Recombinant factor VIII Fc fusion protein drives regulatory macrophage polarization

Abstract

The main complication of replacement therapy with factor in hemophilia A (HemA) is the formation of inhibitors (neutralizing anti-factor VIII [FVIII] antibodies) in ∼30% of severe HemA patients. Because these inhibitors render replacement FVIII treatment essentially ineffective, preventing or eliminating them is of top priority in disease management. The extended half-life recombinant FVIII Fc fusion protein (rFVIIIFc) is an approved therapy for HemA patients. In addition, it has been reported that rFVIIIFc may induce tolerance to FVIII more readily than FVIII alone in HemA patients that have developed inhibitors. Given that the immunoglobulin G1 Fc region has the potential to interact with immune cells expressing Fc receptors (FcRs) and thereby affect the immune response to rFVIII, we investigated how human macrophages, expressing both FcRs and receptors reported to bind FVIII, respond to rFVIIIFc. We show herein that rFVIIIFc, but not rFVIII, uniquely skews macrophages toward an alternatively activated regulatory phenotype. rFVIIIFc initiates signaling events that result in morphological changes, as well as a specific gene expression and metabolic profile that is characteristic of the regulatory type Mox/M2-like macrophages. Further, these changes are dependent on rFVIIIFc-FcR interactions. Our findings elucidate mechanisms of potential immunomodulatory properties of rFVIIIFc.

Graphical abstract

Figure 1.

rFVIIIFc induces Fcγ receptor internalization and signaling in an Fc-dependent fashion. (A) Forward scatter (FSC)/side scatter (SSC) pseudocolor plots of macrophages treated with IgG1, rFVIII, or rFVIIIFc (each at 200 nM) for 24 hours, illustrating the size change of the cells upon rFVIIIFc treatment. Numbers in FSC/SSC pseudocolor plots indicate the percentage of smaller and larger cell populations. One representative experiment is shown. (B) Graph shows cumulative data on macrophage size based on the percentages of low-FSC and high-FSC cell populations, observed by flow cytometry (n = 12). Significance indicated compared with untreated cells. (C) Macrophages (n = 4) were treated with HRP-ICs as positive control, IgG1 as negative control, rFVIII, or rFVIIIFc for 24 hours, and the cell surface expression of the FcγRs CD16, CD32, and CD64 was measured by flow cytometry. Downregulation of the FcγRs from the cell surface indicates internalization. (D) Macrophages (n = 10) were treated with HRP-IC, IgG1, rFVIII, or rFVIIIFc for 15 minutes, and the phosphorylation of Syk was measured using the Meso Scale Diagnostics (MSD) platform. Upregulation of pSyk levels shows direct FcγR engagement by HRP-IC and rFVIIIFc. (E) Mutant rFVIIIFc molecules unable to bind the FcRn (FcRn mutant) or the FcγRs (FcγR mutant) were used to treat macrophages (n = 4) along with the wild-type rFVIIIFc (WT), IgG1, and rFVIII for 30 minutes, and Syk phosphorylation was measured using the MSD platform. Downregulation of pSyk after FcRn and FcγR mutant treatment compared with WT rFVIIIFc shows the role of these receptors in signal transduction. Mean ± SE; *P ≤ .05, ***P ≤ .005.

Figure 2.

rFVIIIFc treatment does not activate macrophages. (A) Macrophages (n = 3) were treated with IgG1, rFVIII, or rFVIIIFc for 24 hours, and the cell surface expression of the costimulatory/activation molecules CD40 and CD80 were measured by flow cytometry. The absence of significant molecule upregulation shows no classical activation of macrophages upon rFVIIIFc treatment. (B) Macrophages (n = 8) were treated with HRP-IC, IgG1, rFVIII, or rFVIIIFc for 24 hours, and the production of interleukin-1β (IL-1β), IL-6, IL-8, IL-10, and tumor necrosis factor α (TNFα) cytokines were measured from cell supernatants using multiplex enzyme-linked immunosorbent assay. Compared with the proinflammatory activation signal from HRP-IC, rFVIIIFc treatment of macrophages did not significantly upregulate proinflammatory cytokine production (error bars and significance not shown for graph clarity). RNA and protein levels of the alternatively activated macrophage markers mannose receptor CD206 (n = 7) (C) and NRP1 (n = 3) (D) were measured by quantitative PCR (qPCR) and flow cytometry, respectively. (E) Macrophages (n = 3) were treated with IgG1, rFVIII, or rFVIIIFc for 1, 5, or 30 minutes, and phosphorylated SHIP1 was measured using the MSD platform. Significant elevation of pSHIP1 levels after rFVIIIFc treatment compared with IgG1 or rFVIII treatments shows triggering of inhibitory signaling events. Mean ± SE; *P ≤ .05, **P ≤ .01, ***P ≤ .005.

Figure 3.

rFVIIIFc induces specific gene expression pattern in macrophages, resembling regulatory phenotype. Macrophages were treated with IgG1, rFVIII, or rFVIIIFc for 6 hours, and total RNA was isolated for high-throughput RNA sequencing. (A) Venn diagrams show significantly up- and downregulated genes between treatment groups (n = 3). (B) Pathway analysis of the first 200 significantly upregulated genes by rFVIIIFc treatment identifies NRF2 and lipid metabolism pathways as overrepresented processes affected by rFVIIIFc treatment. Validation of gene expression changes initiated by rFVIIIFc: NRF2 pathway–related genes (C), lipid metabolism–related genes (D), downregulated genes (E) (n = 6-15). (F) Macrophages (n = 12) were treated with IgG1, rFVIII, or rFVIIIFc for 24 hours, and the intracellular protein levels of HO-1 were measured by flow cytometry. Expression of HO-1 (G) and PPARγ (H) were followed, after treatment with IgG1, rFVIII, rFVIIIFc, modified rFVIIIFc unable to bind to FcRn (FcRn mutant), or modified rFVIIIFc unable to bind to FcγRs (FcγR mutant) for 6 hours (PPARγ gene expression) or 24 hours (HO-1 protein expression). Macrophages were also pretreated with IgG1 (pre-IgG1) or rFVIII (pre-rFVIII) for 1 hour before 24 hours rFVIIIFc treatment (n = 5). Mean ± SE; *P ≤ .05, **P ≤ .01, ***P ≤ .005.

Figure 4.

rFVIIIFc-treated macrophages exhibit a characteristic metabolic signature, resembling the regulatory phenotype. Metabolite screening was performed on lysates of macrophages (n = 4) treated with IgG1, rFVIII, or rFVIIIFc for 6 hours or 24 hours. (A) Of a total of 578 identified metabolites, 144 were changed significantly between the different treatments. Selected individual metabolites are shown: glycolysis and related metabolism pathways (B), long chain fatty acids (C), and oxidative phosphorylation (D). Mean ± standard deviation; *P ≤ .05. UDP, uracil-diphosphate.

Figure 5.

rFVIIIFc-treated macrophages exhibit elevated mitochondrial activity, bioenergy, and glutathione production. Macrophages were treated with IgG1, rFVIII, or rFVIIIFc for 24 hours. Significantly elevated levels of mitochondrial transmembrane potential (A) and active mitochondria staining (B) were measured from lysates of the rFVIIIFc-treated cells, compared with other treatments (n = 3). None of the treatments altered the total mitochondrial mass (C) indicating that rFVIIIFc-educated macrophages specifically upregulate their oxidative phosphorylation capacity (n = 3). Significantly elevated production of ATP (D, n = 5) and GSH (E, n = 3; F, n = 5) of rFVIIIFc-treated macrophages was measured compared with other treatments. (G) Endogenous PPARγ ligands 13-hydroxyoctadecadienoic acid (13-HODE) and 9-HODE were produced by rFVIIIFc-treated macrophages (n = 3). (H) Metabolism status of rFVIIIFc-treated macrophages is illustrated. Mean ± SE; *P ≤ .05.

Figure 6.

HemA macrophages respond to rFVIIIFc treatment similarly to healthy individuals; plasma samples from individuals with hemophilia exhibit compromised antioxidant function. Macrophages from healthy donors and inhibitor-negative HemA patients were treated with IgG1, rFVIII, or rFVIIIFc for 6 hours, and RNA was isolated. Gene expression changes of HO-1 (n = 16 healthy donors and n = 7 HemA patients) (A) and PPARγ (n = 15 healthy donors and n = 7 HemA patients) (B) were measured by qPCR. (C) Antioxidative capacity of healthy vs inhibitor-negative vs inhibitor-positive HemA plasma samples (n = 27 each) shows significant impairment in hemophilia patients. Mean ± SE; *P ≤ .05, **P ≤ .01, ***P ≤ .005.