Stefan Wüller

Cross-regulation of cytokine signalling: Pro-inflammatory cytokines restrict IL-6 signalling through receptor internalisation and degradation

The inflammatory response involves a complex interplay of different cytokines which act in an auto- or paracrine manner to induce the so-called acute phase response. Cytokines are known to crosstalk on multiple levels, for instance by regulating the mRNA stability of targeted cytokines through activation of the p38-MAPK pathway. In our study we discovered a new mechanism that answers the long-standing question how pro-inflammatory cytokines and environmental stress restrict immediate signalling of interleukin (IL)-6-type cytokines. We show that p38, activated by IL-1β, TNFα or environmental stress, impairs IL-6-induced JAK/STAT signalling through phosphorylation of the common cytokine receptor subunit gp130 and its subsequent internalisation and degradation. We identify MK2 as the kinase that phosphorylates serine 782 in the cytoplasmic part of gp130. Consequently, inhibition of p38 or MK2, deletion of MK2 or mutation of crucial amino acids within the MK2 target site or the di-leucine internalisation motif blocks receptor depletion and restores IL-6-dependent STAT activation as well as gene induction. Hence, a novel negative crosstalk mechanism for cytokine signalling is described, where cytokine receptor turnover is regulated in trans by pro-inflammatory cytokines and stress stimuli to coordinate the inflammatory response.

SOCS-mediated downregulation of mutant Jak2 (V617F, T875N and K539L) counteracts cytokine-independent signaling

Recently, mutations in the gene of Janus kinase 2 (Jak2) were discovered in patients suffering from chronic myeloproliferative disorders (MPD) and leukemia. As suppressors of cytokine signaling (SOCS) proteins are potent feedback inhibitors of Jak-mediated signaling, we investigated their role in signal transduction through constitutively active Jak2 mutants. We selected two mutants, Jak2-V617F and Jak2-K539L, found in patients with MPDs and Jak2-T875N identified in acute megakaryoblastic leukemia. We found SOCS family members to be induced through Jak2-V617F in human leukemia cell lines expressing the mutant allele and in stable HEK transfectants inducibly expressing constitutively active Jak2 mutants. SOCS proteins were recruited to the membrane and bound to the constitutively active Jaks. In contrast to wild-type Jak2, the mutant proteins were constitutively ubiquitinated and degraded through the proteasome. Taken together, we show a SOCS-mediated downregulation of the constitutively active, disease-associated mutant Jak2 proteins. Furthermore, a threshold level of mutant Jak expression has to be overcome to allow full cytokine-independent constitutive activation of signaling proteins, which may explain progression to homozygocity in MPDs as well as gene amplification in severe phenotypes and leukemia.

A role of nitric oxide in neurite outgrowth of neuroblastoma cells triggered by mevastatin or serum reduction

Neuroblastoma cell lines are commonly used as a model to study neuronal differentiation as they retain the capacity to differentiate into a neuronal-like phenotype. It is of great medical interest to understand the signalling pathways biasing differentiation versus proliferation. Neuroblastoma cells differentiate in response to serum reduction or addition of the cholesterol synthesis inhibitor mevastatin. The responsible pathways are not well characterized. In Neuro2a neuroblastoma cells, we found that mevastatin and serum withdrawal triggered the production of nitric oxide (NO). In addition, the differentiation of Neuro2a cells and the activation of Akt/PKB triggered by serum withdrawal could be blocked by addition of the NO synthetase (NOS) inhibitor l-NAME. Moreover, mevastatin and serum withdrawal rapidly increased the expression of the neuronal NOS isoform nNOS. However, addition of an NO donor SNP per se did not trigger neurite outgrowth. Taken together, we report for the first time a role of NO in neurite outgrowth of neuroblastoma cells triggered by mevastatin or serum reduction.

An Unusual Insertion in Jak2 Is Crucial for Kinase Activity and Differentially Affects Cytokine Responses

The Janus kinases, Jaks, constitutively associate with the cytoplasmic region of cytokine receptors and play an important role in a multitude of biological processes. Jak2 dysfunction has been implicated in myeloproliferative diseases and leukemia. Although Jaks were studied extensively for many years, the molecular mechanism of Jak activation upon cytokine stimulation of cells is still incompletely understood. In this study, we investigated the importance of an unusual insertion located within the kinase domain in Jak2. We found that the deletion of this insertion, which we named the Jak-specific insertion (JSI), totally abrogates Jak2 autophosphorylation. We further point mutated four residues within the JSI that are conserved in all Jak family members. Three of these mutants showed abrogated or reduced autophosphorylation, whereas the fourth displayed increased autophosphorylation. We found that the phosphorylation state of these mutants is not influenced by other domains of the kinase. Our data further suggest that the JSI is not required for the negative regulation of kinase activity by the suppressor of cytokine signaling proteins, SOCS. Most importantly, we show that mutations in this region differentially affect IFN-γ and erythropoietin signal transduction. Taken together, the dramatic effects on the phosphorylation status of Jak2 as well as the differential effects on the signaling via different cytokines highlight the importance of this unusual region for the catalytic activity of Jaks.

Development of an IL-6 Inhibitor Based on the Functional Analysis of Murine IL-6Rα1

Dysregulated cytokine production contributes to inflammatory and proliferative diseases. Therefore, inhibition of proinflammatory mediators such as TNF, IL-1, and IL-6 is of great clinical relevance. Actual strategies are aimed at preventing receptor activation through sequestration of the ligand. Here we describe the development of an inhibitor of murine IL-6 based on fused receptor fragments. Molecular modeling-guided analysis of the murine IL-6Ralpha revealed that mutations in the Ig-like domain D1 severely affect protein function, although D1 is not directly involved in the ligand-binding interface. The resulting single chain IL-6 inhibitor (mIL-6-RFP) consisting of domains D1-D3 of mgp130, a flexible linker, and domains D1-D3 of mIL-6Ralpha is a highly potent and specific IL-6 inhibitor. mIL-6-RFP will permit further characterization of the role of IL-6 in various disease models and could ultimately lead to anti-IL-6 therapy.

Landmark-based navigation in complex buildings

Growing numbers of mobile devices in our daily life and their capability to fulfill challenging computational tasks raise the question about new application fields beyond well-established tasks. While outdoor navigation became a standard task for many mobile devices, indoor positioning and navigation is still in the research and development stage. Even though complex buildings require aided guidance for visitors, todays mobile hardware is not able to deliver a reliable indoor navigation system. In this paper we describe a novel information system for indoor navigation in complex buildings. Users are guided through the building by using images of the surroundings and textual instructions. We avoid hardware-based user positioning due to its known drawbacks. Instead, users are involved into the navigation process and complete missing information through recognized context and logical constrains of their surroundings. The human navigation process based on recognition of certain unique locations and visual clues is the foundation of this work. The proposed system is universally applicable without restrictions on navigation devices or existing hardware in the building.

A secure two-party bartering protocol using privacy-preserving interval operations

Bartering plays a significant role in todays global economy-both between individuals as well as in B2B settings. However, aside from lacking automation, todays bartering solutions and supporting platforms typically neglect the privacy needs of their users. In this paper, we present a novel two-party protocol which addresses these shortcomings. The new protocol automatically determines whether the desired and offered commodities and quantities overlap in such a way that both parties are willing to barter. Throughout the protocol, the commodities and quantities as specified by the parties are kept private. We show that the protocol is privacy-preserving in the semi-honest model. As main building blocks, the new bartering protocol uses a novel privacy-preserving protocol for selecting a random subinterval out of the overlap of two intervals as well as a newly-developed secure protocol for input-symmetric strong conditional oblivious transfer.

The p38‐Mediated Rapid Down‐Regulation of Cell Surface gp130 Expression Impairs Interleukin‐6 Signaling in the Synovial Fluid of Juvenile Idiopathic Arthritis Patients

Interleukin‐6 (IL‐6) signaling plays an important proinflammatory role, but this role is restricted by regulatory mechanisms that, for example, reduce the cell surface availability of the signal‐transducing chain of the IL‐6 receptor, gp130. The aim of this study was to determine whether the inflammatory environment in arthritic joints has an impact on monocytic gp130 surface expression and the extent to which regulatory processes in the synovial fluid (SF) can be reproduced in an in vitro model.

Privacy-preserving conditional random selection

In this paper, we introduce a new primitive - referred to as conditional random selection. This new primitive allows the random selection of a data record from the subset of data records that meet a specified condition. We present a new privacy-preserving protocol that implements the new primitive and is secure in the semi-honest model. At its core, it uses newly developed protocols for oblivious shuffling, oblivious swapping, and privacy-preserving less than comparison on binary values with shared output. We show the relevance of conditional random selection in various application scenarios.

Privacy-Preserving Multi-Party Bartering Secure Against Active Adversaries

Both B2B bartering as well as bartering between individuals is increasingly facilitated through online platforms. However, these platforms lack automation and neglect the privacy of their users by leaking crucial information about their offers and demands. It is in this context that we introduce the first privacy-preserving two-party bartering protocol which is secure against active attackers. As main building blocks, our bartering protocol uses novel protocols operating on common encrypted input for securely selecting an element out of multiple elements, securely selecting a random element out of an interval, and obliviously shrinking an interval which are of independent interest.