| Multiple sclerosis（MS）is a chronic immune-mediated central nervous system disease.During the inflammatory reaction,lymphocyte infiltration can lead to demyelination and axonal injury of brain and spinal cord.It was listed as a rare disease in China,and thus,it is urgent to conduct in-depth studies.First,the pathogenesis of MS involves abnormalities in many intracellular signaling pathways.The TAM（Tyro3,Axl,and Mer）receptor,a subfamily of receptor tyrosine kinases,is a key molecule that regulates immune homeostasis.Therefore,in-depth investigation of the negative regulatory mechanism of TAM receptors in the inflammatory response in the brain tissue of experimental autoimmune encephalomyelitis（EAE）models,especially in microglia,could help identify new therapeutic targets for MS.Second,the gut microbiota has been a focus of research.Currently,there is no relevant study on the relationship between gut microbiota disturbance and negative regulation of the immune response by TAM receptors in MS.Therefore,we further studied the characteristics of the changes in and mechanism of action of gut microbiota disturbance during the occurrence and development of MS.This study will provide basic research to more effectively determine how to interrupt relapse of MS and assist immune-correction therapy for MS in clinical practice.Objective1.To investigate changes in TAM-toll-like receptors（TLRs）-suppressor of cytokine signaling（SOCS）pathway-related proteins and inflammation factors in brain tissues through the regulation of TAM receptor levels in EAE mice and to discover new therapeutic targets by analyzing the negative immunoregulatory mechanism of TAM receptors in MS.2.To analyze the changes in gut microbiota of EAE,identify strains that play a major role in the course of the disease,and investigate the correlation of the metabolites of gut microbiota and the inflammatory response of EAE mice as well as the underlying mechanism.3.To detect TAM-TLRs-SOCS pathway-related proteins by regulating TAM receptors in activated BV2 microglial,identify the major inflammatory factors associated with negative immunoregulation in microglial,and subsequently investigate the effects of short-chain fatty acids（SCFAs）on TAM receptors and inflammatory targets of action.MethodsPart I:Fifty C57BL/6 mice were randomly divided into a normal control group,an EAE group,and low-,medium-,and high-dose rapamycin groups based on a random number table.Heterozygous Axl gene knockout mice were obtained using clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR associated protein 9 technology.DNA was extracted,and 10 homozygous Axl-/-mice were identified.The EAE models were established,and the body weight and neurological function scores were continuously recorded.Brain tissues were collected from mice at day 21 for hematoxylin and eosin（HE）staining,immunohistochemistry,immunofluorescence,and Weil’s myelin staining.Tyro3,Axl,Mer,and SOCS in brain tissues were analyzed by Western Blotting and real-time quantitative reverse transcription polymerase chain reaction（RT-PCR）.Enzyme-linked immunosorbent assay（ELISA）were used to detect interferon gamma（IFN-γ）and interleukin 17（IL-17）.Part II:Fecal samples were collected from mice in the normal and EAE groups on day 7,14,21,and 30,with three replicates at each time point.The genomic DNA of fecal bacteria was extracted for amplification.The 16S r RNAV3/V4 region of the sample DNA was sequenced,and the ribosomal database project（RDP）classifier,a na?ve Bayesian classifier,was used for taxonomic analysis.IFN-γand IL-17 levels were detected by ELISA.Part III:An inflammatory activation model using BV2 microglial cells was induced by lipopolysaccharide（LPS）.A lentiviral packaging system targeting the Axl gene was constructed to transfect BV2 cells.Stably transfected strains were selected and validated by RT-PCR.The effects of acetic acid,propionic acid,butyric acid,and a mixture of the three substances on cell viability were detected using the Cell Counting Kit-8（CCK-8）.The cells were divided into normal,PBS,Rapamycin,SCFAs,Axl-/-+SCFAs,and Growth arrest specific 6（Gas6）pretreatment groups.Immunofluorescence was performed to detect TAM receptor expression on BV2microglial.The expression levels of the TLR3 and TLR4 proteins were detected by Western blotting.The m RNA levels of Tyro3,Axl,Mer,and SOCS were detected by RT-PCR.The supernatant of the cell culture medium was collected,and three replicates of each sample were examined.Antibody microarray technology was used to screen differentially expressed proteins and enriched pathways in the Axl-/-+SCFAs and SCFAs group.ResultsPart I:1.Identification of homozygous Axl-/-mice based on DNA.The neurological deficit symptoms of mice in the EAE group peaked at 20.3±0.14 days after immunization,and the body weight decreased;lymphocyte infiltration was observed in brain and spinal cord tissues,and demyelination was evident based on Weil’s myelin staining.The symptoms of mice in the Axl-/-group peaked on day 16.6±0.55and were significantly more severe than those in the EAE group,and the body weight decreased significantly（P<0.01）in this group.The inflammatory responses in the brain and spinal cord tissues in the Axl-/-group were significantly more severe than those in the EAE group.2.Based on laser confocal microscopy,Axl and Mer were mainly expressed in neurons and microglial,while Tyro3 was mainly expressed in neurons.TAM receptors were highly expressed in the rapamycin group and weakly expressed in the Axl-/-group.The number of FOXP3+regulatory T（Treg）cells was significantly increased in the rapamycin group compared with the EAE group（P<0.01）.It significantly decreased in the Axl-/-group compared with the EAE group.The integrated optical density（IOD）of myelin basic protein（MBP）in the EAE group was significantly lower than that of the normal control group;the IOD of MBP in the rapamycin group was higher than that in the EAE group;the IOD of MBP in the Axl-/-group was significantly lower than that in the EAE group（P<0.05）.3.Medium-dose rapamycin significantly improved the neurological deficits of the EAE group（P<0.05）,and there was no significant difference in neurological function scores between the high-dose group and the medium-dose group（P>0.05）.Low-dose rapamycin did not improve the neurological function score.4.The levels of Tyro3,Axl,and Merm RNA were significantly increased in the medium-dose rapamycin group compared with the EAE group on day 21（P<0.05）.TLR3 and TLR4 were decreased,and the SOCS1 and SOCS3 were increased;the Axl-/-group showed the opposite results.IFN-γand IL-17 were significantly increased in the Axl-/-group compared with the EAE group.5.Correlation analysis:the Tyro3,Axl,and Mer on day 21 were negatively correlated with neurological function scores in the EAE group.Part II:1.The species abundances of Alistipes,Blautialuti,and Lachnospiraceae（NK4A136 group）were significantly different between the normal group and the EAE group（P<0.05）on day 14.Species abundances of Allobaculum,Eubacterium,and Helicobacter were significantly different between the two groups on day 30.According to the linear discriminant analysis effect size（LEf Se）analysis,Odoribacter played a major role on day 21.2.Compared with the normal group,the species abundances of Prevotella＿NK3B31＿group in the EAE group decreased on day 14 and 30.It is the main strain producing SCFAs.3.IFN-γand IL-17 peaked on day 21 in the EAE group,and the levels were significantly different from those on day 14 and day 30（P<0.05）.Part III:1.A recombinant lentivirus that silenced the Axl gene was successfully constructed and transfected into BV2 microglial.According to the CCK-8 results,the concentration ratio of the SCFAs mixture was 20mmol/l acetic acid,1.0mmol/l butyric acid,and 1.0mmol/l propionic acid.2.Immunofluorescence staining showed that Axl and Mer were expressed in the cell membrane and cytoplasm of BV2 microglial cells,with the less expression of Tyro3.Rapamycin and the mixture of SCFAs alleviated the inflammatory response in BV2 microglial.The Western blotting and RT-PCR results showed that the expression levels of Tyro3,Axl,and Mer were significantly increased,TLR3 and TLR4 were significantly decreased,and the changes were more significant in the SCFAs group.SOCS was most significantly increased in the rapamycin group.TLR3 and TLR4expression levels were decreased,and the SOCS levels were increased in the Axl-/-+SCFAs group,compared with the SCFAs group.No significant difference was identified between the Gas6 pretreatment group and the SCFAs mixture group（P>0.05）.3.There were 24 differentially proteins between the SCFAs group and the Axl-/-+SCFAs group in the supernatant of concentrated BV2 microglial,which was analyzed by antibody microarray technology.These proteins included 10 upregulated proteins and 14 downregulated proteins.Based on the Gene Ontology（GO）enrichment analysis,the biological function and molecular function of these proteins were mainly related to inflammatory factors,and the Kyoto Encyclopedia of Genes and Genomes（KEGG）signaling pathway analysis showed that these proteins were mainly involved in the inflammatory signaling pathway.Conclusion1.The EAE models were made in mice with Axl gene knockout,and the survival rate of the mice significantly decreased.TAM receptors can negatively regulate immune responses in the brain tissue of EAE mice and activate inflammatory BV2 microglial cells through the TAM-TLRs-SOCS pathway,subsequently inhibiting the secretion of the inflammatory factors（IFN-γand IL-17）and reducing inflammatory demyelination.2.The abundances of the gut microbiota in the EAE group changed.The abundance of the Prevotellaceae-NK3B31 group was lower than that of the normal control group,which may be the key bacterial genus that causes demyelination in MS.Prevotellaceae is the main stain which produce SCFAs.3.A certain proportion of the SCFAs mixture could alleviate the inflammatory response in BV2 microglial cells through the TAM-TLRs-SOCS pathway and may play a role through regulation of inflammatory factors such as tumor necrosis factor（TNF）-ɑ,IL-16,intercellular adhesion molecule 1（ICAM-1）,and Eotaxin-2 after silencing the Axl gene.The relationship of SCFAs,Axl and chemokine-related pathways is worth in-depth research in animals.4.The SCFAs is the important metabolites involved in the inflammatory demyelination of MS.Rapamycin can exert the immunosuppressive effects by upregulating TAM receptors. |
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