Critical role for Spi-C 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 function1. Red pulp macrophages are a distinct splenic subset involved in removing senescent red blood cells2. Transcription factors such as PU.1 and C/EBPα play general roles in myelomonocytic development3,4, but the transcriptional basis for producing tissue macrophage subsets remains unknown. Here we show that Spi-C, 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. Spi-C−/− mice exhibit 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 hemoglobin and iron regulation. Spi-C−/− 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.

Bone marrow monocytes were purified by cell sorting for CD11b + Ly6C hi cells using CD11b-PE and Ly6C-FITC, excluding CD11b + Ly6C int neutrophils by gating. The final purity was >92% CD11b + Ly6C hi cells. Conventional splenic DCs were purified by cell sorting for CD11c hi F4/80 lo/cells using CD11c-PE and F4/80-FITC, excluding F4/80 hi red pulp macrophages by gating. The final purity was >95% CD11c hi F4/80 lo/cells. Red pulp macrophages were purified by cell sorting for F4/80 hi autofluorescent cells using F4/80-FITC, excluding CD11c hi DCs by gating. The final purity was >90% F4/80 hi autofluorescent cells. RNA was isolated using RNeasy kits (Qiagen) and biotinylated cRNA probes were prepared using one or two cycle target preparation kits (Affymetrix). Global gene expression analysis was performed using mouse expression 430 2.0 arrays (Affymetrix). Data were normalized and expression values were modeled using DNA-Chip Analyzer (dChip) 2 .
Quantitative real-time PCR. For gene expression analysis, total RNA and cDNA were prepared from various cell types with the RNeasy Mini Kit (Qiagen) and Superscript III reverse transcriptase (Invitrogen). For real-time PCR, the relative standard curve method, SYBR Green PCR master mix and an ABI7000 machine (Applied Biosystems) were used according to the manufacturer's instructions. PCR conditions were 2 min at 50 °C and 10 min at 95 °C, followed by 40 two-step cycles consisting of 15 s at 95 °C and 1 min at 60 °C. The values were normalized to that of HPRT by dividing the average copy number of the respective transcript sample.

Serum immunoglobulins.
Basal serum immunoglobulin levels were quantified by ELISA using the SBA Clonotyping System/HRP (SouthernBiotech).

Retroviral constructs and infection.
Murine Spi-C cDNA was amplified from C57BL/6 splenocyte RNA using Superscript III (Invitrogen) and oligonucleotides (5'-Spi-C-Bgl II, AGAGATCTGCAACCCAAGACTCTTCAATTC; and 3'-Spi-C-XhoI, CCACTCGAGAGGCAAGAAGCTGGGGTCA). To generate the Spi-C-RV retroviral plasmid, the Spi-C PCR product was digested with BglII and XhoI, and cloned into BglII/XhoI-digested GFP-RV 3 . Retroviral vectors were packaged using Phoenix E cells as described 3  μF, divided equally among three wells, and harvested 20 h later and assayed for Luciferase activity measured and normalized using CMV-Renilla as described 3 .
Competitions used 50-fold excess unlabeled annealed probes added 15 min prior to labeled probe. No complexes were found to form using the ECR1 Ets probe taken from another potential Ets GGAA consensus located upstream in the VCAM-1 promoter located at -1490 to -1479. Oligonucleotide sequences used were: Spi-C -/-(-/-) mice. Intercrosses of heterozygous Spi-C +/mice was found to generate genotypes at the following frequencies (30%, +/+; 60%, +/-; 9%, -/-; n=551), indicating that some loss of Spi-C -/embryos is occurring before birth. b, Bone marrow cells from Spi-C +/+ or Spi-C null/null mice were stained for B220, CD43, and either BP-1 and CD24, or IgM and IgD, to identify B cell development subsets as described 9 . No differences were seen in the percentages of B220 + CD43 + (A-C) or B220 + CD43 -(D-F) fractions between Spi-C +/+ or Neo-deleted-Spi-C null/null bone marrow cells. The percentages were similar between the subsets of B220 + CD43 + cells;

Supplementary Figure 4. B cell and DC development are normal in Spi-C -/mice.
a, Spleen cells were stained for B220 and IgM to identify B-1 B cells (IgM + B220 lo/-) and B-2 B cells (IgM + B220 hi ). b, Spleen cells were stained for CD11b and CD11c to identify conventional DC (CD11c hi ). c, CD11c low CD11bcells in b were gated and examined for expression of B220 and Gr-1 to identify plasmacytoid DC (CD11c lo Cd11b -B220 + Gr1 + ). Numbers shown are the percentage of live cells within the indicated gate. d, Spleen cells from Spi-C +/+ or Spi-C -/mice were stained with antibodies to B220, CD23 and AA4.1. Similar development of mature (B220 + AA4.1 -) B cells and immature (B220 + , AA4.1 + ) B cells is evident between Spi-C +/+ and Spi-C -/mice (upper panels).

Supplementary Figure 8. Spi-C -/mice have increased splenic iron stores. a,
Spi-C -/mice show iron accumulation in spleen. Tissue iron levels in spleen (left panel) and liver (right panel) of Spi-C +/+ (white bars) and Spi-C -/-(black bars) maintained on a standard diet (mean ± SD, n=4) (d). b, Serum iron levels were determined for Spi-C +/+ (white) and Spi-C -/-(black) female mice maintained on a standard diet. c and d, Perl's Prussian blue stain for ferric iron in the spleens(c) and liver (d) of 32-week old Spi-C +/+ and Spi-C -/female mice fed a standard diet. Figure 9. Spleen histology in Spi-C -/mice. a, Spleen of Spi-C +/+ and Spi-C -/mice were stained with hematoxylin and eosin. Increased hemosiderin is evident in the red pulp regions of Spi-C -/spleens. b, Electron microscopy of spleen