Ning Xu Landén

MiR-146a Negatively Regulates TLR2-Induced Inflammatory Responses in Keratinocytes

Keratinocytes represent the first line of defense against pathogens in the skin and have important roles in initiating and regulating inflammation during infection and autoimmunity. Here we investigated the role of miR-146a in the regulation of the innate immune response of keratinocytes. Toll-like receptor 2 (TLR2) stimulation of primary human keratinocytes resulted in an NF-κB- and mitogen-activated protein kinase-dependent upregulation of miR-146a expression, which was surprisingly long lasting, contrasting with the rapid and transient induction of inflammatory mediators. Overexpression of miR-146a significantly suppressed the production of IL-8, CCL20, and tumor necrosis factor-α, which functionally suppressed the chemotactic attraction of neutrophils by keratinocytes. Inhibition of endogenous miR-146a induced the production of inflammatory mediators even in nonstimulated keratinocytes, and potentiated the effect of TLR2 stimulation. Transcriptomic profiling revealed that miR-146a suppresses the expression of a large number of immune-related genes in keratinocytes. MiR-146a downregulated interleukin-1 receptor-associated kinase 1 and TNF receptor-associated factor 6, two key adapter molecules downstream of TLR signaling, and suppressed NF-κB promoter-binding activity as shown by promoter luciferase experiments. Together, these data identify miR-146a as a regulatory element in keratinocyte innate immunity, which prevents the production of inflammatory mediators under homeostatic conditions and serves as a potent negative feedback regulator after TLR2 stimulation.

MicroRNA-31 Promotes Skin Wound Healing by Enhancing Keratinocyte Proliferation and Migration

Wound healing is a basic biological process restoring the integrity of the skin. The role of microRNAs during this process remains largely unexplored. By using an in vivo human skin wound healing model, we show here that the expression of miR-31 is gradually upregulated in wound edge keratinocytes in the inflammatory (1 day after injury) through the proliferative phase (7 days after injury) in comparison with intact skin. In human primary keratinocytes, overexpression of miR-31 promoted cell proliferation and migration, whereas inhibition of miR-31 had the opposite effects. Moreover, we identified epithelial membrane protein 1 (EMP-1) as a direct target of miR-31 in keratinocytes. The expression of EMP-1 in the skin was negatively correlated with the level of miR-31 during wound healing. Silencing of EMP-1 mimicked the effects of overexpression of miR-31 on keratinocyte proliferation and migration, indicating that EMP-1 is a critical target mediating the functions of miR-31 in keratinocytes. Finally, we demonstrated that transforming growth factor-β2, which is highly expressed in skin wounds, upregulated miR-31 expression in keratinocytes. Collectively, we identify miR-31 as a key regulator for promoting keratinocyte proliferation and migration during wound healing.

MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

Wound healing is a complex process that is characterized by an initial inflammatory phase followed by a proliferative phase. This transition is a critical regulatory point; however, the factors that mediate this process are not fully understood. Here, we evaluated microRNAs (miRs) in skin wound healing and characterized the dynamic change of the miRNome in human skin wounds. miR-132 was highly upregulated during the inflammatory phase of wound repair, predominantly expressed in epidermal keratinocytes, and peaked in the subsequent proliferative phase. TGF-β1 and TGF-β2 induced miR-132 expression in keratinocytes, and transcriptome analysis of these cells revealed that miR-132 regulates a large number of immune response- and cell cycle-related genes. In keratinocytes, miR-132 decreased the production of chemokines and the capability to attract leukocytes by suppressing the NF-κB pathway. Conversely, miR-132 increased activity of the STAT3 and ERK pathways, thereby promoting keratinocyte growth. Silencing of the miR-132 target heparin-binding EGF-like growth factor (HB-EGF) phenocopied miR-132 overexpression in keratinocytes. Using mouse and human ex vivo wound models, we found that miR-132 blockade delayed healing, which was accompanied by severe inflammation and deficient keratinocyte proliferation. Together, our results indicate that miR-132 is a critical regulator of skin wound healing that facilitates the transition from the inflammatory to the proliferative phase.

MicroRNA-31 Is Overexpressed in Cutaneous Squamous Cell Carcinoma and Regulates Cell Motility and Colony Formation Ability of Tumor Cells

Cutaneous squamous cell carcinoma (cSCC) is a malignancy of epidermal keratinocytes that is responsible for approximately 20% of skin cancer-related death yearly. We have previously compared the microRNA (miRNA) expression profile of cSCC to healthy skin and found the dysregulation of miRNAs in human cSCC. In this study we show that miR-31 is overexpressed in cSCC (n = 68) compared to healthy skin (n = 34) and precancerous skin lesions (actinic keratosis, n = 12). LNA in situ hybridization revealed that miR-31 was specifically up-regulated in tumor cells. Mechanistic studies of inhibition of endogenous miR-31 in human metastatic cSCC cells revealed suppressed migration, invasion and colony forming ability, whereas overexpression of miR-31 induced these phenotypes. These results indicate that miR-31 regulates cancer-associated phenotypes of cSCC and identify miR-31 as a potential target for cSCC treatment.

Therapeutic Effect of Intravenous Infusion of Perfluorocarbon Emulsion on LPS-Induced Acute Lung Injury in Rats

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) are the leading causes of death in critical care. Despite extensive efforts in research and clinical medicine, mortality remains high in these diseases. Perfluorocarbon (PFC), a chemical compound known as liquid ventilation medium, is capable of dissolving large amounts of physiologically important gases (mainly oxygen and carbon dioxide). In this study we aimed to investigate the effect of intravenous infusion of PFC emulsion on lipopolysaccharide (LPS) induced ALI in rats and elucidate its mechanism of action. Forty two Wistar rats were randomly divided into three groups: 6 rats were treated with saline solution by intratracheal instillation (control group), 18 rats were treated with LPS by intratracheal instillation (LPS group) and the other 18 rats received PFC through femoral vein prior to LPS instillation (LPS+PFC group). The rats in the control group were sacrificed 6 hours later after saline instillation. At 2, 4 and 6 hours of exposure to LPS, 6 rats in the LPS group and 6 rats in LPS+PFC group were sacrificed at each time point. By analyzing pulmonary pathology, partial pressure of oxygen in the blood (PaO2) and lung wet-dry weight ratio (W/D) of each rat, we found that intravenous infusion of PFC significantly alleviated acute lung injury induced by LPS. Moreover, we showed that the expression of pulmonary myeloperoxidase (MPO), intercellular adhesion molecule-1 (ICAM-1) of endothelial cells and CD11b of polymorphonuclear neutrophils (PMN) induced by LPS were significantly decreased by PFC treatment in vivo. Our results indicate that intravenous infusion of PFC inhibits the infiltration of PMNs into lung tissue, which has been shown as the core pathogenesis of ALI/ARDS. Thus, our study provides a theoretical foundation for using intravenous infusion of PFC to prevent and treat ALI/ARDS in clinical practice.

Activation of Toll‐like receptors alters the microRNA expression profile of keratinocytes

Keratinocytes recognize invading pathogens by various receptors, among them Toll‐like receptors (TLRs), and provide the first line of defense in skin immunity. The role of microRNAs in this important defense mechanism has not been explored yet. Our aim was to identify microRNAs involved in the innate immune response of keratinocytes. MicroRNA expression profiling revealed that the TLR2 ligand zymosan, the TLR3 ligand poly(I:C) or the TLR5 ligand flagellin significantly altered the microRNA expression in keratinocytes. The regulation of microRNAs was concentration‐dependent and it could be neutralized by siRNAs specific for TLR2, TLR3 and TLR5, respectively, confirming the specificity of the TLR response. Interestingly, one microRNA, miR‐146a, was strongly induced by all studied TLR ligands, while other microRNAs were regulated in a TLR‐ or time point‐specific manner. These findings suggest an important role for microRNAs in the innate immune response of keratinocytes and provide a basis for further investigations.

MicroRNA-146a suppresses IL-17–mediated skin inflammation and is genetically associated with psoriasis

Background: Psoriasis is an immune‐mediated inflammatory skin disease with a strong genetic background in which activation of IL‐17 signaling is central in the pathogenesis. Little has been known about the role of noncoding RNAs, including microRNAs (miRNAs), in predisposition to the disease. Objective: We sought to investigate the genetic association of single nucleotide polymorphisms in microRNA‐146a (miR‐146a) to psoriasis and to explore its function in the initiation and resolution of the disease. Methods: Analysis of the genetic association of miR‐146a rs2910164 and psoriasis was carried out on 1546 patients with psoriasis and 1526 control subjects. The role of miR‐146a in patients with psoriasis was assessed by using miR‐146a−/− mice in conjunction with the imiquimod‐induced mouse model of psoriasis. The severity of psoriasis‐like skin inflammation was evaluated at morphologic, histologic, and molecular levels. miR‐146a was ectopically overexpressed and inhibited in keratinocytes treated with IL‐17. Synthetic miR‐146a was injected intradermally into mice. Results: Here we report protective association of a functional polymorphism in the miR‐146a precursor (rs2910164). Genetic deficiency in miR‐146a leads to earlier onset and exacerbated pathology of skin inflammation, with increased expression of IL‐17–induced keratinocyte‐derived inflammatory mediators, epidermal hyperproliferation, and increased neutrophil infiltration. Moreover, miR‐146a–deficient mice do not resolve inflammation after discontinuation of imiquimod challenge. The overexpression of miR‐146a suppressed, whereas its inhibition enhanced, IL‐17–driven inflammation in keratinocytes. Functionally, miR‐146a impairs the neutrophil chemoattractant capacity of keratinocytes. Finally, delivery of miR‐146a mimics into the skin leads to amelioration of psoriasiform skin inflammation, decreased epidermal proliferation, and neutrophil infiltration. Conclusions: Our results define a crucial role for miR‐146a in modulating IL‐17–driven inflammation in the skin.

New insights into T cells and their signature cytokines in atopic dermatitis

An imbalance of the adaptive immune system mediated by various T cells plays a pivotal role in the pathogenesis of atopic dermatitis (AD). Traditionally, sustained exposure of pathogens tailors immune responses and drives the development of specialized T helper (Th) 2‐bias cytokine environment. The increasing understanding of T cell biology has refreshed the roles of classical Th2 responses and regulatory T cells in the development of AD. In particular, the identification of novel CD4+ T cell subsets such as Th9, Th17, and Th22 cells provide further interpretation of immunological mechanisms underlying AD. In this report, we reviewed the functional roles of CD4+ T cell subsets and their derived signature cytokines in AD. We focused on important discoveries of the contribution of CD4+ T cell cytokines to immunomodulation in AD, and particularly, highlighted the multiple consequences of immune dysregulation on the barrier defect of the skin. We subsequently discussed the flexibility and plasticity of these T cells in vivo in terms of cytokine production. T cells involved in innate immunity were also mentioned. Taking the pivotal and varied roles of T‐cell subpopulations and the functional cytokine milieus into account, T cell targeting therapy may thus open up new opportunities for mechanism‐based therapeutic intervention.

Psoriasis Skin Inflammation-Induced microRNA-26b Targets NCEH1 in Underlying Subcutaneous Adipose Tissue

Psoriasis is an immune-mediated inflammatory disease, which is associated with a high risk of developing systemic comorbidities, such as obesity, cardiovascular disease, and diabetes mellitus. However, the mechanistic links between psoriatic skin inflammation and systemic comorbidities remain largely unknown. MicroRNAs (miRNAs) are recently discovered gene regulators that play important roles in psoriasis skin inflammation. In this study we aimed to explore whether the skin inflammation in psoriasis affects miRNA expression of the underlying subcutaneous adipose tissue and whether this may be a link between psoriasis and comorbidities. To this end, we compared the miRNA expression profile of subcutaneous adipose tissue underneath lesional and nonlesional psoriatic skin. We further validated the differential expression of several miRNAs and characterized their expression patterns in different cell types present in subcutaneous adipose tissue. We focused on miR-26b-5p, which was highly up-regulated in subcutaneous adipose tissue underneath lesional psoriasis skin. We showed that it targets and down-regulates neutral cholesterol ester hydrolase 1, an enzyme essential for cholesterol efflux, in monocytes/macrophages, adipocytes, vascular endothelial cells, and fibroblasts. We conclude that this miRNA may serve as a mechanistic link between psoriatic skin inflammation and its systemic comorbidities.

MicroRNAs in skin wound healing

Wound healing is a fundamental physiological progress to keep the integrity of the skin. The transition from inflammation to proliferation is a critical step during skin wound repair process. Impairment of this transition has been known as a common dominator in the pathophysiology of chronic non-healing wounds. MicroRNAs (miRNAs) are short non-coding RNAs regulating gene expression. Emerging evidence has revealed that miRNAs play important roles in both normal skin wound healing and in the pathogenesis of chronic wounds. We focus on the miRNAs regulating the inflammation-proliferation transition during wound healing and propose that these miRNAs may be promising targets for development of more effective wound therapy.