Glucocorticoid Receptor in T Cells Mediates Protection From

Glucocorticoid receptor in T cells mediates protection PNAS PLUS from autoimmunity in pregnancy

Jan Broder Englera, Nina Kursawea, María Emilia Solanob, Kostas Patasa, Sabine Wehrmanna, Nina Heckmanna, Fred Lühderc, Holger M. Reichardtd, Petra Clara Arckb, Stefan M. Golda,e, and Manuel A. Friesea,1

aInstitut für Neuroimmunologie und Multiple Sklerose, Zentrum für Molekulare Neurobiologie Hamburg, Universitätsklinikum Hamburg-Eppendorf, 20251 Hamburg, Germany; bExperimentelle Feto-Maternale Medizin, Klinik für Geburtshilfe und Pränatalmedizin, Universitätsklinikum Hamburg-Eppendorf, 20251 Hamburg, Germany; cInstitut für Neuroimmunologie und Institut für Multiple Sklerose Forschung, Universitätsmedizin Göttingen, 37073 Goettingen, Germany; dInstitut für Zelluläre und Molekulare Immunologie, Universitätsmedizin Göttingen, 37075 Goettingen, Germany; and eKlinik für Psychiatrie und Psychotherapie, Campus Benjamin Franklin, Charité Universitätsmedizin, 12203 Berlin, Germany

Edited by Philippa Marrack, Howard Hughes Medical Institute, National Jewish Health, Denver, CO, and approved December 6, 2016 (received for review October 17, 2016) Pregnancy is one of the strongest inducers of immunological toler- immunological tolerance (18, 22). Depletion of Tregs results in fetal ance. Disease activity of many autoimmune diseases including mul- loss (23). Pregnancy-related hormonal signals, including estrogens tiple sclerosis (MS) is temporarily suppressed by pregnancy, but little and progesterone (1, 24, 25), appear to support Treg proportion is known about the underlying molecular mechanisms. Here, we in- and function, although their exact mode of action remains in- vestigated the endocrine regulation of conventional and regulatory completely understood. T cells (Tregs) during reproduction. In vitro, we found the pregnancy In the context of autoimmunity, Tregs are essential in suppressing hormone progesterone to robustly increase Treg frequencies via autoreactive responses. Foxp3 deficiency results in generalized au- promiscuous binding to the glucocorticoid receptor (GR) in T cells. In toimmune inflammation evident in scurfy mice (2–4, 26) and in vivo, T-cell–specific GR deletion in pregnant animals undergoing ex- patients suffering from immunodysregulation polyendocrinopathy perimental autoimmune encephalomyelitis (EAE), the animal model enteropathy X-linked syndrome (IPEX) (5, 27). Beyond that, of MS, resulted in a reduced Treg increase and a selective loss of quantitative or functional Treg impairment has been described in a pregnancy-induced protection, whereas reproductive success was un- number of autoimmune diseases, including systemic lupus eryth- affected. Our data imply that steroid hormones can shift the immu- ematosus (6, 7, 28, 29), rheumatoid arthritis (8, 30, 31), and type I nological balance in favor of Tregs via differential engagement of the diabetes (9, 10, 32). In MS, Tregs were reported to possess di- GR in T cells. This newly defined mechanism confers protection from minished suppressive potential (11, 33, 34) and decreased expres- autoimmunity during pregnancy and represents a potential target for future therapy. sion of Foxp3 and immunosuppressive cytotoxic T lymphocyte antigen 4 (CTLA-4) (12, 35–37). multiple sclerosis | autoimmunity | pregnancy | Treg | steroid hormones However, it is still unknown which pregnancy-related changes account for the enhanced control of autoreactive responses. Here, we sought to unravel the molecular mechanisms that confer the eproduction is fundamental to the maintenance and evolu- beneficial effect of pregnancy on autoimmunity. By studying the tion of species. To ensure successful pregnancy, mothers R impact of pregnancy and pregnancy hormones on T-cell subsets have to establish robust immunological tolerance toward the semi- including Tregs, we provide evidence that differential sensitivity to allogeneic conceptus providing a secure niche for fetal development. Multiple mechanisms have evolved to prevent fetus-directed immune glucocorticoid receptor (GR) activity is a hitherto unrecognized responses and alloreactive infiltration of the fetomaternal interface (1). These include creating a privileged local microenvironment that Significance hampers T-cell priming and infiltration (2–4) but also imply global modulation of the immune system by pregnancy hormones and the Reproduction in placental mammals relies on potent control of shedding of fetal antigen into the mothers circulation (5). the mother’s immune system to not attack the developing fetus. Intriguingly, pregnancy is also well known to suppress the in- As a bystander effect, pregnancy also potently suppresses the flammatory activity of a number of cell-mediated autoimmune dis- activity of the autoimmune disease multiple sclerosis (MS). Here, eases, including rheumatoid arthritis (6, 7), autoimmune hepatitis we report that T cells are able to directly sense progesterone via (8), and multiple sclerosis (MS) (9, 10). However, this beneficial their glucocorticoid receptor (GR), resulting in an enrichment of effect is limited to the period of gestation and usually followed by a regulatory T cells (Tregs). By using an MS animal model, we found rebound of disease activity postpartum. In the case of MS, third that the presence of the GR in T cells is essential to increase Tregs trimester pregnancy leads to a remarkable reduction of the MS and confer the protective effect of pregnancy, but not for relapse rate (11), which exceeds the effects of most currently avail- maintaining the pregnancy itself. Better understanding of this able disease-modifying drugs. Similarly, pregnancy as well as treat- tolerogenic pathway might yield more specific therapeutic means ment with pregnancy hormones protect rodents from experimental to steer the immunological balance in transplantation, cancer, autoimmune encephalomyelitis (EAE), a widely used animal model and autoimmunity. of MS (12) in both SJL/J and C57BL/6 mice (13–16), underpinning an interaction between pregnancy-related immune and endocrine Author contributions: J.B.E., S.M.G., and M.A.F. designed research; J.B.E., N.K., M.E.S., K.P., S.W., and N.H. performed research; F.L., H.M.R., and P.C.A. contributed new adaptations and central nervous system (CNS) autoimmunity (17). reagents/analytic tools; J.B.E., N.K., P.C.A., and M.A.F. analyzed data; and J.B.E. and The sensitive balance between conventional effector T cells M.A.F. wrote the paper. (Tcons) and regulatory T cells (Tregs) has transpired as a common The authors declare no conflict of interest. theme that connects reproductive biology and autoimmunity on a This article is a PNAS Direct Submission. – mechanistic level (18 21). Tregs are characterized by the tran- Freely available online through the PNAS open access option. scription factor forkhead box P3 (Foxp3) and effectively control 1To whom correspondence should be addressed. Email: [email protected] effector responses mounted by Tcons in response to antigen-specific hamburg.de.

activation. In the context of pregnancy, Tregs were shown to expand This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. INFLAMMATION IMMUNOLOGY AND to safeguard against fetal rejection by establishing antigen-directed 1073/pnas.1617115114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1617115114 PNAS | Published online January 3, 2017 | E181–E190 Downloaded by guest on October 5, 2021 mechanism shaping the T-cell compartment. By targeted GR antigen and the systemic challenge with fetal antigen following pla- deletion in T cells, we show a selective disruption of pregnancy- cental perfusion, respectively. In line with this observation, the first induced protection from autoimmunity. expansion was limited to the local paraaortic lymph nodes, whereas the second was also detectable in regional inguinal lymph nodes and Results systemically in the spleen (Fig. 1E). Additionally, late pregnancy Treg Dynamics During Pregnancy. We first characterized the relative Tregs consistently showed increased expression of the immunosup- abundance and phenotype of Tcons and Tregs throughout preg- pressive molecule CTLA-4 in all assessed organs, rendering them nancy and postpartum time points (Fig. 1A). Because the presence potentially more effective in controlling effector responses (Fig. 1E). + − of fetuses that genetically differ from the mother is a prerequisite CD4 Foxp3 conventional T cells (Tcons) showed only one pro- of robust induction of immune tolerance (13–15,18),weperformed liferative burst in early pregnancy (E2.5) that was accompanied by allogeneic matings of C57BL/6 females with BALB/c males. We increased CTLA-4 expression, whereas their proliferative activity analyzed cells from paraaortic lymph nodes (LNs), inguinal lymph appeared to be tightly controlled at later time points (Fig. S1). nodes, and spleen to get insight into local, regional, and systemic Together, the Treg response commenced in early gestation changes, respectively (Fig. 1B). The acquired data indicated that with a local proliferative burst and generalized toward systemic Treg expansion was most pronounced in the paraaortic lymph nodes + + compartments in late gestation. Late gestational Tregs showed that drain the fetomaternal interface. Frequencies of CD4 Foxp3 increased expression of CTLA-4, potentially supporting their Tregs started to increase from middle to late pregnancy (embryonic suppressive function. days, E10.5–E18.5) and stayed at elevated levels as long as postpartum day 30 (PP30) (Fig. 1 C and D). We observed two pro- T-Cell Intrinsic Sensing of Progesterone Mediates Treg Enrichment in liferative bursts of Tregs in early (E2.5) and late (E18.5) pregnancy Vitro. Progesterone is an essential steroid hormone for successful by Ki67 staining, coinciding with the first contact to paternal sperm pregnancy outcome that peaks at late gestation (18–21, 38, 39),

ABPregnancy Mating Early Mid Late Spleen

BALB/c ♂ Progesterone Aorta serum levels Post partum Para-aortic LN Pregnant uterus C57BL/6 ♀ × Delivery Inguinal LN

Non-preg E2.5 E10.5 E18.5 PP5 PP10 PP30

C Para-aortic LN Non-preg E2.5 E10.5 E18.5 PP5 PP10 PP30

11.9 11.8 13.6 18.9 18.3 16.2 15.1 Foxp3

CD4 D Para-aortic LN Inguinal LN Spleen 25 20 20 ** ** ** ** ** ** 20 * ** ** ** 15 cells (%) cells (%) cells 15 (%) cells + + * + 15 10 10 10 5 5 5 Treg among CD4 Treg among CD4 Treg among CD4 0 0 0

E2.5 PP5 E2.5 PP5 E2.5 PP5 E10.5E18.5 PP10PP30 E10.5E18.5 PP10PP30 E10.5E18.5 PP10PP30 Non-preg Non-preg Non-preg E Para-aortic LN Inguinal LN Spleen 60 60 60 ** ** Fig. 1. Treg dynamics during pregnancy. Flow 40 ** 40 ** 40 cytometry analysis of paraaortic LNs, inguinal LNs, and spleen at indicated pregnancy (E2.5–E18.5) and postpartum (PP5–PP30) time points. Gray shaded areas among Treg (%) among Treg (%) 20 20 among Treg (%) 20 + + + represent pregnancy. (A) Time course of allogeneic Ki67 Ki67 Ki67 mating, sample collection, and progesterone serum 0 0 0 levels. (B) Overview of harvested lymphoid tissue in 100 100 100 * ** relation to pregnant uterus. (C)Representativedot ** ** plots of Treg frequency in paraaortic LNs. (D)Quanti- 90 90 * 90 fication of Treg frequency in indicated tissues. Each 80 80 80 dot represents the result from one individual mouse among Treg (%) among Treg (%) among Treg (%) = + + + (n 7, 8, 8, 8, 6, 6, and 4 per time point). (E) Pheno-

70 70 A-4 70 typic characterization of Tregs. Data are pooled from LA-4 T multiple experimental days. Statistical analyses in D CTLA-4 C CTL 60 60 60 and E were performed by one-way ANOVA with E2.5 PP5 E2.5 PP5 E2.5 PP5 ’ E10.5E18.5 PP10PP30 E10.5E18.5 PP10PP30 E10.5E18.5 PP10PP30 Bonferroni s post hoc test in comparison with non- Non-preg Non-preg Non-preg pregnant control mice. *P < 0.05; **P < 0.01.

E182 | www.pnas.org/cgi/doi/10.1073/pnas.1617115114 Engler et al. Downloaded by guest on October 5, 2021 shortly before we observed a substantial Treg expansion (Fig. receptor-mediated rather than a pleiotropic effect. Importantly, PNAS PLUS 1A). Additionally, progesterone has been shown to have bene- RU486 alone as well as the pregnancy hormone estradiol had no ficial effects in EAE (18, 22, 40) and to expand Treg frequencies effect on Treg frequencies (Fig. S2A). At the same time, we upon in vivo treatment (24). Therefore, we tested the ability of observed increased cell death in progesterone-treated cultures progesterone to influence the ratio of Tregs and Tcons in sple- paralleling the enrichment of Tregs in a dose-dependent manner nocyte cultures. Indeed, treatment with progesterone at doses (Fig. 2 A–C and Fig. S2B). Prolonged cultivation amplified the – – close to late gestational serum levels (∼90 ng·mL 1) readily in- effect (Fig. 2D), whereas 1 μg·ml 1 RU486 was under most + + creased the frequency of Foxp3 Tregs among live CD4 T cells conditions sufficient for a complete blockade (Fig. 2B). Be- from ∼5% in vehicle-treated cultures to ∼15% after 48 h (Fig. 2 cause splenocyte cultures contain different immune cell types A and B). We could inhibit this effect by the steroid hormone that could in principle be involved in sensing progesterone and receptor antagonist mifepristone (RU486), suggesting a defined mediating this finding, we sought to define the target cells of

ABEtOH P4 P4+RU486 25 EtOH P4 ** Viable CD4+ cells Viable CD4+ cells Viable CD4+ cells 20 P4+RU486 ** ** cells (%) 15

5.9 16.5 7.1 + 4 * 10 Treg among Treg Foxp3 5 viable CD 0 CD4 100 CD4+ cells CD4+ cells CD4+ cells 80 ** ****

cells (%) 60

+ ** 7.1 23.7 10.1 4 * * 40 CD

SSC-A Cell death 20 among 0 Cell death dye 0 7 15 30 60 12025050010002000 P4 ng/ml CDEEtOH P4 20 20 P4+RU486 8 R2 = 0.8693 ** ** cells (%) cells (%) +

cells (%) P < 0.0001 + + 15 4 15 ** 6 CD 10 10 ** 4 iable v 5 5 2 – 0 0 0 Fig. 2. T-cell intrinsic sensing of progesterone medi- 0 50 100 reg among T Treg among viable CD4 Treg 24 h 48 h 72 h 96 h ates Treg enrichment in vitro. (A) Splenocytes were Treg among viable CD4 Treg P4 + – Cell death among CD4 cells (%) Incubation time EtOH cultured for 48 h in the presence of 300 ng·mL 1 pro- –1 P4+RU486 gesterone (P4), 1 μg·mL mifepristone (RU486), vehicle F EtOH P4 P4+RU486 control ethanol (EtOH), or indicated combinations. Cultures were analyzed for Treg frequency and cell 7.9 9.5 7.6 10 ** ** 6 ** ** death by flow cytometry. (B) Dose titration of spleno-

) cytes cultured and analyzed as described in A.(C) Cor- + 8 relation of Treg frequency and cell death among CD4

Annexin V 4 among cells (% cells from dose titration experiment in B.(D)Time

6 + among course of splenocytes cultured and analyzed as de- 4 p3 + 2 scribed in A.(E) Purified CD4 cells were cultured (0.2 ×

aCas 6 AnnexinV+ 2 10 cells per well) and analyzed as described in A. viable CD4+ cells (%) 2.9 5.3 3.7 viable CD4+ (F) Splenocytes were cultured as in A but analyzed after 0 aCasp3 0 6 h for apoptosis markers Annexin V and aCasp3. P4 P4 EtOH EtOH (G) Splenocytes were cultured in the presence of cas-

CD4 P4+RU486 P4+RU486 pase 3 inhibitor Z-DEVD-FMK or vehicle control (DMSO) andanalyzedasinA. Dot plots in A are representative EtOH P4 G DMSO of at least three independently analyzed animals. Data Z-DEVD-FMK in B are pooled from five independent experiments with one mouse per experiment (total n = 5). Data in 4.8 12.1 15 DMSO * C show one representative animal out of five (all ani- cells (%)

+ mals are shown Fig. S2B). Data in D show results of one 4 10 experiment (n = 5). Data in E show one representative CD experiment out of two (each n = 4). Data in F are Foxp3 pooled from two independent experiments (total 4.5 7.6 5 = Z-DEVD-FMK n 8). Data in G are pooled from three independent experiments (total n = 10). Statistical analysis was per- among viable 0 formed by linear regression in A,two-wayANOVAin INFLAMMATION Treg P4 B, D,andG, and one-way ANOVA in E and F,allwith CD4 EtOH Bonferroni’s post hoc test. *P < 0.05; **P < 0.01. IMMUNOLOGY AND

Engler et al. PNAS | Published online January 3, 2017 | E183 Downloaded by guest on October 5, 2021 progesterone. To get a first impression, we repeated the assay Progesterone Acts via Promiscuous Binding to the Glucocorticoid + with purified CD4 T cells. Because the progesterone-induced Receptor. Our data suggested the existence of a RU486-blockable enrichment of Treg cells was preserved in these experiments (P = receptor in T cells that is engaged by progesterone and induces 0.0003, Fig. 2E), we concluded a direct action of progesterone on an enrichment of Tregs in vitro. To explore potential molecular + CD4 T cells. targets of progesterone, we next performed gene expression analyses Because cell death was increased in progesterone-treated of different steroid receptors in isolated Tregs and Tcons throughout cultures, we further analyzed Annexin V and activated cas- pregnancy, including the progesterone receptor (PR encoded by + pase 3 (aCasp3) to test whether CD4 T cells were specifi- Nr3c3/Pgr), the glucocorticoid receptor (GR encoded by Nr3c1)and cally driven into apoptosis. Indeed, both markers showed an the estrogen receptor α (ER encoded by Nr3a1/Esr1). Notably, Pgr increase in apoptosis after progesterone treatment, which was mRNA was practically absent in all conditions, whereas Nr3c1 abolished in the presence of RU486 (Fig. 2F). More impor- and Esr1 mRNA could be reliably detected (Fig. 3A). Because tantly, treatment with the caspase 3 inhibitor Z-DEVD-FMK steroid hormones are known to possess promiscuous binding ac- reduced Treg frequencies in progesterone-treated cultures tivity to other steroid receptors (41), we reasoned that progesterone (Fig. 2G), indicating that apoptosis was directly driving Treg might signal via the GR as an alternative receptor. To test this enrichment. hypothesis, we challenged splenocytes with either norgestrel (NOR) Thus, T-cell–intrinsic sensing of progesterone via a RU486- or dexamethasone (DEX), possessing high affinity to the PR or blockable receptor resulted in a shift of the immunological balance GR, respectively. Although applied at lower concentrations, DEX in favor of Tregs. This effect was driven by the induction of apo- treatment showed a much stronger enrichment of Tregs than NOR + ptosis and consecutive cell death in the CD4 T-cell compartment. treatment (P = 0.0009, Fig. 3A), supporting our hypothesis.

Pgr Nr3c1 Esr1 A 0.10 2.0 0.20 Tcon Tcon Tcon Treg Treg Treg 0.08 1.5 level 0.15 A 0.06

1.0 mRN 0.10 0.04 tive 0.5 0.02 0.05 Relative mRNA level Relative mRNA level 2/5 5/5 3/6 Rela n.d. n.d. n.d. 0 0 0 Non-preg E18.5 PP5 Non-preg E18.5 PP5 Non-preg E18.5 PP5 BC ** 60 GRfl/fl 80 GRfl/fl ** GRfl/fl;Lck-Cre GRfl/fl;Lck-Cre cells (%) 10 + 60 40 40 5 20 20 % viable splenocytes among 0 % viable splenocytes among 0 0

Treg among viable CD4 DCs Treg EtOHNOR DEX cells cells T B cells T cells T cells T NK cells + + Neutrophils Fig. 3. Progesterone acts via promiscuous binding to NOR+RU486DEX+RU486 Macrophages CD4 CD8 the glucocorticoid receptor. (A) Relative mRNA levels D EtOH P4 DEX GRfl/fl of progesterone receptor (Pgr), glucocorticoid re- fl/fl α 7.4 12.8 17.6 GR ;Lck-Cre ceptor (Nr3c1), and estrogen receptor (Esr1)inTcons 25 and Tregs from nonpregnant (n = 5), pregnant (E18.5; GRfl/fl ** = = cells (%) 20 n 5), and postpartum (PP5; n 6) mice. mRNA was + quantified by real-time PCR and normalized to Tbp. 15 ** (B) Splenocytes were cultured for 48 h in the presence · –1 · –1 ∼ −9 Foxp3 10 of 300 ng mL NOR, 500 pg mL ( 10 M) DEX, –1 7.9 7.4 7.4 1 μg·mL mifepristone (RU486), vehicle control etha- GRfl/fl;Lck-Cre 5 nol (EtOH), or indicated combinations. Cultures were 0 analyzed for Treg frequency by flow cytometry. (C) Immune cell composition of splenocytes from T-cell– Treg among viable CD4 viable among Treg P4 fl/fl EtOH DEX specific glucocorticoid receptor knockout mice (GR ; CD4 fl/fl P4+RU486 Lck-Cre) and controls (GR ) was analyzed by flow DEX+RU486 cytometry. (D and E) Splenocytes of GRfl/fl;Lck-Cre and E EtOH P4 DEX fl/fl GR GRfl/fl mice were cultured for 48 h in the presence of fl/fl GR ;Lck-Cre –1 –1 −9 40 300 ng·mL progesterone (P4), 500 pg·mL (∼10 M) – ** DEX, 1 μg·mL 1 mifepristone (RU486), vehicle control fl/fl 5.7 23.0 31.0 GR cells (%) 30 + ethanol (EtOH), or indicated combinations. Cultures 4 ** were analyzed for Treg frequency (D) and cell death CD 20 (E). Data in A are pooled from multiple experimental =

SSC-A days. Data in B show results for one experiment (n 4). 10 Data in C–E show one representative experiment out GRfl/fl;Lck-Cre = 5.2 8.2 8.7 of two (n 4 per group). Statistical analysis was per- 0 formed by two-way ANOVA in A and C–E and one-way Cell death among P4 ’ < EtOH DEX ANOVA in B, all with Bonferroni s post hoc test. *P 0.05; **P < 0.01; n.d. = not detected; x/y = number of Cell death dye P4+RU486 DEX+RU486 samples with signal.

E184 | www.pnas.org/cgi/doi/10.1073/pnas.1617115114 Engler et al. Downloaded by guest on October 5, 2021 To definitely pinpoint a contribution of GR engagement to sitivity of Tcons in comparison with Tregs. For example, it is con- PNAS PLUS the observed Treg enrichment, we made use of a T-cell–specific ceivable that Tcons are more sensitive to glucocorticoid-induced GR knockout mouse. In these GRfl/fl;Lck-Cre mice the T-cell–restricted cell death, whereas Tregs are in comparison steroid resistant and lymphocyte-specific protein tyrosine kinase (Lck) promoter drives hence accumulate. This notion was supported by our observation the expression of a Cre recombinase, which leads to the excision of that cell death dye-positive cells were exclusively present in the the essential exon 3 from the GR locus, thereby disrupting GR Tcon but not in the Treg compartment (Fig. 4A). To further sub- function specifically in T cells (42–44). After ruling out a priori stantiate this hypothesis, we isolated Tregs and Tcons and cultured differences in their immune cell composition (Fig. 3C), we cul- them separately in the presence of increasing doses of DEX as a tured splenocytes from GRfl/fl;Lck-Cre knockout and GRfl/fl control potent GR stimulus. Whereas we observed more robust proliferative animals in the presence of either progesterone or DEX. Strikingly, responses of Tcons at low concentrations of DEX (P = 0.0380), Treg enrichment and cell death induction were completely abol- increasing GR stimulation resulted in a proliferative advantage for ished in the GRfl/fl;Lck-Cre knockout cultures, regardless of treat- Tregs (P = 0.0017, Fig. 4B). Similarly, induction of cell death was ment with either progesterone or DEX (Fig. 3 D and E). This more pronounced in Tcons than in Tregs when cultured under complete rescue in the GR knockout cultures also applied to the nonproliferating conditions (P < 0.0001, Fig. 4C). These findings + induction of apoptosis (Fig. S3 A and B), whereas Treg enrichment support the idea that differential steroid sensitivity in the CD4 by both progesterone and DEX was still readily observed in pro- T-cell compartment accounts for an enrichment of the more re- gesterone receptor knockout cultures (Fig. S3C). In line with our sistant Tregs in situations of increased GR activity. previous experiments, these data support a direct action of pro- To check whether steroid levels during pregnancy are sufficient + gesterone on the GR in T cells to mediate Treg enrichment in to induce transcriptional activity of the GR in CD4 T cells in vivo, vitro. To test the effect of GR engagement on T cells in vivo, we we isolated Tregs and Tcons from nonpregnant and E18.5 mice treated wild-type animals with a single i.p. injection of 100 μgDEX and analyzed gene expression of the glucocorticoid-induced leu- and analyzed lymph nodes and spleen 24 h later. Markedly, Treg cine zipper (Gilz), encoded by Tsc22d3. We selected Gilz because frequencies were strongly increased by DEX in all assessed organs its transcription is directly induced by binding of the GR to the (Fig. S4A), whereas this effect was blocked in the GRfl/fl;Lck-Cre Gilz promoter (45, 46), it mediates the antiproliferative activity of mice (Fig. S4B). glucocorticoids (47), and can be used as a surrogate marker for We conclude that Treg enrichment upon steroid challenge was GR activity. Gilz expression was significantly induced by late mediated via the GR in T cells, whereas we found no indication pregnancy in Tcons (P = 0.0207) but not in Tregs (P = 0.2496, for an involvement of the PR. Fig. 4D). Additionally, GR signaling was more active in Tcons irrespective of pregnancy, further supporting the notion that those Tregs Are More Resistant to GR Challenge than Tcons. Because cells might be a priori more susceptible to steroid challenge. progesterone treatment was paralleled by increased cell death in Taken together, Tregs showed a higher resistance to glucocor- + CD4 T cells (Fig. 2 A and B), the increased Treg-to-Tcon ratio ticoid challenges under both proliferative and nonproliferative observed in vitro could be explained by an increased steroid sen- conditions. This finding is consistent with a positive selection of

A EtOH P4 P4+RU486

7.2 32.4 9.7 100 Tcon Tcon ** Fig. 4. Tregs are more resistant to GR challenge than Treg ** 80 ** Tcons. (A) Splenocytes were cultured for 48 h in the –1 –1

th (%) th · μ · 60 ** presence of 300 ng mL progesterone (P4), 1 g mL ** mifepristone (RU486), vehicle control ethanol (EtOH), 40

Cell dea or indicated combinations. Cultures were analyzed for

Cell death dye 1.47 2.77 1.77 20 cell death dye positivity inside Tcon and Treg pop- Treg 0 ulations. (B) Proliferation response curves of Tregs and 0 7 15 30 60 120250500 Tcons. Cells were isolated and cultured separately in 10002000 –1 P4 ng/ml thepresenceofirradiatedfeedercells,1μg·mL anti- – CD3 antibody, 50 U·mL 1 recombinant murine IL-2, Foxp3 and indicated DEX concentrations. Proliferation was assessed by [3H]-thymidine incorporation and nor- B Curve top value Curve bottom value malized to vehicle control (EtOH). Curve fit, curve top, * ** 1.2 0.25 and curve bottom values were computed. (C)Tregs 1.0 Tcon n n and Tcons were isolated and cultured separately in the

Treg 1.1 io 0.20 – tio tion μ · 1 at presence of DEX, 1 g mL mifepristone (RU486), ve- ra r e era f 1.0 0.15 hicle control ethanol (EtOH), or indicated combina- 0.5 0.9 0.10 tions. Dead cells were identified by propidium iodide ve proli ve prolife i i t positivity and cell death was calculated as fold change 0.8 0.05 Rela Relative prolif Relat relative to vehicle control (EtOH). (D)RelativemRNA 0 0.7 0 levels of Gilz (encoded by Tsc22d3)inTconsandTregs −12−11−10−9−8−7−6 Tcon Treg Tcon Treg log M DEX from nonpregnant (n = 5) and pregnant (E18.5; n = 5) Tsc22d3 mice. mRNA was quantified by real-time PCR and CD4 h incubation time 6 h incubation time EtOH ** normalized to Tbp.DatainA are reanalyzed from Fig. 3 3 ** 10−9 MDEX 5 * non-preg −8 * preg 2B (total n = 5). Data in B are pooled from three in- ** 10 MDEX 10−8 MDEX+RU486 4 dependent experiments (total n = 4). Data in C are 2 2 3 pooled from three independent experiments (total =

e mRNA level n 3). Data in D are pooled from multiple experi-

v 2 1 1 mental days (n = 5 per group). Statistical analyses were

Relati 1 performed by two-way ANOVA with Bonferroni’spost Cell fold change death Cell fold change death

’ < INFLAMMATION 0 0 0 hoc test in A, C,andD and Student s t test in B.*P Tcon Treg Tcon Treg Tcon Treg 0.05; **P < 0.01. IMMUNOLOGY AND

Engler et al. PNAS | Published online January 3, 2017 | E185 Downloaded by guest on October 5, 2021 Tregs as the underlying mechanism for increased Treg frequencies CNS autoimmunity, we performed allogeneic matings of GRfl/fl; after GR stimulation. The GR pathway appeared to be engaged Lck-Cre knockout and GRfl/fl control animals and induced EAE on during pregnancy in vivo, because the GR target gene Gilz was gestational day E7.5 ± 1. We observed no differences in the dis- induced in Tcons at E18.5. ease incidence (Fig. 6A). However, as expected from our initial experiments, pregnancy resulted in a protection of GRfl/fl control Late Pregnancy Temporarily Protects from CNS Autoimmunity. Next, mice. By contrast, the EAE protection was abolished in pregnant we aimed to unravel whether GR activity in T cells is responsible GRfl/fl;Lck-Cre knockout mice (P = 0.003, Fig. 6B), whereas we for the beneficial effect of pregnancy on CNS autoimmunity. To found no evidence for an a priori exacerbated EAE in GRfl/fl;Lck- this end, we first established a model of pregnancy protection from Cre knockout animals when comparing the nonpregnant groups EAE in C57BL/6 mice. We performed allogeneic matings with (Fig. 6 B–D). Thus, protection from EAE in pregnant mice BALB/c males and immunized at gestational day E7.5 ± 1, en- depended on the presence of the GR in T cells. Additionally, Treg suring an estimated onset of disease at the beginning of the third frequencies in pregnant GRfl/fl;Lck-Cre knockout animals with trimester of pregnancy—the time point with the strongest pro- EAE (E18.5) were reduced in comparison with pregnant EAE tective effects in MS and EAE (9, 13). We expected the delivery of controls in paraaortic lymph nodes (P = 0.0144, Fig. 6E), sug- the pups around day 12 after immunization and included non- gesting an impaired Treg expansion. Although pregnancy still pregnant females as controls (Fig. 5A). Whereas the disease in- showed a tendency to increase Tregs in GRfl/fl;Lck-Cre mice, this cidence of both groups was indistinguishable (P = 0.5681, Fig. 5B), did not reach statistical significance (P = 0.1023, Fig. 6E). pregnant animals were protected until the day of delivery. Imme- To investigate the possibility that a failure in fetomaternal tol- diately after birth of the pups, the dams succumbed to an exag- erance with consecutive fetal loss could have caused this break- gerated disease course associated with increased motor disability down of EAE protection as a secondary effect, we killed GRfl/fl; and higher mortality, reminiscent of the overshooting disease ac- Lck-Cre mice and controls at E13.5–14.5 and analyzed re- tivity observed in MS patients postpartum (Fig. 5 C–E)(9,11). productive outcome parameters, including fetal loss and pla- Because motor function defects assessed during EAE can be cental morphology. However, all assessed parameters, including attributed to inflammatory injury of responsible neurons and their Treg frequencies, were unchanged in GRfl/fl;Lck-Cre mice (Figs. projections (48, 49), we next assessed the infiltration of immune S5 and S6). Thus, fetomaternal tolerance appeared to be more cells into the CNS during the protective period (at day 10) by flow stable than pregnancy-induced autoantigen tolerance. cytometry. At this time point, pregnant animals were clinically asymptomatic (Fig. 5F) and showed half the absolute number of Discussion immune cells in the CNS (P = 0.0089, Fig. 5G), suggesting a pe- In the present study, we have in detail characterized shifts in the ripheral control of encephalitogenic cells, whereas particularly T-cell compartment during pregnancy with a specific focus on T cells and dendritic cells (DCs) were reduced in pregnant EAE the immunological balance of Tcons and Tregs and the endo- brains (Fig. 5H). crine mechanisms orchestrating them. Our findings indicate that pregnancy-related steroid hormones are directly sensed by The GR in T Cells Mediates EAE Protection During Pregnancy but Is T cells and mediate a relative enrichment of Tregs via engage- Dispensable for Reproductive Success. To assess the biological role ment of the GR. Intriguingly, our conditional GR knockout of GR in T cells in mediating the protective effect of pregnancy in model demonstrated that steroid hormone sensing by T cells is

AB1.0 BALB/c ♂ Pregnant E7.5 ± 1 0.8 × EAE Expected Expected induction EAE onset delivery 0.6 C57BL/6 ♀ Third trimester 0.4

Disease incidence 0.2 Non-pregnant Day 0 Day 7 Day 12 C57BL/6 ♀ 0 Non-preg Preg CDE** 4 Delivery 20 100

t 80 3 15

se * 60

2 of on 10

clinical score 40 Day

1 5 Percent survival Mean Non-preg 20 Non-preg Fig. 5. Late pregnancy temporarily protects from Preg ** Preg CNS autoimmunity. (A) Experimental setup of preg- 0 0 0 5 10152025 Non-preg Preg 0 5 10 15 20 25 nancy EAE. Disease incidence (B), clinical course (C), Days after immunization Days after immunization day of onset (D), and survival (E) are shown for nonpregnant (n = 17) and pregnant (n = 12) animals. FG H Gray shaded areas represent pregnancy. (F–H) Non- ) ) ) 5 6 10 1.5 5 ** = = * ** Non-preg ** Non-preg pregnant (n 4) and pregnant (n 8) animals were × 10 3 × 10 8 ( × 10 Preg ** 2 Preg killed on day 10 after EAE induction. Cumulative 1.0 clinical score (F) and absolute numbers of immune 6 2 cells (G) and immune cell subpopulations in the CNS 4 1 (H) were assessed by flow cytometry. Data in B–E are ive clinical score 0.5 1 pooled from four independent experiments. Data in 2 F–H show one representative experiment out of Cumulat Immune cells in CNS ( Immune cells in CNS 0 ( Immune cells in CNS 0 0 0 two. Statistical analyses were performed by Fisher’s reg ’ Preg Preg DCs cells cells cells T exact test in B, two-way ANOVA with Bonferroni s B cellsT T T NK cells + + T cells Non-preg Non-preg Microglia post hoc test in C and H, Student’s t test in D, F,and Neutrophils Macrophages CD4 CD8 G, and χ2 test in E.*P < 0.05; **P < 0.01.

E186 | www.pnas.org/cgi/doi/10.1073/pnas.1617115114 Engler et al. Downloaded by guest on October 5, 2021 PNAS PLUS A BCDGRfl/fl GRfl/fl GRfl/fl GRfl/fl preg GRfl/fl preg GRfl/fl preg fl/fl fl/fl fl/fl 1.0 GR ;Lck-Cre ** GR ;Lck-Cre GR ;Lck-Cre GRfl/fl;Lck-Cre preg GRfl/fl;Lck-Cre preg GRfl/fl;Lck-Cre preg 0.8 4 25

e 100 0.6 20 3 0.4

onset 15 f Diseasecidence in 0.2 2 50 10

0 Day o 1 Percent survival Fig. 6. The GR in T cells mediates EAE protection fl/fl Mean clinical scor 5 preg preg during pregnancy. (A–D) EAE was induced in non- GRfl/fl ;Lck-Cre 0 0 fl/fl fl/fl 0 pregnant and pregnant GR (n = 20 and n = 18, re- GR 0 5 10 15 ;Lck-Cre 51015 fl/fl GRfl/fl Days after immunization Days after immunization spectively) and nonpregnant and pregnant GR ;Lck- GR Cre mice (n = 21 and n = 14, respectively). Disease Para-aortic LN E GRfl/fl incidence (A), clinical course (B), day of onset (C), and Non-preg Preg GRfl/fl preg survival (D) are shown. Gray shaded areas represent GRfl/fl;Lck-Cre pregnancy. (E) Treg frequency in nonpregnant and fl/fl GR ;Lck-Cre preg pregnant GRfl/fl (n = 10 and n = 14, respectively) and 9.8 18.2 GRfl/fl Para-aortic LN Inguinal LN Spleen nonpregnant and pregnant GRfl/fl;Lck-Cre mice (n = 5 30 * 30 30 and n = 16, respectively) treated as in A–D were killed at E18.5 and analyzed by flow cytometry. Data are cells (%) Foxp3 – + 20 20 20 pooled from four independent experiments in A D and three independent experiments in E.Statistical fl/fl GR ;Lck-Cre 10.9 14.3 analyses were performed by Fisher’s exact test in A; 10 10 10 two-way ANOVA in B; one-way ANOVA in C and E,all with Bonferroni’s post hoc test; and χ2 test in D.*P < reg among CD4 CD4 T 0 0 0 0.05; **P < 0.01.

crucial for conferring protection from maternal autoimmunity in Additionally, progesterone was shown to drive naïve human cord late gestation. However, establishment of fetomaternal tolerance blood cells but not adult peripheral T cells into suppressive Tregs itself, reflected by reproductive success, did not depend on the while impeding their differentiation into TH17 cells (25). presence of GR in T cells. Thus, we addressed the questions of whether progesterone is Although Treg-driven tolerance has been shown to be fetal capable of shifting the balance in the T-cell compartment in favor of antigen specific in the first place (22), it has also been noted that Tregs and whether we could set up a stable assay to decipher the Tregs are able to suppress T-cell responses to unrelated antigens underlying molecular mechanism. Indeed, we observed a robust in the form of a bystander suppression (50). This can have detri- increase of Treg frequencies after treating splenocyte cultures for – mental consequences as Treg expansion leaves pregnant mice 48 h with 300 ng·mL 1 progesterone, which corresponds to ap- more prone to infections with certain pathogens including Listeria proximately three times the serum level present at E16.5 (38). We and Salmonella species, whereas Treg depletion has been shown consider this level close to the physiological range, as we would to restore host defense (23). Thus, enhanced Treg-driven control anticipate similar local levels close to the fetomaternal interface. of effector T-cell responses in pregnancy represents a promising To our surprise however, we found no evidence for PR expres- + target when studying tolerogenic off-target effects, as it might be sion in CD4 T cells on the mRNA level, representing the primary the case in pregnancy-induced reduction of autoimmunity. progesterone target. This finding is in agreement with data from the Herein, we characterized Tregs and Tcons in C57BL/6 females Immunological Genome Project (54) providing Pgr expression levels matedtoBALB/cmalesandobservedamarkedlategestational that do not exceed the detection background. By a series of ex- Treg expansion in the local paraaortic LNs that directly drain the periments, we could finally attribute the effect to a cross engage- uterus and constitute the place of fetal-specific T-cell priming ment of the GR in T cells. The strongest piece of evidence in this (2, 51). Similar findings have been reported by other groups pio- regard is the complete abrogation of Treg enrichment in cultures neering the investigation of Tregs in the context of reproductive from T-cell–restricted GR knockout mice (GRfl/fl;Lck-Cre). This biology (18, 23, 52). Notably, increased Treg frequencies persisted evidence suggests that T cells sense progesterone via their in- even after delivery. This finding is in concordance with a study tracellular GR, leading to an increase of Treg frequencies. Being using a mating system with known paternal antigen to show that engagedintimesofincreasedsteroid serum levels, this finding rep- fetal-specific Tregs can persist as regulatory memory cells that resents a so-far unrecognized tolerogenic pathway that might shape readily reassemble in a consecutive pregnancy (22). Beyond that, the immunological balance during pregnancy. Clearly, this mecha- we identified proliferative bursts in Tregs that appear to reflect a nism is not restricted to progesterone, but rather includes the action local priming event driven by male seminal fluid (51) in early of corticosterone and other steroid hormones with agonistic potential gestation and a systemic challenge to fetus-derived antigens shed on the GR that equally increase during the course of gestation (55). into the mother’s circulation (52, 53) in late gestation. Mechanistically, we found that Tregs are more resistant to However, the most dramatic changes in Treg frequency and steroid challenges in comparison with Tcons, thus possessing a phenotype at E18.5 were directly preceded by the peak pro- survival advantage in times of increased steroid levels. Hence, the gesterone levels present around E16.5 (38). A number of studies observed enrichment of Tregs is likely the result of a selection of have implied that progesterone can support Tregs and dampen resistant Tregs, whereas sensitive Tcons succumbed to glucocor- effector responses, without providing a clear molecular mecha- ticoid-induced cell death. This interpretation is also in agreement + nism. These include observations that s.c. progesterone-releasing with increasing rates of apoptosis and cell death in CD4 cells implants at the onset of disease ameliorate the course of EAE in paralleling the Treg enrichment in dose and time-course experi- C57BL/6 mice, however, without altering the systemic frequency ments. Importantly, targeted inhibition of apoptosis reduced the of Tregs (40). In contrast, another study reported a substantial Treg enrichment, further supporting this notion. Whereas apoptosis

increase of uterine and systemic Treg frequencies when treating induction in leukocytes by DEX is well established (56), there have INFLAMMATION ovariectomized mice with s.c. injection of progesterone (24). only been incidental reports for progesterone (57). Furthermore, IMMUNOLOGY AND

Engler et al. PNAS | Published online January 3, 2017 | E187 Downloaded by guest on October 5, 2021 we observed increased Gilz expression in Tcons isolated from preg- Wildtype T cells GR-deficient T cells nant animals, indicating that GR activity is induced during pregnancy in these cells, but to a lesser extent in the more resistant Tregs. Tcon Treg Tcon Treg To investigate the relevance of this mechanism for the amelioration of autoimmunity during in vivo pregnancy, we estab- GR engagement No GR engagement lished a mouse model in which pregnant animals are protected from EAE until delivery. Strikingly, whereas the protection Selection of resistant Treg conferred by pregnancy is among the strongest beneficial regu- No selection lators of autoimmunity, this effect was completely abrogated in GRfl/fl;Lck-Cre knockout animals, supporting a central role of GR signaling in ensuring proper maintenance of pregnancy- Relative Treg No Treg enrichment related CNS-autoantigen tolerance. Accordingly, the Treg expansion enrichement in paraaortic LNs present in pregnant controls was diminished in pregnant GRfl/fl;Lck-Cre knockout animals during EAE. Our results extend previous evidence supporting the crucial role of intact GR signaling in T cells for the control of neuroinflammation (58). It has EAE pregnancy in mice with GR-deficient T cells been also reported that deletion of the GR in T cells abolishes the Successful reproduction Impaired Treg expansion Loss of protection therapeutic effect of dexamethasone in mice (59, 60), whereas from CNS autoimmunity overexpression of the GR in thymocytes ameliorates EAE in a rat model (61). Here, we show that pregnancy endogenously employs this pathway to exert its protective effects on maternal autoimmunity. Additionally, we provide evidence that the GR in T cells is of particular importance for mediating amelioration of maternal au- Fig. 7. Differential steroid sensitivity as a mechanism of tolerance in- toimmune responses but not for ensuring fetomaternal tolerance as duction. Upon GR engagement steroid-resistant Tregs are positively selected deletion of the GR in T cells completely abrogated the protective and hence accumulate. After targeted GR disruption in T cells, Tcons and effect of pregnancy on EAE but had no impact on any assessed Tregs are equally resistant to GR engagement, thus the enrichment of Treg is measures of reproduction. Notably, also Treg expansion found to abolished. In pregnant EAE animals harboring GR-deficient T cells, re- be disturbed in GRfl/fl;Lck-Cre knockout animals in EAE preg- production is still functional. However, Tregs increase and pregnancy-asso- nancy was unaltered in mid term pregnancy without EAE. These ciated protection from autoimmune disease activity is impaired. findings likely reflect the high stability of reproduction and Treg expansion, which are ensured by redundant mechanisms in the (66) were kept under specific pathogen-free conditions in the Central Ani- absence of inflammatory challenges (1, 5). In line with this re- mal Facility at the University Medical Center Hamburg-Eppendorf. Age- and dundancy, it usually takes more than one hit to cause severe re- sex-matched adult animals (10–20 wk) were used in all experiments. productive failure, as evidenced by a number of mutant mice with largely unaltered reproductive success (1). This result is likely due Dexamethasone Treatment. Mice were treated with one i.p. injection of to high evolutionary pressure on successful species propagation via 100 μg(∼5 mg per kilogram of body weight) dexamethasone (Fortecortin reproduction (62). In contrast, controlling chronic inflammation in Inject, MerckSerono) in PBS or vehicle control (PBS) and killed 24 h later. autoimmune diseases seems to be a more fragile side effect that requires all reproductive tolerance mechanisms to fully operate. Allogeneic Mating. Age-matched female mice were primed with housing Some limitations of this study have to be considered. Whereas material from fertile BALB/c males for 1 wk and then mated for three con- our data clearly demonstrate that GR signaling in T cells is in- secutive nights. Successfully mated females where identified by the presence of dispensable for pregnancy protection from EAE, this does not rule a vaginal plug, separated, and weighted daily to confirm pregnancy. The day of plug was considered gestational day 0.5 (E0.5). In experiments including out additional protective mechanisms of pregnancy exerted by postpartum time points, pups were separated and killed at the day of delivery. other factors such as estrogens, which have shown promise in clinical trials in MS (63–65). Furthermore, because we show that EAE Induction. Mice were immunized s.c. with 200 μgMOG35–55 peptide – successful reproduction does not critically rely on GR signaling in (Schafer-N) in complete Freund’s adjuvant (Difco) containing 4 mg·mL 1 T cells, it needs to be further elucidated on what basis differential Mycobacterium tuberculosis (Difco). In addition, 200 ng pertussis toxin (Calbio- effects on reproductive and autoimmune tolerance are mediated. chem) was injected i.v. on the day of immunization and 48 h later. Animals were Summarizing our findings, we provide evidence that differential scored daily for clinical signs by the following system: 0, no clinical deficits; 1, tail steroid sensitivity of Tregs and Tcons represents a so-far un- weakness; 2, hind limb paresis; 3, partial hind limb paralysis; 3.5, full hind limb paralysis; 4, full hind limb paralysis and forelimb paresis; and 5, premorbid or recognized tolerance mechanism that might be engaged in times ≥ of high steroid levels, as present during gestation (Fig. 7). We dead. Animals reaching a clinical score 4 had to be killed according to the regulations of the Animal Welfare Act. The last observed score of euthanized or show that this pathway works via promiscuous binding of pro- dead animals was carried forward for statistical analysis. The cumulative clinical gesterone to the GR in T cells and is able to enrich Tregs in vitro. score represents the sum of the daily scores given to an animal over time. In- Furthermore, we found evidence that GR signaling is in fact op- vestigators were blinded for genotype during the experiment. erative in Tcons during pregnancy in vivo, whereas T-cell–specific GR deletion resulted in a loss of pregnancy-induced protection Immune Cell Isolation from Lymphoid Organs and CNS. Mice were killed by from EAE and reduced Treg frequencies in uterus-draining LNs. inhalation of CO2 and lymph nodes and spleens were harvested with sterile However, further work is needed to disentangle the molecular instruments into ice-cold PBS. Single-cell suspensions were prepared by ho- basis of the differential sensitivity of Tregs and Tcons. Better mogenization through a 40-μm cell strainer, cells were pelleted by centri- × understanding the T-cell population-specific prerequisites that fugation (300 g, 10 min, 4 °C), and splenic erythrocytes were lysed in communicate this differential sensitivity holds the promise to yield red blood cell lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH = 7.4) for 5 min at 4 °C. Cells were washed with PBS and used in follow-up more specific therapeutic means to steer the immunological bal- applications. For isolation of CNS-infiltrating leukocytes, mice were in- ance in transplantation, cancer, and autoimmunity. tracardially perfused with ice-cold PBS immediately after killing to remove blood from intracranial vessels. Brain and spinal cord were prepared with Materials and Methods sterile instruments, minced with a scalpel, and incubated with agitation in – Mice. C57BL/6 wild-type mice (The Jackson Laboratory) and previously de- RPMI medium 1640 (PAA) containing 1 mg·mL 1 collagenase A (Roche) and − − scribed GRfl/fl;Lck-Cre (Nr3c1tm2GSc;Tg(Lck-cre)1Cwi) mice (42–44) and Pgr / mice 0.1 mg·mL–1 DNaseI (Roche) for 60 min at 37 °C. Tissue was triturated

E188 | www.pnas.org/cgi/doi/10.1073/pnas.1617115114 Engler et al. Downloaded by guest on October 5, 2021 through a 40-μm cell strainer and washed with PBS (300 × g, 10 min, 4 °C). hamster anti-mouse CD3 antibody (BioLegend, clone 145–2C11) in the PNAS PLUS – Homogenized tissue was resuspended in 30% isotonic Percoll (GE Health- presence of 50 U·mL 1 recombinant murine IL-2 (eBioscience) and increasing care) and carefully underlaid with 78% isotonic Percoll. After gradient concentrations of dexamethasone. All experimental conditions were plated centrifugation (1,500 × g, 30 min, 4 °C) CNS-infiltrating immune cells were in technical triplicates and adjusted to a final volume of 200 μL per well. recovered from the gradient interphase and washed twice in ice-cold PBS After 48 h, cells were pulsed with 1 μCi [3H]-thymidine (Amersham) per before staining for flow cytometry. well for 16 h. Then, cells were harvested and spotted on filter mats with a Harvester 96 MACH III M (Tomtec). Incorporated activity per well was Flow Cytometry Analysis. Single-cell suspensions were stained in the presence assessed in counts per minute (cpm) in a beta counter (1450 Microbeta, of TruStain Fc receptor block (BioLegend) with monoclonal antibodies Perkin-Elmer) and proliferation was calculated relative to the mean of directed against CD3e (PerCP-Cy5.5, BioLegend, clone 145–2C11), CD4 (PE- nondexamethasone-treated control wells. Curve fit and curve statistics Cy7, eBioscience, clone GK1.5), CD8a (Pacific blue, BioLegend, clone 53– were computed using Prism 6 software (Graphpad) for Macintosh. 6.7), CD11b (FITC, BioLegend, clone M1/70), CD11c (PE-Cy7, eBioscience, clone N418), CD25 (Alexa Fluor 488 and APC, eBioscience, clone PC61.5), Cell Death Assay. Tregs and Tcons were isolated by MACS and cultured CD45 (APC-Cy7, BioLegend, clone 30-F11), CD45R (V500, BD Horizon, clone separately in complete medium at 1 × 105 cells per well in 96-well round RA3-6B), Ctla4 (APC, eBioscience, clone UC10-4B9), Foxp3 (PE and eFluor bottom plates. Dexamethasone, mifepristone (RU486), or vehicle control 450, eBioscience, clone FJK-16s), Ly6G (V450, BD Biosciences, clone 1A8), ethanol were added at indicated concentrations. Dead cells were identified NK1.1 (PE, eBioscience, clone PK136), aCasp3 (FITC, BD Biosciences, clone by positivity for propidium iodide (BioLegend) after 4 h and 6 h culture time. C92-605), and Ki67 (PE, BD Biosciences, clone B56) and acquired on a LSR II Cell death fold change was calculated relative to vehicle control. FACS analyzer (BD). In indicated experiments, dead cells were labeled before surface staining with Fixable Dead Cell Stain Kit (Life Technologies). Gene Expression Analysis. RNA was purified using RNeasy Mini Kit (Qiagen) For intracellular staining of Foxp3, Ctla4, and Ki67, the Foxp3 Staining and reverse transcribed to cDNA with RevertAid H Minus First Strand cDNA Buffer Set (eBioscience) was used according to the manufacturer’sin- Synthesis Kit (Fermentas) according to the manufacturer’s instructions. Gene structions. For detection of apoptosis, the Active Caspase-3 Apoptosis Kit expression was analyzed by real-time PCR performed in an ABI Prism 7900 (BD Biosciences) and FITC Annexin V Apoptosis Detection Kit (BioLegend) HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan Gene were used according to the manufacturer’s instructions. The absolute cell counts of CD45+ CNS-infiltrating leukocytes were determined by using Expression Assays (Life Technologies) for the following targets: estrogen TrueCount tubes (BD Biosciences). Data analysis was performed with receptor (Esr1, Mm00433149_m1), GR (Nr3c1, Mm00433832_m1), PR (Pgr, Mm00435628_m1), Gilz (Tsc22d3, Mm01306210_g1), and TATA-box binding FlowJo v10 analysis software (TreeStar) for Macintosh. Tregs were identi- −Δ + + + – CT fied as CD4 Foxp3 and Tcons as CD4 Foxp3 cells. Additional immune cell protein (Tbp, Mm00446971_m1). Gene expression was calculated as 2 populations from splenocytes and CNS-infiltrating cells were identified as relative to Tbp as endogenous control. previously reported (49). Histomorphological Analyses of Placental Tissue. Paraffin-embedded placental μ Isolation of T-Cell Populations. Pooled single-cell suspensions from inguinal, tissue was cut into 3- to 6- m histological sections at the midsagittal plane using axillary, brachial, paraaortic lymph nodes, and spleen were subjected to mag- a microtome (Leica). Tissue sections were deparaffinized, rinsed in distilled netic-associated cell sorting (MACS) using the CD4+ T Cell Isolation Kit (Miltenyi water, and dehydrated twice in ethanol (70%). Masson-Goldner Trichrome + + Biotec) or CD4 CD25 Regulatory T Cell Isolation Kit (Miltenyi Biotec) according Staining Kit (VWR International) was used according to the manufacturer’sin- to the manufacturer’s instructions. Purity of isolated cells was routinely above structions to visualize the morphologically different areas of placental tissue. 80% for CD4 T cells and above 90% for Tregs and Tcons. Briefly, tissue sections were stepwise stained with Weigert’s iron hematoxylin, azophloxine staining solution, phosphotungstic acid orange G, and light-green Steroid Assay. Isolated splenocytes were cultured in complete medium (RPMI SF solution. Finally, the tissue was dehydrated and mounted using Eukitt me- – 1640, 10% FCS, 50 μM 2-mercaptoethanol, 100 U·mL 1 penicillin/streptomycin) dium (O. Kindler). Image acquisition was performed using a slide scanner (Mirax at a cell density of 0.5 × 106 cells per well in a 96-well round bottom plate for a Midi, Zeiss). Areas of junctional zone and labyrinth zones were quantified using period of 48 h, unless otherwise stated. Progesterone, DEX, D(–)norgestrel, the program MiraxViewer. estradiol (E2), mifepristone (RU486), or vehicle control ethanol (all Sigma) were added at indicated concentrations. In selected experiments, cells were pre- Statistics. Data were analyzed using Prism 6 software (GraphPad) for Mac- incubated with 10 μM caspase 3 inhibitor Z-DEVD-FMK (BD Biosciences) for 30 intosh and are presented as mean values ± SEM. All n values refer to the min. Four to six replicate wells were pooled per condition and subjected to number of biological replicates, that is, individual mice. Differences between antibody staining for flow cytometry analysis of viable cells. two experimental groups were determined by unpaired, two-tailed Stu- dent’s t test. Comparison of three or more groups was performed by one- Apoptosis Assay. Isolated splenocytes were cultured in complete medium at a way analysis of variance (ANOVA) with Bonferroni’s post hoc test. Statistical cell density of 0.5 × 106 cells per well in a 96-well round bottom plate for a analysis comparing two groups under multiple conditions or over time was period of 6 h. Progesterone, DEX, mifepristone (RU486), or vehicle control performed by two-way ANOVA with Bonferroni’s post hoc test. In two-way ethanol (all Sigma) were added at indicated concentrations. Four to six ANOVA analysis of EAE disease courses, the P value for group × time in- replicate wells were pooled per condition and subjected to Annexin V and teraction is reported. Differences in disease incidence and survival were aCasp3 staining for flow cytometry analysis of viable cells. assessed by Fisher’s exact test and χ2 test, respectively. Significant results are indicated by asterisks: *P < 0.05; **P < 0.01. Proliferation Assay. T-cell–depleted nonproliferative feeder cells were gen- erated by adding 100 μL LowTox Rabbit Complement M (Cedarlane) and 5 μL Study Approval. All animal care and experimental procedures were carried 6 of rat anti-mouse CD90.2 (BioLegend, clone30-H12) to 30 × 10 wild-type out according to institutional guidelines and conformed to requirements of splenocytes resuspended in 900 μL HBSS medium (PAA). After incubation the German Animal Welfare Act. Ethical approvals were obtained from the with mild agitation for 30 min at 37 °C, cells were washed twice with ice-cold State Authority of Hamburg, Germany (approval G12/012). HBSS (300 × g, 10 min, 4 °C), irradiated with 35 Gy in a Biobeam 2000 gamma irradiator (Eckert & Ziegler), washed twice with ice-cold HBSS, and used as ACKNOWLEDGMENTS. We thank Kristin Thiele for breeding and providing irradiated feeder cells. Tregs and Tcons were isolated by MACS as described the Pgr−/− mice. This work was supported by grants (to M.A.F. and S.M.G.) 4 above and cultured separately in complete medium at 5 × 10 cells per well from the Forschungs- und Wissenschaftsstiftung Hamburg and Deutsche in the presence of 5 × 104 irradiated feeder cells per well in 96-well round Forschungsgemeinschaft (FR1720/8-1 and GO1357/8-1, KFO296 Fetomaternal – bottom plates. Cultures were stimulated for proliferation with 1 μg·mL 1 Immune Cross-Talk).

1. Arck PC, Hecher K (2013) Fetomaternal immune cross-talk and its consequences for 4. Nancy P, et al. (2012) Chemokine gene silencing in decidual stromal cells limits T cell maternal and offspring’s health. Nat Med 19(5):548–556. access to the maternal-fetal interface. Science 336(6086):1317–1321. 2. Collins MK, Tay C-S, Erlebacher A (2009) Dendritic cell entrapment within the preg- 5. Erlebacher A (2013) Mechanisms of T cell tolerance towards the allogeneic fetus. Nat nant uterus inhibits immune surveillance of the maternal/fetal interface in mice. J Clin Rev Immunol 13(1):23–33. Invest 119(7):2062–2073. 6. de Man YA, Dolhain RJEM, van de Geijn FE, Willemsen SP, Hazes JMW (2008) Disease

3. Blois SM, et al. (2007) A pivotal role for galectin-1 in fetomaternal tolerance. Nat Med activity of rheumatoid arthritis during pregnancy: Results from a nationwide pro- INFLAMMATION IMMUNOLOGY AND 13(12):1450–1457. spective study. Arthritis Rheum 59(9):1241–1248.

Engler et al. PNAS | Published online January 3, 2017 | E189 Downloaded by guest on October 5, 2021 7. Förger F, et al. (2008) Pregnancy induces numerical and functional changes of 37. Venken K, et al. (2008) Compromised CD4+ CD25(high) regulatory T-cell function in CD4+CD25 high regulatory T cells in patients with rheumatoid arthritis. Ann Rheum Dis patients with relapsing-remitting multiple sclerosis is correlated with a reduced fre- 67(7):984–990. quency of FOXP3-positive cells and reduced FOXP3 expression at the single-cell level. 8. Buchel E, Van Steenbergen W, Nevens F, Fevery J (2002) Improvement of autoimmune Immunology 123(1):79–89. hepatitis during pregnancy followed by flare-up after delivery. Am J Gastroenterol 38. Holinka CF, Tseng YC, Finch CE (1979) Reproductive aging in C57BL/6J mice: Plasma 97(12):3160–3165. progesterone, viable embryos and resorption frequency throughout pregnancy. Biol 9. Vukusic S, et al.; Pregnancy in Multiple Sclerosis Group (2004) Pregnancy and multiple Reprod 20(5):1201–1211. sclerosis (the PRIMS study): Clinical predictors of post-partum relapse. Brain 127(Pt 6): 39. Solano ME, et al. (2015) Progesterone and HMOX-1 promote fetal growth by CD8+ 1353–1360. T cell modulation. J Clin Invest 125(4):1726–1738. 10. Confavreux C, Hutchinson M, Hours MM, Cortinovis-Tourniaire P, Moreau T; Preg- 40. Yates MA, et al. (2010) Progesterone treatment reduces disease severity and increases IL- nancy in Multiple Sclerosis Group (1998) Rate of pregnancy-related relapse in multiple 10 in experimental autoimmune encephalomyelitis. J Neuroimmunol 220(1–2):136–139. sclerosis. N Engl J Med 339(5):285–291. 41. Ojasoo T, Doré JC, Gilbert J, Raynaud JP (1988) Binding of steroids to the progestin 11. Finkelsztejn A, Brooks JBB, Paschoal FM, Jr, Fragoso YD (2011) What can we really tell and glucocorticoid receptors analyzed by correspondence analysis. J Med Chem 31(6): women with multiple sclerosis regarding pregnancy? A systematic review and meta- 1160–1169. analysis of the literature. BJOG 118(7):790–797. 42. Tronche F, et al. (1999) Disruption of the glucocorticoid receptor gene in the nervous 12. Dendrou CA, Fugger L, Friese MA (2015) Immunopathology of multiple sclerosis. Nat system results in reduced anxiety. Nat Genet 23(1):99–103. Rev Immunol 15(9):545–558. 43. Orban PC, Chui D, Marth JD (1992) Tissue- and site-specific DNA recombination in 13. Langer-Gould A, Garren H, Slansky A, Ruiz PJ, Steinman L (2002) Late pregnancy transgenic mice. Proc Natl Acad Sci USA 89(15):6861–6865. suppresses relapses in experimental autoimmune encephalomyelitis: Evidence for a 44. Baumann S, et al. (2005) Glucocorticoids inhibit activation-induced cell death (AICD) suppressive pregnancy-related serum factor. J Immunol 169(2):1084–1091. via direct DNA-dependent repression of the CD95 ligand gene by a glucocorticoid 14. Gatson NN, et al. (2011) Induction of pregnancy during established EAE halts progres- receptor dimer. Blood 106(2):617–625. sion of CNS autoimmune injury via pregnancy-specific serum factors. J Neuroimmunol 45. John S, et al. (2011) Chromatin accessibility pre-determines glucocorticoid receptor 230(1–2):105–113. binding patterns. Nat Genet 43(3):264–268. 15. McClain MA, et al. (2007) Pregnancy suppresses experimental autoimmune enceph- 46. van der Laan S, et al. (2008) Chromatin immunoprecipitation scanning identifies alomyelitis through immunoregulatory cytokine production. J Immunol 179(12): glucocorticoid receptor binding regions in the proximal promoter of a ubiquitously 8146–8152. expressed glucocorticoid target gene in brain. J Neurochem 106(6):2515–2523. 16. Voskuhl RR, Palaszynski K (2001) Sex hormones in experimental autoimmune en- 47. Ayroldi E, et al. (2007) GILZ mediates the antiproliferative activity of glucocorticoids cephalomyelitis: Implications for multiple sclerosis. Neuroscientist 7(3):258–270. by negative regulation of Ras signaling. J Clin Invest 117(6):1605–1615. 17. Gold SM, Voskuhl RR (2016) Pregnancy and multiple sclerosis: From molecular 48. Nikic I, et al. (2011) A reversible form of axon damage in experimental autoimmune mechanisms to clinical application. Semin Immunopathol 38(6):709–718. encephalomyelitis and multiple sclerosis. Nat Med 17(4):495–499. 18. Aluvihare VR, Kallikourdis M, Betz AG (2004) Regulatory T cells mediate maternal 49. Schattling B, et al. (2012) TRPM4 cation channel mediates axonal and neuronal de- tolerance to the fetus. Nat Immunol 5(3):266–271. generation in experimental autoimmune encephalomyelitis and multiple sclerosis. 19. Zenclussen AC, et al. (2006) Regulatory T cells induce a privileged tolerant microen- Nat Med 18(12):1805–1811. vironment at the fetal-maternal interface. Eur J Immunol 36(1):82–94. 50. Kahn DA, Baltimore D (2010) Pregnancy induces a fetal antigen-specific maternal 20. Sakaguchi S (2005) Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells T regulatory cell response that contributes to tolerance. Proc Natl Acad Sci USA in immunological tolerance to self and non-self. Nat Immunol 6(4):345–352. 107(20):9299–9304. 21. Patas K, Engler JB, Friese MA, Gold SM (2013) Pregnancy and multiple sclerosis: Feto- 51. Robertson SA, et al. (2009) Seminal fluid drives expansion of the CD4+CD25+ T reg- maternal immune cross talk and its implications for disease activity. J Reprod Immunol ulatory cell pool and induces tolerance to paternal alloantigens in mice. Biol Reprod 97(1):140–146. 80(5):1036–1045. 22. Rowe JH, Ertelt JM, Xin L, Way SS (2012) Pregnancy imprints regulatory memory that 52. Moldenhauer LM, et al. (2009) Cross-presentation of male seminal fluid antigens sustains anergy to fetal antigen. Nature 490(7418):102–106. elicits T cell activation to initiate the female immune response to pregnancy. 23. Rowe JH, Ertelt JM, Aguilera MN, Farrar MA, Way SS (2011) Foxp3(+) regulatory T cell J Immunol 182(12):8080–8093. expansion required for sustaining pregnancy compromises host defense against 53. Erlebacher A, Vencato D, Price KA, Zhang D, Glimcher LH (2007) Constraints in anti- prenatal bacterial pathogens. Cell Host Microbe 10(1):54–64. gen presentation severely restrict T cell recognition of the allogeneic fetus. J Clin 24. Mao G, et al. (2010) Progesterone increases systemic and local uterine proportions of Invest 117(5):1399–1411. CD4+CD25+ Treg cells during midterm pregnancy in mice. Endocrinology 151(11): 54. Heng TSP, Painter MW; Immunological Genome Project Consortium (2008) The Im- 5477–5488. munological Genome Project: Networks of gene expression in immune cells. Nat 25. Lee JH, Ulrich B, Cho J, Park J, Kim CH (2011) Progesterone promotes differentiation Immunol 9(10):1091–1094. of human cord blood fetal T cells into T regulatory cells but suppresses their differ- 55. Barlow SM, Morrison PJ, Sullivan FM (1973) Plasma corticosterone levels during entiation into Th17 cells. J Immunol 187(4):1778–1787. pregnancy in the mouse. Br J Pharmacol 48(2):346P. 26. Wildin RS, et al. (2001) X-linked neonatal diabetes mellitus, enteropathy and endo- 56. Lépine S, Sulpice J-C, Giraud F (2005) Signaling pathways involved in glucocorticoid- crinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet 27(1):18–20. induced apoptosis of thymocytes. Crit Rev Immunol 25(4):263–288. 27. Bennett CL, et al. (2001) The immune dysregulation, polyendocrinopathy, enteropa- 57. Hoffman-Goetz L, Fietsch CL (2002) Lymphocyte apoptosis in ovariectomized mice thy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 27(1): given progesterone and voluntary exercise. J Sports Med Phys Fitness 42(4):481–487. 20–21. 58. Gold SM, et al. (2012) Dynamic development of glucocorticoid resistance during au- 28. Humrich JY, et al. (2010) Homeostatic imbalance of regulatory and effector T cells due toimmune neuroinflammation. J Clin Endocrinol Metab 97(8):E1402–E1410. to IL-2 deprivation amplifies murine lupus. Proc Natl Acad Sci USA 107(1):204–209. 59. Wüst S, et al. (2008) Peripheral T cells are the therapeutic targets of glucocorticoids in 29. Engler JB, et al. (2011) Unmasking of autoreactive CD4 T cells by depletion of CD25 experimental autoimmune encephalomyelitis. J Immunol 180(12):8434–8443. regulatory T cells in systemic lupus erythematosus. Ann Rheum Dis 70(12):2176–2183. 60. Schweingruber N, et al. (2014) Chemokine-mediated redirection of T cells constitutes 30. Nie H, et al. (2013) Phosphorylation of FOXP3 controls regulatory T cell function and is a critical mechanism of glucocorticoid therapy in autoimmune CNS responses. Acta inhibited by TNF-α in rheumatoid arthritis. Nat Med 19(3):322–328. Neuropathol 127(5):713–729. 31. Munoz-Suano A, Kallikourdis M, Sarris M, Betz AG (2012) Regulatory T cells protect 61. van den Brandt J, et al. (2007) Enhanced glucocorticoid receptor signaling in T cells from autoimmune arthritis during pregnancy. J Autoimmun 38(2–3):J103–J108. impacts thymocyte apoptosis and adaptive immune responses. Am J Pathol 170(3): 32. Baca Jones C, et al. (2014) Regulatory T cells control diabetes without compromising 1041–1053. acute anti-viral defense. Clin Immunol 153(2):298–307. 62. Andersen KG, Nissen JK, Betz AG (2012) Comparative genomics reveals key gain-of- 33. Viglietta V, Baecher-Allan C, Weiner HL, Hafler DA (2004) Loss of functional sup- function events in Foxp3 during regulatory T cell evolution. Front Immunol 3:113. pression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J Exp 63. Sicotte NL, et al. (2002) Treatment of multiple sclerosis with the pregnancy hormone Med 199(7):971–979. estriol. Ann Neurol 52(4):421–428. 34. Carbone F, et al. (2014) Regulatory T cell proliferative potential is impaired in human 64. Pozzilli C, et al. (2015) Oral contraceptives combined with interferon β in multiple autoimmune disease. Nat Med 20(1):69–74. sclerosis. Neurol Neuroimmunol Neuroinflamm 2(4):e120. 35. Sellebjerg F, Krakauer M, Khademi M, Olsson T, Sørensen PS (2012) FOXP3, CBLB and 65. Voskuhl RR, et al. (2016) Estriol combined with glatiramer acetate for women with ITCH gene expression and cytotoxic T lymphocyte antigen 4 expression on CD4(+) relapsing-remitting multiple sclerosis: A randomised, placebo-controlled, phase 2 tri- CD25(high) T cells in multiple sclerosis. Clin Exp Immunol 170(2):149–155. al. Lancet Neurol 15(1):35–46. 36. Huan J, et al. (2005) Decreased FOXP3 levels in multiple sclerosis patients. J Neurosci 66. Lydon JP, et al. (1995) Mice lacking progesterone receptor exhibit pleiotropic re- Res 81(1):45–52. productive abnormalities. Genes Dev 9(18):2266–2278.

E190 | www.pnas.org/cgi/doi/10.1073/pnas.1617115114 Engler et al. Downloaded by guest on October 5, 2021