Modulation of the skin microbiome in cutaneous T-cell lymphoma delays tumour growth and increases survival in the murine EL4 model.

Cutaneous T-cell lymphomas (CTCL) are a group of lymphoproliferative disorders of skin-homing T cells causing chronic inflammation. These disorders cause impairment of the immune environment, which leads to severe infections and/or sepsis due to dysbiosis. In this study, we elucidated the host-microbial interaction in CTCL that occurs during the phototherapeutic treatment regime and determined whether modulation of the skin microbiota could beneficially affect the course of CTCL. EL4 T-cell lymphoma cells were intradermally grafted on the back of C57BL/6 mice. Animals were treated with conventional therapeutics such as psoralen + UVA (PUVA) or UVB in the presence or absence of topical antibiotic treatment (neomycin, bacitracin, and polymyxin B sulphate) as an adjuvant. Microbial colonisation of the skin was assessed to correlate with disease severity and tumour growth. Triple antibiotic treatment significantly delayed tumour occurrence (p = 0.026), which prolonged the survival of the mice (p = 0.033). Allocation to phototherapeutic agents PUVA, UVB, or none of these, along with antibiotic intervention, reduced the tumour growth significantly (p = 0.0327, p ≤ 0.0001, p ≤ 0.0001 respectively). The beta diversity indices calculated using the Bray-Curtis model showed that the microbial population significantly differed after antibiotic treatment (p = 0.001). Upon modulating the skin microbiome by antibiotic treatment, we saw an increase in commensal Clostridium species, e.g., Lachnospiraceae sp. (p = 0.0008), Ruminococcaceae sp. (p = 0.0001)., Blautia sp. (p = 0.007) and a significant reduction in facultative pathogens Corynebacterium sp. (p = 0.0009), Pelomonas sp. (p = 0.0306), Streptococcus sp. (p ≥ 0.0001), Pseudomonas sp. (p = 0.0358), and Cutibacterium sp. (p = 0.0237). Intriguingly, we observed a significant decrease in Staphylococcus aureus frequency (p = 0.0001) but an increase in the overall detection frequency of the Staphylococcus genus, indicating that antibiotic treatment helped regain the microbial balance and increased the number of non-pathogenic Staphylococcus populations. These study findings show that modulating microbiota by topical antibiotic treatment helps to restore microbial balance by diminishing the numbers of pathogenic microbes, which, in turn, reduces chronic inflammation, delays tumour growth, and increases survival rates in our CTCL model. These findings support the rationale to modulate the microbial milieu during the disease course of CTCL and indicate its therapeutic potential.

Epithelial dendritic cells vs. Langerhans cells: Implications for mucosal vaccines.

Next-generation vaccines may be delivered via the skin and mucosa. The stratified squamous epithelium (SSE) represents the outermost layer of the skin (epidermis) and type II mucosa (epithelium). Langerhans cells (LCs) have been considered the sole antigen-presenting cells (APCs) to inhabit the SSE; however, it is now clear that dendritic cells (DCs) are also present. Importantly, there are functional differences in how LCs and DCs take up and process pathogens as well as their ability to activate and polarize T cells, though whether DCs participate in neuroimmune interactions like LCs is yet to be elucidated. A correct definition and functional characterization of APCs in the skin and anogenital tissues are of utmost importance for the design of better vaccines and blocking pathogen transmission. Here, we provide a historical perspective on the evolution of our understanding of the APCs that inhabit the SSE, including a detailed review of the most recent literature.

Macrophage immunophenotypes in Jorge Lobo's disease and lepromatous leprosy- A comparative study.

Jorge Lobo's disease (JLD) and lepromatous leprosy (LL) share several clinical, histological and immunological features, especially a deficiency in the cellular immune response. Macrophages participate in innate and adaptive inflammatory immune responses, as well as in tissue regeneration and repair. Macrophage function deficiency results in maintenance of diseases. M1 macrophages produce pro-inflammatory mediators and M2 produce anti-inflammatory cytokines. To better understand JLD and LL pathogenesis, we studied the immunophenotype profile of macrophage subtypes in 52 JLD skin lesions, in comparison with 16 LL samples, using a panmacrophage (CD68) antibody and selective immunohistochemical markers for M1 (iNOS) and M2 (CD163, CD204) responses, HAM56 (resident/fixed macrophage) and MAC 387 (recently infiltrating macrophage) antibodies. We found no differences between the groups regarding the density of the CD163, CD204, MAC387+ immunostained cells, including iNOS, considered a M1 marker. But HAM56+ cell density was higher in LL samples. By comparing the M2 and M1 immunomarkers in each disease separately, some other differences were found. Our results reinforce a higher M2 response in JLD and LL patients, depicting predominant production of anti-inflammatory cytokines, but also some distinction in degree of macrophage activation. Significant amounts of iNOS + macrophages take part in the immune milieu of both LL and JLD samples, displaying impaired microbicidal activity, like alternatively activated M2 cells.

Inflammasome Activation and IL-1ß Release Triggered by Nanosecond Pulsed Electric Fields in Murine Innate Immune Cells and Skin.

Although electric field-induced cell membrane permeabilization (electroporation) is used in a wide range of clinical applications from cancer therapy to cardiac ablation, the cellular- and molecular-level details of the processes that determine the success or failure of these treatments are poorly understood. Nanosecond pulsed electric field (nsPEF)-based tumor therapies are known to have an immune component, but whether and how immune cells sense the electroporative damage and respond to it have not been demonstrated. Damage- and pathogen-associated stresses drive inflammation via activation of cytosolic multiprotein platforms known as inflammasomes. The assembly of inflammasome complexes triggers caspase-1-dependent secretion of IL-1ß and in many settings a form of cell death called pyroptosis. In this study we tested the hypothesis that the nsPEF damage is sensed intracellularly by the NLRP3 inflammasome. We found that 200-ns PEFs induced aggregation of the inflammasome adaptor protein ASC, activation of caspase-1, and triggered IL-1ß release in multiple innate immune cell types (J774A.1 macrophages, bone marrow-derived macrophages, and dendritic cells) and in vivo in mouse skin. Efflux of potassium from the permeabilized cell plasma membrane was partially responsible for nsPEF-induced inflammasome activation. Based on results from experiments using both the NRLP3-specific inhibitor MCC950 and NLRP3 knockout cells, we propose that the damage created by nsPEFs generates a set of stimuli for the inflammasome and that more than one sensor can drive IL-1ß release in response to electrical pulse stimulation. This study shows, to our knowledge, for the first time, that PEFs activate the inflammasome, suggesting that this pathway alarms the immune system after treatment.

Divergent molecular networks program functionally distinct CD8+ skin-resident memory T cells.

Skin-resident CD8+ T cells include distinct interferon-γ-producing [tissue-resident memory T type 1 (TRM1)] and interleukin-17 (IL-17)-producing (TRM17) subsets that differentially contribute to immune responses. However, whether these populations use common mechanisms to establish tissue residence is unknown. In this work, we show that TRM1 and TRM17 cells navigate divergent trajectories to acquire tissue residency in the skin. TRM1 cells depend on a T-bet-Hobit-IL-15 axis, whereas TRM17 cells develop independently of these factors. Instead, c-Maf commands a tissue-resident program in TRM17 cells parallel to that induced by Hobit in TRM1 cells, with an ICOS-c-Maf-IL-7 axis pivotal to TRM17 cell commitment. Accordingly, by targeting this pathway, skin TRM17 cells can be ablated without compromising their TRM1 counterparts. Thus, skin-resident T cells rely on distinct molecular circuitries, which can be exploited to strategically modulate local immunity.

PD-1 maintains CD8 T cell tolerance towards cutaneous neoantigens.

The peripheral T cell repertoire of healthy individuals contains self-reactive T cells1,2. Checkpoint receptors such as PD-1 are thought to enable the induction of peripheral tolerance by deletion or anergy of self-reactive CD8 T cells3-10. However, this model is challenged by the high frequency of immune-related adverse events in patients with cancer who have been treated with checkpoint inhibitors11. Here we developed a mouse model in which skin-specific expression of T cell antigens in the epidermis caused local infiltration of antigen-specific CD8 T cells with an effector gene-expression profile. In this setting, PD-1 enabled the maintenance of skin tolerance by preventing tissue-infiltrating antigen-specific effector CD8 T cells from (1) acquiring a fully functional, pathogenic differentiation state, (2) secreting significant amounts of effector molecules, and (3) gaining access to epidermal antigen-expressing cells. In the absence of PD-1, epidermal antigen-expressing cells were eliminated by antigen-specific CD8 T cells, resulting in local pathology. Transcriptomic analysis of skin biopsies from two patients with cutaneous lichenoid immune-related adverse events showed the presence of clonally expanded effector CD8 T cells in both lesional and non-lesional skin. Thus, our data support a model of peripheral T cell tolerance in which PD-1 allows antigen-specific effector CD8 T cells to co-exist with antigen-expressing cells in tissues without immunopathology.

Thermal imprinting during embryogenesis modifies skin repair in juvenile European sea bass (Dicentrarchus labrax).

Fish skin is a multifunctional tissue that develops during embryogenesis, a developmental stage highly susceptible to epigenetic marks. In this study, the impact of egg incubation temperature on the regeneration of a cutaneous wound caused by scale removal in juvenile European sea bass was evaluated. Sea bass eggs were incubated at 11, 13.5 and 16 °C until hatching and then were reared at a common temperature until 9 months when the skin was damaged and sampled at 0, 1 and 3 days after scale removal and compared to the intact skin from the other flank. Skin damage elicited an immediate significant (p < 0.001) up-regulation of pcna in fish from eggs incubated at higher temperatures. In fish from eggs incubated at 11 °C there was a significant (p < 0.001) up-regulation of krt2 compared to fish from higher thermal backgrounds 1 day after skin damage. Damaged epidermis was regenerated after 3 days in all fish irrespective of the thermal background, but in fish from eggs incubated at 11 °C the epidermis was significantly (p < 0.01) thinner compared to other groups, had less goblet cells and less melanomacrophages. The thickness of the dermis increased during regeneration of wounded skin irrespective of the thermal background and by 3 days was significantly (p < 0.01) thicker than the dermis from the intact flank. The expression of genes for ECM remodelling (mmp9, colXα, col1α1, sparc, and angptl2b) and innate immunity (lyg1, lalba, sod1, csf-1r and pparγ) changed during regeneration but were not affected by egg thermal regime. Overall, the results indicate that thermal imprinting of eggs modifies the damage-repair response in juvenile sea bass skin.

N-Linked Glycans Shape Skin Immune Responses during Arthritis and Myositis after Intradermal Infection with Ross River Virus.

Arthritogenic alphaviruses are mosquito-borne arboviruses that include several re-emerging human pathogens, including the chikungunya (CHIKV), Ross River (RRV), Mayaro (MAYV), and o'nyong-nyong (ONNV) virus. Arboviruses are transmitted via a mosquito bite to the skin. Herein, we describe intradermal RRV infection in a mouse model that replicates the arthritis and myositis seen in humans with Ross River virus disease (RRVD). We show that skin infection with RRV results in the recruitment of inflammatory monocytes and neutrophils, which together with dendritic cells migrate to draining lymph nodes (LN) of the skin. Neutrophils and monocytes are productively infected and traffic virus from the skin to LN. We show that viral envelope N-linked glycosylation is a key determinant of skin immune responses and disease severity. RRV grown in mammalian cells elicited robust early antiviral responses in the skin, while RRV grown in mosquito cells stimulated poorer early antiviral responses. We used glycan mass spectrometry to characterize the glycan profile of mosquito and mammalian cell-derived RRV, showing deglycosylation of the RRV E2 glycoprotein is associated with curtailed skin immune responses and reduced disease following intradermal infection. Altogether, our findings demonstrate skin infection with an arthritogenic alphavirus leads to musculoskeletal disease and envelope glycoprotein glycosylation shapes disease outcome. IMPORTANCE Arthritogenic alphaviruses are transmitted via mosquito bites through the skin, potentially causing debilitating diseases. Our understanding of how viral infection starts in the skin and how virus systemically disseminates to cause disease remains limited. Intradermal arbovirus infection described herein results in musculoskeletal pathology, which is dependent on viral envelope N-linked glycosylation. As such, intradermal infection route provides new insights into how arboviruses cause disease and could be extended to future investigations of skin immune responses following infection with other re-emerging arboviruses.

Lck signaling inhibition causes improvement in clinical features of psoriatic inflammation through reduction in inflammatory cytokines in CD4+ T cells in imiquimod mouse model.

Psoriasis is a chronic dermal inflammatory disease with a world-wide prevalence in which different immune/non-immune cells, e.g. T cells, macrophages, neutrophils, and keratinocytes play a decisive role. These immune cells interact among themselves by releasing multiple mediators which eventually cause characteristic psoriatic plaques in the skin. T cells are reported to be significant contributors to psoriatic inflammation through release of multiple cytokines which are controlled by several kinases, one of them being Lymphocyte-specific protein tyrosine kinase (Lck). Lck has been reported to be crucial for expression/production of several key inflammatory cytokines though modulation of several other kinases/transcription factors in T cells. Therefore, in this investigation, effect of Lck inhibitor, A-770041 was examined on PLCγ, p38MAPK, NFATc1, NFkB and STAT3, TNF-α, IFN-γ, Foxp3, IL-17A, in CD4+ T cells in imiquimod (IMQ)-induced psoriatic inflammation in mice. Results from the present study exhibit that p-Lck expression is enhanced in CD4+ T cells of IMQ-treated mice which is concomitant with enhanced clinical features of psoriatic inflammation (ear/back skin thickness, MPO activity, acanthosis/leukocytic infiltration) and molecular parameters (enhanced expression of p-Lck, p-PLCγ, p-p38-MAPK, NFATc1, p-NFkB, TNF-α, IFN-γ, p-STAT3, and IL-17A in CD4+ T cells). Inhibition of Lck signaling led to amelioration of clinical features of psoriasis through attenuation of Th1/Th17 immune responses and upregulation of Treg cells in IMQ-treated mice. In summary, current investigations reveal that Lck signaling is a crucial executor of inflammatory signaling in CD4+ T cells in the context of psoriatic inflammation. Therefore, Lck inhibition may be pursued as an effective strategy to counteract psoriatic inflammation.

Suppression of IL-12/IL-23 p40 subunit in the skin and blood of psoriasis patients by Tofacitinib is dependent on active interferon-γ signaling in dendritic cells: Implications for the treatment of psoriasis and interferon-driven diseases.

Interleukin (IL)-12 and IL-23 are pro-inflammatory cytokines produced by dendritic cells (DCs) and associated with Psoriasis (Pso) and Psoriatic Arthritis (PsA) pathogenesis. Tofacitinib, a Janus kinase inhibitor, effectively suppresses inflammatory cascades downstream the IL-12/IL-23 axis in Pso and PsA patients. Here, we investigated whether Tofacitinib directly regulates IL-12/IL-23 production in DCs, and how this regulation reflects responses to Tofacitinib in Pso patients. We treated monocyte-derived dendritic cells and myeloid dendritic cells with Tofacitinib and stimulated cells with either lipopolysaccharide (LPS) or a combination of LPS and IFN-γ. We assessed gene expression by qPCR, obtained skin microarray and blood Olink data and clinical parameters of Pso patients treated with Tofacitinib from public data sets. Our results indicate that in DCs co-stimulated with LPS and IFN-γ, but not with LPS alone, Tofacitinib leads to the decreased expression of IL-23/IL-12 shared subunit IL12B (p40). In Tofacitinib-treated Pso patients, IL-12 expression and psoriasis area and severity index (PASI) are significantly reduced in patients with higher IFN-γ at baseline. These findings demonstrate for the first time that Tofacitinib suppresses IL-23/IL-12 shared subunit IL12B in DCs upon active IFN-γ signaling, and that Pso patients with higher IFN-γ baseline levels display improved clinical response after Tofacitinib treatment.

CD38 Expression by Circulating and Skin-Infiltrating Lymphocytes from Sezary Syndrome Patients: A Flow Cytometry and Immunohistochemistry Study.

BACKGROUND: Reports on the expression of CD38 in Sézary syndrome (SS), erythrodermic primary cutaneous T cell lymphoma with leukemic involvement, are limited. The aim of the present study is the analysis of the expression of CD38 by skin-infiltrating mononuclear cells and circulating T lymphocytes in a cohort of SS patients. METHODS: SS patients diagnosed since 1985 in our clinic were retrospectively analyzed for CD38 expression in biopsy and blood samples by immunohistochemistry and flow cytometry, respectively. RESULTS: SS patients show a predominant CD38-negative phenotype on both skin and blood. A subgroup of patients was found expressing CD38 (12 cases) in either the skin (>25% cell infiltrate) or blood (CD4+CD38+ >50%), among whom 4 in the blood, 7 in the skin, and 1 in both blood and skin. CONCLUSION: The implications of these observations may be twofold: the relevance in basic science is related to a potential role in immune defense regulation, whilst in perspective CD38 may become a target for antibody therapy, considering the availability of different anti-CD38 monoclonal antibodies.

Histopathological and Clinical Analysis of Skin Rashes in Children With Multisystem Inflammatory Syndrome Associated With COVID-19.

INTRODUCTION: A new entity, which occurs a few weeks after SARS-CoV-2 infection and resembling incomplete Kawasaki disease or toxic shock syndrome, has been defined and named multisystem inflammatory syndrome (MIS-C) associated with COVID-19 in children. The aim of our study was to describe histopathological characteristics of skin lesions of MIS-C patients to reveal whether there is a relationship between histopathological features and clinical manifestations. MATERIALS AND METHODS: Seventeen who had skin involvement of 57 patients who were diagnosed with MIS-C between December 2020 and February 2021 were included in this prospective study. Demographic information, laboratory findings, and patients' managements were recorded. Skin biopsies were taken simultaneously of each patient. Formalin-fixed, paraffin-embedded skin samples were examined microscopically. RESULTS: The rate of skin rash was 30% in patients with MIS-C and was predominantly the maculopapular type. The anatomical distribution of the rash was evaluated as localized in 10 and generalized in 7 patients. In patients with myocarditis, C-reactive protein and fibrinogen were found to be significantly higher, and lymphocyte and albumin values were found to be low. Herpes-like inclusions were found in the microscopic examination of 2 patients with a history of zona zoster in themselves or in their mother. There was a significant difference between keratinocyte necrosis and some clinical parameters. DISCUSSION: Localized skin lesions appear to be associated with a more severe inflammatory.

Human skin-resident host T cells can persist long term after allogeneic stem cell transplantation and maintain recirculation potential.

Tissue-resident memory T cells (TRM) have recently emerged as crucial cellular players for host defense in a wide variety of tissues and barrier sites. Insights into the maintenance and regulatory checkpoints of human TRM cells remain scarce, especially due to the difficulties associated with tracking T cells through time and space in humans. We therefore sought to identify and characterize skin-resident T cells in humans defined by their long-term in situ lodgment. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) preceded by myeloablative chemotherapy unmasked long-term sequestration of host T cell subsets in human skin despite complete donor T cell chimerism in the blood. Single-cell chimerism analysis paired with single-cell transcriptional profiling comprehensively characterized these bona fide long-term skin-resident T cells and revealed differential tissue maintenance for distinct T cell subsets, specific TRM cell markers such as galectin-3, but also tissue exit potential with retention of the transcriptomic TRM cell identity. Analysis of 26 allo-HSCT patients revealed profound interindividual variation in the tissue maintenance of host skin T cells. The long-term persistence of host skin T cells in a subset of these patients did not correlate with the development of chronic GvHD. Our data exemplify the power of exploiting a clinical situation as a proof of concept for the existence of bona fide human skin TRM cells and reveal long-term persistence of host T cells in a peripheral tissue but not in the circulation or bone marrow in a subset of allo-HSCT patients.

The cGAS-STING pathway drives type I IFN immunopathology in COVID-19.

COVID-19, which is caused by infection with SARS-CoV-2, is characterized by lung pathology and extrapulmonary complications1,2. Type I interferons (IFNs) have an essential role in the pathogenesis of COVID-19 (refs 3-5). Although rapid induction of type I IFNs limits virus propagation, a sustained increase in the levels of type I IFNs in the late phase of the infection is associated with aberrant inflammation and poor clinical outcome5-17. Here we show that the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, which controls immunity to cytosolic DNA, is a critical driver of aberrant type I IFN responses in COVID-19 (ref. 18). Profiling COVID-19 skin manifestations, we uncover a STING-dependent type I IFN signature that is primarily mediated by macrophages adjacent to areas of endothelial cell damage. Moreover, cGAS-STING activity was detected in lung samples from patients with COVID-19 with prominent tissue destruction, and was associated with type I IFN responses. A lung-on-chip model revealed that, in addition to macrophages, infection with SARS-CoV-2 activates cGAS-STING signalling in endothelial cells through mitochondrial DNA release, which leads to cell death and type I IFN production. In mice, pharmacological inhibition of STING reduces severe lung inflammation induced by SARS-CoV-2 and improves disease outcome. Collectively, our study establishes a mechanistic basis of pathological type I IFN responses in COVID-19 and reveals a principle for the development of host-directed therapeutics.

Potential effects of shift work on skin autoimmune diseases.

Shift work is associated with systemic chronic inflammation, impaired host and tumor defense and dysregulated immune responses to harmless antigens such as allergens or auto-antigens. Thus, shift workers are at higher risk to develop a systemic autoimmune disease and circadian disruption with sleep impairment seem to be the key underlying mechanisms. Presumably, disturbances of the sleep-wake cycle also drive skin-specific autoimmune diseases, but epidemiological and experimental evidence so far is scarce. This review summarizes the effects of shift work, circadian misalignment, poor sleep, and the effect of potential hormonal mediators such as stress mediators or melatonin on skin barrier functions and on innate and adaptive skin immunity. Human studies as well as animal models were considered. We will also address advantages and potential pitfalls in animal models of shift work, and possible confounders that could drive skin autoimmune diseases in shift workers such as adverse lifestyle habits and psychosocial influences. Finally, we will outline feasible countermeasures that may reduce the risk of systemic and skin autoimmunity in shift workers, as well as treatment options and highlight outstanding questions that should be addressed in future studies.

Mutant p53 reactivator SLMP53-2 hinders ultraviolet B radiation-induced skin carcinogenesis.

The growing incidence of skin cancer (SC) has prompted the search for additional preventive strategies to counteract this global health concern. Mutant p53 (mutp53), particularly with ultraviolet radiation (UVR) signature, has emerged as a promising target for SC prevention based on its key role in skin carcinogenesis. Herein, the preventive activity of our previously disclosed mutp53 reactivator SLMP53-2 against UVR-induced SC was investigated. The pre-treatment of keratinocyte HaCaT cells with SLMP53-2, before UVB exposure, depleted mutp53 protein levels with restoration of wild-type-like p53 DNA-binding ability and subsequent transcriptional activity. SLMP53-2 increased cell survival by promoting G1-phase cell cycle arrest, while reducing UVB-induced apoptosis through inhibition of c-Jun N-terminal kinase (JNK) activity. SLMP53-2 also protected cells from reactive oxygen species and oxidative damage induced by UVB. Moreover, it enhanced DNA repair through upregulation of nucleotide excision repair pathway and depletion of UVB-induced DNA damage, as evidenced by a reduction of DNA in comet tails, γH2AX staining and cyclobutane pyrimidine dimers (CPD) levels. SLMP53-2 further suppressed UVB-induced inflammation by inhibiting the nuclear translocation and DNA-binding ability of NF-κB, and promoted the expression of key players involved in keratinocytes differentiation. Consistently, the topical application of SLMP53-2 in mice skin, prior to UVB irradiation, reduced cell death and DNA damage. It also decreased the expression of inflammatory-related proteins and promoted cell differentiation, in UVB-exposed mice skin. Notably, SLMP53-2 did not show signs of skin toxicity for cumulative topical use. Overall, these results support a promising protective activity of SLMP53-2 against UVB-induced SC.

MiR-1294 suppresses ROS-dependent inflammatory response in atopic dermatitis via restraining STAT3/NF-κB pathway.

Atopic dermatitis (AD) is a common inflammatory skin disorder that affects children and adults. Despite the pathology of AD involves in immune dysfunction and epidermal barrier function destruction has been found, the mechanism of immune activation and barrier damage remain largely unknown. In the present study, The TNF-α/IFN-γ-stimulated HaCaTs, organotypic AD-like 3D skin equivalents and AD-like mouse model were constructed. The mRNA, histological morphology, protein levels, cytokines were detected by real-time quantitative polymerasechain reaction (RT-qPCR), hematoxylin and eosin (H & E) staining, Immunohistochemistry (IHC), immunoblotting, immunofluorescence (IF) staining, and enzyme linked immunosorbent assay (ELISA), respectively. Cell viability, cell cycle, and apoptosis were respectively calculated using a Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and flow cytometry. A dual-luciferase reporter gene system was used to investigate the relationship between miR-1294 and STAT3. Compared with the control group, the expression of miR-1294 decreased in TNF-α/IFN-γ-stimulated HaCaTs (P < 0.001), AD-like skin model, and AD-like mouse model (P < 0.001). Moreover, STAT3 was documented as a direct target of miR-1294. Inflammation (P < 0.05) and epidermal barrier function destruction (P < 0.05) in AD was suppressed by overexpression of miR-1294 but enhanced by STAT3 upregulation and its downstream NF-κB pathway. We also found miR-1294 upregulation inhibited inflammation and epidermal barrier function destruction via targeting STAT3 to suppress NF-κB pathway activation in AD.

Analysis of Macrophages and Peptidergic Fibers in the Skin of Patients With Painful Diabetic Polyneuropathy.

BACKGROUND AND OBJECTIVES: The mechanisms of pain in patients with diabetic polyneuropathy are unknown. Studies have suggested a role of inflammation and increased neuropeptides peripherally in pain generation. This study examined the possible skin markers of painful diabetic polyneuropathy (P-DPN): macrophages, substance P (SP), and calcitonin gene-related peptide (CGRP). METHODS: The participants were included from a large Danish cross-sectional clinical study of type 2 diabetes. We diagnosed definite diabetic polyneuropathy using the Toronto criteria and used the Neuropathic Pain Special Interest Group classification for defining P-DPN. We included 60 skin biopsies from patients with diabetic polyneuropathy-30 with P-DPN and 30 with nonpainful diabetic polyneuropathy (NP-DPN)-and 30 biopsies from healthy controls of similar age and sex. The biopsies were stained using PGP 9.5, IbA1, and SP and CGRP primary markers. RESULTS: There was increased macrophage density in patients with P-DPN (8.0%) compared with that in patients with NP-DPN (5.1%, p < 0.001), and there was increased macrophage density in patients with NP-DPN (5.1%) compared with that in healthy controls (3.1%, p < 0.001). When controlling for neuropathy severity, body mass index, age, and sex, there was still a difference in macrophage density between patients with P-DPN and patients with NP-DPN. Patients with P-DPN had higher median nerve fiber length density (274.5 and 155 mm-2 for SP and CGRP, respectively) compared with patients with NP-DPN (176 and 121 mm-2 for SP and CGRP, respectively, p = 0.009 and 0.04) and healthy controls (185.5 and 121.5 mm-2 for SP and CGRP, respectively), whereas there was no difference between patients with NP-DPN and controls without diabetes (p = 0.64 and 0.49, respectively). The difference between P-DPN and NP-DPN for SP and CGRP was significant only in female patients, although a trend was seen in male patients. DISCUSSION: The findings point to a possible involvement of the innate immune system in the pathogenesis of neuropathic pain in patients with DPN, although markers of activated macrophages were not measured in this study.