Inflammatory and Cytotoxic Activities of Abietane Terpenoids from Nepeta bracteata Benth.

Nepeta bracteata Benth. is used clinically to treat tracheal inflammation, coughs, asthma, colds, fevers, adverse urination, and other symptoms, along with functions in clearing heat and removing dampness. However, there have been few studies characterizing the material basis of its efficacy. Therefore, the aim of this study was to screen for compounds with anti-inflammatory activities in N. bracteata Benth. Using silica gel, ODS C18, and Sephadex LH-20 column chromatography, as well as semipreparative HPLC, 10 compounds were separated from N. bracteata Benth. extract, including four new diterpenoids (1–4), one amide alkaloid (5), and five known diterpenoids (6–10). The structures of all the isolates were elucidated by HR-ESI-MS, NMR, and CD analyses. Using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, we investigated the anti-inflammatory activities of compounds 1–10. It is worth noting that all were able to inhibit nitric oxide (NO) production with IC50 values < 50 μM and little effect on RAW 264.7 macrophage viability. Compounds 2 and 4 displayed remarkable inhibition with IC50 values of 19.2 and 18.8 μM, respectively. Meanwhile, screening on HCT-8 cells demonstrated that compounds 2 and 4 also had moderate cytotoxic activities with IC50 values of 36.3 and 41.4 μM, respectively, which is related to their anti-inflammatory effects.

Milk lymphocyte profile and macrophage functions: new insights into the immunity of the mammary gland in quarters infected with Corynebacterium bovis

Backgrounds The present study explored the viability of bovine milk macrophages, their intracellular production of reactive oxygen and nitrogen species (RONS), and their phagocytosis of Staphylococcus aureus, as well as the profile of lymphocytes, from healthy udder quarters and udder quarters infected by Corynebacterium bovis. The study included 28 healthy udder quarters from 12 dairy cows and 20 udder quarters infected by C. bovis from 10 dairy cows. The percentages of macrophages and lymphocytes were identified by flow cytometry using monoclonal antibodies. Macrophage viability, RONS production, and S. aureus phagocytosis were evaluated by flow cytometry. Results Milk samples from quarters infected with C. bovis showed a lower percentage of macrophages but an increased number of milk macrophages per mL and a higher percentage of macrophages that produced intracellular RONS and phagocytosed S. aureus. No effect of C. bovis infection on macrophage viability was found. Udder quarters infected by C. bovis showed a higher percentage of T cells and CD4+ T lymphocytes, but no effect was found on the percentage of CD8+ CD4− T, CD8− CD4− T, or B lymphocytes. Conclusions Thus, our results corroborate, at least in part, the finding that intramammary infections by C. bovis may offer protection against intramammary infections by major pathogens.

Milk Lymphocyte Profile and Macrophage Functions: New Insights Into the Immunity of the Mammary Gland in Quarters Infected With Corynebacterium Bovis

BackgroundsThe present study explored the viability of milk macrophages, their intracellular production of reactive oxygen and nitrogen species (RONS), and their phagocytosis of S. aureus, as well as the profile of lymphocytes, from healthy udder quarters and udder quarters infected by Corynebacterium bovis. The study included 28 healthy udder quarters from 12 dairy cows and 20 udder quarters infected by C. bovis from 10 dairy cows. The percentages of macrophages and lymphocytes were identified by flow cytometry using monoclonal antibodies. Macrophage viability, RONS production, and S. aureus phagocytosis were evaluated by flow cytometry. ResultsMilk samples from quarters infected with C. bovis showed a lower percentage of macrophages but an increased number of milk macrophages per mL and a higher percentage of macrophages that produced intracellular RONS and phagocytosed S. aureus. No effect of C. bovis infection on macrophage viability was found. Udder quarters infected by C. bovis showed a higher percentage of T cells and CD4+ T lymphocytes, but no effect was found on the percentage of CD8+ CD4- T, CD8- CD4- T, or B lymphocytes. ConclusionsThus, our results corroborate, at least in part, the finding that intramammary infections by C. bovis may offer protection against intramammary infections by major pathogens.

Prkaa1 Metabolically Regulates Monocyte/Macrophage Recruitment and Viability in Diet-Induced Murine Metabolic Disorders

Myeloid cells, including monocytes/macrophages, primarily rely on glucose and lipid metabolism to provide the energy and metabolites needed for their functions and survival. AMP-activated protein kinase (AMPK, its gene is PRKA for human, Prka for rodent) is a key metabolic sensor that regulates many metabolic pathways. We studied recruitment and viability of Prkaa1-deficient myeloid cells in mice and the phenotype of these mice in the context of cardio-metabolic diseases. We found that the deficiency of Prkaa1 in myeloid cells downregulated genes for glucose and lipid metabolism, compromised glucose and lipid metabolism of macrophages, and suppressed their recruitment to adipose, liver and arterial vessel walls. The viability of macrophages in the above tissues/organs was also decreased. These cellular alterations resulted in decreases in body weight, insulin resistance, and lipid accumulation in liver of mice fed with a high fat diet, and reduced the size of atherosclerotic lesions of mice fed with a Western diet. Our results indicate that AMPKα1/PRKAA1-regulated metabolism supports monocyte recruitment and macrophage viability, contributing to the development of diet-induced metabolic disorders including diabetes and atherosclerosis.

Photodynamic activity of Photogem® in Leishmania promastigotes and infected macrophages

Objectives: This study aimed to evaluate the effect of photodynamic therapy (PDT) with Photogem® in promastigotes of Leishmania braziliensis and Leishmania major, and in infected macrophages. Materials & methods: The following parameters were analyzed: Photogem® internalization, mitochondrial activity, viability, tubulin marking and morphological alterations in promastigotes and viability in infected macrophages. Results: Photogem® accumulated in the cytosol and adhered to the flagellum. Changes were observed in the mitochondrial activity in groups maintained in the dark, with no viability alteration. After PDT, viability decreased up to 80%, and morphology was affected. Conclusion: The results point out that PDT with Photogem® can reduce parasite and macrophage viability.

Oxidized LDL Disrupts Metabolism and Inhibits Macrophage Survival by Activating a miR-9/Drp1/Mitochondrial Fission Signaling Pathway

Mitochondrial dysfunction is associated with macrophage damage, but the role of mitochondrial fission in macrophage cholesterol metabolism is not fully understood. In this study, we explored the influences of miR-9 and mitochondrial fission on macrophage viability and cholesterol metabolism. Macrophages were incubated with oxidized low-density lipoprotein (ox-LDL) in vitro, after which mitochondrial fission, cell viability, and cholesterol metabolism were examined using qPCR, ELISAs, and immunofluorescence. ox-LDL treatment significantly increased Drp1-associated mitochondrial fission. Transfection of Drp1 siRNA significantly reduced cell death, attenuated oxidative stress, and inhibited inflammatory responses in ox-LDL-treated macrophages. Interestingly, inhibition of Drp1-related mitochondrial fission also improved cholesterol metabolism by balancing the transcription of cholesterol influx/efflux enzymes. We also found that miR-9 was downregulated in ox-LDL-treated macrophages, and administration of a miR-9 mimic decreased Drp1 transcription and mitochondrial fission, as well as its effects. These results indicate that signaling via the novel miR-9/Drp1/mitochondrial fission axis is a key determinant of macrophage viability and cholesterol metabolism.

Annexin A1 Regulates NLRP3 Inflammasome Activation and Modifies Lipid Release Profile in Isolated Peritoneal Macrophages

Annexin A1 (AnxA1) is a potent anti-inflammatory protein that downregulates proinflammatory cytokine release. This study evaluated the role of AnxA1 in the regulation of NLRP3 inflammasome activation and lipid release by starch-elicited murine peritoneal macrophages. C57bl/6 wild-type (WT) and AnxA1-null (AnxA1-/-) mice received an intraperitoneal injection of 1.5% starch solution for macrophage recruitment. NLRP3 was activated by priming cells with lipopolysaccharide for 3 h, followed by nigericin (1 h) or ATP (30 min) incubation. As expected, nigericin and ATP administration decreased elicited peritoneal macrophage viability and induced IL-1β release, more pronounced in the AnxA1-/- cells than in the control peritoneal macrophages. In addition, nigericin-activated AnxA1-/- macrophages showed increased levels of NLRP3, while points of co-localization of the AnxA1 protein and NLRP3 inflammasome were detected in WT cells, as demonstrated by ultrastructural analysis. The lipidomic analysis showed a pronounced release of prostaglandins in nigericin-stimulated WT peritoneal macrophages, while ceramides were detected in AnxA1-/- cell supernatants. Different eicosanoid profiles were detected for both genotypes, and our results suggest that endogenous AnxA1 regulates the NLRP3-derived IL-1β and lipid mediator release in macrophages.