Role of IL-6 in the resolution of pancreatitis in obese mice

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1 Artile Role of IL-6 in the resolution of pnretitis in oese mie Mri Pini,* Dvin H. Rhoes,* Krl J. Cstellnos,* Anrew R. Hll, Roert J. Cy, Rohini Chennuri, Eileen F. Gry, n Gimil Fntuzzi*, Deprtments of *Kinesiology n Nutrition n Pthology, University of Illinois t Chigo, Chigo, Illinois, USA; n Deprtment of Surgery, University of Cliforni, Sn Frniso, Cliforni, USA RECEIVED DECEMER 7, ; REVISED FERUARY 8, ; ACCEPTED FERUARY 4,. DOI:.89/jl.67 ASTRACT Oesity inreses severity of ute pnretitis n risk of pnreti ner. Pnretitis n oesity re ssoite with elevte IL-6, ytokine involve in inflmmtion n tumorigenesis. We stuie the role of IL-6 in the response of len n oese mie to pnretitis inue y IL- IL-8. Len n iet-inue oese (DIO) WT n IL-6 KO mie n o/o mie pretrete with nti-il-6 ntioies were evlute t Dys, 7, n 5 fter inution of pnretitis. Prolonge elevtion of IL-6 in serum n viserl ipose tissue ws oserve in DIO versus len WT mie, wheres irulting sil-6r eline in DIO ut not len mie with pnretitis. The severe inflmmtion n lethlity of DIO mie were lso oserve in IL-6 KO mie. However, the elye resolution of neutrophil infiltrtion; sustine proution of CXCL, CXCL, n CCL; prolonge tivtion of STAT-3; n inution of MMP-7 in the pnres, s well s heightene inution of serum mylse A of DIO mie, were lunte signifintly in DIO IL-6 KO mie. In DIO mie, proution of OPN n TIMP- ws inrese for prolonge perio, n this ws meite y IL-6 in the liver ut not the pnres. Results otine in IL-6 KO mie were onfirme in o/o mie pretrete with nti-il-6 ntioies. In onlusion, IL-6 oes not ontriute to the inrese severity of pnretitis of oese mie ut prtiiptes in elye reovery from ute inflmmtion n my fvor evelopment of protumorigeni environment through prolonge tivtion of STAT-3, inution of MMP-7, n sustine proution of hemokines. J. Leuko. iol. 9: ;. Introution Oesity is ssoite with inrese severity of ute pnretitis (AP) []. However, the mehnisms linking oesity to inrese severity of AP re not known: some suggeste ftors Arevitions: ALT lnine minotrnsferse, AP ute pnretitis, DIO iet-inue oesity, KO knokout, MMP-7 mtrix metlloproteinse-7, OPN osteopontin, p phospho, PC pnreti ner, s solule, SAA serum myloi A, VAT viserl ipose tissue inlue ugmente nerosis of peripnreti ft, ftty pnres, ltertions in the inflmmtory network, n reue respirtory exursion []. Oesity lso inreses the risk of rinogenesis, inluing pnreti ner (PC), whih hs the highest se-ftlity rte of ny mjor ner [3 5]. The ext mehnisms linking oesity to inrese PC risk hve not een eluite, lthough insulin resistne n unerlying hroni inflmmtion re likely involve [6]. IL-6 n its min signling pthwy, STAT-3, hve een implite in AP n PC [7 9]. IL-6 plys ustive role in moels of PC through lssil n trns-signling events, whih le to tivtion of STAT-3 in infiltrting n prenhyml ells [, ]. One of the mehnisms linking STAT-3 to PC is inution of MMP-7 []. In ptients with PC, serum levels of MMP-7 orrelte with metstti isese n survivl [, 3]. In mouse moels of PC, MMP-7 is up-regulte in the pnres, n its efiieny prevents or elys isese, perhps s result of resistne to poptosis in metplsti uts [, 3]. IL-6 my lso prtiipte in up-regultion of other meitors tht re elevte in PC ptients [4], suh s SAA, OPN, TIMP- [5 7], n hemokines [8], whih regulte tumor-stroml intertions in experimentl moels of PC [9]. At vrine with knowlege out the ustive involvement of IL-6 in PC, the mehnisti role of this ytokine in AP remins to e lrifie. In humns, IL-6 is one of the most urte preitors of AP severity, n onstitutively tivte STAT-3 is present in lymphoytes of AP ptients [7, 9, ]. Proution of IL-6 is inrese in humn n experimentl AP, with oesity further inresing IL-6 levels [ 3]. Despite its involvement in the pthogenesis of severl hroni inflmmtory isorers n ner, IL-6 is lso ritil nti-inflmmtory ytokine tht promotes resolution of inflmmtion [8]. Whether IL-6 exerts enefiil or etrimentl roles in AP remins unler. Eviene otine in the erulein moel of AP is ontroversil, with enefiil n eleterious effets reporte with loking of IL-6 [4, 5]. Moreover, it is unknown whether elevte proution of IL-6 meites the enhne. Corresponene: Deprtment of Kinesiology n Nutrition, University of Illinois t Chigo, 99 W. Tylor St., MC57, Chigo, IL 66, USA. E-mil: gimil@ui.eu 74-54//9-957 Soiety for Leukoyte iology Volume 9, June Journl of Leukoyte iology 957

2 severity of AP oserve in oesity [,, 6 8]. Furthermore, proution of IL-6 y ipose tissue in oesity n y musle uring exerise hs pprently opposite effets on moultion of insulin sensitivity [9, 3]. Therefore, the speifi role of this ytokine in regulting inflmmtion n metolism is likely to e highly ontext-epenent. In the present report, we use the IL- IL-8 moel of pnretitis to eluite the role of IL-6 in AP severity in len n oese mie [, 3, 3]. We lso etermine whether oesity promotes signling pthwys tht hve een implite in pnreti enorinom inution n progression through sustine proution of IL-6. Our results emonstrte tht IL-6 oes not meite the ute severity of pnreti inflmmtion in the IL- IL-8 moel. However, heightene proution of IL-6 in oese mie meites prolonge inution of STAT-3, MMP-7, n hemokines, whih my fvor evelopment of protumorigeni environment when hronilly up-regulte. MATERIALS AND METHODS Animls Animl protools were pprove y the Animl Cre n Use Committee of the University of Illinois t Chigo. For inution of DIO, mle WT n IL-6 KO C57L6 mie were fe high-ft iet (6 Kl% ft; Reserh Diets, New runswik, NJ, USA) liitum for 3 weeks, eginning t 4 weeks of ge, wheres groups of WT n IL-6 KO ontrol groups reeive stnr how iet for the sme perio. Leptin-efiient, oese o/o mie n their len littermtes fe stnr how iet were exmine t 7 8 weeks of ge. All mie were from The Jkson Lortory (r Hror, ME, USA). Inution of AP Murine ril- n ril-8 (R&D Systems, Minnepolis, MN, USA) were ministere i.p. t 5 ng/mouse n 75 ng/mouse, respetively, t 4 h intervls, for totl of two injetions, s esrie previously []. For neutrliztion experiments, WT n o/o mie reeive neutrlizing nti-il-6 ntioy ( mg/mouse; iolegen, Sn Diego, CA, USA), previously shown to neutrlize IL-6 tivity [3, 33], t the sme time of the first injetion of IL- IL-8. Mie were euthnize t, 7, or 5 ys fter the seon injetion of IL- IL-8. Control groups reeive vehile n were euthnize y fter the seon injetion. Severity of VAT nerosis ws quntifie mrosopilly s (sent), (few pinhe-size neroti res without retropnreti nerosis), (moertely extene neroti res with moerte/extensive retropnreti nerosis), n 3 (extensive res of nerosis with extensive retropnreti nerosis). loo ws ollete t time of euthnsi n serum prepre. Pnres, liver, n VAT otine from non-neroti n neroti res were frozen immeitely in liqui nitrogen n store t 7 C for susequent proessing or fixe in formlin for histologil evlution. Misellneous mesurements Levels of IL-6, sil-6r, leptin, OPN, SAA, n TIMP- were mesure using ELISA kits from R&D Systems, eiosiene (Sn Diego, CA, USA), n Invitrogen (Crls, CA, USA). Serum mylse, ALT, n lium levels were mesure using kits from Teo Dignostis (Anheim, CA, USA). Pnres homogentes were prepre y homogenizing tissue in ell lysis uffer (Cell Signling Tehnology, Dnvers, MA, USA), followe y sonition. Protein onentrtion ws juste to mg/ml for mesurement of CXCL, CXCL, CCL, OPN, n TIMP- using ELISA kits from R&D Systems n to. mg/ml for evlution of Tyr75-pSTAT-3 levels using PthSn ssy (Cell Signling Tehnology). Histologil nlysis n immunohistohemistry For histologil ssessment, the pnres ws fixe in % uffere formlin n setions stine with H&E for soring y two pthologists (R.J.C. n R.C.), line to the experimentl groups, using soring system esrie previously [3]. For immunohistohemistry, ntioies irete ginst rt nti-mouse Ly6G, rt nti-mouse F4/8 (D iosienes, Sn Diego, CA, USA), rit nti-tyr75-pstat-3, or rit nti-mouse MMP-7 (Cell Signling Tehnology) were use. RNA expression nlysis Totl RNA ws isolte from VAT, otine from res ontining neroti n sponifie tissue (neroti VAT) n from VAT ollete t sites istnt from nerotis res (non-neroti VAT), s well s from liver using Trizol, n reverse trnsrie. Gene expression of IL-6 n SAA- ws ssesse y rel-time RT-PCR using the TqMn system n primers from Applie iosystems (Foster City, CA, USA). Reltive expression ws lulte using the omprtive threshol metho fter normlizing for expression of GAPDH. Sttistil nlysis Dt re expresse s men sem. Sttistil signifine of ifferenes ws etermine y ANOVA s pproprite. The Kpln-Meyer metho ws use for nlysis of survivl t. Sttistil nlyses were performe using MeCl softwre (elgium). RESULTS Inrese IL-6 levels in DIO mie with AP We emonstrte previously tht ministrtion of IL- IL-8 inues AP in mie, with more severe n prolonge isese in DIO n o/o mie ompre with their len ontrols [, 3]. In this moel of AP, irulting levels of IFN- re omprle in len n DIO mie, wheres inution of the STAT-3-tivting ytokines IL-6, IL-, n IL- is signifintly higher in oese ompre with len mie [, 3]. In greement with these previous t, we foun signifintly higher n prolonge elevtion of serum IL-6 in DIO mie ompre with len mie (Fig. A). Wheres len mie h ner-seline levels of IL-6 t 5 ys fter the injetion of IL- IL-8, DIO mie h pg/ml serum IL-6. Severl eleterious tivities of IL-6 re meite through its ility to trns-signl y ining to its solule reeptor (sil-6r) n tivte ells, suh s the enothelium, whih o not express memrne-oun IL-6R [8]. Cirulting levels of sil-6r were omprle in len n DIO ontrol mie. Aministrtion of IL- IL-8 i not signifintly lter serum sil-6r levels in len mie, wheres signifint reution ws oserve in DIO mie t Dys n 7, with return to seline y Dy 5 (Fig. ). We emonstrte previously tht the pnres signifintly ontriutes to proution of IL-6 in response to ministrtion of IL- IL-8 [3]. As inite in Fig., VAT lso ontriute to proution of IL-6 uring AP in len n DIO mie. A -fol inrese in IL-6 mrna expression ws oserve in VAT of len mie t Dy post-ap, with levels returning to seline y Dy 7 (Fig. C). Compre with len 958 Journl of Leukoyte iology Volume 9, June

3 Pini et l. Oesity n IL-6 in pnreti inflmmtion IL-6 (pg/ml) A sil-6r (ng/ml) C Reltive fol inrese D sore Serum IL-6 8, len DIO,, len DIO non-neroti VAT DIO neroti VAT ontrol Dy Dy7 Dy5, len DIO, C Dy Dy 7 Dy 5 Serum sil-6r VAT IL-6, VAT nerosis, C Dy Dy 7 Dy 5, [3, 3] for exmples of mro- n mirosopi VAT nerosis in o/o mie). Ares of VAT, whih inlue neroti tissue, s evlute y the presene of mrosopi sponifition, expresse the highest IL-6 mrna levels, with sustine high levels even t Dy 5 post-ap (Fig. C). IL-6 oes not meite the inrese AP severity of oese mie To evlute whether elevte proution of IL-6 in oese mie uses the inrese isese severity oserve in DIO n o/o mie [, 3], we use len n n IL-6 KO mie, s well s genetilly oese o/o mie pretrete with nti-il-6 ntioies. Aministrtion of IL- IL-8 use no lethlity in len mie (Fig. A). We foun omprle lethlity (3 4%) in n DIO IL-6 KO mie (Fig. A). In len WT n IL-6 KO mie, AP i not lter oy A % Survivl W (g) Len WT Len IL-6 KO DIO IL-6 KO C Dy Dy 7 Dy 5 Figure. IL-6 n sil-6r levels in DIO mie with AP. Len n DIO WT mie reeive two injetions of vehile or IL- IL-8. loo n VAT were otine t the inite time-points. (A n ) Cirulting IL-6 (A) n sil-6r () levels in len (lk rs) n DIO (white rs) WT mie. C, Control. (C) Expression of IL-6 mrna in VAT from len WT mie (lk rs) n non-neroti (white rs) n neroti (hthe rs) VAT from mie. Dt in C re expresse s fol inrese over len ontrol mie. (D) VAT nerosis sore in len (lk rs) n DIO (white rs) mie. Dt re men sem of four to seven mie/group., P. versus respetive ontrol;, P. versus len t the sme time-point;, P.5 versus non-neroti VAT t the sme time-point. mie, non-neroti VAT of DIO mie h signifintly higher n prolonge inrese in IL-6 expression, with n lmost 3-fol inrese t Dy post-ap n n -fol inrese on Dy 7 post-ap. Neroti VAT ws only present in DIO mie with AP (Fig. D n Supplementl Fig. ; see refs. C leptin (ng/ml) Len WT Len IL-6 KO DIO IL-6 KO ys Figure. Survivl, oy weight, n serum leptin in len n n IL-6 KO mie. Len n n IL-6 KO mie reeive two injetions of vehile or IL- IL-8. (A) Kpln-Meyer urves for survivl. () oy weight (W). (C) Serum leptin levels. Dt re men sem of mie/group., P. versus respetive time. Volume 9, June Journl of Leukoyte iology 959

4 weight or serum leptin levels uring the inution or the resolution phse. In mrke ontrst, AP inue signifint n omprle oy weight loss, ompnie y reue leptin levels, in n DIO IL-6 KO mie (Fig. n C). In o/o mie pretrete with nti-il-6 ntioies, neutrliztion of IL-6 i not signifintly lter lethlity rtes or oy weight loss inue y AP (Supplementl Fig. A n ). We verifie tht the ntioy effetively neutrlize IL-6 tivity in vivo y mesuring its ility to suppress inution of the ute-phse protein SAA (Supplementl Tle ). Injetion of IL- IL-8 inue eem, inr ell nerosis, n ft nerosis in ll mie (Fig. 3A C n J). In len mie, these prmeters of histologil mge were iminishe t Dys 7 n 5 fter inution of AP. In shrp ontrst, in n DIO Figure 3. Pnreti tissue mge n inution of SAA in len n n IL-6 KO mie with AP. Len n n IL-6 KO mie reeive two injetions of vehile or IL- IL-8. Pnres, liver, n loo were otine t the inite time-points. Quntifition of eem (A), inr nerosis (), n ft nerosis (C) in the pnres of len WT (lk rs), len IL-6 KO (gry rs), (white rs), n DIO IL-6 KO (hthe rs) mie. Levels of mylse (D), ALT (E), lium (F), IL- (G), n SAA (H) were mesure in serum. mrna expression of SAA ws mesure in liver (I). (J) Representtive smples of H&E-stine setions of the pnres. Originl mgnifition, /.3. Dt re men sem of four to seven mie/group., P. versus respetive ontrol group;, P. versus len groups t the sme time-point;, P.5 versus t the sme time point;, P.5 versus len WT t the sme time-point. A sore ore s C ore s J Control Dy 3 Eem D 3 Ainr nerosis 3 Ft nerosis C Dy Dy 7 Dy 5 ylse (U/L) Amy ALT (U/L) 5 E F (mg/l) C 5 6 ALT 4 Len WT Clium 8 4 Amylse C Dy Dy 7 Dy 5 DIO KO IL L- (pg/ml) SAA A (µg/ml) G 4 3 IL- C Dy Dy 7 Dy 5 H Serum SAA 4 3 I Fol inu ution 3 Liver SAA C Dy Dy 7 Dy 5 Len KO Len WT Len KO DIO KO y 5 Dy 7 Dy 7 96 Journl of Leukoyte iology Volume 9, June

5 Pini et l. Oesity n IL-6 in pnreti inflmmtion IL-6 KO mie, we foun prolonge pnreti mge with inrese eem, inr ell nerosis, n ft nerosis (Fig. 3A C n J). Thus, prolonge histopthologil mge in DIO mie ourre in n IL-6-inepenent mnner. As seen previously with ministrtion of IL- IL-8 [, 3], t Dy, serum mylse n ALT inrese in eh group, wheres serum lium erese signifintly only in DIO mie, irrespetive of genotype (Fig. 3D F). Similrly, ministrtion of nti-il-6 ntioies i not signifintly moify the effet of IL- IL-8 on serum mylse n lium levels in o/o mie (Supplementl Tle ). In ontrst, inution of SAA ws reue signifintly t the mrna n protein level in len n DIO IL-6 KO ompre with WT mie (Fig. 3H n I). Inution of SAA in IL-6 KO mie with AP, lthough of lower mgnitue thn tht seen in WT mie, ws still signifint ompre with helthy ontrols, possily s result of proution of IL- (Fig. 3G), ytokine tht is inrese uring AP inue y IL- IL-8 n tht is known to inue SAA expression [, 34]. In summry, IL-6 i not meite the inrese lethlity, oy weight loss, or tissue mge oserve in DIO mie with AP ut ws ritil meitor for inution of SAA. Delye resolution of the neutrophil infiltrte in the pnres of oese mie with AP is meite y IL-6 A omprle inflmmtory infiltrte ws present in the pnres of len WT,, len IL-6 KO, n DIO IL-6 KO t Dy (Fig. 4A). Thus, the initil reruitment of inflmmtory ells ws unffete y DIO or IL-6. However, in mie, the egree of infiltrtion ontinue to inrese t Dys 7 n 5, wheres it stilize in eh other group, inluing DIO IL-6 KO mie. At Dys 7 n 5, the neutrophil infiltrte of mie ws signifintly higher ompre with the other groups, wheres the mononuler infiltrte ws not ffete signifintly y IL-6 efiieny (Fig. 4 D). Immunohistohemistry for Ly6G, whih ientifies neutrophils, onfirme the presene of signifint neutrophil infiltrte in mie ut not DIO IL-6 KO mie t Dy 7, wheres no signifint ifferenes etween the two groups were oserve t the sme timepoint for F4/8 mrophges (Supplementl Fig. 3A). Thus, in ontrst to initil reruitment, prolonge retention/reruitment of neutrophils ws epenent on DIO n IL-6. Chemokine levels in pnreti homogentes were inrese in len mie t Dy fter AP, ut CXCL ws inrese more rmtilly in n DIO IL-6 KO mie (Fig. 4E G). The kinetis of hemokine levels prllele the elye resolution of the inflmmtory infiltrte of mie. In ft, levels of CXCL n CXCL remine signifintly elevte in the pnres of ut not DIO IL-6 KO mie, up to 7 n 5 ys post-ap, respetively (Fig. 4E n F). Pnreti levels of CCL were omprle in eh group t Dy n remine elevte only in mie t Dys 7 n 5 (Fig. 4G). In o/o mie, neutrliztion of IL-6 signifintly reue CXCL, CXCL, n CCL levels t Dy, ut the effet ws lost t Dy 7 (Supplementl Fig. 3), possily s result of ntioy lerne. A sore sore C sore D sore 3 Inflmmtory infiltrte C Dy Dy 7 Dy 5 3 NE C Dy Dy 7 Dy 5 3 MΦ C Dy Dy 7 Dy 5 3 LY C Dy Dy 7 Dy 5 Prolonge STAT-3 tivtion in the pnres of oese mie with AP is meite y IL-6 To verify whether the inrese n prolonge proution of IL-6 in oese mie with AP ws ssoite with tivtion of STAT-3 in the pnres, we quntifie levels of Tyr75- pstat-3. A signifint inrese in pstat-3 levels ws oserve t Dy in homogentes of pnres otine from eh group of mie injete with IL- IL-8 ompre with tht seen in mie without AP (ontrols), with the highest levels oserve in mie n signifintly lower levels in IL-6 KO mie ompre with WT mie (Fig. 5A). Resiul tivtion of STAT-3 in IL-6 KO mie oul e the onsequene of inution of IL- (Fig. 3G), STAT-3-tivting ytokine whose reeptor is present on inr ells [35]. Ativte STAT-3 returne to seline y Dy 7 in len WT, len IL-6 KO, n DIO IL-6 KO mie. In mie, tivte STAT-3 remine elevte up to Dy 5 fter AP. E (pg/m ml) F (pg/m ml) G (pg/ml) 8 CXCL CL 6 4 C Dy Dy 7 Dy CXCL 3 5 CCL 5 5 C Dy Dy 7 Dy 5 C Dy Dy 7 Dy 5 Len WT Len KO DIO KO Figure 4. Inflmmtory infiltrte in the pnres of len n n IL-6 KO mie with AP. Len n n IL-6 KO mie reeive two injetions of vehile or IL- IL-8. The pnres ws otine t the inite time-points. Quntifition of the totl inflmmtory infiltrte (A), neutrophils (NE; ), mrophges (M ; C), n lymphoytes (LY; D) y histologil nlysis. Levels of the hemokines CXCL (E), CXCL (F), n CCL (G) in pnreti homogentes of len WT (lk rs), len IL-6 KO (gry rs), (white rs), n DIO IL-6 KO (hthe rs) mie. Dt re men sem of four to seven mie/group., P. versus respetive ontrol group;, P. versus len groups t the sme time-point;, P.5 versus t the sme time-point. Volume 9, June Journl of Leukoyte iology 96

6 A As t 45/ pstat3 pstat-3 C Dy Dy 7 Dy 5 Len WT Len WT Len KO Len KO DIO KO DIO KO Figure 5. Ativtion of STAT-3 n inution of MMP-7 in the pnres of len n n IL-6 KO mie with AP. Len n n IL-6 KO mie reeive two injetions of vehile or IL- IL-8. The pnres ws otine t the inite time-points. (A) Quntifition of Tyr75-pSTAT-3 in pnreti homogentes of len WT (lk rs), len IL-6 KO (gry rs), (white rs), n DIO IL-6 KO (hthe rs) mie. Dt re men sem of four to seven mie/group., P. versus respetive ontrol group;, P. versus len groups t the sme time-point;, P.5 versus t the sme time-point;, P.5 versus len WT t the sme time-point. () Representtive smples of immunohistohemistry for Tyr75-pSTAT-3. (C) Representtive smples of immunohistohemistry for MMP-7. Originl mgnifition, /.5; originl rs, m. Dy 5 Dy 7 Dy Control C MMP7 Dy 5 Dy 7 Dy Control Len WT Len KO DIO KO To onfirm the fining tht the AP-inue inrese in tivte STAT-3 ws epenent on IL-6, we use o/o mie. Signifintly higher levels of tivte STAT-3 were oserve in the pnres of o/o ompre with WT mie t Dys n 7 postinution of AP (Supplementl Fig. 4A). Neutrliztion of IL-6 reue levels of tivte STAT-3 in o/o mie t Dy ut not Dy Journl of Leukoyte iology Volume 9, June

7 Pini et l. Oesity n IL-6 in pnreti inflmmtion Immunohistohemistry for pstat-3 onfirme results of the PthSn ssy. Nuler stining for pstat-3 ourre in inr ells n infiltrting inflmmtory ells t Dy fter inution of AP in ll groups (Fig. 5). However, more pronoune stining ws present in WT versus IL-6 KO mie. At Dy, some utl ells were lso pstat-3-positive in len n mie (Supplementl Fig. 4), wheres enorine ells (Supplementl Fig. 4C) n the ileum (Supplementl Fig. 4D) remine pstat-3-negtive. At Dy 7, suset of infiltrting ells ws still positive for pstat-3 in the pnres of len WT mie (Supplementl Fig. 4E), wheres inr ells were no longer positive in this group (Fig. 5). In ontrst, in mie, strong immunoretivity for pstat-3 ws still present t Dy 7 in inr n inflmmtory ells (Fig. 5 n Supplementl Fig. 4F). y Dy 5, pstat-3 immunoretivity susie in eh group exept mie. Thus, the prolonge inrese in immunoretive pstat-3 in the pnres of DIO mie injete with IL- IL-8 epens on IL-6. Inution of MMP-7, OPN, n TIMP- Up-regultion of MMP-7 in pnreti uts hs een uslly linke to evelopment of PC [, 3]. A signifint inution of MMP-7 ws oserve in pnreti uts of len n DIO WT mie t Dy, with lower inution in IL-6 KO mie (Fig. 5C). At Dy 7, the numer of immunoretive MMP-7 ells shrply inrese in mie, wheres tissue from eh other group ws negtive or only fintly positive. At Dy 5, MMP-7-positive utl ells were still present in the pnres of mie n were sent in eh other group. Elevte levels of OPN n TIMP- re present in ptients with PC n hroni pnretitis, onition tht fvors evelopment of PC [4, 36 39]. Aministrtion of IL- IL-8 signifintly inrese serum OPN levels in len n mie t Dy in n IL-6-epenent mnner n with more pronoune inrese in oese nimls (Fig. 6A). y Dy 7, mie were the only group in whih serum OPN levels were still elevte signifintly. Hepti OPN expression prllele the results of irulting levels n thus, ws inrese in n IL-6-epenent mnner (Fig. 6). In ontrst, omprle inution of OPN ws oserve in pnreti homogentes from eh group t Dy (Fig. 6C), with levels remining signifintly elevte in DIO mie, irrespetive of genotype, until Dy 7. Similr to OPN, hepti TIMP- expression ws epenent on IL-6 n enhne y oesity (Fig. 6E), wheres inution of TIMP- in the pnres ws IL-6-inepenent, with omprle inution in eh group t Dy n oth DIO groups hving signifintly higher TIMP- levels t Dy 7 reltive to len mie (Fig. 6F). At vrine with OPN, irulting levels of TIMP- my hve resulte from omintion of hepti n pnreti soures, with signifintly higher serum TIMP- levels in DIO groups t Dys n 7 ut no signifint ifferenes etween n IL-6 KO mie until Dy 5 (Fig. 6D). DISCUSSION As we emonstrte previously, the injetion of IL- IL-8 inues typil roust pnretitis with elevte mylse levels n histologil mge in len n oese nimls [, 3]. In len mie, AP resolves rpily, with return to norml histology y Dy 7, wheres more severe n prolonge isese, ompnie y elevte levels of IL-6 n ute-phse proteins, is oserve in oese mie [, 3], thus proviing n exellent moel to stuy the effet of oesity in AP. The novel fining of the urrent report is efining the role of the elevte proution of the pleiotropi ytokine IL-6 in oese mie with AP. We emonstrte tht high IL-6 in oesity prolongs inflmmtion n lters the resolution of pnreti mge oserve in len mie through sustine tivtion of STAT-3 in the pnres, elye lerne of the neutrophil infiltrte, n up-regultion of CXC n CC hemokines, MMP-7, SAA, OPN, n TIMP-. However, IL-6 oes not meite the heightene ute isese severity n lethlity of oese mie with AP inue y IL- IL-8. Comprison of DIO WT n IL-6 KO mie lso points to issoition etween resolution of tissue mge n kinetis of neutrophil lerng/ml ng/ml A Serum Liver Pnres 4 OPN C 6. OPN OPN D 4 TIMP C Dy Dy 7 Dy 5 Fol inution Fol inution 6 4 E TIMP ng/ml ng/ml TIMP.. C Dy Dy 7 Dy 5 C Dy Dy 7 Dy 5 Len WT Len KO DIO KO F Figure 6. Levels of OPN n TIMP- in len n n IL-6 KO mie with AP. Len n n IL-6 KO mie reeive two injetions of vehile or IL- IL-8. Pnres, liver, n loo were otine t the inite timepoints. Levels of OPN n TIMP- in serum (A n D), mrna expression in liver ( n E), n protein levels in homogentes of pnres (C n F) of len WT (lk rs), len IL-6 KO (gry rs), (white rs), n DIO IL-6 KO (hthe rs) mie. Dt re men sem of four to seven mie/group., P. versus respetive ontrol group;, P. versus len groups t the sme time-point;, P.5 versus t the sme time-point;, P.5 versus len WT t the sme time-point. Volume 9, June Journl of Leukoyte iology 963

8 ne n STAT-3 tivtion, whih requires further investigtion. Moreover, our t onfirm tht oesity, rther thn leptin efiieny, meites inrese isese severity in this moel of AP, s inrese lethlity n elye resolution were oserve in DIO n o/o mie [, 3, 3]. Furthermore, ge oes not pper to ply mjor moultory role, s omprle isese severity ws oserve in 7-week-ol DIO mie n 7-week-ol o/o mie. However, evlution of ge mie is neessry to ientify the ontriution of senesense in etermining AP outomes, n issue of potentil linil relevne [4]. Eviene inites tht IL-6 is mong the most urte preitors of isese severity in AP [7]. The present report emonstrtes tht ipose tissue is n importnt ontriutor to the sustine proution of IL-6 in oese nimls with AP. In prtiulr, res of VAT ontining neroti n sponifie tissue, whih re only oserve in oese nimls, express high levels of IL-6, up to 5 ys postinution of AP. These t re in greement with results otine in the rt moel of turoholte-inue AP, in whih neroti VAT proues high levels of TNF- n other meitors [4]. Although we i not iretly ress the speifi soure of IL-6 in VAT, it is likely tht ipose tissue t the orer etween neroti n nonneroti res hs the highest inflmmtory potentil, s emonstrte in the turoholte moel [4]. It is lso worth noting tht VAT nerosis n sustine inflmmtion persiste up to 5 ys in DIO mie with AP, thus mirroring the linil sitution n unerlying the relevne of IL- IL-8-inue AP to moel humn isese. Thus, the presene of n expne VAT not only ontriutes to the seline hroni inflmmtion of oesity [4] ut lso tively prtiiptes in the systemi inflmmtory response to pnreti mge. However, whether sustine proution of IL-6 in severe AP ontriutes to isese pthogenesis or represents frustrte ttempt t lunting inflmmtion remine unler. Experimentl results otine using the erulein moel in len mie re ontroversil [4, 5], wheres the intertion of oesity n IL-6 in AP severity h not een explore. The present results inite tht IL-6 oes not meite the inrese isese severity n lethlity of oese mie in the moel of IL- IL-8-inue AP, lthough it moultes inution of the ute-phse protein SAA. Oesity is ssoite with elye resolution of the neutrophil infiltrte in response to peritonel inflmmtion [43, 44], lthough less ler-ut effets hve een oserve in moels of pulmonry inflmmtion [45, 46]. Our t emonstrte tht oesity les to elye resolution of the pnreti inflmmtory infiltrte in response to IL- IL-8. Elevte intrpnreti levels of CXCL, CXCL, n CCL likely ontriute to the prolonge influx of inflmmtory ells in the pnres of DIO mie, ut ftors suh s reue lerne of poptoti ells y mrophges of oese nimls might lso prtiipte [47]. Our results lso emonstrte tht IL-6 meites the sustine neutrophil infiltrte n hemokine proution in the pnres of DIO mie with AP n tht ute neutrliztion of IL-6 with ntioies suppresses intrpnreti hemokine levels. This is in greement with reports emonstrting reue neutrophil infiltrte n suppresse proution of CXCL n CXCL in IL-6 KO mie uner vrious experimentl onitions [48 5]. Stimultion with IL-6 les to inution of CCL through tivtion of STAT-3 [5]. Although lssilly onsiere mononuler ell-ttrting hemokine, CCL is involve in neutrophil reruitment n elye poptosis [5, 5, 5]. Thus, up-regultion of CCL my lso ontriute to the prolonge neutrophil infiltrte oserve in DIO mie. In ition to meiting reruitment of inflmmtory ells, hemokines, prtiulrly the CXC fmily, re involve in the pthogenesis of experimentl PC y moulting tumor-stroml intertions [9]. Therefore, sustine proution of CXC hemokines in the pnres of oese mie my fvor evelopment of tumorigeni environment, t lest in prt through IL-6. Trns-signling events meite y sil-6r hve een implite in severl of the effets of hronilly elevte IL-6 levels, inluing moultion of the neutrophil-monoyte trnsition [8]. Our t emonstrte tht oesity i not signifintly ffet serum sil-6r levels, wheres signifint reution in irulting sil-6r ws oserve in DIO mie with AP, perhps s n ttempt to limit exessive IL-6 trns-signling. These t re in greement with reports showing lk of effet of oesity ut reue sil-6r levels in women with polyysti ovry synrome, onition ssoite with elevte serum IL-6 [53]. Chroni tivtion of STAT-3 hs een uslly linke to tumorigenesis in severl orgns, inluing the pnres [8]. Here, we report tht ministrtion of IL- IL-8 les to tivtion of STAT-3 in inr n infiltrting ells in n IL-6- epenent mnner n tht oesity les to prolonge tivtion of this signling pthwy. Pnreti tivtion of STAT-3 plys ritil role t eh stge of tumorigenesis in the Krs moel of PC [ ]. However, our t inite tht pnreti inflmmtion superimpose on oesity les to prolonge tivtion of STAT-3 even in the sene of Krs muttions, thus possily generting n environment onuive to pnreti tumorigenesis if sustine over long perios. This onept is in greement with the role of oesity s tumor promoter in heptoellulr rinom [54]. Ativtion of STAT-3 is ompnie y inution of MMP-7 in utl ells, gin in n IL-6-epenent mnner []. Up-regultion of MMP-7 is STAT-3-meite response, whih regultes inr-to-utl metplsi, tumor size, n metstsis in mouse moels n orreltes with metstti isese n survivl in humns with PC [, 3]. In ition to up-regultion of MMP-7, we emonstrte the presene of elevte levels of SAA, OPN, n TIMP- in DIO mie with AP. Cirulting levels of these ftors re elevte in PC, lthough speifiity remins hllenge [4, 36]. Despite the unler involvement of these meitors in the pthogenesis of PC, their onomitnt upregultion, together with pnreti tivtion of STAT-3 n inution of MMP-7, supports the rgument tht oesity fvors evelopment of tumorigeni environment fter pnreti insult. Inution of SAA y IL- IL-8 is lmost exlusively IL-6-epenent, in greement with results otine in mouse moels of oesity n sterile inflmmtion ut t vrine with inution y miroil stimuli [7, 54, 55], suggesting loser reltionship of the 964 Journl of Leukoyte iology Volume 9, June

9 Pini et l. Oesity n IL-6 in pnreti inflmmtion IL- IL-8 moel with moels of tissue mge rther thn moels of sepsis or infetion. Regultion of hepti expression of OPN n TIMP- prllels tht of SAA, in tht it is enhne y oesity in n IL-6-epenent mnner. In ontrst, lol proution of OPN n TIMP- in the pnres is IL-6- inepenent, suggesting ifferentil regultion tht eserves further investigtion. In onlusion, oesity exertes isese severity in response to ministrtion of IL- IL-8 in n IL-6-inepenent mnner, espite elevte n sustine proution of IL-6 in DIO mie from pnres n VAT. However, in oese mie IL-6 is mjor ftor in prolonging inflmmtion n ltering the resolution from pnreti mge, possily fvoring evelopment of miroenvironment onuive to tumorigenesis. However, ution shoul e use when intepreting these results, s limittion of our stuy is the use of moel of ute rther thn hroni pnretitis, whih woul hve h more iret relevne to the pthogenesis of PC. AUTHORSHIP M.P., D.H.R., K.J.C., n A.R.H. performe n inteprete experiments n helpe rft the mnusript. R.J.C. n R.C. evlute histologil smples n helpe rft the mnusript. E.F.G. helpe rft the mnusript n intepret results. G.F. plnne n irete the stuies performe n inteprete experiments, wrote the mnusript, n provie funing through NIH grnts. ACKNOWLEDGMENTS This stuy ws supporte y NIH grnts DK8338 to G.F. n DK8787-3S3 to E.F.G. REFERENCES. Evns, A. C., Pphristou, G. I., Whitom, D. C. () Oesity n the risk of severe ute pnretitis. Minerv Gstroenterol. Dietol. 56, Steer, M. () Pnretitis severity: who lls the shots? Gstroenterology, Misonneuve, P., Lowenfels, A.. () Epiemiology of pnreti ner: n upte. Dig. Dis. 8, Zyromski, N. J., White, P.. () Pnreti ner in oesity: epiemiology, linil oservtions, n si mehnisms. Antiner Agents Me. Chem., Aune, D., Greenwoo, D. C., Chn, D. S., Vieir, R., Vieir, A. R., Nvrro Rosenltt, D. A., Ce, J. E., urley, V. J., Nort, T. 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Mtsur, T., Mit, A., Minmi, K., Hosook, T., Kitzw, S., Tkhshi, K., Tmori, Y., Yokoi, N., Wtne, M., Mtsuo, E., Nishimur, O., Seino, S. () PGRN is key ipokine meiting high ft ietinue insulin resistne n oesity through IL-6 in ipose tissue. Cell Met. 5, Nvin, S., Ahry, C., DeLny, J. P., Orlihenko, L. S., ty, C. J., Shiv, S. S., Durgmpui, C., Krlsson, J. M., Lee, K., e, K. T., Furln, A., ehri, J., Liu, S., MHle, T., Nihols, L., Pphristou, G. I., Yv, D., Singh, V. P. () Lipotoxiity uses multisystem orgn filure n exertes ute pnretitis in oesity. Si. Trnsl. Me. 3, 7r. 3. Rieemnn, N. C., Neff, T. A., Guo, R. F., ernki, K. D., Lues, I. J., Srm, J. V., Lmris, J. D., Wr, P. A. (3) Protetive effets of IL-6 Volume 9, June Journl of Leukoyte iology 965

10 loke in sepsis re linke to reue C5 reeptor expression. J. Immunol. 7, Yen, D., Cheung, J., Sheerens, H., Poulet, F., MClnhn, T., MKenzie,., Kleinshek, M. A., Owyng, A., Mttson, J., lumenshein, W., Murphy, E., Sthe, M., Cu, D. J., Kstelein, R. A., Rennik, D. (6) IL-3 is essentil for T ell-meite olitis n promotes inflmmtion vi IL-7 n IL-6. J. Clin. Invest. 6, Misse, D., Yssel, H., Trttoni, D., Olet, C., Lo Cputo, S., Mzzott, F., Pene, J., Gonzlez, J. P., Clerii, M., Ves, F. (7) IL- prtiiptes in n innte nti-hiv- host-resistne network through ute-phse protein inution. J. Immunol. 78, Xie, M. H., Aggrwl, S., Ho, W. H., Foster, J., Zhng, Z., Stinson, J., Woo, W. I., Gor, A. D., Gurney, A. L. () Interleukin (IL)-, novel humn ytokine tht signls through the interferon reeptorrelte proteins CRF-4 n IL-R. J. iol. Chem. 75, unger, S., Luert, T., Rolik, U. J., Hermnn, J. K. () Serum iomrkers for improve ignosti of pnreti ner: urrent overview. J. Cner Res. Clin. Onol. 37, Sullivn, J., lir, L., Alnjr, A., Aziz, T., Chipitsyn, G., Gong, Q., Yeo, C. J., Arft, H. A. () Expression n regultion of niotine reeptor n osteopontin isoforms in humn pnreti utl enorinom. Histol. Histopthol. 6, Sullivn, J., lir, L., Alnjr, A., Aziz, T., Ng, C. Y., Chipitsyn, G., Gong, Q., Witkiewiz, A., Weer, G. F., Denhrt, D. T., Yeo, C. J., Arft, H. A. (9) Expression of prometstti splie vrint of osteopontin, OPNC, in humn pnreti utl enorinom. Surgery 46, Pn, S., Chen, R., Crispin, D. A., My, D., Stevens, T., MIntosh, M. W., ronner, M. P., Ziogs, A., Anton-Culver, H., rentnll, T. A. () Protein ltertions ssoite with pnreti ner n hroni pnretitis foun in humn plsm using glol quntittive proteomis profiling. J. Proteome Res., Xin, M. J., Chen, H., Luo,., Sun, J.. (8) Severe ute pnretitis in the elerly: etiology n linil hrteristis. Worl J. Gstroenterol. 4, Frno-Pons, N., Ge-Sorli, S., Clos, D. () Relese of inflmmtory meitors y ipose tissue uring ute pnretitis. J. Pthol., Fntuzzi, G. (5) Aipose tissue, ipokines, n inflmmtion. J. Allergy Clin. Immunol. 5, Pini, M., Fntuzzi, G. () Enhne proution of IL-7A uring zymosn-inue peritonitis in oese mie. J. Leuko. iol. 87, Pini, M., Gove, M. E., Sennello, J. A., vn l, J. W., Chn, L., Fntuzzi, G. (8) Role n regultion of ipokines uring zymosn-inue peritonel inflmmtion in mie. Enorinology 49, Lng, J. E., Willims, E. S., Mizger, J. P., Shore, S. A. (8) Effet of oesity on pulmonry inflmmtion inue y ute ozone exposure: role of interleukin-6. Am. J. Physiol. Lung Cell. Mol. Physiol. 94, L3 L. 46. Shore, S. A., Lng, J. E., Kshr, D. I., Lu, F. L., Verout, N. G., Si, H., Willims, E. S., Terry, R. D., Lee, A., Johnston, R. A. (9) Pulmonry responses to suute ozone exposure in oese vs. len mie. J. Appl. Physiol. 7, Li, S., Sun, Y., Ling, C. P., Thorp, E.., Hn, S., Jehle, A. W., Srswthi, V., Prigen,., Knter, J. E., Li, R., Welh, C. L., Hsty, A. H., ornfelt, K. E., reslow, J. L., Ts, I., Tll, A. R. (9) Defetive phgoytosis of poptoti ells y mrophges in therosleroti lesions of o/o mie n reversl y fish oil iet. Cir. Res. 5, Cole, N., Krokenerger, M., o, S., egley, K. W., Husn, A. J., Willox, M. () Effets of exogenous interleukin-6 uring Pseuomons eruginos ornel infetion. Infet. Immun. 69, Fenton, R. R., Molesworth-Kenyon, S., Okes, J. E., Lush, R. N. () Linkge of IL-6 with neutrophil hemottrtnt expression in virusinue oulr inflmmtion. Invest. Ophthlmol. Vis. Si. 43, Rijnevel, A. W., vn en Doelsteen, G. P., Florquin, S., Stnifor, T. J., Speelmn, P., vn Alphen, L., vn er Poll, T. () Roles of interleukin-6 n mrophge inflmmtory protein- in pneumolysininue lung inflmmtion in mie. J. Infet. Dis. 85, Romno, M., Sironi, M., Tonitti, C., Polentrutti, N., Frusell, P., Ghezzi, P., Fggioni, R., Luini, W., vn Hinsergh, V., Sozzni, S., ussolino, F., Poli, V., Cilierto, G., Mntovni, A. (997) Role of IL-6 n its solule reeptor in inution of hemokines n leukoyte reruitment. Immunity 6, Dio, H., Liu, X., Chen, Y., Xu, W., Co, H., Kohnw, M., Li, L. () Osteopontin expression n reltion to streptool isese severity in mie. Sn. J. Infet. Dis. 43, Nikoljuk, A., Kowlsk, I., Krzewsk-Kupzewsk, M., Amsk, A., Otziomek, E., Wolzynski, S., Kinlsk, I., Gorsk, M., Strzkowski, M. () Serum solule glyoprotein 3 onentrtion is inversely relte to insulin sensitivity in women with polyysti ovry synrome. Dietes 59, Prk, E. J., Lee, J. H., Yu, G. Y., He, G., Ali, S. R., Holzer, R. G., Osterreiher, C. H., Tkhshi, H., Krin, M. () Dietry n geneti oesity promote liver inflmmtion n tumorigenesis y enhning IL-6 n TNF expression. Cell 4, Fttori, E., Cppelletti, M., Cost, P., Sellitto, C., Cntoni, L., Crelli, M., Fggioni, R., Fntuzzi, G., Ghezzi, P., Poli, V. (994) Defetive inflmmtory response in interleukin 6-efiient mie. J. Exp. Me. 8, KEY WORDS: STAT-3 MMP-7 hemokines ytokines inflmmtion 966 Journl of Leukoyte iology Volume 9, June