Complement C5a Induces Pro-inflammatory Microvesicle Shedding in Severely Injured Patients.

Ebru Karasu,Florian Gebhard,Jörg Köhl,Christoph Q Schmidt,Miriam Kalbitz,Rebecca Halbgebauer,Stephanie Kellermann,Julia Demmelmaier,Markus S Huber-Lang,Karlheinz Holzmann

doi:10.3389/fimmu.2020.01789

Abstract

Initially underestimated as platelet dust, extracellular vesicles are continuously gaining interest in the field of inflammation. Various studies addressing inflammatory diseases have shown that microvesicles (MVs) originating from different cell types are systemic transport vehicles carrying distinct cargoes to modulate immune responses. In this study, we focused on the clinical setting of multiple trauma, which is characterized by activation and dysfunction of both, the fluid-phase and the cellular component of innate immunity. Given the sensitivity of neutrophils for the complement anaphylatoxin C5a, we hypothesized that increased C5a production induces alterations in MV shedding of neutrophils resulting in neutrophil dysfunction that fuels posttraumatic inflammation. In a mono-centered prospective clinical study with polytraumatized patients, we found significantly increased granulocyte-derived MVs containing the C5a receptor (C5aR1, CD88) on their surface. This finding was accompanied by a concomitant loss of C5aR1 on granulocytes indicative of an impaired cellular chemotactic and pro-inflammatory neutrophil functions. Furthermore, in vitro exposure of human neutrophils (from healthy volunteers) to C5a significantly increased MV shedding and C5aR1 loss on neutrophils, which could be blocked using the C5aR1 antagonist PMX53. Mechanistic analyses revealed that the interaction between C5aR1 signaling and the small GTPase Arf6 acts as a molecular switch for MV shedding. When neutrophil derived, C5a-induced MV were exposed to a complex ex vivo whole blood model significant pro-inflammatory properties (NADPH activity, ROS and MPO generation) of the MVs became evident. C5a-induced MVs activated resting neutrophils and significantly induced IL-6 secretion. These data suggest a novel role of the C5a-C5aR1 axis: C5a-induced MV shedding from neutrophils results in decreased C5aR1 surface expression on the one hand, on the other hand it leads to profound inflammatory signals which likely are both key drivers of the neutrophil dysfunction which is regularly observed in patients suffering from multiple traumatic injuries.

Highlights

In patients with severe injury, sepsis, and septic shock [1, 2] early and excessive complement activation may be responsible for subsequent innate immune dysfunction [1, 3, 4]
We showed a significant reduction of the complement anaphylatoxin 5a receptor 1 (C5aR1) on neutrophils from septic shock patients and simultaneously increased plasma levels of a circulating form of C5aR1, suggesting that cells lose their receptors through MV shedding [3]
We focused on the anaphylatoxin C5a in MV shedding, because (i) increased concentrations are present early after multiple trauma and correlates with clinical severity, and (ii) the C5a-C5aR1 axis has already been shown to lead to a paralyzed innate immune response but underlying mechanisms remain mostly unexplained

Summary

Introduction

In patients with severe injury, sepsis, and septic shock [1, 2] early and excessive complement activation may be responsible for subsequent innate immune dysfunction [1, 3, 4]. Neutrophils as first-line cellular defense of innate immunity showed major changes in their function driven by an imbalanced complement activity. Equipped with a broad receptor repertoire for complement activation products including the anaphylatoxin receptors as well as membrane-bound complement regulators, neutrophils quickly sense micro-environmental changes. Our previous study showed altered expression profiles of complement receptors and membrane-bound complement regulators on immune cells from polytraumatized patients [5]. Neutrophils, as first cellular line of defense, constitutively express C5aR1 [9], which represents the major receptor for the anaphylatoxin. C5aR1 dysfunction negatively influences cellular effector functions and is associated with poor clinical outcome in sepsis patients [3, 13,14,15]

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