Fas-mediated Cell Death Research Articles

AMBRA1 controls the translation of immune-specific genes in T lymphocytes

T cell receptor (TCR) engagement causes a global cellular response that entrains signaling pathways, cell cycle regulation, and cell death. The molecular regulation of mRNA translation in these processes is poorly understood. Using a whole-genome CRISPR screen for regulators of CD95 (FAS/APO-1)-mediated T cell death, we identified AMBRA1, a protein previously studied for its roles in autophagy, E3 ubiquitin ligase activity, and cyclin regulation. T cells lacking AMBRA1 resisted FAS-mediated cell death by down-regulating FAS expression at the translational level. We show that AMBRA1 is a vital regulator of ribosome protein biosynthesis and ribosome loading on select mRNAs, whereby it plays a key role in balancing TCR signaling with cell cycle regulation pathways. We also found that AMBRA1 itself is translationally controlled by TCR stimulation via the CD28-PI3K-mTORC1-EIF4F pathway. Together, these findings shed light on the molecular control of translation after T cell activation and implicate AMBRA1 as a translational regulator governing TCR signaling, cell cycle progression, and T cell death.

Necroptosis in alveolar epithelial cells drives lung inflammation and injury caused by SARS-CoV-2 infection

COVID-19, caused by SARS-CoV-2 infection, results in irreversible or fatal lung injury. We assumed that necroptosis of virus-infected alveolar epithelial cells (AEC) could promote local inflammation and further lung injury in COVID-19. Since CD8+ lymphocytes induced AEC cell death via cytotoxic molecules such as FAS ligands, we examined the involvement of FAS-mediated cell death in COVID-19 patients and murine COVID-19 model. We identified the occurrence of necroptosis and subsequent release of HMGB1 in the admitted patients with COVID-19. In the mouse model of COVID-19, lung inflammation and injury were attenuated in Fas-deficient mice compared to Fas-intact mice. The infection enhanced Type I interferon-inducible genes in both groups, while inflammasome-associated genes were specifically upregulated in Fas-intact mice. The treatment with necroptosis inhibitor, Nec1s, improved survival rate, lung injury, and systemic inflammation. SARS-CoV-2 induced necroptosis causes cytokine induction and lung damage, and its inhibition could be a novel therapeutic strategy for COVID-19.

Systemic Treatment with Fas-Blocking Peptide Attenuates Apoptosis in Brain Ischemia.

Apoptosis plays a crucial role in neuronal injury, with substantial evidence implicating Fas-mediated cell death as a key factor in ischemic strokes. To address this, inhibition of Fas-signaling has emerged as a promising strategy in preventing neuronal cell death and alleviating brain ischemia. However, the challenge of overcoming the blood-brain barrier (BBB) hampers the effective delivery of therapeutic drugs to the central nervous system (CNS). In this study, we employed a 30 amino acid-long leptin peptide to facilitate BBB penetration. By conjugating the leptin peptide with a Fas-blocking peptide (FBP) using polyethylene glycol (PEG), we achieved specific accumulation in the Fas-expressing infarction region of the brain following systemic administration. Notably, administration in leptin receptor-deficient db/db mice demonstrated that leptin facilitated the delivery of FBP peptide. We found that the systemic administration of leptin-PEG-FBP effectively inhibited Fas-mediated apoptosis in the ischemic region, resulting in a significant reduction of neuronal cell death, decreased infarct volumes, and accelerated recovery. Importantly, neither leptin nor PEG-FBP influenced apoptotic signaling in brain ischemia. Here, we demonstrate that the systemic delivery of leptin-PEG-FBP presents a promising and viable strategy for treating cerebral ischemic stroke. Our approach not only highlights the therapeutic potential but also emphasizes the importance of overcoming BBB challenges to advance treatments for neurological disorders.

AMBRA1 control immune-specific gene expression in T lymphocytes

Abstract Autoimmune lymphoproliferative syndrome (ALPS) is a disease characterized by the dysregulation of cell death in lymphocytes, resulting in symptoms such as hepatomegaly, splenomegaly, and anemia. While most patients with ALPS have a mutation in the FAS gene or within the FAS pathway, which helps mediate cell death, some patients who present with ALPS-like symptoms do not have known causative gene mutations. To identify other genes that confer resistance to cell death, and thus may play a role in diseases such as ALPS, we performed a whole genome CRISPR screen of Fas-mediated cell death and identified AMBRA1, a protein previously studied in autophagy and E3 ubiquitin ligase activity. We verified the screen results and indeed found that AMBRA1 KO cells resist Fas-mediated cell death. To identify the mechanism of how AMBRA1 contributes to cell death resistance, we performed cycloheximide chase assays, qPCR, and proteomic analysis and confirmed that AMBRA1 regulates FAS expression. Furthermore, we found that AMBRA1 itself can be upregulated by TCR stimulation through the CD28-PI3K-mTORC1 pathway. Mass spectrometry analysis and subsequent experiments also confirmed that AMBRA1 regulates other immune genes. Together, these data establish a novel function for AMBRA1 as a critical regulator in T cells and unveil new insights into how AMBRA1 may contribute to ALPS and dysregulated cell death in other immune diseases. This research was supported by the Intramural Research Program of the NIH.

S163: EFFICACY OF JAK 1/2 INHIBITION IN MURINE IMMUNE BONE MARROW FAILURE

Background: Immune aplastic anemia (AA) is a severe blood disorder characterized by cytotoxic T-lymphocyte mediated stem cell destruction. Therapies including hematopoietic stem cell transplant and immunosuppression are effective but entail costs and risks, and are not effective or possible in all patients. The Janus Kinase (JAK) 1/2 inhibitor ruxolitinib (RUX) suppresses cytotoxic T cell activation and inhibits production of interferon gamma and tumor necrosis factor alpha in models of graft-versus-host disease. Aims: Assess RUX in murine immune AA for potential therapeutic benefit. Methods: In a murine major histocompatibility complex mismatched C67BL/6(B6) to CByB6F1 lymph node (LN) cell infusion AA model, and a C.B10 minor histocompatibility antigen mismatched B6 to C.B10 LN cell infusion AA model, RUX was administered as a food additive (Rux-chow), which achieves therapeutic levels in 48-72 hours after administration. Control BMF mice received chow without RUX. Animals were fed with Rux-chow two days before LN cell infusion as a prophylaxis (BMF+RUXD-2), or two days after LN cell infusion as therapy (BMF+RUXD+2). Recipient mice were either bled and euthanized at day 14 following LN infusion to collect tissues or were kept for 56 days to record animal survival. Blood counts were measured biweekly. Samples for flow cytometry, histology, and gene expression assays were collected. Results: In both AA murine models, RUX attenuated bone marrow hypoplasia, ameliorated peripheral blood pancytopenia, and prevented mortality when used either prophylactically or therapeutically. Treated mice had significantly higher blood counts (neutrophils, red blood cells, hemoglobin, platelets) as well as bone marrow (BM) and RBM (residual BM, excluding T-cells), and cellularity relative to control BMF mice. RUX suppressed infiltration, proliferation and activation of effector T cells in the bone marrow and mitigated Fas-mediated apoptotic destruction of target hematopoietic cells. All mice who received RUX were alive at 56 days while control BMF mice all died (Figure 1). With the exception of two mice with low RBC at day 56, discontinuing Rux-chow at day 28 or day 42 did not affect animal blood counts measurements, nor survival. Gene expression in mice who received RUX revealed downregulated T cell function and JAK/STAT pathway-related genes (Stat1, Stat3, Stat4, Fas, Ly6a, Infg, Gzmb, Gzma, Gzmk, Infgr1, Il2rb, Il2rg, and Lag3). On network analysis of differentially expressed genes in downregulated pathways, Stat1 and Ifn-g genes were at the center of the network, connecting immune responses and cell cycle pathways. When toxicity was assessed, RUX exerted modest suppression of lymphoid and erythroid hematopoiesis in normal and irradiated CByB6F1 mice, but the drug showed impressive clinical efficacy despite this. Image:Summary/Conclusion: RUX showed striking therapeutic efficacy, improving blood counts and prolonging survival in two different BMF murine models. In patients, clonal T cell expansion and activation, IFN-g and TNF-α upregulation, and Fas mediated cell death, lead to severe BM destruction and the development of AA. Our study demonstrated that JAK 1/2 inhibition with RUX produced its suppressive effects by inhibiting T cell activation, reducing inflammatory cytokine secretion, and limiting FasL/Fas-mediated BM destruction. Given these results, JAK 1/2 inhibition with RUX is an exciting and novel potential therapy for BMF patients based on a well characterized mechanism of action.

MHC class II molecules on pancreatic cancer cells indicate a potential for neo-antigen-based immunotherapy

ABSTRACT MHC class II expression is a hallmark of professional antigen-presenting cells and key to the induction of CD4+ T helper cells. We found that these molecules are ectopically expressed on tumor cells in a large proportion of patients with pancreatic ductal adenocarcinoma (PDAC) and on several PDAC cell lines. In contrast to the previous reports that tumoral expression of MHC-II in melanoma enabled tumor cells to evade immunosurveillance, the expression of MHC-II on PDAC cells neither protected cancer cells from Fas-mediated cell death nor caused T-cell suppression by engagement with its ligand LAG-3 on activated T-cells. In fact and surprisingly, the MHC-II/LAG-3 pathway contributed to CD4+ and CD8+ T-cell cytotoxicity toward MHC-II-positive PDAC cells. By combining bioinformatic tools and cell-based assays, we identified a number of immunogenic neo-antigens that can be presented by the patients’ HLA class II alleles. Furthermore, CD4+ T-cells stimulated with neo-antigens were capable of recognizing and killing a human PDAC cell line expressing the mutated genes. To expand this approach to a larger number of PDAC patients, we show that co-treatment with IFN-γ and/or MEK/HDAC inhibitors induced tumoral MHC-II expression on MHC-II-negative tumors that are IFN-γ-resistant. Taken together, our data point to the possibility of harnessing MHC-II expression on PDAC cells for neo-antigen-based immunotherapy.

Bacterial inhibition of Fas-mediated killing promotes neuroinvasion and persistence.

Infections of the central nervous system are among the most serious infections1,2, but the mechanisms by which pathogens access the brain remain poorly understood. The model microorganism Listeria monocytogenes (Lm) is a major foodborne pathogen that causes neurolisteriosis, one of the deadliest infections of the central nervous system3,4. Although immunosuppression is a well-established host risk factor for neurolisteriosis3,5, little is known about the bacterial factors that underlie the neuroinvasion of Lm. Here we develop a clinically relevant experimental model of neurolisteriosis, using hypervirulent neuroinvasive strains6 inoculated in a humanized mouse model of infection7, and we show that the bacterial surfaceprotein InlB protects infected monocytes from Fas-mediated cell death by CD8+ T cells in a manner that depends on c-Met, PI3 kinase and FLIP. This blockade of specific anti-Lmcellular immune killing lengthens the lifespan of infected monocytes, and thereby favours the transfer of Lm from infected monocytes to the brain. The intracellular niche that is created by InlB-mediated cell-autonomous immune resistance also promotes Lm faecal shedding, which accounts for the selection of InlB as a core virulence gene of Lm. We have uncovered a specific mechanism by which a bacterial pathogen confers an increased lifespan to the cells it infects by rendering them resistant to cell-mediated immunity. This promotes the persistence of Lm within the host, its dissemination to the central nervous system and its transmission.

Neurorestorative Effects of a Novel Fas-Associated Factor 1 Inhibitor in the MPTP Model: An [18F]FE-PE2I Positron Emission Tomography Analysis Study.

Fas-associated factor 1 (FAF1), a Fas-binding protein, is implicated in neuronal cell death in Parkinson’s disease (PD). We examined the effects of a novel FAF1 inhibitor, KM-819, in dopaminergic neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model using [18F]FE-PE2I positron emission tomography (PET). The MPTP model was generated with subacute MPTP treatment (20 mg/kg/day, i.p.) for 5 consecutive days in C57bl/6J mice. This study included three groups: the control group (treatment with saline only), the MPTP model group with KM-819 treatment (20 mg/kg/day p.o.) for 6 days, and the MPTP model group without KM-819 treatment. [18F]FE-PE2I PET studies were conducted in the same animals before and after MPTP with or without KM-819 treatment to monitor changes in striatal dopamine transporter activity indicated by non-displaceable binding potential (BPND) of [18F]FE-PE2I, and the expression levels of tyrosine hydroxylase were assessed using immunohistochemistry before and after KM-819 treatment. After MPTP injection, decreased striatal BPND was observed in the MPTP model group compared with the control group. Striatal BPND increased in the MPTP model group with KM-819 treatment, but not in the MPTP model group without KM-819 treatment. The tyrosine hydroxylase expression levels also significantly increased in the MPTP model group with KM-819 treatment compared with the control group. This study indicates that inhibition of the Fas-mediated cell death pathway by KM-819 has neurorestorative effects in striatal dopamine neurons in the MPTP model. Further studies would be needed to investigate the potential of KM-819 as a therapeutic drug for PD treatment.

Dephosphorylation of Fas-ligand and caveolin-1 is a prerequisite step in Fas-ligand - caveolin-1 complex formation and cell death stimulation

Fas-ligand/CD178 belongs to the TNF family proteins and is the well-characterized inducer of cell death. We showed previously that the interaction of Fas-ligand and caveolin-1 is necessary for Fas-ligand translocation to rafts, and the subsequent induction of Fas-ligand-dependent cell death. Both molecules can undergo phosphorylation, however the role of the phosphorylation state of Fas-ligand and caveolin-1 in their physical association, and consequently in of Fas - mediated cell death induction is currently unknown. In this study, we show that in control cells Fas-ligand interaction with caveolin-1 is not observed, and both molecules are phosphorylated. The intracellular part of Fas-ligand was shown to form a complex with p59Fyn-kinase. Upon cell death activation, the expression and activity of p59Fyn-kinase decreases substantially, leading to the disruption of Fas-ligand - p59Fyn-kinase association, dephosphorylation of Fas-ligand and caveolin-1, and formation of a complex between them (Fas-ligand – caveolin-1). The analysis of the effects of kinase and phosphatase inhibitors revealed that phosphorylation of Fas-ligand and caveolin-1 at tyrosine residues suppressed Fas-mediated cell death. Thus, dephosphorylation of Fas-ligand and caveolin-1 is critical for triggering Fas-ligand-mediated apoptotic pathway and cell death execution.

Phenotypic alterations in liver cancer cells induced by mechanochemical disruption

Hepatocellular carcinoma (HCC) is a highly fatal disease recognized as a growing global health crisis worldwide. Currently, no curative treatment is available for early-to-intermediate stage HCC, characterized by large and/or multifocal tumors. If left untreated, HCC rapidly progresses to a lethal stage due to favorable conditions for metastatic spread. Mechanochemical disruption of cellular structures can potentially induce phenotypic alterations in surviving tumor cells that prevent HCC progression. In this paper, HCC response to mechanical vibration via high-intensity focused ultrasound and a chemical disruptive agent (ethanol) was examined in vitro and in vivo. Our analysis revealed that mechanochemical disruption caused a significant overproduction of reactive oxygen species (ROS) in multiple HCC cell lines (HepG2, PLC/PRF/5, and Hep3B). This led to a decrease in cell viability and long-term proliferation due to increased expression and activity of death receptors TNFR1 and Fas. The cells that survived mechanochemical disruption had a reduced expression of cancer stem cell markers (CD133, CD90, CD49f) and a diminished colony-forming ability. Mechanochemical disruption also impeded HCC migration and their adhesion to vascular endothelium, two critical processes in hematogenous metastasis. The HCC transformation to a non-tumorigenic phenotype post mechanochemical disruption was confirmed by a lack of tumor spheroid formation in vitro and complete tumor regression in vivo. These results show that mechanochemical disruption inhibits uncontrolled proliferation and reduces tumorigenicity and aggressiveness of HCC cells through ROS overproduction and associated activation of TNF- and Fas-mediated cell death signaling. Our study identifies a novel curative therapeutic approach that can prevent the development of aggressive HCC phenotypes.

FAIM: An Antagonist of Fas-Killing and Beyond.

Fas Apoptosis Inhibitory Molecule (FAIM) is an anti-apoptotic protein that is up-regulated in B cell receptor (BCR)-activated B cells and confers upon them resistance to Fas-mediated cell death. Faim has two alternatively spliced isoforms, with the short isoform ubiquitously expressed in various tissues and the long isoform mainly found in the nervous tissues. FAIM is evolutionarily conserved but does not share any significant primary sequence homology with any known protein. The function of FAIM has been extensively studied in the past 20 years, with its primary role being ascribed to be anti-apoptotic. In addition, several other functions of FAIM were also discovered in different physiological and pathological conditions, such as cell growth, metabolism, Alzheimer’s disease and tumorigenesis. However, the detailed molecular mechanisms underlying FAIM’s role in these conditions remain unknown. In this review, we summarize comprehensively the functions of FAIM in these different contexts and discuss its potential as a diagnostic, prognostic or therapeutic target.

Wide Profiling of Circulating MicroRNAs in Spinocerebellar Ataxia Type 7

Spinocerebellar ataxia type 7 (SCA7), a neurodegenerative disease characterized by cerebellar ataxia and retinal degeneration, is caused by a CAG repeat expansion in the ATXN7 gene coding region. Disease onset and progression are highly variable between patients, thus identification of specific/sensitive biomarkers that can improve the monitoring of disease progression is an immediate need. Because altered expression of circulating microRNAs (miRNAs) has been shown in various neurological diseases, they could be useful biomarkers for SCA7. In this study, we showed, to our knowledge for the first time, the expression profile of circulating miRNAs in SCA7. Using the TaqMan profiling low density array (TLDA), we found 71 differentially expressed miRNAs in the plasma of SCA7 patients, compared with healthy controls. The reliability of TLDA data was validated independently by quantitative real-time polymerase chain reaction in an independent cohort of patients and controls. We identified four validated miRNAs that possesses the diagnostic value to discriminate between healthy controls and patients (hsa-let-7a-5p, hsa-let7e-5p, hsa-miR-18a-5p, and hsa-miR-30b-5p). The target genes of these four miRNAs were significantly enriched in cellular processes that are relevant to central nervous system function, including Fas-mediated cell-death, heparansulfate biosynthesis, and soluble-N-ethylmaleimide-sensitive factor activating protein receptor pathways. Finally, we identify a signature of four miRNAs associated with disease severity that discriminate between early onset and adult onset, highlighting their potential utility to surveillance disease progression. In summary, circulating miRNAs might provide accessible biomarkers for disease stage and progression and help to identify novel cellular processes involved in SCA7.

Resistance to Fas-Mediated Apoptosis Does Not Correlate to Structural Alterations or Expression Changes of the Death Receptor in Papillary Thyroid Carcinomas

Background: Malignant cells exhibit significant resistance to FAS-mediated cell death, through different processes, including FAS mutations, soluble FAS expression, or FAS transcriptional dysregulation by P53, eventually escaping from immune surveillance. Since thyroid carcinomas were shown to be resistant to FAS-mediated apoptosis, we investigated the above mechanisms in thyroid carcinoma samples. Methods: Thirty-seven thyroid carcinoma samples were analyzed for mutations in FAS exon 9 and TP53 exons 5–8 and protein expression by means of immunohistochemistry. Moreover, thyroid carcinoma mRNA samples were subjected to reverse transcription – PCR, to evaluate the relative expression of transmembrane FAS versus its soluble form. Results: Analysis revealed indications for TP53 mutations in the anaplastic carcinomas, but not in the other thyroid specimens examined for TP53 or FAS exon 9 mutations. FAS receptor expression was observed in almost all thyroid specimens (97%) with significant up-regulation in papillary carcinomas. P53 nuclear staining was observed only in anaplastic carcinomas. Full-length FAS mRNA was detected in all specimens examined, with soluble FAS mRNA being either absent or present in very low amounts. Conclusions: Our results denote that FAS death domain or TP53 DNA-binding domain mutations, down-regulation of FAS receptor expression, or expression of FAS soluble isoform are not responsible for the seeming inhibition of FAS-mediated apoptosis in papillary thyroid carcinoma cells.

Nuclear pore complex-mediated modulation of TCR signaling is required for naïve CD4+ T cell homeostasis.

Nuclear pore complexes (NPCs) are channels connecting the nucleus with the cytoplasm. We report that loss of the tissue-specific NPC component Nup210 causes a severe deficit of naïve CD4+ T cells. Nup210-deficient CD4+ T lymphocytes develop normally but fail to survive in the periphery. The decreased survival results from both an impaired ability to transmit tonic TCR signals and increased levels of Fas, which sensitize Nup210−/− naïve CD4+ T cells to Fas-mediated cell death. Mechanistically, Nup210 regulates these processes by modulating the expression of Caveolin-2 (Cav2) and cJun at the nuclear periphery. While the TCR-dependent and CD4+ T cell-specific upregulation of Cav2 is critical for proximal TCR signaling, cJun expression is required for STAT3-dependent repression of Fas. Our results uncover an unexpected role for Nup210 as a cell-intrinsic regulator of TCR signaling and T cell homeostasis; and expose NPCs as key players in the adaptive immune system.

Impact of peripheral immune status on central molecular responses to facial nerve axotomy

When facial nerve axotomy (FNA) is performed on immunodeficient recombinase activating gene-2 knockout (RAG-2−/−) mice, there is greater facial motoneuron (FMN) death relative to wild type (WT) mice. Reconstituting RAG-2−/− mice with whole splenocytes rescues FMN survival after FNA, and CD4+ T cells specifically drive immune-mediated neuroprotection. Evidence suggests that immunodysregulation may contribute to motoneuron death in amyotrophic lateral sclerosis (ALS). Immunoreconstitution of RAG-2−/− mice with lymphocytes from the mutant superoxide dismutase (mSOD1) mouse model of ALS revealed that the mSOD1 whole splenocyte environment suppresses mSOD1 CD4+ T cell-mediated neuroprotection after FNA. The objective of the current study was to characterize the effect of CD4+ T cells on the central molecular response to FNA and then identify if mSOD1 whole splenocytes blocked these regulatory pathways.Gene expression profiles of the axotomized facial motor nucleus were assessed from RAG-2−/− mice immunoreconstituted with either CD4+ T cells or whole splenocytes from WT or mSOD1 donors. The findings indicate that immunodeficient mice have suppressed glial activation after axotomy, and cell transfer of WT CD4+ T cells rescues microenvironment responses. Additionally, mSOD1 whole splenocyte recipients exhibit an increased astrocyte activation response to FNA. In RAG-2−/− + mSOD1 whole splenocyte mice, an elevation of motoneuron-specific Fas cell death pathways is also observed. Altogether, these findings suggest that mSOD1 whole splenocytes do not suppress mSOD1 CD4+ T cell regulation of the microenvironment, and instead, mSOD1 whole splenocytes may promote motoneuron death by either promoting a neurotoxic astrocyte phenotype or inducing Fas-mediated cell death pathways. This study demonstrates that peripheral immune status significantly affects central responses to nerve injury. Future studies will elucidate the mechanisms by which mSOD1 whole splenocytes promote cell death and if inhibiting this mechanism can preserve motoneuron survival in injury and disease.

Microbiota promotes systemic T-cell survival through suppression of an apoptotic factor

Symbiotic microbes impact the severity of a variety of diseases through regulation of T-cell development. However, little is known regarding the molecular mechanisms by which this is accomplished. Here we report that a secreted factor, Erdr1, is regulated by the microbiota to control T-cell apoptosis. Erdr1 expression was identified by transcriptome analysis to be elevated in splenic T cells from germfree and antibiotic-treated mice. Suppression of Erdr1 depends on detection of circulating microbial products by Toll-like receptors on T cells, and this regulation is conserved in human T cells. Erdr1 was found to function as an autocrine factor to induce apoptosis through caspase 3. Consistent with elevated levels of Erdr1, germfree mice have increased splenic T-cell apoptosis. RNA sequencing of Erdr1-overexpressing cells identified the up-regulation of genes involved in Fas-mediated cell death, and Erdr1 fails to induce apoptosis in Fas-deficient cells. Importantly, forced changes in Erdr1 expression levels dictate the survival of auto-reactive T cells and the clinical outcome of neuro-inflammatory autoimmune disease. Cellular survival is a fundamental feature regulating appropriate immune responses. We have identified a mechanism whereby the host integrates signals from the microbiota to control T-cell apoptosis, making regulation of Erdr1 a potential therapeutic target for autoimmune disease.

Protective Effect of Met12, a Small Peptide Inhibitor of Fas, on the Retinal Pigment Epithelium and Photoreceptor After Sodium Iodate Injury

PurposeA major problem in macular degeneration is the inability to reduce RPE and photoreceptor death. These cells die by necroptosis and apoptosis, respectively, but the upstream activator(s) of these death pathways is unknown. In this study, we use the sodium iodate (NaIO3) model of oxidative stress to test the hypothesis that activation of the Fas receptor contributes to the death of the RPE and photoreceptors.MethodsSodium iodate was injected in Brown-Norway rats via femoral vein injection. Both in vivo (fundus photography, optical coherence tomography, and fluorescein angiography) and ex vivo (histology, immunohistochemistry, Western blot, and RT-PCR) analyses of the RPE and retina were conducted at baseline, as well as at various times post NaIO3 injection. The ability of intravitreal injection of Met12, a small peptide inhibitor of the Fas receptor, to prevent RPE and photoreceptor cell death was assessed.ResultsInjection of NaIO3 led to Fas-mediated activation of both necroptosis and apoptosis in the RPE and photoreceptors, respectively. This was accompanied by a significant increase in the number of microglia/macrophages in the outer retina. Met12 significantly reduced the activation of the Fas-mediated death pathways, resulting in reduced RPE and photoreceptor death and a decreased immune response.ConclusionsOur results demonstrate that NaIO3 activates Fas-mediated cell death, both in the RPE and photoreceptor, and that a small peptide antagonist of the Fas receptor, Met12, significantly reduces the extent of this cell death. These findings suggest a role for Fas inhibition to protect the RPE and photoreceptors from death due to oxidative stress.

Autoimmune lymphoproliferative syndrome caused by homozygous FAS mutations with normal or residual protein expression

Autoimmune lymphoproliferative syndrome caused by homozygous FAS mutations with normal or residual protein expression

Graft-Derived IL-6 Amplifies Proliferation and Survival of Effector T Cells That Drive Alloimmune-Mediated Vascular Rejection.

IL-6 is an inflammatory cytokine that controls effector T cell responses but the mechanisms by which it controls allogeneic immune responses and vascular rejection that leads to transplant arteriosclerosis (TA) are poorly understood. We have examined the mechanism by which IL-6 contributes to the pathogenesis of vascular rejection and TA using a murine aortic interposition model of vascular rejection. The absence of IL-6 production from artery graft cells reduced the development of vascular rejection and arteriosclerotic thickening. There was no apparent effect of donor-derived IL-6 on endothelial cell integrity or on the intimal accumulation of smooth muscle cells, macrophages, and anti-donor antibodies. However, reduced vascular pathology in IL-6 artery grafts was accompanied by a significant reduction in the accumulation of CD4 and CD8 T cells. Further, the absence of graft-derived IL-6 resulted in a significant decrease in the activation and proliferation of alloreactive CD4 and CD8 T cells after transplantation as well as in a marked increase in cell death of effector T cells. Alloreactive effector T cells that expanded in the absence of IL-6 were also more susceptible to Fas-mediated activation-induced cell death in vitro. Finally, systemic neutralization of IL-6R did not reduce arteriosclerotic thickening but reduced endothelial integrity in allograft arteries, indicating differential effects of specific elimination of IL-6 in graft cells and systemic IL-6 neutralization. Donor-derived IL-6 amplifies the expansion of allogeneic T cell responses that cause vascular rejection and TA by increasing T cell proliferation and preventing Fas-mediated T cell death.

The Interaction of CD154 with the α5β1 Integrin Inhibits Fas-Induced T Cell Death.

CD154, a critical regulator of the immune response, is usually associated with chronic inflammatory, autoimmune diseases as well as malignant disorders. In addition to its classical receptor CD40, CD154 is capable of binding other receptors, members of the integrin family, the αIIbβ3, αMβ2 and α5β1. Given the role attributed to integrins and particularly the β1 integrins in inhibiting apoptotic events in normal as well as malignant T cells, we were highly interested in investigating the role of the CD154/α5β1 interaction in promoting survival of malignant T cells contributing as such to tumor development and/or propagation. To support our hypothesis, we first show that soluble CD154 binds to the T-cell acute lymphoblastic leukemia cell line, Jurkat E6.1 in a α5β1-dependent manner. Binding of soluble CD154 to α5β1 integrin of Jurkat cells leads to the activation of key survival proteins, including the p38 and ERK1/2 mitogen-activated protein kinases (MAPKs), phosphoinositide 3 kinase (PI-3K), and Akt. Interestingly, soluble CD154 significantly inhibits Fas-mediated apoptosis in T cell leukemia-lymphoma cell lines, Jurkat E6.1 and HUT78 cells, an important hallmark of T cell survival during malignancy progression. These anti-apoptotic effects were mainly mediated by the activation of the PI-3K/Akt pathway but also involved the p38 and the ERK1/2 MAPKs cascades. Our data also demonstrated that the CD154-triggered inhibition of the Fas-mediated cell death response was dependent on a suppression of caspase-8 cleavage, but independent of de novo protein synthesis or alterations in Fas expression on cell surface. Together, our results highlight the impact of the CD154/α5β1 interaction in T cell function/survival and identify novel targets for the treatment of malignant disorders, particularly of T cell origin.