Epigenetic alteration of the genome has been shown to provide palliative effects in mouse models of certain human autoimmune diseases. We have investigated whether chromatin remodeling could provide protection against autoimmune diabetes in NOD mice. Treatment of female mice during the transition from prediabetic to diabetic stage (18-24 weeks of age) with the well-characterized histone deacetylase inhibitor, trichostatin A effectively reduced the incidence of diabetes. However, similar treatment of overtly diabetic mice during the same time period failed to reverse the disease. Protection against diabetes was accompanied by histone hyperacetylation in pancreas and spleen, enhanced frequency of CD4(+) CD62L(+) cells in the spleen, reduction in cellular infiltration of islets, restoration of normoglycemia and glucose-induced insulin release by beta cells. Activation of splenic T lymphocytes derived from protected mice in vitro with pharmacological agents that bypass the antigen receptor or immobilized anti-CD3 antibody resulted in enhanced expression of Ifng mRNA and protein without altering the expression of Il4, Il17, Il18, Inos and Tnfa genes nor the secretion of IL-2, IL-4, IL-17 and TNF-α proteins. Consistently, expression of the transcription factor involved in Ifng transcription, Tbet/Tbx21 but not Gata3 and Rorgt, respectively, required for the transcription of Il4 and Il17, was upregulated in activated splenocytes of protected mice. These results indicate that chromatin remodeling can lead to amelioration of diabetes by using multiple mechanisms including differential gene transcription. Thus, epigenetic modulation could be a novel therapeutic approach to block the transition from benign to frank diabetes.

Asthma is a chronic inflammatory disease of the airways. It is characterized by allergic airway inflammation, airway remodelling, and airway hyperresponsiveness (AHR). Asthma patients, in particular those with chronic or severe asthma, have airway remodelling that is associated with the accumulation of extracellular matrix (ECM) proteins, such as collagens. Fibulin-1 (Fbln1) is an important ECM protein that stabilizes collagen and other ECM proteins. The level of Fbln1c, one of the four Fbln1 variants, which predominates in both humans and mice, is increased in the serum and airways fluids in asthma but its function is unclear. We show that the level of Fbln1c was increased in the lungs of mice with house dust mite (HDM)-induced chronic allergic airway disease (AAD). Genetic deletion of Fbln1c and therapeutic inhibition of Fbln1c in mice with chronic AAD reduced airway collagen deposition, and protected against AHR. Fbln1c-deficient (Fbln1c-/- ) mice had reduced mucin (MUC) 5 AC levels, but not MUC5B levels, in the airways as compared with wild-type (WT) mice. Fbln1c interacted with fibronectin and periostin that was linked to collagen deposition around the small airways. Fbln1c-/- mice with AAD also had reduced numbers of α-smooth muscle actin-positive cells around the airways and reduced airway contractility as compared with WT mice. After HDM challenge, these mice also had fewer airway inflammatory cells, reduced interleukin (IL)-5, IL-13, IL-33, tumour necrosis factor (TNF) and CXCL1 levels in the lungs, and reduced IL-5, IL-33 and TNF levels in lung-draining lymph nodes. Therapeutic targeting of Fbln1c reduced the numbers of GATA3-positive Th2 cells in the lymph nodes and lungs after chronic HDM challenge. Treatment also reduced the secretion of IL-5 and IL-13 from co-cultured dendritic cells and T cells restimulated with HDM extract. Human epithelial cells cultured with Fbln1c peptide produced more CXCL1 mRNA than medium-treated controls. Our data show that Fbln1c may be a therapeutic target in chronic asthma. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

BACKGROUND

Regulatory dendritic cell (DC) markers, such as C1Q, are upregulated in PBMCs of patients with grass pollen allergy exhibiting clinical benefit during allergen immunotherapy (AIT).

OBJECTIVE

We sought to define markers differentially expressed in human monocyte-derived DCs differentiated toward a proallergic (DCs driving the differentiation of TH2 cells [DC2s]) phenotype and investigate whether changes in such markers in the blood correlate with AIT efficacy.

METHODS

Transcriptomes and proteomes of monocyte-derived DCs polarized toward DCs driving the differentiation of TH1 cells (DC1s), DC2s, or DCs driving the differentiation of regulatory T cells (DCreg cells) profiles were compared by using genome-wide cDNA microarrays and label-free quantitative proteomics, respectively. Markers differentially regulated in DC2s and DCreg cells were assessed by means of quantitative PCR in PBMCs from 80 patients with grass pollen allergy before and after 2 or 4 months of sublingual AIT in parallel with rhinoconjunctivitis symptom scores.

RESULTS

We identified 20 and 26 new genes/proteins overexpressed in DC2s and DCreg cells, respectively. At an individual patient level, DC2-associated markers, such as CD141, GATA3, OX40 ligand, and receptor-interacting serine/threonine-protein kinase 4 (RIPK4), were downregulated after a 4-month sublingual AIT course concomitantly with an upregulation of DCreg cell-associated markers, including complement C1q subcomponent subunit A (C1QA), FcγRIIIA, ferritin light chain (FTL), and solute carrier organic anion transporter family member 2B1 (SLCO2B1), in the blood of clinical responders as opposed to nonresponders. Changes in such markers were better correlated with clinical benefit than alterations of allergen-specific CD4(+) T-cell or IgG responses.

CONCLUSIONS

A combination of 5 markers predominantly expressed by blood DCs (ie, C1Q and CD141) or shared with lymphoid cells (ie, FcγRIIIA, GATA3, and RIPK4) reflecting changes in the balance of regulatory/proallergic responses in peripheral blood can be used as early as after 2 months to monitor the early onset of AIT efficacy.

The transcription factors T-bet and GATA3 are important reciprocal determinants of Th1 and Th2 T helper cell differentiation. Recent evidence suggests that these factors may affect airway immunopathology in asthma.

OBJECTIVE

To quantify and functionally characterize the intramyocardial T-cells in endomyocardial biopsies (EMBs) from patients presenting with acute myocarditis (AMC) and dilated cardiomyopathy (DCM).

RESULTS

Expression of genes characterizing Th1 [interferon (IFN)γ, Tbet-1, Eomesodermin, interleukin (IL)-27], Th2 (IL-4, IL-5, GATA3), Th17 (IL-17), regulatory [regulatory T-cells (Treg); FoxP3, TGFβ, IL-10], anergic (GRAIL), and cytotoxic T-cells (CTLs: Perforin, Granulysin, Granzyme A), as well as of functional T-cell receptor Vbeta (TRBV) families were investigated in EMBs from AMC patients (n= 58) and DCM patients (n= 34) by pre-amplified real-time reverse transcription-polymerase chain reaction. These data were compared with EMBs from n= 19 controls. Expression of CD3d, CD3z, and TRBC (T-cell receptor beta constant region) were associated with the immunohistological diagnosis of inflammatory cardiomyopathy (DCMi). In EMBs from DCM patients with increased CD3d expression, significantly increased markers of Th1 (IFNγ, T-bet, Eomesodermin), regulatory T-cells (Treg; FoxP3, TGFβ), and cytotoxic T-cells (CTLs: Perforin, Granulysin, Granzyme A) were present, while no differential polarization of T-cells was found in EMBs form AMC patients. A differential dominance of distinct functional TRBV families was associated with different cardiotropic viruses: TRBV 11 and 24 with Parvovirus B19; TRBV4, 10 and 28 with human herpes virus type 6; and TRBV14 for Coxsackie virus, respectively.

CONCLUSIONS

The T-cell infiltrates in human DCMi are characterized by differential expression of functional T-cell markers indicating Th1, Treg, and CTLs, while no major role could be confirmed for Th17. The virus-associated differential TRBV dominance suggests an antiviral specificity of virus-induced T-cell responses in human DCMi.

Differential prognostic roles of Androgen Receptor (AR) have been proposed in breast cancer (BC) depending on tumour oestrogen receptor (ER) status. This study aimed to evaluate the prognostic and/or predictive significance of AR expression in invasive BC. In this study AR expression was studied on a large (n = 1141) consecutive series of early-stage (I-III) BC using tissue microarray and immunohistochemistry (IHC). AR mRNA expression was assessed in a subset of cases. The prognostic impact of AR mRNA expression was externally validated using the online BC gene expression data sets (n = 25 data sets, 4078 patients). Nuclear AR IHC expression was significantly associated with features of good prognosis including older age, smaller tumour size, lower grade and lobular histology particularly in the ER-positive tumours. AR was associated with ER-related markers GATA3, FOXa1, RERG and BEX1. Negative association was observed with HER2, p53, Ki67, TK1, CD71 and AGTR1. AR Overexpression was associated with longer survival (p < 0.001), independent of tumour size, grade, stage [p = 0.033, hazard ratio (HR) = 0.80 95 % CI = 0.64-0.98]. Similar associations were maintained in ER+ tumours in univariate and multivariate analysis (p < 0.01) both in patients with and without adjuvant endocrine or chemotherapy. AR mRNA expression showed significant association with tumour grade, molecular subtypes, and longer 10 and 15 years survival in luminal BC. In the external validation cohorts, AR gene expression data were associated with improved patients' outcome (p < 0.001, HR = 0.84, 95 % CI 0.79-0.90). AR is not only an independent prognostic factor in ER-positive luminal BC but is also expressed in ER-negative tumours. AR could act as a molecular target in patients with ER-positive disease predicting response to adjuvant therapy.

Sphingosine 1-phosphate (S1P) is a natural lipid mediator that regulates immune cell traffic, Ab production, and T cell cytokine generation by mechanisms that enhance Th2 activities. Responses to S1P are controlled principally by the diverse expression patterns of its receptors in different cells. In T cells, the type 1 (S1P(1)) and type 4 (S1P(4)) G protein-coupled receptors are predominant. S1P(1) mainly transduces effects on T cell migration and trafficking, whereas S1P(4) transduces immunosuppression via its effects on T cell proliferation and cytokine production. Using T cell-specific S1P(1) transgenic (TG) mice, we investigated the regulatory effects of the S1P-S1P(1) axis on T cell cytokine production. The production of IL-4, but not IL-2 or IFN-gamma, was significantly up-regulated >10-fold in activated CD4 T cells from S1P(1) TG mice compared with those from wild-type mice. Quantitative real-time PCR analysis revealed that IL-4 up-regulation was initiated at the mRNA level as early as 4 h after T cell activation. The up-regulation of IL-4 mRNA was mediated by c-Maf, Jun B, and Gata3 as demonstrated by increases in their protein expression and DNA-binding activities. In contrast, the expression and DNA-binding activities of T-bet, FosB, C-Fos, Jun D, Fra-1, Fra-2, and c-Jun all were identical in wild-type and TG CD4 T cells. Immunological assays showed that increased IL-4 levels induced greater production of IgE. Thus, the S1P-S1P(1) axis specifically up-regulates c-Maf, Jun B, and Gata3, which consequently enhance IL-4 production that may lead to a Th2 phenotype.

The epidermis is maintained by epidermal stem cells (ESCs) that reside in distinct niches and contribute to homeostasis and wound closure. Keratinocytes at the nonhealing edges of venous ulcers (VUs) are healing-incompetent, hyperproliferative, and nonmigratory, suggesting deregulation of ESCs. To date, genes which regulate ESC niches have been studied in mice only. Utilizing microarray analysis of VU nonhealing edges, we identified changes in expression of genes harboring regulation of ESCs and their fate. In a prospective clinical study of 10 VUs, we confirmed suppression of the bone morphogenetic protein receptor (BMPR) and GATA binding protein 3 (GATA3) as well as inhibitors of DNA-binding proteins 2 and 4 (ID2 and ID4). We also found decreased levels of phosphorylated glycogen synthase kinase 3 (GSK3), nuclear presence of β-catenin, and overexpression of its transcriptional target, c-myc, indicating activation of the Wnt pathway. Additionally, we found down-regulation of leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1), a gene important for maintaining ESCs in a quiescent state, and absence of keratin 15 (K15), a marker of the basal stem cell compartment suggesting local depletion of ESCs. Our study shows that loss of genes important for regulation of ESCs and their fate along with activation of β-catenin and c-myc in the VU may contribute to ESC deprivation and a hyperproliferative, nonmigratory healing incapable wound edge.

Complex insertions and deletions (indels) are formed by simultaneously deleting and inserting DNA fragments of different sizes at a common genomic location. Here we present a systematic analysis of somatic complex indels in the coding sequences of samples from over 8,000 cancer cases using Pindel-C. We discovered 285 complex indels in cancer-associated genes (such as PIK3R1, TP53, ARID1A, GATA3 and KMT2D) in approximately 3.5% of cases analyzed; nearly all instances of complex indels were overlooked (81.1%) or misannotated (17.6%) in previous reports of 2,199 samples. In-frame complex indels are enriched in PIK3R1 and EGFR, whereas frameshifts are prevalent in VHL, GATA3, TP53, ARID1A, PTEN and ATRX. Furthermore, complex indels display strong tissue specificity (such as VHL in kidney cancer samples and GATA3 in breast cancer samples). Finally, structural analyses support findings of previously missed, but potentially druggable, mutations in the EGFR, MET and KIT oncogenes. This study indicates the critical importance of improving complex indel discovery and interpretation in medical research.

A favorable interplay between cancer cells and the tumor microenvironment (TME) facilitates the outgrowth of metastatic tumors. Because of the distinct initiating processes between primary and metastatic tumors, we investigate the differences in tumor-associated macrophages (TAMs) from primary and metastatic cancers. Here we show that dual expression of M1 and M2 markers is noted in TAMs from primary tumors, whereas predominant expression of M2 markers is shown in metastatic TAMs. At metastatic sites, TAMs secrete interleukin-35 (IL-35) to facilitate metastatic colonization through activation of JAK2-STAT6-GATA3 signaling to reverse epithelial-mesenchymal transition (EMT) in cancer cells. In primary tumors, inflammation-induced EMT upregulates IL12Rβ2, a subunit of the IL-35 receptor, in cancer cells to help them respond to IL-35 during metastasis. Neutralization of IL-35 or knockout of IL-35 in macrophages reduces metastatic colonization. These results indicate the distinct TMEs of primary and metastatic tumors and provide potential targets for intercepting metastasis.

Senescence or biological aging impacts a vast variety of molecular and cellular processes. To date, it is unknown whether CD4(+) Th cells display an age-dependent bias for development into specific subpopulations. In this study, we show the appearance of a distinct CD4(+) T cell subset expressing IL-4 at an early stage of development in infant adenoids and cord blood that is lost during aging. We identified by flow cytometric, fluorescent microscopic, immunoblot, and mass spectrometric analysis a population of CD4(+) T cells that expressed an unglycosylated isoform of IL-4. This T cell subpopulation was found in neonatal but not in adult CD4(+) T cells. Furthermore, we show that the mRNA of the Th2 master transcription factor GATA3 is preferentially expressed in neonatal CD4(+) T cells. The Th2 phenotype of the IL-4(+)CD4(+) T cells could be reinforced in the presence of TGF-β. Although the IL-4(+)CD4(+) T cells most likely originate from CD31(+)CD4(+) T recent thymic emigrants, CD31 was downregulated prior to secretion of IL-4. Notably, the secretion of IL-4 requires a so far unidentified trigger in neonatal T cells. This emphasizes that cytokine expression and secretion are differentially regulated processes. Our data support the hypothesis of an endogenously poised cytokine profile in neonates and suggest a link between cytokine production and the developmental stage of an organism. The determination of the IL-4 isoform-expressing cells in humans might allow the identification of Th2 precursor cells, which could provide novel intervention strategies directed against Th2-driven immunopathologies such as allergies.

Histone deacetylase (HDAC) inhibitor treatments can augment the anti-tumor effects of retinoids in renal cancer cells. We studied the effects of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and 13-cis retinoic acid (cRA) on two human renal cell carcinoma (RCC) lines. Cells were cultured in the presence of each drug for six days to determine the responses to monotherapy and to combination therapy. The proliferation of SKRC06 was inhibited with cRA treatment; the proliferation of SKRC39 was not. However, both RCC lines were sensitive to growth inhibition by dibutyryl cyclic AMP, with or without 13-cis RA. SAHA alone also reduced cell proliferation in both cell lines. To identify the alterations in gene expression that correlate with the responsiveness to treatment, gene microarray analyses were performed. Several retinoid-regulated genes exhibited much higher mRNA levels in SKRC06 than in SKRC39, even in the absence of drugs; these included crabp2, rargamma and cyp26A1. Combination treatment of cells with both SAHA and cRA induced several transcripts with known anti-cancer/immunomodulatory effects, including dhrs9, gata3, il1beta, phlda1, txk and vhl. Immunostaining confirmed the decreased expression of gata3 in human RCC specimens compared to normal kidney. Together, our results show that treatment of RCC with cRA and/or SAHA increases the expression of several genes and gene families that result in reduced cell proliferation.

NK cells differentiate into either NK1 or NK2 cells that produce IFN-gamma or IL-5 and IL-13, respectively. Little is known, however, about the molecular mechanisms that control NK1 and NK2 cell differentiation. To address these questions, we established an in vitro mouse NK1/NK2 cell differentiation culture system. For NK1/NK2 cell differentiation, initial stimulation with PMA and ionomycin was required. The in vitro differentiated NK2 cells produced IL-5 and IL-13, but the levels were 20 times lower than those of Th2 or T cytotoxic (Tc)2 cells. No detectable IL-4 was produced. Freshly prepared NK cells express IL-2Rbeta, IL-2RgammaC, and IL-4Ralpha. After stimulation with PMA and ionomycin, NK cells expressed IL-2Ralpha. NK1 cells displayed higher cytotoxic activity against Yac-1 target cells. The levels of GATA3 protein in developing NK2 cells were approximately one-sixth of those in Th2 cells. Both NK1 and NK2 cells expressed large amounts of repressor of GATA, the levels of which were equivalent to CD8 Tc1 and Tc2 cells and significantly higher than those in Th2 cells. The levels of histone hyperacetylation of the IL-4 and IL-13 gene loci in NK2 cells were very low and equivalent to those in naive CD4 T cells. The production of IL-5 and IL-13 in NK2 cells was found to be STAT6 dependent. Thus, similar to Th2 cells, NK2 cell development is dependent on STAT6, and the low level expression of GATA3 and the high level expression of repressor of GATA may influence the unique type 2 cytokine production profiles of NK2 cells.

Preplacodal ectoderm (PPE) and neural crest (NC) are specified at the interface of neural and nonneural ectoderm and together contribute to the peripheral nervous system in all vertebrates. Bmp activates early steps for both fates during late blastula stage. Low Bmp activates expression of transcription factors Tfap2a and Tfap2c in the lateral neural plate, thereby specifying neural crest fate. Elevated Bmp establishes preplacodal competence throughout the ventral ectoderm by coinducing Tfap2a, Tfap2c, Foxi1 and Gata3. PPE specification occurs later at the end of gastrulation and requires complete attenuation of Bmp, yet expression of PPE competence factors continues well past gastrulation. Here we show that competence factors positively regulate each other's expression during gastrulation, forming a self-sustaining network that operates independently of Bmp. Misexpression of Tfap2a in embryos blocked for Bmp from late blastula stage can restore development of both PPE and NC. However, Tfap2a alone is not sufficient to activate any other competence factors nor does it rescue individual placodes. On the other hand, misexpression of any two competence factors in Bmp-blocked embryos can activate the entire transcription factor network and support the development of NC, PPE and some individual placodes. We also show that while these factors are partially redundant with respect to PPE specification, they later provide non-redundant functions needed for development of specific placodes. Thus, we have identified a gene regulatory network that coordinates development of NC, PPE and individual placodes in zebrafish.

Epithelioid mesotheliomas and breast carcinomas can present a variety of morphologic patterns. Because of this, breast carcinomas that metastasize to the pleura and lung may be confused with mesotheliomas. The aim of the present study is to compare the immunohistochemical markers currently available for the diagnosis of these 2 malignancies and to determine the best panel of markers that can be used to assist in discriminating between them. Sixty epithelioid mesotheliomas and 80 breast carcinomas (40 triple negative and 40 estrogen receptor positive) were investigated for expression of the positive mesothelioma markers calretinin, keratin 5/6, mesothelin, podoplanin, thrombomodulin, and WT1; the positive carcinoma marker claudin 4; and the breast-associated markers gross cystic disease fluid protein 15 (GCDFP-15), mammaglobin, and GATA3. All of the epithelioid mesotheliomas reacted for calretinin and keratin 5/6, 93% for WT1; 88% for podoplanin; 77% for thrombomodulin; 23% for GATA3; and 0% for claudin 4, GCDFP-15, and mammaglobin, respectively. Of the triple-negative breast carcinomas, 100% expressed claudin 4; 5%, keratin 5/6; 30%, GATA3; 18%, mammaglobin; 15%, GCDFP-15; 56%, mesothelin; 38%, calretinin; 18%, thrombomodulin; 5%, WT1; and 3%, podoplanin. Among the estrogen receptor-positive breast carcinomas, 100% were claudin 4 and GATA3 positive; 70% expressed GCDFP-15; 63%, mammaglobin; 13%, calretinin; 13%, thrombomodulin; 8%, WT1; 5%, keratin 5/6; 3%, mesothelin; and 0%, podoplanin. It is concluded that podoplanin and WT1 are the best positive mesothelioma markers for differentiating epithelioid mesotheliomas from breast carcinomas. An accurate differential diagnosis can be reached with the use of these two markers in combination with the breast-associated markers GCDFP-15, mammaglobin, and GATA3.

OBJECTIVE

Clinical data suggest that the estrogen receptor (ER) contributes to chemotherapeutic responsiveness. However, ER status alone is not consistently predictive. In this study, we used a microarray approach to find novel ER-related genes that predicted chemotherapy responses, with the hope of providing a robust multi-variable prediction method.

METHODS

One hundred and ten patients with stages II and III breast cancer were included. They received four preoperative cycles of a weekly PCb (paclitaxel plus carboplatin) regimen. A total of 55 training cases were used for marker discovery and for identification of any ER-related genes that may have been associated with a chemotherapeutic response ("training cases"). The other 55 patients were available as an independent validation set ("validation cases") to test, using immunohistochemistry (IHC).

RESULTS

In the training set, 20 significantly differentially expressed genes were identified. Among these 20 genes, TFF1, ESR1, GATA3 and TFF3 were found to be ER-related. Among 55 independent validation cases, univariate analysis indicated that clinical variables and ER-related genes were all significantly associated with pCR. It was shown that the pCR rate was as high as 80% when these five factors were all negative. In contrast, these five factors were all positive in seven of nine chemo-resistant patients.

CONCLUSIONS

In conjunction with levels of ER-related genes, expression of ER protein may provide important predictive outcomes for responses to neoadjuvant chemotherapy and may allow for the identification of a subgroup of patients who could significantly benefit from chemotherapy (or who may be resistant to it).

BACKGROUND

Dietary zinc supplementation may help to promote growth, boost the immune system, protect against diabetes, and aid recovery from diarrhoea. We exploited the zebrafish (Danio rerio) gill as a unique vertebrate ion transporting epithelium model to study the time-dependent regulatory networks of gene-expression leading to homeostatic control during zinc supplementation. This organ forms a conduit for zinc uptake whilst exhibiting conservation of zinc trafficking components.

RESULTS

Fish were maintained with either zinc supplemented water (4.0 μM) and diet (2023 mg zinc kg-1) or water and diet containing Zn2+ at 0.25 μM and 233 mg zinc kg-1, respectively. Gill tissues were harvested at five time points (8 hours to 14 days) and transcriptome changes analysed in quintuplicate using a 16 K microarray with results anchored to gill Zn2+ influx and whole body nutrient composition (protein, carbohydrate, lipid, elements). The number of regulated genes increased up to day 7 but declined as the fish acclimated. In total 525 genes were regulated (having a fold-change more than 1.8 fold change and an adjusted P-value less than 0.1 which is controlling a 10% False discovery rate, FDR) by zinc supplementation, but little overlap was observed between genes regulated at successive time-points. Many genes displayed cyclic expression, typical for homeostatic control mechanisms. Annotation enrichment analysis revealed strong overrepresentation of "transcription factors", with specific association evident with "steroid hormone receptors". A suite of genes linked to "development" were also statistically overrepresented. More specifically, early regulation of genes was linked to a few key transcription factors (e.g. Mtf1, Jun, Stat1, Ppara, Gata3) and was followed by hedgehog and bone morphogenic protein signalling.

CONCLUSIONS

The results suggest that zinc supplementation reactivated developmental pathways in the gill and stimulated stem cell differentiation, a response likely reflecting gill remodelling in response to its altered environment. This provides insight to the role of zinc during cell differentiation and illustrates the critical nature of maintaining zinc status. The study also highlights the importance of temporal transcriptomics analysis in order resolve the discrete elements of biological processes, such as zinc acclimation.

GATA3 has been identified as a master regulator of T helper cells, as well as being important for early thymic progenitors and T-cell commitment. However, Gata3 expression initiates already at the hematopoietic stem cell (HSC) level, implicating a potential role also in the regulation of HSCs. Herein we used a conditional Gata3 knockout strategy in which Gata3 expression was completely deleted from the earliest stage of embryonic hematopoietic development after emergence of HSCs from hemogenic endothelium. Through a detailed analysis of HSCs at the phenotypic and functional level, we demonstrate that steady-state levels of HSCs are normal in Gata3(fl/fl)Vav-Cre(tg/+) mice. Moreover, through long-term primary and secondary transplantation experiments, we also unequivocally demonstrate that Gata3 has a redundant role in post-transplantation HSC self-renewal.

Previously, a modest association was observed between the progesterone receptor +331G/A gene variant and breast cancer risk. Here, in a larger sample of breast cancer cases and controls (n = 1,322/n = 1,953) nested in the Nurses' Health Study cohort, we confirm a significant association (odds ratio, 1.41; 95% confidence interval, 1.10-1.79) and suggest a molecular model. The association of the +331G/A variant with breast cancer was particularly strong among obese women (body mass index>> 30; odds ratio, 2.87; 95% confidence interval, 1.40-5.90). To help understand the molecular mechanism by which this variant may predispose women to breast cancer, we identified nearby transcription factor binding sites. This search predicted a binding site for the GATA family of transcriptional regulators adjacent to this hPR polymorphism. Importantly, we found GATA3, GATA4, and GATA6 are expressed in normal breast tissue and two breast cancer cell lines, whereas GATA5 is minimally expressed in normal mammary tissue and more strongly expressed in two breast cancer cell lines. This finding was relevant because GATA5 bound the site adjacent to the +331G/A polymorphism, and activated the hPR (-711 to +822)-luciferase reporter plasmid in breast cancer cells. Overexpression of GATA5 increased expression of the endogenous hPR transcript, and GATA5 more strongly activated an hPR promoter construct encoding the PR-B isoform. Finally, hPR promoter constructs including the +331A were more strongly activated by GATA5 than constructs including +331G. Our findings suggest that GATA5 interacts with the +331G/A polymorphism to stimulate hPR-B expression in mammary cells, which may contribute to breast cancer susceptibility.

BACKGROUND

Cashmere growth is a seasonal and cyclic phenomenon under the control of photoperiod and multiple stimulatory and inhibitory signals. Beyond relevant coding genes, microRNA (miRNA) and long non coding RNA (lncRNA) play an indispensable role in hair follicle (HF) development and skin homeostasis. Furthermore, the influence of lncRNA upon miRNA function is also rapidly emerging. However, little is known about miRNAs, lncRNAs and their functions as well as their interactions on cashmere development and cycling.

RESULTS

Here, based on lncRNA and miRNA high-throughput sequencing and bioinformatics analysis, we have identified 1108 lncRNAs and 541 miRNAs in cashmere goat skin during anagen and telogen. Compared with telogen, 1388 coding genes, 41 lncRNAs and 15 miRNAs were upregulated, while 1104 coding genes, 157 lncRNAs and 8 miRNAs were downregulated in anagen (adjusted P-value ≤0.05 and relative fold-change ≥2). Subsequently, we investigated the impact of lncRNAs on their target genes in cis and trans, indicating that these lncRNAs are functionally conserved during HF development and cycling. Furthermore, miRNA-mRNA and miRNA-lncRNA interaction were identified through the bioinformatics algorithm miRanda, then the ceRNA networks, miR-221-5p-lnc_000679-WNT3, miR-34a-lnc_000181-GATA3 and miR-214-3p-lnc_000344-SMAD3, were constructed under defined rules, to illustrate their roles in cashmere goat HF biology.

CONCLUSIONS

The present study provides a resource for lncRNA, miRNA and mRNA studies in cashmere cycling and development. We also demonstrate potential ceRNA regulatory networks in cashmere goat HF cycling for the first time. It expands our knowledge about lncRNA and miRNA biology as well as contributes to the annotation of the goat genome.

OBJECTIVE

To explore the potential role of thymic stromal lymphopoietin (TSLP) and its downstream molecules in the development of ocular allergic inflammation using a short ragweed (SRW)-induced mouse model of allergic conjunctivitis (AC).

METHODS

BALB/c mice were topically challenged with SRW pollen after they were sensitized with SRW in the footpad. After the last SRW challenge, the corneal epithelium, conjunctiva, and cervical lymph nodes were harvested for total RNA extraction and gene expression by RT and real-time PCR, and whole eye globes were collected to make cryosections for immunohistochemical staining.

RESULTS

Repeated topical challenges with SRW allergen generated typical signs of AC in mice. Compared with the untreated controls, TSLP mRNA expression and immunoreactivity were significantly increased in the corneal and conjunctival epithelia of SRW-induced AC mice. CD11c(+) and OX40L(+) immunoreactive cells largely infiltrated the conjunctiva with increased mRNA levels of CD11c, TSLPR, and OX40L detected in the corneal epithelium, conjunctiva, and cervical lymph nodes. CD4(+) Th2 cell infiltration was evidenced by increased levels of mRNA and immunoreactivity of CD4, IL-4, IL-5, and IL-13 in the ocular surface, mainly in the conjunctiva, accompanied by increased expression of OX40, STAT6, and GATA3, in AC mice. The maturation of immature DCs was observed with the use of TSLP containing conditioned media from corneal epithelial cultures exposed to polyI:C, which stimulates TSLP production.

CONCLUSIONS

This study provides new findings regarding the role of local mucosal epithelial cells in the initiation of ocular allergic inflammation by producing a novel proallergic cytokine, TSLP, which activates dendritic cells to prime Th2 differentiation and allergic inflammation through the TSLP-TSLPR and OX40L-OX40 signaling pathway.

The mammalian target of rapamycin (mTOR) controls T-cell differentiation in response to polarizing cytokines. We previously found that mTOR blockade by rapamycin (RAPA) delays the G1-S cell cycle transition and lymphocyte proliferation. Here, we report that both mTOR complex 1 and mTOR complex 2 are readily activated following TCR/CD28 engagement and are critical for early expression of Ifng, Il4 and Foxp3, and for effector T cell differentiation in the absence of polarizing cytokines. While inhibition of mTOR complex 1 and cell division were evident at low doses of RAPA, inhibition of mTOR complex 2, Ifng, Il4 and Foxp3 expression, and T-cell polarization required higher doses and more prolonged treatments. We found that while T-bet and GATA3 were readily induced following TCR/CD28 engagement, administration of RAPA delayed their expression, and interfered with the loss of DNA methylation within Ifng and Il4 promoter regions. In contrast, RAPA prevented activation-dependent DNA methylation of the Foxp3 promoter favoring Foxp3 expression. As a result, RAPA-cultured cells lacked immediate effector functions and instead were enriched for IL-2+ cells. We propose that mTOR-signaling, by timing the expression of critical transcription factors and DNA methylation of proximal promoter regions, regulates transcriptional competence at immunologically relevant sites and hence lymphocyte differentiation.

USP21 is a ubiquitin specific protease that catalyzes protein deubiquitination, however the identification of its physiological substrates remains challenging. USP21 is known to deubiquitinate transcription factor GATA3 and death-domain kinase RIPK1 in vitro, however the in vivo settings where this regulation plays a biologically significant role remain unknown. In order to determine whether USP21 is an essential and non-redundant regulator of GATA3 or RIPK1 activity in vivo, we characterized Usp21-deficient mice, focusing on mouse viability and development, hematopoietic stem cell function, and lymphocyte differentiation. The Usp21-knockout mice were found to be viable and fertile, with no significant dysmorphology, in contrast to the GATA3 and RIPK1 knockout lines that exhibit embryonic or perinatal lethality. Loss of USP21 also had no effect on hematopoietic stem cell function, lymphocyte development, or the responses of antigen presenting cells to TLR and TNFR stimulation. GATA3 levels in hematopoietic stem cells or T lymphocytes remained unchanged. We observed that aged Usp21-knockout mice exhibited spontaneous T cell activation, however this was not linked to altered GATA3 levels in the affected cells. The contrast in the phenotype of the Usp21-knockout line with the previously characterized GATA3 and RIPK1 knockout mice strongly indicates that USP21 is redundant for the regulation of GATA3 and RIPK1 activity during mouse development, in hematopoietic stem cells, and in lymphocyte differentiation. The Usp21-deficient mouse line characterized in this study may serve as a useful tool for the future characterization of USP21 physiological functions.

Induced pluripotent stem cells (iPSCs) undergo extensive nuclear reprogramming and are generally indistinguishable from embryonic stem cells (ESCs) in their functional capacity and transcriptome and DNA methylation profiles. However, direct conversion of cells from one lineage to another often yields incompletely reprogrammed, functionally compromised cells, raising the question of whether pluripotency is required to achieve a high degree of nuclear reprogramming. Here, we show that transient expression of Gata3, Eomes, and Tfap2c in mouse fibroblasts induces stable, transgene-independent trophoblast stem-like cells (iTSCs). iTSCs possess transcriptional profiles highly similar to blastocyst-derived TSCs, with comparable methylation and H3K27ac patterns and genome-wide H2A.X deposition. iTSCs generate trophoectodermal lineages upon differentiation, form hemorrhagic lesions, and contribute to developing placentas in chimera assays, indicating a high degree of nuclear reprogramming, with no evidence of passage through a transient pluripotent state. Together, these data demonstrate that extensive nuclear reprogramming can be achieved independently of pluripotency.