Mast Cell Differentiation Research Articles

Identification of Biological and Pharmaceutical Mast Cell‐ and Basophil‐Related Targets

Identification of Biological and Pharmaceutical Mast Cell‐ and Basophil‐Related Targets

TET2 Regulates Mast Cell Differentiation and Proliferation through Catalytic and Non-catalytic Activities

Dioxygenases of the TET family impact genome functions by converting 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC). Here, we identified TET2 as a crucial regulator of mast cell differentiation and proliferation. In the absence of TET2, mast cells showed disrupted gene expression and altered genome-wide 5hmC deposition, especially at enhancers and in the proximity of downregulated genes. Impaired differentiation of Tet2-ablated cells could be relieved or further exacerbated by modulating the activity of other TET family members, and mechanistically it could be linked to the dysregulated expression of C/EBP family transcription factors. Conversely, the marked increase in proliferation induced by the loss of TET2 could be rescued exclusively by re-expression of wild-type or catalytically inactive TET2. Our data indicate that, in the absence of TET2, mast cell differentiation is under the control of compensatory mechanisms mediated by other TET family members, while proliferation is strictly dependent on TET2 expression.

GATA2 is critical for mast cell differentiation and maintenance

Abstract The GATA binding protein 2 (GATA2) plays critical roles in hematopoietic stem cell survival and proliferation, granulocyte–monocyte progenitor differentiation, and basophil and mast cell differentiation. However, precise roles of GATA2 in mast cell differentiation and maintenance have not been delineated. We identified GATA2 is essential for pre-basophil and mast cell progenitors (pre-BMPs) to differentiate into mast cells. We found that GATA2 is critical in maintaining the expression of a wide range of genes that are important for performing mast cell functions. To determine the in vivo role of GATA2 in mast cell development, we generated connective tissue mast cell-specific Gata2 deficient mice. We found that Gata2 was required for connective tissue mast cell development. These mice failed to develop IgE-mediated anaphylaxis. GATA2 regulated the expression of Hdc gene, which is critical for IgE-mediated anaphylaxis, by binding to regulatory regions of the Hdc gene to promote its transcription. Our study reveals GATA2 is critical for mast cell differentiation and maintenance.

Transcriptional regulation of mast cell and basophil lineage commitment

Basophils and mast cells have long been known to play critical roles in allergic disease and in immunity against parasitic infection. Accumulated evidence also supports that basophils and mast cells have important roles in immune regulations, host defense against bacteria and viruses, and autoimmune diseases. However, origin and molecular regulation of basophil and mast cell differentiation remain incompletely understood. In this review, we focus on recent advances in the understanding of origin and molecular regulation of mouse and human basophil and mast cell development. A more complete understanding of how basophils and mast cells develop at the molecular level will lead to development of interventions that are more effective in achieving long-term success.

Mast cell differentiation: still open questions?

In this issue of Blood, Dahlin et al report on a minor circulating human mast cell (MC) progenitor cell population (lineage-negative [Lin−]/CD34hi/CD117int/hi/high-affinity immunoglobulin E receptor-positive [FcεRI+]), with an immature MC-like appearance, which is present in the peripheral blood (PB) of healthy individuals and of asthma subjects well controlled by treatment or with reduced lung function.

Wnt-β-Catenin Signaling Promotes the Maturation of Mast Cells.

Mast cells play an important role in the pathogenesis of allergic diseases. Immature mast cells migrate into peripheral tissues from the bone marrow and undergo complete maturation. Interestingly, mast cells have characteristics similar to hematopoietic stem cells (HSCs), such as self-renewal and c-kit expression. In HSCs, Wnt signaling is involved in their maintenance and differentiation. On the other hand, the relation between Wnt signaling and mast cell differentiation is poorly understood. To study whether Wnt signals play a role in the maturation of mast cells, we studied the effect of Wnt proteins on mast cell maturation of bone marrow-derived mast cells (BMMCs). The expression levels of CD81 protein and histidine decarboxylase mRNA and activity of mast cell-specific protease were all elevated in BMMCs treated with Wnt5a. In addition, Wnt5a induced the expression of Axin2 and TCF mRNA in BMMCs. These results showed that Wnt5a could promote the maturation of mast cells via the canonical Wnt signaling pathway and provide important insights into the molecular mechanisms underlying the differentiation of mast cells.

Novel characterisation of mast cell phenotypes from peripheral blood mononuclear cells in chronic fatigue syndrome/myalgic encephalomyelitis patients.

Mast cells (MCs) mediate inflammation through neuropeptides and cytokines, along with histamine and reactive oxygen species (ROS). Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) is an illness characterized by an unexplained disabling fatigue with multiple physiological impairments as well as dysregulated cytokine profiles. To determine mast cell phenotypes in isolated human PBMCs, in healthy controls and in CFS/ME patients. Second, determine receptor expression of RAGE and its ligand high mobility group box 1 protein (HMGB1). Moderately severe CFS/ME patients (n=12, mean age 39.25 ± SD3.52 years), severe CFS/ME patients (n=6, mean age 43.00 ± SD4.02 years) and healthy controls (n=13, mean age 42.69 ± SD3.87 years) were included in this study. CFS/ME patients were classified according to the 2011 International Consensus Criteria. LSRFortessa X-20 Flow cytometry was used for the identification of phenotypic peripheral mast cell population in PBMCs using an exclusion marker Lin2 cocktail (anti-CD3, anti-CD14, anti-CD19, anti-CD20 and anti-CD56) and inclusion markers (CD117, CD34, FCεRI, chymase, HLA-DR and CD154) following comparative investigation. HMGB1 and soluble RAGE expression in plasma was measured by sandwich ELISA assay. There was a significant increase in CD117⁺CD34⁺FCεRI-chymase- mast cell populations in moderate and severe CFS/ME patients compared with healthy controls. There was a significant increase in CD40 ligand and MHC-II receptors on differentiated mast cell populations in the severe CFS/ME compared with healthy controls and moderate CFS/ME. There were no significant differences between groups for HMGB1 and sRAGE. This preliminary study investigates mast cell phenotypes from PBMCs in healthy controls. We report significant increase of naïve MCs in moderate and severe CFS/ME patients compared with healthy controls. Moreover, a significant increase in CD40 ligand and MHC-II receptors on differentiated mast cells in severe CFS/ME patients. Peripheral MCs may be present in CFS/ME pathology however, further investigation to determine their role is required.

Novel Functional Roles for Ten-Eleven-Translocation 2 (Tet2) in Normal and Leukemic Growth of Mast Cells

KIT receptor signaling plays an important role in mast cell development. Gain-of-function mutations in KIT receptor have been identified in human diseases including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM) and acute myeloid leukemia (AML). Although KIT mutations found in GIST are sensitive to imatinib, KIT mutation (KITD816V) found in 90% of SM patients is imatinib-resistant and currently no therapies are available to treat the human diseases associated with this mutation. Our recent studies have identified Ten-Eleven-Translocation 2 (TET2) mutations in ~23% of SM patients and are associated with poor prognosis and overall survival. TET2 is a methylcytosine dioxygenase that plays a vital role in active DNA demethylation. Recent studies suggest that patients with mutations in TET2 and KITD816V develop more aggressive form of mastocytosis with worse prognosis. Although it is known that TET2 and KITD816V cooperate in SM patients, it is not clear how they cooperate with each other and what is the physiologic role of TET2 in normal mast cell development. We show that loss of Tet2 results in impaired maturation of mast cells in vivo and in bone marrow-derived mast cells (BMMC) compared to WT controls, which is associated with reduction in 5-hmc levels compared to WT BMMCs. We also observed reduction in the expression of mast cell-specific genesincluding Mast cell proteinase-5 (MCP-5), Mast cell proteinase-6 (MCP-6) and Carboxypeptidase A (CPA). To determine the mechanism behind altered mast cell differentiation in Tet2-/- BMMCs, we performed RNA-seq analysis in WT and Tet2-/- mast cells and observed altered expression of various genes involved in development of mast cells including Kit, FcεR1, Mitf, Notch, and Myc. We further confirmed altered expression of Mitf, Gata-2, and PU.1 in Tet2-/- BMMCs compared to WT BMMCs by western blotting. Since Tet2 regulates DNA demethylation, we tested whether altered BMMC differentiation in Tet2-/- mice is due to enhanced DNA methylation. We treated WT or Tet2-/- BM cells for 3 weeks with vehicle or 5-azacytidine (hypomethylating agent) and analyzed mast cell differentiation. Treatment with 5-azacytidine completely corrected the defective mast cell differentiation in Tet2-/- cells to WT levels. These results suggest that Tet2 plays a significant role in mast cell differentiation by regulating the expression of critical transcription factors including Mitf, Gata-2 and PU.1. We next analyzed the growth of Tet2-/- BMMCs in response to cytokines. Tet2-deficient BMMCs show enhanced cytokine mediated growth compared to WT BMMCs. Hyper-proliferation of Tet2-/- BMMCs is associated with reduced expression of tumor suppressor, PTEN, whose promoter is hypermethylated and a concomitant increase in the activation of the PI3K/AKT pathway. Since loss of function TET2 mutations have been observed in SM patients in addition to KITD816V mutation, we tested whether loss of Tet2 cooperates with KIT mutation in vitro and in vivo. Tet2-deficiency or knockdown in conjunction with the expression of KIT mutation resulted in significantly enhanced growth compared to cells bearing KIT mutation alone or lacking Tet2 expression. Likewise in human mastocytosis xenograft model, significantly enlarged tumors were observed in NSG mice transplanted with human mastocytosis cell line bearing the KITD816V mutation (HMC1.2) and knockdown of TET2 compared to HMC1.2 cells bearing only the KITD816V mutation. The cooperation between loss of Tet2 and KIT mutation was associated with further increase in PI3K/AKT activation and pharmacologic inhibitor treatment with a PI3K inhibitor GDC-0941 (Pan PI3K), but not TGX221 (p110β-specific) or IC87114 (p110δ-specific), significantly reduced the hyper-proliferation of Tet2-/- BMMCs and cell lines as well as primary BM blasts derived from SM patients bearing the KITD816V mutation. Consistently, combined loss of p110α and p110δ subunits of PI3K resulted in the most profound growth repression in oncogenic KIT bearing BM cells, but did not correct altered differentiation in Tet2-/- BMMCs. Taken together our results suggest that combinational therapy involving 5-azacytidine (which corrects the impaired mast cell differentiation) and PI3K inhibitor (which corrects the excessive proliferation) is a better therapeutic option for treating human mastocytosis patients bearing TET2 and KIT mutations. DisclosuresNo relevant conflicts of interest to declare.

A Constitutively-Active ABL Family Kinase, TEL-ARG, Induces Lethal Mastocytosis through Sensitizing Hematopoietic Stem/Progenitor Cells to IL-3

ABL family kinases, ABL1 (ABL) and ABL2 (ARG), share functional domains such as SH2-, SH3- and kinase domains, and are highly homologous except their C-terminal domain. Fusions to TEL (ETV6), TEL-ABL and TEL-ARG, are constitutively-active kinases and have been reported in rare cases of human CML, AML or ALL. Although TEL-ABL is involved in leukemogenesis, the role of TEL-ARG has not been elucidated because this fusion protein has been always accompanied with other major translocations, such as PML-RARα.We have previously shown that although their kinase activities are comparable, TEL-ABL strongly transforms Ba/F3 cells, while TEL-ARG has a much lower transforming activity, and these differences are attributed to their distinct C-terminal domain (Okuda K and Hirai H, Open Journal of Blood Diseases 2013). At the last ASH annual meeting, we have shown that TEL-ABL induces myeloid leukemia in a short latency, whereas TEL-ARG induces lethal mastocytosis in a long latency in a mouse bone marrow (BM) transplantation model (Abstract number #2368, ASH 2014). Here we investigated the clonogenicity of mastocytosis and explored the detailed mechanism underlying the onset of mastocytosis induced by TEL-ARG.First, we performed a serial transplantation experiment to evaluate mastocytosis-initiating capacity of TEL-ARG-expressing cells. Hematopoietic stem/progenitor cells (HSPCs) from 5-FU-treated mice were retrovirally transduced with TEL-ARG and transplanted to the first recipient mice. BM cells from moribund mice due to mastocytosis were transplanted to the sublethally irradiated second recipients. On day 219 after transplantation, we detected mast cells circulating in the peripheral blood of these two recipients, and observed severe pancytopenia and body weight loss in one of them. In this mouse, mast cells engulfing blood cells were accumulated in the BM and spleen, and subcutaneous tissues were massively infiltrated by mast cells, all of which were characteristics of mastocytosis observed in the first recipients. These results indicate that TEL-ARG confers mastocytosis-initiating capacity on HSPCs.Next, we focused on the mechanisms why TEL-ARG induces mastocytosis, whereas TEL-ABL induces myeloid leukemia. HSPCs from 5-FU-treated mice were retrovirally transduced with TEL-ABL or TEL-ARG, and subjected to the in vitro mast cell differentiation assay in the presence of WEHI-conditioned medium, as a source of IL-3 (Figure). IL-3 enhanced differentiation and proliferation of empty-virus-transduced HSPCs toward mast cells in a dose-dependent manner. TEL-ARG induced mast cell differentiation in the absence of IL-3 to some extent, and IL-3 markedly increased mast cell number even at a lower concentration. TEL-ARG-expressing mast cells continue to proliferate for more than 4 months maintaining their phenotype as mast cells. In contrast, IL-3 did not enhance mast cell differentiation but support myeloid differentiation of TEL-ABL-expressing HSPCs. These data suggest that while TEL-ABL induces myeloid differentiation, TEL-ARG strongly promotes differentiation toward mast cells through sensitizing HSPCs to IL-3, an important factor for differentiation, survival and proliferation of mast cells. Furthermore, these results might account for differences in the phenotypes of diseases induced by TEL-ABL (myeloid leukemia) or TEL-ARG (mastocytosis).In conclusions, TEL-ABL strongly induces myeloid-skewed differentiation, whereas TEL-ARG promotes mast cell differentiation through increasing sensitivity to IL-3 and induces clonal mast cell disease. We are currently investigating the molecular mechanisms by which they activate distinct differentiation pathways toward myeloid cells or mast cells. We believe that further exploration of the underlying mechanisms will deepen our understanding of the molecular basis for ABL kinase-mediated leukemogenesis as well as mast cell disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Editorial introductions

Editorial introductions

이중배양법에 따른 Lactococcus lactis의 아토피 유발인자 억제 효과 증대

We analyzed whether lactic acid bacteria could control the expression of IL-4 and IL-13 in activated mast cells and whether these bacteria could inhibit the activity of transcription factors such as GATA-1, GATA-2, NF-AT1, NF-AT2, and NF-κB p65. We previously described a technique for identification of lactic acid bacteria with anti-atopy functionality by confirming increased expression of CD4+/CD25+/foxp3+ in T cells. We also confirmed that a double-culture method increased the antibacterial activity of these lactic acid bacteria against Staphylococcus aureus (S. aureus). In the present study, we characterized the effect of lactic acid bacteria cultured by this double-culture method on inhibition of allergic inflammatory reactions of RBL-2H3 mast cells, a cellular model of atopic dermatitis. The strongest anti-allergic effects of the lactic acid bacteria were seen in the following order: Lactococcus lactis broth cultured with medium containing Lactobacillus plantarum culture supernatant > Lc. lactis > Lc. lactis broth cultured with medium containing Lb. plantarum culture supernatant > Lb. plantarum. Thus, Lc. lactis cultured in medium containing Lb. plantarum culture supernatant had the strongest inhibitory effect on the differentiation of mast cells during allergic reactions, which may be mediated through the selective regulation of expression of relevant genes.

GATA2 is critical for the maintenance of cellular identity in differentiated mast cells derived from mouse bone marrow

AbstractGATA2 plays a crucial role for the mast cell fate decision. We herein demonstrate that GATA2 is also required for the maintenance of the cellular identity in committed mast cells derived from mouse bone marrow (BMMCs). The deletion of the GATA2 DNA binding domain (GATA2ΔCF) in BMMCs resulted in a loss of the mast cell phenotype and an increase in the number of CD11b- and/or Ly6G/C-positive cells. These cells showed the ability to differentiate into macrophage- and neutrophil-like cells but not into eosinophils. Although the mRNA levels of basophil-specific genes were elevated, CD49b, a representative basophil marker, never appeared on these cells. GATA2 ablation led to a significant upregulation of C/EBPα, and forced expression of C/EBPα in wild-type BMMCs phenocopied the GATA2ΔCF cells. Interestingly, simultaneous deletion of the Gata2 and Cebpa genes in BMMCs restored the aberrant increases of CD11b and Ly6G/C while retaining the reduced c-Kit expression. Chromatin immunoprecipitation assays indicated that GATA2 directly binds to the +37-kb region of the Cebpa gene and thereby inhibits the RUNX1 and PU.1 binding to the neighboring region. Upregulation of C/EBPα following the loss of GATA2 was not observed in cultured mast cells derived from peritoneal fluid, whereas the repression of c-Kit and other mast cell-specific genes were observed in these cells. Collectively, these results indicate that GATA2 maintains cellular identity by preventing Cebpa gene activation in a subpopulation of mast cells, whereas it plays a fundamental role as a positive regulator of mast cell-specific genes throughout development of this cell lineage.

The STAT5-GATA2 pathway is critical in basophil and mast cell differentiation and maintenance.

Transcription factor GATA binding protein 2 (GATA2) plays critical roles in hematopoietic stem cell survival and proliferation, granulocyte-monocyte progenitor differentiation, and basophil and mast cell differentiation. However, precise roles of GATA2 in basophil and mast cell differentiation and maintenance have not been delineated. We have identified GATA2 as an essential transcription factor in differentiation of newly identified common basophil and mast cell progenitors into basophils and mast cells. We observed Gata2 haploinsufficiency for mast cell differentiation, but not for basophil differentiation. We examined the precise role of GATA2 in maintaining the expression of a wide range of genes that are important for performing basophil or mast cell functions. The effects of GATA2 on gene expression were broadly based. We demonstrated that GATA2 was required for maintaining Fcer1a mRNA and FcεRIα protein expression on both basophils and mast cells, as well as for maintaining Kit mRNA and c-Kit protein expression on mast cells. GATA2 was required for histamine synthesis and was also critical for Il4 mRNA expression in basophils and Il13 mRNA expression in mast cells. We demonstrate a STAT5-GATA2 connection, showing that the STAT5 transcription factor directly bound to the promoter and an intronic region of the Gata2 gene. Overexpression of the Gata2 gene was sufficient to direct basophil and mast cell differentiation in the absence of the Stat5 gene. Our study reveals that the STAT5-GATA2 pathway is critical for basophil and mast cell differentiation and maintenance.

The role of macrophage migration inhibitory factor in mast cell-stimulated fibroblast proliferation and collagen production.

Current clinical and translational studies have shown that mast cell plays a pivotal role in multiple fibrotic diseases including scleroderma. However, the lack of mature human mast cell culture model exhibits a major obstacle for further dissection of cytokines and signaling molecules required for mast cell mediated fibrosis in various diseases. Macrophage Migration Inhibitory Factor is a mast cell released pro-inflammatory cytokine which is deregulated in scleroderma patients and is also involved in non-scleroderma related fibrosis. In the current study, we successfully generated a practical and reliable human mast cell culture system with bone marrow CD34+ hematopietic precursors. The derivative mast cell is normal in terms of both morphology and function as manifested by normal degranulation. More importantly, we were able to show mast cell conditioned medium as well as MIF supplementation augments fibroblast proliferation and collagen synthesis. This positive regulatory effect of mast cell conditioned medium can be dampened by MIF antibody. In addition, MIF-knockdown significantly inhibits pro-fibrotic activities of CD34+ hematopietic precursor derived mast cells. These data strongly suggest that mast cell released MIF is required for mast cell mediated fibrogenic activities. The current manuscript seems to be the first mechanistic report showing the significance of MIF in mast cell mediated fibrosis, which may pave the way for the development of potential MIF-targeted therapy for fibrotic diseases to a further extent. Moreover, we strongly believe mast cell culture and differentiation model as well as corresponding genetic manipulation methodology will be helpful in characterizing novel mast cell based therapeutic targets.

Subthreshold IKK activation modulates the effector functions of primary mast cells and allows specific targeting of transformed mast cells.

Mast cell differentiation and proliferation depends on IL-3. IL-3 induces the activation of MAP-kinases and STATs and consequently induces proliferation and survival. Dysregulation of IL-3 signaling pathways also contribute to inflammation and tumorigenesis. We show here that IL-3 induces a SFK- and Ca²⁺-dependent activation of the inhibitor of κB kinases 2 (IKK2) which results in mast cell proliferation and survival but does not induce IκBα-degradation and NFκB activation. Therefore we propose the term "subthreshold IKK activation".This subthreshold IKK activation also primes mast cells for enhanced responsiveness to IL-33R signaling. Consequently, co-stimulation with IL-3 and IL-33 increases IKK activation and massively enhances cytokine production induced by IL-33.We further reveal that in neoplastic mast cells expressing constitutively active Ras, subthreshold IKK activation is associated with uncontrolled proliferation. Consequently, pharmacological IKK inhibition reduces tumor growth selectively by inducing apoptosis in vivo.Together, subthreshold IKK activation is crucial to mediate the full IL-33-induced effector functions in primary mast cells and to mediate uncontrolled proliferation of neoplastic mast cells. Thus, IKK2 is a new molecularly defined target structure.

The role of Lin28b in myeloid and mast cell differentiation and mast cell malignancy.

Mast cells (MCs) are critical components of the innate immune system and important for host defense, allergy, autoimmunity, tissue regeneration and tumor progression. Dysregulated MC development leads to systemic mastocytosis (SM), a clinically variable but often devastating family of hematologic disorders. Here we report that induced expression of Lin28, a heterochronic gene and pluripotency factor implicated in driving a fetal hematopoietic program, caused MC accumulation in adult mice in target organs such as the skin and peritoneal cavity. In vitro assays revealed a skewing of myeloid commitment in LIN28B-expressing hematopoietic progenitors, with increased levels of LIN28B in common myeloid and basophil-MC progenitors altering gene expression patterns to favor cell fate choices that enhanced MC specification. In addition, LIN28B-induced MCs appeared phenotypically and functionally immature, and in vitro assays suggested a slowing of MC terminal differentiation in the context of LIN28B upregulation. Finally, interrogation of human MC leukemia samples revealed upregulation of LIN28B in abnormal MCs from patients with SM. This work identifies Lin28 as a novel regulator of innate immune function and a new protein of interest in MC disease.

Activated Mast Cells Produce Soluble ST2, a Decoy Receptor for IL-33

Interleukin (IL)-33 mediates inflammatory responses in allergic and autoimmune diseases. IL-33 acts through its membrane bound receptor, ST2. A soluble spliced variant of ST2 (sST2) that lacks the ST2 cytosolic and transmembrane domains is thought to act as a decoy receptor to neutralize IL-33 activity. sST2 and IL-33 are elevated in many inflammatory diseases and serum levels of sST2 appear to correlate with disease severity. We investigated whether mast cells produce, and are a significant source of sST2. Production of sST2 and IL-33 by stimulated mast cells derived from human peripheral blood CD34+ cells and mouse bone marrow was measured by qPCR, ELISA and Western blotting. A mouse model of passive systemic anaphylaxis was used to investigate sST2 production during anaphylaxis. Antigen and IL-33 evoked substantial release of sST2 from mouse and human mast cells and did so synergistically when added together or in combination with stem cell factor, a growth factor that is critical for mast cell differentiation, growth, survival and homing. Rapid release of sST2 into circulation was also apparent during systemic anaphylaxis in mice. Human mast cells failed to generate IL-33 and IL-33 produced by mouse bone marrow-derived mast cells was retained within the cells. Mast cells are a significant source of sST2, which may serve as a modulator of exogenous IL-33 activity and, in some circumstances, serve as a possible biomarker of allergic inflammation.

Knockdown of the antiapoptotic Bcl-2 family member A1/Bfl-1 protects mice from anaphylaxis.

Many forms of hypersensitivity reactions and allergic responses depend on deregulated mast cell activity. Several reports suggested that the antiapoptotic Bcl-2 family protein Bcl2a1/Bfl-1/A1 plays a critical role in mast cell survival upon activation. However, its in vivo relevance is poorly understood because of quadruplication of the Bcl2a1 gene locus in mice, hindering conventional knockout studies. In this study, we used a mouse model allowing traceable constitutive knockdown of all A1 isoforms expressed in the hematopoietic system by RNA interference. Knockdown of A1 reduced mast cell numbers in the skin and impaired connective tissue-like mast cell survival upon FcεRI-mediated activation in vitro. In contrast, A1 was dispensable for mucosa-like mast cell differentiation and survival. Moreover, knockdown of A1 prevented IgE-mediated passive systemic and cutaneous anaphylaxis in vivo. Our findings demonstrate that A1 is essential for the homeostasis of connective tissue mast cells, identifying A1 as a possible therapeutic target for therapy of certain types of mast cell-driven allergy symptoms.

Transcription factor IRF8 plays a critical role in the development of murine basophils and mast cells

AbstractBasophils and mast cells play critical roles in host defense against pathogens and allergic disorders. However, the molecular mechanism by which these cells are generated is not completely understood. Here we demonstrate that interferon regulatory factor-8 (IRF8), a transcription factor essential for the development of several myeloid lineages, also regulates basophil and mast cell development. Irf8−/− mice displayed a severe reduction in basophil counts, which was accounted for by the absence of pre-basophil and mast cell progenitors (pre-BMPs). Although Irf8−/− mice retained peripheral tissue mast cells, remaining progenitors from Irf8−/− mice including granulocyte progenitors (GPs) were unable to efficiently generate either basophils or mast cells, indicating that IRF8 also contributes to the development of mast cells. IRF8 appeared to function at the GP stage, because IRF8 was expressed in GPs, but not in basophils, mast cells, and basophil/mast cell-restricted progenitor cells. Furthermore, we demonstrate that GATA2, a transcription factor known to promote basophil and mast cell differentiation, acts downstream of IRF8. These results shed light on the pathways and mechanism underlying the development of basophils and mast cells.

Altered MicroRNA Expression Profiles in Activated Mast Cells Following IgE-FcεRI Cross-Linking with Antigen

Background/Aims: MicroRNAs (miRNAs) are critical regulators of immune responses and immunologic disorders. However, little is known about miRNA expression and function during mast cell differentiation, proliferation and activation. This study aimed to determine the miRNA expression profiles in mast cells stimulated by immunoglobulin E (IgE) and antigen and to analyze the potential functions of specific miRNAs. Methods: Bone marrow-derived mast cells (BMMCs) generated from differentiated mouse bone marrow cells were untreated (Unstimu) or stimulated with IgE-antigen complexes for 1 h or 6 h (Stimu). The miRNA profiles were evaluated by miRNA microarray. MiRNA target gene prediction and enrichment analyses were performed using bioinformatics. Results: Seven significantly up-regulated and 10 down-regulated miRNAs were identified in the 1 h Stimu group relative to the Unstimu group (fold change>2; P<0.05). Of 8 miRNAs randomly selected from the 17 identified, the expression levels of 6 were confirmed by quantitative real-time PCR (qRT-PCR). The potential target genes of several candidate miRNAs were enriched in FcεRI signaling, response to stimulus and cellular exocytosis. Conclusion: The expression of many miRNAs changes following IgE-FcεRI cross-linking in activated mast cells, and these miRNAs probably play key regulatory roles in core signaling pathways and biological behaviors. Evaluating the functions of these characteristic miRNAs will further our understanding of IgE-associated allergic disease pathogenesis and the development of therapeutic strategies.