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Signaling in B cells via Toll-like receptors
Signaling in B cells via Toll-like receptors Stanford L Peng Toll-like receptors (TLRs) and their ligands have emerged as important regulators of immunity, relevant to a wide range of effector responses from vaccination to autoimmunity. The most well-studied ligands of TLRs expressed on B cells include the lipopolysaccharides (for TLR4) and CpG-containing DNAs (for TLR9), which induce and/or co-stimulate B cells to undergo proliferation, class switching and differentiation into antibodysecreting cells. Recent developments in this area include advancements into our understanding of the role of these receptor pathways in B cells, and in particular the relevance of TLR9, which has received substantial attention as both a Th1-like inflammatory immunomodulator and a pathogenic co-stimulator of autoreactive B cell responses. Addresses Departments of Internal Medicine and Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8045, St. Louis, Missouri 63110, USA Corresponding author: Peng, Stanford L ([email protected])
Current Opinion in Immunology 2005, 17:230–236 This review comes from a themed issue on Lymphocyte activation Edited by Gail A Bishop and Jonathan R Lamb Available online 9th April 2005 0952-7915/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2005.03.003
Introduction The Toll-like receptors (TLRs) include at least 11 type I integral membrane glycoproteins, and are members of a larger superfamily that includes the IL-1 receptors [1]. TLRs recognize a diverse array of ligands, including pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharides (LPS), RNAs and DNAs, which are widely recognized by immune cells, including cells of both adaptive and innate lineages. In the past few years, several studies have specifically examined the expression of TLRs in B cells, primarily in human tonsillar or peripheral blood populations (Table 1). Although most studies agree that mRNAs corresponding to all TLRs can be detected in B cells, significant expression is only generally agreed upon at this time for TLR1 and TLR6–10, all of which are upregulated during activation; for example, in response to B cell receptor (BCR) or CD40 ligation, CpG oligonucleotide exposure in vitro, and as seen in activated B cell subsets ex vivo. B cells are Current Opinion in Immunology 2005, 17:230–236
now particularly well recognized for their expression of the TLR9 and TLR10, which are induced in response to BCR stimulation and appear to predominate in activated and/or memory populations [2,3]. This spectrum of inducible expression of the TLRs has been generally presumed to extend to rodent B cells; however, species differences clearly exist, as naı¨ve murine B cells are known to express TLR4 and undergo proliferation and plasmacytoid differentiation in vitro in response to LPS exposure, in contrast to human B cells, which seem to lack significant TLR4 expression, at least in the naı¨ve resting state [4]. As such, the vast majority of studies involving TLRs in B cells have focused upon TLR9 and its ligands (hypomethylated CpG-containing DNAs); nonetheless, several rodent-based studies have addressed TLR4 and its predominant ligand, LPS. This review focuses particularly upon recent advances regarding TLR9 signaling in B cells; for an overview of TLRs in general, their ligands and functional importance during global immune responses, the reader is directed to one of many excellent recent reviews (e.g. [1,5,6]).
An overview of the signaling pathways of TLR4 and TLR9 The signaling mechanisms of both the TLR4 and TLR9 pathways have been studied extensively (Figures 1 and 2). TLR4 is expressed on the cell surface in complex with the MD-2 molecule, and this heterodimer participates in LPS recognition to initiate intracellular signaling by at least two major adaptor pathways. These pathways include the TIRAP–MyD88 pathway, which regulates rapid NF-kB activation and related inflammatory cytokine production, and the TRIF–TRAM pathway, which regulates the activation of interferon regulatory factor (IRF)-3 and the subsequent induction of type I interferons and co-stimulatory molecules (reviewed in [1]). By contrast, TLR9 is expressed in the endoplasmic reticulum and is recruited to endosomal/lysosomal compartments after stimulation with CpG DNAs, activating the MyD88 pathway without TIRAP, culminating in NF-kB activation [7]. Interestingly, some studies have preliminarily suggested that TRIF also provides an additional MyD88-independent pathway for TLR9 signaling [8]. These pathways have primarily been elucidated in macrophages, dendritic cells and/or cultured cell lines. Relatively few investigations have specifically confirmed the details of these pathways in B cells, although they are likely to be relevant as they have been observed in multiple cell lineages. It is worth noting, however, that, www.sciencedirect.com
Signaling in B cells via TLRs Peng 231
Table 1 Expression of Toll-like receptors on human B cells Compared to Receptor
Naı¨ve/Resting
Activated
PMN
DC
Mono
TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR7 TLR8 TLR9 TLR10 TLR11
+ +/ +/ +/ +/ + + + + + ND
++ +/ +/ +/ +/ +++ +++ ++ ++++ ++++ ND
+++ +++ ND +++ ND +++ ND ND ND ND ND
ND ND + ND ++ ND ND +++ ND ND ND
ND ND ND ND ND ND +++ ND ND ND ND
Relative expression levels of the indicated Toll-like receptors on human B cells are indicated. +++, strong expression; ++, moderate expression; +, low but definite expression; +/ , expression at a barely detectable or functionally controversial level. Naı¨ve/Resting, CD19+CD27 or high-density peripheral blood or tonsillar B cells; activated, memory (CD19+CD27+) or intermediate-density peripheral blood or tonsillar B cells. Where data are available, expression levels are compared with neutrophils (PMN), dendritic cells (DC) or monocytes/macrophages (Mono). Summarized from references [2–4,29,42,43]. ND, not determined.
unlike macrophages and dendritic cells, B cells also utilize a heterodimer consisting of RP105 and MD-1, which are structurally related to TLR4 and MD-2, to recognize and respond to LPS (reviewed in [9]). Although B cells deficient in either TLR4 or MD-2 do not respond to LPS at all, cells deficient in either RP105 or MD-1 are hyporesponsive to LPS, suggesting that the RP105–MD-1 heterodimer plays a uniquely important role in B cells by enhancing TLR4dependent LPS responses. Indeed, patients with systemic lupus erythematosus, dematomyositis or Sjo¨ gren’s syndrome possess an unusually high percentages of RP105negative B cell populations, which are associated with activation and accentuated production of antibodies and autoantibodies [9,10]. As such, it is interesting to consider that, in addition to such apparently lineage-specific LPS signaling features as RP105 and MD-1, B cells might possess additional lineage-specific signaling molecules that participate in the recognition of LPS and/or other PAMPs, such as CpG, which might play specific roles in pathogenic states of humoral and/or B cell related immunity.
Effects of TLR9 signaling in B cells The TLR9 pathway continues to receive growing attention as a consequence of the potential relevance of unmethylated CpG-containing DNAs to the pathogenesis of autoimmune diseases, as well as in immunomodulatory therapeutic strategies [11].
can form complexes with hypomethylated CpG-containing DNAs, and then bind both to rheumatoid factor (antiIgG) B cells through their BCR via direct IgG binding, and to TLR9 via binding of the co-complexed DNA, resulting in activation [12]. These hypomethylated CpG DNAs have traditionally been thought to derive from environmental pathogens, suggesting a need for a non-self trigger for autoreactive B cell activation; however, mammalian DNAs contain rare hypomethylated CpG regions that are capable of co-stimulating autoreactive B cells. Therefore, sources of self DNA, such as apoptotic cells or necrotic debris, can potentially function in this situation as truly autoreactive stimuli in such immune complexes [13]. Furthermore, anti-DNA B cells themselves, by being able to bind DNAs through both their BCR and TLR9, might be directly co-stimulated by hypomethylated CpG DNA motifs without a need for immune complexes (Figure 2). In this sense, hypomethylated CpG motifs, whether or not in the form of immune complexes, can initiate and/or promote systemic humoral autoimmunity by preventing or promoting the breakage of tolerance of autoreactive B cells. It is, however, important to note that, at least in the antilysozyme transgenic model of B cell tolerance (in which anti-lysozyme transgenic B cells are rendered anergic by transgenic soluble lysozyme), CpG DNAs can break tolerance, inducing proliferation of, and immunoglobulin secretion by, autoreactive cells in vitro, but do not appear to be capable of inducing pathogenic disease [14,15]. This insufficiency has been attributed to the observation that anergic B cells have uncoupled the BCR from a calcineurin-dependent signaling pathway, leading to continuous ERK signaling, which inhibits CpG-induced plasma cell differentiation [14]. As such, additional stimuli, which could simply consist of excess antigen [15], are probably required to cooperate with CpG DNAs to break tolerance fully and result in pathogenic humoral immunity. Perhaps this tolerance breakdown, in part, involves the physically close coupling of BCR–TLR9 interactions that could be conferred by IgG–CpG DNA-containing immune complexes upon rheumatoid factor B cells, or by CpG DNAs alone upon anti-DNA B cells, resulting in a supramolecular activation complex. Alternatively, such immune complexes could also activate complement and/or Fc receptors, providing additional co-stimulation to the B cells, although there is less evidence for such mechanisms [12]. Thus, it remains unclear if TLR9 signals are necessary or single-handedly sufficient to both induce autoimmunity and propagate the response to full-blown pathogenic humoral immunity in vivo, although their ability to promote autoreactive B cell responses, at least to some degree, seems clear.
TLR9 signaling in B cell autoimmunity
There now exists strong evidence that TLR9 activation can co-stimulate autoreactive B cells, thereby breaking tolerance. For instance, anti-DNA IgG autoantibodies www.sciencedirect.com
Immunomodulatory effects of TLR9 signaling in B cells
CpG DNAs can furthermore skew immune responses towards a Th1-like phenotype, enhancing, for example, Current Opinion in Immunology 2005, 17:230–236
232 Lymphocyte activation
Figure 1
LBP LPS
TLR4– MD-2
RP105– MD-1
LPS LPS
TRIF
TRAM
TIRAP
MyD88
CD14 Cytoplasm
PKR
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TBK1
IKK-ε
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MKK3/6
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MKK4
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STAT1 IRF-3
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IFN-inducible genes
Inflammatory gene transcription
TAK1
AP1
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Current Opinion in Immunology
A proposed model for TLR4 signaling in B cells. Extracellular lipopolysaccharide (LPS) is bound by LPS-binding protein (LBP), recognized by CD14, and brought in proximity to the TLR4–MD-2 and RP105–MD-1 heterodimers. TLR4 initiates signaling via at least two adaptor pathways: TIRAP–MyD88 and TRIF–TRAM. The TIRAP–MyD88 pathway elicits an IRAK–TRAF6–TAK1 pathway. TAK1 activates NIK, which activates the IKK complex, which in turn phosphorylates the IkB proteins that normally sequester NF-kB proteins in the cytoplasm. TAK1 also activates the JNK and p38 MAPK pathways, leading to the activation of AP-1 complexes. Both the NF-kB and AP-1 proteins enter the nucleus where they activate target genes involved in inflammation, particularly cytokines. The TRIF–TRAM pathway results in activation of the atypical IKKs IKK-e and TBK1, as well as the IFN-inducible PKR [44]. Together, these kinases result in both IkB phosphorylation and NF-kB activation, enhancing the transcription of inflammatory cytokines, as well as the activation of STAT1 and IRF-3, which regulate the expression of IFN-inducible genes. Abbreviations: AP-1, activating protein; IkB, inhibitor of NF-kB; IKK, IkB kinase; IRAK, IL-1R-associated kinase; IRF, interferon regulatory factor; NIK, NF-kB-inducing kinase; PKR, double-stranded RNA-dependent protein kinase; STAT, signaling transducer and activator of transcription; TAB, TAK1-binding protein; TAK1, TGF-b-activated kinase; TBK1, TRAF-family-member-associated NF-kB activator-binding kinase 1; TIRAP, TIR-domain-containing adaptor protein; TRAF, TNF-receptor-associated factor; TRAM, TRIF-related adaptor molecule; TRIF, TIR-domain-containing adaptor protein inducing IFN-b.
IFN-g and IL-12 responses in vivo [16]. In such studies, CpG treatments are associated with the preferential production of ‘Th1-like’ immunoglobulin isotypes, such as the IFN-g-related IgG2a in mice. Previously, such effects upon B cells were presumed to reflect the indirect effect of skewing towards and/or preferential survival of Th1 cells in the helper T cell compartment, but recent studies indicate that CpG DNAs might have direct effects upon murine B cells, inducing and/or promoting class switching to ‘Th1-like’ isotypes, such as IgG2a, IgG2b and IgG3, Current Opinion in Immunology 2005, 17:230–236
while suppressing the production of the ‘Th2-like’ (IL-4related) isotypes IgG1 and IgE [17–19]. Interestingly, the Th1-like effect requires the classical MyD88 adaptor pathway and, at least for IgG2a class switching, requires the Th1-related T-box transcription factor T-bet, but the effect of CpG DNAs on IgG1 and IgE production appears to be at least partially MyD88 independent [17,19]. Thus, alternative pathways for CpG–TLR9 signaling are likely to exist, perhaps involving TRIF [8], and participate in the regulation of B cell responses; www.sciencedirect.com
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Figure 2
CpG-containing DNA
anti-DNA BCR Late endosome/ lysosome
Early endosome
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TLR9 ERK IRAK
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TLR9 TAB1 TAK1 TAB2
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AF
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Activation proliferation Ig secretion
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IκB Current Opinion in Immunology
A proposed model for TLR9 signaling in B cells. Environmental CpG-containing DNAs (mammalian or non-mammalian) are directly endocytosed or internalized after binding to anti-DNA BCRs. TLR9, normally generated and found in the endoplasmic reticulum, encounters CpG DNAs in lysosomal/late endosomal compartments, where it initiates a signaling cascade via MyD88–IRAK–TRAF6–TAK1, similar to TLR4 (Figure 1 but without TIRAP. TAK1 activates NIK, which activates the IKK complex, which in turn phosphorylates the IkB proteins that normally sequester NF-kB proteins in the cytoplasm. TAK1 also activates the JNK and p38 MAPK pathways, leading to the activation of AP-1 complexes. Both the NF-kB and AP-1 proteins enter the nucleus where they activate target genes involved in B cell activation, proliferation, and immunoglobulin (Ig) production. In anergic B cells, such as anti-DNA B cells in non-autoimmune hosts, BCR signaling is dissociated from activation such that BCR signaling alone results in suboptimal NF-kB activation, perhaps related to tonic ERK activation. TLR9 ligation can bypass and/or co-stimulate this mechanism, leading to autoreactive B cell activation. Abbreviations: AP-1, activating protein; BCR, B cell receptor; IkB, inhibitor of NF-kB; IKK, IkB kinase; IRAK, IL-1R-associated kinase; NIK, NF-kB-inducing kinase; TAK, TGF-b-activated kinase; TRAF, TNF-receptor-associated factor; TIRAP, TIR-domain-containing adaptor protein.
nonetheless, CpG-mediated immunomodulation could obviously be particularly beneficial in Th2- or IgE-related inflammation, such as asthma or allergy. Studies of TLR9 in human B cells have revealed analogous observations, but with several caveats. In humans, CpG DNAs can also induce class switching to IgG1, IgG2 and IgG3, which are distantly related to the murine IgG2a, IgG2b and IgG3 isotypes; however, significant immunoglobulin secretion requires additional signals through the BCR and dendritic cell derived B cell activating factor www.sciencedirect.com
(BAFF; also called TALL-1, BLyS, THANK, zTNF4), indicating that CpG DNAs are less potent in human than in murine B cells [20]. In addition, whereas CpG-containing DNAs stimulate essentially all murine B cells, most studies have indicated that they preferentially stimulate activated and/or memory human B cells, as opposed to naı¨ve cells, reflecting the preferential expression of TLR9 on activated cells (Table 1). Indeed, in one study, BCR triggering was required to render naı¨ve human B cells responsive to CpG DNAs, whereas memory B cells were responsive to CpG DNAs alone [3]. Other studies have Current Opinion in Immunology 2005, 17:230–236
234 Lymphocyte activation
indicated that, whereas CpG DNAs alone can induce the expression of co-stimulatory molecules and chemokine receptors such as CXCR3 in human B cells, CpG DNAs require synergistic cooperation with CD40 signals and/or simultaneous culture with plasmacytoid dendritic cells to induce secretion of immunoglobulins or cytokines such as IL-6 and IL-10 [21–23], or to promote Th1 development by helper T cells [4]. Thus, although CpG DNAs are highly potent B cell immunomodulators in murine systems, their application to immunomodulation in humans, for example, in the treatment of asthma or allergy, might require additional manipulations to yield a robust effect.
Effects of other Toll-like receptor signals on B cells Although the ability of rodent B cells to respond to LPS by proliferation, class switching, immunoglobulin secretion and plasmacytoid differentiation has been extensively described, the TLR4 pathway has generally received less attention than TLR9, and human B cells have classically been considered LPS-unresponsive due to an absence of TLR4 (Table 1; [2,24]). Still, many studies have suggested that TLR4 expressed on B cells recognizes not only classical LPS but also a growing list of pathogenic antigens, including viral proteins and parasitic heat shock proteins [6,25,26], although other studies suggest that contaminating endotoxins might have confounded these findings [27,28]. Furthermore, some studies have indicated that TLR4 expression can in fact be induced in human B cells by specific stimuli, such as IL-4 [29], suggesting that human B cells might in fact be susceptible to TLR4 signaling under certain conditions. Thus, multiple environmental pathogens can exert physiologically important immunomodulatory effects on B cells via TLR4, presumably promoting activation and differentiation. Other TLRs similarly seem likely to affect B cell function both in humans and rodents in vivo. TLR2 ligands, such as neisserial porins, lipoproteins and glycoinositolphospholipids, have been shown to activate B cells, generally acting as mitogens [30–32]. Also, the TLR7-binding guanosine analogs can stimulate human B cells [33], suggesting that B cells can be stimulated by the physiological ligands of TLR7, the single-stranded RNAs [34– 36]. The physiological effects of such ligands on B cells, however, remain incompletely characterized.
Conclusions TLR ligands, particularly CpG-containing DNAs and LPS, have emerged as critical modulators of B cell effector function, promoting proliferation, plasmacytoid differentiation and class switching, as well as autoimmunityor Th1-related inflammation. However, questions remain regarding the applicability of these findings to immunomodulatory strategies in vivo – both in rodents and in humans. For instance, species-specific differences in Current Opinion in Immunology 2005, 17:230–236
optimal CpG sequences suggest differential regulation and/or recognition of TLR9 binding and/or signaling [11,37], and it has become clear that several ‘classes’ of stimulatory CpG oligonucleotides (ODNs) exist: CpG-A ODNs, which strongly induce IFN-a in plasmacytoid dendritic cells (PDCs) and potently activate NK cells; CpG-B ODNs, which potently activate NK and B cells but not PDCs; and CpG-C ODNs, which combine the effects of both [38,39]. On the one hand, these findings suggest the presence of yet more TLRs or co-receptors that specify CpG recognition, but simultaneously suggest on the other hand that the discovery of specific CpG sequences in murine B cells might not directly translate to humans and/or in vivo. Indeed, repeated CpG administration to mice surprisingly results in immunodeficiency due to lymphoid follicle destruction [40]. Thus, the translation of TLR ligands to immunotherapy should probably proceed with cautious enthusiasm. Although the studies summarized here strongly suggest that TLR ligands can break B cell tolerance, primarily stimulating and/or co-stimulating otherwise anergic autoreactive B cells, the normally constant exposure of the majority of mammals to environmental TLR ligands (e.g. in the form of bacterial LPS or CpG-containing DNAs) without the development of autoimmunity suggests the presence of protective mechanisms in B cells to prevent such adverse outcomes in vivo. One strategy, at least in murine B cells, appears to involve the desensitization of signaling pathways, such as Erk in anergic BCR cascades [14]. Another strategy, demonstrated in human B cells, appears to involve the differential expression of TLRs, conferring TLR ligand responsiveness only to activated/ memory B cells that have previously undergone antigen selection [41]. However, naı¨ve murine B cells can be activated by LPS alone, and both naı¨ve human and murine B cells can be stimulated by ligation of both the BCR and TLR9 [3,12,14].These mechanisms, therefore, seem insufficient to explain the resistance of B cells to TLR-induced autoimmunity in vivo. Future studies will hopefully elucidate the precise means by which TLRs modulate B cell activation versus tolerance.
Acknowledgements This work was supported in part by the Siteman Cancer, Rheumatic Diseases, Diabetes Research and Training, and the Digestive Diseases Research Core Centers of the Washington University School of Medicine, as well as grants from the National Institutes of health (AI061478 and AI057471), and the Lupus Research Institute. SLP is supported in part by an Arthritis Investigator Award from the Arthritis Foundation.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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20. He B, Qiao X, Cerutti A: CpG DNA induces IgG class switch DNA recombination by activating human B cells through an innate pathway that requires TLR9 and cooperates with IL-10. J Immunol 2004, 173:4479-4491. Here, human B cells are also demonstrated to respond to CpG DNAs by undergoing class switch recombination to IgG1, IgG2 and IgG3 by a mechanism that cooperates with IL-10. Interestingly, significant immunoglobulin secretion appears to require additional factors, such as BAFF and BCR signaling. 21. Gantner F, Hermann P, Nakashima K, Matsukawa S, Sakai K, Bacon KB: CD40-dependent and -independent activation of human tonsil B cells by CpG oligodeoxynucleotides. Eur J Immunol 2003, 33:1576-1585. 22. Kato A, Ogasawara T, Homma T, Batchelor J, Imai S, Wakiguchi H, Saito H, Matsumoto K: CpG oligodeoxynucleotides directly induce CXCR3 chemokines in human B cells. Biochem Biophys Res Commun 2004, 320:1139-1147. 23. Poeck H, Wagner M, Battiany J, Rothenfusser S, Wellisch D, Hornung V, Jahrsdorfer B, Giese T, Endres S, Hartmann G: Plasmacytoid dendritic cells, antigen, and CpG-C license human B cells for plasma cell differentiation and immunoglobulin production in the absence of T-cell help. Blood 2004, 103:3058-3064. The requirements for plasmacytoid differentiation in human B cells by CpG-C oligonucleotides are demonstrated to require also BCR ligation and plasmacytoid dendritic cells. 24. Muzio M, Bosisio D, Polentarutti N, D’Amico G, Stoppacciaro A, Mancinelli R, van’t Veer C, Penton-Rol G, Ruco LP, Allavena P et al.: Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J Immunol 2000, 164:5998-6004. 25. Aosai F, Chen M, Kang HK, Mun HS, Norose K, Piao LX, Kobayashi M, Takeuchi O, Akira S, Yano A: Toxoplasma gondiiderived heat shock protein HSP70 functions as a B cell mitogen. Cell Stress Chaperones 2002, 7:357-364. 26. Rassa JC, Meyers JL, Zhang Y, Kudaravalli R, Ross SR: Murine retroviruses activate B cells via interaction with toll-like receptor 4. Proc Natl Acad Sci USA 2002, 99:2281-2286. 27. Bausinger H, Lipsker D, Ziylan U, Manie S, Briand JP, Cazenave JP, Muller S, Haeuw JF, Ravanat C, de la Salle H et al.: Endotoxin-free heat-shock protein 70 fails to induce APC activation. Eur J Immunol 2002, 32:3708-3713. See annotation to [28]. 28. Gao B, Tsan MF: Endotoxin contamination in recombinant human heat shock protein 70 (Hsp70) preparation is responsible for the induction of tumor necrosis factor alpha release by murine macrophages. J Biol Chem 2003, 278:174-179. Current Opinion in Immunology 2005, 17:230–236
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This study and [27] demonstrate that, at least for some heat shock proteins, contaminating endotoxin accounts for their TLR stimulatory activities. 29. Mita Y, Dobashi K, Endou K, Kawata T, Shimizu Y, Nakazawa T, Mori M: Toll-like receptor 4 surface expression on human monocytes and B cells is modulated by IL-2 and IL-4. Immunol Lett 2002, 81:71-75. 30. Massari P, Henneke P, Ho Y, Latz E, Golenbock DT, Wetzler LM: Cutting edge: Immune stimulation by neisserial porins is toll-like receptor 2 and MyD88 dependent. J Immunol 2002, 168:1533-1537. 31. Hovav AH, Davidovitch L, Nussbaum G, Mullerad J, Fishman Y, Bercovier H: Mitogenicity of the recombinant mycobacterial 27-kilodalton lipoprotein is not connected to its antiprotective effect. Infect Immun 2004, 72:3383-3390. 32. Previato JO, Wait R, Jones C, DosReis GA, Todeschini AR, Heise N, Previato LM: Glycoinositolphospholipid from Trypanosoma cruzi: structure, biosynthesis and immunobiology. Adv Parasitol 2004, 56:1-41. 33. Lee J, Chuang TH, Redecke V, She L, Pitha PM, Carson DA, Raz E, Cottam HB: Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of Toll-like receptor 7. Proc Natl Acad Sci USA 2003, 100:6646-6651. 34. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C: Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 2004, 303:1529-1531. 35. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, Lipford G, Wagner H, Bauer S: Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004, 303:1526-1529. 36. Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC, Gale NW, Iwasaki A, Flavell RA: Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc Natl Acad Sci USA 2004, 101:5598-5603. 37. Bauer S, Kirschning CJ, Hacker H, Redecke V, Hausmann S, Akira S, Wagner H, Lipford GB: Human TLR9 confers responsiveness
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to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA 2001, 98:9237-9242. 38. Vollmer J, Weeratna R, Payette P, Jurk M, Schetter C, Laucht M, Wader T, Tluk S, Liu M, Davis HL et al.: Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. Eur J Immunol 2004, 34:251-262. 39. Jurk M, Schulte B, Kritzler A, Noll B, Uhlmann E, Wader T, Schetter C, Krieg AM, Vollmer J: C-Class CpG ODN: sequence requirements and characterization of immunostimulatory activities on mRNA level. Immunobiology 2004, 209:141-154. 40. Heikenwalder M, Polymenidou M, Junt T, Sigurdson C, Wagner H, Akira S, Zinkernagel R, Aguzzi A: Lymphoid follicle destruction and immunosuppression after repeated CpG oligodeoxynucleotide administration. Nat Med 2004, 10:187-192. Continuous CpG oligonucleotide administration to mice surprisingly induces an immunodeficient state, altering lymphoid organ morphology and inducing multifocal liver necrosis. 41. Pasare C, Medzhitov R: Toll-like receptors: balancing host resistance with immune tolerance. Curr Opin Immunol 2003, 15:677-682. 42. Hornung V, Rothenfusser S, Britsch S, Krug A, Jahrsdorfer B, Giese T, Endres S, Hartmann G: Quantitative expression of tolllike receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol 2002, 168:4531-4537. 43. Zarember KA, Godowski PJ: Tissue expression of human Tolllike receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol 2002, 168:554-561. 44. Hsu LC, Park JM, Zhang K, Luo JL, Maeda S, Kaufman RJ, Eckmann L, Guiney DG, Karin M: The protein kinase PKR is required for macrophage apoptosis after activation of Toll-like receptor 4. Nature 2004, 428:341-345.
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Current Opinion in Immunology 2005, 17:230–236 This review comes from a themed issue on Lymphocyte activation Edited by Gail A Bishop and Jonathan R Lamb Available online 9th April 2005 0952-7915/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2005.03.003
Introduction The Toll-like receptors (TLRs) include at least 11 type I integral membrane glycoproteins, and are members of a larger superfamily that includes the IL-1 receptors [1]. TLRs recognize a diverse array of ligands, including pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharides (LPS), RNAs and DNAs, which are widely recognized by immune cells, including cells of both adaptive and innate lineages. In the past few years, several studies have specifically examined the expression of TLRs in B cells, primarily in human tonsillar or peripheral blood populations (Table 1). Although most studies agree that mRNAs corresponding to all TLRs can be detected in B cells, significant expression is only generally agreed upon at this time for TLR1 and TLR6–10, all of which are upregulated during activation; for example, in response to B cell receptor (BCR) or CD40 ligation, CpG oligonucleotide exposure in vitro, and as seen in activated B cell subsets ex vivo. B cells are Current Opinion in Immunology 2005, 17:230–236
now particularly well recognized for their expression of the TLR9 and TLR10, which are induced in response to BCR stimulation and appear to predominate in activated and/or memory populations [2,3]. This spectrum of inducible expression of the TLRs has been generally presumed to extend to rodent B cells; however, species differences clearly exist, as naı¨ve murine B cells are known to express TLR4 and undergo proliferation and plasmacytoid differentiation in vitro in response to LPS exposure, in contrast to human B cells, which seem to lack significant TLR4 expression, at least in the naı¨ve resting state [4]. As such, the vast majority of studies involving TLRs in B cells have focused upon TLR9 and its ligands (hypomethylated CpG-containing DNAs); nonetheless, several rodent-based studies have addressed TLR4 and its predominant ligand, LPS. This review focuses particularly upon recent advances regarding TLR9 signaling in B cells; for an overview of TLRs in general, their ligands and functional importance during global immune responses, the reader is directed to one of many excellent recent reviews (e.g. [1,5,6]).
An overview of the signaling pathways of TLR4 and TLR9 The signaling mechanisms of both the TLR4 and TLR9 pathways have been studied extensively (Figures 1 and 2). TLR4 is expressed on the cell surface in complex with the MD-2 molecule, and this heterodimer participates in LPS recognition to initiate intracellular signaling by at least two major adaptor pathways. These pathways include the TIRAP–MyD88 pathway, which regulates rapid NF-kB activation and related inflammatory cytokine production, and the TRIF–TRAM pathway, which regulates the activation of interferon regulatory factor (IRF)-3 and the subsequent induction of type I interferons and co-stimulatory molecules (reviewed in [1]). By contrast, TLR9 is expressed in the endoplasmic reticulum and is recruited to endosomal/lysosomal compartments after stimulation with CpG DNAs, activating the MyD88 pathway without TIRAP, culminating in NF-kB activation [7]. Interestingly, some studies have preliminarily suggested that TRIF also provides an additional MyD88-independent pathway for TLR9 signaling [8]. These pathways have primarily been elucidated in macrophages, dendritic cells and/or cultured cell lines. Relatively few investigations have specifically confirmed the details of these pathways in B cells, although they are likely to be relevant as they have been observed in multiple cell lineages. It is worth noting, however, that, www.sciencedirect.com
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Table 1 Expression of Toll-like receptors on human B cells Compared to Receptor
Naı¨ve/Resting
Activated
PMN
DC
Mono
TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR7 TLR8 TLR9 TLR10 TLR11
+ +/ +/ +/ +/ + + + + + ND
++ +/ +/ +/ +/ +++ +++ ++ ++++ ++++ ND
+++ +++ ND +++ ND +++ ND ND ND ND ND
ND ND + ND ++ ND ND +++ ND ND ND
ND ND ND ND ND ND +++ ND ND ND ND
Relative expression levels of the indicated Toll-like receptors on human B cells are indicated. +++, strong expression; ++, moderate expression; +, low but definite expression; +/ , expression at a barely detectable or functionally controversial level. Naı¨ve/Resting, CD19+CD27 or high-density peripheral blood or tonsillar B cells; activated, memory (CD19+CD27+) or intermediate-density peripheral blood or tonsillar B cells. Where data are available, expression levels are compared with neutrophils (PMN), dendritic cells (DC) or monocytes/macrophages (Mono). Summarized from references [2–4,29,42,43]. ND, not determined.
unlike macrophages and dendritic cells, B cells also utilize a heterodimer consisting of RP105 and MD-1, which are structurally related to TLR4 and MD-2, to recognize and respond to LPS (reviewed in [9]). Although B cells deficient in either TLR4 or MD-2 do not respond to LPS at all, cells deficient in either RP105 or MD-1 are hyporesponsive to LPS, suggesting that the RP105–MD-1 heterodimer plays a uniquely important role in B cells by enhancing TLR4dependent LPS responses. Indeed, patients with systemic lupus erythematosus, dematomyositis or Sjo¨ gren’s syndrome possess an unusually high percentages of RP105negative B cell populations, which are associated with activation and accentuated production of antibodies and autoantibodies [9,10]. As such, it is interesting to consider that, in addition to such apparently lineage-specific LPS signaling features as RP105 and MD-1, B cells might possess additional lineage-specific signaling molecules that participate in the recognition of LPS and/or other PAMPs, such as CpG, which might play specific roles in pathogenic states of humoral and/or B cell related immunity.
Effects of TLR9 signaling in B cells The TLR9 pathway continues to receive growing attention as a consequence of the potential relevance of unmethylated CpG-containing DNAs to the pathogenesis of autoimmune diseases, as well as in immunomodulatory therapeutic strategies [11].
can form complexes with hypomethylated CpG-containing DNAs, and then bind both to rheumatoid factor (antiIgG) B cells through their BCR via direct IgG binding, and to TLR9 via binding of the co-complexed DNA, resulting in activation [12]. These hypomethylated CpG DNAs have traditionally been thought to derive from environmental pathogens, suggesting a need for a non-self trigger for autoreactive B cell activation; however, mammalian DNAs contain rare hypomethylated CpG regions that are capable of co-stimulating autoreactive B cells. Therefore, sources of self DNA, such as apoptotic cells or necrotic debris, can potentially function in this situation as truly autoreactive stimuli in such immune complexes [13]. Furthermore, anti-DNA B cells themselves, by being able to bind DNAs through both their BCR and TLR9, might be directly co-stimulated by hypomethylated CpG DNA motifs without a need for immune complexes (Figure 2). In this sense, hypomethylated CpG motifs, whether or not in the form of immune complexes, can initiate and/or promote systemic humoral autoimmunity by preventing or promoting the breakage of tolerance of autoreactive B cells. It is, however, important to note that, at least in the antilysozyme transgenic model of B cell tolerance (in which anti-lysozyme transgenic B cells are rendered anergic by transgenic soluble lysozyme), CpG DNAs can break tolerance, inducing proliferation of, and immunoglobulin secretion by, autoreactive cells in vitro, but do not appear to be capable of inducing pathogenic disease [14,15]. This insufficiency has been attributed to the observation that anergic B cells have uncoupled the BCR from a calcineurin-dependent signaling pathway, leading to continuous ERK signaling, which inhibits CpG-induced plasma cell differentiation [14]. As such, additional stimuli, which could simply consist of excess antigen [15], are probably required to cooperate with CpG DNAs to break tolerance fully and result in pathogenic humoral immunity. Perhaps this tolerance breakdown, in part, involves the physically close coupling of BCR–TLR9 interactions that could be conferred by IgG–CpG DNA-containing immune complexes upon rheumatoid factor B cells, or by CpG DNAs alone upon anti-DNA B cells, resulting in a supramolecular activation complex. Alternatively, such immune complexes could also activate complement and/or Fc receptors, providing additional co-stimulation to the B cells, although there is less evidence for such mechanisms [12]. Thus, it remains unclear if TLR9 signals are necessary or single-handedly sufficient to both induce autoimmunity and propagate the response to full-blown pathogenic humoral immunity in vivo, although their ability to promote autoreactive B cell responses, at least to some degree, seems clear.
TLR9 signaling in B cell autoimmunity
There now exists strong evidence that TLR9 activation can co-stimulate autoreactive B cells, thereby breaking tolerance. For instance, anti-DNA IgG autoantibodies www.sciencedirect.com
Immunomodulatory effects of TLR9 signaling in B cells
CpG DNAs can furthermore skew immune responses towards a Th1-like phenotype, enhancing, for example, Current Opinion in Immunology 2005, 17:230–236
232 Lymphocyte activation
Figure 1
LBP LPS
TLR4– MD-2
RP105– MD-1
LPS LPS
TRIF
TRAM
TIRAP
MyD88
CD14 Cytoplasm
PKR
?
PKR
TBK1
IKK-ε
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JNK1/2
STAT1 IRF-3
NF-κB/AP1
IFN-inducible genes
Inflammatory gene transcription
TAK1
AP1
Nucleus
Current Opinion in Immunology
A proposed model for TLR4 signaling in B cells. Extracellular lipopolysaccharide (LPS) is bound by LPS-binding protein (LBP), recognized by CD14, and brought in proximity to the TLR4–MD-2 and RP105–MD-1 heterodimers. TLR4 initiates signaling via at least two adaptor pathways: TIRAP–MyD88 and TRIF–TRAM. The TIRAP–MyD88 pathway elicits an IRAK–TRAF6–TAK1 pathway. TAK1 activates NIK, which activates the IKK complex, which in turn phosphorylates the IkB proteins that normally sequester NF-kB proteins in the cytoplasm. TAK1 also activates the JNK and p38 MAPK pathways, leading to the activation of AP-1 complexes. Both the NF-kB and AP-1 proteins enter the nucleus where they activate target genes involved in inflammation, particularly cytokines. The TRIF–TRAM pathway results in activation of the atypical IKKs IKK-e and TBK1, as well as the IFN-inducible PKR [44]. Together, these kinases result in both IkB phosphorylation and NF-kB activation, enhancing the transcription of inflammatory cytokines, as well as the activation of STAT1 and IRF-3, which regulate the expression of IFN-inducible genes. Abbreviations: AP-1, activating protein; IkB, inhibitor of NF-kB; IKK, IkB kinase; IRAK, IL-1R-associated kinase; IRF, interferon regulatory factor; NIK, NF-kB-inducing kinase; PKR, double-stranded RNA-dependent protein kinase; STAT, signaling transducer and activator of transcription; TAB, TAK1-binding protein; TAK1, TGF-b-activated kinase; TBK1, TRAF-family-member-associated NF-kB activator-binding kinase 1; TIRAP, TIR-domain-containing adaptor protein; TRAF, TNF-receptor-associated factor; TRAM, TRIF-related adaptor molecule; TRIF, TIR-domain-containing adaptor protein inducing IFN-b.
IFN-g and IL-12 responses in vivo [16]. In such studies, CpG treatments are associated with the preferential production of ‘Th1-like’ immunoglobulin isotypes, such as the IFN-g-related IgG2a in mice. Previously, such effects upon B cells were presumed to reflect the indirect effect of skewing towards and/or preferential survival of Th1 cells in the helper T cell compartment, but recent studies indicate that CpG DNAs might have direct effects upon murine B cells, inducing and/or promoting class switching to ‘Th1-like’ isotypes, such as IgG2a, IgG2b and IgG3, Current Opinion in Immunology 2005, 17:230–236
while suppressing the production of the ‘Th2-like’ (IL-4related) isotypes IgG1 and IgE [17–19]. Interestingly, the Th1-like effect requires the classical MyD88 adaptor pathway and, at least for IgG2a class switching, requires the Th1-related T-box transcription factor T-bet, but the effect of CpG DNAs on IgG1 and IgE production appears to be at least partially MyD88 independent [17,19]. Thus, alternative pathways for CpG–TLR9 signaling are likely to exist, perhaps involving TRIF [8], and participate in the regulation of B cell responses; www.sciencedirect.com
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Figure 2
CpG-containing DNA
anti-DNA BCR Late endosome/ lysosome
Early endosome
Cytoplasm
TLR9 ERK IRAK
MyD88
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Nucleus
TLR9 TAB1 TAK1 TAB2
TR
AF
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Activation proliferation Ig secretion
NF-κB NIK
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IκB Current Opinion in Immunology
A proposed model for TLR9 signaling in B cells. Environmental CpG-containing DNAs (mammalian or non-mammalian) are directly endocytosed or internalized after binding to anti-DNA BCRs. TLR9, normally generated and found in the endoplasmic reticulum, encounters CpG DNAs in lysosomal/late endosomal compartments, where it initiates a signaling cascade via MyD88–IRAK–TRAF6–TAK1, similar to TLR4 (Figure 1 but without TIRAP. TAK1 activates NIK, which activates the IKK complex, which in turn phosphorylates the IkB proteins that normally sequester NF-kB proteins in the cytoplasm. TAK1 also activates the JNK and p38 MAPK pathways, leading to the activation of AP-1 complexes. Both the NF-kB and AP-1 proteins enter the nucleus where they activate target genes involved in B cell activation, proliferation, and immunoglobulin (Ig) production. In anergic B cells, such as anti-DNA B cells in non-autoimmune hosts, BCR signaling is dissociated from activation such that BCR signaling alone results in suboptimal NF-kB activation, perhaps related to tonic ERK activation. TLR9 ligation can bypass and/or co-stimulate this mechanism, leading to autoreactive B cell activation. Abbreviations: AP-1, activating protein; BCR, B cell receptor; IkB, inhibitor of NF-kB; IKK, IkB kinase; IRAK, IL-1R-associated kinase; NIK, NF-kB-inducing kinase; TAK, TGF-b-activated kinase; TRAF, TNF-receptor-associated factor; TIRAP, TIR-domain-containing adaptor protein.
nonetheless, CpG-mediated immunomodulation could obviously be particularly beneficial in Th2- or IgE-related inflammation, such as asthma or allergy. Studies of TLR9 in human B cells have revealed analogous observations, but with several caveats. In humans, CpG DNAs can also induce class switching to IgG1, IgG2 and IgG3, which are distantly related to the murine IgG2a, IgG2b and IgG3 isotypes; however, significant immunoglobulin secretion requires additional signals through the BCR and dendritic cell derived B cell activating factor www.sciencedirect.com
(BAFF; also called TALL-1, BLyS, THANK, zTNF4), indicating that CpG DNAs are less potent in human than in murine B cells [20]. In addition, whereas CpG-containing DNAs stimulate essentially all murine B cells, most studies have indicated that they preferentially stimulate activated and/or memory human B cells, as opposed to naı¨ve cells, reflecting the preferential expression of TLR9 on activated cells (Table 1). Indeed, in one study, BCR triggering was required to render naı¨ve human B cells responsive to CpG DNAs, whereas memory B cells were responsive to CpG DNAs alone [3]. Other studies have Current Opinion in Immunology 2005, 17:230–236
234 Lymphocyte activation
indicated that, whereas CpG DNAs alone can induce the expression of co-stimulatory molecules and chemokine receptors such as CXCR3 in human B cells, CpG DNAs require synergistic cooperation with CD40 signals and/or simultaneous culture with plasmacytoid dendritic cells to induce secretion of immunoglobulins or cytokines such as IL-6 and IL-10 [21–23], or to promote Th1 development by helper T cells [4]. Thus, although CpG DNAs are highly potent B cell immunomodulators in murine systems, their application to immunomodulation in humans, for example, in the treatment of asthma or allergy, might require additional manipulations to yield a robust effect.
Effects of other Toll-like receptor signals on B cells Although the ability of rodent B cells to respond to LPS by proliferation, class switching, immunoglobulin secretion and plasmacytoid differentiation has been extensively described, the TLR4 pathway has generally received less attention than TLR9, and human B cells have classically been considered LPS-unresponsive due to an absence of TLR4 (Table 1; [2,24]). Still, many studies have suggested that TLR4 expressed on B cells recognizes not only classical LPS but also a growing list of pathogenic antigens, including viral proteins and parasitic heat shock proteins [6,25,26], although other studies suggest that contaminating endotoxins might have confounded these findings [27,28]. Furthermore, some studies have indicated that TLR4 expression can in fact be induced in human B cells by specific stimuli, such as IL-4 [29], suggesting that human B cells might in fact be susceptible to TLR4 signaling under certain conditions. Thus, multiple environmental pathogens can exert physiologically important immunomodulatory effects on B cells via TLR4, presumably promoting activation and differentiation. Other TLRs similarly seem likely to affect B cell function both in humans and rodents in vivo. TLR2 ligands, such as neisserial porins, lipoproteins and glycoinositolphospholipids, have been shown to activate B cells, generally acting as mitogens [30–32]. Also, the TLR7-binding guanosine analogs can stimulate human B cells [33], suggesting that B cells can be stimulated by the physiological ligands of TLR7, the single-stranded RNAs [34– 36]. The physiological effects of such ligands on B cells, however, remain incompletely characterized.
Conclusions TLR ligands, particularly CpG-containing DNAs and LPS, have emerged as critical modulators of B cell effector function, promoting proliferation, plasmacytoid differentiation and class switching, as well as autoimmunityor Th1-related inflammation. However, questions remain regarding the applicability of these findings to immunomodulatory strategies in vivo – both in rodents and in humans. For instance, species-specific differences in Current Opinion in Immunology 2005, 17:230–236
optimal CpG sequences suggest differential regulation and/or recognition of TLR9 binding and/or signaling [11,37], and it has become clear that several ‘classes’ of stimulatory CpG oligonucleotides (ODNs) exist: CpG-A ODNs, which strongly induce IFN-a in plasmacytoid dendritic cells (PDCs) and potently activate NK cells; CpG-B ODNs, which potently activate NK and B cells but not PDCs; and CpG-C ODNs, which combine the effects of both [38,39]. On the one hand, these findings suggest the presence of yet more TLRs or co-receptors that specify CpG recognition, but simultaneously suggest on the other hand that the discovery of specific CpG sequences in murine B cells might not directly translate to humans and/or in vivo. Indeed, repeated CpG administration to mice surprisingly results in immunodeficiency due to lymphoid follicle destruction [40]. Thus, the translation of TLR ligands to immunotherapy should probably proceed with cautious enthusiasm. Although the studies summarized here strongly suggest that TLR ligands can break B cell tolerance, primarily stimulating and/or co-stimulating otherwise anergic autoreactive B cells, the normally constant exposure of the majority of mammals to environmental TLR ligands (e.g. in the form of bacterial LPS or CpG-containing DNAs) without the development of autoimmunity suggests the presence of protective mechanisms in B cells to prevent such adverse outcomes in vivo. One strategy, at least in murine B cells, appears to involve the desensitization of signaling pathways, such as Erk in anergic BCR cascades [14]. Another strategy, demonstrated in human B cells, appears to involve the differential expression of TLRs, conferring TLR ligand responsiveness only to activated/ memory B cells that have previously undergone antigen selection [41]. However, naı¨ve murine B cells can be activated by LPS alone, and both naı¨ve human and murine B cells can be stimulated by ligation of both the BCR and TLR9 [3,12,14].These mechanisms, therefore, seem insufficient to explain the resistance of B cells to TLR-induced autoimmunity in vivo. Future studies will hopefully elucidate the precise means by which TLRs modulate B cell activation versus tolerance.
Acknowledgements This work was supported in part by the Siteman Cancer, Rheumatic Diseases, Diabetes Research and Training, and the Digestive Diseases Research Core Centers of the Washington University School of Medicine, as well as grants from the National Institutes of health (AI061478 and AI057471), and the Lupus Research Institute. SLP is supported in part by an Arthritis Investigator Award from the Arthritis Foundation.
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