FasL-mediated Apoptosis Research Articles

Modulating alloreactive immune responses by controlled dual delivery of Fas and IL-2R agonists using a lipocoacervate platform

Abstract Allogeneic islet transplantation is an effective treatment for type 1 diabetes (T1D). Graft rejection is controlled by immunosuppressive drugs that have adverse effects. An imbalance between Teffs and Tregs is the primary cause of T1D and graft rejection. Activated Teff cells express Fas and are sensitive to FasL-mediated apoptosis. Treg cells are relatively refractive to apoptosis and expand in response to IL-2. Thus, a combination of Fas and IL-2R agonists has the potential to modulate alloreactive responses for sustained graft survival. Given the off-target effects of systemic delivery of biologics, we established a lipocoacervate formulation consisting of a biodegradable polycation, PEAD, and polyanion heparin to form coacervate for controlled delivery of a novel Fas agonist, SA-FasL, and IL-2. Release kinetics and activities of proteins were assessed in vitro. The immunomodulatory efficacy of protein-loaded lipocoacervate was assessed in vivo. IL-2 and SA-FasL proteins showed a steady release over 30 days in vitro. IL-2 released on day 14 had minimal activity loss, whereas FasL showed ~20% activity loss on day 9. Treatment with SA-FasL/IL-2-loaded lipocoacervate modulated in vivo alloreactive T cell responses, resulting in an increased Treg/Teff cell ratio. In conclusion, lipocoacervate is an effective platform for controlled and sustained release of SA-FasL and IL-2 biologics to modulate allo and autoreactive T cells with significant therapeutic potential for T1D.

TP induces hepatic intolerance to FasL-mediated hepatocyte apoptosis by inhibiting XIAP

Triptolide (TP) is extracted from the traditional Chinese medicine Tripterygium wilfordii Hook. F. (TWHF). Its severe toxic side effects, especially hepatotoxicity, have limited the clinical application of TP-related drugs. In this study, we investigated the mechanism of the hepatotoxic effects of TP from the perspective that TP inhibited the expression of the pro-survival protein X-linked inhibitor of apoptosis protein (XIAP) and enhanced FasL-mediated apoptosis of hepatocytes. TP and CD95/Fas antibody (Jo-2) were administered by gavage to C57BL/6 mice for 7 consecutive days. After co-administration of TP and Jo-2, mouse livers showed large areas of necrosis and apoptosis and significantly increased Caspase-3 activity. KEGG pathway enrichment analysis indicated that TP may cause the development of liver injury through the apoptotic signaling pathway. Proteinprotein interaction networks showed that XIAP played an essential role in this process. TP reduced the protein expression of XIAP after combination treatment with Jo-2/FasL in vivo/in vitro. TP and FasL co-stimulation significantly increased microRNA-137 (miR-137) levels in AML12 cells, while inhibition of miR-137 expression induced a rebound in XIAP protein expression. In conclusion, TP presensitizes hepatocytes and enhances the sensitivity of hepatocytes to the Fas/FasL pathway by inhibiting the protein expression of XIAP, leading to hepatocyte apoptosis.

Exploiting Fas-mediated autocrine apoptosis of donor alloreactive T cells as an effective approach to prevent acute graft-versus-host disease in a humanized mouse model

Abstract Acute GVHD initiated by donor T cells is a barrier to allogeneic bone marrow transplantation. Pan T cell elimination from the graft is associated with failure of stem cell engraftment and delayed immune reconstitution. T cells upregulate the Fas receptor on their surface following activation and become susceptible to FasL-mediated apoptosis. The objective of this study was to target Fas-mediated apoptosis for the preferential elimination of alloreactive T cells. We previously reported a novel form of FasL protein chimeric with streptavidin (SA-FasL). Mouse and human lymphocytes were surface modified with biotin, followed by engineering with the SA-FasL protein taking advantage of biotin's high affinity to SA. SA-FasL-engineered cells were assessed for autocrine apoptosis of alloreactive T cells in vitro and in vivo. NSG mice transplanted with 10 7human PBMC were used as an acute GVHD xenogeneic model. SA-FasL induced autocrine apoptosis in alloreactive T cells in vitro and in vivo. Mice transplanted with SA-FasL-engineered PBMC had significantly lower numbers of human T cells in the spleen and liver analyzed five days post-transplantation compared to the recipients of SA-engineered cells. Recipients of SA-engineered PBMC developed acute GVHD with a median survival time of 15 days. In marked contrast, sixty percent of SA-FasL-engineered PBMCs recipients survived for a 60-day observation period and showed minimal clinical GVHD scores. Transient display of SA-FasL protein on the surface of donor cells provides a practical and clinically applicable effective approach to prevent acute GVHD with significant translational potential.

Interferon-λ contributes to endometrial receptivity.

IFN-λs participate in the fetal-maternal immune interaction, involving in immune regulation, uterine receptivity, cell migration and adhesion, and endometrium apoptosis. Our study helps to elucidate the underlying causes of the IFN-λs deficiency to spontaneous pregnancy loss in women. Immunotherapy has been commonly used to prevent recurrent pregnancy loss in women with inadequate uterus receptivity or immunological imbalance. Many immune regulators are now identified as having crucial roles at the embryo-maternal interface. However, the clinical efficacy of immunity-related markers during the peri-implantation period remains to be explored in depth. Here, we demonstrated that endometrial expression of interferon-λ (IFN-λ), regarded as a newer class of interferons, is aberrantly lower in women who suffered from recurrent implantation failure than that in fertile control. We further uncovered genetic and biochemical evidence that IFN-λ is induced directly by estrogen in the endometrial cells, and IFN-λ pathway may play multiple roles involving the inflammatory response, uterine receptivity, cell migration, and blastocyst adhesion. Furthermore, we indicated IFN-λ lessens the sensitivity of endometrium to FASL-mediated apoptosis. In addition to uncovering this IFN-λ as a novel nonredundant regulator that participates in the fetal-maternal immune interaction, our study helps to elucidate the underlying causes of spontaneous pregnancy loss in women.

SETD2 Deficiency Promotes Inflammatory Bowel Disease via Oxidative Stress and FasL-Mediated Apoptosis

SETD2 Deficiency Promotes Inflammatory Bowel Disease via Oxidative Stress and FasL-Mediated Apoptosis

Vitamin E Attenuates FasL-Induced Apoptotic Death of Dendritic Cells Through PI3K Signalling

Vitamin E Attenuates FasL-Induced Apoptotic Death of Dendritic Cells Through PI3K Signalling

The cleft palate candidate gene BAG6 supports FoxO1 acetylation to promote FasL-mediated apoptosis during palate fusion

BackgroundCleft palate is a common craniofacial defect, which occurs when the palate fails to fuse during development. During fusion, the palatal shelves migrate towards the embryonic midline to form a seam. Apoptotic elimination of medial edge epithelium (MEE) cells along this seam is required for the completion of palate fusion. MethodsWhole exome sequencing (WES) of six Chinese cleft palate families was applied to identify novel cleft palate-associated gene variants. Palatal fusion and immunofluorescence studies were performed in a murine palatal shelf organ culture model. Gene and protein expression were analyzed by qPCR and immunoblotting in murine MEE cells during seam formation in vivo. Mechanistic immunoprecipitation studies were performed in murine MEE cells in vitro. ResultsWES identified Bcl-2 associated anthanogene 6 (BAG6) as a novel cleft palate-associated gene. In murine MEE cells, we discovered upregulation of Bag6 and the transcription factor forkhead box protein O1 (FoxO1) during seam formation in vivo. Using a palatal shelf organ culture model, we demonstrate that nuclear-localized Bag6 enhances MEE cell apoptosis by promoting p300's acetylation of FoxO1, thereby promoting transcription of the pro-apoptotic Fas ligand (FasL). Subsequent gain- and loss-of-function studies in the organ culture model demonstrated that FasL is required for Bag6/acFoxO1-mediated activation of pro-apoptotic Bax/caspase-3 signaling, MEE apoptosis, and palate fusion. Palatal shelf contact was shown to enhance Bag6 nuclear localization and upregulate nuclear acFoxO1 in MEE cells. ConclusionsThese findings demonstrate that nuclear-localized Bag6 and p300 co-operatively enhance FoxO1 acetylation to promote FasL-mediated MEE apoptosis during palate fusion.

Treg sensitivity to FasL and relative IL-2 deprivation drive idiopathic aplastic anemia immune dysfunction

AbstractIdiopathic aplastic anemia (AA) has 2 key characteristics: an autoimmune response against hematopoietic stem/progenitor cells and regulatory T-cells (Tregs) deficiency. We have previously demonstrated reduction in a specific subpopulation of Treg in AA, which predicts response to immunosuppression. The aims of the present study were to define mechanisms of Treg subpopulation imbalance and identify potential for therapeutic intervention. We have identified 2 mechanisms that lead to skewed Treg composition in AA: first, FasL-mediated apoptosis on ligand interaction; and, second, relative interleukin-2 (IL-2) deprivation. We have shown that IL-2 augmentation can overcome these mechanisms. Interestingly, when high concentrations of IL-2 were used for in vitro Treg expansion cultures, AA Tregs were able to expand. The expanded populations expressed a high level of p-BCL-2, which makes them resistant to apoptosis. Using a xenograft mouse model, the function and stability of expanded AA Tregs were tested. We have shown that these Tregs were able to suppress the macroscopic clinical features and tissue manifestations of T-cell–mediated graft-versus-host disease. These Tregs maintained their suppressive properties as well as their phenotype in a highly inflammatory environment. Our findings provide an insight into the mechanisms of Treg reduction in AA. We have identified novel targets with potential for therapeutic interventions. Supplementation of ex vivo expansion cultures of Tregs with high concentrations of IL-2 or delivery of IL-2 directly to patients could improve clinical outcomes in addition to standard immunosuppressive therapy.

Differential Transcription of CD95 in Abdominal and Lower‐Body Subcutaneous Fat Depot–Implications for Regional Differences in Adipose Tissue Function

BackgroundAbdominal (ABD) and lower‐body (LB) fat depots exert opposing effects on cardiometabolic risk factors. However, molecular mechanisms underlying these differences are not completely understood. Studies suggest an inflammatory role of CD95 in obesity‐associated adipose tissue inflammation and dysfunction. Also, leptin deficiency is associated with increased CD95 expression.ObjectiveTo examine the expression of CD95 in the ABD and LB fat depots and to investigate leptin‐dependent CD95 regulation.MethodsTranscription of CD95 and other adipokines was determined in paired subcutaneous abdominal (ABD) and lower‐body (femoral, LB) fat samples obtained from 29 healthy individuals (10 females, age: 29.4 ± 7.6 years, BMI: 24 ± 4.1 kg/m2). The underlying mechanisms and down‐stream effects of altered CD95 expression were evaluated using differentiated human white preadipocytes (dHWP).ResultsReduced mRNA expression of CD95 was apparent in adipose tissue obtained from LB fat depot [ABD: 4.30 (13.91 – 0.06), LB: 2.49 (10.88 – 0.04), p = 0.01]. mRNA expression of MCP1 [ABD: 0.51 (1.35 – 0.34), LB: 0.51 (1.21 – 0.28), p = 0.51], CCL5 [ABD: 6.60 (43.45 – 1.40), LB: 4.44 (11.28 – 1.27), p = 0.20], TNFα [ABD: 1.10 (6.14 – 0.03), LB: 0.07 (5.84 – 0.03), p = 0.30], and leptin [ABD: 21.86 (31.33 – 14.6), LB: 27.23 (41.69 – 20.35), p = 0.08] was not different between the two fat depots. A positive relationship between CD95 mRNA and other inflammatory cytokines was evident in both ABD (MCP1: ρ = 0.79, p < 0.001; CCL5: ρ = 0.83, p < 0.001; TNFα: ρ = 0.87, p < 0.001) and LB fat depot (MCP1: ρ = 0.87, p < 0.001; CCL5: ρ = 0.78, p < 0.001; TNFα: ρ = 0.83, p < 0.001). Furthermore, leptin‐dependent decreases in CD95 mRNA (p = 0.04) and protein expression (p = 0.03) was seen in dHWP. Also, 24 hour pre‐treatment of dHWP with leptin protected cells from FasL (p = 0.03) induced apoptosis.ConclusionsCompared to ABD adipose tissue, LB fat has reduced transcription of CD95. Additionally, expression of CD95 mRNA was associated with increased expression of inflammatory cytokines in both ABD and LB subcutaneous fat. Moreover, we show that leptin may mediate decreases in CD95 expression thereby protecting adipose tissue from FasL mediated apoptosis. Our studies suggest that the decreased CD95 mRNA expression in LB fat depot may be related to higher localized leptin expression and possibly contribute to depot specific alterations in adipose tissue function and consequent cardiometabolic risk.Support or Funding InformationPrachi Singh is supported by AHA grant‐in‐Aid 17GRNT33660138.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Selection of NFκB Inhibitors to Block Inflammation and Induce Sensitisation to FasL-Induced Apoptosis in HNSCC Cell Lines Is Critical for Their Use as a Prospective Cancer Therapy.

Inflammation is a central aspect of tumour biology and can contribute significantly to both the origination and progression of tumours. The NFκB pathway is one of the most important signal transduction pathways in inflammation and is, therefore, an excellent target for cancer therapy. In this work, we examined the influence of four NFκB inhibitors—Cortisol, MLN4924, QNZ and TPCA1—on proliferation, inflammation and sensitisation to apoptosis mediated by the death ligand FasL in the HNSCC cell lines PCI1, PCI9, PCI13, PCI52 and SCC25 and in the human dermal keratinocyte cell line HaCaT. We found that the selection of the inhibitor is critical to ensure that cells do not respond by inducing counteracting activities in the context of cancer therapy, e.g., the extreme IL-8 induction mediated by MLN4924 or FasL resistance mediated by Cortisol. However, TPCA1 was qualified by this in vitro study as an excellent therapeutic mediator in HNSCC by four positive qualities: (1) proliferation was inhibited at low μM-range concentrations; (2) TNFα-induced IL-8 secretion was blocked; (3) HNSCC cells were sensitized to TNFα-induced cell death; and (4) FasL-mediated apoptosis was not disrupted.

SU125MODELLING THE IMPACT OF SCHIZOPHRENIA GWAS LOCI DURING HUMAN BRAIN DEVELOPMENT USING SPATIO-TEMPORAL GENE EXPRESSION DATA

Cleft palate is a common craniofacial defect, which occurs when the palate fails to fuse during development. During fusion, the palatal shelves migrate towards the embryonic midline to form a seam. Apoptotic elimination of medial edge epithelium (MEE) cells along this seam is required for the completion of palate fusion.Whole exome sequencing (WES) of six Chinese cleft palate families was applied to identify novel cleft palate-associated gene variants. Palatal fusion and immunofluorescence studies were performed in a murine palatal shelf organ culture model. Gene and protein expression were analyzed by qPCR and immunoblotting in murine MEE cells during seam formation in vivo. Mechanistic immunoprecipitation studies were performed in murine MEE cells in vitro.WES identified Bcl-2 associated anthanogene 6 (BAG6) as a novel cleft palate-associated gene. In murine MEE cells, we discovered upregulation of Bag6 and the transcription factor forkhead box protein O1 (FoxO1) during seam formation in vivo. Using a palatal shelf organ culture model, we demonstrate that nuclear-localized Bag6 enhances MEE cell apoptosis by promoting p300's acetylation of FoxO1, thereby promoting transcription of the pro-apoptotic Fas ligand (FasL). Subsequent gain- and loss-of-function studies in the organ culture model demonstrated that FasL is required for Bag6/acFoxO1-mediated activation of pro-apoptotic Bax/caspase-3 signaling, MEE apoptosis, and palate fusion. Palatal shelf contact was shown to enhance Bag6 nuclear localization and upregulate nuclear acFoxO1 in MEE cells.These findings demonstrate that nuclear-localized Bag6 and p300 co-operatively enhance FoxO1 acetylation to promote FasL-mediated MEE apoptosis during palate fusion.

Immunomodulation with SA-FasL-Engineered Microgels Achieves Long-Term Survival of Allogeneic Islet Grafts

Introduction and Objective Allogenic islet transplantation has shown efficacy in the clinic for the treatment of type 1 diabetes. However, sustained survival of allogeneic islet grafts requires chronic immunosuppression that has significant adverse effects. T effector (Teff) cells recognizing and responding to allograft antigens are the major culprit of graft rejection. Upon activation, Teff cells upregulate Fas death receptor on their surface and become sensitive to FasL-mediated apoptosis. Fas pathway, therefore, presents an important target for immunomodulation to block alloreactive responses with significant therapeutic potential. Herein, we assessed the efficacy of PEG microgels engineered with a novel form of FasL chimeric with a modified form of core streptavidin, SA-FasL, in achieving sustained survival of allogeneic islet grafts in the absence of chronic immunosuppression. Materials and Methods Microgels presenting biotin on their surface were produced by reacting biotin-PEG-thiol with a maleimide-terminated 4-arm poly(ethylene) glycol macromer, and generating 200 μm diameter microgels crosslinked with dithiothreitol. Biotinylated microgels were engineered with SA-FasL (1 μg/103 microgels) taking the advantage of the high affinity interaction between biotin and streptavidin. The apoptotic activity of SA-FasL-engineered microgels was tested on mouse A20 B cell lymphocytes in vitro. The immunomodulatory function of microgels for the prevention of graft rejection was tested by mixing ~500 BALB/c naïve islets with 1000 SA-FasL-engineered microgels and transplanting under the kidney capsule of streptozotocin-induced diabetic C57BL/6 recipients. A group of recipients were also treated transiently with rapamycin (0.2 mg/kg daily for 15 days starting the day of transplantation). Results and Discussion Biotin-PEG microgels were efficiently coupled with SA-FasL, and showed a dose-dependent apoptotic activity in A20 cells. Co-transplantation of SA-FasL-engineered microgels with naïve islets led to prolonged survival of grafts as compared with the control group (islets + unmodified microgels) with 20% surviving for a 200-day observation period. Transient use of low dose rapamycin, enhanced survival to > 90% of the grafts. In marked contrast, only 20% of islets co-transplanted with PEG microgels, and short-course rapamycin, survived long-term. Importantly, CD4+CD25+FoxP3+ Treg cells were required for graft survival as depletion of this cell population on day 50 post-transplantation resulted in prompt rejection. Conclusion These results provide strong proof-of-efficacy and feasibility for the use of SA-FasL-engineered microgels as an off-the-shelf product for the modulation of alloreactive responses with significant therapeutic potential. Funded in part by NIH (grants R21EB020107, R21AI113348, 185 R56AI121281, and F30AR069472) and the Juvenile Diabetes Research Foundation (2-SRA-186 2014-287-Q-R).

The siRNA silencing of DcR3 expression induces Fas ligand-mediated apoptosis in HepG2 cells.

Dysfunctional Fas ligand (FasL) may inhibit the apoptosis of tumor cells. FasL contains two receptors, Fas and Decoy Receptor 3 (DcR3). DcR3 competitively binds to FasL over Fas, resulting in the inhibition of FasL-mediated apoptosis. Therefore, it was suggested that the downregulation of DcR3 expression enhances FasL-mediated apoptosis. In the current study, the expression of DcR3 was silenced in liver cancer HepG2 cells in order to study the effect of FasL on HepG2 cell activity and invasiveness. DcR3 siRNA knockdown HepG2 cells (KD), DcR3 blank plasmid control HepG2 cells and wild-type HepG2 cells (WT) were treated with FasL (10 ng/ml). Flow cytometry was used to detect changes in the cell cycle and apoptosis. MTS, clonogenic, wound healing and Transwell assays were performed to examine changes in cell activity, proliferation, migration and invasiveness. Reverse transcription polymerase chain reaction and western blot analysis were performed to measure the expression of DcR3, matrix metallopeptidase 9 (MMP9), vascular endothelial growth factor (VEGF)-C and VEGF-D. The results demonstrated that, compared with WT cells, the proportion of KD cells in the G2/M phase decreased following treatment with FasL. KD cells were more sensitive to FasL-induced apoptosis. Following treatment with FasL, the activity and proliferation, migration and invasion of KD cells were reduced, and the expression of MMP9, VEGF-C and VEGF-D decreased. Furthermore, it was demonstrated that DcR3 is involved in the proliferation and invasion of HepG2 cells, and this mechanism may be associated with the regulatory effect of the expression of MMP9, VEGF-C and VEGF-D; however, the exact mechanism of action remains unclear. FollowingDcR3 silencing, FasL-mediated apoptosis increased in HepG2 cells. Therefore, DcR3 combined with FasL may be a potential target for the treatment of liver cancer.

Aspirin cooperates with p300 to activate the acetylation of H3K9 and promote FasL-mediated apoptosis of cancer stem-like cells in colorectal cancer

Cancer stem-like cells (CSCs) have been proposed as a key driving force of tumor growth and relapse in colorectal cancer (CRC), and therefore, they are promising targets for cancer therapy. Epidemiological evidence has suggested that the daily use of aspirin reduces overall mortality of CRC and the risk of distant metastasis. We investigated the effect and mechanism of aspirin on CSCs in CRC.Methods: The ratio of CSCs was analyzed after aspirin treatment both in a cell model and patient samples. Chemically modified aspirin and immunoprecipitation were adopted to detect the target proteins of aspirin. A locus-specific light-inducible epigenetic modification system based on CRISPR technology was constructed to verify the causal relationship in these molecular events. In vivo characterization was performed in a xenograft model.Results: We found that aspirin induces apoptosis in enriched colorectal CSCs, inhibits tumor progression, and enhances the anti-neoplastic effects of chemotherapeutic agents. Furthermore, aspirin directly interacts with p300 in the nucleus, promotes H3K9 acetylation, activates FasL expression, and induces apoptosis in colorectal CSCs. Notably, these effects of aspirin are absent in non-CSCs since H3K9 is hypermethylated in non-CSCs and the effects are not induced by other NSAIDs. In addition, aspirin can suppress oxaliplatin-enriched CSCs and serve as an adjuvant therapy.Conclusions: Taken together, we revealed a unique epigenetic and cox-independent pathway (p300-AcH3K9-FasL axis) by which aspirin eliminates colorectal CSCs. These findings establish an innovative framework of the therapeutic significance of aspirin.

Changes in the cellular microRNA profile by the intracellular expression of HIV-1 Tat regulator: A potential mechanism for resistance to apoptosis and impaired proliferation in HIV-1 infected CD4+ T cells.

HIV-1 induces changes in the miRNA expression profile of infected CD4+ T cells that could improve viral replication. HIV-1 regulator Tat modifies the cellular gene expression and has been appointed as an RNA silencing suppressor. Tat is a 101-residue protein codified by two exons that regulates the elongation of viral transcripts. The first exon of Tat (amino acids 1–72) forms the transcriptionally active protein Tat72, but the presence of the second exon (amino acids 73–101) results in a more competent regulatory protein (Tat101) with additional functions. Intracellular, full-length Tat101 induces functional and morphological changes in CD4+ T cells that contribute to HIV-1 pathogenesis such as delay in T-cell proliferation and protection against FasL-mediated apoptosis. But the precise mechanism by which Tat produces these changes remains unknown. We analyzed how the stable expression of intracellular Tat101 and Tat72 modified the miRNA expression profile in Jurkat cells and if this correlated with changes in apoptotic pathways and cell cycle observed in Tat-expressing cells. Specifically, the enhanced expression of hsa-miR-21 and hsa-miR-222 in Jurkat-Tat101 cells was associated with the reduced expression of target mRNAs encoding proteins related to apoptosis and cell cycle such as PTEN, PDCD4 and CDKN1B. We developed Jurkat cells with stable expression of hsa-miR-21 or hsa-miR-222 and observed a similar pattern to Jurkat-Tat101 in resistance to FasL-mediated apoptosis, cell cycle arrest in G2/M and altered cell morphology. Consequently, upregulation of hsa-miR-21 and hsa-miR-222 by Tat may contribute to protect against apoptosis and to anergy observed in HIV-infected CD4+ T cells.

Liver receptor homolog-1 (NR5a2) regulates CD95/Fas ligand transcription and associated T-cell effector functions

CD95/Fas ligand (FasL) is a cell death-promoting member of the tumor necrosis factor family with important functions in the regulation of T-cell homeostasis and cytotoxicity. In T cells, FasL expression is tightly regulated on a transcriptional level involving a complex set of different transcription factors. The orphan nuclear receptor liver receptor homolog-1 (LRH-1/NR5a2) is involved in the regulation of development, lipid metabolism and proliferation and is predominantly expressed in epithelial tissues. However, its expression in T lymphocytes has never been reported so far. Based on in silico analysis, we identified potential LRH-1 binding sites within the FASLG promoter. Here, we report that LRH-1 is expressed in primary and secondary lymphatic tissues, as well as in CD4+ and CD8+ T cells. LRH-1 directly binds to its binding sites in the FASLG promoter, and thereby drives FASLG promoter activity. Mutations in the LRH-1 binding sites reduce FASLG promoter activity. Pharmacological inhibition of LRH-1 decreases activation-induced FasL mRNA expression, as well as FasL-mediated activation-induced T-cell apoptosis and T-cell cytotoxicity. In a mouse model of Concanavalin A-induced and FasL-mediated hepatitis pharmacological inhibition of LRH-1 resulted in decreased hepatic FasL expression and a significant reduction of liver damage. In summary, these data show for the first time LRH-1 expression in T cells, its role in FASLG transcription and the potential of pharmacological inhibition of LRH-1 in the treatment of FasL-mediated immunopathologies.

Exploration of Fas S-Nitrosylation by the Biotin Switch Assay.

S-nitrosylation is the covalent attachment of nitric oxide radical to the thiol side chain of cysteine. The death receptor Fas/CD95 can be S-nitrosylated in cancer cell lines by NO donors or iNOS activation. This posttranslational modification (PTM) induces Fas aggregation into lipid rafts and enhances FasL-mediated signaling and apoptosis. In this report, we describe the detection of Fas S-nitrosylation by the most commonly used method, the biotin switch assay (BSA) technique, that allows the detection of this very labile covalent modification in cells or tissues. Briefly, this technique relies on the ability of ascorbate to reduce the covalent bond between the NO radical and the protein, allowing the exchange of the NO radical with a thiol reactive biotin-HPDP. The biotinylated proteins are then easily purified by using NeutrAvidin resin, separated by SDS-PAGE resolution and analyzed by Western blotting.

Balance between IL-3 and type Iinterferons and their interrelationship with FasL dictates lifespan and effector functions of human basophils.

In contrast to eosinophils and neutrophils, the regulation of the lifespan of human basophils is poorly defined, with the exception of the potent anti-apoptotic effect of IL-3 that also promotes pro-inflammatory effector functions and phenotypic changes. Type I IFNs (IFN-α, IFN-β), which are well known for their anti-viral activities, have the capacity to inhibit allergic inflammation. To elucidate whether type I IFNs have the potential to abrogate the lifespan and/or effector functions of human basophils. We cultured human basophils, and for comparison, eosinophils and neutrophils, with IL-3, interferons, FasL and TRAIL, alone or in combination, and studied cell survival, effector functions and signalling pathways involved. Despite an identical pattern of early signalling in basophils, eosinophils and neutrophils in response to different types of interferons, only basophils displayed enhanced apoptosis after type I IFN treatment. IFN-γ prolonged survival of eosinophils but did not affect the lifespan of basophils. IFN-α-mediated apoptosis required STAT1-STAT2 heterodimers and the contribution of constitutive p38 MAPK activity. Whereas the death ligands FasL and TRAIL-induced apoptosis in basophils per se, IFN-α-mediated apoptosis did neither involve autocrine TRAIL signalling nor did it sensitize basophils to FasL-induced apoptosis. However, IFN-α and FasL displayed an additive effect in killing basophils. Interestingly, IL-3, which protected basophils from IFN-α-, TRAIL- or FasL-mediated apoptosis, did not completely block the additive effect of combined IFN-α and FasL treatment. Moreover, we demonstrate that IFN-α suppressed IL-3-induced release of IL-8 and IL-13. In contrast to IFN-α-mediated apoptosis, these inhibitory effects of IFN-α were not dependent on p38 MAPK signalling. Our study defines the unique and granulocyte-type-specific inhibitory and pro-apoptotic function of type I IFNs and their cooperation with death ligands in human blood basophils, which may be relevant for the anti-allergic properties of type I IFNs.

Idiopathic pulmonary fibrosis fibroblasts become resistant to Fas ligand-dependent apoptosis via the alteration of decoy receptor 3.

Idiopathic pulmonary fibrosis (IPF) is an irreversible lethal lung disease with an unknown etiology. IPF patients' lung fibroblasts express inappropriately high Akt activity, protecting them in response to an apoptosis-inducing type I collagen matrix. FasL, a ligand for Fas, is known to be increased in the lung tissues of patients with IPF, implicated with the progression of IPF. Expression of Decoy Receptor3 (DcR3), which binds to FasL, thereby subsequently suppressing the FasL-Fas-dependent apoptotic pathway, is frequently altered in various human disease. However, the role of DcR3 in IPF fibroblasts in regulating their viability has not been examined. We found that enhanced DcR3 expression exists in the majority of IPF fibroblasts on collagen matrices, resulting in the protection of IPF fibroblasts from FasL-induced apoptosis. Abnormally high Akt activity suppresses GSK-3β function, thereby accumulating the nuclear factor of activated T-cells cytoplasmic1 (NFATc1) in the nucleus, increasing DcR3 expression in IPF fibroblasts. This alteration protects IPF cells from FasL-induced apoptosis on collagen. However, the inhibition of Akt or NFATc1 decreases DcR3 mRNA and protein levels, which sensitizes IPF fibroblasts to FasL-mediated apoptosis. Furthermore, enhanced DcR3 and NFATc1 expression is mainly present in myofibroblasts in the fibroblastic foci of lung tissues derived from IPF patients. Our results showed that when IPF cells interact with collagen matrix, aberrantly activated Akt increases DcR3 expression via GSK-3β-NFATc1 and protects IPF cells from the FasL-dependent apoptotic pathway. These findings suggest that the inhibition of DcR3 function may be an effective approach for sensitizing IPF fibroblasts in response to FasL, limiting the progression of lung fibrosis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Knockdown of the putative Lifeguard homologue CG3814 in neurons of Drosophila melanogaster.

Lifeguard is an integral transmembrane protein that modulates FasL-mediated apoptosis by interfering with the activation of caspase 8. It is evolutionarily conserved, with homologues present in plants, nematodes, zebra fish, frog, chicken, mouse, monkey, and human. The Lifeguard homologue in Drosophila, CG3814, contains the Bax inhibitor-1 family motif of unknown function. Downregulation of Lifeguard disrupts cellular homeostasis and disease by sensitizing neurons to FasL-mediated apoptosis. We used bioinformatic analyses to identify CG3814, a putative homologue of Lifeguard, and knocked down CG3814/LFG expression under the control of the Dopa decarboxylase (Ddc-Gal4) transgene in Drosophila melanogaster neurons to investigate whether it possesses neuroprotective activity. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons resulted in a shortened lifespan and impaired locomotor ability, phenotypes that are strongly associated with the degeneration and loss of dopaminergic neurons. Lifeguard interacts with anti-apoptotic Bcl-2 proteins and possibly pro-apoptotic proteins to exert its neuroprotective function. The co-expression of Buffy, the sole anti-apoptotic Bcl-2 gene family member in Drosophila, and CG3814/LFG by stable inducible RNA interference, suppresses the shortened lifespan and the premature age-dependent loss in climbing ability. Suppression of CG3814/LFG in the Drosophila eye reduces the number of ommatidia and increases disruption of the ommatidial array. Overexpression of Buffy, along with the knockdown of CG3814/LFG, counteracts the eye phenotypes. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons in Drosophila diminishes its neuroprotective ability and results in a shortened lifespan and loss of climbing ability, phenotypes that are improved upon overexpression of the pro-survival Buffy.