Microvesicles in disease

On activation cells shed vesicles from their surface by the process of ectocytosis. These micro-vesicles or Ectosomes express phosphatidyl-serine (PS) on their surface, which can elicit a range of biological effects from immune-suppression to the activation of the clotting and complement pathways. While the full consequence of this phenomena is still subject of intensive research, it is clear that microvesicles, despite their size, can profoundly impact the immune system.
The aim of this thesis was to study clinically relevant scenarios where micro-vesicle release alters the course of inflammation. The conditions studied were:
1. The release of neutrophil microvesicles (PMN-Ecto) and spontaneous resolution of Gout.
Gout is an inflammatory arthritis caused by mono-sodium urate crystals (MSU) precipitating in joints. When attempting to clear MSU, resident macrophages activate their inflammasome complex and release large amounts of highly inflammatory cytokine IL-1b. One of the intriguing features of gout is that acute attacks resolve spontaneously and leave minimal residual damage to the joint. The mechanism driving the resolution of gout is still poorly understood. We hypothesized that the early release of PMN-Ecto during acute gout contains the inflammatory response to MSU.
In a murine peritonitis model of gout we found that the release of IL-1b following stimulation with MSU relied on the generation of C5a by MSU itself. Neutrophils infiltrating the peritoneum in response to C5a released phosphatidylserine (PS)-positive ectosomes early on in the course of inflammation. Treatment of the peritoneum with these PMN-Ecto in turn resulted in suppression of IL-1b release and translated in a decrease of PMN influx. Ectosome-mediated suppression could be reproduced using PS-expressing liposomes and required the presence of the PS-receptor MerTK. Finally, PMN-Ecto from joint aspirates of patients with gouty arthritis had similar anti-inflammatory properties. Ectosome-mediated control of inflammasome-driven inflammation is a compelling concept of autoregulation initiated early on during PMN activation in gout.
2. Platelet microvesicles (PLT-Ecto) and sub-clinical disseminated intravascular coagulopathy (DIC) during anti-thymoglobulin therapy (ATG).
Anti-Thymo-Globulins (ATG) are T-cell depleting antibodies used for hematopoietic stem cell (HSCT) and kidney (NTX) transplant patients as an immuno-suppressant to prevention graft vs. host disease (GvHD). Although designed to target T-cell, ATG recognizes a wide range of epitopes, including those present on platelets. Consequently the rapid infusion of ATG frequently cause side effects that are associated with the overt activation of the clotting system called DIC. The mechanism relating ATG therapy to DIC remains unclear. We hypothesized that ATG induces DIC by the release of pro-coagulant PLT-Ecto.
We could demonstrate that ATG induces platelet activation, degranulation and release of PLT-Ecto. The shedding of PLT-Ecto required the presence of complement and could be inhibited by an anti-C5 blocking antibody. PLT-Ecto exhibited high pro-thrombinase activity. The blocking of C5 during ATG treatment of platelets therefore indirectly inhibited thrombin generation. In HSCT and NTx patients, ATG treatment resulted in elevated levels of d-dimer and thrombin-anti-thrombin complexes indicating heightened state of clotting. Furthermore, we could confirm the presence of PLT-Ecto in patients plasma and ATG dose dependent complement fragment deposition on PLT-Ecto.
In conclusion, ATG induced complement dependent release of pro-coagulant PLT-Ecto and the use of anti-C5 antibody may prove to be beneficial in preventing thrombotic complications during ATG therapy.
3. Red Blood cell microvesicles (RBC-Ecto) and transfusion related complication.
Transfusion of RBC is known to exacerbate systemic inflammation and increase morbidity and mortality of critically ill patients. The adverse effect of RBC transfusions has been attributed to changes that occur during extended periods of RBC storage. The ageing process of RBC is accompanied by the shedding of RBC-Ecto, which accumulate in storage and are transfused along with RBC. We hypothesized that RBC-Ecto derived from aged RBCs amplify systemic inflammation and contribute to some of the deleterious effects of aged blood transfusions.
We established a murine transfusion model using RBC-Ecto purified from aged erythrocytes. While the injection of RBC-Ecto into healthy mice had no effect, transfusion of RBC-Ecto amplified systemic inflammation in endotoxemic mice. These effects were mediated by a thrombin depenedent activation of C5 complement factor on the surface of RBC-Ecto. Consequently, the treatment of mice with a thrombin anticoagulant prior to transfusion alleviated the proinflammatory effects of RBC-Ecto. The inhibition of thrombin in vivo lead to suppresion of systemic C5a generation, inflammatory cytokine release and subsequently lung neutrophil sequestration.
In conclusion, we could identify RBC-Ecto as potential mediators of transfusion related morbidity. RBC-Ecto mediated their effects by sequentially activating the coagulation and complement system, which then aggrevated systemic inflammation.