The Role And The Mechanism Of Microglial Derived Extracellular Vesicles In Retinitis Pigmentosa Complicated Cataract

Backgrounds:Retinitis pigmentosa(RP)is a chronic,progressive,and inherited disease of the retina.The role of inflammation in RP has received much attention in recent years.Micro-inflammatory reaction in RP has been found not only in the retina,but also in aqueous humor,vitreous and even in peripheral blood,suggesting that RP patients have a chronic and persistent micro-inflammatory environment in the eye and whole system.However,the production of this micro-inflammation is still unclear.Activated microglia in the retina of RP promotes the neuroinflammatory response and apoptosis of photoreceptor cells,and plays an important role in the progression of RP.It has been reported that the extracellular vesicles(EVs)secreted by activated microglia can promote the dissemination of micro-inflammation in the brain.In view of this result,we speculate that there is micro-inflammation in the retina of RP patients.The activated microglia could transmit inflammatory messages through EVs secreted by them,and maintain intraocular inflammatory environment,participating in complicated changes in the eye,and systemic inflammatory reaction which may result in the increase of NLR value(neutrophil count to lymphocyte count ratio)in peripheral blood.Objectives:To explore the correlation between the ocular manifestations of RP complicated with cataract and the systemic inflammation marker of NLR in the peripheral blood.To verify the existence of intraocular and systemic micro-inflammatory reaction in RP mice.To clarify the role and mechanism of EVs secreted by activated microglia in maintaining intraocular and systemic micro-inflammatory environment,and explore the mechanism of intraocular changes in RP caused by activated microglia derived EVs.Methods:1.Correlations between NLR and clinical manifestations of RP complicated with cataractThe clinical data of RP patients complicated with cataract and age-and sex-matched age-related cataract(ARC)patients were collected from department of ophthalmology in our hospital.These patients all underwent phacoemulsification combined with intraocular lens implantation from January 1,2008 to June 1,2018.The differences in type of cataract,axial length(AL),zonular status,retinal thickness and NLR value between the two groups were compared,and the correlation between clinical manifestations of RP complicated with cataract and NLR was analyzed.2.EVs secreted by activated microglia mediates the intraocular and systemic micro-inflammatory environment in RP patients(1)To verify the activated microglia and inflammatory reaction in vivo and in vitroThe microglial cell line,BV2 cells were stimulated by lipopolysaccharide(LPS),and the morphological changes of the cells were observed under an inverted microscope.Rd/b6,the RP mouse model,and normal control mouse C57 were employed.The changes of retinal thickness of Rd/b6 and C57 mice at different ages were observed on paraffin section of retinal tissues.The activation and localization of microglia in retina were observed by Ibal immuno-staining.The activated microglia was labeled by CD68 immuno-staining.The retinal function was evaluated by electroretinogram(ERG),and the expressions of inflammatory factors and inflammation-related miRNAs in BV2 cells and retinal tissues were detected by Western blot,RT-qPCR and immunofluorescence staining.(2)To verify the intraocular and systemic micro-inflammatory responses in Rd/b6 mice and their effects on eyesThe expressions of inflammatory factors in the retina,Collagen I and MMP-2 in the scleral tissue and EMT marker protein in the lens of Rd/b6 and C57 mice were detected by Western blot.Lens paraffin sections were used to observe the distribution of LECs.The expression levels of IL-6,IL-8,and CCL-2 in peripheral blood of mice were detected by ELISA,and the NLR value of blood was detected by complete blood cell count.(3)To verify the inflammatory effects of EVs secreted by activated microglia in vivo and in vitroLPS-EVs and Control-EVs were extracted by ultracentrifugation,and the expressions of inflammatory factors and inflammation-related miRNA in EVs were detected by RT-qPCR.After 24h treatment of these two EVs,the expressions of the above RNAs and Iba1 in BV2 cells were detected.Intra-vitreous injection of LPS-EVs was performed on Rd/b6 and C57 mice.The activated microglia was labeled by immunofluorescence staining of Ibal and CD68.The expressions of inflammatory cytokines and Iba1 in the retina,Collagen I and MMP-2 in the scleral tissues and EMT markers in the lens were detected by Western blot and RT-qPCR.The changes of NLR values in peripheral blood of mice were compared at the time of before-injection and 1-week-after-injection.3.The effects and mechanisms of EVs secreted by activated microglia on EMT of lens epithelial cells(LECs)(1)EVs up-taken by LECs and translocation in the eyeLPS-EVs were labeled with PKH26 and co-cultured with LECs for 24h.The distribution of EVs in cells was observed under confocal microscopy.EVs were labeled with DIR and injected into the vitreous cavity of Rd/b6 mice.In living imaging of small animals was performed 48 hours later to observe the distribution of EVs in the eye in vivo and in vitro.(2)The effect of LPS-EVS on EMT of LECsLECs were stimulated by LPS-EVs and Control-EVs,respectively.After 24h,the expressions of EMT markers and the molecules in TGF-β1/Smad signaling pathway were detected by Western blot and RT-qPCR.Cell migration ability was detected by cell scratch assay and Transwell assay.(3)Effect of miR-155-5p in LPS-EVs on EMT of LECsLPS-stimulated BV2 cells were transfected with miR-155-5p inhibitor or miR-155-5p mimics,respectively,to knock down or overexpress miR-155-5p in LPS-EVs.LECs were treated with these EVs for 24h.The same experiments were performed as in method Ⅲ(2).Results:1.Correlations of NLR and clinical manifestations of RP complicated with cataractThe type of cataracts between the RP group and ARC group was significant different(P<0.001).PSC was the most common type of cataract in the RP group,while cortical cataract was the one in the ARC group.The percentage of ultra-long AL(AL>30 mm)in RP patients was higher than that in ARC group,although there was no significant difference in AL distribution between the two groups(P=0.235).Zonular weakness was more likely to occur in RP patients than in ARC patients,and even more likely to occur in RP patients with longer AL.Retinal thickness of RP group was significantly lower than that in ARC group.The absence rates of ELM,IZ,and EZ in RP group were significantly higher than those in ARC group(P<0.001).NLR value in RP group was significantly higher than that in ARC group(P=0.029).NLR increased with the severities of subcapsular opacification(P<0.05)and zonular weakness(P<0.05).RP patients with better BCVA(Log MAR≤1)had lower NLR values than those with poorer BCVA(Log MAR>1,P=0.048).NLR was significantly correlated with visual field parameters such as MD(r=-0.356,P=0.049)and VFI(r=-0.772,P=0.005),but not with PSD.The value of NLR≥1.36 predicted higher degrees(>P1)of PSC(P=0.002,95%CI,0.672-0.934).NLR≥2.12 predicted zonular weakness before cataract surgery(P=0.002,95%CI,0.665-0.928),and NLR>1.51 could be used as an auxiliary method to predict BCVA(P=0.015,95%CI,0.540-0.793).2.EVs secreted by activated microglia mediated intraocular and systemic micro-inflammation in RP mice(1)Activation of microglia and inflammatory reaction in vivo and in vitroAfter stimulation with LPS,the BV2 cells expressed higher levels of inflammatory cytokines,IL-1,IL-6,CCL-2,TNF-α,iNOS,and IL-8,and inflammatory related miRNAs,such as miR-155-5p,miR-146a-5p and miR-21a-5p,and Iba1,comparing with those in the resting BV2 cells.In Rd/b6 mice,the expressions of the above inflammatory cytokines and Iba1 in the retina were higher than those in C57 mice.In Rd/b6 mice at P14,Iba1 positive microglia migrated to the outer retina,and the number of CD68 positive microglia was at peak.At P21,the number of microglia in the outer retina decreased,and the number of CD68 positive microglia decreased and remained stable.The retinal thickness of Rd/b6 mice decreased gradually with age(all were P<0.001).Electroretinogram indicated rapid deterioration of retinal function in Rd/b6 mice at day P14.(2)Intraocular and systemic micro-inflammatory reaction in Rd/b6 mice and their effects on intraocular tissuesComparing with C57 mice,in Rd/b6 mice,the expressions of IL-1β,IL-6,CCL-2,TNF-α and IL-8 in aqueous humor were significantly increased;the expression of Collagen I was reduced(P<0.05),while the level of MMP-2 increased(P<0.01)in the sclera;the expression of E-cadherin was decreased(P<0.001),while the expressions ofα-SMA(P<0.01)and Fibronectin(P<0.001)were increased in the lens.LECs polarity disappeared,the tight junctions between cells were broken,and large number of nucleated cells appeared in the posterior capsules in adult Rd/b6 mice.The levels of IL-8,IL-6,and CCL-2 in the peripheral blood of Rd/b6 mice were increased(P<0.001,P<0.01,P<0.001),and NLR value was also significantly higher than that in C57 mice(P<0.001).(3)Proinflammatory effects of LPS-EVs in vivo and in vitro:In LPS-EVs,the inflammatory factors and inflammatory related miRNAs were higher than those in Control-EVs.After BV2 cells were treated with LPS-EVs,the expressions of IL-1β,IL-6,IL-8,miR-155-5p,miR-146a-5p,miR-21a-5p and Iba1 were significantly higher than those in NC group and Control-EVs group,and miR-155-5p was the most up-regulated miRNA(P<0.001).After injection of LPS-EVs into the vitreous cavity of the right eye of C57 and Rd/b6 mice,the number of Iba1-positive and CD68-positive microglia increased,and the protein expressions of IL-1β,IL-8 and Ibal in the retinas of both kinds of mice were increased.The expression of Collagen I reduced,but MMP-2 significantly increased in the sclera.The expressions of Fibronectin and α-SMA in the lens significantly increased,while E-cadherin significantly decreased.The NLR values of peripheral blood were significantly higher than those before LPS-EVs injection,and the NLR in Rd/b6 mice was significantly higher than that in C57 mice.3.The effects and mechanisms of EVs secreted by activated microglia on EMT of LECs(1)EVs were up-taken by LECs and translocated in the eyePKH26 labeled LPS-EVs were observed in LECs and located around the nucleus.DIR labeled EVs moved into the anterior chamber from the vitreous in both C57 and Rd/b6 mice.(2)The effect of LPS-EVs on EMT of LECsIn LECs treated with LPS-EVs,the expression of E-cadherin decreased,while the expressions of α-SMA and Fibronectin increased,and the migration ability of LECs enhanced.The expressions of TGF-β1,Smad2 and p-Smad2 were up-regulated.(3)Effect of miR-155-5p in LPS-EVs on EMT of LECs:The upregulation of miR-155-5p in LPS-EVs by transfection with miR-155-5p mimics inhibited the expression of E-cadherin in LECs,promoted the expression ofα-SMA and Fibronectin,and enhanced cell migration.The expressions of TGF-β1,Smad2 and p-Smad2 were also up-regulated.Downregulation of miR-155-5p in LPS-EVs by transfection with miR-155-5p inhibitor could reverse the above changes in LECs.Conclusions:The severity of the clinical manifestations of RP patients complicated with cataract closely related with the inflammation.NLR can predict some clinical manifestations and their severity in RP complicated with cataract.Micro-inflammatory reactions exist in retina,eyes,and whole system of RP mice.EVs secreted by activated microglia in the retina of RP,containing inflammatory mediators,are important substances for the transmission of inflammatory mediators and maintenance of intraocular and systemic micro-inflammatory environments.EVs derived from activated microglia could act on intraocular tissue which is far away from the retina,for instance,lens and sclera,and induce EMT of LECs and degradation of scleral collagen,participating in formation of ocular complications of RP.