RESUMEN
Mammalian pregnancy is a complex phenomenon allowing the maternal immune system to support its allogeneic fetus. Physiological pathways protecting the fetus from rejection are thought to be comparable with those leading to allograft acceptance. Heme oxygenase (HO)-1 is known to protect locally against rejection in transplantation models due to its anti-oxidant, anti-inflammatory and cytoprotective functions. Based on previous data on low HO-1 levels in placenta from mice undergoing abortion, we hypothesized that an up-regulation of HO-1 during pregnancy would avoid fetal rejection in the murine abortion combination CBA/J x DBA/2J, using BALB/c-mated CBA/J as normal controls. We injected pregnant mice undergoing abortion with 1 x 10(5) PFU of an adenoviral vector containing HO-1 and GFP (AdHO-1/GFP), and compared the pregnancy outcome with PBS- or 1 x 10(5) AdEGFP-treated abortion-prone mice and with PBS-treated normal pregnant mice. The abortion rate diminished significantly after adenoviral gene transfer of AdHO-1/GFP. The systemic and local IL-4/IFN-gamma ratio was augmented in AdHO-1-treated mice compared to abortion-prone mice. Interestingly, the HO-1 treatment up-regulated the ratio IL-10/TNF-alpha in spleen but not in decidual lymphocytes. HO-1-treated mice further showed diminished apoptosis rate and increased Bag-1 mRNA levels at the materno-fetal interface. Thus, we propose HO-1 as a key regulator of pregnancy success. HO-1 would exert its action by locally up-regulating the Th2/Th1 cytokine ratio and by further protecting tissues from apoptosis.
Asunto(s)
Aborto Espontáneo/prevención & control , Terapia Genética , Hemo-Oxigenasa 1/genética , Aborto Espontáneo/inmunología , Adenoviridae/genética , Animales , Apoptosis , Citocinas/biosíntesis , ADN/análisis , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Femenino , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Hígado/química , Masculino , Ratones , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Placenta/química , Placenta/citología , Embarazo , Resultado del Embarazo , ARN Mensajero/metabolismo , Células Th2/inmunología , Factores de Transcripción/análisis , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
The mechanisms underlying immune tolerance during pregnancy are poorly understood. In this regard, Treg seem to play an important role in mediating maternal tolerance to the fetus. We proposed a crucial role of T regulatory cells (Treg) in avoiding immunological rejection of the fetus after observing diminished number and function of Treg in abortion-prone mice. We further confirmed the protective role of Treg during pregnancy by transferring pregnancy-induced Treg into abortion-prone mice, which prevented rejection. Here, we analyzed the mechanisms involved in Treg-mediated protection. As expected, Treg therapy prevented abortion, while expanding the peripheral and thymic Treg population. Surprisingly, the decidual levels of the Th1 cytokines IFN-gamma and TNF-alpha were not diminished after therapy. Interestingly, the mRNA levels of leukemia inhibitory factor, TGF-beta and heme oxygenase-1 at the fetal-maternal interface were dramatically up-regulated after Treg transfer, while the levels of indolamine 2,3-dioxygenase remained unchanged. Our data suggest that Treg treatment can not prevent T cell infiltration or high Th1 levels but is able to create a privileged tolerant microenvironment at the fetal-maternal interface, further shedding light onto the molecular mechanisms involved in pregnancy tolerance.
Asunto(s)
Decidua/inmunología , Feto/inmunología , Tolerancia Inmunológica , Preñez/inmunología , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Citocinas/biosíntesis , Citocinas/inmunología , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Ratones , Neuropilina-1/inmunología , Neuropilina-1/metabolismo , Embarazo , ARN Mensajero/análisis , Receptores de Interleucina-2/inmunología , Receptores de Interleucina-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Tolerance mechanisms allowing pregnancy success resemble those involved in allograft acceptance. Heme oxygenase (HO) is a tissue-protective molecule, which allows graft acceptance and is known to have antiapoptotic effects on several cell types. We previously reported down-regulated levels of HO-1 and HO-2 in placenta from allopregnant mice undergoing abortion. In this study, we analyzed whether the up-regulation of HO-1 by cobalt-protoporphyrin (Co-PP) during implantation window can rescue mice from abortion. Induction of HO-1 by Co-PP treatment prevented fetal rejection, whereas the down-regulation of HOs by zinc-protoporphyrin application boosted abortion. The beneficial effect of HO-1 induction was not related to a local shift to Th2-profile or to a change in the NO system. Interestingly, the expression of the antiapoptotic/cytoprotective molecule Bag-1 as well as the levels of neuropilin-1, a novel marker for T regulatory cells, were up-regulated after Co-PP treatment. Our data strongly support a very important role for HO-1 in fetal allotolerance and suggest that HO-1 might be protective by up-regulating tissue protective molecules, i.e., Bag-1, and by activating T regulatory cells rather than by changing the local cytokine profile.
Asunto(s)
Aborto Espontáneo/metabolismo , Proteínas Portadoras/metabolismo , Hemo-Oxigenasa 1/fisiología , Intercambio Materno-Fetal/fisiología , Neuropilina-1/metabolismo , Aborto Espontáneo/prevención & control , Animales , Biomarcadores , Proteínas de Unión al ADN , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Óxido Nítrico Sintasa de Tipo III/genética , Embarazo , Protoporfirinas/farmacología , Linfocitos T Reguladores/metabolismo , Células Th2/metabolismo , Factores de Transcripción , Regulación hacia Arriba/fisiologíaRESUMEN
Mammalian pregnancy is thought to be a state of immunological tolerance. The mechanisms underlying this phenomenon are still poorly understood. Here, we determined whether an inappropriate function of T regulatory (Treg) cells is involved in the pathogenesis of spontaneous abortion. We evaluated spleen and decidual lymphocytes from CBA/J mice undergoing immunological abortion (DBA/2J-mated) or having normal pregnancy (BALB/c-mated) on day 14 of gestation for ex vivo cytokine production after PMA or paternal antigen (alloantigen) stimulation. Treg activity was characterized by quantifying CD4(+)CD25(+) cells, foxp3 expression, and interleukin-10 secretion. Decidual lymphocytes from abortion CBA/J mice contained a significantly higher frequency of interferon-gamma-producing T cells specific for paternal antigens compared to those from normal pregnancy (7.8% versus 2.7%, P < 0.05). Compared to virgin CBA/J females, normal pregnant mice showed strongly elevated numbers of CD4(+)CD25(+) and interleukin-10(+) Treg cells in the thymus whereas significantly lower frequencies of Treg cells were observed in abortion mice. Very interestingly, CD4(+)CD25(+) Treg cells from normal pregnant and nonpregnant CBA/J mice could inhibit both proliferation and interferon-gamma secretion of lymphocytes from abortion mice in vitro whereas in vivo prevention of fetal rejection could only be achieved after adoptive transfer of Treg cells from normal pregnant mice. Our data suggest that pregnancy-induced Treg cells play a vital role in maternal tolerance to the allogeneic fetus.
Asunto(s)
Aborto Espontáneo/inmunología , Traslado Adoptivo , Antígenos CD4/biosíntesis , Receptores de Interleucina-2/biosíntesis , Linfocitos T/citología , Linfocitos T/inmunología , Animales , Western Blotting , Proliferación Celular , Técnicas de Cocultivo , Citocinas/biosíntesis , Cartilla de ADN/química , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Padre , Femenino , Citometría de Flujo , Factores de Transcripción Forkhead , Inmunohistoquímica , Interleucina-10/biosíntesis , Interleucina-10/metabolismo , Linfocitos/citología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Embarazo , Preñez , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timo/metabolismo , Regulación hacia ArribaRESUMEN
The allogeneic fetus has been considered to be an allograft and the tolerance mechanisms involved in pregnancy maintenance resemble those leading to graft acceptance. Up-regulation of Heme Oxygenase-1 (HO-1) promotes graft acceptance. Additionally, HO-1 has been proposed to have tissue-protective properties. Previous studies reported the presence of HO-1 and HO-2 in mammalian placenta and postulated a protective role for HO during pregnancy. Here, we analyze HO-1 and HO-2 expression at the feto-maternal interface from normal pregnant and abortion mice and correlate these findings with inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expression as well as with Th1/Th2 cytokine production by immune cells. DBA/2-mated CBA/J females undergoing abortion and BALB/c-mated CBA/J females having normal pregnancies were included in our study. The mice received no treatment. On day 14 of pregnancy, the mice were sacrificed, the abortion rate was calculated and the ex vivo Th1/Th2 production by decidual immune cells was analyzed by flow cytometry. The expression of HO-1 and HO-2, iNOS and eNOS was analyzed by immunohistochemistry (IHC) and Western blot in placenta samples. The Th1/Th2 cytokines ratio was augmented in decidua from abortion mice. We further observed a significant down-regulation of HO-1, HO-2, iNOS and eNOS molecules in placental tissue from mice undergoing abortion when compared to normal pregnant mice. Since we found diminished HOs and nitric oxide synthase (NOS) levels at the feto-maternal interface from abortion mice when compared to normal pregnant mice, which were further associated with increased Th1/Th2 cytokine production, we propose HO as a putative therapeutic target in immunological abortions. Up-regulation of HO-1 or HO-2 would favour the Th2-cytokine production, which could avoid abortion onset.