1α-hydroxylase Adoptive Gene Therapy Ameliorates DSS-induced Colitis Without Causing Hypercalcemia in Mice

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1 The Americn Society of Gene & Cell Therpy originl rticle Trgeted 25-hydroxyvitmin 1α-hydroxylse Adoptive Gene Therpy Ameliortes DSS-induced Colitis Without Cusing Hyperclcemi in Mice Bo Li 1, Dvid J Bylink 1, Michel H Wlter 1, Kin-Hing Willim Lu 1,2, Xinmei Meng 1, Jun Wng 3, Andriy Cherks 1,4, Xiolei Tng 1 nd Xuezhong Qin 1,2 1 Deprtment of Medicine, Lom Lind University, Lom Lind, Cliforni, USA; 2 Musculoskeletl Disese Center, Jerry L. Pettis Memoril Veterns Affirs Medicl Center, Lom Lind, Cliforni, USA; 3 Deprtment of Pthology nd Humn Antomy, Lom Lind University, Lom Lind, Cliforni, USA; 4 Deprtment of Medicine, Lviv Stte College of Physicl Culture, Lviv, Ukrine Systemic 1,25(OH) 2 tretment meliorting murine inflmmtory bowel diseses (IBD) could not be pplied to ptients becuse of hyperclcemi. We tested the hypothesis tht incresing 1,25(OH) 2 synthesis loclly by trgeting delivery of the 1α-hydroxylse gene (27B1) to the inflmed bowel would meliorte IBD without cusing hyperclcemi. Our trgeting strtegy is the use of CD11b + /Gr1 + monocytes s the cell vehicle nd mcrophge-specific promoter (Mc1) to control 27B1 expression. The CD11b + /Gr1 + monocytes migrted initilly to inflmed colon nd some helthy tissues in dextrn sulfte sodium (DSS) colitis mice; however, only the migrtion of monocytes to the inflmed colon ws sustined. Adoptive trnsfer of Gr1 + monocytes did not cuse heptic injury. Infusion of Mc1-27B1-modified monocytes incresed body weight gin, survivl, nd colon length, nd expedited mucosl regenertion. Expression of pthogenic Th17 nd Th1 cytokines (interleukin (IL)-17 nd interferon (IFN)-α) ws decresed, while expression of protective Th2 cytokines (IL-5 nd IL-13) ws incresed, by the tretment. This therpy lso enhnced tight junction gene expression in the colon. No hyperclcemi occurred following this therpy. In conclusion, we hve for the first time obtined proof-of-principle evidence for novel monocyte-bsed doptive 27B1 gene therpy using mouse IBD model. This strtegy could be developed into novel therpy for IBD nd other utoimmune diseses. Received 14 My 214; ccepted 24 September 214; dvnce online publiction 25 November 214. doi:1.138/mt INTRODUCTION Ulcertive colitis (UC) nd Crohn s disese re the two mjor inflmmtory bowel diseses (IBD), which ffect more thn 3.6 million people in the United Sttes nd Europe lone. 1,2 The incidence of IBD, especilly UC, hs significntly incresed in the trditionlly low-risk res, such s Asi nd southern Europe. 3 IBD is not only common disese but lso reoccurring nd incurble, leding to significnt morbidity nd even deth t dvnced stges. Current IBD mngements include the use of nti-inflmmtory drugs (5-minoslicylic cid), immuno-suppressnts (glucocorticoids, zthioprine, nd 6-mercptopurine), nd the most recently biologicl drugs, such s nti-tumor necrosis fctor-α monoclonl ntibody (infliximb). 4 These drugs hve limittions due to their vrious severe side effects. 3 6 Of prticulr concern is the use of nti-immunity-bsed drugs becuse they cn increse the risk of serious infection tht hs lredy been problem in IBD ptients. Thus, n effective nd sfe therpy for IBD remins to be n importnt unmet need. IBD hs been viewed s n utoimmune disese, in which mucosl immune system shows n berrnt response towrds luminl ntigens, such s dietry fctors nd/or commensl bcteri, in geneticlly susceptible individuls. 7 Thus, gents with both nti-immune nd nti-microbil properties re likely to be better cndidtes for IBD tretment thn those with either function lone. One of the gents flling into this ctegory is 1,25(OH) 2, the ctive form of vitmin. Although the clssicl function of this hormone is to regulte clcium homeostsis, studies in the pst two decdes hve provided compelling evidence for n importnt role of 1,25(OH) 2 in suppressing utoimmune inflmmtion. 8 1 It ppers tht 1,25(OH) 2 exerts this ction vi regulting the development/function of essentilly ll types of cells involved in innte nd doptive immunity ,25(OH) 2, unlike trditionl immunosuppressive molecules, does not promote, but in fct my reduce, infection by promoting ntimicrobil peptide production Moreover, it improves mucosl brrier function, which is compromised in IBD, by enhncing synthesis of tight junction proteins The therpeutic benefits of 1,25(OH) 2 in utoimmune disorders ws first demonstrted in 1991 in mice with experimentl utoimmune encephlomyelitis (EAE), n niml model of humn multiple sclerosis. 22 Subsequently, systemic tretment with 1,25(OH) 2 ws shown to be effective in tretment of IBD in mice Unfortuntely, the optiml therpeutic efficcy could not be chieved unless hyperclcemic doses of 1,25(OH) 2 were utilized, regrdless of the types of the diseses. 14,16,22 25,27 Thus, until Correspondence: Xuezhong Qin, Division of Regenertive Medicine, Deprtment of Medicine, School of Medicine, Lom Lind University, Anderson Street, Lom Lind, Cliforni, USA. E-mil: xqin@llu.edu Moleculr Therpy vol. 23 no. 2, feb

2 Monocyte-bsed 27B1 Gene Therpy for IBD The Americn Society of Gene & Cell Therpy the issue of hyperclcemi is stisfctorily ddressed, this promising therpy is unlikely to be used in clinicl settings. One wy to chieve the therpeutic benefit but lso circumvent the systemic hyperclcemic dverse effect of 1,25(OH) 2 is to increse the synthesis of 1,25(OH) 2 loclly in the inflmed bowel. It is known tht the rte of 1,25(OH) 2 synthesis in the body is determined by the hydroxyltion of 25(OH) in the kidney s well s in extr-renl tissues; rection tht is ctlyzed by the 1α-hydroxylse encoded by the 27B1 gene. 28 We resoned tht, if strtegy could be developed to trget expression of this enzyme loclly to the inflmed bowel, therpeuticlly dequte concentrtion of 1,25(OH) 2 could be obtined loclly to tret intestinl lesions without cusing hyperclcemi. To test the forementioned hypothesis, we developed strtegy to trget the 1α-hydroxylse gene bsed on the physiologicl phenomen tht mcrophges re recruited to the inflmed bowel, nd tht the Mc1 promoter is ctivted in the recruited ctivted mcrophges. We selected subtype of monocytes/mcrophges, which specificlly migrte to inflmed tissues, nd engineered these cells to overexpress the 1α-hydroxylse gene under the control of the promoter of the Mc1 gene, which is expressed t high level upon mcrophge ctivtion. 29 We found tht single doptive trnsfer of the 27B1 gene-modified monocytes/mcrophges effectively meliorted cute IBD induced by the dextrn sulfte sodium (DSS) insult in mice. Importntly, this therpy did not cuse hyperclcemi s it occurs during systemic 1,25(OH) 2 tretment. RESULTS Development of novel strtegy to trget expression of 27B1 loclly t the site of inflmed bowel Development of the doptive 27B1 gene therpy requires t lest three key elements: (i) n optiml cell vehicle to deliver the 27B1 gene to the inflmed bowel, (ii) suitble promoter to restrict the 27B1 expression in ctivted mcrophges, nd (iii) production of dequte mounts of 1,25(OH) 2 in the 27B1 gene-modified cells. We selected the CD11b + /Gr1 + monocytes s the cell vehicle becuse previous study hs shown tht Gr1 +, but not Gr1, monocytes preferbly trffic to inflmed tissues in other disese model. 3 In ddition, our pilot experiments in the DSS-colitis model hve confirmed tht there ws inefficient recruitment of Gr1 monocytes to the inflmed colon (dt not shown). Although CD11b + /Gr1 + monocytes were ble to improve the DSS-induced colitis nd CD14 + blood monocytes were recruited to the inflmed bowel of IBD ptients, 31,32 distribution of CD11b + /Gr1 + monocytes nd their specificity of trfficking to the inflmed colon hve not yet been fully investigted in the IBD model. To ddress this importnt issue, we injected fluorescence-lbeled CD11b + /Gr1 + monocytes nd evluted their tissue distribution profile by fluorescence microscopy. We chose to use fr-red dye to lbel the monocytes, becuse this fluorescent dye hs essentilly no uto fluorescence in frozen tissue sections nd hs been used to evlute trfficking of mesenchyml stem cells in mice with DSS-induced colitis. 33 At 1 dy postinjection, lbeled monocytes were present not only in the inflmed colon but lso in the lung, kidney, spleen, nd liver of the injected nimls (Figure 1). Interestingly, no lbeled cells were detected in the duodenum, which is the primry site of ctive C bsorption, nd which is not inflmed following the DSS insult (Figure 1). At 3 dys post-cell injection, substntil mounts of cells remined in the inflmed colon, while those lbeled monocytes, which initilly migrted to other tissues, ll disppered (Figure 1b). These dt suggest tht CD11b + /Gr1 + monocytes require n inflmmtory environment to survive or engrft, thereby representing suitble cell vehicle for use to trget 27B1 gene expression t the inflmed bowel. It hs previously been reported tht heptic dmges in mice treted with crbon tetrchloride ws ttenuted in CCR2 knockout mice, which hd reduced number of Gr1 + monocytes in the injured liver. 34 These dt suggest indirectly tht G1 + monocyte my enhnce liver dmge under certin conditions. Thus, we lso evluted if doptive trnsfer of CD11b + /Gr1 + monocytes could potentilly cuse liver dmge in mice with DSS-induced colitis. Tretment of mice with DSS lone or together with the monocyte trnsfer hd no significnt effect on liver expression of inflmmtory cytokines such s IL-1β nd IL-6 (Supplementry Figure S1,b). Expression of tumor necrosis fctor-α nd IFNα in liver ws very low nd did not differ significntly mong tretment groups (dt not shown). The ctivity of serum lnine minotrnsferse (ALT), commonly used serum mrker of liver dmge, ws lso not ffected by either the DSS tretment or the monocyte infusion (Supplementry Figure S1c). Collectively, our dt suggest tht the CD11b + /Gr1 + monocytes purified under our defined conditions re not likely to hve negtive effects on the liver function in mice of this cute IBD model. While the use of inflmmtion-specific CD11b + /Gr1 + monocytes s the cell vehicle my be dequte to trget expression of exogenous 27B1 gene to the inflmed bowel, the use of tissue-specific promoter, such s the Mc1 promoter, which is highly ctive only in ctivted mcrophges 35 cn further restrict 27B1 expression to ctivted mcrophges. The ctivity nd specificity of the Mc1 promoter to drive mrker gene green fluorescent protein () expression ws ssessed in vivo using mice trnsplnted with hemtopoietic stem cells (HSCs) tht hd been trnsduced with lentivirl vector expressing the gene driven by the Mc1 promoter (Figure 2). As nticipted, the mjority of peritonel cells of these mice re mcrophges, nd they expressed Mc1-driven (dt not shown). Further ctivtion of these mcrophges by tretment with phorbol 12-myristte 13-cette in vitro yielded much stronger expression of Mc1-driven (Figure 2b). On the other hnd, phorbol 12-myristte 13-cette-treted spleen cells, which contin donor HSCs-derived monocytes, showed very low level of expression (Figure 2b). To further evlute the reltive ctivity of the Mc1 promoter in mcrophges versus resting monocytes, Gr1 + monocytes isolted from bone mrrow trnsplnts were treted with mcrophge colony-stimulting fctor (M-CSF) to induce differentition of monocytes to M2 mcrophges. The level of signl ws very wek in resting monocytes (dt not shown) or t dy 3 following M-CSF tretment (Figure 2c). However, t dy 7, when monocytes were fully differentited into M2 mcrophges, the expression ws clerly evident. These dt suggest tht the ctivity of the Mc1 promoter is wek in monocytes, but it becomes highly ctivted fter the monocytes re differentited into mcrophges vol. 23 no. 2 feb. 215

3 The Americn Society of Gene & Cell Therpy Dy 1 Monocyte-bsed 27B1 Gene Therpy for IBD b Dy 3 Figure 1 Engrftment of CD11b+/Gr1+ monocytes t the inflmed colon of mice with dextrn sulfte sodium (DSS)-induced colitis. ( nd b) DSS colitis in 8-week-old C57BL/6 mice ws induced s described in Mterils nd Methods. Gr1+ monocytes were isolted from C57BL/6 mice, lbeled with fr-red dye nd injected, vi til vein, into colitis mice (5 16 cells per mouse) t dy 7 of the DSS tretment. At dy 8 or 1 (1 or 3 dys post-cell infusion), mice were perfused with phosphte-buffered sline nd the tissue smples were collected for imging. () Ex vivo imging of fr-red dye lbeled monocytes in frozen tissue sections t dy 1 post-cell infusion. (b) Ex vivo imging of fr-red dye lbeled monocytes in frozen tissue sections t dy 3 post-cell infusion. Arrows indicte lbeled monocytes in orgns of injected mice. Imges show 1 originl mgnifictions. Adoptive trnsfer of Gr1+ monocytes or M2 mcrophges overexpressing 27B1 effectively meliorted DSS-colitis without cusing hyperclcemi We next evluted if the gene trgeting strtegy developed bove could deliver the therpeutic 27B1 gene to the inflmed colon to tret DSS-induced colitis. Becuse lentivirl vectors trnsduce Gr1+ monocytes very poorly nd ineffectively, we chose n lterntive pproch to obtin proof-of-principle evidence for the efficcy of our therpy by using monocytes isolted from bone mrrow trnsplnts overexpressing 27B1 under the control of the Mc1 promoter s the cell vehicle. The design of this experiment ws illustrted in Figure 3. Sc1+ HSCs were trnsduced with either Mc1-27B1-PGK- lentivirl vector (therpeutic vector) or Mc1--PGK-mCherry (control vector). fluorescence-ctivted cell sorting (FACS) nlysis reveled tht ~7% of HSCs were trnsduced by the lentivirl vectors (Supplementry Figure S2). Five weeks fter trnsplnttion with the trnsduced HSCs, CD11b+Gr1+ monocytes were isolted from bone mrrow of the trnsplnt recipients. Essentilly ll of the purified monocytes were positive for both CD11b nd Gr1 (~97%, Supplementry Figure S2b) nd more thn 6% of Moleculr Therpy vol. 23 no. 2 feb. 215 these monocytes expressed the trnsgene (Supplementry Figure S2c). Adoptive trnsfer of Mc1-27B1-expressing Gr1+ monocytes obtined from bone mrrow trnsplnts not only prevented further loss in body weight but lso regined some of the lost body weight during the disese induction phse (Figure 3b). Approximtely 2% of DSS-colitis mice receiving no tretment or infused with -expressing control monocytes died before the end of the experiment (Figure 3c). In contrst, no mortlity occurred in mice treted with Mc1-27B1-expressing monocytes (Figure 3c). There ws lso significnt increse in colon length in mice treted with Mc1-27B1-expressing monocytes compred to mice receiving no tretment or Mc1-expressing monocytes (Figure 3d; Supplementry Figure S3), indicting tht the tretment lso effectively reversed the DSS-induced shrinkge of the colon. Importntly, serum C level did not differ significntly mong the test groups (Figure 3e). Hemtoxylin nd eosin (H&E) stining of the colon crosssections reveled robust regenertion of the lost crypts, ccompnied by ttenuted inflmmtory cell infiltrtion (Figure 3f; lower mgnifiction is shown in Supplementry Figure S3b). Accordingly, histologicl score nd mucosl regenertion index 341

4 Monocyte-bsed 27B1 Gene Therpy for IBD The Americn Society of Gene & Cell Therpy b Splenocytes + PMA Peritonel mcrophges + PMA c Monocytes + M-CSF Monocytes + M-CSF Dy 3 Dy 7 Mc1- Mc1- Non-fluorescent Non-fluorescent Figure 2 Mc1 promoter ws ble to yield high levels of trnsgene expression in ctivted mcrophges. () The lentivirl vector contins centrl polypurine trct (cppt) for efficient nucler entry, nd self-inctivting deletion in the U3 region of the LTRs. The cis-cting prtil Gg sequence (Gg) nd Rev-response element (RRE) sequences re lso indicted. WPRE (woodchuck heptitis post-trnscriptionl regultory element) element is included to enhnce mrker gene expression. (b) Sc1 + HSCs were isolted from bone mrrow cells of C57BL/6 mice, trnsduced with the lentivirl vector Mc1--PGK-mCherry, nd injected vi til vein into recipient mice. Five weeks post-bone mrrow trnsplnttion, peritonel mcrophges nd splenocytes were isolted nd further treted with 5 ng/ml phorbol 12-myristte 13-cette for 16 hours. Expression of the mrker gene,, ws visulized under fluorescent microscopy ( 2 mgnifiction). (c) Bone mrrow CD11b + Gr1 + monocytes were isolted from the bone mrrow trnsplnts nd then treted with mcrophge colony-stimulting fctor for 7 dys. Expression of the mrker gene ws visulized under fluorescent microscopy t dy 3 nd dy 7 ( 2 mgnifiction). were significntly improved by the therpy (Figure 3g,h). Mice with DSS-induced colitis infused with Mc1--expressing monocytes showed only moderte improvement in body weight loss (Figure 3b). Moreover, this cell vehicle tretment lone (without the Mc1-27B1 trnsgene) produced little benefits in terms of reducing mortlity (Figure 3c), reducing DSS-induced shrinkge of colon (Figure 3d), or enhncing mucosl regenertion (Figure 3f h; Supplementry Figure S3,b). After estblishing the proof-of-principle for our therpy using Mc1-27B1-expressing monocytes generted by the bone mrrow trnsplnttion pproch, we sought to develop cliniclly relevnt strtegy to engineer monocytes to overexpress Mc1-27B1 for the IBD tretment. We found tht, in contrst to monocytes in suspension, ttched mcrophges generted from Gr1 + monocytes treted with M-CSF were efficiently trnsduced with lentivil vectors (~7%, Supplementry Figure S4). FACS nlysis reveled tht ~85% of the mcrophges remined Gr1-positive (Supplementry Figure S4b). While level of CD11b expression remined unchnged (Supplementry Figure S4c), expression of the M2 mcrophge mrker, CD26, in these mcrophges ws high (Supplementry Figure S4d). This result indictes tht the mcrophges prepred under our defined conditions belong to the immunosuppressive M2 mcrophges. Interestingly, compred to monocytes, these M2 mcrophges expressed high levels of chemokine receptor 2 (CCR2) (Supplementry Figure S4e), which is essentil for the homing of monocytes to inflmmtory sites. 36 Next, experiments were performed to evlute if M2 mcrophges trnsduced with the Mc1-27B1 lentivirl vector ws ble to meliorte DSS-induced colitis. M2 mcrophges trnsduced with the Mc1-27B1 lentivirl vector led to high levels of 27B1 expression nd the consequent synthesis of lrge mounts of 1,25(OH) 2 from the substrte, 25(OH) (Figure 4,b). Similr to the tretment with Mc1-27B1-expressing Gr1 + monocytes, single doptive trnsfer of Mc1-27B1 gene modified M2 mcrophges to mice with DSS-induced colitis (Supplementry Figure S5) prevented further loss in body weight nd reduced mortlity without significntly elevting serum C levels (Supplementry Figure S5b d). These benefits were ccompnied with remrkble improvement in histopthology (Figure 4c e; low-mgnifiction dt re shown in Supplementry Figure S5e). Notbly, while doptive trnsfer of Mc1--expressing control M2 mcrophges lone, reduced further body weight loss modertely (Supplementry Figure S5b), it did not significntly improve the mucosl histopthology (Figure 4c e). These results indicte tht M2 mcrophges cn be used in plce of Gr1 + monocytes s the cell vehicle for our therpy. Evidence tht Mc1-27B1-expressing monocytes intercted with immune cells nd epithelil cells to reduce inflmmtion nd to increse mucosl regenertion Towrd understnding the mechnism by which doptive trnsfer of Mc1-27B1-expressing monocytes meliortes IBD, we evluted the effect of recruitment of Mc1-27B1-expressing monocytes to inflmmtory sites on immune cells nd epithelil cells. The pro-inflmmtory cytokines produced by infiltrting vol. 23 no. 2 feb. 215

5 The Americn Society of Gene & Cell Therpy Monocyte-bsed 27B1 Gene Therpy for IBD C57BL/6 mouse 1Gy Irrdition Til vein injection 4h lter 3% DSS 7 dys Clodrosome t dy 5 Lenti-Mc--HSCs Lenti-Mc--HSCs 5 weeks Bone mrrow Gr1 + monoctyes Til vein injection - Dy 7 Wter 6 dys Scrificed for nlysis b c d e *** *** 14 * ** *** ** Control Control 7. 8 * 6 DSS DSS DSS+ DSS+ 4 2 DSS+ DSS Body weight chnge (%) Survivl rte (%) Colon length (cm) Serum clcium (mg/dl) Dys post-dss tretment Dys post-dss tretment 5. Control DSS Control DSS f Control DSS DSS + DSS + g h *** *** * 5 2. *** *** Histologicl score Index of mucosl regenertion Control DSS Control DSS Figure 3 Adoptive trnsfer of CD11b + /Gr1 + monocytes overexpressing 27B1 meliorted dextrn sulfte sodium (DSS)-induced colitis without cusing hyperclcemi. () Six-week-old C57BL/6 mice were trnsplnted with Sc1 + HSCs trnsduced with the Mc1- (Mc1- h27b1-pgk-mcherry) or the Mc1- (Mc1--PGK-mCherry) lentivirl vector. At the ge of 8 weeks, ech recipient mouse ws treted with DSS to induce colitis. At dy 7 of the DSS tretment, mice were injected vi til vein with Gr1 + monocytes tht were isolted from the bone mrrow trnsplnts. (b) Body weights were recorded during disese induction nd recovery period. (c) Survivl rte ws recorded dily. (d) At dy 13 (6 dys post-monocytes infusion), mice were scrificed nd the colon length ws mesured. (e) Serum concentrtions of clcium were mesured 6 dys post-monocytes infusion. Dt from one of the two independent experiments re shown s mens ± SEM (n = 5 7). (f) Representtive H&E stining of distl colon cross-sections. All imges re shown t 2 mgnifiction. (g) Histologicl score ws mesured in cross-sections of the distl colon. Dt re presented s mens ± SEM (n = 1 12) pooled from two independent experiments. (h) Mucosl regenertion index ws determined t cross-sections of the distl colon (Mteril nd Methods). Dt of the representtive of the two independent experiments re presented s mens ± SEM (n = 4 5). *P <.5, **P <.1, or ***P <.1 versus DSS-treted mice. Mo, monocytes. mcrophges nd dendritic cells (tumor necrosis fctor-α, IL-1β, IL-6, IL-12, nd IL-23) were ll incresed drmticlly by the DSS insult nd these increses were significntly suppressed by doptive trnsfer of the Mc1-27B1-expressing monocytes (Figure 5). Similrly, doptive trnsfer of Mc1-27B1- expressing monocytes significntly suppressed the DSS-induced Moleculr Therpy vol. 23 no. 2 feb

6 Monocyte-bsed 27B1 Gene Therpy for IBD The Americn Society of Gene & Cell Therpy 8 *** b 6, 27B1 mrna level (fold) ,25 (OH) 2 (pg/ml) 4, 2, c LV: Mc1- Control Mc1- LV: Mc1- Mc1-25 (OH)D: + + DSS DSS + M2 - DSS + M2 - d *** e Histologicl score *** N.S. Index of mucosl regenertion *** ** Control DSS M2 - M2 - Control DSS M2 - M2 - Figure 4 Adoptive trnsfer of CD11b + /Gr1 + M2 mcrophges overexpressing 27B1 improved IBD pthology. ( nd b) M2 mcrophges were trnsduced with the indicted lentivirl vectors nd were incubted with 2.5 μmol/l 25(OH) for 2 hours (Mterils nd Methods). () Humn 27B1 mrna level ws mesured by rel-time qrt-pcr. Dt re shown s mens ± SEM (n = 4). ***P <.1. (b) 1,25(OH) 2 concentrtion in the conditioned medi pooled from triplicte smples ws mesured by rdioimmunossy. (c) Colitis induction in 8-week-old C57BL/6 mice nd M2 mcrophges doptive trnsfer were performed described in Mteril nd Methods. On dy 13 (6 dys post-mcrophges infusion), mice were scrificed, nd the colon smples were collected. Representtive H&E stining imges of the frozen distl colon sections were shown. All imges re shown t 2 mgnifiction. (d) Histologicl score of colon sections ws determined s described in Mterils nd Methods. Dt re presented s mens ± SEM (n = 5). (e) Mucosl regenertion index ws determined s described in Mteril nd Methods. Dt re presented s mens ± SEM (n = 4 5). **P <.1, or ***P <.1 versus dextrn sulfte sodium-treted mice. M2Ø, M2 mcrophges. expression of pthogenic Th1 nd Th17 cytokines (IFN-α nd IL-17) (Figure 5b,c). Infusion of control Mc1--expressing monocytes lone did not significntly reduce the expression of inflmmtory cytokines except for IL-6 nd IFN-α (Figure 5,b). The expression of protective Th2 cytokines (IL-4, IL-5, nd IL-13) in both the colon (Figure 6) nd lymph nodes (Figure 6b) ws incresed significntly by infusion of Mc1-27B1- expressing monocytes but not the Mc1--expressing monocytes. However, in contrst to the other Th2 cytokines exmined, the expression of IL-1 ws incresed (rther thn decresed) in the inflmed colon nd this increse ws ttenuted by the Mc1-27B1 monocyte trnsfer (Figure 6). We lso evluted the effect of Mc1-27B1 monocyte infusion on gene expression of cludin-1 nd zonul occludens protein-1 (ZO-1), which re importnt epithelil tight junction proteins. Consistent with the results obtined using systemic 1,25(OH) 2 tretment, 2 doptive trnsfer of Mc1-27B1 monocytes significntly incresed the mrna levels of these two genes (Figure 7,b). Next, we determined whether the incresed expression of the tight junction genes in the inflmed colon is secondry effect resulting from the recovery of colon inflmmtion or direct effect of the loclly produced 1,25(OH) 2 on epithelil cells. Tretment of Cco-2 epithelil cells with DSS reduced mrna level of these two genes. Such decrese ws either completely or prtilly bolished by the 1,25(OH) 2 tretment (Figure 7c). Consistent with this dt, tretment of Cco-2 cell monolyers with 1,25(OH) 2 functionlly prevented the DSS tretment-induced monolyer lekge (Figure 7d). Thus, it is very vol. 23 no. 2 feb. 215

7 The Americn Society of Gene & Cell Therpy Monocyte-bsed 27B1 Gene Therpy for IBD *** TNF-α * 1, 8 6 * IL-1 β * 6, 4, ** IL-6 * ** Proinflmmtory cytokines mrna levels (fold) Control DSS IL Control DSS 4 IL-23 3 ** * ** * 2 2, Control DSS 1 1 Control DSS Control DSS b Th1 cytokine mrna (fold) 5 4 *** IFN-γ ** * c Th17 cytokine mrna (fold) IL-17 ** ** Control DSS Control DSS Figure 5 Adoptive trnsfer of CD11b + /Gr1 + monocytes overexpressing 27B1 reduced the expression of inflmmtory cytokines in the colon of mice with dextrn sulfte sodium (DSS)-induced colitis. The experimentl design ws described in Figure 3. () Rel-time quntittive reverse trnscription polymerse chin rection (qrt-pcr) nlysis of the mrna levels of proinflmmtory cytokines produced minly by mcrophges/dendritic cells. (b) Rel-time qrt-pcr nlysis of the mrna level of IFN-γ, pthogenic Th1-specific cytokine in the colon. (c) Rel-time qrt- PCR nlysis of the mrna level of IL-17, pthogenic Th17-specific cytokine in the colon. Dt of representtive experiment of two independent experiments re presented s mens ± SEM (n = 4 6). *P <.5, **P <.1, or ***P <.1 versus DSS-treted mice. Mo, monocytes. likely tht 1,25(OH) 2 produced loclly by Mc1-27B1- expressing monocytes could promote mucosl regenertion in prt through direct ctions on epithelil cells to promote expression of tight junction genes. DISCUSSION The therpeutic benefit of 1,25(OH) 2 in tretment of utoimmune inflmmtory diseses ws estblished in niml models more thn two decdes go However, little progress hs been mde towrd trnsltion of this invention to the tretment of humn diseses, due to lck of strtegy to overcome the ttending hyperclcemi. Herein, we hve obtined proof-of-principle evidence for novel biologic therpy, which is 1,25(OH) 2 -bsed but is entirely different from the systemic 1,25(OH) 2 tretment. This therpy, which involves doptive trnsfer of inflmmtionspecific monocytes engineered to loclly synthesize 1,25(OH) 2, ws cpble of meliorte IBD without cusing hyperclcemi. The proof-of-principle evidence for this experimentl therpy ws obtined using the DSS-induced colitis mouse IBD model tht fithfully reproduces mny of the immunologicl disturbnces observed in humn IBD. 37 Recent dt suggest tht microbiot plys n importnt role in mintining homeostsis in the Moleculr Therpy vol. 23 no. 2 feb

8 Monocyte-bsed 27B1 Gene Therpy for IBD The Americn Society of Gene & Cell Therpy b Figure 6 Adoptive trnsfer of CD11b + /Gr1 + monocytes overexpressing 27B1 significntly incresed expression of the protective Th2 cytokines. The experimentl design ws described in Figure 3. Colon nd lymph node smples were collected 6 dys post-monocytes infusion. () mrna levels of Th2 cytokines in colon were mesured by rel-time quntittive reverse trnscription polymerse chin rection (qrt-pcr). (b) mrna levels of Th2 cytokines in lymph nodes were mesured by rel-time qrt-pcr. Dt re presented s mens ± SEM (n = 4 6). *P <.5, **P <.1 versus dextrn sulfte sodium-treted mice. Mo, monocytes. gut nd dysbiosis is often ssocited with humn IBD. 38 In this regrd, lthough DSS-induced colitis my not represent the entire pthogenic process of humn IBD, the DSS-induced colitis fithfully simultes the biologicl consequences s result of dysbiosis nd is therefore highly relevnt to humn IBD. The gross nd locl pthologicl chnges in DSS-induced colitis include hemtochezi, body weight loss, colon shrinkge, mucosl ulcers, nd inflmmtory cells infiltrtion. 37 All of these pthologies were remrkbly improved by single i.v. infusion of CD11b + /Gr1 + monocytes overexpressing 27B1 controlled by the Mc1 promoter. Zhng et l. 31 reported recently tht CD11b + / Gr1 + monocytes were induced in the spleen of mice with DSSinduced colitis, nd tht doptive trnsfer of splenic CD11b + / Gr1 + cells derived from the disesed mice improved DSS-induced colitis. 31 These monocytes re referred to inflmmtory monocytes with immunosuppressing function nd cn be quickly recruited into inflmmtion sites. In this regrd, the biologicl chrcteristics nd function of CD11b + /Gr1 + monocytes re not unique to the DSS-induced colitis model. We found tht infusion of CD11b + /Gr1 + monocytes overexpressing modertely reduced body weight loss nd mrginlly improved colon histologicl score. However, these monocytes did not significntly increse colon length or reduce mortlity under our experimentl conditions. In contrst, tretment with CD11b + /Gr1 + monocytes overexpressing 27B1 significntly improved the mjority of disese prmeters with the most pronounced effect observed on crypt sprouting. The incomplete restortion of mucosl rchitecture is likely due to the extremely severe mucosl dmges obtined using our protocol s well s the short durtion of the tretment. It is importnt to point out tht the therpeutic benefit might hve been underestimted, s some severely disesed colitis mice nd colitis mice treted with mrker gene-expressing monocytes lone did not survive nd were excluded from histopthologicl nlysis. Tken together, our findings clerly demonstrte tht the combintion of CD11b + / Gr1 + monocytes cell therpy nd 27b1 gene therpy could effectively meliorte DSS-induced colitis. Pst studies suggest tht Th1 nd Th17 dptive immune responses ply mjor role in the pthogenesis of DSS-induced colitis. 39 The gut is unique tissue, in tht it hs developed its own complex immune system, the gut-ssocited lymphoid tissue (GALT). 4 The GALT consists of Peyer s ptches (the lymphoid ggregtes in the submucos) nd mesenteric lymph nodes (MLNs) in the intestinl wll. 4 As our therpy is locl, it vol. 23 no. 2 feb. 215

9 The Americn Society of Gene & Cell Therpy Monocyte-bsed 27B1 Gene Therpy for IBD 8 N.S. b Cludin-1 ZO *** ** * mrna level in colon (fold) mrna level in colon (fold).8.4 Control DSS Control DSS c mrna level (fold) Control Vd DSS DSS+1,25D *** *** *** ** d Permebility control s 1% N.S. ** ** ZO-1 Cludin-1 Control 1,25D DSS DSS+1,25D Figure 7 Evidence tht doptive trnsfer of CD11b + /Gr1 + monocytes overexpressing 27B1 meliorted dextrn sulfte sodium (DSS)- induced colitis in prt through restoring epithelil brrier function. ( nd b) The experimentl design ws described in Figure 3. Colon tissues were collected 6 dys post-monocytes infusion. (,b) The mrna level of two tight junction genes, cludin-1 nd ZO-1, in colon ws mesured by rel-time quntittive reverse trnscription polymerse chin rection (qrt-pcr). Dt re presented s mens ± SEM (n = 4 6). (c) Monolyers of Cco-2 cells were pretreted with or without 1 nmol/l 1,25(OH) 2 for 48 hours. 5% DSS ws then dded to the medi for 1 hours before the nlysis of gene expression. The mrna level of ZO-1 nd cludin-1 in cultured epithelil cells ws determined by rel-time qrt-pcr. Dt re presented s mens ± SEM (n = 4). (d) The effects of 1,25(OH) 2 on prcellulr permebility of epithelil cell monolyers. Epithelil monolyers were obtined by culturing Cco-2 cells on filters in trnswells nd were treted s described in pnel c. The permebility to fluorescein isothiocynteconjugted dextrn is presented s % of the vlue in cells treted with DSS only. Dt re presented s mens ± SEM (n = 3). *P <.5, **P <.1, or ***P <.1 versus DSS-treted group. is importnt to exmine if recruited Mc1-27B1-expressing monocytes intercted with immune cells in the inflmed colon. Our results demonstrte tht the expression of protective T cell subtype (Th2) cytokines ws incresed wheres the expression of the pthogenic effector T cell (Th1 nd Th17) cytokines ws decresed in the colon of colitis mice treted with 27B1 genemodified monocytes (Figures 5 nd 6). Interestingly, the colonic expression of the nti-inflmmtory cytokine IL-1 ws decresed rther thn incresed following the doptive trnsfer of Mc1-27B1-expressing monocytes in mice with DSS-induced colitis. It is well known tht IL-1 is n immunoregultory cytokine tht efficiently blocks the in vivo production of proinflmmtory cytokines in colitis. IL-1-deficient mice spontneously develop chronic colitis due to n berrnt immune response to commensl bcteri. 41 However, severl studies hve shown tht the production of IL-1 significntly incresed in the colon of mice with DSSinduced colitis nd peked t lte stges of the disese. 39,42,43 In ddition, production of IL-1 by lmin propri mononucler cells ws significntly enhnced in colon of mice with DSSinduced colitis. 44 Thus, our findings tht colonic expression of IL-1 ws incresed upon the DSS insult re consistent with previous reports. These dt suggest tht endogenous IL-1 my prticipte in self-regultory circuit tht countercts the inflmmtory process in this niml model. Moreover, neutrlizing IL-1 modestly ugmented tissue dmge in DSS-induced colitis, suggesting tht the endogenous IL-1 is indequte to suppress DSS-induced colitis in the presence of excessive mounts of inflmmtory cytokines. 42 Tken together, the therpy developed in our study does not seem to ct directly vi modulting the locl production of IL-1 in the inflmed colon. The reduced colonic expression of IL-1 following this therpy could be the secondry effect resulting from the recovery of colonic inflmmtion. Overll, our findings support our premise tht this therpy ttenutes intestinl inflmmtion in prt through skewing the development of T cell differentition loclly in the colon in fvor of nti-inflmmtion nd immunologicl tolernce (Figure 8). Moleculr Therpy vol. 23 no. 2 feb

10 Monocyte-bsed 27B1 Gene Therpy for IBD The Americn Society of Gene & Cell Therpy Figure 8 A proposed model of the mechnism by which doptive 27B1 gene therpy cts to meliorte dextrn sulfte sodiuminduced colitis. Infused exogenous Gr1 + monocytes or M2 mcrophges overexpressing 27B1 guided by the Mc1 promoter re recruited to the inflmed colon, where they re ctivted to express exogenous 27B1 to synthesize 1,25(OH) 2 loclly. 1,25(OH) 2 produced by the ctivted exogenous mcrophges then cts on T cells to cuse switch from the pthogenic Th1-Th17 response to the protective Th2 response. 1,25(OH) 2 lso cts on infiltrting mcrophges leding to reduced expression of proinflmmtory cytokines. As result of the reduced inflmmtion, mucosl regenertion is expedited. In ddition, 1,25(OH) 2 lso promotes mucosl regenertion vi incresing epithelil tight junction protein synthesis nd restoring epithelil brrier function. Intestinl brrier breching contributes to further ggrvtion of the disese s result of bcteril infection. 45 Therefore, mintining the mucosl brrier function nd integrity could provide potentil benefits in the tretment of IBD. 46 An importnt determinnt of the mucosl brrier function/integrity is the synthesis of epithelil tight junction proteins. It is possible tht incresed expression of these two junction proteins by our therpy is secondry effect of mucosl recovery. However, our finding tht 1,25(OH) 2 tretment of epithelil cells ttenuted the negtive effect of DSS on the expression of tight junction gene nd the integrity of the epithelil monolyer (Figure 7) supports our model tht 1,25(OH) 2 produced by the Mc1-27B1- expressing monocytes cts directly on proliferting epithelil cells to promote tight junction gene expression (Figure 8). The therpeutic benefit of the doptive 27B1 gene therpy is not unexpected, s systemic 1,25(OH) 2 tretment is known to meliorte DSS-induced colitis, nd our therpy is designed to promote locl 1,25(OH) 2 synthesis in the inflmed colon. The significnt impct of our study is tht, while this therpy is t lest s effective s the systemic 1,25(OH) 2 therpy, it does not cuse hyperclcemi, which is significnt sfety issue tht cretes severe short- nd long-term helth problems. 24 The successful elimintion of hyperclcemi is direct result of our crefully designed 27B1 gene trgeting strtegy. We found tht CD11b + Gr1 + monocytes do not trffic to duodenum tht is not inflmed in mice with DSS-induced colitis (Figure 1). Becuse duodenum is one of the primry sites of ctive clcium bsorption, the inbility of the Mc1-27B1-expressing monocytes to migrte to this region of the gut helps to void lrge increse in the intestinl clcium bsorption, which is the primry cuse of 1,25(OH) 2 - induced hyperclcemi. We did observe tht the Gr1 + monocytes migrted to other tissues; however, their presence is trnsient nd is unlikely to yield significnt dverse side effects (Figure 1). On the other hnd, sustined presence of G1 + positive monocytes ws seen in the inflmed colon. Pst studies, using CCR2 knockout mice, hve clerly demonstrted tht CCR2 vi binding to its lignd MCP-1 present in the inflmed sites is essentil for recruitment of Gr + positive monocytes to the inflmed tissues. 47 The high level of CCR2 expression in both CD11b + Gr1 + monocytes nd their derived mcrophges tht we observed could explin the reltive specificity of the CD11b + Gr1 + monocytes homing to inflmed colon. The use of Mc1 promoter to control 27B1 expression my hve lso contributed to the prevention of hyperclcemi. Mc1 promoter ws only wekly ctive in monocytes but its ctivity ws significntly incresed in differentited/ctivted mcrophges 35 (Figure 2b). Therefore, the use of the Mc1 promoter my hve served s secondry mechnism to ensure tht ctive expression of the exogenous 27B1 occurs only fter infused monocytes/mcrophges hve become ctivted mcrophges in the locl intestinl lesion. We cknowledge tht our therpy is still in its infnt stge nd the long-term sfety nd efficcy of this novel therpy needs to be thoroughly evluted before it cn be dvnced to trnsltionl studies. One of the concerns of using CD11 + /Gr1 + monocytes or their M2 mcrophges derivtives s the cell vehicle could be tht these cells re immunotolerigenic, thereby exhibiting generl immunosuppressive ctivity. Such ctivities could potentilly led to suppression of the immune system tht my increse the chnces of infection or tumor growth. It is possible tht such potentil risk could be prevented or minimized by the 1,25(OH) 2 synthesized by the Mc1-27B1-expressing mcrophges. 1,25(OH) 2 is regultor nd not merely suppressor of the immune system. 1,25(OH) 2 cn enhnce production of ntibcteril peptides to fight ginst pthogenic bcteri. Also, 1,25(OH) 2 hs been shown to effectively tret vrious cncers in both experimentl nimls nd humns. 48 Our ide tht combintion of M2 mcrophge nd 27B1 could be reltively sfer thn M2 mcrophges lone or other immunosuppressnts need to be confirmed experimentlly. MATERIALS AND METHODS Preprtion of plsmid constructs. The 1.6-kb C-terminlly Myc-tgged humn 27B1 cdna ws mplified by polymerse chin rection (PCR) using plsmid contining the humn 27B1 cdna (OriGene, Rockville, MD). The mplified h27b1 cdna frgment with 5 KOZAK ribosome entry sequence nd the 1.7-kb humn Mc1 promoter were cloned into the prrl-sin.cppt.pgk-.wpre lentivirl vector (Addgene, Cmbridge, MA), in which the cytomeglovirus (CMV) promoter hd been deleted. The resulting construct ws designted the Mc1--PGK-. This bicistronic plsmid expresses 27B1 tht vol. 23 no. 2 feb. 215

11 The Americn Society of Gene & Cell Therpy Monocyte-bsed 27B1 Gene Therpy for IBD is controlled by the mcrophge-specific Mc1 promoter nd tht is controlled by the tissue nonspecific PGK promoter. Similrly, bicistronic vector expressing under the control of the Mc1 promoter nd mcherry under the control of PGK promoter (Mc1--PGK-mCherry) ws lso prepred s control. Lentivirl vectors. Lentivirl vectors were produced in HEK-293T cells s previously described. 49 Briefly, HEK-293T cells were trnsfected with the indicted trnsgene plsmids together with CMV-VSVG nd PAX2 plsmids using clcium phosphte method. Superntnts were collected 48 hours fter trnsient trnsfection nd virl prticles were concentrted by centrifugtion for 24 hours t 6, g t 4 C. After removl of the superntnt, virl prticles were resuspended in phosphte-buffered sline contining 5% glycerol nd stored t 8 C. The biologicl titers were ~5 1 7 prticles/ml, s determined by FACS nlysis of + cells in 293T cells trnsduced with vrious doses of the concentrted vectors. Animls. Femle C57BL/6 mice were purchsed from the Jckson Lbortory (Br Hrbor, ME). Animls were kept in pthogen-free environments t Lom Lind University Animl Cre Fcility nd ll experiments were performed ccording to protocols pproved by the Institutionl Animl Cre nd Use Committee t the Lom Lind University. Induction of DSS colitis. Colitis ws induced in C57BL/6 mice by providing the nimls with 3% DSS (MP Biomedicls, moleculr weight = 36 5 kd, OH) in drinking wter for 7 dys d libitum. Mortlity nd body weight of ech niml were recorded dily. Preprtion of bone mrrow monocytes nd mcrophges. After lysis of the red blood cells, bone mrrow nucleted cells were collected nd used for isoltion of CD11b + /Gr1 + monocytes using the MACS technology. Gr1 + cells were positively selected using APC-conjugted nti-gr1 ntibody nd nti-apc MicroBeds (Miltenyi Biotec, Auburn, CA). To obtin M2 mcrophges, the isolted bone mrrow CD11b + /Gr1 + monocytes were cultured in RPMI medium 164 (Invitrogen, Grnd Islnd, NY) contining 1% fetl bovine serum (Invitrogen) nd 4 ng/ml of mouse M-CSF (R&D Systems, Minnepolis, MN) for 5 dys. Adoptive trnsfer of monocytes or mcrophges. Mice were injected i.v. with 1 μl clodrosome-contining liposoml clodronte (Encpsul NnoSciences, Nshville, TN) to deplete endogenous monocytes on dy 5 post-dss tretment. For monocytes doptive trnsfer, CD11b + /Gr1 + monocytes, isolted from bone mrrow cells, were injected vi til vein into mice with DSS-induced colitis 2 dys lter. For mcrophges doptive trnsfer, M2 mcrophges were trnsduced with indicted lentivirl vectors twice t multiplicity of infection of 3 nd injected vi til vein into mice on dy 7 of the DSS tretment. Ex vivo fluorescent imging. Bone mrrow CD11b + /Gr1 + monocytes were lbeled with 1 ìmol/l CellTrce Fr Red (DDAO-SE; Moleculr Probes, Invitrogen) ccording to the mnufcturer s protocol nd previous report. 33 Lbeled monocytes were injected into mice with DSSinduced colitis vi til vein (5 1 6 cells/mouse), which ws pretreted the Clodrosome. Mice were scrificed. The lungs, spleens, livers, kidneys, colon, nd duodenum were isolted from the treted mice nd fixed with 4% prformldehyde. Frozen tissue sections were prepred, counter stined with 4-6-dimidino-2-phenylindole, nd observed with fluorescent microscope (Crl Zeiss, Thornwood, NY) equipped with ner infrred wvelength filter. Bone mrrow trnsplnttion. Sc1-positive HSCs were isolted from bone mrrow cells using Sc-1 MicroBeds kit (Miltenyi Biotec). Cells were cultured in Iscove s modified Dulbecco s medium (Invitrogen) contining 1% fetl bovine serum, humn TPO (R&D Systems), mouse SCF (R&D Systems), humn FL (R&D Systems), humn G-CSF (R&D Systems), nd humn IL-3 (R&D Systems), ech t 1 ng/ml. After overnight culture, cells were trnsduced with the indicted lentivirl vectors t n multiplicity of infection of 3. After 24 hours, cells were trnsduced gin nd hrvested for trnsplnttion. Five-week-old femle C57BL/6 mice were injected vi til vein with trnsduced Sc1-positive HSCs (1 million cells per mouse) 2 hours fter receiving lethl dose of gmm irrdition (1Gy). Engrftment ws ssessed by FACS nlysis of the percentge of + cells in peripherl blood mononucler cells 4 5 weeks post-trnsplnttion. Preprtion of peritonel mcrophges. Mice were intrperitonelly injected with 2 ml of 4% thioglycolte medium (Difco, Detroit, MI). Animls were scrificed 3 dys lter, nd thioglycollte-elicited peritonel mcrophges were hrvested by lvge of the peritonel cvity. Cells were seeded in 24-well pltes (2 1 6 cells/well) nd llowed to dhere to the culture plte for 4 hours t 37 C. After gentle rinsing to remove nondherent cells, the dherent cells were collected s peritonel mcrophges for in vitro experiments. Flow cytometry. Trnsduced Sc1-positive cells or peripherl blood mononucler cells were wshed with phosphte-buffered sline nd nlyzed for expression on FACSAri II flow cytometer (BD Biosciences, Sn Jose, CA). Fluorochrome-conjugted ntibodies specific for mouse CD11b, Gr1, or F4/8 were purchsed from BD or ebioscience (Sn Diego, CA). Cells were stined with respective fluorochrome-conjugted ntibodies, nd then nlyzed by flow cytometer. In vitro evlution of 27B1 trnsgene expression nd ctivity. M2 mcrophges were trnsduced with either Mc1-27B1 or Mc1- lentivirl vector t multiplicity of infection of 3. After 2 dys, the cells were seeded t density of cells/ml in 12-well plte, nd the finl concentrtion of 2.5 μmol/l of 25(OH) (Sigm-Aldrich, St Louis, MO) ws dded. After 2 hours incubtion, cells were hrvested for totl RNA isoltion, nd the reltive mrna level of CPY27B1 ws mesured by rel-time PCR. 1,25(OH) 2 concentrtion in the conditioned medi ws determined by rdioimmunossy by the Hertlnd Assys (Ames, IA). Histologicl nlysis. After flushing with cold phosphte-buffered sline, the distl colon tissues were fixed in 4% prformldehyde. Eight micrometers-thick frozen sections were stined with H&E (Sigm-Aldrich). Sections were exmined blindly nd scored by pthologist ccording to widely used criteri s previously described. 5 Mucosl regenertion index ws defined s the length of epithelil lyer of the regenerted mucos divided by the length of musculris mucos. ALT ctivity ssy. The serum level of ALT ws mesured using the ALT Activity Assy Kit (Sigm-Aldrich) ccording to the mnufcturer s protocol. Mesurement of serum clcium. Serum clcium levels were mesured using the Clcium Colorimetric Assy Kit (BioVision, Milpits, CA). Cco-2 cell culture nd prcellulr permebility ssy. Humn colonic Cco-2 epithelil cell lines, obtined from Americn Type Culture Collection (Mnsss, VA), were cultured in Dulbecco's modified Egle's medium supplemented with 1% fetl bovine serum, 2 mmol/l glutmine, 1 μg/ml penicillin, nd 1 μg/ml streptomycin in humidified tmosphere contining 5% CO 2 t 37 C. Cco-2 cell monolyer ws incubted with or without 1 nmol/l 1,25(OH) 2 for 48 hours, nd 5% DSS ws then dded to the medium for 1 hours. Prcellulr permebility ws determined by mesuring the flux of fluorescein isothiocynte-conjugted dextrn (4 kd, Sigm-Aldrich) cross Cco-2 monolyers. Briefly, monolyers were gently wshed with Hnk's blnced slt solution twice nd trnsferred to 6 ml Hnk's blnced slt solution. Apicl chmber were gently spirted nd replced with 1 ìl of 1 mg/ml fluorescein isothiocynte-conjugted dextrn in Hnk's blnced slt solution. The monolyers were incubted t 37 C for 2 hours. A volume of 1 ìl smple ws removed from the bsl chmber, nd the fluorescence ws mesured Moleculr Therpy vol. 23 no. 2 feb

12 Monocyte-bsed 27B1 Gene Therpy for IBD The Americn Society of Gene & Cell Therpy using fluorescent plte reder (excittion 485 nm, emission 52 nm, Synergy HT; Bio-Tek Instruments, Winooski, VT). Quntittive rel-time RT-PCR. Totl RNA ws isolted from cell nd tissue smples using RNesy mini kit (Qigen, Vlenci, CA). cdna ws prepred using the superscript III cdna synthesis kit (Invitrogen), ccording to instructions. Rel-time PCR ws performed with SYBR Green PCR Mster Mix (Applied Biosystems, Crlsbd, CA) in 75 Fst Rel Time PCR System (Applied Biosystems). The dt were normlized to GAPDH mrna s reference nd presented s fold chnge reltive to control smples. Specific primers for ech gene of interest were given in Supplementry Tble S1. Sttisticl nlysis. Results re expressed s men ± SEM nd sttisticlly nlyzed by Student s T-test or one-wy nlysis of vrince. A vlue of P <.5 ws considered sttisticlly significnt. SUPPLEMENTARY MATERIAL Figure S1. Adoptive trnsfer of CD11b + /Gr1 + monocytes did not cuse heptic injury in mice with DSS-induced colitis. Figure S2. Trnsduction efficiency of Sc1 + HSCs with lentivirl vector, nd engrftment efficiency of exogenous HSCs in recipient mice. Figure S3. Adoptive trnsfer of CD11b + /Gr1 + monocytes overexpressing 27B1 decresed DSS-induced colonic inflmmtion, nd reduced shrinkge nd improved overll integrity of the colon of mice with DSS-induced colitis. Figure S4. Chrcteriztion of Gr1 + monocyte-derived M2 mcrophges. Figure S5. Adoptive trnsfer of CD11b + /Gr1 + M2 mcrophges overexpressing 27B1 incresed body weight gin without cusing hyperclcemi in mice with DSS-induced colitis. Tble S1. Rel-time qrt-pcr primer sets used in this study. ACKNOWLEDGMENTS We thnk Deb Chndr for chrcteriztion of the DSS-induced colitis model, Ftim Rjllh for ssistnce with lentivirus production nd the niml work, Penelope Grci for excellent technicl help, nd Peter Gifford for his technicl support in cryostt. 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