{"paper_id":"64fab125-9e92-40a5-958f-54680b3914fb","body_text":"1\nVol.:(0123456789)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports\nSteroid hormone bioavailability \nis controlled by the lymphatic \nsystem\nRahel Klossner1,2,3, Michael Groessl1,3, Nadine Schumacher1, Michaela Fux4, \nGeneviève Escher1,3, Sophia Verouti1,3, Heidi Jamin1,3, Bruno Vogt1,3, \nMarkus G. Mohaupt2,5,6 & Carine Gennari‑Moser1,3*\nThe steroid hormone progesterone accounts for immune tolerance in pregnancy. Enhanced \nprogesterone metabolism to 6α‑OH‑pregnanolone occurs in complicated pregnancies such as in \npreeclampsia with preterm delivery or intrauterine growth restriction, and in cancer. As lymphatic \nendothelial cells (LECs) promote tumor immunity, we hypothesized that human LECs modify \nprogesterone bioavailability. Primary human LECs and mice lymph nodes were incubated with \nprogesterone and progesterone metabolism was analyzed by thin layer chromatography and liquid \nchromatography‑mass spectrometry. Expression of steroidogenic enzymes, down‑stream signal \nand steroid hormone receptors was assessed by Real‑time PCR. The placental cell line HTR‑8/SV neo \nwas used as reference. The impact of the progesterone metabolites of interest was investigated on \nthe immune system by fluorescence‑activated cell sorting analysis. LECs metabolize progesterone \nto 6α‑OH‑pregnanolone and reactivate progesterone from a precursor. LECs highly express \n17β‑hydroxysteroid dehydrogenase 2 and are therefore antiandrogenic and antiestrogenic. LECs \nexpress several steroid hormone receptors and PIBF1. Progesterone and its metabolites reduced \nTNF‑α and IFN‑γ production in CD4+ and CD8+ T cells. LECs modify progesterone bioavailability and \nare a target of steroid hormones. Given the global area represented by LECs, they might have a critical \nimmunomodulatory control in pregnancy and cancer.\nThe lymphatic system, especially lymphatic endothelial cells (LECs) are thought to play a crucial role in tumor \nimmunity. For example, tumorigenic cells travelling inside lymphatic vessels towards sentinel lymph nodes are \nnot being recognized as foreign and an appropriate immune response is missing, leading to tumor  survival1. \nAdditionally, the expression and release of the lymphangiogenic factor VEGF-C by tumors is linked to metasta-\nsis, poor prognosis and immune  tolerance2. LECs are, like dendritic cells, antigen-presenting cells that mediate \nsystemic peripheral immune  tolerance3,4. Upon presentation of self-antigens by LECs, T-cells undergo apoptosis \nand LECs therefore contribute to immune tolerance. LECs also promote immune tolerance when tumorigenic \nantigens are presented to T-cells. T-cells, which were activated by LECs, become more rapidly apoptotic as T-cells \nactivated by mature dendritic  cells5. Though preventing autoimmune reactions and promoting immune tolerance \nduring pregnancy, this behavior may favor tumor development. LECs in the local microenvironment of a tumor \nmight therefore be a target for  immunomodulation5.\nDuring pregnancy, immune tolerance is required and linked to high progesterone  levels6,7. This immunomod-\nulatory effect of progesterone is mediated via upregulation of the lymphocyte-derived progesterone-induced \nblocking factor (PIBF)8–13. Thus, in pregnant  women14 and cancer  patients15, there is an increased progesterone \nmetabolism.\nThe first step in progesterone metabolism leads to the formation of active 5α-dihydroprogesterone (5α-\nDHP), which is further metabolized into the 6α-OH-pregnanolones (5α-pregnan-3α/β,6α-diol-20-one) and \nallopregnane-3,20-diol (5α-pregnan-3β,20α-diol). The metabolism of 5α-DHP is extrahepatic, as the final step \nof 6α-OH-pregnanolones and allopregnane-3,20-diol formation is not catalyzed by the cytochrome P450 steroid \n6α-hydroxylase expressed in the  liver16. Horwitz et al. proposed an intracellular metabolism of progesterone \nOPEN\n1Department of Nephrology and Hypertension, University of Bern, 3010 Bern, Switzerland. 2Department of \nMedicine, Lindenhofgruppe, 3006 Bern, Switzerland. 3Department for BioMedical Research, University of Bern, \n3010 Bern, Switzerland. 4Department for Clinical Chemistry, Inselspital, 3010 Bern, Switzerland. 5Campus SLB, \nSitem, 3010 Bern, Switzerland. 6Division of Child Health, Obstetrics and Gynecology, University of Nottingham, \nNottingham NG5 1PB, UK. *email: carine.gennari@dbmr.unibe.ch\n\n2\nVol:.(1234567890)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports/\ninto the 6α-OH-pregnanolones involving 3 different enzymes: 5α-reductase (SRD5A1-3), 3β-hydroxysteroid \ndehydrogenases (HSD3B1/2), and a 6α-hydroxylase17.\nSeveral reports have demonstrated that the 5α- and 5β-DHP metabolites are biologically active in \n pregnancy18–22, but the mechanism leading to these metabolites remains to be elucidated. Allopregnanolone, \nthe metabolite down-stream of 5α-DHP , stimulates proliferation and migration in the ovarian cancer cell line \nIGROV-115 and in  glioblastoma23.\nSeveral enzymes accounting for steroid hormone metabolism, specifically progesterone metabolism, have \nbeen involved in cancer. These are the 6α-hydroxylase, the 5α-reductase, the 3β-hydroxysteroid dehydrogenase \nand 17β-hydroxysteroid dehydrogenase 2 (HSD17B2)24–29. The progesterone metabolite 5α-DHP produced by the \nenzyme 5α-reductase, is a target for prostate cancer and glioblastoma  therapy30–33. Interestingly, HSD17B2 also \nexerts a high 20α-hydroxysteroid dehydrogenase  activity34 and can convert the weak 20α-hydroxyprogesterone \n(20α-OHP) into  progesterone35–38. Many compounds targeting HSD17B2 activity in various cancers are under \n investigation39.\nThe hypothesis of this investigation is that human LECs modify the bioavailability of progesterone. To prove \nthis, first, progesterone metabolism was assessed in primary human LECs isolated from lymph nodes (HLEC) \nand results were confirmed in LECs isolated from lymphatic skin vessels (dLEC); second, the resulting proges-\nterone metabolite was identified and characterized by LC–MS; third, the enzymatic steps involved were clarified; \nfourth, human cell culture results were matched to ex-vivo mouse lymph node; fifth, the impact of the found \nprogesterone metabolites 5α-dihydroprogesterone (5α-DHP) and 6α-OH-pregnanolone (6α-OH-Pregn) was \ntested on immune cells. The placental cell line HTR-8/SVneo was used as a control and reference for the experi-\nments with lymphatic endothelial cells.\nResults\nProduction of de‑novo steroid hormones from cholesterol in HLEC and dLEC. HLECs and \ndLECs were cultured in steroid-free medium and incubated with 3H-cholesterol to quantify the de-novo synthe-\nsis of progesterone, corticosterone, cortisol, testosterone and estradiol (HLEC), and of progesterone, 11-deoxy-\ncortisol, corticosterone, aldosterone and cortisol (dLEC), after an incubation time of 24 h. No significant de-novo \nsteroid hormone production could be detected out of the precursor cholesterol by thin layer chromatography \n(TLC) in HLEC (Supplementary Fig. 1a online) and dLEC (Supplementary Fig. 1b online).\nCharacterisation of progesterone metabolism in HLEC, dLEC and HTR‑8/SVneo by TLC. HLECs, \ndLECs and HTR-8/SVneo cells were cultured in steroid-free medium and incubated with 14C-progesterone for \n4 h/8 h/24 h/48 h (HLEC/dLEC) or 1 h/4 h/8 h/24 h (HTR-8/SVneo). Steroids were extracted from superna-\ntants and the metabolites separated by TLC. Progesterone was time-dependently and significantly converted to \none major steroid hormone, 6α-OH-pregnanolone, in all three cell lines with a production rate of 19.3% ± 3.6 \n(24 h) and 31.7% ± 6.3 (48 h) in HLEC, 13.6% ± 2.5 (24 h) and 23.9% ± 4.2 (48 h) in dLEC and 21.0% ± 11.5 (8 h) \nand 42.7% ± 17.1 (24  h) in HTR-8/SV neo (Fig.  1). One-way ANOV A, Dunnett’s multiple comparisons test. \nMean ± SD, n = 5.\nCharacterization of 6α‑OH‑pregnanolone in HLEC, dLEC and HTR‑8/SVneo by LC–MS. In \norder to validate the results obtained by TLC, steroidomic analysis of cell supernatants was performed using \nLC–MS. Samples were measured in untargeted mode and investigated for peaks that showed strongest signal \nincrease over the time course experiment. Subsequently, the accurate mass of peaks of interest was compared \nwith the theoretical value for 6α-OH-pregnanolone and both, fragmentation spectrum and retention time, were \ncompared to an authentic standard (Figs. 2 and 3). The LC–MS data clearly confirms the production of 6α-OH-\npregnanolone from progesterone as obtained by TLC.\nGene expression analysis in HLEC, dLEC and HTR‑8/SVneo. HLECs, dLECs and HTR-8/SV neo \ncells were cultured as described in methods. RNA extraction and Real-time PCR was conducted using assay on \ndemand primers or primers and probes from the Roche library (Tables  1, 2). Cyclophilin A served as endog-\nenous control. \nThe mRNA expression of enzymes involved in progesterone metabolism (Table 3) was quantified first. HLEC \nand dLEC express high levels of SRD5A1, SRD5A3, AKR1C1, AKR1C2, AKR1C3, but they do not express \nSRD5A2, AKR1D1, and AKR1C4. No expression of HSD3B1/2 was found (Ct > 35). HTR-8/SV neo express high \nlevels of SRD5A1, SRD5A3, AKR1C2 and AKR1C3, but rather low levels of SRD5A2 and AKR1C1. AKR1D1 \nand AKR1C4 as well as HSD3B1/2 are not expressed in HTR-8/SV neo (Ct > 35).\nNext, the mRNA expression of the progesterone regulated gene (PIBF1) and of steroid hormone receptors \nin HLEC, dLEC and HTR-8/SV neo was assessed (Table  4). All three cell lines significantly express PIBF1 as \nwell as the glucocorticoid (NR3C1) and the mineralocorticoid receptor (NR3C2), the two membrane bound \nprogesterone receptors type 1 and 2 (PGRMC1 and 2) and the estrogen receptor (GPER1). They show no or very \nlow expression of the nuclear progesterone receptor (NR3C3).\nSteroidogenic enzymes involved in de-novo steroid hormone production were assessed thereafter \n(Table 5). HLEC and dLEC express no CYP17A1, CYP21A2, CYP11B1, CYP11B2, HSD11B1 and no HSD11B2, \nbut they highly express HSD17B2. HTR-8/SV neo express no CYP17A1, CYP21A2, CYP11B1, CYP11B2, and \nno HSD11B1 but they express HSD11B2 and HSD17B2.\nAssessment of the mRNA expression of steroidogenic proteins involved in de-novo steroid hormone produc-\ntion (Table 6) revealed no significant expression of StAR in HLEC and dLEC, while FDXR, FDX1 and NR5A2 \nwere strongly expressed. HTR-8/SV neo significantly expressed StAR, FDXR, FDX1 and NR5A2.\n\n3\nVol.:(0123456789)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports/\nDerived cascade of progesterone metabolism in HLEC, dLEC and HTR‑8/SV neo. Based on our \nfindings by Real-time PCR, LC–MS and Thin layer chromatography, progesterone is metabolized via 5α-DHP \nand allopregnanolone to 6α-OH-pregnanolone in HLECs, dLECs and HTR-8/SVneo (Fig.  4). Furthermore, \nHLEC and dLEC efficiently back-convert 20α-hydroxyprogesterone to progesterone due to high HSD17B2 \nexpression.\nInhibition of 6α‑OH‑pregnanolone formation in HLEC, dLEC and HTR‑8/SV neo using the \nSRD5A1‑3 inhibitor dutasteride. HLECs, dLECs and HTR-8/SV neo cells were cultured and incubated \nfor 24 h with the 5α-reductase inhibitor dutasteride at 2 different concentrations,  10−5  M and  10−6  M for HLEC \nand HTR-8/SV neo and  10−6   M and  10−8   M for dLEC. Supernatants were analyzed by TLC to measure the \nconversion of 14C-progesterone to 6α-OH-pregnanolone (Fig.  5). Dutasteride significantly inhibited 6α-OH-\npregnanolone formation at both concentrations in all three cell lines. In untreated HLECs (DMSO) 39.2% ± 6.3% \n6α-OH-pregnanolone was produced from progesterone (Fig. 5a). This production was reduced to 6.3% ± 5.8%, \nrespectively 6.2% ± 4.7% with dutasteride 10−5 M and 10−6 M. A similar inhibitory effect by dutasteride was \nobserved in dLECs (Fig.  5b) and HTR-8/SV neo cells (Fig.  5c). In dLECs, production of 6α-OH-pregnanolone \nwent from 14.5% ± 0.9% to 4.1% ± 0.6% with dutasteride 10−6 M and to 5.7% ± 0.2% with dutasteride 10−8 M. \nSimilarly, HTR-8/SV neo cells produced 32.1% ± 9.1% 6α-OH-pregnanolone (DMSO), and this was reduced \nto 3.0% ± 2.7% with dutasteride 10−5 M and to 3.7% ± 2.9% with dutasteride 10−6 M. The involvement of the \nSRD5A1-3 for the production of 6α-OH-pregnanolone was confirmed by enzymatic activity and RT-PCR. One-\nway ANOV A, Dunnett’s multiple comparisons test. Mean ± SD, n = 3.\nFigure 1.  Characterization of progesterone metabolism in HLEC, dLEC and HTR8/SV neo by TLC. \nMetabolism of progesterone in HLEC, dLEC and HTR-8/SV neo. (a) Characteristic phosphorimager pictures \nof time-dependent conversion of progesterone to a main metabolite, 6α-OH-pregnanolone. Cell-free controls \nwere run for time point 0 h (HLEC, dLEC and HTR-8/SV neo), for time point 24 h (HTR-8/SVneo) and for time \npoint 48 h (HLEC and dLEC). (b) Densitometric quantification of all performed experiments. Progesterone \nwas time-dependently and significantly converted to 6α-OH-pregnanolone in all three cell lines. Data were \nnormalized to condition 0 h = 100% progesterone. One-way ANOV A, Dunnett’s multiple comparisons test, \nn = 5. Production of 6α-OH-pregnanolone: HLEC: 8 h-24 h ***p < 0.0001, 24 h-48 h **p = 0.0002. dLEC: 8 h-24 h \n** p = 0.0026, 24 h-48 h ** p = 0.0020. HTR-8/SV neo: 1 h-24 h ** p = 0.0002. Green rectangle/black column: \nsubstrate (progesterone). Red rectangle/white column: product (6α-OH-pregnanolone). * p < 0.05, ** p < 0.01, \n*** p < 0.0001, ns = not significant.\n\n4\nVol:.(1234567890)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports/\nFigure 2.  Identification of 6α-OH-pregnanolone by LC–MS/MS in HTR-8/SV neo cells: LC–MS trace of m/z \n317.2469 corresponding to a sum formula of  C21H34O3 in an authentic standard (a) and in HTR-8/SV neo cells \n(b). MS/MS fragment spectra recorded at LC–MS peak maximum for an authentic standard (c) and in HTR-8/\nSVneo cells (d).\nFigure 3.  Identification of 6α-OH-pregnanolone by LC–MS/MS in HLEC and dLEC cells. LC–MS trace of m/z \n317.2469 corresponding to a sum formula of  C21H34O3 in an authentic standard (a), in HLEC cells (b) and in \ndLEC cells (c). MS/MS fragment spectra recorded at LC–MS peak maximum in an authentic standard (d), in \nHLEC cells (e) and in dLEC cells (f).\n\n5\nVol.:(0123456789)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports/\nDetection of HSD17B2 protein and its activity in HLEC, dLEC and HTR‑8/SV neo. Proteomics \nanalysis revealed a strong expression of HSD17B2 protein in HLEC and dLEC. HTR-8/SV neo also expressed \nHSD17B2, but much weaker than the LECs. HSD17B2 activity was next quantified by LC–MS by measuring the \nconversion of testosterone to androstenedione, of 17β-estradiol to estrone, of androstenediol to dehydroepian-\ndrosterone (DHEA) and DHEA-S and of 20α-OHP to progesterone. There was a significant HSD17B2 activity, \nboth in HLEC (Fig.  6) and dLEC (data not shown). LECs, completely converted testosterone, 17β-estradiol, \nandrostenediol and 20α-OHP to androstenedione, estrone, DHEA/DHEA-S and progesterone within 24  h.   \nTable 1.  Human primers and probes used in Real-time PCR.\nGene Accession number Primers Probe Amplicon size (nt)\nSRD5A1 NM_001324322.1\n5′-ttg gag aaa tca tgg agt ggt-3’\n37 141\n5′-act ctt caa att tcc gga ggt a-3’\nSRD5A2 NM_000348.3\n5′-cag cta cag gat tcc aca agg-3’\n50 72\n5′-tca atg atc tca ccg agg aa-3’\nSRD5A3 NM_024592.4\n5′-ggc ttc atg gtt tgc tca g-3’\n24 96\n5′-gca gcc aca gaa ata cta gca c-3’\nAKR1D1 NM_001190906.1\n5′-cca aaa tga aca cga agt tgg-3’\n7 108\n5′-aca tga ttt gta gcc cat agc tt-3’\nAKR1C1 NM_001353.5\n5′-cat gcc tgt cct ggg att t-3’\n49 109\n5′-aga atc aat atg gcg gaa gc-3’\nAKR1C2 NM_001354.5\n5′-cta tgc gcc tgc aga ggt-3’\n31 114\n5′-acc tgc tcc tca tta ttg taa aca t-3’\nAKR1C3 NM_003739.5\n5′-cat tgg ggt gtc aaa ctt ca-3’\n27 112\n5′-ccg gtt gaa ata cgg atg ac-3’\nAKR1C4 NM_001818.3\n5′-agg tga gac gcc act acc aa-3’\n53 96\n5′-tcc tta cac ttc tcc atg acc tc-3’\nHSD3B1 NM_000862.2\n5′-atc atc cgc ctc ttg gtg-3’\n17 114\n5′-cag ctt ggt ctt gtt ctg ga-3’\nHSD3B2 NM_000198.3\n5′-ctt gga caa ggc ctt cag ac-3’\n50 78\n5′-tca agt aca gtc agc ttg gtc ct-3’\nHSD17B2 NM_002153.2\n5′-gcc aag aat tgt tac ctg tgg-3’\n8 88\n5′-tcc aga tac ttg cac aaa gca-3’\nHSD11B1 NM_001206741.1\n5′-tct gtg ttc ttg gcc tca tag a-3’\n8 75\n5′-gag ctg ctt gca tat gga cta tc-3’\nFDXR NM_001258015.2\n5′-tcc tac tga ccc cac ctg ag-3’\n8 77\n5′-tcg ac tct gcc tca gta cac c-3’\nNR5A2 NM_205860.2\n5′-ccg aca agt ggt aca tgg aa-3’\n61 88\n5′-tcc ggc ttg tga tgc tat ta-3’\nTable 2.  Human assay on demand primers (Applied Biosystems) used in Real-time PCR.\nGene # Cat Nr\nPIBF1 Hs00197131_m1\nNR3C1 Hs00353740_m1\nNR3C2 Hs01031809_m1\nNR3C3 Hs01556702_m1\nPGRMC1 Hs009998344_m1\nPGRMC2 Hs01128672_m1\nGPER1 Hs00173506_m1\nCYP17A1 Hs01124136_m1\nCYP21A2 Hs00416901_g1\nCYP11B1 Hs01596404_m1\nCYP11B2 Hs01597732_m1\nHSD11B2 Hs00388669_m1\nStAR Hs00986559_g1\nFDX1 Hs01070067_g1\n\n6\nVol:.(1234567890)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports/\nTable 3.  Gene expression analysis in HLEC, dLEC and HTR-8/SVneo. mRNA expression of enzymes involved \nin progesterone metabolism in HLEC, dLEC and HTR-8/SV neo. Expression levels are shown as ct values \nper 12.5 ng cDNA. Y ellow: significant expression (ct < 35). Blue: no or very low expression (ct ≥ 35). SRD5A1 \n(5α-reductase isoform 1), SRD5A2 (5α-reductase isoform 2), SRD5A3 (5α-reductase isoform 3), AKR1D1 \n(5β-reductase), AKR1C1 (20α-HSD), AKR1C2 (3α-HSD3), AKR1C3 (HSD17B5), AKR1C4 (3α-HSD1), \nHSD3B1, HSD3B2. Appropriate positive and negative controls were run.\n SRD5A1 SRD5A2 SRD5A3 AKR1D1 AKR1C1 AKR1C2 AKR1C3 AKR1C4 HSD3B1 HSD3B2\nHLEC 28 38 26 > 40 31 26 25 > 40 > 40 > 40 \ndLEC 28 38 26 > 40 31 29 25 > 40 > 40 > 40 \nHTR-8/ \nSV neo 26 35 27 > 40 35 33 30 > 40 35 34 \nTable 4.  Gene expression analysis in HLEC, dLEC and HTR-8/SVneo. mRNA expression of PIBF1, and \nof steroid hormone receptors in HLEC, dLEC and HTR-8/SV neo. Expression levels are shown as ct values \nper 12.5 ng cDNA. Y ellow: significant expression (ct < 35). Blue: no or very low expression (ct ≥ 35). PIBF1 \n(progesterone induced blocking factor 1), NR3C1 (glucocorticoid receptor), NR3C2 (mineralocorticoid \nreceptor), NR3C3 (progesterone receptor), PGRMC1 (progesterone receptor membrane component 1), \nPGRMC2 (progesterone receptor membrane component 2), GPER1 (G protein-coupled estrogen receptor 1). \nAppropriate positive and negative controls were run.\n PIBF1 NR3C1 \n(GR) \nNR3C2 \n(MR) \nNR3C3 \n(PR) PGRMC1 PGRMC2 GPER1 \nHLEC 26 26 28 35 27 25 31 \ndLEC 26 26 30 > 40 27 26 34 \nHTR-8/ \nSV neo 25 24 33 36 26 24 32 \nTable 5.  Gene expression analysis in HLEC, dLEC and HTR-8/SVneo. mRNA expression of steroidogenic \nenzymes involved in de-novo steroid hormone production in HLEC, dLEC and HTR-8/SV neo. Expression \nlevels are shown as ct values per 12.5 ng cDNA. Y ellow: significant expression (ct < 35). Blue: no or very low \nexpression (ct ≥ 35). CYP17A1 (steroid-17α-hydroxylase), CYP21A2 (steroid-21-hydroxylase), CYP11B1 \n(steroid-11β-hydroxylase), CYP11B2 (aldosterone synthase), HSD11B1 (11β-hydroxysteroid-dehydrogenase \n1), HSD11B2 (11β-hydroxysteroid-dehydrogenase 2), HSD17B2 (17β-hydroxysteroid dehydrogenase 2). \nAppropriate positive and negative controls were run.\n CYP17A1 CYP21A2 CYP11B1 CYP11B2 HSD11B1 HSD11B2 HSD17B2 \nHLEC > 40  37 > 40 > 40 36 35 23 \ndLEC > 40 38 > 40 > 40 > 40 36 23 \nHTR-8/ \nSV neo 38 35 > 40 > 40 38 31 32 \n\n7\nVol.:(0123456789)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports/\nIn HLECs, testosterone (0 h: 1000 ± 0; 4 h: 318.7 ± 160.6, 24 h: 10.3 ± 14.9) was time-dependently converted to \nandrostenedione (0 h: 1.4 ± 1.0; 4 h: 776.6 ± 148.1; 24 h: 899.9 ± 114.4) (Fig. 6a); 17β-estradiol (0 h: 1000 ± 0; 4 h: \n205.1 ± 159.7; 24 h: 7.7 ± 15.3) to estrone (0 h: 6.5 ± 4.4; 4 h: 659.4 ± 263.9; 24 h: 955.5 ± 62.0) (Fig. 6b); Andros-\ntenediol (0 h: 1000 ± 0; 4 h: 816.5 ± 217.2; 24 h: 14.3 ± 6.5) to DHEA (0 h: 2.2 ± 2.3; 4 h: 375.0 ± 277.6; 24 h: \n946.3 ± 55.8) (Fig. 6c) and 20α-OHP (0 h: 1000 ± 0; 4 h: 618.9 ± 116.6; 24 h: 101.9 ± 31.9) to progesterone (0 h: \n39.0 ± 51.2; 4 h: 542.3 ± 166.6; 24 h: 929.6 ± 104.6) (Fig. 6d). One-way ANOV A, Dunnett’s multiple comparisons \ntest. Mean ± SD, n = 3.\nProgesterone metabolism in mouse lymph nodes. Mouse lymph nodes, isolated from male mice \n(n = 7), were incubated for 24  h and 48  h with 14C-progesterone. Progesterone metabolism was analysed in \nsupernatants as described. Adrenal gland was used as positive control. Progesterone was converted into 7 differ-\nent metabolites in all lymph nodes. The progesterone metabolism pattern was different as compared to the one \nfound in the adrenal gland, confirming progesterone metabolism and not de-novo steroid hormone production \n(Fig. 7).\nTable 6.  Gene expression analysis in HLEC, dLEC and HTR-8/SVneo. mRNA expression of steroidogenic \nproteins involved in de-novo steroid hormone production in HLEC, dLEC and HTR-8/SV neo. Expression levels \nare shown as ct values per 12.5 ng cDNA. Y ellow: significant expression (ct < 35). Blue: no or very low expression \n(ct ≥ 35). CYP17A1 (steroid-17α-hydroxylase), CYP21A2 (steroid-21-hydroxylase), CYP11B1 (steroid-11β-\nhydroxylase), CYP11B2 (aldosterone synthase), HSD11B1 (11β-hydroxysteroid-dehydrogenase 1), HSD11B2 \n(11β-hydroxysteroid-dehydrogenase 2), HSD17B2 (17β-hydroxysteroid dehydrogenase 2). Appropriate positive \nand negative controls were run.\n StAR FDXR FDX1 NR5A2 \n(LRH-1) \nHLEC 36 27 23 25 \ndLEC 37 28 23 25 \nHTR-8/ \nSV neo 33 26 24 30 \nFigure 4.  Putative progesterone metabolism pathway in HLECs, dLECs and HTR-8/SV neo. Based on \nour mRNA, proteomics, TLC and LC–MS data, we propose the following pathway in green to take place. \nThe pathway with the bulky arrows down to 6α-OH-pregnanolone is favored over the pathway to the \n20α-hydroxyprogesterone in HLECs, dLECs and HTR-8/SV neo. HLEC and dLEC highly express HSD17B2 and \nefficiently back-convert 20α-hydroxyprogesterone to progesterone.\n\n8\nVol:.(1234567890)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports/\nAll assessed steroid hormones, enzymes and steroidogenic factors in this manuscript are summarized in \nSupplementary Fig. 2 online in a schematic view.\nImmune response of CD4+ and CD8+ T cells upon stimulation with progesterone metabo‑\nlites. PBMCs of three different healthy female donors were isolated and pre-incubated with the steroid hor-\nmones progesterone, 5α-DHP , 6α-OH-pregnanolone, and with dexamethasone (Dexa) as positive control for 6 h \nbefore activation. Solvent control of all steroid hormones was EtOH. Activation of T cells was thereafter induced \nby PMA/Ionomycin. TNF-α and IFN-γ production was assessed 24 h later using FACS analysis. Progesterone \nand Dexa, strongly decreased TNF-α positive CD4+ (Fig. 8a) and CD8+ (Fig. 8b) T cell populations. 5α-DHP \nand 6α-OH-pregnanolone reduced the percentage of TNF-α positive CD4+ T cell subpopulations only in donor \n3, while both progesterone metabolites reduced the percentage of TNF-α positive CD8+ T cells in all three \ndonors (Fig. 8). Dexa strongly decreased IFN-γ positive CD4+ (Fig. 9a) and CD8+ (Fig. 9b) T cell populations. \nProgesterone reduced % of IFN-γ positive CD8+ T cells in all three donors, while it inhibited IFN-γ positive \nCD4+ T cells only in donor 1. The progesterone metabolites 5α-DHP and 6α-OH-pregnanolone showed a strong \neffect in CD8+ T cells of donor 3, where both steroids decreased IFN-γ positive CD8+ T cells by 20% (Fig. 9).\nFigure 5.  Inhibitory effect of dutasteride on 5α-reductase (SRD5A1-3) in HLECs, dLECs and HTR-8/SV neo. \nCharacteristic phorphorimager pictures of (a) HLEC, (b) dLEC, (c) HTR-8/SV neo and their densitometry (d) \nof three independent experiments. Cells were cultured without (lane 1) and with (lane 2 and 3) the indicated \nconcentration of dutasteride and 14C-progesterone. Lane 4 is the cell-free control, where 14C-progesterone was \nincubated for 24 h without cells. HLEC and HTR-8/SV neo: (1) no dutasteride = DMSO control, (2) dutasteride \n10−5 M, (3) dutasteride 10−6 M, (4) no cells; dLEC: (1) no dutasteride, (2) dutasteride 10−6 M, (3) dutasteride \n10−8 M, (4) no cells. Progesterone was significantly metabolized to 6α-OH-pregnanolone in the DMSO control, \nwhile dutasteride significantly inhibited the conversion of progesterone to 6α-OH-pregnanolone in all three \ncell lines. Data were normalized to condition 0 h = 100% progesterone. One-way ANOV A, Dunnett’s multiple \ncomparisons test, n = 3. HLEC: DMSO control *** p < 0.0001; dutasteride 10−5 M *** p < 0.0001; dutasteride \n10−6 M *** p < 0.0001. dLEC: DMSO control *** p < 0.0001; dutasteride 10−6 M ** p = 0.0001; dutasteride 10−8 \nM ** p = 0.0006. HTR-8/SV neo: DMSO control *** p < 0.0001; dutasteride 10−5 M ** p = 0.0005; dutasteride \n10−6 M ** p = 0.0006. Green rectangle/black column: substrate (progesterone). Red rectangle/white column: \nproduct (6α-OH-pregnanolone). * p < 0.05, ** p < 0.01, *** p < 0.0001, ns = not significant.\n\n9\nVol.:(0123456789)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports/\nDiscussion\nTo our knowledge, this is the first report showing detailed steroid hormone metabolism in the lymphatic sys-\ntem. The lymphatic endothelium could be identified as an important regulator of steroid hormone bioavail-\nability, yet also as a target of steroid hormones by expressing the respective steroid hormone receptors. De-novo \nsteroid hormone synthesis from cholesterol is absent in lymphatic tissue, which is consistent with the lack of \nsteroidogenic acute regulatory protein (StAR) in LECs. However, FDXR, FDX1 and LRH-1, genes involved \nin  steroidogenesis40–42, are expressed in LECs. Progesterone, a steroid hormone critical in regulating immune \nresponses and pregnancy, is intensely metabolized. In primary human LECs derived either from lymph nodes \nor from dermal lymphatic vessels, progesterone is mainly converted to a single metabolite, which was identified \nas 6α-OH-pregnanolone (5α-pregnan-3α, 6α-diol-20-one). Enzymatic steps involved are the two isoforms of \nthe 5α-reductase, SRD5A1 and SRD5A3, the isoform 1 of 3α-HSD (AKR1C1) and a 6α-hydroxylase. Besides \nconverting progesterone to the down-stream metabolites 5α-dihydroprogesterone (5α-DHP), allopregnanolone \nand 6α-OH-pregnanolone, HLECs and dLECs were able to reactivate progesterone from the less potent metabo-\nlite 20α-OHP . Further steroidogenic activity of HLEC and dLEC included deactivation of testosterone and \n17β-estradiol to androstenedione and estrone as well as androstenediol to DHEA and DHEA-S.\nThese findings are pivotal in understanding the progesterone-dependent immunomodulation observed earlier \nby various  groups7,10,11,43. The data show that progesterone is not only metabolized to the primary and active \nmetabolite 5α-DHP which plays a critical role in  pregnancy44, but is also restored from 20α-OHP .\nFigure 6.  Activity of HSD17B2 in HLECs. Time-dependent conversion of testosterone to androstenedione, \n17β-estradiol to estrone, androstenediol to DHEA-S and DHEA as well as 20α-hydroxyprogesterone to \nprogesterone was assessed by LC–MS. Data were normalized to condition at 0 h = 1000 nM = 10−6 M for each \ncompound. Y-axis shows steroid hormone concentrations in nM. Testosterone was significantly converted to \nandrostenedione (testosterone 0 h/4 h/24 h: *** p < 0.0001/** p = 0.0019; androstenedione 0 h/4 h/24 h: *** \np < 0.0001/ns). 17β-estradiol was significantly converted to estrone (17β-estradiol 0 h/4 h/24 h: *** p < 0.0001/\nns; estrone 0 h/4 h/24 h: *** p < 0.0001/* p = 0.01). Androstenediol was significantly converted to DHEA \n(androstenediol 0 h/4 h/24 h: ns/*** p < 0.0001; DHEA 0 h/4 h/24 h: ** p = 0.0087/** p = 0.0002). 20α-OHP was \nsignificantly converted to progesterone (20α-OHP 0 h/4 h/24 h: *** p < 0.0001/*** p < 0.0001; progesterone 0 \nh/4 h/24 h: *** p < 0.0001/*** p < 0.0001). One-way ANOV A, Dunnett’s multiple comparisons test, n = 3. Black \nrectangle/column: substrate. White rectangle/column: product. * p < 0.05, ** p < 0.01, *** p < 0.0001, ns = not \nsignificant.\n\n10\nVol:.(1234567890)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports/\nProgesterone metabolism to 6α-OH-pregnanolone and the reactivation of progesterone from 20α-OHP by \nLECs serves to adapt immune tolerance and to control local and/or systemic progesterone bioavailability in high \nprogesterone conditions as found during pregnancy. As the exact mechanism for this phenomenon is unknown \nand given the huge surface of the lymphatic endothelium throughout the body, it is supposable that local pro -\ngesterone metabolism in LECs could be the key regulator in preventing cancer cells from being destroyed.\nThe direct immunosuppressive role of progesterone in both reproduction and tumor progression was impres-\nsively shown by Szekeres-Bartho and  Polgar45. Immunosuppression by progesterone is mediated via the proges-\nterone regulated gene  PIBF18,11,13,46, which has now also been found to be highly expressed in LECs. A reduced \nexpression of PIBF in threatened  pregnancies47–51 and an increased production of PIBF in  cancer52,53 are clearly \nlinked to a failed immune modulation. Production of TNF-α and IFN-γ in activated CD4+ and CD8+ T cells \nfrom 3 different donors was modulated upon stimulation with progesterone and its metabolites. It needs to be \ninvestigated if the interaction of LECs and the immune cells is PIBF1 regulated.\nSteroid hormone effector mechanisms such as the glucocorticoid receptor (GR), the mineralocorticoid recep-\ntor (MR), the G protein-coupled estrogen receptor 1 (GPER1), and the progesterone receptor membrane com-\nponents PGRMC1 and PGRMC2 are present in HLECs and dLECs, while no expression of nuclear progesterone \nreceptors (PRA and PRB) was found.\nMost of the known anti-inflammatory effects of progesterone are transmitted through the GR, with proges-\nterone binding to the GR even though progesterone can also bind to  PGRMCs54,55. PGRMCs were overexpressed \nin the maternal–fetal interface and in the embryonic/fetal trophectoderm in  pregnancy56,57 as well as in T cells \nduring  pregnancy58,59. Since many steroid hormone receptors are expressed by immune  cells12,60–62 it is most likely \nthat the LECs not only have an autocrine, but also a paracrine role in controlling immune responses through the \naction of steroid hormones via binding to a receptor.\nThe serum concentrations of progesterone are according to the literature much too low to support the concept \nof a generalized  immunosuppression63,64 and are only sufficient to inhibit peripheral natural killer cell activity \nin normal pregnant women. The presence of lymphatic vessels throughout the body, together with the immu -\nnosuppressive role of the placenta, might be a much better explanation for the systemic immunosuppression.\nThe mononuclear phagocyte system stimulates VEGF-C release and regulates thereby  lymphangiogenesis65. \nHigh progesterone levels seem to coincide with high VEGF-C  expression66. Activated macrophages and mono-\ncytes releasing VEGF-C thus enhance LEC growth and could well contribute to immune regulatory mechanisms \nvia progesterone.\nFigure 7.  Progesterone metabolism in male mice lymph nodes and adrenal gland. Phosphorimager picture \nof TLC showing time-dependent conversion of progesterone to down-stream metabolites in lymph nodes. For \npositive control, adrenal glands are used under similar experimental conditions. Tissue-free controls were run \nfor both time points. Line 1 at timepoint 24 h and line 2 at 48 h were entire lymph nodes, while all other lymph \nnodes were cut in half. The figure shows results of 7 isolated lymph nodes. Each lymph node was from a separate \nmouse. * p < 0.05, ** p < 0.01, *** p < 0.0001, ns = not significant.\n\n11\nVol.:(0123456789)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports/\nIn 2009, Zhang et  al. found, that in isolated mature adipocytes progesterone was converted to \n20α-hydroxyprogesterone as the main metabolite, most likely through the activity of aldo–keto reductases \nAKR1C1, AKR1C2 and AKR1C3 (20α-HSD, 3α-HSD type 3 and HSD17B5),  respectively67. Even though HLECs \nand dLECs express the same enzymes, 6α-OH-pregnanolone was found instead as major metabolite of progester-\none, and not 20α-hydroxyprogesterone. That LECs favour the 5α-reductase/3α-HSD pathway over the 20α-HSD \npathway might be explained by the different tissues investigated and eventually the limitations of some cofactors.\nThe strength of this study is the thorough analysis of different steroid hormone metabolites using state-of-the-\nart technology. It shows enzyme expression on mRNA and on protein level, as well as functional assays including \ninhibitory experiments. All results are referred to our self-designed and validated model of a positive control, \nthe placental cell line HTR-8/SV neo. Since fast, significant and similar progesterone metabolism was found in \nHTR-8/SV neo cells, they were used as a model of reference for all experiments. Taking a placental cell line as \nmodel is obvious, as the placenta is the primary organ exposed to high progesterone levels. A direct immunologic \neffect of the lymphatic progesterone metabolites on CD4+ and CD8+ T cells could be shown as well.\nInvestigation of further immunomodulatory effects of the lymphatic progesterone metabolism and proges -\nterone reactivation described herein on immune and cancer cells is the goal of future studies.\nVerification of our observations in an in vivo model is difficult due to the systemic presence of steroid \nhormones, further complicated by the delicate structure of lymphatic vessels, therefore precluding differential \nconcentration measurements. But, the capacity of steroid hormone metabolism and reactivation in lymph nodes \nisolated from both sexes, including pregnancy, should be explored. Additionally, future detailed assessment \nin patients treated with the 5α-reductase inhibitors dutasteride or finasteride could be investigated with more \naccuracy.\nIn conclusion, we identified and characterized the lymphatic system as major steroid hormone metabolizing \ntissue, which is additionally able to reactivate progesterone from 20α-OHP and therefore adapts progesterone \navailability. This is important in high progesterone conditions such as in pregnancy in order to adjust local \nimmune tolerance. The impact of these findings on cancer and pregnancy research might be meaningful and \ncould provide new targets for the treatment of cancer and pregnancy related diseases. Furthermore, these results \ncould also help to elucidate disease mechanisms in autoimmunity and allergy.\nFigure 8.  TNF-α production in CD4+ and CD8+ T cells upon stimulation with progesterone metabolites. \nProgesterone (Prog), 5α-dihydroprogesterone (5α-DHP), 6α-hydroxypregnanolone (6α-OH-Pregn) and \ndexamethasone (Dexa), were added at a concentration of  10−3  M to the PBMCs obtained from 3 different female \ndonors for 24 h. 6 h after first steroid hormone contact, PBMCs were activated with PMA/Ionomycin. TNF-α \npositive CD4+ and CD8+ T cells were counted by FACS analysis. The y-axis shows the % of CD4+ (a) and \nCD8+ (b) T cells positively staining for TNF-α. * p < 0.05, ** p < 0.01, *** p < 0.0001, ns = not significant.\n\n12\nVol:.(1234567890)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports/\nMethods\nMaterial and cell lines. Collagen I coated petridishes were from Corning (Milian, Nesselnbach, Swit-\nzerland). The primary HLEC (Cat. No. 2500, Lots. 19,399, 19,394 and 19,415) were from Sciencell (Chemie \nBrunschwig, Basel, Switzerland) and the primary human dLEC (CC-2810, Lots. 4F3029 and 4F3037) were from \nLonza (Ruwag, Bettlach, Switzerland) or from PromoCell Heidelberg, Germany (C-12217, Lot 431Z021.3). Pri-\nmary cells were obtained from female donors. The HTR-8/SVneo cells were a gift from Charles H. Graham \n(Queen’s University, Kingston, Ontario, Canada). The vascular cell basal medium (PCS-100-030) and the sup-\nplementary factors (PCS-100-041) were from ATCC (LGC Standards GmbH, Wesel, Germany). The Endothelial \nCell Growth Medium EGM-2 (CC-3162), including EBM-2 Basal Medium (CC-3156) and EGM-2 Supplements \n(CC-4176), was from Lonza (Ruwag, Bettlach, Switzerland). The PromoCell MV-2 medium with supplements \n(C-22121) was from PromoCell (Heidelberg, Germany). Medium RPMI 1640 (Cat.Nr. 21,875) was from Gibco \n(ThermoFisher Scientific, Reinach, Switzerland). Penicillin/streptomycin (P/S, Cat. No.  0503) and FBS (Cat. \nNo. 0025) were from Sciencell (Chemie Brunschwig, Basel, Switzerland). 3H-cholesterol (ART-0255), 3H-pro-\ngesterone (ART-0795) and 14C-progesterone (ARC-1398) were from American radiolabeled chemicals (ARC) \nInc. (Anawa Trading SA, Wangen ZH, Switzerland). Scintillation solution Optiphase Supermix (Cat. No. 1200-\n439) was from Perkin Elmer Schweiz AG (Schwerzenbach, Schweiz). The SRD5A-inhibitor dutasteride (SML \n1221) was purchased from Sigma-Aldrich (Buchs, Switzerland).\nCholesterol, progesterone, and all steroid hormone standards for LC–MS and TLC analysis were purchased \nfrom Steraloids, Inc. (Newport, RI, USA). Pre-coated TLC plates SIL G25  UV254 (REF 809,023) and pre-coated \nTLC sheets Polygram SIL G/UV254 (REF 805,023) were from Macherey–Nagel GmbH, Düren, Germany. Dichlo-\nromethane (1.06050), methanol (1.06009) and choloroform (1.02445) were from Merck Millipore, Zug, Swit-\nzerland. Microbeta 2 Microplate counter 2450 was from Perkin Elmer Schweiz AG (Schwerzenbach, Schweiz). \nPhosphorimager Typhoon FLA 7000 and the Storage Phosphor Screen (BAS-IP TR 2040 E Tritium Screen) were \nfrom GE Healthcare Life Sciences, Glattbrugg, Switzerland. Prime Script RT Reagent Kit Cat. RR037A was from \nTakara Bio Europe (Saint-Germain-en-Laye, France).\nFigure 9.  IFN-γ production in CD4+ and CD8+ T cells upon stimulation with progesterone metabolites. \nProgesterone (Prog), 5α-dihydroprogesterone (5α-DHP), 6α-hydroxypregnanolone (6α-OH-Pregn) and \ndexamethasone (Dexa), were added at a concentration of  10−3  M to the PBMCs of 3 different female donors \nfor 24 h. 6 h after first steroid hormone contact, PBMCs were activated with PMA/Ionomycin. IFN-γ positive \nCD4+ and CD8+ T cells were counted by FACS analysis. The y-axis shows the % of CD4+ (a) and CD8+ (b) T \ncells positively staining for IFN-γ. * p < 0.05, ** p < 0.01, *** p < 0.0001, ns = not significant.\n\n13\nVol.:(0123456789)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports/\nFor LC–MS analysis, a Vanquish UHPLC (equipped with an ACQUITY UPLC HSS T3 Column, 100 Å, \n1.8 µm, 1 mm X 100 mm column; Waters, Switzerland) was coupled to a Q Exactive Plus Orbitrap (both Thermo \nFisher Scientific, Reinach, Switzerland). Separation was achieved using gradient elution over 11 min using water \nand methanol both supplemented with 0.1% formic acid (all Sigma-Aldrich, Buchs, Switzerland) as mobile \nphases. Data analysis was performed using TraceFinder 4.1 (Thermo Fisher Scientific, Reinach, Switzerland). \nAll steroids analyzed by LC–MS and their systematic names are listed in Table 7.\nPrimer pairs for SRD5A1, SRD5A2, SRD5A3, AKR1D1, AKR1C1, AKR1C2, AKR1C3, AKR1C4, HSD3B1, \nHSD3B2, HSD17B2, HSD11B1, FDXR, and NR5A2 (Table  1) were designed by means of the Universal Pro-\nbeLibrary Assay Design Center software from Roche. All primers were intron-spanning and were synthesized \nby Microsynth AG (Balgach, Switzerland). Universal ProbeLibrary hydrolysis probes were purchased from \nSigma-Aldrich (Buchs, Switzerland). Assay on demand primers for Cyclophilin A (4326316E), PIBF1, NR3C1, \nNR3C2, NR3C3, PGRMC1, PGRMC2, GPER1, CYP17A1, CYP21A2, CYP11B1, CYP11B2, HSD11B2, StAR, \nFDX1 (Table 2), and the 7500 Fast Real-time PCR system machine were from Applied Biosystems (Thermo \nFisher Scientific, Reinach, Switzerland).\nThe MS part was done by the Proteomic Mass Spectrometry Core Facility PMSCF at DBMR in Bern, \nSwitzerland.\nFor FACS analysis, GolgiPlug, anti-CD3 V500 (clone SK7), anti-CD4 PerCP (clone SK3) and anti-CD8 \nPE-Cy7 (clone SK1) were purchased from BD Biosciences. The fixable Live/Dead dye was obtained from Invit-\nrogen. Anti-TNF-α AF647 (clone Mab11) and anti-IFN-γ BV421 (clone 4sB3) and their matched isotype controls \nwere bought from Biolegend. PMA/Ionomycin was obtained from Sigma Aldrich.\nAll methods used in this manuscript were carried out in accordance with relevant guidelines and regulations.\nCell culture. Primary HLECs and dLECs were cultured in collagen I coated cell ware. Medium for HLECs \nwas the vascular cell basal medium from ATCC with all supplementary factors but without cortisol (hydro-\ncortisone). FBS concentration was 5%. Medium for dLECs was the EBM-2 Basal Medium from Lonza with all \nEGM-2 supplements but without cortisol. FBS concentration was 2%. Primary HLECs and dLECs were used up \nto passage 6.\nHTR-8/SV neo cells were cultured in RPMI 1640 5% FBS.\nIn order to minimize steroid hormone contamination in our medium or FBS, all experiments were performed \nin cortisol-free medium containing 5% or 2% of charcoal treated (ct) FBS.\nTable 7.  List of all steroids analyzed or mentioned in this study with their short and systematic name.\nClass Short name Systematic name\nProgesterones\nPregnenolone 5-Pregnen-3β-ol-20-one\nProgesterone 4-Pregnen-3, 20-dione\n17OH-Progesterone 4-Pregnen-17-ol-3, 20-dione\n20α-Hydroxyprogesterone 4-Pregnen-20α-ol-3-one\n5α-Dihydroprogesterone 5α-Pregnan-3, 20-dione\n5β-Dihydroprogesterone 5β-Pregnan-3, 20-dione\nPregnanolone 5β-Pregnan-3α-ol-20-one\nEpipregnanolone 5β-Pregnan-3β-ol-20-one\nAllopregnanolone 5α-Pregnan-3α-ol-20-one\nIsopregnanolone 5α-Pregnan-3β-ol-20-one\n6α-OH-Pregnanolone 5α-Pregnan-3α, 6α-diol-20-one\n6α-OH-Epipregnanolone 5α-Pregnan-3β,6α-diol-20-one\nAllopregnane-3,20-diol 5α-pregnan-3β,20α-diol\nMineralocorticoids\n11-deoxycorticosterone 4-Pregnen-21-ol-3, 20-dione\nCorticosterone 4-Pregnen-11β, 21-diol-3, 20-dione\nAldosterone 4-Pregnen-11β, 21-diol-3, 18, 20-trione\nEstrogens\n17β-Estradiol 1,3,5(10)-estratriene-3,17β-diol\nEstriol 1,3,5(10)-estratriene-3,16α,17β-triol\nGlucocorticoids\n11-deoxycortisol 4-Pregnen-17α,21-diol-3,20-dione\nCortisone 17,21-dihydroxy-4-pregnene-3,11,20-trione\nCortisol 11β,17,21-trihydroxy-4-pregnene-3,20-dione\nAndrogens\nAndrosterone 3α-hydroxy-5α-androstan-17-one\nEtiocholanolone 3α-Hydroxy-5β-androstan-17-one\nDehydroepiandrosterone 5-androsten-3β-ol-17-one\nDehydroepiandrosterone-sulphate 5-androsten-3β-ol-17-one sulphate\n5α-Dihydrotestosterone 17β-hydroxy-5α-androstan-3-one\nTestosterone 17β-hydroxy-4-androsten-3-one\nAndrostenedione 4-androsten-3, 17-dione\n\n14\nVol:.(1234567890)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports/\nPBMCs were cultured in RPMI1640 containing 10% FBS after isolation. During experiments, PBMCs were \nmaintained in steroid-free RPMI1640.\nThin layer chromatography. 14C-progesterone and 3H-cholesterol as substrate for HLEC and dLEC. HLECs \nand dLECs were cultured in collagen I coated 6-well plates with medium containing 5% or 2% ct FBS for 24 h. \nAfter 24 h cells were washed twice with DPBS and 0.05 μCi 14C-progesterone or 1 μCi 3H-cholesterol were added \nin steroid free medium. Cell culture supernatant was collected into glass vials at the indicated time points. 5 ml \nethyl acetate was added and glass vials were vortexed for exactly 1 min. Thereafter, supernatants were stored \nat − 20 °C until phase separation. The upper phase was transferred into new glass vials and evaporated under a \nnitrogen stream at 56 °C. The pellet was suspended in 30ul of cold progesterone dissolved in EtOH (10 mg/ml) \nfor the 14C-experiments and in 30ul of the cold mixture cholesterol, progesterone, corticosterone, cortisol, tes-\ntosterone and estradiol (all 10 mg/ml EtOH) for the 3H-cholesterol experiments in HLEC. For the 3H-cholesterol \nexperiments in dLEC, the pellet was dissolved in 30ul of the cold mixture of cholesterol, progesterone, 11-de-\noxycortisol, corticosterone, aldosterone and cortisol (all 10 mg/ml EtOH). A 10ul aliquot of each sample was \nloaded on TLC glass plates or sheets. Running medium was dichloromethane, methanol,  H2O (150:10:1). For \nMicrobeta 2 measurements, spots were visualized and marked under the UV lamp. Marked spots were scratched \ninto scintillation vials. 3.5 ml of scintillation fluid was added and vials were counted in a Microbeta 2. Individual \nsteroid hormones were run on the same plate to allow for localization of the steroid hormones. A control without \ncells (0 h/48 h for HLEC and dLEC; 0 h/24 h for HTR-8/SV neo) was loaded as baseline/background. Counts are \ndisplayed in counts per minute (CPM = CCPM1). Conversion of 14C-progesterone to 6α-OH-pregnanolone or of \n3H-cholesterol to progesterone, corticosterone, cortisol, testosterone and estradiol (HLEC), or of 3H-cholesterol \nto progesterone, 11-deoxycortisol, corticosterone, aldosterone and cortisol (dLEC), was calculated. For visuali-\nzation by a phorphorimager, TLC sheets were exposed to a Storage Phosphor Screen. Spots were visualized by \nthe Phosphorimager Typhoon FLA 7000. Individual steroid hormones were run on the same sheet to allow for \nlocalization of the steroid hormones. A control without cells (0 h/48 h for HLEC and dLEC; 0 h/24 h for HTR-\n8/SV neo) was loaded as baseline/background. Quantification was done using ImageJ software. All conversion \nrates were compared to the controls without cells. Progesterone substrate availability at timepoint 0 h (control \nwithout cells) was taken as 100% for all experiments.\nSRD5A-inhibition with dutasteride. HLECs and dLECs were cultured as described above and incubated with \ndutasteride dissolved in DMSO and used at a final concentration of  10−5  M and  10−6  M for HLECs and HTR-8/\nSV neo and  10−6   M and  10−8   M for dLECs. Total incubation time was 24  h. Conversion of progesterone to \n6a-OH-pregnanolone was quantified by TLC.\n14C-progesterone as substrate for HTR-8/SV neo. HTR-8/SV neo were cultured in RPMI 1640 medium contain-\ning 5% ct FBS for 24 h. After 24 h cells were washed twice with DPBS and 0.05 μCi 14C-progesterone was added \nin steroid free medium. Cell culture supernatant was collected into glass vials at timepoints 1 h, 4 h, 8 h and 24 h. \n5 ml ethyl acetate was added and glass vials were vortexed for exactly 1 h. The rest of the procedure was done \nexactly as described above for the HLEC and dLEC.\nReal‑time PCR. mRNA expression of the progesterone metabolizing enzymes (SRD5A1, SRD5A2, SRD5A3, \nAKR1D1, AKR1C1, AKR1C2, AKR1C3, AKR1C4, HSD3B1, HSD3B2), of PIBF1, of the steroid hormone recep-\ntors (NR3C1, NR3C2, NR3C3, PGRMC1, PGRMC2, GPER1) of the steroidogenic enzymes (CYP17A1, CYP21A2, \nCYP11B1, CYP11B2, HSD11B1, HSD11B2, HSD17B2) and of the steroidogenic proteins StAR, FDXR, FDX1, \nNR5A2 in HLEC, dLEC and HTR-8/SV neo.\nHLECs and dLECs were cultured in collagen I coated 6-well plates with medium containing 5% or 2% FBS \nfor 24 h. HTR-8/SV neo were cultured in RPMI 1640 medium containing 5% FBS for 24 h. After the incubation \ntime total extraction of RNA was performed using the Trizol method. RNA was reverse transcribed using the \nPrime Script RT Reagent kit from Takara. Real-time PCR was performed using assay on demand primers or \nprimers and probes designed with the Roche Library in order to detect the following genes: SRD5A1, SRD5A2, \nSRD5A3, AKR1D1, AKR1C1, AKR1C2, AKR1C3, AKR1C4, HSD3B1, HSD3B2, PIBF1, NR3C1, NR3C2, NR3C3, \nPGRMC1, PGRMC2, GPER1, CYP17A1, CYP21A2, CYP11B1, CYP11B2, HSD11B1, HSD11B2, HSD17B2, StAR, \nFDXR, FDX1, and NR5A2 (Tables  1, 2). Cyclophilin A served as endogenous control. Assays were performed \nin duplicates.\nProteomics. HLEC and dLEC were cultured in their medium containing 5% or 2% FBS. Upon confluency, \ncells were washed 2 × with DPBS, detached with trypsine/EDTA (1 × concentrated) and centrifuged at 800 rpm. \nCell pellet was washed 2 × with DPBS and the dried pellet was lysed in 8 M UREA buffer containing a protease \ninhibitor cocktail. Protein amount was measured by the Qubit Protein Assay. The MS part was done by the Pro-\nteomic Mass Spectrometry Core Facility PMSCF using standard procedure.\nLC–MS. HLECs and dLECs were cultured in collagen I coated 6-well plates with medium containing \n5% ct FBS for 24  h. After 24  h cells were washed twice with DPBS and the cold testosterone, 17β-estradiol, \n5α-dihydroprogesterone and androstenediol were added separately in steroid-free medium at a concentration of \n 10−6  M. A time-course experiment was performed with incubation times 0 h as baseline, 4 h and 24 h for HLEC \nand 0 h and 24 h for dLEC. Steroids were extracted from the supernatant by solid phase extraction using Oasis \nHLB SPE plates (Waters, Switzerland). Remaining substrate concentrations and metabolites were assessed using \n\n15\nVol.:(0123456789)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports/\nLC–MS. 20 uL of the extract were injected and separation was achieved using gradient elution over 11 min using \nwater and methanol both supplemented with 0.1% formic acid as mobile phases. 6α-OH-pregnanolone was \nidentified based on accurate mass measurement, comparison of MS/MS spectra and retention times between the \nanalyte in the samples and an authentic standard. Data was normalized to condition at 0 h = 1000 nM =  10−6  M \nfor each compound.\nMouse lymph nodes and adrenals. Animal experimentation was approved by the Ethics Committee for \nAnimal Experiments of the Veterinary Administration of the Canton of Berne, Switzerland and conformed to \nthe rules of the Swiss Federal Act on Animal Protection (BE58/19). C57/Bl6 mice were bred according to the \nrules in the central animal facility of the university of Bern. They were maintained under 12-h dark–light cycles \nwith unrestricted access to food and water. Males used in experiment were fed for 6–8 weeks with regular chow \ndiet. On the day of sacrifice, lymph nodes and adrenal glands were isolated and put on ice until arrival at the \nlab. Thereafter, organs were washed in PBS and put into warm, steroid-free medium (PCS-100-030) containing \n5% ct FBS. The fat was dissected, and the adrenal gland, and half of the lymph nodes were cut in half. The rest \nof the lymph nodes were uncut and contained a capsule. Every lymph node (cut or entirely), and the adrenal \ngland were placed in 48-well plates and incubated with 500ul of steroid-free medium containing 5% ct FBS and \n0.05 µCi 14C-progesterone for 24 h or 48 h at 37 °C. For controls, medium from wells incubated under the same \nconditions and incubation time but without tissue was used. After 24 h and 48 h supernatants were collected into \nglass vials. 5 ml ethyl acetate was added and vials were vortexed exactly for 1 min. The next steps were performed \nas described in the section thin layer chromatography. Products of progesterone metabolism were visualized \nwith a Phosphorimager.\nFACS. Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy, non-pregnant female \ndonors using Ficoll gradient centrifugation. After isolation, PBMCs were kept for 30 min in RPMI1640 medium \ncontaining 10  mM HEPES, 10% heat-inactivated FBS and 100  μg/ml Streptomycin, 100U/ml penicillin (all \nfrom Gibco, Life Technologies). After 30  min, PBMCs were stimulated with progesterone, 5α-DHP , 6α-OH-\npregnanolone and dexamethasone for 6 h (priming). Control medium was RPMI1640 containing EtOH, the \nsolvent of the steroid hormones. Final concentration of all steroid hormones on PBMCs was  10−3  M. After the \n6 h pre-incubation period with steroid hormones, lymphocytes were activated with 15 ng/ml PMA and 1 μg/ml \nIonomycin. Simultaneously, GolgiPlug was added in order to inhibit cytokine release. After a total incubation \nperiod of 24 h, PBMCs were stained for CD3, CD4, CD8, IFN-γ and TNF-α and analyzed by FACS as follows: \nCells were stained with fixable Live/Dead dye for 30 min one ice. Subsequently cells were washed using PBS and \nfixed by adding Medium A from the Fix & Perm kit (Invitrogen) for 15 min at RT. Afterwards cells were washed \nand the permeabilization Medium B and the antibody cocktail containing anti-CD3, anti-CD4, anti-CD8, anti-\nIFN-γ and anti-TNF-α was added. Cells were incubated for 20 min, washed and resuspended in PBS contain-\ning 2% heat-inactivated FBS, 0.05% sodium azide. Data was acquired using a FACSCanto II (BD Biosciences). \nMatched isotype controls were used for anti-IFN-γ and anti-TNF-α Percentage of IFN-γ and TNFα positive cells \nwas determined in alive cells, which were CD3 positive.\nThe % of CD4+ and CD8+ T cells owning the intracellular cytokines IFN-γ and TNF-α is shown in Fig.  8 \n(TNF-α) and Fig. 9 (IFN-γ).\nStatistics. All experiments were performed at least 3 times. Pictures in figures are unprocessed. Representa-\ntive blots are shown. Densitometry was performed for all phosphorimager pictures using Image J software. \nConversion and production rates are displayed as mean ± SD using one-way ANOV A with Dunnetts multiple \ncomparison test. Significance was assigned at p < 0.05. TLC experiments were additionally controlled by CCPM1 \nmeasurements in a microbeta2 instrument. For TaqMan results ct values were calculated as mean of all experi-\nments. All statistical analyses were performed using GraphPad PRISM version 8 (PRISM, USA).\nEquipment and settings. For the phosphorimager pictures in Figs. 1, 5 and 7 the image acquisition tool Image J \nwas used. No manipulations have been made to the pictures.\nData availability\nAll data generated or analysed during this study are included in this published article (and its supplementary \ninformation files online).\nReceived: 28 July 2020; Accepted: 13 April 2021\nReferences\n 1. Swartz, M. A. & Lund, A. W . Lymphatic and interstitial flow in the tumour microenvironment: linking mechanobiology with \nimmunity. Nat. Rev. Cancer 12(3), 210–219 (2012).\n 2. Lund, A. W . et al. VEGF-C promotes immune tolerance in B16 melanomas and cross-presentation of tumor antigen by lymph \nnode lymphatics. Cell Rep. 1(3), 191–199 (2012).\n 3. Cohen, J. N. et al. Lymph node-resident lymphatic endothelial cells mediate peripheral tolerance via Aire-independent direct \nantigen presentation. J. Exp. 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Differential expression of vascular endothelial growth factor angiogenic factors in different endometrial compart-\nments in women who have an elevated progesterone level before oocyte retrieval, during in vitro fertilization-embryo transfer \ntreatment. Fertil. Steril. 104(4), 1030–1036 (2015).\n 67. Zhang, Y . et al. Progesterone metabolism in adipose cells. Mol. Cell Endocrinol. 298(1–2), 76–83 (2009).\nAcknowledgements\nWe thank Sofia Bachmann and the Centre of Laboratory Medicine, Inselspital Bern for their help with the \nimmunologic experiments, and Sophie Braga Lagache and Natasha Buchs for the proteomics analysis. CGM has \ndrawn Figure 4 and Supplementary Figure 2.\nAuthor contributions\nR.K. performed experiments and reviewed the manuscript. M.G. designed the steroidomic analysis, analyzed the \nLC–MS results and edited the manuscript, N.S. performed the LC–MS experiments, M.F . conducted the FACS \nexperiments and contributed to scientific discussions, G.E. and S.V . provided the mouse lymph nodes and adrenal \nglands and edited the manuscript. H.J. performed experiments. B.V . reviewed the manuscript and contributed to \nscientific discussions. M.G.M. supported with scientific discussions, edited and reviewed the manuscript. C.G.M. \ndesigned and analyzed the experiments, performed some and wrote the manuscript.\nFunding\nThis research was supported by the Swiss National Science Foundation (personal grant 32-135596 to MGM) and \nB. Vogt is supported by «Fonds pour la recherche thérapeutique, Lausanne, Switzerland».\nCompeting interests \nThe authors declare no competing interests.\nAdditional information\nSupplementary Information The online version contains supplementary material available at https:// doi. org/ \n10. 1038/ s41598- 021- 88508-w.\nCorrespondence and requests for materials should be addressed to C.G.-M.\nReprints and permissions information is available at www.nature.com/reprints.\nPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and \ninstitutional affiliations.\n\n18\nVol:.(1234567890)Scientific Reports |         (2021) 11:9666  | https://doi.org/10.1038/s41598-021-88508-w\nwww.nature.com/scientificreports/\nOpen Access  This article is licensed under a Creative Commons Attribution 4.0 International \nLicense, which permits use, sharing, adaptation, distribution and reproduction in any medium or \nformat, as long as you give appropriate credit to the original author(s) and the source, provide a link to the \nCreative Commons licence, and indicate if changes were made. The images or other third party material in this \narticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the \nmaterial. If material is not included in the article’s Creative Commons licence and your intended use is not \npermitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from \nthe copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.\n© The Author(s) 2021","source_license":"CC0","license_restricted":false}