Is Lack of “Education” a Mechanism Driving Loss of Tolerance in Crohn's Disease?

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Iliev ID, Spadoni I, Mileti E, et al. (Department of Experimental Oncology, European Institute of Oncology, Milan, Italy). Human intestinal epithelial cells promote the differentiation of tolerogenic dendritic cells. Gut 2009;58:1481–1489.

The interaction of dendritic cells (DC) with the intestinal epithelium has been talked about extensively during the last years. As the group of Maria Rescigno and others have shown, intestinal epithelial cells (IEC) may send key protolerogenic signals to local DC, thus promoting gut homeostasis and maintenance of tolerance. This phenomenon is referred to as “education” of DC by IEC, and promotes a tolerogenic phenotype of DC and their ability to drive the differentiation of regulatory T cells (T_reg). Importantly, as discussed here, a failure to receive adequate “education” may lead to lack of tolerance. This could represent yet another mechanism involved in Crohn's disease pathogenesis.

In this study, Rescigno et al show that human IEC, both Caco-2 and primary isolated, educate peripheral monocyte-derived DC to induce the differentiation of T_reg cells, supporting their hypothesis that tissue resident DC are shaped by the environment. When naïve T cells are co-cultured with IEC-conditioned DC there is an increase in FOXP3 mRNA and in the percentage of Foxp3-expressing cells. Moreover, these Foxp3⁺ cells are able to inhibit the proliferation of effector T cells, supporting the true suppressive nature of this de novo–formed T_reg cells.

To elucidate the mechanisms that IECs use to “educate” DC, the authors test the role of retinoic acid (RA), transforming growth factor (TGF)-β and thymic stromal lymphopoietin (TSLP), all of them factors that can be released by the epithelium and that, at least in murine models, have been shown to induce tolerance and/or inhibit Th1 responses (J Exp Med 2007;204:1757–1764; Nature 2005;436:1181–1185; Nat Immunol 2005;6:507–514). Indeed, neutralization of either TGF-β, RA, or TSLP leads to reduced induction of T_reg by IEC-conditioned DC. However, exogenous administration of each factor to DC could only induce Foxp3 differentiation at high, nonphysiologic concentrations.

Having described the mechanisms that educate DC to promote a tolerogenic response, the authors show that primary IECs isolated from Crohn's disease patients express significantly less mRNA for TSLP, TGF-β, and ALDH1A1 (an enzyme involved in RA metabolism). In agreement with this observation, DC conditioned with supernatants from Crohn's disease IECs have a significantly reduced ability to induce the differentiation of Foxp3⁺ cells. This, as they suggest, could represent a mechanism driving loss of tolerance and development of intestinal inflammation in Crohn's disease patients.

Recent papers in mice and humans have identified CD103⁺ (αE integrin subunit) as a marker of tolerogenic gut DC. Murine CD103⁺ DC from mesenteric lymph nodes (MLN) and small intestine have been shown to induce the expression of Foxp3 and the gut-homing receptor CCR9 on T cells (J Exp Med 2007;204:1757–1764; J Exp Med 2008;205:2139–2149). No published data are available, to the extent of our knowledge, in isolated human intestinal DC. Nonetheless, to analyze the importance of the CD103 marker on gut-derived DC in humans Iliev et al isolated CD11c⁺HLA-DR⁺lin⁻ DC from MLN and sort them into CD103⁺ and CD103⁻ cells. The authors describe several differences between the 2 populations of MLN DC: (1) CD103⁺ express higher CCR7, suggesting that these cells could represent an influx from the lamina propria to the lymph nodes; (2) CD103⁺ DC induce the differentiation of T_reg cells more efficiently than CD103⁻ DC; and (3) CD103⁺ DC present a reduced ability to differentiate T helper (Th)1 effector cells. Overall, the authors conclude, that CD103 is indeed a marker in human for tolerance-promoting DC.

Nonetheless, it remains to be assessed how DC acquire expression of CD103 in vivo. As they show in vitro, RA and Caco2 supernatant can only induce CD103 expression on monocyte-derived DC when incubated during differentiation of CD14⁺ monocytes, but had no effect on already differentiated DC.

In summary, these results show that soluble factors released by human IECs play a role in achieving the tolerogenic phenotype displayed by intestinal DC. Moreover, they provide evidence from human primary MLN DC that the specialized tolerogenic phenotype is owned by the CCR7⁺CD103⁺ population, potentially migrating from the intestine after acquiring a tolerogenic education by IEC. Importantly, the authors are not only providing evidence on the cross-talk between IEC and DC, but also pointing to deregulated signals coming from IEC as a possible mechanism driving Crohn's disease.

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Comment 

The so-called crosstalk between IEC and DC, which here is referred to as the “education” of DC by neighboring IECs, has emerged in the last 10 years as a key process in regulating intestinal homeostasis. Rescigno herself in a pioneering work (Nat Immunol 2001;2:361–367) elegantly described the close contact of the epithelium and a subset of DC in the lamina propria extending their protrusions to the luminal site and sensing bacteria. Parallel work from other labs demonstrated that DC from the gut-associated tissues are specialized in imprinting T cells with a gut-homing (CCR9⁺α4β7⁺; Nature 2003;424:88–93; J Exp Med 2003;198:963–969) and regulatory (Foxp3⁺) phenotype (J Exp Med 2007;204:1757–1764; J Exp Med 2007;204:1765–1774; J Exp Med 2007;204:1775–1785). More recently, these 2 observations were put together and it was demonstrated both in mice and humans that IEC indeed participate in “educating” neighboring DC with a tolerogenic phenotype (Mucosal Immunol 2009;2:340–350; Gut 2009;58:1481–1489).

The mechanisms involved in transmission of such tolerogenic phenotype by IECs include production of TSLP, RA, and TGF-β. Eliminating either one of these from the supernatant of cultured IECs negatively affects education of DC; however, none of these factors on its own (unless at high and nonphysiologic concentrations) can induce a tolerogenic state on DC. This suggests that synergistic effects may take place (although the authors do not explore this possibility), or even that alternative, still undescribed, IEC-derived factors are playing a role. Overall, despite the fact that some questions remain unanswered, this recent work contributes to further our understanding of IEC-DC crosstalk in the context of the intestinal mucosa and, even more important, it points toward a new mechanism driving loss of tolerance and intestinal inflammation in Crohn's disease patients.

An earlier report by the same group demonstrated that IEC from Crohn's patients produce lower amounts of TSLP (Nat Immunol 2005;6:507–514). In the present paper, they also show a defect in expression of TGFB1 and ALDH1A1 genes, as well as TSLP, in noninvolved areas of the mucosa. In agreement with this observation, IEC supernatants from these patients are much less efficient in driving the differentiation of Foxp3⁺ T_reg cells compared with that of healthy individuals. Although these are important novel observations, the question of their physiologic relevance remains unanswered. An attractive hypothesis, as suggested by Iliev et al is that the epithelial cells in the intestinal mucosa of Crohn's disease patients would not sufficiently “educate” DC to drive tolerogenic responses as a result of this defect. Because the IEC in this study were obtained from the noninvolved mucosa of patients this may represent an innate defect, and not the result of active ongoing inflammation. If that is the case, one would expect to detect lower number and/or impaired function of Foxp3⁺ T_reg in the uninvolved intestinal mucosa of Crohn's disease patients. The gut is a well-known site of T_reg differentiation outside the thymus, and the implications of defective T_reg numbers and/or function on intestinal homeostasis are well-described in mice and to a lesser extent in humans (Annu Rev Immunol 2009;27:313–338). Therefore, it makes sense to assume that impaired T_reg differentiation in the gut would negatively affect intestinal homeostasis and foster development of intestinal inflammation. Nonetheless, studies addressing this issue in humans are still inconclusive. Whereas an increase in the number of Foxp3⁺ cells has been reported in the inflamed mucosa of IBD patients, no convincing data exist on Foxp3⁺ CD4⁺ cell numbers in quiescent or noninvolved areas of these patients (Annu Rev Immunol 2009;27:313–338). However, a decrease in Foxp3⁺ regulatory CD8⁺ cells has been reported in the intestinal mucosa of Crohn's disease patients and it has been linked to a defective IEC function (J Immunol 2005;174:5814–5822). Therefore, an interesting possibility that needs to be considered is that, owing to an innate defect in IEC, there is a decrease in the number and function of Foxp3⁺ cells present in the intestine, which could constitute a mechanism of deregulated immune responses to commensal flora.

The hypothesis the authors propose may have potential therapeutic implications and it would support strategies that specifically promote development of tolerance in the gut, by either increasing the production or signaling of factors such as TGF-β and TSLP or by directly administering tolerogenic DCs to these patients. TGF-β is a pleiotropic cytokine with powerful functions in both tolerance and immunity. It can be produced by an array of different cells in the intestinal mucosa with strictly regulated synthesis and activation. As this paper shows, TGF-β produced by the epithelium acts on DC and induces phosphorylation of SMAD2 that in turn activates the production of TGF-β by DC themselves. Other cells, such as T_reg and fibroblasts can also produce this cytokine under certain conditions. Despite its well-known protolerogenic, regulatory, and regenerating functions, TGF-β, whose expression is highly up-regulated during intestinal inflammation, is a key factor mediating colorectal cancer development. Therefore, strategies that up-regulate the release or signaling of TGF-β have to be looked at cautiously. On the other hand, TSLP seems like a more feasible target. TSLP is known to induce T_reg cells in the thymus through its action on DCs (Nature 2005;436:1181–1185) and to reduce Th1 responses (Nat Immunol 2005;6:507–514). In the intestine under steady-state conditions, TSLP produced by IEC influences DC to prime Th2 responses and as shown here it may play a role in educating human DC to drive T_reg differentiation. However, a lot remains to be resolved on the role of this cytokine in mucosal homeostasis. As discussed, the addition of high, nonphysiologic, doses of TSLP is necessary to induce T_reg conversion and studies in mice show that TSLPR^−/−DC have the same ability as wt DC to drive T_reg differentiation in response to IEC supernatant conditioning. Moreover, no information is available on how TSLP production by IEC or other stromal cells is regulated during intestinal inflammation.

Finally, an important issue that remains unresolved is the role of CD103⁺ DC in human intestinal mucosa and how it is affected by disease. Iliev et al suggest that the tolerogenic CD103⁺ CCR7⁺ population in MLN may well represent a migrating subset originated in the gut; however, direct evidence of this would be very hard to obtain in humans. In mice, different populations of DC and macrophages have been described in the lamina propria including CD103⁺ tolerogenic DC, interdigitating CX3CR1⁺ cells and CD11b⁺F4/80⁺CD11c⁻ macrophages (Nat Immunol 2007;8:1086–1094). According to Iliev et al, tolerogenic DC are irreversibly differentiated and would not respond to pathogenic bacteria or inflammatory stimuli. In a previous report, Rescigno's laboratory had already demonstrated that challenge of “educated” DC with a pro-Th1 stimulus like S typhimurium, does not trigger the expected pro-inflammatory response (Nat Immunol 2005;6:507–514). They speculate that the responsibility of generating an efficient immune response when a pathogen or another foreign harmful antigen is present falls on newly recruited monocytes or DC. The authors propose a population of noneducated CCR6⁺DC from Peyer's patches that could act as a nearby and readily to act population (J Exp Med 2007;204:2253–2257). In that regard, studies in humans have identified CD14⁺ cells expressing macrophages in colonic samples of Crohn's disease patients. This population, almost inexistent in the colon of healthy individuals, releases high amounts of interleukin-23 and tumor necrosis factor-α that in turn promote the expansion of interferon-γ–producing T cells (J Clin Invest 2008;118:2269–2280), and could represent “noneducated” DC subsets with higher capacity to prime Th1 responses. Therefore, apart from the role of protolerogenic DC in the gut inducing T_reg differentiation and preventing lose of tolerance, newly recruited noneducated DC and/or macrophages in response to infection or other stimuli may also play a role in development of Crohn's disease. Also important, the stability of tolerogenic DC when not in the presence of a protolerogenic environment or in response to activation is relevant to the use of tolerogenic DC as therapy in clinically active Crohn's disease patients.

Overall, the line of research discussed herein has the potential to open new avenues of therapeutic intervention and once again points to defects in epithelial cell function as potentially responsible to drive intestinal inflammation. Little by little, contributions like this help to unravel the many and finely regulated cellular interactions that take place in the intestinal mucosa immune system.