Exploding the necroptotic bubble

The apoptotic death of cells is accompanied by the exposure of “eat-me” signals that serve to prevent necrotic degradation of apoptotic cells, and thereby prevent inflammation, promote resolution of immune responses, and stimulate tissue repair. These “eat-me” signals include the exposure of phosphatidylserine (PS) on the outer plasma membrane during the early stages of apoptosis as well as on the surface of apoptotic bodies, plasma membrane vesicles that are shed during the later stages of cell death. In our recent publication (PLoS Biol. 15(6):e2002711), we describe similar ‘eat-me’ and ‘find-me’ signals present during necroptosis, challenging some of our common assumptions about regulated forms of lytic death

Adaptive immune response to BNT162b2 mRNA vaccine in immunocompromised adolescent patients

Protective immunity against COVID-19 is orchestrated by an intricate network of innate and adaptive anti-viral immune responses. Several vaccines have been rapidly developed to combat the destructive effects of COVID-19, which initiate an immunological cascade that results in the generation of neutralizing antibodies and effector T cells towards the SARS-CoV-2 spike protein. Developing optimal vaccine-induced anti-SARS- CoV-2 protective immunity depends on a fully competent immune response. Some evidence was gathered on the effects of vaccination outcomes in immunocompromised adult individuals. Nonetheless, protective immunity elicited by the Pfizer Biontech BNT162b2 vaccine in immunocompromised adolescents received less attention and was mainly focused on the antibody response and their neutralization potential. The overall immune response, including T-cell activities, was largely understudied. In this study, we characterized the immune response of vaccinated immunocompromised adolescents. We found that immunocompromised adolescents, which may fail to elicit a humoral response and develop antibodies, may still develop cellular T-cell immunity towards SARS-CoV-2 infections. Furthermore, most immunocompromised adolescents due to genetic disorders or drugs (Kidney and liver transplantation) still develop either humoral, cellular or both arms of immunity towards SARS-CoV-2 infections. We also demonstrate that most patients could mount a cellular or humoral response even after six months post 2nd vaccination. The findings that adolescents immunocompromised patients respond to some extent to vaccination are promising. Finally, they question the necessity for additional vaccination boosting regimens for this population who are not at high risk for severe disease, without further testing of their post-vaccination immune status

Phosphatidylserine externalization, "necroptotic bodies" release, and phagocytosis during necroptosis.

Necroptosis is a regulated, nonapoptotic form of cell death initiated by receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL) proteins. It is considered to be a form of regulated necrosis, and, by lacking the "find me" and "eat me" signals that are a feature of apoptosis, necroptosis is considered to be inflammatory. One such "eat me" signal observed during apoptosis is the exposure of phosphatidylserine (PS) on the outer plasma membrane. Here, we demonstrate that necroptotic cells also expose PS after phosphorylated mixed lineage kinase-like (pMLKL) translocation to the membrane. Necroptotic cells that expose PS release extracellular vesicles containing proteins and pMLKL to their surroundings. Furthermore, inhibition of pMLKL after PS exposure can reverse the process of necroptosis and restore cell viability. Finally, externalization of PS by necroptotic cells drives recognition and phagocytosis, and this may limit the inflammatory response to this nonapoptotic form of cell death. The exposure of PS to the outer membrane and to extracellular vesicles is therefore a feature of necroptotic cell death and may serve to provide an immunologically-silent window by generating specific "find me" and "eat me" signals

Combined acetyl-11-keto-β-boswellic acid and radiation treatment inhibited glioblastoma tumor cells.

Glioblastoma multiforme (GBM) is the most common and most aggressive subtype of malignant gliomas. The current standard of care for newly diagnosed GBM patients involves maximal surgical debulking, followed by radiation therapy and temozolomide chemotherapy. Despite the advances in GBM therapy, its outcome remains poor with a median survival of less than two years. This poor outcome is partly due to the ability of GBM tumors to acquire adaptive resistance to therapy and in particular to radiation. One of the mechanisms contributing to GBM tumor progression and resistance is an aberrant activation of NF-ĸB, a family of inducible transcription factors that play a pivotal role in regulation of many immune, inflammatory and carcinogenic responses. Acetyl-11-keto-β-boswellic acid (AKBA) is a pentacyclic terpenoid extracted from the gum Ayurvedic therapeutic plant Boswellia serrata. AKBA is anti-inflammatory agent that exhibits potent cytotoxic activities against various types of tumors including GBM. One of the mechanisms underlying AKBA anti-tumor activity is its ability to modulate the NF-ĸB signaling pathway. The present study investigated in vitro and in vivo the effect of combining AKBA with ionizing radiation in the treatment of GBM and assessed AKBA anti-tumor activity and radio-enhancing potential. The effect of AKBA and/or radiation on the survival of cultured glioblastoma cancer cells was evaluated by XTT assay. The mode of interaction of treatments tested was calculated using CalcuSyn software. Inducing of apoptosis following AKBA treatment was evaluated using flow cytometry. The effect of combined treatment on the expression of PARP protein was analysed by Western blot assay. Ectopic (subcutaneous) GBM model in nude mice was used for the evaluation of the effect of combined treatment on tumor growth. Immunohistochemical analysis of formalin-fixed paraffin-embedded tumor sections was used to assess treatment-related changes in Ki-67, CD31, p53, Bcl-2 and NF-ĸB-inhibitor IĸB-α. AKBA treatment was found to inhibit the survival of all four tested cell lines in a dose dependent manner. The combined treatment resulted in a more significant inhibitory effect compared to the effect of treatment with radiation alone. A synergistic effect was detected in some of the tested cell lines. Flow cytometric analysis with Annexin V-FITC/PI double staining of AKBA treated cells indicated induction of apoptosis. AKBA apoptotic activity was also confirmed by PARP cleavage detected by Western blot analysis. The combined treatment suppressed tumor growth in vivo compared to no treatment and each treatment alone. Immunohistochemical analysis showed anti-angiogenic and anti-proliferative activity of AKBA in vivo. It also demonstrated a decrease in p53 nuclear staining and in Bcl-2 staining and an increase in IĸB-α staining following AKBA treatment both alone and in combination with radiotherapy. In this study, we demonstrated that AKBA exerts potent anti-proliferative and apoptotic activity, and significantly inhibits both the survival of glioblastoma cells in vitro and the growth of tumors generated by these cells. Combination of AKBA with radiotherapy was found to inhibit factors which involved in cell death regulation, tumor progression and radioresistence, therefore it may serve as a novel approach for GBM patients

Generation and Characterization of Novel Local and Metastatic Human Neuroblastoma Variants12

Neuroblastoma (NB) is the most commonly occurring solid tumor in children. The disease usually arises in the adrenal medulla, and it is characterized by a remarkable heterogeneity in its progression. Most NB patients with an advanced disease have massive bone marrow infiltration at diagnosis. Lung metastasis represents a widely disseminated stage and is typically considered to be a terminal event. Much like other malignancies, NB progression is a complex, multistep process. The expression, function, and significance of the various factors involved in NB progression must be studied in relevant in vivo and in vitro models. Currently, models consisting of metastatic and nonmetastatic cell variants of the same genetic background exist for several types of cancer; however, none exists for NB. In the present study, we describe the generation of a NB metastasis model. SH-SY5Y and MHH-NB-11 NB cells were inoculated orthotopically into the adrenal glands of athymic nude mice. Neuroblastoma cells metastasizing to the lungs were isolated from mice bearing adrenal tumors. Lung metastatic variants were generated by repeated cycles of in vivo passage. Characterization of these variants included cellular morphology and immunophenotyping in vitro, aggressiveness in vivo, and various biologic parameters in vitro. The NB metastatic variant in each model displayed unique properties, and both metastatic variants demonstrated a metastatic phenotype in vivo. These reproducible models of human NB metastasis will serve as an unlimited source of transcriptomic and proteomic material. Such models can facilitate future studies on NB metastasis and the identification of novel NB biomarkers and targets for therapy

Detection of A5 single-positive cells during necroptosis.

<p><b>(A–C)</b> L929 cells were stimulated for either (i) apoptosis (TS), necroptosis (TSZ), or (ii) left untreated (Con). Where indicated, RIPK1 (Nec-1s) and RIPK3 (gsk872) inhibitors were added to the cells 30 minutes prior to TSZ stimulation. <b>(A)</b> 10⁶ cells were harvested 3 hours after stimulation of cell death, and cell death key factors pMLKL and CC3 were detected using western blot. <b>(B)</b> Cell viability was measured at different time points after stimulation of cell death using A5/PI staining and then analyzed by flow cytometry (mean ± SD). <b>(C)</b> Example single A5-FITC-positive necroptotic cells, imaged using live microscopy. <b>(D–F)</b> U937 cells were stimulated for either (i) apoptosis (TS), necroptosis (TSZ) or (ii) left untreated (Con). Where indicated, RIPK1 (Nec-1s), RIPK3 (gsk872) and pMLKL (NSA) inhibitors were added to the cells 30 minutes prior to TSZ stimulation. <b>(D)</b> Cell viability was measured at different time points after stimulation of cell death using A5/PI staining and then analyzed by flow cytometry (mean ± SD). <b>(E)</b> Example smoothed flow cytometry density plots. <b>(F)</b> Example single A5-FITC-positive necroptotic cells, imaged using live microscopy. <b>(G–H)</b> 3 x 10⁴ HaCaT cells per well in 96-well plate were stimulated, and PI and A5-FITC were added. The plate was placed on IncuCyteZOOM apparatus and 2 images per well were recorded every 30–45 minutes. <b>(G)</b> Normalized PI- or A5-positive objects per image. <b>(H)</b> Example single A5-positive necroptotic cells, imaged using IncuCyteZOOM apparatus. <b>(I)</b> HaCaT cells were harvested, and the cell death key factors pMLKL and CC3 were detected using western blot. Representative data are shown from 1 of at least 2 independent experiments. All raw data for the data summarized under this Fig can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002711#pbio.2002711.s008" target="_blank">S1 Data</a>. A5, annexin V, CC3, cleaved caspase 3; Con, control; FITC, fluorescein isothiocyanate; MLKL, mixed lineage kinase domain-like; Nec-1s, necroptotic inhibitor of RIPK1; NSA, necrosulfonamide; PI, propidium iodide; pMLKL, phosphorylated mixed lineage kinase-like; RIPK1, receptor-interacting protein kinase-1; RIPK3, receptor-interacting protein kinase-3; Sd, protein ladder; SD, standard deviation; TS, TNFα + SMAC mimetic; TSZ, TNFα + SMAC mimetic + zVAD.</p

Necroptotic A5 single-positive cell death requires pMLKL membrane translocation.

<p>U937 cells were stimulated for necroptosis using TNFα, birinapant (SMAC mimetic) and zVAD (TBZ). After 2 hours, cells were treated with necroptotic inhibitors RIPK1 (Nec-1s), RIPK3 (gsk872), and pMLKL (NSA), or left untreated (Con). <b>(A)</b> Cell viability was measured at different time points after stimulation of cell death using A5/PI staining and then analyzed by flow cytometry (mean ± SD). <b>(B)</b> Cell counts were measured using the Attune NxT flow cytometer (mean ± SD). Statistic comparisons between total cell numbers in each group to total cell numbers at the 2 hour time point in the control group was carried using ANOVA, followed by a Tukey’s multiple comparison test, * <i>p</i> < <i>0</i>.<i>05</i>. Data are representative of 1 experiment from at least 3 independent ones. <b>(C–F)</b> U937 cells were stimulated as in (A) for 2 hours, prior to A5/PI staining. Cells were sorted into live cells (double-negative) or PS-exposed-necroptotic (A5-positive PI-negative). <b>(C)</b> Necroptotic cell death key factor pMLKL was detected in the different sorted populations using western blot. Cell viability was measured at different time points after sorting in the A5⁻PI⁻ <b>(D)</b> and A5⁺PI⁻ <b>(E)</b> populations, using A5/PI staining, and then analyzed by flow cytometry (mean ± SD). Where indicated, RIPK1 (Nec-1s), RIPK3 (gsk872), and pMLKL (NSA) inhibitors were added to the collection tubes. Dashed lines represent the state of cells prior to the addition of inhibitors. Data are taken from 3 independent experiments. <b>(E)</b> U937 cells were stimulated and sorted into PS-exposed-necroptotic (A5-positive PI-negative) as above. Viable cells (A5⁻PI⁻) were measured and counted at 6 days after sorting. Data are taken from 2 independent experiments. (mean ± SD). Statistic comparisons between total cell numbers was carried using parametric paired Student <i>t</i> test. All raw data for the data summarized under this Fig can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002711#pbio.2002711.s012" target="_blank">S5 Data</a>. A5, annexin V; Con, control; Nec-1s; necroptotic inhibitor of RIPK1; MLKL, mixed lineage kinase domain-like; NSA; necrosulfonamide; PI; propidium iodide; pMLKL; phosphorylated mixed lineage kinase-like protein; PS, phosphatidylserine; RIPK1, receptor-interacting protein kinase-1; RIPK3, receptor-interacting protein kinase-3; SD, standard deviation; SMAC, second mitochondrial-derived activator of caspase; Std, protein ladder; TBZ, TNFα + birinapant + zVAD; zVAD, Z-VAD-FMK.</p

Phagocytosis of PS-exposed-necroptotic cells.

<p><b>(A)</b> U937 cells were first stained with CFSE prior to stimulation for apoptosis and necroptosis using a combination of TNFα, birinapant (SMAC mimetic), and zVAD. PS exposure was tested every 30 minutes until exposure reached 40% in both the apoptotic and necroptotic samples (determined by A5/PI staining). Cells were washed twice and resuspended in DMEM before adding on IFN-γ treated BMDMs at a 2.5:1 ratio. Phagocytosis was analyzed by flow cytometry (mean ± SD). BMDMs with or without addition of live cells served as negative controls. Data are taken from 3 independent experiments. <b>(B)</b> U937 cells treated as above were added on TG peritoneal macrophages at a 2.5:1 ratio, and phagocytosis was analyzed by flow cytometry (mean ± SD). <b>(C)</b> Viability of the U937 cells, which were used as target to (B), is shown. TG peritoneal macrophages with no addition of cells or with addition of live cells served as negative controls. <b>(D–I)</b> Supernatants from the phagocytic TG peritoneal macrophages from (B) were collected and then analyzed for cytokines and chemokines using ELISAs. Statistic comparisons between each injected target cells were carried using ANOVA, followed by a Tukey’s multiple comparison test, * <i>p < 0</i>.<i>05</i>. <b>(J–K)</b> U937 cells were left untreated or stimulated for apoptosis or necroptosis. PS exposure was tested every 30 minutes until reaching 40% in apoptosis and necroptotic samples (determined by A5/PI staining). 2 x 10⁶ cells in 100 μl per mouse were IP injected. One hour after injection, CD11b peritoneal cells were analyzed for CFSE fluorescence <b>(J)</b> and phagocytosis of CFSE target cells <b>(K)</b> by flow cytometry (<i>N</i> = 4, mean ± sem). Statistic comparisons between each injected target cells were carried using ANOVA, followed by a Tukey’s multiple comparison test, * <i>p < 0</i>.<i>05</i>. <b>(L)</b> Illustration of competitive in vivo phagocytosis assay. CFSE- or Hoescht-stained L929 cells were left untreated or stimulated for necroptosis (TSZ). PS exposure was tested every 30 minutes until reaching 40% in necroptotic samples (determined by A5/PI staining). A 1:1 ratio mix of live and necroptotic cells was IP injected (total of 2 x 10⁶ cells in 100 μl per mouse). (M) One hour after injection, F4/80 peritoneal cells were analyzed for phagocytosis of the live and/or necroptotic cells by flow cytometry (<i>N</i> = 4, mean ± sem). Statistic comparisons between each injected target cells populations were carried using ANOVA, followed by a Tukey’s multiple comparison test, * <i>p < 0</i>.<i>05</i>. All raw data for the data summarized under this Fig can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002711#pbio.2002711.s013" target="_blank">S6 Data</a>. A5, annexin V; BMDM, bone marrow-derived macrophages; CFSE, carboxyfluorescein succinimidyl ester; DMEM, dulbecco’s modified eagle’s medium; IP, intraperitoneally; PI, propidium iodide; MFI, mean fluorescence intensity; PS, phosphatidylserine; SD, standard deviation; SMAC, second mitochondrial-derived activator of caspase; TG, thioglycolate; zVAD, Z-VAD-FMK.</p

Necroptotic cells expose PS to the outer plasma membrane prior to its permeabilization.

<p>U937 cells were stimulated for either (i) apoptosis (TS), necroptosis (TSZ), or (ii) left untreated (Con). <b>(A–D)</b> Cell viability was measured at different time points after stimulation of cell death by flow cytometry using <b>(A)</b> A5 and LD, <b>(B)</b> A5- and PI-staining, <b>(C)</b> A5, LD, and PI or <b>(D)</b> A5, Z, and PI triple staining. <b>(E)</b> Four hours after stimulation of apoptosis (TB) or necroptosis (TBZ), U937 cells were triple stained with A5, Z, and PI, and analyzed by imagestream flow cytometry. Example imagestream flow plots are shown, brightness contrast for each channel between each population was identical. Representative data are shown from 1 of at least 2 independent experiments that were carried out. All raw data for the data summarized under this Fig can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002711#pbio.2002711.s009" target="_blank">S2 Data</a>. A5, annexin V; BF, bright field; Con, control; LD, LiveDead; PI, propidium iodide; PS, phosphatidylserine; SSC, side scatter; TB, TNFα + birinapant; TBZ, TNFα + birinapant + zVAD; TS, TNFα + SMAC mimetic; TSZ, TNFα + SMAC mimetic + zVAD; Z, Zombie.</p