In this report, we demonstrate multiple genetic deficiencies in immune regulation result in fulminant ulcerative colitis that is distinct from and more severe than the disease induced by either of the single deficiencies alone. In addition to documenting these pathologic findings, we further show that disease in dnKO mice is characterized by elevated proinflammatory cytokines, can be induced in recipient animals by transfer of dnKO CD4+ T cells, is responsive to combined neutralization of IFNγ and TNFα, and can be completely inhibited through treatment with high-dose oral ciprofloxacin and metronidazole.
Previous work has shown that the inhibitory cytokines IL-10 and TGFβ, both known to control immune homeostasis, are associated with protection from colitis in murine models of IBD [7
]. We crossed dnTGFβRII mice, which have significantly attenuated TGFβRII signaling in T cells, with IL-10R2−/−
mice. The resulting dnKO mice showed a highly reproducible, diffuse, and severe ulcerative colitis that occurred in a predictable time frame. At the same time point at which dnKO mice developed severe disease and died, all controls including WT, dnTGFβRII, and IL-10R2−/−
mice demonstrated little to no pathology. Similarly, work in IL-10−/−
mice has demonstrated differential colitis severity in a C3H/HeJBir versus a C57BL/6J background and linked the differences to a region on Chromosome 3, referred to as the cytokine deficiency-induced colitis susceptibility 1 (Cdcs1) [10
]. These findings all support the idea that IBD is a complex disease influenced by multiple interacting host genetic factors.
Because both dnTGFβRII and IL-10R2 signaling are important for various aspects of immune regulation, the disease observed in dnKO mice was likely due to a culmination of several dysregulated parameters. The recent generation of T cell specific TGFβRII knockout mice revealed that this defect alone was sufficient to induce rapid, multiorgan autoimmunity. These mice demonstrated alterations in Th1 T cell differentiation, NKT cell development, and impaired maintenance, but not generation, of Foxp3 regulatory T cells [36
]. Similarly, in dnKO mice, regulatory T cells were present at similar percentages to controls, suggesting that regulatory T cell development was also intact in these mice. Furthermore, dnKO T regulatory cells were able to inhibit naive T cell proliferation in vitro, suggesting that they still are able to function in this context. Regulation of a T cell transfer model of colitis has previously been demonstrated to depend on TGFβ responsiveness by pathogenic naive T cells [31
], emphasizing the importance of this cytokine in disease inhibition. We showed that CD4+
T cells from dnTGFβRII mice could also transfer disease, but found that the additional loss of IL-10R2 signaling increased the severity of disease above that conferred by cells in which only TGFβRII signaling was disrupted. These studies, however, did not address whether the loss of IL-10R2 and TGFβRII signaling caused dysregulation of the naive or regulatory T cell subsets found within the CD4+
In addition to impaired TGFβRII signaling, IL-10R2 signaling was completely absent in dnKO mice. As a result, these mice were unresponsive to IL-10, which normally inhibits antigen-presenting cell function and may play a role in converting naive CD4+
T cells to those with regulatory potential [12
]. The importance of IL-10 in regulation of colitis was initially demonstrated in IL-10−/−
mice that developed spontaneous enterocolitis marked by crypt abnormalities, epithelial hyperplasia, and leukocytic infiltrates [7
]. The generation of IL-10R2−/−
mice recapitulated data from IL-10−/−
mice in that they also spontaneously developed colitis marked by epithelial hyperplasia and leukocytic infiltrates. Interestingly, IL-10 has also been implicated in contributing to epithelial barrier integrity [43
], which would be a critical aspect in limiting intestinal mucosal inflammation.
Because the IL-10R2 is also a redundant receptor for other cytokines, including IL-22, IL-26, IL-28, and IL-29 [44
], the effects of these cytokines on colitis development in dnKO mice could not be ruled out. While little is known about the relevance of most of these cytokines in the context of intestinal immunity, increased levels of IL-22 have been noted in human IBD patients, and IL-22 has also been demonstrated to induce IL-10 expression by colon epithelial cell lines [45
]. However, abrogation of signaling in these cells may be beneficial, as IL-22 has also been shown to induce production of proinflammatory cytokines by colonic subepithelial myofibroblasts and intestinal epithelial cells [45
]. Additionally, previous characterization of IL-10R2−/−
mice did not reveal an increased disease severity compared to what was described for IL-10−/−
]. With the given caveat that there may have been differences in enteric flora, the data from these studies suggest that IL-10, but not the other redundant IL-10R2 cytokines, plays the predominant role in colitis induction and exacerbation.
In addition to potential alterations in APC function, epithelial barrier integrity, and T regulatory-mediated suppression, a major function attributed to both IL-10 and TGFβ is the inhibition of proinflammatory cytokine production [12
]. Excessive production of proinflammatory cytokines has been implicated as a potential factor in human IBD [24
]. For example, CD4+
lamina propria cells from patients with Crohn's disease produced increased levels of IFNγ and TNFα [24
]. Furthermore, neutralization of TNFα via administration of infliximab resulted in clinical benefits for IBD patients and a variety of other such biologic agents are in various stages of development and clinical testing [48
]. Studies in murine colitis models have also shown reduced pathology upon neutralization of IFNγ or TNFα [49
]. In dnKO mice, impairment or loss of both TGFβRII and IL-10R2 signaling resulted in intestinal pathology that was associated with dramatic increases in both T cell activation and IFNγ, TNFα, and IL-6 levels. Depletion of IFNγ and TNFα decreased the extent of pathology seen in 4-wk-old dnKO mice as compared to controls. While anti-IFNγ treatment alone partially ameliorated disease, anti-TNFα treatment alone had little to no impact on intestinal pathology. Interestingly, significant reductions in epithelial hyperplasia were only detected when the two treatments were combined, suggesting that in the dnKO model these two cytokines have redundant roles in inducing hyperplasia. Our data therefore suggest that treatment regimens that effectively neutralize multiple proinflammatory cytokines could result in more successful clinical responses.
In recent years the role of IL-12 versus IL-23 in chronic inflammation has been reevaluated upon the realization that both cytokines share the IL-12p40 subunit [53
]. In both human trials and murine models of colitis, neutralization of the IL-12p40 subunit resulted in decreased pathology [56
]. Recently, a mutation in the IL-23R has been negatively linked with human IBD, suggesting that reduced signaling through this receptor protects against disease [58
]. Additionally, a study by Yen et al. has indicated a role for IL-23, rather than IL-12, in the induction of colitis due to its ability to promote IL-17 production [59
]. However, the proinflammatory effects of IL-23 are not necessarily restricted to its effects on IL-17 producing cells as IL-23 can increase proliferation and IFNγ production by effector/memory T cells [55
] and has been demonstrated to also increase Th1 responses in colitis models [60
]. Because IL-17 secretion was detected in the supernatants of colon explants (unpublished data) from dnKO mice, albeit at lower levels compared to IFNγ and TNFα production, IL-17 and/or IL-23 may account for the residual pathology observed in dnKO mice after neutralization of both IFNγ and TNFα. Regardless, results from our study and others would suggest that large perturbations in immune homeostasis through production of excessive Th1, Th17, or Th2 cytokines all have the ability to cause intestinal pathology.
Several overlapping lines of evidence, primarily from murine models, point to involvement of enteric bacteria in inducing IBD (reviewed in [28
]). For example, intestinal pathology in a number of models including IL-10−/−
mice was diminished in germ-free settings [62
]. Similarly, colonic inflammation due to DSS-induced injury has been shown to be reduced in germ-free and antibiotic-treated mice [22
]. Experiments employing antibiotics to treat murine models of spontaneous colitis have demonstrated variable degrees of success depending on the animal model and the antibiotics used (e.g., [64
]). We chose to employ two orally delivered antibiotics at high dosages: ciprofloxacin, which broadly targets aerobic bacteria, and metronidazole, which is active against anaerobic organisms. Antibiotic treatment resulted in complete inhibition of disease in dnKO mice as demonstrated by survival, weight gain equivalent to controls, absence of macroscopic and microscopic signs of colitis, and near-complete abrogation of proinflammatory cytokine production. These findings dramatically demonstrate that intestinal microbes are required for disease induction in this model of ulcerative colitis.
As discussed previously, current efforts at developing treatments for various forms of IBD, particularly ulcerative colitis, have focused extensively on devising costly new biologic agents, many of which act by specifically targeting cytokines including TNFα and IFNγ [48
]. Our results demonstrate that neutralization of these cytokines, particularly in combination, also produced significant amelioration of colitis in dnKO mice. Although antibiotics, including ciprofloxacin and metronidazole, are sometimes used to treat patients with IBD, research regarding their efficacy remains inadequate. Current clinical consensus holds that they are useful in managing septic complications of IBD and certain aspects of Crohn's disease. However, their utility in ulcerative colitis has not been adequately assessed [30
], and recommendations vary regarding usage in patients with fulminant ulcerative colitis [48
]. We know of no large, blinded, controlled clinical studies examining combined metronidazole and ciprofloxacin treatment in ulcerative colitis. Our results suggest that carefully controlled studies examining the benefits of combined ciprofloxacin and metronidazole in ulcerative colitis may be appropriate.
Overall, our studies support the hypothesis that multiple genetic hits to immune regulation have the ability to produce the most severe forms of IBD. While impairment of either TGFβRII or IL-10R2 signaling alone was sufficient to cause some aspects of disease, the extent of pathology was significantly increased once these deficiencies were compounded. Our data demonstrate that the mechanism was at least in part due to uncontrolled T cell activation resulting in increased proinflammatory cytokine production. We further show that the inciting stimulus for this immune activation is microbial in origin. We propose that this model will be useful in the future to evaluate the mechanism of loss/attenuation of specific mucosal barrier components (e.g., goblet cells, surface enterocyte maturation, and epithelial stem cells) that also occurs in human ulcerative colitis, to evaluate combinatorial therapies directed against the numerous proinflammatory cytokines that are elicited by disregulated leukocytes, and to elucidate the mechanisms by which enteric microbes trigger disease.