Phenotypic comparison and the potential antitumor function of immortalized bone marrow-derived macrophages (iBMDMs).

Introduction: Macrophages are an important component of innate immunity and involved in the immune regulation of multiple diseases. The functional diversity and plasticity make macrophages to exhibit different polarization phenotypes after different stimuli. During tumor progression, the M2-like polarized tumor-associated macrophages (TAMs) promote tumor progression by assisting immune escape, facilitating tumor cell metastasis, and switching tumor angiogenesis. Our previous studies demonstrated that functional remodeling of TAMs through engineered-modifying or gene-editing provides the potential immunotherapy for tumor. However, lack of proliferation capacity and maintained immune memory of infused macrophages restricts the application of macrophage-based therapeutic strategies in the repressive tumor immune microenvironment (TIME). Although J2 retrovirus infection enabled immortalization of bone marrow-derived macrophages (iBMDMs) and facilitated the mechanisms exploration and application, little is known about the phenotypic and functional differences among multi kinds of macrophages. Methods: HE staining was used to detect the biosafety of iBMDMs, and real-time quantitative PCR, immunofluorescence staining, and ELISA were used to detect the polarization response and expression of chemokines in iBMDMs. Flow cytometry, scratch assay, real-time quantitative PCR, and crystal violet staining were used to analyze its phagocytic function, as well as its impact on tumor cell migration, proliferation, and apoptosis. Not only that, the inhibitory effect of iBMDMs on tumor growth was detected through subcutaneous tumor loading, while the tumor tissue was paraffin sectioned and flow cytometry was used to detect its impact on the tumor microenvironment. Results: In this study, we demonstrated iBMDMs exhibited the features of rapid proliferation and long-term survival. We also compared iBMDMs with RAW264.7 cell line and mouse primary BMDMs with in vitro and in vivo experiments, indicating that the iBMDMs could undergo the same polarization response as normal macrophages with no obvious cellular morphology changes after polarization. What's more, iBMDMs owned stronger phagocytosis and pro-apoptosis functions on tumor cells. In addition, M1-polarized iBMDMs could maintain the anti-tumor phenotypes and domesticated the recruited macrophages of receptor mice, which further improved the TIME and repressed tumor growth. Discussion: iBMDMs can serve as a good object for the function and mechanism study of macrophages and the optional source of macrophage immunotherapy.

Myeloid-specific blockade of notch signaling alleviates dopaminergic neurodegeneration in Parkinson's disease by dominantly regulating resident microglia activation through NF-κB signaling.

Yolk sac-derived microglia and peripheral monocyte-derived macrophages play a key role during Parkinson's disease (PD) progression. However, the regulatory mechanism of microglia/macrophage activation and function in PD pathogenesis remains unclear. Recombination signal-binding protein Jκ (RBP-J)-mediated Notch signaling regulates macrophage development and activation. In this study, with an 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) hydrochloride-induced acute murine PD model, we found that Notch signaling was activated in amoeboid microglia accompanied by a decrease in tyrosine hydroxylase (TH)-positive neurons. Furthermore, using myeloid-specific RBP-J knockout (RBP-JcKO) mice combined with a PD model, our results showed that myeloid-specific disruption of RBP-J alleviated dopaminergic neurodegeneration and improved locomotor activity. Fluorescence-activated cell sorting (FACS) analysis showed that the number of infiltrated inflammatory macrophages and activated major histocompatibility complex (MHC) II+ microglia decreased in RBP-JcKO mice compared with control mice. Moreover, to block monocyte recruitment by using chemokine (C-C motif) receptor 2 (CCR2) knockout mice, the effect of RBP-J deficiency on dopaminergic neurodegeneration was not affected, indicating that Notch signaling might regulate neuroinflammation independent of CCR2+ monocyte infiltration. Notably, when microglia were depleted with the PLX5622 formulated diet, we found that myeloid-specific RBP-J knockout resulted in more TH+ neurons and fewer activated microglia. Ex vitro experiments demonstrated that RBP-J deficiency in microglia might reduce inflammatory factor secretion, TH+ neuron apoptosis, and p65 nuclear translocation. Collectively, our study first revealed that RBP-J-mediated Notch signaling might participate in PD progression by mainly regulating microglia activation through nuclear factor kappa-B (NF-κB) signaling.

Babesia gibsoni Whole-Genome Sequencing, Assembling, Annotation, and Comparative Analysis.

The intracellular protozoan parasite Babesia gibsoni infects canine erythrocytes and causes babesiosis. The hazards to animal health have increased due to the rise of B. gibsoni infections and medication resistance. However, the lack of high-quality full-genome sequencing sets has expanded the obstacles to the development of pathogeneses, drugs, and vaccines. In this study, the whole genome of B. gibsoni was sequenced, assembled, and annotated. The genomic size of B. gibsoni was 7.94 Mbp in total. Four chromosomes with the size of 0.69 Mb, 2.10 Mb, 2.77 Mb, and 2.38 Mb, respectively, 1 apicoplast (28.4 Kb), and 1 mitochondrion (5.9 Kb) were confirmed. KEGG analysis revealed 2,641 putative proteins enriched on 316 pathways, and GO analysis showed 7,571 annotations of the nuclear genome in total. Synteny analysis showed a high correlation between B. gibsoni and B. bovis. A new divergent point of B. gibsoni occurred around 297.7 million years ago, which was earlier than that of B. bovis, B. ovata, and B. bigemina. Orthology analysis revealed 22 and 32 unique genes compared to several Babesia spp. and apicomplexan species. The metabolic pathways of B.gibsoni were characterized, pointing to a minimal size of the genome. A species-specific secretory protein SA1 and 19 homologous genes were identified. Selected specific proteins, including apetala 2 (AP2) factor, invasion-related proteins BgAMA-1 and BgRON2, and rhoptry function proteins BgWH_04g00700 were predicted, visualized, and modeled. Overall, whole-genome sequencing provided molecular-level support for the diagnosis, prevention, clinical treatment, and further research of B. gibsoni. IMPORTANCE The whole genome of B. gibsoni was first sequenced, annotated, and disclosed. The key part of genome composition, four chromosomes, was comparatively analyzed for the first time. A full-scale phylogeny evolution analysis based on the whole-genome-wide data of B. gibsoni was performed, and a new divergent point on the evolutionary path was revealed. In previous reports, molecular studies were often limited by incomplete genomic data, especially in key areas like life cycle regulation, metabolism, and host-pathogen interaction. With the whole-genome sequencing of B. gibsoni, we provide useful genetic data to encourage the exploration of new terrain and make it feasible to resolve the theoretical and practical problems of babesiosis.

Notch-mediated lactate metabolism regulates MDSC development through the Hes1/MCT2/c-Jun axis.

Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) play critical roles in tumorigenesis. However, the mechanisms underlying MDSC and TAM development and function remain unclear. In this study, we find that myeloid-specific activation of Notch/RBP-J signaling downregulates lactate transporter MCT2 transcription via its downstream molecule Hes1, leading to reduced intracellular lactate levels, blunted granulocytic MDSC (G-MDSC) differentiation, and enhanced TAM maturation. We identify c-Jun as a novel intracellular sensor of lactate in myeloid cells using liquid-chromatography-mass spectrometry (LC-MS) followed by CRISPR-Cas9-mediated gene disruption. Meanwhile, lactate interacts with c-Jun to protect from FBW7 ubiquitin-ligase-mediated degradation. Activation of Notch signaling and blockade of lactate import repress tumor progression by remodeling myeloid development. Consistently, the relationship between the Notch-MCT2/lactate-c-Jun axis in myeloid cells and tumorigenesis is also confirmed in clinical lung cancer biopsies. Taken together, our current study shows that lactate metabolism regulated by activated Notch signaling might participate in MDSC differentiation and TAM maturation.

Astragaloside IV Alleviates the Experimental DSS-Induced Colitis by Remodeling Macrophage Polarization Through STAT Signaling.

Inflammatory bowel disease (IBD) is characterized by chronic and relapsing intestinal inflammation, which currently lacks safe and effective medicine. Some previous studies indicated that Astragaloside IV (AS-IV), a natural saponin extracted from the traditional Chinese medicine herb Ligusticum chuanxiong, alleviates the experimental colitis symptoms in vitro and in vivo. However, the mechanism of AS-IV on IBD remains unclear. Accumulating evidence suggests that M2-polarized intestinal macrophages play a pivotal role in IBD progression. Here, we found that AS-IV attenuated clinical activity of DSS-induced colitis that mimics human IBD and resulted in the phenotypic transition of macrophages from immature pro-inflammatory macrophages to mature pro-resolving macrophages. In vitro, the phenotype changes of macrophages were observed by qRT-PCR after bone marrow-derived macrophages (BMDMs) were induced to M1/M2 and incubated with AS-IV, respectively. In addition, AS-IV was effective in inhibiting pro-inflammatory macrophages and promoting the pro-resolving macrophages to ameliorate experimental colitis via the regulation of the STAT signaling pathway. Hence, we propose that AS-IV can ameliorate experimental colitis partially by modulating macrophage phenotype by remodeling the STAT signaling, which seems to have an essential function in the ability of AS-IV to alleviate the pathological progress of IBD.

miR-139/PDE2A-Notch1 feedback circuit represses stemness of gliomas by inhibiting Wnt/ß-catenin signaling.

Rationale: The malignant phenotypes of glioblastomas (GBMs) are primarily attributed to glioma stem cells (GSCs). Our previous study and other reports have suggested that both miR-139 and its host gene PDE2A are putative antitumor genes in various cancers. The aim of this study was to investigate the roles and mechanisms of miR-139/PDE2A in GSC modulation. Methods: Clinical samples were used to determine miR-139/PDE2A expression. Patient-derived glioma stem-like cells (PD-GSCs) were stimulated for immunofluorescent staining, sphere formation assays and orthotopic GBM xenograft models. Bioinformatic analysis and further in vitro experiments demonstrated the downstream molecular mechanisms of miR-139 and PDE2A. OX26/CTX-conjugated PEGylated liposome (OCP) was constructed to deliver miR-139 or PDE2A into glioma tissue specifically. Results: We demonstrated that miR-139 was concomitantly transcribed with its host gene PDE2A. Both PDE2A and miR-139 indicated better prognosis of gliomas and were inversely correlated with GSC stemness. PDE2A or miR-139 overexpression suppressed the stemness of PD-GSCs. FZD3 and ß-catenin, which induced Wnt/ß-catenin signaling activation, were identified as targets of miR-139 and mediated the effects of miR-139 on GSCs. Meanwhile, PDE2A suppressed Wnt/ß-catenin signaling by inhibiting cAMP accumulation and GSK-3ß phosphorylation, thereby modulating the self-renewal of PD-GSCs. Notably, Notch1, which is also a target of miR-139, suppressed PDE2A/miR-139 expression directly via downstream Hes1, indicating that miR-139 promoted its own expression by the miR-139-Notch1/Hes1 feedback circuit. Expectedly, targeted overexpression miR-139 or PDE2A in glioma with OCP system significantly repressed the stemness and decelerated glioma progression. Conclusions: Our findings elaborate on the inhibitory functions of PDE2A and miR-139 on GSC stemness and tumorigenesis, which may provide new prognostic markers and therapeutic targets for GBMs.

Numerical Study on the Characteristics of Boger Type Viscoelastic Fluid Flow in a Micro Cross-Slot under Sinusoidal Stimulation.

The cross-slot geometry plays an important role in the study of nonlinear effects of viscoelastic fluids. The flow of viscoelastic fluid in a micro cross-slot with a high channel aspect ratio (AR, the ratio of channel depth to width) can be divided into three types, which are symmetric flow, steady-state asymmetric flow and time-dependent flow under the inlet condition with a constant velocity. However, the flow pattern of a viscoelastic fluid in the cross-slot when a stimulation is applied at inlets has been rarely reported. In this paper, the response of cross-slot flow under an external sinusoidal stimulation is studied by numerical simulations of a two-dimensional model representing the geometry with a maximum limit of AR. For the cases under constant inlet velocity conditions, three different flow patterns occur successively with the increase of Weissenberg number (Wi). For the cases under sinusoidal varying inlet velocity conditions, when the stimulation frequency is far away from the natural frequency of a viscoelastic fluid, the frequency spectrum of velocity fluctuation field shows the characteristics of a fundamental frequency and several harmonics. However, the harmonic frequency disappears when the stimulation frequency is close to the natural frequency of the viscoelastic fluid. Besides, the flow pattern shows spatial symmetry and changes with time. In conclusion, the external stimulation has an effect on the flow pattern of viscoelastic fluid in the 2D micro cross-slot channel, and a resonance occurs when the stimulation frequency is close to the natural frequency of the fluid.

Targeted delivery of miR-99b reprograms tumor-associated macrophage phenotype leading to tumor regression.

BACKGROUND: Accumulating evidence has shown that tumor-associated macrophages (TAMs) play a critical role in tumor progression. Targeting TAMs is a potential strategy for tumor immunotherapy. However, the mechanism underlying the TAM phenotype and function needs to be resolved. Our previous studies have demonstrated that miR-125a can reverse the TAM phenotype toward antitumor. Meanwhile, we have found that miR-125a and miR-99b cluster in the first intron of the same host gene, and are transcribed simultaneously in bone marrow-derived macrophages (BMDMs) following LPS+IFNγ stimulation. However, it remains unclear whether miR-99b by itself can exert an antitumor effect by regulating macrophage phenotype. METHODS: miR-99b and/or miR-125a were delivered into TAMs of orthotopic hepatocellular carcinoma (HCC) or subcutaneous Lewis lung cancer (LLC) mice. The effect of treatment was evaluated by live imaging, TUNEL staining and survival tests. The phenotype of the immune cells was determined by qRT-PCR, ELISA, western blot and FACS. The capability of miR-99b-mediated macrophage phagocytosis and antigen presentation was detected by FACS and immunofluorescence staining. The underlying molecular mechanism was examined by qRT-PCR, reporter assay and western blot, and further verified in the tumor model. The expression of miR-99b and its target genes was determined in TAMs sorted from tumor and adjacent tissues in patients with liver cancer. RESULTS: Targeted delivery of miR-99b and/or miR-125a into TAMs significantly impeded the growth of HCC and LLC, especially after miR-99b delivery. More importantly, the delivery of miR-99b re-educated TAM toward antitumor phenotype with enhanced immune surveillance. Further investigation of mechanisms showed that macrophage-specific overexpression of miR-99b promoted M1 while suppressing M2 macrophage polarization by targeting κB-Ras2 and/or mTOR, respectively. miR-99b-overexpressed M1 macrophage was characterized by stronger capability of phagocytosis and antigen presentation. Additionally, delivery of simTOR or siκB-Ras2 into TAMs inhibited miR-99b antagomir-triggered tumor growth. Finally, miR-99b expression was lower in TAMs of patients with liver cancer than that in adjacent tissues, while the expression of κB-Ras2 and mTOR was reversed. CONCLUSIONS: Our results reveal the mechanism of miR-99b-mediated TAM phenotype, indicating that TAM-targeted delivery of miR-99b is a potential strategy for cancer immunotherapy.

Downregulation of FHL1 protein in glioma inhibits tumor growth through PI3K/AKT signaling.

Human four-and-a-half LIM domains protein 1 (FHL1) is a member of the FHL protein family, which serves an important role in multiple cellular events by interacting with transcription factors using its cysteine-rich zinc finger motifs. A previous study indicated that FHL1 was downregulated in several types of human cancer and served a role as a tumor suppressive gene. The overexpression of FHL1 inhibited tumor cell proliferation. However, to the best of our knowledge, there is no evidence to confirm whether FHL1 affected glioma growth, and the molecular mechanisms through which FHL1 represses tumor development remain unclear. In the present study, the expression level of FHL1 was determined using immunohistochemical staining in 114 tumor specimens from patients with glioma. The results indicated that FHL1 expression was negatively associated with the pathological grade of gliomas. Furthermore, Kaplan-Meier survival curves demonstrated that the patients with an increased FHL1 expression exhibited a significantly longer survival time, suggesting that FHL1 may be a prognostic marker for glioma. The protein level of FHL1 was relatively increased in the U251 glioma cell line compared with that in the U87 cell line. Therefore, FHL1 was knocked down in U251 by siRNA and overexpressed in U87, and it was identified that FHL1 significantly decreased the activation of PI3K/AKT signaling by interacting with AKT. Further experiments verified that FHL1 inhibited the growth of gliomas in vivo by modulating PI3K/AKT signaling. In conclusion, the results of the present study demonstrated that FHL1 suppressed glioma development through PI3K/AKT signaling.

MicroRNA-144 represses gliomas progression and elevates susceptibility to Temozolomide by targeting CAV2 and FGF7.

Malignant gliomas are the most common tumor in central nervous system with poor prognosis. Due to the limitation of histological classification in earlier diagnosis and individualized medicine, it is necessary to combine the molecular signatures and the pathological characteristics of gliomas. Lots of microRNAs presented abnormal expression in gliomas and modulated gliomas development. Exploration the miRNAs profile is helpful for the diagnosis, therapy and prognosis of gliomas. It has been demonstrated that miR-144 plays important roles in solid tumors. However, the detail mechanisms remained unrevealed. In this study, we have demonstrated the level of miR-144 decreased in glioma tissues from patients, especially in gliomas with higher grades. MiR-144 was also validated have lower expression in glioma cell lines compared with cortical neuron cell by using qRT-PCR. The in vitro functional experiment indicated miR-144 improved gliomas progression through repressing proliferation, sensitizing to chemotherapeutics and inhibiting metastasis. We further identified fibroblast growth factor 7 (FGF7) and Caveolin 2 (CAV2) were target genes of miR-144 by luciferase reporter assay and western blotting. The mechanisms study suggested forced FGF7 expression elevated Akt activation and decreased reactive oxygen species (ROS) generation. The MTT and cell cycle assay indicated miR-144 suppressed glioma cells proliferation through modulating FGF mediated Akt signaling pathway. Meanwhile, miR-144 promoted Temozolomide (TMZ) induced apoptosis in glioma cells via increasing ROS production by using FACS. On the other hand, CAV2, as another target of miR-144, accelerated glioma cells migration and invasion via promoting glioma cells EMT progress. Retrieved expression of FGF7 or CAV2 rescued the proliferation and migration function mediated by miR-144. Furthermore, the in vivo experiments in PDX models displayed the anti-tumor function of miR-144, which could be retrieved by overexpression of FGF7 and CAV2. Taken together, these findings indicated miR-144 acted as a potential target against gliomas progression and uncovered a novel regulatory mechanism, which may provide a new therapeutic strategy and prognostic indicator for gliomas.

NOTCH Signaling via WNT Regulates the Proliferation of Alternative, CCR2-Independent Tumor-Associated Macrophages in Hepatocellular Carcinoma.

Tumor-associated macrophages (TAM) play pivotal roles in tumor progression and metastasis, but the contribution and regulation of different macrophage populations remain unclear. Here we show that Notch signaling plays distinct roles in regulating different TAM subsets in hepatocellular carcinoma (HCC). Myeloid-specific NOTCH blockade by conditional disruption of recombination signal binding protein Jκ (RBPj cKO) significantly delayed the growth of subcutaneously inoculated Lewis lung carcinoma (LLC), but accelerated orthotopically inoculated hepatic Hepa1-6 tumors in mice. In contrast to subcutaneous LLC, RBPj cKO significantly increased the number of TAMs in hepatic Hepa1-6 tumors despite impeded differentiation of monocyte-derived TAMs (moTAM). The dominating TAMs in orthotopic HCC manifested properties of Kupffer cells (KC) and hence are tentatively named KC-like TAMs (kclTAM). The increased proliferation of RBPj cKO kclTAMs was maintained even in Ccr2 -/- mice, in which moTAMs were genetically blocked. NOTCH signaling blockade accelerated proliferation of kclTAMs via enhanced ß-catenin-dependent WNT signaling, which also downregulated IL12 and upregulated IL10 expression by kclTAMs likely through c-MYC. In addition, myeloid-specific RBPj cKO facilitated hepatic metastasis of colorectal cancer but suppressed lung metastasis in mice, suggesting that the phenotype of RBPj cKO in promoting tumor growth was liver-specific. In patient-derived HCC biopsies, NOTCH signaling negatively correlated with WNT activation in CD68+ macrophages, which positively correlated with advanced HCC stages. Therefore, NOTCH blockade impedes the differentiation of moTAMs, but upregulates Wnt/ß-catenin signaling to promote the proliferation and protumor cytokine production of kclTAMs, facilitating HCC progression and hepatic metastasis of colorectal cancer. SIGNIFICANCE: These findings highlight the role of NOTCH and WNT signaling in regulating TAMs in hepatocellular carcinoma.

A Vortex-Assisted Dispersive Liquid-Liquid Microextraction Followed by UPLC-MS/MS for Simultaneous Determination of Pesticides and Aflatoxins in Herbal Tea.

A method for detecting the organophosphorus pesticides residue and aflatoxins in China herbal tea has been developed by UPLC-MS/MS coupled with vortex-assisted dispersive liquid-liquid microextraction (DLLME). The extraction conditions for vortex-assisted DLLME extraction were optimized using single-factor experiments and response surface design. The optimum conditions for the experiment were the pH 5.1, 347 µL of chloroform (extraction solvent) and 1614 µL of acetonitrile (dispersive solvent). Under the optimum conditions, the targets were good linearity in the range of 0.1 µg/L⁻25 µg/L and the correlation coefficient above 0.9998. The mean recoveries of all analytes were in the ranged from 70.06%⁻115.65% with RSDs below 8.54%. The detection limits were in the range of 0.001 µg/L⁻0.01µg/L. The proposed method is a fast and effective sample preparation with good enrichment and extraction efficiency, which can simultaneously detect pesticides and aflatoxins in China herbal tea.

Comparison of the Efficacy of Ultra-Mini PCNL, Flexible Ureteroscopy, and Shock Wave Lithotripsy on the Treatment of 1-2 cm Lower Pole Renal Calculi.

OBJECTIVE: To compare the efficacy of new percutaneous technique ("ultra-mini PCNL", UMP), shock wave lithotripsy (SWL) and flexible ureteroscopy (FURS) on the treatment of 1-2 cm lower pole kidney stones, and to determine the advantages and disadvantages of each method. MATERIALS AND METHODS: This prospective study was based on data collected from the files of patients between March 2015 and March 2017. This study recruited a total of 180 patients with single radio-opaque lower caliceal calculi of 1-2 cm. All patients were randomly divided into 3 groups: group A was treated with UMP, group B was treated with FURS by using holmium laser and group C was treated with SWL by using the electromagnetic lithotripter. The average age, sex, size of the stone, the time of operation, the rate of no stone, the time of hospitalization, the rate of retreatment, the cost and the complications of the 3 groups were compared. The success of the operation was defined as no residual stone or < 0.3 cm on computed tomography at 3 months postoperatively. RESULTS: The stone burdens of the groups were equivalent. The re-treatment rate in group C was significantly higher than that in group A and B (30 vs. 1.6%, 5%). The average operating time in group B (93.35 ± 21.64 min) was statistically significantly longer than that in group A and C (68.58 ± 15.82 min, 46.33 ± 5.81 min). Although the time of hospitalization of group A (5.32 ± 1.20 day) was longer than that of group B (3.22 ± 0.52 day) and C (1.08 ± 0.28 day; p < 0.05). The stone-free rate (SFR) in UMP, FURS, SWL were 98, 92, and 73% respectively; the highest SFR was in the UMP group (p < 0.05). The complication rates were evaluated by using the Clavien grading system, which were determined to be 16.67% in UMP, 6.67% in SWL and 8.33% in FURS. In particular, the complications of GI and GII were more common in group A (p < 0.05). CONCLUSIONS: UMP, FURS, and SWL are all safe and effective in the treatment of 1-2 cm lower pole kidney stones. UMP and FURS had a better SFR than SWL, but the time of hospitalization in UMP group was longer and there were more complications in the UMP group. In addition, the operation time of FURS is longer as compared to UMP and SWL, and there is a higher rate of postoperative fever. The invasiveness and cost of SWL were lower than that of UMP and FURS, but the re-treatment rate was higher.

Notch Signaling Modulates Macrophage Polarization and Phagocytosis Through Direct Suppression of Signal Regulatory Protein α Expression.

The Notch pathway plays critical roles in the development and functional modulation of myeloid cells. Previous studies have demonstrated that Notch activation promotes M1 polarization and phagocytosis of macrophages; however, the downstream molecular mechanisms mediating Notch signal remain elusive. In an attempt to identify Notch downstream targets in bone marrow-derived macrophages (BMDMs) using mass spectrometry, the signal regulatory protein α (SIRPα) appeared to respond to knockout of recombination signal-binding protein Jk (RBP-J), the critical transcription factor of Notch pathway, in macrophages. In this study, we validated that Notch activation could repress SIRPα expression likely via the Hes family co-repressors. SIRPα promoted macrophage M2 polarization, which was dependent on the interaction with CD47 and mediated by intracellular signaling through SHP-1. We provided evidence that Notch signal regulated macrophage polarization at least partially through SIRPα. Interestingly, Notch signal regulated macrophage phagocytosis of tumor cells through SIRPα but in a SHP-1-independent way. To access the translational value of our findings, we expressed the extracellular domains of the mouse SIRPα (mSIRPαext) to block the interaction between CD47 and SIRPα. We demonstrated that the soluble mSIRPαext polypeptides could promote M1 polarization and increase phagocytosis of tumor cells by macrophages. Taken together, our results provided new insights into the molecular mechanisms of notch-mediated macrophage polarization and further validated SIRPα as a target for tumor therapy through modulating macrophage polarization and phagocytosis.

Crosstalk between hepatic tumor cells and macrophages via Wnt/ß-catenin signaling promotes M2-like macrophage polarization and reinforces tumor malignant behaviors.

Tumor-associated macrophages (TAMs) are a major component of tumor microenvironment (TME) and play pivotal roles in the progression of hepatocellular carcinoma (HCC). Wnt signaling is evolutionarily conserved and participates in liver tumorigenesis. Several studies have shown that macrophage-derived Wnt ligands can activate Wnt signaling in tumor cells. However, whether Wnt ligands secreted by tumor cells can trigger Wnt signaling in macrophages is still elusive. In this study, we first verified that canonical Wnt/ß-catenin signaling was activated during monocyte-to-macrophage differentiation and in M2-polarized macrophages. Knockdown of ß-catenin in M2 macrophages exhibited stronger antitumor characteristics when cocultured with Hepa1-6 HCC cells in a series of experiments. Activation of Wnt signaling promoted M2 macrophage polarization through c-Myc. Moreover, co-culturing naïve macrophages with Hepa1-6 HCC cells in which Wnt ligands secretion was blocked by knockdown of Wntless inhibited M2 polarization in vitro. Consistently, the growth of HCC tumor orthotopically inoculated with Wntless-silenced Hepa1-6 cells was impeded, and the phenotype of M2-like TAMs was abrogated due to attenuated Wnt/ß-catenin signaling in TAMs, leading to subverted immunosuppressive TME. Finally, we confirmed the correlation between M2 macrophage polarization and nuclear ß-catenin accumulation in CD68+ macrophages in human HCC biopsies. Taken together, our study indicates that tumor cells-derived Wnt ligands stimulate M2-like polarization of TAMs via canonical Wnt/ß-catenin signaling, which results in tumor growth, migration, metastasis, and immunosuppression in HCC. To block Wnts secretion from tumor cells and/or Wnt/ß-catenin signal activation in TAMs may be potential strategy for HCC therapy in future.

The RIO protein kinase-encoding gene Sj-riok-2 is involved in key reproductive processes in Schistosoma japonicum.

BACKGROUND: Schistosomiasis is one of the most prevalent parasitic diseases worldwide and is caused by parasitic trematodes of the genus Schistosoma. The pathogenesis of schistosomiasis is caused by eggs whose production is the consequence of the pairing of schistosomes and the subsequent sexual maturation of the female. Previous studies have demonstrated that protein kinases are involved in processes leading to the male-induced differentiation of the female gonads, ovary and vitellarium. Right open reading frame protein kinase 2 (RIOK-2) is a member of the atypical kinase family and shown in other organisms to be responsible for ribosomal RNA biogenesis and cell-cycle progression, as well as involves in nematode development. However, nothing is known about its functions in any trematode including schistosome. METHODS: We isolated and characterized the riok-2 gene from S. japonicum, and detected the transcriptional profiles of Sj-riok-2 by using real-time PCR and in situ hybridization. RNAi-mediated knockdown of Sj-riok-2 was performed, mitotic activities were detected by EdU incorporation assay and morphological changes on organs were observed by confocal laser scanning microscope (CLSM). RESULTS: In silico analyses of the amino acid sequence of Sj-RIOK-2 revealed typical features of this class of kinases including a winged helix (wHTH) domain and a RIO kinase domain. Sj-riok-2 is transcribed in different developmental stages of S. japonicum, with a higher abundance in adult females and eggs. Localization studies showed that Sj-riok-2 was mainly transcribed in female reproductive organs. Experiments with adult schistosomes in vitro demonstrated that the transcriptional level of Sj-riok-2 was affected by pairing. Knocking down Sj-riok-2 by RNAi reduced cell proliferation in the vitellarium and caused the increased amount of mature oocytes in ovary and an accumulation of eggs within the uterus. CONCLUSIONS: Sj-riok-2 is involved in the reproductive development and maturation of female S. japonicum. Our findings provide first evidence for a pairing-dependent role of Sj-riok-2 in the reproductive development and maturation of female S. japonicum. Thus this study contributes to the understanding of molecular processes controlling reproduction in schistosomes.

miR-148a-3p Mediates Notch Signaling to Promote the Differentiation and M1 Activation of Macrophages.

The Notch pathway plays critical roles in the differentiation and polarized activation of macrophages; however, the downstream molecular mechanisms underlying Notch activity in macrophages remain elusive. Our previous study has identified a group of microRNAs that mediate Notch signaling to regulate macrophage activation and tumor-associated macrophages (TAMs). In this study, we demonstrated that miR-148a-3p functions as a novel downstream molecule of Notch signaling to promote the differentiation of monocytes into macrophages in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF). Meanwhile, miR-148a-3p promoted M1 and inhibited M2 polarization of macrophages upon Notch activation. Macrophages overexpressing miR-148a-3p exhibited enhanced ability to engulf and kill bacteria, which was mediated by excessive production of reactive oxygen species (ROS). Further studies using reporter assay and Western blotting identified Pten as a direct target gene of miR-148a-3p in macrophages. Macrophages overexpressing miR-148a-3p increased their ROS production through the PTEN/AKT pathway, likely to defend against bacterial invasion. Moreover, miR-148a-3p also enhanced M1 macrophage polarization and pro-inflammatory responses through PTEN/AKT-mediated upregulation of NF-κB signaling. In summary, our data establish a novel molecular mechanism by which Notch signaling promotes monocyte differentiation and M1 macrophage activation through miR-148a-3p, and suggest that miR-148a-3p-modified monocytes or macrophages are potential new tools for the treatment of inflammation-related diseases.

Structural and developmental expression of Ss-riok-2, an RIO protein kinase encoding gene of Strongyloides stercoralis.

RIO kinases are essential atypical protein kinases in diverse prokaryotic and eukaryotic organisms, playing significant roles in yeast and humans. However, little is known about their functions in parasitic nematodes. In the present study, we have isolated and characterized the full-length cDNA, gDNA and a putative promoter of a RIOK-2 protein kinase (Ss-RIOK-2) encoding gene (Ss-riok-2) from Strongyloides stercoralis, a medically important parasitic nematode (Order Rhabditida). A three-dimensional structure (3D) model of Ss-RIOK-2 was generated using the Chaetomium thermophilum RIOK-2 protein kinase (Ct-RIOK-2) crystal structure 4GYG as a template. A docking study revealed some critical sites for ATP binding and metal binding. The putative promoter of Ss-riok-2 contains a number of conserved elements. RNAseq analysis revealed the highest levels of the Ss-riok-2 transcript in free-living females and parasitic females. To identify anatomical patterns of Ss-riok-2 expression in S. stercoralis, we observed expression patterns of a transgene construct encoding green fluorescent protein under the Ss-riok-2 promoter in post free-living S. stercoralis. Expression driven by this promoter predominated in intestinal cells. This study demonstrates significant advancement in molecular and cellular biological study of S. stercoralis and of parasitic nematodes generally, and provides a foundation for further functional genomic studies.

Cytotherapy with M1-polarized macrophages ameliorates liver fibrosis by modulating immune microenvironment in mice.

BACKGROUND & AIMS: Macrophages play vital roles in chronic liver injury, and have been tested as a tool for cytotherapy in liver fibrosis. However, macrophages possess ontogenic and functional heterogeneities. Some subsets are pro-fibrotic, whereas others are anti-fibrotic. This study aimed to clarify which macrophage subset is efficient for cytotherapy in liver fibrosis and to elucidate the underlying mechanisms. METHODS: Liver fibrosis was induced in mice by carbon tetrachloride injection or bile duct ligation. Bone-marrow-derived macrophages (BMDMs) were polarized into M0, M1, or M2 macrophages, respectively. BMDMs were infused into mice through the tail vein at different stages of fibrogenesis. Fibrosis progression, hepatic cell populations, and related molecular changes were evaluated. RESULTS: Both M0 and M1 BMDMs significantly ameliorated liver fibrosis, but M1 exhibited stronger therapeutic effects than M0. M2 macrophages were not effective on liver fibrosis. M1 macrophages reduced the number and activation of hepatic stellate cells (HSCs), which could be attributed at least partly to increased HSC apoptosis. M1 macrophages enhanced the recruitment of endogenous macrophages into fibrotic liver, which displayed the phenotype of Ly6Clo restorative macrophages and produced matrix metalloproteinases (MMPs) and hepatic growth factor (HGF) to enhance collagen degradation and hepatocyte proliferation, respectively. M1 macrophages also increased the number of total and activated natural killer (NK) cells in the fibrotic liver, which released TNF-related apoptosis-inducing ligand (TRAIL), inducing HSC apoptosis. CONCLUSIONS: M1 macrophages, which modulate the immune microenvironment to recruit and modify the activation of endogenous macrophages and NK cells, are effective for cytotherapy in experimental liver fibrosis. Lay summary: M1 Bone marrow-derived macrophages (BMDMs) exhibit a stronger therapeutic effect by modulating the hepatic microenvironment to recruit and modify the activation of endogenous macrophages and natural killer (NK) cells, which likely lead to hepatic stellate cells (HSCs) apoptosis and hampered fibrogenesis.

Monogamy relation of multi-qubit systems for squared Tsallis-q entanglement.

Tsallis-q entanglement is a bipartite entanglement measure which is the generalization of entanglement of formation for q tending to 1. We first expand the range of q for the analytic formula of Tsallis-q entanglement. For , we prove the monogamy relation in terms of the squared Tsallis-q entanglement for an arbitrary multi-qubit systems. It is shown that the multipartite entanglement indicator based on squared Tsallis-q entanglement still works well even when the indicator based on the squared concurrence loses its efficacy. We also show that the µ-th power of Tsallis-q entanglement satisfies the monogamy or polygamy inequalities for any three-qubit state.