The Role Of Neuronal Nitric Oxide Synthase In The Regulation Of Macrophage Polarity

Macrophage can differentiate to different functional phenotypes response to various stimuli,namely classically activated macrophage（M1）and alternatively activated macrophage（M2）.Neuronal nitric oxide synthase（n NOS,NOS1）has been domenstrated to be involved in the early polarization of macrophages.However,the regulatory mechanism has not been fully understood.This thesis studies the role of NOS1 in regulating the macrophages polarization towards M1 and M2 phenotypes.The main results are as follows:1.The wild-type（WT）and NOS1^-/-macrophages are polarized towards M2 type through interleukin 4（IL-4）stimulation.Then the m RNA levels of M2 marker genes and protein levels of M2 key regulatory factors are detected by quantitative PCR and western blot respectively.The results show that compared with WT macrophages,the expression of M2 marker genes such as arginase-1（Arg-1）,resistin-like molecules（Fizz1）and chitinase（YM1）in NOS1^-/-macrophages are not significantly changed.Moreover,the expression levels of key regulatory factors such as signal transducer and activator of transcription 6（STAT6）,phosphorylated STAT6（p STAT6）and peroxisome proliferators-activated receptorsγ（PPARγ）have not been altered significantly.Therefore,NOS1 does not participate in the M2 polarization of macrophages.2.The wild-type（WT）and NOS1^-/-macrophages are stimulated to M1 type through lipopolysaccharides（LPS）.RNA sequencing analysis of macrophages at different induction time（0h,2h and 8h）is performed,and some differentially expressed genes are validated by quantitative PCR,which comprehensively reveals the role of NOS1 in M1macrophage polarization from the perspective of transcriptome.The results show that NOS1 deletion only affects the expression of dozens of genes in macrophages in the absence of LPS stimulation.However,after 2 hours and 8 hours of LPS stimulation,NOS1 deletion results in hundreds of differentially expressed genes compared with WT macrophages,which are all involved in inflammatory response and transcription-related clustering.These differentially expressed genes also participate in some signaling pathways that related to the polarization of macrophages to M1 type and inhibit the activity of these pathways.For example,at 2h LPS stimulation,the differentially expressed genes take part in Toll-like receptors,NOD-like receptors and cytokine-cytokine receptor interaction pathway,whereas,the differentially expressed genes stimulated by LPS for 8 hours are involved in Fox O,AMPK signaling pathways.In addition,this study also construct two gene interaction networks based on the relationship among the differential expressed genes 2h and 8h post LPS treatment.At the same time,the expression levels of several differentially expressed genes detect by quantitative PCR are consistent with those of sequencing data.These results indicate that NOS1 deletion can inhibit the polarization of macrophages to M1 phenotype,and that NOS1 affects the polarization of macrophages in different ways over time.