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Cutting Edge: Progesterone Directly Upregulates Vitamin D Receptor Gene Expression for Efficient Regulation of T Cells by Calcitriol This information is current as of September 23, 2021. Shankar Thangamani, Myughoo Kim, Youngmin Son, Xinxin Huang, Heejoo Kim, Jee H. Lee, Jungyoon Cho, Benjamin Ulrich, Hal E. Broxmeyer and Chang H. Kim J Immunol published online 29 December 2014 http://www.jimmunol.org/content/early/2014/12/28/jimmun Downloaded from ol.1401923

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 29, 2014, doi:10.4049/jimmunol.1401923 Th eJournal of Cutting Edge Immunology

Cutting Edge: Progesterone Directly Upregulates Vitamin D Receptor Gene Expression for Efﬁcient Regulation of T Cells by Calcitriol Shankar Thangamani,*,1 Myughoo Kim,*,1 Youngmin Son,* Xinxin Huang,† Heejoo Kim,* Jee H. Lee,* Jungyoon Cho,*x Benjamin Ulrich,* Hal E. Broxmeyer,† and Chang H. Kim*,†,‡, The two nuclear hormone receptor ligands progesterone absorption and UV-induced synthesis in the skin and se- and vitamin D (vit.D) play important roles in regulating quentially activated in the liver and kidneys to become cal- T cells. The mechanism that connects these two hor- citriol, P4 is produced at high levels directly from ovaries and

mones in regulating T cells has not been established. placenta and at low levels in the adrenal gland and the testes Downloaded from In this study, we report that progesterone is a novel in- (12). In a manner similar to vit.D, P4 increases Tregs but ducer of vit.D receptor (VDR) in T cells and makes suppresses inflammatory effector T cells (13). In this study, we report that P4 induces VDR expression in T cells highly sensitive to calcitriol. At the molecular + level, the induction by progesterone is mediated by CD4 Th cells, an effect mediated by P4-induced binding of two progesterone receptor-binding elements in the in- P4 receptor (PR) to canonical PR-binding elements (PREs) in tron region after the first noncoding exon of the human the human VDR (hVDR) gene. VDR induction by P4 allows http://www.jimmunol.org/ VDR gene. Increased expression of VDR by progester- T cells to be more efficiently regulated by calcitriol for enhanced promotion of Tregs but suppression of potentially one allows highly sensitive regulation of T cells by vit.D inflammatory effector T cells. even when vit.D levels are suboptimal. This novel regulatory pathway allows enhanced induction of regula- Materials and Methods tory T cells but suppression of Th1 and Th17 cells by Cell isolation and animal study the two nuclear hormones. The results have significant 2 Cord blood (CB) CD4+CD25 and adult peripheral blood 2 2 2 ramifications in effective regulation of T cells to prevent CD4+CD25 CD45RO CD69 naive CD4+ T cells were isolated as de- by guest on September 23, 2021 adverse immune responses during pregnancy. The scribed before (14). Total spleen CD4+ T cells were isolated from pregnant + Journal of Immunology, 2015, 194: 000–000. mice (15–18 d postcoitus), and naive CD4 T cells were isolated from the spleen of nonpregnant mice as described before (15). Female C57BL/6 mice were injected s.c. with medroxyprogesterone (2 mg; Depo-Provera from Pfizer) and sacrificed 4 d later. All human subject and animal studies were he biologically active form of vitamin D (vit.D; 1,25- approved by Institutional Review Committees at Purdue University. dihydroxyvitamin D3, also called calcitriol) is a nu- T clear hormone ligand mediating its effects mainly In vitro differentiation of T cells and hVDR knockdown with small through the vit.D receptor (VDR)–retinoid X receptor nu- interfering RNA clear hormone receptor system (1). VDR is widely expressed Human naive CD4+ T cells were activated with anti-CD3/28 beads (5 ml/ in the body. Within the immune system, T cells express VDR million cells: Miltenyi Biotec) and IL-2 (25 U/ml) or cultured in Th1/Th17/ and are an important target of calcitriol for immune regula- Treg cytokine conditions in phenol red–free RPMI 1640 supplemented with 10% charcoal/dextran-treated FBS (HyClone) or in vit.D-free X-VIVO 15 tion (2). Calcitriol induces regulatory T cells (Tregs) but medium (Lonza) as described before (14). CB T cells, activated for 24 h with suppresses the generation of effector T cells (3–8). Vit.D anti-CD3/28 beads and human IL-2, were transfected with control or hVDR metabolites inhibit the maturation of dendritic cells and make small interfering RNA (siRNA; 20 pmol/4 million cells, Santa Cruz Bio- them tolerogenic cells (9, 10). Progesterone (P4) is another technology) using an Amaxa Nucleofector (Lonza). Cells were cultured with P4 (2 mg/ml) and/or calcitriol (1–100 nM) for 3 d for VDR mRNA ex- nuclear hormone critical for preparation and maintenance of pression or 5–6 d for the expression of Foxp3, CD25, CD38, latency- pregnancy (11). Whereas vit.D is obtained through dietary associated peptide/TGF-b1, IL-17, and/or IFN-g (13).

*Laboratory of Immunology and Hematopoiesis, Department of Comparative Pathobi- Address correspondence and reprint requests to Prof. Chang H. Kim, Purdue University, ology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907; VPTH 126, 725 Harrison Street, West Lafayette, IN 47907. E-mail address: †Department of Microbiology and Immunology, Indiana University School of Medi- [email protected] cine, Indianapolis, IN 46202; ‡Weldon School of Biomedical Engineering, Purdue x The online version of this article contains supplemental material. University, West Lafayette, IN 47907; and Purdue Center for Cancer Research, Purdue University, West Lafayette, IN 47907 Abbreviations used in this article: CB, cord blood; ChIP, chromatin immunoprecipitation; hVDR, human VDR; P4, progesterone; PB, peripheral blood; PR, P4 receptor; 1S.T. and M.K. are coﬁrst authors. PRE, PR-binding element; siRNA, small interfering RNA; Treg, regulatory T cell; vit.D, Received for publication August 4, 2014. Accepted for publication December 1, 2014. vitamin D; VDR, vitamin D receptor.

This work was supported in part by National Institutes of Health Grants R01AI074745, Ó R01DK076616, 1R01AI080769, and 1S10RR028293 and by a grant from the National Copyright 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 Multiple Sclerosis Society (to C.H.K.).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401923 2 CUTTING EDGE: PROGESTERONE INDUCES VDR IN T CELLS

In vitro T cell suppression assay CB naive CD4+ T cells were stained with CFSE and 5 3 104 cells were added to 96-well plates in the presence of anti-CD3/CD28–coated beads as target cells along with in vitro–differentiated Tregs generated with IL-2 (25 U/ml) and calcitriol (100 nM) and/or P4 (2 mg/ml). CFSE dilution was determined by ﬂow cytometry on day 4. Microarray, RT-PCR, and Western blotting of VDR expression The microarray analysis of CB naive CD4+ T cells was performed previously (13). Quantitative RT-PCR was performed with the primers for human or mouse VDR gene (Supplemental Table I). CB naive CD4+ T cells, activated with anti-CD3/CD28 and IL-2 with or without P4 (2 mg/ml) for 3–5 d, were examined for VDR protein expression with an mAb to hVDR (R&D Sys- tems) and HRP-conjugated anti-mouse IgG (Santa Cruz Biotechnology). Promoter analysis and chromatin immunoprecipitation assay PREs on the hVDR gene were identiﬁed with TESS. A chromatin immunoprecipitation (ChIP) assay was performed as described before with the primers listed in Supplemental Table I (15). CB CD4+ T cells, activated with anti-CD3/CD28–coated beads in the presence of P4 (2 mg/ml) for 3–4 d,

were processed and immunoprecipitated using 4 mg rabbit mAb to human PR Downloaded from (Abnova). Mutagenesis and reporter assay PRE no. 1 (59-AGAACT-39) and PRE no. 2 (59-GGGACA-39) in the regulatory region of VDR gene cloned in pGL3-VDR (+490/21267) (16) were mutated to 59-AAAGGT-39 and 59-GAAGGA-39, respectively, with a site-

directed transformer mutagenesis kit (Clontech Laboratories). Mutant pGL3- http://www.jimmunol.org/ VDR vectors (20 mg) were electrotransfected into MCF-7 cells (310 V, 950 mF; Bio-Rad). The transfected cells were rested overnight, activated with PMA (50 ng/ml) in the presence or absence of P4 (2 mg/ml) for 6 h, and then assayed for luciferase activity with a Synergy HT reader (BioTek). FIGURE 1. P4 induces VDR expression in human CD4+ T cells. (A) Statistical analysis Multiplot microarray data showing P4-regulated genes in CB T cells. (B and Significant differences between indicated groups were determined by a Student C) Expression of hVDR mRNA was determined by quantitative RT-PCR in paired two-tailed t test. cultured human naive CD4+ T cells. CB T cells were used unless indicated otherwise. (D) Expression of VDR protein in cultured CB CD4+ T cells was E Results and Discussion determined by Western blotting. ( ) P4 induces hVDR mRNA expression in by guest on September 23, 2021 P4 induces VDR expression in T cells various cytokine conditions. (F) VDR expression in mouse T cells and uteri. + + Naive CD4 T cells were activated with anti-CD3/28 and IL-2 in charcoal- Analysis of microarray data on human CB naive CD4 T cells treated serum-containing (A and C–E) or vit.D-free medium (B and F) in the activated with P4 (13) revealed that one of the major genes presence or absence of P4 (2 mg/ml unless indicated otherwise) or RU486 induced by P4 is the VDR gene (Fig. 1A). To verify this (100 mg/ml). Combined or representative data from three to five separate finding, CB naive CD4+ T cells were activated in the presence experiments are shown in (B)–(F). *p , 0.05 between indicated groups or of P4, and VDR expression at mRNA and protein levels was between controls. examined. P4 substantially increased the expression of VDR mRNA in activated T cells in a dose-dependent manner into the first exon and intron regions (Fig. 2A). A luciferase (Fig. 1B). Optimal induction occurred at ∼2 mg/ml P4, reporter containing this regulatory region was highly re- concentrations that were detected in placental tissues during sponsive to P4 (Fig. 2B), suggesting that this region contains pregnancy (17). Thus, this concentration range is physiolog- functional PREs. A ChIP assay revealed that only one site ically relevant. The induction of VDR gene expression was (site D) between exon 1A and 1B had a clear PR binding suppressed by RU486, a PR antagonist (Fig. 1C). A similar activity (Fig. 2C). When the two PREs in site D were mu- induction was observed in adult peripheral blood CD4+ tated, the VDR gene promoter activity was largely abolished T cells. P4 also induced the expression of VDR protein in CB (Fig. 2D). T cells (Fig. 1D). The VDR induction by P4 occurred in Treg-, Th1-, and Th17-polarizing cytokine conditions P4-induced VDR potentiates the effect of calcitriol on suppression of (Fig. 1E). The VDR induction by P4 was observed in mouse Th1 and Th17 cells T cells as well (Fig. 1F). It was also increased in spleen CD4+ T cells and uterus in pregnant or progestin-injected mice Vit.D metabolites suppress the generation of Th1 and Th17 (Fig. 1F). These results indicate that P4 induces VDR gene cells (5, 8, 18). VDR directly binds the Ifng gene promoter to expression in T cells in heterogeneous conditions or species. suppress Th1 cells (19) and induce C/EBP protein expression to suppress Th17 cells (8). We found that P4 decreases the P4 responsive elements on the hVDR gene mediate P4-induced VDR effective concentration of calcitriol in suppressing the induc- expression tion of Th1 and Th17 cells (Fig. 3A). Interestingly, the To gain insights into the molecular mechanism of the VDR suppression by calcitriol in the presence of P4 was largely induction by P4, we examined the DNA sequence of the VDR abolished when the VDR gene was knocked down with siRNA gene for the presence of the canonical PREs. We found five (Fig. 3B, 3C). Thus, P4-induced VDR functions to increase PREs in the 59 regulatory region of the VDR gene spanning the sensitivity of T cells to calcitriol. The Journal of Immunology 3

FIGURE 2. PR binding to cis-acting elements in the VDR gene promoter drives gene expression. (A) Putative PREs and ChIP sites in the hVDR gene are shown. (B) A luciferase assay was performed with a reporter vector containing the VDR gene promoter. (C) A ChIP assay was performed to identify PR binding sites in the 59 regulatory region of the hVDR gene. (D) A reporter assay with null mutations in the PRE sites in the VDR gene. A representative dataset of three to four separate Downloaded from experiments is shown. *p , 0.05 between indicated groups. n.d., not detectable. http://www.jimmunol.org/

P4-induced VDR enhances the function of calcitriol in inducing Tregs with calcitriol and P4 were more suppressive than were the by guest on September 23, 2021 We next examined the effect of calcitriol on induction of Tregs Tregs generated with P4 or calcitriol alone (Fig. 4B). The expressing Foxp3, CD38, and/or latency-associated peptide/ Treg-inducing activity of calcitriol in the presence of P4 was TGF-b1 (20, 21) in the presence or absence of P4. P4 in- greatly abolished when the VDR gene was knocked down with creased T cell sensitivity to calcitriol in upregulating the Treg- siRNA (Fig. 4C). associated molecules (Fig. 4A). Moreover, the Tregs generated Our results revealed the presence of a novel regulatory pathway linking P4 and vit.D in regulating T cells. The VDR induced by P4 increases the sensitivity of T cells to calcitriol. This VDR induction in T cells is mediated by two PRE sites after the noncoding first exon of the VDR gene, which contains a number of other cis-acting elements (22). It is known that VDR expression in human T cells is upregulated upon TCR signaling (23), and we think that P4 and calcitriol are prob- ably involved in the induction because culture media in- cluding animal sera generally contain these two nuclear hormones. Vit.D is important for normal function of the reproductive system, and its deficiency is common in pregnant females and is linked to pregnancy complications such as preeclampsia and miscarriage and to defective immune tol- erance in the newborn (24–27). The VDR upregulation may allow T cells to sense low levels of vit.D and become effec- tively regulated to prevent inflammation when vit.D levels FIGURE 3. Impact of P4 and induced VDR on T cell response to calcitriol are decreased in the body. Therefore, the P4-induced VDR in suppression of Th1 and Th17 cells. (A)CBCD4+ naive T cells were acti- in T cells would be important to prevent adverse immune vated in Th1 (IL-2, IL-12, and anti–IL-4) or Th17 (IL-6, IL-21, IL-23, IL-1b, responses involved in pregnancy complications. The regula- TGF-b1, anti–IL-4, and anti–IFN-g) polarization conditions in a vit.D-free tory pathway may be active in other cell types as well because medium containing P4 (2 mg/ml) and/or calcitriol for 5–6 d. (B) Expression it has been observed that VDR expression was increased in of hVDR mRNA after siRNA knockdown. (C) Impact of VDR knockdown on T cell differentiation into effector T cells in response to calcitriol (1 nM) endometrial tumor cells cultured with P4 (28) and in the and/or P4 (2 mg/ml). Pooled data with SEM are shown (n = 4). *p , 0.05 uterus in pregnant females, as determined in this study. In versus controls. summary, our findings reveal a novel role of P4 in expression 4 CUTTING EDGE: PROGESTERONE INDUCES VDR IN T CELLS

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