The expression mechanism of programmed cell death 1 ligand 1 and its role in immunomodulatory ability of mesenchymal stem cells.

Programmed cell death 1 ligand 1 (PD-L1) is an important immunosuppressive molecule, which inhibits the function of T cells and other immune cells by binding to the receptor programmed cell death-1. The PD-L1 expression disorder plays an important role in the occurrence, development, and treatment of sepsis or other inflammatory diseases, and has become an important target for the treatment of these diseases. Mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells with multiple differentiation potential. In recent years, MSCs have been found to have a strong immunosuppressive ability and are used to treat various inflammatory insults caused by hyperimmune diseases. Moreover, PD-L1 is deeply involved in the immunosuppressive events of MSCs and plays an important role in the treatment of various diseases. In this review, we will summarize the main regulatory mechanism of PD-L1 expression, and discuss various biological functions of PD-L1 in the immune regulation of MSCs.

Interferon-gamma and tumor necrosis factor-alpha synergistically enhance the immunosuppressive capacity of human umbilical-cord-derived mesenchymal stem cells by increasing PD-L1 expression.

BACKGROUND: The immunosuppressive capacity of mesenchymal stem cells (MSCs) is dependent on the "license" of several proinflammatory factors to express immunosuppressive factors such as programmed cell death 1 ligand 1 (PD-L1), which determines the clinical therapeutic efficacy of MSCs for inflammatory or immune diseases. In MSCs, interferon-gamma (IFN-γ) is a key inducer of PD-L1 expression, which is synergistically enhanced by tumor necrosis factor-alpha (TNF-α); however, the underlying mechanism is unclear. AIM: To reveal the mechanism of pretreated MSCs express high PD-L1 and explore the application of pretreated MSCs in ulcerative colitis. METHODS: We assessed PD-L1 expression in human umbilical-cord-derived MSCs (hUC-MSCs) induced by IFN-γ and TNF-α, alone or in combination. Additionally, we performed signal pathway inhibitor experiments as well as RNA interference experiments to elucidate the molecular mechanism by which IFN-γ alone or in combination with TNF-α induces PD-L1 expression. Moreover, we used luciferase reporter gene experiments to verify the binding sites of the transcription factors of each signal transduction pathway to the targeted gene promoters. Finally, we evaluated the immunosuppressive capacity of hUC-MSCs treated with IFN-γ and TNF-α in both an in vitro mixed lymphocyte culture assay, and in vivo in mice with dextran sulfate sodium-induced acute colitis. RESULTS: Our results suggest that IFN-γ induction alone upregulates PD-L1 expression in hUC-MSCs while TNF-α alone does not, and that the co-induction of IFN-γ and TNF-α promotes higher expression of PD-L1. IFN-γ induces hUC-MSCs to express PD-L1, in which IFN-γ activates the JAK/STAT1 signaling pathway, up-regulates the expression of the interferon regulatory factor 1 (IRF1) transcription factor, promotes the binding of IRF1 and the PD-L1 gene promoter, and finally promotes PD-L1 mRNA. Although TNF-α alone did not induce PD-L1 expression in hUC-MSCs, the addition of TNF-α significantly enhanced IFN-γ-induced JAK/STAT1/IRF1 activation. TNF-α up-regulated IFN-γ receptor expression through activation of the nuclear factor kappa-B signaling pathway, which significantly enhanced IFN-γ signaling. Finally, co-induced hUC-MSCs have a stronger inhibitory effect on lymphocyte proliferation, and significantly ameliorate weight loss, mucosal damage, inflammatory cell infiltration, and up-regulation of inflammatory factors in colitis mice. CONCLUSION: Overall, our results suggest that IFN-γ and TNF-α enhance both the immunosuppressive ability of hUC-MSCs and their efficacy in ulcerative colitis by synergistically inducing high expression of PD-L1.

Fibroblast-like cells Promote Wound Healing via PD-L1-mediated Inflammation Resolution.

Chronic non-healing wounds fail to progress beyond the inflammatory phase, characterized by a disorder of inflammation resolution. PD-1/PD-L1, a major co-inhibitory checkpoint signaling, plays critical roles in tumor immune surveillance and the occurrence of inflammatory or autoimmune diseases, but its roles in wound healing remains unclear. Here, we described a novel function of PD-L1 in fibroblast-like cells as a positive regulator of wound healing. PD-L1 dynamically expressed on the fibroblast-like cells in the granulation tissue during wound healing to form a wound immunosuppressive microenvironment, modulate macrophages polarization from M1-type to M2-type, and initiates resolution of inflammation, finally accelerate wound healing. Loss of PD-L1 delayed wound healing, especially in mice with LPS-induced severe inflammation. Furthermore, the mainly regulatory mechanism is that combination of FGF-2 and TGF-ß1 promotes PD-L1 translation in fibroblasts through enhancing the eIF4E availability regulated by both PI3K-AKT-mTOR-4EBP1 and p38-ERK-MNK signaling pathways. Our results reveal the positive role of PD-L1 in wound healing, and provide a new strategy for the treatment of chronic wounds.

HDAC2 inhibits EMT-mediated cancer metastasis by downregulating the long noncoding RNA H19 in colorectal cancer.

BACKGROUND: Emerging evidence suggests that epithelial mesenchymal transition (EMT) and epigenetic mechanisms promote metastasis. Histone deacetylases (HDACs) and noncoding RNAs (ncRNAs) are important epigenetic regulators. Here, we elucidated a novel role of histone deacetylase 2 (HDAC2) in regulating EMT and CRC metastasis via ncRNA. METHODS: The expression of HDACs in CRC was analyzed using the public databases and matched primary and metastatic tissues, and CRC cells with different metastatic potentials (DLD1, HCT116, SW480 and SW620). Microarray analysis was used to identify differential genes in parental and HDAC2 knockout CRC cells. EMT and histone modifications were determined using western blot and immunofluorescence. Migration ability was assessed by transwell assay, and metastasis was assessed in vivo using a tail vain injection. Gene expression and regulation was assessed by RT-PCR, chromatin immunoprecipitation and reporter assays. Protein interaction was assessed by immunoprecipitation. Specific siRNAs targeting H19, SP1 and MMP14 were used to validate their role in HDAC2 loss induced EMT and metastasis. RESULTS: Reduced HDAC2 expression was associated with poor prognosis in CRC patients and found in CRC metastasis. HDAC2 deletion or knockdown induced EMT and metastasis by upregulating the long noncoding RNA H19 (LncRNA H19). HDAC2 inhibited LncRNA H19 expression by histone H3K27 deacetylation in its promoter via binding with SP1. LncRNA H19 functioned as a miR-22-3P sponge to increase the expression of MMP14. HDAC2 loss strongly promoted CRC lung metastasis, which was suppressed LncRNA H19 knockdown. CONCLUSION: Our study supports HDAC2 as a CRC metastasis suppressor through the inhibition of EMT and the expression of H19 and MMP14.

Full title: High glucose protects mesenchymal stem cells from metformin-induced apoptosis through the AMPK-mediated mTOR pathway.

Micro- and macro-vascular events are directly associated with hyperglycemia in patients with type 2 diabetes mellitus (T2DM), but whether intensive glucose control decreases the risk of diabetic cardiovascular complications remains uncertain. Many studies have confirmed that impaired quality and quantity of mesenchymal stem cells (MSCs) plays a pathogenic role in diabetes. Our previous study found that the abundance of circulating MSCs was significantly decreased in patients with T2DM, which was correlated with the progression of diabetic complications. In addition, metformin-induced MSC apoptosis is one of the reasons for the decreased quantity of endogenous or exogenous MSCs during intensive glucose control. However, the role of glucose in metformin-induced MSC apoptosis during intensive glucose control in T2DM remains unknown. In this study, we found that metformin induces MSC apoptosis during intensive glucose control, while high glucose (standard glucose control) could significantly reverse its adverse effect in an AMPK-mTOR pathway dependent manner. Thus, our results indicate that the poorer clinical benefit of the intensive glucose control strategy may be related to an adverse effect due to metformin-induced MSC apoptosis during intensive glucose control therapy in patients with T2DM.

Metformin induces apoptosis in mesenchymal stromal cells and dampens their therapeutic efficacy in infarcted myocardium.

BACKGROUND: Cardiovascular complications, especially myocardial infarctions (MIs), are the leading mortality cause in diabetic patients. The transplantation of stem cells into damaged hearts has had considerable success as a treatment for MI, although whether antidiabetic drugs affect the therapeutic efficacy of stem cell transplantation is still unknown. This study aims to understand whether and how metformin, one of the first-line drugs used to treat type 2 diabetes mellitus (T2DM), induces mesenchymal stromal cell (MSC) apoptosis and dampens their cardioprotective effect after transplantation into infarcted hearts. METHODS: A mouse MI model was generated via permanent ligation of the left anterior descending (LAD) coronary artery. MSCs with or without metformin treatment were transplanted after MI in diabetic mice. Echocardiography was used to assess cardiac function and determine cardiac remodeling, and TTC staining was performed to evaluate infarction size. A mouse gavage model was performed to evaluate bone marrow MSCs for flow cytometry assay. RESULTS: Metformin dampened MSC therapeutic efficacy, which increased infarct size and restricted functional cardiac recovery. Specifically, metformin induced the activation of AMP-activated protein kinase (AMPK)-mediated apoptosis through the inhibition of S6K1-Bad-Bcl-xL cell survival signaling, resulting in the upregulated expression of apoptosis-associated proteins and increased MSC apoptosis. Accordingly, counteracting AMPK attenuated metformin-induced apoptosis in MSCs and partially restored their cardioprotective effects in diabetic mice with MI. Furthermore, a decrease in peripheral blood MSCs was found in patients with T2DM who had a metformin medication history. CONCLUSIONS: Our results highlight an unexpected adverse effect of metformin-induced MSC apoptosis through AMPK-mediated mTOR suppression, which is attenuated by an AMPK inhibitor. Moreover, AMPK inhibition may be a novel strategy for enhancing the effectiveness of stem cell therapy after MI in diabetes.

A surgical protocol for bicondylar four-quadrant tibial plateau fractures.

PURPOSE: Bicondylar tibial plateau fractures involving four articular quadrants are severe and complex injuries, and they remain a challenging problem in orthopaedic trauma. The aim of this study was to introduce a new treatment protocol with dual-incision and multi-plate fixation in the floating supine patient position as well as to report the preliminary clinical results. METHODS: From January 2006 to December 2011, 16 consecutive patients with closed bicondylar four-quadrant tibial plateau fractures (Schatzker type VI, OTA/AO 41C2/3) were treated with posteromedial inverted L-shaped and anterolateral incisions. With the posteromedial approach, three quadrants (posteromedial, anteromedial and posterolateral) can be exposed, reduced and fixed with multiple small antiglide plates and short screws in an enclosure pattern. With the anterolateral approach, after articular elevation and bone substitute grafting, a strong locking plate with long screws to the medial cortex is used to raft-buttress the reduced lateral plateau fracture, hold the entire reconstructed tibial condyles together, and contact the condyles with the tibial shaft. All patients were encouraged to exercise knee motion at an early stage. The outcome was evaluated clinically and radiologically after a minimum two-year follow-up. RESULTS: The average operation time was 98 ± 26 minutes (range 70-128) and the average duration of hospitalization was 29 ± 8.6 days (range 20-41). Three cases used five plates, nine cases used four plates, and four cases used three plates. All patients were followed for a mean of 28.7 ± 6.1 months (range 26-38). Fifteen incisions healed initially, while one patient developed a medial wound dehiscence and was successfully managed by debridement. All patients achieved radiological fracture union after an average of 20.2 weeks. At the two-year follow up, the average knee range of motion (ROM) was 98° ± 13.7 (range 88-125°), with a Hospital for Special Surgery (HSS) knee score of 87.7 ± 10.3 (range 75-95), and SMFA score of 21.3 ± 8.6 (range 12-33). CONCLUSION: For bicondylar four-quadrant tibial plateau fractures, the treatment protocol of multiple medial-posterior small plates combined with a lateral strong locking plate through dual incisions can provide stable fracture fixation to allow for early stage rehabilitation. Good clinical outcomes can be anticipated.

Schatzker type IV medial tibial plateau fractures: a computed tomography-based morphological subclassification.

Schatzker type IV medial tibial plateau fractures have an unfavorable prognosis, likely due to the mechanism of injury (fracture-dislocation/subluxation type) and possibly due to the involvement of the posterolateral plateau, which is different from previously thought. The aim of this study was to propose a new subclassification of Schatzker type IV fracture patterns based on 2-dimensional (2-D) computed tomography and three-dimensional (3-D) reconstruction. The authors defined Schatzker type IV medial tibial plateau fractures as AO/OTA 41 type B fractures (partial articular), with partial or total medial plateau involvement, leaving at least the anterolateral quadrant intact. The images of 42 fractures (42 patients) were evaluated. The fractures were further anatomically divided into 2 groups: Group 1 were classic medial unicondylar fractures and Group 2 were more complicated variants involving both condyles, characterized by medial condyle fractures with lateral plateau extension, usually with articular impaction of the centroposterior lateral plateau. Twelve (29%) cases involved only the medial condyle, and 30 (71%) involved both the medial and lateral condyles. Twenty-nine (69%) cases demonstrated posterior coronal fractures. The most common patterns were bicondylar posteromedial plateau fractures with posterolateral quadrant depression (bicondylar posterior fractures: 14 cases, 33%) and total/subtotal medial condyle fractures with posterolateral quadrant depression (13 cases, 31%). The isolated unicondylar posteromedial split fracture was uncommon (2 cases, 5%). Computed tomography-based reconstruction enhances the understanding of fracture anatomy and the relationships between fracture fragments. In Schatzker type IV medial tibial plateau fractures, the involvement of posterolateral quadrants is common.