Calcitriol has been identified in a number of studies as a prominent negative regulator of T helper (Th1)-type immune responses, whereas Th2 responses are not affected or even augmented as indicated by an induction of T1/ST2 (Schmitz et al., 2005; Wang et al., 2005). Although these effects have been preferentially explained by direct effects on lymphocytes, subsequent studies clearly supported a role of calcitriol in modulating monocyte differentiation or dendritic cell (DC) maturation (Griffin et al., 2003). Calcitriol clearly reduced the transition of the innate immune stimulation to an adaptive inflammatory immune response by interfering with signal transducers and activators of transcription, interferon regulatory factor 1/4, and possibly with the molecular elements involved in cross-presentation (Penna and Adorini, 2000; Muthian et al., 2006). With respect to myeloid DC development, the phenotype of calcitriol-conditioned DC reflects an immature status with poor capacity to induce antigen-specific T cell proliferation and a tendency to promote tolerance in vivo (Mellman and Steinman, 2001).

The immunoregulatory properties of calcitriol have been demonstrated in different models of autoimmune diseases (systemic lupus erythematosus, allograft rejection, autoimmune diabetes mellitus, and experimental allergic encephalomyelitis), in which a substantial amelioration was observed following treatment with calcitriol. These data from animal models, either explainable by a skewing effect of calcitriol on the differentiation of DC or a direct activity in lymphocytes, support epidemiologic studies in humans indicating that vitamin D receptor (VDR) genotype and vitamin D status are definite risk factors for autoimmune diseases (Cantorna et al., 2004).

Glucocorticoids (GCs) are effectively applied for the treatment of the general inflammatory sequelae of both Th1- and Th2-mediated disorders, including rheumatoid arthritis and asthma (Wilckens and De, 1997; Karin, 1998). They inhibit both T cells and antigen-presenting cells at the level of proliferation and cytokine production (Blotta et al., 1997). In addition, it was shown that in vitro, a combination of dexamethasone and calcitriol induced high numbers of IL-10-producing T cells (Barrat et al., 2002).

Recent studies of inflammatory bowel diseases (IBDs) encompassing Crohn's disease (CD) and ulcerative colitis suggest that these diseases are attributed to inappropriate and/or excessive responses to antigens present in the normal bacterial microflora. Th1/Th2 cytokine profiles characterize CD as a preferentially Th1 cell-mediated disease initiated by DC-derived IL-12 and/or IL-23 (Uhlig et al., 2006). Although it is not clear which of these DC factors dominates in local CD pathophysiology, recently, Th17 cells have been defined to comprise an IL-23-induced Th cell lineage with a proinflammatory role in autoimmunity and tissue inflammation (Bettelli et al., 2006, 2007). As a model of hapten-induced colitis in mice caused by rectal instillation of trinitrobenzene sulfonic acid (TNBS)-colitis exhibits features comparable with such a Th1 autoimmune process. Consequently, it was demonstrated that agents that block a Th1 response or promote a Th2 profile are reducing disease symptoms and progression (Neurath et al., 1995). Herein, we describe in an in vivo setting the inhibitory potential of calcitriol on Th1 and for the first time on Th17 (IL-17-producing T helper cells) effector functions in corticosteroid-treated mice with TNBS colitis.

Materials and Methods

Mouse Experiments

Male, 8-week old BALB/c mice weighing approximately 20 g were obtained from Charles River Laboratories (Sulzfeld, Germany). All studies were performed under approval of the Ethics Committee of Darmstadt/Hessen (Germany, F133/03) and are in agreement with the guidelines for the proper use of animals in biomedical research. The mice were kept in polycarbonate cages in temperature-controlled rooms with a 12-h light/dark cycle and fed standard mouse chow and tap water. At the end of the experiments, mice were sacrificed by cervical dislocation under isoflurane anesthesia (Forene; Abbott, Wiesbaden, Germany).

Induction of Colitis by the Haptenating Agent TNBS

The haptenating agent TNBS (2,4,6-TNBS; Sigma-Aldrich, Deisenhofen, Germany) was used at a concentration of 2% in 45% ethanol. For induction of colitis TNBS was administered [100 mg/kg body weight (BW)] to slightly anesthetized mice through a 3.5-F catheter carefully inserted into the rectum. The catheter tip was inserted 4 cm proximal to the anal verge. To ensure distribution of the TNBS within the entire colon and cecum, mice were held in a vertical position for 1 min after the instillation of the TNBS enema. Control animals were administered 45% ethanol alone using the same technique.

Administration of Calcitriol and/or Dexamethasone and Study Design

Calcitriol was purchased from Biomol (Hamburg, Germany), dissolved in ethanol at a concentration of 1 × 10^–2 M and kept at –80°C until use. Dexamethasone (D-2915, water-soluble; Sigma-Aldrich) was dissolved in water to prepare a stock solution at a concentration of 6 mg/ml and stored at 4°C. Calcitriol was administered i.p. at a dose of 0.2 μg/kg BW in 0.9% NaCl solution, containing 0.085% Myrj53 (Sigma, Deisenhofen, Germany), the solutions were prepared daily. Dexamethasone was used at doses from 0.6 to 1.2 mg/kg, respectively. Two protocols were used. 1) Acute ongoing colitis, dexamethasone and/or calcitriol were administered i.p. 2 h before the instillation of the TNBS enema and the after 3 days. On day 3 the colon was removed following cervical dislocation under isoflurane anesthesia; and 2) established colitis, dexamethasone and/or calcitriol were administered from days 3 to 5 following the instillation of the TNBS enema. On day 5, the colon was removed.

Analytical Procedure for Determining Serum Calcium and Creatinine

Serum calcium levels were determined after treatment at the end of experiment. The concentration was measured by the calcium-cresolphthalein colorimetric assay according to the manufacturer's instructions (Hitado, Möhnsee Delecke, Germany). Serum creatinine levels were measured using the alkaline picrate method (Hitado).

Assessment of Inflammation and Colitis Severity

Clinical Activity Score of Colitis. For the assessment of the clinical severity of colitis the BW, as well as the stool consistency and rectal bleeding, were examined daily. The clinical activity score of colitis was determined independently by two investigators being unaware of the treatment protocol using a scoring system described previously in detail (Hartmann et al., 2000). In brief, the loss of BW was scored as follows: 0, no weight loss; 1, weight loss of 1 to 5%; 2, weight loss of 5 to 10%; 3, loss of 10 to 20%; and 4, weight loss > 20%. The assessment of diarrhea (stool consistency) was scored as follows: 0, normally formed pellets; 2, pasty and semiformed pellets; and 4, liquid stools. Bleeding was scored as follows: 0, no blood in hemoccult; 2, positive hemoccult; and 4, gross bleeding from the rectum. The results of these scoring parameters were added resulting in a total clinical score ranging from 0 (healthy) to 12 (maximal ill/activity of colitis). The length and weight of the colon were used as indirect markers of disease-associated intestinal wall thickening correlating with the intensity of inflammation.

Macroscopic Scoring System. The assessment of the macroscopic colonic damage was performed using the scoring system of Wallace and Keenan (1990), taking into account the area of inflammation and the presence and absence of ulcers. The criteria for the evaluation of macroscopic damage were based on a semiquantitative scoring system. Features were graded as follows: 0, no ulcer, no inflammation; 1, no ulcer, local hyperemia; 2, ulceration without hyperemia; 3, ulceration and inflammation at one site only; 4, two or more sites of ulceration and inflammation; and 5, ulceration extending more than 2 cm.

Histological Analysis of the Colon. For histological examination, a sample of colonic tissue located precisely 3 cm above the anal canal was obtained from the mice of all treatment groups. The colonic tissues were fixed in 10% neutral buffered formalin and embedded in paraffin for histological analysis. Four-micrometer-sections were deparaffinized with xylene and stained with hematoxylin and eosin using routine techniques. Tissues were graded semiquantitatively from 0 to 5 in a blinded fashion according to previously described criteria (Boirivant et al., 1998).

Measurement of Myeloperoxidase Activity. The MPO activity assay was performed using a modification of the method described by Bradley at al. (1982). The enzyme activity was determined photometrically as the MPO-catalyzed change in absorbance occurring in the redox reaction of 3,3,5,5-tetramethylbenzidine dihydrochloride (TMB-substrate; Sigma-Aldrich) at 650 nm. MPO (Sigma-Aldrich) was used as an internal standard. Values are expressed as MPO units per gram of wet tissue.

Colonic Protein Extraction

Colonic protein extraction was performed using the Active Motif Nuclear cell extraction kit according to the manufacturer's instructions for extraction procedures starting from tissue (Active Motif Nuclear extract kit, Rixensart, Belgium). In brief, the excised colon was washed with ice-cold phosphate-buffered saline and homogenized in ice-cold complete lysis buffer. The homogenates were then incubated on ice for 30 min and centrifuged twice at 4000g at 4°C for 20 min. Aliquots of the resulting extracts were analyzed for their protein content using the Bio-Rad colorimetric assay according to the Bradford method (Bio-Rad Laboratories, Muenchen, Germany) and stored at –80°C until use.

Cytokine Assays

The amount of murine TNFα, interferon-γ, IL-12p70, IL-6, IL-10, TGFβ, IL-1β, and IL-4 in the colonic protein lysates were quantified by commercially available enzyme-linked immunosorbent assay kits (R&D Systems, Abingdon, UK) according to the manufacturer's instructions and adapted to the protein content of the colon tissue sample.

Western Blot Analysis

After addition of sample buffer to the colonic protein extracts and boiling samples at 95°C for 5 min, 150 μg of total protein lysate was separated on a 10% SDS-polyacrylamide gel (T-bet, GATA3, FoxP3, IL-23p19, CTLA4) or 15% (IL-17), respectively. Proteins were transferred onto nitrocellulose membrane (Schleicher and Schuell, Kassel, Germany), and the membrane was blocked for 1 h at room temperature with 3% skim milk in Tris-buffered saline containing 0.05% Tween 20. The level of proteins was assayed using the appropriate primary antibody (mouse FoxP3; eBioscience, San Diego, CA; mouse T-bet sc-21749, rabbit GATA-3 sc-268, and goat IL-23p19 sc-21083; all from Santa Cruz Biotechnology, Santa Cruz, CA; Armenian Hamster CTLA4 Ab; eBioscience; and rat IL-17 MAB271 Ab; R&D Systems) overnight at +4°C. Immunoreactivity was demonstrated by an ECL system (Amersham Pharmacia Biotech, Buckinghamshire, UK) using an appropriate horseradish peroxidase-conjugated secondary ab (NA931, NA934V, NA935, all from Amersham Biosciences Europe, Freiburg, Deutschland; donkey anti-goat sc-2020, Santa Cruz Biotechnology; anti-Armenian Hamster IgG, Rockland, Gilbertsville, PA). Bands were detected after exposure to Hyperfilm-MP (Amersham International plc, Buckinghamshire, UK). Blots were reprobed with actin ab (Santa Cruz Biotechnology). For quantitative analysis, the bands were detected with scanning densitometry using a Desaga CabUVIS scanner and Desaga ProViDoc software (Desaga, Wiesloch, Germany).

Statistical Analysis

All data are expressed as mean ± S.E.M. Clinical activity score of colitis and macroscopic and histological scores were statistically analyzed using the Mann-Whitney U test. Differences in parametric data were determined by the unpaired two-tailed Student's t test (SigmaStat, Chicago, IL). Differences were considered statistically significant with P < 0.05.

Results

Blood Calcium and Creatinine Profile. We measured serum calcium and creatinine levels in mice following treatment with calcitriol and/or dexamethasone. Although there was a trend toward hypercalcemia in mice treated with calcitriol alone in comparison with controls, the treatment with dexamethasone alone and in combination with calcitriol did not cause any significant alterations of serum calcium and creatinine levels (Table 1).

Impact of Calcitriol and/or Dexamethasone on Inflammation and Colitis Severity in Acute and Established Ongoing Th1-Mediated TNBS Colitis. The TNBS instillation in 45% ethanol led to a substantial wasting disease caused by severe diarrhea. Dexamethasone dose dependently (0.6–1.2 mg/kg) led to an improvement of colitis severity. Calcitriol on its own also significantly reduced the colitis intensity in acute TNBS colitis. However, the most clinical benefit with respect to colitis severity was observed following the combined application of dexamethasone and calcitriol, as reflected by weight gain, as well as improvement of clinical, macroscopic, microscopic, and immunological parameters of colitis (Table 2).

Macroscopic inspections of colons resected 3 days after administration of the TNBS enema revealed a striking hyperemia, inflammation, and necrosis compared with the ethanol-treated control groups, which showed only negligible signs of inflammation (Fig. 1, A and B). Strikingly, the combined application of the steroids caused a synergistic improvement, with clinical disease scores falling to 20% of those of the TNBS group. This was associated with a substantial decrease of colitis-mediated hyperemia and inflammation. Histological examinations (Fig. 1, C and D) determined by day 3, in the TNBS group, a transmural inflammation characterized by infiltration of inflammatory cells, predominantly lymphocytes and neutrophils that were associated with ulcerations, loss of goblet cells, and fibrosis throughout the entire colons. The combination of calcitriol and dexamethasone most effectively restored the normal histological appearance of the mucosa and submucosa compared with the TNBS group and the ethanol-treated control group (Fig. 1, C and D). Moreover, when applied 2 days after onset, the combination of dexamethasone and calcitriol still was very potent to ameliorate disease, as indicated by macroscopic and clinical assessment of colitis severity (Fig. 2, A–H; Table 3).

Calcitriol Affects the Differentiation of T Cells in Mice with TNBS Colitis. To integrate our clinical observations following calcitriol treatment with the possible molecular targets and mechanisms in a first set of experiments, we focused on the differentiation of T cells induced by vitamin D. We observed a clear-cut down-regulation of the inflammatory response with a combined steroid treatment including calcitriol (Table 4).

Calcitriol Inhibits the Th1 and Promotes the Th2 Profile in Mice with TNBS Colitis. Th1 lymphocyte differentiation is known to be based on a sequence of cell-intrinsic and exogenous, DC-derived factors, including augmentation of the transcription factor T-bet following activation by DC-derived IL-12. The observed reduction of the Th1 mediators, especially after treatment with the combination of dexamethasone and calcitriol, was further confirmed by immunoblot analysis of the T-bet. The combined administration of calcitriol and dexamethasone led to a significant reduction of T-bet protein expression compared with dexamethasone monotherapy (P < 0.001 versus dexamethasone, Fig. 3, A and B). As a next step to assess a possible role of calcitriol and dexamethasone in promoting the Th2 subset, we analyzed IL-4 production as well as the relevant Th2 lineage commitment factor GATA3. Calcitriol significantly up-regulated IL-4, whereas dexamethasone alone did not cause any significant change in IL-4 concentrations. The combined application of dexamethasone and calcitriol, however, led to a significant and distinct induction of IL-4 (P < 0.05 versus dexamethasone, Fig. 3E). These results were supported by the finding that already calcitriol alone led to a significant 2-fold induction of GATA3 protein expression, whereas the combination of dexamethasone and calcitriol caused a significant 5-fold increase of GATA3 protein expression (P < 0.001 versus dexamethasone, Fig. 3, C and D). In contrast, and further supporting an independent role of calcitriol on GATA3 protein expression, dexamethasone alone at 0.6 mg/kg caused no significant GATA3 up-regulation.

Calcitriol Enhances Regulatory T Cell Functions. Recent investigations clearly indicated that the suppressor cytokines IL-10 and TGFβ are involved in the control of colitis. Herein, calcitriol monotherapy significantly up-regulated IL-10 as well as TGFβ levels, whereas dexamethasone treatment alone failed to cause a significant induction of these cytokines (P < 0.01 dexamethasone versus calcitriol monotherapy). Nevertheless, calcitriol effects were enforced by dexamethasone and led to a significant increase of these regulatory cytokines (IL-10, P < 0.01; TGFβ, P < 0.001 versus dexamethasone, Fig. 4, A and B). As a prototypic transcription factor, FoxP3 plays a unique role for the development and function of CD4⁺CD25⁺ Tregs. In agreement with the IL-10 and TGFβ data shown above, the combined application of dexamethasone and calcitriol led to a significant induction of FoxP3 (P < 0.001 versus dexamethasone, Fig. 4, C and D). Furthermore, within the regulatory T lymphocyte circuit, it was shown that TGFβ may accelerate the expression of CTLA4, and time course studies have suggested that CTLA4-ligation of CD80 shortly after T cell activation enables TGFβ to induce CD4⁺CD25^– cells to express FoxP3 and subsequently develop suppressor activity. As shown in Fig. 4, E and F, calcitriol as well as dexamethasone monotherapy led to a significant induction of CTLA4 expression, whereas the combination most distinctly up-regulated CTLA4.

Calcitriol Affects DC Mediators Responsible for a Proinflammatory Differentiation of T Cells in Mice with TNBS Colitis. To better understand the background of our results obtained so far in this colitis model, we expanded our investigations to the DC mediators IL-12p70 and IL-23p19, which play a pivotal role for the promotion of a proinflammatory differentiation of naive T cells toward Th1 and Th17, respectively. In mice with acute TNBS colitis, IL-12p70 expression was significantly up-regulated. Here, both calcitriol and dexamethasone, when applied as monotherapy, led to a distinct reduction of IL-12p70 production. However, again, the most prominent reduction of IL-12p70 was observed with a combination of dexamethasone and calcitriol (P < 0.01 versus dexamethasone; Fig. 5). For the first time in acute TNBS colitis, we observed an increased expression of IL-23p19. Protein expression of IL-23p19 was clearly inhibited following treatment with calcitriol and/or dexamethasone. In this setting, calcitriol was even more potent to down-regulate IL-23p19 expression, and we observed a significant difference between dexamethasone and calcitriol monotherapy with respect to their inhibitory potential on IL-23p19 (P < 0.05 dexamethasone versus calcitriol monotherapy). Moreover, the combined application of calcitriol and dexamethasone most effectively reduced IL-23p19 expression (Fig. 6, A and B).

A recent investigation revealed that unlike IL-23, IL-6 and TGFβ in combination may be even more potent to induce the differentiation of pathogenic Th17 effector cells. In this study, IL-6 was significantly up-regulated in acute ongoing TNBS colitis. Both dexamethasone and calcitriol significantly down-regulated IL-6, whereas the calcitriol/dexamethasone combination resulted in an strong inhibition of IL-6 expression (Fig. 6E). Th17 effector cells are highly pathogenic and play a pivotal role in the maintenance of inflammation and in mediating tissue destruction. Here, Western blots of IL-17 revealed that, as hinted by calcitriol and dexamethasone combinatory effects on IL-23p19 and IL-6 before, this treatment exhibited the highest inhibitory activity on IL-17 expression in acute TNBS colitis (Fig. 6, C and D).

Discussion

Extending two previous investigations (Daniel et al., 2005, 2006), the data of this in vivo study in an accepted model of Th1-mediated colitis in mice clearly indicates and unravels that combining dexamethasone with calcitriol represents an attractive new immune modulatory treatment regimen of inflammatory disorders, including CD. To our knowledge, here we show for the first time that the complex immune-modulating effects of calcitriol may result from a differential down-regulation of proinflammatory capacities of intestinal DC as assessed by analysis of IL-12p70 as well as IL-23p19. This in turn leads to a moderate induction of a Th2 profile with increased levels of IL-4, which might already counteract Th1 and Th17 effector functions. Remarkably, however, calcitriol on its own, and more effective in combination with dexamethasone, substantially promoted Treg functions with distinct increases of the regulatory set of IL-10, TGFβ, FoxP3, and CTLA4.

Multiple epidemiological studies have indicated that a compromised vitamin D status is not only a well known risk factor for skeletal disorders but also contributes to the pathogenesis of frequent malignant, infectious, chronic inflammatory, and autoimmune disorders (e.g., insulin-dependent diabetes mellitus, inflammatory bowel disease, and multiple sclerosis). Especially among patients with CD, vitamin D deficiency is common, even when the disease is in remission (Cantorna et al., 2004). Furthermore, VDR deficiency (knock-out) was shown to result in severe inflammation of the gastrointestinal tract, pointing toward the prominent role for VDR signaling in the regulation of gut inflammation (Froicu et al., 2003). In the absence of VDR, Th1 cell-driven IBD is more severe, and Th2 cell-driven asthma does not develop (Cantorna et al., 2004). The VDR gene maps to a region on chromosome 12 that has been shown to be linked to IBD by genome screening techniques (Simmons et al., 2000). Analysis of single nucleotide polymorphisms in VDR typed in patients with CD provides preliminary evidence for a genetic association between CD susceptibility and the VDR gene lying within one of the candidate regions determined by linkage analysis (Simmons et al., 2000).

The results provided by our study using a combination of calcitriol and dexamethasone demonstrate a potent inhibition of the DC-derived IL-12p70 followed by a prominent down-regulation of the Th1 inflammatory cytokines profiles in Th1-mediated TNBS colitis. To further support these observations, the combined application of calcitriol and dexamethasone used in our study most evidently led to a down-regulation of T-bet expression, whereas GATA3 protein expression was significantly up-regulated.

To expand the complexity beyond the Th1-Th2 paradigm, IL-23 comprising the p40 subunit of IL-12 and a specific p19 subunit was identified with the p40-p19 complex being secreted by activated DC and macrophages. Initially, only IL-23 was described to be responsible for IL-17 production and subsequent inflammatory diseases including the maintenance and expansion of mucosal inflammation. However, subsequently, a mixture of IL-6 and TGFβ was found to be a potent cocktail, resulting also in the generation of IL-17-producing T cells (Harrington et al., 2005; Hunter, 2005; Bettelli et al., 2007). Remarkably, recent studies toward an orphan retinoid X receptor-related nuclear receptor, retinoid-related orphan receptor gamma, possibly interacting with VDR, revealed an essential IL-6-dependent function for the IL-17 expression of lamina propria T cells (Ivanov et al., 2006). Thus, IL-17-producing CD4⁺ effector cells differentiate along a distinct developmental program and might be antagonized by cytokines produced by Th1 or Th2 cells (interferon γ and IL-4). Here, we observed a remarkable down-regulation of IL23p19 following treatment with calcitriol and dexamethasone, further resulting in a clear inhibition of IL-17 expression. Furthermore, we observed a substantial inhibition of IL-6 expression. Concerning the dual role of TGFβ, it was shown that there is a dichotomy in the generation of Th17 and Treg cells, depending on whether they are activated in the presence of TGFβ or TGFβ plus IL-6 (Bettelli et al., 2006, 2007). Thus, the initial down-regulation of IL-6 we observed after treatment with calcitriol and/or dexamethasone may not only decrease Th17 development but moreover allow TGFβ to induce FoxP3 regulatory T cells, thereby promoting tolerance. Then, in turn, Tregs are also able to suppress Th1 cells as well as Th17 cells (Bettelli et al., 2006, 2007).

Distinct from the discussion above regarding the impact of calcitriol on the Th2 profile, we stress that especially the combination of dexamethasone and calcitriol caused a prominent induction of IL-10 and TGFβ. The functional activity of Tregs has been clearly linked to the expression of IL-10 and TGFβ. FoxP3, which is predominantly expressed in CD4⁺CD25⁺ regulatory T cells, was accepted as the most reliable Treg marker being sufficient for their development and function. TGFβ has been implicated in the conversion of naive CD4⁺CD25^– T cells into CD4⁺CD25⁺ T cells via the induction of FoxP3 (Fontenot et al., 2003; Huber et al., 2004). Recent studies using mice with a reporter for FoxP3 mRNA also indicated that TGFβ may directly influence FoxP3 (Ziegler, 2006). Here, the combined application of calcitriol and dexamethasone enhanced FoxP3 expression accompanied by the above-discussed induction of IL-10 and TGFβ. However, in vitro-generated homogenous populations of IL-10 Tregs obtained by stimulating naive CD4⁺ T cells in the presence of a combination of the anti-inflammatory drugs vitamin D₃ and dexamethasone were shown not to express high levels of FoxP3 (Vieira et al., 2004). Thus, although FoxP3 appears to be important for the development and function of naturally occurring CD4⁺CD25⁺ T cells, in vitro-derived IL-10-secreting Tregs appear to have regulatory functions despite low levels of FoxP3. It seems also important to consider that in vivo Tregs might also be generated via interactions with tolerogenic DC in the periphery.

Here, we report a high expression of FoxP3 following combined application of dexamethasone and calcitriol, arguing that FoxP3 may be involved in multiple lineages of Tregs and can be induced depending on microenvironmental signals such as TGFβ. Supporting this concept, it was recently demonstrated that GC treatment also promotes or initiates the differentiation of effector Tregs in a FoxP3-dependent manner. In addition, this study demonstrates that FoxP3 and IL-10 are functionally linked together in vivo (Karagiannidis et al., 2004). These observations were further underlined by the fact that IL-10 in turn regulates responsiveness to GC. In CD4⁺ T cells, cultures isolated from GC-resistant asthma patients' addition of calcitriol and dexamethasone enhanced IL-10 synthesis to levels observed in GC-sensitive patients, thus restoring GC responsiveness (Xystrakis et al., 2006). Calcitriol significantly counteracted the reduction of GR expression following dexamethasone, and IL-10 up-regulated GR expression by CD4⁺ T cells, suggesting mechanisms whereby these mediators may overcome GC resistance. The possible relevance of this finding was further supported by a study demonstrating that IL-10 may regulate responsiveness to GC by increasing the GR concentration. In contrast, TNFα decreased GR concentration. Changes in the receptor may be one of several mechanisms for altering tissue sensitivity to GC (Bamberger et al., 1996; Franchimont et al., 1999). Induction of IL-10 production in association with the distinct decrease of TNFα following combined application of dexamethasone and calcitriol highlight a potential indirect benefit of these treatment regimens because these results suggest that the balance of TNFα/IL-10 during the course of an inflammatory disease may determine changes in sensitivity to GC. In CD patients, not only GR resistance often represents a critical healthcare problem, but also decreased calcitriol levels seem to trigger the inflammatory status (Cantorna et al., 2004; Peterlik and Cross, 2005). Thus, our results stress that the combined application of dexamethasone and calcitriol may be an appealing future therapeutic option carrying the additional benefit of a restored GC responsiveness, an induction of Tregs, and the production of IL-10.

Finally, calcitriol led to a considerable increase in CTLA4 expression; this increase was even more potent using the combination of calcitriol and dexamethasone. This supports the concept of an inductive potential of calcitriol on Treg functions. These findings are in line with recent reports indicating that CTLA4 engagement can up-regulate IL-10 and TGFβ. Furthermore, FoxP3 has been shown to up-regulate CTLA4 expression (Liu et al., 2003; Zheng et al., 2006). Thus, calcitriol may trigger a TGFβ/CTLA4/FoxP3-positive loop vital for the generation and maintenance of CD4⁺CD25⁺ Tregs in corticosteroid-treated mice with Th1-mediated colitis (Zheng et al., 2006).

In conclusion, we have shown in this mouse model of human CD that calcitriol functions as a promising new therapeutic option for Th1-or Th17-driven IBD, especially when combined with GC. Nevertheless, further studies are needed to assess whether the immune modulatory effects of calcitriol and dexamethasone combinations are indeed synergistic. The beneficial pharmacological profile of calcitriol might help to overcome the clinical refractoriness to GC therapy that is a common feature among IBD patients. Furthermore, its future therapeutic potential might not only result from its complex immunosuppressive capacities on proinflammatory signals of DC also leading to the observed inhibition of Th1 and Th17 effector functions and the distinct promotion of the Th2 profile but might additionally lead to a long-term, highly appreciated induction of regulatory T cell functions, including IL-10 synthesis. These new data regarding the calcitriol activity carefully evaluated in an approved model of CD and supported by two previous investigations may accelerate preparations for a large prospective study with a calcitriol comedication, especially in CD patients with low active vitamin D₃ levels. Such a study may be facilitated by the advent of calcitriol analogs with an activity skewed more than 2 logs toward immune modulation, thus lowering the risk of a perturbation of calcium/phosphate and bone metabolism. With careful preparation, it should be feasible to design a study with meaningful primary and secondary endpoints to the benefit of the CD patients and to solve the ongoing enigma of calcitriol's immune action.