not significantly different in a series of 18 MPM patients receiving immunotherapy with durvalumab alone or with pembrolizumab in combination with chemotherapy, some of whom achieved a partial clinical response. Two established murine mesothelioma cell lines: AB1 and AB22 of sarcomatoid and epithelioid histology, respectively, spontaneously produced high levels of OPN. Silencing of the OPN gene (Spp1) dramatically inhibited tumor growth in vivo in an orthotopic model, indicating that OPN has an important promoting role in the proliferation of MPM cells. Treatment of mice with anti-CD44 mAb, blocking a major OPN receptor, significantly reduced tumor growth in vivo. Conclusion: These results demonstrate that OPN is an endogenous growth factor for mesothelial cells and inhibition of its signaling may be helpful to restrain tumor progression in vivo. These findings have translational potential to improve the therapeutic response of human MPM.

Mannose-modified hyaluronic acid nanocapsules for the targeting of tumor-associated macrophages.

Tumor-associated macrophages (TAMs), a class of immune cells that play a key role in tumor immunosuppression, are recognized as important targets to improve cancer prognosis and treatment. Consequently, the engineering of drug delivery nanocarriers that can reach TAMs has acquired special relevance. This work describes the development and biological evaluation of a panel of hyaluronic acid (HA) nanocapsules (NCs), with different compositions and prepared by different techniques, designed to target macrophages. The results showed that plain HA NCs did not significantly influence the polarization of M0 and M2-like macrophages towards an M1-like pro-inflammatory phenotype; however, the chemical functionalization of HA with mannose (HA-Man) led to a significant increase of NCs uptake by M2 macrophages in vitro and to an improved biodistribution in a MN/MNCA1 fibrosarcoma mouse model with high infiltration of TAMs. These functionalized HA-Man NCs showed a higher accumulation in the tumor compared to non-modified HA NCs. Finally, the pre-administration of the liposomal liver occupying agent Nanoprimer™ further increased the accumulation of the HA-Man NCs in the tumor. This work highlights the promise shown by the HA-Man NCs to target TAMs and thus provides new options for the development of nanomedicine and immunotherapy-based cancer treatments.

In Vitro Methods to Evaluate Macrophage Polarization and Function in Cancer.

Tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer and hinder the anti-tumoral efficacy of most treatments currently applied in the clinic. However, a key feature of macrophages is their phenotypical and functional plasticity, which called their attention as promising targets for therapeutic intervention based on their elimination or reprogramming toward M1-like cytotoxic effector cells, with anti-tumor functions. This polarization status of macrophages can be studied in terms of molecular markers and functional activities, using an appropriate combination of experimental methodologies, both in vitro and in vivo. Here we focus on describing in vitro protocols to isolate primary monocytes from buffy coats and to study macrophage phenotype and function, after exposure to new therapies, by a combination of flow cytometry, RT-PCR, and ELISA analysis. We also provide the methodology to evaluate in vitro the cytotoxic activity of treated macrophages toward cancer cells.

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.

Polymeric nanocapsules loaded with poly(I:C) and resiquimod to reprogram tumor-associated macrophages for the treatment of solid tumors.

Background: In the tumor microenvironment (TME), tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer. Toll-like receptors (TLRs) ligands, such as poly(I:C) or resiquimod (R848) are able to reprogram TAMs towards M1-like antitumor effector cells. The objective of our work has been to develop and evaluate polymeric nanocapsules (NCs) loaded with poly(I:C)+R848, to improve drug stability and systemic toxicity, and evaluate their targeting and therapeutic activity towards TAMs in the TME of solid tumors. Methods: NCs were developed by the solvent displacement and layer-by-layer methodologies and characterized by dynamic light scattering and nanoparticle tracking analysis. Hyaluronic acid (HA) was chemically functionalized with mannose for the coating of the NCs to target TAMs. NCs loaded with TLR ligands were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry, using primary human monocyte-derived macrophages. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry and multispectral immunophenotyping. Results: We have developed polymeric NCs loaded with poly(I:C)+R848. Among a series of 5 lead prototypes, protamine-NCs were selected based on their physicochemical properties (size, charge, stability) and in vitro characterization, showing good biocompatibility on primary macrophages and ability to stimulate their production of T-cell attracting chemokines (CXCL10, CCL5) and to induce M1-like macrophages cytotoxicity towards tumor cells. In mouse tumor models, the intratumoral injection of poly(I:C)+R848-protamine-NCs significantly prevented tumor growth and lung metastasis. In an orthotopic murine lung cancer model, the intravenous administration of poly(I:C)+R848-prot-NCs, coated with an additional layer of HA-mannose to improve TAM-targeting, resulted in good antitumoral efficacy with no apparent systemic toxicity. While no significant alterations were observed in T cell numbers (CD8, CD4 or Treg), TAM-reprogramming in treated mice was confirmed by the relative decrease of interstitial versus alveolar macrophages, having higher CD86 expression but lower CD206 and Arg1 expression in the same cells, in treated mice. Conclusion: Mannose-HA-protamine-NCs loaded with poly(I:C)+R848 successfully reprogram TAMs in vivo, and reduce tumor progression and metastasis spread in mouse tumors.

A PCR-based method for the diagnosis of Enterobius vermicularis in stool samples, specifically designed for clinical application.

Background: Enterobius vermicularis (E. vermicularis) is a nematode that infects up to 200 million people worldwide, despite effective medications being available. Conventional diagnostic tests are hindered by low sensitivity and poor patient compliance. Furthermore, no biomolecular techniques are available for clinical application. The aim of this study was to develop a procedure specifically designed for clinical application to detect E. vermicularis by means of PCR. Materials and methods: Two subject groups were taken into account: a group of 27 infected patients and a control group of 27 healthy subjects. A nested-PCR was performed on fecal samples to detect E. vermicularis. Due to the intrinsic difficulties of the fecal matrix, several countermeasures were adopted to ensure the efficient performance of the method: (a) a large amount of feces for the extraction process (20 g instead of 200 mg); (b) a combination of chemical and physical treatments to grind the fecal matrix; (c) an additional purification process for the negative samples after the first nested-PCR; and (d) the selection of a very specific target region for the PCR. Results: Due to the lack of overlap with other organisms, a sequence of the 5S ribosomal DNA (rDNA) spacer region including the tract SL1 was chosen to design appropriate external and internal primers. The first nested-PCR detected E.vermicularis in 19/27 samples from infected patients. After further purification, 5/8 of the negative samples resulted positive at the second PCR. Conversely, all the samples from healthy controls resulted negative to both PCRs. Sensitivity and specificity of the method were, respectively, 88.9% and 100%. Conclusion: The results prove the high diagnostic accuracy of the proposed method, addressing and overcoming the challenges posed by both conventional tests and PCR-based approaches. Therefore, the method can be proposed for clinical application.

Innate and Adaptive Responses of Intratumoral Immunotherapy with Endosomal Toll-Like Receptor Agonists.

Toll-like receptors (TLRs) are natural initial triggers of innate and adaptive immune responses. With the advent of cancer immunotherapy, nucleic acids engineered as ligands of endosomal TLRs have been investigated for the treatment of solid tumors. Despite promising results, their systemic administration, similarly to other immunotherapies, raises safety issues. To overcome these problems, recent studies have applied the direct injection of endosomal TLR agonists in the tumor and/or draining lymph nodes, achieving high local drug exposure and strong antitumor response. Importantly, intratumoral delivery of TLR agonists showed powerful effects not only against the injected tumors but also often against uninjected lesions (abscopal effects), resulting in some cases in cure and antitumoral immunological memory. Herein, we describe the structure and function of TLRs and their role in the tumor microenvironment. Then, we provide our vision on the potential of intratumor versus systemic delivery or vaccination approaches using TLR agonists, also considering the use of nanoparticles to improve their targeting properties. Finally, we collect the preclinical and clinical studies applying intratumoral injection of TLR agonists as monotherapies or in combination with: (a) other TLR or STING agonists; (b) other immunotherapies; (c) radiotherapy or chemotherapy; (d) targeted therapies.

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.

Intratumoral combination therapy with poly(I:C) and resiquimod synergistically triggers tumor-associated macrophages for effective systemic antitumoral immunity.

BACKGROUND: Tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer and hinder the antitumoral efficacy of most treatments currently applied in the clinic. Previous studies have evaluated the antitumoral immune response triggered by (TLR) agonists, such as poly(I:C), imiquimod (R837) or resiquimod (R848) as monotherapies; however, their combination for the treatment of cancer has not been explored. This study investigates the antitumoral efficacy and the macrophage reprogramming triggered by poly(I:C) combined with R848 or with R837, versus single treatments. METHODS: TLR agonist treatments were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry using primary human and murine M-CSF-differentiated macrophages. Cytotoxic activity of TLR-treated macrophages toward cancer cells was evaluated with an in vitro functional assay by flow cytometry. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry, RT-qPCR, multispectral immunophenotyping, quantitative proteomic experiments, and protein-protein interaction analysis. RESULTS: Results demonstrated the higher efficacy of poly(I:C) combined with R848 versus single treatments or combined with R837 to polarize macrophages toward M1-like antitumor effectors in vitro. In vivo, the intratumoral synergistic combination of poly(I:C)+R848 significantly prevented tumor growth and metastasis in lung cancer and fibrosarcoma immunocompetent murine models. Regressing tumors showed increased infiltration of macrophages with a higher M1:M2 ratio, recruitment of CD4+ and CD8+ T cells, accompanied by a reduction of immunosuppressive CD206+ TAMs and FOXP3+/CD4+ T cells. The depletion of both CD4+ and CD8+ T cells resulted in complete loss of treatment efficacy. Treated mice acquired systemic antitumoral response and resistance to tumor rechallenge mediated by boosted macrophage cytotoxic activity and T-cell proliferation. Proteomic experiments validate the superior activation of innate immunity by poly(I:C)+R848 combination versus single treatments or poly(I:C)+R837, and protein-protein-interaction network analysis reveal the key activation of the STAT1 pathway. DISCUSSION: These findings demonstrate the antitumor immune responses mediated by macrophage activation on local administration of poly(I:C)+R848 combination and support the intratumoral application of this therapy to patients with solid tumors in the clinic.

Structural equation modeling to shed light on the controversial role of climate on the spread of SARS-CoV-2.

Climate seems to influence the spread of SARS-CoV-2, but the findings of the studies performed so far are conflicting. To overcome these issues, we performed a global scale study considering 134,871 virologic-climatic-demographic data (209 countries, first 16 weeks of the pandemic). To analyze the relation among COVID-19, population density, and climate, a theoretical path diagram was hypothesized and tested using structural equation modeling (SEM), a powerful statistical technique for the evaluation of causal assumptions. The results of the analysis showed that both climate and population density significantly influence the spread of COVID-19 (p < 0.001 and p < 0.01, respectively). Overall, climate outweighs population density (path coefficients: climate vs. incidence = 0.18, climate vs. prevalence = 0.11, population density vs. incidence = 0.04, population density vs. prevalence = 0.05). Among the climatic factors, irradiation plays the most relevant role, with a factor-loading of - 0.77, followed by temperature (- 0.56), humidity (0.52), precipitation (0.44), and pressure (0.073); for all p < 0.001. In conclusion, this study demonstrates that climatic factors significantly influence the spread of SARS-CoV-2. However, demographic factors, together with other determinants, can affect the transmission, and their influence may overcome the protective effect of climate, where favourable.

Therapeutic Manipulation of Tumor-associated Macrophages: Facts and Hopes from a Clinical and Translational Perspective.

The stroma of most solid tumors is populated by myeloid cells, which mostly represent macrophages. Tumor-associated macrophages (TAMs), strongly influenced by cancer cell-derived factors, are key drivers of immunosuppression and support tumor growth and spread to distant sites. Their accurate quantification and characterization in the tumor microenvironment are gaining prognostic value: increasing evidence demonstrates their ability to hamper cancer patients' response to chemotherapy, as well as to immunotherapies based on checkpoint inhibition. Therefore, strategies to counteract their negative effects are nowadays gaining momentum at preclinical, translational, and clinical levels. Our knowledge of the biology of TAMs has greatly advanced in the last years; several strategies to target and reprogram their functions to become antitumor effectors have proven successful in experimental preclinical tumor models; on the other hand, few approaches have so far been effectively translated into clinic practice. A growing interest in the therapeutic manipulation of TAMs is evidenced by numerous early-phase clinical trials, which are continuously fueled by new discoveries from basic research. This gives us hope that the targeting and sustained reprogramming of TAMs will be more specific to synergize with current therapies and maximize antitumor responses in patients.

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.

Current Strategies to Target Tumor-Associated-Macrophages to Improve Anti-Tumor Immune Responses.

: Established evidence demonstrates that tumor-infiltrating myeloid cells promote rather than stop-cancer progression. Tumor-associated macrophages (TAMs) are abundantly present at tumor sites, and here they support cancer proliferation and distant spreading, as well as contribute to an immune-suppressive milieu. Their pro-tumor activities hamper the response of cancer patients to conventional therapies, such as chemotherapy or radiotherapy, and also to immunotherapies based on checkpoint inhibition. Active research frontlines of the last years have investigated novel therapeutic strategies aimed at depleting TAMs and/or at reprogramming their tumor-promoting effects, with the goal of re-establishing a favorable immunological anti-tumor response within the tumor tissue. In recent years, numerous clinical trials have included pharmacological strategies to target TAMs alone or in combination with other therapies. This review summarizes the past and current knowledge available on experimental tumor models and human clinical studies targeting TAMs for cancer treatment.