Mesenchymal stem cells alleviate renal injury of lupus nephritis mice by reconstructing the immune microenvironment of renal tissue

Abstract

BACKGROUND

Previous studies have found that umbilical cord mesenchymal stem cells (UCMSCs) have the potential to treat refractory lupus nephritis (LN), but their mechanisms have not been elucidated. One pathological feature of LN is the infiltration of multiple immune cells into renal tissue, forming a local immune microenvironment, which leads to renal pathological damage.

AIM

To demonstrate the changes in the number and proportion of locally infiltrating immune cells in the kidneys of LN mice after UCMSCs treatment.

METHODS

We analyzed the number and location of different immune cell subsets infiltrating in renal tissues using immunohistochemistry and multiplex immunofluorescence; and analyzed the number of regulatory T (Treg) cells in the mouse spleen using flow cytometry. Finally, we conducted in vitro co-culture of UCMSCs and peripheral blood mononuclear cells to detect the effects of UCMSCs on the differentiation of T helper 1 (Th1), Th17, and Treg cells.

RESULTS

In our study, UCMSCs can improve renal function, reduce proteinuria, alleviate renal pathological damage, and inhibit immune cell infiltration into renal tissue. Multiple immunofluorescence staining of renal tissue showed that after treatment with UCMSCs, the number of neutrophils, B cells, T cells, and macrophages decreased, but the proportion of Treg in T cells was upregulated, while the proportion of Th17 decreased. The proportion of M2 cells in macrophages increases, while the proportion of M1 cells decreases. In addition to the change in the proportion of infiltrating immune cells, we also found that infiltrating immune cells mainly gather around renal blood vessels, and there is also immune cell infiltration in the glomerulus and renal interstitium, indicating that immune cells may mainly infiltrate renal tissue through vascular endothelium. We further found that the proportion of Treg cells in the spleen of mice increased after UCMSCs treatment. In vitro experiments, we co-cultured UCMSCs with peripheral blood mononuclear cells and found that UCMSCs have inhibitory effects on Th1 and Th17 differentiation, as well as promoting Treg differentiation.

CONCLUSION

Our study found that after treatment with UCMSCs, the number and proportion of immune cells infiltrating renal tissue changed. It is suggested that UCMSCs may improve LN by reconstructing the local immune microenvironment of the kidneys.

Key Words: Lupus nephritis; Umbilical cord mesenchymal stem cells; Regulatory T; T helper 17; Macrophages

Core Tip: In our study, multiple immunofluorescence staining in renal tissue of lupus nephritis mice showed that after treatment with umbilical cord mesenchymal stem cells (UCMSCs), the proportion of regulatory T (Treg) was upregulated, while T helper 17 (Th17) decreased. The proportion of M2 cells increases, while M1 cells decreases. We further found that the proportion of Treg cells in the spleen of mice increased after UCMSCs treatment. In vitro experiments, we co-cultured UCMSCs with peripheral blood mononuclear cells and found that UCMSCs have inhibitory effects on Th1 and Th17 differentiation, as well as promoting Treg differentiation. It is suggested that UCMSCs may improve lupus nephritis by reconstructing the local immune microenvironment of the kidneys.

INTRODUCTION

Lupus nephritis (LN) occurs in 50% of patients with systemic lupus erythematosus (SLE)[1]. Kidney involvement in SLE has been associated with increased mortality, especially for patients who progress to kidney failure[2]. Despite the continuing development of immunomodulatory agents, the prognosis of LN has not improved substantially in the past decade, with end-stage kidney disease still developing in 22% of patients[3]. Several clinical studies have suggested that mesenchymal stem cells (MSCs) have therapeutic effects on LN[4-6]. However, the underlying molecular mechanisms of these effects are still unclear.

LN is a complicated autoimmune disease marked by an imbalance of immunological reactivity and immune tolerance. In addition to systemic immune cells, local immune cells infiltrating the kidneys are crucial in LN[7,8]. The infiltrating immune cells are mainly composed of macrophages, T and B lymphocytes, which form the immune microenvironment of LN in the local renal tissue. Organ histology and extensive transcriptome analysis of LN showed that immune cell infiltration is associated with renal dysfunction and poor prognosis in renal biopsy samples[9-11]. MSCs have the potential to treat autoimmune diseases by regulating overactive immune responses to restore immune tolerance[12,13]. Previous studies have found that umbilical cord MSCs (UCMSCs) have inhibitory effects on the proliferation of various immune cells in vitro[14,15]. However, it is still unclear how UCMSCs exerts immunomodulatory effects on immune cells infiltrating in renal tissue of LN. Therefore, this study will take MRL/Ipr mice as the research object, analyze the changes in renal tissue infiltrating immune cells, and clarify the regulation of renal local immune microenvironment by UCMSCs treatment.

MATERIALS AND METHODS

Establishment of animal models

Ten-week-old MRL/Ipr mice were used as experimental subjects in this study (Shanghai Slake Laboratory Animal Co., Ltd., Shanghai, China). All animal experiments are carried out in accordance with the license of the Yunnan Province Science and Technology Office (Kunming, Yunnan Province, China), and has obtained approval from the Animal Ethics Committee of Yan’an Hospital Affiliated to Kunming Medical University. All animals were housed in a specific pathogen-free animal room with controlled humidity (50% ± 10%) and free access to water and food under a 12-hour light/dark cycle. All experiments are accord with the Guidelines for Ethical Conductions in the Care and Use of Animals. Every effort was made to minimize animal stress.

Preparation of human UCMSCs

The central laboratory of Yan’an Hospital Affiliated to Kunming Medical University prepared and provided UCMSCs, which is the National Stem Cell Clinical Research Institute in China. Flow cytometric analysis was used to identify cell phenotypes. Major histocompatibility complex class II molecule DR haplotype (HLA-DR) and CD73, CD29, CD45, CD105, CD90, CD79, CD14, and CD34 antibodies for labeling the cell surface of UCMSCs were acquired from BD Bioscience (CA, United States), along with their isotype controls. Furthermore, our research indicated that these UCMSCs may develop into chondrocytes, adipocytes, and osteoblasts (Figure 1).

Figure 1 Umbilical cord mesenchymal stem cells morphology and identification. A: Umbilical cord mesenchymal stem cells (UCMSCs) morphology is displayed at 50 × magnification; B: UCMSCs morphology is displayed at 200 × magnification; C and D: The effective differentiation of UCMSCs into osteoblasts was confirmed by alizarin red staining. At a magnification of 100 × representative pictures of the control group (C) and induction group (D) were captured; E and F: Oil Red O staining was used to successfully differentiate UCMSCs into adipocytes. At a magnification of 100 ×, representative pictures of the control group (E) and induction group (F) were captured; G and H: Alcian blue staining was used to identify the chondrocytes that were successfully differentiated from UCMSCs. At 200 × magnification, representative pictures of the control group (G) and induction group (H) were captured; I-L: Using flow cytometry, the surface markers of UCMSCs, such as CD90, CD105, CD73, CD45, CD34, CD11b, CD19, and HLA-DR, were examined. Of them, more than 99% of the cells were positive for CD90, CD105, and CD73. The other genes’ expression was nearly negligible.

Experimental group

Two groups of MRL/Ipr mice were randomly assigned: The LN group and the UCMSCs group. There were 16 mice altogether, 8 mice in each group. Age-matched C57BL/6 mice served as the normal group (Figure 2A). Every two weeks commencing on the 10^th week, 2 × 10⁵/100 μL of UCMSCs were given via the tail vein to the UCMSCs group, these mice received a total of 3 injections. The normal group received an equivalent volume of normal saline. The mice were sacrificed on the 16^th week. After gathering blood, the serum was separated by centrifugation and kept at -80 °C. Kidneys were removed in order to do pathological investigations and transmission electron microscopy. For western blotting analysis, kidney tissue was promptly frozen at -80 °C. Kidney tissue was preserved in 10% formalin, and for pathological analyses.

Figure 2 Comparing the results of biochemical measurements changes to renal function, serum dsDNA and proteinuria levels. A: Schematic illustration of umbilical cord mesenchymal stem cells treatment in MRL/Lpr mice; B: Serum dsDNA level (U/mL); C: Urinary albumin/urinary creatinine ratio level (mg/mmoL); D: Blood urea nitrogen level (mmol/L); E: Serum creatinine level (μmol/L). All data was expressed as mean ± SEM, ^aP < 0.05 vs lupus nephritis group, ^bP < 0.01 vs lupus nephritis group, ^cP < 0.001 vs lupus nephritis group. LN: Lupus nephritis; UCMSCs: Umbilical cord mesenchymal stem cells; uALB: Urinary albumin; uCr: Urinary creatinine.

Biochemical measurements

Serum creatinine and blood urea nitrogen levels were used to assess renal function. The urinary albumin/creatinine ratio and renal function were assessed using a biochemical device (BECKMAN AU5421, Japan). We used an enzyme-linked immunosorbent assay kit (BioVendor, Brno, Czech Republic) to measure the amounts of serum dsDNA.

Analysis of hematoxylin and eosin, periodic acid-Schiff, and Masson’s trichrome staining and electron microscopy in renal tissue

Renal specimens embedded in paraffin were sliced into 3-μm-thick slices, stained with hematoxylin and eosin, periodic acid-Schiff and Masson’s trichrome, and examined using an Olympus BX-43 microscope. Tiny fragments of renal cortex were embedded in Araldite 618 (Sigma Aldrich, 10951, MO, United States) after being pretreated in 3.5% glutaraldehyde, fixed in 1% osmic acid, and dehydrated using a gradient of acetone and alcohol. Ultrathin slices were studied using a transmission electron microscope (JEM-1011, Japan) after being counterstained with uranyl acetate and lead citrate.

Immunohistochemical analysis

Paraffin-embedded kidney tissue was cut into 4-microns thick sections, followed by antigen removal in hot citrate buffer for 20 minutes. After that, the antigen was blocked in TBST containing 5% bovine serum albumin for 1 hour, followed by an overnight incubation with the first antibody. Tissue sections were incubated at 4 °C with CD3 (Abcam, 1:1000, United Kingdom), CD68 (CST, 1:800, MA, United States) and B220 (eBioscience, 1:50, CA, United States). Subsequently, the sections were incubated with HRP-labeled second antibodies for 30 minutes, followed by DABxian coloration and hematoxylin staining, and then viewed under a microscope after sealing the sections with neutral resin.

Immunofluorescence analysis

The formalin-fixed, paraffin-embedded blocks of renal tissues were cut into 4 μm-thick sections. The slides were heated for at least 1 hour in a dry oven at 60 °C. After dewaxing and antigen retrieval, the sections were incubated with antibodies. The slides were incubated with primary antibodies against CD4 (Abcam, 1:1000, United Kingdom), CD25 (CST, 1:200, MA, United States), FOXP3 (CST, 1:200, MA, United States), interleukin (IL)-17A (Abcam, 1:500, United Kingdom), F4/80 (Abcam, 1:1000, United Kingdom), inducible nitric oxide synthase (Abcam, 1:2000, United Kingdom), CD206 (CST, 1:8000, MA, United States) for 1 hour and then analysed using polymer HRP immunoglobulin G for 10 minutes. Then, the slides were incubated with the next primary antibodies against for 1 hour and visualized using polymer HRP immunoglobulin G for 10 minutes. The same procedure was repeated for staining with and observed under a Zeiss Scanning Microscope (Zeiss AXIOSCAN).

UCMSCs and peripheral blood mononuclear cell coculture

Peripheral blood mononuclear cells (PBMCs) isolated from the blood of healthy volunteers were resuspended at 2 × 10⁷/mL in phosphate buffered saline (PBS). The PBMC were resuspended at 10⁶ cells/mL in RPMI 1640 culture medium. To evaluate the effects of UCMSCs on regulatory T (Treg) differentiation. UCMSCs (2 × 10⁵) were seeded into 12-well culture plates, 1.0 × 10⁵ PBMC were added to each well, and the cells were cocultured for 72 hours at 37 °C. PBMC cultured alone (without UCMSCs) served as controls. After 3 days of co-culture, the suspended PBMC were collected for flow cytometry analysis. To evaluate the impact of UCMSCs on the proliferation of lymphocytes, the PBMC were labeled with Carboxy Fluorescein Succinimidyl Ester (CFSE) for 30 minutes at 37 °C and washed twice with cold PBS, then cultured with phytohemagglutinin-A. The labeled PBMC were seeded into 12-well plates at 10⁶ cells/well. At the same time, UCMSCs (2 × 10⁵) were added to each well for co-culture, after 3 days, the suspended PBMC were collected for flowmetry analysis.

Flow cytometry

The suspended PBMC were incubated with antibodies against T helper 1 (Th1) (PerCP-CD3, FITC-CD8, PE-IFN-γ), Th17 (PerCP-CD3, FITC-CD8, PE-IL-17) and Treg (PerCP-CD4, FITC-CD127, PE-CD25). After the organ tissue was harvested from the mice, the spleens were placed in ice-cold PBS, passed through 100 μm cell strainers to generate single-cell suspensions, which were subsequently centrifuged for 5 minutes at 1200 rpm. Then, the cells were collected and resuspended in PBS to stain for Treg (FITC-CD4, APC-CD25, PE-FOXP3). The stained cells and CFSE-labelled cells were assessed by a flow cytometer (BD Bioscience, CA, United States), and the data were analysed with Flow Jo software. All experiments were repeated three times.

Statistical analysis

The statistical analyses were performed with GraphPad Prism 8.0. The measurement data are presented as the mean ± SD or SEM, One-way analysis of variance (ANOVA) was used for comparisons among multiple groups (^aP < 0.05; ^bP < 0.01; ^cP < 0.001).

RESULTS

UCMSCs therapy can enhance renal function and decrease proteinuria in MRL/Ipr mice

The mice in the LN group demonstrated significant increases in serum creatinine levels, urine albumin/creatinine ratios, and dsDNA levels when compared to those in the normal group. These findings suggest that the LN models were successful. Compared with the LN group, the UCMSCs group had decreased dsDNA levels, urinary/albumin creatinine ratio, and serum creatinine levels. The blood urea nitrogen levels in the UCMSCs group showed a declining trend when compared with the LN group. These findings demonstrated that UCMSCs therapy improved renal function in MRL/Ipr mice and decreased ds-DNA levels and proteinuria (Figure 2).

Renal pathological damage in MRL/Ipr mice can be lessened by UCMSCs therapy

Compared with the normal group, hematoxylin and eosin staining revealed that the LN group had marked glomerular sclerosis, and greater inflammatory cell infiltration. The glomeruli in the LN group had a markedly increased mesangial matrix, as shown by periodic acid-Schiff staining. In the LN group, there was clear glomerular fibrosis as shown by Masson’s trichrome staining. Transmission electron microscopy showed that the LN group had endothelial cell enlargement, evident foot process fusion and thicker glomerular basement membrane. The pathological alterations in the kidney of the LN group further suggested that the LN model in MRL/Ipr mice was successful. Pathological damage to the kidney in the UCMSCs group was noticeably improved, indicating that UCMSCs could alleviate renal pathological injury in LN mice. We found that in the LN group, many immune cells were present in the renal tissue around the blood vessels, glomerulus and renal interstitium. Therefore, we performed immunohistochemical staining for CD3, CD68 and B220 in mouse kidney tissue and found that the infiltrating immune cells in the LN model group mainly included T cells, macrophages, and B cells. However, after treatment with UCMSCs, the infiltrating T cells, macrophages, and B cells were significantly reduced (Figure 3).

Figure 3 Results of renal tissue pathology and electron microscopy. A: Pathological and electron microscopy results of renal tissue. Hematoxylin and eosin staining (1^st line, 400 ×), periodic acid-Schiff staining (2^nd line, 400 ×), Masson’s trichrome staining (3^rd line, 400 ×) and electron microscopy (4^th line, 12000 ×). Normal group (1^st column), lupus nephritis group (2^nd column), and umbilical cord mesenchymal stem cells group (3^rd column) were used; B: Immunohistochemical staining and statistical results of CD3, CD68 and B220 in mice kidney tissue. All data was expressed as mean ± SEM, ^bP < 0.01 vs lupus nephritis group, ^cP < 0.001 vs lupus nephritis group. LN: Lupus nephritis; UCMSCs: Umbilical cord mesenchymal stem cells; HE: Hematoxylin and eosin; PAS: Periodic acid-Schiff.

UCMSCs therapy increases Treg and decreases Th17 numbers in the kidney of MRL/Ipr mice

To analyze the types of T cells infiltrating renal tissue, we used multiplex immunofluorescence detection to determine the quantity and location of Treg and Th17 in mouse renal tissue. We found that after treatment with UCMSCs, the total number of CD4+ immune cells decreased, but the number of Tregs (CD4+CD25+foxp3+) around the blood vessels significantly increased, while the number of Th17 cells were significantly inhibited. These results suggested that UCMSCs may exert local immunosuppressive effects on the kidneys by promoting Treg infiltration and inhibiting Th17 cells (Figure 4A-D). We found that Treg cells mainly infiltrate and increase around blood vessels, so we considered that infiltrating immune cells may enter renal tissue through vascular endothelium. Moreover, we found that the number of Treg in the spleens of mice in the LN group was lower than that in the normal group, while the number of Treg increased after UCMSCs treatment (Figure 4E and F).

Figure 4 Changes in immune cells in the spleens and kidney tissues of MRL/Ipr mice. A-D: Multiple immunofluorescence and quantitative analysis of regulatory T (Treg) (CD4+CD25+Foxp3+) (A and C); multiple immunofluorescence and quantitative analysis of T helper 17 (IL-17A+CD4+) (B and D); E: Flow cytometric analysis of the percentages of Treg (CD4+CD25+Foxp3+) in the spleen; F: Quantitative analysis of the percentages of Treg in the spleen. All data were expressed as mean ± SEM, ^aP < 0.05 vs lupus nephritis group, ^bP < 0.01 vs lupus nephritis group. LN: Lupus nephritis; UCMSCs: Umbilical cord mesenchymal stem cells; IL: Interleukin.

UCMSCs therapy increases M2 and decreases M1 numbers in the kidney of MRL/Ipr mice

To analyze the types of macrophages infiltrating renal tissue, we used multiplex immunofluorescence detection to determine the quantity and location of M1 and M2 in mice renal tissue. We found that after treatment with UCMSCs, the total number of F4/80+ macrophages decreased, but the number of M2 cells around the blood vessels showed an increasing trend, while the number of M1 cells showed an inhibitory trend. These results suggest that UCMSCs may exert effects on the local kidney by promoting M2 while inhibiting M1 infiltration (Figure 5).

Figure 5 Macrophage infiltration in mice kidney tissue. A: Multiplex immunofluorescence and quantitative analysis of M1 macrophages (F4/80+ iNOS+); B: Multiplex immunofluorescence and quantitative analysis of M2 macrophages (F4/80+ CD206+). LN: Lupus nephritis; UCMSCs: Umbilical cord mesenchymal stem cells; iNOS: Inducible nitric oxide synthase.

UCMSCs inhibit Th1 and Th17 proliferation and increase Treg numbers in vitro

In vitro experiments, results indicate that UCMSCs can play an immunosuppressive role by promoting the proliferation of Treg and inhibiting the Th17. After co-culture of UCMSCs and PBMC, flow cytometry revealed that the proportion of CFSE-labelled lymphocytes was significantly decreased, and flow cytometry analysis revealed that the number of Treg cells increased significantly, while the number of Th1 and Th17 cells decreased significantly (Figure 6).

Figure 6 Changes in the percentages of immune cells after coculture of umbilical cord mesenchymal stem cells and peripheral blood mononuclear cells. A: Flow cytometry was used to analyse the proliferation of Carboxy Fluorescein Succinimidyl Ester-labelled lymphocytes and changes in the percentages of T helper 1 (TH1), TH17, and regulatory T cells after coculture of umbilical cord mesenchymal stem cells and peripheral blood mononuclear cell; B: Quantitative analysis of the percentages of TH1 cells, TH17 cells and regulatory T after co-culture of umbilical cord mesenchymal stem cells and peripheral blood mononuclear cell. All data was expressed as mean ± SEM, ^aP < 0.05 vs lupus nephritis group, ^cP < 0.001 vs lupus nephritis group. TH1: T helper 1; Treg: Regulatory T; IFN: Interferon; IL: Interleukin; CFSE: Carboxy Fluorescein Succinimidyl Ester; UCMSCs: Umbilical cord mesenchymal stem cells.

DISCUSSION

In this study, we found that UCMSCs had the potential to treat LN, which was consistent with other studies[16]. Numerous previous studies showed that multiple types of immune cells infiltrated in the renal tissue of patients with LN, forming a local immune microenvironment[17]. Our study found that after treatment with UCMSCs, the infiltration of T cells, B cells, and macrophages in the renal tissue of LN mice decreased. We found that after UCMSCs treatment, the total number of CD4+ T cells decreased, but the proportion of Treg increased, while the proportion of Th17 decreased. We also found that macrophages in the renal tissue were significantly inhibited after UCMSCs treatment. Moreover, the proportion of M1 cells showed a downward trend, while the proportion of M2 cells showed an upward trend. These findings indicated that UCMSCs treatment could regulate the quantity and proportion of locally infiltrated cells in the renal tissue and alleviated renal pathological damage by reconstructing the renal microenvironment.

Treg played a key role in the regulation of immune tolerance, characterized by their immunosuppressive function. In addition to inhibiting the proliferation of effector T cells, Treg maintained tolerance to self-antigens and regulated immunity through direct cell-to-cell contact, regulation of antigen presenting cells, and secretion of immunosuppressive cytokines[18]. Th17 secreted pro-inflammatory cytokines such as IL-17A, IL-17F, IL-21 and IL-22, which promoted the inflammatory response and exacerbated tissue damage[19]. Patients with active SLE were characterized by a deficiency of Treg in the peripheral blood, an elevated level of Th17, and a decreased Treg/Th17 ratio[20]. The imbalance of Treg/Th17 seemed to be an important determinant of SLE disease activity and the degree of inflammation[21]. Mice lacking Treg exhibited characteristic symptoms of producing a large amount of dsDNA[22]. The number of Treg in the blood of LN patients was significantly lower than that in healthy individuals, and there was an inverse relationship between the number of Treg and clinical disease activity[23]. Another study showed that after glucocorticoid treatment in LN patients, the infiltration of Treg in the renal tissue increased[24]. Previous reports found the correlation between the level of Th17 cells and the disease activity of LN[25]. Some studies showed that the level of Th17 in the peripheral blood of LN patients was elevated, which was related to disease activity[26]. Our study found that UCMSCs treatment inhibited the number of infiltrated CD4+ cells in the renal tissue, and also exerted local immunosuppressive effects by increasing the proportion of Treg and inhibited the proportion of Th17. Li et al[27] also found, through flow cytometry detection of Treg and Th17 cells in the renal tissue, that UCMSCs had the effect of inhibiting Th17 cells and promoting Treg cells, which was similar to our results, although the research methods were different.

Macrophages were one of the main infiltrating cells in the kidneys of patients with LN. Different macrophage subtypes had different effects on the development and progression of LN. An abnormal M1/M2 balance in the immune microenvironment could trigger or exacerbate the immune response by affecting the inflammatory reaction[28]. M1 macrophages produced pro-inflammatory mediators including tumor necrosis factor α, IL-6, and IL-1β, while M2 macrophages produced anti-inflammatory factors, including IL-10. In addition, M2 macrophages contributed to tissue remodeling[29]. Previous studies showed that the imbalance of M1/M2 macrophage polarization was closely related to the pathogenesis of SLE, and regulating the polarization direction of macrophages could improve autoimmune diseases[30,31]. Our study found that UCMSCs treatment significantly inhibited the number of infiltrated macrophages in the renal tissue, and the proportion of M2 showed an upward trend, thus promoting the repair of renal tissue. Scholars found that UCMSCs-derived exosomes could promote M2 polarization in vitro and increase the number of M2 cells and inhibit M1 cells in the spleen of lupus mice[32]. Although they did not study the macrophages in the renal tissue, their findings indicated that UCMSCs-derived exosomes had the function of regulating macrophage polarization.

This study also found that the infiltrating immune cells in the renal tissue of LN mice were mainly located around blood vessels, and also were distributed in the glomeruli and renal interstitium. This indicated that the infiltrating immune cells in the kidneys of LN mice migrated from blood vessels to the kidneys through the chemokine signaling axis. Therefore, we used flow cytometry to detect Treg in the spleen tissue of mice and found that after UCMSCs treatment, the number of Treg in the spleen of LN mice increased. Sun et al[32] found that after intravenous injection of UCMSCs-derived exosomes in MRL/Lpr mice, the infiltration of Treg cells in the spleen increased. Chemokines played a role in the mechanism of renal immune cell infiltration. Researchers showed that exosomes from MSCs promoted the polarization of inflammatory macrophages and recruited Treg cells by secreting cysteine-cysteine motif chemokine ligand 20[33]. Exploring the mechanism of chemokines on renal tissue immune cell infiltration will also be of great significance for the treatment of LN.

A case report found that after autologous hematopoietic stem cell and MSCs transplantation, the clinical symptoms of SLE patients were relieved, and the SLEDAI score decreased[34]. In addition, the number of Treg cells in PBMC increased after transplantation. Our in vitro experiment, we co-cultured UCMSCs with PBMC and found that UCMSCs could exert lymphocyte-inhibitory effects in vitro by increasing the number of Treg and reducing the number of Th1 and Th17 cells. The results are consistent with our in vivo experiments, indicating that UCMSCs may improve LN by regulating the proportion and quantity of immune cells.

However, the mechanisms underlying UCMSC-mediated regulation of Treg and M2 macrophage polarization remain to be elucidated, which was a limitation of this study. Some studies showed that MSCs-derived exosomes can improve the functional stability of Treg by activating autophagy and the signal transducer and activation of transcription 5 signaling pathway[35]. Other researchers showed that MSCs were involved in T-cell activation and Treg differentiation through mitochondrial transfer[36]. Other studies have found that the IL4I1 derived from UCMSCs plays a crucial role in reshaping the immune microenvironment of LN mice[37]. Studies have demonstrated that IL4I1 has the ability to drive naive T cells to differentiate into induced Treg cells[38]. Some scholars have reported that IL-27 upregulates indoleamine 2,3-dioxygenase in MSCs through the Janus kinase 1-signal transducer and activator of transcription 1 signaling pathway, promoting the transformation of MSCs into an anti-inflammatory (MSC2) phenotype. These indoleamine 2,3-dioxygenase-expressing MSC2 cells produce metabolites of tryptophan - kynurenine and kynurenic acid, which bind to the aryl hydrocarbon receptors within the cells. This interaction stimulates the increase of anti-inflammatory factor tumor necrosis factor-alpha-stimulated gene/protein-6 and induces the differentiation of Treg cells. Notably, in a mouse LN model, IL-27-conditioned MSC2 has a superior therapeutic effect compared to conventional MSCs[39]. The research on the mechanism of MSCs regulate local immune cell in renal tissue may potentially facilitate the development of new types of MSCs with enhanced immune regulatory functions for the treatment of LN, which is of great significance for future clinical applications.

In the future, the specific mechanism of how UCMSCs reconstruct the renal tissue immune microenvironment can be clarified through single cell sequencing and spatial transcriptome sequencing of renal tissue. Then, discover new targets for regulating immune cells, and enhance the immune regulatory function of MSCs or their derived exosomes through engineered modification. By conducting in vivo experiments to confirm its therapeutic effect and safety on the lupus mouse model, a foundation was laid for subsequent clinical research. UCMSCs have great potential for application in the treatment of LN, but there are still some challenges in clinical practice, such as cell quality control and standardization, as well as the evaluation of long-term safety and efficacy, which require further research.

CONCLUSION

This study found that UCMSCs treatment could significantly inhibit the infiltration of immune cells in the renal tissue. More interestingly, UCMSCs could change the proportion of cells in the local renal microenvironment. By upregulating the proportion of Treg cells and M2 cells, UCMSCs reconstructed the renal tissue immune microenvironment and exerted immunosuppressive and damage repair effects locally in the kidneys.

ACKNOWLEDGEMENTS

We would like to acknowledge all authors for their contributions to this article.