The nuclear factor-κB (NF-κB) pathway has many com-ponents that positively or negatively regulate its activity (for recent comprehensive reviews, see REFS 1,2). The NF-κB family of transcription factors consists of five members in mammalian cells: RELA (also known as p65), REL (also known as c-REL), RELB, p50 and p52, which can form homodimers or heterodimers. In resting cells, NF-κB dimers are normally kept in an inactive state by association with proteins of the inhibitor of NF-κB (IκB) family, which includes IκBα, IκBβ, IκBγ, IκBε, the p105 and p100 precursors of p50 and p52, respectively, and the atypical members B cell lymphoma 3 (BCL-3), IκBζ and IκBNS (also known as IκBδ). Activation of NF-κB is controlled by the IκB kinase (IKK) complex, which consists of two catalytic subunits — IKKα (also known as IKK1) and IKKβ (also known as IKK2)3–6 — and the regulatory subunit NEMO (NF-κB essential modulator; also known as IKKγ)7,8. Following cell stim-ulation, the IKK complex phosphorylates IκB proteins on specific serine residues, which induces their subse-quent polyubiquitylation and proteasomal degradation, allowing NF-κB dimers to accumulate in the nucleus and activate gene transcription (FIG. 1).

Two distinct NF-κB activation pathways have been described. The canonical (classical) NF-κB pathway is activated by a large number of stimuli, including pro-inflammatory cytokines, bacterial and viral products, and also stress-inducing stimuli such as γ-radiation, ultraviolet light and oxidative stress. These stimuli induce the degradation of IκBα and the nuclear accumulation

of mainly p50–RELA dimers, which regulate the expres-sion of immunoregulatory and anti-apoptotic genes. The non-canonical (alternative) NF-κB pathway is acti-vated by receptors that are involved in lymphoid tissue organogenesis and lymphocyte development, such as the lymphotoxin-β receptor and the receptor for B cell- activating factor (BAFF), which induce processing of p100 to p52 and the nuclear accumulation of p52–RELB dimers9. Genetic studies have revealed specific and redundant functions of IKK subunits in activating the two NF-κB pathways. Activation of the non-canonical NF-κB pathway requires IKKα and is independent of IKKβ and NEMO9–11. Activation of canonical NF-κB signalling requires NEMO7,12–14 and mainly relies on IKKβ14–17, although IKKα can induce IκB phosphorylation and canonical NF-κB activity in IKKβ-deficient cells18,19.

Its unique properties make the NF-κB pathway a cen-tral regulator of cellular responses in physiology and dis-ease. NF-κB is rapidly activated in response to a wide range of stress-inducing stimuli, which provides the cell with a sensitive and quick detection system for potential threats in its microenvironment. NF-κB also determines cellu-lar responses by translating upstream signals into a rapid reprogramming of gene expression. On the one hand, NF-κB promotes the expression of pro-inflammatory genes that are important for the activation of immune responses, including genes encoding cytokines, chemo-kines, adhesion molecules and also enzymes and mol-ecules with microbicidal activity20,21. On the other hand, NF-κB protects cells from apoptosis22–24 by inducing the

Institute of Genetics, Centre for Molecular Medicine (CMMC), and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Zülpicher Strasse 47, 50674 Cologne, Germany.e-mail: pasparakis@uni-koeln.dedoi:10.1038/nri2655

Regulation of tissue homeostasis by NF‑κB signalling: implications for inflammatory diseasesManolis Pasparakis

Abstract | The nuclear factor-κB (NF-κB) signalling pathway regulates immune responses and is implicated in the pathogenesis of many inflammatory diseases. Given the well established pro-inflammatory functions of NF-κB, inhibition of this pathway would be expected to have anti-inflammatory effects. However, recent studies in mouse models have led to surprising and provocative results, as NF-κB inhibition in epithelial cells resulted in the spontaneous development of severe chronic inflammatory conditions. These findings indicate that NF-κB signalling acts in non-immune cells to control the maintenance of tissue immune homeostasis. This Review discusses the mechanisms by which NF-κB activity in non-immune cells regulates tissue immune homeostasis and prevents the pathogenesis of inflammatory diseases.

t i s s u e - s p e c i f i c i m m u n e r e s p o n s e s

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expression of anti-apoptotic proteins, including BCL-XL25,

FLICE-like inhibitory protein (FLIP)26,27 and members of the inhibitor of apoptosis (IAP) family28,29. In addition, NF-κB also protects cells from necrosis by inhibiting the accumulation of reactive oxygen species (ROS)30 through the transcriptional upregulation of proteins with antioxi-dant functions, such as manganese superoxide dismutase (MnSOD) and ferritin heavy chain31. So, NF-κB has a dual function by helping cells to survive and at the same time eliciting an appropriate response to protect the organism from infection and injury.

NF-κB has been implicated in the pathogenesis of many inflammation-related diseases. Studies in genetic mouse models have addressed the role of NF-κB in vivo and have provided invaluable information about the function of NF-κB signalling in physiology and disease. In general, genetic mouse models allow researchers to study the role of NF-κB by either inhibiting or increas-ing its activity32. Inhibition of NF-κB can be achieved by the removal of Rel family proteins or their activa-tors, such as the IKK subunits, or by the overexpres-sion of degradation-resistant IκB proteins (referred to here as IκB-DR) that are unresponsive to regulation by IKK. Increased NF-κB activity can be achieved by the removal of NF-κB inhibitors (such as IκB proteins, A20 or CyLD) or by the overexpression of NF-κB activators (for example, constitutively active IKKβ). ubiquitous inhibition of NF-κB activity is embryonic lethal12–17,33, and the ubiquitous removal of NF-κB inhibitors results in severe inflammation and early postnatal death34–36; therefore, cell-specific manipulation is required to study the function of NF-κB in adult tissues.

As expected, genetic manipulations that lead to increased NF-κB activity often trigger inflammation- related pathologies34,36–38, confirming the strong pro-inflammatory function of NF-κB. Genetic manipulations that inhibit NF-κB were expected to attenuate inflam-mation and protect mice from inflammation-related pathologies. Indeed, several studies confirmed that NF-κB inhibition has anti-inflammatory effects in vivo39–43, prompting intense interest in the develop ment of NF-κB inhibitors for the treatment of inflammatory diseases. However, several recent studies have surprisingly shown that NF-κB inhibition in non-immune, epithelial or parenchymal cells triggers the spontaneous develop-ment of severe inflammatory conditions. These appar-ently paradoxical and provocative findings highlight the multi-faceted roles of NF-κB signalling and indicate that NF-κB inhibition in certain tissues might have pro-inflammatory effects by disrupting physiological immune homeostasis. This Review focuses on those provocative studies in which the inhibition of NF-κB activity in non-immune cells surprisingly resulted in the spontaneous development of severe inflammatory conditions and discusses the mechanisms by which NF-κB activity in epi-thelial or parenchymal cells might control physiological tissue immune homeo stasis. I also discuss the potential implications of these findings for our understanding of the role of NF-κB in the pathogenesis of chronic inflam-matory diseases in humans. Although they are highly relevant to our understanding of the in vivo function of

NF-κB, owing to space limitations I do not discuss studies in which NF-κB was targeted in immune cells or studies in which genetic manipulations that induce increased activation of NF-κB resulted in pathological conditions.

epithelial nf-κB in skin homeostasisThe skin is a complex and highly specialized organ, the main function of which is to provide a barrier that prevents water loss and protects the body from adverse environmental agents, but it also has important immu-nological properties. The skin is composed of two main compartments, the epidermis and the dermis, which communicate extensively to establish, maintain and restore homeostasis. The epidermis consists mainly of keratinocytes, whereas the dermis is rich in fibroblasts and contains large numbers of immune cells (macro-phages, dendritic cells and mast cells). Disturbance of skin immune homeostasis causes chronic inflammatory

Figure 1 | Schematic depiction of the canonical NF‑κB signalling pathway. Nuclear factor-κB (NF-κB) dimers are normally sequestered in the cytoplasm of resting cells by association with inhibitor of NF-κB (IκB) proteins. Pro-inflammatory signals stimulate receptors belonging to the tumour necrosis factor receptor (TNFR) or interleukin-1 (IL-1)/Toll-like receptor (TLR) superfamilies, which activate the IκB kinase (IKK) complex. The IKK complex phosphorylates IκB proteins on specific serine residues, thereby triggering their polyubiquitylation (polyUb) and proteasome-dependent degradation. Removal of IκB allows NF-κB dimers to accumulate in the nucleus, where they bind to NF-κB sites on the promoters of target genes and activate the transcription of genes with mainly pro-inflammatory and anti-apoptotic functions. NEMO, NF-κB essential modulator.

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diseases such as psoriasis. Although these diseases were initially thought to be caused by immune system abnor-malities, more recently the role of epidermal keratinocytes as an important factor in the pathogenesis of inflamma-tory skin disorders has received increased attention44.

Studies in genetic mouse models have provided evi-dence that NF-κB signalling in epidermal keratinocytes has a crucial role in the maintenance of skin immune homeostasis. Mice with keratinocyte (epidermis)-specific ablation of IKKβ (IKKβEKO mice) were normal when born but three to four days after birth developed a severe inflammatory skin disease, which was charac-terized by increased cytokine expression, immune cell infiltration and epidermal hyperplasia, and died before the age of 10 days45. The development of skin lesions was dependent on signalling through tumour necrosis factor receptor 1 (TNFR1), as IKKβEKOTnfr1–/– animals remained healthy throughout their life45. Skin inflam-mation in IKKβEKO mice was not affected by the absence of T cells45 or by impaired neutrophil recruitment46. By contrast, the elimination of macrophages by subcutane-ous administration of clodronated liposomes prevented the development of skin lesions46. Therefore, epidermis-specific ablation of IKKβ results in inflammatory skin lesions that depend on TNFR1 signalling and require the presence of macrophages but not neutrophils or T cells.

TAK1EKO mice with epidermis-specific ablation of transforming growth factor-β-activated kinase 1 (TAK1), a kinase that is essential for the activation of NF-κB and mitogen-activated protein kinase (MAPK) signalling47–49, also developed a TNF-dependent inflammatory skin disease, providing additional evidence that the blockade of canonical NF-κB activity in epidermal keratinocytes triggers skin inflammation50,51. Increased production of ROS triggering the death of TAK1-deficient keratino-cytes was proposed to contribute to the development of skin inflammation in TAK1EKO mice51. However, in that study the authors suggested that TAK1 controls ROS production in keratinocytes through JuN and not through NF-κB51. Mice expressing a degradation-resistant form of IκBα specifically in epidermal keratinocytes (k5-IκBα-DR mice) also developed skin inflammation and epidermal hyperplasia shortly after birth52. These mice survived to adulthood but had chronic skin lesions that resulted in the development of squamous cell carci-noma (SCC)52. Subsequent studies showed that crossing the k5-IκBα-DR mice onto a Tnfr1–/– background fully prevented the development of skin inflammation, epi-dermal hyperplasia and SCC53, which shows that TNFR1 signalling is essential for the pathogenesis of skin lesions in the k5-IκBα-DR mice, similarly to the IKKβEKO mice and TAK1EKO mice. Another study also reported that transgenic expression of IκBα-DR in the epider-mis resulted in epidermal hyperplasia and early post-natal death, although the role of inflammation was not addressed in this model54. Curiously, mice with epidermal keratinocyte-restricted ablation of RELA did not develop skin inflammation37. Skin lacking both RELA and REL developed TNF-dependent inflammatory lesions and epidermal hyperplasia after transplantation into immuno-deficient recombination-activating gene 1 (Rag1)-knockout

animals55, which indicates that RELA and REL might be functionally redundant in mediating the crucial homeostatic function of NF-κB in the epidermis.

The discovery that mutations disrupting the X-linked gene encoding NEMO in humans cause incontinentia pigmenti provided evidence that NF-κB inhibition in keratinocytes causes skin inflammation in humans as well as mice56. Incontinentia pigmenti is a genetic disease characterized by male embryonic lethality and by the development of a complex range of pathologies involv-ing mainly the skin and its appendages in heterozygous females57. The skin pathology in patients with inconti-nentia pigmenti starts shortly after birth with the appear-ance of inflammatory skin lesions that progress through consecutive stages to verrucous, hyperpigmented and finally atrophic hypopigmented skin in adulthood57. NEMO-deficient mice reproduced the incontinentia pig-menti phenotype, showing male embryonic lethality and the development of inflammatory skin lesions in hetero-zygous females12,14. Moreover, epidermis-specific ablation of NEMO (in NEMOEKO mice) resulted in skin inflam-mation and early postnatal death58. NEMOEKOTnfr1–/– mice did not show signs of skin inflammation in young age but developed skin lesions 3–4 months after birth, showing that lack of TNFR1 could delay but not fully rescue skin lesion development in this model58.

Collectively, these studies showed that NF-κB inhibi-tion in epidermal keratinocytes spontaneously triggers a strong inflammatory response in the skin of both mice and humans, establishing a crucial role for NF-κB sig-nalling in epidermal keratinocytes in the maintenance of skin immune homeostasis. However, the molecular mechanisms by which NF-κB inhibition in keratinocytes causes skin inflammation remain poorly understood. Considering the well-established pro-survival function of NF-κB, one plausible explanation could be that the increased death of keratinocytes with inhibited NF-κB activity triggers an inflammatory response. The presence of increased numbers of dying keratinocytes in the skin of NEMOEKO mice and TAK1EKO mice before the devel-opment of strong skin inflammation is consistent with this hypothesis50,58. However, the epidermis of IKKβEKO mice and k5-IκBα-DR mice did not contain increased numbers of dying keratinocytes during the early stages of disease initiation45,52, which argues against a pri-mary role for keratinocyte death as a causative factor triggering skin inflammation in these mice. Moreover, IKKβEKO mice did not have skin barrier defects45, which indicates that inflammation was not caused by impaired barrier function of the skin. Increased expression of interleukin-1β (IL-1β) was observed in the epidermis of both IKKβEKO mice and NEMOEKO mice45,58, which indi-cates that IL-1β production by NF-κB-deficient keratino-cytes could trigger skin inflammation. This hypothesis remains to be experimentally tested by crossing these mice to mice deficient for IL-1β or the IL-1 receptor. However, IL-1 receptor deficiency failed to prevent skin inflammation and SCC in k5-IκBα-DR mice53, which indicates that IL-1 signalling might not have a major role in the pathogenesis of skin lesions resulting from epidermis-specific inhibition of NF-κB.

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Genetic experiments have provided compelling evidence that TNFR1 signalling is required for the development of skin inflammation in mice with epidermis- specific inhibition of NF-κB. In these studies, TNF was found to be expressed mainly by myeloid cells in the der-mis, which suggests that the increased TNF production is a response of innate immune cells in the dermis to signals originating from the NF-κB-deficient epidermis45. Given its multi-faceted roles, TNF could cause skin inflam-mation by acting on many cell types. Reconstitution of lethally irradiated k5-IκBα-DRTnfr1–/– mice with Tnfr1+/– bone marrow failed to trigger skin inflammation, which indicates that TNFR1 signalling in radiation-resistant non-haematopoietic cells is crucial for skin lesion patho-genesis in this model53. An important open question is whether TNFR1 signals through keratinocytes or through non-epithelial cells such as fibroblasts or endothelial cells trigger skin inflammation. Mouse models allowing cell-specific manipulation of TNFR1 signalling will be required to address this issue.

Interestingly, skin inflammation starts shortly after birth in mice with epidermis-specific inhibition of NF-κB and in patients with incontinentia pigmenti, although the impairment in NF-κB activation is already present in utero. Inducible NF-κB inhibition in the epi-dermis of adult mice also resulted in skin inflamma-tion58–60, which argues against a role for developmental processes that occur in the early postnatal period in triggering the skin lesions. Alternatively, exposure of the skin to environmental factors could provide the trigger for skin inflammation. Germ-free k5-IκBα-DR mice have been reported to develop skin inflammation61, which indicates that skin bacteria are not required for lesion development. However, the skin is routinely exposed to various stresses that could challenge the NF-κB-inhibited epidermis. For example, environmen-tal antigens, temperature changes, mechanical stress or even exposure to the air could provide such signals. A recent study showed that the oxygen-sensing proper-ties of keratinocytes are important for the regulation of systemic hypoxic responses62. Given the established role of NF-κB in controlling cellular responses to oxida-tive stress30,31,63 and hypoxia64, the inhibition of NF-κB might trigger skin inflammation by altering the response of epidermal keratinocytes to atmospheric oxygen. However, in the absence of supporting experimental evidence, the contribution of environmental stress fac-tors to the development of skin inflammation resulting from epidermis-restricted NF-κB inhibition remains purely hypothetical.

The maintenance of skin homeostasis depends on an extensive cross-talk between the epidermis and the der-mis65. NF-κB inhibition in the epidermis could disrupt the communication between keratinocytes and dermal cells, thereby triggering skin inflammation. For example, the expression of keratinocyte-derived factors that are essential for the maintenance of skin homeostasis could be under the control of NF-κB signalling. Alternatively, factors produced by dermal cells might act on the epi-dermis by activating NF-κB signalling in keratinocytes to control homeostasis.

Although the cellular and molecular mechanisms by which NF-κB inhibition in the epidermis disturbs skin immune homeostasis and results in skin inflammation remain elusive, it seems that impaired NF-κB signalling in keratinocytes elicits a response that has many similari-ties to the wound-healing reaction (FIG. 2). It is probable that the NF-κB-deficient epidermis releases stress sig-nals that are interpreted by stromal and immune cells in the dermis as an injury, eliciting a wound-healing response. Although the important differences between human and mouse skin make a direct comparison dif-ficult, the inflammatory skin lesions that develop in mice with epidermis-restricted NF-κB inhibition have similarities to the lesions observed in human inflam-matory skin diseases such as psoriasis. The efficacy of TNF-targeted therapies in psoriasis66,67, which is consist-ent with the important role of TNF in the development of skin inflammation in mice with epidermis-specific NF-κB inhibition, further emphasizes that the mecha-nisms responsible for skin lesions in the mouse models discussed here could be relevant to the pathogenesis of human inflammatory skin diseases. So, elucidating the mechanisms by which NF-κB signalling in the epidermis controls skin immune homeostasis might also lead to a better understanding of the mechanisms causing human inflammatory skin diseases and possibly indicate new possibilities for therapeutic intervention.

epithelial nf-κB in gut homeostasisThe gastrointestinal lumen is host to a very large number of commensal bacteria, and the intestinal mucosa con-tains the highest number of immune cells in the body. Mechanisms to regulate mucosal immune responses to commensal bacteria are therefore essential for the main-tenance of intestinal immune homeostasis, disturbance of which can result in severe chronic inflammatory bowel disease (IBD)68. The intestinal epithelium forms a single cell layer covering the luminal surface of the gut and has an important function in the maintenance of intestinal immune homeostasis. By forming a mechani-cal barrier and producing antimicrobial peptides, intesti-nal epithelial cells (IECs) prevent bacteria from invading the mucosa where they could encounter immune cells and induce inflammation. In addition, IECs have been proposed to carry out immunoregulatory functions by producing factors that act on immune cells to modulate their activity.

NF-κB activation has been detected in the mucosa of patients with IBD, which indicates that NF-κB sig-nalling might contribute to disease pathogenesis69,70. Pharmacological inhibition of NF-κB had protective effects in mouse models of colitis, which supports a path-ogenic role for NF-κB in gut inflammation71–73. However, recent studies that addressed the cell-specific function of NF-κB signalling in the intestine have revealed a more complex picture and provided evidence that NF-κB activation in IECs is essential for the maintenance of intestinal immune homeostasis. Mice with IEC-specific ablation of NEMO (NEMOIEC–KO mice) spontaneously developed severe chronic colitis, manifesting with diar-rhoea, intestinal bleeding and weight loss74. The disease

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started locally in the colon of 2–3-week-old mice with the death of IECs in a small number of crypts, which led to disruption of the epithelial barrier and the invasion of luminal bacteria into the mucosa, triggering a strong inflammatory response that progressed to severe chronic colitis affecting the entire colon, but sparing the caecum and the small intestine (FIG. 3). In addition to forming a physical barrier, IECs produce several antimicrobial peptides, such as defensins, that are thought to protect the epithelium from bacterial colonization75. Impaired expression of human β-defensin 2 (HBD2; also known as DEFB2) has been associated with increased suscepti-bility to colonic Crohn’s disease76, which indicates that decreased antibacterial defence could be implicated in the pathogenesis of intestinal inflammation. Decreased expression of mouse β-defensin 3 (MBD3; which is

homologous to HBD2) was detected in the colon of NEMOIEC–KO mice74, which indicates that impaired expression of defensins could also contribute to lesion development in these animals.

Clinical studies in humans carrying mutations in the X-linked gene encoding NEMO have shown that, in addition to several other symptoms including severe immunodeficiency, osteopetrosis and skin defects, these patients often suffer from colitis77,78. Allogeneic haemat-opoietic stem cell transplantation (HSCT) was reported to correct the immune deficiency and osteopetrosis. However, many of these patients continued to suffer from colitis after HSCT, which suggests that impaired NF-κB signalling in non-haematopoietic cells is respon-sible for the susceptibility to colitis77,78. Interestingly, in some cases, HSCT worsened or even triggered colitis

Figure 2 | NF‑κB inhibition in epidermal keratinocytes disturbs skin immune homeostasis. a | Normal skin homeostasis is maintained by an extensive cross-talk between epidermal keratinocytes and stromal and immune cells in the dermis. b | Inhibition of nuclear factor-κB (NF-κB) in mouse epidermis disturbs skin homeostasis and triggers tumour necrosis factor (TNF)-dependent skin inflammation, epidermal hyperplasia and the development of squamous cell carcinoma. Although the molecular mechanisms by which NF-κB signalling in keratinocytes controls skin homeostasis remain poorly understood, it is possible that NF-κB inhibition compromises the response of the epidermis to environmental challenges or disturbs the communication between the epidermis and the dermis, triggering an inflammatory response that resembles a wound-healing reaction. This response does not require the adaptive immune system and is driven mainly by macrophages.

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in patients who did not suffer from intestinal compli-cations before transplantation77, which shows that the correction of immunodeficiency could aggravate pre-existing disease or even cause colitis in patients with NEMO mutations. HSCT in these patients creates an imbalance in NF-κB signalling, such that stromal and epithelial cells have impaired NF-κB activity in the context of normal NF-κB signalling in the immune system. On the basis of the findings in the NEMOIEC–KO mouse model, it is reasonable to assume that NF-κB deficiency in the intestinal epithelium is responsible for the pathogenesis of colitis in patients with NEMO muta-tions. So, elucidating the mechanisms causing colitis in NEMOIEC–KO mice will have profound implications for the better understanding, and hopefully the treatment, of colitis in human patients with NEMO mutations.

NEMOIEC–KO mice did not develop intestinal inflam-mation when bred onto a MyD88 (myeloid differen-tiation primary-response protein 88)-deficient genetic background, which indicates that signalling through Toll-like receptors (TLRs), probably activated by the recognition of intestinal bacteria, might be involved in causing colitis74. However, MyD88 is an essential adap-tor molecule for signalling downstream not only of most

TLRs but also of the IL-1 receptor79–81. So, further experi-ments are required to address the specific contributions of bacteria, and TLR and IL-1 receptor signalling, in the pathogenesis of colitis in this model. Accumulating evidence supports an important role for TLR signal-ling in the maintenance of immune homeostasis in the intestine82–87. Although a comprehensive discussion of the mechanisms by which TLR signalling affects intesti-nal homeostasis is outside the scope of this Review, it is important to discuss the potential involvement of TLR signalling in the mechanisms by which NF-κB signalling in epithelial cells controls immune homeostasis in the colon. Several studies using MyD88- or TLR-deficient mice indicated that signalling initiated by commensal bacteria prevents excessive colon inflammation82,83,85,86. Furthermore, TLR signalling in IECs was proposed to be important for the maintenance of the epithelial bar-rier and intestinal immune homeostasis83,87,88. Therefore, it is possible that TLR signalling induces NF-κB acti-vation in IECs to prevent colon inflammation. By contrast, adoptive transfer experiments indicated that MyD88-dependent signalling protects mice from colitis induced by the administration of dextran sodium sul-phate (DSS) by acting in haematopoietic cells and not in IECs84. Elucidating the cell-specific TLR- and MyD88-dependent functions will be essential to fully understand the mechanisms by which these pathways interact with NF-κB to regulate intestinal homeostasis.

The cellular specificity of the pathogenic MyD88-dependent signals that induce colitis in NEMOIEC–KO mice remains elusive. Bacteria might induce MyD88-dependent TLR signalling on mucosal innate immune cells, thereby activating cytokine expression and trig-gering colon inflammation. Alternatively, NF-κB inhibition could result in the dysregulation of MyD88-dependent TLR signalling in IECs, resulting in loss of tolerance to commensal bacteria, which in turn causes intestinal inflammation. TLR signalling has also been shown to induce apoptosis in IECs89, an effect that could be increased by NF-κB deficiency, leading to disruption of the epithelial barrier. Elucidating the mechanisms by which MyD88-dependent signalling causes disease pathogenesis in NEMOIEC–KO mice will require further experiments in mouse models with cell-specific targeting of MyD88.

TNF was shown to be involved in the pathogenesis of colitis in NEMOIEC–KO mice, as crossing these mice with TNFR1- or TNF-deficient mice inhibited disease development74. The mechanisms by which TNF exerts its pathogenic effects in NEMOIEC–KO mice remain elu-sive. TNF is a potent inducer of inflammation and has an important role in the development of intestinal inflam-mation not only in mouse models but also in human patients, in whom TNF-targeted therapy is highly effec-tive for the treatment of IBD90,91. TNF could orchestrate the inflammatory response in the colon of NEMOIEC–KO mice by acting on stromal, endothelial or immune cells to induce the expression of pro-inflammatory cytokines and chemokines, leading to the recruitment and activa-tion of inflammatory cells. A recent study showing that TNFR1 expression by mesenchymal cells is required

Figure 3 | NF‑κB inhibition in intestinal epithelial cells disturbs intestinal immune homeostasis and causes chronic colitis. The intestinal epithelium has an essential role in the maintenance of immune homeostasis in the gut. By forming a mechanical barrier and expressing antimicrobial peptides (such as defensins), intestinal epithelial cells (IECs) prevent commensal bacteria from invading the mucosa (left hand side). Nuclear factor-κB (NF-κB) inhibition results in increased death of IECs and decreased expression of antimicrobial peptides, thereby compromising the epithelial barrier and allowing commensal bacteria to invade the colonic mucosa, which triggers severe chronic colitis in a MYD88- and tumour necrosis factor receptor 1 (TNFR1)-dependent manner (right hand side). TLR, Toll-like receptor.

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to induce intestinal (as well as joint) inflammation in a TNF-dependent mouse model92 raises the possibility that TNF-dependent signalling in stromal cells might also be involved in causing colitis in NEMOIEC–KO mice. Alternatively, TNF might act on the epithelium to induce apoptosis in NF-κB-deficient IECs, resulting in disrup-tion of the epithelial barrier, bacterial translocation into the mucosa and inflammation. Experiments involv-ing cell-specific targeting of TNFR1 signalling will be required to address the cellular specificity and molecular mechanisms by which TNF acts to induce colitis in mice with epithelium-specific inhibition of NF-κB.

Mice with IEC-specific ablation of TAK1 (TAK1IEC–KO mice) developed a severe intestinal phenotype involving both the small intestine and the colon and died within one day after birth93,94. widespread apoptosis of IECs in the entire intestine was observed in these mice, which indicates that TAK1 deficiency might cause colitis by sen-sitizing IECs to death. Crossing of TAK1IEC–KO mice with Tnfr1–/– mice could rescue the severe intestinal disease of perinatal TAK1IEC–KO mice, although some (40–50%) of the TAK1IEC–KOTnfr1–/– mice had increased IEC apopto-sis and developed ileitis and colitis three to four weeks after birth, which indicates that TNFR1-independent mechanisms are involved in the later stages of colitis development in these mice. The more severe phenotype of TAK1IEC–KO mice compared with NEMOIEC–KO mice could be explained by the essential role of TAK1 not only for NF-κB signalling but also for MAPK signal-ling47–49, which might also be important for the survival of IECs. Considering the phenotype of TAK1IEC–KO mice, it is interesting that NF-κB inhibition in IECs triggered inflammation in the colon but not the small intestine. The different populations of bacteria present in the colon compared with the small intestine95–97 might be responsi-ble for providing pathogenic signals only in the colon in mice with IEC-specific NF-κB inhibition. Alternatively, NF-κB might have a different role in the epithelium of the colon compared with the small intestine, where other TAK1-dependent pathways (such as MAPK signalling) might also contribute to the maintenance of epithelial homeostasis.

In contrast to NEMO deficiency, ablation of IKKα or IKKβ in IECs did not cause spontaneous intesti-nal inflammation in mice39,74. This result was surpris-ing, considering that IKKβ is thought to be the main kinase mediating canonical NF-κB activation by pro-inflammatory signals, and it indicated that IKKα might compensate for the loss of IKKβ. Indeed, mice lacking both IKKα and IKKβ in IECs developed spontaneous colitis similarly to NEMOIEC–KO mice, which shows that IKKα and IKKβ are functionally redundant in inducing canonical NF-κB signalling in IECs and protecting the colon from inflammation74. Moreover, mice with IEC-specific ablation of RELA (RELAIEC–KO mice) also did not develop spontaneous colitis, although 10–15% of these animals were reported to develop intestinal pathology shortly after birth and died within 4 weeks98. The fact that RELAIEC–KO mice do not develop colitis similarly to the NEMOIEC–KO mice or IKKαIEC–KOIKKβIEC–KO mice indicates that other members of the NF-κB family can

compensate for the lack of RELA in IECs. The generation of mice with combined deficiency of different NF-κB proteins in IECs will be required to address potential functional redundancies between members of the Rel protein family.

Although epithelial cell-specific ablation of IKKβ or RELA did not result in spontaneous intestinal inflam-mation owing to the only partial NF-κB inhibition that was achieved, these animals had increased sensitivity to disease in a model of DSS-induced colitis, which triggers colon inflammation by provoking IEC death and the dis-ruption of the epithelial barrier98,99. IKKβIEC–KO mice were also sensitive to radiation-induced intestinal injury100, and they had increased epithelial destruction but less systemic inflammation after ischaemia–reperfusion of the intestine101. So, although partial inhibition of canoni-cal NF-κB signalling in IECs did not trigger spontaneous disease, it did sensitize epithelial cells to injury. A recent study also provided evidence that epithelial NF-κB con-trols adaptive immune responses in the gut after infec-tion with the parasitic worm Trichuris muris102. when infected with this gut parasite, IKKβIEC–KO mice failed to elicit a protective T helper 2 (TH2) cell response and developed severe TH1 cell-mediated intestinal inflamma-tion102. The immunoregulatory cytokine thymic stromal lymphopoietin (TSLP), which is expressed mainly by epi-thelial cells and is induced by NF-κB signalling103,104, was shown to be essential for a protective TH2 cell response after infection with T. muris102. Decreased levels of TSLP were expressed in the intestine of IKKβIEC–KO mice, which suggests that epithelial IKKβ deficiency prevented the expression of TSLP by IECs, resulting in the develop-ment of pathogenic TH1 cell-mediated inflammation instead of a protective TH2 cell response after T. muris infection. So, NF-κB carries out different functions in IECs that are important for the maintenance of intes-tinal immune homeostasis: NF-κB signalling preserves the normal epithelial barrier under steady state condi-tions and after injury (FIG. 3), and it also controls mucosal adaptive immune responses by inducing the expression of epithelial cell-derived immunoregulatory cytokines such as TSLP.

Hepatocyte nf-κB in liver homeostasisThe first indication that NF-κB signalling is important for hepatocytes came from the analysis of mice lacking RELA, which died in utero as a result of TNF-induced degeneration of the fetal liver33,105,106. IKKβ- or NEMO-deficient mice had the same phenotype12–17, which shows that impaired canonical NF-κB activity sensitizes embry-onic hepatocytes to TNF-mediated toxicity. More recent studies showed that NF-κB activation is also required to protect the adult liver from acute injury caused by in vivo challenge with TNF or lipopolysaccharide (LPS). Mice lacking NEMO or both IKKα and IKKβ in liver paren-chymal cells (LPCs; including hepatocytes and bile duct epithelial cells) had increased sensitivity to TNF-induced liver damage19,107. Interestingly, mice with LPC-specific ablation of IKKα or IKKβ alone were not sensitive to TNF-induced LPC apoptosis19,108,109, which shows that the two IκB kinases are functionally redundant in protecting

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Hepatocyte Dyinghepatocyte

Kupffer cellIntracellular

PRR

PAMPs andtoxic substances

Endothelial cellwith fenestrae

Compensatory hepatocyteproliferation, inflammationand hepatocellular carcinoma

Anti-apoptoticfactors

NF-κBNF-κB

ROS

TLR orother PRR

Death receptor

Death-inducingligand

Sinusoidallumen

Cytokines

the liver from the effects of TNF. Moreover, LPC-specific ablation of RELA110 or overexpression of IκBα-DR111 also sensitized the liver to TNF-induced damage.

In addition to sensitizing hepatocytes to acute TNF-induced injury, NF-κB inhibition in LPCs also affected normal homeostasis in the adult liver. Mice with LPC-specific ablation of NEMO (NEMOLPC–KO mice) devel-oped spontaneous chronic hepatitis characterized by increased hepatocyte apoptosis, liver inflammation, steatosis and compensatory hepatocyte proliferation, together with the activation of liver progenitor cells107. This chronic liver condition resulted in the development of hepatocellular carcinoma (HCC) in NEMOLPC–KO mice. LPC-specific ablation of FADD (FAS-associated via death domain), an adaptor molecule that is essen-tial for death receptor-induced apoptosis112, or feed-ing with the antioxidant butyl-hydroxy-anisol (BHA), could prevent spontaneous hepatocyte apoptosis, liver inflammation, steatosis and hepatocyte proliferation in NEMOLPC–KO mice, showing that the increased death of hepatocytes is the initiating event that triggers chronic

liver disease in these mice. So, LPC-specific deficiency of NEMO causes chronic hepatitis and HCC by sensitiz-ing hepatocytes to death receptor-induced and oxidative stress-dependent toxicity. The initiating events and the molecular mechanisms that trigger spontaneous hepa-tocyte apoptosis in NEMOLPC–KO mice remain unclear at present. LPS, draining from the intestine to the liver through the portal vein, might be involved by induc-ing the TLR-dependent expression of death receptor ligands such as TNF, CD95L (also known as FASL) and TRAIL. Alternatively, other as-yet-unidentified stress-inducing stimuli might induce the death of NEMO-deficient LPCs, thereby triggering liver inflammation and chronic hepatitis (FIG. 4).

Surprisingly, mice with combined ablation of IKKα and IKKβ in LPCs did not recapitulate the liver pheno-type of NEMOLPC–KO mice, but developed a different liver disease characterized by the inflammatory destruction of the small portal bile ducts and severe cholestasis, resulting in the premature death of most animals before the age of 6 months19. This phenotype also developed in mice with combined ablation of NEMO and IKKα, which indicates that IKKα mediates additional functions that are impor-tant for liver homeostasis. These IKKα-specific functions could be mediated by non-canonical NF-κB signalling or by NF-κB-independent pathways, and they seem to be related to the maintenance of the hepatobiliary barrier.

In contrast to NEMO or combined IKKα and IKKβ deficiency, ablation of IKKα, IKKβ or RELA, or expres-sion of IκBα-DR, did not cause spontaneous liver pathol-ogy in mice19,107,108,110,111,113. The most probable explanation for this difference is that the respective genetic manipula-tions only partially inhibited instead of completely blocked NF-κB activation, which was only achieved by ablation of NEMO or both IKKα and IKKβ. However, although it did not affect normal liver homeostasis, partial NF-κB inhibition could sensitize the liver to damage induced by different challenges, such as concanavalin A-induced hepatitis108 or bacterial infection113, whereas it had a protective effect after ischaemia–reperfusion109 or in a model of chemically induced liver carcinogenesis114.

contrasting effects of nf-κB inhibitionAs discussed above, the inhibition of NF-κB activity in skin and intestinal epithelial cells and in liver parenchy-mal cells results in the spontaneous development of severe chronic inflammatory conditions. These effects seem to be tissue specific, as NF-κB inhibition in other organs did not have a similar effect. NF-κB inhibition in the central nervous system (CNS) by conditional ablation of NEMO, IKKα or IKKβ in CNS parenchymal cells did not result in spontaneous pathology, but instead protected mice from autoimmune encephalomyelitis and from ischaemia-induced neuronal death115,116. Also, NF-κB inhibition in skeletal muscle by ablation of IKKβ or overexpression of IκBα-DR did not disturb normal homeostasis, whereas it had protective effects in models of muscle injury38,41,117,118. Furthermore, NF-κB inhibition in exocrine or endocrine pancreas by the overexpression of IκBα-DR or a dominant- negative form of IKKβ, or the ablation of IKKβ or NEMO did not trigger the development of spontaneous

Figure 4 | NF‑κB inhibition in liver parenchymal cells causes hepatitis and hepatocellular carcinoma. Cells in the liver are constantly exposed to toxic substances that need to be detoxified and also to microbial pathogen-associated molecular patterns (PAMPs) that originate from the intestine and reach the liver through the portal circulation. These toxic components can damage liver parenchymal cells (hepatocytes) and also activate Kupffer cells to secrete pro-inflammatory cytokines. Nuclear factor-κB (NF-κB) activation provides survival signals that protect hepatocytes from toxic compounds and also from cytokines secreted by Kupffer cells. NF-κB inhibition sensitizes hepatocytes to death-inducing signals, triggering liver inflammation and compensatory hepatocyte proliferation, and ultimately resulting in the development of hepatocellular carcinoma. PRR, pattern recognition receptor; ROS, reactive oxygen species; TLR, Toll-like receptor.

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p50 RELA

Canonical NF-κB

Survival,pro-inflammatory factor expression and host defence

Pro-inflammatory cytokines

Environmental insults,infection and injury

Innate immune cellEpithelial, parenchymalor stromal cell

Survival andmaintenance of tissue homeostasis

IKKα IKKβ

NEMO

NEMO

IKKα IKKβ

p50 RELA

Canonical NF-κB

inflammation in this tissue119,120 (M. A. Ermolaeva and M.P., unpublished observations). So, NF-κB inhibition could disturb tissue homeostasis and cause spontaneous inflammatory disease in the skin, intestine and liver, but not in the CNS, pancreas or skeletal muscle. Although the mechanisms determining the effect of NF-κB inhibition in each of these tissues remain to be fully elucidated, it is intriguing to note an important difference between tis-sues in which NF-κB inhibition resulted in inflammation and tissues in which it did not. The cell types in which NF-κB inhibition triggered inflammation are in direct contact with the environment (such as skin and intes-tinal epithelial cells) or are exposed to toxic substances or microbial products that have entered the circulation and need to be removed (hepatocytes). By contrast, cells in which NF-κB inhibition did not trigger spontaneous inflammation are located in relatively protected tissues that are normally not in contact with the environment or with toxic substances (such as the CNS, skeletal muscle and pancreas). One exception seems to be lung epithelial cells, in which NF-κB inhibition by IKKβ ablation did not cause spontaneous inflammation40, despite the fact that the lung epithelium is constantly exposed to challenges from inhaled substances, particles and microorganisms. However, IKKβ ablation results in only partial NF-κB inhibition that might not be suf-ficient to trigger spontaneous pathology in the lungs, an effect that has been described in the gut epithelium and in the liver74,107. Therefore, the complete blockade of NF-κB activity by ablation of NEMO or of both IKKα and IKKβ in the lung epithelium might have a more severe effect and cause disease. This hypothesis remains to be tested experimentally.

closing remarksIt is tempting to speculate that NF-κB signalling has evolved to carry out a special function in cells that are exposed to environmental insults, which is crucial for the maintenance of immune homeostasis in the respec-tive tissues. when NF-κB activity in these cells is within a normal range, tissue homeostasis is maintained. However, inhibition (but also increased activation) of NF-κB in these cells triggers an immune response, pre-sumably with the aim of defending the organism from a perceived injury or infection. The molecular mecha-nisms by which NF-κB carries out this function need to be further characterized. However, NF-κB activity seems to regulate the responses of these cells to stress caused by exposure to environmental or toxic insults or to local inflammatory challenges caused by increased cytokine expression at epithelial surfaces and in the liver. Innate immune cells that are present in these tissues, such as macrophages and dendritic cells in the skin and intes-tine and Kupffer cells in the liver, could be responsible for triggering such local inflammatory reactions as they are constantly exposed to invading microorganisms and their products, or to toxic substances and stress-inducing factors from the environment. It is reasonable to assume that these tissues are, under physiological conditions, in a permanent state of sub-clinical inflammation caused by the constant exposure of local innate immune cells to activating stimuli from the environment. In this con-text, NF-κB could serve a dual function, by providing both immune and non-immune cells with the capacity to respond to dangerous insults and, at the same time, protecting epithelial and stromal cells from both toxic challenges and the inflammation they induce by acti-vating local innate immune responses (FIG. 5). In this scenario, a balanced NF-κB response maintains tis-sue integrity and homeostasis and also the ability of the innate immune system to react rapidly to poten-tial threats. An imbalance in NF-κB signalling, such as when NF-κB is inhibited in epithelial or parenchymal cells but not in immune cells (as occurs in the mouse models discussed here), triggers chronic inflammatory conditions. So, NF-κB deficiency in epithelial or paren-chymal cells provides the trigger, but NF-κB-competent immune cells drive the inflammatory response. Clinical studies in human patients with NEMO mutations have provided evidence that the mechanisms by which NF-κB signalling in non-immune cells controls tissue immune homeostasis, at least in the skin and the colon, are similar in humans and mice56,77,78.

Strategies targeting NF-κB undoubtedly have great potential for the treatment of inflammatory diseases. However, the potentially detrimental side effects of NF-κB blockade seem to be a major problem in bring-ing NF-κB inhibitors to the clinic. Studies in animal models such as those discussed here will be invaluable for a better understanding of the mechanisms by which NF-κB activity controls normal tissue homeostasis and protects the organism from disease, which will be essen-tial for the design of therapeutic protocols that block the pathogenic effects of NF-κB without hindering its beneficial functions.

Figure 5 | Balanced NF‑κB signalling is essential for the maintenance of immune homeostasis. In tissues that are exposed to environmental and toxic challenges, nuclear factor-κB (NF-κB) activity in both immune and non-immune cells is important for the maintenance of physiological tissue homeostasis. Danger signals activate innate immune cells by inducing canonical NF-κB activation, resulting in the expression of pro-inflammatory cytokines. NF-κB activation protects non-immune cells from damage caused by environmental insults and from inflammatory cytokines expressed by immune cells, thereby preserving tissue integrity and homeostasis.

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AcknowledgementsI am indebted to all the present and past members of my labo‑ratory for making this work possible. Work in my laboratory is supported by funding from the University of Cologne (Germany), the Human Frontier Science Program, the Deutsche Forschungsgemeinschaft and the European Union.

DAtABAsesOMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIMincontinentia pigmentiUniProtKB: http://ca.expasy.org/sprotA20 | BCL-3 | BCL-X

L | CYLD | FADD | ferritin heavy chain |

FLIP | HBD2 | IκBα | IκBβ | IκBε | IκBζ | IκBNS | IKKα | IKKβ | IL-1β | MBD3 | MnSOD | MYD88 | NEMO | p100 | p105 | REL | RELA | RELB | TAK1 | TNF | TNFR1 | TSLP

furtHer informAtionManolis Pasparakis’ laboratory: http://www.genetik.uni-koeln.de/groups/Pasparakis/

All lINkS Are ActIve IN the oNlINe pdF

R E V I E W S

788 | NOvEMBER 2009 | vOLuME 9 www.nature.com/reviews/immunol

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