Pro-inflammatory effector Th cells transmigrate through anti-inflammatory environments into the murine fetus

Abstract

The presence of maternal DNA or even maternal cells within the offspring (microchimerism) has been reported for many fetal tissues, including the liver, heart, and spleen. Microchimerism is believed to be involved in the pathogenesis of autoimmune diseases; however, the cellular origin of this phenomenon remains unknown. Here, we determined whether differentiated T lymphocytes could transmigrate through the immunosuppressive environment of the placenta to reach the fetus. In vitro-differentiated effector/memory Th1 and Th17 cells from OVA_323–339–specific TCR^tg T cells of OT-II mice were adoptively transferred (i.v.) into the tail veins of pregnant Ly5.1 mice at d15 and d19 of gestation. Mice were then sacrificed 40 h after adoptive cell transfer. Using radioactive labeling of T cells with sodium chromate [Cr⁵¹] prior to adoptive transfer, we observed that homing of pro-inflammatory Th cells was equally efficient in both pregnant and non-pregnant mice. Transmigration of Th1- and Th17-like cells through the highly immunosuppressive environment of the placenta into the fetus was significantly enhanced in experimental mice compared to control mice (P < 0.0001). In addition, a substantial amount of effector Th cells accumulated in the placenta. Finally, we found that treatment with Pertussis Toxin resulted in a 3-fold increase in the transmigration of effector Th17 cells into the fetus (P < 0.0001).

When pro-inflammatory Th1-or Th17-like cells were injected into syngeneic mothers, almost all of the fetuses analyzed exhibited radioactivity, suggesting that transmigration of effector T cells occurs frequently. Our results suggest the possibility of novel roles for these maternal effector cells in the pathogenesis or reduction of disease.

Introduction

During healthy pregnancy, the maternal organism develops tolerance and anti-inflammatory mechanisms. Additionally, the placenta functions as a physical and immunosuppressive barrier between mother and fetus, providing an immunoprivileged site for fetal development within the uterus. To prevent rejection of the semi-allogeneic tissues and spontaneous abortion of the fetus, the maternal immune system is equipped with several tolerance mechanisms. These mechanisms include the production of immunosuppressive asymmetric IgG Abs (AAbs) that link with paternally inherited antigens [1], expression of tryptophan metabolism enzymes, such as Indoleamine-2,3-dioxygenase, at the maternal-fetal interface that prevent immunological rejection of fetal allografts [2], and expression of non-classical MHC molecules, such as HLA-G on trophoblasts, that function to circumvent immune responses that would target the fetus as an allogeneic graft [3]. Additionally, two models suggest that T cells are directed to avoid immune responses against the fetus. One group claims that a suppressive immune environment at the placenta occurs due to expression of Th2 and anti-inflammatory cytokines, such as IL-10, IL-4, and TGFβ, in the decidua [4]. Another proposes that an increase of regulatory T cells (Tregs) in the maternal lymph vessels [5] and at the placenta [6] functions to prevent adverse immune reactions during pregnancy. Because only some immune deficiencies are associated with adverse pregnancy outcome, it is likely that several mechanisms play together to ensure positive pregnancy outcomes.

Accumulating evidence indicates that the barrier between mother and fetus is not as tight as previously believed. Because the placenta is selectively permeable and allows for diffusion, active transport, or pinocytosis [7], a transfer of circulating maternal cells into the blood of the fetus may be common during pregnancy and could explain the appearance of foreign DNA (microchimerism) within the offspring. In recent years, it has been shown that maternal hematopoietic cells persist within fetal organs such as the liver, lung, heart, adrenal gland, kidney, blood, and placenta [8], that maternal cells are present in heart tissue of male neonates with neonatal lupus syndrome [9], and that maternal cells are able to enter the fetal circulation and migrate to newborn tissues [10]. In mice, previous studies described a bi-directional cell transmigration through the placenta no matter whether maternal mice and their offspring were syngeneic or congeneic [11], [12]. In addition, the frequency of detected cells in the placenta increased during the progression of pregnancy [12]. It has been reported that specific DNA of noninherited maternal antigen (NIMA)-carrying cells is present in lymphoid and nonlymphoid tissue of neonatal and adult mice depending on the level of maternal-fetal histocompatibility [13]. Interestingly, maternal cells presenting noninherited MHC antigens in fetus and newborns increase suppression of their NIMA-allospecific T cells leading to the development of organ transplant tolerance in adult mice [14]. Furthermore, tolerance to maternal noninherited antigens is explained with maternal microchimerism detected in fetal heart, lung, liver, lymph nodes, and blood [15], [16].

Until now, diverse subsets of pro-and anti-inflammatory CD4⁺ T cell types including pro-inflammatory Th1 and Th17 cells have been described. Naïve CD4⁺ T lymphocytes can be differentiated both in and ex vivo into Th1 or Th17 cells depending on a defined cytokine milieu. Th1 cells are known to produce IFNγ as a key cytokine in the cellular immunity process and to mediate acute graft-versus-host-disease [17]. These cells also function in the induction and progression of autoimmune diseases, such as Rheumatoid Arthritis [18]. Th17 cells producing IL-17A, IL-17F, and to a lesser extent, tumor necrosis factor (TNF), IL-6, and IL-22 [19], have been demonstrated to play a critical role in the pathogenesis of autoimmune diseases, such as Rheumatoid Arthritis [20], [21]. Additionally, these cells have been demonstrated to play a role in inflammatory bowel disease [22]. Interestingly, several studies have demonstrated that a predominance of Th1-type immunity correlates with an increase in spontaneous abortion [23] and pre-eclampsia [24], while data from another study describe an increased prevalence of Th17 cells in the peripheral blood and decidua of patients with unexplained recurrent spontaneous abortion [25]. So far, it is not known whether mature pro-inflammatory effector T cells transmigrate during pregnancy into the fetus and whether either the altered environment during pregnancy within the maternal organism or the local immunosuppressive effects at the placenta functions to prevent their transmigration.

Our study provides the first example of adoptive transfer of pro-inflammatory Th1 and Th17 cells into the anti-inflammatory environment of pregnant mice. Based on recent studies of maternal cell transmigration, we demonstrate that terminally differentiated Th1 and Th17 cells are able to pass through the placenta and to persist in the fetus. Additionally, we analyze the influence of mild, asymptomatic inflammation induced by Pertussis Toxin on the transmigration of pro-inflammatory effector Th cells.