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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...