Jian-Sheng Zhou

Adoptive transfer of M2 macrophages promotes locomotor recovery in adult rats after spinal cord injury

Classically activated pro-inflammatory (M1) and alternatively activated anti-inflammatory (M2) macrophages populate the local microenvironment after spinal cord injury (SCI). The former type is neurotoxic while the latter has positive effects on neuroregeneration and is less toxic. In addition, while the M1 macrophage response is rapidly induced and sustained, M2 induction is transient. A promising strategy for the repair of SCI is to increase the fraction of M2 cells and prolong their residence time. This study investigated the effect of M2 macrophages induced from bone marrow-derived macrophages on the local microenvironment and their possible role in neuroprotection after SCI. M2 macrophages produced anti-inflammatory cytokines such as interleukin (IL)-10 and transforming growth factor β and infiltrated into the injured spinal cord, stimulated M2 and helper T (Th)2 cells, and produced high levels of IL-10 and -13 at the site of injury. M2 cell transfer decreased spinal cord lesion volume and resulted in increased myelination of axons and preservation of neurons. This was accompanied by significant locomotor improvement as revealed by Basso, Beattie and Bresnahan locomotor rating scale, grid walk and footprint analyses. These results indicate that M2 adoptive transfer has beneficial effects for the injured spinal cord, in which the increased number of M2 macrophages causes a shift in the immunological response from Th1- to Th2-dominated through the production of anti-inflammatory cytokines, which in turn induces the polarization of local microglia and/or macrophages to the M2 subtype, and creates a local microenvironment that is conducive to the rescue of residual myelin and neurons and preservation of neuronal function.

Effects of Olig2-overexpressing neural stem cells and myelin basic protein-activated T cells on recovery from spinal cord injury.

Neural stem cell (NSC) transplantation is a major focus of current research for treatment of spinal cord injury (SCI). However, it is very important to promote the survival and differentiation of NSCs into myelinating oligodendrocytes (OLs). In this study, myelin basic protein-activated T (MBP-T) cells were passively immunized to improve the SCI microenvironment. Olig2-overexpressing NSCs were infected with a lentivirus carrying the enhanced green fluorescent protein (GFP) reporter gene to generate Olig2-GFP-NSCs that were transplanted into the injured site to differentiate into OLs. Transferred MBP-T cells infiltrated the injured spinal cord, produced neurotrophic factors, and induced the differentiation of resident microglia and/or infiltrating blood monocytes into an “alternatively activated” anti-inflammatory macrophage phenotype by producing interleukin-13. As a result, the survival of transplanted NSCs increased fivefold in MBP-T cell-transferred rats compared with that of the vehicle-treated control. In addition, the differentiation of MBP-positive OLs increased 12-fold in Olig2-GFP-NSC-transplanted rats compared with that of GFP-NSC-transplanted controls. In the MBP-T cell and Olig2-GFP-NSC combined group, the number of OL-remyelinated axons significantly increased compared with those of all other groups. However, a significant decrease in spinal cord lesion volume and an increase in spared myelin and behavioral recovery were observed in Olig2-NSC- and NSC-transplanted MBP-T cell groups. Collectively, these results suggest that MBP-T cell adoptive immunotherapy combined with NSC transplantation has a synergistic effect on histological and behavioral improvement after traumatic SCI. Although Olig2 overexpression enhances OL differentiation and myelination, the effect on functional recovery may be surpassed by MBP-T cells.

Temporal kinetics of macrophage polarization in the injured rat spinal cord

Local activated macrophages derived from infiltrating monocytes play an important role in the damage and repair process of spinal cord injury (SCI). The present study investigates the dynamic change of classically activated proinflammatory (M1) and alternatively activated anti‐inflammatory (M2) cells in a rat model with contusive SCI by flow cytometry (FCM) and immunohistochemistry. The macrophage subsets were immunophenotyped by using antibodies against cluster of differentiation (CD)−68, C‐C chemokine receptor type 7 (CCR7), CD163, and arginase 1 (Arg1). The CD68+CD163– and CD68+CCR7+ cells were determined to be M1 subsets, whereas the CD68+CD163+ and CD68+Arg1+ cell subpopulations represented M2 cells. The subsets of macrophages in the injured spinal cord at 1, 3, 5, 7, 14, and 28 days postinjury (dpi) were examined. In the sham‐opened spinal cord, few M1 or M2 cells were found. After SCI, the phenotypes of both M1 and M2 cells were rapidly induced. However, M1 cells were detected and maintained at a high level for up to 28 dpi (the longest time evaluated in this study). In contrast, M2 cells were transiently detected at high levels before 7 dpi and returned to preinjury levels at 14 dpi. These results indicate that M1 cell response is rapidly induced and sustained, whereas M2 induction is transient after SCI in rat. Increasing the fraction of M2 cells and prolonging their residence time in the injured local microenvironment is a promising strategy for the repair of SCI.

Cyclosporin A increases recovery after spinal cord injury but does not improve myelination by oligodendrocyte progenitor cell transplantation

BackgroundTransplantation of oligodendrocyte precursor cells (OPCs) is an attractive therapy for demyelinating diseases. Cyclosporin A (CsA) is one of the foremost immunosuppressive agents and has widespread use in tissue and cell transplantation. However, whether CsA affects survival and differentiation of engrafted OPCs in vivo is unknown. In this study, the effect of CsA on morphological, functional and immunological aspects, as well as survival and differentiation of engrafted OPCs in injured spinal cord was explored.ResultsWe transplanted green fluorescent protein (GFP) expressed OPCs (GFP-OPCs) into injured spinal cords of rats treated with or without CsA (10 mg/kg). Two weeks after cell transplantation, more GFP-positive cells were found in CsA-treated rats than that in vehicle-treated ones. However, the engrafted cells mostly differentiated into astrocytes, but not oligodendrocytes in both groups. In the CsA-treated group, a significant decrease in spinal cord lesion volume along with increase in spared myelin and neurons were found compared to the control group. Such histological improvement correlated well with an increase in behavioral recovery. Further study suggested that CsA treatment could inhibit infiltration of T cells and activation of resident microglia and/or macrophages derived from infiltrating monocytes in injured spinal cords, which contributes to the survival of engrafted OPCs and repair of spinal cord injury (SCI).ConclusionsThese results collectively indicate that CsA can promote the survival of engrafted OPCs in injured spinal cords, but has no effect on their differentiation. The engrafted cells mostly differentiated into astrocytes, but not oligodendrocytes. The beneficial effect of CsA on SCI and the survival of engrafted cells may be attributed to its neuroprotective effect.

PDGF-AA Mediates B104CM-Induced Oligodendrocyte Precursor Cell Differentiation of Embryonic Neural Stem Cells Through Erk, PI3K, and p38 Signaling

The conditioned medium from B104 neuroblastoma cells (B104CM) induces neural stem cells (NSCs) to differentiate into OPCs in vitro, which indicates that certain factor(s) contained within the B104CM must give instructional signals that direct OPC differentiation of NSCs. However, the OPC-inductive factor(s) present within the B104CM has not been well identified yet. Platelet-derived growth factor AA (PDGF-AA) was not only known to be a potent mitogen for OPC proliferation but also to act as a regulator of oligodendrocyte differentiation from multipotent embryonic NSCs. This raises the possibility that B104CM induces OPC differentiation of NSCs through secretion of PDGF-AA. In the present study, we detected the expression of PDGF-AA mRNA in B104 cells and the high level of PDGF-AA protein in B104CM. Most importantly, B104CM-induced OPC differentiation of NSCs could be completely blocked by AG1295, a specific inhibitor of PDGFR signal pathway, suggesting that the PDGF-AA in B104CM is the key factor that induces NSCs to differentiate into OPCs. Moreover, such B104CM-induced OPC differentiation appears to be mediated by the extracellular signal-regulated kinases 1 and 2 (Erk1/2), phosphatidylinositol-3 kinase (PI3K), and p38 signal pathway because B104CM elicited the activation of Erk1/2, PI3K, and p38, which could be markedly blocked by U0126, LY294002, and SB203580, several specific inhibitors of these signal pathway, respectively. These inhibitors also abolished OPC differentiation of NSCs completely. Together our study suggests that PDGF-AA contained in B104CM is the key regulating molecule that instructs OPC differentiation from embryonic NSCs through the activation of Erk, PI3K, and p38 signal pathway in vitro.

Effects of adenoviral vector expressing hIGF-1 on apoptosis in nucleus pulposus cells in vitro

The excessive apoptosis of cells of the nucleus pulposus may plays an important role in intervertebral disc (IVD) degeneration. It has been shown that the pro-inflammatory cytokine tumour necrosis factor (TNF)-α can induce disc cell apoptosis. Insulin-like growth factor (IGF)-1 can promote nucleus pulposus cell proliferation; however, whether or not IGF-1 inhibits TNF-α-induced apoptosis in the nucleus pulposus has not yet been elucidated. In this study, our objective was to create a potentially therapeutic viral vector, which could be used to achieve the enforced expression of IGF-1 in rabbit nucleus pulposus cells. Furthermore, we investigated the ability of IGF-1 to reverse TNF-α-induced apoptosis in cells of the nucleus pulposus. Isolated nucleus pulposus cells were cultured to a confluent monolayer, digested with collagenase Ⅱ and purified using trypsin and differential adhesion methods. Nucleus pulposus cells were positively identified using type Ⅱ collagen immunohistochemistry. Following transfection with adenoviral vectors engineered to overexpress recombinant human IGF-1 (Ad-hIGF-1) or TNF-α, the cells were observed under a light microscope. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) and flow cytometry (FCM) were used to assess the rate of apoptosis. The Ad-hIGF-1 viral vector was effectively transduced into the nucleus pulposus cells and increased IGF-1 expression as confirmed by RT-PCR and western blot analysis. In the TNF-α-treated group, a large number of apoptotic cells was observed that exhibited morphological changes associated with this form of cell death. Minimal apoptosis was observed in the Ad-hIGF-1-treated group and the control group showed no obvious signs of apoptosis. TUNEL assay revealed that the rate of apoptosis in the Ad-hIGF-1 group was significantly reduced compared with the TNF-α-treated group (P<0.01). This result was confirmed using FCM. The rate of apoptosis was also significantly increased in the TNF-α-exposed cells compared with the control group (P<0.01). Our findings strongly suggest that the adenoviral vector expressing hIGF-1 can successfully infect nucleus pulposus cells in vitro and effectively enhance the expression of IGF-1. In addition, IGF-1 reversed the TNF-α -induced apoptosis of nucleus pulposus cells. Thus, Ad-hIGF-1 may be useful in the development of clinical interventions for disc degeneration.

PDGF-AA and bFGF mediate B104CM-induced proliferation of oligodendrocyte precursor cells

The conditioned medium from B104 neuroblastoma cells (B104CM) induces proliferation of οligodendrocyte precursor cells (OPCs) in vitro, which indicates that certain factors contained within B104CM give instructional signals that direct the proliferation of OPCs. However, the OPC-proliferative factors present in B104CM have yet to be identified. Platelet-derived growth factor AA (PDGF-AA), basic fibroblast growth factor (bFGF) and insulin-like growth factor-1 (IGF-1) have been reported to act as potent mitogens for OPC proliferation. This raises the possibility that B104CM induces proliferation of OPCs through secretion of PDGF‑AA, bFGF and/or IGF-1. In the present study, we detected the expression and levels of PDGF-AA, bFGF and IGF-1 in B104 cells and B104CM, and observed the expression of their receptors in OPCs. The results indicated that these growth factors were expressed in B104 cells and B104CM. All 3 receptors, PDGFR, FGFR2 and IGF-1R, were also detected in OPCs. Furthermore, B104CM-stimulated OPC proliferation could be markedly decreased by both AG1295 (an inhibitor of PDGFR) and PD173074 (an inhibitor of FGFR). However, the inhibition of IGF-1R with AG1204 did not affect the proliferation of OPCs. Our study suggests that the PDGF-AA and bFGF in B104CM are 2 key factors that stimulate OPC proliferation.

Inhibitory effects of forkhead box L1 gene on osteosarcoma growth through the induction of cell cycle arrest and apoptosis

Forkhead box L1 (FOXL1), which is considered a novel candidate tumor suppressor, inhibits proliferation and invasion in certain types of cancer. However, the regulation and function of FOXL1 in osteosarcoma remains unclear. The expression of FOXL1 gene in osteosarcoma tissues and cell lines was examined using RT-PCR, immunohistochemistry and western blot analysis. pcDNA-FOXL1 carrying full-length FOXL1 cDNA was constructed to upregulate the level of FOXL1 expression in osteosarcoma cell lines. The proliferation, cell cycle and apoptosis of osteosarcoma cells in vitro or in vivo were examined following transfection with pcDNA-FOXL1. In addition, the expression of p21, p27, cytochrome c and caspase-3, which may be involved in the regulation of the cell cycle and apoptosis was examined using western blot analysis. The results showed that FOXL1 expression was downregulated in osteosarcoma tissues and cell lines. Loss or downregulation of FOXL1 was associated with poor prognosis. Ectopic FOXL1 expression inhibited cell proliferation in vitro and in vivo. The ectopic FOXL1 expression increased the expression of p21 and p27, which induced G1 arrest in the U-2 OS cells. In addition, the ectopic FOXL1 expression induced cytochrome c release between mitochondria and cytoplasm, which disrupted the mitochondrial transmembrane potential and triggered intrinsic pathway apoptosis. In conclusion, the downregulation of FOXL1 expression was associated with osteosarcoma cell growth. Restoration of FOXL1 gene expression by gene therapy may have a therapeutic potential for patients with osteosarcoma.

Olig1 and ID4 interactions in living cells visualized by bimolecular fluorescence complementation technique.

Olig1, a member of class B basic-helix-loop-helix (bHLH), plays key roles in early oligodendrocyte specification. Inhibitors of DNA binding (Id) is another sub-class of HLH proteins, act as dominant-negative regulators of bHLH proteins, which can form heterodimers with class A or B bHLH proteins, but lack the critical basic DNA binding domain. Id4 was recently found to interact with olig1 and inhibit oligodendrocyte differentiation. However, there still no direct evidence to reveal the spatial and temporal interaction of olig1 and ID4 in living cells. In this study, we performed bimolecular fluorescence complementation (BiFC) analysis to further characterize the distinct subcellular localization of olig1, ID4 and their dimer in living SW1116 cells. To examine the subcellular localization of olig1 and ID4 by themselves, the olig1-EGFP or ID4-DsRed2 fusion proteins were also expressed in SW1116 cells, respectively. As predicted, the olig1-EGFP fusion proteins were located in the nucleus, and ID4-DsRed2 fusion proteins were located in the cytoplasm. When olig1-EGFP and ID4-DsRed2 fusion proteins were co-expressed, the green and red signals were co-located in the cytoplasm. Using BiFC, the strong BiFC signals could be detected in pBiFC-olig1VN173 and pBiFC-ID4VC155 co-transfected cells and the fluorescence signal was located in the cytoplasm. These results collectively confirmed that olig1 and ID4 could interact and form dimer in living cells, and ID4 could block the transport of olig1 from cytoplasm to nucleus.

JNK is necessary for oligodendrocyte precursor cell proliferation induced by the conditioned medium from B104 neuroblastoma cells.

The conditioned medium from B104 neuroblastoma cells (B104CM) induces proliferation of oligodendrocyte progenitor cells (OPCs) in vitro. Our previous study showed that phosphorylation of extracellular signal-regulated protein kinase(s), not PI3K or p38, is key to B104CM-induced OPC proliferation. However, whether there are still other signaling pathways that are also involved in B104CM-induced proliferation remains unknown. In the present study, we evaluated the implication of c-Jun N-terminal kinase (JNK) signaling pathway in the B104CM-induced proliferation of OPCs using the specific inhibitor of JNK. We provided convincing evidence for the first time that the phosphorylation of JNK is necessary for OPC proliferation induced by B104CM in vitro. Moreover, the activation of JNK results in subsequent expressions of c-jun, c-fos, and c-myc, which initiates proliferation of OPCs. Collectively, these results suggest that JNK is also necessary for B104CM-induced OPC proliferation.