Spontaneously Apoptotic Cells In Therapeutic Stem Cell Preparation Exert Immunomodulatory Effects Through Release Of Phosphatidylserine

Mesenchymal stem cells（MSCs）are multipotent adult stem cells and can differentiate into a variety of mesenchymal cells,including bone cells,cartilage cells,adipose cells,and skeletal muscle cells.MSCs-mediated immunomodulation has been harnessed for the treatment of human diseases,but its underlying mechanisms have not been fully understood.However,via homing and tracking MSCs,recent studies have found that MSCs are short-lived after transplantation into recipients because of continuous compressive mechanical stress,lung capillaries,harsh microenvironment,and gradual loss.For instance,only a few living MSCs have been observed in the injured brain,liver,or spinal cord.Most MSCs died of apoptosis in a short time in the recipient.However,it is unclear whether these dead MSCs（DMSCs）play an immunomodulatory role in vivo.From the reported clinical trials data,we knew that the nonviable MSCs of injected MSCs were about 5%～50%.It has been reported that apoptotic cells showed immunoregulatory effects.Therefore,the above evidences made us hypothesize that nonviable MSCs might also play a role in the therapeutic effects of the MSCs through regulating the local immune-microenvironment.Therefore,in this study,we administered living MSCs and dead MSCs to mice of four injury mice models,including ConA-and CCl₄-induced liver injury,LPS-induced lung injury and spinal cord injury（SCI）,to evaluate the therapeutic effects of DMSCs and further investigated the underlying mechanisms.MSCs were isolated from the femur and tibia of C57 mice aged 2～3 weeks.The isolated MSCs exhibited long-spindle shape and were positive for CD29,CD44,and Sca-1 but negative for CD45,CD11b,and CD31 identified by flow cytometry.The cells that died spontaneously during the culture of MSCs at 4～6 generations were collected.The dead cells were identified by trypan blue staining and flow cytometry with cell death fluorescent dye,namely dead MSCs（DMSCs）.The DMSCs were apoptotic rather than necrotic identified by cleaved caspase 3 and cathepsin B staining.We calculated the percentage of DMSCs by the flow cytometry and found that there were about 5%DMSCs in our MSC therapeutic preparation.Namely,1×10⁶ MSCs contained 5×10⁴ DMSCs.Mice were injected with ConA or CCl₄ injection,followed by intravenous injection with 1×10⁶ MSCs（containing 5×10⁴ DMSCs）or 5×10⁴ DMSCs,1×10⁵DMSCs,2.5×10⁵ DMSCs,5×10⁵ DMSCs and 1×10⁶ DMSCs,respectively.The mice without any treatment were Control group.Livers were obtained 12 h after ConA injection or 24 h after CCl₄ injection.We observed that the liver of PBS group seemed brownish-red with obvious hemorrhagic focus（in ConA model）or pale with granular sense（in CCl₄ model）.And the H&E staining results showed that the structure of hepatic lobule was broken with a range of liver necrotic area compared with PBS group.In addition,levels of serum ALT,AST and pro-inflammatory factors IL-6,IFN-γ,TNF-αand TUNEL positive cell number in PBS group were significantly increased.It is interesting to find that treatment with the DMSCs alone,even with the equal number of DMSCs（5×10⁴）in our DMSCs-containing MSCs preparation,is as effective as DMSCs-containing MSCs（1×10⁶ MSCs）in improving the structure of hepatic lobule,decreased the range of necrotic area,reduced the level of serum ALT,AST and had fewer apoptotic hepatocytes.In addition,DMSCs reduced the levels of IL-6,IFN-γ,and TNF-αin serum and increased IL-10 and HGF levels in hepatic tissues.More importantly,both DMSCs and MSCs improved the mice survival compared with PBS group.Taken together,the above results showed that DMSCs,even existed in a small fraction in our MSC therapeutic preparation,displayed similar hepatoprotective properties as MSCs in alleviating ConA-and CCl₄-induced liver injury.In order to better clarify that DMSCs played protective effects as MSCs in tissue injury,we constructed a LPS induced lung injury model,and then injected PBS via tail vein,1×10⁶ MSCs（containing 5×10⁴ DMSCs）or 5×10⁴ DMSCs.We observed that the lung of PBS group had larger pulmonary edema and obvious hemorrhage compared with NS group.And inflammatory infiltrates,interalveolar septal thickening,and interstitial edema were observed in PBS group.The injection of both 1×10⁶ MSCs（containing 5×10⁴ DMSCs）and 5×10⁴ DMSCs attenuated lung damages and reduced the histopathology score of lung sections.Next,we established a SCI model to determine the role of MSCs and DMSCs.PBS,1×10⁶ MSCs（containing 5×10⁴ DMSCs）or 5×10⁴ DMSCs were injected into the mice after SCI.The movement of lower limbs after spinal cord injury was observed by Basso mouse scale（BMS）.The results showed that there were no differences in BMS scores among PBS,MSC and DMSC groups at 3 days after SCI.Transplantation of MSCs and DMSCs showed a protective role in SCI mice for increasing the BMS score of SCI mice at day 21 and 28.The above results indicated that DMSCs had similar protective effects to MSCs in protecting tissues from damages.The previous studies reported that most injected MSCs were trapped in lung and died of apoptosis within few hours.In order to reveal the intrinsic relationship that why DMSCs have similar protective effects to MSCs,we isolated GFP labeled MSCs（GFP-MSCs）from GFP mice,and transferred GFP-MSCs into mice to observe whether GFP-MSCs went through apoptosis within few hours.And the results showed that GFP-MSCs in lung and liver tissues stayed viable after 0.5 h.However,MSCs were stained as cleaved caspase 3-positive after 2 h to 4 h as observed by fluorescence microscopy.Additionally,the numbers of living MSCs in the lung and liver were remarkably declined 12 h and 24 h after GFP-MSCs injection,indicating that most transplanted GFP-MSCs died within 12 h.The above results suggest that injected MSCs went through apoptosis.Since the most prominent characteristics of apoptosis cells are phospholipid rearrangement and exposure and capsizing of phosphatidylserine（PS）,we then determined the levels of lipids after injection of 1×10⁶ MSCs（containing 5×10⁴ DMSCs）and 5×10⁴ DMSCs at 0.5 h or 4 h in vivo.We found that lipid abundance varies with the injection time and cell types as analyzed by Principal component analysis（PCA）.It is interesting to find that,one of the representative markers of apoptosis,PS,has intensively changed after MSCs infusion.Specifically,higher levels of lipids,including PS（18:0/16:1）-H,PS（18:0/18:1）-H,and PS（18:0/22:6）-H were observed in both DMSC-0.5 h and MSC-0.5 h groups,and a significant uplift was observed in MSC-4 h group compared with Control group.Furthermore,the levels of PS（18:0/22:5）-H,PS（37:2）-H,and PS（39:1）-H raised significantly 4 h after MSCs injection.The above results indicated that the injection of MSCs released PS into the blood and the amount of lipids released correlated with the number of transferred cells.More importantly,we prepared PS liposomes（PSLs）to mimic the membrane-located PS and control liposomes without PS（Phosphatidylcholine liposomes,PCLs）to further characterize the function of PS.Interestingly,we found that intravenous injection of PSLs exhibited hepatoprotective effects with reduced necrotic area,more apoptotic hepatocytes,improved liver function and animal survival.Moreover,consistent with MSCs and DMSCs,PSLs also increased the levels of HGF and IL-10 and decreased the levels of proinflammatory cytokines,including IL-6,IFN-γ,and TNF-α.Besides,PSLs also reduced LPS-induced lung injury.Taken together,the above results suggested that PS played a protective role in modulating acute liver injury and lung injury similar to that of MSCs and DMSCs.Next,we investigated what receptors were responsible for the therapeutic effect of PSLs.Previous studies have reported that TAM receptor family including Tyro3,Axl and MerTK,are the main receptors for PS recognition expressed on monocytes/macrophages.To clarify whether MSCs,DMSCs and PSLs protect against tissue injury through TAM receptors,we used LDC1267 to selectively inhibit MerTK,Tyro3 and Axl.LDC1267（30 mg/kg）was injected intraperitoneally 30 min before ConA injection,and the same volume of solvent（Vehicle Group）was injected as control.We observed that administration of MSCs,DMSCs and PSLs showed reduced necrotic areas and lower levels of AST and ALT in mice compared to Vehicle combined with PBS group.However,pretreatment of mice with LDC1267 efficiently blocked the protective effects of MSCs,DMSCs and PSLs,indicating that TAM receptors are crucial in mediating the therapeutic effects of MSCs,DMSCs and PSLs.Further,we detected the expression level of three receptors.Notably,the administration of MSCs,DMSCs and PSLs enhanced the m RNA level of MerTK,whereas with no significant changes in the levels Tyro3 and Axl.We then used MerTK knockout mice(MerTK^-/-group)to evaluate the therapeutic effects,and B6/129 mice with the same genotype background were used as control（WT group）.We found that MSCs,DMSCs or PSLs improved pathological appearances,decreased necrotic areas,AST and ALT levels compared with PBS group in WT mice.In addition,levels of p-p38 MAPK and IL-10 were significantly increased while the level of p-NF-κB p65decreased in MSC,DMSC and PSL groups compared with PBS and PCL groups in WT mice.However,MSCs,DMSCs or PSLs had little effect in attenuating the liver injury in MerTK^-/-mice,and failed to promote the survival in MerTK^-/-mice while compared with that of the PBS group.And there were no significant differences in p-p38 MAPK,IL-10 or p-NF-κB p65 levels among the groups in MerTK^-/-mice.In summary,the above results suggested that MSCs,DMSCs and PSLs ameliorated liver injury through MerTK signaling.To further understand which population of immune cells were effective in the process of liver protection,we evaluated the activation of CD4⁺T cells（identified as CD45⁺CD3⁺CD4⁺）,CD8⁺T cells（identified as CD45⁺CD3⁺CD8⁺）,and natural killer（NK）cells（CD45⁺CD3^-NK1.1⁺）in WT and MerTK^-/-mice.The infiltration of neutrophils（identified as CD45⁺CD11b⁺Ly-6G⁺）,monocyte-derived macrophages(MoMF,identified as CD45⁺CD11b⁺Ly-6G^-F4/80^low),and kupffer cells（KCs,identified as CD45⁺CD11b^-Ly-6G^-F4/80⁺）in liver tissues were also characterized.In contrast to mice treated with PBS,mice treated with MSCs,DMSCs or PSLs showed a significant reduction in the percentages of activated NK cells and infiltrated neutrophils.There were no significant differences in the percentages of activated CD4⁺T cells,CD8⁺T cells though,the percentage of Ly-6C^high MoMF(identified as CD45⁺CD11b⁺Ly-6G^-F4/80^lowLy-6C^high)and number of Ly-6C^highIL-10-producing MoMF were significantly increased in MSC,DMSC and PSL groups compared to that of PBS group.Moreover,these changes were reversed in MerTK^-/-mice.These results suggested that MSCs,DMSCs and PSLs played a protective role in liver by regulating the immune microenvironment in liver tissue through MerTK.Since the Ly-6C^high MoMF population increased sharply in the mice of MSC,DMSC or PSL groups,we further explored their roles in the immunomodulation of ConA-induced ALI and we used CCR2^-/-mice to verify whether Ly-6C^high MoMF infiltration is dependent on CCR2.We found that MSCs,DMSCs and PSLs attenuated liver injury compared to PBS in WT mice,but these effects were eliminated in CCR2^-/-mice.Moreover,the percentages of activated NK cells,infiltrated neutrophils,Ly-6C^high MoMF or IL-10 producing Ly-6C^high MoMF showed no significant differences among the groups.These results suggested that CCR2 plays a role in the MSCs,DMSCs and PSLs mediated reduced neutrophils infiltration and promoted Ly-6C^highMoMF recruitment.Next,we found that the treatment of MSCs,DMSCs and PSLs decreased the percentages of F4/80⁺i NOS⁺macrophages（M1 macrophages）and increased the percentages of F4/80⁺CD206⁺macrophages（M2 macrophages）.The detected m RNA levels of i NOS and CD206 in liver tissues were also consistent with the IF results.Moreover,MSCs,DMSCs and PSLs induced higher level of IL-10 in macrophages.In a word,the above results suggested that MSCs,DMSCs and PSLs could promote monocytes/macrophages polarization into M2 phenotypes with enhanced IL-10 secretion.In conclusion,our data elucidated that the apoptotic MSCs（DMSCs）in MSCs preparation play an important role in attenuating the tissue injury by exposing PS.PS from the DMSCs activates MerTK and increases the IL-10-producing Ly-6C^highMoMF recruitment through CCR2.In addition to the decreased neutrophil infiltration,the inhibition of NK cell activation,PS from DMSCs also induces macrophage polarization towards M2 phenotype and promotes IL-10 production.These results provide new insight into how MSC-based therapies function and exert an immunomodulating property in the acute tissue injury model.