TMPRSS6 Inhibition Rapidly Reverses Liver Iron Overload and Prevents an Increase of Splenic Pro-Inflammatory Macrophages in a Mouse Model of Beta-Thalassemia

Beta-thalassemia is a genetic disorder arising from loss-of-function mutations in the beta-globin gene, leading to ineffective erythropoiesis, elevated red blood cell (RBC) turnover, and organ iron overload. Iron overloads contributed to liver fibrosis, type 2 diabetes, cancer, and cardiac toxicities. The transmembrane serine protease 6 (TMPRSS6) negatively regulates hepcidin, the main regulator of iron homeostasis. Individuals with homozygous loss-of-function mutations in the TMPRSS6 gene have iron-restricted/iron-deficiency anemia and show high levels of circulating hepcidin. We have shown that inhibition of TMPRSS6 using a monoclonal antibody (REGN7999) rapidly reverses liver iron loading and splenomegaly in a mouse model of beta-thalassemia (Hbb^th3/+) by using non-invasive MRI. Here we found that Hbb^th3/+ mice had significantly higher R2* values (WT 0.09 vs. Hbb^th3/+ 0.11 ms^-1) at the beginning of the study. With REGN7999 treatment, the liver iron content was significantly reduced within 31 days after the first injection. Spleen volume, which was significantly increased in Hbb^th3/+ mice at the beginning of the study, was normalized 17 days after REGN7999 injection, 2 weeks before we observed changes in liver iron loading. Interestingly, we found that splenic iron concentration in Hbb^th3/+ mice treated with REGN7999 remained as high as isotype injected controls. We hypothesized that the liver iron remains trapped within the macrophages in the spleen, due to high hepcidin levels and reduced ferroportin expression. We investigated, if the increased iron in these macrophages induces a pro-inflammatory polarization, which can contribute to chronic inflammation. We injected 5-7-week-old Hbb^th3/+ mice with REGN7999 or isotype control and measured splenic macrophage polarization as well as serum cytokine levels after 4 weeks of treatment. Hbb^th3/+ mice controls showed an increase in tissue resident macrophages (F4/80 positive), which was normalized with REGN7999 treatment. We measured the ratio of inducible Nitric Oxide Synthase (iNOS) and Arginase 1 (Arg 1) to determine if these macrophages are pro- or anti-inflammatory polarized. Hbb^th3/+ mice showed an increase of the iNOS/Arg 1 ratio when compared to wild-type mice, suggesting a pro-inflammatory profile. REGN7999 treatment reduced this ratio, suggesting a reduction of pro-inflammatory macrophages. In support of this, we found a significant reduction of Granulocyte/Macrophage-colony-stimulating-factor in the serum of Hbb^th3/+ mice treated with REGN7999.

Taken together, these data suggest that splenic macrophages are not more pro-inflammatory in response to TMPRSS6 inhibition, despite iron load status.

Lob:Regeneron Pharmaceuticals, Inc.: Current Employment. Noakes:Regeneron Pharmaceuticals, Inc.: Current Employment. Preda:Regeneron Pharmaceuticals, Inc.: Current Employment. Ivanova:Regeneron Pharmaceuticals, Inc.: Current Employment. Crowell:Regeneron Pharmaceuticals, Inc.: Current Employment. Economides:Regeneron Pharmaceuticals, Inc.: Current Employment. Hatsell:Regeneron Pharmaceuticals, Inc.: Current Employment.