Human umbilical cord-derived mesenchymal stem cells ameliorate experimental colitis by normalization of gut microbiota

doi:10.21203/rs.3.rs-1617255/v1

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Research Article

Human umbilical cord-derived mesenchymal stem cells ameliorate experimental colitis by normalization of gut microbiota

https://doi.org/10.21203/rs.3.rs-1617255/v1

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Background

Crohn's disease (CD) is a chronic nonspecific inflammatory bowel disease. Current CD therapeutics cannot fundamentally change the natural course of CD and therefore it is of great significance to find new treatment strategy for CD. Preclinical and clinical studies have shown that mesenchymal stromal cells (MSCs) are a promising therapeutic approach, However，the mechanism of MSC alleviating CD and how MSC affects gut microbes is still unclear and needs to be further elucidated.

Methods

We used 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce experimental colitis in mice, and analyzed the microbiota in the fecal samples, which come from the control group, the TNBS group and the TNBS+MSC group with fecal 16S rDNA sequencing. Subsequent analysis of alpha and beta diversity were all performed based on the rarified data. PICRUStII analysis was subsequently performed from 16S rRNA gene sequences to infer gut microbiome functions.

Results

MSCs treatment improved the TNBS-induced colitis by increasing survival rates, relieving symptoms. A distinct bacterial signature was found in the TNBS group that differed from the TNBS+MSC group and controls. MSC alleviated the increased activities of sulfur and riboflavin metabolism.Meanwhile some metabolisms such as biosynthesis of amino acids，lysine biosynthesis, sphingolipid metabolism，secondary bile acid biosynthesis were decreased in the TNBS group when compared with the control group and the TNBS+MSC group.

Conclusions

MSC treatment could modulate the dysregulated metabolism pathways in mice with colitis, restoring the abnormal microbiota function to a normal situation as the control group.

Mesenchymal stem cells

Crohn's disease

TNBS

16S rRNA gene sequences

gut microbiota

metabolisms

Crohn’s disease (CD)is a subtype of inflammatory bowel disease (IBD) characterized by chronic recurrent colonic mucosal inflammation.In CD the inflammation can be found throughout the gastrointestinal tract and often extends deeper into the colonic tissue layer. Inflammatory bowel disease has become a global disease with accelerating incidence^[1]. Newly industrialized countries/regions in Asia and Latin America are in the acceleration in incidence stage. For the reason that in newly industrialized countries their societies have become more westernized. Developing countries are in the Emergence stage, newly industrialized countries are in the acceleration in incidence stage, and western regions are in the compounding prevalence stage^[2]. In western countries although incidence is stabilising, burden remains high as prevalence surpasses 0·3%^[3]. So far, there is no effective cure for Crohn’s disease and the recurrence rate is high. Inflammatory bowel disease is a very complex and costly disease, and these data highlight the need for prevention research and health-care system innovations.

In the last decade,mesenchymal stem cells (MSC) have emerged as promising therapeutic options for many incurable diseases^[4]. Further interest in MSC has been raised by the observation that they exhibit profound due to attractive features of plasticity, tropism for inflamed tissues, and a high immunomodulatory potential. The MSC is a non-hemopoietic cell capable of self-renewal and secreting antibacterial peptides and cytokines. In theory, they can be used as a potential treatment for CD ^{[5, 6]}。The 2,4,6-trinitrobenzenesulfonicacid (TNBS)-induced colitis model shared most features with human CD. Preclinical animal experiments have also fully confirmed that MSC significantly improve clinical abnormalities in TNBS-induced colitis mice and reducing lesion scores and inflammation in the gut. However,the mechanism of MSC alleviating CD is still unclear and needs to be further elucidated.

The human gut microbiota which composed of trillions of individual microbes has adapted to the uniquely diverse environments found in the intestine. Gut microbes are an essential part of the microbiota ecosystem. They outnumber human cells by tenfold^[7]. In recent years, with the continuous progress of the theoretical research of gut microbiota and the gradual maturity of molecular technology, the relationship between CD and gut microbiota has continuously shown new hot spots. The involvement of the gut microbiota in the maintenance of the gut ecosystem is two-fold: it educates host immune cells and protects the host from pathogens. However, when healthy microbial composition and function are disrupted (dysbiosis), the dysbiotic gut microbiota can trigger the initiation and development of various GI diseases, including inflammatory bowel disease (IBD)^[8]. Experiments on enteric microbiota from IBD patients have revealed that a decreased abundance of the Firmicutes and Bacteroidetes phyla with an increased abundance of Proteobacteria is significantly correlated to the severity of IBD, and genera of the Firmicutes phylum reduced in IBD patients^{[9, 10]} Therefore, we aimed to investigate the pathogenic connection between the MSC, the progression of IBD, and the enteric microbiota using a mouse model of 2,4,6-trinitrobenzene-sulfonic acid- (TNBS-) induced colitis.

Mice

BALB/c mice were purchased from the Model Animal Research Center, Nanjing University (Nanjing, Jiangsu, China). All animals used for in vivo studies were 8-week-old male. BALB/c mice were randomly allocated to each group. All animal protocols were reviewed and approved by the Sun Yat-sen University Institutional Animal Care and Use Committee.

Fresh human umbilical cords are obtained from newborns with parental consent and collected on PBS at 4 ° C.The rope was washed twice with PBS to remove any remaining blood.The rinsed cords were cut into 10-mm3 pieces and placed in type I collagenase with hyaluronidase (0.1%) containing CaC12 (3 mM) for digestion at 37°C for 4 h. The specimens were transferred to DMEM (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Pan Biotech, Aidenbach, Germany) at 37°C in a humidified atmosphere with 5% CO2. The medium in the primary culture was changed 3 days later, and nonadherent cells were removed. After that, the culture medium was refreshed every 4 days. Following the appearance of colonies of fibroblast-like cells after 14 days, the cells were trypsinized and transferred to a new flask for further expansion. We incubated the MSCs with 0.05% trypsin-EDTA at 37°C and stored them at 80°C for further analysis. For the collection of umbilical cords, the Human Ethics Committee of the Third Affiliated Hospital at Sun Yat-sen University approved the project. Written informed consent was obtained from all participants.

CytoFLEX flow cytometers (Beckman Coulter) were used for Flow Cytometry, and CytoExpert software (Beckman Coulter) for data analysis.Anti-human CD90-FITC (Catalog #IM1839U), Anti-human CD19-FITC(Catalog #A07768), Anti-human CD11b-FITC(Catalog #IM0530), Anti-human HLA-DR -FITC(Catalog #IM1638U)), Anti-human CD34-FITC(Catalog #IM1870), Anti-human CD45-FITC(Catalog #AO7782),CD105-PE(Catalog #B76299), Anti-human CD73-PE (Catalog #B76299)were purchased from Beckman Coulter.

hUC-MSCs were seeded into 24-well plates for osteogenic differentiation and cultured for 12 hours at a density of 6 × 10⁴ cells per well. In the following days, the media was changed to osteogenic differentiation medium (Biological Industries) and the media were refreshed every 3 days. Alizarin red solution was used to stain the induced cells.

In order to differentiate hUC-MSCs into adipocytes, we seeded them in 24-well plates and cultured them for 12 hours at a density of 6 × 10⁴ cells per well. Following that, the medium was replaced by the adipogenic differentiation medium (Biological Industries ) for 21 days. The medium was then refreshed every 3 days. According to the instructions, oil red O was applied to the induced cells.

For the colitis experiment, the backs of 8-week-old male BALB/c mice were smeared with 150Μl of pre-sensitization solution (TNBS; Sigma) 7 days before colitis was induced. We divided the mice into groups and allowed them to fast (but still allow them to drink freely) for 24 hours. Our previous study described the procedures for inducing colitis and treating it with MSCs ^[11].The parameters of body weight loss, diarrhea, and survival were recorded daily for 7 days. 3 days after TNBS injection (the peak of the disease), the length of the colon was measured from the cecum to the anus. The colons were examined for macroscopic damage based on our previous investigation ^[11].

The control mice received only 50% ethanol. MSCs were used for cell transplantation in passages 3–5. After instilling TNBS, BALB/c mice were either treated with the control medium or with 10⁶ MSCs per mouse 2 hours later.

Animals were monitored for body weight loss, stool consistency, the presence of blood on the anus or in the stool and survival every day for a total of 7days. The baseline data were collected before instillation of TNBS. Disease activity and histologic scores were evaluated as previously described^[12]. For disease activity, a score system containing percentage of weight loss, stool consistency, and fecal occult blood test was used ^{[13, 14]}. For histopathology analysis, a colon specimen from the middle part (1 cm to the anus to cecum) was fixed in 10% buffered formalin phosphate and then embedded in paraffin. Sections were stained with hematoxylin and eosin, and inflammation was graded from 0–4 as follows, in a blinded fashion: 0, no signs of inflammation; 1, low leukocyte infiltration; 2, moderate leukocyte infiltration; 3, high leukocyteinfiltration, moderate fibrosis, high vascular density, thickening of the colon wall, moderate goblet cell loss and focal loss of crypts; and 4, transmural infiltrations, massive loss of goblet cells, extensive fibrosis, and diffuse loss of crypts.

Following deparaffinization and rehydration, the tissue sections are placed in a repair box filled with citric acid(PH6.0) antigen retrieval buffer and heated in a microwave oven to allow antigen retrieval. Then, the slides were transferred into an antigen retrieval solution and incubated with a primary antibody overnight at 4°C.After incubation with a secondary antibody at room temperature for 1 hour, 3,3-diaminobenzidine was used. After counterstaining with hematoxylin, optical microscopy was used to examine the sections. The primary antibodies including rabbit anti-human zonula occludens-1 (ZO-1, 1 : 150), and rabbit anti-human Occludin (1 : 100). The antibodies were purchased from Wuhan servicebio technology CO.,LTD. The stained sections were read by a microscope and quantified using the Image-Pro Plus (IPP) 6.0 software. Quantified results of Occludin and ZO-1 expression presented as the mean density.The mean densities of 5 randomly selected fields at in each group.

Feces of mice were collected by sterile tubes and were immediately stored at -80℃ before use. Microbial community genomic DNA was extracted from samples using the E.Z.N.A.® soil DNA Kit(Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer’s instructions. The concentration and purity of DNA samples were determined using NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE).

The hypervariable region V3-V4 of the bacterial16S rRNA gene were amplified with primer pairs338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R(5'-GGACTACHVGGGTWTCTAAT-3') by an ABIGeneAmp®9700 PCR thermocycler (ABI, CA, USA). The PCR amplification of 16S rRNA gene was performed as follows:initial denaturation at 95 ℃ for 3 min, followed by 27 cycles of denaturing at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s and extension at 72 ℃for 45 s, and single extension at 72 ℃ for 10 min, and end at 4 ℃. The PCR mixtures contain 5 × TransStartFastPfu buffer 4 µL, 2.5 mM dNTPs 2 µL, forward primer (5 µM) 0.8 µL, reverse primer (5 µM) 0.8 µL, TransStartFastPfu DNA Polymerase 0.4 µL, template DNA 10 ng, and finally ddH2O up to 20 µL. PCR reactions were performed in triplicate. The PCR product was extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences,Union City, CA, USA) according to manufacturer’s instructions and quantified using Quantus™ Fluorometer (Promega, USA).

The purified amplicons were pooled equimolarly and sequenced on an Illumina MiSeq PE300/NovaSeq PE250 platform (Illumina, San Diego,USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database

The raw 16S rRNA gene sequencing reads were demultiplexed, quality-filtered by fastpversion 0.20.0[15]and merged byFLASHversion 1.2.7[16]with the following criteria: (i) the 300 bp reads were truncated at any site receiving an average quality score of < 20 over a 50 bp sliding window, and the truncated reads shorter than 50 bp were discarded, reads containing ambiguous characters were also discarded;(ii) only overlapping sequences longer than10 bp were assembled according to their overlapped sequence. The maximum mismatch ratio of overlap region is 0.2. Reads that could not be assembled were discarded; (iii) Samples were distinguished according to the barcode and primers, and the sequence direction was adjusted, exact barcode matching, 2 nucleotide mismatch in primer matching.

Operational taxonomic units (OTUs) with 97% similarity cutoff[17] were clustered using UPARSE version7.1, and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed by RDP Classifierversion 2.2[18]against the 16S rRNA database (eg. Silva v138) using confidence threshold of 0.7.

Observed-species, Sob, Shannon and Shannoneven are used to evaluate the complexity of species diversity. By performing LEfSe, we identified the features contributing to the most variation between control and treatment groups (LDA > 3). Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUStII) was further used for genome prediction of microbial communities in this study (Microbiota Sequencing)’ and then performed function categorization according to KEGG Orthology. STAMP3 was used for functional profiling^[19].

Statistical Analysis

Data in this study were analyzed using IBM SPSS 21.0 software. All results were expressed as mean ± SD. Statistical comparison was made by two-tailed Student’s

t test between two groups or one-way ANOVA for multi-group comparison. P < 0.05 was considered significant. Analyses and graphs were performed using GraphPad Prism version5.01.

Identification of hUC-MSCs phenotype

The hUC-MSCs showed a fibroblast-like morphology, expressed certain antigens (CD90,CD105 and CD73, [≥95 % positive]) (Figure S1a,g,h), and lacked hematopoietic lineage markers (CD11b, CD34, CD19 ,HLA-DR and CD45, [≤2 %positive]). MSCs showed a spindle-shaped and fibroblast-like morphology(Figure S1 i).After certain inducing environments of osteogenesis and adiposis, mineral accumulation and bone nodules formation was identified by alizarin red staining(Figure S1 j),hUC-MSCs formed numerous neutral lipid droplets in the cytoplasm identified by Oil Red O staining (Figure S1 k).,

Figure S1 Surface markers expression on human umbilical cord-derived MSCs at passage 3 analyzed using flow cytometry(a.CD90,b.CD19,c.CD11b,d.HLA-DR,e.CD34,f.CD45,g.CD105,h.CD73), i. fibroblast-like morphology of MSCs, j.Osteogenic differentiation (alizarin red staining ) k.Adipogenic differentiation (oil red staining);

hUC-MSCs alleviate TNBS-induced colitis

Mice treated with TNBS developed severe symptoms, including bloody diarrhea, rectal prolapse, ancolitis, and sustained weight loss. Similar to control mice, hUC-MSC-treated mice had rapidly recovered body weight loss and milder inflammation. (Figure 1a) .Disease activity index (DAI) score is an indicator of the severity of colitis, is based on the results including weight loss, stool features and fecal occult blood. The DAI score for the TNBS group was significantly increased compared with the control group, while the DAI scores following treatment with MSC were significantly reduced compared with the TNBS group.( Figure 1b). The colon tissue of the control group is normal in length, uniform in thickness, transparent and smooth, as well as light yellowish white. It is evident that the colon of the model group becomes thicker and shorter. Numerous ulcers and erosions are observed in the area 2-4 cm from the anus.The colon injury in the hUC-MSCs group was significantly reduced, mostly manifested as scattered congestion and erosion.（Figure 1 c）. Histopathological staining was used to evaluate the histological characteristics of the colon samples. The results indicated that mice maintained an integrated normal colonic structure in the control group, but mice with TNBS-induced colitis exhibited significant inflammatory cell infiltration, loss of crypts, destruction of the mucosal layer, and edema (Figure 1e). In contrast, TNBS-induced colitis in mice treated with hUC-MSCs exhibited mild inflammation. ZO-1 and Occludin are important TJ proteins that maintain the integrity of intestinal mucosal barrier and play an important role in maintaining intestinal permeability.Immunohistochemistry was used to determine tissue distribution of occludin, and ZO-1 in the colon of the mice. Immunohistochemistry showed positive staining for occludin, and ZO-1 in the colon of control group.However, the expression of these two proteins in the colon of TNBS-induced colitis group was barely detected. TNBS-induced colitis in mice treated with hUC-MSCs express mild occluding and ZO-1 level.( Figure 1g,h)

Alpha and Beta Diversity Among the Three groups

Pan/Core analysis is used to annotate OTU species taxonomy and count the corresponding abundance information of each OTU annotation . When species curve is flat, it indicated that the numbers of samples are sufficient for sequencing.So Pan/Core analysis can be used to evaluate if the numbers of sample is sufficient for sequencing. Pan/Core analysis shows that species curve is flat（Figure 2a，Figure 2b）It is indicated that the numbers of samples are sufficient for sequencing. Meanwhile rarefaction curve is already reached stable values at the current sequencing.It is indicated that the sequencing depth covered rare new phylotypes (Figure 3c). The Sob diversity index indices that the TNBS group was significantly lower than control and TNBS+MSC group (P < 0.05 and P < 0.05, respectively) Figure 2b.However,the Shannon and Shannoneven diversity index indices of the TNBS group was significantly lower than control and TNBS+MSC group (P < 0.05 and P < 0.05, respectively, Figure 2e). PCoA plot based on weighted UniFrac distance analysis was used to evaluate beta diversity. As shown in Figure2g , an apparent clustering pattern was identiﬁed for the red, blue and green points, which represented the control group,the TNBS group and the TNBS+MSC group, respective. ANOSIM analysis suggested that the bacterial microﬂora composition of difference between groups was greater than that within groups (P=0.001)( Figure 2h). Venn diagrams were constructed to evaluate the number and identity of the shared species among the control group, TNBS group and TNBS+MSC group. The Venn analysis revealed that the OTUs were shared by three groups (Figure 2i).

Gut microbiota Differences Among the Three groups

Phylogenetic analysis of the gut microbiome from the phylum to the species level was performed on the samples using standard operational taxonomic unit (OTU) classification. A distinct bacterial signature was found in the TNBS + Vehicle mice that differed from the TNBS+MSC group and controls. TNBS group particularly enrich of mostly bacteria in phylum proteobacteria. At the phylum level, mice with colitis had decreased abundance in the phylum Bacteriodetes , Firmicutes and Tenericutes compared with control or MSC mice ( Figure 3 a,b,c). A similar lineage pattern was seen at the genus level, as Escherichia-Shigella, Citrobacter were enriched in

TNBS + Vehicle mice,.At the genus level, mice with colitis had decreased abundance in the Tenericutes，Lactobacillus, Ruminococcus.(Figure3 d,e,f). We used LEfSe to identify the specific bacterial phylotypes that were altered by TNBS administration and MSC treatment. To investigate the effects of the experimental colitis acting on the microbiome, the differentiated taxon between the control group and the TNBS group was detected. The cladogram representative of the structure and their dominant bacteria were shown in Figure 4, In comparisons of the control group versus the TNBS groups, linear discriminant analysis (LDA) scores and the corresponding cladogram showed that colitis mice were most enriched in Proteobacteria phylum which include Gammaproteobacteria class, Enterobacteriaceae order , Enterobacteriales family, Escherichia_Shigella genus , Citrobacter genus. However, control group had higher enrichment of Bacteroidetes phylum which include Bacteroidia class,Bacteroidales order, Muribaculaceae family, norank_f_Muribaculaceae genus and Tenericutes phylum which include Mollicutes class, Anaeroplasmatales order, Anaeroplasmataceae family, Anaerotruncus genus. Moreover, to investigate the effects of the MSC on the microbiome, the differentiated taxon between the TNBS+MSC group and the TNBS group was assessed(Figure 5). TNBS group had higher enrichment of Proteobacteria phylum which include Gammaproteobacteria class, Enterobacteriaceae order , Enterobacteriales family, Escherichia_Shigella genus , Citrobacter genus. However, TNBS+MSC group had higher enrichment of Firmicutes phylum which include Bacilli class, Lactobacillales order, Lactobacillaceae family, Lactobacillus genus.

Microbial Metabolic Functions Associated with MSC Treatment in TNBS-Induced Colitis

To investigate the gut microbiome functions related to the TNBS administration and MSC treatment, we adopted PICRUStII to infer putative metagenomes from 16S rRNA gene profiles. STAMP was used to identify microbially relevant functions linked with the TNBS administration of MSC. These pathways were further analyzed in KEGG level 3. This analysis permitted a comparison of the differences in the functional profiles among all groups and revealed pathways that were significantly different among control, TNBS and TNBS+MSC group (Figure 6. The increased metabolisms were presented in the TNBS group when compared with the control group and the TNBS+MSC group. As a result, MSC decreased the increased activities of sulfur metabolism and riboflavin metabolism (Figure 6a）. While some metabolisms were decreased in the TNBS group when compared with the control group and the TNBS+MSC group，such as biosynthesis of amino acids，lysine biosynthesis, sphingolipid metabolism，secondary bile acid biosynthesis (Figure6b).

The human body is colonized by a large number of microbes coexisting peacefully with their host. The most colonized site is the gastrointestinal tract. More than 70% of all the microbes in the human body are to be found in the colon. The microorganism population is 10 times larger of the total number of our somatic and germ cells. Two bacterial phyla, making up more than 90% of the microbial cells, dominate the healthy adult intestine: Firmicutes and Bacteroidetes^[20]. The microbiota acts as a barrier from pathogens, exerts important metabolic functions, and regulates the inflammatory response by stimulating the immune system. ^[21]. The composition of the gut microbiota exhibits broad inter-individual and intra-individual variability^[22] and is thought to be a crucial determinant of host susceptibility to several diseases, including IBD.A key feature of IBD is alteration of the composition of the gut microbiota (known as dysbiosis)^[23]; however, the precise role of dysbiosis in disease remains poorly understood. Dysbiosis can destroy these mutualistic relationships, and influence host physiology, compromising human health status^[24].

The pathogenesis of IBD in genetically susceptible hosts has been proposed to begin with a breakdown of the intestinal epithelial barrier, followed by a disproportionate immune response to the enteric microbiota, which results in a loss of intestinal homeostasis^[25]. Such dysbiosis in the intestinal symbionts of IBD patients can be indicated by the low-level alpha diversity and manifests as a decrease in many beneficial bacteria including Firmicutes and Bacteroidetes, while an increase abundance of Proteobacteria respectively^{[26, 27]}.

Similar to previous studies, the current results showed that TNBS treatment caused microbiota dysbiosis by reducing alpha-diversity. At phylum level the TNBS group was characterized by an enrichment in Proteobacteria while an reduction in Firmicutes and Bacteroidetes. The distribution of bacterial flora TNBS-induced colitis mice is similar to that of CD patients. Here we demonstrated that MSC administration significantly ameliorated loss of body weight and intestinal inflammation and decreased DAI in TNBS-induced colitis mice. After MSC administration, the phylum Bacteroidetes, Firmicutes and Tenericutes were up-regulated and the phylum Proteobacteria was down-regulated.

Linear discriminant analysis effect size (LefSe) was performed to identify any key microbial biomarkers that might differentiate both the disease and MSC treated states. In comparisons of the control versus TNBS groups, linear discriminant analysis (LDA) scores (Fig. 4) and the corresponding cladogram showed that colitis mice were most enriched in Proteobacteria phylum which include Gammaproteobacteria class, Enterobacteriaceae order, Enterobacteriales family, Escherichia_Shigella genus, Citrobacter genus.However, the control mice had higher enrichment of Muribaculaceae, Bacteroidetes, Firmicutes. When comparing TNBS with TNBS + MSC group, Firmicutes was found to have the highest LDA score and enrichment in the MSC treated mice. (Fig. 5)

Collectively, these data show that a combination of Proteobacteria phylum which include Gammaproteobacteria class, Enterobacteriaceae order, Enterobacteriales family, Escherichia_Shigella genus, Citrobacter genus could serve as potential biomarkers of colitis. Whereas effective treatment with MSC was characterized by an enrichment in Firmicutes and Bacteroidetes.MSC can modulate the gut microbiome during colitis in such a way that it prevents the overgrowth of potential pathogenic bacteria.

The potential mechanism may be associated with the gut microbiota and host metabolism interaction as gut bacteria often target host metabolism, which further drive immune activation and chronic inflammation. Some pathways and genes are overrepresented and underrepresented in the TNBS group.Based on functional analysis, it is evident that activities of sulfur and riboflavin metabolism have increased,while the biosynthesis of amino acids, lysine biosynthesis, sphingolipid metabolism, secondary bile acid biosynthesis have declined.

The TNBS group has an enhanced capacity for managing oxidative stress, a feature of the inflammatory environment, as indicated by incremental riboflavin and sulfur metabolism in the TNBS group by comparing with the control group and the TNBS + MSC group. Oxidative stress increases in response to inflammation, which allows the microbiota to maintain homeostasis. Thus, the increases in sulfur and riboflavin metabolism may reflect a mechanism by which the gut microbiome addresses the oxidative stress caused by inflammation.

IBD was also associated with a decrease in amino acid synthesis according to previous research^[27]. Biosynthesis of amino acids and lysine biosynthesis were reduced in TNBS group. Under states of inflammation, bacteria typically have a weak ability to produce their own nutrients^[28]. The metabolism of amino acids was greatly perturbed. Genes for the metabolism and synthesis of nearly all amino acids (particularly lysine) declined in abundance.

The sphingolipid metabolism have declined.in TNBS group. It is known that sphingolipids play multiple roles in the healthy gut, including membrane structural components in intestinal cells and assigning molecules involved in cell fate decisions^[29].Previous research suggested that sphingolipid metabolism might be disrupted in IBD, causing an accumulation of compounds that promote inflammatory responses^[30].

The secondary bile acid biosynthesis have declined. Secondary bile acids (SBAs) are produced by bacteria from primary bile acids (PBAs), which have mainly anti-inflammatory effects. It has been found that IBD patients have disrupted BAs metabolism, with fecal BAs pools skewed toward lower SBAs than healthy controls^[31]. Which was in agreement with our results.

In all, the functional variation of microbiota among the control group, the TNBS group, and the TNBS + MSC group may indicate that MSC treatment could modulate the metabolism pathways in mice with colitis, restoring the abnormal microbiota function to a normal situation as the control group.

The case could reveal that the perturbations in bacterial composition were associated with the gut microbiome function. In all, the functional variation of microbiota among the control group, the TNBS group, and the TNBS + MSC group may indicate that MSC treatment could modulate the metabolism pathways in mice with colitis, restoring the abnormal microbiota function to a normalsituation as the control group

In summary, TNBS treatment induced colonic injury and inflammatory response in mice.MSC exhibited a protective role via repairing of intestinal mucosal barrier.MSC could notably restore the alpha diversity and reverse the alterations of the abundance of the gut microbiota in the mice treated with TNBS. The mechanism might be associated with gut microbiota as MSC improved gut microbiota communities in TNBS-induced murine colitis. The present study provided evidence that MSC has the potential to be used for the prevention and treatment of IBD.

hUC-MSCs

Human umbilical cord-derived mesenchymal stem cells

Crohn's disease

TNBS

2,4,6-trinitrobenzenesulfonic acid

OUT

operational taxonomic unit

ZO-1

zonula occludens-1

IBD

inflammatory bowel disease

LEfSe

Linear discriminant analysis effect size

LDA

linear discriminant analysis

PICRUSt

Phylogenetic Investigation of Communities by Reconstruction of Unobserved States

DAI

Disease activity index

IPP

Image-Pro Plus

PCoA

principal co-ordinates analysis

PBAs

primary bile acids

SBAs

Secondary bile acids

Ethics approval and consent to participate

All experiments were performed in accordance with the institutional guidelines of Third Affiliated Hospital of Sun Yat-sen University. Use of animals in this study was approved by Institutional Animal Care and Use Committee of Sun Yat-sen University. Informed consent was obtained from all subjects and/or their legal guardian(s) under the ethical committee of the Third Affiliated Hospital of Sun Yat-sen University.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

We thank all members of the Qi Zhang's laboratory for their support.

Authors’ contributions

FY designed experiments, performed experiments, analyzed data, and wrote the paper. BBN, LL and XMZ analyzed data, and edited the paper. YYC, HZL and JXL collected human umbilical cord samples, performed experiments, and edited the paper.QLL, XFZ, RXX,YZH performed animal experiments. FPH,QZ, XGZ and WJC designed the study, analyzed data, and edited the paper. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Qi Zhang([email protected])，Xiaoguang Zou ([email protected]) or Wenjie Chen ([email protected])

Funding

This work was supported by project funded by China Postdoctoral Science Foundation(2019M653904XB),Natural Science Foundation of Xinjiang Uyghur Autonomous Region(2020D01C006),Tianshan Cedar Project Science and Technology Department of Xinjiang Uygur Autonomous Region(2018XS23),Science and Technology Projects in Guangdong Province(2018YJ007),The provincial and ministerial joint construction of the open project of the State Key Laboratory of High Incidence and Prevention in Central Asia ( SKL-HIDCA-2020-10),National Natural Science Foundation of China (81870449, 82170674, 51933011)

Author information

Fan Yang,Beibei Ni and Qiuli Liu are co-first authors.

Affiliations

1.Xinjiang Medical University, 393 Xinyi Road, Xinshi District, Ürümqi, 830011, China

Fan Yang

2.Department of Infectious Diseases, The First People’s Hospital of Kashi, (The Affiliated Kashi Hospital of Sun Yat-Sen University), 66 Yingbin Road, Kashi, 844000, China

Fan Yang

3.Biotherapy Centre, The Third Affiliated Hospital, Sun Yat-Sen University, 600# Tianhe Road, Guangzhou, 510630, China

Fan Yang，Qiuli Liu，Xiaofan Zheng，Jianxi Lu， Xiaoyan Chen，Huizhu Lin，Ruixuan Xu，Yizhan He，Qi Zhang，Wenjie Chen

4.Cell-Gene Therapy Translational Medicine Research Centre, The Third Affiliated Hospital, Sun Yat-Sen University, 600# Tianhe Road, Guangzhou, 510630, China

Beibei Ni，Jianxi Lu ，Qi Zhang，Wenjie Chen

5. Department of Respiratory and Critical Care Medicine, The First People’s Hospital of Kashi, (The Affiliated Kashi Hospital of Sun Yat-Sen University), 66 Yingbin Road, Kashi, 844000, China

Li Li，Xuemei Zhong，Xiaoguang Zou

6. Department of Infectious Diseases, The Eighth Affiliated Hospital, Sun Yat-Sen University, Shennan Zhong Road #3025, Futian District, Shenzhen, 518033, Guangdong, China

Fangping He

Availability of data and materials

All data and materials generated or used during the study are available from the corresponding authors upon

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FigureS1.png
Surface markers expression on human umbilical cord-derived MSCs at passage 3 analyzed using flow cytometry(a.CD90,b.CD19,c.CD11b,d.HLA-DR,e.CD34,f.CD45,g.CD105,h.CD73), i. fibroblast-like morphology of MSCs, j.Osteogenic differentiation (alizarin red staining ) k.Adipogenic differentiation (oil red staining);

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Editorial decision: Major Revision
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08 May, 2022
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Human umbilical cord-derived mesenchymal stem cells ameliorate experimental colitis by normalization of gut microbiota

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