Research Article Effects of Vitamin B6 Deficiency on the Composition and Functional Potential of T Cell Populations

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1 Hindwi Journl of Immunology Reserch Volume 217, rticle ID , 12 pges Reserch rticle Effects of Vitmin 6 Deficiency on the Composition nd Functionl Potentil of T Cell Popultions ingjun Qin, 1,2 Shnqi Shen, 2 Jinhu Zhng, 2 nd Pu Jing 2 1 Deprtment of Nutrition nd Helth, Reserch Centre of iomedicl Technology Co. Ltd., Yncheng Voctionl Institute of Helth Sciences (YIHS), Jingsu 2245, Chin 2 Reserch Center for Food Sfety nd Nutrition, Key Lb of Urbn griculture (South), or S. Luh Food Sfety Reserch Center, School of griculture & iology, Shnghi Jio Tong University, Shnghi 224, Chin Correspondence should be ddressed to Pu Jing; pjing@sjtu.edu.cn Received 18 November 216; Revised 21 Jnury 217; ccepted 14 Februry 217; Published 6 Mrch 217 cdemic Editor: Xio-Feng Yng Copyright 217 ingjun Qin et l. This is n open ccess rticle distributed under the Cretive Commons ttribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. The immune system is criticl in preventing infection nd cncer, nd mlnutrition cn weken different spects of the immune system to undermine immunity. Previous studies suggested tht vitmin 6 deficiency could decrese serum ntibody production with concomitnt increse in IL4 expression. However, evidence on whether vitmin 6 deficiency would impir immune cell differentition, cytokines secretion, nd signl molecule expression involved in JK/STT signling pthwy to regulte immune response remins lrgely unknown. The im of this study is to investigte the effects of vitmin 6 deficiency on the immune system through nlysis of T lymphocyte differentition, IL-2, IL-4, nd INF-γ secretion, nd SOCS-1 nd T-bet gene trnscription. We generted vitmin 6-deficient mouse model vi vitmin 6-depletion diet. The results showed tht vitmin 6 deficiency retrds growth, inhibits lymphocyte prolifertion, nd interferes with its differentition. fter Con stimultion, vitmin 6 deficiency led to decrese in IL-2 nd increse in IL-4 but hd no influence on IFN-γ. Rel-time PCR nlysis showed tht vitmin 6 deficiency downregulted T-bet nd upregulted SOCS-1 trnscription. This study suggested tht vitmin 6 deficiency influenced the immunity in orgnisms. Menwhile, the pproprite supplement of vitmin 6 could benefit immunity of the orgnism. 1. Introduction The immune system s role is crucil in prevention nd control of pthogenic infection s well s vrious cncers [1]. Menwhile, the nturl ging process, mlnutrition, nd incresed stress brought upon by the fst-pced urbn lifestyle hve been demonstrted to decrese immunity [2 4]. mong these fctors, the effect of mlnutrition on immunity hs been widely investigted on children in developing countries, people with eting-disorder problems, nd the elderly [5 7]. Close to one billion people suffer from vrying degrees of mlnutrition s result of insufficient food or food lck of micronutrients [4]. Vitmin 6 deficiency is very common phenomenon, especilly mong women of childbering ge s well s the elderly [8 1]. Vitmin 6 is n intriguing micronutrient tht medites numerous metbolic processes in vivo including mino cid metbolism, gluconeogenesis, lipid metbolism, nd nervous system development nd functioning. Vitmin 6 hs been implicted in the regultion of immune responses tht re ssocited with wide rnge of diseses, including inflmmtion [11] nd vrious cncers [1, 12, 13]. Previous studies suggested tht vitmin 6 deficiency could impir immune responses. Kumr nd xelrod found tht the serum ntibody (IgG, IgM) production in vitmin 6 deficient mice decresed fter got erythrocyte immune stimultion nd could be recovered to norml level fter short-term of vitmin 6 supplementtion [14]. Doke et l. found tht the vitmin 6 deficient mice produces specific IgE ntibody compred to norml controls fter the dinitrophenylted ovlbumin (DNP-OV) immune stimultion [15]. The level of IL-4, n essentil fctor for IgE synthesis, ws significntly higher in the vitmin 6 deficient mice thn in the norml controls, while the level of IL-2 in deficient groups ws significntly lower thn in the norml controls [15]. Subsequently, vitmin 6 supplementtion in

2 2 Journl of Immunology Reserch Tble 1: Diet composition of vitmin 6 deficient mouse model. Diet composition Minerl/Kg diet Vitmin/Kg diet Sugr 4% MgCl 6H 2 O 17 g Vitmin 14 IU Csein 2% KCl 5 g Vitmin D 15 IU Strch 18% NCl 6 g Vitmin E 12 IU α-cellulose 8% FeCl mg Vitmin 1 13 mg Oil 5% MnCl 4 4H 2 O 27 mg Vitmin 2 12 mg Minerl 3.5% CuSO 4 5H 2 O 4 mg Vitmin 6 /12/12 mg Vitmin 1% ZnCl 2 63 mg Folic cid 6. mg Methionine.3% Sn.2 mg Nicin 45 mg Choline itrtrte.2% Pntothenic cid 17 mg iotin.1 mg Vitmin K 3. mg Vitmin 6 ws given s pyridoxine (PN) nd its content ws different in different diets. The diet ws strictly defined s Non-V6 Diet, Norml-V6 Diet, nd Excessive-V6 Diet, which contined, 12 mg, nd 12 mg of vitmin 6 per kg diet, respectively. The Excessive-V6 Diet ws mixed with 1 times vitmin 6 of the dily recommended intke. the deficient groups resulted in the serum ntibody (IgG) nd nti-dnp IgE recovering to the sme levels s those in the norml controls [15]. Further reserch showed tht excessive vitmin 6 (6 mg/1 g diet) supplementtion could inhibit the production of the nti-ov ntibody IgE nd IgG1 due to the suppression of heptic cthepsin ctivity by vitmin 6 [16]. Therefore, modertion of vitmin 6 might medite immune signl trnsduction or regulte immune cell differentition nd cytokine production together with other signl molecules to rech n immune homeostsis. Jnus tyrosine kinse/signl trnsducer nd ctivtor of trnscription (JK/STT) signling pthwy exists in lmost ll cytokine signling pthwys [17]. Mny intrcellulr cytokines, including the colony-stimulting fctor, interleukins (ILs), interferons (IFNs), erythropoietin (Epo), ndthrombopoietin(tpo),cnbindwithtypeioriicytokine receptors to trnsduce the signl into the nucleus vi the JK/STT signling pthwy [18]. These cytokines cn further induce the expression of downstrem genes nd regulte series of biologicl effects including immune response nd cell growth [18]. Suppressor of cytokine signling (SOCS) is cytokine signl trnsduction suppressor in the JK/STT signling pthwy [19]. SOCS-1 could inhibit the differentition of the IFN-γ-expression in cells or terminte IFN-γ signl trnsduction to block the signling [2]. The expression of IL-1b, IL-2, nd IL-2R ws suppressed in CD4-lymphocytes in the vitmin 6 deficient mice [21] due to decresed serine hydroxymethyl trnsferse (SHMT) ctivity in the bsence of vitmin 6, resulting in the reduction of one crbon unit nd the blocking of mrn synthesis, which in turn ffects the gene expression. However, IL-4 expression incresed in vitmin 6 deficient mice [15]. This ws opposite to the decline of gene expression cused by the reduction of one crbon unit. Therefore, this study minly investigted the effect of vitmin 6 deficiency on the composition nd functionl potentil of T cell popultions through nlysis of pyridoxine 5 -phosphte (PLP) nd xnthurenic cid (X) plsm level, lymphocyte prolifertion nd differentition, cytokines expression, nd SOCS-1 nd T-bet trnscription. 2. Mterils nd Methods 2.1. Mterils nd Chemicls. The mle L/c mice were purchsed from Shnghi Slc Lbortory niml Center. The diet contined sugr, fiber, slt, nd oil tht were ll foodgrde nd purchsed from uchn supermrket (Shnghi, Chin). The micronutrients, such s vitmins, D, E, 1, 2, 6, 12, nd K1 nd folic cid, were provided by Shnghi Xinyi Medicl Co. Ltd. (Shnghi, Chin). Nicotinic cid nd pntothenic cid were purchsed from the Genery iotech (Shnghi, Chin). Selenium nd iotin were from Swnson (Frgo, ND, US). Other chemicls were ll chemiclly pure nd provided by Sinophrm Chemicl Regent (Shnghi, Chin). Deionized wter ws supplied s the drink for mice Ethics Considertions. niml study ws crried out strictly in ccordnce with the Guidelines for the Cre nd Use of Lbortory nimls of Shnghi Jio Tong University. The protocol ws pproved by Shnghi Municipl Lbortory niml Mngement Office, Shnghi Municipl Science nd Technology Commission (Permit Number: 11ZR14162) Construction of Vitmin 6 Deficient Mouse Model. In order to investigte the influence of vitmin 6 deficiency on the immune system nd the subsequent remedition by vitmin 6 supplementtion, three-week-old L\c mice, weighingnvergeof1g,wererndomlydividedinto 4 dietry groups, nmely, the control group (), the deficiency group (), the recovery group (), nd the excess group (). Ech group contined 7 mice. Theywerehousedinthestndrdcgeswith12hlight/12h drk cycle. The temperture nd humidity in the cges were controlled t 24 ± 1 Cnd6± 5%, respectively. The diet formul (Tble 1) ws bsed on the previous studies by Miller & umnn [22], Doke et l. [15], nd

3 Journl of Immunology Reserch 3 Inubushi et l. [16]. Minerl substnces nd vitmin demnds werebsedonthestndrd Lbortoryniml-Nutrients for Formul Feed (G , Chin). The vitmins listed in Tble 1 were ground into powders nd mixed with other diet ingredients to form dily diet. ll the diets contined the sme composition, except vitmin 6. The diet ws strictly defined s Non-V6 Diet, Norml-V6 Diet, nd Excessive-V6 Diet, which contin, 12 mg, nd 12 mg of vitmin 6 per kg diets, respectively. The Norml-V6 Diet contined the dily recommended intke (DRI) of vitmin 6 (12 mg), while the Excessive-V6 Diet contined 1 times the vitmin 6 in the Norml-V6 Diet. The control group () ws fed the Norml-V6 Diet throughout the experiment, nd the other three groups were deprived of vitmin 6 by being fed with Non-V6 Diet for the first 5 weeks. Following this, the deficiency group () continued to tke the Non-V6 Diet, nd the recovery group () ws chnged to the Norml-V6 Diet, while the excess group () took the Excessive- V6 Diet until the end of the experiment. Diet nd wter were given d libitum. Wter contined the sme composition of vitmins s the diet Weight Mesurement. To estimte the effect of vitmin 6 deficiency on growth, the body weight of the mice ws mesured weekly. bsic growth curve of the mice ws constructed nd the differences mong the 4 groups were comprtively nlyzed Determintion of Xnthurenic cid nd Pyridoxl 5 - Phosphte in Plsm by LC-MS/MS. To confirm the success of the vitmin 6 deficient mouse model, X ( metbolic intermedite of tryptophn) nd PLP (the ctive form of vitmin 6), plsm levels were ssyed using ultr performnceliquidchromtogrphy(cquityuplcsystem, M, US) linked with triple qudrupole mss spectrometer of SCIEX Triple Qud 55 LC-MS/MS System ( SCIEX, Toronto, Ontrio, Cnd) in the Instrumentl nlysis Center of Shnghi Jio Tong University. lood smples were treted ccording to the description by Midttun et l. [23]. riefly, orbitl blood of mice ws collected into tubes, which were heprinized for 1 dy prior to scrifiction to prevent cogultion. Then, plsm ws crefully pipetted into nother microtube on ice fter being centrifuged t 2g for 1 min t 4 Cndstoredt 8 C. Sixty microliters of blood plsm ws deproteinized for 6 min by dding n equl volume of 6 g/l ice-cold trichlorocetic cid (TC), followed by centrifugtion t 58g for 15 min t 4 Cto remove the dentured protein. The superntnt (6 μl) ws removed to new tube to be dried by blowing gseous nitrogen. Finlly, methnol ws dded to finl volume of 2 μl todissolvethetretedsmple.1ng/mlmixture of X nd PLP (Sigm, St. Louis, MO, US) ws used s stndrd, respectively. Zorbx stble-bond C8 reversedphse column (8 Å, 3.5 μm, mm; gilent) equipped with similr gurd column (8 Å, 5 μm, mm; gilent) ws used with mobile phse consisting of solution (65 mmol/l cetic cid), solution (1 mmol/l heptfluorobutyric cid in ), nd solution C (9% cetonitrile in wter) t flow rte of 1.3 ml/min. X nd PLP were identified on the bsis of m/z rtio nd retention time. There ws no cross-tlk between ion pirs from those different nlytes. Quntifiction ws by linerity grdient curve; R 2 ws.9988 (.34 1 ng/ml) nd.9975 (.34 1 ng/ml), respectively, for X nd PLP Totl Spleen Lymphocytes Isoltion. Totl spleen lymphocytes were isolted for further cellulr experiments. Mouse spleen lymphocytes were isolted from the 4 groups of mice by using stndrd protocol [24] with the Cppel LSM lymphocyte seprtion medium (MP iomedicls Solon, OH, US). The mice were scrificed vi euthnsi nd plced into 7% ethnol for disinfection. The spleen ws removed quickly with n septic technique nd wshed thoroughly with RPMI-164 medium (Gibco, Invitrogen, Crlsbd, C, US). The spleen cells were then relesed by being ground on 2 mesh grids nd resuspended in 5 ml RMPI-164 medium. The cells were then trnsferred to 15 ml tube nd n equl volume of LSM ws dded. fter centrifuging t 13g for 2 min with slow ccelertion nd slow decelertion, the middle lyer of spleen lymphocytes ws pipetted crefully nd resuspended in RPMI-164 medium supplemented with 1% fetl bovine serum (FS) (iologicl Industries, eit HEmek, Isrel). Spleen lymphocytes count ndvibilityweressyedbytrypnbluepriortofinlplting. The cell concentrtion ws djusted to /ml. The suspension ws then seeded into 96-well pltes nd incubted t 37 C in humidified tmosphere (5% CO 2 ). fter 2 h incubtion, hlf of the murine spleen lymphocytes were incubted with 5 μg/ml concnvlin (Con, type IV; Sigm, St. Louis, MO, US) to stimulte immune rections, such s T lymphocyte differentition, prolifertion ndcytokineproduction,ndimmunereltedgenemrn trnscription. Cells without Con stimultion were set s control, where equl volume of RMPI-164 medium ws dded s substitute Lymphocyte Prolifertion ssy. To estimte the effect of different doses of vitmin 6 on immunomodultory potentil, spleen lymphocyte prolifertion ws ssessed using WST-8 Cell Counting Kit-8 (eyotime, shnghi, Chin) fter 72-hour stimultion with Con. Ten microliters of CCK-8 solution ws dded to ech well wy from light nd incubted for nother 2 h under the sme conditions. The bsorbnce t 45 nm ws determined by the Multiskn GO Microplte spectrophotometer (Thermo, US) with three technicl repets. Prolifertion response ws expressed s stimultion index (SI) clculted s the rtio of the men OD 45 vlue of the Con-stimulted cells to the men OD 45 vlue of the medium lone-stimulted cells T Lymphocyte Differentition ssy. To investigte the trend of T lymphocyte differentition influenced by the different doses of vitmin 6 in diet fter immune stimultion, the prolifertion of the CD3+ cells ws estimted to represent

4 4 Journl of Immunology Reserch ody weight (g) Feeding time (week) Growth rte Feeding time (week) control deficiency recovery powered control deficiency recovery powered () (b) Figure 1: ody weight nd body weight gin rte of L/c mice fed with the experiment diets. Growth rte ws clculted using the formul: Growth rte =(ody weight n week ody weight week )/ody weight week. Ech column represents the men ± SEM (n =7)with three independent experiments. Men vlues with sterisk () ttched indicte significnt difference (P <.5) mong the groups t the sme time point. tht of totl T lymphocytes, nd percentges of two min T- lymphocyte cell subsets ssocited with immunomodultion, CD4+ (helper T lymphocytes) nd CD8+ (cytotoxic T lymphocytes), were estimted by detecting their cell surfce specific glycoproteins CD4 nd CD8 molecule, respectively, using n ELIS ssy kit (Shnghi XinRn iologicl, Shnghi, Chin) ccording to Frnke et l. s description [25] with some modifictions. ll kit regents were llowed to rech room temperture prior to usge. The bsorbnce ws determined t 45 nm by the Microplte spectrophotometer (Multiskn GO; Thermo Scientific, Wlthm, M, US). The stndrd curves of CD3, CD4, nd CD8 were estblished by plotting the U/mL concentrtions versus bsorbnce vlues ofthestndrdwells.thecurveswereusedtoquntify the concentrtion of CD3, CD4, nd CD8 in cell culture superntes Cytokine IL-2, IL-4, nd IFN-γ Secretion Levels. To investigte the secretion of cytokines relted to immune regultion, such s interleukin-2 (IL-2), interleukin-4 (IL-4), nd interferon-γ (IFN-γ), mouse IL-2/IL-4/IFN-γ ELIS sets (eioscience, Sn Diego, C, US) were used to mesure the IL-2, IL-4, nd IFN-γ production following the mnufcturer s recommendtion. Ech experiment ws performnce with three technicl repets nlysis of SOCS-1 nd T-bet Gene Trnscriptionl Level by Rel-Time PCR. To estimte the trnscriptionl levels of SOCS-1 nd T-bet genes involved in the JK/STT immunomodultion signling pthwy, totl RN ws extrcted from the T lymphocyte from 4 groups of mice with or without Con stimultion using the RNesy Mini Kit (Qigen, Snt Clrit, C, US) by following the mnufcturer s recommendtion. The ReverTr ce--first- Strnd cdn synthesis kit (Toyobo, Jpn) ws employed to synthesize the oligo (dt) primed first-strnd cdn with.3 mg RN s templtes. Quntittive rel-time-pcr (qrt-pcr) nlysis ws performed using SYR Premix EX Tq (TKR, Jpn) on io-rd CFX96 Touch Rel- Time PCR Detection System (iord, Hercules, C) s described by Ding et l. [26] with the exception of the nneling temperture t 58 C when using the primers: SOCS-1 (forwrd: 5 -TCCGTTCCGGCGCTCCG-3, reverse: 5 -CTCCGCGCTCGGGC-3 )ndt-bet (forwrd: 5 -GCCGGGCCGCTTTTG-3,reverse:5 - GCGTCTCTGGGTCCTTGT-3 ). Gene GPDH ws used s reference gene with primers GPDH (forwrd: 5 -CCTGGGGGTGGG-3,reverse:5 -C- GTTGTCTGGTGCC-3 ). Three biologicl replictes were used, ech with three technicl repets Sttisticl nlysis. Seven replictions with three independent experiments were performed. Dt ws reported s men ± SEM or SD. One-wy NOV nd LSD test t the level of.5 were used to identify differences in mens. Correltions mong the indices of vitmin -6 sttus were nlyzed using two-tiled Person correltion coefficient. Sttistics nlyses were crried out using SPSS for Windows (version rel. 1.5, 1999, SPSS Inc., Chicgo, IL, US). 3. Results 3.1. Vitmin 6 Deficiency Retrds Growth Rte in Mice. Previous studies showed tht vitmin 6 deficiency could reduce body weight of orgnisms [15]. Four types of diets were formulted to investigte the effect of vitmin 6 deficiency ndrepletiononthegrowthofmicendtheirweight-forge growth curve ws estblished (Figure 1()). t the 5-week time point, the end of vitmin 6 deficiency, the vitmin 6 deficient diet-3 groups grew significntly slower thn the control group (+V+) (P <.5) (Figure 1(b)). t the 8- week time point, the vitmin 6 deficient group () still grew significntly slower thn the other 3 groups (,

5 Journl of Immunology Reserch 5 Tble 2: Plsm xnthurenic cid (X) nd pyridoxl-5 -phosphte (PLP) concentrtions t the end of the vitmin 6-deficiency stge () nd the other three stges of vitmin 6: norml (), recovery (), nd excess (). Plsm X (ng/ml) Plsm PLP (pg/ml) ± ± ± 28.7 b ND ± ± ± ± Dt represent the men ± SD of seven replictes with three independent experiments. Significnt difference ws indicted with different letters (P <.5). Cpcity of lymphocyte prolifertion Stimultion index Control Con SI, nd ) (P <.5), mong which there ws no significnt difference (P >.5) Plsm Concentrtions of PLP nd X. For further confirming vitmin 6 sttus in orgnisms, plsm levels of PLP nd X were nlyzed by LC-MS/MS. In the group, the plsm PLP level ws not detected (Tble 2). The plsmplplevelsintheother3groupsincresedfollowing the order of > >, suggesting tht the mice could bsorb nd store vitmin 6 to compenste for the mlnutrition cused by 28-dy depletion. However, the 12 mg vitmin 6/kg diet intke did not improve the plsm PLP concentrtion 1 times more thn 12 mg/kg diet vitmin 6 intke. It might be due to the fct tht 12 mg/kg diet of Vitmin 6 seriously exceeded the recommended dietry llownce (RD) nd the bsorption of nutrients displyed sturtion kinetics [27]. Menwhile, the men vlue of plsm X levels in the group ( ng/ml) ws significntly higher thn tht in,, nd group (P <.5) (Tble 2). Even though there ws certin mount of PLP in the plsm, X ws still detected, presenting the sme tendency with Miller nd umnn s study [22]. The subsequent urinry X level begn to reduce when chnging to vitmin 6-repletion diet nd reched the lowest point fter 4 dys of recovery feeding, suggesting tht the recovery from the dysfunction cused by vitmin 6 deficiency tkes time Vitmin 6 Deficiency Wek Cpcity of Lymphocyte Prolifertion. The effect of vitmin 6 on the prolifertion cpcity of lymphocytes ws estimted using the stimultion index (SI) vi Con stimultion. Con ws chosen for T lymphocytes stimultion, while PH is used for lymphocytes. The results showed tht vitmin 6 deficiency slightly reduced the prolifertion of lymphocytes by 3.37% compred with the group (Figure 2), lthough there were no significnt differences in the SI mong groups of (SI, ), (SI, ), nd (SI, 1.27). fter supplementtion of norml dose of vitmin 6 (12 mg/kg diet, group) for 35 d, the prolifertion of lymphocytes ws recovered to nd ws significntly stronger thn tht of the other three groups (P <.5). However, the excessive supplementtion (12 mg/kg diet, group) Figure 2: Cpcity of lymphocyte prolifertion. The stimultion index (SI) ws clculted using the following formul prolifertion of T lymphocytes incubted with Con stimultion divided by prolifertion of T lymphocytes incubted without Con stimultion. Ech column dt represents men vlues of seven replictions with three independent experiments. SI index with denotes significnt differences mong the groups (P <.5). did not significntly improve the lymphocyte prolifertion (P >.5) Vitmin 6 Deficiency Inhibits CD4 T Lymphocyte Differentition fter Con Stimultion. The result showed tht CD3+cellsrepresentingtotlTlymphocytesin group ws not significntly different from the other 3 groups of,, nd without Con stimultion (Figure 3()), lthough it ws less thn nd group. Moreover, without Con stimultion, CD4+ nd T- helper cell in the groups of (73.63), (74.66), nd (69.39)werelllessthnthoseofthegroup (82.2), nd CD8+ nd cytotoxic T lymphocyte in groups (26.37), (25.34), nd (3.64) were more thn those in the group of (17.89) (Figures 3(b), 3(c), nd 3(d)). Menwhile, the rtio of CD4/CD8 in group ws significntly less thn tht in group (P <.5) (Figure 3(d)). However, CD3 in group decresed significntly compred with tht in group fter Con stimultion (P <.5) (Figure 3()). In the nd groups, the levels of CD3+ were more thn tht in group but less thn tht in group (Figure 3()). Menwhile, rtio of CD4/CD8 ws decresed in,, nd groups (Figure 3(d)), suggesting tht the reduction of immunity ws minly contributed by the reduction of CD3 prolifertion nd the impirment of T cell differentition cused by vitmin 6 deficiency Effect of Vitmin 6 Deficiency on Cytokines IL-2\IL- 4\IFN-γ Secretion Level. Without Con stimultion, IL-2 secretions of T lymphocytes from the four groups were ll t low level (Figure 4()). However, fter Con stimultion, IL-2 secretion levels in the group were higher thn the group, lthough the difference ws not significnt

6 6 Journl of Immunology Reserch The level of CD Without Con stimultion With Con stimultion The level of CD Without Con stimultion With Con stimultion () (b) The level of CD Without Con stimultion With Con stimultion,, Percentge of CD4 nd CD8 subsets (%) Without Con stimultion With Con stimultion The rtio of CD4/CD8 (c) CD8 CD4 CD4/CD8 (d) Figure 3: Level of T lymphocyte cell with or without Con stimultion ((), (b), nd (c)) nd differentition of CD4+ nd CD8+ subsets with or without Con stimultion (d). Clcultion ws bsed on the percentge of concentrtion. Ech group dt represents men vlues of seven replictions with three independent experiments. Column mrked with different lphbets denotes significnt differences mong the groups (P <.5). (P >.5). IL-2 secretion levels in the nd groups were significntly higher thn tht of the other two groups (P <.5), but excessive vitmin 6 supplementtion did not promote the IL-2 secretion much more thn norml diet. Without Con stimultion, the IL-4 secretion levels of four groups were low. fter Con stimultion, IL-4 secretion levels significntly improved (P <.5) inthefour groups nd incresed much more in group thn in nd groups but less thn in group (Figure 4(b)). On the other hnd, without Con stimultion, IFN- γ production in the primitive spleen lymphocyte from the four groups ws t low levels, lthough the group ws higher thn the other 3 groups (Figure 4(c)). fter stimultion, IFN-γ incresed significntly in ll experiment groups nd with no significnt difference observed between the tretments (P >.5) Down- nd Upregultion of Gene SOCS-1 nd T-bet ssocited with JK/STT Signling Pthwy. The results of rel-time PCR nlysis showed tht trnscriptionl levels of SOCS-1 were higher in vitmin 6 deficient mice thn in the other three treted mice groups (Figure 5()), consistent with the expression level of the IFN-γ expression (Figure 4(c)). fter Con stimultion, the trnscriptionl level of SOCS- 1 ws significntly downregulted. There ws no significnt difference mong,, nd group (P >.5), while the trnscriptionl level of SOCS-1 ws significntly higher in group thn in the other three tretment groups (P <.5) lthough no significnt difference of IFN-γ level mong these four groups ws observed. Trnscriptionl levels of T-bet in the four groups were ll low without Con stimultion. However, it ws upregulted fter Con stimultion nd improved more significntly in the V6 group thn in the nd groups (P <.5), ll of which were significntly higher thn in group (P <.5) (Figure 5(b)) Correltions between the Vitmin 6 Sttus nd Immune Responses. Person correltion coefficients were performed to understnd the reltionship between the plsm PLP

7 Journl of Immunology Reserch 7 IL-2 level (pg/ml) Control Con C IL-4 level (pg/ml) Control Con C () (b) IFN-훾 level (pg/ml) b Control b Con (c) Figure 4: Cytokines IL-2 (), IL-4 (b), nd IFN-γ (c) secretion level of different vitmin 6 diet mice splenocytes fter Con stimultion. 5 μg/ml Con ws dded to stimulte the splenocyte, while the control received the sme volume of RMPI164 medium. fter 48 h of incubtion, cytokine levels in the culture medium were mesured by ELIS method. Ech column represents the men ± SD of seven replictions with three independent experiments. Columns mrked with different letters re significntly different (P <.5) mong different diet mouse. ndimmuneresponseindictors(tble3).eforecon stimultion, plsm PLP levels were inversely ssocited with plsm X level, CD4+ T lymphocyte numbers, IL- 2 expression levels, nd SOCS1 trnscriptionl level but positively correlted with spleen lymphocyte prolifertion, the rtio of CD4/CD8, nd IL-4 nd IFN-γ expression level. However, fter Con stimultion, plsm PLP concentrtion hd positive correltion with IL-2 expression level, nd T- bet trnscription plyed more importnt role thn SOCS-1 trnscription did. This lso indictes tht vitmin 6 plys n importntroleinimmunomodultion. 4. Discussion Vitmin 6 is n importnt micronutrient for our helth, nd its deficiency could weken immunity [14 16], including decrese of the serum ntibody nd IL-2 production nd increse of the level of IL-4. We constructed vitmin 6-deficient mouse model vi vitmin 6-depletion diet nd found tht mice in group grew significntly slower thn the group (P <.5) t the 5-week time point, the end of vitmin 6 deficiency. In this study, wter ws supplied sufficiently nd givendlibitum,sothelossofmouseweightwsnotffected by dehydrtion. Riordn et l., [28] lso found tht vitmin 6 deficiency reduces the rt growth to bout.49 times the norml, possibly ttributble to the reduced rts ppetite cused by vitmin 6 deficiency. In this study, we observed no sttisticl chnge in food consumption in vitmin 6 supplementtion nd deficiency groups, which indicted tht the vitmin 6 deficiency might slow the growth of mice. Lewick et l. [29] lso demonstrted tht vitmin 6 supplementtion cn sttisticlly increse the verge weight of rts fed with protein deficiency diets, difference of 1.5 times in comprison to the nonsupplemented group fter 3 dys of study. lso, supplementtion of vitmin 6 could help orgnisms recover some of the temporry weight loss cused

8 8 Journl of Immunology Reserch SOCS b b b T-bet C C Control Con Control Con () (b) Figure 5: Rel-time RN results. mrn SOCS-1 (), T-bet (b) expression level of different vitmin 6 diet mice splenocytes fter Con stimultion. 5 μg/ml Con ws dded to stimulte the splenocyte, while the control received the sme volume of RMPI164 medium. fter 24 h of incubtion, mrn ws extrcted nd reversed. Ech column represents the men ± SD (n = 7) with three independent experiments. Columns mrked with different letters re significntly different (P <.5) mong different diet mouse. by vitmin 6 deficiency, lthough, t the end of feeding, no significntdifferenceinfinlweightmongllgroupsws observed (P >.5), which greed with the result obtined in rts by Miller et l. [3]. Vitmin 6 is cofctor in enzymtic rections involved in tryptophn metbolism, nd X is one of the metbolites. The suboptiml vitmin 6 deficiency could cuse ccumultion of X in plsm nd urine [22, 31, 32]. PLP is the ctive form of vitmin 6 in vivo. PLP nd X re usully considered s biomrkers to estimte the vitmin 6 biovilbility in plsm to evlute vitmin 6 sttus in orgnisms. The plsm PLP nd the significntly highest level of X were detected in the groups. Correltion nlysis lso showed tht the plsm PLP level ws negtively correlted to plsm X level with Person correltion coefficient of.618 (Tble 3). So, it indicted tht the vitmin 6- deficient mouse model ws successfully estblished, which ws in greement with the prior reserches [2, 31, 32]. The vitmin 6 deficiency could slightly reduce lymphocyte prolifertion cpcity. There ws wek correltion between PLP nd SI with Person correltion coefficient of.283, greeing with the study by Willis-Crr tht vitmin 6-deficient diet could reduce the number of T lymphocytes duetonimpiredimmunefunctioninresponsetocon stimultion [33]. Cheng et l. reported reltively wek correltion between plsm PLP level nd immune cells, nd 1 mg Vitmin 6 dily supplementtion did not show more improvement of immune function in severely ill ptients thn the 5 mg vitmin 6 supplementtion dily [34]. Sun lsofoundthtmorethn7mgvitmin6/kgofbody weight of vitmin 6 supplementtion cnnot improve the prolifertion cpcity of lymphocytes in mice nymore but rther suppressed it [35]. We lso found tht supplement of vitmin 6 with norml diet doses could significntly recover the lymphocyte prolifertion cpcity (P <.5), but the excessive supplementtion did not (P >.5). It indicted tht supplementtion of vitmin 6 cn recover the impired immunity cused by short-term vitmin 6 deficiency, but the excessive supplementtion might not do much better. The supplementtion of vitmin 6 could ffect the differentition of immture T cells to mture T cells [7, 36], nd it could increse immune responsiveness of T cells but not tht of cells [33]. CD3+ plys role by prticipting in the ssembly nd stbility of the T cell receptor/cd3 complex on mture T lymphocyte nd trnsducing the immune signl [37, 38]. Therefore, no significnt reduction of CD3 in groups indicted tht vitmin 6 deficiency would not impir the immune signl trnsduction bility significntly.however,significntdecreseofcd3in group encountering Con stimultion indicted tht the immune responsiveness of mice reduced (P <.5) (Figure 3()), suggesting tht vitmin 6 plys n importnt role in cellmedited immunity. Menwhile, the rtio of CD4/CD8, mrker of immune dysfunction [39], in group ws significntly less thn tht in group (P <.5) (Figure 3(b)). CD4+ plys n importnt role in regulting immune functions, nd CD8+ is minly responsible for removloftrgetcellsbykillingthemdirectly[4].therefore, it lso suggested tht vitmin 6 deficiency cused declineinimmunomodultoryctivityencounteringcon stimultion nd 5-week supplement of vitmin 6 did not significntly recover it (P >.5). It lso indicted tht the supplementtion of vitmin 6 could recover from thedeclinedimmunitycusedbyshort-termvitmin6 deficiency but not completely. CD4+ T-helper cells plyed n importnt role in regulting immune functions. It cn be induced into two types of T-helper cells: Th1 nd Th2, which cn ctivte cytokine

9 Journl of Immunology Reserch 9 Tble 3: Correltions between plsm PLP level in mice nd indices of immune responses. () PLP X SI CD4 CD8 CD4/CD8 IL-2 IL-4 IFN-γ SOCS-1 T-bet PLP 1. X SI CD CD CD4/CD IL IL IFN-γ SOCS T-bet (b) PLP X SI ΔCD4 ΔCD8 ΔCD4/ΔCD8 ΔIL-2 ΔIL-4 ΔIFN-γ ΔSOCS-1 ΔT-bet PLP 1. X SI ΔCD ΔCD ΔCD4/ΔCD ΔIL ΔIL ΔIFN-γ ΔSOCS ΔT-bet The correltionswereclcultedsperson scorreltion coefficientmtrix. CD, clusterofdifferentition; IFN-γ, interferon gmm; IL-2, interleukin-2; IL-4, interleukin-4; PLP, pyridoxl 5 -phosphte; SOCS-1, suppressor of cytokine signling 1; T-bet, T-box trnscription fctor; X, xnthurenic cid; Δ mens the indictor ws detected fter Con stimultion. P <.5. P <.1. IL-2 nd IL-4, respectively [41 43]. esides being produced predominntly by nturl killer (NK) nd nturl killer T (NKT) cells s prt of the innte immune response, IFN- γ is lso produced by CD4+ Th1 nd CD8+ cytotoxic T lymphocyte (CTL) nd effector T cells once ntigen-specific immunity develops [44]. With Con stimultion, vitmin 6 supplementtion with norml diet dose did not recover IL-2 secretion. Excessive vitmin 6 supplementtion did not promote the IL-2 secretion much more thn norml diet either. It greed with the report by Doke et l. tht vitmin 6-depletion decresed IL-2 secretion nd vitmin 6-repletion with dose of 7. mg/1 g diet did not recover its secretion level more thn vitmin 6-repletion with dose.7 mg/1 g diet [15]. study conducted in elderly dults lso showed tht IL-2 production is significntly reduced by vitmin -6 depletion (61% decrese), while the norml dose of vitmin 6 supplementtion could not reverse tht chnge ndil-2productioninonlythreeofthesubjectsincresed by consuming lrge mounts of vitmin -6 during the lst 4 dys of the reserch [36]. IL-4 production of lymphocytes in vitmin 6 deficient mice ws promoted more thn tht in vitmin 6 norml mice, which exhibited trend similr to tht obtined by Doke et l. [15]. The vitmin 6 deficiency did not chnge IFN-γ production, which ws lso described in study by Kjeldby et l. [8]. They found tht vitmin 6 deficiency vi the feeding of 4-deoxypyridoxine, potent ntgonist of vitmin 6 coenzyme, does not impir the production of IFN-γ in mice infected with Trichinell spirlis [8]. Collectively, these results demonstrted tht norml levels of vitmin 6 re enough to meet the requirement of communiction between immune cells, especilly in Th-1 response. Jnus tyrosine kinse/signl trnsducer nd ctivtor oftrnscription(jk-stt)signlingpthwyexistsin lmost ll cytokine signling pthwys, through which some cytokines conduct the immunomodultion [18]. Cytokines cn induce the signling cscdes simultneously nd there is n ntgonistic system to terminte or ttenute the signl to rech homeostsis. The suppressor of cytokine signling (SOCS) cn suppress the cytokine signl trnsduction

10 1 Journl of Immunology Reserch through the JK/STT signling pthwy [19]. Mrine et l. [2] stted tht SOCS-1 my inhibit the differentition of cells in which IFN-γ ws expressed or terminte the IFN-γ signl trnsduction. In this study, without Con stimultion, the trnscriptionl levels of SOCS-1 negtively correlted with IFN-γ expression levels, with correltion coefficient of.712. fter Con stimultion, it lso exhibited negtive correltion between the trnscriptionl level of SOCS-1 nd IFN-γ expression level, with the correltion coefficient of.758. lso, the trnscriptionl levels of SOCS- 1 were positively correlted with the percentge of CD4+ T- helper cells, with the correltion coefficient of.651, which mightbeduetothefctthttheifn-γ level showed negtive correltion with the percentge of CD4+ T-helper cells, with the correltion coefficient of.972. Therefore, it my be concluded tht SOCS1 inhibited the differentition of cells to influence IFN-γ expression before encountering n immunostimultion, but it would suppress IFN-γ expression levels to terminte its signl trnsduction fter stimultion, which supported Mrine et l. s hypothesis [2]. dditionlly, positive correltion ws exhibited with IFN-γ expressed in CD4+ Th1 of the group nd groups, suggestingthtt-betctedinnupregultionroleincytokinesinduced signling [45]. IFN-γ, potent ctivtor of cell-medited immunity, could ctivte the JK-STT signling pthwy through interction with the cytokine receptor, resulting in the induction of SOCS1 nd T-bet [46]. SOCS1 cn bind to the JKs using its SH2 domin nd inhibit its ctlytic ctivity of inducing STT1 phosphoryltion to inhibit signling. Nevertheless, T-bet, member of the T-box fmily of trnscription fctors, hs been shown to promote TH1 development nd IFN-γ production [45]. lthough T-bet cn be expressed in ll these cell types, it ws more prominent in CD4+ s it ws required for control of IFN-γ production in CD4+ cells, which is not needed in CD8 cells [45, 46]. Hence, it is understndble tht the CD8+ cells did not chnge much; menwhile the IFN-γ incresed fter Con stimultion (Figures 3(c) nd 4(c)). Tken together, the results showed tht vitmin 6 deficiency wekened the immune response through ffecting T lymphocyte differentition nd prolifertion nd IFN-γ expression nd further ffecting SOCS1 nd T-bet trnscription, which were involved in JK/STT signling pthwy. 5. Conclusions This study successfully estblished vitmin 6 deficiency nd recovery using L/c mouse model. Results demonstrted tht vitmin 6 deficiency influenced the immune system in three wys: (1) downregultion of SOCS-1 expression, s well s upregultion of T-bet expression, (2) suppression of T lymphocyte differentition, nd (3) decresed levels of IL-2 secretion nd incresed levels of IL-4 secretion. pproprite supplementtion of vitmin 6 could recover the impired immunity cused by short-term vitmin 6 deficiency. Further studies re needed to deeply investigte the effects of vitmin 6 deficiency on JK/STT signling pthwy. Competing Interests The uthors declre tht they hve no competing interests. cknowledgments The uthors would like to thnk Professor Zho Ynyun t Oregon Stte University for her generous ssistnce nd editingskillsswellstheinstrumentlnlysiscenterof Shnghi Jio Tong University for nlysis of PLP nd X, Professor Yu Yn s Lb for the cell culture, nd Lv Yingfng for the mice mngement. This study ws finncilly supported by the Shnghi Municipl Nturl Science Foundtion (Grnt no. 11ZR14162). 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