The immunomodulatory effects of bone marrow-derived mesenchymal stem cells on lymphocyte in spleens of aging rats

Objective: To investigate the effects of bone marrow-derived mesenchymal stem cells (BMSCs) on the proliferation and secretion of IgM, IgG and IL-2 in spleen lymphocytes (L) of aging rats. Methods: BMSCs were isolated by the whole bone marrow adherence method and characterized. A rat model of aging was produced by daily subcutaneous injection of D-galactose into the back of the neck. Rat spleen lymphocyte isolate kit to isolate spleen lymphocytes from aging rats and young rats. In vitro , the co-culture system of BMSCs and aging rats lymphocytes was established, and under the induction of mitogen LPS and ConA, the proliferative activity of lymphocytes in each group was detected by CCK-8 assay, the levels of IgM and IgG in the culture supernatant of each group was detected by ELISA, and the IL-2 radioimmunoassay kits were used to detect the content of IL-2 in the supernatant of each group. Results: (1) The isolated adherent cells showed the characteristics of BMSCs, including spindle-shaped morphology, high expression of CD29, CD44, low expression of CD34 and CD45, and osteogenic/adipogenic ability. (2) Under LPS induction, lymphocyte proliferative activity and secretion of immunoglobulin IgG were reduced in the aging group compared with the young group, and co-culture with BMSCs reversed this trend. (3) Under ConA induction, the IL-2 content of BMSCs co-cultured with aging lymphocytes was higher than that of aging lymphocytes alone ( P < 0.0001); the IL-2 content of CsA co-cultured with aging lymphocytes was lower than that of aging lymphocytes alone ( P < 0.0001). Conclusion: BMSCs have immunomodulatory effects on the spleen lymphocytes of aging rats in vitro .


Introduction
Aging is a normal and complex physiological phenomenon in the life process, and its essence is cellular senescence and the decline of body functions. The aging of the immune system, which maintains the homeostasis of the internal environment and against foreign pathogens, is not only the result of the aging of the body but also one of the reasons causing organismal aging [1]. Immunosenescence, defined as the impact of age on the immune system, is characterized by structural and functional changes that affect the innate and adaptive immune system [2]. T cells and B cells (collectively known as lymphocytes) are important component of the adaptive immune system and undergo a series of changes in the aging process [3].
Mesenchymal stem cells (MSCs) are a class of mesoderm-derived nonhematopoietic stem cells with self-renewal and multidirectional differentiation potential. Numerous studies have shown that transplantation of MSCs is a viable therapeutic option to stop immunosenescence because these cells have excellent immunomodulatory function and low immunogenicity [4]. However, the specific mechanism of interaction between MSCs and immune cells of aging organisms is not well understood. At present, bone marrow-derived mesenchymal stem cells (BMSCs) have the most comprehensive and in-depth research on the regulation of immunity, and are the most widely used in preclinical and clinical research [5]. Therefore, in this experiment, D-galactose was used to prepare an aging rat model, and the effects of BMSCs on the proliferation capacity and secretion of immunoglobulin (Ig) and IL-2 in spleen lymphocyte (L) of aging rats under in vitro co-culture conditions were observed, and the immunomodulatory effect of BMSCs on spleen lymphocytes of D-galactose-induced rats was preliminarily investigated.

Materials and methods
Rats and aging model construction 6-week-old SPF-grade young male SD rats weighing 180-220 g were injected subcutaneously with D-galactose at a dose of 400 mg/kg/d for 4 months to prepare an aging rat model. Company. Anti-rat-CD34FITC, anti-rat-CD29FITC, anti-rat-CD44FITC, and anti-rat-CD45FITC were obtained from Santa Cruz Biotechnology (BD). D(+)-galactose biochemical reagent (Shanghai Sinopharm Company). Rat immunoglobulin ELISA detection kit (IgG) (enzyme exemption, MM-0064R2). Rat immunoglobulin (IgM) ELISA Assay Kit (Enzyme Immunity, MM-0065R2). The levels of serum malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were measured by the thiobarbituric acid (TBA) method and the xanthine oxidation (XTO) method, respectively. IL-2 Release Kit was purchased from Shanghai Jianglai Biotechnology Co. Ltd.

Isolation of BMSCs
Bilateral femurs and tibias of 6-week-old SD rats were isolated under aseptic conditions, the muscle was removed to fully expose the bone marrow cavity, the bone marrow cavity was rinsed repeatedly with DMEM medium, the rinsing fluid was collected, the supernatant was discarded after centrifugation; the cells were resuspended by adding DMEM medium containing 10% fetal bovine serum, and incubated at 37°C in a 5% CO 2 incubator. When the primary cells reached 80% of wall growth, the primary cells were labeled as P0, and the first subcultured cells were labeled P1, and so on. P3 cells were digested by trypsin and prepared into cell suspensions, and FITC-labeled rabbit anti-rat CD34, CD29, CD44, and CD45 monoclonal antibodies, or negative controls were added and incubated for 30 min at room temperature and protected from light. The cell surface markers were detected by flow cytometry. The cells were cultured in lipogenic and osteogenic differentiation induction medium for 2~3 weeks. Adipogenic and osteogenic differentiation was assessed by Oil Red O and Alizarin Red staining, respectively [6].

Isolation of spleen lymphocytes from aging rats and young rats
Aged and young SD rats were executed by cervical dislocation, the spleen was aseptically removed, and the cell suspension was collected after complete grinding. Take a 15 mL centrifuge tube, add the same amount of separation solution as the spleen tissue cell suspension, carefully aspirate the spleen tissue cell suspension with a pipette and spread it on the surface of the separation solution, centrifuge at 500 g for 30 min at room temperature. After centrifugation, the tube was divided into four layers from top to bottom: the first was the dilution layer; the second was the annular milky white lymphocyte layer; the third was the clear separation layer; and the fourth was the erythrocyte layer. The second layer was carefully aspirated and washed with 10 mL of cell washing solution. After centrifugation (250 g for 10 min), the supernatants were discarded and cells were resuspended with PBS.

Co-culture system I
The experiment was divided into three groups: (1) Young lymphocytes group; (2) Aging lymphocytes group; (3) Aging lymphocytes + BMSCs (co-culture group). 1.0 × 10 6 lymphocytes of the youth group or senescence group were added in each well of the 24-well plate; 1.0 × 10 6 lymphocytes of the senescence group were inoculated in the lower chamber of the co-culture group, and 1.0 × 10 5 BMSCs were inoculated in the upper chamber (co-culture ratio 10:1). To stimulate B lymphocyte proliferation, LPS at a final concentration of 5 μg/ml was added simultaneously to each group, and three replicate wells were set up. After incubation in an incubator at 37°C, 5% CO 2 , and saturated humidity for 72 h, the supernatants of each culture group were separated to detect the contents of IgG and IgM in the supernatants with ELISA kits, and the cells of each culture group were collected to detect the proliferative activity of splenic lymphocytes with CCK8.

Co-culture system II
The experiment was divided into three groups: (1) Aging lymphocytes group; (2) Aging lymphocytes + BMSCs (co-culture group); (3) Aging lymphocytes + CsA (co-culture group). 1.0 × 10 6 aging lymphocytes were inoculated in the lower chamber of each group in a 24-well plate, and 1.0 × 10 5 BMSCs or cyclosporine A (CsA) at a final concentration of 5 μg/mL were added to the upper chamber of the co-culture group, respectively. To stimulate the proliferation of T lymphocytes cell proliferation, ConA at a final concentration of 5 μg/ml was added to all three groups simultaneously, and three replicate wells were set up. The supernatants of each group were separated after co-culture for 48 h, and the levels of IL-2 in the supernatants were measured according to the instructions of the IL-2 Release Kit.

Statistical analysis
All the data were analyzed and processed by SPSS 25.0 statistical software, and the measurement data were expressed as mean ± standard deviation ( x ± s), the independent sample t-test was used for comparison between the two groups, one-way ANOVA was used for the comparison between the three groups, followed by LSD-t test for the two-pair comparison between the three groups. Submit a manuscript: https://www.tmrjournals.com/bmec

Isolation and identification of BMSCs
BMSCs cultured by the whole bone marrow apposition method, some cells were seen to be adherent to the wall after 24 h. Some small colonies were observed to form after 3~4 d. The primary cells grew in a long spindle shape with multiple clonal colonies, and partial fusion of clonal colonies could be observed in 7~14 days. The BMSCs, after passaging, were faster to adhere to the wall than the P0 cells, and the cells were completely adherent to the wall in 24 h. The morphology was similar to that of the P0 cells and the BMSCs were uniformly distributed with a single shuttle shape after passaging ( Figure 1A). The flow cytometry results showed that CD34 and CD45 were negatively expressed and CD29 and CD44 were positively expressed on BMSCs ( Figure 1D-G). The third-generation BMSCs were stained with Oil Red O at 14 d of lipogenesis induction, and a large number of bead-like lipid droplets of different sizes were stained red under an inverted microscope, which was positive for Oil Red O ( Figure 1B); Alizarin Red staining was performed at 21 d of osteogenesis induction, and the formation of red mineralized nodules was evident under an inverted microscope, which was positive for Alizarin Red staining ( Figure 1C).

Evaluation of aging rat model and isolation of spleen lymphocytes in aging rats
Before the start of the experiment, the rats in each group had normal coat color, diet, activity and stool, and their mental state was good. Compared with the control group, the model group had a yellow coat, reduced diet, slow activity, acceptable mental state, increased weight, and unformed stool, which indicated that the model rats showed signs of aging. Compared with the normal control group, the activity of SOD in the serum of the model group was significantly lower (Figure 2A, P < 0.0001) and the MDA content was significantly higher ( Figure 2B, P < 0.0001). After the completion of modeling, the spleen lymphocytes of senescent rats were aseptically isolated and observed under an inverted microscope, which showed that the spleen lymphocytes of senescent rats were growing in suspension, with round or oval cells, good refractive index, and relatively uniform morphology and size, mostly B and T lymphocytes.

Effects of BMSCs on the proliferation of spleen lymphocytes and secretion of immunoglobulins IgG、IgM and IL-2 in aging rats
Mitogen LPS mainly induced B lymphocyte proliferation. Compared with the young lymphocytes group, lymphocytes in the aging group showed significantly lower proliferative activity and secretion of immunoglobulin IgG, and co-culture with BMSCs significantly reversed this trend ( Figure 3A-B, P < 0.0001); Compared with the young lymphocytes group, the content of IgM secreted by lymphocytes in the aging group was slightly higher, but the difference was not statistically significant ( Figure 3C, P > 0.05). Mitogenic ConA mainly induced the proliferation of T lymphocytes, and the IL-2 content in the group of BMSCs co-cultured with aging lymphocytes was higher than that in the group of aging lymphocytes alone (P < 0.0001), the IL-2 content in the CsA and aging lymphocytes co-culture group was lower than that in the aging lymphocytes alone culture group (P < 0.0001) ( Figure 3D, P < 0.0001).

Discussion
The immunological hypothesis of aging was first proposed in the 1960s, suggesting that the immune system is fundamentally involved in the aging process of normal vertebrates and is one of the major regulatory systems of the aging process. Aging is also reflected in the decline of immune cells, which is closely related to infections and autoimmune diseases [7]. Aging T cells have a limited proliferative capacity, mainly due to shortened telomere length and loss of telomerase, resulting in less IL-2 production and further reducing the proliferation of these cells [8]. B lymphocytes are produced in the bone marrow and mature in the spleen. With aging, fewer B-lymphocytes are produced in the bone marrow and the diversity of receptors decreases. The number of high-affinity protective antibodies produced decreases, which reduces the responsiveness of the body to infection and vaccination [9].
Numerous studies have shown the potential of MSCs in restoring the aging immune system, and MSCs can effectively reduce inflammatory markers, restore the T-cell pool, and improve the histopathology of inflammatory diseases [4]. Previous studies by our group have shown that BMSCs transplantation could improve the spleen and thymus structure and lymphocyte proliferation activity of aging rats to a certain extent, thereby significantly enhancing the immune activity of aging rats and slowing down immune aging [6,10]. To further investigate how BMSCs regulate the immune function of immune cells in the spleen of senescent rats, we established a D-galactose-induced senescence rat model, extracted senescent rat spleen lymphocytes, and co-cultured bone marrow-derived MSCs with spleen lymphocytes in vitro to observe the immunomodulatory effects of bone marrow-derived MSCs on senescent rat spleen lymphocytes under the stimulation of mitogens LPS and ConA. Submit a manuscript: https://www.tmrjournals.com/bmec MSCs can exert immunomodulatory effects by interacting directly with immune cells or by secretion of several types of paracrine cytokines [11]. MSCs have unique immunomodulatory effects on some immune cells, such as T-lymphocytes, B-lymphocytes, natural killer cells and dendritic cells [12]. Numerous experiments have shown that MSCs can significantly inhibit the activation, proliferation and cytokine secretion of T lymphocytes in vitro, significantly upregulate the Treg ratio, and their immunomodulatory effects are mostly manifested as immunosuppression, and their immunosuppressive effects need to be obtained in exposure to inflammatory rings such as IFN-γ [13]. However, the interaction between MSCs and B lymphocytes is also influenced by various factors such as the culture medium, the source of B cells and MSCs, the maturity of B cells, and other immune cells in the culture system to promote or inhibit proliferation, differentiation, and promotion or inhibition of Ig secretion [14].
Ji Yueru et al. demonstrated that human umbilical cord-derived MSCs have the ability to promote B lymphocyte proliferation and terminal differentiation as well as secretion of immunoglobulins and that this immunomodulatory effect is achieved through soluble cytokines [15]. Similar to the above experiment, the present experiment also demonstrated that the proliferative activity and secretion of immunoglobulin IgG of lymphocytes in the aging group were significantly lower than those in the young group under LPS induction, and co-culture with BMSCs could significantly reverse this trend. Immunoglobulin M (IgM) is the earliest antibody in the initial humoral immune response. IgG is the main antibody produced by the humoral immune response again, with high affinity, widely distributed in the body, with important immune effects, which is one of the main substances of the body to fight infection. Antibody class switch recombination (CSR) refers to the process in which B lymphocytes alter the immunoglobulin (Ig) heavy chain constant region to allow Ig isotypes to switch from IgM to IgG, IgA, or IgE [16]. The CSR process of the mammalian IgH occurs in mature B cells in the germinal centers of the lymph nodes, spleen, and Peyer patches of the gastrointestinal tract. Without CSR, humans and other mammals would only produce IgM and IgD, which severely limits the effectiveness of the humoral immune response against infection [17]. CSR from IgM to IgG in humoral immune responses, producing optimal Ig class is essential for clearance of invading pathogens, and mitogen-stimulated splenic B lymphocyte CSR from aged mice is reduced [18]. In combination with the present study, we found that IgG secretion of aging lymphocytes in the group co-cultured with BMSCs was significantly increased compared with that of aging lymphocytes cultured alone, and there was no statistically significant difference in IgM secretion, suggesting that BMSCs may also achieve immunomodulation of aging splenic lymphocytes by affecting CSR.
Cyclosporine A (CsA), a calcium-regulated phosphatase inhibitor, selectively inhibits T lymphocyte-mediated IL-2 production, thereby suppressing the immune response. The activation and proliferation of T cells during antigen recognition is dependent on the interaction between IL-2 and its receptor. Thymic involution decreases the production of naive T cells and then leads to an age-related decrease in the synthesis of IL-2, which affects the development and proliferation of lymphocytes. IL-2 is an important immune-enhancing factor, and its reduced level is an important molecular mechanism of immunosenescence [19]. This study showed that CsA significantly inhibited IL-2 secretion in senescent spleen lymphocytes activated by ConA, while BMSCs increased IL-2 secretion. To some extent, BMSCs exerted immunomodulatory effects by influencing IL-2 secretion by senescent rat T lymphocytes. Since the spleen monocyte suspension used in this experiment contains not only lymphocytes but also a small number of NK cells, monocytes and dendritic cells, and different immune cells may interact with each other, the immunomodulatory effects of BMSCs on T and B lymphocytes in aging rats can be further explored later with immunomagnetic beads or flow-sorting of specific lymphocyte subtypes.

Conclusion
The results showed that co-culture of bone marrow-derived MSCs with lymphocytes from senescent rats increased the number of lymphocytes and promoted antibody class switching (CSR), immunoglobulin secretion (especially IgG) and IL-2 secretion, indicating a degree of repair and reversal of immune senescence by bone marrow-derived MSCs. MSCs are widely used in the therapeutic exploration and clinical practice of autoimmune diseases such as SLE, the rheumatoid arthritis and inflammatory diseases such as acute kidney injury, inflammatory bowel disease and COVID-19 because of their weak immunogenicity and strong differentiation and repair ability [20]. If the immunomodulatory effect of MSCs on spleen lymphocytes or other immune cells in aging organisms can be further confirmed, this will open a new door for the basic experiments and clinical application of MSCs in regenerative medicine, geriatric medicine, immunology and other aspects.