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Modulation of T Lymphocyte Function by the Pregnane X Sandrine Dubrac, Andreas Elentner, Susanne Ebner, Jutta Horejs-Hoeck and Matthias Schmuth This information is current as of October 2, 2021. J Immunol 2010; 184:2949-2957; Prepublished online 19 February 2010; doi: 10.4049/jimmunol.0902151 http://www.jimmunol.org/content/184/6/2949 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2010/02/15/jimmunol.090215 Material 1.DC1 References This article cites 42 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/184/6/2949.full#ref-list-1 http://www.jimmunol.org/

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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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Modulation of T Lymphocyte Function by the Pregnane X Receptor

Sandrine Dubrac,* Andreas Elentner,* Susanne Ebner,*,† Jutta Horejs-Hoeck,‡ and Matthias Schmuth*

The pregnane X receptor (PXR) is a ligand-activated regulating central to and hormone in the liver. Previous reports indicated that PXR is expressed in PBMC, but the role of PXR in immune cells remains unknown. In this paper, we report increased PXR expression in mouse and human T lymphocytes upon immune activation. Furthermore, phar- macologic activation of PXR inhibits T lymphocyte proliferation and anergizes T lymphocytes by decreasing the expression of CD25 and IFN-g and decreasing phosphorylated NF-kB and MEK1/2. Although these effects are preceded by an increase of suppressor of cytokine signaling 1, a master switch for IFN-g expression, in a PXR-dependent manner, T-bet expression remains unchanged. Conversely, PXR-deficient mice exhibit an exaggerated T lymphocyte proliferation and increased CD25 expression. Furthermore, Downloaded from PXR-deficient lymphocytes produce more IFN-g and less of the anti-inflammatory cytokine IL-10. In summary, these results reveal a novel immune-regulatory role of PXR in T lymphocytes and identify suppressor of cytokine signaling 1 as an early signal in PXR-mediated T lymphocyte suppression. The Journal of Immunology, 2010, 184: 2949–2957.

he pregnane X receptor (PXR) is a member of the nuclear CD19+ B lymphocytes, and CD14+ monocytes in humans (6, 7).

receptor superfamily that includes the , retinoid, Several studies showed that , a human PXR activator, http://www.jimmunol.org/ T and orphan receptors. PXR is activated by a broad range of suppresses both humoral and cellular immunity and identified ri- compounds, including , bile acids, and a wide variety of fampicin as a powerful immunosuppressive drug (8–12). Sub- including , rifampicin, 16a car- sequently, rifampicin was successfully used as an effective bonitrile (PCN), and RU-486 (1). PXR mediates the detoxification antipsoriatic drug (13–15). As drug metabolism and the immune of endogenous and exogenous compounds and prevents toxic ac- system are intertwined, recent work shed initial light on the mo- cumulation of metabolites within cells (2). DNA polymorphisms lecular aspects of this interaction. A reciprocal repression between within the PXR have been linked to interindividual differ- PXR and NF-kB was shown (16, 17). In the colon, PXR-mediated ences in drug responsiveness (3, 4). repression of NF-kB target genes appears to be a critical mecha-

PXR acts as a transcription factor. Once activated, PXR heter- nism by which PXR activation decreases inflammation (17). by guest on October 2, 2021 odimerizes with the (RXR), binds to regulatory Whereas PXR activation inhibits NF-kB activation, conversely, DNA sequences in the nucleus, and modulates transcription of p65 NF-kB disrupts the binding between PXR and RXR a to DNA genes involved in the oxidation, conjugation, and export of and inhibits the transactivation of target genes (18, 19). In the compounds from cells. These include the cytochrome P-450 present work, we set out to delineate the molecular mechanism and the multiple drug resistance gene (MDR1/ABCB1), involved in the control of T lymphocyte function by PXR. among many others (1, 2, 5). Notably, cytochrome P-450 enzymes are responsible for the metabolism of .50% of current pre- Materials and Methods scription drugs (1). Reagents and Abs PXR is primarily expressed in the gastrointestinal tract and liver. LPS, PCN, RU-486, rifampicin, PMA, and BrdU were purchased from However, recent reports showed that PXR is expressed in immune Sigma-Aldrich (St Louis, MO); oligodeoxynucleotide containing embedded + + cells, that is, PBMC, including CD4 , CD8 T lymphocytes, unmethylated CpG motifs (CpG) from Coleypharma (Wellesley, MA); recombinant mouse-TSPL from R&D Systems (Minneapolis, MN); and peptidoglycan (PGN) from InvivoGen (San Jose, CA). Directly labeled primary mAb specific for mouse CD4 (clone GK 1.5), CD25 (clone PC61), *Department of Dermatology and Venereology, Innsbruck Medical University; †Oncotyrol, Center for Personalized Cancer Medicine, Innsbruck; and ‡Institute for IL-10-PE (clone JES5-16E3), and IFN-g–FITC (clone XMG1.2) were , University of Salzburg, Salzburg, Austria purchased from BD Pharmingen (San Diego, CA). Alexa Fluor 488 con- jugate anti-BrdU was purchased from Invitrogen (Carlsbad, CA). Anti– Received for publication July 16, 2009. Accepted for publication January 5, 2010. phospho-p65 NF-kB (ser536), phospho-SAPK/JNK (Thr183/Tyr185), phos- This work was supported by Grant FWF P-16990 from the Austrian Science Fund (to pho-MEK1/2 (ser217/221), phospho-p38 MAPK (Thr180/Tyr182), and M.S.) and by Grant MFI-4301 from the Medical University of Innsbruck (to S.D. and cleaved caspase 3 were purchased from Cell Signaling (Danvers, MA); A.E.). anti-mouse T-bet and anti-human PXR (clone H4417) from Perseus Pro- Address correspondence and reprint requests to Dr. Sandrine Dubrac and Dr. Matthias teomics (Tokyo, Japan); anti-mouse suppressor of cytokine signaling 1 Schmuth, Department of Dermatology and Venereology, Innsbruck Medical Univer- (SOCS-1) from Imgenex (San Diego, CA); anti-human SOCS-1 from Gene sity, Anichstrasse 35, A-6020 Innsbruck, Austria. E-mail addresses: sandrine.dubra- Tex (Irvine, CA); anti–b-actin from Sigma-Aldrich; biotinylated anti- [email protected] and [email protected] rabbit Abs and streptavidin-APC from Amersham (GE Healthcare, U.K.); The online version of this article contains supplemental material. and annexin V from Bender Medsystems (Vienna, Austria). mAb specific Abbreviations used in this paper: PCN, pregnenolone 16a carbonitrile; PGN, pepti- for human CD14 (clone mC5-3), CD16 (clone 3G8), CD56 (clone N- doglycan; PXR, pregnane X receptor; RXR, retinoid X receptor; SOCS-1, suppressor CAM), CD19 (clone Leu-12), CD235a (clone GA-R2), CD123 (clone of cytokine signaling 1. 9F5), CD36 (clone CB38), and HLA-DR (clone L243) were purchased from BD Biosciences (San Jose, CA) and CD45 RO (clone UCHL1) from Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 Dako (Glostrup, Denmark). www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902151 2950 ROLE OF PXR IN T LYMPHOCYTES

Animals primers (synthesized by Microsynth, Balgach, Switzerland) specific for mouse TATA binding , PXR, CYP3A11, SULT2B1, IFN-g, and Mice of the inbred strains C57BL/6 and BALB/c were purchased from SOCS-1 mRNA molecules and for human ribosomal protein 18S, TATA Charles River Laboratories (Sulzfeld, Germany), maintained on a standard +/2 binding protein, SOCS-1, and PXR mRNA molecules were selected using chow diet, and used at 2–3 mo of age. PXR mice were bred on a C57BL/ Primer Express software (Applied Biosystems, Foster City, CA). 6 background as described previously (20). All animal experiments were carried out according to governmental guidelines. Luciferase assay Preparation of cells and cell culture A pLUC vector containing a 1433-bp DNA fragment of the human SOCS-1 promoterwasusedaspreviouslydescribed(23).Jurkatcellswerecotransfected T cell isolation. T lymphocytes were isolated from mouse lymph nodes and with 2 mg pLUC-HuSOCS-1-promoter and 0.2 mg pLUC-GL4 (Promega, spleens as described earlier and further purified by negative selection (21). Madison, WI) complexed with PlasFect reagent (1:4) (Bioline, Taunton, MA), Purity was 98 6 1% as determined by FACS analysis. Human PBMC were according to the manufacturer’s directions. After overnight incubation, 10 mM obtained from donors after informed consent in accordance with the rifampicin or vehicle was added to the medium for 24 additional hours. Lu- Declaration of Helsinki. Naive CD3+ T lymphocytes were isolated using ciferase activity was measured using the Dual-Glo Luciferase Assay System a panning technique, as described previously (22). The purity of isolated from Promega, according to the manufacturer’s directions. CD3+ cell population was .90%, as determined by FACS analysis. Jurkat cells. Jurkat T leukemia cells (clone E6-1) derived from a human MLRs acute T cell leukemia were obtained from the American Type Culture Collection (Manassas, VA). Epidermal dendritic cell-containing interphases (21) were cocultured with allogenic PXR2/2 and PXR+/+ T lymphocytes (3 3 105 per well) at var- Epidermal cell isolation. Epidermal cells were isolated by trypsinization, as ious ratios for 4 d, and proliferation was measured by [3H]thymidine in- described earlier (21). corporation for the last 16 h (activity, 4mCi/ml = 148,000 Bq/ml; New FACS analysis England Nuclear/PerkinElmer, Boston, MA). Downloaded from Specimens were analyzed on a FACScalibur (BD Biosciences). Nonviable Statistical analysis cells were excluded by 7-aminoactinomycin D (Sigma-Aldrich) uptake Results are expressed as mean 6 SEM. Data were analyzed using a paired when fixation of cells was not required for analysis. After fixation and or an unpaired Student t test as appropriate. permeabilization with a cell permeabilization kit (Fix&Perm, An der Grub Bio-Research, Kaumberg, Austria) for 15 min at room temperature, the expression of phosphoproteins and T-bet was assessed on spleen cells by Results incubation with the first-step Ab, followed by staining with biotinylated http://www.jimmunol.org/ Abs (listed above) and then with streptavidin-APC. T lymphocytes were Increased PXR expression in activated T lymphocytes counterstained with an anti–CD3-FITC mAb. Recent reports indicated PXR expression in human CD4+, CD8+ + + Cytokine detection T lymphocytes, CD19 B lymphocytes, and CD14 monocytes (6, 7), but not in bone marrow-derived mouse macrophages (24). InvivoproductionofcytokinesbyTlymphocyteswasdeterminedasdescribed Because the functional significance of PXR expression in these earlier (21). Alternatively, cells were isolated from spleens by homogeniza- tion and stimulated for 48 h. Cells were cultured for 4.5 h with 1 mg/ml cells remained unknown, we stimulated mouse T lymphocytes Brefeldin A (BD Pharmingen) to prevent cytokine secretion. Then, cells were with various inflammatory stimuli known to activate T lympho- fixed, permeabilized, and stained with mAb, as described above. Highly cytes and assessed PXR expression. We used anti-CD3/CD28, purified T lymphocytes were cultured, supernatants were collected and

which cross-links TCR or PMA, activating the protein kinase C by guest on October 2, 2021 pooled, and cytokine concentrations were determined by multianalyte profiler ELISArray Kit from SuperArray Bioscience Corporation (Frederick, MD). pathway. In addition, we have combined PMA or anti-CD3/CD28 with TLR ligands, such as LPS, CpG, and PGN. LPS and CpG are In vivo proliferation TLR4 and TLR9 ligands, respectively, inducing a Th1 response in In vivo proliferation of T lymphocytes was determined by BrdU in- T cells. In contrast, PGN is a TLR2 ligand and induces a Th2 corporation. Mouse ear skin was topically treated with 20 ml 1% 2,4,6-tri- response. We used these different stimulation mixtures to establish nitro-1-chlorobenzene picryl chloride (Kodak Eastman, Rochester, NY) at whether PXR upregulation is specific to one type of stimulation or, day 0. At day 5, mice were injected i.p. with 0.8 mg BrdU. At day 7, cells in contrast, if PXR upregulation is a general event that occurs in were isolated from lymph nodes by collagenase P digestion, as described earlier (21), and stained according to the manufacturer´s instruction. all activated T cells independent of the nature of the stimulus. Real-time PCR analysis revealed a significant increase in PXR Immunocytochemical analysis mRNA transcript upon T lymphocyte activation (Fig. 1A, Sup- Cytospins of highly purified human T lymphocytes were fixed in acetone for plemental Fig. 1). Moreover, the combination of PMA or anti- 5 min. Immunostaining was performed with anti-human PXR mAb or CD3/CD28 with LPS and/or CpG (Th1) revealed additional ef- isotype control for 1 h at 37˚C, using biotinylated sheep anti-mouse Ig fects on PXR expression that were not present with PGN (Th2) (Amersham Biosciences, Piscataway, NJ) in 1% BSA followed by a streptavidin Alexa-Fluor 594 conjugate (Molecular Probes, Eugene, OR). (Fig. 1A, Supplemental Fig. 1A). We also observed a parallel The staining was visualized with a 403 objective using an Olympus BX60 upregulation of cyp3a11, a known PXR downstream gene (1) (Fig. epifluorescence microscope (Olympus, Melville, NY). 1B). A similar PXR induction was found in human PMA-activated T lymphocytes derived from PBMC (Fig. 1C) and in Jurkat cells Western blot analysis following different inflammatory stimuli (Fig. 1D). Time course T lymphocytes were lysed in radioimmunoprecipitation assay lysis buffer in expression of PXR shows that PXR expression is upregulated the presence of phosphatase and protease inhibitors (Pierce, Rockford, IL). beginning 18 h postactivation of mouse T lymphocytes or 30 h Electrophoresis for SOCS-1 was performed under reducing conditions, using DTT. Endogenous were detected with primary Abs, as listed postactivation of Jurkat cells (Supplemental Fig. 1B, 1C). Im- above, and with Alexa A680-conjugated anti-rabbit secondary Ab (Invi- munohistochemistry showed that in unstimulated resting and trogen, Carlsbad, CA). Blots were then scanned with a LI-COR Biosciences PMA-stimulated human T lymphocytes, PXR mainly localizes to (Lincoln, NE) analyzer. the cytoplasmic and perinuclear compartment (Fig. 1E). RNA quantification PXR activation inhibits T cell proliferation, CD25 and IFN-g Total RNA was extracted using TRIzol (Life Technologies, Carlsbad, CA), expression in vitro and random primed cDNA was prepared (Superscript II RNase H-reverse transcriptase; Life Technologies, Vienna, Austria). Quantitative PCR We next determined the effects of PXR activation on T cell function. analysis was performed by real-time PCR (ABI PRISM 7700 sequence We first asked whether the immunogenic potential of T lymphocytes detector; Applied Biosystems, Vienna, Austria). Sequences for probes and is modulated by PXR in vitro by assessing the ability of mouse The Journal of Immunology 2951

A

B C Downloaded from http://www.jimmunol.org/

D E by guest on October 2, 2021

FIGURE 1. PXR expression is increased in activated T lymphocytes. A and B, Quantitative PCR analyses were performed to measure the expression of PXR (A) or CYP3A11 (B), using real-time PCR with the TaqMan PCR master mix from Applied Biosystems. Random-primed cDNA was prepared from total RNA isolated from highly purified unstimulated or stimulated mouse T lymphocytes. Activation stimuli included the following: anti-mouse CD3/ CD28 (2 mg/ml), anti-mouse CD3/CD28 (2 mg/ml) + CpG (5mg/ml), PMA (100 ng/ml), PMA (50 ng/ml) + LPS (50 ng/ml) + CpG (5mg/ml), or PMA (50 ng/ml) + PGN (1mg/ml). RNA levels were measured after 48 h; n = 3. Data were analyzed using a Student t test. pp , 0.05; ppp , 0.005, showing differences between activated and unstimulated mouse T lymphocytes. C, Quantitative PCR analyses were performed on random-primed cDNA prepared from total RNA isolated from highly purified unstimulated or PMA-stimulated human T lymphocytes (100 ng/ml PMA, for 24 h); n = 4. Data were analyzed using a paired t test. pp , 0.05. D, Quantitative PCR analyses were performed on random-primed cDNA prepared from total RNA isolated from Jurkat cells incubated for 48 h prior to RNA isolation in the presence of anti-human CD3/CD28 (2 mg/ml) or PMA (100 ng/ml). Data were analyzed using a Student t test. pppp , 0.001, showing differences between activated and unstimulated Jurkat cells. E, Immunohistochemistry showing the expression of PXR in PMA-stimulated and unstimulated (resting) human CD3+ T lymphocytes. Cytospins were prepared and stained for PXR expression, as described in Materials and Methods. The staining was visualized with a 340 objective. Negative controls were stained with isotype control. Nuclei were visualized by DAPI staining.

T lymphocytes to proliferate upon treatment with the mouse PXR lymphocytes to proliferate (Fig. 2A, 2B) without affecting their activators PCN and RU-486. We found that, in a PXR-dependent viability (Supplemental Fig. 3). These results indicate that PXR manner, PXR activation dramatically decreased the ability of T ligands modulate the immunogenicity of T lymphocytes in a PXR- 2952 ROLE OF PXR IN T LYMPHOCYTES Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. PXR activation inhibits T cell proliferation and IFN-g production and expression in vitro. A, Purified mouse T lymphocytes isolated from PXR+/+ mouse spleen were cultured in the presence of 50 ng/ml PMA + 50 ng/ml LPS + 5mg/ml CpG combined with vehicle or increasing concentration of PCN or RU-486 and pulsed with [3H]thymidine for 48 h, n = 3–6. Data were analyzed using a Student t test. pp , 0.05; ppp , 0.005. B, Purified mouse T lymphocytes isolated from PXR2/2 mouse spleen were cultured in the presence of 50 ng/ml PMA + 50 ng/ml LPS + 5 mg/ml CpG combined with vehicle or PCN or RU-486 and pulsed with [3H]thymidine for 48 h; n = 3–6. Data are expressed as percentage of controls, that is, vehicle-treated T lymphocytes, and were analyzed using a Student t test. C, Expression of CD25 on wild-type or PXR2/2 mouse T lymphocyte cell surface after activation with 50 ng/ml PMA + 50 ng/ml LPS + 5 mg/ml CpG for 24 h, in the presence of 10 mM PCN or RU-486 or vehicle, was analyzed by flow cytometry; n = 3. Representative results are shown. The dashed black line designates DMSO-treated cells, the dark gray line PCN, and the light gray line the RU-486 treated cells; the dotted gray line indicates the isotype control. D, Expression of CD25 expressed as percentage of positive cells on Jurkat cell. Cell surface was stimulated with anti- human CD3/CD28 for 48 h in the presence of DMSO, rifampicin, or RU-486; n = 3. Data were analyzed with a one-way ANOVA followed by a Newman– Keuls post hoc test; n =3.E, Production of IFN-g by wild-type or PXR2/2 mouse T lymphocytes stimulated with anti-CD3/CD28, combined with vehicle, PCN, or RU-486 for 48 h, was analyzed by flow cytometry; n = 3. Numbers in quadrants show percentage of CD4+ IFN-g+ cells. F, IFN-g mRNA was quantified by real-time PCR analysis with the TaqMan PCR master mix from Applied Biosystems. Purified wild-type mouse T lymphocytes were PMA stimulated overnight in the presence of PCN or vehicle. Random-primed cDNAs were prepared from total RNA isolated from highly purified mouse T cells; n = 5. Data were analyzed using a Student t test. Representative results are shown. dependent manner and independent of apoptotic effects. To further combination with the PXR activators PCN or RU-486, or vehicle delineate the immune-regulatory role of PXR, we treated spleen- (1). PXR activation resulted in decreased expression of the cell derived mouse T lymphocytes with various inflammatory stimuli in surface activation marker CD25 (Fig. 2C, Supplemental Fig. 2), The Journal of Immunology 2953 whereas it did not alter the viability of activated spleen cells production in activated splenocytes. PCN and RU-486 treatment (Supplemental Fig. 3). 7-Aminoactinomycin D+ cells were ex- decreased T lymphocyte-derived IFN-g in a PXR-dependent cluded from these analyses, ensuring that the effects of PXR ac- manner (Fig. 2E, 2F). Notably, PXR2/2 T lymphocytes produced tivity on T lymphocyte function did not depend on cell viability. more IFN-g than did wild types after in vitro stimulation with anti- Furthermore, a lack of effect in PXR activator-treated PXR2/2 cells CD3/CD28 (Fig. 2E). A multianalyte cytokine profiling ELISA showed that the decrease was mediated by PXR (Fig. 2C). Similar assay revealed that production of IL-10 and TNF-a by T lympho- results were observed in a human T cell line; that is, rifampicin and cytes treated with PCN (10 mM) or DMSO was very low and did not RU-486 reduced the expression of CD25 in Jurkat T cells (Fig. 2D). exhibit significant changes except a 4-fold decrease in IFN-g in Finally, we tested whether PXR activation modulated cytokine PCN-treated T lymphocytes, as compared with vehicle controls Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. PXR deficiency targets T lymphocyte function. A, Purified T lymphocytes isolated from PXR2/2 and littermate control mice were cocultured with graded doses of allogenic epidermal dendritic cells for 72 h and then pulsed with [3H]thymidine for 16 h, as described in Materials and Methods; n = 3. Data were analyzed using a Student t test; pp , 0.05. CPM values for T cells in the absence of epidermal dendritic cells were consistently below 2000. B, In vivo proliferation of CD4+ T lymphocytes in PXR2/2 and littermate control mice measured by BrdU incorporation; n = 4. Data were analyzed using a Student t test. C, Flow cytometry analysis showing the production of IFN-g by CD4+ T lymphocytes in the skin draining lymph nodes of PXR2/2 and littermate control mice 4 d after skin sensitization with 2,4,6-trinitro-1-chlorobenzene picryl chloride; n = 4; representative plots are shown. Numbers in quadrants show the percentage of CD4+ IFN-g+ cells. D, Production of IL-10 by T lymphocytes stimulated with 2 mg/ml anti-mouse CD3/CD28 for 48 h, isolated from PXR2/2 or littermate control mice and analyzed by flow cytometry, as described in Materials and Methods; n = 3. Data were analyzed with a Student t test. E, Expression of CD25 on PXR2/2 or littermate control T lymphocyte cell surface isolated from spleens and stimulated with anti-mouse CD3/CD28 for 24 h was analyzed by flow cytometry, as described in Materials and Methods; n = 3. The black line designates PXR2/2 cells, the gray line PXR+/+ cells, and the dotted gray line the isotype control. 2954 ROLE OF PXR IN T LYMPHOCYTES

(Supplemental Fig. 4). Together, these results indicate that PXR level of phosphorylated p38 MAPK and JNK/SAPK in activated ligand treatment compromised T lymphocyte function by sup- T lymphocytes was not significantly altered by PCN treatment at pressing CD25 expression and IFN-g production. any time point (data not shown). Recently, it was reported that LXR activation decreases T lymphocyte proliferation by inducing PXR deficiency activates T lymphocytes SULT2B1, which would decrease cholesterol availability, a limit- We next investigated whether T lymphocyte function is altered in ing factor for T lymphocyte proliferation (29). Therefore, we as- 2/2 PXR-deficient cells. We found that PXR T lymphocyte pro- sessed the expression of this in our experimental liferation was significantly increased when compared with litter- conditions. PXR activation did not affect the expression of mate controls in vitro (Fig. 3A) and in vivo (Fig. 3B). To SULT2B1 after 18 and 24 h of cell culture, nor that of cleaved determine, whether PXR deficiency resulted in altered cytokine caspase 3 after 48 h, showing that PXR activation does not alter production in activated T lymphocytes, we performed intracellular cholesterol availability or level of the proapoptotic protein cleaved 2/2 cytokine staining, which demonstrated that PXR T lympho- caspase 3 in proliferating T lymphocytes (data not shown). In cytes produced more IFN-g (Fig. 3C) and less IL-10 (Fig. 3D). In conclusion, PXR activation alters T lymphocyte proliferation, 2/2 addition, spleen-derived PXR T lymphocytes mildly activated presumably owing to inhibition of MEK1/2 and the p65-NF-kB + by 50 ng/ml PMA exhibited a higher percentage of CD25 cells pathways. (PXR+/+, 21.9% 6 3.9%, n = 3; PXR2/2, 45.5% 6 1.2%, n =3, PXR regulates SOCS-1 but not T-bet p = 0.004). We also observed increased CD25 expression upon anti-CD3/CD28 stimulation (Fig. 3E). The altered T lymphocyte Because T-bet and SOCS-1 are master switches of IFN-g pro- phenotype in PXR-deficient animals was not observed under duction by T lymphocytes (30–32), we next determined whether baseline conditions. Increased T lymphocyte activation only be- PXR activation alters T-bet and SOCS-1 expression. T lympho- Downloaded from came apparent upon stimulation. Additional immune parameters, cytes were stimulated for various time periods in the presence of including the size, cellularity, and architecture of spleen and PCN or vehicle. Whereas T-bet was not altered by PXR activation lymph nodes, were not altered in PXR-deficient mice at baseline (Fig. 5A), SOCS-1 was increased in PCN-treated T lymphocytes (data not shown). These results demonstrate that PXR deficiency (Fig. 5B) in a PXR-dependent manner (Fig. 5C), suggesting leads to a hyperresponsiveness of T lymphocytes to inflammatory a regulation of SOCS-1 expression by PXR. Concomitant acti- stimuli. nomycin D treatment blocked the PCN-induced increase in SOCS- http://www.jimmunol.org/ 1, indicating transcriptional regulation (Fig. 5C). Moreover, we PXR activation inhibits MEK1/2 and NF-kB signaling assessed the amount of SOCS-1 transcripts in activated T lym- Activation of NF-kB and MAPKs produces important signals for phocytes by quantitative PCR following treatment with vehicle or T lymphocyte proliferation (25–28). Therefore, we next assessed PCN. We found that PCN increased the amount of SOCS-1 mRNA changes in phospho-p65 NF-kB, phospho-SAPK/JNK, phospho- (∼3.6 times) after 1 h (Fig. 5D). Furthermore, in Jurkat cells, MEK1/2, and phospho-p38 MAPK expression in activated SOCS-1 expression culminated 2–3 h after CD3/CD28 stimulation T lymphocytes treated with the PXR activator PCN or vehicle for (Supplemental Fig. 6A) and PXR activation maintained high levels various time periods. Our results show that the level of phos- of SOCS-1 (Supplemental Fig. 6B). Again, to ensure that PXR phorylated p65 NF-kB and MEK1/2 in activated T lymphocytes was required, this experiment was also done in PXR2/2 by guest on October 2, 2021 was decreased by PCN treatment after 24 h of cell culture (Fig. T lymphocytes. In the absence of PXR, the ability of PCN to 4B, Supplemental Fig. 5A) in a PXR-dependent manner (Fig. 4B), induce SOCS-1 was lost (Fig. 5D). Because these data strongly but not at earlier time points (15–60 min, Fig. 4A). Similar results supported that PCN-induced SOCS-1 expression was directly were found in Jurkat cells (Supplemental Fig. 5B). In contrast, the mediated by PXR, we transfected Jurkat cells with pLUC

FIGURE 4. PXR activation inhibits MEK1/2 and NF-kB signaling. T lym- phocytes isolated from PXR2/2 or lit- termate control mouse spleens were stimulated in the presence of vehicle or 10 mM PCN. Amount of phosphoproteins in T lymphocytes was determined by flow cytometry, and T lymphocytes were counterstained with an anti-mouse CD3 mAb. A, Time course expression of phosphorylated p65 NF-kB and MEK1/2. Two independent experiments were done. Results are expressed as percentage of controls 15 min poststimulation. B, Level of phosphorylated p65 NF-kB and MEK1/2 24 h poststimulation; n = 3–8. Data were analyzed using a Student t test. The Journal of Immunology 2955

FIGURE 5. PXR regulates SOCS-1 but not T-bet expression. A, Expres- sion of T-bet by mouse T lymphocytes stimulated with 50 ng/ml PMA + 50 ng/ml LPS + 5mg/ml CpG for 6 and 24 h in the presence of vehicle or 10 mM PCN was analyzed by flow cytometry. Data were analyzed using a Student t test; n = 3–4. B, Expres- sion of SOCS-1 by mouse T lympho- cytes stimulated with PMA + LPS + CpG, as in A, for 60 or 120 min. in the presence of PCN or vehicle was ana- lyzed by Western blotting (80 mg protein/lane), as described in Materi- als and Methods; n = 3. Lysate from Jurkat cells (50 mg protein/lane)

transfected with SOCS-1 was used as Downloaded from positive control. C, Expression of SOCS-1 by PXR2/2 or PXR+/+ T lymphocytes stimulated with PMA + LPS + 5 CpG, as in A, for 2 h in the presence of vehicle, PCN, or PCN + 5 mg/ml actinomycin D was analyzed by Western blotting (80 mg protein/ http://www.jimmunol.org/ lane), as described in Materials and Methods. Independent experiments were carried out in duplicate. Lysate from Jurkat cells transfected with SOCS-1 was used as positive control. D, Expression of SOCS-1 mRNA by PXR+/+ or PXR2/2 T lymphocytes stimulated with PMA + LPS + CpG, as in A, for 60 min in the presence of by guest on October 2, 2021 PCN or vehicle was determined by quantitative PCR analysis; n =3.E, PXR inducibility by SOCS-1 pro- moter luciferase reporter assay in transiently transfected Jurkat cells. Cells were treated with vehicle or 10 mM rifampicin. Values are the mean of two independent experiments done in triplicate. Data were normalized to the vehicle-treated values and analyzed using a Student t test. ppp , 0.01.

containing the human SOCS-1 promoter sequence. We cultivated in the adaptive immune response. The combination of PXR ago- cells in the presence of vehicle or the human PXR activator ri- nists with PXR2/2 animals allowed us to dissect the role of PXR fampicin for 24 h, and we observed a significant increase in - in T lymphocytes in detail. ative luciferase activity following rifampicin treatment when We demonstrate that pharmacologic activation of PXR inhibits compared with vehicle (Fig. 5E). Together, these results identify T lymphocyte function. PXR agonists inhibit the expression of SOCS-1 as an early signal in PXR-mediated suppression of T CD25, a T lymphocyte activation marker, as well as synthesis and lymphocyte function. production of the Th1 cytokine IFN-g by T lymphocytes in a PXR- dependent manner. Moreover, pharmacologic PXR activation Discussion dramatically reduces the ability of T lymphocytes to proliferate in By regulating the transcription of a variety of genes, PXR is response to a strong immune stimulation. Combined with prior a powerful integrator of endogenous and exogenous metabolic and publications reporting immunosuppressive effects of rifampicin xenobiotic cues. The reports of PXR expression in T lymphocytes (8–12, 33, 34), there is now accumulating evidence that PXR (6, 7) and our initial observation of a marked increase following activation suppresses T lymphocyte function. Notably, the possi- immune activation (Fig. 1) prompted us to further explore its role ble involvement of the in mediating the 2956 ROLE OF PXR IN T LYMPHOCYTES immunosuppressive effects of PXR activators (35) was previously skin disease (13–15). The present study, by demonstrating dismissed (36). a downregulation of IFN-g production and a decrease in NF-kB Because PXR is mostly known for regulating drug metabolism, and MEK1/2 preceded by increased SOCS-1 in activated T lym- our results raise the question of how PXR exerts its anti-in- phocytes upon PXR activation, can explain, at least in part, such flammatory effects. Recent reports demonstrated a mutual re- beneficial use of rifampicin in psoriasis. pression between PXR and NF-kB (16). Indeed, in hepatocytes PXR activation decreased the transcriptional activity of NF-kB, Acknowledgments and reciprocally NF-kB activation inhibited both PXR activity and We thank Daniel Hebenstreit for the human SOCS-1 promoter pLUC. We CYP3A4 (16). Moreover, inhibition of NF-kB kindly thank Dr. Christine Heufler for providing lysates of Jurkat cells activity by IkBaM was able to rescue repressed PXR activity and transfected with SOCS-1 and Dr. Christiane Ruedl for the gift of X63- to enhance PXR-mediated CYP3A4 expression (16). In our work, Ag8 cells. Mouse breeding was supported by Dr. Franz Koch. We are in- we additionally show that PCN decreases the amount of active debted to Markus Forstner for excellent technical assistance, and we thank NF-kB and MEK1/2 in T lymphocytes and that this requires the Drs. Nikolaus Romani, Christine Heufler, and Hartmut Glossmann for presence of PXR because PCN does not decrease levels of active valuable discussions. NF-kB and MEK1/2 in PXR2/2 T lymphocytes. It is possible that the anergy of T lymphocytes induced by PXR activation originates Disclosures from the inhibition of both NF-kB and MEK1/2. We observed The authors have no financial conflicts of interest. these effects at late (24 h) rather than early (15–60 min) time points after T lymphocyte activation. Similarly, Zhou et al. (16)

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