the journal of rheumatology volume 76, no. d-hormone and ... · the journal of rheumatology volume...

The Journal of Rheumatology Volume 76, no.

D-hormone and the immune system.

Margherita T Cantorna and Brett D Mahon

http://www.jrheum.org/content/76/11J Rheumatol 2005;76;11-20

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D-Hormone and the Immune System

The discovery of the vitamin D receptor (VDR) in thecells of the immune system and the fact that activateddendritic cells produce the vitamin D hormone1 suggestedthat vitamin D could have immunoregulatory properties.VDR, a member of the nuclear hormone receptor super-family, was identified in mononuclear cells, dendritic cells,antigen-presenting cells, and activated T lymphocytes.

A physiological role for vitamin D in the immune sys-tem is suggested by the presence of the VDR in primarylymphoid organs. The primary lymphoid organs (bonemarrow and thymus) are the centers where the immunesystem develops and differentiates2,3. However, VDRknockout (KO) mice have normal thymuses, normalmyelopoiesis of the bone marrow, and no overt abnor-malities in other immune system compartments4.Recently it has been shown that when activated, the VDRknockout mouse has overactive and inflammatory Tcells; moreover, in animals susceptible to inflammatorybowel disease (IBD), this results in a fulminating form ofIBD5. The function of VDR in the primary lymphoid tis-sues is not known, but arguably there is a role of the D-hormone in regulating the processes occurring there.

VITAMIN D AND AUTOIMMUNITYAutoimmune diseases are diseases where the immune sys-tem’s ability to discriminate between self - and non-selftissue fails. People with diseases like multiple sclerosis(MS), arthritis, and IBD have T cells that target self anddrive the immune system to induce inflammation in theperipheral tissues. The causes of the inappropriateimmune attacks are not known; however, it is clear thatboth genetic and environmental factors contribute to theetiology of these diseases.

T cells have been shown to be central for the pathologyof autoimmune disease. Specifically, type 1 helper (Th1)cells, which secrete interferon-γ (IFN-γ) and tumor necro-sis factor-α (TNF-α) have been shown to transferautoimmune disease in mice. Treatments that can direct-ly or indirectly block Th1 cell function are effective forsuppressing autoimmunity. Type 2 helper cells (Th2)secrete interleukin 4 (IL-4), which inhibits the differenti-ation of Th1 cells. Other regulatory T cells produce trans-forming growth factor-β1 (TGF-β1) or IL-10, which alsoinhibit Th1 effector cell function.

Vitamin D status has been linked to autoimmune dis-eases in humans. Recently a large population study(Nurses Health Study I and II) showed that women in thehighest quintile of vitamin D intake had a 40% reducedrate of developing MS6. Similarly, vitamin D intake wasinversely associated with rheumatoid arthritis in theWomen’s Iowa Health Study, which contained data from29,368 women7. Experimentally it has been shown thatvitamin D deficiency exacerbates both IBD and MS inanimals8,9. Further, D-hormone has been shown to sup-press experimental MS and IBD in mice8,9. Interestingly,D-hormone has been shown to effectively inhibit autoim-munity even when animals were vitamin D sufficient.

MARGHERITA T. CANTORNA and BRETT D. MAHON

ABSTRACT. D-hormone [1,25(OH)2 D3] is an important immune system regulator that has been shown to inhibit devel-opment of autoimmune diseases including experimental inflammatory bowel disease (IBD), rheumatoidarthritis (RA), multiple sclerosis (MS), and type 1 diabetes. Paradoxically, other immune mediated diseases(experimental asthma) and immunity to infectious organisms were not found to be affected by D-hormonetreatment. The effectiveness of D-hormone treatment of autoimmune diseases is due to inhibition of thedevelopment and function of Th1 cells and the induction of other Th cells including Th2 cells. We reportresults of microarray analysis of colons from D-hormone treated mice with experimental IBD. Two hundredthirty-nine genes were inhibited and 298 genes were upregulated in the colon by D-hormone treatment ofmice with IBD. Of interest was the D-hormone mediated inhibition of 3 tumor necrosis factor-α (TNF-α,lipopolysaccharide-induced TNF-α factor, and TNF receptor) related genes in the colon. It is likely that theeffectiveness of D-hormone treatment of experimental autoimmunity is due in part to the inhibition of theTNF family of genes. D-hormone is a selective regulator of the immune system, and the outcome of D-hor-mone treatment depends on the nature (infectious disease, asthma, autoimmune disease, etc.) of the immuneresponse. (J Rheumatol 2005;32 Suppl 76:11-20)

Key Indexing Terms:VITAMIN D RECEPTORS IMMUNE SYSTEM TUMOR NECROSIS FACTORCALCITRIOL ANIMAL DISEASE MODELS

From the Department of Nutritional Sciences, Pennsylvania StateUniversity, University Park, Pennsylvania, USA.

Supported by Crohn’s and Colitis Foundation of America, SeniorResearch Award, and NIH-NINDS 1R01 NS38888-01A4.

M.T. Cantorna, PhD, Assistant Professor of Nutrition Immunology;B.D. Mahon, PhD.

Address reprint requests to Dr. M.T. Cantorna, Department ofNutritional Sciences, 126 S. Henderson Bldg., University Park, PA 16802.E-mail: [email protected]

Cantorna and Mahon: D-hormone and the immune system 11






different mice (Tables 2 and 3). The inclusion criteria forgenes reported were (1) the gene was up- or downregulat-ed 2-fold or more in one of the experiments, (2) the genewas up- or downregulated by 1.3-fold or more in the sec-ond experiment in the same direction as the first, and (3)the microarray spot for the gene had a combined medianfluorescent intensity (median green fluorescence medianbackground + median red fluorescence median back-ground) of more than 100 on both array replicates. Twohundred thirty-nine downregulated (Table 2) and 298upregulated (Table 3) potential targets of the D-hormonewere identified using these criteria.

Table 4 shows the results for 6 genes. The expression ofVDR is known to be positively regulated by the D-hor-mone. As expected, D-hormone treatment of the IL-10KO mouse increased the expression of VDR mRNA inthe colons of D-hormone treated mice. Interestingly,calmodulin and calcium binding protein A6 (calcyclin) areexpressed in colonic tissue, and it seems reasonable thatvitamin D would increase expression of these calcium reg-ulators. Three genes (TNF-α, lipopolysaccharide-inducedTNF-α factor, and TNF receptor) involved in the regula-tion of TNF-α were inhibited by D-hormone in thecolons of IL-10 KO mice. The inhibition of these TNF-αrelated genes by D-hormone correlated with decreasedseverity of colitis in D-hormone treated IL-10 KO mice.Elevated TNF-α secretion has been shown to play a rolein Crohn’s disease, and treatments that inhibit TNF-αsecretion have been shown to be effective for treating IBDin humans.

D-HORMONE SELECTIVELY REGULATESIMMUNE FUNCTIONClearly, D-hormone is a potent suppressor of autoim-mune diseases. Based on the ability of the D-hormone tosuppress autoimmunity and to prolong allograft survival,scientists have labeled it “immunosuppressive”. However,the effect of the D-hormone on immune function has notbeen shown to be broadly immunosuppressive. D-hor-mone treatment was tested for an effect on the ability ofa host to fight an infection with Candida albicans orHerpes simplex virus15. Mice were treated with the D-hormone at levels previously shown to be effective at pro-longing allograft transplants15. As controls, mice were

IN VIVO TARGETS OF D-HORMONEIn vivo targets of the D-hormone in experimental MSinclude a number of inflammatory cytokines (Table 1). D-hormone treatment of mice with experimental MS result-ed in inhibition of lymphocyte accumulation in the lymphnodes10. Mice treated with the D-hormone had fewer cellssecreting TNF-α and IFN-γ10. In addition, D-hormonetreatment increased the level of IL-4 and TGF-1 producedin the animals10. The ability to produce IL-4 is importantfor the function of D-hormone, since D-hormone treat-ment was ineffective for suppressing experimental MS inmice that were deficient in IL-4 (IL-4 KO, Table 1)11. Asexpected, D-hormone was ineffective at inhibiting experi-mental MS in VDR KO animals12. Vitamin D deficiencyaccelerated the development of IBD in IL-10 KO mice,and colitis symptoms in IL-10 KO mice were suppressedby D-hormone treatment (Table 1)9. Conversely, the coli-tis that develops in IL-2 KO mice was unaffected by vita-min D status13. In addition, colitis in IL-2 KO mice wasnot suppressed by D-hormone treatment (Table 1)13. Thenet result of D-hormone treatment in experimentalautoimmunity includes decreased symptoms, which paral-lel the reduced production of Th1 associated cytokinesand TNF-α, and a concomitant increase in cytokines thatcorrespond to the resolution of inflammation anddecreased severity of autoimmunity. IL-4 and IL-2 pro-duction have been shown to be crucial for the effectivenessof D-hormone in experimental autoimmunity. Overall,the action of D-hormone in vivo functions to suppressautoimmunity by inhibiting Th1 cell associated responsesand increasing Th2 and other regulatory T cell processes.

NOVEL D-HORMONE TARGETS IN THE COLONOF MICE WITH EXPERIMENTAL IBDWhen D-hormone targets were probed in experimentalIBD, results were consistent with previous findings:Untreated vitamin D deficient IL-10 KO mice developedmore severe IBD, as shown by larger small intestine/bodyweight ratios (9.0 ± 0.6%) compared to the D-hormonetreated IL-10 KO mice (6.5 ± 0.7%). Based on the highexpression of TNF-α in the colons of IL-10 KO mice(data not shown), microarray analysis was done usingcolon tissue. Microarray analysis was performed exactlyas described14 and repeated once using total RNA from

Genotype Effects of D-hormone Experimental Models ReferencesTested

Wildtype Suppressed MS, lupus, IBD, 5,8,9,16,17arthritis, type-1 diabetes

IL-4 KO Reduced MS 11

VDR KO No effect MS 12

IL-2 KO No effect IBD 13

IL-10 KO Suppressed IBD 9

Table 1. In vivo effects of D-hormone in mice with targeted gene deletions.

12 The Journal of Rheumatology 2005, Volume 32 Supplement 76






Table 2. Genes downregulated by D-hormone in whole colon tissue of IL-10 knockout mice. *The ratio is the inverse of the average of the medianintensities of red fluorescence over green fluorescence. Negative ratios represent the factor by which genes were inhibited by D-hormone treat-ment. All values are the means of 2 independent experiments.







Table 3. Genes upregulated by D-hormone in whole colon tissue of IL-10 knockout mice. *The ratio is the average of the median intensities of redfluorescence over green fluorescence. Positive ratios represent the factor by which genes were increased by D-hormone treatment. All values are themeans of 2 independent experiments.







either not treated or treated with the immunosuppressivedrug cyclosporin A. The mice treated with cyclosporin Ashowed reduced survival following C. albicans and H.simplex infections15. Conversely, the D-hormone treatedmice were not different from the untreated controls intheir ability to survive either the C. albicans or the H.simplex infections15. D-hormone did not alter the abilityof the host to mount an immune response to either C.albicans or H. simplex.

Asthma is a disease that is driven by Th2 cells respond-ing to environmental antigens. Based on the ability of D-hormone to upregulate the Th2 cell response, we hypoth-esized that D-hormone might exacerbate experimentalasthma. Two strains of mice were treated with D-hor-mone and then induced to develop experimental asthma.There was no effect of D-hormone on the inflammationand epithelial hyperplasia in the lungs of mice with asth-ma. Interestingly, VDR KO mice were also induced to

develop asthma; however, asthma failed to develop in thelungs of these mice. The absence of inflammation in thelungs of VDR KO mice induced to develop experimentalasthma suggests an important role for vitamin D signal-ing in the development of inflammation in the lungs. TheD-hormone, however, had no effect on the severity ofexperimental asthma in mice. Together these data suggestthat the D-hormone is a selective regulator of theimmune system.

CONCLUSIONSThe in vivo effect of vitamin D status on immune functiondepends on the nature of the immune challenge. Themost dramatic effects of D-hormone on the immune sys-tem seem to be in the control of Th1-driven autoimmu-nity. D-hormone had no effect on the ability of the hostto fight infections with C. albicans and H. simplex. Inaddition, Th2-driven asthma was not affected by D-hor-


Gene Experiment 1 Experiment 2Intensity* Ratio** Intensity Ratio

TNF-α 414 –2.1 309 –2.7LPS-induced TNF-α factor# 4267 –1.7 4516 –1.9TNF receptor 3494 –1.7 163 –3.1VDR 745 2.6 422 1.9Calmodulin 4663 1.7 11878 3.6Calcium Binding Protein A6 97601 2.3 39160 4.7

* Combined median red and green intensities.** The ratio is either the ratio of green fluorescence over red fluorescence (positive values)or the inverse (negative values). Positive ratios represent genes that are activated in the presence of D-hormone and negative ratios represent genes that are repressed in thepresence of D-hormone.

# LPS-induced TNF-α factor was reanalyzed using quantitative real-time PCR.The expressionof this gene was 4 to 461 times lower in colons of D-hormone treated IL-10 KO mice (n=4) com-pared to the D–control (n=4) colons. The microarray underestimated the efficacy of D-hormoneto inhibit LPS-induced TNF-α.

Table 4. D-hormone targets 3 TNF-α related genes in the colon of mice with experimental IBD.






mone treatment. The vitamin D or VDR-deficient hosthas elevated Th1 cell responses and diminished Th2 asso-ciated responses. In the absence of the VDR, Th1-drivenIBD is more severe and Th2-driven asthma does notdevelop. The evidence suggests a model where the effec-tiveness of D-hormone treatment of autoimmune dis-eases comes as a result of the inhibition of the develop-ment and function of Th1 cells and the induction ofother CD4+ T cells including Th2 cells. The mechanismsunderlying the paradoxical effects of D-hormone onautoimmune diseases and lack of effect on experimentalasthma and infectious host resistance are still not known.

Glucocorticoids are broadly immunosuppressive drugscommonly used to treat a variety of diseases includingautoimmune diseases. Autoimmune patients are at an ele-vated risk of developing osteoporosis as a result of theincreased inflammation and glucocorticoid use. Studieshave shown that D-hormone can increase bone mineraldensity and improve other bone markers in patients withIBD and MS. Clearly, there are direct effects of the D-hormone on bone health. It is also possible that the D-hormone mediates a reduction in inflammation that indi-rectly inhibits bone disease by halting further bonedestruction. Little is known about the effect of vitamin Dsupplementation or D-hormone treatment on the severi-ty of human autoimmune diseases. However, the poten-tial benefits of D-hormone treatment for patients withautoimmune diseases include a reduction in glucocorti-coid use, a reduction in the symptoms of their disease,and increased bone mineral density.

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13. Bemiss CJ, Mahon BD, Henry A, Weaver V, Cantorna MT.Interleukin-2 is one of the targets of 1,25-dihydroxyvitamin D3 inthe immune system. Arch Biochem Biophys 2002;402:249-54.

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