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 colon. Moreover, the signs of colitis were dramatically attenuated by IL-16 depletion using anti–IL-16 Abs. In vivo IL-16 administration induced remarkable intestinal inflammation with typical ulcerative colitis–like 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-16–induced 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-16–based therapies for IBD.

Inflammatory bowel disease (IBD) is a chronic autoimmune-relevant disorder with a high risk of developing colon cancer that has a sustained worldwide increasing incidence and continues to be a global healthcare problem (1). Normally, IBD comprises two major forms, namely, ulcerative colitis (UC) and Crohn's disease (CD), with several common pathological features such as inflammatory cell infiltration, ulceration, hyperplasia of crypt cells, edema, goblet cell loss, and granulomas, which affect the gastrointestinal tract and colonic mucosa (1, 2). However, UC is characterized by diffused superficial inflammatory changes in the mucosa and submucosa that are mainly limited to the colon, whereas CD affects any part of the gastrointestinal tract by noncontinuous transmural inflammation that commonly affects the terminal ileum (1, 3). Although the clinical and pathological features of IBD have been well studied in the past years, its etiology and pathogenesis remain poorly understood. IBD is considered the outcome of inappropriate and continual inflammatory responses to commensal microbe imbalance and some environmental factors in a genetically susceptible host, which involve the extremely complicated cross-regulation of innate and adaptive immunity (3, 4). However, the detailed intercellular and molecular mechanisms underlying the IBD process remain elusive.

Recently, several susceptible genes, cytokines, and cytokine-signaling regulatory factors have been identified to contribute to the pathogenesis of IBD (59). For example, the single Ig IL-1R–related molecule controls the homeostasis of the colon by maintaining microbial tolerance (10). TNFR-associated factor-2 prevents the spontaneous development of colitis by prohibiting colonic epithelial cells from undergoing apoptosis (11). Functional alteration of nucleotide-binding oligomerization domain–containing protein 2 increases the association of intestinal bacteria with the epithelium (12, 13). The knockout of leucine-rich repeat kinase 2, which is closely related to susceptibility to CD, may cause exacerbated colitis (14). Furthermore, the TNF-like cytokine TL1A reportedly drives adaptive immune-mediated ileitis (15, 16), and several ILs such as IL-2, IL-10, IL-12, and IL-23 are involved in IBD, which indicates an imbalance between proinflammatory and anti-inflammatory influences (17, 18). However, the exact effects of these cytokines on the mechanism of IBD development are still undetermined.

Several previous epidemiological investigations have shown that IL-16 is considerably increased at both the mRNA and protein levels in pathologic intestinal tissues and the systemic circulation of patients with UC and CD compared with healthy control individuals (19, 20). Neutralization of IL-16 reportedly attenuates colitis-like inflammation in an experimental mouse model (21). Substantial increases in the number of IL-16+ mast cells have been observed in active CD (22). Concordantly, an association was also observed between polymorphism of the IL-16 gene promoter and CD (23). Hence, IL-16 may play an important role in the pathogenesis of IBD. However, direct functional evidence is still needed to clarify this implication. In the present study, we elucidate the involvement of IL-16 in the pathology of IBD in a fish model.

IL-16 was initially identified as a lymphocyte chemoattractant factor secreted from human PBMCs (24, 25). IL-16 is now thought to be a multifunctional cytokine and is the only known single PDZ domain protein secreted by human PBMCs (26, 27). Aside from its wide range of effects on cellular activities, such as upregulation of the high-affinity IL-2 receptor (CD25), induction of cells to reset into the G1 phase, inhibition of Ag-specific proliferation, and maintenance of Ag nonspecific proliferative properties, numerous investigations have implied that IL-16 is involved in the exacerbation of infectious and autoimmune inflammatory diseases, such as atopic dermatitis, IBD, systemic lupus erythematosus, multiple sclerosis, and neurodegenerative disorders, as well as viral and bacterial infections (26). Nonetheless, distinct evidence that reveals the mechanism of IL-16 in the direct regulation of disease progression is still limited. In the present study, we found that IL-16 is able to induce colitis by upregulating the expression of the oligopeptide transporter member 1 (PepT1) molecule on epithelial cells of the colon in a Tetraodon nigroviridis fish model. To our knowledge, this is the first detailed report that shows the involvement of IL-16 in the pathogenesis of UC, which indicates a new function of IL-16 and provides new insight into the molecular mechanism of IBD. Additionally, fish may become a novel animal model for the study and drug screening of IBD. Our findings should help in understanding the involvement of IL-16 in basic biological processes of disease pathways and even the functional involvement of this cytokine in the evolutionary history of vertebrates.

One-year-old spotted green pufferfish, T. nigroviridis, of both sexes, which were 4–5 cm in body length and weighed ∼3–5 g, were kept in running water at 25–28°C and fed with frozen red worms twice a day. The fish were held in the laboratory for at least 2 wk to allow them to acclimatize to the new environment and evaluate their overall health. Only healthy fish, determined by their general appearance and level of activity, were used in the study.

The colon-like cell line SW480 and HeLa cells were maintained in DMEM (HyClone Laboratories) and IMDM (HyClone Laboratories) supplemented with 10% FBS (PAA Laboratories), 100 U/ml penicillin (Life Technologies BRL), and 100 μg/ml streptomycin (Life Technologies BRL) at 37°C in 5% CO2. At near confluence, the cells were subcultured in dishes at a density of 1 × 104 cells/cm2.

Total RNA was extracted from the Tetraodon intestinal tissues using TRIzol reagent (Invitrogen) and reverse-transcribed into first single-stranded cDNA (Avian Myeloblastosis Virus RT-PCR Kit [version 3.0], TaKaRa Bio, Shiga, Japan). The full-length Tetraodon PepT1 (TnPepT1) cDNA was generated using RT-PCR and RACE-PCR with primers (shown in Supplemental Table I) designed according to the homolog sequence predicted from genome databases (University of California, Santa Cruz and National Center for Biotechnology Information) using the human PepT1 (SLC15A1) sequence (NP_005064) as a query and compiled by the Genscan and BLAST software programs (28). The encoding sequence of Tetraodon IL-16 (TnIL-16) was amplified using primers designed according to our previous report (29). The PCR products were purified using a gel extraction kit (Qiagen), inserted into the pGEM-T EASY vector (Promega), and transformed into competent Escherichia coli TOP10 cells (Invitrogen). The plasmid DNA was purified using a plasmid Miniprep kit (Qiagen) and sequenced on an ABI 3730 sequencer (Invitrogen). The comparative gene map positions and organizations, phylogenetic alignment, and potential structures of the PepT1 proteins were determined using databases and software programs as previously described (3033).

For distribution analysis, tissues samples from the intestines, spleen, liver, kidneys, skin, gills, muscles, brain, and heart were carefully collected from the healthy fish. For intestine-specific expression analysis, colon-like tissues were collected after dextran sodium sulfate (DSS) or IL-16 challenge. Total RNA was extracted and reversed-transcribed into cDNA as described above. The PCR amplifications were performed in 10 μl reaction mixtures containing 0.3 μl DNA template, 1 μl forward and reverse primers (shown in Supplemental Table I; 10 μM), 5 μl Taq polymerase mixture (TaKaRa Bio), and 3.7 μl double distilled H2O. The cycling protocol consisted of an initial denaturing cycle at 94°C for 4 min followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, with a final elongation step at 72°C for 10 min.

The encoding sequence of the extracellular region of TnPepT1 (TnPepT1-ex) was inserted into pET28a and pcDNA6 between the EcoRI and XhoI sites, respectively, to construct a prokaryotic expression vector (pET28a-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 encoding sequence was inserted into pET28a and pET41a between the EcoRI and XhoI sites, respectively, to construct two prokaryotic expression vectors (pET28a-TnIL-16 and pET41a-TnIL-16) where in the N terminus of TnIL-16 was fused with a His-tag or a GST-tag plus a His-tag. The constructed vectors were verified by double digestion with EcoRI and XhoI and further confirmed by DNA sequencing.

The pET28a-TnPepT1-ex, pET28a-TnIL-16, pET41a-TnIL-16, and pET-41a (for GST-tagged protein expression) plasmids were separately transformed into E. colI BL21 (DE3) cells. A single colony was inoculated into 100 ml Luria–Bertani (LB) medium with kanamycin (50 μg/ml). The inoculated Luria–Bertani medium was incubated in a shaking incubator at 37°C, 200 rpm until the OD600 value reached 0.6. Then isopropyl β-d-thiogalactoside 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 recombinant soluble TnIL-16 and GST-tag proteins were purified using Ni-NTA agarose affinity chromatography according to the QIAexpressionist manual (Qiagen). The recombinant TnPepT1-ex and TnIL-16 proteins from the inclusion bodies without GST-tags were purified by a similar protocol for polyclonal Ab preparation.

Six-week-old male New Zealand White rabbits weighing ∼1.5 kg were immunized with 500 μg purified recombinant TnPepT1-ex and TnIL-16 proteins, respectively, in CFA on the 1st day and IFA on the 3rd, 28th, and 35th days (34). One week after the final immunization, blood was collected from the rabbits when the Ab titers were >1/60,000 as determined by a microplate-based ELISA using the same TnPepT1-ex and TnIL-16 proteins adsorbed onto the solid phase. The Abs were initially purified into an IgG isotype using an affinity protein A-agarose column and further purified via an immunosorbent-based protocol using recombinant proteins absorbed onto the nitrocellulose membrane phase (Qiagen). Western blot and Ag-specific ELISA were performed to characterize the specificities of the Abs to TnPepT1 and TnIL-16.

The expression levels of TnPepT1, IL-1β, IL-6, IL-10, TNF-α, and IFN-γ transcripts were quantified through real-time PCR on a Mastercycler ep realplex machine (Eppendorf, Hamburg, Germany) using an SYBR Premix Ex Taq kit (TaKaRa Bio) according to the manufacturers’ instructions. Briefly, all real-time PCR reactions were performed in a total reaction volume of 10 μl. The experimental protocol consisted of the following: 1) 40 cycles of amplification at 95°C for 30 s, and then at 55–60°C (depending on the different genes) for 20 s; 2) melting curve analysis at 95°C for 15 s, 65°C for 15 s, 65°C drop to 95°C in 20 min and 95°C for 15 s; and 3) cooling at 40°C for 30 s. The total amount of mRNA was normalized to endogenous β-actin mRNA. The PCR primers are shown in Supplemental Table I. The relative gene expression was analyzed using the 2−ΔΔCt method (35). In all cases, each PCR trial was performed with triplicate samples and repeated at least three times.

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 inhibitors (Roche) at pH 8.0. The lysates were then boiled in sample buffer containing 2% SDS and 20% glycerol for 5 min. The samples containing 50 μg tissue proteins were separated using 10% SDS-PAGE and transferred onto 0.45-μm polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA) for 2 h at 4°C. The blots were blocked for 1 h in 5% nonfat dry milk at room temperature. After washing with TBST buffer, the blots were incubated overnight at 4°C with rabbit anti–TnPepT1-ex Ab at 1:1000 dilution. After washing three times for 30 min in TBST buffer, the blots were further incubated with HRP-conjugated anti-rabbit Abs at 1:5000 dilution for 1 h at room temperature. The blots were washed three times for 30 min in TBST buffer and were then visualized using ECL.

Each intestinal sample was fixed overnight in 10% buffered formalin, dehydrated using graded ethanol, vitrified with dimethylbenzene, and embedded in paraffin. Then, 5-μm sections were stained with H&E-safranin and examined under light microscopy (Zeiss Axiostar Plus) in a blinded manner. The gross inflammation index was assessed by histopathological scoring according to items shown in Table I. For inflammatory cell analysis, 5-μm frozen sections of intestinal samples were stained with Wright–Giemsa (Thermo Scientific) or a peroxidase staining kit (Merck Millipore) following the manufacturers’ instructions.

Table I.
Histopathological scoring standards
Observation ItemEvaluation Standards
OccurrenceScore
Infiltration of inflammatory cells None 
Mild 
Medium 
Severe 
Depth of inflammatory cell infiltration None 
Mucosa 
Muscularis propria 
Serosa 
Crypt Normal 
Occasionally vanishing 
Part vanishing 
Diffusedly vanishing 
Edema None 
Existence 
Mucoprotein decrease None 
Mild 
Severe 
Hemorrhage None 
Existence 
Pseudomembrane None 
Existence 
Observation ItemEvaluation Standards
OccurrenceScore
Infiltration of inflammatory cells None 
Mild 
Medium 
Severe 
Depth of inflammatory cell infiltration None 
Mucosa 
Muscularis propria 
Serosa 
Crypt Normal 
Occasionally vanishing 
Part vanishing 
Diffusedly vanishing 
Edema None 
Existence 
Mucoprotein decrease None 
Mild 
Severe 
Hemorrhage None 
Existence 
Pseudomembrane None 
Existence 

Paraffinized tissue sections were deparaffinized, hydrated in an ethyl alcohol gradient, and blocked with 5% BSA for 30 min. After washing twice with PBS, the sections were incubated with the primary Abs (rabbit anti-myeloperoxidase [MPO] or rabbit anti-TnPepT1) for 1.5 h at 37°C. Afterward, the sections were washed three times with PBS and incubated with HRP- or FITC-conjugated anti-rabbit IgG Abs for 1 h at 37°C. For immunohistochemical staining of MPO, the sections were further stained with H&E for 10 min at room temperature. The sections were washed three times with PBS and observed under a light or fluorescence microscope (Zeiss Axiostar Plus).

The DSS-induced colitis was prepared according to a previously reported method (36). Briefly, the experimental fish were fed twice daily with radiation-sterilized frozen red worms at ∼3% of their body weight. Then, they were administered with 500 μl 5% DSS (molecular mass of 36–50 kDa; MP Biomedicals) via oral perfusion once a day just before the second feeding time to ensure that the intestines of the fish were empty when they received the drug. The DSS administration was performed for 7 consecutive days to induce colitis. At the same time, fish in the control groups were given the same amount of mock PBS.

The plasma IL-16 levels in the DSS-induced Tetraodon colitis model were determined via ELISA and Western blot. For ELISA, serum samples were diluted 10-fold using coating buffer (1.59 g/l Na2CO3 and 2.94 g/l NaHCO3) and absorbed into the solid phase of the ELISA plate (100 μl/pore). Then, 2% BSA containing 0.1% Tween 20 was used as blocking solution. The plate was then incubated for 2 h with rabbit anti–IL-16 Abs at 37°C. HRP-conjugated anti-rabbit IgG Abs at 1:10,000 dilution were used as the secondary Ab. After washing the plate five times with TBST, freshly prepared substrate solution (100 μl/well) was added into the plate. Then, 2 M H2SO4 (50 μl/well) was added to the plate to stop the reaction until the color of the reactant solution changed. The absorbance (at 450 nm and at 630 nm) was detected using an absorbance microplate reader (BioTek Instruments model ExL800). Western blot analysis was performed as described above.

An in vivo IL-16 depletion assay was performed using anti–TnIL-16 Abs according to a previous study to evaluate the effects of IL-16 in IBD progression (21). Briefly, the fish were injected i.p. with the anti–TnIL-16 Abs (50 μg/fish) 1 d before DSS challenge and during the whole DSS treatment period (once daily before the DSS treatment). In parallel, the same amounts of nonspecific Abs (rabbit IgG) were administered to another group of DSS-treated fish as the negative control. Then, the activity of heme-containing enzyme MPO was determined to examine the severity of colitis.

Colon-like intestines were cleaned, weighed, and homogenized in 50 mM potassium phosphate buffer on ice and centrifuged at 12,000 × g for 15 min at 4°C. The precipitates were suspended in 300 μl 50 mM hexadecyltrimethylammonium bromide and 700 μl potassium phosphate buffer. Then, the precipitates were ultrasonicated for 20 s, frozen at −80°C for 5 min, and thawed to room temperature. After repeating the procedure twice, the samples were centrifuged at 12,000 × g for 10 min at 4°C and then the suspension was collected. The suspension (100 μl) was placed into a colorimetric cuvette with 2.9 ml potassium phosphate buffer containing 0.167 mg/ml O-dianisidine dihydrochloride and 0.165 mM H2O2. Then, the change in absorbance was measured continually every 30 s at 460 nm using a UV spectrophotometer (SP2100, Shanghai Spectrum Instruments, Shanghai, China) (37). One unit of MPO activity was defined as the quantity of enzyme that converts 1 μmol hydrogen peroxide into water within 1 min at room temperature and is expressed as units per gram of tissue (37).

An in vivo administration assay was performed to confirm that IL-16 plays a role in intestinal inflammation. The fish were initially fed radiation-sterilized frozen red worms twice daily at ∼3% of their body weight. Then, the fish were administered recombinant TnIL-16 protein via i.m. injection under the dorsal fin at a dosage gradient of 0.1, 0.5, 1, 5, and 10 μg per fish and a time gradient of 1, 2, 4, 7, 14, and 21 d after injection. In parallel, a GST-tag protein–injected group (5 μg/fish; this dose was much higher than the amount contained in the soluble IL-16-GST fusion protein) was devised to eliminate the interference of the GST-tagged protein, and a PBS-injected group was set as the negative control. Then, the colon-like intestines were collected to evaluate the extent of inflammation in terms of MPO activity, histologic changes, and inflammatory cytokine expression.

IL-16–induced TnPepT1 upregulation was examined both in vivo and in vitro. For the in vivo assay, the fish were administered TnIL-16 (at dosage and time gradients as described above) and their colon-like intestines were collected for PepT1 examination using real-time PCR, Western blot, and immunofluorescence staining as described above. For the in vitro assay, SW480 cells were stimulated with different TnIL-16 concentrations (25, 50, and 100 ng/ml). Then, the cells were collected for PepT1 expression analysis using real-time PCR.

The colon-like intestines were washed with PBS and carefully everted to expose the mucosa. Then, Kerbs buffer was poured into the enteric cavity and two ends of the intestine were ligated. The everted intestines were soaked in 1 ml fMLF (Sangon Biotech) solution (1 μM) and shaken at 100 rpm for 15 min at 28°C to allow sufficient transportation. In parallel, similarly prepared tissues from PBS-injected fish were set as the control. Subsequently, the concentration of the remaining fMLF in the solution was determined by HPLC following previously described methods (38). Briefly, 25 μl sample was injected into an HPLC system (Shimadzu VP-ODS column with 5-μm particle size, 150 × 4.6 mm) once at 30°C. Acetonitrile was 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 a concentration gradient was used to establish a standard curve. The comparative transport activity of TnPepT1 was calculated as follows: ΔfMLF concentration/tissue weight.

Small interfering RNA (siRNA) specific for TnPepT1 was designed using siRNA template design tools (Dharmacon and Invitrogen RNAi designer) (39). Four TnPepT1 sequence-targeted siRNAs (Supplemental Table I) were selected to screen the activity. DNA oligonucleotides for hairpin RNA expression (synthesized by Invitrogen) were dissolved in double distilled H2O at 100 mM. Then, equimolar amounts of sense and antisense strands were mixed with annealing buffer (10× buffer stock, 100 mM Tris-HCl [pH 7.5], 10 mM EDTA, and 1 M NaCl) and the mixture (10 μl) was incubated at 90°C for 4 min, 70°C for 10 min, and then cooled to 10°C. The pSUPER vector (pSUPER.retro.puro; OligoEngine) was digested with HindIII and BglII, and the annealed oligonucleotides were ligated into pSUPER downstream of the H1 promoter to generate four constructs (pSUPER-TnPepT1-siRNA-142, -220, -363, and -453) with different siRNAs. The pSUPER-TnPepT1-siRNA constructs or the control pSUPER were cotransfected with pcDNA6-TnPepT1 (4 μg DNA in serum-free, antibiotic-free Opti-MEM I medium using Lipofectamine 2000; Invitrogen) into HeLa cells in six-well 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 after transfection, the cells were lysed for total RNA extraction. The specificity and efficiency of siRNA-targeted TnPepT1 was determined using real-time PCR.

In vivo knockdown of TnPepT1 was performed using a pSUPER vector-based siRNA deliver system that was recently applied in the gut system (40). Briefly, after IL-16 administration, the vector or the plasmid with high efficiency of siRNA against TnPepT1 (pSUPER-TnPepT1-siRNA-142) mixed with Lipofectamine 2000 was administered s.c. under the dorsal fin at 12 μg (∼3 μg/g body weight) per injection into each fish for five consecutive days. In parallel, the control group was injected with control vector. Total RNA was extracted from the colon-like intestines and was reverse-transcribed into cDNA. Real-time PCR was conducted to evaluate the efficiency of the in vivo TnPepT1 suppression.

All experiments were replicated at least three times. The sample number of each group was 10 fish with equal average body weights. The data are expressed as means ± SD. Statistical evaluation of the differences between values from different experimental groups was performed with multiple Student t tests. A p value < 0.05 was considered statistically significant.

It has been reported that DSS treatment leads to superficial inflammatory changes in the mucosa and submucosa of colon tissues in mouse models with various histopathological features similar to that seen in human UC (36, 4145). Therefore, the DSS-induced colitis model has become one of the most widely used experimental models for human UC research (42). In this study, to reconfirm the increased IL-16 level in clinical epidemiological investigations and to explore the possible role of IL-16 in intestinal bowel disease by a fish model, we generated a DSS-induced intestine inflammation model in Tetraodon. Histological analysis showed that the structure of Tetraodon intestines is generally similar to that of mammals. Different intestinal segments exhibited distinct differences. For example, the villi in the middle intestinal segment of Tetraodon were relatively long and the striated border was distinct. These characteristics are similar to those in mammalian small intestines (Fig. 1B, 1C). Similarly, the posterior intestinal segment consisted of pillar and goblet cells with considerably shorter villi, similar to the colon of mammals; thus, we called it the colon-like intestine of Tetraodon (Fig. 1D). After DSS administration, intestinal hyperemia and swelling were anatomically observed. Histological analysis showed that the lamina propria and submucosa of the colon-like segment were clearly damaged, and a significant number of inflammatory cells, including lymphocytes, neutrophilic granulocytes, and eosinophilic granulocytes, were observed in the mucosa (Figs. 1H, 2B, 2D, 2F). However, the structure of the middle intestines seemed to be slightly injured, but no apparent damage or inflammatory infiltration was observed in the mucosa of the front segments, although the villi structure exhibited slight irregularities (Fig. 1E–G). Furthermore, the severity of the intestinal inflammation was also determined based on histopathological scoring and MPO activity. The histopathological scoring of each intestinal segment was performed according to various items shown in Table I. The result showed that among all of the intestinal segments tested, the most severe inflammation occurs in colon-like tissues (Fig. 1I). MPO is one of the key components of inflammatory granulocytes such as neutrophils that accumulate at inflammation sites and play a major role in inflammatory pathology (46, 47). Thus, MPO is a valuable biomarker for determining inflammatory cell infiltration and the degree of inflammation in IBD pathology (46, 47). As expected, our results show that the MPO activity of the colon-like intestine in the DSS-treated groups was significantly increased (p < 0.01; Fig. 2G). Given that the DSS-induced Tetraodon intestine inflammation was mainly restricted to the colon-like intestines, and the most striking inflammatory changes were superficial and limited to the mucosa and submucosa, all of which resembled the histopathological aspects of DSS-induced UC-like injury in mice, as well as UC in humans, we termed it DSS-induced Tetraodon UC-like colitis, thereby providing further insights into the conservation of intestinal systems. Recently, zebrafish has been suggested as a good model for investigating the possible susceptibility genes and host–microbial interactions in IBD (4850). It is thus anticipated that fish may become another model animal that is swimming into the view of IBD research.

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 Tetraodon 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 μm (except for the partially enlarged images at the bottom of (D) and (H), 10 μm). (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 ± SEM. **p < 0.01 as compared with control groups.

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 Tetraodon 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 μm (except for the partially enlarged images at the bottom of (D) and (H), 10 μm). (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 ± SEM. **p < 0.01 as compared with control groups.

Close modal
FIGURE 2.

DSS-induced intestinal inflammation is accompanied by a significant increase in IL-16 level. (A and B) Wright–Giemsa (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 μm. (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 β-actin was used as the internal reference gene. Values are expressed as means ± SEM. *p < 0.05, **p < 0.01 compared with the control groups.

FIGURE 2.

DSS-induced intestinal inflammation is accompanied by a significant increase in IL-16 level. (A and B) Wright–Giemsa (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 μm. (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 β-actin was used as the internal reference gene. Values are expressed as means ± SEM. *p < 0.05, **p < 0.01 compared with the control groups.

Close modal

It has been reported that IL-16 expression in the colonic mucosa of IBD patients increased compared with the healthy controls (19). To determine whether a similar mechanism occurs in the Tetraodon colitis model, the plasma IL-16 concentrations in fish with DSS-induced colitis were measured. As expected, the IL-16 concentrations were significantly increased 5-fold (p < 0.01) in the serum of the DSS-treated fish compared with the PBS-treated controls (Fig. 2H). This result is in accordance with the results of the human epidemiological investigation. To confirm that IL-16 affects colitis progression, a depletion assay was performed using anti–TnIL-16 Abs. The Abs were administered once a day to optimally deplete TnIL-16 during the DSS treatment. MPO activity was used to determine the severity of inflammation. The results show that the MPO activity was significantly (p < 0.05) decreased in the affected fish compared with the nondepleted and the negative IgG-administered controls (Fig. 2I). Dramatically, the TnPepT1 expression was also significantly decreased (p < 0.05) in the colon-like intestines accompanied by depletion of TnIL-16 (Fig. 2J). These findings suggest that IL-16 may largely contribute to the pathophysiology of IBD, and that PepT1 might be closely involved in this process.

The aforementioned experiments provided preliminary observations of the positive correlation between IL-16 and colitis. Thus, an in vivo administration assay was performed to provide further evidence. Recombinant TnIL-16 was administered into fish at different doses for different periods. Inflammation of the colon-like intestines was determined based on histological changes, MPO activity, and expression of proinflammatory cytokines. As expected, administration of TnIL-16 to Tetraodon induced typical inflammatory intestinal injury with the pathological features seen in the DSS-induced model (Fig. 3). Histopathological analysis showed that the colon-like intestines developed severe hyperemia and swelling, and the intestinal walls were severely damaged. The villi in the colon-like intestines were blunted and broadened with epithelial exfoliation and fibrinous exudation (Fig. 3C). Decreased goblet cells and interstitial edema were clearly observed in the colonic mucosa. Dilatation of vessels was associated with general infiltration of inflammatory cells, including lymphocytes, neutrophilic granulocytes, and eosinophilic granulocytes in the lamina propria and submucosa (Figs. 3C, 3D, 4C, 4F, 4I). Furthermore, other histopathological features, such as granulomas with macrophage aggregation, crypt microabscesses, and cryptitis, were also observed. The histopathological scoring (Table I), which estimated the severity of inflammation, was determined from various observation items and based on overall merit. The TnIL-16 treatment groups obtained a score of 7.0 (Fig. 3E), which was similar to the score of the DSS-administered fish (i.e., 7.7).

FIGURE 3.

IL-16–induced 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 μg/fish) fish (B) showed normal intestinal wall features 7 d after treatment. Histological sections of the colon-like intestines 7 d after treatment with 5 μg IL-16 per fish (C, D) show serious colon wall damage. Fibrinous exudation associated with general infiltration of inflammatory cells (lymphocytes, neutrophils, and eosinophilic granulocytes) in the lamina propria and submucosa (C), granulomas (D), and epithelial exfoliation are shown. Scale bars, left panels of (A)–(D), 100 μm; right panels of (A)–(D), 10 μm. Typical inflammatory cells are indicated as follows: ▴, neutrophils; ↑, lymphocytes; *, eosinophils. (E) Histopathological scoring of the control, GST-treated, and IL-16–treated 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 representative of IL-16–treated fish. The fish in the control groups were treated with mock PBS. Values are expressed as means ± SEM. **p < 0.01 compared with the control group.

FIGURE 3.

IL-16–induced 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 μg/fish) fish (B) showed normal intestinal wall features 7 d after treatment. Histological sections of the colon-like intestines 7 d after treatment with 5 μg IL-16 per fish (C, D) show serious colon wall damage. Fibrinous exudation associated with general infiltration of inflammatory cells (lymphocytes, neutrophils, and eosinophilic granulocytes) in the lamina propria and submucosa (C), granulomas (D), and epithelial exfoliation are shown. Scale bars, left panels of (A)–(D), 100 μm; right panels of (A)–(D), 10 μm. Typical inflammatory cells are indicated as follows: ▴, neutrophils; ↑, lymphocytes; *, eosinophils. (E) Histopathological scoring of the control, GST-treated, and IL-16–treated 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 representative of IL-16–treated fish. The fish in the control groups were treated with mock PBS. Values are expressed as means ± SEM. **p < 0.01 compared with the control group.

Close modal
FIGURE 4.

IL-16 administration leads to significant inflammatory cell infiltration in the colon-like intestines of Tetraodon. (A)–(C) Wright–Giemsa (WG) staining of the frozen sections of control, GST-treated, and IL-16–treated Tetraodon colon-like intestines. (D)–(F) Peroxidase (POX) staining of the frozen sections of control, GST-treated and IL-16–treated Tetraodon colon-like intestines showing granulocyte infiltration. (G)–(I) Immunohistochemical staining of MPO in paraffin sections of control, GST-treated, and IL-16–treated 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-16–treated fish. Typical inflammatory cells are indicated as follows: ▴, neutrophils; ↑, lymphocytes; *, eosinophils; ★, granulocytes. Scale bars, 10 μm. Fish in the IL-16 and GST groups were treated with 5 μg IL-16 or GST per fish. Fish in the control groups were treated with mock PBS. The analyses were performed 7 d after treatment.

FIGURE 4.

IL-16 administration leads to significant inflammatory cell infiltration in the colon-like intestines of Tetraodon. (A)–(C) Wright–Giemsa (WG) staining of the frozen sections of control, GST-treated, and IL-16–treated Tetraodon colon-like intestines. (D)–(F) Peroxidase (POX) staining of the frozen sections of control, GST-treated and IL-16–treated Tetraodon colon-like intestines showing granulocyte infiltration. (G)–(I) Immunohistochemical staining of MPO in paraffin sections of control, GST-treated, and IL-16–treated 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-16–treated fish. Typical inflammatory cells are indicated as follows: ▴, neutrophils; ↑, lymphocytes; *, eosinophils; ★, granulocytes. Scale bars, 10 μm. Fish in the IL-16 and GST groups were treated with 5 μg IL-16 or GST per fish. Fish in the control groups were treated with mock PBS. The analyses were performed 7 d after treatment.

Close modal

Quantitative MPO activity analysis showed that the enzyme significantly increased (p < 0.05 and p < 0.01) with increasing TnIL-16 concentration (on the day 7). A 3- to 11-fold elevation was observed in the groups stimulated with 0.1, 0.5, 1, 5, and 10 μg TnIL-16 (per fish). The effects of increasing TnIL-16 concentration plateaued at 10 μg per fish (Fig. 5A). However, only a slight increase in MPO activity was observed in the GST-tag–injected control group. This result may be attributed to the GST protein, which acts as an Ag and mildly stimulates the fish immune system. MPO activity was also significantly increased (p < 0.05 and p < 0.01) in colitis tissues with time (1 d to 21 d) during the progression of intestinal inflammation (Fig. 6A). A slight increase in MPO activity was observed 1 d after induction (5 μg/fish). The MPO activity was highest on day 7, at 12-fold higher than that of the negative controls. However, the MPO activity stopped increasing 14 d after treatment. Thus, the severity of inflammation induced by TnIL-16 was dose- and time-dependent.

FIGURE 5.

Dosage-dependent IL-16–induced intestinal inflammation. (A) MPO activity of the colon-like intestines of the control, GST-treated, and IL-16–treated Tetraodon. Values are expressed as means ± SEM. Additionally, the mRNA levels of inflammatory cytokines in the colon-like intestines were examined by real-time PCR: (B) IL-1β, (C) IL-6, (D) IFN-γ, (E) TNF-α, (F) IL-10. β-Actin was used as the internal reference gene. Values are expressed as means ± 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).

FIGURE 5.

Dosage-dependent IL-16–induced intestinal inflammation. (A) MPO activity of the colon-like intestines of the control, GST-treated, and IL-16–treated Tetraodon. Values are expressed as means ± SEM. Additionally, the mRNA levels of inflammatory cytokines in the colon-like intestines were examined by real-time PCR: (B) IL-1β, (C) IL-6, (D) IFN-γ, (E) TNF-α, (F) IL-10. β-Actin was used as the internal reference gene. Values are expressed as means ± 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).

Close modal
FIGURE 6.

Time-dependent IL-16–induced intestinal inflammation. (A) MPO activity of the colon-like intestines of the GST-treated (5 μg/fish) and IL-16–treated (5 μg/fish) Tetraodon at different time points after administration. Values are expressed as means ± 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-1β, (C) IL-6, (D) IFN-γ, (E) TNF-α, (F) IL-10. β-Actin was used as the internal reference gene. Values are expressed as means ± SEM. The quantitative PCR value is the average of three replicates. *p < 0.05, **p < 0.01 versus values on day 0 (n = 10).

FIGURE 6.

Time-dependent IL-16–induced intestinal inflammation. (A) MPO activity of the colon-like intestines of the GST-treated (5 μg/fish) and IL-16–treated (5 μg/fish) Tetraodon at different time points after administration. Values are expressed as means ± 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-1β, (C) IL-6, (D) IFN-γ, (E) TNF-α, (F) IL-10. β-Actin was used as the internal reference gene. Values are expressed as means ± SEM. The quantitative PCR value is the average of three replicates. *p < 0.05, **p < 0.01 versus values on day 0 (n = 10).

Close modal

The proinflammatory cytokines in intestines with colitis were also examined. IL-1β, IL-6, IFN-γ, and TNF-α were all upregulated in response to TnIL-16 stimulation (Fig. 5B–E), although IFN-γ and TNF-α showed only slight increases under the low-dose treatment (0.1 μg/fish) (Fig. 5D, 5E). The TnIL-16–induced proinflammatory cytokine expressions were also dose- and time-dependent. The fish treated with 5 μg TnIL-16 for 4–7 d developed the most serious inflammation by overall assessment. Notably, the expression of anti-inflammatory cytokine IL-10, an important marker for IBD that is inhibited during inflammation, was significantly decreased (p < 0.01) even under the low-dose treatment (0.5 μg/fish) (Fig. 5F). On the first day after treatment, the IL-10 expression decreased by 40% compared with the control groups that received GST-tag or the mock PBS (Fig. 6F), whereas the other proinflammatory cytokines were not increased at this point. However, all of the proinflammatory cytokines were upregulated and reached their peak on day 4–7 after treatment (Fig. 6B–E). This process was accompanied by consistent decreases in IL-10 expression at different points. Collectively, the results clearly show that IL-16 could induce significant colon inflammation in Tetraodon. The pathological features of IL-16–induced intestinal inflammation resembled that of DSS-induced colitis in both Tetraodon and mouse models (3). Therefore, the nature of the IL-16–induced fish intestinal inflammation could also be considered UC-like colitis.

PepT1 plays a crucial role in IBD development by accelerating the transportation of the fMLF peptide, an important signal for the infiltration of inflammatory cells (51). Therefore, a TnIL-16–induced PepT1 expression assay was performed both in vivo and in vitro to confirm that TnIL-16 induces colitis through TnPepT1 upregulation in the colon-like intestines. For this purpose, PepT1 was initially identified in Tetraodon. The results showed that the TnPepT1 cDNA contains a 2178-bp open reading frame that encodes a predicted polypeptide with 725 aa, a 90-bp 5′ untranslated region, and a 60-bp 3′ untranslated region (Supplemental Fig. 1; GenBank accession no. JX177494; http://www.ncbi.nlm.nih.gov/genbank/). The TnPepT1 gene is located within a 9208-bp genomic fragment on chromosome 3 with 23 exons. The structural organization of the gene conforms to that of other species such as humans and mice (Supplemental Fig. 2A). The genes adjacent to the TnPepT1 locus, such as MBNL2, RAP2A, FARP1, DOCK9, and STK24, were all also found in identical order on human chromosome 13 and mouse chromosome 14, which shows the high conservation of genome synteny of PepT1 molecules (Supplemental Fig. 2B). Phylogenetic analysis showed that TnPepT1 was clustered with PepT1 family members (Supplemental Fig. 2C). The TnPepT1 protein was predicted to have 12 transmembrane domains and an extracellular region between the 9th and 10th transmembrane domains, which is also highly conserved in the PepT1 proteins in different species (52). Multiple alignment analysis showed that TnPepT1 shares an overall sequence identity of 55–61% with other species, and the sequence identity of the transmembrane domains was even much higher (Supplemental Fig. 3). The results suggests that key functional motifs, such as the first four N-terminal and the seventh and ninth transmembrane domains, which are required for substrate binding and transporter functionality (53), are completely conserved in many vertebrate species from fish to mammals.

To determine whether fish PepT1 has a tissue-specific distribution similar to that in mammals, various tissues were collected from Tetraodon. The intestinal tissues were divided into several segments for more precise determination. The TnPepT1 mRNA transcripts in healthy fish (without DSS and IL-16 stimulation) were highest in the middle intestines, but were at very low or undetectable levels in other tissues (Fig. 7). Importantly, TnPepT1 expression was scarcely detected in the posterior intestines under homeostatic conditions without DSS and IL-16 stimulation, whereas the expression was significantly upregulated under inflammatory conditions. Therefore, TnPepT1 is specifically distributed in the middle intestines (equivalently to the small intestines of mammals), which further suggests that the PepT1 in fish functionally corresponds to the intestinal PepT1 in mammals.

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 β-actin genes, and products from livers, spleens, posterior intestines, 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 β-actin expression. Values are expressed as means ± SEM. The quantitative PCR value is the average of three replicates, each of which included 10 fish.

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 β-actin genes, and products from livers, spleens, posterior intestines, 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 β-actin expression. Values are expressed as means ± SEM. The quantitative PCR value is the average of three replicates, each of which included 10 fish.

Close modal

The TnIL-16–induced PepT1 expression was determined at the mRNA and protein levels by real-time PCR, Western blot analysis, and immunofluorescence staining, respectively. The real-time PCR showed that in vivo TnIL-16 administration significantly induces TnPepT1 expression in the colon-like intestines in a dose- and time-dependent manner. Treatment with 5 μg and with 10 μg per fish for 7 d induced a 30-fold increase in TnPepT1 expression in the TnIL-16–injected groups compared with the control groups (Fig. 8A). The TnPepT1 expression was consistently elevated during the first 7 d of stimulation (5 μg/fish), which persisted for 14 d (Fig. 8B). Similar results were observed in the Western blot analysis (Fig. 8C). Immunofluorescence staining provided further evidence that abundant PepT1 proteins are expressed on the epithelium of the colon-like intestines 7 d after TnIL-16 induction (5 μg/fish; Fig. 8D). For a more accurate determination, an in vitro stimulation assay was performed on a human colon-derived SW480 cell line. A previous study in our laboratory showed that secreted IL-16 is highly conserved between human and Tetraodon (29). Therefore, TnIL-16 was used as a stimulant. Accordingly, PepT1 expression was significantly induced by TnIL-16 in a dose-dependent manner (Fig. 8E). This result provides direct evidence that IL-16 was able to trigger PepT1 expression. In summary, the IL-16–induced upregulation of PepT1 expression in the colon-like intestines corresponds to the development of inflammation.

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 μg 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 μm. (E) PepT1 mRNA level in SW480 cells after IL-16 stimulation at different doses, analyzed by real-time PCR. (F) IL-16–mediated 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-16–treated 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 ± SEM. The quantitative PCR value is the average of three replicates, and β-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].

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 μg 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 μm. (E) PepT1 mRNA level in SW480 cells after IL-16 stimulation at different doses, analyzed by real-time PCR. (F) IL-16–mediated 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-16–treated 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 ± SEM. The quantitative PCR value is the average of three replicates, and β-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].

Close modal

As mentioned above, PepT1-mediated fMLF transport plays an important role in triggering colon inflammation. Therefore, we determined whether the upregulated PepT1 facilitates fMLF transport in the colon-like intestine using an in vitro fMLF perfusion experiment. The colon-like intestines were collected from the fish 7 d after stimulation with an optimized TnIL-16 concentration (5 μg/fish). The transported fMLF was detected by HPLC analysis. After incubating the intestines in Kerbs buffer at 28°C for 15 min, the fMLF concentration in the incubation solution was significantly decreased (p < 0.05) compared with the control intestines stimulated with the mock PBS, as reflected by the elution peak area (Fig. 8F). The comparative transport activity of TnPepT1 in the TnIL-16–induced colon-like intestine was 11.8 c/g (ΔfMLF concentration per gram tissue weight), whereas that in the control intestine was only 5.7 c/g. These results clearly indicate that PepT1 upregulation in the colon-like intestines largely facilitates the transport of fMLF, which contributes to the subsequent colitis.

The role of PepT1 in IL-16–induced colitis was further investigated using an in vivo TnPepT1 knockdown assay in Tetraodon to evaluate whether knockdown of TnPepT1 attenuates IL-16–induced colitis. A pSUPER vector system was developed to deliver the siRNA against TnPepT1 following a method previously described for a mouse model (40). Four siRNAs from predicted siRNAs that target different regions of TnPepT1 were selected for functional assessment (Supplemental Table I). Of the four generated constructs (pSUPER-TnPepT1-siRNA-142, -220, -363, and -453), pSUPER-TnPepT1-siRNA-142 was proved to be the most effective (>75%) in inducing TnPepT1 mRNA degradation (Fig. 9A). Thus, this construct was used in the subsequent experiment. At 12 h after TnIL-16 injection (5 μg/fish in 100 μl PBS), the fish were administered pSUPER-TnPepT1-siRNA-142 once a day for 5 consecutive days. On day 7 after administration, real-time PCR and Western blot analysis were performed to detect TnPepT1 suppression in the colon-like intestines. The MPO activity was also examined to evaluate the colon inflammation. As expected, the TnIL-16–induced TnPepT1 upregulation in the colon-like intestines was significantly suppressed (p < 0.01) in the siRNA-treated groups compared with the control vector group (Fig. 9B, 9C). Accordingly, the TnIL-16–induced increase in MPO activity in the colon-like intestines was significantly decreased (p < 0.01) in the siRNA-treated groups, suggesting that the inflammation induced by TnIL-16 could be dramatically attenuated by the knockdown of TnPepT1 (Fig. 9D). This result was further supported by the histopathological analysis. The signs of inflammatory injury to the colon-like intestines, such as colon wall damage, vessel dilatation, structure disorder of villi, inflammatory cell infiltration, decline of goblet cells, interstitial edema, crypt microabscess, and cryptitis, were all markedly alleviated by siRNA treatment (Fig. 9E–H). These observations provide definite support to the role of TnPepT1 in TnIL-16–induced colitis.

FIGURE 9.

Intestinal inflammation could be significantly attenuated by in vivo PepT1 knockdown in the IL-16–induced 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) Histopathological scoring of the control, IL-16 plus control vector–treated, and IL-16 plus pSUPER-siRNA–treated 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 histopathological features presented are representative of the fish population. Fish in control groups were treated with mock PBS. For (A), (B), (D), and (E), values are expressed as means ± SEM. The quantitative PCR value is the average of three replicates, each of which included 10 fish, and β-actin was used as the internal reference gene. **p < 0.01 versus values in the IL-16 plus vector-treated groups. (F)–(H) Histological sections of the Tetraodon colon-like intestine stained with H&E-safranin. After siRNA treatment (H), the intestinal mucosa damage and inflammatory cell infiltration were greatly improved, 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 μm; lower panels of (F)–(H), 10 μm. Typical inflammatory cells are indicated as follows: ▴, neutrophils; ↑, lymphocytes; *, eosinophils.

FIGURE 9.

Intestinal inflammation could be significantly attenuated by in vivo PepT1 knockdown in the IL-16–induced 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) Histopathological scoring of the control, IL-16 plus control vector–treated, and IL-16 plus pSUPER-siRNA–treated 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 histopathological features presented are representative of the fish population. Fish in control groups were treated with mock PBS. For (A), (B), (D), and (E), values are expressed as means ± SEM. The quantitative PCR value is the average of three replicates, each of which included 10 fish, and β-actin was used as the internal reference gene. **p < 0.01 versus values in the IL-16 plus vector-treated groups. (F)–(H) Histological sections of the Tetraodon colon-like intestine stained with H&E-safranin. After siRNA treatment (H), the intestinal mucosa damage and inflammatory cell infiltration were greatly improved, 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 μm; lower panels of (F)–(H), 10 μm. Typical inflammatory cells are indicated as follows: ▴, neutrophils; ↑, lymphocytes; *, eosinophils.

Close modal

Animal models are powerful tools for exploring major questions in the life sciences. For example, in investigation on human IBD, various animal models of colonic inflammation simulate certain important immunological and histopathological aspects of IBD that are difficult to address in humans, such as pathophysiological mechanisms in the early phases of colitis (41). Aside from mammalian models, establishing new model systems is of considerable importance for understanding critical disease pathways and providing long-term scientific robustness. In recent years, experimental fish models have attracted considerable interest of scientists because of the growing percentage of known fish species that contribute to research. Hence, fish models have been incorporated into the scientific mainstream (54). Increasing numbers of researchers are engaged in using fish species as nonmammalian models to study human diseases and basic biological processes at the cellular and molecular levels. Among them, zebrafish and pufferfish (such as T. nigroviridis and Fugu rubripes) are the two most attractive and most commonly used fish species because of their several advantages. For example, the optical clarity of embryos and larvae of zebrafish provides real-time visualization of developmental processes (55). The genome of Tetraodon, which is the smallest among the known vertebrates (∼350 Mb), is very similar to that of humans and other mammals in terms of genome catalogs and gene structures (56, 57). Therefore, Tetraodon is an excellent model for comparative genomic studies, which facilitates the understanding of common genetic elements associated with stress responses, disease progression, and basic physiological mechanisms and behavior, all of which may involve both environmental and hereditary components (58). Actually, zebrafish and pufferfish have been used in various research areas, including developmental biology, immunology, physiology, biochemistry, genetics, and evolutionary biology. Particularly, these fish species have greatly contributed to investigations that revealed the onset and the course of pathological processes as well as the molecular mechanisms of many human diseases, such as hematopoietic disorders, kidney diseases, and cancers (4850, 55, 5866). Recently, zebrafish has been successfully used to study the susceptibility genes and host/microbe interactions in IBD (4850). In the present study, Tetraodon showed again its advantage for uncovering the molecular mechanisms underlying IBD occurrence. We think that fish models, as complementary to mammalian models, will greatly benefit the cross-species understanding of IBD pathology from fish to mammals as a whole.

The proposed role of IL-16 in the pathogenesis of IBD, particularly the connection between IL-16 and PepT1 during disease progression, was investigated using the Tetraodon model. Previously, some investigations showed that the uptake of fMLF, a major N-formylated peptide from bacteria in the human colonic lumen (67, 68), contributes to the progression of IBD (69). The transportation of fMLF is undertaken by PepT1, an exclusive proton-coupled oligopeptide transporter in the brush border of the small intestinal epithelium. Normally, PepT1 is an essential contributor to the transport of digested protein products (dipeptides and tripeptides) and a variety of peptidomimetic substances but not tetrapeptides or free amino acids, using the energy generated from the inwardly directed transmembrane proton gradient (52, 70). Physiologically, PepT1 is expressed restrictively in small intestines that have low bacterial populations (71, 72). However, PepT1 is minimally expressed or absent in healthy colons that have high bacterial populations, a condition that minimizes the intracellular uptake of bacterial peptides (70, 72, 73). However, abnormal PepT1 expression has been observed in the colonic epithelia under pathological conditions (74), which may trigger downstream proinflammatory effects, such as NF-κB activation and MHC class I expression, and this abnormal expression has a close relationship with the progression of intestinal inflammation (6870, 74). Although the aforementioned observations suggest that PepT1 upregulation in the colon might be a new mechanism for colonic mucosa damage by mediating fMLF transport in IBD (51, 70, 72), the exact molecular mechanism by which PepT1 expression is induced remains unknown.

In the present study, IL-16 was found to be an important inducer of PepT1 expression in colonic tissues. It induces colonic inflammation through upregulating PepT1 in the colon, thereby increasing fMLF transport, which triggers the downstream inflammatory pathways. To our knowledge, this is the first study on the relationship between IL-16 and PepT1 in the pathogenesis of IBD. Several experiments in the present study support this hypothesis. First, the IL-16 level was significantly increased in the DSS-induced colitis model, which was accompanied by significant PepT1 elevation in the colon tissues. In contrast, depletion of IL-16 with anti–TnIL-16 Abs inhibited the occurrence of colitis, which provides initial insights into the role of IL-16 in colitis. Second, administration of recombinant IL-16 to fish induces a series of pathological changes in the colon-like intestines, including severe tissue structural damage, significant elevations in MPO activity, and upregulation of proinflammatory cytokines such as IL-1β, IL-6, IFN-γ, and TNF-α. These findings show that IL-16 can induce colonic inflammation with pathological features similar to those of a typical DSS-induced colitis model, which provides direct evidence that IL-16 is indeed an important inducer of colitis, especially UC-like intestinal inflammation. Third, IL-16 administration to fish also induces PepT1 expression in the colon tissues, accompanied by inflammatory injury to the colon, as described above. In this experiment, IL-1β, a representative proinflammatory cytokines, was used as a control to exclude the possible nonspecific role of other inflammatory cytokines, and the results showed that IL-1β has no significant effect on TnPepT1 expression (data not shown). A similar result was observed in an in vitro assay using a colon-derived cell line under IL-16 stimulation. Furthermore, upregulated PepT1 expression in the colon facilitated the transport of fMLF into colon tissues, which is a crucial factor thought to result in the development of IBD. These results provide both in vivo and in vitro evidence that IL-16 is able to trigger PepT1 expression in the colon, thereby leading to fMLF-elicited inflammation. Finally, a significant alleviation of intestinal inflammation was observed using in vivo PepT1 knockdown in the IL-16–induced colitis model. This result strongly supports the observation that the IL-16–induced colonic inflammation is caused by PepT1 upregulation. This cytokine-mediated upregulation of PepT1 expression might be a key mechanism underlying IBD development. Thus, the present study provides new insights into the pathogenesis of IBD and into the development of anti-inflammatory therapy for IBD. Furthermore, it provides a better understanding of the functional characteristics of IL-16 in colon inflammation and the molecular basis for bowel disease.

Our previous studies have shown that TnIL-16 shares a number of similar characteristics with its mammalian counterpart in terms of gene organization, amino acid sequence, functional motifs or domains, molecular structure, protein cleavage site, and chemoattractant function (29, 75). Thus, IL-16 might be a highly conserved IL family member, which is conservative in both structure and function from fish to mammals throughout vertebrate evolution. Therefore, the role of IL-16 in IBD pathogenesis may be universal among vertebrate species. Alternatively, TnPepT1 also shares highly conserved characteristics with mammalian PepT1 homologs, including similar gene organizations, amino acid sequences, 12 transmembrane regions, functional motifs, protein structure, tissue distribution pattern, and transporter function (53, 71, 76). Hence, PepT1 is also an evolutionarily conserved transporter molecule in the gut system from fish to mammals. This finding implies that the digestive tracts of vertebrates have similar basic structures and functions. These two observations suggest that the mechanism of IL-16–induced PepT1-mediated colitis may be a common nature underlying gut pathophysiology among different vertebrate species, which indicates the evolutionarily conserved biological role of IL-16 in inflammatory disease. Therefore, the conclusion drawn from the present research on Tetraodon fish may also be, at least partly, extrapolated to the pathogenesis of human IBD. The fish model may become a promising platform for developing preventive and therapeutic interventions for IBD complications. Furthermore, IL-16 may also be a novel biomarker for IBD development. We hope that these findings will not only greatly contribute to the current knowledge on IBD, leading to a better understanding of the molecular basis of this disease by which an IL-16–based therapy could be developed, but also provide insights into the evolutionary role of ILs from fish to mammals. However, further investigations are still needed to clarify the detailed molecular mechanisms of IL-16 in the development of IBD, such as the identification of IL-16–mediated signaling pathways related to PepT1 upregulation, as well as the regulatory networks in which complex interactions between IL-16 and other cytokines may occur.

This work was supported by National Basic Research Program of China (973) Grants 2012CB114404 and 2012CB114402, Hi-Tech Research and Development Program of China (863) Grant 2012AA092202, National Natural Science Foundation of China Grants 31072234, 31172436, 31272691, and 30871936, and by Program for Key Innovative Research Team of Zhejiang Province Grant 2010R50026.

The online version of this article contains supplemental material.

Abbreviations used in this article:

CD

Crohn's disease

DSS

dextran sodium sulfate

IBD

inflammatory bowel disease

MPO

myeloperoxidase

PepT1

oligopeptide transporter member 1

siRNA

small interfering RNA

TnIL-16

Tetraodon IL-16

TnPepT1

Tetraodon oligopeptide transporter member 1

TnPepT1-ex

extracellular region of Tetraodon oligopeptide transporter member 1

UC

ulcerative colitis.

1
Xavier
R. J.
,
Podolsky
D. K.
.
2007
.
Unravelling the pathogenesis of inflammatory bowel disease.
Nature
448
:
427
434
.
2
Kaser
A.
,
Zeissig
S.
,
Blumberg
R. S.
.
2010
.
Inflammatory bowel disease.
Annu. Rev. Immunol.
28
:
573
621
.
3
Khor
B.
,
Gardet
A.
,
Xavier
R. J.
.
2011
.
Genetics and pathogenesis of inflammatory bowel disease.
Nature
474
:
307
317
.
4
Siegmund
B.
,
Zeitz
M.
.
2011
.
Innate and adaptive immunity in inflammatory bowel disease.
World J. Gastroenterol.
17
:
3178
3183
.
5
Duerr
R. H.
,
Taylor
K. D.
,
Brant
S. R.
,
Rioux
J. D.
,
Silverberg
M. S.
,
Daly
M. J.
,
Steinhart
A. H.
,
Abraham
C.
,
Regueiro
M.
,
Griffiths
A.
, et al
.
2006
.
A genome-wide association study identifies IL23R as an inflammatory bowel disease gene.
Science
314
:
1461
1463
.
6
Hampe
J.
,
Franke
A.
,
Rosenstiel
P.
,
Till
A.
,
Teuber
M.
,
Huse
K.
,
Albrecht
M.
,
Mayr
G.
,
De La Vega
F. M.
,
Briggs
J.
, et al
.
2007
.
A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1.
Nat. Genet.
39
:
207
211
.
7
Peltekova
V. D.
,
Wintle
R. F.
,
Rubin
L. A.
,
Amos
C. I.
,
Huang
Q.
,
Gu
X.
,
Newman
B.
,
Van Oene
M.
,
Cescon
D.
,
Greenberg
G.
, et al
.
2004
.
Functional variants of OCTN cation transporter genes are associated with Crohn disease.
Nat. Genet.
36
:
471
475
.
8
Rioux
J. D.
,
Daly
M. J.
,
Silverberg
M. S.
,
Lindblad
K.
,
Steinhart
H.
,
Cohen
Z.
,
Delmonte
T.
,
Kocher
K.
,
Miller
K.
,
Guschwan
S.
, et al
.
2001
.
Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn disease.
Nat. Genet.
29
:
223
228
.
9
Rioux
J. D.
,
Xavier
R. J.
,
Taylor
K. D.
,
Silverberg
M. S.
,
Goyette
P.
,
Huett
A.
,
Green
T.
,
Kuballa
P.
,
Barmada
M. M.
,
Datta
L. W.
, et al
.
2007
.
Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis.
Nat. Genet.
39
:
596
604
.
10
Xiao
H.
,
Gulen
M. F.
,
Qin
J.
,
Yao
J.
,
Bulek
K.
,
Kish
D.
,
Altuntas
C. Z.
,
Wald
D.
,
Ma
C.
,
Zhou
H.
, et al
.
2007
.
The Toll-interleukin-1 receptor member SIGIRR regulates colonic epithelial homeostasis, inflammation, and tumorigenesis.
Immunity
26
:
461
475
.
11
Piao
J. H.
,
Hasegawa
M.
,
Heissig
B.
,
Hattori
K.
,
Takeda
K.
,
Iwakura
Y.
,
Okumura
K.
,
Inohara
N.
,
Nakano
H.
.
2011
.
Tumor necrosis factor receptor-associated factor (TRAF) 2 controls homeostasis of the colon to prevent spontaneous development of murine inflammatory bowel disease.
J. Biol. Chem.
286
:
17879
17888
.
12
Swidsinski
A.
,
Ladhoff
A.
,
Pernthaler
A.
,
Swidsinski
S.
,
Loening-Baucke
V.
,
Ortner
M.
,
Weber
J.
,
Hoffmann
U.
,
Schreiber
S.
,
Dietel
M.
,
Lochs
H.
.
2002
.
Mucosal flora in inflammatory bowel disease.
Gastroenterology
122
:
44
54
.
13
Petnicki-Ocwieja
T.
,
Hrncir
T.
,
Liu
Y. J.
,
Biswas
A.
,
Hudcovic
T.
,
Tlaskalova-Hogenova
H.
,
Kobayashi
K. S.
.
2009
.
Nod2 is required for the regulation of commensal microbiota in the intestine.
Proc. Natl. Acad. Sci. USA
106
:
15813
15818
.
14
Liu
Z.
,
Lenardo
M. J.
.
2012
.
The role of LRRK2 in inflammatory bowel disease.
Cell Res.
22
:
1092
1094
.
15
Bamias
G.
,
Mishina
M.
,
Nyce
M.
,
Ross
W. G.
,
Kollias
G.
,
Rivera-Nieves
J.
,
Pizarro
T. T.
,
Cominelli
F.
.
2006
.
Role of TL1A and its receptor DR3 in two models of chronic murine ileitis.
Proc. Natl. Acad. Sci. USA
103
:
8441
8446
.
16
Takedatsu
H.
,
Michelsen
K. S.
,
Wei
B.
,
Landers
C. J.
,
Thomas
L. S.
,
Dhall
D.
,
Braun
J.
,
Targan
S. R.
.
2008
.
TL1A (TNFSF15) regulates the development of chronic colitis by modulating both T-helper 1 and T-helper 17 activation.
Gastroenterology
135
:
552
567
.
17
Rogler
G.
,
Andus
T.
.
1998
.
Cytokines in inflammatory bowel disease.
World J. Surg.
22
:
382
389
.
18
Danese
S.
2012
.
New therapies for inflammatory bowel disease: from the bench to the bedside.
Gut
61
:
918
932
.
19
Seegert
D.
,
Rosenstiel
P.
,
Pfahler
H.
,
Pfefferkorn
P.
,
Nikolaus
S.
,
Schreiber
S.
.
2001
.
Increased expression of IL-16 in inflammatory bowel disease.
Gut
48
:
326
332
.
20
Gao
L. B.
,
Rao
L.
,
Wang
Y. Y.
,
Liang
W. B.
,
Li
C.
,
Xue
H.
,
Zhou
B.
,
Sun
H.
,
Li
Y.
,
Lv
M. L.
, et al
.
2009
.
The association of interleukin-16 polymorphisms with IL-16 serum levels and risk of colorectal and gastric cancer.
Carcinogenesis
30
:
295
299
.
21
Keates
A. C.
,
Castagliuolo
I.
,
Cruickshank
W. W.
,
Qiu
B.
,
Arseneau
K. O.
,
Brazer
W.
,
Kelly
C. P.
.
2000
.
Interleukin 16 is up-regulated in Crohn’s disease and participates in TNBS colitis in mice.
Gastroenterology
119
:
972
982
.
22
Middel
P.
,
Reich
K.
,
Polzien
F.
,
Blaschke
V.
,
Hemmerlein
B.
,
Herms
J.
,
Korabiowska
M.
,
Radzun
H. J.
.
2001
.
Interleukin 16 expression and phenotype of interleukin 16 producing cells in Crohn’s disease.
Gut
49
:
795
803
.
23
Glas
J.
,
Török
H. P.
,
Unterhuber
H.
,
Radlmayr
M.
,
Folwaczny
C.
.
2003
.
The −295T-to-C promoter polymorphism of the IL-16 gene is associated with Crohn’s disease.
Clin. Immunol.
106
:
197
200
.
24
Cruikshank
W.
,
Center
D. M.
.
1982
.
Modulation of lymphocyte migration by human lymphokines. II. Purification of a lymphotactic factor (LCF).
J. Immunol.
128
:
2569
2574
.
25
Center
D. M.
,
Cruikshank
W.
.
1982
.
Modulation of lymphocyte migration by human lymphokines. I. Identification and characterization of chemoattractant activity for lymphocytes from mitogen-stimulated mononuclear cells.
J. Immunol.
128
:
2563
2568
.
26
Glass
W. G.
,
Sarisky
R. T.
,
Vecchio
A. M.
.
2006
.
Not-so-sweet sixteen: the role of IL-16 in infectious and immune-mediated inflammatory diseases.
J. Interferon Cytokine Res.
26
:
511
520
.
27
Mühlhahn
P.
,
Zweckstetter
M.
,
Georgescu
J.
,
Ciosto
C.
,
Renner
C.
,
Lanzendörfer
M.
,
Lang
K.
,
Ambrosius
D.
,
Baier
M.
,
Kurth
R.
,
Holak
T. A.
.
1998
.
Structure of interleukin 16 resembles a PDZ domain with an occluded peptide binding site.
Nat. Struct. Biol.
5
:
682
686
.
28
Burge
C.
,
Karlin
S.
.
1997
.
Prediction of complete gene structures in human genomic DNA.
J. Mol. Biol.
268
:
78
94
.
29
Wen
Y.
,
Shao
J. Z.
,
Xiang
L. X.
,
Fang
W.
.
2006
.
Cloning, characterization and expression analysis of two Tetraodon nigroviridis interleukin-16 isoform genes.
Comp. Biochem. Physiol. B Biochem. Mol. Biol.
144
:
159
166
.
30
Schultz
J.
,
Milpetz
F.
,
Bork
P.
,
Ponting
C. P.
.
1998
.
SMART, a simple modular architecture research tool: identification of signaling domains.
Proc. Natl. Acad. Sci. USA
95
:
5857
5864
.
31
Thompson
J. D.
,
Higgins
D. G.
,
Gibson
T. J.
.
1994
.
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res.
22
:
4673
4680
.
32
Kumar
S.
,
Tamura
K.
,
Nei
M.
.
2004
.
MEGA3: integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment.
Brief. Bioinform.
5
:
150
163
.
33
Saitou
N.
,
Nei
M.
.
1987
.
The neighbor-joining method: a new method for reconstructing phylogenetic trees.
Mol. Biol. Evol.
4
:
406
425
.
34
Hu
Y. X.
,
Guo
J. Y.
,
Shen
L.
,
Chen
Y.
,
Zhang
Z. C.
,
Zhang
Y. L.
.
2002
.
Get effective polyclonal antisera in one month.
Cell Res.
12
:
157
160
.
35
Livak
K. J.
,
Schmittgen
T. D.
.
2001
.
Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC(T) method.
Methods
25
:
402
408
.
36
Wirtz
S.
,
Neufert
C.
,
Weigmann
B.
,
Neurath
M. F.
.
2007
.
Chemically induced mouse models of intestinal inflammation.
Nat. Protoc.
2
:
541
546
.
37
Krawisz
J. E.
,
Sharon
P.
,
Stenson
W. F.
.
1984
.
Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. Assessment of inflammation in rat and hamster models.
Gastroenterology
87
:
1344
1350
.
38
Liutkeviciute
Z.
,
Lukinavicius
G.
,
Masevicius
V.
,
Daujotyte
D.
,
Klimasauskas
S.
.
2009
.
Cytosine-5-methyltransferases add aldehydes to DNA.
Nat. Chem. Biol.
5
:
400
402
.
39
Elbashir
S. M.
,
Harborth
J.
,
Weber
K.
,
Tuschl
T.
.
2002
.
Analysis of gene function in somatic mammalian cells using small interfering RNAs.
Methods
26
:
199
213
.
40
Lee
J. H.
,
Cho
E. S.
,
Kim
M. Y.
,
Seo
Y. W.
,
Kho
D. H.
,
Chung
I. J.
,
Kook
H.
,
Kim
N. S.
,
Ahn
K. Y.
,
Kim
K. K.
.
2005
.
Suppression of progression and metastasis of established colon tumors in mice by intravenous delivery of short interfering RNA targeting KITENIN, a metastasis-enhancing protein.
Cancer Res.
65
:
8993
9003
.
41
Wirtz
S.
,
Neurath
M. F.
.
2007
.
Mouse models of inflammatory bowel disease.
Adv. Drug Deliv. Rev.
59
:
1073
1083
.
42
Kim
J. J.
,
Shajib
M. S.
,
Manocha
M. M.
,
Khan
W. I.
.
2012
.
Investigating intestinal inflammation in DSS-induced model of IBD.
J. Vis. Exp.
60
:
3678
.
43
Fang
K.
,
Bruce
M.
,
Pattillo
C. B.
,
Zhang
S.
,
Stone
R.
 II
,
Clifford
J.
,
Kevil
C. G.
.
2011
.
Temporal genomewide expression profiling of DSS colitis reveals novel inflammatory and angiogenesis genes similar to ulcerative colitis.
Physiol. Genomics
43
:
43
56
.
44
Yan
Y.
,
Kolachala
V.
,
Dalmasso
G.
,
Nguyen
H.
,
Laroui
H.
,
Sitaraman
S. V.
,
Merlin
D.
.
2009
.
Temporal and spatial analysis of clinical and molecular parameters in dextran sodium sulfate induced colitis.
PLoS ONE
4
:
e6073
.
45
Okayasu
I.
,
Hatakeyama
S.
,
Yamada
M.
,
Ohkusa
T.
,
Inagaki
Y.
,
Nakaya
R.
.
1990
.
A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice.
Gastroenterology
98
:
694
702
.
46
Arnhold
J.
,
Flemmig
J.
.
2010
.
Human myeloperoxidase in innate and acquired immunity.
Arch. Biochem. Biophys.
500
:
92
106
.
47
van der Veen
B. S.
,
de Winther
M. P.
,
Heeringa
P.
.
2009
.
Myeloperoxidase: molecular mechanisms of action and their relevance to human health and disease.
Antioxid. Redox Signal.
11
:
2899
2937
.
48
Oehlers
S. H.
,
Flores
M. V.
,
Hall
C. J.
,
Swift
S.
,
Crosier
K. E.
,
Crosier
P. S.
.
2011
.
The inflammatory bowel disease (IBD) susceptibility genes NOD1 and NOD2 have conserved anti-bacterial roles in zebrafish.
Dis. Model. Mech.
4
:
832
841
.
49
Love
D. R.
,
Lan
C. C.
,
Dodd
A.
,
Shelling
A. N.
,
McNabb
W. C.
,
Ferguson
L. R.
.
2007
.
Modeling inflammatory bowel disease: the zebrafish as a way forward.
Expert Rev. Mol. Diagn.
7
:
177
193
.
50
Oehlers
S. H.
,
Flores
M. V.
,
Okuda
K. S.
,
Hall
C. J.
,
Crosier
K. E.
,
Crosier
P. S.
.
2011
.
A chemical enterocolitis model in zebrafish larvae that is dependent on microbiota and responsive to pharmacological agents.
Dev. Dyn.
240
:
288
298
.
51
Buyse
M.
,
Tsocas
A.
,
Walker
F.
,
Merlin
D.
,
Bado
A.
.
2002
.
PepT1-mediated fMLP transport induces intestinal inflammation in vivo.
Am. J. Physiol. Cell Physiol.
283
:
C1795
C1800
.
52
Daniel
H.
,
Spanier
B.
,
Kottra
G.
,
Weitz
D.
.
2006
.
From bacteria to man: archaic proton-dependent peptide transporters at work.
Physiology (Bethesda)
21
:
93
102
.
53
Daniel
H.
,
Kottra
G.
.
2004
.
The proton oligopeptide cotransporter family SLC15 in physiology and pharmacology.
Pflugers Arch.
447
:
610
618
.
54
Wolf
J. C.
,
Wolfe
M. J.
.
2003
.
Good laboratory practice considerations in the use of fish models.
Toxicol. Pathol.
31
(
Suppl
):
53
57
.
55
Lieschke
G. J.
,
Currie
P. D.
.
2007
.
Animal models of human disease: zebrafish swim into view.
Nat. Rev. Genet.
8
:
353
367
.
56
Roest Crollius
H.
,
Jaillon
O.
,
Bernot
A.
,
Dasilva
C.
,
Bouneau
L.
,
Fischer
C.
,
Fizames
C.
,
Wincker
P.
,
Brottier
P.
,
Quétier
F.
, et al
.
2000
.
Estimate of human gene number provided by genome-wide analysis using Tetraodon nigroviridis DNA sequence.
Nat. Genet.
25
:
235
238
.
57
Roest Crollius
H.
,
Jaillon
O.
,
Dasilva
C.
,
Ozouf-Costaz
C.
,
Fizames
C.
,
Fischer
C.
,
Bouneau
L.
,
Billault
A.
,
Quetier
F.
,
Saurin
W.
, et al
.
2000
.
Characterization and repeat analysis of the compact genome of the freshwater pufferfish Tetraodon nigroviridis.
Genome Res.
10
:
939
949
.
58
Walter
R. B.
2001
.
Introduction: aquaria fish models of human disease.
Mar. Biotechnol. (NY)
3
(
Suppl. 1
):
S1
S2
.
59
Bahary
N.
,
Zon
L. I.
.
1998
.
Use of the zebrafish (Danio rerio) to define hematopoiesis.
Stem Cells
16
:
89
98
.
60
Bui
P.
,
Bagherie-Lachidan
M.
,
Kelly
S. P.
.
2010
.
Cortisol differentially alters claudin isoforms in cultured puffer fish gill epithelia.
Mol. Cell. Endocrinol.
317
:
120
126
.
61
Clelland
E. S.
,
Bui
P.
,
Bagherie-Lachidan
M.
,
Kelly
S. P.
.
2010
.
Spatial and salinity-induced alterations in claudin-3 isoform mRNA along the gastrointestinal tract of the pufferfish Tetraodon nigroviridis.
Comp. Biochem. Physiol. A Mol. Integr. Physiol.
155
:
154
163
.
62
Drummond
I. A.
,
Majumdar
A.
,
Hentschel
H.
,
Elger
M.
,
Solnica-Krezel
L.
,
Schier
A. F.
,
Neuhauss
S. C.
,
Stemple
D. L.
,
Zwartkruis
F.
,
Rangini
Z.
, et al
.
1998
.
Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function.
Development
125
:
4655
4667
.
63
Feitsma
H.
,
Cuppen
E.
.
2008
.
Zebrafish as a cancer model.
Mol. Cancer Res.
6
:
685
694
.
64
Ingham
P. W.
2009
.
The power of the zebrafish for disease analysis.
Hum. Mol. Genet.
18
(
R1
):
R107
R112
.
65
Sepulcre
M. P.
,
Alcaraz-Pérez
F.
,
López-Muñoz
A.
,
Roca
F. J.
,
Meseguer
J.
,
Cayuela
M. L.
,
Mulero
V.
.
2009
.
Evolution of lipopolysaccharide (LPS) recognition and signaling: fish TLR4 does not recognize LPS and negatively regulates NF-κB activation.
J. Immunol.
182
:
1836
1845
.
66
Weinstein
B. M.
,
Schier
A. F.
,
Abdelilah
S.
,
Malicki
J.
,
Solnica-Krezel
L.
,
Stemple
D. L.
,
Stainier
D. Y.
,
Zwartkruis
F.
,
Driever
W.
,
Fishman
M. C.
.
1996
.
Hematopoietic mutations in the zebrafish.
Development
123
:
303
309
.
67
Marasco
W. A.
,
Phan
S. H.
,
Krutzsch
H.
,
Showell
H. J.
,
Feltner
D. E.
,
Nairn
R.
,
Becker
E. L.
,
Ward
P. A.
.
1984
.
Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli.
J. Biol. Chem.
259
:
5430
5439
.
68
Chadwick
V. S.
,
Mellor
D. M.
,
Myers
D. B.
,
Selden
A. C.
,
Keshavarzian
A.
,
Broom
M. F.
,
Hobson
C. H.
.
1988
.
Production of peptides inducing chemotaxis and lysosomal enzyme release in human neutrophils by intestinal bacteria in vitro and in vivo.
Scand. J. Gastroenterol.
23
:
121
128
.
69
Adibi
S. A.
2003
.
Regulation of expression of the intestinal oligopeptide transporter (Pept-1) in health and disease.
Am. J. Physiol. Gastrointest. Liver Physiol.
285
:
G779
G788
.
70
Ingersoll
S. A.
,
Ayyadurai
S.
,
Charania
M. A.
,
Laroui
H.
,
Yan
Y.
,
Merlin
D.
.
2012
.
The role and pathophysiological relevance of membrane transporter PepT1 in intestinal inflammation and inflammatory bowel disease.
Am. J. Physiol. Gastrointest. Liver Physiol.
302
:
G484
G492
.
71
Liang
R.
,
Fei
Y. J.
,
Prasad
P. D.
,
Ramamoorthy
S.
,
Han
H.
,
Yang-Feng
T. L.
,
Hediger
M. A.
,
Ganapathy
V.
,
Leibach
F. H.
.
1995
.
Human intestinal H+/peptide cotransporter. Cloning, functional expression, and chromosomal localization.
J. Biol. Chem.
270
:
6456
6463
.
72
Shi
B.
,
Song
D.
,
Xue
H.
,
Li
N.
,
Li
J.
.
2006
.
PepT1 mediates colon damage by transporting fMLP in rats with bowel resection.
J. Surg. Res.
136
:
38
44
.
73
Ogihara
H.
,
Saito
H.
,
Shin
B. C.
,
Terado
T.
,
Takenoshita
S.
,
Nagamachi
Y.
,
Inui
K.
,
Takata
K.
.
1996
.
Immuno-localization of H+/peptide cotransporter in rat digestive tract.
Biochem. Biophys. Res. Commun.
220
:
848
852
.
74
Merlin
D.
,
Si-Tahar
M.
,
Sitaraman
S. V.
,
Eastburn
K.
,
Williams
I.
,
Liu
X.
,
Hediger
M. A.
,
Madara
J. L.
.
2001
.
Colonic epithelial hPepT1 expression occurs in inflammatory bowel disease: transport of bacterial peptides influences expression of MHC class 1 molecules.
Gastroenterology
120
:
1666
1679
.
75
Wen
Y.
,
Fang
W.
,
Xiang
L. X.
,
Pan
R. L.
,
Shao
J. Z.
.
2011
.
Identification of Treg-like cells in Tetraodon: insight into the origin of regulatory T subsets during early vertebrate evolution.
Cell. Mol. Life Sci.
68
:
2615
2626
.
76
Steiner
H. Y.
,
Naider
F.
,
Becker
J. M.
.
1995
.
The PTR family: a new group of peptide transporters.
Mol. Microbiol.
16
:
825
834
.

The authors have no financial conflicts of interest.