Complement receptor C3aR1 controls neutrophil mobilization following spinal cord injury through physiological antagonism of CXCR2
Faith H. Brennan, Trisha Jogia, Ellen R. Gillespie, Linda V. Blomster, Xaria X. Li, Bianca Nowlan, Gail M. Williams, Esther Jacobson, Geoff W. Osborne, Frederic A. Meunier, Stephen M. Taylor, Kate E. Campbell, Kelli P.A. MacDonald, Jean-Pierre Levesque, Trent M. Woodruff, Marc J. Ruitenberg
Faith H. Brennan, Trisha Jogia, Ellen R. Gillespie, Linda V. Blomster, Xaria X. Li, Bianca Nowlan, Gail M. Williams, Esther Jacobson, Geoff W. Osborne, Frederic A. Meunier, Stephen M. Taylor, Kate E. Campbell, Kelli P.A. MacDonald, Jean-Pierre Levesque, Trent M. Woodruff, Marc J. Ruitenberg
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Research Article Inflammation Neuroscience

Complement receptor C3aR1 controls neutrophil mobilization following spinal cord injury through physiological antagonism of CXCR2

Abstract

Traumatic spinal cord injury (SCI) triggers an acute-phase response that leads to systemic inflammation and rapid mobilization of bone marrow (BM) neutrophils into the blood. These mobilized neutrophils then accumulate in visceral organs and the injured spinal cord where they cause inflammatory tissue damage. The receptor for complement activation product 3a, C3aR1, has been implicated in negatively regulating the BM neutrophil response to tissue injury. However, the mechanism via which C3aR1 controls BM neutrophil mobilization, and also its influence over SCI outcomes, are unknown. Here, we show that the C3a/C3aR1 axis exerts neuroprotection in SCI by acting as a physiological antagonist against neutrophil chemotactic signals. We show that C3aR1 engages phosphatase and tensin homolog (PTEN), a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, to restrain C-X-C chemokine receptor type 2–driven BM neutrophil mobilization following trauma. These findings are of direct clinical significance as lower circulating neutrophil numbers at presentation were identified as a marker for improved recovery in human SCI. Our work thus identifies C3aR1 and its downstream intermediary, PTEN, as therapeutic targets to broadly inhibit neutrophil mobilization/recruitment following tissue injury and reduce inflammatory pathology.

Authors

Faith H. Brennan, Trisha Jogia, Ellen R. Gillespie, Linda V. Blomster, Xaria X. Li, Bianca Nowlan, Gail M. Williams, Esther Jacobson, Geoff W. Osborne, Frederic A. Meunier, Stephen M. Taylor, Kate E. Campbell, Kelli P.A. MacDonald, Jean-Pierre Levesque, Trent M. Woodruff, Marc J. Ruitenberg

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Figure 8

C3aR1 controls the responsiveness of BM neutrophils to chemotactic signals.

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C3aR1 controls the responsiveness of BM neutrophils to chemotactic signa...
(A) Chemotaxis assays of Ly6G-enriched BM neutrophils toward CXCL1. Note that CXCL1-induced cell migration is selectively abolished in WT mice with C3aR1 coactivation (E7 treatment) and/or CXCR2 antagonism (CXCR2-A). Error bars are SEM (n = 5–7 per group). **P < 0.01; ***P < 0.001 by 2-way ANOVA with Dunnett’s post hoc test against baseline (i.e., no CXCL1; dashed line). (B) Working model as to how C3aR1 might regulate CXCL1/CXCR2–mediated BM neutrophil chemotaxis, and the site of action for the various inhibitors used. (C) Time course showing the percentage of BM neutrophils staining positive for phosphorylated ERK (p-ERK) and PTEN following E7 exposure, or phosphorylated AKT (p-AKT) after CXCL1 stimulation. (D–F) Overlays showing the geometric mean fluorescence intensity (MFI) for p-AKT staining in BM neutrophils at baseline (unstimulated) and under test conditions (D). Exposure to CXCL1, but not E7, increases both the MFI (E) and overall percentage (F) of p-AKT+ BM neutrophils. C3aR1 agonism with E7 prevents these CXCL1-induced changes in AKT phosphorylation/positivity. (G–I) Overlays showing the MFI for p-ERK staining in BM neutrophils at baseline (unstimulated) and under test conditions (G). Activation of C3aR1 and CXCR2 with E7 and CXCL1, respectively, (or a combination thereof) increases both the MFI (H) and overall percentage (I) of p-ERK+ BM neutrophils. (J–L) Overlays showing PTEN MFI in BM neutrophils at baseline (unstimulated) and under test conditions (J). Activation of C3aR1 with E7 increases both the MFI (K) and overall percentage of PTEN+ BM neutrophils (L). Error bars for E, F, H, I, K, and L are SEM (n = 9 per group). *P < 0.05; **P < 0.01 by 1-way ANOVA with Dunnett’s post hoc test compared with unstimulated controls. (M) PTEN inhibition with VO-OHpic (100 nM) overcomes the E7/C3aR1-mediated inhibition of BM neutrophil responsiveness to CXCL1. Treatment with wortmannin (100 nM) confirmed a key role of the PI3K/AKT pathway in mediating the chemotactic response of BM neutrophils to CXCL1. Error bars are SEM (n = 3–8 per group). **P < 0.01; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA with Dunnett’s post hoc test versus baseline (i.e., no CXCL1; dashed line). (N) Administration of VO-OHpic to WT SCI mice leads to increased mobilization/circulating neutrophil numbers (2 hours after surgery; data are from 3 independent experiments). Error bars are SEM (n = 5 per group). *P < 0.05 by 2-tailed Student’s t test.