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Keywords:

  • colitis;
  • genetically modified mice;
  • immunoregulatory molecules

Abstract

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES

Abnormal response to enteric microflora is a critical factor driving bowel inflammation in patients with inflammatory bowel disease (IBD). Mice with genetically engineered mutations have played a central role in both formulating this hypothesis and elucidating the mechanism that normally protect the host from excessive inflammation within the bowel. One emerging theme is the importance of regulation within the innate immune system in protecting from microflora-driven pathology. In this review, I describe how genetically engineered mice have played a crucial role in shaping our conceptual understanding of pathways that regulate the development of chronic bowel inflammation, and furthermore, explore data derived from the study of genetically engineered mice that implicates the fundamental importance of regulation within the innate immune system in the control of this process.

(Inflamm Bowel Dis 2007)


MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES

In 1993 there were stunning back-to-back reports in the journal Cell describing the development of colitis in mice that lacked key players in the immune response, including IL-2, IL-10, class II major histocompatibility complex, and the α and β chains of the T-cell receptor1–3 (Table 1). Interestingly, while disease in these models was observed in mice housed in conventional facilities, it was reported that IL-2-deficient mice housed in a germ-free facility did not develop the clinical or histological signs of colitis. This effect of germ-free environment on the development of colitis in IL-2-deficient mice has been confirmed in additional studies.4, 5 Likewise, IL-10-deficient mice that were housed in specific pathogen-free (SPF) conditions developed less severe disease than mice housed in conventional animals facilities.2 Furthermore, a later study indicated that IL-10-deficient mice housed in germ-free conditions did not develop clinical or histological signs of colitis for up to at least 6 months of age.6 These studies clearly suggested that defects in immune regulation may be central to the development of IBD, and furthermore that microflora likely have a critical role in stimulating the abnormal inflammatory response in susceptible hosts. These insights have been verified in many models and unraveling the details of these observations has been one of the main focuses of this field for the past decade.

Table 1. Immune Models Used in the Study of Colitis
StrainMajor Immune Defect
IL-2-deficientDefect in the development of thymically derived regulatory T cells
IL-10-deficientDefect in the function of thymically derived regulatory T cells
scidMarked reduction in mature T and B cells
RAG-deficientComplete absence of mature T and B cells
TCRα-deficientAberrant development of mature αβ T cells

INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES

While the experiments described above suggest that microflora play a critical role in the development of experimental colitis in susceptible hosts, the exact nature of the organisms responsible for the development of colitis is controversial and has remained somewhat elusive. Colitis in many susceptible models has been strongly associated with colonization of the cecum, colon, and biliary tract with several different Helicobacter species.7–10 One of these enterohepatic species, H. hepaticus, was originally isolated from A/J mice that were noted to have a transmissible chronic active hepatitis.11Helicobacter hepaticus was found to be present within the normal flora of many conventional mouse facilities12 and its presence was strongly correlated with the development of colitis in a number of mouse models. Targeted infection experiments demonstrated that H. hepaticus or related species can induce colitis in scid, IL-10-deficient, as well as TCRα-deficient mice.13–16 RAG-deficient (RAG) mice nursed by H. hepaticus-positive foster mothers developed colitis while littermates raised by H. hepaticus-negative foster mothers did not.17 However, it has been demonstrated that enterohepatic Helicobacter species are not required for the development of colitis in susceptible hosts. Germ-free IL-10-deficient mice rapidly developed colitis when transferred to an SPF environment free of known Helicobacter species.6, 18 Furthermore, while mono-association of germ-free A/J mice with H. hepaticus induced mild colitis 33 weeks to 2 years after infection,19 there was minimal colitis observed for up to 16 weeks in germ-free IL-10-deficient mice infected with H. hepaticus,18 despite the rapid development of colitis in a control group of germ-free IL10-deficient mice infected with H. hepaticus-free SPF flora. In these studies, persistent H. hepaticus colonization was demonstrated in mice inoculated with H. hepaticus, despite the absence of disease. Finally, several more recent studies have demonstrated that mono-association of germ-free IL-10-deficient mice with Enterococcus faecelis20, 21 was able to induce moderately severe distal colitis, while in 1 of these studies (but not the other) E. coli was able to induce moderate disease in the more proximal bowel.21 These studies demonstrate that while specific flora can clearly have a role in the induction and manifestations of disease, the ability of microflora to induce inflammation in susceptible hosts is not limited to a single family of bacteria. Identifying the mechanisms through which microflora induce inflammation in susceptible hosts remains an important challenge for this field.

ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES

Experiments examining the ability of subpopulations of T cells to induce or inhibit the development of colitis have provided a framework with which to decipher the mechanisms that regulate microflora-induced colitis (Table 2). Powrie and others demonstrated that CD4+ T cells expressing high levels of CD45RB have the ability to induce colitis after adoptive transfer into lymphocyte deficient hosts.22–24 In contrast, it was shown that colitis induced by the adoptive transfer of CD4+ CD45RBhigh T cells can be inhibited by the concomitant transfer of CD4+ T cells that express low levels of CD45RB.24 These cells are referred to as regulatory T cells or Tregs, and a highly overlapping population is defined by cell surface expression of CD25.25, 26 These CD4+ CD25+ T regulatory cells have also been shown to express the transcription factor Foxp3.27, 28 With this framework in place, it has been possible to ask detailed questions regarding the factors that facilitate T-cell-induced pathology in the bowel, as well as the factors that normally prevent this pathogenic process.

Table 2. T-Cell Subsets Used in Models of Murine Colitis
DescriptionCell Surface MarkersFunction
Naive peripheral T cellCD4+CD45RBhighInduce colitis upon transfer into lymphocyte deficient hosts
Thymically derived regulatory T cellCD4+CD45RBlowCD25+1. Inhibits colitis induced by transfer of CD4+CD45RBhigh cells into lymphocyte-deficient hosts
  2. Inhibits colitis induced by H. hepaticus infection of lymphocyte-deficient hosts

CD4+ CD45RBhigh T cells are predominantly naïve thymically derived T cells that differentiate into colitigenic effector cells after transfer into lymphocyte-deficient hosts. It appears that these effector cells are responding to bacterial antigens rather than endogenous or food-related antigen. Germ-free scid mice are resistant to the development of colitis after adoptive transfer of CD4+ CD45RBhigh cells.29, 30 In addition, H. hepaticus infected scid mice developed more severe colitis after adoptive transfer of CD4+ CD45RBhigh cells than noninfected scid mice.16 Furthermore, in a substrain of C3H/HeJ mice (C3H/HeJBir) that develop spontaneous colitis, peripheral T cells could be induced to proliferate when stimulated with antigen-presenting cells (APCs) pulsed with a lysate of cecal bacteria antigens, but not when stimulated with APCs stimulated with lysates of syngeneic mouse epithelial lines or food antigen.31 When CD4+ T cells from C3H/HeJBir mice were activated by cecal bacterial antigens they were able to induce colitis when transferred into scid recipients, while CD4+ T cells from C3H/HeJBir mice activated by anti-CD3 were not.31 Taken together, these data strongly suggest that effector CD4+ T cells are activated to induce colitis in response to bacterial antigens.

Initial studies strongly suggested that the pathogenic T-cell response involved in mediating bacterially driven colitis was predominantly Th1-like in nature (Table 3). CD4+ T cells isolated from the colon of scid mice that received CD4+ CD45RBhigh T cells produce high levels of IFN-γ, but little IL-4 or IL-10 after stimulation in vitro with anti-CD3, and in this model mice were partially protected from weight loss by anti-IFN-γ or anti-tumor necrosis factor (TNF) antibody.32Helicobacter hepaticus-induced intestinal pathology in IL-10-deficient mice is reduced by treatment with anti-IL-12 and anti-IFN-γ antibody.13 Furthermore, STAT-4-deficient CD4+ CD45RBhigh T cells lacking the ability to respond to IL-12 induce much reduced disease compared to wildtype (WT) CD4+ CD45RBhigh T cells after adoptive transfer into RAG mice.33 While these studies strongly implicate a Th1-like response in disease development, there is evidence that Th2 cells are also capable of mediating colitis in response to microbial antigens. Transfer of DO11.10 RAG2−/− ovalbumin (OVA)-specific T cells polarized either in the Th1 direction or Th2 direction in vitro were able to induce colitis after adoptive transfer into RAG hosts that were colonized with OVA-expressing E. coli.34 Disease was dependent on bacterially expressed OVA, and while there was some difference in pathological manifestations, overall severity was very similar between groups that received Th1 and Th2 cells.34

Table 3. Effector T-Cell Lineages Implicated in the Development of Microflora-driven Colitis
LineageHallmarkInducer
  • a

    IL-23 is not strictly required for differentiation of Th17 cells but may be necessary for induction of high-level IL-17 secretion.

Th1Ability to secrete IFN-γIL-12 p70
Th2Ability to secrete IL-4, 5, 13IL-4
Th17Ability to secrete IL-17IL-6, TGF-β, IL-23a

In addition to Th1 and Th2 effector lineages, a third effector CD4 cell lineage characterized by expression of IL-17 has recently been characterized.35 Differentiation of these Th17 cells from naïve precursors appears to depend on TGF-β and IL-6.36–38 Interestingly, it has been reported that IL-23, a dimer of IL-12 p40 and p19, strongly enhances the effector function of Th17 cells.39 As IL-23 shares the p40 subunit with IL-12 p70 (a dimer of p40 and p35) it was recognized that experiments using anti-p40 antibodies or p40-deficient mice would have interfered with the function of IL-23, as well as IL-12 p70, and therefore IL-23 could be responsible for some activities previously attributed to IL-12 p70. A series of recent articles have addressed these issues. Mice doubly deficient for IL-10 and p19 (thus lacking IL-23) were protected from the development of spontaneous colitis observed in mice lacking IL-10 alone, while mice doubly deficient in IL-10 and p35 (thus lacking IL12-p70) were not.40 Mice lacking IL-10 alone expressed higher levels of IL-17 mRNA in the colon than mice lacking both IL-10 and p19, and also demonstrated a higher percentage of IL-17 expressing memory T cells. In addition, it has been demonstrated that the ability of CD4+ CD45RBhigh T cells to induce colitis in RAG mice is compromised in the absence of p19 but unaffected by the absence of p35, implicating IL-23 but not IL-12 p70 in the development of colitis.41 Consistent with this observation, mice lacking the IL-17 receptor were noted to be resistant to acute trinitrobenzene sulfonic acid (TNBS)-induced colitis.42 IL-23 was also required for the induction of mucosal pathology in RAG mice treated with antibody directed at CD40 ligand. However, disease in this model was shown to be independent of microflora.43 These studies strongly suggest that the IL-23/IL-17 axis plays a critical role in driving mucosal inflammation. In contrast, it has been reported that mice lacking p19 develop more severe pathology in models of dextran sulfate sodium (DSS) and TNBS-induced colitis, possibly as the result of increased expression of IL-12 p70,44 although the role of microflora in these experiments was not addressed. Interestingly, recent evidence from other systems suggests that IL-23 and IL-17 appear to be involved in protection from the extracellular pathogens Klebsiella pneumoniae45, 46 and C. rodentia.38 Underscoring the importance of IL-23 in the regulating of mucosal inflammation is a recent report describing a highly significant association between Crohn's disease and the gene for the IL-23 receptor.47

ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES

While it is clear that activation of CD4+ T plays a central role in the development of colitis in many models, examination of studies with microflora-driven models suggest that T cells are not always essential for the development of inflammatory responses in the lower bowel. In fact, initial studies describing the association of enterohepatic Helicobacter infection with the development of spontaneous colitis in several lymphocyte-deficient mouse strains such as scid and nude mice7–9 suggested that in certain situations innate inflammatory responses (here defined as responses that occur in animals lacking lymphocytes) were sufficient to induce the clinical signs of colitis in the complete absence of T cells. While the study of Cahill et al,16 cited above, suggested that the presence of CD4+ CD45RBhigh T cells could exacerbate colitis in H. hepaticus-infected scid mice, an interesting finding of that study is that targeted infection of scid mice in the absence of adoptively transferred T cells also induced a relatively robust colitis. In addition, it was shown that targeted infection of scid mice with H. bilis could induce colitis.14 While it has been noted that scid mice can exhibit some low levels of T cell production, mice lacking the gene for RAG-2, which do not exhibit any residual T-cell development, were also shown to develop colitis when vertically infected with H. hepaticus.17 Subsequent studies in several laboratories have verified that targeted infection of RAG-2-deficient mice with H. hepaticus can induce the development of colitis.48–50 Interestingly, it appears that the innate inflammatory response to H. hepaticus is considerably more robust in RAG mice on the 129SvEv background than on either the BALB/C or C57BL/6 background, although the basis for this discrepancy is not understood (unpubl. data).

As is the case in the T-cell-dependent models of colitis described above, it appears that IL-12/IL-23 and TNF also play essential roles in the development of the H. hepaticus-induced innate inflammatory response. Treatment of RAG mice from the time of infection with depleting antibodies directed at either IL-12 p40 or TNF ameliorated disease.48 Further, RAG mice lacking p40 are completely resistant to colitis induced by H. hepaticus, and those lacking p19 are partially resistant, while those lacking p35 are as sensitive as RAG mice.51 This suggests a predominant role for IL-23 rather than IL-12 p70. It has also been reported that RAG-2 mice lacking IL-7 are resistant to H. hepaticus-induced inflammation,17 although the basis for this observation has not been delineated. While IL-23 and TNF are important effector molecules for the development of microflora-induced inflammation both in T-cell-dependent and innate models, it appears likely that even in the T-cell-dependent models production of these molecules by the innate branch of the immune system is essential. IL-12 p40 (a component of IL-12 p70 and IL-23) is made predominantly by antigen-presenting cells of the innate immune system and production has not been detected within T cells themselves.52 Furthermore, while TNF can be made by many cell types, including Th1 cells, it has been shown that TNF-deficient CD4+ CD45RBhigh T cells are as capable as WT CD4+ CD45RBhigh T cells to induce colitis after adoptive transfer into RAG-2-deficient hosts, whereas WT CD4+ CD45RBhigh T cells are unable to induce colitis when transferred into TNF-deficient RAG-2-deficient hosts.53 Similarly, while it has been shown that depletion of IFN-γ with antibodies can inhibit development of disease induced by adoptive transfer of CD4+ CD45RBhigh cells,32 IFN-γ-deficient T cells are capable of inducing disease when adoptively transferred into RAG hosts,33 suggesting that production of IFN-γ by cells of the innate immune system rather than T cells may be involved in disease pathogenesis. Taken together, these results strongly suggest that the ability of microflora to induce inflammatory gene expression within the innate immune system is essential for the development of colitis in both T-cell-dependent and independent models, and thus understanding the regulation of microflora-induced cytokine expression within the innate immune compartment of the bowel is crucial for defining relevant pathogenic mechanisms.

REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES

The studies described above suggest that both innate and CD4+ T-cell-mediated effector mechanisms are involved in driving bowel inflammation in response to microflora. Interestingly, it appears that CD4+ CD25+ Treg cells have a central role in inhibiting both of these effector mechanisms. Early experiments from the Powrie group demonstrated that CD4+ CD45RBlow T cells could inhibit colitis induced by the adoptive transfer of CD4+ CD45RBhigh T cells.24 It has since been shown that CD4+ CD45RBlow cells selected for expression of CD25 inhibit colitis induced by CD4+ CD45RBhigh cells considerably more efficiently than CD4+ CD45RBlow CD25 cells.54 In addition, adoptive transfer of these cells into RAG hosts can inhibit the ability of H. hepaticus to induce colitis,48, 49 indicating that Treg cells can directly inhibit the innate inflammatory response to infection with inflammatory microflora. Thus, while CD4+ CD25+ T cells can inhibit the proliferation of CD4+ CD45RBhigh T cells in vitro,25, 26 it is not clear whether CD4+ CD25+ T cells have a direct effect on the ability of CD4+ CD45RBhigh T cells to induce colitis or work indirectly by inhibiting the innate inflammatory response to microflora.

MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES

In many colitis susceptible models there appears to be a defect in the development of CD4+ CD25+ T cells. One of the factors that has been shown to be central for the development of Treg cells is the transcription factor Foxp3. It was observed that that the mutant mouse strain scurfy, which carries a defect in the gene for Foxp3, exhibit a similar spectrum of autoimmune phenomena to mice depleted of CD4+ CD25+ Treg cells.27, 28 Naïve Treg cells express Foxp3, and expression of Foxp3 in CD4+ CD45RBhigh CD25 T cells is able to induce cell surface expression of CD25.27, 28 These Foxp3-transduced T cells can inhibit the ability of CD4+ CD45RBhigh CD25 T cells to induce colitis after adoptive transfer into lymphocyte-deficient hosts.27, 28 CD4+ T cells that express CD25 can be detected in mice lacking Foxp3; however, these cells exhibit no ability to suppress T-cell proliferation in vitro. Because CD25 can be induced on naïve CD4+ CD25 T cells after activation it was hypothesized that the CD4+ CD25+ T cells in Foxp3-deficient mice might represent activated cells from the CD4+ CD25 pool, rather than nonfunctional regulatory cells. In a series of elegant experiments, mixed bone marrow chimeric animals were produced in which lethally irradiated mice were reconstituted with a mixture of bone marrow harvested from WT B6 Ly5.1 congenic animals and Foxp3-deficient mice.27 These mice showed no signs of inflammatory disease, and while both WT B6 Ly5.1+ and Foxp3-deficient bone marrow cells contributed to the CD4+ CD25 T-cell compartment, the CD4+ CD25+ regulatory T-cells compartment consisted solely of cells derived from the WT B6 Ly5.1 bone marrow. Furthermore, inflammatory disease in scurfy mice could be rescued by the adoptive transfer of WT CD4+ CD25+ regulatory T cells.27 These experiments definitively establish that Foxp3 is required for the development of CD4+ CD25+ regulatory T cells.

MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES

While mutations that interfere with the development or survival of regulatory T cells clearly establish the importance of these cells in preventing colitis, they do not lead to insights into the factors vital for their regulatory function. Based on their susceptibility to colitis, it was hypothesized that IL-10-deficient mice might have a defect in the development or function of regulatory T cells. Mice lacking IL-10 were reported to have normal levels of CD4+ CD45RBlow cells.55 To test the regulatory function of these cells, the ability of IL-10-deficient CD4+ CD45RBlow T cells to inhibit colitis induced by CD4+ CD45RBhigh cells was evaluated. IL-10-deficient CD4+ CD45RBlow cells were completely unable to inhibit colitis.55 To determine whether IL-10 was necessary for regulatory T cells to directly inhibit the innate response to H. hepaticus, several studies compared the ability of WT and IL-10-deficient Tregs to inhibit H. hepaticus-induced lower bowel inflammation in RAG-deficient mice.48, 49, 56 While WT Tregs completely inhibited H. hepaticus-induced disease, IL-10-deficient Tregs were unable to inhibit. These experiments confirm that IL-10 is required for the function of regulatory T cells, and furthermore that IL-10 produced by regulatory T cells directly inhibits the ability of microflora to induce innate inflammation within the lower bowel.

The second molecule that has been strongly suggested to be involved in the function of regulatory T cells is TGF-β. TGF-β is widely expressed and can be detected on the surface of regulatory T cells.57 TGF-β1-deficient mice rapidly develop a systemic inflammatory syndrome that includes colitis.58, 59 Furthermore, it has been reported that mice lacking Smad3, one of the primary transducers of signals from the TGF-β receptor develop signs of colitis after targeted infection with H. hepaticus.60 Antibody to TGF-β interferes with the ability of CD4+ CD25+ regulatory T cells to prevent colitis induced by adoptive transfer of CD4+ CD45RBhigh T cells.54 TGF-β appears to directly inhibit the inflammatory potential of CD4+ T cells, as mice expressing a dominant negative mutant of the TGF-β type II receptor (TGF-β dnRII) specifically within these cells or mice with a T-cell-specific deletion of the TGF-β type II receptor develop systemic inflammatory syndrome including colitis,61–63 and WT CD4+ CD25+ regulatory T cells are unable to inhibit the ability of CD4+ CD45RBhigh T cells expressing TGF-β dnRII to induce colitis after adoptive transfer into RAG host.64 CD4+ CD25+ T cells can be detected in mice lacking TGF-β1. Therefore, to determine whether TGF-β expression by regulatory T cells is required to inhibit colitis, CD4+ CD25+ T cells were isolated from young TGF-β1-deficient mice and their ability to inhibit colitis induced by CD4+ CD45RBhigh T cells was evaluated. CD4+ CD25+ T cells isolated from TGF-β1-deficient mice were unable to inhibit colitis, suggesting that inhibition by regulatory T cells requires the intrinsic expression of TGF-β.65

However, whether expression of TGF-β by regulatory T cells is required for suppressive function has been somewhat controversial, and it has been argued that regulatory T cells do not need to express TGF-β to mediate suppressive functions.66 Furthermore, one complicating factor in the experiment described in the previous paragraph is that in addition to functioning as a marker for T regulatory cells, CD25 is also a T cell activation marker. Therefore, it is possible, as observed in Foxp3-deficient mice,27 that because of the very early immune activation observed in the absence of TGF-β1, activated CD4+ T cells are entering the CD25+ pool and altering the balance of activity. Furthermore, an independent study has demonstrated that CD4+ CD25+ T cells isolated from TGF-β1-deficient mice expressing the DO11.10 TCR specific for ovalbumin can inhibit colitis induced by CD4+ CD45RBhigh T cells.64 The discrepancy between this result and the previously described result may be because the development of autoreactive T cells in TGF-β1-deficient mice is delayed by the presence of the transgenic TCR. Interestingly, the ability of CD4+ CD25+ T cells isolated from TGF-β1-deficient mice expressing the DO11.10 TCR to inhibit colitis is abrogated by the administration of anti-TGF-β1 antibody.64 Taken together, these studies are consistent with a model in which TGF-β1 is not absolutely required for the development of CD4+ CD25+ T cells, nor is intrinsic expression of TGF-β1 by CD4+ CD25+ T cells required for regulatory function. Rather, it appears that expression of TGF-β1 by a source other than CD4+ CD25+ T cells is required to sensitize CD4+ CD45RBhigh T cells to the inhibitory effects of CD4+ CD25+ T cells.

ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES

The observation that IL-10 has a central role in regulating the innate inflammatory response to microflora suggests that the response to IL-10 within cells of the innate immune system is critical in preventing excessive inflammatory gene expression. It has been thought that myeloid cells are a primary target of IL-10, and that a key mediator of the effects of IL-10 is STAT3,67 which is activated in response to IL-10 receptor ligation.68 STAT3-deficient mice are not viable,69 therefore to determine whether activation of STAT3 within myeloid cells plays a critical role in the inhibition of colitis, mice with a cell-type-specific disruption of STAT3 in macrophages and neutrophils were produced.70 The ability of IL-10 to inhibit LPS induced expression of TNF was completely abrogated in macrophages derived from these mice. Furthermore, mice lacking STAT3 in macrophages and neutrophils developed spontaneous enterocolitis at 20 weeks of age. Splenic T cells from these mice secreted higher amounts of IFN-γ after stimulation with anti-CD3 than splenocytes from control animals, and peritoneal macrophages isolated from these mice secreted higher levels of IL-12 after LPS treatment than control cells.70 Mice that lack STAT3 specifically in all bone marrow cells have also been described and these mice develop a severe enterocolitis 4–6 weeks after birth.71 These experiments strongly suggest that one of the primary targets of IL-10 action necessary to prevent colitis are myeloid cells, indicating that controlling the inflammatory potential of these cells is critical to prevent pathogenic inflammation in the lower bowel. Interestingly, it has also been suggested that IL-10 can have direct effects on nonmyeloid cells including epithelial cells. Thus, while it is clear that the inhibitory effects of IL-10 on myeloid cells are essential to prevent colitis, IL-10 could have important inhibitory functions on epithelial cells as well. This question could be addressed with mice with epithelial cell-specific disruption of STAT3, although to our knowledge such mice have not yet been reported.

The mechanisms by which IL-10-activated STAT-3 limits inflammatory gene expression within myeloid cells have proven somewhat difficult to understand. In one study, while the ability of LPS to induce IL-12 p40 in IL-10-deficient bone marrow-derived macrophages depended on NF-κB activation, there was no difference in IκB degradation, or Rel A phosphorylation between WT and IL-10-deficient macrophages.72 However, chromatin immunoprecipitation (ChIP) assays demonstrated increased binding of both c-Rel and p65 to the endogenous IL-12 p40 promoter in LPS-stimulated IL-10-deficient macrophages compared to LPS-stimulated WT controls.72 As previous studies have shown that c-Rel is required for LPS-induced expression of IL-12 p40 in macrophages,73 these observations suggest that IL-10, and therefore likely STAT3, may inhibit IL-12 p40 expression by inhibiting recruitment of c-Rel to the IL-12 p40 promoter. The mechanism that underlies this phenomenon has not been reported. In contrast, other investigators have not been able to observe recruitment of c-Rel to the IL-12 p40 promoter using ChIP assays.74

ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES

In addition to IL-10, TGF-β may also have important inhibitory roles within the innate immune system. However, the cellular and molecular basis for inhibition of the innate immune response by TGF-β is much less clear. While mice that lack TGFβ1 develop a systemic inflammatory syndrome,58, 59 it has been reported that mice doubly deficient for RAG and TGF-β1 (TGFβ/RAG) do not.75 These doubly deficient mice have been reported to have an increased rate of colonic adenocarcinoma compared to mice lacking the RAG gene alone. The development of carcinoma appears to be dependent on the presence of colonic microflora because germ-free TGFβ/RAG mice did not develop cancer.75 Interestingly, exposure of mice from this germ-free colony to SPF flora that contained H. hepaticus restored the development of cancer, while reconstitution with SPF flora that did not contain H. hepaticus was unable to reconstitute cancer.75 Despite these differences in the rate of development of adenocarcinoma between RAG and TGFβ/RAG mice in that study, the authors report that both strains appear to develop mild colitis in SPF flora without a significant difference in the severity of disease between strains. However, formal data comparing the severity of inflammation in these 2 strains was not reported.

In an alternative approach to examine the role of TGF-β signaling within the innate immune system, mice expressing a dominant negative mutant of the TGF-β type II receptor (TGF-β dnRII) under the expression of the intestinal trefoil factor were generated and shown to predominantly express within intestinal epithelial cells.76 It was reported that these mice spontaneously develop colitis when housed in a conventional barrier but not when housed in an SPF barrier. However, the severity of colitis was not quantitatively evaluated. These mice did develop more severe colitis than WT mice after challenge with DSS in their drinking water.76 While the role of flora in the DSS-induced inflammatory process is not clearly defined, it does appear likely that TGF-β signaling is required within intestinal epithelial cells to inhibit the development of colitis.

While TGF-β can strongly influence the gene expression profiles of epithelial cells, inhibition of proinflammatory cytokine expression has not been uniformly observed. In one study examining an intestinal epithelial cell line it was demonstrated that TGF-β treatment partially inhibits the ability of E. feacalis to induce IL-6 and IP-10.77 This inhibition of inflammatory gene expression was accompanied by a decrease in E. feacalis-induced binding of phospho-RelA to the endogenous IL-6 promoter. The alteration in DNA binding was accompanied by TGF-β and E. feacalis-dependent downregulation of TLR2, which was speculated to have a causative role in the inhibition of NF-κB-dependent gene expression observed after TGF-β treatment.77 These data, in combination with the observation that TGF-β dnRII expression within intestinal epithelia sensitize mice to colitis, suggest that TGF-β signaling within the epithelia is crucial to inhibit excessive inflammatory responses to microflora. However, whether TGF-β also plays an important role in inhibiting inflammatory gene expression within myeloid cells of the innate immune system has yet to be clearly addressed. Thus, whether IL-10 and TGF-β are working in collaboration to inhibit inflammatory gene expression within a specific innate immune cell such as epithelia or myeloid cells, or alternatively are targeting different cells, remains an open question.

KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES

Experiments described above indicate that IL-10 and TGF-β are critical inhibitors of the inflammatory response to colonic microflora. Furthermore, it appears that inhibition of the innate response to colonic microflora plays a central role in the function of regulatory T cells. Therefore, it has been of increasing interest to evaluate the factors that regulate the innate response to inflammatory microflora (Table 4). It has been hypothesized that the activation of the NF-κB family of transcription factors plays a predominant role in the activation of innate inflammatory responses in the lower bowel. The NF-κB family consists of 5 members, p50, p65, c-Rel, p52, and RelB. While p65 and c-Rel have been implicated in the induction of inflammatory responses, there is some evidence that p50 may in fact have inhibitory roles (see below). The hypothesis that NF-κB activation is involved in the inflammatory response to microflora has been bolstered by studies showing increased NF-κB binding activity in mouse models of colitis as well as patients with IBD.78, 79 In addition, many of the successful therapeutic modalities have been shown to inhibit NF-κB activity,80 and targeting NF-κB with antisense or decoy oligonucleotides can inhibit colitis.79, 81 The importance of NF-κB activity in driving inflammation within the innate immune system has been demonstrated by mice engineered to lack the TNF-inducible factor A20.82 Embryo fibroblasts derived from these mice demonstrate prolonged NF-κB activation in response to TNF, and macrophages derived from these mice exhibit enhanced NF-κB-dependent gene expression after LPS challenge.82, 83 A20-deficient mice develop a multiorgan inflammatory syndrome that includes a pronounced colitis. Colitis observed in these mice is a property of the innate immune system, as mice that are doubly deficient for both A20 and RAG develop disease.82 While the role of intestinal microflora in these animals has not been directly tested, these studies suggest that NF-κB activation plays a central role in inducing the innate inflammatory response in the lower bowel. These studies are also consistent with those demonstrating that mice engineered to overexpress TNF develop a marked inflammatory syndrome that includes colitis.84

Table 4. Key Signaling Molecules that Regulate the Innate Inflammatory Response to Colonic Microflora
NameFunctionPhenotype of KO Mice
  • a

    See text for details.

A20Feedback inhibitor of NF-κB activationDefect within hematopoietic cells of the innate immune system increases susceptibility to colitis
STAT3Mediator of IL-10-induced inhibition of inflammatory gene expressionDefect within hematopoietic cells of the innate immune system increases susceptibility to colitis
p50/p105Component of NF-κBDefect within hematopoietic cells of the innate immune system increases susceptibility to colitis
NOD2Induces NF-κB activity in response to muramyl dipeptideKnock-out-No increase in sensitivity to colitis yet reporteda; Knock-in of disease associated allele demonstrates increased sensitivity to DSS colitisa

THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES

It has been suggested that the ability of both IL-10 and TGF-β to inhibit the function of NF-κB may be critical to preventing colitis. Interestingly, it appears that the p50 subunit of NF-κB itself may also have a critical role in inhibiting microflora-induced inflammation in the lower bowel. Mice lacking the p50 subunit of NF-κB develop colitis when infected with H. hepaticus,85 and sensitivity to the development of colitis is the result of a defect within the innate immune system.50 Recent experiments have shown that this defect appears to be the result of a failure to respond appropriately to the inhibitory effects of IL-10.86 Furthermore, data derived from radiation chimeras suggest that the inability to respond to IL-10 represents a defect within myeloid cells of the innate immune system.86 Thus, the p50 subunit of NF-κB may have a critical inhibitory role in facilitating suppression of microflora-induced inflammatory gene expression by IL-10. Whether p50/p105 functions to interfere with the activity of other NF-κB subunits with proinflammatory activity, such as c-Rel, is under active investigation.

ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES

The studies described above suggest that genes that regulate NF-κB function may have critical roles in regulating the development of colitis. It is now well documented that there are several families of proteins that have the ability to sense the presence of pathogen-associated molecular patterns (PAMPs) and activate NF-κB. Therefore, a critical question is whether activation of these sensors plays an important role in driving intestinal inflammation associated with the presence of microflora. Members of the TLR family are present at the cell surface and within endosomes. With the possible exception of TLR3, all TLRs have the capacity to recruit the adapter protein Myd88, through homotypic TIR domain interactions. Myd88 in turn interacts with IRAK4, which induces the activation of NF-κB.87 To examine the role of TLR signaling in the induction of colitis, colitis-susceptible strains have been bred to those lacking key components of TLR signaling. Colitis in mice lacking STAT3 within myeloid cells is abrogated in the absence of TLR4.88 Furthermore, mice lacking IL-10 and Myd88 are resistant to the development of spontaneous colitis.89 These results suggest that TLR signaling is necessary for the development of spontaneous colitis in these models. However, TLR signaling does not appear essential in all models. Despite a loss of function mutation in TLR4, the C3H/HeJBir strain is susceptible to the development of spontaneous colitis and this is exacerbated by the absence of IL-10.90, 91 In addition, while the absence of Myd88 inhibits spontaneous colitis in IL-10-deficient mice, it does not interfere with the development of spontaneous colitis in mice lacking IL-2,89 which had previously been documented to require the presence of endogenous microflora. Thus, while TLR signaling appears to play an essential role in some situations, it is clear that some pathways are independent of Myd88 and TLR4. Whether this is the result of redundancies between TLRs or TIR adapter proteins is not completely clear, although the absence of another TIR adapter TRIF, that functions downstream of TLR3 and TLR4, in addition to Myd88, did not alter the development of colitis in mice lacking IL-2.89

PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES

In addition to the TLR family, which is able to sense PAMPs at the cell surface and within endosomes, there are several related families of cytosolic proteins that appear to function as intracellular pathogen sensors.92 One such gene that has garnered a large amount of attention is NOD2 (CARD15).93 NOD2 is a member of the NOD-leucine-rich repeat (LRR) family that has been shown to have critical roles in regulating human inflammatory conditions.92 Certain mutations in NOD2 are strongly associated with the development of Crohn's disease (CD).94, 95 Individuals with a single disease-associated allele have a 2–4-fold increased risk of developing CD, while homozygous or compound heterozygotes have a 15–40-fold increased risk of CD. It is now known that NOD2 functions as an intracellular receptor for muramyl dipeptide (MDP), a component of peptidoglycan, and activates NF-κB through interaction of N-terminal CARD domains with the RICK protein kinase, an upstream activator of the IKK complex.93, 96, 97 Alleles associated with CD are confined to the C-terminal LRR domain of NOD2,98 and initial studies suggested that disease-associated alleles exhibit a defect in the ability to activate NF-κB.95, 98 Because patients with IBD have been shown to exhibit increased activation of NF-κB,78 it is somewhat paradoxical that mutations that interfere with NF-κB activation predisposes to the development of colitis, and there has been some controversy regarding the possible mechanisms through which alterations in NOD2 contribute to an overexuberant inflammatory response. Thus, the phenotype of mice with engineered mutations in NOD2 was eagerly anticipated.

To date, 3 mouse lines with targeted mutations in NOD2 have been generated.99–101 Mice with a complete knockout of NOD2 are developmentally normal and have not yet been reported to develop spontaneous colitis.99–100 Macrophages and splenocytes from these mice produced normal levels of IL-12 p40 after stimulation with LPS, but as expected exhibit a defect in expression of IL-12 p40 in response to MDP,99, 100, 101 and a defect in the ability of MDP to synergize with LPS to induce IL-12 p40 and IL-6.100 In 1 mouse line it has been reported that the absence of NOD2 increases the ability of peptidoglycan (PGN) (a TLR2 ligand that also contains the MDP motif) to induce expression of IL-12 p40.102 Consistent with a role for NOD2 in controlling IL-12 p40 expression, APCs derived from these mice produced increased amounts of IL-12 p40 when cocultured with PGN and antigen-specific T cells, compared to APCs derived from WT counterparts.103 Furthermore, adoptively transferred T cells specific for ovalbumin were able to induce a transient colitis in these NOD2-deficient but not WT mice after intrarectal administration of an E. coli strain engineered to express the ovalbumin epitope recognized by the transferred T cells. Interestingly, the development of this transient colitis was completely abrogated in the absence of TLR2, suggesting that the ability of NOD2 to inhibit TLR2-specific responses may play an important role in inhibiting the development of T-cell-mediated lower bowel inflammation.103 Despite the inherent attractiveness of such a model of NOD2 function, other authors have observed no difference in the expression of IL-12 p40 after stimulation of NOD2-deficient macrophages with PGN.100, 101

Rather than a primary defect in the regulation of inflammatory gene expression, it has been argued that NOD2-deficient mice may exhibit increased susceptibility to bacterial pathogenesis.100 Adult mice deficient in NOD2 were also shown to be highly susceptible to infection with Listeria monocytogenes when organisms were administered intragastrically by gavage, but not when administered i.v. or i.p.100 Sensitivity to entrically administered Listeria in NOD2-deficient mice was associated with a defect in the expression of a subset of cryptdins, antibacterial proteins preferentially produced by Paneth cells of the small intestine.100 In support of the hypothesis that a defect in cryptidin expression could lead to increased bacterial pathogenesis, mice that lack all mature cryptdins as the result of the absence of matrilysin, a matrix metalloproteinase that localizes to Paneth cells, exhibit increased numbers of E. coli within the small bowel 2 hours after oral challenge, and increased mortality after oral challenge with a virulent strain of Salmonella typhimurium.104 A role for NOD2 in regulating bacterial pathogenesis is supported by experiments demonstrating that overexpression of WT NOD2 but not a disease associated allele can protect intestinal epithelial cells from Salmonella infection.105 These observations lead to the hypothesis that a failure to control bacterial pathogenesis in NOD2-deficient mice might secondarily result in excessive inflammation within the bowel.100, 105

In addition to the 2 lines of NOD2-deficient mice that have been described, a line of mice have been produced in which the WT alleles of NOD2 have been replaced by an allele (Nod22939iC) that is equivalent to the most common disease-associated allele found in CD patients.101 Macrophages derived from these mice exhibit increased NF-κB activation in response to MDP and elevated secretion of IL-1β. Interestingly, these mice developed more severe colitis in response to DSS than WT controls, although the differences between genotypes was relatively modest. Colitis in both WT and mutant mice could be abrogated by treatment with IL-1 receptor antagonist. Based on these observation, the authors suggested that the mutant NOD2 allele in these mice may in fact be operating as a gain of function, possibly leading to increased IL-1β processing, facilitated by interaction of the N-terminal CARD domains of NOD2 with caspase-1 (ICE, IL-1 converting enzyme), an enzyme required for the conversion of IL-1β to its active form.101, 106

Thus, experiments in mice with engineered mutations in NOD2 have yielded conflicting results. Interestingly, the observation that these mice do not spontaneously develop colitis suggests that whatever the role of NOD2, it does not appear to play an essential role in preventing the development of spontaneous bowel inflammation. However, the microflora status of the individual facilities housing these animals has not been specified, and it is possible that these mice have not been exposed to microflora with high inflammatory potential, such as enterohepatic Helicobacter species. Furthermore, as genetic background can strongly influence the susceptibility to microflora-induced colitis, it is possible that breeding mice with NOD2 mutations onto backgrounds with higher intrinsic susceptibility to colitis such as 129SvEv, C3H/HeJ, or IL-10-deficient could be revealing.

PERSPECTIVE

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES

Mice with genetically engineered mutations have played a critical role in elucidating the pathways that control the response to inflammatory enteric microflora. These observations strongly suggest that the response of the cells of the innate immune system to inhibitory factors, including IL-10, play a central role in preventing excessive inflammatory responses within the bowel. As there is no evidence that patients with IBD exhibit defects in the production of the inhibitory factors themselves, and treatment with IL-10 has been unsuccessful,107, 108 this might suggest that defects within the innate immune system of patients could interfere with the response to inhibitory factors, and play a role in sensitizing patients to inflammation. In support of this concept, expression of NOD2, alleles of which strongly sensitize to the development of CD, appears restricted to cells of the innate immune system. Furthermore, altered function of NF-κB within cells of the innate immune system is a recurrent theme in multiple models that exhibit increased sensitivity to colitis. Therefore, we suggest that future research directed toward understanding pathways within the innate immune system that are involved in limiting the inflammatory response to enteric microflora could lead to fundamental advances in our understanding of the pathogenesis of inflammatory bowel diseases.

REFERENCES

  1. Top of page
  2. Abstract
  3. MICE LACKING KEY IMMUNOREGULATORY MOLECULES ARE SENSITIVE TO MICROFLORA-DRIVEN COLITIS
  4. INFLAMMATORY MICROFLORA CAN DRIVE THE DEVELOPMENT OF COLITIS IN SUSCEPTIBLE HOSTS
  5. ROLE OF T-CELL SUBSETS IN REGULATING THE DEVELOPMENT OF COLITIS
  6. ROLE OF THE INNATE IMMUNE SYSTEM IN THE DEVELOMENT OF MICROFLORA-INDUCED COLITIS
  7. REGULATORY T CELLS CAN INHIBIT BOTH INNATE AND ACQUIRED EFFECTOR MECHANISMS
  8. MICE WITH DEFECTS IN THE DEVELOPMENT OF REGULATORY T CELLS ARE SUSCEPTIBLE TO COLITIS
  9. MICE WITH DEFECTS IN THE FUNCTION OF REGULATORY T CELLS DEVELOP COLITIS
  10. ROLE OF IL-10 IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  11. ROLE OF TGF-β IN REGULATING THE INNATE INFLAMMATORY RESPONSE
  12. KEY FACTORS THAT REGULATE THE INNATE INFLAMMATORY RESPONSE TO MICROFLORA
  13. THE p50/p105 SUBUNIT OF NF-κB IS NECESSARY TO PREVENT AN EXCESSIVE INNATE RESPONSE TO COLONIC MICROFLORA
  14. ROLE OF BACTERIAL SENSORS IN ACTIVATING THE INNATE RESPONSE TO COLONIC MICROFLORA
  15. PHENOTYPE OF MICE WITH ENGINEERED MUTATIONS IN THE NOD2 GENE
  16. PERSPECTIVE
  17. REFERENCES