Gram-negative anaerobes elicit a robust keratinocytes immune response with potential insights into HS pathogenesis.

Hidradenitis Suppurativa (HS) is a chronic autoinflammatory skin disease with activated keratinocytes, tunnel formation and a complex immune infiltrate in tissue. The HS microbiome is polymicrobial with an abundance of commensal gram-positive facultative (GPs) Staphylococcus species and gram-negative anaerobic (GNA) bacteria like Prevotella, Fusobacterium and Porphyromonas with increasing predominance of GNAs with disease severity. We sought to define the keratinocyte response to bacteria commonly isolated from HS lesions to probe pathogenic relationships between HS and the microbiome. Type strains of Prevotella nigrescens, Prevotella melaninogenica, Prevotella intermedia, Prevotella asaccharolytica, Fusobacterium nucleatum, as well as Staphylococcus aureus and the normal skin commensal Staphylococcus epidermidis were heat-killed and co-incubated with normal human keratinocytes. RNA was collected and analysed using RNAseq and RT-qPCR. The supernatant was collected from cell culture for protein quantification. Transcriptomic profiles between HS clinical samples and stimulated keratinocytes were compared. Co-staining of patient HS frozen sections was used to localize bacteria in lesions. A mouse intradermal injection model was used to investigate early immune recruitment. TLR4 and JAK inhibitors were used to investigate mechanistic avenues of bacterial response inhibition. GNAs, especially F. nucleatum, stimulated vastly higher CXCL8, IL17C, CCL20, IL6, TNF and IL36γ transcription in normal skin keratinocytes than the GPs S. epidermidis and S. aureus. Using RNAseq, we found that F. nucleatum (and Prevotella) strongly induced the IL-17 pathway in keratinocytes and overlapped with transcriptome profiles of HS patient clinical samples. Bacteria were juxtaposed to activated keratinocytes in vivo, and F. nucleatum strongly recruited murine neutrophil and macrophage migration. Both the TLR4 and pan-JAK inhibitors reduced cytokine production. Detailed transcriptomic profiling of healthy skin keratinocytes exposed to GNAs prevalent in HS revealed a potent, extensive inflammatory response vastly stronger than GPs. GNAs stimulated HS-relevant genes, including many genes in the IL-17 response pathway, and were significantly associated with HS tissue transcriptomes. The close association of activated keratinocytes with bacteria in HS lesions and innate infiltration in murine skin cemented GNA pathogenic potential. These novel mechanistic insights could drive future targeted therapies.

Regulatory Mechanism of the IL-33-IL-37 Axis via Aryl Hydrocarbon Receptor in Atopic Dermatitis and Psoriasis.

Interleukin (IL)-33 and IL-37 have been identified as novel cytokines involved in various inflammatory diseases. However, their specific roles remain largely unknown. Recent studies have shown that IL-33, which triggers inflammation, and IL-37, which suppresses it, cooperatively regulate the balance between inflammation and anti-inflammation. IL-33 and IL-37 are also deeply involved in the pathogenesis of inflammatory skin diseases such as atopic dermatitis (AD) and psoriasis. Furthermore, a signaling pathway by which aryl hydrocarbon receptor (AHR), a receptor for dioxins, regulates the expression of IL-33 and IL-37 has been revealed. Here, we outline recent findings on the mechanisms regulating IL-33 and IL-37 expression in AD and psoriasis. IL-33 expression is partially dependent on mitogen-activated protein kinase (MAPK) activation, and IL-37 has a role in suppressing MAPK in human keratinocytes. Furthermore, IL-33 downregulates skin barrier function proteins including filaggrin and loricrin, thereby downregulating the expression of IL-37, which colocalizes with these proteins. This leads to an imbalance of the IL-33-IL-37 axis, involving increased IL-33 and decreased IL-37, which may be associated with the pathogenesis of AD and psoriasis. Therefore, AHR-mediated regulation of the IL-33-IL-37 axis may lead to new therapeutic strategies for the treatment of AD and psoriasis.

PDE4 inhibition by difamilast regulates filaggrin and loricrin expression via keratinocyte proline-rich protein in human keratinocytes.

BACKGROUND: Difamilast, a topical phosphodiesterase 4 (PDE4) inhibitor, has been shown to be effective for treating atopic dermatitis (AD), but the molecular mechanism involved is unclear. Since skin barrier dysfunction including reduced expression of filaggrin (FLG) and loricrin (LOR) contributes to AD development, difamilast treatment may be able to improve this dysfunction. PDE4 inhibition increases transcriptional activity of cAMP-responsive element binding protein (CREB). Therefore, we hypothesized that difamilast may affect FLG and LOR expression via CREB in human keratinocytes. OBJECTIVE: To elucidate the mechanism by which difamilast regulates FLG and LOR expression via CREB in human keratinocytes. METHODS: We analyzed normal human epidermal keratinocytes (NHEKs) treated with difamilast. RESULTS: We observed increases of intracellular cAMP levels and CREB phosphorylation in difamilast (5 µM)-treated NHEKs. Next, we found that difamilast treatment increased mRNA and protein levels of FLG and LOR in NHEKs. Since reduced expression of keratinocyte proline-rich protein (KPRP) is reported to be involved in skin barrier dysfunction in AD, we examined KPRP expression in difamilast-treated NHEKs. We found that difamilast treatment increased mRNA and protein levels of KPRP in NHEKs. Furthermore, KPRP knockdown using siRNA transfection abolished the upregulation of FLG and LOR in difamilast-treated NHEKs. Finally, CREB knockdown canceled the upregulation of FLG, LOR, and KPRP in difamilast-treated NHEKs, indicating that PDE4 inhibition by difamilast treatment positively regulates FLG and LOR expression via the CREB-KPRP axis in NHEKs. CONCLUSION: These findings may provide further guidance for therapeutic strategies in the treatment of AD using difamilast.

Novel Therapeutic Targets for the Treatment of Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that significantly impacts quality of life. The pathogenesis of AD is a complex combination of skin barrier dysfunction, type II immune response, and pruritus. Progress in the understanding of the immunological mechanisms of AD has led to the recognition of multiple novel therapeutic targets. For systemic therapy, new biologic agents that target IL-13, IL-22, IL-33, the IL-23/IL-17 axis, and OX40-OX40L are being developed. Binding of type II cytokines to their receptors activates Janus kinase (JAK) and its downstream signal, namely signal transduction and activator of transcription (STAT). JAK inhibitors block the activation of the JAK-STAT pathway, thereby blocking the signaling pathways mediated by type II cytokines. In addition to oral JAK inhibitors, histamine H4 receptor antagonists are under investigation as small-molecule compounds. For topical therapy, JAK inhibitors, aryl hydrocarbon receptor modulators, and phosphodiesterase-4 inhibitors are being approved. Microbiome modulation is also being examined for the treatment of AD. This review outlines current and future directions for novel therapies of AD that are currently being investigated in clinical trials, focusing on their mechanisms of action and efficacy. This supports the accumulation of data on advanced treatments for AD in the new era of precision medicine.

Cathelicidin Antimicrobial Peptide LL37 Induces Toll-Like Receptor 8 and Amplifies IL-36γ and IL-17C in Human Keratinocytes.

LL37 is produced by skin injury and bacterial infection and plays an important role in the early stages of psoriasis. In particular, the intracellular receptors toll-like receptors (TLR)3, TLR7, TLR8, and TLR9 are thought to be involved in the pathogenesis of psoriasis in conjunction with LL37, but the interaction between TLR7/8 and LL37 in keratinocytes (KCs) remains unclear. This study aimed to clarify the relationship between LL37 and TLR7/8 in KCs and their involvement in the pathogenetic pathways seen in psoriasis using cultured KCs and skin samples of patients with psoriasis. TLR7/8 was induced by LL37 in KCs. TLR8 but not TLR7 functionally induced many psoriasis-related molecules, whereas IL-17C was not altered by the blockade of TLR7/8. Although costimulation of LL37 with self-RNA/DNA did not show any interaction, LL37 itself would promote psoriasis-related genes. IL-36 receptor antagonistic antibody suppressed IL-17C induced by LL37. In psoriatic epidermis, LL37, TLRs, IL-17C, and IL-36γ expressions were increased and coexpressed with each other. Thus, we concluded that LL37 activates TLR8 in KCs and induces IL-17C through the induction of IL-36γ. Regulation of TLR8 or LL37 in KCs could be a potential therapeutic strategy for psoriatic inflammation.

The Dawn of a New Era in Atopic Dermatitis Treatment.

Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases, and the condition is typified by barrier dysfunction and immune dysregulation. Recent studies have characterized various phenotypes and endotypes of AD and elucidated the mechanism. Numerous topical and systemic narrow targeting therapies for AD have been developed according to these findings. Topical medications, including Janus kinase (JAK) inhibitors, phosphodiesterase 4 inhibitors, and the aryl hydrocarbon receptor agonist tapinarof, are effective and safe for AD compared to topical corticosteroids. Oral JAK inhibitors and monoclonal antibodies targeting interleukin (IL)-4, IL-13, IL-31, IL-33, OX40, thymic stromal lymphopoietin, and sphingosine 1-phosphate signaling have displayed outstanding efficacy against moderate-to-severe AD. We are currently in a new era of AD treatment.

OVOL2-Mediated ZEB1 Downregulation May Prevent Promotion of Actinic Keratosis to Cutaneous Squamous Cell Carcinoma.

Progression of actinic keratosis (AK) to cutaneous squamous cell carcinoma (cSCC) is rare. Most cases of AK remain as intraepidermal lesions, owing to the suppression of the epithelial-to-mesenchymal transition (EMT). Ovo-like transcriptional repressor 1 (OVOL1) and ovo-like zinc finger 2 (OVOL2) are important modulators of EMT in some tumors, but their roles in skin tumors remain elusive. This study elucidated the roles of OVOL1/2 in AK and cSCC using 30 AK/30 cSCC clinical samples, and an A431 human SCC cell line using immunohistochemistry and molecular biological approaches. Immunohistochemically, OVOL1/2 were upregulated in AK and downregulated in cSCC. Meanwhile, EMT-related factors, vimentin and zinc finger E-box binding homeobox 1 (ZEB1) were downregulated in AK and upregulated in cSCC. Moreover, ZEB1 expression was higher in tumors in which OVOL2 expression was low. Thus, we observed an inverse association between OVOL2 and ZEB1 expression in AK and cSCC. Although knockdown of OVOL1 or OVOL2 increased the mRNA and protein levels of ZEB1, only OVOL2 knockdown increased the invasive ability of A431. In conclusion, OVOL2 inhibits ZEB1 expression and may inhibit the promotion of AK into cSCC. OVOL2/ZEB1 axis may be a potential target for preventing the development of cSCC.

Serum squamous cell carcinoma antigen (SCCA)-2 correlates with clinical severity of pediatric atopic dermatitis in Ishigaki cohort.

BACKGROUND: We sometimes encounter difficulties in assessing the severity of pediatric atopic dermatitis (AD) using currently available biomarkers such as thymus and activation-regulated chemokine (TARC) due to the higher baseline values in non-AD children. Recent case control studies have indicated the usefulness of squamous cell carcinoma antigens (SCCAs) in pediatric and adult AD. Notably, SCCAs are induced by IL-4 and IL-13, vital Th2 cytokines that play important roles in AD etiology. OBJECTIVES: Relatively low prevalence and mild disease severity of pediatric AD are observed in our Ishigaki cohort presumably due to the moisturising subtropical climate, which could conversely mean possible higher allergic potential of non-AD subjects towards AD. Thus, the purpose of this study was to further investigate the feasibility of using SCCAs together with TARC and periostin as biomarkers for pediatric AD even in the Ishigaki cohort. METHODS: We enrolled 1459 nursery school children and identified 96 as having AD through 2009-2011. As statistical analyses, we performed Student's t-test, correlation analysis, and receiver and operating characteristic (ROC) analysis. RESULTS: Serum SCCA1, SCCA2, periostin and TARC levels were all significantly increased in AD compared with those in non-AD, but only serum SCCA2 showed a significant increase in AD when assessed in each age group or in subgroup analysis. Among the biomarkers tested, serum SCCA2 also showed the best correlations with clinical AD severity and TARC and showed the best diagnosability for AD in ROC analysis. CONCLUSIONS: SCCA2 is a potent biomarker for pediatric AD in the Ishigaki cohort.

Cyto/chemokine profile of in vitro scratched keratinocyte model: Implications of significant upregulation of CCL20, CXCL8 and IL36G in Koebner phenomenon.

BACKGROUND: Scratch injury induces Koebner phenomenon in psoriasis. Smoking is also a risk factor for psoriasis. Keratinocytes can produce various psoriasis-related molecules including TNF, IL1 A, IL1B, IL6, IL12B, IL17C, IL23 A, IL36 A, IL36B, IL36 G, CXCL1, CXCL2, CXCL8, CXCL9, CXCL10, CCL20, IFNB, and CAMP. However, the scratch-induced molecular profiling remains elusive. OBJECTIVE: To profile the induction pattern of above-mentioned psoriasis-related and keratinocyte-derived molecules by scratch injury in the presence or absence of anti-psoriatic drugs or benzo[a]pyrene, a major environmental pollutant of tobacco smoke. METHODS: Confluent normal human keratinocytes were scratched and molecules were assayed by qRT-PCR, ELISA and Western blotting with or without drugs and benzo[a]pyrene. RESULTS: Among the 18 molecules, the scratch injury on a confluent keratinocyte sheet significantly and selectively upregulated the mRNA expression of four cyto/chemokines, CXCL8, CCL20, IL36G, and TNF, in a scratch-line-number-dependent manner under either low- or high-calcium condition. However, significant protein secretion was only demonstrated for CXCL8 and CCL20. The IL36 G protein was not secreted, but its intracellular level was significantly upregulated by scratch injury, whereas neither the secretion nor the intracellular level of TNF protein was affected by scratch injury. Dexamethasone, but not maxacalcitol nor the phosphodiesterase 4 inhibitor apremilast, partially inhibited the CXCL8 and CCL20 secretion. Benzo[a]pyrene significantly and synergistically enhanced the scratch-induced CCL20 secretion that may explain why smoking is a risk factor for psoriasis. CONCLUSION: CCL20 and to a less extent CXCL8 may play a key role in triggering the Koebner phenomenon after scratch injury to keratinocytes.

Cholesterol sulfate is a DOCK2 inhibitor that mediates tissue-specific immune evasion in the eye.

Although immune responses are essential to protect the body from infection, they can also harm tissues. Certain tissues and organs, including the eye, constitute specialized microenvironments that locally inhibit immune reactivity. Dedicator of cytokinesis protein 2 (DOCK2) is a Rac-specific guanine nucleotide exchange factor (GEF) that is predominantly found in hematopoietic cells. DOCK2 plays a key role in immune surveillance because it is essential for the activation and migration of leukocytes. DOCK2 mutations cause severe immunodeficiency in humans. We found that DOCK2-mediated Rac activation and leukocyte migration were effectively inhibited by cholesterol sulfate (CS), but not by cholesterol or other sulfated steroids. CS bound to the catalytic domain of DOCK2 and suppressed its GEF activity. Mass spectrometric quantification revealed that CS was most abundantly produced in the Harderian gland, which provides the lipids that form the oily layer of the tear film. Sulfation of cholesterol is mediated by the sulfotransferases SULT2B1b and, to a lesser extent, SULT2B1a, which are produced from the same gene through alternative splicing. By genetically inactivating Sult2b1, we showed that the lack of CS in mice augmented ultraviolet- and antigen-induced ocular surface inflammation, which was suppressed by administration of eye drops containing CS. Thus, CS is a naturally occurring DOCK2 inhibitor and contributes to the generation of the immunosuppressive microenvironment in the eye.

Tryptophan photo-product FICZ upregulates AHR/MEK/ERK-mediated MMP1 expression: Implications in anti-fibrotic phototherapy.

BACKGROUND: Scleroderma is caused by aberrant transforming growth factor-ß signaling. The degradation of extracellular matrix proteins is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Ultraviolet (UV) radiation has been a therapy for scleroderma. 6-Formylindolo[3,2-b]carbazole (FICZ), an endogenous aryl hydrocarbon receptor (AHR) ligand, is a tryptophan metabolite generated by UV exposure. Nonetheless, whether FICZ regulates MMPs and TIMPs has not been investigated. OBJECTIVE: To elucidate the regulatory roles of FICZ in the expression of MMPs and TIMPs in normal human dermal fibroblasts (NHDFs). METHODS: Quantitative real-time polymerase chain reaction was performed to determine the expression of MMPs or TIMPs in the NHDFs treated with FICZ or UVB. The MMPs levels were measured by enzyme-linked immunosorbent assay. The actions of FICZ on MMPs were analyzed using AHR-knockdown NHDFs or selective inhibitors of mitogen-activated protein kinases (MAPKs). Microtubule-associated protein kinase (MEK) and extracellular signal-regulated kinase (ERK) phosphorylation was examined by western blotting. RESULTS: UVB increased the mRNA and protein levels of MMP1 and MMP3 in NHDFs, while FICZ upregulated those of MMP1, but not MMP3. The effects of FICZ on TIMPs were negligible. FICZ increased MMP1 expression in an AHR-dependent manner. The FICZ-induced MMP1 upregulation was ameliorated with MEK/ERK inhibitors, whereas the effects of UVB were canceled with c-Jun N-terminal kinase (JNK) and p38-MAPK as well as MEK/ERK inhibitors. FICZ-induced ERK phosphorylation is dependent on AHR. CONCLUSION: FICZ contributes to the UV-mediated anti-fibrotic effects via the AHR/MEK/ERK signal pathway in NHDFs. FICZ is a potential therapeutic agent for scleroderma.

Highlighting Interleukin-36 Signalling in Plaque Psoriasis and Pustular Psoriasis.

Plaque psoriasis and pustular psoriasis are overlapping, but distinct, disorders. The therapeutic response to biologics supports the pivotal role of the tumour necrosis alpha (TNF-?)/ interleukin (IL)-23/IL-17/IL-22 axis in the pathogenesis of these disorders. Recently, functional activation of the IL-36 receptor (IL-36R) was discovered to be another driving force in the pathogenesis of psoriasis. This was first highlighted by the discovery that a loss-of-function mutation of the IL-36R antagonist (IL-36Ra) causes pustular psoriasis. Although the TNF-?/IL-23/IL-17/IL-22 axis and the functional activation of IL-36R are fundamentally involved in plaque psoriasis and pustular psoriasis, respectively, the 2 pathways are closely related and mutually reinforced, resulting in full-blown clinical manifestations. This review summarizes current topics on how IL-36 agonists (IL-36?, IL-36?, IL-36?) signal IL-36R, the pathological expression of IL-36 agonists and IL-36Ra in plaque and pustular psoriatic lesions, and the cross-talk between the TNF-?/IL-23/IL-17/IL-22 axis and the functional activation of IL-36R in the epidermal milieu.

The transcription factor EPAS1 links DOCK8 deficiency to atopic skin inflammation via IL-31 induction.

Mutations of DOCK8 in humans cause a combined immunodeficiency characterized by atopic dermatitis with high serum IgE levels. However, the molecular link between DOCK8 deficiency and atopic skin inflammation is unknown. Here we show that CD4+ T cells from DOCK8-deficient mice produce large amounts of IL-31, a major pruritogen associated with atopic dermatitis. IL-31 induction critically depends on the transcription factor EPAS1, and its conditional deletion in CD4+ T cells abrogates skin disease development in DOCK8-deficient mice. Although EPAS1 is known to form a complex with aryl hydrocarbon receptor nuclear translocator (ARNT) and control hypoxic responses, EPAS1-mediated Il31 promoter activation is independent of ARNT, but in collaboration with SP1. On the other hand, we find that DOCK8 is an adaptor and negative regulator of nuclear translocation of EPAS1. Thus, EPAS1 links DOCK8 deficiency to atopic skin inflammation via IL-31 induction in CD4+ T cells.

Levels of immunoglobulin E specific to the major food allergen and chemokine (C-C motif) ligand (CCL)17/thymus and activation regulated chemokine and CCL22/macrophage-derived chemokine in infantile atopic dermatitis on Ishigaki Island.

Atopic dermatitis (AD) is a multifactorial T-helper (Th)2-mediated skin disease frequently associated with elevated serum immunoglobulin (Ig)E and food allergy is also a Th2- and IgE-mediated adverse immunological reaction. Our previous study indicated the relation of egg allergy history and disease severity of AD. Thus, the purpose of the study was to investigate the levels of IgE specific to major food allergens (egg, milk, wheat) and Th2 chemokines (chemokine [C-C motif] ligand [CCL]17/thymus and activation regulated chemokine [TARC] and CCL22/macrophage-derived chemokine [MDC]) and the relationship between them. A total of 743 nursery school children were enrolled. Dermatologist-based physical examination and a questionnaire survey were also conducted. Significantly increased levels of disease severity markers (CCL17/TARC and CCL22/MDC) were confirmed in children with AD. The levels of CCL22/MDC in all of the children were markedly high compared with those reported in adults. IgE specific to egg white, ovomucoid, wheat and mite antigen were significantly higher in the AD group than in the non-AD group. Among them, IgE specific to egg allergens were well associated with disease severity markers, and IgE specific to ovomucoid seemed particularly well correlated with the presence of egg allergy history. In conclusion, the markedly high level of CCL22/MDC in children as compared with those reported in adults may partly explain the AD-prone nature of children and their spontaneous remission afterwards. Mild but significant correlation of IgE specific to egg allergens and Th2 chemokines may explain correlation of disease severity and comorbidity of egg allergy in our previous study.