Project description:Study the effect of PARP-14 and its activity on Th2 differentiation ChIP seq was performed on Th2 differentiated cells isolated from PARP-14 +/+ and PARP-14 -/- treated with or without PJ34
Project description:Naive CD4+ T cells, sorted from PBMCs of grass-fed beef cattle, were stimulated with anti-bovine CD3 under Th2 differentiation conditions with or without Ostertagia ostertagi (OO) protein extract. Th1 differentiation conditions were included for comparison. The effect of recombinant bovine IL-4 (rbIL-4) and weakening TCR signal strength on Th2 differentiation were also tested. Flow cytometry, qPCR and proteomic assay were performed to analyze the differentiated cells.
The majority of differentiated cells expressed IFNgamma (a hallmark cytokine for Th1) and a small percentage of cells expressed both IFNgamma and IL-4 (a hallmark cytokine for Th2), indicating the presence of Th0 cells, as previously reported in cattle. While weakening TCR signal strength reduced Th2 cell expansion, adding rbIL-4 or OO protein extract enhanced Th2 cell expansion but without significant changes in IFNgamma and IL-4 expression. Proteomic data predicted that, as in mice and humans, bovine Th2 differentiation results from three upstream stimulation with CD3, CD28, and IL-4, which were inhibited when OO protein extract was added into the Th2 differentiation media. Importantly, protein profiling indicated that the addition of rbIL-4 enhanced IL-4 signaling but inhibited IL-12 signaling. Soon, we are planning to explore if other cytokines like IL-10 can be applied to optimize this Th2 differentiation in vitro.
Naive bovine CD4+ T cells differentiate into a mixed population containing a high percentage of IFNgamma producing cells and a small percentage of Th0 cells. Furthermore, bovine Th2 differentiation is sensitive to regulation such as by OO or rbIL-4.
Project description:PARP-14, a member of the poly ADP-ribose polymerase super family, promotes T helper cell 2 (Th2) differentiation by regulating interleukin-4 (IL-4) and STAT6-dependent transcription. Yet, whether PARP-14 globally impacts gene regulation has not been determined. In this report, using an RNA pol II ChIP-seq approach, we identify genes in Th2 cells that are regulated by PARP-14, and either dependent or independent of ADP-ribosyltransferase catalytic activity. Our data demonstrate that PARP-14 enhances the expression of Th2 genes as it represses the expression of Th1-associated genes. Among the relevant targets are Signal Transducer and Activator of Transcription genes required for polarizing Th1 and Th2 cells. To define a mechanism for PARP-14 function, we use an informatics approach to identify putative PARP-14 DNA binding sites. Two putative PARP-14 binding motifs are identified in multiple Th2 cytokine genes, and we demonstrate that PARP-14 interacts with each motif using in vitro binding assays. Taken together our results indicate that PARP-14 is an important factor for T helper cell differentiation and it binds to specific DNA sequences to mediate its function.
Project description:Poly(ADP-ribose) polymerase-2 (PARP-2) is acknowledged as a DNA repair enzyme; however, recently metabolic properties had been attributed to it. Hereby, we examined the metabolic consequences of PARP-2 ablation in liver. Microarray analysis of PARP-2 knockdown HepG2 cells revealed the dysregulation of lipid and cholesterol metabolism genes. Induction of cholesterol biosynthesis genes stemmed from the enhanced expression of sterol-regulatory element binding protein (SREBP)-1. We revealed that PARP-2 is a suppressor of the SREBP-1 promoter, therefore ablation of PARP-2 induces SREBP-1 expression and consequently cholesterol synthesis. PARP-2-/- mice had higher SREBP-1 expression that was translated into enhanced hepatic and serum cholesterol levels. PARP-2 silencing was performed employing shPARP-2 (small hairpin) and scPARP-2 (scrambled) shRNA by lentiviral delivery (Sigma) using 40 MOI lentiviruses coding shRNA sequence against PARP-2.
Project description:Adenylate cyclase signaling pathway is suggested to be a key regulator of immune system functions. However, specific effects of cyclic adenosine monophosphate on T helper cell differentiation and functions are unclear. Involvement of cAMP in Th cell differentiation program, in particular development of Th1, Th2, and Th17 subsets, was evaluated employing forskolin (FSK), a labdane diterpene well known as AC activator.