PD-L1 Expression Is Increased in LPS-Induced Acute Respiratory Distress Syndrome by PI3K-AKT-Egr-1/C/EBPδ Signaling Pathway.

The role of programmed death ligand 1 (PD-L1) has been extensively investigated in adaptive immune system. However, increasing data show that innate immune responses are also affected by the immune checkpoint molecule. It has been demonstrated that regulation of PD-L1 signaling in macrophages may be a potential therapeutic method for acute respiratory distress syndrome (ARDS). However, the PD-L1 expression pattern in local macrophages and whole lung tissues remains mysterious, hindering optimization of the potential treatment program. Therefore, we aim to determine the PD-L1 expression pattern during ARDS. Our findings show that PD-L1 levels are markedly increased in lipopolysaccharide (LPS)-stimulated lung tissues, which might be attributable to an increase in the gene expression by immune cells, including macrophages and neutrophils. In vitro experiments are performed to explore the mechanism involved in LPS-induced PD-L1 production. We find that PD-L1 generation is controlled by transcription factors early growth response 1 (Egr-1) and CCAAT/enhancer binding protein delta (C/EBPδ). Strikingly, PD-L1 production is enhanced by phosphoinositide-3 kinase (PI3K)-protein kinase B (AKT) signaling pathway via up-regulation of Egr-1 and C/EBPδ expressions. Additionally, we observe that expressions of Egr-1 and C/EBPδ mutually reinforce each other. Moreover, we observe that PD-L1 is protective for ARDS due to its regulatory role in macrophage-associated inflammatory response. In summary, during LPS-induced ARDS, PD-L1 expression, which is beneficial for the disease, is increased via the PI3K-AKT1-Egr-1/C/EBPδ signaling pathway, providing theoretical basis for application of methods controlling PD-L1 signaling in macrophages for ARDS treatment in clinic.

IgG immune complex-induced acute lung injury is ameliorated by cAMP via down-regulation of C/EBP- and AP-1-mediated transcriptions.

BACKGROUND: Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) are life threatening pulmonary diseases, and we are now lack of effective therapeutic methods. Inflammatory responses are essential for initiating ALI/ARDS. Thus, ameliorating inflammatory reaction might be beneficial for treatment of the disease. There are increasing data that phosphodiesterase-4 (PDE4)-selective inhibitors, which may elevate cellular cyclic adenosine 3', 5'-monophosphate (cAMP) level, could suppress inflammation. However, whether they could be used to treat IgG immune complex (IgG-IC)-associated ALI has not been determined. METHODS: ALI is induced by treating mice with airway deposition of IgG immune complexes. Cellular cAMP concentrations are elevated by treating mice or macrophages with Rolipram/Roflumilast. The degree of pulmonary injury is reflected by lung permeability, leukocyte accumulation, histological change and expressions of pro-inflammatory mediators. 6-Bnz-cAMP and H-89 are used to regulate protein kinase A (PKA) activity, and 8-pCPT-2'-O-Me-cAMP is applied to activate exchange proteins directly activated by cAMP (Epac). Gene expressions are analyzed by real-time PCR, ELISA or Western blot. CCAAT/enhancer binding protein (C/EBP) and activation protein 1 (AP-1) transcription activities are estimated by measuring the luciferase productions. RESULTS: IgG-IC-induced ALI is attenuated by the PDE4-selective inhibitor, which is due to reduced expressions of cytokine and chemokines. Interestingly, we find that cAMP downstream effector molecule PKA but not Epac is involved in negative regulation of IgG-IC-mediated pro-inflammatory mediators' productions. Mechanistically, activation of cAMP-PKA signal axis leads to inactivation of MAPK pathway, resulting in a decrease in C/EBP- and AP-1-mediated transcriptions of pro-inflammatory mediators. CONCLUSIONS: Our data demonstrate, for the first time, that cAMP-PKA signal is involved in down-regulation of IgG-IC-associated inflammatory responses via down-regulating MAPK activation, which is critical for transcriptional activities of C/EBP and AP-1. Collectively, our experiments provide theoretical base for the potential application of PDE4-selective inhibitor to clinic for treatment of IgG-IC-related acute lung injury.

Material Stiffness in Cooperation with Macrophage Paracrine Signals Determines the Tenogenic Differentiation of Mesenchymal Stem Cells.

Stiffness is an important physical property of biomaterials that determines stem cell fate. Guiding stem cell differentiation via stiffness modulation has been considered in tissue engineering. However, the mechanism by which material stiffness regulates stem cell differentiation into the tendon lineage remains controversial. Increasing evidence demonstrates that immune cells interact with implanted biomaterials and regulate stem cell behaviors via paracrine signaling; however, the role of this mechanism in tendon differentiation is not clear. In this study, polydimethylsiloxane (PDMS) substrates with different stiffnesses are developed, and the tenogenic differentiation of mesenchymal stem cells (MSCs) exposed to different stiffnesses and macrophage paracrine signals is investigated. The results reveal that lower stiffnesses facilitates tenogenic differentiation of MSCs, while macrophage paracrine signals at these stiffnesses suppress the differentiation. When exposed to these two stimuli, MSCs still exhibit enhanced tendon differentiation, which is further elucidated by global proteomic analysis. Following subcutaneous implantation in rats for 2 weeks, soft biomaterial induces only low inflammation and promotes tendon-like tissue formation. In conclusion, the study demonstrates that soft, rather than stiff, material has a greater potential to guide tenogenic differentiation of stem cells, which provides comprehensive evidence for optimized bioactive scaffold design in tendon tissue engineering.

Thymosin α-1 in cancer therapy: Immunoregulation and potential applications.

Thymosin α-1 (Tα-1) is an immunomodulating polypeptide of 28 amino acids, which was the first peptide isolated from thymic tissue and has been widely used for the treatment of viral infections, immunodeficiencies, and especially malignancies. Tα-1 stimulates both innate and adaptive immune responses, and its regulation of innate immune cells and adaptive immune cells varies under different disease conditions. Pleiotropic regulation of immune cells by Tα-1 depends on activation of Toll-like receptors and its downstream signaling pathways in various immune microenvironments. For treatment of malignancies, the combination of Tα-1 and chemotherapy has a strong synergistic effect by enhancing the anti-tumor immune response. On the basis of the pleiotropic effect of Tα-1 on immune cells and the promising results of preclinical studies, Tα-1 may be a favorable immunomodulator to enhance the curative effect and decrease immune-related adverse events of immune checkpoint inhibitors to develop novel cancer therapies.

Tumor cell-released LC3-positive EVs promote lung metastasis of breast cancer through enhancing premetastatic niche formation.

Most breast cancer-related deaths are caused by metastasis in vital organs including the lungs. Development of supportive metastatic microenvironments, referred to as premetastatic niches (PMNs), in certain distant organs before arrival of metastatic cells, is critical in metastasis. However, the mechanisms of PMN formation are not fully clear. Here, we demonstrated that chemoattractant C-C motif chemokine ligand 2 (CCL2) could be stimulated by heat shock protein 60 (HSP60) on the surface of murine 4 T1 breast cancer cell-released LC3+ extracellular vesicles (LC3+ EVs) via the TLR2-MyD88-NF-κB signal cascade in lung fibroblasts, which subsequently promoted lung PMN formation through recruiting monocytes and suppressing T cell function. Consistently, reduction of LC3+ EV release or HSP60 level or neutralization of CCL2 markedly attenuated PMN formation and lung metastasis. Furthermore, the number of circulating LC3+ EVs and HSP60 level on LC3+ EVs in the plasma of breast cancer patients were positively correlated with disease progression and lung metastasis, which might have potential value as biomarkers of lung metastasis in breast cancer patients (AUC = 0.898, 0.694, respectively). These findings illuminate a novel mechanism of PMN formation and might provide therapeutic targets for anti-metastasis therapy for patients with breast cancer.

Thymosin α-1 Reverses M2 Polarization of Tumor-Associated Macrophages during Efferocytosis.

The immunologic effects of chemotherapy-induced tumor cell death are not completely understood. Accumulating evidence suggests that phagocytic clearance of apoptotic tumor cells, also known as efferocytosis, is an immunologically silent process, thus maintaining an immunosuppressive tumor microenvironment (TME). Here we report that, in the breast tumor microenvironment, thymosin α-1 (Tα-1) significantly reverses M2 polarization of IL10-producing tumor-associated macrophages (TAM) during efferocytosis induced by apoptotic cells. Mechanistically, Tα-1, which bound to phosphatidylserine on the surface of apoptotic tumor cells and was internalized by macrophages, triggered the activation of SH2-containing inositol 5'-phosphatase 1 (SHIP1) through the lysosomal Toll-like receptor 7 (TLR7)/MyD88 pathway, subsequently resulting in dephosphorylation of efferocytosis-activated TBK1 and reduction of efferocytosis-induced IL10. Tα-1 combined with epirubicin chemotherapy markedly suppressed tumor growth in an in vivo breast cancer model by reducing macrophage-derived IL10 and enhancing the number and function of tumor-infiltrating CD4+ and CD8+ T cells. In conclusion, Tα-1 improved the curative effect of chemotherapy by reversing M2 polarization of efferocytosis-activated macrophages, suggesting that Tα-1 injection immediately after chemotherapy may contribute to highly synergistic antitumor effects in patients with breast cancer. SIGNIFICANCE: Thymosin α-1 improves the curative effect of chemotherapy by reversing efferocytosis-induced M2 polarization of macrophages via activation of a TLR7/SHIP1 axis.

The Crucial Role of PPARγ-Egr-1-Pro-Inflammatory Mediators Axis in IgG Immune Complex-Induced Acute Lung Injury.

Background: The ligand-activated transcription factor peroxisome proliferator-activated receptor (PPAR) γ plays crucial roles in diverse biological processes including cellular metabolism, differentiation, development, and immune response. However, during IgG immune complex (IgG-IC)-induced acute lung inflammation, its expression and function in the pulmonary tissue remains unknown. Objectives: The study is designed to determine the effect of PPARγ on IgG-IC-triggered acute lung inflammation, and the underlying mechanisms, which might provide theoretical basis for therapy of acute lung inflammation. Setting: Department of Pathogenic Biology and Immunology, Medical School of Southeast University. Subjects: Mice with down-regulated/up-regulated PPARγ activity or down-regulation of Early growth response protein 1 (Egr-1) expression, and the corresponding controls. Interventions: Acute lung inflammation is induced in the mice by airway deposition of IgG-IC. Activation of PPARγ is achieved by using its agonist Rosiglitazone or adenoviral vectors that could mediate overexpression of PPARγ. PPARγ activity is suppressed by application of its antagonist GW9662 or shRNA. Egr-1 expression is down-regulated by using the gene specific shRNA. Measures and Main Results: We find that during IgG-IC-induced acute lung inflammation, PPARγ expression at both RNA and protein levels is repressed, which is consistent with the results obtained from macrophages treated with IgG-IC. Furthermore, both in vivo and in vitro data show that PPARγ activation reduces IgG-IC-mediated pro-inflammatory mediators' production, thereby alleviating lung injury. In terms of mechanism, we observe that the generation of Egr-1 elicited by IgG-IC is inhibited by PPARγ. As an important transcription factor, Egr-1 transcription is substantially increased by IgG-IC in both in vivo and in vitro studies, leading to augmented protein expression, thus amplifying IgG-IC-triggered expressions of inflammatory factors via association with their promoters. Conclusion: During IgG-IC-stimulated acute lung inflammation, PPARγ activation can relieve the inflammatory response by suppressing the expression of its downstream target Egr-1 that directly binds to the promoter regions of several inflammation-associated genes. Therefore, regulation of PPARγ-Egr-1-pro-inflammatory mediators axis by PPARγ agonist Rosiglitazone may represent a novel strategy for blockade of acute lung injury.

C/EBPγ is a critical negative regulator of LPS-/IgG immune complex-induced acute lung injury through the downregulation of C/EBPß-/C/EBPδ-dependent C/EBP transcription activation.

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome are life-threatening diseases. Despite recent advances in intensive care medicine, the mortality is still as high as 50%, which stems from our insufficient understanding of the underlying mechanisms of the diseases. The roles of C/EBPß and C/EBPδ have been extensively investigated in LPS- and IgG immune complexes-stimulated acute lung injury. However, the effect of C/EBPγ, belonging to the same family as C/EBPß and C/EBPδ, on ALI has not been elucidated. Our previous data have shown that during LPS-/IgG immune complexes-induced ALI, the DNA binding activities of C/EBPγ are obviously reduced. In the present study, we determine whether ALI induced by LPS and IgG immune complexes is affected by C/EBPγ. We find that adenovirus-mediated C/EBPγ expression in the lung tissue alleviates LPS-/IgG immune complexes-stimulated acute pulmonary damage through reducing vascular permeability changes and recruitment of neutrophils into alveolar spaces, which might be linked to a decrease in the production of pro-inflammatory mediators, such as TNF-α and IL-6. Moreover, our data obtained from macrophages in vitro are consistent with the in vivo results. In terms of mechanisms, C/EBPγ might inhibit LPS-/IgG immune complexes-mediated inflammation via alleviating C/EBPß and C/EBPδ transcription activities as reflected by luciferase assays. However, the NF-κB-dependent production of pro-inflammatory mediators is not affected by C/EBPγ. Taken together, C/EBPγ suppresses LPS- and IgG immune complexes-induced pro-inflammatory mediators' production through the downregulation of C/EBP but not NF-κB activation, leading to the subsequent attenuation of ALI. Collectively, our data provide an insight into the critical role of C/EBPγ in acute lung injury.

Involvement of multiple transcription factors in regulation of IL-ß-induced MCP-1 expression in alveolar type II epithelial cells.

During acute lung injury, a large number of monocytes are recruited into the pulmonary tissue, which is mainly mediated by local production of monocyte chemotactic protein 1 (MCP-1). As an essential component of the lung tissues, alveolar type II epithelial cells are one of the major sources of MCP-1. Therefore, uncovering the mechanism whereby MCP-1 production is regulated in the alveolar type II cells will provide a pivotal theoretical basis for clinical intervention in acute lung injury. In the current study, we find that there is a κB binding site in the MCP-1 promoter region, and mutation of the site leads to reduced production of MCP-1 in alveolar type II epithelial cells. In contrast, overexpression of NF-κB p65 significantly increases MCP-1 expression. Furthermore, we elucidate that IKKα/ß-NF-κB p65 signaling pathway and phosphorylation of serine 534 in NF-κB p65 are required for the maximal expression of MCP-1. Also, Activator protein 1 (AP-1) site in the promoter region and JNK1/2-c-Jun signaling are required for MCP-1 generation in alveolar type II epithelial cells. Moreover, a CCAAT/enhancer-binding protein (C/EBP) element is identified in the MCP-1 promoter region through the point mutation technique, and further experiments demonstrate that both C/EBPß and C/EBPδ are involved in basic and IL-1ß-mediated MCP-1 expression. Of note, specificity protein 1-Sp1 expression is not changed in alveolar type II epithelial cells incubated with IL-1ß, but it still control MCP-1 production by binding to the consensus sequence in the promoter region. More importantly, we find that the results derived from the cell line-MLE-12 cells and primary cells are consistent. Taken together, our data provide insights into the molecular mechanism how MCP-1 expression in inflammatory alveolar type II epithelial cells is regulated at transcription level.

TNF-α induction of IL-6 in alveolar type II epithelial cells: Contributions of JNK/c-Jun/AP-1 element, C/EBPδ/C/EBP binding site and IKK/NF-κB p65/κB site.

Although participation of IL-6 in lung inflammation has been widely elucidated, the transcriptional regulation of its generation in alveolar type II cells stimulated by TNF-α remain unclear. Here, we find that TNF-α significantly induces IL-6 production, and TNF-α induction of IL-6 is mainly regulated at transcriptional level. Upon stimulated by TNF-α, Activator Protein-1 (AP-1)-mediated transcriptional activity is apparently increased in alveolar type II epithelial cells, which might be derived from elevated phosphorylation of JNK and subsequent activation of c-Jun. Either down-regulation of c-Jun or the AP-1 site mutation leads to significant reduction of IL-6 expression. In contrast, ectopic expression of c-Jun notably increases IL-6 generation. So, c-Jun, one of the AP-1 family members, plays a pivotal role in TNF-α-induced IL-6 generation. CCAAT/enhancer binding protein δ (C/EBPδ) expression is significantly amplified by TNF-α, which may contribute to the rise of C/EBP activity in alveolar type II cells. C/EBPδ shRNA treatment results in attenuation of IL-6 expression in the cells, which is consistent with data by introduction of mutations into the C/EBP site in the promoter. However, overexpression of C/EBPδ greatly increases the IL-6 promoter activity. In addition, data regarding another transactivator in the family-C/EBPß show that it does not affect IL-6 production. We also find that the IKK/NF-κB p65 pathway is activated in TNF-α-treated alveolar type II epithelial cells, and plays an essential role in positive regulation of IL-6 expression in TNF-α-treated alveolar type II epithelial cells via knockdown or forced expression of NF-κB p65, or elimination of κB sites in the IL-6 promoter. Notably, IL-6 promoter-driven luciferase production in primary alveolar type II epithelial cells can also be increased by the ectopic expression of c-Jun, C/EBPδ, and NF-κB p65, respectively. Collectively, our data provide insights into molecular mechanism involved in IL-6 expression in alveolar type II epithelial cells on TNF-α treatment, which provides a theoretical basis for specific inhibition of IL-6 production at the transcriptional level.

Protective Effect of Quercetin in LPS-Induced Murine Acute Lung Injury Mediated by cAMP-Epac Pathway.

Quercetin (Que) as an abundant flavonol element possesses potent antioxidative properties and has protective effect in lipopolysaccharide (LPS)-induced acute lung injury (ALI), but the specific mechanism is still unclear, so we investigated the effect of Que from in vivo and in vitro studies and the related mechanism of cAMP-PKA/Epac pathway. The results in mice suggested that Que can inhibit the release of inflammatory cytokine, block neutrophil recruitment, and decrease the albumin leakage in dose-dependent manners. At the same time, Que can increase the cAMP content of lung tissue, and Epac content, except PKA. The results in epithelial cell (MLE-12) suggested that Que also can inhibit the inflammatory mediators keratinocyte-derived chemokines release after LPS stimulation; Epac inhibitor ESI-09 functionally antagonizes the inhibitory effect of Que; meanwhile, PKA inhibitor H89 functionally enhances the inhibitory effect of Que. Overexpression of Epac1 in MLE-12 suggested that Epac1 enhance the effect of Que. All those results suggested that the protective effect of quercetin in ALI is involved in cAMP-Epac pathway.

Activation of Epac alleviates inflammation and vascular leakage in LPS-induced acute murine lung injury.

Exchange protein directly activated by cAMP (Epac) is an important molecule in cAMP signal transduction, but the effect of Epac on lipopolysaccharide (LPS)-induced acute lung injury (ALI) is unclear. In this study, we treated in vitro and in vivo models with the Epac activator 8CPT to determine the effect and related mechanisms of Epac. The in vitro results indicate that 8CPT inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) release from mouse macrophages (MH-S), whereas the protein kinase A (PKA) activator 6BnZ has no effect. Furthermore, Epac over-expression can significantly suppress TNF-α release from LPS induced MH-S cell, while Epac siRNA can slightly increase TNF-α release. Moreover, 8CPT reduces LPS-induced microvascular permeability in human pulmonary microvascular endothelial cells (HPMVECs), whereas the PKA activator 6BnZ has no effect. In mice with LPS-induced ALI, 8CPT significantly reduces LPS-induced inflammatory cytokine release, neutrophil recruitment, and albumin leakage. LPS simultaneously decreases the Epac but not the PKA levels. However, 8CPT reverses the decreased Epac levels. Furthermore, the mechanism involves the small GTPase Rac1/2 but not the mitogen-activated protein kinase (MAPK) pathway. Thus, Epac activation reduces inflammation and microvascular permeability in LPS-induced lung injury and an Epac activator represents a novel choice for the early therapy of ALI.

2-Methoxyestradiol protects against IgG immune complex-induced acute lung injury by blocking NF-κB and CCAAT/enhancer-binding protein ß activities.

Increasing evidences indicate that 2-Methoxyestradiol (2ME2) plays an essential role in protecting against inflammatory responses. However, its effect on IgG immune complex (IC)-induced acute lung injury (ALI) remains enigmatic. In the study, by using i.p. administration of 2ME2, we evaluated its influence on IgG IC-induced pulmonary injury in mice. We found that during IgG IC-induced ALI, mice treated by 2ME2 displayed a substantial decrease in vascular permeability and neutrophil influx (represented by myeloperoxidase activity) when compared with their counterparts receiving vehicle treatment. Furthermore, 2ME2 treatment significantly decreased pro-inflammatory mediator production and inflammatory cell, especially neutrophil accumulation in bronchoalveolar lavage fluids (BALFs) upon IgG IC stimulation. In vitro, IgG IC-triggered inflammatory mediator production was markedly down-regulated by 2ME2 in macrophages. Moreover, we verified that the activation of the transcription factors, NF-κB and CCAAT/enhancer-binding protein (C/EBP) ß, were inhibited by 2ME2 in IgG IC-challenged macrophages. We demonstrated that alleviation of NF-κB-dependent transcription might be associated with reduced phosphorylation of NF-κB p65, and reduction of C/EBP activation was directly linked to its expression. In addition, we discovered that IgG IC-stimulated phosphorylation of both p38 MAPK and ERK1/2 was alleviated by 2ME2. These data indicated a novel strategy for blockade of IgG IC-induced inflammatory activities.

Bigelovii A Protects against Lipopolysaccharide-Induced Acute Lung Injury by Blocking NF-κB and CCAAT/Enhancer-Binding Protein δ Pathways.

Optimal methods are applied to acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS), but the mortality rate is still high. Accordingly, further studies dedicated to identify novel therapeutic approaches to ALI are urgently needed. Bigelovii A is a new natural product and may exhibit anti-inflammatory activity. Therefore, we sought to investigate its effect on lipopolysaccharide- (LPS-) induced ALI and the underlying mechanisms. We found that LPS-induced ALI was significantly alleviated by Bigelovii A treatment, characterized by reduction of proinflammatory mediator production, neutrophil infiltration, and lung permeability. Furthermore, Bigelovii A also downregulated LPS-stimulated inflammatory mediator expressions in vitro. Moreover, both NF-κB and CCAAT/enhancer-binding protein δ (C/EBPδ) activation were obviously attenuated by Bigelovii A treatment. Additionally, phosphorylation of both p38 MAPK and ERK1/2 (upstream signals of C/EBPδ activation) in response to LPS challenge was also inhibited by Bigelovii A. Therefore, Bigelovii A could attenuate LPS-induced inflammation by suppression of NF-κB, inflammatory mediators, and p38 MAPK/ERK1/2-C/EBPδ, inflammatory mediators signaling pathways, which provide a novel theoretical basis for the possible application of Bigelovii A in clinic.

Cancer stem cell vaccine expressing ESAT-6-gpi and IL-21 inhibits melanoma growth and metastases.

Tumor vaccines may induce antitumor efficacy, however, weak immunogenicity of tumor antigens is one of the prime obstacles for excitation of the antitumor immune responses. Therefore, strategies that enhance immunogenicity of tumor vaccines are of particular interest. In this study, a novel melanoma B16F10 CD133(+)CD44(+) cancer stem cell (CSC) vaccine expressing 6 kDa early secreted antigenic target (ESAT-6) in the glycosylphosphatidylinositol (GPI)-anchored form and secreting interleukin (IL)-21 was developed. Its anti-melanoma efficacy and mechanisms were investigated in mice. The results demonstrated that the B16F10-ESAT-6-gpi/IL-21 CD133(+)CD44(+) CSC vaccine exhibited enhanced anti-melanoma efficacy as determined by inhibited melanoma growth, prolonged survival of melanoma bearing mice. The anti-melanoma immunity was associated with elevated levels of serum anti-ESAT-6 and interferon (IFN)-γ as well as increased cytotoxic activities of natural killer cells, splenocytes, and complement dependent cytotoxicity. Furthermore, this CSC-based vaccine apparently inhibited melanoma lung metastasis by decreasing the level of Vimentin while increasing the level of E-cadherin expression, suggesting an inhibited epithelial mesenchymal transition. Thus, the B16F10-ESAT-6-gpi/IL-21 CD133(+)CD44(+) CSC vaccine may be used to reactivate the anti-tumor immunity and for treatment of melanoma.

Effect of targeted ovarian cancer immunotherapy using ovarian cancer stem cell vaccine.

BACKGROUND: Accumulating evidence has shown that different immunotherapies for ovarian cancer might overcome barriers to resistance to standard chemotherapy. The vaccine immunotherapy may be a useful one addition to conditional chemotherapy regimens. The present study investigated the use of vaccine of ovarian cancer stem cells (CSCs) to inhibit ovarian cancer growth. METHODS: CD117(+)CD44(+)CSCs were isolated from human epithelial ovarian cancer (EOC) SKOV3 cell line by using a magnetic-activated cell sorting system. Pre-inactivated CD117(+)CD44(+)CSC vaccine was vacccinated into athymic nude mice three times, and then the mice were challenged subcutaneously with SKOV3 cells. The anti-tumor efficacy of CSC vaccine was envaluated by in vivo tumorigenicity, immune efficient analysis by flow cytometer, and enzyme-linked immunosorbent assays, respectively. RESULTS: The CD117(+) CD44(+)CSC vaccine increased anti-ovarian cancer efficacy in that it depressed ovarian cancer growth in the athymic nude mice. Vaccination resulted in enhanced serum IFN-γ, decreased TGF-ß levels, and increased cytotoxic activity of natural killer cells in the CD117(+) CD44(+)CSC vaccine immunized mice. Moreover, the CSC-based vaccine significantly reduced the CD117(+)CD44(+)CSC as well as the aldehyde dehydrogenase 1 positive cell populations in the ovarian cancer tissues in the xenograft mice. CONCLUSION: The present study provided the first evidence that human SKOV3 CD117(+) CD44(+)CSC-based vaccine may induce the anti-ovarian cancer immunity against tumor growth by reducing the CD117(+)CD44(+)CSC population.

Protective Role of Rho Guanosine Diphosphate Dissociation Inhibitor, Ly-GDI, in Pulmonary Alveolitis.

Growing evidences indicate that Ly-GDI, an inhibitory protein of Rho GTPases, plays an essential role in regulating actin cytoskeletal alteration which is indispensible for the process such as phagocytosis. However, the role of Ly-GDI in inflammation remains largely unknown. In the current study, we found that Ly-GDI expression was significantly decreased in the IgG immune complex-injured lungs. To determine if Ly-GDI might regulate the lung inflammatory response, we constructed adenovirus vectors that could mediate ectopic expression of Ly-GDI (Adeno-Ly-GDI). In vivo mouse lung expression of Ly-GDI resulted in a significant attenuation of IgG immune complex-induced lung injury, which was due to the decreased pulmonary permeability and lung inflammatory cells, especially neutrophil accumulation. Upon IgG immune complex deposition, mice with Ly-GDI over-expression in the lungs produced significant less inflammatory mediators (TNF-α, IL-6, MCP-1, and MIP-1α) in bronchoalveolar lavage fluid when compared control mice receiving airway injection of Adeno-GFP. Mechanically, IgG immune complex-induced NF-κB activity was markedly suppressed by Ly-GDI in both alveolar macrophages and lungs as measured by luciferase assay and electrophoretic mobility shift assay. These findings suggest that Ly-GDI is a critical regulator of inflammatory injury after deposition of IgG immune complexes and that it negatively regulates the lung NF-κB activity.

Suppressors of cytokine signaling 3 is essential for FcγR-mediated inflammatory response via enhancing CCAAT/enhancer-binding protein δ transcriptional activity in macrophages.

Compelling evidence indicates that suppressor of cytokine signaling 3 (SOCS3) plays a pivotal regulatory role in inflammation. However, the function of SOCS3 in inflammatory responses mediated by Fcγ receptor (FcγR) remains largely unknown. In the current study, we found that SOCS3 expression was greatly enhanced in peritoneal macrophages treated with IgG immune complex (IgG IC). By over-expressing SOCS3 in macrophages, we observed that SOCS3 promoted IgG immune complex-induced production of inflammatory mediators, including IL-6, TNF-α, MIP-2, and MIP-1α. In contrast, SOCS3-defective peritoneal macrophages generated less inflammatory cytokines and chemokines when compared with their wild type counterparts during IgG IC-induced inflammatory responses. We further demonstrated that CCAAT/enhancer-binding protein (C/EBP) δ transcription factor was the major downstream target of SOCS3 in macrophages. These data suggested that SOCS3 was an inflammatory enhancer in IgG IC-treated macrophages by increasing C/EBPδ activity. To elucidate the role for myeloid-derived SOCS3 in IgG IC-induced inflammation in vivo, LysM-cre SOCS3(fl/fl) mice lacking SOCS3 in macrophages and neutrophils were generated. We found that SOCS3 deficiency greatly alleviated IgG IC-induced generation of pro-inflammatory mediators in lungs, consistent with the in vitro data. Our current findings may provide a new theoretical basis for designing drugs for treatment of IgG IC-associated diseases.

Protective actions of aspirin-triggered (17R) resolvin D1 and its analogue, 17R-hydroxy-19-para-fluorophenoxy-resolvin D1 methyl ester, in C5a-dependent IgG immune complex-induced inflammation and lung injury.

Increasing evidence suggests that the novel anti-inflammatory and proresolving mediators such as the resolvins play an important role during inflammation. However, the functions of these lipid mediators in immune complex-induced lung injury remain unknown. In this study, we determined the role of aspirin-triggered resolvin D1 (AT-RvD1) and its metabolically stable analog, 17R-hydroxy-19-para-fluorophenoxy-resolvin D1 methyl ester (p-RvD1), in IgG immune complex-induced inflammatory responses in myeloid cells and injury in the lung. We show that lung vascular permeability in the AT-RvD1- or p-RvD1-treated mice was significantly reduced when compared with values in mice receiving control vesicle during the injury. Furthermore, i.v. administration of either AT-RvD1 or p-RvD1 caused significant decreases in the bronchoalveolar lavage fluid contents of neutrophils, inflammatory cytokines, and chemokines. Of interest, AT-RvD1 or p-RvD1 significantly reduced bronchoalveolar lavage fluid complement C5a level. By EMSA, we demonstrate that IgG immune complex-induced activation of NF-κB and C/EBPß transcription factors in the lung was significantly inhibited by AT-RvD1 and p-RvD1. Moreover, AT-RvD1 dramatically mitigates IgG immune complex-induced NF-κB and C/EBP activity in alveolar macrophages. Also, secretion of TNF-α, IL-6, keratinocyte cell-derived chemokine, and MIP-1α from IgG immune complex-stimulated alveolar macrophages or neutrophils was significantly decreased by AT-RvD1. These results suggest a new approach to the blocking of immune complex-induced inflammation.