Platelet Factor 4 Enhances Endothelial Cell Antimicrobial Activity in Vitro and Promotes Bacterial Clearance In Vivo, Improving Sepsis Outcomes

Introduction: In this study, we investigated mechanism(s) by which the platelet-specific chemokine, platelet factor 4 (PF4), interacts with bacteria to enhance host-defense function and improve outcome in sepsis.

Sepsis, a life-threatening dysregulated response to infection, is the leading cause of mortality worldwide for which there are no targeted treatments. Upon sensing pathogens, platelets release high concentrations of PF4, the most abundant protein in platelet α granules. Due to its strong positive charge, following release, PF4 is rapidly sequestered from the circulation by binding to polyanionic glycosaminoglycans on the surface of leukocytes and endothelial glycocalyx. PF4 also interacts specifically with CXCR3B, a chemokine receptor that is constitutively expressed by ECs, and forms non-specific electrostatic interactions with anionic polymers in the bacterial cell wall.

We previously found that PF4 improves outcomes in the murine lipopolysaccharide endotoxemia model and enhances in vitro bacterial capture by neutrophil extracellular traps. We now show that PF4-coating of bacteria reduces systemic coagulopathy, limits bacterial dissemination, and improves survival in a murine model of polymicrobial sepsis, in part through the novel mechanism of enhancing the antimicrobial function of endothelial cells (ECs).

Methods: Effects of PF4 on EC killing of live bacteria: HUVECs were exposed to live Escherichia (E) coli pre-treated with PF4 (0-25µg/mL) for 5hr. Every hour, ECs were washed and treated with 1% penicillin-streptomycin to remove excess unbound bacteria, and cell lysates were plated on agar plates overnight to count colony forming units (CFUs). To assess the role of receptor CXCR3 in facilitating PF4-mediated bacteria uptake, experiments were repeated in HUVECs pre-treated with small molecule inhibitor (10mM AMG-487; 1hr) prior to E coli ± PF4 (0-25µg/mL) exposure.

Visualizing PF4-mediated bacterial clearance in vivo: Wildtype (WT) and PF4^-/- mice were intravenously (IV) infused with fluorescent E coli (8x10¹¹ CFU) and subjected to lung confocal intravital imaging. Liver and spleen residual CFUs were quantified at 15min and 2hr post E coli infusion.

Effects of PF4 on polymicrobial sepsis outcomes: Contents from the cecum (cecal slurry, CS) of WT C57BL6 donor mice (100mg/mL) were pre-treated with vehicle or PF4 (100µg; 20min) and injected IV into WT or PF4^-/- recipient mice to induce immediate bacteremia. Animals were evaluated for sepsis severity for up to 96hr, and survival analysis was performed using the Mantel-Cox test.

A separate cohort of WT and PF4^-/- mice was given IV CS (400mg/mL) pre-incubated with vehicle or PF4 (8µg; 20min). Blood was drawn at 24hr post-CS infusion and subjected to complete blood count to assess thrombocytopenia. Thrombin anti-thrombin (TAT) and VWF levels were measured by ELISAs in plasma as indicators of intravascular coagulopathy and endothelial activation. Plasma was also subjected to multiplex Olink analyses for inflammatory markers. To assess bacterial load, blood and liver, spleen, and lung homogenates were plated on agar plates to count for CFUs.

Results: PF4 increased E coli uptake by HUVECs at 2hr but reduced intracellular E coli proliferation at 3-5hr. Such enhanced bacterial uptake effects by PF4 was prevented by blocking CXCR3. Lung intravital microscopy demonstrate that following E coli infusion, PF4^-/- mice had accelerated platelet accumulation in the pulmonary microvasculature and impaired bacterial clearance as compared to WT mice. In the CS model of polymicrobial sepsis, PF4 reduced sepsis severity and mortality in both WT and PF4 ^-/- mice, and decreased bacterial load in blood, lung, and liver 24hr post-CS inoculation. WT mice also exhibited reduced leukopenia, thrombocytopenia, TAT, VWF, and inflammatory cytokine levels 24hr post-CS challenge.

Conclusion: In vitro: PF4 crosslinks E coli, initially enhancing bacterial uptake by ECs via CXCR3, but prevent subsequent intracellular proliferation. In vivo: PF4 promotes bacterial clearance while dampening coagulopathy and inflammation, leading to improved outcomes in polymicrobial sepsis.

These studies suggest that PF4 binding to bacteria may be an important host defense mechanism. The ability of PF4-based therapeutics to enhance clearance of microbes merits further translational studies in sepsis.