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  1. 1. Transforming growth factor-b ‘reprograms’ the differentiation of T helper 2 cells and promotes an interleukin 9–producing subset Eman abd el-raouf ahmed
  2. 2. The content Back ground information
  3. 3. IL-9 is a multifunctional cytokine, belong to a family of cytokines. IL-9 was initially reported as a T cell growth factor in mice. It is now known to target multiple cell types. It plays an important role in the expansion and recruitment of mast cells in response to intestinal nematode infection or during autoimmune encephalomyelitis. It is also known to act on various cell types known to be involved in asthma including T cells, B cells, mast cells, eosinophils, neutrophils, and epithelial cells. IL 9 can promote the expression of TGF-beta in lipopolysaccharide induced monocytes and macrophages. IL-9 is also known to play important roles in conditions including airway inflammation,
  4. 4. IL-9 signals through the heterodimeric receptor composed of a specific chain (IL-9R) and a gamma chain (IL2RG), which is shared between IL-2, IL-4, IL-7, IL15 and IL-21. The IL-9R and IL-2RG associates with JAK1 and JAK3 respectively. Receptor engagement results in JAK1- JAK3 cross phosphorylation and activation of the JAK proteins which leads to the activation of Signal transducer and activator of transcription (STAT-1, STAT-3 and STAT-5) and Insulin receptor substrate 1 and 2 (IRS1 and IRS2)/PI3K cascades. IL-9 stimulation also results in the activation of MEK/ERK signaling cascade.
  5. 5. General pathway
  6. 6. T-bet pathway
  7. 7. METHODS Mice. Transgenic CD4dnTGFbRII mice29 and 4get mice9 on a B6 background, as well as wild-type B6 mice, were kept in specific pathogen–free conditions, and all animal experiments were done according to institutional guidelines (National Institute for Medical Research Ethical Review Panel) and UK Home Office regulations. Spleens from Stat6–/– mice on a BALB/c background were provided by C. Watson
  8. 8. In vitro T cell differentiation and cytokine analysis. Naive T cells (CD4+CD25–CD44lo) sorted by flow cytometry were cultured in Iscove’s modified Dulbecco’s medium (Sigma) supplemented with 5% (vol/vol) FCS, L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml) and mercaptoethanol (50 nM; all from Sigma) in the presence of anti-CD3 (1 mg/ml; 2C11) and anti-CD28 (10 mg/ml; 37.51; both plate bound). Cytokines for effector cell differentiation were as follows (all from Invitrogen): TH1, IL-12 (3 ng/ml); TH2, IL-4 (10 ng/ml) and anti-TGF-b (5 mg/ml; 1D11); iTreg, TGF-b (5 ng/ml) and anti-IL-4 (5 mg/ml; 11B11); TH-17, TGF-b (1 ng/ml), IL-6 (20 ng/ml) and anti-IL-4 (5 mg/ml); and ‘TH-9’, IL-4 (10 ng/ml) and TGF-b (1 ng/ml). Cells were cultured for 5 d and then restimulated for 4 h with phorbol dibutyrate and ionomycin (both at 500 ng/ml) in the presence of brefeldin A (1 mg/ml) before intracellular staining of cytokines. For intracellular staining of IL-9, goat anti–mouse IL-9 was affinity-purified (0.5 mg) and was labeled with Alexa Fluor 488 (Invitrogen) or fluorescein isothiocyanate according to the manufacturer’s instructions. For ELISA of mouse IL-9, the mouse IL-9–specific monoclonal antibody MM9C1 (ref. 12) was used as the capture antibody and goat anti-IL-9 was used for development.
  9. 9. Quantitative analysis of mRNA for cytokines and transcription factors. RNA was extracted with TRIzol and 1-bromo-3-chloro-propane (Sigma) and was reverse-transcribed with oligo(dT)16 (Applied Biosystems) according to the manufacturer’s protocol. The cDNA served as template for amplification of target genes, as well as of the ‘housekeeping’ gene Hprt1 (encoding hypoxanthine guanine phosphoribosyl transferase), by real-time PCR with TaqMan Gene Expression sssays (Applied Biosystems), universal PCR Master Mix (Applied Biosystems) and the ABI-PRISM 7900 sequence-detection system (Applied Biosystems). Expression of target genes was calculated with by comparative method for relative quantification after normalization to Hprt1 expression.
  10. 10. Infection with T muris. T. muris was maintained as described31. Mice were infected with 150 embryonated eggs by oral gavage on day 0, and the number of adult worms in the cecum was assessed 30 d later. Cecal tissue was fixed in Carnoy’s fluid and was histologically processed by standard methods; sections 5 mm in thickness were stained for mucosal mast cells (0.5% (vol/vol) toluidine blue). Mucosal mast cells per 20 randomly selected cecal crypt units were counted by light microscopy from at least two sections per mouse
  11. 11. references /research_group/paphos/App59.pdf