Dataset Information

Platelets modulate multiple markers of neutrophil function in response to in vitro Toll-like receptor stimulation.

ABSTRACT: INTRODUCTION:In addition to their role in facilitating leukocyte-mediated inflammation, platelets can dampen leukocyte pro-inflammatory responses in some contexts. Consequently, platelets are increasingly appreciated as regulators of inflammation. Together, platelets and neutrophils play a role in inflammation through Toll-like receptor (TLR) expression, although we do not fully understand how platelets shape neutrophil responses to TLR stimulation. Here, we aimed to determine the extent to which platelets can modulate neutrophil function in response to in vitro stimulation with TLR4, TLR2/1, and TLR2/6 agonists. METHODS:Neutrophils from 10 healthy individuals were cultured alone or with autologous platelets. Neutrophils ± platelets were left unstimulated or were stimulated with 1 or 100 ng/mL lipopolysaccharide (LPS; a TLR4 agonist), Pam3CSK4 (a TLR2/1 agonist) and fibroblast-stimulating lipopeptide (FSL)-1 (a TLR2/6 agonist). Neutrophil activation and phagocytic activity were assessed by flow cytometry, and elastase and interleukin-8 secretion were assessed by ELISA. RESULTS:The addition of platelets attenuated neutrophil CD66b and CD11b expression in response to various doses of Pam3CSK4 and FSL-1. Furthermore, platelet co-culture was associated with higher CD62L expression (indicating reduced CD62L shedding) in response to these TLR agonists. Platelets also reduced elastase secretion in unstimulated cultures and in response to low-dose TLR stimulation. Conversely, platelet co-culture increased neutrophil phagocytosis in unstimulated cultures and in response to low-dose Pam3CSK4 and FSL-1. Platelets also increased IL-8 secretion in response to low-dose LPS. CONCLUSION:Platelets are complex immunomodulators that can attenuate some, and simultaneously augment other, neutrophil functions. This modulation can occur both in the absence and presence of TLR stimulation.

SUBMITTER: Hally KE

PROVIDER: S-EPMC6776355 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Platelets modulate multiple markers of neutrophil function in response to in vitro Toll-like receptor stimulation.

Hally Kathryn E KE Bird Georgina K GK La Flamme Anne C AC Harding Scott A SA Larsen Peter D PD

PloS one 20191003 10

<h4>Introduction</h4>In addition to their role in facilitating leukocyte-mediated inflammation, platelets can dampen leukocyte pro-inflammatory responses in some contexts. Consequently, platelets are increasingly appreciated as regulators of inflammation. Together, platelets and neutrophils play a role in inflammation through Toll-like receptor (TLR) expression, although we do not fully understand how platelets shape neutrophil responses to TLR stimulation. Here, we aimed to determine the extent ...[more]

PMID: 31581214

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Project description:Neonates rely on their innate immune system, and neutrophils in particular, to recognize and combat life-threatening bacterial infections. Pretreatment with lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 agonist, improves survival to polymicrobial sepsis in neonatal mice by enhancing neutrophil recruitment. To understand the response of human neonatal neutrophils to TLR4 stimulation, ex vivo spontaneous neutrophil migration, neutrophil transcriptomics, and cytokine production in the presence and absence of LPS were measured directly from whole blood of adults, term neonates, and preterm neonates. Spontaneous neutrophil migration was measured on novel microfluidic devices with time-lapse imaging for 10 h. Genome-wide neutrophil transcriptomics and plasma cytokine concentrations were also determined. Preterm neonates had significantly fewer spontaneously migrating neutrophils at baseline, and both term and preterm neonates had decreased neutrophil velocity, compared to adults. In the presence of LPS stimulation, the number of spontaneously migrating neutrophils was reduced in preterm neonates compared to term neonates and adults. Neutrophil velocity was not significantly different among groups with LPS stimulation. Preterm neonates upregulated expression of genes associated with the recruitment and response of neutrophils following LPS stimulation, but failed to upregulate the expression of genes associated with antimicrobial and antiviral responses. Plasma levels of IL-1β, IL-6, IL-8, MIP-1α, and TNF-α increased in response to LPS stimulation in all groups, but IL-10 was increased only in term and preterm neonates. In conclusion, age-specific changes in spontaneous neutrophil migration counts are not affected by LPS despite changes in gene expression and cytokine production. KEY MESSAGES:Preterm neonates have reduced spontaneous neutrophil migration compared to term neonates and adults in the absence and presence of TLR4 stimulation. Preterm and term neonates have reduced neutrophil velocities compared to adults in the absence of TLR4 stimulation but no difference in the presence of TLR4 stimulation. Unique transcriptomic response to TLR4 stimulation is observed in neutrophils from preterm neonates, term neonates, and adults. TLR4 stimulation produces an age-specific cytokine response.

Project description:ObjectiveThrombocytopenia is associated with many viral infections suggesting virions interact with and affect platelets. Consistently, viral particles are seen inside platelets, and platelet activation markers are detected in viremic patients. In this article, we sought mechanistic insights into these virion/platelet interactions by examining how platelets endocytose, traffic, and are activated by a model virion. Approach and Results: Using fluorescently tagged HIV-1 pseudovirions, 3-dimensional structured illumination microscopy, and transgenic mouse models, we probed the interactions between platelets and virions. Mouse platelets used known endocytic machinery, that is, dynamin, VAMP (vesicle-associated membrane protein)-3, and Arf6 (ADP-ribosylation factor 6), to take up and traffic HIV-1 pseudovirions. Endocytosed HIV-1 pseudovirions trafficked through early (Rab4+) and late endosomes (Rab7+), and then to an LC3+ (microtubule-associated protein 1A/1B-light chain 3) compartment. Incubation with virions induced IRAK4 (interleukin 1 receptor-associated kinase 4), Akt (protein kinase B), and IKK (IκB kinase) activation, granule secretion, and platelet-leukocyte aggregate formation. This activation required TLRs (Toll-like receptors) and MyD88 (myeloid differentiation primary response protein 88) but was less extensive and slower than activation with thrombin. In vivo, HIV-1 pseudovirions injection led to virion uptake and platelet activation, as measured by IKK activation, platelet-leukocyte aggregate formation, and mild thrombocytopenia. All were decreased in VAMP-3-/- and, megakaryocyte/platelet-specific, Arf6-/- mice. Similar platelet activation profiles (increased platelet-leukocyte aggregates, plasma platelet factor 4, and phospho-IκBα) were detected in newly diagnosed and antiretroviral therapy-controlled HIV-1+ patients.ConclusionsCollectively, our data provide mechanistic insights into the cell biology of how platelets endocytose and process virions. We propose a mechanism by which platelets sample the circulation and respond to potential pathogens that they take up.

Dataset Information

Platelets modulate multiple markers of neutrophil function in response to in vitro Toll-like receptor stimulation.

Publications

Platelets modulate multiple markers of neutrophil function in response to in vitro Toll-like receptor stimulation.

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