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. 2020 Mar 13:8:131.
doi: 10.3389/fcell.2020.00131. eCollection 2020.

Human Umbilical Mesenchymal Stem Cells Display Therapeutic Potential in Rheumatoid Arthritis by Regulating Interactions Between Immunity and Gut Microbiota via the Aryl Hydrocarbon Receptor

Xiaoya Li  1   2   3 , Cheng Lu  4 , Danping Fan  2   3 , Xiangchen Lu  2   5 , Ya Xia  2   5 , Hongyan Zhao  6 , Huihui Xu  6 , Yongliang Zhu  7 , Jingtao Li  8 , Honglin Liu  2 , Cheng Xiao  1   2   3
Affiliations

Human Umbilical Mesenchymal Stem Cells Display Therapeutic Potential in Rheumatoid Arthritis by Regulating Interactions Between Immunity and Gut Microbiota V体育安卓版 - via the Aryl Hydrocarbon Receptor

Xiaoya Li et al. Front Cell Dev Biol. .

Abstract

Background: Rheumatoid arthritis (RA) is an autoimmune disease that may be associated with gut microbiota via the aryl hydrocarbon receptor (AhR). Human umbilical mesenchymal stem cells (HUMSCs) have therapeutic potential against RA, but the underlying mechanism has not been fully elucidated VSports手机版. The purpose of this study was to explore the mechanism of action of HUMSCs in rats with collagen-induced arthritis (CIA). .

Method: HUMSCs (1 × 106) were transplanted into each rat with CIA. The tissue localization of HUMSCs and the therapeutic effects in the ankles were assessed V体育安卓版. The immune status and expression of immune-related genes and proteins in related lymphoid tissues were subsequently tested. Furthermore, the levels of immune-related factors in serum and the changes in gut microbiota in the ileum were detected, and the levels of indole and their derivatives in plasma and the levels of AhR in the ileum were evaluated. .

Results: HUMSCs homed to the popliteal lymph node (PLN), mesenteric lymph node (MLN), ankle cartilage, and ileum mucosa in rats with CIA. The transplantation of HUMSCs reduced the pathology scores and the degree of bone damage in the ankles. The immune status of T regulatory cells (Tregs) and T helper (Th)17 cells and the gene expression levels of interleukin (IL)-10, transforming growth factor (TGF)-β1, and IL-17A were altered in the PLN, which is the lymph tissue closest to the nidus, and the MLN, which is one of the gut-associated lymphoid tissues (GALTs). The proportion and function of B cells, Tregs, and Th17 cells were regulated in other GALTs, namely, Peyer's patches and the lamina propria. The gene expression of TGF-β1 and IL-17A and protein expression of IL-10, TGF-β1, IL-17A, IL-22, and immunoglobulin A (IgA) were modulated in the ileum, and the serum levels of IL-10, TGF-β1, IL-17A, IL-1β, and tumor necrosis factor (TNF)-α were regulated in the rats with CIA. The relative abundances of the genera Bacteroides and Bacillus were increased in the HUMSCs-treated rat with CIA; in addition, the levels of indole, indoleacetic acid, and indole-3-lactic acid were consistently upregulated, and this upregulation was accompanied by increases in AhR gene and protein expression. V体育ios版.

Conclusion: Our study demonstrates that HUMSCs play a therapeutic role in rats with CIA by regulating the interactions between host immunity and gut microbiota via the AhR VSports最新版本. .

Keywords: aryl hydrocarbon receptor; gut microbiota; host immunity; human umbilical mesenchymal stem cells; rheumatoid arthritis. V体育平台登录.

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Figures (VSports注册入口)

FIGURE 1
FIGURE 1
Characteristics of human umbilical mesenchymal stem cells (HUMSCs). The cells were labeled with APC, Allophycocyanin; FITC, Fluoresein Isothiocyanate; PerCP-cy5-5, Peridinin-ChlorophyII-Protein Complex-Cyanin 5-5; PE, Phycoerythrin-conjugated anti-HUMSC surface markers and analyzed by flow cytometry.
FIGURE 2
FIGURE 2
Therapeutic effect of human umbilical mesenchymal stem cells (HUMSCs) transplantation on the rat model of collagen-induced arthritis (CIA). The rats were injected with HUMSCs via the tail vein (1 × 106 cells/rat, only once) or orally administered methotrexate (MTX; 1.5 mg/kg, twice a week) or pure water (both the control and CIA groups) for 28 days beginning on day 10 after primary immunization, and the arthritic index (AI) and inflammation score were then evaluated. (A) Representative gross lesions of the ankle joints after model development and treatments. (B) The arthritis severity was assessed every 3 days. Line plots show the AI score of all the groups. (C) Representative pathological sections of the ankle joints subjected to H&E staining. Scale bar = 200 μm. (D) The degree of inflammation in the ankle joints was scored according to lymphocyte infiltration, synovial hyperplasia, pannus, cartilage damage, and joint destruction based on H&E staining N = 8. The data are presented as the mean ± SEM. ##P < 0.01 compared with the Control group, *P < 0.05 compared with the CIA group. Control group = normal control group, CIA group = model group, MTX treatment group = positive control group, HUMSCs group = HUMSCs transplantation group.
FIGURE 3
FIGURE 3
Representative images of human umbilical mesenchymal stem cells (HUMSCs) in the different tissues examined. (A) Representative immunofluorescence images of NuMA protein expression in the ankle, popliteal lymph node (PLN), mesenteric lymph node (MLN), and ileum. (B) Representative immunohistochemical images of NuMA protein expression in the ankle, PLN, MLN, and ileum (magnification, 20×).
FIGURE 4
FIGURE 4
Bone-protective effect of human umbilical mesenchymal stem cells (HUMSCs) transplantation in the ankle joints of rats with collagen-induced arthritis (CIA). Three-dimensional images of the left ankle joints and paws were reconstructed and analyzed based on micro-CT scans. The osteoclasts in the ankle joints were detected and counted after tartrate-resistant acid phosphatase (TRACP) staining. (A) Representative three-dimensional (3D) images of the ankle joints and paws taken from above and the side. (B) Bar plots showing bone volume (BV), bone surface (BS), and ratio of bone surface to bone volume (BS/BV). (C) Representative images of ankle joints osteoclasts detected by TRACP staining. (D) Bar plots showing the mean numbers of osteoclasts in the ankle joints. The data are presented as the mean ± SEM N = 8. #P < 0.05 compared with the Control group, ##P < 0.01 compared with the Control group, *P < 0.05 compared with the CIA group.
FIGURE 5
FIGURE 5
Human umbilical mesenchymal stem cells (HUMSCs) transplantation-mediated regulation of the percentage of T regulatory cells (Tregs) and T helper (Th)17 cells and related gene expression in the popliteal lymph node (PLN) of rats with collagen-induced arthritis (CIA). The percentages of Tregs and Th17 cells were detected by flow cytometry, and the RNA expression levels of interleukin (IL)-10, transforming growth factor (TGF)-β1, and IL-17A were determined by quantitative real-time PCR (Q-PCR). (A,B) Representative flow cytometry images of Tregs and Th17 cells in the PLN. (C) Bar plots showing the percentages of Tregs and Th17 cells. (D) Bar plots showing the mean relative RNA expression levels of IL-10, TGF-β1, and IL-17A. The data are presented as the mean ± SEM N = 8. #P < 0.05 compared with the Control group, ##P < 0.01 compared with the Control group, *P < 0.05 compared with the CIA group, **P < 0.01 compared with the CIA group.
FIGURE 6
FIGURE 6
Human umbilical mesenchymal stem cells (HUMSCs) transplantation-mediated regulation of cytokines in the collagen-induced arthritis (CIA) rats. The serum cytokine levels were measured by Luminex assays. Bar plots showing the mean levels of interleukin (IL)-10, transforming growth factor (TGF)-β1, IL-17A, IL-1β, and tumor necrosis factor (TNF)-α in all the groups N = 8. The data are presented as the mean ± SEM. #P < 0.05 compared with the Control group, ##P < 0.01 compared with the Control group, *P < 0.05 compared with the CIA group.
FIGURE 7
FIGURE 7
Human umbilical mesenchymal stem cells (HUMSCs) modulate the immune status of the mesenteric lymph node (MLN) and ileum. The interleukin (IL)-10, IL-17A, and transforming growth factor (TGF)-β1 mRNA expression levels in the MLN and the IL-17A and TGF-β1 mRNA expression levels in the ileum were detected by quantitative real-time PCR (Q-PCR). The protein expression of IL-10, IL-17A, TGF-β1, IL-22, and immunoglobulin A (IgA) was determined by immunohistochemistry (IHC) assay. (A) Gene expression of IL-10, IL-17A, and TGF-β1 in the MLN. (B) Gene expression of IL-17A and TGF-β1 in the ileum. (C) Representative images of the ileum obtained by IHC (20×). (D) Protein expression of IL-10, IL-17A, TGF-β1, IL-22, and IgA in the ileum N = 8. The data are presented as the mean ± SEM. #P < 0.05 compared with the Control group, ##P < 0.01 compared with the Control group, *P < 0.05 compared with the CIA group, **P < 0.01 compared with the CIA group.
FIGURE 8
FIGURE 8
Changes in the gut microbiota in collagen-induced arthritis (CIA), methotrexate (MTX), and human umbilical mesenchymal stem cells (HUMSCs) groups. The alpha diversity, beta diversity, and the typical bacterial genera and species were obtained by 16S sequencing. (A–C) Bar plots showing the mean Chao1 and Shannon diversity indices and the operational taxonomic units (OTUs) of all groups. (D) Relative abundance of the top 10 most abundant phyla in each sample. (E) The differences in the intestinal microbial communities among the groups were assessed by partial least square discriminant analysis (PLS-DA). (F) Taxonomical differences among the groups at the different levels. The dominant bacteria were determined by Lefse analyses. (G) Relative abundances among the groups at the genus and species levels N = 8. The data in panels (A–C) are presented as the medians [interquartile range (IQR)]. #P < 0.05 compared with the Control group, ##P < 0.01 compared with the Control group, *P < 0.05 compared with the CIA group, **P < 0.01 compared with the CIA group.
FIGURE 9
FIGURE 9
Levels of indole and its derivatives in the plasma and aryl hydrocarbon receptor (AhR) expression in the ileum. The levels of indole, indoleacetic acid (IAA), and indole-3-lactic acid (ILA) in the plasma were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the mRNA and protein expression levels of AhR were determined by quantitative real-time PCR (Q-PCR) and immunohistochemistry (IHC), respectively. (A) Bar plots showing the mean levels of indole, IAA, and ILA in all the groups. (B) AhR gene expression in the ileum. (C) Representative images of the ileum obtained by IHC (20×). (D) AhR protein expression in the ileum N = 8. The data are presented as the mean ± SEM. #P < 0.05 compared with the Control group, ##P < 0.01 compared with the Control group, *P < 0.05 compared with the CIA group, **P < 0.01 compared with the CIA group.
FIGURE 10
FIGURE 10
Mechanism of HUMSCs therapy in CIA rat. CIA, collagen-induced arthritis; HUMSCs, Human umbilical mesenchymal stem cells; IAA, indoleacetic acid; ILA, indole-3-lactic acid; SIgA, secretory immunoglobulin A; MLN, mesenteric lymph node; AhR, aryl hydrocarbon receptor; Treg, T regulatory cell; Th17, T helper 17 cell; OC, osteoclast; PLN, popliteal lymph node.
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