The significance of TIM-3 and Galectin-9 expression during pregnancy in a mouse model
Matyas Meggyes
1, Tamas Palkovics
1, Barbara Bogar
1, Aliz Barakonyi
1, Eva Miko
1, Zsolt Illes
2, Julia Szekeres
1, Laszlo Szereday
1Department of Medical Microbiology and Immunology, University of Pecs, Medical School, Pecs, Hungary
1Department of Neurology, University of Pecs, Medical School, Pecs, Hungary
2IMMUNOLOGICAL BACKGROUND
Fig. 1. The structure of Tim-3 and Galectin-9 molecules
MATERIALS AND METHODS
RESULTS
Flow cytometry
Our results show that the expression of TIM-3 by decidual CD8, CD4, and g/d T cells is higher than by the peripheral blood lymphocytes.
Spongiotrophoblast
Immunohistochemistry
We could successfully detect the presence of Galectin-9 on the trophoblast by immunohistochemistry.
We hypothesize that the expression of Galectin-9 by the trophoblast and of elevated TIM-3 on decidual responder cells may contribute to the regulation of Th1 immunity and tolerance induction.
Functional tests are planned to investigate the ultimate outcome of these interaction. All of the data obtained by animal experiments will be extrapolated to human studies.
DISCUSSION
Considering allograft rejection as a basic feature of the immune system, the human pregnancy is an immunological paradox where the semi-allogeneic fetus is not rejected. Multiple mechanisms are involved in peripheral and local tolerance induction that prevents fetal rejection while maintaining competent immune surveillance and protection.
The T-cell immunoglobulin domain and mucin domain (TIM) family is a relatively newly described group of molecules with a conserved structure and important immunological functions. A growing body of evidence supports the critical role of different TIM molecules as modulators of the immune response in transplant tolerance. Since pregnancy and childbirth shares several immunological features with allograft transplantation tolerance and rejection, understanding the tremendous data obtained by investigating the role of Galectin-9/TIM-3 pathway during transplantation let us emphasize the importance of this pathway in reproductive immunology. TIM-3 was the first, and is presently the only, surface molecule that can specifically identify Th1 cells in both mice and humans. Identification of Galectin-9 as a ligand for TIM-3 has established the Galectin-9/TIM-3 pathway as an important regulator of Th1 immunity and tolerance induction.
Two weeks pregnant BALB-C mice were used and sacrificed by cervical dislocation.
Spleen and uterine horns were isolated.
Placentas were separated from the fetuses and the decidua was carefully removed from the placentae with microforceps. After that decidual cells were digested with collagenase in 37°C.
Lymphocytes were separated in Ficoll Paque gradient.
Periphery Decidua
Cell isolation
Fig. 4. Ficoll Paque gradient
1. Animal model 2. Flow cytometry 3. Immunohistochemistry
Isolated peripheral and decidual cells were stained with anti mouse monoclonal antibodies. The cells were measured with a BD FACSCalibur flow cytometer and evaluated by FCS express.
Primer antibody: a-mouse Galectin-9 Secunder antibody: a-rabbit HRPO Stain: AEC (amino-ethyl-carbasol)
Fig. 2. Pregnant BALB-C mice
Fig. 3. Isolated fetuses and resorption
Fig. 5. Immunohistochemical staining
We examined the Galectin-9 expression by real time PCR and immunohistochemistry in the two weeks old pregnant BALB-C mice placentae and defined the TIM-3 surface expression in the isolated decidual and peripheric lymphocytes subpopulation by flow cytometric analyses.
Fig. 7. Galectin-9 expression by the mouse placenta, indirect immunhistochemical staining Fig. 6. TIM-3 expression by peripheral and decidual lymphocytes (flow cytometric analysis)
4. Real Time PCR
Isolated placentas and resorptions were homogenized and frozen in -80°C.
After thawing Galectin-9 mRNS expression was detected by a Corbett Research Real Time PCR.
Real Time PCR
Galectin-9 mRNA levels by the fetal resorptions were 4x lower compared to healthy placentae.
Habilitas Mentor Program
Research was supported by the
TÁMOP 4.2.2/B-10/1-2010-0029 „Development of a complex educational assistence/suport system for talented students and prospective researches at the Pecs University project.
TÁMOP 4.2.2/B-10/1-2010-0029
Mononuclear cells
15LEP CD4FITC TIM1PE TIM3AP.001
01-Jun-11 TIM-3 APC
100 101 102 103 104
SSC-Height
0 256 512 768 1024
0,4%
16DEC CD4FITC TIM1PE TIM3AP.002
16-Jun-11 TIM-3 APC
100 101 102 103 104
SSC-Height
0 256 512 768 1024
5,37%
16LEP CD49b TIM1 CD3 TIM3.001
16-Jun-11 TIM-3 APC
100 101 102 103 104
SSC-Height
0 256 512 768 1024
6,15%
15DEC CD49b TIM1 TIM3.002
01-Jun-11 TIM-3 APC
100 101 102 103 104
SSC-Height
0 256 512 768 1024
6,98%
2LEP CD8FIT TIM1PE TIM3APC.002
25-Feb-10 TIM-3 APC
100 101 102 103 104
SSC-Height
0 256 512 768 1024
0,33%
6DEC CD8FIT TIM1PE TIM3APC.002
03-Mar-10 TIM-3 APC
100 101 102 103 104
SSC-Height
0 256 512 768 1024
12,23%
5LEP gdFITC TIM1PE TIM3APC.001
02-Mar-10 TIM-3 APC
100 101 102 103 104
SSC-Height
0 256 512 768 1024
2,23%
16DEC gdFITC TIM1PE TIM3APC.002
16-Jun-11 TIM-3 APC
100 101 102 103 104
SSC-Height
0 256 512 768 1024
8,39%
Decidual lymphocytes
TIM-3 expression by CD4+ cells
TIM-3 expression by NK cells TIM-3 expression
by NK cells TIM-3 expression
by CD8+ cells
TIM-3 expression by CD4+ cells
TIM-3 expression by CD8+ cells
TIM-3 expression
by g/d T cells TIM-3 expression
by g/d T cells
AIM OF STUDIES
The aim of our study was to investigate the possible role of Galectin-9/TIM-3 pathway in the immunregulation during pregnancy.
Fetal resorption Healthy fetuses
Group comparisons were made using non-parametric Mann-Whitney U-test. Differences were accepted as significant at a level of p<0.05.
12
10
8
6
4
2
0 NS
TIM3 expression
n=8 n=8
TIM3 expression
30
20
10
0
21 28
p<0,001
n=8 n=8
CD4+ cells CD8+ cells gd+ cells NK cells
20
10
0
8 28
p<0,02
TIM3 expression
n=8 n=8
10
8
6
4
2
0
p<0,001
TIM3 expression
n=8 n=8
Peripheral lymphocytes
Acknowledgement
This work was supported by grants from Hungarian National Research Fund (OTKA-NNF 78790), Pecs University Research Found (34039/KA-OTKA/11-19 and 11-14)
TÁMOP-4.2.1. B-10/2/KONV-2010-0002
