The Mechanism Of 17β-estradiol In Promoting Female Trained Immunity Against Early Sepsis

Sepsis,as a common clinical systemic inflammatory response caused by infection,is usually owing to bacterial infection.At the same time,fungi,viruses and parasites can also cause sepsis.The occurrence of sepsis can lead to a very high mortality rate of30%-50%thus makes sepsis one of the diseases with the highest mortality rate except cancer.Even with the development of modern medicine,the clinical mortality rate of sepsis has not been maintained at a low level.When the body is in a state of sepsis,the main organs will be damaged.Many key biomarkers of sepsis have been discovered and explored.Among them,the damage of kidney,liver and lung is a vital feature and is accompanied by increment of kidney bacterial load,serum transaminase and lactic acid.The entire occurrence and development of sepsis cannot be simply classified as pro-or anti-inflammatory.Sepsis includes early systemic inflammatory response syndrome（SIRS）and later compensatory anti-inflammatory response syndrome（CARS）stage.The SIRS stage can be considered as early sepsis.Promotion of inflammatory response in this stage is beneficial for the body to fight infection.In CARS,many patients cannot alleviate the inflammatory response and died from cytokine storm caused by overexpression of inflammatory cytokines attacking their own tissues and organs.Therefore,sepsis is an infectious disease in SIRS and an autoimmune disease in CARS.The research on these two periods of sepsis is completely different.The commonly used sepsis model in research is achieved by intestinal puncture or direct intraperitoneal injection of E.coli and LPS（a substance that exists on the cell wall of gram-negative bacteria）in mice.In addition,some studies indicated that sepsis has obvious gender differences,and females have significant lower severity and mortality in sepsis than males after infection.Based on this phenomenon,even though most studies on gender differences in sepsis focus on chromosomal and genetic differences,there are still some studies that attribute the gender differences in sepsis to different hormone levels in males and females.Estrogen is mainly divided into two categories:estrogen and progesterone.Among them,as the most vital estrogen in female,exploring the mechanism of estradiol（E2）in females against sepsis is of great importance in clinical practice and also provides a possible method to improve the treatment of male sepsis clinically.In the meanwhile,exploring the gender differences in diseases is a significant basic and prospective research of precision medicine,which is of great significance for understanding the physiological processes of the human body and discussing personalized medical solutions.The immune system can be divided into innate immunity without specificity and acquired immunity with specificity and memory.However,the concept of trained immunity discovered in 2011 broke this traditional cognition.Trained immunity is a novel immune method that occurs in innate immune cells,especially macrophages.When macrophages are stimulated by the first antigen stimulation（such asβ-glucan）,a series of biological changes will occur including increased glycolysis,epigenetic changes,and activation of AKT-m TOR signaling pathway.When encountering a second antigen stimulation（not necessarily the same as the first antigen type）,macrophages will produce a faster and stronger immunological response,thereby helping the body to resist subsequent various infections.It is suggested that trained immunity can promote innate immune cells to acquire“immune memory”ability similar to that in acquired immunity,which can produce more timely and stronger response to a wider range of subsequent bacterial infections.During the process of fighting sepsis,the discovery of trained immunity provides a new idea and treatment method for scientific research and clinical practice.Since macrophages mainly protect against infection in the body by secreting inflammatory cytokines and phagocytosing pathogens,the current research on trained immunity of macrophage mainly focused on its promotion of inflammatory cytokines,however,it has not been studied whether trained immunity can enhance pathogens-elimination ability by regulating macrophage polarization or facilitating phagocytosis.In addition,whether E2 participates in this process remains to be further explored.For this reason,we established mouse trained immunity and sepsis model by two intraperitoneal（i.p.）injection ofβ-glucan,E.coli or LPS into female,male and ovariectomized（OVX）C57BL/B mice respectively;at the same time in vitro trained immunity and sepsis model was also set up in macrophage cell lines RAW264.7,J774A.1 and bone marrow-derived primary macrophages（BMDMs）.We confirmed that female mice have better sepsis resistance in trained immunity than males and this advantage is closely related to the higher E2 level in female mice than that of males;analyzed the polarization of mouse peripheral blood macrophages,macrophage cell lines and BMDMs in the process of sepsis resistance after trained immunity and the mechanism of E2 involved in regulating polarization;In addition,we also explored the expression of reactive oxygen species（ROS）and NADPH oxidase 2（NOX2）in the important organs of female and male mice after trained immunity and described the mechanism by which E2 promotes LC3B-related phagocytosis（LAP）of macrophages by up-regulating the NOX2-RUBICON signaling pathway to relieve the symptoms of sepsis.The specific results of this research are as follows:1.The better sepsis resistant ability of female mice after trained immunity than males is closely related to the higher E2 level in females:We firstly established trained immunity and sepsis model in ten-week-old male and female mice by i.p.injection ofβ-glucan and E.coli or E.coli alone.Comparing female and male mice,the data showed that the damage to the main organs of females in either the sepsis model or the trained immunity sepsis model is significantly less than that of the males:the medical scores of HE slices that characterize lung injury in females are better than males;The burden of E.coli in kidney is less than males,the levels of aspartate aminotransferase（AST）and alanine aminotransferase（ALT）in serum,which characterize liver damage,are lower than males;in addition,the serum lactate content,an important biomarker of sepsis,is also lower than males.In the models above,the E2 content in female serum was much higher than males.All these indicated that females have better resistance than males.For the most vital organ for estrogen production is ovary,in order to further confirm that higher E2 level in females is the reason for better resistant ability,a batch of OVX mice were obtained by operating six-week-old female mice and established trained immunity and sepsis model at ten weeks of age.The results showed that when E2 level in the serum of OVX mice was significantly reduced,the pathological score of lung HE section was worse than female mice;the burden of E.coli in the kidney was significantly higher than that of females;the levels of ALT and AST in the serum were also significantly higher than female.These results indicated that OVX mice were weeker than female mice in fighting sepsis after trained immunity,suggesting that better sepsis resistant ability is closely related to E2.2.E2 can facilitate sepsis resistance by promoting macrophage M1polarization and inhibiting their M2 polarization in trained immunity:In order to explore the specific process of trained immunity involved in to fight sepsis,and because macrophages secrete inflammatory cytokines to kill pathogens,we tested female and male mice in trained immunity and sepsis models,the content of inflammatory cytokines in females was found to be significantly higher than males in serum IL-6 and TNFα.Furthermore,by detecting the ratio of macrophages in the spleen of female and male mice in the model,it was found that the ratio of female CD11b+F4/80+macrophages in the E.coli group and trained immunity+E.coli group was significantly higher than that of male;by detecting macrophage ratio in peripheral blood mononuclear cell（PBMC）,we found that proportion of F4/80+i NOS+M1macrophages in female PBMCs in E.coli group and TI+E.coli group was significantly higher than males.In addition,by examining F4/80+CD206+M2 macrophage ratio in PBMC,it was found that female is lower than that of males.These results showed that females can gather more macrophages in the spleen in early sepsis,on the other hand,their macrophages will significantly polarize to M1 and secrete more inflammatory cells.The ratio of M2 macrophages is also significantly lower than that of males.In order to further confirm the role of E2 in macrophage polarization after trained immunity,we further examined the levels of inflammatory cytokines in the serum of OVX mice and found that IL-6 and TNFαwere significantly lower than female mice.The proportion of F4/80+CD206+M2 macrophage in PBMC was higher than that of females.In vitro,female F4/80+i NOS+M1 BMDM ratio was much higher than males in LPS group and TI+LPS group,and the F4/80+CD206+M2 BMDM was significantly lower than males.By adding 50n M estradiol,it is found that E2 can further activate expression of trained immunity signal molecules,upregulate IL-6 and TNFαexpression,not only increase M1 BMDM but also reduce M2 BMDM.In RAW264.7 and J774A.1,by establishing an in vitro trained immunity model,E2 can further facilitate TNFαand IL-1βm RNA expression.These results indicated that estradiol can resist sepsis by promoting the polarization of macrophages to M1 and inhibiting M2 polarization.3.Estradiol regulates macrophage polarization in trained immunity by inhibiting Rel B nuclear translocation:The NFκB signaling pathway is closely related to cytokines expression and macrophage polarization.It is reported that as an important transcription factor of NFκB family,Rel B can increase expression of M2-related genes.Since E2 can significantly down-regulate the synthesis of IL-10 and IL-4 m RNA in RAW264.7,this effect can be eliminated when the expression of estrogen receptorα（ERα）is silenced.Through immunofluorescence and detection of Rel B expression in the nucleus of RAW264.7,it was found that E2 inhibits the entry of Rel B into the nucleus but has no effect on P65distribution.The protein level of Rel B in RAW264.7 nucleus can be significantly reduced by E2 and restored by knocking down ERα.These results indicated that estradiol can inhibit the entry of Rel B into the nucleus through ERα,thereby regulating macrophage polarization.4.Estradiol promotes LC3B-associated phagocytosis in trained immunity of macrophage against sepsis:When macrophages fight against sepsis,in addition to killing pathogens by polarizing to M1 and secreting inflammatory cytokines,they can also kill and digest pathogens through autophagy and upregulate ROS production.ROS and NOX2,which produce ROS,are vital signaling molecules for non-classical autophagy namely LAP.In order to detect the effect of estradiol and trained immunity on LAP,we tested the mouse trained immunity and sepsis model.Female mice had lower ALT and AST levels in LPS and TI+LPS groups.The ROS level in the female lungs and livers in E.coli group and TI+E.coli group is higher than males.Furthermore,in E.coli and TI+E.coli group,the number of phagocytes and NOX2 expression in female lungs,liver and kidney were significantly higher than males.In OVX mice,the lung and liver ROS levels were significantly lower than female mice;and the number of phagocytes in the lungs,liver and kidney of OVX mice were also lower than female mice.In vitro,E2can up-regulate the number of phagocytosed E.coli particles,hydrogen peroxide and ROS levels in RAW264.7 and J774A.1.E2 can also increase NOX2 subunits gp91^phoxand p47^phox in macrophage and reduce m RNA levels of SOD1,GPX1,GPX2 and Catalase which degrade ROS.Adding NOX2 inhibitor DPI to the in vivo and in vitro trained immunity and sepsis model will significantly inhibit sepsis resistant and phagocytic ability of macrophage.In RAW264.7,J774A.1 and BMDM,E2 can up-regulate macrophage ROS production,phagocytic ability,and facilitate the expression and co-localization of LAP-related proteins.We also found that E2 can promote RUBICON expression and phagocytic ability of macrophage through ERαwhile silencing ERαdisappears the up-regulation effect.These results indicated that E2 can resist sepsis by promoting LAP process mediated by macrophage NOX2-RUBICON in trained immunity through ERα.To sum up:（1）We verified that female mice were better than males in fighting sepsis after trained immunity and is closely related to higher levels of estradiol;（2）We found that estradiol in trained immunity can promote macrophage M1 polarization and inhibit M2 polarization to resist sepsis;（3）We clarified estradiol regulates macrophage polarization by inhibiting Rel B nucleus translocation;（4）We also revealed that estradiol can up-regulate LAP process mediated by NOX2-RUBICON in trained immunity through ERαto resist sepsis.