Abstract
Background
The efficacy of human umbilical cord mesenchymal stem cell (hUC-MSC) transplantation in treating systemic lupus erythematosus (SLE) has been confirmed by small-scale clinical trials. However, these trials focused on severe or refractory SLE, while few studies focused on mild SLE. Therefore, this study focused on the therapeutic effects of hUC-MSC transplantation in early-stage or mild MRL/lpr lupus model mice.
Methods
Commercially available hUC-MSCs were transplanted into 8-week-old MRL/lpr mice by tail vein injection. Flow cytometry was used to analyze B cells and their subsets in the peripheral blood. Further, plasma inflammatory factors, autoantibodies, and plasma biochemical indices were detected using protein chip technology and ELISA kits. In addition, pathological staining and immunofluorescence were performed to detect kidney injury in mice.
Results
hUC-MSC transplantation did not affect the mice’s body weight, and both middle and high dose hUC-MSC transplantation (MD and HD group) actually reduced spleen weight. hUC-MSC transplantation significantly decreased the proportion of plasmablasts (PB), IgG1− PB, IgG1+ PB, IgG1+ memory B (MB) cells, IgG1+ DN MB, and IgG1+ SP MB cells. The hUC-MSC transplantation had significantly reduced plasma levels of inflammatory factors, such as TNF-α, IFN-γ, IL-6, and IL-13. Pathological staining showed that the infiltration of glomerular inflammatory cells was significantly reduced and that the level of glomerular fibrosis was significantly alleviated in hUC-MSC-transplanted mice. Immunofluorescence assays showed that the deposition of IgG and IgM antibodies in the kidneys of hUC-MSC-transplanted mice was significantly lower than in the control.
Conclusion
hUC-MSC transplantation could inhibit the proliferation and differentiation of peripheral blood B cells in the early-stage of MRL/lpr mice, thereby alleviating the plasma inflammatory environment in mice, leading to kidney injury remission. The study provides a new and feasible strategy for SLE treatment.
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Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the aberrant activation of B cells and autoantibody production [1]. The pathogenic autoantibodies combine with the corresponding autoantigens; they are then deposited in the skin, joints, glomeruli, and other parts of the body leading to multiple organs and system damage (e.g., lupus nephritis; LN) [2,Blood sample collection and processing Blood sample collection was performed by cardiac puncture after anesthesia at 14 weeks old. The blood samples were collected with EDTA anticoagulant tube and centrifuged at 3000 rpm for 10 min to obtain the plasma, the precipitation is blood cells for flow cytometry (FCM) analysis, and the supernatant is plasma which was immediately stored at − 80 °C. After centrifugation of mice's peripheral blood, red blood cells were lysed using a lysis buffer (BD PharmLyse Lysis Buffer; BD Biosciences, San Diego, CA, USA) to obtain a single-cell suspension. The single-cell suspension was incubated with Purified anti-mouse CD16/32 Antibody (Catalog No. 158002, BioLegend, San Diego, CA, USA) to block nonspecific antibody binding. Subsequently, a cocktail of fluorescence-conjugated antibodies, including CD19, CD138, and PD-L2, was added for staining. Following staining, the cells were fixed with 4% PFA and analyzed using the FACS Celesta™ Flow Cytometer (BD Biosciences, San Diego, CA, USA). The analysis of B cell subsets and the gate scheme in flow cytometry were performed according to previous reports (Fig. 3A) [23, 24]. Furthermore, to evaluate cell surface PD-L1 expression, hUC-MSCs were stained with Alexa Fluor 647-conjugated PD-L1 antibody (Additional file 1: Fig. S1A). All flow cytometry data were analyzed using FlowJo 10.8.1 software (FlowJo, Ashland, OR, USA). For mice lymphocyte-derived single cells, the samples were down-sampled to 30,000 cells per sample. The detailed list of fluorescence-conjugated antibodies used for flow cytometry can be found in Additional file 2: Table S1.
Plasma levels of Antinuclear Antibodies (ANA), anti-dsDNA antibodies, and complement 3 (C3) were measured using a Mouse ANA Total Ig ELISA Kit (Catalog No. 5210, Alpha Diagnostic, San Antonio, Texas, USA), a Mouse anti-dsDNA Antibodies Total Ig ELISA Kit (Catalog No. 5110, Alpha Diagnostic, San Antonio, Texas, USA), and a Mouse C3 ELISA Kit (Catalog No. 6270, Alpha Diagnostic, San Antonio, Texas, USA), respectively, according to the manufacturer’s instructions. Plasma levels of inflammatory cytokines, including interferon gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-10, and IL-13, were assayed using a Milliplex® MAP kit (Millipore, Billerica, MA, USA), according to the manufacturer’s recommendations. Plasma levels of TGF-β1 were assayed using a Mouse TGF-β1 ELISA kit (Catalog No. VAL611, Novus Biologicals, USA) according to the manufacturer’s instructions. Plasma biochemical parameters, including albumin/globulin (A/G) ratio, glycocholic acid (CG), triglyceride (TG), and total bile acid (TBA), were measured using an autoanalyzer (Cobas 8000, Roche, Switzerland). The MRL/lpr mice (14 weeks old) fasted for 12 h, and the total body weight was measured before sacrifice. Next, the spleens were dissected and weighed. The spleen index is presented as spleen/body weight. Kidneys were harvested at the time of sacrifice and fixed with 4% paraformaldehyde in PBS. The kidneys were then embedded in paraffin and sectioned (1.5 μm). Sections were stained with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and Masson’s trichrome stain, following a blind assessment by an experienced pathologist. The quantification of renal histopathological changes was performed as previously described in reference [45]. The study co-cultured BM-MSCs with NZB/NZW F1 mouse PC in vitro and found that MSCs enhanced the viability and function of PC and promoted the production of IgG antibodies [45]; however, the mechanism was unclear. These results further illustrate the potential proinflammatory risk of MSCs, and the impact of this change on disease progression remains to be further verified. On the other hand, the evaluation of renal function and renal pathology found that the 24-h urine protein content of lupus mice in the MD and HD groups were lower than the Ctrl. The results of the renal pathological staining and immunofluorescence analysis showed that the renal pathology of mice in the MD and HD groups had an improvement compared to the Ctrl; these results are similar to those of previous reports. Transplantation of BM-MSCs derived from B6 mice into NZB/NZW F1 lupus model mice did not contribute to disease progression and did not affect autoantibody production, proteinuria levels, or mortality, but did improve renal pathology and reduce lymphocytic infiltration [46]. The results demonstrate that MSC transplantation could reduce the deposition of IgG antibodies in the kidney and alleviate kidney damage to a certain extent. However, hUC-MSC transplantation may not have a repair effect on kidney damage. Still, hUC-MSC transplantation in the early-stage of the disease can reduce the risk of kidney injury secondary to SLE. Further, it has a specific preventive and protective effect on the kidney. The reasons for the inefficacy of MSC transplantation are still unclear, and it is a critical question that impedes the clinical application of MSCs. Therefore, it is a promising strategy to maintain MSCs viability and protect against oxidative stress by preconditioning or modification in vitro, such as improvement of culture methods [47], growth factor or cytokine stimulation [48], hypoxia induction [49], and genetic modifications [50, 51] to enhance its immunosuppressive functions [52]. However, these modification strategies are still in the basic experimental stage and require more evidential support and rigorous clinical trials. Therefore, further research is being done to explore the autophagy activation of hUC-MSC transplantation in SLE treatments, as well as trying to modify MSCs from a new perspective to enhance their immunosuppressive effects and reduce proinflammatory side effects.Flow cytometry (FCM)
Enzyme-linked immunosorbent assay (ELISA)
Determination of plasma cytokine levels
Determination of biochemical parameters
Spleen index measurement
Pathology assessment of kidneys
Conclusion
This study comprehensively analyzed the altered frequency of peripheral B cell subsets by FCM. Further, it analyzed inflammatory cytokine plasma levels by protein chip technology and their changes associated with hUC-MSC transplantation in MRL/lpr mice. This study found that hUC-MSC transplantation partially alleviated disease progression and exerted a protective effect on the kidneys, the MD had the best effect, and LD and HD also had a weak effect, but some parameters in the HD group were contrary to expectations, indicating that higher concentration of hUC-MSC transplantation may cause opposite outcomes. Furthermore, the lack of a comparison with advanced lupus mice makes it impossible to determine whether hUC-MSC transplantation is better in the early stages of the disease than in the advanced stages. Therefore, further research is required on the immunotherapy effects of hUC-MSCs modification on SLE and whether hUC-MSC transplantation in the early-stage of the disease can effectively delay disease progression and maintain extended low activity.
Availability of data and materials
The data that supports the fundings of this study are available on request from the authors.
Abbreviations
- A/G ration:
-
Albumin/globulin ratio
- ACR:
-
Albumin/creatinine ratio
- ANA:
-
Anti-nuclear antibodies
- BUN:
-
Blood urea nitrogen
- CG:
-
Glycocholic acid
- Ctrl:
-
Control
- HD:
-
High dose
- hUC-MSCs:
-
Human umbilical cord mesenchymal stem cells
- IFN-γ:
-
Interferon gamma
- IL:
-
Interleukin
- LD:
-
Low dose
- MB:
-
Memory B cells
- MD:
-
Middle dose
- P:
-
Inorganic phosphorus
- PB:
-
Plasmablasts
- PC:
-
Plasma cells
- SLE:
-
Systemic lupus erythematosus
- TBA:
-
Total bile acid
- TG:
-
Triglyceride
- TGF-β1:
-
Transforming growth factor-beta l
- TNF-α:
-
Tumor necrosis factor-alpha
- UA:
-
Uric acid
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Acknowledgments
We acknowledgement Dr. Minjie Zhang, Dr. Hongluan Wu and Dr. Lin Ye for the treatment of animal models.
Funding
This study was supported by National Natural Science Foundation of China (No. 82070757, 82270770) (QP), Guangdong Provincial Key Laboratory of Autophagy and Major Chronic Noncommunicable Diseases (2022B1212030003) (HL), The Stem Cell Preclinical Research Projects of the Affiliated Hospital of Guangdong Medical University (HL), Guangdong Basic and Applied Basic Research Foundation Enterprise Joint Fund (No. 2022A1515220028) (FG), Affiliated Hospital of Guangdong Medical University “Clinical Medicine +” CnTech Co-operation Project (No. CLP2021B007) (FG), Zhanjiang Science and Technology Project (Competitive) (No. 2021A05060) (FG).
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FG, QRP, HL, and QJP conceived and designed the study and wrote the manuscript. FG, QRP, and TC performed most experiments and statistical analyses. SZL, SML, AL, SC, JC, and ZX repeated some experiments and provide experimental support. HS, LY, and CY helped with the revised manuscript preparation and editing manuscript. FG, HL, and QJP supervised the project. All authors reviewed the manuscript and approved the final version of the manuscript.
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Ethics approval for the experiments was obtained from the laboratory animal ethical committee (LAEC) of Guangdong Medical University (license no. GDY2103031). The Ethics approval document entitled “Mechanism study of human umbilical cord mesenchymal stem cells alleviating the progression of lupus nephritis” was approved on August 25, 2021. All experiments were performed following relevant laws and institutional guidelines of the laboratory animal ethical committee (LAEC) of Guangdong Medical University. This study did not involve human subjects.
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Additional file 1: Figure S1.
A. MSCs have the capability to interact with B cells via the PD-1/PD-L1 pathway. The flow cytometry results demonstrated that MSCs exhibited a PD-L1 positivity rate exceeding 98% (a). In the spleens of 14-week-old MRL/lpr mice, CD19 and PD-1 exhibited colocalization, presenting as yellow fluorescence (b). DAPI emitted blue fluorescence, CD19 emitted red fluorescence, and PD-1 emitted green fluorescence, scale bar: 50 μm. B. Distribution of spleen T cell subsets, including Tfh cells (CXCR5-positive, green fluorescence) (a), Th1 cells (IFN-γ-positive, green fluorescence), Th2 cells (GATA3-positive, green fluorescence), Th17 cells (IL-17-positive, green fluorescence) and Treg cells (FoxP3-positive, green fluorescence). Blue fluorescence represents DAPI staining, scale bar: 50 μm. The number of mice is n=5.
Additional file 2: Table S1.
Antibodies used in the study.
Additional file 3: Figure S2.
hUC-MSC transplantation effects on spleen weight of 14-week-old MRL/lpr mice. Spleen weight ratio of mice in the MSC transplantation groups (MD and HD groups) showed a decrease compared with the Ctrl. (Ctrl: control, LD: low dose, MD: middle dose, HD: high dose). Compare the distance measured by two rulers of different units. The result indicated that the distance measurement was the same. The number of mice is n = 5.
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Guo, F., Pan, Q., Chen, T. et al. hUC-MSC transplantation therapy effects on lupus-prone MRL/lpr mice at early disease stages. Stem Cell Res Ther 14, 211 (2023). https://doi.org/10.1186/s13287-023-03432-2
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DOI: https://doi.org/10.1186/s13287-023-03432-2