title: Effect of tyrosine hydroxylase gene silencing in CD4+ T lymphocytes on differentiation and function of helper T cells.
abstract: OBJECTIVE
  We explored effect of gene silencing of tyrosine hydroxylase (TH), a rate-limiting enzyme for synthesis of catecholamines (CAs), in CD4+ T cells on differentiation and function of helper T (Th) cells to provide more evidence for functional significance of lymphocyte-derived CAs.
  
  
  METHODS
  CD4+ T lymphocytes were isolated and purified from the mesenteric lymph nodes of mice. Recombinant TH miRNA expression vector (pcDNA6.2-GW/EmGFPmiR-TH) was constructed and transfected into concanavalin A (Con A)-activated CD4+ T lymphocytes using nucleofection technology. After incubated for 48 h, these cells were detected for TH gene and protein expression and CA content. Simultaneously, percentage of interferon-γ (IFN-γ)- and interleukin-4 (IL-4)-producing cells and levels of IL-2, IFN-γ, tumor necrosis factor (TNF), IL-4 and IL-5 in culture supernatants of Con A-stimulated CD4+ T cells were examined by flow cytometric analysis.
  
  
  RESULTS
  CD4+ T lymphocytes with TH RNAi expressed less TH mRNA and protein and synthesized less CAs including norepinephrine, epinephrine and dopamine than control cells with mock transfection. The silencing of TH gene in CD4+ T lymphocytes reduced percentage of IL-4-producing cells and elevated ratio of IFN-γ-producing cells to IL-4-producing cells, although it did not alter proportion of IFN-γ-producing cells. The Th1 cytokines, IL-2, IFN-γ and TNF, were increased, but the Th2 cytokines, IL-4 and IL-5, were decreased in the culture supernatants of Con A-stimulated CD4+ T lymphocytes that were transfected with TH miRNA.
  
  
  CONCLUSIONS
  TH gene silencing attenuates TH expression and CA synthesis in CD4+ T lymphocytes and promotes polarization of differentiation and function towards Th1 cells.
authors.affiliation: Department of Physiology, School of Medicine, Nantong University, Nantong, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.forename: Xiao-Qin
authors.initials: XQ
authors.lastname: Wang
authors.forename: Yu-Ping
authors.initials: YP
authors.lastname: Peng
authors.forename: Yi-Hua
authors.initials: YH
authors.lastname: Qiu
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000201:enzymology* / Q000276:immunology*; D002395:Catecholamines / Q000096:biosynthesis; D002454:Cell Differentiation / Q000276:immunology; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic / Q000276:immunology*; D020868:Gene Silencing / Q000502:physiology*; D007371:Interferon-gamma / Q000378:metabolism; D015847:Interleukin-4 / Q000378:metabolism; D008198:Lymph Nodes / Q000166:cytology; D051379:Mice; D018417:Th1 Cells / Q000201:enzymology / Q000276:immunology; D018418:Th2 Cells / Q000201:enzymology / Q000276:immunology; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
chemical_list: D002395:Catecholamines; D015847:Interleukin-4; D007371:Interferon-gamma; D014446:Tyrosine 3-Monooxygenase