The Investigation Of Adipose-derived Mesenchymal Stem Cells In Treating Rabbit With Induced Autoimmune Dacryoadenitis

Objective Dry eye syndrome(DES)is one of the most common ocular diseases in clinical affecting the quality of life of over 9 million people in United States.The patients with dry eye associated with Sjogren's syndrome or ocular graft-versus-host disease suffered from the functional and professional disability.To date,the limited effective therapies are based on altering the symptom and decelerating the progression of DES.Mesenchymal stem cells(MSCs)are capable of highly self-renewal and have multi-differentiated capacity.MSCs are under investigation for the treatment of autoimmune disease due to their ability of low immunogenicity and potential immunosuppression.Adipose-derived mesenchymal stem cells(ADSCs)outcompete bone marrow-derived mesenchymal stem cells(BMSCs)in terms of easy material ability,abundant source,rapid proliferation and low immunogenicity,so they have been the research hotspot in cell therapy and an important tool for treating autoimmune disorder.In the study,our purposes are to investigate the efficacy of ADSCs in treatment of rabbit with induced autoimmune dacryoadentitis and pursue the possible mechanism by studying the Treg cells,the associated cytokines and transcription factors in vitro and vivo,so as to provide the guidelines for the treatment of autoimmune DES.Methods 1.The isolated lacrimal gland epithelium cells were irradiated and co-cultured with equal numbers of autologous peripheral blood lymphocytes,the lymphocytes proliferation index was studied by measurement of bromodeoxyuridine(BrdU)incorporation using a cell proliferation kit.Five days later,the activated lymphocytes were harvested and injected via ear margin vein to induce dacryoadenitis.Clinical examination were performed before and every two weeks after injection.Lacrimal gland and conjunctiva were collected for histopathology examination and the expression level of IL-1?,IL-6,IL-10,IL-17,IL-18,MMP-2,MMP-9,IFN-? and TNF-? were detected by real-time qPCR in lacrimal gland.2.ADSCs were obtained by digesting the fat tissue with collagenase type ?.ADSCs(P3)were tested for the ability to differentiate into adipocyte and osteoblast lineages under distinct induction conditions and measured the expression level of cell surface markers CD29,CD34,CD44,CD45,CD73 and CD90 by RT-PCR.Lymphocyte proliferation was measured by flow cytometry of carboxyfjrorescein succinimidyl ester(CFSE)-labelled lymphocytes.Then the percentage of Treg and the expression levels of gene related with Th1/Th17/Treg were detected by FCM and real-time qPCR.3.The first injection of ADSCs or PBS was performed at one day after adoptive transfer of activated PBLs,the next injections were performed every other day.Clinical examinations were performed every two weeks for six weeks after first ADSCs administration.Lacrimal gland and conjunctiva were collected for histopathology examination and the expression level of IL-1?,IL-6,IL-10,IL-17,IL-18,T-bet,RORC,MMP-2,MMP-9,IFN-?,TGF-? and TNF-? was detected by real-time qPCR in lacrimal gland.The lymphocyte proliferation index was studied by measurement of bromodeoxyuridine(BrdU)incorporation using a cell proliferation kit.Then the percentages of Treg in lacrimal glands and spleens were detected by FCM.Results 1.The lacrimal gland epithelial cells could activate and stimulate the proliferation of autologous peripheral blood lymphocytes,and proliferation index was 3.63.The activated lymphocytes injected via ear margin vein could induce rabbit autoimmune dacryoadenitis.The rabbits showed the decreased tear production,shorten tear break-up time,abnormal corneal staining,large infiltration in lacrimal gland and conjunctiva.The mRNA expression levels of IL-1?,IL-6,IL-10,IL-17,MMP-2,MMP-9,IFN-? and TNF-? in lacrimal gland of dry eye group were significantly increased than normal group,while no changes in expression of IL-18.2.ADSCs were able to grow adhering to the wall with a shape of long fusiform or polygon,arrange in swirl and differentiate into adipocytes and osteoblasts,which were verified by Oil Red-O and Alizarin red staining.ADSCs were positive for the MSC markers CD29,CD44,CD73 and CD90,and negative for hematopoietic markers CD34 and CD45.The lymphocytes cultured in the presence of ADSCs showed a clear reduction in lymphocytes proliferation in a dose-dependent manner.ADSCs significantly inhibited the expression of IL-17,IFN-?,TNF-?,but enhanced the expression of anti-inflammatory cytokines IL-10 and TGF-?.The percentage of Treg cells was significantly higher in ADSCs treated group than that in the control group.3.Clinical symptoms showed the improvement after ADSCs administration The H&E-stained sections revealed that the lacrimal glands of ADSCs treated group had less immune cell infiltration,whereas those from untreated group were heavily infiltrated and showed clusters of abnormal epithelial cells.The proliferation rates of PBLs from ADSCs treated group were significantly lower compared with that from untreated group.ADSCs treated group had significant lower mRNA expression of IL-17,RORC,T-bet and IFN-? than untreated group suggest that ADSCs suppressed both Th1 and Th17 responses concurrently in vivo.The percentages of Treg cells were greatly higher in the spleens and lacrimal glands of ADSCs treated rabbits than that of untreated group.ADSCs injection significantly reduced the expression of inflammatory mediators,including IL-6,IL-1?,TGF-?,MMP-2,MMP-9,while it increased the expression of the anti-inflammatory cytokine IL-10 in the lacrimal glands of autoimmune dacryoadenitis rabbits.However,ADSCs treatment had no effect on the mRNA expression of IL-18 and TNF-?.Conclusions Injection of the autologous peripheral blood lymphocytes activated by lacrimal gland epithelial cells could induce the stable rabbit models of autoimmune dacryoadenitis.The occurrence of dry eye is probably related to the imbalanced T cell subsets,and IL-1?,IL-6,IL-10,IL-17,IL-18,IFN-?,TNF-?,MMP-2 and MMP-9 may participate in the obstruction and the secretion deficiency of the lacrimal gland.ADSCs transplantation can alleviated autoimmune dacryoadenitis by suppressing the T cell activation and differentiation of Th1/Th17 cells,promoting the Treg proliferation and regulating the unbalance of Th17/Treg.