scholarly journals Effect of probiotics on the viability of porcine and human neutrophils in vitro

2017 ◽  
Vol 62 (No. 12) ◽  
pp. 637-646
Author(s):  
T. Sustrova ◽  
P. Ondrackova ◽  
L. Leva ◽  
M. Kolarova ◽  
P. Kulich ◽  
...  
Keyword(s):  
Tissue Regeneration ◽  
Lactobacillus Rhamnosus ◽  
Bifidobacterium Bifidum ◽  
Human Neutrophils ◽  
Neutrophil Apoptosis ◽  
Large White ◽  
Healthy Human ◽  
Lactate Dehydrogenase Activity ◽  
Late Apoptosis

Lactobacillus, Bifidobacterium and Enterococcus cultures are increasingly used as probiotics for humans and pigs. The aim of this study was to investigate if co-cultivation of porcine and human neutrophils with probiotics can lead to increased apoptosis in vitro. Ten adult Large white pigs and 10 healthy human donors were used in this study. Neutrophils were isolated by dextran sedimentation and cultivated with and without the lactic acid bacteria Bifidobacterium bifidum, Lactobacillus rhamnosus, and Enterococcus faecium for 2, 4, 24 and 48 h. Early and late apoptosis was measured using flow cytometry, and cell lysis was detected based on lactate dehydrogenase activity (LDH). A significant (P < 0.05; P < 0.01) increase in apoptotic neutrophils and LDH was observed at 24 h and 48 h in vitro. All probiotics exerted their greatest effects on the early apoptosis of porcine neutrophils, while the effects of L. rhamnosus were most pronounced on late apoptosis and those of B. bifidum on LDH release of human neutrophils. The increased neutrophil apoptosis caused by probiotic bacteria can be beneficial for more efficient efferocytosis and faster resolution of inflammation and tissue regeneration. In conclusion, we have demonstrated that the interaction of B. bifidum, L. rhamnosus, and E. faecium with human and porcine neutrophils leads to their apoptosis.

Involvement of a ferroprotein sensor in hypoxia-mediated inhibition of neutrophil apoptosis

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 3008-3016 ◽  
Author(s):  
Katy I. Mecklenburgh ◽  
Sarah R. Walmsley ◽  
Andrew S. Cowburn ◽  
Michael Wiesener ◽  
Benjamin J. Reed ◽  
...  
Keyword(s):  
Oxygen Sensing ◽  
Hypoxia Inducible Factor ◽  
Glucose Deprivation ◽  
Cell Types ◽  
Human Neutrophils ◽  
Neutrophil Apoptosis ◽  
Low Oxygen ◽  
Sensing Mechanism ◽  
Survival Effect

Neutrophil apoptosis represents a major mechanism involved in the resolution of acute inflammation. In contrast to the effect of hypoxia observed in many other cell types, oxygen deprivation, as we have shown, causes a profound but reversible delay in the rate of constitutive apoptosis in human neutrophils when aged in vitro. This effect was mimicked by exposing cells to 2 structurally unrelated iron-chelating agents, desferrioxamine (DFO) and hydroxypyridines (CP-94), and it appeared specific for hypoxia in that no modulation of apoptosis was observed with mitochondrial electron transport inhibitors, glucose deprivation, or heat shock. The involvement of chelatable iron in the oxygen-sensing mechanism was confirmed by the abolition of the DFO and CP-94 survival effect by Fe2+ ions. Although hypoxia inducible factor-1α (HIF-1α) mRNA was identified in freshly isolated neutrophils, HIF-1α protein was only detected in neutrophils incubated under hypoxic conditions or in the presence of DFO. Moreover, studies with cyclohexamide demonstrated that the survival effect of hypoxia was fully dependent on continuing protein synthesis. These results indicate that the neutrophil has a ferroprotein oxygen-sensing mechanism identical to that for erythropoietin regulation and results in HIF-1α up-regulation and profound but reversible inhibition of neutrophil apoptosis. This finding may have important implications for the resolution of granulocytic inflammation at sites of low-oxygen tension.

scholarly journals Flow Cytometric Evaluation of Human Neutrophil Apoptosis During Nitric Oxide Generation In Vitro: The Role of Exogenous Antioxidants

2005 ◽  
Vol 2005 (2) ◽  
pp. 81-87 ◽  
Author(s):  
Zofia Sulowska ◽  
Ewa Majewska ◽  
Magdalena Klink ◽  
Malgorzata Banasik ◽  
Henryk Tchórzewski
Keyword(s):  
Nitric Oxide ◽  
Human Neutrophil ◽  
Flow Cytometric Analysis ◽  
Human Neutrophils ◽  
Neutrophil Apoptosis ◽  
No Donor ◽  
No Donors ◽  
Flow Cytometric

Among numerous inflammatory mediators a nitric oxide molecule is supposed to be important in the modulation of neutrophil survival in vivo and in vitro. The effect of exogenous supply of NO donors such as SNP, SIN-1, and GEA-3162 on the course of human neutrophil apoptosis and the role of extracellular antioxidants in this process was investigated. Isolated from peripheral blood, neutrophils were cultured in the presence or absence of NO donor compounds and antioxidants for 8, 12, and20hours. Apoptosis of neutrophils was determined in vitro by flow cytometric analysis of cellular DNA content and Annexin V protein binding to the cell surface. Exposure of human neutrophils to GEA-3162 and SIN-1 significantly accelerates and enhances their apoptosis in vitro in a time-dependent fashion. In the presence of SNP, intensification of apoptosis has not been revealed until12hours after the culture. The inhibition of GEA-3162- and SIN-1-mediated neutrophil apoptosis by superoxide dismutase (SOD) but not by catalase (CAT) was observed. Our results show that SOD and CAT can protect neutrophils against NO-donors-induced apoptosis and suggest that the interaction of NO and oxygen metabolites signals may determine the destructive or protective role of NO donor compounds during apoptotic neutrophil death.

scholarly journals Regulation of Neutrophil Apoptosis by Tumor Necrosis Factor-α: Requirement for TNFR55 and TNFR75 for Induction of Apoptosis In Vitro

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2772-2783 ◽  
Author(s):  
Joanna Murray ◽  
Jeffrey A.J. Barbara ◽  
Sarah A. Dunkley ◽  
Angel F. Lopez ◽  
Xaveer Van Ostade ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Α ◽  
Human Neutrophils ◽  
Receptor Subtypes ◽  
Neutrophil Apoptosis ◽  
Tnf Α ◽  
Factor Α ◽  
Necrosis Factor

Abstract Granulocyte apoptosis is an important mechanism underlying the removal of redundant neutrophils from an inflammatory focus. The ability of many proinflammatory agents to impede this event suggests that such agents act not only in a priming or secretagogue capacity but also increase neutrophil longevity by delaying apoptosis. We have examined whether this hypothesis holds true for all neutrophil priming agents, in particular tumor necrosis factor-α (TNF-α), which has been variably reported to either induce, delay, or have no effect on neutrophil apoptosis. After 20 hours coincubation TNF-α inhibited neutrophil apoptosis; however, more detailed analysis demonstrated its ability to promote apoptosis in a subpopulation of cells at earlier (2 to 8 hours) times. Formyl-Met-Leu-Phe, platelet-activating factor, inositol hexakisphosphate, lipopolysaccharide, leukotriene B4 , and granulocyte-macrophage colony-stimulating factor all inhibited apoptosis at 6 and 20 hours. The early proapoptotic effect of TNF-α was concentration-dependent (EC50 2.8 ng/mL), abolished by TNF-α neutralizing antibody, and was not associated with any change in cell viability or recovery. Of relevance to the inflamed site, the ability of TNF-α to accelerate apoptosis was lost if neutrophils were primed with 1 μmol/L PAF or aged for 6 hours before TNF-α addition. The TNFR55-selective TNF-α mutants (E146K, R32W-S86T) induced neutrophil apoptosis but with a potency 14-fold lower than wild-type TNF-α. Although the TNFR75-selective mutant (D143F ) did not induce apoptosis, blocking antibodies to both receptor subtypes abolished TNF-α–stimulated apoptosis. Hence, TNF-α has the unique ability to induce apoptosis in human neutrophils via a mechanism where TNFR75 facilitates the dominant TNFR55 death effect. This may be an important mechanism controlling neutrophil longevity and clearance in vivo.

scholarly journals Regulation of Neutrophil Apoptosis by Tumor Necrosis Factor-α: Requirement for TNFR55 and TNFR75 for Induction of Apoptosis In Vitro

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2772-2783 ◽  
Author(s):  
Joanna Murray ◽  
Jeffrey A.J. Barbara ◽  
Sarah A. Dunkley ◽  
Angel F. Lopez ◽  
Xaveer Van Ostade ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Α ◽  
Human Neutrophils ◽  
Receptor Subtypes ◽  
Neutrophil Apoptosis ◽  
Tnf Α ◽  
Factor Α ◽  
Necrosis Factor

Granulocyte apoptosis is an important mechanism underlying the removal of redundant neutrophils from an inflammatory focus. The ability of many proinflammatory agents to impede this event suggests that such agents act not only in a priming or secretagogue capacity but also increase neutrophil longevity by delaying apoptosis. We have examined whether this hypothesis holds true for all neutrophil priming agents, in particular tumor necrosis factor-α (TNF-α), which has been variably reported to either induce, delay, or have no effect on neutrophil apoptosis. After 20 hours coincubation TNF-α inhibited neutrophil apoptosis; however, more detailed analysis demonstrated its ability to promote apoptosis in a subpopulation of cells at earlier (2 to 8 hours) times. Formyl-Met-Leu-Phe, platelet-activating factor, inositol hexakisphosphate, lipopolysaccharide, leukotriene B4 , and granulocyte-macrophage colony-stimulating factor all inhibited apoptosis at 6 and 20 hours. The early proapoptotic effect of TNF-α was concentration-dependent (EC50 2.8 ng/mL), abolished by TNF-α neutralizing antibody, and was not associated with any change in cell viability or recovery. Of relevance to the inflamed site, the ability of TNF-α to accelerate apoptosis was lost if neutrophils were primed with 1 μmol/L PAF or aged for 6 hours before TNF-α addition. The TNFR55-selective TNF-α mutants (E146K, R32W-S86T) induced neutrophil apoptosis but with a potency 14-fold lower than wild-type TNF-α. Although the TNFR75-selective mutant (D143F ) did not induce apoptosis, blocking antibodies to both receptor subtypes abolished TNF-α–stimulated apoptosis. Hence, TNF-α has the unique ability to induce apoptosis in human neutrophils via a mechanism where TNFR75 facilitates the dominant TNFR55 death effect. This may be an important mechanism controlling neutrophil longevity and clearance in vivo.

scholarly journals p38-MAPK Signals Survival by Phosphorylation of Caspase-8 and Caspase-3 in Human Neutrophils

2004 ◽  
Vol 199 (4) ◽  
pp. 449-458 ◽  
Author(s):  
Maria Alvarado-Kristensson ◽  
Fredrik Melander ◽  
Karin Leandersson ◽  
Lars Rönnstrand ◽  
Christer Wernstedt ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Caspase 3 ◽  
Mitogen Activated Protein Kinase ◽  
Human Neutrophils ◽  
Caspase 8 ◽  
Neutrophil Apoptosis ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Phosphopeptide Mapping

Neutrophil apoptosis occurs both in the bloodstream and in the tissue and is considered essential for the resolution of an inflammatory process. Here, we show that p38–mitogen-activated protein kinase (MAPK) associates to caspase-8 and caspase-3 during neutrophil apoptosis and that p38-MAPK activity, previously shown to be a survival signal in these primary cells, correlates with the levels of caspase-8 and caspase-3 phosphorylation. In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3. Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively. Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition. Consequently, p38-MAPK can directly phosphorylate and inhibit the activities of caspase-8 and caspase-3 and thereby hinder neutrophil apoptosis, and, in so doing, regulate the inflammatory response.

scholarly journals Mesenchymal Stem Cell-Conditioned Medium Induces Neutrophil Apoptosis Associated with Inhibition of the NF-κB Pathway in Endotoxin-Induced Acute Lung Injury

2019 ◽  
Vol 20 (9) ◽  
pp. 2208 ◽  
Author(s):  
Vincent Yi-Fong Su ◽  
Chi-Shiuan Lin ◽  
Shih-Chieh Hung ◽  
Kuang-Yao Yang
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Conditioned Medium ◽  
Mouse Lung ◽  
Human Neutrophils ◽  
Neutrophil Apoptosis ◽  
Neutrophil Accumulation ◽  
Mobility Shift

The immunomodulatory effects of mesenchymal stem cells (MSCs) are established. However, the effects of MSCs on neutrophil survival in acute lung injury (ALI) remain unclear. The goal of this study was to investigate the effect of an MSC-conditioned medium (MSC-CM) on neutrophil apoptosis in endotoxin-induced ALI. In this study, an MSC-CM was delivered via tail vein injection to wild-type male C57BL/6 mice 4 h after an intratracheal injection of lipopolysaccharide (LPS). Twenty-four hours later, bronchoalveolar lavage fluid (BALF) and lung tissue were collected to perform histology, immunohistochemistry, apoptosis assay of neutrophil, enzyme-linked immunosorbent assays, and an electrophoretic mobility shift assay. Human neutrophils were also collected from patients with sepsis-induced acute respiratory distress syndrome (ARDS). Human neutrophils were treated in vitro with LPS, with or without subsequent MSC-CM co-treatment, and were then analyzed. Administration of the MSC-CM resulted in a significant attenuation of histopathological changes, the levels of interleukin-6 and macrophage inflammatory protein 2, and neutrophil accumulation in mouse lung tissues of LPS-induced ALI. Additionally, MSC-CM therapy enhanced the apoptosis of BALF neutrophils and reduced the expression of the anti-apoptotic molecules, Bcl-xL and Mcl-1, both in vivo and in vitro experiments. Furthermore, phosphorylated and total levels of nuclear factor (NF)-κB p65 were reduced in lung tissues from LPS + MSC-CM mice. Human MSC-CM also reduced the activity levels of NF-κB and matrix metalloproteinase-9 in the human neutrophils from ARDS patients. Thus, the results of this study suggest that the MSC-CM attenuated LPS-induced ALI by inducing neutrophil apoptosis, associated with inhibition of the NF-κB pathway.

scholarly journals Lactobacillus Rhamnosus GG and Bifidobacterium Bifidum TMC3115 Can Affect the Development of Hippocampal Neurons Cultured in Vitro in a Strain-dependent Manner (P20-009-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Ruyue Cheng ◽  
Fang He ◽  
Yugang Jiang
Keyword(s):  
Hippocampal Neurons ◽  
Lactobacillus Rhamnosus ◽  
Bifidobacterium Bifidum ◽  
Neuronal Morphology ◽  
Dependent Manner ◽  
Lactobacillus Rhamnosus Gg ◽  
Creb Protein ◽  
Protein Levels ◽  
Strain Dependent

Abstract Objectives This study aimed to examine whether probiotics could morphologically or physiologically influence hippocampal neuron development. Methods Hippocampal neurons cultured in vitro were exposed to live or heat-inactivated Lactobacillus rhamnosus GG (LGG), or live or heat-inactivated Bifidobacterium bifidum TMC3115 (TMC3115), for either 6 or 24 h. Neuron viability was then tested using the methyl thiazolyl tetrazolium assay. Neuronal morphological changes and drebrin (DRB) and synaptophysin (SYP) protein levels were monitored using immunofluorescence. And the levels of DRB, SYP, and brain-derived neurotrophic factor (BDNF), and cAMP-response element binding protein (CREB) mRNA were detected using RT-PCR. The BDNF, CREB and phosphorylated-CREB (P-CREB) protein levels were detected by ELISA or Western blot assays. Results We found exposure to probiotics could enhance neuron viability, although no significant differences were found in neuronal morphology among the groups following exposure to the test bacteria. However, the synapse development-related proteins, DRB and SYP, as well as BDNF and P-CREB protein levels, were significantly altered in this specific culture system. Conclusions These results demonstrated that LGG and TMC3115 exposure can affect neuronal viability, along with synaptic and brain function development, in a strain-dependent manner, which may also be closely associated with the physiological and cultural conditions of each strain, The up-regulated P-CREB protein level may be one of the underlying mechanisms by which the tested bacteria, especially live TMC3115 following exposure for 24 h, are able to regulate neuronal BDNF protein production. Further studies are needed to explore other possible effects probiotic exposure may have on hippocampal neurons, as well as the corresponding mechanisms that underlie them. Funding Sources This work was funded by the National Natural Science Foundation of China (Grant number 81872606).

Functional responses of human neutrophils to sodium sulfite (Na2SO3) in vitro

1998 ◽  
Vol 17 (11) ◽  
pp. 600-605 ◽  
Author(s):  
Pierrette Labbé ◽  
Martin Pelletier ◽  
Felix O Omara ◽  
Denis Girard
Keyword(s):  
Gene Expression ◽  
Sodium Sulfite ◽  
Pulmonary Inflammation ◽  
Superoxide Production ◽  
Human Neutrophils ◽  
Neutrophil Apoptosis ◽  
Dependent Manner ◽  
Functional Responses ◽  
Concentration Dependent Manner

An influx of neutrophils into the airways is a common feature observed during pulmonary inflammation induced by air pollutants, including sulfur dioxide and sulfates. In the present study focusing on the in vitro interactions of sodium sulfite (Na2SO3) with human neutrophils, we confirm results indicating that this sulfite induces superoxide production (O27) by itself. We demonstrated that this response can occur more rapidly than previously reported (within 5 min), and that Na2SO3 can act as a priming agent, in a concentration-dependent fashion, to the bacterial tripeptide N-formyl-methionineleucine-phenylalanine (fMLP) by increasing O27 production. In addition, our results show that Na2SO3 induces gene expression in human neutrophils in a concentration-dependent manner as assessed by incorporation of 5-[3H] uridine into total RNA. However, it does not induce cell shape changes. We also demonstrated that Na2SO3 does not modulate neutrophil apoptosis nor reverse the well-known delaying effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on apoptosis. We conclude that Na2SO3 acts rapidly on neutrophil physiology, within a few minutes with respect to superoxide production, and a few hours (4 h) with respect to gene expression without altering a biological process such as the rate of apoptosis evaluated after a long period of incubation (20 h). We further conclude that Na2SO3-induced production of O27 does not drive neutrophils to undergo apoptosis, a mechanism known to occur in other conditions. Therefore, the potential toxicity of Na2SO3 during pulmonary inflammation or lung-associated diseases may be related to its ability to induce superoxide production without altering neutrophil apoptosis rate.

scholarly journals Pseudomonas expression of an oxygen sensing prolyl hydroxylase homologue regulates neutrophil host responses in vitro and in vivo

2017 ◽  
Vol 2 ◽  
pp. 104 ◽  
Author(s):  
Rebecca S. Dickinson ◽  
Fiona Murphy ◽  
Catherine Doherty ◽  
Sam Williams ◽  
Ananda Mirchandani ◽  
...  
Keyword(s):  
Active Sites ◽  
Hypoxia Inducible Factor ◽  
Elongation Factor ◽  
Prolyl Hydroxylase ◽  
Human Neutrophils ◽  
Neutrophil Apoptosis ◽  
Wild Type ◽  
Prolyl Hydroxylases

Background: Pseudomonas species are adapted to evade innate immune responses and can persist at sites of relative tissue hypoxia, including the mucus-plugged airways of patients with cystic fibrosis and bronchiectasis.  The ability of these bacteria to directly sense and respond to changes in local oxygen availability is in part consequent upon expression of the 2-oxoglutarate oxygenase, Pseudomonas prolyl hydroxylase (PPHD), which acts on elongation factor Tu (EF-Tu), and is homologous with the human hypoxia inducible factor (HIF) prolyl hydroxylases. We report that PPHD expression regulates the neutrophil response to acute pseudomonal infection. Methods: In vitro co-culture experiments were performed with human neutrophils and PPHD-deficient and wild-type bacteria and supernatants, with viable neutrophil counts determined by flow cytometry. In vivo consequences of infection with PPHD deficient P. aeruginosa were determined in an acute pneumonia mouse model following intra-tracheal challenge. Results: Supernatants of PPHD-deficient bacterial cultures contained higher concentrations of the phenazine exotoxin pyocyanin and induced greater acceleration of neutrophil apoptosis than wild-type PAO1 supernatants in vitro.  In vivo infection with PPHD mutants compared to wild-type PAO1 controls resulted in increased levels of neutrophil apoptosis and impaired control of infection, with higher numbers of P. aeruginosa recovered from the lungs of mice infected with the PPHD-deficient strain.  This resulted in an overall increase in mortality in mice infected with the PPHD-deficient strain. Conclusions: Our data show that Pseudomonas expression of its prolyl hydroxylase influences the outcome of host-pathogen interactions in vitro and in vivo, demonstrating the importance of considering how both host and pathogen adaptations to hypoxia together define outcomes of infection. Given that inhibitors for the HIF prolyl hydroxylases are in late stage trials for the treatment of anaemia and that the active sites of PPHD and human HIF prolyl hydroxylases are closely related, the results are of current clinical interest.

Sign in / Sign up

Export Citation Format

Share Document