Platelet TLR4 knock out does not affect bacterial clearance in polymicrobial sepsis and ameliorates endotoxemic shock
Author(s):
Hui Zhou; Meihong Deng; Neal Matthew; Melanie Scott; Timothy Billiar, University of Pittsburgh
Background: Platelets express the cell surface LPS receptor, toll-like receptor 4 (TLR4). In addition, TLR4 on platelets is known to contribute to PMN net formation. However, the role of platelet TLR4 on host responses to polymicrobial intra-abdominal sepsis is not known.
Hypothesis: We hypothesize that platelet TLR4 would contribute to bacterial clearance in the setting of abdominal sepsis.
Methods: We constructed platelet specific TLR4 knockout mice by crossing TLR4 flox mice with mice expressing the Cre recombinase driven by PF4. We have previously confirmed platelet-specific TLR4 deletion in this strain. We assessed bleeding time and other coagulation parameters as well as the response of the mice to a systemic LPS injection. We also carried out cecal ligation and puncture (CLP) without antibiotics in control (TLR4 flox mice) and platelet-specific TLR4 knockout.
Results: Platelet-specific TLR4 knockout mice demonstrated increased bleeding time; however, normal prothrombin, activated partial thromboplastin time and thrombin time. In ex vivo studies, TLR4 knockout platelets demonstrated suppressed platelet aggregation confirming the role of TLR4 in platelet responses. Platelet-specific TLR4 knockout mice injected with LPS exhibited a marked reduction in circulating IL-6 and IL-1β levels at 6 and 18 hours. Furthermore, the circulating platelet number, WBC, and total neutrophil numbers were higher in the platelet-specific TLR4 knockout mice demonstrating the role of platelet TLR4 in regulating systemic responses to LPS. In contrast to LPS treatment, platelet-specific TLR4 knockout mice subjected to CLP demonstrated no change in bacterial counts in the peritoneum or blood indicating that platelet TLR4 was not required for bacterial clearance in the setting of intra-abdominal polymicrobial sepsis.
Conclusions: TLR4 on platelets plays an important role in regulating inflammation in response to TLR4 agonists, such as LPS. However, TLR4 on platelets is not required for normal bacterial clearance mechanisms in the setting of CLP. This is most likely explained by the redundancy in functions of pattern recognition receptors on platelets for anti-microbial responses.