Therapeutic effects of mesenchymal stem cells administered at later phase of recurrent experimental autoimmune uveitis

AIM：To test the therapeutic effects of delayed treatment of mesenchymal stem cells（MSCs） in recurrent experimental autoimmune uveitis（r EAU）.METHODS： The efficacy of different regimens of MSC administration in r EAU were tested by evaluation of clinical and pathological intraocular inflammation,as well as retinal structural and functional integrity using optical coherence tomography（OCT） and electroretinogram（ERG）.The retinal sections were also immunostained with antibodies to glial fibrillary acidic protein（GFAP）and rhodopsin（RHO）. RESULTS： Delayed treatment of MSCs effectively alleviated the severity of intraocular inflammation with relative intact of outer retinal structure and function.Moreover,double therapies with longer interval led to an even better clinical evaluation,as well as a trend of decrease in relapse and amelioration of retinal function.MSC therapies also effectively reduced GFAP expression and increased RHO expression in the retina.CONCLUSION： MSC administration can effectively treat developed diseases of rEAU,and multiple therapies can provide additional therapeutic benefits.

The interaction of dendritic cells and γδ T cells promotes the activation of γδ T cells in experimental autoimmune uveitis

Uveitis is a severe inflammatory disease that can cause visual impairment.Recently,activatedγδT cells were proved to play a central role in the development of experimental autoimmune uveitis(EAU).However,the mechanism underlyingγδT cell activation in EAU is incompletely known.In this study,we determined the percentage changes in and the phenotypes ofγδT cells and dendritic cells(DCs)obtained from the spleens of immunized C57BL/6(B6)mice,an animal model of EAU.We found that the number ofγδT cells and DCs obviously increased during the inflammation phase of EAU(days 16-20 of our experiment),and that during this time,γδT cells expressed high levels of CD69 and the integrin lymphocyte function-associated antigen-1(LF A-1)and secreted high levels of interleukin(IL)-17A.Moreover,DCs obtained during this phase expressed high levels of CD80,CD83,CD86,and intracellular cell adhesion molecule-1(ICAM-1).Furthermore,we studied the interaction between DCs andγδT cells by using flow cytometry and confocal microscopy in order to determine whether DCs affectedγδT-cell activation in vitro.Co-cultures of the two types of cells showed that DCs induced high levels of CD69,LFA-1,and I-17A inγδT cells.Imaging studies revealed contact between the DCs andγδT cells.This interaction was mediated by the accumulation of ICAM-1 and LFA-1 at the interface of DCs-γδT cells.Thus,the activation ofγδT cells in EAU was promoted by DCs interacting withγδT cells.

Cytotoxic effect of specific T cells from mice with experimental autoimmune uveitis on murine photoreceptor cells

AIM:To investigate the cytotoxic effect of specific T cells from mice with experimental autoimmune uveitis(EAU)as well as their secreted interferon(IFN)-γand interleukin(IL)-17A on murine photoreceptor(661 W)cells.METHODS:An EAU model was established in female mice by injection of interphotoreceptor retinoid binding protein(IRBP)emulsion supplemented with complete Freund’s adjuvant(CFA)and Mycobacterium tuberculosis(TB).On day 12 after induction of EAU,specific T cells from spleen and lymph node tissues were isolated and cultured for 4 d and the levels of IFN-γand IL-17A in the supernatants were determined by enzyme-linked immunosorbent assays(ELISAs).T cells and their supernatants were added to 661 W cells to observe the alteration of cell morphology;IFN-γand IL-17A were separately added to 661 W cells to observe the effect of IFN-γand IL-17A on cell proliferation.RESULTS:The levels of IFN-γand IL-17A in the T cell supernatants were 1568.64±38.79 pg/m L and 1456.57±46.98 pg/mL,respectively.The supernatants apparently inhibited 661 W cell proliferation(P<0.05).T cells could also attach to the surface of 661 W cells,and IFN-γshowed a more serious cytotoxic effect on 661 W cells than IL-17A,inhibiting cell proliferation(P<0.01).CONCLUSION:IFN-γand IL-17A from T cells of EAU mice model can exert cytotoxic effects on murine photoreceptor cell proliferation,and IFN-γshows more serious cytotoxic effects on murine photoreceptor cells than IL-17A.

Influence of Yanyankang Powder (眼炎康散) on Th1/Th2 in Rats with Experimental Autoimmune Uveitis

Objective： To study the influence of Yanyankang Powder （眼炎康散） on Th1/Th2 in rats with experimental autoimmune uveitis （EAU）. Methods： The EAU models were induced in Lewis rats by immunization with interphotoreceptor retinoid binding protein （IRBP） 1177-1191 in complete Freund＇s adjuvant. The rats were randomly divided into 3 groups： a model control group, a Yanyankang group, and a prednisone group, 9 rats in each group. The model control group was intervened with saline solution by gavage. The Yanyankang group was intervened with Yanyankang Powder 4 g/（kg·day） by gavage. The prednisone group were intervened with prednisone acetate tablets 5 mg/（kg·d） by gavage. All groups were intervened after immunization once every 2 days for 18 days and monitored by slit-lamp biomicroscopy daily until day 18. The levels of gamma interferon （INF-γ） and interleukin-10 （IL-10） in the supernatants of T cells were determined by enzyme-linked immunosorbent assay. Polymerase chain reaction （PCR） technology was used for measuring Thl and Th2 related cytokine mRNA expressions. Results： Slighter intraocular inflammation was found in the Yanyankang group and the prednisone group than the control group. The levels of the IFN-γ and IL-10 in the supernatants of the spleen lymph node cells were 382.33±6.30, 155.87±4.46 μg/L in the Yanyankang group and 270.93 ± 7.76, 265.32 ± 11.88 μg/L in the prednisone group. Both had significant differences compared with the control group （941.53± 8.59, 20.67± 4.65 μg/L; P=0.01）. The PCR results showed the same tendency. Conclusion： Yanyankang Powder showed favorable effects in the rats with EAU by influencing the function of Th1 and Th2 cells.

Aging weakens Th17 cell pathogenicity and ameliorates experimental autoimmune uveitis in mice

Aging-induced changes in the immune system are associated with a higher incidence of infection and vaccination failure.Lymph nodes,which filter the lymph to identify and fight infections,play a central role in this process.However,careful characterization of the impact of aging on lymph nodes and associated autoimmune diseases is lacking.We combined single-cell RNA sequencing(scRNA-seq)with flow cytometry to delineate the immune cell atlas of cervical draining lymph nodes(CDLNs)of both young and old mice with or without experimental autoimmune uveitis(EAU).We found extensive and complicated changes in the cellular constituents of CDLNs during aging.When confronted with autoimmune challenges,old mice developed milder EAU compared to young mice.Within this EAU process,we highlighted that the pathogenicity of T helper 17 cells(Th17)was dampened,as shown by reduced GM-CSF secretion in old mice.The mitigated secretion of GMCSF contributed to alleviation of IL-23 secretion by antigen-presenting cells(APCs)and may,in turn,weaken APCs’effects on facilitating the pathogenicity of Th17 cells.Meanwhile,our study further unveiled that aging downregulated GM-CSF secretion through reducing both the transcript and protein levels of IL-23R in Th17 cells from CDLNs.Overall,aging altered immune cell responses,especially through toning down Th17 cells,counteracting EAU challenge in old mice.

Preventive effect of chrysin on experimental autoimmune uveitis triggered by injection of human IRBP peptide 1-20 in mice

Uveitis is a common cause of blindness worldwide.Experimental autoimmune uveitis(EAU)is an animal model of noninfectious uveitis.Chrysin(5,7-dihydroxyflavone)is a member of the flavonoid family and has anti-inflammatory effects.We immunized C57BL/6J mice with human interphotoreceptor retinoid-binding protein peptide 1–20 to induce EAU.Chrysin was administered intragastrically at 25 mg/kg daily to the chrysin-treated mice from 3 days before immunization to 21 days after immunization.Vehicle was administered to the mice in the control group according to the same protocol.Lower clinical and histopathological scores,increased integrity of the blood–retinal barrier(BRB)and higher expression of tight junction proteins were observed in the chrysin-treated mice.Chrysin significantly decreased the proportions of Th1,Th17 and CD4^(+)CD3^(+)CD62L^(+)Th0 cells,and increased the proportion of Treg cells.Both macrophage infiltration and the expression of inducible nitric oxide synthase in the retina were efficiently inhibited by chrysin treatment.In chrysin-treated mice,the expression of interferon-γ,interleukin(IL)-17A,IL-6,IL-1βand tumor necrosis factor-αwas reduced in the retina,whereas higher levels of transforming growth factor-βwere detected.Furthermore,NF-κBp65 was downregulated after chrysin treatment.In conclusion,as an anti-inflammatory molecule,chrysin exerts a preventive effect on EAU by modulating the balance among helper T-cell subsets and suppressing ocular inflammation,thereby maintaining the integrity of the BRB.