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(Journal of Leukocyte Biology. 2008;83:1476-1483.)
© 2008 by Society for Leukocyte Biology

Survival and function of human thymic dendritic cells are dependent on autocrine Hedgehog signaling

Alberto Varas*,1, Carmen Hernández-López*, Jaris Valencia*, Silvia Mattavelli{dagger}, Victor G. Martínez*, Laura Hidalgo*, Cruz Gutiérrez-Frías*, Agustín G. Zapata{dagger}, Rosa Sacedón* and Angeles Vicente*

* Department of Cell Biology, Faculties of Medicine and
{dagger} Biology, Complutense University, Madrid, Spain

1Correspondence: Department of Cell Biology, Faculty of Medicine, Avda. Complutense s/n, Ciudad Universitaria, Complutense University, 28040 Madrid, Spain. E-mail: avaras{at}bio.ucm.es


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ABSTRACT
 
The Hedgehog (Hh) family of signaling molecules functions in the development of numerous tissues during embryogenesis and has also been involved in adult self-renewing tissues. Recent results have demonstrated that the different components of the Hh signaling pathway are expressed in the human thymus. In this study, we investigate whether thymic dendritic cells (DCs) are cell targets for Hh signaling. Both components of the Hh receptor, Patched and Smoothened, as well as other Hh-binding proteins with modulating functions, are expressed by human thymic DCs. The expression of Gli1, Gli2, and Gli3 transcription factors suggests that the Hh signaling pathway is active in thymic DCs, and approximately one-half of thymic DCs produces Sonic Hh (Shh). The culture of thymic DCs with Shh protects them from apoptosis [similarly to CD40 ligand (CD40L)], and these antiapoptotic effects are related to an up-regulation of Bcl-2 and Bcl-XL protein expression. The addition of the Hh pathway inhibitor, cyclopamine, decreases DC viability and impairs their allostimulatory function in vitro. In addition, the blockade of the Hh signaling pathway by cyclopamine treatment abrogates the up-regulation of HLA-DR, CD86, CD80, and CD83 expression induced by CD40L on thymic DCs. Finally, we also show that after activation with CD40L thymic DCs down-regulate the expression of Hh receptor components as well as Shh production. Taken together, these results suggest that the survival and function of thymic DCs are regulated by an autocrine Hh signaling.

Key Words: thymus • cyclopamine • Patched • Smoothened


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INTRODUCTION
 
The Hedgehog (Hh) signaling pathway plays critical roles in vertebrate and invertebrate embryogenesis, determining cell fate and patterning during the development of many organs, as well as regulating survival and proliferation of a number of different cell types [1 , 2 ]. The importance of this pathway is emphasized by the fact that mutations leading to an aberrant Hh pathway can result in severe developmental defects and cancers in humans [3 4 5 6 ]. In mammals, three proteins have been described to compose the Hh family: Sonic (Shh), Indian (Ihh), and Desert Hh (Dhh), and Shh is the best studied [1 , 7 ]. Hh proteins undergo extensive post-translational modifications before being secreted, and Hh signaling is initiated by binding of Hh proteins to the 12-span transmembrane receptor Patched (Ptc), which in the absence of ligand, represses signaling by inhibiting the seven-transmembrane protein Smoothened (Smo). Upon ligand binding, the repressive activity of Ptc is abrogated, allowing Smo to transduce the signal toward the nucleus by a complex mechanism [7 8 9 ]. At the end of the Hh signaling pathway are the members of the Gli family of transcription factors, Gli1, Gli2, and Gli3. The specific functions of the Gli proteins are still poorly understood, although all of the activities demonstrated for Gli1 are positive, whereas Gli2 and Gli3 mainly function as positive and negative transcriptional regulators, respectively [10 11 12 ].

The different components of the Hh signaling pathway have been described to be expressed in the thymus [13 14 15 ] and to play an important role regulating the proliferation and differentiation of thymocyte precursors [13 , 14 , 16 17 18 19 ]. In humans, the three mammalian Hh proteins are produced by thymic epithelial cells. Shh-producing epithelial cells are restricted to the thymic subcapsula and medulla, and Ihh- and Dhh-expressing epithelial cells appear distributed throughout the thymic parenchyma. The requisite Hh receptors, Ptc and Smo, are expressed mainly by immature thymocytes and also by thymic epithelial cells located in the cortex and medulla. The Gli transcription factors are differentially expressed by the different thymic cell subpopulations [15 ]. The culture of CD34+ T cell precursors in the presence of recombinant Shh protein induces their survival. It also inhibits their IL-7-mediated proliferation and differentiation by down-regulating CD127/IL-7R expression and inhibiting IL-7-dependent STAT5 phosphorylation, indicating that Shh contributes to maintain the intrathymic progenitor cell population [17 ]. However, nothing has been described about the expression of the Hh signaling pathway components on human thymic dendritic cells (DCs) or about a possible role for Hh signaling in thymic DC function. Thymic DCs are specialized APCs, mainly localized in the medulla and corticomedullary junction, which are able to present self-antigens to developing thymocytes and play a pivotal role in thymocyte-negative selection and central tolerance induction [20 21 22 ]. We report in this study that thymic DCs express all the molecular machinery required to respond to Hh proteins. In addition, we show that thymic DCs produce Shh, which functions autocrinally to regulate their survival and functionality.


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MATERIALS AND METHODS
 
Isolation of human thymic DCs
Human thymus samples from patients, aged 1 month to 5 years, undergoing corrective cardiac surgery, were obtained and used according to the guidelines of the Medical Ethics Commission of La Zarzuela and Madrid-Monte Príncipe Hospitals (Madrid, Spain). Informed consent was provided according to the Declaration of Helsinki. Thymuses were dissected free of surrounding connective tissue and then gently disrupted with a potter homogenizer until completely disaggregated. Thymic cell suspensions were first depleted of CD2+ thymocytes by using the SRBC rosseting technique, and the recovered cells were then depleted of T, B, myeloid, and NK cells and thymic progenitors by treatment with anti-CD3, anti-CD7, anti-CD19, anti-CD14, anti-CD34, and anti-CD56 (all from BD Biosciences, San Jose, CA, USA) bound to sheep anti-mouse, Ig-coated magnetic beads (Dynal, Oslo, Norway), as described previously [23 ]. The purity of the recovered DCs was greater than 95%, and the cell surface phenotype of the resulting DC population is shown in Figure 1A .


Figure 1
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  		Figure 1. Human thymic DCs express Hh signaling pathway components. (A) Representative flow cytometry profile of thymic DCs isolated as described in Materials and Methods. Dashed histograms indicate background fluorescence. Dot-plots show forward (FSC)/side-scatter (SSC) profiles of the total thymocyte population and purified thymic DC population. (B) Surface expression of Hh receptors on thymic DCs. The open histograms represent the expression of Ptc and Smo on thymic DCs, and solid histograms indicate background staining. The percentages of positive cells are shown in each histogram. Dot-plot shows the correlated expression of Ptc and Smo on isolated thymic DCs. (C) RT-PCR analysis of the expression of different components of the Hh signaling pathway on total thymus and isolated thymic DCs. Band sizes are indicated. H2O served as negative control. Gas1, Growth arrest-specific gene 1; Hip, Hh-interacting protein. (D) Open histograms show the cytoplasmic expression of the Shh protein on thymic DCs, as well as the surface expression of the Smo receptor of Shh-producing and nonproducing thymic DCs. The percentages of positive cells are indicated in each histogram. Data are representative of five to nine independent experiments.

RT-PCR analysis
RNA isolation was performed using a Strataprep Total RNA Miniprep kit (Stratagene Cloning Systems, La Jolla, CA, USA), including a DNase I digestion step, as recommended by the supplier, to avoid genomic DNA contamination. Total cDNA was synthesized with Superscript II RT polymerase (Invitrogen, Grand Island, NY, USA), according to the instructions of the commercial supplier, and then used as a target in the PCR amplifications, which were performed using specific primer sets described previously [15 , 24 ]. All PCR reactions were performed on a Mastercycler gradient machine (Eppendorf, Hamburg, Germany) using AmpliTaqGold DNA polymerase (Applied Biosystems, Foster City, CA, USA) under the following conditions: 3 min at 94°C, 40 cycles of 45 s at 94°C, 45 s of each particular T annealing, and 45 s at 72°C, followed by 10 min at 72°C. PCR products were resolved on a 2% agarose gel, and the measured sizes were as expected.

Culture of isolated thymic DCs
Purified thymic DCs (1x105) were cultured in 96-well flat-bottom culture plates in 0.2 ml AIM V serum-free medium (Invitrogen) in the absence or presence of cyclopamine (5 µM), a cell-permeable inhibitor of the Hh signaling pathway (generously gifted by Dr. William Gaffield, Western Regional Research Center, Albany, CA, USA); tomatidine (5 µM), a steroidal alkaloid that structurally resembles cyclopamine but lacks the capacity to inhibit Hh signaling (Calbiochem, Nottingham, UK); recombinant human CD40 ligand (CD40L; 10 µg/ml; R&D Systems, Minneapolis, MN, USA); and modified recombinant human Shh (kindly provided by Curis, Cambridge, MA, USA; endotoxin level: <1 EU/mg Shh protein). This Shh protein was octylated for high activity. In vitro modification of N-Shh (aa 25–199), with a lipofilic group on the N-terminal cysteine, significantly increases the specific activity (>30-fold) of Escherichia coli-derived N-Shh as measured by activation of Hh signal transduction in cultured cells. Octyl N-Shh is a hydrophobically modified version of the Shh signaling protein generated by coupling N-octyl-malemide to the N-terminal cysteine of bacterially derived N-Shh. This modification represents a simple and efficient way to generate high, specific-activity Shh protein from bacterially expressed Shh [25 ]. After different periods of culture at 37°C in a 5% CO2–in-air incubator, cells were harvested, counted, and processed for viability analysis and flow cytometry stainings.

MLR assays
Thymic DCs were used at different numbers (10 cells to 15x103 cells) as stimulators for resting allogeneic T cells (2x105) isolated from peripheral blood. The cultures were performed in 96-well flat-bottom culture plates, using 0.2 ml AIM V supplemented with 2% human serum. After 3 days at 37°C in a 5% CO2–in-air incubator, the cultures were pulsed for 12 h with 10 µM BrdU. A specific kit from Roche Diagnostics (Barcelona, Spain), BrdU Labeling and Detection Kit III, was used to measure BrdU incorporation into newly synthesized DNA. Briefly, the labeling medium was removed, and cells were dried (2 h at 60°C), fixed in ethanol in HCl (0.5 M) for 30 min at –20°C, treated with nucleases (30 min at 37°C), and then incubated with peroxidase-conjugated Fab fragments of mouse anti-BrdU (30 min at 37°C). The peroxidase reaction was developed with 2,2'-azinobis-(3-ethylbenzthiazoline sulphonic acid) substrate, and the sample absorbance was measured using an ELISA reader (ELX800MB, Bio-Tek Instruments, Winooski, VT, USA) at 405 nm with a reference wavelength at 492 nm.

Flow cytometry
The following mAb conjugated with FITC, PE, or Cychrome were used for flow cytometric analysis: CD4 (SK3), CD13 (R3-242), CD33 (WM53), CD40 (5C3), CD80 (BB1), CD86 (2331), and HLA-DR (G46-6) from BD Biosciences and CD83 (HB15A) from Coulter-Immunotech (Beckman-Coulter, Marseille, France). The extracellular domains of Hh receptors were detected with PE-conjugated anti-Smo (N-19) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-Ptc1 antibodies (Abcam, Cambridge, UK) followed by FITC- or PE-conjugated, multiadsorbed F(ab')2 fragments of donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories; West Grove, PA, USA). Two- and three-color immunofluorescence stainings were performed by incubating the cells in PBS containing 1% FCS and 0.1% NaN3 in the presence of saturating amounts of fluorochrome-conjugated antibodies for 30 min at 4°C. For the intracellular stainings and according to the manufacturer’s instructions, cells were treated with Cytofix/Cytoperm solution (BD Biosciences) for 20 min at 4°C, washed with Perm/Wash buffer (BD Biosciences), and stained with PE-conjugated anti-human Bcl-2 mAb (BD Biosciences), anti-human Bcl-XL and anti-human Bim mAb (both from Chemicon, Temecula, CA, USA), and anti-Shh C-terminal peptide antibody (R&D Systems), followed by FITC- or PE-conjugated, multiadsorbed F(ab')2 fragments of donkey anti-mouse, anti-rat, or anti-goat IgG, respectively—all of them diluted in Perm/Wash buffer. Cell viability was estimated by propidium iodide staining. Analyses were conducted in a FACSCalibur flow cytometer (BD Biosciences) and a three-laser BD LSR flow cytometer (BD Biosciences) from the Centro de Microscopía y Citometría, Complutense University (Madrid, Spain).


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RESULTS
 
Expression of Hh signaling pathway components in human thymic DCs
To determine whether DCs are targets for Hh proteins in the human thymus, the cell surface expression of the Hh receptor components was analyzed by flow cytometry. As shown in Figure 1B , 40–60% of thymic DCs were positive for Ptc, the Hh-binding subunit, and 20–45% expressed Smo, the signaling component of the Hh receptor. Moreover, the majority of Smo-expressing DCs coexpressed Ptc in the plasma membrane (Fig. 1B) .

The expression of Hh receptors was confirmed by RT-PCR, as Ptc- and Smo-encoding RNAs were detected in purified thymic DCs (Fig. 1C) . Likewise, the thymic DC population expressed RNAs for Ptc2, a homologue of the Ptc receptor [26 ], and other Hh-binding proteins with modulating functions [27 , 28 ], such as Hip and Gas1 (Fig. 1C) . We also found that thymic DCs expressed specific RNAs for Gli1, Gli2, and Gli3 transcription factors (Fig. 1C) , suggesting that the Hh signaling pathway is active in DCs from human thymus.

As Ptc2 expression seems to be associated with Hh-producing cells [29 ], we assessed the expression of RNAs encoding the mammalian Hh proteins and showed that Shh RNA transcripts were expressed in the thymus DC population (Fig. 1C) . The intracytoplasmic expression of Shh protein was analyzed further by flow cytometry, demonstrating that the proportion of Shh-producing DCs was 50–60% (Fig. 1D) . We could find no correlation between Smo expression and Shh production in thymic DCs, as a similar percentage of Shh-producing and nonproducing DCs expressed the Smo receptor (Fig. 1D) .

Shh is a thymic DC survival factor
The relevance of Hh signaling in thymic DCs was assessed further by culturing DCs in serum-free medium supplemented with Shh and evaluating the cell viability. After 24 h, DCs treated with different doses of Shh were always protected from spontaneous cell death compared with untreated cells (Fig. 2A ). The viability of control thymic DCs progressively decreased in culture, and by Day 3, a high proportion of DCs was dead. In contrast, the presence of Shh significantly prevented DC apoptosis during the whole culture period, being the highest effect detected on Day 3 (Fig. 2B) . These antiapoptotic effects were comparable with those induced by CD40 ligation (Fig. 2B) .


Figure 2
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  		Figure 2. Shh enhances the survival of thymic DCs (A), which were cultured in serum-free medium alone or supplemented with different doses of Shh (number of input cells, 105). After 24 h, cells were recovered, counted, and stained with propidium iodide. Viable cells were defined as propidium iodide-negative. Data represent the mean (±SD) of three independent experiments, including three cultures per point (*, P≤0.05, by t-test). Ctrl, Control. (B) Thymic DCs were cultured for several days in the absence or presence of Shh (100 ng/ml), cyclopamine (Cyclop; 5 µM), and CD40L (10 µg/ml). Cell viability was assessed daily by propidium iodide staining. Data represent the mean (±SD) of three to four independent experiments. (C) The expression of Bcl-2, Bcl-XL, and Bim proteins was determined by flow cytometry in untreated and Shh-treated thymic DCs. Dashed lines represent background staining. The percentages of positive cells are indicated in each histogram.

As thymic DCs produce Hh proteins and express Hh receptors, DCs were also treated with cyclopamine, a steroidal alkaloid that binds directly to Smo and induces a conformational shift leading to Smo inactivation and Hh signaling pathway inhibition [30 ]. The presence of cyclopamine decreased the proportion of viable DCs, and only ~10% of viable cells could be recovered after 3 days of culture. In comparison, the proportion of cell survival in control and Shh-treated cultures was two and six times higher, respectively (Fig. 2B) .

It has been previously shown that Bcl-2 and Bcl-XL expression is up-regulated in DCs after CD40 triggering, and this correlates to an increased cell survival [31 32 33 ]. To determine whether Shh-dependent stimulation of DC survival was also accompanied by an increased expression of Bcl-2 and Bcl-XL, we analyzed by flow cytometry the expression of antiapoptotic proteins as well as the expression of the proapoptotic Bcl-2 family member Bim, also described to be involved in the regulation of DC survival [32 ]. We found that Shh treatment led to an up-regulation of Bcl-2 and Bcl-XL protein expression (Fig. 2C) . No change was seen in Bim expression (Fig. 2C) . These results suggest that Shh, in addition to CD40L, influences thymic DC survival by regulating the expression levels of antiapoptotic Bcl-2 family members.

Inhibition of Hh signaling impairs thymic DC allostimulatory capacity
To evaluate the role of Hh proteins on DC functionality, we analyzed the effects of blocking endogenous Shh signaling in the allostimulatory capacity of DCs. Thymic DCs were pretreated overnight with cyclopamine or the control steroidal alkaloid tomatidine, and equivalent numbers of viable cyclopamine- and tomatidine-treated or untreated DCs were used in a MLR. The MLR assays revealed a strong suppression of allogeneic stimulatory function of cyclopamine-treated DCs. As shown in Figure 3 , when 7.5 x 103 cyclopamine-treated DCs were added, proliferation was similar to that observed when only 1.8 x 103 tomatidine-treated or untreated control DCs were used as stimulatory cells.


Figure 3
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  		Figure 3. T cell stimulatory function of cyclopamine-treated thymic DCs is strongly decreased. Thymic DCs were cultured overnight in the absence or presence of cyclopamine or the control steroidal alkaloid tomatidine and used at different numbers as stimulators for resting allogenic T cells. After 3 days, the cultures were pulsed for 12 h with BrdU. A specific kit was used to measure BrdU incorporation into newly synthesized DNA. Full details are given in Materials and Methods. Results are the means (±SD) of the pooled data from five to seven experiments, each with three cultures per point.

Hh signaling blockade inhibits DC activation induced by CD40 ligation
Previous reports have pointed out that resting and activated peripheral T cells produce Shh [34 , 35 ]. To avoid the possible effects of T cell-derived Shh on DC function when using MLR assays, we analyzed the effects of cyclopamine pretreatment on CD40-dependent DC activation. As expected, the addition of CD40L to control DC cultures induced a strong up-regulation of the expression levels of HLA-DR and the costimulatory molecules CD86 and CD80. In addition, all DCs acquired CD83 expression (Fig. 4A ). Interestingly, the blockade of the Hh signaling pathway by cyclopamine pretreatment abrogated the up-regulation of HLA-DR, CD86, CD80, as well as CD83 expression induced by CD40L on thymic DCs (Fig. 4A) . Likewise, cyclopamine also inhibited the survival-promoting effect of CD40L on thymic DCs (Fig. 4B) . Therefore, these results suggest that thymic DC activation and survival are dependent on Shh signaling.


Figure 4
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  		Figure 4. Blockade of Hh signaling inhibits DC survival and activation induced by CD40 ligation. (A) Thymic DCs were cultured overnight in the absence or presence of cyclopamine, washed, and cultured for 48 h more in the absence or presence of CD40L. The expression of HLA-DR, CD40, CD80, CD86, and CD83 was then analyzed by flow cytometry. The mean fluorescence intensities or the percentages of positive cells are indicated in the histograms. (B) Thymic DCs were cultured for several days in the absence or presence of CD40L and cyclopamine plus CD40L. Cell viability was assessed daily by propidium iodide staining. Data represent the mean (±SD) of two to three independent experiments.

Activated DCs down-regulate Hh receptor expression and Shh production
Finally, we analyzed whether the expression of the Hh receptor changes upon DC activation. Our data showed that stimulation by CD40L notably down-regulated the expression levels of the Hh receptor components (Fig. 5 ). The proportion of Ptc-expressing DCs was also reduced after activation (Fig. 5) . Likewise, when the cytoplasmic expression of Shh was assessed, we found that the proportion of Shh-producing thymic DCs decreased after CD40 ligation (Fig. 5) , suggesting that Shh production is down-regulated in activated DCs.


Figure 5
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  		Figure 5. Down-regulation of the Hh signaling pathway in activated thymic DCs, which were cultured for 24 h in the absence or presence of CD40L, and then the surface expression of Ptc and Smo receptors and the intracellular expression of Shh were determined by flow cytometry. The percentages of positive cells and their mean fluorescence intensities are indicated in the histograms.


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DISCUSSION
 
The data presented in this paper further extend our investigation about the role of Hh proteins in the human thymus. Previously, we reported that the components of the Hh signaling pathway exhibit a complex intrathymic expression pattern involving thymic epithelial cells and thymocytes located in the different thymic compartments [15 ]. Likewise, we showed that Hh proteins function in the maintenance of intrathymic CD34+ precursor cell populations [17 ]. Here, we provide evidence that the activity of other key components of the thymus gland, the thymic DCs, is also regulated by the Hh signaling pathway. Thymic DCs express the two components of the Hh receptor, Ptc and Smo, as well as the three members of the Gli protein family. These transcription factors exhibit distinct, although overlapping, functions, and whereas Gli1 is exclusively a Hh-dependent activator of Hh targets, Gli2 and Gli3 may function as positive or negative regulators of transcription, although Gli2 acts primarily as a transcriptional activator, and Gli3 functions mainly as a repressor [10 , 11 ]. In addition, thymic DCs express Ptc2, which seems to have functions related but also distinct from its homologue Ptc [36 , 37 ]. These findings suggest the occurrence of complex Hh signaling responses in thymic DCs.

The expression of the Hh-binding proteins Hip and Gas1 by thymic DCs indicates that they have the ability to modulate their own Hh responses, as well as those that take place in neighboring Hh-responsive thymic cells. Hip is a membrane protein, positively regulated by Hh signaling, which competes with Ptc for binding the Hh ligands. Hip does not regulate Smo activity but exclusively sequesters the ligand, consequently attenuating the Hh signaling and forming a negative-feedback loop with Hh ligands [28 , 38 ]. Gas1 is a glycosylphosphatidylinositol-linked membrane glycoprotein, which controls Hh ligand availability by binding to the ligand, and acts as a Hh pathway antagonist [27 ]. The existence of a significant proportion of thymic DCs, which express Ptc but not Smo at the plasma membrane, suggests that Ptc alone could also participate in the modulation of Hh responses, as apart from repressing Smo signaling, it can function sequestering and internalizing Hh proteins, which limits their availability and creates a barrier to their further movement [39 , 40 ]. The possibility that this Hh internalization is linked to signal transduction has also been proposed [40 ].

Thymic DCs also synthesize and secrete Shh and constitute together with medullary and subcapsular epithelial cells the main source of Shh in the thymus [15 ]. Until now, it is unclear whether Shh establishes a functional gradient in the human thymus, as described in other tissues [1 , 41 ]. Nevertheless, it is likely that the differential Shh production and the different modulatory Hh-binding proteins, expressed by thymic DCs and epithelial cells [15 ], contribute to the appearance of distinct thymic microenvironments, where various Shh concentrations can differentially affect, quantitative and qualitatively, distinct Hh-sensitive thymic cell populations.

We also demonstrate that Shh produced by thymic DCs is used autocrinally to increase their survival. Although many authors have described that different factors, including TNF-related activation-induced cytokine (TRANCE), IL-12, IL-15, GM-CSF, TLR ligands, and CD40 ligation, influence the viability of peripheral DCs [31 32 33 , 42 , 43 ], to our knowledge, only one report has previously investigated this issue with thymic DCs. Vasilijic et al. [44 ] described that the addition of GM-CSF to rat thymic DC cultures inhibited their apoptosis and in correlation, up-regulated bcl-2 expression. Our results demonstrate that Shh also promotes thymic DC survival, inducing the up-regulation of the antiapoptotic bcl-2 and bcl-XL proteins. The survival-promoting effects of Shh have been reported in other different cell types [17 , 34 , 45 46 47 ], and interestingly, bcl-2 has been described recently as a Hh target gene, whose transcription is regulated by Gli1 and mainly Gli2 [48 , 49 ]. Although as pointed out above, the regulation of thymic DC survival has been poorly investigated, several authors, using different experimental approaches [50 51 52 ], have studied the lifespan of DCs in the thymus gland. All of these reports agree to conclude that thymic DCs exhibit a rapid turnover of 2–3 weeks, and we here propose that during that time period, Shh could be a main factor involved in promoting thymic DC survival in an autocrine manner.

Thymic DCs play an important role presenting self-antigens and inducing apoptotic death of potentially autoreactive developing T cells [21 , 22 ]. This process requires DC–thymocyte contacts, clearly mediated by CD40–CD40L interactions [53 , 54 ], which induce the survival and activation of DCs [55 56 57 ]. An important finding of this work is that an autocrine Shh signaling is required for the CD40-induced survival and activation of thymic DCs, as shown by the fact that the Hh signaling inhibitor cyclopamine abrogates the increase in thymic DC viability and the up-regulation of HLA-DR, CD80, CD86, and CD83 cell markers induced by CD40 ligation. Also in support, the allostimulatory capacity of thymic DCs is notably impaired after blocking Hh signaling with cyclopamine. In contrast, Rowbotham et al. [58 ] have reported that the intrathymic negative selection of a transgenic TCR was increased in fetal and neonatal Shh–/– mice. The apparent discrepancy between these results could reflect a differential involvement of Shh signaling in the first processes of deletion of autoreactive thymocytes occurring in the perinatal period, and those taking place in the steady-state adult thymus. Alternatively, species-specific differences in Shh requirement during intrathymic-negative selection could also explain the different results.

The survival of activated thymic DCs and therefore, their functionality must be finely controlled. As described with peripheral DCs, their limited lifespan would be controlled through the maintenance, activation, or silencing of several survival signals [32 , 59 60 61 ]. Our results also show that activated thymic DCs exhibit a decreased autocrine Shh production as well as a reduced expression of Hh receptors. This down-regulation of Hh signaling would drive to a decrease in thymic DC longevity and would constitute an important mechanism for controlling the turnover and functionality of thymic DCs.

In the future, it would be interesting to examine whether the Hh signaling pathway also regulates the lifespan of peripheral DCs and therefore, whether Hh proteins could be used to control the efficacy of DCs used as vaccines.


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ACKNOWLEDGEMENTS
 
This work was supported by grants BFU2006-00651/BMC and BFU2004-03132 from the Ministerio de Educación y Ciencia, RD06/0010/0003 from the Instituto de Salud Carlos III, and GR85/06-910552 from the Universidad Complutense y Comunidad Autónoma de Madrid. We thank Curis for providing human Shh protein, Dr. W. Gaffield for the gift of cyclopamine, and Dr. F. Villagrá and the Pediatric Cardiosurgery Units from Hospital La Zarzuela and Hospital Madrid-Montepríncipe for the thymus samples.

Received November 26, 2007; revised January 24, 2008; accepted February 13, 2008.


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S. V. Outram, A. L. Hager-Theodorides, D. K. Shah, N. J. Rowbotham, E. Drakopoulou, S. E. Ross, B. Lanske, J. T. Dessens, and T. Crompton
Indian hedgehog (Ihh) both promotes and restricts thymocyte differentiation
Blood, March 5, 2009; 113(10): 2217 - 2228.
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