EUFOREA Rhinology Research Forum 2016: report of the brainstorming sessions on needs and priorities in rhinitis and rhinosinusitis.

The first European Rhinology Research Forum organized by the European Forum for Research and Education in Allergy and Airway Diseases (EUFOREA) was held in the Royal Academy of Medicine in Brussels on 17th and 18th November 2016, in collaboration with the European Rhinologic Society (ERS) and the Global Allergy and Asthma European Network (GA2LEN). One hundred and thirty participants (medical doctors from different specialties, researchers, as well as patients and industry representatives) from 27 countries took part in the multiple perspective discussions including brainstorming sessions on care pathways and research needs in rhinitis and rhinosinusitis. The debates started with an overview of the current state of the art, including weaknesses and strengths of the current practices, followed by the identification of essential research needs, thoroughly integrated in the context of Precision Medicine (PM), with personalized care, prediction of success of treatment, participation of the patient and prevention of disease as key principles for improving current clinical practices. This report provides a concise summary of the outcomes of the brainstorming sessions of the European Rhinology Research Forum 2016.

The adjuvant-like activity of staphylococcal enterotoxin B in a murine asthma model is independent of IL-1R signaling.

BACKGROUND: Staphylococcal enterotoxin B (SEB) is a superantigen known to be a modulator of chronic airway inflammation in mice and humans, yet little is known about the mechanisms that regulate its interaction with the innate immune system. We investigated this mechanism in a murine model of allergic airway inflammation induced by OVA (ovalbumin) in the presence of SEB. METHODS: Superantigen-induced allergic inflammation was studied in IL-1R knockout (KO) mice exposed to OVA+SEB. Multicolor flow cytometry was used to analyze the inflammatory cell profile in airways and lymph nodes. Production of IL-4, IL-5, IL-10, and IL-13 in lymph nodes was assessed by Luminex technology. RESULTS: In wild-type mice, endonasal instillation of OVA+SEB induced a pulmonary inflammation, characterized by an increase in the number of eosinophils, T cells, and dendritic cells and in the production of Th2 cytokines and OVA-specific IgE. In IL-1R KO mice exposed to OVA+SEB, attraction of CD4+ cells and production of Th2 cytokines were reduced. However, knocking out IL-1R did not affect any of the features of allergic airway inflammation, such as bronchial eosinophilia, OVA-specific IgE production and goblet cell metaplasia. CONCLUSION: We provide new insights into the mechanisms of airways allergy development in the presence of bacterial superantigen. The asthma features induced by OVA+SEB, such as bronchial eosinophilia, goblet cell proliferation, production of OVA-specific IgE and increase in inflammatory dendritic cells, are IL-1R independent. Yet, IL-1R signaling is crucial for CD4 cell accumulation and Th2 cytokine production.

Alternatively activated macrophages and impaired phagocytosis of S. aureus in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by biased Th2 inflammation and CRS without nasal polyps (CRSsNP) by a Th1 immune response. Colonization by Staphylococcus aureus is increased in CRSwNP. We aimed to determine macrophage phenotypes in nasal mucosa of CRSwNP and CRSsNP and to examine phagocytosis of S. aureus in these pathologies. METHODS: Macrophage phenotyping was performed by immunohistochemical staining on nasal mucosa sections from 28 patients; in addition flow cytometry analysis was performed. Tissue homogenate protein levels of IFN-γ, IL-5, IL-6, IL-1ß, TGF-ß, eosinophil cationic protein (ECP) and total IgE were analyzed and correlated with macrophage subtypes. Phagocytosis of S. aureus was analyzed by flow cytometry. Survival of S. aureus in Thp1 cells in the presence of polarizing cytokines was studied in vitro. RESULTS: By immunohistochemical analysis more M2 macrophages were present in CRSwNP than in CRSsNP. This also was positively correlated with increased levels of IL-5, ECP and locally produced IgE and decreased levels of IL-6, IL-1ß and IFN-γ. FACS analysis of dissociated nasal tissue confirmed the presence of increased numbers of M2 macrophages (CD206(+) HLADR(+) CD14(+) CD11c(+) CD20(-) ) in CRSwNP as compared to controls, while the number of M1 macrophages (CD206(-) HLADR(+) CD14(+) CD11c(int) CD16(-) CD20(-) ) was not different. Phagocytosis of S. aureus by human tissue derived macrophages was reduced in CRSwNP as compared to macrophages from the control inferior turbinates. CONCLUSIONS: Decreased phagocytosis of S. aureus and an M2 activation phenotype in CRSwNP could potentially contribute to persistence of chronic inflammation in CRSwNP.

Staphylococcus aureus enterotoxin B facilitates allergic sensitization in experimental asthma.

BACKGROUND: Staphylococcus aureus Enterotoxin B (SEB) has immunomodulatory effects in allergic airway disease. The potential contribution of SEB to the sensitization process to allergens remains obscure. OBJECTIVE: In order to study the effects of staphylococcal-derived toxins on the sensitization to ovalbumin (OVA) and induction of allergic airway inflammation, we have combined the nasal application of OVA with different toxins. METHODS: Nasal applications of OVA and saline, SEA, SEB, toxic shock syndrome toxin (TSST)-1, protein A or lipopolysaccharide (LPS) were performed on alternate days from day 0 till 12. On day 14, mice were killed for the evaluation of OVA-specific IgE, cytokine production by mediastinal lymph node (MLN) cells and bronchial hyperreactivity (BHR) to inhaled metacholine. The effect of SEB on dendritic cell (DC) migration and maturation, and on T cell proliferation was evaluated. RESULTS: Concomitant endonasal application of OVA and SEB resulted in OVA-specific IgE production, whereas this was not found with SEA, TSST-1, protein A, LPS or OVA alone. Increased DC maturation and migration to the draining lymph nodes were observed in OVA/SEB mice, as well as an increased T cell proliferation. Bronchial inflammation with an influx of eosinophils and lymphocytes was demonstrated in OVA/SEB mice, together with hyperresponsiveness and the production of IL-4, IL-5, IL-10 and IL-13 by MLN stimulated with OVA. CONCLUSIONS: Our data demonstrate that SEB facilitates sensitization to OVA and consecutive bronchial inflammation with features of allergic asthma. This is likely due to augmentation of DC migration and maturation, as well as the allergen-specific T cell proliferation upon concomitant OVA and SEB application.

Characterization of the in vivo immune network of IDO, tryptophan metabolism, PD-L1, and CTLA-4 in circulating immune cells in melanoma.

In melanoma, both the induction of immunosuppression by tumor cells and the inflammatory antitumor response can induce an upregulation of counter-regulatory mechanisms such as indoleamine 2,3-dioxygenase (IDO), programmed death-ligand 1 (PD-L1) and CTLA-4+ regulatory T-cells (Tregs) in the tumor microenvironment. Even though these immunosuppressive mediators are targets for immunotherapy, research investigating their expression in the peripheral blood is lacking. We therefore, performed flow cytometry on PBMCs of stage I-IV melanoma patients. IDO expression was detected in plasmacytoid dendritic cells (pDC) and monocytic myeloid-derived suppressor cells (mMDSC), and increased in advanced disease stage (p = 0.027). Tryptophan breakdown confirmed the functional activity of IDO and was linked with increased PD-L1+ cytotoxic T-cells (p = 0.009), relative lymphopenia (p = 0.036), and a higher mDC/pDC ratio (p = 0.002). High levels of circulating PD-L1+ cytotoxic T-cells were associated with increased CTLA-4 expression by Tregs (p = 0.005) and MDSC levels (p = 0.033). This illustrates that counter-regulatory immune mechanisms in melanoma should be considered as one interrelated signaling network. Moreover, both increased PD-L1+ T-cells and CTLA-4 expression in Tregs conferred a negative prognosis, indicating their in vivo relevance. Remarkably, circulating CTLA-4, IDO, and pDC levels were altered according to prior invasion of the sentinel lymph node and IDO expression in the sentinel was associated with more IDO+ PBMCs. We conclude that the expression of IDO, PD-L1, and CTLA-4 in the peripheral blood of melanoma patients is strongly interconnected, associated with advanced disease and negative outcome, independent of disease stage. Combination treatments targeting several of these markers are therefore likely to exert a synergistic response.

Many faces of DAMPs in cancer therapy.

A new concept of immunogenic cell death (ICD) has recently been proposed. The immunogenic characteristics of this cell death mode are mediated mainly by molecules called 'damage-associated molecular patterns' (DAMPs), most of which are recognized by pattern recognition receptors. Some DAMPs are actively emitted by cells undergoing ICD (e.g. calreticulin (CRT) and adenosine triphosphate (ATP)), whereas others are emitted passively (e.g. high-mobility group box 1 protein (HMGB1)). Recent studies have demonstrated that these DAMPs play a beneficial role in anti-cancer therapy by interacting with the immune system. The molecular pathways involved in translocation of CRT to the cell surface and secretion of ATP from tumor cells undergoing ICD are being elucidated. However, it has also been shown that the same DAMPs could contribute to progression of cancer and promote resistance to anticancer treatments. In this review, we will critically evaluate the beneficial and detrimental roles of DAMPs in cancer therapy, focusing mainly on CRT, ATP and HMGB1.

TLR-2 and TLR-9 are sensors of apoptosis in a mouse model of doxorubicin-induced acute inflammation.

Anthracycline antibiotics are inducers of an immunogenic form of apoptosis that has immunostimulatory properties because of the release of damage-associated molecular patterns. To study the mechanisms used by the innate immune system to sense this immunogenic form of cell death, we established an in vivo model of cell death induced by intraperitoneal injection of doxorubicin, a prototype of anthracyclines. The acute sterile inflammation in this model is characterized by rapid influx of neutrophils and increased levels of IL-6 and monocyte chemotactic protein-1. We demonstrate that acute inflammation induced by doxorubicin is associated with apoptosis of monocytes/macrophages and that it is specific for doxorubicin, an immunogenic chemotherapeutic. Further, the inflammatory response is significantly reduced in mice deficient in myeloid differentiation primary response gene 88 (MyD88), TLR-2 or TLR-9. Importantly, a TLR-9 antagonist reduces the recruitment of neutrophils induced by doxorubicin. By contrast, the acute inflammatory response is not affected in TRIF(Lps2) mutant mice and in TLR-3, TLR-4 and caspase-1 knockout mice, which shows that the inflammasome does not have a major role in doxorubicin-induced acute inflammation. Our findings provide important new insights into how the innate immune system senses immunogenic apoptotic cells and clearly demonstrate that the TLR-2/TLR-9-MyD88 signaling pathways have a central role in initiating the acute inflammatory response to this immunogenic form of apoptosis.

Phosphatidylserine exposure during early primary necrosis (oncosis) in JB6 cells as evidenced by immunogold labeling technique.

Apoptotic cell death is characterized by the early exposure of phosphatidylserine (PS) at the outer surface of the plasma membrane. The aim of the present study was to examine whether PS exposure also occurs during oncosis (early primary necrosis) and to localize PS at the subcellular level, applying a pre-embedding immunogold labeling technique with biotin conjugated annexin V. The issue was addressed by using caspase-8 deficient, Bcl-2 overexpressing JB6 cells, which die by oncosis when stimulated with synthetic dsRNA. We observed by fluorescence microscopy that oncotic cells with preserved plasma membrane integrity showed PS exposure (annexin+/propidium iodide-). The data was confirmed on the ultrastructural level and PS was localized in oncosis at the outer leaflet of the continuous plasma membrane with preserved trilamellar structure. In postoncotic necrotic cells the immunogold labels were found on the plasma membrane and on the intracellular membranes of the cells, which underwent plasma membrane disruption. In conclusion, this study reveals that PS externalization occurs not only in apoptosis but also in oncosis at least in our cell model system.