The small molecule inhibitor BX-795 uncouples IL-2 production from inhibition of Th2 inflammation and induces CD4+ T cells resembling iTreg

BackgroundTreg cells have been shown to be an important part of immune-homeostasis and IL-2 which is produced upon T cell receptor (TCR)-dependent activation of T lymphocytes has been demonstrated to critically participate in Treg development.ObjectiveTo evaluate small molecule inhibitors (SMI) for the identification of novel IL-2/Treg enhancing compounds.Materials and methodsWe used TCR-dependent and allergen-specific cytokine secretion of human and mouse T cells, next generation messenger ribonucleic acid sequencing (RNA-Seq) and two different models of allergic airway inflammation to examine lead SMI-compounds.ResultsWe show here that the reported 3-phosphoinositide dependent kinase-1 (PDK1) SMI BX-795 increased IL-2 in culture supernatants of Jurkat E6-1 T cells, human peripheral blood mononuclear cells (hPBMC) and allergen-specific mouse T cells upon TCR-dependent and allergen-specific stimulation while concomitantly inhibiting Th2 cytokine secretion. RNA-Seq revealed that the presence of BX-795 during allergen-specific activation of T cells induces a bona fide Treg cell type highly similar to iTreg but lacking Foxp3 expression. When applied in mugwort pollen and house dust mite extract-based models of airway inflammation, BX-795 significantly inhibited Th2 inflammation including expression of Th2 signature transcription factors and cytokines and influx into the lungs of type 2-associated inflammatory cells such as eosinophils.ConclusionsBX-795 potently uncouples IL-2 production from Th2 inflammation and induces Th-IL-2 cells, which highly resemble induced (i)Tregs. Thus, BX-795 may be a useful new compound for the treatment of allergic diseases

A novel tool for the rapid isolation and sensitive multiplex analysis of lipid raft components with single cell resolution

Die "lipid rafts"-Theorie beschreibt eine heterogene Klasse dynamisch organisierter Mikrodomänen der Zellmembran mit fundamentaler Bedeutung für zelluläre Homöostase, Differenzierung und Signalgebung. Moleküle und Dynamiken der "lipid rafts"-Domänen werden mittels sehr unterschiedlicher Methoden untersucht: diese reichen von Phasentrennungsanalyse über biochemische Fraktionierung von Detergens-resistenten Membranfraktionen bis zur hochauflösenden Nanoskopie lebender Zellen. Detergens-basierte Standardprotokolle zur Membranfraktionierung liefern wichtige quantitative Informationen zur Membranorganisation, benötigen jedoch einige Tage und Dutzende oder Hunderte Millionen Zellen an Ausgangsmaterial, was die Analyse seltener Zellen und heterogener Zellpopulation erschwert oder gänzlich ausschließt. Weiters wird die biologisch relevante Information einzelner Zellen zerstört. Im Zuge dieser Arbeit entwickelten wir eine robuste und dabei rasch durchführbare, Detergens-basierte Methode um multiple "lipid rafts"-assoziierte Membrankomponenten simultan und hochsensitiv mittels Durchflusszytometrie auf der Ebene einzelner Zellen zu quantifizieren. Unser einfach zu implementierender Ansatz ermöglicht die präzise Erfassung von "lipid rafts"-Molekülen und deren Dynamiken in Immunzellen, wenngleich er zur Analyse von Signalgebung und Differenzierung praktisch aller in Laboratorien untersuchter Säugetierzellen eingesetzt werden kann. Überdies ist unser Protokoll besonders zur Analyse von peripher mit "lipid rafts" assoziierten Komponenten und komplexer biologischer Proben geeignet.The lipid rafts concept describes a heterogeneous class of dynamic cell membrane microdomains that are integral to cell homeostasis, differentiation and signaling. Fundamentally different techniques are employed to study lipid raft molecules and dynamics, including phase separation analysis, biochemical fractionation of detergent-resistant components, and live-cell super-resolution imaging. While current day-long approaches based on detergent-resistance of membrane fractions can provide a valuable and quantitative assessment of membrane order, they require 10 7 or more cells for readout - which excludes rare cells or heterogeneous cell populations from comprehensive analysis - and destroy single cell information. We thus developed a rapid and reliable detergent- and flow cytometry-based method, which allows for multidimensional and sensitive high throughput quantitation of lipid raft-associated components with single cell resolution. As demonstrated, our easy-to-implement approach enables - but is not limited to - the precise assessment of lipid raft components and their dynamics associated with immune cell signaling and differentiation. It is uniquely suited for the accurate assessment of weakly raft-associated factors and complex biological samples.submitted by Philipp SchatzlmaierAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2015OeBB(VLID)171571

TLR4/CD14/MD2 Revealed as the Limited Toll-like Receptor Complex for <i>Chlamydia trachomatis</i>-Induced NF-κB Signaling

Chlamydia trachomatis (Ct) is the most common cause of genital tract infections as well as preventable blindness worldwide. Pattern recognition receptors such as toll-like receptors (TLRs) represent the initial step in recognizing pathogenic microorganisms and are crucial for the initiation of an appropriate immune response. However, our understanding of TLR-signaling in Chlamydia-infected immune cells is incomplete. For a better comprehension of pathological inflammatory responses, robust models for interrogating TLR-signaling upon chlamydial infections are needed. To analyze the TLR response, we developed and utilized a highly sensitive and selective fluorescent transcriptional cellular reporter system to measure the activity of the transcription factor NF-κB. Upon incubation of the reporter cells with different preparations of Ct, we were able to pinpoint which components of TLRs are involved in the recognition of Ct. We identified CD14 associated with unique characteristics of different serovars as the crucial factor of the TLR4/CD14/MD2 complex for Ct-mediated activation of the NF-κB pathway. Furthermore, we found the TLR4/CD14/MD2 complex to be decisive for the uptake of Ct-derived lipopolysaccharides but not for infection and replication of Ct. Imaging flow cytometry provided information about inclusion formation in myeloid- as well as lymphocytic cells and was highest for Ct L2 with at least 25% of inclusion forming cells. Ct E inclusion formation was eminent in Jurkat cells without CD14 expression (11.1%). Thus, our model enables to determine Ct uptake and signal induction by pinpointing individual components of the recognition and signaling pathways to better understand the immune response towards infectious pathogens