Heme Oxygenase-1 Ameliorates Dextran Sulfate Sodium-induced Acute Murine Colitis by Regulating Th17/Treg Cell Balance

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn''s disease, is a group of autoimmune diseases characterized by nonspecific inflammation in the gastrointestinal tract. Recent investigations suggest that activation of Th17 cells and/or deficiency of regulatory T cells (Treg) is involved in the pathogenesis of IBD. Heme oxygenase (HO)-1 is a protein with a wide range of anti-inflammatory and immune regulatory function, which exerts significantly protective roles in various T cell-mediated diseases. In this study, we aim to explore the immunological regulation of HO-1 in the dextran sulfate sodium-induced model of experimental murine colitis. BALB/c mice were administered 4% dextran sulfate sodium orally; some mice were intraperitoneally pretreated with HO-1 inducer hemin or HO-1 inhibitor stannum protoporphyrin IX. The results show that hemin enhances the colonic expression of HO-1 and significantly ameliorates the symptoms of colitis with improved histological changes, accompanied by a decreased proportion of Th17 cells and increased number of Tregs in mesenteric lymph node and spleen. Moreover, induction of HO-1 down-regulates retinoic acid-related orphan receptor γt expression and IL-17A levels, while promoting Treg-related forkhead box p3 (Foxp3) expression and IL-10 levels in colon. Further study in vitro revealed that up-regulated HO-1 switched the naive T cells to Tregs when cultured under a Th17-inducing environment, which involved in IL-6R blockade. Therefore, HO-1 may exhibit anti-inflammatory activity in the murine model of acute experimental colitis via regulating the balance between Th17 and Treg cells, thus providing a possible novel therapeutic target in IBD.     

PARP Inhibition Attenuates Acute Kidney Allograft Rejection by Suppressing Cell Death Pathways and Activating PI-3K-Akt Cascade

Background

Novel immunosuppressive therapy facilitates long term allograft survival, but acute tubular necrosis and ischemia-reperfusion during transplantation can compromise allograft function. These processes are related to oxidative stress which activates poly- (ADP-ribose) polymerase (PARP) contributing to the activation of cell death pathways. Here we raised the possibility that PARP inhibition curbs cell death pathways and shifts kinase signaling to improved graft survival.

Methods Findings

In an acute rat kidney rejection model, we provided evidence that the PARP inhibitor 4-hydroxy-quinazoline (4OHQ) attenuates rejection processes initiated oxidative/nitrosative stress, nuclear poly-ADP-ribosylation and the disintegration of the tubulo-interstitial structures. The PARP inhibitor attenuated rejection processes induced pro-apoptotic pathways by increasing Bcl-2/Bax ratio and suppressing pro-apoptotic t-Bid levels. In transplanted kidneys, the cell death inducing JNK1/2 is normally activated, but PARP inhibition suppressed this activation with having only modest effects on ERK1/2 and p38 MAP kinases. In untreated transplanted kidneys, no significant alterations were detected in the cytoprotective PI-3K-Akt pathway, but the PARP inhibitor significantly activated Akt (by S473 phosphorylation) and suppressed GSK-3β, as well as activated acute NF-kappaB activation contributing to graft protection.

Conclusion

These data show the protective role of PARP inhibition on graft survival by attenuating poly-ADP-ribosylation, oxidative stress, suppressing pro-apoptotic and increasing anti-apoptotic protein level, and by shifting MAP kinases and PI-3-K-Akt pathways to cytoprotective direction. Thus, addition of PARP inhibitors to standard immunosuppressive therapies during kidney transplantation may provide increased protection to prolong graft survival.     

The Glycosylated Rv1860 Protein of Mycobacterium tuberculosis Inhibits Dendritic Cell Mediated TH1 and TH17 Polarization of T Cells and Abrogates Protective Immunity Conferred by BCG

We previously reported interferon gamma secretion by human CD4⁺ and CD8⁺ T cells in response to recombinant E. coli-expressed Rv1860 protein of Mycobacterium tuberculosis (MTB) as well as protection of guinea pigs against a challenge with virulent MTB following prime-boost immunization with DNA vaccine and poxvirus expressing Rv1860. In contrast, a Statens Serum Institute Mycobacterium bovis BCG (BCG-SSI) recombinant expressing MTB Rv1860 (BCG-TB1860) showed loss of protective ability compared to the parent BCG strain expressing the control GFP protein (BCG-GFP). Since Rv1860 is a secreted mannosylated protein of MTB and BCG, we investigated the effect of BCG-TB1860 on innate immunity. Relative to BCG-GFP, BCG-TB1860 effected a significant near total reduction both in secretion of cytokines IL-2, IL-12p40, IL-12p70, TNF-α, IL-6 and IL-10, and up regulation of co-stimulatory molecules MHC-II, CD40, CD54, CD80 and CD86 by infected bone marrow derived dendritic cells (BMDC), while leaving secreted levels of TGF-β unchanged. These effects were mimicked by BCG-TB1860His which carried a 6-Histidine tag at the C-terminus of Rv1860, killed sonicated preparations of BCG-TB1860 and purified H37Rv-derived Rv1860 glycoprotein added to BCG-GFP, but not by E. coli-expressed recombinant Rv1860. Most importantly, BMDC exposed to BCG-TB1860 failed to polarize allogeneic as well as syngeneic T cells to secrete IFN-γ and IL-17 relative to BCG-GFP. Splenocytes from mice infected with BCG-SSI showed significantly less proliferation and secretion of IL-2, IFN-γ and IL-17, but secreted higher levels of IL-10 in response to in vitro restimulation with BCG-TB1860 compared to BCG-GFP. Spleens from mice infected with BCG-TB1860 also harboured significantly fewer DC expressing MHC-II, IL-12, IL-2 and TNF-α compared to mice infected with BCG-GFP. Glycoproteins of MTB, through their deleterious effects on DC may thus contribute to suppress the generation of a TH1- and TH17-dominated adaptive immune response that is vital for protection against tuberculosis.     

Inhibition of Cancer Cell Migration and Invasion through Suppressing the Wnt1-mediating Signal Pathway by G-quadruplex Structure Stabilizers

WNT1 encodes a multifunctional signaling glycoprotein that is highly expressed in several malignant tumors. Patients with Wnt1-positive cancer are usually related to advanced metastasis. Here, we found that a stretch of G-rich sequences located at the WNT1 promoter region is capable of forming G-quadruplex structures. The addition of G-quadruplex structure stabilizers, BMVC and BMVC4, raises the melting temperature of the oligonucleotide formed by the WNT1 promoter G-rich sequences. Significantly, the expression of WNT1 was repressed by BMVC or BMVC4 in a G-quadruplex-dependent manner, suggesting that they can be used to modulate WNT1 expression. The role of G-quadruplex stabilizers on Wnt1-mediated cancer migration and invasion was further analyzed. The protein levels of β-catenin, a mediator of the Wnt-mediated signaling pathway, and the downstream targets MMP7 and survivin were down-regulated upon BMVC or BMVC4 treatments. Moreover, the migration and invasion activities of cancer cells were inhibited by BMVC and BMVC4, and the inhibitory effects can be reversed by WNT1-overexpression. Thus the Wnt1 expression and its downstream signaling pathways can be regulated through the G-quadruplex sequences located at its promoter region. These findings provide a novel approach for future drug development to inhibit migration and invasion of cancer cells.     

The Inhibition of Human Tumor Cell Proliferation by RNase Pol,a Member of the RNase T1 Family,from Pleurotus ostreatus

RNase Po1 is a guanylic acid-specific ribonuclease (a RNase T1 family RNase) from Pleurotus ostreatus. We determined the cDNA sequence encoding RNase Po1 and expressed RNase Po1 in Escherichia coli. A comparison of the enzymatic properties of RNase Po1 and RNase T1 indicated that the optimum temperature for RNase Po1 activity was 20 °C higher than that for RNase T1. An MTT assay indicated that RNase Po1 inhibits the proliferation of human neuroblastoma cells (IMR-32 and SK-N-SH) and human leukemia cells (Jurkat and HL-60). Furthermore, Hoechst 33342 staining showed morphological changes in HL-60 cells due to RNase Po1, and flow cytometry indicated the appearance of a sub-G1 cell population. The extent of these changes was dependent on the concentration of RNase Pol. We suggest that RNase Po1 induces apoptosis in tumor cells.     

Regulation of Th1/Th2 and Th17/Treg by pDC/mDC imbalance in primary immune thrombocytopenia

This study investigates the regulatory effect of plasmacytoid dendritic cells (pDC)/myeloid dendritic cells (mDC) imbalance on balance of Th1/Th2 and Th17/Treg in primary immune thrombocytopenia (ITP). A total of 30 untreated ITP patients and 20 healthy controls were recruited. Compared with healthy control, the pDC proportion of ITP patients was significantly reduced (P = 0.004), while the mDC proportion was not significantly changed (P = 0.681), resulting in a decrease in the pDC/mDC ratio (P = 0.001). Additionally, compared with controls, serum levels of interleukin (IL)-6, IL-12, and IL-23 were increased in ITP patients (P < 0.001), and mRNA levels of IL-12p40, IL-12p35, and IL-23p19 were also increased (P =0.014, P = 0.043, P < 0.001). Compared with the healthy control, the proportion of Th1 and Th17 cells in ITP patients increased (P = 0.001, P = 0.031). Serum levels of interferon gamma (IFN-γ) and IL-17 in ITP patients also increased (P = 0.025, P = 0.005). Furthermore, T-bet and RORγt mRNA levels were increased in peripheral blood of ITP patients (P = 0.018, P < 0.001). Correspondingly, the proportion of Th2 and Treg cells decreased (P = 0.007, P < 0.001), along with a decrease in serum IL-4 and transforming growth factor beta (TGF-β) (P = 0.028, P = 0.042), and an increase in GATA-3 mRNA (P < 0.001). However, there was no significant difference in Foxp3 mRNA levels (P = 0.587). Pearson correlation analysis showed that the proportion of total dendritic cells (DCs) was positively correlated with IL-12 (r = 0.526, P = 0.003) and IL-23 (r = 0.501, P = 0.005) in ITP patients. Th1/Th2 ratio, IFN-γ, and IL-12 levels were negatively correlated with platelet counts (r = −0.494, P = 0.009; r = –0.415, P = 0.028; r = –0.492, P = 0.032). However, IL-23 was positively correlated with IL-17 (r = 0.489, P = 0.006) and negatively correlated with platelet count (r = –0.564, P = 0.001). The ratio of IL-6 and Th17 cells was negatively correlated with platelet count (r = –0.443, P = 0.014; r = –0.471, P = 0.011). The imbalance of pDC/mDC and the increase of IL-6, IL-12, and IL-23 lead to the increased differentiation of CD4+ T cells into Th1 and Th17 cells, which might be the important mechanisms underlying the imbalance of Th1/Th2 and Th17/Treg in ITP patients.     

Interleukin 7 Plays a Role in T Lymphocyte Apoptosis Inhibition Driven by Mesenchymal Stem Cell without Favoring Proliferation and Cytokines Secretion

Since 2004, when a case report describing the use of human mesenchymal stem cells (hMSCs) infusion as a therapy for GVHD after bone marrow transplantation, a new perspective in MSC function emerged. Since then hMSCs immunomodulatory potential became the target of several studies. Although great progress has been made in our understanding of hMSCs, their effect on T cell remains obscure. Our study has confirmed the already described effect of hMSCs on lymphocytes proliferation and survival. We also show that the impairment of lymphocyte proliferation and apoptosis is contact-independent and occurs in a prostaglandin-independent manner. A potential correlation between IL-7 and hMSCs effect is suggested, as we observed an increase in IL-7 receptors (CD127) on lymphocyte membrane in MSC presence. Additionally, blocking IL-7 in hMSCs-lymphocytes co-cultures increased lymphocytes apoptosis and we also have demonstrated that hMSCs are able to produce this interleukin. Moreover, we found that during Th1/Th17 differentiation in vitro, hMSCs presence leads to Th1/Th17 cells with reduced capacity of INF-y and IL-17 secretion respectively, regardless of having several pro-inflammatory cytokines in culture. We did not confirm an increment of Treg in these cultures, but a reduced percentage of INF-y/IL-17 secreting cells was observed, suggesting that the ratio between anti and pro-inflammatory cells changed. This changed ratio is very important to GvHD therapy and links hMSCs to an anti-inflammatory role. Taken together, our findings provide important preliminary results on the lymphocyte pathway modulated by MSCs and may contribute for developing novel treatments and therapeutic targets for GvHD and others autoimmune diseases.     

MiR‐133a‐3p Inhibits Oral Squamous Cell Carcinoma (OSCC) Proliferation and Invasion by Suppressing COL1A1

The aim of our study was to investigate the effects of miR‐133a‐3p on human oral squamous cell carcinoma (OSCC) cells by regulating gene COL1A1. OSCC tissues, adjacent tongue epithelial tissues, the immortalized oral epithelial cell line HIOEC, and OSCC cell lines (CAL‐27, TCA‐8113, SCC‐4, SCC‐9, and SCC‐15) were used in this research. Quantitative real‐time PCR (RT‐qPCR) was employed to determine the expression of miR‐133a‐3p and COL1A1. Dual luciferase reporter gene assay and Western blot were applied to verify the binding relationship between miR‐133a‐3p and COL1A1. Functional assays were also conducted in this study, including CCK‐8 assay, colony formation assay, flow cytometry analysis as well as Transwell assay. MiR‐133a‐3p was found low‐expressed both in OSCC tissues and cells lines compared with normal tissues and cell line, respectively, whereas COL1A1 was just the opposite. The over‐expression of miR‐133a‐3p or the down‐regulation of COL1A1 suppressed the proliferation, invasion, and mitosis of OSCC cells, whereas simultaneous down‐regulation of miR‐133a‐3p and up‐regulation of COL1A1 led to no significant alteration of cell activities. MiR‐133a‐3p could inhibit the proliferation and migration of OSCC cells through directly targeting COL1A1 and reducing its expression. J. Cell. Biochem. 119: 338–346, 2018. © 2017 Wiley Periodicals, Inc.     

Exendin-4 Prevents Vascular Smooth Muscle Cell Proliferation and Migration by Angiotensin II via the Inhibition of ERK1/2 and JNK Signaling Pathways

Angiotensin II (Ang II) is a main pathophysiological culprit peptide for hypertension and atherosclerosis by causing vascular smooth muscle cell (VSMC) proliferation and migration. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, is currently used for the treatment of type-2 diabetes, and is believed to have beneficial effects for cardiovascular diseases. However, the vascular protective mechanisms of GLP-1 receptor agonists remain largely unexplained. In the present study, we examined the effect of exendin-4 on Ang II-induced proliferation and migration of cultured rat aortic smooth muscle cells (RASMC). The major findings of the present study are as follows: (1) Ang II caused a phenotypic switch of RASMC from contractile type to synthetic proliferative type cells; (2) Ang II caused concentration-dependent RASMC proliferation, which was significantly inhibited by the pretreatment with exendin-4; (3) Ang II caused concentration-dependent RASMC migration, which was effectively inhibited by the pretreatment with exendin-4; (4) exendin-4 inhibited Ang II-induced phosphorylation of ERK1/2 and JNK in a pre-incubation time-dependent manner; and (5) U0126 (an ERK1/2 kinase inhibitor) and SP600125 (a JNK inhibitor) also inhibited both RASMC proliferation and migration induced by Ang II stimulation. These results suggest that exendin-4 prevented Ang II-induced VSMC proliferation and migration through the inhibition of ERK1/2 and JNK phosphorylation caused by Ang II stimulation. This indicates that GLP-1 receptor agonists should be considered for use in the treatment of cardiovascular diseases in addition to their current use in the treatment of diabetes mellitus.     

SAHA抑制间充质干细胞C3H10T1/2的增殖与成脂分化

本文研究组蛋白去乙酰化酶抑制剂辛二酰苯胺异羟肟酸（suberoylanilide hydroxamic acid,SAHA）对间充质干细胞(mesenchymal stem cells, MSCs) C3H10T1/2增殖和成脂分化的影响及其可能的作用机制.用Western印迹验证SAHA对细胞内蛋白乙酰化的影响;用MTT和流式细胞术检测细胞活性和细胞周期;利用油红O染色检测细胞成脂分化,实时定量PCR检测PPARγ2和成脂分化标志物Fabp4、perilipin以及adipoq mRNA的转录.Western印迹结果显示,SAHA可促进细胞内蛋白的乙酰化.MTT和流式细胞术结果显示,SAHA对C3H10T1/2细胞活性的抑制呈浓度依赖性,随着SAHA浓度增加,细胞形态趋向于展平,并将细胞周期抑制在G0/G1期;SAHA可呈浓度依赖性抑制C3H10T1/2 细胞的成脂分化作用.同时实时定量PCR结果显示,SAHA抑制成脂关键转录因子PPARγ2,脂肪因子Fabp4、perilipin和adipoq mRNA的转录.综上所述,SAHA可影响间充质干细胞C3H10T1/2细胞形态,并呈剂量依赖性地抑制其增殖和成脂分化.     

EGR1/2 Inhibits Papillary Thyroid Carcinoma Cell Growth by Suppressing the Expression of PTEN and BAX

Biochemical Genetics - Early growth response (EGR) proteins have been reported to be involved in cell growth and apoptosis in a variety of cancer types and could inhibit tumor development. However,...