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Halide Tuna,* Rita G. Avdiushko,* Vishal J. Sindhava,† Leia Wedlund,* Charlotte S. Kaetzel,* ...... tion by naturally occurring TNF/iNOS-producing dendritic cells.
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Regulation of the mucosal phenotype in dendritic cells by PPAR!: role of tissue microenvironment Halide Tuna,* Rita G. Avdiushko,* Vishal J. Sindhava,† Leia Wedlund,* Charlotte S. Kaetzel,* Alan M. Kaplan,* Subbarao Bondada,* and Donald A. Cohen*,1 *University of Kentucky, College of Medicine, Department of Microbiology, Immunology and Molecular Genetics, Lexington, Kentucky, USA; and †University of Pennsylvania, Perelman School of Medicine, Department of Pathology and Laboratory Medicine, Philadelphia, Pennsylvania, USA RECEIVED JULY 25, 2013; REVISED OCTOBER 28, 2013; ACCEPTED OCTOBER 28, 2013. DOI: 10.1189/jlb.0713408

ABSTRACT Mucosal DCs play a critical role in tissue homeostasis. Several stimuli can induce a mucosal phenotype; however, molecular pathways that regulate development of mucosal DC function are relatively unknown. This study sought to determine whether PPAR! contributes to the development of the “mucosal” phenotype in mouse DCs. Experiments demonstrated that PPAR! activation in BMDCs induced an immunosuppressive phenotype in which BMDCs had reduced expression of MHC class II and costimulatory molecules, increased IL-10 secretion, and reduced the ability to induce CD4 T cell proliferation. Activation of PPAR! enhanced the ability of BMDC to polarize CD4 T cells toward iTregs and to induce T cell expression of the mucosal homing receptor, CCR9. Activation of PPAR! increased the ability of BMDCs to induce T cell-independent IgA production in B cells. BMDCs from PPAR!!DC mice displayed enhanced expression of costimulatory molecules, enhanced proinflammatory cytokine production, and decreased IL-10 synthesis. Contrary to the inflammatory BMDC phenotype in vitro, PPAR!!DC mice showed no change in the frequency or phenotype of mDC in the colon. In contrast, mDCs in the lungs were increased significantly in PPAR!!DC mice. A modest increase in colitis severity

Abbreviations: 9cRA#9-cis-retinoic acid, A#anti-sense, APRIL#a proliferation-inducing ligand, BAFF#B cell-activating factor, BLys#B lymphocyte stimulator, BM#bone marrow, BMDC#bone marrow-derived DC, DAI#disease activity index, DSS#dextran sulfate sodium, FoxP3#forkhead box P3, HGF#hepatocyte growth factor, IEC#intestinal epithelial cell, iTreg#induced regulatory T cell, KO#knockout, LP#lamina propria, LysM#lysozyme M, M"#macrophage, mDC#myeloid DC, MLN#mesenteric LN, PAP#pulmonary alveolar proteinosis, PPAR!#peroxisome proliferator-activated receptor !, PPAR!!DC#mice with a Cre-Lox conditional deletion of peroxisome proliferator-activated receptor ! in CD11c" cells, RA#retinoic acid, Rosi#rosiglitazone, RXR#retinoid X receptor, S#sense, SEA#staphylococcal enterotoxin A, Treg#regulatory T cell, TSLP#thymic stromal lymphopoietin, VDR#vitamin D receptor, VIP#vasoactive intestinal peptide The online version of this paper, found at www.jleukbio.org, includes supplemental information.

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was observed in DSS-treated PPAR!!DC mice compared with control. These results indicate that PPAR! activation induces a mucosal phenotype in mDCs and that loss of PPAR! promotes an inflammatory phenotype. However, the intestinal microenvironment in vivo can maintain the mucosal DC phenotype of via PPAR!-independent mechanisms. J. Leukoc. Biol. 95: 471– 485; 2014.

Introduction Mucosal DCs play a critical role in regulation of immune homeostasis in the gut and lungs and represent the only cells in the intestinal tract capable of effectively inducing an adaptive immune response to commensal or pathogenic microorganisms [1]. Effective regulation of the adaptive immune system is evident in the intestinal tract by the finely tuned adaptive responses to commensal microorganisms, in which excessive entry of bacteria into submucosal tissue is prevented without elimination of valuable luminal commensal organisms. This dampened response to commensals must be ready to expand rapidly toward strong, protective responses when pathogenic microorganisms gain access to the intestinal tract. Similarly, mucosal DCs of the lung normally prevent development of tissue-destructive responses to inhaled innocuous particles but remain poised to respond vigorously to infectious microorganisms [2]. The understanding of the biological and molecular mechanisms that regulate the mucosal phenotype of DCs is important if targeted therapies are to be developed to reverse uncontrolled adaptive immune responses toward commensal microorganisms that occur during inflammatory bowel diseases or against inhaled innocuous particles. We have shown previously in mice that depletion of CD11c" mDCs, beginning before induction of DSS colitis, leads to more severe intestinal disease, illustrating an important immunoregulatory role for resident mDCs in the intestines [3].

1. Correspondence: Dept. of Microbiology, Immunology and Molecular Genetics, University of Kentucky, 800 Rose St., Room MS419, Lexington, KY 40536-0298, USA. E-mail: [email protected]

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Studies from other labs have demonstrated a number of biological mechanisms that can induce this immunoregulatory phenotype in vivo in the intestine and in vitro in BMDCs and monocyte-derived DCs. For example, functional differences between intestinal and splenic mDCs [4, 5] point toward a role of the intestinal microenvironment in inducing and maintaining a mucosal DC phenotype. CD103" DCs of the LP are much more effective than splenic DCs at inducing Tregs in vitro. Addition of exogenous TGF-# and RA was shown to increase Treg development induced by splenic mDCs [4], suggesting that the presence of these molecules in the intestine contributes to development of a mucosal phenotype in mDCs. Similarly, splenic DCs do not induce T cell-independent IgA production by B cells, as can be induced by CD103" LP DCs. However, supplementing splenic DCs with exogenous RA enhances their ability to induce T cell-independent IgA synthesis [6], suggesting that RA production by mucosal DCs is a characteristic imposed by the intestinal microenvironment. Several cytokines and growth factors, including IL-10, IL-6, TGF-#, and HGF, as well as VIP and agonists for the VDR, have been shown to induce an immunoregulatory phenotype in mDCs [7–13]. In addition,TSLP, which is expressed by IECs and by intestinal DCs in response to commensal bacteria [14], has been shown to activate Th2-inducing mDCs and enhance their ability to drive IgA production and Treg development [15, 16]. Thus, there are a number of diverse pathways to induce a “mucosal phenotype” in mDCs. Whether any or all of these different pathways converge on a common molecular pathway is unknown. PPAR! is a ligand-activated nuclear hormone receptor that functions as a transcription factor to regulate genes having PPAR! response elements. PPAR! serves important homeostatic functions, including regulation of adipogenesis and glucose metabolism [17, 18]; however, PPAR! is also expressed at high constitutive levels in the intestinal tract and lung within epithelial and innate-immune cells, where it serves primarily an anti-inflammatory role [19 –23]. Activation of intestinal PPAR! can reduce the severity of colitis in humans and experimental animals, and it has been shown that PPAR! in IECs and M" can contribute to the protective effects [24 –27]. However, a protective role for PPAR! in intestinal and lung DCs is less clear. PPAR! is commonly activated by several factors known to reduce the severity of colitis [28, 29], as well as factors that induce a regulatory phenotype in DCs. For example, recent studies have shown that there is cross-talk between VDR and PPAR!, in that activation of VDR by the vitamin D analog 1,25-dihydroxyvitamin D3 enhances PPAR! expression in some tumor cell lines [30], and this cross-talk may be mediated by physical interactions between VDR and PPAR! [31]. In addition, the promoter of mouse PPAR!1 has been shown to contain a VDR response element [32]. Other studies have shown functional links between STAT3 and PPAR!. Most of these studies have shown that PPAR! agonists can inhibit the activation of STAT3; however, several intriguing studies demonstrated that activation of PPAR! can also be downstream of STAT3 activation. Studies by Wang et al. [33] have shown that activation of STAT3 can lead to the activation of 472 Journal of Leukocyte Biology

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PPAR! during adipogensis. Similarly, inhibition of STAT3 by protein inhibitor of activated STAT3 in 3T3-L1 preadipocytes suppressed expression of PPAR! mRNA [34], suggesting that STAT3 can regulate the activity of PPAR!. Moreover, IL-10, IL-6, HGF, and VIP (all of which induce a mucosal phenotype) use STAT3 in signal transduction [35– 39]. Thus, it is reasonable to consider that PPAR! could be a point of convergence for several pathways that can induce the immunosuppressive phenotype seen in resident DCs in mucosal tissues. The goal of this study was to determine whether PPAR! activity and expression contributed to the development of the mucosal phenotype in mDCs of the intestine and lungs. Experiments in BMDCs indicated that activation of PPAR! induced a more immunoregulatory phenotype, in which BMDCs had reduced expression of MHC II and costimulatory molecules and reduced the ability to induce proliferation of naive CD4 T cells. Activation of PPAR! in BMDCs caused a decrease of proinflammatory and an increase of anti-inflammatory cytokine synthesis. BMDCs with activated PPAR! were able to induce expression by CD4 T cells of the mucosal homing receptor, CCR9, to drive iTreg development and to induce T cell-independent IgA production in naive B cells. BMDCs from PPAR!!DC mice had a more inflammatory phenotype, including increased expression of costimulatory molecules, enhanced proinflammatory cytokine production, and decreased IL-10. In contrast to the inflammatory phenotype observed in vitro in PPAR!!DC BMDCs, mDCs, in the colons of PPAR!!DC mice, maintained a near-normal phenotype, suggesting that PPAR!independent mechanisms exist in the intestine, which also contribute to maintenance of mucosal phenotype and function in intestinal mDCs. However, the frequency of mDCs in the lungs was increased significantly in PPAR!!DC mice. These results indicate that activation of PPAR! in BMDCs is sufficient to induce a mucosal phenotype and suggest that PPAR! contributes to the development of mucosal DC phenotype and function. However, compensatory mechanisms appear to function in the intestines, which partially maintain the immunoregulatory functions of DCs in the absence of PPAR! expression.

MATERIALS AND METHODS

Mice C57BL/6-specific pathogen-free female mice (6 – 8 weeks of age) were purchased from Harlan Laboratories (Indianapolis, IN, USA). Transgenic mice expressing loxP sites flanking Exons 1 and 2 of PPAR! (B6.129-Ppargtm2Rev/J) and transgenic mice expressing Cre recombinase under control of the CD11c promoter {C57BL/6J-Tg(Itgax-cre,EGFP)4097Ach/J (Line 1) and [B6.Cg-Tg(Itgax-cre)1-1Reiz/J (Line 2)]} were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All mice were housed in microisolator cages (five/cage), provided sterile water and food ad libitum, and maintained by the Department of Laboratory Animal Resources at the University of Kentucky (Lexington, KY, USA). All animal studies were approved by the Institutional Animal Care and Use Committee and were carried out in accordance with the Animal Welfare Act.

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Generation of mice with a targeted deletion of PPAR! in CD11c" cells To generate mouse lines in which PPAR! was deleted in mDCs (PPAR!!DC), female PPAR!-floxed mice (B6.129-Ppargtm2Rev/J; PPARgflox/flox) [40] were crossed with two different CD11c-Cre strains of mice: (1) CD11cCre-EGFP [C57BL/6J-Tg(Itgax-cre,-EGFP)4097Ach/J (Jax #007567)] and (2) CD11c-Cre [B6.Cg-Tg(Itgax-cre)1-1Reiz/J (Jax #008068)]. Two lines of offspring mice were generated in which PPAR! was deleted in CD11c" cells: PPAR!!DC/Line 1 and PPAR!!DC/Line 2. F1 mice were backcrossed to generate homozygous and heterozygous KO and littermate controls, depending on the breeding scheme used. Offspring were genotyped by PCR on tail-snip DNA for expression of floxed PPAR! and Cre recombinase transgenes using the following primer sets: (1) PPAR!: (S) 5=-TGTAATGGAAGGGCAAAAGG-3=; (A) 5=-TGGCTTCCAGTGCATAAGTT-3= (product size: WT, 200 bp; floxed, 230 bp). (2) Cre recombinase: (S) 5=-ACC TGA AGA TGT TCG CGA TTA TCT-3=; (A) 5=-ACC GTC AGT ACG TGA GAT ATC TT-3= (product size, 420 bp). Excision of floxed PPAR! Exons 1 and 2 in BMDCs was detected by PCR using the following PPAR! primer set that spanned Exons 1– 4: (S) 5=-GTCACGTTCTGACAGGACTGTGTCAC-3=; (A) 5=-TATCACTGGAGATCTCCGCCAACAGC-3= (product size, WT allele, 700 bp; floxed allele, 300 bp).

Preparation of BMDCs BMDCs were generated from normal C57BL/6 or PPAR!!DC mice, as described previously [41]. Briefly, a single-cell suspension of BM was obtained by flushing femur and tibia bones, and 4 $ 106 BM cells were cultured for 9 days in 100 $ 15 mm bacterial grade petri dishes (BD Falcon; Becton Dickinson, Franklin Lakes, NJ, USA) in 10 mL RPMI, supplemented with 10% FBS (RPMI-10%) plus 20% conditioned medium from GM-CSF-transfected F10.9 cells, murine B16 melanoma cells (DC medium) [42]. BMDCs were supplemented with fresh DC medium on Days 3 and 6, and on Day 9, adherent and nonadherent fraction of cells were harvested and pooled by scraping with a rubber policeman. Cells were suspended in fresh RPMI10% and then treated with appropriate ligands as indicated. On Day 9, cultures routinely contained %85% CD11c" DCs.

Ligand activation of nuclear receptors Day 9 BMDCs were plated at 1 $ 106 cells/mL in six-well tissue-culturetreated plates (BD Falcon; Becton Dickinson). For activation of PPAR! and its requisite heterodimer partner, RXR, BMDCs were incubated for 24 h in RPMI-10% containing 10 $M Rosi (Cayman Chemical, Ann Harbor, MI, USA) and 1 $M 9cRA (Sigma-Aldrich, St. Louis, MO, USA). Control cells were cultured in the presence of diluent only, 0.01% DMSO. To obtain “mature” BMDCs, LPS (Escherichia coli 0111:B4; Sigma-Aldrich) was added at the time of ligand treatment at a final concentration of 1 $g/mL. BMDCs were harvested at indicated time-points and washed three times by centrifugation in fresh RPMI-10% to remove any residual receptor ligand.

T cell proliferation and polarization For analysis of T cell proliferation, single-cell suspensions were isolated from spleen and pooled LNs of naive mice. CD4" T cells were then purified by magnetic sorting using CD4" T cell isolation kits (Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer’s instructions. Purified CD4" T cells (1$105 cells/well) were cultured in 96-well tissue culturetreated plates (BD Falcon; Becton Dickinson) in the presence of a decreasing number of BMDCs and in the presence of 1 ng/mL SEA (Sigma-Aldrich). Purified anti-mouse IL-10R (Clone 1B1.3a; BD PharMingen, San Diego, CA, USA) was added to indicated wells at a concentration of 1 $g/ ml. Cells were incubated for 72 h at 37°C and pulsed with 5 $Ci/mL 3Hthymidine (PerkinElmer, Waltham, MA, USA) during the last 4 h of incubation. Cells were harvested onto 96-well Unifilter Plates (PerkinElmer), and the incorporation of 3H isotope into DNA was quantified on a TopCount scintillation counter (PerkinElmer).

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For analysis of T cell polarization, purified CD4 T cells (1$106 cells/ well) were added to six-well tissue culture-treated plates (BD Falcon; Becton Dickinson), which were coated previously with anti-CD3 (1 $g/mL; BioLegend, San Diego, CA, USA) for 2 h at 37°C. BMDCs (cultured overnight with the appropriate ligands) were cocultured with the T cells at a concentration of 1 $ 105 cells/well. Human rTGF-# (BioLegend) and human rIL-2 (National Cancer Institute, Bethesda, MD, USA) were added to each well at a final concentration of 5 ng/mL and 100 IU/mL, respectively. Cocultures were incubated at 37°C for 6 days. Aliquots of each culture were evaluated for T cell chemokine receptor expression by flow cytometry. The remaining cells were restimulated with PMA (10 ng/mL; Sigma-Aldrich) and ionomycin (1 $M; Calbiochem, La Jolla, CA, USA) for 24 h at 37°C. Brefeldin A (BioLegend) was added at a concentration of 5 $g/ml during the last 4 h, and cells were then evaluated by flow cytometry for intracellular expression of IFN-! and FoxP3, as described below.

Flow cytometry All antibodies for flow cytometry were purchased from BioLegend unless stated otherwise. All antibodies were used at saturating concentrations, as suggested by the manufacturer. Cells were evaluated for surface molecule expression, as described previously [41]. Briefly, FcRs on cells were blocked for 15 min on ice with anti-Fc!R mAb (anti-mouse CD16/32; BioLegend) in flow cytometry buffer (1% BSA (Sigma-Aldrich) and 0.01% sodium azide (Sigma-Aldrich). Cells were then stained for 30 min on ice with antibodies specific for the following surface markers: CD11c, CD80, CD86, MHC II, or CCR9. For intracellular expression of IFN-! and FoxP3, CD4" T cells were harvested from 6-day BMDC:naive CD4 T cell cocultures and restimulated for 24 h at 37°C in RPMI-5% (RPMI containing 5% FBS, 50 $M 2-ME), plus PMA (10 ng/mL) and ionomycin (1 $M; Calbiochem). Brefeldin-A (BioLegend) was added to the cultures (5 $g/ml) during the last 4 h of incubation to block protein secretion and allow intracellular cytokine accumulation. Cells were washed and stained for surface markers, as described above, and then fixed and permeabilized with commercial immunocytochemistry staining kits, according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA). Fixed and permeabilized cells were then stained for intracellular IFN-! and FoxP3 (eBioscience) for 30 min at 4°C, washed by centrifugation with permeabilization buffer, and resuspended in flow cytometry buffer for analysis on a FACSCalibur cell analyzer (BD Biosciences, San Jose, CA, USA).

B cell proliferation and IgA production B cells were purified from spleens of C57BL/6 mice by magnetic sorting using a B220-positive selection kit (Miltenyi Biotec). Purified B cells (1$105 cells/well) were cultured in RPMI-5%, containing LPS (2.5 $g/mL) in the presence of decreasing numbers of BMDCs. Cocultures were incubated for 48 h at 37°C and then pulsed with 5 $Ci/mL 3H-thymidine during the last 4 h of culture. Incorporation of 3H isotope into DNA was quantified on a TopCount scintillation counter (PerkinElmer). To assay for antibody secretion, purified B cells (1$105 cells/well) were plated in 96-well tissue culture-treated plates in the presence of LPS (2.5 $g/mL) and the indicated number of BMDCs. Cocultures were incubated for 5 days at 37°C. Supernatants were evaluated for total IgA by ELISA in anti-mouse IgA (Bethyl Laboratories, Montgomery, TX, USA)-coated microtiter plates, as described previously [43]. IgA in culture supernatants was captured by overnight incubation, followed by detection with anti-mouse IgA-HRP (Bethyl Laboratories). Known concentrations of purified mouse IgA were included as a standard.

Statistical analyses Significant differences in measured outcomes in response to genotype differences and/or specific treatments were determined by repeated-measures ANOVA or factorial ANOVA, with post hoc Fisher’s protected least-significant difference test or Student’s t-test. Statistical analyses were conducted using StatView software (SAS Institute, Cary, NC, USA).

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RESULTS

PPAR! activation promotes an immunoregulatory phenotype in BMDCs During homeostasis, mucosal DCs are typically found in an immature state characterized by low-level expression of MHC II and costimulatory molecules. The immature DC phenotype is also characteristic of immunoregulatory DCs, which are poorly stimulatory for naive CD4 T cells but efficient inducers of Treg differentiation [44 – 46]. To determine whether activation of PPAR! promotes an immature immunoregulatory phenotype, BMDCs were treated in vitro with PPAR! agonists and evaluated for expression of mucosal mDC phenotype markers. As full activation of genes containing PPAR response elements requires DNA binding of PPAR! and its requisite heterodimer partner RXR, BMDCs were treated in vitro with Rosi, a synthetic PPAR! agonist, and 9cRA, an agonist for RXR. Normal C57BL/6 BMDCs were treated with Rosi plus 9cRA or DMSO diluent control in the presence or absence of LPS (to induce maturation) for 24 h. BMDCs were analyzed by flow cytometry for expression of MHC II and costimulatory molecules, CD86 and CD80, and culture supernatants were assayed by ELISA for the presence of anti-inflammatory cytokine IL-10 and proinflammatory cytokines IL-6 and TNF-%. As expected, BMDCs treated with LPS (mature), regardless of PPAR! activation status, had markedly higher expression of CD80, CD86, and MHC II compared with cells not treated with LPS (immature; Fig. 1A). However, treatment of samples with LPS in the pres-

ence of Rosi/9cRA resulted in a significantly decreased expression of CD80 and CD86 compared with LPS-treated DMSO controls, indicating a more immature phenotype. Although the difference in MHC II expression was not statistically significant, LPS-treated, PPAR!-activated BMDCs consistently showed a trend toward lower expression levels of MHC II compared with the LPS-treated DMSO controls. In the absence of LPS stimulation, BMDCs secreted nondetectable amounts of IL-6, TNF-%, and IL-10; however, activation of PPAR! induced low levels of IL-10. In LPS-matured BMDCs, activation of PPAR! caused a significant increase in IL-10 release compared with DMSO control treatment (Fig. 1B). Expression of IL-6 and TNF-% trended toward lower levels following PPAR! activation.

PPAR! activation promotes mucosal DC-like functions in BMDCs The ability of mucosal mDCs under homeostatic conditions to drive Treg differentiation in naive CD4 T cells is a critical feature of their immunoregulatory armament in the intestinal tract. However, mucosal mDCs also possess a unique ability to present antigens in their native form directly to naive B cells to induce IgA synthesis in a T cell-independent fashion [47, 48]. The ability of PPAR! activation to affect the ability of BMDCs to support CD4 T cell proliferation was evaluated with normal splenic CD4 T cells cultured in the presence of SEA (Fig. 2). Purified CD4 T cells in the absence of added BMDCs proliferated poorly to SEA, as optimal proliferation requires a

Figure 1. Activation of PPAR! induces an immature phenotype in BMDCs. BMDCs from normal C57Bl/6 mice were matured with 1 $g/mL LPS for 24 h in the presence of 10 $M Rosi and 1 $M 9cRA (Rosi/9cRA) or DMSO control. Culture supernatants (n#3) were assayed by ELISA for IL-10, TNF-%, and IL-6 (B). Cells were harvested (n#3) and assayed by flow cytometry for expression of CD80, CD86, and MHC II (A). *P & 0.05 Student’s t-test between DMSO- and Rosi/9cRA-treated samples. Data are representative of two to three independent experiments.

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Figure 2. BMDC with activated PPAR! have reduced ability to induce T cell proliferation. BMDCs from normal C57Bl/6 mice were cultured for 24 h in the presence of 10 $M Rosi and 1 $M 9cRA or DMSO control and in the presence of 1 $g/mL LPS. Splenic CD4" T cells from normal C57Bl/6 mice (1$105 cells/well) were cultured with BMDCs for 3 days at a ratio of 8:1 or 16:1 in the presence of SEA. Purified anti-mouse IL-10R antibody (Clone 1B1.3a; BD PharMingen) was added to the indicated wells at a final concentration of 1 $g/ml. Samples were pulsed with 5 $Ci/mL 3H-thymidine for the last 4 h of culture and analyzed for proliferation. Results are presented as mean ' se of triplicates. *P & 0.01 [twoway ANOVA with post hoc test (Fisher’s protected least-significant difference)] DMSO LPS- and Rosi/9cRA LPS-treated samples. Data are representative of three independent experiments.

source of MHC II" APCs [49]. The addition of LPS-matured, DMSO-treated BMDCs enhanced proliferation of CD4 T cell in response to SEA in a dose-dependent manner. LPS-matured BMDCs with activated PPAR! were significantly less efficient in promoting CD4 T cell proliferation at all BMDC:T cell ratios (P&0.01), consistent with their immature phenotype. The reduced ability to support CD4 T cell proliferation appeared a result of IL-10 production, as the addition of anti-mouse IL10R antibody (Clone 1B1.3a; BD PharMingen) completely restored their ability to induce T cell proliferation. IL-10 from PPAR!-activated BMDCs likely caused the T cell inhibition, although we cannot exclude IL-10 release from T cells in the cultures. T cell polarization cultures were set up to determine whether activation of PPAR! altered the capacity of BMDCs to induce Th1 and Tregs. DMSO-treated or PPAR!-activated immature or LPS-matured BMDCs were incubated with splenic CD4 T cells for 6 days in anti-CD3-coated culture plates in the presence of exogenous TGF-# (5 ng/ml) plus rIL-2 (100 IU/ml). To analyze intracellular expression of IFN-! and Foxp3, T cells from Day 6 cocultures were stimulated with PMA and ionomycin for 24 h, with Brefeldin A added during the final 4 h of culture to allow intracellular accumulation of IFN-!. T cells cultured with immature or LPS-matured, PPAR!-activated BMDCs had a significantly higher expression of FoxP3 transcription factor compared with the T cells cultured with DMSO control BMDCs (Fig. 3A). The increase in number of FoxP3" T cells was a result of an increased T cell differentiation rather than stabilization of Treg survival, as the www.jleukbio.org

Figure 3. PPAR!-activated BMDCs induce a regulatory mucosal phenotype in T cells. Splenic CD4" T cells from C57Bl/6 mice (1$106 cells/well) were cultured with C57Bl/6 BMDCs at a ratio of 10: 1 in the presence of plate-bound 1 $g/mL anti-CD3, 100 IU/mL rIL-2, and 5 ng/mL TGF-# for 6 days. For analysis of intracellular Foxp3 and IFN-! (A and B), Day 6 cultures were stimulated with 10 ng/mL PMA and 1 $M ionomycin in the presence of Brefeldin A (5 $g/ml) to promote intracellular accumulation of IFN-!. FoxP3 and IFN-! expression was determined by flow cytometry. CCR9 expression on T cells (C) was determined by flow cytometry. Results are presented as mean percentage of CD4" T cells ' se of triplicates. *P & 0.05 Student’s t-test between DMSO- and Rosi/9cRA-treated samples. Data are representative of two independent experiments.

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average percentage (n#9 mice) of FoxP3 in freshly isolated CD4 T cells from normal C57Bl/6 mice was only 5.4% ' 0.5% (data not shown). In contrast, T cells cultured with PPAR!-activated BMDCs (immature and LPS-matured) had a lower percentage of IFN-!-positive cells compared with those cocultured with control BMDCs (Fig. 3B), indicating that BMDCs with activated PPAR! functioned in vitro in a manner similar to mucosal mDCs with a greater capacity to drive naive CD4 T cells toward Treg development. To evaluate changes in expression of chemokine receptors, T cells from Day 6 CD4 T cell:BMDC cocultures were evaluated by flow cytometry for expression of CCR5, CCR7, and CCR9. Expression of CCR9 is the primary chemokine receptor for migration to mucosal tissue sites [50]. However, there is evidence that CCR5 can be used for migration to intestinal tissues under some conditions [50, 51], whereas CCR7 expression allows migration to the peripheral LNs [52]. All three receptors were detected at low levels on cocultured T cells. The percentage of CD4 T cells expressing CCR5 and CCR7 did not differ between T cells cocultured

with PPAR!-activated or DMSO-treated control BMDCs (unpublished results). In contrast, a significantly greater fraction of T cells cocultured with PPAR!-activated, LPS-matured BMDCs expressed CCR9 compared with the T cells treated with DMSOtreated control BMDCs (Fig. 3C), indicating a shift toward T cells with mucosal tissue-homing potential. Interestingly, immature BMDCs treated with Rosi/9cRA were not able to increase expression of CCR9. We and others have shown that immature BMDCs can inhibit proliferation of B cells [43, 53] and that normal intestinal mDCs, which have an immature phenotype, are effective at inducing T cell-independent IgA production by B cells [48, 54]. To determine whether activation of PPAR! induces similar functions in BMDCs, normal splenic B cells were activated with LPS and cocultured with immature or LPS-matured BMDCs for 2 days in vitro. As reported by us previously [43], immature BMDCs potently inhibited LPS-induced proliferation of naive B cells (Fig. 4A) to a greater extent than did mature

Figure 4. PPAR!-activated BMDCs inhibit B cell proliferation and promote IgA production. BMDCs from normal C57Bl/6 mice were cultured for 24 h in the presence of 10 $M Rosi and 1 $M 9cRA or DMSO control with (LPS-matured BMDC) or without (immature BMDC) the addition of 1 $g/mL LPS. Splenic C57Bl/6 B cells were cultured with immature (A) or LPS-matured (B) BMDCs at decreasing numbers in the presence of exogenous LPS (2.5 $g/mL) for 2 days. Samples were pulsed with 5 $Ci/mL 3H-thymidine for the last 4 h of culture and analyzed for proliferation. Results are presented as mean ' se of triplicates. Data are representative of three independent experiments. Similarly prepared samples (C; immature) and (D; LPS-matured) were cultured for 5 days, and supernatants were analyzed for the presence of IgA by ELISA. Results are presented as mean ' se of triplicates. *P & 0.05 Student’s t-test between DMSO- and Rosi/9cRA-treated samples. Data are representative of two independent experiments.

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BMDCs (Fig. 4B). PPAR!-activated, LPS-treated BMDCs, which have a more immature phenotype, had a significantly greater ability to inhibit B cell proliferation compared with LPSmatured, DMSO-treated BMDCs (Fig. 4B). The addition of blocking antibodies to IL-10R did not reverse this inhibition, whereas neutralizing antibody to TGF-# only partially blocked B cell inhibition (unpublished results), suggesting a possible dependency on cell contact, as reported previously for immature BMDCs [43]. To investigate whether PPAR! activation provided BMDCs with an ability to promote IgA production in B cells, naive B cells were activated with LPS and cocultured with immature or LPS-matured BMDCs for 5 days. Culture supernatants were collected and quantified for IgA content by ELISA (Fig. 4C and D). Supernatants isolated from B cells stimulated with immature BMDCs contained low but detectable concentrations of IgA, and IgA levels were significantly higher in cultures containing the greatest number of PPAR!-activated BMDCs (Fig. 4C). B cells stimulated with PPAR!-activated, mature BMDCs also had a significantly greater ability to induce T cell-independent IgA production in vitro, even at concentrations eightfold lower than immature BMDCs (Fig. 4D), consistent with a more mucosal DC phenotype being induced following activation of PPAR!. The ability of mDCs to induce T cell-independent IgA production is dependent on secretion of several factors, including BAFF (BLyS) and APRIL and TGF-# [55]. Expression of BAFF and APRIL was evaluated by immunoblot in immature and LPS-matured BMDCs during activation of PPAR!. Treatment with Rosi did not alter the expression of BAFF and APRIL protein significantly in immature or LPS-matured BMDCs (unpublished results), suggesting that protein levels of BAFF and APRIL are unaffected by PPAR! activity in BMDCs. These results indicate that activation of PPAR! in BMDCs induces functional abilities that are consistent with those seen in mucosal mDCs, including an ability to promote Treg and inhibit Th1 cell development and the ability to promote T cellindependent IgA synthesis by B cells.

Selective deletion of PPAR! in CD11c" cells promotes a more mature, inflammatory phenotype in BMDCs As part of studies to investigate the role of PPAR! in mDCs in vivo, conditional deletion transgenic mouse lines were developed in which PPAR! was selectively deleted in CD11c" cells. PPAR!-floxed mice [40] were crossed with two different CD11c-Cre strains of mice: (1) CD11c-Cre-EGFP [56] and (2) CD11c-Cre [57]. Two lines of offspring mice were generated in which PPAR! was deleted in CD11c" cells: (1) PPAR!!DC (Line 1) and (2) PPAR!!DC (Line 2). It should be noted that Line 2 was generated and analyzed at a later time, as the original Line 1 was lost during initial studies. PCR analysis of total RNA from BMDCs of homozygous, heterozygous, and WT littermates showed that Lines 1 and 2 had the expected patterns for the PPAR! gene (Fig. 5, top and middle). WT littermates expressed only the 700-bp WT allele, and homozygous offspring expressed only the 300-bp excised mutant allele, whereas BMDCs from heterozygous mice expressed both alleles. As Exons 1 and 2 are excised, the mutant alleles cannot www.jleukbio.org

Figure 5. Expression of truncated PPAR! gene in BMDCs from PPAR!#DC mice. BMDCs from Line 1 (top panel) and line 2 (middle panel) were generated. RNA was isolated from Day 9 BMDCs, reversetranscribed into cDNA, and amplified for expression of PPAR!. RNA was also isolated from IECs (bottom panel) of the colons of normal CD57Bl/6 mice for analysis of PPAR! gene expression. The PPAR! primers amplify a region spanning Exons 1– 4, which encompasses the loxP sites that flank Exons 1 and 2 of the PPAR! gene. Amplicons (700 and 300 bp) indicate expression of the full-size or truncated PPAR! gene, respectively. Data are representative of two independent studies.

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synthesize functional PPAR! protein [40]. As a control, PCR analysis was performed on RNA from IECs isolated from colons of Line 2 (Fig. 5, bottom), which demonstrated that excision of PPAR! did not occur in cells that do not express CD11c. Expression of the costimulatory molecules, CD80 and CD86, was evaluated on untreated and LPS-stimulated BMDCs from PPAR!!DC mice (Fig. 6A and B). BMDCs from homozygous PPAR!!DC mice (PPAR!(/() of Lines 1 and 2 expressed greater levels of CD80 and CD86 compared with littermate controls (PPAR!"/"), although enhanced expression by immature BMDCs (no LPS) was not evident in BMDCs from Line 2. Heterozygous BMDCs (PPAR!"/() tended to display an intermediate increase in expression. Significantly enhanced expression of MHC II was observed in LPS-matured PPAR!null BMDCs of Line 2 compared with WT littermates (Fig. 6C), but these effects were not evident in BMDCs from Line 1. BMDCs from PPAR!!DC mice were also evaluated for expression of TNF-% and IL-10. LPS-stimulated BMDCs from Line 1 homozygous PPAR!!DC mice produced significantly more TNF-% than BMDCs from WT littermates (Fig. 6D). Line 2 PPAR!!DC BMDCs also showed increased TNF-% production, but the differences did not reach statistical significance. In contrast, the level of IL-10 induced in BMDCs by LPS was significantly lower in PPAR!!DC BMDCs from Lines 1 and 2 compared with control BMDCs (Fig. 6E). Note that endogenous levels of IL-10 that were observed in control BMDCs were undetectable in PPAR!!DC BMDCs. Overall, these results indicate that PPAR! deletion enhanced expression of MHC II and costimulatory molecules on BMDCs; however, effects were generally more pronounced in Line 2 BMDCs. Deletion of PPAR! in BMDCs generally caused a more inflammatory cytokine expression pattern, such that LPS induced greater amounts of TNF-% and reduced levels of IL-10 compared with WT BMDCs. It may appear from a comparison of results for Lines 1 and 2 that differences exist between Lines 1 and 2 in magnitude and pattern of expression of MHC II and costimulatory molecules and cytokines. In multiple studies, PPAR! deletion always demonstrated a more inflammatory phenotype in BMDCs; however, the mean fluorescent intensity varied between experiments. We do not think that these differences reflect stable phenotypic differences between Lines 1 and 2; however, we cannot exclude this possibility. Phenotypic differences between mouse lines with Cre-Lox deletion of the same gene have been observed previously and have been shown to be a result of incomplete Cre-mediated excision [58]. We did observe equal levels of PPAR! gene deletion in both lines (Fig. 5), suggesting that differences in PPAR! gene excision do not account for these phenotypic differences. To determine the ability of BMDCs from PPAR!!DC mice to act as APCs, OVA-specific CD4" T cells from OT-II mice were cultured for 3 days in the presence or absence of OVA-pulsed BMDCs from homozygous PPAR!!DC mice (Line 1) or WT littermate controls and evaluated for proliferation by 3H-thymidine incorporation (Fig. 7). OVA-specific T cell proliferation was enhanced significantly by BMDCs with a PPAR! deletion at all BMDC:T cell ratios compared with BMDCs with WT expression, indicating that the elevated expression of MHC II 478 Journal of Leukocyte Biology

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and costimulatory molecules likely enhanced antigen-presentation capacity by BMDCs from PPAR!!DC mice.

PPAR!#DC mice develop enhanced DSS colitis Given that deletion of the PPAR! gene in CD11c" cells caused BMDCs to display a more inflammatory phenotype in vitro, colitis studies were performed in PPAR!!DC mice to determine if this phenotype affected the development of colitis. DSS-induced colitis is typically considered to be mediated by innaterather than adaptive-immune cells, where intestinal M" play a prominent role. However, recent reports have shown that Th17 cells accumulate in colons of DSS-treated mice [59] and may contribute to the pathogenesis of DSS colitis [60, 61]. Moreover, our lab has shown that depletion of resident CD11c" mDCs significantly enhanced the severity of DSS colitis [3]. In separate studies, Lines 1 and 2 PPAR!!DC mice were placed on 3% or 4% DSS, respectively, in their drinking water and monitored for the development and severity of colitis (Fig. 8). Homozygous Line 1 PPAR!!DC mice developed colitis significantly more rapidly (P#0.0272) and to greater severity (P#0.0362) than DSS-treated control mice, as indicated by the DAI (Fig. 8, upper). In contrast, colitis in Line 2 developed more slowly, and disease severity was not statistically greater in PPAR!!DC mice. The only time-point showing a significant difference was Day 9, the last day of the study (Fig. 8, lower). In repeat studies with both lines of PPAR!!DC mice, it was clear that colitis was only slightly more severe, as indicated by an elevated DAI, in PPAR!!DC mice compared with WT control mice; however, statistically significant differences were generally seen only at late time-points. Colons from mice killed at the completion of DSS colitis studies were also scored for histologic disease severity (Fig. 9), as described by us [3]. Histopathologic disease scores in Line 2 mice with DSS colitis were elevated significantly in PPAR!!DC mice compared with WT controls. In repeat DSS colitis studies in Lines 1 and 2, histopathology scores always trended toward more severe disease in colons from PPAR!!DC mice; however, the difference between KO and control mice did not always achieve statistical significance.

Intestinal microenvironment can override functional changes in DCs of PPAR!#DC mice The discrepancy between the increased inflammatory phenotype observed in vitro with PPAR!!DC BMDCs and the relatively minor increase of DSS colitis severity in vivo in PPAR!!DC mice led to a more in-depth analysis of myeloid cells in the intestinal tract. Flow cytometric analysis of the percentage of mDCs and M" in colons and MLNs from Line 2 mice indicated no significant difference in the frequency of these myeloid cell populations between PPAR!!DC and WT control mice, as well as no difference in the level of MHC II expression on these cell types (Table 1). The levels of MHC II expression on mDCs (CD11b", MHC II") and M"s (CD11b", MHC IIneg-low) in MLN of PPAR!!DC also did not differ from WT control mice (unpublished results). As a tissue representative of another mucosal tissue site, we also analyzed myeloid cells from the lung from Line 2 mice (Fig. 10). Flow cytometwww.jleukbio.org

Tuna et al. PPAR! regulates mucosal phenotype in myeloid DCs

Figure 6. BMDCs from PPAR!#DC mice have a more proinflammatory phenotype. BMDCs from homozygous KO, heterozygous KO, and littermate controls of Lines 1 and 2 were treated with the indicated concentrations of LPS for 24 h. Expression levels of CD80, CD86, and MHC II were analyzed by flow cytometry to determine mean fluorescent intensity (A–C). Secretion of TNF-% and IL-10 proteins by Lines 1 and 2 BMDCs (D and E) was evaluated by ELISA in culture supernatants of BMDCs treated with LPS (1 $g/ml) for 24 h or left untreated. *P & 0.05 Student’s t-test. Data are representative of two independent experiments.

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the ability to induce Treg and inhibit Th1/Th17 development by CD4 T cells, promote IgA synthesis by B cells, and secrete anti-inflammatory mediators such as IL-10, TGF-#, and indoleamine 2,3-dioxygenase. An anti-inflammatory/tolerogenic phenotype is not limited to DCs of the intestinal tract, as DCs in other mucosal tissues, such as the lung, express a similar mucosal phenotype [62– 64]. In spite of a commonality of function seen in mucosal tissue DCs, it is intriguing that such a diverse number of stimuli have been shown to induce the mucosal phenotype in DCs. This includes factors, such as IL10, IL-6, TGF-#, vitamin D metabolites, HGF, and VIP [7–11].

Figure 7. BMDCs from PPAR!#DC mice are more potent APCs. BMDCs were generated from homozygous KO, heterozygous KO, and littermate controls of Line 1 and were pulsed for 60 min at 37°C with purified OVA (100 $g/ml) and then cocultured for 3 days with splenic OT-II T cells (1$105 cells/well). Samples were pulsed with 5 $Ci/mL 3H-thymidine for the last 4 h of culture and analyzed for proliferation. *P & 0.01 [two-way ANOVA with post hoc test (Fisher’s protected least-significant difference)].

ric analysis of CD11b and CD11c expression in the CD45" fraction identified four distinct cell populations expressing these markers in the lung. Cell Subset 1 (CD11bhigh, CD11chigh), which was defined as mDCs, was increased significantly in PPAR!!DC mice compared with littermate controls. In contrast, Subset 2 (CD11blow, CD11chigh), which was defined as alveolar M"s, was reduced significantly in PPAR!!DC mice. Note that Subsets 3 and 4 (presumably containing interstitial M"s) did not differ between KO and control mice. Similar gating on cells from the MLN and colon (Fig. 10) identified a CD11bhigh, CD11chigh mDC population (Quadrant 1) and a CD11bhigh, CD11clow M" subset (Quadrant 3), in which no change in cell frequency was observed in Table 1. These results suggest that the mucosal tissue microenvironment in the intestine and lung may be different, as indicated by differential effects on CD11c" cells from PPAR!!DC mice. Moreover, the microenvironment of the colon may express redundant mechanisms that compensate for the loss of PPAR! expression in CD11c" cells to maintain mucosal phenotype and function of CD11c" mDCs and that these mechanisms may be mucosal tissue-specific.

DISCUSSION DCs of the intestinal tract exist primarily as two different subsets, “conventional” mDCs and plasmacytoid DCs. A number of additional subsets are found within the conventional DC subset, based on expression of several markers, including CD11b, CD8, CD103, and CX3CR1. However, a common feature of all conventional intestinal DCs during homeostasis is a so-called mucosal phenotype, which can be defined as an immature DC with anti-inflammatory and tolerogenic functions, including 480 Journal of Leukocyte Biology

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Figure 8. DSS colitis is more severe in PPAR!#DC mice. Mice were administered 3% DSS (Line 1, n#4 mice/treatment) or 4% DSS (Line 2, n#5 mice/treatment) in drinking water. DAI was calculated daily, based on scores obtained from weight loss, stool consistency, and rectal bleeding for each mouse. *P & 0.05 Student’s t-test. Repeated-measures ANOVA of DAI in Line 1 indicated that there was a significant effect of genotype across all of the days of the study (P#0.0362) and that there was a significant interaction between genotype and time (P#0.0272), indicating that the rate of change in DAI with time was greater for KO mice compared with controls. Repeated-measures ANOVA of DAI in Line 2 indicated no significant differences between genotypes. Data are representative of two to three experiments for each mouse line.

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Tuna et al. PPAR! regulates mucosal phenotype in myeloid DCs

synthesis of RA. It is interesting that curcumin has been shown in numerous studies to mediate its anti-inflammatory effects via activation of PPAR!, implicating a role for PPAR! in the curcumin-induced mucosal phenotype [70]. Results from this study demonstrate that activation of PPAR! in BMDCs is sufficient to induce features of mucosal DCs. Treatment of BMDCs with the PPAR! agonist Rosi, plus the RXR agonist 9cRA, promoted an immature phenotype with reduced MHC II, CD80, and CD86 expression and a reduced capacity to drive CD4 T cell proliferation. Moreover, Rosi plus 9cRA also instilled BMDCs with the capacity to polarize naive CD4 T cells toward Foxp3" Tregs and away from IFN-!" Th1 cells, as well as an enhanced ability to induce T cell-independent IgA synthesis in B cells. Note that costimulation with 9cRA was chosen for these studies, as RXR is the requisite heterodimeric partner for PPAR!, and coactivation of RXR is thought to be required for full DNA-binding activity of PPAR!. However, we have observed during these studies that treatment of BMDCs with Rosi alone was also able to induce a mucosal phenotype (unpublished results), suggesting that endogenous levels of RXR activation are sufficient to observe effects by Rosi. The anti-inflammatory functions of PPAR! in intestinal cell subsets have been studied previously in M" and epithelial cells. Studies by Shah et al. [71] and Hontecillas et al. [72] each used LysM-Cre mice to delete PPAR! from M" and other myeloid cells. In both studies, KO mice showed enhanced susceptibility to DSS colitis, displaying greater loss of body weight and increased rectal bleeding and diarrhea. A Cre-Lox approach also was used by Adachi et al. [27] to generate mice with a deletion of PPAR! from villin" IECs. The severity of DSS colitis in these conditional KO mice was increased comFigure 9. Colon histopathology is more severe in DSS treated in PPAR!#DC mice. Colons were removed at the termination of studies from mice killed, and tissue sections for Lines 1 and 2 were prepared and stained with H&E. Sections were scored in a blinded manner for histopathologic disease. *P & 0.05 Student’s t-test. Data are representative of at least three studies.

Whether these diverse stimuli converge onto a common pathway to activate the mucosal phenotype is not fully understood. Studies by several labs have demonstrated an important role for RAs for development of a mucosal phenotype in DCs [65, 66]. Exposure of BMDCs or monocyte-derived DCs to RAs was able to induce expression of CCR9 and TGF-# and enhance their ability to induce Treg polarization in T cells and IgA synthesis in B cells. In addition, induction of a mucosal DC phenotype by IL-4 is associated with IL-4-mediated induction of retinaldehyde dehydrogenase activity [67]. Importantly, studies have shown that alternative activation of M" by IL-4 is dependent on IL-4-mediated induction of PPAR! [68]. IL-4 has been shown to activate PPAR! in a variety of cell types, which suggests that the mucosal phenotype induced in DCs by IL-4 also could be dependent on PPAR!. Additional studies by Cong et al. [69] have demonstrated that treatment with curcumin also was able to induce a mucosal phenotype in BMDCs, including www.jleukbio.org

TABLE 1. Flow Cytometric Analysis of mDCs and M$ in the Intestines of PPAR!#DC Mice Colon "

MLN

"

Percent mDCs (CD11b , CD11c ) PPAR! KO 1.42 ' 0.22 5.00 ' 0.21 Littermate 1.86 ' 0.40 5.57 ' 0.67 Percent M" (CD11b", CD11cneg) PPAR! KO 90.37 ' 1.07 85.50 ' 0.76 Littermate 85.40 ' 2.29 82.23 ' 1.61 MHC II MFI (mDCs) 2382 ' 781 4534 ' 188 PPAR! KO 2382 ' 781 4534 ' 188 Littermate 3296 ' 592 3924 ' 244 MHC II MFI (M") PPAR! KO 535 ' 155 1073 ' 40 Littermate 672 ' 115 1042 ' 156 Colons and MLNs were isolated from Line 2 PPAR!!DC and littermate control mice (n#3/group). Single-cell suspensions were analyzed by flow cytometry for expression of CD45, CD11b, CD11c, and MHC II. The percentage of mDCs (CD45", CD11b", CD11c") and M" (CD45", CD11b", CD11cneg) was determined by gating on the CD45", CD11b" subset and then analyzing for the percentage of CD11c" and CD11cneg fractions. The mean fluorescent intensity (MFI) of MHC II expression for mDCs and M" was determined on each CD11c" and CD11cneg fraction. Data are representative of two independent experiments.

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Figure 10. CD11c" cell subsets are altered in the lungs of PPAR!#DC mice. Lungs, colons, and MLNs were isolated from Line 2 PPAR!!DC, and WT littermate mice and single-cell suspensions were stained for CD11b, CD11c, and CD45 expression. Four populations of CD45" cells were identified in each tissue based on differences in expression levels of CD11c and CD11b (upper panel, dot plots). Differences in myeloid cell populations of the lung (lower panel, n#3 mice/group) were represented as percent of CD45" cells ' se of triplicates. Data are representative of two independent experiments. *P & 0.05 Student’s t-test.

pared with littermate control mice. Thus, PPAR! has an antiinflammatory role in at least three different intestinal cell types: mDCs, M"s, and IECs. Whether the mechanisms by which these different cell types mediate PPAR!-dependent anti-inflammatory function are the same is unknown. Studies in vitro on ligand-mediated activation of PPAR! have shown that proinflammatory cytokine secretion is reduced, and antiinflammatory cytokines are increased following PPAR! activation in M"s, mDCs, and IECs [73, 74]. The extent to which deletion of PPAR! in mDCs enhanced the severity of DSS colitis in our studies was less robust than expected in PPAR!!DC mice, given the enhanced inflammatory phenotype seen in vitro in PPAR!!DC BMDCs. In addition, the increase in severity of colitis in PPAR!!DC mice generally appeared less than in other published studies where PPAR! was deleted in M"s or IECs. However, even when PPAR! was deleted in M"s or IECs, the overall increase in colitis severity was relatively small compared with the effects of PPAR! in M"s or IECs in vitro in multiple studies. Thus, it appears that compensatory mechanisms strive to maintain a regulatory mucosal phenotype when PPAR! activity is diminished in intestinal M"s, IECs, or mDCs. A number of different 482 Journal of Leukocyte Biology

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mechanisms have been described that could have maintained regulatory functions in mDCs of PPAR!!DC mice in our studies. First of all, intestinal mDCs are a rare population compared with the number of M"s and IECs, both of which secrete immunoregulatory factors that could have induced a mucosal mDC phenotype in the absence of PPAR!. Intestinal M"s not only constitutively secrete IL-10 [75] but also have been shown to synthesize RA [76]. Either of these factors can induce a regulatory phenotype in mDCs, which could override the effect of PPAR! deletion. IECs also produce IL-10, TGF-#, and RA but also constitutively produce TSLP, and TSLP, produced by IECs, has been shown to induce tolerogenic functions in mDCs [14]. Secondly, the commensal bacteria in the gut have been shown in multiple studies to induce regulatory activity directly in mDCs [77–79]. Thus, immunoregulatory factors released by more abundant M"s and IECs and/or direct interactions with commensal microorganisms could potentially maintain a mucosal phenotype in intestinal mDCs in the absence of PPAR! expression. If PPAR!-negative DCs are responding to exogenous regulatory factors being produced by other intestinal cells, induction of the mucosal phenotype must be occurring in a PPAR!-independent manner. Other www.jleukbio.org

Tuna et al. PPAR! regulates mucosal phenotype in myeloid DCs

isoforms of PPAR (PPAR% and PPAR#/') have also been shown to mediate anti-inflammatory effects in vivo and in vitro [80]. It is possible that these related transcription factors may play a compensatory role in the intestinal tract of PPAR!!DC mice. The relatively minor changes that appeared in CD11c" cells of the intestine were mirrored by the striking differences that occurred in the lungs of PPAR!!DC mice. Whatever mechanisms are present in the intestine to maintain mucosal DC phenotype and function in the absence of PPAR! expression, similar mechanisms apparently are not operational in the mucosal environment of the lung. At the present time, we do not know if these changes in lung mDCs affect immune responses to pulmonary pathogens. Additional studies are necessary to characterize cellular and functional changes in the lungs of PPAR!!DC mice. However, it is clear that phenotypic alterations in the lungs of PPAR!!DC mice differ substantially from results of a study published previously by Baker et al. [81], in which PPAR! was deleted in CD11b" cells. A similar Cre-Lox approach was used to delete PPAR!, except that Cre was driven by the murine LysM promoter, which led to deletion of PPAR! in myeloid cell populations. These LysM-PPAR! KO mice developed features of PAP with accumulation of large, foamy alveolar M" that stained positive for lipid-laden vesicles. We have analyzed lung tissues of our PPAR!!DC mice up to 1 year of age and have not observed the presence of foamy M" in the alveoli or interstitial tissues. Histological tissue sections from PPAR!!DC mice appear similar to WT littermates with no evidence of material accumulating in alveoli (Supplemental Fig. 1).The reason for the different findings between these two studies is not known; however, the LysM promoter used by Baker et al. [81] to target Cre recombinase expression is expressed in several additional subsets of myeloid cells [82], including CD11cneg interstitial M"s, in addition to CD11c" alveolar M"s and mDCs. As interstitial lung M"s in our PPAR!!DC mice express normal levels of PPAR!, the lack of a PAP phenotype suggests that loss of PPAR! in interstitial M"s may play a role in the development of PAP. Additional studies are necessary to resolve how these related myeloid cell types in the lung are differentially regulated by PPAR!. In conclusion, these studies have demonstrated that activation of PPAR! in mDCs is sufficient to induce functions that are characteristic of mucosal mDCs. However, it is unlikely that PPAR! alone is responsible for inducing the mucosal phenotype in mDCs, as results suggest that additional mechanisms are likely present in vivo, which partially override the inflammatory effects seen in DCs that have a PPAR! deletion. This suggests that multiple levels of immunoregulation are present in the intestinal tract to maintain tolerogenic and anti-inflammatory functions in mDCs.

ACKNOWLEDGMENTS These studies were funded by a grant from the Crohn’s and Colitis Foundation of America. We thank the UK Flow Cytometry & Cell Sorting Core Facility, which is supported, in part, by the Office of the Vice President for Research, the Markey Cancer Center, and a National Cancer Institute Center Core www.jleukbio.org

Support Grant (P30 CA177558) to the University of Kentucky Markey Cancer Center.

AUTHORSHIP H.T., R.G.A., V.J.S., and L.W. each performed studies presented in this manuscript. D.A.C., S.B., C.S.K., and A.M.K. each made significant contributions to the design, analysis, and interpretation of studies in this manuscript.

DISCLOSURES

The authors declare no conflict of interest.

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KEY WORDS: homeostasis ! immunoregulation ! colitis

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