Cultures of Human Skin Mast Cells, an Attractive In Vitro Model for Studies of Human Mast Cell Biology.

Studies of mast cell biology are dependent on relevant and validated in vitro models. Here, we present detailed information concerning the phenotype of both freshly isolated human skin mast cells (MCs) and of in vitro cultures of these cells that were obtained by analyzing their total transcriptome. Transcript levels of MC-related granule proteins and transcription factors were found to be remarkably stable over a 3-week culture period. Relatively modest changes were also seen for important cell surface receptors including the high-affinity receptor for IgE, FCER1A, the low-affinity receptor for IgG, FCGR2A, and the receptor for stem cell factor, KIT. FCGR2A was the only Fc receptor for IgG expressed by these cells. The IgE receptor increased by 2-5-fold and an approximately 10-fold reduction in the expression of FCGR2A was observed most likely due to the cytokines, SCF and IL-4, used for expanding the cells. Comparisons of the present transcriptome against previously reported transcriptomes of mouse peritoneal MCs and mouse bone marrow-derived MCs (BMMCs) revealed both similarities and major differences. Strikingly, cathepsin G was the most highly expressed granule protease in human skin MCs, in contrast to the almost total absence of this protease in both mouse MCs. Transcript levels for the majority of cell surface receptors were also very low compared to the granule proteases in both mouse and human MCs, with a difference of almost two orders of magnitude. An almost total absence of T-cell granzymes was observed in human skin MCs, indicating that granzymes have no or only a minor role in human MC biology. Ex vivo skin MCs expressed high levels of selective immediate early genes and transcripts of heat shock proteins. In validation experiments, we determined that this expression was an inherent property of the cells and not the result of the isolation process. Three to four weeks in culture results in an induction of cell growth-related genes accompanying their expansion by 6-10-fold, which increases the number of cells for in vitro experiments. Collectively, we show that cultured human skin MCs resemble their ex vivo equivalents in many respects and are a more relevant in vitro model compared to mouse BMMCs for studies of MC biology, in particular human MC biology.

Synergism between IL-33 and MRGPRX2/FcεRI Is Primarily Due to the Complementation of Signaling Modules, and Only Modestly Supplemented by Prolonged Activation of Selected Kinases.

Skin mast cells (MCs) express high levels of MRGPRX2, FcεRI, and ST2, and vigorously respond to their ligands when triggered individually. IL-33/ST2 also potently synergizes with other receptors, but the molecular underpinnings are poorly understood. Human skin-derived MCs were stimulated via different receptors individually or jointly in the presence/absence of selective inhibitors. TNF was quantified by ELISA. Signaling cascades were studied by immunoblot. TNF was stimulated by FcεRI ≈ ST2 > MRGPRX2. Surprisingly, neither FcεRI nor MRGPRX2 stimulation elicited NF-κB activation (IκB degradation, p65 phosphorylation) in stark contrast to IL-33. Accordingly, TNF production did not depend on NF-κB in FcεRI- or MRGPRX2-stimulated MCs, but did well so downstream of ST2. Conversely, ERK1/2 and PI3K were the crucial modules upon FcεRI/MRGPRX2 stimulation, while p38 was key to the IL-33-elicited route. The different signaling prerequisites were mirrored by their activation patterns with potent pERK/pAKT after FcεRI/MRGPRX2, but preferential induction of pp38/NF-κB downstream of ST2. FcεRI/MRGPRX2 strongly synergized with IL-33, and some synergy was still observed upon inhibition of each module (ERK1/2, JNK, p38, PI3K, NF-κB). IL-33's contribution to synergism was owed to p38 > JNK > NF-κB, while the partner receptor contributed through ERK > PI3K ≈ JNK. Concurrent IL-33 led to slightly prolonged pERK (downstream of MRGPRX2) or pAKT (activated by FcεRI), while the IL-33-elicited modules (pp38/NF-κB) remained unaffected by co-stimulation of FcεRI/MRGPRX2. Collectively, the strong synergistic activity of IL-33 primarily results from the complementation of highly distinct modules following co-activation of the partner receptor rather than by altered signal strength of the same modules.

Clorfl86/RHEX Is a Negative Regulator of SCF/KIT Signaling in Human Skin Mast Cells.

Mast cells (MCs) are key effector cells in allergic and inflammatory diseases, and the SCF/KIT axis regulates most aspects of the cells' biology. Using terminally differentiated skin MCs, we recently reported on proteome-wide phosphorylation changes initiated by KIT dimerization. C1orf186/RHEX was revealed as one of the proteins to become heavily phosphorylated. Its function in MCs is undefined and only some information is available for erythroblasts. Using public databases and our own data, we now report that RHEX exhibits highly restricted expression with a clear dominance in MCs. While expression is most pronounced in mature MCs, RHEX is also abundant in immature/transformed MC cell lines (HMC-1, LAD2), suggesting early expression with further increase during differentiation. Using RHEX-selective RNA interference, we reveal that RHEX unexpectedly acts as a negative regulator of SCF-supported skin MC survival. This finding is substantiated by RHEX's interference with KIT signal transduction, whereby ERK1/2 and p38 both were more strongly activated when RHEX was attenuated. Comparing RHEX and capicua (a recently identified repressor) revealed that each protein preferentially suppresses other signaling modules elicited by KIT. Induction of immediate-early genes strictly requires ERK1/2 in SCF-triggered MCs; we now demonstrate that RHEX diminution translates to this downstream event, and thereby enhances NR4A2, JUNB, and EGR1 induction. Collectively, our study reveals RHEX as a repressor of KIT signaling and function in MCs. As an abundant and selective lineage marker, RHEX may have various roles in the lineage, and the provided framework will enable future work on its involvement in other crucial processes.

CREB Is Activated by the SCF/KIT Axis in a Partially ERK-Dependent Manner and Orchestrates Survival and the Induction of Immediate Early Genes in Human Skin Mast Cells.

cAMP response element binding protein (CREB) functions as a prototypical stimulus-inducible transcription factor (TF) that initiates multiple cellular changes in response to activation. Despite pronounced expression in mast cells (MCs), CREB function is surprisingly ill-defined in the lineage. Skin MCs (skMCs) are critical effector cells in acute allergic and pseudo-allergic settings, and they contribute to various chronic dermatoses such as urticaria, atopic dermatitis, allergic contact dermatitis, psoriasis, prurigo, rosacea and others. Using MCs of skin origin, we demonstrate herein that CREB is rapidly phosphorylated on serine-133 upon SCF-mediated KIT dimerization. Phosphorylation initiated by the SCF/KIT axis required intrinsic KIT kinase activity and partially depended on ERK1/2, but not on other kinases such as p38, JNK, PI3K or PKA. CREB was constitutively nuclear, where phosphorylation occurred. Interestingly, ERK did not translocate to the nucleus upon SCF activation of skMCs, but a fraction was present in the nucleus at baseline, and phosphorylation was prompted in the cytoplasm and nucleus in situ. CREB was required for SCF-facilitated survival, as demonstrated with the CREB-selective inhibitor 666-15. Knock-down of CREB by RNA interference duplicated CREB's anti-apoptotic function. On comparison with other modules (PI3K, p38 and MEK/ERK), CREB was equal or more potent at survival promotion. SCF efficiently induces immediate early genes (IEGs) in skMCs (FOS, JUNB and NR4A2). We now demonstrate that CREB is an essential partaker in this induction. Collectively, the ancient TF CREB is a crucial component of skMCs, where it operates as an effector of the SCF/KIT axis, orchestrating IEG induction and lifespan.

CREB Is Indispensable to KIT Function in Human Skin Mast Cells-A Positive Feedback Loop between CREB and KIT Orchestrates Skin Mast Cell Fate.

Skin mast cells (MCs) are critical effector cells in acute allergic reactions, and they contribute to chronic dermatoses like urticaria and atopic and contact dermatitis. KIT represents the cells' crucial receptor tyrosine kinase, which orchestrates proliferation, survival, and functional programs throughout the lifespan. cAMP response element binding protein (CREB), an evolutionarily well-conserved transcription factor (TF), regulates multiple cellular programs, but its function in MCs is poorly understood. We recently reported that CREB is an effector of the SCF (Stem Cell Factor)/KIT axis. Here, we ask whether CREB may also act upstream of KIT to orchestrate its functioning. Primary human MCs were isolated from skin and cultured in SCF+IL-4 (Interleukin-4). Pharmacological inhibition (666-15) and RNA interference served to manipulate CREB function. We studied KIT expression using flow cytometry and RT-qPCR, KIT-mediated signaling using immunoblotting, and cell survival using scatterplot and caspase-3 activity. The proliferation and cycle phases were quantified following BrdU incorporation. Transient CREB perturbation resulted in reduced KIT expression. Conversely, microphthalmia transcription factor (MITF) was unnecessary for KIT maintenance. KIT attenuation secondary to CREB was associated with heavily impaired KIT functional outputs, like anti-apoptosis and cell cycle progression. Likewise, KIT-elicited phosphorylation of ERK1/2 (Extracellular Signal-Regulated Kinase 1/2), AKT, and STAT5 (Signal Transducer and Activator of Transcription) was substantially diminished upon CREB inhibition. Surprisingly, the longer-term interference of CREB led to complete cell elimination, in a way surpassing KIT inhibition. Collectively, we reveal CREB as non-redundant in MCs, with its absence being incompatible with skin MCs' existence. Since SCF/KIT regulates CREB activity and, vice versa, CREB is required for KIT function, a positive feedforward loop between these elements dictates skin MCs' fate.

How "Neuronal" Are Human Skin Mast Cells?

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.

ß-arrestin-1 and ß-arrestin-2 Restrain MRGPRX2-Triggered Degranulation and ERK1/2 Activation in Human Skin Mast Cells.

As a novel receptor that efficiently elicits degranulation upon binding to one of its numerous ligands, MRGPRX2 has moved to the center of attention in mast cell (MC) research. Indeed, MRGPRX2 is believed to be a major component of pseudo-allergic reactions to drugs and of neuropeptide-elicited MC activation in skin diseases alike. MRGPRX2 signals via G proteins which organize downstream events ultimately leading to granule discharge. Skin MCs require both PI3K and ERK1/2 cascades for efficient exocytosis. ß-arrestins act as opponents of G proteins and lead to signal termination with or without subsequent internalization. We recently demonstrated that ligand-induced internalization of MRGPRX2 requires the action of ß-arrestin-1, but not of ß-arrestin-2. Here, by using RNA interference, we find that both isoforms counter skin MC degranulation elicited by three MRGPRX2 agonists but not by FcεRI-aggregation. Analyzing whether this occurs through MRGPRX2 stabilization under ß-arrestin attenuation, we find that reduction of ß-arrestin-1 indeed leads to increased MRGPRX2 abundance, while this is not observed for ß-arrestin-2. This led us speculate that ß-arrestin-2 is involved in signal termination without cellular uptake of MRGPRX2. This was indeed found to be the case, whereby interference with ß-arrestin-2 has an even stronger positive effect on ERK1/2 phosphorylation compared to ß-arrestin-1 perturbation. Neither ß-arrestin-1 nor ß-arrestin-2 had an impact on AKT phosphorylation nor affected signaling via the canonical FcεRI-dependent route. We conclude that in skin MCs, ß-arrestin-2 is chiefly involved in signal termination, whereas ß-arrestin-1 exerts its effects by controlling MRGPRX2 abundance.

The SCF/KIT axis in human mast cells: Capicua acts as potent KIT repressor and ERK predominates PI3K.

BACKGROUND: 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. METHODS: 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. RESULTS: ≈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. CONCLUSION: 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.

Myeloid PHD2 deficiency accelerates neointima formation via Hif-1α.

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 PHD2fl/fl, PHD2fl/fl/Hif1αfl/fl and PHD2fl/fl/Hif2αfl/fl to get myeloid specific knockout of PHD2 and the Hif-α subunits. Denudation of the femoral artery was performed and animals were fed a western type diet afterwards with analysis of neointima formation 5 and 35 days after denudation. Increased neointima formation in myeloid PHD2 knockouts was observed, which was blunted by double-knockout of PHD2 and Hif1α whereas double knockout of PHD2 and Hif-2α showed comparable lesions to the PHD2 knockouts. Macrophage infiltration was comparable to the neointima formation, suggesting a more inflammatory reaction, and was accompanied by increased intimal VEGF-A expression. Collagen-content inversely correlated to the extent of neointima formation suggesting a destabilization of the plaque. This effect might be triggered by macrophage polarization. Therefore, in vitro results showed a distinct expression pattern in differentially polarized macrophages with high expression of Hif-1α, VEGF and MMP-1 in proinflammatory M1 macrophages. In conclusion, the results show that myeloid Hif-1α is involved in neointima hyperplasia. Our in vivo and in vitro data reveal a central role for this transcription factor in driving plaque-vascularization accompanied by matrix-degradation leading to plaque destabilization.

MRGPRX2-Mediated Degranulation of Human Skin Mast Cells Requires the Operation of Gαi, Gαq, Ca++ Channels, ERK1/2 and PI3K-Interconnection between Early and Late Signaling.

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 ß-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.

Mast cells instruct keratinocytes to produce thymic stromal lymphopoietin: Relevance of the tryptase/protease-activated receptor 2 axis.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) promotes TH2 inflammation and is deeply intertwined with inflammatory dermatoses like atopic dermatitis. The mechanisms regulating TSLP are poorly defined. OBJECTIVE: We investigated whether and by what mechanisms mast cells (MCs) foster TSLP responses in the cutaneous environment. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Cytokine Stimulation by MRGPRX2 Occurs with Lower Potency than by FcεRI Aggregation but with Similar Dependence on the Extracellular Signal-Regulated Kinase 1/2 Module in Human Skin Mast Cells.

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.

Cytokines Stimulated by IL-33 in Human Skin Mast Cells: Involvement of NF-κB and p38 at Distinct Levels and Potent Co-Operation with FcεRI and MRGPRX2.

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-1ß, 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.

HIF2α is a direct regulator of neutrophil motility.

Orchestrated recruitment of neutrophils to inflamed tissue is essential during the initiation of inflammation. Inflamed areas are usually hypoxic, and adaptation to reduced oxygen pressure is typically mediated by hypoxia pathway proteins. However, it remains unclear how these factors influence the migration of neutrophils to and at the site of inflammation during their transmigration through the blood-endothelial cell barrier, as well as their motility in the interstitial space. Here, we reveal that activation of hypoxia-inducible factor 2 (HIF2α) as a result of a deficiency in 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 RNA sequencing 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 novel PHD2-HIF2α-RhoA axis is vital to the initial stages of inflammation because it promotes neutrophil movement through highly confined tissue landscapes.

Thymic Stromal Lymphopoietin Promotes MRGPRX2-Triggered Degranulation of Skin Mast Cells in a STAT5-Dependent Manner with Further Support from JNK.

Thymic stromal lymphopoietin (TSLP) is released by epithelial cells following disturbed homeostasis to act as "alarmin" 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., FcεRI-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 FcεRI-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 FcεRI-triggered MC activation. The relevance of TSLP, MCs and MRGPRX2 to pruritis and atopic skin pathology indicates broad repercussions of the identified connection.

MRGPRX2 Is the Codeine Receptor of Human Skin Mast Cells: Desensitization through ß-Arrestin and Lack of Correlation with the FcεRI Pathway.

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 ß-arrestin-1‒dependent manner. Codeine-triggered ß-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.

Clinal variation in investment into reproduction versus maintenance suggests a 'pace-of-life' syndrome in a widespread butterfly.

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.

Rapidly Switchable Universal CAR-T Cells for Treatment of CD123-Positive Leukemia.

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).

Regulation of sirtuin expression in autoimmune neuroinflammation: Induction of SIRT1 in oligodendrocyte progenitor cells.

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.