Necrotic Bone Fluid Suppresses Energy Metabolism of Porcine PBMC-Derived Macrophages In Vitro

Legg-Calvé-Perthes disease is a juvenile ischemic osteonecrosis (ON) of the femoral head. A disruption of blood supply to the femoral head produces extensive cell death and necrotic debris. Macrophages are innate immune cells recruited to the necrotic bone to orchestrate the repair process. However, the role macrophages play in the ON repair process is still not elucidated. The purpose of this study was to determine the effect of artificial necrotic bone fluid (NBF) on porcine peripheral blood mononuclear cell (PBMC)-derived macrophages. Monocytes were positively selected by CD14 MicroBeads from pig PBMCs. After maturation, cells were treated with no stimulant (Con), LPS + IFNγ (M1), IL4 + IL13 (M2), or NBF. All culture supernatants and cells were harvested for ELISA, Western blot, FACS, RT-qPCR and bulk RNAseq. The Western blot and ELISA showed that only the M1 condition elevated the protein level of pro-inflammatory cytokines. The FACS results indicated that percentage of CD8086+ (M1 marker) cells was significantly lower in the M2 vs. other conditions, whereas the relative median fluorescence intensity of CD8086 was significantly higher in the M1 vs. other conditions. The NBF did not show any significant change compared to the Con. mRNA analysis showed significantly increased IL1β and IL8 expression in the M1 vs. Con scenario. TNFα expression was significantly decreased in the M2 vs. Con scenario. Interestingly, the NBF did not induce pro-inflammatory gene expression. For bulk RNAseq, the Gene Set Enrichment Analyses of the M1-stimulated cells revealed the enrichment of pro-inflammatory gene sets. For the M2, most of the enriched categories were related to the down-regulation of inflammation. For the NBF, the most enriched categories were related to the down-regulation of protein translation and mitochondrial metabolism. We further confirmed the suppressive effects of NBF on macrophage functions using Seahorse Cell Mito Stress Tests, ¹³C-glucose metabolic flux analysis, mitochondrial ROS detection via MitoSOX^TM staining, and phagocytosis assay. Taken together, these results revealed that the artificial NBF down-regulates the overall cellular activity and energy metabolism of macrophages.