Aktivierung eosinophiler Granulozyten-Zytokinfreisetzung und Überlebenssignale

Eosinophile Granulozyten wurden erstmals im Jahr 1879 von Paul Ehrlich beschrieben. Ihre Beteiligung an allergischen Reaktionen und ihre Rolle bei der Abwehr von Wurmparasiten ist seit langem bekannt. Wissenschaftliche Arbeiten in den letzten Jahren haben weitere Funktionen von eosinophilen Granulozyten abseits immunologischer Reaktionen beschrieben. Eosinophile Granulozyten sind in der Regulation physiologischer Prozesse involviert z.B.: Glukosehomöostase, Darmhomöostase und Gewebereparatur. Einige Studien weisen außerdem auf eine Beteiligung von Eosinophilen bei der Tumorabstoßung hin. Eosinophile Granulozyten sind daher potenzielle Ziele therapeutischer Interventionen in verschiedenen klinischen Bereichen. Um effektive zielgerichtete Therapien zu entwickeln, ist ein tiefgreifendes Verständnis der Mechanismen, die das Überleben und die Zytokinfreisetzung eosinophiler Granulozyten regulieren, entscheidend. Ein Ziel dieser Arbeit war es, die Rolle von Signaltransduktionswegen bei der Verarbeitung von Überlebenssignalen durch die Zytokine IL-5 und GM-CSF darzulegen. Die vorliegende Arbeit demonstriert eine wichtige Rolle des NF-κB Signalweges für das Überleben eosinophiler Granulozyten. Es konnte gezeigt werden, dass NF-κB entscheidend zur Regulation des anti-apoptotischen Moleküls Bcl-xL in Eosinophilen beiträgt. Die Signalwege p38-MAPK und STAT5 sind an der Regulation der IL-5 vermittelten Differenzierung von Eosinophilen aus den im Knochenmark residierenden Vorläufer-Zellen beteiligt. Ein zweiter Schwerpunkt der vorliegenden Arbeit war es, die Reaktion von eosinophilen Granulozyten auf das Alarmin IL-33 zu untersuchen. Die hier präsentierten Daten zeigen, dass IL-33 die GM-CSF Produktion von Eosinophilen stimuliert. Blockade des GM-CSF-Rezeptors sowie GM-CSF neutralisierende Antikörper haben gezeigt, dass IL-33 vermittelte Überlebenssignale über autokrin produziertes GM-CSF wirken. Neben GM-CSF stimuliert IL-33 auch die Freisetzung der Zytokine IL-4 und IL-13. Hier zeigte sich, dass die Antikörper-vermittelte Kreuzvernetzung des Sialylsäure-erkennenden Lektins Siglec-F amplifikatorisch auf die durch IL-33 initiierte IL-4/13 Freisetzung wirkt. Weiterhin fördert Siglec-F Stimulation die Proliferation von eosinophilen Vorläufer-Zellen in Knochenmarkskulturen. Somit wurden erstmals aktivierende Funktionen von Siglec-F für eosinophile Granulozyten beschrieben. Abschließend war es Ziel, ein Maus-Modell zu etablieren, in dem die Rolle des von Eosinophilen sekretierten IL-4 und IL-13 ermittelt werden kann. Aspergillus fumigatus löst bei wiederholter Einatmung von Sporen eine Lungeneosinophilie aus, die durch weitere Symptome allergischer Reaktionen gekennzeichnet ist. Daher wurde als Teil dieser Arbeit ein Maus-Modell für die als allergische bronchopulmonale Aspergillose (ABPA) bezeichnete Pneumonie etabliert. Die Ergebnisse dieser Arbeit könnten zur Entwicklung neuer therapeutischer Strategien zur Linderung von Symptomen eosinophil-vermittelter Erkrankungen dienen.In 1879 Paul Ehrlich noted the appearance of cells in the blood that contain granula visualized by eosin staining: the birth of the eosinophil. Eosinophils have long been considered as terminal effector cells in allergic asthma as well as during worm infections. Research in the last decade has uncovered so far unexpected new roles for eosinophils in metabolic homeostasis and tissue repair responses. There is also emerging evidence for a contribution of eosinophils in gut homeostasis and cancer modulation. In summary, eosinophils are attractive targets for therapeutic interventions in extending clinical areas. Hence, deeper knowledge of these cells is required to develop efficient therapies. The first aim of this study was to unravel the molecular mechanism behind eosinophil survival. The cytokines IL-5 and GM-CSF turned out to be capable of rescuing eosinophil from spontaneous apoptosis. This work demonstrates an important role for the NF-κB signaling pathway behind IL-5 and GM-CSF induced eosinophil survival while the STAT5 and p38-MAPK pathway are rather involved in IL-5-triggered eosinophil development. Furthermore, this study reveals a critical role for the NF-κB-regulated anti-apoptotic protein Bcl-xL as executer of pro-survival signals. A second aim was to characterize the response of eosinophils to the alarmin IL-33. The data obtained here demonstrate an IL-33-initiated anti-apoptotic program in eosinophils that operates via autocrine GM-CSF release. Furthermore, IL-33-stimulated eosinophils release the cytokines IL-4 and IL-13. In addition, this study revealed that IL-33-induced cytokine release is amplified by cross-linking of the sialic acid-recognizing lectin Siglec-F. Surprisingly, Siglec-F crosslinking also enhances the proliferation of eosinophil precursor cells. Siglec-F has been regarded as apoptosis inducing inhibitory receptor on eosinophils. Hence, this work proposes a new activatory role for the characteristic eosinophil marker Siglec-F. Finally, the Aspergillus fumigatus infection protocol was established as model for allergic lung inflammation for future studies to demonstrate a relevance of eosinophil-derived IL-4/13 in vivo

IL-33-Induced Cytokine Secretion and Survival of Mouse Eosinophils Is Promoted by Autocrine GM-CSF

<div><p>Eosinophils are major effector cells during allergic responses and helminth infections. Recent studies further highlight eosinophils as important players in many other biological processes. Therefore it is important to understand how these cells can be modulated in terms of survival and effector function. In the present study we investigated how eosinophils respond to the alarmin IL-33. We show that IL-33 promotes eosinophil survival in a ST2- and MyD88-dependent manner. IL-33-mediated protection from apoptosis was dependent on autocrine GM-CSF release. In addition, GM-CSF increased the IL-33-induced secretion of IL-4 and IL-13 from eosinophils. Unexpectedly, this effect was further enhanced by cross-linking of Siglec-F, a proposed inhibitory and apopotosis-inducing receptor on eosinophils. Co-culture experiments with eosinophils and macrophages revealed that the IL-33-induced release of IL-4 and IL-13 from eosinophils was required for differentiation of alternatively activated macrophages (AAMs). The differentiation of AAMs could be further increased in the presence of GM-CSF. These results indicate that cross-talk between Siglec-F and the receptors for IL-33, LPS and GM-CSF plays an important role for efficient secretion of IL-4 and IL-13. Deciphering the molecular details of this cross-talk could lead to the development of new therapeutic option to treat eosinophil-associated diseases.</p></div

IL-33 mediates eosinophil survival via autocrine GM-CSF production.

<p>(A) BMDE were cultured in medium containing 10 ng/ml IL-5 and indicated concentrations of IL-33 and LPS for 24 hours. Supernatants were analyzed by GM-CSF-specific ELISA. Bar graph shows the mean + SD from one of two independent experiments (n = 4;). (B) BMDE were stimulated with the indicated cytokines in the absence (black bars) or presence of a isotype control antibody (dark grey bars) or GM-CSF receptor blocking antibody (light grey bars). After 72 hours cells were analyzed by flow cytometry to detect Annexin V⁺ cells. Bars show the mean + SEM of apoptotic cells from three independent experiments (n = 6; ** p<0.01). (C) Sort-purified BMDE (100% purity, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163751#pone.0163751.s001" target="_blank">S1 Fig</a>) were left untreated (black bar) or stimulated with IL-33 in the presence of isotype control antibody (dark grey bar) or anti-GM-CSF-R antibody (light grey bar). After 72 hours cells were analyzed by flow cytometry to detect Annexin V⁺ cells. Bars show the mean + SEM of apoptotic cells from two experiments (n = 4; * p<0.05; ** p<0.01). (D) BMDE were treated as described above and harvested after 24 hours to analyze Bcl-x_L transcripts by quantitative RT-PCR. Bar graphs show mean + SEM Bcl-x_L mRNA expression levels normalized to HPRT1 mRNA levels with data pooled from two independent experiments (n = 6; * p<0.05).</p

IL-33 activated eosinophils mediate alternative activation of macrophages.

<p>(A) and (B) BMDM generated from wild-type BALB/c mice were co-cultured with BMDE from either wild-type (black bars) or IL-4/IL-13-deficient BALB/c mice (grey bars) for 24 hours in the presence of the indicated cytokines. Quantitative RT- PCR was performed to analyze Relm-α and Arginase-1 expression as established markers for alternative activation of macrophages. Data are presented as normalized expression to PBGD. Bars show the mean + SD (n = 3) from one of three independent experiments with similar results. (C) and (D) only supernatants of IL-5+IL-33+GM-CSF stimulated BMDE from WT (black bars) or IL-4/IL-13-deficient mice (grey bars) were added to BMDM for 24 hours before quantitative RT-PCR was performed. Bars show the mean + SD (n = 3) from one experiment.</p

IL-33 mediates eosinophil survival that depends on ST2 and MyD88.

<p>BMDE from wild-type (WT), ST2-deficient (ST2^-/-) and MyD88-deficient (MyD88^-/-) mice were cultured in presence of 10 ng/ml IL-5 or IL-33 or were left untreated for 72 hours. Cells were harvested and analyzed by flow cytometry to detect apoptotic cells. (A) Dot plots show representative AnnexinV/PI stainings for each experimental group. (B) Bars show the mean + SEM of apoptotic cells from indicated mice. Data are pooled from two independent experiments (n = 5–6; *** p<0.001). (C) BMDE from wild-type mice were stimulated with the indicated cytokines or 10 μg/ml LPS. Control samples were left untreated (w/o). After 72 hours cells were analyzed by flow cytometry to detect Annexin V/PI positive cells. Bars show the mean + SEM of Annexin V⁺ cells pooled from 2–3 independent experiments (n = 8–10; *** p<0.001).</p

GM-CSF, LPS and Siglec-F signaling enhance IL-33-induced secretion of IL-4 and IL-13 from eosinophils.

<p>(A) BMDE were cultured with IL-5 and indicated concentrations of IL-33 and LPS. (B) BMDE were cultured in 10 ng/ml IL-5 and IL-33 in the presence or absence of 10 ng/ml GM-CSF. In addition, anti-Siglec-F, isotype control antibody or no antibodies were added to the culture. (C) BMDE were cultured in 10 ng/ml IL-5 and IL-33. In addition, the p38 kinase was blocked with 5 μM SB203580 and DMSO was used as solvent control. All supernatants were analyzed at 24 hours after onset of culture by IL-4 and IL-13 specific ELISAs. Bars show the mean + SD from one of two independent experiments with similar results (A; n = 4) the mean + SD from one of three independent experiments (B; n = 4) and mean + SEM pooled from two independent experiments (C; n = 8) *p<0.05; **p<0.01; *** p<0.001).</p

Wetland Roofs as an Attractive Option for Decentralized Water Management and Air Conditioning Enhancement in Growing Cities—A Review

While constructed wetlands have become established for the decentralized treatment of wastewater and rainwater, wetland roofs have only been built in isolated cases up to now. The historical development of wetland roofs is described here on the basis of a survey of literature and patents, and the increasing interest in this ecotechnology around the world is presented. In particular, this article describes the potential for using wetland roofs and examines experience with applications in decentralized water management in urban environments and for climate regulation in buildings. Wetland roofs are suitable as a green-blue technology for the future&mdash;particularly in cities with an acute shortage of unoccupied ground-level sites&mdash;for the decentralized treatment of wastewater streams of various origins. Positive &ldquo;side effects&rdquo; such as nearly complete stormwater retention and the improvement of climates in buildings and their surroundings, coupled with an increase in biodiversity, make wetland roofs an ideal multi-functional technology for urban areas

High Salt Inhibits Tumor Growth by Enhancing Anti-tumor Immunity

Excess salt intake could affect the immune system by shifting the immune cell balance toward a pro-inflammatory state. Since this shift of the immune balance is thought to be beneficial in anti-cancer immunity, we tested the impact of high salt diets on tumor growth in mice. Here we show that high salt significantly inhibited tumor growth in two independent murine tumor transplantation models. Although high salt fed tumor-bearing mice showed alterations in T cell populations, the effect seemed to be largely independent of adaptive immune cells. In contrast, depletion of myeloid-derived suppressor cells (MDSCs) significantly reverted the inhibitory effect on tumor growth. In line with this, high salt conditions almost completely blocked murine MDSC function in vitro. Importantly, similar effects were observed in human MDSCs isolated from cancer patients. Thus, high salt conditions seem to inhibit tumor growth by enabling more pronounced anti-tumor immunity through the functional modulation of MDSCs. Our findings might have critical relevance for cancer immunotherapy.MKi was supported by a Ph.D. grant from the Research Foundation Flanders (FWO), Belgium. MKl was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (640116), by a SALK-grant from the government of Flanders, Belgium and by an Odysseus-grant of the FWO, Belgium.cancer; dietary factor; MDSC; cancer immunotherapy; sodium chloride (dietary