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

Abstract

IntroductionIn 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.MethodsNeutrophils 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.ResultsThe 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.ConclusionPlatelets 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.

Revisiting Platelets and Toll-Like Receptors (TLRs): At the Interface of Vascular Immunity and Thrombosis

“…In an example of the juxtaposition of their roles in inflammation, platelets aggregate with neutrophils to propagate vascular damage in acute lung injury [ 63 , 64 ] but these cells can also temporally produces pro-resolving mediators to reduce lung inflammation [ 65 ]. It is now appreciated that platelets can have both pro-and anti-inflammatory effects, perhaps in a context-specific [ 66 , 67 ] and sequential [ 65 , 68 ] manner.…”

From Classical to Unconventional: The Immune Receptors Facilitating Platelet Responses to Infection and Inflammation

“…When platelets from TLR2 knockout mice were incubated with wild type neutrophils, mice platelets are unable to form aggregates with neutrophils in response to periodontopathogens [ 62 ]. In an in vitro study, the incubation of human platelets, stimulated using TLR2 agonist Pam3CSK4, with human neutrophils with were found to enhance neutrophil phagocytosis as indicated by the internalization of FITC conjugated latex beads [ 65 ]. On the contrary, markers of neutrophil activation such as CD11b were found to decrease upon co-culture with stimulated platelets [ 65 ].…”

“…In an in vitro study, the incubation of human platelets, stimulated using TLR2 agonist Pam3CSK4, with human neutrophils with were found to enhance neutrophil phagocytosis as indicated by the internalization of FITC conjugated latex beads [ 65 ]. On the contrary, markers of neutrophil activation such as CD11b were found to decrease upon co-culture with stimulated platelets [ 65 ]. The opposing effects of increasing phagocytosis while inhibiting neutrophil activation indicates that platelet TLRs can play an immunomodulatory role [ 65 ].…”

“…On the contrary, markers of neutrophil activation such as CD11b were found to decrease upon co-culture with stimulated platelets [ 65 ]. The opposing effects of increasing phagocytosis while inhibiting neutrophil activation indicates that platelet TLRs can play an immunomodulatory role [ 65 ].…”

The Immune Nature of Platelets Revisited

“…Intriguingly, platelet TLR4 can both inhibit and augment neutrophil responses. Co-culture of platelets with neutrophils and TLR4 agonists increased neutrophil CD62L expression, phagocytosis and IL-8 secretion but reduced CD62L shedding and elastase secretion [ 43 ]. It is now clear that platelet TLR4 is critical for orchestrating neutrophil responses against invading pathogens and the source of LPS determines their ultimate effect on leukocytes.…”