International Immunopharmacology 17 (2013) 744–751

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International Immunopharmacology

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1,25-dihydroxyvitamin D3-3-bromoacetate, a novel vitamin D analoginduces immunosuppression through PI3K/Akt/mTOR signaling cascade

Ananya Datta-Mitra a,b,1, Anupam Mitra b,c,1, Rahul Ray d,Siba P. Raychaudhuri a,b, Smriti Kundu-Raychaudhuri a,b,⁎a Department of Internal Medicine/Rheumatology, Allergy and Clinical Immunology, School of Medicine, University of California Davis, Davis, CA 95616, USAb VA Medical Centre Sacramento, Mather, CA 95655, USAc Department of Dermatology, School of Medicine, University of California, Davis, Sacramento, CA 95817, USAd Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA

⁎ Corresponding author at: VA Medical Center SacramMedicine/Rheumatology, Allergy and Clinical ImmunoloUniversity of California, 10535 Hospital Way, Bldg noTel.: +1 650 575 6186; fax: +1 413 771 6419.

E-mail address: smray@ucdavis.edu (S. Kundu-Raycha1 Both the authors equally contributed.

1567-5769/$ – see front matter. Published by Elsevier B.Vhttp://dx.doi.org/10.1016/j.intimp.2013.08.009

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 3 April 2013Received in revised form 15 August 2013Accepted 16 August 2013Available online 13 September 2013

Keywords:1α,25-dihydroxyvitamin D3-3-bromoacetateT cellsAkt/mTORProliferation

Purpose: The molecular mechanism responsible for the immunomodulatory effect of 1,25-dihydroxyvitamin D3(Vit-D) is still notwell elucidated. Unavoidable systemic toxicity of Vit-D has encouraged to developmore potentand less toxic Vit-D analogs, such as 1,25-dihydroxyvitaminD3-3-bromoacetate (BE). Our aimwas to explore theimmunosuppressive effect of BE and its molecular mechanism in autoimmune diseases.Method: Magnetically sorted CD3+ T cells (T cells) from PBMCs of psoriasis and autoimmune arthritis patientswere cultured with/without BE and Vit-D followed by proliferation (MTT, CFSE dilution assays) and apoptosisassays (annexin V). Immunoblot was performed to determine the signaling cascade responsible for theantiproliferative effect.Results: In MTT assay, BE (OD: 0.64 ± 0.08) markedly inhibited the anti-CD3/CD28 stimulated proliferation ofT cells (OD: 1.8 ± 0.30, p b 0.001) and at equivalent doses, the inhibitory effect was more than that of Vit-D

(OD: 0.91 ± 0.11, p b 0.05). The antiproliferative effect of BE was extended to activated CD4+ and CD8+ mem-ory T cells (CD45RA−CD11a+) without much effect on the naïve T cells. BE induced more apoptosis of T cells(45.01 ± 4.27%, p b 0.01) compared to untreated cells (3.45 ± 1.8%), and the proapoptotic effect wasmarkedly more than that of Vit-D (26.1 ± 2.05%, p b 0.05). BE effectively inhibited the anti-CD3/CD28-inducedphosphorylation of Akt and mTOR and in both, BE showed more potency than Vit-D (p b 0.05).Conclusion: Topical Vit-D is being used successfully in psoriasis for years. However, its potency is less comparedto topical corticosteroids. The de novo BE showed significantlymore immunosuppression than conventional Vit-D and the immunosuppressive effect is PI3K/Akt/mTOR dependent. Our results indicate that BE could be aneffective therapeutic agent for psoriasis and other T-cell-mediated autoimmune diseases.

Published by Elsevier B.V.

1. Introduction

The association of 1α,25-dihydroxyvitamin D3 [referred to as Vit-Dthroughout themanuscript]with bonehomeostasis and calciummetab-olism is quite well known; however, the extra renal synthesis of Vit-Dby macrophages and other immune cells have rediscovered the immu-nomodulatory role of Vit-D. Vit-D has been shown to control the growthand differentiation of both innate and acquired immune cells, as well asinfluence their activity such as cytokine production [1–3]. Currentresearch has been aimed at identifying its therapeutic potential in auto-immune and inflammatory diseases [4–8]. The functions of Vit-D are

ento, Department of Internalgy, Davis School of Medicine,. 807, Mather, CA 95655, USA.

udhuri).

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mediated by interaction of Vit-D receptor (VDR) with retinoid X re-ceptor followed by activation of VDR-responsive genes (VDRE),resulting in inhibition of proliferation and promotion of differentia-tion of cells [9,10]. Originally, the therapeutic potential of Vit-D asan antiproliferative agent has been shown in different cancers[11,12]; however, the inherent calcemic toxicity at pharmacologicaldoses limits its use as an anticancer agent [13,14]. Among the auto-immune diseases, Vit-D analogue (Calcipotriol) has been shownsome efficacy as a topical agent in psoriasis but not all patients re-spond to this therapy and addition of topical steroid gives a betteroutcome than Calcipotriol alone [15,16]. These studies led to devel-opment of more potent and less toxic analogues of Vit-D. In this con-text, our research group had developed an affinity labeling analogof Vit-D, 1α,25-dihydroxyvitamin D3-3-bromoacetate (henceforthreferred as BE throughout the text), which covalently binds toVDR-ligand binding domain (VDR-LBD), thus physically protectedfrom catabolic enzymes, resulting in increased half-life but withcapacity to activate gene transcription similar to Vit-D [17]. Studies

Fig. 1. Dose–response studies of equimolar concentration of Vit-D and BE in CD3+ T cells.Magnetically sorted CD3+ T cells were cultured with or without anti-CD3/CD28 and Vit-D(10−9 M to 10−5 M) or BE (10−9 M to 10−5 M) for 5 days. Cell proliferation wasmeasured by MTT assay. All experiments were done in triplicate. Bar diagram representsthe dose-dependent antimitotic effect of Vit-D and BE. At equimolar concentrations, BEshowed significantly more antimitotic effect than Vit-D. Both the compound showedmaximum antimitotic effect with minimum toxicity at 10−6 M concentration. Resultswere expressed as mean ± SEM. Non-parametric unpaired test (Mann–Whitney U test)was done to determine statistical significance. *p b 0.01.

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have shown that BE is amore potent antiproliferative agent compared toVit-D in keratinocytes [18] and in human cancer cell lines, such as pros-tate [19–21], kidney [22], pancreas [23] and neuroblastoma [24]. Also,BE found to be relatively safer than systemically administered Vit-D [22].

One of the well-known targets of Vit-D is the T lymphocytes, whichexpress VDR extensively [25–28]. Expression of VDR gets upregulatedon T-cell activation, indicating a relationship between Vit-D and adap-tive immunity. Several in vitro and in vivo studies have illustrated that,both in human and animal models of autoimmune diseases, not onlyVit-D suppressed Th1 and Th17 cytokine responses [4,29,30] but alsoenhanced the polarization to Th2 and regulatory T-cell (Treg) pheno-types [31–33]. Extended evidence suggests that Vit-D also affects themigration and localization of CD4+ T cells in response to physiologicaland inflammatory stimuli [34,35]. Moreover, Vit-D was found to inhibitthe production of proinflammatory cytokines like tumor necrosisfactor-α (TNF-α), interleukin-17 (IL-17), and interferon-γ (IFN-γ) andstimulate expression of anti-inflammatory cytokines like IL-4, IL-5 andIL-10 by peripheral blood mononuclear cells (PBMCs) [4,36,37].

Autoimmune diseases are characterized by loss of homeostasis ofthe immune system, resulting in aberrant function of the key effectorcells including helper (CD4+), cytotoxic (CD8+) T lymphocytes, Treg,B lymphocytes, and natural killer (NK) cells [38]. Among the autoim-mune diseases, psoriasis is themost prevalent one followed by rheuma-toid arthritis (RA) and others [39]. Psoriasis and RA are autoimmunediseases of the skin and joints respectively, involving a complex in-terplay of genetic, environmental and immunologic factors [40–42].Briefly, the role of different subsets of T lymphocytes and its cytokineis well documented in psoriasis, and there is mixed leukocytic infiltra-tion: CD4+ T lymphocytes infiltrating dermis and CD8+ T lymphocytesinfiltrating the epidermis [43–46]. In RA, activated T lymphocytes havebeen thought to drive the synovial inflammation and thus contributesignificantly in its pathogenesis [47–49]. The in-house Vit-D analogue,BE, has been shown to have potent antiproliferative effect in in vitropsoriasis disease model [50] as well as in different malignant celllines [19–24]; however, the immunosuppressive effect of BE andthe molecular mechanism behind this effect has not been exploredtill date. Thus, in this study, we explored the immunosuppressive ef-fect of BE and compared it with that of Vit-D in psoriasis and RA pa-tients. Moreover, we dissected out the molecular mechanism of theimmunosuppressive effect of BE in autoimmune diseases.

2. Materials and methods

2.1. Materials

Calcitriol (Vit-D) and the de novo Vit-D analog BE are kind giftfrom Dr. Rahul Ray (Vitamin D, Skin and Bone Research Laboratory,Boston University School of Medicine, Boston, Massachusetts). Wecarried out in vitro dose–response studies on magnetically sortedCD3+ T cells (n = 3) with different concentrations of Vit-D and BE(10−9 to 10−5 M) according to the previous reports [17,22,23]. Weobserved optimum response at 10−6 M (Fig. 1). Therefore, we used10−6 M for subsequent experiments. All other chemicals were pur-chased from Sigma-Aldrich (St. Louis, MO, USA), if not mentionedseparately.

2.2. Cell culture

PBMCs were collected from psoriasis (n = 5) and rheumatoid ar-thritis (n = 7) patients after obtaining IRB approved informed consent.All the recruited patients were not on any DMARDs or biologics for atleast 6 weeks before inclusion in this study. Two of the RA patientsand one of the psoriasis patient were on oral steroid (b7.5 mg/day).CD3+ T cells were sorted magnetically from the PBMCs using negativeT-cell enrichment kit (Stem Cell Technologies, Vancouver, BC Canada)as per manufacturer's instructions. The purity of the sorted CD3+ T

cells was found to be ~80% as determined by flow cytometry. Thecells were cultured in Roswell Park Memorial Institute medium(RPMI) 1640 (Cellgro Manassas, VA, USA).

2.3. Cell proliferation assays

Cell proliferation was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and carboxyfluoresceinsuccinimidyl ester (CFSE) dilution assay.

2.3.1. MTT assayThe 24-well tissue culture plates were pre-coated with anti-human

CD3 and CD28 antibody cocktail (5 μg/ml each) for 4 h at 37 °C/5%CO2, followed by addition of 5 × 105 CD3+ T cells in presence or ab-sence of BE and Vit-D and incubated for 5 days. MTT assay was doneaccording to our standardized protocol [51].

2.3.2. CFSE dilution assay1 × 105 CD3+ T cells were stained with CFSE (Molecular probes,

Eugene, OR, USA) reagent followed by treatment with anti-CD3/CD28cocktail, BE and Vit-D, incubated at 37 °C/5% CO2 for 5 days. On the5th day, cells were stained with anti-human primary antibodies forCD3, CD4, CD8, CD45RA and CD11a (BD Biosciences, San Jose, CA, USA).CFSE dilution was assessed by flow cytometry (LSRFortessaTM cell ana-lyzer, BD, San Jose, CA, USA) and data analyzed using FlowJo software(Tree Star, Ashland, OR, USA). Briefly, the parent population is repre-sented by a single peak (orange color) in the FlowJo software. Thecells proliferating from the parent population, i.e., the daughter popula-tion, are represented by several peaks (pink color). Thus, the number ofdaughter population (pink peak) aswell as number of cells in each peakwill represent the proliferative activity.

2.4. Apoptosis assay

CD3+ T cells were treated with or without BE and Vit-D for 5 daysat 37 °C/5% CO2. Staurosporine (Cell Signaling, Danvers, MA, USA)-induced apoptosis was used as a positive control [52,53]; 1 μM of

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Staurosporine was used for this assay. At the end of incubation, cellswere collected, washed and incubated on ice for 15 min with annexinV (BD Pharmingen, San Diego, CA, USA). Propidium iodide (1 μL) (BDPharmingen, SanDiego, CA, USA)was added just before data acquisitionin flow cytometer (FACS CaliburTM, BD, San Jose, CA, USA). Data wereanalyzed using FlowJo software.

2.5. Expression of pAkt, pmTOR in PBMCs

CD3+ T cells (1 × 106) were plated in 24-well culture plate with orwithout anti-CD3/CD28 cocktail, BE and Vit-D, incubated overnight at37 °C. Cell lysates were prepared as described in earlier studies [54].Briefly, an equal amount of protein (30 μg) for each lysatewas subjectedto 10% (for pAkt and α-tubulin) and 6% (for pmTOR, total mTOR) SDS–PAGE. We used pAkt (Ser473) at 1:250, α-tubulin at 1:500, pmTOR(Ser2448) at 1:200 and total mTOR at 1:500 dilutions. Membrane wasthen washed and incubated with 1:5000 dilution of peroxidase-conjugated goat anti-rabbit IgG Fc fragment specific antibody(Jackson Immunoresearch Labs, USA). All primary antibodies werepurchased from Cell Signaling Technology (Danvers, MA, USA). Thebandswere analyzed for their density using Image J software (NIH, Be-thesda, MD, USA). Results were expressed as relative intensity (RI:intensity of each band was adjusted to that of α-tubulin).

2.6. Statistical analysis

All experiments were done in triplicate. Results were expressed asmean ± SEM. For CFSE dilution assay, the percentage of divided cellsof each treatment group was determined using the FlowJo software.For apoptosis assay, the percentage of cells in the annexin V+PI−quad-rant (early apoptosis) was considered for statistical analysis. Statisticalanalysis to determine the differences in cell proliferation, apoptosisand protein expression between different treatment groups weredone using the Graph Pad Prism software, version 5.0 (Graph PadSoftware Inc, San Diego, CA, USA). Non-parametric unpaired test(Mann–Whitney U test) was used to determine statistical signifi-cance. A p value of b0.05 was considered statistically significant.

3. Result

3.1. BE showed significantly more antiproliferative effect than Vit-D on Tcells and its subpopulation

In MTT assay, physiological stimulus (anti-CD3/CD28 antibodycocktail) induced significant proliferation of CD3+ T cells as comparedto unstimulated cells (OD: 1.8 ± 0.30 vs. 0.9 ± 0.11, p b 0.01). BE(OD: 0.64 ± 0.08, p b 0.001) and Vit-D (OD: 0.92 ± 0.11, p b 0.01)both showed significant antiproliferative effect compared to anti-CD3/CD28 (OD: 1.8 ± 0.30). The antiproliferative effect of BE (OD: 0.64 ±0.08) was significantly more than that of Vit-D (OD: 0.92 ± 0.11,p b 0.05) (Fig. 2A). Similar results were observed in the CFSE dilutionassay; the proliferation of CD3+ T cells in terms of % divided cells wassignificantly reduced with BE (4.75 ± 0.61%, p b 0.001) and Vit-D(13.14 ± 1.94%, p b 0.01) compared to anti-CD3/CD28 stimulatedcells (21.18 ± 1.27%). Here also, BE showed significantly moreantiproliferative effect than Vit-D (p b 0.01) (Fig. 2B and C).

In CFSE dilution assay, we found that incubation of CD3+ Tcells with physiological stimulus (anti-CD3/CD28 cocktail) induced sig-nificant proliferation of CD11a+CD45RA− (activated memory T cell)subset of both CD4+ (21.53 ± 2.03%, p b 0.001) and CD8+ T cells(35.16 ± 4.62, p = 0.0004) as compared to unstimulated cells(4.33 ± 1.11% and 2.16 ± 0.33% respectively). BE showed signifi-cant antiproliferative effect on activated memory helper T cells(CD4+CD11a+CD45RA−, 5.8 ± 1.45%, p b 0.001) and cytotoxic/suppressor T cells (CD8+CD11a+CD45RA−, 3.51 ± 1.69%, p = 0.0004)compared to CD3/CD28 stimulation (21.53 ± 2.03% and 35.16 ±

4.62%, respectively). Vit-D also showed significant antiproliferativeeffect on activated memory helper T cells (CD4+CD11a+CD45RA−,8.26 ± 0.78%, p b 0.001) and memory cytotoxic/suppressor T cells(CD8+CD11a+CD45RA−, 9.45 ± 2.02%, p = 0.002) as compared toonly CD3/CD28 stimulation (21.53 ± 2.03% and 35.16 ± 4.62%,respectively) (Fig. 3). In activated memory cytotoxic/suppressor Tcells, BE (3.51 ± 1.69%, p b 0.05) showed significantly more inhibi-tion than Vit-D (9.45 ± 2.02%). In activated memory helper T cells,the antiproliferative effect of BE was more than Vit-D, but thedifference was not statistically significant. Neither BE nor Vit-Dshowed significant antiproliferative effect on the naïve helper andcytotoxic/suppressor T cells (CD45RA+CD11a−).

3.2. BE induced significantly more apoptosis than Vit-D in T cells

We performed annexin V assay to determine apoptosis of T cellswith BE, which may account for its antiproliferative effect. Our analysisshowed that almost 90% (90.24 ± 3.70%) CD3+ T cells were live in theuntreated group. BE (45.01 ± 4.27%, p b 0.01) significantly inducedearly apoptosis (annexin V+PI−) of CD3+ T cells compared to untreatedcells (3.28 ± 0.17%). Vit-D also induced apoptosis in approximately26% (26.10 ± 2.05%, p b 0.01) (Fig. 4A and B), but the apoptosis of Tcells with BE (45.01 ± 4.27%, p b 0.05) was significantly more thanthat of Vit-D (26.10 ± 2.05%) at the same dosage levels. Staurosporine(53.82 ± 1.83%), a positive control also induced significant early apo-ptosis of CD3+ T cells.

3.3. Antimitotic effect of BE in T cells is dependent on PI3K/Akt/mTORsignaling cascade

To explore themolecularmechanism of BE as an antimitotic agent inCD3+ T cells, we examined the activation status of the PI3K/Akt/mTORsignaling pathway. We observed that in CD3+ T cells, anti-CD3/CD28stimulus induced marked phosphorylation of Akt (RI: 1.44 ± 0.17,p b 0.05) relative to unstimulated cells (RI: 1). BE (RI: 0.78 ± 0.04,p b 0.05) and Vit-D (RI: 0.98 ± 0.02, p b 0.05) significantly decreasedthe expression of pAkt compared to CD3/CD28 stimulated cells(1.44 ± 0.17). BE inhibited the phosphorylation of Akt (pAkt) signifi-cantly more than Vit-D (p b 0.05). We also determined the effect of BEand Vit-D on phosphorylation of mTOR, which is a downstreamprotein of PI3K/Akt/mTOR cascade. Here also we observed that BE(RI: 0.54 ± 0.01, p b 0.01) and Vit-D (RI: 0.67 ± 0.02, p b 0.01) effec-tively inhibited the phosphorylation of mTOR in CD3+ T cells comparedto anti-CD3/CD28 stimulated media (RI: 1.25 ± 0.07). BE showedsignificantly more inhibition of pmTOR than Vit-D (p b 0.05) (Fig. 5Aand B).

4. Discussion

Historically, 1α,25-dihydroxyvitamin D3 (Vit-D) has been associ-ated with calcium-phosphate homeostasis and bonemetabolism andconsidered as a risk factor for musculoskeletal disorders [6,55].Eventually, it was established that Vit-D has pleiotropic effect, andits contribution to health and disease extends far beyond just bonehealth. The antiproliferative and prodifferentiative effect of Vit-D indifferent cancers encouraged to study this agent as a potent antican-cer drug; however, its systemic toxicity at pharmacological doseshas restricted the clinical application [11,13,14,56,57]. This led tosearch for a more potent Vit-D analogue with low toxicity. In accor-dance with this approach, our research group has developed 1α,25-dihydroxyvitamin D3-3-bromoacetate [1,25(OH)2D3-3-BE] (BE),an affinity labeling analog of Vit-D,which showedmore antiproliferativeeffect in different cancers as well as in an in vitro model of psoriasiscompared to that of Vit-D [20,22–24,50]. Moreover, in vivo studieswith BE did not show significant change in body weight and serum cal-cium level compared to untreated control, denoting lack of significant

Fig. 2. BE inhibited proliferation of CD3+ T cells. Magnetically sorted CD3+ T cells were treated in presence or absence of anti-CD3/CD28, BE (10−6 M) or Vit-D (10−6 M) either aloneor in combination for 5 days. Cell proliferation was measured by (A) MTT assay and (B,C) CFSE dilution assay. (A) Bar diagram showing significant proliferation (OD) of CD3+ T cellswith anti-CD3/CD28 antibody cocktail stimulation, which is effectively inhibited by both 1α,25-dihydroxyvitamin D3-3-bromoacetate (BE) and 1α,25-dihydroxyvitamin D3 (Vit-D). BEwas more antimitotic than Vit-D. (B) Similar results were observed in CFSE dilution assay (% divided cells). (C) A representative CFSE dilution assay showing more proliferation (numberof pink peaks) of CD3+ T cells with anti-CD3/CD28 stimulation compared to untreated cells. BE and Vit-D effectively inhibited the anti-CD3/CD28 stimulation-induced CD3+ T-cellproliferation. Results were expressed asmean ± SEM. Each assay was repeated for ten times and each experimentwas done in triplicates Non-parametric unpaired test (Mann–WhitneyU test) was done to determine statistical significance.

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systemic toxicity [20,22]. The immunosuppressive effect of the in-houseVit-D analogue, BE, has not been elucidated till date.

In addition to its role in cancer biology, lately the pleiotropic effect ofVit-D on immune responses is gaining more recognition. In early andlate 90s, studies found the expression of VDR in immune cells and itsupregulation on activation of those cells [25–28]. The immunomodula-tory role of Vit-D is further strengthen by several studies, which showedthat Vit-D inhibits the proinflammatory activity of Th1/Th17 cells, pro-motes the activity of Th2 cells and increases the frequency and functionof Treg cells resulting in its anti-inflammatory effect [4,29–31,33,58,59].Lately, studies have found a negative correlation between serum Vit-Dlevel, and occurrence and severity of autoimmune diseases [60–65].Keeping this in mind, in this study we evaluated the immunosuppres-sive effect of the novel Vit-D analogue, BE, and dissected out the signal-ing cascade behind its immunosuppressive effect.

To determine the immunosuppressive effect of BE, we evaluated theantiproliferative effect of BE on CD3+ T cells, isolated frompsoriasis andrheumatoid arthritis (RA) patients.We observed that BE significantlyinhibited physiological stimulus (anti-CD3/CD28) induced CD3+ T-cell proliferation (Fig. 2). Previously, few studies have shown theantiproliferative effect of Vit-D on T cells [58,66–68]. Herein, we ob-served that the T-cell antimitotic effect of BE is significantly morethan that of Vit-D, which is in line with BE's more anticancer effect

[20,22]. Therapeutic targeting of CD4+ T cells in autoimmune diseaseshas led to improvement of disease activity, but with side effects of per-sistent CD4+ T-cell depletion [69–72], including increase susceptibilityto infections. To our best knowledge, to date, only one study showedthat memory T cells are more sensitive to the antimitotic effect of Vit-D compared to naïve T cells [67]. Thus, after observing themore antimi-totic effect of BE on activatedCD3+ T cells compared toVit-D,we soughtto determine the differential effect on naïve andmemory T cells. In CFSEdilution assay, we found that BE significantly inhibited the proliferationof activated memory helper (CD4+CD11a+CD45RA−) and cytotoxic/suppressor (CD8+CD11a+CD45RA−) T cells (Fig. 3). Vit-D also exertedsignificant antimitotic effect on activated memory helper and cytotoxicT cells, which is in line with the previous report [67]. Here also, weobserved more inhibitory effect with BE compared to Vit-D (Fig. 3).One of the intriguing finding of this study is that BE did not have anysignificant effect on the proliferation of the naive cells. This might beexplained by the fact that the expression of Vit-D receptor (VDR) aswell as 1-α-hydroxylase [the enzyme responsible for conversionof circulating 25(OH)D3 into bioactive 1,25(OH)2D3] is upregulatedin activated T cells [73,74]. Therefore, this finding might be of thera-peutic significance because the naïve T cells are spared from theantimitotic effect of BE, which could be beneficial in combatinginfections.

Fig. 3.Antiproliferative effect of BE on activated CD4+ and CD8+ memory T cells. Sorted CD3+ T cells were incubated in presence or absence of anti-CD3/CD28 antibody cocktail,Vit-D (10−6 M) and BE (10−6 M) either alone or in combination for 5 days. (A) Gating strategy for CD4+, CD8+, CD45RA− and CD11a− shown. (B) A representative CFSE dilutionassay showing inhibition of anti-CD3/CD28 induced proliferation of activated memory helper T cells (CD4+CD11a+CD45RA−) with BE and Vit-D. (C) A bar diagram showingsignificant inhibition of anti-CD3/CD28 induced proliferation of activated memory helper T cells with BE and Vit-D. (D) A representative CFSE dilution assay showing inhibitionof anti-CD3/CD28 induced proliferation of activated memory CD8+ T cells with BE and Vit-D. (E) A bar diagram showing significant inhibition of anti-CD3/CD28 induced prolif-eration of activated memory CD8+ T cells. Data were analyzed using FlowJo software and results were expressed in % divided cells. CFSE dilution assays were repeated ten timesand each experiment was done in triplicates Non-parametric unpaired test (Mann–Whitney U test) was done to determine statistical significance.

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Next, we sought to determinewhether the antimitotic effect of BE isdue to induction of apoptosis in CD3+ T cells. We found that BE is apotent proapoptotic agent in CD3+ T cells isolated from psoriasis andRA patients (Fig. 4). Vit-D also induced apoptosis in those CD3+ Tcells. The proapoptotic effect of BE outweighs that of Vit-D (Fig. 4).This might be an explanation to the fact that BE is a more antimitoticcompared to Vit-D (Fig. 2). Moreover, in autoimmune diseases, suchas psoriasis and RA, the activated pathogenic T cells become resistantto apoptosis and thus perpetuate the disease process. In vitro andin vivo studies have shown that induction of apoptosis of pathogenic Tcells lessens the disease severity [75,76]. Thus, the novel Vit-D analogue,BE, indicates to have therapeutic implications in autoimmune diseases.

After observing the potent immunosuppressive effect of the in-house Vit-D analogue, BE, we sought to elucidate the signaling pathwayresponsible behind these effects. PI3K/Akt/mTOR signaling cascade con-tributes a key role in cell proliferation/survival, and in this cascade, PI3Kand protein kinase B (Akt) are the key upstreammolecules that link theligation of receptors to the phosphorylation and activation state ofmTOR [77–80]. Recently, our research group has shown that IL-17 andIL-22, two relevant cytokines of RA and psoriasis, induced proliferationof synovial fibroblasts (FLS) and keratinocytes through PI3K/Akt/mTORpathway [50,81,82]. Further, a recent publication has shown that BE in-hibits the phosphorylation of Akt in renal cancer [22]. In line with this,we wanted to see whether the antiproliferative effect of BE in CD3+ T

Fig. 4. BE is a more potent proapoptotic agent in T cells compared to Vit-D. Sorted CD3+ T cells were cultured with or without Vit-D (10−6 M) or BE (10−6 M) for 5 days. Staurosporine(1uM)was used as a positive control. Cellswere stainedwith annexinV and propidium iodide and data analyseswere done using FlowJo software. (A) A representative dot plot showing %early apoptotic cells (Q3, annexin v+PI−) with different treatments (BE, Vit-D and Staurosporine) as compared to untreated control. Six experiments were done in triplicates. (B) A bardiagram showing significantly increase % early apoptotic cells (annexin+ PI−) with different treatments (BE, Vit-D and Staurosporine). Results were expressed as mean ±SEM. Non-parametric unpaired test (Mann–Whitney U test) was done to determine statistical significance.

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cells is also mediated through PI3K/Akt/mTOR cascade or not. We ob-served that BE effectively inhibited the anti-CD3/CD28 induced phos-phorylation of Akt and its downstream protein mTOR in lysates ofCD3+ T cells (Fig. 5). The inhibitory effect of BE on pAkt and pmTORwas significantly more than that of Vit-D.

5. Conclusion

Herein, we established that the novel Vit-D analogue, BE, is amore potent immunosuppressive than the equimolar dose of Vit-D.The immunosuppressive effect of BE is attributed to its antimitoticand proapoptotic effect on activated memory T cells, sparing the naïveT cells. This may have a therapeutic implication to treat autoimmunediseases with BE. Further preclinical and clinical studies of BE are

Fig. 5. Immunosuppressive effect of BE is mediated through PI3K/Akt/mTOR pathway. CDVit-D (10−6 M) and BE (10−6 M) either alone or in combination for 5 days. (A) A represe(pAkt) and mTOR (pmTOR). (B) A bar diagram showing relative intensity (RI) of pAkt anunpaired test (Mann–Whitney U test) was done to determine statistical significance.

required to confirm its in vivo efficacy as a potent immunosuppres-sive agent.

Conflicts of interest

Authors declare no conflict of interest.

Acknowledgements

The project described was supported by a VA Merit grant,I01CX000201. Contents do not necessarily represent the views ofthe Department of Veterans Affairs or the United States Government.

3+ T cells were cultured in presence or absence of anti-CD3/CD28 antibody cocktail,ntative western blot showing that BE and Vit-D inhibited the phosphorylation of Aktd pmTOR in CD3+ T cells. Results were expressed as mean ± SEM. Non-parametric

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