Type: Oral
Session: 201. Granulocytes, Monocytes, and Macrophages: From Inflammation to Hemophagocytic Lymphohistiocytosis
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research
Methods. 8-10 weeks-old, sex-matched Stk25-/- (KO) and Stk25+/+ wild-type (WT) littermate mice, Stk25fl/fl, Stk25fl/fl-Vav-iCre+ and Stk25fl/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, Stk25fl/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 KO BM 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.
Disclosures: No relevant conflicts of interest to declare.