Atopic Dermatitis Skin Lesions Research Articles

Interleukin-13 inhibition by tralokinumab reduces inducible T-cell costimulator-positive innate lymphoid cells in skin lesions of atopic dermatitis.

Despite the low frequency of skin ILCs and the limited number of samples analyzed in this study, our data indicate that ICOS+ ILCs express IL-13Rα1 and that the density of ICOS+ ILCs decreased four weeks after initiation of treatment with tralokinumab.

IL-31–generating network in atopic dermatitis comprising macrophages, basophils, thymic stromal lymphopoietin, and periostin

IL-31 is a type 2 cytokine involved in the itch sensation in atopic dermatitis (AD). The cellular origins of IL-31 are generally considered to be TH2 cells. Macrophages have also been implicated as cellular sources of IL-31. We sought to determine the expression of IL-31 by macrophages and to elucidate the productive mechanisms and contributions to itch in AD skin lesions. Expression of IL-31 by macrophages, expressions of thymic stromal lymphopoietin (TSLP) and periostin, and presence of infiltrating basophils in human AD lesions were examined through immunofluorescent staining, and correlations were assessed. Furthermore, mechanisms of inducing IL-31-expressing macrophages were analyzed in an MC903-induced murine model for AD invivo and in mouse peritoneal macrophages exvivo. A significant population of IL-31+ cells in human AD lesions was that of CD68+ cells expressing CD163, an M2 macrophage marker. The number of IL-31+/CD68+ cells correlated with epidermal TSLP, dermal periostin, and the number of dermal-infiltrating basophils. In the MC903-induced murine AD model, significant scratching behaviors with enhanced expressions of TSLP and periostin were observed, accompanied by massive infiltration of basophils and IL-31+/MOMA-2+/Arg-1+ cells. Blockade of IL-31 signaling with anti-IL-31RA antibody or direct depletion of macrophages by clodronate resulted in attenuation of scratching behaviors. To effectively reduce lesional IL-31+ macrophages and itch, basophil depletion was essential in combination with TSLP- and periostin-signal blocking. Murine peritoneal macrophages produced IL-31 when stimulated with TSLP, periostin, and basophils. A network comprising IL-31-expressing macrophages, TSLP, periostin, and basophils plays a significant role in AD itch.

176 C10orf99/2610528A11Rik induces keratinocyte proinflammatory response and regulates lipid metabolism and barrier formation of the skin

The protective machinery of the skin opposes external agents by barrier and innate and adaptive immunity. The local interplay in the epithelial-immune microenvironment (EIME) contributes to the situation-specific protective/inflammatory responses, and its dysregulation generates inflammatory loops in atopic dermatitis and psoriasis. Keratinocytes are the key player in the interplay and inflammatory loops in the EIME. However, a common regulator of keratinocyte response in this machinery has not yet been identified. To address this issue, we analyzed skin specimens from four animal models for atopic dermatitis by microarray. We found that expression levels of one orphan gene, 2610528A11Rik, were up in all the models. Protein expression levels of the human ortholog, C10orf99, were much higher in the lesional skin of atopic dermatitis or psoriasis than in healthy skin. RNA-Seq analyses of normal human epidermal keratinocytes (NHEKs) transfected with C10orf99-expressing plasmids revealed their increased proinflammatory transcriptional response, and gene ontology analyses suggested the highest relation to “Lipid/cholesterol biosynthesis,” “Cadherin/cell adhesion,” and “Mitochondrion.” Furthermore, transcription factor (TF) enrichment analysis found that only one pathway activating SREBP1, a TF regulating sterol synthesis, was significantly impaired in C10orf99-expressing NHEKs. Moreover, treatment of 3D-cultured human epidermis with C10orf99 synthetic peptides during its stratification downregulated the expression levels of filaggrin and loricrin in a dose-dependent manner. Our results suggest that 2610528A11Rik/C10orf99 can be a master regulator that induces keratinocyte protective response and regulates barrier formation of the skin.

Stratum corneum cytokine levels in mycosis fungoides.

Mycosis fungoides (MF) is characterised by malignant CD4+ T-cell infiltrates in the skin. The functional characteristics of the malignant T cells and their interaction with the tumor immune microenvironment is largely unknown. We performed tape stripping of the stratum corneum (SC), a non-invasive technique, to gain insight into the cytokine secretion patterns in MF skin lesions. In addition, we assessed whether the SC cytokine profile of MF lesions is distinct from that of atopic dermatitis (AD) lesions. We compared nine cytokine levels in 20 patients with MF, 10 patients with AD and 10 healthy controls. In patients with MF and AD, lesional SC levels of IL-8 and MMP9 were significantly higher than in non-lesional SC and in healthy controls. VEGFα was significantly higher in lesional MF and AD skin than in healthy controls. The SC levels of IL-1α were significantly lower in MF and AD lesions than in healthy controls. There was no specific cytokine profile or inflammation pattern that could reliably distinguish MF from AD. In conclusion, in lesional SC of MF patients, pro-inflammatory cytokines can be detected. As a diagnostic method, tape stripping of lesional SC cannot discriminate MF skin from AD skin.

Metabolomic Differences between the Skin and Blood Sera of Atopic Dermatitis and Psoriasis.

Atopic dermatitis (AD) and psoriasis (PS) are common chronic inflammatory dermatoses. Although the differences at the intercellular and intracellular signaling level between AD and PS are well described, the resulting differences at the metabolism level have not yet been systematically analyzed. We compared the metabolomic profiles of the lesional skin, non-lesional skin and blood sera of AD and PS. Skin biopsies from 15 patients with AD, 20 patients with PS and 17 controls were collected, and 25 patients with AD, 55 patients with PS and 63 controls were recruited for the blood serum analysis. Serum and skin samples were analyzed using a targeted approach to find the concentrations of 188 metabolites and their ratios. A total of 19 metabolites differed in the comparison of lesional skins, one metabolite in non-lesional skins and 5 metabolites in blood sera. Although we found several metabolomic similarities between PS and AD, clear differences were outlined. Sphingomyelins were elevated in lesional skin of AD, implying a deficient barrier function. Increased levels of phosphatidylcholines, carnitines and asymmetric dimethylarginine in PS lesional skin and carnitines amino acids in the PS serum pointed to elevated cell proliferation. The comparison of the metabolomic profiles of AD and PS skin and sera outlined distinct patterns that were well correlated with the differences in the pathogenetic mechanisms of these two chronic inflammatory dermatoses.

Staphylococcus aureus Biofilm Inhibiting Activity of Advanced Glycation Endproduct Crosslink Breaking and Glycation Inhibiting Compounds.

Staphylococcus aureus is a Gram-positive bacterium that plays a role in the pathogenesis of skin lesions in diabetes mellitus, atopic dermatitis, and psoriasis, all of which are associated with elevated non-enzymatic glycation biomarkers. The production of biofilm protects resident bacteria from host immune defenses and antibiotic interventions, prolonging pathogen survival, and risking recurrence after treatment. Glycated proteins formed from keratin and glucose induce biofilm formation in S. aureus, promoting dysbiosis and increasing pathogenicity. In this study, several glycation-inhibiting and advanced glycation endproduct (AGE) crosslink-breaking compounds were assayed for their ability to inhibit glycated keratin-induced biofilm formation as preliminary screening for clinical testing candidates. Ascorbic acid, astaxanthin, clove extract, n-phenacylthiazolium bromide, and rosemary extract were examined in an in vitro static biofilm model with S. aureus strain ATCC 12600. Near complete biofilm inhibition was achieved with astaxanthin (ED50 = 0.060 mg/mL), clove extract (ED50 = 0.0087 mg/mL), n-phenacylthiazolium bromide (ED50 = 5.3 mg/mL), and rosemary extract (ED50 = 1.5 mg/mL). The dosage necessary for biofilm inhibition was not significantly correlated with growth inhibition (R2 = 0.055. p = 0.49). Anti-glycation and AGE breaking compounds with biofilm inhibitory activity are ideal candidates for treatment of S. aureus dysbiosis and skin infection that is associated with elevated skin glycation.

RNA sequencing reveals the transcriptome profile of the atopic prurigo nodularis with severe itching.

Prurigo nodularis (PN), characterized by inevitable chronicity and severe pruritus, is most frequently associated with atopy compared with other origins. However, the skin transcriptomic profiling of PN arising from atopic dermatitis (AD), so-called atopic PN (APN), remains unclear. We sought to explore the cutaneous transcriptome of APN with severe pruritus and compare it with classic AD. RNA sequencing was performed on the lesional skin from 13 APN to 11 AD patients with severe pruritus (itch numerical rating scale score ≥ 7) and normal skin from 11 healthy subjects. Quantitative real-time polymerase chain reaction and immunochemistry were used for validation. We detected 1085 and 1984 differentially expressed genes (DEGs) in lesional APN skin and lesional AD skin versus normal skin, respectively. In total, 142 itch/inflammation-related DEGs were identified. Itch/inflammation-related DEGs, such as IL-6, IL-10, IL-13, oncostatin M, and IL-4 receptor, had elevated gene transcript levels in both diseases. The itch/inflammation-related DEGs that increased only in APN were mainly neuroactive molecules, while many inflammatory mediators such as T helper 22-related genes were found to be increased only in AD. Both disorders showed mixed Th1/Th2/Th17 polarisation and impaired skin barrier. In contrast to AD, M1/M2 macrophage activation, tumor necrosis factor production, fibrosis, revascularization and neural dysregulation are unique features of APN. The study findings broaden our understanding of the pathogenesis underlying APN, which provides insights into novel pathogenesis with potential therapeutic implications.

A Comparison for Type 2 Cytokines and Lesional Inflammatory Infiltrations in Bullous Pemphigoid and Atopic Dermatitis.

Bullous pemphigoid (BP) and atopic dermatitis (AD) are both type 2 inflammatory skin diseases with similar clinical features. Thymic stromal lymphopoietin (TSLP) is an epithelial-derived cytokine which is upregulated in AD. However, the expression of TSLP in BP and the correlation between TSLP and inflammatory infiltrations have not been fully studied. To characterize the serum Th2 cytokines level and Th2 inflammatory cell infiltrations in BP and AD. To study TSLP levels in serum, blister fluids and expression in lesional skin in patients with BP and AD. TSLP level in serum and blister fluids was measured by enzyme-linked immunosorbent assay (ELISA). Inflammatory cells (CD4+ T cells, CD8+ T cells, CD1a+ cells, eosinophils and mast cells) were stained immunohistochemically and quantified by image analysis. TSLP level was significantly increased in blister fluids of BP and was highly expressed in lesional skin of BP and AD. Serum levels of IL-6, IL-4, IL-22, IFN-γ and thymic activation regulates chemokines (TARC) were significantly higher in patients with BP and AD than in healthy controls. CD4+ T cells, CD8+ T cells and CD1a+ cells were significantly more in upper dermis of BP and AD lesions. Eosinophils were found more in BP lesions while mast cells were found more in AD lesions than in healthy controls. A distinct correlation was found between TSLP levels and the intensities of CD4+ T cells, CD1a+ cells infiltrations. TSLP was significantly higher in blister fluids and skin lesions of BP, suggesting that it might contribute to the pathogenesis of BP. BP exhibited a similar type 2 immune response and a slight difference in cells infiltrations with AD.

Phellopterin alleviates atopic dermatitis-like inflammation and suppresses IL-4-induced STAT3 activation in keratinocytes

Anti-inflammation medication is one of the most important treatment for people with atopic dermatitis (AD) which presents persistent type 2 inflammation in skin lesions. Interaction between activated keratinocytes and immune cells in AD skin lesions amplifies inflammatory signaling by augmenting production of cytokines, such as keratinocyte-derived thymic stromal lymphopoietin (TSLP) and interleukin-33 (IL-33). Phellopterin is a bioactive compound isolated from ethanol extract of Angelica dahurica root which has been traditionally used for AD therapy in China. In the present study, we showed that Phellopterin possessed anti-type 2 inflammation activity and alleviated AD-like phenotypes including reduction in serum immunoglobulin E (IgE) levels and infiltration of eosinophils and mast cells in the AD-like skin lesions. Further molecular analysis found that Phellopterin suppressed phosphorylation of signal transducer and activator of transcription 3 (STAT3) at Tyr705, and the expression of TSLP and IL-33 in epidermal keratinocytes of AD-like lesions. In vitro studies in cultured human keratinocytes demonstrated that STAT3 was required for interleukin-4 (IL-4)-induced overexpression of TSLP and IL-33. Phellopterin inhibited IL-4-induced activation of STAT3, which leaded to suppress the STAT3-mediated transcription of TSLP and IL-33. Our study suggested that Phellopterin is an active compound with bioactivities of anti- type 2 inflammation and STAT3 inactivation, thus allowing to be a promising candidate for AD topical therapy.

The Sera levels of Interleukin-13 in Patients with Atopic Dermatitis in Thi-Qar province

The Interleukin-13 (IL-13) is a component of the innate immune system that is primarily produced by Th2 cells and is found in high concentrations in skin lesions of atopic dermatitis. The study comprised 90 people of both genders, 45 of whom were patients who attending to Al-Nassirya teaching Hospital, Dermatology, during a period between November 2021 to March 2022, and the same number served as controls. Blood samples were obtained from both groups, and the serum levels of IL-13 were estimated using an ELISA-ready kit.The findings of the current study referred that young adult, and the females gender were as more effective in Atopic Dermatitis, andthe female to male ratio was 1.14: 1. On the other hand, the results indicated there were elevated sera levels of IL-13 in AD patients in contrast to the controls group. Also, there is no significant difference in IL-13 sera levels among patients groups regarding to severity of AD P˃0.05). we concluded from a significant difference between the healthy control group and patients that IL-13involved in the pathogenesisof AD

The Genetics of Eczema Herpeticum.

Eczema herpeticum (EH) is a viral skin infection caused by herpes simplex virus (HSV) superimposed on eczematous skin lesions in atopic dermatitis (AD). Though the pathogenesis of EH has yet to be fully elucidated, the fact that EH is relatively rare despite a majority of adults showing serologic evidence of HSV exposure points to a genetic component predisposing to the disease. A number of genetic variants have been isolated in EH which may help distinguish a subgroup of patients susceptible to developing the condition. These unique genetic characteristics include deficiencies in skin barrier function and hydration, as well as at the level of the immune system. In this review, we summarize the genetic findings associated with EH identified to date. Isolating genetic markers in EH will be instrumental in stratifying patients at risk for EH and will have significant implications in the development and tailoring of targeted therapies.

33557 Dupilumab treatment restores skin barrier function and improves clinical and patient reported outcomes in adults and adolescents with moderate to severe atopic dermatitis

Objective: To determine the effect of dupilumab on skin barrier function in adults and adolescents with moderate to severe atopic dermatitis (AD). Methods: Transepidermal water loss (TEWL) was measured before and after skin tape stripping (STS) in AD lesional skin (n = 26) over 16 weeks of dupilumab therapy, and matched healthy control skin (n = 26) in an Open Label Exploratory study (BALISTAD [NCT04447417]). TEWL before and after STS was assessed repeatedly between baseline and Week 16. Change from baseline in TEWL was analyzed using a mixed model, adjusting for age, gender, and lesion location. Secondary and exploratory endpoints included clinical, and patient reported outcomes. Photographic documentation was also obtained during the study. Results: The mean TEWL in AD lesions was significantly reduced from baseline, (both before STS and following 5/10/15/20 STS) ranging from 47.2, 62.5, 73.0, 80.4, and 88.0 g/m2 x h at baseline to 23.6, 28.3, 34.3, 42.9, and 49.2 g/m2 x h at Week 16, respectively; representing 48% to 57% reduction (P < .0001). By week 16, the mean distribution of TEWL in AD patients was close to matched healthy controls. Clinical and patient reported outcomes (EASI, SCORAD, peak pruritus NRS, sleep quality NRS, POEM and DLQI/CDLQI) significantly improved from baseline (P < .0001 for all endpoints except CDLQI [P < .01]). Conclusions: Dupilumab treatment leads to significant improvement in TEWL in AD lesional skin, demonstrating restoration of epidermal barrier function and improvement in signs and symptoms of AD.

405 Atopic dermatitis patients have decreased epidermal innervation but increased neuronal substance p expression

Severe pruritus in atopic dermatitis (AD) significantly impairs patients’ quality of life, primarily due to relentless itching. Sensory nerves in the epidermis are important in mediating sensation of itch, but there is conflicting evidence whether epidermal nerve density is increased or decreased in lesional skin of AD. Here we measured nerve length, branching, and neurotransmitter expression in epidermis of healthy controls and lesional and non-lesional skin of AD patients. Four millimeter punch biopsies were taken from skin of 8 healthy adult control patients and 8 lesional and non-lesional skin of patients with moderate-to-severe AD.

Bitter taste receptor T2R38 is expressed on skin-infiltrating lymphocytes and regulates lymphocyte migration

Bitter taste receptors (T2Rs) are G protein-coupled receptors involved in the perception of bitter taste on the tongue. In humans, T2Rs have been found in several sites outside the oral cavity. Although T2R38 has been reported to be expressed on peripheral lymphocytes, it is poorly understood whether T2R38 plays immunological roles in inflammatory skin diseases such as atopic dermatitis (AD). Then, we first confirmed that T2R38 gene expression was higher in lesional skin of AD subjects than healthy controls. Furthermore, skin T2R38 expression levels were correlated with serum thymus and activation-regulated chemokine and IgE levels in AD patients. In lesional skin of AD, section staining revealed that CD3+ T cells in the dermis were T2R38 positive. In addition, flow cytometry analysis showed T2R38 expression in skin T cells. Migration assays using T2R38-transduced Jurkat T cell leukemia cells revealed that T2R38 agonists exerted a dose-dependent migration inhibitory effect. Moreover, skin tissue extracts, as well as supernatants of cultured HaCaT keratinocytes, caused T2R38-dependent migration inhibition, indicating that there should be an endogenous ligand for T2R38 in the skin epidermis. These findings implicate T2R38 as a migratory inhibitory receptor on the skin-infiltrating lymphocytes and as a therapeutic target for allergic/inflammatory skin diseases.

Immunological Pathomechanisms of Spongiotic Dermatitis in Skin Lesions of Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic pruritic skin disease with a complex pathogenesis underlying its heterogeneous clinical phenotypes and endotypes. The skin manifestation of AD reflects the cytokine milieu of a type-2-dominant immunity axis induced by genetic predisposition, innate immunity dysregulation, epidermal barrier defects, and allergic inflammation. However, the detailed pathomechanism of eczematous dermatitis, which is the principal characteristic of AD, remains unclear. This review examines previous studies demonstrating research progress in this area and considers the immunological pathomechanism of “spongiotic dermatitis”, which is the histopathological hallmark of eczematous dermatitis. Studies in this field have revealed the importance of IgE-mediated delayed-type hypersensitivity, the Fas/Fas-ligand system, and cell-mediated cytotoxicity in inducing the apoptosis of keratinocytes in spongiotic dermatitis. Recent studies have demonstrated that, together with infiltrating CD4 T cells, IgE-expressing dendritic cells (i.e., inflammatory dendritic epidermal cells and Langerhans cells) that capture specific allergens (i.e., house dust mites) are present in the spongiotic epidermis of lichenified eczema in patients with IgE-allergic AD. These findings suggest that IgE-mediated delayed-type hypersensitivity plays a pivotal role in the pathogenesis of spongiotic dermatitis in the skin lesions of AD.

Inhibitory effect of Isatis tinctoria L. water extract on DNCB-induced atopic dermatitis in BALB/c mice and HaCaT cells.

BackgroundIsatis tinctoria L (PLG) is a medicinal herb from the roots of Isatis indigotica Fort (Family Cruciferae). Previous studies have shown that PLG has anti-inflammatory and therapeutic effects against conditions such as acute and chronic hepatitis, various respiratory inflammations, and cancer. The purpose of this study was to define the pharmacological effects of PLG on inflammatory reactions and skin hyperkeratosis, which are the main symptoms of atopic dermatitis (AD), in vivo and in vitro.MethodsFor the AD in vivo experiment, 2,4-dinitrochlorobenzene (DNCB) induction and oral administration of PLG were performed on male BALB/c mice for four weeks. For in vitro experiments, keratinocytes were activated using TNF-α/IFN-γ in cultured human keratinocyte (HaCaT) cells. PLG inhibited inflammatory chemokine production and blocked the nuclear translocation of NF-κB p65 in activated keratinocytes.ResultsAs a result of oral administration of PLG, dermis and epidermis thickening, as well as eosinophil and mast cell infiltration, were attenuated in AD skin lesions. In addition, the levels of immunoglobulin E (IgE), pro-inflammatory cytokines, and the MAPK/NF-κB signaling pathway were decreased in serum and dorsal skin tissues. Furthermore, PLG inhibited inflammatory chemokine production and blocked the nuclear translocation of NF-κB p65 in activated keratinocytes. In addition, epigoitrin and adenosine, the standard compounds of PLG, were identified as candidate AD compounds.ConclusionsThese results indicate that PLG is a potent therapeutic agent for attenuating symptoms of AD.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13020-022-00624-5.

Immune‐modulating Effects of Low Carbohydrate Ketogenic Foods in Healthy Canines

Ketogenic foods (KF) limit digestible carbohydrate but contain high fat, and have antioxidant and anti‐inflammatory effects as well as improving mitochondrial function. Beta hydroxybutyrate (BHB), one of the ketone bodies, has been reported to reduce pro‐inflammatory NLRP3 inflammasome, as well chemokines including CXCL2 and CCL5 in cultures. We assessed the immune‐modulating effects of two low carbohydrate (LoCHO) foods varying in protein and fat, and compared their effects to a food replete with carbohydrate (CON) in healthy canines. This study was approved by IACUC and the Animal Welfare Committee, Hills PNC. Macronutrients (P/F/C; % energy) for the foods were: CON (23/34/35), LoCHO1 (53/39/8) and LoCHO2 (27/67/5). We previously reported that LoCHO2 significantly increased circulating BHB compared to both CON and LoCHO1 although LoCHO1 did not increase BHB above that found when dogs consumed CON food. In the present study, dogs were fed control food (CON; ketogenic ratio (KR 0.46) followed by LoCHO1 (KR: 0.97), then LoCHO2 (KR: 1.63) or LoCHO2 followed by LoCHO1. Each food was fed for 5 weeks, with collections in the 5th week; 15 weeks feeding total. Gene expression for inflammatory cytokines and receptors from whole blood collected in PAXgene RNA tubes from 10 dogs was assessed using Canine RT2 Profiler PCR array. When compared to CON there was a significant decrease in several pro‐inflammatory cytokines/chemokines in both LoCHO1 and LoCHO2 groups including CCL1, CCL8, CCL13, CCL17,CCL24, CX3CL1, CXCR1, IL‐10RA, IL‐17A, IL‐1RN, IL‐5, IL‐9, and SPP1 (all p&lt;0.05). Interestingly, a subset of inflammatory proteins that decreased in LoCHO1, but not in LoCHO2, included IL‐33, IL‐6R, Il‐7, IL‐8, NAMPT, and TNFRSF11B. In contrast, the decrease in inflammatory markers in LoCHO2, but not in LoCHO1, included C5, CSF3, IFN‐γ, IL‐3, IL‐10RB, IL‐17C, TNFSF13, TNFSF13B, and TNFSF14. Decreased levels of selected cytokines indicate the ability of both low‐carbohydrate foods to exert an anti‐inflammatory effect and provide a strong rationale for testing its efficacy in dogs with inflammatory conditions. For instance, cytokines including IL‐7 and IL‐8 are increased in chronic inflammatory conditions such as colitis, dermatitis, as well as in autoimmune conditions. Increased IL‐33 is also associated with chronic lesional skin of atopic dermatitis in canines and these conditions might benefit from LoCHO1 food. LoCHO2 food decreased complement component 5 (C5) and blockade of C5 attenuates renal dysfunction including glomerulopathy. LoCHO2 also decreased TNFSF13, a member of the tumor necrosis factor superfamily, highly expressed in tumors, and associated with poor prognosis, indicating its potential as a nutritional intervention strategy for cancer. Taken together, our results indicate that both LoCHO1 and LoCHO2 foods might be important as part of immune‐modulating therapeutic nutritional strategies to reduce inflammation or inflammatory pathways to maintain health in canines.

Expression of Staphylococcus aureus Virulence Factors in Atopic Dermatitis

Atopic dermatitis (AD) is a skin inflammatory disease in which the opportunistic pathogen Staphylococcus aureus is prevalent and abundant. S. aureus harbors several secreted virulence factors that have well-studied functions in infection models, but it is unclear whether these extracellular microbial factors are relevant in the context of AD. To address this question, we designed a culture-independent method to detect and quantify S. aureus virulence factors expressed at the skin sites. We utilized RNase-H‒dependent multiplex PCR for preamplification of reverse-transcribed RNA extracted from tape strips of patients with AD sampled at skin sites with differing severity and assessed the expression of a panel of S. aureus virulence factors using qPCR. We observed an increase in viable S. aureus abundance on sites with increased severity of disease, and many virulence factors were expressed at the AD skin sites. Surprisingly, we did not observe any significant upregulation of the virulence factors at the lesional sites compared with those at the nonlesional control. Overall, we utilized a robust assay to directly detect and quantify viable S. aureus and its associated virulence factors at the site of AD skin lesions. This method can be extended to study the expression of skin microbial genes at the sites of various dermatological conditions.

Influence of the Th1 Cytokine Environment on CCL5 Production from Langerhans Cells.

In the chronic skin lesions of atopic dermatitis (AD), T helper type 1 (Th1) cells appear in addition to Th2 cells, but the role played by Th1 cells in skin inflammation during the chronic phase remains unknown. Here we examined CCL5 production from Langerhans cells (LCs) in the Th1 cytokine environment. LCs were generated from mouse bone marrow cells, then stimulated with anti-CD40 antibody and the Th1 cytokine, interferon (IFN)-γ. Their CCL5 production was then measured. In addition, the LCs were incubated with naïve CD4+ T cells from a DO 11.10 TCR Tg mouse in the presence of ovalbumin peptide for 5 d, and their IFN-γ, interleukin-4 and CCL5 production was then measured. When LCs were stimulated with the anti-CD40 antibody in the presence of IFN-γ, significant levels of CCL5 production were confirmed. Furthermore, when LCs presented antigen to Th cells in the Th1 cytokine environment, significant levels of CCL5 production were induced. This CCL5 production was associated with IFN-γ activity and CD40L expression by Th cells in the culture. Our present data suggest that LCs augment CCL5 production by responding to IFN-γ while presenting antigen to Th cells, and that this augmentation of CCL5 production would likely contribute to infiltration of eosinophils and other Th1 cells into skin lesions, followed by expansion of chronic inflammation in the skin.

Single-cell transcriptome profile of mouse skin undergoing antigen-driven allergic inflammation recapitulates findings in atopic dermatitis skin lesions

Allergic skin inflammation elicited in mice by epicutaneous (EC) sensitization with antigen shares characteristics with human atopic dermatitis (AD). We characterized gene expression by single cells in mouse skin undergoing antigen-driven allergic inflammation and compared the results with findings in AD skin lesions. Mice were EC sensitized by application of ovalbumin (OVA) or saline to tape-stripped skin. Single-cell RNA sequencing was performed on skin cells 12 days later. Flow cytometry analysis was performed to validate results. Sequencing identified 7 nonhematopoietic and 6 hematopoietic cell subsets in EC-sensitized mouse skin. OVA sensitization resulted in the expansion in the skin of T cells, dendritic cells, macrophages, mast cells/basophils, fibroblasts, and myocytes cell clusters, and in upregulation of TH2 cytokine gene expression in CD4+ T cells and mast cells/basophils. Genes differentially expressed in OVA-sensitized skin included genes important for inflammation in dendritic cells and macrophages, collagen deposition, and leukocyte migration in fibroblasts, chemotaxis in endothelial cells and skin barrier integrity, and differentiation in KCs-findings that recapitulate those in AD skin lesions. Unexpectedly, mast cells/basophils, rather than T cells, were the major source of Il4 and ll13 in OVA-sensitized mouse skin. In addition, our results suggest novel pathways in fibroblast and endothelial cells that may contribute to allergic skin inflammation. The gene expression profile of single cells in mouse skin undergoing antigen-driven shares many features with that in AD skin lesions and unveils novel pathways that may be involved in allergic skin inflammation.