Eosinophils as potential biomarkers in respiratory viral infections.

Eosinophils are bone marrow-derived granulocytes that, under homeostatic conditions, account for as much as 1-3% of peripheral blood leukocytes. During inflammation, eosinophils can rapidly expand and infiltrate inflamed tissues, guided by cytokines and alarmins (such as IL-33), adhesion molecules and chemokines. Eosinophils play a prominent role in allergic asthma and parasitic infections. Nonetheless, they participate in the immune response against respiratory viruses such as respiratory syncytial virus and influenza. Notably, respiratory viruses are associated with asthma exacerbation. Eosinophils release several molecules endowed with antiviral activity, including cationic proteins, RNases and reactive oxygen and nitrogen species. On the other hand, eosinophils release several cytokines involved in homeostasis maintenance and Th2-related inflammation. In the context of SARS-CoV-2 infection, emerging evidence indicates that eosinophils can represent possible blood-based biomarkers for diagnosis, prognosis, and severity prediction of disease. In particular, eosinopenia seems to be an indicator of severity among patients with COVID-19, whereas an increased eosinophil count is associated with a better prognosis, including a lower incidence of complications and mortality. In the present review, we provide an overview of the role and plasticity of eosinophils focusing on various respiratory viral infections and in the context of viral and allergic disease comorbidities. We will discuss the potential utility of eosinophils as prognostic/predictive immune biomarkers in emerging respiratory viral diseases, particularly COVID-19. Finally, we will revisit some of the relevant methods and tools that have contributed to the advances in the dissection of various eosinophil subsets in different pathological settings for future biomarker definition.

Mechanisms of Action of Ozone Therapy in Emerging Viral Diseases: Immunomodulatory Effects and Therapeutic Advantages With Reference to SARS-CoV-2.

Medical oxygen-ozone (O2-O3) is a successful therapeutic approach accounting on the assessed beneficial action of ozone in the range 30-45 µg/ml (expanded range 10-80 µg/ml according to different protocols), as in this dosage range ozone is able to trigger a cellular hormetic response via the modulating activity of reactive oxygen species (ROS), as signaling molecules. The ozone-dependent ROS-mediated fatty acid oxidation leads to the formation of lipid ozonization products (LOPs), which act as signal transducers by triggering ROS signaling and therefore mitohormetic processes. These processes ultimately activate survival mechanisms at a cellular level, such as the Nrf2/Keap1/ARE system activation, the AMPK/FOXO/mTOR/Sir1 pathway and the Nrf2/NF-kB cross talk. Furthermore, indirectly, via these pathways, LOPs trigger the HIF-1α pathway, the HO-1 signaling and the NO/iNOS biochemical machinery. Ozone-driven shift of cytokine activation pathways, from pro-inflammatory to anti-inflammatory immediately afterwards, also exert direct immunoregulatory effects on regulatory T lymphocytes as well as on the intestinal microbiota, which in turn can affect immune response thus influencing the progression of the disease. In this review, we will describe the biological and biochemical mechanisms of action of ozone therapy with the aim of evaluating both positive and critical aspects of ozone use as a therapeutic adjuvant in the light of emerging viral infections, such as SARS-CoV-2 and microbiome-associated disorders related to SARS-CoV-2.

Anti-Tumorigenic Activities of IL-33: A Mechanistic Insight.

Interleukin-33 (IL-33) is an epithelial-derived cytokine that can be released upon tissue damage, stress, or infection, acting as an alarmin for the immune system. IL-33 has long been studied in the context of Th2-related immunopathologies, such as allergic diseases and parasitic infections. However, its capacity to stimulate also Th1-type of immune responses is now well established. IL-33 binds to its specific receptor ST2 expressed by most immune cell populations, modulating a variety of responses. In cancer immunity, IL-33 can display both pro-tumoral and anti-tumoral functions, depending on the specific microenvironment. Recent findings indicate that IL-33 can effectively stimulate immune effector cells (NK and CD8+ T cells), eosinophils, basophils and type 2 innate lymphoid cells (ILC2) promoting direct and indirect anti-tumoral activities. In this review, we summarize the most recent advances on anti-tumor immune mechanisms operated by IL-33, including the modulation of immune checkpoint molecules, with the aim to understand its potential as a therapeutic target in cancer.

Corrigendum to "Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring".

[This corrects the article DOI: 10.1155/2020/1938704.].

Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring.

BACKGROUND: Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. METHODS: The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. RESULTS: Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. CONCLUSION: Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.

IL-33 restricts tumor growth and inhibits pulmonary metastasis in melanoma-bearing mice through eosinophils.

The alarmin IL-33 is an IL-1 family member that stimulates pleiotropic immune reactions depending on the target tissue and microenvironmental factors. In this study, we have investigated the role of IL-33/ST2 axis in antitumor response to melanoma. Injection of IL-33 in mice-bearing subcutaneous B16.F10 melanoma resulted in significant tumor growth delay. This effect was associated with intratumoral accumulation of CD8+ T cells and eosinophils, decrease of immunosuppressive myeloid cells, and a mixed Th1/Th2 cytokine expression pattern with local and systemic activation of CD8+ T and NK cells. Moreover, intranasal administration of IL-33 determined ST2-dependent eosinophil recruitment in the lung that prevented the onset of pulmonary metastasis after intravenous injection of melanoma cells. Accordingly, ST2-deficient mice developed pulmonary metastasis at higher extent than wild-type counterparts, associated with lower eosinophil frequencies in the lung. Of note, depletion of eosinophils by in vivo treatment with anti-Siglec-F antibody abolished the ability of IL-33 to both restrict primary tumor growth and metastasis formation. Finally, we show that IL-33 is able to activate eosinophils resulting in efficient killing of target melanoma cells, suggesting a direct antitumor activity of eosinophils following IL-33 treatment. Our results advocate for an eosinophil-mediated antitumoral function of IL-33 against melanoma, thus opening perspectives for novel cancer immunotherapy strategies.

Cancer cell-autonomous contribution of type I interferon signaling to the efficacy of chemotherapy.

Some of the anti-neoplastic effects of anthracyclines in mice originate from the induction of innate and T cell-mediated anticancer immune responses. Here we demonstrate that anthracyclines stimulate the rapid production of type I interferons (IFNs) by malignant cells after activation of the endosomal pattern recognition receptor Toll-like receptor 3 (TLR3). By binding to IFN-α and IFN-ß receptors (IFNARs) on neoplastic cells, type I IFNs trigger autocrine and paracrine circuitries that result in the release of chemokine (C-X-C motif) ligand 10 (CXCL10). Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or Cxcl10, respectively, was artificially supplied. Moreover, a type I IFN-related signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast carcinoma characterized by poor prognosis. Our data suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such 'viral mimicry' constitutes a hallmark of successful chemotherapy.

APC activation by IFN-alpha decreases regulatory T cell and enhances Th cell functions.

Type I IFNs are central to a vast array of immunological functions. Their early induction in innate immune responses provides one of the most important priming mechanisms for the subsequent establishment of adaptive immunity. The outcome is either promotion or inhibition of these responses, but the conditions under which one or the other prevails remain to be defined. The main objective of the current study was to determine the involvement of IFN-alpha on murine CD4(+)CD25(-) Th cell activation, as well as to define the role played by this cytokine on CD4(+)CD25(+) regulatory T (Treg) cell proliferation and function. Although IFN-alpha promotes CD4(+)CD25(-) Th cells coincubated with APCs to produce large amounts of IL-2, the ability of these cells to respond to IL-2 proliferative effects is prevented. Moreover, in medium supplemented with IFN-alpha, IL-2-induced CD4(+)CD25(+) Treg cell proliferation is inhibited. Notably, IFN-alpha also leads to a decrease of the CD4(+)CD25(+) Treg cell suppressive activity. Altogether, these findings indicate that through a direct effect on APC activation and by affecting CD4(+)CD25(+) Treg cell-mediated suppression, IFN-alpha sustains and drives CD4(+)CD25(-) Th cell activation.

Type I interferons as vaccine adjuvants against infectious diseases and cancer.

Presently, new attention is given to type I interferons (IFNs) as essential factors linking innate and adaptive immunity. Several studies provided evidence about the importance of IFN-alpha in the differentiation of the Th1 subset, in the generation and activity of cytotoxic T lymphocytes, in the enhancement of a primary antibody response and in the activation of dendritic cells. Owing to their immunomodulatory properties, type I IFNs can represent good candidates to be used as adjuvants for vaccination. In the present review, we summarize recent studies in humans and in animal models, suggesting a possible application of type I IFNs as adjuvants for the development of more effective vaccines against infectious diseases and cancer.

Cyclophosphamide enhances the antitumor efficacy of adoptively transferred immune cells through the induction of cytokine expression, B-cell and T-cell homeostatic proliferation, and specific tumor infiltration.

PURPOSE: Immunotherapy is a promising antitumor strategy, which can be successfully combined with current anticancer treatments, as suggested by recent studies showing the paradoxical chemotherapy-induced enhancement of the immune response. The purpose of the present work is to dissect the biological events induced by chemotherapy that cooperate with immunotherapy in the success of the combined treatment against cancer. In particular, we focused on the following: (a) cyclophosphamide-induced modulation of several cytokines, (b) homeostatic proliferation of adoptively transferred lymphocytes, and (c) homing of transferred lymphocytes to secondary lymphoid organs and tumor mass. EXPERIMENTAL DESIGN: Here, we used the adoptive transfer of tumor-immune cells after cyclophosphamide treatment of tumor-bearing mice as a model to elucidate the mechanisms by which cyclophosphamide can render the immune lymphocytes competent to induce tumor rejection. RESULTS: The transfer of antitumor immunity was found to be dependent on CD4(+) T cells and on the cooperation of adoptively transferred cells with the host immune system. Of note, tumor-immune lymphocytes migrated specifically to the tumor only in mice pretreated with cyclophosphamide. Cyclophosphamide treatment also promoted homeostatic proliferation/activation of transferred B and T lymphocytes. Optimal therapeutic responses to the transfer of immune cells were associated with the cyclophosphamide-mediated induction of a "cytokine storm" [including granulocyte macrophage colony-stimulating factor, interleukin (IL)-1beta, IL-7, IL-15, IL-2, IL-21, and IFN-gamma], occurring during the "rebound phase" after drug-induced lymphodepletion. CONCLUSIONS: The ensemble of these data provides a new rationale for combining immunotherapy and chemotherapy to induce an effective antitumor response in cancer patients.

A pre-S/S CHO-derived hepatitis B virus vaccine protects woodchucks from WHV productive infection.

We evaluated whether a non-adjuvanted vaccine derived from Chinese hamster ovary cells was capable of providing protection against woodchuck hepatitis virus (WHV). Three woodchucks were vaccinated with four 50-microg doses and challenged with a previously characterized virus isolate (WHV197). In all three animals, titre levels of antibodies against hepatitis B surface antigens (anti-HBs) exceeded 10 mIU/ml, peaking at 150 mIU/ml. Challenge resulted in productive acute infection in the two non-vaccinated woodchucks yet in none of the vaccinated woodchucks. In the vaccinated animals, there was evidence of abortive infection. The results demonstrate that a human vaccine is able to protect woodchucks from WHV infection.

In vivo transmission and dynamics of deleted genomes after experimental infection of woodchuck hepatitis B virus in adult animals.

The presence of Deleted Genomes has been shown in a number of viral models including Hepadnaviridae. The analysis of woodchuck hepatitis B virus (WHV) population after experimental infection of woodchuck 197 (W197) with WHV7-PI inoculum revealed the presence of two Deleted Genomes: DG600 lacking a 1330 bp region (Core/Polymerase/PreS1) and DG900 showing a deletion of 869 nts (Pol/PreS/S). These mutants were also present in WHV7-PI. The successive WHV experimental infections in adult animals were performed using W197-w7 inoculum containing DG600 and DG900. Infections were divided into three groups presenting different patterns of viral replication, different presence of markers, occurrence of variants and persistence of infection. The first group displayed 2-3 weeks viremic phase and WHV-DNA titres of 10-30 ng/ml; the second a longer viremic phase (8-9 weeks) and higher WHV-DNA titres (up to 78 ng/ml). In contrast, the third group exhibited lifetime presence of WHV-DNA and WHVeAg in serum and viral replication in liver. The Deleted Genomes were transmitted in the newly infected animals with the same genomic organization. DG600 was persistently found only in chronically infected woodchuck, whereas a different pattern of presence was described for DG900. The characterization of these classes of deleted mutants in woodchuck-WHV model raises new questions on the link between DGs and persistent infections.