| Sepsis was previously defined as fatal organ dysfunction caused by host reaction disorder caused by infection.In recent years,the definition of sepsis has been updated clinically.It is believed that the immune response to infection is out of balance,which leads to life-threatening organ dysfunction.The organ dysfunction caused by the immune response imbalance is manifested as physiological and biochemical abnormalities at the cellular level.In other words,it means that the host’s immune response to the infectious agent is out of control,which leads to excessive inflammatory response,which in turn leads to immune failure,multiple organ dysfunction or failure,and ultimately death.Patients with severe trauma,shock,infection and major surgical operations are prone to infection,which leads to unstable homeostasis and potentially fatal risks.Clinical data show that early diagnosis of sepsis and effective treatment are the key to improve the survival rate of patients.The mortality rate of sepsis is still as high as 20%,although fluid resuscitation,timely removal of infection foci,early use of antibiotics and "cluster therapy" supported by organ function are the cornerstones of sepsis treatment.It is generally believed that sepsis is an important organ that is easy to induce liver injury and is often characterized by acute liver injury.In addition,the mortality of septic patients with early liver dysfunction is also increased.In recent years,it has been found that effective regulation of liver immune cells and cytokines produced by them can realize early diagnosis and treatment of sepsis,especially many studies believe that macrophages play a crucial role in liver injury caused by sepsis.Therefore,how to regulate macrophages to improve liver injury induced by sepsis has become a scientific issue that should be paid attention to at present.Recent studies have found that nano-iron oxide can be used as a carrier for diagnosis and treatment of sepsis.Some nano-iron oxides have antibacterial and anti-inflammatory effects,which can be used as markers for diagnosising sepsis.Treatment of LPS-induced inflammatory mouse models with albumin modified nano-iron oxide loaded drugs has been found to reduce infiltration of monocytes/macrophages and neutrophils.Recently,it was found that a kind of superparamagnetic iron oxide particle（SPIONs）—Ferumoxytol approved by the FDA can induce the polarization of macrophages in tumors.We also found that SPIONs can induce macrophage polarization,alleviate liver injury induced by LPS in septic mice modle,and inhibit infiltration of inflammatory cells such as macrophages into the liver.However,the mechanism of how SPIONs regulates macrophages to affect septic liver injury is still unknown.At present,it is believed that mesenchymal stem cells（MSCs）can regulate immune response to inhibit inflammation and promote injury repair by directly interacting with immune cells or paracrine a variety of cytokines.Studies have shown that MSCs can also inhibit the proliferation and function of pro-inflammatory macrophages and promote the production of anti-inflammatory macrophages by secreting transforming growth factor（TGF-β,prostaglandin E（PGE）2,indoleamine 2,3 dioxygenase（IDO）and exosomes.We also found earlier that MSCs can improve the symptoms of septic mice model induced by cecum ligation and reduce the mortality of mice.However,the mechanism by which nano-iron oxide-—SPIONs affects MSCs and then regulates macrophages to affect sepsis is still not completely clear.Therefore,this study explored the mechanism of nano-iron oxide（SPIONs）regulating macrophages to alleviate septic liver injury,analyzed the effect of SPIONs on MSCs,and revealed the mechanism of SPIONs-labeled MSCs regulating macrophages to affect sepsis.1.SPIONS can alleviate liver injury caused by sepsis through up-regulating IL-10 secreted by macrophages.In order to evaluate the effect of SPIONs on liver injury,we established LPS-induced septic mouse model and treated it by tail vein infusion of SPIONs in 4 hours when the model was successfully established.After 48 hours,the serum and liver tissues of mice were sampled,and the changes of iron content,inflammatory factors in serum and macrophages in liver were analyzed.The experimental results showed that iron content in liver of LPS-induced septic mice decreased,while SPIONs treatment could increase iron content in liver of mice.Liver pathological results show that compared with the control group,LPS-induced sepsis mouse model liver has a large number of inflammatory cell infiltration,balloon degeneration and punctate necrosis of hepatocytes,while SPIONs can significantly alleviate the above symptoms.Serological analysis showed that the levels of aspartate aminotransferase（AST）and alanine aminotransferase（ALT）in serum of septic mice were significantly increased,while the levels of ALT and AST in serum of SPIONs treated mice were significantly decreased.These results suggest that SPIONs may reduce liver injury in septic mice by restoring disordered iron homeostasis.Furthermore,the flow cytometry results showed that CD11b+F4/80+macrophages in the liver of the model group increased significantly,but SPIONs treatment could reduce the production of inflammatory macrophages and significantly increase the number of IL-10 positive macrophages.In addition,Treg cells in liver of mice in each treatment group had no obvious changes.ELISA results showed that the levels of IL-6 and TNF-α in serum of model group mice increased significantly,while SPIONs treatment could reduce the levels of the above markers.It is noteworthy in the experiment that SPIONs significantly increased the level of serum IL-10,suggesting that IL-10 produced by macrophages may have an effect on alleviating liver injury.In order to determine the role of IL-10 produced by macrophages in sepsis-induced liver injury,we repeated the above model experiment on normal mice and mice with IL-10 gene deficiency（IL-10-/-）,respectively,and found that SPIONs no longer plays a role in alleviating sepsis-induced injury.In vitro studies have also shown that SPIONs can promote the level of IL-10 in macrophages.These results show that SPIONs’s effect of alleviating liver injury depends on the cytokine IL-10 produced by macrophages.2.SPIONs induced macrophages to up-regulate IL-10 expression depends on activation of Cav1-Notchl/HES1 signaling pathway.Autophagy is an internal equilibrium mechanism for eukaryotic cells to maintain homeostasis.Recent studies have found that autophagy is activated in the early stage of sepsis and then inhibited in the pathophysiological process,and activated autophagy can improve organ damage.Different nanoparticles can induce autophagy of a variety of cells such as macrophages,and activation of autophagy can affect metabolic reprogramming of macrophage IL-10.However,the mechanism of SPIONs regulating macrophage expression of IL-10 through autophagy is still unclear.In order to reveal the mechanism of SPIONs inducing macrophages to produce IL-10,RAW 264.7 cells and bone marrow-induced macrophages（BMDMs）were treated with autophagy inhibitor Baf-A1.The results showed that inhibiting autophagy of macrophages could significantly reduce the expression level of IL-10.It is known that cell autophagy is regulated by various signal pathways such as NOTCh,PI3K-Akt-mTOR RC1 and AMPK-mTOR.Our previous research show that Notch-Hes-1 pathway can induce P62 protein to control TLR7-mediated autophagy death of macrophages.In order to reveal the related pathways in regulation of autophagy,we inhibit the signal transduction of macrophage Notch pathway and find that IL-10 level is also significantly reduced,suggesting that Notchl/HES1 signal pathway may be related to the production of macrophage anti-inflammatory factor IL-10.In order to further verify this phenomenon,RAW 264.7 cells treated with SPIONs at different concentrations and times were harvested.Western-blot results showed that SPIONs could increase the expression of Beclinl and LC3B proteins in a time and dose-dependent manner.In addition,immunofluorescence results showed that SPIONs could significantly increase the number of punctate aggregates in macrophages.Furthermore,we confirmed that SPIONs can induce autophagy of macrophages in the presence of LPS.Studies have shown that caveolin-1（Cav1）,expressed in macrophages,has regulatory effects on lipid transport,cell membrane transport and intracellular signal transduction,and participates in the regulation of a variety of cellular physiology and pathology.We used protein imprinting and immunofluorescence methods to explore how SPIONs entered macrophages.The results showed that SPIONs could up-regulate the expression of Cav1 in macrophages.Atomic absorption spectrometry showed that iron uptake of macrophages after knockout of Cav1 decreased,suggesting that Cav1 may be related to the uptake of SPIONs by macrophages.In order to clarify the exact function of Cav1,we overexpress or knock out Cav1 protein of macrophages.the results show that the high/low expression of Cav1 is indeed closely related to the increase/decrease of autophagy of macrophages caused by SPIONs.Further inhibition/interference of receptor HES1 of Notch pathway can significantly reduce the induction of macrophage autophagy by SPIONs.the above data indicate that Cav1-Notchl/HES1 signal participates in the regulation of macrophage autophagy induced by SPIONs.The above research results reveal a new mechanism of SPIONs inducing macrophage autophagy,that is,SPIONs promotes IL-10 production in macrophages by activating the Cav1-Notchl/HES1 signaling pathway,thus inhibiting LPS-induced sepsis and liver injury,suggesting SPIONs may be a potential therapeutic drug for sepsis and liver injury.3.SPIONS-labeled MSC can effectively improve sepsis by regulating macrophage polarization to M2.Earlier we proved that SPIONs treatment alone can regulate macrophages to relieve sepsis and liver injury.In a large number of clinical trials,MSCs has been widely used in the treatment of autoimmune diseases,sepsis and other diseases.It is generally believed that MSCs can interact with macrophages through direct contact and paracrine in an inflammatory environment to inhibit the polarization of M1-type macrophages and promote the polarization of M2-type macrophages.IL-10 secreted by M2-type macrophages has immunoregulation,promoting tissue repair and inhibiting inflammation.Here we will discuss the regulatory effects of SPIONs-MSCs on immune response and inflammation.First of all,we determined by pre-experiment that 200 ug/ml SPIONs did not significantly affect the basic characteristics of MSCs derived from human umbilical cord,such as adherence,surface markers（positive expression of CD73,CD90 and CD 105;;Negative expression of CD 19,CD31 and HLA-DR）as well as cell viability and apoptosis.In order to investigate the immunosuppressive function of SPIONs-MSCs in vivo,we established a sepsis model induced by cecum ligation and puncture（CLP）in mice.MSCs or SPIONs-MSCs were infused through caudal vein 4 hours after surgery for treatment.Serum and liver tissues of mice were obtained at 2 and 7 days respectively for detection.The results showed that there were obvious brown deposits stained with Prussian blue in the liver of mice treated with SPIONs-MSCs.Immunofluorescence detection shows that both MSCs treatment group and SPIONs-MSCs treatment group can find the co-localization of human CD90 protein and mouse F4/80 protein,and the co-localization point in SPIONs-MSCs treatment group is more than that in MSCs group,and the duration is longer.These results indicate that SPIONs-MSCs can reach the liver of mice and interact with or be engulfed by liver macrophages.Then we detected the changes of inflammatory factors in serum and liver macrophages,and found that SPIONs-MSCs treatment group improved sepsis symptoms more significantly than MSCs group.The main manifestations were increased survival rate of mice,decreased levels of serum inflammatory factors TNF-α and IL-6,increased levels of serum anti-inflammatory factor IL-10,decreased concentrations of serum glutamic pyruvic transaminase（ALT）and glutamic oxaloacetic transaminase（AST）,and improved liver injury.In order to further verify the effect of macrophages on sepsis,we firstly used the method of intravenous injection of clodronate disodium liposome into the tail of mice to remove the whole body macrophages of mice,and then constructed the mouse sepsis model again.After 48 hours,relevant tissues were obtained and inflammatory indexes were detected.The results showed that the repair effect of SPIONs-MSCs on septic liver injury in mice was significantly reduced after macrophages of septic mice were removed.The results of flow cytometry and immunofluorescence showed that SPIONs-MSCs treatment could effectively reduce the proportion of M1-type macrophages in liver tissue and promote the up-regulation of M2-type macrophages compared with MSC group alone.The detection results of LPS-induced sepsis model and CLP model are similar.These results suggest that SPIONs pretreatment may enhance the immunosuppressive function of MSCs.In order to determine the effect of SPIONs pretreated MSCs on the immune regulation function of macrophages,we co-cultured MSCs and macrophages RAW264.7.Flow cytometry results show that SPIONs-MSCs can significantly increase the proportion of M2 macrophages,significantly reduce the proportion of M1 macrophages,significantly inhibit the expression of M1 markers TNF-α and iNOS at mRNA and protein levels,and promote the expression of M2 markers IL-10 and Arg-1.These results indicate that SPIONs-MSCs can effectively alleviate sepsis and liver injury by inducing polarization of M2 macrophages.4.SPIONS-labeled MSC can regulate macrophage polarization due to TRAF1 protein expression of MSCs.In order to explain the mechanism of SPIONs regulating MSCs,we analyzed the changes of MSC gene expression profile after SPIONs pretreatment by gene chip.The results showed that there were more than 20 receptor protein genes with significantly high expression in TNF signaling pathway,of which TRAF1 had the highest expression.It has been reported that TRAF protein can be associated with various receptors of TNFR superfamily and mediate related signal transduction,in which TRAF1 and TRAF2 form heterodimer complexes which are necessary for TNF-αmediated activation of mitogen-activated protein kinase 8/JNK and nuclear factor-κB.In the mouse sepsis model treated by MSCs derived from bone marrow,TNF-αproduced by LPS stimulating macrophage activation can activate TNFR-1 receptor-NF-κB pathway of MSCs,thus up-regulating COX2 and promoting PGE2 secretion.PGE2 in turn induces macrophage polarization to M2 type,accompanied by large amount of IL-10 expression.However,the role of TRAF1 in the regulation of macrophages by MSC is still unclear.Therefore,we treated MSC with 200 ug/ml SPIONs,and found that SPIONs significantly promoted the high expression of TRAF1,but did not affect the expression of TRAF2.Western blot analysis showed that SPIONs significantly activated phosphorylation of p65 protein and increase of cox2 and IL-10 in TRAF1 downstream signal pathway,suggesting SPIONs may activate NF-kB signal pathway through TRAF1.In order to prove that SPIONs can activate NF-kb pathway by up-regulating TRAF1 and further promote the immunoregulation of MSCs,we knocked out TRAF1 of MSCs and co-cultured with macrophages.The results show that the expression level of Cox2 and IL-10 and other cytokines in MSCs after SPIONs pretreatment is significantly reduced,and the number of M2-type macrophages is reduced.In order to confirm the above results,we established a septic mouse model.After MSCs knocked out TRAF1（SITAF 1）were used for treatment,it was found that compared with SPIONs-MSCs treatment group,SPIONS-MSCS-SITAF 1 treatment group had a lower protective effect and a lower proportion of promoting polarization of M2-type macrophages.These results indicate that the therapeutic function of SPIONs-MSCs depends on TRAF1 protein.In addition,some studies have reported that 1（Heme oxygenase 1（HO-1）can play anti-inflammatory,cell protection and induced tolerance functions.HO-1 modified MSCs can effectively enhance the anti-apoptosis characteristics of MSCs after transplantation into the body under complex environment.Therefore,we examined the changes of MSC status and HO-1 in LPS environment.The results showed that LPS significantly decreased MSC vitality and HO-1 expression,while SPIONs treatment significantly increased HO-1 expression of MSCs.Interference experiments confirmed that SPIONs can enhance the viability of MSCs by promoting HO-1 expression.These results suggest that SPIONs can not only enhance the regulatory immune function of MSCs,but also prolong the survival time of MSCs in vivo.In summary,1）The study found a new mechanism of SPIONs inducing macrophage autophagy,namely SPIONs promotes IL-10 production in macrophages by activating the Cav1-Notchl/HES1 signaling pathway,thus alleviating LPS-induced sepsis and liver injury.2）The study found that SPIONs pretreated MSCs have significant effects on promoting macrophage polarization to M2 type and improving sepsis and liver injury in mice.3)This study reveals that the enhancement of immune regulation function of MSCs pretreated by SPIONs is a new mechanism that depends on TRAF1 expression.In addition,SPIONs can enhance the viability of MSCs by promoting HO-1 expression. |
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