Interleukin-3 stabilizes CD124/IL-4α surface expression in mast cells via Tyk2 and STAT6.

Interleukin (IL)-4 signals can modulate mast cells, which express the IL-4Rα chain. The IL-4Rα can heterodimerise with the common γ-chain and utilizes JAK1 and JAK2 for signal transduction, while complexes of IL-4Rα with IL-13Rα1 subunit mediates signals via JAK2 and Tyk2. Here, we report that IL-3 is an essential factor for the continuous expression of the IL-4Rα chain on mast cells, which did not express the IL-13Rα1 chain. We demonstrate that the signals induced by IL-3 important for IL-4Rα expression are mediated by Tyk2 and STAT6 activation and the subsequent maintenance of HSP90 levels. In line with that, inhibition of either Tyk2, STAT6 or HSP90 impaired the IL-3-induced IL-4Rα upregulation. Consequently, the IL-3 maintained IL-4Rα surface expression via Tyk2 is essential for the costimulatory effect of IL-4 on the IL-33-induced production of IL-6 and IL-13.

The Heat Shock Protein 90 (HSP90) Is Required for the IL-33-Induced Cytokine Production in Mast Cells (MCs).

The alarmin interleukin-33 (IL-33) is released upon cell stress and damage in peripheral tissues. The receptor for IL-33 is the Toll/Interleukin-1 receptor (TIR)-family member T1/ST2 (the IL-33R), which is highly and constitutively expressed on MCs. The sensing of IL-33 by MCs induces the MyD88-TAK1-IKK2-dependent activation of p65/RelA and MAP-kinases, which mediate the production of pro-inflammatory cytokines and amplify FcεRI-mediated MC-effector functions and the resulting allergic reactions. Therefore, the investigation of IL-33-induced signaling is of interest for developing therapeutic interventions effective against allergic reactions. Importantly, beside the release of IL-33, heat shock proteins (HSPs) are upregulated during allergic reactions. This maintains the biological functions of signaling molecules and/or cytokines but unfortunately also strengthens the severity of inflammatory reactions. Here, we demonstrate that HSP90 does not support the IL-33-induced and MyD88-TAK1-IKK2-dependent activation of p65/RelA and of mitogen-activated protein (MAP)-kinases. We found that HSP90 acts downstream of these signaling pathways, mediates the stability of produced cytokine mRNAs, and therefore facilitates the resulting cytokine production. These data show that IL-33 enables MCs to perform an effective cytokine production by the upregulation of HSP90. Consequently, HSP90 might be an attractive therapeutic target for blocking IL-33-mediated inflammatory reactions.

ATP/IL-33-triggered hyperactivation of mast cells results in an amplified production of pro-inflammatory cytokines and eicosanoids.

IL-33 and ATP are alarmins, which are released upon damage of cellular barriers or are actively secreted upon cell stress. Due to high-density expression of the IL-33 receptor T1/ST2 (IL-33R), and the ATP receptor P2X7, mast cells (MCs) are one of the first highly sensitive sentinels recognizing released IL-33 or ATP in damaged peripheral tissues. Whereas IL-33 induces the MyD88-dependent activation of the TAK1-IKK2-NF-κB signalling, ATP induces the Ca2+ -dependent activation of NFAT. Thereby, each signal alone only induces a moderate production of pro-inflammatory cytokines and lipid mediators (LMs). However, MCs, which simultaneously sense (co-sensing) IL-33 and ATP, display an enhanced and prolonged activation of the TAK1-IKK2-NF-κB signalling pathway. This resulted in a massive production of pro-inflammatory cytokines such as IL-2, IL-4, IL-6 and GM-CSF as well as of arachidonic acid-derived cyclooxygenase (COX)-mediated pro-inflammatory prostaglandins (PGs) and thromboxanes (TXs), hallmarks of strong MC activation. Collectively, these data show that co-sensing of ATP and IL-33 results in hyperactivation of MCs, which resembles to MC activation induced by IgE-mediated crosslinking of the FcεRI. Therefore, the IL-33/IL-33R and/or the ATP/P2X7 signalling axis are attractive targets for therapeutical intervention of diseases associated with the loss of integrity of cellular barriers such as allergic and infectious respiratory reactions.

Biomimetic reconstruction of the hematopoietic stem cell niche for in vitro amplification of human hematopoietic stem cells.

Hematopoietic stem cell transplantation is successfully applied since the late 1950s; however, its efficacy still needs to be increased. A promising strategy is to transplant high numbers of pluripotent hematopoietic stem cells (HSCs). Therefore, an improved ex vivo culture system that supports proliferation and maintains HSC pluripotency would override possible limitations in cell numbers gained from donors. To model the natural HSC niche in vitro, we optimized the HSC medium composition with a panel of cytokines and valproic acid and used an artificial 3D bone marrow-like scaffold made of polydimethylsiloxane (PDMS). This 3D scaffold offered a suitable platform to amplify human HSCs in vitro and, simultaneously, to support their viability, multipotency and ability for self-renewal. Silicon oxide-covering of PDMS structures further improved amplification of CD34+ cells, although the conservation of naïve HSCs was better on non-covered 3D PDMS. Finally, we found that HSC cultivated on non-covered 3D PDMS generated most pluripotent colonies within colony forming unit assays. In conclusion, by combining biological and biotechnological approaches, we optimized in vitro HSCs culture conditions, resulting in improved amplification, multipotency maintenance and vitality of HSCs.

The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies.

This article describes the generation of the Human Combinatorial Antibody Library HuCAL GOLD. HuCAL GOLD is a synthetic human Fab library based on the HuCAL concept with all six complementarity-determining regions (CDRs) diversified according to the sequence and length variability of naturally rearranged human antibodies. The human antibody repertoire was analyzed in-depth, and individual CDR libraries were designed and generated for each CDR and each antibody family. Trinucleotide mixtures were used to synthesize the CDR libraries in order to ensure a high quality within HuCAL GOLD, and a beta-lactamase selection system was employed to eliminate frame-shifted clones after successive cloning of the CDR libraries. With these methods, a large, high-quality library with more than 10 billion functional Fab fragments was achieved. By using CysDisplay, the antibody fragments are displayed on the tip of the phage via a disulfide bridge between the phage coat protein pIII and the heavy chain of the antibody fragment. Efficient elution of specific phages is possible by adding reducing agents. HuCAL GOLD was challenged with a variety of different antigens and proved to be a reliable source of high-affinity human antibodies with best affinities in the picomolar range, thus functioning as an excellent source of antibodies for research, diagnostic, and therapeutic applications. Furthermore, the data presented in this article demonstrate that CysDisplay is a robust and broadly applicable display technology even for high-throughput applications.