Immune response to cold exposure: Role of γδ T cells and TLR2-mediated inflammation.

The mammalian body possesses remarkable adaptability to cold exposure, involving intricate adjustments in cellular metabolism, ultimately leading to thermogenesis. However, cold-induced stress can impact immune response, primarily through noradrenaline-mediated pathways. In our study, we utilized a rat model subjected to short-term or long-term mild cold exposure to investigate systemic immune response during the cold acclimation. To provide human relevance, we included a group of regular cold swimmers in our study. Our research revealed complex relationship between cold exposure, neural signaling, immune response, and thermogenic regulation. One-day cold exposure triggered stress response, including cytokine production in white adipose tissue, subsequently activating brown adipose tissue, and inducing thermogenesis. We further studied systemic immune response, including the proportion of leukocytes and cytokines production. Interestingly, γδ T cells emerged as possible regulators in the broader systemic response, suggesting their possible contribution in the dynamic process of cold adaptation. We employed RNA-seq to gain further insights into the mechanisms by which γδ T cells participate in the response to cold. Additionally, we challenged rats exposed to cold with the Toll-like receptor 2 agonist, showing significant modulation of immune response. These findings significantly contribute to understanding of the physiological acclimation that occur in response to cold exposure.

Xenogeneic Sertoli cells modulate immune response in an evolutionary distant mouse model through the production of interleukin-10 and PD-1 ligands expression.

BACKGROUND: Immunomodulatory mechanisms of Sertoli cells (SCs) during phylogeny have not been described previously. This study attempted to reveal mechanisms of SC immune modulation in an evolutionary distant host. METHODS: The interaction of the SC cell line derived from Xenopus tropicalis (XtSC) with murine immune cells was studied in vivo and in vitro. The changes in the cytokine production, the intracellular and surface molecules expression on murine immune cells were evaluated after co-culturing with XtSCs. Migration of XtSCs in mouse recipients after intravenous application and subsequent changes in spleen and the testicular immune environment were determined by flow cytometry. RESULTS: The in vitro co-culture model was established, allowing the study of XtSCs interaction with murine immune cells. Intracellular staining of interleukin (IL-)10 revealed a significant increase in its expression in macrophages and B cells co-cultured with XtSCs, compared to both unstimulated cells and xenogeneic control. On the contrary, a significant decrease in Th lymphocytes expressing interferon-gamma was observed. The expression of both PD-1 ligands (PD-L1 and PD-L2) was upregulated on the macrophage surfaces after co-culture with XtSCs, but not with the controls. XtSCs migrated specifically to testes when administered intravenously and modulated systemic and local testicular microenvironment; this was detected by the expression of molecules associated with suppressive phenotype by CD45+ cells in both spleen and testes. CONCLUSION: We have demonstrated for the first time that SCs can migrate and modulate immune response in a phylogenetically distant host. It was further observed that SCs induce expression of molecules associated with immunosuppression, such as IL-10 and PD-1 ligands.

Interleukin-10 production by B cells is regulated by cytokines, but independently of GATA-3 or FoxP3 expression.

The knowledge of mechanisms of regulation of IL-10 production by B cells remains still very limited. We show here that highly purified mouse B cells stimulated with LPS produce significant levels of IL-10, but Bregs in our model do not express detectable level of either Foxp3 or GATA-3. Nevertheless, IL-10 production by B cells is regulated by cytokines. In activated B cells, IL-10 production was significantly enhanced by IFN-γ and decreased in the presence of IL-4 or TGF-ß. These findings are in sharp contrast with the observations in T cells, where IL-10 production correlates with GATA-3 or FoxP3 expression, and the cytokines regulate IL-10 production in a reverse manner than in activated B cells. These results thus show that the production of IL-10 by Bregs is regulated by cytokines independently of the expression of GATA-3 and FoxP3, which is clearly different from GATA-3-dependent IL-10 production by activated Th2 cells and FoxP3 expression in IL-10-producing Tregs.

Kinetics of Helios(+) and Helios(-) T regulatory cell subsets in the circulation of healthy pregnant women.

Regulatory T cells (Tregs) play a critical role in the maintenance of a pregnancy. While the kinetics of the number of peripheral blood Tregs has been satisfactorily described in mouse models, analysis of these cell populations in human pregnancy is complicated by high variability in the quantity of Tregs and inconsistencies in the markers used for detecting different types of Treg. In the light of this, we set out to investigate the kinetics of various types of Treg, including CD45RA, GARP and PD-1(+) Tregs, in the peripheral blood of pregnant women in the first, second and third trimester, and at the time of delivery. Tregs, defined as a CD4(+)CD25(++)CD127(dim)Foxp3(+) population of leucocytes, were detected using flow cytometry. Natural thymus-derived Tregs and induced Tregs in the peripheral blood were distinguished by the expression or absence of a Helios marker, respectively. Our results showed that during normal pregnancy the sizes of various Treg subpopulations varied across women and also in an individual woman did not remain constant but varied significantly, most notable being the decrease observed at the time of delivery. Helios(-) cells were significantly less frequent in the peripheral blood of healthy pregnant women than Helios(+) cells, and the majority of Tregs were Helios(+)PD-1(+) Tregs.

Cyclosporine A promotes the therapeutic effect of mesenchymal stem cells on transplantation reaction.

The successful application of mesenchymal stem cells (MSCs) remains a major challenge in stem cell therapy. Currently, several in vitro studies have indicated potentially beneficial interactions of MSCs with immunosuppressive drugs. These interactions can be even more complex in vivo, and it is in this setting that we investigate the effect of MSCs in combination with Cyclosporine A (CsA) on transplantation reaction and allogeneic cell survival. Using an in vivo mouse model, we found that CsA significantly promoted the survival of MSCs in various organs and tissues of the recipients. In addition, compared to treatment with CsA or MSCs alone, the survival of transplanted allogeneic cells was significantly improved after the combined application of MSCs with CsA. We further observed that the combinatory treatment suppressed immune response to the alloantigen challenge and modulated the immune balance by harnessing proinflammatory CD4+T-bet+ and CD4+RORγt+ cell subsets. These changes were accompanied by a significant decrease in IL-17 production along with an elevated level of IL-10. Co-cultivation of purified naive CD4+ cells with peritoneal macrophages isolated from mice treated with MSCs and CsA revealed that MSC-educated macrophages play an important role in the immunomodulatory effect observed on distinct T-cell subpopulations. Taken together, our findings suggest that CsA promotes MSC survival in vivo and that the therapeutic efficacy of the combination of MSCs with CsA is superior to each monotherapy. This combinatory treatment thus represents a promising approach to reducing immunosuppressant dosage while maintaining or even improving the outcome of therapy.

Distinct cytokines balance the development of regulatory T cells and interleukin-10-producing regulatory B cells.

Regulatory T cells have been well described and the factors regulating their development and function have been identified. Recently, a growing body of evidence has documented the existence of interleukin-10 (IL-10) -producing B cells, which are called regulatory B10 cells. These cells attenuate autoimmune, inflammatory and transplantation reactions, and the main mechanism of their inhibitory action is the production of IL-10. We show that the production of IL-10 by lipopolysaccharide-stimulated B cells is significantly enhanced by IL-12 and interferon-γ and negatively regulated by IL-21 and transforming growth factor-ß. In addition, exogenous IL-10 also inhibits B-cell proliferation and the expression of the IL-10 gene in lipopolysaccharide-stimulated B cells. The negative autoregulation of IL-10 production is supported by the observation that the inclusion of anti-IL-10 receptor monoclonal antibody enhances IL-10 production and the proliferation of activated B cells. The effects of cytokines on IL-10 production by B10 cells did not correlate with their effects on B-cell proliferation or on IL-10 production by T cells or macrophages. The cytokine-induced changes in IL-10 production occurred on the level of IL-10 gene expression, as confirmed by increased or decreased IL-10 mRNA expression in the presence of a particular cytokine. The regulatory cytokines modulate the number of IL-10-producing cells rather than augmenting or decreasing the secretion of IL-10 on a single-cell level. Altogether these data show that the production of IL-10 by B cells is under the strict regulatory control of cytokines and that individual cytokines differentially regulate the development and activity of regulatory T cells and IL-10-producing regulatory B cells.

Lipopolysaccharide pretreatment increases the sensitivity of the TRPV1 channel and promotes an anti-inflammatory phenotype of capsaicin-activated macrophages.

BACKGROUND: The transient receptor potential vanilloid 1 (TRPV1) is well-established in neuronal function, yet its role in immune reactions remains enigmatic. The conflicting data on its inflammatory role, suggesting both pro-inflammatory and anti-inflammatory effects upon TRPV1 stimulation in immune cells, adds complexity. To unravel TRPV1 immunomodulatory mechanisms, we investigated how the TRPV1 agonist capsaicin influences lipopolysaccharide (LPS)-induced pro-inflammatory macrophage phenotypes. RESULTS: Changes in the surface molecules, cytokine production, and signaling cascades linked to the phenotype of M1 or M2 macrophages of the J774 macrophage cell line and bone marrow-derived macrophages, treated with capsaicin before or after the LPS-induced inflammatory reaction were determined. The functional capacity of macrophages was also assessed by infecting the stimulated macrophages with the intracellular parasite Leishmania mexicana. CONCLUSION: Our findings reveal that TRPV1 activation yields distinct macrophage responses influenced by the inflammatory context. LPS pre-treatment followed by capsaicin activation prompted increased calcium influx, accompanied by a shift toward an anti-inflammatory M2b-like polarization state.

Immunoregulatory properties of mouse limbal stem cells.

Stem cells have been demonstrated in nearly all adult mammalian tissues and play a vital role in their physiological renewal and healing after injury. Due to their irreplaceable role in tissue repair, these cells had to develop mechanisms protecting them from deleterious inflammatory immune reactions and ensuring their increased resistance to various apoptosis-inducing agents. In this study, we demonstrate that a population of mouse limbal cells highly enriched for cells expressing markers and characteristics of limbal stem cells (LSCs) suppresses in a dose-dependent manner the proliferation of lymphocytes elicited by mitogens or TCR-triggering and significantly inhibits the production of proinflammatory cytokines by activated T cells. The suppression was mediated by soluble factor(s) and did not affect early cell activation. LSCs were even more suppressive than mesenchymal stem cells or natural regulatory T cells. In addition, the cells expressing markers and characteristics of LSC had significantly higher levels of mRNA for Fas ligand and for the antiapoptotic molecules Mcl-1, XIAP, and survivin than other limbal cell populations. LSCs were also more resistant to staurosporin-induced apoptotic cell death and to cell-mediated cytotoxic reaction than other limbal cells. Collectively, these results suggest that SC isolated from fresh adult limbal tissue possess immunomodulatory properties and inhibit proinflammatory immune reactions. Simultaneously, these cells express high levels of mRNA for antiapoptotic molecules, which can protect them against cell-mediated cytotoxic reactions and various apoptosis-inducing agents.

The Inability of Ex Vivo Expanded Mesenchymal Stem/Stromal Cells to Survive in Newborn Mice and to Induce Transplantation Tolerance.

An encounter of the developing immune system with an antigen results in the induction of immunological areactivity to this antigen. In the case of transplantation antigens, the application of allogeneic hematopoietic cells induces a state of neonatal transplantation tolerance. This tolerance depends on the establishment of cellular chimerism, when allogeneic cells survive in the neonatally treated recipient. Since mesenchymal stem/stromal cells (MSCs) have been shown to have low immunogenicity and often survive in allogeneic recipients, we attempted to use these cells for induction of transplantation tolerance. Newborn (less than 24 h old) C57BL/6 mice were injected intraperitoneally with 5 × 106 adipose tissue-derived MSCs isolated from allogeneic donors and the fate and survival of these cells were monitored. The impact of MSC application on the proportion of cell populations of the immune system and immunological reactivity was assessed. In addition, the survival of skin allografts in neonatally treated recipients was tested. We found that in vitro expanded MSCs did not survive in neonatal recipients, and the living MSCs were not detected few days after their application. Furthermore, there were no significant changes in the proportion of individual immune cell populations including CD4+ cell lineages, but we detected an apparent shift to the production of Th1 cytokines IL-2 and IFN-γ in neonatally treated mice. However, skin allografts in the MSC-treated recipients were promptly rejected. These results therefore show that in vitro expanded MSCs do not survive in neonatal recipients, but induce a cytokine imbalance without induction of transplantation tolerance.

The Altered Migration and Distribution of Systemically Administered Mesenchymal Stem Cells in Morphine-Treated Recipients.

Mesenchymal stem cells (MSCs) have the ability to migrate to the site of injury or inflammation, and to contribute to the healing process. Since patients treated with MSCs are often users of analgesic drugs, to relieve their uncomfortable pain associated with the tissue disorder, there is a possibility of negative effects of these drugs on the migration of endogenous and exogenous MSCs. Therefore, we tested the impact of acute and chronic treatment with morphine on the migration and organ distribution of exogenous adipose tissue-derived MSCs in mouse models. Firstly, we showed that the incubation of MSCs with morphine significantly reduced the expression of adhesive molecules CD44 (HCAM), CD54 (ICAM-1) and CD106 (VCAM-1) on MSCs. Using a model of systemic administration of MSCs labeled with vital dye PKH26 and by the application of flow cytometry to detect living CD45-PKH26+ cells, we found a decreased number of labeled MSCs in the lung, spleen and bone marrow, and a significantly increased number of MSCs in the liver of morphine-treated recipients. A skin allograft model was used to study the effects of morphine on the migration of exogenous MSCs to the superficial wound. Intraperitoneally administered MSCs migrated preferentially to the wound site, and this migration was significantly decreased in the morphine-treated recipients. The present results showed that morphine significantly influences the distribution of exogenous MSCs in the body, and decreases their migration to the site of injury.

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

It is becoming increasingly evident that selecting an optimal source of mesenchymal stromal 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 confirmed. 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 profile of canonical MSC markers on the SC surface. Besides, SCs rescued T helper (Th) cells from undergoing apoptosis, promoted the anti-inflammatory phenotype of these cells, but did not regulate Th cell proliferation. MSCs impaired the Th17-mediated response; on the other hand, SCs suppressed the inflammatory polarisation in general. SCs induced M2 macrophage polarisation more effectively than MSCs. For the first 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-inflammatory and protective effects, especially in the therapies for diseases associated with testicular tissue inflammation.

Loci controlling lymphocyte production of interferon c after alloantigen stimulation in vitro and their co-localization with genes controlling lymphocyte infiltration of tumors and tumor susceptibility.

Low infiltration of lymphocytes into cancers is associated with poor prognosis, but the reasons why some patients exhibit a low and others a high infiltration of tumors are unknown. Previously we mapped four loci (Lynf1­Lynf4) controlling lymphocyte infiltration of mouse lung tumors. These loci do not encode any of the molecules that are involved in traffic of lymphocytes. Here we report a genetic relationship between these loci and the control of production of IFNγ in allogeneic mixed lymphocyte cultures (MLC). We found that IFNγ production by lymphocytes of O20/A mice is lower than by lymphocytes of OcB-9/Dem mice (both H2pz) stimulated in MLC by irradiated splenocytes of C57BL/10SnPh (H2b) or BALB/ cHeA (H2d) mice, or by ConA. IFNγ production in MLCs of individual (O20 9 OcB-9)F2mice stimulated by irradiated C57BL/10 splenocytes and genotyped for microsatellite markers revealed four IFNγ-controlling loci (Cypr4-Cypr7), each of which is closely linked with one of the four Lynf loci and with a cluster of susceptibility genes for different tumors. This suggests that inherited differences in certain lymphocyte responses may modify their propensity to infiltrate tumors and their capacity to affect tumor growth.

Distinct regulatory roles of transforming growth factor-beta and interleukin-4 in the development and maintenance of natural and induced CD4+ CD25+ Foxp3+ regulatory T cells.

The development and function of CD4(+) CD25(+) Foxp3(+) regulatory T cells (Tregs) are strictly regulated by cytokines. Here we show that transforming growth factor-beta (TGF-beta) and interleukin-4 (IL-4) play a crucial and antagonistic role in the development of Tregs. Additionally, these cytokines also have distinct effects on the maintenance of natural (nTregs) and antigen-induced (iTregs) Tregs. Using double-staining and tracking of proliferation of purified and carboxyflourescein succinimidyl ester (CFSE)-labelled mouse T-cell subpopulations we demonstrated that CD4(+) CD25(+) Foxp3(+) iTregs develop upon alloantigenic stimulation in the presence of TGF-beta exclusively from CD4(+) CD25(-) Foxp3(-) precursors. Both the induction of Foxp3 expression and Treg proliferation were prevented when the cells were stimulated in the presence of IL-4. By contrast, nTregs did not proliferate in the presence of the antigen and TGF-beta, and partially lost their Foxp3 expression. IL-4 not only prevented the development of iTregs, but also down-regulated the level of Foxp3 mRNA and decreased the number of Foxp3(+) cells in a population of iTregs. Further analyses proved that IL-4 decreased the expression of Foxp3 only in a population of iTregs, whereas it substantially supported the survival of nTregs. Functional experiments showed that Tregs induced in the presence of alloantigen and TGF-beta inhibited, on a per-cell basis, cell proliferation comparably to nTregs, and their suppressive capacity was not modulated by IL-4. These data suggest that TGF-beta and IL-4 differentially regulate the development of Tregs and distinctly sustain Foxp3 expression and the number of nTregs and iTregs, but have no influence on the suppressive activity of Tregs on a per-cell basis.

Cytokine interplay among the diseased retina, inflammatory cells and mesenchymal stem cells - a clue to stem cell-based therapy.

Retinal degenerative disorders, such as diabetic retinopathy, retinitis pigmentosa, age-related macular degeneration or glaucoma, represent the most common causes of loss of vision and blindness. In spite of intensive research, treatment options to prevent, stop or cure these diseases are limited. Newer therapeutic approaches are offered by stem cell-based therapy. To date, various types of stem cells have been evaluated in a range of models. Among them, mesenchymal stem/stromal cells (MSCs) derived from bone marrow or adipose tissue and used as autologous cells have been proposed to have the potential to attenuate the negative manifestations of retinal diseases. MSCs delivered to the vicinity of the diseased retina can exert local anti-inflammatory and repair-promoting/regenerative effects on retinal cells. However, MSCs also produce numerous factors that could have negative impacts on retinal regeneration. The secretory activity of MSCs is strongly influenced by the cytokine environment. Therefore, the interactions among the molecules produced by the diseased retina, cytokines secreted by inflammatory cells and factors produced by MSCs will decide the development and propagation of retinal diseases. Here we discuss the interactions among cytokines and other factors in the environment of the diseased retina treated by MSCs, and we present results supporting immunoregulatory and trophic roles of molecules secreted in the vicinity of the retina during MSC-based therapy.

The interconnection between cytokeratin and cell membrane-bound ß-catenin in Sertoli cells derived from juvenile Xenopus tropicalis testes.

Sertoli cells (SCs) play a central role in the determination of male sex during embryogenesis and spermatogenesis in adulthood. Failure in SC development is responsible for male sterility and testicular cancer. Before the onset of puberty, SCs are immature and differ considerably from mature cells in post-pubertal individuals regarding their morphology and biochemical activity. The major intermediate filament (IF) in mature SCs is vimentin, anchoring germ cells to the seminiferous epithelium. The collapse of vimentin has resulted in the disintegration of seminiferous epithelium and subsequent germ cell apoptosis. However, another IF, cytokeratin (CK) is observed only transiently in immature SCs in many species. Nevertheless, its function in SC differentiation is poorly understood. We examined the interconnection between CK and cell junctions using membrane ß-catenin as a marker during testicular development in the Xenopus tropicalis model. Immunohistochemistry on juvenile (5 months old) testes revealed co-expression of CK, membrane ß-catenin and E-cadherin. Adult (3-year-old males) samples confirmed only E-cadherin expression; CK and ß-catenin were lost. To study the interconnection between CK and ß-catenin-based cell junctions, the culture of immature SCs (here called XtiSCs) was employed. Suppression of CK by acrylamide in XtiSCs led to breakdown of membrane-bound ß-catenin but not F-actin and ß-tubulin or cell-adhesion proteins (focal adhesion kinase and integrin ß1). In contrast to the obvious dependence of membrane ß-catenin on CK stability, the detachment of ß-catenin from the plasma membrane via uncoupling of cadherins by Ca2+ chelator EGTA had no effect on CK integrity. Interestingly, CHIR99021, a GSK3 inhibitor, also suppressed the CK network, resulting in the inhibition of XtiSCs cell-to-cell contacts and testicular development in juvenile frogs. This study suggests a novel role of CK in the retention of ß-catenin-based junctions in immature SCs, and thus provides structural support for seminiferous tubule formation and germ cell development.

Erratum to "Epithelial-Mesenchymal Transition Promotes the Differentiation Potential of Xenopus tropicalis Immature Sertoli Cells".

[This corrects the article DOI: 10.1155/2019/8387478.].

Epithelial-Mesenchymal Transition Promotes the Differentiation Potential of Xenopus tropicalis Immature Sertoli Cells.

Epithelial-mesenchymal transition (EMT) is a fundamental process in embryonic development by which sessile epithelial cells are converted into migratory mesenchymal cells. Our laboratory has been successful in the establishment of Xenopus tropicalis immature Sertoli cells (XtiSCs) with the restricted differentiation potential. The aim of this study is the determination of factors responsible for EMT activation in XtiSCs and stemness window acquisition where cells possess the broadest differentiation potential. For this purpose, we tested three potent EMT inducers-GSK-3 inhibitor (CHIR99021), FGF2, and/or TGF-ß1 ligand. XtiSCs underwent full EMT after 3-day treatment with CHIR99021 and partial EMT with FGF2 but not with TGF-ß1. The morphological change of CHIR-treated XtiSCs to the typical spindle-like cell shape was associated with the upregulation of mesenchymal markers and the downregulation of epithelial markers. Moreover, only CHIR-treated XtiSCs were able to differentiate into chondrocytes in vitro and cardiomyocytes in vivo. Interestingly, EMT-shifted cells could migrate towards cancer cells (HeLa) in vitro and to the injury site in vivo. The results provide a better understanding of signaling pathways underlying the generation of testis-derived stem cells.

The Immunomodulatory Potential of Mesenchymal Stem Cells in a Retinal Inflammatory Environment.

Retinal degenerative disorders are characterized by a local upregulation of inflammatory factors, infiltration with cells of the immune system, a vascular dysfunction and by the damage of retinal cells. There is still a lack of treatment protocols for these diseases. Mesenchymal stem cell (MSC)-based therapy using immunoregulatory, regenerative and differentiating properties of MSCs offers a promising treatment option. In this study, we analyzed the immunomodulatory properties of mouse bone marrow-derived MSCs after their intravitreal delivery to the inflammatory environment in the eye, caused by the application of pro-inflammatory cytokines IL-1ß, TNF-α and IFN-γ. The intravitreal administration of these cytokines induces an increased expression of pro-inflammatory molecules such as IL-1α, IL-6, inducible nitric oxide synthase, TNF-α and vascular endothelial growth factor in the retina. However, a significant decrease in the expression of genes for all these pro-inflammatory molecules was observed after the intravitreal injection of MSCs. We further showed that an increased infiltration of the retina with immune cells, mainly with macrophages, which was observed after pro-inflammatory cytokine application, was significantly reduced after the intravitreal application of MSCs. The similar immunosuppressive effects of MSCs were also demonstrated in vitro in cultures of cytokine-stimulated retinal explants and MSCs. Overall, the results show that intravitreal application of MSCs inhibits the early retinal inflammation caused by pro-inflammatory cytokines, and propose MSCs as a promising candidate for stem cell-based therapy of retinal degenerative diseases.

Immunomodulatory Properties of Bone Marrow Mesenchymal Stem Cells from Patients with Amyotrophic Lateral Sclerosis and Healthy Donors.

Pathogenesis of amyotrophic lateral sclerosis (ALS) involves several mechanisms resulting in a shift from a neuroprotective to a neurotoxic immune reaction. A promising tool for ALS treatment is represented by mesenchymal stem cells (MSCs), which possess both regenerative potential and immunomodulatory properties. In this study, we aimed to compare the immunomodulatory properties of MSCs isolated from the bone marrow of patients suffering from ALS and healthy donors. Moreover, the influence of proinflammatory cytokines on the immunoregulatory functions of MSCs was also evaluated. We found that MSCs from ALS patients and healthy donors comparably affected mitogen-stimulated peripheral blood mononuclear cells and reduced the percentage of T helper (Th)1, Th17 and CD8+CD25+ lymphocytes. These MSCs also equally increased the percentage of Th2 and CD4+FOXP3+ T lymphocytes. On the other hand, MSCs from ALS patients decreased more strongly the production of tumour necrosis factor-α than MSCs from healthy donors, but this difference was abrogated in the case of MSCs stimulated with cytokines. Significant differences between cytokine-treated MSCs from ALS patients and healthy donors were detected in the effects on the percentage of CD8+CD25+ and CD4+FOXP3+ T lymphocytes. In general, treatment of MSCs with cytokines results in a potentiation of their effects, but in the case of MSCs from ALS patients, it causes stagnation or even restriction of some of their immunomodulatory properties. We conclude that MSCs from ALS patients exert comparable immunomodulatory effects to MSCs from healthy donors, but respond differently to stimulation with proinflammatory cytokines. Graphical Abstract Treatment of mesenchymal stem cells (MSCs) with cytokines results in a potentiation of their effects, but in the case of MSCs from amyotrophic lateral sclerosis (ALS) patients, it causes stagnation (an equal reduction of the percentage of CD8+CD25+ T lymphocytes) or even restriction (no increase of proportion of CD4+FOXP3+ T lymphocytes) of some of their immunomodulatory properties. It means that MSCs from ALS patients exert comparable immunomodulatory effects to MSCs from healthy donors, but respond differently to stimulation with proinflammatory cytokines.

The effect of clinically relevant doses of immunosuppressive drugs on human mesenchymal stem cells.

Immunosuppressive drugs are used to suppress graft rejection after transplantation and for the treatment of various diseases. The main limitations of their use in clinical settings are severe side effects, therefore alternative approaches are desirable. In this respect, mesenchymal stem cells (MSCs) possess a regenerative and immunomodulatory capacity that has generated considerable interest for their use in cell-based therapy. Currently, MSCs are tested in many clinical trials, including the treatment of diseases which require simultaneous immunosuppressive treatment. Since the molecular targets of immunosuppressive drugs are also present in MSCs, we investigated whether immunosuppressive drugs interact with the activity of MSCs. Human MSCs isolated from the bone marrow (BM) or adipose tissue (AT) were cultured in the presence of clinical doses of five widely used immunosuppressive drugs (cyclosporine A, mycophenolate mofetil, rapamycin, prednisone and dexamethasone), and the influence of these drugs on several factors related to the immunosuppressive properties of MSCs, including the expression of immunomodulatory enzymes, various growth factors, cytokines, chemokines, adhesion molecules and proapoptotic ligands, was assessed. Glucocorticoids, especially dexamethasone, showed the most prominent effects on both types of MSCs and suppressed the expression of the majority of the factors that were tested. A significant increase of hepatocyte growth factor production in AT-MSCs and of indoleamine 2,3-dioxygenase expression in both types of MSCs were the only exceptions. In conclusion, clinically relevant doses of inhibitors of calcineurin, mTOR and IMPDH and glucocorticoids interfere with MSC functions, but do not restrain their immunosuppressive properties. These findings should be taken into account before preparing immunosuppressive strategies combining the use of immunosuppressive drugs and MSCs.