Phagocytosis Of Apoptotic Cells By Mesenchymal Stem Cells In The Treatment Of SLE

Backround:Defective clearance of apoptotic cells(ACs)results in the accumulation of ACs in systemic lupus erythematosus(SLE).Accumulated ACs are prone to progress to secondary necrosis,which then exposes autoantigens,leading to the breakdown of self-tolerance and tissue damage.Mesenchymal stem cells(MSCs)exhibit promising therapeutic effects on SLE.Our previous studies showed that MSC transplantation reduced apoptotic T cell numbers and enhanced the phagocytic function of macrophages(Mφ)in patients with SLE.However,whether MSCs phagocytose ACs and the underlying effector mechanism in the treatment of SLE remain unknown.Objectives:To examine the phagocytic function of MSCs and investigate the immunosuppressive capacity and therapeutic effects of AC-engulfed MSCs(AC-MSCs)in SLE.Methods:Umbilical cord(UC)MSCs were co-cultured with ACs,and the in vitro engulfment of ACs by MSCs was either detected by flow cytometry(FCM)or observed under confocal laser scanning microscope.ACs and MSCs labeled with different fluorescent dyes were infused into mice to observe in vivo phagocytosis of ACs by MSCs.Using recombinant Annexin V to block phosphatidylserine(PS)or small interfering RNA(siRNA)to knock down milk fat globule epidermal growth factor 8(MFG-E8),the mechanisms underlying AC engulfment by MSCs were investigated.The phagocytic function of bone marrow(BM)MSCs from SLE patients and pristaneinduced lupus mice was also assessed.The suppressive abilities of MSCs and ACMSCs on CD4+T cell proliferation were compared.Peripheral blood mononuclear cells(PBMCs)from HC were also cultured in MSC conditioned medium(MCM)or ACMSC conditioned medium(ACMCM),and then CD4+T cell proliferation was detected.Soluble factors including prostaglandin E2(PGE2)in the supernatants of MSCs and AC-MSCs,as well as in the mouse peritoneal lavage fluids(PLF)were determined by enzyme-linked immunosorbent assay(ELISA).Real time polymerase chain reaction(real time PCR)and western blotting(WB)analyses were performed to assess the expression of cyclooxygenase 2(COX2).COX2 inhibitors and siRNA transfection were utilized to determine the function of COX2/PGE2 in AC-MSCs mediated suppression of CD4+T cell proliferation.Moreover,signaling pathways involved in the activation of COX2/PGE2 were also investigated by WB analyses.PGE metabolites(PGEM)in the plasma of SLE patients were measured before and 24 hours after MSC transplantation respectively.Furthermore,MSCs,AC-MSCs and COX2 knock-down MSCs and AC-MSCs were respectively infused into MRL/lpr mice for assessing their effects on lupus symptoms and disease progression.Results:MSCs were found to engulf ACs both in vitro and in vivo.PS blocking and MFG-E8 knock-down by siRNA transfection significantly impaired the phagocytosis of ACs by MSCs.BM-MSCs from SLE patients and pristane-induced lupus mice showed reduced AC uptake.Compared to MSCs,AC-MSCs showed enhanced suppressive effects on CD4+T cell proliferation.Besides,ACMCM remarkably inhibited CD4+T cell proliferation,whereas MCM could not.PGE2 level in the supernatant of AC-MSCs was markedly higher than that of MSCs.Similarly,in vivo phagocytosis of ACs by MSCs resulted in elevated PGE2 levels in mouse PLF.Notably,COX2 expressions also increased in AC-MSCs.In addition,COX2 inhibition and COX2 knock-down experiments showed that phagocytosis of ACs enhanced the suppressive function of MSCs through COX2/PGE2 pathway.Further cell signaling assays demonstrated that Rac1 and NF-kB mediated the activation of COX2/PGE2.In patients with SLE,the plasma PGEM levels increased significantly in those with reduced apoptotic T cells after MSC transplantation.In MRL/lpr mice,AC-MSCs treatment led to significantly higher survival rate than PBS infusion.Moreover,ACMSCs decreased proteinuria levels as early as one week after infusion.The serum dsDNA antibody levels and plasma cells in both spleen and renal draining lymph node in AC-MSCs treated mice decreased significantly compared with PBS treated group.Both of MSCs and AC-MSCs reduced IgG and C3 deposits and increased CD206+ Mφin kidneys,whereas COX2 knock-down MSCs and AC-MSCs could not.Conclusion:These results demonstrate that MSCs can phagocytose ACs in SLE.ACengulfed MSCs possess greater immnosuppressive ability via increasing PGE2 production to alleviate SLE.Our findings may help develop new therapeutic strategies for MSC transplantation in treating SLE.