Dissecting the complement pathway in hepatic injury and regeneration with a novel protective strategy

Abstract

Liver resection is commonly performed under ischemic conditions, resulting in two types of insult to the remnant liver: ischemia reperfusion injury (IRI) and loss of liver mass. Complement inhibition is recognized as a potential therapeutic modality for IRI, but early complement activation products are also essential for liver regeneration. We describe a novel site-targeted murine complement inhibitor, CR2-CD59, which specifically inhibits the terminal membrane attack complex (MAC), and we use this protein to investigate the complement-dependent balance between liver injury and regeneration in a clinical setting of pharmacological inhibition. CR2-CD59 did not impact in vivo generation of C3 and C5 activation products but was as effective as the C3 activation inhibitor CR2-Crry at ameliorating hepatic IRI, indicating that the MAC is the principle mediator of hepatic IRI. Furthermore, unlike C3 or C5 inhibition, CR2-CD59 was not only protective but significantly enhanced hepatocyte proliferation after partial hepatectomy, including when combined with ischemia and reperfusion. Remarkably, CR2-CD59 also enhanced regeneration after 90% hepatectomy and improved long-term survival from 0 to 70%. CR2-CD59 functioned by increasing hepatic TNF and IL-6 levels with associated STAT3 and Akt activation, and by preventing mitochondrial depolarization and allowing recovery of ATP stores.

Figure 1.

In vitro characterization of CR2-CD59. (A) Flow cytometric analysis of CR2-CD59 binding to C3-opsonized CHO cells. Antibody-sensitized CHO cells were incubated with C6^−/− mouse serum followed by incubation with CR2-CD59 (thick black trace) or PBS (dark gray trace). CR2-CD59 was also incubated with unopsonized cells, either without antibody (light gray trace) or without serum (thin black trace). Shown is a representative of 3 separate experiments. (B) CR2-CD59 inhibition of complement-mediated RBC lysis. Antibody-sensitized chicken RBCs were incubated with either CR2-Crry or CR2-CD59 in the presence of mouse serum. (C) Effect of CR2-CD59 on C3 activation. Activated zymosan particles were incubated with mouse serum and increasing doses of CR2-Crry or CR2-CD59, and C3 deposition on particles detected by flow cytometry. Data are presented as mean ± SEM (n = 2–3) and are representative of 2–4 independent experiments.

Figure 2.

Effect of CR2-CD59 and CR2-Crry on outcomes after hepatic IRI. Mice were subjected to 30 min of total warm ischemia and treated with PBS, CR2-Crry, or CR2-CD59 upon reperfusion. (A) Histological quantification of hepatic necrosis and injury in H&E-stained sections, scored on a scale of 0–3. Samples obtained 6 h after reperfusion. (B) Serum ALT levels in samples obtained 6 h after reperfusion. (C) Hepatic tissue levels of MPO measured 24 h after reperfusion by ELISA. (D) Hepatic tissue levels of IL-18 measured 24 h after reperfusion by ELISA. (E) Hepatic C5a levels 24 h after reperfusion. All ELISA measurements were normalized to total protein content. For A–E, results are expressed as mean ± SEM (n = 4–9) and are representative of 2–3 independent experiments; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns = not significant, assessed by ANOVA. (F) C3d and MAC deposition 24 h after reperfusion. Shown are representative images of immunostained liver sections, with 4 animals analyzed per group. Bars, 20 µM.

Figure 3.

Biodistribution and localization of CR2-CD59 after hepatic IRI. (A) Biodistribution of CR2-CD59 after IRI. ¹²⁵I-labeled CR2-CD59 was injected into mice immediately after IR or after a sham operation, and tissue distribution of radiolabel was assessed after 2 h reperfusion. Shown are the combined results from 3 independent experiments of 1 mouse per group (mean ± SEM); *, P < 0.05 versus all other samples, as assessed by ANOVA. (B) Hepatic localization of C3d and CR2-CD59 after IRI. CR2-CD59 was injected into WT or CD59^−/− mice immediately after IR or after a sham operation, and livers were isolated after 2 h reperfusion. Serial sections were prepared and analyzed by immunohistochemistry for C3d and CR2-CD59 binding. Representative images, n = 3. Bars, 20 µM.

Figure 4.

Effect of complement inhibition on outcomes after 70% PHx. Mice were treated with PBS, CR2-Crry, or CR2-CD59 immediately after surgery, and the following determinations were made 48 h after surgery. (A) Serum ALT. (B) Histological quantification of hepatic necrosis and injury in H&E-stained sections, scored on a scale of 0–3. (C) Liver weight restitution (D) Assessment of liver regeneration by BrdU incorporation, detected by immunohistochemistry and expressed as % positive cells in 10 high-power fields. Results are expressed as mean ± SEM (n = 4–6) and are representative of 3 independent experiments; ***, P < 0.001; *, P < 0.05, assessed by ANOVA. (E) Effect of CR2-Crry and CR2-CD59 on complement activation. C3d and MAC deposition was analyzed in sections prepared from livers isolated 48 h after surgery. Shown are representative images of immunostained liver sections, with 4 animals analyzed per group. Bars, 20 µM.

Figure 5.

Effect of C3aRA, anti-C5 mAb, and CR2-CD59 treatment on outcome after 70 PHx. Mice were treated with C3aRA or anti-C5 mAb, with or without CR2-CD59 co-treatment, and the following determinations were made 48 h after surgery. (A) Serum ALT. (B) Histological quantification of hepatic necrosis and injury in H&E-stained sections (C) Assessment of liver regeneration by BrdU incorporation, detected by immunohistochemistry and expressed as % positive cells in 10 high-power fields. (D) Survival. Data are combined from 3 independent experiments (n = 8). Results for A–C are expressed as mean ± SEM. ^#, P > 0.05: no significant difference between any complement inhibitor–treated group, assessed by ANOVA.

Figure 6.

Effect of complement inhibition on outcomes in a model incorporating both IRI and 70% PHx. Mice were treated with PBS, CR2-Crry, or CR2-CD59 immediately after combined surgery. (A) Serum ALT levels. (B) Histological quantification of hepatic necrosis and injury in H&E-stained sections. (C) Hepatic tissue levels of MPO. (D) Assessment of liver regeneration by BrdU incorporation. (E) Liver weight restitution. (F) Mouse survival. All determinations were made 48 h after surgery. (G–J) Hepatic and serum cytokines were measured by ELISA. (G) Hepatic IL-6 levels 3 h after surgery. (H) Hepatic TNF levels 3 h after surgery. (I) Serum IL-6 levels 6 h after surgery. (J) Serum TNF levels 6 h after surgery. Results are expressed as mean ± SEM (n = 4–6) and are representative of 3 independent experiments; ***, P < 0.001; **, P < 0.01, assessed by ANOVA.

Figure 7.

CR2-CD59 protects against acute hepatic failure after 90% PHx. Mice were treated with PBS, CR2-Crry, or CR2-CD59 immediately after 90% PHx. (A) Serum ALT levels 6 h after surgery. (B) Histological quantification of hepatic necrosis and injury in H&E-stained sections, scored on a scale of 0–3. (C) Assessment of liver regeneration by BrdU incorporation, detected by immunohistochemistry and expressed as % positive cells counted in 10 high-power fields, determined 24 h after surgery. Results are expressed as mean ± SEM (n = 6) and are representative of 3 independent experiments; ***, P < 0.00; **, P < 0.01; *, P < 0.05, as assessed by ANOVA. (D) Survival analysis over a 7-d period after surgery. Difference between the CR2-CD59–treated group and CR2-Crry– or PBS-treated groups was statistically significant as determined by the Kaplan-Meier test (P < 0.05). Combined data from 2 independent experiments (n = 12).

Figure 8.

Systemic and local cytokine production after 90% PHx. Mice were treated with PBS, CR2-Crry, or CR2-CD59 immediately after 90% PHx. Hepatic and serum cytokines were measured by ELISA, normalized to total protein content. (A) Hepatic IL-6 levels 3 h after surgery. (B) Hepatic TNF levels 3 h after surgery. (C) Serum IL-6 levels 6 h after surgery. (D) Serum TNF levels 6 h after surgery. Results are expressed as mean ± SEM (n = 4–6) and are representative of 3 independent experiments; ***, P < 0.00, as assessed by ANOVA.

Figure 9.

Stat3 and Akt activation occurs in CR2-CD59–treated mice but not CR2-Crry–treated mice after 90% PHx. Mice were treated with PBS, CR2-Crry, or CR2-CD59 immediately after 90% PHx. (A) Representative Western blot of STAT3, p-STAT3, Akt, and p-Akt in liver homogenate at 3 h after surgery. (B) Densitometry. Results are expressed as mean ± SEM (n = 4–6) and are representative of 3 independent experiments; ***, P < 0.001, as assessed by ANOVA.

Figure 10.

CR2-CD59 treatment prevents mitochondrial depolarization and promotes recovery of ATP stores after 90% PHx. Mice underwent 90% PHx or sham operation, and resected mice were treated with either PBS or CR2-CD59 immediately after surgery. For intravital microscopy study, live animals were infused intravascularly with Rh123 fluorescence and PI (live/dead) stain into the carotid artery 2 h after resection. (A) Representative confocal image of hepatic Rh123 (green) and PI (red). Bars, 10 µM. (B) Percentage of hepatocytes with depolarized mitochondria in 10 high-power fields. Shown are the combined results from 4 independent experiments of 1 mouse per group (mean ± SEM). (C) ATP content in liver tissue taken at different time points after 90% PHx. Results are expressed as mean ± SEM (n = 4–6) and are representative of 3 independent experiments; ***, P < 0.001; **, P < 0.01, as assessed by ANOVA.