The Alarmin IL-33 Promotes Regulatory T Cell Function in the Intestine

Foxp3+ regulatory T cells are abundant in the intestine where they prevent dysregulated inflammatory responses to self and environmental stimuli. It is now appreciated that Treg cells acquire tissue-specific adaptations that facilitate their survival and function1; however, key host factors controlling the Treg response in the intestine are poorly understood. IL-1 family member IL-33 is constitutively expressed in epithelial cells at barrier sites2 where it functions as an endogenous danger signal or alarmin following tissue damage3. Recent studies in humans have described high levels of IL-33 in inflamed lesions of inflammatory bowel disease (IBD) patients4-7 suggesting a role for this cytokine in the pathogenesis of IBD. In the intestine, both protective and pathologic roles for IL-33 have been described in murine models of acute colitis8-11 but its contribution to chronic inflammation remains ill defined. Here we show that the IL-33 receptor ST2 is preferentially expressed on colonic Treg (cTreg) cells, where it promotes Treg function and adaptation to the inflammatory environment. IL-33 signaling into T cells stimulates Treg responses in several ways. Firstly, it enhances transforming growth factor-β1 (TGF-β1) mediated differentiation of Treg cells and secondly, it provides a necessary signal for Treg accumulation and maintenance in inflamed tissues. Strikingly, IL-23, a key pro-inflammatory cytokine in the pathogenesis of IBD, restrained Treg responses through inhibition of IL-33 responsiveness. These results demonstrate a hitherto unrecognized link between an endogenous mediator of tissue damage and a major anti-inflammatory pathway, and suggest that the balance between IL-33 and IL-23 may be a key controller of intestinal immune responses.

contribution to chronic inflammation remains ill defined. Here we show that the IL-33 receptor ST2 is preferentially expressed on colonic T reg (cT reg ) cells, where it promotes T reg function and adaptation to the inflammatory environment. IL-33 signaling into T cells stimulates T reg responses in several ways. Firstly, it enhances transforming growth factor-β1 (TGF-β 1 ) mediated differentiation of T reg cells and secondly, it provides a necessary signal for T reg accumulation and maintenance in inflamed tissues. Strikingly, IL-23, a key pro-inflammatory cytokine in the pathogenesis of IBD, restrained T reg responses through inhibition of IL-33 responsiveness. These results demonstrate a hitherto unrecognized link between an endogenous mediator of tissue damage and a major anti-inflammatory pathway, and suggest that the balance between IL-33 and IL-23 may be a key controller of intestinal immune responses.
To identify potential tissue-specific modulators of cT reg cells, we compared the mRNA expression profiles of mesenteric lymph node (MLN) and cT reg cells. We identified St2, the transcript coding for the IL-33 receptor 12 , as one of the top differentially upregulated genes in cT reg cells (Fig. 1a,b). Flow-cytometric analysis confirmed selective enrichment of ST2 + T reg cells in the colon (Fig. 1c) and these cells expressed high levels of the activation markers KLRG1, CD103 and OX40 (Fig. 1d). Analysis of Helios expression revealed that ST2 + T reg cells are a heterogeneous population containing thymus-derived T regulatory (tT reg ) cells as well as peripherally generated Helios − T reg cells (Fig. 1e) 13 . A significant proportion of intestinal Foxp3 + T reg cells co-express the transcription factor GATA3 [14][15][16] and GATA3 is known to regulate ST2 expression in Th2 cells 17 . Indeed, ST2 expression was largely restricted to GATA3-expressing cT reg cells (Fig. 1e) and selective ablation of GATA3 in Foxp3-expressing cells, using Gata3 f/f -Foxp3-Cre mice 15 , caused a marked reduction of ST2 protein levels (Fig. 1f).
Given that ST2 + T reg cells are prominent in the colon we postulated that IL-33 may modulate in vitro-induced T reg -cell (iT reg ) differentiation. To test this, we sort-purified naïve CD4 + T cells from Foxp3 gfp reporter mice and activated them in the presence of TGF-β 1 . Notably, both Gata3 and St2 expression were induced under iT reg -differentiation conditions (Extended Data Fig. 1). Addition of IL-33 to iT reg cultures significantly increased both the percent and total number of Foxp3-expressing cells but had no effect on Foxp3 expression in the absence of TGF-β 1 (Fig. 2a). The presence of IL-33 in iT reg cultures did not affect induction of Th2 cytokines or expression of Th1 and Th17-associated transcription factors Tbx21 and Rorc (Extended Data Fig. 1), suggesting that IL-33 preferentially regulates Foxp3 expression. Thus, our data indicate that the alarmin IL-33 is a novel co-factor in TGF-β 1mediated iT reg generation.
GATA3 is highly expressed in ST2 + T reg cells (Fig. 1e) and IL-33 has been shown to activate GATA3 in Th2 cells 18 as well as innate lymphoid cells 19 . Consistent with this notion, we observed serine phosphorylation of GATA3 upon acute stimulation of iT reg cells with IL-33 (Fig. 2b). The Foxp3 locus contains putative GATA3-binding sites within its promoter and intragenic conserved noncoding sequences (CNS) 1-3 14 . To investigate whether IL-33 influences the binding of GATA3 to any of these elements in iT reg cells, we performed chromatin immunoprecipitation (ChIP) followed by quantitative PCR. Acute stimulation of iT reg cells with IL-33 induced GATA3 recruitment to the Foxp3 promoter but not CNS1, 2 or 3 (Fig. 2c). In addition, RNA polymerase II (Pol II) was recruited to the Foxp3 promoter upon IL-33 stimulation (Fig. 2d) suggesting that IL-33 directly regulates Foxp3 expression through activation and recruitment of GATA3 to the Foxp3 promoter. In Th2 cells, GATA3 has been shown to promote St2 gene expression by binding to an enhancer element located 12-kb upstream of the St2 transcription start site 17 . Consistent with this we detected recruitment of GATA3 to the St2 enhancer upon acute stimulation of iT reg cells with IL-33 and this correlated with RNA Pol II enrichment at the St2 promoter (Extended Data Fig. 2). Thus, in addition to its role in Foxp3 induction, IL-33 also promoted its own receptor expression in iT reg cells through direct transcriptional regulation of the St2 locus providing an amplification loop for further enhancement of iT reg -cell differentiation.
Next we focused on thymus-derived T reg cells, which constitute a significant proportion of ST2 + cT reg cells (Fig. 1e). In line with published reports 20,21 administration of recombinant IL-33 led to a significant increase in the frequency and total number of splenic T reg cells (Extended Data Fig. 3a,b) and these IL-33-elicted T reg cells expressed higher levels of Foxp3 and ST2 (Extended Data Fig. 3c,d). Further analysis of proliferation marker Ki67 showed IL-33 induced proliferation in splenic T reg cells but not T effector cells (Extended Data Fig. 3e). To examine if IL-33 acts directly on T reg cells we injected IL-33 into chimeric mice containing a mixture of WT and St2 −/− hematopoietic cells. In this setting, the proliferative capacity of St2 −/− T reg cells was significantly impaired (Extended Data Fig.  3f), suggesting that IL-33 acts directly on tT reg cells to promote their proliferation and accumulation in vivo. This is further supported by the finding that sort-purified splenic T reg cells cultured in the presence of IL-33 expressed higher levels of ST2, showed a more activated phenotype and expressed increased amounts of Foxp3 protein (Fig. 2e). In addition, acute stimulation of TCR-activated splenic T reg cells with IL-33 induced serine phosphorylation of GATA3 (Fig. 2f) further demonstrating that IL-33 acts directly on tT reg cells.
To assess the impact of IL-33 on the T reg response during intestinal inflammation we induced chronic colitis by infection with Helicobacter hepaticus and administration of an IL-10R blocking antibody 22 (Extended Data Fig. 4a). We detected an increase in IL-33 protein levels in colon explant cultures and its expression kinetics mirrored that of IL-23, which is essential for the development of intestinal inflammation in this model (Extended Data Fig. 4b). Consistent with its pattern of expression, IL-33 protein levels were elevated in colonic intestinal epithelial cells isolated from the inflamed gut (Extended Data Fig. 4c-d).
Interestingly, the onset of intestinal pathology correlated with a marked increase of soluble ST2 (sST2), which is produced primarily by colonic stromal cells (Extended Data Fig. 4b, ef). sST2 is thought to limit IL-33 bioavailability by acting as a decoy receptor 23 and is increased in patients with active IBD 6,24 suggesting that the chronic inflammatory tissue environment may antagonize IL-33 activity. Despite high levels of sST2, analysis of chimeric mice showed that accumulation of St2 −/− T reg cells in the colon but not the spleen was significantly impaired during the peak of intestinal inflammation (Fig. 2g). In addition, colonic St2 −/− T reg cells expressed lower levels of Foxp3 protein on a per cell basis as compared to their WT counterparts (Fig. 2h). Together, these observations indicate that the alarmin IL-33 acts in a cell-intrinsic manner to promote the tissue-specific accumulation and stability of the T reg phenotype in the intestine under inflammatory conditions. Furthermore, high levels of sST2 during chronic intestinal inflammation may represent a mechanism to further perpetuate pathogenic responses by limiting IL-33-driven T reg accumulation.
We next sought to compare the suppressive capacity of WT and St2 −/− T reg cells. St2 −/− T reg cells inhibited T cell proliferation to the same extent as WT T reg cells in vitro (Extended Data Fig. 5) and addition of IL-33 did not enhance WT T reg suppressor function. We then tested the ability of St2 −/− T reg to protect from colitis induced by adoptive transfer of naïve CD4 + T cells. Interestingly, ST2 was highly expressed on WT T reg cells upon T cell transfer pointing toward a potential role of ST2 in modulating T reg function in this model (Extended Data Fig. 6a). Indeed, St2 −/− T reg cells were significantly impaired in their ability to prevent colonic inflammation and cellular infiltration (Fig. 3a,b), demonstrating that IL-33 signaling into T reg cells is important for their suppressive function in vivo. Analysis of WT or St2 −/− T reg cells two weeks post transfer, prior to the onset of intestinal pathology, showed similar proliferative capacity and Foxp3 expression between groups (Fig. 3c). The ratio of T effector cells (CD45.1 + RB hi progeny) to T reg cells (CD45.1 − Foxp3 + T reg progeny) was also similar ( Fig. 3d) suggesting that IL-33 signaling in T reg cells is dispensable for their ability to expand and index with effector cells in the lymphopenic host at this time point. By contrast, analysis at 8 weeks post-transfer showed that progeny of St2 −/− T reg cells contained a significantly lower proportion of Foxp3 + cells and expressed significantly less Foxp3 on a per cell basis suggesting they had lost Foxp3 expression (Fig.  3e,h). Under these circumstances the ratio of T effector/T reg cells and the total number of T effector cells (CD45.1 + RB hi progeny) was markedly increased in recipients of St2 −/− T reg cells (Fig. 3f,g). Importantly, ST2-deficient T reg cells did not themselves acquire the capacity to produce inflammatory cytokines (Extended Data Fig. 6c). Perturbations of Foxp3 expression have been shown to affect T reg cell function [25][26][27] and our data indicate that IL-33 signaling in T reg cells contributes to the maintenance of Foxp3 expression under inflammatory stress, enabling T reg cells to compete in the inflammatory niche and control the intestinal effector T cell response.
Next we sought to integrate our observations with existing pro-inflammatory pathways. IL-23 is a pivotal mediator of intestinal inflammation and polymorphisms in the IL23R locus are associated with increased susceptibility to IBD in humans 28 . We previously showed that IL-23 promotes intestinal inflammation in part through inhibition of iT reg cell differentiation 29,30 . However, the mechanism by which IL-23 blocked iT reg generation remained undefined. Interestingly, whole transcriptome analysis of IL-23 target genes in colonic effector CD4 + T cells revealed that IL-23 inhibits expression of Gata3 and St2 (Extended Data Fig. 7). Based on this observation we hypothesized that IL-23 might limit T cell responsiveness to IL-33. Indeed, the co-factor activity of IL-33 on TGF-β 1 -mediated Foxp3 induction in vitro was completely abrogated in the presence of IL-23 (Fig. 4a). Notably, addition of IL-23 prevented induction of Gata3 and St2 mRNA under iT reg differentiation conditions (Fig. 4b) resulting in reduced ST2 protein expression (Fig. 4c). Consequently, acute stimulation of IL-23-exposed iT reg cells with IL-33 did not lead to recruitment of GATA3 to the ST2 enhancer (Extended Data Fig. 8). We observed a similar role for IL-23 in limiting T reg ST2 expression during bacterially driven intestinal inflammation (Extended Data Fig. 9). Collectively our data indicate that IL-23 inhibits iT reg differentiation by regulating T cell responsiveness to IL-33.
We previously showed that IL-23 restrains T reg cells in vivo because naïve T cell transfer into Il23a −/− Rag1 −/− recipients resulted in increased T reg differentiation 29 . Therefore, we hypothesized that enhanced responsiveness to IL-33 may contribute to increased iT reg differentiation in Il23a −/− Rag1 −/− hosts. To test this we transferred WT or St2 −/− naïve T cells into Il23a −/− Rag1 −/− hosts and monitored iT reg generation. Indeed, ST2-deficient T cells were significantly impaired in their ability to differentiate into T reg cells (Fig. 4d) and this correlated with a significant increase in intestinal pathology (Fig. 4e). Importantly, ST2deficiency had minor effects on in vivo differentiation of Th1, Th2 or Th17 cells (Fig. 4f) further supporting the notion that the increased colitogenic potential of St2 −/− CD4 + T cells is a consequence of deficient iT reg differentiation rather than dysregulated effector T cell responses. In summary, our data strongly suggest that IL-33 is a major factor responsible for driving iT reg differentiation in the absence of IL-23.
Finally, we investigated whether IL-23 can interfere with ST2 signaling in T reg cells. Interestingly, sort-purified ST2 + T reg cells from the colon expressed detectable levels of Il23r (Extended Data Fig. 10). Indeed, exposure of TCR-activated tT reg cells to IL-23 completely abolished IL-33-mediated GATA3 phosphorylation (Fig. 4g). Furthermore, IL-33 preferentially induced genes co-regulated by Foxp3 and GATA3 16 and this was completely abrogated in the presence of IL-23 (Fig. 4h). Addition of a specific inhibitor of STAT3, the main transcription factor downstream of IL-23 signaling, reversed this inhibitory effect of IL-23 (Fig. 4i). Together our data suggest that IL-23 inhibits ST2 signal transduction and expression of a distinct set of GATA3 regulated genes in tT reg cells.
Our results identify a new function for IL-33 as an important link between inflammationdriven tissue damage and the local intestinal T reg cell response. We show that colonic T reg cells are poised to respond to the release of IL-33 upon tissue damage through selective expression of ST2 and that signaling through this pathway plays an essential role in their capacity to adapt to the inflammatory tissue environment and restrain intestinal inflammation. The ability of IL-33 to amplify regulatory networks in response to tissue injury may represent a more general mechanism by which alarmins limit immune-mediated damage to self at barrier tissues. Strikingly, IL-23 limits this regulatory mechanism through inhibition of T reg cell responsiveness to IL-33 suggesting that the balance between IL-23 and IL-33 may be a major determinant of the outcome of intestinal immune responses.

Method Summary
A description of the in vitro assays and details of in vivo models can be found in the full Methods.

Mice
Wild-type C57BL/6, congenic B6.SJL-Cd45.1, C57BL/6 Il23r −/− , C57BL/6 Rag1 −/− , C57BL/6 Il23a −/− Rag1 −/− and Foxp3 gfp reporter mice 31,32 were bred and maintained under specific pathogen-free conditions in accredited animal facilities at the University of Oxford. Where indicated, mice were i.p. injected with recombinant IL-33 (1 μg / injection; Biolegend) for 5 consecutive days and sacrificed 24 h after the last injection. Gata3 f/f -Foxp3Cre mice were kept at the NIH and experiments were performed at the NIH. Spleen, MLN and bone marrow from C57BL/6 St2 −/− mice were obtained from Padraic Fallon (Trinity College Dublin, Ireland) or Max Loehning (Charité -University Medicine Berlin, Germany). All procedures were conducted in accordance with the UK Scientific Procedures Act of 1986. Mice were negative for Helicobacter spp. and other known intestinal pathogens, were age and sex-matched and more than 6 weeks old when first used. Both female and male mice were used in experiments. Wherever possible preliminary experiments were performed to determine requirements for sample size, taking into account resources available and ethical, reductionist animal use. Generally, each mouse of the different experimental groups is reported. Exclusions criteria such as, inadequate staining or low cell yield due to technical problems, were pre-determined. Animals were assigned randomly to experimental groups. Each cage contained animals of all the different experimental groups.

Induction of Helicobacter hepaticus/anti-IL-10R colitis
Mice were fed 1×10 8 cfu Helicobacter hepaticus by oral gavage with a 22G curved needle on day 0, day 1 and day 2 of the experiment. In addition mice received 1mg of an anti-IL-10R blocking antibody by i.p. injection once weekly starting at the day of Helicobacter hepaticus infection.

Histological assessment of intestinal inflammation
Proximal, mid, and distal colon samples were fixed in buffered 10% formalin solution. Paraffin embedded sections were cut (5 mm) and stained with hematoxylin and eosin, and inflammation was scored in a blinded fashion using a previously described scoring system 34 .

Isolation of leukocyte subpopulations and flow cytometry
Cell suspensions from spleen, MLN, and the lamina propria were prepared as described previously 35 and first incubated with anti-CD16/CD32 (eBioscience) to prevent nonspecific binding. Single cell suspensions were stained with antibodies against CD4, CD25, TCR-β, CD45.1, CD45.2, CD103, OX40, IL-17A, IFN-γ, IL-13, Foxp3, GATA3, Helios, (all from eBioscience), RORγt, Ki67, (BD Biosciences) and CD45RB, KLRG1 (all from Biolegend) and anti-ST2 conjugated to biotin (mdBioproducts). Intracellular staining was performed as follows: cells were restimulated for 4 h as previously described 36 , washed and incubated with anti-CD16/CD32. Cells were washed and stained for surface markers indicated above and fixed in eBioscience Fix/Perm buffer, followed by permeabilization in eBioscience permeabilization buffer for 1 h in the presence of antibodies. Cells were acquired with a BD LSRII and analysis was performed with FlowJo (Tree Star) software.

T reg cell suppression assay
CD25 + CD4 + T reg cells were sorted by flow cytometry from WT and St2 −/− mice. CD25 − CD62L + CD44 − CD4 + T cells (responder cells) were sorted from St2 −/− mice. Antigen presenting cells (APCs) were isolated by magnetic bead separation from splenocytes and LN cells from St2 −/− mice. T responder cells were labeled with Violet cell trace and plated together with the sorted Tregs at ratios of 1:1 and 1:3 together with irradiated APCs in the presence of anti-CD3 (1μg/ml). IL-33 was added at 30 ng/ml. After 4 days proliferation of T responder cells was measured by flow cytometry.

Colon explant cultures
Organ explants were prepared as previously described 36 and cultured overnight in complete media. Cytokine levels in the supernatants were determined by ELISA IL-23 and IL-33 (eBioscience) and soluble ST2 (R&D), and concentrations were normalized to the weight of the explants.

Isolation and culture of colonic stromal cells
Colonic tissue was digested and cells from the 30:40% Percoll gradient interface harvested and washed twice in PBS containing 2% BSA. Cells were plated at 10×10 6 cells/ml in complete DMEM containing 40μg/ml Gentamycin (Sigma). After 24-48 hours, non-adherent cells were removed by vigorous washing, and media replaced. Adherent intestinal stromal cells were cultured for 10 days until confluent. Cells were then plated at 1×10 6 cells/ml for 48 hours and the concentration of sST2 in supernatants was determined by ELISA (R&D Systems).

qPCR and microarray preparation
RNA was extracted according to the manufacturers' protocol (RNeasy, Qiagen) and cDNA synthesis was performed using Superscript III reverse transcription and Oligo dT primers (both from Invitrogen). qPCR reactions were performed using TaqMan Gene Expression Assays and normalized to HPRT (all from Applied Biosystems). Alternatively, qPCR reactions were performed using SYBR green PCR SensiMix (Quantace) with primers from 37 . For soluble ST2 the following primers were used: sST2 forward 5′tcgaaatgaaagttccagca -3′ and sST2 reverse 5′-tgtgtgagggacactccttac -3′ Samples were assayed in duplicate on a Bio-Rad CFX96 RT-qPCR machine and differences were calculated using the 2 ΔC(t) method.
For microarray gene expression analysis RNA was extracted using the RNAqueous-Micro total RNA Isolation Kit or RiboPure RNA Purification Kit (both Ambion). For RNA amplification and labelling the TargetAmp2-Round Biotin-aRNA Amplification Kit 3.0 (Epicentre, Illumina) or the Illumina TotalPrep RNA Amplification Kit (Ambion) were used and RNA quality was assessed using the Agilent 2100 Bioanalyzer and only samples with RNA integrity number (RIN) values above 7 were used for further processing. Biotinylated cRNA was hybridised to MouseWG-6 v2.0 Expression BeadChip (Illumina). Hybridisations to BeadChips and data acquisition were performed by the microarray core facility at the Wellcome Trust Center for Human Genetics, Oxford. Microarray analysis was performed using GeneSpring GX12 software (Agilent). Data was normalised using 75% percentile shift normalisation algorithm and baseline transformed to the median of all samples. Statistical significance was determined using an unpaired t-test followed by Benjamini Hochberg false discovery rate multiple testing correction. P value cut-off was set to 0.05.

Statistical analysis
Where appropriate Student's t-test was used. For comparison of more than two groups a one-way ANOVA followed by a Bonferroni multiple comparison test was performed. All statistical analysis was calculated in Prism (GraphPad). Differences were considered to be statistically significant when P ≤ 0.05.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material. e, Representative plots of T reg cells cultured with anti-CD3/CD28 plus indicated cytokines and analyzed after 3 days. Data are representative of three independent experiments. f, T reg cells were cultured with anti-CD3/CD28 for 24h followed by stimulation with IL-33. Blots are representative of three independent experiments. g, Mixed chimaeras were generated containing WT and St2 −/− bone marrow cells. Reconstituted mice were analyzed at steady state or two weeks after infection with Helicobacter hepaticus and anti-IL-10R treatment (inflamed). Absolute numbers of WT or St2 −/− T reg cells in steady state (n = 3) and inflamed (n = 6) hosts (mean ± s.e.m). h, Analysis of Foxp3 expression in T reg cells from inflamed chimaeric hosts presented as geometric mean fluorescence intensity (gMFI). *P<0.05, **P<0.01 *** P<0.001 as calculated by 1way-ANOVA with Bonferroni post-test or paired Students' t-test. e, C57BL/6 Rag1 −/− mice were injected as in (a) and sacrificed at 8 weeks post injection. Representative plots are gated on colonic T reg cell progeny (CD45.1 − ). f, Ratio of RB hi T cell progeny (CD45.1 + ) to WT or St2 −/− T reg cell progeny (CD45.1 − ) in the colon from mice in (e), (mean ± s.e.m). g, Absolute numbers of RB hi T cell progeny (CD45.1 + ) in the colon from mice in (e), (mean ± s.e.m). h, Analysis of Foxp3 expression in colonic Foxp3 + CD45.1 − T reg cells presented as gMFI (mean ± s.e.m). Results are representative of two independent experiments. *P<0.05 **P<0.01 *** P<0.001 **** P<0.0001 as calculated by 1way-ANOVA with Bonferroni post-test or Students' t-test. a, Naïve CD4 + T cells were cultured with anti-CD3/CD28 plus TGF-β 1 as well as indicated cytokines and the frequencies of Foxp3 + T cells determined after 3 days later (mean ± s.e.m. of three independent experiments). b, Naïve CD4 + T cells were cultured with anti-CD3/ CD28 plus indicated cytokines for 48h. Data are from one experiment representative of two (mean ± s.d.). c, Naïve CD4 + T cells were cultured as indicated. Representative blots of two independent experiments are shown. d, C57BL/6 Il23a −/− Rag1 −/− mice were injected with CD45RB hi WT (n = 9) or St2 −/− (n = 10) T cells. Mice were sacrificed after 6-8 weeks post transfer and frequencies of Foxp3 + CD4 + T cells in colon are shown. e, Colitis scores for mice in (d). f, Expression of indicated cytokines by colonic CD4 + T cells from mice in (d).
g, T reg cells were cultured with anti-CD3/CD28 for 24h followed by stimulation with IL-33 in the presence or absence of IL-23. Representative blots of two independent experiments are shown. h, T reg cells were cultured in the presence of anti-CD3/CD28 for 24h and mRNA expression of indicated genes measured following stimulation with IL-33 for 45 minutes in the presence or absence of IL-23 (mean ± s.e.m. of three independent experiments). i, T reg cells were cultured with anti-CD3/CD28 for 24h and representative blots of two independent experiments are shown. *P<0.05, **P<0.01 *** P<0.001 as calculated by 1way-ANOVA with Bonferroni post-test or Students' t-test.