Neutrophil Isolation From Nonhuman Species

Siemsen, Daniel W.; Schepetkin, Igor A.; Kirpotina, Liliya N.; Lei, Benfang; Quinn, Mark T.

doi:10.1007/978-1-59745-467-4_3

Cited by 62 publications

(46 citation statements)

References 14 publications

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“…Blood was collected from healthy horses during the slaughter process (and immediately upon death) in tubes containing 3 mM EDTA anticoagulant. Equine neutrophils were isolated using 70 and 85% Percoll gradients as described44.…”

Section: Methodsmentioning

confidence: 99%

Identification of LukPQ, a novel, equid-adapted leukocidin of Staphylococcus aureus

Koop

Vrieling

Storisteanu

et al. 2017

Sci Rep

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Bicomponent pore-forming leukocidins are a family of potent toxins secreted by Staphylococcus aureus, which target white blood cells preferentially and consist of an S- and an F-component. The S-component recognizes a receptor on the host cell, enabling high-affinity binding to the cell surface, after which the toxins form a pore that penetrates the cell lipid bilayer. Until now, six different leukocidins have been described, some of which are host and cell specific. Here, we identify and characterise a novel S. aureus leukocidin; LukPQ. LukPQ is encoded on a 45 kb prophage (ΦSaeq1) found in six different clonal lineages, almost exclusively in strains cultured from equids. We show that LukPQ is a potent and specific killer of equine neutrophils and identify equine-CXCRA and CXCR2 as its target receptors. Although the S-component (LukP) is highly similar to the S-component of LukED, the species specificity of LukPQ and LukED differs. By forming non-canonical toxin pairs, we identify that the F-component contributes to the observed host tropism of LukPQ, thereby challenging the current paradigm that leukocidin specificity is driven solely by the S-component.

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Section: Methodsmentioning

confidence: 99%

Identification of LukPQ, a novel, equid-adapted leukocidin of Staphylococcus aureus

Koop

Vrieling

Storisteanu

et al. 2017

Sci Rep

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“…Erythrocytes were removed by dextran sedimentation and PMNs were separated from peripheral blood mononuclear cells using Histopaque-1077® (Sigma Aldrich) followed by water lysis of remaining erythrocytes under endotoxin-free conditions (<25.0 pg/ml) as described [11]. Bone marrow-derived mouse neutrophils were isolated from BALB/c mice using a Percoll gradient and resuspended in RPMI + 5 mM HEPES media [12]. PMN phagocytosis was synchronized by centrifuging PMNs and bacteria at 380 × g for 8 minutes at 4°C [11].…”

Section: Methodsmentioning

confidence: 99%

The Role of Innate Immunity in Promoting SaeR/S-Mediated Virulence in Staphylococcus aureus

et al. 2013

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The ability of Staphylococcus aureus to infect tissues is dependent on precise control of virulence through gene-regulatory systems. While the SaeR/S two-component system has been shown to be a major regulator of S. aureus virulence, the influence of the host environment on SaeR/S-regulated genes (saeR/S targets) remains incompletely defined. Using QuantiGene 2.0 transcriptional assays, we examined expression of genes with the SaeR binding site in USA300 exposed to human and mouse neutrophils and host-derived peptides and during subcutaneous skin infection. We found that only some of the saeR/S targets, as opposed to the entire SaeR/S virulon, were activated within 5 and 10 min of interacting with human neutrophils as well as α-defensin. Furthermore, mouse neutrophils promoted transcription of saeR/S targets despite lacking α-defensin, and the murine skin environment elicited a distinctive expression profile of saeR/S targets. These findings indicate that saeR/S-mediated transcription is unique to and dependent on specific host stimuli. By using isogenic USA300ΔsaeR/S and USA300Δagr knockout strains, we also determined that SaeR/S is the major regulator of virulence factors, while Agr, a quorum-sensing two-component system, has moderate influence on transcription of the saeR/S targets under the tested physiological conditions.

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“…Neutrophils were isolated from the blood using dextran sedimentation, followed by Histopaque 1077 gradient separation and hypotonic lysis of red blood cells, as described previously [57]. Isolated neutrophils were washed twice and resuspended in HBSS without Ca 2+ and Mg 2+ for Ca 2+ mobilization or with Ca 2+ and Mg 2+ for chemotaxis measurement.…”

Section: Methodsmentioning

confidence: 99%

“…The change in absorbance at 460 nm (ΔA 460 ) was recorded with time with a SPECTRA max 384 Plus spectrophotometer (Molecular Devices). The myeloperoxidase activity, ΔA 460 /min, was converted into the number of neutrophils using a stand curve of myeloperoxidase activities versus known numbers of murine neutrophils, which were isolated from the bone marrow of mice, as previously described [57].…”

Section: Methodsmentioning

confidence: 99%

Group A Streptococcus Secreted Esterase Hydrolyzes Platelet-Activating Factor to Impede Neutrophil Recruitment and Facilitate Innate Immune Evasion

Mengyao

Zhu

et al. 2012

PLoS Pathog

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The innate immune system is the first line of host defense against invading organisms. Thus, pathogens have developed virulence mechanisms to evade the innate immune system. Here, we report a novel means for inhibition of neutrophil recruitment by Group A Streptococcus (GAS). Deletion of the secreted esterase gene (designated sse ) in M1T1 GAS strains with (MGAS5005) and without (MGAS2221) a null covS mutation enhances neutrophil ingress to infection sites in the skin of mice. In trans expression of SsE in MGAS2221 reduces neutrophil recruitment and enhances skin invasion. The sse deletion mutant of MGAS5005 (Δ sse MGAS5005 ) is more efficiently cleared from skin than the parent strain. SsE hydrolyzes the sn-2 ester bond of platelet-activating factor (PAF), converting biologically active PAF into inactive lyso-PAF. K M and k cat of SsE for hydrolysis of 2-thio-PAF were similar to those of the human plasma PAF acetylhydrolase. Treatment of PAF with SsE abolishes the capacity of PAF to induce activation and chemotaxis of human neutrophils. More importantly, PAF receptor-deficient mice significantly reduce neutrophil infiltration to the site of Δ sse MGAS5005 infection. These findings identify the first secreted PAF acetylhydrolase of bacterial pathogens and support a novel GAS evasion mechanism that reduces phagocyte recruitment to sites of infection by inactivating PAF, providing a new paradigm for bacterial evasion of neutrophil responses.

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Neutrophil Isolation From Nonhuman Species

Cited by 62 publications

References 14 publications

Identification of LukPQ, a novel, equid-adapted leukocidin of Staphylococcus aureus

Identification of LukPQ, a novel, equid-adapted leukocidin of Staphylococcus aureus

The Role of Innate Immunity in Promoting SaeR/S-Mediated Virulence in Staphylococcus aureus

Group A Streptococcus Secreted Esterase Hydrolyzes Platelet-Activating Factor to Impede Neutrophil Recruitment and Facilitate Innate Immune Evasion

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