Sertoli Cells Possess Immunomodulatory Properties and the Ability of Mitochondrial Transfer Similar to Mesenchymal Stem Cells

It is becoming increasingly evident that selecting an optimal source of mesenchymal stem cells (MSCs) is crucial for the successful outcome of MSC-based therapies. During the search for cells with potent regenerative properties, Sertoli cells (SCs) have been proven to modulate immune response in both in vitro and in vivo models. Based on morphological properties and expression of surface markers, it has been suggested that SCs could be a kind of MSCs, however, this hypothesis has not been fully conrmed. Therefore, we compared several parameters of MSCs and SCs, with the aim to evaluate the therapeutic potential of SCs in regenerative medicine. We showed that SCs successfully underwent osteogenic, chondrogenic and adipogenic differentiation and determined the expression prole of canonical MSC markers on the SC surface. Besides, SCs rescued T helper (Th) cells from undergoing apoptosis, promoted the anti-inammatory phenotype of these cells, but did not regulate Th cell proliferation. MSCs impaired the Th17-mediated response; on the other hand, SCs suppressed the inammatory polarisation in general. SCs induced M2 macrophage polarisation more effectively than MSCs. For the rst time, we demonstrated here the ability of SCs to transfer mitochondria to immune cells. Our results indicate that SCs are a type of MSCs and modulate the reactivity of the immune system. Therefore, we suggest that SCs are promising candidates for application in regenerative medicine due to their anti-inammatory and protective effects, especially in the therapies for diseases associated with testicular tissue inammation. Spleen cells (1 × 10 6 cells/ml) were cultured in a volume of 1 ml of complete RPMI 1640 medium in 24-well tissue culture plates stimulated with ConA (1.25 µg/ml) for 48 h in the presence or absence of SCs or MSCs (SCs/MSCs:spleen cells ratio 1:10 or 1:20). To analyze intracellular cytokine production, Phorbol 12-Myristate 13-Acetate (PMA, 20 ng/ml, Sigma-Aldrich), Ionomycin (500 ng/ml, Sigma-Aldrich), Brefeldin A (5 µg/ml, eBioscience) were added to the cultures for at least 4.5 h of the 48 h incubation period. Cells were harvested, washed with PBS/0.5% BSA and incubated for 30 min on ice with Alexa Fluor 700-labeled mAb anti-CD45 (clone 30-F11, BioLegend), FITC-labeled mAb anti-CD4 (clone GK1.5, BD Pharmingen) and Live/Dead Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientic) for staining of dead cells. Cells were then xed and permeabilized using a Fixation and Permeabilization Kit (eBioscience) according to the manufacturer's instructions. The cells were intracellularly stained for 30 min with PE-labeled mAb anti-TNFα (clone MP6-XT22, eBiosciece), APC-labeled mAb anti-IL-2 (clone JES6-5H4, eBioscience), APC-labeled mAb anti-IL-17A (clone eBio17B7, eBioscience). A total of 40 000 cells were analyzed after exclusion of dead cells and debris. Data were collected using LSR II cytometer (BD Bioscience) and analyzed using GateLogic 400.2A software (Invai). Representativedot plots illustrating the gating strategy are shown in Supplementary Figure S3.


Introduction
Stem cells are recently intensively investigated for the therapy of various diseases. Results obtained from many clinical studies proved the safety of the application of MSCs isolated from various tissues, but clinical outcomes of MSC transplantation still do not meet expectations [1]. Therefore, new approaches are studied, and the search for stem cells with better regenerative and immunomodulatory capacities continues. As MSCs from different tissue differ in their properties [2], the choice of an optimal source of MSCs with the best healing effect is critical to obtain favourable results in the therapy [3,4]. Promising candidates in this regard include Sertoli cells (SCs).
In the speci c environment of testes, SCs have been described to possess a similar biological function as MSCs. Adult SCs have been previously considered terminally differentiated cells with the only function to protect and nourish spermatogonial stem cells. However, this opinion has been challenged, as it was proved that adult SCs could regain their proliferation potential after transplantation [5]. According to the current knowledge, the primary function of SCs is, in addition to nourishing and supporting germ cells, to protect them from immune destruction and form blood-testis barrier and immune privilege. Besides, it has been documented that SCs provide support and a tolerogenic environment for co-transplanted cells even across immunological barriers in various in vivo models [6].
Recently, it has been proposed that SCs could be a kind of MSCs. Chikhovskaya et al. [7] demonstrated that somatic testicular cell cultures form colonies resembling MSCs. SCs also possess a phenotype similar to MSCs, including the ability to undergo differentiation along mesodermal lineages [8] and the expression of MSC-like surface markers [9]. These studies suggested that SCs could be a stem cell population, but this hypothesis has not been con rmed.
An essential property of MSCs is the ability to modulate immune response [10]. Signi cant immunosuppressive properties of SCs and their ability to promote cell growth have also been described in this regard [11], but mechanisms of the suppressive effect of SCs remain unclear. Recent studies have shown that the suppressive ability of SCs, similarly to MSCs, depends on the used model. SCs modulate the reactivity of the innate immune system, including the induction of an alternative M2 phenotype of macrophages and suppression of the co-stimulatory abilities of dendritic cells [12]. The effect of SCs on T-cell responses, particularly the shift towards Th2 and regulatory T cell (Treg) type of immune response, has been demonstrated, as documented by the upregulation of IL-10 and TGFβ production, as well as the increased Treg number in the periphery [13]. Expression of molecules participating in the tolerance, including PD-L1, FasL and indoleamine 2,3-dioxygenase, also plays an essential role in SC-mediated immunomodulation [14]. Furthermore, SCs have been a potent immunoregulatory tool in in vivo models of diabetes, neurodegenerative diseases and transplantation [8,[15][16][17].
The mitochondrial transfer has been described as one of the mechanisms, which MSCs use to support anti-in ammatory conditions and cell survival [18]. This mechanism has been identi ed as a critical pathway for the Th17 to Treg switch [19]. In various models, MSCs transferred mitochondria to cardiomyocytes, bronchial epithelial cells or cortical neurons [20][21][22]. The mitochondrial transfer has never been described in SCs; however, connexin43, which is one of the key proteins in this process, is a major protein in forming tight junctions and blood-testis barriers by SCs [23].
The ability of SCs to promote cell growth, their bene cial anti-in ammatory effects and the protection of co-transplanted tissue, together with the fact that SCs can be easily isolated from patient testicular biopsies performed routinely by fertility clinics, made them promising candidates in the eld of tissue repair and regeneration. Besides, due to the non-immunogenic properties of SCs, allogeneic cells isolated from a donor with healthy SCs can be used. Several groups reported that transplantation of MSCs or their products could restore spermatogenesis and fertility in various models [24][25][26]. Con rmation of SC stemness and further elucidation of molecular aspects of immunomodulation, differentiation and mechanisms of their action may enable new approaches for their application with the aim to support the regeneration of testicular damage. Therefore, the objective of this study was to verify the stem properties of SCs and compare them with those of MSCs, including their immunomodulatory potential. We also examined the ability of mitochondrial transfer as one of the mechanisms by which MSCs and SCs could provide protection of tissue from acute damage.

Materials And Methods
Female BALB/c mice (spleen cell isolation) at the age of 8-12 weeks and male BALB/c mice (SC isolation) at the age of 3 weeks were obtained from the breeding unit of the Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.
The present study was approved by the Animal Ethics Committee of Charles University, and all experimental procedures were performed following the guidelines for the care and use of laboratory animals.

Isolation of Adipose-Derived MSCs
Adipose-derived MSCs were isolated from inguinal fat pads of BALB/c mice as we have described [27], cultured in Dulbecco's modi ed Eagle medium (DMEM, PAA Laboratories, Pasching, Austria) supplemented with 10% FBS (Sigma-Aldrich Corporation, St. Louis, MO, USA), antibiotics (100 mg/ml of streptomycin, 100 U/ml of penicillin) and 10 mM Hepes buffer, and maintained in culture as adherent monolayers. Cells between passages 3 and 5 were used in the experiments.

Isolation of Sertoli Cells
Brie y, testes were decapsulated with tweezers and digested with Collagenase II and DNase I in PBS, 20 min in shaking bath (32°C), centrifuged (10 min, 800g) and ltered through 70 µm and then through 40 µm cell strainer. Cells on the 40 µm cell strainer were washed out by centrifugation (10 min, 800g). The cell suspension was plated on DSA (lectin from Datura Stramonium, Sigma-Aldrich) coated ask, washed 1 h after plating with warm DMEM medium and cultured in a complete DMEM medium supplemented with glutamine, LIF (0.1 ng/ml, Peprotech Rocky Hill, NJ, USA) and FSH (0.5 ng/ml, Sigma-Aldrich) for 3 weeks with regular exchange of medium, then passaged twice a week. Cells were maintained in culture as adherent monolayers, and between passages 3-6 were used in the experiments. Macrophages were prepared by washing the peritoneal cavity of unstimulated BALB/c mice as we described elsewhere [28], and cells (5 × 10 5 cells/ml) were plated in 24-well tissue culture plates (Nunc, Roskilde, Denmark) in a volume of 1 ml of RPMI-1640 medium supplemented with 10% FBS (Sigma-Aldrich), antibiotics (100 mg/ml of streptomycin, 100 U/ml of penicillin) and 10 mM Hepes buffer  Table S1.

Osteogenic, Chondrogenic and Adipogenic Differentiation of SCs and MSCs
SCs or MSCs were cultivated in DMEM medium to ensure mid-log growth phase con uence (60-80%). Then cells were gently harvested, and depending on the type of differentiation, were seeded into multiwell plates or Petri dishes. Cells underwent osteogenic differentiation using StemPro® Osteogenesis Differentiation Kit (Thermo Fisher Scienti c), according to the manufacturer's instructions. Osteocytes were stained with 2% Alizarin Red S solution (Sigma-Aldrich) for 15 min. A micromass culture was generated from SCs or MSCs and cultured in media prepared from StemPro® Chondrogenesis Differentiation Kit (Thermo Fisher Scienti c) to induce chondrogenic differentiation. Droplets with a volume of 5 µl of the cell suspension were seeded in the centre of the 6-well plate well to generate the micromass. After incubation for 2 h under high humidity conditions, chondrogenesis media was added to the cultures. Chondrocytes were detected using 1% Alcian Blue solution on day 21. Adipogenesis was After washing with PBS/0.5% BSA, cells were stained for Annexin V using Annexin V detection kit according to the manufacturer's protocol (Apronex, Jesenice, Czech Republic). Dead cells were excluded using Hoechst 33258 (Sigma-Aldrich), added 15 min before ow cytometry analysis. Data were collected using LSR II cytometer (BD Bioscience) and analyzed using GateLogic 400.2A software (Invai). Representative dot plots illustrating the gating strategy are shown in Supplementary Figure

Statistical Analysis
For the statistical analysis, the program The Prism (GraphPad Software, San Diego, CA, USA) was used. The results are expressed as the mean ± standard error (SE). The statistical signi cance of differences between individual groups was calculated using one-way analysis of variance (ANOVA) and Tukey post hoc test. P values less than 0.05 were considered statistically signi cant.

SCs Modulate CD4 + T Cell Proliferation, Apoptosis and Phenotype
The anti-in ammatory effect of MSCs on T cells is well described [30]. Therefore, we measured several parameters, including proliferation, apoptosis and Treg/Th17 ratio and compared the effects of SCs with those of MSCs. MSCs suppress the proliferation of CD4 + cells induced by ConA. Expression of a nuclear protein Ki67 was downregulated on CD4 + cells after co-culture with MSCs; the suppression by SCs was less pronounced (Fig. 3A). SCs protected activated CD4 + cells from apoptosis, revealed by the presence of phosphatidylserine on the cell surface using Annexin V similarly to MSCs (Fig. 3B). As shown in Fig. 3C, SCs promoted CD4 + T cell phenotype switch to anti-in ammatory. Treg/Th 17 ratio showed only a tendency to increase in the presence of MSCs in the culture, while in the presence of SCs, this ratio increased signi cantly.

SCs Suppress the Production of In ammatory Cytokine by Activated CD4 + T Cells
To further determine the activation of T cells cultivated in the presence of SCs, spleen cells were stimulated with ConA for 48 h in the presence of SCs or MSCs. The intracellular levels of selected cytokines were determined by ow cytometry. As shown in Fig. 4, the percentage of CD4 + TNFα + (Fig. 4A) and CD4 + IL-2 + (Fig. 4B) cells was decreased in a dose-dependent manner after cultivation with SCs and MSCs (Fig. 4B). This effect was more pronounced in the presence of SCs. A signi cant decrease in the proportion of CD4 + IL-17 + cells was observed in spleen cells stimulated with ConA in the presence of MSCs; cultivation with SCs has no signi cant effect on the intracellular level of IL-17.

The Effect of SCs on Peritoneal Macrophages
To further investigate the potential of SCs to modulate the phenotype of immune cells, cells isolated from the peritoneal cavity of mice were cultivated in the presence or absence of SCs or MSCs for 48 h. Macrophages can change their phenotype between pro-(M1) and anti-in ammatory (M2) type according to the cytokine microenvironment [31]. As shown in Fig. 5, the percentage of F4/80 + cells positive for CD206, a marker of M2 macrophages, was signi cantly increased. In the presence of MSCs in the culture, no signi cant changes were detected.

The Ability of SCs to Transfer Mitochondria to Immune Cells
It is known that MSCs can transfer mitochondria to various types of cells and thus modulate their metabolism or phenotype [32,33]. Therefore, we investigated the possibility of whether SCs are also able to transfer mitochondria to immune cells. Spleen cells were cultivated in the presence or absence of SCs or MSCs and stained for mitochondria with MiTT. As shown in Figs. 6A and 6B, SCs possess a similar capacity to transfer mitochondria to immune cells as have MSCs, as determined by the percentage of CD45 + MiTT + cells in co-cultures of spleen cells with MSCs or SCs. Figure 6C shows uorescence microscopy images of this experiment.

Discussion
MSCs are currently studied in many areas of regenerative medicine and developmental biology. It is increasingly apparent that the choice of the appropriate type of MSCs is crucial to the favourable outcome of therapy. Until now, MSCs isolated from various tissues have been used in cell-based therapies to promote the repair of testicular damage or treatment of male infertility. Although the results of these studies have been promising, further research in this area is needed [24,34,35]. In this regard, the use of cells possessing immunosuppressive properties, which occur naturally in affected testicular tissue, could be bene cial. SCs has been for a long time supposed to be nourishing cells providing support for germ cells and creating a blood-testis barrier. Nowadays, this simple view has been expanded. SCs have been described to provide testicular immune privilege, regulate immune response and play an essential role in modulating the phenotype of immune cells, changing the environment within testes from tolerogenic into in ammatory in the presence of infection [36][37][38]. However, the immunoregulatory properties of SCs were tested mainly in co-transplantation studies [6,15,39]. A better de nition and understanding of the stem capabilities of SCs will allow the extension of their therapeutic use for the regeneration of testicular tissues and the treatment of many other diseases.
Two previous studies suggested that SCs are kind of MSCs. However, these studies were based mainly on their morphological properties [7] and the ability to differentiate into key cell types of mesenchymal origin [9]. On the SC surface, we con rmed the expression of MSC-speci c markers and the absence of hematopoietic markers as de ned by Dominici et al. [29]. We further focused on the immunomodulatory abilities of SCs. MSCs induce arrest of the T cell cycle and thus suppress proliferation and apoptosis of these cells [40,41], and promote polarisation into anti-in ammatory T cell populations [42,43]. SCs demonstrated similar properties, although they differed in their expression. The ability of SCs to inhibit proliferation was less pronounced than that of MSC's; on the other hand, they increased the Treg /Th17 ratio even more than MSCs.
MSCs have been described to alter cytokine production by various immune cells populations [44,45]. In this study, we have shown that SCs signi cantly suppressed the production of pro-in ammatory cytokine TNFα, and the down-regulation of IL-2 production by SCs was even more pronounced. On the other hand, the suppression of IL-17 was not signi cant in the case of SCs. According to our data, both cell types modulate cytokine production and suppress in ammation. However, MSCs seem to be more effective in suppressing Th17 response, while SCs suppress in ammation or activation of CD4 + T cells by downregulating the production of two key cytokines IL-2 and TNFα, respectively.
The effect of SCs on innate immune cells has been documented but never compared to MSCs before. The polarisation of macrophage, from pro-in ammatory M1 to wound-healing M2 population, depends greatly on the microenvironment and external stimuli [31]; macrophages could acquire alternative M2 phenotype also in the presence of MSCs [48,49]. According to our data, SCs were more effective in the induction of this tolerogenic phenotype, as shown by up-regulation of CD206 molecule on their surface, which may subsequently extend their ability to regulate T cell immune responses. In the testes, the interplay between SCs and other cell populations, especially macrophages, is crucial for maintaining the optimal conditions for spermatogenesis and immune privilege function [6,50]; this could be the reason why SCs showed better e cacy in inducing the M2 macrophage phenotype.
The direct link between a metabolic con guration of immune cells and their phenotype and function is well known and vastly studied [51]. The master regulators of the metabolic setup are mitochondria, and the transfer of this organelle between different cell types was reported as an inductor of a phenotype switch, polarisation or protection of recipient cells [52]. For example, the transfer of mitochondria from MSCs to T cells triggers Treg differentiation and repression of Th17 cells [19,32]. MSCs modulate macrophage phagocytosis and activity also via mitochondrial transfer, both in vitro and in vivo [33,53]. We showed here for the rst time the ability of SCs to transfer mitochondria to other cell types. The percentage of CD45 + immune cells, which acquired mitochondria from SCs and MSCs was similar. This study con rms the previously stated hypothesis about mesenchymal origin and stem cell properties of SCs. We have shown that SCs differentiate into key cell types of mesenchymal origin and express some MSCs-like markers. Regarding their immunomodulatory properties, SCs are similar to MSCs, but differ in several aspects, which we assume can be attributed to their speci c natural niche in the organism. SCs are in close contact with various cells in testes, provide support for germ cells, but also are responsible for a protective immune environment. In this respect, the mitochondrial transfer could be one of the regulatory mechanisms. The obtained data support the possibility of applying SCs in various therapeutic approaches, especially testicular in ammation, orchitis or impaired spermatogenesis due to exposure to chemotherapy or radiotherapy. A better understanding of the effects of SCs and their capacity to down-regulate in ammation in vivo will be a crucial step for their implementation in cellbased therapy.

Availability of Data and Materials
The authors con rm that all data and materials support the published claims and comply with eld standards.

Author Contributions
All authors contributed to this study. MK and BP designed the study. BP, DV, VS, MHa, MHl and TT performed the experiments, MK, BP, and VH wrote the paper; all authors read and approved the nal manuscript.

Funding
This study was supported by grant No. 970120 from the Grant Agency of Charles University, grant No. 19-02290S from the Grant Agency of the Czech Republic, grant No. NU21-08-00488 from Ministry of Health of the Czech Republic and the Charles University programs 4EU+/20/F4/29, SVV 260435 and 20604315 PROGRES Q43.

Ethics Approval
This study was carried out in strict accordance with the Act No. 246/1992 Coll., on the protection of animals against cruelty, the basic law related to animal protection governing the activities of all the state authorities of animal protection in the Czech Republic, such as the Ministry of Agriculture, including the Central Commission for Animal Welfare, and the veterinary administration authorities. The authorization to use experimental animals was issued to the Faculty of Science, Charles University, 37428/2019-MZE-18134.

Con ict of Interest
The authors declare that they have no con ict of interest. Figure 1 Expression of genes characteristic for individual testicular cell populations. SCs did not express markers of germ cells and Leydig cells, but they expressed Sox9 (a marker of SCs), CD44, Vimentin (markers of MSCs) and Acta2 (actin alpha 2, testis associated marker). Data from one of two similar experiments are shown. O, Alcian Blue or Alizarin Red S, respectively. Representative images of differentiated MSCs and SCs are shown (C).    The transfer of mitochondria from MSCs and SCs to CD45+ immune cells. MSCs and SCs were stained by MiTT and cultivated with spleen cells for 3 h. The transfer of MiTT positive mitochondria originated from MSCs or SCs into CD45+ cells was evaluated by ow cytometry (A). One representative of the dot plot analysis from 3 independent experiments is presented (B). The mitochondrial transfer was also