Histamine-releasing factor in severe asthma and rhinovirus-associated asthma exacerbation.

BACKGROUND: Histamine-releasing factor (HRF) is implicated in allergic diseases. We previously showed its pathogenic role in murine models of asthma. OBJECTIVE: We aim to present data analysis from 3 separate human samples (sera samples from asthmatic patients, nasal washings from rhinovirus [RV]-infected individuals, and sera samples from patients with RV-induced asthma exacerbation) and 1 mouse sample to investigate correlates of HRF function in asthma and virus-induced asthma exacerbations. METHODS: Total IgE and HRF-reactive IgE/IgG as well as HRF in sera from patients with mild/moderate asthma or severe asthma (SA) and healthy controls (HCs) were quantified by ELISA. HRF secretion in culture media from RV-infected adenovirus-12 SV40 hybrid virus transformed human bronchial epithelial cells and in nasal washings from experimentally RV-infected subjects was analyzed by Western blotting. HRF-reactive IgE/IgG levels in longitudinal serum samples from patients with asthma exacerbations were also quantified. RESULTS: HRF-reactive IgE and total IgE levels were higher in patients with SA than in HCs, whereas HRF-reactive IgG (and IgG1) level was lower in asthmatic patients versus HCs. In comparison with HRF-reactive IgElow asthmatic patients, HRF-reactive IgEhigh asthmatic patients had a tendency to release more tryptase and prostaglandin D2 on anti-IgE stimulation of bronchoalveolar lavage cells. RV infection induced HRF secretion from adenovirus-12 SV40 hybrid virus transformed bronchial epithelial cells, and intranasal RV infection of human subjects induced increased HRF secretion in nasal washes. Asthmatic patients had higher levels of HRF-reactive IgE at the time of asthma exacerbations associated with RV infection, compared with those after the resolution. This phenomenon was not seen in asthma exacerbations without viral infections. CONCLUSIONS: HRF-reactive IgE is higher in patients with SA. RV infection induces HRF secretion from respiratory epithelial cells both in vitro and in vivo. These results suggest the role of HRF in asthma severity and RV-induced asthma exacerbation.

Histamine-Releasing Factor Is a Novel Alarmin Induced by House Dust Mite Allergen, Cytokines, and Cell Death.

Histamine-releasing factor (HRF) is a multifunctional protein with fundamental intracellular functions controlling cell survival and proliferation. HRF is also secreted during allergic reactions and promotes IgE-mediated activation of mast cells and basophils. In this study, we investigated HRF secretion and its relevance to airway inflammation. HRF monomers were constitutively secreted from BEAS-2B human bronchial epithelial cells (HBECs) and converted to oligomers over the course of culture. Stimulation with house dust mite (HDM) extract increased HRF secretion substantially. Several cytokines involved in asthma pathogenesis showed moderate effects on HRF secretion but dramatically enhanced HDM-induced HRF secretion. HDM-induced HRF secretion from BEAS-2B cells and normal HBECs proceeded via TLR2. Consistent with this, multiple TLR2 ligands, including Der p 2, Der p 5, Der p 13, and Der p 21, induced HRF secretion. Der p 10 (tropomyosin) also promoted HRF secretion. Cell death or incubation with adenosine and ATP, compounds released upon cell death, also enhanced HRF secretion. Furthermore, intranasal administration of recombinant HRF elicited robust airway inflammation in HDM-sensitized mice in an FcεRI-dependent manner. Therefore, we conclude that HRF is a novel alarmin that promotes allergic airway inflammation.

PU.1 and IRF8 Modulate Activation of NLRP3 Inflammasome via Regulating Its Expression in Human Macrophages.

NLRP3 inflammasomes play crucial roles in the initiation of host defense by converting pro-Caspase-1 to mature Caspase-1, which in turn processes immature IL-1ß and IL-18 into their biologically active forms. Although NLRP3 expression is restricted to monocytic lineages such as monocytes, macrophages, and dendritic cells, the mechanisms determining the lineage-specific expression of NLRP3 remain largely unknown. In this study, we investigated the transcription factors involved in cell-type-specific transcription of NLRP3. We found that a distal, rather than a proximal, promoter of human NLRP3 was predominantly used in the human monocytic cell lines and macrophages. Reporter analysis showed that an Ets/IRF composite element (EICE) at -309/-300 and an Ets motif at +5/+8 were critical for transcriptional activity of the distal promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that two transcription factors, PU.1 and IRF8, both of which play essential roles in development and gene expression of the monocytic lineage, were bound to the EICE site, whereas PU.1 alone was bound to the Ets site. Knockdown of PU.1 and/or IRF8 mediated by small interfering RNA downregulated expression of NLRP3 and related molecules and markedly diminished the LPS-induced release of IL-1ß in THP-1, suggesting that activity of the NLRP3 inflammasome was suppressed by knockdown of PU.1 and IRF8. Taken together, these results indicate that PU.1 and IRF8 are involved in the monocytic lineage-specific expression of NLRP3 by binding to regulatory elements within its promoter and that PU.1 and IRF8 are potential targets for regulating the activity of the NLRP3 inflammasome.

Pterostilbene reduces colonic inflammation by suppressing dendritic cell activation and promoting regulatory T cell development.

Dendritic cells (DCs) and T cells play important roles in immune regulation, and modulating their function is an approach for developing preventive or therapeutic strategies against immune disorders. Herein, the effect of pterostilbene (PSB) (3',5'-dimethoxy-resveratrol)-a resveratrol-related polyphenol found in blueberries-on immune regulation was evaluated. Using an in vitro co-culture system, PSB was found to exert the strongest inhibitory effect among all tested resveratrol derivatives on DC-mediated T cell proliferation; moreover, PSB treatment decreased the Th1 and Th17 populations and increased the regulatory T cell (Treg) population. Upon co-stimulation with anti-CD3 and anti-CD28 antibodies, PSB inhibited CD4+ T cell proliferation and differentiation into Th1 cells. Additionally, PSB acted on DCs to suppress the lipopolysaccharide-induced transactivation of genes encoding antigen presentation-related molecules and inflammatory cytokines by attenuating the DNA-binding ability of the transcription factor PU.1. Furthermore, PSB promoted DC-mediated Foxp3+ Treg differentiation, and PU.1 knockdown increased DC-induced Treg activity. Oral administration of PSB alleviated the symptoms of dextran sulfate sodium-induced colitis and decreased tumor necrosis factor-α expression in mice. Thus, PSB treatment ameliorates colonic inflammation.

The anti-inflammatory effect of the gut lactic acid bacteria-generated metabolite 10-oxo-cis-6,trans-11-octadecadienoic acid on monocytes.

We evaluated the effect of gut bacterial metabolites of polyunsaturated fatty acids on inflammation and found that 10-oxo-cis-6,trans-11-octadecadienoic acid (γKetoC) strikingly suppressed LPS-induced IL-6 release from bone marrow-derived macrophages (BMMs), which was accompanied by reduced mRNA expression of Il6, TNF, and Il1b. γKetoC decreased the cAMP concentration in BMMs, suggesting that γKetoC stimulated G protein-coupled receptors. A Gq agonist significantly suppressed LPS-induced IL-6 expression in BMMs, whereas a Gi inhibitor partially abrogated γKetoC-mediated IL-6 suppression. Cytosolic Ca2+ was markedly increased by γKetoC, which was partly but not fully abrogated by an ion channel inhibitor. Taken together, these data suggest that γKetoC suppresses inflammatory cytokine expression in macrophages primarily through Gq and partially through Gi. γKetoC suppressed osteoclast development and IL-6 expression in synovial fibroblasts from rheumatoid arthritis (RA) patients, suggesting the beneficial effect of γKetoC on the prevention or treatment of RA.

Dual Roles of PU.1 in the Expression of PD-L2: Direct Transactivation with IRF4 and Indirect Epigenetic Regulation.

PD-L2, which has been identified as a PD-1 ligand, is specifically expressed in dendritic cells (DCs) and macrophages. The transcription factors that determine the cell type-specific expression of PD-L2 are largely unknown, although PD-1 and its ligands, which have been shown to play important roles in T cell suppression, have been vigorously analyzed in the field of cancer immunology. To reveal the mechanism by which Pdcd1lg2 gene expression is regulated, we focused on DCs, which play key roles in innate and acquired immunity. The knockdown of the hematopoietic cell-specific transcription factors PU.1 and IRF4 decreased PD-L2 expression in GM-CSF-induced mouse bone marrow-derived DCs. Chromatin immunoprecipitation assays, luciferase assays, and electrophoretic mobility shift assays demonstrated that PU.1 and IRF4 bound directly to the Pdcd1lg2 gene via an Ets-IRF composite element sequence and coordinately transactivated the Pdcd1lg2 gene. Furthermore, PU.1 knockdown reduced the histone acetylation of the Pdcd1lg2 gene. The knockdown of the typical histone acetyltransferase p300, which has been reported to interact with PU.1, decreased the expression and H3K27 acetylation of the Pdcd1lg2 gene. GM-CSF stimulation upregulated the Pdcd1lg2 gene expression, which was accompanied by an increase in PU.1 binding and histone acetylation in Flt3L-generated mouse bone marrow-derived DCs. The involvement of PU.1, IRF4, and p300 were also observed in mouse splenic DCs. Overall, these results indicate that PU.1 positively regulates Pdcd1lg2 gene expression as a transactivator and an epigenetic regulator in DCs.

PU.1 regulates Ccr7 gene expression by binding to its promoter in naïve CD4+ T cells.

C-C chemokine receptor type 7 (CCR7) is expressed on naïve T cells, B cells, and activated dendritic cells (DCs). We previously demonstrated that the transcription factor PU.1/Spi1 positively regulates the expression of CCR7 in DCs. In the present study, we investigated the role of PU.1 in CCR7 expression in T cells. To confirm whether PU.1 is involved in the expression of CCR7, we conducted a ChIP assay in various T cells purified from splenocytes and thymocytes and found that PU.1 binds to the Ccr7 promoter-proximal region in spleen naïve CD4+ T cells, but not in thymocytes. Small interfering RNA-mediated PU.1 knockdown resulted in decreased CCR7 expression in spleen naïve CD4+ T cells. Compared to naïve CD4+ T cells, Spi1 and Ccr7 mRNA levels decreased in Th1 and Th2 cells, in which PU.1 did not bind to the Ccr7 promoter, suggesting that CCR7 expression decreases due to the dissociation of PU.1 from the Ccr7 promoter during the development of effector T cells from naïve T cells. Collectively, we concluded that CCR7 expression level correlates with the binding level of PU.1 to the Ccr7 promoter and PU.1 acts as a transcriptional activator of the Ccr7 gene in naïve CD4+ T cells.

Cooperative Regulation of the Mucosal Mast Cell-Specific Protease Genes Mcpt1 and Mcpt2 by GATA and Smad Transcription Factors.

Mouse mast cell proteases (mMCP)-1 and -2 are specifically expressed in mucosal mast cells (MCs). However, the transcriptional regulation mechanism of the Mcpt1 and Mcpt2 genes induced in mucosal MCs is largely unknown. In the current study, we found that TGF-ß stimulation drastically induced upregulation of Mcpt1 and Mcpt2 mRNA in mouse bone marrow-derived MCs (BMMCs). TGF-ß-induced expression of Mcpt1 and Mcpt2 was markedly suppressed by transfection with small interfering RNA targeting Smad2 or Smad4 and moderately reduced by Smad3 small interfering RNA. We next examined the roles of the hematopoietic cell-specific transcription factors GATA1 and GATA2 in the expression of Mcpt1 and Mcpt2 and demonstrated that knockdown of GATA1 and GATA2 reduced the mRNA levels of Mcpt1 and Mcpt2 in BMMCs. The recruitment of GATA2 and acetylation of histone H4 of the highly conserved GATA-Smad motifs, which were localized in the distal regions of the Mcpt1 and Mcpt2 genes, were markedly increased by TGF-ß stimulation, whereas the level of GATA2 binding to the proximal GATA motif was not affected by TGF-ß. A reporter assay showed that TGF-ß stimulation upregulated GATA2-mediated transactivation activity in a GATA-Smad motif-dependent manner. We also observed that GATA2 and Smad4 interacted in TGF-ß-stimulated BMMCs via immunoprecipitation and Western blotting analysis. Taken together, these results demonstrate that TGF-ß induced mMCP-1 and -2 expression by accelerating the recruitment of GATA2 to the proximal regions of the Mcpt1 and Mcpt2 genes in mucosal MCs.

Suppressive role of PPARγ in the IgE-dependent activation of mast cells.

Mast cells (MCs) play a central role in IgE-dependent immune responses. PPARγ is a nuclear receptor that is essential for adipocyte differentiation and insulin sensitivity. Although PPARγ is expressed in activated MCs, the effect of PPARγ suppression in IgE-mediated activation of MCs is largely unknown. In the current study, we evaluated the effect of PPARγ knockdown on the function of IgE plus antigen (Ag)-stimulated MCs using siRNA-transfected bone marrow-derived MCs (BMMCs). We found that the mRNA expression level of cytokines in IgE/Ag-stimulated BMMCs was significantly increased in PPARγ knockdown BMMCs, and IgE/Ag-mediated degranulation and the protein production level of TNF-α was moderately increased by PPARγ knockdown, whereas the cell surface expression level of FcεRI was not affected by PPARγ knockdown. Oral administration of pioglitazone (PPARγ agonist) significantly suppressed body temperature change of mice in passive systemic anaphylaxis, supporting the inhibitory functions of PPARγ in IgE/Ag-dependent activation of MCs in vivo. IgE-mediated up-regulation of mRNA levels of Ptgs2 (encoding COX-2) was drastically enhanced in PPARγ knockdown BMMCs. Although several prostaglandin (PG) derivatives are known to be ligands for PPARγ, treatment with a COX inhibitor, acetyl salicylic acid, up-regulated the IgE-mediated increase of Il13, Tnf and Ptgs2 mRNA levels in a synergistic manner with PPARγ siRNA. Knockdown of COX-1 and/or COX-2 by siRNA showed that suppression of IgE/Ag-mediated activation was mainly dependent on COX-1. Taken together, these results indicate that PPARγ suppresses IgE/Ag-induced transactivation of cytokine genes and the Ptgs2 gene in MCs in a manner distinguishable from that of PGs.

Histamine-Releasing Factor, a New Therapeutic Target in Allergic Diseases.

Histamine-releasing activities on human basophils have been studied as potential allergy-causing agents for four decades. An IgE-dependent histamine-releasing factor (HRF) was recently shown to interact with a subset of immunoglobulins. Peptides or recombinant proteins that block the interactions between HRF and IgE have emerged as promising anti-allergic therapeutics, as administration of them prevented or ameliorated type 2 inflammation in animal models of allergic diseases such as asthma and food allergy. Basic and clinical studies support the notion that HRF amplifies IgE-mediated activation of mast cells and basophils. We discuss how secreted HRF promotes allergic inflammation in vitro and in vivo complex disease settings.

PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

C-C chemokine receptor type 7 (CCR7) is essential for migration of dendritic cells (DCs) to draining lymph nodes. PU.1/Spi1 is a transcription factor playing a critical role in the gene regulation of DCs. PU.1 knockdown decreased the expression of CCR7 in bone marrow-derived DCs and subsequently attenuated migration in vitro and in vivo. Reporter assays, EMSA, and chromatin immunoprecipitation assays revealed that PU.1 binds to the most proximal Ets motif of the Ccr7 promoter, which is involved in transcriptional activation. The CCR7 expression level, which was higher in the programmed cell death 1 ligand 2 (PD-L2)+ population than in the PD-L2- population and was markedly suppressed by TGF-ß treatment, coincided with the binding level of PU.1 to the Ccr7 promoter. The PU.1 binding level in CCR7high mesenteric lymph nodes DCs was higher than in other DC subtypes. The involvement of PU.1 in the expression of the CCR7 gene was also observed in human DCs. We conclude that PU.1 plays a pivotal role in DC migration by transactivating the CCR7 gene via the Ets motif in the promoter in both humans and mice.-Yashiro, T., Takeuchi, H., Nakamura, S., Tanabe, A., Hara, M., Uchida, K., Okumura, K., Kasakura, K., Nishiyama, C. PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

A transcription factor PU.1 is critical for Ccl22 gene expression in dendritic cells and macrophages.

The chemokine CCL22 is predominantly produced by dendritic cells (DCs) and macrophages. CCL22 acts on CCR4-expressing cells including Th2 and Treg. Although a correlation between the CCL22-CCR4 axis and allergic diseases has been established, the mechanism of monocyte lineage-specific Ccl22 gene expression is largely unknown. In the current study, we investigated transcriptional regulation of the Ccl22 gene in DCs and macrophages. Using reporter assays, we identified the critical cis-enhancing elements at 21/-18 and -10/-4 in the Ccl22 promoter. Electrophoretic mobility shift assays proved that transcription factor PU.1 directly binds to the cis-elements. Knockdown of PU.1 markedly decreased Ccl22 expression in bone marrow-derived DCs (BMDCs) and BM macrophages (BMDMs). Chromatin immunoprecipitation assays revealed that PU.1 bound to the Ccl22 promoter in not only BMDCs and BMDMs, but also splenic DCs and peritoneal macrophages. LPS stimulation increased the amount of PU.1 recruited to the promoter, accompanied by upregulation of the Ccl22 mRNA level, which was diminished by Spi1 knockdown. We identified similar cis-elements on the human CCL22 promoter, which were bound with PU.1 in human monocytes. Taken together, these findings indicate that PU.1 transactivates the Ccl22 gene in DCs and macrophages by directly binding to the two elements in the promoter.

The Transcription Factors PU.1 and IRF4 Determine Dendritic Cell-Specific Expression of RALDH2.

RALDH2 expressed in dendritic cells (DCs) plays a critical role in the development of regulatory T cells in mesenteric lymph nodes. Despite the importance of RALDH2 in intestinal immunity, little is known about the mechanism of DC-specific expression of RALDH2. In the current study, we focused on the hematopoietic cell-specific transcription factors PU.1 and IRF4 as the determinants of Aldh1a2 gene expression. The mRNA level of Aldh1a2, and subsequently the enzyme activity, were decreased by knockdown of PU.1 and IRF4 in bone marrow-derived DCs (BMDCs) of BALB/c mice. Chromatin immunoprecipitation assays showed that PU.1 and IRF4 bound to the Aldh1a2 gene ∼2 kb upstream from the transcription start site in BMDCs. A reporter assay and an EMSA revealed that the Aldh1a2 promoter was synergistically transactivated by a heterodimer composed with PU.1 and IRF4 via the EICE motif at -1961/-1952 of the gene. The effect of small interfering RNAs for Spi1 and Irf4 and specific binding of PU.1 and IRF4 on the Aldh1a2 gene were also observed in DCs freshly isolated from spleen and mesenteric lymph nodes, respectively. GM-CSF stimulation upregulated the Aldh1a2 transcription in Flt3 ligand-generated BMDCs, in which the IRF4 expression and the PU.1 recruitment to the Aldh1a2 promoter were enhanced. We conclude that PU.1 and IRF4 are transactivators of the Aldh1a2 gene in vitro and ex vivo.

The effect of PU.1 knockdown on gene expression and function of mast cells.

PU.1 is a hematopoietic cell-specific transcription factor. In the current study, we investigated the role of PU.1 in the gene expression and the function of mouse mast cells (MCs) in vitro and in vivo. When PU.1 siRNA was introduced into bone marrow-derived MCs (BMMCs), IgE-mediated activation was reduced, and the Syk and FcεRIß mRNA levels were significantly decreased. As the regulatory mechanism of the Syk gene is largely unknown, we performed promoter analysis and found that PU.1 transactivated the Syk promoter through direct binding to a cis-element in the 5'-untranslated region. The involvement of PU.1 in the Syk promoter was also observed in mouse dendritic cells and human MCs, suggesting that the relationship between PU.1 and Syk is common in mammals and in hematopoietic lineages. When antigen was administrated intravenously after the transfusion of siRNA-transfected BMMCs in the mouse footpad, the footpad thickening was significantly suppressed by PU.1 knockdown. Finally, administration of the immunomodulator pomalidomide suppressed passive systemic anaphylaxis of mice. Taken together, these results indicate that PU.1 knockdown might be an efficacious strategy for the prevention of MC-mediated allergic diseases.

The hematopoietic cell-specific transcription factor PU.1 is critical for expression of CD11c.

PU.1 is a hematopoietic cell-specific transcription factor belonging to the Ets family, which plays an important role in the development of dendritic cells (DCs). CD11c (encoded by Itgax) is well established as a characteristic marker of hematopoietic lineages including DCs. In the present study, we analyzed the role of PU.1 (encoded by Spi-1) in the expression of CD11c. When small interfering RNA (siRNA) for Spi-1 was introduced into bone marrow-derived DCs (BMDCs), the mRNA level and cell surface expression of CD11c were dramatically reduced. Using reporter assays, the TTCC sequence at -56/-53 was identified to be critical for PU.1-mediated activation of the promoter. An EMSA showed that PU.1 directly bound to this region. ChIP assays demonstrated that a significant amount of PU.1 bound to this region on chromosomal DNA in BMDCs, which was decreased in LPS-stimulated BMDCs in accordance with the reduced levels of mRNAs of Itgax and Spi-1, and the histone acetylation degree. Enforced expression of exogenous PU.1 induced the expression of the CD11c protein on the cell surface of mast cells, whereas control transfectants rarely expressed CD11c. Quantitative RT-PCR also showed that the expression of a transcription factor Irf4, which is a partner molecule of PU.1, was reduced in PU.1-knocked down BMDCs. IRF4 transactivated the Itgax gene in a synergistic manner with PU.1. Taken together, these results indicate that PU.1 functions as a positive regulator of CD11c gene expression by directly binding to the Itgax promoter and through transactivation of the Irf4 gene.

Role of PU.1 in MHC Class II Expression via CIITA Transcription in Plasmacytoid Dendritic Cells.

The cofactor CIITA is a master regulator of MHC class II expression and several transcription factors regulating the cell type-specific expression of CIITA have been identified. Although the MHC class II expression in plasmacytoid dendritic cells (pDCs) is also mediated by CIITA, the transcription factors involved in the CIITA expression in pDCs are largely unknown. In the present study, we analyzed the role of a hematopoietic lineage-specific transcription factor, PU.1, in CIITA transcription in pDCs. The introduction of PU.1 siRNA into mouse pDCs and a human pDC cell line, CAL-1, reduced the mRNA levels of MHC class II and CIITA. When the binding of PU.1 to the 3rd promoter of CIITA (pIII) in CAL-1 and mouse pDCs was analyzed by a chromatin immunoprecipitation assay, a significant amount of PU.1 binding to the pIII was detected, which was definitely decreased in PU.1 siRNA-transfected cells. Reporter assays showed that PU.1 knockdown reduced the pIII promoter activity and that three Ets-motifs in the human pIII promoter were candidates of cis-enhancing elements. By electrophoretic mobility shift assays, it was confirmed that two Ets-motifs, GGAA (-181/-178) and AGAA (-114/-111), among three candidates, were directly bound with PU.1. When mouse pDCs and CAL-1 cells were stimulated by GM-CSF, mRNA levels of PU.1, pIII-driven CIITA, total CIITA, MHC class II, and the amount of PU.1 binding to pIII were significantly increased. The GM-CSF-mediated up-regulation of these mRNAs was canceled in PU.1 siRNA-introduced cells. Taking these results together, we conclude that PU.1 transactivates the pIII through direct binding to Ets-motifs in the promoter in pDCs.

Involvement of PU.1 in NFATc1 promoter function in osteoclast development.

BACKGROUND: The transcription factors NFATc1 and PU.1 play important roles in osteoclast development. NFATc1 and PU.1 transactivate osteoclast-specific gene expression and a deficiency in NFATc1 or PU.1 genes causes osteopetrosis due to an insufficient development of osteoclasts. However, the existence of cross-regulation between NFATc1 and PU.1 is largely unknown. In the present study, the role of PU.1 in NFATc1 expression was investigated. METHODS: Osteoclasts were generated from mouse bone marrow cells. PU.1 knockdown was performed with siRNA introduction. The mRNA levels in siRNA-introduced cells were determined by quantitative RT-PCR. The involvement of PU.1 in the NFATc1 promoter was analyzed by using a chromatin immunoprecipitation (ChIP) assay and a reporter assay. Retrovirus vector was used for enforced expression of PU.1. RESULTS: Introduction of PU.1 siRNA into bone marrow-derived osteoclasts resulted in a decrease in NFATc1 mRNA level. A ChIP assay showed that PU.1 bound to the NFATc1 promoter in osteoclasts. NFATc1 promoter activity was reduced in PU.1 knockdown cells as assessed by a reporter assay. PU.1 siRNA introduction also downregulated the expression of osteoclast-specific genes and tartrate resistant acid phosphatase (TRAP) activity. Enforced expression of PU.1 using a retrovirus vector increased NFATc1 expression and TRAP activity. When NFATc1 expression was knocked down by using siRNA, the induction of osteoclast-specific genes and TRAP-positive cells was suppressed without affecting the expression level of PU.1. CONCLUSIONS: These results indicate that PU.1 is involved in osteoclast development by transactivating NFATc1 expression via direct binding to the NFATc1 promoter.

C/EBPα controls mast cell function.

CCAAT/enhancer binding protein alpha (C/EBPα) is a transcription factor that influences immune cell fate and differentiation. However, the effect of C/EBPα on mast cells is not fully understood. In this study, we showed that C/EBPα suppressed granule formation in mast cells and increased macrophage inflammatory protein (MIP)-2 production from mast cells upon bacterial stimulation. These results indicate that C/EBPα regulates the balance between the allergic response and the innate immune response of mast cells. Furthermore, we showed that stimulation of mast cells with the Lactobacillus casei JCM1134(T) strain during late differentiation up-regulated C/EBPα expression in differentiated mast cells. This suggests that intestinal commensal bacteria modulate C/EBPα expression and thereby regulate mast cell function.

Critical Roles for PU.1, GATA1, and GATA2 in the expression of human FcεRI on mast cells: PU.1 and GATA1 transactivate FCER1A, and GATA2 transactivates FCER1A and MS4A2.

The high-affinity IgE receptor, FcεRI, which is composed of α-, ß-, and γ-chains, plays an important role in IgE-mediated allergic responses. In the current study, involvement of the transcription factors, PU.1, GATA1, and GATA2, in the expression of FcεRI on human mast cells was investigated. Transfection of small interfering RNAs (siRNAs) against PU.1, GATA1, and GATA2 into the human mast cell line, LAD2, caused significant downregulation of cell surface expression of FcεRI. Quantification of the mRNA levels revealed that PU.1, GATA1, and GATA2 siRNAs suppressed the α transcript, whereas the amount of ß mRNA was reduced in only GATA2 siRNA transfectants. In contrast, γ mRNA levels were not affected by any of the knockdowns. Chromatin immunoprecipitation assay showed that significant amounts of PU.1, GATA1, and GATA2 bind to the promoter region of FCER1A (encoding FcεRIα) and that GATA2 binds to the promoter of MS4A2 (encoding FcεRIß). Luciferase assay and EMSA showed that GATA2 transactivates the MS4A2 promoter via direct binding. These knockdowns of transcription factors also suppressed the IgE-mediated degranulation activity of LAD2. Similarly, all three knockdowns suppressed FcεRI expression in primary mast cells, especially PU.1 siRNA and GATA2 siRNA, which target FcεRIα and FcεRIß, respectively. From these results, we conclude that PU.1 and GATA1 are involved in FcεRIα transcription through recruitment to its promoter, whereas GATA2 positively regulates FcεRIß transcription. Suppression of these transcription factors leads to downregulation of FcεRI expression and IgE-mediated degranulation activity. Our findings will contribute to the development of new therapeutic approaches for FcεRI-mediated allergic diseases.

GATA2 is a critical transactivator for the human IL1RL1/ST2 promoter in mast cells/basophils: opposing roles for GATA2 and GATA1 in human IL1RL1/ST2 gene expression.

The IL1RL1/ST2 gene encodes a receptor for IL-33. Signaling from IL1RL1/ST2 induced by IL-33 binding was recently identified as a modulator of the Th2 response. The target cells for IL-33 are restricted in some hematopoietic lineages, including mast cells, basophils, eosinophils, Th2 cells, natural killer cells, and dendritic cells. To clarify the molecular mechanisms of cell type-specific IL1RL1/ST2 expression in mast cells and basophils, transcriptional regulation of the human IL1RL1/ST2 promoter was investigated using the mast cell line LAD2 and the basophilic cell line KU812. Reporter assays suggested that two GATA motifs just upstream of the transcription start site in the ST2 promoter are critical for transcriptional activity. These two GATA motifs possess the capacity to bind GATA1 and GATA2 in EMSA. ChIP assay showed that GATA2, but not GATA1, bound to the ST2 promoter in LAD2 cells and that histone H3 at the ST2 promoter was acetylated in LAD2 cells, whereas binding of GATA1 and GATA2 to the ST2 promoter was detected in KU812 cells. Knockdown of GATA2 mRNA by siRNA reduced ST2 mRNA levels in KU812 and LAD2 cells and ST2 protein levels in LAD2 cells; in contrast, GATA1 siRNA transfection up-regulated ST2 mRNA levels in KU812 cells. The ST2 promoter was transactivated by GATA2 and repressed by GATA1 in coexpression analysis. When these siRNAs were introduced into human peripheral blood basophils, GATA2 siRNA reduced ST2 mRNA, whereas GATA1 siRNA up-regulated ST2 mRNA. These results indicate that GATA2 and GATA1 positively and negatively control human ST2 gene transcription, respectively.