**2.1 Phases of ACD**

Three temporally distinct phases can be identified in ACD: the **sensitization phase**, the **elicitation phase** and the **resolution phase**.

The **sensitization phase,** also referred to as the afferent phase or induction phase, occurs at the first skin contact with a hapten (Fig. 1A). This phase lasts 10–15 days in humans and 5–7 days in mice. Most of employed haptens induce local inflammation by acting on innate immunity receptors (see section 4.1). Activation of the skin innate immunity induces the production of mediators (IL-18, IL-1, TNF-, ATP, PGE2, LTB4, ROS, histamine, CCL20) by resident skin cells. These mediators are able to induce the recruitment, migration and activation of cutaneous antigen presenting cells (APC). The skin contains a dense network of APC constituted by two phenotypically and spatially distinct subsets: Langerhans cells (LC), which are located in the epidermal layer of the skin, and dermal DCs (dDC). In addition to skin resident APC, circulating CCR2+ monocytes are rapidly recruited from blood to skin and locally differentiate into dendritic cells. Once activated, APC phagocyte haptenated proteins and migrate from the skin to the para-cortical area of draining lymph nodes (Kripke et al., 1990). There, APC present haptenated peptides through MHC classes I and II molecules at their cell surface (Weltzien et al., 1996) to hapten-specific CD8+ and CD4+ T lymphocytes thus inducing their priming and expansion (Bour et al., 1995 ; Xu H et al., 1996 ; Krasteva et al., 1998). Primed hapten-specific T cells can then emigrate from the lymph nodes and enter the blood from which they recirculate to different tissues including the skin.

The **elicitation phase**, also referred to as the efferent or challenge phase, occurs after subsequent challenges with the same hapten to which a host has been previously sensitized (Fig. 1B). This efferent phase of CHS takes place within 72 hours upon exposure to the allergen in humans and 24 to 48 hours in mice. Once more, haptenated peptides are uptaken by skin APC which present to hapten specific primed T cells patrolling in the skin. As this process directly takes place on the site of inflammation and as T cells activated at this stage are already primed cells, the elicitation phase develops extremely rapidly.

Activated T cells produce effector cytokines, notably IFN- and IL-17 (see section 4.2), which activate skin resident cells which in turn secrete cytokines and chemokines pertaining cell recruitment at exposure site. Continuous recruitment of circulating cells then leads to polymorphous cellular infiltrate and persistence of the inflammatory reaction over several days.

The **resolution phase**, during which the inflammatory response progressively disappears, then follows. This phase presumably results from down-regulating mechanisms including passive processes, such as the progressive disappearance of the hapten from the epidermis, as well as active cellular processes, such as the intervention of regulatory T cells (see section 5).

responsible for human ACD. Importantly recent studies have reproduced the conclusions drawn with strong allergens by using weak haptens (Vocanson et al., 2006; Vocanson et al.,

Three temporally distinct phases can be identified in ACD: the **sensitization phase**, the

The **sensitization phase,** also referred to as the afferent phase or induction phase, occurs at the first skin contact with a hapten (Fig. 1A). This phase lasts 10–15 days in humans and 5–7 days in mice. Most of employed haptens induce local inflammation by acting on innate immunity receptors (see section 4.1). Activation of the skin innate immunity induces the production of mediators (IL-18, IL-1, TNF-, ATP, PGE2, LTB4, ROS, histamine, CCL20) by resident skin cells. These mediators are able to induce the recruitment, migration and activation of cutaneous antigen presenting cells (APC). The skin contains a dense network of APC constituted by two phenotypically and spatially distinct subsets: Langerhans cells (LC), which are located in the epidermal layer of the skin, and dermal DCs (dDC). In addition to skin resident APC, circulating CCR2+ monocytes are rapidly recruited from blood to skin and locally differentiate into dendritic cells. Once activated, APC phagocyte haptenated proteins and migrate from the skin to the para-cortical area of draining lymph nodes (Kripke et al., 1990). There, APC present haptenated peptides through MHC classes I and II molecules at their cell surface (Weltzien et al., 1996) to hapten-specific CD8+ and CD4+ T lymphocytes thus inducing their priming and expansion (Bour et al., 1995 ; Xu H et al., 1996 ; Krasteva et al., 1998). Primed hapten-specific T cells can then emigrate from the lymph nodes and enter the

2009) which are more close to those encountered in clinical practice.

blood from which they recirculate to different tissues including the skin.

The **elicitation phase**, also referred to as the efferent or challenge phase, occurs after subsequent challenges with the same hapten to which a host has been previously sensitized (Fig. 1B). This efferent phase of CHS takes place within 72 hours upon exposure to the allergen in humans and 24 to 48 hours in mice. Once more, haptenated peptides are uptaken by skin APC which present to hapten specific primed T cells patrolling in the skin. As this process directly takes place on the site of inflammation and as T cells activated at this stage are already primed cells, the elicitation phase develops extremely

Activated T cells produce effector cytokines, notably IFN- and IL-17 (see section 4.2), which activate skin resident cells which in turn secrete cytokines and chemokines pertaining cell recruitment at exposure site. Continuous recruitment of circulating cells then leads to polymorphous cellular infiltrate and persistence of the inflammatory

The **resolution phase**, during which the inflammatory response progressively disappears, then follows. This phase presumably results from down-regulating mechanisms including passive processes, such as the progressive disappearance of the hapten from the epidermis, as well as active cellular processes, such as the intervention of regulatory T

**2.1 Phases of ACD** 

rapidly.

reaction over several days.

cells (see section 5).

**elicitation phase** and the **resolution phase**.

Fig. 1. Schematic representation of the pathophysiology of the different phases of allergic contact dermatitis. (A) the sensitization phase (B) the elicitation phase (C) the resolution phase.

#### **2.2 Experimental protocol**

Experimental protocol for ACD induction is graphically summarized in Figure 2. For sensitization, mice are painted at day 0 on the shaved back (or abdomen) with 100 μl of DNFB 0.5% in a 4:1 mixture of acetone and olive oil. Mice are challenged 6 days later by application of 20 μl DNFB 0.2% in olive oil (10 μl to each side of one ear). Ear thickness is measured with a digital calliper before challenge and at 24-48 hours after treatment. Ear swelling is calculated by subtracting the value recorded for vehicle-control ear from the hapten-applied ear. Histological examination can be performed in order to confirm changes in ear thickness and to quantify cellular infiltration. Moreover, lymphocytes infiltrating

Animal Models of Contact Dermatitis 27

processes. Importantly, using an experimental murine model of primary CHS induced by a single DNFB or FITC painting on mice ears without subsequent challenge, Saint-Mezard and colleagues were able to show that the pathophysiology of one-step ICD is identical to that of two-step classical ACD, involving the same effector and down-regulating immune mechanisms (Saint-Mezard et al., 2003). Remarkably, ICD responses were strictly dosedependent and reproducibly more important in C57Bl/6 mice than in BALB mice (Bonneville et al., 2007). More importantly, Bonneville et al. demonstrated an interrelationship between ICD and ACD and showed that upon hapten rechallenge the intensity of ACD reaction is proportional to the magnitude of the former ICD response

Experimental protocol for ICD induction is graphically summarized in Figure 3. Naive mice are exposed at day 0 to a single application of 20 µl of DNFB 0.5% in a 4:1 mixture of acetone and olive oil applied on the left ear, while the same volume of vehicle alone is applied on the right ear as a control. Six days after ear sensitization, ear thickness is measured with a

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

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

digital calliper. Analysis can be performed similarly to ACD model (see section 2.1).

**4. Effector mechanisms in allergic contact dermatitis** 

feature depends on their ability to activate the innate immune system.

(Bonneville et al., 2007).

**3.1 Experimental protocol** 

**4.1 Role of innate immunity** 

inflamed skin can be investigated by FACS analysis after isolation. In figure 4 we provide a detailed protocol to isolate lymphocytes from skin.

Fig. 2. Allergic contact dermatitis experimental protocol.
