Decoy nanoparticles bearing native C5a receptors as a new approach to inhibit complement-mediated neutrophil activation

Abstract

Complement-activated neutrophils are integrally involved in many pathological conditions as well as in dampening the efficacy of cell-based therapies. Mesenchymal stem cells (MSCs) hold promise for regenerative medicine and inflammatory disease therapy, but current MSC-based therapies still require further improvements to ensure success. We recently reported that immediately upon delivery to the bloodstream, MSCs activate complement to produce C5a, which binds to its receptor, C5aR, on neutrophils and thus activates these cells to damage MSCs. Thus, blocking this C5a-C5aR interaction should yield improvements in MSC survival and treatment efficacy. In this project, we developed decoy nanoparticles with surface displaying native C5aR by coating membrane vesicles derived from macrophages expressing high levels of C5aR onto poly(lactic-co-glycolic acid) (PLGA) cores. These C5aR-displaying decoy nanoparticles effectively inhibited neutrophil activation and thus reduced sequential injury to MSCs upon exposure to blood both in vitro and in vivo. Consequently, survival and treatment potency of the MSCs were significantly improved by these decoy nanoparticles. This finding suggests that the C5aR-displaying decoy nanoparticles represent a unique approach toward improving current MSC-based therapies. Additionally, these decoy nanoparticles can be useful as a new reagent for the treatment of other pathological conditions that involve C5a-C5aR signaling.

Statement of Significance

Complement C5aR has been implied in the pathogenesis of many disorders and is emerging as a new target for the development of therapeutics. So far all the inhibitors of C5aR are either biologicals or small compounds with various shortcomings. Since C5aR is a G-protein coupled receptor that features a multi-loop binding interface with its ligand, C5a, soluble forms of C5aR as decoys for cell surface C5aR are unlikely. We believe this is the first evidence suggesting that C5aR decoy nanoparticles can be developed to treat various C5aR-mediated pathological conditions.

Introduction

Mesenchymal stem cells (MSCs) comprise a population of multipotent adult stem cells found in many tissues, including the bone marrow, adipose, and umbilical cord [1]. These cells retain the ability to differentiate into mesenchymal cells (e.g., osteoblasts, adipocytes, and chondrocytes) [2], and potentially some other non-mesenchymal cells [3], [4], [5]. Therefore, MSCs could be used in regenerative medicine. In addition, MSCs are highly immunosuppressive and act through multiple mechanisms to potently inhibit both the adaptive and innate immune systems [6]. Consequently, intense clinical-stage research is currently directed toward the development of new MSC-based therapies for the treatment of various inflammatory diseases, including graft-versus-host disease, diabetes mellitus, multiple sclerosis, and stroke [7]. Despite these efforts and promising pre-clinical data, however, no MSC-based therapy has yet received approval from the US Food and Drug Administration.

One significant issue of the MSC-based therapies is the short half-life of the administered MSCs in vivo [8]. MSCs are thought to be able to escape host immune surveillance. Therefore, MSCs propagated from a single donor are commonly used to treat other patients. After administration, MSC trend to migrate to the site of inflammation in vivo, however, both animal and human studies have frequently found the rapid disappearance of the administrated MSCs, suggesting that these foreign cells are recognized and cleared by the host [8], [9]. Indeed, we and others have reported that despite the apparent tolerance of the host adaptive immune system, the administered MSCs are immediately recognized and attacked by complement, a key component of the innate immune system, leading to reduced MSC survival and function [10], [11], [12], [13], [14]. We further showed that activated complement not only directly damages MSCs by forming membrane attack complexes (MAC) [12], but also by releasing the anaphylatoxins C3a and C5a [11], which are known as potent promoters of inflammation. Particularly, C5a binds to its receptor C5aR on neutrophils, the most abundant leukocytes in the blood, and subsequently activates these cells to attack MSCs through an oxidative burst [11]. Notably, we demonstrated significant improvements in MSC survival and immune regulatory potency in vivo after depleting neutrophils with monoclonal antibodies or inhibiting local complement activation on MSCs thus the production of anaphylatoxins by “painting” complement inhibitors onto MSCs [11]. These findings suggest that current MSC-based therapies could effectively be improved by targeting C5a–C5aR interactions to suppress complement-mediated neutrophil activation.

Previous attempts to use soluble forms of receptors as decoys to block interactions between cell surface receptors and their ligands have been successful. For example, Aflibercept, which has been approved to treat diseases that integrally involve vascular endothelial growth factor (VEGF) signaling, is a soluble form of the VEGF receptor (VEGFR) containing the native extracellular domains sufficient for high-affinity competitive binding of VEGF [15]. Accordingly, aflibercept serves as a decoy to block VEGF-mediated signaling. By contrast, C5aR is a G-protein-coupled receptor (GPCR) that spans the cell membrane 7 times and use multiple extracellular loops to interact with C5a [16]. Therefore, C5aR-based decoys to prevent C5a-mediated signaling would be unlikely to involve soluble forms of this receptor.

In this report, we describe the development of nanoparticles displaying native C5aR on their surfaces. We found that these nanoparticles function as decoys to block interactions between C5a and C5aR, thereby significantly reducing neutrophil activation after MSC administration and markedly improving the survival and functionality of the MSCs.