Cellular & Molecular Immunology (2014), 1–3 ß 2014 CSI and USTC. All rights reserved 1672-7681/14 $32.00 www.nature.com/cmi
SHORT COMMUNICATION
Seminal vesicle fluid ameliorates experimental autoimmune encephalomyelitis by increasing FoxP31 regulatory T-cell numbers Nafiseh Pakravan1, Ameneh Ghaffarinia2, Cyrus Jalili3, Farhad Riazi-Rad4, Marzieh Tajedini5 and Ali Mostafaie2 Previous studies have shown that insemination elicits the expansion of the natural FoxP31 regulatory T cell (Treg) pool in female reproductive tissue during mating. Further research demonstrated that this effect was due to seminal vesicle fluid. Indeed, seminal vesicle fluid causes differential recruitment of Tregs into different organs. The recruitment of Tregs into the brain occurs at the lowest rate of all studied organs. In addition, Tregs are present at a lower frequency and/or have a reduced function in multiple sclerosis. These observations prompted us to investigate how intra-CSF administration of seminal vesicle fluid affects experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Our results suggest that seminal vesicle fluid contains all of the ligands required to activate the Treg pool and exhibits an anti-inflammatory effect on organs outside the reproductive system. Cellular & Molecular Immunology advance online publication, 22 September 2014; doi:10.1038/cmi.2014.88
In 2004, it was demonstrated that changes in female reproductive tissue due to insemination result in immunological priming to paternal antigens and influence pregnancy outcome. During mating, exposure to seminal fluid elicits a tolerant state to paternal allo-antigens by expanding the regulatory T cell (Treg) pool. Notably, mating with males with surgically removed seminal vesicle glands was shown to fail to induce such effects.1 Evidence of antigen-specific Treg responses2–4 suggested that the expansion of the Treg pool occurs in response to seminal vesicle fluid.6 Further research has revealed differential recruitment of Tregs into different tissues, including the liver, kidney, heart, spleen, lymph nodes, mucosal tissues, blood, skin, thymus and brain, of which recruitment to the brain was the lowest.1 Owing to these effects on Treg expansion and the lower recruitment to the brain, we administered seminal vesicle fluid via an intra-CSF route in a Lewis rat model of experimental autoimmune encephalomyelitis (EAE) and investigated its effect on Treg expansion and clinical disease course. We induced EAE in Lewis rats and treated them with saline, 0.1 mg/ml chymotrypsin or seminal vesicle fluid (liquefied using chymotrypsin). Then, IFN-c, IL-4, IL-17 and FoxP3 expression were estimated using a two-step real-time
PCR method on samples from the brain, spinal cord and deep cervical lymph node (Supplementary Information). Our EAE Lewis rat model exhibited an acute monophasic disease, with clinical signs beginning on day 7 and reached the peak of the disease on day 14 post-induction. Intra-CSF injection of seminal vesicle fluid, 0.1 mg/ml chymotrypsin or saline was performed on day 7 after EAE induction. Changing trends in clinical signs in seminal vesicle fluid-treated animals were markedly different from those in the animals treated with 0.1 mg/ml chymotrypsin or saline (Figure 1a). The maximum mean clinical scores during the course of study were 1.46, 1.41 and 0.41, in saline-, 0.1 mg/ml chymotrypsin- and seminal vesicle fluid-treated animals, respectively. Changes in EAE-associated gene expression were estimated during the peak of the disease (day 14 after EAE immunization). Because EAE is a T cell-mediated disease, we investigated the changes in IFN-c, IL-4, IL-17 and FoxP3 expression as representative of Th1, Th2, Th17 and natural Treg populations, respectively (Figure 1b). Seminal vesicle fluid administration resulted in a significant decreased in IFN-c expression in the brain compared with saline-treated animals. In addition, IFN-c expression was
1 Department of Microbiology and Immunology, Medical School, Alborz University of Medical Sciences, Karaj, Iran; 2Department of Immunology, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran; 3Department of Anatomy, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran; 4Department of Immunology, Pasture Institute of Iran, Tehran, Iran and 5Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran Correspondence: Dr N Pakravan, Department of Microbiology and Immunology, Medical School, Alborz University of Medical Sciences, Karaj, Iran. E-mail: [email protected] Received: 7 August 2014; Revised: 18 August 2014; Accepted: 18 August 2014
Short Communication
2
a
6 Saline
Seminal Vesicle Fluid
0.1mg/ml Chymotrypsin
Clinical Score
5 4 3 2 1 0 0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
30 25 20 15
* * ** ** * **
10
*
*
5 0
IL-17 relative expression
* 15
* **
*
10
* **
5 0
Sal
ine
sin
/ml
g .1m
0
p otry
m
chy
Sem Brain
ina
l Ve
eF sicl
luid
* **
0.7
0.16
160
*
0.6
140 120
0.5
100
0.4
80 0.3
60
* **
0.2
40
0.1
20 0
0
6
* * ** **
0.12
5 4 3
0.08
* **
0.04 0
1 0
Sal
ine
sin
/ml
g .1m
0 Spinal Cord (S.C)
2
p otry
m
chy
Sem
ina
l Ve
eF sicl
luid
FoxP3 relative expression in DCLN
20
IL-4 relative expression in Brain and S.C
IFN-g relative expression
35
IL-4 relative expression in DCLN
b
FoxP3 relative expression in Brain and S.C
Days After EAE Induction
Deep Cervical Lymph Node (DCLN)
Figure 1 Effect of treatment with seminal vesicle fluid (liquefied using 0.1 mg/ml chymotrypsin) (??&??), 0.1 mg/ml chymotrypsin (-m-), or saline (-- --) on the clinical score. Female Lewis rats were immunized subcutaneously with a suspension of guinea pig spinal cord and CFA. Intra-CSF injection of seminal vesicle fluid, 0.1 mg/ml chymotrypsin or saline was performed on day 7 (onset of EAE). Clinical scores were measured daily from the day of disease induction (a). Comparison of IFN-c, IL-17, IL-4 and FoxP3 expression (b) in the brain, SC and DCLN. The animals were treated via an intra-CSF route with saline-, 0.1 mg/ml chymotrypsin- or seminal vesicle fluid on day 7 after disease induction. The animals were euthanized on day 14 after EAE induction, at the peak of the disease. The quantification of each gene was normalized against the reference gene beta-actin. IL-4 and FoxP3 levels in the DCLN are shown on the secondary vertical axis because of the difference in scale. The data are presented as the mean6s.d. *Significantly different from the saline-treated animals. **Significantly different from the 0.1 mg/ml chymotrypsin-treated animals. CFA, complete Freund’s adjuvant; DCLN, deep cervical lymph node; EAE, experimental autoimmune encephalomyelitis; SC, spinal cord.
N
significantly decreased in animals treated with 0.1 mg/ml chymotrypsin compared with saline- and seminal vesicle fluidtreated animals. IL-17 expression was also determined because it is a critical cytokine in EAE. Injecting animals with seminal vesicle fluid significantly downregulated IL-17 expression compared with the saline- and 0.1 mg/ml chymotrypsin-treated animals. In contrast, IL-17 expression in the 0.1 mg/ml chymotrypsin-treated animals was markedly increased compared with in saline-treated animals. To further explore the mechanism of Cellular & Molecular Immunology
the decreased clinical score upon seminal vesicle fluid treatment, the expression of IL-4 and FoxP3 was investigated.5,6 FoxP3 is indicative of naturally occurring Tregs which have an anti-inflammatory effect.5 IL-4 is also indicative of the Th2 response and exhibits an ameliorating effect on EAE.6 No significant differences were observed in IL-4 expression between the groups treated with saline, 0.1 mg/ml chymotrypsin, or seminal vesicle fluid. Regarding FoxP3, significant upregulation of FoxP3 expression was observed in the seminal
Short Communication
3
vesicle fluid-treated animals compared with the 0.1 mg/ml chymotrypsin- and saline-treated animals, suggesting Treg upregulation upon seminal vesicle fluid treatment. No significant differences in FoxP3 expression were observed between the groups of animals treated with 0.1 mg/ml chymotrypsin or saline. The seminal vesicle fluid- and 0.1 mg/ml chymotrypsintreated rats exhibited a significant decrease in IFN-c expression in the spinal cord compared with the saline-treated animals. However, IFN-c levels in the seminal vesicle fluid-treated animals were significantly higher than those of the 0.1 mg/ml chymotrypsin-treated animals. Seminal vesicle fluid treatment markedly attenuated IL-17 expression compared with the 0.1 mg/ ml chymotrypsin- and saline-treated animals. However, no significant differences were observed between the saline- and 0.1 mg/ ml chymotrypsin-treated animals in terms of IL-17 expression. The levels of IL-4, a Th2 cytokine, in the seminal vesicle fluid- and 0.1 mg/ml chymotrypsin-treated animals were similar to those in the saline-treated group. FoxP3 expression was significantly elevated only in the animals treated with seminal vesicle fluid compared with the saline- and 0.1 mg/ml chymotrypsintreated animals. However, FoxP3 expression was not significantly different between the 0.1 mg/ml chymotrypsin- and salinetreated animals. Seminal vesicle fluid treatment resulted in significant suppression of IFN-c expression in the DCLNs (drainage lymph nodes of central nervous system7) compared with saline and 0.1 mg/ml chymotrypsin treatment. In addition, IL-17 mRNA levels were significantly decreased in the DCLNs of the seminal vesicle fluid-treated animals compared with the saline- and 0.1 mg/ml chymotrypsin-treated animals. Additionally, IL-17 expression was significantly stimulated in the 0.1 mg/ml chymotrypsin-treated animals compared with the saline-treated group. In contrast, IL-4 expression was significantly increased upon treatment with seminal vesicle fluid or 0.1 mg/ml chymotrypsin compared with saline treatment. Treatment with 0.1 mg/ml chymotrypsin resulted in significant stimulation of IL-4 expression compared with treatment with seminal vesicle fluid. A significant increase in FoxP3 mRNA levels upon seminal vesicle fluid treatment was observed compared with saline or 0.1 mg/ml chymotrypsin treatment. No significant differences in FoxP3 mRNA levels were observed between the saline- and 0.1 mg/ml chymotrypsin-treated animals. Our results suggest that seminal vesicle fluid contains all of the ligands required to activate the natural Treg pool and has anti-inflammatory effects in organs outside the reproductive system. Seminal vesicle fluid can increase Treg numbers in the central nervous system, specifically at the inflammatory foci (i.e., the spinal cord), and these effects are accompanied by a decrease in clinical signs. Our observations are consistent with the unexplained preferential targeting of inflammation to the spinal cord in EAE.8 The Treg expansion in response to seminal
vesicle fluid appears to be affected by a distal effect, as observed in the brain during mating.1 Presumably, local seminal vesicle fluid administration by intra-CSF injection can ameliorate this distal effect, as observed in this study. Interestingly, seminal vesicle fluid from male rats more than 410 g (about 3–4 months of age) exhibited this healing effect. However, seminal vesicle fluid obtained from younger rats did not exhibit this effect on Treg expansion. Seminal vesicle fluid administration seems to cause a ‘pseudo-mating state’ in the target organ. This study was performed at the gene expression level and represents the initial step in developing a hypothesis regarding the action of seminal vesicle fluid in the central nervous system and organs outside the female reproductive system. Further optimization of the route, dose and regimen of administration may improve the beneficial effect of seminal vesicle fluid. COMPETING INTERESTS The authors have no competing interests. ACKNOWLEDGEMENTS The authors wish to express their gratitude to Dr Reza Khodarahmi and Dr Mitra Bakhtiari for their useful advice and valuable discussions. Sincere thanks are due to Professor Nariman Mosaffa for her invaluable support and discussions. This work was supported by the Deputy of Research, Kermanshah University of Medical Sciences, under reference No. 90276. Supplementary Information accompanies the paper on Cellular & Molecular Immunology’s website. (http://www.nature.com/cmi).
1 Johansson M, Bromfield JJ, Jasper MJ, Robertson SA. Semen activates the female immune response during early pregnancy in mice. Immunology 2004; 112: 290–300. 2 Kallikourdis M, Andersen KG, Welch KA, Betz AG. Alloantigenenhanced accumulation of CCR51 ‘effector’ regulatory T cells in the gravid uterus. Proc Natl Acad Sci USA 2007; 104: 594–599. 3 Zhao JX, Zeng YY, Liu Y. Fetal alloantigen is responsible for the expansion of the CD4 1 CD25 1 regulatory T cell pool during pregnancy. J Reprod Immunol 2007; 75: 71–81. 4 Guerin LR, Moldenhauer LM, Prins JR, Bromfield JJ, Hayball JD, Robertson SA. Seminal fluid regulates accumulation of FOXP31 regulatory T cells in the preimplantation mouse uterus through expanding the FOXP31 cell pool and CCL19-mediated recruitment. Biol Reprod 2011; 85: 397–408. 5 Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD41CD251 T regulatory cells. Nat Immunol 2003; 4: 337–342. 6 Bitan M, Weiss L, Reibstein I, Zeira M, Fellig Y, Slavin S et al. Heparanase upregulates Th2 cytokines, ameliorating experimental autoimmune encephalitis. Mol Immunol 2010; 47: 1890–1898. 7 Okamoto Y, Yamashita J, Hasegawa M, Fujisawa H, Yamashima T, Hashimoto T et al. Cervical lymph nodes play the role of regional lymph nodes in brain tumour immunity in rats. Neuropathol Appl Neurobiol 1999; 25: 113–122. 8 Constantinescu CS, Farooqi N, O’Brien K, Gran B. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br J Pharmacol 2011; 164: 1079–1106.
Cellular & Molecular Immunology
SHORT COMMUNICATION
Seminal vesicle fluid ameliorates experimental autoimmune encephalomyelitis by increasing FoxP31 regulatory T-cell numbers Nafiseh Pakravan1, Ameneh Ghaffarinia2, Cyrus Jalili3, Farhad Riazi-Rad4, Marzieh Tajedini5 and Ali Mostafaie2 Previous studies have shown that insemination elicits the expansion of the natural FoxP31 regulatory T cell (Treg) pool in female reproductive tissue during mating. Further research demonstrated that this effect was due to seminal vesicle fluid. Indeed, seminal vesicle fluid causes differential recruitment of Tregs into different organs. The recruitment of Tregs into the brain occurs at the lowest rate of all studied organs. In addition, Tregs are present at a lower frequency and/or have a reduced function in multiple sclerosis. These observations prompted us to investigate how intra-CSF administration of seminal vesicle fluid affects experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Our results suggest that seminal vesicle fluid contains all of the ligands required to activate the Treg pool and exhibits an anti-inflammatory effect on organs outside the reproductive system. Cellular & Molecular Immunology advance online publication, 22 September 2014; doi:10.1038/cmi.2014.88
In 2004, it was demonstrated that changes in female reproductive tissue due to insemination result in immunological priming to paternal antigens and influence pregnancy outcome. During mating, exposure to seminal fluid elicits a tolerant state to paternal allo-antigens by expanding the regulatory T cell (Treg) pool. Notably, mating with males with surgically removed seminal vesicle glands was shown to fail to induce such effects.1 Evidence of antigen-specific Treg responses2–4 suggested that the expansion of the Treg pool occurs in response to seminal vesicle fluid.6 Further research has revealed differential recruitment of Tregs into different tissues, including the liver, kidney, heart, spleen, lymph nodes, mucosal tissues, blood, skin, thymus and brain, of which recruitment to the brain was the lowest.1 Owing to these effects on Treg expansion and the lower recruitment to the brain, we administered seminal vesicle fluid via an intra-CSF route in a Lewis rat model of experimental autoimmune encephalomyelitis (EAE) and investigated its effect on Treg expansion and clinical disease course. We induced EAE in Lewis rats and treated them with saline, 0.1 mg/ml chymotrypsin or seminal vesicle fluid (liquefied using chymotrypsin). Then, IFN-c, IL-4, IL-17 and FoxP3 expression were estimated using a two-step real-time
PCR method on samples from the brain, spinal cord and deep cervical lymph node (Supplementary Information). Our EAE Lewis rat model exhibited an acute monophasic disease, with clinical signs beginning on day 7 and reached the peak of the disease on day 14 post-induction. Intra-CSF injection of seminal vesicle fluid, 0.1 mg/ml chymotrypsin or saline was performed on day 7 after EAE induction. Changing trends in clinical signs in seminal vesicle fluid-treated animals were markedly different from those in the animals treated with 0.1 mg/ml chymotrypsin or saline (Figure 1a). The maximum mean clinical scores during the course of study were 1.46, 1.41 and 0.41, in saline-, 0.1 mg/ml chymotrypsin- and seminal vesicle fluid-treated animals, respectively. Changes in EAE-associated gene expression were estimated during the peak of the disease (day 14 after EAE immunization). Because EAE is a T cell-mediated disease, we investigated the changes in IFN-c, IL-4, IL-17 and FoxP3 expression as representative of Th1, Th2, Th17 and natural Treg populations, respectively (Figure 1b). Seminal vesicle fluid administration resulted in a significant decreased in IFN-c expression in the brain compared with saline-treated animals. In addition, IFN-c expression was
1 Department of Microbiology and Immunology, Medical School, Alborz University of Medical Sciences, Karaj, Iran; 2Department of Immunology, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran; 3Department of Anatomy, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran; 4Department of Immunology, Pasture Institute of Iran, Tehran, Iran and 5Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran Correspondence: Dr N Pakravan, Department of Microbiology and Immunology, Medical School, Alborz University of Medical Sciences, Karaj, Iran. E-mail: [email protected] Received: 7 August 2014; Revised: 18 August 2014; Accepted: 18 August 2014
Short Communication
2
a
6 Saline
Seminal Vesicle Fluid
0.1mg/ml Chymotrypsin
Clinical Score
5 4 3 2 1 0 0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
30 25 20 15
* * ** ** * **
10
*
*
5 0
IL-17 relative expression
* 15
* **
*
10
* **
5 0
Sal
ine
sin
/ml
g .1m
0
p otry
m
chy
Sem Brain
ina
l Ve
eF sicl
luid
* **
0.7
0.16
160
*
0.6
140 120
0.5
100
0.4
80 0.3
60
* **
0.2
40
0.1
20 0
0
6
* * ** **
0.12
5 4 3
0.08
* **
0.04 0
1 0
Sal
ine
sin
/ml
g .1m
0 Spinal Cord (S.C)
2
p otry
m
chy
Sem
ina
l Ve
eF sicl
luid
FoxP3 relative expression in DCLN
20
IL-4 relative expression in Brain and S.C
IFN-g relative expression
35
IL-4 relative expression in DCLN
b
FoxP3 relative expression in Brain and S.C
Days After EAE Induction
Deep Cervical Lymph Node (DCLN)
Figure 1 Effect of treatment with seminal vesicle fluid (liquefied using 0.1 mg/ml chymotrypsin) (??&??), 0.1 mg/ml chymotrypsin (-m-), or saline (-- --) on the clinical score. Female Lewis rats were immunized subcutaneously with a suspension of guinea pig spinal cord and CFA. Intra-CSF injection of seminal vesicle fluid, 0.1 mg/ml chymotrypsin or saline was performed on day 7 (onset of EAE). Clinical scores were measured daily from the day of disease induction (a). Comparison of IFN-c, IL-17, IL-4 and FoxP3 expression (b) in the brain, SC and DCLN. The animals were treated via an intra-CSF route with saline-, 0.1 mg/ml chymotrypsin- or seminal vesicle fluid on day 7 after disease induction. The animals were euthanized on day 14 after EAE induction, at the peak of the disease. The quantification of each gene was normalized against the reference gene beta-actin. IL-4 and FoxP3 levels in the DCLN are shown on the secondary vertical axis because of the difference in scale. The data are presented as the mean6s.d. *Significantly different from the saline-treated animals. **Significantly different from the 0.1 mg/ml chymotrypsin-treated animals. CFA, complete Freund’s adjuvant; DCLN, deep cervical lymph node; EAE, experimental autoimmune encephalomyelitis; SC, spinal cord.
N
significantly decreased in animals treated with 0.1 mg/ml chymotrypsin compared with saline- and seminal vesicle fluidtreated animals. IL-17 expression was also determined because it is a critical cytokine in EAE. Injecting animals with seminal vesicle fluid significantly downregulated IL-17 expression compared with the saline- and 0.1 mg/ml chymotrypsin-treated animals. In contrast, IL-17 expression in the 0.1 mg/ml chymotrypsin-treated animals was markedly increased compared with in saline-treated animals. To further explore the mechanism of Cellular & Molecular Immunology
the decreased clinical score upon seminal vesicle fluid treatment, the expression of IL-4 and FoxP3 was investigated.5,6 FoxP3 is indicative of naturally occurring Tregs which have an anti-inflammatory effect.5 IL-4 is also indicative of the Th2 response and exhibits an ameliorating effect on EAE.6 No significant differences were observed in IL-4 expression between the groups treated with saline, 0.1 mg/ml chymotrypsin, or seminal vesicle fluid. Regarding FoxP3, significant upregulation of FoxP3 expression was observed in the seminal
Short Communication
3
vesicle fluid-treated animals compared with the 0.1 mg/ml chymotrypsin- and saline-treated animals, suggesting Treg upregulation upon seminal vesicle fluid treatment. No significant differences in FoxP3 expression were observed between the groups of animals treated with 0.1 mg/ml chymotrypsin or saline. The seminal vesicle fluid- and 0.1 mg/ml chymotrypsintreated rats exhibited a significant decrease in IFN-c expression in the spinal cord compared with the saline-treated animals. However, IFN-c levels in the seminal vesicle fluid-treated animals were significantly higher than those of the 0.1 mg/ml chymotrypsin-treated animals. Seminal vesicle fluid treatment markedly attenuated IL-17 expression compared with the 0.1 mg/ ml chymotrypsin- and saline-treated animals. However, no significant differences were observed between the saline- and 0.1 mg/ ml chymotrypsin-treated animals in terms of IL-17 expression. The levels of IL-4, a Th2 cytokine, in the seminal vesicle fluid- and 0.1 mg/ml chymotrypsin-treated animals were similar to those in the saline-treated group. FoxP3 expression was significantly elevated only in the animals treated with seminal vesicle fluid compared with the saline- and 0.1 mg/ml chymotrypsintreated animals. However, FoxP3 expression was not significantly different between the 0.1 mg/ml chymotrypsin- and salinetreated animals. Seminal vesicle fluid treatment resulted in significant suppression of IFN-c expression in the DCLNs (drainage lymph nodes of central nervous system7) compared with saline and 0.1 mg/ml chymotrypsin treatment. In addition, IL-17 mRNA levels were significantly decreased in the DCLNs of the seminal vesicle fluid-treated animals compared with the saline- and 0.1 mg/ml chymotrypsin-treated animals. Additionally, IL-17 expression was significantly stimulated in the 0.1 mg/ml chymotrypsin-treated animals compared with the saline-treated group. In contrast, IL-4 expression was significantly increased upon treatment with seminal vesicle fluid or 0.1 mg/ml chymotrypsin compared with saline treatment. Treatment with 0.1 mg/ml chymotrypsin resulted in significant stimulation of IL-4 expression compared with treatment with seminal vesicle fluid. A significant increase in FoxP3 mRNA levels upon seminal vesicle fluid treatment was observed compared with saline or 0.1 mg/ml chymotrypsin treatment. No significant differences in FoxP3 mRNA levels were observed between the saline- and 0.1 mg/ml chymotrypsin-treated animals. Our results suggest that seminal vesicle fluid contains all of the ligands required to activate the natural Treg pool and has anti-inflammatory effects in organs outside the reproductive system. Seminal vesicle fluid can increase Treg numbers in the central nervous system, specifically at the inflammatory foci (i.e., the spinal cord), and these effects are accompanied by a decrease in clinical signs. Our observations are consistent with the unexplained preferential targeting of inflammation to the spinal cord in EAE.8 The Treg expansion in response to seminal
vesicle fluid appears to be affected by a distal effect, as observed in the brain during mating.1 Presumably, local seminal vesicle fluid administration by intra-CSF injection can ameliorate this distal effect, as observed in this study. Interestingly, seminal vesicle fluid from male rats more than 410 g (about 3–4 months of age) exhibited this healing effect. However, seminal vesicle fluid obtained from younger rats did not exhibit this effect on Treg expansion. Seminal vesicle fluid administration seems to cause a ‘pseudo-mating state’ in the target organ. This study was performed at the gene expression level and represents the initial step in developing a hypothesis regarding the action of seminal vesicle fluid in the central nervous system and organs outside the female reproductive system. Further optimization of the route, dose and regimen of administration may improve the beneficial effect of seminal vesicle fluid. COMPETING INTERESTS The authors have no competing interests. ACKNOWLEDGEMENTS The authors wish to express their gratitude to Dr Reza Khodarahmi and Dr Mitra Bakhtiari for their useful advice and valuable discussions. Sincere thanks are due to Professor Nariman Mosaffa for her invaluable support and discussions. This work was supported by the Deputy of Research, Kermanshah University of Medical Sciences, under reference No. 90276. Supplementary Information accompanies the paper on Cellular & Molecular Immunology’s website. (http://www.nature.com/cmi).
1 Johansson M, Bromfield JJ, Jasper MJ, Robertson SA. Semen activates the female immune response during early pregnancy in mice. Immunology 2004; 112: 290–300. 2 Kallikourdis M, Andersen KG, Welch KA, Betz AG. Alloantigenenhanced accumulation of CCR51 ‘effector’ regulatory T cells in the gravid uterus. Proc Natl Acad Sci USA 2007; 104: 594–599. 3 Zhao JX, Zeng YY, Liu Y. Fetal alloantigen is responsible for the expansion of the CD4 1 CD25 1 regulatory T cell pool during pregnancy. J Reprod Immunol 2007; 75: 71–81. 4 Guerin LR, Moldenhauer LM, Prins JR, Bromfield JJ, Hayball JD, Robertson SA. Seminal fluid regulates accumulation of FOXP31 regulatory T cells in the preimplantation mouse uterus through expanding the FOXP31 cell pool and CCL19-mediated recruitment. Biol Reprod 2011; 85: 397–408. 5 Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD41CD251 T regulatory cells. Nat Immunol 2003; 4: 337–342. 6 Bitan M, Weiss L, Reibstein I, Zeira M, Fellig Y, Slavin S et al. Heparanase upregulates Th2 cytokines, ameliorating experimental autoimmune encephalitis. Mol Immunol 2010; 47: 1890–1898. 7 Okamoto Y, Yamashita J, Hasegawa M, Fujisawa H, Yamashima T, Hashimoto T et al. Cervical lymph nodes play the role of regional lymph nodes in brain tumour immunity in rats. Neuropathol Appl Neurobiol 1999; 25: 113–122. 8 Constantinescu CS, Farooqi N, O’Brien K, Gran B. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br J Pharmacol 2011; 164: 1079–1106.
Cellular & Molecular Immunology
