immunomodulatory effects of Saccharomyces cerevisiae Fermentation Product

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1 Originl Reserch pulished: 13 Mrch 17 doi: /fvets immunomodultory effects of Scchromyces cerevisie Fermenttion Product supplementtion on immune gene expression nd lymphocyte Distriution in immune Orgns in Broilers Wen K. Chou 1, Jungwoo Prk 1, John B. Crey 1 *, Don R. McIntyre nd Luc R. Berghmn 1,3 1 Deprtment of Poultry Science, Texs A&M University, College Sttion, TX, USA, Dimond V Mills, Cedr Rpids, IA, USA, 3 Deprtment of Veterinry Pthoiology, Texs A&M University, College Sttion, TX, USA Edited y: Pul Wigley, University of Liverpool, UK Reviewed y: Sherry Lyton, Vetnco, Argentin Christi Swggerty, United Sttes Deprtment of Agriculture, USA *Correspondence: John B. Crey jcrey@poultry.tmu.edu Specilty section: This rticle ws sumitted to Veterinry Infectious Diseses, section of the journl Frontiers in Veterinry Science Received: 1 Decemer 16 Accepted: 7 Ferury 17 Pulished: 13 Mrch 17 Cittion: Chou WK, Prk J, Crey JB, McIntyre DR nd Berghmn LR (17) Immunomodultory Effects of Scchromyces cerevisie Fermenttion Product Supplementtion on Immune Gene Expression nd Lymphocyte Distriution in Immune Orgns in Broilers. Front. Vet. Sci. 4:37. doi: /fvets A study ws conducted to evlute the moleculr nd cellulr immunomodultory effects of Scchromyces cerevisie fermenttion product (Originl XPC, Dimond V) in roilers. Our l hs previously demonstrted tht roilers fed XPC generte fster nd stronger ntigen-specific humorl immune responses to Newcstle disese virus (NDV) vccintion. This study ims t investigting the mechnism ehind this incresed immunocompetence. One-dy-old roilers were rndomly ssigned to one of two tretments: 1.5 kg/ton S. cerevisie fermenttion product (XPC tretment group) or control diet. Birds were vccinted ginst NDV on dy 1 (B1 strin) nd dy 1 (LSot strin) post-htch. Innte nd dptive immune-relted gene expression profiles in centrl (thymus nd urs of Fricius) nd peripherl (spleen) immune orgns were investigted t 14 nd 8 dys of ge y qpcr rry. Fold chnges lrger thn 1. (P <.5) etween treted nd control were considered significnt. Lymphocyte supopultions in centrl nd peripherl immune orgns nd lood leukocytes were nlyzed y flow cytometry t 14, 1, 8, nd 4 dys of ge. In the spleen, Th1 immune responses nd ntivirl genes, such s IFN-γ, nd its downstrem genes signl trnsducer nd ctivtor of trnscription (STAT4) nd NFκB, were significntly upregulted in the treted group y 14 dys of ge. In the thymus, genes elonging to different functionl groups were influenced t different time points. Cytokine genes ssocited with lymphocyte mturtion, differentition, nd prolifertion, such s IL-1R, IL-, nd IL-15 were significntly upregulted in the treted group y 8 dys of ge. Genes preferentilly expressed in the medull of the thymus nd mture thymocytes, such s Myxovirus resistnce gene 1, interferon regultory fctor-1, interferon regultory fctor-7, nd STAT1, were upregulted in the irds supplemented with XPC. Birds supplemented with XPC hd significntly higher percentges of CD3 +, CD4 +, nd CD8 + T-cells in the thymus t dy 8 of ge, indicting production of more Frontiers in Veterinry Science 1

2 mture T-cells, which ws consistent with gene expression results. Results suggest tht XPC supplementtion primes roilers to ecome more immunocompetent, without compromising growth performnce. Keywords: Scchromyces cerevisie fermenttion product, immunomodultion, qpcr, spleen, thymus INTRODUCTION Over the pst five decdes, production efficiency including rpid growth, superior feed conversion, nd high processing yield in the poultry industry hve incresed drmticlly due to genetic selection (1, ). Unfortuntely, genetic selection for performnce trits my result in negtive impcts on immunity (1, 3). In the modern poultry industry, intensive production nd confined housing conditions increse the risks of exposure to pthogens nd stress fctors (4). Therefore, severl tools, including vccintion, iosecurity, ntiiotic growth promoters, nd nutritionl immunomodultors, hve een developed in order to mintin the lnce etween growth performnce nd the niml s helth nd welfre. Recently, concerns over development of ntimicroil resistnce from nimls to humns led to chnges in legisltion regrding the use of ntiiotics in poultry diets. Removl of ntiiotic growth promoters hs ecome desirle in light of evolving consumer demnd. Thus, efforts hve een focused on development of nturl immunomodultors. The chrcteristics of nturl immunomodultors hve een discussed in detil (4, 5). Recently, the immunologicl effects of Scchromyces cerevisie fermenttion product (Originl XPC, Dimond V; referred s XPC in the following context) hve een reported in severl studies. Go et l. (6) reported tht supplementtion with XPC not only resulted in etter verge dily weight gin nd feed conversion rtes through the improvement of intestinl morphology ut lso stimulted oth innte nd humorl immunity. They demonstrted serum lysozyme nd ntigen-specific ntiody titers significntly incresed when roiler diets were supplemented with XPC. In the follow-up study, Go et l. (7) further reported roilers whose diets were supplemented with XPC were le to recover ody weight lost due to Eimeri tenell infection. XPC supplementtion lso incresed T-cell repertoires in the spleen nd peripherl lood, indicting n upregultion of cellulr immunity in the XPC supplemented roilers. Similrly, Lensing et l. (8) report supplementtion with XPC decresed the severity of lesions fter Eimeri mxim chllenge. Despite the lck of evidence indicting direct nticoccidin effect of dietry XPC, it hs een suggested tht XPC cn help lnce the immune system of roilers in n indirect mnner. Supplementtion with XPC lso prevented sorption of fltoxin in the intestinl trct of chickens (9). Tken together, these studies suggest tht XPC supplementtion is le to lnce nd modulte the immune system without compromising growth performnce in roilers. Our previous study lso demonstrted tht dietry supplementtion with XPC in roilers not only significntly improved feed conversion rte ut lso ccelerted the estlishment of Newcstle disese virus (NDV)-specific humorl immune response when compred with non-supplemented roilers (1). While the eneficil effects of XPC supplementtion on roiler immunity hve een convincingly documented, the impct of supplementtion on the immune cell repertoire nd gene expression ptterns in immune orgns of roilers, especilly fter chllenge, hs yet to e ddressed. In the current mnuscript, we further discuss the effect of dietry XPC on cell-medited immunity (CMI) y mesuring the lymphocyte repertoire in immune orgns (urs, thymus, nd spleen) nd peripherl lood on 14, 1, 8, nd 4 dys of ge using four-color flow cytometry. Also, gene expression profiling in immune orgns (urs, thymus, nd spleen) ws performed t 14 nd 8 dys of ge using the innte nd dptive immune response RT Profiler PCR rry (SABiosciences, Frederick, MD, USA). This rry profiles 84 immune-relted genes functionlly grouped into innte, dptive, humorl immunity, inflmmtory response, nd defense response ginst cteri nd viruses, which provides comprehensive overview of the effects of XPC supplementtion on n extensive rry of the irds immune prmeters upon chllenge with live-ttenuted NDV vccine. MATERIALS AND METHODS Experimentl Design One hundred fifty roilers (Ross 78, Avigen, Huntsville, AL, USA) were rndomly ssigned into control nd treted groups t 1 dy of ge. The dietry tretments consisted of (Control) nd 1.5 kg/metric ton of S. cerevisie fermenttion product (XPC; Originl XPC, Dimond V, Cedr Rpids, IA, USA). Strter, grower, nd finisher complete diets (Tle 1) were mnufctured t the Texs A&M University Poultry Reserch, Teching nd Extension Center. All the irds were vccinted with 3 μl of live-ttenuted NDV B1 strin vccine (Meril, Duluth, GA, USA) on dy 1 of ge nd oosted with live-ttenuted NDV LSot strin vccine (Meril, Duluth, GA, USA) on 1 dys of ge vi eye drop. In order to ssess the effects of XPC on roiler CMI, immune orgns nd peripherl lood mononucler cells (PBMC) were collected every 7 dys (except for 35 dys of ge) from s erly s 14 to 4 dys of ge (the end point of the experiment). In ddition, gene expression profiling of immune orgns ws performed t 14 nd 8 dys of ge. All reserch on nimls reported in this mnuscript ws pproved y the Institutionl Animl Cre nd Use Committee of Texs A&M University. Smple Collection In order to evlute the effect of dietry XPC on the lymphocyte repertoire in roilers, five rndomly selected chickens from ech group were euthnized nd immune orgns (urs, thymus, spleen, nd lood) were hrvested t 14, 1, 8, nd 4 dys of ge. Hlf of ech orgn ws processed for further lymphocyte isoltion, Frontiers in Veterinry Science

3 Tle 1 Experimentl diets nd nutrient composition. Strter ( 14 dys) Grower (15 8 dys) Finisher (9 4 dys) Ingredients, % Corn, yellow grin Soyen mel, dehulled solvent Bio Phos 16/1 P Limestone Slt, plin Minerl mix dl-methionine Lysine Vitmin premix Ft, A/V lend Originl XPC c Nutrient composition Crude protein, % ME, kcl/kg 3,48 3,55 3,151 Crude ft, % Lysine, % Methionine (M), % M + Cysteine, % Tryptophn, %.6.3. Threonine, % Arginine, % Vline, % Clcium, % Aville phosphorus, % Sodium, %..1. Chloride, % Trce minerl premix dded t this rte yields mg mngnese, 55. mg zinc, 6.4 mg iron, 4.4 mg copper, 1.5 mg iodine,.5 mg selenium, minimum of 6.7 mg clcium, nd mximum of 8.69 mg clcium per kilogrm of diet. The crrier is clcium cronte, nd the premix contins less thn 1% minerl oil. Vitmin premix dded t this rte yields 11,3 IU vitmin A, 3,858 IU vitmin D3, 46 IU vitmin E,.165 mg B1, mg rioflvin, mg nicin,.1 mg d- pntothenic cid, mg choline, 1.47 mg mendione, 1.75 mg folic cid, 7.17 mg pyroxidine,.94 mg thimine,.55 mg iotin per kilogrm diet. The crrier is ground rice hulls. c Dimond V Originl XPC, Cedr Rpids, IA, USA. while the other hlf ws stored in RNAlter (Life Technologies, Inc., Crlsd, CA, USA) for gene expression profiling (1 mg tissue smple to 1 ml stiliztion solution rtio). RNAlter immersed smples were stored t C until further nlysis. Single Cell Suspension Preprtion nd Lymphocyte Isoltion Lymphocytes from spleen, urs, nd thymus were prepred ccording to previous pulished protocols with minor modifictions (11). Briefly, freshly isolted immune orgns were pssed through 7 μm mesh cell striners nd lymphocytes, including mononucler cells, were isolted y density grdient sedimenttion using Ficoll-Hypque (Histopque-177, Sigm-Aldrich, St. Louis, MO, USA). Isolted single cell suspensions were kept on ice until they were immunofluorescently stined for surfce mrkers. Similr procedures were pplied to PBMC isoltion (11). Briefly, 3 ml of peripherl lood ws collected from the rchil vein in sodium citrte-contining tues. PBMC were further isolted y density grdient sedimenttion method s previously descried. Isolted PBMC were kept on ice until immunofluorescent stining ws performed. Lymphocyte Suset Anlysis y Flow Cytometry All the primry monoclonl ntiodies were purchsed from Southern Biotech (Birminghm, AL, USA). Freshly prepred single cell suspensions were treted with 5 mg/ml of mouse whole molecule IgG (Jckson Immunoreserch, West Grove, PA, USA) on ice for 3 min to void non-specific Fc receptor inding. Fourcolor immunostining with FITC-conjugted-nti-CD3 (.5 mg/ ml), R-PE-conjugted nti-cd4 (.1 mg/ml), Cy5-conjugted nti-cd8 (.1 mg/ml), nd iotinylted Bu-1 (.5 mg/ml) were dpted for the current study. Single-color stining ws used for compenstion of overlpping spectr. After stining with dye-conjugted primry ntiodies, ll smples were centrifuged t 1,5 g for 5 min t 4 C nd wshed three times with fluorescence-ctivted cell sorting (FACS) uffer (1% FBS nd.1% sodium zide in PBS, ph 7.4). After wshing,.5 μg/ml of Pc- Blue conjugted streptvidin (Jckson ImmunoReserch) ws dded nd incuted in the drk for 15 min t 4 C, then wshed times with FACS uffer. The stined smples were re-suspended with FACS uffer nd fixed with % (w/v) phosphte-uffered prformldehyde. Dt were cquired on BD FACSAri II cell sorter system (BD Biosciences, Sn Jose, CA, USA) nd nlyzed with FlowJo version softwre (Tree Str, Ashlnd, OR, USA). Results were nlyzed y performing the Student s t-test etween control nd XPC group using JMP softwre (SAS Institute, Cry, NC, USA). Sttisticl significnce ws determined t P <.5. Gene Expression Anlysis y Quntittive PCR Arry Totl RNA ws extrcted from urs, thymus, nd spleen using the Trizol method ccording to mnufcturer s instructions. Totl RNA smples were then converted into cdna using the RT First Strnd Kit (SABiosciences, Frederick, MD, USA). cdna ws then dded to the RT SYBR Green Mster Mix (SABiosciences, Frederick, MD, USA). All mixtures were pplied on the Chicken Innte & Adptive Immune Responses RT Profiler PCR Arry (SABiosciences, Frederick, MD, USA). All steps were done ccording to mnufcturer s instructions for the ABI 79HT Fst Rel Time PCR System. The dt were nlyzed using Excel-sed PCR rry Dt Anlysis Templtes. Gene expression differences etween control nd XPC group with P-vlue <.5 nd Fold Chnge (FC) > 1. were considered s significntly influenced y XPC supplementtion. RESULTS The Effect of S. cerevisie Fermenttion Product Supplementtion on Cellulr Immunity Prmeters: T- nd B-Cell Repertoire in Immune Orgns As shown in Figure 1, XPC supplementtion significntly (P <.5) incresed the T-cell repertoire (CD3 +, CD4 +, nd CD8 + T-cells) in the thymus t 8 dys of ge, 7 dys post-ndv vccine Frontiers in Veterinry Science 3

4 A 8 % CD3+ in the thymus C dy dy 14 14dy dy 1 1dy dy 8 8dy dy 4 4 Control XPC B D Figure 1 Lymphocyte supopultions in thymus t 14, 1, 8, nd 4 dys of ge. Percentge of (A) CD3 + ; (B) CD4 + ; (C) CD8 + ; (D) Bu-1 + lymphocytes in control (drk column) nd XPC-supplemented (gry column) roilers (1.5 kg/ton; Dimond V Originl XPC, Cedr Rpids, IA, USA). n = 5 per tretment per dy. Dt re presented s group mens ± SEM. Different letters indicte significnt differences (P <.5). oost. However, t 1 nd 4 dys of ge, only numericlly higher percentges of CD3 +, CD4 +, nd CD8 + T-cells were oserved in the thymus of XPC supplemented roilers (Figure 1). The effect of dietry XPC on lymphocyte supopultions in peripherl lood ws not significnt (Figure ). However, the proportionl chnge in T-cell mrker (CD3 nd CD4) stining ptterns mirrors NDV-specific ntiody titers (1) tht were mesured in the sme irds. The proportion of positive stining T-cells drmticlly incresed (P <.5) t 14 nd 8 dys of ge nd decresed t 1 nd 4 dys of ge. In the spleen, dietry XPC did not significntly chnge T-cell supopultions. As shown in Figure 3, irds supplemented with XPC hd significntly (P <.5) higher percentge of CD8 + T-cells positive stining t 1 dys of ge. The numer of Bu-1 + B-cells ws significntly (P <.5) higher in the spleen of irds supplemented with XPC t 14 dys of ge. As shown in Figure 4, the supplemented roilers hd significntly (P <.5) higher percentge of CD4 + nd CD8 + T-cells t 1 dys of ge in the urs. The numer of Bu-1 + B-cells in the urs ws lso significntly (P <.5) higher t 4 dys of ge. Gene Expression Anlysis Using Quntittive PCR Arry In order to mesure the effects of XPC feed dditive on the host response to live NDV vccine strin in vivo, qpcr rry covering 84 immune-ssocited genes ws performed. Tles 4 summrize the significntly modulted (P-vlue <.5 nd FC > 1.) genes in the thymus, spleen, nd urs of Fricius of the controls nd the XPC-supplemented irds. As shown in Figure 5, immune-ssocited gene expression ptterns were different etween centrl nd peripherl immune orgns. At 14 dys of ge, the spleen ws the mjor immune orgn tht ws significntly modulted y XPC. There were 14 genes significntly (P <.5) modulted in the spleen. On the other hnd, only two nd three genes were significntly (P <.5) modulted in the urs nd thymus, respectively (Figure 5). Interestingly, t 8 dys of ge (1 week post-oost of the vccintion), the pttern of gene expression chnged entirely. Thymus ecme the mjor immune orgn tht ws significntly modulted y XPC supplemented. Seventeen genes were significntly (P <.5) modulted in the thymus. However, only nd 4 genes were significntly (P <.5) regulted in the spleen nd urs, respectively (Figure 5). Genes Differentilly Expressed in the Immune Orgns of XPC-Supplemented Broilers t 14 Dys of At 14 dys of ge, weeks fter the primry NDV vccintion, only three nd two genes were significntly (P <.5) modulted in the thymus nd urs of Fricius of the XPC group when Frontiers in Veterinry Science 4

5 A %CD4+ in the lood B 8 Dy 14 Dy 1 Dy 8 Dy 4 C 8 % of CD8+ in the lood Dy 14 Dy 1 Dy 8 Dy 4 D % Bu-1+ in the lood 8 Dy 14 Dy 1 Dy 8 Dy 4 Figure Lymphocyte supopultions in lood t 14, 1, 8, nd 4 dys of ge. Percentge of (A) CD3 + ; (B) CD4 + ; (C) CD8 + ; (D) Bu-1 + lymphocytes in control (drk column) nd XPC-supplemented (gry column) roilers (1.5 kg/ton; Dimond V Originl XPC, Cedr Rpids, IA, USA). n = 5 per tretment per dy. Dt re presented s group mens ± SEM. Different letters indicte significnt differences (P <.5). compred with control groups, respectively (Tles nd 4). The most ffected upregulted gene in the thymus t 14 dys of ge ws Integrin β (ITGB) (Tle ). Conversely, two genes, mitogen-ctivted protein kinse (MAP kinse) nd myeloid differentition primry response gene (MyD88), were downregulted in the thymus t 14 dys of ge (Tle ). As shown in Tle 3, our results reveled 14 genes in the spleen tht were significntly (P <.5) ffected in the treted group on 14 dys of ge, which involved three different functionl ctegories: innte immunity, ntivirl, nd pro-inflmmtory. The most ffected gene ffected y XPC supplementtion, complement component 3 (C3), ws sevenfold upregulted over the control group. C3 lso plys role in B-cell ctivtion nd is thought to fcilitte dptive immunity (1, 13). Severl germline-encoded innte immune sensors re mong the significntly modified genes of the XPC group, including toll-like receptors (TLRs), interferon induced with helicse C domin 1 (IFIH1), nd nucleotide-inding oligomeriztion domin contining 1 (NOD1) (Tle 3). IFN-stimulted genes nd trnscription fctors, such s interferon (lph, et, nd omeg) receptor 1 (IFNAR1), IFN-γ, STAT4, nd NFκB, were lso upregulted in the spleen of XPC-supplemented roilers. Two genes involved in poptosis, Cspse 8 nd poptosisrelted cysteine peptidse (CASP8), nd Fs TNF receptor superfmily, memer 6 (FAS) were lso upregulted in the spleen of the XPC group t 14 dys of ge. Gene of cytokine IL-17, produced y T-helper (T h) 17 cells, ws lso significntly upregulted in the spleen of XPC-supplemented roilers t 14 dys of ge (Tle 3). Genes Differentilly Expressed in Immune Orgns of XPC Supplemented Broilers t 8 Dys of At dy 8 of ge (1 week post-oost of the vccintion), 17 (15 up- nd downregulted) nd 4 ( up- nd downregulted) genes were differentilly expressed (P <.5) in the thymus nd urs of Fricius, respectively, in XPC-supplemented roilers when compred with control (Tles nd 4). As shown in Tle, severl IFNs, IFN-stimulted genes, nd trnscription fctors [such s IFIH1, IFNAR1, Myxovirus resistnce 1 (Mx1), Interferon regultory fctor (IRF) 1, IRF7, nd STAT1] were significntly (P <.5) upregulted in the thymus of the XPC group t 8 dys of ges. Severl cytokines tht contriute to lymphocyte prolifertion nd differentition (including IL-1R1, IL-, IL-1, nd IL-15) were lso significntly (P <.5) upregulted in the thymus of roilers supplemented with dietry XPC t 8 dys of ge (Tle ). Two genes [Chemokine (C X C motif) lignd 1 (stroml cell-derived fctor 1) CXCL1 nd CD14] were significntly (P <.5) downregulted in the thymus of the XPC group t 8 dys of ge when compred with the control group. CXCL1 is chemokine produced y stroml cells nd plys pivotl role in the migrtion nd locliztion of developing T-cells within different zones of the thymus (Tle ). DISCUSSION In the current study, the live-ttenuted NDV vccintion model on roilers ws dopted. The effect of S. cerevisie fermenttion Frontiers in Veterinry Science 5

6 A 8 %CD3+ in the spleen C dy 14 dy 1 dy 8 dy 4 Con XPC B 8 %CD4+ in the spleen D 8 dy 14 dy 1 dy 8 dy 4 % CD8+ in the spleen 8 dy 14 dy 1 dy 8 dy 4 Control XPC %Bu-1+ in the spleen dy 14 dy 1 dy 8 dy 4 Figure 3 Lymphocyte supopultions in spleen t 14, 1, 8, nd 4 dys of ge. Percentge of (A) CD3 + ; (B) CD4 + ; (C) CD8 + ; (D) Bu-1 + lymphocytes in control (drk column) nd XPC-supplemented (gry column) roilers (1.5 kg/ton; Dimond V Originl XPC, Cedr Rpids, IA, USA). n = 5 per tretment per dy. Dt re presented s group mens ± SEM. Different letters indicte significnt differences (P <.5). product (Originl XPC, Dimond V) s nturl immunomodultor on centrl nd peripherl immune orgns ws evluted t the cellulr nd moleculr levels. The differentilly expressed genes (P <.5) t 14 nd 8 dys of ge were grouped into different functionl groups. Interestingly, severl distinct modes of ction were oserved. Severl germline-encoded innte immune sensors were ffected y XPC supplementtion. Pthogens tht invde host re initilly recognized y the innte immune system through germlineencoded pttern recognition receptor (PRRs). TLRs, NOD-like receptors, nd RIG-I-like proteins (RLRs) re mjor clsses of PRRs with key roles in immune homeostsis nd defense ginst infections (14 16). Stimultion of PRRs y pthogen-ssocited moleculr ptterns plys crucil role in shping the profile of the susequent dptive immune responses. In the current study, our results reveled tht weeks post primry vccintion with the NDV B1 strin, stronger expression of TLR-, NOD-1, nd IFIH-1 ws mesured in the spleen of XPC-supplemented roilers. Yitrek et l. (17) reported roilers fed.% yest-derived mcromolecule supplements showed TLR nd TLR4 gene upregultion in the spleen t 4 dys of ge when compred with control. Our current findings revel tht when chllenged with live-ttenuted NDV vccine, roilers fed XPC significntly upregulted TLR, ut not TLR4. This inconsistency could result from the NDV vccine chllenge. TLR3, NOD-1, nd IFIH-1 re common host sentinel proteins for NDV (18). In this study, NOD-1 nd IFIH-1 were significntly upregulted in the spleen weeks post primry vccintion in the XPC group. TLR3 ws lso upregulted despite not reching sttisticl significnce (P =.7). In the chicken, TLR is widely distriuted in the hert, liver, gizzrd, muscle, spleen, cecl tonsils, urs of Fricius, nd liver (19). TLR hs lso een detected y RT-PCR in heterophils, monocytes, mcrophges, B-cells, nd T-cells (, 1). The ligtion of TLR with vrious lignds cn trigger different immunologicl events (). In mmmls, zymosn, S. cerevisie cell wll derivte, is recognized y TLR nd induces peripherl tolerogenic responses vi induction of regultory ntigen-presenting cells [APCs; (, 3)]. Thus, the upregultion of TLR y XPC supplementtion in roilers my contriute to mintining the lnce of the immune system nd preventing innte immunity over-rection in the host. In the current study, NOD1 nd MDA-5 in the spleen were le to recognize NDV through CARD-dependent recruitment of RIP, driving ctivtion of MAP kinse nd NF-κB. These oservtions re in line with results from previous studies (18). At 14 dys of ge, oth type I (IFN-α, P =.7 nd IFN-β, P =.7) nd type II (IFN-γ) IFN responses were detected y qpcr rry in the spleen of XPC-supplemented roilers. These cytokines my prticipte in priming nturl killer (NK) cells, inducing mturtion of Th1 cells, nd virl clernce (4, 5). Frontiers in Veterinry Science 6

7 A %CD3+ in the urs C %CD8+ in the urs dy 14 dy 1 dy 8 dy 4 dy 14 dy 1 dy 8 dy 4 B D %Bu-1+ in the urs dy 14 dy 1 dy 8 dy 4 dy 14 dy 1 dy 8 dy 4 Figure 4 Lymphocyte supopultions in urs t 14, 1, 8, nd 4 dys of ge. Percentge of (A) CD3 + ; (B) CD4 + ; (C) CD8 + ; (D) Bu-1 + lymphocytes in control (drk column) nd XPC-supplemented (gry column) roilers (1.5 kg/ton; Dimond V Originl XPC, Cedr Rpids, IA, USA). n = 5 per tretment per dy. Dt re presented s group mens ± SEM. Different letters indicte significnt differences (P <.5). IFN-γ is lso involved in leukocyte ttrction, regultion of B-cell immunogloulin production, nd clss-switching (6). These results re consistent with the recent report y Yitrek et l. (17) where the uthors showed yest-derived mcromolecules significntly enhnced IFN-γ in spleen compred with non-supplemented control irds. However, the up-strem signl interferon regultory fctor-1 nd 7 were not significntly ltered in the spleen of XPC-supplemented roilers. The downstrem effectors of IFN ction include Mx1 nd MyD88, nd those genes were not consistently modulted (P >.5). We lso noticed tht IL-17, T h17 cell-produced cytokine, ws significntly upregulted in the spleen of XPC-supplemented roilers. T h17 cells re importnt in clering pthogens during host defense rections (7). IL-17 is lso fctor tht contriutes to the formtion of germinl centers of lymphoid follicles (8), which is consistent with our flow cytometry dt tht XPC-supplemented roilers hve higher percentges of Bu-1 + B-cells in the spleen t 14 dys of ge. These results suggest tht XPC-supplemented roilers hve higher seline expression of IFNs nd cn keep the innte immune system in stnd-y mode. As consequence, supplemented roilers re cple of responding to stimuli more rpidly thn their non-treted counterprts. In our previous study (1), XPC supplementtion ws le to estlish roust NDV-specific humorl immunity ginst n NDV vccine chllenge t 8 dys of ge, which is 1 week erlier thn control irds. Since the spleen is the most importnt peripherl lymphoid orgn in chickens, we expected to oserve more conspicuous effects immune-ssocited genes t this time point. Interestingly, our gene expression dt reveled tht t the sme time point, more genes were ffected in the thymus nd not in the spleen. It is possile tht the timing of tht smpling point ws not optiml. Rue et l. (9) reported tht virulent NDV-CA chllenge elicited strong innte immune response within 7 h in the spleen of specific pthogen-free chicken (9). Despite roilers in the current study receiving live-ttenuted NDV LSot strin ooster, fter 7 dys the cute effects of the virl chllenge my e over. The gene expression profile in the thymus of XPC-supplemented roilers t 8 dys of ge, in spite of the fct tht no significnt IFN regultion ws oserved, showed enhnced expression of the type I IFN receptor. The ctivtion of type I IFN receptor results in the induction of IRF 1 nd 7 (P =.56) required for regultion of the downstrem interferon stimultory gene. In this study, the expression of Mx1 gene, importnt for resistnce ginst virl infection, nd STAT1 were significntly induced. IFNs re fmily of cytokines tht ply centrl role in innte immunity to viruses. In the thymus, IFNs re constitutively expressed in the medull nd hve direct impct on the development of T-cells in the thymus (16). The significntly upregulted IFN-ssocited genes my contriute Frontiers in Veterinry Science 7

8 Tle Genes significntly up- or downregulted in the thymus with supplementtion of XPC., Thymus Dy 14 Dy 8 Gene Fold regultion (XPC vs. control) P-vlue Fold regultion (XPC vs. control) P-vlue CD CD CD CXCL FAS IFIH IFNAR IL IL IL1R IL IL8L IRF ITGB Mitogen-ctivted protein kinse 8 MX Myeloid differentition primry response gene 88 PTGS STAT TLR Dimond V Originl XPC, Cedr Rpids, IA, USA. Supplemented in the diet t (control) or 1.5 kg/ton of feed. Fold regultion is the fold chnge (upregultion s positive vlue or downregultion s negtive vlue) of genes in irds supplemented with XPC compred to control. A lnk vlue indictes no differences etween the two groups of irds. to innte immune responses nd lso my shpe the repertoire of T-cells in the thymus, which is consistent with our flow cytometric dt. In the present study, severl cytokines were significntly upregulted in the thymus of XPC-supplemented roilers t 8 dy of ge. In mmmls, IL-1R expression in the thymus is restricted on immture T-cells. However, the function of IL-1R in vin species is still unknown (3). IL- is importnt for the production, function, nd homeostsis of CD4 + CD5 + T reg cells. It is lso potent T-cell growth fctor during the initition of immune responses (31, 3). IL-1 is pleiotropic cytokine. The expression of IL-1 plys criticl role in limiting immune response to pthogens, including downregultion of T h1 cytokines, MHC-molecules, nd co-stimultory molecules on mcrophges (33). Tken together, the significnt upregultion of IL- nd IL-1 in the thymus of supplemented roilers t 8 dys of ge indictes XPC promotes the developmentl production of suppressive T reg cells nd suppresses the innte immune response, thus contriuting to the proportionl, i.e., trnsient nture, of the innte immune responses induced y NDV vccine chllenge. IL-15, cytokine essentil for NK cell development, functionl mturtion nd ctivtion ws significntly upregulted in supplemented roilers. This result is in concert with previous studies showing XPC tretment induced ctivtion mrkers CD69 nd CD5 on CD3 + CD56 + NK cells in mmmls (34). CXCL1 is Tle 3 Genes significntly upregulted in the spleen with supplementtion of XPC., Spleen Dy 14 Dy 8 Gene Tle 4 Genes significntly up- or downregulted in the urs with supplementtion of XPC., Burs Dy 14 Dy 8 Gene Fold regultion (treted vs. control) Fold regultion (treted vs. control) P-vlue P-vlue Fold regultion (treted vs. control) Fold regultion (treted vs. control) P-vlue C CASP CRP FAS FASLG IFIH IFNAR IFNG IL17C LY Mitogen-ctivted protein kinse 8 MBL NFKB NOD Signl trnsducer nd ctivtor of trnscription 4 TLR Dimond V Originl XPC, Cedr Rpids, IA, USA. Supplemented in the diet t (Control) or 1.5 kg/ton of feed. Fold regultion is the fold chnge (upregultion s positive vlue or downregultion s negtive vlue) of genes in irds supplemented with XPC compred to control. A lnk vlue indictes no differences etween the two groups of irds. P-vlue CASP CD CRP.5.93 CSF JAK TLR Dimond V Originl XPC, Cedr Rpids, IA, USA. Supplemented in the diet t (control) or 1.5 kg/ton of feed. Fold regultion is the fold chnge (upregultion s positive vlue or downregultion s negtive vlue) of genes in irds supplemented with XPC compred to control. A lnk vlue indictes no differences etween the two groups of irds. produced y thymus stroml cells in the cortex nd ply pivotl role guiding the trffic of T-cell precursors during erly development (35, 36). The downregultion of CXCL1 in the XPC group indictes XPC supplement ccelerted the development of T-cells compred with the control group. This result is lso consistent with our flow cytometric dt showing significntly more CD4 + nd CD8 + T-cells in the thymus of the XPC group t 8 dys of ge. CMI is crucil fctor for development of protection nd virl clernce s well s vccine-induced protective immunity in mmmls nd chickens (37). The supopultions of T-cells, Frontiers in Veterinry Science 8

9 # of genes significntly regulted Spleen Burs Thymus Orgns Dy 14 Dy 8 Figure 5 Numer of genes significntly regulted etween XPC feed dditive group nd control in immune orgns on the host response to live Newcstle disese virus vccine strin in vivo. Immune-ssocited gene expression pttern in spleen, urs, nd thymus etween XPC nd control groups mesure y qpcr rry on dys 14 nd 8 of ge. Fold regultion more thn 1. nd P <.5 ws considered s significntly regulted. including cytokine-secreting CD4 + T-helper cells nd CD8 + cytotoxic T-cells (CTL), constitute the min components of the CMI responses. Our dt indicte tht dietry XPC incresed the repertoire of oth effector T-cells [T-helper (CD4 + ) nd Cytotoxic (CD8 + )] in the thymus 1 week fter NDV vccine chllenge. The thymus hs een shown to e sensitive immune orgn following exposure to immune toxins nd endogenous corticosteroids, especilly the cortex T-cells. An increse in lymphocyte numers in the thymus is usully response to ntigenic stimultion, nd the cell popultion is mixed in contrst to the more homogeneous neoplstic popultion. The thymus is primry lymphoid orgn, le to generte mture T-cells tht eventully colonize T-cell-dependent res in secondry lymphoid orgns, nd is essentil for peripherl T-cell renewl. Higher percentges of effector T-cells in the thymus signify more diverse T-cell repertoire to id in the host s fight ginst pthogens. Therefore, XPC-supplemented roilers re more cple of estlishing protective immunity thnks to ntigen-specific T-cell expnsion, further fcilitting susequent prolifertion nd differentition of CD4 + T h cells nd CD8 + CTL. In the current study, the output of nïve T-cells my hve contriuted to the NDV vccine-induced humorl immune response. These results re consistent with our previous report tht NDV-specific ntiody titers pek 1 week post-oost [8 dys of ge; (1)]. We expect mtured nïve T-cells in the thymus to migrte to the periphery nd to contriute to the shping of the lymphocyte distriution in circultion nd spleen. However, no differences etween supplemented roiler nd non-supplemented control were oserved in the percentge of effector T-cells nd B-cells in the circultion cross ll the experiment smpling time points. As previously mentioned, smpling 7 dys post-oost my e too lte for mesuring the dynmic chnge of lymphocyte supopultions in the circultion nd the spleen. Also, our dt show tht inclusion of XPC into the feed significntly (P <.5) incresed the percentge of Bu-1 + B-cells in the spleen t 14 dys of ge. Bu-1 mrker is expressed on erly nd mture B cells, except plsm cells (38). Plsm cells re effector B-cells tht cn secrete ntiodies. Our current study suggests tht the higher percentge of Bu-1 + B-cells in the spleen indictes XPC promotes the growth nd mturtion of B-cells in the spleen. These results re consistent with our previous finding tht supplemented roilers hve more white pulp in the spleen t 14 dys of ge (1). Furthermore, supplementtion with XPC significntly incresed the percentge of CD4 + nd CD8 + in the urs t 1 dys of ge. Also, significntly higher percentges of Bu-1 + B-cells were mesured in the supplemented group t 4 dys of ge. The urs of Fricius is unique primry lymphoid orgn where B-cell lymphopoiesis tkes plce (39). Higher B-cell prolifertion rtes were seen in the supplemented group, indicting more diverse B-cell repertoire. This might lso contriute to enhncement of the humorl immune response. Only very limited resident T-cells reside in the uninfected urs. How the inclusion of XPC ffects the T-cell popultion in the urs t 1 dys of ge nd how it contriutes to the immune response ginst NDV vccine requires further investigtion. Our current study suggests inclusion of XPC in the feed modultes more thn one spect of the coordinted immune response to NDV vccine chllenge through ctivtion of genes involved in the innte immune response nd modertion of potentilly excessive immune responses. More importntly, XPC triggers the innte immune system mildly, therey priming the defense mechnisms, thus shortening the time for estlishing the dptive immune response. ETHICS STATEMENT This study ws performed t Texs A&M University Poultry Reserch, Teching nd Extension Center (TAMUPRC) nd ws conducted under TAMU pproved niml use protocol IACUC AUTHOR CONTRIBUTIONS WC ws responsile for mjority of the l work, dt nlysis nd interprettion. JP ws the grdute student in the project nd provided ird cre nd l work s well s some dt nlysis. JC ws CO-PI nd provided project oversight, experimentl design, dt nlysis, nd interprettion. DM provided technicl detils of the product, ssisted with experimentl design. LB ws CO-PI nd provided experimentl design, dt nlysis, nd interprettion. FUNDING The uthors declre tht this study received funding from Dimond V Mills, Cedr Rpids, Iow. The funder ws not involved in the study design or collection, nlysis, or interprettion of the dt. Frontiers in Veterinry Science 9

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Poult Sci (8) 87(): doi:1.338/ps Kim IJ, You SK, Kim H, Yeh H, Shrm JM. Chrcteristics of ursl T lymphocytes induced y infectious ursl disese virus. J Virol () 74(19): doi:1.118/jvi Conflict of Interest Sttement: The uthors declre tht the reserch ws conducted in the sence of ny commercil or finncil reltionships tht could e construed s potentil conflict of interest. Copyright 17 Chou, Prk, Crey, McIntyre nd Berghmn. This is n open-ccess rticle distriuted under the terms of the Cretive Commons Attriution License (CC BY). The use, distriution or reproduction in other forums is permitted, provided the originl uthor(s) or licensor re credited nd tht the originl puliction in this journl is cited, in ccordnce with ccepted cdemic prctice. No use, distriution or reproduction is permitted which does not comply with these terms. Frontiers in Veterinry Science 1