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1 IL-16 Induces Intestinal Inflammation via PepT1 Upregulation in a Pufferfish Model: New Insights into the Molecular Mechanism of Inflammatory Bowel Disease This information is current as of June 23, 2017. Ping Wang, Yi-qi Lu, Yi Wen, Dai-yong Yu, Liang Ge, Wei-ren Dong, Li-xin Xiang and Jian-zhong Shao J Immunol published online 1 July 2013 http://www.jimmunol.org/content/early/2013/06/27/jimmun ol.1202598 Downloaded from http://www.jimmunol.org/ by guest on June 23, 2017 Supplementary http://www.jimmunol.org/content/suppl/2013/06/28/jimmunol.120259 Material 8.DC1 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

2 Published July 1, 2013, doi:10.4049/jimmunol.1202598 The Journal of Immunology IL-16 Induces Intestinal Inflammation via PepT1 Upregulation in a Pufferfish Model: New Insights into the Molecular Mechanism of Inflammatory Bowel Disease Ping Wang, Yi-qi Lu, Yi Wen, Dai-yong Yu, Liang Ge, Wei-ren Dong, Li-xin Xiang, and Jian-zhong Shao Inflammatory bowel disease (IBD) has long been a worldwide health care problem with a persistently increasing incidence. Although its clinical features have been well described, its etiology and pathogenesis remain unclear. IL-16 is a chemoattractant cytokine with various effects on cellular activities and diseases. However, the involvement of IL-16 in IBD remains poorly understood. In this study, to our knowledge we report for the first time the mechanism by which IL-16 induces intestinal inflammation by upregulating the expression of oligopeptide transporter member 1 (PepT1) in a Tetraodon nigroviridis fish model. The dextran sodium sulfate induced colitis model in this species revealed that IL-16 levels significantly increase accompanied by elevations in PepT1 in the Downloaded from http://www.jimmunol.org/ by guest on June 23, 2017 colon. Moreover, the signs of colitis were dramatically attenuated by IL-16 depletion using antiIL-16 Abs. In vivo IL-16 administration induced remarkable intestinal inflammation with typical ulcerative colitislike features, including histologic damage, inflammatory cell infiltration, increased myeloperoxidase activity, and proinflammatory cytokines expression, which corresponded with significant PepT1 upregulation in the colon. The IL-16induced PepT1 expression and its upregulated fMLF transport were also demonstrated in vitro. To our knowledge, our study provides the first evidence of the connection between IL- 16 and PepT1, which provides new insights into the molecular mechanism underlying IBD development. Additionally, this study suggests that fish species are an attractive model for studying IBD. By providing a better understanding of IL-16 biology from fish to mammals, this study should aid the development of IL-16based therapies for IBD. The Journal of Immunology, 2013, 191: 000000. I nflammatory bowel disease (IBD) is a chronic autoimmune- whereas CD affects any part of the gastrointestinal tract by non- relevant disorder with a high risk of developing colon cancer continuous transmural inflammation that commonly affects the that has a sustained worldwide increasing incidence and terminal ileum (1, 3). Although the clinical and pathological continues to be a global healthcare problem (1). Normally, IBD features of IBD have been well studied in the past years, its eti- comprises two major forms, namely, ulcerative colitis (UC) and ology and pathogenesis remain poorly understood. IBD is con- Crohns disease (CD), with several common pathological features sidered the outcome of inappropriate and continual inflammatory such as inflammatory cell infiltration, ulceration, hyperplasia of responses to commensal microbe imbalance and some environ- crypt cells, edema, goblet cell loss, and granulomas, which affect mental factors in a genetically susceptible host, which involve the the gastrointestinal tract and colonic mucosa (1, 2). However, UC extremely complicated cross-regulation of innate and adaptive is characterized by diffused superficial inflammatory changes in immunity (3, 4). However, the detailed intercellular and molecular the mucosa and submucosa that are mainly limited to the colon, mechanisms underlying the IBD process remain elusive. Recently, several susceptible genes, cytokines, and cytokine- signaling regulatory factors have been identified to contribute to College of Life Sciences, Zhejiang University, Hangzhou 310058, Peoples Republic the pathogenesis of IBD (59). For example, the single Ig IL-1R of China; Key Laboratory for Cell and Gene Engineering of Zhejiang Province, related molecule controls the homeostasis of the colon by main- Hangzhou 310058, Peoples Republic of China; and Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou 310058, Peo- taining microbial tolerance (10). TNFR-associated factor-2 pre- ples Republic of China vents the spontaneous development of colitis by prohibiting colonic Received for publication September 18, 2012. Accepted for publication June 2, 2013. epithelial cells from undergoing apoptosis (11). Functional alter- This work was supported by National Basic Research Program of China (973) Grants ation of nucleotide-binding oligomerization domaincontaining 2012CB114404 and 2012CB114402, Hi-Tech Research and Development Program protein 2 increases the association of intestinal bacteria with the of China (863) Grant 2012AA092202, National Natural Science Foundation of China Grants 31072234, 31172436, 31272691, and 30871936, and by Program for Key epithelium (12, 13). The knockout of leucine-rich repeat kinase 2, Innovative Research Team of Zhejiang Province Grant 2010R50026. which is closely related to susceptibility to CD, may cause ex- Address correspondence and reprint requests to Prof. Jian-zhong Shao and Assoc. acerbated colitis (14). Furthermore, the TNF-like cytokine TL1A Prof. Li-xin Xiang, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, reportedly drives adaptive immune-mediated ileitis (15, 16), and Peoples Republic of China. E-mail addresses: [email protected] (J.-z.S.) and [email protected] (L.-x.X.) several ILs such as IL-2, IL-10, IL-12, and IL-23 are involved in IBD, which indicates an imbalance between proinflammatory and The online version of this article contains supplemental material. anti-inflammatory influences (17, 18). However, the exact effects Abbreviations used in this article: CD, Crohns disease; DSS, dextran sodium sulfate; IBD, inflammatory bowel disease; MPO, myeloperoxidase; PepT1, oligopeptide of these cytokines on the mechanism of IBD development are still transporter member 1; siRNA, small interfering RNA; TnIL-16, Tetraodon IL-16; undetermined. TnPepT1, Tetraodon oligopeptide transporter member 1; TnPepT1-ex, extracellular Several previous epidemiological investigations have shown that region of Tetraodon oligopeptide transporter member 1; UC, ulcerative colitis. IL-16 is considerably increased at both the mRNA and protein Copyright 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 levels in pathologic intestinal tissues and the systemic circulation of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202598

3 2 IL-16 INDUCES COLITIS THROUGH UPREGULATING PepT1 patients with UC and CD compared with healthy control indi- the pGEM-T EASY vector (Promega), and transformed into competent viduals (19, 20). Neutralization of IL-16 reportedly attenuates Escherichia coli TOP10 cells (Invitrogen). The plasmid DNA was purified using a plasmid Miniprep kit (Qiagen) and sequenced on an ABI 3730 colitis-like inflammation in an experimental mouse model (21). sequencer (Invitrogen). The comparative gene map positions and organ- Substantial increases in the number of IL-16+ mast cells have been izations, phylogenetic alignment, and potential structures of the PepT1 observed in active CD (22). Concordantly, an association was also proteins were determined using databases and software programs as observed between polymorphism of the IL-16 gene promoter and previously described (3033). CD (23). Hence, IL-16 may play an important role in the patho- Distribution and expression analysis genesis of IBD. However, direct functional evidence is still needed For distribution analysis, tissues samples from the intestines, spleen, liver, to clarify this implication. In the present study, we elucidate the kidneys, skin, gills, muscles, brain, and heart were carefully collected from involvement of IL-16 in the pathology of IBD in a fish model. the healthy fish. For intestine-specific expression analysis, colon-like tissues IL-16 was initially identified as a lymphocyte chemoattractant were collected after dextran sodium sulfate (DSS) or IL-16 challenge. Total factor secreted from human PBMCs (24, 25). IL-16 is now thought RNA was extracted and reversed-transcribed into cDNA as described above. to be a multifunctional cytokine and is the only known single PDZ The PCR amplifications were performed in 10 ml reaction mixtures con- taining 0.3 ml DNA template, 1 ml forward and reverse primers (shown in domain protein secreted by human PBMCs (26, 27). Aside from its Supplemental Table I; 10 mM), 5 ml Taq polymerase mixture (TaKaRa wide range of effects on cellular activities, such as upregulation Bio), and 3.7 ml double distilled H2O. The cycling protocol consisted of an of the high-affinity IL-2 receptor (CD25), induction of cells to initial denaturing cycle at 94C for 4 min followed by 35 cycles of 94C for reset into the G1 phase, inhibition of Ag-specific proliferation, and 30 s, 58C for 30 s, and 72C for 30 s, with a final elongation step at 72C for 10 min. maintenance of Ag nonspecific proliferative properties, numerous investigations have implied that IL-16 is involved in the exacer- Plasmid constructions bation of infectious and autoimmune inflammatory diseases, such Downloaded from http://www.jimmunol.org/ by guest on June 23, 2017 The encoding sequence of the extracellular region of TnPepT1 (TnPepT1- as atopic dermatitis, IBD, systemic lupus erythematosus, multiple ex) was inserted into pET28a and pcDNA6 between the EcoRI and XhoI sclerosis, and neurodegenerative disorders, as well as viral and sites, respectively, to construct a prokaryotic expression vector (pET28a- bacterial infections (26). Nonetheless, distinct evidence that reveals TnPepT1-ex) and a eukaryotic expression vector (pc6-TnPepT1-ex) where in the N terminus of TnPepT1-ex was fused with a His-tag. The TnIL-16 the mechanism of IL-16 in the direct regulation of disease pro- encoding sequence was inserted into pET28a and pET41a between the gression is still limited. In the present study, we found that IL-16 EcoRI and XhoI sites, respectively, to construct two prokaryotic expression is able to induce colitis by upregulating the expression of the vectors (pET28a-TnIL-16 and pET41a-TnIL-16) where in the N terminus oligopeptide transporter member 1 (PepT1) molecule on epithelial of TnIL-16 was fused with a His-tag or a GST-tag plus a His-tag. The cells of the colon in a Tetraodon nigroviridis fish model. To our constructed vectors were verified by double digestion with EcoRI and XhoI and further confirmed by DNA sequencing. knowledge, this is the first detailed report that shows the involve- ment of IL-16 in the pathogenesis of UC, which indicates a new Recombinant protein preparation function of IL-16 and provides new insight into the molecular The pET28a-TnPepT1-ex, pET28a-TnIL-16, pET41a-TnIL-16, and pET- mechanism of IBD. Additionally, fish may become a novel animal 41a (for GST-tagged protein expression) plasmids were separately trans- model for the study and drug screening of IBD. Our findings should formed into E. colI BL21 (DE3) cells. A single colony was inoculated into help in understanding the involvement of IL-16 in basic biological 100 ml LuriaBertani (LB) medium with kanamycin (50 mg/ml). The in- oculated LuriaBertani medium was incubated in a shaking incubator at processes of disease pathways and even the functional involvement 37C, 200 rpm until the OD600 value reached 0.6. Then isopropyl b-D-thio- of this cytokine in the evolutionary history of vertebrates. galactoside was added to a final concentration of 1 mM and the incubation was continued for 6 h. The protein expression levels were assessed using 10% SDS-PAGE followed by Coomassie brilliant blue R250 staining. The Materials and Methods recombinant soluble TnIL-16 and GST-tag proteins were purified using Ni- Experimental fish NTA agarose affinity chromatography according to the QIAexpressionist One-year-old spotted green pufferfish, T. nigroviridis, of both sexes, which manual (Qiagen). The recombinant TnPepT1-ex and TnIL-16 proteins from were 45 cm in body length and weighed 35 g, were kept in running the inclusion bodies without GST-tags were purified by a similar protocol water at 2528C and fed with frozen red worms twice a day. The fish were for polyclonal Ab preparation. held in the laboratory for at least 2 wk to allow them to acclimatize to the Polyclonal Ab preparation new environment and evaluate their overall health. Only healthy fish, de- termined by their general appearance and level of activity, were used in the Six-week-old male New Zealand White rabbits weighing 1.5 kg were study. immunized with 500 mg purified recombinant TnPepT1-ex and TnIL-16 proteins, respectively, in CFA on the 1st day and IFA on the 3rd, 28th, and Cell cultures 35th days (34). One week after the final immunization, blood was collected The colon-like cell line SW480 and HeLa cells were maintained in DMEM from the rabbits when the Ab titers were .1/60,000 as determined by (HyClone Laboratories) and IMDM (HyClone Laboratories) supplemented a microplate-based ELISA using the same TnPepT1-ex and TnIL-16 pro- with 10% FBS (PAA Laboratories), 100 U/ml penicillin (Life Technologies teins adsorbed onto the solid phase. The Abs were initially purified into an BRL), and 100 mg/ml streptomycin (Life Technologies BRL) at 37C in IgG isotype using an affinity protein A-agarose column and further purified 5% CO2. At near confluence, the cells were subcultured in dishes at a via an immunosorbent-based protocol using recombinant proteins absor- density of 1 3 104 cells/cm2. bed onto the nitrocellulose membrane phase (Qiagen). Western blot and Ag-specific ELISA were performed to characterize the specificities of the Molecular cloning Abs to TnPepT1 and TnIL-16. Total RNA was extracted from the Tetraodon intestinal tissues using TRIzol Real-time PCR reagent (Invitrogen) and reverse-transcribed into first single-stranded cDNA (Avian Myeloblastosis Virus RT-PCR Kit [version 3.0], TaKaRa Bio, Shiga, The expression levels of TnPepT1, IL-1b, IL-6, IL-10, TNF-a, and IFN-g Japan). The full-length Tetraodon PepT1 (TnPepT1) cDNA was generated transcripts were quantified through real-time PCR on a Mastercycler ep using RT-PCR and RACE-PCR with primers (shown in Supplemental Table realplex machine (Eppendorf, Hamburg, Germany) using an SYBR Premix I) designed according to the homolog sequence predicted from genome Ex Taq kit (TaKaRa Bio) according to the manufacturers instructions. databases (University of California, Santa Cruz and National Center for Briefly, all real-time PCR reactions were performed in a total reaction Biotechnology Information) using the human PepT1 (SLC15A1) sequence volume of 10 ml. The experimental protocol consisted of the following: 1) (NP_005064) as a query and compiled by the Genscan and BLAST software 40 cycles of amplification at 95C for 30 s, and then at 5560C (depending programs (28). The encoding sequence of Tetraodon IL-16 (TnIL-16) was on the different genes) for 20 s; 2) melting curve analysis at 95C for 15 s, amplified using primers designed according to our previous report (29). The 65C for 15 s, 65C drop to 95C in 20 min and 95C for 15 s; and 3) cooling PCR products were purified using a gel extraction kit (Qiagen), inserted into at 40C for 30 s. The total amount of mRNA was normalized to endogenous

4 The Journal of Immunology 3 b-actin mRNA. The PCR primers are shown in Supplemental Table I. The kDa; MP Biomedicals) via oral perfusion once a day just before the second relative gene expression was analyzed using the 22DDCt method (35). In all feeding time to ensure that the intestines of the fish were empty when they cases, each PCR trial was performed with triplicate samples and repeated received the drug. The DSS administration was performed for 7 consec- at least three times. utive days to induce colitis. At the same time, fish in the control groups were given the same amount of mock PBS. Western blot Examination of IL-16 levels Tissue samples were lysed with 1% (w/v) Triton X-100 in 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% sodium deoxycholate, and protease The plasma IL-16 levels in the DSS-induced Tetraodon colitis model were inhibitors (Roche) at pH 8.0. The lysates were then boiled in sample buffer determined via ELISA and Western blot. For ELISA, serum samples were containing 2% SDS and 20% glycerol for 5 min. The samples containing 50 diluted 10-fold using coating buffer (1.59 g/l Na2CO3 and 2.94 g/l mg tissue proteins were separated using 10% SDS-PAGE and transferred NaHCO3) and absorbed into the solid phase of the ELISA plate (100 ml/ onto 0.45-mm polyvinylidene difluoride membranes (Bio-Rad Laborato- pore). Then, 2% BSA containing 0.1% Tween 20 was used as blocking ries, Hercules, CA) for 2 h at 4C. The blots were blocked for 1 h in 5% solution. The plate was then incubated for 2 h with rabbit antiIL-16 Abs nonfat dry milk at room temperature. After washing with TBST buffer, the at 37C. HRP-conjugated anti-rabbit IgG Abs at 1:10,000 dilution were blots were incubated overnight at 4C with rabbit antiTnPepT1-ex Ab at used as the secondary Ab. After washing the plate five times with TBST, 1:1000 dilution. After washing three times for 30 min in TBST buffer, freshly prepared substrate solution (100 ml/well) was added into the plate. the blots were further incubated with HRP-conjugated anti-rabbit Abs at Then, 2 M H2SO4 (50 ml/well) was added to the plate to stop the reaction 1:5000 dilution for 1 h at room temperature. The blots were washed three until the color of the reactant solution changed. The absorbance (at 450 nm times for 30 min in TBST buffer and were then visualized using ECL. and at 630 nm) was detected using an absorbance microplate reader (BioTek Instruments model ExL800). Western blot analysis was performed as Histological examination described above. Each intestinal sample was fixed overnight in 10% buffered formalin, dehydrated using graded ethanol, vitrified with dimethylbenzene, and em- Depletion of IL-16 using Abs bedded in paraffin. Then, 5-mm sections were stained with H&E-safranin and Downloaded from http://www.jimmunol.org/ by guest on June 23, 2017 An in vivo IL-16 depletion assay was performed using antiTnIL-16 Abs examined under light microscopy (Zeiss Axiostar Plus) in a blinded manner. according to a previous study to evaluate the effects of IL-16 in IBD pro- The gross inflammation index was assessed by histopathological scoring gression (21). Briefly, the fish were injected i.p. with the antiTnIL-16 Abs according to items shown in Table I. For inflammatory cell analysis, 5-mm (50 mg/fish) 1 d before DSS challenge and during the whole DSS treatment frozen sections of intestinal samples were stained with WrightGiemsa period (once daily before the DSS treatment). In parallel, the same amounts (Thermo Scientific) or a peroxidase staining kit (Merck Millipore) following of nonspecific Abs (rabbit IgG) were administered to another group of DSS- the manufacturers instructions. treated fish as the negative control. Then, the activity of heme-containing Immunochemistry and immunofluorescence staining enzyme MPO was determined to examine the severity of colitis. Paraffinized tissue sections were deparaffinized, hydrated in an ethyl alco- MPO activity assay hol gradient, and blocked with 5% BSA for 30 min. After washing twice Colon-like intestines were cleaned, weighed, and homogenized in 50 mM with PBS, the sections were incubated with the primary Abs (rabbit anti- potassium phosphate buffer on ice and centrifuged at 12,000 3 g for 15 min myeloperoxidase [MPO] or rabbit anti-TnPepT1) for 1.5 h at 37C. Af- at 4C. The precipitates were suspended in 300 ml 50 mM hexadecyl- terward, the sections were washed three times with PBS and incubated trimethylammonium bromide and 700 ml potassium phosphate buffer. Then, with HRP- or FITC-conjugated anti-rabbit IgG Abs for 1 h at 37C. For the precipitates were ultrasonicated for 20 s, frozen at 280C for 5 min, and immunohistochemical staining of MPO, the sections were further stained thawed to room temperature. After repeating the procedure twice, the with H&E for 10 min at room temperature. The sections were washed three samples were centrifuged at 12,000 3 g for 10 min at 4C and then the times with PBS and observed under a light or fluorescence microscope suspension was collected. The suspension (100 ml) was placed into a col- (Zeiss Axiostar Plus). orimetric cuvette with 2.9 ml potassium phosphate buffer containing 0.167 mg/ml O-dianisidine dihydrochloride and 0.165 mM H2O2. Then, the DSS-induced Tetraodon colitis model change in absorbance was measured continually every 30 s at 460 nm using The DSS-induced colitis was prepared according to a previously reported a UV spectrophotometer (SP2100, Shanghai Spectrum Instruments, Shanghai, method (36). Briefly, the experimental fish were fed twice daily with China) (37). One unit of MPO activity was defined as the quantity of en- radiation-sterilized frozen red worms at 3% of their body weight. Then, zyme that converts 1 mmol hydrogen peroxide into water within 1 min at they were administered with 500 ml 5% DSS (molecular mass of 3650 room temperature and is expressed as units per gram of tissue (37). IL-16induced colitis Table I. Histopathological scoring standards An in vivo administration assay was performed to confirm that IL-16 plays a role in intestinal inflammation. The fish were initially fed radiation- Evaluation Standards sterilized frozen red worms twice daily at 3% of their body weight. Then, the fish were administered recombinant TnIL-16 protein via i.m. Observation Item Occurrence Score injection under the dorsal fin at a dosage gradient of 0.1, 0.5, 1, 5, and 10 Infiltration of inflammatory cells None 0 mg per fish and a time gradient of 1, 2, 4, 7, 14, and 21 d after injection. In Mild 1 parallel, a GST-tag proteininjected group (5 mg/fish; this dose was much Medium 2 higher than the amount contained in the soluble IL-16-GST fusion protein) Severe 3 was devised to eliminate the interference of the GST-tagged protein, and Depth of inflammatory cell None 0 a PBS-injected group was set as the negative control. Then, the colon-like infiltration Mucosa 1 intestines were collected to evaluate the extent of inflammation in terms of Muscularis propria 2 MPO activity, histologic changes, and inflammatory cytokine expression. Serosa 3 IL-16 induces PepT1 upregulation Crypt Normal 0 Occasionally vanishing 1 IL-16induced TnPepT1 upregulation was examined both in vivo and Part vanishing 2 in vitro. For the in vivo assay, the fish were administered TnIL-16 (at Diffusedly vanishing 3 dosage and time gradients as described above) and their colon-like intes- Edema None 0 tines were collected for PepT1 examination using real-time PCR, Western Existence 1 blot, and immunofluorescence staining as described above. For the in vitro Mucoprotein decrease None 0 assay, SW480 cells were stimulated with different TnIL-16 concentrations Mild 1 (25, 50, and 100 ng/ml). Then, the cells were collected for PepT1 ex- Severe 2 pression analysis using real-time PCR. Hemorrhage None 0 Existence 1 fMLF transportation assay Pseudomembrane None 0 The colon-like intestines were washed with PBS and carefully everted to Existence 1 expose the mucosa. Then, Kerbs buffer was poured into the enteric cavity

5 4 IL-16 INDUCES COLITIS THROUGH UPREGULATING PepT1 and two ends of the intestine were ligated. The everted intestines were (Fig. 1B, 1C). Similarly, the posterior intestinal segment consisted soaked in 1 ml fMLF (Sangon Biotech) solution (1 mM) and shaken at 100 of pillar and goblet cells with considerably shorter villi, similar to rpm for 15 min at 28C to allow sufficient transportation. In parallel, sim- ilarly prepared tissues from PBS-injected fish were set as the control. the colon of mammals; thus, we called it the colon-like intestine of Subsequently, the concentration of the remaining fMLF in the solution was Tetraodon (Fig. 1D). After DSS administration, intestinal hyper- determined by HPLC following previously described methods (38). Briefly, emia and swelling were anatomically observed. Histological anal- 25 ml sample was injected into an HPLC system (Shimadzu VP-ODS col- ysis showed that the lamina propria and submucosa of the colon-like umn with 5-mm particle size, 150 3 4.6 mm) once at 30C. Acetonitrile was segment were clearly damaged, and a significant number of in- used as the mobile phase at a flow rate of 1 ml/min. The fMLF was detected at 196 and 212 nm for 10 min. Pure fMLF (Sangon Biotech) diluted into flammatory cells, including lymphocytes, neutrophilic granulocytes, a concentration gradient was used to establish a standard curve. The com- and eosinophilic granulocytes, were observed in the mucosa (Figs. parative transport activity of TnPepT1 was calculated as follows: DfMLF 1H, 2B, 2D, 2F). However, the structure of the middle intestines concentration/tissue weight. seemed to be slightly injured, but no apparent damage or inflam- Small interfering RNA screening against TnPepT1 matory infiltration was observed in the mucosa of the front seg- Small interfering RNA (siRNA) specific for TnPepT1 was designed using ments, although the villi structure exhibited slight irregularities (Fig. siRNA template design tools (Dharmacon and Invitrogen RNAi designer) 1EG). Furthermore, the severity of the intestinal inflammation was (39). Four TnPepT1 sequence-targeted siRNAs (Supplemental Table I) were also determined based on histopathological scoring and MPO ac- selected to screen the activity. DNA oligonucleotides for hairpin RNA ex- tivity. The histopathological scoring of each intestinal segment was pression (synthesized by Invitrogen) were dissolved in double distilled H2O performed according to various items shown in Table I. The result at 100 mM. Then, equimolar amounts of sense and antisense strands were mixed with annealing buffer (103 buffer stock, 100 mM Tris-HCl [pH 7.5], showed that among all of the intestinal segments tested, the most 10 mM EDTA, and 1 M NaCl) and the mixture (10 ml) was incubated at severe inflammation occurs in colon-like tissues (Fig. 1I). MPO is 90C for 4 min, 70C for 10 min, and then cooled to 10C. The pSUPER one of the key components of inflammatory granulocytes such as Downloaded from http://www.jimmunol.org/ by guest on June 23, 2017 vector (pSUPER.retro.puro; OligoEngine) was digested with HindIII and neutrophils that accumulate at inflammation sites and play a major BglII, and the annealed oligonucleotides were ligated into pSUPER down- stream of the H1 promoter to generate four constructs (pSUPER-TnPepT1- role in inflammatory pathology (46, 47). Thus, MPO is a valuable siRNA-142, -220, -363, and -453) with different siRNAs. The pSUPER- biomarker for determining inflammatory cell infiltration and the TnPepT1-siRNA constructs or the control pSUPER were cotransfected with degree of inflammation in IBD pathology (46, 47). As expected, our pcDNA6-TnPepT1 (4 mg DNA in serum-free, antibiotic-free Opti-MEM I results show that the MPO activity of the colon-like intestine in the medium using Lipofectamine 2000; Invitrogen) into HeLa cells in six-well DSS-treated groups was significantly increased (p , 0.01; Fig. 2G). plates. At 5 h after transfection, the cells were washed and the medium was replaced with antibiotic-free DMEM, supplemented with 10% FBS. At 48 h Given that the DSS-induced Tetraodon intestine inflammation was after transfection, the cells were lysed for total RNA extraction. The spec- mainly restricted to the colon-like intestines, and the most striking ificity and efficiency of siRNA-targeted TnPepT1 was determined using inflammatory changes were superficial and limited to the mucosa and real-time PCR. submucosa, all of which resembled the histopathological aspects of In vivo knockdown of TnPepT1 DSS-induced UC-like injury in mice, as well as UC in humans, we In vivo knockdown of TnPepT1 was performed using a pSUPER vector- termed it DSS-induced Tetraodon UC-like colitis, thereby providing based siRNA deliver system that was recently applied in the gut system (40). further insights into the conservation of intestinal systems. Recently, Briefly, after IL-16 administration, the vector or the plasmid with high ef- zebrafish has been suggested as a good model for investigating the ficiency of siRNA against TnPepT1 (pSUPER-TnPepT1-siRNA-142) mixed possible susceptibility genes and hostmicrobial interactions in IBD with Lipofectamine 2000 was administered s.c. under the dorsal fin at 12 mg (4850). It is thus anticipated that fish may become another model (3 mg/g body weight) per injection into each fish for five consecutive days. In parallel, the control group was injected with control vector. Total RNA animal that is swimming into the view of IBD research. was extracted from the colon-like intestines and was reverse-transcribed into cDNA. Real-time PCR was conducted to evaluate the efficiency of the Effect of IL-16 in DSS-induced Tetraodon colitis model in vivo TnPepT1 suppression. It has been reported that IL-16 expression in the colonic mucosa of Statistical analysis IBD patients increased compared with the healthy controls (19). To All experiments were replicated at least three times. The sample number of determine whether a similar mechanism occurs in the Tetraodon each group was 10 fish with equal average body weights. The data are colitis model, the plasma IL-16 concentrations in fish with DSS- expressed as means 6 SD. Statistical evaluation of the differences between induced colitis were measured. As expected, the IL-16 concen- values from different experimental groups was performed with multiple trations were significantly increased 5-fold (p , 0.01) in the serum Student t tests. A p value , 0.05 was considered statistically significant. of the DSS-treated fish compared with the PBS-treated controls (Fig. 2H). This result is in accordance with the results of the human ep- Results idemiological investigation. To confirm that IL-16 affects colitis DSS-induced Tetraodon colitis model progression, a depletion assay was performed using antiTnIL-16 It has been reported that DSS treatment leads to superficial in- Abs. The Abs were administered once a day to optimally deplete flammatory changes in the mucosa and submucosa of colon tissues in TnIL-16 during the DSS treatment. MPO activity was used to de- mouse models with various histopathological features similar to that termine the severity of inflammation. The results show that the MPO seen in human UC (36, 4145). Therefore, the DSS-induced colitis activity was significantly (p , 0.05) decreased in the affected fish model has become one of the most widely used experimental compared with the nondepleted and the negative IgG-administered models for human UC research (42). In this study, to reconfirm the controls (Fig. 2I). Dramatically, the TnPepT1 expression was also increased IL-16 level in clinical epidemiological investigations and significantly decreased (p , 0.05) in the colon-like intestines ac- to explore the possible role of IL-16 in intestinal bowel disease by companied by depletion of TnIL-16 (Fig. 2J). These findings suggest a fish model, we generated a DSS-induced intestine inflammation that IL-16 may largely contribute to the pathophysiology of IBD, model in Tetraodon. Histological analysis showed that the structure and that PepT1 might be closely involved in this process. of Tetraodon intestines is generally similar to that of mammals. Different intestinal segments exhibited distinct differences. For IL-16 induces colitis in Tetraodon example, the villi in the middle intestinal segment of Tetraodon The aforementioned experiments provided preliminary observa- were relatively long and the striated border was distinct. These tions of the positive correlation between IL-16 and colitis. Thus, an characteristics are similar to those in mammalian small intestines in vivo administration assay was performed to provide further

6 The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on June 23, 2017 FIGURE 1. DSS administration induced intestinal inflammation in Tetraodon. Staining of Tetraodon intestinal tissue sections with H&E-safranin: (A) anterior segment of healthy Tetraodon intestines; (B) middle-anterior segment of healthy Tetraodon intestines; (C) middle-posterior segment of healthy Tet- raodon intestines; (D) posterior segment of healthy Tetraodon intestines; (E) anterior segment of DSS-treated Teraodon intestines; (F) middle-anterior segment of DSS-treated Tetraodon intestines; (G) middle-posterior segment of DSS-treated Tetraodon intestines; (H) posterior segment of DSS-treated Tetraodon intestines. Scale bars, 100 mm (except for the partially enlarged images at the bottom of (D) and (H), 10 mm). (A)(D) show that the histological structure of the Tetraodon intestines is very similar to that of mammals; (H) shows the damage to the lamina propria and submucosa of the colon-like intestine; a significant number of infiltrating inflammatory cells (neutrophils, lymphocytes, and eosinophilic granulocytes) are shown in the partially enlarged image below. Typical inflammatory cells are indicated as follows: , neutrophils; , lymphocytes; *, eosinophils. (I) Histopathological scoring of healthy and DSS-treated Tetraodon intestinal segments. Statistical results are given as the average of 50 visual yields with 10 fish in each group, which also indicate that the histopathological features presented are representative of DSS-treated fish. Values are expressed as means 6 SEM. **p , 0.01 as compared with control groups. evidence. Recombinant TnIL-16 was administered into fish at inflammatory intestinal injury with the pathological features seen different doses for different periods. Inflammation of the colon- in the DSS-induced model (Fig. 3). Histopathological analysis like intestines was determined based on histological changes, showed that the colon-like intestines developed severe hyperemia MPO activity, and expression of proinflammatory cytokines. As and swelling, and the intestinal walls were severely damaged. The expected, administration of TnIL-16 to Tetraodon induced typical villi in the colon-like intestines were blunted and broadened with

7 6 IL-16 INDUCES COLITIS THROUGH UPREGULATING PepT1 Downloaded from http://www.jimmunol.org/ by guest on June 23, 2017 FIGURE 2. DSS-induced intestinal inflammation is accompanied by a significant increase in IL-16 level. (A and B) WrightGiemsa (WG) staining of the frozen sections of control and DSS-treated Tetraodon colon-like intestines, respectively. (C and D) Peroxidase (POX) staining of the frozen sections of control and DSS-treated Tetraodon colon-like intestines, respectively, showing granulocyte infiltration. (E and F) Immunohistochemical staining of MPO in paraffin sections of control and DSS-treated Tetraodon colon-like intestines, respectively, showing neutrophil infiltration. Scale bars, 10 mm. (B), (D), and (F) show inflammatory cell infiltration in the mucosa of colon-like intestines in DSS-treated fish. Typical inflammatory cells are indicated as follows: , neutrophils; , lymphocytes; *, eosinophils; , granulocytes. (G) MPO activity of the colon-like intestines in control and DSS-treated Tetraodon. (H) ELISA and Western blot analysis of IL-16 levels in serum from control and DSS-treated Tetraodon. (I) MPO activity of the Tetraodon colon-like intestines in the IL-16 depletion experiment. (J) PepT1 mRNA levels in the IL-16 depletion experiment. For (G)(J), each group contained 10 fish. Fish in control groups were treated with mock PBS. The quantitative PCR values are the average of three replicates, and b-actin was used as the internal reference gene. Values are expressed as means 6 SEM. *p , 0.05, **p , 0.01 compared with the control groups. epithelial exfoliation and fibrinous exudation (Fig. 3C). Decreased also observed. The histopathological scoring (Table I), which es- goblet cells and interstitial edema were clearly observed in the timated the severity of inflammation, was determined from various colonic mucosa. Dilatation of vessels was associated with general observation items and based on overall merit. The TnIL-16 treat- infiltration of inflammatory cells, including lymphocytes, neutro- ment groups obtained a score of 7.0 (Fig. 3E), which was similar philic granulocytes, and eosinophilic granulocytes in the lamina to the score of the DSS-administered fish (i.e., 7.7). propria and submucosa (Figs. 3C, 3D, 4C, 4F, 4I). Furthermore, Quantitative MPO activity analysis showed that the enzyme other histopathological features, such as granulomas with mac- significantly increased (p , 0.05 and p , 0.01) with increasing rophage aggregation, crypt microabscesses, and cryptitis, were TnIL-16 concentration (on the day 7). A 3- to 11-fold elevation

8 The Journal of Immunology 7 FIGURE 3. IL-16induced injury to the colon-like intestines of Tetraodon. The histological sections were stained with H&E-safranin. PBS-treated control fish (A) and GST-treated (5 mg/fish) fish (B) showed normal intestinal wall features 7 d after treatment. Histological Downloaded from http://www.jimmunol.org/ by guest on June 23, 2017 sections of the colon-like intestines 7 d after treatment with 5 mg IL-16 per fish (C, D) show serious colon wall damage. Fibrinous exudation associated with general infiltration of inflammatory cells (lymphocytes, neu- trophils, and eosinophilic granulocytes) in the lamina propria and submucosa (C), granulomas (D), and epi- thelial exfoliation are shown. Scale bars, left panels of (A)(D), 100 mm; right panels of (A)(D), 10 mm. Typical inflammatory cells are indicated as follows: , neutrophils; , lymphocytes; *, eosinophils. (E) Histo- pathological scoring of the control, GST-treated, and IL-16treated Tetraodon colon-like intestinal segments. Statistical results are given as the average of 50 visual yields with 10 fish for each group, which also indicates that the histopathological features presented are repre- sentative of IL-16treated fish. The fish in the control groups were treated with mock PBS. Values are expressed as means 6 SEM. **p , 0.01 compared with the control group. was observed in the groups stimulated with 0.1, 0.5, 1, 5, and 10 fish). The MPO activity was highest on day 7, at 12-fold higher mg TnIL-16 (per fish). The effects of increasing TnIL-16 con- than that of the negative controls. However, the MPO activity centration plateaued at 10 mg per fish (Fig. 5A). However, only stopped increasing 14 d after treatment. Thus, the severity of in- a slight increase in MPO activity was observed in the GST-tag flammation induced by TnIL-16 was dose- and time-dependent. injected control group. This result may be attributed to the GST The proinflammatory cytokines in intestines with colitis were protein, which acts as an Ag and mildly stimulates the fish im- also examined. IL-1b, IL-6, IFN-g, and TNF-a were all upregu- mune system. MPO activity was also significantly increased (p , lated in response to TnIL-16 stimulation (Fig. 5BE), although 0.05 and p , 0.01) in colitis tissues with time (1 d to 21 d) during IFN-g and TNF-a showed only slight increases under the low- the progression of intestinal inflammation (Fig. 6A). A slight in- dose treatment (0.1 mg/fish) (Fig. 5D, 5E). The TnIL-16induced crease in MPO activity was observed 1 d after induction (5 mg/ proinflammatory cytokine expressions were also dose- and time-

9 8 IL-16 INDUCES COLITIS THROUGH UPREGULATING PepT1 FIGURE 4. IL-16 administration leads to significant inflammatory cell infiltration in the colon-like intestines of Tetraodon. (A)(C) WrightGiemsa (WG) staining of the frozen sections of control, GST-treated, and IL-16 treated Tetraodon colon-like intestines. (D)(F) Peroxi- dase (POX) staining of the frozen sections of control, GST-treated and IL-16treated Tetraodon colon-like intestines showing granulocyte infiltration. (G)(I) Im- munohistochemical staining of MPO in paraffin sections of control, GST-treated, and IL-16treated Tetraodon colon-like intestines showing neutrophil infiltration. (C), (F), and (I) show inflammatory cell infiltration in the mucosa of the colon-like intestines of IL-16treated fish. Typical inflammatory cells are indicated as fol- lows: , neutrophils; , lymphocytes; *, eosinophils; , granulocytes. Scale bars, 10 mm. Fish in the IL-16 and Downloaded from http://www.jimmunol.org/ by guest on June 23, 2017 GST groups were treated with 5 mg IL-16 or GST per fish. Fish in the control groups were treated with mock PBS. The analyses were performed 7 d after treatment. dependent. The fish treated with 5 mg TnIL-16 for 47 d developed RAP2A, FARP1, DOCK9, and STK24, were all also found in the most serious inflammation by overall assessment. Notably, the identical order on human chromosome 13 and mouse chromosome expression of anti-inflammatory cytokine IL-10, an important marker 14, which shows the high conservation of genome synteny of PepT1 for IBD that is inhibited during inflammation, was significantly de- molecules (Supplemental Fig. 2B). Phylogenetic analysis showed creased (p , 0.01) even under the low-dose treatment (0.5 mg/fish) that TnPepT1 was clustered with PepT1 family members (Supple- (Fig. 5F). On the first day after treatment, the IL-10 expression de- mental Fig. 2C). The TnPepT1 protein was predicted to have 12 creased by 40% compared with the control groups that received GST- transmembrane domains and an extracellular region between the 9th tag or the mock PBS (Fig. 6F), whereas the other proinflammatory and 10th transmembrane domains, which is also highly conserved cytokines were not increased at this point. However, all of the in the PepT1 proteins in different species (52). Multiple alignment proinflammatory cytokines were upregulated and reached their peak analysis showed that TnPepT1 shares an overall sequence identity of on day 47 after treatment (Fig. 6BE). This process was accom- 5561% with other species, and the sequence identity of the trans- panied by consistent decreases in IL-10 expression at different points. membrane domains was even much higher (Supplemental Fig. 3). Collectively, the results clearly show that IL-16 could induce sig- The results suggests that key functional motifs, such as the first four nificant colon inflammation in Tetraodon. The pathological features N-terminal and the seventh and ninth transmembrane domains, of IL-16induced intestinal inflammation resembled that of DSS- which are required for substrate binding and transporter functionality induced colitis in both Tetraodon and mouse models (3). There- (53), are completely conserved in many vertebrate species from fish fore, the nature of the IL-16induced fish intestinal inflammation to mammals. could also be considered UC-like colitis. To determine whether fish PepT1 has a tissue-specific distribution similar to that in mammals, various tissues were collected from TnIL-16 induces TnPepT1 expression in colon-like intestine Tetraodon. The intestinal tissues were divided into several segments PepT1 plays a crucial role in IBD development by accelerating the for more precise determination. The TnPepT1 mRNA transcripts in transportation of the fMLF peptide, an important signal for the in- healthy fish (without DSS and IL-16 stimulation) were highest in the filtration of inflammatory cells (51). Therefore, a TnIL-16induced middle intestines, but were at very low or undetectable levels in PepT1 expression assay was performed both in vivo and in vitro to other tissues (Fig. 7). Importantly, TnPepT1 expression was scarcely confirm that TnIL-16 induces colitis through TnPepT1 upregulation detected in the posterior intestines under homeostatic conditions in the colon-like intestines. For this purpose, PepT1 was initially without DSS and IL-16 stimulation, whereas the expression was identified in Tetraodon. The results showed that the TnPepT1 cDNA significantly upregulated under inflammatory conditions. Therefore, contains a 2178-bp open reading frame that encodes a predicted TnPepT1 is specifically distributed in the middle intestines (equiv- polypeptide with 725 aa, a 90-bp 59 untranslated region, and a 60-bp alently to the small intestines of mammals), which further suggests 39 untranslated region (Supplemental Fig. 1; GenBank accession no. that the PepT1 in fish functionally corresponds to the intestinal JX177494; http://www.ncbi.nlm.nih.gov/genbank/). The TnPepT1 PepT1 in mammals. gene is located within a 9208-bp genomic fragment on chromosome The TnIL-16induced PepT1 expression was determined at 3 with 23 exons. The structural organization of the gene conforms to the mRNA and protein levels by real-time PCR, Western blot anal- that of other species such as humans and mice (Supplemental Fig. ysis, and immunofluorescence staining, respectively. The real- 2A). The genes adjacent to the TnPepT1 locus, such as MBNL2, time PCR showed that in vivo TnIL-16 administration significantly

10 The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on June 23, 2017 FIGURE 5. Dosage-dependent IL-16induced intestinal inflammation. (A) MPO activity of the colon-like intestines of the control, GST-treated, and IL- 16treated Tetraodon. Values are expressed as means 6 SEM. Additionally, the mRNA levels of inflammatory cytokines in the colon-like intestines were examined by real-time PCR: (B) IL-1b, (C) IL-6, (D) IFN-g, (E) TNF-a, (F) IL-10. b-Actin was used as the internal reference gene. Values are expressed as means 6 SEM. The quantitative PCR value is the average of three replicates. Fish in the control groups were treated with mock PBS. All analyses were performed 7 d after administration. *p , 0.05, **p , 0.01 versus values in the control group (n = 10). induces TnPepT1 expression in the colon-like intestines in a dose- Therefore, TnIL-16 was used as a stimulant. Accordingly, PepT1 and time-dependent manner. Treatment with 5 mg and with 10 mg expression was significantly induced by TnIL-16 in a dose-dependent per fish for 7 d induced a 30-fold increase in TnPepT1 expression in manner (Fig. 8E). This result provides direct evidence that IL-16 the TnIL-16injected groups compared with the control groups was able to trigger PepT1 expression. In summary, the IL-16induced (Fig. 8A). The TnPepT1 expression was consistently elevated upregulation of PepT1 expression in the colon-like intestines cor- during the first 7 d of stimulation (5 mg/fish), which persisted for responds to the development of inflammation. 14 d (Fig. 8B). Similar results were observed in the Western blot analysis (Fig. 8C). Immunofluorescence staining provided further IL-16induced PepT1 facilitates fMLF transport evidence that abundant PepT1 proteins are expressed on the epi- As mentioned above, PepT1-mediated fMLF transport plays an thelium of the colon-like intestines 7 d after TnIL-16 induction (5 important role in triggering colon inflammation. Therefore, we mg/fish; Fig. 8D). For a more accurate determination, an in vitro determined whether the upregulated PepT1 facilitates fMLF trans- stimulation assay was performed on a human colon-derived SW480 port in the colon-like intestine using an in vitro fMLF perfusion cell line. A previous study in our laboratory showed that secreted experiment. The colon-like intestines were collected from the fish IL-16 is highly conserved between human and Tetraodon (29). 7 d after stimulation with an optimized TnIL-16 concentration (5

11 10 IL-16 INDUCES COLITIS THROUGH UPREGULATING PepT1 Downloaded from http://www.jimmunol.org/ by guest on June 23, 2017 FIGURE 6. Time-dependent IL-16induced intestinal inflammation. (A) MPO activity of the colon-like intestines of the GST-treated (5 mg/fish) and IL- 16treated (5 mg/fish) Tetraodon at different time points after administration. Values are expressed as means 6 SEM. Additionally, the mRNA levels of inflammatory cytokines in the colon-like intestines of the groups mentioned above were examined using real-time PCR: (B) IL-1b, (C) IL-6, (D) IFN-g, (E) TNF-a, (F) IL-10. b-Actin was used as the internal reference gene. Values are expressed as means 6 SEM. The quantitative PCR value is the average of three replicates. *p , 0.05, **p , 0.01 versus values on day 0 (n = 10). mg/fish). The transported fMLF was detected by HPLC analysis. evaluate whether knockdown of TnPepT1 attenuates IL-16in- After incubating the intestines in Kerbs buffer at 28C for 15 min, duced colitis. A pSUPER vector system was developed to deliver the fMLF concentration in the incubation solution was significantly the siRNA against TnPepT1 following a method previously de- decreased (p , 0.05) compared with the control intestines stimu- scribed for a mouse model (40). Four siRNAs from predicted lated with the mock PBS, as reflected by the elution peak area (Fig. siRNAs that target different regions of TnPepT1 were selected for 8F). The comparative transport activity of TnPepT1 in the TnIL-16 functional assessment (Supplemental Table I). Of the four gener- induced colon-like intestine was 11.8 c/g (DfMLF concentration ated constructs (pSUPER-TnPepT1-siRNA-142, -220, -363, and per gram tissue weight), whereas that in the control intestine was -453), pSUPER-TnPepT1-siRNA-142 was proved to be the most only 5.7 c/g. These results clearly indicate that PepT1 upregulation effective (.75%) in inducing TnPepT1 mRNA degradation (Fig. in the colon-like intestines largely facilitates the transport of fMLF, 9A). Thus, this construct was used in the subsequent experiment. which contributes to the subsequent colitis. At 12 h after TnIL-16 injection (5 mg/fish in 100 ml PBS), the fish In vivo knockdown of PepT1 attenuated IL-16induced colitis were administered pSUPER-TnPepT1-siRNA-142 once a day for The role of PepT1 in IL-16induced colitis was further investi- 5 consecutive days. On day 7 after administration, real-time PCR gated using an in vivo TnPepT1 knockdown assay in Tetraodon to and Western blot analysis were performed to detect TnPepT1

12 The Journal of Immunology 11 FIGURE 7. Tissue distribution and expression detection of Tetraodon PepT1. (A) RT-PCR analysis illustrating the distribution of PepT1 in healthy Tetraodon. The PCR was performed for 35 cycles using specific primers for PepT1 and b-actin genes, and products from livers, spleens, posterior intes- tines, middle intestines, head kidneys, heart, brains, gills, skins, and muscles were loaded from left to right. (B) PepT1 expressions levels in different Tetraodon tissue were measured using real-time PCR and are shown relative to b-actin expression. Values are expressed as means 6 SEM. The quantitative PCR value is the average of three replicates, each of which included 10 fish. Downloaded from http://www.jimmunol.org/ by guest on June 23, 2017 suppression in the colon-like intestines. The MPO activity was vertebrates (350 Mb), is very similar to that of humans and other also examined to evaluate the colon inflammation. As expected, mammals in terms of genome catalogs and gene structures (56, 57). the TnIL-16induced TnPepT1 upregulation in the colon-like Therefore, Tetraodon is an excellent model for comparative geno- intestines was significantly suppressed (p , 0.01) in the siRNA- mic studies, which facilitates the understanding of common genetic treated groups compared with the control vector group (Fig. 9B, elements associated with stress responses, disease progression, and 9C). Accordingly, the TnIL-16induced increase in MPO activity basic physiological mechanisms and behavior, all of which may in the colon-like intestines was significantly decreased (p , 0.01) involve both environmental and hereditary components (58). Ac- in the siRNA-treated groups, suggesting that the inflammation tually, zebrafish and pufferfish have been used in various research induced by TnIL-16 could be dramatically attenuated by the areas, including developmental biology, immunology, physiology, knockdown of TnPepT1 (Fig. 9D). This result was further sup- biochemistry, genetics, and evolutionary biology. Particularly, these ported by the histopathological analysis. The signs of inflamma- fish species have greatly contributed to investigations that revealed tory injury to the colon-like intestines, such as colon wall damage, the onset and the course of pathological processes as well as the vessel dilatation, structure disorder of villi, inflammatory cell in- molecular mechanisms of many human diseases, such as hemato- filtration, decline of goblet cells, interstitial edema, crypt micro- poietic disorders, kidney diseases, and cancers (4850, 55, 5866). abscess, and cryptitis, were all markedly alleviated by siRNA Recently, zebrafish has been successfully used to study the sus- treatment (Fig. 9EH). These observations provide definite sup- ceptibility genes and host/microbe interactions in IBD (4850). In port to the role of TnPepT1 in TnIL-16induced colitis. the present study, Tetraodon showed again its advantage for un- covering the molecular mechanisms underlying IBD occurrence. Discussion We think that fish models, as complementary to mammalian models, Animal models are powerful tools for exploring major questions will greatly benefit the cross-species understanding of IBD pathol- in the life sciences. For example, in investigation on human IBD, ogy from fish to mammals as a whole. various animal models of colonic inflammation simulate certain The proposed role of IL-16 in the pathogenesis of IBD, par- important immunological and histopathological aspects of IBD ticularly the connection between IL-16 and PepT1 during disease that are difficult to address in humans, such as pathophysiological progression, was investigated using the Tetraodon model. Previ- mechanisms in the early phases of colitis (41). Aside from mam- ously, some investigations showed that the uptake of fMLF, a malian models, establishing new model systems is of considerable major N-formylated peptide from bacteria in the human colonic importance for understanding critical disease pathways and pro- lumen (67, 68), contributes to the progression of IBD (69). The viding long-term scientific robustness. In recent years, experimental transportation of fMLF is undertaken by PepT1, an exclusive fish models have attracted considerable interest of scientists because proton-coupled oligopeptide transporter in the brush border of the of the growing percentage of known fish species that contribute to small intestinal epithelium. Normally, PepT1 is an essential con- research. Hence, fish models have been incorporated into the sci- tributor to the transport of digested protein products (dipeptides entific mainstream (54). Increasing numbers of researchers are en- and tripeptides) and a variety of peptidomimetic substances but gaged in using fish species as nonmammalian models to study not tetrapeptides or free amino acids, using the energy generated human diseases and basic biological processes at the cellular and from the inwardly directed transmembrane proton gradient (52, molecular levels. Among them, zebrafish and pufferfish (such as T. 70). Physiologically, PepT1 is expressed restrictively in small nigroviridis and Fugu rubripes) are the two most attractive and most intestines that have low bacterial populations (71, 72). However, commonly used fish species because of their several advantages. For PepT1 is minimally expressed or absent in healthy colons that example, the optical clarity of embryos and larvae of zebrafish have high bacterial populations, a condition that minimizes the provides real-time visualization of developmental processes (55). intracellular uptake of bacterial peptides (70, 72, 73). However, The genome of Tetraodon, which is the smallest among the known abnormal PepT1 expression has been observed in the colonic

13 12 IL-16 INDUCES COLITIS THROUGH UPREGULATING PepT1 Downloaded from http://www.jimmunol.org/ by guest on June 23, 2017 FIGURE 8. IL-16 upregulates PepT1 expression and its transport of fMLF. (A) TnPepT1 mRNA levels 7 d after treatment with different doses of IL-16. (B) TnPepT1 mRNA levels at different time points after IL-16 or GST treatment at 5 mg per fish. (C) Western blot analysis of TnPepT1 expression in IL-16 treated intestines at different doses (7 d after treatment). (D) TnPepT1 expression in the colon-like intestine examined under immunofluorescence staining. Scale bars, 50 mm. (E) PepT1 mRNA level in SW480 cells after IL-16 stimulation at different doses, analyzed by real-time PCR. (F) IL-16mediated PepT1 upregulation enhanced the transport capacity of the intestines. After in vitro fMLF incubation, a significant elevation in fMLF transportation was observed in the colon-like intestines of the IL-16treated fish. The peaks on the top left corner are the results of the HPLC analysis of the concentration of fMLF that remained in the solution; bigger peak areas indicate higher concentrations. Values are expressed as means 6 SEM. The quantitative PCR value is the average of three replicates, and b-actin was used as the internal reference gene. For the in vivo experiments, each group included 10 fish. *p , 0.05, **p , 0.01 versus values in the control group [for (B), versus values on day 0]. epithelia under pathological conditions (74), which may trigger for colonic mucosa damage by mediating fMLF transport in IBD downstream proinflammatory effects, such as NF-kB activation (51, 70, 72), the exact molecular mechanism by which PepT1 and MHC class I expression, and this abnormal expression has expression is induced remains unknown. a close relationship with the progression of intestinal inflammation In the present study, IL-16 was found to be an important inducer (6870, 74). Although the aforementioned observations suggest of PepT1 expression in colonic tissues. It induces colonic in- that PepT1 upregulation in the colon might be a new mechanism flammation through upregulating PepT1 in the colon, thereby in-

14 The Journal of Immunology 13 FIGURE 9. Intestinal inflammation could be significantly attenuated by in vivo PepT1 knockdown in the IL-16induced colitis model. (A) Screening of effective siRNA. The HeLa cells were cotransfected with pSUPER-TnPepT1- siRNA or the control plasmid (pSUPER) together with the overexpression plasmid pc6-TnPepT1. The efficiency of the siRNA was detected using real-time PCR. (B and C) After in vivo TnPepT1- siRNA treatment, the PepT1 expression was remarkably suppressed both at the mRNA and protein levels. (D) MPO activity of the colon- like intestines of Tetraodon (n = 10). (E) His- topathological scoring of the control, IL-16 plus control vectortreated, and IL-16 plus pSUPER- siRNAtreated Tetraodon colon-like intestinal segments. Statistical results are shown as the average scores of 50 visual yields with 10 fish each group, which also indicates that the his- topathological features presented are repre- Downloaded from http://www.jimmunol.org/ by guest on June 23, 2017 sentative of the fish population. Fish in control groups were treated with mock PBS. For (A), (B), (D), and (E), values are expressed as means 6 SEM. The quantitative PCR value is the av- erage of three replicates, each of which included 10 fish, and b-actin was used as the internal reference gene. **p , 0.01 versus values in the IL-16 plus vector-treated groups. (F)(H) Histo- logical sections of the Tetraodon colon-like in- testine stained with H&E-safranin. After siRNA treatment (H), the intestinal mucosa damage and inflammatory cell infiltration were greatly im- proved, compared with the group treated with IL- 16 and vector (G). The fish in the control group (F) were treated with the same amount of mock PBS instead of IL-16. Scale bars, upper panels of (F)(H), 100 mm; lower panels of (F)(H), 10 mm. Typical inflammatory cells are indicated as follows: , neutrophils; , lymphocytes; *, eosi- nophils. creasing fMLF transport, which triggers the downstream inflam- IL-1b has no significant effect on TnPepT1 expression (data not matory pathways. To our knowledge, this is the first study on the shown). A similar result was observed in an in vitro assay using relationship between IL-16 and PepT1 in the pathogenesis of IBD. a colon-derived cell line under IL-16 stimulation. Furthermore, Several experiments in the present study support this hypothesis. upregulated PepT1 expression in the colon facilitated the transport First, the IL-16 level was significantly increased in the DSS- of fMLF into colon tissues, which is a crucial factor thought induced colitis model, which was accompanied by significant to result in the development of IBD. These results provide both PepT1 elevation in the colon tissues. In contrast, depletion of IL-16 in vivo and in vitro evidence that IL-16 is able to trigger PepT1 with antiTnIL-16 Abs inhibited the occurrence of colitis, which expression in the colon, thereby leading to fMLF-elicited inflam- provides initial insights into the role of IL-16 in colitis. Second, mation. Finally, a significant alleviation of intestinal inflammation administration of recombinant IL-16 to fish induces a series of was observed using in vivo PepT1 knockdown in the IL-16in- pathological changes in the colon-like intestines, including severe duced colitis model. This result strongly supports the observation tissue structural damage, significant elevations in MPO activity, that the IL-16induced colonic inflammation is caused by PepT1 and upregulation of proinflammatory cytokines such as IL-1b, IL- upregulation. This cytokine-mediated upregulation of PepT1 ex- 6, IFN-g, and TNF-a. These findings show that IL-16 can induce pression might be a key mechanism underlying IBD development. colonic inflammation with pathological features similar to those of Thus, the present study provides new insights into the pathogenesis a typical DSS-induced colitis model, which provides direct evi- of IBD and into the development of anti-inflammatory therapy for dence that IL-16 is indeed an important inducer of colitis, espe- IBD. Furthermore, it provides a better understanding of the func- cially UC-like intestinal inflammation. Third, IL-16 administration tional characteristics of IL-16 in colon inflammation and the mo- to fish also induces PepT1 expression in the colon tissues, ac- lecular basis for bowel disease. companied by inflammatory injury to the colon, as described Our previous studies have shown that TnIL-16 shares a number above. In this experiment, IL-1b, a representative proinflammatory of similar characteristics with its mammalian counterpart in terms cytokines, was used as a control to exclude the possible nonspecific of gene organization, amino acid sequence, functional motifs or role of other inflammatory cytokines, and the results showed that domains, molecular structure, protein cleavage site, and chemo-

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