FasL Research Articles

Zonisamide improves Fas/FasL-mediated apoptosis and inflammation in a degenerative cervical myelopathy rat model

Degenerative cervical myelopathy (DCM) is a severe condition of the spinal cord caused by chronic compression. However, no studies to date have examined the effects of zonisamide (ZNS) on DCM via the Fas/FasL-mediated pathway. The aim of this study was to investigate the effects of ZNS on a DCM rat model and to explore the potential mechanisms. First, 40 adult Sprague–Dawley rats were used to establish the DCM rat model and were individually divided into four groups: the Sham group, DCM model group (DCM), ZNS group (DCM model rats treated with ZNS, 30 mg/kg/day), and ZNS + CD95 group (DCM model rats treated with ZNS and CD95). Histopathology injury and cell apoptosis, Fas and Fas ligand (FasL) expression and Fas/FasL relative protein levels were detected by hematoxylin and eosin staining, TUNEL assay, and immunofluorescence and western blotting, respectively. The results of our study demonstrated that ZNS could promote motor recovery while reversing histopathological injury and cell apoptosis in DCM rats. Moreover, Iba-1, Fas and FasL expression in DCM rats was decreased, accompanied by a decrease in cleaved caspase-3/caspase-3, cleaved caspase-8/caspase-8, cleaved caspase-9/caspase-9, cleaved caspase-10/caspase-10 and B-cell lymphoma-2 (Bcl-2)/Bcl-2 associated X (Bax) levels. All these results revealed that ZNS attenuates DCM injury in a rat model via the regulation of Fas and FasL signaling. Our study indicated that ZNS had beneficial effects on DCM and thus provided a novel theoretical approach for subsequent academic and clinical research on DCM injury.

Protective Effect of Vitamin D against Hepatic Molecular Apoptosis Caused by a High-Fat Diet in Rats

The protective effects of vitamin D (VitD) in different diseases were studied. The liver is of great interest, especially with the presence of VitD receptors. A high-fat diet (HFD) is associated with many diseases, including liver injury. Consumption of saturated fatty acids triggers hepatic apoptosis and is associated with increased inflammation. We aimed in this study to investigate the protective effects of VitD on hepatic molecular apoptotic changes in response to an HFD in rats. Forty male Wistar albino rats were used and divided into four groups: control, HFD, control + VitD, and VitD-supplemented HFD (HFD + VitD) groups. After six months, the rats were sacrificed, and the livers were removed. RNA was extracted from liver tissues and used for the quantitative real-time RT-PCR of different genes: B-cell lymphoma/leukemia-2 (BCL2), BCL-2-associated X protein (Bax), Fas cell surface death receptor (FAS), FAS ligand (FASL), and tumor necrosis factor α (TNF-α). The results showed that an HFD increased the expression of the pro-apoptotic genes Bax, FAS, and FASL, and reduced the expression of the anti-apoptotic gene BCL2. Interestingly, a VitD-supplemented HFD significantly increased the BCL2 expression and decreased the expression of all pro-apoptotic genes and TNFα. In conclusion, VitD has a protective role against hepatic molecular apoptotic changes in response to an HFD.

Revisiting autoimmune lymphoproliferative syndrome caused by Fas ligand mutations

Fas ligand (FasL) is expressed by activated T cells and induces death in target cells upon binding to Fas. Loss-of-function FAS or FASLG mutations cause autoimmune-lymphoproliferative syndrome (ALPS) characterized by expanded double-negative T cells (DNT) and elevated serum biomarkers. While most ALPS patients carry heterozygous FAS mutations, FASLG mutations are rare and usually biallelic. Only 2 heterozygous variants were reported, associated with an atypical clinical phenotype. We revisited the significance of heterozygous FASLG mutations as a cause of ALPS. Clinical features and biomarkers were analyzed in 24 individuals with homozygous or heterozygous FASLG variants predicted to be deleterious. Cytotoxicity assays were performed with patient T cells and biochemical assays with recombinant FasL. Homozygous FASLG variants abrogated cytotoxicity and resulted in early-onset severe ALPS with elevated DNT, raised vitamin B12, and usually no soluble FasL. In contrast, heterozygous variants affected FasL function by reducing expression, impairing trimerization, or preventing Fas binding. However, they were not associated with elevated DNT and vitamin B12, and they did not affect FasL-mediated cytotoxicity. The dominant-negative effects of previously published variants could not be confirmed. Even Y166C, causing loss of Fas binding with a dominant-negative effect in biochemical assays, did not impair cellular cytotoxicity or cause vitamin B12 and DNT elevation. Heterozygous loss-of-function mutations are better tolerated for FASLG than for FAS, which may explain the low frequency of ALPS-FASLG.

PCSK9 facilitates melanoma pathogenesis via a network regulating tumor immunity

BackgroundPCSK9 regulates cholesterol homeostasis and promotes tumorigenesis. However, the relevance of these two actions and the mechanisms underlying PCSK9’s oncogenic roles in melanoma and other cancers remain unclear.MethodsPCSK9’s association with melanoma was analysed using the TCGA dataset. Empty vector (EV), PCSK9, gain-of-function (D374Y), and loss-of-function (Q152H) PCSK9 mutant were stably-expressed in murine melanoma B16 cells and studied for impact on B16 cell-derived oncogenesis in vitro and in vivo using syngeneic C57BL/6 and Pcsk9−/− mice. Intratumoral accumulation of cholesterol was determined. RNA-seq was performed on individual tumor types. Differentially-expressed genes (DEGs) were derived from the comparisons of B16 PCSK9, B16 D374Y, or B16 Q152H tumors to B16 EV allografts and analysed for pathway alterations.ResultsPCSK9 expression and its network negatively correlated with the survival probability of patients with melanoma. PCSK9 promoted B16 cell proliferation, migration, and growth in soft agar in vitro, formation of tumors in C57BL/6 mice in vivo, and accumulation of intratumoral cholesterol in a manner reflecting its regulation of the low-density lipoprotein receptor (LDLR): Q152H, EV, PCSK9, and D374Y. Tumor-associated T cells, CD8 + T cells, and NK cells were significantly increased in D374Y tumors along with upregulations of multiple immune checkpoints, IFNγ, and 143 genes associated with T cell dysfunction. Overlap of 36 genes between the D374Y DEGs and the PCSK9 DEGs predicted poor prognosis of melanoma and resistance to immune checkpoint blockade (ICB) therapy. CYTH4, DENND1C, AOAH, TBC1D10C, EPSTI1, GIMAP7, and FASL (FAS ligand) were novel predictors of ICB therapy and displayed high level of correlations with multiple immune checkpoints in melanoma and across 30 human cancers. We observed FAS ligand being among the most robust biomarkers of ICB treatment and constructed two novel and effective multigene panels predicting response to ICB therapy. The profiles of allografts produced by B16 EV, PCSK9, D374Y, and Q152H remained comparable in C57BL/6 and Pcsk9−/− mice.ConclusionsTumor-derived PCSK9 plays a critical role in melanoma pathogenesis. PCSK9’s oncogenic actions are associated with intratumoral cholesterol accumulation. PCSK9 systemically affects the immune system, contributing to melanoma immune evasion. Novel biomarkers derived from the PCSK9-network effectively predicted ICB therapy responses.

Improved colorectal cancer screening by adding noninvasive serum-based biomarkers

Background Colorectal cancer (CRC) is one of the most common causes of cancer-related morbidity and mortality worldwide. The most widely used CRC diagnostic tests either noninvasive or invasive are limited by insufficient performance or patient compliance. Profiling inflammatory mediators with carcinogenic roles could aid in CRC diagnosis. We aimed to use multiplex bead assay in evaluating the utility of serum eotaxin-1, macrophage-inflammatory protein-1β (MIP-1β), granulocyte colony-stimulating factor (G-CSF), vascular endothelial growth factor A (VEGF-A), and Fas ligand (FasL) as potential biomarkers in CRC. Patients and methods The study was conducted on 87 patients. Based on colonoscopy findings, patients were divided into 35 CRC and 52 nonmalignant patients (nine with colon polyp, 24 with colitis, and 19 with normal mucosa). Multiplex assay was used for measuring the studied biomarkers. Results The median values of eotaxin-1, MIP-1β, G-CSF, and VEGF-A were significantly higher in patients with CRC compared with the nonmalignant group. The area under the receiver operating characteristics curve for eotaxin-1, MIP-1β, G-CSF, and VEGF-A was 0.863. The area under the ROC for occult blood in stool was only 0.597. Moreover, significantly higher levels of G-CSF and VEGF-A were found in patients with CRC than the precancerous colon polyp group. Conclusions Serum profiling of eotaxin-1, MIP-1β, G-CSF, and VEGF-A could be used as potential biomarkers in early CRC diagnosis with better discriminatory power than stool occult blood and may increase occult blood performance, thus reducing false-positives rates and unneeded colonoscopy. Multiplexing bead technology represents a promising approach for CRC screening and diagnosis.

P055: Association of high FASLG gene copy number in Burkitt lymphoma with reduced survival probabilities

P055: Association of high FASLG gene copy number in Burkitt lymphoma with reduced survival probabilities

Effectiveness of a novel gene nanotherapy based on putrescine for cancer treatment.

Gene therapy has long been proposed for cancer treatment. However, the use of therapeutic nucleic acids presents several limitations such as enzymatic degradation, rapid clearance, and poor cellular uptake and efficiency. In this work we propose the use of putrescine, a precursor for higher polyamine biosynthesis for the preparation of cationic nanosystems for cancer gene therapy. We have formulated and characterized putrescine-sphingomyelin nanosystems (PSN) and studied their endocytic pathway and intracellular trafficking in cancer cells. After loading a plasmid DNA (pDNA) encoding the apoptotic Fas Ligand (FasL), we proved their therapeutic activity by measuring the cell death rate after treatment of MDA-MB-231 cells. We have also used xenografted zebrafish embryos as a first in vivo approach to demonstrate the efficacy of the proposed PSN-pDNA formulation in a more complex model. Finally, intratumoral and intraperitoneal administration to mice-bearing MDA-MB-231 xenografts resulted in a significant decrease in tumour cell growth, highlighting the potential of the developed gene therapy nanoformulation for the treatment of triple negative breast cancer.

Large granular lymphocytic leukemia and its association with immune dysregulation

Large granular lymphocytic (LGL) leukemia is a chronic lymphoproliferative disease of cytotoxic T cells or NK cells with LGL morphology and frequently complicated cytopenia and/or different autoimmune diseases, which often require medical interventions, although LGL leukemia itself is seldom lethal. Immunologic dysregulations in LGL leukemia contribute to the development of complications, for example, neutropenia with the involvement of Fas ligand system and, in pure red cell aplasia, which is a common complication among the patients of East Asian origin, impairing erythroid developments by cytotoxic T cells. Rheumatoid arthritis (RA) is the most prevalent nonhematological consequence, and Felty syndrome, a rare form of RA, and T-LGL leukemia have a lot in common. When patients have LGL leukemia-associated complications, immunosuppressive medication is a mainstay of treatment. Characteristic mutational features in STAT3, STAT5B, CCL22, and other genes in specific subtypes of LGL leukemia have been detected, that would be associated with immunologically mediated molecular pathogenesis in LGL leukemia, and these new findings may help in creating optimal diagnostic approaches or novel therapies for LGL leukemia.

Immunity status and expression of molecular markers (ICAM-1, CD5, CD25, CD95) on lymphocytes of patients with recurrent anterior uveitis complicated by macular edema.

Treatment of macular edema in uveitis is a key goal of treatment, because this complication remains a potential therapeutic problem for specialists. Examination was carried out in 1-2 groups - 50 persons with uncomplicated recurrent anterior uveitis (AU) in the stage of relapse or remission - and 3-4 groups - 26 persons with recurrent AU complicated by uveitic macular edema (UME) in the stage of relapse or remission of AU. Control group - 27 healthy volunteers. All patients underwent an ophthalmologic examination, using OCT (Spectralis HRA + OCT (Heidelberg Engineering)). The state of cellular and humoral immunity and the expression of activation markers on blood lymphocytes in all patients were assessed. An immunohistocytochemical analysis using monoclonal antibodies (the peroxidase-anti-peroxidase method) was employed to assess the expression of lymphocyte activation markers. The monoclonal antibody panel (MCAP) for immunophenotyping included antibodies reacting with CD5, CD54 (ICAM-1), CD25, and CD95 (FAS) antigens. Immunophenotyping was performed using immunohistochemistry. In the patients with AU + UME, the parameters of the T-cell link of the immune response were increased and the parameters of humoral immunity (CD19, IgA, IgG) were decreased in comparison with the uncomplicated AU patients. The absolute and relative numbers of natural killer cells in the peripheral blood were higher, and the relative numbers of phagocytic neutrophils were lower in the group of anterior uveitis with UME than in uncomplicated AU. The absolute and relative expression levels of ICAM-1 (CD54) and the absolute expression level of CD5 on peripheral blood lymphocytes were increased in relapse at patients with AU + UME compared with uncomplicated AU. The expression levels of CD25 and CD95 were not different in these groups. Activation of cellular immunity markers ICAM-1 and CD5 seems to play a role in uveitic macular edema. The expression levels of CD25 and CD95 were not significantly different in these groups.

Assessment Of Soluble Fas Ligand In Patients With Chronic Myeloid Leukemia

Background: Soluble FasL (sFasL) generating from Membrane-bound FasL (mFasL) cleaved by matrix metalloproteinases (MMPs). sFasL inhibits the apoptotic and inflammatory activity of mFasL because of sFasL competes with the mFasL and binds to Fas. To evaluate serum soluble Fas ligand (sFasL) level in patients with chronic myeloid leukemia.
 Materials and Method: Serum levels of sFasL were measured by ELISA method after venous blood was collected from 56 CML patients (newly diagnosed and optimally treated) and 28 healthy subjects as control group.
 Results: There were no significant increases in serum sFasL patient compared to healthy control with P=0.07. When the mean sFasL concentration was obviously highest in newly diagnosed (216.9pg/ml) followed by healthy control (152.5pg/ml) and lowest in optimally treated (147.7 pg/ml).
 Conclusion: Production of sFasL in tumor patients may be a key mechanism to inhibit Fas-mediated apoptosis.

Differential Regulation of MMPs, Apoptosis and Cell Proliferation by the Cannabinoid Receptors CB1 and CB2 in Vascular Smooth Muscle Cells and Cardiac Myocytes.

Cannabinoids (CB) are implicated in cardiovascular diseases via the two main receptor subtypes CB1R and CB2R. This study investigated whether cannabinoids regulate the activity of matrix metalloproteases (MMP-2, MMP-9) in vascular smooth muscle cells (VSMCs) and in cells of cardiac origin (H9c2 cell line). The influence of CB1- and CB2 receptor stimulation or inhibition on cell proliferation, apoptosis and glucose uptake was also evaluated. We used four compounds that activate or block CB receptors: arachidonyl-2-chloroethylamide (ACEA)-CB1R agonist, rimonabant-CB1R antagonist, John W. Huffman (JWH133)-CB2R agonist and CB2R antagonist-6-Iodopravadoline (AM630). Treatment of cells with the CB2R agonist JWH133 decreased cytokine activated secretion of proMMP-2, MMP-2 and MMP-9, reduced Fas ligand and caspase-3-mediated apoptosis, normalized the expression of TGF-beta1 and prevented cytokine-induced increase in glucose uptake into the cell. CB1R inhibition with rimonabant showed similar protective properties as the CB2R agonist JWH133, but to a lesser extent. In conclusion, CB1R and CB2R exert opposite effects on cell glucose uptake, proteolysis and apoptosis in both VSMCs and H9c2 cells. The CB2R agonist JWH133 demonstrated the highest protective properties. These findings may pave the way to a new treatment of cardiovascular diseases, especially those associated with extracellular matrix degradation.

Serum Soluble Fas Levels and Incidence of Liver Cancer in Nested Case-Control Study.

Soluble Fas (sFas) plays various roles in carcinogenesis and tumor dissemination by preventing apoptosis via binding to Fas ligand. We analyzed associations of serum sFas levels with the incidence of liver cancer in a prospective case-control study nested in the Japan Collaborative Cohort Study. A baseline survey was conducted from 1988, with blood samples obtained from 39,242 subjects. Patients diagnosed with liver cancer were regarded as cases. Two or three controls were selected and matched for sex, age, and geographic area. Conditional logistic regression was used to estimate ORs for cancer incidence associated with sFas. This study contained 86 cases and 249 controls. After controlling for alcohol intake, body mass index, smoking, and hepatitis viral infection, participants with high sFas showed elevated risk of cancer (Ptrend = 0.003) and the third tertile of sFas showed a higher risk compared with the first tertile [OR, 3.53; 95% confidence interval (CI), 1.28-9.69]. In hepatocellular carcinoma, high sFas was associated with elevated risk (Ptrend < 0.001). In men and the elderly, subjects in the highest tertiles showed higher cancer risk. Limiting subjects to those followed for 3 years, high sFas was related to liver cancer risk (Ptrend = 0.033) and the third tertile showed a higher risk compared with the first (OR, 2.94; 95% CI, 0.94-9.14). High serum sFas may be related to future risk of liver cancer. Our findings highlight this biomarker for further analysis in pooled investigations with different/larger prospective cohorts.

A Systematic Review on Current Trends in the Treatment of Chronic Hepatitis B to Predict Disease Remission and Relapse.

Despite decreasing the prevalence of chronic hepatitis B (CHB), it is still a major health care challenge. Current antiviral regimens aim to suppress hepatitis B virus (HBV) deoxyribonucleic acid (DNA) activity to prevent the risk of hepatic decompensation, liver cirrhosis, and hepatocellular carcinoma (HCC). Currently, pegylated interferon (Peg-IFN) and nucleos(t)ide analogs (NA) are the first-line choices of drugs. Peg-IFN is now discontinued due to its mode of application and side effects. NA is used once daily to suppress HBV DNA activity but has little effect on covalently closed circular DNA (cccDNA), so continuous long-term therapy is required to suppress HBV DNA. Due to this effect, disease remission, relapse, and even clinical flare are common phenomena after the end of treatment (EOT). This review aimed to analyze the current regimens for treating chronic hepatitis B. Their mode of action, duration of treatment, and events after stopping therapy. The review was performed using the preferred reporting items for systematic reviews and meta-analysis (PRISMA) 2020 guidelines. A search was undertaken in PubMed, PubMed Central, Google Scholar, and ScienceDirect. Screening of articles was carried out to find relevant and appropriate articles. Articles were then quality-checked before inclusion. Our analysis showed that long-term finite therapy with nucleoside analogs could improve clinical outcomes and suppress viral DNA activity. However, a functional cure, loss of hepatitis B surface antigen (HBsAg), is rarely achieved. The decision to end treatment depends on quantitative HBsAg level (qHBsAg), alanine aminotransferase (ALT), HBV DNA (deoxyribonucleic acid), hepatitis B e antigen (HBeAg), and fibrosis assessment. It is concluded that patients with HBeAg negative without cirrhosis can be easily withdrawn from treatment if they have long-term viral remission and a high HBsAg loss rate. However, patients with positive HBeAg should continue treatment because there is a high chance of disease relapse and even acute flare. To predict whether patients will benefit from EOT, some immunomodulatory markers are studied, including interleukin (IL-20, IL-8), fas ligand (FASGL), and IFN gamma. Although these factors are reliable, none pose an independent effect on disease remission. Combination therapy (IFN alpha + oral nucleoside analogs) is promising but has clinical shortcomings.

The BCL-2 Inhibitor Venetoclax Augments Immune Effector Function Mediated by Fas Ligand, TRAIL, and Perforin/Granzyme B, Resulting in Reduced Plasma Viremia and Decreased HIV Reservoir Size during Acute HIV Infection in a Humanized Mouse Model.

The BCL-2 prosurvival protein is implicated in HIV persistence and is a potential therapeutic target for HIV eradication efforts. We now know that cells harboring HIV are preferentially enriched for high BCL-2 expression, enabling their survival, and that the BCL-2 inhibitor venetoclax promotes the death of actively replicating HIV-infected cells in vitro and ex vivo. Herein, we assess the effect of venetoclax on immune clearance of infected cells and show that BCL-2 inhibition significantly enhances target cell killing induced by Fas ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), and perforin/granzyme B and synergistically enhances autologous NK (natural killer) and CD8 cells' killing of target cells. In a humanized mouse model of acute HIV infection, venetoclax monotherapy significantly decreases plasma viremia and normalizes CD4:CD8 ratios, and results in more mice with undetectable provirus levels than control. In this model, treatment was associated with leukopenia, as has been described clinically in patients receiving venetoclax for other indications. These data confirm meaningful anti-HIV effects of venetoclax during HIV infection but suggest that venetoclax use should be combined with ART (antiretroviral therapy) to reduce toxicity. IMPORTANCE This study is the first to examine the applicability of BCL-2 inhibition in the setting of active HIV infection in vivo. Furthermore, this study demonstrates that venetoclax significantly enhances target cell killing induced by Fas ligand, TRAIL, and perforin/granzyme B and synergistically enhances autologous NK and CD8 cells' killing of target cells.

FasL is a catabolic factor in alveolar bone homeostasis.

Fas ligand (FasL) belongs to the tumour necrosis factor superfamily regulating bone turnover, inflammation, and apoptosis. The appendicular and axial skeleton phenotype of mature Faslgld mice has been reported. The impact of FasL on the alveolar bone providing support for the teeth at mature stages under healthy and induced inflammatory conditions remains unknown. We performed a phenotypical analysis of mice carrying the homozygous Faslgld mutation and wild-type (WT) mice (C57BL/6) under healthy conditions and upon ligature-induced periodontitis. After 12 days, micro-computed tomography analysis revealed the distance between the cement enamel junction and the alveolar bone crest. Additional structural parameters, such as the bone volume fraction (BV/TV) and the periodontal ligament space volume, were measured. Histological analyses were performed to visualize the catabolic changes at the defect site. Healthy Faslgld mice were found to have more periodontal bone than their WT littermates. Faslgld had no significant effect on inflammatory osteolysis compared to WT controls with ligatures. Histology revealed eroded surfaces at the root and in the inter-proximal bone in both strains. Our findings suggest that FasL is a catabolic factor in alveolar bone homeostasis but it does not affect the inflammatory osteolysis.

016 PC111, a novel, First In Class, human anti-FasL antibody for the treatment of Pemphigus: a translational study

Pemphigus is a rare and debilitating autoimmune blistering disease due to keratinocyte cell-cell detachment (acantholysis). Treatments focus on immune suppression but are often associated with severe side effects, slow onset of action and frequent clinical relapses; therefore, innovative and non-immunosuppressive therapies are needed to provide rapid, safer and long-lasting responses. Patients’ autoantibodies (PVIgG) are fundamental for initiating the pathologic mechanisms of pemphigus. We have recently demonstrated that soluble Fas Ligand (sFasL), that is massively up-regulated and released from keratinocytes upon PVIgG binding, is essential for blister formation.

Platelets Bind Senescent Red Blood Cells and Mediate Their Clearance from the Circulation

The bloodstream of mice and humans contain consistent proportions of red blood cells (RBC) which are bound to platelets (1-2% and 0.1-0.3%, respectively). This study identifies senescent RBC as the main constituent of platelet-RBC complexes (Plt-RBC) and their importance in RBC clearance. RBC have a defined circulation lifespan and upon reaching senescence are eliminated by phagocytes, thereby ensuring homeostasis and preventing accumulation of dead cells and toxic hemolytic byproducts. Senescent RBC are characterized by cellular and molecular changes, including reduced deformability, externalization of membrane phosphatidylserine (PS), and altered expression of membrane proteins such Fas ligand (CD95; upregulated) and integrin associated protein (CD47; downregulated), which act as pro-phagocytic signals. Similar changes to deformability and membrane composition occur in RBC compromised by sickle cell disease or Plasmodium infection. These pathologic cells also display a 10x higher platelet-binding capacity which is related to disease pathogenesis. Thus, we investigated how platelets may interact with senescent RBC and the purpose of Plt-RBC. Platelet binding to senescent RBC was studied using mice pulse-labelled with biotin-NHS, which enabled tracking of RBC age and clearance over time. The biotin+ RBC remaining for longer times represented older cells; the oldest detected were aged ≥58 days consistent with the reported maximal RBC lifespan in mice. During the first 45 days of observation Plt-RBC were similar in biotin+ versus newly replenished biotin- RBC (1.6-2.2%), but were significantly higher in biotin+ cells detected at 52 and 58 days (average ± SD: 6.7 ± 5.1% and 15 ± 8.1%, respectively; p < 0.001). These observations were replicated when biotin-NHS was replaced with CFSE. The aged RBC and Plt-RBC also contained significantly increased PS and CD95 compared to the replenished and non-complexed cells, although CD47 levels were not different. Flow cytometry imaging revealed Plt-RBC were composed of single RBC bound by 1-2 platelets. Collectively this indicated that platelets preferentially bind senescent RBC. To investigate if platelet binding affected senescent RBC clearance, platelet-depleting antibodies (anti-GPIbα or anti-GPIIb/IIIa) were given to temporarily reduce Plt-RBC when biotin+ Plt-RBC levels first began to diverge (Day 46-49 in multiple experiments). The treatment caused a maximal 10-fold reduction in Plt-RBC levels sustained for 2-3 days, and a marked retention of biotin+ RBC which coincided with Plt-RBC reductions. Consistent with these results, the clearance of transfused RBC in platelet-depleted mice was also significantly inhibited compared to control mice. Erythrophagocytes in the spleen, but not the liver, from the platelet-depleted mice also contained fewer transfused cells, and erythrophagocytes containing platelets were observed using flow cytometry and microscopy. Thus, platelets are important for the clearance and/or phagocytosis of both senescent and transfused RBC. We also studied the Plt-RBC clearance mechanism by transfusing ex vivo generated and labelled Plt-RBC and RBC. The Plt-RBC were cleared at least 5x faster than RBC (p=0.0014) suggesting that once formed, their lifespan in the circulation is transient. Similar transfusions in mice depleted of phagocytes (using clodronate liposomes) impeded Plt-RBC clearance, indicating a phagocyte-dependent pathway for their removal. An additional mechanistic insight, Plt-RBC displayed an increased retention in a microsphiltration assay system, indicating platelet binding may hinder the passage of cells through the microcirculation. Our studies have thus revealed an unexpected contribution by platelets in senescent RBC clearance. In our proposed model (Figure 1) senescent RBC have an enhanced ability to bind platelets, possibly due to upregulated PS and CD95, which accelerates their removal via splenic retention and phagocytosis. Platelets may also present additional pro-phagocytic signals to stimulate erythrophagocytosis. A normal platelet count provides surplus platelets to maintain RBC clearance, but in conditions of acute platelet loss, reduction of plt-RBC may cause accumulation of senescent RBC to pathological levels. Conversely, conditions where more RBC bind platelets such as in malarial infection will increase plt-RBC and result in thrombocytopenia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Amiodarone-drove XBP1s aggravates endoplasmic reticulum stress and apoptosis in Hashimoto’s thyroiditis through regulating LINC00842/miR-214/FASL axis

Hashimoto’s thyroiditis (HT) is an organ-specific autoimmune disease, that eventually lead to hypothyroidism. XBP1s is an endoplasmic reticulum stress related protein and participates in the pathogenesis of several diseases. Nevertheless, the potential role of XBP1s in amiodarone (AMIO)-treated HT patients remains unknown. In this study, AMIO aggravated the endoplasmic reticulum stress responses in HT patients and thyroid epithelial follicular cells. Moreover, MTT assay and flow cytometry analysis revealed that knockdown of XBP1s suppressed AMIO-induced thyroid epithelial follicular cells apoptosis. Mechanically, the Chromatin Immunoprecipitation (ChIP) and luciferase activity assay proved that XBP1s enhanced LINC00842 expression in HT patients and thyroid epithelial follicular cells via binding to LINC00842 promoter. LINC00842 functioned as a miR-214 sponge in HT patients and thyroid epithelial follicular cells. Besides, LINC00842 up-regulated Fas ligand (FASL) expression via inhibition of miR-214. In rescue experiments, overexpression of FASL reversed shXBP1s-induced suppression of cell apoptosis in AMIO-treated thyroid epithelial follicular cells. These findings concluded that AMIO-drove XBP1s aggravated endoplasmic reticulum stress and apoptosis in HT via modulating LINC00842/miR-214/FASL axis, providing a new sight for the therapeutic strategy of AMIO-induced HT.

Impaired intestinal physical barrier of on-growing grass carp by deficient iron under Aeromonas hydrophila infection

This research discovered the impacts of dietary iron on intestine health of on-growing grass carp (Ctenopharyngodon idella). Six hundred and thirty grass carp (242.32 ± 0.58 g) were fed different diets containing 12.15, 35.38, 63.47, 86.43, 111.09, 136.37 and 73.50 mg iron/kg, where the iron source first six groups was ferrous fumarate, and the last group was ferrous sulfate. Feeding trial lasted for sixty days. Thereafter, a fourteen days' challenge test was carried out through Aeromonas hydrophila infection. Previous study showed that the optimum iron level of on-growing grass carp based on per weight gain was estimated to be 75.65 mg/kg. Results from the present study displayed that in contrast to optimum iron level, deficient iron downregulated gene expression of tight junction complexes, Apoptosis regulator Bcl-2 (Bcl-2), MCL1 apoptosis regulator BCL2 family member (Mcl-1), inhibitor of apoptosis protein (IAP) and NF-E2-related factor 2 (Nrf2). It also reduced activities and gene expression of antioxidant enzymes, and upregulated gene expression of apoptosis-related cysteine peptidase CASP (caspase) -2, -7, -8, -9, apoptotic protease activating factor 1 (Apaf-1), Apoptosis regulator Bax (Bax) (in MI and DI), Fas ligand (FasL), p38 mitogen activated protein kinase (p38MAPK), Kelch like ECH associated protein 1a (Keap1a), Keap1b, claudin-12 and myosin light chain kinase (MLCK). In addition, deficient iron raised malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) generation in intestine of grass carp (P < 0.05). These data demonstrated that deficient iron is harmful to intestinal physical barrier of fish. Besides, the efficiency of ferrous fumarate on intestinal physical barrier function was better than ferrous sulfate in fish. At last, based on MDA contents in MI and DI, the iron requirements were 83.40 and 80.20 mg/kg diet (ferrous fumarate as sources), respectively.

Anti-Apoptotic c-FLIP Reduces the Anti-Tumour Activity of Chimeric Antigen Receptor T Cells.

Simple SummaryChimeric antigen receptor (CAR) T cell treatment is a promising adoptive cell therapy that utilises CAR-expressing primary T cells to target specific tumour antigens. Since its first approval in 2017, the FDA has approved six CAR T cell therapies for blood cancer treatment. Despite their success in treating B cell malignancies, solid-tumour-directed CAR T cells face multifactorial challenges, often resulting in a lack of anti-tumour response in tumour elimination. The aim of this study was to evaluate the potency of expressing anti-apoptotic gene FLICE-like inhibitory protein p43 (c-FLIPp43) in Her2-CAR T cells to resist activation-induced cell death (AICD). This study confirmed the anti-apoptotic activity of c-FLIPp43. However, the expression of c-FLIPp43 reduced the CAR activity in a Her-2 breast cancer xenograft model relative to the control Her2-CAR T cells. This work provides insight into the role of c-FLIP in T cells and its impact on anti-tumour immunity in CAR T cells.CAR T cell treatment of solid tumours is limited by poor persistence partly due to CD95 ligand (CD95L)-induced apoptosis. Both T cells and cells within the tumour microenvironment (TME) may express CD95L, triggering apoptosis in CD95-receptor-positive CAR T cells. Tonic signalling of CAR T cells may also increase CD95-dependent AICD. Because the intracellular protein c-FLIP protects T cells from AICD, we expressed c-FLIPp43 within a Her-2 targeted CAR cassette and evaluated the potential of c-FLIPp43 through in vitro functional assays and in vivo tumour-bearing xenograft model. cFLIP expression protected against CD95L-induced cell death in the Jurkat T cell lines. However, in primary human CAR T cells containing CAR-CD28 domains, c-FLIPp43 overexpression had minimal additional impact on resistance to CD95L-induded cell death. In vitro cytotoxicity against a breast cancer tumour cell line was not altered by c-FLIPp43 expression, but the expression of c-FLIPp43 in Her2-CAR T cells lowered interferon-γ secretion, without markedly affecting IL-2 levels, and c-FLIPp43-Her2-CAR T cells showed reduced anti-tumour activity in immunodeficient mice with breast cancer. The findings of this study provide a new understanding of the effects of controlling extrinsic apoptosis pathway suppression in CAR T cells, suggesting that c-FLIPp43 expression reduces anti-tumour immunity through the modulation of effector T cell pathways.