The results presented in this study identify a novel role of CCL25/CCR9 interactions in the pathogenesis of a well-established acute colitis model mediated by oral administration of DSS. In our study, DSS specifically targets the large intestine, but not the small intestine, and we show that CCL25 expression is upregulated after acute large intestinal inflammation. When studying CCR9−/−
mice with the DSS model, we found that the large intestinal mucosa displays characteristics of enhanced tissue inflammation resulting in exacerbated IBD symptoms and a delay in recovery from tissue injury. Thus, our findings are of high relevance for clinical care of human IBD as they point to potential detrimental effects of CCR9-blocking therapy for patients suffering from inflammation in the large intestine, as commonly observed in ulcerative colitis. This suggests that blocking CCL25/CCR9 interactions in ulcerative colitis patients may lead to an inefficiency in resolving intestinal inflammation or, at worst case, even exacerbated IBD symptoms. An oral CCR9 antagonist (CCX282-B) is currently used in a Phase II clinical trial for the treatment of Crohn's disease and has shown efficacy in the treatment of patients with moderate to severe small bowel Crohn's disease 
. This compound has shown potent inhibition of CCL25-mediated chemotaxis of CCR9-expressing cells 
. However, contradicting results have emerged in the role of CCL25/CCR9 interactions in the experimental small intestinal inflammation of the TNFΔare
mouse model that mimics human Crohn's Disease. Walters et al. showed that the orally bioavailable CCR9 antagonist CCX282-B can ameliorate, in one single experiment, the severity of small intestinal chronic inflammation of TNFΔare
. Apostolaki et al. demonstrated that the development of intestinal inflammation in the TNFΔare
mouse model is critically dependent on β7 integrin-mediated T-lymphocyte recruitment, while the function of the CCL25/CCR9 axis in T-cell specific mucosal recruitment appears dispensable in this model 
. The fact that Walters et al. did not provide the CCR9 antagonist orally but subcutaneously may explain such discrepancy. Oral administration of CCR9 antagonists should thus be evaluated in animal models of large intestinal inflammation.
In the SAMP1/Yit chronic ileitis mouse model that shares similarities with the TNFΔare
mouse model, Matsuzaki et al. showed that antibodies against the α4β7-integrin ligand MAdCAM-1 significantly inhibited adhesion of T lymphocytes to microvessels of the terminal ileum of SAMP1/Yit mice, and led to a strong chronic ileitis suppressive effect 
. Although antibody blockade of CCL25/CCR9 interactions ameliorated early, but not late, SAMP1/Yit chronic murine ileitis 
, it would be very interesting to test whether the CCR9 antagonist CCX282-B and/or CCR9-deficiency in the SAMP1/Yit ileitis model can ameliorate small intestinal chronic inflammation as well. The fact that these studies were focused on small intestinal but not large intestinal inflammation, made CCL25 and CCR9 attractive candidates for the treatment of Crohn's disease but not ulcerative colitis. However, prior to this study, no work has been presented on CCL25/CCR9 interactions in an experimental acute colitis mouse model mediated by DSS exposure.
Our data on the gastrointestinal expression pattern of CCL25 support the findings of Stenstad et al. showing expression of this chemokine in the colon 
. To date, only low expression levels at steady state were described. We show here that inflammation increases CCL25 expression in the large intestinal mucosa. It has been described that the small intestinal mucosa reacts to injection of the inflammatory cytokine TNFα with rapid upregulation of CCL25 
. Our data suggest that the colonic mucosa reacts similarly to inflammation, presumably to attract CCR9-positive leukocytes to participate in the mucosal immune response.
The most compelling argument for a physiologic role of CCL25/CCR9 interactions during an inflammatory response in the colon is derived from the increased susceptibility to colitis and the high mortality rate observed in CCR9−/− animals. Increased IBD scores, more pronounced weight loss, a delay in recovery from acute inflammation and the inability to regain initial body weight during the experimental time period support the critical role for CCL25/CCR9 dependent trafficking events during the acute and recovery phase of DSS colitis. The altered microenvironment of the large intestine appears to favor CCL25/CCR9-independent migration of pro-inflammatory leukocytes in CCR9−/− animals. This situation is highly relevant for our understanding of the therapeutic effects of CCR9-antagonists as this type of medication could result in a comparable scenario in human IBD patients.
To date, targeting of chemokine receptors such as CCR2, CCR5, CCR6, CXCR3, CXCR4 and CX3CR1 by gene disruption or antagonistic peptides has been found to protect animals from DSS colitis 
. The mechanism commonly proposed is that interference with chemokine receptor function is beneficial, because these receptors are potentially pro-inflammatory. In contrast, we show that CCR9−/−
animals respond with exacerbated IBD symptoms. To our knowledge, this is the first report to show that interference with a chemokine/receptor pair aggravates IBD symptoms. As a mechanism, we propose that CCL25/CCR9 interactions are critical components of an anti-inflammatory tissue response in the colon.
The interpretation of our results as a consequence of an impaired anti-inflammatory response in CCR9−/−
animal is supported by our data in two ways: the pronounced accumulation of activated inflammatory macrophages and the increased expression of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the large intestinal mucosa. Expression of the latter cytokines is not only described for animal models of colitis 
but also for human IBD 
, underlining the relevance of the murine model for human disease. Unlike other models of murine colitis 
, our model shows that the absence of CCR9 does not change the immune profile of the inflammatory response. Only higher levels of Th1- and Th17-type inflammatory cytokines, but no Th2-type immune responses, were detected. Additionally, high levels of IL-17A and IL-17F were detected as part of the aggravated immunophenotype of CCR9−/−
mice both at the mRNA level and the protein level. As IL-17 is considerably increased in chronic IBD patients 
, it is conceivable that the CCL25/CCR9 interactions are playing a physiological role during the development of the chronic disease symptoms as well. Melgar et al.
showed that a single cycle of high-dose DSS can induce chronic inflammation over time in wild type C57Bl/6 mice 
. Along this line, we have preliminary evidence that a single cycle of low-dose DSS is sufficient in CCR9−/−
C57Bl/6 animals to induce chronic IBD (data not shown). Further investigation will determine whether CCL25/CCR9 interactions play a role in DSS chronic colitis.
The delayed recovery from acute colitis is further characterized by an accumulation of pDCs and a change of the pDC/cDC ratio in the gut-associated lymphoid tissue in CCR9−/−
mice. pDCs have been described as immune suppressive APCs 
that can induce systemic tolerance to T cell-mediated delayed-type hypersensitivity after oral sensitization 
. pDCs are also discussed as regulators of DSS colitis 
. However, Hadeiba et al. described a significant difference in the ability of pDCs subsets to promote tolerance 
. In fact, CCR9 expression delineates a tolerogenic pDC subset while CCR9-negative pDCs were unable to prevent T cell activation/proliferation. Our results also suggest the concerted action of two functionally distinct pDCs subsets: a CCR9-positive tolerogenic pDC subset that regulates the inflammatory response during and after DSS colitis and a CCR9-negative predominantly inflammatory pDC subset. When regulatory pDCs lose CCR9 expression due to genetic manipulation, they also lose their ability to migrate in response to CCL25 upregulation in the colon and become trapped in the MLN. Alternatively, CCR9 expression by itself could influence the tolerogenic potential of these pDCs. One might even argue that the subset of CCR9-positive regulatory pDCs is underrated in WT animals, as these cells are likely to escape detection by flow cytometry once they have reached large intestinal LP. These cells likely down-regulate their surface chemokine receptors in response to CCL25. The CCR9-negative pDCs appear inflammatory in our model as demonstrated by the correlation of exacerbated IBD symptoms with the expansion of this pDC subset. In CCR9−/−
mice, the altered microenvironment might also favor CCL25/CCR9-independent tissue homing of inflammatory pDCs. Additionally, the impaired balance of DCs subpopulations may contribute to the enhanced activation of the immune system and the failure to recover from tissue injury in the absence of physiological CCL25/CCR9 interactions. It now will be important to study if the impaired ratio of inflammatory and regulatory pDCs, the expanded macrophage population or both cell types regulate the exacerbated IBD symptoms in CCR9−/−
animals. The question whether this phenotype is actually exacerbated IBD or rather an impaired ability to recover from acute large intestinal inflammation also requires further analysis.
Prevention of the CD45RBhigh
CD4+ T cell transfer colitis model in immuno-compromised animals can be achieved by cotransferring Regulatory T cells (Tregs) 
. Given the fact that CCR9-expressing pDCs are inducers of T regulatory cell functions, further examination of Tregs distribution and function would determine whether Tregs play a role in DSS colitis recovery and whether or not it is CCL25/CCR9 dependent.
In summary, our study shows that impairment of CCL25/CCR9-interaction has profound negative effects on the regulation of the local inflammatory immune response. While a phase II clinical study with a CCR9-inhibitor suggests a therapeutic benefit for Crohn's disease patients 
, our results strongly suggest that this form of therapeutic intervention could have detrimental effects when offered to patients that suffer from ulcerative large intestinal-specific inflammation.