The receptor for complement component C3a mediates protection from intestinal ischemia-reperfusion injuries by inhibiting neutrophil mobilization

Abstract

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.

Keywords: bone-marrow; intestine; leukocyte; therapeutic.

Fig. 1.

Intestinal IR up-regulates C3a, neutrophil mobilizers, and circulating neutrophil numbers. Intestinal ischemia was induced by clamping the superior mesenteric artery in WT mice for 30 min, followed by reperfusion for 0, 30, 60, 90 or 150 min. (A, C, D, and E) Levels of C3a/C3a-desArg (A), G-CSF (C), KC (CXCL1/IL-8) (D), and SDF-1 (CXCL12) (E) were increased at endpoint IR, as measured by ELISA. (B) Circulating neutrophil numbers also increased progressively during the reperfusion phase. n = 6–8 per time point. Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 compared with preischemia (−30 min).

Fig. 2.

Genetic ablation of C3aR exacerbates intestinal injury after IR. (A–C) Representative H&E staining of cross-sections of ileum from WT sham-operated (A), WT IR (B), and C3aR^−/− IR (C) mice, demonstrating considerably increased damage to villi after IR in the absence of C3aR. Arrows indicate swelling of villi, and bars indicate approximate length of villi. (D–F) Representative TUNEL-stained ileum sections from WT sham-operated (D), WT IR (E), and C3aR^−/− IR (F) mice, demonstrating increased apoptosis (arrows) with IR, which was worsened in C3aR^−/− mice. (G and H) Histopathological scores of H&E-stained intestinal sections (G) and TUNEL staining intensity (H), showing worsened mucosal injury and elevated apoptosis in C3aR^−/− mice compared with WT mice after IR injury. (I) Intestinal ALP (Left) and hemoglobin (Right) levels were also significantly higher in C3aR^−/− mice compared with WT mice after IR. n = 4–6 mice/group for sham-operated, n = 10 mice/group for IR. Data are mean ± SEM. *P < 0.05; **P < 0.01. (Scale bars: 50 μm in A–C; 20 μm in D–F.)

Fig. 3.

Elimination of C3aR increases neutrophil infiltration after intestinal IR. (A) IR increased intestinal MPO activity in WT mice and even more significantly in C3aR^−/− mice. (B) Representative sections of ileum showing infiltrating neutrophils in the villi of small intestine (red/pink, arrows). Note the enhanced IR-induced neutrophil infiltration in C3aR^−/− mice. (C) Quantitative counts of the stained cells per villus, showing significant increases in C3aR^−/− mice compared with WT mice after intestinal IR. n = 6–12/group. Data in A and C are mean ± SEM. *P < 0.05; **P < 0.01. n.s., not significant (P > 0.05). (Scale bar: 20 μm.)

Fig. 4.

Elimination of C3aR increases granulocytic neutrophil mobilization in response to intestinal IR or human G-CSF infusion. (A) Quantitative counts showing a significant elevation of circulating neutrophils with C3aR deficiency after a 150-min reperfusion period following intestinal IR. (B) Only neutrophils were selectively increased in C3aR^−/− mice in response to IR. (C and D) Representative forward/side scatter (FSC/SSC) plots showing the major blood leukocyte populations (i.e., granulocytes, monocytes, and lymphocytes) in WT (C) and C3aR^−/− (D) mice after intestinal ischemia and 150 min of reperfusion. Note the increase in granulocytic neutrophils in C3aR^−/− mice. (E) C3aR^−/− mice also mobilized more neutrophils into the circulation in response to recombinant G-CSF infusion. n = 7–14 mice/group. Data in A, B, and E are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. n.s., not significant (P > 0.05).

Fig. 5.

Blockade of C5aR reduces intestinal IR injury in C3aR^−/− mice. C3aR^−/− mice were treated with the selective C5aR antagonist PMX53 (1 mg/kg i.p.) or vehicle [5% (wt/vol) glucose] before intestinal ischemia. (A and B) Histopathological scores (A) and intestinal MPO activity (B) were significantly reduced in C3aR^−/− IR mice after PMX53 treatment. (C) In contrast, the number of circulating neutrophils was not altered by C5aR antagonism. n = 7–11 mice/group. Data are mean ± SEM. ***P < 0.001. n.s., not significant (P > 0.05).

Fig. 6.

Specific pharmacologic activation of C3aR reduces neutrophil mobilization, infiltration, and tissue injury after intestinal IR. (A) Peritoneal macrophages (pϕ cells) from WT mice showed a strong calcium mobilization response after the addition of the C3a agonist WWGKKYRASKLGLAR (1 μM); no such response was seen in C3aR^−/− macrophages. (B–E) WT or C3aR^−/− mice were treated with WWGKKYRASKLGLAR (10 μg/kg i.v.) or vehicle (saline) before intestinal ischemia. Note that the numbers of circulating neutrophils (B), histopathological scores (C), intestinal ALP levels (D), and intestinal MPO activity (E) were all significantly reduced in mice treated with the C3a agonist. n = 9–12 mice/group. Data are mean ± SEM. *P < 0.05; **P < 0.01.