The Role And Mechanism Of SIRT2 In The Pro-Inflammatory Polarization Of Alveolar Macrophages In Cold-exposed Mice

Respiratory tract infection is one of the most common acute diseases in the world,and cold is an important factor to induce respiratory diseases for various organisms.Alveolar macrophages(AMs)are the subset of macrophages that colonizes the lung and plays a "barrier" and "sentinel" role in maintaining lung homeostasis.AMs phenotype is the "switch" that determines the mode and intensity of immune response.When the organisms are stimulated by endogenous or exogenous,AMs can be polarized into M1 pro-inflammatory phenotype and M2 anti-inflammatory phenotype.At the same time,they secrete a variety of cytokines which exert pro-inflammatory or antiinflammatory functions in order to maintain the orderly progress of lung physiological functions.So,how are AMs affected after cold exposure?What are the complex regulatory mechanisms?Therefore,it is of great significance to deeply explore the internal relationship between cold exposure and the changes of AMs phenotype or functional.This is of great significance to reduce the risk of respiratory diseases induced by cold exposure in livestock and poultry and then to ensure the health of livestock and poultry.In order to clarify the effect of cold exposure on the phenotype and function of mouse’s AMs,this study firstly treated 5-week-old C57BL/6 mice with a 5-day,random 3 hours 4℃ cold exposure from 8:00 to 20:00 every day.The lungs and primary AMs were harvested after the cold exposure period.The phenotype and function of AMs were detected by ATAC-Seq(Assay for Transposase-Accessible Chromatin with high throughput sequencing,ATAC-seq),RNA-Seq(RNA Sequencing,RNA-seq),flow cytometry and so on.The results showed that the mRNA expressions of M1 macrophage on surface markers(F4/80,CD86),protein markers(iNOS)and related cytokines(IL-1β and TNF-α)were significantly increased after cold exposure(P<0.001),but phagocytosis was significantly reduced(P<0.001).At the same time,lung section results showed inflammatory cell infiltrates into the lung tissue after cold exposure.The biological processes that enriched by the annotated genes at the peak of ATAC-Seq after cold exposure were mostly related to the production of inflammatory factors,and the enriched pathways were mostly related to cell proliferation and glucose metabolism.The results of gene enrichment analysis of differentially expressed genes by RNA-seq showed that AMs oxidative phosphorylation(OXPHOS)pathway was down-regulated after cold exposure.In addition,the oxygen consumption rate(OCR)of AMs after cold exposure was significantly decreased(P<0.001),and the extracellular dynamic acidification efficiency(ECAR)was significantly increased(P<0.001).The results confirmed that cold exposure induced metabolic reprogramming in mouse AMs,that is,the glucose metabolic pathway was switched from oxidative phosphorylation to aerobic glycolysis.Which promoted the pro-inflammatory polarization of AMs.Silent regulator 2(SIRT2)is an important energy sensor in eukaryotic cells.As an important deacetylase,according to the changes of intracellular energy metabolism,SIRT2 can regulate a variety of transcription factors and transcriptional regulatory proteins through its deacetylation function.It converts the changes of energy metabolism into biological regulatory signals,and then participates in the regulation of cell stress response,metabolism and apoptosis.The previous work showed that acetylation was involved in regulating the polarization of microglia in the hippocampus of cold-exposed mice,and aerobic glycolysis mainly occurred in the cytoplasm.Therefore,based on the above,in order to explore whether the SIRT2 is involved in regulating the effect of cold exposure on AMs or not,and how it plays a regulatory role,the immunoblotting,immunoprecipitation tandem mass spectrometry(IP-MS)and other techniques were used to detect the AMs.The results showed that the mRNA,protein and expression of SIRT2 in AMs decreased significantly after cold exposure(P<0.05).IP-MS screened out the PKF1(Phosphofructokinase-1,PFK1),a key rate-limiting enzyme in glycolysis,which may potentially interact with SIRT2.IP-MS also screened out that K614 was acetylated.At the same time,the protein expression of PFK1 decreased significantly(P<0.001),but the level of acetylation modification increased.Therefore,we speculate that SIRT2 may interferes the acetylation modification of PFK1 to regulate the metabolic reprogramming of AMs,which thereby driving the pro-inflammatory polarization of AMs.In order to verify the above speculation,based on the research model of LPSinduced M1-type polarization of mouse alveolar macrophage cell set MH-S,we systematically explore the regulatory effect of sirt2 mediated acetylation of PFK1 on the pro-inflammatory polarization of mouse alveolar macrophages and its related mechanisms with seahouse cell respiration energy metabolism analysis,coimmunoprecipitation,immunofluorescence and other techniques.The results showed that SIRT2 may be involved in regulating the mRNA expression of IL-1β and TNF-αin MH-S cells and OCR.At the same time,SIRT2 interacts with PFK1 and mediates its ubiquitination degradation with regulating the level of acetylation modification of PFK1.When lysine at K614 site of PFK1 was mutated to arginine(simulated deacetylation),the regulatory effect of SIRT2 on acetylation modification level of PFK1 was eliminated.That affects the ubiquitination modification of PFK1 and alleviate the reduction of mRNA expression of IL-1β and TNF-α in MH-S cells and OCR.However,the results were reversed if we mutate lysine at K614 site of PFK1 to glutamine(simulated acetylation).Based on the above results,it was determined that SIRT2 mediates ubiquitination and degradation of PFK1 by interfering with the acetylation modification level of PFK1,thus regulating the glucose metabolism pathway of MH-S cells,and then affecting the polarization of MH-S cells.Next,in order to further verify the important regulatory role of SIRT2 on the phenotype and function of AMs under cold exposure conditions.We took SIRT2-KO mice as research objects and collected lungs and AMs after cold exposure.The experimental results were verified by targeted glycometabonomics and western blot.The results showed that SIRT2 knockout could promote the reduction of PFK1 protein expression in AMs after cold exposure,and up-regulate the acetylation and ubiquitination levels of PFK1.Meanwhile,SIRT2 deletion aggravated the attenuation of the OXPHOS pathway in AMs after cold exposure and so did the pro-inflammatory differentiation of AMs.In addition,SIRT2 deficiency aggravated the decrease of phagocytic activity of AMs after cold exposure,and also aggravated the inflammatory reaction of lung after cold exposure.This suggests that SIRT2 has an important regulatory role in the effects of cold exposure on the phenotype and function of AMs.Taken together,under cold exposure conditions,SIRT2 promotes PFK1 ubiquitin degradation by interfering with its acetylation modification,and mediates the occurrence of aerobic glycolysis,and promotes the pro-inflammatory polarization of AMs.This study analyzes the internal relationship between cold exposure and AMs phenotype and functional changes from the perspective of immune metabolism,as well as the important regulatory role of SIRT2,which provides a scientific basis for the mechanism of cold exposure easily triggering respiratory diseases in livestock and poultry.And it has important theoretical significance and practical application value for the prevention of cold exposure-induced injury.