Effect of depletion of NLRC3 on glycolytic genes of macrophage with LPS treatment

The impaired aerobic glycolysis of monocyte/macrophage are the main features in sepsis that encountered immunosuppression. NLRC3, an inhibitory sensor, can diminish the Toll-like receptor 4 pathways of macrophages which is associated with sepsis-induced immunosuppression, but whether NLRC3 plays key role in regulating the immunosuppression of macrophage via interfering glycolysis still unclear. The aim of this study was to characterize the transcriptome alterations by which NLRC3 loss, and further investigate the role of the glycolysis in the macrophage with the development of sepsis. Bone marrow-derived macrophages (BMDMs) was isolated from the femurs and tibias of the NLRC3 WT (LysM-Cre-NLRC3fl/fl) and NLRC3?Mac (LysM-Cre+ NLRC3fl/fl) mice and then was treated with or without LPS for 12 hours, and the macrophages mRNA profiles of five-weeks-old NLRC3 WT mice (WT macrophages) and of five-weeks-old NLRC3?Mac mice (NLRC3-/- macrophages) were generated by deep sequencing, in triplicate, using Illumina 6000. Overall design: Bone marrow-derived macrophages (BMDMs) was isolated from the femurs and tibias of the NLRC3 WT (LysM-Cre-NLRC3fl/fl) and NLRC3?Mac (LysM-Cre+ NLRC3fl/fl) mice and then treated with or without LPS for 12 hours.

单核细胞/巨噬细胞的有氧糖酵解受损，是伴发免疫抑制的脓毒症的核心特征。NLRC3作为一种抑制性感受器，可削弱巨噬细胞的Toll样受体4（Toll-like receptor 4）通路，该通路与脓毒症诱导的免疫抑制密切相关，但NLRC3是否通过干预糖酵解过程调控巨噬细胞免疫抑制，目前仍不明确。本研究旨在阐明NLRC3缺失所介导的转录组改变，并进一步探究糖酵解在脓毒症进程中巨噬细胞内的调控作用。本研究从NLRC3野生型（NLRC3 WT，LysM-Cre-NLRC3fl/fl）及NLRC3巨噬细胞特异性敲除（NLRC3ΔMac，LysM-Cre+ NLRC3fl/fl）小鼠的股骨与胫骨中分离骨髓来源巨噬细胞（Bone marrow-derived macrophages, BMDMs），随后分别用脂多糖（Lipopolysaccharide, LPS）处理或不处理，培养12小时；采用Illumina 6000测序平台，对5周龄NLRC3野生型小鼠来源巨噬细胞（WT巨噬细胞）及NLRC3ΔMac小鼠来源巨噬细胞（NLRC3-/-巨噬细胞）的mRNA进行深度测序，每组设置3次生物学重复。实验整体设计：从NLRC3野生型（NLRC3 WT，LysM-Cre-NLRC3fl/fl）及NLRC3巨噬细胞特异性敲除（NLRC3ΔMac，LysM-Cre+ NLRC3fl/fl）小鼠的股骨与胫骨中分离骨髓来源巨噬细胞（BMDMs），经脂多糖（LPS）处理或不处理后培养12小时。