Effects Of Parental Uveitis On The Development And Uveitis Susceptibility Of Offspring In Experimental Autoimmune Uveitis (EAU) Model

Background and objectiveUveitis is a common ocular inflammatory disease in the world,which,also known as uveitis,is a general term for inflammation of iris,ciliary body and telachorioidea.Broadly,uveitis includes inflammation of the whole eyeball.According to the pathogenesis,uveitis can be divided into infective uveitis and non-infective uveitis.Non-infectious uveitis is the main type of uveitis,accounting for more than 70%in China.The pathogenesis of uveitis is related to the autoimmune disorder caused by the interaction between heredity and environment.In particular,human clinical studies have found that the mother with uveitis will have a child with uveitis at a certain age,suggesting that the uveitis from mother can be passed on to her offspring.The clinical investigation and research on the inflammatory enteritis of autoimmune diseases found that the children born to the parents with the disease were more likely to have inflammatory enteritis than the children born to the single parents with the disease,suggesting that the parents with the autoimmune diseases had a greater impact on the probability children with this disease.As an autoimmune disease related to multi-organ,whether and how non-infective uveitis from their parents affects their offspring has not been reported.Experimental autoimmune uveitis（EAU）is a T-cell mediated autoimmune disease induced by retinal antigens in susceptible animals.As a disease animal model simulating human non-infective uveitis,it is widely used to study the pathogenesis,mechanism,prevention and treatment of human uveitis.The commonly used EAU model animals include rat,mouse,tree shrew and others,and the mouse EAU includes B10RIII mouse and C57BL/6 inbred line.Among them,B10RIII mice are the most susceptible animals to uveitis,while C57BL/6 mice are only moderately susceptible animals to uveitis.In addition,the characteristics of EAU induced by B10RIII mice are similar to those of human uveitis in terms of pathological manifestations and immune pathogenesis,which is the most commonly used classical EAU model.Based on the research background mentioned above,IRBP_161-180antigen peptide was used to immunize B10RIII adult male and female mice.On the day of immunization,the male and female mice were mated and propagated in cages to evaluate the effects of parental uveitis on the tissue and organ development in offspring mice.The skin,eye tissues and spleen of the affected offspring mice were selected for histopathological sectioning or RNA extraction,and the effects of parental uveitis on the pathomorphology of the affected tissues were evaluated by hematoxylin eosin staining（H&E）and transcriptome sequencing（RNA SEQ）,and the biological processes,metabolic pathways and signaling pathways of gene function enrichment in the offspring mice affected by uveitis were screened.Finally,we immunized the offspring mice affected by uveitis with IRBP_161-180 antigen to evaluate whether the parental uveitis affected the susceptibility of the offspring mice to uveitis and its mechanism.PartⅠEffects of parental EAU on the growth and development of offspring and the differential gene expression profile of the affected tissuesMethodsIn order to induce uveitis in both parents,the classical immune induction method was used to immunize the adult male and female B10RIII mice:50μg of IRBP_161-180 peptide PBS solution and complete Freund’s adjuvant（CFA）were emulsified in the proportion of 1:1,and the mice were immunized by subcutaneous injection with 50μL in the left and right thighs and 100μL in the back of the tail respectively.The occurrence and progression of uveitis in immunized parental mice were observed by ophthalmic ophthalmoscopy.On the same day after immunization,2 male and 3 female mice were caged to allow mating and reproduction.The next morning,the female mice were examined for the presence of vaginal suppository,which was considered as the first day of pregnancy.On the next day,the changes of hair coat and open eyes of newborn mice were observed,and the effects of parental uveitis on the growth and development of newborn mice was evaluated.On the 29th day after the birth of the offspring mice,three offspring mice with abnormal appearance were selected and their skin,eyes and spleen tissues were taken for histopathological sectioning and the RNA sequence（RNA SEQ）was performed after RNA extraction.The effects of parental uveitis on gene expression profile of the above-mentioned affected tissues was evaluated.The differentially expressed genes affecting the skin,eye tissue and spleen of the offspring mice by parental uveitis,as well as the biological process,metabolic pathway and signal pathway of functional enrichment of differential genes were screened.Real-time fluorescence quantitative Q-PCR was used to verify the differential genes selected by transcriptome sequencing.In the above experiment,the normal mice of the same age born from the parents of B10RIII mice without antigen immunity were also set as the normal control group.ResultsThe EAU model was successfully established in the parent mice of the experimental group by ophthalmoscope observation.The parental mice began to show signs of inflammation 7 or 8 days after immunization and the inflammation reached its peak on the 14th day,showing pupillary defects,iris congestion and corneal opacity.Then the inflammation gradually decreased,and dropped to a lower level on the 21st day.Therefore,the new born mice born during the period of inflammatory activity from 7 to 20 days after immunization were regarded as F1 mice,while the new born mice born during the period of inflammatory transition from 21 days after immunization were regarded as F2 mice.Through the general observation,it was found that the F1 mice born in the period of inflammatory had hair drop on the 14th day after birth,which lasted until the 39th day after birth.At the same time,it was found that the mice were delayed in opening their eyes,and the spleen was significantly enlarged at the time of sampling 29 days after birth,while no obvious hair abnormalities were observed in F2 mice born in the resting phase of inflammation compared with the healthy mice in the control group.The skin tissue RNA did not meet the quality standard of RNA SEQ sequencing and was excluded from the subsequent sequencing experiment.The results of RNA SEQ sequencing showed that the clustering of spleen and eye tissues between the experimental group and the control group showed that the correlation coefficient of samples from different tissues was low,while the correlation coefficient of samples from the same tissue group was high;the analysis of differential genes showed that there were 393 differentially expressed genes in eye tissues,including 363 up-regulated genes and 30 down regulated genes.The main differentially expressed genes ontology GO classification was enriched in586 biological processes,111 cell components,110 molecular functions,and the top three GO classification were successively the immune system process,muscle system process and cytoskeleton protein connection;The most relevant processes of immune system were immune response,external stimulus response,myeloid cell response,antigen treatment and presentation,T cell activation,and immune effects.More interestingly,the expression of the effector Th17 transcription factor retinoic acid orphan receptor-α（RORα）was significantly increased in the affected offspring.The GO classification of the spleen and the eye was enriched in the process of immune system,immune response,innate immune response,external stimulation response,regulation and development of multicellular biological processes.The results of Q-PCR showed that the expression patterns of 8 different genes in the eyes of mice in the experimental group and the control group were consistent with the corresponding RNA SEQ sequencing results,indicating the accuracy of RNA SEQ sequencing results.ConclusionEAU post-propagation was successfully established in B10RIII mice induced by IRBP_161-180 antigen immunization.Parental EAU can lead to hair loss,delayed eye opening,and enlarged spleen in the offspring of pregnant mice in the inflammatory phase,and these abnormalities are related to abnormal expression of genes related to hair follicle development and differentiation,genes related to eye muscle activity,and immune-related genes.Most importantly,the spleen tissue and eye tissue differential gene GO classification were most significantly co-enriched in the immune system process,suggesting that the parental uveitis has the most significant effects on the immune system process of the offspring conceived in the inflammatory period.Part Ⅱ:Effects of p parental uveitis on the susceptibility of offspring to uveitisMethodsIt can be seen from the above study that parental uveitis significantly affects the immune system process of offspring mice.Therefore,whether these immune system processes affected by parental uveitis can further affect the susceptibility of offspring to autoimmune uveitis remains to be further studied.Therefore,to further evaluate the effects of parental uveitis on the susceptibility of offspring to uveitis,the F1 mice and F2mice,which were pregnant in the inflammatory activity phase and in the inflammatory regression phase of their EAU parents,were injected subcutaneously with emulsions containing 50μg,25μg and 5μg IRBP_161-18061-180 peptides and 200μl of CFA to induce EAU in F1 mice and F2mice,respectively.At the same time,EAU control was set up for the healthy offspring mice with the above three doses induced from the parents of the unimmunized antigen peptide.On the 7th,14th,21st and35th day after immunization,the development process and clinical scores of inflammation in the anterior segment and fundus were observed by slit lamp microscopy system and ophthalmoscope;on the 14th and 35th day after immunization,the eyeballs of mice were taken for HE staining to evaluate the histopathological changes and histopathological scores of inflammation in the ocular tissues;on the 14th and 35th day after immunization,the spleen and peripheral lymph nodes were isolated by nylon wool column method to obtain antigen presenting cells and T lymphocytes.The effects of parental uveitis on IRBP antigen-specific T lymphocyte proliferation in offspring EAU mice was detected by MTT cell proliferation kit.Finally,at 35 days after immunization,spleen and peripheral lymph nodes were taken and isolated by nylon wool column method to obtain antigen presenting cells and T lymphocytes.ELISA method was used to evaluate the effects of parental uveitis on the production of inflammatory cytokine il-17 by IRBP antigen-specific T lymphocytes stimulated by antigen presenting cells in daughter EAU mice.ResultsThe inflammation occurred in F1 and F2 progenies induced by IRBP_161-18061-180 antigen peptide with low dose（5μg）and medium dose（25μg）on the 8th day after immunization,reached the peak of inflammation on the 14th day,and then the severity of inflammation gradually decreased,maintaining a moderate level of inflammation;the clinical manifestations in the anterior end of eye were mainly corneal clouding,conjunctiva and or ciliary hyperemia,hypopyon,irregular pupil and pupil defect,etc.;fundus observation showed optic disc inflammation,vasculitis,retinal tissue inflammation and severe retinal structural damage;the main histopathological changes were mainly manifested as extensive infiltration of inflammatory cells in retina and choroid,disorder of retinal structure folding,formation of granuloma in retina,and exudation of cellulose in subretinal cavity.EAU was not observed in normal offspring mice induced by IRBP_161-180 antigen peptide with low dose（5μg）,but in normal offspring mice induced by IRBP_161-180 antigen peptide medium dose（25μg）,inflammation appeared on the 11th or 12th day after immunization,then the severity of inflammation gradually increased to medium level,peaked on the 28th day,and then decreased to low level.Compared with EAU of normal control offspring mice induced by corresponding dose,the clinical scores in F1 offspring mouse EAU group were significantly increased（P=0.0041,corresponding to F1 offspring mouse EAU）induced by IRBP161-180 antigen peptide medium dose（5μg）.However,in the EAU induced by 50μg IRBP161-180 peptide in the high-dose group,the EAU of the F1 subgroup and the control mice showed inflammation on the 8th day after immunization.There was no significant difference between the score and histopathological score（P>0.05）.In IRBP_161-180 peptide-induced EAU,the clinical scores of F1 mice were significantly increased,and the clinical scores of F2 mice showed an increase trend;in the pathological score,the EAU induced by IRBP_161-180antigen peptide with low dose（5μg）and medium dose（25μg）showed a similar rule with the clinical score.However,in the EAU induced by IRBP_161-18061-180 antigen peptide with high dose（50μg）,EAU in F1 mice group and control mice appeared inflammation on the 8th day after immunization,and their progression rules,clinical manifestations and histopathological characteristics were similar.There was no significant difference in clinical score and histopathological score of inflammation（P>0.05）.The results of lymphocyte proliferation assay by MTT assay showed compared with the EAU control group of normal offspring mice,10μg IRBP_161-180 significantly induced lymphocyte proliferation in the EAU of F1 mice immune-induced by 5,25 and 50μg（P=0.0024,P=0.0019,P=0.039,followed by 10μg IRBP161-180 antigen stimulation low-dose,medium-dose and high-dose groups）;20μg IRBP_161-180significantly induced lymphocyte proliferation in the EAU of F1 mice immune-induced by 5and 50μg（P=5.79e-6,P=0.0041,P=0.011,followed by 20μg IRBP161-180 antigen stimulation low-dose,medium-dose and high-dose groups）;the results of cell supernatant detection by ELISA showed that compared with the EAU control group of normal offspring mice,10μg IRBP_161-180（P=0.0018,P=0.000059;corresponding to 5μg and 25μg of EAU,respectively）and 20μg IRBP_161-180（P=0.0021,P=0.0033;corresponding to 5μg and 25μg of EAU,respectively）in EAU of F1 mice stimulated the production of the effective Th17 cell-specific inflammatory cytokine IL-17,which was significantly increased by T lymphocytes.10μg and 20μg IRBP_161-180IRBP_161-180 in EAU of F2 mice immunized with 5μg and 25μg did not significantly stimulate the production of IL-17 by T lymphocytes（P>0.05）.ConclusionBoth parents with uveitis increased the susceptibility of pregnant offspring mice to uveitis under the active inflammation and submissive inflammation at the same time,but the degree of EAU inflammation induced by low dose antigen exposure was higher.In addition,the susceptibility may be related to theion of T lymphocyte proliferation and the abnormal increase of IL-17 in offspring mice induced by parental uveitis.