IL-33-induced keratoconjunctivitis is mediated by group 2 innate lymphoid cells in mice.

BACKGROUND: Interleukin-33 (IL-33) is involved in type 2 innate immunity by inducing type 2 cytokines, such as IL-5 and IL-13, through the activation of group 2 innate lymphoid cells (ILC2s) or T helper 2 (Th2) cells. We previously reported that mice overexpressing IL-33 (IL-33Tg) in the cornea and conjunctiva spontaneously develop atopic keratoconjunctivitis-like inflammation. Despite previous studies, it is not fully understood what types of immune cells contribute to the disease process of IL-33-induced keratoconjunctivitis. METHODS: To defect Th2 cells, IL-33Tg mice were crossed with Rag2KO mice. To defect ILC2s, IL-33Tg mice received bone marrow transplantations from B6.C3(Cg)-Rorasg/J mice that lacked ILC2. Immunostaining techniques were used to determine where ILC2 is distributed in the cornea and conjunctiva. We analyzed the transcriptomes of ILC2 from the conjunctiva by using single-cell RNA-seq analysis. To investigate whether tacrolimus reduces type 2 cytokine production by ILC2, ILC2 was cultured with tacrolimus, and the percentage of cytokine-producing ILC2 was examined. To investigate whether tacrolimus can inhibit IL-33-induced keratoconjunctivitis in vivo, IL-33Tg mice were treated with tacrolimus eye drops. RESULTS: ILC2 infiltrated the conjunctival epithelium and subepithelial tissue. Keratoconjunctivitis developed spontaneously in Rag2KO/IL-33Tg mice, but keratoconjunctivitis was abolished in IL-33Tg mice lacking ILC2. ILC2 was not a uniform cluster but a heterogeneous cluster. Tacrolimus inhibited cytokine production from ILC2s in vitro, and tacrolimus eye drops inhibited keratoconjunctivitis in IL-33Tg mice in vivo. CONCLUSIONS: ILC2 plays a pivotal role in IL-33-induced keratoconjunctivitis in mice.

Single-cell RNA sequencing of mycosis fungoides reveals a cluster of actively proliferating lymphocytes.

A 70-year-old man's chronic erythematous skin lesion in the extremity had developed into a tumour one year before his first visit at our hospital. A biopsy showed atypical lymphocyte-like cells, and immunostaining identified atypical cells as CD3+, CD4+, CD5+ and FOXP3+. Single-cell RNA sequencing (scRNA-seq) analysis using BD Rhapsody revealed the higher expression of CD3, CD4, CD5 and FOXP3 genes in a group of cells that highly expressed genes, such as PCNA, in the S/M phase, which is in agreement with immunofluorescence staining results. The use of scRNA-seq analysis data is expected to promote personalised medicine for cutaneous lymphoma.

Alarmins/stressorins and immune dysregulation in intractable skin disorders.

Unlike other barrier epithelia of internal organs, the stratified squamous epithelium of the skin is always exposed to the external environment. However, the robust barrier structure and function of the skin are highly resistant against external insults so as to not easily allow foreign invasions. Upon sensing danger signals, the innate immunity system is promptly activated. This process is mediated by alarmins, which are released passively from damaged cells. Nuclear alarmins or stressorins are actively released from intact cells in response to various cellular stresses. Alarmins/stressorins are deeply involved in the disease processes of chronic skin disorders of an unknown cause, such as rosacea, psoriasis, and atopic dermatitis. Furthermore, alarmins/stressorins are also induced in the congenital skin disorders of ichthyosis and keratoderma due to defective keratinization. Studies on alarmin activation and its downstream pathways may help develop novel therapeutic agents for intractable skin disorders.

Relationship between YKL-40 and pulmonary arterial hypertension in systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is an intractable connective tissue disease that causes skin and organ fibrosis. Interstitial lung disease (ILD) and pulmonary arterial hypertension (PAH) affect its prognosis. YKL-40 protein impacts inflammation and tissue remodeling. Therefore, we evaluated the utility of YKL-40 blood levels in identifying patients with SSc complicated by PAH, as confirmed by immunohistochemistry (IHC) examination. METHODS: We retrospectively analyzed 78 patients with SSc and performed IHC on 7 normal and 7 SSc skin samples in the Japanese population. Age-adjusted YKL-40 serum levels were analyzed. RESULTS: YKL-40 age percentile was significantly elevated in SSc patients. There was no difference between patients with SSc with and without ILD and PAH. YKL-40 age percentile was greater in patients with PAH complication. YKL-40 immunostaining was negative in normal skin and prominent in the subcutaneous vascular wall of all SSc samples. Receiver operating characteristic (ROC) curve analysis indicated that YKL-40 age percentile correctly differentiated between patients with and without PAH with a sensitivity of 80% and a specificity of 94.1%. CONCLUSION: A higher YKL-40 level with PAH may be reflective of angiogenesis due to capillary injury in SSc. YKL-40 may offer a useful and easily applicable diagnostic biomarker of SSc complicated with PAH.

Characterization of TG2 and TG1-TG2 double knock-out mouse epidermis.

Transglutaminases (TGs) are a family of enzymes that catalyse the formation of isopeptide bonds between the γ-carboxamide groups of glutamine residues and the ε-amino groups of lysine residues leading to cross-linking reactions among proteins. Four members, TG1, TG2, TG3, and TG5, of the nine mammalian enzymes are expressed in the skin. TG1, TG3 and TG5 crosslinking properties are fundamental for cornified envelope assembly. In contrast, the role of TG2 in keratinization has never been studied at biochemical level in vivo. In this study, taking advantage of the TG2 knock-out (KO) and TG1 heterozygous mice, we generated and characterized the epidermis of TG1-TG2 double knock-out (DKO) mice. We performed morphological analysis of the epidermis and evaluation of the expression of differentiation markers. In addition, we performed analysis of the amino acid composition from isolated corneocytes. We found a significant change in amino acid composition in TG1KO cornified cell envelopes (CEs) while TG2KO amino acid composition was similar to wild-type CEs. Our results confirm a key role of TG1 in skin differentiation and CE assembly and demonstrate that TG2 is not essential for CE assembly and skin formation.

Skin-specific expression of IL-33 activates group 2 innate lymphoid cells and elicits atopic dermatitis-like inflammation in mice.

Transgenic mice expressing the mouse interleukin 33 (IL-33) gene driven by a keratin 14 promoter were generated. The skin-selective expression of the IL-33 gene was enhanced, and intense immunofluorescence for IL-33 was evident in the nuclei of the epidermis. Spontaneous itchy dermatitis developed in those mice at 6-8 wk of age in specific pathogen-free conditions. In the lesional skin, the epidermis was thickened and the eosinophils were infiltrated with increased expression of the eosinophil peroxidase and major basic protein genes. Mast cells were also abundant there, and blood histamine and total IgE levels were high. Those phenotypes closely resemble the features of atopic dermatitis. In peripheral blood and lesional skin, IL-5, IL-13, regulated upon activation, normally T-expressed, and presumably secreted (RANTES)/CCL5, and Eotaxin 1/CCL11 were increased, whereas TNF-α, IFN-γ, and thymic stromal lymphopoietin (TSLP) were unaltered. Furthermore, the proportion of group 2 innate lymphoid cells (ILC2s), which produce IL-5, were significantly increased in the lesional skin, peripheral blood, and regional lymph nodes. The dermatitis with eosinophil infiltration was improved by the administration of an anti-IL-5 antibody. These results suggest that the expression of IL-33 in the skin activates an immune response involving ILC2 and that this process might play a crucial role in the pathogenesis of allergic inflammation that is characteristic of atopic dermatitis.

Exploration of Metabolite Biomarkers to Predict the Efficacy of Dupilumab Treatment for Atopic Dermatitis.

Dupilumab (DUP) is the first biological agent used treating atopic dermatitis (AD). Notwithstanding its high cost, the type of patient group for which the drug is effective remains unclear. In this retrospective study, we aimed to identify novel and reliable biomarkers which can be measured before DUP administration and to predict the efficacy of DUP. Serum samples from 19 patients with AD treated with DUP were analysed by metabolome analysis using gas chromatography-mass spectrometry. Total 148 metabolites were detected, and the relative values of the metabolites were compared between the patient group that achieved 75% improvement in Eczema Area and Severity Index 16 weeks after administration of DUP (high responders: HR; n = 11) and that did not (low responders: LR; n = 8). The HR and LR groups had significant differences in the relative values of the eight metabolites (lactic acid, alanine, glyceric acid, fumaric acid, nonanoic acid, ribose, sorbitol, and ornithine), with ribose emerging as the best. Furthermore, we evaluated the serum concentrations of ribose and found that ribose may be a useful metabolite biomarker for predicting the efficacy of DUP in AD.

Markedly elevated serum levels of calcium-binding S100A8/A9 proteins in psoriatic arthritis are due to activated monocytes/macrophages.

BACKGROUND: Serum levels of S100A8/A9 may correlate with disease activity in psoriasis. OBJECTIVE: We sought to elucidate the association of serum levels of S100A8/A9 heterodimers with the clinical subtypes of psoriasis and the major cell source. METHODS: Serum samples were collected from patients with psoriasis vulgaris (n = 30), psoriatic arthritis (PA) (n = 16), pustular psoriasis (n = 24), and atopic dermatitis (n = 14) and from healthy control subjects (n = 21). Serum concentrations of S100A8/A9 were measured, and the expression levels were examined in psoriatic lesions. The messenger RNA levels were quantified in circulating monocytes and neutrophils. RESULTS: Serum levels of S100A8/A9 were significantly increased in all subtypes of psoriasis as compared with healthy controls and atopic dermatitis. Among the psoriatic subtypes, PA and pustular psoriasis showed remarkably high concentrations of S100A8/A9 heterodimers. The higher serum levels were associated with the presence of articular symptoms, but not significantly correlated with body surface areas of psoriatic lesions. S100A8 was expressed by both keratinocytes and infiltrating mononuclear cells, whereas S100A9 was predominantly expressed by neutrophils. The expression levels of S100A8 and S100A9 messenger RNA in monocytes were increased by approximately 2.25- and 1.91-fold in PA, respectively, whereas no significant increase was observed in psoriasis vulgaris and pustular psoriasis. LIMITATIONS: Difficulty in acquisition of clinical and laboratory samples in untreated patients, and of a sufficient number of subjects, were limitations. CONCLUSIONS: Although serum levels of S100A8/A9 were increased in all types of psoriasis examined, patients with PA had higher levels of S100A8/A9, probably because of an activated monocyte/macrophage system.

Dupilumab Effects on Innate Lymphoid Cell and Helper T Cell Populations in Patients with Atopic Dermatitis.

Group 2 innate lymphoid cells (ILCs) are thought to contribute to the pathogenesis of atopic dermatitis (AD). IL-4 stimulates T helper type 2 (Th2) cells and ILC2s to proliferate and produce cytokines. Dupilumab, an antibody against the IL-4 receptor, is used in AD therapy. We speculated that its efficacy might involve blocking the activation of Th2 cells and ILC2s via IL-4. Here, we examined circulating Th2 cells and ILC2s in 27 Japanese patients with AD before and after the administration of dupilumab. Between 0 and 4 months after dupilumab administration, the percentages of Th2 cells and ILC2s were decreased. Notably, ILC2/3 ratio was decreased after dupilumab treatment. Interestingly, ILC2/3 ratio before dupilumab treatment were significantly higher in high responders than in low responders to dupilumab. To resolve the molecular signatures of the Th2 and ILC2s in AD, we sorted CD4+ T cells and ILCs from peripheral blood and analyzed their transcriptomes using the BD Rhapsody Single-cell RNA sequencing system. Between 0 and 4 months after dupilumab administration, the Th2 and ILC2 cluster gene signatures were downregulated. Thus, dupilumab might improve dermatitis by suppressing the Th2 cell and ILC2 populations and altering the Th2 and ILC2 repertoire in patients with AD.

Effectiveness and safety of tacrolimus ointment combined with dupilumab for patients with atopic dermatitis in real-world clinical practice.

Atopic dermatitis (AD) is the most common inflammatory skin disease affecting people of all age groups worldwide. To our knowledge, there are currently no studies estimating the effectiveness of tacrolimus ointment and dupilumab as a combination therapy for AD. Thus, here we describe the effectiveness and safety of tacrolimus ointment in combination with dupilumab for facial rashes in patients with AD. Overall, we included 109 patients who newly received dupilumab from April 2018 to July 2020 in the Dermatology Department of Hyogo College of Medicine Hospital. Of them, 60 patients were treated with tacrolimus ointment. Specifically, of the 60 patients, 40 were treated with dupilumab in combination with tacrolimus ointment and topical steroids, whereas the remaining 20 were prescribed tacrolimus ointment alone and were further analyzed. The analysis showed that the combination does not cause serious side-effects at high frequency. The patients showed rapid improvement of facial dermatitis along with systemic dermatitis, and the rate of improvement of head/neck Eczema Area and Severity Index (EASI) score significantly correlated with the rate of improvement of overall EASI score. In addition, there was no complication of herpes simplex observed in these 20 patients. Thus, tacrolimus ointment combined with dupilumab is an effective and safe treatment option for facial AD.

Comprehensive stratum corneum ceramide profiling reveals reduced acylceramides in ichthyosis patient with CERS3 mutations.

The stratum corneum (SC) of the epidermis acts as a skin permeability barrier, and abnormalities in SC formation lead to several skin disorders. Lipids, especially the epidermis-specific ceramide classes ω-O-acylceramides (acylceramides) and protein-bound ceramides, are essential for skin barrier formation. Ceramide synthase 3 (CERS3) is involved in the synthesis of acylceramides and protein-bound ceramides, and CERS3 mutations cause autosomal recessive congenital ichthyosis. In the present study, we measured ceramide synthase activity and performed comprehensive SC ceramide profiling in an ichthyosis patient with compound heterozygous CERS3 mutations: nonsense mutation p.Arg75* and missense mutation p.Arg229His. The activity of p.Arg75* and p.Arg229His mutant CERS3 proteins was reduced to 4% and 56%, respectively, of the wild-type protein. In the patient's SC, acylceramide levels were greatly reduced, but the levels of protein-bound ceramides remained almost unchanged. Non-acylated ceramide levels were also affected in the patient; in particular, the levels of ceramides composed of sphingosine and non-hydroxy or α-hydroxy fatty acid were substantially higher than in healthy controls. These results suggest that a reduction in acylceramide levels alone leads to ichthyosis. Although protein-bound ceramides are synthesized from acylceramides, levels of acylceramides and protein-bound ceramides are not necessarily correlated.

Monitoring Cellular Movement with Photoconvertible Fluorescent Protein and Single-Cell RNA Sequencing Reveals Cutaneous Group 2 Innate Lymphoid Cell Subtypes, Circulating ILC2 and Skin-Resident ILC2.

We previously generated a transgenic mouse line expressing skin-specific IL-33 (IL33tg mice) and showed that IL-33 elicits group 2 innate lymphoid cell (ILC2)-dependent atopic dermatitis-like skin inflammation. ILC2s are believed to be tissue-resident cells under steady-state conditions, but the dynamics of ILC2 migration are not fully understood. We sorted ILC2s from the skin and draining lymph nodes of IL33tg mice and analyzed their transcriptomes using the single-cell RNA sequencing technique, which revealed that the skin ILC2s had split into two clusters: circulating ILC2 and skin-resident ILC2. The circulating ILC2s expressed H2-related major histocompatibility complex class II genes. Conversely, the skin-resident ILC2s demonstrated increased mRNA expression of the ICOS, IL-5, and IL-13. Next, we tracked ILC2 migration using IL33tg-Kikume Green-Red mice. Exposing the IL33tg-Kikume Green-Red mice's inflamed skin to violet light allowed us to label the circulating ILC2s in their skin and track the ILC2 migration from the skin to the draining lymph nodes. Cutaneous local innate responses could transition to systemic type 2 responses by migrating the activated ILC2s from the skin into the draining lymph node. Conversely, the skin-resident ILC2s produced a large number of cytokines. Thus, the skin ILC2s turned out to be a heterogeneous cell population.

Tetracyclines modulate protease-activated receptor 2-mediated proinflammatory reactions in epidermal keratinocytes.

In addition to their antibiotic effects, tetracyclines have anti-inflammatory action that is often beneficial in the control of inflammatory skin disorders. In this study, we examined the effects of tetracycline (TET) and two of its derivatives, doxycycline (DOX) and minocycline (MIN), on the production of interleukin-8 (IL-8) elicited by the activation of protease-activated receptor 2 (PAR2) in normal human epidermal keratinocytes (NHEK). In NHEK, the production of IL-8 stimulated by an agonist peptide of PAR2, SLIGKIV-NH(2), at 100 microM was significantly reduced by TET, DOX, or MIN at 5 and 10 microM, concentrations that are noncytotoxic. The tumor necrosis factor alpha (TNF-alpha)-induced production of IL-8 was synergistically augmented by SLIGKIV-NH(2), and that synergistic increase in the production of IL-8 was suppressed by 100 nM PAR2-specific small interfering RNA. It was also suppressed by TET, DOX, or MIN but not by the 14-membered-ring macrolide antibiotics erythromycin, roxithromycin, and clarithromycin, which also have anti-inflammatory activities, at 10 microM. These results suggest that tetracyclines attenuate the PAR2-IL-8 axis in keratinocytes and thereby effectively modulate proinflammatory responses in the skin.

IL-33-Induced Atopic Dermatitis-Like Inflammation in Mice Is Mediated by Group 2 Innate Lymphoid Cells in Concert with Basophils.

IL-33 is a proinflammatory cytokine that plays a pivotal role in allergic disorders. In a transgenic mouse expressing IL-33 driven by a keratin-14 promoter (IL33tg), atopic dermatitis (AD)-like inflammation develops spontaneously with the activation of group 2 innate lymphoid cells (ILC2s). However, it remains unknown how effector cells, such as T helper type 2 cells, ILC2s, and basophils, contribute to the inflammatory process induced by IL-33. To address the question, we examined the phenotype of IL33tg mice lacking each of these cells. AD-like inflammation still developed in Rag2KO IL33tg mice lacking T and B cells; in contrast, when ILC2s were depleted in IL33tg mice via bone marrow transplantation from ILC2-lacking, RAR-related orphan receptor alpha-deficient mice, the development of AD-like inflammation was almost completely suppressed. Basophils were accumulated in the inflamed skin of IL33tg mice, and AD-like inflammation was alleviated by the conditional depletion of basophils using anti-FcεRIα antibodies or a Bas-TRECK transgenic mouse system. In these basophil-depleted IL33tg skins, ILC2s were decreased, and cytokines and chemokines such as IL-5, IL-13, and CCL5 were reduced. From these results, we suggest that IL-33-induced AD-like inflammation is dependent on innate immune responses that are mediated by ILC2s in concert with basophils.