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1.
Cell ; 187(19): 5316-5335.e28, 2024 Sep 19.
Article in English | MEDLINE | ID: mdl-39096902

ABSTRACT

Neutrophils are sentinel immune cells with essential roles for antimicrobial defense. Most of our knowledge on neutrophil tissue navigation derived from wounding and infection models, whereas allergic conditions remained largely neglected. Here, we analyzed allergen-challenged mouse tissues and discovered that degranulating mast cells (MCs) trap living neutrophils inside them. MCs release the attractant leukotriene B4 to re-route neutrophils toward them, thus exploiting a chemotactic system that neutrophils normally use for intercellular communication. After MC intracellular trap (MIT) formation, neutrophils die, but their undigested material remains inside MC vacuoles over days. MCs benefit from MIT formation, increasing their functional and metabolic fitness. Additionally, they are more pro-inflammatory and can exocytose active neutrophilic compounds with a time delay (nexocytosis), eliciting a type 1 interferon response in surrounding macrophages. Together, our study highlights neutrophil trapping and nexocytosis as MC-mediated processes, which may relay neutrophilic features over the course of chronic allergic inflammation.


Subject(s)
Inflammation , Mast Cells , Mice, Inbred C57BL , Neutrophils , Animals , Mast Cells/metabolism , Mast Cells/immunology , Neutrophils/metabolism , Neutrophils/immunology , Mice , Inflammation/metabolism , Inflammation/immunology , Inflammation/pathology , Leukotriene B4/metabolism , Signal Transduction , Cell Degranulation , Macrophages/metabolism , Macrophages/immunology , Extracellular Traps/metabolism , Male , Female
2.
Cell ; 179(2): 417-431.e19, 2019 Oct 03.
Article in English | MEDLINE | ID: mdl-31585081

ABSTRACT

Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.


Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Asthma/therapy , Mast Cells/enzymology , Mast Cells/immunology , Tryptases/antagonists & inhibitors , Tryptases/immunology , Adolescent , Allosteric Regulation/immunology , Animals , Cell Line , Female , Humans , Macaca fascicularis , Male , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, SCID , Rabbits
4.
Nature ; 543(7644): 199-204, 2017 03 09.
Article in English | MEDLINE | ID: mdl-28241135

ABSTRACT

Long non-coding RNAs (lncRNAs) are largely heterogeneous and functionally uncharacterized. Here, using FANTOM5 cap analysis of gene expression (CAGE) data, we integrate multiple transcript collections to generate a comprehensive atlas of 27,919 human lncRNA genes with high-confidence 5' ends and expression profiles across 1,829 samples from the major human primary cell types and tissues. Genomic and epigenomic classification of these lncRNAs reveals that most intergenic lncRNAs originate from enhancers rather than from promoters. Incorporating genetic and expression data, we show that lncRNAs overlapping trait-associated single nucleotide polymorphisms are specifically expressed in cell types relevant to the traits, implicating these lncRNAs in multiple diseases. We further demonstrate that lncRNAs overlapping expression quantitative trait loci (eQTL)-associated single nucleotide polymorphisms of messenger RNAs are co-expressed with the corresponding messenger RNAs, suggesting their potential roles in transcriptional regulation. Combining these findings with conservation data, we identify 19,175 potentially functional lncRNAs in the human genome.


Subject(s)
Databases, Genetic , RNA, Long Noncoding/chemistry , RNA, Long Noncoding/genetics , Transcriptome/genetics , Cells, Cultured , Conserved Sequence/genetics , Datasets as Topic , Enhancer Elements, Genetic/genetics , Epigenesis, Genetic , Gene Expression Profiling , Gene Expression Regulation , Genome, Human/genetics , Genome-Wide Association Study , Genomics , Humans , Internet , Molecular Sequence Annotation , Organ Specificity/genetics , Polymorphism, Single Nucleotide , Promoter Regions, Genetic/genetics , Quantitative Trait Loci/genetics , RNA Stability , RNA, Messenger/genetics
5.
J Allergy Clin Immunol ; 149(6): 2053-2061.e6, 2022 06.
Article in English | MEDLINE | ID: mdl-35240143

ABSTRACT

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.


Subject(s)
Dermatitis, Atopic , Receptor, PAR-2 , Animals , Chymases/metabolism , Cytokines/metabolism , Histamine/metabolism , Humans , Keratinocytes/metabolism , Mast Cells/metabolism , Mice , Receptor, PAR-2/genetics , Receptor, PAR-2/metabolism , Tryptases/metabolism , Thymic Stromal Lymphopoietin
6.
Int J Mol Sci ; 24(4)2023 Feb 18.
Article in English | MEDLINE | ID: mdl-36835547

ABSTRACT

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.


Subject(s)
Cyclic AMP Response Element-Binding Protein , Genes, Immediate-Early , Humans , Cyclic AMP Response Element-Binding Protein/metabolism , Mast Cells/metabolism , Phosphorylation , Phosphatidylinositol 3-Kinases/metabolism
7.
Allergy ; 77(11): 3337-3349, 2022 11.
Article in English | MEDLINE | ID: mdl-35652819

ABSTRACT

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.


Subject(s)
Mast Cells , Stem Cell Factor , Humans , Chromatography, Liquid , Cytokines/metabolism , Mast Cells/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-kit/genetics , Stem Cell Factor/pharmacology , Stem Cell Factor/metabolism , Tandem Mass Spectrometry , Extracellular Signal-Regulated MAP Kinases/metabolism
8.
J Allergy Clin Immunol ; 147(2): 456-469, 2021 02.
Article in English | MEDLINE | ID: mdl-33071069

ABSTRACT

The Mas-related G protein-coupled receptor X2 (MRGPRX2) is a multiligand receptor responding to various exogenous and endogenous stimuli. Being highly expressed on skin mast cells, MRGPRX2 triggers their degranulation and release of proinflammatory mediators, and it promotes multicellular signaling cascades, such as itch induction and transmission in sensory neurons. The expression of MRGPRX2 by skin mast cells and the levels of the MRGPRX2 agonists (eg, substance P, major basic protein, eosinophil peroxidase) are upregulated in the serum and/or skin of patients with inflammatory and pruritic skin diseases, such as chronic spontaneous urticaria or atopic dermatitis. Therefore, MRGPRX2 and its agonists might be potential biomarkers for the progression of cutaneous inflammatory diseases and the response to treatment. In addition, they may represent promising targets for prevention and treatment of signs and symptoms in patients with skin diseases or drug reactions. To assess this possibility, this review explores the role and relevance of MRGPRX2 and its activators in cutaneous inflammatory disorders and chronic pruritus.


Subject(s)
Chronic Urticaria/immunology , Dermatitis, Atopic/immunology , Nerve Tissue Proteins/immunology , Pruritus/immunology , Receptors, G-Protein-Coupled/immunology , Receptors, Neuropeptide/immunology , Animals , Chronic Urticaria/metabolism , Dermatitis, Atopic/metabolism , Humans , Hypersensitivity, Immediate/immunology , Hypersensitivity, Immediate/metabolism , Mast Cells/immunology , Mast Cells/metabolism , Nerve Tissue Proteins/metabolism , Pruritus/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Neuropeptide/metabolism
9.
Int J Mol Sci ; 23(18)2022 Sep 17.
Article in English | MEDLINE | ID: mdl-36142795

ABSTRACT

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.


Subject(s)
Mast Cells , Neural Cell Adhesion Molecule L1 , Adaptor Proteins, Signal Transducing/metabolism , Animals , Dopamine/metabolism , Histamine/metabolism , Histocompatibility Antigens/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Humans , LIM Domain Proteins/metabolism , Mast Cells/metabolism , Mice , Neural Cell Adhesion Molecule L1/metabolism , Serotonin/metabolism , Skin/metabolism , Transcription Factors/metabolism
10.
Allergol Int ; 71(3): 310-317, 2022 Jul.
Article in English | MEDLINE | ID: mdl-35662539

ABSTRACT

In October 2021, researchers from the German Society of Allergy and Clinical Immunology (DGAKI) and from the Japanese Society of Allergology (JSA) focused their attention on the pathological conditions and modifiers of various allergic diseases. Topics included 1) the pathophysiology of IgE/mast cell-mediated allergic diseases; 2) the diagnosis and prevention of IgE/mast cell-mediated diseases; 3) the pathophysiology, diagnosis, and treatment of eosinophilic airway diseases; and 4) host-pathogen interaction and allergic diseases. This report summarizes the panel discussions, which highlighted the importance of recognizing the diversity of genetics, immunological mechanisms, and modifying factors underlying allergic diseases.


Subject(s)
Hypersensitivity , Immunoglobulin E , Humans , Hypersensitivity/drug therapy , Hypersensitivity/therapy
11.
J Allergy Clin Immunol ; 146(6): 1387-1396.e13, 2020 12.
Article in English | MEDLINE | ID: mdl-32407837

ABSTRACT

BACKGROUND: Reduced levels of prostaglandin E2 (PGE2) contribute to aspirin-induced hypersensitivity. COX inhibitors are also frequent cofactors in anaphylaxis. Whether alterations in the PGE2 system contribute to anaphylaxis independently of COX inhibitor intake is unclear. OBJECTIVE: Our aim was to test the hypothesis that relative PGE2 deficiency predisposes to anaphylaxis. METHODS: Sera from 48 patients with anaphylaxis and 27 healthy subjects were analyzed for PGE2 levels and correlated against severity; 9α,11ß-PGF2 and PGI2 metabolites were measured for control purposes. PGE2 stabilization by 15-hydroxyprostaglandin dehydrogenase inhibitor or EP2 or EP4 receptor agonists were used in a murine model of passive systemic anaphylaxis. FcεRI-triggered mediator release was determined in bone marrow-derived cultured mast cells (MCs) and human skin-derived MCs. Signaling was studied by Western blot analysis. RESULTS: Patients with anaphylaxis were characterized by markedly reduced PGE2 levels vis-à-vis healthy subjects, whereas prostacyclin metabolite levels were diminished only weakly, and 9α,11ß-PGF2 levels conversely increased. PGE2 was negatively correlated with severity. Lower PGE2 levels and higher susceptibility to anaphylaxis were also found in C57BL/6 mice vis-à-vis in Balb/c mice. Stabilization of PGE2 level by 15-hydroxyprostaglandin dehydrogenase inhibitor protected mice against anaphylaxis. Exogenous PGE2 attenuated bone marrow-derived cultured MC activation through EP2 and EP4 receptors. EP2 and EP4 agonism also curbed FcεRI-mediated degranulation of human MCs. Mechanistically, PGE2 interfered with the phosphorylation of phospholipase C gamma-1 and extracellular signal-regulated kinase. CONCLUSIONS: Homeostatic levels of PGE2 attenuate MC activation via EP2/EP4 and protect against anaphylaxis. Relative deficiency of PGE2 predisposes to anaphylaxis in humans and mice, whereas PGE2 stabilization protects against anaphylactic reactions.


Subject(s)
Anaphylaxis/immunology , Dinoprostone/deficiency , Mast Cells/immunology , Anaphylaxis/pathology , Animals , Dinoprostone/immunology , Disease Susceptibility/immunology , Extracellular Signal-Regulated MAP Kinases/immunology , Humans , Mast Cells/pathology , Mice , Mice, Inbred BALB C , Phospholipase C gamma/immunology , Receptors, Prostaglandin E, EP2 Subtype/immunology , Receptors, Prostaglandin E, EP4 Subtype/immunology , Severity of Illness Index
12.
Int J Mol Sci ; 22(7)2021 Mar 30.
Article in English | MEDLINE | ID: mdl-33808264

ABSTRACT

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.


Subject(s)
Cytokines/immunology , Interleukin-33/pharmacology , Mast Cells/drug effects , Nerve Tissue Proteins/immunology , Receptors, G-Protein-Coupled/immunology , Receptors, IgE/immunology , Receptors, Neuropeptide/immunology , Skin/drug effects , p38 Mitogen-Activated Protein Kinases/immunology , Foreskin/cytology , Humans , Interleukin-33/immunology , JNK Mitogen-Activated Protein Kinases/metabolism , Male , Mast Cells/cytology , Mast Cells/immunology , Phosphorylation , Primary Cell Culture , Signal Transduction/drug effects , Skin/immunology
13.
Exp Dermatol ; 29(11): 1104-1111, 2020 11.
Article in English | MEDLINE | ID: mdl-32866307

ABSTRACT

The discovery of MRGPRX2 marks an important change in MC biology, explaining non-IgE-mediated clinical phenomena relying on MCs. As receptor for multiple drugs, MRGPRX2 is crucial to drug-induced hypersensitivity. However, not only drugs, but also endogenous mediators like neuropeptides and host defense peptides activate MRGPRX2, suggesting its broad impact in cutaneous pathophysiology. Here, we give a brief overview of MRGPRX2 and its regulation by microenvironmental stimuli, which support MCs and can be altered in skin disorders, and briefly touch on the functional programs elicited by MRGPRX2 ligation. Studies in Mrgprb2-deficient mice (the murine ortholog) help illuminate MRGPRX2's function in health and disease. Recent advances in this model support the long-suspected operational unit between MCs and nerves, with MRGPRX2 being a vital component. Based on the limited evidence for a major contribution of FcεRI/IgE-activated MCs to atopic dermatitis (AD), we develop the hypothesis that MRGPRX2 constitutes the missing link connecting MCs and AD, at least in selected endotypes. Support comes from the multifold changes in the MC-neuronal system of AD skin (eg greater density of MCs and closer connections between MCs and nerves, increased PAR-2/Substance P). We theorize that these deregulations suffice to initiate AD, but external triggers, many of which activating MRGPRX2 themselves (eg Staphylococcus aureus) further feed into the loop. Itch, the most burdensome hallmark of AD, is mostly non-histaminergic but tryptase-dependent, and tryptase is preferentially released upon MRGPRX2 activation. Because MRGPRX2 is a very active research field, some of the existing gaps are likely to be closed soon.


Subject(s)
Dermatitis, Atopic/metabolism , Mast Cells/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Neuropeptide/metabolism , Animals , Cell Communication , Cellular Microenvironment , Humans , Mast Cells/physiology , Nerve Tissue Proteins/physiology , Neurons/physiology , Receptors, G-Protein-Coupled/physiology , Receptors, Neuropeptide/physiology
14.
Nature ; 507(7493): 462-70, 2014 Mar 27.
Article in English | MEDLINE | ID: mdl-24670764

ABSTRACT

Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.


Subject(s)
Atlases as Topic , Molecular Sequence Annotation , Promoter Regions, Genetic/genetics , Transcriptome/genetics , Animals , Cell Line , Cells, Cultured , Cluster Analysis , Conserved Sequence/genetics , Gene Expression Regulation/genetics , Gene Regulatory Networks/genetics , Genes, Essential/genetics , Genome/genetics , Humans , Mice , Open Reading Frames/genetics , Organ Specificity , RNA, Messenger/analysis , RNA, Messenger/genetics , Transcription Factors/metabolism , Transcription Initiation Site , Transcription, Genetic/genetics
16.
Exp Dermatol ; 27(11): 1298-1303, 2018 11.
Article in English | MEDLINE | ID: mdl-30091263

ABSTRACT

MRGPRX2 was recently uncovered as the "missing link" in clinically relevant mast cell (MC) activation explaining previously puzzling phenomena. It is the receptor for various endogenous ligands and exogenous compounds alike, whose binding evokes rapid degranulation much like allergen-mediated exocytosis. While the perceivable outcomes are similar, the two activation routes differ regarding mechanism and regulation. We recently reported that acute SCF administration curbs responses evoked by MRGPRX2 in human skin MCs. Maintenance of MCs in culture requires the presence of MC supportive factors and renders the cells functionally and molecularly unequal to ex vivo counterparts. Here, we asked whether expansion in culture impacts the pseudo-allergic route, and if so, what contribution SCF and IL-4 play in this scenario. We report that the in vitro micromilieu dampens (but does not erase) pseudo-allergic responses and that this is accompanied by strongly reduced MRGPRX2 expression. Withdrawal of SCF or IL-4 individually, but most potently of both collectively, partially reinstates the MRGPRX2 pathway, revealing that SCF and IL-4 make negative adjustments to the pseudo-allergic pathway. Under all conditions, the FcεRI-triggered route showed the inverse pattern of regulation, substantiating that allergic and pseudo-allergic MC activation can obey opposite rules, hinting at possible competition between them.


Subject(s)
Cell Degranulation/drug effects , Interleukin-4/pharmacology , Mast Cells/immunology , Nerve Tissue Proteins/genetics , Receptors, G-Protein-Coupled/genetics , Receptors, IgE/genetics , Receptors, Neuropeptide/genetics , Stem Cell Factor/pharmacology , Adaptive Immunity/genetics , Cell Degranulation/genetics , Cells, Cultured , Gene Expression/drug effects , Histamine/metabolism , Humans , Ligands , Male , Mast Cells/metabolism , Nerve Tissue Proteins/metabolism , RNA, Messenger/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, IgE/metabolism , Receptors, Neuropeptide/metabolism , Signal Transduction , Skin/cytology
17.
Eur J Immunol ; 46(7): 1727-36, 2016 07.
Article in English | MEDLINE | ID: mdl-27111856

ABSTRACT

Food allergy is a common health problem and can cause anaphylaxis. Avoidance of the offending food allergen is still the mainstay therapeutic approach. In this study, we investigated the role of plasma cell reduction by proteasome inhibition in a murine model of food allergy and examined the impact of this treatment on the systemic and local immune response. For this purpose, intestinal anaphylaxis was induced in BALB/c mice with the food allergen hazelnut, in conjunction with different adjuvants (alum and Staphylococcal enterotoxin B SEB) and different administration routes (oral and intraperitoneal). In both models, allergy symptoms were observed, but the clinical severity was more pronounced in the hazelnut-alum model than in the hazelnut-SEB model. Accordingly, allergen-specific immunoglobulin E (IgE) against hazelnut was detectable, and mast cell protease-1 in serum was increased after allergen provocation. Treatment with the proteasome inhibitor bortezomib reduced plasma cells and resulted in an abolishment of hazelnut allergen-specific IgE, which was associated with amelioration of clinical symptoms as well as a significant decrease in both CD19(+) and follicular B lymphocytes. Our data demonstrate the importance of allergen-specific IgE in food allergy and point to B cells as potential therapeutic targets for its treatment.


Subject(s)
Allergens/immunology , Anaphylaxis/immunology , Bortezomib/pharmacology , Corylus/adverse effects , Food Hypersensitivity/immunology , Intestines/immunology , Anaphylaxis/diagnosis , Anaphylaxis/drug therapy , Animals , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Bortezomib/administration & dosage , Disease Models, Animal , Female , Food Hypersensitivity/diagnosis , Food Hypersensitivity/drug therapy , Immunoglobulin E/blood , Immunoglobulin E/immunology , Intestines/drug effects , Intestines/pathology , Mice , Mice, Inbred BALB C , Proteasome Inhibitors/administration & dosage , Proteasome Inhibitors/pharmacology , Severity of Illness Index
18.
Exp Dermatol ; 26(11): 1136-1139, 2017 11.
Article in English | MEDLINE | ID: mdl-28418623

ABSTRACT

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.


Subject(s)
Gene Knockdown Techniques/methods , Mast Cells , RNA, Small Interfering/genetics , Cell Survival , Gene Expression , Humans , RNA Interference , Skin/cytology , Transfection
20.
Int Arch Allergy Immunol ; 172(1): 20-26, 2017.
Article in English | MEDLINE | ID: mdl-28219062

ABSTRACT

BACKGROUND: The clinical efficacy and safety of allergoid immunotherapy have been demonstrated in clinical trials. However, simultaneous monitoring of the immunological changes by allergoids versus allergens in the cells of the same individual has not been extensively performed, and the impact of concurrent Toll-like receptor 4 (TLR4) ligation has not been specified. METHODS: Three types of birch allergen were utilized: glutaraldehyde-treated allergoid (extract A), the same allergoid plus monophosphoryl lipid A (MPL), i.e., TLR4 ligand (extract A*), and native allergen (extract B). Antigen-specific responses after the in vitro stimulation of blood cells with the extracts were assessed by studying costimulatory receptors on the B cell surface by flow cytometry, cytokine responses by ELISA, and CD63 and CD203c upregulation (basophil activation test) in allergic versus nonallergic subjects. RESULTS: HLA-DR selectively increased upon allergen or allergoid treatment in the allergic group only. The extract types elicited similar cytokine responses, with IL-6 and IL-10 production detected only in certain atopic subjects. The allergoids revealed a strong reduction (100- to <10,000-fold) in basophil activation versus native allergen. Reactivity was undetectable in the basophils from nonallergic subjects. CONCLUSION: The allergenicity of the allergoid employed was sharply reduced when compared to the native allergen, while its immunogenicity was largely retained, especially in the presence of MPL. We also provide further evidence that allergic and nonallergic individuals show preexisting differences in their immune repertoires.


Subject(s)
Adjuvants, Immunologic/therapeutic use , Allergens/immunology , Betula/immunology , Desensitization, Immunologic/methods , Lipid A/analogs & derivatives , Plant Extracts/immunology , Pollen/immunology , Rhinitis, Allergic, Seasonal/immunology , Allergoids , Basophils/immunology , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , HLA-DR Antigens/immunology , Humans , Immunotherapy/methods , Interleukin-10/metabolism , Interleukin-6/metabolism , Lipid A/immunology , Lipid A/therapeutic use , Phosphoric Diester Hydrolases/biosynthesis , Pyrophosphatases/biosynthesis , Tetraspanin 30/biosynthesis , Toll-Like Receptor 4/immunology
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