Symposium

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doi: 10.1111/j.1365-2796.2008.01953.x

NF-jB in inammatory bowel disease

I. Atreya, R. Atreya & M. F. Neurath
From the Institute of Molecular Medicine and I. Medical Clinic, University of Mainz, Mainz, Germany

Abstract. Atreya I, Atreya R, Neurath MF (Institute of Molecular Medicine and I. Medical Clinic, University of Mainz, Germany). NF-jB in inammatory bowel disease (Review). J Intern Med 2008; 263: 591596. Apart from genetic and environmental factors, the mucosal immune system of the gut plays a central role in the pathogenesis of inammatory bowel disease (IBD). In the healthy gut, the mucosal immune system ensures the balance between pro- and antiinammatory mediators and thereby allows an effective defence against luminal pathogens but at the same time prevents an overwhelming immune reaction directed against the huge amount of harmless luminal antigens (for example, components of food or nonpathological bacteria). In both entities of IBD (Crohns disease and ulcerative colitis) this immunological balance is severely impaired and shifted towards the pro-inammatory side. The chronic muco-

sal inammation in IBD is caused by hyperactivation of effector immune cells, which produce high levels of pro-inammatory cytokines like tumour necrosis factor-a, interleukin-6 and interferon-c, resulting in colonic tissue damage. The nuclear transcription factor kappaB (NF-jB) was identied as one of the key regulators in this immunological setting. Its activation is markedly induced in IBD patients and through its ability to promote the expression of various proinammatory genes, NF-jB strongly inuences the course of mucosal inammation. Considering the different cell-type specic effects which are mediated by NF-jB, this review aims at describing the complex role of NF-jB in IBD and discusses existing pharmacological attempts to block the activation of NF-jB to develop new therapeutic strategies in IBD. Keywords: gastroenterology, inammation.

Activation of NF-jB
In general, the family of nuclear transcription factor kappaB (NF-jB) proteins consists of ve different members, which are namely p65 (RelA), c-Rel, RelB, p50 and p52. These proteins are all characterized by a structurally conserved N-terminal 300 amino acid region containing specic domains, which allow dimerization, nuclear localization and DNA-binding [13]. Amongst these members of the NF-jB family, only p65, c-Rel and RelB are directly able to activate the transcription of target genes. The transcriptional capacity of p50 and p52, which are initially synthesized as large precursors called p105 and p100, are dependent on dimerization with p65, c-Rel or RelB [2, 3]. In unstimulated cells, the majority of NF-jB dimers are inactivated and retained in the cytoplasm

by association with small inhibitory molecules called IjBa, IjBb or IjB [4]. These inhibitors of NF-jB are able to mask the motif within the amino acid sequence of NF-jB, which is responsible for the nuclear localization of NF-jB. In addition, IjBa is also able to enter the nucleus by itself and subsequently mediates the blockade of DNA-binding of NF-jB and promotes the nuclear export of NF-jB [4]. To activate NF-jB, there exist two different intracellular pathways the classic and the alternative pathway where both result in the release of NF-jB from its inhibitors and in the nuclear localization of NF-jB [57]. Classic activation of NF-jB can be initiated by a broad panel of different stimuli including bacterial cell wall components like lipopolysaccharide, pro-inammatory cytokines like tumour necrosis factor (TNF)-a or interleukin (IL)-1, viruses and DNA 2008 Blackwell Publishing Ltd

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damaging agents [6]. These triggering substances are able to induce intracellular signalling cascades, resulting in a subsequent activation of the IjB kinase (IKK) complex. The IKK complex is composed of two catalytic subunits IKKa and IKKb as well as a regulatory protein, named NFkappaB essential modulator (NEMO). Whilst NEMO serves as an essential adaptor molecule, both catalytic subunits are able to phosphorylate specic serine residues within the IjB molecules [1, 8, 9]. Phosphorylation of NF-jB-bound IjB, mainly mediated by IKKb in case of classic NFjB activation, subsequently initiates the proteosomal degradation of IjB and nally allows the nuclear localization of NF-jB [7, 10]. Some inducers of the classical NF-jB pathway (for example the TNF receptor family member CD40) are additionally able to trigger the alternative NF-jB pathway, which aims at post-translational processing of the p100 precursor to mature p52 [3, 7]. In contrast to the classical pathway, NEMO is not absolutely required for alternative NFjB activation and instead of IKKb, IKKa is of indispensable importance [3, 7]. Irrespective of the signalling cascade, which leads to its activation, activated and translocated NF-jB dimers in the nucleus are able to interact with regulatory NF-jB elements in promoters and enhancers, thereby inducing the expression of NF-jB target genes [11]. Generally, genes which are regulated by the transcriptional activity of NF-jB can be categorized into four functional groups: inammatory and immunoregulatory genes, cell cycle regulating genes, anti-apoptotic genes and genes that encode negative regulators of NF-jB (autoinhibitory feedback loop) [5].

patients showed augmented levels of NF-jB p65 [12]. Analysing the activation status of NF-jB in this setting, by immunouorescence staining of activated p65 in biopsies, clearly demonstrated that NF-jB is not just expressed but is also in a state of activation in mucosal macrophages and epithelial cells in IBD patients. Interestingly, the amount of activated NF-jB correlated signicantly with the severity of intestinal inammation [13]. In addition to macrophages and epithelial cells, lamina propria broblasts are also assumed to play a NF-jB mediated pro-inammatory role in IBD [14]. Because of the unequal role and function of macrophages, epithelial cells and broblasts within the mucosal immune system and very broad panel of genes which are controlled by NF-jB, it is necessary to carefully regard the different cellspecic effects of NF-jB which contribute to the pathogenesis of IBD. In IBD patients, the increased NF-jB expression in mucosal macrophages is accompanied by an increased capacity of these cells to produce and secrete TNF-a, IL-1 and IL-6 [12]. This nding nicely reects the central function of NF-jB in monocytes, which is the induction and control of pro-inammatory cytokines. Beside TNF-a, IL-1 and IL-6, NF-jB is also able to regulate the expression of IL-12 and IL-23 [15, 16]. To some extent, these pro-inammatory cytokines are directly involved in the mucosal tissue damage typically occuring in IBD. For example, TNF-a and IL-1 mediated upregulation of matrix metalloproteinase production results in severe damage of the extracellular matrix and mucosal degradation [17, 18]. But predominantly NF-jB-induced cytokines are responsible for further stimulation, activation and differentiation of lamina propria immune cells, resulting in the perpetuation of mucosal inammation. Especially, the differentiation of T-helper (Th)1 cells which are of pivotal importance for the pathogenesis of IBD [17, 19] is strongly driven by the major Th1-inducing cytokine IL-12, but is also supported by IL-23 and TNF-a [17, 1921]. As an additional feature, NF-jBinduced TNF-a is in turn able to potentiate the activation of NF-jB, thereby providing a kind of positive feedback [18].

Cell-specic role of NF-jB in IBD

Dysregulated cytokine production and signalling mechanisms by intestinal epithelial cells, lymphocytes and macrophages have been implicated in the pathogenesis of inammatory bowel disease (IBD), and the transcription factor NF-jB turned out to be one of the major regulatory components in this complex scenario. Obviously the expression and activation of NFjB is strongly induced in the inamed gut of IBD patients. Especially macrophages and epithelial cells isolated from inamed gut specimens from IBD 592

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Whilst NF-jB in macrophages is regarded as an explicit pro-inammatory player in the setting of IBD, its role in epithelial cells turned out to be more controversial. IL-6-induced NF-jB activation in colonic epithelial cells could be demonstrated to be associated with an increased epithelial expression of intercellular adhesion molecule-1 which plays a critical role in the recruitment of neutrophil granulocytes to the site of inammation [22]. So obviously, NF-jB is clearly involved in some pro-inammatory epithelial signalling cascades, but on the other hand, there exist recent data from different experimental models in knockout mice, which clearly demonstrate an antiinammatory overall function of NF-jB in colonic epithelial cells [23, 24]. For example, intestinal epithelial cell-specic inhibition of NF-jB through conditional ablation of NEMO caused a spontaneous development of severe chronic intestinal inammation in mice. NEMO-decient intestinal epithelial cells in this model revealed an increased rate of apoptosis and a decreased production of antimicrobial peptides, subsequently resulting in an impaired integrity of the epithelial barrier and in an enhanced mucosal immune response, triggered by invading bacteria [23]. Furthermore, mice with an intestinal epithelial cell-specic deletion of IKK-b showed a reduced expression of the epithelial cell restricted cytokine thymic stromal lymphopoetin, an impaired development of a pathogen specic Th2 response and therefore an exacerbated production of pro-inammatory Th1 cytokines after parasite infection occurred [24]. Both these models of intestinal epithelial cell-specic NF-jB inhibition clearly demonstrated a critical regulatory role of NF-jB for epithelial integrity and for the intestinal immune homeostasis [23, 24]. In colonic lamina propria broblasts, NF-jB activation can be initiated via T cell expressed CD40L. CD40L was shown to interact with the CD40 receptor, expressed on the surface of colonic broblast, and thereby to induce the activation of NF-jB. Subsequently, NF-jB activation in broblasts resulted in an increased expression of cytokines like IL-8, IL-6 and the monocyte chemotactic protein [14]. In this way, colonic broblasts showed the capacity to participate in a NF-jB-dependent manner in the immunopathogenesis of IBD [14].

This broad panel of different cell-specic ways in which NF-jB inuences numerous immunological processes within the intestinal mucosa, clearly demonstrates the central involvement of this transcription factor in the development, maintenance and chronication of IBD.

Blockade of NF-jB activation as a therapeutic strategy in IBD

As early studies revealed that trinitrobenzene sulphonic acid (TNBS)-induced colitis could successfully be treated by local administration of p65 antisense oligonucleotides [12], the NF-jB pathway soon became an attractive target for therapeutic interventions in IBD. Many of the already established immunosuppressive drugs in IBD like corticosteroids, sulfasalazine, methotrexate and anti-TNF-a antibodies are known to mediate their anti-inammatory effects at least partly via inhibition of NF-jB activity [2529]. For example, corticosteroids are able to induce an increased expression of IjBa, which in turn retains NF-jB in the cytoplasm and interact physically with p65, thereby preventing the transactivation of NF-jB [25, 26, 30]. In agreement with these data, colonic mononuclear-, epithelial- and endothelial cells from glucocorticoid-treated IBD patients showed signicantly lower nuclear NF-jBp65 levels than cells from untreated patients [26]. Whereas all these established drugs do not target NFjB specically, many efforts have been made during the last years to develop selective inhibitors of NFjB. Beside the direct inhibition of NF-jB expression, driven for example by specic antisense oligonucleotides [12], there are further steps within the NF-jB activating signalling cascade, which represent promising targets for pharmacological inhibition of NF-jB. One possible strategy which has already been tested successfully in experimental mouse models of colitis is the direct targeting of the DNA-binding activity of specic NF-jB proteins by decoy oligonucleotides. It could be demonstrated by Fichtner-Feigl et al. [31] that local treatment with NF-jB specic decoy oligonucleotides resulted in an amelioration of chronic

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TNBS colitis in mice accompanied by reduced production of pro-inammatory cytokines and a diminished development of brosis. Alternative attempts are aimed at the specic inhibition of nuclear import systems to prevent the translocation of NF-jB into the nucleus, target the IKK complex or try to stabilize IjB proteins by developing ubiquitylation or proteasome inhibitors [1]. The importance of proteasomal degradation within the NF-jB activating pathway for the pathogenesis of Crohns disease was underlined by the nding that the subunit composition and the proteolytic function of proteasomes differ between patients with Crohns disease, ulcerative colitis and healthy controls. Proteasomes isolated from patients with Crohns disease showed an enhanced capacity to process the inactive p105 precursor towards active p50 [32]. So obviously, the distinct proteasome subunits critically inuence the severity of NFjB-mediated inammation in IBD. Correspondingly, MG-341, a selective inhibitor of 26S proteasomes, which can be administered orally has been proven to attenuate colonic inammation in vivo [33]. Although NF-jB is one of the key regulators in the immunological setting of IBD and therefore appears as a very promising target for therapeutic intervention in IBD, it is nevertheless important to remember that NF-jB is also involved in normal cell physiology [1]. For example, NF-jB activation is essential for physiological development of lymphocytes [4] and is critically involved in the mediation of effective host defence against bacterial infections [34]. With regard to possible systemic effects of therapeutic NF-jB inhibition, it should also be mentioned that blockade of NF-jB activation in murine hepatocytes was associated with a spontaneous development of hepatocellular carcinoma [35]. Therefore to avoid severe side effects, special attention should be paid to attempts which allow a local inhibition of NF-jB restricted to the immune cells within the inamed colonic mucosa. NF-jB antisense oligonucleotides which were administered directly into the colon resulted in a signicant reduction of TNBS-colitis in mice, without mediating any detectable side effects in other organs [12]. By carefully targeting specic NF-jB subunits or signalling components that are particularly involved in the 594

Fig. 1 Targets for the therapeutical inhibition of nuclear transcriptional factor kappa B (NF-jB). During the classic activation of NF-jB, the activated IjB kinase (IKK) complex mediates the phosphorylation of NF-jB-bound IjB, thereby initiating the proteasomal degradation of IjB and the subsequent release and nuclear translocation of NF-jB dimers. Within the nucleus, activated NF-jB mediates its transcriptional activity via interaction with regulatory NFjB elements of different promoter regions. Because of this signalling cascade, different targets for therapeutical inhibition of NF-jB activity exist: 1. Targeting of the IKK complex by specic inhibitors of IKKb; 2. Stabilizing of IjB proteins by inhibitors of ubiquitylation or of proteasomal degradation; 3. Direct inhibition of NF-jB expression by specic antisense oligonucleotides; 4. Inhibition of nuclear import systems to block nuclear translocation of NF-jB; 5. Targeting of the DNA-binding activity of NF-jB proteins by specic decoy oligonucleotides; 6. Inhibition of proteosomal IjB degradation.

pathogenesis of IBD, it might be possible to further minimize systemic toxic effects [1]. Considering these principles of local administration and high specicity, the therapeutical targeting of NF-jB activation probably represents a promising tool for future therapy of IBD.

Conict of interest statement

No conict of interest was declared.

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