Statin-mediated reduction in mitochondrial cholesterol primes an anti-inflammatory response in macrophages by upregulating Jmjd3.

Statins are known to be anti-inflammatory, but the mechanism remains poorly understood. Here, we show that macrophages, either treated with statin in vitro or from statin-treated mice, have reduced cholesterol levels and higher expression of Jmjd3, a H3K27me3 demethylase. We provide evidence that lowering cholesterol levels in macrophages suppresses the adenosine triphosphate (ATP) synthase in the inner mitochondrial membrane and changes the proton gradient in the mitochondria. This activates nuclear factor kappa-B (NF-κB) and Jmjd3 expression, which removes the repressive marker H3K27me3. Accordingly, the epigenome is altered by the cholesterol reduction. When subsequently challenged by the inflammatory stimulus lipopolysaccharide (M1), macrophages, either treated with statins in vitro or isolated from statin-fed mice, express lower levels proinflammatory cytokines than controls, while augmenting anti-inflammatory Il10 expression. On the other hand, when macrophages are alternatively activated by IL-4 (M2), statins promote the expression of Arg1, Ym1, and Mrc1. The enhanced expression is correlated with the statin-induced removal of H3K27me3 from these genes prior to activation. In addition, Jmjd3 and its demethylase activity are necessary for cholesterol to modulate both M1 and M2 activation. We conclude that upregulation of Jmjd3 is a key event for the anti-inflammatory function of statins on macrophages.

Pro- and Anti-Inflammatory Cytokines in the Context of NK Cell-Trophoblast Interactions.

During pregnancy, uterine NK cells interact with trophoblast cells. In addition to contact interactions, uterine NK cells are influenced by cytokines, which are secreted by the cells of the decidua microenvironment. Cytokines can affect the phenotypic characteristics of NK cells and change their functional activity. An imbalance of pro- and anti-inflammatory signals can lead to the development of reproductive pathology. The aim of this study was to assess the effects of cytokines on NK cells in the presence of trophoblast cells in an in vitro model. We used TNFα, IFNγ, TGFß and IL-10; the NK-92 cell line; and peripheral blood NK cells (pNKs) from healthy, non-pregnant women. For trophoblast cells, the JEG-3 cell line was used. In the monoculture of NK-92 cells, TNFα caused a decrease in CD56 expression. In the coculture of NK cells with JEG-3 cells, TNFα increased the expression of NKG2C and NKG2A by NK-92 cells. Under the influence of TGFß, the expression of CD56 increased and the expression of NKp30 decreased in the monoculture. After the preliminary cultivation of NK-92 cells in the presence of TGFß, their cytotoxicity increased. In the case of adding TGFß to the PBMC culture, as well as coculturing PBMCs and JEG-3 cells, the expression of CD56 and NKp44 by pNK cells was reduced. The differences in the effects of TGFß in the model using NK-92 cells and pNK cells may be associated with the possible influence of monocytes or other lymphoid cells from the mononuclear fraction.

NK-92 cells change their phenotype and function when cocultured with IL-15, IL-18 and trophoblast cells.

NK cell development is affected by their cellular microenvironment and cytokines, including IL-15 and IL-18. NK cells can differentiate in secondary lymphoid organs, liver and within the uterus in close contact with trophoblast cells. The aim was to evaluate changes in the NK cell phenotype and function in the presence of IL-15, IL-18 and JEG-3, a trophoblast cell line. When cocultured with JEG-3 cells, IL-15 caused an increase in the number of NKG2D+ NK-92 cells and the intensity of CD127 expression. IL-18 stimulates an increase in the amount of NKp44+ NK-92 cells and in the intensity of NKp44 expression by pNK in the presence of trophoblast cells. NK-92 cell cytotoxic activity against JEG-3 cells increased only in presence of IL-18. Data on changes in the cytotoxic activity of NK-92 cells against JEG-3 cells in the presence of IL-15 and IL-18 indicate the modulation of NK cell function both by the cytokine microenvironment and directly by target cells. IL-15 and IL-18 were present in conditioned media (CM) from 1st and 3rd trimester placentas. In the presence of 1st trimester CM and JEG-3 cells, NK-92 cells showed an increase in the intensity of NKG2D expression. In the presence of 3rd trimester CM and JEG-3 cells, a decrease in the expression of NKG2D by NK-92 cells was observed. Thus, culturing of NK-92 cells with JEG-3 trophoblast cells stimulated a pronounced change in the NK cell phenotype, bringing it closer to the decidual NK cell-like phenotype.

Effects of cobalt and chromium ions on glycolytic flux and the stabilization of hypoxia-inducible factor-1α in macrophages in vitro.

Implant wear and corrosion have been associated with adverse tissue reactions that can lead to implant failure. Wear and corrosion products are therefore of great clinical concern. For example, Co2+ and Cr3+ originating from CoCrMo-based implants have been shown to induce a proinflammatory response in macrophages in vitro. Previous studies have also shown that the polarization of macrophages by some proinflammatory stimuli is associated with a hypoxia-inducible factor-1α (HIF-1α)-dependent metabolic shift from oxidative phosphorylation (OXPHOS) towards glycolysis. However, the potential of Co2+ and Cr3+ to induce this metabolic shift, which plays a determining role in the proinflammatory response of macrophages, remains largely unexplored. We recently demonstrated that Co2+ , but not Cr3+ , increased oxidative stress and decreased OXPHOS in RAW 264.7 murine macrophages. In the present study, we analyzed the effects of Co2+ and Cr3+ on glycolytic flux and HIF-1α stabilization in the same experimental model. Cells were exposed to 6 to 24 ppm Co2+ or 50 to 250 ppm Cr3+ . Glycolytic flux was determined by analyzing extracellular flux and lactate production, while HIF-1α stabilization was analyzed by immunoblotting. Results showed that Co2+ , and to a lesser extent Cr3+ , increased glycolytic flux; however, only Co2+ acted through HIF-1α stabilization. Overall, these results, together with our previous results showing that Co2+ increases oxidative stress and decreases OXPHOS, suggest that Co2+ (but not Cr3+ ) can induce a HIF-1α-dependent metabolic shift from OXPHOS towards glycolysis in macrophages. This metabolic shift may play an early and pivotal role in the inflammatory response induced by Co2+ in the periprosthetic environment.

The uteroplacental contact zone cytokine influence on NK cell cytotoxicity to trophoblasts.

OBJECTIVE: The present study was to estimate the role of cytokines for trophoblast death in NK cells presence. METHODS: This study involves assessment of NK-92 line NK cell cytotoxic activity against JEG-3 line cells, in presence of cytokines. We also assessed the effect of secretory placenta products on NK cell cytotoxic activity toward JEG-3 line cells. RESULTS: Uteroplacental contact zone cytokines are able to enhance trophoblast mortality both by themselves in case of IL-1ß, IL-6, IFNγ, IL-4, TGFß, bFGF, and also through increasing the cytotoxic potential of NK cells in case of IL-1ß, IFNγ, IL-8, TGFß, and GM-CSF. PLGF decreases NK cell cytotoxicity for trophoblasts. Secretory products of first trimester placenta enhance NK cell cytotoxic potential for trophoblasts. CONCLUSIONS: Cytokines of the uteroplacental contact zone can appear a mechanism ensuring trophoblast mortality dynamics throughout pregnancy.

Effects of cobalt and chromium ions on oxidative stress and energy metabolism in macrophages in vitro.

Cobalt and chromium ions released from cobalt-chromium-molybdenum (CoCrMo)-based implants are a potential health concern, especially since both ions have been shown to induce oxidative stress in macrophages, the predominant immune cells in periprosthetic tissues. Ions of other transition metals (Cd, Ni) have been reported to inhibit the activity of mitochondrial enzymes in the electron transport chain. However, the effects of Co and Cr ions on the energy metabolism of macrophages remain largely unknown. The objective of the present study was to analyze the effects of Co2+ and Cr3+ on oxidative stress and energy metabolism in macrophages in vitro. RAW 264.7 murine macrophages were exposed to 6-18 ppm Co2+ or 50-150 ppm Cr3+ . Results showed a significant increase in two markers of oxidative stress, reactive oxygen species level and protein carbonyl content, with increasing concentrations of Co2+ , but not with Cr3+ . In addition, oxygen consumption rates (OCR; measured using an extracellular flux analyzer) showed significant decreases in both mitochondrial respiration and non-mitochondrial oxygen consumption with increasing concentrations of Co2+ , but not with Cr3+ . OCR results further showed that Co2+ , but not Cr3+ , induced mitochondrial dysfunction, including a decrease in oxidative phosphorylation capacity. Overall, this study suggests that mitochondrial dysfunction may contribute to Co2+ -induced oxidative stress in macrophages, and thereby to the inflammatory response observed in periprosthetic tissues. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3178-3187, 2018.

The chromosomal passenger complex activates Polo kinase at centromeres.

The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules) requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. Our study concerns the coordination of the activities of these two kinases in vivo. We show that INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC), which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. We show here that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity) and is critical for Polo function at kinetochores. Our results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation.

Independent modulation of the kinase and polo-box activities of Cdc5 protein unravels unique roles in the maintenance of genome stability.

Polo-like kinases (PLKs) are evolutionarily conserved kinases essential for cell cycle regulation. These kinases are characterized by the presence of a C-terminal phosphopeptide-interaction domain, the polo-box domain (PBD). How the functional domains of PLKs work together to promote cell division is not understood. To address this, we performed a genetic screen to identify mutations that independently modulate the kinase and PBD activities of yeast PLK/Cdc5. This screen identified a mutagenic hotspot in the F-helix region of Cdc5 kinase domain that allows one to control kinase activity in vivo. These mutations can be systematically engineered into other major eukaryotic cell cycle kinases to similarly regulate their activity in live cells. Here, using this approach, we show that the kinase activity of Cdc5 can promote the execution of several stages of mitosis independently of PBD activity. In particular, we observe that the activation of Cdc14 and execution of mitotic exit are uniquely sensitive to the modulation of Cdc5 kinase activity. In contrast, PBD-defective mutants are capable of completing mitosis but are unable to maintain spindle pole body integrity. Consistent with this defect, PBD-deficient cells progressively double the size of their genome and ultimately lose genome integrity. Collectively, these results highlight the specific contributions of Cdc5 functional domains to cell division and reveal unexpected mechanisms controlling spindle pole body behavior and genome stability.