Steven R. Presnell

Cutting Edge: MicroRNA-181 Promotes Human NK Cell Development by Regulating Notch Signaling

MicroRNAs (miRs) have recently been identified as important regulators of gene expression at the posttranscriptional level. Although it has clearly been established that miRs influence the ontogeny of several immune cell lineages, the role of individual miRs during NK cell development has not been described. In this study, we show that miR-181 expression levels have a profound impact on the development of human NK cells from CD34+ hematopoietic progenitor cells and IFN-γ production in primary CD56+ NK cells. We also demonstrate that nemo-like kinase (NLK), an inhibitor of Notch signaling, is a target of miR-181 in NK cells, and knockdown of NLK mirrors the developmental effect of miR-181 overexpression. We conclude that miR-181 promotes NK cell development, at least in part, through the suppression of NLK, providing an important link between miRs and Notch signaling.

Proteasome regulation of ULBP1 transcription.

Killer lymphocytes recognize stress-activated NKG2D ligands on tumors. We examined NKG2D ligand expression in head and neck squamous cell carcinoma (HNSCC) cells and other cell lines. HNSCC cells typically expressed MHC class I chain-related gene A (MICA), MICB, UL16-binding protein (ULBP)2, and ULBP3, but they were uniformly negative for cell surface ULBP1 and ULBP4. We then studied how cancer treatments affected NKG2D ligand expression. NKG2D ligand expression was not changed by most cancer-relevant treatments. However, bortezomib and other proteasome inhibitor drugs with distinct mechanisms of action dramatically and specifically up-regulated HNSCC ULBP1 mRNA and cell surface protein. Proteasome inhibition also increased RNA for ULBP1 and other NKG2D ligands in nontransformed human keratinocytes. Proteasome inhibitor drugs increased ULBP1 transcription by acting at a site in the 522-bp ULBP1 promoter. Although the DNA damage response pathways mediated by ATM (ataxia-telangiectasia, mutated) and ATR (ATM and Rad3-related) signaling had been reported to up-regulate NKG2D ligand expression, we found that ULBP1 up-regulation was not inhibited by caffeine and wortmannin, inhibitors of ATM/ATR signaling. ULBP1 expression in HNSCC cells was not increased by several ATM/ATR activating treatments, including bleomycin, cisplatin, aphidicolin, and hydroxyurea. Ionizing radiation caused ATM activation in HNSCC cells, but high-level ULBP1 expression was not induced by gamma radiation or UV radiation. Thus, ATM/ATR signaling was neither necessary nor sufficient for high-level ULBP1 expression in human HNSCC cell lines and could not account for the proteasome effect. The selective induction of ULBP1 expression by proteasome inhibitor drugs, along with variable NKG2D ligand expression by human tumor cells, indicates that NKG2D ligand genes are independently regulated.

Human NK Cells Proliferate and Die In Vivo More Rapidly than T Cells in Healthy Young and Elderly Adults

NK cells are essential for health, yet little is known about human NK turnover in vivo. In both young and elderly women, all NK subsets proliferated and died more rapidly than T cells. CD56bright NK cells proliferated rapidly but died relatively slowly, suggesting that proliferating CD56bright cells differentiate into CD56dim NK cells in vivo. The relationship between CD56dim and CD56bright proliferating cells indicates that proliferating CD56dim cells both self-renew and are derived from proliferating CD56bright NK cells. Our data suggest that some dying CD56dim cells become CD16+CD56− NK cells and that CD16−CD56low NK cells respond rapidly to cellular and cytokine stimulation. We propose a model in which all NK cell subsets are in dynamic flux. About half of CD56dim NK cells expressed CD57, which was weakly associated with low proliferation. Surprisingly, CD57 expression was associated with higher proliferation rates in both CD8+ and CD8− T cells. Therefore, CD57 is not a reliable marker of senescent, nonproliferative T cells in vivo. NKG2A expression declined with age on both NK cells and T cells. Killer cell Ig-like receptor expression increased with age on T cells but not on NK cells. Although the percentage of CD56bright NK cells declined with age and the percentage of CD56dim NK cells increased with age, there were no significant age-related proliferation or apoptosis differences for these two populations or for total NK cells. In vivo human NK cell turnover is rapid in both young and elderly adults.

Folate deficiency, mismatch repair-dependent apoptosis, and human disease

The vitamin that is most commonly deficient in the American diet is folate. Severe folate deficiency in humans is known to cause megaloblastic anemia and developmental defects, and is associated with an increased incidence of several forms of human cancer. Although the exact mechanisms by which this vitamin deficiency may cause these diseases are not known at the present time, recent work has shown that folate deficiency also causes genomic instability and programmed cell death (or apoptosis). Additionally, it is known that the DNA mismatch repair pathway mediates folate deficiency-induced apoptosis. This review will first describe work suggesting that folate deficiency causes genomic instability and apoptosis, then discuss possible mechanisms by which the mismatch repair pathway could trigger folate deficiency-induced apoptosis, which has either protective or destructive effects on tissue.

The effect of sex on immune cells in healthy aging: Elderly women have more robust natural killer lymphocytes than do elderly men.

Immune gender differences have been reported, but are little studied in elderly humans. We compared monocyte and lymphocyte subsets, along with soluble immune mediators in healthy men and women over the age of 70. We also measured natural killer (NK) lymphocyte cytotoxic granule exocytosis, chemokine synthesis, and cytokine synthesis in response to a variety of stimuli. Elderly women had significantly more circulating B cells than men, whereas men had more CD4 central memory T cells and higher monocyte levels. Plasma adiponectin levels were higher in women, plasma retinol-binding protein 4 levels were higher in men, but there were no significant gender differences in C-reactive protein, IL-15, or sphingosine-1-phosphate. Women had a higher ratio of immature CD56(bright) NK cells to mature CD56(dim) NK cells, indicating a gender difference in NK cell maturation in the elderly. Comparing sexes, female mature NK cells had more vigorous cytotoxic granule responses to K562 leukemia cells and IFN-γ responses to NKp46 crosslinking. Moreover, female NK cells were more likely to produce MIP-1β in response to a variety of stimuli. These data show that gender influences NK cell activity in elderly humans.

Differential Transcription Factor Use by the KIR2DL4 Promoter under Constitutive and IL-2/15–Treated Conditions

KIR2DL4 is unique among human KIR genes in expression, cellular localization, structure, and function, yet the transcription factors required for its expression have not been identified. Using mutagenesis, EMSA, and cotransfection assays, we identified two redundant Runx binding sites in the 2DL4 promoter as essential for constitutive 2DL4 transcription, with contributions by a cyclic AMP response element (CRE) and initiator elements. IL-2– and IL-15–stimulated human NK cell lines increased 2DL4 promoter activity, which required functional Runx, CRE, and Ets sites. Chromatin immunoprecipitation experiments show that Runx3 and Ets1 bind the 2DL4 promoter in situ. 2DL4 promoter activity had similar transcription factor requirements in T cells. Runx, CRE, and Ets binding motifs are present in 2DL4 promoters from across primate species, but other postulated transcription factor binding sites are not preserved. Differences between 2DL4 and clonally restricted KIR promoters suggest a model that explains the unique 2DL4 expression pattern in human NK cells.

Human natural killer cell microRNA: differential expression of MIR181A1B1 and MIR181A2B2 genes encoding identical mature microRNAs

Natural killer (NK) and T lymphocytes share many properties, yet only NK cells respond rapidly to infection and cancer without pre-activation. We found that few microRNAs (miRNAs) differed significantly between human NK and T cells. Among those miRNAs, miR-181a and miR-181b levels rose during NK cell differentiation. Prior studies indicate that miR-181a and miR-181b are critical for human NK cell development and are co-transcribed from genes on chromosome 1 (MIR181A1B1) and on chromosome 9 (MIR181A2B2). We mapped human MIR181A1B1 and MIR181A2B2 transcription start sites to 78.3 kb and 34.0 kb upstream of the mature miRNAs, generating predominantly unspliced transcripts of 80–127 kb and ~60 kb, respectively. Unlike mouse thymocytes, human T cells expressed both MIR181A1B1 and MIR181A2B2. We tested the hypothesis that NK cells differentially transcribe the two genes during development and in response to immune regulatory cytokines. During NK-cell differentiation, MIR181A2B2 expression rose markedly and exceeded that of MIR181A1B1. TGF-β treatment increased NK-cell MIR181A2B2 transcription, whereas IL-2, IL-15 and IL-12/IL-18 treatments upregulated MIR181A1B1. The MIR181A2B2 promoter was strongly transactivated by SMAD3 and SMAD4 transcription factors, suggesting that TGF-β signaling upregulates MIR181A2B2 expression, at least in part, through SMAD-dependent promoter activation.

Data correlations between gender, cytomegalovirus infection and T cells, NK cells, and soluble immune mediators in elderly humans.

We describe a cohort of 50 elderly subjects, age at least 70 years. We present gender-specific findings in T lymphocyte markers and soluble immune mediators. We show the correlation between cytomegalovirus infection status with CD56dim NK cell responses to a variety of stimuli and with CD56bright/CD56dim NK cell ratio. We also present the correlation of retinol binding protein (RBP)−4 plasma levels with NK cell responses and we explore the relationship between gender and adiponectin, 25(OH)D (vitamin D), and RBP4 in affecting CD56dim NK cell responses. These data are discussed in Al-Attar et al. (2016) [1].

Human Body Composition and Immunity: Visceral Adipose Tissue Produces IL-15 and Muscle Strength Inversely Correlates with NK Cell Function in Elderly Humans

Natural killer (NK) lymphocyte-mediated cytotoxicity and cytokine secretion control infections and cancers, but these crucial activities decline with age. NK cell development, homeostasis, and function require IL-15 and its chaperone, IL-15 receptor alpha (IL-15Rα). Macrophages and dendritic cells (DC) are major sources of these proteins. We had previously postulated that additional IL-15 and IL-15Rα is made by skeletal muscle and adipose tissue. These sources may be important in aging, when IL-15-producing immune cells decline. NK cells circulate through adipose tissue, where they may be exposed to local IL-15. The objectives of this work were to determine (1) if human muscle, subcutaneous adipose tissue (SAT), and visceral adipose tissue (VAT) are sources of IL-15 and IL-15 Rα, and (2) whether any of these tissues correlate with NK cell activity in elderly humans. We first investigated IL-15 and IL-15Rα RNA expression in paired muscle and SAT biopsies from healthy human subjects. Both tissues expressed these transcripts, but IL-15Rα RNA levels were higher in SAT than in skeletal muscle. We also investigated tissue obtained from surgeries and found that SAT and VAT expressed equivalent amounts of IL-15 and IL-15Rα RNA, respectively. Furthermore, stromal vascular fraction cells expressed more IL-15 RNA than did adipocytes. To test if these findings related to circulating IL-15 protein and NK cell function, we tested 50 healthy adults aged > 70 years old. Plasma IL-15 levels significantly correlated with abdominal VAT mass in the entire cohort and in non-obese subjects. However, plasma IL-15 levels did not correlate with skeletal muscle cross-sectional area and correlated inversely with muscle strength. Plasma IL-15 did correlate with NK cell cytotoxic granule exocytosis and with CCL4 (MIP-1β) production in response to NKp46-crosslinking. Additionally, NK cell responses to K562 leukemia cells correlated inversely with muscle strength. With aging, immune function declines while infections, cancers, and deaths increase. We propose that VAT-derived IL-15 and IL-15Rα is a compensatory NK cell support mechanism in elderly humans.

IL-2/IL-15 activate the human clonally restricted KIR3DL1 reverse promoter

Killer cell immunoglobulin-like receptors (KIRs) are expressed in a clonally restricted manner by human natural killer (NK) cells and allow detection of aberrant cells with low major histocompatibility complex class I levels. Clonally restricted KIR transcription is maintained by demethylation of the proximal promoter. Antisense transcripts also arise from this promoter and may enforce silencing of nonexpressed methylated KIR alleles in NK cells. Here we show that interleukin (IL)-2 and IL-15, cytokines critical for NK cell development and maintenance, greatly stimulated KIR3DL1 reverse promoter activity, but not forward promoter activity. Activated STAT5 was both necessary and sufficient for this effect and bound to the promoter in NK cells that expressed KIR3DL1 or were poised for expression. A systematic investigation of the KIR3DL1 reverse promoter showed significant differences from the forward promoter, with STAT and YY1 sites having relatively greater roles in regulating reverse proximal promoter activity. On the basis of our data, we propose a new role for antisense transcripts in the initiation of KIR gene expression during NK cell development.