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IgE-mediated degranulation of mast cells (MCs) provides rapid protection against environmental hazards, including animal venoms. A fraction of tissue-resident MCs intimately associates with blood vessels. These perivascular MCs were reported to extend projections into the vessel lumen and to be the first MCs to acquire intravenously injected IgE, suggesting that IgE loading of MCs depends on their vascular association. We sought to elucidate the molecular basis of the MC–blood vessel interaction and to determine its relevance for IgE-mediated immune responses. We selectively inactivated the Itgb1 gene, encoding the β1 chain of integrin adhesion molecules (ITGB1), in MCs by conditional gene targeting in mice. We analyzed skin MCs for blood vessel association, surface IgE density, and capability to bind circulating antibody specific for MC surface molecules, as well as in vivo responses to antigen administered via different routes. Lack of ITGB1 expression severely compromised MC–blood vessel association. ITGB1-deficient MCs showed normal densities of surface IgE but reduced binding of intravenously injected antibodies. While their capacity to degranulate in response to IgE ligation in vivo was unimpaired, anaphylactic responses to antigen circulating in the vasculature were largely abolished. ITGB1-mediated association of MCs with blood vessels is key for MC immune surveillance of blood vessel content, but is dispensable for slow steady-state loading of endogenous IgE onto tissue-resident MCs. [ABSTRACT FROM AUTHOR]

Mast cells (MCs) are capable of executing powerful inflammatory response programs triggered by surface IgE cross-linking or through pattern recognition receptors. The question of how MCs contribute to human disease has been intensely investigated and stimulated much controversy. Correlative evidence comes from human studies, pointing to pathogenetic or protective MC functions in patients with atopic conditions, autoimmune disorders, type 2 diabetes, chronic urticaria, mastocytosis, and cancer. Experiments in MC-deficient mice underpinned key roles for MCs in patients with IgE-mediated allergic conditions. Important pathogenetic MC contributions to other inflammatory and neoplastic conditions were suggested by studies in traditional KIT mutant MC-deficient mouse strains. However, many of these findings were not reproduced in more recently developed improved mouse models of MC deficiency, largely ruling out roles for MCs in mouse models for autoimmune disease, diabetes, and cancer. We discuss limitations of studies in mice and human subjects and provide suggestions for how they can be overcome, such as through the development of specific and selective MC-targeted treatments. [ABSTRACT FROM AUTHOR]

Background Histamine is a critical mediator of IgE/mast cell–mediated anaphylaxis. Histamine is synthesized by decarboxylating the amino acid histidine, a reaction catalyzed by the histidine decarboxylase (Hdc) gene–encoded enzyme HDC. However, regulation of the Hdc gene in mast cells is poorly understood. Objective We sought to investigate the in vivo regulation of IgE/mast cell–mediated anaphylaxis by the transcription factors GATA2 and microphthalmia-associated transcription factor (MITF) and the mechanisms by which GATA2 and MITF regulate Hdc gene expression in mouse and human mast cells. Methods Mice deficient in the transcription factors Gata2 , aryl hydrocarbon receptor (Ahr), aryl hydrocarbon receptor repressor (Ahrr), or basic helix-loop-helix family member E40 (Bhlhe40) were assessed for anaphylactic reactions. Chromatin immunoprecipitation sequencing analysis identified putative Hdc enhancers. Luciferase reporter transcription assay confirmed enhancer activities of putative enhancers in the Hdc gene. The short hairpin RNA knockdown approach was used to determine the role of MITF in regulating mouse and human HDC gene expression. Results Connective tissue mast cell–specific Gata2 -deficient mice did not have IgE/mast cell-mediated anaphylaxis. GATA2 induced the expression of Mitf , Ahr , Ahrr , and Bhlhe40 in mast cells. MITF, but not AHR, AHRR, or BHLHE40, was required for anaphylaxis. MITF bound to an enhancer located 8.8 kb upstream of the transcription start site of the Hdc gene and directed enhancer activity. MITF overexpression largely restored Hdc gene expression in the Gata2 -deficient mast cells. In the human mast cell line LAD2, MITF was required for the HDC gene expression and histamine synthesis. Conclusion The transcription factors GATA2 and MITF regulate Hdc gene expression in mast cells and are required for IgE/mast cell–mediated anaphylaxis. Graphical abstract [ABSTRACT FROM AUTHOR]

All three members of the Orai family of cation channels–Orai1, Orai2 and Orai3–are integral membrane proteins that can form store-operated Ca 2+ channels resembling endogenous calcium release-activated channels (CRAC) in many aspects. Loss of function studies in human and murine models revealed many functions of Orai1 proteins not only for Ca 2+ homeostasis, but also for cellular and systemic functions in many cell types. By contrast, the knowledge regarding the contribution of Orai2 and Orai3 proteins in these processes is sparse. In this study, we report the generation of mouse models with targeted inactivation of the Orai2 gene to study Orai2 function in peritoneal mast cells (PMC), a classical cell model for CRAC channels and Ca 2+ -dependent exocytosis of inflammatory mediators. We show that the Ca 2+ rise triggered by agonists acting on high-affinity Fc receptors for IgE or on MAS-related G protein-coupled receptors is significantly increased in Orai2-deficient mast cells. Ca 2+ entry triggered by depletion of intracellular stores (SOCE) is also increased in Orai2 −/− PMCs at high (2 mM) extracellular Ca 2+ concentration, whereas SOCE is largely reduced upon re-addtion of lower (0.1 mM) Ca 2+ concentration. Likewise, the density of CRAC currents, Ca 2+ -dependent mast cell degranulation, and mast cell-mediated anaphylaxis are intensified in Orai2-deficient mice. These results show that the presence of Orai2 proteins limits receptor-evoked Ca 2+ transients, store-operated Ca 2+ entry (SOCE) as well as degranulation of murine peritoneal mast cells but also raise the idea that Orai2 proteins contribute to Ca 2+ entry in connective tissue type mast cells in discrete operation modes depending on the availability of calcium ions in the extracellular space. [ABSTRACT FROM AUTHOR]

High numbers of mast cells populate the stroma of many types of neoplasms, including human papilloma virus-induced benign and malignant tumors in man and mouse. Equipped with numerous pattern recognition receptors and capable of executing important pro-inflammatory responses, mast cells are considered innate sentinels that significantly impact tumor biology. Mast cells were reported to promote human papilloma virus (HPV)-induced epithelial hyperproliferation and neo-angiogenesis in an HPV-driven mouse model of skin cancer. We analyzed HPV-induced epithelial hyperplasia and squamous cell carcinoma formation, as well as growth of tumors inoculated into the dermis, in mice lacking skin mast cells. Unexpectedly, the absence of mast cells had no effect on HPV-induced epithelial growth or angiogenesis, on growth kinetics of inoculated tumors, or on the immunological tumor micro-milieu. Thus, the conspicuous recruitment of mast cells into tumor tissues cannot necessarily be equated with important mast cell functions in tumor growth. [ABSTRACT FROM AUTHOR]

Summary The immunoglobulin E (IgE)-mediated mast cell (MC) response is central to the pathogenesis of type I allergy and asthma. IκB kinase 2 (IKK2) was reported to couple IgE-induced signals to MC degranulation by phosphorylating the SNARE protein SNAP23. We investigated MC responses in mice with MC-specific inactivation of IKK2 or NF-κB essential modulator (NEMO), or animals with MC-specific expression of a mutant, constitutively active IKK2. We show that the IgE-induced late-phase cytokine response is reduced in mice lacking IKK2 or NEMO in MCs. However, anaphylactic in vivo responses of these animals are not different from those of control mice, and in vitro IKK2-deficient MCs readily phosphorylate SNAP23 and degranulate similarly to control cells in response to allergen or calcium ionophore. Constitutive overactivation of the NF-κB pathway has only slight effects on allergen-triggered MC responses. Thus, IKK2 is dispensable for MC degranulation, and the important question how IgE-induced signals trigger MC vesicle fusion remains open. [ABSTRACT FROM AUTHOR]

Summary: Atopic dermatitis (AD) is a chronic inflammatory skin disease. Here, we show that phospholipase C-β3 (PLC-β3)-deficient mice spontaneously develop AD-like skin lesions and more severe allergen-induced dermatitis than wild-type mice. Mast cells were required for both AD models and remarkably increased in the skin of Plcb3 −/− mice because of the increased Stat5 and reduced SHP-1 activities. Mast cell-specific deletion of Stat5 gene ameliorated allergen-induced dermatitis, whereas that of Shp1 gene encoding Stat5-inactivating SHP-1 exacerbated it. PLC-β3 regulates the expression of periostin in fibroblasts and TSLP in keratinocytes, two proteins critically involved in AD pathogenesis. Furthermore, polymorphisms in PLCB3, SHP1, STAT5A, and STAT5B genes were associated with human AD. Mast cell expression of PLC-β3 was inversely correlated with that of phospho-STAT5, and increased mast cells with high levels of phospho-STAT5 were found in lesional skin of some AD patients. Therefore, STAT5 regulatory mechanisms in mast cells are important for AD pathogenesis. [Copyright &y& Elsevier]

Summary: Aicardi-Goutières syndrome (AGS), a hereditary autoimmune disease, clinically and biochemically overlaps with systemic lupus erythematosus (SLE) and, like SLE, is characterized by spontaneous type I interferon (IFN) production. The finding that defects of intracellular nucleases cause AGS led to the concept that intracellular accumulation of nucleic acids triggers inappropriate production of type I IFN and autoimmunity. AGS can also be caused by defects of SAMHD1, a 3′ exonuclease and deoxynucleotide (dNTP) triphosphohydrolase. Human SAMHD1 is an HIV-1 restriction factor that hydrolyzes dNTPs and decreases their concentration below the levels required for retroviral reverse transcription. We show in gene-targeted mice that also mouse SAMHD1 reduces cellular dNTP concentrations and restricts retroviral replication in lymphocytes, macrophages, and dendritic cells. Importantly, the absence of SAMHD1 triggered IFN-β-dependent transcriptional upregulation of type I IFN-inducible genes in various cell types indicative of spontaneous IFN production. SAMHD1-deficient mice may be instrumental for elucidating the mechanisms that trigger pathogenic type I IFN responses in AGS and SLE. [Copyright &y& Elsevier]

Abstract: Social identity theory has been applied to many organizational contexts, including family businesses. However, the current study is one of the first to explore the organizational identification of non-family member employees. Based on previous research, it seems likely that, for family business employees, organizational identification mediates the relationship between organizational justice, homophily, and commitment. This study proposes a model of identification for family business employees based on these considerations. Although the current study did not confirm the proposed model, an alternative model is discussed. [ABSTRACT FROM AUTHOR]

Summary: Students admitted to the solo singing education at the University of Music Dresden, Germany have been submitted to a detailed physical examination of a variety of factors with relevance to voice function since 1959. In the years 1959–1991, this scheme of examinations included X-ray profiles of the singers'' vocal tracts. This material of 132 X-rays of voice professionals was used to investigate different laryngeal morphological measures and their relation to vocal fold length. Further, the study aimed to investigate if there are consistent anatomical differences between singers of different voice classifications. The study design used was a retrospective analysis. Vocal fold length could be measured in 29 of these singer subjects directly. These data showed a strong correlation with the anterior-posterior diameter of the subglottis and the trachea as well as with the distance from the anterior contour of the thyroid cartilage to the anterior contour of the spine. These relations were used in an attempt to predict the 132 singers'' vocal fold lengths. The results revealed a clear covariation between predicted vocal fold length and voice classification. Anterior-posterior subglottic-tracheal diameter yielded mean vocal fold lengths of 14.9, 16.0, 16.6, 18.4, 19.5, and 20.9mm for sopranos, mezzo-sopranos, altos, tenors, baritones, and basses, respectively. The data support the assumption that there are consistent anatomical laryngeal differences between singers of different voice classifications, which are of relevance to pitch range and timbre of the voice. [Copyright &y& Elsevier]

Wound healing is a coordinated process that initially relies on pro-inflammatory macrophages, followed by a pro-resolution function of these cells. Changes in cellular metabolism likely dictate these distinct activities, but the nature of these changes has been unclear. Here, we profiled early- versus late-stage skin wound macrophages in mice at both the transcriptional and functional levels. We found that glycolytic metabolism in the early phase is not sufficient to ensure productive repair. Instead, by combining conditional disruption of the electron transport chain with deletion of mitochondrial aspartyl-tRNA synthetase, followed by single-cell sequencing analysis, we found that a subpopulation of early-stage wound macrophages are marked by mitochondrial ROS (mtROS) production and HIF1α stabilization, which ultimately drives a pro-angiogenic program essential for timely healing. In contrast, late-phase, pro-resolving wound macrophages are marked by IL-4Rα-mediated mitochondrial respiration and mitohormesis. Collectively, we identify changes in mitochondrial metabolism as a critical control mechanism for macrophage effector functions during wound healing. [Display omitted] • Early-stage, inflammatory versus late-stage, pro-resolving wound macrophages are profiled • Pro-inflammatory wound macrophages are marked by mtROS production and HIF1α stabilization • This molecular process is required for proper vascularization during wound repair • Pro-resolving macrophages are marked by mitochondrial respiration and mitohormesis How mitochondrial metabolism contributes to the early-stage, pro-inflammatory versus late-stage, pro-resolution functions of macrophages during wound healing requires further investigation. Here, Willenborg et al. show that effective wound healing requires the production of mtROS in early-stage wound macrophages to promote proper vascularization, while late-stage wound macrophages are dependent on OXPHOS and mitohormesis. [ABSTRACT FROM AUTHOR]

Pro-inflammatory caspase-1 is a key player in innate immunity. Caspase-1 processes interleukin (IL)-1β and IL-18 to their mature forms and triggers pyroptosis. These caspase-1 functions are linked to its enzymatic activity. However, loss-of-function missense mutations in CASP1 do not prevent autoinflammation in patients, despite decreased IL-1β production. In vitro data suggest that enzymatically inactive caspase-1 drives inflammation via enhanced nuclear factor κB (NF-κB) activation, independent of IL-1β processing. Here, we report two mouse models of enzymatically inactive caspase-1-C284A, demonstrating the relevance of this pathway in vivo. In contrast to Casp1 −/− mice, caspase-1-C284A mice show pronounced hypothermia and increased levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-6 when challenged with lipopolysaccharide (LPS). Caspase-1-C284A signaling is RIP2 dependent and mediated by TNF-α but independent of the NLRP3 inflammasome. LPS-stimulated whole blood from patients carrying loss-of-function missense mutations in CASP1 secretes higher amounts of TNF-α. Taken together, these results reveal non-canonical caspase-1 signaling in vivo. • Enzymatically inactive caspase-1-C284A induces pro-inflammatory signaling in vivo • Caspase-1-C284A-mediated inflammation requires RIP2-dependent TNF-α secretion • Caspase-1-C284A signaling is independent from NLRP3 inflammasome and IL-1 cytokines Reinke et al. show that enzymatically inactive caspase-1-C284A mediates non-canonical caspase-1 signaling. This pathway is RIP2 dependent and mediated by TNF-α but independent from IL-1 cytokines. [ABSTRACT FROM AUTHOR]

Perturbation of epidermal barrier formation will profoundly compromise overall skin function, leading to a dry and scaly, ichthyosis-like skin phenotype that is the hallmark of a broad range of skin diseases, including ichthyosis, atopic dermatitis, and a multitude of clinical eczema variants. An overarching molecular mechanism that orchestrates the multitude of factors controlling epidermal barrier formation and homeostasis remains to be elucidated. Here we highlight a specific role of mammalian target of rapamycin complex 2 (mTORC2) signaling in epidermal barrier formation. Epidermal mTORC2 signaling was specifically disrupted by deleting rapamycin-insensitive companion of target of rapamycin (Rictor) , encoding an essential subunit of mTORC2 in mouse epidermis (epidermis-specific homozygous Rictor deletion [RicEKO] mice). Epidermal structure and barrier function were investigated through a combination of gene expression, biochemical, morphological and functional analysis in RicEKO and control mice. RicEKO newborns displayed an ichthyosis-like phenotype characterized by dysregulated epidermal de novo lipid synthesis, altered lipid lamellae structure, and aberrant filaggrin (FLG) processing. Despite a compensatory transcriptional epidermal repair response, the protective epidermal function was impaired in RicEKO mice, as revealed by increased transepidermal water loss, enhanced corneocyte fragility, decreased dendritic epidermal T cells, and an exaggerated percutaneous immune response. Restoration of Akt-Ser473 phosphorylation in mTORC2-deficient keratinocytes through expression of constitutive Akt rescued FLG processing. Our findings reveal a critical metabolic signaling relay of barrier formation in which epidermal mTORC2 activity controls FLG processing and de novo epidermal lipid synthesis during cornification. Our findings provide novel mechanistic insights into epidermal barrier formation and could open up new therapeutic opportunities to restore defective epidermal barrier conditions. [ABSTRACT FROM AUTHOR]

Background: Studies with c-kit mutant mast cell (MC)–deficient mice and antibody-mediated depletion of basophils suggest that both MCs and basophils can contribute to peanut-induced anaphylaxis (PIA). However, interpretation of data obtained by using such approaches is complicated because c-kit mutant mice have several phenotypic abnormalities in addition to MC deficiency and because basophil-depleting antibodies can also react with MCs. Objective: We analyzed (1) the changes in the features of PIA in mice after the selective and inducible ablation of MCs or basophils and (2) the possible importance of effector cells other than MCs and basophils in the PIA response. Methods: Wild-type and various mutant mice were orally sensitized with peanut extract and cholera toxin weekly for 4 weeks and challenged intraperitoneally with peanut extract 2 weeks later. Results: Peanut-challenged, MC-deficient Kit W-sh/W-sh mice had reduced immediate hypothermia, as well as a late-phase decrease in body temperature that was abrogated by antibody-mediated depletion of neutrophils. Diphtheria toxin–mediated selective depletion of MCs or basophils in Mcpt5-Cre;iDTR and Mcpt8 DTR mice, respectively, and treatment of wild-type mice with the basophil-depleting antibody Ba103 significantly reduced peanut-induced hypothermia. Non–c-kit mutant MC- and basophil-deficient Cpa3-Cre;Mcl-1 fl/fl mice had reduced but still significant responses to peanut. Conclusion: Inducible and selective ablation of MCs or basophils in non–c-kit mutant mice can significantly reduce PIA, but partial responses to peanut can still be observed in the virtual absence of both cell types. The neutrophilia in Kit W-sh/W-sh mice might influence the responses of these mice in this PIA model. [Copyright &y& Elsevier]

Background & Aims: Chronic infection with the bacterial pathogen Helicobacter pylori causes gastric disorders, ranging from chronic gastritis to gastric adenocarcinoma. Only a subset of infected persons will develop overt disease; most remains asymptomatic despite lifelong colonization. This study aims to elucidate the differential susceptibility to H pylori that is found both across and within populations. Methods: We have established a C57BL/6 mouse model of H pylori infection with a strain that is capable of delivering the virulence factor cytotoxin-associated gene A (CagA) into host cells through the activity of a Cag-pathogenicity island–encoded type IV secretion system. Results: Mice infected at 5–6 weeks of age with CagA+ H pylori rapidly develop gastritis, gastric atrophy, epithelial hyperplasia, and metaplasia in a type IV secretion system–dependent manner. In contrast, mice infected during the neonatal period with the same strain are protected from preneoplastic lesions. Their protection results from the development of H pylori–specific peripheral immunologic tolerance, which requires transforming growth factor-β signaling and is mediated by long-lived, inducible regulatory T cells, and which controls the local CD4+ T-cell responses that trigger premalignant transformation. Tolerance to H pylori develops in the neonatal period because of a biased ratio of T-regulatory to T-effector cells and is favored by prolonged low-dose exposure to antigen. Conclusions: Using a novel CagA+ H pylori infection model, we report here that the development of tolerance to H pylori protects from gastric cancer precursor lesions. The age at initial infection may thus account for the differential susceptibility of infected persons to H pylori–associated disease manifestations. [Copyright &y& Elsevier]

Conclusive evidence for the impact of mast cells (MCs) in skin repair is still lacking. Studies in mice examining the role of MC function in the physiology and pathology of skin regenerative processes have obtained contradictory results. To clarify the specific role of MCs in regenerative conditions, here we used a recently developed genetic mouse model that allows conditional MC ablation to examine MC-specific functions in skin. This mouse model is based on the cell type-specific expression of Cre recombinase in connective tissue-type MCs under control of the Mcpt5 promoter and the Cre-inducible diphtheria toxin receptor-mediated cell lineage ablation by diphtheria toxin. In response to excisional skin injury, genetic ablation of MCs did not affect the kinetics of reepithelialization, the formation of vascularized granulation tissue, or scar formation. Furthermore, genetic ablation of MCs failed to prevent the development of skin fibrosis upon bleomycin challenge. The amount of deposited collagen and the biochemistry of collagen fibril crosslinks within fibrotic lesions were comparable in MC-depleted and control mice. Collectively, our findings strongly suggest that significant reduction of MC numbers does not affect skin wound healing and bleomycin-induced fibrosis in mice, and provide to our knowledge previously unreported insight in the long-debated contribution of MCs in skin regenerative processes. [ABSTRACT FROM AUTHOR]

The article discusses a study on the deregulated type 1 IFN response in TREX1-associated familial chilblain lupus. The study looks into the role of type I IFN in the pathogenesis of SLE and AGS. It also reveals that the finding of spontaneous type 1 IFN-dependent cutaneous pathology in TREX1-deficient mice illustrates common pathogenetic pathways in cutaneous as well as systemic TREX1-associated lupus erythematosus.

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy and, in most cases, is of pro- or pre-B cell origin (B-ALL). The receptor tyrosine kinase KIT is expressed by hematopoietic stem and precursor cells. Gain-of-function mutations of KIT cause systemic mastocytosis, which is characterized by abnormal accumulations of mast cells. We previously reported a mouse model of mastocytosis based on conditional expression of a constitutively active Kit protein. Half of these animals developed leukemic disease of B-lineage origin. Herein, we report that this condition bears striking similarities to human B-ALL. The immuno-phenotype of the leukemic cells was compatible with a pro-B-cell origin, as was the finding of immunoglobulin heavy-chain gene rearrangements in all cases, whereas light-chain loci were mostly not rearranged. Leukemogenesis was independent of pre-B-cell receptor expression. Primary leukemic cells and permanent cell lines derived from these were serially transplantable and rapidly killed the recipients. In a few animals, the leukemia was of T-cell origin with abnormal CD4/8 double-positive T-cell precursors dominating in the circulation. In summary, we report a novel ALL mouse model that may prove useful for in vivo drug testing and identification of novel oncogenic mutations and principles. [ABSTRACT FROM AUTHOR]

Aim NMDP high resolution (HR) testing programs require typing for common or well documented (CWD) null alleles in alignment with Mack et al., Tissue Antigens . 2013: 81(4): 194–203 (CWD 2.0). The aim of this study was to determine the frequency of CWD 2.0 Class I null alleles in NMDP HR typing programs and whether these alleles occur outside of their expected haplotypes. Methods Labs performing HR typing routinely test exons 2–4, then additional regions depending on where a CWD null allele polymorphism is located. Typing methods include SSO, SBT, and NGS. HR Class I typing results from historic adult donor registry samples and CBU samples performed in 2013–2015 were queried for reports of CWD null alleles. Results Four different Class I CWD null alleles were reported a total of 35 times in 41,864 tests. B∗51:11N is expected in the A∗02∼DRB1∗04 haplotype but was observed in A∗03:01∼C∗07:02∼DRB1∗04:02. C∗04:09N was observed with its most common haplotype A∗23:01∼B∗44:03∼DRB1∗07:01 in 53% of cases and with B∗44 in 100% of cases. The common A∗02:53N was not reported; it occurs in up to 0.10% of Chinese populations. The remainder of the CWD nulls were not observed. G group frequency is the null allele frequency within the two digit G group typing. Conclusion Although the frequency of Class I CWD null alleles in the queried HLA results is low, they may warrant continued routine typing. Failure to identify a null allele results in a hidden mismatch between donor and patient. However, lab costs increase due to the additional typing required to detect polymorphisms located outside of exons 2–3. As exon 7 is not routinely tested, typing for C∗04:09N only in the presence of B∗44 may be a future consideration for NMDP HR contracts and policies. [ABSTRACT FROM AUTHOR]