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Keywords:

  • contact hypersensitivity;
  • dendritic cells;
  • skin;
  • tolerance;
  • vitamin D

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The active form of vitamin D, 1,25-hydroxyvitamin D3 [1,25(OH)2D3] is produced in skin following exposure to sunlight. It is also used topically to control inflammatory skin diseases by stimulating keratinocyte differentiation and suppressing immune responses. Administration of 1,25(OH)2D3 to the skin of mice increases the capacity of CD4+ CD25+ (Foxp3+) regulatory T cells residing in the skin-draining lymph nodes (SDLN) to suppress immune responses. We hypothesized that dendritic cells (DC) may migrate from the skin to the lymph nodes to regulate T-cell function. Increased proportions of skin-derived DC (CD11c+ ClassII+ DEC-205hi CD8lo) cells were detected in the SDLN 18 hr after topical 1,25(OH)2D3 treatment of mouse skin. The capacity of DC from the SDLN to take up, process and present antigen to co-cultured T cells was not modified following topical 1,25(OH)2D3. However, CD11c+ cells from the SDLN of 1,25(OH)2D3-treated mice induced a significantly smaller ear-swelling response in a T helper type 1/17-mediated model of contact hypersensitivity. CD4+ CD25+ cells isolated from the ear-draining lymph nodes (EDLN) of mice that received ear injections of CD11c+ cells from donor mice topically treated with 1,25(OH)2D3 more potently suppressed effector cell proliferation. In addition, EDLN cells from recipients of CD11c+ cells from 1,25(OH)2D3-treated mice produced increased interleukin-4 levels. The CD11c+ cells from the SDLN of mice treated with topical 1,25(OH)2D3 expressed increased levels of indoleamine 2,3-dioxygenase messenger RNA, a molecule by which topical 1,25(OH)2D3 may enhance the ability of DC to control the suppressive function of CD4+ CD25+ cells.


Abbreviations:
1,25(OH)2D3

1,25-dihydroxyvitamin D3

DC

dendritic cell

DNBS

dinitrobenzylsulphonic acid

DNFB

2,4-dinitrofluorobenzene

EDLN

ear-draining lymph nodes

FACS

fluorescence-activated cell sorting

FITC

fluorescein isothiocyanate

IDO

indoleamine 2,3-dioxygenase

IFN

interferon

IL

interleukin

mAb

monoclonal antibody

OVA

ovalbumin

RANKL

receptor activator of nuclear factor-κB ligand

SDLN

skin-draining lymph nodes

TGF

transforming growth factor

Th1

T helper type 1

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Interest in the involvement of vitamin D in various body systems and disease settings has recently increased with observations that deficiency in this hormone is an emerging health problem in many countries. With ultraviolet B (UVB) irradiation (290–315 nm), 7-dehydrocholesterol in skin is converted into pre-vitamin D3. This molecule then isomerizes with body heat into vitamin D3. Vitamin D binding protein transports much of the vitamin D3 to the liver and kidneys for further hydroxylation into 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active form of vitamin D. Various skin cells including melanocytes and keratinocytes also possess the enzymatic machinery to produce 1,25(OH)2D3 where local skin concentrations of 2–5 nm can be achieved following UVB irradiation.1,2 Recent investigations have identified that 1,25(OH)2D3 is important within the skin immune system.

The function of various immune cells located in the skin and draining lymphatic tissue can be modified by 1,25(OH)2D3 including the Langerhans cells,3 mast cells4,5 and regulatory T cells.6,7 Skin homing receptors (such as CCR10) can be up-regulated on 1,25(OH)2D3-stimulated T cells.8 Topically applied 1,25(OH)2D3 is also an effective treatment for skin inflammatory diseases such as psoriasis.9 1,25(OH)2D3 reduces morbidity in the skin through direct effects on keratinocyte proliferation and differentiation in psoriatic lesions (reviewed in ref. 9) and also through immunoregulation (reviewed in ref. 10). The ability of topical 1,25(OH)2D3 (or analogues) to modify immune responses has been demonstrated in both mice7,11 and humans,12,13 where this treatment suppresses contact hypersensitivity responses. Topical 1,25(OH)2D3 may also be effective in controlling allergic diseases including asthma, where subcutaneous immunotherapies incorporating 1,25(OH)2D3 reduce respiratory inflammation during allergic airway disease.14

We recently described how topical application of 1,25(OH)2D3 or UVB irradiation increases the suppressive ability of CD4+ CD25+ Foxp3+ cells isolated from the skin-draining lymph nodes (SDLN).6 The pathway by which topical 1,25(OH)2D3 controls regulatory T cells in the SDLN is unknown. A candidate skin cell, which could be responsible for directly interacting with regulatory T cells in the SDLN is the dendritic cell (DC). Indeed, topical 1,25(OH)2D3 (or an analogue) reduces the number of MHC class II+ cells in the skin of treated mice,7,11 suggesting that these cells could leave the skin following 1,25(OH)2D3 treatment. In other studies, incorporation of 1,25(OH)2D3 (100 ng) into a subcutaneous vaccine consisting of diphtheria and alum promoted the migration of cutaneous DC to secondary lymphoid sites such as the Peyer’s patches.15 This study details new investigations into the ability of topical 1,25(OH)2D3 to modulate DC activity in the skin and the SDLN. Topical 1,25(OH)2D3 altered the phenotypic profile of DC in the SDLN. The ability of CD11c+ cells from the SDLN to prime T helper type 1 (Th1)/Th17-driven contact-hypersensitivity responses was subverted by earlier skin treatment with 1,25(OH)2D3, with reduced responses linked with an increased suppressive activity of CD4+ CD25+ regulatory cells and enhanced Th2 responses.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Mice

Female 8-week-old BALB/c mice were purchased from the Animal Resources Centre (Murdoch, Western Australia, Australia). All experiments were performed according to the ethical guidelines of the National Health and Medical Research Council of Australia and with approval from the Telethon Institute for Child Health Research Animal Ethics Committee.

Topical vitamin D application

A 100-μl aliquot containing 125 ng 1,25(OH)2D3 (Sigma Chemical Company, St. Louis, MO, USA) diluted in ethanol, propylene glycol and water mixed at a 2 : 1 : 1 ratio was painted onto a clean-shaven 8-cm2 area of dorsal skin surface of mice as described previously.6

Flow cytometric analysis of skin- or ear-draining lymph node cells

The SDLN (inguinal, axillary and brachial) or ear-draining lymph nodes (EDLN, auricular) were removed from mice, pooled within experimental groups and physically disaggregated. Staining of surface antigens was performed as previously described.6 An intracellular staining kit (eBiosciences, San Diego, CA, USA) was used to determine intracellular Foxp3 and interleukin-10 (IL-10) expression. At least 10 000 cells of interest were collected using either the fluorescence-activated cell sorter (FACS) LSRII or FACS Calibur (BD Biosciences, Heidelberg, Germany) flow cytometers. Data were analysed using FlowJo software (Treestar, Ashland, OR, USA).

Measuring in vitro antigen uptake and processing by skin-derived DC

Eighteen hours after topical 1,25(OH)2D3 treatment, the SDLN were removed and a single-cell suspension was generated by using a sterile scalpel blade to mince the lymph nodes, which were then digested with collagenase (type 4, 1 mg/ml, Worthington Biochemical, Lakewood, NJ, USA) and DNase (type 1, 0·1 mg/ml, Sigma) for 30 min at 37° with 5% CO2. Digested lymph nodes were filtered and cells were treated for 5 min at 4° with anti-CD16/CD32 monoclonal antbodies (mAb; BD Biosciences), and then DQ-OVA (Molecular Probes, Invitrogen Australia, Mulgrave, Australia 10–10000 ng/ml) or Alexa488–ovalbumin (OVA) (Molecular Probes, 10–1000 ng/ml) for 2 hr at 37° or 4°. Alexa488-OVA is used as a measure of antigen uptake.16 Upon cleavage by proteolytic activity within cells, the fluorochrome DQ fluoresces and is used as a measure of antigen processing.16 These processes occur at 37°. Surface molecules on DC in the lymph nodes were detected by incubating cells with mAb as described above.

Purification of CD11c+ cells

Eighteen hours after topical 1,25(OH)2D3 treatment, the SDLN were removed and a single-cell suspension was generated as described above. CD11c microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were used with an AutoMACS cell separator (Miltenyi Biotec) to positively select for CD11c+ cells. Flow cytometry was used to determine that the purity of live isolated CD11c+ cells was routinely > 90%. The expression of activation markers such as CD86 on the CD11c+ cells was not modified by this positive selection process (data not shown).

Measuring in vitro antigen presentation by skin-derived DC

The fluorescent antigen, 0·5% fluorescein isothiocyanate (FITC; Sigma, diluted 1 : 1 in acetone : dibutylphthalate) was painted onto the skin of mice 30 min after topical treatment. The SDLN were again removed 18 hr after topical 1,25(OH)2D3 treatment with FITC+ CD11c+ cells sorted to 80–90% purity using a FACSAria cell sorter. These cells were co-cultured with CD4+ T cells purified from the SDLN of mice administered 0·5% FITC 7 days earlier in complete RPMI-1640 (RPMI medium; Gibco, Auckland, New Zealand) with 10% fetal calf serum, 2 mm l-glutamine, 50 μm 2-mercaptoethanol and 5 mg/ml gentamicin). For all co-cultures, methyl-[3H]thymidine (10 μl (0·25 mCi)/well; Amersham Pharmacia Biotech, Piscataway, NJ) was added after 72 hr at 37° with 5% CO2 and cells were harvested at 96 hr. Culture supernatants were also obtained at 96 hr.

Cytokine detection in tissue culture supernatants

Interleukin-2, IL-4, IL-5, IL-10, IL-17 and interferon-γ (IFN-γ) were detected using rat anti-mouse IL-2, IL-4, IL-5, IL-10, IL-17 or IFN-γ enzyme-linked immunosorbent assay capture and detection mAb (BD Biosciences) in a dissociation-enhanced time-resolved fluorescence immunoassay with Europium as the label (sensitivity 25 pg/ml). Recombinant mouse IL-2, IL-4, IL-5, IL-10, IL-17 or IFN-γ (BD Biosciences) were used as the standards.

In vivo priming assay

Eighteen hours after topical skin treatment, CD11c+ cells were purified from the SDLN of treated mice and 105 cells in 20 μl 0·9% saline were adoptively transferred into the ear pinnae of naive BALB/c mice. CD11c+ cells were loaded with antigen before adoptive transfer by incubating cells with 1 mm dinitrobenzylsulphonic acid (DNBS, Sigma) for 30 min at 37°. Cells were washed three times before the transfer. The ears of some mice received a saline injection only to control for the non-specific inflammatory effects of transferring fluid into the ears. Seven days after the injection of cells, an ear-swelling response was elicited by painting 10 μl 0·2% 2,5-dinitrofluorobenzene (DNFB, Sigma, diluted in acetone) onto each ear pinna. An additional group of mice that received saline only were also challenged with DNFB. Forty-eight hours after the ear challenge with DNFB, the ear thickness was measured in a blinded manner, as previously described.17

Culturing ear-draining lymph node cells

Forty-eight hours after ear challenge with 0·2% DNFB, the EDLN were removed and cells were cultured at 105 cells/200 μl per well (six replicates per treatment) in complete RPMI medium. DNBS was added to cultures at a concentration of 1 mm. Methyl-[3H]thymidine was added after 72 hr at 37° with 5% CO2 and cells were harvested at 96 hr. Culture supernatants were also obtained at 96 hr.

Regulation of proliferation by CD4+ CD25+ cells from the EDLN

Forty-eight hours after ear challenge with 0·2% DNFB, CD4+ CD25+ cells (≥ 95%, as determined using flow cytometry) were purified from the EDLN of recipient mice by using the CD4+ CD25+ regulatory T-cell isolation kit (Miltenyi Biotec). Responder CD4+ CD25 cells (≥ 95% pure) were purified from the EDLN of mice who received CD11c+ cells from vehicle-treated mice also by using the CD4+ CD25+ regulatory T-cell isolation kit (Miltenyi Biotec) and then labelled with 5 μm CFSE (Molecular Probes). CD4 cells isolated from the same mice were used as an antigen-presenting cell population. CD4+ CD25 cells (105 cells/200 μl per well) were co-cultured with CD4 cells at a constant ratio of 1 : 20 with 1 mm DNBS in complete RPMI. CD4+ CD25+ cells were then cultured at ratios of 1 : 1 or 1 : 10 with the CD4+ CD25 responder cell population. After incubation at 37° for 92 hr with 5% CO2, supernatants were harvested and cells were washed and stained with phycoerythrin-Cy5-conjugated anti-CD4 mAb (BD Biosciences). In some experiments anti-IL-10 mAb (BD Biosciences, 1 μg/ml) was added to the co-cultures. Data were analysed using the proliferation algorithm for FlowJo (version 8.7.3) on viable cells, which were gated according to forward- and side-scatter properties and CD4 expression with 50 000 cells collected on the flow cytometer. The percentage of total cells that had divided was determined by using measurements of the number of viable CD4+ CFSElo cells.

Measuring messenger RNA in CD11c+ cells

Eighteen hours after topical skin treatment, CD11c+ cells were purified from the SDLN and ≥ 5 × 105 cells were snap-frozen in 350 μl buffer RLT plus (Qiagen, Doncaster, Vic, Australia). RNA was extracted using the RNAeasy plus minikit (Qiagen) according to the manufacturer’s instructions. Complementary DNA was reversed transcribed from the RNA samples using the Quantitect kit (Qiagen). Using primers designed in-house, a real-time polymerase chain reaction (PCR) was then performed as previously described18 using 2× RT2 qPCR Master Mix (SABiosciences, Frederick, MD, USA). The PCR was performed using the standard two-step cycling conditions on the ABI 7000 SDS (Applied Biosystems, Foster City, CA, USA). Melting curve analysis was used to assess the specificity of the assay. Expression levels were determined by a standard curve created from serial dilutions of the PCR product and normalized to the reference gene eukaryotic translation elongation factor 1α. Primer pairs were as follows: eukaryotic translation elongation factor 1α, 5′-CTGGAGCCAAGTGCTAATATGCC-3′ and 5′-GCCAGGCTTG-AGAACACC AGTC-3; CCL22 5′-GGTCCCTATGGTG-CCAATG-3′ and 5′-TTATCAAAACAACGCCAGGC-3′; indoleamine 2,3-dioxygenase (IDO) 5′-AGGCTGGCAAA-GAATCTCCT-3′ and 5′-AATGACAAACTCACGGACT-GG-3′; IFN-γ; 5′-TCAAGTGGCATAGATGTGGAAGAA-3′ and 5′-TGGCTCTGCAGGATTTTCATG-3′; IL-10, 5′-GG-TTGCCAAGCCTTATCGGA-3′ and 5′-ACCTGCTCCACT-GCCTTGCT-3′; IL-12p35 5′-TGGACCTGCCAGGTGTCTTAG-3′ and 5′-CAATGTGCTGGTTTGGTCCC-3′; IL-12p40 5′-AAGGAACAGTGGGTGTCCAG-3′ and 5′-GGCAAAC-CAGGAGATGGTTA-3′; relB; 5′-TCCGCGCCCGAGCTA-3′ and 5′-GCCAAAGCCGTTCTCCTTAA-3′ transforming growth factor-β (TGF-β), 5′-CACTGATACGCCTGAGTG-3′ and 5′-GTGAGCGCTGAATCGAAA-3′.

Statistical analyses

Data were compared using Student’s t test with the Prism statistical analysis programs for Macintosh (v5.0a). Differences were considered to be significant at P-values <0·05.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Topical 1,25(OH)2D3 alters the phenotypic profile of DC in the SDLN

The SDLN were removed from mice topically treated up to 48 hr earlier with vehicle or 1,25(OH)2D3. The total number and viability of SDLN cells was not modified by topical 1,25(OH)2D3 (data not shown). The profile of DC populations in the SDLN was determined using the cell surface markers CD11c and MHC class II to differentiate DC (CD11chi MHC class IIhi) from other lymph node cells (Fig. 1a). Populations of DC were further classified into populations expressing high and low levels of DEC205 and CD8 (Fig. 1a); markers used previously to distinguish skin-derived from lymph node-derived DC populations in the SDLN.19 At 18 hr after treatment with 1,25(OH)2D3, there were significant increases in the proportions of DC (CD11chi MHC class IIhi cells) in the SDLN that were DEC205+ CD8+ (by 50%) or DEC205+ CD8 cells (by 28%; Fig. 1b). These differences persisted until 24 hr, but resolved by 48 hr post-skin-treatment (data not shown). DEC205+ CD8 cells comprise a mixture of skin-derived DC, whereas DEC205+ CD8+ cells express Langerin, and may belong to a recently identified subpopulation of DC in the SDLN (reviewed in ref. 20).

image

Figure 1.  Topical vitamin D3 [1,25(OH)2D3] modifies the phenotype of dendritic cell (DC) populations in the skin-draining lymph nodes (SDLN). In (a), the proportions of CD11chi MHC classIIhi cells differentiated according to the expression of CD8 and DEC-205 were determined in the SDLN of mice after topical vehicle or 1,25(OH)2D3 application. In (b) the proportion of CD11chi MHC classIIhi cells that were also DEC205 CD8+, DEC205+ CD8+, DEC205+ CD8 or DEC205 CD8 in the SDLN 18 hr after topical vehicle or 1,25(OH)2D3 treatment is shown for nine individual mice per treatment from three repeated experiments (mean ± SEM, *P < 0.05).

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Topical 1,25(OH)2D3 down-regulates CD86 expression by some SDLN DC

The expression of CD40, CD80, CD86, B7-H3 and B7-H4 by the different SDLN DC subpopulations was examined at 18 hours post-skin-treatment. CD8 and DEC205 were again used to distinguish four DC subpopulations in the SDLN, where the DEC205+ CD8 DC expressed the greatest quantity of each surface marker, except for B7-H3 (Fig. 2a, and data not shown). However, there was no change in the expression of CD40, CD80 or B7-H3 (data not shown) on any DC population from the SDLN with topical 1,25(OH)2D3 treatment. The expression of CD86 was down-regulated on DEC205+ CD8+ and DEC205+ CD8 cells at 18 hr post-topical 1,25(OH)2D3 (Fig. 2a), while B7-H4 was up-regulated on DEC205+ CD8+ cells (data not shown).

image

Figure 2.  Topical vitamin D3 [1,25(OH)2D3] reduces the expression of CD86 on dendritic cell (DC) populations in the skin-draining lymph nodes (SDLN) but not the ability of DC from the SDLN to uptake or process antigen in vitro. The geometric mean fluorescent intensity (GMFI) of (a) CD86 on DC (CD11chi MHC ClassIIhi) populations distinguished by the expression of DEC205 and/or CD8. Results are shown for nine individual mice per treatment from three repeated experiments (mean ± SEM, *P < 0.05). In (b–d), the ability of different DC populations from the SDLN to take-up or process antigen was assessed using ovalbumin (OVA) conjugated to the fluorescent molecules Alexa488 or DQ. Cells that take-up OVA-Alexa will also fluoresce when, during antigen processing, the usually quenched molecule DQ will begin to fluoresce. In (b), examples of OVA-Alexa+ and DQ-OVA+ cells are shown relative to a negative control (no OVA). The proportions of CD11chi MHC classIIhi DEC205+ CD8 cells that expressed OVA-Alexa or DQ-OVA are shown when SDLN cells from mice treated 18 hr earlier with either vehicle or 1,25(OH)2D3 were incubated at 37° for 2 hr with various concentrations of (c) OVA-Alexa or (d) DQ-OVA, respectively.

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Topical 1,25(OH)2D3 does not modify the in vitro antigen uptake, processing or presenting capacity of SDLN DC

The ability of these SDLN DC populations to take-up and process antigen was assessed using fluorescently tagged OVA proteins. Eighteen hours after skin treatment, SDLN cells were incubated with various concentrations of Alexa488-OVA or DQ-OVA, and the extents of antigen uptake and processing were determined, respectively. Examples of cells that had taken-up antigen (Alexa488-OVA+) or processed antigen (DQ-OVA+) are shown relative to a negative (no OVA) control in Fig. 2(b). The ability of DEC205+ CD8 DC (or any of the other DC populations) in the SDLN to either take-up (Fig. 2c) or process (Fig. 2d) antigen was not modified by topical 1,25(OH)2D3. In a further assay, there was no difference in the ability of CD11c+ cells purified from the SDLN 18 hr after treatment with either vehicle or 1,25(OH)2D3, to present OVA peptide to co-cultured OVA-TCR+ CD4+ T cells (from DO11.10 mice) as no differences in the proliferation or cytokine production (IFN-γ and IL-10, data not shown) by co-cultured T cells was observed.

In an alternative assay, to test the ability of DC in the skin to take-up and process antigen in vivo, the fluorescent antigen FITC was applied to the skin of mice 30 min after topical 1,25(OH)2D3 treatment. Eighteen hours after topical 1,25(OH)2D3, the proportion of FITC+ CD11c+ cells was assessed in the SDLN of mice. In Fig. 3(a), an example of FITC expression by CD11c+ cells is depicted in mice treated (or not) with FITC 18 hr earlier. The proportion of CD11c+ FITC+ cells in the SDLN was not modified by topical 1,25(OH)2D3 (data not shown). The expression of CD86 (Fig. 3b) and CD80, but not of CD40 (data not shown), was down-regulated on CD11c+ FITC+ cells from mice treated topically with 1,25(OH)2D3. However, there was no difference in the capacity of CD11c+ FITC+ cells sorted from the SDLN 18 hr after topical vehicle or 1,25(OH)2D3 to induce the proliferation of co-cultured CD4+ T cells from FITC-sensitized mice (Fig. 3c). Together, these data indicate that the functional abilities of DC from the SDLN to take-up and process antigen both in vitro and in vivo were not modified by topical 1,25(OH)2D3.

image

Figure 3.  Topical vitamin D3 [1,25(OH)2D3] does not modify the ability of skin dendritic cells (DC) to take-up antigen in vivo. To assess the ability of DC in the skin of topically treated mice to take-up antigen in vivo, the fluorescent antigen, fluorescein isothiocyanate (FITC), was applied (0.5%) to the skin of mice treated 30 min earlier with vehicle or 1,25(OH)2D3. In (a), FITC+ CD11c+ cells were identified in the skin-draining lymph nodes (SDLN) of mice 18 hr after skin treatment as compared with mice untreated with FITC (no FITC). In (b), the expression of CD86 on CD11c+ FITC cells and CD11c+ FITC+ cells for six individual mice per treatment from two repeated experiments (mean + SEM, *P < 0.05) is shown. In (c), the ability of CD11c+ FITC+ cells sorted from the SDLN 18 hr after topical treatment to induce the proliferation of co-cultured CD4+ T cells from FITC-sensitized mice is shown (mean ± SEM, for six replicate wells per treatment).

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The in vivo priming capacity of CD11c+ cells from the SDLN of mice topically treated with 1,25(OH)2D3 is compromised

Our results to date suggested that DC populations in the SDLN of 1,25(OH)2D3-treated mice exhibit minor phenotypic differences with no change in their in vitro abilities to present antigen. An in vivo priming assay was used to further investigate the functional ability of these cells after topical application of 1,25(OH)2D3. Eighteen hours later, CD11c+ cells were purified from the SDLN and labelled with DNBS (Fig. 4a), a water-soluble analogue of the hapten, DNFB. Then, 105 cells were adoptively transferred via subcutaneous injection into the ear pinnae of naive recipient mice (Fig. 4a), with another group of mice receiving a saline injection as a control. Seven days after ear injection, the ear pinnae were challenged with 0·2% DNFB to induce ear-swelling, which was measured 48 hr later (Fig. 4a). Significant ear-swelling was observed in mice that received CD11c+ cells from the SDLN of vehicle-treated donor animals (Fig. 4b). This response was significantly reduced (by 62%, relative to challenge-only controls) in recipients of CD11c+ cells from 1,25(OH)2D3-treated mice (Fig. 4b). The number of cells recovered from the EDLN cells was proportional to the extent of ear-swelling, such that the number of cells in recipients of CD11c+ cells from 1,25(OH)2D3-treated mice was significantly reduced relative to the vehicle treatment (Fig. 4c). Together, these observations indicate that topical 1,25(OH)2D3 treatment compromised the ability of CD11c+ cells in the SDLN to prime an immune response in vivo.

image

Figure 4.  Topical vitamin D3 [1,25(OH)2D3] reduces the in vivo priming ability of CD11c+ cells from the SDLN. In (a), the ability of CD11c+ cells from the SDLN of mice treated topically with 1,25(OH)2D3 to prime immune responses in vivo was assessed. 18 hr after topical treatment, the SDLN were removed and CD11c+ cells were purified and labelled with 1 mm DNBS. Cells (105) were then adoptively transferred (by subcutaneous injection) into the ear pinnae of naive mice, with a group of mice receiving saline only. After 7 days, ear pinnae of mice were challenged with 10 μl 0.2% DNFB (in acetone), with ear-swelling measured 48 hr later. In (b), the ear-swelling response 48 hr after ear challenge with DNFB is shown for recipients of CD11c+ cells from the skin-draining lymph nodes (SDLN) of vehicle-treated (Vehicle CD11c+) or 1,25(OH)2D3-treated (VitD CD11c+) mice. Responses are also shown for recipients of saline only (Saline) and mice that were only challenged with DNFB (Challenge only) (mean + SEM, n = 5 mice/treatment, *P < 0.05). In (c), the number of ear-draining lymph node (EDLN) cells determined 48 hr after ear challenge of recipient mice are shown, with results for the Vehicle CD11c+, VitD CD11c+ and Challenge only treatments described above (mean ± SEM, pooled EDLN cells from n = 5 mice/treatment for four or five repeated experiments, *P < 0.05).

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CD11c+ cells from the SDLN of 1,25(OH)2D3-treated mice skew immune responses towards a Th2 phenotype

The EDLN cells were removed from recipient mice 48 hr after ear challenge (Fig. 4a) and the capacity of these cells to secrete various cytokines was assessed by culture for 96 hr, with or without DNBS. Although the secretion of IL-2, IL5, IL-10 and IL-17 by EDLN cells was not modified, IL-4 levels were significantly enhanced in the supernatants of EDLN cells from recipients of CD11c+ cells from 1,25(OH)2D3-treated mice (Fig. 5, with data not shown for IL-2 or IL-5). When DNBS was included in the culture, IFN-γ levels were reduced in supernatants of EDLN from recipients of CD11c+ cells from 1,25(OH)2D3-treated mice. No difference in the proliferation of EDLN cells over this 96-hr time–course was detected for EDLN from recipients of CD11c+ cells from vehicle- or 1,25(OH)2D3-treated mice (data not shown). Hence, the reduced ability of CD11c+ cells from 1,25(OH)2D3-treated mice to prime immune responses in vivo may be partly the result of a switch towards a Th2 phenotype.

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Figure 5.  Topical vitamin D3 [1,25(OH)2D3] enhances interleukin-4 (IL-4) secretion by ear-draining lymph node (EDLN) cells from recipients of CD11c+ cells from the SDLN. The EDLN cells were removed from the recipients of CD11c+ cells and from vehicle-treated or 1,25(OH)2D3-treated mice (as described in Fig. 4a) 48 hr after ear challenge and cultured for 96 hr with and without 1 mm DNBS. Concentrations of IL-4, IL-10, IL-17 and interferon-γ (IFN-γ) in supernatants are shown for results pooled from two individual experiments (mean + SEM, pooled EDLN cells from n = 5 mice/treatment for three replicate wells/treatment, *P < 0.05).

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CD11c+ cells from the SDLN of 1,25(OH)2D3-treated mice increase the regulatory activity of CD4+ CD25+  cells in vivo

To determine if the reduced priming ability of CD11c+ from 1,25(OH)2D3-treated mice was the result of an effect on regulatory T cells, EDLN were removed 48 hr after ear challenge (Fig. 4a). There was no difference in the number of CD4+ CD25+ Foxp3+ cells identified in the EDLN, with 3·9% ± 0·1% and 3·9% ± 0·3% (mean ± SEM, n = 3 repeat experiments) in recipients of CD11c+ cells from vehicle or 1,25(OH)2D3-treated mice, respectively. There was also no difference in mean fluorescence intensity of Foxp3 expressed by CD4+ CD25+ cells in the EDLN of the vehicle or 1,25(OH)2D3 treatments, with most of the cells (≥ 90%) expressing Foxp3 (data not shown). To examine the suppressive activity of CD4+ cell populations, CD4+ CD25+ and CD25+ CD25 cells were isolated from the EDLN 48 hr after ear challenge. The ability of these cells to modulate the proliferation of a responding cell population was determined by incubating them at various ratios with CFSE-labelled responder cells together with CD4 cells (for antigen presentation) and DNBS. The dilution of CFSE in the responder cells in the cultures was determined after 96 hr of culture. CD4+ CD25+ cells from the EDLN of recipients of CD11c+ cells had a greater suppressive capacity than CD4+ CD25+ cells from mice challenged with DNFB (no cell transfer, Fig. 6). Previous topical 1,25(OH)2D3 treatment further increased the capacity of CD4+ CD25+ cells isolated from the EDLN to suppress the proliferation of co-cultured responder cells (Fig. 6). The proliferation of responder cells was not affected by CD4+ CD25 cells from either experimental recipient mice (Fig. 6). The increased ability of CD4+ CD25+ cells from recipients of CD11c+ cells from 1,25(OH)2D3-treated mice was not dependent on IL-10 because the inclusion of anti-IL-10 mAb in these co-cultures did not prevent their enhanced ability to suppress responder cell proliferation (data not shown). Therefore, CD11c+ cells from the SDLN of mice treated topically with 1,25(OH)2D3 increase the suppressive activity of CD4+ CD25+ cells in recipient mice.

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Figure 6.  Topical vitamin D3 [1,25(OH)2D3] enhances the suppressive ability of CD4+ CD25+ cells from the ear-draining lymph nodes (EDLN) of recipients of CD11c+ cells from the skin-draining lymph nodes (SDLN). The EDLN cells from the recipients of CD11c+ cells from vehicle-treated or 1,25(OH)2D3-treated or challenge-only mice (as described in Fig. 4a), were removed 48 hr after ear challenge. CD4+ CD25+ or CD4+ CD25 cells were purified from the EDLN and cultured with a CFSE-labelled CD4+ CD25 responding T-cell population, CD4 antigen-presenting cells and 1 mm DNBS for 96 hr. CD4+ CD25+ or CD4+ CD25 cells were cultured with the responding cells at a ratio of 1 : 1 or 1 : 10. The proliferation of the responding cells as determined by dilution of CFSE is shown as the proportion of CD4+ CFSElo cells. Results were pooled from three repeated experiments (mean + SEM, pooled EDLN cells from n = 5 mice/treatment for three replicate wells/treatment, *P < 0.05).

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Topical 1,25(OH)2D3 enhances indoleamine 2,3-dioxygenase messenger RNA expression by CD11c+ cells in the SDLN

To investigate a potential mechanism by which topical 1,25(OH)2D3 compromises the ability of CD11c+ cells to prime immune responses in vivo, the expression of eight messenger RNAs (mRNAs) was assessed, including CCL22, IDO, IFN-γ, IL-10, IL-12p35, IL-12p40, relB and TGF-β. Of these eight mRNAs investigated for cells from three independent experiments, only that for IDO was significantly up-regulated in CD11c+ cells isolated from the SDLN of mice treated topically with 1,25(OH)2D3 (Fig. 7).

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Figure 7.  Topical vitamin D3 [1,25(OH)2D3] increases indoleamine 2,3-dioxygenase messenger RNA (IDO mRNA) expression in CD11c+ cells from the skin-draining lymph nodes (SDLN). CD11c+ cells were purified from the SDLN of mice 18 hr after topical treatment with vehicle or 1,25(OH)2D3 and the expression of mRNAs for eight genes was determined as shown relative to the house-keeping gene Eef1α. Results are from three repeated experiments (mean + SEM, pooled CD11c+ cells from n = 10 mice/treatment, *P < 0.05).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In this study, a single topical application of 1,25(OH)2D3 modified the immunostimulatory capacity of DC residing in the SDLN. Eighteen hours after topical 1,25(OH)2D3, CD11c+ DC from the SDLN primed a significantly smaller ear-swelling response in recipient mice, and enhanced the suppressive capacity of CD4+ CD25+ regulatory T cells. These results provide a mechanism for the suppressive effects of topical 1,25(OH)2D3 on immune responses, where DCs prime a reduced response and alter the function of regulatory T cells residing in the SDLN. We also identify a candidate immunomodulatory molecule, IDO, which may be expressed by 1,25(OH)2D3-modified DC to affect T-cell function. These are novel findings, which for the first time link experimentally the effects of 1,25(OH)2D3 with DC function and regulatory T-cell activity in vivo.

In vitro stimulation with 1,25(OH)2D3 can induce human or murine DC to acquire ‘tolerogenic’ qualities with reduced abilities to secrete IL-12 and to present antigen to co-cultured T cells, and enhanced capacities to induce regulatory T cells (reviewed in ref. 21). These observations are complemented by recent findings, where many gene targets identified in 1,25(OH)2D3-treated DC are associated with a tolerogenic phenotype.22 Although the effects of 1,25(OH)2D3 on DC in vivo are less well described, similar tolerogenic effects have been observed with 1,25(OH)2D3-modified DC indirectly promoting allograft survival, suppressing contact hypersensitivity responses, reducing allergic airway disease and enhancing T regulatory cell activity.7,11,14,23 By transferring the CD11c+ cells from 1,25(OH)2D3-treated mice into naive recipient mice, we have confirmed these earlier observations, and formalized a direct link between the action of 1,25(OH)2D3 on DC function and subsequent regulation of CD4+ CD25+ cells.

While topical 1,25(OH)2D3 modulated the function of DC upon adoptive transfer in vivo, we did not observe any significant changes in the ability of CD11c+ cells from the SDLN to take-up, process or present antigen in vitro, even when the antigen (FITC) was painted onto skin immediately following 1,25(OH)2D3 application. Furthermore, co-stimulatory molecule expression was only modestly down-regulated on skin-derived DC populations in the SDLN by topical 1,25(OH)2D3. These observations are reminiscent of those published recently where there was no difference in antigen (FITC) carriage or co-stimulatory molecule expression by DC from the skin of vitamin D3-deficient or -replete mice.24 In our studies, we did not define the skin-derived DC population affected by topical 1,25(OH)2D3. SDLN-resident and not skin-derived DC could be the target cells. However, topical 1,25(OH)2D3 affected the numbers and phenotype (e.g. CD86 expression) of multiple DC subtypes (i.e. DEC205+ CD8+ and DEC205+ CD8) in the SDLN suggesting that more than one type of DC may be affected by topical 1,25(OH)2D3. The expression of CD8 and DEC-205 on DC in the SDLN was similar to that described previously for mice in steady-state conditions.18 However, the co-expression of DEC-205 and CD8 on DC may be different in other lymphoid organ or tissue sites such as the spleen25 or following antigenic stimulation.

This study identified IDO as a candidate molecule by which CD11c+ cells from 1,25(OH)2D3-treated mice may increase regulatory T-cell activity in 1,25(OH)2D3-treated mice. The expression of a panel of genes was examined, as they have previously been linked with a tolerogenic DC phenotype (CCL22,26 IDO,27 relB,28 IL-10, TGF-β, reviewed in ref. 21) or an immunostimulatory phenotype (IL-12p35, IL-12p40, reviewed in ref. 21) with the expression of IFN-γ mRNA also determined as a negative control. The mRNA of IDO was the only mRNA of this eight gene panel to be significantly regulated in CD11c+ cells isolated from the SDLN of mice topically treated with 1,25(OH)2D3. However, previous studies of IDO expression by human cells cultured with 1,25(OH)2D3 have had conflicting results. Increased IDO enzymatic activity and enhanced IDO mRNA expression was detected in human CD4+ T cells cultured with 1,25(OH)2D3.27 In contrast, human DC differentiated from peripheral blood monocytes in the presence of 1,25(OH)2D3 expressed an immature phenotype with an enhanced capacity to generate CD4+ T cells with regulatory activity, but they had reduced IDO enzymatic activity.29 Perhaps the difference between the findings of Pedersen et al.29 and our own is the result of the presence of 1,25(OH)2D3 during DC differentiation in vitro, as opposed to an acute dose of 1,25(OH)2D3 administered to the skin in vivo, where the presence of other cells and mediators could affect skin DC phenotype and function. In addition, our results appear to be independent of changes in relB mRNA expression (reviewed in ref. 28).

Others have postulated that the increased expression of receptor activator of nuclear factor-κB ligand (RANKL) by 1,25(OH)2D3-exposed keratinocytes may be responsible for modifying the downstream activity of epidermal DC and regulatory T cells.7,30 Following topical administration of a 1,25(OH)2D3 analogue or UV irradiation of skin, keratinocytes up-regulate RANKL expression.7,30 The DC from mice with keratinocytes over-expressing RANKL have an increased propensity to stimulate the proliferation of co-cultured regulatory T cells, although the suppressive activity of these cells was not modified on a per cell basis.30 In contrast, following topical 1,25(OH)2D3, we observed that DC from the SDLN have an increased ability to promote the suppressive activity, but not the numbers of regulatory T cells.6 It is therefore unclear what role exists in our studies for increased RANKL expression by 1,25(OH)2D3-exposed keratinocytes to promote the tolerogenic potential of DC.

The 1,25(OH)2D3 can regulate Th2 immune responses in vitro. However, controversy exists as to how 1,25(OH)2D3 regulates IL-4 secretion and GATA-3 expression during Th2 differentiation of CD4+ T cells.31–33 From our studies, DC affected by 1,25(OH)2D3in vivo possess the capacity to switch immune responses in the EDLN of recipient mice towards a Th2 type environment, as demonstrated by the increased IL-4 levels. Our observations are similar to those detected in a model of hapten-induced colitis, where 1,25(OH)2D3 (0·2 μg/kg intraperitoneally) reduced the severity of colitis and up-regulated Th2 markers like IL-4 and GATA-3 in the colons of treated mice.34

In conclusion, topical 1,25(OH)2D3 modulates the activity of SDLN-resident CD4+ CD25+ cells through its tolerogenic effects on DC. These cells also promote a Th2 immune environment and perhaps through an IDO-dependent mechanism suppress Th1/17-driven immune responses. Our results support the continued use of 1,25(OH)2D3 (or its analogues) as a topical treatment of inflammatory skin diseases, for the induction of a tolerogenic environment to control or limit the severity of immune disease.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Dr Michelle Tourigny for her cell-sorting expertise and are grateful to the Cancer Council of Western Australia, the National Health and Medical Research Council of Australia and the University of Western Australia for funding this research.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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