**4. The role of the Nrf2-KEAP1 pathway in skin sensitization**

Besides triggering the innate immune response, *in vitro* studies have shown that exposure to skin sensitizers induced oxidative stress in keratinocytes and dendritic cells (Matsue et al., 2003; Mehrotra et al., 2005). Particularly the antioxidant response Nuclear factor (erythroidderived 2)-like 2 (Nrf2)-Keap1 pathway has been identified to play an important role in sensitization. The importance of this pathway has been shown in several microarray studies, in which gene expression analysis revealed that genes downstream of Nrf2 were highly upregulated in sensitizer exposed keratinocytes and dendritic cells (Ade et al., 2009; Python et al., 2009; Vandebriel et al., 2010). The relevance of Nrf2 for skin sensitization has been shown in Nrf2 deficient mice. The ear swelling in response to DNFB and oxazolone was reduced but not completely prevented in old (but not young) Nrf2 deficient mice. Furthermore, in these old mice it was shown that IFN- but not IL-4 production was absent, indicating that Nrf2 plays a role in the type 1 T cell response (Kim et al., 2008).

Under physiological conditions, the transcription factor (Nrf2) is bound to the sensory protein Keap1 (Figure 3). This complex promotes Cul-3 mediated ubiquitination and subsequent degradation of Nrf2. In response to oxidative stress, the highly reactive Cys residues of Keap1 are activated and Nrf2 is released (Niture et al., 2009). The free Nrf2 translocates into the nucleus and forms a complex with small MAF (F, G and K) molecules. This complex then recognizes the antioxidant responsive elements (ARE) in the promoter region of several genes, such as the cytoprotective heme oxygenase 1 (HMOX1), the phase II detoxification protein NAD(P)H quinine oxidoreductase (NQO1) and several genes involved in glutathione regulation. It is thought that skin sensitizers, which are known to be reactive to cysteine, are able to bind to the cysteine residues of Keap1 and thereby facilitate the release of Nrf2 (Motohashi & Yamamoto, 2004; Natsch, 2009). Although the majority of sensitizers can indeed bind to the cysteine residues, there are some that preferentially bind to lysine residues and these do not activate Nrf2 (Gerberick et al., 2007b; Natsch, 2009).

with R-848, a TLR7 ligand (Gunzer et al., 2005). Pretreatment with the TLR9 ligand CpG ODN enhanced ear swelling in response to DNFB, but it was shown that the site of exposure to CpG ODN is important. When the site of hapten exposure was not the same as the CpG administration site, no effect on skin sensitization was observed, illustrating that co-existing inflammatory signaling in the same skin area is needed to enhance the response (Akiba et al., 2004). There is evidence that a reduced skin barrier function, caused by mutations in the filaggrin gene, increases the sensitization rates to nickel (Novak et al., 2008; Metz & Maurer, 2009). Possibly, the impaired barrier function leads to more pathogen exposure and increased TLR activation. On the other hand, this mutation might also lead to increased skin

In general, when mice are exposed to a hapten together with PAMPs, the ACD response is enhanced, which is most likely due to the increased activation of the innate immune system. In the skin, multiple danger signals are produced in response to the PAMPs thereby facilitating the innate immune response and subsequent adaptive immune response. Therefore, it is possible that concurrent hapten and pathogen exposure leads to increased

Besides triggering the innate immune response, *in vitro* studies have shown that exposure to skin sensitizers induced oxidative stress in keratinocytes and dendritic cells (Matsue et al., 2003; Mehrotra et al., 2005). Particularly the antioxidant response Nuclear factor (erythroidderived 2)-like 2 (Nrf2)-Keap1 pathway has been identified to play an important role in sensitization. The importance of this pathway has been shown in several microarray studies, in which gene expression analysis revealed that genes downstream of Nrf2 were highly upregulated in sensitizer exposed keratinocytes and dendritic cells (Ade et al., 2009; Python et al., 2009; Vandebriel et al., 2010). The relevance of Nrf2 for skin sensitization has been shown in Nrf2 deficient mice. The ear swelling in response to DNFB and oxazolone was reduced but not completely prevented in old (but not young) Nrf2 deficient mice. Furthermore, in these old mice it was shown that IFN- but not IL-4 production was absent,

Under physiological conditions, the transcription factor (Nrf2) is bound to the sensory protein Keap1 (Figure 3). This complex promotes Cul-3 mediated ubiquitination and subsequent degradation of Nrf2. In response to oxidative stress, the highly reactive Cys residues of Keap1 are activated and Nrf2 is released (Niture et al., 2009). The free Nrf2 translocates into the nucleus and forms a complex with small MAF (F, G and K) molecules. This complex then recognizes the antioxidant responsive elements (ARE) in the promoter region of several genes, such as the cytoprotective heme oxygenase 1 (HMOX1), the phase II detoxification protein NAD(P)H quinine oxidoreductase (NQO1) and several genes involved in glutathione regulation. It is thought that skin sensitizers, which are known to be reactive to cysteine, are able to bind to the cysteine residues of Keap1 and thereby facilitate the release of Nrf2 (Motohashi & Yamamoto, 2004; Natsch, 2009). Although the majority of sensitizers can indeed bind to the cysteine residues, there are some that preferentially bind to lysine residues and these do not activate Nrf2 (Gerberick et al., 2007b; Natsch, 2009).

penetration of the haptens thereby increasing the bioavailability in the skin.

**4. The role of the Nrf2-KEAP1 pathway in skin sensitization** 

indicating that Nrf2 plays a role in the type 1 T cell response (Kim et al., 2008).

risk of sensitization.

Fig. 3. The Nrf2-Keap1 pathway (adapted from biocarta, available at: http://www.biocarta.com/pathfiles/h\_ARENRF2PATHWAY.asp)

It can be hypothesized that Nrf2 plays different roles in skin sensitization. First, Nrf2 activation is a result of oxidative stress induced by sensitizer exposure. Second, Nrf2 is involved in the regulation of the immune response, since several genes that are under Nrf2 control have been shown to have immunological effects. For example, upregulation of HMOX1 has been shown to inhibit the maturation of dendritic cells, thereby reducing T cell activation. Furthermore, HMOX1 induces an increased expression of the anti-inflammatory cytokine IL-10 (Listopad et al., 2007). Nrf2 has been shown to be important in attenuating different inflammatory responses (Kim et al., 2010). In Nrf2 deficient mice, the severity of disease in a colitis model was aggravated and this was attributed to increased levels of IL1- , IL-6, IL12p40, and TNF-, (Khor et al., 2006). In old mice deficient of Nrf2, skin sensitization was less pronounced, meaning that Nrf2 is essential for the skin sensitization process (Kim et al., 2008). Hence, Nrf2 has an important role in regulating immune responses.

Since skin sensitization involves different cell types, signaling pathways, cytokines and chemokines it might be possible that there is a direct link between Nrf2 activation and TLR activation. Nrf2 is involved in protection against oxidative stress and the induction of antioxidants. Activation of this pathway could affect redox-sensitive factors associated with TLR activation, such as NF-κB (Kim et al., 2010) and chemokines (Sozzani et al., 2005). For example, in cells exposed to LPS or nickel, the levels of thioredoxin-1 (Trx-1), an enzyme that is regulated by Nrf2, were elevated. It is postulated that the production of reactive

Keratinocytes, Innate Immunity and Allergic Contact Dermatitis - Opportunities

**predict the sensitizing potential of chemicals** 

solubility (Coquette et al., 2003) .

for the Development of *In Vitro* Assays to Predict the Sensitizing Potential of Chemicals 49

**5. Opportunities for the development of** *in vitro* **keratinocyte-based assays to** 

In recent years many efforts have been made to develop cell-based assays able to identify skin sensitizers and to distinguish them from irritants. In keratinocyte-based assays, cytokine induction or gene expression profiles were assessed to find predictive biomarkers or pathways. In the human keratinocyte cell line HaCaT it was shown that intracellular IL-18 was significantly upregulated after exposure to four sensitizers and not after exposure to irritants (Van Och et al., 2005). Similarly, in the keratinocyte cell line NCTC2544 IL-18 was found to be predictive for skin sensitizers, and to distinguish them from respiratory sensitizers and irritants. Prohaptens require metabolic activation to become sensitizers were included in this assay and could be identified as well, indicating that these cells have sufficient metabolic activity (Corsini et al., 2009). IL-18 production is dependent on caspase-1 and requires the inflammasome activation. The relevance of signal transduction pathways in skin sensitizer induced IL-18 production was demonstrated using selective inhibitors and revealed a role for oxidative stress, NF-κB and p38 MAPK activation (Van Och et al., 2005; Corsini et al., 2009; Galbiati et al., 2011). In a reconstructed human epidermis model cytokine profiling was used to discriminate skin sensitizers from irritants. Five sensitizers and three irritants were tested in this 3D model and it was shown that sensitizers induce IL-8 production and secrete only low levels of IL-1. In contrast, irritants induce high levels of IL-1 and only low levels of IL-8. With this limited number of substances the ratio of IL-8/ IL-1 could be used to distinguish sensitizers from irritants. The benefits of using a 3D skin model are the possibilities to test topical formulations and compounds with low water

Tools that can be used to identify biomarkers for specific toxic effects, such as skin sensitization, include "omics" technologies, such as transcriptomics (measuring mRNA expression) and proteomics (measuring protein expression in tissues or cells). Transcriptomics has been used in the HaCaT cell line to find genes that are able to distinguish between sensitizers and irritants. After exposure to eight sensitizers and six irritants, pathway analysis showed that the Nrf2 pathway was significantly affected by sensitizers while it was not triggered by irritants. In addition, a set of 13 genes was identified that could predict the sensitizing potential of chemicals with 73% accuracy (Vandebriel et al., 2010). Further research is needed to confirm the accuracy of this gene list when more substances are used. The relevance of Nrf2 for skin sensitization was confirmed

using primary keratinocytes exposed to two skin sensitizers (Yoshikawa et al., 2010)

assay was reproducible between the laboratories (Natsch et al., 2011).

The importance of Nrf2 in skin sensitization has resulted in the development of a reporter cell line from the HaCaT cell line, the KeratinoSens assay (Natsch, 2009). The promoter sequence of the Nrf2 dependent human gene AKR1C2, coding for an aldo-keto reductase, was placed before the gene encoding luciferase. The principle of this assay is that exposure to skin sensitizers leads to activation of the Nrf2 pathway and luciferase expression. In this assay, 67 substances (sensitizers, non-sensitizers and irritants) were tested. A fold increase of 1.5 in luciferase expression was used to identify skin sensitizers and it was shown that accuracy of this assay was 85.1% (Natsch, 2009; Emter et al., 2010). In a ring study with five laboratories it was shown that the KeratinoSens assay was easy transferable to other laboratories. The accuracy of the assay was tested with 28 blinded test compounds and the

oxygen species following TLR4 activation causes Nrf2 translocation and transcription of the target genes (Listopad et al., 2007; Rushworth et al., 2008). Evidence for a direct link between Nrf2 and TLR was found in *in vitro* studies in macrophages. It was shown that Nrf2 activation by LPS was dependent on MyD88 but independent of the production of reactive oxygen species, indicating a second induction mechanism for Nrf2. It was speculated that MyD88 dependent signaling induced via TLR4 leads to activation of Nrf2 in order to regulate the inflammatory response (Kim et al., 2011). It remains unclear if there is a link between Nrf2 and TLR activation in skin sensitization and more research is needed to better understand the underlying mechanisms of skin sensitization.

Fig. 4. Schematic overview of innate immune responses induced by sensitizers. Hapten exposure leads to degradation of hyaluronic acid (HA) in the skin and the HA fragments act as endogenous ligands for TLR activation. Downstream signaling pathways such as p38 MAPK are activated leading to NF-kB activation and release of pro-inflammatory cytokines (IL-6, IL-8, TNF- in the skin. At the same time, stress induced by haptens activates the NLRP3 inflammasome leading to caspase-1 activation and processing pro-IL-1 and pro-IL18 and subsequent release of IL-1 and IL-18. Together, this cascade of signaling pathways results in the essential factors needed for the development of an adaptive immune response to the skin sensitizers. On the other hand, the hapten exposure activates the Nrf2 pathway through generation of ROS and binding to Keap1, releasing Nrf2. Leading to production of antioxidants, affecting the redox balance. Redox-sensitive signaling pathways, such as p38 MAPK, NF-B and chemokine production are attenuated in an attempt to reduce the inflammatory response in the skin and prevent skin sensitization (adapted from Martin et al., 2011).

oxygen species following TLR4 activation causes Nrf2 translocation and transcription of the target genes (Listopad et al., 2007; Rushworth et al., 2008). Evidence for a direct link between Nrf2 and TLR was found in *in vitro* studies in macrophages. It was shown that Nrf2 activation by LPS was dependent on MyD88 but independent of the production of reactive oxygen species, indicating a second induction mechanism for Nrf2. It was speculated that MyD88 dependent signaling induced via TLR4 leads to activation of Nrf2 in order to regulate the inflammatory response (Kim et al., 2011). It remains unclear if there is a link between Nrf2 and TLR activation in skin sensitization and more research is needed to better

Fig. 4. Schematic overview of innate immune responses induced by sensitizers. Hapten exposure leads to degradation of hyaluronic acid (HA) in the skin and the HA fragments act as endogenous ligands for TLR activation. Downstream signaling pathways such as p38 MAPK are activated leading to NF-kB activation and release of pro-inflammatory cytokines (IL-6, IL-8, TNF- in the skin. At the same time, stress induced by haptens activates the NLRP3 inflammasome leading to caspase-1 activation and processing pro-IL-1 and pro-IL18 and subsequent release of IL-1 and IL-18. Together, this cascade of signaling pathways results in the essential factors needed for the development of an adaptive immune response to the skin sensitizers. On the other hand, the hapten exposure activates the Nrf2 pathway through generation of ROS and binding to Keap1, releasing Nrf2. Leading to production of antioxidants, affecting the redox balance. Redox-sensitive signaling pathways, such as p38 MAPK, NF-B and chemokine production are attenuated in an attempt to reduce the inflammatory response in the skin and prevent skin sensitization (adapted from Martin et

al., 2011).

understand the underlying mechanisms of skin sensitization.
