IRF3 Research Articles

Retroviral integration site analysis identifies ICSBP as a collaborating tumor suppressor gene in NUP98-TOP1-induced leukemia

The NUP98-TOP1 fusion gene is one of 18 distinct translocations identified in acute myeloid leukemia involving the N-terminal portion of the nucleoporin NUP98. We previously reported that expression of NUP98-TOP in murine bone marrow induces a lethal, transplantable leukemia. However, the long latency suggests the in vivo acquisition of additional mutations and/or time required for clonal outgrowth of rare transformed cells arising from the collaboration of NUP98-TOP1 and a cooperating event. The aim of this study was to test whether retroviral insertional mutagenesis contributes to disease onset and whether integration site analysis can identify collaborating genes. The genomic sites of retroviral integration in NUP98-TOP1-induced leukemic mice were analyzed. This screen identified a proviral integration that disrupts expression of the Interferon consensus sequence binding protein (ICSBP) tumor suppressor gene. Intriguingly, an ICSBP deficiency induces a chronic myeloid leukemia-like disease in mice and its reduced expression has been observed in several human leukemias. To ascertain whether an ISCBP deficiency collaborates with NUP98-TOP1 in leukemogenesis, we expressed NUP98-TOP1 in ICSBP(-/-) bone marrow. The in vivo myeloproliferation induced by NUP98-TOP1 was markedly exaggerated with the ICSBP(-/-) deficiency. Moreover, NUP98-TOP1/ICSBP(-/-) mice had a reduced survival compared with NUP98-TOP1/ICSBP(+/+) mice. These results reveal the novel finding of collaboration between the ICSBP tumor suppressor gene and NUP98-TOP1 in leukemogenesis. Moreover they further illustrate the power of retroviral integration site analysis for identifying novel cooperating tumor suppressor genes.

ICSBP/IRF-8 differentially regulates antigen uptake during dendritic-cell development and affects antigen presentation to CD4+ T cells

Interferon consensus sequence-binding protein (ICSBP)/interferon regulatory factor 8 (IRF-8) is a transcription factor that plays critical roles in the differentiation of defined dendritic-cell (DC) populations and in the immune response to many pathogens. In this study, we show that splenic DCs (s-DCs) from ICSBP(-/-) mice are markedly defective in their ability to capture and to present exogenous antigens (Ags) to naive CD4(+) T lymphocytes. We found that CD8alpha(+) DCs and, to a lesser extent, CD8alpha(-) DCs from ICSBP(-/-) mice are impaired at internalizing Ags, either through a receptor-mediated pathway or by macropinocytosis, in spite of having a more immature phenotype than their wild-type (WT) counterparts. These features reflected a greatly impaired ability of ICSBP(-/-) s-DCs to present injected soluble ovalbumin (OVA) to OVA-specific CD4(+) T cells in vivo. Conversely, bone marrow (BM)-derived DCs from ICSBP(-/-) mice, in keeping with their immature phenotype, exhibited higher endocytic activity than WT cells. However, Ag-loaded ICSBP(-/-) BM-DCs were defective in priming Ag-specific CD4(+) T lymphocytes and failed to induce a contact hypersensitivity (CHS) response when injected into competent WT hosts. Together, these results indicate that, throughout the developmental program of DCs, ICSBP differentially controls Ag uptake and MHC class II (MHC-II) presentation affecting both functions only in differentiated peripheral DCs.

Genetic Predisposition of Responsiveness to Therapy for Chronic Hepatitis C

A combination of interferon-alpha (IFN-alpha) and ribavirin has been the choice for treating chronic hepatitis C (CHC) patients. It achieves an overall sustained response rate of approximately 50%; however, the treatment takes 6-12 months and often brings significant adverse reactions to some patients. It would therefore be beneficial to include a pretreatment evaluation in order to maximize the efficacy. In addition to viral genotypes, we hypothesize that patient genotypes might also be useful for the prediction of treatment response. We retrospectively analyzed the genetic differences of CHC patients that are associated with IFN/ribavirin responses. The DNA polymorphisms among 195 sustained responders and 122 nonresponders of CHC patients of Taiwanese origin were compared. Statistical and algorithmic methods were used to select the genes associated with drug response and single nucleotide polymorphisms (SNPs) that permitted the construction of a predictive model. Association studies and haplotype reconstruction revealed selection of seven genes: adenosine deaminase, RNA-specific (ADAR), caspase 5, apoptosis-related cysteine peptidase (CASP5), fibroblast growth factor 1 (FGF1), interferon consensus sequence binding protein 1 (ICSBP1), interferon-induced protein 44 (IFI44), transporter 2, ATP-binding cassette, subfamily B (TAP2) and transforming growth factor, beta receptor associated protein 1 (TGFBRAP1) for the responsiveness trait. Based on confirmed linkage disequilibrium block in the population, a minimal set of 26 SNPs in the seven selected genes was inferred. To predict treatment outcome, a multiple logistic regression model was constructed using susceptible genotypes of SNPs. The performance of the resultant model had a sensitivity of 68.2% and specificity of 60.7% on 317 CHC patients treated with IFN-combined therapy. In addition, a prediction model with both the host genetic and viral genotype information was also constructed which enhanced the performance with a sensitivity of 80.7% and specificity of 67.2%. A genetic model was constructed to predict outcomes of the combination therapy in CHC patients with high sensitivity and specificity. Results also provide a possible process of selecting targets for predicting treatment outcomes and the basis for developing pharmacogenetic tests.

Rapid and sensitive detection of CpG-methylation using methyl-binding (MB)-PCR

Methylation of CpG islands is associated with transcriptional repression and, in cancer, leads to the abnormal silencing of tumor suppressor genes. We have developed a novel technique for detecting CpG-methylated DNA termed methyl-binding (MB)-PCR. This technique utilizes a recombinant protein with high affinity for CpG-methylated DNA that is coated onto the walls of a PCR vessel and selectively captures methylated DNA fragments from a mixture of genomic DNA. The retention and, hence, the degree of methylation of a specific DNA fragment (e.g. a CpG island promoter of a specific gene) is detected in the same tube by gene-specific PCR. MB-PCR does not require bisulfite treatment or methylation-sensitive restriction and provides a quick, simple and extremely sensitive technique allowing the detection of methylated DNA, in particular in tumor tissue or tumor cells from limited samples. Using this novel approach, we determined the methylation status of several established and candidate tumor suppressor genes and identified the ICSBP gene, encoding the myeloid and B-cell-specific transcription factor interferon consensus sequence-binding protein, as a target for aberrant hypermethylation in acute myeloid leukemia.

The Icsbp locus is a common proviral insertion site in mature B-cell lymphomas/plasmacytomas induced by exogenous murine leukemia virus

ICSBP (interferon consensus sequence binding protein)/IRF8 (interferon regulatory factor 8) is an interferon gamma-inducible transcription factor expressed predominantly in hematopoietic cells, and down-regulation of this factor has been observed in chronic myelogenous leukemia and acute myeloid leukemia in man. By screening about 1200 murine leukemia virus (MLV)-induced lymphomas, we found proviral insertions at the Icsbp locus in 14 tumors, 13 of which were mature B-cell lymphomas or plasmacytomas. Only one was a T-cell lymphoma, although such tumors constituted about half of the samples screened. This indicates that the Icsbp locus can play a specific role in the development of mature B-lineage malignancies. Two proviral insertions in the last Icsbp exon were found to act by a poly(A)-insertion mechanism. The remaining insertions were found within or outside Icsbp. Since our results showed expression of Icsbp RNA and protein in all end-stage tumor samples, a simple tumor suppressor function of ICSBP is not likely. Interestingly, proviral insertions at Icsbp have not been reported from previous extensive screenings of mature B-cell lymphomas induced by endogenous MLVs. We propose that ICSBP might be involved in an early modulation of an immune response to exogenous MLVs that might also play a role in proliferation of the mature B-cell lymphomas.

Differential expression of inducible nitric oxide synthase and IL-12 between peritoneal and splenic macrophages stimulated with LPS plus IFN-γ is associated with the activation of extracellular signal-related kinase

Resident peritoneal macrophages (pMphi) are found deficient in T cell-stimulating capacity compared with the competent splenic macrophages (sMphi). Macrophages (Mphi)-derived nitric oxide (NO) and IL-12 have been shown to play crucial roles in the interaction between Mphi and T cells. To further understand differential functions between pMphi and sMphi, we focused on the production of NO and IL-12 from LPS plus IFN-gamma-activated Mphi. We demonstrated the differential expression of inducible nitric oxide synthase (iNOS) and IL-12 in pMphi and sMphi with LPS plus IFN-gamma stimulation. pMphi produced high level of NO but low level of IL-12, whereas sMphi produced high level of IL-12 but no NO. Furthermore, we demonstrated that there were no differences in IFN-gamma-induced signal transducer and activator of transcription-1 activation and consequent interferon regulatory factor-1 and interferon consensus sequence-binding protein up-regulation between pMphi and sMphi. Likewise, p38 mitogen-activated protein kinase was activated by LPS with identical kinetics in both pMphi and sMphi. However, LPS-induced extracellular signal-regulated kinase (ERK) activation was prolonged in pMphi comparing with sMphi. Moreover, we demonstrated, using inhibitor selective for ERK cascade (PD98059), that the prolonged ERK activation contributed a positive signal for iNOS expression and a negative signal for IL-12p40 expression in resident pMphi. In addition, anti-IL-10-neutralizing antibody plus indomethacin could abrogate the inhibitory effects of endogenous IL-10 and prostaglandin E2 on the production of IL-12 by resident pMphi possibly through suppressing ERK activation. Taken together, profound difference in ERK activation may account for differential LPS plus IFN-gamma responsiveness between pMphi and sMphi. High production of NO and low production of IL-12 by pMphi may contribute to its deficiency in T cell-stimulating capacity.

Crosstalk Between IFN-γ and TLR

Zhao et al. noted that when peritoneal macrophages were exposed to both interferon-γ (IFN-γ) and a ligand for Toll-like receptors (TLRs) such as lipopolysaccharide (LPS), the ligand for TLR-4, the expression and secretion of proinflammatory mediators was enhanced compared with that in cells exposed to either class of ligand alone. Synergism was also observed in an interleukin-12 (IL-12) reporter gene assay between IFN-γ and ligands for TLR-2 [peptidoglycan (PGN)] and TLR-3 (polyI:C). The activation of the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways by LPS was enhanced by application of both IFN-γ and LPS, whereas activation of the nuclear factor κB (NF-κB) pathway by LPS was not changed by the addition of IFN-γ. LPS stimulated an increase in the abundance of interferon regulator factor 8 (IRF8), and cells deficient for IRF8 showed lower production of inflammatory mediators and were deficient in ERK and JNK activation in response to LPS, PGN, or polyI:C. Introduction of IRF8 or an IRF8 mutant that lacked DNA binding activity into the knockout cells restored the activation of ERK by LPS. IRF8 and TRAF6, a ubiquitin ligase involved in TLR signaling, coimmunoprecipitated from either transfected cells or LPS-stimulated peritoneal macrophages. In transfected cells, IRF8 enhanced ubiquitination of TRAF6, and TRAF6 ubiquitination was reduced in the IRF8 knockout cells. Thus, in addition to its role in stimulating gene expression in response to IFN-γ, IRF8 also enhances signaling through the TLR family of receptors by interacting with and stimulating TRAF6. J. Zhao, H. J. Kong, H. Li, B. Huang, M. Yang, C. Zhu, M. Bogunovic, F. Zheng, L. Mayer, K. Ozato, J. Unkeless, H. Xiong, IRF-8/interferon (IFN) consensus sequence-binding protein is involved in Toll-like receptor (TLR) signaling and contributes to the cross-talk between TLR and IFN-γ signaling pathways. J. Biol. Chem. 281 , 10073-10080 (2006). [Abstract] [Full Text]

Identification of IRF-8 and IRF-1 target genes in activated macrophages

Interferon regulatory factor 1 (IRF-1) and IRF-8, also known as interferon consensus sequence binding protein (ICSBP), are important regulators of macrophage differentiation and function. These factors exert their activities through the formation of heterocomplexes. As such, they are coactivators of various interferon-inducible genes in macrophages. To gain better insights into the involvement of these two transcription factors in the onset of the innate immune response and to identify their regulatory network in activated macrophages, DNA microarray was employed. Changes in the expression profile were analyzed in peritoneal macrophages from wild type mice and compared to IRF-1 and IRF-8 null mice, before and following 4 h exposure to IFN-γ and LPS. The expression pattern of 265 genes was significantly changed (up/down) in peritoneal macrophages extracted from wild type mice following treatment with IFN-γ and LPS, while no changes in the expression levels of these genes were observed in samples of the same cell-type from both IRF-1 and IRF-8 null mice. Among these putative target genes, numerous genes are involved in macrophage activity during inflammation. The expression profile of 10 of them was further examined by quantitative RT-PCR. In addition, the promoter regions of three of the identified genes were analyzed by reporter gene assay for the ability to respond to IRF-1 and IRF-8. Together, our results suggest that both IRF-1 and IRF-8 are involved in the transcriptional regulation of these genes. We therefore suggest a broader role for IRF-1 and IRF-8 in macrophages differentiation and maturation, being important inflammatory mediators.

Toward the identification of a tolerogenic signature in IDO-competent dendritic cells

AbstractAlthough much is known about the transcriptional profiles of dendritic cells (DCs) during maturation, the molecular switches critical for the induction of a tolerogenic program in DC subsets are still obscure. We examined the gene-expression profiles of murine splenic CD8+ DCs rendered highly tolerogenic by interferon-γ (IFN-γ), which activates the enzyme indoleamine 2,3-dioxygenase (IDO, encoded by Indo) and thus initiates the immunosuppressive pathway of tryptophan catabolism. By examining the expression of a series of relevant genes in IDO+ compared with IDO- DCs, we found consistent and selective association of the IDO-competent phenotype with down-modulation of the Tyrobp gene, encoding the signaling adapter DAP12, which typically associates with activating receptors. Down-modulation of Tyrobp involved IFN consensus sequence binding protein (ICSBP), a transcription factor also known as IRF-8. In murine and human monocyte-derived DCs, silencing DAP12 expression imparted IDO functional competence to IDO- cells, whereas silencing IRF-8 in IDO+ counterparts abolished IDO expression and function. Thus, IRF-8 is required in tolerogenic DCs for the positive regulation of Indo and the negative regulation of Tyrobp. Overall, these studies reveal the occurrence of a simple and evolutionarily conserved code in the control of tolerance by an ancestral metabolic enzyme.

IRF-8/Interferon (IFN) Consensus Sequence-binding Protein Is Involved in Toll-like Receptor (TLR) Signaling and Contributes to the Cross-talk between TLR and IFN-γ Signaling Pathways

Toll-like receptor (TLR) and interferon-gamma (IFN-gamma) signaling pathways are important for both innate and adaptive immune responses. However, the cross-talk between these two signaling pathways is incompletely understood. Here we show that IFN-gamma and LPS synergistically induce the expression of proinflammatory factors, including interleukin-1 (IL-1), IL-6, IL-12, NO, and tumor necrosis factor-alpha (TNF-alpha). Comparable synergism was observed between IFN-gamma and peptidoglycan (PGN; a TLR2 ligand) and poly(I:C) (a TLR3 ligand) in the induction of IL-12 promoter activity. IFN-gamma enhanced lipopolysaccharide (LPS)-induced ERK and JNK phosphorylation but had no effect on LPS-induced NF-kappaB activation. Interestingly, we found that IRF-8-/- macrophages were impaired in the activation of LPS-induced ERK and JNK and the production of proinflammatory cytokines induced by LPS or IFN-gamma plus LPS. Retroviral transduction of IRF-8 into IRF-8-/- macrophages rescued ERK and JNK activation. Furthermore, co-immunoprecipitation experiments show that IRF-8 physically interacts with TRAF6 at a binding site between amino acid residues 356 and 305 of IRF-8. Transfection of IRF-8 enhanced TRAF6 ubiquitination, which is consistent with a physical interaction of IRF-8 with TRAF6. Taken together, the results suggest that the interaction of IRF-8 with TRAF6 modulates TLR signaling and may contribute to the cross-talk between IFN-gamma and TLR signal pathways.

Polymorphism discovery and association analyses of the interferon genes in type 1 diabetes.

BackgroundThe aetiology of the autoimmune disease type 1 diabetes (T1D) involves many genetic and environmental factors. Evidence suggests that innate immune responses, including the action of interferons, may also play a role in the initiation and/or pathogenic process of autoimmunity. In the present report, we have adopted a linkage disequilibrium (LD) mapping approach to test for an association between T1D and three regions encompassing 13 interferon alpha (IFNA) genes, interferon omega-1 (IFNW1), interferon beta-1 (IFNB1), interferon gamma (IFNG) and the interferon consensus-sequence binding protein 1 (ICSBP1).ResultsWe identified 238 variants, most, single nucleotide polymorphisms (SNPs), by sequencing IFNA, IFNB1, IFNW1 and ICSBP1, 98 of which where novel when compared to dbSNP build 124. We used polymorphisms identified in the SeattleSNP database for INFG. A set of tag SNPs was selected for each of the interferon and interferon-related genes to test for an association between T1D and this complex gene family. A total of 45 tag SNPs were selected and genotyped in a collection of 472 multiplex families.ConclusionWe have developed informative sets of SNPs for the interferon and interferon related genes. No statistical evidence of a major association between T1D and any of the interferon and interferon related genes tested was found.

Interferon-γ induces regression of epithelial cell carcinoma: critical roles of IRF-1 and ICSBP transcription factors

We have developed an epithelial cell carcinoma model for studying efficacy of IFNgamma gene therapy and have identified components of IFNgamma-signaling pathway responsible for its direct anti-tumor actions. The tumor results from ectopic expression of SV40 Large T-Antigen (SV40 T-Ag) oncogene in lens of transgenic mouse (alphaT3) and complete regression of the tumor is induced by targeting expression of IFNgamma into malignant lens cells. Inflammatory cells are absent in lens of alphaT3 or DT (co-expressing IFNgamma and SV40-T-Antigen) mice and the transformed lens cells are non-immunogenic, suggesting non-involvement of immunologic cells. We show that IFNgamma has direct growth-inhibitory effects on tumor cells, induces death of tumor cells by apoptosis and that these effects are mediated by two transcription factors, IRF-1 (interferon-regulatory factor-1) and ICSBP (interferon-consensus sequence-binding protein) induced by IFNgamma. Furthermore, stable transfection with ICSBP or IRF-1 construct inhibits lens carcinoma cell growth by upregulating Caspase-1, p21(WAF1) and p27 expression. In contrast, tumor progression in alphaT3 lens correlates with inhibition of IRF-1 and ICSBP expression. Our results suggest that IFNgamma gene therapy maybe effective in malignant diseases for which DNA tumor viruses are etiologic agents and that antitumor actions of IRF-1/ICSBP can be exploited therapeutically to circumvent adverse clinical effects associated with IFN therapy.

Cellular and Molecular Mechanisms of the Selective Regulation of IL-12 Production by 12/15-Lipoxygenase

IL-12 drives type I immune responses and can mediate chronic inflammation that leads to host defense as well as disease. Recently, we discovered a novel role for 12/15-lipoxygenase (12/15-LO) in mediating IL-12p40 expression in atherosclerotic plaque and in isolated macrophages. We now demonstrate that 12/15-LO regulates IL-12 family cytokine production in a cell-type and stimulus-restricted fashion. LPS-stimulated elicited peritoneal macrophages derived from 12/15-LO-deficient (Alox15) mice produced reduced IL-12 and IL-23 levels, but comparable amounts of several other inflammatory mediators tested. Furthermore, LPS stimulation triggered an increase in wild-type macrophage 12/15-LO activity, whereas pharmacological inhibition of 12/15-LO activity suppressed LPS-induced IL-12 production in wild-type macrophages. 12/15-LO-deficient macrophages also produced reduced levels of IL-12 in response to TLR2 stimulation, but not in response to CpG (TLR9) or CD40/CD40L-mediated activation. In contrast to our previous finding of reduced IL-12 production in the setting of atherosclerosis, we found that comparable IL-12 levels were produced in Alox15 and wild-type mice during an acute response to LPS in vivo. This paradox may be explained by normal production of IL-12 by 12/15-LO-deficient neutrophils and dendritic cells, which are major sources of IL-12 during acute inflammation. Finally, we detected selectively decreased association of the transcription factors IFN consensus sequence binding protein and NF-kappaB with the IL-12p40 promoter in 12/15-LO-deficient macrophages. Taken together, these findings reveal a highly selective pathway to IL-12 production that may prove a useful target in chronic inflammation while sparing the acute response to infection.

ICSBP Activates Transcription of the Gene Encoding Neurofibromin 1 in Differentiating Myeloid Cells.

Previous studies in murine models and human myeloid malignancies indicate that the interferon consensus sequence binding protein (ICSBP) functions as a leukemia tumor suppressor. Previously identified ICSBP target genes include genes encoding gp91phox and p67phox (components of the phagocyte respiratory burst oxidase), the Toll like receptor 4, and other genes involved in mediating the phagocyte inflammatory response. Transcription of these target genes involves interaction of ICSBP with PU.1 and/or interferon regulatory factor 1 (IRF1), and is restricted to mature phagocytic cells. These results predict that ICSBP-deficiency would be associated with terminal differentiation block and immune dysfunction. However, the phenotype of ICSBP −/− mice is dominated by development of a myeloproliferative disorder which evolves to acute myeloid leukemia. Therefore, we used the chromatin immuno-precipitation technique to identify novel ICSBP target genes which might be involved in the ICSBP tumor suppressor effect. We identified the gene encoding neurofibromin 1 (the NF1 gene) as an ICSBP-target-gene. We found that ICSBP activates NF1 transcription during cytokine induced differentiation of myeloid progenitor cells. Because Nf1 protein has ras-GAP activity, increased Nf1-expression down regulates cytokine-induced ras activity, resulting in proliferation arrest during myeloid cell differentiation. In the current studies, we investigate the mechanism by which ICSBP activates NF1-transcription. We find that ICSBP is part of a multi protein complex that binds to and activates a cis element in the NF1 gene. This complex also includes the ets protein PU.1 and interferon regulatory factor 2 (IRF2). We find that tyrosine phosphorylation of ICSBP in response to hematopoietic cytokines is required for assembly of this transcriptional activation complex and therefore NF1-transcription. Identification of NF1 as an ICSBP target gene represents a mechanism for ICSBP-leukemia-suppression. These studies also provide the first indication of the importance of NF1-transcriptional regulation to the control of cytokine-induced proliferation in differentiating myeloid cells.

Immune-Mediated Protection Against BCR/ABL-Induced Leukemia: A Common Pathway Shared between IRF8/ICSBP and IFN alpha and beta.

Most Chronic Myeloid Leukemia (CML) patients are treated with the targeted agent imatinib (Gleevec), but cytogenetic remission is rarely achieved and the only cure remains bone marrow transplantation. The mainstay of therapy prior to imatinib was interferon (IFN) alpha, which induces cytogenic remission in a minority of patients. Because clinical response to IFN alpha correlates with immune system reactivity against leukemic clones, immunotherapy may be an important adjunct to imatinib. ICSBP (Interferon Consensus Sequence Binding Protein) is a tumor suppressor of CML. Ectopic expression of ICSBP in Bcr-Abl transformed cells prevents the formation of a lethal leukemia, and we have shown that this anti-leukemic effect is mediated by a long-lasting and potent CD8+ response against unknown epitopes on the leukemic cells. We have begun to characterize the immune response against CML induced by ICSBP and to understand its relationship to the anti-leukemic effects of IFN alpha. IFN alpha and IFN beta can substitute for ICSBP expression in inducing potent anti-leukemic immunity against BCR-ABL transformed cells. We could find little evidence for the direct regulation of the IFN genes by ICSBP; thus we have favored the alternative hypothesis that ICSBP and IFNs alpha and beta activate a common pathway that ultimately triggers an immune-mediated anti-leukemic response. To test this hypothesis, we compared the transcriptional profiles of BaF3 cells to those overexpressing Bcr-Abl, ICSBP, or both (unpublished data). Our data confirm the reported effects of ICSBP on genes that regulate proliferation and apoptosis in cells overexpressing Bcr-Abl (e.g. cyclin D2). In addition, our analysis revealed an interesting new mechanism through which ICSBP may stimulate an anti-leukemic immune response and Bcr-Abl expressing cells may escape immune detection. IFN alpha and beta appear to share the mechanism induced by ICSBP. Our data confirm the differential expression of several known (e.g. Proteinase 3) as well as new potential protein epitopes of the anti-leukemic immune response induced by ICSBP and the IFNs. Our in vivo experiments should shed further light on the role of the immune regulatory genes we have identified in the immune response to CML.

Transcription Factor ICSBP/IRF8 Regulates B Cell Development at Multiple Checkpoints.

The generation of lymphoid and myeloid lineage cells from hematopoietic stem cells is controlled by multiple transcription factors regulating distinct developmental and functional aspects. Interferon consensus sequence binding protein (ICSBP)/interferon regulatory factor 8 (IRF8) is a transcription factor known to regulate the differentiation of macrophages, granulocytes, and dendritic cells. Our recent findings that IRF8 transcripts and protein are highly expressed in germinal center (GC) B cells suggest that IFR8 may also play a role in normal B cell development. In IRF8 deficient mice, the number of early B lineage cells (pre-pro-B) was reduced by 5-fold, indicating a defect in early B lineage commitment. While the numbers of late pre-B and immature B cells were moderately reduced (~2-fold), recirculating mature B cells were almost undetectable in the bone marrow of mutant mice. This deficiency in early stage B cells is correlated with increased expression of PU.1, a crucial transcription factor for myeloid and lymphoid lineage specification. Interestingly, the number of splenic transitional 1 (T1) cells was slightly increased but the numbers of T2 and follicular (FO) B-2 cells were moderately decreased in mutant mice. This indicates that positive selection of T2 cells into the mature B-2 pool is regulated by IRF8. The marginal zone (MZ) B cell and peritoneal CD11b+ B-1b cell compartments were also slightly expanded in IRF8 knockout mice. Overall, these results provide compelling evidence that IRF8 regulates B cell differentiation and function at multiple stages.

Identification of target genes and a unique cis element regulated by IRF-8 in developing macrophages

AbstractInterferon regulatory factor-8 (IRF-8)/interferon consensus sequence–binding protein (ICSBP) is a transcription factor that controls myeloid-cell development. Microarray gene expression analysis of Irf-8-/- myeloid progenitor cells expressing an IRF-8/estrogen receptor chimera (which differentiate into macrophages after addition of estradiol) was used to identify 69 genes altered by IRF-8 during early differentiation (62 up-regulated and 7 down-regulated). Among them, 4 lysosomal/endosomal enzyme-related genes (cystatin C, cathepsin C, lysozyme, and prosaposin) did not require de novo protein synthesis for induction, suggesting that they were direct targets of IRF-8. We developed a reporter assay system employing a self-inactivating retrovirus and analyzed the cystatin C and cathepsin C promoters. We found that a unique cis element mediates IRF-8–induced activation of both promoters. Similar elements were also found in other IRF-8 target genes with a consensus sequence (GAAANN[N]GGAA) comprising a core IRF-binding motif and an Ets-binding motif; this sequence is similar but distinct from the previously reported Ets/IRF composite element. Chromatin immunoprecipitation assays demonstrated that IRF-8 and the PU.1 Ets transcription factor bind to this element in vivo. Collectively, these data indicate that IRF-8 stimulates transcription of target genes through a novel cis element to specify macrophage differentiation.

BCR/ABL Promotes Dendritic Cell–Mediated Natural Killer Cell Activation

BCR/ABL fusion gene, encoding a paradigmatic tyrosine kinase involved in chronic myelogenous leukemia (CML), can modulate the expression of genes involved in natural killer (NK) cell target recognition. Recent reports outline the role of allogeneic antileukemic NK effectors in the graft-versus-leukemia effect but the regulation of NK cell activation in the setting of graft-versus-leukemia effect remains unknown. Here we show that dendritic cells derived from monocytes of CML patients are selectively endowed with NK cell stimulatory capacity in vitro. We further show, using a gene transfer approach in mouse bone marrow progenitors, that ABL/ABL is necessary to promote dendritic cell-mediated NK cell activation. The dendritic cell/NK cell cross-talk in ABL/ABL-induced CML seems unique because JunB or IFN consensus sequence binding protein loss of functions, associated with other myeloproliferative disorders, do not promote dendritic cell-mediated NK cell activation. NK cell activation by leukemic dendritic cells involves NKG2D activating receptors and is blocked by imatinib mesylate. Indeed, ABL/ABL translocation enhances the expression levels of the NKG2D ligands on dendritic cells, which is counteracted by imatinib mesylate. Altogether, the clonal ABL/ABL dendritic cells display the unique and selective ability to activate NK cells and may participate in the NK cell control of CML. This study also highlights the deleterious role of imatinib mesylate at the dendritic cell level for NK cell activation.

Heat‐shock response down‐regulates interleukin‐18 expression in murine peritoneal macrophages

The heat-shock response is a self-defence mechanism that protects cells and organisms from a wide range of harmful stresses. Recent studies revealed that it involved the regulation of cytokine expression. Interleukin-18 (IL-18) is an important cytokine in mediating immune response. We studied interferon-gamma (IFN-gamma)-induced IL-18 expression in heat-shock-treated murine peritoneal macrophages. Our results showed that the heat-shock response significantly inhibited the expression of IFN-gamma-induced pro-inflammatory cytokine IL-18. Interferon consensus sequence binding protein (ICSBP) is a transcription factor that binds to the promoter of IL-18 and regulates the transcription of IL-18. Further research on the down-regulation mechanism showed that the DNA-binding activity of ICSBP was greatly reduced by the heat shock response. These results suggest that the inhibitory effect of heat-shock response on IL-18 production in IFN-gamma-stimulated macrophages is related to the suppression of the binding activity of ICSBP.

LPS regulates a set of genes in primary murine macrophages by antagonising CSF-1 action

We previously reported that bacterial products such as LPS and CpG DNA down-modulated cell surface levels of the Colony Stimulating Factor (CSF)-1 receptor (CSF-1R) on primary murine macrophages in an all-or-nothing manner. Here we show that the ability of bacterial products to down-modulate the CSF-1R rendered bone marrow-derived macrophages (BMM) unresponsive to CSF-1 as assessed by Akt and ERK1/2 phosphorylation. Using toll-like receptor ( tlr) 9 as a model CSF-1-repressed gene, we show that LPS induced tlr9 expression in BMM only when CSF-1 was present, suggesting that LPS relieves CSF-1-mediated inhibition to induce gene expression. Using cDNA micro-arrays, we identified a cluster of similarly CSF-1 repressed genes in BMM. By real time PCR we confirmed that the expression of a selection of these genes, including integral membrane protein 2B ( itm2b), receptor activity-modifying protein 2 ( ramp2) and macrophage-specific gene 1 ( mpg-1), were repressed by CSF-1 and were induced by LPS only in the presence of CSF-1. This pattern of gene regulation was also apparent in thioglycollate-elicited peritoneal macrophages (TEPM). LPS also counteracted CSF-1 action to induce mRNA expression of a number of transcription factors including interferon consensus sequence binding protein 1 (Icsbp1), suggesting that this mechanism leads to transcriptional reprogramming in macrophages. Since the majority of in vitro studies on macrophage biology do not include CSF-1, these genes represent a set of previously uncharacterised LPS-inducible genes. This study identifies a new mechanism of macrophage activation, in which LPS (and other toll-like receptor agonists) regulate gene expression by switching off the CSF-1R signal. This finding also provides a biological relevance to the well-documented ability of macrophage activators to down-modulate surface expression of the CSF-1R.