Wnt signaling antagonist DKK1, a secreted glycoprotein, expresses in many species, including human derived stromal cells and mouse colonic tissues [26, 41]. DKK1 possesses a conservative gene sequence41 and its expression in stromal cells can be down-regulated by IL-1β or up-regulated by glucocorticoid stimulation . Relying on competing with Wnt-ligands for receptors, DKK1 can uniquely inhibit the Wnt/β-catenin signaling transduction, which exhibits anti-inflammatory effects in the development of ulcer colitis . Therefore, in present study, we hypothesized that ERCs, like other stromal cells, could also secrete DKK1, which blocks Wnt mediated anti-inflammatory signal transduction, and thus weakening the immunoregulation of ERCs. Furthermore, we could optimize the therapeutic effect of ERCs on colitis by downregulating the expression of DKK1.
To carry out the current study, we determined DKK1 levels in different groups, including unmodified ERC group, GC-treated ERC group and IL-1β-treated ERC group. Our results showed that ERCs indeed secret a considerable amount of DKK1, just like MSCs26, and the expression in different groups could be markedly changed after ERCs stimulated by IL-1β or glucocorticoid (GC), not only at the protein level but also at the level of RNA.
Next, we carried out the following experiments in vivo and revealed that in DKK1low-ERC group, clinical characters and pathological manifestations of colitis were strikingly ameliorated, in which the immune-regulatory effects of ERCs may be enhanced by low DKK1 production, as a result of Wnt/β-catenin pathway being activated [34, 43]. Comparing with that in DKK1high-ERC (pretreated with GC) group, we confirmed that DKK1 really plays a vital role in affecting therapeutic function of ERCs on experimental colitis and this action may be associated with the activation of Wnt/β-catenin pathway.
As we know, the balance between CD4+T cells (Th1/Th2, Th17 and Tregs) is essential for sustaining the intestinal homeostasis and closely involved in the development of UC . In present study, we analyzed the CD4+T cell population changes in splenocytes by FACS, and found that, Th1 and Th17 populations in DKK1low-ERC (ERCs treated with IL-1β) group, were the lowest among experimental groups, while the populations of Th2 and Tregs in DKK1low-ERC group presented the highest. These data indicated that DKK1, also participates in the immune-regulatory function of ERCs, and it can affect the immunosuppressive effects of ERCs on CD4+T cells.
Among immunocytes activated, macrophages and DC cells, were also revealed acting a role in the development of DSS-induced colitis . Macrophages, were illustrated with a diverse plasticity, and its differentiation can be driven by the surrounding settings . M1 phenotype cells, which are known as pro-inflammatory macrophages, have the ability to generate interleukin (IL)-1β, tumor necrosis factor alpha (TNF-α), and nitric oxide (NO) to exacerbate the inflammation . While, M2 phenotype cells exhibit immune-regulatory and anti-inflammatory effects by producing IL-10, which could promote tissue repairs by its negative immune regulation ability . DCs, the major part of antigen-presenting cells (APCs), can exert its effects by costimulatory molecules (MHC class II and CD86) expressed on their cytomembranes , promoting the migration of immune cells into damaged colons, and then result in inducing T cell-mediated immune response.
Thus, in the present study, we evaluated the population changes of macrophages and DCs of splenocytes. The results showed that the number of mature DCs (CD11c+MHCII+ or CD11c+CD86+) in DKK1low-ERC group was significantly lower than that of other groups. Meanwhile, the population of M2 phenotype macrophages (CD68+CD206+) rised up to the highest level among those of experimental groups. Taking together, these observations suggested that DKK1, secreted by ERCs, negatively affect the immunomodulatory effects of ERCs on macrophages and DCs. In addition, when we downregulate the DKK1 expression of ERCs, this immunoregulatory mechanism tend to be active and exert effective therapeutic roles in DSS-induced colitis.
Accumulating documents have illustrated that inflammatory mediators in colons, such as IL-4, IL-10, SOD, IFN-γ, TNF-α, COX-2, MPO and iNOs, which were produced by infiltrating immune cells, macrophages and epithelial cells, orchestrated the pathogenesis of ulcer colitis temporally and specially [50, 51]. The changed inflammatory mediator profiles, induced by administration of DSS solution, contribute to promoting the immunocyte proliferations or differentiations, disrupting the balance between the immune system and antigens, and thus ultimately render the inflammatory cascade and the destruction in the colonic tissues [52, 53]. ERCs, injected into UC paradigms, were reported to have an ability to ameliorate production and accumulation of inflammatory mediators in colons, and thus polish up the immunomodulatory properties in relieving the local tissue injury . But, whether down-regulating DKK1 expression in ERCs, could also modulate the imbalance of inflammatory mediator profiles, and exit a much more provoking effect has not been illustrated till now. Therefore, in our present study, we determined different inflammatory mediators in colonic tissues and try to address the relationship between DKK1low-ERC treatment and inflammatory mediator productions.
As shown in results (Fig. 5), we found a significant higher expression of IL-4 and IL-10 in DKK1low ERC group, both at the protein and RNA level. Correspondingly, a lower expression of IL-4 and IL-10 in the DKK1high-ERC group was witnessed, compared with that in unmodified ERC group. These changes indicated that DKK1, secreted by ERCs, could negatively modulate IL-4 and IL-10 profiles in colons. As known, IL-4 is an anti-inflammatory cytokine which was illustrated with plenty of protective effects in colons, through the IL-4/signal transducer and activator of transcription 6 (Stat6) signaling pathway . The increased IL-4 assists in inducing T helper 2 cell (Th2) responses, inhibiting Th17 cell development and polarizing macrophages toward M2 phenotype [54–56], and thus prompts its ability in maintaining the mucosal surface integrity, modulating intestinal immune responses and harboring the goblet cell functions . Similar with IL-4, IL-10 was also reported to act as an anti-inflammatory factor in the pathogenesis of UC . The expression of IL-10 was reported with the ability in suppressing the antigen presentations and the synthesis of pro-inflammatory cytokines. Meanwhile, its high expression could also inhibit T cell activation and function , and thus attenuate UC development.
IFN-γ, another pro-inflammatory factor, could exacerbate the inflammations cascade in colitis, which was enhanced by DSS administration and decreased by ERC treatment . IFN-γ has been recorded that it could cause intestinal barrier impairments and detriments in colons via pathological T cell effects . In our current study, we determined that the expression of IFN-γ was markedly decreased in DKK1low-ERC group, while raised in DKK1high-ERC group (compared with that in unmodified ERC group). It suggested that DKK1 could contribute to the ERC-mediated immune regulation by reducing the production of IFN-γ in colons.
In addition, we also measure the relevant inflammatory cytokines and inducible enzymes at the transcriptional level, which reflected the degree of local tissue inflammation. TNF-α, mainly generated by macrophages and neutrophils, is reported with overexpression in UC patients’ colons and correlated with the intensity and severity of mucosal inflammation . MPO is a significant peroxidase produced by neutrophils, and recognized as a criterion to reflect the activations of neutrophils. MPO activity could indicate the activity of inflammatory bowel disease (IBD), acclaimed by Chadwick et al . COX-2 and iNOs, which are inducible enzymes and produced by macrophages and epithelial cells, were observed accumulating in inflammation sites and involved in UC pathogenesis . In the present study, we witnessed there was a lower mRNA expression of these factors (TNF-α, IFN-γ, MPO, COX-2 and iNOs) in DKK1low-ERC group. While, when we exerted the DKK1high-ERC treatment, these pro-inflammatory mediators tended to be elevate straightly.
In addition, we also tested the superoxide dismutases (SOD) mRNA expression in different groups. SOD, which is regarded as a critical antioxidant enzyme, could catalyze the dismutation of toxic superoxides and scavenge superoxide radicals . In our present study, we found that in DKK1low-ERC group, the SOD level presented the highest level when compared with other groups. Taking together, by analyzing inflammatory cytokines and inducible enzymes changes, we believed that downregulating the DKK1 secretion of ERCs indeed alleviated inflammatory mediator expressions in the pathogenesis of UC.
Wnt/β-catenin signaling was recorded with anti-inflammatory effects in the development of ulcer colitis . As previous studies described, with the activating of canonical Wnt signaling, β-catenin would accumulate in cytoplasm and ultimately regulates the expressions of Wnt-related genes . But, in absence of Wnt-ligand stimulus, cytosolic β-catenin will be degraded after rounds of ubiquitination and phosphorylation. Thus, we measured the β-catenin expressions, both at the protein level and the RNA level, to investigate the activity of canonical Wnt pathway. As shown in Fig. 7, β-catenin production was significantly increased in DKK1low-ERC group, not only in colons but also in spleens. Intriguingly, high expression level of β-catenin was in accordance with the powerful immune regulation ability of DKK1low-ERCs exhibiting in colitis. Thus, we concluded that reducing DKK1 expression in ERCs could weaken the antagonistic effect of DKK1 on Wnt signaling, and then the Wnt β-catenin pathway could be activated and β-catenin expression would be promoted, so as to improve the immunoregulatory effect of ERCs and optimize their therapeutic effects in the process of ulcer colitis.