Review

Oncogene (2006) 25, 6758–6780. doi:10.1038/sj.onc.1209943

NF-kappaB and the immune response

M S Hayden1, A P West1 and S Ghosh1,2

  1. 1Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA
  2. 2Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USA

Correspondence: Professor S Ghosh, Yale University School of Medicine, Department of Immunobiology, 300 Cedar Street, New Haven, CT 06510, USA. E-mail: sankar.ghosh@yale.edu

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Abstract

One of the primary physiological roles of nuclear factor-kappa B (NF-kappaB) is in the immune system. In particular, NF-kappaB family members control the transcription of cytokines and antimicrobial effectors as well as genes that regulate cellular differentiation, survival and proliferation, thereby regulating various aspects of innate and adaptive immune responses. In addition, NF-kappaB also contributes to the development and survival of the cells and tissues that carry out immune responses in mammals. This review, therefore, describes the role of the NF-kappaB pathway in the development and functioning of the immune system.

Keywords:

NF-kappaB, T-cell receptor, B-cell receptor, inflammation, TLR, hematopoiesis

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Introduction

The discovery and characterization of the nuclear factor-kappa B (NF-kappaB) family of transcription factors resulted from studies in two major areas of research: immunology and cancer biology. Although the role of NF-kappaB in cancer biology is becoming progressively better established, historically much of our current knowledge of NF-kappaB resulted from efforts directed at understanding the regulation and function of the immune response. In keeping with the critical role played by NF-kappaB in different areas of immunology, numerous excellent reviews have been published covering the role of NF-kappaB in Toll-like receptor (TLR) and antigen receptor (AgR) signaling, lymphoid organogenesis and hematopoiesis (Mebius, 2003; Bonizzi and Karin, 2004; Hayden and Ghosh, 2004; Lin and Wang, 2004; Siebenlist et al., 2005; Akira et al., 2006). This review will, therefore, attempt to provide a more comprehensive, if less detailed, review of the diverse functions of NF-kappaB in immunology, with the goal of illuminating how it is that so much in immunology seems to revolve around this family of transcription factors.

Considered broadly, mammalian immune responses can be divided into innate and adaptive responses. The immune response begins with the host recognizing the presence of foreign pathogens, followed by responses at the cellular, tissue and organismal levels, that ultimately lead to the clearance of the pathogen. As such, immune responses can be broken down into individual signal transduction events through which changes in the extracellular environment elicit altered gene expression at the cellular level. In a remarkable number of instances, NF-kappaB is the transcription factor that mediates these transcriptional changes. The gene products characteristic of early events in immune responses include cytokines and other soluble factors that propagate and elaborate the initial recognition event. The activation and modulation of NF-kappaB is also a common target of these factors. Thus, in a surprising number of situations NF-kappaB mediates the critical changes that are characteristic of innate and adaptive immune responses.

In mammals, the NF-kappaB family is composed of five related transcription factors: p50, p52, RelA (aka p65), c-Rel and RelB (see Gilmore, 2006). These transcription factors are related through an N-terminal DNA-binding/dimerization domain, called the Rel homology domain, through which they can form homodimers and heterodimers, which bind to a variety of related target DNA sequences called kappaB sites to modulate gene expression. RelA, c-Rel and RelB also contain C-terminal transcription activation domains (TADs), which enable them to activate target gene expression. In contrast, p50 and p52 do not contain C-terminal transactivation domains; therefore, p50 and p52 homodimers can repress transcription unless they are bound to a protein containing a TAD, such as Bcl-3. Alternatively, p50 and p52 often form heterodimers with RelA, c-Rel or RelB and act as transcriptional activating dimers.

In most cells, NF-kappaB complexes are inactive, residing primarily in the cytoplasm in a complex with any of the family of inhibitory IkappaB proteins. When the pathway is activated, the IkappaB protein is degraded and the NF-kappaB complex enters the nucleus to modulate target gene expression. In almost all cases, the common step in this activating process is mediated by an IkappaB kinase (IKK) complex, which phosphorylates IkappaB and targets it for proteasomal degradation (see Scheidereit, 2006). The IKK complex consists of two catalytically active kinases (IKKalpha and IKKbeta) and a regulatory scaffold protein, NEMO. In what is called the canonical (or classical) pathway, IKKbeta and NEMO are required for the activation of complexes such as p50/RelA, p50/c-Rel, etc., whereas IKKalpha is relatively dispensable. Conversely, in the non-canonical (or alternative) pathway IKKalpha alone controls the activation of complexes that are inhibited by the IkappaB protein p100. These two NF-kappaB pathways can be activated by overlapping but distinct sets of stimuli, and also target activation/repression of overlapping but distinct sets of target genes. One of the most conserved functions of the NF-kappaB signaling pathway is the regulation of the immune system; indeed, NF-kappaB is even the primary regulator of innate immunity in insects such as Drosophila and mosquitoes (see Minakhina and Steward, 2006). This review focuses on the vast role played by NF-kappaB in mammalian immunity.

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Development and formation of the immune system

The mammalian immune system consists of a functionally linked group of anatomically disparate tissues and cell types. The dispersed cellular components of the immune system that arise from the bone marrow receive much of the attention in immunology and the study of NF-kappaB has likewise focused on its role in leukocytes. However, lymphoid organs that facilitate coordination and dissemination of immune responses carried out by immune cells are also key sites of NF-kappaB function. Therefore, whereas this section is largely concerned with the role of NF-kappaB in hematopoiesis, the role of NF-kappaB in lymphoid organogenesis is also discussed briefly.

NF-kappaB and hematopoiesis

Most cells of the immune system are subject to rapid turnover. This process requires the regulation of the competing forces of cell proliferation and cell death – processes heavily influenced by NF-kappaB-regulated genes. Bone marrow-derived hematopoietic cells in particular are subject to high levels of turnover and consequently are particularly sensitive to changes in rates of apoptosis or proliferation. Likewise, during immune responses immune cells selectively undergo rapid expansion that must be resolved by targeted cell death. Although the role of NF-kappaB in development and homeostasis of hematopoietic cells has focused largely on B-cell and T-cell maturation, it is likely that as our understanding increases of the pathways responsible for the development of natural killer (NK) cells, dendritic cells (DCs), macrophages, etc. our appreciation for the role of NF-kappaB in the biology of these cell types will also expand.

Hematopoietic components of the immune system include cells of the lymphoid, myeloid and granulocytic lineages. These lineages give rise to T cells, B cells, monocytes, macrophages, DCs (both myeloid and lymphoid), NK cells, basophils, eosinophils, neutrophils and mast cells (Figure 1). Many cells of the body can contribute to immune responses; however, these bone marrow-derived cells are the core constituents of both the innate and adaptive immune responses. Although NF-kappaB generally plays a prosurvival role in these cells, its function during hematopoiesis is far more nuanced than one might expect. In the current review, we limit our discussion to those instances where the role for NF-kappaB is illustrative of its broader functions in the immune system.

Figure 1.
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Schematic of NF-kappaB in hematopoiesis. Red arrows indicate stages in which NF-kappaB activation is thought to contribute negatively and green arrows indicate a positive function in the development of the indicated lineages. Curved arrows indicate examples in which NF-kappaB contributes to the survival of the mature cell population, either in the resting state or during immune responses. Gray arrows indicate developmental events for which NF-kappaB plays no role or for which the role of NF-kappaB has not being clearly demonstrated. See the text for details.

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Before delving into specific aspects of NF-kappaB function in hematopoiesis, it is worthwhile to discuss the shortcomings of the experimental approaches that are used. For example, embryonic lethality of RelA knockout mice prevents straightforward analysis of the hematopoietic events that are relevant to adult animals. In other instances, severe defects in lymphoid organogenesis in the absence of NF-kappaB make it difficult to determine whether the observed defects are intrinsic to the hematopoietic lineage or are due to alterations in the relevant organ, for example, stromal tissues, within which hematopoietic development occurs. In some instances, such as for RelA or IKKbeta knockouts, embryonic lethality can be rescued by deletion of the tumor necrosis factor-receptor (TNF-R) or tumor necrosis factor-alpha (TNFalpha), which permits analysis of hematopoiesis in these mice, but potentially distorts certain aspects of the hematopoietic pathway. Similar concerns apply to adoptive transfer experiments that can be influenced by the cytokine milieu to which transferred cells are exposed. In each case, therefore, one must ask whether the defect exhibited by a cell lacking some component of the NF-kappaB pathway is relevant to the course of normal hematopoiesis or simply to the experimental system being employed. Finally, there are numerous instances where one NF-kappaB family member can complement the function of another member, or alternatively, where the absence of one family member impedes the function or expression of other family members. Nevertheless, despite these limitations, genetic analysis has unequivocally illustrated a key role for NF-kappaB in the development and survival of hematopoietic cells.

NF-kappaB in development of innate immune cells

DC development is largely dependent on canonical NF-kappaB complexes, although a particular subset appears to require only the non-canonical RelB containing NF-kappaB complexes. RelB is known to facilitate the development of DCs (Burkly et al., 1995; Weih et al., 1995); specifically development of CD8alpha-, but not CD8alpha+, DCs (Wu et al., 1998). Conversely, double knockout studies have shown that canonical p50/RelA complexes are required for the development of both CD8alpha+ and CD8alpha- DCs, but not other myeloid and lymphoid lineages, most likely by mediating the response of DCs to TNFalpha (Ouaaz et al., 2002; Abe et al., 2003). Survival of DCs in the periphery following activation tends to be short, but can be prolonged upon CD40L expression on T cells. CD40L activates both the canonical and non-canonical NF-kappaB pathways and hence DCs deficient in both p50 and c-Rel, or DCs overexpressing a mutant super-repressor form of IkappaBalpha, demonstrate significantly decreased survival (Ouaaz et al., 2002; Kriehuber et al., 2005).

IkappaBalpha knockout mice display robust granulocytosis (Beg et al., 1995), and suggest an antiapoptotic role for NF-kappaB during granulocyte development. Likewise, NF-kappaB has an antiapoptotic role in mature granulocytes. For example, neutrophils, which undergo daily turnover and rapidly apoptose in vitro, exhibit accelerated apoptosis as well as sensitization to proapoptotic stimuli following NF-kappaB inhibition. Unlike lymphocytes, which are relatively long-lived in the absence of activation, protection from apoptosis in neutrophils is more important during the inflammatory response than in homeostasis. Indeed, many TLR ligands increase neutrophil survival in vitro, likely due to NF-kappaB- mediated expression of antiapoptotic genes (Francois et al., 2005). Although neutrophils are capable of activating NF-kappaB in response to many pro-inflammatory stimuli (McDonald, 2004), they lack p52 and RelB (McDonald et al., 1997), the very subunits that are crucial for the maintenance of long-lived lymphocytes. Thus in the case of neutrophils, NF-kappaB fulfills its predicted role as a prosurvival and proinflammatory factor.

The general granulocytosis observed in IkappaBalpha knockout mice suggested that an antiapoptotic role could be broadly assigned to NF-kappaB in this lineage. However, chimeras generated with cells from ikba-/-ikbe-/-mice (i.e., lacking IkappaBalpha and IkappaBalt epsilon) instead display a modest defect in both myelopoiesis and granulopoiesis of transferred cells (Goudeau et al., 2003), and a pronounced defect in NK cells and lymphoid lineages (Samson et al., 2004). Therefore, elevated levels of NF-kappaB activity in these cells appears to exert a proapoptotic effect. Thus, it appears that the role of NF-kappaB in granulopoiesis is selective and cell-type-specific. Furthermore, as described for lymphocyte development (below), the requirement for individual NF-kappaB subunits is not uniform at different developmental stages.

NF-kappaB in development of B and T cells

As in cells of the innate immune system, NF-kappaB is vital for the development and function of adaptive immune cells (Siebenlist et al., 2005). Although lymphocytes may exhibit great longevity in the periphery, their selection in the bone marrow and thymus is characterized by a high rate of apoptosis. As a consequence, the antiapoptotic properties of NF-kappaB play a key role in lymphopoiesis. The centrality of its antiapoptotic function is supported in part by the demonstration that most of the requirements for NF-kappaB during T-cell development can be overcome by transgenic expression of the prototypical antiapoptotic factor Bcl-2 (Sentman et al., 1991). The necessity of NF-kappaB for lymphopoiesis is strikingly illustrated in human genetic diseases wherein the gene encoding NEMO is inactivated by mutation (see Courtois and Gilmore, 2006). Because the NEMO gene is located on the X-chromosome, it is usually subject to random inactivation in individual cells in females. However, in female patients who are heterozygous for a mutant version of NEMO all peripheral lymphocytes possess an intact NEMO gene, rather than the 50% predicted by random inactivation, suggesting that in the absence of NEMO-dependent NF-kappaB signaling, B and T cells fail to develop.

The effects of NEMO inactivation in both mice and humans solidify the role of NF-kappaB in lymphopoiesis, even though the details by which NF-kappaB functions in this process remain obscure. NF-kappaB plays diverse roles in lymphocyte development that can be grouped according to when in development it functions – that is, before, during or after pre-AgR signaling. Although no single NF-kappaB subunit knockout mouse has as severe of a phenotype as NEMO deficiencies with regard to the generation of mature lymphocytes, double knockouts demonstrate that the antiapoptotic function of NF-kappaB is important in the maturation and survival of lymphocytes. For example, loss of both of the canonical NF-kappaB family members p50 and RelA, or both RelA and c-Rel, halts development early in lymphopoiesis, before expression of the pre-AgRs (Horwitz et al., 1997; Grossmann et al., 1999), suggesting that NF-kappaB is involved in the expression of antiapoptotic factors required for early lymphoid cell survival in response to proapoptotic stimuli (Figure 2). In fact, early CD34+ bone marrow cells can activate NF-kappaB in response to TNFalpha, and in these cells NF-kappaB acts as a prosurvival factor (Pyatt et al., 1999).

Figure 2.
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NF-kappaB function during lymphopoiesis. NF-kappaB plays a prosurvival role in common lymphoid precursor cells that give rise to B- and T-cell lineages. B-cell development occurs in the bone marrow, where NF-kappaB protects pre-B cells from proapoptotic stimuli including TNFalpha. Signaling to NF-kappaB through the pre-B-cell receptor mediates survival of pre-B-cells that then undergo light chain recombination to produce a functional B-cell receptor. Expression of BCR leads to NF-kappaB-dependent differentiation into immature B cells. High levels of BCR signaling, that is, through recognition of self-antigen, results in negative selection through the loss of NF-kappaB activity. Transitional B cells exit the bone marrow and migrate to the spleen where they mature and differentiate, a process that also requires NF-kappaB. T-cell development occurs following migration of precursor cells into the thymus. Stimulation of NF-kappaB through pre-TCRalpha provides a prosurvival signal allowing recombination of the TCRalpha chain and maturation to the DP stage. Optimal signaling through the TCRalpha/beta complex induces NF-kappaB-dependent survival pathways, whereas a failure to signal or high-level signaling results in death by neglect or negative selection, respectively. NF-kappaB activity is required for the maintenance of long-lived B and T cells.

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Evidence suggests that expression of the pre-AgR leads to survival signals that depend, at least in part, on NF-kappaB. For example, pre-T-cell receptor (pre-TCR) expression in double negative (DN; CD8-CD4-) thymocytes coincides with high levels of NF-kappaB activity, and NF-kappaB activity at this stage is necessary for DN survival and maturation (Figure 2). Therefore, enforced IKKbeta activation eliminates the requirement for TCR recombination, whereas inhibition of NF-kappaB by expression of an IkappaBalpha super-repressor decreases DN thymocyte maturation and survival (Voll et al., 2000). Signaling through the pre-B-cell receptor (pre-BCR) also likely induces antiapoptotic signals through NF-kappaB. Consequently, the reduced pre-B-cell population seen upon expression of the IkappaBalpha super-repressor in bone marrow cells can be rescued by overexpression of the antiapoptotic NF-kappaB target gene Bcl-XL (Feng et al., 2004; Jimi et al., 2005). However, whereas evidence points toward NF-kappaB-mediated production of antiapoptotic factors it remains unclear how NF-kappaB is activated downstream of the pre-AgR.

Selection of DP (double positive; CD4+CD8+) thymocytes depends on the ability of their TCR to recognize peptide:MHC (major histocompatibility complex) complexes. Thymocytes that express a TCR that is unable to bind MHC die in a process termed 'death by neglect', whereas those that bind peptide:MHC are either positively or negatively selected depending on the strength of this interaction. Thymocytes that bind self-peptide:MHC with very high affinity are likely to be self-reactive, and hence are deleted through negative selection. Thus, only DP thymocytes that recognize self-peptide:MHC with an affinity that falls within a defined range are positively selected to become single-positive T cells. Somewhat counterintuitively, it appears that NF-kappaB functions in both positive and negative selection of thymocytes. During negative selection, NF-kappaB facilitates the induction of apoptosis following high-affinity TCR ligation (Hettmann et al., 1999; Mora et al., 2001b), perhaps by facilitating expression of proapoptotic genes and the consequent sensitization to proapoptotic signals (French et al., 1996; Kishimoto et al., 1998). The role of NF-kappaB in positive selection of thymocytes is more in keeping with the better-established role of NF-kappaB as an inducer of antiapoptotic genes. Unlike in thymocytes, however, NF-kappaB functions as a prosurvival factor during negative selection of B cells. Immature B cells display constitutive NF-kappaB activity that is down-regulated following BCR ligation (Wu et al., 1996). Decreased NF-kappaB activity might then sensitize these cells to proapoptotic signals. Interestingly, some signaling components required for NF-kappaB activation in mature B and T cells can be genetically disrupted without affecting their development, suggesting that pathways leading to activation of NF-kappaB in developing B or T cells differ significantly from the pathways engaged following AgR ligation in mature lymphocytes.

Following positive and negative selection, DP thymocytes must make a lineage commitment and become single positive (SP) thymocytes (CD4+CD8- or CD4-CD8+), which shortly thereafter emigrate from the thymus. Analyses of kappaB-site luciferase transgenic reporter mice have shown that CD8 SP cells have significantly higher levels of NF-kappaB activity than CD4 SP thymocytes (Voll et al., 2000). Conversely, the antiapoptotic factor Bcl-2 is more highly expressed in CD4 than CD8 cells, suggesting that CD8 SP thymocytes are more dependent on NF-kappaB for survival. However, during the course of the development from the DP stage to emigration from the thymus, both DP and SP lineages require NF-kappaB. Targeted deletion of floxed-NEMO using cd4-promoter-driven Cre recombinase expression, or overexpression of kinase dead IKKbeta, results in loss of mature peripheral T cells (Schmidt-Supprian et al., 2004). These data strongly suggest that NF-kappaB activation is required for late stages of T-cell development; however, ikkb-/-,tnfr1-/- double knockouts, ikkb-/- chimeras or cd4-Cre IKKbeta conditional knockouts are not defective in the production of naïve T cells (Senftleben et al., 2001b; Schmidt-Supprian et al., 2004), suggesting a requirement for NEMO but not IKKbeta.

Immature B cells exit the bone marrow, becoming transitional B cells, and complete development into either follicular or marginal zone B cells. NF-kappaB-regulated expression of prosurvival factors is important to these final steps of B-cell development (Grossmann et al., 2000). Interestingly, the activation of NF-kappaB in late B-cell maturation is the result of signaling by both canonical and non-canonical NF-kappaB pathways. Thus deficiency in NEMO, IKKalpha or IKKbeta decreases the numbers of mature B cells (Kaisho et al., 2001; Senftleben et al., 2001b; Pasparakis et al., 2002). Likewise, either p50/p52 or RelA/c-Rel double knockout progenitor cells are defective in their ability to mature beyond the transitional B-cell stage (Franzoso et al., 1997; Grossmann et al., 1999). A requirement for both the canonical and non-canonical NF-kappaB pathways may explain why deletion of p50 and p52 produces a more complete block in B-cell development than loss of RelA and c-Rel. Although both canonical and non-canonical NF-kappaB pathways are functional during B-cell development, recent work (Batten et al., 2000; Schiemann et al., 2001; Claudio et al., 2002) has underscored the importance of BAFF ligation in selectively activating the non-canonical NF-kappaB pathway and the consequent expression of antiapoptotic Bcl-2 family members in transitional B cells (see Mackay et al., 2003). Indeed, BAFF knockout mice exhibit a complete failure of transitional B-cell maturation, which mirrors that seen in Bcl-XL knockout mice (Motoyama et al., 1995; Gross et al., 2001; Schiemann et al., 2001). Thus, only those knockouts that target both the canonical and non-canonical NF-kappaB pathways have an effect that approximates the phenotype seen in BAFF or Bcl-XL deficiency.

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NF-kappaB and lymphoid organogenesis

In addition to its role in the development of cells that directly mediate immune responses, NF-kappaB also plays an important role in the development and function of primary and secondary lymphoid tissues. Primary (central) lymphoid organs include the bone marrow and thymus whereas secondary (peripheral) lymphoid organs include lymph nodes (LNs), Peyer's patches, mucosal-associated lymphoid tissue (MALT) and the spleen. Among the primary lymphoid organs, the bone marrow remains active throughout life, whereas thymic activity dwindles with the onset of adulthood. The secondary lymphoid tissues are associated with the maintenance and activation of mature lymphocytes, and provide an environment within which the interaction of lymphocytes and other leukocytes can be carefully orchestrated. Although there is clearly a role for NF-kappaB in the development and regulation of bone, this role has not yet been clearly correlated with effects on hematopoiesis (Jimi and Ghosh, 2005). Careful anatomical and histological examination of NF-kappaB-deficient mice has resulted in NF-kappaB being assigned an increasingly prominent role in lymphoid organogenesis.

Secondary lymphoid organs

The secondary lymphoid organs have highly characteristic structural features that are crucial to the development and activation of lymphocytes. Analysis of the role of NF-kappaB in lymphoid organogenesis in knockouts has been complicated by the necessity of interfering with the TNF response to rescue the lethality associated with NF-kappaB deficiency. The initial events of lymphoid organogenesis involve the association of lymphotoxin (LT)alpha1beta2-expressing hematopoietic cells and vascular cell adhesion molecule-1 (VCAM1)-expressing stromal cells (for a review see Mebius, 2003). This interaction initiates a positive feedback-signaling loop in which NF-kappaB plays a prominent role (Figure 3). Cytokines implicated in this signaling loop – LTalpha1beta2, RANKL (receptor activator of NF-kappaB ligand) and TNFalpha – are known to activate NF-kappaB. Also, mediators of lymphoid organogenesis and homeostasis, such as the adhesion molecules ICAM (intercellular adhesion molecule), VCAM, PNAd (peripheral node addressin), GlyCAM-1 (glycosylation-dependent cell adhesion molecule) and MadCAM (mucosal addressin cellular adhesion molecule), cytokines including TNFalpha, and organogenic chemokines such as CXCL12 (GRO/MIP-2), CXCL13 (BLC), CCL19 (ELC) and CCL21 (SLC), are regulated by NF-kappaB.

Figure 3.
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NF-kappaB function in the early events of lymphoid organogenesis. NF-kappaB is a vital part of the positive feedback loop between hematopoietic and stromal cells that comprises the early events of lymphoid organogenesis. LTalpha1beta2-expressing hematopoietic cells induce production of VCAM-1 through the canonical NF-kappaB pathway and chemokines through the non-canonical (IKKalpha-dependent) pathway in LTbetaR-expressing stromal cells. Stromal expression of chemokines induces the upregulation of integrins (alpha4beta1) on hematopoietic cells resulting in increased recruitment of LTalpha1beta2-expressing cells and signaling through stromal LTbetaR. RANKL stimulation of NF-kappaB through TRAF6 is also crucial for the upregulation of LTalpha1beta2 in hematopoietic cells.

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Lymphoid organogenesis exhibits distinct requirements for both the canonical and non-canonical NF-kappaB pathways. Signaling through TNF-R, LTbetaR and RANK activates canonical RelA-containing complexes and, hence, it is not surprising that rela-/-/tnfr1-/- double knockout mice lack Peyer's patches and LNs and exhibit disorganized spleens (Alcamo et al., 2002). The requirement for RelA in development of these tissues lies with the stromal cells and is likely due to a combination of effects: regulation of apoptosis (e.g., that induced by TNF); regulation of expression of organogenic factors including VCAM and LTalpha1beta2; and enhancement of the non-canonical p52/RelB pathway through the LTbetaR signaling pathway.

Several lines of evidence highlight the importance of the non-canonical pathway and activation of p52/RelB complexes in LN development. Mice with a point mutation in nik (aly/aly mice) lack multiple secondary lymphoid organs (Miyawaki et al., 1994; Koike et al., 1996; Shinkura et al., 1999) and share several phenotypic similarities with lymphotoxin and IKKalpha single knockout animals (Mebius, 2003; Bonizzi and Karin, 2004). p52/RelB, which is activated downstream of NIK and IKKalpha, is thought to be the primary transcriptional mediator of several key organogenic factors including CXCL12, CXCL13, CCL19, CCL21 and MadCAM-1 (Yilmaz et al., 2003). The p52 single knockout lacks normal B-cell follicles, germinal centers (GCs) and Peyer's patch development (Caamano et al., 1998; Franzoso et al., 1998; Paxian et al., 2002); RelB is likewise also required for Peyer's patch development (Yilmaz et al., 2003). Although LN development occurs in RelB knockout mice, the nodes are small at birth and are resorbed perinatally. In addition to LTbetaR, knockouts of RANK, which likewise signals through the non-canonical pathway, also lack peripheral LNs (Dougall et al., 1999).

Splenic architecture is crucial for B-cell development as well as for the initiation and maturation of B-cell responses. The spleen is divided histologically into white and red pulp zones. Macrophages in the red pulp are responsible for destroying erythrocytes that are damaged or have reached the end of their lifespan. The white pulp is populated by splenic lymphocytes and consists of B-cell follicles and T-cell zones. Splenic architecture allows for dynamic changes, most notably in the formation of GCs, during the initiation and maturation of B-cell responses. Multiple NF-kappaB knockouts exhibit defects in some aspect of splenic architecture; however, as for other lymphoid organs, the analysis of splenic architecture has been complicated by defects that occur upon deletion of TNF-R used to rescue the embryonic lethality. Nevertheless, there has been considerable progress in deciphering the role of NF-kappaB family members in development and maintenance of splenic architecture. Mice in which RelA has been targeted for deletion exhibit aberrant segregation of B- and T-cell areas and defects in one particular macrophage population, the metallophilic marginal zone macrophages. In addition, rela-/-/tnfr1-/- spleens have a more pronounced defect in GC generation following immunization, than do tnrf1-/- mice (Alcamo et al., 2002). However, it is worth emphasizing that defects observed in tnfr1-/- animals may, in fact, be due to changes in RelA-dependent responses, and it is possible that the role of the RelA/canonical NF-kappaB pathway in the spleen is underappreciated.

The importance of the non-canonical pathway in the spleen has been observed in multiple circumstances. Mice in which non-canonical pathway components, RelB, NIK or IKKalpha, have been inactivated demonstrate severe defects in splenic architecture, similar to that seen in ltbr-/- spleens. These defects largely reflect deficiencies in splenic stromal cells (Miyawaki et al., 1994; Koike et al., 1996). Mice deficient in the non-canonical pathway fail to segregate B-cell–T-cell zones and FDC networks, and they fail to form GCs following immunization. Marginal zone macrophages, which line the border between red and white pulp areas, are also absent or disorganized in RelB, p52, NIK or IKKalpha knockouts (Franzoso et al., 1998; Weih et al., 2001). Some splenic defects are also attributable to effects on hematopoietic cells. For example, the presence of metallophilic marginal zone macrophages depends on p52 (Franzoso et al., 1997). Finally, knockout of the atypical IkappaB family member BCL-3 also leads to alterations in lymphoid architecture that are reminiscent of those seen in the absence of p52, with which BCL-3 forms a transcriptionally active complex. BCL-3 knockout mice lack splenic GCs, and although they exhibit normal serum antibody levels, they fail to develop antigen-specific humoral responses (Sha et al., 1995; Caamano et al., 1996; Franzoso et al., 1997; Schwarz et al., 1997).

In summary, both the canonical and non-canonical NF-kappaB pathways are required for the development of most secondary lymphoid organs. However, the role of the non-canonical pathway, as assessed by examining mice deficient for IKKalpha, p52, NIK or RelB, is especially important both during organogenesis and maintenance of splenic architecture. Recent data suggest that the non-canonical pathway is also important in thymic development and organization (Burkly et al., 1995; Weih et al., 1995; Kajiura et al., 2004; Kinoshita et al., 2006). However, it is important to note that canonical NF-kappaB pathway function in these events may be underappreciated owing to embryonic lethality and complicated by the defects introduced by crossing them onto the tnfr-/- background. Nevertheless, our understanding of non-canonical pathway function in secondary lymphoid organs is consistent with the ability of RelB-containing complexes to regulate genes encoding key organogenic chemokines and adhesion molecules that direct leukocyte trafficking. The functional consequences of defects in these processes are severe and have direct ramifications for the host's ability to mount a robust immune response. Alterations in lymphoid architecture likewise impede the initiation of the adaptive response as well as the fine-tuning of this response through processes such as B-cell affinity maturation.

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Role of NF-kappaB in the innate response

Pattern recognition receptors

To activate an appropriate immune response, the host must first recognize the presence of pathogens. This discrimination between self and non-self is an absolute requirement for the initiation of effector functions, such as the secretion of cytokines and antimicrobial peptides, carried out by the cells of the innate immune system. A number of pattern recognition receptors (PRRs) have evolved to recognize microbial invaders. These PPRs include TLRs, members of the CATERPILLAR/NOD family of cytoplasmic receptors, scavenger receptors and the complement system. Although epithelial cells are frequently the first to encounter pathogens, they are also constantly exposed to non-pathogenic microbes. Therefore, whereas a variety of TLRs are differentially expressed in epidermis, gut, pulmonary, urinary and reproductive epithelium, in many cases it is thought that both TLR expression and responsiveness is tightly controlled in these cells. For example, keratinocytes upregulate TLRs expression and responsiveness following transforming growth factor-alpha (TGFalpha) exposure (Miller et al., 2005); renal epithelial cells increase expression of TLR2 and TLR4 in response to IFNitalic gamma or TNFalpha (Wolfs et al., 2002) and intestinal epithelial cells have been shown to alter TLR expression under inflammatory conditions (Mueller et al., 2006). However, sentinel cells of the innate immune system, particularly tissue resident DCs and macrophages, express a more complete complement of PRRs, and thus are likely to bear the largest portion of the burden in the earliest events of pathogen recognition.

Toll-like receptors

TLRs are evolutionarily conserved PRRs that recognize unique, essential molecules characteristic of various classes of microbes (Akira et al., 2006). The function of TLRs as arbitrators of self/non-self discrimination highlights their central role in innate immunity as well as in the initiation of the adaptive immune response. The 11 characterized mammalian TLRs have varied tissue distribution and serve as recognition receptors for pathogen-associated molecular patterns (PAMPs) present on bacteria, viruses, fungi and parasites. Perhaps due to the multimeric nature of the TLR extracellular domain (ED), which consists of multiple leucine-rich repeats (LRRs), several receptors are capable of recognizing more than one microbial molecule (Figure 4 and below). Heterodimerization of some TLRs and the use of co-receptors (e.g., CD14 and MD-2) further expand the repertoire of PAMPs recognized. As we shall see below, the ability of TLRs to distinguish between pathogen types is translated into appropriate innate and adaptive responses through the selective activation of NF-kappaB and other inducible transcription factors. Significant progress has been made over the past few years in deciphering the relevant signaling pathways that operate downstream of TLRs in particular.

Figure 4.
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PRRs that signal to NF-kappaB and their cognate ligands. TLRs 3, 7, 8, 9 and 11 have been reported to exhibit endosomal or intracellular localization whereas NOD1, NOD2, RIG-I and MDA5 function in the cytoplasm.

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TLR signaling to NF-kappaB

Ligand binding to TLRs is just now beginning to be understood at the molecular level. Extracellular LRRs bind to ligand and, either through receptor oligomerization and/or induction of a conformational change across the plasma membrane, induce the recruitment/activation of adapter proteins through the Toll/IL-1 Receptor (TIR) domain. These adapters lead to the activation of canonical IKKbeta-dependent complexes, degradation of IkappaBalpha and IkappaBbeta, and liberation of, primarily, RelA and c-Rel containing NF-kappaB complexes. TLR signaling to NF-kappaB is divided into two pathways: those that are MyD88 (myeloid differentiation primary response gene 88)-dependent and those that are MyD88-independent (Figure 5). We will base our discussion primarily on signaling events emanating from TLR4, which despite having the most complex downstream pathways is the most thoroughly studied TLR. Clear differences exist in signaling from other TLRs as noted throughout our discussion, and it is likely that further specializations will become apparent as individual TLR signaling pathways are investigated more thoroughly.

Figure 5.
Figure 5 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

PRR signaling to NF-kappaB. Signaling through LPS/TLR4 via the MyD88-dependent (b) and TRIF-dependent (a) pathways converge on IKK activation through TRAFs. Signaling through dsRNA/RIG-I (c) proceeds through ISP1 to IKKi/TBK1 and through RIP1 to IKK. Signaling from NOD to NF-kappaB (d) is thought to involve oligomerization of RIP2 and activation of IKK through induced proximity. See the text for details.

Full figure and legend (226K)

MyD88-dependent signaling to NF-kappaB
 

TLR4 signaling is relatively unique amongst TLRs in that the effector adaptors are one step removed from the receptor. For example, MyD88 recruitment to the receptor complex depends upon the TIR-domain containing adapter protein (TIRAP, also known as Mal) (Fitzgerald et al., 2001; Horng et al., 2002; Yamamoto et al., 2002). TLR2 also requires TIRAP to bridge MyD88 to the receptor; however it is believed that other MyD88-utilizing TLRs directly recruit MyD88. Recent reports suggest that the requirement for these intermediatory adapters is related to localization of the TLRs to certain domains in the plasma membrane (Kagan and Medzhitov, 2006; Rowe et al., 2006). The N-terminal domain of MyD88 contains a death domain (DD) that recruits the DD-containing serine/threonine kinase interleukin-1-associated kinase-4 (IRAK-4). IRAK-4 and IRAK-1 form an active complex capable of recruiting the TNF receptor-associated factor TRAF6 (Figure 5a). The link between TRAF6 and the IKK complex remains somewhat enigmatic, although a few key players are known. The kinase TAK1 (TGFbeta-activated kinase-1) is required for NF-kappaB, as well as AP-1 and extracellular signal-related kinase (ERK), activation downstream of MyD88 (Sato et al., 2005; Shim et al., 2005). Although it is widely accepted that ubiquitination is a key switch at this crucial step of NF-kappaB activation, considerable work at the molecular level remains to be done to understand how ubiquitination leads to activation.

In addition to TAK1, another protein, termed ECSIT (evolutionarily conserved signaling intermediate in Toll pathways), was identified because of its interaction with TRAF6. ECSIT binds to TRAF6 and is required for TLR and interleulin-1 (IL-1) signaling, but not TNF-signaling (Kopp et al., 1999; Xiao et al., 2003). Although these studies suggested that ECSIT functions by recruiting and activating the kinase MEKK1 (mitogen activated protein kinase or ERK kinase (MEK) kinase 1) (Kopp et al., 1999; Xiao et al., 2003), the role of MEKK1 in TLR signaling remains unclear (Xia et al., 2000; Yujiri et al., 2000). MEKK3-deficient cells, however, do not transcribe IL-6 following TLR4 or IL-1R stimulation and exhibit delayed and weakened NF-kappaB DNA binding following lipopolysaccharide (LPS) stimulation (Huang et al., 2004). ECSIT interacts with both TAK1 and MEKK3 (AP West and S Ghosh, unpublished observations) and it is, therefore, possible that ECSIT exerts its role in TLR signaling by modulating the function of TAK1 and/or MEKK3.

TRIF-dependent signaling to NF-kappaB

Somewhat unexpectedly, when exposed to LPS, MyD88-/- cells display partial NF-kappaB activation, albeit with slower kinetics than in wild-type cells (Kawai et al., 1999). When cells are stimulated through TLR3 and TLR4, TRIF (TICAM-1), a TIR-domain containing adapter, mediates activation of NF-kappaB in the absence of MyD88 (Oshiumi et al., 2003). Furthermore, in the case of TLR3, all downstream signaling appears to be TRIF-dependent. In TLR4 signaling, TRIF is required for late-phase NF-kappaB and IRF3 responses, but is not required for activation of JNK (Yamamoto et al., 2003). TRIF signaling to NF-kappaB and IRF3 also appear to be separately regulated (Figure 5b). Signaling to IRF-3 occurs through two divergent members of the IKK family, IKKi (IKKalt epsilon) and TBK1 (T2K) (Fitzgerald et al., 2003; Sharma et al., 2003); however, neither kinase is required for NF-kappaB activation by LPS or TNFalpha (Hemmi et al., 2004; McWhirter et al., 2004). Furthermore, reconstitution of trif-/- cells with mutant TRIF lacking the TRAF-binding domain selectively restores induction of IRF3 but not NF-kappaB. Increasingly, therefore, it appears that the events leading from TRIF to IKK activation share a common set of intermediates as seen in other NF-kappaB activation pathways.

TRIF interacts with receptor interacting protein (RIP)1 and RIP3 through their RIP homotypic interaction motif (RHIM), and rip1-/- embryonic fibroblasts have decreased NF-kappaB activation following TLR3-poly(I:C) signaling (Meylan et al., 2004). Finally, another TIR-domain containing adapter TRAM (TRIF-related adapter molecule) functions upstream of TRIF in MyD88-independent signaling from TLR4. TRAM is required for IRF3 activation and for the delayed phase of NF-kappaB activation following TLR4 engagement. TLR4-induced IRAK activation by MyD88, however, is unaffected by the absence of TRAM and TRAM does not function in TLR3 TRIF-dependent signaling pathways (Fitzgerald et al., 2003; Yamamoto et al., 2003). Therefore, it appears that TRAM is only needed for TRIF signaling downstream of TLR4. Adding further complexity, it has recently been suggested that TLR4, but not TLR3, TRIF-dependent NF-kappaB activation is largely due to IRF3-induced TNFalpha rather than to direct signaling to IKK (Covert et al., 2005; Werner et al., 2005). Although the applicability of these finding to other cell types is, as of yet, unclear, these results may be explained by differences in the recruitment of TRIF to the receptor; that is, by TRAM in the case of TLR4 versus directly to TLR3, resulting in changes in the availability of TRAF binding site or the availability of additional signaling intermediates at distinct subcellular localizations.

Negative regulation of TLR signaling

Inflammatory responses are built upon waves of cytokine production and positive feedback mechanisms. As a result, tight control must be placed on the initiation and maintenance of these responses. Multiple negative feedback loops have been described that involve proteins that are induced or activated upon TLR signaling. In a number of instances, the target of these regulatory mechanisms is the IRAK family of proteins. For example, IRAK-M (IRAK3) inhibits signaling to TRAF6 by fixing IRAK-1/4 to the TLR/MyD88 signaling complex; irakm-/- knockouts exhibit enhanced signaling to NF-kappaB (Kobayashi et al., 2002). Tollip, an adapter protein constitutively associated with IRAK, is phosphorylated and dissociates following IRAK4 activation (Burns et al., 2000; Zhang and Ghosh, 2002). Negative regulation of TLR signaling by Tollip in the intestinal epithelium may prevent inflammatory responses to commensal bacteria (Melmed et al., 2003). However, Tollip-deficient cells demonstrate only minor defects in the production of NF-kappaB-regulated cytokines (Didierlaurent et al., 2006). Therefore, it is unclear whether Tollip indeed functions as initially thought. SIGIRR (TIR8), a member of the IL-1R family, binds to Toll/IL-1 receptors, IRAK and TRAF6 and may also function by inhibiting the association of IRAK with TLRs (Thomassen et al., 1999; Wald et al., 2003). SIGIRR deficiency yields prolonged activation of NF-kappaB by Toll/IL-1 stimulation consistent with a regulatory function. Interestingly, SIGIRR is also highly expressed in epithelial cells, suggesting that it too may suppress signaling at sites of constitutive microbial exposure. Finally, suppressor of cytokine signaling-1 (SOCS-1) has been reported to negatively regulate LPS signaling to NF-kappaB and socs1-/- mice exhibit an inflammatory phenotype that is consistent with this prediction (Kinjyo et al., 2002; Nakagawa et al., 2002). SOCS-1 may function by directly targeting TIRAP/Mal, and selectively inhibit TLR4 signaling through the TIRAP/Mal/MyD88 pathway (Mansell et al., 2006).

In addition to these TLR-specific regulators of signaling to NF-kappaB, other proteins function to control the extent and duration of NF-kappaB activation. These factors both set thresholds for activation and help to prevent uncontrolled, and potentially deleterious, innate immune responses. The broad array of PAMPs recognized by the TLR system affords the host the ability to mount responses against many pathogens. Nevertheless, for some pathogens, TLRs alone are not sufficient, and some physical spaces, most notably the cytosol, are not effectively monitored by TLRs.

Cytoplasmic PRRs that activate NF-kappaB

PRRs that recognize bacterial PAMPs are expressed at the plasma membrane or with LRRs projecting into the lumen of vesicles that are topologically related to the extracellular space. However, in such a system, intracellular pathogens are uniquely protected from detection. Furthermore, viral infection and the resulting induction of interferon occurs in many cells that do not express the full panoply of antiviral TLRs – suggesting that other PRRs must be at work. In fact, cells do have at their disposal families of cytoplasmic PRRs that are capable of activating NF-kappaB and other transcriptional mediators of the innate immune response. Interestingly, many of these PRRs contain caspase activation and recruitment domains (CARDs) that are required for activation of NF-kappaB following ligand binding. Here, we provide a brief description of two classes of cytoplasmic PRRs – CARD-containing members of the CATEPILLAR and DExD/H-box helicase families.

CATEPILLER-NODs
 

Nucleotide oligomerization domain proteins (NOD) 1 and 2 are part of a large family termed the CATEPILLER family, which is named for CARD, transcription enhancer, R (purine)-binding, pyrin, lots of leucine repeats (Figure 4). The NOD-LRR subfamily is typified by the presence of LRRs and nucleotide oligomerization domains. NOD1, NOD2 and IPAF have CARDs and can signal to NF-kappaB (for a review see Inohara and Nunez, 2003). NOD1 recognizes a peptidoglycan containing meso-diaminopimelic acid (meso-DAP) and induces NF-kappaB through a canonical pathway that includes activation of IKKbeta. NOD2 recognizes muramyl dipeptide, a ubiquitous component of nearly all bacterial cell walls. Relatively, few signaling intermediates downstream of NOD-LRRs are known; however, there is growing evidence that the CARD-containing kinase RIP2 (RICK) is required for NF-kappaB activation. Intriguingly, the ATP-binding cassette of both NOD1 and NOD2 is needed for signaling (Tanabe et al., 2004). RIP2 binds to NEMO and therefore is thought to directly mediate activation of the IKK complex by induced proximity (Inohara et al., 2000). In this model, ligand-dependent oligomerization of NOD-LRRs, which is dependent on the ATP-binding cassette, leads to a scaffold containing multiple RIP2 molecules, which allows trans-autophosphorylation of neighboring IKK complexes (Figure 5d).

Retinoic acid inducible gene I and melanoma differentiation-associated gene 5

Two members of the DExD/H-box RNA helicase family stand out because of the presence of N-terminal CARD domains. Retinoic acid inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are RNA helicase-containing cytoplasmic proteins. The RNA helicase domains of RIG-I and MDA5 bind directly to double-stranded RNA (dsRNA) and induce production of type I interferons (Kang et al., 2002; Andrejeva et al., 2004; Yoneyama et al., 2004). Upon binding to dsRNA, representing either the viral genome or viral replication intermediate, RIG-I and MDA5 induce the activation of IRF3 and NF-kappaB. Interestingly, initiation of these signaling cascade is abrogated by point mutations in the Walker-type ATP-binding site, suggesting that their ATPase activity is required for signaling (Yoneyama et al., 2004). The link between these two proteins and NF-kappaB remains somewhat unclear (Figure 5c); however overexpression of the N-terminal CARD domain alone is sufficient to induce signaling. Recently, a CARD-containing protein, variably named CARDIF, IPS1, MAVS and VISA, has been implicated downstream of RIG-I; however, the link between this protein and IKKbeta is unclear (Kawai et al., 2005; Meylan et al., 2005; Seth et al., 2005; Xu et al., 2005). It appears, however, that there are similarities to TRIF mediated signaling, in that RIG-I activation of NF-kappaB requires FADD and RIP-1 (Balachandran et al., 2004; Yoneyama et al., 2005). Recently, it was shown that RIG-I and MDA5 differentially recognize various groups of RNA viruses and are thus critical for a robust antiviral response (Kato et al., 2006).

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Pathogen recognition in innate immunity

Bacterial recognition

Pathogens recognized by PRRs can be categorized as bacterial, viral or eukaryotic. In each of these categories, PAMPs have been described that more or less fit with existing hypotheses of how pathogen recognition by the innate immune system should occur (Janeway, 1989). Both in terms of accessibility and uniqueness to prokaryotes, the bacterial cell well is a logical source of PAMPs for TLRs and other PRRs.

LPS was originally thought to be the ligand for TLR2, but subsequent studies revealed that contaminating bacterial lipoprotein in LPS preparations is the actual ligand (Wetzler, 2003). TLR2 also mediates responses to several Gram-positive bacterial cell wall components as well as Staphylococcus aureus peptidoglycan (Takeuchi et al., 2000). Additional work has shown that TLR2 is involved in the recognition of a wide range of microbial products and generally functions as a heterodimer with either TLR1 or TLR6 (Ozinsky et al., 2000; Wyllie et al., 2000). The TLR2/TLR1 heterodimer recognizes a variety of lipoproteins, including those from mycobacteria and meningococci (Takeuchi et al., 2002; Wetzler, 2003), whereas the TLR2/TLR6 complex recognizes mycoplasma lipoproteins and peptidoglycan (Takeuchi et al., 2001). Recent reports have demonstrated that triacylated lipoproteins from bacteria are preferentially recognized by the TLR1/TLR2 complex, whereas diacylated lipoproteins are recognized by the TLR2/TLR6 complex (Takeuchi et al., 2002). However, additional TLR2 ligands do not require TLR1 or TLR6 for signaling, implying that TLR2 recognizes some ligands as a homodimer or heterodimer with other non-TLR molecules. Such TLR2 ligands include the Gram-positive cell wall component lipoteichoic acid; the mycobacterial cell wall component lipoarabinomannan; atypical LPS produced by Legionella, Leptospira interrogans, Porphyromonas gingivitis and Bordetella; and porins present in the outer membrane of Neisseria (Massari et al., 2003; Wetzler, 2003).

The TLR4 ligand LPS, a glycolipid component of the outer membrane of Gram-negative bacteria, is the most thoroughly studied and the most potent TLR ligand known. Trace amounts of LPS activate the innate immune system via TLR4, leading to the production of numerous proinflammatory mediators, such as TNFalpha, IL-1 and IL-6. TLR4-mediated responses to LPS require CD14 and MD-2. Other bacterial TLR4 ligands include Lipid A analogs (Lien et al., 2001) and mycobacterial components (Means et al., 1999).

TLR5 recognizes flagellin, a protein component of Gram-negative bacterial flagella and virulence factor for multiple human pathogens (Hayashi et al., 2001). In light of the fact that it was thought that proteins would be too mutable to serve as PAMPs, it is notable that TLR5 recognizes a highly conserved, central core structure of flagellin that is essential for protofilament assembly (Smith et al., 2003). Interestingly, the TLR5 recognition site is masked in the filamentous flagellar structure, thus indicating that TLR5 recognizes only monomeric flagellin (Smith et al., 2003). Furthermore, flagellin appears to bind directly to TLR5 at residues 386–407, as TLR5 mutants lacking this domain are unable to interact with flagellin in biochemical assays (Mizel et al., 2003). Recent articles have demonstrated TLR5-independent recognition of cytosolic Salmonella typhimurium flagellin via Ipaf, a member of the NOD-LRR family (Franchi et al., 2006; Miao et al., 2006). Ipaf-mediated recognition of cytosolic flagellin induces caspase-1 activation and subsequent IL-1beta secretion by macrophages. TLR11 recognizes a protein PAMP that is present on uropathogenic Escherichia coli (Zhang et al., 2004). Although the identity of this ligand is unknown, its ability to stimulate in a TLR11-dependent manner is destroyed by proteinase treatment.

Conserved differences in bacterial nucleic acid structures can also be recognized by the innate immune system. TLR9 recognizes bacterial DNA containing unmethylated CpG motifs, and TLR9-deficient mice are not responsive to CpG DNA challenge (Hemmi et al., 2000). The low frequency and high rate of methylation of CpG motifs prevent recognition of mammalian DNA by TLR9 under physiological circumstances. A recent report indicated that the intracellular, endosomal restriction of TLR9 is critical for discriminating between self and nonself DNA, as host DNA, unlike microbial DNA, does not usually enter the endosomal compartment (Barton et al., 2006).

Viral recognition

Although viruses are composed entirely of host products they, nevertheless, have unique components that readily serve as PAMPs. Nucleic acids are also key viral PAMPs, and are recognized by TLRs 3, 7, 8 and 9, as well as by cytoplasmic receptors of the RIG family (as described above). TLR3 recognizes dsRNA, a common viral replicative intermediate (Alexopoulou et al., 2001). TLR3 signaling results in the activation of NF-kappaB and IRF3, ultimately leading to the production of antiviral molecules, such as type I interferons (IFN-alpha/beta) (Alexopoulou et al., 2001). The importance of RIG-I- and MDA5-mediated viral recognition is further supported by gene-targeting experiments demonstrating that TLR3 and its adaptor TRIF are not required for type I IFN production in some virally infected cells, such as fibroblasts and conventional DCs (Honda et al., 2003). However, plasmacytoid DCs exclusively utilize TLR3/TRIF signaling for type I IFN production in response to RNA viruses and poly(I:C) (Kato et al., 2005).

Although initially found to recognize synthetic antiviral compounds, namely imidazoquinolines, the azoquinoline R-848 and loxoribine (Hemmi et al., 2002; Jurk et al., 2002), TLR7 and human TLR8 are now known to recognize guanosine- or uridine-rich single-stranded RNA derived from RNA viruses (Diebold et al., 2004; Heil et al., 2004; Lund et al., 2004). Interestingly, mammalian RNA, which contains many modified nucleosides, is significantly less stimulatory via TLRs 7 and 8 than bacterial RNA, suggesting that nucleoside modification allows mammals to distinguish between endogenous and pathogen-derived RNA (Kariko et al., 2005). Similar to TLR3, engagement of these receptors leads to the production of type I IFNs.

TLR9 recognizes viral CpG sequences and induces the induction of IFN-alpha (Takeshita et al., 2001; Lund et al., 2003; Krug et al., 2004). However, as membrane restriction prevents TLRs from sampling the cytosol where much of the viral life cycle occurs, cytosolic PRRs provide comprehensive innate immune recognition. For example, recognition of cytoplasmic dsDNA leading to NF-kappaB activation and type I interferon production has also been reported, although the relevant receptor has not yet been identified (Ishii et al., 2006; Stetson and Medzhitov, 2006). This receptor(s) is predicted to be important for type I IFN production in response to viruses and intracellular pathogens, such as Listeria monocytogenes and Shigella flexneri. Finally, there have been some reports suggesting that certain viral proteins function as PAMPs. For example, TLR4 may recognize respiratory syncytial virus (RSV) F protein (Kurt-Jones et al., 2000).

Recognition of other pathogens

MyD88-deficient cells demonstrate that many fungal species are capable of activating TLR pathways, although the receptors have not always been identified. TLR4 has been shown to recognize Aspergillus hyphae (Mambula et al., 2002), and Cryptococcus neoformans capsular polysaccharide (Shoham et al., 2001). TLR2 and TLR6 are required for recognition of yeast zymosan, whereas TLR4 is thought to recognize certain yeast mannans (for a review see Levitz, 2004). The identification of parasite PAMPs has been more elusive, and their existence is somewhat controversial. However, TLR2 heterodimers reportedly recognize various parasite GPI-anchored proteins and glycoinositolphospholipids from the parasitic protozoa Trypanosoma cruzi (Campos et al., 2001). Some TLR knockout mice have been shown to have variable defects in their ability to defend against various parasites (for a review see Gazzinelli et al., 2004). Recently, TLR9 has been reported to recognize the malarial pigment hemozoin, a byproduct of heme metabolism in infected erythrocytes (Coban et al., 2005) whereas TLR11 recognizes a profilin-like protein that is conserved in apicomplexan parasites including Toxoplasma gondii (Yarovinsky et al., 2005).

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Immediate antimicrobial responses

PRRs initiate a complex series of events following exposure to certain microbial components: the first is the mounting of immediate antimicrobial responses at the cellular level. This is an effective and evolutionarily conserved function of PRRs, and one in which NF-kappaB has an important role. The liberation of products with direct antimicrobial activity occurs early at sites of pathogen entry. TLR ligation is at least partly responsible for the NF-kappaB-dependent expression of defensins – cationic peptides that exert direct bactericidal activity by inducing membrane permeabilization. Small intestinal Paneth cells, for example, release large amounts of alpha-defensins into the intestinal lumen following exposure to a variety of bacteria/bacterial products (Ayabe et al., 2000). The production of antimicrobial nitrogen and oxygen species, which are acutely toxic to a variety of microbes, augments the activity of antimicrobial peptides. Production of nitric oxide is mediated in part by inducible nitric oxide synthase (iNOS), which is partially regulated by NF-kappaB. Consequently, iNOS production results from TLR or NOD-LRR ligation by PAMPs.

Much of the early innate response has been demonstrated to depend on the canonical NF-kappaB pathway. Thus, rela-/-/tnfr1-/- double knockout mice have increased susceptibility to bacterial infection (Alcamo et al., 2001). Likewise, B cells from p50-/- mice do not respond efficiently to LPS, emphasizing the importance of p50-containing complexes, that is, p50/RelA, p50/p50/BCL-3 and p50/c-Rel, in TLR signaling (Sha et al., 1995). As might be expected, TNFR/IKKbeta double knockouts show a more pronounced defect in innate responses owing to the more complete block in canonical NF-kappaB pathways, and succumb to infection more rapidly than rela-/-/tnfr1-/- mice (Li et al., 1999a, 1999b; Senftleben et al., 2001b). Furthermore, MEFs from nemo-/- mice do not exhibit NF-kappaB activation by LPS or IL-1 (Rudolph et al., 2000). Therefore, activation of NF-kappaB responsive genes by the innate immune system depends on NEMO and likely progresses through the canonical NF-kappaB signaling pathway.

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Inflammation

There is a staggering amount of literature that correlates NF-kappaB activation with inflammation in a wide array of diseases and animal models. There are, likewise, numerous studies using gene targeting and inhibitors of NF-kappaB that have established that NF-kappaB plays a causative role in inflammatory processes. We have already discussed the role of NF-kappaB in the survival of leukocytes, and how this role is particularly important during the responses that include inflammation. Here, we briefly discuss a few of the additional ways in which NF-kappaB regulates inflammation. Inflammation begins with epithelial or stromal cells of the infected tissue or tissue resident hematopoietic cells such as mast cells or DCs recognizing an inflammatory stimulus and propagating proinflammatory signals. These signals lead to the recruitment and activation of effector cells, initially neutrophils and later macrophages and other leukocytes, resulting in the tissue changes characteristic of inflammation – rubor, calor, dolor and tumor (redness, heat, pain and swelling, respectively).

As for the immediate antimicrobial products discussed above, NF-kappaB is responsible for the transcription of the genes encoding many proinflammatory cytokines and chemokines. One important early target of these effectors is the vascular endothelium. Changes in vascular endothelial cells both recruit circulating leukocytes and provide them with a means of exiting the vasculature into the infected tissue. NF-kappaB regulates the expression of adhesion molecules, both on leukocytes and endothelial cells, which allow the extravasation of leukocytes from the circulation to the site of infection (Eck et al., 1993). Indeed, RelA-deficient mice display a severe defect in the recruitment of circulating leukocytes to sites of inflammation (Alcamo et al., 2001).

Recruited neutrophils are the key mediators of local inflammation and NF-kappaB is important for the survival of these cells, which must function in relatively toxic conditions (Ward et al., 1999). NF-kappaB is important for the production of the enzymes that generate prostaglandins and reactive oxygen species (e.g., iNOS and Cox, both NF-kappaB target genes) and may, furthermore, be involved in the signaling induced by prostaglandins (Poligone and Baldwin, 2001; Catley et al., 2003). NF-kappaB has also been implicated in the response to leukotrienes, which like prostaglandins are short-lived paracrine effectors, although it is unclear whether this represents a direct signaling event. Finally, matrix metalloproteinases (MMPs) also are crucial mediators of local inflammation and leukocyte chemotaxis; and their expression is also regulated by NF-kappaB (Vincenti et al., 1998; Vincenti and Brinckerhoff, 2002; Lai et al., 2003).

The pathway from pathogen recognition to proinflammatory cytokine production demonstrates a particular reliance on NF-kappaB. The immediate targets of NF-kappaB-dependent proinflammatory cytokines, such as TNFalpha, tend to be receptors that, in turn, activate NF-kappaB. Therefore, NF-kappaB is crucial to the propagation and elaboration of cytokine responses. TNFalpha is particularly important for both local and systemic inflammation, and it is a potent and well-studied inducer of NF-kappaB.

TNF-R superfamily signaling

The TNF-R superfamily is remarkably diverse with more than two-dozen receptors and nearly as many ligands that are variably expressed throughout the body. However, despite the physiological diversity of responses, in most cases signaling converges on the activation of NF-kappaB and AP-1. NF-kappaB activation in response to TNF signaling induces expression of antiapoptotic genes such as cIAP1/2 and Bcl-XL (see Dutta et al., 2006).

TNF family receptors lack intrinsic enzymatic activity. Instead, signaling is achieved by recruitment of intracellular adapter molecules that associate with the cytoplasmic tail of the TNF-R in a signal-dependent manner (Figure 6a). The recruitment of TNF-R1 to membrane microdomains, referred to as lipid rafts, with subsequent assembly of the signaling complex, is necessary for signaling to NF-kappaB and prevention of apoptosis (Hueber, 2003; Legler et al., 2003). Ligation of TNF-R1 by trimeric TNFalpha causes aggregation of the receptor allowing binding of the TNF-R-associated death domain protein (TRADD). TRADD subsequently recruits adapter molecules including TRAF2 (Hsu et al., 1996); however, TRAF2 and TRAF5 appear to play redundant roles in TNF signaling to NF-kappaB. TRAF2 or TRAF5-deficient mice have intact TNF activation of NF-kappaB, whereas TRAF2/5 double knockout cells have substantially reduced TNF-induced IKK activation (Yeh et al., 1997; Nakano et al., 1999; Tada et al., 2001). TRAFs may either recruit the IKK complex directly (Devin et al., 2001) or indirectly through the serine/threonine kinase, RIP1. RIP1 can also interact independently with TRADD and is an essential adapter for TNF-induced NF-kappaB activation and protection from apoptosis (Hsu et al., 1996; Ting et al., 1996; Kelliher et al., 1998; Devin et al., 2000). Upon ubiquitination, RIP1 can bind directly to NEMO and recruit IKK independent of TRAF2 (Zhang et al., 2000). Signaling downstream of RIP1 requires TAK1 for the activation of IKK (Sato et al., 2000; Shim et al., 2005). Whether TAK1 directly activates IKK or this process proceeds through an intermediary such as MEKK3 is not yet clear (Takaesu et al., 2003; Li et al., 2006). However, it does not appear that TAK1 is involved in the activation of the non-canonical NF-kappaB pathway (Shim et al., 2005).

Figure 6.
Figure 6 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

TNF receptor superfamily signaling to NF-kappaB. Activation of the canonical NF-kappaB pathway downstream of TNF-RI is initiated by trimerization through ligand binding and recruitment of FADD, TRADD, RIP1 and TRAF2/5 to the receptor (a). TAK1 and MEKK3 are subsequently recruited to the receptor complex through RIP1, and with TRAF2/5, mediate the activation of IKK. The phosphorylation and degradation of classical IkappaBs require IKKbeta and NEMO. The non-canonical pathway is mediated by IKKalpha through NIK (b). In the resting state, NIK is inactivated/degraded through an interaction with TRAF3. Upon stimulation, TRAF3 is inactivated/degraded resulting in the accumulation of NIK, activation of IKKalpha, phosphorylation of p100 and liberation of p100-inhibited NF-kappaB complexes. Simultaneous activation of the canonical NF-kappaB pathway through either TRAF2, 5, or 6 also commonly occurs downstream of receptors that activate the non-canonical pathway. See the text for additional details.

Full figure and legend (139K)

The non-canonical NF-kappaB pathway is unique in that it is independent of IKKbeta and NEMO and instead requires IKKalpha which is phosphorylated by NF-kappaB inducing kinase (NIK) (Xiao et al., 2001; Senftleben et al., 2001a; Claudio et al., 2002; Dejardin et al., 2002; see Scheidereit, 2006). The key question concerning signaling by these stimuli is how they are channeled to NIK and IKKalpha, even though their receptor signaling domains resemble those of other TNF family members (Figure 6b). Because RANKL, BAFF and CD40L may also activate the canonical pathway through TRAF2/6, it would appear that the intracellular signaling domain of these receptors possess additional sequence motifs that allow their signaling to NIK. This function is mediated by TRAF3, which interacts with these receptors. TRAF3 negatively regulates NIK, and undergoes signal-dependent degradation resulting in the activation of the non-canonical pathway (Liao et al., 2004).

Resolution of inflammation may also involve NF-kappaB

Resolution of inflammation and subsequent tissue repair is a crucial event, and its failure is a common source of pathology. It is believed that reversal of inflammation is an active process that is as complex as the inflammatory response itself, and involves numerous pathways that are not all directly relevant to NF-kappaB (Serhan and Savill, 2005). Although the traditional view of NF-kappaB would lead one to imagine that it would primarily function by being turned off during the resolution phase of inflammation, recent work has suggested that NF-kappaB also has a more active role.

During acute inflammation, there are multiple negative feedback pathways that help to rein in inflammatory responses. It has long been known that cells such as macrophages become resistant to repeated proinflammatory stimuli. BCL-3, which is induced late following LPS stimulation, in combination with p50 dimers has been shown to have a role in the inhibition of repeated LPS responses in macrophages, a phenomenon also referred to as LPS tolerance (Wessells et al., 2004). Furthermore by selectively affecting chromatin remodeling, BCL-3 mediates repression of proinflammatory genes, but also facilitates expression of the anti-inflammatory gene IL-10. NF-kappaB p50 also appears to negatively regulate IFNitalic gamma production and proliferation by NK cells (Tato et al., 2006).

In addition to these and other negative feedback pathways, it was recently found that inhibition of NF-kappaB during the resolution phase can prolong the inflammatory process and prevent proper tissue repair (Lawrence et al., 2001). It was subsequently found that IKKalpha-deficient mice display increased inflammatory responses in models of local and systemic inflammation (Lawrence et al., 2005). Macrophages, in particular, show increased production of proinflammatory chemokines and cytokines in the absence of IKKalpha (Lawrence et al., 2005; Li et al., 2005). It was suggested from these studies that IKKalpha negatively regulates proinflammatory gene expression, perhaps through mediating degradation of RelA and c-Rel following macrophage activation by LPS.

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Initiation of adaptive responses

Although innate responses alone can bring about potent antimicrobial activities, alerting and activating the adaptive immune system remains a crucial step for robust and durable immune responses. This process is largely mediated by activation and maturation of antigen-presenting cells (APCs), which can, in turn instruct T and B cells to carry out the adaptive response.

DC maturation mediated by pathogen recognition is crucial for the initiation of the adaptive immune response. To activate naïve T cells, DCs must undergo multiple changes. First, DCs must gain the ability to interact with T cells by changing their chemokine receptor expression and migrating into lymphoid tissues. Second, DCs must alter their antigen processing machinery to favor the presentation of pathogen epitopes on MHC. Third, APCs must upregulate the expression of costimulatory molecules B7.1/B7.2 (or CD80/CD86), which are regulated by NF-kappaB and ligate CD28 providing the second signal necessary to induce T-cell activation. Finally, as progress is made in exploring these events it is becoming increasingly clear that the responses to different pathogens are tailored based on the distribution of PRRs in different cell types and the ability of different cell types to, in turn, interact with T cells in a biasing manner (Iwasaki and Medzhitov, 2004).

Maturation of DCs following viral infection depends on nucleic acid-binding PRRs, including both TLRs and cytoplasmic RIG family molecules. Indeed, DC maturation during viral infection occurs normally in the absence of MyD88 or TLR3, as reported previously (Lopez et al., 2003). Bacterial responses are either mediated through TLRs – DCs express TLRs 1, 2, 5 and 6 – or other classes of PRRs. Murine CD8alpha+ DCs, which tend to induce TH1 responses important in clearance of viral and parasitic infections, express TLR1, 2, 6, 9 and 11. In the absence of TLR11, for example, mice fail to mount a TH1 response against T. gondii, owing to the failure of CD8alpha+ DCs to recognize the TLR11 ligand (Yarovinsky et al., 2005). It remains unclear whether the ability of distinct TLR ligands to induce TH1 versus TH2 responses is intrinsic to the specific TLR/ligand, dose of ligand or the cell type within which this activation occurs; evidence to date points towards the latter. Even less clear are the roles of other PRRs in APC maturation and the initiation of adaptive responses.

Finally, there is the question of the role of innate recognition of pathogens by lymphocytes themselves. Recently, it has been suggested that TLR signaling in B cells is also required for optimal response to certain antigens (Pasare and Medzhitov, 2005). Both B and T cells express TLRs, although exactly which TLRs are expressed is debatable. At the level of mRNA expression, human peripheral B cells express TLR1, 2, 4, 6 and 9 (Hornung et al., 2002).

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Role of NF-kappaB in the adaptive response

AgR signaling to NF-kappaB

The hallmark of the adaptive immune response is antigen specificity. In the section on hematopoiesis, we discussed how NF-kappaB plays an important role in the selection of lymphocytes bearing somatically generating AgRs. Signaling through these antigen-specific B-cell and T-cell receptors is therefore the central event of the adaptive immune response. Activation of NF-kappaB downstream of BCR and TCR ligation facilitates antigen-specific proliferation and maturation of lymphocytes into effector cells. Signaling through these two AgRs appears to be functionally analogous, although some of the components utilized differ.

BCR and TCR signaling are analogous in many aspects, in particular with respect to the activation of NF-kappaB (Figure 7). The T-cell receptor complex consists of alpha/beta subunits that are associated with the CD3 protein heterodimers italic gamma/alt epsilon, delta/alt epsilon, and either eta/eta, eta/zeta or zeta/zeta, for a total of eight membrane proteins. The BCR is, likewise, a multiprotein complex consisting of the surface immunoglobulin receptor associated with a heterodimer of Igalpha and Igbeta. The AgR complexes associate with Src family tyrosine kinases (SFKs), Lck and Fyn in T cells and Lyn in B cells, which phosphorylate immunoreceptor tyrosine activation motifs (ITAMs) on CD3 and Igalpha/beta chains. The cytoplasmic tyrosine kinases ZAP70 or Syk are then recruited via SH2 domains to the phosphorylated ITAMs and initiate activation of the IP3 and Ras family pathways. The IKK complex is rapidly recruited to the immunological synapse and can be colocalized to the TCR (Khoshnan et al., 2000; Weil et al., 2003). In ZAP-70-deficient T cells, NF-kappaB activation can be rescued by directly targeting a chimeric NEMO to the immunological synapse, suggesting that signaling downstream of ZAP-70 may largely function for IKK recruitment (Weil et al., 2003).

Figure 7.
Figure 7 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

AgR signaling to NF-kappaB. Binding of TCR to peptide:MHC and co-stimulation through CD28:B7 interaction activates NF-kappaB (a). Through multiple steps, initial phosphorylation events at the TCR complex lead to PI3K activation and recruitment of PDK1. PDK1, in turn, mediates recruitment of the IKK complex through PKCtheta and the CARMA1 directly. PKCtheta phosphorylates CARMA1 leading to the activation of IKK through BCL10, MALT1 and TRAF6. Binding to TI antigens or TD antigens in the presence of co-stimulatory cytokines results in the activation of NF-kappaB through the BCR complex (b). Receptor proximal events of BCR activation are highly analogous to those of the TCR. Although the role of PDK1 in B-cell signaling is not established, BCR signaling to NF-kappaB requires PKCbeta as well as the CBM complex to achieve activation of IKK.

Full figure and legend (214K)

The signaling pathway from the receptor to NF-kappaB requires PKCtheta (PKCbeta in B cells), CARMA1/CARD11, BCL10 and MALT1 (Sun et al., 2000; Ruland et al., 2001, 2003; Saijo et al., 2002; Hara et al., 2003; Ruefli-Brasse et al., 2003;). Although there has been some controversy, available data suggest that PKCtheta is largely essential for activation of NF-kappaB via T-cell stimulation (Sun et al., 2000; Pfeifhofer et al., 2003) and can mediate the activation of IKK (Lin et al., 2000). PKCtheta is specifically recruited to the immunological synapse; although how PKCtheta but not other PKC isoforms is selectively recruited remains a mystery. In B cells PKCbeta is, likewise, required for recruitment of the IKK complex to lipid rafts following BCR ligation (Su et al., 2002). PKCtheta is capable of directly interacting with the IKK complex in primary T cells (Khoshnan et al., 2000) and might, therefore, function by bringing IKK to the receptor complex and into proximity with other essential components in this pathway; namely CARMA1, BCL10 and MALT1 (collectively known as the CBM complex). Recently, studies have provided evidence that the protein kinase PDK1 recruits PKCtheta and the IKK complex to lipid rafts. In addition, PDK1 can simultaneously recruit the CBM complex through binding to CARMA1 (Lee et al., 2005). The induced proximity of PKC and the CBM may allow phosphorylation of CARMA1 by PKCtheta/beta (Matsumoto et al., 2005; Sommer et al., 2005). Interestingly, T-cell-specific PDK1 conditional deletion results in a defect in T-cell development, preventing the production of peripheral T cells (Hinton et al., 2004). On the other hand, PCKtheta knockouts do not display defects in thymocyte development.

The CBM complex is essential for both AgR signaling in mature B and T cells. What is surprising however, as mentioned above, is the lack of a role for this complex in developing lymphocytes, and by extrapolation signaling through the pre-AgRs. The MAGUK family protein CARMA1 is required for activation of NF-kappaB in T cells following TCR ligation, but its loss has no effect on the development of thymocyte (Gaide et al., 2002; Egawa et al., 2003; Hara et al., 2003). Similarly, BCL10 is critical for NF-kappaB activation via the BCR and TCR, yet normal numbers of peripheral T cells are seen in BCL10 knockouts, and no clear defects in B-cell development is observed (Ruland et al., 2001). BCL10 interacts with CARMA1 leading to BCL10 phosphorylation, although CARMA1 lacks kinase activity (Bertin et al., 2001; Gaide et al., 2001).

Interestingly, genetic evidence of a role for RIP2 has recently been reported in T-cell signaling (Ruefli-Brasse et al., 2004). RIP2 associates with BCL10 and is necessary for TCR-induced BCL10 phosphorylation and IKK activation. It is not yet clear how TCR signaling regulates RIP2 and, in turn, how RIP2 might affect IKK activity. BCL10 oligomerization has been implicated in IKK activation through a process that involves ubiquitination of NEMO (Zhou et al., 2004). This ubiquitination event appears to be mediated by MALT1, and perhaps TRAF6, although no TCR signaling deficits have been reported in TRAF6-deficient mice (Lomaga et al., 1999; Sun et al., 2004). If this is true however, it is possible that the effect is mediated through the kinase TAK1, which functions in IKK activation downstream of TRAF6 in other pathways. However, B cells in which TAK1 has been conditionally inactivated have normal BCR signaling to NF-kappaB. More work must be carried out with TAK1 conditional knockouts in both the BCR and TCR pathways to address the role of this kinase.

T-cell responses mediated by NF-kappaB

To become activated, naïve T cells must receive two distinct signals: antigen-specific and co-stimulatory. Antigen-specific activation signals emanate from the binding of the TCR to cognate antigenic peptides presented in the binding cleft of MHC. Co-stimulatory signaling is provided through ligation of CD28 by B7 molecules expressed on activated APCs. Stimulation of naïve T cells results in the production of IL-2, which is necessary for their proliferation and survival. These activated naïve T cell blasts proliferate rapidly and simultaneously undergo differentiation into effector cells. In the case of TH cells, proliferation leads to differentiation into immature effector cells, TH0, which subsequently differentiate into TH1 or TH2 cells depending on the predominant cytokine milieu. CD8 T cells are likewise activated by professional APCs, although they may receive secondary signals from activated TH1 cells. The unavailability of CD8 conditional knockouts, and the selective loss of CD8 cells in the absence of NF-kappaB activity has prevented a thorough characterization of the role of NF-kappaB in these cells.

Rapidly proliferating activated T cells rely on NF-kappaB activity for protection from apoptosis as well as for the production of cytokines supporting proliferation and differentiation. As expected, inhibition of NF-kappaB in activated T cells facilitates progression towards AICD or apoptosis (Ivanov and Nikolic-Zugic, 1997; Jeremias et al., 1998). Indeed, stimulation of RelA-deficient naïve T cells induces cell death (Wan and DeGregori, 2003). Peripheral T cells lacking c-Rel do not undergo apoptosis, but nevertheless, fail to proliferate in response to typical mitogenic stimuli (Köntgen et al., 1995), and both RelA and c-Rel containing complexes accumulate in the nucleus following TCR/CD28 stimulation (Ghosh et al., 1993). Interestingly, c-Rel-deficient T cells appear to have a defect in TH1 proliferation and production of IFNitalic gamma, indicating a selective role for NF-kappaB family members in TH1/TH2 differentiation, independent of that mediated by the innate response.

Multiple transcriptional activators and repressors regulate expression of IL-2. Among these, members of the NF-kappaB family play multiple roles. In naïve T cells, which do not express IL-2, repressive p50 homodimers are found associated with the IL-2 promoter (Grundstrom et al., 2004). Failure of T-cell proliferative responses in c-Rel knockout mice is attributable to a failure to produce IL-2 (Köntgen et al., 1995). In naïve T cells, c-Rel is responsible for mediating chromatin remodeling across the IL-2 locus following CD3/CD28 co-stimulation (Rao et al., 2003). Naïve T cells can be primed by exposure to inflammatory cytokines such that they generate a more robust response to CD3/CD28 co-stimulation. Overexpression of an IkappaBalpha super-repressor suggested that NF-kappaB is required for this priming event in T cells (Mora et al., 2001a). More recent data indicate that c-Rel is necessary for naïve helper T-cell priming by pro-inflammatory cytokines elicited following stimulation with TLR ligands (Banerjee et al., 2005). NF-kappaB RelA-containing complexes, on the other hand, appear to function more traditionally in mediating transactivation of IL-2 gene expression, and overexpression of RelA with c-Jun can overcome the requirement for co-stimulation in naïve T cells (Parra et al., 1998). However, as discussed below, these complexes may also be the targets of negative regulation following T-cell differentiation.

Recent work in TH1/TH2 differentiation has focused on the induction of specific transcription factors in these two effector cell types – T-bet and GATA3, respectively. Interestingly, mice lacking p50 are unable to mount an asthma-like airway TH2 response, and do not induce GATA-3 expression during T-cell stimulation under TH2 differentiating conditions (Das et al., 2001). Consistent with this finding, BCL-3-deficient T cells also fail to undergo TH2 differentiation. Furthermore, BCL-3 can induce expression of a reporter gene from a gata-3 promoter, suggesting that p50/BCL-3 complexes are crucial for TH2 differentiation (Corn et al., 2005). Conversely, the same authors found that RelB-deficient T cells are deficient in TH1 differentiation and IFNitalic gamma production, and show decreased expression of T-bet; likely through a failure to upregulate STAT4, which functions in signaling from IFN to T-bet induction. Therefore, it appears that NF-kappaB activation during TCR stimulation may render cells competent for both proliferative and differentiating stimuli.

As TH cells differentiate into TH1 or TH2, they decrease their expression of IL-2 and, instead, become dependent on TH1 and TH2 cytokines (e.g., IFNitalic gamma and IL-4). As a corollary, NF-kappaB transactivation of the IL-2 gene is repressed. Direct binding of T-bet to p65 that is associated with the IL-2 gene enhancer may mediate the repression of IL-2 production in TH1 cells (Hwang et al., 2005). Alternatively, in TH2 cells the lack of IL-2 transcription may be due to the decreased levels of RelA activation in TH2 cells (Lederer et al., 1994).

B-cell responses mediated by NF-kappaB

B-cell responses can be classified into two groups: thymus-dependent (TD) or thymus-independent (TI). In response to T-dependent antigens, B cells require co-stimulatory signaling from TH cells expressing CD40L and cytokines, such as IL-4. B cells from individuals with a mutation in CD40L are unable to undergo class switch recombination in response to T-dependent antigens (Aruffo et al., 1993). Signaling through CD40 activates both canonical and non-canonical NF-kappaB pathways, although it is unclear which is operative in the response to T-dependent antigens. For example, whereas B cells from p52-/- mice mount inadequate humoral responses to various T-dependent antigens, they exhibit a normal response following adoptive transfer into rag-1-/- mice – indicating that this deficit is not intrinsic to B cells (Franzoso et al., 1998). Furthermore, B cells from relB-/- mice, although crippled in their proliferative response, undergo normal IgM secretion and class switching in response to various stimuli (Snapper et al., 1996a). Therefore, non-canonical NF-kappaB pathway activation downstream of CD40 is probably not required for class switching during T-dependent antigen responses.

Analysis of the canonical NF-kappaB pathway is complicated by more generalized defects in lymphocyte response owing to the requirement for this pathway in AgR signaling. Nevertheless, whether downstream of CD40 or other stimuli, evidence supports canonical pathway activation in the process of class switch recombination. Following adoptive transfer, B cells from rela-/- mice exhibit markedly diminished class switching, despite a modest loss of lymphocyte proliferation following various stimuli (Doi et al., 1997). Likewise, c-Rel-deficient mice, or mice lacking the c-Rel C-terminal transactivation domain, fail to generate a productive humoral immune response suggesting a requirement for c-Rel in class switch recombination (Köntgen et al., 1995; Zelazowski et al., 1997; Carrasco et al., 1998). B cells from nfkb1-/- mice exhibit decreased proliferation in response to mitogenic stimulation, and p50/RelA double knockout B cells exhibit greater defects in proliferation and class switching (Snapper et al., 1996b; Horwitz et al., 1999). Therefore, analyses of knockout animals suggest that the canonical NF-kappaB pathway likely has a role in maturation of the B-cell response in addition to directly mediating proliferative responses following BCR ligation.

As discussed above, signaling through TLRs has an important role in the initiation of the adaptive immune response via APCs of the innate immune system. In recent years, however, there has also been increasing interest in the ability of TLR signaling to directly modulate the adaptive response. For example, it has been observed that homeostatic polyclonal activation of B cells, which results in the so-called serological memory, that is, detectable antibody to antigens that are no longer present in the host, can be induced/maintained by TLR ligation (Bernasconi et al., 2002). Analogously, TLR2 is upregulated in CD4+ T cells following TCR stimulation, and TLR2 ligands may thus provide an activation/maintenance signal in these cells (Komai-Koma et al., 2004). That signaling through TLRs in these aspects of B-cell responses requires NF-kappaB seems likely, but has yet to be demonstrated.

TI antigens have an intrinsic ability to activate B-cell responses in the absence of T-cell help by acting as B-cell mitogens, for example by acting as TLR ligands or by binding with high avidity to the BCR through repetitive structural features. In such cases, it is expected that B-cell responses are more dependent on members of the canonical pathway that have well-documented roles in TLR signaling, or BCR signaling (as discussed above). For example, c-Rel-deficient B cells are highly sensitive to apoptosis following BCR cross-linking (Grumont et al., 1998, 1999; Owyang et al., 2001). As mentioned above, p50 and p50/pRelA double knockout B cells are deficient in responses to TI stimulation. Likewise, IKKbeta-deficient B cells fail to mount TI or TD responses (Li et al., 2003). These IKKbeta-deficient B cells also exhibit increased spontaneous apoptosis, suggesting that NF-kappaB is important in survival of B cells.

Maintenance and memory: a role for NF-kappaB in lymphoid cell survival

Lymphocyte homeostasis is dependent on the survival of mature lymphocytes in addition to replenishment of the peripheral lymphocyte pool through lymphopoiesis. Consequently, there is increasing interest in the possible role of NF-kappaB in the survival of mature lymphocytes. It is widely accepted that lymphocyte survival is mediated through tonic stimulation downstream of the AgR, as well as certain cytokine receptors. As discussed above, genetic targeting experiments support an important role for NF-kappaB family members in lymphocyte survival. Naïve T cells require continued contact with MHC:self-peptides, most likely expressed on lymphoid DCs, to generate the tonic TCR signal that is essential for continued survival. Survival of memory cells, on the other hand, is independent of continued contact with self-peptide:MHC complexes.

B cells are formed at a far higher rate than T cells and therefore B cells also undergo a significantly higher rate of turnover. Nonetheless, B cells too require maintenance signals to achieve peripheral homeostasis. The AgR on B cells most likely provides a basal level of signaling, albeit independent of the presence of antigen, which is required for maintenance of mature B cells. Not surprisingly, B cells from RelA-/-, p100-/-, p105-/- and c-Rel-/- mice display increased sensitivity to apoptosis and/or decreased survival ex vivo (Grumont et al., 1998; Claudio et al., 2002; Prendes et al., 2003).

In large part, these defects appear to be due to a loss of BCR signaling, as demonstrated in an elegant study that demonstrated that deletion of the BCR from mature B cells led to a complete loss of the peripheral B-cell pool (Kraus et al., 2004). Most likely this was due to the loss of signaling to NF-kappaB in these cells because loss of IKKbeta, NEMO or components of the CBM complex in mature B cells also results in a complete loss of peripheral B cells (Pasparakis et al., 2002; Li et al., 2003; Thome, 2004).

The non-canonical NF-kappaB pathway is also relevant to B-cell survival, as the loss of IKKalpha results in striking defects in B-cell survival (Kaisho et al., 2001; Senftleben et al., 2001a). However, rather than acting downstream of tonic BCR signaling, recent studies have implicated signaling from BAFFR in this aspect of the B lymphocyte survival (reviewed in Mackay et al., 2003). Together, these data suggest that a subset of Bcl-2 family members, for example, the antiapoptotic factor A1, are regulated by p52/RelB-containing complexes and are necessary for the maintenance of mature B cells.

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Concluding remarks

NF-kappaB was originally described as a transcriptional regulator in the adaptive immune response; however, subsequent studies have revealed its importance in hematopoiesis, lymphoid organogenesis and innate immunity. Investigations of mice with targeted deletions and mutations have shed considerable light on the role of NF-kappaB in lymphoid organogenesis. For example, characterization of the aly/aly mouse in part led to the discovery of the non-canonical NF-kappaB pathway and its role in organogenesis. Progress continues to be made in understanding AgR signaling to NF-kappaB, and this work has led to the appreciation of regulatory ubiquitination events in IKK activation (see reviews by Perkins, 2006; Scheidereit, 2006). The specificity of the requirement for the CBM complex and PKCtheta/beta in signaling by T-cell and B-cell receptors opens the door to the development of equally specific NF-kappaB inhibitors. However, there remain fundamental gaps in our understanding of how these components mediate IKK activation and the possible role of regulatory ubiquitination in this process.

The role of NF-kappaB in hematopoiesis remains partially defined, although there is little doubt about its importance. Genetic targeting in mice has allowed the enumeration of multiple steps in hematopoiesis at which various NF-kappaB pathway components are required; however, a cohesive picture of how NF-kappaB functions in these steps remains elusive. For example, whereas we know that canonical NF-kappaB activity is required in early lymphopoiesis, it is unclear whether this is in the regulation of TNFalpha production or in mediating a survival signal in the developing lymphocyte. NF-kappaB is required in the process of positive and negative selection, but again, its mechanism(s) of action remains poorly defined. Progress using in vitro systems for studying lymphocyte development may allow a more rigorous assessment of NF-kappaB function in these processes. Likewise, advances in conditional gene targeting approaches and the generation of animals in which defective versions of NF-kappaB genes have been knocked-in may help to overcome the problems of embryonic lethality and functional redundancy that have sometimes made existing studies difficult to interpret.

T- and B-cell responses require NF-kappaB as a prosurvival factor as well as for the regulation of genes involved in differentiation to effector cells. More recently, a limited number of studies have suggested a role for PRRs in lymphocyte activation, and the role of NF-kappaB in this process remains to be elucidated. The role of NF-kappaB in the differentiation of TH cells is incompletely understood, and requires further clarification. We also know very little about the role of NF-kappaB in CD8+ activation, differentiation and function; progress in this area awaits development of better tools for genetically manipulating this cell population. Upon successful clearance of pathogen, regulation of NF-kappaB allows resolution of the response and facilitates the development of memory cells. To date, relatively little is known about the role of NF-kappaB in memory cells, although recent advances in identifying and characterizing memory precursors bodes well for future progress in this area.

In summary, research to date has highlighted the importance of NF-kappaB in regulating genes that prevent apoptosis and that promote differentiation and development in cells of the innate and adaptive immune systems. A large body of work has elucidated many of the molecular mechanisms governing regulation of NF-kappaB by engagement of innate or lymphocyte antigen-specific receptors. In turn, these data provide a trove of information that will likely prove useful in attempts to manipulate the immune system to prevent and treat disease.

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Acknowledgements

Research in the authors' laboratory was supported by grants from the National Institutes of Health (to SG). MSH was supported by NIH/National Institute of General Medical Sciences Medical Scientist Training Grant GM07205.

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