MSC-Ex Mitigates Colitis Via The Modulation Of The Gut Metagenomics-Metabolomics-Bile Acid-Activated Receptor Axis

Background: Inflammatory bowel disease（IBD）,a chronic gut immune dysregulation and dysbiosis condition which affects individuals’ quality of life,is rapidly increasing in global incidence.Traditional treatment strategies rely on frequent administration of high doses of medications such as non-steroidal anti-inflammatory drugs,antibiotics,biologics,and immunomodulators,which often have short-term efficacy and cytotoxic effects.Over the past few decades,IBD has become an important health issue with dramatic advances in its therapeutic approaches including the introduction of targeted biologic therapies,and the optimization of older therapies.Regardless of the progress made,no ideal treatment regimen is available for IBD.Among the promising biological therapies for IBD is mesenchymal stem cells（MSCs）and its derived exosomes（MSC-Ex）.MSC-Ex as cell-free therapy,is capable of shuttling various proteins,messenger RNA（m RNA）and micro RNAs（mi RNAs）to modulate the activity of recipient cells,and has been demonstrated to be more efficient,safe,and convenient to store and transport compared to MSCs.In the IBD microenvironment,these nanovesicles,not only regulate the immune components but also the gut microbiome,thus capable of influencing the level of host’s cytokines,metagenomics,and metabolomics.Bile acids and their receptors form a crucial component of the chemical communications between the intestinal microbiota and the host immune system.Alterations in bile acid pool affects intestinal homeostasis and enhance immune dysfunction in IBD,making bile acid receptors an interesting therapeutic target in IBD.To date,there is no published data on the modulatory effect of MSC-Ex on the gut metagenomics,metabolomics,and bile acid receptors.This study aimed at assessing the metagenomics and metabolomics profile of IBD in both human and animal model.The regulatory effect of MSC-Ex on the gut bacteria composition and diversity,metabolites,and their related functions and pathways was explored.The effect of MSC-Ex on key inflammatory and anti-inflammatory cytokines,as well as clinical signs of IBD was also examined in mice model.Method: A total of 30 fecal samples were collected from 15 IBD patients and 15 healthy controls for 16 S r RNA gene sequencing of metagenomics and ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry（UHPLC/Q-TOF-MS）detection of metabolomics.The colon tissues of 10 IBD patients and 10 healthy controls were also obtained for farnesoid X receptor（FXR）gene expression analysis via q RT-PCR.Dextran sulfate sodium（DSS）-induced IBD model in BABL/C mice was established,consisting of three groups;normal control group,DSS group,and MSC-Ex group.Post administration of MSC-Ex,the therapeutic effect was evaluated by means of mouse body weight,fecal traits,colon/spleen gross examination,colon tissue hematoxylin and eosin（H＆E）staining,and cytokine analysis using q RT-PCR.Mice faecal samples were obtained for metagenomics and metabolomics analysis.The regulatory effect of MSC-Ex on the gut bacteria,metabolites,and related functions/pathways were examined by splicing,filtering,and de-chimera the raw data to obtain effective data,from which analytical tools such as Anosim,Adonis,LEFs E,STAMP,PCA,PCo A,KEGG,and COG were employed in analyzing the outcome.The effect of MSC-Ex on bile acid-activated receptors FXR,TGR5,and CYP7A1 was also investigated in vivo and in vitro by q RT-PCR,and FXR selected for further confirmation through western blot and immunohistochemistry（IHC）.Results: IBD patients had severe perturbation of gut bacteria community composition,diversity,and metabolites compared to healthy individuals.The two most abundance phyla in humans（Bacteroidetes and Firmicutes）were decreased while disease-associated phyla such as Proteobacteria and Actinobacteria were increased.‘Healthy bacteria’ including Ruminococcaceae UCG-002,UCG-005,UCG-010,Dialister,and Alistipes were reduced.DSS-induced colitis mice revealed a similar profile of altered Bacteroidetes and Firmicutes with increased abundance of disease-causing bacteria among others including Proteobacteria,Epsilonbacteraeota,Actinobacteria,Patescibacteria,and Tenericutes,but reduced ‘healthy bacteria’ such as Lactobacillus reuteri,uncultured Bacteroidales bacterium,Lactobacillus reuteri,and Lactobacillus gasseri.A total of 3146 metabolites were detected out of which 135 were differentially expressed between IBD and healthy controls.Similarly,in the animal model,1383 metabolite were identified with 156 differentially expressed between the DSS-induced colitis group and the normal control mice.The IBD group had increased functional indication of immune system diseases,infectious diseases,cancer,and other significantly affected pathways including primary bile acid biosynthesis,vitamin digestion and absorption,protein digestion and absorption,ABC transporters,basal cell carcinoma,glutathione metabolism,and ferroptosis.MSC-Ex treatment significantly restored colon tissue microscopic structural integrity,recovered colon length and spleens size,maintained body weight,reduced DAI,decreased pro-inflammatory cytokines（IL-1β,IL-6,TNF-α）and increased the anti-inflammatory cytokine IL-10 in mice.MSCEx improved the gut microbiota composition by enhancing OTUs structure,restoring colitis induced reduction in α-diversity,increasing the abundance of ‘healthy’ gut bacteria,decreasing disease-associated bacteria,decreasing detrimental functions,enhances other vital cellular functions,and reducing colitis-induced alterations in gut metabolomics profile in mice.For the first time,we demonstrated that MSC-Ex potently regulated the bile acid-activated receptors FXR,TGR5,and CYP7A1,and further confirmed the regulatory effect on FXR using western blot and IHC analyses.Conclusion: There is severe alteration in the gut metagenomics and metabolomics in both human IBD and animal model,with significantly reduced expression of FXR and TGR5.MSC-Ex relieves IBD by restoring the gut bacteria community structure and diversity,metabolomics profile,and vital cellular functions in mice.The mitigation effect of MSC-Ex in colitis is likely related to its ability to potently regulate the bile acid receptors FXR,TGR5,and CYP7A1.Thus,we demonstrate for the first time that MSC-Ex mitigates colitis in mice by modulating the gut metagenomics-metabolomics-bile acid activated receptor axis.