C3a is a key complement activation fragment, yet its neutrophil-expressed receptor (C3aR) still has no clearly defined role. In this study, we used a neutrophil-dependent mouse model of intestinal ischemia-reperfusion (IR) injury to explore the role of C3aR in acute tissue injuries. C3aR deficiency worsened intestinal injury, which corresponded with increased numbers of tissue-infiltrating neutrophils. Circulating neutrophils were significantly increased in C3aR −/− mice after intestinal ischemia, and C3aR −/− mice also mobilized more circulating neutrophils after granulocyte colony-stimulating factor infusion compared with WT mice, indicating a specific role for C3aR in constraining neutrophil mobilization in response to intestinal injury. In support of this role, C3aR −/− mice reconstituted with WT bone marrow reversed IR pathology back to WT levels. Complement C5a receptor (C5aR) antagonism in C3aR −/− mice also rectified the worsened pathology after intestinal IR injury but had no effect on circulating neutrophils, highlighting the opposing roles of C3a and C5a in disease pathogenesis. Finally, we found that using a potent C3a agonist to activate C3aR in vivo reduced neutrophil mobilization and ameliorated intestinal IR pathology in WT, but not C3aR −/− , mice. This study identifies a role for C3aR in regulating neutrophil mobilization after acute intestinal injury and highlights C3aR agonism as a potential treatment option for acute, neutrophil-driven pathologies.

This study investigated the role of the complement activation fragment C5a in secondary pathology following contusive spinal cord injury (SCI). C5arϪ/ Ϫ mice, which lack the signaling receptor for C5a, displayed signs of improved locomotor recovery and reduced inflammation during the first week of SCI compared with wild-type mice. Intriguingly, the early signs of improved recovery in C5ar Ϫ/ Ϫ mice deteriorated from day 14 onward, with absence of C5aR ultimately leading to poorer functional outcomes, larger lesion volumes, reduced myelin content, and more widespread inflammation at 35 d SCI. Pharmacological blockade of C5aR with a selective antagonist (C5aR-A) during the first 7 d after SCI improved recovery compared with vehicle-treated mice, and this phenotype was sustained up to 35 d after injury. Consistent with observations made in C5ar Ϫ/ Ϫ mice, these improvements were, however, lost if C5aR-A administration was continued into the more chronic phase of SCI. Signaling through the C5a-C5aR axis thus appears injurious in the acute period but serves a protective and/or reparative role in the post-acute phase of SCI. Further experiments in bone marrow chimeric mice suggested that the dual and opposing roles of C5aR on SCI outcomes primarily relate to its expression on CNS-resident cells and not infiltrating leukocytes. Additional in vivo and in vitro studies provided direct evidence that C5aR signaling is required during the postacute phase for astrocyte hyperplasia, hypertrophy, and glial scar formation. Collectively, these findings highlight the complexity of the inflammatory response to SCI and emphasize the importance of optimizing the timing of therapeutic interventions.

The complement system, a major component of the innate immune system, is becoming increasingly recognised as a key participant in physiology and disease. The awareness that immunological mediators support various aspects of both normal central nervous system (CNS) function and pathology has led to a renaissance of complement research in neuroscience. Various studies have revealed particularly novel findings on the wide-ranging involvement of complement in neural development, synapse elimination and maturation of neural networks, as well as the progression of pathology in a range of chronic neurodegenerative disorders, and more recently, neurotraumatic events, where rapid disruption of neuronal homeostasis potently triggers complement activation. The purpose of this review is to summarise recent findings on complement activation and acquired brain or spinal cord injury, i.e. ischaemic-reperfusion injury or stroke, traumatic brain injury (TBI) and spinal cord injury (SCI), highlighting the potential for complement-targeted therapeutics to alleviate the devastating consequences of these neurological conditions.

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Neutrophil infiltration after insult is a prominent feature of both human and experimental traumatic spinal cord injury (SCI) (8-10), and presence of these cells at the lesion site is thought to contribute considerably to secondary inflammatory pathology and thus worse outcomes (11-14). Relatively little is known, however, about the molecular mechanisms orchestrating neutrophil mobilization and recruitment to the injured spinal cord, and to the best of our knowledge no study to date has explored a role for C3aR1 in this pathology. Here, we first delineated a role for C3a/C3aR1 in SCI by comparing the recovery of wild-type (WT) and C3ar1-/-(i.e., knockout) mice from blunt spinal cord trauma, one of the most common types of SCI in humans (15). Because C3aR1 is expressed by cells of myeloid (16, 17) and central nervous system (CNS) origin (18, 19), we also used BM chimera approaches to disentangle peripheral from central C3a/C3aR1 roles in relation to SCI outcomes. We then employed a variety of genetic and pharmacological approaches, including in vitro and in vivo functional assays, antibody-mediated neutrophil depletion, and chemotaxis assays to demonstrate that C3aR1 engages phosphatase and tensin homolog (PTEN) to negatively regulate granulocyte egress from the BM into the circulation in response to inflammatory stimuli. These findings are significant from a therapeutic perspective, as a greater number of circulating neutrophils in the blood was associated with worse outcomes in both mouse and human SCI. Results SCI leads to C3a generation, leukocyte infiltration, and elevated C3aR1 expression. To begin exploring a role for C3a in SCI, we first assessed the time course of its generation. C3a levels in the mouse spinal cord rapidly increased following injury (Figure 1A), and they were significantly elevated over sham-operated controls at 6, 12, and 24 hours after surgery (>5-fold increase; P < 0.001). Plasma C3a levels also rose sharply within 30 minutes of SCI, and remained elevated over sham-operated controls for at least 1 day after SCI (Figure 1B). Select key comparisons of C3a levels in plasma and spinal cord samples of C3ar1-/mice yielded similar results, suggesting a similar magnitude of complement system activation between genotypes (2 hours after surgery: C3ar1-/plasma 7.93 ± 1.63 μg/ml vs. WT plasma 6.94 ± 0.90 μg/ml, n = 4-5 per genotype, P = 0.51; C3ar1-/spinal cord 0.76 ± 0.10 pg/μg vs. WT spinal cord 0.68 ± 0.08 pg/μg, n = 4 per genotype, P = 0.58). Widespread C3aR1 staining was observed at and around the site of SCI, and on a variety of cell types. In the acute phase, C3aR1-expressing Ly6B.2 + and CD11b + cells were abundant at and around the lesion site at 1 day after injury (Figure 1, C and D), a time point that coincides with peak neutrophil recruitment (20). The majority of infiltrating Ly6B.2 + cells were genuine neutrophils, as little overlap was observed between Ly6B.2 staining and GFP + cells of monocytic lineage in Cx3cr1 gfp/+ mice at this time point (Figure 1E). Overall, these findings ar...

This study examined the sensitivity of ultra-high field (16.4 T) diffusion tensor imaging (DTI; 70 μm in-plane resolution, 1mm slice thickness) to evaluate the spatiotemporal development of severe mid-thoracic contusive spinal cord injury (SCI) in mice. In vivo imaging was performed prior to SCI, then again at 2h, 1 day, 3 days, 7 days, and 30 days post-SCI using a Bruker 16.4 T small animal nuclear magnetic resonance spectrometer. Cross-sectional spinal cord areas were measured in axial slices and various DTI parameters, i.e. fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (λ||) and radial diffusivity (λ⊥), were calculated for the total spared white matter (WM), ventral funiculi (VF), lateral funiculi (LF) and dorsal columns (DCs) and then correlated with histopathology. Cross-sectional area measurements revealed significant atrophy (32% reduction) of the injured spinal cord at the lesion epicentre in the chronic phase of injury. Analysis of diffusion tensor parameters further showed that tissue integrity was most severely affected in the DCs, i.e. the site of immediate impact, which demonstrated a rapid and permanent decrease in FA and λ||. In contrast, DTI parameters for the ventrolateral white matter changed more gradually with time, suggesting that these regions are undergoing more delayed degeneration in a manner that may be amenable to therapeutic intervention. Of all the DTI parameters, λ⊥ was most closely correlated to myelin content whereas changes in FA and λ|| appeared more indicative of axonal integrity, Wallerian degeneration and associated presence of macrophages. We conclude that longitudinal DTI at 16.4T provides a clinically relevant, objective measure for assessing white matter pathology following contusive SCI in mice that may aid the translation of putative neuroprotective strategies into the clinic.

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Traumatic spinal cord injury (SCI) triggers a neuro-inflammatory response dominated by tissue-resident microglia and monocyte derived macrophages (MDMs). Since activated microglia and MDMs are morphologically identical and express similar phenotypic markers in vivo, identifying injury responses specifically coordinated by microglia has historically been challenging. Here, we pharmacologically depleted microglia and use anatomical, histopathological, tract tracing, bulk and single cell RNA sequencing to reveal the cellular and molecular responses to SCI controlled by microglia. We show that microglia are vital for SCI recovery and coordinate injury responses in CNS-resident glia and infiltrating leukocytes. Depleting microglia exacerbates tissue damage and worsens functional recovery. Conversely, restoring select microglia-dependent signaling axes, identified through sequencing data, in microglia depleted mice prevents secondary damage and promotes recovery. Additional bioinformatics analyses reveal that optimal repair after SCI might be achieved by co-opting key ligand-receptor interactions between microglia, astrocytes and MDMs.