Molecular Basis for Detection of Invading Pathogens in the Brain
Jeppe Falsig,l* Johan van Beek,2 Corinna Hermann,3 and Marcel Leise
I Institute of N europathology, Universitae tsspital Z urich, Zuerich, Switzerland 2Experimemal Pharn1acology, Merck Serono International S.A., Geneva, Switzerland 30epartment of Natural Sciences, University of Konstanz, Konstanz, Germany
Classical immunology textbooks have described the their response, be it adaptive or innate. This is partially central nervous system as an immune-privileged site, achieved by the release of pro- and antiinflammatory Le., as devoid of inflammatory and host-vs.-graft immu cytokines and chemokines capable of attracting periph noreactions. This view has been refined, since we now eral immune cells (Heppner et aI., 2005; Owens et aI., know that hematopoietic cells infiltrate the CNS under 2001). In addition, CNS cells can regulate the survival certain circumstances and that CNS-resident cells are of infiltrating cells once they have entered the brain pa capable of launching an innate immune response. renchyma. A more accurate term would be to describe Microglia cells express an extensive repertoire of pat the CNS as an "immune-attenuated" site. References tern-recognition receptors and act as sentinels surveil on the function of CNS-infiltrating immune cells can be ling the CNS for possible damage or infection. Astro found elsewhere (Becher et aI. , 2006; Simard et aI., cytes are the most abundant cell type in the brain, and 2006; Bechmann et al., 2007). Here, we describe how they are capable of launching a strong supportive CNS-resident cells detect microorganisms and initiate an innate immune response. Novel findings show that both innate immune response. astrocytes and, surprisingly, even neurons express pat tern-recognition receptors. Activation of these recep PATTERN RECOGNITION RECEPTORS tors leads to a functional response, indicating that cells Pattern recognition receptors (PRRs; Fig. 1) are a other than microglia are capable of initiating a primary group of evolutionarily conserved receptor families capa innate immune response against CNS-invading patho ble of detecting pathogen-associated molecular patterns gens. Here, we put these findings into context with (P AMPs). P AMPs are molecular structures with certain what has been learned from recent in vitro and in vivo physicochemical characteristics, usually in a repetitive/ experiments about the initiation of an innate immune polymeric form, that are recognized by PRR instead of response in the brain. chemically defined epitopes (Medzhitov and Janeway, 2002; Akira et al., 2006). T he most studied PRRs are the Key words: pathogen-associated molecular patterns; Toll-like-receptors (TLRs). Other membrane PRRs are pattern recognition receptors; microglia; CNS; astrocytes the scavenger receptor fami ly, the receptor for advanced glycation-endproducts (RAGE), and C-type lectin recep Historically, the CNS was described as an tors (Stahl and Ezekowitz, 1998; Murphy et al., 2005). immune-privileged site based on the tolerance of the T he complement system comprises another type of brain to a skin graft (Medawar, 1948). In addition, the brain appeared to lack a draining lymphatic system, com bined with the presence of a blood- brain barrier and a Contract grant sponsor: Zentrum fiir Neurowissenschaften Zlirich (ZNZ; low expression of m'\ior histocompatibility complex to J. F.); Contract grant sponsor: Desiree and N iels Ydc Foundation (to (MHC) molecules on C NS-resident cells. In recent J.F.); Contract grant sponsor: Henny Sophie C lausen og m0belarkitekt years, this view has been altered dramatically, and we Axel C lausens Foundation (to J. F.); Contract grant sponsor: Ivan Nielsens now know that petipheral immune cells can migrate Foundation (to J.F.). into the brain and affect the course of CNS diseases or *Correspondence to: Jeppe Falsig, Institute of Neuropathology, Universi even be major players in neuropathological events taersspital ZU rich, Schmclzberglitrasse 12, 8091 Zuerich, Switzerland. (Becher et aI., 2006; Simard et aI., 2006; Priller et aI., E-mail: [email protected] 2006). Cell infiltration is often limited to the vessel walls or perivascular spaces (Bechmann et aI., 2007), and CNS-residing cells regulate the entry of blood-borne immune cells into the brain parenchyma and modulate 1435
C-Type Lectins
Endosome Cytosol
L--_____,
NODs and NALPs RNA Helicases TLR-7/8 '------., (RIG-1/MDA-5)
Fig. 1. Different types of pathogen- recognition receptors and their bound to pathogens, as pathogen recognition receptors; C-type lec subcellular localization. Toll-like receptors (TLRs) are found on the tins recognize mainly sugar polymers and represent a heterogeneous, cell surface and on the membrane of intracellular organelles, where growing class of receptors; nucleotide-binding oligomerization do they recognize internalized PAM Ps; scavenger receptors are a hetero main (NOD) proteins comprise different NOD members, including geneous group of receptor f:1milies (types A- H) characterized by the NALP, and recognize intracellular bacterial products in the cytosol; unifying property to bind to oxidized LDL. CD36 is an example of a also involved in intracellular recognition are RNA- helicase related B-type scavenger receptor; complement receptors (complement R) proteins such as retinoic- inducible protein- 1 (RIG- 1)/ melanoma dif act together with their primary ligand, activated complement f:1ctors ferentiation-associated protein- 5 (MDA-5).
extracellular PRRs, where the membrane signaling com nizes profliin-like molecules from Toxoplasma gondii (for a ponents (i.e., the complement receptors) are dissociated more extensive review on TLR ligands see Akira et al., from the detection and effector component (soluble com 2006, and Fig. 2) . Some TLRs display high ligand selec plement factors). In addition, several intracellular PRRs tivity. TLR3 recognizes viral double-stranded RNA; have been identified, capable of detecting P AMPs in dif TLR7 and TLR8 recognize single stranded vIral RNA; ferent intracellular compartments (Meylan et al., 2006). and TLR9 recognizes viral DNA and bacterial nonme thylated epG-containing DNA motifs. Other receptors are much less selective in their ligand recognition. TLR2 The TLRs and TLR4 recognize a wide variety of lipids, glycolipids, Ten members of the TLR family (Medzhitov and and envelope proteins. They can even be stimulated by Janeway, 2002) have been identified in humans and 12 host-derived molecules such as chaperones or S100 pro in mice (Akira et a!., 2006). Toll, the initial member of teins (Akira et a!., 2006) and may thus take a role in the TLR family, was first identified in Drosophila mela chronic neurodegeneration and regenerative processes in nogaster as a type-l receptor with an intracellular domain the brain (Rolls et al., 2007; Tang et a!., 2007). similar to that of the interleukin-l receptor. Mutation of TLRs are associated as hetero- and homodimers in the Toll receptor and the downstream signaling compo the absence of ligands, interacting via their extracellular nents cactus and dorsal (homologues of IKE and NF-KE) domains (Ozinsky et a!., 2000; Mizel et a!., 2003) . The led to an increased susceptibility to fungal infections and cellular response to stimulation with a given ligand decreased survival upon fungal infection (Lemaitre et a!., depends strongly on the TLR receptor composition. 1996). TLRs are glycosylated type-l integral membrane Activation of homodimerized TLR4 triggers a signaling proteins with a molecular mass ranging from 90 to 115 cascade that ultimately leads to the production of tumor kDa. TLRs can be divided into subgroups based on the necrosis factor (TNF) in macrophages. In contrast, acti molecules that they recognize. For instance, TLR1, vation of homodimerized TLR1, TLR2, or TLR6 does TLR2, TLR4, and TLR6 recognize modified lipids; not. However, stimulation of heterogeneous receptor TLR5 recognizes bacterial flagellin; and TLRll recog- complexes consisting of TLR2 dimerized with either 1436
I/) •.,J '"Tl () a. r Peptidoglycan products c (J) o -i Fungal constituents; en to or "0 ~ -0 I ' :3. "C (gram-positive and -negative) -0 1.0 G> ;;0 ;;0 (f) OJ ' Q,)Q) C z Z n OJ p-glycan u-mannan c:>< ~ 0 '< () ::JQ) ns » » "j"'~ tn S' z r ' EO. iE-OAP MOP » -0 r zymosan HIV ::i -0 _0EE LTA u ------_ ....------.. ------~-- o~ Q.I/) -(1) C (1)-E= :!~ IMALT1-8c1101 tn en Intra-cellular receptors Toll-like receptors C-type Lectins Fig. 2. Pathogen recognition receptors couple pathogen- derived in cytosine and guanine bases; UBP, unspecified products of uropa ligands to the cellula r signal transduction machinery. The receptors thogenic bacteria; CARD, caspase recruitment domain; TIR, Toll/ are shown with their cognate ligands. The protein domain essential IL- l R domain; ITAM, immunoreceptor tyrosine-based activation for signal transduction is indicated in the bottom part of the recep motif; SYK, LYN, FYN (common to all C- type lectins), receptor tors. For the IT AM-containing rece ptors, some examples of essential linked tyrosine kinases; MALT1-Bcll0, mucosa associated lymphoid transducers are shown. MDP, muramyl dipeptide; LPS, lipopolysac tisslle lymphoma translocation protein-B cell lymphoma 10; Fey re charide; LP, lipopeptide; LTA, hpoteichoic acid; iE-DAP, gamma ceptor, complement receptor; ds/ss, double stranded/single stranded. D-glutamyl-mesodiaminopimclic acid; CpG, demethylated DNA rich TLR1 or TLR6 does lead to the production of TNF ("rough") activates MyD88/TlRAP in a CD14-inde (Ozinsky et al., 2000). TLRs are expressed predomi pendent manner Giang et aI., 2005; Fig. 3). nantly on the cell surface, but the nucleic acid-detecting Activation of TLR2 and TLR4 eventually leads to receptors TLR3, TLR7, TLR8, and TLR9 are also the downstream activation of NF-KB and MAP kinases highly expressed on endosomes. The ratio of relative (Akira et al., 2006; O'Neill, 2006; and references therein). surface to endosomal expression varies for different cell NF-KB and MAP kinases (see Fig, 4) are master regula types (Ozinsky et aI., 2000; Akira et aI. , 2006) . tors of innate and adaptive immunity as well as cell sur vival. MyD88 is a vital signaling component for most TLRs (except for TLR3), but the MyD88-TLR bridging TLR Signal Transduction molecule TlRAP is dispensable for MyD88 recruitment The complexity of TLR signaling is increased by to TLR5, -7, -8, and -9 (Oshiumi et aI., 2003). the fact that other coreceptors are involved in the bind TLR4 Myd88-independent signaling is mediated ing and signaling of PAMPs. Lipopolysaccharide (LPS), a via TRIF using TRAM as a bridging molecule, whereas cell-wall constituent of gram-negative bacteria, is a phar TLR3 functions through TRIF in a TRAM-independ macological ligand of TLR4. In a physiological context, ent fashion (Yamamoto et aI., 2003). This ultimately LPS is initially bound in the plasma by LPS binding pro leads to IRF-3 and IRF-7 phosphorylation (Hacker tein (LBP) and from there transferred to a glycosylphos et aI., 2006). IRF3 and IRF7 are essential for the pro phatidylinositol (GPI)-anchored molecule (CD14) ex duction of type-1 interferons (IFN), potent antiviral fac pressed on the surface of macrophages. An intracellular tors (Honda et aI., 2005). In addition, IFN-regulatory signaling cascade is subsequently initiated through a re factor 5 (IRF5), a downstream MyD88-regulated tran ceptor complex consisting of TLR4 and the coreceptor scription factor, is essential for interleukin (IL)-6, IL-12, MD-2 (Nagai et aI., 2002; Akashi et aI., 2003; Fig. 3). and TNF expression (Takaoka et aI., 2005). TLR signaling requires various intracellular adaptor molecules to activate divergent pathways (Fig. 4) . The normal form of LPS (termed "smooth") activates both TLRs on Microglia MyD88/TlRAP and TRIF/TRAM in a CD14-depend Microglia express most of the known TLR recep ent manner, whereas the mutant short-form of LPS tor repertoire as well as the adaptor molecules required 1437 TLR9 ligands release nitric oxide in a synergistic fashion. This observation may be important for an effective host 'Rough' LPS defense, in that individual microorganisms express ligands for multiple TLR receptors. Peptidoglycans (PGN) or LT A from Staphylococcus aureus may activate microglia via TLR2, but TLR2 expression is not essen tial for recognition of the intact gram-positive bacteria (Kielian et aI. , 2005b) . Deletion of TLR2 led to a decrease in the pro inflammatory cytokine production in Staphylococcus aureus-infected CNS tissue. Despite this vi ability, the number of brain abscesses and the titer of gram-positive bacteria are not affected in TLR2-defi cient mice (Kielian et aI., 2005a). Inadvertent Functional Consequences of TLR NF-kB/MAPK response IFN response Activation on Microglia In vitro, activation of microglia can result in Fig. 3. Example for signali ng pathways triggered by gram-negative li bystander toxicity of cocultured neurons or oligodendro popolysaccharide. 1: LPS binds to LPS-binding protein (LOP) in so cytes (Lehnardt et aI., 2002, 2003, 2006; Iliev et aI., lution and then to the co-receptor CD14. 2: The complex binds to TLR4, which transmits signals via its cytoplasmic Toll /IL- l R do 2004). Intracerebroventricular administration of LPS main (TIR). MD-2 is required as a further coreceptor, whereas MD- leads to an increased leukocyte recruitment to the brain 1 has an inhibitory function. 3: MyD88-adaptor-like = TIR-associ via CD14-TLR4 activation in both an MyD88-depend ated protein (TlRAP/MAL) binds to the TLR-TIR domain and ent and an MyD88-independent fashion (H. Zhou et aI., attracts MyD88. 4: MyD88 is a template for attraction of further 2006) . Intracerebral injection of LPS causes rapid and adaptors and activation of kinases involved in the triggering of NF progressive demyelination and neurotoxicity via TLR4 KB and MAP kinase cascades. 5: TRIF-related adaptor molecule (Lehnardt et aI., 2002; Nadeau et aI., 2003). LPS (TRAM) can also bind directly the TLR-TIR and attract TIR- do induced neurodegeneration is partially actuated by TNF main-containing adaptor protein-inducing IFN- 13 (TRIF). 6: This but is reduced by an endogenous glucocorticoid response leads to activation of the interferon- response f:1ctor 3 (IRF3) and in the CNS, which suggests that endogenous mecha triggering of an interferon response. 7: Rough (=shortened) LPS does not require CD14 to induce the pathway triggered by MyD88, nisms exist for keeping the innate immune response in whereas CD14 is necessary for TRIF mediated signaling. [Color check (Nadeau et al., 2003). Peripheral LPS administra figure can be viewed in the online issue, which is avai lable at tion, used as a model for sepsis, also induces a strong www.interscience.wiley.com.] and chronic pro inflammatory CNS response. Despite the delivery of LPS to the periphery, this response requires the expression of TLR4 in the CNS compartment for downstream TLR signaling (Bsibi et aI., . 2002; Kielian (Chakravarty and Herkenham, 2005) . Eventually, such et al ., 2002; Olson and Miller, 2004; Jack et aI., 2005; mice develop a delayed, progressive loss of dopaminergic Mishra et aI., 2006; Town et aI., 2006) . Activation of neurons in the substantia nigra, showing that peripheral microglia leads to an increased expression of TLRs (Kie infections have the capability to induce or augment lian et aI., 2002; Olson and Miller, 2004; Jack et aI., CNS pathology (Qin et aI., 2007) . Roles of the TLR 2005) . These observations are well in line with the driven innate immune response, beyond pathogen recog expected role of microglia as the main cell type responsi nition, are suggested by reports that TLR expression on ble for the primary defense against pathogens entering the microglia promotes the clearance of [3-amyloid peptide brain. Already in a simplified model, using only one arti in amyloid precursor protein (APP) transgenic mice and ficial ligand, TLR- induced microglia response typically in vitro and that stimulation of TLR9 antagonizes gli leads to the production of antiviral/bacterial molecules oma formation (Tahara et aI., 2006; EI Andaloussi et aI., such as TNF, IFN-[3, inducible nitric oxide synthetase, 2006). and a multitude of chemokines and pro- and antiinflam matory interleukins, as well as cell proliferation (Olson and Miller, 2004; Lund et al., 2006). Microglia react to a TLRs on Astrocytes single specific stimulation of either TLR2 or TLR4 by Within the last few years, it has become apparent lipoteichoic acid (LTA) or LPS, respectively, with the that nonmyeloid CNS-residing cells express TLRs. release of proinflammatory cytokines (Lund et aI., 2005). Astrocytes express constitutively high levels of TLR3 Costimulation of microglia with concentrations of and the adaptor molecules required for TLR signaling two or more TLR ligands that individually lead to a (Farina et aI., 2005; Jack et aI., 2005). As opposed to submaximal stimulus can induce a synergistic response microglia, which mainly express TLR3 in intracellular (Ebert et aI., 2005). Microglia stimulated with combina compartments, astrocytes appear to have both a high in tions of two different TLR2 or TLR4 ligands or combi tracellular and a high surface expression of TLR3. This nations of TLR2/TLR4, TLR4/TLR9, and TLR2/ possibly reflects the fact that astrocytes are not professio- 1438 TLR4/4 TLR112 TLR2I6 TLR51? TLR7/B TLR91? TLR31? -l _ d...... --'Q) ...... t ! m r. ~~ I ...... ~~ ...J ...... ~ ...... IT:': I ...... ~ • ...I ..~~~F I .. ,...... mm ...... m...... /~ ..:.: .'.' ...... m ...... ~m ...... i T:AF I NODs ~ ~:~k" '.. rT.A" ,TBK1 I ~ OJ i RAF6 MKK -§:§ : 1 IK . : ~ 1...... ±...... i...... · ~t'lI.. ~.... · . _.~~.a . \ ...... ~...... ; ...... Q ~ Ci. (I) ~ ~ lnf1am mation. 1 ~ ,r::~ r,;:; < ~ IFN I .;:: \"IRF3 ) ~ Cell survival ~ .. ~ ~~gen;:;spons ve ~ -- r+ IFN responsive genes ~ Host defence r+"lnflammalion ~ IL6, II1 2, TNF .. . ~Type - 1 IFNs ~~ ~~~~ Fig. 4. Inflammatory responses triggered by TLR and NOD. Upon proteins, RICK, [RAK kinases, TANK-binding kinase- l (TI3Kl), ligand activation, receptors recruit one or several adap tor proteins to MAP kinases, and inhibitor of NF- KB kinases (IKK). Ultimately, the their cytoplas mic domain (TIR fo r TLRs, CARDs for NOD). TRIF signal transduction results in th e activation of transcription f.1 ctors and MyD88 are important platforms for th e activation of signalin g that trigger diflcrent panels of inflanmlatory/host response genes. complexes and cascades involvi ng further adaptors, such as TRAF nal phagocytes and thus would require a high surface of astrocytes from an infection in vitro. For instance, expression of TLR3 for an efficient detection of extrac poly(I:C) induces the production of an antiviral mole ellular pathogens. Whether receptor localization has any cule, Viperinl cig5, that inhibited the replication of a functional consequences is unknown, but astrocytes pseudotyped human immunodeficiency virus (HIV) viral respond strongly to TLR3 stimulation in vitro (Car particle in astrocytes (Rivieccio et al., 2006). Poly(I:C) pentier et al., 2005; Farina et al ., 2005; Jack et al., 2005; induced viperin expression was prevented with IFN- I3- Scumpia et al., 2005; Bsibsi et al., 2006) . Stimulation of neutralizing antibodies, by inhibiting NF-Kl3 and IRF3, TLR3 with the dsRNA- mimetic TLR3 ligand polyino and by a specific viperin knockdown using RNA inter sinic-cytidyli c acid [poly(I: C)J leads to a proinflammatory ference. In all cases, a decrease in viperin expression cor response with many similarities to a microglial TLR related with an increased viral titter (Rivieccio et al., response. Upon TLR-3 activation, TNF, IFN-I3, induci 2006). Theiler's murine encephalomyelitis virus (TMEV), ble nitric oxide synthetase, and a multitude of chemo an experimental model for a virus-mediated demyelinating kines and pro- and antiinflammatory ILs as well as IFN disease, induces a proinfianunatory response in TMEV regulated antiviral molecules are induced (Scumpia et al., infected astrocytes. In astrocytes devoid of TLR3, this 2005; Bsibsi et al., 2006; So et al ., 2006). A similar response is reduced, but the lack of an infiammatOlY response is observed when astrocytes are activated by response does not affect the replication of the virus cytokines Oohn et al., 2005; Falsig et al., 2006). Stimula within astrocytes (So et al., 2006). tion of astrocytes with poly(I:C) decreases glutamate Comparing a response of cells infected with a virus uptake by astrocytes, suggesting that an astrocytic re and cells treated with purified TLR ligands has certain sponse to poly(I:C) may indirectly affect neuronal func drawbacks. One of the main concerns associated with tion and viability (Scumpia et al., 2005). In a different using purified TLR ligands as an experimental tool is experimental setup, conditioned medium from poly(I:C) the purity and specificity of the li gands. Impurities can treated astrocytes was found to be neuroprotective for often be found in LPS preparations (Werts et al ., 2001), cortical brain slice cultures (Bsibsi et al., 2006). and many other ligand preparations have in the past of The main role of astrocyte TLRs may li e in viral ten been contaminated with LPS. Even when a ligand defense, insofar as poly(I:C) treatment triggers protection preparation is pure, the ligand is not necessarily specific 1439 chemotaxis mediators to the site of infection/damage (Babcock et al., 2003; Marella et aI., 2005). In the previously mentioned in adhesionf inflammation vivo studies, astrocytes were possibly activated indirectly migration Extracellular matrix through microglia, indicated by the fact that astrocytes (EeM) modulation were also strongly activated by LPS, a TLR4 ligand that phagocytosis is relatively microglia specific (Town et a!., 2006). Low host-defence! levels of TLR4 expression on astrocytes in vitro have cell survival T cell recruitment been described throughout the literature, but in vivo pathways pathogen acute phase! astrocytes do not appear to express TLR4 (Lehnardt recognition metabolic change et a!., 2002; Ma et aI., 2006; Mishra et aI., 2006). TLR4 activation by LPS leads to highly variable Fig. 5. Downstream events ofTLR signaling. functional astrocytic responses, possibly because of vary ing preactivation states of astrocyte cultures, and different states of TLR up-regulation by the preactivation (Lee for a given receptor. Poly(I:C), a ligand commonly used et aI., 1993; Bowman et aI., 2003; Esen et al., 2004; to study TLR3 function, stimulates TLR3 when applied Carpentier et aI., 2005; Farina et aI., 2005; Bsibsi et al., extracellularly. On the other hand, cytoplasmic delivery 2006). For instance, rat astrocytes have been r ~ ported to of poly(I:C) stimulates the activation of protein kinase R respond strongly to LPS but also to be devoid of recep (PKR) and a novel PRR, retinoic acid-inducible gene I tor expression and LPS binding (Chung and Benveniste, (RIG-I; Gunnery and Mathews, 1998; Matsumoto et aI., 1990; Lehnardt et aI., 2002). Human astrocytes have 2002; Yoneyama et aI., 2004) . This is substantiated by been reported to show an increased mRNA expression the observation that poly(I:C) induces a robust, although in response to LPS stimulation but to a much lesser reduced, microglial cytokine response in TLR3-/ extend than the response shown after poly(I:C) stimula mice compared with wild type (wt; Town et aI., 2006). tion (Bsibsi et aI., 2006) . Concerning the production of Discussion of the signaling of TLR in astrocytes is polypeptide mediators (e.g., cytokines), astrocytes were still controversial, because pharmacological tools used to found either to be unresponsive to LPS stimulation (Lee dissect signaling cascades mostly lack absolute specificity, et aI., 1993) or to release less than 10% of IL-6 com and knockouts may induce compensatory responses pared with that released by poly(I:C)-stimulated astro allowing for alternative signaling pathways. For instance, cytes (Farina et al., 2005). Also, reports of TLR4-medi poly(I:C) activates PKR in astrocytes, and pharmacologi ated functional responses on murine astrocytes vary cal inhibition of PKR was shown to block the enormously. In our hands, we see no effect of LPS stim poly(I:C)-mediated production of nitric oxide and cyto ulation on purified astrocytes from C57BLl6 mice kines. The authors suggested that PKR acted as a down (Falsig et aI., 2004). Other groups reported effects on stream effector of a membrane receptor (likely TLR3; murine astrocytes (Bowman et aI., 2003; Esen et aI., Scumpia et aI., 2005). Recently, this hypothesis was dis 2004; Carpentier et aI., 2005), but none of the studies puted by showing that PKR - / - astrocytes respond showed that . the LPS-induced effects are dependent on normally to poly(I:C) treatment, suggesting that PKR TLR4 expression on astrocytes. Furthermore, two of the does .not play a significant role in TLR3 signaling in studies show maximal astrocyte activation at concentra astrocytes (Carpentier et aI., 2007). Also, not all signaling tions of 0.01 or 1 ng/ml of LPS. These concentrations observations correlate with functional consequences. For are at least 1,000-fold below what is needed to activate instance, TMEV activation of astrocytes was shown to microglia via TLR maximally and thus point to a differ be dependent on PKR (not on TLR3). However, no ent mechanism on the astrocytes of these studies (Lee difference in viral replication was detected between wt et al., 1993; Bowman et a!., 2003; Esen et aI., 2004). and PKR-null cells (Carpentier et al., 2007; Fig. 5). The expression pattern of multiple TLR receptors is modulated by pro inflammatory stimulation of micro Functional Implication of Astrocyte TLR glia and astrocytes. Especially TLR2 expression on astro Receptors cytes is induced by stimulation with TLR3 ligands and Little is known about the importance of TLR3 cytokines in vitro (Bowman et aI., 2003; Olson and expression on astrocytes in vivo. Intracerebral or periph Miller, 2004; Carpentier et aI., 2005; Farina et al., 2005; eral administration of poly(I:C) leads to a strong activa Jack et aI., 2005; Bsibi et aI., 2006; Falsig et a!., 2006) as tion of microglia and astrocytes. These findings well as during a parasitic infection and experimental prompted the authors to suggest that poly(I:C) activates autoimmune encephalomyelitis in vivo (Zekki et aI., astrocytes in vivo (Zhang et al., 2005; Town et aI., 2002; Mishra et aI., 2006). Few reports of TLR2 func 2006) . A pitfall of these studies is that many microglia tion on astrocytes have been reported, so whether the derived factors strongly affect the activation state of other up-regulation of TLR2 has any functional consequences CNS residing cells Oohn et aI., 2005) . Microglial release is at this point unknown. One report suggests that pure of TNF and IL-l induces a strong production of cyto astrocyte cultures depleted of microglia using a gliotoxin kines and chemokines in astrocytes in vivo, leading to can respond strongly to TLR2 stimulation induced by the attraction of microglia and peripheral immune cells Staphylococcus aureus or its cell wall constituent PGN 1440 (Esen et al., 2004). In addition, soluble CD14 was tive TLR3, causing a dramatically decreased cellular IFN recently reported to activate astrocytes through TLR2 response to HSV - 1 and vesicular stomatitis virus. The (Bsibsi et aI., 2007) . However, the role of TLR2 in mutation did not affect the cellular response to a host of astrocyte ac tivation requires further characterization. other viral infections, showing that the TLR3-mediated TLR5 and TLR9 expression has been reported on astro natural immunity to HSV-1 and VSV is selective. The cytes in vitro and TLR6 and TLR7 in vivo, but func authors suggest that the defects in the patients deficient tional roles still have to be elucidated (Bsibsi et al., 2002; in TLR3 signaling involves an intrinsic defect affecting Mishra et aI., 2006). CNS-residing cells, underscoring the importance of future research into the function of TLR3 in CNS im munity (S .Y. Zhang et aI., 2007). The fact that only one TLRs on Oligodendrocytes and Neurons report exists regarding the role of TLRs in protection of Not much is known about TLR expression on oli the CNS from pathogens points to a high degree of godendrocytes, but mRNA expression for TLR2 and functional redundancy between different extracellular TLR3 and immunoreactivity for TLR2 have been and intracellular PRR systems, especially when complex described (Bsibi et aI. , 2002; Lehnardt et al., 2006). pathogens with multiple PAMPs are to be recognized. Recently, TLR3 expression on Purkinje neurons in the cerebellar cortex of post-mortem tissues from patients suffering from chronic or infectious neuropathies was c-Type Lectin Receptors as reported Oackson et al., 2006) . In addition, rabies virus Membrane-Associated PRRs with TLR3 stimulation with poly(I:C) was shown to The C-type lectin receptors are a family of carbo induce the release of IFN- 13 in neurons (a terminally dif hydrate-recognizing PRRs. T he most well-studied ferentiated human cell line; Prehaud et al., 2005). Even member of the C-type lectins is the mannose receptor more surprising, TLR8 expression was demonstrated on (MR; Stahl and Ezekowitz, 1998). The MR is capable embryonic and early postnatal neurons in vivo by immu of recognizing gram-positive and· gram-negative bacteria, nocytochemistry and in situ hybridization, suggesting a parasites, mycobacteria, and yeasts and has been impli role for TLRs in neurodevelopment (Ma et aI. , 2006) . It cated in pathogen uptake and inflammatory responses was shown that TLR8 is expressed on cortical neurons (Stahl and Ezekowitz, 1998). in vitro and that stimulation with the TLR8 ligand R - MR protein expression was convincingly shown in 848 leads to apoptosis and inhibition of neurite out primary cultures of human astrocytes, in murine and rat growth independent of NF-KB ac tivation (Ma et al., microglia, and in murine astrocytes and neurons in vivo 2006). It will be exciting to see whether primary cul (Marzolo et al., 1999; Burudi and Regnier-Vigouroux, tures of neurons can produce IFN- 13 in response to 2001; Zimmer et al., 2003; Liu et aI., 2004). However, TLR activation and whether this has any implication for in other studies, MR expression was found to be re protection against foreign pathogens. stricted to perivascular, meningeal, and choroid plexus TLR3 is expressed broadly in inflamed CNS tissue, macrophages (Fabriek et al., 2005; Galea et aI. , 2005). and so far it appears to be the main, if not the only, The MR binds the highly mannosylated envelope glyco TLR receptor expressed at physiologically relevant levels protein gp120 and serves as a CD4-independent HIV- l on nonmicroglia cells in the noninjured postnatal CNS. entry receptor on astrocytes (Liu et aI., 2004). Astro This is consistent with the fac t that microglia are the cyte- HIV-1 interaction induces MMP-2 through the main, but not exclusive, sentinel of the C NS. MR, showing that the receptor is functional (L6pez Herrera et al., 2005). Surprisingly, MR expression on microglia is down-regulated by proinflammatory stimula Functional Implication of TLRs in Mammalian tion with LPS or IFN-'Y and up-regulated by stimulation Host Response with IL-4 or treatment with dexamethasone (Marzolo It is important to note that the contribution of et aI., 1999; Zimmer et aI., 2003). This is inconsistent TLR expression to protection against infections in the with a role of MR in an inflanunatory setting, and CNS is currently unclear. Until recently, there was not a indeed it has been shown in several infection models single report showing that the loss of TLR expression in that MR does not playa crucial tole in fungal infections the brain parenchyma affects the replication of a patho in vivo (Lee et aI. , 2003) . Macrophage uptake of the gen or the survival of the infected host. One report sug fungal pathogen Candida albicans was inhibited by l3 -glu gests that TLRs [or other poly(I:C) receptorsJ can medi can and not by a -mannan (an MR antagonist), suggest ate a protective CNS response to vesicular stomatitis vi ing that fungal uptake was regulated by another l3 -glu rus when hyperstimulated with poly(I:C) (De Clercq can-sensitive PRR (Lee et aI., 2003). Recently, dectin-1 and De Somer, 1971). Excitingly, it has now been was identified as this receptor, and it was shown that reported that some patients suffering from herpes simplex dectin-deficient mice display severe deficits in their anti encephalitis, caused by the neuroinvasion of herpes sim fungal response (Taylor et al., 2007) . Dectin-1 and dec plex virus 1 (HSV-1), express a dominant-negative tin-2 are C-type lectins important for the . recognition mutation in the TLR3 gene (S .Y. Zhang et al., 2007) . and inunune response to fungal cell wall constituents This mutation leads to the expression of dominant-nega- such as l3-glucans (Dostert and Tschopp, 2007). How- 1441 ever, under resting conditions, no dectins are expressed et al" 2002; Alarcon et al" 2005; Nakamura et aI., in the brain (Reid et al" 2004; Taylor et aI., 2005), 2006), Astrocytes express SR-BI, SR-MARCO, and Dectin expression is up-regulated by proinflanunatory SR-PSOX (CXCL16; Alarcon et aI., 2005; Ludwig stimuli, Thus, it is possible that the receptors can be up et aI., 2005), SRs appear to mediate inflammation in regulated in inflamed CNS tissue, but this is yet to be neurodegenerative diseases (EI Khoury et aI., 2003), but shown (Taylor et aI., 2005), the exact role of SRs in CNS infections is at present not Dendritic-cell-specific C-type ICAM3 grabbing well characterized, nonintegrin (DC-SIGN)/CD163 is a C-type lectin PRR that is capable of detecting a multitude of patho Structure and Signal Transduction of NOD gens, including virus, bacteria, fungus, parasites, and Proteins as Intracellular Receptors tumors (T. Zhou et aI., 2006), Both DC-SIGN and dec The cytoplasmic family of nucleotide-binding oli tin have been reported to modulate TLR signaling (T. gomerization domain (NOD) proteins comprises impor Zhou et aI., 2006; Dostert and Tschopp, 2007), DC tant pattern recognition receptors for intracellular SIGN is commonly used as a marker for perivascular immune defense, The NODs are all characterized by macrophages (sometimes described as CNS-residing den three structural domains: 1) a carboxy (C)-terminal do dritic cells) in CNS tissue of humans, mice, and mon main with multiple leucine-rich repeats (LRR) that rec keys (Kim et al" 2006), The receptor functions as an ognizes ligands; 2) a central nucleotide binding site HIV-1 entry receptor, and the receptor is up-regulated (NBS or Nacht domain), which is important for self-oli in a few microglia and giant nucleated cells after an gomerization and possess ATPase activity; and 3) an HIV-l infection in vivo (Kim et al" 2006), In addition, amino (N)-tenninal effector motif, the CARD domain, DC-SIGN is up-regulated in microglia and foamy mac which is composed of protein-protein interaction cas rophages in the brains of mice suffering from experimen settes leading to the activation of NF-KB and/or caspases tal autoimmune encephalomyelitis (EAE; Fabriek et aI., upon NOD stimulation (Chamaillard et al" 2003a; Mar 2005), Another PRR C-type lectin, Dec 205/CD205, tinon and Tschopp, 2005), Two prominent NOD family displays a CNS expression pattern similar to that of DC members, NODi (also called CARD 4) and NOD2 (also SIGN (Rosicarelli et al" 2005) , Few studies have investi called CARD15) trigger activation of NF-KB and subse gated a possible protective role of C-type lectins in CNS quent induction of inflammatory mediators, Only infections, recently has an exclusive role of the RICK/RIP2 kinase for coupling of NOD to NF- KB been shown in RICK Scavenger Receptors null mice, displaying a defective response toward NODi and NOD2 agonists (Park et aI., 2007), Scavenger receptors (SR) are a diverse group of Both NODs are expressed in the cytosol, where membrane proteins that share the capacity to bind modi they recognize muropeptides, building blocks of bacterial fied low-density lipoproteins, The SRs can be broadly cell wall PGNs (Chamaillard et aI., 2003b; Girardin divided into eight different classes (A-H), and members et aI., 2003a,c), NODi was initially falsely described as of several classes have been shown to bind and mediate an LPS receptor (Inohara et al" 2001), as a result of uptake or respond to a variety of pathogens (Murphy muropeptide contamination of an LPS preparation, et aI., 2005), Class A receptor SR-A mediates phagocy NOD2 recognizes as minimal active motif the muramyl tosis of gram-positive and gram-negative bacteria and dipeptide (MDP), which is present in both gram-nega clearance of endotoxin, SR macrophage receptor with tive and gram-positive PGN, NOD2, therefore, can be collagenous structure (SR-MARCO) binds bacteria, and regarded as a broad bacterial sensor (Girardin et aI., SR with C-type lectin (SRCL) mediates uptake of yeasts 2003b) , After phagocytosis and treatment by lysosomal and gram-positive and gram-negative bacteria (Murphy proteases, muropeptides may become available to intra et aI., 2005, and references therein), SR-BJ binds LPS, cellular proteins, but the precise route remains to be and mutations or polymorphisms of the class B receptor defined (Carneiro et aI., 2004), Besides NOD, the family CD36 are associated with protection against cerebral of NOD-like intracellular receptors also contains malaria (Omi et aI., 2003; Murphy et aI., 2005), CD36 NALPl-3 and other members, but information on their is expressed by microglia and mediates a ~-amyloid role in neural cells is not yet available, The same applies induced inflammatory microglia response (El Khoury to RIG-like helicases, another family of cytosolic PRR, et aI., 2003) , CD36-deficient mice are hypersusceptible to Staphylococcus aureus infection, and it was found that CD36 is a receptor for microbial diacylglycerides that Interplay of NOD and TLR signal via TLR2/6 (Hoebe et aI., 2005), In contrast, the Independently of the cell type used, all studies con response to triacyl lipoproteins requires the presence of sistently describe a strong synergistic activity of NOD TLR1/TLR2 and CD14, but not CD36 (Triantafilou and TLR (for review see Traub et aI., 2006) , In the case et aI., 2006), Other scavenger receptors expressed or of the TLR4 ligand LPS, NOD stimulation can result in induced by inflammatory stimuli on microglia are SR a shift of the dose-response curve by a factor of 1,000 AIISRAII, SR-MARCO, SR-BI, SRCL, and receptor (Traub et aI., 2004) , It has to be noted that synergistic for advanced glycation endpoints (RAGE; Husemann actions of TLR and NOD ligands already occur with 1442 low doses of stimuli. Although we do not know in More recently, complement has been shown to playa which phase of an infection such synergy can develop, key role in eliciting and modulating adaptive immunity synergistic pathogenic stimuli through different immune (Carroll, 2004; Hawlisch and Kohl, 2006; X. Zhang receptors might be the rule rather than the exception. et aI., 2007) . The brain has been shown to synthesize a full and functional complement system (Morgan and Role of NOD for Neural Cells Gasque, 1996) . The individual components are produced Murine astrocytes as well as murine microglia cells from diverse cell types, such as neurons, astrocytes, and constitutively express functional NOD2 protein, which microglia. There is now compelling evidence that com is up-regulated by stimulation with Borrelia hurgdoiferi or plement activation in the brain is a double-edged sword, Neisseria meningitides or by stimulation with TLR ligands in that it can exert detrimental or beneficial effects (Sterka and Marriott, 2006; Sterka et aI., 2006). Further depending on the pathophysiological context (Barnum, more, both astrocytes and microglia express RICK ki 2002; van Beek et aI., 2003). More specifically, the nase, which is crucial for NOD signaling (Park et aI., complement system has recently emerged as one of the 2007) . Peripheral administration of the NOD2 ligand key players in innate immune recognition and clearance MDP, as with ligands for TLR3, TLR4, and TLR7/8, of pathogens and toxic entities by resident cells of the leads to proliferation of spinal cord cells in vivo, which human brain (Gasque, 2003; Hauwell et aI., 2005). is accompanied by activation of microglia cells (Su et aI., Complement can be activated through three dis 2005). MDP stimulation of rat astrocytes alone results in tinct routes: the classical, alternative, and lectin path a mild release of nitric oxide, which is significantly ways. Ultimately, activation of the entire complement increased by stimulation with a ligand of TLR1I2, cascade leads to the formation of the membrane attack TLR4, or TLR6 (Guo et aI., 2006). The expression of complex (MAC), which forms a pore in the phosholipid the ionotropic purinergic P2X4 receptor is regulated in bilayer of cells. The classical complement. pathway is a similar fashion, but the most pronounced effect was activated primarily by the interaction of Clq with anti observed for MDP-induced TNF release, which was body-antigen complexes, C-reactive protein, and serum markedly increased in presence of any of the TLR amyloid P protein of the pentraxin family, or ligands (TLRl- 9) ligands investigated. However, the observed exposed on the surface of pathogens (Storrs et aI., 1983; effects appeared to be additive rather than synergistic. Gewurz et aI., 1993). Furthermore, Clq binds neuropa Whether NOD2 signaling is protective or rather detri thologically relevant ligands including f3-amyloid fibrils mental in an infection scenario and whether there are and apoptotic neurons in vitro (Tenner, 1999; Tacnet immune-modulating influences of TLR signaling remain Delorme et aI., 2001; Cole et aI., 2006), suggesting an to be determined. important role for Clq as sensor molecule that facilitates the clearance of a large variety of danger entities from Indirect PRRs the CNS (Hauwel et aI., 2005). The initiation of the alternative pathway does not In addition to the recognition of P AMPs by mem require the presence of immune complexes and leads to brane and intracellular receptors, nature has devised a the deposition of C3 fragments on target microorgan different recognition strategy. Recognition may occur by isms. A third activation route involves mannose binding soluble proteins, and these proteins, bound to their tar lectin (MBL) , a lectin homologous to Clq that recog get, are then recognized by dedicated recognition recep nizes polysaccharide structures of bacteria, fungi, viruses, tors. Two major systems using this principle are the and parasites (Turner, 2003) as well as apoptotic cells complement system and the immunoglobulin system. (Ogden et aI., 2001) and triggers the complement path The latter bridges the gap between the innate and the way independently of both the classical and the alterna adoptive immune system. Pathogen antigens can be rec tive activation pathways. Activation of the three comple ognized by millions of different antibodies, which are ment pathways generates the fragments C3b and finally then in turn recognized by a small number of immuno iC3b, two opsonins that flag the target cell to promote globulin receptors, which are also found on microglia phagocytosis. cells (Peress et aI., 1993), the most prominent being the Fc-gamma receptors. The complement system involves several levels of recognition and will therefore be Complement Receptors Involved in Phagocytosis described in more detail. The complement receptor CR3 (CDllb/CD18) recognizes C3b and iC3b and is expressed by microglia COMPLEMENT: A UNIQUE INNATE IMMUNE (Perry et aI., 1985; Akiyama and McGeer, 1990). Addi SENSOR FOR DANGER SIGNALS tionally, CR3 directly recognizes pathogen surface mole The complement system is a central effector arm of cules such as LPS and beta-glucan (Ross, 2002) and the innate immune response consisting of some 30 fluid mediates the elimination of degenerated myelin by macro phase and cell-membrane-associated proteins involved in phages and microglia (Rotshenker, 2003). The comple the recognition and killing of invading pathogens, the ment receptor CRl (CD35) also binds to Clq, C3b, iC3b, clearance of immune complexes and apoptotic cells, and and MBL and, as such, has been implicated in phagocytic the mediation of inflammation (Frank and Fries, 1991). and complement regulatory activities (Krych-Goldberg 1443 and Atkinson, 2001), whereas the complement receptors pathogenesis of acquired immune deficiency syndrome C3aR and CSaR have different immunomodulatory (AIDS)-associated neurological disorders (Speth et aI., functions. 2002). Complement synthesis by astrocytes is dramati cally increased after infection with HIV. This observa Regulation of Complement Activation tion is in line with data showing elevated complement To protect "self' cells against bystander lysis, host in cerebrospinal fluid of patients with HIV infection cells express a battery of secreted or membrane-bound Oongen et al., 2000) . Complement ac tivation may thus complement inhibitors. Membrane-associated comple provide first-line brain immune defense by triggering ment inhibitors include membrane cofactor protein lysis of virions and infected cells (Speth et aI., 2007). In (MCP = CD46), decay-accelerating factor (DAF = turn, HIV, either opsonized by complement or immu CDSS), and CDS9. Notably, an inhibitor of comple nocomplexed, may bind to complement receptors ment activation termed "complement receptor-related expressed on the surface of astrocytes and microglia, thus protein y" (Crry) is expressed on rodent, but not on favoring their infection, as observed in the periphery human, cell membranes. Crry is broadly distributed and (Montefiori et aI., 1996). Additionally, viruses can enter is a functional and structural analog of human DAF and the brain by interacting with specific complement recep MCP. Wyss-Coray and colleagues (2002) demonstrated tors expressed by neurons and glial cells: DAF for enter that complement activation reduces the accumulation or oviruses and echoviruses, CD21 for Epstein-Barr virus, promotes the clearance of [3 -amyloid plaques in trans emphasizing the critical role of the complement system genic mice overexpressing the human amyloid precurser in virus-induced encephalitis (Hauwell et aI., 2005). protein (hAPP) . Transgenic hAPP mice coexpressing a soluble form of the complement inhibitor Crry under SUMMARY AND OUTLOOK the glial fibrillary acidic protein promoter displayed Many receptors associated with the detection of increased [3-amyloid deposits and more prominent neu pathogens and the initiation of an immune response in rodegeneration than what is found in age-matched mice the periphery are also expressed within the brain. The (Wyss-Coray et aI., 2002). recent observation that PRR receptors are expressed not only by microglia but also by other CNS-residing cells Complement and CNS Bacterial Infection suggests a role of these cells in pathogen detection. It Complement protein levels have been found to be will be exciting to find out whether these cells play a elevated in the cerebrospinal fluid of patients with bacte major role in initiating a protective CNS innate immune rial meningitis (Stahel et aI., 1997). Furthennore, defi response and which PRRs contribute to it. 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