Human and Pathogen Derived Ndpks Act As Novel Damps and PAMPs to Drive Leukemia Cell Survival and Progression through Signaling Via the TLR4-Mediated Alternative NLRP3 Inflammasome Pathway

Elevated plasma levels of the nucleoside diphosphate kinase (NDPK) NM23-H1 are associated with poorer prognosis in acute myeloid leukemia (AML). We previously demonstrated that leukemic blasts release NM23-H1, which binds to more differentiated myeloid cells inducing their secretion of inflammatory cytokines, including IL-1β, that promote survival and proliferation of leukemic blasts1. Both AML and myelodysplastic syndrome (MDS) patients are prone to infections due to impaired hematopoiesis that is worsened by treatment. NDPKs are highly evolutionarily conserved raising the possibility that bacterial/fungal NDPKs could mediate the same survival effect on malignant AML/MDS blasts and exacerbate disease progression. To test this, we generated recombinant NDPKs (rNDPKs) from bacteria and fungi associated with common infections in these patients (E. coli, S. aureus, S. pneumoniae, K. pneumoniae, C. albicans). Cytokine production and survival responses of primary AMLs to these proteins were indistinguishable from their response to rNM23-H1. This activity was independent of NDPK enzyme activity since mutant rNM23-H1 and bacterial and fungal rNDPKs with impaired oligomerization, kinase or exonuclease activity elicited the same cytokine and survival response. Toll like receptors (TLRs) are the major family of human DAMP/PAMP receptors and IL-1β secretion is closely associated with TLR-4 mediated activation of the NLRP3 inflammasome in monocytes. We therefore postulated that NM23-H1 and pathogen derived NDPKs act as novel damage- and pathogen- associated molecular pattern (DAMP, PAMP) molecules. We confirmed that fluorescently labelled rNM23-H1 and S. pneumoniae rNDPK bound selectively to monocytes in peripheral blood. Using in vitro generated monocytes (vitamin D3 differentiated THP-1 cells) we demonstrated that both wild type and mutant rNM23-H1 and bacterial/fungal rNDPKs induced activation of caspase-1 and cleavage of pro-IL-1β into its active form. Secretion of IL-1β was inhibited by antagonists/inhibitors of TLR4, NLRP3 and caspase-1 indicating the involvement of the TLR4-NLRP3 inflammasome axis is mediating the NDPK response. Unlike the canonical NLRP3-inflammasome pathway that leads to monocyte cell death by pyroptosis, rNM23-H1 and rNDPKs did not lead to cell death indicating that rNDPKs are responsible for the activation of the alternative inflammasome. In our earlier studies, and those of others, we demonstrated that not all AML primary samples responsed to NM23-H1 in vitro. We have observed that non responders to NM23-H1 also do not respond to pathogen derived rNDPKs. In contrast, we have observed uniform responses in terms of cytokine release in all normal peripheral blood. We hence hypothesized that the non-rNDPK-responding AML samples may reflect the absence of monocytes in culture. To test this, we generated conditioned media using normal donor leukocytes, in presence or absence of a TLR-4 antagonist to inhibit the IL-1β production. The conditioned media was then used to culture primary AML samples, in parallel with rNDPK in unconditioned media. All the samples analyzed showed increased survival in rNDPK conditioned media even whilst some did not respond to rNDPK in unconditioned media. In summary, our data demonstrate for the first time that NM23-H1 and bacterial/fungal NDPKs are novel DAMPs/PAMPS that signal via TLR4 in monocytes. We further demonstrate that this interaction results in activation of the alternative NLRP3 inflammasome and subsequent cleavage and secretion of IL-1β without death by pyroptosis. Our data showing that bacterial/fungal NDPKs can promote survival of AML blasts indicates that rather than just being a consequence of AML associated immunosuppression, infections may drive the progression and AML. These findings have important implications in the clinical management of AML and its precursor myelodysplastic syndromes (MDS). Lilly AJ, et al.Cancer Res. 2011;71(3):1177-86.Gaidt MM, et al. Immunity. 2016;44(4):833-46 Disclosures Drayson: Abingdon Health: Consultancy, Equity Ownership.

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Suppression of NLRP3 Inflammasome, Pyroptosis, and Cell Death by NIM811 in Rotenone-Exposed Cells as an in vitro Model of Parkinson’s Disease

Neurodegenerative Diseases ◽

10.1159/000511207 ◽

2020 ◽

pp. 1-11

Author(s):

Minghao Zhang ◽

Qingping He ◽

Guisheng Chen ◽

P. Andy Li

Keyword(s):

Cell Death ◽

Nlrp3 Inflammasome ◽

Mitochondrial Membrane ◽

In Vitro Model ◽

Ros Production ◽

Double Staining ◽

Caspase 1 ◽

Vitro Model ◽

Pass Through

Background: Parkinson’s disease (PD) is characterized by the selective death of dopaminergic neurons in the substantia nigra. Recently, NLRP3 inflammasome and pyroptosis were found to be associated with PD. Cyclosporine A (CsA), an immunosuppressant, reduces neuronal death in PD. However, CsA could hardly pass through the blood-brain barrier (BBB) and high dose is associated with severe side effects and toxicity. N-methyl-4-isoleucine-cyclosporine (NIM811) is a CsA derivate that can pass through the BBB. However, little is known about its effect on PD. Objective: The objectives of this study were to explore the mechanism of rotenone-induced cell damage and to examine the protective effects of NIM811 on the neurotoxicity of a Parkinson-like in vitro model induced by rotenone. Methods: Murine hippocampal HT22 cells were cultured with the mitochondrial complex I inhibitor rotenone, a widely used pesticide that has been used for many years as a tool to induce a PD model in vitro and in vivo and proven to be reproducible. NIM811 was added to the culture media 3 h prior to the rotenone incubation. Cell viability was determined by resazurin assay, reactive oxygen species (ROS) production by dihydroethidine (DHE), and mitochondrial membrane potential by tetramethyl rhodamine methyl ester (TMRM). TUNEL and caspase-1 immunofluorescent double staining was used to detect pyroptosis. NLRP3, caspase-1, pro-caspase-1, GSDMD, and interleukin-18 (IL-18) were measured using Western blotting after 24 h of rotenone incubation. The reactivity of interleukin-1β (IL-1β) was determined by ELISA. Results: Our results demonstrated that rotenone caused more than 40% of cell death, increased ROS production, and reduced mitochondrial membrane potential, while NIM811 reversed these alterations. Immunofluorescent double staining showed that rotenone increased the percentage of caspase-1 and TUNEL double-labelled cells, an indication of pyroptosis, after 24 h of incubation. The protein expression of NLRP3, caspase-1, pro-caspase-1, GSDMD, IL-18, and IL-1β was significantly increased after 24 h of rotenone incubation. NIM811 suppressed rotenone-induced pyroptosis and downregulated the protein expression of NLRP3, caspase-1, pro-caspase-1, GSDMD, IL-1β, and IL-18. Conclusion: These results provide evidence that rotenone activates the NLRP3 inflammomere and induces pyroptosis. NIM811 protects the cell from rotenone-induced damage and inhibits NLRP3 inflammasome and pyroptosis. NIM811 might serve as a potential therapeutic drug in the treatment of PD.

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FRI0001 NEUTROPHILS IN GRANULOMATOSIS WITH POLYANGIITIS DISPLAY FEATURES OF PYROPTOSIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.4523 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 572.2-572

Author(s):

A. Kerstein-Staehle ◽

N. Leinung ◽

J. Meyer ◽

S. Pitann ◽

A. Müller ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Nlrp3 Inflammasome ◽

Peripheral Blood ◽

Granulomatosis With Polyangiitis ◽

Granulomatous Inflammation ◽

Clinical Aspects ◽

Inflammasome Activation ◽

Caspase 1 ◽

Active Caspase

Background:Granulomatosis with polyangiitis (GPA) is characterized by extravascular necrotizing granulomatous inflammation and systemic ANCA – associated (AAV) vasculitis with neutrophils as a key player in the pathogenesis (1). We and others have shown that neutrophil-related cell death mechanisms contribute to chronic inflammatory processes in AAV (2, 3). Recently, another form of inflammatory cell death primarily described in monocytes called pyroptosis was also discovered in neutrophils (4). A cardinal feature of pyroptosis is the activation of the NLRP3 inflammasome, a sensor of different pathogen- and damage-associated molecular patterns (PAMP, DAMP), following caspase-1-mediated processing and secretion of IL-1beta (5).Objectives:The aim of this study was to investigate, if neutrophils from GPA patients express pyroptosis-related components NLRP3, active caspase 1 and cleaved IL-1beta.Methods:Polymorphonuclear leukocytes (PMN) were isolated from peripheral blood of GPA patients and healthy controls (HC) (n = 10 each). Expression of NLRP3, inactive/active caspase 1 and active IL-1beta was determined by western blot. In addition, peripheral blood mononuclear cells (PBMC) were isolated from GPA and HC. mRNA expression ofnlrp3andil1bwas determined by qPCR. To exclude false-positive results by contamination with monocytes we performed flow cytometry analysis of whole blood samples with markers CD3, CD14, CD15, CD66b and NLRP3.Results:PMN from GPA patients showed markedly increased expression of NLRP3, active caspase 1 and active IL-1beta compared to HC. In contrast, there was no difference between GPA and HC on the mRNA level of neithernlrp3noril1bin PBMC. In addition, we confirmed by flow cytometry increased expression of NLRP3 in PMN from GPA, but not in monocytes.Conclusion:Here we provide evidence, that neutrophils from GPA undergo pyroptosis, demonstrated by increased NLRP3, active caspase 1 expression as well as IL-1beta processing. Neutrophils are present in high numbers at the site of granulomatous lesions of inflamed tissue in GPA and IL-1beta is increased in GPA sera (2). Therefore, neutrophils represent a potential source of IL-1beta in GPA. Given the fact that GPA-associated features such as massive release of necrosis-related DAMP or microbial agents such asStaphylococcus aureus(6) can activate the NLRP3-inflammasome, we identified here a potential relevant mechanism of neutrophils contributing to chronic inflammation of GPA.References:[1]Jennette, J.C., and Falk, R.J. (2014). Pathogenesis of antineutrophil cytoplasmic autoantibody-mediated disease. Nat. Rev. Rheumatol.10, 463–473.[2]Millet, A., Martin, K.R., Bonnefoy, F., Saas, P., Mocek, J., Alkan, M., Terrier, B.,Kerstein,A., Tamassia, N., Satyanarayanan, S.K., et al. (2015). Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis. J. Clin. Invest. 125, 4107–4121.[3]Schreiber, A., Rousselle, A., Becker, J.U., von Mässenhausen, A., Linkermann, A., and Kettritz, R. (2017). Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis. Proc. Natl. Acad. Sci. 201708247.[4]Tourneur, L., and Witko-Sarsat, V. (2019). Inflammasome activation: Neutrophils go their own way. J. Leukoc. Biol.105, 433–436.[5]Bergsbaken, T., Fink, S.L., and Cookson, B.T. (2009). Pyroptosis: Host cell death and inflammation. Nat. Rev. Microbiol.7, 99–109.[6]Lamprecht, P.,Kerstein, A., Klapa, S., Schinke, S., Karsten, C.M., Yu, X., Ehlers, M., Epplen, J.T., Holl-Ulrich, K., Wiech, T., et al. (2018). Pathogenetic and Clinical Aspects of Anti-Neutrophil Cytoplasmic Autoantibody-Associated Vasculitides. Front. Immunol.9, 1–10.Disclosure of Interests:Anja Kerstein-Staehle: None declared, Nadja Leinung: None declared, Jannik Meyer: None declared, Silke Pitann: None declared, Antje Müller: None declared, Gabriela Riemekasten Consultant of: Cell Trend GmbH, Janssen, Actelion, Boehringer Ingelheim, Speakers bureau: Actelion, Novartis, Janssen, Roche, GlaxoSmithKline, Boehringer Ingelheim, Pfizer, Peter Lamprecht: None declared

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Polyphyllin VI Induces Caspase-1-Mediated Pyroptosis via the Induction of ROS/NF-κB/NLRP3/GSDMD Signal Axis in Non-Small Cell Lung Cancer

Cancers ◽

10.3390/cancers12010193 ◽

2020 ◽

Vol 12 (1) ◽

pp. 193 ◽

Cited By ~ 11

Author(s):

Jin-Feng Teng ◽

Qi-Bing Mei ◽

Xiao-Gang Zhou ◽

Yong Tang ◽

Rui Xiong ◽

...

Keyword(s):

Lung Cancer ◽

Cell Death ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Nlrp3 Inflammasome ◽

Small Cell ◽

Small Cell Lung ◽

Caspase 1 ◽

Vitro Effect

Trillium tschonoskii Maxim (TTM), a traditional Chinese medicine, has been demonstrated to have a potent anti-tumor effect. Recently, polyphyllin VI (PPVI), a main saponin isolated from TTM, was reported by us to significantly suppress the proliferation of non-small cell lung cancer (NSCLC) via the induction of apoptosis and autophagy in vitro and in vivo. In this study, we further found that the NLRP3 inflammasome was activated in PPVI administrated A549-bearing athymic nude mice. As is known to us, pyroptosis is an inflammatory form of caspase-1-dependent programmed cell death that plays an important role in cancer. By using A549 and H1299 cells, the in vitro effect and action mechanism by which PPVI induces activation of the NLRP3 inflammasome in NSCLC were investigated. The anti-proliferative effect of PPVI in A549 and H1299 cells was firstly measured and validated by MTT assay. The activation of the NLRP3 inflammasome was detected by using Hoechst33324/PI staining, flow cytometry analysis and real-time live cell imaging methods. We found that PPVI significantly increased the percentage of cells with PI signal in A549 and H1299, and the dynamic change in cell morphology and the process of cell death of A549 cells indicated that PPVI induced an apoptosis-to-pyroptosis switch, and, ultimately, lytic cell death. In addition, belnacasan (VX-765), an inhibitor of caspase-1, could remarkably decrease the pyroptotic cell death of PPVI-treated A549 and H1299 cells. Moreover, by detecting the expression of NLRP3, ASC, caspase-1, IL-1β, IL-18 and GSDMD in A549 and h1299 cells using Western blotting, immunofluorescence imaging and flow cytometric analysis, measuring the caspase-1 activity using colorimetric assay, and quantifying the cytokines level of IL-1β and IL-18 using ELISA, the NLRP3 inflammasome was found to be activated in a dose manner, while VX-765 and necrosulfonamide (NSA), an inhibitor of GSDMD, could inhibit PPVI-induced activation of the NLRP3 inflammasome. Furthermore, the mechanism study found that PPVI could activate the NF-κB signaling pathway via increasing reactive oxygen species (ROS) levels in A549 and H1299 cells, and N-acetyl-L-cysteine (NAC), a scavenger of ROS, remarkably inhibited the cell death, and the activation of NF-κB and the NLRP3 inflammasome in PPVI-treated A549 and H1299 cells. Taken together, these data suggested that PPVI-induced, caspase-1-mediated pyroptosis via the induction of the ROS/NF-κB/NLRP3/GSDMD signal axis in NSCLC, which further clarified the mechanism of PPVI in the inhibition of NSCLC, and thereby provided a possibility for PPVI to serve as a novel therapeutic agent for NSCLC in the future.

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Dimethyl Fumarate Alleviates NLRP3 Inflammasome Activation in Microglia and Sickness Behavior in LPS-Challenged Mice

Frontiers in Immunology ◽

10.3389/fimmu.2021.737065 ◽

2021 ◽

Vol 12 ◽

Author(s):

Bora Tastan ◽

Burak I. Arioz ◽

Kemal Ugur Tufekci ◽

Emre Tarakcioglu ◽

Ceren Perihan Gonul ◽

...

Keyword(s):

Cell Death ◽

Nlrp3 Inflammasome ◽

Forced Swim Test ◽

Sickness Behavior ◽

Dimethyl Fumarate ◽

Inflammasome Activation ◽

Caspase 1 ◽

Nlrp3 Inflammasome Activation

NLRP3 inflammasome activation contributes to several pathogenic conditions, including lipopolysaccharide (LPS)-induced sickness behavior characterized by reduced mobility and depressive behaviors. Dimethyl fumarate (DMF) is an immunomodulatory and anti-oxidative molecule commonly used for the symptomatic treatment of multiple sclerosis and psoriasis. In this study, we investigated the potential use of DMF against microglial NLRP3 inflammasome activation both in vitro and in vivo. For in vitro studies, LPS- and ATP-stimulated N9 microglial cells were used to induce NLRP3 inflammasome activation. DMF’s effects on inflammasome markers, pyroptotic cell death, ROS formation, and Nrf2/NF-κB pathways were assessed. For in vivo studies, 12–14 weeks-old male BALB/c mice were treated with LPS, DMF + LPS and ML385 + DMF + LPS. Behavioral tests including open field, forced swim test, and tail suspension test were carried out to see changes in lipopolysaccharide-induced sickness behavior. Furthermore, NLRP3 and Caspase-1 expression in isolated microglia were determined by immunostaining. Here we demonstrated that DMF ameliorated LPS and ATP-induced NLRP3 inflammasome activation by reducing IL-1β, IL-18, caspase-1, and NLRP3 levels, reactive oxygen species formation and damage, and inhibiting pyroptotic cell death in N9 murine microglia via Nrf2/NF-κB pathways. DMF also improved LPS-induced sickness behavior in male mice and decreased caspase-1/NLRP3 levels via Nrf2 activation. Additionally, we showed that DMF pretreatment decreased miR-146a and miR-155 both in vivo and in vitro. Our results proved the effectiveness of DMF on the amelioration of microglial NLRP3 inflammasome activation. We anticipate that this study will provide the foundation consideration for further studies aiming to suppress NLRP3 inflammasome activation associated with in many diseases and a better understanding of its underlying mechanisms.

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The Human Leukemic HL-60 Cell Line: An in Vitro Model for Cell Death Endpoint Identification

Alternatives to Laboratory Animals ◽

10.1177/026119299602400419 ◽

1996 ◽

Vol 24 (4) ◽

pp. 581-587

Author(s):

Cristiana Zanetti ◽

Arrnalaura Stammati ◽

Orazio Sapora ◽

Flavia Zucco

Keyword(s):

Cell Death ◽

Cell Line ◽

In Vitro Model ◽

Leukemia Cell ◽

Promyelocytic Leukemia ◽

Leukemia Cell Line ◽

Cell Type ◽

Vitro Model ◽

Promyelocytic Leukemia Cell Line

The aim of this study was to investigate the endpoints related to cell death, either necrosis or apoptosis, induced by four chemicals in the promyelocytic leukemia cell line, HL-60. Cell morphology, DNA fragmentation, cytofluorimetric analysis and oxygen consumption were used to classify the type of cell death observed. In our analysis, we found that not all the selected parameters reproduced the differences observed in the cell death caused by the four chemicals tested. As cell death is a very complex phenomenon, several factors should be taken into account (cell type, exposure time and chemical concentration), if chemicals are to be classified according to differences in the mechanisms more directly involved in cell death.

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The Essential Oil of Artemisia argyi H.Lév. and Vaniot Attenuates NLRP3 Inflammasome Activation in THP-1 Cells

Frontiers in Pharmacology ◽

10.3389/fphar.2021.712907 ◽

2021 ◽

Vol 12 ◽

Author(s):

Pengxiao Chen ◽

Qi Bai ◽

Yanting Wu ◽

Qiongzhen Zeng ◽

Xiaowei Song ◽

...

Keyword(s):

Essential Oil ◽

Nlrp3 Inflammasome ◽

Therapeutic Potential ◽

Inflammasome Activation ◽

Caspase 1 ◽

Artemisia Argyi ◽

Nlrp3 Inflammasome Activation ◽

Long Time

Artemisia argyi H. Lév. and Vaniot is a traditional medical herb that has been used for a long time in China and other Asian counties. Essential oil is the main active fraction of Artemisia argyi H. Lév. and Vaniot, and its anti-inflammatory potential has been observed in vitro and in vivo. Here, we found that the essential oil of Artemisia argyi H. Lév. and Vaniot (EOAA) inhibited monosodium urate (MSU)- and nigericin-induced NLRP3 inflammasome activation. EOAA suppressed caspase-1 and IL-1β processing and pyroptosis. NF-κB p65 phosphorylation and translocation were also inhibited. In addition, EOAA suppressed nigericin-induced NLRP3 inflammasome activation without blocking ASC oligomerization, suggesting that it may inhibit NLRP3 inflammasome activation by preventing caspase-1 processing. Our study thus indicates that EOAA inhibits NLRP3 inflammasome activation and has therapeutic potential against NLRP3-driven diseases.

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A novel leukocyte differentiation antigen: two monoclonal antibodies TM2 and TM3 define a 120-kd molecule present on neutrophils, monocytes, platelets, and activated lymphoblasts

Blood ◽

10.1182/blood.v66.4.873.bloodjournal664873 ◽

1985 ◽

Vol 66 (4) ◽

pp. 873-881

Author(s):

H Ohto ◽

H Maeda ◽

Y Shibata ◽

RF Chen ◽

Y Ozaki ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Peripheral Blood ◽

Leukemia Cell ◽

Cell Extract ◽

Peripheral Blood Lymphocytes ◽

Leukemia Cell Line ◽

Neutrophilic Granulocytes ◽

Blood Monocytes ◽

Normal Peripheral Blood ◽

Unique Cell

We produced two hybridomas by fusion of mouse myeloma cells with splenocytes from a mouse immunized with the THP-1 human monocytoid leukemia cell line. Two cloned hybridoma cell lines, designated as TM2 and TM3, were obtained. They secreted antibodies against a unique cell surface antigen expressed on all normal peripheral blood monocytes, neutrophilic granulocytes, platelets, and mitogen-induced lymphoblasts, some cells from patients with immature-type lymphoid leukemias. However, the antibodies reacted neither with large numbers of peripheral blood lymphocytes nor with red cells. Cross-blocking studies showed that these monoclonal antibodies recognized the same or a nearly positioned antigen epitope. Immunoprecipitation of THP-1 cell extract with TM2 or TM3 under reducing and nonreducing conditions yielded a specific band of mol wt equal to 120,000 daltons. This determinant appeared to be involved in granulocyte chemotaxis, since neutrophilic granulocytes exposed to TM2 or TM3 showed a significant decrease in chemotaxis toward endotoxin-activated serum. These two monoclonal antibodies did not affect O2- release or luminol-dependent chemiluminescence of neutrophils. Moreover, they did not alter platelet aggregation induced by thrombin. TM2 and TM3 will provide a new reagent in defining the linkage between lymphoid and myeloid differentiation and intermyeloid development.

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Astragaloside IV Suppresses High Glucose-Induced NLRP3 Inflammasome Activation by Inhibiting TLR4/NF-κB and CaSR

Mediators of Inflammation ◽

10.1155/2019/1082497 ◽

2019 ◽

Vol 2019 ◽

pp. 1-16 ◽

Cited By ~ 10

Author(s):

Bin Leng ◽

Yingjie Zhang ◽

Xinran Liu ◽

Zhen Zhang ◽

Yang Liu ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

High Glucose ◽

Inflammasome Activation ◽

Astragaloside Iv ◽

Caspase 1 ◽

Endothelial Inflammation ◽

Nlrp3 Inflammasome Activation ◽

Anti Inflammatory

Long-term exposure to high glucose induces vascular endothelial inflammation that can result in cardiovascular disease. Astragaloside IV (As-IV) is widely used for anti-inflammatory treatment of cardiovascular diseases. However, its mechanism of action is still not fully understood. In this study, we investigated the effect of As-IV on high glucose-induced endothelial inflammation and explored its possible mechanisms. In vivo, As-IV (40 and 80 mg/kg/d) was orally administered to rats for 8 weeks after a single intraperitoneal injection of streptozotocin (STZ, 65 mg/kg). In vitro, human umbilical vein endothelial cells (HUVECs) were treated with high glucose (33 mM glucose) in the presence or absence of As-IV, NPS2143 (CaSR inhibitor), BAY 11-7082 (NF-κB p65 inhibitor), and INF39 (NLRP3 inhibitor), and overexpression of CaSR was induced by infection of CaSR-overexpressing lentiviral vectors to further discuss the anti-inflammatory property of As-IV. The results showed that high glucose increased the expression of interleukin-18 (IL-18), interleukin-1β (IL-1β), NLRP3, caspase-1, and ASC, as well as the protein level of TLR4, nucleus p65, and CaSR. As-IV can reverse these changes in vivo and in vitro. Meanwhile, NPS2143, BAY 11-7082, and INF39 could significantly abolish the high glucose-enhanced NLRP3, ASC, caspase-1, IL-18, and IL-1β expression in vitro. In addition, both NPS2143 and BAY 11-7082 attenuated high glucose-induced upregulation of NLRP3, ASC, caspase-1, IL-18, and IL-1β expression. In conclusion, this study suggested that As-IV could inhibit high glucose-induced NLRP3 inflammasome activation and subsequent secretion of proinflammatory cytokines via inhibiting TLR4/NF-κB signaling pathway and CaSR, which provides new insights into the anti-inflammatory activity of As-IV.

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Expression of CD10 by Human T Cells That Undergo Apoptosis Both In Vitro and In Vivo

Blood ◽

10.1182/blood.v94.9.3067 ◽

1999 ◽

Vol 94 (9) ◽

pp. 3067-3076 ◽

Cited By ~ 48

Author(s):

Giovanna Cutrona ◽

Nicolò Leanza ◽

Massimo Ulivi ◽

Giovanni Melioli ◽

Vito L. Burgio ◽

...

Keyword(s):

T Cells ◽

Peripheral Blood ◽

Ex Vivo ◽

Lymphoid Cells ◽

Normal Peripheral Blood ◽

Apoptotic Process ◽

Cd10 Expression ◽

T Cell Lines

Abstract This study shows that human postthymic T cells express CD10 when undergoing apoptosis, irrespective of the signal responsible for initiating the apoptotic process. Cells from continuous T-cell lines did not normally express CD10, but became CD10+ when induced into apoptosis by human immunodeficiency virus (HIV) infection and exposure to CD95 monoclonal antibody, etoposide, or staurosporin. Inhibitors of caspases blocked apoptosis and CD10 expression. Both CD4+ and CD8+ T cells purified from normal peripheral blood expressed CD10 on apoptotic induction. CD10 was newly synthesized by the apoptosing cells because its expression was inhibited by exposure to cycloheximide and CD10 mRNA became detectable by reverse transcription-polymerase chain reaction in T cells cultured under conditions favoring apoptosis. To show CD10 on T cells apoptosing in vivo, lymph node and peripheral blood T cells from HIV+ subjects were used. These suspensions were composed of a substantial, although variable, proportion of apoptosing T cells that consistently expressed CD10. In contrast, CD10+ as well as spontaneously apoptosing T cells were virtually absent in peripheral blood from normal individuals. Collectively, these observations indicate that CD10 may represent a reliable marker for identifying and isolating apoptosing T cells in vitro and ex vivo and possibly suggest novel functions for surface CD10 in the apoptotic process of lymphoid cells.

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Shiga Toxins Activate the NLRP3 Inflammasome Pathway To Promote Both Production of the Proinflammatory Cytokine Interleukin-1β and Apoptotic Cell Death

Infection and Immunity ◽

10.1128/iai.01095-15 ◽

2015 ◽

Vol 84 (1) ◽

pp. 172-186 ◽

Cited By ~ 28

Author(s):

Moo-Seung Lee ◽

Haenaem Kwon ◽

Eun-Young Lee ◽

Dong-Jae Kim ◽

Jong-Hwan Park ◽

...

Keyword(s):

Cell Death ◽

Nlrp3 Inflammasome ◽

Apoptotic Cell ◽

Apoptotic Cell Death ◽

Interleukin 1Β ◽

Dependent Manner ◽

Shiga Toxins ◽

Caspase 1 ◽

Immune Signaling Pathway ◽

Tnf Α

Shiga toxin (Stx)-mediated immune responses, including the production of the proinflammatory cytokines tumor necrosis-α (TNF-α) and interleukin-1β (IL-1β), may exacerbate vascular damage and accelerate lethality. However, the immune signaling pathway activated in response to Stx is not well understood. Here, we demonstrate that enzymatically active Stx, which leads to ribotoxic stress, triggers NLRP3 inflammasome-dependent caspase-1 activation and IL-1β secretion in differentiated macrophage-like THP-1 (D-THP-1) cells. The treatment of cells with a chemical inhibitor of glycosphingolipid biosynthesis, which suppresses the expression of the Stx receptor globotriaosylceramide and subsequent endocytosis of the toxin, substantially blocked activation of the NLRP3 inflammasome and processing of caspase-1 and IL-1β. Processing and release of both caspase-1 and IL-1β were significantly reduced or abolished in Stx-intoxicated D-THP-1 cells in which the expression of NLRP3 or ASC was stably knocked down. Furthermore, Stx mediated the activation of caspases involved in apoptosis in an NLRP3- or ASC-dependent manner. In Stx-intoxicated cells, the NLRP3 inflammasome triggered the activation of caspase-8/3, leading to the initiation of apoptosis, in addition to caspase-1-dependent pyroptotic cell death. Taken together, these results suggest that Stxs trigger the NLRP3 inflammasome pathway to release proinflammatory IL-1β as well as to promote apoptotic cell death.

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