Stem cells can be divided into three main categories: ESC (embryonic stem cells), iPSC (induced pluripotent stem cells) and MSC. ESC represents the inner cell mass of the blastocyst and possesses a pluripotent differentiation capacity. However, undifferentiated ESC can form teratomas and malignant teratocarcinoma in vivo, which is of high risk for direct clinical treatment. Moreover, ESC has the ability to form all three layers, so its use may raise clinical ethical issues. In the process of cell subculture, the iPSC could transfer more passages than ESC. The passage ability of iPSC was higher than ESC. However, clinical application of iPSC also has the risk of causing teratomas and malignant teratocarcinoma. Compared with ESC and iPSC, MSCs are easier to isolate and preserve, have a lower risk of clinical tumorigenesis, and do not raise ethical issues (1).

MSCs have immunomodulatory properties and tissue regeneration capabilities (2, 3). MSCs are present in various tissues, including bone marrow, adipose tissue, amniotic membrane and amniotic fluid, placenta and fetal tissue, umbilical cord tissue, endometrium, blood and synovial fluid. Because MSCs have the potential to differentiate into osteoblasts, chondrocytes, adipocytes and other mesoderms and have immunoregulatory functions, they have great application prospects in regenerative medicine research and the treatment of immune diseases (4, 5). However, MSCs can exhibit different morphological and physiological characteristics in different culture environments, and the currently prepared MSCs are heterogeneous mixed cell populations (3, 6). The International Society for Cellular Therapy has provided the minimum necessary criteria for a cell to be defined as an MSC: CD105, CD73, and CD90 positive and CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR negative, with the ability to differentiate into osteoblasts, adipocytes and chondrocytes in vitro (7, 8). Therefore, the definition of the pro-inflammatory and anti-inflammatory subpopulations of MSCs still needs to be explored in depth.

In recent years, many preclinical and clinical studies have conducted extensive investigations on the therapeutic potential and safety of MSCs in terms of immune regulation and the regenerative repair of diseases (9–12). The anti-inflammatory and immunomodulatory functions of MSCs have potent therapeutic effects on immunity/inflammation and autoimmune diseases (11, 12). MSCs have a wide range of immunomodulatory capabilities and can affect adaptive and innate immune responses. Therefore, MSCs are also considered a type of effective immune regulatory cell and are one of the best candidate cells for the treatment of inflammatory and autoimmune diseases. Recent studies have also shown that MSCs can interact with components in the body's immune microenvironment such as Toll-like receptors (TLRs) and cytokines. Through these interactions, MSCs can exhibit anti-inflammatory or pro-inflammatory effects (13–15). This dual immunomodulatory property of MSCs may be the reason for the differences between the results of preclinical and clinical studies and the significant difference in the therapeutic effects of MSCs among different studies. Therefore, the immunomodulatory properties of MSCs need to be studied in depth in order to optimize MSC-based treatment regimens.

Similar to macrophages, which can polarize into pro-inflammatory M1 or anti-inflammatory M2 phenotypes, MSCs can exhibit two phenotypes, pro-inflammatory MSC1 or anti-inflammatory MSC2, as a result of different inflammatory environments (16, 17). The pro-inflammatory MSC1 phenotype seems to help establish an appropriate early inflammatory response, while the anti-inflammatory MSC2 phenotype helps to inhibit the inflammatory response. MSC1 can secrete more pro-inflammatory cytokines, such as interleukin 6 (IL-6) and IL-8, to promote the activation of T cells, and MSC2 can secrete more anti-inflammatory cytokines, such as indoleamine-2,3-dioxygenase (IDO), IL-10, and prostaglandin E2 (PGE2), to inhibit the activation of T cells. Elucidating the molecular mechanisms that regulate the phenotypic transition of MSC1 and MSC2 and adopting specific methods and strategies to enhance the immunosuppressive ability of MSCs will have important significance for the future clinical application of MSCs in the treatment of immune/inflammatory diseases.

Although MSCs were first reported to be derived from bone marrow, a number of studies have reported similar cell types in a wide range of tissues, e.g., umbilical cord blood, placenta, adipose tissue, amniotic fluid, dental tissue, skin, hair follicles and tonsils. At present, most of MSCs used are bone marrow (BM-MSCs), umbilical cord (UC-MSCs), umbilical cord blood (CB-MSCs), placenta (P-MSCs) and adipose tissue (A-MSCs). Studies have found that MSCs from different sources do have different functions and characteristics, and therefore different therapeutic effects. P-MSCs showed a slight increase in growth, BM- and A-MSCs possess the highest capacity for self-renewal and differentiation potential in multiple lineages, whereas P-MSCs have the least functionality as stem cells of those which were tested. UC-MSCs were shown to express superior clonogenicity, migration, and paracrine capacities in vitro, as well as less senescence when compared with BM-MSCs. At present, UC-MSCs, BM-MSCs and A-MSCs are mainly used in inducing MSC2 (18).

Studies have found that the activation of TLRs, the inflammatory cytokine microenvironment and the activation of glycolysis in the metabolic reprogramming play important roles in the immunosuppressive functional transformation of MSCs (Figure 1). MSCs can be activated by pathogen-associated molecular patterns (PAMPs). PAMPs activate MSCs by binding to pattern recognition receptors (PRRs) on MSCs (19, 20). MSCs express multiple TLRs; furthermore, their ability to migrate, invade, and secrete immunomodulatory factors is also strongly influenced by specific TLR agonists. The activation of different types of TLRs can drive the transformation of MSCs into the MSC1 or MSC2 phenotype. Pro-inflammatory cytokines such as IFN-γ, TNF, and IL-1β can enhance the immunosuppressive function of MSCs (21). A pro-inflammatory environment, e.g., the presence of INF-γ and IL-6, can induce MSCs to transform into anti-inflammatory MSC2 and secrete the anti-inflammatory factor PGE2 (22). Studies have also confirmed that the metabolic changes in MSCs are related to their immunomodulatory functions (23). The metabolic transformation of MSCs to aerobic glycolysis can significantly regulate the immunomodulatory properties of MSCs by regulating the production of IDO, thereby enhancing T cell inhibition and anti-inflammatory effects (24).

In this paper, we will focus on the role of TLR activation, inflammatory cytokines, and metabolic reprogramming, as well as the correlation among them, in enhancing the immunosuppressive function of MSCs to identify optimal strategies and methods to promote the transformation of MSCs into MSC2 (Figure 1, Table 1).

Inflammation plays a vital role in kidney diseases. Therefore, improving the ability of MSCs to control the inflammatory progression of kidney injury tissues is a focus of research on the application of MSCs in immune-related nephropathy. Although MSCs currently generate good results related to the treatment of immune-related nephropathy (such as systemic lupus erythematosus nephritis, Thy-1 nephritis, and renal ischemia–reperfusion, etc.), there are also studies that indicate that the efficacy of MSCs in the treatment of immune-related nephropathy is inconsistent. To address the controversy regarding the therapeutic effects of MSCs, many studies have found that inducing the transformation of MSCs into anti-inflammatory MSCs can improve the therapeutic effects.

Changes in the metabolic pattern of MSCs can affect the changes in their immunosuppressive function. Jang et al. found that in a systemic lupus erythematosus nephritis model, metformin enhanced the immunomodulatory potential of adipose-derived MSCs through STAT1, thereby improving the progression of lupus nephritis (25). Metformin directly activated AMPK to promote the expression of a series of energy metabolism-related genes, i.e., metformin induced glycolysis by activating AMPK, thereby changing cellular metabolic reprogramming (67). In addition, Ishiuchi's study found that serum-free culture and hypoxic pretreatment synergistically enhanced the therapeutic effects of MSCs on renal fibrosis and that hypoxia transformed cellular metabolism into glycolysis. Therefore, it is speculated that the production of anti-inflammatory MSCs can be induced by regulating the metabolic reprogramming of MSCs (26).

Studies have found that many inflammatory factors (such as IFN-γ, TNF-α, and IL-17A) can induce the production of anti-inflammatory MSCs. Kanai found that IFN-γ preconditioning enhanced the anti-fibrosis ability of MSCs in rats with ischemia–reperfusion injury (IRI) and unilateral ureteral obstruction (27). Bai found that IL-17A pretreatment of bone marrow MSCs improved their immunosuppressive ability and increased the percentage of Treg through the COX-2/PGE2 pathway, thereby enhancing the efficacy of MSCs in IRI-induced acute kidney injury (AKI) mice (28). Xu et al. found that IL-37 overexpression in MSCs improved the immunosuppressive effects of MSCs in systemic lupus erythematosus (29). In addition, some small molecule compounds can also induce the production of anti-inflammatory MSCs. In a study by Deng, a small molecule compound, chlorzoxazone (CZ), was screened to induce the formation of an anti-inflammatory MSC2 phenotype in MSCs; CZ enhanced the immunosuppressive ability of MSCs and more effectively reduced the inflammatory infiltration of renal tissues and glomerular fibrin-like necrosis in Thy-1 nephritis, thereby improving renal function (30) (Table 1).

Mesenchymal stem cells (MSCs) have been used to treat various immune-related diseases due to their immunomodulatory function. MSCs can regulate the progression of immune inflammation by secreting anti-inflammatory cytokines (such as IDO, PEG2, IL-10, etc.). However, mesenchymal stem cells currently used for the treatment is a mixed, undefined, heterogeneous population of cells, resulting in inconsistent clinical outcomes. MSCs can be transformed into pro-inflammatory MSC1 or anti-inflammatory MSC2 phenotype. In most immune-related diseases, the enhanced transformation from MSC into MSC2 is required to better inhibit immune inflammation. In the newly added (Table 1), we summarize how to promote the transformation of MSC into the anti-inflammatory phenotype MSC2 and its application in immune-related diseases. The anti-inflammatory function of MSC can be increased by cytokines (IFN-γ, TGF-β, IL-17A), metabolic reprogramming (Metformin, hypoxia) and TLR3 excitation (Poly(I:C)). The MSCs with the increased anti-inflammatory function mainly secretes more anti-inflammatory factors (IDO, PGE2) to inhibit the proliferation of T cells, reduce the infiltration of inflammatory cell, regulate the differentiation of T cells to reduce inflammation (such as ratio of Th1 and Th17 decreases, while Treg increases).

At present, MSCs have been widely reported in the treatment of renal diseases, while MSC2 has rarely been studied in the treatment of immune-related nephropathy. However, in other diseases, many studies have confirmed that the therapeutic effect of MSC2 is stronger than untreated MSC (31, 33–38) (Table 1).

Studies on the immunomodulatory function of MSCs mainly focus on three aspects: 1. the association between the activation of different TLRs in MSCs and the MSC1/MSC2 phenotypes; 2. the effects of inflammation and related pathways on the anti-inflammatory effects of MSCs; and 3. the role of MSC metabolic reprogramming in the induction of MSC anti-inflammatory functions. Recent studies have confirmed that the above three aspects can all affect the pro-inflammatory and anti-inflammatory phenotypes of MSCs. However, whether the three are independent or partially dependent on each other or whether there is a unified pathway connecting the three remains to be studied. There may be a synergistic effect among the three factors; the effect of the combination may be higher than that of a single factor, and different combinations of each may be an effective strategy to enhance the immunosuppressive function of MSCs. Regulation of the key components of the inflammation-induced MSC2 pathway and metabolic reprogramming-induced MSC2 pathway, for example the PI3Kα and STAT1 pathways in the former and AMPK, HIF1-α, and PPARβ in the latter, can also enhance the immunosuppressive effect of MSCs.

The controversy regarding the efficacy of clinical stem cell application is largely due to the inconsistent definition of MSCs. Currently, the International Society for Cellular Therapy only provides the minimum criteria for a cell to be defined as an MSC. Similar to macrophages, MSCs have heterogeneity and plasticity, i.e., MSC1 (pro-inflammatory) and MSC2 (anti-inflammatory) phenotypes. Therefore, the minimum criteria can not accurately define MSCs. Studies on specific surface markers or morphological shape of MSC2 have just started, and there are no universally recognized specific surface markers on MSC2. To stably enhance the immunosuppressive function of MSCs, the relationship among the three phenotypes requires further clarification, and better methods and strategies for defining the MSC1/MSC2 phenotypes need to be developed.

MSC2s have been used for the treatment of a variety of immune-related nephropathies and have been shown to be safe and effective. However, most have only been validated in animal models; clinical trials are lacking. Therefore, it is necessary to conduct more in-depth mechanistic studies on enhancing the immunosuppressive function of MSCs to better define MSCs and their subpopulations, especially MSC2s. The ultimate goal is to develop an internationally recognized standard for the definition of the MSC1/MSC2 phenotypes to facilitate the clinical application of MSCs.

Both authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

This study was supported by a grant (No. 2020YFA0113004) from the National Key Research and Development Program of China and a grant (No. 81830060) from the National Natural Science Foundation of China.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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