Intestinal Tissue-derived Exosomes Promoting M1 Macrophage Polarization In Intestinal Ischemia-reperfusion Induced Liver Injury In Mice

Background:Intestine ischemia/reperfusion is one of the main cause of multiple organ dysfunction or failure in perioperative patients,which is associated with exacerbation of intestinal injury itself and progressive distal organ impairment,especially liver injury.The precise molecular mechanisms involved have not been fully elucidated despite intensive investigation,as much effective strategies for its prevention is still lacking.Exosomes are extracellular vesicles composed of a phospholipid bilayer and enclosed content such as proteins and nucleotides;they participate in intercellular communications by transferring their cargo from donor cells into the target cells.However,whether intestinal tissue-derived exosomes is involved in intestinal ischemia-reperfusion induced liver injury is not clear.Macrophage,as an essential component of innate immunity,has emerged as a key role in multiple liver diseases.Activated macrophages are defined as classically activated or M1 type and alternatively activated or M2 type.M1 type macrophages are a group of pro-inflammatory heterogeneous macrophages that present antigens,initiate and promote tissue inflammation and damage.At present,there is only one literature report that HMGB1-associated necroptosis and Kupffer cells M1 polarization underlies remote liver injury induced by intestinal ischemia/reperfusion.Therefore,We hypothesized that exosomes derived from intestinal tissue after intestinal ischemia reperfusion are endogenous signals leading to acute liver injury by M1 macrophage activation.Objective:To explore whether EXOs released following intestinal ischemia/reperfusion are endogenous signals leading to acute liver injury by M1 macrophage polarization.Methods:(1)The effects of blocking exosome release on intestinal I/R liver injury in mice.Adult male C57/BL6 mice were randomly divided into three groups:sham group,IR group and GW4869 group.In sham group,SMA was isolated but not occluded;in IR group,SMA was occluded for 1h followed by 6h of reperfusion;the mice in EXOs inhibitor group(GW4869 group)was subcutaneously injected with GW4869(2.5mg/kg)24h prior to intestinal ischemia and at the onset of intestinal reperfusion,and the rest procedures were performed as the method described in IR group.The 3-day survival rate of the mice was recorded,and the degree of damage to liver and intestinal mucosa was evaluated according to Suzuki’s classification and Chiu’s scoring.Moreover,serum ALT and AST levels were measured.(2)The isolation,purification and identification of exosomes from mice intestinal tissues in a mouse model of intestinal I/R.The morphology of isolated exosomes was identified by transmission electron microscopy(TEM).The size distribution of exosomes were measured using nanoparticle tracking analysis.The surface markers of exosomes,CD9,CD63 and CD81 were identified by Western-blot analysis.(3)The effects of intestinal tissue-derived exosomes injection on liver injury in mice.Adult male C57/BL6 mice were randomly divided into three groups:PBS group,EXO-S group and EXO-IR group.The mice in PBS group were injected with 200μl PBS via tail vein;the mice in EXO-S group and EXO-IR group were injected with 200μl EXOs(100μg by protein quantification)derived from intestinal tissues in the sham or IR group via tail vein respectively.After 6 hours,the mice were sacrificed in order to harvest their liver and blood.The degree of damage to liver was evaluated according to Suzuki’s classification,the serum ALT and AST levels were measured.(4)The effects of intestinal I/R and exosome from mice intestinal tissues in a mouse model of intestinal I/R on the phenotype of liver macrophages.Adult male C57/BL6 mice were randomly divided into three groups:sham group,IR group and GW4869 group,and then the procedures were performed as the method described in part 1.Localization of F4/80 in the liver with immunohistochemistry staining was used to determine macrophage infiltration.Additionally,the expression of CD86(M1 Marker)and CD206(M2 Marker)in hepatic macrophages was detected by flow cytometry,and the mRNA expression of iNOS(M1 Marker),Arg-1(M2 Marker),IL-1β,IL-6 and CCL-2 was detected using qRT-PCR.Adult male C57/BL6 mice were randomly divided into three groups:PBS group,EXO-S group and EXO-IR group,and then the procedures were performed as the method described in part 3.Localization of F4/80 in the liver with immunohistochemistry staining was used to determine macrophage infiltration.Immunofluorescence staining of frozen liver sections was utilized to observe fluorescence co-localization of the PKH26-labeled exosomes and FITC-F4/80 labeled hepatic macrophages.The expression of CD86 and CD206 in hepatic macrophages was detected by flow cytometry,and the mRNA expression of iNOS,Arg-1,IL-1β,IL-6,and CCL-2 was detected with qRT-PCR.Raw264.7 macrophages were divided into three groups:control group(Crtl group),EXO-S group and EXO-IR group.The Crtl group,where cells were not processed;EXO-S group and EXO-IR group,where EXOs(10μg by protein quantification)derived from intestinal tissues in the sham or IR group were added into a cell culture medium for 12h.Co-localization of the PKH67-labeled exosomes and PKH26-labeled Raw264.7 cells was observed using fluorescence microscopy,and the expression of CD86 and CD206 in Raw264.7 macrophages was analyzaed by flow cytometry.Furthermore,the mRNA expression of iNOS,Arg-1,IL-1β,IL-6,and TNF-α was detected with qRT-PCR.Results:(1)Compared to the sham group and GW4869 group,the 3-day survival rate of mice in IR group was found to be significantly reduced.Severe damage of the liver and intestinal mucosa was observed in the IR group.The pathological scores in the IR group were significantly higher than those in the sham group and GW4869 group;Moreover,the serum ALT and AST levels dramatically increased in the IR group compared with the sham group and GW4869 group.Compared to the IR group,the 3-day survival rate of mice in the GW4869 group was observed to be significantly increased.The liver and intestinal mucosa histopathological scores in the GW4869 group were significantly decreased compared with the IR group;the levels of ALT and AST were lower in the GW4869 group than those in the IR group.These data indicate that inhibition of exosomes release could attenuate liver injury induced by intestinal I/R.(2)TEM showed typical cup-shaped structure and size of exosomes purified from mice intestinal tissues as detected by phosphotungstic acid staining.The average size of the vesicles were about 100nm.Western blot analysis showed that these exosomes were positive for CD9,CD63 and CD81.These results affirm that exosomes derived from intestinal tissue were isolated successfully.(3)There is no significant difference in the liver pathological score and the levels of ALT and AST between the PBS group and EXO-S group.However,the liver pathological score and the levels of ALT and AST in EXO-IR group were markly higher when compared with the PBS group and EXO-S group.These results suggested that exosomes from mice intestinal tissues in a mouse model of intestinal I/R could damage the liver of mice.(4)Compared to the sham group and GW4869 group,the infiltration of F4/80-positive macrophages in the IR group were significantly increased.Flow cytometry analysis showed the percentage of CD86+(M1-type)macrophages in IR group increased markedly compared with the sham group and GW4869 group,while no significant difference was observed for the percentage of CD206+(M2-type)macrophages among the three groups.The mRNA expression levels of M1-specific cytokines,such as iNOS,IL-6,IL-1β,CCL-2 in IR group were also significantly higher than those in the sham group and GW4869 group.In contrast,the mRNA expression level of M2 macrophage marker Arg-1 in IR group was decreased.However,the infiltration of F4/80-positive macrophages in the GW4869 group were significantly decreased compared with the IR group.The percentage of CD86+macrophages in GW4869 group decreased markedly compared with the IR group.The mRNA expression levels of M1-specific cytokines in GW4869 group were also significantly lower than those in the IR group,the mRNA expression level of M2 macrophage marker Arg-1 in GW4869 group was increased.Our results suggest that M1 polarization of hepatic macrophages is involved in the occurrence of intestinal I/R liver injury and inhibiting exosomes release can reduce M1 polarization of hepatic macrophages and the expression of pro-inflammatory cytokines.EXOs derived from intestinal tissues in the sham or IR group were mainly uptaken by macrophages.There is no significant difference in the macrophage polarization,and the expression of inflammatory cytokines between the PBS group and EXO-S group.However,the infiltration of F4/80-positive macrophages and the percentage of CD86+macrophages in the EXO-IR group were significantly increased compared with the EXO-S group.The mRNA expression levels of M1-specific cytokines were also significantly higher than those in the EXO-S group,the mRNA expression level of M2 macrophage marker Arg-1 in GW4869 group was decreased.Our results suggest that exosomes from mice intestinal tissues in a mouse model of intestinal I/R promote M1 polarization and pro-inflammatory cytokines expression in hepatic macrophages.EXOs derived from intestinal tissues in the sham or IR group were efficiently engulfed by Raw264.7 macrophages.There is no significant difference in the macrophage polarization,and the expression of inflammatory cytokines between the Ctrl group and EXO-S group.However,the percentage of CD86+macrophages and M1 macrophage marker gene in the EXO-IR group were significantly increased compared with the EXO-S group.There is no significant difference in the percentage of CD206+macrophages and M2 macrophage marker gene between the EXO-IR and EXO-S group.The result of cell experiments further confirmed that exosomes from mice intestinal tissues in a mouse model of intestinal I/R promote M1 polarization and pro-inflammatory cytokines expression in Raw264.7 macrophages.Conclusion:1.Exosomes derived from intestinal tissue were isolated successfully.2.Intestinal tissue-derived exosomes are involved in the occurrence of intestinal I/R liver injury,which is mainly mediated by the M1 type macrophages polarization in the liver.This study provides a novel idea for the prevention and treatment of intestinal I/R injury.