Serine Threonine Protein Kinase 25 (STK25) Is a New Mediator of Myelopoiesis and Myeloid Cell Effector Function(s) during Injury and Inflammation

Introduction. Blood cell production within the bone marrow (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. At homeostasis, HSPCs and their resulting mature leukocytes continuously egress out of the BM into the circulation; however, upon inflammation/stress, they undergo rapid mobilization. Homeostasis of myeloid cells is particularly critical as they orchestrate early immune responses to resolve acute inflammatory responses. 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. The molecules and mechanisms that regulate blood cell production, retention, and mobilization are not entirely understood. We recently identified serine threonine protein kinase 25 (STK25) as a new modulator of stress hematopoiesis and myeloid effector function. STK25 is a member of the mammalian sterile 20-like (MST) kinase family and is primarily known to participate in autophagy, cell polarity, apoptosis, and migration, and is thus considered a potential therapeutic target in cancer.

Methods. 8-10 weeks-old, sex-matched Stk25^-/-(KO) and Stk25^+/+wild-type (WT) littermate mice, Stk25^fl/fl, Stk25^fl/fl-Vav-iCre⁺and Stk25^fl/fl-Vav-iCre^-mice were immunophenotyped (blood, spleen, BM) by flow cytometric analysis and cell blood counts (CBCs) at homeostasis and in response to inflammation (emergency myelopoiesis). HSPC proliferation and differentiation were assessed by flow cytometric analysis, CFU assays and scRNAseq analysis. In vitro chemotaxis assays were performed with blood and BM neutrophils. Phagocytosis was measured by fluorescence bead uptake; neutrophil extracellular traps (NETs) and degranulation were quantified by flow cytometric analysis and ELISA. In vivo models of LPS-induced acute respiratory distress syndrome (ARDS), thioglycolate-induced peritonitis and wound healing were used to examine myeloid cell differentiation, trafficking and effector function.

Results. Immunophenotyping revealed a significant reduction in the numbers and percentages of blood monocytes and neutrophils within KO mice; all other cell types were unchanged. Reductions were conserved in the spleen and BM of KO mice. Further, Stk25^fl/fl-Vav-iCre⁺mice phenocopied the defect in blood monocytes and neutrophils, supporting a cell autonomous role for STK25 in myelopoiesis. Given the observed defects in blood and BM, we measured the relative amounts of megakaryocyte-erythroid progenitors (MEP), granulocyte-monocyte progenitors (GMP) and common myeloid progenitors (CMP), and multipotent progenitors (MPP) in the BM of WT and KO mice. At homeostasis, we observed significant reductions in LK and LSK populations within KO BM, and significant reductions in GMP, LT-HSC, ST-HSC (MPP1), and MPP3 that differentiate into granulocytes and monocytes. We also examined granulopoiesis and found significant reductions in NeP-1 neutrophil progenitors with limited effects on NeP-2. Functional analyses of KOBM and blood myeloid cells identified defective BM egress, in part due to a significant retention of CXCR4⁺CD11b⁺Ly6G⁺ neutrophils in the BM of KO mice as compared to WT, reduced trafficking of these cells to inflammatory stimuli, reduced phagocytosis of pathogenic stimuli, and reduced NET formation that is required to immobilize and kill invading microorganisms. Results from scRNAseq support these functional findings and lend insight into mechanism(s). Last, KO mice had defective wound healing, with a significant reduction in the infiltration of inflammatory myeloid cells.

Conclusions. Together, results identify STK25 as a new factor involved in granulocyte-monocyte lineage decision during homeostasis and inflammation. Results also support a role for STK25 in essential myeloid effector functions that are vital for wound healing and inflammation.

No relevant conflicts of interest to declare.