Anticoagulants impact on innate immune responses and bacterial survival in whole blood models of Neisseria meningitidis infection

Strobel, Lea; Johswich, Kay

doi:10.1038/s41598-018-28583-8

Cited by 32 publications

(51 citation statements)

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“…Limitations of the present study stem from the use of heparinized blood samples and plasma storage at −20°C. Heparin has been shown to partially inhibit complement activity at concentrations of 20 USP/ml and higher (Strobel & Johswich, 2018). However, heparin is the preferred anticoagulant for most chelonians, including box turtles, due to the occurrence of hemolysis in whole blood samples anticoagulated with EDTA (Heatley & Russell, 2010; Muro, Cuenca, Pastor, Vinas, & Lavin, 1998).…”

Section: Discussionmentioning

confidence: 99%

“…While our findings may be biased by the use of heparinized samples, they are comparable to the existing chelonian innate immune function literature, which also relies heavily upon this anticoagulant. Other anticoagulants are also problematic for innate immune function testing, for example, EDTA and citrate‐based anticoagulants inhibit complement activation, and can decrease the survival of bacteria used in bacterial killing assays (Strobel & Johswich, 2018). Hirudin‐based anticoagulants are preferred in mammalian complement studies due to minimal impacts on complement activity and bacterial survival (Strobel & Johswich, 2018).…”

Section: Discussionmentioning

confidence: 99%

“…Other anticoagulants are also problematic for innate immune function testing, for example, EDTA and citrate‐based anticoagulants inhibit complement activation, and can decrease the survival of bacteria used in bacterial killing assays (Strobel & Johswich, 2018). Hirudin‐based anticoagulants are preferred in mammalian complement studies due to minimal impacts on complement activity and bacterial survival (Strobel & Johswich, 2018). However, these anticoagulants have not yet been investigated in chelonians, and the suitability of hirudin for inhibiting clot formation and preserving complement function in this taxon is unknown.…”

Section: Discussionmentioning

confidence: 99%

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Plasma antibacterial activities in ornate (Terrapene ornata) and eastern box turtles (Terrapene carolina)

et al. 2020

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Chelonians are one of the most imperiled vertebrate taxa and many species are increasingly threatened by disease, however, the immune response in this group is understudied. We quantified the innate immune response of eastern (Terrapene carolina; EBT) and ornate (Terrapene ornate; OBT) box turtles using plasma antibacterial activity assays. Plasma from both species abolished or significantly reduced the growth of all eight bacterial species evaluated, including Salmonella typhimurium, Escherichia coli, Enterobacter cloacae, Citrobacter freundi, Bacillus subtilis, Staphylococcus epidermidis, and Staphylococcus aureus. Bactericidal capacity was greater in OBT compared to EBT, and OBT plasma retained high antibacterial activities at a broader temperature range (20-40°C) compared to EBT (30-40°C). Plasma antibacterial activity was abolished following treatment with heat, protease, and ethylenediaminetetraacetic acid, indicating that complement is likely responsible for the observed effects. Further characterization of the box turtle immune response may provide insight into the importance of infectious diseases for species conservation, enabling the development of more efficient and effective population management strategies. K E Y W O R D S box turtle, chelonian, innate immunity, reptile, Terrapene

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Plasma antibacterial activities in ornate (Terrapene ornata) and eastern box turtles (Terrapene carolina)

et al. 2020

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“…In fact, unbridled neutrophil activation is deemed detrimental during sepsis, and this has been particularly attributed to the C5a/C5aR1 axis [17,48]. In order to determine differences in neutrophil responses in the ATRdeficient mouse strains, we used a hirudin-based whole blood model for ex vivo infection, as this anticoagulant does not interfere with complement activation or Nme growth [49,50]. Upon infection with Nme, the neutrophils showed an oxidative burst response (Figure 3(a)), degranulation ( Figure 3(b)), and they engulfed GFP-expressing Nme (Figure 3(c)).…”

Section: C3ar C5ar1 and C5ar2 Impact Similarly On Cytokine Response mentioning

confidence: 99%

“…Mice were euthanized by CO 2 inhalation and whole blood was drawn via cardiac puncture using hirudin monovettes (525 antithrombin units/ml; Sarstedt). Hirudin was chosen as anticoagulant since it does not interfere with complement or with Nme viability [50]. Mouse whole blood samples were infected with 10 7 CFU/ml of Nme; 100 nM PMA (Sigma Aldrich/ Merck) served as a positive control, whereas PBS was used as a negative control.…”

Section: Nme Sepsis Experiments Using Antagonists and Agonistsmentioning

confidence: 99%

Distinct roles of the anaphylatoxin receptors C3aR, C5aR1 and C5aR2 in experimental meningococcal infections

et al. 2019

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The complement system is pivotal in the defense against invasive disease caused by Neisseria meningitidis (Nme, meningococcus), particularly via the membrane attack complex. Complement activation liberates the anaphylatoxins C3a and C5a, which activate three distinct G-protein coupled receptors, C3aR, C5aR1 and C5aR2 (anaphylatoxin receptors, ATRs). We recently discovered that C5aR1 exacerbates the course of the disease, revealing a downside of complement in Nme sepsis. Here, we compared the roles of all three ATRs during mouse nasal colonization, intraperitoneal infection and human whole blood infection with Nme. Deficiency of complement or ATRs did not alter nasal colonization, but significantly affected invasive disease: Compared to WT mice, the disease was aggravated in C3ar −/mice, whereas C5ar1 −/and C5ar2 −/mice showed increased resistance to meningococcal sepsis. Surprisingly, deletion of either of the ATRs resulted in lower cytokine/chemokine responses, irrespective of the different susceptibilities of the mice. This was similar in ex vivo human whole blood infection using ATR inhibitors. Neutrophil responses to Nme were reduced in C5ar1 −/mouse blood. Upon stimulation with C5a plus Nme, mouse macrophages displayed reduced phosphorylation of ERK1/2, when C5aR1 or C5aR2 were ablated or inhibited, suggesting that both C5a-receptors prime an initial macrophage response to Nme. Finally, in vivo blockade of C5aR1 alone (PMX205) or along with C5aR2 (A8 Δ71−73) resulted in ameliorated disease, whereas neither antagonizing C3aR (SB290157) nor its activation with a "super-agonist" peptide (WWGKKYRASKLGLAR) demonstrated a benefit. Thus, C5aR1 and C5aR2 augment disease pathology and are interesting targets for treatment, whereas C3aR is protective in experimental meningococcal sepsis.

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A Two‐Stage Flow Cytometry Strategy to Distinguish Single Cells from Doublets in Heterogeneous Cell Mixtures and Improve Cell Cluster Identification: Application to Human Monocyte Subpopulations

2021

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Flow cytometry is a powerful method, widely used to identify cell types present in tissues, to describe their phenotypes, and to purify cells for functional analyses. As a single cell technique, flow cytometry relies on identifying and excluding cell doublets and aggregates present in samples in the initial gating steps. This identification is based on detection of events generating electrical pulses falling outside of linear variations of pulse height, width, and area in a singlet population with increasing cell sizes. In heterogeneous cell mixtures, however, with cell types varying extensively in size and granularity, exclusion of doublets has the risk of removing single cells that co‐localize with doublets of another cell type. This is particularly the case when doublets of a smaller cell type overlap with large cells of a distinct, larger cell type. Here, we describe a gating method to reduce this risk. In this protocol, initial gating steps aim to segregate cells according to physical characteristics (such as size and granularity) and gene expression properties in order to obtain more homogeneous cell clusters. Doublet exclusion is then performed separately in each cluster, minimizing the risk of confusion between single cells and doublets. To illustrate this protocol, human blood monocytes are separated and analyzed. By implementing this protocol, we were able to reveal the existence of a population of large monocytes previously unrecognized using conventional gating strategies. In subsequent functional assays, we have shown that this novel population exhibits unique inflammatory responses, highlighting the need and pertinence of this approach to identify and characterize infrequent—yet functionally relevant—cell populations present in complex cell mixtures. © 2021 Wiley Periodicals LLC. Basic Protocol: Distinguishing single cells from doublets in heterogeneous cell mixtures by flow cytometry

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Anticoagulants impact on innate immune responses and bacterial survival in whole blood models of Neisseria meningitidis infection

Cited by 32 publications

References 59 publications

Plasma antibacterial activities in ornate (Terrapene ornata) and eastern box turtles (Terrapene carolina)

Plasma antibacterial activities in ornate (Terrapene ornata) and eastern box turtles (Terrapene carolina)

Distinct roles of the anaphylatoxin receptors C3aR, C5aR1 and C5aR2 in experimental meningococcal infections

A Two‐Stage Flow Cytometry Strategy to Distinguish Single Cells from Doublets in Heterogeneous Cell Mixtures and Improve Cell Cluster Identification: Application to Human Monocyte Subpopulations

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