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Mast cells are highly versatile in terms of their mode of activation by a host of stimuli and their ability to flexibly release a plethora of biologically highly active mediators. Within the immune system, mast cells can best be designated as an active nexus interlinking innate and adaptive immunity. Here we try to draw an arc from initiation of acute inflammatory reactions to microbial pathogens to development of adaptive immunity and allergies. This multifaceted nature of mast cells is made possible by interaction with multiple cell types of immunologic and nonimmunologic origin. Examples for the former include neutrophils, eosinophils, T cells, and professional antigen-presenting cells. These interactions allow mast cells to orchestrate inflammatory innate reactions and complex adaptive immunity, including the pathogenesis of allergies. Important partners of nonimmunologic origin include cells of the sensory neuronal system. The intimate association between mast cells and sensory nerve fibers allows bidirectional communication, leading to neurogenic inflammation. Evidence is accumulating that this mast cell/nerve crosstalk is of pathophysiologic relevance in patients with allergic diseases, such as asthma.

BackgroundWith increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these “off-the-shelf” therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function.MethodsEffectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others.ResultsOur results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NK-mediated specific lysis of tumor cells was maintained at stable levels for three days post-irradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30.ConclusionsThe presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application.

The airways are constantly exposed to a multitude of inhaled particles and, as such, require a finely tuned discrimination between harmful or potentially threatening stimuli, and discrete responses to maintain homeostasis. Both the immune and nervous systems have the ability to sense environmental (and internal) signals, to integrate the obtained information and to initiate a protective reaction. Lung immunity and innervation are known to be individually involved in these processes, but it is becoming clear that they can also influence one another via a multitude of complex mechanisms. Here, we specifically describe how sensory innervation affects airways immunity with a focus on pathological conditions such as asthma or infections, describing cellular and molecular mechanisms, and highlighting potentially novel therapeutic targets.

Long non-coding RNAs (lncRNAs) have potential as novel therapeutic targets in cardiovascular diseases, but detailed information about the intercellular lncRNA shuttling mechanisms in the heart is lacking. Here, we report an important novel crosstalk between cardiomyocytes and fibroblasts mediated by the transfer of lncRNA-enriched extracellular vesicles (EVs) in the context of cardiac ischemia. lncRNA profiling identified two hypoxia-sensitive lncRNAs: ENSMUST00000122745 was predominantly found in small EVs, whereas lncRNA Neat1 was enriched in large EVs in vitro and in vivo. Vesicles were taken up by fibroblasts, triggering expression of profibrotic genes. In addition, lncRNA Neat1 was transcriptionally regulated by P53 under basal conditions and by HIF2A during hypoxia. The function of Neat1 was further elucidated in vitro and in vivo. Silencing of Neat1 in vitro revealed that Neat1 was indispensable for fibroblast and cardiomyocyte survival and affected fibroblast functions (reduced migration capacity, stalled cell cycle, and decreased expression of fibrotic genes). Of translational importance, genetic loss of Neat1 in vivo resulted in an impaired heart function after myocardial infarction highlighting its translational relevance., Graphical Abstract

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), and interleukin 1 alpha (IL-1alpha) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

The cellular serine protease TMPRSS2, a member of the type II transmembrane serine protease (TTSP) family, cleaves and activates the hemagglutinin of influenza A viruses (FLUAV) in cell culture and is essential for spread of diverse FLUAV in mice. Non-human primates (NHP), in particular rhesus and cynomolgus macaques, serve as animal models for influenza and experimental FLUAV infection of common marmosets has recently also been reported. However, it is currently unknown whether the NHP orthologues of human TMPRSS2 cleave and activate FLUAV hemagglutinin and contribute to viral spread in respiratory tissue. Here, we cloned and functionally analyzed the macaque and marmoset orthologues of human TMPRSS2. In addition, we analyzed the macaque orthologues of human TMPRSS4 and HAT, which also belong to the TTSP family. We found that all NHP orthologues of human TMPRSS2, TMPRSS4 and HAT cleave and activate HA upon directed expression and provide evidence that endogenous TMPRSS2 is expressed in the respiratory epithelium of rhesus macaques. Finally, we demonstrate that a serine protease inhibitor active against TMPRSS2 suppresses FLUAV spread in precision-cut lung slices of human, macaque and marmoset origin. These results indicate that FLUAV depends on serine protease activity for spread in diverse NHP and in humans. Moreover, our findings suggest that macaques and marmosets may serve as models to study FLUAV activation by TMPRSS2 in human patients. peerReviewed