Kazumi Kasakura

A TLR2 Ligand Suppresses Allergic Inflammatory Reactions by Acting Directly on Mast Cells

Background: Although much attention has been focused on the anti-allergic effects of probiotics, their mode of action is not fully understood. Mast cells, which play a central role in inducing allergic inflammation, are potential targets of probiotics given the recent discovery that they express Toll-like receptors (TLRs), the pattern recognition receptors for microbial components. In this study, we examined whether allergic reactions of mast cells are modulated by stimulation through TLR2. Methods: The effects on mast cells of the synthetic TLR2 ligand Pam3CSK4 and Bifidobacterium pseudocatenulatum JCM 7041 were evaluated in vitro. Furthermore, the effects of Pam3CSK4 on mast cell-induced increase in vascular permeability in vivo were investigated by employing mast cell-deficient W/Wv mice into which IgE-sensitized mouse bone marrow-derived mast cells were transferred. Results: Pam3CSK4 and Bifidobacterium pseudocatenulatum JCM 7041 suppressed degranulation of IgE-sensitized mast cells upon antigen stimulation in vitro. Pam3CSK4 also suppressed leukotriene C4 production triggered by engagement of the high-affinity IgE receptor, FcεRI. Intracellular Ca2+ mobilization and phosphorylation of Erk were suppressed by pretreatment with Pam3CSK4, suggesting that the TLR2 ligand suppresses activation of mast cells by interrupting FcεRI-mediated intracellular signaling. Pam3CSK4 treatment of bone marrow-derived mast cells reduced the increase in vascular permeability in recipient W/Wv mice upon intravenous injection of antigen; the decrease was by about half, in a TLR-dependent manner. Conclusion: Collectively, these results demonstrate that the FcεRI-mediated inflammatory responses of mast cells are suppressed by stimulation through TLR2, suggesting that probiotics exert potential anti-allergic effects, at least in part, through direct effects on mast cells.

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

Background: The IL1RL1/ST2 gene encodes the receptor for IL-33, which is important for Th2 responses. Results: GATA2 knockdown reduced the expression of human IL1RL1/ST2 in KU812 and LAD2 cells and in human primary peripheral basophils. Conclusion: GATA2, but not GATA1, is a critical transcription factor for expression of human IL1RL1/ST2 in mast cell/basophil lineages. Significance: GATA2 and GATA1 exhibit distinctive roles in the expression of human IL1RL1/ST2. 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.

Commensal bacteria promote migration of mast cells into the intestine.

Mast cells differentiate from hematopoietic stem cells in the bone marrow and migrate via the circulation to peripheral tissues, where they play a pivotal role in induction of both innate and adaptive immune responses. In this study, the effect of intestinal commensal bacteria on the migration of mast cells into the intestine was investigated. Histochemical analyses showed that germ-free (GF) mice had lower mast cell densities in the small intestine than normal mice. It was also shown that GF mice had lower mast cell proportion out of lamina propria leukocytes in the small intestine and higher mast cell percentages in the blood than normal mice by flow cytometry. These results indicate that migration of mast cells from the blood to the intestine is promoted by intestinal commensal bacteria. In addition, MyD88⁻/⁻ mice had lower densities of intestinal mast cells than CV mice, suggesting that the promotive effect of commensals is, at least in part, TLR-dependent. The ligands of CXC chemokine receptor 2 (CXCR2), which is critical for homing of mast cells to the intestine, were expressed higher in intestinal tissues and in intestinal epithelial cells (IECs) of normal mice than in those of GF or MyD88⁻/⁻ mice. Collectively, it is suggested that commensals promote migration of mast cells into the intestine through the induction of CXCR2 ligands from IECs in a TLR-dependent manner.

Commensal bacteria directly suppress in vitro degranulation of mast cells in a MyD88-independent manner

The intestine harbors a substantial number of commensal bacteria that provide considerable benefits to the host. Epidemiologic studies have identified associations between alterations in the composition of the intestinal microbiota and the development of allergic disease. However, the cellular and molecular mechanisms underlying these effects remain to be determined. Here, we show that heat-killed commensal bacteria suppressed degranulation of mast cells in vitro in a MyD88-independent manner. In particular, Enterococcus faecalis showed the strongest suppression of degranulation through partial inhibition of Ca2+ signaling upon the high affinity IgE receptor (FcεRI) cross-linking. Graphical Abstract Commensal bacteria, especially Enterococcus faecalis, suppress degranulation of mast cells upon IgE/antigen stimulation in vitro.

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-, β-, 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 β 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 FcεRIβ). 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 FcεRIβ, 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 FcεRIβ 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.

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 JCM1134T 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.

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.

The Orphan Nuclear Receptor NR4A3 Is Involved in the Function of Dendritic Cells

NR4A3/NOR1 belongs to the NR4A subfamily of the nuclear hormone receptor superfamily, which is activated in a ligand-independent manner. To examine the role of NR4A3 in gene expression of dendritic cells (DCs), we introduced NR4A3 small interfering RNA (siRNA) into bone marrow–derived DCs and determined the expression levels of mRNA and proteins of cytokines, cell surface molecules, NF-κB signaling–related proteins, and transcription factors. The expression level of NR4A3 was markedly upregulated by TLR-mediated stimulation in DCs. NR4A3 knockdown significantly suppressed LPS, CpG, or poly(I:C)-mediated upregulation of CD80, CD86, IL-10, IL-6, and IL-12. Proliferation and IL-2 production levels of T cells cocultured with NR4A3 knocked-down DCs were significantly lower than that of T cells cocultured with control DCs. Furthermore, the expression of IKKβ, IRF4, and IRF8 was significantly decreased in NR4A3 siRNA-introduced bone marrow–derived DCs. The knockdown experiments using siRNAs for IKKβ, IRF4, and/or IRF8 indicated that LPS-induced upregulation of IL-10 and IL-6 was reduced in IKKβ knocked-down cells, and that the upregulation of IL-12 was suppressed by the knockdown of IRF4 and IRF8. Taken together, these results indicate that NR4A3 is involved in TLR-mediated activation and gene expression of DCs.

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.