Codonopsis pilosula-derived glycopeptide dCP1 promotes the polarization of tumor-associated macrophage from M2-like to M1 phenotype.

The majority of the immune cell population in the tumor microenvironment (TME) consists of tumor-associated macrophages (TAM), which are the main players in coordinating tumor-associated inflammation. TAM has a high plasticity and is divided into two main phenotypes, pro-inflammatory M1 type and anti-inflammatory M2 type, with tumor-suppressive and tumor-promoting functions, respectively. Considering the beneficial effects of M1 macrophages for anti-tumor and the high plasticity of macrophages, the conversion of M2 TAM to M1 TAM is feasible and positive for tumor treatment. This study sought to evaluate whether the glycopeptide derived from simulated digested Codonopsis pilosula extracts could regulate the polarization of M2-like TAM toward the M1 phenotype and the potential regulatory mechanisms. The results showed that after glycopeptide dCP1 treatment, the mRNA relative expression levels of some M2 phenotype marker genes in M2-like TAM in simulated TME were reduced, and the relative expression levels of M1 phenotype marker genes and inflammatory factor genes were increased. Analysis of RNA-Seq of M2-like TAM after glycopeptide dCP1 intervention showed that the gene sets such as glycolysis, which is associated with macrophage polarization in the M1 phenotype, were significantly up-regulated, whereas those of gene sets such as IL-6-JAK-STAT3 pathway, which is associated with polarization in the M2 phenotype, were significantly down-regulated. Moreover, PCA analysis and Pearson's correlation also indicated that M2-like TAM polarized toward the M1 phenotype at the transcriptional level after treatment with the glycopeptide dCP1. Lipid metabolomics was used to further explore the efficacy of the glycopeptide dCP1 in regulating the polarization of M2-like TAM to the M1 phenotype. It was found that the lipid metabolite profiles in dCP1-treated M2-like TAM showed M1 phenotype macrophage lipid metabolism profiles compared with blank M2-like TAM. Analysis of the key differential lipid metabolites revealed that the interconversion between phosphatidylcholine (PC) and diacylglycerol (DG) metabolites may be the central reaction of the glycopeptide dCP1 in regulating the conversion of M2-like TAM to the M1 phenotype. The above results suggest that the glycopeptide dCP1 has the efficacy to regulate the polarization of M2-like TAM to M1 phenotype in simulated TME.

Elesclomol-Copper Nanoparticles Overcome Multidrug Resistance in Cancer Cells.

Elesclomol (ES), a copper-binding ionophore, forms an ES-Cu complex with copper ions (Cu(II)). ES-Cu has been proven to induce mitochondrial oxidative stress and copper-dependent cell death (cuprotosis). However, ES-Cu is poorly water-soluble, and its delivery to various cancer cells is a challenge. Herein, we designed a d-α-tocopherol polyethylene glycol 1000 succinate/chondroitin sulfate-cholic acid (TPGS/CS-CA)-based micellar nanoparticle for delivering the ES-Cu complex to various cancer cell lines to demonstrate its efficacy as an anticancer agent. The ES-Cu nanoparticles exerted high encapsulation efficiency and excellent serum stability. The anticancer efficacy of ES-Cu nanoparticles was evaluated in various drug-sensitive cell lines (DU145, PC3, and A549) and drug-resistant cell lines (DU145TXR, PC3TXR, and A549TXR). The results showed that ES-Cu nanoparticles exerted potent anticancer activities in both drug-sensitive and drug-resistant cell lines. The Western blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), and molecular docking results suggested that ES-Cu is not a substrate for P glycoprotein (P-gp), which is an efflux transporter potentially causing multidrug resistance (MDR) in cancer cells. ES-Cu nanoparticles could bypass P-gp without compromising their activity, indicating that they may overcome MDR in cancer cells and provide a novel therapeutic strategy. Additionally, the extracellular matrix of ES-Cu nanoparticles-pretreated drug-resistant cells could polarize Raw 264.7 macrophages into the M1 phenotype. Therefore, our TPGS/CS-CA-based ES-Cu nanoparticles provide an effective method of delivering the ES-Cu complex, a promising strategy to overcome MDR in cancer therapy with potential immune response stimulation.

Roles of prokineticin 2 in electroconvulsive shock-induced memory impairment via regulation of phenotype polarization in astrocytes.

Electroconvulsive shock (ECT) is the most effective treatment for depression but can impair learning and memory. ECT is increasingly being shown to activate astrocytes and induce neuroinflammation, resulting in cognitive decline. Activated astrocytes can differentiate into two subtypes, A1-type astrocytes and A2-type astrocytes. Regarding cognitive function, neurotoxic A1 astrocytes and neuroprotective A2 astrocytes may exhibit opposite effects. Specifically, prokineticin 2 (PK2) functions as an essential mediator of inflammation and induces a selective A2-protective phenotype in astrocytes. This study aimed to clarify how PK2 promotes improved learning memory following electroconvulsive shock (ECS). As part of the study, rats were modeled using chronic unpredictable mild stress. Behavioral experiments were conducted to assess their cognitive abilities and depression-like behaviors. Western blot was used to determine the expression of PK2. Immunohistochemical and electron microscopy analyses of the hippocampal CA1 region were conducted to study the activation of astrocyte subtypes and synaptic ultrastructure, respectively. In this study, rats' spatial learning and memory impairment began to improve as activated A1-subtype astrocytes gradually decreased, and PK2 and A2 phenotype activation peaked on the third day after ECS. PKRA7 (PK2 antagonist) inhibits A2-type astrocyte activation partially and suppresses spatial learning and memory improvement. Collectively, our findings support that PK2 may induce a selective modulation of astrocytic polarization to a protective phenotype to promote learning and memory improvement after ECS.

Effects of simulated digestion in vitro on the structure and macrophages activation of fucoidan from Sargassum fusiforme.

Molecular size and spatial structure affect the bioactivities of polysaccharides. SFF is a fucoidan extracted from Sargassum fusiforme. The possible changes of SFF affected by gastrointestinal tract and subsequently changes of its physicochemical property or its bioactivity have yet to be systematically investigated. Our results showed that DSFF, the gastrointestinal digestion product of SFF, has increased reducing sugar content, increased proportion of low molecular weight components, and a more clustered island-like morphology. Both SFF and DSFF activate RAW 264.7 macrophages evidenced by the increasing level of NO, intracellular ROS, and macrophages cytokines. Further investigation showed that DSFF induced M1 phenotype polarization in RAW 264.7 cells. DSFF also showed stronger macrophage activation and phenotype polarization than SFF. Our present work showed that SFF could be digested by simulated gastrointestinal environment in vitro and the digested product DSFF showed higher efficiency in macrophages activation and phenotype polarization.

Haimufang decoction, a Chinese medicine formula for lung cancer, arrests cell cycle, stimulates apoptosis in NCI-H1975 cells, and induces M1 polarization in RAW 264.7 macrophage cells.

BACKGROUND: Lung cancer has the highest morbidity and mortality in the world and novel treatment strategies are still needed. Haimufang decoction (HMF) is a patented clinical prescription of traditional Chinese medicine for lung cancer treatment. HMF is composed of four herbs and has been applied clinically in advanced cancer patients. However, its therapeutic mechanisms are still unclear. This study aims to elucidate the possible mechanisms of HMF for the treatment of lung cancer. METHODS: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was applied for evaluating the proliferative effect of HMF in lung cancer cells and monocyte macrophage RAW264.7 cells. Flow cytometer was used to detect the effects of HMF on cell cycle and apoptosis, and western blotting was employed to explore the potential apoptotic mechanisms of HMF on lung cancer cells. For immunomodulatory effect, co-culture system was used to detect the activation of macrophage RAW264.7 cells when treated with HMF, and neutral red assay was used to measure the effect of HMF on the phagocytosis of the activated macrophages. Enzyme linked immunosorbent assay, flow cytometer, and immunofluorescence staining method were employed for the investigation on the underlying mechanisms of the immunomodulatory effect on RAW264.7 induced by HMF. RESULTS: HMF inhibited the proliferation, induced S phase cell cycle arrest, and stimulated apoptosis in lung cancer NCI-H1975 cells, while had negligible cytotoxicity on macrophage RAW264.7 cells. Moreover, HMF could activate macrophage RAW264.7 cells and promote the inhibition activity of RAW264.7 cells against lung cancer cells. And also, HMF activated macrophages and increased their phagocytic activity in a concentration-dependent manner. HMF increased the expression of macrophage activation marker CD40, the level of nitric oxide, the generation of intracellular reactive oxygen species, as well as M1 macrophages cytokines including tumor necrosis factor-α, interleukin-1ß, interleukin 12 p70, and interleukin 6. Further investigation showed that HMF induced M1 but not M2 phenotype polarization in RAW264.7 cells. CONCLUSIONS: HMF can mainly exert anticancer activity via (1) cytotoxicity to human lung cancer cells by proliferation inhibition, cell cycle arrest, and apoptosis induction; and also via (2) immunomodulation via macrophage cells activation and M1 phenotype polarization induction.

Roles of Prokineticin 2 in Subarachnoid Hemorrhage-Induced Early Brain Injury via Regulation of Phenotype Polarization in Astrocytes.

Previous studies have postulated that neuroinflammation can induce two different types of reactive astrocytes, A1 and A2. A1 astrocytes may be harmful, whereas A2 astrocytes may be protective. Specifically, prokineticin 2 (PK2) has been shown to regulate neuron-astrocyte signaling mechanism by promoting an alternative A2-protective phenotype in astrocytes. This study aimed to examine the role of PK2 in early brain injury (EBI) caused by subarachnoid hemorrhage (SAH). SAH-induced astrocytic activation was confirmed by Western blotting. We confirmed C3 and PTX3 as appropriate reactivity markers for discriminating A1 and A2 astrocytes, respectively. We also observed SAH-induced astrocytic activation in SAH patients. The increase of PK2 in neurons after SAH in both humans and rats suggested a possible relationship between PK2 and SAH pathology. PK2 knockdown promoted an A1 astrocytic phenotype with upregulation of neurodegenerative indicators, while intravascular injection of recombinant PK2 (rPK2) promoted A2 astrocytic phenotype and reduced SAH-induced neuronal injury and behavioral dysfunction. Finally, we identified that tumor necrosis factor alpha (TNF-α) was sufficient to elevate the protein level of PK2 in neurons and enhance astrocytic activation in vitro. Moreover, rPK2 selectively promoted astrocytic polarization to an A2 phenotype under a TNF-α stimulus and induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), suggesting that SAH-induced increases in PK2 may function as an endogenous mechanism for self-repair. Collectively, our findings support that enhancing PK2 expression or administration of rPK2 may induce a selective modulation of astrocytic polarization to a protective phenotype following SAH-like stimuli.

XIAOPI Formula Inhibits Breast Cancer Stem Cells via Suppressing Tumor-Associated Macrophages/C-X-C Motif Chemokine Ligand 1 Pathway.

Macrophages are the most abundant stromal cells associated with the host immune system in multiple malignancies including breast cancer. With proven clinical efficacy and no noticeable adverse effects, XIAOPI formula (XPS) has been approved for breast hyperplasia treatment by the State Food and Drug Administration of China (SFDA) in 2018. The existing knowledge about the anti-breast cancer activities and mechanisms of XPS has been very limited. The present study aimed to investigate whether XPS could exert an anti-breast cancer effect by regulating tumor-associated macrophages (TAMs) in tumor microenvironment. Herein, breast cancer cells and TAMs were co-cultured using the transwell co-culture system to simulate the coexistence of them. XPS could significantly inhibit the proliferation, colony formation, breast cancer stem cells (CSCs) subpopulation, mammosphere formation abilities as well as stemness-related genes expression in both human and mouse breast cancer cells in the co-culture system. Additionally, XPS could suppress M2 phenotype polarization as well as C-X-C motif chemokine ligand 1 (CXCL1) expression and secretion of TAMs. Notably, further mechanistic explorations verified TAMs/CXCL1 as the critical target of XPS in inhibiting breast CSCs self-renewal in the co-culture system as the exogenous CXCL1 administration could abrogate the inhibitory effect of XPS on breast CSCs self-renewal. More importantly, XPS significantly inhibited mammary tumor growth, breast CSCs subpopulation, and TAMs/CXCL1 activity in mouse 4T1-Luc xenografts in vivo without any detectable side effects. Taken together, this study not only uncovers the immunomodulatory mechanism of XPS in treating breast cancer but also sheds novel insights into TAMs/CXCL1 as a potential molecular target for breast CSCs elimination.

Phenotype and Secretome of Monocyte-Derived Macrophages Interacting with Mesenchymal Stromal Cells under Conditions of Hypoxic Stress.

We studied the effect of short-term hypoxic stress on the phenotypic polarization of monocyte-derived macrophages and their secretory activity during interaction with mesenchymal stromal cells. In the presence of mesenchymal stromal cells, monocyte-derived macrophages exhibited the signs of M2 polarization, which was evidenced by increased expression of CD206 and CD163 markers, as well as increased transcription and translation of IL-6. Short-term hypoxic stress promoted a shift of macrophage phenotype from inflammatory M1 towards anti-inflammatory M2 in monoculture and co-culture with mesenchymal stromal cells. In addition to the immunoregulatory action, mesenchymal stromal cells demonstrated stromal activity and maintained high viability of monocyte-derived macrophages.

Human Placental Hofbauer Cells Maintain an Anti-inflammatory M2 Phenotype despite the Presence of Gestational Diabetes Mellitus.

BACKGROUND: Hofbauer cells (HBCs) are macrophages of the feto-placental unit. Despite the general view that these cells have an anti-inflammatory M2 phenotype, recent studies have claimed that pregnancy pathologies-e.g., gestational diabetes mellitus (GDM)-cause a switch from an M2 to an M1 pro-inflammatory phenotype in HBCs. The pilot-study presented here challenges this claim, showing that HBCs maintain anti-inflammatory properties in spite of the hyperglycemic, low-grade inflammatory environment of GDM. METHODS: HBCs were isolated from placentae of healthy women (N = 5) and women with GDM (N = 6) diagnosed in the second trimester. FACS was used to measure surface markers associated with either M1 or M2 phenotype on the cells. In addition, placental tissue sections were subjected to immune histochemical imaging to assess the phenotype within the tissue context. Supernatant from control and GDM HBCs was collected at defined time points and used in a multiplex ELISA-on-beads approach to assess secretion of cytokines, chemokines, and growth factors. The effect of HBC cell culture supernatant on placental endothelial activation was investigated. RESULTS: FACS and immune staining showed that, indeed, M2 markers, such as CD206 and CD209, are increased in HBCs isolated from GDM placentae. Also, the M1 marker CD86 was increased, but only by trend. Secretion of numerous cytokines, chemokines and growth factors was not changed; pro-inflammatory interleukin (IL)-1ß and IL-6 release form GDM HBC was increased but not significant. Exposure to GDM HBC supernatant did not induce cell adhesion molecules (VCAM-1, selectins, vascular endothelial-cadherin) in placental endothelial cells compared to supernatant from control HBCs, an induction of intracellular adhesion molecule 1 was observed however. CONCLUSION: Our study-although performed in a small set of patients-shows that placental macrophages maintain their anti-inflammatory, tissue remodeling M2 phenotype even in pregnancies affected by gestational diabetes. This consistent phenotype might be important for propagation of maternal tolerance toward the fetus and for protection of the fetus from a low-grade inflammatory environment.

Macrophage in chronic kidney disease.

Chronic kidney disease (CKD) has become a major health problem worldwide. This review describes the role of macrophages in CKD and highlights the importance of anti-inflammatory M2 macrophage activation in both renal fibrosis and wound healing processes. Furthermore, the mechanisms by which M2 macrophages induce renal repair and regeneration are still under debate and currently demand more attention. The M1/M2 macrophage balance is related to the renal microenvironment and could influence CKD progression. In fact, an inflammatory renal environment and M2 plasticity can be the major hurdles to establishing macrophage cell-based therapies in CKD. M2 macrophage cell-based therapy is promising if the M2 phenotype remains stable and is 'fixed' by in vitro manipulation. However, a greater understanding of phenotype polarization is still required. Moreover, better strategies and targets to induce reparative macrophages in vivo should guide future investigations in order to abate kidney diseases.