Harmine Alleviated Sepsis-Induced Cardiac Dysfunction by Modulating Macrophage Polarization via the STAT/MAPK/NF-κB Pathway

Sepsis is a dysregulated systemic inflammatory response that often leads to cardiac dysfunction, which is termed sepsis-induced cardiomyopathy (SIC). Harmine, a natural β-carboline alkaloid compound, has been shown to exert pharmacological effects on several diseases. Here, we investigated whether harmine protected against SIC development and the underlying mechanisms. In vitro, the expression of the M1 phenotype markers iNOS and COX-2 was increased in RAW 264.7 cells stimulated with lipopolysaccharide (LPS), but this effect was reversed by the harmine intervention. Furthermore, LPS-induced increases in the levels of inflammatory cytokines, including IL-1β, IL-6, TNF-α, iNOS, COX-2, PGE2 and TXB2, generated by macrophages were suppressed when the cells were pretreated with harmine. Meanwhile, our findings showed that harmine administration effectively attenuated inflammation and apoptosis in H9c2 cells in the proinflammatory environment produced by macrophages, as evidenced by reductions in NLRP3 and cleaved caspase 3 levels and the p-NF-κB/NF-κB ratio. The western blot results indicated that the mechanisms underlying harmine-mediated inhibition of M1 polarization might be associated with suppression of STAT1/3, NF-κB and MAPK activation. Furthermore, an LPS injection induced cardiac dysfunction and decreased the survival rate of mice, which were alleviated by harmine treatment, and the relevant mechanism was possibly attributed to a drug-induced attenuation of the inflammatory and apoptotic processes in cardiomyocytes. Collectively, these results implied that harmine treatment protected against SIC by suppressing M1 phenotypic polarization and inflammation in macrophages.

MiR-200c-3p inhibits LPS-induced M1 polarization of BV2 cells by targeting RIP2

Background Microglia are important immune cells, which can be induced by lipopolysaccharide (LPS) into M1 phenotype that express pro-inflammatory cytokines. Some studies have shown that microRNAs play critical roles in microglial activation. Objective This study was designed to investigate the role of miR-200c-3p in regulating inflammatory responses of LPS-treated BV2 cells. Methods The expression of miR-200c-3p in BV2 cells was detected by real-time PCR. Receptor-interacting protein 2 (RIP2) was predicted as a target gene of miR-200c-3p. Their relationship was verified by dual-luciferase reporter assay. The function of miR-200c-3p and RIP2 in microglial polarization and NF-κB signaling was further evaluated. Results LPS treatment reduced miR-200c-3p expression in a dose-dependent and time-dependent manner in BV2 cells. LPS treatment increased the expression of M1 phenotype markers inducible nitric oxide synthase (iNOS) and major histocompatibility complex class (MHC)-II, promoted the release of pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, and enhanced the nuclear translocation and phosphorylation of nuclear factor-kappaB (NF-κB) p65. Reversely, miR-200c-3p mimics down-regulated the levels of these inflammatory factors. Furthermore, RIP2 was identified to be a direct target of miR-200c-3p. RIP2 knockdown had a similar effect to miR-200c-3p mimics. Overexpression of RIP2 eliminated the inhibitory effect of miR-200c-3p on LPS-induced M1 polarization and NF-κB activation in BV2 cells. Conclusions MiR-200c-3p mimics suppressed LPS-induced microglial M1 polarization and NF-κB activation by targeting RIP2. MiR-200c-3p/RIP2 might be a potential therapeutic target for the treatment of neuroinflammation-associated diseases.

Effect of Ethyl Acetate Fraction of Marsilea crenata Presl. Leaf Extract on Major Histocompatibility Complex Class II Expression in Microglial HMC3 Cell Lines

Phytoestrogens are plant-derived chemical substances that have estrogen-like structures or estrogenic functions. Deficiency of estrogen in human brain causes neuroinflammation characterized by increase of major histocompatibility complex class II (MHC II) expression as a marker of M1 phenotype in microglia. Recent research found phytoestrogen compounds in Marsilea crenata Presl. The aim of this study was to investigate the effect of ethyl acetate fraction of Marsilea crenata Presl. leaf extract in MHC II expression of microglial HMC3 cell lines, for resolution of inflammation and tissue repair. The fractions were given at concentrations of 62.5, 125, and 250 ppm to microglia, that had been previously induced by IFNγ 10 ng for 24 hours to stimulate the cells into M1 phenotype. Genistein as phytoestrogen was given at a concentration of 50 μM as positive control. Expression of MHC II was analyzed using immunocytochemistry method. Result showed reduction in MHC II expression of microglial cells, which indicated the activity of all extracts and, showed that 250 ppm of the fraction showed the strongest effect with MHC II value expression of 148.632 AU, and ED50 of 1,590 ppm. It was concluded from the study, that ethyl acetate fraction of Marsilea crenata Presl. leaves has antineuroinflammation effect.

Group 1 Innate Lymphocyte-Derived IFN-γ Regulates Macrophage Alternative Activation In Colon Cancer

Background: Tumor-associated macrophage (TAM) is an important innate immune cell-subset in tumor microenvironment, and that is also a pivotal orchestrator of tumor-promoting inflammation and tumor progression. Evidence proved that TAMs are up-regulated in a great number of cancers, and most of them are alternative activated M2 phenotype, which greatly promote the progress of cancer diseases. Group 1 innate lymphocytes including conventional NK cells and type 1 innate lymphocytes (ILC1s), are abundant in intestinal tissue, and characterized by expressing transcription factor T-bet and secreting interferon (IFN)-γ, which can promote the macrophage to classically activated anti-tumor M1 phenotype. However, the relationship between these two cell subsets remains unclear in colon cancer. Methods: Flow cytometry was used to detect the percentage of M1 phenotype macrophage, M2 phenotype macrophage and group 1 innate lymphocytes in colon cancer tissue and paracancer healthy colon tissue of AOM/DSS-induced colon cancer mice model. In vitro isolating group 1 innate lymphocytes and inducing bone marrow-derived macrophage to detect the cross-talk when co-cultured. Adoptively transfer or blocking group 1 innate lymphocytes in vivo to investigate the role of group 1 innate lymphocytes on tumor-infiltrating macrophage and the tumor growth. Results: We found that M1 phenotype macrophage and group 1 innate lymphocytes were down-regulated in colon cancer tissue, and they were positively correlated. Group 1 innate lymphocytes promoted macrophage to classically activated M1 phenotype in vitro, and that could be blocked by anti-IFN-γ. In vivo results showed that the administration of group 1 innate lymphocytes-blocking antibody anti-NK1.1 could decrease the number of M1 phenotype macrophage in tumor tissue of MC38 tumor-bearing mice and promote the tumor growth, while adoptively transferring group 1 innate lymphocytes led to tumor-inhibiting and level of M1 phenotype macrophage up-regulating in MC38 tumor-bearing mice. Conclusions: Our studies preliminarily prove that group 1 innate lymphocytes promote the alternative activation of M1 macrophage by secreting IFN-γ to inhibit the progress of colon cancer for the first time, which may provide an insight in the immunotherapy of colon cancer.

IN VITRO ANTI-NEUROINFLAMMATORY EFFECT OF GENISTEIN (4',5,7-TRIHYDROXYISOFLAVONE) ON MICROGLIA HMC3 CELL LINE, AND IN SILICO EVALUATION OF ITS INTERACTION WITH ESTROGEN RECEPTOR-β

Objective: This study was aimed to evaluate the role of genistein or 4',5,7-trihydroxyisoflavone as a phytoestrogen in the treatment of estrogen deficiency-induced neuroinflammatory. The specific objectives of this study were to determine the anti-neuroinflammatory effect of genistein through measurement of MHC II and Arg1 expressions on microglia HMC3 cell line, as well as to prove that the effect occurs in an ER-dependent manner, through the measurement of free-ERβ expression. Methods: The cells were cultured in 24-well microplates, induced with 10 ng IFN-γ, and incubated for 24 h to activate the cell to M1 phenotype which has pro-inflammatory characteristics. Genistein with a concentration of 50 μM was added to the cells. The expression of MHC II, Arg1, and free-ERβ as markers was tested through an immunocytochemistry method and measured using the CLSM instrument. In silico approach was also conducted to determine the interaction between genistein and ERβ, compared to 17β-estradiol. Genistein structure was prepared with Avogadro 1.0.1, and molecular docking was done using PyRx 0.8 software. Biovia Discovery Studio Visualizer 2016 was used to visualize the structure of genistein against 3OLS protein. The physicochemical characteristics of genistein were analyzed using the SwissADME web tool. Results: Genistein can decrease MHC II expression and increase Arg1 expression in microglia HMC3 cells compared to negative controls (p<0.005), with expression value of 472.577±26.701 AU and 114.299±6.578 AU. But, genistein cannot decrease the free-ERβ expression in cells (p<0.005). The results of in silico analysis showed that genistein is an ERβ agonist. Conclusion: Genistein shows anti-neuroinflammatory effects by decreasing the MHC II expression and increasing Arg1 expression in the microglia HMC3 cell line. However, this effect does not occur through the binding of genistein to ERβ, but it is likely to occur through the binding of genistein with other types of ER.

Diethyl Succinate Modulates Microglial Polarization and Activation by Reducing Mitochondrial Fission and Cellular ROS

Succinate is a metabolite in the tricarboxylic acid cycle (TCA) which plays a central role in mitochondrial activity. Excess succinate is known to be transported out of the cytosol, where it activates a succinate receptor (SUCNR1) to enhance inflammation through macrophages in various contexts. In addition, the intracellular role of succinate beyond an intermediate metabolite and prior to its extracellular release is also important to the polarization of macrophages. However, the role of succinate in microglial cells has not been characterized. Lipopolysaccharide (LPS) stimulates the elevation of intracellular succinate levels. To reveal the function of intracellular succinate associated with LPS-stimulated inflammatory response in microglial cells, we assessed the levels of ROS, cytokine production and mitochondrial fission in the primary microglia pretreated with cell-permeable diethyl succinate mimicking increased intracellular succinate. Our results suggest that elevated intracellular succinate exerts a protective role in the primary microglia by preventing their conversion into the pro-inflammatory M1 phenotype induced by LPS. This protective effect is SUCNR1-independent and mediated by reduced mitochondrial fission and cellular ROS production.

Gold nanoclusters conjugated berberine reduce inflammation and alleviate neuronal apoptosis by mediating M2 polarization for spinal cord injury repair

Spinal cord injury (SCI) leads to nerve cell apoptosis and loss of motor function. Herein, excessive activation of the M1 phenotype macrophages/microglia is found to be the main reason for the poor prognosis of SCI, but the selective activation phenotype (M2) macrophages/microglia facilitates the recovery of SCI. Thereafter, we used gold nanoclusters loaded berberine (BRB-AuNCs) to reduce inflammation by inhibiting the activation of M1 phenotype macrophages/microglia, which simultaneously inhibited neuronal apoptosis after SCI. In vitro and in vivo experiments showed that BRB-AuNCs reduced M1 protein marker CD86, increased M2 protein marker CD206, reduced inflammation and apoptotic cytokines (IL-1β, IL-6, TNF-α, Cleaved Caspase-3, Bax). These results indicate that BRB-AuNCs have excellent anti-inflammatory and anti-apoptotic effects by inducing the polarization of macrophages/microglia from M1 phenotype to M2 phenotype. Thereafter, the motor functions of SCI rats were significantly improved after treatment with BRB-AuNCs. This work not only provides a new way for the treatment of SCI but also broadens BRB utilization strategies.

Tilapia Piscidin 4 (TP4) Reprograms M1 Macrophages to M2 Phenotypes in Cell Models of Gardnerella vaginalis-Induced Vaginosis

Gardnerella vaginalis is associated with bacterial vaginosis (BV). The virulence factors produced by G. vaginalis are known to stimulate vaginal mucosal immune response, which is largely driven by activated macrophages. While Tilapia piscidin 4 (TP4), an antimicrobial peptide isolated from Nile tilapia, is known to display a broad range of antibacterial functions, it is unclear whether TP4 can affect macrophage polarization in the context of BV. In this study, we used the culture supernatants from G. vaginalis to stimulate differentiation of THP-1 and RAW264.7 cells to an M1 phenotype. The treatment activated the NF-κB/STAT1 signaling pathway, induced reactive nitrogen and oxygen species, and upregulated inflammatory mediators. We then treated the induced M1 macrophages directly with a non-toxic dose of TP4 or co-cultured the M1 macrophages with TP4-treated vaginal epithelial VK2 cells. The results showed that TP4 could not only decrease pro-inflammatory mediators in the M1 macrophages, but it also enriched markers of M2 macrophages. Further, we found that direct treatment with TP4 switched M1 macrophages toward a resolving M2c phenotype via the MAPK/ERK pathway and IL-10-STAT3 signaling. Conversely, tissue repair M2a macrophages were induced by TP4-treated VK2 cells; TP4 upregulated TSG-6 in VK2 cells, which subsequently activated STAT6 and M2a-related gene expression in the macrophages. In conclusion, our results imply that TP4 may be able to attenuate the virulence of G. vaginalis by inducing resolving M2c and tissue repair M2a macrophage polarizations, suggesting a novel strategy for BV therapy.

Cancer cell membrane-coated nanoparticles for bimodal imaging-guided photothermal therapy and docetaxel-enhanced immunotherapy against cancer

Background Mono-therapeutic modality has limitations in combating metastatic lesions with complications. Although emerging immunotherapy exhibits preliminary success, solid tumors are usually immunosuppressive, leading to ineffective antitumor immune responses and immunotherapeutic resistance. The rational combination of several therapeutic modalities may potentially become a new therapeutic strategy to effectively combat cancer. Results Poly lactic-co-glycolic acid (PLGA, 50 mg) nanospheres were constructed with photothermal transduction agents (PTAs)-Prussian blue (PB, 2.98 mg) encapsulated in the core and chemotherapeutic docetaxel (DTX, 4.18 mg)/ immune adjuvant-imiquimod (R837, 1.57 mg) loaded in the shell. Tumor cell membranes were further coated outside PLGA nanospheres (designated “M@P-PDR”), which acted as “Nano-targeted cells” to actively accumulate in tumor sites, and were guided/monitored by photoacoustic (PA)/ magnetic resonance (MR) imaging. Upon laser irradiation, photothermal effects were triggered. Combined with DTX, PTT induced in situ tumor eradication. Assisted by the immune adjuvant R837, the maturation rate of DCs increased by 4.34-fold compared with that of the control. In addition, DTX polarized M2-phenotype tumor-associated macrophages (TAMs) to M1-phenotype, relieving the immunosuppressive TME. The proportion of M2-TAMs decreased from 68.57% to 32.80%, and the proportion of M1-TAMs increased from 37.02% to 70.81%. Integrating the above processes, the infiltration of cytotoxic T lymphocytes (CTLs) increased from 17.33% (control) to 35.5%. Primary tumors and metastasis were significantly inhibited when treated with “Nano-targeted cells”-based cocktail therapy. Conclusion “Nano-targeted cells”-based therapeutic cocktail therapy is a promising approach to promote tumor regression and counter metastasis/recurrence. Graphical Abstract