The Epidermis as an Adjuvant - Core

REVIEW

The Epidermis as an Adjuvant Danuta Gutowska-Owsiak1 and Graham S. Ogg1 It is now clear that the epidermis has an active role in local immune responses in the skin. Keratinocytes are involved early in inflammation by providing first-line innate mechanisms and, in addition, can contribute to adaptive immune responses that may be associated with clinical disease. Moreover, keratinocytes are capable of enhancing and shaping the outcome of inflammation in response to stimuli and promoting particular types of immune bias. Through understanding the underlying mechanisms, the role of keratinocytes in disease pathogenesis will be further defined, which is likely to lead to the identification of potential targets for prophylactic or therapeutic intervention. Journal of Investigative Dermatology (2012) 132, 940–948; doi:10.1038/ jid.2011.398; published online 5 January 2012

INNATE RECOGNITION MECHANISMS OF KERATINOCYTES (KCs) The epidermis provides a structural barrier for many external challenges, yet is also able to provide active signals related to barrier compromise and distinguish different types of challenges. Through these mechanisms, KCs can significantly influence the resultant innate and adaptive immune responses. For example, KCs are known to express Fc receptors such as FcgRI, FcgRII, and FcgRIII (Tigalonowa et al., 1990; Cauza et al., 2002), as well as complement receptors, mannose receptor (Szolnoky et al., 2001), and other molecules that potentially aid internalization such as a5b1 fibronectin–binding integrin (Pellegrini et al., 1992). Binding to these results in effective endocytosis and subsequent killing of bacteria, fungi, and viruses, as documented for a variety of pathogens, including Staphylococcus aureus (Kisich et al., 2007), Candida albicans (Csato et al., 1986; Szolnoky et al., 2001), and Herpes simplex virus (Nicola et al., 2005). In addition, KCs express a spectrum of innate recognition receptors that can screen the external 1

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK Correspondence: Graham S. Ogg, MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, UK. E-mail: [email protected] Abbreviations: DC, dendritic cell; KC, keratinocyte; LC, Langerhans cell; NK, natural killer; TLR, Toll-like receptor; Treg, T-regulatory cell; TSLP, thymic stromal lymphopoietin.

Received 2 July 2011; revised 14 October 2011; accepted 24 October 2011; published online 5 January 2012

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environment and provide proinflammatory and chemotactic signals to immune cells in a case of pathogen invasion. Various studies have reported that KCs also express Tolllike receptors (TLRs) 1–6 and 10 (Baker et al., 2003; Mempel et al., 2003; Pivarcsi et al., 2003, 2004; Flacher et al., 2006; Lebre et al., 2007; de Koning et al., 2010), as well as TLR4associated CD14 and MD-2 proteins (Kollisch et al., 2005) in culture and in the skin. In addition, some studies also showed the presence of TLR7, 8, and 9 (Mempel et al., 2003; Miller et al., 2005; Flacher et al., 2006; Lebre et al., 2007) in these cells. Moreover, TLR expression can be further upregulated by certain pathogens, e.g., Mycobacteria (Pivarcsi et al., 2003; Lee et al., 2009), Malassezzia (Baroni et al., 2006), Propionibacterium (Jugeau et al., 2005), viral stimulation (Lebre et al., 2003; Kalali et al., 2008), and various pathogenderived products (Pivarcsi et al., 2003; Kobayashi et al., 2009). Cytokines such as transforming growth factor-a and IFN-a (Miller et al., 2005; Prens et al., 2008) also enhance TLR levels in KCs in vitro. In agreement with this, increased expression of certain TLRs has been observed in inflammatory and infectious skin diseases such as psoriasis, acne, atopic eczema, and Varicella–Zoster virus infection (Baker et al., 2003; Lai and Gallo, 2008). In addition to the TLRs, it has been recently documented that KCs express other receptors that recognize conserved molecular patterns, e.g., NOD-like receptors. Both NOD 1 and 2 seem to be constitutively present in these cells (Harder and Nunez, 2009; Kobayashi et al., 2009). NOD1 can be further upregulated under stimulation with cytokines, especially IFN-g (Harder and Nunez, 2009). Relevance of the pathway was additionally demonstrated by detection of IL-8 release from KCs during the response to infection with NOD1-activating bacteria, Pseudomonas aeruginosa (Harder and Nunez, 2009). Expression and function of NOD2 receptor in the epidermis was investigated in the context of antimicrobial function by Voss et al. (2006). This study demonstrated that the activation of the NOD2 pathway results in HBD-2 production (Voss et al., 2006). On the basis of a murine model, Kim et al. (2008) suggested that transforming growth factor–activated kinase 1 is an essential signaling molecule in the NOD2 pathway and important for maintenance of epidermal homeostasis (Omori et al., 2006). C-type lectins comprise another group of conserved pattern receptors that may have a role in epidermal recognition of microbes. In the steady state, KCs express low levels or appear negative for the expression of the bestcharacterized member of this group, dectin-1 (Lee et al., 2009; de Koning et al., 2010). However, the expression of dectin-1 is inducible in KCs on exposure to bacteria and the receptor aids pathogen internalization, as demonstrated in & 2012 The Society for Investigative Dermatology

D Gutowska-Owsiak and GS Ogg Role of Keratinocytes

Mycobacterium ulcerans infection (Kobayashi et al., 2009). The same study reported that the stimulation by b-glucan itself can also upregulate its own receptor (Kobayashi et al., 2009), which suggests a possibility that dectin-1 could be induced upon exposure to various fungal species. Very recently, KCs were also tested for the expression of members of the family of RIG-like helicase receptors that are able to detect viral single-stranded RNA and double-stranded RNA (Kalali et al., 2008). Kalali et al. (2008) demonstrated that human KCs constitutively express low levels of RIG-I, MDA-5, and protein kinase R and that these virus-sensing receptors can be upregulated by several-fold following exposure to poly I:C. The authors also highlighted the importance of protein kinase R as a central antiviral mechanism for double-stranded RNA stimulation, controlling a broad spectrum of pathways activated during viral invasion (Kalali et al., 2008). Inflammatory conditions are also likely to influence the recognition of double-stranded RNA by RIG-like helicase receptors in KCs, as demonstrated under IFN-g stimulation (Prens et al., 2008). Enhanced expression of molecules belonging to this group of pattern recognition receptors has been reported in various skin conditions (Prens et al., 2008; Zahn et al., 2011), which suggests relevance to the inflammatory state. In agreement with this, upregulation of the receptors under IFN-a/g stimulation in the in vitro model of psoriasis has been reported (Racz et al., 2011). Interestingly, this overexpression was demonstrated to diminish under UVB exposure (Racz et al., 2011), known to dampen the inflammation in psoriatic patients. Finally, the notion of RNF114, recently reported as a novel target gene in psoriasis by genome-wide association studies (Capon et al., 2008), as a regulator of RIG-I/MDA5 signaling in KCs, suggests that these viral sensors are key mediators during epithelial autoimmune inflammation (Bijlmakers et al., 2011). As shown above, considerable evidence points to the role of KCs in early detection of pathogens characterized by the presence of conserved molecular patterns, such as viral nucleic acids, lipopolysaccharide, flagellin, and zymosan, and so on. Sensing incoming threats enables the epidermis to react as the first line of response that involves, depending on the agonist, induction of chemotactic mediators, such as IL-8, CCL2, CCL20, CCL27, CXCL16, CXCL9, and CXCL10 (Pivarcsi et al., 2003; Kollisch et al., 2005; Miller et al., 2005; Lebre et al., 2007; Tohyama et al., 2007; Lee et al., 2009; Niebuhr et al., 2010). In addition, proinflammatory tumor necrosis factor (TNF)-a, type I IFN, IL-1a, IL-6, IL-18, defensins, cathelicidin (LL-37), and thymic stromal lymphopoietin (TSLP) secretion can be also observed (Lebre et al., 2007; Prens et al., 2008; Kinoshita et al., 2009; Kobayashi et al., 2009). These key mediators help promote recruitment of neutrophils, monocytes, dendritic cells (DCs), and various subsets of lymphocytes to the epidermis. Several studies have observed links between pathways of innate systems of microbial recognition in KCs. For example, dectin-1 expression can be modulated by TLR2 stimulation (Kobayashi et al., 2009; Lee et al., 2009). Moreover, very recently, Muller-Anstett et al. (2010) have also reported that

Staphylococcal peptidoglycan is able to stimulate both NOD2 and TLR2 receptor in mice. These examples indicate that there is a degree of cross-talk between innate pathways in KCs. It has also been reported that levels of TLR2, NOD-2, and dectin-1 are amplified by histamine stimulation (Kobayashi et al., 2009). This synergism between pattern recognition receptors and histamine suggests that there could be positive feedback between mechanisms of innate immunity and inflammation, especially during allergic-type responses in the skin. This could potentially result in an increased reactivity to pathogens, e.g., in the skin of atopic individuals. Finally, very intriguing data come from the recent study of Trompette et al. (2009), who revealed that apart from structural mimicry between MD-2 molecules and house dust mite antigen Der p2 (Derewenda et al., 2002; Kim et al., 2007; Ohto et al., 2007), there is also a strong functional similarity, resulting in nonspecific adjuvant-like effect of the antigen via innate immune pathways. Taken together, it is clear that KCs are well equipped in mechanisms that support detection of conserved molecules, which can be enhanced further as the cells encounter pathogens. Activation of these pathways results in cytokine and chemokine release, independent from epidermal or dermal immune cells. Moreover, any further interactions between the innate pathways of microbial recognition are likely to additionally enhance the induction of proinflammatory factors, which further supports adjuvant-like properties of the epidermis. This suggests that KCs have an important role during the innate phase of immune responses to invading pathogens as they react by an early wave of chemokine and cytokine release and attract key effector immune cells. DIRECT ACTIVATION OF ANTIGEN-SPECIFIC T CELLS Various studies documented that KCs do not normally express the co-stimulatory molecules CD80/86 (Black et al., 2007), and thus they are unlikely to prime naı¨ve T cells in a steady state (Gaspari et al., 1988; Otten et al., 1996; Laning et al., 1997). Consistently, early reports have indicated that classII–mediated presentation of antigen by KCs results in tolerance or anergy of T cells (Gaspari et al., 1988; Bal et al., 1990). However, the upregulation of molecules involved in the stimulation of T cells by KCs has been observed in multiple human skin diseases, which implies that during inflammatory states, KCs may be able to stimulate antigen-experienced T cells. However, most authors agree that they are unlikely to be able to prime new T-cell responses, a characteristic of conventional antigen-presenting cells (APCs) such as DCs. Specifically, it has been demonstrated that under certain inflammatory conditions, KCs can upregulate both major histocompatibility complex class I and II molecules (Basham et al., 1985; Bieber et al., 1989; Banno et al., 2003; Wittmann et al., 2005; Ardern-Jones et al., 2007), as well as various molecules required for effective major histocompatibility complex peptide loading (Albanesi et al., 1998). In addition, IL-4 exposure has been described to induce co-stimulatory molecules (Junghans et al., 1996), whereas stimulation with IFN-g and, to a lower extent TNF-a, www.jidonline.org

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promotes ICAM-1 expression (Little et al., 1998; Ardern-Jones et al., 2007; Black et al., 2007). The upregulation of ICAM-1 has been also observed when KCs were exposed to some sensitizing agents and oxidative stress (Little et al., 1998). ICAM-1 seems to be able to provide important co-stimulation (Gaglia et al., 2000). In agreement with this, KCs pretreated with IFN-g can efficiently stimulate CD4 þ antigen–specific T cells that have already encountered their cognate antigen (Mutis et al., 1993; Ardern-Jones et al., 2007; Black et al., 2007). Interestingly, Goodman et al. (1994) determined differences in cytokine secretion patterns between T cells stimulated by IFN-g–exposed KCs or peripheral blood mononuclear cells. These data demonstrated that when KCs are involved in stimulating T cells, a bias toward differential T-helper effector function can be observed. Lebre et al. (2007) reported that TLR stimulation of KCs extends further than the release of inflammatory mediators and attracting immune cells to the site of infection. According to this study, TLR ligands, namely poly I:C, flagellin, and lipopolysaccharide, can additionally upregulate expression of class I and class II major histocompatibility complex molecules, along with ICAM-1 and CD40, on KCs (Lebre et al., 2007). This further suggests the possibility of direct interaction of KCs and T cells during adaptive phase of immune response to common pathogens. The mechanisms that allow KCs to directly stimulate T cells have been investigated in the context of S. aureus exposure (Strange et al., 1994; Ardern-Jones et al., 2007). Although only a small percentage of the general population has been reported to be either colonized or infected by the bacteria, the prevalence increases to up to 90% in patients suffering from atopic skin disease (Goh et al., 1997; Chiu et al., 2009; Gomes et al., 2011). First, Strange et al. (1994) observed KC-mediated activation of allogeneic T cells in the presence of IFN-g and staphylo–enterotoxin B that was reduced by the addition of HLA-DR antibodies. However, as staphylo–enterotoxin B represents nonspecific superantigen-type reactivity, relevance of this finding to specific antigen-driven activation was difficult to assess. This problem has been subsequently resolved by Ardern-Jones et al. (2007). In this experimental system, KC-mediated presentation of house dust mite antigen Der p1 to specific CD4 þ T cells significantly increased when the cells were first pretreated with supernatant of staphylo–enterotoxin B–exposed peripheral blood mononuclear cell cultures (Ardern-Jones et al., 2007). The study showed that soluble factors (particularly IFN-g), released from immune cells following exposure to the bacteria, could greatly enhance the T-cell stimulation capabilities of KCs and result in adaptive immune responses to unrelated antigens (including allergens). Although this feature of KCs has only been documented in the context of S. aureus infection, it is plausible that the exposure to other species of either commensal or pathogenic flora could result in a similar switch of antigen-presenting function by KCs. In addition, it is possible that direct or indirect effects of KCs on regulatory T-cell induction or function may be important in influencing the eventual inflammatory milieu, but this has been relatively understudied (Figure 1). 942


UVB

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Figure 1. Direct and indirect effects of keratinocytes on innate and adaptive immune responses. DC, dendritic cell; LC, Langerhans cell; NK, natural killer; Treg, T-regulatory cell; TSLP, thymic stromal lymphopoietin.

NONCLASSICAL PRESENTATION BY KCs Apart from involvement in the classical presentation of peptides, KCs could also be capable of facilitating nonclassical pathways, such as presentation of glycolipids via CD1 molecules. The expression of CD1d in normal skin is largely limited to cells of the upper epidermal layers and hair follicle (Bonish et al., 2000; Adly et al., 2005; Fishelevich et al., 2006). However, CD1d is overexpressed (Bonish et al., 2000; Zhao et al., 2008) and can also be present in lower strata in psoriatic skin, which seems to correspond to the observed in vitro effect of IFN-g on its expression (Bonish et al., 2000). Bonish et al. (2000) have also documented that CD1d could be induced in normal KCs by mechanical trauma (tape stripping) and, interestingly, by exposure to poison ivy. CD1d-expressing cultured KCs are able to activate natural killer T cells for cytokine release in vitro, particularly when exposed to IFN-g. This indicates that these cells are potentially capable of presentation of lipid-derived ligands, in the context of both infection and autoimmune inflammation. In contrast, however, despite close proximity, KCs were not able to directly stimulate natural killer T cells during allergic contact dermatitis (Gober et al., 2008). Keratinocytes of the stratified epidermis were also demonstrated to express CD1a, especially in suprabasal regions of atopic skin (Bieber et al., 1989; Furue et al., 1992), but it is not clear whether this represents de novo KC synthesis or acquisition of CD1a from neighboring Langerhans cells. Interestingly, this molecule seems to be a potential target for the newly reported Th22 cells that express a


skin-homing phenotype (Duhen et al., 2009). Although it has been shown that this population is able to respond to the CD1a-mediated presentation by DCs (de Jong et al., 2010), whether these KCs are also able to activate/prime this population remains currently unknown. The expression and functional relevance of stress-induced HLA-related antigens have not been extensively studied in the epidermis. Reports showed that MICA is expressed by human KCs (Zwirner et al., 1998, 1999). In a small study, Molinero et al. (2003) has documented that the expression of this molecule is increased in seborrheic dermatitis. Interestingly, the genetic association with MICA locus has been identified in psoriasis (Zwirner et al., 1998; Romphruk et al., 2004; Pollock et al., 2011). Although these studies suggest an involvement of the molecule in inflammatory process in the skin, the cellular contribution has not yet been identified. KCs CAN INDIRECTLY INFLUENCE IMMUNE RESPONSES Apart from any direct induction of T-cell effector function, activated KCs seem to be also involved in the generation of adaptive responses by affecting APCs in the skin. Initial suggestions that these cells may indeed have a role in the process came from the study by Pastore et al. (1997), who documented the promoting influence of KC-derived GM-CSF on the differentiation of monocytes into DCs and their ability to stimulate naı¨ve T cells. There are some conflicting data regarding the effect of house dust mite antigens on the induction of GM-CSF and IL-8. One study suggested that in contrast to cells of bronchial epithelium, KCs do not express these cytokines (Mascia et al., 2002). However, the release of GM-CSF from KCs has been observed following exposure to Der p1 and Der f1 antigens (Ogawa et al., 2008). Activation of pattern recognition pathways due to stimulation with bacterial products results in the induction of chemokines and cytokines in KCs, as mentioned earlier in this review. In addition, synthesis of the inflammatory derivatives of arachidonic acid is also increased (Eberhard et al., 2002). Exposure of KCs to various Malassezia species also induces a broad spectrum of mediators, including proinflammatory, Th2- and Th17-promoting cytokines (Watanabe et al., 2001; Ishibashi et al., 2006, 2011; Thomas et al., 2008); the profile of response varies depending on the species. Interestingly, the direct interaction involving contact of KCs and fungal cells is required, as Malassezia culture supernatant did not mediate the effect (Watanabe et al., 2001). Moreover, the secretion seems to be enhanced if the yeast is decapsulated (Thomas et al., 2008). Although IL-1a is constitutively produced by KCs (Luger et al., 1981; Sauder et al., 1982; Kupper et al., 1986; Little et al., 1998), it can be further upregulated in response to colonization with Malassezia or bacterial species: Staphylococcus and Propionibacterium (Walters et al., 1995), as well as following epidermal barrier disruption (Wood et al., 1996). Early reports suggested that IL-1a represents the majority or all of IL-1 activity derived from KCs owing to the inability of these cells to effectively process pro-IL-1b into the active form (Kupper et al., 1986; Cooper et al., 1990;

McKenzie et al., 1990; Mizutani et al., 1991). However, others reported the release of IL-1b form by these cells (Watanabe et al., 2001). The functional effects of IL-1 in the skin are broad as many cell types express IL-1 receptors. For example, murine models imply that IL-1a modulates class II expression and function of Langerhans’ cells (Lundqvist and Back, 1990). In addition, overexpression of IL-1a in the epidermis seems to have a significant effect on the recruitment of immune cells, as well as enhancing both T-cell and antibody responses to vaccinia virus (Tian et al., 2008). Moreover, it has been also reported that the epicutaneous sensitization in animal model of contact hypersensitivity is largely dependent on this cytokine, which is required for the activation of contact allergen–specific T cells (Nakae et al., 2001). Keratinocytes also express TNF-a, which seems to be further induced by exposure to lipopolysaccharide (Kock et al., 1990) and UVB (Kock et al., 1990; Schwarz et al., 1995). Early murine studies by Kimber and Cumberbatch, (1992) reported the effect of TNF-a on induction of Langerhans cell migration and their accumulation in the lymph nodes (Cumberbatch and Kimber, 1995). This was also confirmed later by Stoitzner et al. (1999) who detected similar augmentation of the migratory properties in both Langerhans and dermal DCs. More recently, this cytokine has been reported to be involved in the generation of immature Langerhans’ cells from CD34 þ precursors (Arrighi et al., 2003). The role of the cytokine during inflammatory process in the skin has been well documented (Kristensen et al., 1993; Ettehadi et al., 1994; Junghans et al., 1998; Kerstan et al., 2011) and further supported by clinical effectiveness of the anti-TNF treatment in psoriasis (Leonardi et al., 2003; Papp et al., 2005; Marble et al., 2007; Tyring et al., 2007; van de Kerkhof et al., 2008). In 2003, Lebre et al. (2003) investigated the indirect influence of KCs on DC maturation and T-cell polarization in the context of antiviral responses. The study showed that KCs that sense the presence of viral products are able to induce DC maturation in a TNF-a/IL-1b–dependent mode (Lebre et al., 2003). Furthermore, they have also documented that virally activated KCs acquire the ability to indirectly influence the function of activated T cells. Specifically, the presence of type I IFNs and IL-18 in the supernatants from cultures of double-stranded RNA-stimulated KCs influenced IL-12p70 secretion from immature DCs, which in turn induced strong Th1 bias in T cells during their priming (Lebre et al., 2003). A study by Soumelis et al. (2002) showed that TSLP exerts multiple effects on peripheral blood–isolated DCs during their activation and maturation, as well as promotes their survival. Specifically, under TSLP stimulation, DCs produced high levels of CCL17 (TARC) and CCL22 that attract CCR4expressing Th2 cells to the site of inflammation. Interestingly, T cells that were primed by TSLP-stimulated DCs produce a distinct spectrum of cytokines characterized by Th2 bias (including IL-13, IL-5 and IL-4) and increased TNF-a levels. In contrast, this type of stimulation resulted in a reduced release of Th1/Th1-promoting cytokines: IFN-g, IL-12p70, as well as IL-1b and the regulatory IL-10 (Soumelis et al., 2002). As the www.jidonline.org

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authors observed high expression of TSLP in KCs, particularly in atopic dermatitis, as well as in activated mast cells, these findings are particularly relevant to the Th2-type responses in allergic skin inflammation. Subsequently, additional groups have reported findings that support the role of KC-derived TSLP as a Th2-promoting cytokine in the skin. Ito et al. (2005) determined a critical role of OX40 ligation in the TSLP-mediated instructing of T cells to become Th2 committed. Subsequently, Wang et al. (2006) published a study that suggested that TSLP-stimulated bloodderived DCs are involved in inducing Th2 bias not only during priming of naı¨ve cells but also during the maintenance phase of central memory cells and further T-cell polarization. In the following year, Ebner et al. (2007) brought the context directly to the skin when they investigated the effects of the cytokine on freshly isolated Langerhans cells and reported very similar findings; Langerhans cell survival, proliferation, and maturation were enhanced under the stimulation. In addition, the profile of secreted cytokines was parallel to those that Soumelis et al. (2002) observed for the peripheral blood DCs, which suggests that the effect of TSLP is not limited to a given antigen-presenting subset in the blood, but that potentially also APCs in the skin can induce Th2-skewed polarization of naı¨ve T cells. As an additional finding, Ebner et al. (2007) also presented direct evidence implying enhanced migratory properties of Langerhans cells under TSLP stimulation. However, TSLP-mediated impact on innate cells has also been recognized. The study published by Allakhverdi et al. (2007) determined its role in direct activation and induction of proinflammatory and Th2 cytokines, as well as in the release of chemokines from mast cells. The magnitude of the adaptive responses in the skin is also likely to be altered by the milieu that influences KCs. Indeed, this was shown a few years later by Kinoshita et al. (2009) who studied the effect of various cytokines on the TLSP release from KCs following TLR3 stimulation. This study demonstrated an enhancing effect of IL-4, IL-13, type I IFNs, and TNF-a, as well as a downregulating influence of IFN-g, transforming growth factor-b, or IL-17 on the expression and release of this cytokine. Moreover, the integrity of the epidermis seems to be an important factor in the induction of TSLP under TLR3 stimulation as well, as recently documented by Lee et al. (2011) in a filaggrin knockdown model of viral infection. These two studies imply an existence of a positive feedback loop between viral infections and compromised integrity of epidermal barrier on one side and allergic skin inflammation on the other. Another epidermis-derived cytokine that seems to influence antigen-specific adaptive responses is a recently characterized member of the IL-17 family, IL-25 (IL-17E). Keratinocytes have been shown to constitutively express IL25 transcript and protein (Wang et al., 2007; Corrigan et al., 2011). It has been documented in animal studies that stimulation with the cytokine also results in the promotion of Th2 responses (Fort et al., 2001; Pan et al., 2001; Angkasekwinai et al., 2007). Recently, it has been proposed that it is an indirect mechanism and that the cytokine functions by enhancing the Th2 responses driven by TSLP944


stimulated APCs (Wang et al., 2007), a possible explanation of the induction of the cytokine bias observed in mice. Finally, a novel IL-1-like cytokine, the ‘‘alarmin’’ IL-33, has been detected as a constitutively expressed product present in KCs (Moussion et al., 2008) and upregulated in atopic and psoriatic plaques. The cytokine also seems to have similar functional properties as it seems to promote mainly proinflammatory and Th2-type responses from a spectrum of various cell populations, including antigen-specific–committed Th2 cells (Schmitz et al., 2005; Smithgall et al., 2008). Moreover, it promotes survival of mast cells and, interestingly, their activation independent of IgE cross-linking (Iikura et al., 2007; Hueber et al., 2011). It is also plausible that the aforementioned cytokines also directly affect APCs in the skin, as well as other cells including nuocytes, a novel population of immune cells that seems to respond to IL-25 and IL-33 stimulation by secretion of Th2 cytokines (Neill et al., 2010). These possibilities, however, remain to be formally proven. Keratinocytes are also capable of directing the response toward a Th1-type bias, by inducing IFN-g release from T cells, monocytes and macrophages. For example, IL-18, also constitutively expressed by KCs of the basal epidermal layer and upregulated in psoriatic skin possesses this property (Kohka et al., 1998; Naik et al., 1999). Moreover, the cytokine has been reported to influence Langerhans cell migration in mouse models of contact hypersensitivity (Cumberbatch et al., 2001; Plitz et al., 2003). Taken together, these findings suggest that KCs are a potent source of cytokines that target immune cells, leading to the amplification of local innate and adaptive inflammatory responses. IMPLICATIONS FOR VACCINE DEVELOPMENT As discussed above, KCs are not just bystanders, but immunologically active cells that are able to participate at various stages of the immune response. In fact, the properties of the epidermis described above, including an ability to augment inflammatory state and modify its direction, are also the properties that are characteristics of adjuvants. This carries some important implications for the generation of vaccination strategies, especially regarding the feasibility of relatively recently proposed transcutaneous immunization. Differential antigen delivery can significantly influence the resultant immune response (Estcourt et al., 2005). Human studies documented that immunogenicity against enterotoxigenic strains of Escherichia coli (Glenn et al., 2000, 2007; Guerena-Burgueno et al., 2002; Yu et al., 2002; Frech et al., 2008), flu (Van Kampen et al., 2005), and measles (Etchart et al., 2007) could be induced by transcutaneous immunization. In murine models, efficacy has been reported against tetanus (Shi et al., 2001; Ishii et al., 2008; Matsuo et al., 2011), diphtheria (Matsuo et al., 2011), Japanese encephalitis virus (Cheng et al., 2009), adenovirus (Ishii et al., 2008) and various flu strains (Guebre-Xabier et al., 2004; Garg et al., 2007; Mkrtichyan et al., 2008). The efficacy in inducing systemic humoral and cellular responses has been proven (Guerena-Burgueno et al., 2002;


Yu et al., 2002; Matsuo et al., 2011). In addition, delivery of antigens via such a formulation has been shown to enhance Langerhans cell migration to the draining lymph nodes (Ishii et al., 2008). Specific-IgG titers seem to correlate with measured transepidermal water loss after barrier-disrupting treatment preceding application of the patch (Glenn et al., 2007). Although one can argue that this is a result of increased absorption of active vaccine constituents (antigens, pharmaceutical adjuvants) through the epidermis into dermal layer, it can also be related to the enhanced antigen presentation and secretory capacity of mechanically stimulated KCs. GuebreXabier et al. (2003) have defined a synergistic role of a transepidermal patch (used as an immunostimulant) and subcutaneous injection in mice. This was also reproduced in a human influenza trial in elderly patients (Frech et al., 2005). It is difficult to assess how much of the in vivo systemic effect induced by the patches in the aforementioned studies is due to the adjuvant role of KCs as compared with other cells of the skin. An intriguing study has been also recently published by Liu et al. (2010). The authors of this work aimed to compare the immunogenicity of an antigen administered through the superficially injured skin (skin scarification) with other commonly used delivery routes. Interestingly, they have observed superior efficacy of this method of administration in the induction of both T- and B-cell responses to poxviruses, as well as in the cutaneous melanoma challenge in mice. Certainly, as KCs have the ability to potentiate stimulation of inflammatory pathways, the epidermis could contribute to the modulation of the observed responses, supporting its adjuvant role. In summary, the epidermis is a rich source of cytokines and chemokines that can influence the innate and adaptive immune responses. It is clear that KCs can directly interact with immune cells and indirectly influence function through effects on cutaneous DCs. The underlying mechanisms will be important in understanding disease pathogenesis, as well as informing new approaches for antigen delivery through the skin for vaccination or therapy. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We are grateful for funding from the MRC and the NIHR Biomedical Research Centre Programme.

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