Ahmad Al-Attar

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.

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.

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.

Psychosocial resources, aging, and natural killer cell terminal maturity

Psychosocial factors may influence aspects of immunological aging. The present study tested the hypothesis that psychosocial resources correlate with the expression of the cell surface maker CD57 on natural killer (NK) immune cells. CD57 is a marker of terminal maturation and senescence in this cell subset. The study further tested the relative contribution of specific resources in the social, psychological, financial, and status-skill domains, given the potential differential value of different resources for younger and older adults, and the contribution of relative versus absolute resources. Younger (n = 38) and older (n = 34) women completed measures of relative and absolute resources and had blood drawn. Examined both between groups and within the older women, older age and fewer total relative resources were associated with more CD57 expression on NK cells. One SD in resources was the equivalent of 5 years of aging among the older women. Among the specific resource types, a preponderance of financial resources, both relative and absolute, was associated with less CD57 expression on NK cells, and these relationships did not significantly vary between younger and older women. There was no evidence that depressive symptoms mediated the effects of resources on CD57 expression on NK cells. These findings provide support for the hypothesis that the sense that one has substantial resources, particularly with regard to finances and possessions, may retard age-associated aspects of the microenvironment in which NK cells develop and mature, independent of effects on distress, and this process may begin in younger adulthood.

Alloantibody to a Bw4 epitope in a Bw4+B*27: 05 patient.

Background Alloantibodies to the Bw4 epitope are known to be heterogeneous, but it is widely assumed that anti-Bw4 alloantibodies arise only in individuals who do not express a Bw4 epitope. Methods Bw4 expression was confirmed by DNA sequence analysis. Anti-Bw4 reactivity was confirmed by absorption with transfected cells. Results A Bw4+ (B*27:05 or B*27:13) patient expressed antibody that bound all Bw4+ human leukocyte antigen-A and human leukocyte antigen-B antigens tested, except B*27:05 and B*44:02. Serum absorbed with B*51:01-transfected HYM2.C1R cells left only reactivity to B17 (B57, B58), but not to any other Bw4+ antigens. Conclusion A Bw4+ patient made antibody to a Bw4 epitope. This finding indicates that apparent anti-Bw4 or anti-Bw6 antibody should not be ignored even in patients who express a common Bw4+ or Bw6+ antigen, respectively.

Activation of Notch-1 in oral epithelial cells by P. gingivalis triggers the expression of the antimicrobial protein PLA 2 -IIA

P. gingivalis (Pg) is an oral pathogen with the ability to induce oral dysbiosis and periodontal disease. Nevertheless, the mechanisms by which mucosal responses to the oral microbiota in the presence of specific pathogens such as Pg could abrogate the host-microbe symbiotic relationship leading to periodontitis remain unclear. Herein, we identified the Notch-1/PLA2-IIA axis as a new molecular pathway through which Pg could be specifically modulating oral epithelial antimicrobial and inflammatory responses. Pg activated Notch-1, and inhibition or silencing of Notch-1 completely abrogated Pg-induced PLA2-IIA in oral epithelial cells (OECs). Activation of Notch-1 and PLA2-IIA production were associated with Pg-produced gingipains. Other oral Gram-positive and Gram-negative species failed to induce similar responses. Pg enhanced OEC antimicrobial activity through PLA2-IIA. Increased Notch-1 activation correlated with higher PLA2-IIA gingival expression and changes in the abundance of specific oral bacteria phyla during periodontal disease. Oral bacterial species exhibited differential antimicrobial susceptibility to PLA2-IIA. These findings support previous evidence suggesting an important role for epithelial Notch-1 activation and PLA2-IIA production during health and disease at mucosal surfaces, and provide new mechanistic information concerning the regulation of epithelial antimicrobial and pro-inflammatory responses modulated by oral pathogenic bacteria associated with periodontal disease.

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.