EGFR-MEK1/2 cascade negatively regulates bactericidal function of bone marrow macrophages in mice with Staphylococcus aureus osteomyelitis

Ojectives: To further discover the mechanism of EGFR-MEK1/2 activation might suppress the production of mitochondrial reactive oxygen species (mtROS) upon Staphylococcus aureus (S. aureus) challenge. Methods: Bone marrow derived macrophages (BMDMs) were isolated from C57BL/6 mice (8-10 weeks old) and cultured for 7days. On day 8, cells in S.aureus-infected group were infected with S. aureus at multiplicity of infection (MOI) of 10 for 1 h, and then treated with 20 μg/mL lysostaphin and 50 μg/mL gentamicin to remove extracellular bacteria. After being washed with PBS for three times, Cells were then cultured for additional 12 h. Afterwards, cells were washed with PBS, lysed in Trizol, and sent for next-generation sequencing. RNA was quality-assessed with Agilent 2100 using RNA 6000 Nano kit, with RNA integrity number above 9 for library construction. The total RNA samples from three independent experiments were used for library construction according to the protocol of Illumina NovaSeq 6000 platform and then sequenced on a Thermal Cycler using HighSensivity DNA assay Kit. The FASTq sequencing files were aligned to the mouse reference genome (GRCm38, mm10) using STAR aligner for further analysis. The resulting gene profiles were analyzed using limma package (3.54.0) to determine differentially expressed genes (DEGs) between S. aureus infected BMDMs and control ones. Gene oncology (GO) analysis of DEGs (p value < 0.05, and │Log2(fold change)│>1) was performed by clusterProfiler package (Version 4.7.1) in R platform (Version 4.2.3). Results: GO analysis of DEGs associated with cellular response to environmental stimulus and regulation of DNA damage checkpoint showed that Map3k20, Chek2 and Fbxo4 were significantly down-regulated. Chek2 is a central effector of DNA damage response, mediating DNA checkpoints activation under stress conditions.