| ObjectiveDry eye disease is a common immune-inflammation mediated ocular disorder.Sj?gren’s syndrome(SS)is one of the clinical chronic and refractory autoimmune dry eye diseases,even developing vision-threatening complications such as corneal melt or perforation.To date,there is no treatment proven effective to cure this slow-evolving disease.Mesenchymal stem cells(MSCs)have been proposed as a promising therapeutic alternative for inflammatory and autoimmune diseases due to their low immunogenicity and immunoregulation function.However,the underlying mechanisms of MSC treatment applied in autoimmune dry eye diseases are not fully understood.Human umbilical cord mesenchymal stem cells(hUC-MSCs)have low immunogenicity,high accessibility,great proliferation capability and few ethical constraints,thus offering the best option for the MSC-based therapy.In this study,we aim to investigate the therapeutic effects of hUC-MSCs on rabbit autoimmune dry eye induced by transfer of in vitro activated peripheral blood lymphocytes and explore the underlying mechanisms,so as to provide the guidelines for the hUC-MSC treatment in clinical autoimmune dry eye and other immune-related ocular diseases.Methods1.The hUC-MSCs were isolated from fresh human umbilical cords by the sequent digestion of collagenase II and trypsin,followed by adherent culture methods.When the adherent monolayer cells reached 80%to 90%confluence,they were trypsinized and subcultured.hUC-MSCs at passage 3 were characterized by cell morphylogy,MSC surface markers measured by real-time PCR(RT-PCR)and multi-differentiation potentials into adipocytes and osteoblasts.2.27 rabbits were randomly divided into the healthy group(n=9),the untreated group(n=9)and the hUC-MSCs treated group(n=9).Rabbit model of autoimmune dry eye was established by transfer of in vitro activated peripheral blood lymphocytes via ear margin vein.At the second week after model induction,rabbits in the hUC-MSCs treated group were injected with 1×10~7hUC-MSCs(suspended in 1mL 1×PBS)via ear margin vein for 5 consecutive days.As a control,rabbits in the untreated group were injected with 1mL 1×PBS.Clinical ocular surface assessments were conducted on day0 for baseline and every 2 weeks after model induction.After 8-week observation,rabbits were sacrificed,and the lacrimal glands and conjunctiva were collected for hematoxylin and eosin(H&E)staining.Quantitative real-time PCR(qRT-PCR)was performed to detect the mRNA expression of M1/M2 macrophage phenotypic molecular and related cytokine genes.Besides,the protein level of the M2 macrophage phenotypic molecular Arg1 was detected using Western blot analysis.3.In vitro,activated peripheral blood mononuclear cells(PBMCs)from rabbit autoimmune dry eye model or LPS+IFN-γ-stimulated THP-1 cells were incubated with or without hUC-MSCs in a trans-well system.After co-culture,the mRNA expression of M1/M2 macrophage markers in PBMCs and THP-1 cells was analyzed by qRT-PCR,the percentage of Arg1 positive cells in THP-1 cells was detected using immunofluorescence staining,and the protein expression of Arg1,M1 macrophage phenotypic molecular i NOS,and phosphorylated Akt(p-AKT)in macrophages was examined by Western blot analysis.Results1.hUC-MSCs exhibited a fusiform adherent growth,arranged in bundles or swirl,and were capable of differentiating into osteoblasts and adipocytes under appropriate inductive culture conditions.Phenotypic assays showed that these cells strongly expressed MSC surface markers CD44,CD73,CD105 and CD29,but not CD45,CD34and HLA-DR.2.hUC-MSC injection obviously improved the tear production and tear stability,meanwhile decreased the corneal fluorescein staining.Besides,the histopathological findings showed that hUC-MSC treatment significantly diminished immune cell infiltrates and structure damage in lacrimal glands and conjunctiva.qRT-PCR analysis revealed that the mRNA expression of M2 macrophage markers Arg1,CD206,IL-10,TGF-βand IL-4 was remarkably lower in lacrimal glands of the dry eye group than that of the healthy group.Administration of hUC-MSCs significantly increased the mRNA level of these M2 macrophage markers,while down-regulated the expression of pro-inflammatory mediators including TNF-α,IL-1β,IL-6,IFN-γ,GM-CSF,MCP-1,MMP-2 and MMP-9 mRNA(all at P<0.05).There was no statistical difference in the expression of i NOS(M1 macrophage phenotypic molecular)between the dry eye group and the hUC-MSC treated group.In addition,the Arg1 protein level in lacrimal glands was significantly up-regulated by hUC-MSC treatment(P<0.05).3.After hUC-MSC co-culture in a trans-well system,the expression of M2macrophage markers Arg1,CD206,IL-10 and IL-4 in PBMCs was significantly increased,whereas the mRNA level of M1 macrophage-associated genes iNOS,TNF-αand IL-6 was decreased(all at P<0.05).The expression of IL-1βand TGF-βmRNA between two groups had no statistical difference.Besides,the increased protein level of Arg1 was detected in the hUC-MSC group compared to the control group.Similarly,the results from qRT-PCR,Western blot analysis and immunofluorescent staining demonstrated that hUC-MSCs could convert THP-1-derived M1 macrophages into an anti-inflammatory M2 phenotype through paracrine mechanism.Additionally,hUC-MSC co-culture obviously enhanced the expression of p-AKT in the LPS+IFN-γ-stimulated macrophages.After blockage of AKT by 10μM LY294002,a specific pharmacological inhibitor of PI3K/AKT,the upregulated expression of Arg1 protein in the stimulated macrophages induced by hUC-MSCs was partially abolished(all at P<0.05).ConclusionsIntravenous injection of hUC-MSCs efficiently attenuated the clinical severity,suppressed inflammation and diminished lacrimal structure destruction in autoimmune dry eye,and this therapeutic effect may be partly ascribed to regulating local immune milieu in lacrimal glands and thus promoting macrophages into an anti-inflammatory M2 state.hUC-MSCs may induce M2 macrophage polarization via activating AKT pathway in a paracrine manner. |
PDF Full Text Request