Yaoying Zeng

Quantitative Proteomics Analysis of Chondrogenic Differentiation of C3H10T1/2 Mesenchymal Stem Cells by iTRAQ Labeling Coupled with On-line Two-dimensional LC/MS/MS

The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.

TLR3-Involved Modulation of Pregnancy Tolerance in Double-Stranded RNA-Stimulated NOD/SCID Mice

This study aims to extend understanding of the relationship between TLR3-involved cell signaling and dsRNA-induced embryo resorption. Upon stimulation of dsRNA, the resorption rate of embryos was boosted dramatically in syngeneic mating BALB/c mice, but not significantly influenced in syngeneic mating NOD/SCID mice. Accordingly, there was an enhanced cell surface expression of TLR3 on placental CD45+ cells derived from BALB/c mice, concomitant with both increased percentages of CD45+CD80+ cells and CD8α+CD80+ cells in flow cytometric analysis. In addition, both increased IL-2 and decreased IL-10 expression could be observed in CD45+ cell group in the intracellular detection by flow cytometry. In contrast, no such trends were observed in NOD/SCID model, and its resorption rate of embryos was kept at a low level throughout pregnancy. Neutralizing Abs against TLR3 could abrogate the embryo rejection induced by dsRNA in BALB/c mice, and simultaneously could reduce the CD80+ percentage in the CD45+ cell group. These results indicate that the interaction between dsRNA and TLR3 may be involved in the mobilization of CD45+CD80+ and CD8α+CD80+ cells, followed by the up-regulation of IL-2 and down-regulation of IL-10 expression at the feto-maternal interface, and finally resulting in embryo rejection. The relatively low responsiveness of NOD/SCID mice may be one of the reasons why these mice appeared to be resistant to dsRNA-induced embryo resorption.

Icaritin exhibits anti-inflammatory effects in the mouse peritoneal macrophages and peritonitis model.

Icaritin, an intestinal metabolite of prenylflavonoids from Herba Epimedii, has been known to regulate many cellular processes. The purpose of this study was to investigate the protective effects of icaritin on inflammation in lipopolysaccharide (LPS) stimulated mouse peritoneal macrophages in vitro and zymosan induced peritonitis model in vivo. The release of Nitric oxide (NO) was measured by a Griess reagent system. The phagocytosis, the expression of CD69, the production of inflammatory cytokines and the leukocytes numbers were determined by flow cytometry. The Ca(2+) influx was recorded by confocal microscopy. The phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) was determined by Western blot. The results showed that icaritin significantly inhibited the NO, IL-6, IL-10 TNF-α, and MCP-1 production both in vitro and in vivo. Icaritin efficiently diminished the uptake of nonopsonized pHrodo™-labeled Escherichia coli bacteria on the LPS-stimulated macrophages. In addition, icaritin significantly inhibited the expression of CD69 on CD11b(+) macrophages. Icaritin pretreatment significantly inhibited the elevation of intracellular Ca(2+) induced by LPS. Furthermore, icaritin markedly decreased phospho-p38 and JNK protein expression in LPS-stimulated mouse peritoneal macrophages. In vivo study, it was also observed that icaritin prolonged survival of peritonitis mice, and inhibited massive leukocyte influx into the peritoneal cavity. These results suggest that icaritin possesses significant anti-inflammatory effects that may be mediated through the regulation of inflammatory cytokines and phosphorylation of p38 and JNK.

Calcium influx blocked by SK&F 96365 modulates the LPS plus IFN-γ-induced inflammatory response in murine peritoneal macrophages

A rise in intracellular Ca(2+) ([Ca(2+)](i)) is crucial for the activation of macrophages, however, the mechanisms and consequences of this [Ca(2+)](i) increase remain unclear. This study investigated the role of calcium in mouse peritoneal macrophages stimulated with LPS plus IFN-γ by using the store-operated Ca(2+) channel (SOCC) blocker SK&F 96365. Our results showed that SK&F 96365 pretreatment significantly inhibited the elevation of [Ca(2+)](i) induced by ionomycin, thapsigargin, and LPS plus IFN-γ, respectively. Phagocytosis analyzing results showed that SK&F 96365 efficiently diminished the uptake of nonopsonized 1 μM yellow-green beads or pHrodo™-labeled Escherichia coli bacteria both on the resting and LPS plus IFN-γ-stimulated macrophages. In addition, SK&F 96365 significantly inhibited the LPS plus IFN-γ-induced brisk uptake of NO and ROS. The CBA analyzing results showed that SK&F 96365 pretreatment efficiently inhibited the production of LPS plus IFN-γ-induced inflammatory cytokines of IL-6, MCP-1, TNF, INF-γ, and IL-10. However, SK&F 96365 pretreatment did not inhibit but augment the production of LPS plus IFN-γ-induced IL-1β secretion. Furthermore, SK&F 96365 pretreatment inhibited the LPS plus IFN-γ-induced translocation of NF-κB to the nucleus, and induced a decrease in mitochondrial membrane potential (ΔΨm) in LPS plus IFN-γ-activated macrophages. This study provides insight into the role of calcium in the activation of peritoneal macrophages induced by LPS plus IFN-γ, and blocking the calcium influx by SK&F 96365 exhibited a domain inhibitory effect on the LPS plus IFN-γ-induced inflammatory response in macrophages.

Topographical and Biological Evidence Revealed FTY720-Mediated Anergy-Polarization of Mouse Bone Marrow-Derived Dendritic Cells In Vitro

Abnormal inflammations are central therapeutic targets in numerous infectious and autoimmune diseases. Dendritic cells (DCs) are involved in these inflammations, serving as both antigen presenters and proinflammatory cytokine providers. As an immuno-suppressor applied to the therapies of multiple sclerosis and allograft transplantation, fingolimod (FTY720) was shown to affect DC migration and its crosstalk with T cells. We posit FTY720 can induce an anergy-polarized phenotype switch on DCs in vitro, especially upon endotoxic activation. A lipopolysaccharide (LPS)-induced mouse bone marrow-derived dendritic cell (BMDC) activation model was employed to test FTY720-induced phenotypic changes on immature and mature DCs. Specifically, methods for morphology, nanostructure, cytokine production, phagocytosis, endocytosis and specific antigen presentation studies were used. FTY720 induced significant alterations of surface markers, as well as decline of shape indices, cell volume, surface roughness in LPS-activated mature BMDCs. These phenotypic, morphological and topographical changes were accompanied by FTY720-mediated down-regulation of proinflammatory cytokines, including IL-6, TNF-α, IL-12 and MCP-1. Together with suppressed nitric oxide (NO) production and CCR7 transcription in FTY720-treated BMDCs with or without LPS activation, an inhibitory mechanism of NO and cytokine reciprocal activation was suggested. This implication was supported by the impaired phagocytotic, endocytotic and specific antigen presentation abilities observed in the FTY720-treated BMDCs. In conclusion, we demonstrated FTY720 can induce anergy-polarization in both immature and LPS-activated mature BMDCs. A possible mechanism is FTY720-mediated reciprocal suppression on the intrinsic activation pathway and cytokine production with endpoint exhibitions on phagocytosis, endocytosis, antigen presentation as well as cellular morphology and topography.

Bererine induces peripheral lymphocytes immune regulations to realize its neuroprotective effects in the cerebral ischemia/reperfusion mice

Previous studies have shown that Bererine (Ber) has significant neuroprotective effects and the present article aimed to investigate its mechanism from the aspect of peripheral immune system. We evaluated the effects of Ber 24 h after cerebral ischemia/reperfusion (I/R) on histologic injury in BALB/C mice and NOD-SCID (severe combined immunodeficient) mice lacking T and B cells. Infarct volume, neurological scores and brain water content were strikingly reduced by Ber in BALB/C mice versus NOD-SCID mice. Which suggested that Ber induces peripheral immune regulations to realize its neuroprotective effects in the cerebral I/R mice. Then we tested the lymphocytes from BALB/C lymph nodes (LNs) with their survival, activation, proliferation, cell cycle, apoptosis and differentiation induced by cytokine secretion to provide direct evidences that Ber realized its neuroprotective effects by regulating I/R-induced peripheral lymphocytes early immunoactivation and following immunotolerance and to better understand the importance of peripheral immune system following I/R insults.

Endogenous HIV-1 Vpr-mediated apoptosis and proteome alteration of human T-cell leukemia virus-1 transformed C8166 cells

HIV-1 viral protein R (Vpr) can induce cell cycle arrest and cell death, and may be beneficial in cancer therapy to suppress malignantly proliferative cell types, such as adult T-cell leukemia (ATL) cells. In this study, we examined the feasibility of employing the HIV-vpr gene, via targeted gene transfer, as a potential new therapy to kill ATL cells. We infected C8166 cells with a recombinant adenovirus carrying both vpr and GFP genes (rAd-vpr), as well as the vector control virus (rAd-vector). G2/M phase cell cycle arrest was observed in the rAd-vpr infected cells. Typical characteristics of apoptosis were detected in rAd-vpr infected cells, including sub-diploid peak exhibition in DNA content assay, the Hoechst 33342 accumulation, apoptotic body formation, mitochondrial membrane potential and plasma membrane integrity loss. The proteomic assay revealed apoptosis related protein changes, exhibiting the regulation of caspase-3 activity indicator proteins (vimentin and Rho GDP-dissociation inhibitor 2), mitochondrial protein (prohibitin) and other regulatory proteins. In addition, the up-regulation of anti-inflammatory redox protein, thioredoxin, was identified in the rAd-vpr infected group. Further supporting these findings, the increase of caspase 3&7 activity in the rAd-vpr infected group was observed. In conclusion, endogenous Vpr is able to kill HTLV-1 transformed C8166 cells, and may avoid the risks of inducing severe inflammatory responses through apoptosis-inducing and anti-inflammatory activities.

The anti-inflammatory effect of the SOCC blocker SK&F 96365 on mouse lymphocytes after stimulation by Con A or PMA/ionomycin.

SK&F 96365, 51-(beta-[3-(p-methoxyphenyl)-propyloxy]-p-methoxyphenethyl)-1H-imidazole hydrochloride, has emerged as a useful pharmacological tool in the study of store-operated Ca²⁺ entry (SOCE). But the precise molecular mechanism and effect of SK&F 96365 on mouse lymphocytes are still not well determined. This study investigated the pharmacological profile of SK&F 96365 on mouse lymphocytes stimulated by mitogen concanavalin A (Con A) or by a combination of a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA) and a calcium ionophore, ionomycin in vitro. Our results showed that SK&F 96365 pre-treatment diminished the cytosolic calcium rise on lymphocytes induced by ionomycin, PMA/ionomycin, and thapsigargin (TG), respectively. CFDA-SE staining results showed that SK&F 96365 (5-20 μM) inhibited both Con A- and PMA/ionomycin-induced lymphocytes proliferation in a time- and dose-dependent manner. Upon the same stimulation, SK&F 96365 inhibited the expression of CD69 and CD25 on CD3⁺ T lymphocytes in a dose-dependent manner. The cell cycle analyzing results showed that SK&F 96365 caused a G0/G1 phase cell cycle arrest on both Con A- and PMA/ionomycin-activated lymphocytes in a dose-dependent manner. In addition, SK&F 96365 induced a decrease in mitochondrial membrane potential (ΔΨm) and promoted mitochondrial permeability transition (MPT) in both Con A- and PMA/ionomycin-activated lymphocytes. Furthermore, SK&F 96365 significantly inhibited the production of proinflammatory cytokines (interferon (IFN)-γ and tumor necrosis factor (TNF)), and the anti-inflammatory cytokine (IL-10) on both Con A- and PMA/ionomycin-activated lymphocytes. SK&F 96365 did not induce a statistically significant increase in levels of proinflammatory IL-6 and monocyte chemoattractant protein-1 (MCP-1) but of IL-12p70 upon the stimulation of Con A, whereas these three cytokines were markedly inhibited by it upon the stimulation of PMA/ionomycin. This finding revealed that SK&F 96365 exhibited an anti-inflammatory effect on mouse lymphocytes both upon the stimulation of Con A and PMA/ionomycin, and the precise mechanism of SK&F 96365 inhibiting Con A-activated lymphocytes proliferation is different from PMA/ionomycin.

Indoleamine 2,3-dioxygenase (IDO) downregulates the cell surface expression of the CD4 molecule.

Indoleamine 2,3-dioxygenase (IDO) has been implicated in preventing the fetus from undergoing maternal T cell-mediated immune responses, yet the mechanism underlying these kinds of IDO-mediated immune responses has not been fully elucidated. Since the CD4 molecule plays a central role in the onset and regulation of antigen-specific immune responses, and T cell is sensitive in the absence of tryptophan, we hypothesize that IDO may reduce cell surface CD4 expression. To test this hypothesis, an adenoviral vector-based construct IDO-EGFP was generated and the effect of IDO-EGFP on CD4 expression was determined on recombinant adenoviral infected C8166 and MT-2 cells, by flow cytometry and/or Western blot analysis. The results revealed a significant downregulation of cell membrane CD4 in pAd-IDOEGFP infected cells when compared to that of mock-infected cells or infection with empty vector pAd-EGFP. Further experiments disclosed that either an addition of tryptophan or IDO inhibitor could partly restore CD4 expression in pAd-IDOEGFP infected C8166 cells. Our findings suggest that downregulation of CD4 by IDO might be one of the mechanisms through which IDO regulates T cell-mediated immune responses.

FTY720 mediates activation suppression and G 0/G 1 cell cycle arrest in a concanavalin A-induced mouse lymphocyte pan-activation model

ObjectiveFTY720 is a potent drug for multiple sclerosis treatment. To biologically address its possible applications to more generalized diseases with aberrant inflammation, we are testing whether FTY720 can function as a lymphocyte cell cycle blocker and activation suppressor via a concanavalin A (ConA)-mediated mouse lymphocyte pan-activation model.MethodsMouse lymphocytes were obtained from lymph nodes and subjected to ConA and/or FTY720 treatment. Cell viability was assayed by MTT and mitochondrial assays. Early and late activation, cell cycle, proliferation and intracellular Ca2+ concentration ([Ca2+]i) were analyzed by flow cytometry.ResultsAt concentrations of less than 500xa0nM, FTY720 significantly down-regulated both CD69 and CD25 expressions of T cells, as well as inhibiting proliferation of activated lymphocytes. In addition, FTY720 blocked the ConA-induced mitogenesis, exhibiting lymphocyte G0/G1 phase cell cycle arrest with significant reduction of cells in S and G2/M phases. Meanwhile, a significant decline in [Ca2+]i was observed. The correlation of [Ca2+]i and cell cycle arrest were validated by employing a [Ca2+]i inhibitor SK&F 96365 and testing with and without FTY720 treatment.ConclusionWe demonstrated that FTY720 induces G0/G1 phase cell cycle arrest, resulting in proliferation inhibition upon lymphocyte pan-activation, which may be related to reduction of overall intracellular Ca2+ load.