**2. Innate immune responses: Toll-like receptors and other pattern recognition receptors**

The induction of innate signaling pathways by skin sensitizers in keratinocytes is believed to be a crucial factor in skin sensitization and a requirement for activation of Langerhans cells and dendritic cells and subsequent T cell priming (Martin et al., 2011). Studies have demonstrated that human primary keratinocytes express mRNA for Toll-like receptors (TLRs), such as TLR 1, 2, 3, 4, 5 and 9 (Kollisch et al., 2005; Son et al., 2006), which are important receptors of the innate immune system. Upon TLR activation, keratinocytes are able to produce a range of cytokines and chemokines, which allows for attraction of other immune cells, such as dendritic cells (Lebre et al., 2007).

TLRs are the first family to be identified of the germ-line encoded pattern recognition receptors (PRR). These receptors are involved in the first line of defense against pathogens. To date, 10 different TLRs have been identified in humans (Boehme & Compton, 2004). The TLRs are known to recognize various pathogen associated molecular patterns (PAMPs), which are conserved and essential molecules of pathogens. Some well-known examples are lipopolysaccharide (LPS) that is recognized by TLR4, double-stranded RNA recognized by TLR3 and lipopeptides recognized by TLR2 (Hosogi et al., 2004; Lebre et al., 2007; Kumar et al., 2009). The primary function of TLRs is the initial recognition of pathogenic microorganisms and subsequent activation of the innate immune response. Most pathogens express multiple PAMPs and are thus recognized by multiple TLRs. Activated TLRs will promote the phagocytosis of pathogens in innate immune cells such as macrophages and induce a respiratory burst, production of ROS and RNS, to neutralize pathogens. Combined, this promotes the presentation of pathogen specific peptides to cells of the adaptive immune system. Furthermore, the secreted reactive oxygen species can act as signaling molecules

Keratinocytes, Innate Immunity and Allergic Contact Dermatitis - Opportunities

for the Development of *In Vitro* Assays to Predict the Sensitizing Potential of Chemicals 43

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

The importance of TLRs in skin sensitization has been shown *in vivo* by using different types of TLR knockout mice. In the majority of these experiments, the ear swelling response was measured as a read-out for contact hypersensitivity responses. Mice deficient of TLR2 had reduced ear swelling after challenge with the skin sensitizer oxazolone and these mice were unable to launch an effector Th1 response (Jin et al., 2009). The importance of TLR2 in skin sensitization was confirmed by Martin et al (2008), showing a reduced ear swelling in response to the skin sensitizers trinitrochlorobenzene (TNCB), oxazolone and fluorescein isothiocyanate (FITC). Skin sensitization to these compounds was completely prevented in the combined absence of TLR2 and TLR4, indicating that for full induction of sensitization both TLRs are required (Martin et al., 2008). More evidence for the importance of TLRs in skin sensitization was found in experiments in mice deficient for the TLR adaptor molecule MyD88. These mice were unable to mount an ear swelling response to dinitrofluorobenzene (DNFB), which was explained by an impaired upregulation of CD86 on dendritic cells, leading to a reduced activation of hapten specific T cells. In contrast, mice lacking TLR2, TLR4, TLR6 or TLR9 had no impaired ear swelling in response to DNFB. Mice deficient of

MyD88.

**3. The role of TLRs in skin sensitization** 

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., 2011).

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