This document summarizes research on interleukin-9 (IL-9), a multifunctional cytokine that plays important roles in conditions like airway inflammation and asthma. The study found that transforming growth factor-beta (TGF-β) can "reprogram" the differentiation of T helper 2 cells and promote a IL-9-producing T cell subset. The researchers investigated IL-9 signaling pathways and used mouse models to examine the effects of IL-9 on intestinal nematode infection and autoimmune encephalomyelitis. They analyzed gene expression and cytokine production from T cells cultured under various conditions to identify factors that induce IL-9 production.

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. The content
Back ground information

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. 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.

9. General pathway

10. T-bet pathway

14. 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

15. 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.

16. 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.

17. 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

18. references
http://www.fao.org/ag/againfo/commissions/docs
/research_group/paphos/App59.pdf