session

85 - Oral Communication
Mechanisms of Disease II
Feb. 26, 2021, 1:45 p.m. - 3:15 p.m., Lausanne

Abstract

2
Microvascular immune cell recruitment regulating development of immunothrombosis in systemic bacterial infection
T. Mueller, S. Meister, M. Thakur, M. Wohlrab, S. Bortoluzzi, N. Jensen, M. Rossner, M. Schmidt-Supprian, S. Massberg, B. Engelmann, Presenter: T. Mueller (Munich)

Background and Objective
During systemic bacterial infection coagulation is activated within the microcirculation of infected organs. The immune cells regulating intravascular fibrin formation and immunothrombosis are insufficiently characterized.
Methods
Wild type (WT) mice, Jalpha18-/- mice, Valpha14-Jalpha18 mice and bhlhe40/bhlhe41 double knock out (DKO) mice were infected with E.Coli. Kinetic profiles of classical monocytes (Ly6C+,Ly6G-), non-classical monocytes (CX3CR1+,Ly6C-), neutrophils (Ly6G+), B1a cells (CD19+,CD5+), NKT cells (CD1d+) and Th17 cells (CD4+,RORgammat+) were studied specifically at the site of infection. In parallel, association of tissue factor (TF) and uPAR with immune cells was visualized. Immunohistochemistry was also performed to analyze fibrin formation and its association with GFP+ E.Coli in liver microvessels.
Results
The earliest recruited cells were identified via tetramer staining as NKT cells. Classical monocytes and neutrophils were recruited rapidly whereas non-classical monocytes were immobilized with a delay. T helper cells and B cells appeared early after infection and increased over time. T helper cells represented largely Th17 cells, while most B cells were B1a cells. Intravascular fibrin formation and vessel occlusions peaked at 3 and 6h post infection. Bacteria were mostly embedded in fibrin rich areas. Proteins initiating fibrin formation (TF) or promoting its degradation (uPAR) could be detected on several recruited immune cells. We found TF to be associated at an early time point mainly with NKT cells, as well as with neutrophils and classical monocytes. uPAR was associated with B1a cells, especially during the peak of fibrin formation. In Jalpha18-/- mice with greatly reduced levels of NKT cells, microcirculatory coagulation activation was reduced. Depletion of classical monocytes decreased fibrin formation to a similar extent. In Valpha14-Jalpha18 mice with raised NKT levels, fibrin deposition and vessel occlusions were augmented. In bhlhe40/41 DKO mice, a model for impaired B1a production, the amount of fibrin formation was unchanged compared to the WT mice.
Conclusion
Immunothrombosis development is crucially promoted by the sequential recruitment of NKT cells, classical monocytes and neutrophils. NKT cells immobilized in the liver microcirculation express TF and initiate intravascular coagulation. B1a cells appear to be irrelevant for fibrin homeostasis.
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