Abstract ORIGINAL ARTICLE. Basic Study. Yaron Ilan, Ami Ben Ya'acov, Yehudit Shabbat, Svetlana Gingis-Velitski, Einat Almon, Yoseph Shaaltiel

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1 Submit Mnuscript: Help Desk: DOI: /wjg.v22.i World J Gstroenterol 216 October 21; 22(39): ISSN (print) ISSN (online) 216 Bishideng Publishing Group Inc. All rights reserved. ORIGINAL ARTICLE Bsic Study Orl dministrtion of non-bsorbble plnt cellexpressed recombinnt nti-tnf fusion protein induces immunomodultory effects nd llevites nonlcoholic stetoheptitis Yron Iln, Ami Ben Y'cov, Yehudit Shbbt, Svetln Gingis-Velitski, Eint Almon, Yoseph Shltiel Yron Iln, Ami Ben Y'cov, Yehudit Shbbt, Gstroenterology nd Liver Units, Deprtment of Medicine, Hdssh Hebrew University Medicl Center, Jeruslem IL9112, Isrel Svetln Gingis-Velitski, Eint Almon, Yoseph Shltiel, Protlix Biotherpeutics, Crmiel 21, Isrel Author contributions: All uthors contributed to the conception, experiments, writing nd finl pprovl of the mnuscript. Supported by Protlix Biotherpeutics nd The Romn-Epstein Liver Reserch Foundtion (in prt). Conflict-of-interest sttement: Iln Y is consultnt for Protlix, Gingis-Velitski S, Almon E, Shltiel Y re employees of Protlix. The study ws supported in prt by Protlix, Isrel. Open-Access: This rticle is n open-ccess rticle which ws selected by n in-house editor nd fully peer-reviewed by externl reviewers. It is distributed in ccordnce with the Cretive Commons Attribution Non Commercil (CC BY-NC 4.) license, which permits others to distribute, remix, dpt, build upon this work non-commercilly, nd license their derivtive works on different terms, provided the originl work is properly cited nd the use is non-commercil. See: licenses/by-nc/4./ Mnuscript source: Unsolicited mnuscript Correspondence to: Yron Iln, MD, Gstroenterology nd Liver Units, Deprtment of Medicine, Hdssh Hebrew University Medicl Center, Ein-Krem, POB 12, Jeruslem IL9112, Isrel. iln@hdssh.org.il Telephone: Received: My 25, 216 Peer-review strted: My 27, 216 First decision: July 13, 216 Revised: July 21, 216 Accepted: August 5, 216 Article in press: August 5, 216 Published online: October 21, 216 Abstrct AIM To evlute the immunomodultory effect of orl dministrtion of in the high-ft diet model. METHODS For 22 wk, C57BL/6 HFD-fed mice received dily orl tretments with BY-2 cells expressing recombinnt ntitumor necrosis fctor lph fusion protein (). Mice were followed for serum liver enzyme nd triglyceride levels, liver histology nd intrheptic nd systemic FACS. RESULTS The orlly dministered non-bsorbble ws biologiclly ctive. Altered distribution of CD4+CD25+FoxP3+ between the liver nd spleen nd n increse in the intrsplenic-to-intrheptic CD4+CD25+FoxP3+ rtio nd decrese in the intrsplenic-to-intrheptic CD8+CD25+FoxP3+ rtio were observed. An increse in intrheptic NKT cells nd decrese in the intrsplenic-to-intrheptic NKT rtio were noted. Assessment of the CD4-to-CD8 rtios showed sequestrtion of CD8+ lymphocytes in the liver. These effects were ssocited with decrese in serum triglyceride levels, decrese in the sprtte minotrnsferse levels, serum glucose levels, nd HOMA-IR score. A decrese in heptic triglycerides content ws observed in the high dose-treted mice. CONCLUSION Orlly dministered shows biologicl ctivity nd exerts n immunomodultory effect, lleviting liver 876 October 21, 216 Volume 22 Issue 39

2 Iln Y et l. Orl nti TNF fusion protein for heptitis dmge. The dt suggest tht my provide n orl immunotherpy for nonlcoholic stetoheptitis. Key words: Nonlcoholic stetoheptitis; Nonlcoholic ftty liver disese; Tumor necrosis lph; Enbrel; Dibetes The Author(s) 216. Published by Bishideng Publishing Group Inc. All rights reserved. Core tip: The BY-2 plnt cell-expressed recombinnt nti-tumor necrosis fctor lph (TNF) fusion protein () tht consists of the soluble form of the humn TNF receptor fused to the Fc component of humn IgG1 domin ws orlly dministered in high-ft diet model. Altered distribution of CD4+CD25+FoxP3+ nd decrese in the intrsplenic-to-intrheptic CD8+CD25+FoxP3+ rtio were observed. These effects were ssocited with decrese in serum triglyceride levels, decrese in the sprtte minotrnsferse levels, serum glucose levels, nd HOMA-IR score. A decrese in heptic triglycerides content ws observed in the high dose-treted mice. The dt suggest tht my provide n orl immunotherpy for nonlcoholic stetoheptitis. Iln Y, Ben Y'cov A, Shbbt Y, Gingis-Velitski S, Almon E, Shltiel Y. Orl dministrtion of non-bsorbble plnt cell-expressed recombinnt nti-tnf fusion protein induces immunomodultory effects nd llevites nonlcoholic stetoheptitis. World J Gstroenterol 216; 22(39): Avilble from: URL: v22/i39/876.htm DOI: i INTRODUCTION A chronic inflmmtory stte nd dysfunction of metbolic-inflmmtory signling re involved in the development of vrious spects of non-lcoholic stetoheptitis (NASH) [1,2]. Severl downstrem signling pthwys, which provide the crosstlk between inflmmtory nd metbolic signling, hve been identified. The pro-inflmmtory xis consisting of the nucler trnscription fctor NF-kpp B nd its upstrem kinse IKK-bet is one pthwy responsible for nutritionlly induced inflmmtion [3]. c-jun N-terminl kinse nd I kpp bet kinse (I kpp K) hve been identified s meditors of insulin resistnce (IR) [4,5]. These pthwys re ctivted long with other signls by tumor necrosis fctor lph (TNF-α) [6]. The importnce of TNF in humn nd niml ftty liver diseses, which were induced both by genetic mnipultion nd by overnutrition, hs been demonstrted [7]. Neutrliztion of TNF-α ctivity improves IR nd ftty liver disese in nimls. Adiponectin is potent TNF-neutrlizing nd nti-inflmmtory dipocytokine. Both in vitro nd in vivo studies showed its importnce in countercting inflmmtion nd IR [8,9]. Some of the nti-inflmmtory effects of diponectin re medited by suppression of TNF synthesis nd the promotion of nti-inflmmtory cytokines, including IL-1 nd the IL-1 receptor ntgonist [7]. The NLRP6 nd NLRP3 inflmmsomes nd the effector protein IL-18 negtively regulte NASH progression [1]. Inflmmsome deficiency-ssocited chnges in the configurtion of the gut microbiot re ssocited with excerbted heptic stetosis nd inflmmtion through influx of TLR4 nd TLR9 gonists into the portl circultion, leding to the enhnced heptic TNF-α expression tht drives NASH progression [1]. A recent met-nlysis showed difference in the TNF-α-238 genotype distribution between nonlcoholic ftty liver disese (NAFLD) ptients nd controls, suggesting tht polymorphism t position-238 is risk fctor for NAFLD [11]. Incresed gut permebility long with bcteril trnsloction (BT) nd incresed lipopolyscchride (LPS) levels hve been described in ptients with NASH [12,13]. BT t mesenteric lymph nodes leds to lymphocyte ctivtion [14]. The TNF-lph mrna expression in liver tissue is significntly higher in ptients with NASH nd correltes with the increse in the plsm levels of LPS binding protein. The development of biologicl gents tht trget TNF hve mrkedly chnged the therpeutic pproch to inflmmtory diseses [15]. Pentoxifylline, n nti- TNF-α gent ws shown to reduce sprtte minotrnsferse (AST) nd lnine minotrnsferse (ALT) levels nd to improve liver histologicl scores in ptients with NAFLD/NASH [16]. Prenterl dministrtion of recombinnt nti-tnf proteins lowers disese ctivity nd, in some ptients, induces remission. A report described NASH ptient who experienced rpid normliztion of liver biochemistry during tretment for n ssocited rheumtoid rthritis with the humnized nti-tnf-lph ntibody dlimumb [17]. Etnercept is recombinnt, dimeric, soluble tumor necrosis fctor receptor fusion protein tht blocks only soluble TNF but not membrne-bound TNF. Prenterl dministrtion of etnercept is being used for rheumtoid rthritis, juvenile rheumtoid rthritis, psoritic rthritis, psorisis, nd nkylosing spondylitis [15,18]. Prenterlly dministered TNF ntgonists re generlly well tolerted but crry risk of side effects. Ares of concern include opportunistic nd non-opportunistic infections, vccintion, neurologicl complictions, heptotoxicity, hemtologicl side effects, mlignncies, infusion rections nd utoimmunity. Contrindictions, such s hert filure nd cute infectious diseses, re lso of concern [19]. The immunosuppressive cpcity of these gents necessittes rigorous long-term sfety followup, nd the potentil risks of their use should lwys be tken into considertion [19,2]. Orl delivery of therpeutic proteins is mjor gol when developing new therpeutic modlities. The BY-2 plnt cell-expressed recombinnt nti-tnf fusion protein 8761 October 21, 216 Volume 22 Issue 39

3 Iln Y et l. Orl nti TNF fusion protein for heptitis () consisting of the soluble form of the humn TNF receptor (TNFR) fused to the Fc component of humn IgG1 domin cn be orlly dministered, nd hs n mino cid sequence tht is identicl to tht of Enbrel. The im of the present study ws to determine the immunomodultory effect of orl dministrtion of plnt cells expressing in n niml model of NASH. MATERIALS AND METHODS Animls Mle C57BL/6 mice (11-12-wk-old) were obtined from Hrln Lbortories (Jeruslem, Isrel) nd mintined in the Animl Core of the Hdssh-Hebrew University Medicl School. Mice were dministered stndrd lbortory chow nd wter d libitum nd kept on 12 h light/drk cycle. Animl experiments were crried out ccording to the guidelines of the Hebrew University- Hdssh Institutionl Committee for the Cre nd Use of Lbortory Animls nd with the committee s pprovl. Mice were fed high-ft diet (HFD, Hrln, TD88137; 42% of the clories re from ft) from dy until their scrifice t 24 wk. Experimentl groups Four groups of C57BL/6 mice, n = 1 ech, were orlly fed three times week for 24 wk with one of the following t volume of 35 μl: phosphtebuffered sline (PBS, group A), BY-2 cells t 28.8 mg of BY- (mock cells, group B), 2.88 mg (.5 μg TNF) of BY+ (group C), or 2.88 mg (1 μg nti-tnf) of BY+ (group D). Fresh preprtions were mde before ech dministrtion. Assessment of the effect of orl on the systemic immune system The immunomodultory effect of ws determined by FACS nlysis nd serum cytokines. Isoltion of splenocytes nd heptic lymphocytes: Spleens nd livers were kept in RPMI-164 supplemented with FCS. Spleens were crushed through 7 μm nylon cell striner [21] nd centrifuged (125 rpm for 7 min) to remove debris. Red blood cells were lysed with 1 ml of cold 155 mmol/l mmonium chloride lysis buffer nd immeditely centrifuged (125 rpm for 3 min). The splenocytes were then wshed nd suspended in 1 ml of RPMI + FCS. Any remining connective tissue ws removed. The vibility, s ssessed using trypn blue stining, ws bove 9%. For the isoltion of heptic lymphocytes, livers were first crushed through stinless mesh (size 6, Sigm), nd the cell suspension ws plced in 5 ml tube for 5 min so tht the cell debris could form pellet. The cell suspension ws slowly underlid with 1 ml of Lymphoprep (Ficoll, Axis-Shield PoC AS, Oslo, Norwy) in 5 ml tube. The tubes were then centrifuged t 18 rpm for 18 min. Cells t the interfce were collected nd trnsferred to new tube tht ws centrifuged gin t 18 rpm for 1 min. The resulting pellet of heptocyte-depleted lymphocytes ws suspended in finl volume of 25 μl, pproximtely intrheptic lymphocytes were recovered per mouse liver. Flow cytometry: Flow cytometry ws performed on splenocytes nd heptic lymphocytes, which were suspended in 1 ml of FACS buffer (PBS + 1% BSA +.1% sodium zide). The cells were stined with the ntibodies for CD8, CD4, CD25, Foxp3, CD3, nd NK 1.1. Flow cytometry ws performed using LSR-II, nd nd FSC express softwre. Cytokine mesurement: Serum TNF-α levels were mesured in ech niml using commercil kits, ccording to the mnufcturer s instructions (Quntikine, R&D Systems, Minnepolis, MN, United Sttes). Assessment of the effect of orl on the lipid profile, glucose intolernce nd liver dmge Liver enzymes: Serum ws obtined from individul mice. The serum AST nd ALT levels were determined using n utomtic nlyzer. Triglyceride levels in the serum were mesured using stndrd kits. Fsting serum glucose nd insulin levels were mesured on dy 1 nd t week 24. Glucose tolernce test: Glucose tolernce test (GTT) ws performed on ll nimls in ll groups t weeks 8 nd 24. Histologicl exmintion of the liver: Prffinembedded liver sections were prepred from ech mouse. The livers were cut into 4-5 μm thin slices nd stined with hemtoxylin-eosin. A blinded pthologist exmined the tissues using light microscopy to score for morphologicl nd histopthologicl chnges tht re chrcteristic of NAS. Trichome stining ws used to evlute liver fibrosis. The mximl score for stetosis (= 3) ws ssigned for greter thn 66%. The mximl score for lobulr inflmmtion (= 3) ws ssigned for > 4 foci/ 2, nd heptocyte bllooning (= 2) ws ssigned for mny cells/prominent bllooning. The mximl NAS score is simple rithmetic combintion of ll three fetures ( = 8). Heptic triglycerides content: Accumultion of intrcellulr triglycerides (TGs) within the liver ws quntified using modifiction of the Folch method. TGs were extrcted from liquots of snp-frozen livers nd then ssyed. Triglyceride determintion ws performed spectrophotometriclly using GPO-Trinder 8762 October 21, 216 Volume 22 Issue 39

4 Iln Y et l. Orl nti TNF fusion protein for heptitis kit (Sigm, Rehovot, Isrel), nd the levels were normlized to the protein content in the homogente. Sttisticl nlysis Sttisticl nlysis ws performed using Student s t test. A P vlue less thn.5 ws considered significnt. RESULTS Orl dministrtion of non-bsorbble exerted n immunomodultory effect Orl dministrtion of ltered the distribution of CD4+CD25+FoxP3+ Tregs. Figure 1A shows the significnt decrese in intrheptic Tregs in the high dose-treted mice from the 3.53% vlue in the controls to 2.48% (P <.5). Figure 1B shows the significnt effect on intrsplenic Tregs only for BY2 (-) treted mice from 18.4% to 13.73% (P <.1). A reduction trend ws noted for the high dose-treted group (15.49%). The intrsplenic-to-intrheptic CD4+CD25+FoxP3+ Treg rtio incresed significntly in the high dose PRX- 16-treted mice, s seen in Figure 1C, from 5.12 in controls to 6.25 (P <.5). The dt indicte n effect of PRX16 on the redistribution of CD4+CD25+FoxP3+ Tregs. Orl dministrtion of lso ltered the distribution of CD8+CD25+FoxP3+ Tregs. Figure 1D shows significnt increse in intrheptic CD8+CD25+FoxP3+ Tregs in the BY2 (-) nd high dosetreted mice. The levels incresed to.6% nd.86%, respectively, compred with.21% in the controls (P <.5). Figure 1E shows no sttisticlly significnt effect on intrsplenic CD8+CD25+FoxP3+ Tregs. The intrsplenic-to-intrheptic CD8+CD25+FoxP3+ Treg rtio ws significntly ltered by the tretments, s shown in Figure 1F. The rtio ws 9.8 in the controls nd decresed to 2.4 nd 4 in the BY2 (-)- nd high dose-treted mice while incresing to 16.2 in the low dose-treted mice (P <.5 compred with controls). Orl dministrtion of ltered the distribution of NKT (CD3+NK1.1+) lymphocytes. Figure 1G shows n increse in intrheptic NKT cells in the high dose-treted mice, with levels incresing 3.58% compred 2.31% in controls (P <.5). Figure 1H shows significnt decrese in the intrsplenic NKT cells in the BY2(-)-treted mice, in which the levels decresed to.42% compred with.82% in controls (P <.5). The intrsplenic-to-intrheptic NKT rtio significntly decresed in ll treted groups, s shown in Figure 1I. The levels decresed to.15,.24 nd.16 for the BY2(-)-treted nd low dose nd high dose PRX16-treted mice, respectively, compred with.35 in the controls (P <.5 vs controls). To determine the effect of tretment on lymphocyte trpping in the liver, the CD4/CD8 lymphocyte rtio ws clculted. The splenic CD4/CD8 rtio ws 1.62, 1.48, 1.56 nd 1.46 for the controls, BY2 (-)-treted nd low nd high dose PRX16-treted mice, respectively. The heptic CD4/CD8 rtio ws.73,.72,.82, nd.68 for the controls, BY2(-)- treted nd low nd high dose PRX16-treted mice, respectively. Figure 1J shows the rtio between the splenic nd heptic CD4/CD8 rtios. For ll treted groups, decrese in the rtio ws found: 2.6, 1.9, nd 2.15 for the BY2(-)-treted nd the low nd high dose PRX16-treted mice compred with 2.2 for the controls (P <.5 for low dose PRX16 compred with controls). The dt suggest tht the tretment is ssocited with sequestrtion of CD8+ lymphocytes in the liver. Orl dministrtion of ws ssocited with mild increse in serum TNF-α levels. The levels incresed to 11.53, 13.85, nd 1.25 pg/ml for BY2 (-)-treted nd low nd high dose PRX16-treted mice, respectively, compred with 7.7 pg/ml in the controls (P <.5 for low dose PRX16 compred with controls). The dt support the notion tht the observed nti-inflmmtory effect is not ssocited with reduction in TNF-α levels. Orl dministrtion of non-bsorbble exerted beneficil effect on the liver, glucose nd lipid metbolism in NASH During the 24 wk of the experiment, the weight doubled for the mice in ll groups. The verge weight gin ws 82.5%, 83.3%, 81.8%, nd 89.1% for the controls, BY2(-)-treted nd low nd high dose PRX16-treted mice, respectively (P = NS). The dt support the notion tht the beneficil effect on the liver, glucose, nd lipid metbolism ws independent of weight. Both dosges of exerted beneficil effect on the serum TGs t week 24, s shown in Figure 2A. The serum triglyceride levels decresed to 186 nd 124 mg/dl for the low nd high PRX16-treted groups compred with 26 mg/dl for untreted controls (P <.1 for high dose vs controls). Figure 2B shows tht orl dministrtion of the high dose of decresed the AST levels t week 24. The AST levels were 297 compred with 496 IU for the high dose PRX16 vs the controls, respectively (P =.6). No significnt effects on the ALT levels were observed. A beneficil effect of orl PRX16 on fsting glucose levels ws noted. At week 5, significnt decrese in serum glucose levels ws observed for the lower dosetreted PRX 16 with levels of 88, 76 nd 86 mg% for the BY2(-)-treted nd low nd high dose PRX16- treted mice, respectively, compred with 14 mg% in the controls (P <.5 for low dose vs controls). Figure 3A shows the beneficil effect of the tretments t the end of tril, with reduction in the glucose levels to 12, 13, nd 12 mg% for the BY2(-)-treted nd low nd high dose PRX16-treted mice, respectively, compred with 119 mg% in controls. Figure 3B shows the beneficil effect of the tretment on the delt of 8763 October 21, 216 Volume 22 Issue 39

5 Iln Y et l. Orl nti TNF fusion protein for heptitis A B % Heptic CD4+CD25+FoxP Sline BY2 (-) % Splenic CD4+CD25+FoxP b Sline BY2 (-) C D Splenic/heptic CD4+CD25+FoxP3+ rtio Sline BY2 (-) % Heptic CD8+CD25+FoxP Sline BY2 (-) E F % Splenic CD8+CD25+FoxP Sline BY2 (-) Splenic/heptic CD8+CD25+FoxP3+ rtio Sline BY2 (-) G H Heptic NKT cells Sline BY2 (-) Splenic NKT cells (% gted) Sline BY2 (-) 8764 October 21, 216 Volume 22 Issue 39

6 Iln Y et l. Orl nti TNF fusion protein for heptitis I J Splenic/heptic NKT cells rtio Sline BY2 (-) Splenic/heptic CD4+/CD8+ rtio Sline BY2 (-) Figure 1 FACS nlysis ws performed on lymphocytes hrvested from the spleens nd livers of ll mice from the experimentl nd control groups. The results were compred for the following subsets of cells. A: Intrheptic CD4+CD25+FoxP3+ lymphocytes; B: Intrsplenic CD4+CD25+FoxP3+ lymphocytes; C: The intrsplenic-to-intrheptic CD4+CD25+FoxP3+ rtio ws clculted; D: Intrheptic CD8+CD25+FoxP3+ lymphocytes; E: Intrsplenic CD8+CD25+FoxP3+ lymphocytes; F: The intrsplenic-to-intrheptic CD8+CD25+FoxP3+ rtio ws clculted; G: Intrheptic NKT lymphocytes; H: Intrsplenic NKT lymphocytes; I: The intrsplenic-to-intrheptic NKT rtio ws clculted; J: To determine the effect of the tretment on the lymphocyte trpping in the liver, the CD4/CD8 lymphocyte rtio ws clculted in the spleen nd in the liver. The rtio between the splenic nd heptic CD4/CD8 rtios ws clculted. P <.5, b P <.1. A B Serum TG (mg/dl) AST (IU) Sline BY2 (-) (MOCK) Sline BY2 (-) Figure 2 Serum levels of triglycerides (A) nd sprtte minotrnsferse (B) were mesured in ll mice from the experimentl nd control groups. AST: Asprtte minotrnsferse. P <.5. the increse in fsting insulin levels between week nd 24. A trend for reduction in the verge increse in insulin levels between weeks nd 24 ws noted in ll treted mice. The verge chnge in insulin levels ws 2.69,.92, nd 2.77 pg/ml for BY2 (-)-treted nd low nd high dose PRX16-treted mice, respectively, compred with 3.6 pg/ml in controls. Figure 3C shows the effect of orl PRX16 on the HOMA-IR. A reduction trend for reduction to 1.9 ws observed for mice in the low dose PRX16-treted group compred with the 1.25 vlue in the controls. No significnt differences were noted between groups in the orl GTT performed t weeks 7 nd 22 of the study. A decrese in heptic TG content ws observed in the high dose-treted mice, s shown in Figure 4. The triglyceride content ws reduced to 23.54% per g liver for the high dose -treted mice compred with 33.98% for the controls (P =.3). DISCUSSION Orlly dministered plnt cells expressing recombinnt nti-tnf fusion protein show biologicl ctivity nd exert n immunomodultory effect, lleviting the liver dmge in the HFD model. The pthogenesis of NASH involves number of immune mechnisms [22]. Immunomodultory tretments hve been suggested to ply role in lleviting the disese [1]. The results of the present study show tht orl dministrtion of non-bsorbble exerted n immunomodultory effect. Orl dministrtion of ws ssocited with n increse in the intrsplenic-to-intrheptic CD4+CD25+FoxP3+ rtio, suggesting tht the drug genertes signl in the gut tht promotes regultory cells in the periphery, which cn ccount for its nti-inflmmtory effects [23]. Resident Treg cells in dipose tissue modulte metbolism nd glucose homeostsis [24]. Interctions 8765 October 21, 216 Volume 22 Issue 39

7 CHOMA IR Iln Y et l. Orl nti TNF fusion protein for heptitis A B Glucose (mg%) Sline BY2 (-) Delt -24 wk serum insulin (µg/l) Sline BY2 (-) Sline BY2 (-) Figure 3 A beneficil effect of orl PRX16 on fsting glucose levels ws noted. Serum glucose levels were mesured t the end of the study in ll mice (A); the delt between the verge serum insulin levels on dy nd the end of tril ws clculted (B); HOMA-IR ws clculted for ll mice t the end of the study (C). 6 5 mg TG/gr Liv (%) Sline BY2 (-) Figure 4 The liver triglyceride content ws mesured nd clculted per liver weight to determine the percentge of ft in the mouse livers. P =.3. mong leptin, Treg cells nd dipose tissue re potentil trgets for therpeutic interventions [25]. The Treg frequency is lower in TNF-α trnsgenic mice thn in wild-type mice [26]. In humns, nti-tnfα therpy hs clinicl effect ssocited with promotion of Treg number nd function. Anti-TNFα therpy incresed the Treg proportion nd suppressed effector T cells in ptients with rthritis [27]. Orl dministrtion of lso ltered the distribution of CD8+CD25+FoxP3+ Tregs. CD8+CD25+FoxP3+ Tregs were suggested to be importnt in the induction of the systemic ntiinflmmtory effects medited by severl immunomodultory gents [28,29] nd to suppress inflmmtion in severl immune-medited disorders [3]. The dt suggest dose dependency of the observed immune system effect, which my underlie some of the differences noted in the clinicl effects. Orl dministrtion of ltered the distribution of NKT (CD3+NK1.1+) lymphocytes. The intrsplenic-to-intrheptic NKT rtio significntly decresed in ll treted groups. NKT cells ply 8766 October 21, 216 Volume 22 Issue 39

8 Iln Y et l. Orl nti TNF fusion protein for heptitis regultory role tht helps to prevent diet-induced obesity nd metbolic dysfunction [31]. A reduction in the number nd n ltered function of intrheptic NKT lymphocytes hve been reported in leptindeficient ob/ob mice, murine model for NASH [32-35]. NKT cells were suggested to be importnt in the regultion of immune-medited disorders [36,37], nd ltertion of their distribution ws shown to be relevnt in immunomodultion [38-42]. Our results further support regultory role for NKT cells in the crosstlk between metbolism nd the immune system [31]. The rtio between the splenic nd heptic CD4/ CD8 rtios decresed for ll treted groups, supporting the notion tht the tretment is ssocited with sequestrtion of CD8+ lymphocytes in the liver. The liver is site t which poptotic CD8+ cells ccumulte during the clernce phse of peripherl immune responses [43]. It serves s grveyrd for T cells ctivted in the periphery [44]. The liver ws shown to be n importnt site for CD8+ ccumultion during tolernce induction in process tht ws independent of NK cells [45]. Our results support the bility of to promote CD8 lymphocytes in the liver during n ctive systemic inflmmtory process. Orl dministrtion of ws ssocited with mild increse in serum TNF-α levels, suggesting tht the nti-inflmmtory effects re independent of this cytokine. is not bsorbed, nd its immunomodultory effect is therefore ssocited with biologicl ctivity in the gut. A similr effect ws reported for the orl dministrtion of the non-bsorbble nti- CD3 [46-49] nd delyed relese 6 mercptopurine [5], ll of which generte similr signl in the gut which lters the systemic immune system. Orl dministrtion of PRX16 ws ssocited with reduction in the serum triglyceride, glucose, insulin, nd AST levels. A decrese in heptic TG content ws observed in the high dose-treted mice. Tken together, our dt suggest tht the profound immunomodultory effect of orl PRX16 is ssocited with improvement in the metbolic syndrome in the HFD model. Etnercept is TNFR2-Fc fusion protein tht blocks only soluble TNF but not membrne-bound TNF [51]. Prenterl dministrtion of this compound hs been successfully used in the tretment of rheumtoid rthritis nd severl other immunomodultory tretments. The dt of the present study support n immunomodultory effect of orlly dministered. Severl immunomodultory gents exert different effect on the systemic immune system when dministered orlly compred with during prenterl dministrtion [52-56]. Their locl effect on the gut is different thn when the drug is dministered prentlly. These compounds use the inherit bility of the immune system of the gut to systemiclly promote Tregs [54,55] nd to exert potent nti-inflmmtory effects [54,55]. These effects re not ssocited with generlized immune suppression. Adjuvnts were suggested to be importnt for ugmenting the effect of orlly dministered immunomodultory gents [48,49,56]. The plnt cell wll, which is composed of cellulose, serves to protect the ctive molecule from cid-bse chnges in the stomch. The dt of the present study suggest tht the plnt cell wll lso serves s n immune djuvnt in the gut. Orl dministrtion of mock cells, BY2 (-), which included vehicle lone, did exert some immunomodultory effects. Previous studies showed tht orl dministrtion of BY-2 cells expressing llevited immunemedited liver injury in the Concnvlin A immunemedited heptitis model [57]. Similrly, in the TNBS colitis model, orl dministrtion of BY-2 plnt cells expressing resulted in decrese in the weight loss ssocited with immune-medited colitis, long with improvement in bowel histology [58]. In summry, orlly dministered plnt cells expressing recombinnt nti-tnf fusion protein show biologicl ctivity when dministered orlly. It lters the systemic immune environment nd ffects both the intrheptic nd splenic regultory T lymphocyte nd NKT cell subsets. These chnges were ssocited with the observed beneficil effects on the metbolic syndrome in the HFD model. As non-bsorbble gent, PRX16 my serve s potent immunomodultory gent tht lcks immunosuppressive properties. COMMENTS Bckground The BY-2 plnt cell-expressed recombinnt nti-tnf fusion protein () tht consists of the soluble form of the humn TNF receptor (TNFR) fused to the Fc component of humn IgG1 domin ws orlly dministered. The im of the study ws to evlute the immunomodultory effect of orl dministrtion of in the high-ft diet (HFD) model of non-lcoholic stetoheptitis (NASH). Reserch frontiers consisting of the soluble form of the humn TNFR fused to the Fc component of humn IgG1 domin cn be orlly dministered, nd hs n mino cid sequence tht is identicl to tht of Enbrel. Innovtions nd brekthroughs For 22 wk, C57BL/6 HFD-fed mice received dily orl tretments with BY-2 cells expressing. Orlly dministered shows biologicl ctivity nd exerts n immunomodultory effect, lleviting liver dmge. The dt suggest tht my provide n orl immunotherpy for NASH. The orlly dministered non-bsorbble ws biologiclly ctive. Altered distribution of CD4+CD25+FoxP3+ between the liver nd spleen nd n increse in the intrsplenic-to-intrheptic CD4+CD25+FoxP3+ rtio nd decrese in the intrsplenic-to-intrheptic CD8+CD25+FoxP3+ rtio were observed. An increse in intrheptic NKT cells nd decrese in the intrsplenic-to-intrheptic NKT rtio were noted. Assessment of the CD4- to-cd8 rtios showed sequestrtion of CD8+ lymphocytes in the liver. These effects were ssocited with decrese in serum triglyceride levels, decrese in the sprtte minotrnsferse levels, serum glucose levels, nd HOMA-IR score. A decrese in heptic TG content ws observed in the high dose-treted mice. Applictions is biologiclly ctive when dministered orlly. It lters the systemic immune environment nd ffects both the intrheptic nd splenic regultory 8767 October 21, 216 Volume 22 Issue 39

9 Iln Y et l. Orl nti TNF fusion protein for heptitis T lymphocyte nd NKT cell subsets. These chnges were ssocited with the observed beneficil effects on the metbolic syndrome in the HFD model of NASH. As non-bsorbble gent, PRX16 my serve s sfe nd potent immunomodultory gent tht lcks immunosuppressive properties. Terminology A chronic inflmmtory stte nd dysfunction of metbolic-inflmmtory signling re involved in the development of vrious spects of NASH. Orl delivery of therpeutic proteins is mjor gol when developing new therpeutic modlities. Peer-review This is pre-clinicl tril tht shows the potentil beneficil effect of orl dministrtion of in model for NASH. Further clinicl trils re required for showing its effects in ptients with type 2 dibetes nd NASH. REFERENCES 1 Iln Y. Immune therpy for nonlcoholic stetoheptitis: re we there yet? J Clin Gstroenterol 213; 47: [PMID: DOI: 1.197/MCG.b13e dc] 2 Hotmisligil GS. Inflmmtion nd metbolic disorders. 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Heptology 213; 751 Suppl 58: 564A 58 Shltiel Y, BYA, Shbbt Y, Zolotrov L, Gingis-Velitski S, Almon E, Aviezer D, Iln Y. A novel method for nti-tnf bsedorl immunotherpy: Orl dministrtion of plnt cell-expressed recombinnt nti-tnf fusion protein for treting of Crohn s disese. Gstroenterology 214; 5 Suppl 1: S91 [DOI: 1.116/ S16-585(14) ] P- Reviewer: Peltec S, Sherif ZA, Strom SC S- Editor: Yu J L- Editor: A E- Editor: Zhng FF 8769 October 21, 216 Volume 22 Issue 39

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