Role for SpiC in the development of red pulp macrophages and splenic iron homeostasis

Tissue macrophages comprise a heterogeneous group of cell types differing in location, surface markers and function. Red pulp macrophages are a distinct splenic subset involved in removing senescent red blood cells. Transcription factors such as PU.1 (also known as Sfpi1) and C/EBPa (Cebpa) have general roles in myelomonocytic development, but the transcriptional basis for producing tissue macrophage subsets remains unknown. Here we show that Spi-C (encoded by Spic), a PU.1-related transcription factor, selectively controls the development of red pulp macrophages. Spi-C is highly expressed in red pulp macrophages, but not monocytes, dendritic cells or other tissue macrophages. Spic mice have a cell-autonomous defect in the development of red pulp macrophages that is corrected by retroviral Spi-C expression in bone marrow cells, but have normal monocyte and other macrophage subsets. Red pulp macrophages highly express genes involved in capturing circulating haemoglobin and in iron regulation. Spic mice show normal trapping of red blood cells in the spleen, but fail to phagocytose these red blood cells efficiently, and develop an iron overload localized selectively to splenic red pulp. Thus, Spi-C controls development of red pulp macrophages required for red blood cell recycling and iron homeostasis. Spi-C belongs to the Spi subfamily of Ets transcription factors, which includes PU.1 and Spi-B, and was initially reported to be expressed in B cells. We compared expression of PU.1, Spi-B and Spi-C across a panel of immune cells such as B cells, bone marrow monocytes, dendritic cells and several types of resident tissue macrophages, including red pulp macrophages (RPMs; Fig. 1a, Supplementary Fig. 1). PU.1 was broadly expressed and Spi-B was predominantly restricted to B cells. In contrast, Spi-C was highly expressed in RPMs, expressed at lower levels in B cells, and essentially absent in other cells. To test for a role of Spi-C in RPMs and B cells, we generated mice lacking Spic expression by gene targeting (Supplementary Fig. 2). Male and female Spic mice are fertile and healthy, with a normal lifespan, but are born at a somewhat lower than expected Mendelian frequency (Supplementary Fig. 2). We also generated Spic mice, in which the neomycin cassette was deleted from the targeted Spic allele (Supplementary Fig. 2). RPMs have been defined as F4/80CD68CD11b cells with strong autofluorescence. Both Spic mice and Spic mice showed a phenotype characterized by the selective loss of RPMs (Fig. 1b and c, Supplementary Fig. 3a), but showed no abnormalities in the development of B cells, conventional dendritic cells, plasmacytoid dendritic cells (Supplementary Figs 3 and 4, and Supplementary Table 1) or T cells (Supplementary Fig. 5), and had normal serum immunoglobulin levels (Supplementary Fig. 4f). We confirmed the loss of RPMs by immunohistochemical analysis, finding an almost complete loss of F4/80 cells in the splenic red pulp (Fig. 2a). In contrast, SIGN-R1 (also known as CD209a)-expressing marginal zone macrophages and MOMA-1 (also known as Siglec1)-expressing metallophilic marginal zone macrophages were present normally in the spleens of Spic mice (Fig. 2a). Spic mice had normal F4/80 tissue macrophages in peritoneum and liver, normal macrophage and dendritic cell progenitors in bone marrow, and normal monocytes in bone marrow and blood (Supplementary Fig. 6). Loss of RPMs could result either from a cell-autonomous defect or from the loss of a required extrinsic component such as a bone marrow or splenic stromal cell. To distinguish these possibilities, we generated haematopoietic chimaeras by transplanting bone marrow cells from CD45.2 Spic or Spic mice into irradiated congenic CD45.1