Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent activator of Ca 2؉ release from intracellular stores known today. Although recent reports have suggested an important function of NAADP in human T lymphocytes, direct evidence for receptor-induced formation of NAADP is yet missing in these cells. Thus, we developed a highly sensitive and specific enzyme assay capable of quantifying low fmol amounts of NAADP. In unstimulated T cells, the NAADP concentration amounted to 4.4 ؎ 1.6 nM (0.055 ؎ 0.028 pmol/mg of protein). Stimulation of the cells via the T cell receptor/CD3 complex resulted in biphasic elevation kinetics of cellular NAADP levels and was characterized by a bell-shaped concentration-response curve for NAADP. In contrast, the NAADP concentration was elevated neither upon activation of the ADPribose/TRPM2 channel Ca 2؉ signaling system nor by an increase of the intracellular Ca 2؉ concentration upon thapsigargin stimulation. T cell receptor/CD3 complex-mediated NAADP formation was dependent on the activity of tyrosine kinases because genistein completely blocked NAADP elevation. Thus, we propose a regulated formation of NAADP upon specific stimulation of the T cell receptor/CD3 complex, suggesting a function of NAADP as a Ca 2؉ -mobilizing second messenger during T cell activation.During a rise of the intracellular Ca 2ϩ concentration, Ca 2ϩ either enters the cell from the extracellular space or is released from intracellular stores. The latter process is regulated by an expanding group of intracellular messengers (1, 2) including D-myo-inositol 1,4,5-trisphosphate (InsP 3 ), 3 cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). In addition, Ca 2ϩ itself co-modulates Ca 2ϩ release via both InsP 3 receptors and ryanodine receptors (RyR).Clapper et al. (3) reported a Ca 2ϩ releasing activity of a contamination in commercially available NADP preparations. Nearly a decade later, this contamination was identified as NAADP, which turned out to be the most potent Ca 2ϩ -mobilizing compound in sea urchin egg homogenates (4). NAADP is active in a variety of cells, ranging from plants to animals (for reviews, see Refs. 5-7). Interestingly the functional properties of NAADP-induced Ca 2ϩ release in sea urchin egg homogenates are different from the systems activated by InsP 3 or cADPR (4). Moreover because extracellular stimulation induces increases in intracellular NAADP levels, a second messenger function for NAADP has been proposed in a few experimental systems (for reviews, see Refs. 8 and 9), including murine pancreatic beta cells (10), murine pancreatic acinar cells (11), and arterial smooth muscle cells (12). However, the determination of NAADP is still difficult because the data published so far were obtained by a radioligand binding assay requiring sea urchin egg homogenates as well as 32 P-labeled NAADP (10 -14).The molecular identity of the NAADP receptor is still controversial. Although a specific receptor has been proposed in sea urchin eggs (4, 15,...

Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytometry, proteomic analysis and microRNA profiling we describe the morphology, size, density, protein composition and microRNA content of human thymic exosomes. The thymic exosomes share characteristics with previously described exosomes such as antigen presentation molecules, but they also exhibit thymus specific features regarding surface markers, protein content and microRNA profile. Interestingly, thymic exosomes carry proteins that have a tissue restricted expression in the periphery which may suggest a role in T cell selection and the induction of central tolerance. We speculate that thymic exosomes may provide the means for intercellular information exchange necessary for negative selection and regulatory T cell formation of the developing thymocytes within the human thymic medulla.

Altered DNA methylation patterns in CD4+ T-cells indicate the importance of epigenetic mechanisms in inflammatory diseases. However, the identification of these alterations is complicated by the heterogeneity of most inflammatory diseases. Seasonal allergic rhinitis (SAR) is an optimal disease model for the study of DNA methylation because of its well-defined phenotype and etiology. We generated genome-wide DNA methylation (Npatients = 8, Ncontrols = 8) and gene expression (Npatients = 9, Ncontrols = 10) profiles of CD4+ T-cells from SAR patients and healthy controls using Illumina's HumanMethylation450 and HT-12 microarrays, respectively. DNA methylation profiles clearly and robustly distinguished SAR patients from controls, during and outside the pollen season. In agreement with previously published studies, gene expression profiles of the same samples failed to separate patients and controls. Separation by methylation (Npatients = 12, Ncontrols = 12), but not by gene expression (Npatients = 21, Ncontrols = 21) was also observed in an in vitro model system in which purified PBMCs from patients and healthy controls were challenged with allergen. We observed changes in the proportions of memory T-cell populations between patients (Npatients = 35) and controls (Ncontrols = 12), which could explain the observed difference in DNA methylation. Our data highlight the potential of epigenomics in the stratification of immune disease and represents the first successful molecular classification of SAR using CD4+ T cells.

The identification of diagnostic markers and therapeutic candidate genes in common diseases is complicated by the involvement of thousands of genes. We hypothesized that genes co-regulated with a key gene in allergy, IL13, would form a module that could help to identify candidate genes. We identified a T helper 2 (TH2) cell module by small interfering RNA–mediated knockdown of 25 putative IL13-regulating transcription factors followed by expression profiling. The module contained candidate genes whose diagnostic potential was supported by clinical studies. Functional studies of human TH2 cells as well as mouse models of allergy showed that deletion of one of the genes, S100A4, resulted in decreased signs of allergy including TH2 cell activation, humoral immunity, and infiltration of effector cells. Specifically, dendritic cells required S100A4 for activating T cells. Treatment with an anti-S100A4 antibody resulted in decreased signs of allergy in the mouse model as well as in allergen-challenged T cells from allergic patients. This strategy, which may be generally applicable to complex diseases, identified and validated an important diagnostic and therapeutic candidate gene in allergy.

Malassezia is the dominant fungus in the human skin mycobiome and is associated with common skin disorders including atopic eczema (AE)/dermatitis. Recently, it was found that Malassezia sympodialis secretes nanosized exosome-like vesicles, designated MalaEx, that carry allergens and can induce inflammatory cytokine responses. Extracellular vesicles from different cell-types including fungi have been found to deliver functional RNAs to recipient cells. In this study we assessed the presence of small RNAs in MalaEx and addressed if the levels of these RNAs differ when M. sympodialis is cultured at normal human skin pH versus the elevated pH present on the skin of patients with AE. The total number and the protein concentration of the released MalaEx harvested after 48 h culture did not differ significantly between the two pH conditions nor did the size of the vesicles. From small RNA sequence data, we identified a set of reads with well-defined start and stop positions, in a length range of 16 to 22 nucleotides consistently present in the MalaEx. The levels of small RNAs were not significantly differentially expressed between the two different pH conditions indicating that they are not influenced by the elevated pH level observed on the AE skin.Extracellular vesicles (EV) are released not only from different mammalian cell-types but also from microorganisms and parasites and have the capacity to transfer complex biological information [1][2][3][4][5] . Various types of EV ranging in size from 20 nm to 1,000 nm in diameter have been described and are classified mainly on their mechanisms of biogenesis and their physiological functions 1,6 . Those designated exosomes are nanosized vesicles of 50-100 nm which are released extracellularly after fusion of multicellular endosomes with the cell membrane, whereas microvesicles (MV) are larger vesicles (100-1,000 nm) generated through outward budding of the plasma membrane 1,5 . Gram-negative bacteria produce MV by outward budding of the outer membrane and these vesicles are therefore referred to as outer membrane vesicles (OMV) with a diameter in the range of 20-500 nm 6 . Exosomes can be detected in body fluids such as urine, bronchoalveolar lavage fluid (BAL), breast milk and serum 7 . The functions of exosomes include immunoregulatory mechanisms such as modulation of antigen presentation, immune activation, immune suppression, immune surveillance and intercellular communication 6 . EV from microorganisms with thick cell walls, such as Gram-positive bacteria and fungi, have been associated with cytotoxicity, the invasion of host cells, and the transfer of virulence factors 2 . As seen with exosomes 1,8 , fungal EV have been observed to deliver functional messenger (m)RNAs and micro (mi)RNA-like RNAs to recipient cells 9,10 . miRNAs are small non-coding RNAs with a length between 20 and 22 nucleotides (nt) 11 . They are spliced from precursor sequences that form the stable hairpin necessary for transportation from the nucleus to the cytoplasm. After the miRNA has been c...

a b s t r a c tThe role of the multifunctional enzyme CD38 in formation of the Ca 2+ -mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) was investigated. Gene silencing of CD38 did neither inhibit NAADP synthesis in intact Jurkat T cells nor in thymus or spleen obtained from CD38 knock out mice. In vitro, both NAADP formation by base-exchange and degradation to 2-phospho adenosine diphosphoribose were efficiently decreased. Thus in vivo CD38 appears to be a NAADP degrading rather than a NAADP forming enzyme, perhaps avoiding desensitizing NAADP levels in intact cells.

DNA methylation changes may predispose becoming IgE-sensitized to allergens. We analyzed whether DNA methylation in peripheral blood mononuclear cells (PBMC) is associated with IgE sensitization at 5 years of age (5Y). DNA methylation was measured in 288 PBMC samples from 74 mother/child pairs from the birth cohort ALADDIN (Assessment of Lifestyle and Allergic Disease During INfancy) using the HumanMethylation450BeadChip (Illumina). PBMCs were obtained from the mothers during pregnancy and from their children in cord blood, at 2 years and 5Y. DNA methylation levels at each time point were compared between children with and without IgE sensitization to allergens at 5Y. For replication, CpG sites associated with IgE sensitization in ALADDIN were evaluated in whole blood DNA of 256 children, 4 years old, from the BAMSE (Swedish abbreviation for Children, Allergy, Milieu, Stockholm, Epidemiology) cohort. We found 34 differentially methylated regions (DMRs) associated with IgE sensitization to airborne allergens and 38 DMRs associated with sensitization to food allergens in children at 5Y (Sidak p ≤ 0.05). Genes associated with airborne sensitization were enriched in the pathway of endocytosis, while genes associated with food sensitization were enriched in focal adhesion, the bacterial invasion of epithelial cells, and leukocyte migration. Furthermore, 25 DMRs in maternal PBMCs were associated with IgE sensitization to airborne allergens in their children at 5Y, which were functionally annotated to the mTOR (mammalian Target of Rapamycin) signaling pathway. This study supports that DNA methylation is associated with IgE sensitization early in life and revealed new candidate genes for atopy. Moreover, our study provides evidence that maternal DNA methylation levels are associated with IgE sensitization in the child supporting early in utero effects on atopy predisposition.

Background The transcription factor (TF) IRF4 is involved in the regulation of Th1, Th2, Th9 and Th17 cells, and animal studies have indicated an important role in allergy. However, IRF4 and its target genes have not been examined in human allergy. Methods IRF4 and its target genes were examined in allergen-challenged CD4+ cells from patients with IAR using combined gene expression microarrays and chromatin immunoprecipitation chips (ChIP-chips), computational target prediction and RNAi knockdowns. Results IRF4 increased in allergen-challenged CD4+ cells from patients with IAR and functional studies supported its role in Th2 cell activation. IRF4 ChIP-chip showed that IRF4 regulated a large number of genes relevant for Th cell differentiation. However, neither Th1 nor Th2 cytokines were the direct targets of IRF4. To examine if IRF4 induced Th2 cytokines via one or more down-stream TFs, we combined gene expression microarrays, ChIP-chips and computational target prediction and found a putative intermediary TF, namely ETS1 in allergen-challenged CD4+ cells from allergic patients. ETS1 increased significantly in allergen-challenged CD4+ cells from patients compared to controls. Gene expression microarrays before and after ETS1 RNAi knockdown showed that ETS1 induced Th2 cytokines as well as disease-related pathways. Conclusions Increased expression of IRF4 in allergen-challenged CD4+ cells from patients with intermittent allergic rhinitis, leads to activation of a complex transcriptional program, including Th2 cytokines.