Selective targeting of dipeptidyl-peptidase 4 (DPP-4) positive senescent chondrocyte ameliorates osteoarthritis progression.

Senescent cells increase in many tissues with age and induce age-related pathologies, including osteoarthritis (OA). Senescent chondrocytes (SnCs) are found in OA cartilage, and the clearance of those chondrocytes prevents OA progression. However, targeting SnCs is challenging due to the absence of a senescent chondrocyte-specific marker. Therefore, we used flow cytometry to screen and select senescent chondrocyte surface markers and cross-validated with published transcriptomic data. Chondrocytes expressing dipeptidyl peptidase-4 (DPP-4), the selected senescent chondrocyte-specific marker, had multiple senescence phenotypes, such as increased senescence-associated-galactosidase, p16, p21, and senescence-associated secretory phenotype expression, and showed OA chondrocyte phenotypes. To examine the effects of DPP-4 inhibition on DPP-4+ SnCs, sitagliptin, a DPP-4 inhibitor, was treated in vitro. As a result, DPP-4 inhibition selectively eliminates DPP-4+ SnCs without affecting DPP-4- chondrocytes. To assess in vivo therapeutic efficacy of targeting DPP-4+ SnCs, three known senolytics (ABT263, 17DMAG, and metformin) and sitagliptin were comparatively verified in a DMM-induced rat OA model. Sitagliptin treatment specifically and effectively eliminated DPP-4+ SnCs, compared to the other three senolytics. Furthermore, Intra-articular sitagliptin injection to the rat OA model increased collagen type II and proteoglycan expression and physical functions and decreased cartilage destruction, subchondral bone plate thickness and MMP13 expression, leading to the amelioration of OA phenotypes. Collectively, OARSI score was lowest in the sitagliptin treatment group. Taken together, we verified DPP-4 as a surface marker for SnCs and suggested that the selective targeting of DPP-4+ chondrocytes could be a promising strategy to prevent OA progression.

High-glutathione mesenchymal stem cells isolated using the FreSHtracer probe enhance cartilage regeneration in a rabbit chondral defect model.

BACKGROUND: Mesenchymal stem cells (MSCs) are a promising cell source for cartilage regeneration. However, the function of MSC can vary according to cell culture conditions, donor age, and heterogeneity of the MSC population, resulting in unregulated MSC quality control. To overcome these limitations, we previously developed a fluorescent real-time thiol tracer (FreSHtracer) that monitors cellular levels of glutathione (GSH), which are known to be closely associated with stem cell function. In this study, we investigated whether using FreSHtracer could selectively separate high-functioning MSCs based on GSH levels and evaluated the chondrogenic potential of MSCs with high GSH levels to repair cartilage defects in vivo. METHODS: Flow cytometry was conducted on FreSHtracer-loaded MSCs to select cells according to their GSH levels. To determine the function of FreSHtracer-isolated MSCs, mRNA expression, migration, and CFU assays were conducted. The MSCs underwent chondrogenic differentiation, followed by analysis of chondrogenic-related gene expression. For in vivo assessment, MSCs with different cellular GSH levels or cell culture densities were injected in a rabbit chondral defect model, followed by histological analysis of cartilage-regenerated defect sites. RESULTS: FreSHtracer successfully isolated MSCs according to GSH levels. MSCs with high cellular GSH levels showed enhanced MSC function, including stem cell marker mRNA expression, migration, CFU, and oxidant resistance. Regardless of the stem cell tissue source, FreSHtracer selectively isolated MSCs with high GSH levels and high functionality. The in vitro chondrogenic potential was the highest in pellets generated by MSCs with high GSH levels, with increased ECM formation and chondrogenic marker expression. Furthermore, the MSCs' function was dependent on cell culture conditions, with relatively higher cell culture densities resulting in higher GSH levels. In vivo, improved cartilage repair was achieved by articular injection of MSCs with high levels of cellular GSH and MSCs cultured under high-density conditions, as confirmed by Collagen type 2 IHC, Safranin-O staining and O'Driscoll scores showing that more hyaline cartilage was formed on the defects. CONCLUSION: FreSHtracer selectively isolates highly functional MSCs that have enhanced in vitro chondrogenesis and in vivo hyaline cartilage regeneration, which can ultimately overcome the current limitations of MSC therapy.

Modulation of bioactive calcium phosphate micro/nanoparticle size and shape during in situ synthesis of photo-crosslinkable gelatin methacryloyl based nanocomposite hydrogels for 3D bioprinting and tissue engineering.

BACKGROUND: The gelatin-methacryloyl (GelMA) polymer suffers shape fidelity and structural stability issues during 3D bioprinting for bone tissue engineering while homogeneous mixing of reinforcing nanoparticles is always under debate. METHOD: In this study, amorphous calcium phosphates micro/nanoparticles (CNP) incorporated GelMA is synthesized by developing specific sites for gelatin structure-based nucleation and stabilization in a one-pot processing. The process ensures homogenous distribution of CNPs while different concentrations of gelatin control their growth and morphologies. After micro/nanoparticles synthesis in the gelatin matrix, methacrylation is carried out to prepare homogeneously distributed CNP-reinforced gelatin methacryloyl (CNP GelMA) polymer. After synthesis of CNP and CNP GelMA gel, the properties of photo-crosslinked 3D bioprinting scaffolds were compared with those of the conventionally fabricated ones. RESULTS: The shape (spindle to spherical) and size (1.753 µm to 296 nm) of the micro/nanoparticles in the GelMA matrix are modulated by adjusting the gelatin concentrations during the synthesis. UV cross-linked CNP GelMA (using Irgacure 2955) has significantly improved mechanical (three times compressive strength), 3D printability (160 layers, 2 cm self-standing 3D printed height) and biological properties (cell supportiveness with osteogenic differentiation). The photo-crosslinking becomes faster due to better methacrylation, facilitating continuous 3D bioprinting or printing. CONCLUSION: For 3D bioprinting using GelMA like photo cross-linkable polymers, where structural stability and homogeneous control of nanoparticles are major concerns, CNP GelMA is beneficial for even bone tissue regeneration within short period.

Low-dose irradiation could mitigate osteoarthritis progression via anti-inflammatory action that modulates mitochondrial function.

PURPOSE: The use of low-dose radiation therapy (LDRT) for osteoarthritis (OA) are rarely implemented, except in some European regions. Its clinical effects are controversial but little is known about how LDRT affects actual disease progression. We conducted a preclinical study to reveal the potential underlying mechanisms related to its disease modifying abilities. METHODS AND MATERIALS: Using primary cultured human chondrocytes and synovium-derived cells obtained from OA patients, the effects of LDRT were measured by quantitative real-time PCR, western blotting, and mRNA sequencing. For in vivo validation, a surgically-induced isolated OA model was used after anterior cruciate ligament transection or surgical destabilization of the medial meniscus. RESULTS: LDRT decreased the expression of pro-inflammatory factor matrix metalloproteinase 13 (MMP13) in chondrocytes. By contrast, collagen type 2 (COL2) protein expression was increased. LDRT induced large transcriptomic changes in both chondrocytes/synoviocytes, especially in mitochondrial activities. Gene set variation analysis demonstrated inverted U-shaped response in several categories, such as mitochondrial unfolded protein responses and extracellular matrix interactions. Growth differentiation factor 15 (GDF15), which is a mitohormetic signaling factor, was increased after LDRT and mediated the anti-inflammatory effects. Aggrecan was increased in synoviocyte's medium and TNF-α was decreased in chondrocyte's medium after LDRT. Conversely, knockdown of GDF15 did not result in decreased MMP13 expression by LDRT. Next, OA rats treated with LDRT exhibited a decreased OA severity when compared with the no-irradiation group at 10 weeks post-surgery (mean OARSI score 3.7 in 0 Gy, 2.8 in 0.5 Gy, and 1.8 in 1 Gy; p = 0.003). Osteoclast activity was significantly reduced in the LDRT group. CONCLUSIONS: Taken together, these data show that LDRT could mitigate osteoarthritis progression by exerting its anti-inflammatory effects via mitochondrial function modulation.

TGFß1-Induced Transglutaminase-2 Triggers Catabolic Response in Osteoarthritic Chondrocytes by Modulating MMP-13.

BACKGROUND: Transforming growth factor beta 1 (TGFß1) plays an essential role in maintaining cartilage homeostasis. TGFß1 is known to upregulate anabolic processes in articular cartilage, but the role of TGFß1 in chondrocyte catabolism remains unclear. Thus, we examined whether TGFß1 increases catabolic processes in the osteoarthritic joint via transglutaminase 2 (TG2). In this study, we investigated whether interplay between TGFß1 and TG2 mediates chondrocyte catabolism and cartilage degeneration in osteoarthritis. METHODS: To investigate the role of TGFß1 and TG2 in osteoarthritis, we performed immunostaining to measure the levels of TGFß1 and TG2 in 6 human non-osteoarthritic and 16 osteoarthritic joints. We conducted quantitative reverse transcription polymerase chain reaction and western blot analysis to investigate the relationship between TGFß1 and TG2 in chondrocytes and determined whether TG2 regulates the expressions of matrix metalloproteinase (MMP)-13, type II, and type X collagen. We also examined the extent of cartilage degradation after performing anterior cruciate ligament transection (ACLT) and destabilization of the medial meniscus (DMM) surgery in TG2 knock-out mice. RESULTS: We confirmed the overexpression of TGFß1 and TG2 in human osteoarthritic cartilage compared with non-osteoarthritic cartilage. TGFß1 treatment significantly increased the expression of TG2 via p38 and ERK activation. TGFß1-induced TG2 also elevated the level of MMP-13 and type X collagen via NF-κB activation in chondrocytes. Cartilage damage after ACLT and DMM surgery was less severe in TG2 knock-out mice compared with wild-type mice. CONCLUSION: TGFß1 modulated catabolic processes in chondrocytes in a TG2-dependent manner. TGFß1-induced TG2 might be the therapeutic target for treating cartilage degeneration and osteoarthritis.

Enhancement of Cartilage Regeneration of Synovial Stem Cells/Hydrogel by Using Transglutaminase-4.

Although mesenchymal stem cells (MSCs) transplantation is reportedly a promising strategy for repairing damaged articular cartilage, MSCs-based cartilage tissue engineering has numerous limitations, including poor implanted cell adhesion, phenotypic alteration of cells, regulation of mechanical properties, and engraftment rates after implantation. This study aimed to investigate the efficacy of transplantation of synovium-derived mesenchymal stem cells (SDSCs) encapsulated in a hyaluronic acid/collagen/fibrinogen (HA/COL/FG) composite gel by supplementing recombinant human transglutaminase 4 (rhTG-4) in treating osteochondral defects. RhTG-4 treatment induced the expression of integrin ß1 and dynamic actin fiber, enhancing SDSCs adhesion to fibronectin. Supplementation of rhTG-4 significantly induced the proliferation of SDSCs encapsulated in the HA/COL/FG composite gel and increased the hardness of the extracellular matrix. Furthermore, supplementation of rhTG-4 significantly upregulated aggrecan and type II collagen mRNA. Pretreatment with integrin ß1 siRNA markedly suppressed TG4-induced actin remodeling, activation mitogen-activated protein kinase (MAPK), and eventually the chondrogenesis-related genes. Moreover, transplantation of SDSCs encapsulated in HA/COL/FG/rhTG-4 composite gel in vivo yielded reconstructed tissue resembling native hyaline cartilage. These data suggest that rhTG-4 enhances cartilage regeneration of the SDSCs encapsulated in hydrogel in rabbits. Impact statement In this study, we investigated the effects of recombinant human transglutaminase 4 on the ability of synovium-derived mesenchymal stem cells encapsulated in a hyaluronic acid/collagen/fibrinogen composite gel to repair osteochondral defects. We believe that our study makes a significant contribution to the literature because it explores a method of improving an existing modality to mediate tissue repair.

Effect of Storage Conditions and Activation on Growth Factor Concentration in Platelet-Rich Plasma.

This study aimed to evaluate growth factor concentration in platelet-rich plasma (PRP) (leukocyte-rich PRP) based on storage temperature, duration of storage, and method of activation. PRP samples were stored at 24℃ (room temperature group), 4℃ (refrigerator group), and -70℃ (deep-freezer group). In each temperature, four aliquots were prepared based on the time of analysis (immediately, 1, 3, and 7 days after preparation). After storage, concentrations of platelet-derived growth factor-AA (PDGF-AA), transforming growth factor-ß (TGF-ß), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and fibroblast growth factor-basic (FGF-B) were assessed with/without activation using Quantikine colorimetric sandwich immunoassay kits. PRP was activated with 10% Triton-X for PDGF-AA, VEGF, FGF-B, IGF-1 measurement and sonication for TGF-ß1 measurement. Without activation, PDGF-AA concentration was highest on day 7 in the room temperature group. With activation, the concentration of PDGF-AA was constant over the observation period at all temperatures. Without activation, the TGF-ß1 concentration remained negligible over the observation period at all temperatures. However, with activation, TGF-ß1 gradually increased to its highest concentration on day 7 at all temperatures. Over the observation period, VEGF and IGF-1 concentrations were constant with and without activation at all temperatures. Without activation, FGF-B concentration increased, with the highest concentration observed on day 7 in the deep-freezer group. With activation, FGF-B concentration decreased after day 1 in the room temperature group. Growth factor concentration in PRP differed significantly based on storage temperature, duration of storage, and method of activation. Appropriate storage conditions and activation are important to optimize its effects on desired clinical outcomes. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:777-784, 2020.

Correction to: Hypoxic condition enhances chondrogenesis in synovium-derived mesenchymal stem cells.

[This corrects the article DOI: 10.1186/s40824-018-0134-x.].

Hypoxic condition enhances chondrogenesis in synovium-derived mesenchymal stem cells.

BACKGROUND: The chondrogenic differentiation of mesenchymal stem cells (MSCs) is regulated by many factors, including oxygen tensions, growth factors, and cytokines. Evidences have suggested that low oxygen tension seems to be an important regulatory factor in the proliferation and chondrogenic differentiation in various MSCs. Recent studies report that synovium-derived mesenchymal stem cells (SDSCs) are a potential source of stem cells for the repair of articular cartilage defects. But, the effect of low oxygen tension on the proliferation and chondrogenic differentiation in SDSCs has not characterized. In this study, we investigated the effects of hypoxia on proliferation and chondrogenesis in SDSCs. METHOD: SDSCs were isolated from patients with osteoarthritis at total knee replacement. To determine the effect of oxygen tension on proliferation and colony-forming characteristics of SDSCs, A colony-forming unit (CFU) assay and cell counting-based proliferation assay were performed under normoxic (21% oxygen) or hypoxic (5% oxygen). For in vitro chondrogenic differentiation, SDSCs were concentrated to form pellets and subjected to conditions appropriate for chondrogenic differentiation under normoxia and hypoxia, followed by the analysis for the expression of genes and proteins of chondrogenesis. qRT-PCR, histological assay, and glycosoaminoglycan assays were determined to assess chondrogenesis. RESULTS: Low oxygen condition significantly increased proliferation and colony-forming characteristics of SDSCs compared to that of SDSCs under normoxic culture. Similar pellet size and weight were found for chondrogensis period under hypoxia and normoxia condition. The mRNA expression of types II collagen, aggrecan, and the transcription factor SOX9 was increased under hypoxia condition. Histological sections stained with Safranin-O demonstrated that hypoxic conditions had increased proteoglycan synthesis. Immunohistochemistry for types II collagen demonstrated that hypoxic culture of SDSCs increased type II collagen expression. In addition, GAG deposition was significantly higher in hypoxia compared with normoxia at 21 days of differentiation. CONCLUSION: These findings show that hypoxia condition has an important role in regulating the synthesis ECM matrix by SDSCs as they undergo chondrogenesis. This has important implications for cartilage tissue engineering applications of SDSCs.

HIF-1α-mediated BMP6 down-regulation leads to hyperproliferation and abnormal differentiation of keratinocytes in vitro.

Hypoxia-inducible factor-1α (HIF-1α) has been reported to be up-regulated in psoriatic epidermis, resulting in increased proliferation and abnormal differentiation of human keratinocytes (KCs). However, the role of HIF-1α in psoriatic epidermis, which is mainly composed of KCs, is poorly understood. Here, we show that morphogenic protein 6 (BMP6) is down-regulated when HIF-1α is upregulated in patients with psoriasis skin lesions. HIF-1α overexpression in primary human KCs promoted proliferation and inhibited terminal differentiation. Furthermore, HIF1-α repressed the expression of BMP6 by binding directly to the hypoxia-response element (HRE) in the BMP6 promotor region, which shows that BMP6 is a novel target gene of HIF-1α. We also found that HIF-1α-mediated BMP6 suppression could alter the proliferation status by modulating the expression levels of cell cycle regulatory proteins and also affect the early differentiation of KCs. Therefore, we suggest that HIF-1α-dependent BMP6 suppression has a critical role in the induction of hyper-proliferation and abnormal differentiation in psoriatic KCs.

Mitochondrial reprogramming via ATP5H loss promotes multimodal cancer therapy resistance.

The host immune system plays a pivotal role in the emergence of tumor cells that are refractory to multiple clinical interventions including immunotherapy, chemotherapy, and radiotherapy. Here, we examined the molecular mechanisms by which the immune system triggers cross-resistance to these interventions. By examining the biological changes in murine and tumor cells subjected to sequential rounds of in vitro or in vivo immune selection via cognate cytotoxic T lymphocytes, we found that multimodality resistance arises through a core metabolic reprogramming pathway instigated by epigenetic loss of the ATP synthase subunit ATP5H, which leads to ROS accumulation and HIF-1α stabilization under normoxia. Furthermore, this pathway confers to tumor cells a stem-like and invasive phenotype. In vivo delivery of antioxidants reverses these phenotypic changes and resensitizes tumor cells to therapy. ATP5H loss in the tumor is strongly linked to failure of therapy, disease progression, and poor survival in patients with cancer. Collectively, our results reveal a mechanism underlying immune-driven multimodality resistance to cancer therapy and demonstrate that rational targeting of mitochondrial metabolic reprogramming in tumor cells may overcome this resistance. We believe these results hold important implications for the clinical management of cancer.

RIP4 upregulates CCL20 expression through STAT3 signalling in cultured keratinocytes.

The receptor-interacting protein kinase 4 (RIP4), a serine/threonine kinase, is an important modulator of epidermal growth and cutaneous inflammation. We found that RIP4 expression was significantly increased in the lesional skin of psoriasis. However, the role and regulatory mechanism of RIP4 in psoriasis have not been characterized. After treatment with IL-17, RIP4 mRNA and protein levels were increased in HaCaT cells. IL-17 also activated the RIP4 promoter. To understand the functional role of RIP4 in keratinocyte and to investigate the genes regulated by RIP4, RNA-based microarray analysis was performed. Among immune response-related genes, CCL20 expression was significantly changed by RIP4. To identify RIP4-interacting protein, an immunoprecipitation assay was performed. As a result, STAT3 was identified as a new protein that interacts with RIP4. The interaction of RIP4 and STAT3 enhanced STAT3 phosphorylation. In addition, the transcriptional activity of STAT3 induced by RIP4 regulated IL-17-mediated CCL20 expression in HaCaT cells. Taken together, these findings indicate that IL-17 increased RIP4-mediated STAT3 phosphorylation by directly interacting with STAT3. Thus, transcriptional activation of STAT3 promotes the expression of CCL20. Thus, activations of these signalling pathways by RIP4 may contribute to epithermal inflammation in psoriatic keratinocytes.

IL-33 down-regulates CLDN1 expression through the ERK/STAT3 pathway in keratinocytes.

BACKGROUND: Tight junctions (TJs) have important roles in skin barrier function. The TJ protein claudin-1 (CLDN1) is decreased in atopic dermatitis (AD). However, little is known about the mechanism of CLDN1 down-expression. OBJECTIVE: To elucidate the effect of IL-33 on CLDN1 expression in keratinocytes. METHODS: Normal human epidermal keratinocytes (NHEKs) and human skin equivalent models (HSEMs) were cultured in vitro in the presence of IL-33. Production of CLDN1, signal transducer and activator of transcription 3 (STAT3) and Mitogen-activated protein kinases (MAPK) expression were measured by real-time PCR, western blot and immunofluorescence assay. MAPK inhibitors and small interfering RNA were used to confirm the signal pathway of STAT3 and CLDN1. Barrier function was measured by transepithelial electric resistance (TEER) and FITC-dextran flux assays. Electrophoretic Mobility Shift Assay was used to detect STAT3 transcriptional activity. RESULTS: Levels of CLDN1 expression were reduced in the epidermis of AD-model mice overexpressing IL-33. IL-33 down-regulated the expression of CLDN1 mRNA and protein in NHEKs and HSEMs. IL-33 attenuated transepithelial electric resistance and induced FITC-dextran flux in NHEKs. The IL-33 suppressed CLDN1 expression was regulated by an extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3). STAT3 suppressed CLDN1 expression by direct binding to the promoters. CONCLUSION: IL-33 may down-regulate CLDN1 expression through the ERK/STAT3 pathway in keratinocytes.

Direct Coculture of Human Chondrocytes and Synovium-Derived Stem Cells Enhances In Vitro Chondrogenesis.

OBJECTIVES: Coculture of chondrocytes and mesenchymal stem cells (MSCs) has been developed as a strategy to overcome the dedifferentiation of chondrocytes during in vitro expansion in autologous chondrocyte transplantation. Synovium-derived stem cells (SDSCs) can be a promising cell source for coculture due to their superior chondrogenic potential compared to other MSCs and easy accessibility without donor site morbidity. However, studies on coculture of chondrocytes and SDSCs are very limited. The aim of this study was to investigate whether direct coculture of human chondrocytes and SDSCs could enhance chondrogenesis compared to monoculture of each cell. MATERIALS AND METHODS: In this experimental study, passage 2 chondrocytes and SDSCs were directly cocultured using different ratios of chondrocytes to SDSCs (3:1, 1:1, or 1:3). glycosaminoglycan (GAG) synthetic activity was assessed using GAG assays and Safranin-O staining. Expression of chondrogenesis-related genes (collagen types I, II, X, Aggrecan, and Sox-9) were analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry staining. RESULTS: GAG/DNA ratios in 1:1 and 1:3 coculture groups were significantly increased compared to those in the chondrocyte and SDSC monoculture groups. Type II collagen and SOX-9 were significantly upregulated in the 1:1 coculture group compared to those in the chondrocyte and SDSC monoculture groups. On the other hand, osteogenic marker (type I collagen) and hypertrophic marker (type X collagen) were significantly downregulated in the coculture groups compared to those in the SDSC monoculture group. CONCLUSIONS: Direct coculture of human chondrocytes and SDSCs significantly enhanced chondrogenic potential, especially at a ratio of 1:1, compared to chondrocyte or SDSC monocultures.

Intracellular ROS levels determine the apoptotic potential of keratinocyte by Quantum Dot via blockade of AKT Phosphorylation.

Quantum dots (QDs) have shown great potential for biomedical use in a broad range including diagnostic agents. However, the regulatory mechanism of dermal toxicity is poorly understood. In this study, we investigated how QDs-induced apoptosis is regulated in human keratinocytes. We also examined the effect of carboxylic acid-coated QDs (QD 565 and QD 655) on reactive oxygen species (ROS) production and apoptosis-related cellular signalling. The viability of keratinocyte was inhibited by two types of QDs in a concentration-dependent manner. QDs induce ROS production and blockade of AKT phosphorylation. Moreover, the cleavage of AKT-dependent pro-apoptotic proteins such as poly (ADP-ribose) polymerase, caspases-3 and caspases-9 was significantly increased. We also found that a decrease in cellular ROS level by ROS scavenger, N-acetylcysteine (NAC), resulting in the abolishment of QDs-induced AKT de-phosphorylation and cellular apoptosis. Interestingly, QD 655 had a more cytotoxic effect including oxidative stress and AKT-dependent apoptosis than QD 565. In addition, QD 655 had the cytotoxic potential in the human skin equivalent model (HSEM). These data show that QD-induced intracellular ROS levels may be an important parameter in QD-induced apoptosis. These findings from this study indicate that intracellular ROS levels might determine the apoptotic potential of keratinocyte by QD via blockade of AKT phosphorylation.

TSLP Down-Regulates S100A7 and ß-Defensin 2 Via the JAK2/STAT3-Dependent Mechanism.

Elevated T-helper type 2 cytokines in atopic skin, such as IL-4 and IL-13, were thought to be responsible for an impaired expression of antimicrobial proteins, which may contribute to the increased susceptibility to skin infections in patients with atopic dermatitis. In this study, the relationship between thymic stromal lymphopoietin and antimicrobial proteins and the involved molecular pathway was defined in normal human epidermal keratinocytes and human skin equivalent model. Stimulation of normal human epidermal keratinocytes with thymic stromal lymphopoietin decreased both mRNA and levels of S100A7 and human ß-defensin 2 in a dose-dependent manner, and the regulation was JAK2/STAT3-dependent. Thymic stromal lymphopoietin decreased the antimicrobial protein expression, even in the presence of IL-17, which is their strong inducer. STAT3 directly regulated the S100A7 and human ß-defensin 2 promoters in normal human epidermal keratinocytes. Immunohistochemically, lesional atopic skin stained more intensely with phospho-STAT3 compared with healthy control. Our results show that up-regulated thymic stromal lymphopoietin may contribute to the deficiency of antimicrobial proteins in atopic dermatitis, including S100A7 and human ß-defensin 2, by a JAK2/STAT3-dependent mechanism and that STAT3/Sin3a might directly control the transcriptional activity of the antimicrobial protein promoters in normal human epidermal keratinocytes. Taken together, a key role of the JAK2/STAT3/Sin3a signaling pathway in thymic stromal lymphopoietin-mediated immune response in normal human epidermal keratinocytes might give us clues to understanding the pathological signal transductions in atopic dermatitis.