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

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

Keratinocytes, Innate Immunity and Allergic Contact Dermatitis - Opportunities

role in Langerhans migration.

using TLR4, 7 and 9 ligands.

for the Development of *In Vitro* Assays to Predict the Sensitizing Potential of Chemicals 45

TLRs are not the only PRR family members that play a role in the development of ACD, NOD-Like receptors (NLR) have also been implicated, especially their ability to form or activate inflammasomes containing caspase-1 is important. Ear swelling responses to DNFB or oxazolone were decreased significantly in caspase-1 knock out mice when compared to wild type mice (Antonopoulos et al., 2001). In addition, migration of Langerhans cells was evaluated in wildtype and caspase-1 deficient mice. It was shown that in the absence of caspase-1 the migration of Langerhans cells was impaired, but the maturation of Langerhans cells was not affected. In these mice, Langerhans cells migration could be restored when IL1-, but not TNF-were intradermally injected, indicating that these mice were unable to produce IL1-, which is dependent on caspase-1 (Antonopoulos et al., 2001; Cumberbatch et al., 2001). IL1- has been shown to have a marked effect on skin sensitization as well, since ear swelling in response to TNBS was impaired in IL1- deficient mice and not in IL1- deficient mice (Nakae et al., 2001). Whereas IL1- is mainly produced by Langerhans cells, keratinocytes are the main source of IL1-. These studies show that IL1- is required in the induction of skin sensitization, whereas IL1- plays an important

The NLRP3 inflammasome can be assembled due to the activation of the P2X7 receptor. This receptor on the cell membrane recognizes extracellular ATP, which is a damage-associated molecular pattern. Mice deficient of P2X7 had impaired ear swelling responses after exposure to TNCB and oxazolone. The ear swelling response was restored when a potent P2X7-independent NLRP3 activator was applied. To determine whether the triggering of NLRP3 via P2X7 is specific to sensitizers remains to be determined (Weber et al., 2010).

Importantly, the activation of inflammasomes seems to be an effect that is not specific to sensitizers. Other chemicals, such as irritants can also induce cellular stress and activate the inflammasome. The same stress that causes activation of inflammasomes could also lead to the degradation of hyaluronic acid and thus activation of TLR. Hence, this illustrates that NLRP3 inflammasome activation is not limited to skin sensitization and that this pathway

In the initiation of skin sensitization, innate immune responses play an important role and it has been shown that skin sensitizers are able to trigger this pathway via PRR members. In reality it is possible that humans with an existing skin inflammation are exposed to haptens. This inflammation leads to microbial danger signals in the skin which could possibly aggravate the immune response induced by skin sensitizers. Evidence for this was found in *in vivo* studies in which the effects of PAMPs on ACD development were studied in mice

In C57BL6 and C3H/HeN mice that were prior to sensitization exposed to the TLR4 ligand LPS by intradermal ear injection the ear swelling response to DNFB was increased. In the TLR4 deficient C3H/HeJ strain, co-exposure with LPS did not enhance the ear swelling response, providing evidence for a crucial role of TLR4 activation (Yokoi et al., 2009). The dose required for sensitization to DNFB was reduced a 100-fold when mice were pretreated

cannot be used for the identification of skin sensitizing properties.

**3.2 Effects of co-exposure to PAMPs on skin sensitization** 

**3.1 The role of Nod-like receptors and the inflammasome in skin sensitization** 

the adaptor molecule TRIF could be sensitized to DNFB, indicating that TLR3, using only TRIF for signal transduction, is not involved in skin sensitization (Klekotka et al., 2010).

More evidence for the importance of TLR in skin sensitization has recently been found for nickel, one of the most prevalent human sensitizers. Remarkably, nickel is a false-negative in the mouse LLNA. This paradox was recently explained by the discovery that nickel interacts directly with non-conserved histidine residues in human but not mouse TLR4, thereby activating the innate immune system and driving the development of ACD (Schmidt et al., 2010). Taken together, these data show that especially TLR 2 and 4 play an important role in mounting a full immune response to skin sensitizers.

Evidence for an involvement of the signaling pathway p38 MAPK in skin sensitization further underpin the relevance for TLR. p38 MAPK are enzymes that play an important role in the signal transduction of TLR (Mehrotra et al., 2007). In mice treated with the specific p38 MAPK inhibitor SB202190 the ear swelling in response to DNFB was impaired (Takanami-Ohnishi et al., 2002). Further evidence for the importance of p38 MAPK was found in *in vitro* studies. In dendritic cells exposed to DNFB, DNCB or nickel, the signaling pathways p38 MAPK and extracellular signal-regulated kinase (ERK) were activated (Matos et al., 2005; Miyazawa et al., 2008). In the keratinocyte cell line NCTC2544 IL-18 production induced by skin sensitizers was greatly decreased after addition of a specific p38 MAPK inhibitor, SB203580 (Galbiati et al., 2011). Similar results were obtained in an experiment using the monocytic THP-1 cell line (Mitjans et al., 2010). These signaling pathways are essential for the further maturation of dendritic cells and the activation of hapten-specific T cells, since they trigger the production of cytokines such as TNF- IL-6, IL-12 and IL-18 in keratinocytes or dendritic cells and are essential in the upregulation of costimulatory molecules on the surface of the dendritic cells (Antonios et al., 2009; Antonios et al., 2010).

It is not clear which ligands trigger TLR activation after exposure to skin sensitizers. It has been shown that ACD can develop in germ-free mice; hence danger signals from pathogenic microbes are not required for sensitization. This indicates that the presence of haptens is sufficient for TLR activation and presumably endogenous TLR ligands are involved (Martin et al., 2008). Several endogenous danger signals can be formed by the breakdown of extracellular matrix under influence of oxidative stress induced by sensitizers. These fragments can be recognized by TLR and initiate the innate immune response. In addition, NF-κB regulates hyaluronidases that degrade hyaluronic acid. Transcription of these enzymes can therefore be a result of earlier TLR activation, eventually strengthening the TLR activation in the skin (Martin et al., 2011). Another possible endogenous danger signal is uric acid, which can be released due to damage to the skin. Mice that were exposed to a combination of TNCB, uric acid crystals and an uricase inhibitor have increased sensitization. Uric acid has been shown to activate the NLRP3 inflammasome, thereby facilitating cytokine production (Liu et al., 2007). Other endogenous danger signals that have been linked to skin sensitization include the heat-shock proteins 27 and 70, which are also recognized by TLR4. Neutralizing antibodies for these heat shock proteins resulted in an impaired ear swelling in response to DNFB. In addition, the cytokine profile shifted from a Th1 to a Th2 repertoire (Yusuf et al., 2009).

the adaptor molecule TRIF could be sensitized to DNFB, indicating that TLR3, using only TRIF for signal transduction, is not involved in skin sensitization (Klekotka et al., 2010).

More evidence for the importance of TLR in skin sensitization has recently been found for nickel, one of the most prevalent human sensitizers. Remarkably, nickel is a false-negative in the mouse LLNA. This paradox was recently explained by the discovery that nickel interacts directly with non-conserved histidine residues in human but not mouse TLR4, thereby activating the innate immune system and driving the development of ACD (Schmidt et al., 2010). Taken together, these data show that especially TLR 2 and 4 play an important role in

Evidence for an involvement of the signaling pathway p38 MAPK in skin sensitization further underpin the relevance for TLR. p38 MAPK are enzymes that play an important role in the signal transduction of TLR (Mehrotra et al., 2007). In mice treated with the specific p38 MAPK inhibitor SB202190 the ear swelling in response to DNFB was impaired (Takanami-Ohnishi et al., 2002). Further evidence for the importance of p38 MAPK was found in *in vitro* studies. In dendritic cells exposed to DNFB, DNCB or nickel, the signaling pathways p38 MAPK and extracellular signal-regulated kinase (ERK) were activated (Matos et al., 2005; Miyazawa et al., 2008). In the keratinocyte cell line NCTC2544 IL-18 production induced by skin sensitizers was greatly decreased after addition of a specific p38 MAPK inhibitor, SB203580 (Galbiati et al., 2011). Similar results were obtained in an experiment using the monocytic THP-1 cell line (Mitjans et al., 2010). These signaling pathways are essential for the further maturation of dendritic cells and the activation of hapten-specific T cells, since they trigger the production of cytokines such as TNF- IL-6, IL-12 and IL-18 in keratinocytes or dendritic cells and are essential in the upregulation of costimulatory molecules on the surface of the dendritic cells (Antonios

It is not clear which ligands trigger TLR activation after exposure to skin sensitizers. It has been shown that ACD can develop in germ-free mice; hence danger signals from pathogenic microbes are not required for sensitization. This indicates that the presence of haptens is sufficient for TLR activation and presumably endogenous TLR ligands are involved (Martin et al., 2008). Several endogenous danger signals can be formed by the breakdown of extracellular matrix under influence of oxidative stress induced by sensitizers. These fragments can be recognized by TLR and initiate the innate immune response. In addition, NF-κB regulates hyaluronidases that degrade hyaluronic acid. Transcription of these enzymes can therefore be a result of earlier TLR activation, eventually strengthening the TLR activation in the skin (Martin et al., 2011). Another possible endogenous danger signal is uric acid, which can be released due to damage to the skin. Mice that were exposed to a combination of TNCB, uric acid crystals and an uricase inhibitor have increased sensitization. Uric acid has been shown to activate the NLRP3 inflammasome, thereby facilitating cytokine production (Liu et al., 2007). Other endogenous danger signals that have been linked to skin sensitization include the heat-shock proteins 27 and 70, which are also recognized by TLR4. Neutralizing antibodies for these heat shock proteins resulted in an impaired ear swelling in response to DNFB. In addition, the cytokine profile shifted from

mounting a full immune response to skin sensitizers.

et al., 2009; Antonios et al., 2010).

a Th1 to a Th2 repertoire (Yusuf et al., 2009).

#### **3.1 The role of Nod-like receptors and the inflammasome in skin sensitization**

TLRs are not the only PRR family members that play a role in the development of ACD, NOD-Like receptors (NLR) have also been implicated, especially their ability to form or activate inflammasomes containing caspase-1 is important. Ear swelling responses to DNFB or oxazolone were decreased significantly in caspase-1 knock out mice when compared to wild type mice (Antonopoulos et al., 2001). In addition, migration of Langerhans cells was evaluated in wildtype and caspase-1 deficient mice. It was shown that in the absence of caspase-1 the migration of Langerhans cells was impaired, but the maturation of Langerhans cells was not affected. In these mice, Langerhans cells migration could be restored when IL1-, but not TNF-were intradermally injected, indicating that these mice were unable to produce IL1-, which is dependent on caspase-1 (Antonopoulos et al., 2001; Cumberbatch et al., 2001). IL1- has been shown to have a marked effect on skin sensitization as well, since ear swelling in response to TNBS was impaired in IL1- deficient mice and not in IL1- deficient mice (Nakae et al., 2001). Whereas IL1- is mainly produced by Langerhans cells, keratinocytes are the main source of IL1-. These studies show that IL1- is required in the induction of skin sensitization, whereas IL1- plays an important role in Langerhans migration.

The NLRP3 inflammasome can be assembled due to the activation of the P2X7 receptor. This receptor on the cell membrane recognizes extracellular ATP, which is a damage-associated molecular pattern. Mice deficient of P2X7 had impaired ear swelling responses after exposure to TNCB and oxazolone. The ear swelling response was restored when a potent P2X7-independent NLRP3 activator was applied. To determine whether the triggering of NLRP3 via P2X7 is specific to sensitizers remains to be determined (Weber et al., 2010).

Importantly, the activation of inflammasomes seems to be an effect that is not specific to sensitizers. Other chemicals, such as irritants can also induce cellular stress and activate the inflammasome. The same stress that causes activation of inflammasomes could also lead to the degradation of hyaluronic acid and thus activation of TLR. Hence, this illustrates that NLRP3 inflammasome activation is not limited to skin sensitization and that this pathway cannot be used for the identification of skin sensitizing properties.

#### **3.2 Effects of co-exposure to PAMPs on skin sensitization**

In the initiation of skin sensitization, innate immune responses play an important role and it has been shown that skin sensitizers are able to trigger this pathway via PRR members. In reality it is possible that humans with an existing skin inflammation are exposed to haptens. This inflammation leads to microbial danger signals in the skin which could possibly aggravate the immune response induced by skin sensitizers. Evidence for this was found in *in vivo* studies in which the effects of PAMPs on ACD development were studied in mice using TLR4, 7 and 9 ligands.

In C57BL6 and C3H/HeN mice that were prior to sensitization exposed to the TLR4 ligand LPS by intradermal ear injection the ear swelling response to DNFB was increased. In the TLR4 deficient C3H/HeJ strain, co-exposure with LPS did not enhance the ear swelling response, providing evidence for a crucial role of TLR4 activation (Yokoi et al., 2009). The dose required for sensitization to DNFB was reduced a 100-fold when mice were pretreated

Keratinocytes, Innate Immunity and Allergic Contact Dermatitis - Opportunities

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

responses.

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

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

for the Development of *In Vitro* Assays to Predict the Sensitizing Potential of Chemicals 47

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 penetration of the haptens thereby increasing the bioavailability in the 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 risk of sensitization.
