The Ontogeny of a Neutrophil: Mechanisms of Granulopoiesis and Homeostasis.

Comprising the majority of leukocytes in humans, neutrophils are the first immune cells to respond to inflammatory or infectious etiologies and are crucial participants in the proper functioning of both innate and adaptive immune responses. From their initial appearance in the liver, thymus, and spleen at around the eighth week of human gestation to their generation in large numbers in the bone marrow at the end of term gestation, the differentiation of the pluripotent hematopoietic stem cell into a mature, segmented neutrophil is a highly controlled process where the transcriptional regulators C/EBP-α and C/EBP-ε play a vital role. Recent advances in neutrophil biology have clarified the life cycle of these cells and revealed striking differences between neonatal and adult neutrophils based on fetal maturation and environmental factors. Here we detail neutrophil ontogeny, granulopoiesis, and neutrophil homeostasis and highlight important differences between neonatal and adult neutrophil populations.

Review: Transcriptional Regulation of CD4+ T Cell Differentiation in Experimentally Induced Arthritis and Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic inflammation of the joint synovium and infiltration by activated inflammatory cells. CD4+ T cells form a large proportion of the inflammatory cells invading the synovial tissue, and are involved in the RA pathologic process. In general, CD4+ T cells differentiate into various T helper cell subsets and acquire the functional properties to respond to specific pathogens, and also mediate some autoimmune disorders such as RA. Because the differentiation of T helper cell subsets is determined by the expression of specific transcription factors in response to the cytokine environment, these transcription factors are considered to have a role in the pathology of RA. Treg cells control an excess of T cell-mediated immune response, and the transcription factor FoxP3 is critical for the differentiation and function of Treg cells. Treg cell dysfunction can result in the development of systemic autoimmunity. In this review, we summarize how the expression of transcription factors modulates T helper cell immune responses and the development of autoimmune diseases, especially in RA. Understanding the role of transcription factors in the pathogenesis of autoimmunity may lead to novel therapeutic strategies to control the differentiation and function of both T helper cells and Treg cells.

LPS injection reprograms the expression and the 3' UTR of a CAP gene by alternative polyadenylation and the formation of a GAIT element in Ciona intestinalis.

The diversification of cellular functions is one of the major characteristics of multicellular organisms which allow cells to modulate their gene expression, leading to the formation of transcripts and proteins with different functions and concentrations in response to different stimuli. CAP genes represent a widespread family of proteins belonging to the cysteine-rich secretory protein, antigen 5 and pathogenesis-related 1 superfamily which, it has been proposed, play key roles in the infection process and the modulation of immune responses in host animals. The ascidian Ciona intestinalis represents a group of proto-chordates with an exclusively innate immune system that has been widely studied in the field of comparative and developmental immunology. Using this biological system, we describe the identification of a novel APA mechanism by which an intronic polyadenylation signal is activated by LPS injection, leading to the formation of a shorter CAP mRNA capable of expressing the first CAP exon plus 19 amino acid residues whose sequence is contained within the first intron of the annotated gene. Furthermore, such an APA event causes the expression of a translational controlling cis-acting GAIT element which is not present in the previously isolated CAP isoform and identified in the 3'-UTR of other immune-related genes, suggesting an intriguing scenario in which both transcriptional and post-transcriptional control mechanisms are involved in the activation of the CAP gene during inflammatory response in C. intestinalis.

Enhancer sequence variants and transcription-factor deregulation synergize to construct pathogenic regulatory circuits in B-cell lymphoma.

Most B-cell lymphomas arise in the germinal center (GC), where humoral immune responses evolve from potentially oncogenic cycles of mutation, proliferation, and clonal selection. Although lymphoma gene expression diverges significantly from GC B cells, underlying mechanisms that alter the activities of corresponding regulatory elements (REs) remain elusive. Here we define the complete pathogenic circuitry of human follicular lymphoma (FL), which activates or decommissions REs from normal GC B cells and commandeers enhancers from other lineages. Moreover, independent sets of transcription factors, whose expression was deregulated in FL, targeted commandeered versus decommissioned REs. Our approach revealed two distinct subtypes of low-grade FL, whose pathogenic circuitries resembled GC B or activated B cells. FL-altered enhancers also were enriched for sequence variants, including somatic mutations, which disrupt transcription-factor binding and expression of circuit-linked genes. Thus, the pathogenic regulatory circuitry of FL reveals distinct genetic and epigenetic etiologies for GC B-cell transformation.

IFN-α directly promotes programmed cell death-1 transcription and limits the duration of T cell-mediated immunity.

Programmed cell death-1 (PD-1) is an inhibitory coreceptor for T lymphocytes that provides feedback inhibition of T cell activation. Although PD-1's expression on T cells is known to be activation dependent, the factors that determine the timing, intensity, and duration of PD-1 expression in immune reactions are not fully understood. To address this question, we performed a fine mapping analysis of a conserved 5'-flanking region of the PD-1 gene and identified a putative IFN stimulation response element, which was responsible for PD-1 transcription in the 2B4.11 T cell line. Consistent with this finding, activation by IFN-α enhanced both the induction and maintenance of PD-1 expression on TCR-engaged primary mouse T cells through an association IFN-responsive factor 9 (IRF9) to the IFN stimulation response element. Furthermore, PD-1 expression on Ag-specific CD8(+) T cells was augmented by IFN-α in vivo. We propose that strong innate inflammatory responses promote primary T cell activation and their differentiation into effector cells, but also cause an attenuated T cell response in sustained immune reactions, at least partially through type I IFN-mediated PD-1 transcription. Based on this idea, we demonstrate that IFN-α administration in combination with PD-1 blockade in tumor-bearing mice effectively augments the antitumor immunity, and we propose this as a novel and rational approach for cancer immunotherapy.

Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells.

The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non-DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex-binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs.

Immunoglobulin genes and their transcriptional control in teleosts.

Immunoglobulin (Ig), which exists only in jawed vertebrates, is one of the most important molecules in adaptive immunity. In the last two decades, many teleost Ig genes have been identified by in silico data mining from the enormous gene and EST databases of many fish species. In this review, the organization of Ig gene segments, the expressed Ig isotypes and their transcriptional controls are discussed. The Ig heavy chain (IgH) locus in teleosts encodes the variable (V), the diversity (D), the joining (J) segments and three different isotypic constant (C) regions including Cµ, Cδ, and Cζ/τ genes, and is organized as a "translocon" type like the IgH loci of higher vertebrates. In contrast, the Ig light (L) chain locus is arranged in a "multicluster" or repeating set of VL, JL, and CL segments. The IgL chains have four isotypes; two κ L1/G and L3/F), σ (L2) and λ. The transcription of IgH genes in teleosts is regulated by a VH promoter and the Eµ3' enhancer, which both function in a B cell-specific manner. The location of the IgH locus, structure and transcriptional function of the Eµ3' enhancer are important to our understanding of the evolutional changes that have occurred in the IgH gene locus.

Bcl11b represses a mature T-cell gene expression program in immature CD4(+)CD8(+) thymocytes.

Bcl11b is a transcription factor that, within the hematopoietic system, is expressed specifically in T cells. Although Bcl11b is required for T-cell differentiation in newborn Bcl11b-null mice, and for positive selection in the adult thymus of mice bearing a T-cell-targeted deletion, the gene network regulated by Bcl11b in T cells is unclear. We report herein that Bcl11b is a bifunctional transcriptional regulator, which is required for the correct expression of approximately 1000 genes in CD4(+)CD8(+)CD3(lo) double-positive (DP) thymocytes. Bcl11b-deficient DP cells displayed a gene expression program associated with mature CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes, including upregulation of key transcriptional regulators, such as Zbtb7b and Runx3. Bcl11b interacted with regulatory regions of many dysregulated genes, suggesting a direct role in the transcriptional regulation of these genes. However, inappropriate expression of lineage-associated genes did not result in enhanced differentiation, as deletion of Bcl11b in DP cells prevented development of SP thymocytes, and that of canonical NKT cells. These data establish Bcl11b as a crucial transcriptional regulator in thymocytes, in which Bcl11b functions to prevent the premature expression of genes fundamental to the SP and NKT cell differentiation programs.

Distal regions of the human IFNG locus direct cell type-specific expression.

Genes, such as IFNG, which are expressed in multiple cell lineages of the immune system, may employ a common set of regulatory elements to direct transcription in multiple cell types or individual regulatory elements to direct expression in individual cell lineages. By employing a bacterial artificial chromosome transgenic system, we demonstrate that IFNG employs unique regulatory elements to achieve lineage-specific transcriptional control. Specifically, a one 1-kb element 30 kb upstream of IFNG activates transcription in T cells and NKT cells but not in NK cells. This distal regulatory element is a Runx3 binding site in Th1 cells and is needed for RNA polymerase II recruitment to IFNG, but it is not absolutely required for histone acetylation of the IFNG locus. These results support a model whereby IFNG uses cis-regulatory elements with cell type-restricted function.

PU.1 binds to a distal regulatory element that is necessary for B cell-specific expression of CIITA.

The transcriptional coactivator CIITA regulates MHC class II genes. In the mouse, CIITA is expressed from three distinct promoters (pI, pIII, and pIV) in a developmental and cell type-specific manner with pIII being responsible for B lymphocyte-specific expression. Although the promoter proximal sequences that regulate CIITA in B cells have been described, nothing is known about additional distal elements that may regulate its expression in B cells. Sequence homology comparisons, DNase I hypersensitivity assays, and histone modification analysis revealed a potential regulatory element located 11 kb upstream of pIII. Deletion of this element, termed hypersensitive site 1 (HSS1), in a bacterial artificial chromosome encoding the entire CIITA locus and surrounding genes, resulted in a complete loss of CIITA expression from the bacterial artificial chromosome following transfection into B cells. HSS1 and pIII displayed open chromatin architecture features in B cell but not in plasma cell lines, which are silenced for CIITA expression. PU.1 was found to bind HSS1 and pIII in B cells but not in plasma cells. Depletion of PU.1 by short hairpin RNA reduced CIITA expression. Chromatin conformation capture assays showed that HSS1 interacted directly with pIII in B cells and that PU.1 was important for this interaction. These results provide evidence that HSS1 is required for B cell-specific expression of CIITA and that HSS1 functions by interacting with pIII, forming a long-distance chromatin loop that is partly mediated through PU.1.

PU.1 positively regulates GATA-1 expression in mast cells.

Coexpression of PU.1 and GATA-1 is required for proper specification of the mast cell lineage; however, in the myeloid and erythroid lineages, PU.1 and GATA-1 are functionally antagonistic. In this study, we report a transcriptional network in which PU.1 positively regulates GATA-1 expression in mast cell development. We isolated a variant mRNA isoform of GATA-1 in murine mast cells that is significantly upregulated during mast cell differentiation. This isoform contains an alternatively spliced first exon (IB) that is distinct from the first exon (IE) incorporated in the major erythroid mRNA transcript. In contrast to erythroid and megakaryocyte cells, in mast cells we show that PU.1 and GATA-2 predominantly occupy potential cis-regulatory elements in the IB exon region in vivo. Using reporter assays, we identify an enhancer flanking the IB exon that is activated by PU.1. Furthermore, we observe that in PU.1(-/-) fetal liver cells, low levels of the IE GATA-1 isoform is expressed, but the variant IB isoform is absent. Reintroduction of PU.1 restores variant IB isoform and upregulates total GATA-1 protein expression, which is concurrent with mast cell differentiation. Our results are consistent with a transcriptional hierarchy in which PU.1, possibly in concert with GATA-2, activates GATA-1 expression in mast cells in a pathway distinct from that seen in the erythroid and megakaryocytic lineages.

Mechanisms of self-inactivation in anergic T cells

Las células T autorreactivas que escapan al proceso de selección negativa en el timo han de ser inactivadas o eliminadas en la periferia. En respuesta a una estimulación parcial o subóptima, las células T se vuelvenanérgicas e incapaces de proliferar y producir citocinas en respuesta aencuentros posteriores con el antígeno. Las señales mediadas por calcio tienen un papel importante en la inducción de anergia, por medio de la activación de un programa de auto-inactivación intrínseco a la célula dependiente de calcio/calcineurina/NFAT. Esta revisión se centra en la descripción de nuestros conocimientos actuales acerca de los mecanismos reguladores de la expresión de un programa de expresión génica específico dela anergia de las células T, y cómo las proteínas codificadas por esos genesimponen un estado funcional falta de respuesta a nuevos estimulos. Estose lleva a cabo mediante la localización y la modulación de la actividadde sucesos cruciales para la activación de las células T, incluyendo fenó-menos como la atenuación de las señales del receptor de linfocitos T (TCR)y la inhibición de la transcripción de citocinas (AU)

Self-reactive T cells that escape negative selection in the thymus mustbe inactivated or eliminated in the periphery. In response to a partial orsuboptimal stimulation, T cells become anergic and unable to proliferate and express cytokines in response subsequent re-encounters with antigen. Calcium signaling plays a central role in the induction of anergy, causing the activation of a calcium/calcineurin/NFAT-dependent cell-intrinsic program of self-inactivation. This review will focus on our currentknowledge on the mechanisms that regulate the expression of an anergyspecific program of gene expression in T cells, and how the proteins encoded by those genes impose a state of functional unresponsiveness by targeting and modulating the activity of crucial events required for the activation of T cells, which include downregulation of TCR signaling andinhibition of cytokine transcription (AU)

In vivo redundant function of the 3' IgH regulatory element HS3b in the mouse.

In the mouse, the regulatory region located at the 3' end of the IgH locus includes four transcriptional enhancers: HS3a, HS1-2, HS3b, and HS4; the first three lie in a quasi-palindromic structure. Although the upstream elements HS3a and HS1-2 proved dispensable for Ig expression and class switch recombination (CSR), the joint deletion of HS3b and HS4 led to a consistent decrease in IgH expression in resting B cells and to a major CSR defect. Within this pair of distal enhancers, it was questionable whether HS3b and HS4 could be considered individually as elements critical for IgH expression and/or CSR. Studies in HS4-deficient mice recently revealed the role of HS4 as restricted to Igmicro-chain expression from the pre-B to the mature B cell stage and left HS3b as the last candidate for CSR regulation. Our present study finally invalidates the hypothesis that CSR could mostly rely on HS3b itself. B cells from HS3b-deficient animals undergo normal proliferation, germline transcription, and CSR upon in vitro stimulation with LPS; in vivo Ag-specific responses are not affected. In conclusion, our study highlights a major effect of the global ambiance of the IgH locus; enhancers demonstrated as being strongly synergistic in transgenes turn out to be redundant in their endogenous context.

Early signal protein expression profiles in basophils: a population study.

IgE-mediated histamine release from peripheral blood basophils is highly variable within the general population. Recent studies have shown that the ability of anti-IgE antibody to induce release can be predicted reasonably well by knowing the level of syk expression in the cells. The current study expands a previous survey to include 14 additional early elements known to be involved in activation and deactivation of basophils and showed that with the exception of syk, the variance of expression of 19 other elements (lyn, fyn, csk, cbp/PAG, CIN85, Bob1, c-cbl, SHIP1, SHIP2, p85alpha, p110delta, btk, PLCgamma1, PLCgamma2, SHP-1, PTEN, SOS2, CRACM1, and IL-3Ralpha) was narrow despite a broad range of functional capability in the basophils under study. With syk as the only element with high variance and well-correlated to maximum histamine release and cellular sensitivity, this survey examined the expression levels of two proteins thought to regulate syk expression: Bob1/OCA-B and CIN85. Expression of CIN85 was not correlated to syk expression, but Bob1 expression was negatively correlated to expression of syk and maximum histamine release. However, the expected behavior for this protein should have been as a protector of post-translational syk loss and therefore, positively correlated. Previous studies suggested that post-translational control mechanisms regulated syk expression. However, in this study, steady-state mRNA levels for syk in resting basophils showed a correlation with syk protein expression levels (r=0.593). It is concluded that with the exception of syk expression, the expression of 19 early signaling elements is tightly regulated and that a component of the regulation of syk may be related to control of transcription or processing of syk mRNA.

CTCF-independent, but not CTCF-dependent, elements significantly contribute to TCR-alpha locus control region activity.

The mouse TCRalpha/TCRdelta/Dad1 gene locus bears a locus control region (LCR) that drives high-level, position-independent, thymic transgene expression in chromatin. It achieves this through DNA sequences that enhance transcription and protect transgene expression from integration site-dependent position effects. The former activity maps to a classical enhancer region (Ealpha). In contrast, the elements supporting the latter capacity that suppresses position effects are incompletely understood. Such elements likely play important roles in their native locus and may resemble insulator/boundary sequences. Insulators support enhancer blocking and/or chromatin barrier activity. Most vertebrate enhancer-blocking insulators are dependent on the CTCF transcription factor and its cognate DNA binding site. However, studies have also revealed CTCF-independent enhancer blocking and barrier insulator activity in the vertebrate genome. The TCRalpha LCR contains a CTCF-dependent and multiple CTCF-independent enhancer-blocking regions whose roles in LCR activity are unknown. Using randomly integrated reporter transgenes in mice, we find that the CTCF region plays a very minor role in LCR function. In contrast, we report the in vivo function of two additional downstream elements located in the region of the LCR that supports CTCF-independent enhancer-blocking activity in cell culture. Internal deletion of either of these elements significantly impairs LCR activity. These results reveal that the position-effect suppression region of the TCRalpha LCR harbors an array of CTCF-independent, positive-acting gene regulatory elements, some of which share characteristics with barrier-type insulators. These elements may help manage the separate regulatory programs of the TCRalpha and Dad1 genes.

Comprehensive epigenetic profiling identifies multiple distal regulatory elements directing transcription of the gene encoding interferon-gamma.

Unlike the well defined T helper type 2 cytokine locus, little is known about the regulatory elements that govern the expression of Ifng, which encodes the 'signature' T helper type 1 cytokine interferon-gamma. Here our evolutionary analysis showed that the mouse Ifng locus diverged from the ancestral locus as a result of structural rearrangements producing deletion of the neighboring gene encoding interleukin 26 and disrupting synteny 57 kilobases upstream of Ifng. Proximal to that disruption, we identified by high-resolution mapping many regions with CD4+ T cell subset-specific epigenetic modifications. A subset of those regions represented enhancers, whereas others blocked the activity of upstream enhancers or insulated Ifng from neighboring chromatin. Our findings suggest that proper expression of Ifng is maintained through the collective action of multiple distal regulatory elements present in a region of about 100 kilobases flanking Ifng.

A distal conserved sequence element controls Ifng gene expression by T cells and NK cells.

Chromatin dynamics that regulate Ifng gene expression are incompletely understood. By using cross-species comparative sequence analyses, we have identified conserved noncoding sequences (CNSs) upstream of the Ifng gene, one of which, located -22 kb from the transcriptional start site, contains clustered consensus binding sequences of transcription factors that function in T cell differentiation. CNS-22 was uniquely associated with histone modifications typical of accessible chromatin in both T helper 1 (Th1) and Th2 cells and demonstrated significant and selective T-bet (T-box transcription factor expressed in T cells, Tbx21)-dependent binding and enhancer activity in Th1 cells. Deletion of CNS-22 in the context of an Ifng reporter transgene ablated T cell receptor-dependent and -independent Ifng expression in Th1 effectors and similarly blocked expression by cytotoxic T lymphocytes and natural killer cells. Thus, a single distal element may be essential for Ifng gene expression by both innate and adaptive immune effector cell lineages.

Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS).

A major goal in genomics is to understand how genes are regulated in different tissues, stages of development, diseases, and species. Mapping DNase I hypersensitive (HS) sites within nuclear chromatin is a powerful and well-established method of identifying many different types of regulatory elements, but in the past it has been limited to analysis of single loci. We have recently described a protocol to generate a genome-wide library of DNase HS sites. Here, we report high-throughput analysis, using massively parallel signature sequencing (MPSS), of 230,000 tags from a DNase library generated from quiescent human CD4+ T cells. Of the tags that uniquely map to the genome, we identified 14,190 clusters of sequences that group within close proximity to each other. By using a real-time PCR strategy, we determined that the majority of these clusters represent valid DNase HS sites. Approximately 80% of these DNase HS sites uniquely map within one or more annotated regions of the genome believed to contain regulatory elements, including regions 2 kb upstream of genes, CpG islands, and highly conserved sequences. Most DNase HS sites identified in CD4+ T cells are also HS in CD8+ T cells, B cells, hepatocytes, human umbilical vein endothelial cells (HUVECs), and HeLa cells. However, approximately 10% of the DNase HS sites are lymphocyte specific, indicating that this procedure can identify gene regulatory elements that control cell type specificity. This strategy, which can be applied to any cell line or tissue, will enable a better understanding of how chromatin structure dictates cell function and fate.

Spontaneous neutrophil apoptosis and regulation of cell survival by granulocyte macrophage-colony stimulating factor.

Polymorphonuclear leukocytes (PMNs or neutrophils) are the most prominent cellular component of the innate immune system in humans and produce an array of potent cytotoxic molecules. It is important that neutrophils undergo constitutive (spontaneous) apoptosis as a mechanism to facilitate normal cell turnover and immune system homeostasis. Conversely, several proinflammatory cytokines, including granulocyte macrophage-colony stimulating factor (GM-CSF), prolong neutrophil survival. The molecular mechanisms that regulate PMN apoptosis or survival remain incompletely defined. To that end, we compared global gene expression in human neutrophils during spontaneous apoptosis with that in cells cultured with human GM-CSF. Genes encoding proteins that inhibit apoptosis, such as myeloid cell leukemia sequence 1, caspase 8 and Fas-associated via death domain-like apoptosis regulator (CFLAR), B cell chronic lymphocytic leukemia/lymphoma 2 (BCL2)/adenovirus E1B 19 kDa-interacting protein 2 (BNIP2), and serum/glucocorticoid-regulated kinase (SGK), were down-regulated coincident with neutrophil apoptosis. In contrast, those encoding apoptosis inhibitor 5, BCL2-like 1, BNIP2, CFLAR, SGK, and tumor necrosis factor alpha-induced protein 8 were up-regulated in PMNs cultured with GM-CSF. Correspondingly, GM-CSF delayed PMN apoptosis (P<0.03), increased cell viability (P<0.03), and prolonged neutrophil phagocytic capacity (P<0.05). Prolonged functional capacity was paralleled by striking up-regulation of proinflammatory genes and proteins, including CD14, CD24, CD66, and human leukocyte antigen-DR. In addition, expression of SGK protein diminished during PMN apoptosis but was restored by culture with GM-CSF, suggesting SGK is involved in leukocyte survival. These studies provide a global view of the molecular events that regulate neutrophil survival and apoptosis.