Mapping the immune cell landscape of severe atopic dermatitis by single-cell RNA-seq.

BACKGROUND: Efforts to profile atopic dermatitis (AD) tissues have intensified, yet comprehensive analysis of systemic immune landscapes in severe AD remains crucial. METHODS: Employing single-cell RNA sequencing, we analyzed over 300,000 peripheral blood mononuclear cells from 12 severe AD patients (Eczema area and severity index (EASI) > 21) and six healthy controls. RESULTS: Results revealed significant immune cell shifts in AD patients, including increased Th2 cell abundance, reduced NK cell clusters with compromised cytotoxicity, and correlated Type 2 innate lymphoid cell proportions with disease severity. Moreover, unique monocyte clusters reflecting activated innate immunity emerged in very severe AD (EASI > 30). While overall dendritic cells (DCs) counts decreased, a distinct Th2-priming subset termed "Th2_DC" correlated strongly with disease severity, validated across skin tissue data, and flow cytometry with additional independent severe AD samples. Beyond the recognized role of Th2 adaptive immunity, our findings highlight significant innate immune cell alterations in severe AD, implicating their roles in disease pathogenesis and therapeutic potentials. CONCLUSION: Apart from the widely recognized role of Th2 adaptive immunity in AD pathogenesis, alterations in innate immune cells and impaired cytotoxic cells have also been observed in severe AD. The impact of these alterations on disease pathogenesis and the effectiveness of potential therapeutic targets requires further investigation.

Long-term control of diabetes by tofacitinib-based immunosuppressive regimen after allo islet transplantation in diabetic rhesus monkeys that rejected previously transplanted porcine islets.

Porcine islet xenotransplantation has been highlighted as an alternative to allo islet transplantation. Despite the remarkable progress that has been made in porcine-islet pre-clinical studies in nonhuman primates, immunological tolerance to porcine islets has not been achieved to date. Therefore, allo islet transplantation could be required after the failure of porcine islet xenotransplantation. Here, we report the long-term control of diabetes by allogeneic pancreatic islet transplantation in diabetic rhesus monkeys that rejected previously transplanted porcine islets. Four diabetic male rhesus monkeys received the porcine islets and then allo islets (5700-19 000 IEQ/kg) were re-transplanted for a short or long period after the first xeno islet rejection. The recipient monkeys were treated with an immunosuppressive regimen consisting of ATG, humira, and anakinra for induction, and sirolimus and tofacitinib for maintenance therapy. The graft survival days of allo islets in these monkeys were >440, 395, >273, and 127, respectively, similar to that in allo islet transplanted cynomolgus monkeys that received the same immunosuppressive regimen without xeno sensitization. Taken together, it is likely that prior islet xenotransplantation does not affect the survival of subsequent allo islets under clinically applicable immunosuppressants.

Spatial transcriptomic analysis of tumour-immune cell interactions in melanoma arising from congenital melanocytic nevus.

BACKGROUND: Studies on the interaction between tumour-infiltrating immune cells (TIICs) and tumour cells in melanoma arising from congenital melanocytic nevus (CMN) are lacking. OBJECTIVE: The aim of this study was to determine the intratumoral immune landscape of TIICs and tumour cells during invasion and metastasis. METHODS: Tissue specimens were obtained from patients with melanoma originating from CMN. Differential gene expression in melanoma cells and TIICs during invasion and metastasis was determined using spatial transcriptomics. RESULTS: As invasion depth increased, the expression of LGALS3, known to induce tumour-driven immunosuppression, increased in melanoma cells. In T cells, the expression of genes that inhibit T-cell activation increased with increasing invasion depth. In macrophages, the expression of genes related to the anti-inflammatory M2 phenotype was upregulated with increasing invasion depth. Compared to primary tumour cells, melanoma cells in metastatic lesions showed upregulated expression of genes associated with cancer immune evasion, including AXL and EPHA2, which impede T-cell recruitment, and BST2, associated with M2 polarization. Furthermore, T cells showed increased expression of genes related to immunosuppression, and macrophages exhibited increased expression of genes associated with the M2 phenotype. CONCLUSIONS: The interaction between melanomas arising from CMN and TIICs may be important for tumour progression and metastasis.

Detecting T-cell receptor clonality in patients with severe atopic dermatitis refractory to dupilumab.

BACKGROUND: Trials and real-life studies demonstrated clinically meaningful improvements of disease activity in the majority of patients with moderate to severe atopic dermatitis (AD) treated with the anti-IL-4RA-antibody dupilumab. However, misdiagnosis or confounding skin diseases in particular cutaneous T-cell lymphoma (CTCL) may lead to inadequate response. OBJECTIVE: To investigate the clinical and pathological features of patients with AD who showed insufficient response to dupilumab. METHODS: We reviewed the medical records of 371 patients treated with dupilumab for severe AD. Insufficient response was defined as failure to achieve an improvement of the eczema area severity index (EASI) of at least 50% (EASI-50) at Week 16 and of 75% (EASI-75) at Week 52. Among 46 patients with insufficient response, 35 patients consented to a re-evaluation including a full physical exam, biopsies and laboratory assessments including immunohistochemistry and T-cell receptor gene rearrangement analysis to differentiate CTCL. RESULTS: Of the 371 patients treated with dupilumab, 46 (12.3%) patients showed insufficient response to dupilumab. Of these, 35 underwent further evaluation, and 19 (54.2% of inadequate responders) were finally diagnosed with mycosis fungoides (MF). In these patients, transition to or addition of conventional MF treatment led to clinical improvements. CONCLUSIONS: Insufficient response to dupilumab treatment may help uncover early MF on an existing AD background.

Systemic Inflammatory Proteomic Biomarkers in Atopic Dermatitis: Exploring Potential Indicators for Disease Severity.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory cutaneous disorder, that emerges from intricate interplays among genetic predisposition, immune dysregulation, environmental factors, and compromised skin barrier. Understanding the inflammatory pathway in AD is important due to its fundamental role in the pathogenesis of AD. This study aimed to explore the diverse spectrum of proteins linked to the inflammation of AD and the relationship between systemic biomarkers and clinical severity in AD. METHODS: We examined the blood samples from 48 patients with AD and 48 healthy controls (HCs) using the Proximity Extension Assay (Olink). Differentially expressed proteins (DEPs) were identified and Pearson correlation analysis was conducted to determine systemic proteomic biomarkers associated with severity of AD. RESULTS: A total of 29 DEPs were significantly up-regulated and 2 DEPs were significantly down-regulated in AD compared with the HC. The MCP-4, IL-18, MCP-3, TNFRSF9, and IL-17C were the top 5 highest DEPs associated with the severity of AD. CONCLUSION: Our study sheds light on the intricate network of inflammatory proteins in AD and their potential implications for disease severity. Our results indicate that these systemic inflammatory proteins could be valuable for assessing AD severity and enhancing our understanding of the disease's complexity and its potential management strategies.

Single-cell transcriptomics applied to emigrating cells from psoriasis elucidate pathogenic versus regulatory immune cell subsets.

BACKGROUND: In previous human skin single-cell data, inflammatory cells constituted only a small fraction of the overall cell population, such that functional subsets were difficult to ascertain. OBJECTIVE: Our aims were to overcome the aforesaid limitation by applying single-cell transcriptomics to emigrating cells from skin and elucidate ex vivo gene expression profiles of pathogenic versus regulatory immune cell subsets in the skin of individuals with psoriasis. METHODS: We harvested emigrating cells from human psoriasis skin after incubation in culture medium without enzyme digestion or cell sorting and analyzed cells with single-cell RNA sequencing and flow cytometry simultaneously. RESULTS: Unsupervised clustering of harvested cells from psoriasis skin and control skin identified natural killer cells, T-cell subsets, dendritic cell subsets, melanocytes, and keratinocytes in different layers. Comparison between psoriasis cells and control cells within each cluster revealed that (1) cutaneous type 17 T cells display highly differing transcriptome profiles depending on IL-17A versus IL-17F expression and IFN-γ versus IL-10 expression; (2) semimature dendritic cells are regulatory dendritic cells with high IL-10 expression, but a subset of semimature dendritic cells expresses IL-23A and IL-36G in psoriasis; and (3) CCL27-CCR10 interaction is potentially impaired in psoriasis because of decreased CCL27 expression in basal keratinocytes. CONCLUSION: We propose that single-cell transcriptomics applied to emigrating cells from human skin provides an innovative study platform to compare gene expression profiles of heterogenous immune cells in various inflammatory skin diseases.

Mild atopic dermatitis lacks systemic inflammation and shows reduced nonlesional skin abnormalities.

BACKGROUND: Molecular studies in atopic dermatitis (AD) are largely restricted to patients with moderate-to-severe disease. OBJECTIVE: Our aim was to evaluate skin and blood abnormalities in mild, moderate, and severe AD. METHODS: Skin and blood samples were obtained from 61 patients with AD (20 with mild or limited disease, 17 with moderate disease, and 24 with severe disease) and 20 healthy subjects. Immune and barrier markers were measured in lesional, nonlesional, and healthy skin by quantitative real-time PCR and immunohistochemistry, and in blood by using the OLINK proteomic assay. RESULTS: Cellular markers of epidermal hyperplasia and T-cell/dendritic cell infiltration were increased in AD tissues of all patients in all severity groups versus in those of controls, whereas downstream TH2 cell-, TH22 cell-, TH1 cell-, and TH17 cell-related mediators demonstrated incremental elevations with increasing disease severity, in both lesional and nonlesional skin. Whereas the levels of the TH2 (IL13, CCL17, and CCL26) and TH22 (IL-22) cytokines were significantly elevated in both AD lesional and nonlesional skin of all patients regardless of the severity of their disease, patients with mild or limited AD showed increases in their levels of TH1 cell (IFNG, CXCL9, and CXCL10) and TH17 cell (IL-17A, CCL20, and CXCL1) markers in lesional but not nonlesional skin. Regulatory T-cell-related mediators (IL-10 and FOXP3) were comparably upregulated in all groups, without displaying the severity-based gradient in other immune axes. Unsupervised clustering aligned samples along a severity spectrum, where nonlesional mild or limited AD skin clustered with the samples from healthy controls. Furthermore, whereas the blood profiles of patients with moderate and severe AD showed gradual increases in the levels of TH1 cell-, TH2 cell-, and TH17 cell-related and atherosclerosis and/or cardiovascular risk (CCL7, FGF21, and IGFBP1) proteins, the blood profiles of patients with mild or limited AD lacked significant differences from those of the controls. CONCLUSION: Mild and limited AD show high levels of TH2/TH22 cell activation that is primarily localized to skin lesions and lacks the systemic inflammation of moderate and severe disease.

Long-term control of diabetes in a nonhuman primate by two separate transplantations of porcine adult islets under immunosuppression.

Porcine islet transplantation is an alternative to allo-islet transplantation. Retransplantation of islets is a routine clinical practice in islet allotransplantation in immunosuppressed recipients and will most likely be required in islet xenotransplantation in immunosuppressed recipients. We examined whether a second infusion of porcine islets could restore normoglycemia and further evaluated the efficacy of a clinically available immunosuppression regimen including anti-thymocyte globulin for induction; belimumab, sirolimus, and tofacitinib for maintenance and adalimumab, anakinra, IVIg, and tocilizumab for inflammation control in a pig to nonhuman primate transplantation setting. Of note, all nonhuman primates were normoglycemic after the retransplantation of porcine islets without induction therapy. Graft survival was >100 days for all 3 recipients, and 1 of the 3 monkeys showed insulin independence for >237 days. Serious lymphodepletion was not observed, and rhesus cytomegalovirus reactivation was controlled without any serious adverse effects throughout the observation period in all recipients. These results support the clinical applicability of additional infusions of porcine islets. The maintenance immunosuppression regimen we used could protect the reinfused islets from acute rejection.

High-dimensional analysis defines multicytokine T-cell subsets and supports a role for IL-21 in atopic dermatitis.

BACKGROUND: Flow cytometry is a well-accepted approach for immune profiling; however, its value is restricted by the limited number of markers that can be analyzed simultaneously. Mass cytometry/CyTOF offers broad-scale immune characterization integrating large number of parameters. While partial blood phenotyping was reported in atopic dermatitis (AD), patients' comprehensive profiling, critical for leveraging new targeted treatments, is not available. IL-21 may be involved in inflammatory skin diseases but its role in AD is not well established. METHODS: We studied T-cell polarization in the blood of 20 moderate-to-severe AD and 15 controls. Using CyTOF and an unsupervised analysis, we measured the frequencies and mean metal intensities of activated polar CD4+ /CD8+ T-cell subsets. Immunohistochemistry, immunofluorescence, and qRT-PCR were used to analyze skin samples. RESULTS: Examining 24 surface, intracellular markers, and transcription factors, we identified six CD4+ and five CD8+ T-cell metaclusters. A CD4+ skin-homing IL-13+ monocytokine and a novel IL-13+ IL-21+ multicytokine metaclusters were increased in AD vs. controls (p < .01). While IL-13 signature characterized both clusters, levels were significantly higher in the IL-21+ group. Both clusters correlated with AD severity (r = 0.49, p = .029). Manual gating corroborated these results and identified additional multicytokine subsets in AD. Immunohistochemistry and immunofluorescence, validated by mRNA expression, displayed significantly increasedIL-21 counts and colocalization with IL-13/IL-4R in AD skin. CONCLUSION: A multicytokine signature characterizes moderate-to-severe AD, possibly explaining partial therapeutic responses to one cytokine targeting, particularly in severe patients. Prominent IL-21 signature in blood and skin hints for a potential pathogenic role of IL-21 in AD.

Novel Immunomodulatory Approaches for Porcine Islet Xenotransplantation.

PURPOSE OF REVIEW: Porcine islet xenotransplantation is a promising alternative to overcome the shortage of organ donors. For the successful application of islet xenotransplantation, robust immune/inflammatory responses against porcine islets should be thoroughly controlled. Over the last few decades, there have been numerous attempts to surmount xenogeneic immune barriers. In this review, we summarize the current progress in immunomodulatory therapy for the clinical application of porcine islet xenotransplantation. RECENT FINDINGS: Long-term graft survival of porcine islets was achieved by using anti-CD154 Ab-based regimens in a preclinical non-human primate (NHP) model. However, owing to a serious complication of thromboembolism in clinical trials, the development of an anti-CD154 Ab-sparing immunosuppressant procedure is required. The efficacy of new immunosuppressive practices that employ anti-CD40 Abs or other immunosuppressive reagents has been tested in a NHP model to realize their utility in porcine islet xenotransplantation. The recent progress in the development of immunomodulatory approaches, including the immunosuppressive regimen, which enables long-term graft survival in a pig-to-non-human primate islet xenotransplantation model, with their potential clinical applicability was reviewed.

Long-term porcine islet graft survival in diabetic non-human primates treated with clinically available immunosuppressants.

BACKGROUND: Although pancreatic islet transplantation is becoming an effective therapeutic option for patients with type 1 diabetes (T1D) who suffer from a substantially impaired awareness of hypoglycemia, its application is limited due to the lack of donors. Thus, pig-to-human islet xenotransplantation has been regarded as a promising alternative due to the unlimited number of "donor organs." Long-term xenogeneic islet graft survival in pig-to-non-human primate (NHP) models has mainly been achieved by administering the anti-CD154 mAb-based immunosuppressant regimen. Since the anti-CD154 mAb treatment has been associated with unexpected fatal thromboembolic complications in clinical trials, the establishment of a new immunosuppressant regimen that is able to be directly applied in clinical trials is an urgent need. METHODS: We assessed an immunosuppressant regimen composed of clinically available agents at porcine islet transplantation in consecutive diabetic NHPs. RESULTS: Porcine islet graft survival in consecutive diabetic NHPs (n = 7; >222, >200, 181, 89, 62, 55, and 34 days) without severe adverse events. CONCLUSION: We believe that our study could contribute greatly to the initiation of islet xenotransplantation clinical trials.

Duration of Oral Antibiotics Administration for Cetuximab-Induced Acneiform Eruption.

BACKGROUND: Acneiform eruption is the most common cutaneous adverse event associated with cetuximab. As it can affect quality of life and adversely affect chemotherapy schedule, additional medical care is required. OBJECTIVES: To investigate the adherence to and the duration of antibiotic administration to treat cetuximab-induced acneiform eruption. METHODS: Medical data of patients who were referred to the Department of Dermatology were reviewed from January 2013 to June 2018. Dermatologists assessed the severity of acneiform eruption and prescribed tetracycline-class antibiotics according to the severity every 2 or 4 weeks. We investigated the duration and amount of oral antibiotic administration and analyzed the factors that may affect the control of acneiform eruption statistically. RESULTS: A total of 207 of 267 patients referred to the Department of Dermatology showed acneiform eruption; 124 patients were treated with minocycline, 34 patients with doxycycline, 27 patients with both, and 22 patients with topical agents. The mean duration of oral antibiotic medication was 82.7 days. A statistical analysis of the factors that prolonged the use of antibiotics for more than 90 days showed that male and younger age were risk factors. Shorter time interval from starting cetuximab to starting antibiotics was associated with longer duration of antibiotic use, statistically. CONCLUSIONS: Cetuximab-induced acneiform eruption can be well controlled with tetracycline-class antibiotics in about 3 months. It can last longer in male and younger patients. The sooner and the more severe it appears, the longer it can last.

Effect of photobiomodulation therapy on radiodermatitis in a mouse model: an experimental animal study.

This study aimed to evaluate the effect of photobiomodulation (PBM) for prevention of radiodermatitis in an irradiated mouse model and compare the efficacy of PBM using 633- or 830-nm wavelengths. Irradiated mice were randomly distributed into three groups: A (633 nm), B (830 nm), and C (without PBM). On post-irradiation days 7 and 21, we compared acute damage and recovery in treated skin samples to non-irradiated skin using H&E, Masson's trichrome, anti-CD45 and PCNA immunohistochemistry, and a TUNEL assay. Grade 3 radiodermatitis was evident only in group C. Compared with that in group C, the skin in groups A and B had significantly less epidermal hyperplasia, inflammatory cell infiltration, and thinner dermis on day 7 and less inflammatory cell infiltration, fewer apoptotic cells, and thinner dermis on day 21. However, there was no significant difference between groups A and B. This study indicates PBM could prevent severe radiodermatitis by reducing epidermal and dermal damage, inflammation, and apoptosis. There was no difference in PBM efficacy between the 633- and 830-nm wavelengths.

Single-cell transcriptome analysis of human skin identifies novel fibroblast subpopulation and enrichment of immune subsets in atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a prevalent inflammatory skin disease with a complex pathogenesis involving immune cell and epidermal abnormalities. Despite whole tissue biopsy studies that have advanced the mechanistic understanding of AD, single cell-based molecular alterations are largely unknown. OBJECTIVE: Our aims were to construct a detailed, high-resolution atlas of cell populations and assess variability in cell composition and cell-specific gene expression in the skin of patients with AD versus in controls. METHODS: We performed single-cell RNA sequencing on skin biopsy specimens from 5 patients with AD (4 lesional samples and 5 nonlesional samples) and 7 healthy control subjects, using 10× Genomics. RESULTS: We created transcriptomic profiles for 39,042 AD (lesional and nonlesional) and healthy skin cells. Fibroblasts demonstrated a novel COL6A5+COL18A1+ subpopulation that was unique to lesional AD and expressed CCL2 and CCL19 cytokines. A corresponding LAMP3+ dendritic cell (DC) population that expressed the CCL19 receptor CCR7 was also unique to AD lesions, illustrating a potential role for fibroblast signaling to immune cells. The lesional AD samples were characterized by expansion of inflammatory DCs (CD1A+FCER1A+) and tissue-resident memory T cells (CD69+CD103+). The frequencies of type 2 (IL13+)/type 22 (IL22+) T cells were higher than those of type 1 (IFNG+) in lesional AD, whereas this ratio was slightly diminished in nonlesional AD and further diminished in controls. CONCLUSION: AD lesions were characterized by expanded type 2/type 22 T cells and inflammatory DCs, and by a unique inflammatory fibroblast that may interact with immune cells to regulate lymphoid cell organization and type 2 inflammation.

Evolution of pathologic T-cell subsets in patients with atopic dermatitis from infancy to adulthood.

BACKGROUND: The circulating immune phenotype was defined in adults and young children with early atopic dermatitis (AD), but chronologic changes in the blood of infants and children with AD through adolescence have not been explored. OBJECTIVE: We sought to compare immune activation and cytokine polarization in the blood of 0- to 5-year-old (n = 39), 6- to 11-year-old (n = 26), 12- to 17-year-old (n = 21) and 18-year-old or older (n = 43) patients with AD versus age-matched control subjects. METHODS: Flow cytometry was used to measure IFN-γ, IL-9, IL-13, IL-17, and IL-22 cytokine levels in CD4+/CD8+ T cells, with inducible costimulator molecule and HLA-DR defining midterm and long-term T-cell activation, respectively, within skin-homing/cutaneous lymphocyte antigen (CLA)+ versus systemic/CLA- T cells. Unsupervised clustering differentiated patients based on their blood biomarker frequencies. RESULTS: Although CLA+ TH1 frequencies were significantly lower in infants with AD versus all older patients (P < .01), frequencies of CLA+ TH2 T cells were similarly expanded across all AD age groups compared with control subjects (P < .05). After infancy, CLA- TH2 frequencies were increased in patients with AD in all age groups, suggesting systemic immune activation with disease chronicity. IL-22 frequencies serially increased from normal levels in infants to highly significant levels in adolescents and adults compared with levels in respective control subjects (P < .01). Unsupervised clustering aligned the AD profiles along an age-related spectrum from infancy to adulthood (eg, inducible costimulator molecule and IL-22). CONCLUSIONS: The adult AD phenotype is achieved only in adulthood. Unique cytokine signatures characterizing individual pediatric endotypes might require age-specific therapies. Future longitudinal studies, comparing the profile of patients with cleared versus persistent pediatric AD, might define age-specific changes that predict AD clearance.

CD4+ /CD8+ T-cell ratio correlates with the graft fate in pig-to-non-human primate islet xenotransplantation.

BACKGROUND: Xenogeneic islet transplantation using porcine pancreata has been a promising option for substituting human islet transplantation. Moreover, recent advances in pre-clinical results have put islet xenotransplantation closer to the possibility of clinical application. While preparing for the era of clinical xenotransplantation, developing non-invasive immune monitoring method which could predict the graft fate could benefit the patient. However, there are few reports showing predictive immune parameters associated with the fate of the graft in islet xenotransplantation. METHODS: The absolute number and ratio of T-cell subsets have been measured via flow cytometry from the peripheral blood of 16 rhesus monkeys before and after porcine islet xenotransplantation. The correlation between the graft survival and the absolute number or ratio of T cells was retrospectively analyzed. RESULTS: The ratio of CD4+ versus CD8+ T cells was significantly reduced due to CD8+ effector memory cells' increase. Correlation analyses revealed that CD4+ /CD8+ , CD4+ /CD8+ naïve, CD4+ naïve/CD8+ naïve, and CD4+ central memory/CD8+ naïve cell ratios negatively correlated with the duration of graft survival. Conversely, further analyses discovered strong, positive correlation of CD4+ /CD8+ cell ratios within the early graft-rejected monkeys (≤60 days). CONCLUSIONS: This retrospective study demonstrated that CD4+ /CD8+ ratios correlated with graft survival, especially in recipients which rejected the graft in early post-transplantation periods. CD4+ /CD8+ ratios could be used as a surrogate marker to predict the graft fate in pig-to-NHP islet xenotransplantation.

Blood endotyping distinguishes the profile of vitiligo from that of other inflammatory and autoimmune skin diseases.

BACKGROUND: Peripheral blood skin-homing/cutaneous lymphocyte antigen (CLA)+ T cells emerge as biomarkers of cutaneous immune activation in patients with inflammatory skin diseases (atopic dermatitis [AD] and alopecia areata [AA]). However, blood phenotyping across these subsets is not yet available in patients with vitiligo. OBJECTIVE: We sought to measure cytokine production by circulating skin-homing (CLA+) versus systemic (CLA-) "polar" CD4+/CD8+ ratio and activated T-cell subsets in patients with vitiligo compared with patients with AA, AD, or psoriasis and control subjects. METHODS: Flow cytometry was used to measure levels of the cytokines IFN-γ, IL-13, IL-9, IL-17, and IL-22 in CD4+/CD8+ T cells in the blood of 19 patients with moderate-to-severe nonsegmental/generalized vitiligo, moderate-to-severe AA (n = 32), psoriasis (n = 24), or AD (n = 43) and control subjects (n = 30). Unsupervised clustering differentiated subjects into groups based on cellular frequencies. RESULTS: Patients with Vitiligo showed the highest CLA+/CLA- TH1/type 1 cytotoxic T-cell polarization, with parallel TH2/TH9/TH17/TH22 level increases to levels often greater than those seen in patients with AA, AD, or psoriasis (P < .05). Total regulatory T-cell counts were lower in patients with vitiligo than in control subjects and patients with AD or psoriasis (P < .001). Vitiligo severity correlated with levels of multiple cytokines (P < .1), whereas duration was linked with IFN-γ and IL-17 levels (P < .04). Patients and control subjects grouped into separate clusters based on blood biomarkers. CONCLUSIONS: Vitiligo is characterized by a multicytokine polarization among circulating skin-homing and systemic subsets, which differentiates it from other inflammatory/autoimmune skin diseases. Future targeted therapies should delineate the relative contribution of each cytokine axis to disease perpetuation.

Oral Janus kinase/SYK inhibition (ASN002) suppresses inflammation and improves epidermal barrier markers in patients with atopic dermatitis.

BACKGROUND: Moderate-to-severe atopic dermatitis (AD) has been associated with significant disease burden and systemic abnormalities and often requires systemic treatments. Currently, safe and effective oral systemic treatments for moderate-to-severe AD are not yet available. ASN002 is an oral inhibitor of the Janus kinase/spleen tyrosine kinase signaling pathways, targeting several cytokine axes (TH2/TH22/TH17/TH1) and epidermal differentiation. OBJECTIVE: We sought to evaluate the effect of ASN002 on the cellular and molecular biomarker profile of patients with moderate-to-severe AD and to correlate changes in biomarkers to improvements in clinical severity measures and pruritus. METHODS: Thirty-six patients with moderate-to-severe AD were randomized to groups with dose escalation of ASN002 (20, 40, and 80 mg) and a placebo group. Skin biopsy specimens were performed at baseline, day 15, and day 29. Gene expression studies were conducted by using microarray and quantitative RT-PCR, and cellular infiltrates and protein expression were studied by using immunohistochemistry. RESULTS: ASN002 reversed the lesional skin transcriptome toward a nonlesional phenotype. It also rapidly and significantly suppressed key inflammatory pathways implicated in AD pathogenesis, including TH2 (IL4 receptor [IL4R], IL13, CCL13/monocyte chemoattractant protein 4, CCL17/thymus and activation-regulated chemokine, CCL18/pulmonary and activation-regulated chemokine, CCL22/macrophage-derived chemokine, and CCL26/eotaxin-3), TH17/TH22 (lipocalins, PI3/elafin, CCL20, S100A7/S100A8/S100A9, and IL36G/IL36RN), and TH1 (IFNG, CXCL9/CXCL11, and MX1) axes and barrier-related measures (filaggrin [FLG] and CLDN23). Significant improvements in AD gene signatures were observed predominantly in the 40- and 80-mg groups. Smaller and largely nonsignificant molecular changes were seen in the 20-mg and placebo groups. CONCLUSION: The Janus kinase/spleen tyrosine kinase inhibitor ASN002 significantly suppressed key AD inflammatory pathways, corresponding to clinical response. ASN002 might be an effective novel therapeutic agent for moderate-to-severe AD.

High mobility group box 1 secretion blockade results in the reduction of early pancreatic islet graft loss.

Pancreatic islet transplantation has been known as the best cure for patients suffering from severe type 1 diabetes mellitus (T1DM). Despite meaningful advances in human allogeneic islet transplantation field, significant amounts of islet loss in early post-transplantation periods is still a big concern for clinicians. One of the major factors determining the fate of the islets is the danger-associated molecular patterns (DAMPs) secreted by activated immune cells or islets themselves under hypoxic stress. High mobility group box 1 (HMGB1) protein is one of the best characterized DAMP molecules associated with islets. HMGB1 is known to be passively released by transplanted murine islet cells after taking damages from cytokines, reactive oxygen species, and other DAMPS, and the released HMGB1 harms neighboring islet cells by interacting with receptors expressed on murine islets such as toll-like receptor 2 (TLR2) and TLR4, thereby forming a vicious cycle. Here, we show that a small molecule inhibitor inflachromene (ICM) was capable of blocking the secretion of HMGB1 from murine islet cells during the normoxic and hypoxic post-isolation period. Notably, the treatment of ICM during the islet isolation process resulted in decreased HMGB1 levels during the subsequent cell culture. ICM's in vivo efficacy was evaluated in murine syngeneic islet transplantation model, and it significantly reduced the serum and graft level of HMGB1. Ultimately, the intraperitoneal administration of ICM prevented the loss of marginal-mass islet grafts and reversed the diabetes in mice.

Peri-graft porcine-specific CD4+ FoxP3+ regulatory T cells by CD40-CD154 blockade prevented the rejection of porcine islet graft in diabetic mice.

BACKGROUND: Anti-CD154 monoclonal antibody (mAb) treatment has been known to have potential to induce immune tolerance in organ transplantation. Several studies have suggested the involvement of CD4+ regulatory T cells (Treg s) in xeno-immune tolerance. However, the characteristics of Treg s and the mechanisms of their regulatory functions in islet xenotransplantation have not been clearly defined. METHOD: Adult porcine islet cells were isolated and purified, and were transplanted under the kidney capsule of diabetic C57BL/6J mice with the administration of 0.5 mg/mouse of anti-CD154 mAb on 0, 1, 3, 5, and 7 days post-transplantation (DPT). The graft survival was monitored by blood glucose level. The islet graft and recipients' cells were analyzed by immunohistochemistry (IHC), flow cytometry, enzyme-linked immunosorbent spot (ELISPOT) assay, and mixed-lymphocyte reaction. RESULTS: Short-term anti-CD154 mAb monotherapy enabled the porcine islet graft to survive indefinitely in diabetic mice (n = 18). Immunohistochemical staining showed significantly higher ratio of CD4+ Foxp3+ Treg s in the peri-graft site, but not in the spleen and kidney-draining lymph node of the recipient mice. Depletion of Treg s evoked graft rejection, and adoptive transfer of Treg s from anti-CD154 mAb-treated recipients provided protection to the graft from rejection. These Treg s showed more potent suppressive capacity than those from the untreated control and were found to be porcine antigen-specific. CONCLUSIONS: In this study, we showed that anti-CD154 mAb monotherapy resulted in indefinite porcine islet graft survival in mice. The porcine-specific CD4+ Foxp3+ Treg s in the peri-graft site played the critical role in the protection of islet graft from rejection, which suggests a prospective immunosuppressive strategy for islet xenotransplantation.