Nestin+ Cells Isolated From The Peritoneum Attenuate Peritoneal Fibrosis Via Suppressing IL-33 /ST2 Signaling

The intermediate lament protein Nestin, a specic marker for multifunctional, multilineage progenitor cells, is regulated during the progression of reparative and reactive brosis in the liver, heart and kidney. However, whether Nestin regulates peritoneal brosis via suppressing IL-33/ST-2 signaling is unclear. Methods High-glucose peritoneal dialysis solution (PDS) or TGF-β1 was administered to a mouse peritoneal dialysis (PD) model to induce peritoneal brosis (PF) in vivo and to human peritoneal mesothelial cells (HPMCs) in vitro to stimulate accumulation of extracellular matrix (ECM). Nestin + cells isolated from the peritoneum of Nestin-green uorescent protein (GFP) transgenic mice were transplanted in vivo, and adeno-associated virus (AAV)-RNAi was used to silence IL-33 in vitro.


Introduction
Peritoneal dialysis (PD) has been widely used as an effective treatment for end-stage renal disease (ESRD) in which the peritoneal mesothelium acts as a physical barrier with the capacity to trap bacteria and molecules [1,2]. However, the prolonged exposure of peritoneal mesothelial cells (PMCs) in the peritoneal membrane to glucose-based PD solution (PDS) can cause PMC damage and loss, peritoneal brosis (PF), ultra ltration failure and eventual discontinuation of PD [3][4][5]. Several therapeutic strategies for the treatment of PF, including the use of pharmacological strategies and the transplantation of mesenchymal stem cells to inhibit the mesothelial-to-mesenchymal transition (EMT), have shown promise [6][7][8]. However, these therapies cannot prevent PMC loss, which plays a critical role in causing PF [9]. Previous studies have suggested that PMCs showed strong labeling with antibodies against Nestin, which is a speci c marker for multifunctional, multilineage progenitor cells, indicating that these cells represent a "young", not entirely differentiated cell population [10]. The intermediate lament protein Nestin is widely used as a marker of neural stem cells [11]. More importantly, Nestin is also expressed in some adult stem/progenitor cell populations, indicating that Nestin might be a common marker of multipotent stem cells [12]. Under speci c damage conditions, upregulation of Nestin expression was found in tubular cells, podocytes, interstitial cells and brotic rat hearts [13][14][15]. More importantly, Nestin + cells isolated from the kidney showed the characteristics of MSCs, which can repair acute kidney injury [16].
IL-33 is expressed in various tissues and cells in the human body, including endothelial cells, bronchial cells, and intestinal epithelial cells [17]. Recent reports have demonstrated that IL-33 promotes ST-2dependent organ tissue brosis in several animal models, including lung [18], pancreas [19], and liver models [20].
However, the ability of Nestin + cells isolated from the peritoneum to regulate PF has not been evaluated to our knowledge. In this study, cells isolated from the peritoneum of Nestin-GFP transgenic mice were transplanted into C57BL/6 wild-type mice, and the adeno-associated virus (AAV)-RNAi-IL-33 was used to infect to HPMCs. The effects and mechanism of Nestin + cells were investigated in an established PF model.

Materials And Methods
The mice were purchased from Wenzhou Medical University Laboratory Animal Center (Wenzhou, China) and all of the animal work had taken place there. The mice had free access to a standard rodent diet and tap water and were anaesthetized with 40 mg/kg pentobarbital sodium through an intraperitoneal injection before sacri ce. This study has been carried out in accordance with the World Medical Association Declaration of Helsinki, and that all subjects provided written informed consent.

Animal studies
Homozygous transgenic mice that expressed enhanced GFP under the control of a Nestin promoter (Nestin-GFP, on the C57BL/6 genetic background) were provided by Cyagen Biosciences. We used male C57BL/6 mice at 8 weeks of age. C57BL/6 mice were divided into three groups: the control group, which received intraperitoneal administration of normal saline; the PD model mouse group without treatment, which received an intraperitoneal administration of PDS (4.25% Dianeal; Deer eld, IL, Baxter, USA) at 10 ml/100 g -1 ·d -1 for 4 weeks only; and the treatment group, PD model mice transplanted with Nestin + cells.
A peritoneal equilibration test (PET) was applied to evaluate the peritoneal permeability function 28 days after modeling. The mice were instilled with PDS at 10 ml/100 g body weight before being killed. After 30 min, the peritoneal uid was removed [8], and orbital sinus blood samples were collected. Peritoneal solute transport was calculated by D30/D0 and D/P urea . D30 was de ned as the glucose concentration in the dialysate sample at 30 min, and D0 was de ned as the initial dialysate glucose concentration. D indicates the dialysate urea concentration, and P urea indicates the plasma urea concentration. Then, the parietal peritoneum, omentum, and diaphragm were carefully dissected for use in western blots and immuno uorescence staining.
Isolation and Nestin-GFP + cell transplantation The peritoneum was dissected from 2-week-old Nestin-GFP mice and cut into small pieces. The PMCs were then dissociated from the visceral peritoneum by digestion with 0.25% pancreatin at 37 °C for 15 min, and Eagle's medium (DMEM)/F12 (Invitrogen) containing 10% fetal bovine serum (FBS, Gibco) was added to stop the pancreatin activity. The samples were centrifuged at 1000 rpm for 5 min at 4 °C, and the supernatant was aspirated. The centrifugal sediment was resuspended and passed through a 70 μm strainer, which resulted in single cells. Flow cytometry was used to sort these PMCs expressing GFP from the suspension.
In the treatment group, after 1 week of intraperitoneal injection of PDS, Nestin-GFP + PMCs (2×10 6 cells) were suspended in 2.5 ml of 4.25% PDS and injected intraperitoneally. At 28 days of injection, the mice were sacri ced, and the PDE, parietal peritoneum and omentum were collected.

Culture of PMCs from PDE and PMC lines
Human PMCs (HPMCs) harvested from PDE from patients with ESRD who underwent placement of PD catheters for less than 1 month were used for the culture. PDE was drained into a 50 ml centrifuge tube and centrifuged at 1000 rpm for 5 min. Cell pellets were suspended in Dulbecco's modi ed Eagle's medium/F12 (DMEM/F12, Gibco, NY, USA) with 10% fetal bovine serum (FBS, Gibco, NY, USA) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin, Gibco, NY, USA). Then, suspended cells were seeded into a 25-cm 2 culture ask (Corning, Armonk, NY, USA).
Several antibodies were used to examine every batch of initially isolated mesothelial cells to ensure that they were positive for the mesothelial markers cytokeratin 18 (CK 18, Abcam, Cambridge, MA, USA) and vimentin (Abcam, Cambridge, MA, USA). HPMCs were used at passages 3-5. The HPMC line (HMrSV5) was cultured in the same medium mentioned above.
The HPMCs were divided into four groups: a normal control group, with a normal glucose concentration (17.5 mM); a group treated with transforming growth factor β1 (TGF-β1, MCE, NJ, UAS) for varying durations (6, 12, 24 and 48 h) to identify the optimal time, after which the most effective concentrations were determined (1, 5, and 10 nM); and a group with AAV-RNAi-Vector and AAV-RNAi-IL-33 infection. At the end of the treatments, the cells were collected and subjected to western blot analyses and immuno uorescence staining.

Immuno uorescence
Histology and immuno uorescence staining of 4-μm para n sections from the anterior or posterior peritoneal tissues were performed as described previously [21]. For identi cation of the HPMCs, the cells Hematoxylin-eosin staining and Masson's staining.
The parietal peritoneum specimens from the 3 groups of mice were soaked in paraformaldehyde solution and then prepared for hematoxylin-eosin and Masson's staining as previously described [22].

Western blot analysis
Sample collection and immunoblotting were performed as previously described [21]. Total RNA from the peritoneum samples collected from different week-old Nestin-GFP mice was extracted using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) according to the manufacturer's instructions. Real-time PCR for Nestin and β-actin mRNA expression was performed using the FastStart Universal SYBR Green Master (ROX) kit (Roche, Basel, Switzerland) with an ABI Prism 7900HT Sequence Detection System (Life Technology, Carlsbad, CA, USA) as previously described [21].
Cytokine assays IL-33 concentrations were determined in cell culture supernatants and PDS by ELISAs following the manufacturer's instructions (Elabscience Biotechnology, Wuhan, China).

Statistical analysis
The results are presented as the mean ± SD for continuous variables. Comparisons were made using analysis of variance followed by Tukey's post hoc test. SPSS 20.0 (SPSS, Inc., Chicago, IL, USA) was used to analyze the data. A p-value < 0.05 was considered statistically signi cant.

Results
Identi cation of cells isolated from PDE as HPMCs PDE was derived from 4 uremic patients who underwent peritoneal dialysis therapy in one month. Among these 4 patients, 1 had gout, 1 had chronic glomerulonephritis, and 2 showed hypertensive nephropathy.
In PDE-derived cultures, PMCs detached from the PDE generally grew well in DMEM/F12 medium containing 10% FBS. Suspended cells adhered to the dish within 1 day of seeding. Active proliferation occurred if the cell number was su cient for the neighboring adherent cells to be in contact with each other. A con uent monolayer was reached at approximately 1 week, comprising passage 1. Cells isolated from the PDE presented a stellate or fusiform appearance with good transparency prior to con uency ( Figure 1A) and developed a typical cobblestone appearance after 3 passages ( Figure 1B). The phenotype of the PMCs was con rmed by immuno uorescence staining. The immuno uorescence study revealed that a proportion of the cells derived from the PDE showed positive staining in their cytoplasm with both the anti-vimentin and anti-CK 18 antibodies ( Figure 1C). PDE-derived HPMCs could be passaged for more than 20 generations.
Nestin is expressed in the PMCs and the peritoneum As a representative of Nestin expression, GFP uorescence was observed in the peritoneum of Nestin-GFP transgenic mice via microscopic examination ( Figure 1D). Nestin was expressed on the HPMCs derived from the PDE ( Figure 1E) and HMrSV5, as shown by immuno uorescence ( Figure 1F). The Nestin mRNA levels were also observed using RT-PCR analyses of the peritoneum of 1-, 2-, 4-, and 12-week-old mice ( Figure 1G). Thus, Nestin is continuously present in neonates and adults in the peritoneum.

Effects of differentTGF-β1concentrations and durations on IL-33 secretion in HPMCs
HPMCs were stimulated by different concentrations of TGF-β1 (0, 1, 5, and 10 nM) for 24 h. The levels of IL-33 increased signi cantly when HPMCs (both from PDE and HMrSV5) were stimulated with 5 nM and 10 nM ( Figure 5A, B) compared with those of the control group. Then, HPMCs were stimulated with TGF-β1 for different durations (6, 12, 24 and 48 h) at a concentration of 5 nM. The levels of IL-33 showed the strongest changes in HPMCs stimulated with TGF-β1 at 5 nM for 48 h compared with those of the control group ( Figure 5C, D). Thus, a concentration of 5 nM and a duration of 48 h were selected for further use.

Downregulation of IL-33 expressionincreases TGF-β1-induced EMT of HPMCs
After HPMCs were stimulated with 5 ng/ml TGF-β1 for 48 h, western blot ( Figure 6) and immuno uorescence analyses ( Figure 7A, B and C) showed that the levels of α-SMA and Col-I were signi cantly increased compared with those of the control group. Furthermore, in the AAV-RNAi-IL-33 group, α-SMA and Col-I were decreased signi cantly compared with those of the AAV-RNAi-vector group. There were no signi cant changes in the levels of α-SMA and Col-I between the HPMCs incubated with TGF-β1 only and cells incubated with the AAV-RNAi-vector.
Downregulation of IL-33 expressionupregulates TGF-β1 induced IL-33/ST2 signaling in HPMCs HPMCs from PDE were infected with AAV-RNAi-vector and AAV-RNAi-IL-33 and then stimulated with TGF-β1 at 5 ng/ml for 48 h. Compared to the control group, the levels of IL-33 ( Figure 6, 7B) and ST-2 ( Figure  6) were signi cantly increased following TGF-β1 stimulation. There were no signi cant changes in the levels of IL-33 and ST-2 between the HPMCs incubated with TGF-β1 only and the cells incubated with the AAV-RNAi-vector. AAV-RNAi-IL-33 resulted in a stronger decrease in these levels than AAV-RNAi-vector ( Figure 6).

Discussion
This study provided the rst evidence that Nestin + cells isolated from the peritoneum have a clear Nestin is widely known as a marker of neural stem cells [12]. More importantly, this protein labels adult proliferating and stem/progenitor cell populations, indicating that Nestin is a speci c marker of multifunctional progenitor cells [12]. Rats PMCs showed strong labeling with this antibody [10]. Similarly, in the present study, both HPMCs and mouse peritoneum strongly expressed Nestin, indicating that these cells represent a 'young', not entirely differentiated cell population. We also con rmed that the cultured PMCs from the PDE grew well even after 20 passages, which implied that these cells possessed a strong proliferative ability. Nestin expression was upregulated in cardiac, [15] renal [23] and lung ( [24]) brosis.
However, few studies have investigated the effects of Nestin + PMCs on PF. Furthermore, we prospectively isolated Nestin + cells from the peritoneum based on the GFP uorescence intensity of Nestin-GFP mice.
Excitingly, Nestin + cells could suppress the expression of Col I and α-SMA and reduce the functional impairments of the peritoneal membrane in mice with PF, eventually leading to delayed PF progression.
Therefore, Nestin + cells isolated from the peritoneum can exert a protective effect on high-glucose PDSinduced PF.
IL-33 is a major cytokine in the modulation of brosis [25]. Furthermore, recent reports have con rmed that IL-33 promotes ST-2-dependent organ tissue brosis, such as lung [26], liver [27], and kidney brosis [28]. Conversely, IL-33 prevented global myocardial periarteriolar brosis in pressure-overloaded left ventricles [29]. However, whether and how this cytokine can modulate PF remains unclear. To address the possible mechanism of PF induced by PDS, we detected the levels of IL-33 in different groups and found that secretion of IL-33 was obviously increased in mice with PF, indicating that IL-33 positively correlates with the extent of PF. Furthermore, Nestin + cells could reduce the secretion of IL-33, eventually leading to the delayed progression of PF. In addition, downregulation of Nestin expression decreased TGF-β1induced EMT in HPMCs. Therefore, Nestin + PMCs could protect against high-glucose PDS-induced PF by depressing IL-33 secretion.

Consent for publication
Not applicable.

Availability of data and materials
The datasets generated for this study are available on request from the corresponding author. Figure 1 1A. Representative light microscopic features of cells isolated from PDE. Cells presented a stellate or fusiform appearance with good transparency at passage 0. Figure 1B. Cells isolated from PDE showed a typical cobblestone appearance at passage 3. Figure 1C.   Figure 2A, B. The thickness of the submesothelial compact zone increased along with its cellularity until day 28 in PF mice injected with PDS. Cell density and thickening of the zone were suppressed in mice treated with Nestin+ cells. *P < 0.05, **P < 0.01, ***P < 0.001 versus PF mice injected with PDS. Abbreviations: HE hematoxylin-eosin staining; Con control; PDS peritoneal dialysis solution; CK 18 cytokeratin 18; PF peritoneal brosis; PMC peritoneal mesothelial cells. of glucose from the dialysate and the transport rate of blood urea from the plasma were signi cantly higher in mice given the PDS only than in control mice, whereas these parameters were signi cantly improved in PDS-injected mice treated with Nestin+ cells. The cytokine IL-33 was signi cantly increased in the PDE of mice with PDS-induced PF (C). Treatment with Nestin+ cells signi cantly suppressed the secretion of IL-33 compared with treatment with PDS only. **P < 0.01, ***P < 0.001 versus PF mice injected with PDS. Abbreviations: PMC peritoneal mesothelial cells; Con control; PDS peritoneal dialysis solution; PF peritoneal brosis.

Figure 6
Downregulation of IL-33 expression decreased the TGF-β1-induced EMT of HPMCs, as shown by western blots. The levels of IL-33 decreased signi cantly after AAV-RNAi-IL-33 interference, as shown by western blots. The levels of α-SMA, Col-I and ST-2 were signi cantly increased following TGF-β1 stimulation compared with those of the control group. Furthermore, in the AAV-RNAi-IL-33 group, these proteins were decreased signi cantly compared with those of the AAV-RNAi-vector group. *P < 0.05 versus the control group, ▲P < 0.05 versus the AAV-RNAi-control group, △P < 0.05 versus the TGF-β1 group. Abbreviations: HPMCs human peritoneal mesothelial cells; TGF-β1, transforming growth factor β1; Col I, collagen I; α-SMA, α-smooth muscle actin; Con, control; AAV, adeno-associated virus. Downregulation of IL-33 expression increased the levels of Col I and α-SMA and IL-33 secretion in HPMCs. The levels of α-SMA and Col-I were signi cantly increased following TGF-β1 stimulation compared with those of the control group. Furthermore, in the AAV-RNAi-IL-33 group, these proteins were decreased signi cantly compared with those of the AAV-RNAi-vector group. Abbreviations: TGF-β1,