STAT3 Protects Hematopoietic Stem and Progenitor Cell Function in Non-Inflamed Conditions

The transcriptional regulator STAT3 has well established anti-inflammatory functions in mature myeloid cells by preventing excessive pro-inflammatory factor production in response to Toll-like receptor agonists or the gut microbiota. However, this role has precluded understanding of STAT3 function in maintaining hematopoietic stem and progenitor cell (HSPC) homeostasis, as Stat3 deletion in the hematopoietic system leads to systemic inflammation and ineffective hematopoiesis. Therefore, novel experimental approaches are needed to uncouple STAT3 function in HSPCs from the effects of systemic inflammation.

To understand how STAT3 regulates hematopoietic homeostasis in a non-inflamed environment, we developed competitive mixed bone marrow (BM) chimeric mice with conditional Stat3 deletion in approximately 20% of hematopoietic cells (Figure 1a). We demonstrated successful engraftment of test (CD45.2⁺ CreER or CD45.2⁺ CreER Stat3^f/f) and competitor (CD45.1⁺CD45.2⁺) BM cells at a 1:4 ratio, respectively, in lethally-irradiated CD45.1⁺ recipient mice. Then, we induced Stat3 deletion by tamoxifen treatment (Figure 1a). We found a significant impairment in the hematopoietic activity of Stat3-deficient HSPCs, as shown by ~50% reduction of peripheral blood CD45.2⁺ cells after Stat3 deletion, which suggests a crucial role of STAT3 in sustaining hematopoiesis (Figure 1b). Serum cytokine levels and colon pathology scores were similar between mice with Stat3-deficient cells and corresponding CreER controls, which confirms a non-inflammatory environment. We also observed loss of a majority of CD45.2⁺ immune lineages in spleen, colon, and BM upon Stat3 deletion.

To evaluate the self-renewal capability of Stat3-deficient hematopoietic stem cells (HSCs), we performed secondary BM transplantation experiments. These experiments showed that CD45.2⁺Stat3-deficient HSCs could not reconstitute peripheral blood populations. Moreover, we found a striking decline in Stat3-deficient lin^-Sca-1⁺ckit⁺ (LSK) cells and HSCs (LSK CD48^-CD150⁺) in secondary BM transplants (Figure 1c). These results suggests that STAT3 is required to sustain a functional HSC compartment in non-inflamed conditions.

To investigate how loss of STAT3 affects the transcriptional landscape of HSPCs, we performed scRNA-sequencing (scRNA-seq) in phenotypically defined LSKs isolated from primary BM transplant recipients. This analysis identified 12 distinct transcriptional states in the LSK compartment (Figure 1d). Compared with CreER control cells, Stat3-deficient LSKs were enriched in myeloid-related clusters at the expense of lymphoid clusters. Differential expression analysis among the clusters revealed that Stat3-deficient HSCs (Cluster 3) and multipotent progenitors (Cluster 2) upregulated Myc and Interferon (IFN) Alpha Response pathways, including genes involved in cell cycle activation, IFN-stimulated genes (Ifi203, Mndal), and IFN transcriptional regulators (Irf1, Irf2, Irf2bp2) (Figure 1e). In addition, Stat3-deficient LSKs acquired γH2AX modifications over time. Collectively, these data suggest that Stat3 deletion induces excessive DNA damage and intrinsic activation of an IFN transcriptional signature in HSPCs, which leads to impairment of HSC function.

In conclusion, our results reveal a cell-autonomous role for STAT3 in preserving hematopoietic stem and progenitor cell homeostasis, sustaining long-term HSC function, and preventing induction of an intrinsic IFN response and DNA damage (Figure 1f). Our study highlights STAT3-driven protective mechanisms in HSPCs under non-inflammatory conditions.

Colla:Amgen: Research Funding. Watowich:Asylia Therapeutics Inc: Membership on an entity's Board of Directors or advisory committees; Cellino Biotech: Consultancy.