PI3Kinase Alpha and Delta Promote Hematopoietic Stem Activation Under Stress

Hematopoietic stem cells (HSCs) are a rare population in the bone marrow (BM) that mostly exists in a quiescent state. Upon environmental stresses such as infection, inflammatory signals are released and induce HSCs to proliferate to quickly re-establish homeostasis and maintain the blood system. Many hematopoietic growth factors and chemokines signal through PI3K pathway. In hematopoietic cells, 3 Class IA PI3K isoforms are expressed (p110α, β, and δ), each encoded by a different gene. While p110α and p110β are ubiquitously expressed, p110δ is enriched in leukocytes. We previously showed that p110α is dispensable for HSC function (Gritsman et al, J Clin Invest 2014 124:1794-1809), suggesting redundancy between Class I PI3K isoforms in HSCs.

To test for potential redundancy between p110α and p110δ in HSCs, we generated mice with conditional deletion of p110α and germline deletion of p110δ (DKO mice). DKO mice have anemia, leukopenia and decreased BM cellularity. While there is no change in HSC numbers, the number of lymphoid-primed multipotent progenitors (LMPPs) is significantly decreased. In the primary competitive repopulation assay, DKO BM cells fail to reconstitute the B cell lineage, while the myeloid and T cell lineages were relatively preserved. However, in a secondary competitive transplantation setting, we also observed a significant decrease in myeloid reconstitution, suggesting that p110α and p110δ play redundant roles in emergency myelopoiesis. To examine effects of p110α and p110δ deletion on gene expression, we performed microarray analysis of WT, DKO, p110δ-/-, and p110α-/- hematopoietic stem and progenitor cells (HSPCs) after bone marrow transplantation. Gene set enrichment analysis revealed negative enrichment of gene sets associated with inflammatory response pathways in both DKO HSCs and LMPPs. In DKO HSCs, we also observed negative enrichment of gene sets associated with cell cycle progression.

To further examine the roles of p110δ and p110α in the hematopoietic stress response, we injected DKO, p110δ-/-, and WT; Mx1-Cre mice with 5-fluorouracil (5-FU). We observed decreased survival of 5-FU-treated DKO mice associated with impaired hematopoietic recovery, and with the failure of HSCs to enter the cell cycle. Given the important roles of inflammatory signaling pathways in HSC activation and emergency myelopoiesis, we examined the roles of p110α and p110δ in signal transduction in HSPCs in response to IL1β or TNFα. Our phospho-flow cytometry analysis revealed a decrease in p38-MAPK phosphorylation in both p110δ-/- and DKO HSPCs, both at baseline and after stimulation with either IL1β or TNFα. To confirm these results, we stimulated p110δ KO and DKO cKit-enriched bone marrow cells with IL1β or TNFα. We observed a significant decrease in both p38-MAPK phosphorylation and phosphorylation of Akt at Ser473 in DKO cells, but not in p110δ-/- cells, both at baseline and with IL1-β or TNF-α stimulation. This suggests that both p110α and p110δ are required for optimal transduction of IL1β or TNFα in hematopoietic progenitors. Surprisingly, we found that DKO HSPCs can enter the cell cycle normally upon in vivo stimulation with lipopolysaccharide (LPS), which simulates bacterial infection. Our results suggest that p110α and δ act in a redundant fashion to transduce specific inflammatory signals in HSPCs, such as IL1β and TNFα, in response to hematopoietic stress. Our findings have important implications for the use of PI3K inhibitors in combination with chemotherapy and in the setting of infection or inflammation.