Anatomically distinct fibroblast subsets determine skin autoimmune patterns.

The skin serves as a physical barrier and an immunological interface that protects the body from the external environment1-3. Aberrant activation of immune cells can induce common skin autoimmune diseases such as vitiligo, which are often characterized by bilateral symmetric lesions in certain anatomic regions of the body4-6. Understanding what orchestrates the activities of cutaneous immune cells at an organ level is necessary for the treatment of autoimmune diseases. Here we identify subsets of dermal fibroblasts that are responsible for driving patterned autoimmune activity, by using a robust mouse model of vitiligo that is based on the activation of endogenous auto-reactive CD8+ T cells that target epidermal melanocytes. Using a combination of single-cell analysis of skin samples from patients with vitiligo, cell-type-specific genetic knockouts and engraftment experiments, we find that among multiple interferon-γ (IFNγ)-responsive cell types in vitiligo-affected skin, dermal fibroblasts are uniquely required to recruit and activate CD8+ cytotoxic T cells through secreted chemokines. Anatomically distinct human dermal fibroblasts exhibit intrinsic differences in the expression of chemokines in response to IFNγ. In mouse models of vitiligo, regional IFNγ-resistant fibroblasts determine the autoimmune pattern of depigmentation in the skin. Our study identifies anatomically distinct fibroblasts with permissive or repressive IFNγ responses as the key determinant of body-level patterns of lesions in vitiligo, and highlights mesenchymal subpopulations as therapeutic targets for treating autoimmune diseases.

TRPM2-dependent autophagy inhibition exacerbates oxidative stress-induced CXCL16 secretion by keratinocytes in vitiligo.

Vitiligo is a depigmented skin disease due to the destruction of melanocytes. Under oxidative stress, keratinocyte-derived chemokine C-X-C motif ligand 16 (CXCL16) plays a critical role in recruiting CD8+ T cells, which kill melanocytes. Autophagy serves as a protective cell survival mechanism and impairment of autophagy has been linked to increased secretion of the proinflammatory cytokines. However, the role of autophagy in the secretion of CXCL16 under oxidative stress has not been investigated. Herein, we initially found that autophagy was suppressed in both keratinocytes of vitiligo lesions and keratinocytes exposed to oxidative stress in vitro. Autophagy inhibition also promoted CXCL16 secretion. Furthermore, upregulated transient receptor potential cation channel subfamily M member 2 (TRPM2) functioned as an upstream oxidative stress sensor to inhibit autophagy. Moreover, TRPM2-mediated Ca2+ influx activated calpain to shear autophagy related 5 (Atg5) and Atg12-Atg5 conjugate formation was blocked to inhibit autophagy under oxidative stress. More importantly, Atg5 downregulation enhanced the binding of interferon regulatory factor 3 (IRF3) to the CXCL16 promoter region by activating Tank-binding kinase 1 (TBK1), thus promoting CXCL16 secretion. These findings suggested that TRPM2-restrained autophagy promotes CXCL16 secretion via the Atg5-TBK1-IRF3 signaling pathway under oxidative stress. Inhibition of TRPM2 may serve as a potential target for the treatment of vitiligo. © 2024 The Pathological Society of Great Britain and Ireland.

From mice to men: An assessment of preclinical model systems for the study of vitiligo.

Vitiligo is an autoimmune skin disease of multiple etiology, for which there is no complete cure. This chronic depigmentation is characterized by epidermal melanocyte loss, and causes disfigurement and significant psychosocial distress. Mouse models have been extensively employed to further our understanding of complex disease mechanisms in vitiligo, as well as to provide a preclinical platform for clinical interventional research on potential treatment strategies in humans. The current mouse models can be categorized into three groups: spontaneous mouse models, induced mouse models, and transgenic mice. Despite their limitations, these models allow us to understand the pathology processes of vitiligo at molecule, cell, tissue, organ, and system levels, and have been used to test prospective drugs. In this review, we comprehensively evaluate existing murine systems of vitiligo and elucidate their respective characteristics, aiming to offer a panorama for researchers to select the appropriate mouse models for their study.

MIF inhibition alleviates vitiligo progression by suppressing CD8+ T cell activation and proliferation.

In vitiligo, autoreactive CD8+ T cells have been established as the main culprit considering its pathogenic role in mediating epidermal melanocyte-specific destruction. Macrophage migration inhibitory factor (MIF) is a pleiotropic molecule that plays a central role in various immune processes including the activation and proliferation of T cells; but whether MIF is intertwined in vitiligo development and progression and its involvement in aberrantly activated CD8+ T cells remains ill-defined. In this study, we found that MIF was overabundant in vitiligo patients and a mouse model for human vitiligo. Additionally, inhibiting MIF ameliorated the disease progression in vitiligo mice, which manifested as less infiltration of CD8+ T cells and more retention of epidermal melanocytes in the tail skin. More importantly, in vitro experiments indicated that MIF-inhibition suppressed the activation and proliferation of CD8+ T cells from the lymph nodes of vitiligo mice, and the effect extended to CD8+ T cells in peripheral blood mononuclear cells of vitiligo patients. Finally, CD8+ T cells derived from MIF-inhibited vitiligo mice also exhibited an impaired capacity for activation and proliferation. Taken together, our results show that MIF might be clinically targetable in vitiligo treatment, and its inhibition might ameliorate vitiligo progression by suppressing autoreactive CD8+ T cell activation and proliferation. © 2023 The Pathological Society of Great Britain and Ireland.

Self-correction of cycle threshold values by a normal distribution-based process to improve accuracy of quantification in real-time digital PCR.

The digital polymerase chain reaction (dPCR) is a new and developing nucleic acid detection technology with high sensitivity that can realize the absolute quantitative analysis of samples. In order to improve the accuracy of quantitative results, real-time digital PCR emphasizes the kinetic information during amplification to identify prominent abnormal data. However, it is challenging to use a unified standard to accurately classify the amplification curve of each well as negative and positive, due to the interference caused by various factors in the experiment. In this work, a normal distribution-based cycle threshold value self-correcting model (NCSM) was established, which focused on the feature of the cycle threshold values in amplification curves and conducted continuous detection and correction on the whole. The cycle threshold value distribution was closer to the ideal normal distribution to avoid the influence of interference. Thus, the model achieves a more accurate classification between positive and negative results. The corrective process was applied to plasmid samples and resulted in an accuracy improvement from 92 to 99%. The coefficient of variation was below 5% when considering the quantitation of a range between 100 and 10,000 copies. At the same time, by utilizing this model, the distribution of cycle threshold values at the endpoint can be predicted with fewer thermal cycles, which can reduce the cycling time by around 25% while maintaining a consistency of more than 98%. Therefore, using the NCSM can effectively enhance the quantitative accuracy and increase the detection efficiency based on the real-time dPCR platform.

Microstructure Evolution of Reactive Polyurethane Films During In Situ Polyaddition and Film-Formation Processes.

Due to the advantages of low energy consumption, no air and water pollutions, the reactive polyurethane films (RPUFs) are replacing the solvated and waterborne PUFs nowadays, which significantly promotes the green and low-carbon production of PU films. However, the microstructure evolution and in situ film-formation mechanism of RPUFs in solvent-free media are still unclear. Herein, according to time-temperature equivalence principle, the in situ polyaddition and film-formation processes of RPUFs generated by the typical polyaddition of diisocyanate terminated prepolymer (component B) and polyether glycol (component A) are thoroughly investigated at 25 °C. According to the temporal change of viscosity, the RPUFs gradually transfer from liquid to gel and finally to solid state. Further characterizing the molecular weight, hydrogen bonds, crystallinity, gel content, and phase images, the polyaddition and film-formation processes can be divided into three stages as 1) chain extension and microcrystallization; 2) gelation and demicrocrystallization; 3) microphase separation and film-formation. This work promotes the understanding of the microstructure evolution and film-formation mechanism of RPUFs, which can be used as the theoretical guidance for the controllable preparation of high-performance products based on RPUFs.

Serum cytokine profiles predict response to systemic glucocorticoid in active vitiligo.

Introduction: Vitiligo is an immune-related skin disease. Cytokines regulate immune response and inflammation and are involved in the pathogenesis of vitiligo. Aim: To assess the serum levels of pro-inflammatory cytokines pre- and post- systemic glucocorticoid treatment in patients with active vitiligo. Material and methods: We measured serum cytokine levels using the enzyme-linked immunosorbent assay in 31 patients with active vitiligo before and after treatment. All patients received systemic glucocorticoid (compound betamethasone injection) in combination with topical halometasone cream and tacrolimus ointment for 3 months. Twenty healthy controls were also examined. The cytokines measured included TNF-α, IL-1ß, IL-6, IFN-γ, IL-2, IL-17, IL-10, IL-8, and CXCL10. Results: The serum levels of TNF-α, IL-1ß, IL-6, IFN-γ, IL-2, IL-17, IL-8, and CXCL10 were significantly higher, and levels of IL-10 were lower in vitiligo patients compared to controls. Additionally, serum IFN-γ (r = 0.378; p = 0.036), IL-17 (r = 0.426; p = 0.017), and CXCL10 (r = 0.514; p = 0.003) showed a positive correlation with affected body surface area in vitiligo patients. After 3 months of systemic glucocorticoid treatment, the levels of IL-1ß, IFN-γ, IL-2, IL-17, and CXCL10 in responders were significantly decreased and nearly restored to normal levels. The IL-10 level was also increased in response to treatment. In contrast, the non-responder group had persistently high IL-6, IL-17, IL-8, and CXCL10 levels, and negligible changes in TNF-α, IL-1ß, IFN-γ, IL-2, and IL-10. Conclusions: Our study indicated that the levels of inflammatory cytokines were significantly ameliorated in the glucocorticoid responder group. Altered cell-mediated immunity may contribute to the resistance in vitiligo. The cytokines such as TNF-α, IL-1ß, IFN-γ and IL-2 could serve as therapeutic targets for managing glucocorticoid-resistant vitiligo.

Pharmacological inhibition of demethylzeylasteral on JAK-STAT signaling ameliorates vitiligo.

BACKGROUND: The activation of CD8+ T cells and their trafficking to the skin through JAK-STAT signaling play a central role in the development of vitiligo. Thus, targeting this key disease pathway with innovative drugs is an effective strategy for treating vitiligo. Natural products isolated from medicinal herbs are a useful source of novel therapeutics. Demethylzeylasteral (T-96), extracted from Tripterygium wilfordii Hook F, possesses immunosuppressive and anti-inflammatory properties. METHODS: The efficacy of T-96 was tested in our mouse model of vitiligo, and the numbers of CD8+ T cells infiltration and melanocytes remaining in the epidermis were quantified using whole-mount tail staining. Immune regulation of T-96 in CD8+ T cells was evaluated using flow cytometry. Pull-down assay, mass spectrum analysis, molecular docking, knockdown and overexpression approaches were utilized to identify the target proteins of T-96 in CD8+ T cells and keratinocytes. RESULTS: Here, we found that T-96 reduced CD8+ T cell infiltration in the epidermis using whole-mount tail staining and alleviated the extent of depigmentation to a comparable degree of tofacitinib (Tofa) in our vitiligo mouse model. In vitro, T-96 decreased the proliferation, CD69 membrane expression, and IFN-γ, granzyme B, (GzmB), and perforin (PRF) levels in CD8+ T cells isolated from patients with vitiligo. Pull-down assays combined with mass spectrum analysis and molecular docking showed that T-96 interacted with JAK3 in CD8+ T cell lysates. Furthermore, T-96 reduced JAK3 and STAT5 phosphorylation following IL-2 treatment. T-96 could not further reduce IFN-γ, GzmB and PRF expression following JAK3 knockdown or inhibit increased immune effectors expression upon JAK3 overexpression. Additionally, T-96 interacted with JAK2 in IFN-γ-stimulated keratinocytes, inhibiting the activation of JAK2, decreasing the total and phosphorylated protein levels of STAT1, and reducing the production and secretion of CXCL9 and CXCL10. T-96 did not significantly inhibit STAT1 and CXCL9/10 expression following JAK2 knockdown, nor did it suppress upregulated STAT1-CXCL9/10 signaling upon JAK2 overexpression. Finally, T-96 reduced the membrane expression of CXCR3, and the culture supernatants pretreated with T-96 under IFN-γ stressed keratinocytes markedly blocked the migration of CXCR3+CD8+ T cells, similarly to Tofa in vitro. CONCLUSION: Our findings demonstrated that T-96 might have positive therapeutic responses to vitiligo by pharmacologically inhibiting the effector functions and skin trafficking of CD8+ T cells through JAK-STAT signaling.

Improving the Encapsulation Efficiency of Lipase in Molecular Cages and Its Application.

Here, lipase encapsulation is constructed by locking enzyme molecules in nanomolecular cages on the surface of SH-PEI@PVAC magnetic microspheres. To improve the encapsulation efficiency in enzyme loading, the thiol group is efficiently modified on the grafted polyethyleneimine (PEI) using 3-mercaptopropionic acid. N2 adsorption-desorption isotherms reveal the existence of mesoporous molecular cages on the microsphere surface. The robust immobilizing strength of carriers to lipase demonstrates the successful encapsulation of enzymes in nanomolecular cages. The encapsulated lipase shows high enzyme loading (52.9 mg/g) and high activity (51.4 U/mg). Different sizes of molecular cages are established, and the cage size showed important effects on lipase encapsulation. It shows that enzyme loading is low at a small size of molecular cages, which is attributed to that the nanomolecular cage is too small to house lipase. The investigation in lipase conformation suggests that the encapsulated lipase retains its active conformation. Compared with the adsorbed lipase, the encapsulated lipase shows higher thermal stability (4.9 times) and higher resistance to denaturants (5.0 times). Encouragingly, the encapsulated lipase shows high activity and reusability in lipase-catalyzed synthesis of propyl laurate, suggesting the potential application value of encapsulated lipase.

Influences of vitiligo-associated characteristics on the occurrence of diabetes mellitus: Interactive analysis of a cross-sectional study.

The risk of diabetes mellitus (DM) in vitiligo patients is higher than that in non-vitiligo population. Our goal was to explore the influencing factors for DM in vitiligo patients. A matched-pair design of 107 cases with DM and 428 controls without DM was conducted among vitiligo patients in Xijing hospital from January 2010 to October 2021. The baseline characteristics of patients were analysed based on standard descriptive statistics. The vitiligo-associated characteristics were analysed by logistic regression to identify influencing factors of DM. Interaction analysis was performed to explore the additive interactions between vitiligo-associated characteristics and baseline characteristics. After adjustment for the baseline characteristics, the severity of vitiligo [odds ratio (OR) = 2.47, 95% confidence interval (CI): 1.47-4.14] and onset age of vitiligo (OR = 0.98, 95% CI: 0.97-0.99) had a significant correlation with occurrence of DM. The severity of vitiligo had additive interaction with family history of diabetes [relative excess risk due to interaction (RERI) = 132.51 (95% CI: 5.51-1100.20), attributable proportion (AP) = 0.91 (95% CI: 0.17-0.95), synergy index (S) = 11.53 (95% CI: 1.32-100.5)] and with smoking history [RERI = 6.54 (95% CI: 0.67-19.83), AP = 0.64 (95% CI: 0.04-0.80), S = 3.48 (95% CI: 1.17-10.36)]. Earlier onset age of vitiligo and greater BSA involvement might be two independent risk factors for DM in vitiligo patients. Interaction assessment identified the severity of vitiligo as additive interaction factors with diabetes family history and with smoking history for the DM occurrence.

Progress in biological activities and biosynthesis of edible fungi terpenoids.

The edible fungi have both edible and medicinal functions, in which terpenoids are one of the most important active ingredients. Terpenoids possess a wide range of biological activities and show great potential in the pharmaceutical and healthcare industries. In this review, the diverse biological activities of edible fungi terpenoids were summarized with emphasis on the mechanism of anti-cancer and anti-inflammation. Subsequently, this review focuses on advances in knowledge and understanding of the biosynthesis of terpenoids in edible fungi, especially in the generation of sesquiterpenes, diterpenes, and triterpenes. This paper is aim to provide an overview of biological functions and biosynthesis developed for utilizing the terpenoids in edible fungi.

A randomized study of genetic education versus usual care in tumor profiling for advanced cancer in the ECOG-ACRIN Cancer Research Group (EAQ152).

BACKGROUND: Enthusiasm for precision oncology may obscure the psychosocial and ethical considerations associated with the implementation of tumor genetic sequencing. METHODS: Patients with advanced cancer undergoing tumor-only genetic sequencing in the National Cancer Institute Molecular Analysis for Therapy Choice (MATCH) trial were randomized to a web-based genetic education intervention or usual care. The primary outcomes were knowledge, anxiety, depression, and cancer-specific distress collected at baseline (T0), posteducation (T1) and after results (T2). Two-sided, 2-sample t tests and univariate and multivariable generalized linear models were used. RESULTS: Five hundred ninety-four patients (80% from NCI Community Oncology Research Program sites) were randomized to the web intervention (n = 293) or usual care (n = 301) before the receipt of results. Patients in the intervention arm had greater increases in knowledge (P for T1-T0 < .0001; P for T2-T0 = .003), but there were no significant differences in distress outcomes. In unadjusted moderator analyses, there was a decrease in cancer-specific distress among women (T0-T1) in the intervention arm but not among men. Patients with lower health literacy in the intervention arm had greater increases in cancer-specific distress and less decline in general anxiety (T0-T1) and greater increases in depression (T0-T2) in comparison with those receiving usual care. CONCLUSIONS: Web-based genetic education before tumor-only sequencing results increases patient understanding and reduces distress in women. Refinements to the intervention could benefit low-literacy groups and men.

TMEM16F mediated phosphatidylserine exposure and microparticle release on erythrocyte contribute to hypercoagulable state in hyperuricemia.

The link between hyperuricemia (HUA) and the risk of venous thromboembolism (VTE) has been well established. However, the mechanisms of thrombus generation and the effect of HUA on procoagulant activity (PCA) of erythrocytes remain unclear no matter in uremia or hyperuricemia. Here, phosphatidylserine (PS) exposure, microparticles (MPs) release, cytosolic Ca2+, TMEM16F expression, reactive oxygen species (ROS) and lipid peroxidation of erythrocyte were detected by flow cytometer. PCA was assessed by coagulation time, purified coagulation complex and fibrin production assays. The fibrin formation was observed by scanning electron microscopy (SEM). We found that PS exposure, MPs generation, TMEM16F expression and consequent PCA of erythrocyte in HUA patients significantly increased compared to those in healthy volunteers. Furthermore, high UA induced PS exposure, and MPs release of erythrocyte in concentration and time-dependent manners in vitro, which enhanced the PCA of erythrocyte and was inhibited by lactadherin, a PS inhibitor. Additionally, using SEM, we also observed compact fibrin clots with highly-branched networks and thin fibers supported by red blood cells (RBCs) and RBC-derived MPs (RMPs). Importantly, we demonstrated UA enhanced the production of ROS and lipid peroxidation and reduced the generation of glutathione (GSH) of erythrocyte, which enhanced TMEM16F activity and followed PS externalization and RMPs formation. Collectively, these results suggest that Ca2+-dependent TMEM16F activation may be responsible for UA-induced PS exposure and MPs release of RBC, which thereby contribute to the prothrombotic risk in HUA.

Th1-like Treg in vitiligo: An incompetent regulator in immune tolerance.

Vitiligo is an autoimmune skin disease resulting from epidermal melanocyte destruction mediated by CD8+T cells that breach the self-tolerance. Regulatory T cells (Tregs) are critical for keeping the CD8+T cells in check, but the deficiency of Tregs leading to the immune disequilibrium in vitiligo remains undefined. In the present study, we used RNA-sequencing (RNA-seq) to acquire the transcriptome data of Tregs from vitiligo patients and healthy controls, respectively. Further flow cytometry analysis and immunofluorescence assays substantiated the phenotype of Th1-like Tregs in vitiligo. CD8+T cell-/vitiligo serum-Treg co-culture assays and chemotaxis assays were used to functionally examine this subset of Tregs. As a result, RNA-seq, flow cytometry, and immunofluorescence all indicated the transition of bona fide Treg to the Th1-like T-bet+IFN-γ+Treg in vitiligo patients. Besides, these Th1-like Tregs exhibited significantly dampened suppression on the proliferation and activation of CD8+T cells and a markedly higher tendency to be chemoattracted by CXCL10 and CXCL16. More interestingly, vitiligo serum could even elicit bona fide Tregs of healthy controls to adopt the Th1-like phenotype and manifest impaired suppression. To conclude, Tregs from vitiligo patients are functionally disturbed and the Th1-skewed inflammatory microenvironment in the serum of vitiligo patients is responsible for the generation of Th1-like Tregs. We provide a clinical exploitable strategy that in addition to simply replenishing the bona fide Treg or promoting the homing of Treg to the skin, the normalization of the Th1-skewed inflammatory environment in vitiligo patients and targeting the incompetent Th1-like Tregs might be critical in the future treatment of vitiligo.

Mechanisms of melanocyte death in vitiligo.

Vitiligo is an autoimmune depigment disease results from extensive melanocytes destruction. The destruction of melanocyte is thought to be of multifactorial causation. Genome-wide associated studies have identified single-nucleotide polymorphisms in a panel of susceptible loci as risk factors in melanocyte death. But vitiligo onset can't be solely attributed to a susceptive genetic background. Oxidative stress triggered by elevated levels of reactive oxygen species accounts for melanocytic molecular and organelle dysfunction, a minority of melanocyte demise, and melanocyte-specific antigens exposure. Of note, the self-responsive immune function directly contributes to the bulk of melanocyte deaths in vitiligo. The aberrantly heightened innate immunity, type-1-skewed T helper, and incompetent regulatory T cells tip the balance toward autoreaction and CD8+ cytotoxic T lymphocytes finally execute the killing of melanocytes, possibly alarmed by resident memory T cells. In addition to the well-established apoptosis and necrosis, we discuss several death modalities like oxeiptosis, ferroptosis, and necroptosis that are probably employed in melanocyte destruction. This review focuses on the various mechanisms of melanocytic death in vitiligo pathogenesis to demonstrate a panorama of that. We hope to provide new insights into vitiligo pathogenesis and treatment strategies by the review.

Haploidentical hematopoietic cell and kidney transplantation for hematological malignancies and end-stage renal failure.

At Massachusetts General Hospital, we pioneered simultaneous hematopoietic cell (HCT)/kidney transplantation from HLA-identical related donors for the treatment of hematological malignancies with end-stage renal failure. We have now extended this to HLA-haploidentical donors in a pilot trial. Six recipients, 5 of whom were conditioned with fludarabine, cyclophosphamide, and total-body irradiation, underwent combined HCT/kidney transplantation from haploidentical donors; graft-versus-host disease (GVHD) prophylaxis included post-HCT cyclophosphamide, tacrolimus, and mycophenolate mofetil. One patient died as a result of complications of fludarabine neurological toxicity. No neurological toxicity was observed in subsequent patients who received lower fludarabine doses and more intense postfludarabine dialysis. There were no cases of grade 2 to 4 acute GVHD and 1 case of moderate chronic GVHD by 12 months. One patient experienced relapse of multiple myeloma at 30 months after HCT and died 4 years posttransplantation. Overall, 4 of 6 patients remain alive, without disease relapse and with long-term renal rejection-free survival. This trial was registered at www.clinicaltrials.gov as #NCT01758042.

Salmonella Pullorum lacking srfA is attenuated, immunogenic and protective in chickens.

Sallmonella Pullorum is a host-restricted pathogen for poultry and causes severe economic importance in many developing countries. The development of novel vaccines for Salmonella Pullorum is necessary to eradicate the prevalence of the pathogen. In our study, a srfA deletion mutant (C79-13ΔsrfA) of Salmonella Pullorum was constructed, and then the biological characteristics and protective efficacy of the mutant were evaluated. The mutant C79-13ΔsrfA was much less virulent than its parental strain C79-13 in one-day-old HY-line white chickens, immunization with C79-13ΔsrfA (4 × 107 CFU) through oral pathway induced highly specific humoral and cellular immune responses, the growth performance of vaccinated chickens was consistent with that of unvaccinated chickens. The survival percentages of vaccinated chickens reached 90% and 80%, after challenge with Salmonella Pullorum strain C79-13 and Salmonella Gallinarum strain SG9 at 10 days post-immunization (dpi), respectively. Collectively, our results indicate that C79-13ΔsrfA is a live attenuated vaccine candidate.

The role of neutrophil extracellular traps and TLR signaling in skeletal muscle ischemia reperfusion injury.

Ischemia reperfusion (IR) injury results in devastating skeletal muscle fibrosis. Here, we recapitulate this injury with a mouse model of hindlimb IR injury which leads to skeletal muscle fibrosis. Injury resulted in extensive immune infiltration with robust neutrophil extracellular trap (NET) formation in the skeletal muscle, however, direct targeting of NETs via the peptidylarginine deiminase 4 (PAD4) mechanism was insufficient to reduce muscle fibrosis. Circulating levels of IL-10 and TNFα were significantly elevated post injury, indicating toll-like receptor (TLR) signaling may be involved in muscle injury. Administration of hydroxychloroquine (HCQ), a small molecule inhibitor of TLR7/8/9, following injury reduced NET formation, IL-10, and TNFα levels and ultimately mitigated muscle fibrosis and improved myofiber regeneration following IR injury. HCQ treatment decreased fibroadipogenic progenitor cell proliferation and partially inhibited ERK1/2 phosphorylation in the injured tissue, suggesting it may act through a combination of TLR7/8/9 and ERK signaling mechanisms. We demonstrate that treatment with FDA-approved HCQ leads to decreased muscle fibrosis and increased myofiber regeneration following IR injury, suggesting short-term HCQ treatment may be a viable treatment to prevent muscle fibrosis in ischemia reperfusion and traumatic extremity injury.

Activated NLR family pyrin domain containing 3 (NLRP3) inflammasome in keratinocytes promotes cutaneous T-cell response in patients with vitiligo.

BACKGROUND: Keratinocytes can function as innate immune cells under oxidative stress and aggravate the cutaneous T-cell response that undermines melanocytes in the setting of vitiligo. The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a regulator of innate immunity that exists in keratinocytes. However, the role of the NLRP3 inflammasome in the pathogenesis of vitiligo has not been investigated. OBJECTIVE: We sought to explicate the contribution of the activated NLRP3 inflammasome in keratinocytes to the autoimmune response in patients with vitiligo. METHODS: Perilesional and serum samples from patients with vitiligo were collected to examine the status of the NLRP3 inflammasome in the setting of vitiligo. Cultured keratinocytes were treated with H2O2 to investigate the mechanism for NLRP3 inflammasome activation under oxidative stress. Peripheral blood T cells were extracted from patients with vitiligo to explore the influence of the NLRP3 inflammasome on the T-cell response in patients with vitiligo. RESULTS: Expressions of NLRP3 and downstream cytokine IL-1ß were consistently increased in perilesional keratinocytes of patients with vitiligo. Notably, serum IL-1ß levels were increased in patients with vitiligo, correlated with disease activity and severity, and decreased after effective therapy. Furthermore, oxidative stress promoted NLRP3 inflammasome activation in keratinocytes through transient receptor potential cation channel subfamily M member 2 (TRPM2), a redox-sensitive cation channel, which was dependent on TRPM2-mediated calcium influx. More importantly, blocking TRPM2-induced NLRP3 inflammasome activation in keratinocytes impaired chemotaxis for CD8+ T cells and inhibited the production of cytokines in T cells in patients with vitiligo. CONCLUSION: Oxidative stress-induced NLRP3 inflammasome activation in keratinocytes promotes the cutaneous T-cell response, which could be targeted for the treatment of vitiligo.

Long-Term Utilization Patterns of Topical Therapy and Clinical Outcomes of Oral Chronic Graft-versus-Host Disease.

An open-label phase 2 study of topical dexamethasone versus tacrolimus solutions in new-onset oral chronic graft-versus-host disease (cGVHD) revealed the superior efficacy of dexamethasone. The objective of this study was to report long-term patterns of topical therapy utilization and clinical outcomes in this cohort after completing the 30-day trial. A retrospective record review was performed from the date of study completion to January 2017. Topical therapies, systemic immunosuppressive therapies, objective measurements (National Institutes of Health severity score, oral mucosal scores), patient- reported outcomes (dryness, sensitivity, pain), and adverse events were recorded for oral cGVHD-related outpatient visits. Follow-up (FU) periods were defined as FU1 (0-1 month), FU2 (1-3 months), FU3 (3-6 months), FU4 (6-12 months), FU5 (12-18 months), and FU6 (18-24 months). Forty patients (52.5% males, median age, 56 years) completed the clinical trial and were included in the analysis. Topical therapies used were dexamethasone, tacrolimus, clobetasol, or a combination of these agents. At FU1, all 40 patients were receiving topical therapy, which decreased to 54.5% (12 out of 22) at FU6. Clinician-reported oral mucosal scores (0-12) and patient-reported sensitivity scores (0-10) decreased over time from FU1 (median mucosal score, 3; sensitivity, 3) to FU6 (mucosal score, 1; sensitivity, 2). Intralesional steroid therapy was provided to 6 patients for management of refractory oral ulcerations, all within the first year of follow-up. Patients with de novo symptomatic oral cGVHD may require long-term care with topical immunomodulatory therapy for up to 2 years, if not longer. Topical steroid and tacrolimus therapies are safe and effective in managing symptomatic oral cGVHD. Second-line topical therapy for refractory oral cGVHD requires further investigation.