1 C57BL/6 and DBA/1 Mice Differ in Their Response to Supplementation with 1,25D and Paricalcitol

3 Aline Parisoto Missio 1*, Thais Fernanda de Campos Fraga-Silva 1, Larissa Lumi Watanabe Ishikawa 1,

4 Luiza Ayumi Nishiyama Mimura 1, Thais Graziela Donegá França 1, Larissa Ragozo Cardoso de Oliveira

1
5 , Alexandrina Sartori 1 and Sofia Fernanda Gonçalves Zorzella-Pezavento 1

1
7 Department of Microbiology and Immunology, São Paulo State University (UNESP), Institute of

8 Biosciences, Botucatu, São Paulo, Brazil.

*
10 Correspondence: Aline Parisoto Missio, Department of Microbiology and Immunology, São Paulo

11 State University (UNESP), Institute of Biosciences, Rua Dr. Plinio Pinto e Silva, 18618-691, Botucatu,

12 São Paulo, Brazil. (e-mail: alinepm@ibb.unesp.br). Phone: +55 14 38800427 / Fax: +55 14 38800415.

13

14 Author's e-mail address: aline.missio@unesp.br; thaisfragasilva@gmail.com; larissalumi@gmail.com;

15 luizamimura@gmail.com; thais.donega@yahoo.com.br; larissa@ibb.unesp.br;

16 alexandrina.sartori@unesp.br; szorzella@yahoo.com.br.

17

18

19

20

21

22

23

24

25

26

1
27 Abstract

28 Background: Supplementation with active vitamin D (1,25D) is usually recommended to achieve

29 normal levels in subclinical vitamin D deficiency. More recently higher than normal 1,25D levels have

30 been suggested as potentially therapeutic in many clinical conditions including inflammatory

31 pathologies. In this context we compared the immunomodulatory efficacy of 1,25D and paricalcitol

32 administration to C57BL/6 and DBA/1 mice whose diets already included normal levels of 1,25D and

33 calcium. Commented [A1]: Reviewer 3. Text revision.

34 Methods: C57BL/6 and DBA/1 mice were injected with eight doses of 0.1 µg of 1,25D or paricalcitol,

35 delivered every other day during 15 days by intraperitoneal route. Body weight was daily assessed.

36 Cytokine production by cell cultures in vitro stimulated with concanavalin A (ConA) was analyzed by Commented [A2]: Reviewer 3. Text revision.

37 ELISA. The percentages of regulatory T cells (Tregs) expressing Foxp3 and dendritic cells (DCs) were Commented [A3]: Reviewer 3. Text revision.

38 analyzed by flow cytometry. Median fluorescence intensity (MFI) of major histocompatibility complex Commented [A4]: Reviewer 3. Text revision.

39 class II (MHCII) was also evaluated by flow cytometry. Blood samples were collected to assess calcium Commented [A5]: Reviewer 3. Text revision.

40 levels. A second set of similar experiments was performed to evaluate the effect of a higher

41 concentration (0.2 µg) of 1,25D and paricalcitol in DBA/1 mice.

42 Results: In C57BL/6 strain 1,25D decreased body weight and cytokine production by the spleen but did

43 not affect the percentage of DCs and Tregs. However it increased MHC II median fluorescence intensity

44 (MFI) in DCs and also increased serum calcium levels. Paricalcitol did not affect body weight and serum

45 calcium levels but decreased cytokine production. Except body weight, that was downregulated by

46 1,25D, the other parameters were not altered by this dose in the DBA/1 strain. An increased dose of

47 1,25D, but not of paricalcitol, was, however, able to downmodulate the majority of the tested Commented [A6]: Reviewer 3. Text revision.

48 cytokines in this strain. Commented [A7]: Reviewer 3. Text revision.

49 Conclusion: Together, these results show that C57BL/6 and DBA/1 mice differ in their responses to

50 1,25D and paricalcitol supplementation, being DBA/1 more resistant to their effects.

51 Keywords: 1,25D; paricalcitol; immunomodulation; inbred mice

52

2
53 Background

54 Active vitamin D (1,25D) is a fat-soluble vitamin that can be obtained from the diet but that is mainly

55 produced in the skin by the conversion of 7-dehydrocholesterol under ultraviolet light stimulation. Its

56 role in calcium homeostasis, bone growth and prevention of rickets and osteomalacia is known for Commented [A8]: Reviewer 3. Text revision.

[1]
57 over two hundred years . However, its biological functions go beyond these calcemic effects

58 including, for example, regulation of immunological activities, anticancer properties and control of Commented [A9]: Reviewer 3. Text revision.

59 brain development being its deficiency associated with many pathological conditions. Like other Commented [A10]: Reviewer 3. Text revision.
Commented [A11]: Reviewer 3. Text revision.
60 nuclear steroids, this vitamin works through both genomic and nongenomic signaling pathways.

61 However, most of its activities are mediated through 1,25D binding to the nuclear 1,25D receptor

62 (VDR). Upon ligand binding, VDR forms a heterodimer with retinoid X receptor that further binds to Commented [A12]: Reviewer 3. Text revision.

63 1,25D responsive elements controlling the expression of many genes [2]. The possible contribution of Commented [A13]: Reviewer 3. Text revision.

[3]
64 VDR polymorphisms to 1,25D efficacy is being investigated but is still a subject under discussion .

65 Concerning the immune system, both innate and specific immunity are profoundly and oppositely

66 regulated by 1,25D. It promotes innate antimicrobial responses by increasing the production of

67 cathelicidin and defensin beta 4, induction of reactive oxygen intermediates and antibacterial

68 autophagy [4]. On the other hand, its effects on the specific immunity are typically anti-inflammatory Commented [A14]: Reviewer 3. Text revision.

69 and include suppression of Th1 and Th17 differentiation and upregulation of Foxp3 transcription and

70 the consequent differentiation of regulatory T cells (Tregs) [5]. To some extent these effects are related
Commented [A15]: Reviewer 3. Text revision.
[6-8] Commented [A16]: Reviewer 3. 1,25D deficiency was
71 to a blockage on dendritic cells (DCs) differentiation and inhibition of cytokine production . 1,25D
substitute by 25D deficiency.

72 supplementation is considered a simple, safe and inexpensive procedure that is being used to achieve Commented [A17]: Reviewer 3. Text revision.
Commented [A18]: Reviewer 3. Text revision.
73 normal levels in 25D deficiency states. This vitamin is also being tested in supraphysiological amounts Commented [A19]: Reviewer 3. Text revision.
Commented [A20]: Reviewer 3. The reference was changed
74 to investigate its therapeutic potential in certain pathological conditions. Elevated 1,25D doses by another whose supplementation was with 1,25D
Commented [A21]: Reviewer 3. The reference was changed
75 triggered favorable results in bone health[9], metabolic disorders[10] and autoimmune diseases [11-12]. by another whose supplementation was with 1,25D
Commented [A22]: Reviewer 3. 1,25D was substitute by
76 The administration of a given vitamin D dose has been, however, associated with variable serum levels vitamin D
Commented [A23]: Reviewer 3. 1,25D was substitute by
77 of this substance in different patients [13]. Many factors can contribute to this interindividual variability vitamin D

78 in response to vitamin D supplementation, including basal 25D levels, body mass index, seasons, Commented [A24]: Reviewer 3. 1,25D deficiency was
substitute by 25D deficiency.

3
79 calcium and fat diet intake and genetic background [14]. Studies also suggest that this variable response Commented [A25]: Reviewer 3. Text revision.
Commented [A26]: Reviewer 3. Text revision.
80 is significantly influenced by genetic polymorphisms in the 1,25D pathways. It has been demonstrated,

81 for example, that VDR polymorphisms interfere with improvement in metabolic profile associated with

82 1,25D supplementation in type II diabetes mellitus patients[15] and probably also affect prevention of Commented [A27]: Reviewer 3. The reference was changed
by another whose supplementation was with 1,25D
83 advanced colorectal cancer[16].The baseline methylation levels of enzymes involved in vitamin D
Commented [A28]: Reviewer 3. The reference was changed
84 metabolism, such as CYP2R1 and CYP24A1, could also affect the response to vitamin D by another whose supplementation was with 1,25D
Commented [A29]: Reviewer 3. 1,25D was substitute by
85 supplementation [17]
. A classically-described side-effect related to vitamin D supplementation is vitamin D.
Commented [A30]: Reviewer 3. 1,25D was substitute by
86 hypercalcemia. This occurs because absorption of dietary calcium by the intestine is partially regulated vitamin D.

87 by several 1,25D dependent proteins [18]. Vitamin D can be therapeutically applied to other conditions

88 besides its classical use to promote calcium and phosphate absorption. Higher doses of this hormone,

89 that could trigger hypercalcemia, are usually required for these alternative applications. Vitamin D

90 analogues with reduced calcemic activity were, therefore, synthesized. Paricalcitol is a vitamin D Commented [A31]: Review 3. Sentence was rephrased.

91 analogue commercialized by Abbott Laboratories under the trade name Zemplar®. It is usually

[19]
92 indicated for treatment of secondary hyperparathyroidism associated with chronic renal failure .

93 Paricalcitol´s immunomodulatory activities are still being validated but it was demonstrated that this

94 substance is able to impair differentiation of immature dendritic cells (DCs) by significantly decreasing Commented [A32]: Reviewer 3. Text revision.

95 their ability to induce T cell proliferation in response to antigen stimulation [20].

96 Considering that supplementation with 1,25D or paricalcitol has the potential to be used as an

97 adjunct therapy for many inflammatory diseases, the main objective of this investigation was to

98 compare the immunomodulatory effects of supraphysiological amounts of 1,25D in C57BL/6 and

99 DBA/1 mice strains. This comparison in mice strains is important because it will clarify if this variability

100 also occurs in mice and, therefore, if they can be further used to study this phenomenon. The effects Commented [A33]: Reviewer 3. Text revision

101 of 1,25D were also compared with the ones triggered by paricalcitol because this analogue does not

102 trigger hypercalcemia and could be, therefore, more adequate for immunomodulatory purposes.

4
103 The choice of these mice strains was mainly based in their already reported differences related to

104 immunological parameters, pathogen susceptibility and Th1 or Th2- biased polarization. A possible

105 differential response to vitamin D was presumed. Commented [A34]: Review 3. Information about mice
strains choice.
106

107 Materials and Methods

108 Experimental design

109 C57BL/6 and DBA/1 mice were injected with eight doses of 0.1 µg of 1,25D or paricalcitol,

110 delivered every other day during 15 days by intraperitoneal route. Mice injected only with the vehicle

111 (15% absolute ethyl alcohol diluted in water for injection) were used as controls. Body weight was daily

112 assessed. One day after the last dose (16th day) the animals were euthanized and the following

113 parameters were analyzed in the spleen: cytokine production by cell cultures in vitro stimulated with

114 concanavalin A (ConA), percentages of DCs and Tregs expressing Foxp3. Median fluorescence intensity Commented [A35]: Reviewer 3. Text revision.

115 (MFI) of major histocompatibility complex class II (MHCII) was also evaluated. Blood samples were also

116 collected and used to assess calcium levels. A second set of similar experiments was performed to

117 evaluate the effect of a higher concentration (0.2 µg) of 1,25D and paricalcitol in DBA/1 mice. A

118 timeline scheme is showed below to illustrate the experimental design (figure 1).

119

5
120 Figure 1. Experimental design. Animals from each strain were allocated into three experimental

121 groups: 1- control: injected with vehicle used for drug dilution; 2- 1,25D: injected with vitamin D; 3-

122 paricalcitol: injected with this analogue. Animals were injected every other day during 15 days by

123 intraperitoneal route with 0.1 µL containg only the vehicle, the vitamin (0.1 µg) or the analogue (0.1 Commented [A36]: Reviewer 3. Text revision.

124 µg). Body weigh was daily evaluated whereas the other parameters were all assessed one day after

125 last dose.

126 Animals

127 Forty-five C57BL/6 and sixty-one DBA/1 were employed for this investigation. In the first set of

128 experiments, that was done to compare both strain, were used 45 C57BL/6 and 39 DBA/1. In the

129 second step only DBA/1 (n=22) were employed to test the effects of higher vitamin D and paricalcitol

130 supplementation doses. Mice age varied from 9 to 11 weeks old. The number of animals in each Commented [A37]: Review 3. Information about the
number and age of animals.
131 analysis is stated at the legends. Option for females was based on the significantly stronger effect of

132 this vitamin on this gender. Mice received sterilized water and food (Presence®) ad libitum. Animal

133 food already contained physiological levels of this vitamin (4000 IU/Kg). To achieve supraphysiological Commented [A38]: Reviewer 3. Text revision.

134 levels, the animals were intraperitoneally injected, during 15 days at morning, with 0.1 µg of 1,25D or

135 paricalcitol.

136 Reagents

137 1,25D (1α,25-dihydroxyvitamin D3) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and

138 paricalcitol (Zemplar®) was purchased from Abbott Laboratories (Milan, Italy). The following

139 monoclonal antibodies were used in flow cytometric assays: PerCP-labeled anti-mouse CD3 (clone 145-

140 2C11); FITC-labeled anti-mouse CD4 (clone GK1.5); APC-labeled anti-mouse CD25 (clone PC61.5);

141 PerCP-labeled anti-mouse F4/80 (clone BM8); FITC-labeled anti-mouse CD11c (clone N418); APC-

142 labeled anti-mouse MHCII (clone M5/114.15.2); PE-labeled anti-mouse CD86 (clone G11). A forkhead

143 box P3 (Foxp3) PE Staining Set was also used for intracellular staining. All reagents for flow cytometric

144 assays were purchased from eBioscience (San Diego, CA, USA).

145

6
146 Determination of calcium levels

147 Calcium levels were determined using Cálcio Arsenazo III commercial kit (Bioclin, Belo Horizonte, MG,

148 Brazil), according to manufacturer’s instruction. This is a colorimetric endpoint assay in which the

149 calcium reacts with arsenazo III (0.2 mmol/L) in acid conditions forming a blue colored complex whose

150 intensity is proportional to calcium concentration in the sample. Absorbance is measured between 600

151 and 680 nm. Sensitivity is of 0.022 mg/dL and the reaction is linear up to 20 mg/dL. Commented [A39]: Reviewer 1. Information about
determination of calcium.
152

153 Assessment of cytokine production by ELISA

154 One day after the last 1,25D and paricalcitol doses, the animals were euthanized and the spleens

155 removed in aseptic conditions. Spleens were disrupted by gentle meshing through cell strainers

156 (70µm), cells were collected and then were lysed with buffer containing ammonium chloride. After Commented [A40]: Reviewer 1. Information about how
spleen was disrupted.
157 being adjusted to 5.106 cells/ml they were cultured in RPMI medium supplemented with 10% of fetal Commented [A41]: Reviewer 3. Text revision.

158 calf serum and 2 mM of L-glutamine, in the presence of 10 µg/ml of ConA. Cytokine levels were

159 evaluated 48 hours later by enzyme-linked immunosorbent assay in culture supernatants using IL-2

160 (Cat#DY555148), IL-5 (Cat#DY555236), IFN-γ (Cat#DY555138) and IL-10 (Cat#DY555252) OptEIA Sets

161 (BD Biosciences, San Diego, CA, USA) and IL-6 (Cat#DY406), IL-17 (Cat#DY421) and TNF-α (Cat#DY410)

162 DuoSet (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

163

164 Assessment of DCs and Tregs by flow cytometry

165 For Tregs analysis, spleen cells obtained as described above were incubated with 0.2 μg PerCP- Commented [A42]: Reviewer 3. Text revision.

166 Cy5.5-labeled anti-mouse CD3 (145-2C11), 0.25 μg FITC-labeled anti-mouse CD4 (GK1.5), and 0.25 μg Commented [A43]: Reviewer 1. Information about how
spleen was disrupted.
167 APC-labeled anti-mouse CD25 (PC61.5) for 30 min at 4°C. The cells were washed and prepared for

168 intracellular staining with 0.2 μg PE-labeled anti-mouse Foxp3 transcription factor (FJK-16S) according

169 to manufacturer’s instructions (eBiosciences, San Diego, CA, USA). For DCs analysis, spleen cells were

170 incubated with 0.1 μg PerCP-labeled anti-mouse F4/80 (BM8), 0.25 μg FITC-labeled anti-mouse CD11c

171 (N418), 0.03 μg APC-labeled anti-mouse MHCII (MS/114.15.2), and 0.125 μg PE-labeled anti-mouse

7
172 CD86 (GL1) for 30 min at 4°C. The cells were then washed, resuspended in flow cytometry buffer and

173 fixed in 1% paraformaldehyde solution. The cells were analyzed by flow cytometry using FACSCanto II

174 (Becton Dickinson, San Jose, CA, USA) from Institute of Biosciences (UNESP, Botucatu, SP, Brazil) for

175 data acquisition and FlowJo software (TreeStar, Ashland, OR, USA) for Tregs and DCs analyses.

176

177 Statistical Analysis

178 Results were expressed as mean ± standard deviation or median (25-75%). Comparisons between

179 groups were made by One-way ANOVA followed by Tukey’s test for parametric variables and by

180 Kruskal-Wallis followed by Dunn’s test for non-parametric ones. Concerning mice body weight

181 variation, the comparisons were performed by Two-way ANOVA followed by Tukey’s test. Statistical

182 analysis was carried out by using SigmaPlot 12.0 software (Systat Software Inc., San Jose, CA, USA) and Commented [A44]: Reviewer 3. Text revision.

183 p < 0.05 was considered significant. Prism 6.0 software (GraphPad Software Inc., La Jolla, CA, USA) was

184 used for graphic design. Commented [A45]: Reviewer 3. Text revision.

185

186 Results

187 1,25D supplementation decreases body weight in C57BL/6 and DBA/1 mice

188 Low 25D levels are frequently found in obese individuals [21-22], being currently debated if the Commented [A46]: Reviewer 3. 1,25D levels was substitute
by 25D levels.
189 administration of this vitamin could improve changes that are frequently associated with obesity as Commented [A47]: Reviewer 3. Text revision.

190 for example altered glucose metabolism[23]. Both mice strains lost weight but this effect was observed Commented [A48]: Reviewer 3. Text revision.

191 earlier and was more pronounced in the C57BL/6 strain. Body weight variation is shown in figures 2A Commented [A49]: Reviewer 3. Text revision.

192 and 2B for C57BL/6 and DBA/1 mice, respectively. Percentage of total body weight variation at the end

193 of the experiment is illustrated in figure 2C. The results indicate that 1,25D determined a significant

194 body weight loss in both strains. Conversely, paricalcitol administration did not trigger any alteration

195 in the body weight of these two strains (figures 2A, 2B and 2C).

8
196

197 Figure 2. Effect of 1,25D and paricalcitol on body weight and serum calcium levels in C57BL/6 and

198 DBA/1 mice. Mice were intraperitoneally injected with 0.1 µg of 1,25D or paricalcitol during 15

199 alternate days. C57BL/6 body weight (A), DBA/1 body weight (B), weight variation (C) and serum

200 calcium (D). Data presented as mean ± SD of four to six animals per group.*p < 0.05 versus control. Commented [A50]: Reviewer 3. Text revision.

201 Representative results from three and two experiments for body weight and calcium levels,

202 respectively.

203

204 1,25D causes hypercalcemia in C57BL/6 but not in DBA/1 mice

205 1,25D plays a major role in maintaining calcium and phosphate homeostasis. However,

[24]
206 supplementation with elevated 1,25D doses can cause hypercalcemia . To check if the adopted

207 supplementation protocol was altering calcium concentration, this element was quantified in serum

208 samples. As displayed in figure 2D, 1,25D significantly increased calcium levels in C57BL/6 but not in

209 DBA/1 mice. Paricalcitol administration did not affect calcium levels in both strains as illustrated in

210 figure 2D.

211

9
212 Effect of 1,25D supplementation in DCs and Tregs

213 1,25D is able to trigger a strong tolerogenic pathway involving DCs and Tregs. This property is

214 being extensively investigated as a potential strategy to control inflammatory and autoimmune

215 diseases [25-26]. No clear data is available about this pathway in normal subjects. The percentage of DCs,

216 immunophenotyped as F4/80-CD11c+MHCII+CD86+ cells (figure 3A), was not affected by 1,25D or

217 paricalcitol supplementation in C57BL/6 and DBA/1 mice as depicted in figures 3B and 3C, respectively.

218 However, 1,25D significantly increased the median fluorescence intensity of major histocompatibility

219 complex class II expression in C57BL/6 as showed in figure 3D. This effect was not observed in the

220 DBA/1 strain.

221 The percentage of Tregs immunophenotyped as CD3+CD4+CD25hiFoxp3+ cells (figure 3F), was not

222 altered by any of these two substances in C57BL/6 (figure 3G) and DBA/1 mice (figure 3H).

223

224

225

226

10
227

228 Figure 3. Phenotypic analysis of DCs and Tregs in C57BL/6 and DBA/1 mice injected with 1,25D or

229 paricalcitol. Mice were intraperitoneally injected with 0.1 µg of 1,25D or paricalcitol during 15

230 alternate days. Gate strategy for F4/80-CD11c+MHCII+CD86+ DCs (A), percentage of F4/80-

231 CD11c+MHCII+CD86+ cells in C57BL/6 mice (B), percentage of F4/80-CD11c+MHCII+CD86+ cells in DBA/1

232 mice (C), MHCII MFI in C57BL/6 (D), MHCII MFI in DBA/1 (E), gate strategy for CD3+CD4+CD25hiFoxp3+

233 (F), percentage of CD3+CD4+CD25hiFoxp3+ cells in C57BL/6 mice (G) and percentage of

234 CD3+CD4+CD25hiFoxp3+ in DBA/1 mice of spleen samples (H). Data presented as mean ± SD or median

235 (25-75%) of seven to thirteen animals per group. *p < 0.05 versus control. Representative results from

236 three and two experiments were combined for C57BL/6 and DBA/1, respectively.

11
237 1,25D downmodulates cytokine production in C57BL/6 but not in DBA/1 mice

238 Pro-inflammatory cytokines are major mediators of a variety of human diseases, including

[27]
239 autoimmune pathologies and chronic inflammatory states contributing to their initiation and

240 aggravation. A great deal of attention has been focused in substances able to downregulate cytokine

241 production. The adopted short protocol of 1,25D supplementation was able to significantly

242 downmodulate the production of IL-6, TNF-α, IFN-γ, IL-2 and IL-17 production in C57BL/6 mice (figures

243 4A-4E). Paricalcitol triggered a similar but not so pronounced downmodulatory effect. Neither 1,25D

244 nor paricalcitol were able to affect the production of these cytokines in DBA/1 mice. One way to

245 regulate cytokine production by Th1 and Th17 cells is by triggering a counter-regulatory mechanism Commented [A51]: Reviewer 3. Text revision.

246 mediated by anti-inflamatory cytokines such as IL-5 and IL-10 [28]. However, as shown in figure 4F and

247 4G, these two cytokines were also significantly downmodulated in C57BL/6 mice by both, 1,25D and

248 paricalcitol. Again, no effect was detected in these cytokines when spleen cells from DBA/1 mice were

249 tested.

250

251 Figure 4. Effect of 1,25D and paricalcitol on cytokine production by C57BL/6 and DBA/1 mice. Animals

252 were intraperitoneally injected with 0.1 µg of 1,25D or paricalcitol during 15 alternate days. IL-6 (A),

253 TNF-α (B), IFN-γ (C), IL-2 (D), IL-17 (E), IL-5 (F) and IL-10 (G) were measured in spleen cell cultures (5 x

254 106 cells/mL) stimulated with ConA (10 µg/mL). Data presented as mean ± SD or median (25-75%) of

12
255 three to five animals per group. *p < 0.05 versus control. Representative results from two and three

256 experiments for C57BL/6 and DBA/1, respectively.

257

258 DBA/1 mice require higher 1,25D dose than C57BL/6 to be partially immunomodulated

259 To test the possibility that DBA/1 required higher doses of 1,25D and paricalcitol to be

260 immunomodulated, these mice were injected with 0.2 μg of the two substances. As observed in figure Commented [A52]: Reviewer 3. Text revision.

261 5A and 5B, 1,25D, but not paricalcitol, determined body weight loss. These higher doses did not

262 increase serum calcium levels as shown in figure 5C. Except the percentage of DCs that was increased

263 by the 0.2 μg 1,25D dose (figure 5D), the MHCII MFI and the percentage of Tregs were not affected by

264 the higher concentration of 1,25D or paricalcitol as shown in figures (5D, 5E and 5F, respectively). The

265 higher 1,25D dose was however, able to signicantly decrease IL-6, IFN-γ, IL-2 and IL-17 as shown in

266 figure 6.

267

268 Figure 5. Effect of a higher dose of 1,25D or paricalcitol in body weight, calcium, DCs and Tregs from

269 DBA/1 mice. Mice were intraperitoneally injected with 0.2 µg of 1,25D or paricalcitol during 15 Commented [A53]: Reviewer 3. Text revision.

270 alternate days. Body weight (A), weight variation (B), serum calcium (C), percentage of F4/80-

271 CD11c+MHCII+CD86+ DCs in spleen (D), MHCII MFI (E) and percentage of CD3+CD4+CD25hiFoxp3+ Tregs

13
272 in spleen (F). Data presented as mean ± SD or median (25-75%) of three to twelve animals per group.

273 *p < 0.05 versus control. Representative results from two and one experiment for body weight and

274 calcium, respectively. Representative results of cytometry data are from two combined experiments.

275

276 Figure 6. Effect of a higher dose of 1,25D or paricalcitol in DBA/1 mice cytokine production. Mice

277 were intraperitoneally injected with 0.2 µg of 1,25D or paricalcitol during 15 alternate days. IL-6 (A),

278 TNF-α (B), IFN-γ (C), IL-2 (D), IL-17 (E), IL-5 (F) and IL-10 (G) were measured in spleen cell cultures (5 x

279 106 cells/mL) stimulated with ConA (10 µg/mL). Data presented as mean ± SD or median (25-75%) of

280 nine to eleven animals per group. *p< 0.05 versus control. Representative results from two

281 experiments were combined.

282

283 Discussion

284 1,25D is considered an essential hormone that regulates over a thousand genes in the human

285 genome. Its mechanisms of action are finely regulated by genetic and epigenetic modifications [29]. As

286 25D deficiency already reached epidemic proportion worldwide, a lot of effort has been done to define Commented [A54]: Reviewer 3. 1,25D deficiency was
substitute by 25D deficiency.
287 the best supplementation strategies to achieve normal levels in subclinical deficiency and effective Commented [A55]: Reviewer 3. Text revision.

[30-31]
288 levels, usually higher, in many conditions as autoimmune pathologies , inflammatory bowel

289 diseases [32] and allergy [33]. In this scenario, the immunomodulatory ability of 1,25D was compared in

14
290 C57BL/6 and DBA/1 that are mouse strains differing at the MHC. The effect of paricalcitol, that is a

291 selective vitamin D analogue originally prescribed for the treatment of secondary hyperparathyroidism

292 associated with chronic kidney disease, was concomitantly evaluated.

293 The initial supplementation of both strains was done with 0.1 µg of 1,25D or paricalcitol in

[34]
294 alternate days during 15 days, as previously described by Lemire and Archer and recently also

[12, 35]
295 adopted by our research group . 1,25D, but not paricalcitol, significantly decreased the body

296 weight of both strains. This loss was more accentuated and occurred earlier in C57BL/6 mice. Regarding

297 1,25D effect on C57BL/6 mice, this result is similar to our previous findings [12, 35], and is also supported

298 by other reports [36]. This relationship between 25D levels and body weight, mainly considering obesity Commented [A56]: Reviewer 3. 1,25D deficiency was
substitute by 25D deficiency.
299 control, is the focus of much investigation [37-38], and has been analyzed mainly from two perspectives:

300 obesity as a condition that lowers vitamin D levels and, oppositely, the contribution of low 25D levels Commented [A57]: Reviewer 3. 1,25D deficiency was
substitute by 25D deficiency.
301 to obesity development. Considering this last viewpoint, some investigators suggested that this

302 hormone could display anti-obesogenic properties [39]. The therapeutic potential of vitamin D in obesity Commented [A58]: Reviewer 3. 1,25D was substitute by
vitamin D.
303 and its associated alterations are supported by experimental data derived from humans [40] and also Commented [A59]: Reviewer 3. Text revision.

[37, 41]
304 from rodents . It is known, for example, that calcitriol is able to strongly inhibit adipogenesis.

305 Binding of calcitriol to VDR interrupts adipogenesis mainly by down-modulating the expression of

[42]
306 C/EBP-β mRNA and protein . The anti-obesity effect of this vitamin is also Ca+2-dependent. High Commented [A60]: Reviewer 3. Text revision.

307 1,25D and Ca+2 intakes activate mediators of apoptosis in adipose tissue [43]. Recent data also indicate Commented [A61]: Reviewer 3. Text revision.

308 that vitamin D can act directly in the hypothalamus determining an accentuated body reduction by Commented [A62]: Reviewer 3. Text revision.

309 lowering food consumption [22].

310 As expected, 1,25D, but not paricalcitol, determined hypercalcemia in C57BL/6 mice. This is

311 certainly interesting because the rationale for designing 1,25D analogues is exactly to prepare

312 derivatives whose selective activity is directed towards the desired tissue or function. In this sense,

313 paricalcitol and other low-calcemic analogues have received considerable attention over the last

314 decade due to their effective parathyroid suppression whilst avoiding undesirable low bone turnover

[44]
315 . These differential effects of vitamin D and paricalcitol on body weight and calcium levels in C57BL/6

15
316 could be addressed from distinct therapeutic perspectives. Subclinical 25D deficiency is very common Commented [A63]: Reviewer 3. 1,25D deficiency was
substitute by 25D deficiency.
317 and can be associated with hypocalcemia [45-46]. In this case supplementation with the natural hormone

318 (calcitriol) which increases intestinal calcium absorption is recommended. However, there are other

319 conditions as cancer, inflammatory pathologies and obesity and its related comorbidities that have

320 also being associated with 25D deficiency. The potential application of 1,25D as adjunct therapy for Commented [A64]: Reviewer 3. 1,25D deficiency was
substitute by 25D deficiency.
321 these pathologies is already being evaluated in both preclinical tests and clinical trials. However this
Commented [A65]: Reviewer 3. Text revision.
322 approach requires more elevated doses or prolonged therapy what could trigger hypercalcemia. In this

323 case vitamin D analogues, that are endowed with lower calcemic activity, would be more indicated [47- Commented [A66]: Reviewer 3. Text revision.

48]
324 . Differently from C57BL/6, serum calcium levels in DBA/1 animals were not affected by 1,25D.

325 Most of the beneficial effects of 1,25D in inflammatory pathologies have been attributed to its

[49-50]
326 modulatory effects over DCs and Tregs . In this sense, the presence of more immature DCs

327 expressing lower levels of MHCII and a higher frequency of Tregs were expected. However, these

328 alterations were not observed. Oppositely, an even increased level of MHCII expression was associated

329 with 1,25D supplementation in C57BL/6 mice. As far as we know, this finding is being described for the

330 first time. This apparently discrepant result could be, at least partially, due to the adopted

331 experimental model. Differently from usual procedures, supplementation was tested in the absence

332 of any antigenic stimulus. 1,25D dose, route and administration regimens could also affect the results.

333 A few articles give support to this non-standard finding. Sochorova et al. [20] for example, observed that

334 differentiated DCs are much less sensitive to 1,25D treatment than early progenitor cells. Precise

335 timing also seems very relevant. Gambhir et al. [51] demonstrated that certain 1,25D inhibitory activities

336 are triggered only if this binding to VDR occurs before antigenic stimulation. 1,25D and its analogue

337 also did not increase the percentage of Tregs in none of the strains. Even though we had described this

[35]
338 before in a similar in vivo treatment model , this finding is distinct from the majority of the

339 publications in this area. There is growing consensus that 1,25D enhances Tregs differentiation[52-53]. Commented [A67]: Reviewer 3. Reference about vitamin D
and Treg differentiation. The original phrase was
340 Maybe these contradictory findings could be explained by the adopted administration router. substituted.

341 Contrasting with the intraperitoneal route that was not associated with Tregs expansion [12, 35]
, we Commented [A68]: Reviewer 3. Text revision.

16
342 recently observed that epicutaneous application of paricalcitol is associated with a higher frequency

343 of Tregs[11]. This relevant role of the administration route was also recently reinforced by descriptions Commented [A69]: Reviewer 3. Text revision.

344 that epicutaneous and oral route trigger a significant increase in the percentage of Tregs [54-55]. The fact

345 that cytokines were highly down-modulated by 1,25D in C57BL/6 without a simultaneous alteration in

346 DCs phenotype or percentage of Tregs suggests that these two phenomena are occurring Commented [A70]: Reviewer 3. Text revision.

347 independently. 1,25D could act in specific immunity by indirect (maintaining DCs in an immature state)

348 or direct pathways (downregulating cytokine gene transcription). In this scenario, and also considering

349 that no antigen was included to promote activation and migration of DCs, it is possible that 1,25D is

350 affecting only the direct pathway. A possible contribution of other cell subsets with regulatory role

351 cannot be excluded. Commented [A71]: Reviewer 1. Possibility raised by the

reviewer 1.
352 Results concerning cytokine production revealed a striking difference between the two strains. Spleen

353 cell cultures from C57BL/6 mice previously supplemented with 1,25D produced significantly lower

354 levels of pro-inflammatory cytokines upon in vitro stimulation with ConA. Similar findings were already

355 reported by our group [12] and other researchers [56-57]. A similar effect has also been described in cell

356 cultures derived from patients undergoing hemodialysis [58]. However, the ability of spleen cells from

357 DBA/1 to produce pro-inflammatory cytokines was not affected by the same 1,25D supplementation

358 protocol. A similar variation could occur in response to vitamin D supplementation in humans,

[59-60]
359 particularly in the case of autoimmune pathologies , whose purpose is the regulation of the

360 immune system. A comparable downmodulatory effect of paricalcitol was expected and supported by Commented [A72]: Reviewer 3. Sentence was rephrased.

361 literature findings as demonstrated by reduced inflammation in experimental models of peritoneal

362 fibrosis [61] and renal ischemia-reperfusion injury [62]. The results showed that paricalcitol had a similar,

363 even though not so accentuated, immunomodulatory effect in C57BL/6 mice. To test if

364 downmodulation of pro-inflammatory cytokines was associated to a higher production of anti-

365 inflammatory cytokines, the levels of IL-5 and IL-10 were also checked in spleen cell cultures. However,

366 their production was also highly decreased in C57BL/6 mice treated by both, 1,25D and paricalcitol.

367 The effect of 1,25D on anti-inflammatory cytokines is controversial. Similarly to our findings, Bemiss et

17
368 al. [63] demonstrated that in vitro addition of 1,25D significantly reduced IL-10 production. Conversely,

369 other reports showed increased production of these cytokines in the presence of 1,25D

[64-65]
370 supplementation . In addition to differences determined by the employed protocols, these Commented [A73]: Reviewer 3. Text revision.

371 contrasting findings could be related to the state of activation and differentiation of the tested cells as

372 reported by Mahon et al. [66] and Cantorna et al. [67]. It is also possible, due to the absence of a specific

373 antigen in this experimental design, that 1,25D is mediating only a direct effect, that is, decreasing the

[68-69]
374 transcription of cytokine genes as already described for IL-2, IL-17 and IL-10 producing T cells .

375 Surprisingly, the same 1,25D supplementation protocol was not able to downmodulate cytokine

376 production in DBA/1 mice. No differences in gender, age or weight were observed that could explain

377 this differential effect. A possible link of these findings with MHC haplotype or other genetic traits, in

378 special the ones related to 1,25D metabolism, must be considered. C57BL/6 and DBA/1 mice, together

379 with many other mouse strains, are inbred laboratory strains that are strikingly different in terms of

380 immune response due to genetic mutations and polymorphisms [70]. The complex control of cytokines

381 by MHC in humans and mice was already demonstrated by Caruso et al. [71], a long time ago.

382 To investigate if the almost total absence of effects of 1,25D or paricalcitol in DBA/1 mice was due

383 to an insufficient dose, these animals were treated with 0.2 µg of 1,25D or paricalcitol, during 15

384 alternate days. This higher 1,25D dose was able to downmodulate some of the evaluated cytokines.

385 However, unresponsiveness to paricalcitol was maintained even in the presence of this higher

386 analogue dose. The mechanism of this differential response between C57BL/6 and DBA/1 mice

387 certainly should be fully investigated, but considering that 1,25D interaction with its target cells is

388 mediated by VDR, it is possible that this phenomenon is related to differences in VDR expression

389 especially in cells of the immune system. A possible correlation with vitamin D binding protein (VDBP)

390 expression levels is another possibility. This protein has a high affinity for active vitamin D providing a

391 reservoir for this hormone. In this context we could think that DBA/1 mice could express less VDBP.

392 The work of Safadi et al. [72] supports this idea by demonstrating that 1,25D was rapidly metabolized

18
393 by the liver in C57BL/6J mice deficient in VDBP. Interestingly, a recent paper shows that 1,25D serum

394 levels are higher in C57BL/6 than in DBA/1 mice [73].

395 Even though the translation of these findings to clinical practice requires further investigation we

396 believe that they suggest the need of a more rational vitamin D prescription. In this sense we highlight

397 the need to check both, blood vitamin D levels and biological response of each patient during

398 supplementation. We also believe that the analogues need to be carefully compared to vitamin D

399 concerning their uses to control inflammation, kidney pathologies and osteoporosis. Commented [A74]: Reviewer 2. Inclusion of sentences
connecting results with clinical practice.
400

401 Conclusions

402 Three main conclusions can be drawn from this study: 1. C57BL/6 and DBA/1 mice differ in Commented [A75]: Reviewer 3. Text revision.

403 their response to supraphysiological supplementation with 1,25D and paricalcitol; 2. Cytokine

404 production by C57BL/6, but not from DBA/1, was the immunological parameter more intensely

405 downmodulated by 1,25D and its analogue; 3. DBA/1 mice required higher doses of 1,25D and

406 paricalcitol to be slightly immunomodulated.

407 In the context of these findings we believe that further investigation is of paramount

408 importance to reveal if a similar variation also exists in human beings. Another issue that deserves

409 further clarification is the full characterization of vitamin D analogues as immunomodulatory agents.

410 These studies would allow a more rational and appropriate use of 1,25D and its analogues.

411

412 Abbreviations

ConA concanavalin A

DCs dendritic cells

MFI median fluorescence intensity

MHC major histocompatibility complex

MHCII major histocompatibility complex class II

19
 Tregs regulatory T cells

VDBP vitamin D binding protein

VDR vitamin D receptor

413

414 Ethics approval and consent to participate

415 All experimental animal care and handling were performed in accordance with the recommendations

416 in the Guidelines for the Care and Use of Laboratory Animals at São Paulo State University. The animal

417 experimental protocol was approved by Ethics Committee for Animal Experimentation - Institute of

418 Biosciences of Botucatu, approved in February 12th, 2016 (Protocol number 815).

419

420 Consent for publication

421 Not applicable.

422

423 Availability of data and materials

424 The datasets used and/or analyzed during the current study are available from the corresponding Commented [A76]: Reviewer 3. Text revision.

425 author on reasonable request.

426 Competing Interest

427 The authors declare that they have no competing interests.

428

429 Funding

430 This work was supported by São Paulo Research Foundation (FAPESP), grant number 2013/26257-8.

431

432 Authors’ Contributions

433 A.P.M., S.F.G.Z.P. and A.S. conceived and designed the experiments. A.P.M., T.F.C.F.S, L.L.W.I.,

434 L.A.N.M., T.G.D.F., L.R.C.O. and S.F.G.Z.P. performed the experiments. A.P.M., T.F.C.F.S., S.F.G.Z.P. and

435 A.S. analyzed the data. A.P.M., S.F.G.Z.P. and A.S. wrote the paper.

20
436

437 Acknowledgements

438 Not applicable.

439 References Commented [A77]: Reviewer 3. All references have been

revised and corrected.

440 1. Holick, M. F. (2006). Resurrection of vitamin D deficiency and rickets. The Journal of
441 clinical investigation 116 (8), 2062-72.
442 2. Zenata, O.; Vrzal, R. (2017). Fine tuning of vitamin D receptor (VDR) activity by post-
443 transcriptional and post-translational modifications. Oncotarget 8 (21), 35390-35402.
444 3. Valdivielso, J. M.; Fernandez, E. (2006). Vitamin D receptor polymorphisms and
445 diseases. Clinica chimica acta; international journal of clinical chemistry 371 (1-2), 1-12.
446 4. Wei, R.; Christakos, S. (2015). Mechanisms Underlying the Regulation of Innate and
447 Adaptive Immunity by Vitamin D. Nutrients 7 (10), 8251-60.
448 5. Palmer, M. T., et al. (2011). Lineage-specific effects of 1,25-dihydroxyvitamin D(3) on
449 the development of effector CD4 T cells. The Journal of biological chemistry 286 (2), 997-1004.
450 6. Bscheider, M.; Butcher, E. C. (2016). Vitamin D immunoregulation through dendritic
451 cells. Immunology 148 (3), 227-36.
452 7. Ishikawa, L. L., et al. (2014). Immunomodulation in human and experimental arthritis:
453 including vitamin D, helminths and heat-shock proteins. Lupus 23 (6), 577-87.
454 8. Kato, S. (2000). Molecular mechanism of transcriptional control by nuclear vitamin
455 receptors. The British journal of nutrition 84 Suppl 2, S229-33.
456 9. Peppone, L. J., et al. (2010). The efficacy of calcitriol therapy in the management of
457 bone loss and fractures: a qualitative review. Osteoporosis international : a journal established
458 as result of cooperation between the European Foundation for Osteoporosis and the National
459 Osteoporosis Foundation of the USA 21 (7), 1133-49.
460 10. Rustico, S. E., et al. (2015). Calcitriol treatment in metabolic bone disease of
461 prematurity with elevated parathyroid hormone: A preliminary study. Journal of clinical &
462 translational endocrinology 2 (1), 14-20.
463 11. Zorzella-Pezavento, S. F., et al., Experimental autoimmune encephalomyelitis is
464 successfully controlled by epicutaneous administration of MOG plus vitamin D analogue.
465 12. Chiuso-Minicucci, F., et al. (2015). Treatment with Vitamin D/MOG Association
466 Suppresses Experimental Autoimmune Encephalomyelitis. PLoS One 10 (5), e0125836.
467 13. Aloia, J. F., et al. (2008). Vitamin D intake to attain a desired serum 25-hydroxyvitamin
468 D concentration. Am J Clin Nutr 87 (6), 1952-8.
469 14. Mazahery, H.; von Hurst, P. R. (2015). Factors Affecting 25-Hydroxyvitamin D
470 Concentration in Response to Vitamin D Supplementation. Nutrients 7 (7), 5111-42.
471 15. Eftekhari, M. H., et al. (2011). Impact of treatment with oral calcitriol on glucose indices
472 in type 2 diabetes mellitus patients. Asia Pacific journal of clinical nutrition 20 (4), 521-6.
473 16. Ng, K. (2014). Vitamin D for Prevention and Treatment of Colorectal Cancer: What is
474 the Evidence? Current colorectal cancer reports 10 (3), 339-345.
475 17. Zhou, Y., et al. (2014). DNA methylation levels of CYP2R1 and CYP24A1 predict
476 vitamin D response variation. J Steroid Biochem Mol Biol 144 Pt A, 207-14.
477 18. Tebben, P. J., et al. (2016). Vitamin D-Mediated Hypercalcemia: Mechanisms,
478 Diagnosis, and Treatment. Endocrine reviews 37 (5), 521-547.
479 19. Večerić-Haler, Ž., et al. (2016). Comparison of the Pharmacological Effects of
480 Paricalcitol Versus Calcitriol on Secondary Hyperparathyroidism in the Dialysis Population.
481 Ther Apher Dial 20 (3), 261-6.
482 20. Sochorová, K., et al. (2009). Paricalcitol (19-nor-1,25-dihydroxyvitamin D2) and
483 calcitriol (1,25-dihydroxyvitamin D3) exert potent immunomodulatory effects on dendritic cells
484 and inhibit induction of antigen-specific T cells. Clin Immunol 133 (1), 69-77.

21
485 21. Shanmugalingam, T., et al. (2014). Obesity and cancer: the role of vitamin D. BMC
486 cancer 14, 712.
487 22. Sisley, S. R., et al. (2016). &quot;Hypothalamic Vitamin D Improves Glucose
488 Homeostasis and Reduces Weight&quot;. Diabetes 10.2337/db16-0309.
489 23. Mai, S., et al. (2017). Acute Vitamin D(3) Supplementation in Severe Obesity:
490 Evaluation of Multimeric Adiponectin. Nutrients 9 (5):459.
491 24. Ferronato, M. J., et al. (2015). Vitamin D analogue: potent antiproliferative effects on
492 cancer cell lines and lack of hypercalcemic activity. Archiv der Pharmazie 348 (5), 315-29.
493 25. Xie, D. D., et al. (2017). Low vitamin D status is associated with inflammation in patients
494 with prostate cancer. Oncotarget 8 (13), 22076-22085.
495 26. Gordon, J. R., et al. (2014). Regulatory dendritic cells for immunotherapy in
496 immunologic diseases. Front Immunol 5, 7.
497 27. Moudgil, K. D.; Choubey, D. (2011). Cytokines in autoimmunity: role in induction,
498 regulation, and treatment. Journal of interferon & cytokine research : the official journal of the
499 International Society for Interferon and Cytokine Research 31 (10), 695-703.
500 28. Seruga, B., et al. (2008). Cytokines and their relationship to the symptoms and outcome
501 of cancer. Nature reviews. Cancer 8 (11), 887-99.
502 29. Christopher, K. B. (2016). Vitamin D and critical illness outcomes. Curr Opin Crit Care
503 22 (4), 332-8.
504 30. Agmon-Levin, N., et al. (2013). Vitamin D in systemic and organ-specific autoimmune
505 diseases. Clin Rev Allergy Immunol 45 (2), 256-66.
506 31. Arnson, Y., et al. (2007). Vitamin D and autoimmunity: new aetiological and therapeutic
507 considerations. Ann Rheum Dis 66 (9), 1137-42.
508 32. Hlavaty, T., et al. (2015). Vitamin D therapy in inflammatory bowel diseases: who, in
509 what form, and how much? J Crohns Colitis 9 (2), 198-209.
510 33. Muehleisen, B.; Gallo, R. L. (2013). Vitamin D in allergic disease: shedding light on a
511 complex problem. J Allergy Clin Immunol 131 (2), 324-9.
512 34. Lemire, J. M.; Archer, D. C. (1991). 1,25-dihydroxyvitamin D3 prevents the in vivo
513 induction of murine experimental autoimmune encephalomyelitis. The Journal of clinical
514 investigation 87 (3), 1103-7.
515 35. Mimura, L. A., et al. (2016). Association of myelin peptide with vitamin D prevents
516 autoimmune encephalomyelitis development. Neuroscience 317, 130-40.
517 36. Marcotorchino, J., et al. (2014). Vitamin D protects against diet-induced obesity by
518 enhancing fatty acid oxidation. J Nutr Biochem 25 (10), 1077-83.
519 37. Sergeev, I. N. (2015). Vitamin D-Cellular Ca(2+) link to obesity and diabetes. J Steroid
520 Biochem Mol Biol 10.1016/j.jsbmb.2015.11.008.
521 38. Szlagatys-Sidorkiewicz, A., et al. (2017). Long-term effects of vitamin D
522 supplementation in vitamin D deficient obese children participating in an integrated weight-loss
523 programme (a double-blind placebo-controlled study) - rationale for the study design. BMC
524 pediatrics 17 (1), 97.
525 39. Earthman, C. P., et al. (2012). The link between obesity and low circulating 25-
526 hydroxyvitamin D concentrations: considerations and implications. Int J Obes (Lond) 36 (3),
527 387-96.
528 40. Ljunghall, S., et al. (1995). [Vitamin D and osteoporosis]. Nordisk medicin 110 (10),
529 253-7.
530 41. Nameni, G., et al. (2017). The Impact of Vitamin D Supplementation on
531 Neurodegeneration, TNF-alpha Concentration in Hypothalamus, and CSF-to-Plasma Ratio of
532 Insulin in High-Fat-Diet-Induced Obese Rats. Journal of molecular neuroscience : MN 61 (2),
533 247-255.
534 42. Blumberg, J. M., et al. (2006). Complex role of the vitamin D receptor and its ligand in
535 adipogenesis in 3T3-L1 cells. The Journal of biological chemistry 281 (16), 11205-13.
536 43. Sergeev, I. N. (2016). Vitamin D-Cellular Ca2+ link to obesity and diabetes. J Steroid
537 Biochem Mol Biol 164, 326-330.
538 44. Steddon, S. J., et al. (2001). Vitamin D analogues: how do they differ and what is their
539 clinical role? Nephrol Dial Transplant 16 (10), 1965-7.

22
540 45. Yilmaz, B., et al. (2017). Vitamin D levels in newborns and association with neonatal
541 hypocalcemia. The journal of maternal-fetal & neonatal medicine : the official journal of the
542 European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal
543 Societies, the International Society of Perinatal Obstet 10.1080/14767058.2017.1331430, 1-5.
544 46. Khadilkar, A., et al. (2017). Prevention and Treatment of Vitamin D and Calcium
545 Deficiency in Children and Adolescents: Indian Academy of Pediatrics (IAP) Guidelines. Indian
546 pediatrics 54 (7), 567-573.
547 47. Salomon, D. G., et al. (2014). Phosphonate analogues of 1alpha, 25 dihydroxyvitamin
548 D3 are promising candidates for antitumoural therapies. Current topics in medicinal chemistry
549 14 (21), 2408-23.
550 48. Mirzaei, K., et al. (2014). Insulin resistance via modification of PGC1alpha function
551 identifying a possible preventive role of vitamin D analogues in chronic inflammatory state of
552 obesity. A double blind clinical trial study. Minerva Med 105 (1), 63-78.
553 49. Takiishi, T., et al. (2013). Effects of vitamin D on antigen-specific and non-antigen-
554 specific immune modulation: relevance for type 1 diabetes. Pediatr Diabetes 14 (2), 81-9.
555 50. Xie, Z., et al. (2017). 1,25-dihydroxyvitamin D3 -induced dendritic cells suppress
556 experimental autoimmune encephalomyelitis by increasing proportions of the regulatory
557 lymphocytes and reducing T helper type 1 and type 17 cells. Immunology 10.1111/imm.12776.
558 51. Gambhir, V., et al. (2011). Influence of 1,25-dihydroxy vitamin D3 on TLR4-induced
559 activation of antigen presenting cells is dependent on the order of receptor engagement.
560 Immunobiology 216 (9), 988-96.
561 52. Fawaz, L., et al. (2016). Comparative effect of 25(OH)D3 and 1,25(OH)2D3 on Th17
562 cell differentiation. Clin Immunol 166-167, 59-71.
563 53. Kushwah, R.; Hu, J. (2011). Role of dendritic cells in the induction of regulatory T cells.
564 Cell & bioscience 1 (1), 20.
565 54. Tordesillas, L., et al. (2017). Epicutaneous immunotherapy induces gastrointestinal
566 LAP+ regulatory T cells and prevents food-induced anaphylaxis. J Allergy Clin Immunol 139
567 (1), 189-201 e4.
568 55. Rezende, R. M.; Weiner, H. L. (2017). History and mechanisms of oral tolerance.
569 Seminars in immunology 10.1016/j.smim.2017.07.004.
570 56. Kankova, M., et al. (1991). Impairment of cytokine production in mice fed a vitamin D3-
571 deficient diet. Immunology 73 (4), 466-71.
572 57. He, X., et al. (2014). Vitamin D inhibits the occurrence of experimental cerebral malaria
573 in mice by suppressing the host inflammatory response. J Immunol 193 (3), 1314-23.
574 58. Navarro-González, J. F., et al. (2013). Anti-inflammatory profile of paricalcitol in
575 hemodialysis patients: a prospective, open-label, pilot study. J Clin Pharmacol 53 (4), 421-6.
576 59. Marinho, A., et al. (2016). Vitamin D supplementation effects on FoxP3 expression in
577 T cells and FoxP3(+)/IL-17A ratio and clinical course in systemic lupus erythematosus patients:
578 a study in a Portuguese cohort. Immunol Res 10.1007/s12026-016-8829-3.
579 60. Goldsmith, J. R. (2015). Vitamin D as an Immunomodulator: Risks with Deficiencies
580 and Benefits of Supplementation. Healthcare (Basel) 3 (2), 219-32.
581 61. González-Mateo, G. T., et al. (2014). Paricalcitol reduces peritoneal fibrosis in mice
582 through the activation of regulatory T cells and reduction in IL-17 production. PLoS One 9 (10),
583 e108477.
584 62. Hwang, H. S., et al. (2013). Pretreatment with paricalcitol attenuates inflammation in
585 ischemia-reperfusion injury via the up-regulation of cyclooxygenase-2 and prostaglandin E2.
586 Nephrol Dial Transplant 28 (5), 1156-66.
587 63. Bemiss, C. J., et al. (2002). Interleukin-2 is one of the targets of 1,25-dihydroxyvitamin
588 D3 in the immune system. Arch Biochem Biophys 402 (2), 249-54.
589 64. Åivo, J., et al. (2015). Vitamin D3 administration to MS patients leads to increased
590 serum levels of latency activated peptide (LAP) of TGF-beta. J Neuroimmunol 280, 12-5.
591 65. Barker, T., et al. (2015). Supplemental vitamin D increases serum cytokines in those
592 with initially low 25-hydroxyvitamin D: a randomized, double blind, placebo-controlled study.
593 Cytokine 71 (2), 132-8.

23
594 66. Mahon, B. D., et al. (2003). The targets of vitamin D depend on the differentiation and
595 activation status of CD4 positive T cells. J Cell Biochem 89 (5), 922-32.
596 67. Cantorna, M. T., et al. (2015). Vitamin D and 1,25(OH)2D regulation of T cells. Nutrients
597 7 (4), 3011-21.
598 68. Matilainen, J. M., et al. (2010). The number of vitamin D receptor binding sites defines
599 the different vitamin D responsiveness of the CYP24 gene in malignant and normal mammary
600 cells. The Journal of biological chemistry 285 (31), 24174-83.
601 69. Hayes, C. E., et al. (2015). Vitamin D Actions on CD4(+) T Cells in Autoimmune
602 Disease. Front Immunol 6, 100.
603 70. Sellers, R. S., et al. (2012). Immunological variation between inbred laboratory mouse
604 strains: points to consider in phenotyping genetically immunomodified mice. Veterinary
605 pathology 49 (1), 32-43.
606 71. Caruso, C., et al. (1996). Major histocompatibility complex regulation of cytokine
607 production. Journal of interferon & cytokine research : the official journal of the International
608 Society for Interferon and Cytokine Research 16 (12), 983-8.
609 72. Safadi, F. F., et al. (1999). Osteopathy and resistance to vitamin D toxicity in mice null
610 for vitamin D binding protein. The Journal of clinical investigation 103 (2), 239-51.
611 73. Fleet, J. C., et al. (2016). Gene-by-Diet Interactions Affect Serum 1,25-
612 Dihydroxyvitamin D Levels in Male BXD Recombinant Inbred Mice. Endocrinology 157 (2),
613 470-81.
614
615

24