Jürgen Schauber, MD, Department of Dermatology and Allergy, Ludwig-Maximilian University, Frauenlobstr. 9-11, 80337 Munich, Germany, Tel.: +49 (0)89 5160 6388/6010, Fax: +49 (0)89 5160 6389, e-mail: email@example.com
Abstract: Epidermal keratinocytes produce and secrete antimicrobial peptides (AMPs) that subsequently form a chemical shield on the skin surface. Cathelicidins are one family of AMPs in skin with various further immune functions. Consequently, dysfunction of these peptides has been implicated in the pathogenesis of inflammatory skin disease. In particular, the cathelicidin LL-37 is overexpressed in inflamed skin in psoriasis, binds to extracellular self-DNA released from dying cells and converts self-DNA in a potent stimulus for plasmacytoid dendritic cells (pDCs). Subsequently, pDCs secrete type I interferons and trigger an auto-inflammatory cascade. Paradoxically, therapies targeting the vitamin D pathway such as vitamin D analogues or UVB phototherapy ameliorate cutaneous inflammation in psoriasis but strongly induce cathelicidin expression in skin at the same time. Current evidence now suggests that self-DNA present in the cytosol of keratinocytes is also pro-inflammatory active and triggers IL-1β secretion in psoriatic lesions through the AIM2 inflammasome. This time, however, binding of LL-37 to self-DNA neutralizes DNA-mediated inflammation. Hence, cathelicidin LL-37 shows contrasting roles in skin inflammation in psoriasis and might serve as a target for novel therapies for this chronic skin disease.
Antimicrobial peptides: effectors of innate immunity in the skin
One of the skin’s most important functions is to protect our body from hazardous environmental stresses such as microbial pathogens. Various cells exert innate immune functions in the skin: Keratinocytes form a physical and chemical barrier (e.g. with the stratum corneum), and several cells of the innate immune system are present in the skin (e.g. macrophages and neutrophils). In addition, the secretion of small antimicrobial peptides (AMPs) by immune and epithelial cells has been recognized as an essential part of the skin’s primary defense mechanisms (1,2). AMPs are produced by resident skin cells such as keratinocytes, sebocytes and eccrine gland cells (3). In addition, infiltrating immune cells such as neutrophils and natural killer cells contribute to the pool of AMPs in the skin (4–6). AMPs are a very heterogeneous group of peptides that were initially classified according to their antimicrobial activity rather than structural characteristics. To date, several dozens of different cutaneous AMPs are known with the defensins and the cathelicidins as the best studied AMP families (2,7–9).
While some AMPs are expressed constitutively in the skin, the production of others is highly increased in danger situations such as skin injury or infection (10–13). The expression and functions of AMPs are both regulated on the transcriptional and posttranscriptional level. Most AMPs are synthesized as pro-peptides and are activated after proteolytic cleavage from their precursor molecules (14,15). Their biological activity is therefore strongly dependent on the local expression and function of appropriate proteases. This is especially important as most AMPs are positively charged and have membrane interacting activities (including mammalian membranes), and high concentrations could have deleterious effects.
Many AMPs were initially identified for their role in killing microbes and building a chemical shield on the skin. However, in recent years, it became clear that many AMPs are more than sole endogenous antibiotics but also triggers and co-ordinators of innate and adaptive immune reactions (16,17). Based on these additional immune functions, some AMPs were designated ‘alarmins’ to appreciate their role in initiating immune reactions (18). As disturbed production and/or functions of the AMPs could either result in a diminished antimicrobial barrier or could have damaging effects on the epithelial cells, the coordinated control of expression and activation is mandatory.
Cathelicidins are one major family of AMPs in the skin and were among the first mammalian AMPs identified (19,20). In humans, only one cathelicidin has been found and the human cathelicidin gene CAMP locates on chromosome 3 (21). Similar to most AMPs, cathelicidin is synthesized as a pro-peptide that is composed of a conserved N-terminal cathelin domain of approximately 100 amino acids and a C-terminal 37 amino acid sequence with antimicrobial activity. To generate this active cathelicidin peptide, a 37 amino acid residue (LL-37) is cleaved from the precursor by serine proteases of the kallikrein family (14,22). Further processing of LL-37 by the kallikreins leads to smaller cathelicidin peptide fragments with different biological functions (23). In healthy skin, cathelicidin expression is barely detectable. Positive immunostaining can only be found in neutrophil granules and lamellar bodies in keratinocytes (24). However, upon wounding, infection or inflammation cathelicidin expression is strongly induced (19,25–27). Then keratinocytes serve as a major source of cathelicidin and invading neutrophils carry additional loads of cathelicidin to the site of infection.
The mechanisms of cathelicidin regulation in human cells remained unclear for a long time. Eventually, the vitamin D pathway was identified as a regulator of cathelicidin expression in man (28). Vitamin D-responsive elements in the promoter region of the cathelicidin gene were later identified, and subsequent analyses revealed further elements in the vitamin D-mediated cathelicidin expression regulation such as histone acetylation, the role of vitamin D receptor co-activators and the synergistic actions of vitamin D and IL-17 through Act1 and MEK-ERK (10,29,30). The role of vitamin D in the regulation of cathelicidin expression was a surprising result as in conditions with rapid cathelicidin induction, such as skin injury, no sudden change in systemic vitamin D levels was expected. To understand this puzzling observation, one needs to know that vitamin D is activated by multiple hydroxylation steps involving different enzymes: UVB irradiation induces a photochemical process in which the vitamin D precursor 7-dehydrocholesterol (provitamin D3) is converted to calciol (previtamin D3) in the basal and suprabasal layers of the skin (31). Subsequently, in the liver, vitamin D 25-hydroxylase (CYP27A1) hydroxylates calciol to calcidiol and further hydroxylation in the kidney by 25-hydroxyvitamin D3 1-α-hydroxylase (CYP27B1) activates calcidiol to 1,25-dihydroxyvitamin D3 (calcitriol) (32). As keratinocytes express both CYP27A1 and CYP27B1, these cells are able to produce active calcitriol independent of renal and hepatic hydroxylation steps (33). Also, the amount of calcitriol produced by keratinocytes is sufficient to induce cathelicidin in an autocrine loop (34). Furthermore, the expression of keratinocytic CYP27B1 is under control of inflammatory regulators that are active in skin injury or infection such as TGFβ or triggers of Toll-like receptors (TLR) signalling. Thus, CYP27B1 can be activated under these conditions leading to locally increased vitamin D synthesis and subsequent activation of vitamin D-dependent genes such as cathelicidin (12,35).
Structurally, the resulting human cathelicidin peptide LL-37 forms an amphipathic, cationic α-helix in aqueous solutions that permits the incorporation into lipid bilayers, which leads to disruption of microbial membranes, viral envelopes and some fungal structures (36,37). The structure and charge of the cathelicidin peptides is relevant for their antimicrobial but also their immune functions such as cell surface receptor interaction: As an example, LL-37 exerts ‘alarmin’ functions by activating immune cells via the formyl-peptide-like receptor-1 (FPRL-1), G-protein-coupled receptors, the nucleotide receptor P2X7 and TLR signalling (38–40). Additionally, EGF receptor transactivation in keratinocytes, intracellular Ca2+ mobilization as well as induction of chemokine release initiating immune cell migration and neovascularization are known alarmin functions of LL-37 (17,41,42). In this context, LL-37 supports the activities of various cytokines (16,43). Interestingly, the antimicrobial and alarmin functions of cathelicidin peptides such as LL-37 are influenced by the proteolytic processing of the mature peptide, and some LL-37-derived peptide fragments have greater antimicrobial but less immuno-stimulatory capacity (14,44).
The role of cathelicidin in psoriasis pathogenesis
Psoriasis is a common chronic T-cell driven autoimmune skin disease whose pathophysiology is only partly understood (10,45). Approximately, 2% of the general population suffer from psoriasis vulgaris (PSV), the most common form of psoriasis. There are some ethnic differences in prevalence of psoriasis: Japanese have the lowest incidence of psoriasis (0.2%) while inhabitants of the Faroe islands have the highest (2.8%) (46). Clinically, PSV is characterized by red, scaly, raised plaques primarily on the elbows, knees and the scalp. Histologically, because of increased keratinocyte proliferation, the epidermis shows acanthosis, the granular layer is reduced, the rete ridges are elongated, the cornified layer increased (hyperkeratosis) and immune cell infiltrates are present in the dermis and epidermis (45). Genetic factors are strongly involved in psoriasis pathogenesis, and several susceptibility loci have been identified so far (47). Furthermore, psoriasis is associated with different comorbidities such as cardiovascular disease, psoriasis arthritis and depression, and patients suffering from psoriasis often need lifelong treatment.
The initial trigger for the onset of psoriasis remained unknown, but environmental factors play an important role, such as trauma, infection and stress (48). In inflamed skin in psoriasis, dermal dendritic cells and plasmacytoid dendritic cells (pDC) are activated in early stages of the disease and can in turn activate antigen-specific T cells in the draining lymph nodes. Still, the psoriasis-specific autoantigen has not been identified. Activated T cells expressing the homing receptor cutaneous lymphocyte antigen (CLA) then migrate into the epidermis and contribute – together with local cell types – to the specific cytokine micromilieu in the primary psoriatic plaque (49). This psoriasis cytokine milieu is dominated by IFNα secreted from pDCs and IFNγ and TNFα from innate and adaptive immune cells (48).
Physiologically, pDCs sense viral nucleic acids through activation of intracellular TLR 7 and 9 and initiate protective immunity against viruses through the production of type I IFN. Normally, host-derived (self) nucleic acids released by dying cells do not access intracellular TLR7 and 9 compartments. However, in psoriasis, Lande et al. (50) could show that self-nucleic acids gain access to intracellular TLR compartments to trigger high levels of IFNs in pDC when complexed with endogenous cathelicidin peptide LL-37: As a mechanisms, in lesional skin in psoriasis, anionic self-DNA was found complexed to the cationic cathelicidin peptide LL-37, and the LL-37/self-DNA-complex is able to enter pDCs by lipid raft-mediated endocytosis thereby initiating a TLR9/MyD88/IRF7-mediated IFNα response (51,52) (Fig. 1). IFNα in turn drives the following immune reaction by activating self-reactive T cells, which finally results in the formation of a psoriatic lesion (50). In addition, the same authors could then show that LL-37 can also transform self-RNA into a potent stimulus for myeloid dendritic cells (mDC) activation via TLR7 and 8 by a similar mechanism (53). Thus, in psoriasis, extracellular DNA and RNA released from dying cells together with increased LL-37 creates a pro-inflammatory trigger initiating and amplifying cutaneous inflammation.
The cathelicidin paradox in psoriasis
Still, these mechanisms of LL-37 mediated auto-inflammation stand in contrast to long established clinical observations: Topical treatment of PSV patients with vitamin D analogues induces cathelicidin expression in skin and in cultured human keratinocytes, vitamin D analogues increase cathelicidin expression (54,55). At the same time, vitamin D analogues decrease skin inflammation and the production of several key cytokines such as IL-17 (54). Furthermore, narrow-band UVB irradiation ameliorates psoriatic skin inflammation but induces endogenous vitamin D metabolism and subsequent cathelicidin production in epidermal keratinocytes (34,54,56). Thus, anti-inflammatory treatments targeting the vitamin D receptor are associated with increased cathelicidin in psoriatic lesions.
In an effort to elucidate this paradox, further active pro-inflammatory pathways in psoriatic keratinocytes were investigated and IL-1β was found to be strongly increased in involved skin in psoriasis patients (57). IL-1β is another key player in psoriasis pathogenesis and smaller trials targeting IL-1β showed benefits in a subset of PSV patients (58,59). As a mechanism, several studies suggest that IL-1β enhances pathogenic Th17 cell maturation and cytokine production (60,61). Among other cells in skin, epidermal cells secrete IL-1β, and IL-1β produced by keratinocytes fosters further T-cell immune responses (62).
In contrast to other pro-inflammatory signals, IL-1β is transcribed as an immature pro-peptide. Maturation of IL-1β into its biologically active form is then mediated by proteolytic cleavage of the pro-peptide by caspase 1 (63). The trigger for caspase 1 activation is the formation of so called ‘inflammasomes’– multiprotein aggregates that form upon danger-associated or pathogen-associated molecular pattern recognition (64). Several different inflammasome complexes and their triggers are known, and in involved skin in psoriasis, increased caspase 1 activity is found suggesting inflammasome activation (57,64–66).
In a recent study, the expression of the AIM2 inflammasome was found strongly induced in lesional skin in psoriasis patients (57). AIM2 is a cytosolic receptor for double-stranded (ds) DNA and forms an inflammasome after sensing cytosolic DNA (67–69). Unexpectedly, when possible triggers for inflammasome activation in psoriatic skin were investigated, cytosolic DNA in keratinocytes in psoriatic lesions was also detected (57). Physiologically, DNA is compartmentalized in the nucleus or mitochondria but not found in the cytosol. Epidermal barrier disruption, however, triggered the presence of cytosolic DNA and could explain why physical trauma induces skin inflammation in psoriasis patients (58). These clinical observations were then confirmed in vitro when cytosolic dsDNA-activated IL-1β release through the AIM2 inflammasome in primary human keratinocytes (57).
As cathelicidin LL-37 plays a role in psoriatic skin inflammation, forms complexes with nucleic acids and promotes their uptake, it was then tested whether LL-37-mediated uptake of DNA contributes to inflammasome activation (70). Surprisingly, when LL-37 and DNA were delivered together into keratinocytes, IL-1β secretion was completely abolished indicating inhibition of AIM2 inflammasome activity by LL-37. These observations suggested that LL-37 is able to translocate into the cytosol of psoriatic keratinocytes and specifically neutralizes cytosolic DNA thus acting as a physiologic inhibitor of AIM2 activation (Fig. 2).
Summary and perspectives
Eventually, the increasing knowledge of the role of cathelicidin peptide LL-37 in psoriasis pathogenesis and the factors controlling cathelicidin expression in skin could lead to novel therapies (or explain the effect of current therapies): Established therapies for psoriasis include UVB radiation and/or topical treatment with vitamin D analogues, and these pathways directly regulate cathelicidin expression in keratinocytes. Increased epidermal cathelicidin LL-37 could then bind to cytosolic DNA in keratinocytes and block AIM2 inflammasome activation and IL-1β secretion. Before therapy, neutralization of inflammasome activation by binding of epidermal LL-37 to cytosolic DNA is obviously not enough to prevent inflammation in the in vivo situation in psoriatic patients. Thus, specific targeting of these vitamin D-dependent pathways might be a useful strategy to exploit the anti-inflammatory functions of epidermal cathelicidin LL-37 in lesional skin in psoriasis.
At the same time, dermal DNA/LL-37 and RNA/LL-37 complexes that form triggers for pDC activation and the secretion of INFα could serve as another treatment target. These complexes bind to TLRs (TLR7-9) and activate subsequent pro-inflammatory pathways. Inhibition of LL-37-dependent pDC activation in the dermis of lesional skin through, for example, TLR blockade or activation of endogenous TLR pathway inhibitors could further ameliorate cutaneous inflammation in psoriasis.
Possible approaches to target both anti- and pro-inflammatory functions of cathelicidin LL-37 in the pathogenesis of psoriasis definitively warrant further investigation, and it is hoped that it will lead to novel treatment options for this chronic skin disease.