Tài liệu Báo cáo khoa học: Identification and characterization of the transcription factors involved

Identification and characterization of the transcription

factors involved in T-cell development, t-bet, stat6 and

foxp3, within the zebrafish, Danio rerio

Suman Mitra, Ayham Alnabulsi, Chris J. Secombes and Steve Bird

Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, UK

Introduction

Naive CD4+ T-cells, on antigenic stimulation, become

activated, expand and differentiate into different effec￾tor subsets called T-helper (Th) cells. The differentia￾tion of naive T-cells into Th effector cells depends on

a variety of stimuli, such as antigen nature, antigen

dose and the strength and duration of signals through

the T-cell receptor (TCR)–CD3 complex, as well as the

cytokine microenvironment which activates the cellular

signalling pathways [1]. These Th cell subsets are cru￾cial for the induction of the most appropriate immune

response towards a particular pathogen. In mammals,

three types of CD4+ Th effector cell populations exist,

Th1, Th2 and Th17, characterized by their cytokine

repertoire and how they regulate B-cell and T-cell

Keywords

adaptive immunity; fish immunology; T-cells;

transcription factors; zebrafish

Correspondence

S. Bird, Scottish Fish Immunology Research

Centre, School of Biological Sciences,

Zoology Building, University of Aberdeen,

Aberdeen AB24 2TZ, UK

Fax: +44 1224 272396

Tel: +44 1224 272881

E-mail: s.bird@abdn.ac.uk

(Received 25 August 2009, revised

16 October 2009, accepted 27 October

2009)

doi:10.1111/j.1742-4658.2009.07460.x

The discovery of cytokines expressed by T-helper 1 (Th1), Th2, Th17 and

T-regulatory (Treg) cells has prompted speculation that these types of

responses may exist in fish, arising early in vertebrate evolution. In this

investigation, we cloned three zebrafish transcription factors, T-box

expressed in T cells (t-bet), signal transducer and activator of transcription

6 (stat6) and fork-head box p3 (foxp3), in which two transcripts are pres￾ent, that are important in the development of a number of these cell types.

They were found within the zebrafish genome, using a synteny approach in

the case of t-bet and foxp3. Multiple alignments of zebrafish t-bet, stat6

and foxp3 amino acids with known vertebrate homologues revealed regions

of high conservation, subsequently identified to be protein domains impor￾tant in the functioning of these transcription factors. The gene organiza￾tions of zebrafish t-bet and foxp3 were identical to those of the human

genes, with the second foxp3 transcript lacking exons 5, 6, 7 and 8. Zebra￾fish stat6 (21 exons and 20 introns) was slightly different from the human

gene, which contained 22 exons and 21 introns. Immunostimulation of

zebrafish head kidney and spleen cells with phytohaemagglutinin, lipo￾polysaccharide or Poly I:C, showed a correlation between the expression of

t-bet, stat6 and foxp3 with other genes involved in Th and Treg responses

using quantitative PCR. These transcription factors, together with many of

the cytokines that are expressed by different T-cell subtypes, will aid future

investigations into the Th and Treg cell types that exist in teleosts.

Abbreviations

foxp3 ⁄ Foxp3, fork-head box p3; IFN-c, interferon-c; IL, interleukin; LPS, lipopolysaccharide; OSBPL7, oxysterol-binding protein-like 7; PHA,

phytohaemagglutinin; PPP1R3F, protein phosphatase 1, regulatory (inhibitor) subunit 3F; RACE, rapid amplification of cDNA ends;

stat6 ⁄ STAT6, signal transducer and activator of transcription 6; t-bet ⁄ T-bet, T-box expressed in T cells; TCR, T-cell receptor; TGF-b,

transforming growth factor-b; Th, T-helper; Treg, T-regulatory.

128 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS

responses [2]. Th1 cells produce interferon-c (IFN-c)

and lymphotoxin, activating cell-mediated immunity

and providing protection against intracellular patho￾gens and viruses. Th2 cells secrete interleukin-4 (IL-4),

IL-13 and IL-25 (also known as IL-17E), which are

important in the generation of the correct class of

antibodies by B-cells, and for the elimination of

extracellular pathogens, such as helminths and other

extracellular parasites [2]. Th17 is the most recently

identified Th cell subset and secretes pro-inflammatory

cytokines, such as IL-17A, IL-17F, IL-21 and IL-22

[3,4]. Th17 cells play an important role in host defence

against extracellular pathogens, in particular extra￾cellular bacteria, which are not efficiently cleared by

Th1- and Th2-type immunity [5]. Finally, in addition

to Th cells, there is a population of CD4+ T-cells that

is involved in the regulation of Th responses via the

secretion of cytokines, called T-regulatory (Treg) cells,

which help to inhibit harmful immunopathological

responses directed against self- or foreign antigens

[6,7]. The majority of these cells constitutively express

the CD25 cell surface marker and secrete two powerful

anti-inflammatory cytokines: IL-10 and transforming

growth factor-b (TGF-b).

Whether a naive T-cell becomes a Th1, Th2, Th17

or Treg cell is influenced by the cytokines that are pro￾duced within the microenvironment, which, in turn,

influence transcription factors and key signalling path￾ways [8]. Th1 differentiation is initiated by coordinate

signalling through the TCR and cytokine receptors,

for cytokines such as type I and II IFNs or IL-27,

which are associated with STAT1 [9,10]. Activation of

STAT1 induces the transcription factor, T-box

expressed in T cells (T-bet), which is a master regula￾tor of Th1 differentiation [11]. T-bet potentiates the

expression of IFN-c, which, in turn, upregulates the

inducible chain of the IL-12 receptor (IL-12Rb2).

Binding of IL-12 to its receptor induces signalling

through STAT4, which further enhances IFN-c pro￾duction and induces the expression of IL-18Ra, allow￾ing the responsiveness of these now mature Th1 cells

to IL-18 [12]. Th2 differentiation is initiated by TCR

signalling, together with IL-4 receptor signalling via

signal transducer and activator of transcription 6

(STAT6). This, in turn, up-regulates the low-level

expression of GATA3, the master regulator of Th2 dif￾ferentiation [13]. GATA3 autoactivates its own expres￾sion, eventually enabling mature Th2 cells to express

the Th2 cytokine cluster, IL-4, IL-5 and IL-13, as a

result of epigenetic changes [14]. Th1 and Th2 cells

negatively regulate each other’s development. GATA3

suppresses STAT4 and the IL-12Rb2 chain expression,

factors which are critical to the Th1 pathway [15],

whereas IL-27 suppresses Th2 development [16].

Th17 differentiation is slightly more complex

because of differences between mice and humans [17].

In mice, Th17 differentiation is initiated by TCR

signalling, together with TGF-b1 and IL-6 receptor

signalling, which activates STAT3 and induces the

expression of the transcription factor retinoic acid￾related orphan receptor ct. IL-23 also activates STAT3

but, in addition, serves to maintain Th17 cells in vivo.

In contrast, human cells do not require TGF-b1, and

it is IL-1, IL-6 and IL-23 that promote human Th17

differentiation [17]. Lastly, Treg cells are crucial players

in the regulation ⁄suppression of each of the Th

responses and self-reactive T-cells. It is now known

that there is more than one subtype of Treg cells,

although the most important appear to be

CD4+CD25+Foxp3+Treg [18]. These cells are affected

by the transcription factor fork-head box p3 (Foxp3),

whose induction is initiated by TCR signalling,

together with TGF-b1 receptor signalling [19]. Treg

suppressive activity is via IL-10 and TGF-b, although

it remains unclear whether these cytokines are

produced by CD4+CD25+Foxp3+Treg or whether

they induce the production of these cytokines from

another population of cells [20].

To date, our knowledge about the different types of

Th and Treg responses relates to studies performed in

mammals, especially mice and humans [12]. In fish,

there has been a considerable amount of work under￾taken on immunity over the last few decades, and a

large number of genes involved in immune responses

have been discovered. However, although we know a

lot about the innate and inflammatory immune

responses of fish [21], relatively little is known about

the lymphocyte subpopulations involved in the adap￾tive immune responses in fish, and whether Th subsets

exist. Speculation that Th1, Th2, Th17 and Treg

responses may exist in fish, and arose early in verte￾brate evolution, has been prompted by the discovery

of many of the cytokines that are expressed by these

cell types [22,23]. However, it is important to note that

not all the cytokines known in mammals have been

found in fish, and it remains to be determined whether

the regulation of adaptive immunity in fish is similar

to that found in mammals, and if it is equally complex.

In addition, the key transcription factors involved in

driving the differentiation of the naive T-cell to Th1,

Th2, Th17 or Treg cells may exist in fish. In this inves￾tigation, we have identified, for the first time, t-bet and

stat6 in zebrafish and, for the first time in any fish spe￾cies, foxp3. Lastly, we carried out some preliminary

S. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3

FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 129