function as PRR them selves, showing the way by which the innate immunity can be self-‐amplified.
1.2.1 The Pattern-‐recognition receptors
Four main families of PRR are associated with detection of foreign material within a cell: one group of membrane-‐associated receptors and three classes of cytoplasmic PRRs.
Toll-‐like receptors
A first group of PRRs is composed of proteins with a transmembrane domain, an extracellular leucine rich repeat (LRR) region that recognizes specific PAMPs, and an intracellular module containing a TLR/IL-‐1R (TIR) domain that allows them to interact with adaptors molecules for the signal transduction 118.
With ten functional members in humans, the Toll-‐like receptor (TLR) family recognizes a wide variety of PAMPs. Using different adaptor molecules, TLRs bind to many types of molecules including lipids, lipoproteins, proteins and nucleic acids. The members of this family of receptors are located at different cell compartments. Whereas TLRs 1, 2, 4, 5, 6 and 10 are found at the cell surface, TLRs 3, 7, 8, and 9 are located in endosomes 114,119 (figure 6). TLR2 is found in the form of a dimer, either with TLR1, TLR6 or TLR10 mainly detecting PAMPs from Bacteria and fungi 114,120,121. When complexed with TLR1, the dimer recognizes the triacetylated lipoproteins, peptidoglycans and lipopolysaccharides 122, as reviewed by Kawai and Akira 114.
The dimer composed of TLR2 and 6 is responsible for the detection of diacylated lipoproteins 123. The specific molecules recognized by TLR2-‐TLR10 have not been discovered yet 120,121. The other cell surface-‐associated PRRs, TLR4 and 5, were found to bind to lipopolysaccharides (LPS) and flagellin 124, respectively (reviewed in 125).
Within the endosomal compartment, TLRs are responsible for the sensing of foreign nucleic acids. TLR3 was discovered to be activated by a dsDNA synthetic oligonucleotide, the polyinosinic:polycytidylic acid (poly I:C) 114. Whereas TLR7 and 8 recognizes ssRNA, TLR9 binds viral DNA. TLR7 and 9 are located in the endoplasmic reticulum (ER) under resting conditions, but they translocate into the endosomal compartment upon a first TLR-‐ mediated stimulation, for example when TLR4 binds to LPS 126.
A common feature that adaptor molecules used by all TLRs share is a TIR domain that allows TIR-‐TIR interactions with the corresponding receptor 114,127-‐
131. These signal transducers include myeloid differentiation primary response gene 88 (MyD88), TIR domain-‐containing adaptor protein (TIRAP), TIR domain-‐
containing adapter-‐inducing IFNβ (TRIF) and TIR domain-‐containing adapter molecule (TRAM). Whereas TIRAP directs MyD88 towards TLR2, TRAM targets TRIF to TLR4. Both interactions of MyD88 and TRIF with a corresponding TLR dimer will result in the expression of inflammatory cytokines 114,132,133. The TRIF-‐
TRAM-‐TLR complex induces additionally the production of Type I IFN 132. Most TLRs activate signaling leading to the production of inflammatory cytokines via either the MyD88-‐ or the TRIF-‐mediated pathways.
However, TLR4 is an exception because it requires both pathways to induce expression of the corresponding genes 134-‐137.
The cell possesses many means to perform signal transduction, by post-‐
translational modifications.
For example, the phosphorylation of different oligoaminoacid substrates, by protein kinases, produces conformational changes in the molecules, allowing them to interact with other proteins and activate signaling pathways 138.
Another post-‐translational modification involves the addition of monoubiquitin or polyubiquitin (polyUb) chains to target proteins. The best-‐studied types of ubiquitin chains are the ones that are linked via lysine (K) 48 and K63 of
ubiquitin, leading to targeting of the modified protein to the proteasome or to signal transduction complexes, respectively 139.
As review by Schulman and Harper 140, the mechanism of protein ubiquitynation starts with an ubiquitin-‐activating enzyme (E1), which bind to two ubiquitin (Ub) molecules via thioester bonds. In the next step, an ubiquitin-‐conjugating enzyme (E2) recognizes the E1-‐Ub complex and takes over one of the Ub molecules. Finally, an E3 Ub-‐ligase enzyme bound to a specific substrate interacts with the E2, facilitating the catalysis of the ubiquitination of the target protein.
The MyD88-‐induced signaling requires the cooperation of the IL-‐1 receptor-‐
associated kinases (IRAK) 1, 2, 4 and M that interact with tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), which ubiquitylates them with K63-‐
linked polyubiquitin chains in addition to autoubiquitinate itself 114. PolyU chains interact with, from one part TAK-‐1 binding protein 2 (Tab2) and 3 and from another part with the inhibitor of nuclear factor kappa-‐B (NFΚB) kinase gamma (IKKγ), leading eventually to the activation of mitogen-‐activated protein kinase (MAPK)-‐ and NFKB-‐dependent pathways, which will be described later. The engagement of TLR7 and 9 can additionally stimulate TRAF3 in cooperation with TRAF6, in the MyD88 pathway and lead to the activation of IRF7 141. Finally, in some immune cells, TLRs use the MyD88-‐IRAK4 pathway to stimulate IRF5 142.
The TRIF-‐dependent pathway involves the initial activation of the Tab2-‐Tab3-‐
TAK1 complex by the cooperation of TRAF6 and the kinase receptor-‐interacting protein 1 (RIP1) 114. TRIF dimerization can alternatively activate TRAF3 and the TBK1-‐IKKγ kinases leading to the IFN-‐regulatory factor 3 (IRF3) activation and the subsequent production of IFNβ.
Figure 6: Simplified scheme of the role of Toll-‐like receptors in the innate immune signaling. TLR1, 2, 4, 5, 6 and 10 are shown at the cell surface. TLRs 3 and 7-‐9 are depicted on endosomal vesicles. The PAMPs that stimulates each PRR is indicated adjacent to the rectangles that symbolize the extracellular leucine rich repeat (LRR) domains of the TLRs. Sensing of the different PAMPs activate the innate immune cascades AP-‐
1, NF-‐κB, IRF3, IRF7 and/or IRF5, leading to the production of type I IFN and inflammatory cytokines.
Adapted from Van Duin et al., 2006 143.
The cytosolic PRRs
Upon entry of a pathogen within the cell, different types of cytosolic PRRs sense PAMPs. The retinoic acid inducible gene 1 (RIG-‐1)-‐ like family of receptors (RLR) recognizes viral RNA molecules in different conformations 144. Another well-‐studied family of receptors is the NOD-‐like group (NLR), recognizing bacterial products such as peptidoglycans and flagellin 145.
These two first families of receptors activate the innate immune system by the stimulation of the MAPK-‐ and NFκB-‐dependent pathways 145-‐149. Additionally, RLRs are able to activate IRF 3 and/or 7, similar to TLRs 148,150. Some cytosolic DNA-‐sensors were identified recently, such as stimulator of IFN genes protein (STING), IFNγ-‐induced protein 16 (IFI16), the cyclic GMP-‐AMP synthase (cGAS) and the DNA helicases DDX41 and DHX9/DHX36 151-‐154. These proteins mostly
!"#$%&
'()(*+(&,-./(0&
1#2341#25&
1#2341#25&
12678&
9:7$&
signal by activating differentially the IRF3-‐, IRF7-‐ and/or NFKB-‐dependent pathways 151,153,154.
Highlighting the role of the sensing of viral DNA during immunity to retroviruses, HIV-‐1 capsid binds the cellular cyclophilin A and CPSF6 as cofactors to escape to recognition of the reverse-‐transcription products by as yet unidentified cytosolic sensors that would otherwise restrict replication in a type I IFN-‐dependent manner 155.
1.2.2 The innate immune pathways
Two important routes that PRRs use to activate the innate immune response are via the AP-‐1-‐ and the NFκB-‐ mediated signaling (figure 5). The activation of the MAPK-‐dependent pathway is initiated with the stimulation of different MAPK kinase kinases (MAP3Ks) and continues with a cascade of subsequent phosphorylations of a target MAPK kinase (MAP2K) that will in turn act on a specific MAPK 115. Three routes of the MAPK signaling pathway have been extensively studied and are involved in one or both of the pro-‐inflammatory and anti-‐inflammatory processes during the immune response: the extracellular signal-‐regulated kinase (ERK), Jun N-‐terminal Kinase (JNK) and protein of 38 kDa (p38) pathways (figure 7). The corresponding studies are mainly based on the effect of the stimulation of TLRs.
The ERK1 and ERK2 branch involves the activation, upon engagement of a TLR, of the tumor progression locus 2 (TPL2) 156. This MAP3K in turns activate MAPK kinase1 (MKK1) and MKK2. This pathway leads to the production of from one side the pro-‐inflammatory cytokines tumor TNF-‐α and interleukin 1 beta (IL-‐1β), from the other side the anti-‐inflammatory IL-‐10 115,157,158. ERK1 and ERK2 additionally have a repressive action on the expression of IL-‐12 and the two antiviral proteins IFNβ and inducible nitric oxide synthase (iNOS) 158,159.
The stimulation of the complex formed by the MAP3K TAK1 and Tab2/Tab3, results in the phosphorylation of the components of three main pathways. The