S100 proteins govern cellular proliferation, differentiation, apoptosis, calcium homeostasis, energy metabolism, and inflammation. This heterodimeric calcium-binding protein, consisting of the S100A8 and S100A9 subunits, modulates myeloid cell development. S100A9 elevation has been found in myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML)
1–3. Notably, transgenic mice with S100A9 overexpression develop myeloid dysplasia that closely mimics human MDS
2. Haploinsufficiency in Rps14, a human del(5q) MDS-associated gene, leads to impaired erythroid differentiation and triggers overexpression of S100A8/S100A9
3. However, whether endogenous S100A9 is essential for normal or pathological hematopoiesis
in vivo is largely unknown.
We next asked whether S100A9 might play roles in hematological malignant conditions, such as MPN and MDS. We first crossed
S100A9−/− mice with MPN mice (
JAK2V617F/+Vav-Cre)—a model closely resembling human polycythemia vera. The
JAK2V617F mutation attenuated survival regardless of the presence of S100A9 expression (
Figure 1A). Moreover, the splenomegaly in
JAK2V617F mutant mice was not alleviated by S100A9 deletion (
Figure 1B). BM and SP cellularity did not differ between
JAK2V617FVav-Cre and
JAK2V617FS100A9−/−Vav-Cre mice (
Figure 1C). Erythrocytosis promoted by
JAK2V617F mutation was not alleviated by S100A9 depletion, despite a decline in platelet counts observed in
JAK2V617FS100A9−/−Vav-Cre mice (
Figure 1D and Figure S5A–T). This outcome, combined with the previously described role of S100A9 in facilitating the formation of procoagulant platelets through GPIbα
4, underscores S100A9’s role in promoting thrombopoiesis. Subsequently, we conducted lineage analysis through flow cytometry assays. The
JAK2V617F mutant mice exhibited aberrant elevations in both the proportions and cell counts of TER119
+ erythroid cells in the BM and SP (
Figure 1D and Figure S5U). However, the deletion of S100A9 in the presence of
JAK2V617F mutation only slightly ameliorated the absolute number of erythroid cells in the BM but not the SP (
Figure 1D), and the percentages remained unchanged (
Figure S5U). Likewise, myeloid and lymphoid cells in BM or SP showed no differences between
JAK2V617F mutant mice and JAK2/S100A9 double-mutant mice (
Figure 1D and Figure S5V). These results suggest that S100A9 does not substantially contribute to the pathogenesis of
JAK2V617F/+-driven polycythemia. A prior study has indicated that the overexpression of S100A9, triggered by the
Srsf2P95H mutation, hindered polycythemia in
JAK2V617F/+ mice
5. Together, these results indicate that activation, rather than deletion, of S100A9 might be a suitable approach for MPN therapy.
We have previously reported that mice with dual deficiency in mDia1 and MiR-146a (DKO mice) show age-related anemia and ineffective erythropoiesis mimicking human MDS
6. Subsequent single-cell RNA-sequencing analysis of BM nucleated cells revealed enriched S100A9 expression in neutrophils, dendritic cells, fibroblasts, and macrophages (
Figure 1E–G)
7. Specifically, S100A9 was aberrantly overexpressed in B cells, monocytes, stage II neutrophils, T cells, NK cells, neutrophils, and macrophages in DKO mice (
Figure 1G). Moreover, serum S100A9 was significantly higher in DKO mice than controls (
Figure 1H). These results collectively suggested that S100A9 might potentially contribute to MDS pathogenesis. Therefore, we crossed the DKO mice with
S100A9−/− mice to generate
Diaph1−/−Mir146−/−S100A9−/− triple knockout (TKO) mice to interrogate the roles of S100A9 in MDS pathogenesis. Firstly, the survival curves were similar between DKO and TKO mice during aging (
Figure 1I). Accordingly, peripheral blood parameters showed minimal differences between DKO and TKO mice, except for neutrophil counts (
Figure S6A–T). An enlarged SP and extramedullary erythropoiesis were consistently observed in DKO mice and were not alleviated by S100A9 ablation (
Figure 1J–K). Moreover, the loss of BM cellularity in DKO mice was not ameliorated by S100A9 deletion (
Figure 1K). The impaired BM erythropoiesis in DKO animals, as evidenced by the disappearance of varied developmental stages of erythroid cells, was not recovered or ameliorated after S100A9 loss in TKO mice (
Figure 1L and Figure S6U). We observed similar patterns for lymphoid cells (
Figure 1L and Figure S6V, B220
+ and CD3e
+). Notably, in agreement with the diminished peripheral neutrophil counts in TKO mice (
Figure S6R), myelopoiesis was further impaired in DKO mice after S100A9 inactivation (
Figure 1L and Figure S6V, CD11b
+, monocytes/macrophages, and granulocytes). This observation might be attributable to S100A9-mediated modulation of CD11b expression on neutrophils under pathological conditions such as inflammation
8. Secondly, our analysis of the hematopoietic stem progenitor cells (HSPCs) in the BM and SP revealed that the common progenitors (LK, CMP, and GMP), multipotent progenitors (MPP), and hematopoietic stem cells (HSCs), including long-term HSCs (LT-HSCs) and SLAM-LSK were depleted in the BM but markedly accumulated in the SP in aged DKO mice (
Figure 1M–O and Figure S7A–D). Intriguingly, although S100A9 deletion did not restore BM HSPC levels, it substantially decreased splenic HSPC accumulation in DKO mice (
Figure 1N). This finding was further validated through
in vitro colony-forming assays in mononuclear cells (
Figure 1P and Figure S7E–H). Our findings suggested that S100A9 is critical for either HSPC migration from the BM into the SP or the expansion of splenic HSPCs in the context of MDS. Thirdly, aged DKO mice exhibited fibrosis, as evidenced by reticulin staining in BM sections. However, the inactivation of S100A9 scarcely ameliorated the fibrotic conditions in DKO mice (
Figure 1Q and Figure S7I).
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