Use of Flow Cytometry to Evaluate Phagocytosis of Staphylococcus aureus by Human Neutrophils

Boero, Elena; Brinkman, Iris; Juliet, Thessely; Yperen, Eline van; Strijp, Jos A. G. van; Rooijakkers, Suzan H. M.; Kessel, Kok P. M. van

doi:10.3389/fimmu.2021.635825

Cited by 44 publications

(38 citation statements)

References 38 publications

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“…The bacterial strain used in this study was the widely used laboratory strain S. aureus Newman (GenBank accession number AP009351.1 ), the respective green fluorescent protein (GFP)-expressing strain ( Boero et al, 2021 ) and the methicillin-resistant S. aureus (MRSA) strain USA300 ( Berends et al, 2010 ).…”

Section: Methodsmentioning

confidence: 99%

Staphylococcus aureus Infection Influences the Function of Intestinal Cells by Altering the Lipid Raft-Dependent Sorting of Sucrase–Isomaltase

Mergani

Wanes²,

Schecker³

et al. 2021

Front. Cell Dev. Biol.

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Staphylococcus aureus is an important nosocomial and community-acquired facultative intracellular pathogen. Many studies have reported that S. aureus infections are associated with intestinal symptoms, but little is known about the molecular mechanisms implicated in S. aureus-induced alterations of intestinal functions. In this study, we investigated the implication of lipid rafts in the interaction of S. aureus with Caco-2 cells. To assess potential alterations in the lipid raft structure and effects on the hydrolytic function, we utilized sucrase–isomaltase (SI) as the major intestinal α-glucosidase that is associated with and sorted to the apical membrane via lipid rafts. Seven days post-confluent, Caco-2 cells were infected with S. aureus Newman and further incubated for an additional 2 days. After 48 h, the levels of SI expression as well as the enzymatic function of this protein were assessed in the infected versus non-infected cells. Analysis of the sorting behavior of SI to the apical membrane constituted another crucial aspect in studying the effects of S. aureus on Caco-2 cells. For this purpose, the apical membranes or brush border membranes (BBMs; referred to as P2 fraction) were separated in both infected and non-infected cells from the basolateral and intracellular membranes (referred to as P1 fraction) by employing a cationic-based procedure using CaCl2. The data show that there is no significant change in the overall expression levels of SI in the infected versus non-infected cells as assessed by Western blotting analysis using monoclonal anti-SI antibodies. By contrast, a significant decrease in the localization as well as the specific hydrolytic activities of SI toward sucrose and isomaltose (Palatinose) was observed in the BBM (P2 fraction) in Caco-2 cells 48 h post-infection. Concomitantly, the specific SI activities increased in the basolateral membrane/intracellular fraction (P1). Noteworthy, the specific activity of SI in the BBM of infected cells was markedly reduced as compared with that of the non-infected counterparts. The data accumulated from this study strongly suggest that infections with S. aureus influence the final step in the lipid raft-associated trafficking of human SI and thereby may trigger secondary functional gastrointestinal disorders.

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Section: Methodsmentioning

confidence: 99%

Staphylococcus aureus Infection Influences the Function of Intestinal Cells by Altering the Lipid Raft-Dependent Sorting of Sucrase–Isomaltase

Mergani

Wanes²,

Schecker³

et al. 2021

Front. Cell Dev. Biol.

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“…Flow cytometry has become an essential instrument for biomedical research and routine clinical detection of disease diagnosis, prognosis, and treatment monitoring 16 . Recently, the detection of neutrophil phagocytosis by flow cytometry has been proved to be an efficient and objective method 10,17 . Fluorescein isothiocyanate (FITC) or propidium iodide (PI) was used to label inactivated pathogens 18 .…”

Section: Discussionmentioning

confidence: 99%

“… 16 Recently, the detection of neutrophil phagocytosis by flow cytometry has been proved to be an efficient and objective method. 10 , 17 Fluorescein isothiocyanate (FITC) or propidium iodide (PI) was used to label inactivated pathogens. 18 Such methods will change the original biological characteristics of the pathogens and could not truly reflect the interaction between neutrophils and pathogens.…”

Section: Discussionmentioning

confidence: 99%

“…Flow cytometry is a high‐throughput method that can measure tens of thousands of cells in a short time. With the widespread use of flow cytometry, scholars have made a series of new works and attempt to determine the phagocytosis of neutrophils, but they have not established a normal range that can evaluate the phagocytic function 8‐10 . In this study, we developed a simple method to quantify the neutrophil phagocytic function by using whole blood.…”

Section: Introductionmentioning

confidence: 99%

“…With the widespread use of flow cytometry, scholars have made a series of new works and attempt to determine the phagocytosis of neutrophils, but they have not established a normal range that can evaluate the phagocytic function. 8 , 9 , 10 In this study, we developed a simple method to quantify the neutrophil phagocytic function by using whole blood. Furthermore, we established the RIs of neutrophil phagocytosis in healthy adults.…”

Section: Introductionmentioning

confidence: 99%

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Establishment of the reference intervals of whole blood neutrophil phagocytosis by flow cytometry

Yang

Zhang

Yang

et al. 2021

Clinical Laboratory Analysis

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Objective To investigate the reference intervals (RIs) of the whole blood neutrophil phagocytosis by flow cytometry (FCM) and to study the application value of neutrophil phagocytosis in infectious diseases. Methods Pathogens (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923) cultured for 18–24 h were labeled by fluorescence probe carboxyfluorescein diacetate succinimidyl ester (CFDA‐SE), and then incubated with whole blood at 37℃. The phagocytosis of pathogens by neutrophils was detected by flow cytometry, and a reference interval was established. Results In the healthy adults, the reference interval for the neutrophil phagocytosis to Escherichia coli was 46.91%–83.09% and to Staphylococcus aureus was 33.92%–69.48%. This method showed good reproducibility. Neutrophil phagocytosis was negatively correlated with the neutrophil count, neutrophil percentage, and neutrophil‐to‐lymphocyte ratio (NLR, p < 0.05). Conclusion We have successfully established the RIs of neutrophil phagocytosis in whole blood in healthy adults by flow cytometry (FCM), which might be of important clinical value in the diagnosis, treatment, and prognosis of infectious diseases.

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Antibacterial CATH‐2 Peptide Coating to Prevent Bone Implant‐Related Infection

Keikhosravani

Jahanmard

Bollen

et al. 2023

Adv Materials Technologies

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The development of antibacterial coatings is a promising approach to preventing biofilm formation and reducing the overuse of systemic antibiotics. However, widespread antibiotic use has resulted in antibiotic‐resistant bacteria, limiting the efficacy of antibiotic‐based coatings. Herein, an antibacterial coating is developed by layer‐by‐layer (LbL) assembly of two polymers namely PDLG (poly (D,L‐lactide‐co‐glycolide)) and gelatin methacryloyl (GelMA) while chicken cathelicidin‐2 (CATH‐2), a cationic and amphipathic peptide, is loaded between these polymer layers. The electrospray method is used to apply the coatings to achieve efficient peptide loading and durability. The CATH‐2 bactericidal concentration ranges are first identified, followed by a study of their cytotoxicity to human mesenchymal stem cells (hMSCs) and macrophage cell lines. Later, different LbL electrospray coating assemblies loaded with the optimal peptide concentration are sought. Various coating strategies are investigated to identify an LbL coating that exhibits prolonged and biocompatible CATH‐2 release. The resulting CATH‐2‐coated titanium surfaces exhibit strong antibacterial activity against both Staphylococcus aureus and Escherichia coli bacteria for 4 days and are biocompatible with hMSCs and macrophage cells. This coating can be considered as a versatile delivery system platform for the delivery of CATH‐2 peptides while avoiding cytotoxicity, particularly for the prevention of infections associated with implants.

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Use of Flow Cytometry to Evaluate Phagocytosis of Staphylococcus aureus by Human Neutrophils

Cited by 44 publications

References 38 publications

Staphylococcus aureus Infection Influences the Function of Intestinal Cells by Altering the Lipid Raft-Dependent Sorting of Sucrase–Isomaltase

Staphylococcus aureus Infection Influences the Function of Intestinal Cells by Altering the Lipid Raft-Dependent Sorting of Sucrase–Isomaltase

Establishment of the reference intervals of whole blood neutrophil phagocytosis by flow cytometry

Antibacterial CATH‐2 Peptide Coating to Prevent Bone Implant‐Related Infection

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