Nature Reviews Immunology 2008 Aug;8(8):594-606 Wiebke Albrecht, 20.10.2008

1. Nature Reviews Immunology 2008 Aug;8(8):594-606 Wiebke Albrecht, 20.10.2008

2. Plasmacytoid dendritic cells (pDCs): Introduction • first described in the 1950s • plasma-cell morphology • located in the T-cell zone of secondary lymphoid tissue • can produce type I interferons (IFN) in response to CpG-containing DNA, but not to poly I:C • use just one receptor system to sense nucleic acids Soumelis et al. 2006

3. Toll-like receptors (TLR): Introduction • 10 human TLRs known • bind to PAMPs via leucine-rich repeats of the ectodomain • recruitement of cellular adaptors via the cytoplastic TIR domain • TLRs on the cell surface: TLR4, TLR5, TLR1, TLR2,TLR6 • intracellular TLRs: TLR3, TLR7, TLR8, TLR9 PAMP = pathogen-associated molecular patterns TIR = Toll/IL-1 receptor

4. Intracellular signaling • TLR7 and TLR8 respond to G- or U-rich viral ssRNA, some synthetic compounds (imiquimod, R-848) and guanosine analouges • TLR9 responds to unmethylated CpG-containing ssDNA • TLR7, TLR8 and TLR9 signal through the cellular adaptor MyD88, TLR7 and TLR9 strictly depend on MyD88 • TLR3 recognizes viral dsRNA and poly I:C • TLR3 interacts with TRIF and signals through IRF3, TLR3 seems to signal independent of MyD88 MyD88 = myeloid differentiation primary-response gene 88 TRIF = TIR domain-containing adaptor protein inducing IFNβ IRF3 = interferon-regulatory factor 3

5. RIG-I-like-receptor (RLR) signaling pathway • RLR group members: RIG-I, MDA5 and LGP2 • RLR bind to dsRNA poly(rI:rC), poly (rA:rU) and RIG-I additionally to 5‘-triphosphate • RLRs interact with IPS1, which assembles a signaling complex (with TRAF3, TBK1 and IKKε) and signals through IRF3 to produce IFN production RIG-I = retinoic-acid-inducible gene I MDA5 = melanoma differentiation-associated gene 5 LGP2 = laboratory of genetics and physiology IPS1 = IFNB-promoter stimulater 1 TRAF3 = TNF-receptor-associated factor 3 TBK1 = TRAF-family-member-associated NF-κB activator-binding kinase 1 IKKε = inhibitor of NF-κB kinase ε

6. Activation of pDCs • pDCs predominantly express TLR7 and TLR9, which reside in the ER in association with the chaperone gp96 and XBP1 • after exposure to nucleic acid the TLRs translocate to the endosome, which requires UNC93B • type I IFN response in pDCs via the TLR-MyD88 pathway • autophagy possibly facilitates the delivery of viral RNA to the endosome • high expression levels of IRF4, IRF7 and IRF8 in pDCs facilitates rapid IFN response XBP1 = X-box-binding protein 1

7. Signaling of different CpG ODN classes • A-type CpG ODNs (contain a poly G tail; form multimeric aggregates) are retained longer in early endosomes, which allows extented activation of MyD88 and IRF7 • B-type CpG ODNs (monomeric) traffic rapidly through early endosomes into late endosomes/lysosomes •  pDCs detect DNA aggregates in early endosomes, which leads to IRF7 activation and IFN production, while linear DNA is sensed in late endosomes leading to NF-κB activation and TNF and IL-6 production CpG ODNs = CpG oligodeoxynucleotides TNF = tumour-necrosis factor IL-6 = interleukin-6

8. Self DNA sensing by pDCs (1) • release of self DNA into the extracellular environment through necrotic or apoptotic cell death • discrimination between pathogen-derived and self DNA: 1. subcellular localization of TLR facilitates recognition of DNA from pathogens invading the cell, while self DNA fails to enter endosomes 2. high concentration of DNases in the extracellular environment ensures rapid degradation of self DNA 3. in contrast to pathogen DNA, self DNA contains fewer CpG motifs, which are mostly methylated • pDCs were found to be active in several autoimmune diseases (i.e. SLE, psoriasis) • host factors involved in converting self DNA into triggers of pDC activation: LL37, autoantibodies and HMGB1

9. Self DNA sensing by pDCs (2) • the antimicrobial peptide LL37 secreted by dying cells forms aggregates with self DNA fragments, that are protected from degradation and delivered via lipid rafts into early endosomes • HMGB1 binds self DNA-LL37 complexes and promotes their association with TLR9 in early endosomes by binding to RAGE • in SLE, DNA-specific antibodies produced by autoreactive B-cells increase the translocation into endosomes of self DNA-LL37-HMGB1 complexes through FcγRIIA HMGB1 = high-mobility group box 1 protein RAGE = receptor for advanced glycation end-products FcγRIIA = low-affinity Fc receptor for IgG

10. Self RNA sensing by pDCs • limited accessibility of self RNA due to its fast extracellular degradation by RNases • modification of self RNA (poly A tail, nucleotide methylation) contribute to low immunogenicity • uridine-rich or uridine- and guanosine-rich RNA (U/UG-RNA) as well as snRNP (small nuclear ribonucleoprotein) can trigger pDCs to produce type I IFNs through TLR7, when delivered to endosomes by autoantibodies or liposomes

11. Negative regulation of pDC responses • pDCs use a ITAM-regulated, BCR-like pathway to counter-regulate the TLR signalling pathway by inhibiting IFN and cytokine production by pDCs • BDCA2, ILT7 and FcεRIα associate with the γ-chain of FcεRI • human NKp44 and mouse Siglec-H bind to the ITAM-adaptor protein DAP12 • some receptors like FcγRIIA contain intrinsic ITAMs • signalling occurs through the ITAMs and activate a BCR-like pathway involving SRC family PTKs, SYK and the B-cell specific adaptors BLNK and BCAP ITAM = immunoreceptor-based tyrosine activation motif BCR = B-cell receptor BDCA2 = blood DC antigen 2 ILT7 = immunoglobulin-like transcript 7 FcεRIα= high-affinity Fc recector for IgE Siglec-H = sialic-acid-binding immunoglobulin-like lectin H PTK = protein tyrosine kinase; SYK = spleen tyrosine kinase BLNK = B-cell linker; BCAP = B-cell adaptor protein

12. Summary • pDCs are important mediators of antiviral immunity through their ability to produce large amounts of type I IFNs • TLR7 and TLR9 sense viral nucleic acids within early endosomes to activate pDCs • several receptors negatively regulate IFN responses by pDCs for normal homeostasis • sensing of self nucleic acid is excluded by preventing the delivery to early endosomes, but pDCs are also involved in some autoimmune diseases in cases, when self nucleic acid is modified to gain entrance into endosomes