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Supplementary Figures 1-5, Tables 1-2 from Inhibition of HIF Prolyl Hydroxylase-2 Blocks Tumor Growth in Mice through the Antiproliferative Activity of TGFβ

The SCF/KIT axis regulates nearly all aspects of mast cell (MC) biology. A comprehensive view of SCF-triggered phosphorylation dynamics is lacking. The relationship between signaling modules and SCF-supported functions likewise remains ill-defined.Mast cells were isolated from human skin; upon stimulation by SCF, global phosphoproteomic changes were analyzed by LC-MS/MS and selectively validated by immunoblotting. MC survival was inspected by YoPro; BrdU incorporation served to monitor proliferation. Gene expression was quantified by RT-qPCR and cytokines by ELISA. Pharmacological inhibitors were supplemented by ERK1 and/or ERK2 knockdown. CIC translocation and degradation were studied in nuclear and cytoplasmic fractions. CIC's impact on KIT signaling and function was assessed following RNA interference.≈5400 out of ≈10,500 phosphosites experienced regulation by SCF. The MEK/ERK cascade was strongly induced surpassing STAT5 PI3K/Akt p38 JNK. Comparison between MEK/ERK's and PI3K's support of basic programs (apoptosis, proliferation) revealed equipotency between modules. In functional outputs (gene expression, cytokines), ERK was the most influential kinase. OSM and LIF production was identified in skin MCs. Strikingly, SCF triggered massive phosphorylation of a protein not associated with KIT previously: CIC. Phosphorylation was followed by CIC's cytoplasmic appearance and degradation, the latter sensitive to protease but not preoteasome inhibition. Both shuttling and degradation were ERK-dependent. Conversely, CIC-siRNA facilitated KIT signaling, functional outputs, and survival.The SCF/KIT axis shows notable strength in MCs, and MEK/ERK as most prominent module. An inhibitory circuit exists between KIT and CIC. CIC stabilization in MCs may turn out as a therapeutic option to interfere with allergic and MC-driven diseases.

The recent discovery of MRGPRX2 explains mast cell (MC)-dependent symptoms independently of FcεRI-activation. Because of its novelty, signaling cascades triggered by MRGPRX2 are rudimentarily understood, especially in cutaneous MCs, by which MRGPRX2 is chiefly expressed. Here, MCs purified from human skin were used following preculture or ex vivo and stimulated by FcεRI-aggregation or MRGPRX2 agonists (compound 48/80, Substance P) in the presence/absence of inhibitors. Degranulation was assessed by β-hexosaminidase or histamine release. Phosphorylation events were studied by immunoblotting. As a G protein-coupled receptor, MRGPRX2 signals by activating G proteins; however, their nature has remained controversial. In skin MCs, Gαi and Gαq were required for degranulation, but Gαi was clearly more relevant. Ca++ channels were likewise crucial. Downstream, PI3K was essential for granule discharge initiated by MRGPRX2 or FcεRI. ERK1/2 and JNK were additional participants, especially in the allergic route. Addressing possible points of intersection between early and later events, pERK1/2 and pAKT were found to depend on Gαi, further highlighting its significance. Gαq and Ca++ channels made some contributions to the phosphorylation of ERK. Ca++ differentially affected PI3K activation in FcεRI- vis-à-vis MRGPRX2-signaling, as channel inhibition increased pAKT only when triggered via FcεRI. Collectively, our study significantly extends our understanding of the molecular framework behind granule secretion from skin MCs.

The key players of the hypoxic response are the hypoxia-inducible factors (Hif), whose α-subunits are tightly regulated by Prolyl-4-hydroxylases (PHD), predominantly by PHD2. Monocytes/Macrophages are involved in atherosclerosis but also restenosis and were found at hypoxic and sites of the lesion. Little is known about the role of the myeloid PHD2 in atherosclerosis and neointima formation. The study aimed to investigate the consequences of a myeloid deficiency of PHD2 in the process of neointima formation using an arterial denudation model. LysM-cre mice were crossed with PHD2

Mast cells are evolutionarily old cells and the principal effectors in allergic responses and inflammation. They are seeded from the yolk sac during embryogenesis or are derived from hematopoietic progenitors and are therefore related to other leukocyte subsets, even though they form a separate clade in the hematopoietic system. Herein, we systematically bundle information from several recent high-throughput endeavors, especially those comparing MCs with other cell types, and combine such information with knowledge on the genes' functions to reveal groups of neuronal markers specifically expressed by MCs. We focus on recent advances made regarding human tissue MCs, but also refer to studies in mice. In broad terms, genes hyper-expressed in MCs, but largely inactive in other myelocytes, can be classified into subcategories such as traffic/lysosomes (MLPH and RAB27B), the dopamine system (MAOB, DRD2, SLC6A3, and SLC18A2), Ca2+-related entities (CALB2), adhesion molecules (L1CAM and NTM) and, as an overall principle, the transcription factors and modulators of transcriptional activity (LMO4, PBX1, MEIS2, and EHMT2). Their function in MCs is generally unknown but may tentatively be deduced by comparison with other systems. MCs share functions with the nervous system, as they express typical neurotransmitters (histamine and serotonin) and a degranulation machinery that shares features with the neuronal apparatus at the synapse. Therefore, selective overlaps are plausible, and they further highlight the uniqueness of MCs within the myeloid system, as well as when compared with basophils. Apart from investigating their functional implications in MCs, a key question is whether their expression in the lineage is due to the specific reactivation of genes normally silenced in leukocytes or whether the genes are not switched off during mastocytic development from early progenitors.

Chimeric antigen receptor T cells (CAR-T) targeting CD19 or B cell maturation antigen (BCMA) are highly effective against B cell malignancies. However, application of CAR-T to less differentially expressed targets remains a challenge due to lack of tumor-specific antigens and CAR-T controllability. CD123, a highly promising leukemia target, is expressed not only by leukemic and leukemia-initiating cells, but also by myeloid, hematopoietic progenitor, and certain endothelial cells. Thus, CAR-T lacking fine-tuned control mechanisms pose a high toxicity risk. To extend the CAR-T target landscape and widen the therapeutic window, we adapted our rapidly switchable universal CAR-T platform (UniCAR) to target CD123. UniCAR-T efficiently eradicated CD123+ leukemia in vitro and in vivo. Activation, cytolytic response, and cytokine release were strictly dependent on the presence of the CD123-specific targeting module (TM123) with comparable efficacy to CD123-specific CAR-T in vitro. We further demonstrated a pre-clinical proof of concept for the safety-switch mechanism using a hematotoxicity mouse model wherein TM123-redirected UniCAR-T showed reversible toxicity toward hematopoietic cells compared to CD123 CAR-T. In conclusion, UniCAR-T maintain full anti-leukemic efficacy, while ensuring rapid controllability to improve safety and versatility of CD123-directed immunotherapy. The safety and efficacy of UniCAR-T in combination with TM123 will now be assessed in a phase I clinical trial (ClinicalTrials.gov: NCT04230265)., Graphical Abstract, CAR-T cell treatment of acute leukemia beyond CD19 remains challenging. Loff et al. demonstrate that rapidly switchable universal CAR-T cells in combination with soluble CD123-specific adaptors achieve robust anti-leukemic responses, while hematotoxicity is rapidly reversible, enabling safe targeting of such less differentially expressed target antigens and broadening of the therapeutic window.

Thymic stromal lymphopoietin (TSLP) promotes TWe investigated whether and by what mechanisms mast cells (MCs) foster TSLP responses in the cutaneous environment.Ex vivo and in vivo skin MC degranulation was induced by compound 48/80 in wild-type protease-activated receptor 2 (PAR-2)- and MC-deficient mice in the presence or absence of neutralizing antibodies, antagonists, or exogenous mouse MC protease 6 (mMCP6). Primary human keratinocytes and murine skin explants were stimulated with lysates/supernatants of human skin MCs, purified tryptase, or MC lysate diminished of tryptase. Chymase and histamine were also used. TSLP was quantified by ELISA, real-time quantitative PCR, and immunofluorescence staining.Mas-related G protein-coupled receptor X2 (Mrgprb2) activation elicited TSLP in intact skin, mainly in the epidermis. Responses were strictly MC dependent and relied on PAR-2. Complementarily, TSLP was elicited by tryptase in murine skin explants. Exogenous mMCP6 could fully restore responsiveness in MC-deficient murine skin explants. Conversely, PAR-2 knockout mice were unresponsive to mMCP6 while displaying increased responsiveness to other inflammatory pathways, such as IL-1α. Indeed, IL-1α acted in concert with tryptase. In primary human keratinocytes, MC-elicited TSLP generation was likewise abolished by tryptase inhibition or elimination. Chymase and histamine did not affect TSLP production, but histamine triggered IL-6, IL-8, and stem cell factor.MCs communicate with kerationocytes more broadly than hitherto suspected. The tryptase/PAR-2 axis is a crucial component of this cross talk, underlying MC-dependent stimulation of TSLP in neighboring kerationocytes. Interference specifically with MC tryptase may offer a treatment option for disorders initiated or perpetuated by aberrant TSLP, such as atopic dermatitis.

The IL-1 family cytokine IL-33 activates and re-shapes mast cells (MCs), but whether and by what mechanisms it elicits cytokines in MCs from human skin remains poorly understood. The current study found that IL-33 activates CCL1, CCL2, IL-5, IL-8, IL-13, and TNF-α, while IL-1β, IL-6, IL-31, and VEGFA remain unaffected in cutaneous MCs, highlighting that each MC subset responds to IL-33 with a unique cytokine profile. Mechanistically, IL-33 induced the rapid (1–2 min) and durable (2 h) phosphorylation of p38, whereas the phosphorylation of JNK was weaker and more transient. Moreover, the NF-κB pathway was potently activated, as revealed by IκB degradation, increased nuclear abundance of p50/p65, and vigorous phosphorylation of p65. The activation of NF-κB occurred independently of p38 or JNK. The induced transcription of the cytokines selected for further study (CCL1, CCL2, IL-8, TNF-α) was abolished by interference with NF-κB, while p38/JNK had only some cytokine-selective effects. Surprisingly, at the level of the secreted protein products, p38 was nearly as effective as NF-κB for all entities, suggesting post-transcriptional involvement. IL-33 did not only instruct skin MCs to produce selected cytokines, but it also efficiently co-operated with the allergic and pseudo-allergic/neurogenic activation networks in the production of IL-8, TNF-α, CCL1, and CCL2. Synergism was more pronounced at the protein than at the mRNA level and appeared stronger for MRGPRX2 ligands than for FcεRI. Our results underscore the pro-inflammatory nature of an acute IL-33 stimulus and imply that especially in combination with allergens or MRGPRX2 agonists, IL-33 will efficiently amplify skin inflammation and thereby aggravate inflammatory dermatoses.

Thymic stromal lymphopoietin (TSLP) is released by epithelial cells following disturbed homeostasis to act as &ldquo, alarmin&rdquo, and driver of Th2-immunity. Aberrant TSLP expression is a hallmark of atopic diseases, including atopic dermatitis (AD). Mast cells (MCs) are overabundant in AD lesions and show signs of degranulation, but it remains unknown whether TSLP contributes to granule discharge. Degranulation of skin MCs proceeds via two major routes, i.e., Fc&epsilon, RI-dependent (allergic) and MRGPRX2-mediated (pseudo-allergic/neurogenic). Evidence is accumulating that MRGPRX2 may be crucial in the context of skin diseases, including eczema. The current study reveals TSLP as a novel priming factor of human skin MCs. Interestingly, TSLP selectively cooperates with MRGPRX2 to support granule discharge, while it does not impact spontaneous or Fc&epsilon, RI-driven exocytosis. TSLP-assisted histamine liberation triggered by compound 48/80 or Substance P, two canonical MRGPRX2 agonists, was accompanied by an increase in CD107a+ cells (a MC activation marker). The latter process was less potent, however, and detectable only at the later of two time points, suggesting TSLP may prolong opening of the granules. Mechanistically, TSLP elicited phosphorylation of STAT5 and JNK in skin MCs and the reinforced degranulation critically depended on STAT5 activity, while JNK had a contributory role. Results from pharmacological inhibition were confirmed by RNA-interference, whereby silencing of STAT5 completely abolished the priming effect of TSLP on MRGPRX2-mediated degranulation. Collectively, TSLP is the first factor to favor MRGPRX2- over Fc&epsilon, RI-triggered MC activation. The relevance of TSLP, MCs and MRGPRX2 to pruritis and atopic skin pathology indicates broad repercussions of the identified connection.

Orchestrated recruitment of neutrophils to inflamed tissue is essential during initiation of inflammation. Inflamed areas are usually hypoxic, and adaptation to reduced oxygen pressure is typically mediated by hypoxia pathway proteins. However, it is still unclear how these factors influence the migration of neutrophils to and at the site of inflammation either during their transmigration through the blood-endothelial cell barrier, or their motility in the interstitial space. Here, we reveal that activation of the Hypoxia Inducible Factor-2 (HIF2α) due to deficiency of HIF-prolyl hydroxylase domain protein-2 (PHD2) boosts neutrophil migration specifically through highly confined microenvironments. In vivo, the increased migratory capacity of PHD2-deficient neutrophils resulted in massive tissue accumulation in models of acute local inflammation. Using systematic RNAseq analyses and mechanistic approaches, we identified RhoA, a cytoskeleton organizer, as the central downstream factor that mediates HIF2α-dependent neutrophil motility. Thus, we propose that the here identified novel PHD2-HIF2α-RhoA axis is vital to the initial stages of inflammation as it promotes neutrophil movement through highly confined tissue landscapes.

Skin mast cells (MCs) contribute to chronic dermatoses that partially rely on MC-derived cytokines. The discovery of MRGPRX2 explains MC-dependent symptoms independently of FcεRI activation. In this study, we investigated whether MRGPRX2 can elicit cytokines, determined its relative potency versus that of FcεRI, and addressed the underlying mechanisms. MRGPRX2 activation by compound 48/80 or substance P on skin MCs induced TNF-α, IL-8, IL-13, CCL1, and CCL2 protein and mRNA; yet, induction was typically reduced compared with FcεRI crosslinking. Generally, cytokine secretion required de novo synthesis with maximum accumulation at ∼8 hours. Addressing key kinases revealed robust, rapid (1 minute), and lasting (30 minutes) phosphorylation of extracellular signal‒regulated kinase 1/2 after MRGPRX2 ligation, whereas phosphorylated p38 and phosphorylated protein kinase B signals were weaker, and phosphorylated c-Jun N-terminal kinase was hardly detectable. The kinase spectrum after FcεRI aggregation was comparable, but responses were considerably delayed. The MAPK/extracellular signal‒regulated kinase kinase/extracellular signal‒regulated kinase pathway was essential for all cytokines examined, and four inhibitors of this module led to complete suppression. A variable and weaker contribution was found for phosphatidylinositol 3-kinase than for c-Jun N-terminal kinase than for p38. Strikingly, cytokine profiles and signaling prerequisites were similar for MRGPRX2 and FcεRI and were likely mainly dictated by the MC subset. Collectively, in skin MCs, the physiological producers of MRGPRX2, agonist binding elicits cytokines, yet less efficiently than in FcεRI aggregation. MRGPRX2-associated inflammation may thus be less tissue destructive than responses to allergic challenges.

Extreme weather events such as heat waves are predicted to increase in the course of anthropogenic climate change. Widespread species are exposed to a variety of environmental conditions throughout their distribution range, often resulting in local adaptation. Consequently, populations from different regions may vary in their capacity to deal with challenging conditions such as thermal stress. In this study, we investigated clinal variation in body size, fecundity, and oxidative markers along a pan-European latitudinal gradient in the green-veined white butterfly Pieris napi, and additionally gene expression in German individuals. We exposed butterflies from replicated Italian, German, and Swedish populations to cold, control, or hot temperatures for 24 h. Under hot conditions, molecular chaperones were up-regulated, while oxidative damage remained unaffected and levels of the antioxidant glutathione (GSH) were reduced under cold and hot conditions. Thus, the short-term exposure to heat stress did not substantially affect oxidative balance. Moreover, we found decreased body size and fecundity in cooler compared with warmer regions. Interestingly, oxidative damage was lowest in Swedish animals exhibiting (1) high levels of GSH, (2) low early fecundity, and (3) low larval growth rates. These results suggest that Swedish butterflies have a slower life style and invest more strongly into maintenance, while those from warmer regions show the opposite pattern, which may reflect a ‘pace-of-life’ syndrome. Electronic supplementary material The online version of this article (10.1007/s00442-020-04719-4) contains supplementary material, which is available to authorized users.

The social environment experienced by an individual may have striking effects on its trait expression. We show that an increasing number of conspecifics increased male body mass and the probability of success in aggressive interactions, but decreased courtship activity and tended to decrease sperm number in B. anynana butterflies. Thus, males kept at high densities favored maintenance whereas males kept at low-density favored reproduction, indicating prudent strategic investment.

The microbiome may influence disease severity in atopic dermatitis. The skin of atopic dermatitis patients and healthy individuals was sampled in a standardized manner and the microbial composition analysed using next-generation sequencing. Optical density measurements were used to investigate bacterial growth under defined conditions in vitro. Lesional skin from patients with atopic dermatitis had a higher abundance of Staphylococcus aureus and reduced quantities of Propionibacterium acnes and Lawsonella clevelandensis compared with non-lesional skin. The abundance of P. acnes correlated negatively with that of S. aureus (ρ= -0.6501, p < 0.0001). Fermentation products of P. acnes inhibited the growth of S. aureus and S. epidermidis. Serum from patients with atopic dermatitis inhibited the growth of S. aureus to a greater extent than did serum from healthy individuals. These results suggest that selective modification of the skin microbiome could potentially be used as a therapeutic strategy in atopic dermatitis.

Background: Conventional CAR-T cells targeting CD123 in rrAML have achieved objective responses, but led to long-lasting myelosuppression due to expression of CD123 on progenitor cells. UniCAR-T-CD123 is a rapidly switchable two-component CAR-T therapy. An inert universal CAR-T cell (UniCAR-T) is combined with a CD123-specific soluble targeting module with a short half-life (TM123). By administering or withholding the continuous infusion of TM123, the UniCAR-T-cell can be rapidly switched on and off. Within the ongoing Phase IA study in rrAML, we investigated the expansion kinetics of UniCAR-T cells during TM123 administration in peripheral blood and bone marrow as well as cytokine profiles of treated patients. Methods: Prior to administration of autologous UniCAR-T cells, patients received a standard Flu/Cy lymphodepletion regimen at day -5 to -3. TM123 was administered as continuous infusion over 24 days starting at day 0. At day 1 a single dose of UniCAR-T cells was given. Dosing started with 1 x 108 UniCAR-T cells and 0.5 mg TM123 per day in patient 1. Patient 2 received the same TM123 dose and a UniCAR-T dose of 2.5 x 108 cells. Patient 3 received the same cell dose as patient 2 but a higher TM123 dose (1 mg/day). Pharmacokinetic of UniCAR-T and TM123 was determined from peripheral blood and bone marrow by droplet digital PCR and TM123-specific ELISA, respectively. Cytokine levels were measured by microfluidic immunoassay. Results: All 3 patients treated so far achieved an objective response, with one showing a PR and two a CRi. Treatment proved to be tolerable, no DLTs were observed to date and adverse events were mild. Grade 1 CRS (fever) was observed in 2 patients but subsided within 48 h after use of antipyretics. Myelosuppression was observed starting after lymphodepletion, which immediately recovered after TM123 withdrawal on day 24 in all patients, providing evidence for the rapid off-switch of UniCAR-T cells post TM123 administration. UniCAR-T cells expanded in all patients in peripheral blood and bone marrow comparable to data reported for conventional CD123 CAR-T products, and were so far detectable for up to 6 months after administration. Expansion kinetics were TM123-dependent. Patients showed periods of transient increase of IL-6, IFN-γ and TNF-α levels preceding peak expansion and decreasing after termination of TM123 administration. One patient showed additional coinciding momentary elevation of GM-CSF and IL-2. Conclusions: The initial clinical and translational results of UniCAR-T-CD123 represent, to our understanding, a first time evidence for a well-tolerated and effective rapidly switchable CAR-T product. Even though the number of patients treated so far is limited, the data obtained provide clinical proof-of-concept for the opportunity to abrogate side effects by withdrawal of TM123. Enrollment into the Phase IA study is ongoing. Citation Format: Armin Ehninger, Julia Riewaldt, Cordula Gründer, Kristin Franke, Maria Schreiber, Martin Wermke, Sabrina Kraus, Carla Kreissig, Jan Koedam, Michael Pehl, Gerhard Ehninger, Marc Cartellieri. Expansion kinetics and cytokine profiles of UniCAR-T-CD123, a rapidly switchable two-component CAR-T therapy, in patients with relapsed/refractory AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1506.

Codeine stimulates skin mast cells and is therefore used in skin tests and as an inducer of experimental itch. MRGPRX2 responds to various drugs, including opioids, to elicit pseudoallergic reactions, but whether it represents the main opiate receptor of skin mast cells remains unknown. By combining a number of approaches, including the silencing of MRGPRX2, we now report that MRGPRX2 is indeed the dominant codeine receptor of dermal mast cells. Activation by codeine displayed profound subject variability and correlated with secretion elicited by compound 48/80 or substance P but not by FcεRI aggregation. Degranulation by codeine was attenuated by stem cell factor, whereas the opposite was found for FcεRI. Compound 48/80 or codeine alone was able to achieve maximum MRGPRX2 activation. MRGPRX2 was rapidly internalized on codeine binding in a β-arrestin-1‒dependent manner. Codeine-triggered β-arrestin activation was also established by the Tango assay. Prestimulation with MRGPRX2 agonists (but not C3a or FcεRI aggregation) resulted in refractoriness to further stimulation by the same or another MRGPRX2 ligand (cross desensitization). This was duplicated in a cell line (RBL-MRGPRX2). Collectively, codeine degranulates skin mast cells through MRGPRX2, at which it acts as a balanced ligand. It has yet to be determined whether codeine-induced refractoriness could be exploited to desensitize MRGPRX2 to prevent severe pseudoallergic reactions.

Mast cells (MCs) from human skin have been notoriously resistant to gene manipulation, and a method to knock-down gene expression in in situ differentiated MCs is highly desired. The Dharmacon Accell® transfection system proved successful on several "difficult-to-transfect" cells. In the present work, we therefore tested this method on skin-derived MCs using different siRNA entities. The siRNA was readily taken up, followed by pronounced, specific reduction of gene and protein expression. Hence, we present the first efficient technique for the manipulation of gene expression in primary skin MCs ex vivo, which combines high transfection rates with retained cell viability.

Background Thymic stromal lymphopoietin (TSLP) mediates proallergic T helper 2-type responses by acting on leucocytes. Endogenous pathways regulating TSLP production are poorly defined. Objectives To uncover the mechanisms by which skin barrier disruption elicits TSLP production and to delineate the level at which individual mechanistic components may converge. Methods A combination of primary keratinocytes, skin explants and in vivo strategies was employed. Murine skin was tape stripped in the presence of neutralizing antibodies or antagonists. Cells and explants were stimulated with interleukin (IL)-1 and protease-activated receptor 2 agonist (PAR-2-Ag). TSLP levels were quantified by enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction. Chromatin immunoprecipitation and promoter reporter assays were used to examine recruitment and functional activity of nuclear factor kappa B (NF-κB) at the TSLP promoter. Results TSLP induction in mouse skin occurred in a PAR-2- and IL-1-dependent manner. This scenario was duplicated by exogenous IL-1 plus PAR-2-Ag vs. each stimulus alone. Joint activity of PAR-2 and IL-1 was also observed in human keratinocytes. The TSLP promoter was identified as the target of PAR-2/IL-1, whereby PAR-2 activation augmented the recruitment of NF-κB and transcriptional activation over IL-1 alone. Combined treatment showed activity at concentrations of IL-1 unable to elicit NF-κB activity on their own. Conclusions Skin barrier disruption activates the IL-1 and the PAR-2 pathways, which act in concert to activate the TSLP promoter and possibly other inflammatory genes. Awareness of this combined activity may permit a more flexible clinical management by selective targeting of either pathway individually or collectively. What's already known about this topic? Thymic stromal lymphopoietin (TSLP) is rapidly induced upon skin perturbation and mediates proallergic T helper 2-type responses by acting on leucocytes. Endogenous control of TSLP expression is poorly understood, but interleukin (IL)-1 is one regulator in the cutaneous environment In addition to IL-1, protease-activated receptor 2 (PAR-2) organizes central inflammatory pathways in the skin. What does this study add? IL-1 and PAR-2 pathways cooperate in driving TSLP production in mice and humans. Pathway integration occurs at the level of the TSLP promoter through enhanced recruitment and transcriptional activation of nuclear factor kappa B. When PAR-2 is co-stimulated, very low IL-1 levels (inactive by themselves) can induce biologically meaningful responses in the skin environment. What is the translational message? Physical skin irritation results in robust TSLP production by simultaneous activation of PAR-2 and IL-1 pathways.

In multiple sclerosis (MS) regeneration of oligodendrocytes following inflammatory demyelination is limited by the compromised ability of progenitors to repopulate lesioned areas and transition to functionally competent oligodendrocytes. Regarding underlying mechanisms, the involvement of epigenetic processes has been suggested, e.g. the contribution of histone deacetylases (HDAC) known to regulate oligodendrocyte progenitor cell (OPC) differentiation. However, their precise expression patterns, particular of redox-sensitive NAD+ HDACs, remains largely unknown. In this study, we determined the expression and activity of sirtuins, members of the HDAC class III family with a specific focus on SIRT1, previously associated with neurodegenerative, inflammatory and demyelinating disorders of the central nervous system (CNS). By investigating mouse experimental autoimmune encephalomyelitis (EAE), a model for MS, we found that transcription of SIRT1, SIRT2 and SIRT6 was significantly increased in the CNS during chronic disease stages. We confirmed this finding for SIRT1 protein expression and were able to localize upregulated SIRT1 in nuclei of NG2+ or PDGFRα+ OPCs in demyelinated brain lesions. In cultured mouse A2B5+ OPCs blockade of SIRT1 activity by the small molecule compound Ex527 enhanced mitotic activity but did not affect the capacity to differentiate. A similar pattern was detectable in OPCs derived from SIRT1-deficient animals. Taken together, our data suggest that SIRT1 inhibition may help to expand the endogenous pool of OPCs without affecting their differentiation.

Mast cells (MCs) are the principal effector cells of IgE-mediated allergy. IL-33 is released by resident skin cells as alarmin upon tissue damage or allergen contact. Owing to their pronounced receptor expression, MCs are important targets of IL-33 action, but consequences for skin MCs are ill-defined, especially upon chronic exposure to IL-33. Mimicking the inflammatory milieu of skin disorders, we found that persistent exposure to IL-33 (over a 5-week period) strengthened skin MC numbers through accelerated cell-cycle progression and restriction of apoptosis. Conversely, IL-33 attenuated degranulation and FceRI expression, potentially as a feedback to chronic "alarmin" exposure. Interestingly, the negative impact on histamine release was counterbalanced by amplified histamine production. Considering the clinical significance of histamine and scarce information on its regulation, we explored the molecular underpinnings. IL-33 induced swift phosphorylation of p38 and JNK (but not of ERK1/2 or AKT), and stimulated histidine decarboxylase expression. Combining pharmacological inhibition and kinase elimination by Accell-facilitated RNA interference in skin MCs revealed a p38-dependent, but JNK-independent mechanism. Collectively, IL-33 exerts multifaceted effects on cutaneous MCs at a post-maturation stage. The IL-33-skin MC axis may contribute to and balance inflammation in chronic skin disorders.

Rho GTPases play prominent roles in the regulation of cytoskeletal reorganization. Many aspects have been elaborated concerning the individual functions of Rho GTPases in distinct signaling pathways leading to cytoskeletal rearrangements. However, major questions have yet to be answered regarding the integration and the signaling hierarchy of different Rho GTPases in regulating the cytoskeleton in fundamental physiological events like neuronal process differentiation. Here, we investigate the roles of the small GTPases Rac1, Cdc42, and RhoG in defining dendritic tree complexity stimulated by the transmembrane epidermal growth factor family member CALEB/NGC. Combining gain-of-function and loss-of-function analysis in primary hippocampal neurons, we find that Rac1 is essential for CALEB/NGC-mediated dendritic branching. Cdc42 reduces the complexity of dendritic trees. Interestingly, we identify the palmitoylated isoform of Cdc42 to adversely affect dendritic outgrowth and dendritic branching, whereas the prenylated Cdc42 isoform does not. In contrast to Rac1, CALEB/NGC and Cdc42 are not directly interconnected in regulating dendritic tree complexity. Unlike Rac1, the Rac1-related GTPase RhoG reduces the complexity of dendritic trees by acting upstream of CALEB/NGC. Mechanistically, CALEB/NGC activates Rac1, and RhoG reduces the amount of CALEB/NGC that is located at the right site for Rac1 activation at the cell membrane. Thus, Rac1, Cdc42, and RhoG perform very specific and non-redundant functions at different levels of hierarchy in regulating dendritic tree complexity induced by CALEB/NGC. Rho GTPases play a prominent role in dendritic branching. CALEB/NGC is a transmembrane member of the epidermal growth factor (EGF) family that mediates dendritic branching, dependent on Rac1. CALEB/NGC stimulates Rac1 activity. RhoG inhibits CALEB/NGC-mediated dendritic branching by decreasing the amount of CALEB/NGC at the plasma membrane. Palmitoylated, but not prenylated form of the GTPase Cdc42 decreases dendritic branching. CALEB/NGC and Cdc42 are not directly interconnected in regulating dendritic branching. Thus, CALEB/NGC organizes a Rho GTPase signaling module at the plasma membrane for shaping dendritic trees.

The main oxygen sensor hypoxia inducible factor (HIF) prolyl hydroxylase 2 (PHD2) is a critical regulator of tissue homeostasis during erythropoiesis, hematopoietic stem cell maintenance, and wound healing. Recent studies point toward a role for the PHD2-erythropoietin (EPO) axis in the modulation of bone remodeling, even though the studies produced conflicting results. Here, we used a number of mouse strains deficient of PHD2 in different cell types to address the role of PHD2 and its downstream targets HIF-1α and HIF-2α in bone remodeling. Mice deficient for PHD2 in several cell lineages, including EPO-producing cells, osteoblasts, and hematopoietic cells (CD68:cre-PHD2f/f ) displayed a severe reduction of bone density at the distal femur as well as the vertebral body due to impaired bone formation but not bone resorption. Importantly, using osteoblast-specific (Osx:cre-PHD2f/f ) and osteoclast-specific PHD2 knock-out mice (Vav:cre- PHD2f/f ), we show that this effect is independent of the loss of PHD2 in osteoblast and osteoclasts. Using different in vivo and in vitro approaches, we show here that this bone phenotype, including the suppression of bone formation, is directly linked to the stabilization of the α-subunit of HIF-2, and possibly to the subsequent moderate induction of serum EPO, which directly influenced the differentiation and mineralization of osteoblast progenitors resulting in lower bone density. Taken together, our data identify the PHD2:HIF-2α:EPO axis as a so far unknown regulator of osteohematology by controlling bone homeostasis. Further, these data suggest that patients treated with PHD inhibitors or EPO should be monitored with respect to their bone status. © 2016 American Society for Bone and Mineral Research.

Summary The hematopoietic stem cell (HSC) compartment consists of a small pool of cells capable of replenishing all blood cells. Although it is established that the hematopoietic system is assembled as a hierarchical organization under steady-state conditions, emerging evidence suggests that distinct differentiation pathways may exist in response to acute stress. However, it remains unclear how different hematopoietic stem and progenitor cell subpopulations behave under sustained chronic stress. Here, by using adult transgenic mice overexpressing erythropoietin (EPO; Tg6) and a combination of in vivo, in vitro, and deep-sequencing approaches, we found that HSCs respond differentially to chronic erythroid stress compared with their closely related multipotent progenitors (MPPs). Specifically, HSCs exhibit a vastly committed erythroid progenitor profile with enhanced cell division, while MPPs display erythroid and myeloid cell signatures and an accumulation of uncommitted cells. Thus, our results identify HSCs as master regulators of chronic stress erythropoiesis, potentially circumventing the hierarchical differentiation-detour., Highlights • Chronic high EPO drives only HSCs to exhibit an exclusive erythroid progenitor profile • MPPs accumulate as uncommitted cells and display differential signatures, In this article, Wielockx and colleagues reveal that chronic erythropoietic stress induces distinct differences in the behavior of HSCs and their closely related MPPs. In particular, HSCs show greater commitment to an erythroid progenitor profile with heightened cell division, whereas MPPs are characterized by erythroid and immune signatures and an accumulation of uncommitted cells.

Inbreeding is typically detrimental to individual fitness, with negative effects being often exaggerated in stressful environments. However, the causal mechanisms underlying inbreeding depression in general and the often increased susceptibility to stress in particular are not well understood. We here test whether inbreeding interferes with the heat-shock response, comprising an important component of the stress response which may therefore underscore sensitivity to stress. To this end we subjected the tropical butterfly Bicyclus anynana to a full-factorial design with three temperatures and three levels of inbreeding, and measured the expression of heat-shock protein (HSP) 70 via qPCR. HSP70 expression increased after exposure to heat as compared with cold or control conditions. Most strikingly, inbreeding strongly interfered with the heat-shock response, with inbred individuals showing a very weak upregulation of HSP70 only. Our results thus indicate that, in our study organism, interference with the heat-shock response may be one mechanism underlying reduced fitness of inbred individuals, especially when exposed to stressful conditions. However, these indications need to be corroborated using a broader range of different temperatures, genes and taxa.

The potential of advertising to deceive young adolescents is problematic especially when it results in unhealthy food choices. Health warnings are supposed to raise awareness of the risky nature of a food product. However, these warnings compete for consumer’s attention with other advertising components set by marketers, such as product claims, visual frames, and images. To examine perception, attitudes, and behavioural intentions towards an ad, adolescents were exposed with fictitious soft drink advertisements in an experimental design. Hereby, we systematically varied warning labels and visual frames as key design elements of the advertisement. Results suggest that the effects of warnings on attitudes and purchase intention are mitigated by accompanying advertising elements. A single positive visual cue is sufficient to provoke purchase intentions. Overall, distraction from health warnings peaks in the youngest age groups and decreases with age. Findings raise concerns about how public health regulations on advertisements should be designed when the purpose is to inform especially younger adolescents of possible health risks. We discuss several implications for ethical marketing techniques of food products.

Application of autologous T cells genetically engineered to express CD19-specific chimeric antigen receptors (CAR-T) is highly effective in the treatment of B cell malignancies. To this date, application of CAR-T therapy beyond CD19 remains challenging due to the inability to control CAR-T reactivity in patients and the lack of tumor-associated antigens exclusively expressed by malignant cells. The interleukin-3 receptor alpha chain (CD123) is a promising immunotherapeutic target and associated with leukemia-initiating compartments in myeloid- or lymphoid derived diseases. However, in contrast to CD19, CD123 is a precarious target due to its prevalent expression on healthy hematopoietic stem and progenitor cells (HSPC) as well as endothelial cells. Thus, CAR-T lacking any fine-tuned control mechanisms are at risk to cause life threatening toxicities or can only act as bridging therapy to an allogeneic stem cell transplantation. To extend application of CAR-T therapy and safely redirect CAR-engineered T cells to challenging targets such as CD123, a switch-controllable universal CAR-T platform (UniCAR) was recently introduced. The UniCAR system consists of two components: (1) a non-reactive inducible second generation CAR with CD28/CD3ζ stimulation for an inert manipulation of T cells (UniCAR-T) and (2) soluble targeting modules (TM) enabling UniCAR-T reactivity in an antigen-specific manner. Here we provide late stage pre-clinical data for UniCAR-T in combination with a CD123-specific TM (TM123) for treatment of acute leukemia. Primary patient-derived CD123-positive leukemic blasts were efficiently eradicated by TM123-redirected clinical-grade manufactured UniCAR-T in vitro and in vivo. Activation, cytolytic responses and cytokine release were proven to be strictly switch-controlled. Moreover, anti-leukemic responses of UniCAR-T were demonstrated to be comparable to conventional CD123-specific CAR-T in vitro. In contrast to conventional CD123 CAR-T, TM123-redirected UniCAR-T discriminate between CD123high malignant cells and CD123low healthy cells with negligible toxicity towards HSPC in vivo. As 4-1BB mediated co-stimulation is known to enhance CAR-T activity in vivo, a novel CD123-specific targeting module bearing a covalently bound trimeric 4-1BB ligand (4-1BBL) was developed and characterized for co-stimulation at the leukemic site in trans. Specific binding of TM123-4-1BBL was demonstrated against native 4-1BB as well as CD123-positive leukemic blasts. In long-term tumor eradication models, TM123-4-1BBL ameliorated the killing capability of UniCAR-T in vitro. Additionally, the increased hydrodynamic radius of trimeric 4-1BBL-coupled TM123 prolonged plasma half-life and enhanced bioavailability in vivo. In conclusion, UniCAR-T maintain high anti-leukemic efficacy, while adding a sophisticated mechanism for immediate control to improve safety and versatility of CD123-directed CAR-T therapy. Moreover, switching between several TMs from short to moderate plasma half-life allows for an individualized treatment of various leukemic settings while minimizing potential adverse effects. Disclosures Loff: GEMoaB Monoclonals GmbH: Employment. Meyer:Cellex Patient Treatment GmbH: Employment. Dietrich:Cellex Patient Treatment GmbH: Employment. Spehr:Cellex Patient Treatment GmbH: Employment. Julia:Cellex Patient Treatment GmbH: Employment. Gründer:GEMoaB Monoclonals GmbH: Employment. Franke:GEMoaB Monoclonals GmbH: Employment. Bachmann:GEMoaB Monoclonals GmbH: Equity Ownership. Ehninger:Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership. Ehninger:GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Cellex Gesellschaft fuer Zellgewinnung mbH: Equity Ownership. Cartellieri:Cellex Patient Treatment GmbH: Employment.

Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β3-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds.

The tumor microenvironment plays a pivotal role during cancer development and progression. The balance between suppressive and cytotoxic responses of the tumor immune microenvironment has been shown to have a direct effect on the final outcome in various human and experimental tumors. Recently, we demonstrated that the oxygen sensor HIF-prolyl hydroxylase-2 (PHD2) plays a detrimental role in tumor cells, stimulating systemic growth and metastasis in mice. In our current study, we show that the conditional ablation of PHD2 in the hematopoietic system also leads to reduced tumor volume, intriguingly generated by an imbalance between enhanced cell death and improved proliferation of tumor cells. This effect seems to rely on the overall downregulation of protumoral as well as antitumoral cytokines. Using different genetic approaches, we were able to confine this complex phenotype to the crosstalk of PHD2-deficient myeloid cells and T-lymphocytes. Taken together, our findings reveal a multifaceted role for PHD2 in several hematopoietic lineages during tumor development and might have important implications for the development of tumor therapies in the future.

Immune function is greatly affected by biotic and abiotic factors, arguably owing to resource allocation trade-offs. While starvation typically exerts negative effects on immune function, there are two competing hypotheses regarding the impact of population density: a high population density may decrease or increase immune competence, either as a result of reduced energy stores or of augmented resource allocation to minimise infection risk (density-dependent prophylaxis). Against this background, the effects of population (= larval rearing) density and transient food stress on adult immune function and life-history traits in the temperate-zone butterfly Pieris napi (Linnaeus) have been examined. Both a period of larval starvation and high larval rearing density prolonged the development time and reduced the body mass. All immune parameters measured (pro-phenoloxidase and phenoloxidase activity, haemocyte number, encapsulation rate) were also negatively affected by high density, whereas starvation had no effect. We thus found no evidence for density-dependent prophylaxis, but a diminished performance at a high density probably caused by reduced energy stores. The present study reinforces that the maintenance and deployment of an efficient immune system is costly, which may prohibit prophylactic responses to high population densities.

RhoG is a member of the Rho family of small GTPases sharing highest sequence similarity with Rac and Cdc42. Mig-2 and Mtl represent the functional equivalents of RhoG in Caenorhabditis elegans and Drosophila, respectively. RhoG has attracted great interest because it plays a central role in the regulation of cytoskeletal reorganization in various physiological and pathophysiological situations. For example, it is fundamental to phagocytotic processes, is able to regulate gene expression, cell survival and proliferation, and is involved in cell migration and in the invasion of pathogenic bacteria. The activation of Rac1 via an ELMO/Dock180 module has been elaborated to be important for RhoG signaling. Although a stimulatory role for neurite outgrowth in the pheochromocytoma PC12 cell line has been assigned to RhoG, the exact function of this GTPase for the development of the processes of primary neurons remains to be clarified. In this view, we discuss the impact of RhoG on axonal and dendritic differentiation, its role as a conductor of Rac1 and Cdc42 activity and the functional regulation of RhoG expression by the microRNA miR-124.

The induction of DNA double-strand breaks (DSBs) elicits a plethora of responses that redirect many cellular functions to the vital task of repairing the injury, collectively known as the DNA damage response (DDR). We have found that, in the absence of DNA damage, the DSB repair factors RNF8 and BRCA1 are associated with the nucleolus. Shortly after exposure of cells to γ-radiation, RNF8 and BRCA1 translocated from the nucleolus to damage foci, a traffic that was reverted several hours after the damage. RNF8 interacted through its FHA domain with the ribosomal protein RPSA, and knockdown of RPSA caused a depletion of nucleolar RNF8 and BRCA1, suggesting that the interaction of RNF8 with RPSA is critical for the nucleolar localization of these DDR factors. Knockdown of RPSA or RNF8 impaired bulk protein translation, as did γ-irradiation, the latter being partially countered by overexpression of exogenous RNF8. Our results suggest that RNF8 and BRCA1 are anchored to the nucleolus through reversible interactions with RPSA and that, in addition to its known functions in DDR, RNF8 may play a role in protein synthesis, possibly linking the nucleolar exit of this factor to the attenuation of protein synthesis in response to DNA damage. © 2012 Elsevier Inc., M.G. was a recipient of a predoctoral fellowship and F.M. of a Juan de la Cierva postdoctoral fellowship from the Spanish Ministry of Science and Innovation. This study was supported by grants from the Spanish Ministry of Science and Innovation (SAF2008-04136-C02-01 and SAF2011-24686), the Catalan Agency for the Administration of University and Research Grants (AGAUR2009-SGR-1482) and the Biotechnology Reference Network (XeRBa).