BMSCs improve TNBS-induced colitis in rats through inducing Tregs differentiation by expressing PD-L1

Objective Bone marrow-derived mesenchymal stem cells (BMSCs) are a kind of stem cells with high differentiation potential and immunomodulatory ability, which has a broad prospect in the treatment of in�ammatory bowel disease. The aim of this study is to investigate whether BMSCs could improve TNBS-induced colitis in Sprague-Dawley (SD) rats through inducing Tregs differentiation by expressing PD-L1. BMSCs were isolated and identi�ed by �ow cytometry before being transfected with PD-L1 siRNA recombinant lentiviral vector. Then SD rats were randomly divided into 4 groups. Colitis induced by TNBS (sigma Aldrich) except normal group. On the fourth day of modeling, the rats in BMSCs control group and PD-L1 siRNA BMSCs group were injected with corresponding BMSCs through tail vein for 1 week, the dose was 5 × 10 6 cells. The normal group and model group were given the same volume of PBS.


Introduction
Ulcerative colitis (UC), a disease characterized by chronic in ammation and ulceration in colorectum with typical symptoms of recurrent abdominal pain and bloody mucous diarrhea, is diagnosed by colonoscopy and pathological examination. UC, previously considered as a low-risk lesion, has been gradually increasing in incidence in Asia in recent years, and is regarded as an important risk factor for colorectal cancer [1][2][3].Though being studied for several years, the etiology and pathogenesis of UC are still unknown, while most researchers think they are related to environmental factors, genetic susceptibility and immune disorder. Compelling data revealed that the most critical factor contributing to Page 4/20 UC is immune disorder, exactly Intestinal mucosal immune disorder [4]. As we all known, the central part of intestinal immune dysfunction is the imbalance of pro-in ammatory cells and anti-in ammatory cells, which is mainly re ected between regulatory T cell (Treg) and T helper (Th17) cell. Treg, an important anti-in ammatory cell, is proved to play pivotal role in improving UC by activation of forkhead box P3 (FOXP3) and secretion of interleukin-10 (IL-10) [5,6].
Bone marrow-derived mesenchymal stem cells (BMSCs), a kind of non-hematopoietic stem cells existing in bone marrow, are widely used to study of stem cell-based therapy. For their multi-directional differentiation and high proliferation potentials, BMSCs could be induced directly into neural cells, osteoblasts, fat cells and so on under speci c conditions [7,8]. Although they only account for 0.001% − 0.01% in bone marrow monocytes, they can be expanded more than 1 million times or 6 generations in vitro [9]. More and more researches reported that BMSCs show great immunosuppressive potentials and can be used to treat in ammation-mediated diseases including in ammatory bowel disease (IBD) [10]. Our previous study demonstrated that BMSCs can relieve TNBS-induced colitis and raise the percentage of Tregs in blood of rats, so we think that BMSCs alleviated colitis by promoting Tregs differentiation.
PD-L1(also called CD279 or B7-H1), a ligand of programmed cell death-1 (PD-1), is a member of the CD28/B7 superfamily and widely expressed in various organizations [13][14][15]. The PD-1/PD-L1 costimulatory signal is mainly involved in the central and peripheral immune tolerance of CD4 + T cells, and can regulate the balance of effector T cells and Tregs in the progression of multistage autoimmune diseases [15,16]. Some studies show that PD-L1 is the most important factor which is able to induced naive T cells to differentiate into Tregs by inhibiting Akt/mTOR signal pathway [16]. At the same time, BMSCs are reported to inhibit activation and proliferation of CD4 + T cells by PD-1/PD-L1 pathway [17,18]. So based on those ndings above, we hypothesized that BMSCs alleviated colitis of rats model by inducing differentiation of Tregs via PD-L1 expressed on them.

Materials And Methods
Isolation, culture and identi cation of rat BMSCs BMSCs were isolated from 3-week-old healthy male SD rats as described previously. BMSCs were collected from femurs and tibias by ushing marrow cavities and cultured in culture ask using lowglucose complete cell culture medium consisting of α-minimum essential medium (α-MEM; Gibco, Invitrogen Corp., Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco, Invitrogen Corp.). The cells were kept in atmosphere of 5% CO 2 at 37℃. Removing no-adherent cells at every time medium changed and collecting adherent cells using 0.25% trypsin solution (Gibco, Invitrogen Corp.) when passaged, and the passage 2 (P2) BMSCs were used for identi cation and the following experiments. For ow cytometry identi cation, the anti-rat CD-29, CD-90, CD-45, CD-11b antibody (BioLegend, San Diego, CA, USA) were used as surface markers. And the adipogenic and osteogenic differentiation potentials of BMSCs were researched after identi cation as described previously.
Plasmid construction and cell transfection PD-L1 siRNA serial number is NM_001191954. 1 in Gene Bank, and then we designed the synthesis of PD -L1 siRNA according to different sites. Connecting the PD -L1 siRNA gene fragments to GV367 (Genechem, China, Shanghai), 293T cells were co-transfected with GV367 and packaged Plasmid pHelper 1.0 and 2.0 (Genechem) to form recombinant lentivirus. The cells were inoculated on a 6-well plate one day before transfection, 5×10 4 per well, and the complete medium was replaced with 1.9mL serum-free medium half an hour before transfection. At transfection time, the con uence of 2 (P2) BMSCs reached 70%-80% per well, incubated at 37℃ for 4-6h, and then the serum-free medium was replaced with complete medium for further culture. After 48 hours, PCR and western blot were used to detect the transfection e ciency, and Rat-PD-L1-175 was nally determined as the best PD-L1 siRNA. Its gene sequence is 5 '-ggaagacaaggaaguuauuca-3' and 5 '-Aauaacuuccuugucuuccuu-3'.

Induction of UC models and treatment
After an adaptive feeding of one week, forty male rats were randomly assigned to four groups (n=10): normal, model, BMSCs control and PD-L1 siRNA BMSCs. TNBS (Sigma-Aldrich) was used to induce colitis according to Morris et al. On the fourth day of modeling the rats in the BMSCs control group and the PD-L1 siRNA BMSCs group were injected corresponding BMSCs via tail vein at a dose of 5×10 6 cells in 1 ml phosphate-buffered saline (PBS), and the normal group and model group were given the same volume of PBS in the same way. One week after cells injection, all rats were anesthetized and then executed, colons were detached and stored for various studies.

Evaluation of in ammation
From molding, all rats were daily monitored weight loss, stool trait, and degrees of bloody stool to calculate the disease activity index (DAI). After colons were dissected, part of colonic tissue were resected for hematoxylin-eosin (HE), and histological evaluation was performed according to previous description.

Real-time quantitative PCR (RT-PCR)
RT-PCR was used for quantifying the expression of PD-L1, IL-10 mRNA, and the procedure was the same as described previously. All primer sequences are shown in Table 1. Target gene expression was normalized to β-actin and calculated with the 2 -ΔΔCt method.

Flow cytometry
Monocytes were isolated from peripheral blood as described previously. After incubated with anti-CD4 and anti-CD45 (BD Biosciences, San Diego, USA) at 4˚C for 30 min in the dark, the cells were stained with anti-FOXP3 (BD Biosciences) and then analyzed by ow cytometry.

Enzyme-linked immunosorbent assay (ELISA)
To detect the level of IL-10, we collected colon homogenate supernatants for sandwich enzyme-linked immunosorbent assay using rat IL-10 ELISA kit (NeoBioscience, Shenzhen, China) according to the manufacture's protocol.

Statistical analysis
Statistical analysis was performed by SPSS 22.0 software. All data are presented as means ± standard deviation (SD). One-way ANOVA or Dunnett's test (equal variances were not assumed) was used for assessing statistical signi cant, and a value of P 0.05 was regarded as signi cant difference.

Identi cation of BMSCs
To identify the cells' purity and differentiation potential, we used ow cytometry to analyse the bio-marker on their surface and exposed them to speci c medium for verifying pluripotent. The ow cytometric analysis revealed that the cells were positive for CD29 and CD90, the surface markers of bone marrow progenitor cell, but negative for CD11b and CD45, the surface markers of hematopoietic cell (Fig.1A). P2 BMSCs were used for induction, a large number of lipid deposition were shown with Oil Red O staining after adipogenic induction (Fig.1B), and calcium nodules which are showed as red spots with alizarin red staining were observed after osteogenic induction (Fig.1C).

PD-L1 siRNA down-regulated PD-L1 expression in BMSCs
To determine the effect of transfection, uorescent labeling, qRT-PCR and western blotting of PD-L1 were performed in the PD-L1 siRNA BMSCs and the BMSCs control.The expression of GFP in transfected BMSCs was observed under uorescence microscope ( Fig.2A).After 72 hours of plasmid transfection, the levels of PD-L1 mRNA and protein in BMSCs transfected with PD-L1 siRNA were signi cantly lower than those in the control group (Fig.2B).In other words,the plasmid was successfully transfected and did downregulate the expression of PD-L1. To determine whether the homing ability of BMSCs was different after virus transfection,the migration e ciency of BMSCs labeled by GFP in colon tissue was observed by immuno uorescence microscope. The average uorescence intensity of PD-L1 siRNA BMSCs group was 16.346, and that of null-BMSCs group was 16.26. The results showed that there was no signi cant difference in the number of BMSCs colonized in the intestine between the two groups and no effect of virus transfection on homing of BMSCs ( g.2C).

Down-regulation of PD-L1 inhibited remission of TNBS-induced colitis by BMSCs
To evaluate the effect of PD-L1 in TNBS-induced colitis of rats, we monitored weight loss and calculated disease activity index (DAI) daily from molding. Rats in model group suffered the most serious weight loss and their DAI were the highest among all groups. For the cells therapy groups, the weight loss and DAI in the BMSCs control group decreased signi cantly than the PD-L1 siRNA BMSCs group compared with the model group( Fig.3A . As we all known, colonic in ammation leads to colon shortening. The colon length of model group, null-BMSCs group and PD-L1 siRNA BMSCs group were decreased by 21.47%, 5.93% and 13.05% respectively compared with the control group (Fig.3B,C , so the difference between two cells therapy groups was statistical signi cant. Histological analysis showed that there were mucosal erosion and large amount of in ammatory cellular in ltration in rat colon of model group, and cells therapy mitigated these damages and lower the histological score. However, the BMSCs control group indicated less in ammation than PD-L1 siRNA BMSCs group (Fig.3D,E . Taken together, the results reveal that BMSCs inhibit the intestinal in ammation in TNBS-induced rats via PD-L1.

Down-regulation of PD-L1 in BMSCs decreased Tregs in TNBS-induced colitis
To investigate that whether BMSCs promote Tregs differentiation via PD-L1, we analyzed the percentage of Tregs in monouclear cells of spleen and mesenteric lymph nodes. As anti-in ammatory cells, Tregs has been veri ed to decreased in rat model of UC induced by TNBS, and BMSCs transplantation can promote Tregs in UC rats according to our previous studies [12]. Our results showed that we reproduced the results of our previous experiments and PD-L1 inhibition in BMSCs decreased Tregs compared with BMSCs control (Fig.4A, B). What is more, we detected the levels of IL-10, the signature transcription cytokine and effect cytokine of Tregs, in colonic tissue. PD-L1 siRNA BMSCs down-regulated the level of IL10 by PCR and ELISA compared with BMSCs control (Fig.5A). In general, PD-L1 exerts an important role in promoting Tregs differentiation by BMSCs.
Down-regulation of PD-L1 inhibited Akt/mTOR pathway Akt/mTOR pathway is one of the down-stream of PI3K signal channels, and participates in regulating the differentiation of T naive cells, which are induced to Tregs through inhibiting Akt/mTOR pathway. For further veri cation of whether PD-L1 on BMSCs promotes Tregs and relieves in ammation via Akt/mTOR pathway, western blotting of Akt phosphorylation and mTOR phosphorylation were performed. The results display that the levels of phosphor-Akt (Ser473), phosphor-Akt (Thr308) and phosphor-mTOR in the PD-L1 siRNA BMSCs group were obviously increased compared with the BMSCs control group (Fig.5B,C,D). We also detected the level of PTEN (phosphate and tension homology deleted on chromsome ten), which is important for antagonizing PI3K signaling. As is shown in Fig.5E, the expression of PTEN was contrary to the level of Akt/mTOR pathway. Therefore, our results revealed BMSCs induced Tregs by activating Akt/mTOR pathway via PD-L1 in TNBS-treated rats.

Discussions
Ulcerative colitis (UC), together with the crohn's disease (CD), belongs to in ammatory bowel disease (IBD). The typical pathologic manifestations of UC are mucosal ulcer, submucosal edema, lymphocytes in ltration and brosis, which are considered to be related to T cell-mediated immune regulation disorder, though not being fully understood [19,20]. For studying the pathogenesis and therapeutic methods, many animal models are implemented. In this research, we used TNBS to induce colitis of rats, which is the mimic of human UC in both symptoms and pathological processes.
For the past few years, mesenchymal stem cells (MSCs) therapy has attracted lots of attention owing to the immune-regulatory capacity and multi-lineage differentiation ability of MSCs. Currently, the application of MSCs therapy mainly focus on local or systematic transplantation, combining with genetic modi cation and tissue engineering [21][22][23]. And more attractive thing is that BMSCs, one of the most important representations of MSCs, have been proved of signi cant treatment prospect for IBD [12,24]. The locations of IBD are mainly happened in gut, a unique immune organ, and it would be damaged when the balance between pro-in ammatory cells and anti-in ammatory cells was destroyed [25,26]. Some pro-in ammatory cells such as Th1 and Th17, differentiated by naive T cells, exert in uence on inducing and maintaining intestinal in ammation by secreting effector cytokines, while Tregs, another kind of cells derived from Naive T cells, are important anti-in ammatory cells which suppress the function of pro-in ammatory cells [27,28]. Extensive evidence reveals that Tregs decrease in UC, and promoting the differentiation of Tregs alleviates the colitis of animal models [12,29,30]. So the therapeutic effects of both BMSCs and Tregs raise much interests of researchers worldwide. Casiraghi et al. [31]has testi ed that transplantation of MSCs induces Tregs in vivo, and our preliminary experiment shows the percentage of Tregs increased after administration of BMSCs in TNBS-induce colitis [12]. However, some experiments show that transplantation of MSCs enhances tumor growth in some animal model which is considered to be related to their multi-differentiation potential [32]. So the more understanding of complicated interaction between MSCs and Tregs, the greater therapeutic effect we would get, and this study aimed at clarifying possible approaches of regulation between they two based on our previous ndings.
Recently, the functions of PD-1 and its ligand PD-L1 in regulating immunological tolerance and autoimmunity caused signi cant concern of researchers. Several studies have demonstrated that PD-1/PD-L1 pathway exerts pivotal roles in physiological and pathological processes including activation and differentiation of T cells, oncogenesis, and some chronic in ammation [16,[33][34][35]. Both of them are found to expressed on T cells, B cells, macrophages and some dendritic cells (DCs), meanwhile, PD-L1 is found to constitutively expressed on MSCs on murine [36,37]. That is to say, BMSCs might promote the activation of naive T cells and induce them to differentiate into Tregs via PD-1/PD-L1 pathway. In the differentiation process of naive T cells, it is the interaction between PD-1 and PD-L1 that in uences the generation of Tregs [16]. Furthermore, extensive studies indicate that Akt signaling, which is necessary for activaton and proliferation of naive T cells, is dispensible for development and function of Tregs [38][39][40]. During the activation and proliferation of T cells, The binding of PD-1 on the surface of naive T cells and PD-L1 leads to the phosphorylation of ITIM and ITSM on cytoplasmic domain and the recruitment of SHP-1 and SHP-2, SHP-1 and SHP-2 inhibit the activation of PI3K and interdict the phosphorylation of Akt, the down-stream of PI3K [37], and then suppress the Akt/mTOR signaling cascade [41], consequently in uencing on the "molecular switch" in naive T cells to inducing development of Tregs. Simultaneously, the inhibition of PI3K blocks proliferation and survival of T cells, which maintains the function of Tregs [6]. In this study, our data revealed that BMSCs administration inhibited Akt/mTOR pathway, upregulated the expression of PTEN, the inhibitory signal of PI3K, and raised percentage of Tregs, as well as IL-10. However, the results in PD-L1 siRNA BMSCs group were opposite.
In conclusion, BMSCs can improve TNBS-induced colitis by expressing PD-L1, and its mechanism is mainly related to the inhibition of Akt / mTOR pathway which can induce Treg differentiation. PD-L1 is an important target of BMSCs in the treatment of UC.

Declarations
Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.