NF-kB and the Immune Response

REVIEW

NF-jB and the immune response

MS Hayden1, AP West1 and S Ghosh1,2

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

One of the primary physiological roles of nuclear factor-kappa B (NF-jB) is in the immune system. In particular,NF-jB family members control the transcription ofcytokines and antimicrobial effectors as well as genes thatregulate cellular differentiation, survival and proliferation,thereby regulating various aspects of innate and adaptiveimmune responses. In addition, NF-jB also contributes tothe development and survival of the cells and tissues thatcarry out immune responses in mammals. This review,therefore, describes the role of the NF-jB pathway in thedevelopment and functioning of the immune system.Oncogene (2006) 25, 6758–6780. doi:10.1038/sj.onc.1209943

Keywords: NF-kB; T-cell receptor; B-cell receptor;inflammation; TLR; hematopoiesis

Introduction

The discovery and characterization of the nuclearfactor-kappa B (NF-kB) family of transcription factorsresulted from studies in two major areas of research:immunology and cancer biology. Although the role ofNF-kB in cancer biology is becoming progressivelybetter established, historically much of our currentknowledge of NF-kB resulted from efforts directed atunderstanding the regulation and function of theimmune response. In keeping with the critical roleplayed by NF-kB in different areas of immunology,numerous excellent reviews have been published cover-ing the role of NF-kB in Toll-like receptor (TLR) andantigen receptor (AgR) signaling, lymphoid organo-genesis and hematopoiesis (Mebius, 2003; Bonizzi andKarin, 2004; Hayden and Ghosh, 2004; Lin and Wang,2004; Siebenlist et al., 2005; Akira et al., 2006). Thisreview will, therefore, attempt to provide a morecomprehensive, if less detailed, review of the diversefunctions of NF-kB in immunology, with the goal ofilluminating how it is that so much in immunologyseems to revolve around this family of transcriptionfactors.

Considered broadly, mammalian immune responsescan be divided into innate and adaptive responses. Theimmune response begins with the host recognizing thepresence of foreign pathogens, followed by responses atthe cellular, tissue and organismal levels, that ultimatelylead to the clearance of the pathogen. As such, immuneresponses can be broken down into individual signaltransduction events through which changes in theextracellular environment elicit altered gene expressionat the cellular level. In a remarkable number ofinstances, NF-kB is the transcription factor thatmediates these transcriptional changes. The gene pro-ducts characteristic of early events in immune responsesinclude cytokines and other soluble factors that propa-gate and elaborate the initial recognition event. Theactivation and modulation of NF-kB is also a commontarget of these factors. Thus, in a surprising number ofsituations NF-kB mediates the critical changes that arecharacteristic of innate and adaptive immune responses.

In mammals, the NF-kB family is composed of fiverelated transcription factors: p50, p52, RelA (aka p65),c-Rel and RelB (see Gilmore, 2006). These transcriptionfactors are related through an N-terminal DNA-binding/dimerization domain, called the Rel homologydomain, through which they can form homodimersand heterodimers, which bind to a variety of relatedtarget DNA sequences called kB sites to modulategene expression. RelA, c-Rel and RelB also containC-terminal transcription activation domains (TADs),which enable them to activate target gene expression. Incontrast, p50 and p52 do not contain C-terminaltransactivation domains; therefore, p50 and p52homodimers can repress transcription unless they arebound to a protein containing a TAD, such as Bcl-3.Alternatively, p50 and p52 often form heterodimerswith RelA, c-Rel or RelB and act as transcriptionalactivating dimers.

In most cells, NF-kB complexes are inactive, residingprimarily in the cytoplasm in a complex with any of thefamily of inhibitory IkB proteins. When the pathway isactivated, the IkB protein is degraded and the NF-kBcomplex enters the nucleus to modulate target geneexpression. In almost all cases, the common step in thisactivating process is mediated by an IkB kinase (IKK)complex, which phosphorylates IkB and targets it forproteasomal degradation (see Scheidereit, 2006). TheIKK complex consists of two catalytically active kinases(IKKa and IKKb) and a regulatory scaffold protein,NEMO. In what is called the canonical (or classical)

Correspondence: Professor S Ghosh, Yale University School ofMedicine, Department of Immunobiology, 300 Cedar Street, NewHaven, CT 06510, USA.E-mail: [email protected]

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pathway, IKKb and NEMO are required for theactivation of complexes such as p50/RelA, p50/c-Rel,etc., whereas IKKa is relatively dispensable. Conversely,in the non-canonical (or alternative) pathway IKKaalone controls the activation of complexes that areinhibited by the IkB protein p100. These two NF-kBpathways can be activated by overlapping but distinctsets of stimuli, and also target activation/repression ofoverlapping but distinct sets of target genes. One of themost conserved functions of the NF-kB signalingpathway is the regulation of the immune system; indeed,NF-kB is even the primary regulator of innate immunityin insects such as Drosophila and mosquitoes (seeMinakhina and Steward, 2006). This review focuses onthe vast role played by NF-kB in mammalian immunity.

Development and formation of the immune system

The mammalian immune system consists of a function-ally linked group of anatomically disparate tissues andcell types. The dispersed cellular components of theimmune system that arise from the bone marrow receivemuch of the attention in immunology and the study ofNF-kB has likewise focused on its role in leukocytes.However, lymphoid organs that facilitate coordinationand dissemination of immune responses carried out byimmune cells are also key sites of NF-kB function.Therefore, whereas this section is largely concerned withthe role of NF-kB in hematopoiesis, the role of NF-kBin lymphoid organogenesis is also discussed briefly.

NF-kB and hematopoiesisMost cells of the immune system are subject to rapidturnover. This process requires the regulation of thecompeting forces of cell proliferation and cell death –processes heavily influenced by NF-kB-regulated genes.Bone marrow-derived hematopoietic cells in particularare subject to high levels of turnover and consequentlyare particularly sensitive to changes in rates of apoptosisor proliferation. Likewise, during immune responsesimmune cells selectively undergo rapid expansion thatmust be resolved by targeted cell death. Although therole of NF-kB in development and homeostasis ofhematopoietic cells has focused largely on B-cell andT-cell maturation, it is likely that as our understandingincreases of the pathways responsible for the develop-ment of natural killer (NK) cells, dendritic cells (DCs),macrophages, etc. our appreciation for the role of NF-kBin the biology of these cell types will also expand.

Hematopoietic components of the immune systeminclude cells of the lymphoid, myeloid and granulocyticlineages. These lineages give rise to T cells, B cells,monocytes, macrophages, DCs (both myeloid andlymphoid), NK cells, basophils, eosinophils, neutrophilsand mast cells (Figure 1). Many cells of the body cancontribute to immune responses; however, these bonemarrow-derived cells are the core constituents of boththe innate and adaptive immune responses. AlthoughNF-kB generally plays a prosurvival role in these cells,

its function during hematopoiesis is far more nuancedthan one might expect. In the current review, we limitour discussion to those instances where the role forNF-kB is illustrative of its broader functions in theimmune system.

Before delving into specific aspects of NF-kB functionin hematopoiesis, it is worthwhile to discuss the short-comings of the experimental approaches that are used.For example, embryonic lethality of RelA knockoutmice prevents straightforward analysis of the hemato-poietic events that are relevant to adult animals. In otherinstances, severe defects in lymphoid organogenesis inthe absence of NF-kB make it difficult to determinewhether the observed defects are intrinsic to thehematopoietic lineage or are due to alterations in therelevant organ, for example, stromal tissues, withinwhich hematopoietic development occurs. In someinstances, such as for RelA or IKKb knockouts,embryonic lethality can be rescued by deletion of thetumor necrosis factor-receptor (TNF-R) or tumornecrosis factor-alpha (TNFa), which permits analysisof hematopoiesis in these mice, but potentially distortscertain aspects of the hematopoietic pathway. Similarconcerns apply to adoptive transfer experiments thatcan be influenced by the cytokine milieu to whichtransferred cells are exposed. In each case, therefore, onemust ask whether the defect exhibited by a cell lackingsome component of the NF-kB pathway is relevant tothe course of normal hematopoiesis or simply to theexperimental system being employed. Finally, there arenumerous instances where one NF-kB family membercan complement the function of another member, oralternatively, where the absence of one family memberimpedes the function or expression of other familymembers. Nevertheless, despite these limitations, geneticanalysis has unequivocally illustrated a key role forNF-kB in the development and survival of hemato-poietic cells.

NF-kB in development of innate immune cellsDC development is largely dependent on canonicalNF-kB complexes, although a particular subset appearsto require only the non-canonical RelB containingNF-kB complexes. RelB is known to facilitate the de-velopment of DCs (Burkly et al., 1995; Weih et al.,1995); specifically development of CD8a�, but notCD8aþ , DCs (Wu et al., 1998). Conversely, doubleknockout studies have shown that canonical p50/RelAcomplexes are required for the development of bothCD8aþ and CD8a� DCs, but not other myeloid andlymphoid lineages, most likely by mediating theresponse of DCs to TNFa (Ouaaz et al., 2002; Abeet al., 2003). Survival of DCs in the periphery followingactivation tends to be short, but can be prolonged uponCD40L expression on T cells. CD40L activates both thecanonical and non-canonical NF-kB pathways andhence DCs deficient in both p50 and c-Rel, or DCsoverexpressing a mutant super-repressor form of IkBa,demonstrate significantly decreased survival (Ouaazet al., 2002; Kriehuber et al., 2005).

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IkBa knockout mice display robust granulocytosis(Beg et al., 1995), and suggest an antiapoptotic rolefor NF-kB during granulocyte development. Likewise,NF-kB has an antiapoptotic role in mature granulocytes.For example, neutrophils, which undergo daily turnoverand rapidly apoptose in vitro, exhibit acceleratedapoptosis as well as sensitization to proapoptotic stimulifollowing NF-kB inhibition. Unlike lymphocytes, whichare relatively long-lived in the absence of activation,protection from apoptosis in neutrophils is moreimportant during the inflammatory response than inhomeostasis. Indeed, many TLR ligands increaseneutrophil survival in vitro, likely due to NF-kB-mediated expression of antiapoptotic genes (Francoiset al., 2005). Although neutrophils are capable ofactivating NF-kB in response to many pro-inflamma-tory stimuli (McDonald, 2004), they lack p52 and RelB(McDonald et al., 1997), the very subunits that arecrucial for the maintenance of long-lived lymphocytes.Thus in the case of neutrophils, NF-kB fulfills itspredicted role as a prosurvival and proinflammatoryfactor.

The general granulocytosis observed in IkBa knock-out mice suggested that an antiapoptotic role could bebroadly assigned to NF-kB in this lineage. However,chimeras generated with cells from ikba�/�ikbe�/�mice(i.e., lacking IkBa and IkBe) instead display a modestdefect in both myelopoiesis and granulopoiesis oftransferred cells (Goudeau et al., 2003), and a

pronounced defect in NK cells and lymphoid lineages(Samson et al., 2004). Therefore, elevated levels ofNF-kB activity in these cells appears to exert a proapop-totic effect. Thus, it appears that the role of NF-kB ingranulopoiesis is selective and cell-type-specific.Furthermore, as described for lymphocyte development(below), the requirement for individual NF-kB subunitsis not uniform at different developmental stages.

NF-kB in development of B and T cellsAs in cells of the innate immune system, NF-kB is vitalfor the development and function of adaptive immunecells (Siebenlist et al., 2005). Although lymphocytes mayexhibit great longevity in the periphery, their selection inthe bone marrow and thymus is characterized by a highrate of apoptosis. As a consequence, the antiapoptoticproperties of NF-kB play a key role in lymphopoiesis.The centrality of its antiapoptotic function is supportedin part by the demonstration that most of the require-ments for NF-kB during T-cell development can beovercome by transgenic expression of the prototypicalantiapoptotic factor Bcl-2 (Sentman et al., 1991). Thenecessity of NF-kB for lymphopoiesis is strikinglyillustrated in human genetic diseases wherein the geneencoding NEMO is inactivated by mutation (seeCourtois and Gilmore, 2006). Because the NEMO geneis located on the X-chromosome, it is usually subject torandom inactivation in individual cells in females.

Pluripotent HSC

Myeloid Stem Cell Lymphoid Stem Cell

Mast Cell Eosinophil

Granulocyte-MonocyteProgenitor

Platelets

Erythrocytes

Monocyte

BasophilProgenitor

Neutrophil

Macrophage

Basophil

Dendritic Cells

T cell B cell NK cell

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


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However, in female patients who are heterozygous for amutant version of NEMO all peripheral lymphocytespossess an intact NEMO gene, rather than the 50%predicted by random inactivation, suggesting that in theabsence of NEMO-dependent NF-kB signaling, B and Tcells fail to develop.

The effects of NEMO inactivation in both mice andhumans solidify the role of NF-kB in lymphopoiesis,even though the details by which NF-kB functions inthis process remain obscure. NF-kB plays diverse rolesin lymphocyte development that can be groupedaccording to when in development it functions – thatis, before, during or after pre-AgR signaling. Althoughno single NF-kB subunit knockout mouse has as severeof a phenotype as NEMO deficiencies with regard to thegeneration of mature lymphocytes, double knockoutsdemonstrate that the antiapoptotic function of NF-kB isimportant in the maturation and survival of lympho-cytes. For example, loss of both of the canonical NF-kBfamily members p50 and RelA, or both RelA and c-Rel,halts development early in lymphopoiesis, beforeexpression of the pre-AgRs (Horwitz et al., 1997;Grossmann et al., 1999), suggesting that NF-kB isinvolved in the expression of antiapoptotic factorsrequired for early lymphoid cell survival in response toproapoptotic stimuli (Figure 2). In fact, early CD34þbone marrow cells can activate NF-kB in response toTNFa, and in these cells NF-kB acts as a prosurvivalfactor (Pyatt et al., 1999).

Evidence suggests that expression of the pre-AgRleads to survival signals that depend, at least in part, onNF-kB. For example, pre-T-cell receptor (pre-TCR)expression in double negative (DN; CD8�CD4�) thy-mocytes coincides with high levels of NF-kB activity,and NF-kB activity at this stage is necessary for DNsurvival and maturation (Figure 2). Therefore, enforcedIKKb activation eliminates the requirement for TCRrecombination, whereas inhibition of NF-kB by expres-sion of an IkBa super-repressor decreases DN thymo-cyte maturation and survival (Voll et al., 2000).Signaling through the pre-B-cell receptor (pre-BCR)also likely induces antiapoptotic signals through NF-kB.Consequently, the reduced pre-B-cell population seenupon expression of the IkBa super-repressor in bonemarrow cells can be rescued by overexpression of theantiapoptotic NF-kB target gene Bcl-XL (Feng et al.,2004; Jimi et al., 2005). However, whereas evidencepoints toward NF-kB-mediated production of antia-poptotic factors it remains unclear how NF-kB isactivated downstream of the pre-AgR.

Selection of DP (double positive; CD4þCD8þ)thymocytes depends on the ability of their TCR torecognize peptide:MHC (major histocompatibility com-plex) complexes. Thymocytes that express a TCR that isunable to bind MHC die in a process termed ‘death byneglect’, whereas those that bind peptide:MHC areeither positively or negatively selected depending on thestrength of this interaction. Thymocytes that bind self-peptide:MHC with very high affinity are likely to beself-reactive, and hence are deleted through negativeselection. Thus, only DP thymocytes that recognize

self-peptide:MHC with an affinity that falls within adefined range are positively selected to become single-positive T cells. Somewhat counterintuitively, it appearsthat NF-kB functions in both positive and negativeselection of thymocytes. During negative selection,NF-kB facilitates the induction of apoptosis followinghigh-affinity TCR ligation (Hettmann et al., 1999; Moraet al., 2001b), perhaps by facilitating expression ofproapoptotic genes and the consequent sensitization toproapoptotic signals (French et al., 1996; Kishimotoet al., 1998). The role of NF-kB in positive selection ofthymocytes is more in keeping with the better-estab-lished role of NF-kB as an inducer of antiapoptoticgenes. Unlike in thymocytes, however, NF-kB functionsas a prosurvival factor during negative selection ofB cells. Immature B cells display constitutive NF-kBactivity that is down-regulated following BCR ligation

Immature B cell

B cell

Pro-B cell

Pre-B cell

Transitional

CommonLymphoidPrecursor

Naive B cell

Bone Marrow

Spleen

ThymusPeriphery

DN Thymocyte

DP Thymocyte

SP Thymocyte

Naive T cell

Figure 2 NF-kB function during lymphopoiesis. NF-kB plays aprosurvival role in common lymphoid precursor cells that give riseto B- and T-cell lineages. B-cell development occurs in the bonemarrow, where NF-kB protects pre-B cells from proapoptoticstimuli including TNFa. Signaling to NF-kB through the pre-B-cellreceptor mediates survival of pre-B-cells that then undergo lightchain recombination to produce a functional B-cell receptor.Expression of BCR leads to NF-kB-dependent differentiation intoimmature B cells. High levels of BCR signaling, that is, throughrecognition of self-antigen, results in negative selection through theloss of NF-kB activity. Transitional B cells exit the bone marrowand migrate to the spleen where they mature and differentiate, aprocess that also requires NF-kB. T-cell development occursfollowing migration of precursor cells into the thymus. Stimulationof NF-kB through pre-TCRa provides a prosurvival signalallowing recombination of the TCRa chain and maturation tothe DP stage. Optimal signaling through the TCRa/b complexinduces NF-kB-dependent survival pathways, whereas a failureto signal or high-level signaling results in death by neglect ornegative selection, respectively. NF-kB activity is required for themaintenance of long-lived B and T cells.


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(Wu et al., 1996). Decreased NF-kB activity might thensensitize these cells to proapoptotic signals. Interest-ingly, some signaling components required for NF-kBactivation in mature B and T cells can be geneticallydisrupted without affecting their development, suggest-ing that pathways leading to activation of NF-kB indeveloping B or T cells differ significantly from thepathways engaged following AgR ligation in maturelymphocytes.

Following positive and negative selection, DP thy-mocytes must make a lineage commitment and becomesingle positive (SP) thymocytes (CD4þCD8� orCD4�CD8þ ), which shortly thereafter emigrate fromthe thymus. Analyses of kB-site luciferase transgenicreporter mice have shown that CD8 SP cells havesignificantly higher levels of NF-kB activity than CD4SP thymocytes (Voll et al., 2000). Conversely, theantiapoptotic factor Bcl-2 is more highly expressed inCD4 than CD8 cells, suggesting that CD8 SP thymo-cytes are more dependent on NF-kB for survival.However, during the course of the development fromthe DP stage to emigration from the thymus, both DPand SP lineages require NF-kB. Targeted deletion offloxed-NEMO using cd4-promoter-driven Cre recombi-nase expression, or overexpression of kinase deadIKKb, results in loss of mature peripheral T cells(Schmidt-Supprian et al., 2004). These data stronglysuggest that NF-kB activation is required for late stagesof T-cell development; however, ikkb�/�,tnfr1�/� doubleknockouts, ikkb�/� chimeras or cd4-Cre IKKb condi-tional knockouts are not defective in the production ofnaıve T cells (Senftleben et al., 2001b; Schmidt-Supprianet al., 2004), suggesting a requirement for NEMO butnot IKKb.

Immature B cells exit the bone marrow, becomingtransitional B cells, and complete development intoeither follicular or marginal zone B cells. NF-kB-regulated expression of prosurvival factors is importantto these final steps of B-cell development (Grossmannet al., 2000). Interestingly, the activation of NF-kB inlate B-cell maturation is the result of signaling by bothcanonical and non-canonical NF-kB pathways. Thusdeficiency in NEMO, IKKa or IKKb decreases thenumbers 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 knock-out progenitor cells are defective in their ability tomature beyond the transitional B-cell stage (Franzosoet al., 1997; Grossmann et al., 1999). A requirement forboth the canonical and non-canonical NF-kB pathwaysmay explain why deletion of p50 and p52 produces amore complete block in B-cell development than loss ofRelA and c-Rel. Although both canonical and non-canonical NF-kB pathways are functional during B-celldevelopment, recent work (Batten et al., 2000;Schiemann et al., 2001; Claudio et al., 2002) has under-scored the importance of BAFF ligation in selectivelyactivating the non-canonical NF-kB pathway and theconsequent expression of antiapoptotic Bcl-2 familymembers in transitional B cells (see Mackay et al., 2003).Indeed, BAFF knockout mice exhibit a complete failure

of transitional B-cell maturation, which mirrors thatseen in Bcl-XL knockout mice (Motoyama et al., 1995;Gross et al., 2001; Schiemann et al., 2001). Thus, onlythose knockouts that target both the canonical and non-canonical NF-kB pathways have an effect that approx-imates the phenotype seen in BAFF or Bcl-XL

deficiency.

NF-jB and lymphoid organogenesis

In addition to its role in the development of cells thatdirectly mediate immune responses, NF-kB also playsan important role in the development and function ofprimary and secondary lymphoid tissues. Primary(central) lymphoid organs include the bone marrowand thymus whereas secondary (peripheral) lymphoidorgans include lymph nodes (LNs), Peyer’s patches,mucosal-associated lymphoid tissue (MALT) and thespleen. Among the primary lymphoid organs, the bonemarrow remains active throughout life, whereas thymicactivity dwindles with the onset of adulthood. Thesecondary lymphoid tissues are associated with themaintenance and activation of mature lymphocytes, andprovide an environment within which the interaction oflymphocytes and other leukocytes can be carefullyorchestrated. Although there is clearly a role for NF-kBin the development and regulation of bone, this role hasnot yet been clearly correlated with effects on hemato-poiesis (Jimi and Ghosh, 2005). Careful anatomical andhistological examination of NF-kB-deficient mice hasresulted in NF-kB being assigned an increasinglyprominent role in lymphoid organogenesis.

Secondary lymphoid organsThe secondary lymphoid organs have highly charac-teristic structural features that are crucial to thedevelopment and activation of lymphocytes. Analysisof the role of NF-kB in lymphoid organogenesis inknockouts has been complicated by the necessity ofinterfering with the TNF response to rescue the lethalityassociated with NF-kB deficiency. The initial events oflymphoid organogenesis involve the association oflymphotoxin (LT)a1b2-expressing hematopoietic cellsand 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-kB plays a prominent role(Figure 3). Cytokines implicated in this signaling loop –LTa1b2, RANKL (receptor activator of NF-kB ligand)and TNFa – are known to activate NF-kB. Also,mediators of lymphoid organogenesis and homeostasis,such as the adhesion molecules ICAM (intercellularadhesion molecule), VCAM, PNAd (peripheral nodeaddressin), GlyCAM-1 (glycosylation-dependent celladhesion molecule) and MadCAM (mucosal addressincellular adhesion molecule), cytokines including TNFa,and organogenic chemokines such as CXCL12 (GRO/MIP-2), CXCL13 (BLC), CCL19 (ELC) and CCL21(SLC), are regulated by NF-kB.


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Lymphoid organogenesis exhibits distinct requirementsfor both the canonical and non-canonical NF-kBpathways. Signaling through TNF-R, LTbR andRANK activates canonical RelA-containing complexesand, hence, it is not surprising that rela�/�/tnfr1�/�

double knockout mice lack Peyer’s patches and LNsand exhibit disorganized spleens (Alcamo et al., 2002).The requirement for RelA in development of thesetissues lies with the stromal cells and is likely due to acombination of effects: regulation of apoptosis (e.g., thatinduced by TNF); regulation of expression of orga-nogenic factors including VCAM and LTa1b2; and en-hancement of the non-canonical p52/RelB pathwaythrough the LTbR signaling pathway.

Several lines of evidence highlight the importance ofthe non-canonical pathway and activation of p52/RelBcomplexes in LN development. Mice with a pointmutation in nik (aly/aly mice) lack multiple secondarylymphoid organs (Miyawaki et al., 1994; Koike et al.,1996; Shinkura et al., 1999) and share several pheno-typic similarities with lymphotoxin and IKKa singleknockout animals (Mebius, 2003; Bonizzi and Karin,2004). p52/RelB, which is activated downstream of NIKand IKKa, is thought to be the primary transcriptionalmediator of several key organogenic factors includingCXCL12, CXCL13, CCL19, CCL21 and MadCAM-1(Yilmaz et al., 2003). The p52 single knockout lacks

normal B-cell follicles, germinal centers (GCs) andPeyer’s patch development (Caamano et al., 1998;Franzoso et al., 1998; Paxian et al., 2002); RelB islikewise also required for Peyer’s patch development(Yilmaz et al., 2003). Although LN development occursin RelB knockout mice, the nodes are small at birth andare resorbed perinatally. In addition to LTbR, knock-outs of RANK, which likewise signals through the non-canonical pathway, also lack peripheral LNs (Dougallet al., 1999).

Splenic architecture is crucial for B-cell developmentas well as for the initiation and maturation of B-cellresponses. The spleen is divided histologically into whiteand red pulp zones. Macrophages in the red pulp areresponsible for destroying erythrocytes that are da-maged or have reached the end of their lifespan. Thewhite pulp is populated by splenic lymphocytes andconsists of B-cell follicles and T-cell zones. Splenicarchitecture allows for dynamic changes, most notablyin the formation of GCs, during the initiation andmaturation of B-cell responses. Multiple NF-kB knock-outs exhibit defects in some aspect of splenic architec-ture; however, as for other lymphoid organs, theanalysis of splenic architecture has been complicatedby defects that occur upon deletion of TNF-R used torescue the embryonic lethality. Nevertheless, there hasbeen considerable progress in deciphering the role ofNF-kB family members in development and mainte-nance of splenic architecture. Mice in which RelA hasbeen targeted for deletion exhibit aberrant segregationof B- and T-cell areas and defects in one particularmacrophage population, the metallophilic marginalzone macrophages. In addition, rela�/�/tnfr1�/� spleenshave a more pronounced defect in GC generationfollowing immunization, than do tnrf1�/� mice (Alcamoet al., 2002). However, it is worth emphasizing thatdefects observed in tnfr1�/� animals may, in fact, be dueto changes in RelA-dependent responses, and it ispossible that the role of the RelA/canonical NF-kBpathway in the spleen is underappreciated.

The importance of the non-canonical pathway in thespleen has been observed in multiple circumstances.Mice in which non-canonical pathway components,RelB, NIK or IKKa, have been inactivated demonstratesevere defects in splenic architecture, similar to that seenin ltbr�/� spleens. These defects largely reflect deficien-cies in splenic stromal cells (Miyawaki et al., 1994;Koike et al., 1996). Mice deficient in the non-canonicalpathway fail to segregate B-cell–T-cell zones and FDCnetworks, and they fail to form GCs followingimmunization. Marginal zone macrophages, which linethe border between red and white pulp areas, are alsoabsent or disorganized in RelB, p52, NIK or IKKaknockouts (Franzoso et al., 1998; Weih et al., 2001).Some splenic defects are also attributable to effects onhematopoietic cells. For example, the presence ofmetallophilic marginal zone macrophages depends onp52 (Franzoso et al., 1997). Finally, knockout of theatypical IkB family member BCL-3 also leads toalterations in lymphoid architecture that are reminiscentof those seen in the absence of p52, with which BCL-3

Local stromalcell

RANK RANKL

VCAM-1Chemokines

Hematopoieticcell

αα4β1

LTα1β2LTβR

Figure 3 NF-kB function in the early events of lymphoidorganogenesis. NF-kB is a vital part of the positive feedback loopbetween hematopoietic and stromal cells that comprises the earlyevents of lymphoid organogenesis. LTa1b2-expressing hematopoie-tic cells induce production of VCAM-1 through the canonicalNF-kB pathway and chemokines through the non-canonical(IKKa-dependent) pathway in LTbR-expressing stromal cells.Stromal expression of chemokines induces the upregulation ofintegrins (a4b1) on hematopoietic cells resulting in increasedrecruitment of LTa1b2-expressing cells and signaling throughstromal LTbR. RANKL stimulation of NF-kB through TRAF6is also crucial for the upregulation of LTa1b2 in hematopoietic cells.


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forms a transcriptionally active complex. BCL-3 knock-out mice lack splenic GCs, and although they exhibitnormal serum antibody levels, they fail to developantigen-specific humoral responses (Sha et al., 1995;Caamano et al., 1996; Franzoso et al., 1997; Schwarzet al., 1997).

In summary, both the canonical and non-canonicalNF-kB pathways are required for the development ofmost secondary lymphoid organs. However, the role ofthe non-canonical pathway, as assessed by examiningmice deficient for IKKa, p52, NIK or RelB, is especiallyimportant both during organogenesis and maintenanceof splenic architecture. Recent data suggest that thenon-canonical pathway is also important in thymicdevelopment and organization (Burkly et al., 1995; Weihet al., 1995; Kajiura et al., 2004; Kinoshita et al., 2006).However, it is important to note that canonical NF-kBpathway function in these events may be underappre-ciated owing to embryonic lethality and complicated bythe defects introduced by crossing them onto the tnfr�/�

background. Nevertheless, our understanding of non-canonical pathway function in secondary lymphoidorgans is consistent with the ability of RelB-containingcomplexes to regulate genes encoding key organogenicchemokines and adhesion molecules that direct leuko-cyte trafficking. The functional consequences of defectsin these processes are severe and have direct ramifica-tions for the host’s ability to mount a robust immuneresponse. Alterations in lymphoid architecture likewiseimpede the initiation of the adaptive response as well asthe fine-tuning of this response through processes suchas B-cell affinity maturation.

Role of NF-jB in the innate response

Pattern recognition receptorsTo activate an appropriate immune response, the hostmust first recognize the presence of pathogens. Thisdiscrimination between self and non-self is an absoluterequirement for the initiation of effector functions, suchas the secretion of cytokines and antimicrobial peptides,carried out by the cells of the innate immune system. Anumber of pattern recognition receptors (PRRs) haveevolved to recognize microbial invaders. These PPRsinclude TLRs, members of the CATERPILLAR/NODfamily of cytoplasmic receptors, scavenger receptors andthe complement system. Although epithelial cells arefrequently the first to encounter pathogens, they are alsoconstantly exposed to non-pathogenic microbes. There-fore, whereas a variety of TLRs are differentiallyexpressed in epidermis, gut, pulmonary, urinary andreproductive epithelium, in many cases it is thought thatboth TLR expression and responsiveness is tightlycontrolled in these cells. For example, keratinocytesupregulate TLRs expression and responsiveness follow-ing transforming growth factor-alpha (TGFa) exposure(Miller et al., 2005); renal epithelial cells increaseexpression of TLR2 and TLR4 in response to IFNg orTNFa (Wolfs et al., 2002) and intestinal epithelial cells

have been shown to alter TLR expression underinflammatory conditions (Mueller et al., 2006). How-ever, sentinel cells of the innate immune system,particularly tissue resident DCs and macrophages,express a more complete complement of PRRs, andthus are likely to bear the largest portion of the burdenin the earliest events of pathogen recognition.

Toll-like receptorsTLRs are evolutionarily conserved PRRs that recognizeunique, essential molecules characteristic of variousclasses of microbes (Akira et al., 2006). The functionof TLRs as arbitrators of self/non-self discriminationhighlights their central role in innate immunity as well asin the initiation of the adaptive immune response. The11 characterized mammalian TLRs have varied tissuedistribution and serve as recognition receptors forpathogen-associated molecular patterns (PAMPs) pre-sent on bacteria, viruses, fungi and parasites. Perhapsdue to the multimeric nature of the TLR extracellulardomain (ED), which consists of multiple leucine-richrepeats (LRRs), several receptors are capable ofrecognizing more than one microbial molecule (Figure 4and below). Heterodimerization of some TLRs and theuse of co-receptors (e.g., CD14 and MD-2) furtherexpand the repertoire of PAMPs recognized. As we shallsee below, the ability of TLRs to distinguish betweenpathogen types is translated into appropriate innate andadaptive responses through the selective activation ofNF-kB and other inducible transcription factors. Sig-nificant progress has been made over the past few yearsin deciphering the relevant signaling pathways thatoperate downstream of TLRs in particular.

TLR signaling to NF-kBLigand binding to TLRs is just now beginning to beunderstood at the molecular level. Extracellular LRRsbind to ligand and, either through receptor oligomeriza-tion and/or induction of a conformational change acrossthe plasma membrane, induce the recruitment/activa-tion of adapter proteins through the Toll/IL-1 Receptor(TIR) domain. These adapters lead to the activation ofcanonical IKKb-dependent complexes, degradation ofIkBa and IkBb, and liberation of, primarily, RelA andc-Rel containing NF-kB complexes. TLR signaling toNF-kB is divided into two pathways: those that areMyD88 (myeloid differentiation primary response gene88)-dependent and those that are MyD88-independent(Figure 5). We will base our discussion primarily onsignaling events emanating from TLR4, which despitehaving the most complex downstream pathways is themost thoroughly studied TLR. Clear differences exist insignaling from other TLRs as noted throughout ourdiscussion, and it is likely that further specializationswill become apparent as individual TLR signalingpathways are investigated more thoroughly.

MyD88-dependent signaling to NF-kB. TLR4 signalingis relatively unique amongst TLRs in that the effectoradaptors are one step removed from the receptor. For


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example, MyD88 recruitment to the receptor complexdepends upon the TIR-domain containing adapterprotein (TIRAP, also known as Mal) (Fitzgerald et al.,2001; Horng et al., 2002; Yamamoto et al., 2002). TLR2also requires TIRAP to bridge MyD88 to the receptor;however it is believed that other MyD88-utilizing TLRsdirectly recruit MyD88. Recent reports suggest that therequirement for these intermediatory adapters is relatedto localization of the TLRs to certain domains in theplasma membrane (Kagan and Medzhitov, 2006; Roweet al., 2006). The N-terminal domain of MyD88contains a death domain (DD) that recruits the DD-containing serine/threonine kinase interleukin-1-associated kinase-4 (IRAK-4). IRAK-4 and IRAK-1form an active complex capable of recruiting the TNFreceptor-associated factor TRAF6 (Figure 5a). The linkbetween TRAF6 and the IKK complex remains some-what enigmatic, although a few key players are known.The kinase TAK1 (TGFb-activated kinase-1) is requiredfor NF-kB, 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 iswidely accepted that ubiquitination is a key switch atthis crucial step of NF-kB activation, considerable workat the molecular level remains to be done to understandhow 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 withTRAF6. ECSIT binds to TRAF6 and is required forTLR and interleulin-1 (IL-1) signaling, but not TNF-signaling (Kopp et al., 1999; Xiao et al., 2003). Althoughthese studies suggested that ECSIT functions byrecruiting and activating the kinase MEKK1 (mitogenactivated protein kinase or ERK kinase (MEK) kinase1) (Kopp et al., 1999; Xiao et al., 2003), the role ofMEKK1 in TLR signaling remains unclear (Xia et al.,2000; Yujiri et al., 2000). MEKK3-deficient cells,however, do not transcribe IL-6 following TLR4 orIL-1R stimulation and exhibit delayed and weakenedNF-kB DNA binding following lipopolysaccharide(LPS) stimulation (Huang et al., 2004). ECSIT interactswith both TAK1 and MEKK3 (AP West and S Ghosh,unpublished observations) and it is, therefore, possiblethat ECSIT exerts its role in TLR signaling bymodulating the function of TAK1 and/or MEKK3.

TRIF-dependent signaling to NF-kBSomewhat unexpectedly, when exposed to LPS,MyD88�/� cells display partial NF-kB activation, albeitwith slower kinetics than in wild-type cells (Kawai et al.,1999). When cells are stimulated through TLR3 andTLR4, TRIF (TICAM-1), a TIR-domain containingadapter, mediates activation of NF-kB in the absence ofMyD88 (Oshiumi et al., 2003). Furthermore, in the case

Triacyl Lipopeptides

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


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of TLR3, all downstream signaling appears to be TRIF-dependent. In TLR4 signaling, TRIF is required forlate-phase NF-kB and IRF3 responses, but is notrequired for activation of JNK (Yamamoto et al.,2003). TRIF signaling to NF-kB and IRF3 also appearto be separately regulated (Figure 5b). Signaling to IRF-3 occurs through two divergent members of the IKKfamily, IKKi (IKKe) and TBK1 (T2K) (Fitzgerald et al.,2003; Sharma et al., 2003); however, neither kinase isrequired for NF-kB activation by LPS or TNFa(Hemmi et al., 2004; McWhirter et al., 2004). Further-more, reconstitution of trif�/� cells with mutant TRIFlacking the TRAF-binding domain selectively restoresinduction of IRF3 but not NF-kB. Increasingly, there-fore, it appears that the events leading from TRIF toIKK activation share a common set of intermediates asseen in other NF-kB activation pathways.

TRIF interacts with receptor interacting protein(RIP)1 and RIP3 through their RIP homotypic interac-tion motif (RHIM), and rip1�/� embryonic fibroblastshave decreased NF-kB activation following TLR3-poly(I:C) signaling (Meylan et al., 2004). Finally,another TIR-domain containing adapter TRAM

(TRIF-related adapter molecule) functions upstream ofTRIF in MyD88-independent signaling from TLR4.TRAM is required for IRF3 activation and for thedelayed phase of NF-kB activation following TLR4engagement. TLR4-induced IRAK activation byMyD88, however, is unaffected by the absence ofTRAM and TRAM does not function in TLR3 TRIF-dependent signaling pathways (Fitzgerald et al., 2003;Yamamoto et al., 2003). Therefore, it appears thatTRAM is only needed for TRIF signaling downstreamof TLR4. Adding further complexity, it has recentlybeen suggested that TLR4, but not TLR3, TRIF-dependent NF-kB activation is largely due to IRF3-induced TNFa rather than to direct signaling to IKK(Covert et al., 2005; Werner et al., 2005). Althoughthe applicability of these finding to other cell types is, asof yet, unclear, these results may be explained bydifferences in the recruitment of TRIF to thereceptor; that is, by TRAM in the case of TLR4 versusdirectly to TLR3, resulting in changes in the avail-ability of TRAF binding site or the availability ofadditional signaling intermediates at distinct subcellularlocalizations.

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Figure 5 PRR signaling to NF-kB. Signaling through LPS/TLR4 via the MyD88-dependent (b) and TRIF-dependent (a) pathwaysconverge on IKK activation through TRAFs. Signaling through dsRNA/RIG-I (c) proceeds through ISP1 to IKKi/TBK1 and throughRIP1 to IKK. Signaling from NOD to NF-kB (d) is thought to involve oligomerization of RIP2 and activation of IKK throughinduced proximity. See the text for details.


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Negative regulation of TLR signalingInflammatory responses are built upon waves ofcytokine production and positive feedback mechanisms.As a result, tight control must be placed on the initiationand maintenance of these responses. Multiple negativefeedback loops have been described that involveproteins that are induced or activated upon TLRsignaling. In a number of instances, the target of theseregulatory mechanisms is the IRAK family of proteins.For example, IRAK-M (IRAK3) inhibits signaling toTRAF6 by fixing IRAK-1/4 to the TLR/MyD88signaling complex; irakm�/� knockouts exhibit enhancedsignaling to NF-kB (Kobayashi et al., 2002). Tollip, anadapter protein constitutively associated with IRAK, isphosphorylated and dissociates following IRAK4 acti-vation (Burns et al., 2000; Zhang and Ghosh, 2002).Negative regulation of TLR signaling by Tollip in theintestinal epithelium may prevent inflammatory re-sponses to commensal bacteria (Melmed et al., 2003).However, Tollip-deficient cells demonstrate only minordefects in the production of NF-kB-regulated cytokines(Didierlaurent et al., 2006). Therefore, it is unclearwhether Tollip indeed functions as initially thought.SIGIRR (TIR8), a member of the IL-1R family, bindsto Toll/IL-1 receptors, IRAK and TRAF6 and may alsofunction by inhibiting the association of IRAK withTLRs (Thomassen et al., 1999; Wald et al., 2003).SIGIRR deficiency yields prolonged activation ofNF-kB by Toll/IL-1 stimulation consistent with aregulatory function. Interestingly, SIGIRR is also highlyexpressed in epithelial cells, suggesting that it too maysuppress signaling at sites of constitutive microbialexposure. Finally, suppressor of cytokine signaling-1(SOCS-1) has been reported to negatively regulate LPSsignaling to NF-kB and socs1�/� mice exhibit aninflammatory phenotype that is consistent with thisprediction (Kinjyo et al., 2002; Nakagawa et al., 2002).SOCS-1 may function by directly targeting TIRAP/Mal,and selectively inhibit TLR4 signaling through theTIRAP/Mal/MyD88 pathway (Mansell et al., 2006).

In addition to these TLR-specific regulators ofsignaling to NF-kB, other proteins function to controlthe extent and duration of NF-kB activation. Thesefactors both set thresholds for activation and help toprevent uncontrolled, and potentially deleterious, innateimmune responses. The broad array of PAMPs recog-nized by the TLR system affords the host the ability tomount responses against many pathogens. Nevertheless,for some pathogens, TLRs alone are not sufficient, andsome physical spaces, most notably the cytosol, are noteffectively monitored by TLRs.

Cytoplasmic PRRs that activate NF-kBPRRs that recognize bacterial PAMPs are expressed atthe plasma membrane or with LRRs projecting into thelumen of vesicles that are topologically related to theextracellular space. However, in such a system, intra-cellular pathogens are uniquely protected from detec-tion. Furthermore, viral infection and the resultinginduction of interferon occurs in many cells that do not

express the full panoply of antiviral TLRs – suggestingthat other PRRs must be at work. In fact, cells do haveat their disposal families of cytoplasmic PRRs that arecapable of activating NF-kB and other transcriptionalmediators of the innate immune response. Interestingly,many of these PRRs contain caspase activation andrecruitment domains (CARDs) that are required foractivation of NF-kB following ligand binding. Here, weprovide a brief description of two classes of cytoplasmicPRRs – CARD-containing members of the CATEPIL-LAR and DExD/H-box helicase families.

CATEPILLER-NODs. Nucleotide oligomerizationdomain proteins (NOD) 1 and 2 are part of a largefamily termed the CATEPILLER family, which isnamed for CARD, transcription enhancer, R (purine)-binding, pyrin, lots of leucine repeats (Figure 4). TheNOD-LRR subfamily is typified by the presence ofLRRs and nucleotide oligomerization domains. NOD1,NOD2 and IPAF have CARDs and can signal to NF-kB (for a review see Inohara and Nunez, 2003). NOD1recognizes a peptidoglycan containing meso-diaminopi-melic acid (meso-DAP) and induces NF-kB through acanonical pathway that includes activation of IKKb.NOD2 recognizes muramyl dipeptide, a ubiquitouscomponent of nearly all bacterial cell walls. Relatively,few signaling intermediates downstream of NOD-LRRsare known; however, there is growing evidence that theCARD-containing kinase RIP2 (RICK) is required forNF-kB activation. Intriguingly, the ATP-binding cas-sette of both NOD1 and NOD2 is needed for signaling(Tanabe et al., 2004). RIP2 binds to NEMO andtherefore is thought to directly mediate activation ofthe IKK complex by induced proximity (Inohara et al.,2000). In this model, ligand-dependent oligomerizationof NOD-LRRs, which is dependent on the ATP-bindingcassette, leads to a scaffold containing multiple RIP2molecules, which allows trans-autophosphorylation ofneighboring IKK complexes (Figure 5d).

Retinoic acid inducible gene I and melanomadifferentiation-associated gene 5Two members of the DExD/H-box RNA helicase familystand out because of the presence of N-terminal CARDdomains. Retinoic acid inducible gene I (RIG-I) andmelanoma differentiation-associated gene 5 (MDA5) areRNA helicase-containing cytoplasmic proteins. TheRNA helicase domains of RIG-I and MDA5 binddirectly to double-stranded RNA (dsRNA) and induceproduction of type I interferons (Kang et al., 2002;Andrejeva et al., 2004; Yoneyama et al., 2004). Uponbinding to dsRNA, representing either the viral genomeor viral replication intermediate, RIG-I and MDA5induce the activation of IRF3 and NF-kB. Interestingly,initiation of these signaling cascade is abrogated bypoint mutations in the Walker-type ATP-binding site,suggesting that their ATPase activity is required forsignaling (Yoneyama et al., 2004). The link betweenthese two proteins and NF-kB remains somewhatunclear (Figure 5c); however overexpression of the


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N-terminal CARD domain alone is sufficient to inducesignaling. Recently, a CARD-containing protein, vari-ably named CARDIF, IPS1, MAVS and VISA, hasbeen implicated downstream of RIG-I; however, thelink between this protein and IKKb is unclear (Kawaiet al., 2005; Meylan et al., 2005; Seth et al., 2005; Xuet al., 2005). It appears, however, that there aresimilarities to TRIF mediated signaling, in that RIG-Iactivation of NF-kB requires FADD and RIP-1(Balachandran et al., 2004; Yoneyama et al., 2005).Recently, it was shown that RIG-I and MDA5differentially recognize various groups of RNA virusesand are thus critical for a robust antiviral response(Kato et al., 2006).

Pathogen recognition in innate immunity

Bacterial recognitionPathogens recognized by PRRs can be categorized asbacterial, viral or eukaryotic. In each of these categories,PAMPs have been described that more or less fit withexisting hypotheses of how pathogen recognition by theinnate immune system should occur (Janeway, 1989).Both in terms of accessibility and uniqueness toprokaryotes, the bacterial cell well is a logical sourceof PAMPs for TLRs and other PRRs.

LPS was originally thought to be the ligand forTLR2, but subsequent studies revealed that contaminat-ing bacterial lipoprotein in LPS preparations is theactual ligand (Wetzler, 2003). TLR2 also mediatesresponses to several Gram-positive bacterial cell wallcomponents as well as Staphylococcus aureus peptido-glycan (Takeuchi et al., 2000). Additional work hasshown that TLR2 is involved in the recognition of awide range of microbial products and generally func-tions 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 theTLR2/TLR6 complex recognizes mycoplasma lipopro-teins and peptidoglycan (Takeuchi et al., 2001). Recentreports have demonstrated that triacylated lipoproteinsfrom bacteria are preferentially recognized by theTLR1/TLR2 complex, whereas diacylated lipoproteinsare recognized by the TLR2/TLR6 complex (Takeuchiet al., 2002). However, additional TLR2 ligands do notrequire TLR1 or TLR6 for signaling, implying thatTLR2 recognizes some ligands as a homodimer orheterodimer with other non-TLR molecules. Such TLR2ligands include the Gram-positive cell wall componentlipoteichoic acid; the mycobacterial cell wall componentlipoarabinomannan; atypical LPS produced by Legionella,Leptospira interrogans, Porphyromonas gingivitisand Bordetella; and porins present in the outermembrane of Neisseria (Massari et al., 2003; Wetzler,2003).

The TLR4 ligand LPS, a glycolipid component of theouter membrane of Gram-negative bacteria, is the most

thoroughly studied and the most potent TLR ligandknown. Trace amounts of LPS activate the innateimmune system via TLR4, leading to the production ofnumerous proinflammatory mediators, such as TNFa,IL-1 and IL-6. TLR4-mediated responses to LPS requireCD14 and MD-2. Other bacterial TLR4 ligands includeLipid A analogs (Lien et al., 2001) and mycobacterialcomponents (Means et al., 1999).

TLR5 recognizes flagellin, a protein component ofGram-negative bacterial flagella and virulence factor formultiple human pathogens (Hayashi et al., 2001). Inlight of the fact that it was thought that proteins wouldbe too mutable to serve as PAMPs, it is notable thatTLR5 recognizes a highly conserved, central corestructure of flagellin that is essential for protofilamentassembly (Smith et al., 2003). Interestingly, the TLR5recognition site is masked in the filamentous flagellarstructure, thus indicating that TLR5 recognizes onlymonomeric flagellin (Smith et al., 2003). Furthermore,flagellin appears to bind directly to TLR5 at residues386–407, as TLR5 mutants lacking this domain areunable to interact with flagellin in biochemical assays(Mizel et al., 2003). Recent articles have demonstratedTLR5-independent recognition of cytosolic Salmonellatyphimurium flagellin via Ipaf, a member of the NOD-LRR family (Franchi et al., 2006; Miao et al., 2006).Ipaf-mediated recognition of cytosolic flagellin inducescaspase-1 activation and subsequent IL-1b secretion bymacrophages. TLR11 recognizes a protein PAMP that ispresent 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 isdestroyed by proteinase treatment.

Conserved differences in bacterial nucleic acid struc-tures can also be recognized by the innate immunesystem. TLR9 recognizes bacterial DNA containingunmethylated CpG motifs, and TLR9-deficient mice arenot responsive to CpG DNA challenge (Hemmi et al.,2000). The low frequency and high rate of methylationof CpG motifs prevent recognition of mammalian DNAby TLR9 under physiological circumstances. A recentreport indicated that the intracellular, endosomalrestriction of TLR9 is critical for discriminating betweenself and nonself DNA, as host DNA, unlike microbialDNA, does not usually enter the endosomal compart-ment (Barton et al., 2006).

Viral recognitionAlthough viruses are composed entirely of host productsthey, nevertheless, have unique components that readilyserve as PAMPs. Nucleic acids are also key viralPAMPs, and are recognized by TLRs 3, 7, 8 and 9, aswell as by cytoplasmic receptors of the RIG family (asdescribed above). TLR3 recognizes dsRNA, a commonviral replicative intermediate (Alexopoulou et al., 2001).TLR3 signaling results in the activation of NF-kBand IRF3, ultimately leading to the production of anti-viral molecules, such as type I interferons (IFN-a/b)(Alexopoulou et al., 2001). The importance of RIG-I-and MDA5-mediated viral recognition is further


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supported by gene-targeting experiments demonstratingthat TLR3 and its adaptor TRIF are not required fortype I IFN production in some virally infected cells,such as fibroblasts and conventional DCs (Honda et al.,2003). However, plasmacytoid DCs exclusively utilizeTLR3/TRIF signaling for type I IFN production inresponse to RNA viruses and poly(I:C) (Kato et al.,2005).

Although initially found to recognize synthetic anti-viral compounds, namely imidazoquinolines, the azo-quinoline R-848 and loxoribine (Hemmi et al., 2002;Jurk et al., 2002), TLR7 and human TLR8 are nowknown to recognize guanosine- or uridine-rich single-stranded RNA derived from RNA viruses (Dieboldet al., 2004; Heil et al., 2004; Lund et al., 2004).Interestingly, mammalian RNA, which contains manymodified nucleosides, is significantly less stimulatory viaTLRs 7 and 8 than bacterial RNA, suggesting thatnucleoside modification allows mammals to distinguishbetween endogenous and pathogen-derived RNA(Kariko et al., 2005). Similar to TLR3, engagement ofthese receptors leads to the production of type I IFNs.

TLR9 recognizes viral CpG sequences and induces theinduction of IFN-a (Takeshita et al., 2001; Lund et al.,2003; Krug et al., 2004). However, as membranerestriction prevents TLRs from sampling the cytosolwhere much of the viral life cycle occurs, cytosolic PRRsprovide comprehensive innate immune recognition. Forexample, recognition of cytoplasmic dsDNA leading toNF-kB activation and type I interferon production hasalso been reported, although the relevant receptor hasnot yet been identified (Ishii et al., 2006; Stetson andMedzhitov, 2006). This receptor(s) is predicted to beimportant for type I IFN production in response toviruses and intracellular pathogens, such as Listeriamonocytogenes and Shigella flexneri. Finally, there havebeen some reports suggesting that certain viral proteinsfunction as PAMPs. For example, TLR4 may recognizerespiratory syncytial virus (RSV) F protein (Kurt-Joneset al., 2000).

Recognition of other pathogensMyD88-deficient cells demonstrate that many fungalspecies 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 neoformanscapsular polysaccharide (Shoham et al., 2001). TLR2and TLR6 are required for recognition of yeastzymosan, whereas TLR4 is thought to recognize certainyeast mannans (for a review see Levitz, 2004). Theidentification of parasite PAMPs has been more elusive,and their existence is somewhat controversial. However,TLR2 heterodimers reportedly recognize various para-site GPI-anchored proteins and glycoinositolphospholi-pids from the parasitic protozoa Trypanosoma cruzi(Campos et al., 2001). Some TLR knockout mice havebeen shown to have variable defects in their ability todefend against various parasites (for a review seeGazzinelli et al., 2004). Recently, TLR9 has been

reported to recognize the malarial pigment hemozoin,a byproduct of heme metabolism in infected erythro-cytes (Coban et al., 2005) whereas TLR11 recognizes aprofilin-like protein that is conserved in apicomplexanparasites including Toxoplasma gondii (Yarovinskyet al., 2005).

Immediate antimicrobial responses

PRRs initiate a complex series of events followingexposure to certain microbial components: the first is themounting of immediate antimicrobial responses at thecellular level. This is an effective and evolutionarilyconserved function of PRRs, and one in which NF-kBhas an important role. The liberation of products withdirect antimicrobial activity occurs early at sites ofpathogen entry. TLR ligation is at least partly respon-sible for the NF-kB-dependent expression of defensins –cationic peptides that exert direct bactericidal activity byinducing membrane permeabilization. Small intestinalPaneth cells, for example, release large amounts ofa-defensins into the intestinal lumen following exposureto a variety of bacteria/bacterial products (Ayabe et al.,2000). The production of antimicrobial nitrogen andoxygen species, which are acutely toxic to a variety ofmicrobes, augments the activity of antimicrobial pep-tides. Production of nitric oxide is mediated in part byinducible nitric oxide synthase (iNOS), which is partiallyregulated by NF-kB. Consequently, iNOS productionresults from TLR or NOD-LRR ligation by PAMPs.

Much of the early innate response has been demon-strated to depend on the canonical NF-kB pathway.Thus, rela�/�/tnfr1�/� double knockout mice haveincreased susceptibility to bacterial infection (Alcamoet al., 2001). Likewise, B cells from p50�/� mice do notrespond efficiently to LPS, emphasizing the importanceof 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/IKKb doubleknockouts show a more pronounced defect in innateresponses owing to the more complete block incanonical NF-kB pathways, and succumb to infectionmore rapidly than rela�/�/tnfr1�/� mice (Li et al.,1999a, b; Senftleben et al., 2001b). Furthermore, MEFsfrom nemo�/� mice do not exhibit NF-kB activation byLPS or IL-1 (Rudolph et al., 2000). Therefore, activa-tion of NF-kB responsive genes by the innate immunesystem depends on NEMO and likely progressesthrough the canonical NF-kB signaling pathway.

Inflammation

There is a staggering amount of literature that correlatesNF-kB activation with inflammation in a wide array ofdiseases and animal models. There are, likewise,numerous studies using gene targeting and inhibitorsof NF-kB that have established that NF-kB plays acausative role in inflammatory processes. We havealready discussed the role of NF-kB in the survival of


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leukocytes, and how this role is particularly importantduring the responses that include inflammation. Here,we briefly discuss a few of the additional ways in whichNF-kB regulates inflammation. Inflammation beginswith epithelial or stromal cells of the infected tissue ortissue resident hematopoietic cells such as mast cells orDCs recognizing an inflammatory stimulus and propa-gating proinflammatory signals. These signals lead tothe recruitment and activation of effector cells, initiallyneutrophils and later macrophages and other leuko-cytes, resulting in the tissue changes characteristic ofinflammation – rubor, calor, dolor and tumor (redness,heat, pain and swelling, respectively).

As for the immediate antimicrobial products dis-cussed above, NF-kB is responsible for the transcriptionof the genes encoding many proinflammatory cytokinesand chemokines. One important early target of theseeffectors is the vascular endothelium. Changes invascular endothelial cells both recruit circulating leuko-cytes and provide them with a means of exiting thevasculature into the infected tissue. NF-kB regulates theexpression of adhesion molecules, both on leukocytesand endothelial cells, which allow the extravasation ofleukocytes from the circulation to the site of infection(Eck et al., 1993). Indeed, RelA-deficient mice display asevere defect in the recruitment of circulating leukocytesto sites of inflammation (Alcamo et al., 2001).

Recruited neutrophils are the key mediators of localinflammation and NF-kB is important for the survivalof these cells, which must function in relatively toxicconditions (Ward et al., 1999). NF-kB is important forthe production of the enzymes that generate prostaglan-dins and reactive oxygen species (e.g., iNOS and Cox,both NF-kB target genes) and may, furthermore, beinvolved in the signaling induced by prostaglandins(Poligone and Baldwin, 2001; Catley et al., 2003).NF-kB has also been implicated in the response toleukotrienes, which like prostaglandins are short-livedparacrine effectors, although it is unclear whether thisrepresents a direct signaling event. Finally, matrixmetalloproteinases (MMPs) also are crucial mediatorsof local inflammation and leukocyte chemotaxis; andtheir expression is also regulated by NF-kB (Vincentiet al., 1998; Vincenti and Brinckerhoff, 2002; Lai et al.,2003).

The pathway from pathogen recognition to proin-flammatory cytokine production demonstrates a parti-cular reliance on NF-kB. The immediate targets ofNF-kB-dependent proinflammatory cytokines, such asTNFa, tend to be receptors that, in turn, activate NF-kB.Therefore, NF-kB is crucial to the propagation andelaboration of cytokine responses. TNFa is particularlyimportant for both local and systemic inflammation,and it is a potent and well-studied inducer of NF-kB.

TNF-R superfamily signalingThe TNF-R superfamily is remarkably diverse withmore than two-dozen receptors and nearly as manyligands that are variably expressed throughout thebody. However, despite the physiological diversity of

responses, in most cases signaling converges on theactivation of NF-kB and AP-1. NF-kB activation inresponse to TNF signaling induces expression ofantiapoptotic genes such as cIAP1/2 and Bcl-XL (seeDutta et al., 2006).

TNF family receptors lack intrinsic enzymatic acti-vity. Instead, signaling is achieved by recruitment ofintracellular adapter molecules that associate with thecytoplasmic tail of the TNF-R in a signal-dependentmanner (Figure 6a). The recruitment of TNF-R1 tomembrane microdomains, referred to as lipid rafts, withsubsequent assembly of the signaling complex, isnecessary for signaling to NF-kB and prevention ofapoptosis (Hueber, 2003; Legler et al., 2003). Ligation ofTNF-R1 by trimeric TNFa causes aggregation of thereceptor allowing binding of the TNF-R-associateddeath domain protein (TRADD). TRADD subse-quently recruits adapter molecules including TRAF2

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Figure 6 TNF receptor superfamily signaling to NF-kB. Activa-tion of the canonical NF-kB pathway downstream of TNF-RI isinitiated by trimerization through ligand binding and recruitmentof FADD, TRADD, RIP1 and TRAF2/5 to the receptor(a). TAK1 and MEKK3 are subsequently recruited to the receptorcomplex through RIP1, and with TRAF2/5, mediate the activationof IKK. The phosphorylation and degradation of classical IkBsrequire IKKb and NEMO. The non-canonical pathway is mediatedby IKKa 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 ofNIK, activation of IKKa, phosphorylation of p100 and liberationof p100-inhibited NF-kB complexes. Simultaneous activation ofthe canonical NF-kB pathway through either TRAF2, 5, or 6also commonly occurs downstream of receptors that activate thenon-canonical pathway. See the text for additional details.


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(Hsu et al., 1996); however, TRAF2 and TRAF5 appearto play redundant roles in TNF signaling to NF-kB.TRAF2 or TRAF5-deficient mice have intact TNFactivation of NF-kB, whereas TRAF2/5 double knock-out cells have substantially reduced TNF-induced IKKactivation (Yeh et al., 1997; Nakano et al., 1999; Tadaet al., 2001). TRAFs may either recruit the IKKcomplex directly (Devin et al., 2001) or indirectlythrough the serine/threonine kinase, RIP1. RIP1 canalso interact independently with TRADD and is anessential adapter for TNF-induced NF-kB activationand protection from apoptosis (Hsu et al., 1996; Tinget al., 1996; Kelliher et al., 1998; Devin et al., 2000).Upon ubiquitination, RIP1 can bind directly to NEMOand recruit IKK independent of TRAF2 (Zhang et al.,2000). Signaling downstream of RIP1 requires TAK1for the activation of IKK (Sato et al., 2000; Shim et al.,2005). Whether TAK1 directly activates IKK or thisprocess proceeds through an intermediary such asMEKK3 is not yet clear (Takaesu et al., 2003; Liet al., 2006). However, it does not appear that TAK1 isinvolved in the activation of the non-canonical NF-kBpathway (Shim et al., 2005).

The non-canonical NF-kB pathway is unique in thatit is independent of IKKb and NEMO and insteadrequires IKKa which is phosphorylated by NF-kBinducing kinase (NIK) (Xiao et al., 2001; Senftlebenet al., 2001a; Claudio et al., 2002; Dejardin et al., 2002;see Scheidereit, 2006). The key question concerningsignaling by these stimuli is how they are channeled toNIK and IKKa, even though their receptor signalingdomains resemble those of other TNF family members(Figure 6b). Because RANKL, BAFF and CD40L mayalso activate the canonical pathway through TRAF2/6,it would appear that the intracellular signaling domainof these receptors possess additional sequence motifsthat allow their signaling to NIK. This function ismediated by TRAF3, which interacts with thesereceptors. TRAF3 negatively regulates NIK, and under-goes signal-dependent degradation resulting in theactivation of the non-canonical pathway (Liao et al.,2004).

Resolution of inflammation may also involve NF-kBResolution of inflammation and subsequent tissue repairis a crucial event, and its failure is a common source ofpathology. It is believed that reversal of inflammation isan active process that is as complex as the inflammatoryresponse itself, and involves numerous pathways thatare not all directly relevant to NF-kB (Serhan andSavill, 2005). Although the traditional view of NF-kBwould lead one to imagine that it would primarilyfunction by being turned off during the resolution phaseof inflammation, recent work has suggested that NF-kBalso has a more active role.

During acute inflammation, there are multiple nega-tive feedback pathways that help to rein in inflammatoryresponses. It has long been known that cells such asmacrophages become resistant to repeated proinflam-matory stimuli. BCL-3, which is induced late following

LPS stimulation, in combination with p50 dimers hasbeen shown to have a role in the inhibition of repeatedLPS responses in macrophages, a phenomenon alsoreferred to as LPS tolerance (Wessells et al., 2004).Furthermore by selectively affecting chromatin remo-deling, BCL-3 mediates repression of proinflammatorygenes, but also facilitates expression of the anti-inflammatory gene IL-10. NF-kB p50 also appears tonegatively regulate IFNg production and proliferationby NK cells (Tato et al., 2006).

In addition to these and other negative feedbackpathways, it was recently found that inhibition ofNF-kB during the resolution phase can prolong theinflammatory process and prevent proper tissue repair(Lawrence et al., 2001). It was subsequently found thatIKKa-deficient mice display increased inflammatoryresponses in models of local and systemic inflammation(Lawrence et al., 2005). Macrophages, in particular,show increased production of proinflammatory chemo-kines and cytokines in the absence of IKKa (Lawrenceet al., 2005; Li et al., 2005). It was suggested from thesestudies that IKKa negatively regulates proinflammatorygene expression, perhaps through mediating degrada-tion of RelA and c-Rel following macrophage activationby LPS.

Initiation of adaptive responses

Although innate responses alone can bring about potentantimicrobial activities, alerting and activating theadaptive immune system remains a crucial step forrobust and durable immune responses. This process islargely mediated by activation and maturation ofantigen-presenting cells (APCs), which can, in turninstruct T and B cells to carry out the adaptive response.

DC maturation mediated by pathogen recognition iscrucial for the initiation of the adaptive immuneresponse. To activate naıve T cells, DCs must undergomultiple changes. First, DCs must gain the ability tointeract with T cells by changing their chemokinereceptor expression and migrating into lymphoid tissues.Second, DCs must alter their antigen processingmachinery to favor the presentation of pathogenepitopes on MHC. Third, APCs must upregulate theexpression of costimulatory molecules B7.1/B7.2 (orCD80/CD86), which are regulated by NF-kB and ligateCD28 providing the second signal necessary to induceT-cell activation. Finally, as progress is made inexploring these events it is becoming increasingly clearthat the responses to different pathogens are tailoredbased on the distribution of PRRs in different cell typesand the ability of different cell types to, in turn, interactwith T cells in a biasing manner (Iwasaki andMedzhitov, 2004).

Maturation of DCs following viral infection dependson nucleic acid-binding PRRs, including both TLRs andcytoplasmic RIG family molecules. Indeed, DC matura-tion during viral infection occurs normally in theabsence of MyD88 or TLR3, as reported previously


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(Lopez et al., 2003). Bacterial responses are eithermediated through TLRs – DCs express TLRs 1, 2, 5and 6 – or other classes of PRRs. Murine CD8aþ DCs,which tend to induce TH1 responses important inclearance of viral and parasitic infections, expressTLR1, 2, 6, 9 and 11. In the absence of TLR11, forexample, mice fail to mount a TH1 response againstT. gondii, owing to the failure of CD8aþ DCs torecognize the TLR11 ligand (Yarovinsky et al., 2005). Itremains unclear whether the ability of distinct TLRligands to induce TH1 versus TH2 responses is intrinsicto the specific TLR/ligand, dose of ligand or the cell typewithin which this activation occurs; evidence to datepoints towards the latter. Even less clear are the roles ofother PRRs in APC maturation and the initiation ofadaptive responses.

Finally, there is the question of the role of innaterecognition of pathogens by lymphocytes themselves.Recently, it has been suggested that TLR signaling in Bcells is also required for optimal response to certainantigens (Pasare and Medzhitov, 2005). Both B andT cells express TLRs, although exactly which TLRsare expressed is debatable. At the level of mRNA

expression, human peripheral B cells express TLR1, 2, 4,6 and 9 (Hornung et al., 2002).

Role of NF-jB in the adaptive response

AgR signaling to NF-kBThe hallmark of the adaptive immune response isantigen specificity. In the section on hematopoiesis, wediscussed how NF-kB plays an important role in theselection of lymphocytes bearing somatically generatingAgRs. Signaling through these antigen-specific B-celland T-cell receptors is therefore the central event of theadaptive immune response. Activation of NF-kB down-stream of BCR and TCR ligation facilitates antigen-specific proliferation and maturation of lymphocytesinto effector cells. Signaling through these two AgRsappears to be functionally analogous, although some ofthe components utilized differ.

BCR and TCR signaling are analogous in manyaspects, in particular with respect to the activation ofNF-kB (Figure 7). The T-cell receptor complex consists

TCR

RIP2

Ub

GeneTranscription

NEMO

NF- κB

P

P

a

BCR

PDK1

PI3K

BCL10

CARMA1 CARMA1

MALT1

Syk LynZAP70

AgAg

IP3

RasRas

IP3DAG

AP-1

DAG

NFATNFAT

AP-1

Vav

BCL10

MALT1

Vav

P

P

b

P

P

TRAF6

P

Figure 7 AgR signaling to NF-kB. Binding of TCR to peptide:MHC and co-stimulation through CD28:B7 interaction activatesNF-kB (a). Through multiple steps, initial phosphorylation events at the TCR complex lead to PI3K activation and recruitment ofPDK1. PDK1, in turn, mediates recruitment of the IKK complex through PKCy and the CARMA1 directly. PKCy phosphorylatesCARMA1 leading to the activation of IKK through BCL10, MALT1 and TRAF6. Binding to TI antigens or TD antigens in thepresence of co-stimulatory cytokines results in the activation of NF-kB through the BCR complex (b). Receptor proximal events ofBCR activation are highly analogous to those of the TCR. Although the role of PDK1 in B-cell signaling is not established, BCRsignaling to NF-kB requires PKCb as well as the CBM complex to achieve activation of IKK.


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of a/b subunits that are associated with the CD3 proteinheterodimers g/e, d/e, and either Z/Z, Z/z or z/z, for atotal of eight membrane proteins. The BCR is, likewise,a multiprotein complex consisting of the surfaceimmunoglobulin receptor associated with a heterodimerof Iga and Igb. The AgR complexes associate with Srcfamily tyrosine kinases (SFKs), Lck and Fyn in T cellsand Lyn in B cells, which phosphorylate immunoreceptortyrosine activation motifs (ITAMs) on CD3 and Iga/bchains. The cytoplasmic tyrosine kinases ZAP70 or Sykare then recruited via SH2 domains to the phosphory-lated ITAMs and initiate activation of the IP3 and Rasfamily pathways. The IKK complex is rapidly recruitedto the immunological synapse and can be colocalized tothe TCR (Khoshnan et al., 2000; Weil et al., 2003). InZAP-70-deficient T cells, NF-kB activation can berescued by directly targeting a chimeric NEMO to theimmunological synapse, suggesting that signaling down-stream of ZAP-70 may largely function for IKKrecruitment (Weil et al., 2003).

The signaling pathway from the receptor to NF-kBrequires PKCy (PKCb 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 somecontroversy, available data suggest that PKCy is largelyessential for activation of NF-kB via T-cell stimulation(Sun et al., 2000; Pfeifhofer et al., 2003) and can mediatethe activation of IKK (Lin et al., 2000). PKCy isspecifically recruited to the immunological synapse;although how PKCy but not other PKC isoforms isselectively recruited remains a mystery. In B cells PKCbis, likewise, required for recruitment of the IKKcomplex to lipid rafts following BCR ligation (Suet al., 2002). PKCy is capable of directly interactingwith the IKK complex in primary T cells (Khoshnanet al., 2000) and might, therefore, function by bringingIKK to the receptor complex and into proximity withother essential components in this pathway; namelyCARMA1, BCL10 and MALT1 (collectively known asthe CBM complex). Recently, studies have providedevidence that the protein kinase PDK1 recruits PKCyand the IKK complex to lipid rafts. In addition, PDK1can simultaneously recruit the CBM complex throughbinding to CARMA1 (Lee et al., 2005). The inducedproximity of PKC and the CBM may allow phospho-rylation of CARMA1 by PKCy/b (Matsumoto et al.,2005; Sommer et al., 2005). Interestingly, T-cell-specificPDK1 conditional deletion results in a defect in T-celldevelopment, preventing the production of peripheralT cells (Hinton et al., 2004). On the other hand,PCKy knockouts do not display defects in thymocytedevelopment.

The CBM complex is essential for both AgR signalingin mature B and T cells. What is surprising however, asmentioned above, is the lack of a role for this complex indeveloping lymphocytes, and by extrapolation signalingthrough the pre-AgRs. The MAGUK family proteinCARMA1 is required for activation of NF-kB in T cellsfollowing TCR ligation, but its loss has no effect on thedevelopment of thymocyte (Gaide et al., 2002; Egawa

et al., 2003; Hara et al., 2003). Similarly, BCL10 iscritical for NF-kB activation via the BCR and TCR, yetnormal numbers of peripheral T cells are seen in BCL10knockouts, and no clear defects in B-cell development isobserved (Ruland et al., 2001). BCL10 interacts withCARMA1 leading to BCL10 phosphorylation, althoughCARMA1 lacks kinase activity (Bertin et al., 2001;Gaide et al., 2001).

Interestingly, genetic evidence of a role for RIP2 hasrecently been reported in T-cell signaling (Ruefli-Brasseet al., 2004). RIP2 associates with BCL10 and isnecessary for TCR-induced BCL10 phosphorylationand IKK activation. It is not yet clear how TCRsignaling regulates RIP2 and, in turn, how RIP2 mightaffect IKK activity. BCL10 oligomerization has beenimplicated in IKK activation through a process thatinvolves ubiquitination of NEMO (Zhou et al., 2004).This ubiquitination event appears to be mediated byMALT1, and perhaps TRAF6, although no TCRsignaling deficits have been reported in TRAF6-deficientmice (Lomaga et al., 1999; Sun et al., 2004). If this istrue however, it is possible that the effect is mediatedthrough the kinase TAK1, which functions in IKKactivation downstream of TRAF6 in other pathways.However, B cells in which TAK1 has been conditionallyinactivated have normal BCR signaling to NF-kB. Morework must be carried out with TAK1 conditionalknockouts in both the BCR and TCR pathways toaddress the role of this kinase.

T-cell responses mediated by NF-kBTo become activated, naıve T cells must receive twodistinct signals: antigen-specific and co-stimulatory.Antigen-specific activation signals emanate from thebinding of the TCR to cognate antigenic peptidespresented in the binding cleft of MHC. Co-stimulatorysignaling is provided through ligation of CD28 by B7molecules expressed on activated APCs. Stimulation ofnaıve T cells results in the production of IL-2, which isnecessary for their proliferation and survival. Theseactivated naıve T cell blasts proliferate rapidly andsimultaneously undergo differentiation into effectorcells. In the case of TH cells, proliferation leads todifferentiation into immature effector cells, TH0, whichsubsequently differentiate into TH1 or TH2 cellsdepending on the predominant cytokine milieu. CD8 Tcells are likewise activated by professional APCs,although they may receive secondary signals fromactivated TH1 cells. The unavailability of CD8 condi-tional knockouts, and the selective loss of CD8 cells inthe absence of NF-kB activity has prevented a thoroughcharacterization of the role of NF-kB in these cells.

Rapidly proliferating activated T cells rely on NF-kBactivity for protection from apoptosis as well as for theproduction of cytokines supporting proliferation anddifferentiation. As expected, inhibition of NF-kB inactivated T cells facilitates progression towards AICDor apoptosis (Ivanov and Nikolic-Zugic, 1997; Jeremiaset al., 1998). Indeed, stimulation of RelA-deficient naıveT cells induces cell death (Wan and DeGregori, 2003).


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Peripheral T cells lacking c-Rel do not undergoapoptosis, but nevertheless, fail to proliferate inresponse to typical mitogenic stimuli (Kontgen et al.,1995), and both RelA and c-Rel containing complexesaccumulate in the nucleus following TCR/CD28 stimu-lation (Ghosh et al., 1993). Interestingly, c-Rel-deficientT cells appear to have a defect in TH1 proliferationand production of IFNg, indicating a selective role forNF-kB family members in TH1/TH2 differentiation,independent of that mediated by the innate response.

Multiple transcriptional activators and repressorsregulate expression of IL-2. Among these, members ofthe NF-kB family play multiple roles. In naıve T cells,which do not express IL-2, repressive p50 homo-dimers are found associated with the IL-2 promoter(Grundstrom et al., 2004). Failure of T-cell proliferativeresponses in c-Rel knockout mice is attributable to afailure to produce IL-2 (Kontgen et al., 1995). In naıveT cells, c-Rel is responsible for mediating chromatinremodeling across the IL-2 locus following CD3/CD28co-stimulation (Rao et al., 2003). Naıve T cells can beprimed by exposure to inflammatory cytokines such thatthey generate a more robust response to CD3/CD28 co-stimulation. Overexpression of an IkBa super-repressorsuggested that NF-kB is required for this priming eventin T cells (Mora et al., 2001a). More recent data indicatethat c-Rel is necessary for naıve helper T-cell priming bypro-inflammatory cytokines elicited following stimula-tion with TLR ligands (Banerjee et al., 2005). NF-kBRelA-containing complexes, on the other hand, appearto function more traditionally in mediating transactiva-tion of IL-2 gene expression, and overexpression ofRelA with c-Jun can overcome the requirement forco-stimulation in naıve T cells (Parra et al., 1998).However, as discussed below, these complexes may alsobe the targets of negative regulation following T-celldifferentiation.

Recent work in TH1/TH2 differentiation has focusedon the induction of specific transcription factors in thesetwo effector cell types – T-bet and GATA3, respectively.Interestingly, mice lacking p50 are unable to mount anasthma-like airway TH2 response, and do not induceGATA-3 expression during T-cell stimulation underTH2 differentiating conditions (Das et al., 2001).Consistent with this finding, BCL-3-deficient T cellsalso fail to undergo TH2 differentiation. Furthermore,BCL-3 can induce expression of a reporter gene from agata-3 promoter, suggesting that p50/BCL-3 complexesare crucial for TH2 differentiation (Corn et al., 2005).Conversely, the same authors found that RelB-deficientT cells are deficient in TH1 differentiation and IFNgproduction, and show decreased expression of T-bet;likely through a failure to upregulate STAT4, whichfunctions in signaling from IFN to T-bet induction.Therefore, it appears that NF-kB activation duringTCR stimulation may render cells competent for bothproliferative and differentiating stimuli.

As TH cells differentiate into TH1 or TH2, theydecrease their expression of IL-2 and, instead, becomedependent on TH1 and TH2 cytokines (e.g., IFNg andIL-4). As a corollary, NF-kB transactivation of the IL-2

gene is repressed. Direct binding of T-bet to p65 that isassociated with the IL-2 gene enhancer may mediate therepression of IL-2 production in TH1 cells (Hwang et al.,2005). Alternatively, in TH2 cells the lack of IL-2transcription may be due to the decreased levels of RelAactivation in TH2 cells (Lederer et al., 1994).

B-cell responses mediated by NF-kBB-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 CD40Land cytokines, such as IL-4. B cells from individualswith a mutation in CD40L are unable to undergo classswitch recombination in response to T-dependentantigens (Aruffo et al., 1993). Signaling through CD40activates both canonical and non-canonical NF-kBpathways, although it is unclear which is operative inthe response to T-dependent antigens. For example,whereas B cells from p52�/� mice mount inadequatehumoral responses to various T-dependent antigens,they exhibit a normal response following adoptivetransfer into rag-1�/� mice – indicating that this deficitis not intrinsic to B cells (Franzoso et al., 1998).Furthermore, B cells from relB�/� mice, althoughcrippled in their proliferative response, undergo normalIgM secretion and class switching in response to variousstimuli (Snapper et al., 1996a). Therefore, non-canonicalNF-kB pathway activation downstream of CD40 isprobably not required for class switching duringT-dependent antigen responses.

Analysis of the canonical NF-kB pathway is compli-cated by more generalized defects in lymphocyteresponse owing to the requirement for this pathway inAgR signaling. Nevertheless, whether downstream ofCD40 or other stimuli, evidence supports canonicalpathway activation in the process of class switchrecombination. Following adoptive transfer, B cellsfrom rela�/� mice exhibit markedly diminished classswitching, despite a modest loss of lymphocyte proli-feration following various stimuli (Doi et al., 1997).Likewise, c-Rel-deficient mice, or mice lacking the c-RelC-terminal transactivation domain, fail to generate aproductive humoral immune response suggesting arequirement for c-Rel in class switch recombination(Kontgen et al., 1995; Zelazowski et al., 1997; Carrascoet al., 1998). B cells from nfkb1�/� mice exhibit decreasedproliferation in response to mitogenic stimulation, andp50/RelA double knockout B cells exhibit greaterdefects in proliferation and class switching (Snapperet al., 1996b; Horwitz et al., 1999). Therefore, analysesof knockout animals suggest that the canonical NF-kBpathway likely has a role in maturation of the B-cellresponse in addition to directly mediating proliferativeresponses following BCR ligation.

As discussed above, signaling through TLRs has animportant role in the initiation of the adaptive immuneresponse via APCs of the innate immune system. Inrecent years, however, there has also been increasinginterest in the ability of TLR signaling to directly


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modulate the adaptive response. For example, it hasbeen observed that homeostatic polyclonal activation ofB cells, which results in the so-called serologicalmemory, that is, detectable antibody to antigens thatare no longer present in the host, can be induced/maintained by TLR ligation (Bernasconi et al., 2002).Analogously, TLR2 is upregulated in CD4þ T cellsfollowing TCR stimulation, and TLR2 ligandsmay thus provide an activation/maintenance signal inthese cells (Komai-Koma et al., 2004). That signalingthrough TLRs in these aspects of B-cell responsesrequires NF-kB seems likely, but has yet to bedemonstrated.

TI antigens have an intrinsic ability to activate B-cellresponses in the absence of T-cell help by acting as B-cellmitogens, for example by acting as TLR ligands or bybinding with high avidity to the BCR through repetitivestructural features. In such cases, it is expected that B-cellresponses are more dependent on members of thecanonical pathway that have well-documented roles inTLR signaling, or BCR signaling (as discussed above).For example, c-Rel-deficient B cells are highly sensitiveto apoptosis following BCR cross-linking (Grumontet al., 1998, 1999; Owyang et al., 2001). As mentionedabove, p50 and p50/pRelA double knockout B cells aredeficient in responses to TI stimulation. Likewise,IKKb-deficient B cells fail to mount TI or TD responses(Li et al., 2003). These IKKb-deficient B cells alsoexhibit increased spontaneous apoptosis, suggesting thatNF-kB is important in survival of B cells.

Maintenance and memory: a role for NF-kB in lymphoidcell survivalLymphocyte homeostasis is dependent on the survival ofmature lymphocytes in addition to replenishment of theperipheral lymphocyte pool through lymphopoiesis.Consequently, there is increasing interest in the possiblerole of NF-kB in the survival of mature lymphocytes. Itis widely accepted that lymphocyte survival is mediatedthrough tonic stimulation downstream of the AgR, aswell as certain cytokine receptors. As discussed above,genetic targeting experiments support an important rolefor NF-kB family members in lymphocyte survival.Naıve T cells require continued contact with MHC:self-peptides, most likely expressed on lymphoid DCs, togenerate the tonic TCR signal that is essential forcontinued survival. Survival of memory cells, on theother hand, is independent of continued contact withself-peptide:MHC complexes.

B cells are formed at a far higher rate than T cells andtherefore B cells also undergo a significantly higher rateof turnover. Nonetheless, B cells too require mainte-nance signals to achieve peripheral homeostasis. TheAgR on B cells most likely provides a basal level ofsignaling, albeit independent of the presence of antigen,which is required for maintenance of mature B cells. Notsurprisingly, B cells from RelA�/�, p100�/�, p105�/� andc-Rel�/� mice display increased sensitivity to apoptosisand/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 lossof BCR signaling, as demonstrated in an elegant studythat demonstrated that deletion of the BCR frommature B cells led to a complete loss of the peripheralB-cell pool (Kraus et al., 2004). Most likely this was dueto the loss of signaling to NF-kB in these cells becauseloss of IKKb, NEMO or components of the CBMcomplex in mature B cells also results in a complete lossof peripheral B cells (Pasparakis et al., 2002; Li et al.,2003; Thome, 2004).

The non-canonical NF-kB pathway is also relevant toB-cell survival, as the loss of IKKa results in strikingdefects in B-cell survival (Kaisho et al., 2001; Senftlebenet al., 2001a). However, rather than acting downstreamof tonic BCR signaling, recent studies have implicatedsignaling from BAFFR in this aspect of the Blymphocyte survival (reviewed in Mackay et al., 2003).Together, these data suggest that a subset of Bcl-2family members, for example, the antiapoptotic factorA1, are regulated by p52/RelB-containing complexesand are necessary for the maintenance of mature B cells.

Concluding remarks

NF-kB was originally described as a transcriptionalregulator in the adaptive immune response; however,subsequent studies have revealed its importance inhematopoiesis, lymphoid organogenesis and innateimmunity. Investigations of mice with targeted deletionsand mutations have shed considerable light on the roleof NF-kB in lymphoid organogenesis. For example,characterization of the aly/aly mouse in part led to thediscovery of the non-canonical NF-kB pathway and itsrole in organogenesis. Progress continues to be made inunderstanding AgR signaling to NF-kB, and this workhas led to the appreciation of regulatory ubiquitinationevents in IKK activation (see reviews by Perkins, 2006;Scheidereit, 2006). The specificity of the requirement forthe CBM complex and PKCy/b in signaling by T-celland B-cell receptors opens the door to the developmentof equally specific NF-kB inhibitors. However, thereremain fundamental gaps in our understanding of howthese components mediate IKK activation and thepossible role of regulatory ubiquitination in this process.

The role of NF-kB in hematopoiesis remains partiallydefined, although there is little doubt about itsimportance. Genetic targeting in mice has allowed theenumeration of multiple steps in hematopoiesis at whichvarious NF-kB pathway components are required;however, a cohesive picture of how NF-kB functionsin these steps remains elusive. For example, whereas weknow that canonical NF-kB activity is required in earlylymphopoiesis, it is unclear whether this is in theregulation of TNFa production or in mediating asurvival signal in the developing lymphocyte. NF-kB isrequired in the process of positive and negativeselection, but again, its mechanism(s) of action remainspoorly defined. Progress using in vitro systems forstudying lymphocyte development may allow a more


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rigorous assessment of NF-kB function in theseprocesses. Likewise, advances in conditional genetargeting approaches and the generation of animals inwhich defective versions of NF-kB genes have beenknocked-in may help to overcome the problems ofembryonic lethality and functional redundancy thathave sometimes made existing studies difficult tointerpret.

T- and B-cell responses require NF-kB as a prosurvi-val factor as well as for the regulation of genes involvedin differentiation to effector cells. More recently, alimited number of studies have suggested a role forPRRs in lymphocyte activation, and the role of NF-kBin this process remains to be elucidated. The role ofNF-kB in the differentiation of TH cells is incompletelyunderstood, and requires further clarification. We alsoknow very little about the role of NF-kB in CD8þ

activation, differentiation and function; progress in thisarea awaits development of better tools for geneticallymanipulating this cell population. Upon successfulclearance of pathogen, regulation of NF-kB allowsresolution of the response and facilitates the develop-ment of memory cells. To date, relatively little is known

about the role of NF-kB in memory cells, althoughrecent advances in identifying and characterizingmemory precursors bodes well for future progress inthis area.

In summary, research to date has highlighted theimportance of NF-kB in regulating genes that preventapoptosis and that promote differentiation and deve-lopment in cells of the innate and adaptive immunesystems. A large body of work has elucidated many ofthe molecular mechanisms governing regulation ofNF-kB by engagement of innate or lymphocyte anti-gen-specific receptors. In turn, these data provide atrove of information that will likely prove useful inattempts to manipulate the immune system to preventand treat disease.

Acknowledgements

Research in the authors’ laboratory was supported bygrants from the National Institutes of Health (to SG). MSHwas supported by NIH/National Institute of GeneralMedical Sciences Medical Scientist Training GrantGM07205.

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NF-kB and the Immune Response

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