Seminal CD38 Is a Pivotal Regulator for Fetomaternal Tolerance

Seminal CD38 is a pivotal regulator for fetomaternal tolerance

Byung-Ju Kima,b, Yun-Min Choia,b, So-Young Raha,b, Dae-Ryoung Parka,b, Seon-Ah Parka, Yun-Jo Chunga, Seung-Moon Parkc, Jong Kwan Parkd, Kyu Yun Jange, and Uh-Hyun Kima,b,f,1

+ aNational Creative Research Laboratory for Ca2 Signaling Network and Departments of bBiochemistry, dUrology, and ePathology, Chonbuk National University Medical School, Jeonju, 561-180, Korea; cDivision of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, 570-752, Korea; and fInstitute of Cardiovascular Research, Chonbuk National University, Jeonju, 561-180, Korea

Edited by John J. Eppig, The Jackson Laboratory, Bar Harbor, ME, and approved December 22, 2014 (received for review July 16, 2014) A successful pregnancy depends on a complex process that estab- a variety of inflammatory diseases (20–23). For example, recombi- lishes fetomaternal tolerance. Seminal plasma is known to induce nant CD38 inhibits LPS-induced inflammatory signals in mouse maternal immune tolerance to paternal alloantigens, but the seminal macrophages and human DCs through an interaction with CD31 factors that regulate maternal immunity have yet to be characterized. (24, 25). Here, we show that a soluble form of CD38 (sCD38) released from In this study, we found that CD38 is truncated and released seminal vesicles to the seminal plasma plays a crucial role in inducing into the seminal plasma from seminal vesicles (SVs) as a soluble + + + tolerogenic dendritic cells and CD4 forkhead box P3 (Foxp3 )reg- form (sCD38) in humans and mice. This finding prompted us to ulatory T cells (Tregs), thereby enhancing maternal immune tolerance examine whether CD38 plays a role in maternal immune toler- and protecting the semiallogeneic fetus from resorption. The abortion −/− ance during pregnancy. We found that sCD38 present in seminal rate in BALB/c females mated with C57BL/6 Cd38 males was high plasma was crucial for the induction of uterine tDC and Tregs, compared with that in females mated with Cd38+/+ males, and this + which are responsible for the development of the fetomaternal was associated with a reduced proportion of Tregs within the CD4 tolerance. T-cell pool. Direct intravaginal injection of sCD38 to CBA/J pregnant mice at preimplantation increased Tregs and pregnancy rates in mice Results under abortive sonic stress from 48 h after mating until euthanasia.

CD38 Is Truncated and Released from SVs to the Plasma as a Soluble MEDICAL SCIENCES Thus, sCD38 released from seminal vesicles to the seminal plasma acts Form. ADPRC activity was detected in human seminal fluid in as an immunoregulatory factor to protect semiallogeneic fetuses from two proteins with molecular weights of 45 kDa and 37 kDa as maternal immune responses. identified in in-gel assays (Fig. 1A). Notably, the 37 kDa protein bound to a CD38 immunoaffinity column (Fig. 1B, lanes 1 and seminal plasma | CD38 | regulatory T cells | dendritic cells | fetomaternal 3), whereas the 45 kDa protein did not (Fig. 1B, lanes 2 and 4). tolerance This suggests that the 37 kDa protein was a truncated form of CD38, whereas the 45 kDa protein was full-length CD38, which eventy-five percent of pregnancies that are lost represent is encapsulated within prostasomes (13), making the CD38 (45 Sfailure of implantation and are therefore not clinically rec- kDa) physically inaccessible for binding to the affinity column. ognized as pregnancies (1). Recurrent miscarriage (the sponta- Aminoterminal sequencing of the 37 kDa protein identified its neous loss of three or more consecutive pregnancies) is a – cleavage site as Arg58 close to the TM domain of CD38 (Fig. significant health issue for 1 2% of women, with no identifiable 1C). Western blot analysis with two different antibodies against biological cause and no effective treatment. During early stages CD38 peptides (CD3845–57 and CD38171–292) confirmed that this of pregnancy, complex processes help to create a uterine envi- was the case (Fig. 1D). The soluble form, 37 kDa CD38 (sCD38), ronment that is conducive to a successful pregnancy. These was detected in all 19 seminal plasma samples tested (range, include immunological adaptation to the semiallogeneic fetus. Tolerance to paternal alloantigens is critical for successful re- Significance production in placental mammals (2, 3). Many studies have proposed that regulatory T cells (Tregs) play an essential role in the development of fetomaternal tolerance in mice and humans In natural matings, semen delivers spermatozoa and immuno- (4–7). Seminal plasma contains potent immunoregulatory mol- regulatory fluids to the female reproductive tract. Here, a sol- ecules that contribute to the induction of tolerogenic DCs uble form of CD38 (sCD38) is shown to play an important role (tDCs) and ultimately Treg expansion, which is necessary to in facilitating maternal immune tolerance against the fetus by inducing the development of uterine tolerogenic DCs and establish maternal tolerance against paternal antigens (8–10). + + However, the specific molecules in semen that are responsible forkhead box P3 (Foxp3 ) regulatory T cells. Deficiency of for expansion of Tregs and establishment of maternal tolerance sCD38 in seminal fluid increased the rates of loss of allogeneic remain undefined. fetuses, and this loss was rescued by a direct injection of CD38, a mammalian prototype of ADP ribosyl cyclases recombinant sCD38 into the uterus. Thus, seminal sCD38 acts as a pivotal immune suppressor for establishing maternal (ADPRCs), is a type II transmembrane (TM) glycoprotein immune tolerance against the fetus. sCD38 could potentially expressed in many cell types and seminal fluid (11–16). CD38 be used to prevent failed pregnancies. produces calcium-mobilizing second messengers, cyclic ADP ri-

bose, and nicotinic acid adenine dinucleotide phosphate (11, 12). Author contributions: B.-J.K. and U.-H.K. designed research; B.-J.K., Y.-M.C., S.-Y.R., We previously showed that intact CD38 in prostasomes assists D.-R.P., S.-A.P., Y.-J.C., S.-M.P., J.K.P., and K.Y.J. performed research; B.-J.K., K.Y.J., + progesterone-induced sperm Ca2 signaling (13). In addition to and U.-H.K. analyzed data; and B.-J.K. and U.-H.K. wrote the paper. 2+ its enzymatic role for Ca signaling, CD38 may also have a The authors declare no conflict of interest. nonenzymatic role through its interaction with CD31 (17, 18). This article is a PNAS Direct Submission. CD31, a type I TM homophilic or heterophilic receptor, is 1To whom correspondence should be addressed. Email: [email protected]. expressed in endothelial cells and a variety of immune cells (19) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and is involved in attenuating the inflammatory response in 1073/pnas.1413493112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1413493112 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Cd38−/− b A 1 2 B 1 2 3 4 with or wild-type C57BL/6 (H-2 ) male mice. Mice were examined at 12.5 d postcoitum (dpc), at which time both implantation and resorption would be visible. We found that female mice −/− −/− mated with Cd38 males (Cd38 matings) showed higher rates –45 kDa Cd38+/+ –45 kDa –37 kDa of fetal resorption at 12.5 dpc than females mated with –37 kDa

P = 0.015 45-57 171-292 AC40 n.s. C 12 12 P = 0.003 10 10 CD38 ID TM ED 30 8 8 58 20 6 6 sCD38 RFPETV 4 4 10

% Resorption 2

= 9 2 = 13 n= 13 n= n= 9 n= n n= 13 n= n= 9 n= 0 n 0 0 D E site implantation Total -/- -/- -/- Viable implantation site Viable implantation Cd38 Cd38 Cd38 HC HC 45 kDa CD38 42 kDa x 40 x 200 x 400 37 kDa sCD38 B 34 kDa Ib Ib Ib jz Epitope : 45-57 171-292 3 mm jz Fig. 1. Identification of sCD38 in seminal fluid by in-gel ADPRC assay. (A) Visualization of human seminal fluid proteins with ADPRC activity (lanes 1 De and 2 for two healthy volunteers). (B) Coomassie blue staining of proteins Wild-typematings 500 m 100 m 50 m 50 m eluted from a CD38 immunoaffinity column (lane 1) and proteins that passed Ib / jz Ib jz through the column (lane 2). In-gel ADPRC activity assay for the proteins 3 mm Ib matings

eluted from the column (lane 3) and passed through the column (lane 4). (C) -/- jz Upper diagram shows the intracellular domain (ID), TM domain, and extra- De

Cd38 cellular domain (ED) of CD38, and the lower diagram shows the N-terminal 500 m 100 m 50 m 50 m amino acid (aa) sequence (highlighted in red) of sCD38. The segments (aa – – MHCII CD80 P = 0.015 P = 0.04 45 57 and aa 171 292) denoted by green lines represent epitopes of the D 200 60 antibodies used for immunoblotting. The dotted line indicates the cleavage (MFI) 50 (MFI) + 150 site within CD38. (D) Proteins immunoprecipitated from seminal fluid and + 40 prostasome lysates using anti-CD38 antibodies were blotted with antibodies 100 30

– – /CD11c

specific for two different epitopes: amino acids 45 57 and 171 292. HC, + /CD11c 20 50 + heavy chain of Ig. (E) Mouse seminal plasma was collected from B6 females 10 Events MHCII

by uterine lavage within 1 h of mating with B6 intact (CON), vasectomised CD80 0 1 2 3 4 0 1 2 3 4 0 0 (VAS), and SVX males immunoprecipitated with anti-CD38, and the immu- 10 10 10 10 10 10 10 10 10 10 -/- -/- Isotype Wild-type matings Cd38 Cd38 noprecipitates were analyzed for CD38 by Western blotting. Mouse sple- Cd38-/- matings nocyte lysate (SP) and recombinant mouse sCD38 (sCD38) were included in

the experiment as size references of full length and truncated sCD38. E F G P = 0.039 Wild P < 0.05 P = 0.008 + + +

0.5–10.6 μg/mL), including one from a vasectomised individual 14.2 of CD4 of CD4 of CD4

μ + + (7.8 g/mL). 4 + 10 Cd38-/- 3 We next used a mouse model to examine the role of seminal 10 2 CD38 in more detail. We began by asking whether sCD38 was 10 % Foxp3 % % Foxp3 1 % Foxp3 10 present in mouse seminal fluid. Due to technical difficulties in 0 11.7 CD4 10 0 1 2 3 4 obtaining mouse semen, we collected uterine lavage fluid from 10 10 10 10 10 -/- -/- -/- unmated estrous females and mouse SV fluid. sCD38 was not Foxp3 Cd38 Cd38 Cd38 detected in uterine lavage fluid from unmated estrous females, − − whereas sCD38 was present in mouse SV fluid (Fig. S1). To Fig. 2. Pregnancy impairment in female mice mated with Cd38 / male − − identify the organ that secreted the sCD38, we collected uterine mice. Wild-type or Cd38 / C57BL/6 males were mated with BALB/c females, lavage fluid from the female reproductive tract 1 h after mating. and plugged mice were killed at 12 dpc. (A) Resorption rates. (B) Histological Western analysis demonstrated that the uterine lavage fluid evaluation of placental tissue. In wild-type mating, the placenta and the collected from female mice mated with normal or vasectomised junctional zone (jz) area of the placenta located between the labyrinth (lb) and decidual tissues (De) are intact, and all of the cellular components, in- mice contained sCD38 (34 kDa). Mouse splenocytes expressed −/− E cluding trophoblasts (empty arrows), are viable. In Cd38 mating, the only the full-length CD38 (42 kDa) (Fig. 1 ) (26). sCD38 placenta is necrotic (stars), and the junctional zone area shows vascular comigrated with recombinant sCD38 lacking the cytoplasmic and congestion (empty arrow heads), hemorrhage, and inflammatory infiltration TM domains on SDS/PAGE gels (Fig. 1E). By contrast, uterine (arrows). Insets show low-magnification images of the embryos. (C)The lavage fluid collected from females mated with SV-deficient number of total and viable implantation sites in wild-type and Cd38−/− (SVX) males did not contain sCD38. These findings indicate that matings. Bars, mean ± SEM. (D) Phenotypic analysis of CD11c+ uterine DCs − − from wild-type or Cd38 / mating mice at 3.5 dpc. MHC-II and CD80 ex- sCD38 in seminal fluid originates from SV and that the trunca- + tion of CD38 in SV is common to both humans and mice. pression was examined in gated CD11c uterus cells. Mean fluorescent intensities (MFIs) of MHC-II and CD80 were plotted as bar graphs (Right panels). Bars, mean ± SEM; number of mice tested for each experiment, 8–9. Seminal sCD38 Is Crucial for Fetomaternal Tolerance During Pregnancy. + + (E, Left panels) Representative flow cytometric analysis of CD4 Foxp3 T cells As sCD38 was present in mouse seminal fluid, we next investigated in the PALN in wild-type and Cd38−/− matings at 3.5 dpc. Percentage of CD4+ whether seminal sCD38 affected pregnancy, particularly with re- Foxp3+ T cells in the PALN in wild-type and Cd38−/− matings was plotted for spect to fetomaternal immune tolerance. We generated an alloge- statistical evaluation at 3.5 (E, Right panel) and 6.5 dpc (F). (G) Percentage of d + + − − neic pregnancy model based on BALB/c (H-2 ) female mice mated CD4 Foxp3 T cells in the uterus in wild-type and Cd38 / matings at 3.5 dpc.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1413493112 Kim et al. Downloaded by guest on September 29, 2021 males (wild-type matings) (Fig. 2A). In agreement with this finding, suggest that sCD38 directly suppresses the maturation, causing histochemical analysis revealed visible signs of inflammation, in- DCs to maintain an immature phenotype. We also found that cluding thrombosis and massive immune cell infiltration into the upon exposure to LPS, BM-DCsCD38 secreted a higher level of Cd38−/− placentas of matings, none of which were present in wild- IL-10 (TH2 cytokine) and expressed higher levels of TGF-β B type matings (Fig. 2 ). These findings indicate that a lack of CD38 mRNA and protein than did BM-DCcontrol (Fig. 3B). Conversely, in seminal fluid induces embryo resorption, presumably due to the BM-DCsCD38 inhibited the production of both IL-12p70 and impairment of fetomaternal tolerance (3–6). The number of TNF-α (T 1cytokines)(Fig.3C), indicating that sCD38 −/− H implantations in Cd38 matings was similar to that in wild-type −/− skewed DCs toward an anti-inflammatory phenotype. However, matings, whereas the number of surviving fetuses in Cd38 matings was reduced compared with that in wild-type matings (Fig. 2C). To exclude the possibility that mouse pregnancy failures can be ovarian-based due to progesterone insufficiency, we com- A MHCII CD80 CD40 CD11c 79.1 62.0 73.0 87.1 pared maternal plasma progesterone levels at 12.5 dpc, but BM-DCcontrol found no significant difference between the two groups (Fig. S2). This suggests that sCD38 reduces the maternal immune response toward the fetus after implantation, but does not BM-DC 34.6 39.4 42.7 84.9 affect the implantation itself. sCD38 Previous studies suggest that seminal factors promote the gen-

eration of tDCs (9, 10). Therefore, we next examined whether Events 0 1 2 3 4 0 1 2 3 4 0 1 2 3 410 10 10 10 10 0 1 2 3 410 10 10 10 10 seminal sCD38 inhibited the insemination-induced maturation 10 10 10 10 10 10 10 10 10 10 P < 0.001 P < 0.001 of uterine DCs by comparing phenotypic maturation markers on B TGF- −/− uterine DCs in wild-type and Cd38 matings. In agreement -actin −/− P<0.05 with the high resorption rates in Cd38 matings, the uterine + Cd38−/− CD11c cells from mating showed higher levels of 1 (RE) (TGF- 1) -

MHC-II and CD80 expression at 3.5 dpc than those from wild- (pg/ml) IL-10 Relative mRNA type matings (Fig. 2D). Because tDCs promote the generation of TGF Tregs (27), we next asked whether sCD38 was necessary for Tregs expansion. Expression of the transcription factor, Foxp3, is C D E MEDICAL SCIENCES a hallmark of mature Tregs expansion (28). We found that para- P < 0.005 Control sCD38 P < 0.05 −/− P < 0.001 35 Cd38 p-STAT3 aortic lymph nodes (PALNs) in matings were signifi- 30

+ c.p.m.) cantly larger (198 ± 60.4%) and contained more CD4 cells than 3 25 + STAT3 those in wild-type matings at 3.5 dpc. The numbers of Foxp3 P < 0.001 20 −/− T cells in PALNs in Cd38 and wild-type matings were not 15 10 TNF– (pg/ml) different at 3.5 dpc and 6.5 dpc (Fig. S3). However, the per- (pg/ml) IL-12p70 + + −/− centage of CD4 Foxp3 T cells in the PALNs from Cd38 5 Proliferation (10 Proliferation matings was significantly smaller than that from wild-type mat- (RE) p-STAT3 0 ings at both 3.5 (Fig. 2E) and 6.5 dpc (Fig. 2F), indicating that + sCD38 induced Foxp3 Treg-mediated T-cell suppression. This n.s. F BM-DCcontrol BM-DCsCD38 P < 0.01 is consistent with previous reports (29, 30) that Tregs are able to P < 0.01 suppress effector T cells and thereby cause an expansion of + 12 + 10 CD4 cells in the presence of a decreased proportion of Tregs. 4.9 10.8

+ + of CD4 8 + Likewise, the percentage of CD4 Foxp3 T cells in the uterus in Control Cd38−/− 6 matings was also significantly less than that from wild- 4 10 4 type matings at 3.5 dpc (Fig. 2G). By comparison, syngeneic 3 −/− 10 Foxp3 % 2 matings of C57BL/6 females with wild-type and Cd38 males 2 10 4.6 5.6 0 showed no differences in resorption rates, total number of im- 1 + + 10 CD4 Anti-TGF- 0 plantation sites, viable implantation sites, and CD4 Foxp3 10 −/− 0 1 2 3 4 0 1 2 3 4 T-cell population (Fig. S4). In addition, Cd38 females mated 10 10 10 10 1010 10 10 10 10 −/− Foxp3 with C57BL/6 wild-type and Cd38 males showed no difference in fetal loss, indicating that a CD38 deficiency in maternal and/or Fig. 3. sCD38-mediated induction of tDC. (A) DCs were generated from placental tissue does not affect fetal development, at least in mouse BM cells by culture with GM-CSF for 6 d in the absence (BM-DCcontrol, + syngeneic pregnancy (Fig. S4). Thus, by inducing Foxp3 Tregs line histogram) or presence (BM-DCsCD38, filled histogram) of 200 ng/mL μ in females, seminal sCD38 contributes to a successful pregnancy sCD38 and then activated with 1 g/mL LPS for 24 h to induce DC matura- by supporting fetomaternal tolerance. tion; gray histogram, isotype-matched control. Data are representative of nine experiments. (B and C) The levels of cytokines of LPS-stimulated BM-DCs were measured in IL-10, IL12p70, and TNF-α ELISAs. TGF-β1 mRNA and pro- sCD38 Induces Fetomaternal Tolerance Through Expression of tDCs + tein were measured by real-time PCR and immunoblotting, respectively. and Foxp3 Tregs. Because the properties of uterine DC in (Right Bottom) Band intensity of TGF-β1 relative to that of actin. RE, relative pregnant female mice are markedly influenced by seminal expression. (D) Western blot analysis of phosphorylated and total STAT3 in

sCD38, we examined whether sCD38 induced tDC, which is BM-DCcontrol and BM-DCsCD38.(Bottom Panel) Relative band intensity (RE). characterized by expression of immature DC surface phenotype. Data of TGF-β and STAT3 represent the mean ± SD of three experiments. (E) × 4 To this end, we generated DCs by culturing bone marrow (BM) LPS-stimulated BM-DCcontrol and BM-DCsCD38 (2 10 ) were cocultured with + 5 3 cells in vitro with GM-CSF in the absence (BM-DC )or allogeneic CD4 T cells (2 × 10 ), and proliferation was determined by [ H] control ± presence (BM-DC ) of recombinant sCD38 for 6 d. Upon thymidine incorporation. Data represent the mean SD of three in- sCD38 dependent experiments. (F) Representative flow cytometric analysis showing + + LPS stimulation, BM-DCcontrol displayed a typical mature phe- the CD4 Foxp3 T cells in the mixed leukocyte reaction. OVA 323–339 pep- notype, including increased expression of MHC-II, CD80, and + tide-stimulated BM-DCcontrol or BM-DCsCD38 were cocultured with OT-II CD4 A − CD40; however, this was not the case for BM-DCsCD38 (Fig. 3 ). CD25 T cells in the presence or absence of anti–IL-10 or anti–TGF-β mAbs for Moreover, proteinase K-treated recombinant sCD38 was in- 5 d. Bar graph represents mean percentage of Foxp3+ among gated CD4+ capable of suppressing DC maturation (Fig. S5). These data cells. Bars, mean ± SD of three experiments.

Kim et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 no distinct expression levels of indoleamine 2,3-dioxygenase (IDO) A and BM-DCsCD38 and BM-DCcontrol were observed (Fig. S6). Previously, CD31 ligation has been reported to inhibit NF-κB signaling (24, 31). However, sCD38 did not affect NF-κB, Erk, and p38 signaling pathways (Fig. S7). Instead, sCD38 triggered the phosphorylation of a signal transducer and an activator of transcription 3 (STAT3) (Fig. 3D), a transcription factor involved in regulating tDCs (32, 33), indicating that sCD38 induced the differentiation of tDCs via STAT3-dependent pathways. Consistent with this mechanism, BM-DCsCD38 showed impaired activity in + BM-DC

a mixed leukocyte reaction containing allogeneic CD4 Tcells -/- compared with BM-DCcontrol (Fig. 3E). Next, we investigated whether tDCs induced by sCD38 have Cd31 + the potential to induce Foxp3 Tregs. We observed a substantial Cd31-/- BM + + reduction in the percentage of Foxp3 in CD4 cells when sorted Cd31-/- BM OT-II naïve T cells were cocultured with BM-DCcontrol (4.7 ± 0.8%) versus with BM-DCsCD38 (9.9 ± 1.3%) (Fig. 3F). Fur- + thermore, the degree of induction of Foxp3 Tregs by BM- B DCsCD38 was lower in the presence of anti–TGF-β than that in the absence of anti–TGF-β or anti–IL-10 mAbs. When anti–IL- + 10 was present instead of TGF-β, the induction of Foxp3 Tregs by BM-DCsCD38 was not altered. These results indicate that TGF-β released by BM-DCsCD38 was primarily responsible for + the induction of Foxp3 Tregs. Thus, BM-DCsCD38 show features of tDCs, as BM-DCsCD38 are able to induce the differentiation of Tregs, which are crucial for fetomaternal tolerance.

sCD38 Induces the Differentiation of tDCs Through a CD31 Independent Cd31-/- BM Pathway. CD31 has been reported to be a counterreceptor for Cd31-/- BM CD38, and CD38-mediated negative regulation of TLR4 sig- Fig. 4. CD31 is not involved in sCD38-mediated induction of tDC. (A) BM- naling was undertaken by CD31 (24, 25). Thus, we evaluated the − − DCs were produced from BM cells of wild-type and Cd31 / mice by culture possibility of CD31 involvement in the sCD38 effects on sus- in the medium containing GM-CSF for 6 d in the presence (BM-DC )or A sCD38 taining immature phenotypes of BM-DC as seen in Fig. 3 . absence of sCD38 (BM-DCcontrol) and stimulated with 1 μg/mL LPS for 24 h. Although a recent study suggested that DCs lacking CD31 favor Expression of MHC-II and CD80 on BM-DCcontrol and BM-DCsCD38 derived from − − immunogenic potential (31), our data showed that LPS-stimulated wild-type and Cd31 / mice was plotted as line histogram (Left panels). MFIs −/− wild-type BM-DC and Cd31 BM-DC expressed similar of MHC-II and CD80 were plotted as bar graphs (Right panels). Data repre- sCD38 sCD38 ± β levels of MHC-II and CD80 (Fig. 4A). Similarly, upon exposure to sent the mean SD of 15 experiments. (B) The mRNA levels of IL-10, TGF- 1, and IL-12p40 in LPS-stimulated BM-DCcontrol and BM-DCsCD38 derived from LPS even without CD31 expression, mRNA levels of IL-10 and −/− β Cd31 mice. Allogeneic T-cell proliferative responses were measured by TGF- were higher, whereas the mRNA level of IL-12p40 was [3H]thymidine incorporation after coculture with LPS-stimulated BM- −/− lower in sCD38-treated BM-DCsCD38 than those in BM-DCcontrol DCcontrol and BM-DCsCD38 derived from Cd31 mice. Data are expressed ± (Fig. 4B). Regardless of CD31 expression, BM-DCsCD38 maintained as the mean SD of four experiments. weaker allogeneic T-cell proliferative responses than BM-DCcontrol (Fig. 4B). Collectively, these data show that CD31 is not involved in sCD38-driven differentiation of tDCs. Discussion The role of Tregs in fetomaternal tolerance is essential for sCD38 Reduces Stress-Challenged Fetal Resorption by Expansion of normal pregnancy. Studies have shown that the adoptive transfer + + Foxp3 Tregs. Because sCD38 induces tDCs and then Foxp3 of Tregs prevented fetal loss in the embryo resorption model Tregs, we examined whether intravaginal injection of sCD38 (DBA/2J-mated CBA/J female), and the depletion of Tregs led maintained pregnancy in abortion-prone mice under sonic stress to high levels of embryo resorption in the allogeneic pregnancy + (34). Direct injection of sCD38 to the vagina of stress-challenged model (35, 36). Foxp3 Tregs are generated in the thymus and + CBA/J female mice mated with DBA/2J male mice greatly re- periphery from naïve CD4 T cells (5). Interestingly, recent + duced the incidence of fetal resorption (Fig. 5A), although the studies reported that extrathymic Foxp3 Tregs play a pivotal number of fetal implantations was unchanged (Fig. 5B). To ex- role in fetomaternal tolerance and that those memorized fetal- amine the inhibitory effects of sCD38 on the aberrant maturation specific Tregs rapidly expanded to induce tolerance during the + of uterine CD11c cells induced by sonic stress, we characterized subsequent pregnancy (5, 6). Even in the absence of fertilization, + the phenotype of CD11c cells. Sonic stress induced the maturation exposure of the female genital tract to seminal plasma induced + + of uterine CD11c cells with increased levels of MHC-II and CD80 tolerance to the paternal alloantigen via expansion of Foxp3 in pregnant CBA/J females matedwithDBA/2Jmales,compared Tregs (8, 37). Seminal plasma confers DCs with immunoregu- with those in control mice. Administration of sCD38 decreased the latory potential, and these DCs are crucial for embryo implan- + number of phenotypically matured uterine CD11c cells in stress- tation and the generation of Tregs (9, 10, 34, 38). Seminal fluid challenged mice (Fig. 5C). In addition, the PALNs of female mice contains high levels of TGF-β (39, 40) and prostaglandins (41), + injected with sCD38 contained a higher proportion of Foxp3 cells both of which have been proposed to contribute to the induction + + at 6.5. dpc than Foxp3 cells of control females (Fig. 5D). Taken of Foxp3 Tregs and/or the differentiation of tDCs (9, 42). together, the results of the present study show that sCD38 causes However, it has been suggested that other additional molecules + induction of tDCs and Foxp3 cells and thereby protects embryos are involved in the induction of tDCs (9). In the present study, from maternal immune rejection. we demonstrated that sCD38-treated BM-DCs transform into

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1413493112 Kim et al. Downloaded by guest on September 29, 2021 + n.s n.s differentiation, suggesting that the percentage of Foxp3 A P < 0.05 P < 0.05 B 40 8 Tregs may be more important than the sheer number of + 7 Foxp3 Tregs in fetomaternal tolerance. Consistent with

30 6 reports (36) that the contribution of Tregs at the time of early pregnancy impacts fetal survival later on in gestation, our + 5 results showed that the Foxp3 Tregs proportion was higher in per mother 20 −/− n 4 wild-type matings than in Cd38 matings at 3.5 dpc, and this o it

proseR 3 difference persisted through 6.5 dpc. These findings suggest + 10 2 that a consistently high proportion of Foxp3 Tregs, induced Implantation site Implantation Control by sCD38 during the early phase of pregnancy, may play an

% 1 Stress = 10 = 10 important role in fetomaternal tolerance. n n= 8 n= n= 10 n= n= 8 n= n= 10 n= n Stress + sCD38 0 0 Previous studies have reported that maternal inflammation + decreased Foxp3 Treg accumulation, which caused fetal loss, C MHCII P < 0.05 P < 0.05 D 40 P = 0.011 P = 0.013 (MFI) and that the decidua of the surviving fetus exhibited abnormal + 30 spiral artery modification (5). Abnormal maternal inflammation + 20 restricted the growth of surviving fetuses. Abnormal spiral artery /CD11c CD4 + modification in the decidua was associated with complications of 6 10 7 of = = 7

+ pregnancy, including preeclampsia, placental abruption, pre- n n= n= n MHCII 0 term, and fetal loss (43, 44). In addition, immunosuppressive 0 1 2 3 4 P < 0.05 P 10 10 10 10 10 40 < 0.05 function of maternal Tregs in pregnant mice was fetal antigen- %Foxp3 (MFI)

CD80 + 30 specific, and depletion of Tregs led to a reduction in body weight of the surviving embryos (6, 45). In our histological analysis of −/− 20 surviving fetuses from Cd38 matings, although there was /CD11c + 10 6 some variability of individual fetuses, hemorrhage and immune = 7 = n n 0 7 n= CD80 cell infiltration could be observed within the decidua. In some Control cases, spiral arteries clustering in decidua were also observed Stress + Events (Fig. S8A). Moreover, the number of Foxp3 cells within all Stress + sCD38 −/− 100 101 102 103 104 layers of decidua from Cd38 matings was reduced compared Isotype Control with that from wild-type matings (1.57 ± 0.64 vs. 3.36 ± 1.2) (Fig. MEDICAL SCIENCES Stress Stress + sCD38 S8B). Consistent with histological evaluations, the mean weight −/− Fig. 5. Direct injection of sCD38 into the vagina protects fetuses from of fetuses was reduced more in Cd38 matings than in wild- −/− abortion. sCD38 or PBS was injected to the pregnant CBA/J females mated type matings (Fig. S8C). These observations indicate that Cd38 with DBA/2J males intravaginally at 1.5 and 3.5 dpc. Sonic stress was applied matings may show other complications of pregnancy, including low- from 2.5 dpc until sacrifice. (A) Resorption rates and (B) total number of birth-weight babies, besides fetal loss. implantation sites were measured at 12.5 dpc in the mated female mice Seminal plasma interacts not only with epithelial cells of the exposed to sound stress with or without sCD38. Bars, mean ± SEM. (C) + vagina and cervix but also with immune cells such as DCs and Phenotypic analysis of CD11c uterine DCs from nonstressed mating mice injected with PBS (Control), stressed mating mice injected with PBS (Stress), causes the recruitment of DCs and macrophages into the en- and stressed mating mice injected with sCD38 (Stress + sCD38) at 6.5 dpc. dometrium (46, 47). The seminal antigen is processed and dis- Mean fluorescent intensities (MFIs) of MHC-II and CD80 were plotted as bar played on class II MHC cells and transported to draining lymph + + + graphs (Right panels). Bars, mean ± SD. (D) Percentages of CD4 Foxp3 T nodes, activating CD4 T cells and promoting the acquisition of cells in the PALNs of Control, Stress, and Stress + sCD38 were calculated by a regulatory phenotype. Although the present study shows that flow cytometry analysis at 6.5 dpc. allogeneic mating with sCD38-deficient mice causes maturation of uterine DCs and results in fetal resorption, this may be due to + multiple mechanisms exerted by sCD38, for instance, on other tDCs, which in turn promoted the generation of Foxp3 Tregs. A immune cells such as macrophages. Therefore, understanding deficiency of sCD38 in semen led to the immunogenic matura- + the mechanisms by which sCD38 affects immune responses is of tion of uterine DCs and the diminished proportion of Foxp3 utmost importance. Tregs, resulting in fetal loss (Fig. 2). In addition, the adminis- Clement et al. (31) recently showed that CD31 is a coinhibi- tration of sCD38 restored tolerance through the expansion of + tory receptor in the development of tDCs, thus supporting their Foxp3 Tregs and prevented fetal loss. previous finding that the lack of T-cell CD31 signaling increases Regarding fetomaternal tolerance induced by sCD38-medi- – + autoimmune responses (21 23). The disruption of CD31 sig- ated Foxp3 Treg expansion, it is likely to be developed via naling favored immunogenic maturation. Also, ligation of CD31 numerous immunomodulatory mechanisms. The expression of using a homotypic CD31 peptide reduced the expression of MHC-II and costimulatory molecules, which are essential for costimulatory molecules and proinflammatory cytokines and in- tolerogenic ability of DC, was low in DCsCD38. In addition, creased the expression of anti-inflammatory cytokines (31). The DCsCD38 secrete IL-10, which inhibits DC maturation and + role that CD31 plays in inhibitory signaling in effector-adaptive reduces their capacity to stimulate CD4 T-cell–mediated im- immune cells was explained through the action of Src homology munity. DCsCD38 produce TGF-β, which induces the differentiation + 2 domain tyrosine phosphatases SHP-1/SHP-2, which are re- of naïve T cells to Foxp3 Tregs; DCsCD38 inhibit the production of cruited by its cytoplasmic immunoreceptor tyrosine-based in- TH1 cytokines, such as IL-12p70 and TNF-α (Fig. 4). + + hibitory motif (19). The CD31 peptide can also elicit the sig- Although the proportion of Foxp3 Tregs in the CD4 T cell naling pathway (48). Contrary to this report, our data showed −/− −/− was reduced in Cd38 matings (Fig. 2 E and F), the number of that the extent of maturation of Cd31 and wild-type BM-DCs + Foxp3 Tregs was not reduced (Fig. S2). The explanation for this upon stimulation with LPS was similar (Fig. 4A). Moreover, an −/− finding would be that increased mature DCs in Cd38 matings inhibitory signaling on DC maturation by a homotypic CD31 pep- −/− (Fig. 2D) induce naïve T cells to produce IL-2, a paracrine Tregs tide was observed in Cd31 BM-DCs (Fig. S9). These results growth factor. Thus, tDCsCD38 may positively regulate the Treg- suggest that the action of sCD38 in driving the differentiation of effector T-cell balance in favor of Tregs through inhibition tDCs is through an as-yet-unidentified target molecule(s) other + of T-cell–mediated immunity and induction of Foxp3 Treg than CD31.

Kim et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 sCD38 was first found in the amniotic fluid, suggesting that activity assay, CD38 affinity chromatography, and ELISA. Pregnancy in females − − sCD38 levels likely increase under certain conditions (49). mated with wild-type or Cd38 / male mice was determined by checking the sCD38 is present at extraordinarily high concentrations in sem- copulation plug (at 0.5 dpc). Embryo tissue sections from pregnant mice were + inal plasma, but its levels were below the detection limit of our stained with hematoxylin and eosin. Uterine DCs and Foxp3 Tregs from preg- ELISA (250 pg/mL) in normal serum. The immunoregulatory nant mice were analyzed at 3.5 or 6.5 dpc. To assess whether sCD38 induced tDC, role of sCD38 may provide crucial insights into how insemination BM cells were cultured in differentiation medium in the presence or absence of regulates maternal immunity to allow successful pregnancies. sCD38. A mouse model of stress-induced fetal abortion was used to evaluate the Furthermore, this knowledge of the immunoregulatory role of protective effects of sCD38. Details on materials, assays, and experimental pro- sCD38 is important for understanding the function of sCD38 tocol are available in SI Materials and Methods. under pathogenic conditions. Our finding that sCD38 is an im- All data were analyzed using the Student’s t test or ANOVA as appro- portant immune regulator for stimulating Treg cells may inform priate. A P value < 0.05 was considered statistically significant. studies to develop novel treatments for recurrent miscarriage with an immune etiology. ACKNOWLEDGMENTS. We thank Dr. Young June Kim, Dr. Donghee Kim, and Mr. Chansu Park for critical reading of the manuscript. This study was Materials and Methods supported by National Research Foundation Grant 2012R1A3A2026453, funded by the Korean government (Ministry of Science, ICT & Future Plan- The Institutional Review Board approved the collection of semen from normal ning) (to U.-H.K.), and a BK21 grant recipient at Chonbuk National University and vasectomised volunteers. sCD38 was detected by Western blotting, an in-gel (to B.-J.K.)

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