FasL Receptor Research Articles

Immunological Role of Megakaryocytes and Platelets during Influenza A Virus Infection

Influenza viruses pose a serious threat to public health, with severe cases often characterized by lung damage and inflammation. However, the underlying mechanisms of these processes remain poorly understood. This study aimed to investigate the essential role of megakaryocytes (MKs) and platelets (PLTs) in influenza A virus (IAV) infections. Conducted at the Department of Rare Respiratory Diseases, Cystic Fibrosis, and Pulmonology, Nord University Hospital, Marseille, France, the study collected seventy blood samples between October 2018 and March 2019. Samples were obtained from healthy individuals and patients diagnosed with IAV. Messenger RNA was extracted from isolated PLTs and subjected to quantitative real-time-polymerase chain reaction using sets of primers targeting immune marker genes. Western blot analysis was also performed for confirmation, focusing on Fas Ligand (FasL). Results showed that PLTs from IAV-infected individuals expressed the FasL, tumor necrosis factor-related apoptosis-inducing ligand, and Granulysin (GNLY) receptors when activated. Furthermore, an in vitro assay revealed the presence of FasL receptors on infected CMK cell lines. In vivo investigations demonstrated that activated MKs and PLTs in mice also expressed FasL. Interestingly, none of the immune receptors under investigation were found in both MKs and PLTs in mouse model studies. In conclusion, MKs and PLTs play a significant role in influencing immune responses that may help prevent viral spread during infection. However, further examination of their mechanisms of action is warranted. Understanding the involvement of these cells in influenza pathogenesis could offer valuable insights for developing potential therapeutic strategies.

CRISPR activation screen identifies BCL-2 proteins and B3GNT2 as drivers of cancer resistance to T cell-mediated cytotoxicity

The cellular processes that govern tumor resistance to immunotherapy remain poorly understood. To gain insight into these processes, here we perform a genome-scale CRISPR activation screen for genes that enable human melanoma cells to evade cytotoxic T cell killing. Overexpression of four top candidate genes (CD274 (PD-L1), MCL1, JUNB, and B3GNT2) conferred resistance in diverse cancer cell types and mouse xenografts. By investigating the resistance mechanisms, we find that MCL1 and JUNB modulate the mitochondrial apoptosis pathway. JUNB encodes a transcription factor that downregulates FasL and TRAIL receptors, upregulates the MCL1 relative BCL2A1, and activates the NF-κB pathway. B3GNT2 encodes a poly-N-acetyllactosamine synthase that targets >10 ligands and receptors to disrupt interactions between tumor and T cells and reduce T cell activation. Inhibition of candidate genes sensitized tumor models to T cell cytotoxicity. Our results demonstrate that systematic gain-of-function screening can elucidate resistance pathways and identify potential targets for cancer immunotherapy.

Restoring FAS Expression via Lipid-Encapsulated FAS DNA Nanoparticle Delivery Is Sufficient to Suppress Colon Tumor Growth In Vivo.

Simple SummaryA key feature of human colorectal tumor is loss of FAS expression. FAS is the death receptor for FASL of activated T cells. Loss of FAS expression therefore may promote tumor cell immune escape. We aimed at determining whether restoring FAS expression is sufficient to suppress colorectal tumor growth. Mouse and human FAS cDNA was synthesized and encapsulated into cationic lipid nanoparticle DOTAP-Cholesterol to formulate DOTAP-Chol-mFAS and DOTAP-Chol-hFAS, respectively. Restoring FAS expression in metastatic mouse colon-tumor cells enabled FASL-induced elimination of FAS+ tumor cells in vitro and suppressed colon-tumor growth and progression in tumor-bearing mice in vivo. Restoring FAS expression induced FAS receptor auto-oligomerization and tumor cell auto-apoptosis in metastatic human colon-tumor cells in vitro. DOTAP-Chol-hFAS therapy is also sufficient to suppress metastatic human colon tumor xenograft growth in athymic mice. Tumor-selective delivery of FAS DNA nanoparticle is potentially an effective therapy for human colorectal cancer.A hallmark of human colorectal cancer is lost expression of FAS, the death receptor for FASL of cytotoxic T lymphocytes (CTLs). However, it is unknown whether restoring FAS expression alone is sufficient to suppress csolorectal-cancer development. The FAS promoter is hypermethylated and inversely correlated with FAS mRNA level in human colorectal carcinomas. Analysis of single-cell RNA-Seq datasets revealed that FAS is highly expressed in epithelial cells and immune cells but down-regulated in colon-tumor cells in human colorectal-cancer patients. Codon usage-optimized mouse and human FAS cDNA was designed, synthesized, and encapsulated into cationic lipid to formulate nanoparticle DOTAP-Chol-mFAS and DOTAP-Chol-hFAS, respectively. Overexpression of codon usage-optimized FAS in metastatic mouse colon-tumor cells enabled FASL-induced elimination of FAS+ tumor cells in vitro, suppressed colon tumor growth, and increased the survival of tumor-bearing mice in vivo. Overexpression of codon-optimized FAS-induced FAS receptor auto-oligomerization and tumor cell auto-apoptosis in metastatic human colon-tumor cells. DOTAP-Chol-hFAS therapy is also sufficient to suppress metastatic human colon tumor xenograft growth in athymic mice. DOTAP-Chol-mFAS therapy exhibited no significant liver toxicity. Our data determined that tumor-selective delivery of FAS DNA nanoparticles is sufficient for suppression of human colon tumor growth in vivo.

Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2.

To date, no study has demonstrated that soluble Fas ligand (sFasL)-mediated inflammation is regulated via interaction with Fas in vivo. We found that FasL interacts specifically with tumor necrosis factor receptor superfamily (TNFRSF)10B, also known as death receptor (DR)5. Autoantibody-induced arthritis (AIA) was attenuated in FasL (Faslgld/gld)- and soluble FasL (FaslΔs/Δs)-deficient mice, but not in Fas (Faslpr/lpr and Fas-/-)- or membrane FasL (FaslΔm/Δm)-deficient mice, suggesting sFasL promotes inflammation by binding to a Fas-independent receptor. Affinity purification mass spectrometry analysis using human (h) fibroblast-like synovial cells (FLSCs) identified DR5 as one of several proteins that could be the elusive Fas-independent FasL receptor. Subsequent cellular and biochemical analyses revealed that DR5 interacted specifically with recombinant FasL-Fc protein, although the strength of this interaction was approximately 60-fold lower than the affinity between TRAIL and DR5. A microarray assay using joint tissues from mice with arthritis implied that the chemokine CX3CL1 may play an important downstream role of the interaction. The interaction enhanced Cx3cl1 transcription and increased sCX3CL1 production in FLSCs, possibly in an NF-κB-dependent manner. Moreover, the sFasL-DR5 interaction-mediated CX3CL1-CX3CR1 axis initiated and amplified inflammation by enhancing inflammatory cell influx and aggravating inflammation via secondary chemokine production. Blockade of FasL or CX3CR1 attenuated AIA. Therefore, the sFasL-DR5 interaction promotes inflammation and is a potential therapeutic target.

Responsiveness to i.v. immunoglobulin therapy in patients with toxic epidermal necrolysis: A novel pharmaco-immunogenetic concept.

Toxic epidermal necrolysis (TEN) represents a rare drug-induced autoimmune reaction with delayed-type hypersensitivity that initiates the process of developing massive keratinocyte apoptosis, dominantly in the dermoepidermal junction. Although the etiopathophysiology has not yet been fully elucidated, the binding of Fas ligand (FasL, CD95L) to the Fas receptor (CD95) was shown to play a key role in the induction of apoptosis in this syndrome. The knowledge of the role of immunoglobulin G (IgG) in inhibition of Fas-mediated apoptosis contributed to the introduction of i.v. Ig (IVIg) in the therapy of TEN patients. Despite great enthusiasm for this therapy at the end of the 1990s, subsequent studies in various populations and meta-analyses could not unequivocally confirm the efficacy of the IVIg-based treatment concept. Today, therefore, we are faced with the dilemmas of how to adjust therapy of TEN patients most effectively, which patients could benefit from IVIg therapy and what dose of the preparation should be administrated. The ground-breaking question is: do the host genetic profiles influence the responsiveness and side-effects of IVIg therapy in TEN patients? Based on recent pharmacological, immunological and genetic findings, we suggest that the variability of IVIg therapy outcomes in TEN patients may be related to functional variants in Fas, FasL and Fc-γ receptor genes. This novel concept could lead to improved quality of care for patients with TEN, facilitating personalized therapy to reduce mortality.

Toxicology and Pharmacokinetic Studies in Mice and Nonhuman Primates of the Nontoxic, Efficient, Targeted Hexameric FasL: CTLA4-FasL.

Cytotoxic T-lymphocyte antigen 4 (CTLA4)-FasL, a homo-hexameric signal converter protein, is capable of inducing robust apoptosis in malignant cells of the B-cell lineage expressing its cognate B7 and Fas targets, while sparing nonmalignant ones. This fusion protein's striking proapoptotic efficacy stems from its complementary abilities to coordinately activate apoptotic signals and abrogate antiapoptotic ones. A limiting factor in translating FasL or Fas receptor agonists into the clinic has been lethal hepatotoxicity. Here, we establish CTLA4-FasL's in vivo efficacy in multiple murine and xenograft models, for both systemic and subcutaneous tumors. Significantly, good laboratory practice (GLP) toxicology studies in mice indicate that CTLA4-FasL given repeatedly at doses up to five times the effective dose was well-tolerated and resulted in no significant adverse events. An equivalent single dose of CTLA4-FasL administered to nonhuman primates was also well-tolerated, albeit with a moderate dose-dependent leukopenia that was completely reversible. Interestingly, monkey peripheral blood mononuclear cells were more sensitive to CTLA4-FasL-induced apoptosis when tested in vitro. In both species, there was short-term elevation in serum levels of IL6, IL2, and IFNγ, although this was not associated with clinical signs of proinflammatory cytokine release, and further, this cytokine elevation could be completely prevented by dexamethasone premedication. Liver toxicity was not observed in either species, as confirmed by serum liver enzyme levels and histopathologic assessment. In conclusion, CTLA4-FasL emerges from animal model studies as an effective and safe agent for targeted FasL-mediated treatment of B7-expressing aggressive B-cell lymphomas.

T cells genetically engineered to overcome death signaling enhance adoptive cancer immunotherapy.

Across clinical trials, T cell expansion and persistence following adoptive cell transfer (ACT) have correlated with superior patient outcomes. Herein, we undertook a pan-cancer analysis to identify actionable ligand-receptor pairs capable of compromising T cell durability following ACT. We discovered that FASLG, the gene encoding the apoptosis-inducing ligand FasL, is overexpressed within the majority of human tumor microenvironments (TMEs). Further, we uncovered that Fas, the receptor for FasL, is highly expressed on patient-derived T cells used for clinical ACT. We hypothesized that a cognate Fas-FasL interaction within the TME might limit both T cell persistence and antitumor efficacy. We discovered that genetic engineering of Fas variants impaired in the ability to bind FADD functioned as dominant negative receptors (DNRs), preventing FasL-induced apoptosis in Fas-competent T cells. T cells coengineered with a Fas DNR and either a T cell receptor or chimeric antigen receptor exhibited enhanced persistence following ACT, resulting in superior antitumor efficacy against established solid and hematologic cancers. Despite increased longevity, Fas DNR-engineered T cells did not undergo aberrant expansion or mediate autoimmunity. Thus, T cell-intrinsic disruption of Fas signaling through genetic engineering represents a potentially universal strategy to enhance ACT efficacy across a broad range of human malignancies.

Abstract PR05: T-cells engineered to overcome death signaling within the tumor microenvironment enhance adoptive cancer immunotherapy

Abstract Across clinical trials, T-cell expansion and persistence following adoptive cell transfer (ACT) has correlated with superior patient outcomes. Herein, we undertook a pan-cancer analysis across human tumors to identify potentially actionable ligand/receptor pairs that might limit T-cell function and persistence following ACT. We discovered that FASLG, the gene encoding the apoptosis-inducing ligand FasL, is overexpressed within the majority of human tumor microenvironments. Further, we uncovered that Fas, the receptor for FasL, is highly expressed on patient-derived T-cells used for ACT. We hypothesized that a cognate Fas-FasL interaction within the tumor microenvironment might limit both T-cell persistence and antitumor efficacy. We discovered that genetic engineering of Fas variants impaired the ability to bind FADD function as dominant negative receptors (DNRs) in Fas-competent mouse and human T-cells, rescuing cells from FasL-induced apoptosis. Fas DNR-engineered T-cells exhibited enhanced persistence within tumors following ACT, resulting in superior cancer regression and overall survival in solid and hematologic malignancies treated with TCR or CAR-modified cells. Despite enhanced longevity, Fas DNR-engineered T-cells did not undergo aberrant clonal expansion, demonstrating the safety of this approach. Thus, cell-intrinsic “insulation” of T-cells from the negative influence of FasL is a potentially universal strategy to enhance ACT efficacy across a broad range of human malignancies. Citation Format: Christopher A. Klebanoff. T-cells engineered to overcome death signaling within the tumor microenvironment enhance adoptive cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR05.

Quercetin Enhances Ligand-induced Apoptosis in Senescent Idiopathic Pulmonary Fibrosis Fibroblasts and Reduces Lung Fibrosis In Vivo

Although cellular senescence may be a protective mechanism in modulating proliferative capacity, fibroblast senescence is now recognized as a key pathogenic mechanism in idiopathic pulmonary fibrosis (IPF). In aged mice, abundance and persistence of apoptosis-resistant senescent fibroblasts play a central role in nonresolving lung fibrosis after bleomycin challenge. Therefore, we investigated whether quercetin can restore the susceptibility of senescent IPF fibroblasts to proapoptotic stimuli and mitigate bleomycin-induced pulmonary fibrosis in aged mice. Unlike senescent normal lung fibroblasts, IPF lung fibroblasts from patients with stable and rapidly progressing disease were highly resistant to Fas ligand (FasL)-induced and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Senescent IPF fibroblasts exhibited decreased expression of FasL and TRAIL receptors and caveolin-1, as well as increased AKT activation, compared with senescent normal lung fibroblasts. Although quercetin alone was not proapoptotic, it abolished the resistance to FasL- or TRAIL-induced apoptosis in IPF fibroblasts. Mechanistically, quercetin upregulated FasL receptor and caveolin-1 expression and modulated AKT activation. In vivo quercetin reversed bleomycin-induced pulmonary fibrosis and attenuated lethality, weight loss, and the expression of pulmonary senescence markers p21 and p19-ARF and senescence-associated secretory phenotype in aged mice. Collectively, these data indicate that quercetin reverses the resistance to death ligand-induced apoptosis by promoting FasL receptor and caveolin-1 expression and inhibiting AKT activation, thus mitigating the progression of established pulmonary fibrosis in aged mice. Therefore, quercetin may be a viable therapeutic option for IPF and other age-related diseases that progress with the accumulation of senescent fibroblasts.

Killer (FASL regulatory) B cells are present during latent TB and are induced by BCG stimulation in participants with and without latent tuberculosis

Regulatory B cells (Bregs) have been shown to be present during several disease states. The phenotype of the cells is not completely defined and the function of these cells differ between disease. The presence of FASL expressing (killer) B cells during latent and successfully treated TB disease have been shown but whether these cells are similar to regulatory B cells remain unclear. We assessed the receptor expression of FASL/IL5 (killer B cells), CD24/CD38 (regulatory B cells) on whole peripheral blood of participants with untreated active TB and healthy controls. We then isolated B cells from a second cohort of M.tb exposed (Quantiferon (QFN) positive) and unexposed (Quantiferon negative) HIV negative participants, and evaluated the frequency of killer B cells induced following stimulation with BCG and/or CD40 and IL5. Our data reveal no difference in the expression on CD24 and CD38 between participants with active TB and the controls. There was also no difference in the frequency of regulatory B cells measured in the peripheral blood mononuclear cells (PBMC) fraction between latent TB and uninfected controls. We did however notice that regulatory B cells (CD24hiCD38hi) population express the FASL receptor. The expression of killer B cell phenotype (CD178+IL5RA+) was significantly higher in controls compared to those with active TB disease (1,06% vs 0,455%). Furthermore, we found that BCG restimulation significantly induced the FASL/IL5RA B cells but this was only evident in the QFN positive group. Our data suggest that both regulatory and killer B cells are present during latent and active TB disease but that the frequency of these populations are increased during latent disease. We also show that the FASL+IL5RA+ B killer B cells are induced in latent TB infection following BCG restimulation but whether these cells are indicative of protection remains unclear.

In-vitro influence of mycophenolate mofetil (MMF) and Ciclosporin A (CsA) on cytokine induced killer (CIK) cell immunotherapy.

BackgroundCytokine-induced-killer (CIK) cells are a promising immunotherapeutic approach for impending relapse following hematopoietic stem cell transplantation (HSCT). However, there is a high risk for treatment failure associated with severe graft versus host disease (GvHD) necessitating pharmaceutical intervention post-transplant. Whether immunosuppression with mycophenolate mofetil (MMF) or Ciclosporin A (CsA) influences the cytotoxic effect of CIK cell immunotherapy is still an open issue.MethodsCIK cells were generated from PBMC as previously described followed by co-incubation with mycophenolic acid (MPA) or CsA. Proliferation, cytotoxicity and receptor expression were investigated following short- (24 h), intermediate- (3 days) and long-term (7 days) MPA incubation with the intention to simulate the in vivo situation when CIK cells were given to a patient with relevant MPA/CsA plasma levels.ResultsShort-term MPA treatment led to unchanged proliferation capacity and barely had any effect on viability and cytotoxic capability in vitro. The composition of CIK cells with respect to T-, NK-like T- and NK cells remained stable. Intermediate MPA treatment lacked effects on NKG2D, FasL and TRAIL receptor expression, while an influence on proliferation and viability was detectable. Furthermore, long-term treatment significantly impaired proliferation, restricted viability and drastically reduced migration-relevant receptors accompanied by an alteration in the CD4/CD8 ratio. CD3+CD56+ cells upregulated receptors relevant for CIK cell killing and migration, whereas T cells showed the most interference through significant reductions in receptor expression. Interestingly, CsA treatment had no significant influence on CIK cell viability and the cytotoxic potential against K562.ConclusionsOur data indicate that if immunosuppressant therapy is indispensable, efficacy of CIK cells is maintained at least short-term, although more frequent dosing might be necessary.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1024-4) contains supplementary material, which is available to authorized users.

DcR3 gene polymorphisms are associated with sporadic breast infiltrating ductal carcinoma in Northeast Chinese women.

Decoy Receptor 3 (DcR3), also called TNFRSF6β, is a member of the tumor necrosis factor receptor superfamily and is a soluble receptor for FasL. DcR3 is overexpressed in cancers and contributes to tumorigenesis through immune suppression and promotion of angiogenesis. We found that DcR3 is overexpressed in breast infiltrating ductal carcinoma (IDC) cells as compared with normal controls. We also conducted a case-control study analyzing associations of DcR3 polymorphisms with breast IDC risk. Subjects included 531 females with breast IDC and 592 age-matched healthy controls. Four DcR3 single nucleotide polymorphism loci with minor frequencies of more than 5% (rs3208008, rs41309931, rs2297441 and rs1291207) were genotyped using polymerase chain reaction restriction fragment length polymorphism and sequencing. Our results revealed significant differences in rs41309931genotypes and alleles (P < 0.01). Based on Haploview software analysis, the haplotype block Ars3208008 Grs41309931 Grs2297441 Ars1291207 exhibited the highest frequency, but, haplotype blocks Ars3208008 Trs41309931 Grs2297441 Ars1291207 and Crs3208008 Grs41309931 Grs2297441 Ars1291207 were associated with breast IDC risk. This study also detected associations between DcR3 gene polymorphisms and the clinicopathological features of breast IDC, including lymph node metastasis and C-erbB2, P53, estrogen receptor and progesterone receptor status. These data indicate that DcR3 gene polymorphisms are associated with sporadic breast IDC risk in Northeast Chinese females.

Cytotoxic effects of SMAC-mimetic compound LCL161 in head and neck cancer cell lines.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common tumor entities worldwide. Unfortunately, recent drug developments in other fields of oncology have yielded no efficacy in the treatment of oral squamous cell carcinoma. As a new starting point, we investigated the impact of Fas ligand (FasL) and the SMAC-mimetic compound LCL161 in mono- and combination treatment in HNSCC cell lines. Five different cell lines of HNSCC were treated with FasL and LCL161 in mono- and combination treatment. Cytotoxicity was measured via a crystal violet assay. The cell lines were characterized for CD95 (FasL receptor) expression via flow cytometry. The degradation of cellular inhibitor of apoptosis protein 1 (cIAP1) was detected via Western blot. Incubation with FasL led to a significant decrease in three out of five cell lines. Combination treatment with LCL161 enhanced cytotoxicity significantly. Two cell lines were FasL resistant, but one of them could be resensitized with LCL161. In all cell lines, Western blot analysis showed degradation of cIAP1 after LCL161 application. However, one cell line showed only minor vulnerability to the FasL and LCL161 combination. This is the first study investigating combination treatment of FasL and LCL161 in head and neck cancer cell lines. Pro-apoptotic effects of the combination were detected in the majority of the cell lines. Interestingly, one of two FasL-resistant cell lines was sensitive to the combination therapy with FasL and LCL161. SMAC-mimetic compounds show promising results in the treatment of other tumor entities in vitro and might be useful drugs to improve HNSCC therapy.

Role of mitochondria in apoptotic and necroptotic cell death in the developing brain

Hypoxic–ischemic encephalopathy induces secondary brain injury characterized by delayed energy failure. Currently, therapeutic hypothermia is the sole treatment available after severe intrapartum asphyxia in babies and acts to attenuate secondary loss of high energy phosphates improving both short- and long-term outcome. In order to develop the next generation of neuroprotective therapies, we urgently need to understand the underlying molecular mechanisms leading to cell death. Hypoxia–ischemia creates a toxic intracellular environment including accumulation of reactive oxygen/nitrosative species and intracellular calcium after the insult, inducing mitochondrial impairment. More specifically mitochondrial respiration is suppressed and calcium signaling is dysregulated. At a certain threshold, Bax-dependent mitochondrial permeabilization will occur leading to activation of caspase-dependent and apoptosis-inducing factor-dependent apoptotic cell death. In addition, hypoxia–ischemia induces inflammation, which leads to the release of TNF-α, TRAIL, TWEAK, FasL and Toll-like receptor agonists that will activate death receptors on neurons and oligodendroglia. Death receptors trigger apoptotic death via caspase-8 and necroptotic cell death through formation of the necrosome (composed of RIP1, RIP3 and MLKL), both of which converge at the mitochondria.

Mir-590 Is a Novel STAT5 Regulated Oncogenic miRNA and Targets FasL In Acute Myeloid Leukemia

The recent discovery of new molecular lesions with prognostic significance in acute myeloid leukemia (AML) is enhancing our understanding of leukemia biology and our ability to identify new therapeutic targets. Previously, using the unique leukemic myeloid progenitor line AML-193, we profiled IL-3-, GM-CSF-, and G-CSF-regulated miRNA signatures. 301 miRNAs were commonly regulated by these three cytokines, and the most highly induced miRNA was miR-590-5p. Herein, we have attempted to define the functional role and clinical relevance of miR-590 in AML. We first examined the relative miR-590 expression in steady state hematopoiesis and showed it was highest at CD34+ and declined its expression through myeloid lineage differentiation (ANOVA, p<0.0001). To functionally determine the role of increased miR-590 expression, we generated a gain-of-function model in human CD34+ hematopoietic stem cells (HSC) via lentivirus transduction. Increased expression of miR-590 in CD34+ cells resulted in significant increases in CFU-GM colonies, strongly suggesting that dysregulation of miR-590 expression may be myeloproliferative. In AML (n=33) and control (n=9) bone marrow samples, miR-590 expression was determined via RT-qPCR. miR-590-5p expression was highly upregulated in 22 of the samples (67%) compared to control subjects. In silico analysis of the miR-590-5p promoter revealed three potential binding sites for STAT5 (-249, -749, -1499). To functionally determine whether STAT5 directly regulates miR-590-5p expression, we performed a ChIP assay, which showed that STAT5 binds to the -749 region of miR-590-5p promoter. To conclusively determine the STAT5 binding sites, we cloned the miR-590 promoter in a luciferase vector and performed site directed mutagenesis for each potential binding site. This assay confirmed that the -749 binding site was the major STAT5 regulatory site for miR-590 (p<0.002). Importantly, constitutive activation of STAT5 is a hallmark of AML associated with FLT3 mutations, therefore, we set out to determine if specific STAT5 and FLT3 inhibitors could decrease miR-590 expression. We pretreated MV4-11 cells, which harbors the FLT-ITD mutation and has increased STAT5 activation, with 100uM STAT5 inhibitor (N′-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide) for 90 minutes or 100nM FLT3 inhibitor (EMD Millipore, 343020) for 12 hours, both of which resulted in significant inhibition of miR-590-5p expression (p<0.05). To evaluate whether the AML samples with high miR-590 expression also possess elevated phospho-STAT5 or phospho-FLT3 levels, we performed immunohistochemistry analysis on a custom-made tissue microarray. In AML samples with high miR-590 levels, increased activation of FLT3 and STAT5 was observed compared to controls. Since FLT3 mutations result in decreased survival and poorer prognosis in AML, it may be that miR-590-5p plays an important role in the pathology of AML associated with dysregulated FLT3 and STAT5. To understand the complete functional role of miR-590 in AML, the predicted targets need to be identified and validated for their roles in leukemogenesis. Upon molecular screening of several predicted targets, FasL was experimentally found to be a conserved target of miR-590. More specifically, 3'UTR analysis of FasL revealed three potential seed sequences for miR-590 which have been verified experimentally via luciferase assay. Furthermore, significantly increased levels of FasL protein and transcript expression was detected in the MV4-11 cells stably expressing anti-miR-590 compared to control cells. Additionally, we identified the levels of Fas/CD95 (FasL receptor) on AML-193 and MV4-11 cell lines and found these cells had high Fas/CD95 expression on the cell surface as analyzed via flow cytometry. In order to determine the physiological significance of Fas/FasL, these cells were treated with soluble FasL (100ng) for 24 hours and apoptosis was analyzed via Annexin V staining. FasL treatment induced increased apoptosis compared to the untreated cells. Taken together, we have identified miR-590 as a candidate oncomiR that is regulated via the STAT5 pathway and targets FasL to promote cell survival. Thus, our data suggests that further understanding of miR-590's role in AML may lead to development of novel anti-miR-590 therapeutic strategies in AML associated with dysregulated STAT5. Disclosures:No relevant conflicts of interest to declare.

Type-I Interferon is Critical for FasL Expression on Lung Cells to Determine the Severity of Influenza

Infection of influenza A virus in mammals induces hyper lung pneumonia, which often causes lethal diseases. FasL is a specific ligand of Fas, which is a type-I transmembrane protein to induce cell death. Previously, it has been reported that the hyper induction of gene expression associated with Fas signal is observed in lethal influenza A virus infection. More importantly, it was also reported that functional mutation of the FasL gene protects the host against influenza A virus infection. These observations suggest that induction of FasL signal is functionally associated with the severity of influenza. However, regulation of the induction of FasL or Fas by influenza A virus infection is still unknown. Here, we demonstrated that FasL is induced after the viral infection, and inhibition of the Fas/FasL signal by treatment with a recombinant decoy receptor for FasL (Fas-Fc) increases the survival rate of mice after lethal infection of influenza A virus as well as functional mutation of the FasL gene in gld/gld mice. In addition, the induction level of FasL gene expression in the lung was correlated with the severity of influenza. We also showed that a variety of types of cells in the lung express FasL after the viral infection. Furthermore, type-I interferon induced by the viral infection was shown to be critical for induction of FasL protein expression in the lung. These findings suggested that expression of FasL protein induced by type-I IFN on the lung cell surface is critical to determine the severity of influenza.

Enhanced apoptosis in retinal pigment epithelium under inflammatory stimuli and oxidative stress

Age-related macular degeneration (AMD) is a neurodegenerative disease that causes irreversible central vision loss in the elderly. Retinal pigment epithelium (RPE) has been found to be a key component in AMD pathogenesis. The Ccl2(-/-)/Cx3cr1(-/-) (DKO) mouse on Crb1(rd8) background is created as an AMD model, developing AMD-like retinal lesions. Our study aimed to examine RPE apoptosis in DKO mouse and human ARPE-19 cell line. DKO RPE expressed higher apoptotic proteins when compared with age-matched wild type (WT) RPE in physiological conditions. Apoptosis of primary cultured mouse RPE was evaluated under stimulation with lipopolysaccharide for inflammatory stimulation and 2,3,7,8-tetrachlorodibenzo-p-dioxin or H(2)O(2) for oxidative stress. Compared with WT RPE, DKO RPE was more susceptible to Fas ligand (FasL)-mediated apoptosis under both inflammatory and oxidative stress, with less cell viability and higher expression of apoptotic transcripts and proteins. Decreased cell viability was also observed in ARPE-19 cells under each stimulus. Furthermore, we also investigated the anti-apoptotic effects of decoy receptor 3 (DcR3), a decoy receptor for FasL, on ARPE-19 cells under inflammatory and oxidative stress. DcR3 pre-incubated ARPE-19 cells showed decreased apoptosis, with increased cell viability and decreased expression of apoptotic transcripts and proteins under the stimuli. On the contrary, knockdown of DcR3 in ARPE-19 cells showed totally opposite results. Our study demonstrates that FasL-mediated RPE apoptosis may play a pivotal role in AMD pathogenesis.

Ocena ekspresji Fas i FasL na limfocytach krwi obwodowej chorych na raka krtani

The aim of the study was evaluation of Fas and FasL receptors’ expression on peripheral blood lymphocytes in patients with laryngeal cancer. The study was conducted in a group of 62 patients with different clinical stages of the disease. The study material consisted of the peripheral blood obtained from the studied population, from which T lymphocytes were isolated and incubated with monoclonal antibodies. To evaluate antigens’ expression, a FACS Galibur flow cytometre was used. The flow cytometry evaluated cells were labeled with fluorochrome-conjugated monoclonal antibodies. Obtained results were compared with the measurements taken in 20 healthy individuals who constituted the control group. In the conducted study a significant decrease in the Fas and FasL receptors’ expression on CD+ lymphocytes was found in comparison with the expression of these proteins observed in the control group. Moreover, a higher level of Fas expression was observed on the peripheral blood lymphocytes of patients with stage I clinical advancement of laryngeal cancer in comparison with those who had more advanced stages of the disease (Groups II, III, IV), which may be indicative of higher activation and susceptibility of peripheral blood lymphocytes to apoptosis in patients with the early stage of the disease. A negative (reverse) correlation was also found between Fas and FasL receptors’ expression and the organ advancement of laryngeal cancer, which may provide further evidence that together with the progress of the disease the susceptibility of peripheral blood lymphocytes to apoptosis decreases.

Aberrant expression and function of death receptor-3 and death decoy receptor-3 in human cancer

Death receptor-3 (DR3) and death decoy receptor-3 (DcR3) are both members of the tumour necrosis factor receptor (TNFR) superfamily. The TNFR superfamily contains eight death domain-containing receptors, including TNFR1 (also called DR1), Fas (also called DR2), DR3, DR4, DR5, DR6, NGFR and EDAR. Upon the binding of these receptors with their corresponding ligands, the death domain recruits various proteins that mediate both the death and proliferation of cells. Receptor function is negatively regulated by decoy receptors (DcR1, DcR2, DcR3 and OPG). DR3/DcR3 are a pair of positive and negative players with which vascular endothelial growth inhibitor (VEGI) interacts. VEGI has been suggested to be a potential tumour suppressor. The inhibitory effects of VEGI on cancer are manifested in three main areas: a direct effect on cancer cells, an anti-angiogenic effect on endothelial cells, and the stimulation of dendritic cell maturation. A recent study indicated that DR3 may be a new receptor for E-selectin, which has been reported to be associated with cancer metastasis. DcR3 is a soluble receptor, highly expressed in various tumours, which lacks an apparent transmembrane segment, prevents cytokine response through ligand binding and neutralization, and is an inhibitor of apoptosis. DcR3 serves as a decoy receptor for FasL, LIGHT and VEGI. The cytokine LIGHT activates various anti-tumour functions and is expected to be a promising candidate for cancer therapy. Certain tumours may escape FasL-dependent immune-cytotoxic attack by expressing DcR3, which blocks FasL function. DR3/DcR3 play profound roles in regulating cell death and proliferation in cancer. The present review briefly discusses DR3/DcR3 and attempts to elucidate the role of these negative and positive players in cancer.

Increased Killing of Liver NK Cells by Fas/Fas Ligand and NKG2D/NKG2D Ligand Contributes to Hepatocyte Necrosis in Virus-Induced Liver Failure

The role of liver NK cells in virus-induced severe viral hepatitis and, subsequently, hepatic failure is not well defined. In this study, we investigated the role of liver NK cells in the development of hepatocyte necrosis in fulminant hepatic failure (FHF) and acute-on-chronic liver failure (ACLF) because of viral infection. A mouse model of FHF induced by murine hepatitis virus strain 3 (MHV-3) was used to study the role of liver NK cells. Samples from patients with hepatitis B virus-related ACLF (HBV-ACLF) were examined. After MHV-3 infection, the number of NK cells in livers of BALB/cJ mice increased markedly, peaked at 48 h postinfection, and remained at a high level until sacrifice. In peripheral blood, spleen, and bone marrow, this number decreased significantly. Expression of CD69, cytotoxic activity, and intracellular IFN-gamma and TNF-alpha production by liver NK cells at 48 h postinfection were all significantly upregulated. Depletion of NK cells 24 h post-MHV-3 infection increased the mice survival from 0 of 18 (0%) to 4 of 18 (22.2%). Highly activated liver NK cells were cytotoxic to MHV-3-infected hepatocytes and this effect was markedly inhibited by anti-Fas ligand (FasL) plus anti-NKG2D mAbs. Furthermore, the accumulation of hepatic NK cells and increased expression of FasL and natural cytotoxicity receptors (NKp30 and NKp46) on the peripheral NK cells from patients with HBV-ACLF were correlated with disease progression. These results indicate NK cells play a pivotal role in the pathogenesis of FHF and HBV-ACLF, in which process Fas/FasL and NKG2D/NKG2D ligand pathway contribute to the liver NK cell-mediated hepatocyte injury.