Human umbilical cord blood-derived mesenchymal stem cells ameliorate psoriasis-like skin inflammation in mice

Mesenchymal stem cells (MSCs) inhibit the proliferation or activation of lymphocytes, and their inhibitory effects do not require human leukocyte antigen (HLA)-matching because MSCs express low levels of HLA molecules. Therefore, MSCs may be able to regulate immune responses. In this study, we determined whether MSCs could inhibit psoriasis-like skin inflammation in mice. After induction of psoriasis-like skin inflammation using intradermal injection of IL-23 or topical application of imiquimod with or without treatment with MSC, mouse skins were collected, and H&E staining and real-time PCR were performed. IL-23-induced skin inflammation was inhibited when MSCs were injected on day ?1 and day 7. The expression of proinflammatory cytokines such as IL-6, IL-17, and TNF-α was inhibited by MSC injection, and the expression of chemokines such as CCL17, CCL20, and CCL27 was also decreased in mouse skin. We also determined whether MSCs could not only prevent but also treat psoriasis-like skin inflammation in mice. Furthermore, in vitro experiments also showed anti-inflammatory effects of MSCs. Dendritic cells which are co-cultured with MSCs suppressed CD4+ T cell activation and differentiation, which are important for the pathogenesis of psoriasis. These results suggest that MSCs could be useful for treating psoriasis.     

Toll like Receptor 3 & 4 Responses of Human Turbinate Derived Mesenchymal Stem Cells: Stimulation by Double Stranded RNA and Lipopolysaccharide

Background and objectives

Multipotent mesenchymal stromal cells (MSCs) represent a promising cell-based therapy for a number of inflammatory or autoimmune diseases. Herein, Toll like receptor (TLR) expression by MSCs and their immune regulatory roles are investigated. In this study, we investigated the influence of TLR on the immune response, proliferation, and differentiation potential of human turbinated MSC (hTMSC) cultures in vitro.

Subjects and Methods

After isolating hTMSCs from discarded inferior turbinate tissue, FACS analysis was used to assess the expression of TLRs such as TLR2, TLR3, TLR4, and TLR5 in hTMSCs and cell proliferation was assessed using a cell counting kit (CCK)-8. Cytokine and chemokine secretions were analyzed with multiplex immunoassays for IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12, IP-10 (CXCL10), RANTES (CCL5), TNF-a, GM-CSF, and IFN-γ. The differentiation potential of hTMSCs was evaluated in the osteogenic, chondogenic, and adipogeinc media and analyzed by histology and gene expression related to differentiation.

Results

FACS analysis revealed that TLR3 and TLR4 expression consisted of a relatively high percentage of the surface proteins expressed by hTMSCs. The proliferation of hTMSCs was influenced and significantly increased by the presence of TLR4 agonists. In particular, hTMSCs produced a set of cytokines and chemokines and the expression of IL-6, IL-8, IL-12, IP-10 (CXCL10), RANTES (CCL5), TNF-α, and GM-CSF were up-regulated in response to the TLR4 agonist LPS. The osteogenic and adipogeinc differentiation potential of hTMSCs was not affected by TLR agonists.

Conclusions

We conclude that TLR4 stimulation affects TLR expression, proliferation, and the immunomodulation potential of hTMSCs. Understanding the mechanism behind TLR''s influence on hTMSCs and their immunomodulating properties would be useful for providing a novel target to exploit in the improvement of stem cell-based therapeutic strategies.     

Protein kinase C zeta regulates interleukin-8-mediated stromal-derived factor-1 expression and migration of human mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are bone marrow-derived cells with multipotent differentiation capability that are mobilized into the circulation in response to injury and localize to areas of tissue damage including solid tumors. They have the capacity to adopt a phenotype similar to carcinoma-associated fibroblasts (CAFs) and, like CAFs, promote tumor growth. The molecular communication between tumor cells and MSCs has not been well defined. However, MSCs have increased expression of the chemokine stromal-derived factor 1 (SDF-1) when exposed to conditioned medium from tumor cells. Additionally, SDF-1 has been shown to be important in the promotion of tumor growth by CAFs. These data suggest that the SDF-1 signaling axis is a key feature of the tumor microenvironment. In this report, we demonstrate that interleukin 8 (IL-8) induces an increase in SDF-1 expression by MSCs. The increase in SDF-1 expression in response to IL-8 is mediated by the activation of the protein kinase C (PKC) zeta isoform. In a functional assay, activation of PKC is required for in vitro MSC migration in response to tumor conditioned medium. These results indicate that IL-8-mediated SDF-1 production by MSCs requires PKC zeta activation. This signaling pathway provides insight into possible molecular targets for cancer therapy aimed at disrupting the interaction between components of the tumor microenvironment.     

Lysis and phenotypic modulation of mesenchymal stromal cells upon blood contact triggers anti-inflammatory skewing of the peripheral innate immune repertoire

Background aimsMesenchymal stromal cells (MSCs) are used to treat immune-related disorders, including graft-versus-host disease. Upon intravenous infusion, MSCs trigger the instant blood-mediated inflammatory response, resulting in activation of both complement and coagulation cascades, and are rapidly cleared from circulation. Despite no/minimal engraftment, long-term immunoregulatory properties are evident. The aim of this study was to establish the effects of blood exposure on MSC viability and immunomodulatory functions.MethodsHuman, bone marrow derived MSCs were exposed to human plasma +/– heat inactivation or whole blood. MSC number, viability and cellular damage was assessed using the JC-1 mitochondrial depolarization assay and annexin V staining. C3c binding and expression of the inhibitory receptors CD46, CD55 and CD59 and complement receptors C3aR and C5aR were evaluated by flow cytometry. MSCs pre-exposed to plasma were cultured with peripheral blood mononuclear cells (PBMCs) and monocyte subsets characterized by flow cytometry. The PBMC and MSC secretome was assessed using enzyme-linked immunosorbent assays against tumor necrosis factor alpha, interleukin (IL)-6 and IL-10. Monocyte recruitment towards the MSC secretome was evaluated using Boyden chambers and screened for chemotactic factors including monocyte chemoattractant protein (MCP)-1. MSC effects on the peripheral immune repertoire was also evaluated in whole blood by flow cytometry.ResultsPlasma induced rapid lysis of 57% of MSCs, which reduced to 1% lysis with heat inactivation plasma. Of those cells that were not lysed, C3c could be seen bound to the surface of the cells, with a significant swelling of the MSCs and induction of cell death. The MSC secretome reduced monocyte recruitment, in part due to a reduction in MCP-1, and downregulated PBMC tumor necrosis factor alpha secretion while increasing IL-6 levels in the co-culture supernatant. A significant decrease in CD14⁺ monocytes was evident after MSC addition to whole blood alongside a significant increase in IL-6 levels, with those remaining monocytes demonstrating an increase in classical and decrease in non-classical subsets. This was accompanied by a significant increase in both mononuclear and polymorphonuclear myeloid-derived suppressor cells.ConclusionsThis study demonstrates that a significant number of MSCs are rapidly lysed upon contact with blood, with those surviving demonstrating a shift in their phenotype, including a reduction in the secretion of monocyte recruitment factors and an enhanced ability to skew the phenotype of monocytes. Shifts in the innate immune repertoire, towards an immunosuppressive profile, were also evident within whole blood after MSC addition. These findings suggest that exposure to blood components can promote peripheral immunomodulation via multiple mechanisms that persists within the system long after the infused MSCs have been cleared.     

Mesenchymal stem cell–derived conditioned medium attenuate angiotensin II‐induced aortic aneurysm growth by modulating macrophage polarization

Mesenchymal stem cells (MSCs) exhibit therapeutic benefits on aortic aneurysm (AA); however, the molecular mechanisms are not fully understood. The current study aimed to investigate the therapeutic effects and potential mechanisms of murine bone marrow MSC (BM‐MSCs)–derived conditioned medium (MSCs‐CM) on angiotensin II (AngII)‐induced AA in apolipoprotein E‐deficient (apoE^?/?) mice. Murine BM‐MSCs, MSCs‐CM or control medium were intravenously administrated into AngII‐induced AA in apoE^?/? mice. Mice were sacrificed at 2 weeks after injection. BM‐MSCs and MSCs‐CM significantly attenuated matrix metalloproteinase (MMP)‐2 and MMP‐9 expression, aortic elastin degradation and AA growth at the site of AA. These treatments with BM‐MSCs and MSCs‐CM also decreased Ly6c^high monocytes in peripheral blood on day 7 and M1 macrophage infiltration in AA tissues on day 14, whereas they increased M2 macrophages. In addition, BM‐MSCs and MSCs‐CM reduced MCP‐1, IL‐1Ra and IL‐6 expression and increased IL‐10 expression in AA tissues. In vitro, peritoneal macrophages were co‐cultured with BM‐MSCs or fibroblasts as control in a transwell system. The mRNA and protein expression of M2 macrophage markers were evaluated. IL‐6 and IL‐1β were reduced, while IL‐10 was increased in the BM‐MSC systems. The mRNA and protein expression of M2 markers were up‐regulated in the BM‐MSC systems. Furthermore, high concentration of IGF1, VEGF and TGF‐β1 was detected in MSCs‐CM. Our results suggest that MSCs‐CM could prevent AA growth potentially through regulating macrophage polarization. These results may provide a new insight into the mechanisms of BM‐MSCs in the therapy of AA.     

TLR4 Activation Promotes Bone Marrow MSC Proliferation and Osteogenic Differentiation via Wnt3a and Wnt5a Signaling

Mesenchymal stem cells (MSCs) from adult bone marrow maintain their self-renewal ability and the ability to differentiate into osteoblast. Thus, adult bone marrow MSCs play a key role in the regeneration of bone tissue. Previous studies indicated that TLR4 is expressed in MSCs and is critical in regulating the fate decision of MSCs. However, the exact functional role and underlying mechanisms of how TLR4 regulate bone marrow MSC proliferation and differentiation are unclear. Here, we found that activated TLR4 by its ligand LPS promoted the proliferation and osteogenic differentiation of MSCs in vitro. TLR4 activation by LPS also increased cytokine IL-6 and IL-1β production in MSCs. In addition, LPS treatment has no effect on inducing cell death of MSCs. Deletion of TLR4 expression in MSCs completely eliminated the effects of LPS on MSC proliferation, osteogenic differentiation and cytokine production. We also found that the mRNA and protein expression of Wnt3a and Wnt5a, two important factors in regulating MSC fate decision, was upregulated in a TLR4-dependent manner. Silencing Wnt3a with specific siRNA remarkably inhibited TLR4-induced MSC proliferation, while Wnt5a specific siRNA treatment significantly antagonized TLR4-induced MSC osteogenic differentiation. These results together suggested that TLR4 regulates bone marrow MSC proliferation and osteogenic differentiation through Wnt3a and Wnt5a signaling. These finding provide new data to understand the role and the molecular mechanisms of TLR4 in regulating bone marrow MSC functions. These data also provide new insight in developing new therapy in bone regeneration using MSCs by modulating TLR4 and Wnt signaling activity.     

Effect of Short-Term Hypoxic Stress on Immunosuppressive Activity of Perivascular Multipotent Stromal Cells

Multipotent mesenchymal stromal cells (MSCs) are stromal precursors with the capacity to differentiate in osteo-, adipo-, and chondrodirections and participate in repair, regeneration, and immune response. Those abilities, especially immunosuppression, make MSCs a perspective tool for cell therapy and regenerative medicine. Short-term hypoxic stress can occur in damaged tissues and negatively affect MSC capacities to modulate functions of activated peripheral blood mononuclear cells (PBMCs). In the present paper, we evaluated the impact of short-term hypoxic stress (<1% oxygen) on immunosuppressive potential of tissue oxygen (5%) adapted MSCs. At a tissue oxygen level, we detected an increase of the ratio of innate immune cells (natural killers, NK) and a decrease in the ratio of adaptive immune cells (HLA-DR⁺ Т-cells) within floating PBMCs in the presence of MSCs. Additionally, inhibition of T-cell proliferation was observed. Within adhered PBMCs, the ratio of monocytes was higher and the ratio of NK T cells was lower. Short-term hypoxic stress did not affect MSC immunosuppression toward lymphocytes in suspension. Nevertheless, a decrease in percent of monocytes and NK T cells within adhered PBMCs was detected. Thus, hypoxic stress did not influence immunosuppressive activity of MSCs toward floating PBMCs. Attenuation of monocyte adhesion to MSCs upon cell-to-cell interaction may negatively impact the formation of MSCeducated macrophage phenotype with anti-inflammatory activity. In vivo, it may provoke the slowdown of “response to injury” during inflammation.     

Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute

There is great interest in mesenchymal stromal cells (MSCs) for cell-therapy and tissue engineering approaches. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns when used in clinical grade preparations. The aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL), already shown to promote MSC growth, are endowed with biological properties appropriate for cell-therapy approaches. We confirm previously published data showing that MSCs expanded in either FCS or PL display comparable morphology, phenotype, and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. We further extended these data by investigating the immune-regulatory effect of MSCs on the alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to: (i) decrease alloantigen-induced cytotoxic activity; (ii) favor differentiation of CD4+ T-cell subsets expressing a Treg phenotype; (iii) increase early secretion of IL-10 in MLC supernatant, as well as induce a striking augmentation of IL-6 production. As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and reducing early IFNgamma-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by molecular karyotyping and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the immunological functional plasticity of MSCs and suggest that MSCs-PL can be used as an alternative to MSCs-FCS, although these latter cells might be more suitable for preventing/treating alloreactivity-related immune complications.     

An insight into the role of magnesium in the immunomodulatory properties of mesenchymal stem cells

Magnesium (Mg²⁺) is a mineral with the ability to influence cell proliferation and to modulate inflammatory/immune responses, due to its anti-inflammatory properties. In addition, mesenchymal stem cells (MSCs) modulate the function of all major immune cell populations. Knowing that, the current work aimed to investigate the effects of Mg²⁺ enrichment, and its influence on the immunomodulatory capacity of MSCs. Murine C3H/10T1/2 MSCs were cultivated in media with different concentrations of Mg²⁺ (0, 1, 3 and 5 mM), in order to evaluate the effects of Mg²⁺ on MSC immunomodulatory properties, cell proliferation rates, expression of NFκB and STAT-3, production of IL-1β, IL-6, TGF-β, IL-10, PGE2 and NO, and TRPM7 expression. The results showed that TRPM7 is expressed in MSCs, but Mg²⁺, in the way that cells were cultivated, did not affect TRPM7 expression. Additionally, there was no difference in the intracellular concentration of Mg²⁺. Mg²⁺, especially at 5 mM, raised proliferation rates of MSCs, and modulated immune responses by decreasing levels of IL-1β and IL-6, and by increasing levels of IL-10 and PGE2 in cells stimulated with LPS or TNF-α. In addition, MSCs cultured in 5 mM Mg²⁺ expressed lower levels of pNFκB/NFκB and higher levels of pSTAT-3/STAT-3. Furthermore, conditioned media from MSCs reduced lymphocyte and macrophage proliferation, but Mg²⁺ did not affect this parameter. In addition, conditioned media from MSCs cultured at 5 mM of Mg²⁺ modulated the production profile of cytokines, especially of IL-1β and IL-6 in macrophages. In conclusion, Mg²⁺ is able to modulate some immunoregulatory properties of MSCs.     

Transplanted interleukin-4--secreting mesenchymal stromal cells show extended survival and increased bone mineral density in the murine femur

Background

Mesenchymal stromal cell (MSC)–based therapy has great potential to modulate chronic inflammation and enhance tissue regeneration. Crosstalk between MSC-lineage cells and polarized macrophages is critical for bone formation and remodeling in inflammatory bone diseases. However, the translational application of this interaction is limited by the short-term viability of MSCs after cell transplantation.

Gp130 activation by soluble interleukin-6 receptor/interleukin-6 enhances osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells

Interleukin-6 (IL-6) promotes osteodifferentiation in bone-located progenitors; however, it is not known whether this cytokine affects the differentiation of bone marrow-located osteoprogenitors. To address this issue, we prepared human bone marrow-derived mesenchymal stem cells (MSCs), which were characterized by a cell surface phenotype and multipotential nature. It was observed that in the presence of IL-6, MSCs were not differentiated into the osteogenic lineage, as evidenced by a failure to induce alkaline phosphatase activity, an earlier marker of osteodifferentiation. The lack of effect of IL-6 correlates with the observation that MSCs do not express a membrane-bound or soluble IL-6 receptor (sIL-6R). The incompetence of IL-6 was not reversed by the addition of sIL-6R alone or the sIL-6R/IL-6 complex, as it occurs in other IL-6R-negative cells. However, after MSC osteocommittment by dexamethasone, sIL-6R or the sIL-6R/IL-6 complex enhanced alkaline phosphatase activity. The effect of sIL-6R or sIL-6R/IL-6 proved to be dependent on gp130 availability, which is expressed by MSCs, and involves stat-3 phosphorylation. These data suggest that IL-6R deficiency may represent for bone marrow-located mesenchymal progenitors a sort of protective mechanism to escape the osteogenic effect of IL-6, which is produced by the MSC itself as well as by other marrow stromal cells.     

Effects of different mesenchymal stromal cell sources and delivery routes in experimental emphysema

We sought to assess whether the effects of mesenchymal stromal cells (MSC) on lung inflammation and remodeling in experimental emphysema would differ according to MSC source and administration route. Emphysema was induced in C57BL/6 mice by intratracheal (IT) administration of porcine pancreatic elastase (0.1 UI) weekly for 1 month. After the last elastase instillation, saline or MSCs (1×10⁵), isolated from either mouse bone marrow (BM), adipose tissue (AD) or lung tissue (L), were administered intravenously (IV) or IT. After 1 week, mice were euthanized. Regardless of administration route, MSCs from each source yielded: 1) decreased mean linear intercept, neutrophil infiltration, and cell apoptosis; 2) increased elastic fiber content; 3) reduced alveolar epithelial and endothelial cell damage; and 4) decreased keratinocyte-derived chemokine (KC, a mouse analog of interleukin-8) and transforming growth factor-β levels in lung tissue. In contrast with IV, IT MSC administration further reduced alveolar hyperinflation (BM-MSC) and collagen fiber content (BM-MSC and L-MSC). Intravenous administration of BM- and AD-MSCs reduced the number of M1 macrophages and pulmonary hypertension on echocardiography, while increasing vascular endothelial growth factor. Only BM-MSCs (IV > IT) increased the number of M2 macrophages. In conclusion, different MSC sources and administration routes variably reduced elastase-induced lung damage, but IV administration of BM-MSCs resulted in better cardiovascular function and change of the macrophage phenotype from M1 to M2.     

The C10/CCL6 chemokine and CCR1 play critical roles in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 is a potent stimulator of inflammation and tissue remodeling that plays a key role in the pathogenesis of a wide variety of human disorders. To further understand these responses, studies were undertaken to define the role(s) of the chemokine C10/CCL6 in the pathogenesis of IL-13-induced alterations in the murine lung. IL-13 was a very potent stimulator of C10/CCL6 mRNA and protein, and IL-13-induced inflammation, alveolar remodeling, and compliance alterations were markedly ameliorated after C10/CCL6 neutralization. Treatment with anti-C10/CCL6 decreased the levels of mRNA encoding matrix metalloproteinase-2 (MMP-2), MMP-9, and tissue inhibitor of metalloproteinase-4 (TIMP-4) in lungs from wild-type mice. C10/CCL6 neutralization also decreased the ability of IL-13 to stimulate the production of monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, MMP-2, MMP-9, and cathepsins-K, -L, and -S and the ability of IL-13 to inhibit alpha1-antitrypsin. In accord with these findings, a targeted null mutation of CCR1, a putative C10/CCL6 receptor, also decreased IL-13-induced inflammation and alveolar remodeling and caused alterations in chemokines, proteases, and antiproteases comparable to those seen after C10/CCL6 neutralization. These C10/CCL6 and CCR1 manipulations did not alter the production of transgenic IL-13. These studies demonstrate that IL-13 is a potent stimulator of C10/CCL6 and highlight the importance of C10/CCL6 and signaling via CCR1 in the pathogenesis of the IL-13-induced pulmonary phenotype. They also describe a C10/CCL6 target gene cascade in which C10/CCL6 induction is required for optimal IL-13 stimulation of selected chemokines (monocyte chemoattractant protein-1 and MIP-1alpha) and proteases (MMP-2, MMP-9, and cathepsins-K, -L, and -S) and the inhibition of alpha1-antitrypsin.     

Bone marrow-derived mesenchymal stem cells inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia after arterial injury in rats

We investigated whether mesenchymal stem cell (MSC)-based treatment could inhibit neointimal hyperplasia in a rat model of carotid arterial injury and explored potential mechanisms underlying the positive effects of MSC therapy on vascular remodeling/repair. Sprague-Dawley rats underwent balloon injury to their right carotid arteries. After 2 days, we administered cultured MSCs from bone marrow of GFP-transgenic rats (0.8 × 10⁶ cells, n = 10) or vehicle (controls, n = 10) to adventitial sites of the injured arteries. As an additional control, some rats received a higher dose of MSCs by systemic infusion (3 × 10⁶ cells, tail vein; n = 4). Local vascular MSC administration significantly prevented neointimal hyperplasia (intima/media ratio) and reduced the percentage of Ki67 + proliferating cells in arterial walls by 14 days after treatment, despite little evidence of long-term MSC engraftment. Notably, systemic MSC infusion did not alter neointimal formation. By immunohistochemistry, compared with neointimal cells of controls, cells in MSC-treated arteries expressed reduced levels of embryonic myosin heavy chain and RM-4, an inflammatory cell marker. In the presence of platelet-derived growth factor (PDGF-BB), conditioned medium from MSCs increased p27 protein levels and significantly attenuated VSMC proliferation in culture. Furthermore, MSC-conditioned medium suppressed the expression of inflammatory cytokines and RM-4 in PDGF-BB-treated VSMCs. Thus, perivascular administration of MSCs may improve restenosis after vascular injury through paracrine effects that modulate VSMC inflammatory phenotype.     

Cytokine physiognomies of MSCs from varied sources confirm the regenerative commitment post-coculture with activated neutrophils

The interaction of mesenchymal stromal cells (MSCs) with paracrine signals and immunological cells, and their responses and regenerative commitment thereafter, is understudied. In the current investigation, we compared MSCs from the umbilical cord blood (UCB), dental pulp (DP), and liposuction material (LS) on their ability to respond to activated neutrophils. Cytokine profiling (interleukin-1α [IL-1α], IL-2, IL-4, IL-6, IL-8, tumor necrosis factor-α [TNF-α], interferon-γ [IFN-γ], transforming growth factor-β [TGF-β]), cellular proliferation and osteogenic differentiation patterns were assessed. The results showed largely comparable cytokine profiles with higher TNF-α and IFN-γ levels in LSMSCs owing to their mature cellular phenotype. The viability and proliferation between LS/DP/UCB MSCs were comparable in the coculture group, while direct activation of MSCs with lipopolysaccharide (LPS) showed comparable proliferation with significant cell death in UCB MSCs and slightly higher cell death in the other two types of MSC. Furthermore, when MSCs post-neutrophil exposure were induced for osteogenic differentiation, though all the MSCs devoid of the sources differentiated, we observed rapid and significant turnover of DPMSCs positive of osteogenic markers rather than LS and UCB MSCs. We further observed a significant turnover of IL-1α and TGF-β at mRNA and cytokine levels, indicating the commitment of MSCs to differentiate through interacting with immunological cells or bacterial products like neutrophils or LPS, respectively. Taken together, these results suggest that MSCs have more or less similar cytokine responses devoid of their anatomical niche. They readily switch over from the cytokine responsive cell phenotype at the immunological microenvironment to differentiate and regenerate tissue in response to cellular signals.     

Immunomodulatory Activity of a Novel,Synthetic Beta-glucan (β-glu6) in Murine Macrophages and Human Peripheral Blood Mononuclear Cells

Natural β-glucans extracted from plants and fungi have been used in clinical therapies since the late 20th century. However, the heterogeneity of natural β-glucans limits their clinical applicability. We have synthesized β-glu6, which is an analog of the lentinan basic unit, β-(1→6)-branched β-(1→3) glucohexaose, that contains an α-(1→3)-linked bond. We have demonstrated the stimulatory effect of this molecule on the immune response, but the mechanisms by which β-glu6 activates innate immunity have not been elucidated. In this study, murine macrophages and human PBMCs were used to evaluate the immunomodulatory effects of β-glu6. We showed that β-glu6 activated ERK and c-Raf phosphorylation but suppressed the AKT signaling pathway in murine macrophages. Additionally, β-glu6 enhanced the secretion of large levels of cytokines and chemokines, including CD54, IL-1α, IL-1β, IL-16, IL-17, IL-23, IFN-γ, CCL1, CCL3, CCL4, CCL12, CXCL10, tissue inhibitor of metalloproteinase-1 (TIMP-1) and G-CSF in murine macrophages as well as IL-6, CCL2, CCL3, CCL5, CXCL1 and macrophage migration inhibitory factor (MIF) in human PBMCs. In summary, it demonstrates the immunomodulatory activity of β-glu6 in innate immunity.     

The Effects of Co-transplantation of Olfactory Ensheathing Cells and Schwann Cells on Local Inflammation Environment in the Contused Spinal Cord of Rats

Inflammatory response following spinal cord injury (SCI) is important in regulation of the repair process. Olfactory ensheathing cells (OECs) and Schwann cells (SCs) are important donor cells for repairing SCI in different animal models. However, synergistic or complementary effects of co-transplantation of both cells for this purpose have not been extensively investigated. In the present study, we investigated the effects of co-transplantation of OECs and SCs on expression of pro- or anti-inflammatory factor and polarization of macrophages in the injured spinal cord of rats. Mixed cell suspensions containing OECs and SCs were transplanted into the injured site at 7 days after contusion at the vertebral T10 level. Compared with the DMEM, SC, or OEC group, the co-transplantation group had a more extensive distribution of the grafted cells and significantly reduced number of astrocytes, microglia/macrophage infiltration, and expression of chemokines (CCL2 and CCL3) at the injured site. The co-transplantation group also significantly increased arginase⁺/CD206⁺ macrophages (IL-4) and decreased iNOS⁺/CD16/32⁺ macrophages (IFN-γ), which was followed by higher IL-10 and IL-13 and lower IL-6 and TNF-α in their expression levels, a smaller cystic cavity area, and improved motor functions. These results indicate that OEC and SC co-transplantation could promote the shift of the macrophage phenotype from M(IFN-γ) to M(IL-4), reduce inflammatory cell infiltration in the injured site, and regulate inflammatory factors and chemokine expression, which provide a better immune environment for SCI repair.     

Mesenchymal stem/stromal cells stably transduced with an inhibitor of CC chemokine ligand 2 ameliorate bronchopulmonary dysplasia and pulmonary hypertension

Perinatal bronchopulmonary dysplasia (BPD) is defined as lung injury in preterm infants caused by various factors, resulting in serious respiratory dysfunction and high mortality. The administration of mesenchymal stem/stromal cells (MSCs) to treat/prevent BPD has proven to have certain therapeutic effects. However, MSCs can only weakly regulate macrophage function, which is strongly involved in the development of BPD. 7ND-MSCs are MSCs transfected with 7ND, a truncated version of CC chemokine ligand 2 (CCL2) that promotes macrophage activation, using a lentiviral vector. In the present study, we show in a BPD rat model that 7ND-MSC administration, but not MSCs alone, ameliorated the impaired alveolarization evaluated by volume density and surface area in the lung tissue, as well as pulmonary artery remodeling and pulmonary hypertension induced by BPD. In addition, 7ND-MSCs, but not MSCs alone, reduced M1 macrophages and the messenger RNA expressions of interleukin-6 and CCL2 in the lung tissue. Thus, the present study showed the treatment effect of 7ND-MSCs in a BPD rat model, which was more effective than that of MSCs alone.     

Migration potential and gene expression profile of human mesenchymal stem cells induced by CCL25

Recruitment of mesenchymal stem cells (MSC) to tissue damages is a promising approach for in situ tissue regeneration. The physiological mechanisms and regulatory processes of MSC trafficking to injured tissue remain poorly understood. However, the pivotal role of chemokines in MSC recruitment has already been shown.The aim of this study was to determine the migratory potential and the gene expression profile of MSC stimulated with the CC chemokine CCL25 (TECK). Bone marrow derived human MSC were exposed to different doses of CCL25 in a standardized chemotaxis assay. Microarray gene expression profiling and pathway analysis were performed for CCL25 stimulated MSC.Maximum migration of MSC towards CCL25 was observed at 10³ nM. Microarray analysis revealed an induction of molecules directly involved in chemotaxis and homing of bone marrow cells (CXCL1-3, CXCL8, PDE4B), cytoskeletal and membrane reorganisation (CXCL8, PLD1, IGFBP1), cellular polarity (PLD1), and cell movement (CXCL1-3, CXCL6, CXCL8, PTGS2, PDE4B, TGM2). Respective chemokine secretion was confirmed by protein membrane-array analysis. The activation of CXCR2 ligands (CXCL1-3, CXCL5-6, CXCL8) and a LIF-receptor/gp130 ligand (LIF) indicated an involvement of the respective signaling pathways during initiation of chemotaxis and migration.These results suggest CCL25 as a new potential candidate for further in situ regeneration approaches.