Loss of serine threonine protein kinase 25 (STK25) in the bone marrow niche attenuates g/CSF- and TLR7-induced emergency myelopoiesis and mobilization into the periphery

Abstract

Abstract Introduction. Inflammation is the immune system's response to harmful stimuli, such as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). The inflammatory process comprises multiples stages, each of which are triggered and amplified within the bone marrow (BM) niche and in the periphery. Blood cell production within the BM niche is hierarchically organized with hematopoietic stem and progenitor cells (HSPCs) at the top, to ensure continuous production of all blood cells throughout life by both self-renewal and differentiation to multi-potent and blood-lineage committed progenitors. The stages of differentiation allow for homeostatic blood cell maintenance within the niche and a high degree of flexible adaptation to increase blood cell output during inflammation. Importantly, to mount an effective immune response, hematopoiesis is a key component but must be also tightly regulated to produce sufficient blood cells for the resolution of inflammation and avoid unrestrained blood cell production. At homeostasis, HSPCs and their resulting mature leukocytes continuously egress out of the BM into the circulation; however, upon inflammation, they undergo rapid mobilization. The duration and magnitude of inflammation is affected by the efficiency of myeloid precursors to mature into neutrophils and monocytes/macrophages, egress from the BM, migrate into the infected or injured area, and perform downstream effector functions to resolve inflammation. We recently identified serine threonine protein kinase 25 (STK25) as a new mediator of Toll-like receptor (TLR) signaling and as a modulator of inflammation-induced myelopoiesis. Methods. 8-10 weeks-old, sex-matched Stk25-/-(KO) and Stk25+/+wild-type (WT) littermate mice, Stk25fl/fl, Stk25fl/fl-Vav-iCre+,Stk25fl/fl-Vav-iCre-littermate mice, and TLR7.1 and TLR7.1-Stk25-/- littermate mice were immunophenotyped (blood, spleen, BM) by flow cytometric analysis and cell blood counts (CBCs) at homeostasis and during inflammation. In vitro HSPC proliferation and differentiation were assessed by flow cytometric analysis, CFU assays and scRNAseq analysis. In vivo G/CSF injections were used to examine the role of STK25 in neutrophil differentiation within the BM and mobilization to the blood. Results.Immunophenotyping revealed significant homeostatic defects in all three hematopoietic organs – BM, blood and spleen. Significant reductions in both the percentage and absolute counts of monocytes and neutrophils in KO and Stk25fl/fl-Vav-iCre+,as compared to age- and sex-matched WT and Stk25fl/fl-Vav-iCre-littermates were identified, respectively. Flow cytometric analysis of hematopoietic stem cells and lineage-biased multipotent progenitors (MPPs) identified significant defects in the production of short- and long-term HSCs, common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), and MPP3 cells that differentiate into granulocytes and monocytes from the BM of KO and Stk25fl/fl-Vav-iCre+ mice. In two distinct models, one that drives emergency granulopoiesis (G/CSF) and the other that drives emergency myelopoiesis (TLR7), we detected significant defects in myeloid cell expansion, mobilization and differentiation into inflammatory effector subsets. Results from scRNAseq shed light on the mechanism(s) by which STK25 regulates these functions. Conclusions. Together, results identify STK25 as a new factor involved in granulocyte-monocyte lineage decision during homeostasis and inflammation.