Author Manuscript Author Manuscript HHS Public Access Author manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Tricellulin is regulated via interleukin 13 receptor α2, affects macromolecule uptake, and is decreased in ulcerative colitis SM Krug1, C Bojarski2, A Fromm1, IM Lee1, P Dames1, JF Richter3, JR Turner4, M Fromm1,a, and JD Schulzke1,a 1Clinical Physiology, Department of Gastroenterology, Rheumatology and Infectious Diseases, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany 2Department of Gastroenterology, Rheumatology and Infectious Diseases, Charité – Universitätsmedizin, Campus Benjamin Franklin, Berlin, Germany 3Institute for Anatomy II, Jena University Hospital, Jena, Germany 4Departments of Pathology and Medicine (GI), Brigham and Women’s Hospital and Harvard Medical School, Boston, USA Abstract In the two inflammatory bowel diseases, ulcerative colitis (UC) and Crohn’s disease (CD), altered expression of tight junction (TJ) proteins leads to an impaired epithelial barrier including increased uptake of luminal antigens supporting the inflammation. Here we focused on regulation of tricellulin, a protein of the tricellular TJ essential for the barrier against macromolecules, and hypothesized a role in paracellular antigen uptake. We report that tricellulin is downregulated in UC, but not in CD, and that its reduction increases the passage of macromolecules. Using a novel visualization method, passage sites were identified at TJ regions usually sealed by tricellulin. We show that interleukin-13 (IL-13), beyond its known effect on claudin-2, downregulates tricellulin expression. These two effects of IL-13 are regulated by different signaling pathways: The IL-13 receptor α1 upregulates claudin-2, while IL-13 receptor α2 downregulates tricellulin. We suggest to target the α2 receptor in future developments of therapeutical IL-13-based biologicals. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Corresponding author: Jörg-Dieter Schulzke, Address: Clinical Physiology, Department of Gastroenterology, Rheumatology and Infectious Diseases, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin, Germany, joerg.schulzke@charite.de phone: +49 30 450-514531 fax: +49 30 450-514962. aThese authors contributed equally Supplementary Material is linked to the online version of the paper at http://www.nature.com/mi Author Contribution Design of work: SMK, MF, JRT, JDS Data aquiration: SMK, CB, AF, IML. PD Result interpretation: SMK, JFR, JRT,MF, JDS Drafting of manuscript: SMK, MF Revision of manuscript: CB, AF, IML, PD, JFR, JRT, MF, JDS Appoval of final version: SMK, CB, AF, IML, PD, JFR, MF, JDS Disclosure All authors have no conflicts to declare. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Krug et al. Page 2 Introduction The intestinal epithelium consists of a polarized single cell layer with a multi-protein complex, the tight junction (TJ) which strictly regulates paracellular passage of ions, molecules and water. This regulation is determined by transmembrane proteins of the TJ comprising of four families: four-transmembrane TJ proteins include the family of claudins with 2-members in mammals(1), and the tight junction-associated MARVEL proteins (TAMP)(2) containing occludin(3), MARVEL D3(2, 4) and tricellulin(5). Most of the claudins form barriers but some form paracellular ion and water channels(6, 7). Junctional adhesion molecules (JAM(8)) and the most recently described group of angulins(9) span the membrane once. Angulins and tricellulin are located at the tricellular TJ (tTJ), the region where three or more cells meet and their bicellular TJ (bTJ) converge laterally forming a central tube. We have previously shown that tricellulin plays an important role in sealing the tTJ, particularly to macromolecules(10, 11). Integrity of the epithelial barrier is impaired in a variety of pathological conditions, for example in the inflammatory bowel diseases (IBD) Crohn’s disease (CD)(12) and ulcerative colitis (UC)(13). Although pathogenesis of the barrier defect in IBDs is still unclear(14), several studies demonstrate that pro-inflammatory cytokines including TNFα(15), IFNγ(16, 17) and interleukin-13 (IL-13)(18, 19) alter barrier function by changing TJ protein function and expression. For example, IL-13 has been linked as proinflammatory cytokine to UC, stimulating epithelial cell apoptosis and enhancing barrier defects, predominantly by upregulation of the cation-selective paracellular channel claudin-2(18–20). IL-13 is a cytokine which is released by Th2 cells sharing biological properties with IL-4 and both cytokines are involved in immune-mediated processes, where they often act in an manner that opposes Th1-polarized inflammation(21). Not only in UC but also in asthma IL-13 and IL-4 have been reported to work synergistically to enhance inflammatory activity. Two IL-13 receptor complexes are described and the balance between these is believed to regulate both quantitative and qualitative effects of IL-13 signaling(22–25). The first consists of a receptor complex composed of the IL-4 receptor α (IL4Rα) and the IL-13 receptor α1 (IL13Rα1)(26, 27), the second is generated by IL-13 receptor α2 (IL13Rα2). For IL4Rα/IL-13Rα1 signaling, phosphorylation of STAT1(28), STAT6(29), and STAT3(28) have been reported as well as signaling via the MAPK and PI3K pathways(30–32) and have been connected to claudin-2 upregulation by IL-13(33). Two variants of IL13Rα2 which differ in activity have been described, a soluble and a cell surface-bound. The soluble form found in urine and serum of mice(34) is described as a decoy receptor that partially inhibits effects of IL-13(23, 35); function of the human soluble form is more controversial(36, 37). In contrast, the cell surface-bound form of IL13Rα2 stimulates the transcription factor complex activator protein (AP-1)(38, 39), phosphorylates STAT3(40), and enhances MAPK(38), PI3K(41) and ERK1/2 signaling(39), both in rodents and humans. In this study, we analyzed the expression of the TJ protein tricellulin (Tric) in IBD and found it to be downregulated in UC, but not CD. We showed that this altered expression is driven Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Author Manuscript Krug et al. Page 3 by the UC key cytokine IL-13 via IL13Rα2. While IL-13 also upregulates claudin-2 expression, we found that the signaling mechanisms differed from those responsible for tricellulin downregulation. In contrast to increased water and cation flux after claudin-2 upregulation, we found that IL-13-induced suppression of tricellulin led to enhanced paracellular macromolecular uptake which we were able to localize at tTJs. This may represent a novel mechanism for increased luminal uptake of antigens in UC. Results When comparing membrane protein expression levels of tricellulin within sigmoid colon tissue of IBD and control (Ctrl) patients, it was conspicuous that there was profound and substantial reduction of tricellulin (Tric) in UC, but not in CD patients (Fig. 1A+D), comparable to changes reported for claudin-4 (Cldn-4) (42) (Fig. 1C+D). This finding is also in line with GEO expression data, reporting tricellulin (synonym: MARVEL D2) to be downregulated in inflamed UC tissue (GDS3119)(43, 44), but not in CD (GDS560)(45). Claudin-2 (Cldn-2) nearly absent in Ctrl served as control for changes in IBD(46) (Fig. 1B +D). Impedance spectroscopic analysis of UC samples revealed that, although transepithelial resistance (TER) was similar to that of controls, epithelial resistance was decreased, while subepithelial resistance was increased due to inflammatory thickening of the subepithelium due to edema and condensed collagenous fibers (Fig. 1E). Permeabilities for paracellular flux markers as fluorescein and 4 kDa-FITC-dextran (FD4) were increased indicating an impaired paracellular barrier (Fig. 1F). Immunostainings of cryosections (Fig. 2) revealed that in control patients, tricellulin was present all along the crypt and the surface epithelium, while claudin-2 was only detectable in the depths of the crypts. In both IBD, claudin-2 expression was increased and localization was extended to the total length of the crypts and to the surface epithelium. Tricellulin was decreased in UC patients, while in CD patients a shift of localization seemed to occur. While in the crypts the signal seemed to be slightly reduced, an increase of signals was observed in the surface epithelium, which also was extended from the apicolateral regions of the TJ to more basolateral regions. However, these changes in localization did not occur in all CD samples analyzed. To investigate the origin of the UC-specific change in tricellulin expression, the intestinal epithelial cell line HT-29/B6 was treated with cytokines known to be involved in IBD (Fig. S1). IL-13, a major effector in UC (18), not only increased claudin-2 but also decreased tricellulin expression (Fig. 3A, S1). All other cytokines tested affected only claudin-2 (Fig. S1). On mRNA level, tricellulin was reduced to 64±8% (**p<0.01, n=6) and claudin-2 increased to 238±24% of controls (***p<0.001, n=6). mRNA and protein half-lives were not affected, indicating that IL-13 did not affect protein or mRNA stability. At 100 ng/ml, IL-13 had no effect on epithelial apoptotic rate (Tab. S1), measured by TUNEL staining and previously reported (47). IL-13 decreased TER by 62%, while permeability for the macromolecule FD4 was increased from 0.06±0.01 to 0.15±0.01·1076 cm/s (Fig. 3B+C, n=7, ***p<0.001). Comparable results were achieved when tricellulin was downregulated by stable transfection with specific shRNA (Fig. 3D+E), namely increased permeability to FD4 (n=11) and to 10 kDa-FITC dextran (n=4; Fig. 3F, Fig. S2). This provides direct evidence that the observed increase in Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Krug et al. Page 4 macromolecule permeability, i.e. the leak pathway (48), was based on the decrease tricellulin alone, not on alterations of claudin-2 or of occludin, since expression of both was unchanged (Fig. 3D+E). Neither was the expression of angulins affected by tricellulin knockdown (Fig. S3). Together with this, a decrease in TER occurred (Vec: 1325±174 Ω·cm2; shTRIC: 730±64 Ω·cm2; *p<0.05, n=11) which, however, was lower as in the IL-13-treated cells because the TER-decreasing effect of claudin-2 was lacking here. To confirm the cell culture findings for IL-13 and tricellulin in colon epithelium, mice were treated with IL-13 and protein expression of tricellulin as well as functional consequences for the intestine were analyzed by electrophysiological experiments. The chosen dose and duration of the treatment did not lead to obvious changes in the intestinal architecture (Fig. S4A), but a decrease in epithelial resistance was observed indicating impairment of the mucosal barrier, while the subepithelial resistance was unaffected (Fig. S4B, *p<0.05, n=5). Macromolecular permeability for FD4 was doubled in the colon of IL-13-treated mice (Fig. 4A, *p<0.05, n=8). Also, the effect of IL-13 on expression and localization of tricellulin as well as of claudin-2 was reproducible in mice (Fig. 4B+C, *p<0.05, n=7–9; Fig. S4C). To elucidate the signaling of IL-13 involved in tricellulin downregulation several approaches were tested in cell culture. 1 h pretreatment with inhibitory antibodies against the two IL-13 receptors was used to inhibit the interaction of IL-13 (Fig. 5). Importantly, anti-IL13Rα1 was able to block the increase in claudin-2 expression, but not the decrease of tricellulin. In contrast, anti-IL13Rα2 inhibited the decrease of tricellulin, but not the claudin-2 effect (Fig. 5A+B). When analyzing the effect on macromolecule permeability using FD4, the inhibition of IL13Rα1 by the inhibitory antibody had no suppressing effect on the increase of FD4 permeability after treatment with IL-13. When interaction of IL-13 with the IL13Rα2 was inhibited, permeability for FD4 was comparable to untreated cells (Fig. 5C). Also treatment of STAT6−/− mice with IL-13 reduced tricellulin expression, indicating that STAT6 and upstream IL13Rα1 signaling were not involved (Fig. 7A+B). In contrast, claudin-2 expression was not increased in intestinal mucosae from IL-13-treated STAT6−/− mice, providing strong data that support for the conclusion from in vitro data(33) that IL13Rα-induced STAT6 activation regulates claudin-2 expression. These differences implicate the IL13Rα2 signaling pathway, which is independent of STAT6, to operate in tricellulin downregulation. Consistent with this, IL-13 in T84 cells, which do not express IL13Rα2 (Fig. S5C), upregulated claudin-2 expression but did not downregulate tricellulin (Fig. S5A+B). The same was observed when IL13Rα2 was downregulated by stable transfection with specific shRNA (Fig. 6A–D). The knockdown of IL13Rα2 also abolished the macromolecule permeability increase in response to IL-13 (Fig. 6E), supporting the finding of tricellulin downregulation to be involved in this permeability increase. To analyze the signal transduction pathway in more detail, several inhibitors known to be regulated by IL-13(28–33, 38–41) were added before treating the cells with IL-13. For this, protein (Fig. 7C+Fig. S6A) as well as mRNA (Fig. S6B) levels were analyzed. Claudin-2 upregulation was blocked when IL13Rα1-related pathways were inhibited by AS151–499, SAHA (both affecting STAT6), LY294002 (PI3K) or U0126 (MAPK), confirming the findings from the inhibitory antibodies and STAT6−/− mice. In contrast, downregulation of Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Author Manuscript Krug et al. Page 5 tricellulin was inhibited when IL13Rα2 pathways were targeted with JNK V, (JNK), U0126 (MAPK, ERK1/2), or Tanshinone IIa (AP-1). However, not all drugs targeting IL13Rα2 signaling prevented tricellulin downregulation, for example PI3K. Conversely, JAK1/2 inhibition using baricitinib and JAK3 inhibition using JAK3-inhibitor II had no effect on claudin-2 expression, although each is thought to be involved in signaling by the IL4Rα/ IL13Rα1 complex. The data indicate, however, that JAK1 and/or JAK2 are involved in tricellulin regulation. To further delineate signaling, expression and phosphorylation of potential IL13Rα2 signaling intermediates were analyzed. To exclude effects being non-specific for tricellulin, the inhibitor U0126, which was shown to block the tricellulin downregulation and which is supposed to be involved early in the signaling, was used for comparison (Fig. 7D). c-Fos was highly upregulated by IL-13, an effect which was blockable by U0126. All other effects on total protein expression were either not significant or only slightly attenuated by U0126 (Fig. 7E). When comparing the phosphorylation status of the respective proteins (Fig. 7F), it became obvious that c-Fos phosphorylation was not affected by U0126. However, IL-13 alone markedly decreased c-Fos phosphorylation and increased its total expression. For several other intermediates phosphorylation ratios were increased during IL-13-induced tricellulin downregulation; IL-13 increased phosphorylation of FRA-1 and of p44/42, an effect that was inhibited when U0126 was present. Interestingly, IL-13 reduced c-jun phosphorylation at S63, and this effect was also inhibited by U0126. Effects of IL-13 on p38 MAPK and p54 SAPK phosphorylation were not inhibited by U0126 and may therefore be unrelated to tricellulin downregulation. Tricellulin has been reported as barrier-forming TJ protein inhibiting excessive macromolecule passage through the tTJ (10, 11). As IL-13 treatment lead to increased FD4 and FD10 permeability, we investigated the site of macromolecule passage through the bTJ and the tTJ of HT-29/B6 monolayers using the recently developed sandwich assay(49). In controls, i.e. when high amounts of tricellulin are present, no permeation of biotin-labelledTRITC-dextran 10 kDa was observed (Fig. 8, upper panel, A and B). In contrast, in IL-13treated cells tricellulin was reduced and apically added 10 kDa dextran appeared on the basolateral side (Fig. 8, lower panel, A and B). In a merged view (Fig. 8, lower panel, D), the passage site appeared preferentially at tricellular TJs, in rare cases also at TJs where four cells meet. When the cells were additionally incubated with the inhibitor Tanshinone IIa, this passage was strongly diminished (Fig. S7), an effect that we also observed in global analysis of macromolecule permeability performing flux measurements in Ussing chambers (Fig. S8). IL-4 targeting the complex formed by IL13Rα1 and IL4R (Fig. 9) had an effect only on claudin-2 but not on tricellulin expression (Fig. S1). Consistently, we observed no increased passage of the biotin-labelled-TRITC-dextran 10 kDa under these conditions (Fig. S6). In order to demonstrate the localization site of macromolecule passage, representative Zstack images were rendered into three-dimensional movies (Supplementary movie M1). Comparable observations were made using the shTRIC clone (Fig. S7 and Supplementary Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Author Manuscript Krug et al. Page 6 movie M2), supporting that the macromolecule passage may depend on the level of tricellulin present in tTJs. Discussion Tricellulin is downregulated in UC, but not in CD In this study we analyzed the expression of the tricellular TJ (tTJ) protein tricellulin in CD and UC. Though the intestinal barrier is impaired in both CD and UC, we find tricellulin to be downregulated only in UC, indicating UC specific down-regulation, but not in CD. However, the predominant localization of tricellulin was shifted towards the surface epithelium in CD, which might indicate that changes in proteins that regulate the correct localization of tricellulin occur in CD. For example, LSR is known to be predominantly localized within the depths of the crypts, while ILDR-1 can be found along the whole crypt(9). Thus, regulation of tricellulin depends also on other tTJ proteins and might be complex in CD, even if total expression remained unchanged. Elucidation of this regulation and involvement of other components of the tTJ in CD need to be studied in detail and will be object of further examination. IL 13 leads to increased paracellular passage of macromolecules via tricellulin downregulation In human biopsies as well as in mice and in HT-29/B6 monolayers, IL-13-induced tricellulin downregulation resulted in increased macromolecule permeability. Obtaining independent evidence, targeted tricellulin knockdown had the same effect on macromolecule passage, which is not dependent on occludin or claudin-2 because both were unchanged. These findings were consistent with previous results from our and other labs(5, 10, 11, 50). Our results were corroborated by three-dimensional visualization of macromolecules passing IL-13-treated HT-29/B6 cells predominantly at sites where more than two cells meet. Taken together, our data demonstrate that the IL-13-induced permeability increase for macromolecules is directly linked to tricellulin. The mechanistic relation between tricellulin and opening the passage site for macromolecules is unknown yet and will be the target of future studies. In conclusion, tricellulin and the tTJ are critical to maintenance of the paracellular barrier to macromolecular flux in the intestine. In early stages of UC, tricellulin downregulation therefore might already destabilize tricellular and TJ tightness, thereby allowing passage of antigens that may further promote the inflammatory process. IL 13 regulates claudin 2 and tricellulin via different signaling pathways To elucidate the regulation beyond the decreased tricellulin expression in UC, we concentrated on IL-13, which is a key cytokine in UC(18). Treatment with IL-13 increased claudin-2 as described earlier(18–20) and we show here that it also decreased tricellulin expression. This effect was demonstrated in cell cultures as well as in mice treated with IL-13. Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Author Manuscript Krug et al. Page 7 After analyzing the influence of blocking antibodies, knockdown of IL13Rα2, inhibitors of reported signaling pathways and mice lacking STAT6, we found that two pathways are involved and that each had distinct effects. Based on the established signaling cascades of IL-13 (22–29) we conclude that (Fig. 9) (i) claudin-2 expression and trans-tight junction pore pathway flux are upregulated via IL-13 binding to the IL4Rα/IL13Rα1 complex and the subsequent activation of STAT6, PI3K and MAPK. (ii) Tricellulin is downregulated and trans-tight junction leak pathway flux is enhanced via IL-13 binding to IL13Rα2. The subsequent signaling involved ERK1/2, JNK and AP-1. The transcription factor AP-1 may be composed of dimers of proteins belonging to the family of Fos, Jun or ATF proteins. Analysis of the most common AP-1 sub-proteins revealed that in tricellulin downregulation by IL-13, higher expression levels of c-Fos as well as the phosphorylation status of FRA-1 and c-jun (position S63) are involved as inhibition by U0126 affected these alterations as well as the tricellulin decrease. Taken together, IL-13 affects both, the bTJ and the tTJ. Claudin-2 as size-restrictive channel for small cations and water is a main factor in enhancing the high-capacitive pore-pathway, as by upregulation of claudin-2 permeability for Na+ increases. This can also be retraced by TER decrease, as TER depends on ions. The further influence of IL-13 on the tTJ via downregulation of tricellulin affects the leak pathway as permeability for macromolecular solutes is increased. However, as in tight epithelia the occurrence of bicellular conductive channels is low (tight epithelia possess a lower paracellular than transcellular conductance), opening of the tTJ also affects ion permeability. Thus, both effects of IL-13 need to be taken in account when developing new strategies of intervention. Demand of new receptor specific IL 13 based intervention strategies Our findings indicate that the increased macromolecule passage in UC is at least partly caused by tricellulin downregulation and that, among all cytokines tested in HT-29/B6 cells, only IL-13 causes tricellulin downregulation. In contrast, claudin-2 expression can be upregulated by several cytokines involved in IBD. The role of claudin-2 in IBD has been linked to leak flux diarrhea as claudin-2 increases permeability for small cations as well as for water. On the other hand, recent studies on claudin-2-overexpressing mice suggested that claudin-2 also may have protective properties, as these mice were protected against DSSinduced colitis. Besides feasible dilution of the DSS by enhanced paracellular water flux by claudin-2, it might be speculated whether the additionally described lower immune activation and increased colonocyte proliferation in these mice were leading to the protection(51). Increase of claudin-2 and water flux in IBD might in conclusion have two effects, it may contribute to leak-flux diarrhea and by this may serve as a beneficial rinsing of the inflamed mucosa (“enteric tears”(52)). IL-13 had been proposed as key player in both UC and asthma, and great hope has been linked to development of IL-13-focused biologics. However, to date, all approaches have Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Author Manuscript Krug et al. Page 8 failed: (i) Anrukinzumab, an antibody blocking the attachment of IL-13 to IL4Rα by binding to IL-13, showed no significant therapeutic effect in active UC(53), which is not surprising on the basis of our present data, since the binding to IL13Rα2 was unaffected. (ii) A second antibody, tralokinumab which binds and neutralizes IL-13, induced no clinical improvement in UC but at least led to higher clinical remission rates in a subset of patients(54). (iii) A third antibody, GSK69586, was directed against the IL-13 binding to both receptors, but was only tested in asthma patients so far (55, 56). We hypothesize that the earlier attempts to develop biologics targeting IL-13 have failed because they did not differentiate between the two pathways. Targeting solely the IL-13 receptor α1 might be less beneficial or even harmful, because blocking the UC-induced claudin-2 increase hinders the beneficial rinsing of the inflamed mucosa. In contrast, targeting the IL-13 receptor α2, however, would block the UC-induced decrease of tricellulin and by this would restore the barrier against the uptake of inflammatory macromolecules. We suggest that inhibiting the IL-13 receptor α2 pathway may be effective in UC, maybe most effective in early stages or in an attempt to achieve remission, when after healing of gross lesions this macromolecule leak is functionally essential. Therefore new approaches differentiating the two receptors are required for successful treatment. Not less importantly, tricellulin itself might be another potential candidate for intervention, targeting the loss of the macromolecular barrier and at the same time uncontrolled luminal uptake of macromolecular agents, especially in early disease states before gross lesions develop. Methods Patients and Tissue Preparation Biopsies from sigmoid colon were obtained from patients with histologically confirmed active ulcerative colitis (UC, n=20, median age of 34 years, range 20–79 years; 9 female and 11 male) or Crohn’s disease (CD, n=17, median age of 38 years, range 21–63 years; 9 female and 8 male) and from 21 control subjects (Ctrl, median age of 50 years, range 19–75 years; 14 female and 7 male). 17 UC patients received prednisolone (median dose 15 mg/ day, range 5–70 mg/day). Of these, 9 patients also received 5-aminosalicylic acid (1.5–5 g/ day). 3 UC patients only received 5-aminosalicylic acid. Measurements were performed on inflamed specimens without visible erosions/ulcers (histologically mild to moderate inflammation, e.g. Fig. S9). The specimens were spread and glued with histoacryl tissue glue (B Braun, Melsungen, Germany) to a support disk and mounted in Ussing-type chambers with an exposed tissue area of 0.49 cm2. The study design was approved by the local ethics committee (EA4/015/13). Cell culture and transfections The human colon cell line HT-29/B6 exhibits a transepithelial resistance and other basic properties making it an excellent model of colonic epithelia(57). For stable tricellulin knockdown, HT-29/B6 cells were transfected with pLKO.1-puro vector containing a sequence for shRNA targeting tricellulin (TRCN00000-2636NM_144-24.1-988s1c1TRC 1, Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Author Manuscript Krug et al. Page 9 Sigma-Aldrich) or targeting IL13Rα2 (TRCN0000058526_SHCLNG-NM_000640, SigmaAldrich) using Amaxa™ Nucleofector™ Technology. Puromycin-resistant clones were screened for knockdown by western blot. For experimentation, cell monolayers were grown on Millicell-PCF inserts (pore size 3.0 μm, effective area 0.6 cm2, Millipore, Bedford, MA). Confluent cell layers exhibiting transepithelial resistances (TER) above 600 Ω·cm2 were used 7 days after seeding. Cytokine and inhibitor experiments Cells were incubated as listed in table 1 for 24 h (TNFα) or 48 h (all others). Treatment with inhibitors occurred 1 h before IL-13 application. For experiments with inhibitory antibodies, cells were pretreated for 1 h with 5 μg/mL mouse anti-CD213a1/IL13Ra1 (AM31180AF-N, Acris, Herford, Germany) or mouse anti-IL13Ra2 (clone 2E10, Sigma-Aldrich) before addition of IL-13. Treatment of mice with IL 13 BALB/c wild type mice (Charité, Forschungseinrichtungen für experimentelle Medizin, Berlin, Germany) and STAT6−/− mice (Jackson Laboratory, Bar Harbor, USA) were housed, fed and handled under accordance with the rules of institutional animal care. Experiments were approved by the Landesamt für Gesundheit und Soziales Berlin (G 0208/12). 12 – 20 weeks-old female mice were injected intraperitoneally with 3 μg murine IL-13 (BioLegend GmbH, Fell, Germany) diluted in 300 μl PBS with 0.1% BSA or vehicle alone daily for three days. 72 h after the last treatment, mice were sacrificed and colonic tissue was collected for further analysis. Immunofluorescent staining Human biopsies and mouse tissue was cryosectioned and fixed with methanol (−20 °C). After permeabilization with 0.5% Triton-X100 and blocking with 5% goat serum, sections were incubated with primary antibodies against claudin-2 (Invitrogen, Karlsruhe, Germany; 1:200) and tricellulin (Abfinity, Invitrogen; 1:600), followed by washing steps and incubation with the respective secondary antibodies (Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit, each 1:500; Molecular Probes MoBiTec) and DAPI (1:1000). Images were obtained with a confocal laser scanning microscope (LSM 580, Zeiss). Electrophysiological and flux measurements Electrophysiological and flux studies were performed in Ussing chambers under shortcircuit conditions as described earlier.(46) For flux measurements, after apical addition of fluorescein (0.1 mM) or FITC-labeled and dialyzed 4 or 10 kDa dextran (0.4 mM, TdB Consultancy, Sweden) together with basolateral addition of unlabeled dextran of the same size (0.4 mM, Serva, Heidelberg, Germany), basolateral samples were taken at 0, 30, 60, 90, and 120 min. Tracer fluxes and apparent permeabilities were calculated from the amount of FITC-dextran in the basolateral compartment measured fluorometrically (Tecan Infinite M200, Tecan, Switzerland). One-path impedance analysis was performed employing specialized Ussing chambers as described.(58). Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Author Manuscript Krug et al. Page 10 Western blotting, phosphorylation assays and protein stability Cells were washed with ice-cold PBS, scraped from the permeable supports and incubated on ice in lysis buffer containing 10 mM Tris-Cl (pH 7.5), 150 mM NaCl, 0.5% (v/v) Triton X-100, 0.1% (w/v) SDS and protease inhibitors (Complete, Roche). Protein was obtained in the supernatant after a centrifugation at 15.000×g (15 min, 4°C). For phosphorylation assays, cells were lysed at one hour after IL-13 treatment using phospholyis buffer (20 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Trition X-100, 2.5 mM Na4P2O7, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1μg/mL leupeptin, 1mM PMSF). For tissues, samples were homogenized in ice-cold lysis buffer (1 M Tris-Cl pH 7,4, 1 M MgCl2, 0.5 M EDTA, 0.5 M EGTA, protease inhibitors) and pushed through a 1 mL hypodermic syringe with a 0.45 × 10 mm needle. Supernatants of a short centrifugation (1,000 ×g, 5 min, 4°C) were centrifuged (42,100 ×g, 30 min, 4°C) and the resulting pellets containing the membrane protein fraction were resolved in lysis buffer. 10 μg of the protein samples were electrophoresed on a SDS-polyacrylamide gel and then transferred to a PVDF membrane (Perkin Elmer). Proteins were detected by immunoblotting employing primary antibodies against claudin-2 and -4 (Invitrogen, Karlsruhe, Germany), tricellulin (Abfinity, Invitrogen), c-Fos, phopsho-c-Fos, c-jun, phosphor-c-junS63, phosphorc-junS-3, FRA-1, phosphor-FRA1, SAPK/JNK, phosphor-SAPK/JNK, ERK1/2, phosphorERK1/2, p38 and phosphor-p38 (Cell Signaling Technology). For chemiluminescence detection, membranes were washed and incubated with Lumi-Lightplus (Roche). Specific signals were quantified by luminescence imaging (Fusion FX7, Vilber Lourmat, Eberhardzell, Germany) and quantification software (Multi Gauge V2.3, FujiFilm, Japan). For analysis of protein stability, HT-29/B6 cells were cultured in the presence or absence of IL-13, while protein synthesis was blocked by addition of 100 μg/ml cycloheximide (SigmaAldrich) 2 h prior to cytokine treatment. Protein was isolated 0, 2, 8, 12, 24, 30 and 48 h after IL-13 addition, and tricellulin protein expression was evaluated by western blotting. Amounts of tricellulin were plotted against time on a logarithmic scale. The half-life (T1/2) of the protein was calculated from the slope of the linear smoothing function. Isolation of RNA, reverse transcription, qRT PCR and mRNA stability RNA was isolated using the peqGOLD RNAPure (peqLab Biotechnologie GmbH, Erlangen, Germany) according to the manufacturer’s instructions and RNA was quantified by NanoDrop® ND-1000 UV-Vis Spectrophotometer (peqLab Biotechnologie GmbH). 2 μg of total RNA per reaction were reverse-transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Mannheim, Germany). Quantitative RT-PCR of tricellulin (Hs00930631_m1), claudin-2 (Hs00252666_s1) and GAPDH as control were amplified using taqman probes (Applied Biosystems). Differential expression was calculated according to the 2−ΔΔCT method. Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Author Manuscript Krug et al. Page 11 For analysis of mRNA stability, HT-29/B6 cells were cultured in the presence or absence of IL-13, while RNA transcription 1 h in advance was blocked by addition of 7 μg/mL actinomycin D (Sigma-Aldrich). RNA was isolated 0, 6, 12, 24, and 28 h after actinomycin D addition, reversely transcribed, and quantified by RT-PCR. Amounts of the target-specific copies were plotted against time on a logarithmic scale and the half-life (T1/2) of mRNA was calculated. TUNEL staining Cells were fixed in 1% paraformaldehyde and DNA was stained with a TUNEL assay (TdTmediated X-dUTP nick end labeling; Roche, Mannheim, Germany). Labeled nuclei were counted and expressed as a fraction of total DAPI-stained nuclei. Passage visualization For visualization of macromolecule passage, the recently developed sandwich assay(49) was slightly modified. In brief, filter supports (pore size 0.45 μm) were transferred to chilled HEPES-buffered ringer, kept at 4 °C and basally incubated with 15 μM avidin (Lee Biosolutions) for 20 min. Excess avidin was removed by washing. 10 μM biotin-labelled TMR-dextran 10 kDa (Invitrogen) was added apically for 1 h and then carefully removed by washing. Cells were fixed with 2% paraformaldehyde (Electron Microscopy Sciences) and used for subsequent immunofluorescent staining of ZO-1 (BD, Heidelberg, Germany) and tricellulin (Abfinity, Invitrogen). Statistical analysis If not differently stated, data are expressed as mean values ± standard error of the mean (SEM) indicating n as the number of single, independent measurements. Statistical analysis was performed using Student’s t-test with Bonferroni-Holm adjustment for multiple testing or the Mann-Whitney-test for not normally distributed data. p<0.05 was considered significant (*p<0.05, **p<0.01, ***p<0.001). Supplementary Material Refer to Web version on PubMed Central for supplementary material. Acknowledgments This study was supported by grants from the Deutsche Forschungsgemeinschaft (DFG FOR 721/2, KR 3807/171, Schu 559/11-2), from the Sonnenfeld-Stiftung Berlin, and from the US National Institutes of Health (DK061931). Abbreviations AP-1 bTJ CD Cldn activator protein 1 bicellular tight junction Crohn’s disease claudin Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Krug et al. Page 12 ERK1/2 extracellular-signal-regulated kinases 1 and 2 FeNO exhaled nitric oxide FD10 FITC-dextran 10 kDa FD4 FITC-dextran 4 kDa FITC fluorescein isothiocyanate IBD inflammatory bowel disease IL-13 interleukin-13 IL-4 IL13Rα interleukin-4 IL-13 receptor alpha IL4R INFγ IL-4 receptor interferon gamma JAK Janus kinase JAM junctional adhesion molecule JNK c-Jun N-terminal kinase MAPK mitogen-activated protein kinases MARVEL MAL and related proteins for vesicle trafficking and membrane link PI3K phosphatidylinositide 3-kinase Repi epithelial resistance Rsub subepithelial resistance Rt transepithelial resistance (Rt=TER=Repi+Rsub) SAHA suberoylanilide hydroxamic acid SAPK stress-activated kinase STAT signal transducers and activators of transcription TAMP tight junction-associated MARVEL proteins TER TGFβ transepithelial resistance transforming growth factor beta TJ TNFα tight junction tumor necrosis factor alpha Tric tricellulin Mucosal Immunol. 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[PubMed: 1656765] 58. Stockmann M, Gitter AH, Sorgenfrei D, Fromm M, Schulzke JD. Low edge damage container insert that adjusts intestinal forceps biopsies into Ussing chamber systems. Pflugers Archiv: European journal of physiology. 1999; 438(1):107–12. [PubMed: 10370094] Author Manuscript Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Author Manuscript Krug et al. Page 17 Fig. 1. Expression, functional and electrophysiological analysis of human biopsies A. Scatterplot of tricellulin protein expression in sigmoid colon biopsies of Ctrl, CD and UC. Median of Ctrl is set 100%. Tricellulin is downregulated in UC, while in CD no difference to controls occurs (median; Ctrl: 100.0%, n=20; CD: 108.6%, n=17; UC: 53.0%, n=19, ***p<0.001). B. Scatterplot of claudin-2 protein expression in sigmoid colon of Ctrl, CD and UC. Because Ctrl patients show no claudin-2 expression, the median of CD is set 100%. In both diseases claudin-2 is upregulated, however in UC this increase is higher than in CD (**p<0.01). C. Scatterplot of claudin-4 protein expression in sigmoid colon of Ctrl, CD and UC. Median of Ctrl is set 100%. Claudin-4 is downregulated in UC, while in CD no significant difference to Ctrl occurs (Ctrl: 100.0%, n=15; CD: 89.4%, n=11; UC: 32.3%, n=13, ***p<0.001). D. Representative western blot images of sigmoid colon tissue of Ctrl, CD and UC. E. Electrical resistances of sigmoid colon of Ctrl and UC (n=8). While the epithelial resistance (Repi) is decreased in UC (*p<0.05), the resistance of the subepithelial layers (Rsub) is increased (*p<0.05), resulting in an almost unchanged transepithelial resistance (Rt). F. Permeability for the paracellular flux markers fluorescein and 4 kDa-FITC dextran. Both permeabilities are increased in UC (fluorescein: Ctrl: 0.18±0.07·10−6 cm/s, n=6, UC: 1.07±0.41·10−6 cm/s, n=7, *p<0.05; FD4: Ctrl: 0.03±0.01·10−6 cm/s, n=6, UC: 0.26 ± 0.10·10−6 cm/s, n=6, *p<0.05). Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Krug et al. Page 18 Fig. 2. Localization of claudin 2 (red) and tricellulin (green) in human colon biopsies Representative immunofluorescent stainings of cryosections of patient biopsies. While claudin-2 is only expressed within the crypts of control patients (ctrl), it is expressed all along the crypt and surface in CD and UC patients. Tricellulin is present in all areas of the crypt and is also detectable in surface areas of the control patients. In UC, a decreased expression is observed, while in CD patients there seems to be a shift of expression; while in the crypts the signal appears to be reduced, localization within the surface is increased. The merged localizations within the crypt (blue box) and the surface epithelium (yellow) are also shown in higher magnifications. Bar = 50 nm. Author Manuscript Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Krug et al. Page 19 Author Manuscript Author Manuscript Author Manuscript Fig. 3. Expression and functional analysis of intestinal cells A. Representative western blots. IL-13 treatment lead to decreased tricellulin (72±4%,***p<0.001;n=11) and increased claudin-2 (145±14%,**p>0.01;n=7) expression in HT-29/B6 (densitometry Fig. 4E). B. Effect of IL-13 on transepithelial resistance. 48 h incubation with IL-13 decreases Rt in HT-29/B6 (***p<0.001). C. Effect of IL-13 on permeability for 4 kDa-FITC dextran (FD4). 48 h incubation with IL-13 increases permeability to FD4 in HT-29/B6 (***p<0.001) D. Representative western blots. E. Densitometric analysis of protein expression levels in stable shTRIC transfectants in comparison to vector-transfected controls. shTRIC leads to decreased tricellulin expression (60±9%, **p<0.01, n=10), but had no influence on expression of claudin-2 (87±12%, n=5) or occludin (Occl; 100±10%, n=5). F. Permeability for the macromolecular paracellular fluxmarkers 4 kDa-and 10 kDa-FITC dextran. Both permeabilities are increased in shTRIC clones (FD4: **p<0.01; FD10: *p<0.05). Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Krug et al. Page 20 Author Manuscript Author Manuscript Author Manuscript Fig. 4. Functional and expression analysis of mice treated with IL 13 A. Permeability for 4 kDa-FITC dextran. Permeability for FD4 is increased in mice treated with IL-13 (*p<0.05, n=9–12). B. Representative western blots. Treatment of mice with IL-13 results in increased claudin-2 and decreased tricellulin expression. C. Densitometric analysis of protein expression colon tissue of untreated and IL-13-treated mice. After treatment, claudin-2 is increased and tricellulin is decreased (*p<0.05, n=9–12). Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Krug et al. Page 21 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Fig. 5. Analysis of the involvement of IL13 receptors in regulation of tricellulin and claudin 2 A. Densitometric analysis of protein expression of HT-29/B6 cells treated with inhibitory antibodies and IL-13. In HT-29/B6, the inhibitory antibody against IL13 receptor α1 is able to inhibit the IL-13-caused increase of claudin-2, but not the decrease of tricellulin. Tricellulin decrease by IL-13 is inhibited by blocking the IL-13 receptor α 2, while its inhibition has no effect on the claudin-2 increase (n=7–11). B. Representative western blots of the incubation with inhibitory antibodies and IL-13. C. Permeability for 4 kDa-FITC dextran. Permeability for FD4 is increased in HT-29/B6 cells treated with IL-13. This effect was not inhibited by preincubation with the inhibitory antibody against IL13 receptor α1, but was abolished by preincubation with the inhibitory antibody against IL13 receptor α2 (**p<0.01; ***p<0.001, n=4). Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Krug et al. Page 22 Fig. 6. Effects of IL13Rα2 knockdown A. Representative western blots of different clones of HT-29/B6 cells stably transfected with shRNA against IL13Rα2 (shRα2) in comparison with vector-transfected controls. B. Densitometric analysis of tricellulin and IL13Rα2 expression in shRα2 knockdown clones (n=4; **p<0.01). C. Representative western blots of vector controls and shRα2 clones treated with IL-13. D. Densitometric analysis of tricellulin expression in shRα2 and vector control clones in response to IL-13-treatment (n=6; ***p<0.001). E. Permeability for 4 kDa- FITC dextran. Permeability for FD4 is increased in vector controls treated with IL-13, while in IL13Rα2 knockdown, no effect is observed after IL-13 treatment (n=8–13; *p<0.05). Author Manuscript Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Krug et al. Page 23 Author Manuscript Author Manuscript Author Manuscript Fig. 7. Analysis of the signaling pathways regulating tricellulin and claudin 2 A. Representative western blots of STAT6−/− mice treated with IL-13. IL-13 treatment still decreases tricellulin, while claudin-2 expression is at levels of untreated mice due to absence of STAT6 (n=4–6). B. Densitometric analysis of protein expression in colon tissue of untreated and IL-13-treated STAT6−/− mice. After treatment, tricellulin is decreased (**p<0.01), while claudin-2 is unaffected. C. Densitometric analysis of protein expression of HT-29/B6 pretreated with different inhibitors before application of IL-13 (representative blots see Fig. S6, n=4–12). Protein expression levels were normalized to the respective inhibitor treatment without IL-13. Inhibitors targeting STAT6, ERK1/2, MAPK, and PI3K affect the IL-13-caused increase of claudin-2, while inhibitors targeting JAK1 and/or JAK2, JNK, ERK1/2, MAPK and AP-1 inhibit the IL-13-caused decrease of tricellulin (#p<0.05; *p<0.01, n=6–12). D. Representative western blots of total and phosphorylated proteins involved in ERK1/2 and AP-1 signaling. HT-29/B6 cells are either pretreated or not with U0126 before application of IL-13. E. Densitometric analysis of total protein. Expression of cFos, FosB and c-jun is increased by IL-13 (*p<0.001, #p<0.01, ~p<0.05; n=12). U0126 inhibits this increase for c-Fos (IL-13: 265±33%, ***p<0.001; U0126+IL-13: 63±11 %, ***p<0.001 to IL-13). F. Ratio of phosphorylated protein to total protein under influence of IL-13. Phosphorylation levels are increased by IL-13 for FRA-1, p44/42, p38, p54 and SAPK/JNK and decreased for c-Fos and c-jun (S63; IL-13: 50±10%, ***p<0.001; U0126+IL-13: 92±18 %, *p<0.05 to IL-13, n=12). These changes in phosphorylation are Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Krug et al. Page 24 inhibited by U0126 only for FRA-1 (IL-13: 209±41%, *p<0.05; U0126 + IL-13: 92±19 %, *p<0.05 to IL-13, n=12) and p44/42 (ERK1/2; IL-13: 173±30%, *p<0.05; U0126+IL-13: 6±2 %, *p<0.05 to IL-13, n=12) (n=10–12). Author Manuscript Author Manuscript Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Krug et al. Page 25 Fig. 8. Visualization of macromolecule passage sites by sandwich assay Maximum intensity projections and Z-stacks of HT-29/B6 immunofluorescence stainings. One out of four experiments is shown here, all yielding the same result. Cells were either untreated (upper panel) or treated with IL-13 (lower panel) and incubated basolaterally with avidin and then apically with biotin- and TRITC-labelled 10 kDa-dextran (A). Tricellulin (B) and ZO-1 (C) were counterstained for localization of the macromolecular passage. In merged views featuring Z-stacks and enlarged Z-stacks (D), 10 kDa dextran is shown in red, tricellulin in green, and ZO-1 in gray. IL-13 treatment results in decrease of tricellulin, while 10 kDa dextran appears predominantly at sites where more than two cells are in contact. For clearer evaluation of the localization of tricellulin, its signals were increased in contrast, while the ZO-1 signals were decreased in D. Bar = 20 μm. Author Manuscript Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Krug et al. Page 26 Author Manuscript Author Manuscript Author Manuscript Fig. 9. Schematic view of signaling pathways for claudin 2 and tricellulin triggered by IL 13 While claudin-2 expression is regulated via interaction of IL-13 with the IL4Rα/IL13Rα1 complex, tricellulin expression is regulated via interaction of IL-13 with IL13Rα2 and the downstream signaling cascades. Mucosal Immunol. Author manuscript; available in PMC 2017 December 15. Author Manuscript Author Manuscript Krug et al. Cytokine and inhibitor experiments Table. 1 cytokine concentration source TNFα IL-4 INFγ TGFβ1 TGFβ2 IL-13 500 U/mL 100 ng/mL 50 U/mL 10 ng/mL 10 ng/mL 100 ng/mL Peprotech, Hamburg, Germany Peprotech, Hamburg, Germany Peprotech, Hamburg, Germany Peprotech, Hamburg, Germany Peprotech, Hamburg, Germany Peprotech, Hamburg, Germany inhibitor concentration source SAHA Tanshinone Iia LY294002 JAK3-Inhibitor II JNK V U0126 AS151-499 Baricitinib 5 μM 10 μM 10 μM 50 μM 10 μM 10 μM 1 μg/mL 250 nM Sigma-Aldrich, Schnelldorf, Germany Sigma-Aldrich, Schnelldorf, Germany Calbiochem, Darmstadt, Germany Calbiochem, Darmstadt, Germany Calbiochem, Darmstadt, Germany Cell Signaling Technology, Frankfurt am Main, Germany Axon Medchem, Groningen, Netherlands Selleckchem, Munich, Germany Page 27 Author Manuscript Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2017 December 15.