Transcription factors IRF8 and PU.1 are required for follicular B cell development and BCL6-driven germinal center responses.

The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells.

Cutting Edge: Expression of IRF8 in Gastric Epithelial Cells Confers Protective Innate Immunity against Helicobacter pylori Infection.

IFN regulatory factor 8 (IRF8) is expressed in many types of blood cells and plays critical roles in cellular differentiation and function. However, the role of IRF8 in nonhematopoietic systems remains poorly understood. In this study, we provide evidence that IRF8 is a transcriptional modulator of the gastric mucosa necessary for limiting Helicobacter pylori colonization. H. pylori infection significantly upregulated expression of IRF8, which, in turn, promoted IFN-γ expression by gastric epithelial cells. Mice deficient in IRF8 exhibited increased H. pylori colonization and aborted induction of mucosal IFN-γ. Genome-wide analyses of IFN-γ-treated gastric epithelial cells by chromatin immunoprecipitation sequencing and RNA sequencing led to the identification of IRF8 target genes, with many belonging to the IFN-regulated gene family that was observed previously in immune cells. Our results identify the IRF8-IFN-γ circuit as a novel gastric innate immune mechanism in the host defense against infection with H. pylori.

A reporter mouse reveals lineage-specific and heterogeneous expression of IRF8 during lymphoid and myeloid cell differentiation.

The IFN regulatory factor family member 8 (IRF8) regulates differentiation of lymphoid and myeloid lineage cells by promoting or suppressing lineage-specific genes. How IRF8 promotes hematopoietic progenitors to commit to one lineage while preventing the development of alternative lineages is not known. In this study, we report an IRF8-EGFP fusion protein reporter mouse that revealed previously unrecognized patterns of IRF8 expression. Differentiation of hematopoietic stem cells into oligopotent progenitors is associated with progressive increases in IRF8-EGFP expression. However, significant induction of IRF8-EGFP is found in granulocyte-myeloid progenitors and the common lymphoid progenitors but not the megakaryocytic-erythroid progenitors. Surprisingly, IRF8-EGFP identifies three subsets of the seemingly homogeneous granulocyte-myeloid progenitors with an intermediate level of expression of EGFP defining bipotent progenitors that differentiation into either EGFP(hi) monocytic progenitors or EGFP(lo) granulocytic progenitors. Also surprisingly, IRF8-EGFP revealed a highly heterogeneous pre-pro-B population with a fluorescence intensity ranging from background to 4 orders above background. Interestingly, IRF8-EGFP readily distinguishes true B cell committed (EGFP(int)) from those that are noncommitted. Moreover, dendritic cell progenitors expressed extremely high levels of IRF8-EGFP. Taken together, the IRF8-EGFP reporter revealed previously unrecognized subsets with distinct developmental potentials in phenotypically well-defined oligopotent progenitors, providing new insights into the dynamic heterogeneity of developing hematopoietic progenitors.

Characterization of ARF-BP1/HUWE1 interactions with CTCF, MYC, ARF and p53 in MYC-driven B cell neoplasms.

Transcriptional activation of MYC is a hallmark of many B cell lineage neoplasms. MYC provides a constitutive proliferative signal but can also initiate ARF-dependent activation of p53 and apoptosis. The E3 ubiquitin ligase, ARF-BP1, encoded by HUWE1, modulates the activity of both the MYC and the ARF-p53 signaling pathways, prompting us to determine if it is involved in the pathogenesis of MYC-driven B cell lymphomas. ARF-BP1 was expressed at high levels in cell lines from lymphomas with either wild type or mutated p53 but not in ARF-deficient cells. Downregulation of ARF-BP1 resulted in elevated steady state levels of p53, growth arrest and apoptosis. Co-immunoprecipitation studies identified a multiprotein complex comprised of ARF-BP1, ARF, p53, MYC and the multifunctional DNA-binding factor, CTCF, which is involved in the transcriptional regulation of MYC, p53 and ARF. ARF-BP1 bound and ubiquitylated CTCF leading to its proteasomal degradation. ARF-BP1 and CTCF thus appear to be key cofactors linking the MYC proliferative and p53-ARF apoptotic pathways. In addition, ARF-BP1 could be a therapeutic target for MYC-driven B lineage neoplasms, even if p53 is inactive, with inhibition reducing the transcriptional activity of MYC for its target genes and stabilizing the apoptosis-promoting activities of p53.

ATP-degrading ENPP1 is required for survival (or persistence) of long-lived plasma cells.

Survival of antibody-secreting plasma cells (PCs) is vital for sustained antibody production. However, it remains poorly understood how long-lived PCs (LLPCs) are generated and maintained. Here we report that ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is preferentially upregulated in bone marrow LLPCs compared with their splenic short-lived counterparts (SLPCs). We studied ENPP1-deficient mice (Enpp1 -/- ) to determine how the enzyme affects PC biology. Although Enpp1 -/- mice generated normal levels of germinal center B cells and plasmablasts in periphery, they produced significantly reduced numbers of LLPCs following immunization with T-dependent antigens or infection with plasmodium C. chabaudi. Bone marrow chimeric mice showed B cell intrinsic effect of ENPP1 selectively on generation of bone marrow as well as splenic LLPCs. Moreover, Enpp1 -/- PCs took up less glucose and had lower levels of glycolysis than those of wild-type controls. Thus, ENPP1 deficiency confers an energetic disadvantage to PCs for long-term survival and antibody production.

Homeostatic defects in B cells deficient in the E3 ubiquitin ligase ARF-BP1 are restored by enhanced expression of MYC.

The E3 ligase ARF-BP1 governs the balance of life and death decisions by directing the degradation of p53 and enhancing the transcriptional activity of MYC. We find B cells selectively deficient in ARF-BP1 have many defects in developing and mature B cells associated with increased expression of p53 and reduced expression of Myc. Overexpression of Myc results in suppression of p53 and complete reversal of defects induced by ARF-BP1 deficiency. These findings indicate that the dynamic balance between MYC and p53 required for normal B cell maturation and function is finely tuned and critically dependent on the activities of ARF-BP1.