**2.1 Mechanisms of TLR activation**

Several TLRs are located at the cell membrane, while others are in endosomal membranes (Figure 2). All TLRs consist of three domains: a leucine rich repeat domain that recognizes PAMPs, an anchoring transmembrane domain and a cytoplasmatic Toll/interleukin-1 receptor (TIR) domain involved in signal transduction (Kumar et al., 2009; Olaru & Jensen, 2010). Adaptor proteins need to bind to the TIR domain to ensure continuation and amplification of the signal transduction. The majority of TLRs use the myeloid differentiation primary-response gene 88 (MyD88) as adaptor, except TLR3 which uses the TIR-domain containing adaptor protein inducing IFN-β (TRIF). TLR4 can use both MyD88 and TRIF for signal transduction, with MyD88 being involved in the majority of TLR4 mediated processes (Hosogi et al., 2004; Lebre et al., 2007; Trinchieri & Sher, 2007; Kumar et al., 2009).

Through PAMP recognition, the TLRs homodimerize and enable the binding of the adaptor protein. An exception is the TLR2 molecule which forms heterodimers with either TLR1 or TLR6. Following activation of the MyD88 adaptor protein, a signaling cascade involving mitogen-activated protein kinases (MAPK) and ending with the ubiquitination of IκB occurs. This induces the release of NF-κB, resulting in the transcription of pro-inflammatory cytokines such as IFN-, IL1- and CXCL8. After activation of TRIF the resulting signaling cascade ends with the activation of IFN regulatory factors (IRFs) and the induction of type I IFNs. The expression pattern differs for each TLR. The activation of TLR3 by poly I:C generally leads to the production of the largest panel of cytokines and chemokines, including CCL2, CCL20, CCL27, CXCL8, CXCL9, CXCL10, and TNF-α. In comparison, activation of TLR4 using LPS leads to the production of CCL2, CCL20, CXCL8, and TNF-α (Lebre et al., 2007; Olaru & Jensen, 2010).

and have been identified to be important pro-inflammatory mediators. Upon TLR activation, cells start to produce pro-inflammatory cytokines, attracting more immune cells to the site of infection (Kawai & Akira, 2009). In addition, TLRs are able to recognize endogenous danger signals, known as danger-associated molecular patterns (DAMPs). These molecules are released under cellular stress and include components of the extracellular matrix, such as hyaluronic acid and biglycan, heat shock proteins and uric acid crystals (Seong & Matzinger, 2004; Wheeler et al., 2009; Kawai & Akira, 2010; Martin et al.,

Another important family of pattern recognition receptors are the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). These intracellular receptors recognize PAMPs with the same leucine-rich repeat domains that can be found in TLRs. NLRs are activated by PAMPs such as bacterial RNA, flagellin and breakdown products of peptidoglycan (Feldmeyer et al., 2010). In addition, the NLRs are able to recognize DAMPs such as ATP and uric acid (Kawai & Akira, 2009). Upon activation, NOD receptors can activate NF-κB and large protein complexes called inflammasomes (Stutz et al., 2009; Feldmeyer et al., 2010; Latz, 2010). The inflammasomes are required to activate and secrete several cytokines that are expressed as nonfunctional proteins after NF-κB activation. The most important of these are IL-1β and IL-18, which are expressed as pro-IL-1β and pro-IL-

Several TLRs are located at the cell membrane, while others are in endosomal membranes (Figure 2). All TLRs consist of three domains: a leucine rich repeat domain that recognizes PAMPs, an anchoring transmembrane domain and a cytoplasmatic Toll/interleukin-1 receptor (TIR) domain involved in signal transduction (Kumar et al., 2009; Olaru & Jensen, 2010). Adaptor proteins need to bind to the TIR domain to ensure continuation and amplification of the signal transduction. The majority of TLRs use the myeloid differentiation primary-response gene 88 (MyD88) as adaptor, except TLR3 which uses the TIR-domain containing adaptor protein inducing IFN-β (TRIF). TLR4 can use both MyD88 and TRIF for signal transduction, with MyD88 being involved in the majority of TLR4 mediated processes (Hosogi et al., 2004; Lebre et al., 2007; Trinchieri & Sher, 2007; Kumar et

Through PAMP recognition, the TLRs homodimerize and enable the binding of the adaptor protein. An exception is the TLR2 molecule which forms heterodimers with either TLR1 or TLR6. Following activation of the MyD88 adaptor protein, a signaling cascade involving mitogen-activated protein kinases (MAPK) and ending with the ubiquitination of IκB occurs. This induces the release of NF-κB, resulting in the transcription of pro-inflammatory cytokines such as IFN-, IL1- and CXCL8. After activation of TRIF the resulting signaling cascade ends with the activation of IFN regulatory factors (IRFs) and the induction of type I IFNs. The expression pattern differs for each TLR. The activation of TLR3 by poly I:C generally leads to the production of the largest panel of cytokines and chemokines, including CCL2, CCL20, CCL27, CXCL8, CXCL9, CXCL10, and TNF-α. In comparison, activation of TLR4 using LPS leads to the production of CCL2, CCL20, CXCL8, and TNF-α

2011).

al., 2009).

18, respectively (Nestle et al., 2009).

**2.1 Mechanisms of TLR activation** 

(Lebre et al., 2007; Olaru & Jensen, 2010).

Fig. 2. Schematic presentation of TLR localization and signaling pathways (Boehme & Compton, 2004). TLR2 in combination with either TLR1 or TLR6, as well as TLR4, are expressed on the cell surface and use MyD88 as adaptor for cell signaling. This leads to NFκB activation and inflammatory cytokine secretion. In addition to MyD88, TLR4 uses TRIF to activate IRF3 and the IFN pathway. TLRs 3, 7, 8, and 9 typically localize to endosomal membranes, where they detect a variety of nucleic acids. TLR3 utilizes TRIF to activate IRF3. TLRs 7, 8, and 9 trigger inflammatory cytokine secretion and the IFN pathway through MyD88.
