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β 2 -Adrenergic receptors (β 2 -ARs) transduce the effects of (nor)epinephrine on a variety of cell types and act as key mediators of the body’s reaction to stress. β 2 -ARs are also expressed on immune cells and there is ample evidence for their role in immunomodulation. A key regulator of the immune response and a target for regulation by stress-induced signals is the transcription factor Nuclear Factor-kappaB (NF-κB). NF-κB shapes the course of both innate and adaptive immune responses and plays an important role in susceptibility to disease. In this review, we summarise the literature that has been accumulated in the past 20 years on adrenergic modulation of NF-κB function. We here focus on the molecular basis of the reported interactions and show that both physiological and pharmacological triggers of β 2 -ARs intersect with the NF-κB signalling cascade at different levels. Importantly, the action of β 2 -AR-derived signals on NF-κB activity appears to be highly cell type specific and gene selective, providing opportunities for the development of selective NF-κB modulators. [ABSTRACT FROM AUTHOR]

Abstract Type I Interferon (IFN) is widely used for multiple sclerosis (MS) treatment, but its side effects are limiting and its mechanism of action still unknown. Furthermore, 30–50% of MS patients are unresponsive, and IFN can even induce relapses. Fundamental understanding of the cellular target(s) of IFN will help to optimize treatments by reducing side effects and separating beneficial from detrimental effects. To improve clinical systemic IFN usage, we are developing AcTaferons (Activity-on-Target IFNs = AFNs), optimized IFN-based immunocytokines that allow cell-specific targeting. In experimental autoimmune encephalitis (EAE) in mice, high dose WT mIFNα could delay disease, but caused mortality and severe hematological deficits. In contrast, AFN targeted to dendritic cells (DC, via Clec9A) protected without mortality or hematological consequences. Conversely, CD8-targeted AFN did not protect and exacerbated weight loss, indicating the presence of both protective and unfavorable IFN effects in EAE. Comparing Clec9A-, XCR1-and SiglecH-targeting, we found that targeting AFN to plasmacytoid (p) and conventional (c) DC is superior and non-toxic compared to WT mIFN. DC-targeted AFN increased pDC numbers and their tolerogenic potential, evidenced by increased TGFβ and IDO synthesis and regulatory T cell induction. In addition, both regulatory T and B cells produced significantly more immunosuppressive TGFβ and IL-10. In conclusion, specific DC-targeting of IFN activity induces a robust in vivo tolerization, efficiently protecting against EAE, without noticeable side effects. Thus, dissecting positive and negative IFN effects via cell-specific targeting may result in better and safer MS therapy and response rates. Highlights • AcTaferons (AFNs) are optimized immunocytokines, targeting mutated IFN to selected cell types only. • Clec9A-targeted AFN is superior to wild-type IFN for treating EAE in mice. • Clec9A-AFN induces a tolerogenic response in vivo , mostly depending on pDC. • DC-AFN is non-toxic, safe and an alternative for WT IFN as first line treatment to reduce attacks and lesions in MS patients. • DC-AFN offers a simple alternate for ex vivo generated tolDC currently tested for treating MS and other autoimmune diseases. [ABSTRACT FROM AUTHOR]

The regulation of intracellular Ca 2+ signaling is an important aspect of how anti-apoptotic B-cell lymphoma 2 (Bcl-2) proteins regulate cell death and cell survival. At the endoplasmic reticulum (ER) the Bcl-2 homology (BH) 4 domain of Bcl-2 is known to bind to and inhibit both inositol 1,4,5-trisphosphate receptors (IP 3 Rs) and ryanodine receptors (RyRs). Besides this, drugs that target the hydrophobic cleft of Bcl-2 have been reported to deplete ER Ca 2+ stores in an IP 3 R- and RyR-dependent way. This suggests that the hydrophobic cleft of Bcl-2 may also be involved in regulating these ER-located Ca 2+ -release channels. However, the contribution of the hydrophobic cleft on the binding and regulatory properties of Bcl-2 to either IP 3 Rs or RyRs has until now not been studied. Here, the importance of the hydrophobic cleft of Bcl-2 in binding to and inhibiting the RyR was assessed by using a genetic approach based on site-directed mutagenesis of Bcl-2's hydrophobic cleft and a pharmacological approach based on the selective Bcl-2 hydrophobic cleft inhibitor, ABT-199. Both binding assays and single-cell Ca 2+ measurements indicated that RyR binding and the inhibition of RyR-mediated Ca 2+ release by Bcl-2 is independent of its hydrophobic cleft. [ABSTRACT FROM AUTHOR]

Abstract: Leptin is an adipocyte-derived hormone, recognized as a critical mediator of the balance between food intake and energy expenditure by signalling through its functional receptor (Ob-Rb) in the hypothalamus. Structurally, leptin belongs to the long-chain helical cytokine family, and is now known to have pleiotropic functions in both innate and adaptive immunity. The presence of the functional leptin receptor in the lung together with evidence of increased airspace leptin levels arising during pulmonary inflammation, suggests an important role for leptin in lung development, respiratory immune responses and eventually pathogenesis of inflammatory respiratory diseases. The purpose of this article is to review our current understanding of leptin and its functional role on the different resident cell types of the lung in health as well as in the context of three major respiratory conditions being chronic obstructive pulmonary disease (COPD), asthma, and pneumonia. [Copyright &y& Elsevier]

Abstract: Developing agents that target protein–protein interactions (PPIs) is an emerging field in drug discovery. Although this target class has hitherto remained underexplored, it holds exceptional promise related to the large amount of potential PPI targets compared to single protein targets and it offers important opportunities to increase the specificity of therapeutic molecules. While several PPI modulating therapeutics have recently been reported and a number of these are in clinical trial, progress in the field has been hampered by the lack of efficient screening systems. Recently, a number of cellular approaches have been developed that complement classical in vitro screening methods and which exhibit a number of important assets related to the physiological context they provide. Here we discuss the utility of two-hybrid technologies towards high-throughput screening for PPI inhibitors, in particular those that operate in a mammalian cellular background. We review a number of cases where mammalian two-hybrids have been successfully applied to identify small molecule disruptors of PPIs and zoom in further on the MAPPIT (Mammalian Protein–Protein Interaction Trap) technology platform. The value of this approach for drug discovery is illustrated by recent data from MAPPIT-based screening projects. [Copyright &y& Elsevier]

β 2-adrenergic receptor ( β 2-AR) agonists are widely applied in the clinic for treatment of respiratory disease. Although their therapeutic effect mainly results from their spasmolytic activity, β -agonists have also been attributed with anti-inflammatory properties. We studied the effect of β -agonist cotreatment on TNF- α -induced activation of inflammatory signalling in astrocytes, focusing on Nuclear Factor- κ B (NF- κ B), a master regulator of inflammatory gene expression. Unexpectedly, we observed that, unlike other anti-inflammatory drugs, β -agonists did not inhibit the activity of NF- κ B. Instead, β -agonist cotreatment enhanced TNF- α -induced expression of a subset of prototypical NF- κ B target genes. The most pronounced effect was apparent for Interleukin-6 (IL-6) and the CXC chemokines CXCL2 and CXCL3. We established that cooperativity between NF- κ B and β 2-AR-induced transcriptional regulators is at the basis of the synergy. Our current research aims at further defining the molecular players regulating IL-6 transcription using a novel approach combining DNA affinity purification and mass spectrometry. Importantly, the β 2-agonist-induced potentiation of inflammatory responses was also recapitulated in vivo , in rats treated intracerebroventricularly with a combination of TNF- α and clenbuterol. Gene expression changes were furthermore reflected in a redistribution of the leukocyte subsets present in the brain. More specifically, a skewing of the T-cell population towards a double-negative phenotype, and a shift towards neutrophilic expansion in the myeloid subset were apparent. Although functional characterization of the infiltrating leukocytes remains to be done, these results suggest β -agonists can catalyze inflammatory reactions in vivo and warrant further study into their mode of action. [ABSTRACT FROM AUTHOR]

The activating JAK2-V617F mutation is found in many patients suffering from Myeloproliferative neoplasms such as Polycythemia Vera and Essential Thrombocythemia. JAK2-V617F mutation confers cytokine hypersensitivity, constitutive activation of the JAK-STAT pathway, and cytokine-independent growth. We demonstrate uncontrolled membrane binding of the multi-site docking protein Gab1 in JAK2-V617F expressing cells. We elaborated the molecular mechanism and regulation of Gab1-membrane recruitment. In line with the regulatory function of Gab1 on STAT-independent signalling we show that PI3K signalling regulates MAPK activation in JAK2-V617F-positive cells. This cross-regulation of the MAPK pathway by PI3K affects JAK2-V617F-specific target gene induction, erythroid colony formation, and regulates proliferation of JAK2-V617F-positive patient cells in a synergistically manner. In summary, our study demands to consider cross-talk mechanisms as potential new targets. This may increase the specificity for intervention approaches compared to the interference in very upstream signalling elements such as receptor kinases or receptor-associated kinases. [ABSTRACT FROM AUTHOR]

Systemic toxicity currently prevents full clinical application of type I interferons (IFNs) for the treatment of cancers and viral infections. Although the use of IFNs fused to antibodies resulted in a 10-fold increased activity towards targeted cells, off-target side effects experienced by the patients are expected to remain a major obstacle. We present a novel strategy to engineer IFNα with high in vitro and in vivo activity towards cells expressing a specific surface marker while having an up to 1000-fold reduced activity towards non-relevant cells. The concept lies in the use of immunocytokines based on IFN mutants with strongly reduced binding affinity for the IFN receptor. Such a targeted IFN recovers almost maximally its antiviral activity uniquely on the targeted cells and acts to selected cell populations in mice with targeting efficiencies up to 3 orders of magnitude. This novel concept holds promise to revitalize the clinical potential of IFNα and other cytokines. [ABSTRACT FROM AUTHOR]