Blockage of heme oxygenase-1 abrogates the protective effect of regulatory T cells on murine pregnancy and promotes the maturation of dendritic cells.

Regulatory T cells (Treg) play an important role in fetal protection. They expand during normal pregnancy and protect fetal antigens from maternal effector cells. Their effect is associated with the up-regulation of tolerance-associated molecules at the fetal-maternal interface. Among these, Heme Oxygenase-1 (HO-1, coded by Hmox1) is of special importance as its blockage correlates with increased abortion rates and its up-regulation positively affects pregnancy outcome. Here, we aimed to investigate whether the protective effect of Treg is mediated by HO-1 in a mouse model. HO-1 blockage by Zinc Protoporhyrin (ZnPPIX) abrogated the protective effect of Treg transfer. We found that HO-1 is important in maintaining maternal dendritic cells (DCs) in an immature state, which contributes to the expansion of the peripheral Treg population. This brings to light one essential pathway through which Treg mediates the semi-allogeneic fetus tolerance.

Exploring the potential of low doses carbon monoxide as therapy in pregnancy complications.

Heme Oxygenase-1 (HO-1) has been shown to play a pivotal role in pregnancy outcome and its ablation leads to abnormal placentation, intrauterine fetal growth restriction (IUGR) and subsequent intrauterine fetal death. Carbon monoxide (CO) has been found to mimic the protective effects of HO-1 activity, rescuing HO-1-deficient fetuses. This gasotransmitter arises in biological systems during the oxidative catabolism of heme by HO. Here, we explored the potential of CO in preventing IUGR and established the optimal doses and therapeutic time window in a clinically relevant mouse model. We additionally investigated the pathways activated upon CO application in vivo. We established 50 ppm as the best lowest dose of CO necessary to prevent growth restriction being the optimal time frame during days 3 to 8 of mouse pregnancy. CO lead to higher fetal and placental weights and avoided fetal death without showing any pathologic effects. CO breathing further suppressed inflammatory responses, diminished placenta apoptosis and complement deposition and regulated placental angiogenesis. Our results confirm the protective role of the HO-1/CO axis and point this gas as an emerging therapeutic possibility which is worth to further explore.

Regulation of the germinal center reaction by Foxp3+ follicular regulatory T cells.

Follicular helper T (T(FH)) cells participate in humoral responses providing selection signals to germinal center B cells. Recently, expression of CXCR5, PD-1, and the transcription factor Bcl-6 has allowed the identification of T(FH) cells. We found that a proportion of follicular T cells, with phenotypic characteristics of T(FH) cells and expressing Foxp3, are recruited during the course of a germinal center (GC) reaction. These Foxp3(+) cells derive from natural regulatory T cells. To establish the in vivo physiologic importance of Foxp3(+) follicular T cells, we used CXCR5-deficient Foxp3(+) cells, which do not have access to the follicular region. Adoptive cell transfers of CXCR5-deficient Foxp3(+) cells have shown that Foxp3(+) follicular T cells are important regulators of the GC reaction following immunization with a thymus-dependent Ag. Our in vivo data show that Foxp3(+) follicular T cells can limit the magnitude of the GC reaction and also the amount of secreted Ag-specific IgM, IgG1, IgG2b, and IgA. Therefore, Foxp3(+) follicular regulatory T cells appear to combine characteristics of T(FH) and regulatory T cells for the control of humoral immune responses.

Identification of regulatory Foxp3+ invariant NKT cells induced by TGF-beta.

Invariant NKT (iNKT) cells were shown to prevent the onset of experimental autoimmune encephalomyelitis in mice following administration of their specific TCR agonist alpha-galactosylceramide. We found that this protection was associated with the emergence of a Foxp3(+) iNKT cell population in cervical lymph nodes. We demonstrate that the differentiation of these cells is critically dependent on TGF-beta in both mice and humans. Moreover, in vivo generation of Foxp3(+) iNKT cells was observed in the TGF-beta-rich environment of the murine gut. Foxp3(+) iNKT cells displayed a phenotype similar to that of Foxp3(+) regulatory T cells, and they suppress through a contact-dependent, glucocorticoid-induced TNFR-mediated mechanism. Nevertheless, Foxp3(+) iNKT cells retain distinctive NKT cell characteristics, such as promyelocytic leukemia zinc finger protein expression and preferential homing to the liver following adoptive transfer, where they stably maintained Foxp3 expression. Our data thus unveil an unexpected capacity of iNKT cells to acquire regulatory functions that may contribute to the establishment of immunological tolerance.

Mechanisms of action of regulatory T cells specific for paternal antigens during pregnancy.

OBJECTIVE: To investigate whether pregnancy-induced regulatory T cells are generated specifically for paternal antigens or expanded by hormonal changes and to study regulatory T cell-related mechanisms during pregnancy. METHODS: We used murine models of normal, abortion-prone, and pseudopregnancy to characterize regulatory T cells and hormones by methods such as flow cytometry, molecular biology techniques, and chemiluminescence. Antigen specificity was studied in experiments in which animals were vaccinated with paternal antigens or adoptively transferred with regulatory T cells. To analyze regulatory T cell-mediated mechanisms, we used neutralizing antibodies against IL-10 or TGF-beta. RESULTS: Regulatory T cells are activated by male antigens, and minor antigens are protected by linked immunosuppression. Our data exclude the possibility that regulatory T cell expansion during pregnancy is exclusively driven by hormonal changes. An increase in systemic regulatory T cell levels in pseudopregnant females after mating with vasectomized males but not after pseudopregnancy induced mechanically confirms generation of regulatory T cells specific for paternal antigens. As for the mechanisms, neutralizing IL-10 abrogates the protective effect of regulatory T cells, whereas blockage of TGF-beta does not provide the same effect. CONCLUSION: Our data confirm that regulatory T cells act in an antigen-specific manner during pregnancy and strongly suggest that IL-10 is involved in regulatory T cell-mediated protection of the fetus. These data contribute to the knowledge of the basic mechanisms regulating immune tolerance during pregnancy, a major biologic question with important medical implications. LEVEL OF EVIDENCE: II.