Roles of N-linked glycosylation and glycan-binding proteins in placentation: trophoblast infiltration, immunomodulation, angiogenesis, and pathophysiology.

Protein N-linked glycosylation is a structurally diverse post-translational modification that stores biological information in a larger order of magnitude than other post-translational modifications such as phosphorylation, ubiquitination and acetylation. This gives N-glycosylated proteins a diverse range of properties and allows glyco-codes (glycan-related information) to be deciphered by glycan-binding proteins (GBPs). The intervillous space of the placenta is richly populated with membrane-bound and secreted glycoproteins. Evidence exists to suggest that altering the structural nature of their N-glycans can impact several trophoblast functions, which include those related to interactions with decidual cells. This review summarizes trophoblast-related activities influenced by N-glycan-GBP recognition, exploring how different subtypes of trophoblasts actively adapt to characteristics of the decidualized endometrium through cell-specific expression of N-glycosylated proteins, and how these cells receive decidua-derived signals via N-glycan-GBP interactions. We highlight work on how changes in N-glycosylation relates to the success of trophoblast infiltration, interactions of immunomodulators, and uterine angiogenesis. We also discuss studies that suggest aberrant N-glycosylation of trophoblasts may contribute to the pathogenesis of pregnancy complications (e.g. pre-eclampsia, early spontaneous miscarriages and hydatidiform mole). We propose that a more in-depth understanding of how N-glycosylation shapes trophoblast phenotype during early pregnancy has the potential to improve our approach to predicting, diagnosing and alleviating poor maternal/fetal outcomes associated with placental dysfunction.

In Vitro Development of Mouse and Human NK Cells from Hematopoietic Progenitor Cells.

Natural killer (NK) cells are lymphocytes that play an important role at clearing virally infected or cancer cells. Their potential and role in cancer immunotherapy have generated great interest, given the promising results of NK cell adoptive transfer clinical trials. The remaining challenge to bring emerging NK cell immunotherapies to the clinic is to enhance the production of large numbers of functionally competent NK cells ex vivo. Here, we describe two in vitro NK cell development assays using hematopoietic progenitor cells (HPCs), one for human NK cells and one for mouse NK cells. These protocols describe two robust methods that can be utilized for investigation of NK cell development and function.

The transcriptional repressor REV-ERB as a novel target for disease.

REV-ERB is a member of the nuclear receptor superfamily of transcription factors involved in the regulation of many physiological processes, from circadian rhythm, to immune function and metabolism. Accordingly, REV-ERB has been considered as a promising, but difficult drug target for the treatment of numerous diseases. Here, we concisely review current understanding of the function of REV-ERB, modulation by endogenous factors and synthetic ligands, and the involvement of REV-ERB in select human diseases. Particular focus is placed on the medicinal chemistry of synthetic REV-ERB ligands, which demonstrates the need for higher quality ligands to aid in robust validation of this exciting target.

Spontaneous pulmonary hypertension in genetic mouse models of natural killer cell deficiency.

Natural killer (NK) cells are cytotoxic innate lymphoid cells with an established role in the regulation of vascular structure in pregnancy and cancer. Impaired NK cell function has been identified in patients with pulmonary arterial hypertension (PAH), a disease of obstructive vascular remodeling in the lungs, as well as in multiple rodent models of disease. However, the precise contribution of NK cell impairment to the initiation and progression of PAH remains unknown. Here, we report the development of spontaneous pulmonary hypertension in two independent genetic models of NK cell dysfunction, including Nfil3-/- mice, which are deficient in NK cells due to the absence of the NFIL3 transcription factor, and Ncr1-Gfp mice, which lack the NK activating receptor NKp46. Mouse models of NK insufficiency exhibited increased right ventricular systolic pressure and muscularization of the pulmonary arteries in the absence of elevated left ventricular end-diastolic pressure, indicating that the development of pulmonary hypertension was not secondary to left heart dysfunction. In cases of severe NK cell impairment or loss, a subset of mice failed to develop pulmonary hypertension and instead exhibited reduced systemic blood pressure, demonstrating an extension of vascular abnormalities beyond the pulmonary circulation into the systemic vasculature. In both mouse models, the development of PAH was linked to elevated interleukin-23 production, whereas systemic hypotension in Ncr1-Gfp mice was accompanied by a loss of angiopoietin-2. Together, these results support an important role for NK cells in the regulation of pulmonary and systemic vascular function and the pathogenesis of PAH.

Interleukin-2 shapes the cytotoxic T cell proteome and immune environment-sensing programs.

Interleukin-2 (IL-2) and Janus kinases (JAKs) regulate transcriptional programs and protein synthesis to promote the differentiation of effector CD8+ cytotoxic T lymphocytes (CTLs). Using high-resolution mass spectrometry, we generated an in-depth characterization of how IL-2 and JAKs configure the CTL proteome to control CTL function. We found that IL-2 signaling through JAK1 and JAK3 (JAK1/3) increased the abundance of a key subset of proteins to induce the accumulation of critical cytokines and effector molecules in T cells. Moreover, IL-2 maintained the concentration of proteins that support core metabolic processes essential for cellular fitness. One fundamental insight was the dominant role for IL-2 in stimulating effector T cells to detect microenvironmental cues. IL-2-JAK1/3 signaling pathways thus increased the abundance of nutrient transporters, nutrient sensors, and critical oxygen-sensing molecules. These data provide key insights into how IL-2 promotes T cell function and highlight signaling mechanisms and transcription factors that integrate oxygen sensing to transcriptional control of CD8+ T cell differentiation.

Multiple Levels of Control Determine How E4bp4/Nfil3 Regulates NK Cell Development.

The transcription factor E4bp4/Nfil3 has been shown to have a critical role in the development of all innate lymphoid cell types including NK cells. In this study, we show that posttranslational modifications of E4bp4 by either SUMOylation or phosphorylation have profound effects on both E4bp4 function and NK cell development. We examined the activity of E4bp4 mutants lacking posttranslational modifications and found that Notch1 was a novel E4bp4 target gene. We observed that abrogation of Notch signaling impeded NK cell production and the total lack of NK cell development from E4bp4-/- progenitors was completely rescued by short exposure to Notch peptide ligands. This work reveals both novel mechanisms in NK cell development by a transcriptional network including E4bp4 with Notch, and that E4bp4 is a central hub to process extrinsic stimuli.

Murine thymic NK cells: A case of identity.

Just over a decade ago, it was established that NK cells in the thymus do not follow precisely the same developmental pathway as conventional NK cells that develop in the bone marrow. Subsequently, it has emerged that NK cells are one branch of a family of innate lymphoid cells (ILCs). ILC1s and thymic NK cells have, however, sufficient similarities such that questions have been raised about how distinctive each cell type is from the other. In this issue of European Journal of Immunology, Gabrielli et al. [Eur. J. Immunol. 2017. 47: 800-805] make a detailed study of the transcription factor requirements of murine thymic NK cells. They provide a valuable insight into the distinctive identity of thymic NK cells with regard to Tbet, Nfil3, Id2, and Ets1. In addition, they clarify the nature of DX5 expression on NK cells and ILC-like cells in the murine thymus.

Adipose Natural Killer Cells Regulate Adipose Tissue Macrophages to Promote Insulin Resistance in Obesity.

Obesity-induced inflammation mediated by immune cells in adipose tissue appears to participate in the pathogenesis of insulin resistance. We show that natural killer (NK) cells in adipose tissue play an important role. High-fat diet (HFD) increases NK cell numbers and the production of proinflammatory cytokines, notably TNFα, in epididymal, but not subcutaneous, fat depots. When NK cells were depleted either with neutralizing antibodies or genetic ablation in E4bp4(+/-) mice, obesity-induced insulin resistance improved in parallel with decreases in both adipose tissue macrophage (ATM) numbers, and ATM and adipose tissue inflammation. Conversely, expansion of NK cells following IL-15 administration or reconstitution of NK cells into E4bp4(-/-) mice increased both ATM numbers and adipose tissue inflammation and exacerbated HFD-induced insulin resistance. These results indicate that adipose NK cells control ATMs as an upstream regulator potentially by producing proinflammatory mediators, including TNFα, and thereby contribute to the development of obesity-induced insulin resistance.

The Residual Innate Lymphoid Cells in NFIL3-Deficient Mice Support Suboptimal Maternal Adaptations to Pregnancy.

Uterine NK cells are innate lymphoid cells (ILC) that populate the uterus and expand during pregnancy, regulating placental development and fetal growth in humans and mice. We have recently characterized the composition of uterine ILCs (uILCs), some of which require the transcription factor NFIL3, but the extent to which NFIL3-dependent cells support successful reproduction in mice is unknown. By mating Nfil3 (-/-) females with wild-type males, here we show the effects of NFIL3 deficiency in maternal cells on both the changes in uILCs during pregnancy and the downstream consequences on reproduction. Despite the presence of CD49a(+)Eomes(-) uILC1s and the considerable expansion of residual CD49a(+)Eomes(+) tissue-resident NK cells and uILC3s in pregnant Nfil3 (-/-) mice, we found incomplete remodeling of uterine arteries and decidua, placental defects, and fetal growth restriction in litters of normal size. These results show that maternal NFIL3 mediates non-redundant functions in mouse reproduction.

Composition, Development, and Function of Uterine Innate Lymphoid Cells.

Innate lymphoid cells (ILCs), including NK cells, contribute to barrier immunity and tissue homeostasis. In addition to the role of uterine NK cells in placentation and fetal growth, other uterine ILCs (uILCs) are likely to play roles in uterine physiology and pathology. In this article, we report on the composition of uILCs in the endometrium during the luteal phase and in the decidua during early pregnancy. Whereas nonkiller uILC1s and uILC2s are barely detectable in mouse and not detected in humans, a sizeable population of uILC3s is found in human endometrium and decidua, which are mostly NCR(+) and partially overlap with previously described IL-22-producing uterine NK cells. Development of mouse uILC3 is Nfil3 independent, suggesting unique features of uILCs. Indeed, although the cytokine production profile of mouse uILCs recapitulates that described in other tissues, IL-5, IL-17, and IL-22 are constitutively produced by uILC2s and uILC3s. This study lays the foundation to understand how ILCs function in the specialized uterine mucosa, both in tissue homeostasis and barrier immunity and during pregnancy.

ILC Lineage Specification: To Be or Not 11b, That Is the Question.

The transcription factor Bcl11b is important for T cell development and maintaining their phenotype. In this issue of Immunity, Califano et al. (2015) show that Bcl11b has a role in specifying type II innate lymphoid cell (ILC2) identity and blocks their conversion to ILC3s.

Transcriptional control of NK cell differentiation and function.

Natural killer (NK) cells are crucial to mounting an effective immune response. They have a significant role in cancer immunosurveillance and function as a bridge between innate and adaptive immunity. However, until recently, surprisingly little was known about the molecular basis of NK cell development as compared to the impressive body of knowledge on B- and T-cell development. Here we outline the key transcription factors known to influence NK cell development and at what stages they function. The recent progress in understanding allows us to speculate on the nature of the network of interactions between transcription factors that ultimately facilitate the production of mature functional NK cells.

The transcription factor E4bp4/Nfil3 controls commitment to the NK lineage and directly regulates Eomes and Id2 expression.

The transcription factor E4bp4 (Nfil3) is essential for natural killer (NK) cell production. Here, we show that E4bp4 is required at the NK lineage commitment point when NK progenitors develop from common lymphoid progenitors (CLPs) and that E4bp4 must be expressed at the CLP stage for differentiation toward the NK lineage to occur. To elucidate the mechanism by which E4bp4 promotes NK development, we identified a central core of transcription factors that can rescue NK production from E4bp4(-/-) progenitors, suggesting that they act downstream of E4bp4. Among these were Eomes and Id2, which are expressed later in development than E4bp4. E4bp4 binds directly to the regulatory regions of both Eomes and Id2, promoting their transcription. We propose that E4bp4 is required for commitment to the NK lineage and promotes NK development by directly regulating the expression of the downstream transcription factors Eomes and Id2.

Differential requirement for Nfil3 during NK cell development.

NK cells can be grouped into distinct subsets that are localized to different organs and exhibit a different capacity to secrete cytokines and mediate cytotoxicity. Despite these hallmarks that reflect tissue-specific specialization in NK cells, little is known about the factors that control the development of these distinct subsets. The basic leucine zipper transcription factor Nfil3 (E4bp4) is essential for bone marrow-derived NK cell development, but it is not clear whether Nfil3 is equally important for all NK cell subsets or how it induces NK lineage commitment. In this article, we show that Nfil3 is required for the formation of Eomes-expressing NK cells, including conventional medullary and thymic NK cells, whereas TRAIL(+) Eomes(-) NK cells develop independently of Nfil3. Loss of Nfil3 during the development of bone marrow-derived NK cells resulted in reduced expression of Eomes and, conversely, restoration of Eomes expression in Nfil3(-/-) progenitors rescued NK cell development and maturation. Collectively, these findings demonstrate that Nfil3 drives the formation of mature NK cells by inducing Eomes expression and reveal the differential requirements of NK cell subsets for Nfil3.

The transcription factor E4BP4 is not required for extramedullary pathways of NK cell development.

NK cells contribute to antitumor and antiviral immunosurveillance. Their development in the bone marrow (BM) requires the transcription factor E4BP4/NFIL3, but requirements in other organs are less well defined. In this study, we show that CD3(-)NK1.1(+)NKp46(+)CD122(+) NK cells of immature phenotype and expressing low eomesodermin levels are found in thymus, spleen, and liver of E4BP4-deficient mice, whereas numbers of mature, eomesodermin(high) conventional NK cells are drastically reduced. E4BP4-deficient CD44(+)CD25(-) double-negative 1 thymocytes efficiently develop in vitro into NK cells with kinetics, phenotype, and functionality similar to wild-type controls, whereas no NK cells develop from E4BP4-deficient BM precursors. In E4BP4/Rag-1 double-deficient (DKO) mice, NK cells resembling those in Rag-1-deficient controls are found in similar numbers in the thymus and liver. However, NK precursors are reduced in DKO BM, and no NK cells develop from DKO BM progenitors in vitro. DKO thymocyte precursors readily develop into NK cells, but DKO BM transfers into nude recipients and NK cells in E4BP4/Rag-1/IL-7 triple-KO mice indicated thymus-independent NK cell development. In the presence of T cells or E4BP4-sufficient NK cells, DKO NK cells have a selective disadvantage, and thymic and hepatic DKO NK cells show reduced survival when adoptively transferred into lymphopenic hosts. This correlates with higher apoptosis rates and lower responsiveness to IL-15 in vitro. In conclusion, we demonstrate E4BP4-independent development of NK cells of immature phenotype, reduced fitness, short t1/2, and potential extramedullary origin. Our data identify E4BP4-independent NK cell developmental pathways and a role for E4BP4 in NK cell homeostasis.

Recruitment of histone methyltransferase G9a mediates transcriptional repression of Fgf21 gene by E4BP4 protein.

The liver responds to fasting-refeeding cycles by reprogramming expression of metabolic genes. Fasting potently induces one of the key hepatic hormones, fibroblast growth factor 21 (FGF21), to promote lipolysis, fatty acid oxidation, and ketogenesis, whereas refeeding suppresses its expression. We previously reported that the basic leucine zipper transcription factor E4BP4 (E4 binding protein 4) represses Fgf21 expression and disrupts its circadian oscillations in cultured hepatocytes. However, the epigenetic mechanism for E4BP4-dependent suppression of Fgf21 has not yet been addressed. Here we present evidence that histone methyltransferase G9a mediates E4BP4-dependent repression of Fgf21 during refeeding by promoting repressive histone modification. We find that Fgf21 expression is up-regulated in E4bp4 knock-out mouse liver. We demonstrate that the G9a-specific inhibitor BIX01294 abolishes suppression of the Fgf21 promoter activity by E4BP4, whereas overexpression of E4bp4 leads to increased levels of dimethylation of histone 3 lysine 9 (H3K9me2) around the Fgf21 promoter region. Furthermore, we also show that E4BP4 interacts with G9a, and knockdown of G9a blocks repression of Fgf21 promoter activity and expression in cells overexpressing E4bp4. A G9a mutant lacking catalytic activity, due to deletion of the SET domain, fails to inhibit the Fgf21 promoter activity. Importantly, acute hepatic knockdown by adenoviral shRNA targeting G9a abolishes Fgf21 repression by refeeding, concomitant with decreased levels of H3K9me2 around the Fgf21 promoter region. In summary, we show that G9a mediates E4BP4-dependent suppression of hepatic Fgf21 by enhancing histone methylation (H3K9me2) of the Fgf21 promoter.

CD8α+ DCs can be induced in the absence of transcription factors Id2, Nfil3, and Batf3.

Antiviral immunity and cross-presentation is mediated constitutively through CD8α+ and CD103+ DCs. Development of these DC subsets is thought to require the transcription factors Irf8, Id2, Nfil3, and Batf3, although how this network is regulated is poorly defined. We addressed the nature of the differentiation blocks observed in the absence of these factors and found that although all 4 factors are required for CD103+ DC development, only Irf8 is essential for CD8α+ DCs. CD8α+ DCs emerged in the absence of Id2, Nfil3 and Batf3 in short-term bone marrow reconstitution. These "induced" CD8α+ DCs exhibit several hallmarks of classic CD8α+ DCs including the expression of CD24, Tlr3, Xcr1, Clec9A, and the capacity to cross-present soluble, cell-associated antigens and viral antigens even in the absence of Batf3. Collectively, these results uncover a previously undescribed pathway by which CD8α+ DCs emerge independent of Id2, Nfil3, and Batf3, but dependent on Irf8.

E4BP4: an unexpected player in the immune response.

Until recently, the basic leucine zipper transcription factor E4BP4 (also known as NFIL3) was of little interest to immunologists, being best known for its role in regulating circadian rhythm in chick pineal gland. However, characterisation of E4bp4(-/-) mice, independently generated in four different laboratories, has revealed roles for E4BP4 in diverse haematopoietic lineages. E4BP4 is essential for the development of NK cells and CD8α(+) conventional dendritic cells, and is also involved in macrophage activation, polarisation of CD4(+) T cell responses and B cell class switching to IgE. Here, we discuss the role of E4BP4 as a regulator of the immune response and highlight future questions for the field.

Transcriptional control of natural killer cell differentiation and function.

Gene expression can be modulated depending on physiological and developmental requirements. A multitude of regulatory genes, which are organized in interdependent networks, guide development and eventually generate specific phenotypes. Transcription factors (TF) are a key element in the regulatory cascade controlling cell fate and effector functions. In this review, we discuss recent data on the diversity of TF that determine natural killer (NK) cell fate and NK cell function.

AML1/ETO proteins control POU4F1/BRN3A expression and function in t(8;21) acute myeloid leukemia.

A variety of genetic lesions, including chromosomal translocations, internal tandem duplications, and mutations, have been described in acute myeloid leukemia (AML). Expression profiling has shown that chromosomal translocations, in particular, are associated with distinctive patterns of gene expression. AML exhibiting the translocation t(8;21), which fuses the AML1 and ETO genes, has such a characteristic expression profile. One gene whose expression is highly correlated with the presence of the AML1/ETO fusion is POU4F1, which encodes the POU homeodomain transcription factor BRN3A. Here we show using specific siRNA in t(8;21) cells and overexpression studies in progenitor cells that AML1/ETO promotes expression of POU4F1/BRN3A. This effect requires DNA-binding function of AML1/ETO, and accordingly, AML1/ETO is bound to the POU4F1 locus in t(8;21) cells. Functionally, whereas overexpression of Brn3a in murine hematopoietic progenitor cells induces terminal myeloid differentiation, coexpression of AML1/ETO or AML1/ETO9a blocks this effect. Furthermore, Brn3a reduction by shRNA impairs AML1/ETO-induced immortalization of murine progenitors. In summary, we identify POU4F1/BRN3A as a novel potential upregulated AML1/ETO target gene whose dramatically high expression may cooperate with AML1/ETO in t(8;21) cells.