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.

TNF-α Mediated the Disruption of Hepatic Tight Junction Expression in Blood-Biliary Barrier of Colitis via Downregulating PI3K/AKT Signaling Pathway.

Hepatocyte tight junctions (TJ) constituted blood-biliary barrier is the most important hepatic barrier for separating bile from the bloodstream, disruption or dysfunction of TJ barrier is involved in hepatobiliary manifestations of colitis, but the underlying mechanism is still not clear. This study aims to investigate the effect and underlying mechanism of tumor necrosis factor alpha (TNF-α) on hepatic TJ protein expression in blood-biliary barrier and identify its role in the pathogenesis of acute colitis-related cholestasis. Acute colitis rat model was induced by trinitrobenzene sulfonic acid (TNBS) intra-colonic administration. TJs expression of blood-biliary barrier was tested in colitis rats, the serum TNF-α level was also determined in order to elucidate the correlation of TNF-α and TJs. HepaRG cells were used to investigate the effect of TNF-α on TJs, and the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway were also evaluated in rats and TNF-α treated HepaRG cells. Acute colitis was induced in rats at 5 d post TNBS, which is accompanied with cholestasis-like alteration. Serum TNF-α level was increased in colitis rats and positively correlated with the alteration of total bile acids and bilirubin, marked decrease in TJs was found in TNF-α treated HepaRG cells and the rats, down-regulated PI3K/AKT signaling pathway were also identified in TNF-α treated HepaRG cells and the rats. The study concluded that serum TNF-α mediated the down-regulation of PI3K/AKT signaling pathway, which contributed to the reduction of TJ protein expression in acute colitis-related intrahepatic cholestasis. These findings suggest that TNF-α plays an important role in the pathogenesis of intrahepatic cholestasis of colitis.

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.

Molecular hallmarks of breast multiparametric magnetic resonance imaging during neoadjuvant chemotherapy.

PURPOSE: To identify molecular basis of four parameters obtained from dynamic contrast-enhanced magnetic resonance imaging, including functional tumor volume (FTV), longest diameter (LD), sphericity, and contralateral background parenchymal enhancement (BPE). MATERIAL AND METHODS: Pretreatment-available gene expression profiling and different treatment timepoints MRI features were integrated for Spearman correlation analysis. MRI feature-related genes were submitted to hypergeometric distribution-based gene functional enrichment analysis to identify related Kyoto Encyclopedia of Genes and Genomes annotation. Gene set variation analysis was utilized to assess the infiltration of distinct immune cells, which were used to determine relationships between immune phenotypes and medical imaging phenotypes. The clinical significance of MRI and relevant molecular features were analyzed to identify their prediction performance of neoadjuvant chemotherapy (NAC) and prognostic impact. RESULTS: Three hundred and eighty-three patients were included for integrative analysis of MRI features and molecular information. FTV, LD, and sphericity measurements were most positively significantly correlated with proliferation-, signal transmission-, and immune-related pathways, respectively. However, BPE did not show marked correlation relationships with gene expression alteration status. FTV, LD and sphericity all showed significant positively or negatively correlated with some immune-related processes and immune cell infiltration levels. Sphericity decreased at 3 cycles after treatment initiation was also markedly negatively related to baseline sphericity measurements and immune signatures. Its decreased status could act as a predictor for prediction of response to NAC. CONCLUSION: Different MRI features capture different tumor molecular characteristics that could explain their corresponding clinical significance.

Proteomics reveals the potential mechanism of Tanshinone IIA in promoting the Ex Vivo expansion of human bone marrow mesenchymal stem cells.

Introduction: Bone marrow mesenchymal stem cells (BMSCs) are a promising cell type for tissue engineering, however, the application of BMSCs is largely hampered by the limited number harvested from bone marrow cells. The methods or strategies that focused on promoting the capacity of BMSCs expansion ex vivo become more and more important. Tanshinone IIA (Tan IIA), the main active components of Danshen, has been found to promote BMSCs proliferation, but the underlying mechanism is still unclear. The aim of this study is to explore the effect and underlying mechanism of Tan IIA on the expansion capacity of hBMSCs ex vivo. Methods: In this present study, the effect of Tan IIA on the expansion capacity of BMSCs from human was investigated, and quantitative proteome analysis was applied furtherly to identify the differentially expressed proteins (DEPs) and the molecular signaling pathways in Tan IIA-treated hBMSCs. Finally, molecular biology skills were employed to verify the proposed mechanism of Tan IIA in promoting hBMSCs expansion. Results: The results showed that a total of 84 DEPs were identified, of which 51 proteins were upregulated and 33 proteins were downregulated. Besides, Tan IIA could promote hBMSCs proliferation by regulating the progression of S phase via increasing the release of fibroblast growth factor 2 (FGF2), FGF-mediated PI3K/AKT signaling pathways may play an important role in Tan IIA's effect on hBMSCs expansion. Conclusions: This study employed molecular biology skills combined with quantitative proteome analysis, to some extent, clarified the mechanism of Tan IIA's effect on promoting hBMSCs proliferation, and will give a hint that Tan IIA may have the potential to be used for BMSCs applications in cell therapies in the future.

The versatility of macrophage heterogeneity in liver fibrosis.

Liver fibrosis is a highly conserved wound healing response to liver injury, characterized by excessive deposition of extracellular matrix (ECM) in the liver which might lead to loss of normal functions. In most cases, many types of insult could damage hepatic parenchymal cells like hepatocytes and/or cholangiocytes, and persistent injury might lead to initiation of fibrosis. This process is accompanied by amplified inflammatory responses, with immune cells especially macrophages recruited to the site of injury and activated, in order to orchestrate the process of wound healing and tissue repair. In the liver, both resident macrophages and recruited macrophages could activate interstitial cells which are responsible for ECM synthesis by producing a variety of cytokines and chemokines, modulate local microenvironment, and participate in the regulation of fibrosis. In this review, we will focus on the main pathological characteristics of liver fibrosis, as well as the heterogeneity on origin, polarization and functions of hepatic macrophages in the setting of liver fibrosis and their underlying mechanisms, which opens new perspectives for the treatment of liver fibrosis.

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.

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.

Determination of Total and Unbound Meropenem, Imipenem/Cilastatin, and Cefoperazone/Sulbactam in Human Plasma: Application for Therapeutic Drug Monitoring in Critically Ill Patients.

BACKGROUND: Critically ill patients show several pathophysiological alterations that can complicate antibiotic dosing. Hence, there is a strong rationale to individualize anti-infective dosing in these patients by using therapeutic drug monitoring (TDM). The current study aimed to develop and validate a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of total and unbound plasma concentrations of 3 commonly used antibiotics (meropenem, imipenem/cilastatin, and cefoperazone/sulbactam) in the treatment of infections in critically ill patients in China, which could be suitable for routine TDM in hospital laboratories. METHODS: The unbound drug was separated from the bound drug by ultrafiltration. Simple protein precipitation was used for sample preparation. Meropenem, imipenem/cilastatin, cefoperazone/sulbactam, and their corresponding internal standards were then resolved using the Waters CORTECS C18 column. All the compounds were detected using electrospray ionization in the positive/negative ion-switching mode. RESULTS: The calibration curves were linear for all compounds, with correlation coefficients (R) above 0.99 for total concentrations in human plasma and unbound concentrations in the plasma ultrafiltrate. For both total and unbound drugs, the relative errors and intra-assay/interassay relative standard deviations were below 15%. The limit of quantification was 0.05 mcg/mL for both total plasma concentrations and plasma ultrafiltrate concentrations of all compounds. CONCLUSIONS: The method was simple, rapid, and reliable and is currently being used to provide a TDM service to enhance the efficacious use of the 3 antibiotics.

Enterochromaffin cell hyperplasia in the gut: Factors, mechanism and therapeutic clues.

Enterochromaffin (EC) cell is the main cell type that responsible for 5-hydroxytryptamine (5-HT) synthesis, storage and release of the gut. Intestinal 5-HT play a key role in visceral sensation, intestinal motility and permeability, EC cell hyperplasia and increased 5-HT bioavailability in the gut have been found to be involved in the symptoms generation of irritable bowel syndrome and inflammatory bowel disease. EC cells originate from intestinal stem cells, the interaction between proliferation and differentiation signals on intestinal stem cells enable EC cell number to be regulated in a normal level. This review focuses on the impact factors, pathogenesis mechanisms, and therapeutic clues for intestinal EC cells hyperplasia, and showed that EC cell hyperplasia was observed under the condition of physiological stress, intestinal infection or intestinal inflammation, the disordered proliferation and/or differentiation of intestinal stem cells as well as their progenitor cells all contribute to the pathogenesis of intestinal EC cell hyperplasia. The altered intestinal niche, i.e. increased corticotrophin releasing factor (CRF) signal, elevated nerve growth factor (NGF) signal, and Th2-dominant cytokines production, has been found to have close correlation with intestinal EC cell hyperplasia. Currently, CRF receptor antagonist, nuclear factor-κB inhibitor, and NGF receptor neutralizing antibody have been proved useful to attenuate intestinal EC cell hyperplasia, which may provide a promising clue for the therapeutic strategy in EC cell hyperplasia related diseases.

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.

From psychology to physicality: how nerve growth factor transduces early life stress into gastrointestinal motility disorders later in life.

Environmental stressors in early childhood can have a detrimental impact later in life, manifesting in functional gastrointestinal disorders including irritable bowel syndrome (IBS). The phenomenon is also observed in rodents, where neonatal-maternal separation, a model of early life stress, induces phenotypes similar to IBS; however, the underlying mechanisms remain unelucidated. Our recent study provided a mechanism for the pathogenesis in the gut, demonstrating that increased visceral hyperalgesia resulted from the expansion of the intestinal stem cell compartment leading to increased differentiation and proliferation of serotonin (5-hydroxytryptamine/5-HT)-producing enterochromaffin cells. Moreover, it identified nerve growth factor (NGF) as a key mediator of the pathogenesis; surprisingly, it exerts its effect via cross talk with Wnt/ß-catenin signaling. This article addresses the roles of NGF in driving IBS and its potential clinical implications, outstanding questions in how psychological stimuli are transduced into physical phenotypes, as well as future directions of our findings. Abbreviations: 5-HT: 5-hydroxytryptamine/serotonin; BDNF: brain-derived neurotrophic factor; CRF: corticotrophin-releasing factor; EC: enterochromaffin; ENS: enteric nervous system; GI: gastrointestinal; GPCR: G-protein-coupled receptor; IBS (-D): irritable bowel syndrome (diarrhea predominant); LRP5/6: low-density lipoprotein receptor-related protein 5/6; MAPK: mitogen-activated protein kinase; NGF: nerve growth factor; NMS: neonatal-maternal separation; PI3K: phosphoinositode3-kinase; PLCγ: phospholipase c, gamma subtype; TrkA: tropomyosin receptor kinase A.

Early life stress disrupts intestinal homeostasis via NGF-TrkA signaling.

Early childhood is a critical period for development, and early life stress may increase the risk of gastrointestinal diseases including irritable bowel syndrome (IBS). In rodents, neonatal maternal separation (NMS) induces bowel dysfunctions that resemble IBS. However, the underlying mechanisms remain unclear. Here we show that NMS induces expansion of intestinal stem cells (ISCs) and their differentiation toward secretory lineages including enterochromaffin (EC) and Paneth cells, leading to EC hyperplasia, increased serotonin production, and visceral hyperalgesia. This is reversed by inhibition of nerve growth factor (NGF)-mediated tropomyosin receptor kinase A (TrkA) signalling, and treatment with NGF recapitulates the intestinal phenotype of NMS mice in vivo and in mouse intestinal organoids in vitro. Mechanistically, NGF transactivates Wnt/ß-catenin signalling. NGF and serotonin are positively correlated in the sera of diarrhea-predominant IBS patients. Together, our findings provide mechanistic insights into early life stress-induced intestinal changes that may translate into treatments for gastrointestinal diseases.

Quercetin Attenuates Visceral Hypersensitivity and 5-Hydroxytryptamine Availability in Postinflammatory Irritable Bowel Syndrome Rats: Role of Enterochromaffin Cells in the Colon.

Intestinal enterochromaffin (EC) cell hyperplasia and increased 5-hydroxytryptamine (5-HT) availability play key roles in the pathogenesis of abdominal hypersensitivity of irritable bowel syndrome (IBS). This study aims to study the effect of quercetin on visceral pain and 5-HT availability in postinflammatory IBS (PI-IBS) rats. PI-IBS model rats were administered quercetin by gavage at doses of 5, 10, and 20 mg/kg for 14 days. Compared with normal rats, the visceral pain threshold of PI-IBS rats was markedly decreased and the abdominal motor response to colon distension was markedly increased. The EC cell count and 5-HT level, as well as tryptophan hydroxylase (TPH) protein, were all significantly elevated in PI-IBS rats, while the 5-HT reuptake transporter (serotonin transporter) was reduced. Genes that are responsible for enteroendocrine cell differentiation, that is, Ngn3 and pdx1, were significantly increased in the PI-IBS group. Quercetin treatment markedly elevated the pain threshold pressure and decreased the visceral motor response of PI-IBS animals; and EC cell density and 5-HT level, as well as TPH expression, in the PI-IBS group were all reduced by quercetin. Quercetin treatment also significantly reduced colonic expression of Ngn3 and pdx1 of PI-IBS. Findings from the present study indicated that the analgesic effect of quercetin on PI-IBS may result from reduction of 5-HT availability in the colon, and the regulatory role of quercetin in endocrine progenitors may contribute to reduced EC cells.

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 urinary excretion of metformin, ceftizoxime and ofloxacin in high serum creatinine rats: Can creatinine predict renal tubular elimination?

The renal excretion of creatinine and most drugs are the net result of glomerular filtration and tubular secretion, and their tubular secretions are mediated by individual transporters. Thus, we hypothesized that the increase of serum creatinine (SCr) levels attributing to inhibiting tubular transporters but not glomerular filtration rate (GFR) could be used to evaluate the tubular excretion of drugs mediated by identical or partial overlap transporter with creatinine. In this work, we firstly developed the creatinine excretion inhibition model with normal GFR by competitively inhibiting tubular transporters, and investigated the renal excretion of metformin, ceftizoxime and ofloxacin in vivo and in vitro. The results showed that the 24-hour urinary excretion of metformin and ceftizoxime in model rats were decreased by 25% and 17% compared to that in control rats, respectively. The uptake amount and urinary excretion of metformin and ceftizoxime could be inhibited by creatinine in renal cortical slices and isolated kidney perfusion. However, the urinary excretion of ofloxacin was not affected by high SCr. These results showed that the inhibition of tubular creatinine transporters by high SCr resulted to the decrease of urinary excretion of metformin and ceftizoxime, but not ofloxacin, which implied that the increase of SCr could also be used to evaluate the tubular excretion of drugs mediated by identical or partial overlap transporter with creatinine in normal GFR rats.

Multicomponent determination of traditional Chinese medicine preparation yin-zhi-huang injection by LC-MS/MS for screening of its potential bioactive candidates using HepaRG cells.

Yin-zhi-huang (YZH) injection is an injectable multiherbal prescription derived from the ancient Chinese medicine formula of Yin-chen-hao-tang, which is widely used in the clinic for the treatment of jaundice and chronic liver diseases. To date, the systematic study of the components in this multiherbal prescription still lacks suitable analytical methods that are able to simultaneously detect a broad array of components at low concentrations. In this study, a new liquid chromatography-tandem mass spectrometry method using dynamic multiple reaction monitoring mode was developed to determine multiple peaks in traditional Chinese medicine preparation YZH injection. This simple, selective and sensitive method enabled the quantification of 22 components with standard materials with a lower limit of quantification of 1.46-12.5 ng/mL in cell lysates. This method was successfully applied to celluar uptake and binding investigation of components in YZH injection. The results indicated that this strategy might be a useful approach for rapidly screening of the potential bioactive candidates from YZH injection, and the discovered candidates could be used to investigate the pharmacodynamics in further studies.

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.