Tricellulin is regulated via interleukin 13 receptor α2, affects macromolecule uptake, and is decreased in ulcerative colitis

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.


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).
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)(23)(24)(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).
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 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 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 ( 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 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-labelled-TRITC-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 movie M2), supporting that the macromolecule passage may depend on the level of tricellulin present in tTJs.

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)(19)(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.
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)(23)(24)(25)(26)(27)(28)(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 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.

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 cm 2 . The study design was approved by the local ethics committee (EA4/015/13).

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.

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).

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 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 (Sigma-Aldrich) 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 (T 1/2 ) 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.
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 (T 1/2 ) 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.

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.   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). 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).