Interleukin-17A and Keratinocytes in Psoriasis

The excellent clinical efficacy of anti-interleukin 17A (IL-17A) biologics on psoriasis indicates a crucial pathogenic role of IL-17A in this autoinflammatory skin disease. IL-17A accelerates the proliferation of epidermal keratinocytes. Keratinocytes produce a myriad of antimicrobial peptides and chemokines, such as CXCL1, CXCL2, CXCL8, and CCL20. Antimicrobial peptides enhance skin inflammation. IL-17A is capable of upregulating the production of these chemokines and antimicrobial peptides in keratinocytes. CXCL1, CXCL2, and CXCL8 recruit neutrophils and CCL20 chemoattracts IL-17A-producing CCR6+ immune cells, which further contributes to forming an IL-17A-rich milieu. This feed-forward pathogenic process results in characteristic histopathological features, such as epidermal hyperproliferation, intraepidermal neutrophilic microabscess, and dermal CCR6+ cell infiltration. In this review, we focus on IL-17A and keratinocyte interaction regarding psoriasis pathogenesis.

In addition, various murine psoriasis models stress a pivotal role of the IL-23/IL-17A axis in experimental psoriasis [76][77][78][79][80]. Multiple animal studies have indicated that the interaction between IL-17A and keratinocyte is the key issue in the development of psoriasis [76][77][78][79]. In parallel, the importance of IL-17A and keratinocyte interaction is reinforced in patients with psoriasis who are successfully treated with the anti-IL-17A biologic [69]. In this review, we will focus on the multifaceted biological response in keratinocytes stimulated by IL-17A with regard to psoriatic pathogenesis.
In addition to the above-mentioned signaling cascades, IL-17A activates various other signal molecules including signal transducer and activator of transcription 3 (STAT3) in keratinocytes [100]. STAT3 is a very crucial signaling molecule in the development of psoriasis because transgenic mice with keratinocytes expressing a constitutively active Stat3 (K5.Stat3C mice) develop a skin phenotype either spontaneously, or in response to wounding, that closely resembles psoriasis [101]. Moreover, a STAT3 inhibitor STA-21 inhibits the generation of skin lesion in these psoriatic mice [102]. IL-17A is known to activate STAT3 via receptor-interacting protein 4 (RIP4) activation and upregulates the CCL20 expression [103]. IL-17A also upregulates keratin 17 expression via STAT1 and STAT3 activation [104]. IL-6 and IL-22 also play a synergistic role in development of psoriasis with IL-17A [68]. Notably, both IL-6 and IL-22 are potent STAT3 activators [105]. In accordance, biological or natural molecules such as indirubin and its derivatives useful for inactivating STAT3 exhibit therapeutic potential for psoriasis [106] (Figure 2). It reveals that IL-17 and IL-22 promote The ligation of IL-17RA/IL-17RC activates epidermal growth factor receptor (EGFR) directly or by transforming growth factor-α (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) and promotes keratinocyte proliferation. The ligation of IL-17RA/IL-17RC activates various signal transduction molecules, including ERK, p38 MAPK, JNK, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), IκBζ, C/CAAT-enhancer-binding protein β (C/EBPβ), and C/EBPδ. In contrast, the ligation of IL-17RA/IL-17RD preferentially activates JNK and p38 MAPK pathways. IL-17RA/IL-17RD is estimated to transactivate fibroblast growth factor receptor (FGFR); however, this is not conclusive. IL-17RA/IL-17RC signaling stimulates KCs to produce IL-19, which induces the production of keratinocyte growth factor (KGF) from fibroblasts. KGF also enhances the proliferation of KCs. IL-17A also induces the production of antimicrobial peptides, including S100A7, S100A8, S100A9, LL-37, and defensin β 4A (DEFB4A). These antimicrobial peptides amplify the local inflammatory process. Chemokines, such as CCL20, CXCL1, and CXCL8, are also produced from keratinocytes by IL-17RA/IL-17RC ligation. CCL20 is a key chemokine for the recruitment of CCR6 + Th17 cells and group 3 innate lymphoid cells (ILC3). These CCR6 + cells produce large amounts of IL-17A. DEFB4A also exhibits a chemotactic activity by binding to CCR6. CXCL1 and CXCL2 are potent chemoattractants for CXCR2 + neutrophils. Therefore, IL-17A is associated with all of the histopathologic features of psoriasis.
In addition to the above-mentioned signaling cascades, IL-17A activates various other signal molecules including signal transducer and activator of transcription 3 (STAT3) in keratinocytes [100]. STAT3 is a very crucial signaling molecule in the development of psoriasis because transgenic mice with keratinocytes expressing a constitutively active Stat3 (K5.Stat3C mice) develop a skin phenotype either spontaneously, or in response to wounding, that closely resembles psoriasis [101]. Moreover, a STAT3 inhibitor STA-21 inhibits the generation of skin lesion in these psoriatic mice [102]. IL-17A is known to activate STAT3 via receptor-interacting protein 4 (RIP4) activation and upregulates the CCL20 expression [103]. IL-17A also upregulates keratin 17 expression via STAT1 and STAT3 activation [104]. IL-6 and IL-22 also play a synergistic role in development of psoriasis with IL-17A [68]. Notably, both IL-6 and IL-22 are potent STAT3 activators [105]. In accordance, biological or natural molecules such as indirubin and its derivatives useful for inactivating STAT3 exhibit therapeutic potential for psoriasis [106] (Figure 2). It reveals that IL-17 and IL-22 promote keratinocyte stemness and potentiate its regeneration [107]. IL-6 is produced from keratinocytes in response to IL-17A [108]. IL-22 is produced from Th17/22 cells, Th22 cells, and other immune cells [109,110].
keratinocyte stemness and potentiate its regeneration [107]. IL-6 is produced from keratinocytes in response to IL-17A [108]. IL-22 is produced from Th17/22 cells, Th22 cells, and other immune cells [109,110]. In humans, impairment of the IL-17 signal causes infectious diseases, especially by Candida albicans, which is a ubiquitous fungus and commensal yeast of the intestines and skin [96]. Notably, deficiency of the IL17RA, IL17RC, IL17F, or TRAF3IP2 genes is implicated in chronic mucocutaneous candidiasis disease (CMCD), which is characterized by recurrent or persistent infection affecting the nails, skin, and oral and genital mucosae caused by the Candida species, often C. albicans [96,[111][112][113]. Impairment of the IL-17 signal is evident in other immunocompromised inborn errors, including autosomal-dominant hyper IgE syndrome, autosomal dominant STAT1 gain-of-function, autosomal-recessive autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), autosomal-recessive CARD9 deficiency, IL12RB1 deficiency, IL12B deficiency, and RORC deficiency [96]. However, these inborn errors seem to exhibit more complicated immune defects beyond IL-17 dysfunction and manifest CMCD together with other types of infection, including Mycobacterium, Staphylococcus, and viral disorders [96]. Of note, mice lacking Traf3ip2 (Act1) or Il17ra manifest similar clinical phenotype as human CMCD patients lacking IL17RA or IL17RC [114][115][116][117]. A recent murine study by Sparber et al. also indicates that Malassezia infection selectively triggers the IL-17A-induced immune response [118]. These findings indicate a crucial role of IL-17A in anti-fungal immunity in humans and mice.
Mice overexpressing IL-17A in keratinocytes (K14-IL-17A ind/+ mice) exhibit severe psoriasiform skin inflammation and vascular dysfunction in conjunction with infiltration of the vasculature by inflammatory myeloid cells [119]. The K14-IL-17A ind/+ mice acquire the highest local and systemic In humans, impairment of the IL-17 signal causes infectious diseases, especially by Candida albicans, which is a ubiquitous fungus and commensal yeast of the intestines and skin [96]. Notably, deficiency of the IL17RA, IL17RC, IL17F, or TRAF3IP2 genes is implicated in chronic mucocutaneous candidiasis disease (CMCD), which is characterized by recurrent or persistent infection affecting the nails, skin, and oral and genital mucosae caused by the Candida species, often C. albicans [96,[111][112][113]. Impairment of the IL-17 signal is evident in other immunocompromised inborn errors, including autosomal-dominant hyper IgE syndrome, autosomal dominant STAT1 gain-of-function, autosomal-recessive autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), autosomal-recessive CARD9 deficiency, IL12RB1 deficiency, IL12B deficiency, and RORC deficiency [96]. However, these inborn errors seem to exhibit more complicated immune defects beyond IL-17 dysfunction and manifest CMCD together with other types of infection, including Mycobacterium, Staphylococcus, and viral disorders [96]. Of note, mice lacking Traf3ip2 (Act1) or Il17ra manifest similar clinical phenotype as human CMCD patients lacking IL17RA or IL17RC [114][115][116][117]. A recent murine study by Sparber et al. also indicates that Malassezia infection selectively triggers the IL-17A-induced immune response [118]. These findings indicate a crucial role of IL-17A in anti-fungal immunity in humans and mice.
Mice overexpressing IL-17A in keratinocytes (K14-IL-17A ind/+ mice) exhibit severe psoriasiform skin inflammation and vascular dysfunction in conjunction with infiltration of the vasculature by inflammatory myeloid cells [108]. The K14-IL-17A ind/+ mice acquire the highest local and systemic IL-17A levels and exhibit a particularly severe psoriasis-like skin phenotype [108].
Homozygous CD11c-IL-17A ind/ind mice and heterozygous CD11c-IL-17A ind/+ mice show a delayed onset of moderate to severe psoriasis-like skin disease associated with reduced amounts of cutaneous IL-17A compared with K14-IL-17A ind/+ mice. In agreement with elevated skin and a stepwise increase in systemic IL-17A [119], homozygous CD11c-IL-17A ind/ind mice develop earlier and more severe skin lesions, as well as more pronounced vascular inflammation and dysfunction than heterozygous CD11c-IL-17A ind/+ mice [76,119]. These experimental models indicate that IL-17A induces psoriasis-like lesions in a dose-dependent manner, irrespective of its cellular source [76,119].

IL-17A and Psoriasis
In the early 2000s, IL-23 was found to induce the production of IL-17A by activated CD4 + T cells, which were later named Th17 cells [74,120]. These cells express RORγt (RORC) as the master transcription factor [121] and promoted the anti-infectious defense in the mucosa and skin [96], whereas excessive exposure to IL-23 induces their transformation into autoimmune or autoinflammatory immune cells [122,123]. Accordingly, the intradermal injection of IL-23 induces murine psoriasis-like dermatitis with epidermal acanthosis, neutrophil recruitment, and the infiltration of IL-17-producing T cells [78,79,124].
IL-17A alone is likely to be insufficient to evoke a significant inflammatory response and may cooperatively or synergistically accelerate the proinflammatory cascade in combination with other cytokines, such as TNF-α, IL-23, IL-1β, IL-6, IL-22 and transforming growth factor-β (TGF-β) [68,172]. Transcriptomic analysis reveals a clear additive or synergistic gene regulation by IL-17A and TNF-α in human keratinocytes [173]. This gene regulation is likely attributable to two sets of transcription factors: NF-κB and the C/CAAT-enhancer-binding proteins (C/EBP), C/EBPβ, or C/EBPδ [169,173,174]. TNF-α is a strong inducer of active NF-κB, while IL-17A activates C/EBPβ or C/EBPδ and to a lesser extent NF-κB [169,173,174]. Therefore, the IL-17A blockade reduces the expression of these responsive genes to a greater extent than TNF-α inhibition [168].
The C/EBP family members are highly expressed transcription factors in epidermal keratinocytes and sebocytes in mice and humans and accelerate their differentiation [175]. The expression of C/EBPβ protein is located in the upper spinous layer and is strongly upregulated in the lesional skin of psoriasis [169]. Together with the elevated gene expression of CEBPB, the expression of keratinocyte-terminal differentiation genes, such as IVL, FLG2, and TGM1, is upregulated in the lesional skin of psoriasis [169]. In the promoter region of the IVL gene, there is a binding site for C/EBP, and the C/EBP transcription factor is necessary for the appropriate and continuous production of IVL protein [176]. These results suggest that IL-17A accelerates keratinocyte differentiation by increasing C/EBPβ protein in keratinocytes [168,169,173].
Regarding keratinocyte proliferation, IL-17A stimulates keratinocytes to produce IL-19 [173,177]. The combination of IL-17 and TNF-α results in the synergistic expression of IL-19 in keratinocytes. IL-17 alone promotes IL-19 expression by approximately 1.79-fold, and TNF-α alone slightly reduces IL-19 expression, whereas a combination of IL-17 and TNF-α promotes expression by 54.6-fold [173,177]. IL-19 itself promotes keratinocyte migration but not proliferation [178]. However, it activates fibroblasts to produce keratinocyte growth factor, which upregulates keratinocyte proliferation [178]. Additionally, the upregulation of cell cycle-related genes, such as CCNE1, CDCA5, and CDCA25A, suggests a direct contribution of IL-17 to epidermal KC proliferation [169]. The expression of cytokeratin 16 is upregulated in the lesional epidermis of psoriasis patients and in keratinocytes incubated with IL-17A [69,168,179]. The expression of cytokeratin 16 increases the proliferative capacity of keratinocytes via EGFR phosphorylation [180].
Other studies also underpin a possibility that IL-17A activates EGFR [98,181,182]. Chen et al. have demonstrated that IL-17A transactivates EGFR in keratinocyte stem cells [98]. IL-17A accelerates the enzymatic cleavage of amphiregulin, which activates EGFR in airway epithelial cells [181]. In the lesional skin of psoriasis, the expression of EGFR ligands, such as heparin-binding EGF, transforming growth factor-α, and amphiregulin, is overexpressed [183]. Consistent with these notions, the inhibition of EGFR by erlotinib or cetuximab successfully improves severe psoriasis [184][185][186][187]. These studies suggest that EGFR activation may partly explain the IL-17A-mediated upregulation of keratinocyte proliferation.
While most chemokines redundantly bind to multiple receptors, CCL20 has only one known receptor, CCR6 [191]. CCR6 is expressed on dendritic cells [159] and IL-17-producing immune cells, including Th17 [89,143,201], ILC3 [161,162], Tc17 [163,164], and γδ T cells [78,165]. CCR6 is now considered a representative marker for Th17 cells [202,203]. The human psoriatic epidermis expresses abundant CCL20 with the dermal infiltration of CCR6 + dendritic cells and skin-homing T cells [143,158,159]. Although dermal dendritic cells and T cells express CCL20 in the psoriatic dermis [159], the expression of CCL20 is largely confined to the psoriatic epidermis, suggesting that epidermal keratinocytes are the major source of CCL20 production in psoriatic lesions [158,159]. CCL20 is constitutively expressed in cultured keratinocytes [201,204], and its production is upregulated by TNF-α and IL-17A [182,204]. Scratch injury upregulates the gene expression of CCL20, CXCL8, and IL36G, which may be related to the scratch-induced Koebner phenomenon frequently observed in patients with psoriasis [201]. Preclinical studies have revealed that the humanized anti-CCR6 antibody efficiently inhibits the cutaneous infiltration of CCR6 + T cells in human and murine models [205,206]. The development of small molecule inhibitors against CCR6 is also ongoing [207,208]. Targeting the CCL20/CCR6 axis may be a potential therapeutic strategy for the treatment of psoriasis.
LL-37 is an antimicrobial peptide of human cathelicidin that is produced when keratinocytes are injured by a broad range of bacteria, viruses, and fungi [221,222]. In addition to antimicrobial peptide activity, LL-37 exhibits "alarmin" function, affects adenosine triphosphate-receptor P2X7 and Toll-like receptor (TLR) signaling, and EGFR transactivation or intracellular Ca2+ mobilization [223][224][225]. The released LL-37 binds to the infiltrated neutrophils [221]. Neutrophils are a rich source of extracellular DNA due to their neutrophil extracellular traps [226]. Upon stimulation with complexes of host DNA and LL-37, plasmacytoid dendritic cells produce large amounts of IFN-α [227]. Notably, LL-37 induced the proliferation of circulating CD3 + T cells in 24 out of 52 patients with psoriasis (46%) [144]; therefore, LL-37 is effective for autoantigens. In total, 50 LL-37-reactive CD3 + T cells, including both CD4 + and CD8 + T cells, express the skin-homing receptor cutaneous lymphocyte antigen [144]. LL-37 peptides bind to HLA-DR in dendritic cells and are presented to CD4 + T cells, while LL-37 peptides and the HLA-C*0602 complex activate CD8 + T cells [144,228]. The majority of LL-37-reactive CD3 + T cells produce IL-17, and the capacity of their IL-17 production is associated with disease severity [144]. Interestingly, the LL-37-specific IL-17-producing T cells are exclusively CD4 + , whereas the LL-37-specific CD8 + T cells do not produce IL-17 [144]. A recent study by Takahashi et al. revealed that LL-37 can bind to self-RNA and stimulate macrophages to produce IL-6 via a scavenger receptor [229].
DEFB4A is highly expressed in psoriasis plaques and is the most psoriasis-specific antimicrobial peptide [230,231]. In contrast, DEFB4A is expressed at negligible or low levels in normal skin and skin lesions of eczema [230][231][232]. The expression of DEFB4A is upregulated by IL-17A and synergistically by IL-17A and TNF-α [215,220]. The serum levels of DEFB4A are highly specific biomarkers for disease activity in patients with psoriasis [215]. The number of neutrophil extracellular traps increase in psoriasis and upregulate the expression of DEFB4A [226]. Notably, DEFB4A is a functional (non-chemokine) ligand for CCR6 and feasibly attracts Th17 cells [233,234].
Although the biological implications of increased antimicrobial peptides in psoriasis remain obscure, they are intimately associated with IL-17A-rich milieu. Therefore, the upregulated expression of these antimicrobial peptides is rapidly normalized by the neutralization of IL-17A by ixekizumab [168] or secukinumab [69].

Conclusions
IL-17A is a multifunctional cytokine produced from adaptive and innate immune cells, such as Th17 and ILC3s. It orchestrates and promotes the peripheral tissue defense system against microbial insult, especially fungal infection. Psoriasis is a major inflammatory skin disease in which the interaction between IL-17A and epidermal keratinocytes plays a critical pathogenic role. IL-17A stimulates the proliferation of keratinocytes. Keratinocytes also produce a variety of antimicrobial peptides and cytochemokines in response to IL-17A. The antimicrobial peptides further exacerbate skin inflammation. CCL20 produced from IL-17A-stimulated keratinocytes recruits IL-17A-producing Th17 cells and ILC3s and accelerates the feed-forward vicious cycle, which causes fully developed psoriasis. This pathogenetic scheme has been verified with a high clinical efficacy of anti-IL-17A biologics. Therefore, psoriasis is considered an excellent human model of how IL-17A works with target peripheral tissues, and it provides in-depth insight into human autoinflammatory diseases.