Gene Expression Profiling Implicates Attenuation of NFkB Signalling By Regulatory T Cells in Modulating Dendritic Cell Function

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3068-3068
Author(s):  
Lindsay Nicholson ◽  
Emily Mavin ◽  
Lynne Minto ◽  
Julie Irving ◽  
Anne Dickinson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
T Cells ◽  
Regulatory T Cells ◽  
Gene Expression Profiling ◽  
Pathway Analysis ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Antigen Presenting ◽  
Unique Genes

Abstract Dendritic cells (DC) play a key role in the pathogenesis of Graft versus Host Disease (GvHD), a complication of haematopoietic stem cell transplantation and offer an attractive target for therapy. Regulatory T cells (Treg) have a potent immunoregulatory effect on the maturation and the antigen-presenting cell (APC) function of DC and adoptive transfer of Treg is highly efficacious in the induction of tolerance in an experimental model of GvHD and has entered Phase I clinical trials. Several mechanisms of suppression have been proposed, including Treg acting directly on DCs, attenuating their antigen-presenting and co-stimulatory functions by arresting their maturation. However, the molecular basis underpinning these effects in DCs remains ill-defined. We investigated the effect of Treg treatment on DCs by conducting gene expression profiling and confirmed the functional consequences using downstream assays. Immature, mature and Treg-treated DCs were generated from immuno-magnetic isolated monocytes (im-DC, mat-DC and Treg-DC, respectively) and moDC populations were generated using a well-established 6 day culture with GM-CSF and IL-4, followed by 24 hour LPS maturation. Treg were added on day 3 of culture at a 3:1 ratio. All cell populations were harvested on day 7 and sorted via FSC/SSC/CD3neg gating to remove Treg present in the co-culture and control for any changes in gene expression caused by shear stress. Gene expression profiling was carried out using the Illumina HumanHT12 microarray platform. Data was processed using R/Bioconductor workflows and the functional significance of differentially expressed genes was evaluated using Ingenuity Pathway Analysis software. Mat-DC and Treg-DC expression profiles were compared relative to the im-DC for data analysis. Upon LPS treatment, the levels of 1834 unique genes were differentially regulated in mat-DC by at least twofold (862 genes upregulated/972 downregulated) compared to the im-DC counterparts. In the Treg-DC, 1326 unique genes were differentially modulated (633 genes upregulated/693 genes downregulated). Microarray analysis of the CD markers identified a higher expression of the previously identified surface markers CD80, CD83 and CD86 in the mat-DC compared to the Treg-treated counterpart (validated by flow cytometry), confirming the semi-mature phenotype. Novel findings from the dataset include the reduction of the endocytotic-related genes, CD206 and CD209, in the Treg-DCs compared to the im-DC and this reduction manifested functionally in an impaired antigen uptake, as assessed by FITC-Dextran. Additionally, the surface marker, CD38, was downregulated in the Treg-DC compared to the mat-DC, confirmed by flow cytometry. CD38 has been shown to be NFκB-dependent and a marker of maturation in monocyte-derived DCs, further supporting the semi-mature phenotype. Furthermore, CD38 is functionally involved in CD83 expression and IL-12 induction. We assessed IL-12 cytokine secretion by Treg-treated DCs and showed a significantly reduced level of induction compared to mat-DC (p=0.0079). Pathway analysis revealed NFκB-related genes to be downregulated in the Treg-DC compared to the mat-DC. These differentially expressed genes included the TLR-adaptor protein, MYD88, the NFκB subunit, RELB and an inhibitor of NFκB, NFκB1A. This finding, coupled to the importance of NFκB signalling pathway in DC function, prompted us to investigate it at the functional level by measuring levels of phosphorylation of serine 536 of the RelA subunit as a marker of activity in response to LPS stimulation. DC cultured in the absence of Tregs (mat-DC) showed significantly higher levels of Ser536 phosphorylation when compared to those unstimulated cells (im-DC) (p= 0.0018). Concordant with the gene expression data, Treg-treated DCs (Treg-DC), showed a significantly attenuated NFκB activation when compared to their LPS-stimulated DCs counterparts (p = 0.0191), however, signalling was not completely abolished compared to those unstimulated DCs (p= 0.0003). In conclusion, gene expression profiles of Treg-treated DCs are significantly different to their mat-DC and im-DC counterparts. Here, we present the novel finding that Tregs modulate DC function, in part, by attenuation of the NFkB signalling pathway, arresting the DCs at a semi-mature phenotype, as evidenced by expression arrays and functional assays. Disclosures No relevant conflicts of interest to declare.

scholarly journals 256CONSTRUCTION OF A BOVINE CDNA ARRAY TO MONITOR GENE EXPRESSION PROFILES IN BOVINE PREIMPLANTATION EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 248
Author(s):  
C. Wrenzycki ◽  
T. Brambrink ◽  
D. Herrmann ◽  
J.W. Carnwath ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Cdna Array ◽  
Preimplantation Embryos ◽  
Average Insert Size ◽  
Rt Pcr ◽  
Public Data

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

scholarly journals Three Years Follow-Up Study Showed Prenatal Methamphetamine Exposure May Cause Chronic Abnormalities In Transcriptomic Pattern

2021 ◽  
Author(s):  
Arvin Haghighatfard ◽  
Soha Seifollahi ◽  
Pegah Rajabi ◽  
Niloofar Rahmani ◽  
Rojin Ghannadzadeh
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Rna Extraction ◽  
Childbearing Age ◽  
Methamphetamine Use ◽  
Methamphetamine Use Disorder

Abstract Background: The high rate of methamphetamine use disorder among young adults and women of childbearing age makes it imperative to clarify the long-term effects of Methamphetamine exposure on the offspring. Behavioral and cognitive problems had been reported in children with parental Methamphetamine exposure (PME). The present study aimed to assess the acute and chronic effects of PME in molecular regulations and gene expression profiles of children during their first years of life.Methods: All subjects were recruited before birth, and sampling was conducted from the first ten days of birth, twelve months, twenty months, and thirty-six months of age. Finally, 2658 children with PME and 3573 normal children had been finished the follow-up. RNA extraction was operated from blood samples and gene expression profiling was conducted by using the Affymetrix GeneChip Human Genome U133 plus 2.0 Array Platform. Gene expression data were confirmed by Real-time PCR. Results: Gene expression profiling during thirty-six months showed several constant mRNA level alterations in children with PME compared with normal. These genes are involved in several gene ontologies and pathways involved with the immune system, neuronal functions, and bioenergetic metabolism. It seems that Methamphetamine use disorder before and during the pregnancy period may affect the expression profile of children, and these changes could remain years after birth. Affected genes have some similarities with the gene expression patterns of addiction, psychiatric disorders, neurodevelopmental disabilities, and immune deficiencies. Conclusion: Findings may shed light on the molecular effects of prenatal methamphetamine exposure and may lead to new psychological and somatic caring protocols for these children based on their potential abnormalities.

scholarly journals Effectiveness of Gene Expression Profiling for Response Prediction of Rectal Adenocarcinomas to Preoperative Chemoradiotherapy

2005 ◽  
Vol 23 (9) ◽  
pp. 1826-1838 ◽  
Author(s):  
B. Michael Ghadimi ◽  
Marian Grade ◽  
Michael J. Difilippantonio ◽  
Sudhir Varma ◽  
Richard Simon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Response Prediction ◽  
Preoperative Chemoradiotherapy ◽  
Response To Therapy ◽  
Phase Iii ◽  
Correct Prediction

Purpose There is a wide spectrum of tumor responsiveness of rectal adenocarcinomas to preoperative chemoradiotherapy ranging from complete response to complete resistance. This study aimed to investigate whether parallel gene expression profiling of the primary tumor can contribute to stratification of patients into groups of responders or nonresponders. Patients and Methods Pretherapeutic biopsies from 30 locally advanced rectal carcinomas were analyzed for gene expression signatures using microarrays. All patients were participants of a phase III clinical trial (CAO/ARO/AIO-94, German Rectal Cancer Trial) and were randomized to receive a preoperative combined-modality therapy including fluorouracil and radiation. Class comparison was used to identify a set of genes that were differentially expressed between responders and nonresponders as measured by T level downsizing and histopathologic tumor regression grading. Results In an initial set of 23 patients, responders and nonresponders showed significantly different expression levels for 54 genes (P < .001). The ability to predict response to therapy using gene expression profiles was rigorously evaluated using leave-one-out cross-validation. Tumor behavior was correctly predicted in 83% of patients (P = .02). Sensitivity (correct prediction of response) was 78%, and specificity (correct prediction of nonresponse) was 86%, with a positive and negative predictive value of 78% and 86%, respectively. Conclusion Our results suggest that pretherapeutic gene expression profiling may assist in response prediction of rectal adenocarcinomas to preoperative chemoradiotherapy. The implementation of gene expression profiles for treatment stratification and clinical management of cancer patients requires validation in large, independent studies, which are now warranted.

scholarly journals Innate inflammatory gene expression profiling in potential brain-dead donors: detailed investigation of the effect of common corticosteroid therapy

2017 ◽  
Vol 23 (5) ◽  
pp. 440-448 ◽  
Author(s):  
Reza Gholamnezhadjafari ◽  
Nader Tajik ◽  
Reza Falak ◽  
Reza Aflatoonian ◽  
Sanaz Dehghan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Inflammatory Factors ◽  
Control Group ◽  
Organ Donors ◽  
Complement Components ◽  
Tnf Α ◽  
Brain Dead

Our study aimed to assess the influence of common methylprednisolone therapy on innate inflammatory factors in potential brain-dead organ donors (BDDs). The study groups consisted of 50 potential BDDs who received 15 mg/kg/d methylprednisolone and 25 live organ donors (LDs) as control group. Innate immunity gene expression profiling was performed by RT-PCR array. Soluble serum cytokines and chemokines, complement components, heat shock protein 70 (HSP70) and high mobility group box-1 (HMGB1) were measured by ELISA. Surface expression of TLR2 and TLR4 were determined using flow cytometry. Gene expression profiling revealed up-regulation of TLRs 1, 2, 4, 5, 6, 7 and 8, MYD88, NF-κB, NF-κB1A, IRAK1, STAT3, JAK2, TNF-α, IL-1β, CD86 and CD14 in the BDD group. Remarkably, the serum levels of C-reactive protein and HSP70 were considerably higher in the BDD group. In addition, serum amounts of IL-1β, IL-6, TNF-α, HMGB1, HSP70, C3a and C5a, but not IL-8, sCD86 or monocyte chemoattractant protein-1, were significantly increased in the BDD group. Significant differences were observed in flow cytometry analysis of TLR2 and TLR4 between the two groups. In summary, common methylprednisolone therapy in BDDs did not adequately reduce systemic inflammation, which could be due to inadequate doses or inefficient impact on other inflammatory-inducing pathways, for example oxidative stress or production of damage-associated molecules.

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