| 1. |
- Singel, Kelly L., et al.
(författare)
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Mature neutrophils suppress T cell immunity in ovarian cancer microenvironment
- 2019
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Ingår i: JCI Insight. - : American Society for Clinical Investigation. - 2379-3708. ; 4:5
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Tidskriftsartikel (refereegranskat)abstract
- Epithelial ovarian cancer (EOC) often presents with metastases and ascites. Granulocytic myeloid-derived suppressor cells are an immature population that impairs antitumor immunity. Since suppressive granulocytes in the ascites of patients with newly diagnosed EOC were morphologically mature, we hypothesized that PMN were rendered suppressive in the tumor microenvironment (TME). Circulating PMN from patients were not suppressive but acquired a suppressor phenotype (defined as ≥1 log10 reduction of anti-CD3/CD28-stimulated T cell proliferation) after ascites supernatant exposure. Ascites supernatants (20 of 31 supernatants) recapitulated the suppressor phenotype in PMN from healthy donors. T cell proliferation was restored with ascites removal and restimulation. PMN suppressors also inhibited T cell activation and cytokine production. PMN suppressors completely suppressed proliferation in naive, central memory, and effector memory T cells and in engineered tumor antigen-specific cytotoxic T lymphocytes, while antigen-specific cell lysis was unaffected. Inhibition of complement C3 activation and PMN effector functions, including CR3 signaling, protein synthesis, and vesicular trafficking, abrogated the PMN suppressor phenotype. Moreover, malignant effusions from patients with various metastatic cancers also induced the C3-dependent PMN suppressor phenotype. These results point to PMN impairing T cell expansion and activation in the TME and the potential for complement inhibition to abrogate this barrier to antitumor immunity.
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| 2. |
- Singel, Kelly L., et al.
(författare)
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Mitochondrial DNA in the tumor microenvironment activates neutrophils and is associated with worsened outcomes in patients with advanced epithelial ovarian cancer
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesized that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worsened outcomes.Banked ascites supernatants from patients with newly diagnosed advanced EOC were analyzed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELPand ELANE. The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n=68, log-rank, p=0.0178). NETs were detected in resected tumors. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivostimulated T cell proliferation. Increased SELPmRNA expression correlated with worsened overall survival (n=302, Cox model, p=0.02).In this single-center retrospective analysis, ascites mtDNA correlated with worsened PFS in advanced EOC. Our results support mitochondrial and other DAMPs activating neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumor immunity. These pathways are potential prognostic markers and therapeutic targets.
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| 3. |
- Singel, Kelly L., et al.
(författare)
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Mitochondrial DNA in the tumour microenvironment activates neutrophils and is associated with worse outcomes in patients with advanced epithelial ovarian cancer
- 2019
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Ingår i: British Journal of Cancer. - : Nature Publishing Group. - 0007-0920 .- 1532-1827. ; 120:2, s. 207-217
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesised that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worse outcomes.METHODS: Banked ascites supernatants from patients with newly diagnosed advanced EOC were analysed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELP and ELANE.RESULTS: The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n = 68, log-rank, p = 0.0178). NETs were detected in resected tumours. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivo stimulated T cell proliferation. Increased SELP mRNA expression correlated with worse overall survival (n = 302, Cox model, p = 0.02).CONCLUSION: In this single-centre retrospective analysis, ascites mtDNA correlated with worse PFS in advanced EOC. Mitochondrial and other DAMPs in ascites may activate neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumour immunity. These pathways are potential prognostic markers and therapeutic targets.
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| 4. |
- Grimm, Melissa J, et al.
(författare)
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Monocyte- and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice
- 2013
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 190:8, s. 4175-4184
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Tidskriftsartikel (refereegranskat)abstract
- Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was >100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and proinflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate beta-glucans, whereas inflammation in transgenic and wild-type mice was mild and transient. Taken together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation. The Journal of Immunology, 2013, 190: 4175-4184.
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| 5. |
- Almyroudis, Nikolaos G., et al.
(författare)
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NETosis and NADPH oxidase : at the intersection of host defense, inflammation, and injury
- 2013
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 4
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Forskningsöversikt (refereegranskat)abstract
- Neutrophils are armed with both oxidant-dependent and -independent pathways for killing pathogens. Activation of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase constitutes an emergency response to infectious threat and results in the generation of antimicrobial reactive oxidants. In addition, NADPH oxidase activation in neutrophils is linked to activation of granular proteases and generation of neutrophil extracellular traps (NETs). NETosis involves the release of nuclear and granular components that can target extracellular pathogens. NETosis is activated during microbial threat and in certain conditions mimicking sepsis, and can result in both augmented host defense and inflammatory injury. In contrast, apoptosis, the physiological form of neutrophil death, not only leads to non-inflammatory cell death but also contributes to alleviate inflammation. Although there are significant gaps in knowledge regarding the specific contribution of NETs to host defense, we speculate that the coordinated activation of NADPH oxidase and NETosis maximizes microbial killing. Work in engineered mice and limited patient experience point to varying susceptibility of bacterial and fungal pathogens to NADPH oxidase versus NET constituents. Since reactive oxidants and NET constituents can injure host tissue, it is important that these pathways be tightly regulated. Recent work supports a role for NETosis in both acute lung injury and in autoimmunity. Knowledge gained about mechanisms that modulate NETosis may lead to novel therapeutic approaches to limit inflammation-associated injury.
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| 6. |
- Ginley, Brandon G, et al.
(författare)
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Computational detection and quantification of human and mouse neutrophil extracellular traps in flow cytometry and confocal microscopy
- 2017
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Neutrophil extracellular traps (NETs) are extracellular defense mechanisms used by neutrophils, where chromatin is expelled together with histones and granular/cytoplasmic proteins. They have become an immunology hotspot, implicated in infections, but also in a diverse array of diseases such as systemic lupus erythematosus, diabetes, and cancer. However, the precise assessment of in vivo relevance in different disease settings has been hampered by limited tools to quantify occurrence of extracellular traps in experimental models and human samples. To expedite progress towards improved quantitative tools, we have developed computational pipelines to identify extracellular traps from an in vitro human samples visualized using the ImageStream® platform (Millipore Sigma, Darmstadt, Germany), and confocal images of an in vivo mouse disease model of aspergillus fumigatus pneumonia. Our two in vitro methods, tested on n = 363/n =145 images respectively, achieved holdout sensitivity/specificity 0.98/0.93 and 1/0.92. Our unsupervised method for thin lung tissue sections in murine fungal pneumonia achieved sensitivity/specificity 0.99/0.98 in n = 14 images. Our supervised method for thin lung tissue classified NETs with sensitivity/specificity 0.86/0.90. We expect that our approach will be of value for researchers, and have application in infectious and inflammatory diseases.
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| 7. |
- Ginley, Brandon, et al.
(författare)
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Identification and characterization of neutrophil extracellular trap shapes in flow cytometry
- 2017
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Ingår i: Medical Imaging 2017. - : SPIE. - 9781510607255 - 9781510607262
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Konferensbidrag (refereegranskat)abstract
- Neutrophil extracellular trap (NET) formation is an alternate immunologic weapon used mainly by neutrophils. Chromatin backbones fused with proteins derived from granules are shot like projectiles onto foreign invaders. It is thought that this mechanism is highly anti-microbial, aids in preventing bacterial dissemination, is used to break down structures several sizes larger than neutrophils themselves, and may have several more uses yet unknown. NETs have been implied to be involved in a wide array of systemic host immune defenses, including sepsis, autoimmune diseases, and cancer. Existing methods used to visually quantify NETotic versus non-NETotic shapes are extremely time-consuming and subject to user bias. These limitations are obstacles to developing NETs as prognostic biomarkers and therapeutic targets. We propose an automated pipeline for quantitatively detecting neutrophil and NET shapes captured using a flow cytometry-imaging system. Our method uses contrast limited adaptive histogram equalization to improve signal intensity in dimly illuminated NETs. From the contrast improved image, fixed value thresholding is applied to convert the image to binary. Feature extraction is performed on the resulting binary image, by calculating region properties of the resulting foreground structures. Classification of the resulting features is performed using Support Vector Machine. Our method classifies NETs from neutrophils without traps at 0.97/0.96 sensitivity/specificity on n = 387 images, and is 1500X faster than manual classification, per sample. Our method can be extended to rapidly analyze whole-slide immunofluorescence tissue images for NET classification, and has potential to streamline the quantification of NETs for patients with diseases associated with cancer and autoimmunity.
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| 8. |
- Hosseinzadeh, Ava, et al.
(författare)
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Nicotine induces neutrophil extracellular traps
- 2016
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Ingår i: Journal of Leukocyte Biology. - 0741-5400 .- 1938-3673. ; 100:5, s. 1105-1112
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Tidskriftsartikel (refereegranskat)abstract
- NETs serve to ensnare and kill microbial pathogens. However, NETs can at the same time contribute to tissue damage and excessive inflammation. Nicotine is a major toxic agent and has been associated with exacerbated inflammatory diseases. The current study aimed at investigating the role of nicotine, the addictive component of tobacco and electronic cigarettes, on triggering NET formation. We report that nicotine induces neutrophils to release NETs in a dose-dependent manner. Nicotine-induced NET formation is mediated via nicotine acetylcholine receptors, depends on Akt and PAD4 activation, but is Nox2-independent, as demonstrated by pharmacological inhibition of Nox2 and by use of Nox2-deficient mouse neutrophils. These findings demonstrate that nicotine induces NETs, which may in turn contribute to smoking-related diseases.
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| 9. |
- Röhm, Marc, et al.
(författare)
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NADPH Oxidase Promotes Neutrophil Extracellular Trap Formation in Pulmonary Aspergillosis
- 2014
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 82:5, s. 1766-1777
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Tidskriftsartikel (refereegranskat)abstract
- NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wildtype and NADPH oxidase-deficient (p47(phox-/-)) mice which had resolved in wild-type mice by day 5 but progressed in p47(phox-/-) mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47(phox-/-) mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance.
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| 10. |
- Vethanayagam, R. Robert, et al.
(författare)
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Role of NADPH Oxidase versus Neutrophil Proteases in Antimicrobial Host Defense
- 2011
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Ingår i: PLOS ONE. - San Francisco : Public Library of Science. - 1932-6203. ; 6:12, s. e28149-
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Tidskriftsartikel (refereegranskat)abstract
- NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47(phox-/-)) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)(-/-) x cathepsin G (CG)(-/-) mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47(phox-/-) mice, whereas NE(-/-) x CG(-/-) mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens.
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