Pauline Damien

Platelets and infections - complex interactions with bacteria.

Platelets can be considered sentinels of vascular system due to their high number in the circulation and to the range of functional immunoreceptors they express. Platelets express a wide range of potential bacterial receptors, including complement receptors, FcγRII, Toll-like receptors but also integrins conventionally described in the hemostatic response, such as GPIIb–IIIa or GPIb. Bacteria bind these receptors either directly, or indirectly via fibrinogen, fibronectin, the first complement C1q, the von Willebrand Factor, etc. The fate of platelet-bound bacteria is questioned. Several studies reported the ability of activated platelets to internalize bacteria such as Staphylococcus aureus or Porphyromonas gingivalis, though there is no clue on what happens thereafter. Are they sheltered from the immune system in the cytoplasm of platelets or are they lysed? Indeed, while the presence of phagolysosome has not been demonstrated in platelets, they contain antimicrobial peptides that were shown to be efficient on S. aureus. Besides, the fact that bacteria can bind to platelets via receptors involved in hemostasis suggests that they may induce aggregation; this has indeed been described for Streptococcus sanguinis, S. epidermidis, or C. pneumoniae. On the other hand, platelets are able to display an inflammatory response to an infectious triggering. We, and others, have shown that platelet release soluble immunomodulatory factors upon stimulation by bacterial components. Moreover, interactions between bacteria and platelets are not limited to only these two partners. Indeed, platelets are also essential for the formation of neutrophil extracellular traps by neutrophils, resulting in bacterial clearance by trapping bacteria and concentrating antibacterial factors but in enhancing thrombosis. In conclusion, the platelet–bacteria interplay is a complex game; its fine analysis is complicated by the fact that the inflammatory component adds to the aggregation response.

The Inflammatory Role of Platelets via Their TLRs and Siglec Receptors

Platelets are non-nucleated cells that play central roles in the processes of hemostasis, innate immunity, and inflammation; however, several reports show that these distinct functions are more closely linked than initially thought. Platelets express numerous receptors and contain hundreds of secretory products. These receptors and secretory products are instrumental to the platelet functional responses. The capacity of platelets to secrete copious amounts of cytokines, chemokines, and related molecules appears intimately related to the role of the platelet in inflammation. Platelets exhibit non-self-infectious danger detection molecules on their surfaces, including those belonging to the “toll-like receptor” family, as well as pathogen sensors of other natures (Ig- or complement receptors, etc.). These receptors permit platelets to both bind infectious agents and deliver differential signals leading to the secretion of cytokines/chemokines, under the control of specific intracellular regulatory pathways. In contrast, dysfunctional receptors or dysregulation of the intracellular pathway may increase the susceptibility to pathological inflammation. Physiological vs. pathological inflammation is tightly controlled by the sensors of danger expressed in resting, as well as in activated, platelets. These sensors, referred to as pathogen recognition receptors, primarily sense danger signals termed pathogen associated molecular patterns. As platelets are found in inflamed tissues and are involved in auto-immune disorders, it is possible that they can also be stimulated by internal pathogens. In such cases, platelets can also sense danger signals using damage associated molecular patterns (DAMPs). Some of the most significant DAMP family members are the alarmins, to which the Siglec family of molecules belongs. This review examines the role of platelets in anti-infection immunity via their TLRs and Siglec receptors.

Immune-reactive soluble OX40 ligand, soluble CD40 ligand, and interleukin-27 are simultaneously oversecreted in platelet components associated with acute transfusion reactions

Leukoreduction of labile blood components dramatically decreases the frequency of minor, intermediate, and severe adverse events (AEs), referred to as acute transfusion reactions (ATRs), especially after transfusion of platelet components (PCs). The pathophysiology of AEs may result from accumulation of soluble, secreted, platelet (PLT) factors with proinflammatory functions stored in PCs. Thus, several cosynergizing factors associated with PLT accumulation in PCs may contribute to clinically reported ATRs with inflammatory symptoms.

Toll-like receptor 4 signal transduction in platelets: novel pathways

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Highly Active Antiretroviral Therapy Alters Inflammation Linked to Platelet Cytokines in HIV-1–Infected Patients

TO THE EDITOR—Chronic immune activation and persistent inflammation are critical features of human immunodeficiency virus type 1 (HIV-1) infection [1, 2]. For example, CD40 and its soluble ligand (sCD40L) are upregulated upon human immunodeficiency virus (HIV) infection, and their enhanced interaction inhibits production of interferon α by plasmacytoiddendritic cells [3].Recently,Naranbhai et al reported that innate immune activation drivesHIVacquisition, despite the use of a tenofovirmicrobicide gel [4].Theysuggested that dampening inflammation may reduce primary infection. Among the numerous actors involved in immune activation and inflammation during HIV infection, activated platelets are usually underconsidered. However, platelets are the major source of circulating sCD40L, a master immune activator [5]. Platelets are activated by infectious pathogens, including HIV, because they sense infectious danger and secrete adapted panels of cytokines [6]. Levels of inflammatory platelet-derived products are elevated in the plasma of HIV-1 infected patients [7]. We assessed, in an in vitro model, platelet participation in inflammation in HIV-1–infected patients who were or were not receiving highly active antiretroviral therapy (HAART). A total of 41 HIV-positive patients without coinfection who had been receiving stable HAART for at least 1 year and 30 HIV-positive patients who, in accordance with World Health Organization recommendations (ie, their CD4 T-cell count was >350 cells/μL), were not receiving HAART were recruited at our hospital. Written informed consent was obtained for inclusion into the study, which was approved by the French National Agency for the Safety of Medicines and Health Products (study B111011-40). Forty healthy blood donors were recruited as negative controls after signing the donation form, which states that donation of blood does not preclude use of the donation for nontherapeutic purposes (in accordance with the French Public Health Code). Clinical data on patients of different groups are presented in Table 1. Platelet-free and platelet-rich plasma (PRP) samples were prepared as described previously [6, 8]. Freshly isolated platelets were stimulated with thrombin receptor– activating peptide (TRAP)–SFLLRN peptide (50 μg/mL for 30 minutes; SigmaAldrich, Saint-Quentin Fallavier, France). Supernatants were cryopreserved at −80°C until later assay. Levels of soluble CD40L (sCD40L), growth-regulated oncogene (GRO)–α, regulated and normal T-cell expressed and secreted (RANTES), and soluble CD62P (sCD62P) were quantitated in plasma samples and PRP supernatants by Luminex technology (Merck-Millipore, Molsheim, France). Concentrations are expressed as mean values ± SD, and each samplewas assayed in triplicate. The values were compared by the t test or χ2 test for proportions. P < .05 was considered statistically significant. HAART recipients and untreated HIV-1–infected patients had comparable platelet counts, which were significantly lower than those in healthy controls (Table 1). Therefore, concentrations of factors released by platelets were normalized for 3 × 10 platelets. Consistent with other studies [7, 9], we observed that plasma levels of several platelet-associated inflammatory molecules were significantly increased in HIV-positive patients, which supports the participation of platelets in systemic immune activation during HIV infection. Moreover, we found that platelets displayed different responses to TRAP according to the HIV status of patients and whether HAART was implemented. Platelets from HAART-treated patients, and evenmore platelets from untreated patients, released significantly more sCD40L

Altered release of regulated upon activation, normal T-cell expressed and secreted protein from human, normal platelets: contribution of distinct HIV-1MN gp41 peptides.

Platelets can bind HIV and, in turn, be altered in with respect to and function during HIV progression. This study examines the secretion of normal platelets after exposure to recombinant HIV-1MN gp120 or gp41 peptides. There was a modest but significant decrease in regulated upon activation, normal T-cell expressed and secreted protein production in the presence of two out of 10 peptides, which was restored by monoclonal antibodies to gp41. Our data provide novel information on possible primary interactions between platelets and HIV env proteins.

LPS stimulation of purified human platelets is partly dependent on plasma soluble CD14 to secrete their main secreted product, soluble-CD40-Ligand

BackgroundPlatelets are instrumental to primary haemostasis; in addition, as they are central to endothelium vascular repair, they play a role in physiological inflammation. Platelets have also been demonstrated to be key players in innate immunity and inflammation, expressing Toll-like receptors (TLRs) to sense microbial infection and initiate inflammatory responses. They are equipped to decipher distinct signals, to use alternate pathways of signalling through a complete signalosome, despite their lack of a nucleus, and to adjust the innate immune response appropriately for pathogens exhibiting different types of ‘danger’ signals. Previous work has described the two main LPS isoforms-TLR4 activation pathways in purified platelets. However, the precise mechanism of TLR4 signalling in platelets is not completely unravelled, especially how this signalling may occur since platelets do not express CD14, the TLR4 pathophysiological companion for LPS sensing. Thus, we investigated from what source the CD14 molecules required for TLR4 signalling in platelets could come.ResultsHere we show that CD14, required for optimal response to LPS stimulation, is obtained from plasma, but used with restrictive regulation. These data add to the body of evidence that platelets are closer to regulatory cells than to first line defenders. The readout of our experiments is the canonical secreted cytokine-like protein, soluble (s)CD40L, a molecule that is central in physiology and pathology and that is abundantly secreted by platelets from the alpha-granules upon stimulation.ConclusionsWe show that sCD14 from plasma contributes to LPS/TLR4 signalling in platelets to allow significant release of soluble CD40L, thereby elucidating the mechanism of LPS-induced platelet responses and providing new insights for reducing LPS toxicity in the circulation.

Contribution of activated platelets to plasma IL-27 levels

Serum interleukin-27 (IL-27) protein concentration is predictive of bacterial infection in critically ill children. Here we show that upon activation platelets release functional IL-27 that is able to specifically induce B cell activation in vitro. These data highlight the inflammatory role of platelet-derived IL-27 and suggest that platelets could contribute to immune dysregulation in septic patients.

Acetylsalicylic acid differentially limits the activation and expression of cell death markers in human platelets exposed to Staphylococcus aureus strains

Beyond their hemostatic functions, platelets alter their inflammatory response according to the bacterial stimulus. Staphylococcus aureus is associated with exacerbated inflammation and thrombocytopenia, which is associated with poor prognosis during sepsis. Acetylsalicylic acid and statins prevent platelet aggregation and decrease the mortality rate during sepsis. Therefore, we assessed whether these two molecules could reduce in vitro platelet activation and the inflammatory response to S. aureus. Platelets were exposed to clinical strains of S. aureus in the presence or absence of acetylsalicylic acid or fluvastatin. Platelet activation, aggregation, and release of soluble sCD62P, sCD40 Ligand, RANTES and GROα were assessed. Platelet cell death was evaluated by analyzing the mitochondrial membrane potential, phosphatidylserine exposure, platelet microparticle release and caspase-3 activation. All S. aureus strains induced platelet activation but not aggregation and decreased the platelet count, the expression of cell death markers and the release of RANTES and GROα. Acetylsalicylic acid but not fluvastatin limited platelet activation and inflammatory factor release and restored the platelet count by protecting platelets from Staphylococcus-induced expression of cell death markers. This study demonstrates that acetylsalicylic acid limits S. aureus-induced effects on platelets by reducing cell death, revealing new strategies to reduce the platelet contribution to bacteremia-associated inflammation.

NF-κB Links TLR2 and PAR1 to Soluble Immunomodulator Factor Secretion in Human Platelets

The primary toll-like receptor (TLR)-mediated immune cell response pathway common for all TLRs is MyD88-dependent activation of NF-κB, a seminal transcription factor for many chemokines and cytokines. Remarkably, anucleate platelets express the NF-κB machinery, whose role in platelets remains poorly understood. Here, we investigated the contribution of NF-κB in the release of cytokines and serotonin by human platelets, following selective stimulation of TLR2 and protease activated receptor 1 (PAR1), a classical and non-classical pattern-recognition receptor, respectively, able to participate to the innate immune system. We discovered that platelet PAR1 activation drives the process of NF-κB phosphorylation, in contrast to TLR2 activation, which induces a slower phosphorylation process. Conversely, platelet PAR1 and TLR2 activation induces similar ERK1/2, p38, and AKT phosphorylation. Moreover, we found that engagement of platelet TLR2 with its ligand, Pam3CSK4, significantly increases the release of sCD62P, RANTES, and sCD40L; this effect was attenuated by incubating platelets with a blocking anti-TLR2 antibody. This effect appeared selective since no modulation of serotonin secretion was observed following platelet TLR2 activation. Platelet release of sCD62P, RANTES, and sCD40L following TLR2 or PAR1 triggering was abolished in the presence of the NF-κB inhibitor Bay11-7082, while serotonin release following PAR1 activation was significantly decreased. These new findings support the concept that NF-κB is an important player in platelet immunoregulations and functions.