Spleen hypoplasia leads to abnormal stress hematopoiesis in mice with loss of Pbx homeoproteins in splenic mesenchyme

Abstract

The spleen plays critical roles in immunity and also provides a permissive microenvironment for hematopoiesis. Previous studies have reported that the TALE-class homeodomain transcription factor Pbx1 is essential in hematopoietic stem and progenitor cells (HSPCs) for stem cell maintenance and progenitor expansion. However, the role of Pbx1 in the hematopoietic niche has not been investigated. Here we explored the effects that genetic perturbation of the splenic mesenchymal niche has on hematopoiesis upon loss of members of the Pbx family of homeoproteins. Splenic mesenchyme-specific inactivation of Pbx1 (SKO) on a Pbx2- or Pbx3-deficient genetic background (DKO) resulted in abnormal development of the spleen, which is dysmorphic and severely hypoplastic. This phenotype, in turn, affected the number of HSPCs in the fetal and adult spleen at steady state, as well as markedly impairing the kinetics of hematopoietic regeneration in adult mice after sub-lethal and lethal myelosuppressive irradiation. Spleens of mice with compound Pyx deficiency 8 days following sublethal irradiation displayed significant downregulation of multiple cytokine-encoding genes, including KitL/SCF, Cxcl12/SDF-1, IL-3, IL-4, GM-CSF/Csf2 IL-10, and Igf-1, compared with controls. KitL/SCF and Cxcl12/SDF-1 were recently shown to play key roles in the splenic niche in response to various haematopoietic stresses such as myeloablation, blood loss, or pregnancy. Our results demonstrate that, in addition to their intrinsic roles in HSPCs, non-cell autonomous functions of Pbx factors within the splenic niche contribute to the regulation of hematopoiesis, at least in part via the control of KitL/SCF and Cxcl12/SDF-1. Furthermore, our study establishes that abnormal spleen development and hypoplasia have deleterious effects on the efficiency of hematopoietic recovery after bone marrow injury.

Keywords: Cxcl12/SDF-1; GM-CSF; Nkx2-5+  spleen niche KitL/SCF; Pbx transcription factors; extramedullary hematopoiesis; hematopoietic stem cells; spleen hypoplasia.

Figure 1

Spleen mesenchyme specific inactivation of Pbx1 results in hypoplastic embryonic spleens with low hematopoietic progenitor cell content but does not affect in vitro hematopoietic progenitor function. (A) Gross appearance of E17.5 spleens from control and embryos with loss of Pbx1 alone (SKO) or loss of two Pbx genes (DKO) in Nkx2‐5‐positive splenic mesenchyme. Scale bars: 1 mm. (B,C) Methylcellulose colony assays performed with E16.5–17.5 spleen cells. (B) Representative images of BFU‐Es (*) and CFU‐GEMMs (**). Scale bars: 150 μm. (C) Colony counts at day 8 of culture. Histogram shows average data from three independent experiments. (D) Table summarizing total cell numbers (P = 0.016) and numbers of hematopoietic progenitors (P = 0.005) observed in a typical control and SKO spleen. (E) Lineage tracing analysis by flow cytometry in Nkx2‐5 ^Cre/+ ;Rosa ^{YFP /+} embryonic tissues confirms the absence of ectopic Nkx2‐5‐Cre activity in leukocytes. Data are mean percentage + SEM of CD45⁺ YFP ⁺ cells of total CD45⁺ cells collected from six embryos.

Figure 2

Loss of Pbx1 in spleen mesenchyme results in delayed hematopoietic recovery after acute radiation‐induced stress. (A‐C) Periodic sampling of peripheral blood to monitor multilineage hematologic recovery kinetics after increasing times (measured in days). (D) Following sublethal irradiation (IR) for age‐matched control (Ctrl; n = 10), SKO (n = 8), DKO (n = 10) adult mice. Each data point represents mean ± SEM. * P ≤ 0.05; ** P ≤ 0.01. (D) Histogram showing the average + SEM of control (n = 5) and DKO (n = 4) spleens before and 5 or 12 days following irradiation. P < 0.002. (E) H&E staining of spleen sections from 3‐month‐old mice at day 8 post‐irradiation reveals hypocellular WP in DKO spleens (indicated by white arrowheads) as opposed to normal WP histology in control and SKO (light blue arrowheads). Scale bars: 200 μm. (F) Histogram illustrating average bone marrow (BM) cell counts (as total nucleated blood cells) + SEM. Analysis conducted in two femurs per mouse (number of mice analyzed = 5), at the indicated time points. P = 0.04 at steady‐state; P = 0.05 at day (d) 12 post‐irradiation.

Figure 3

DKO spleens form sparse and minute in vivo colony‐forming units (CFU‐S). (A) Light micrographs of CFU‐S colonies at day 12 (CFU‐S₁₂) (highlighted by white arrowheads) in sub‐lethally irradiated control (Ctrl), SKO, and DKO mice. The number of CFU‐S₁₂ colonies was counted after fixation of the spleen and represented by a histogram. The size and number of colonies were significantly reduced in DKO spleens (P = 0.03). Scale bars: 2 mm. (B) H&E stained histological sections of the spleens shown in (A) demonstrate robust stress‐induced HSPC expansion in control and SKO spleens but not in DKOs. Red arrowheads point to CFU‐S_12, some of which are delimited by dashed white lines. Scale bars: 500 μm.

Figure 4

Impaired in vitro hematopoietic progenitor function in Pbx1 ^f/f ;Pbx2 ^+/− ;Nkx2‐5 ^Cre/+ (DKO) mutant spleens compared with controls. (A‐C) Control (Ctrl) and DKO spleens and bone marrow were harvested at the indicated time points (day; d) before or after irradiation. Subsequently, single cell suspensions were prepared and plated on Methocult^™ media containing SCF, IL‐3, IL‐6, EPO, insulin and transferrin. The number of colonies of erythroid (CFU‐E, BFU‐E), myeloid (CFU‐GM, CFU‐M, CFU‐G), and multipotent progenitors (CFU‐GEMM) was counted after 3 or 9 days of culture. n = 3 or 4 per group; error bars indicate mean + SEM. *P < 0.05; **P < 0.01.

Figure 5

Decreased numbers of hematopoietic stem and progenitor cells in Pbx‐deficient splenic mesenchyme. (A–D) Representative FACS analyses are shown for spleen cells from control (Ctrl) and DKO age‐matched, non‐irradiated mice. HSCs (LSK cells) are defined as Lin^– cKit⁺Sca1⁺; LT‐HSCs as Lin⁻ cKit⁺Sca1⁺ CD34⁻; ST‐HSCs^# as Lin⁻ cKit⁺Sca1⁺ CD34⁺ [note: this phenotyping also includes spleen‐specific stress erythroid progenitors (Harandi et al. 2010)]; MEPs as Lin⁻ cKit⁺Sca1⁻ CD34⁻FcγRII/III ⁻; GMPs as Lin⁻ cKit⁺Sca1⁻ CD34⁺FcγRII/III ⁺; CMPs as Lin⁻ cKit⁺Sca1⁻ CD34⁺FcγRII/III ^lo; BFU‐Es as Lin⁻ cKit⁺Sca1⁻ CD71^lo; CFU‐Es as Lin⁻ cKit⁺Sca1⁻ CD71⁺; proerythroblasts as Lin⁻ cKit^loSca1⁻ CD71⁺. (E,F) Flow cytometric quantitation of indicated populations in spleen and BM. Ctrl n = 5, DKO n = 4 per time point. (G) Flow cytometry for spleen erythroid progenitors (Lin⁻Ter119⁻ CD71⁺) at day 8 following irradiation. Top panel: representative contour plots for CD71 vs. Ter119 after gating on lineage negative population. Bottom panel: bar charts displaying the average numbers of erythroid progenitors in Ctrl, SKO, DKO spleens (n = 3 per cohort). Error bars indicate mean + SEM; *P ≤ 0.05; **P < 0.01.

Figure 6

Impaired expression of cytokine‐encoding genes in DKO spleens at steady‐state and during post‐injury hematopoiesis. (A,B) Control (Ctrl; n = 3) and DKO (n = 3) spleens were enzymatically digested to generate single cell suspensions and erythrocytes were lysed, following which RNAs were prepared from the cell pellets. Graphs show differentially expressed genes that code for secreted factors and receptors with known functions in facilitating hematopoietic regeneration. Notably, KitL (also known as SCF) and Cxcl12 are markedly down‐regulated in Pbx mutant spleens. The housekeeping transcript Hprt was used to normalize RT‐qPCR. Error bars indicate mean + SEM; *P < 0.05; **P < 0.01; ***P < 0.001; n.s., non‐significant. (A) Transcripts for 12 factors are downregulated 2‐ to 15‐fold in irradiated Pbx DKO vs. control spleens. (B) Transcripts of seven additional cytokines are downregulated < 2‐fold in irradiated DKO spleens vs. controls.

Figure 7

Pbx‐dependent spleen hypoplasia and morphogenesis defects cause impaired production of cytokines and growth factors in the splenic niche. (A) Normal splenic niche function requires developmental expression of Pbx genes in the Nkx2–5⁺ mesenchyme (Pbx⁺ Mesenchyme). (B) Splenic mesenchyme‐specific inactivation of Pbx1 on Pbx2‐ or Pbx3‐deficient background (Pbx⁻ mesenchyme) results in spleen stromal hypoplasia and morphogenesis defects that impact the niche. The cellular components of the splenic niche are illustrated at the bottom of the cartoon. Our study establishes that genetic perturbation of splenic stromal development due to loss of Pbx factors is associated with impaired expansion and maintenance of HSCs, as well as myeloid and erythroid progenitors, during injury‐induced hematopoietic regeneration. This Pbx‐dependent hematopoietic phenotype results at least in part from the downregulation of genes encoding interleukins, colony‐stimulating factors, KitL and Cxcl12, all of which are essential growth factors for hematopoietic reconstitution after BM damage.