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- MATERIALS AND METHODS
Background: Factors determining the extension and degree of inflammation in the colonic mucosa of patients with ulcerative colitis (UC) are largely unknown, but CD4+CD25high regulatory T cells (Tregs) have been implicated to play an important role in suppressing inflammation. Therefore, the aims of this study were to determine whether colonic Tregs have suppressive effects on colonic effector T cells in UC and to analyze the association between segmental colonic Treg distribution and disease activity. Materials and Methods: The suppressive activity of colonic CD4+CD25high Tregs from patients with active UC was determined in coculture assays measuring proliferation and cytokine production. The frequency of Tregs and the expression of the Treg marker FOXP3 were analyzed with flow cytometry and RT-PCR in isolated cells and the whole mucosa from patients with active and inactive disease, as well as healthy mucosa. Results: Colonic CD4+CD25high T cells from patients with UC suppressed the proliferation and cytokine secretion of colonic effector CD4+ T cells. Healthy controls but not patients with UC had lower Treg frequencies in the sigmoid than in the ascending colon. Patients with UC with active disease had increased frequency of colonic Tregs. The frequency of Tregs was positively correlated with colonic disease activity and serum C-reactive protein. Conclusions: Colonic CD4+CD25high Tregs are able to suppress colonic effector T cell activity in vitro, and the Treg frequency in the inflamed intestine increases with disease activity in patients with active UC. This suggests that Tregs may be outnumbered by other inflammatory cells or that their suppressive activity may be influenced by the in vivo environment.
The extension of ulcerative colitis (UC) is variable; it can be confined to the rectum (proctitis), can involve the colonic mucosa up to the left flexure (proctosigmoiditis), or can extend above the left flexure (extensive colitis). Colitis confined to the distal colon only is characterized by a milder clinical course, and the patients generally are more responsive to therapy than patients with extensive colitis.1 The factors determining the extension and degree of activity of the mucosal inflammation are largely unknown.
Studies in mouse models of experimental colitis have demonstrated that intestinal inflammation not only can be prevented but also can be cured by the presence of CD4+ CD25+ T cells with regulatory properties in the gut mucosa.2–4 Recent reports also have described the occurrence of CD4+ T cells with the phenotype of regulatory cells expressing high levels of CD25 (CD4+CD25high T cells) in the colonic mucosa of healthy individuals and patients with inflammatory bowel disease (IBD).5,6 The CD4+CD25high T cells from inflamed human mucosa maintain the regulatory phenotype after propagation in vitro.7 Freshly isolated and propagated colonic CD4+CD25high T cells from patients with IBD also have been shown to suppress the activity of autologous blood effector cells.5,7 However, although previous studies indicate that colonic effector T cells from patients with IBD are nonresponsive to T cell-mediated suppression,8–10 it is also important to investigate whether colonic effector T cells can be suppressed by CD4+CD25high T cells.
The suppressive effects of CD4+CD25high regulatory T cells (Tregs) are most likely dependent on cell-cell contact, although the mechanisms by which Tregs suppress naïve and effector T cells activity are so far inadequately understood. It has been shown that Tregs need antigen-specific or polyclonal activation via the T cell receptor to exert suppressive activity, at least in vitro.11,12 Recent studies have shown that the FOXP3 gene is important in the development and function13 of CD4+CD25high T cells in both humans and mice14 and that a defective FOXP3 expression generates strong activation of the immune system, resulting in multiorgan autoimmune diseases, including chronic intestinal inflammation.15,16 Furthermore, CD4+CD25high T cells have increased intracellular expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4),17,18 which is a negative regulator of T cell activity.
Even though it has previously been shown that naturally occurring CD4+CD25high T cells are present in the colonic mucosa of patients with IBD, many questions remain concerning the function of the CD4+CD25high T cells and the colonic distribution of the cells in these patients. We therefore investigated the ability of colonic CD4+CD25high T cells from patients with UC to suppress colonic and blood effector/naïve T cells. Furthermore, we examined the segmental distribution of CD4+CD25high T cells and studied the relationship between colonic CD4+CD25high T cell frequency and colonic/systemic disease activity in patients with UC in the exacerbation phase.
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- MATERIALS AND METHODS
The presence of CD4+CD25+ Tregs has been reported to prevent the outbreak of colitis and to cure established intestinal inflammation in experimental animal models of IBD.2–4 Despite the promising data of the putative therapeutic effects of Tregs obtained from such experimental colitis models, the role of Tregs in human IBD is largely unknown. Recently, Makita et al5 demonstrated that colonic Tregs from patients with IBD can suppress the activity of blood effector T cells. However, the important question of whether lamina propria T cells from the inflamed colons of patients with IBD are responsive to Treg-mediated suppression has not previously been addressed. Therefore, in the present study, we analyzed whether colonic effector T cells can be suppressed by colonic Tregs from patients with UC. We found that colonic Tregs are indeed able to suppress both the proliferation and the cytokine production of colonic effector T cells. Thus, our results indicate that Tregs isolated from the site of active inflammation retain their suppressive activity and that effector T cells from inflamed tissues are able to respond to the suppression.
Our study also shows that the patients with UCa have higher frequencies of colonic Tregs relative to patients with UCr and healthy control subjects. Our data are compatible with the work recently presented by Makita et al,5 who reported increased ratios of cells with a Treg phenotype in inflamed lesions of active patients with IBD compared with noninflamed mucosa of the healthy individuals. In addition, we found a strong association between amplified local and systemic activity of the disease and the presence of colonic Treg frequencies. The reason for the insufficient suppression of the intestinal inflammation despite the accumulation of an apparently suppressive colonic Treg pool in patients with UC remains unclear. However, the regulatory effect of the colonic Treg population on colonic effector T cells from patients with UC shown in our study was obtained with high suppressor-to-responder ratios (1:1 and 1:2), and the suppressive effect was lost at lower ratios. In vivo, even higher proportions of Tregs may be required to fully suppress inflammation because cell-to-cell contact is likely to be important for suppression. The beneficial effects of Tregs seen in the murine colitis settings also were obtained by administrating excessive numbers of Tregs.22–24 Thus, although Tregs accumulate in the intestinal mucosa of patients with IBD, the frequency may not be enough to restrain the intestinal inflammation. In support of this hypothesis, Maul et al6 found that patients with diverticulitis harbored significantly increased frequencies of cells with a Treg phenotype in inflamed lesions compared with patients with active IBD.
We also analyzed Treg frequencies in the blood of the different study groups. Consistent with the results presented by Maul et al,6 we found decreased frequencies of blood Treg in patients with UCa relative to patients with UCr. This supports the notion that during exacerbation, Tregs are recruited from the blood to the inflamed tissue. We also found that the T cell receptors of colonic and blood Tregs have similar polyclonal Vβ T cell receptor repertoires. Thus, there is an accumulation of Tregs with a polyclonal Vβ T cell receptor repertoire analogous to that of blood Tregs in the colonic mucosa of patients with UCa. This indicates that the increase in colonic Tregs in inflamed tissue is caused by recruitment from the peripheral Treg pool, not by local expansion of antigen-specific clones in the gut.
We showed that most individuals with healthy colonic mucosa have lower Treg frequencies in the sigmoid compared with the ascending colon. The finding of lower Treg frequencies, and thus a reduced ability to suppress effector T cells responses, at the left side of the colon of healthy individuals may explain at least in part why distal inflammation is typical at the onset of UC. The normal segmental variation of colonic Treg frequencies was lost in patients with established UC, suggesting that more Tregs are recruited to the site at the onset of colitis but that it is not enough to stop disease progression.
Our finding of an accumulation of Tregs able to suppress colonic effector T cells at the site of inflammation is consistent with studies of Tregs in patients with rheumatoid arthritis. Several groups have demonstrated Treg suppression of effector T cells isolated from inflamed joints, and patients with rheumatoid arthritis have increased frequencies of Tregs in inflamed joints relative to blood.25–28 Both UC and rheumatoid arthritis are remitting and relapsing chronic inflammatory disorders that afflict patients from the time of onset throughout the lifespan. Both diseases are characterized by tissue destruction and accumulation of leukocytes in the inflamed organ. Furthermore, arthritis is recognized as an extraintestinal manifestation of patients with UC. It is therefore intriguing to note that these immunologically closely related diseases share the feature of having functional Treg populations but still have insufficient regulation of T cell activity in the inflamed areas.
It cannot be entirely excluded that the suppressive activity of Tregs and their ability to regulate other cells may be influenced by factors present in the local in situ environment that are lacking in the in vitro culture of separated cells. For example, Toll-like receptor 2 (TLR2) triggering, together with T cell receptor signaling, results in a temporal loss of the suppressive Treg capacity that is regained after removal of the TLR2 trigger,29 and TLR8 ligation reverses the suppressive function of Tregs.30 Furthermore, studies have demonstrated that IL-6 produced by TLR-activated dendritic cells renders antigen-specific T cells nonreceptive to Treg suppression.31 However, lipopolysaccharide and flagellin signaling via TLR4 and TLR5, respectively, potently increased the suppressive capacity and enhanced FOXP3 expression of CD4+CD25+ Tregs.32,33 Thus, it is clear that further studies are needed to fully understand the role of factors influencing the capacity of Tregs to control inflammation in vivo.
In conclusion, this article demonstrates that colonic Tregs from patients with UC have a suppressive effect on colonic effector T cells in vitro. However, the frequency of colonic Tregs increases with colonic and systemic disease activity, suggesting that Tregs may be outnumbered by other inflammatory cells or that their suppressive activity may be influenced by the in vivo environment. Nevertheless, our results demonstrate that colonic Tregs from patients with colitis have the potential to downregulate T cell-mediated inflammation, given the right stimuli. Thus, Tregs are important targets for future immunotherapy against colitis, and future trials may be designed to investigate whether transfer of in vitro-propagated Tregs to patients with colitis or enhanced in vivo recruitment, expansion, and/or activation of Tregs may be able to control mucosal inflammation.