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. 2018 Nov 5;2(21):2904–2916. doi: 10.1182/bloodadvances.2018024497

Figure 3.

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.