| BackgroundSepsis,which is defined as a life-threatening organ disorder due to the dysregulated reaction to infections,is the leading cause of death in the world and brings huge health burden to the society.Due to the development of antibiotics and the theory of sepsis during the past decades,the death rate of sepsis has decreased,but there is still more than 10 million people die of sepsis related diseases each year.The undeveloped areas suffer the highest morbidity and mortality of sepsis.In the early stage of sepsis,there will be excessive inflammation in our body,leading to a large number of immune cell apoptosis,and results in immune suppression.And the drugs recommended for septic shock such as norepinephrine and hydrocortisone even aggravate immune suppression.These make immune suppression the major cause of secondary or co-infection of sepsis patients in the ward.Moreover,according to the latest report about the burden of disease shows that more than 4.5 million people died in2019 with drug-resistant bacteria infection,and the WHO predicts that number will reach 10 million in 2050.It’s a great challenge to us both in development and inheritance.Among the infectious diseases,staphylococcus aureus is the secondary most common bacteria,and methicillin-resistant Staphylococcus aureus(MRSA)is one of the top five clinically lethal bacteria,with diverse infection sites and variable symptoms.Although currently available antibiotics for Gram-positive bacteria,such as Vancomycin,Teicoplanin and Linezolid are with good therapeutic effect.However,the side effects of antibiotics could not be ignored,such as the risk of kidney failure and death caused by Vancomycin,and the risk of bone marrow suppression caused by Linezolid.The more antibiotics used will make the "double-edged sword" of antibiotics show more obvious side effects.The current guidelines for sepsis recommend some basic strategies including early usage of sensitive antibiotics,removal of infected lesions,organ support or replacement therapy,and control of primary disease,however there is no specific measure proposed.Also,there is no recommended treatment for immune system.Therefore,it is promising to research on how to improve the immunosuppressive state of patients with sepsis and decrease the use of antibiotics which will results in reducing the side effects and costs.By controlling invading pathogens with the function of phagocytosis as well as antigen-presenting cell to the immune system,macrophages play an important role in immune system.In the condition of sepsis induced immunosuppression,macrophages can’t escape the fate of increased apoptosis,decreased costimulatory signals,and inhibited phagocytosis and cytokine section functions.In addition,the whole immune system was suppressed by immunosuppression,resulting in the decreased co-working immune cells,which further worsened the condition for macrophages.In macrophages,protein modifications play an important role in the process of physiological function.Especially the post-translational modification(PTM)of proteins,which has attracted much attention in recent years,greatly improves protein diversity and enriches protein’s function.Nowadays,many new drugs were designed to binding special structures of proteins,the study of posttranslational modification is helpful for further analysis of the target protein.Crotonylation is a newly discovered post-translational modification of proteins.It is similar to acetylation,but its function and spatial structure are quite different.It is still unknown whether crotonylation of proteins changes will occur in macrophages after infection with MRSA,and whether crotonylation changes will affect the function of macrophages.MethodsBy analyzing the changes of lysine crotonylation in macrophages while infected with MRSA,the potential targets and signal pathways regulating the function of macrophages were sought,and a new discovered crotonylation protein HACD2(3-Hydroxyacyl-Co A Dehydratase 2)was found.Further analysis were performed to identify whether HACD2 involved in the regulation of macrophages.1.Systematic analysis of lysine crotonylation in macrophages after MRSA infection.THP1 cells were used to infect with MRSA with different time period.And western blot was performed to detect the change of crotonylation both in THP1 cells and MRSA.And the phenotype of altered crotonylation in macrophages after MRSA infection was obtained.Also,LC-MS/MS was used to conduct proteomic and modification omics data for the obtained proteins,and GO,KEGG,COG etc.were used to analyze the obtained data,so as to find out the physiological activities that macrophages may participate in and the possible regulatory signaling pathway of crotonylation changes after MRSA infection.A clear spectrum of lysine crotonylation in macrophages after MRSA infection was obtained and its biological information was analyzed.2.The crotonylation of HACD2 regulates macrophage functions.By analyzing the proteome and crotonylation modification omics data of MRSA infected macrophages,HACD2 was changed in both data.In this part,the si-HACD2 RNA was used to interfere HACD2 in RAW264.7 cells,and both control cells and interfered cells were infected with MRSA.For further analysis,RNA extraction,total protein extraction and protein co-immunoprecipitation were performed prior to qPCR,western blot and some other methods to verify that crotonylation of HACD2 regulate the function of macrophages.By those means,we will explore how the crotonylation of HACD2 regulate the function of macrophages.Results1.Systematic analysis of lysine crotonylation in macrophages after MRSA infection.1.1 The lysine crotonylation of total proteins was down regulated after MRSA infection in macrophages,but some increased.1.2 Lysine crotonylation in macrophages after MRSA infection is involved in a variety of inflammatory signaling pathways and metabolic processes.2.The crotonylation of HACD2 regulates macrophage functions.2.1 The expression of HACD2 in macrophages after MRSA infection is down-regulated;However,the crotonylation of HACD2 increased.2.2 HACD2 inhibited IL-10 expression in MRSA infection,but crotonylation enhanced HACD2 promoted IL-10 expression.2.3 In MRSA infection,HACD2 and crotonylation promoted the expression of inflammatory factors;2.4 Mechanism study found that during MRSA infection,crotonylation of HACD2 promoted p-STAT1 expression and engaged in polarization.ConclusionAfter infected with MRSA,crotonylation levels of total proteins in macrophages were decreased,but some proteins were increased,and the altered crotonylation proteins were involved in a variety of cellular activities and signaling pathways related to inflammation and metabolism.In addition,although the expression of HACD2 is down-regulated during MRSA infection,the crotonylation modification is enhanced and promotes the expression of IL-10.Through the application of si-HACD2 cells and the induction of croton acylation overexpression,it is concluded that both HACD2 and crotonylation were involved in the inflammatory function regulation of macrophages during MRSA infection.And engaged in the polarization of macrophages.These discoveries will serve as a basis for further studies on the regulation of macrophage function. |
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