Unraveling The Links Underlying T Cell Exosomal MiRNA And Immune Indicators With Osteoporosis

Osteoporosis(OP)is a systemic skeletal disorder characterized by low bone density and degradation of bone microarchitecture,which in turn leads to decreased bone strength and increased susceptibility to fracture.Dual energy X-ray absorptiometry(DXA)-based measurement of bone mineral density(BMD)at whole-body or focal sites such as the hip(femoral neck and total hip)and spine is a well-established method worldwide for OP diagnosis,prediction of future fracture risk,and dynamic monitoring of patients.OP is known to be a multifactorial,multi-linked insidious process,with relevant pathogenetic studies focusing on genetic,endocrine,metabolic disorders and mechanical factors,while the chronic low-grade inflammatory state that accompanies ageing and estrogen deficiency mediates the indirect involvement and regulation of bone remodelling by a variety of immune cells through direct contact with osteogenic cells or paracrine mechanisms.Studies have shown that microRNAs(miRNAs)encapsulated by exosomes have sufficient biological activity to play a key role in OP progression as a class of participant in intercellular remote communication.In view of this,the present study intends to unfold research contents including the following three chapters from the perspective of osteoimmunology,including the following three chapters:firstly,to investigate the effect of exosomes derived from activated T cells on the differentiation of osteoclast precursor cells;secondly,to screen and experimentally validate the function of miRNAs differentially expressed in exosomes before and after T cell activation;and thirdly,to systematically explore and assess the potential causal association between immune indicators and BMD using a Mendelian randomisation approach.PartⅠObjectiveGiven that a number of studies have reported that aberrantly activated T lymphocytes can regulate the differentiation of osteoclasts through a variety of immune factors and thus participate in the bone remodelling process,we focused on the exosomes before and after T cells(Jurkat)activation to observe the effect on the differentiation phenotype of osteoclast precursor cells and to lay the foundation for further functional and mechanistic related experiments.MethodsIn this study,a human-derived T-lineage Jurkat cell line was proposed as a model.Cell supernatants were obtained,and exosomes were collected and extracted by combined supercentrifugation,membrane ultrafiltration,precipitation and membrane affinity column methods,and analyzed by transmission electron microscopy,Zetasizer nano ZS analyzer,and Western blot(WB)for morphological,size distribution,and molecular phenotyping.The expression of T cell activation markers CD25 and CD69 molecules were detected by flow cytometry and the established Jurkat cell activation system was identified.Exosomes from the activation group(AC Jurkat Exos)and the non-activation group(NC Jurkat Exos)were extracted separately.After the exosomes of both groups were labeled with membrane protein dye PKH67 and then incubated in osteoclast precursor cells(CD14+ monocytes,RAW264.7)to observe the expression and localization of green fluorescence and confirm that it can be endocytosed by osteoclast precursor cells,which were then added to target cells as follows:human peripheral blood CD14+monocytes,human monocyte line THP-1 cells,bone marrow-derived macrophage(BMMs)and mouse macrophage line Raw264.7 for differentiation induction,and their effects on osteoclastogenesis were assessed by tartrateresis-tant acid phosphatase staining(TRAP)staining and F-actin staining.In addition,realtime quantitative reverse transcription PCR(qRT-PCR)was used to examine the expression of osteoclast differentiation related transcriptional regulators:Proto-oncogene protein(cFOS),Nuclear factor of activated T-cells cl(NFATcl)and specific genes:matrix metalloproteinase 9(MMP9),cathepsin K(CTSK)and TRAP at the mRNA level after treatment with NC Jurkat exos and AC Jurkat exos in two commonly used murine osteoclast models.ResultsUsing transmission electron microscopy,the extracted exosomes were found to have a circular vesicle-like structure,with a typical teatro-like bilayer,and the particle size was mainly concentrated around 100 nm.The results of the WB assay showed the expression of the characteristic exosomal proteins CD9 and CD63 as well as TSG101,showing that the physical characteristics and molecular phenotype are consistent with the main features of exosomes.Flow cytometry results showed that the expression of both CD25 and CD69 molecules were significantly increased after Jurkat activation(P<0.05).The results of dye tracing experiments showed that exosomes from both groups could be transported to osteoclast precursor cells,and AC Jurkat exos could be preferentially delivered to humanand murine derived osteoclast precursor cells compared with NC Jurkat exos.The results of TRAP staining and F-actin staining in the four models of osteoclast precursors showed that the AC Jurkat exo group had more trap positive multinucleated osteoclasts with larger size and a significantly higher number of actin rings compared with the empty control group(P<0.05);However,there was no significant difference between the NC Jurkat exos group and the blank control group(P>0.05).In addition,qRT-PCR results showed that the expression of osteoclast differentiation-related genes c-FOS,NFATc1,MMP9,CTSK and TRAP at the mRNA level among the three groups showed similar trends to the osteoclast differentiation phenotype.Conclusions1 We successfully established a T cell activation model and stably prepared exosomal NC Jurkat exos and AC Jurkat exos,respectively,from cell supernatants.2 AC Jurkat exos can promote the differentiation of osteoclast precursor cells.Part ⅡObjectiveBased on the results of Part Ⅰ,we further screened and validated the differentially expressed miRNAs between the two groups of exosomes and focused on the miRNAs of interest to investigate their role in mediating AC Jurkat Exos to promote osteoclast precursor cell differentiation and to initially clarify their biological functions.MethodsTotal RNA was extracted from NC Jurkat Exos and AC Jurkat Exos,and the differentially expressed miRNAs in the two groups of exosomes were detected using efficient microRNA microarray technique,as well as a literature search for miRNAs enriched in the exosomes of human primary T lymphocytes as reported in previous studies,qRT-PCR was performed to validate some of the differentially expressed miRNAs and miRNAs summarized from the literature.The 10 miRNAs selected after in-depth validation were used for target gene prediction and further Gene Ontology(GO)and signaling pathway enrichment analysis(including Kyoto Encyclopedia of Genes and Genomes,KEGG and Wiki)by TargetScan and other databases.The corresponding analog sequences(mimics)were transiently transfected into RAW264.7 and BMMs cells,respectively,and their effects on osteoclast differentiation phenotype were initially observed by TRAP staining and relative quantification of osteoblast differentiation-related genes at the mRNA level.The differentially expressed hsa-miR-802 and hsa-miR-106a-5p were selected for further study,and recombinant plasmids of GV647-miR-802,GV647-miR-106a-5p and their negative controls were constructed respectively.After lentiviral packaging,collection,concentration and titre determination,and infection of Jurkat cells with optimal conditions.And qRT-PCR was applied to detect the expression of miR-802 and miR-106a-5p at the cellular level as well as at the exosomal level.After the successful establishment of two groups of stable cell lines overexpressing miR-802 and miR-106a-5p respectively,the exosomes from the supernatant of the cell culture medium of each group were collected and added directly into RAW264.7 and BMMs,respectively,and the experiments were divided into the following three groups:①Jur-GV647-miR-NC-Exo,②Jur-GV647-miR-106-Exo,③Jur-GV647-miR-802-Exo,and the effects of exosomes from the three groups on the proliferation and differentiation phenotypes of osteoclasts were assessed by CCK8 assay,Trap staining,F-actin staining,and relative quantification of osteoclast differentiation genes at the mRNA level,respectively.In addition,the non-contact co culture system of stable cell lines and BMMs was established with the aid of Transwell chambers,and the same method described above was used to explore the changing trend of their effect on osteoclast differentiation phenotype after being intervened by exosome inhibitor GW4869.The corresponding analog sequences(miR-802 mimic,miR-106a-5p mimic),inhibitor sequences(miR-802 inhibitor,miR-106a-5p inhibitor),as well as negative control sequences(mimic/inhibitor NC)were constructed and transiently transfected to BMMs.The effects of miR-802 and miR-106a-5p on the cellular activity as well as differentiation of BMMs were analyzed in the same way as described above.Target genes closely related to osteoclastogenesis were selected,and their interaction with target genes was futher experimentally verified by qRT-PCR and dual luciferase reporter gene system.ResultsThe results of miRNA microarray analysis showed that 127 miRNAs were differentially expressed in their exosomes before and after Jurkat activation,of which 43 were upregulated and 84 were downregulated.Seven miRNAs selected to be up-regulated with high human mouse homology(FC>1.1,P<0.05)and highly enriched in T cell exosomes derived from the literature were validated in depth by qRT-PCR and found to be significantly enriched in AC Jurkat exos compared to control exos.The results of GO analysis showed that the target genes of the above miRNAs were mainly involved in negative regulation of cell differentiation,development of the immune system,and activation of T cells;The results of the KEGG and Wiki pathway analysis revealed that they were mainly enriched in the phosphatidyl-inositol 3-kinase/serine-threonine kinase(PI3K/AKT)signalling pathway,mitogen-activated protein kinase(MAPK)signalling pathway,actin cytoskeleton regulation and T cell receptor signalling pathway.After overexpression of 10 miRNAs in osteoclast precursor cells,we initially found that all of them enhanced the differentiation ability of osteoclasts in varying degrees.On this basis,hsa-miR-802 and hsa-miR-106a-5p were selected for further in-depth study.Stable cell lines overexpressing these 2 miRNAs were successfully constructed by using lentiviruses(i.e.,Jur-GV647-miR-802 and Jur-GV647-miR-106a-5p),and similar to the cellular level,the corresponding miR-802/106a-5p expression levels were significantly increased in exosomes.After co-incubation of osteoclast precursor cells with each of the three groups of exosomes,cell viability was not significantly affected in any of the JurGV647-miR-106a-5p-Exo and Jur-GV647-miR-802-Exo treated groups compared to the Jur-GV647-miR-NC-Exo treated group,and in addition,it was observed that the differentiation process of osteoclasts was significantly promoted.This trend was found to be partially reversed by the addition of the exosome inhibitor GW4869 to pretreat stable transient strains in the transwell co-culture system(P<0.05).After overexpression or inhibition of the expression of these 2 miRNAs in BMMs by transient transfection,respectively,it was found that,compared with the negative control group,neither cell viability was affected,but osteoclast differentiation capacity could be promoted or inhibited to some extent,respectively.The target genes of miR-802 or miR106a-5p were predicted using online websites,and those closely related to bone metabolism were selected for experimental validation,respectively.The qRT-PCR results indicated that overexpression of miR-802 expression could inhibit the expression of the corresponding target genes:Leucine-rich repeat-containing G protein-coupled receptor-4(LGR4),tumor necrosis factor receptor-associated factor 3(TRAF3),and Ras homolog gene family,member A(RHOA)at the mRNA level,respectively.Similarly,overexpression of miR106a-5p could inhibit the expression of the corresponding target gene,phosphatase and tensin homolog deleted on chromosome ten(PTEN),at the mRNA level,respectively.The results of the dual luciferase reporter gene assay revealed that pmirGL-LGR4-3’-UTR-wt,pmirGL-TRAF3-3’-UTR-wt and pmirGL-RHOA-3’-UTR-wt cotransfected with miR-802 mimic cotransfected with miR-802 mimic resulted in a significant decrease in luciferase activity(P<0.05),while the corresponding mutant-type cotransfected with miR-802 mimic showed no significant change in luciferase activity(P>0.05).Similarly,pmirGL-PTEN-3’UTR-wt cotransfected with miR-106a-5p mimic resulted in a significant decrease in luciferase activity(P<0.05),whereas luciferase activity of the mutant-type was not affected by miR-106a-5p mimic cotransfection(P>0.05).Conclusions1 The miRNA differential expression profiles of exosomes before and after T cell activation were initially constructed,combined with relevant miRNAs gathered from the literature,a total of 10 upregulated miRNAs were screened for enrichment analysis,which may bridge the interaction of bone system and immune system by regulating multiple signaling pathways from immune system that can simultaneously participate in bone metabolism.2 Exosomes derived from Jur-GV647-miR-802 and Jur-GV647-miR-106a-5p could promote the differentiation of osteoclasts into mature osteoclasts by increasing the expression of miR802 and miR-106a-5p in osteoclast precursor cells,respectively,with no significant effect on their viability.3 miR-802 and miR-106a-5p mediate the positive promotion of osteoclastic differentiation and participate in the development of OP by targeting and downregulating the corresponding target genes TRAF3,LGR4,RHOA and PTEN,respectively.Part ⅢObjectiveThe mechanism of how immune factors interact with the skeletal system and thus participate in the development of OP remains elusive.A total of immune indicators,including median fluorescence intensities(MFI),absolute cell counts(AC),relative cell counts(RC)and morphological parameters(MP),were systematically investigated by Mendelian Randomization(MR).The potential causal associations between MFI,absolute cell counts(AC),relative cell counts(RC)and morphological parameters(MP)and BMD at multiple sites may provide new clues and ideas to further enrich the role of the immune system in the regulation of bone metabolic homeostasis for the prevention and treatment of OP.MethodsGWAS data from the databases GWAS Catalog and GEnetic Factors for OSteoporosis Consortium(GEFOS)were selected and downloaded.A two-sample MR study approach was used,with immune indicators as the exposure factor and bone mineral density as the outcome variable.Single nucleotide polymorphism(SNP)(P<1.00E-5,r2<0.1)significantly associated with MFI,RC,AC and MP were screened and extracted as instrumental variables and corresponding information(effect alleles,effect values and standard errors,etc),respectively.Through a series of methods,inverse variance weighting(IVW)as the primary method and three additional MR methods including weighted median(WM),MR-Egger regression and MR pleiotropy residual sum and outlier(MR-PRESSO)were used as a sensitivity analysis method to estimate the causal association between immune indicators and BMD.Inverse MR analysis was also used to avoid the effect of reverse causality and multivariate MR analysis was developed to further identify whether significant exposure factors were independent protective or risk factors for OP.Finally,a multivariate MR framework was used to estimate the mediating effect mediated by monocytes in the immune indicators and BMD pathway,and confidence intervals were estimated using a coefficient product test as well as bootstrap resampling.ResultsUsing genetic variants as predictors,we found causal associations of multiple immune indicators with BMD at nominally significant levels(P<0.05).For MFI traits,the T and B cell panels both had the largest number of significant immune indicators pairs than other panels.CD40,as a molecule expressed by 4 subsets of monocytes,was highlighted due to its consistently positive correlation with BMD at four sites.For both AC and RC traits,immune indicators from the T-cell panel were highlighted,with CD39-positive T-cell subsets in particular being the most frequently observed feature.For MP traits,the most significant association with BMD was SSC-A on CD14+monocyte.After false discovery rate(FDR)correction,we detected 9 MFI-,16 AC-,22 RC-and 5 MPBMD pairs,respectively,all at significant levels(q<0.05).For example,the effects of different activation status subsets of CD4+Treg cells on BMD were all causally significant and were accompanied by different patterns.And sensitivity analyses suggested that the identified immune factors were robust to pleiotropy.Multivariable MR analysis conformed independent causal effect of several immune indicators on BMD.Mediation analyses showed that CD40 on monocytes could mediate multiple immune indicators,in particular,suggestive associations of CD27 on several memory B cells with osteoporosis mediated by CD40 on CD14+ CD16-monocyte.ConclusionsOur study is the first to systematically assess the causal association of peripheral immune indicators with the risk of developing OP,and these findings highlight the diversity of modalities and complexity of mechanisms involved in the regulation of OP development by immune factors.