Animal models of psoriasis

With exception of a few sporadic cases reported in primates (e.g. a rhesus monkey and a cynomolgus monkey), psoriasis is a disease unique to humans and has not been observed in other animals [282,283]. Therefore, the lack of a naturally occurring animal disease that sufficiently resembles the complex pathophysiology of the human disease has hindered research into the pathogenesis of psoriasis and drug development [284]. Moreover, most of

the features of psoriasis are not disease specific; for example, metabolic and biochemical dysregulation is also present in other inflammatory skin diseases, inflammatory mediators are also increased in other disorders, and established therapies are not exclusively effective in psoriasis. For those reasons, an animal model that completely mimics the complex human disease (i.e. chronic skin inflammatory lesions, epidermal hyperproliferation and altered differentiation, marked angiogenesis and a characteristic immune cell infiltrate) and that respond to established therapies, is still not available [285]. Nevertheless, in the past decades numerous mouse models have been identified or created by genetic engineering that show at least some features of the pathogenesis of the disease. One of the main limitations of the mouse models stands with the major differences that exist between mouse and human skin (Figure 8). The fur-covered skin epithelium in mice presents a high density of hairs follicles, whereas the human skin is mostly constituted by interfolicular regions. Mouse epidermis is also considerably thinner than the human, generally comprising only 2 to 3 keratinocytes layers, and has a faster turnover. Furthermore, human skin shows a differential protein expression in the outer root sheet of the hair follicles compared to the interfollicular areas, while mouse skin does not share this feature, neither contain any rete ridges between interfollicular regions [286]. Non-epithelial skin compartments are also significantly different across species. Particularly, the dermis is considerably thicker in human compared to mouse [284]. Likewise, the immune system also differs in human and mice. Mouse skin contains a large fraction of γδ T cells in the epidermis, known as dendritic epidermal cells, while human skin mainly contains T cells bearing α/β receptors that are localized in the dermis [287,288].

Mouse skin also contain several different subtypes of DC, including the CD8⁺ DC and other inflammatory cells that are not found in humans [289]. Nevertheless, mice models are still widely used due to the lack of alternative methods.

The current mouse models of psoriasis are mainly based on three experimental settings:

spontaneous mutations, genetic engineering and xenotransplantation. Some spontaneous mutations, such as the flaky (fsn) skin mice [290], homozygous asebia (Scd1ab/Scd1ab) [291]

and the spontaneous chronic proliferative dermatitis (cpdm/cpdm) [292] mutant mice are associated with psoriasis-like phenotype.

Figure 8. Histological differences between human and mouse skin.

Representative micrographs of adult human and mouse skin. Human skin contains sparse hair follicles that separate wide regions of interfollicular epidermis. By contrast, mouse skin has closely spaced hair follicles throughout (arrows). Human skin has much thicker dermis (D) and epidermis (E), with many more cell layers in the epidermis. Epidermal rete ridges (arrows), common in human epidermis, are absent in mouse skin. Finally, mice contain an entire cutaneous muscle layer, the panniculus carnosus, absent in human (Figure adapted and modified from [293]).

These mutants display some histological features of psoriasis such as acanthosis, increased dermal vascularity, and a dermal infiltrate composed of mast cells and macrophages.

However, the general absence of T cells in the dermal infiltrate and the lack of efficacy of some drugs used in psoriatic patients, suggest that these mice do not recapitulate all pathologic features of psoriasis [284]. Several transgenic and gene knock-out mouse models have been created to perform more specific genotype-phenotype studies. In most of these

suprabasal layer of the epidermis using promoters such as involucrin and keratin genes (e.g.

KRT5, KRT14). This approach has been used to investigate the role numerous cytokines (e.g.

IL-1a, IL-6, INF-g), growth factors (e.g. TGF-b, VEGF, keratinocyte growth factor), adhesion molecules (e.g. b1 integrins) and signal transduction elements (e.g. STAT3) [124,294–299]. The current best approach to study psoriasis in animals may still be humanization of mice through the use of transplanted human skin. These models, in which non-lesional and lesional psoriatic skin is transplanted into immunodeficient mice, are the closest to incorporate genetic, phenotypic and immunopathogenic processes of psoriasis.

Xenotransplantation models are also helpful tools in validating potential new drug targets [300]. However, these models have several limitations: they are technically very difficult, as they require multiple biopsies from patients, and grafting needs to be performed quickly to minimize graft ischemia [284]. Recently, a new model of psoriasiform skin inflammation induced with the topical use of imiquimod has been described [221]. In the next sections, the two in vivo models used in this study (i.e. the imiquimod model and a model of skin inflammation induced by tape-stripping) will be described in further detail.

3.1 Imiquimod model

Imiquimod (IMQ) is a ligand of the TLR7/8 and a potent immune activator, that is used for the topical treatment of genital warts caused by human papilloma virus, actinic keratosis and suprabasal cell carcinomas [301,302]. The anti-viral and anti-tumour efficacy of IMQ relies on its ability to activate the host immune system via activation of TLR expressed on monocytes, macrophages and pDC. TLR activation leads to the production of multiple pro-inflammatory cytokines and chemokines, recruitment of immune cells to the site of application and type I interferon-mediated anti-viral activity [303]. IMQ has been show to induce and exacerbate psoriatic lesions [304,305]. Van der Fits has recently proposed

repeated IMQ application as experimental model for psoriasis. Daily application of imiquimod-containing cream (5%) Aldara^TM, either to the ear or the shaved back skin for 5 or 6 consecutive days induces inflamed scaly lesions resembling plaque type psoriasis.

Moreover, these lesions showed a hyperplastic epidermis, increased keratinocyte proliferation, abnormal differentiation, epidermal accumulation of neutrophils in microabcesses, neoangiogenesis and infiltration of multiple immune cells, including T cells, DC, neutrophils and pDC, suggested phenotypic and histological features of psoriasis. In addition, IMQ application induces transient expression of IL-23, IL-17A, IL-17F and IL-22.

IL-23 and IL-17RA deficient mice are protected from IMQ-induced skin inflammation, suggesting a pivotal role of the IL-23/IL-17 axis in this model [221]. Gene expression analysis also showed that IMQ induces a pattern of significantly dysregulated genes similar to the one found in human psoriasis [306]. Importantly, IMQ-induced gene expression shifts are also consistent with other human skin conditions (e.g. wounds or infections) demonstrating that this model does not uniquely resemble psoriasis [306].

The mechanisms of action of Aldara^TM are complex, and several pathways have been reported to mediate immune activation and inflammation. The primary mode of action of imiquimod is the activation of TLR7 in mice [307] or TLR7 and TLR8 in humans [308]. Binding of IMQ to TLR mediates intracellular activation of several signaling pathways, including NF-kB, JNK, p38 MAPK, STAT1 and STAT3, leading to the transcription of pro-inflammatory molecules, including TNF-a, IL-6, IL-12, GM-CSF, IL-8 and INF-a [307,309,310].

IMQ may also exert broader activities in a TLR-independent manner, even though mechanisms are much less clear. IMQ can activate inflammasome pathway via NLRP3, leading to activation of caspase 1 and production of IL-1β and IL-18 [311], thus activating cells which might not express TLR7/8, such as keratinocytes [312]. In addition, IMQ was reported to act as an antagonist for adenosine receptors, significantly suppressing cAMP

levels [313]. At clinically relevant concentrations, IMQ leads to alterations in the expression of Bcl-2 family members, caspase activity, and causes release of mitochondrial cytochrome c in keratinocytes, all of which may contribute to increased rates of apoptosis and downstream inflammation [314,315].

The vehicle of Aldara^TM has also been reported to have some biological activity. Isostearic acid, one of the major components, induces inflammasome activation and apoptosis in keratinocytes, leading to pro-inflammatory cytokine production and early epidermal changes [316]. Nevertheless, full psoriasiform phenotype induced by Aldara™ is mainly dependent on imiquimod activation of MyD88 signaling [317,318].

In summary, the imiquimod model is a simple and convenient tool to study acute inflammation and dissect the early events during psoriasiform plaque formation. The major limitation of this model is the lack of chronicity and association with comorbidities, such PsA, which are present in a subset of psoriatic patients. Nevertheless, many of the phenotypic, histological and immunological features of the human disease appear to be also present, thus supporting the use of this model to study not only psoriasis but also other inflammatory skin diseases.

3.2 Tape stripping

Tape stripping is a technique that consists on subsequent removal of layers of the stratum corneum by using adhesive tape. The stratum corneum is the outermost layer of the epidermis. It acts as barrier, protecting the organisms against the entry of exogenous substances and loss of water. Therefore, this procedure has become a standard method to access the penetration of topically applied substances, the physiology of the stratum corneum and the regulation of the barrier recover during epidermal wound healing [319]. Additionally, tape stripping can be used as surrogate of mechanical cutaneous injury by scratching in

humans. In mice, tape stripping induces an increase in dermal and epidermal thickening, possibly due to a release of pro-inflammatory cytokines that induce keratinocyte proliferation.

Moreover, it also induces a strong inflammatory response with infiltration of immune cells in the dermis, particularly neutrophils [320,321]. This method has been used to study mild skin injury and inflammation, and to evoke disease in mouse models of atopic dermatitis and psoriasis [124,322,323].