**4.1 Role of innate immunity**

ACD is a delayed type hypersensitivity reaction. Despite being a prototypical T cell mediated response, a role of innate immunity has been pointed out since early studies and has been recently better elucidated. A common feature of contact allergens employed in experimental studies is their local irritancy and their capacity to act as adjuvants. This feature depends on their ability to activate the innate immune system.

Innate immune system cells express pattern recognition receptors (PRRs), germ-line encoded receptors that recognize so-called pathogen-associated molecular patterns (PAMPs), microbial molecular structures such as bacterial or fungal cell wall components, microbial nucleic acids, proteins or sugars. Depending on their localization we can distinguish transmembrane PRRs, such as Toll-like receptors (TLRs) which recognize PAMPs in the extracellular space and/or in phagosomes or endosomes, and cytosolic PRRs, such as nucleotide-binding oligomerization domain containing (Nod)-like receptors (NLR).

Investigating the involvement of TLRs in the development of CHS to contact allergens, Martin and coworkers first revealed a crucial role for TLR2 and TLR4 (Martin et al., 2008). Mice lacking both TLR4 and TLR2 were resistant to TNCB induced CHS, thus establishing a link between hapten-induced inflammation and innate immune-responses. Accordingly, mice deficient for MyD88, a molecule centrally involved in TLRs signaling trunsduction, failed to mount CHS responses to DNFB (Klekotka et al., 2010). Further evidence for a role of TLRs in cutaneous ACD comes from a study on Nickel (Ni(2+)), by far one of the most relevant contact allergens in terms of incidence of contact eczema and sensitization rates. Schmidt et al., identified human TLR4 as the crucial mediator of the innate immune response to Ni(2+) (Schmidt M et al., 2010). After having provided in vitro evidence for the need of TLR4 expression for Ni(2+) induced activation, the authors demonstrated that transgenic expression of hTLR4 in TLR4-deficient mice confers sensitivity of naturally

Animal Models of Contact Dermatitis 29

Production of IL-18, a cytokine with structural similarities to IL-1, is also regulated by NLRP3 inflammasome activation. In vivo studies showed that CHS responses to oxazolone and DNFB were significantly inhibited in mice treated with neutralizing IL-18 Ab (Wang et al., 2002) or which were deficient for either IL-18 (Antonopoulos et al., 2008) or IL-18R (Klekotka et al., 2010). Interestingly CHS could be rescued by local intradermal administration of IL-18 prior to sensitization, in agreement with an implication of IL-18 in

Trying to dissect the relative contribution of IL-1 and IL-18 in ACD development, Antonopoulos et al., further showed that IL-1 but not IL-18 administration was able to rescue the defective CHS response observed in caspase-1-/- mice, which have no functional IL-1 or IL-18 (Antonopoulos et al., 2008). Therefore IL-1 appears to be the main caspase-1-

ACD is a cellular immune reaction which has been identified since early studies as mediated primarily by T cells. Adoptive transfer of T cells from sensitized mice into non-sensitized recipients results in the transfer of sensitization (Moorhead et al., 1978 ; van Loveren et al., 1983). Moreover, T cell depletion before the elicitation phase results in complete abolition of

As ACD is a classical DTH reaction and since cutaneous infiltrates in humans show a clear preponderance of CD4+ T cells, ACD has first being considered to be mediated primarily by CD4 T cells. However, based on contrasting experimental results, the role exerted by different T cell subsets in the physiopathology of the disease has longly been debated.

In some experimental models CD4 T cells have indeed been shown to mediate the allergic response (Miller and Jenkins, 1985 ; Gocinski and Tigelaar 1990 ; Kohler et al., 1995 ; Wang B et al., 2000). However, most studies agree in identifying CD8+ T cells as the main effector compartment in ACD to different haptens (Gocinski and Tigelaar, 1990 ; Bour et al, 1995 ; Xu et al, 1996 ; Bouloc et al, 1998 ; Kehren et al., 1999 ; Martin et al., 2000 ; Akiba et al., 2002 ;

Such evidence pointing to a role of CD8 T cells in ACD has been obtained through several experimental approaches such as in vivo depletion of normal mice with anti-CD4 and anti-CD8 mAbs, use of MHC class I-/- CD8+ T cell-deficient mice and MHC class II-/- CD4+ T cell-deficient mice or adoptive transfer of purified primed CD4+ and/or CD8+ T cells from

Using the antibody depletion model, Gocinski and Tigelaar showed that CD8 depletion prior to DNFB sensitisation led to a substantial reduction of ear swelling upon rechallenge, for the first time (Gocinski and Tigelaar, 1990) pointing to CD8 T cells as major players in ACD. Even more surprisingly, the authors demonstrated that when CD4 T cells were depleted prior to sensitization with DNFB, rather than observing a reduction in ear swelling upon rechallenge, responses were augmented suggesting that CD4 T cells were indeed behaving as downregulatory cells. These conclusions were subsequently confirmed by Bour et al. studying CHS in MHC class I and MHC class II knockout mice, which are deficient in CD8+ and CD4+ T

the afferent phase of the disease.

**4.2 Role of adaptive immunity** 

dependent inducer of inflammation in CHS.

the reaction (Gocinski and Tigelaar, 1990).

Dubois et al., 2003 ; Saint-Mezard et al., 2004).

sensitized mice into naïve recipients.

resistant mice to Ni(2+)-induced CHS. Those results formally demonstrate that Ni(2+) employs TLR4, a signaling component of the antibacterial defense system, to elicit its allergic reactions.

Fig. 3. Irritant contact dermatitis experimental protocol.

NLRP3, also known as NALP3/Cryopyrin/CIAS1/PYPAF1, belongs to the NLR family of PRRs and is activated by a variety of pathogen- and host-derived "danger" signals including: whole pathogens (Candida albicans, Saccharomyces cerevisiae, Staphylococcus aureus, Listeria monocytogenes); pathogen-associated molecules (bacterial pore–forming toxins and malarial hemozoin); environmental irritants (silica, asbestos, ultraviolet light); host-derived "danger-associated molecular patterns" (ATP, glucose, monosodium urate, calcium pyrophosphate dihydrate, amyloid β, hyaluronan); and immune adjuvants (aluminum salt). NLRP3 forms a multi-protein complex, known as the NLRP3 inflammasome, together with the adaptor protein apoptosis-associated specklike protein (ASC) and caspase-1. Inflammasome activation leads to the proteolysis to bioactive form of the proinflammatory cytokines IL-1 and IL-18. Langherans cells and keratinocytes can secrete IL-1 and IL-18 upon exposure to sensitizing agents (Sauder etal., 1984 ; Enk et al., 1993 ; Nail et al., 1999) and these cytokines are crucial for Langherans cells migration to the draining lymph nodes (Cumberbatch et al., 2002). It is therefore not surprising that NLRP3 inflammasome and IL-1/IL-1R signaling are required for ACD development. Shornick and colleagues first demonstrated that IL-1 deficient mice showed defective CHS responses to topically applied TNCB and that this defect could be overcome by local intradermal injection of recombinant IL-1 immediately before antigen application (Shornick et al., 1996). Accordingly, as NLRP3 inflammasome activation is necessary for active IL-1 production, mice lacking either NLRP3, the adaptor protein ASC or caspase-1 showed impaired CHS responses to TNCB and DNFB (Sutterwala et al., 2006 ; Watanabe et al., 2007 ; Antonopoulos et al., 2001). Finally, IL-1R deficiency (Klekotka et al., 2010) or treatment of mice with the IL-1R antagonist (Kondo et al., 1995) efficiently prevented CHS development.

resistant mice to Ni(2+)-induced CHS. Those results formally demonstrate that Ni(2+) employs TLR4, a signaling component of the antibacterial defense system, to elicit its

NLRP3, also known as NALP3/Cryopyrin/CIAS1/PYPAF1, belongs to the NLR family of PRRs and is activated by a variety of pathogen- and host-derived "danger" signals including: whole pathogens (Candida albicans, Saccharomyces cerevisiae, Staphylococcus aureus, Listeria monocytogenes); pathogen-associated molecules (bacterial pore–forming toxins and malarial hemozoin); environmental irritants (silica, asbestos, ultraviolet light); host-derived "danger-associated molecular patterns" (ATP, glucose, monosodium urate, calcium pyrophosphate dihydrate, amyloid β, hyaluronan); and immune adjuvants (aluminum salt). NLRP3 forms a multi-protein complex, known as the NLRP3 inflammasome, together with the adaptor protein apoptosis-associated specklike protein (ASC) and caspase-1. Inflammasome activation leads to the proteolysis to bioactive form of the proinflammatory cytokines IL-1 and IL-18. Langherans cells and keratinocytes can secrete IL-1 and IL-18 upon exposure to sensitizing agents (Sauder etal., 1984 ; Enk et al., 1993 ; Nail et al., 1999) and these cytokines are crucial for Langherans cells migration to the draining lymph nodes (Cumberbatch et al., 2002). It is therefore not surprising that NLRP3 inflammasome and IL-1/IL-1R signaling are required for ACD development. Shornick and colleagues first demonstrated that IL-1 deficient mice showed defective CHS responses to topically applied TNCB and that this defect could be overcome by local intradermal injection of recombinant IL-1 immediately before antigen application (Shornick et al., 1996). Accordingly, as NLRP3 inflammasome activation is necessary for active IL-1 production, mice lacking either NLRP3, the adaptor protein ASC or caspase-1 showed impaired CHS responses to TNCB and DNFB (Sutterwala et al., 2006 ; Watanabe et al., 2007 ; Antonopoulos et al., 2001). Finally, IL-1R deficiency (Klekotka et al., 2010) or treatment of mice with the IL-

1R antagonist (Kondo et al., 1995) efficiently prevented CHS development.

allergic reactions.

Fig. 3. Irritant contact dermatitis experimental protocol.

Production of IL-18, a cytokine with structural similarities to IL-1, is also regulated by NLRP3 inflammasome activation. In vivo studies showed that CHS responses to oxazolone and DNFB were significantly inhibited in mice treated with neutralizing IL-18 Ab (Wang et al., 2002) or which were deficient for either IL-18 (Antonopoulos et al., 2008) or IL-18R (Klekotka et al., 2010). Interestingly CHS could be rescued by local intradermal administration of IL-18 prior to sensitization, in agreement with an implication of IL-18 in the afferent phase of the disease.

Trying to dissect the relative contribution of IL-1 and IL-18 in ACD development, Antonopoulos et al., further showed that IL-1 but not IL-18 administration was able to rescue the defective CHS response observed in caspase-1-/- mice, which have no functional IL-1 or IL-18 (Antonopoulos et al., 2008). Therefore IL-1 appears to be the main caspase-1 dependent inducer of inflammation in CHS.

#### **4.2 Role of adaptive immunity**

ACD is a cellular immune reaction which has been identified since early studies as mediated primarily by T cells. Adoptive transfer of T cells from sensitized mice into non-sensitized recipients results in the transfer of sensitization (Moorhead et al., 1978 ; van Loveren et al., 1983). Moreover, T cell depletion before the elicitation phase results in complete abolition of the reaction (Gocinski and Tigelaar, 1990).

As ACD is a classical DTH reaction and since cutaneous infiltrates in humans show a clear preponderance of CD4+ T cells, ACD has first being considered to be mediated primarily by CD4 T cells. However, based on contrasting experimental results, the role exerted by different T cell subsets in the physiopathology of the disease has longly been debated.

In some experimental models CD4 T cells have indeed been shown to mediate the allergic response (Miller and Jenkins, 1985 ; Gocinski and Tigelaar 1990 ; Kohler et al., 1995 ; Wang B et al., 2000). However, most studies agree in identifying CD8+ T cells as the main effector compartment in ACD to different haptens (Gocinski and Tigelaar, 1990 ; Bour et al, 1995 ; Xu et al, 1996 ; Bouloc et al, 1998 ; Kehren et al., 1999 ; Martin et al., 2000 ; Akiba et al., 2002 ; Dubois et al., 2003 ; Saint-Mezard et al., 2004).

Such evidence pointing to a role of CD8 T cells in ACD has been obtained through several experimental approaches such as in vivo depletion of normal mice with anti-CD4 and anti-CD8 mAbs, use of MHC class I-/- CD8+ T cell-deficient mice and MHC class II-/- CD4+ T cell-deficient mice or adoptive transfer of purified primed CD4+ and/or CD8+ T cells from sensitized mice into naïve recipients.

Using the antibody depletion model, Gocinski and Tigelaar showed that CD8 depletion prior to DNFB sensitisation led to a substantial reduction of ear swelling upon rechallenge, for the first time (Gocinski and Tigelaar, 1990) pointing to CD8 T cells as major players in ACD. Even more surprisingly, the authors demonstrated that when CD4 T cells were depleted prior to sensitization with DNFB, rather than observing a reduction in ear swelling upon rechallenge, responses were augmented suggesting that CD4 T cells were indeed behaving as downregulatory cells. These conclusions were subsequently confirmed by Bour et al. studying CHS in MHC class I and MHC class II knockout mice, which are deficient in CD8+ and CD4+ T

Animal Models of Contact Dermatitis 31

disruption of the IFN-γR2 gene (Lu et al., 1998) or of the IFN-γ encoding gene (Wakabayashi et al., 2005) suppresses CHS responses. However other reports failed to confirm impaired CHS responses in mice with IFN-γ signaling disruption (Saulnier et al., 1995 ; Reeve et al., 1999). Moreover, neutralization of IFN-γ in DNFB sensitized mice before challenge failed to

Searching for other mediatiors of CHS, Nakae et al. reported that CHS was reduced in IL-17 knockout mice compared with wild-type controls (Nakae et al., 2002). Subsequently, IL-17 neutralization in DNFB sensitized mice before challenge has been shown to suppress the elicitation of CHS (He et al., 2006). IL-17 is a well known chemotactic factor and absence of IL-17 signaling inhibited the infiltration of T cells, monocytes/macrophages and granulocytes into hapten-challenged skin tissues. Interestingly, He and coworkers demonstrated that in the DNFB induced CHS model CD8+ T cells represents the major source of IL-17 at the inflammation site. Moreover, CD8+ IL-17-producing T cell subpopulation is distinct from CD8+ IFN-γ-producing T cells and is important in effector functions during the elicitation of CHS. Subsequently the same group extended those results and demonstrated that IL-17 and IFN-γ signaling are both required for optimal elicitation of

Globally, murine studies allowed to identify CD8+ T cells as the major cellular player in ACD pathogenesis and pointed out several molecular pathways which can be taken into

Both sensitization to chemicals and the effector phase of contact allergy are highly regulated events. This task is guaranteed by multiple mechanisms, including antigen-presenting cells and effector T cells apoptosis, production and release of anti-inflammatory mediators and action of a specialized subset of T lymphocytes with down-regulatory properties known as

Regulatory T cells (Treg) are a critical CD4 T cell subset involved in the control of immune homeostasis and in regulation of inflammation (Sakaguchi et al., 2008). Treg represent about 5-10% of the whole T cell compartment and are characterized by the preferential expression of several molecules including CD25, the alpha chain of IL-2 receptor (Sakaguchi et al., 1995), and FOXP3, a transcription factor which is necessary for Treg development and

CD4+CD25+ Treg cells have been implicated in the control CHS responses to haptens in mice. The first evidence of such a role for Treg cells came from a study investigating the mechanisms responsible for the "oral tolerance" phenomenon (Dubois et al., 2003). It was known that DNFB oral administration prior to sensibilisation could induce a tolerant state and prevent the development of ACD (Garside et al., 2001). Desvignes and coworkers showed that tolerance induction was dependent on the presence of CD4 T cells (Desvignes et al., 1996 ; Desvignes et al., 2000) suggesting the implication of a CD4 T cell subset with regulatory characteristics. Using in vivo models of adoptive transfer and antibody depletion of CD4+CD25+ cells, Dubois et al. demonstrated that naturally occurring CD4+CD25+ T cells are instrumental for orally induced tolerance and control hapten-specific CD8+ T cell

CHS by probably acting through distinguished mechanisms (He et al., 2009).

account for the development of immuno-therapeutic strategies.

responses mediating skin inflammation (Dubois et al., 2003).

regulatory T cells.

function (Fontenot et al., 2003).

**5. Regulatory mechanisms in allergic contact dermatitis** 

suppress the elicitation of CHS (He et al., 2006).

cells, respectively. In this system, Class I deficient mice failed to develop DNFB-induced CHS responses while class II deficient mice developed an enhanced CHS reaction (Bour et al., 1995).

An explanation to these phenomena came from the demonstration that upon sensitization with DNFB or oxaloxone hapten-loaded APC migrated to draining lymph nodes from the skin induce the differentiation of IFN--producing effector CD8+ T cells and IL-4/IL-10 producing CD4+ T cells that negatively regulate the response (Xu et al., 1996). Since CD8+ effector T cells primarily exert their function through cytotoxicity, the demonstration of a central role of CD8 T cells in CHS leads to investigate whether cytotoxicity mediated skin inflammation. Kehren et al. showed that mice double deficient in perforin and FasL were able to develop hapten-specific CD8+ T cells in the lymphoid organs but did not show CHS reaction in the skin, thus demonstrating that the CHS reaction is dependent on CD8+ T cells cytotoxic activity (Kehren et al., 1999). Using immunohistochemistry and RT-PCR analysis Akiba and colleagues extended those results by demonstrating that epidermal keratinocytes were the target cells of hapten-specific CD8+ T cells cytotoxicity (Akiba et al., 2002).


Fig. 4. Skin infiltrating lymphocytes preparation for FACS analysis

In addition to the aforementioned role of perforin and FasL, IFN-γ has been shown to play an important role in CHS responses and a defect in IFN-γ signals as a result of the genetic

cells, respectively. In this system, Class I deficient mice failed to develop DNFB-induced CHS responses while class II deficient mice developed an enhanced CHS reaction (Bour et al., 1995). An explanation to these phenomena came from the demonstration that upon sensitization with DNFB or oxaloxone hapten-loaded APC migrated to draining lymph nodes from the skin induce the differentiation of IFN--producing effector CD8+ T cells and IL-4/IL-10 producing CD4+ T cells that negatively regulate the response (Xu et al., 1996). Since CD8+ effector T cells primarily exert their function through cytotoxicity, the demonstration of a central role of CD8 T cells in CHS leads to investigate whether cytotoxicity mediated skin inflammation. Kehren et al. showed that mice double deficient in perforin and FasL were able to develop hapten-specific CD8+ T cells in the lymphoid organs but did not show CHS reaction in the skin, thus demonstrating that the CHS reaction is dependent on CD8+ T cells cytotoxic activity (Kehren et al., 1999). Using immunohistochemistry and RT-PCR analysis Akiba and colleagues extended those results by demonstrating that epidermal keratinocytes

were the target cells of hapten-specific CD8+ T cells cytotoxicity (Akiba et al., 2002).

Fig. 4. Skin infiltrating lymphocytes preparation for FACS analysis

In addition to the aforementioned role of perforin and FasL, IFN-γ has been shown to play an important role in CHS responses and a defect in IFN-γ signals as a result of the genetic disruption of the IFN-γR2 gene (Lu et al., 1998) or of the IFN-γ encoding gene (Wakabayashi et al., 2005) suppresses CHS responses. However other reports failed to confirm impaired CHS responses in mice with IFN-γ signaling disruption (Saulnier et al., 1995 ; Reeve et al., 1999). Moreover, neutralization of IFN-γ in DNFB sensitized mice before challenge failed to suppress the elicitation of CHS (He et al., 2006).

Searching for other mediatiors of CHS, Nakae et al. reported that CHS was reduced in IL-17 knockout mice compared with wild-type controls (Nakae et al., 2002). Subsequently, IL-17 neutralization in DNFB sensitized mice before challenge has been shown to suppress the elicitation of CHS (He et al., 2006). IL-17 is a well known chemotactic factor and absence of IL-17 signaling inhibited the infiltration of T cells, monocytes/macrophages and granulocytes into hapten-challenged skin tissues. Interestingly, He and coworkers demonstrated that in the DNFB induced CHS model CD8+ T cells represents the major source of IL-17 at the inflammation site. Moreover, CD8+ IL-17-producing T cell subpopulation is distinct from CD8+ IFN-γ-producing T cells and is important in effector functions during the elicitation of CHS. Subsequently the same group extended those results and demonstrated that IL-17 and IFN-γ signaling are both required for optimal elicitation of CHS by probably acting through distinguished mechanisms (He et al., 2009).

Globally, murine studies allowed to identify CD8+ T cells as the major cellular player in ACD pathogenesis and pointed out several molecular pathways which can be taken into account for the development of immuno-therapeutic strategies.
