Simvastatin enhances protection against Listeria monocytogenes infection in mice by counteracting Listeria-induced phagosomal escape

Statins are well-known cholesterol lowering drugs targeting HMG-CoA-reductase, reducing the risk of coronary disorders and hypercholesterolemia. Statins are also involved in immunomodulation, which might influence the outcome of bacterial infection. Hence, a possible effect of statin treatment on Listeriosis was explored in mice. Statin treatment prior to subsequent L. monocytogenes infection strikingly reduced bacterial burden in liver and spleen (up to 100-fold) and reduced histopathological lesions. Statin-treatment in infected macrophages resulted in increased IL-12p40 and TNF-α and up to 4-fold reduced bacterial burden within 6 hours post infection, demonstrating a direct effect of statins on limiting bacterial growth in macrophages. Bacterial uptake was normal investigated in microbeads and GFP-expressing Listeria experiments by confocal microscopy. However, intracellular membrane-bound cholesterol level was decreased, as analyzed by cholesterol-dependent filipin staining and cellular lipid extraction. Mevalonate supplementation restored statin-inhibited cholesterol biosynthesis and reverted bacterial growth in Listeria monocytogenes but not in listeriolysin O (LLO)-deficient Listeria . Together, these results suggest that statin pretreatment increases protection against L. monocytogenes infection by reducing membrane cholesterol in macrophages and thereby preventing effectivity of the cholesterol-dependent LLO-mediated phagosomal escape of bacteria

The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA.1 phenotype.

Variant of concern (VOC) Omicron-BA.1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and animal models are urgently needed. Here, we characterize Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in hamsters, ferrets and hACE2-expressing mice, and immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In hamsters, Delta shows dominance over Omicron-BA.1, and in ferrets Omicron-BA.1 infection is abortive. In hACE2-knock-in mice, Delta and a Delta spike clone also show dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naïve K18-hACE2 mice, we observe Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of replication and pathogenicity. Finally, the Omicron-BA.1 spike clone is less well-controlled by mRNA-vaccination in K18-hACE2-mice and becomes more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance

The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA. 1 phenotype

Variant of concern (VOC) Omicron-BA.1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and animal models are urgently needed. Here, we characterize Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in hamsters, ferrets and hACE2-expressing mice, and immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In hamsters, Delta shows dominance over Omicron-BA.1, and in ferrets Omicron-BA.1 infection is abortive. In hACE2-knock-in mice, Delta and a Delta spike clone also show dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naïve K18-hACE2 mice, we observe Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of replication and pathogenicity. Finally, the Omicron-BA.1 spike clone is less well-controlled by mRNA-vaccination in K18-hACE2-mice and becomes more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance

Drought-induced modifications of photosynthetic electron transport in intact leaves: Analysis and use of neural networks as a tool for a rapid non-invasive estimation

AbstractWater deficit is one of the most important environmental factors limiting sustainable crop yields and it requires a reliable tool for fast and precise quantification. In this work we use simultaneously recorded signals of photoinduced prompt fluorescence (PF) and delayed fluorescence (DF) as well as modulated reflection (MR) of light at 820nm for analysis of the changes in the photosynthetic activity in detached bean leaves during drying. Depending on the severity of the water deficit we identify different changes in the primary photosynthetic processes. When the relative water content (RWC) is decreased to 60% there is a parallel decrease in the ratio between the rate of excitation trapping in the Photosystem (PS) II reaction center and the rate of reoxidation of reduced PSII acceptors. A further decrease of RWC to 20% suppresses the electron transfer from the reduced plastoquinone pool to the PSI reaction center. At RWC below values 15%, the reoxidation of the photoreduced primary quinone acceptor of PSII, QA–, is inhibited and at less than 5%, the primary photochemical reactions in PSI and II are inactivated. Using the collected sets of PF, DF and MR signals, we construct and train an artificial neural network, capable of recognizing the RWC in a series of “unknown” samples with a correlation between calculated and gravimetrically determined RWC values of about R2≈0.98. Our results demonstrate that this is a reliable method for determination of RWC in detached leaves and after further development it could be used for quantifying of drought stress of crop plants in situ. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial

Intracellular cholesterol levels and phagocytosis in macrophages in presence of simvastatin.

<p>(A) Representative images of simvastatin ± mevalonate-treated macrophages overnight. Cells were washed and then stained with filipin and (B) fluorescent intensity (arbitrary units) per cell was quantified by Laser Scanning Microscope (LSM) software. Data is shown as intensity from 50-100 cells/group (Original magnification x100). (C) Macrophage cholesterol levels were measured using filipin staining 1 hour after <i>L. monocytogenes</i> infection. (D) Cholesterol content was measured in simvastatin-treated macrophage cell lysates following lipid extraction. (E) Macrophages were treated with either simvastatin ± mevalonate for 24 hours or ± methyl-β-cyclodextrin (MβCD) or with cholesterol for 2 hours, and then incubated with latex beads at MOI=10 to measure phagocytosis (Original magnification x100). Cells were then analyzed for number of internalized beads in each setting. (F) Uptake of GFP-expressing Listeria was measured in simvastatin-treated macrophages at 90 minutes post-infection. (G) Extracellular growth of <i>L. monocytogenes</i> was determined in tryptic soy broth supplemented with indicated concentrations of simvastatin. Results are shown as mean ± SEM of triplicates and are representative of two or three independent experiments, * <i>p</i> < 0.05, ** <i>p</i> < 0.01 versus control.</p

Growth and cytokine profile following <i>L. monocytogenes</i> infection in murine macrophages after simvastatin treatment.

<p>(A) Murine BMDM and (B) RAW264.7 murine macrophage cell line were pretreated with the indicated concentrations of simvastatin, followed by <i>L. monocytogenes</i> infection (MOI=10). Bacterial growth was measured at 6 and 12 hours post-infection. (C) Macrophages were analyzed for statin-mediated cytotoxicity using MTT assay. Following simvastatin treatment and IFN-γ stimulation, macrophages were infected for 12 hours and supernatants were analyzed for the production of (D) IL-12p40, (E) TNF-α, (F) IL-6 and (G) nitric oxide. Results are shown as mean ± SEM of triplicate cultures and are representative of two independent experiments, * <i>p</i> < 0.05, ** <i>p</i> < 0.01 versus control.</p

Effect of pravastatin treatment on <i>L. monocytogenes</i> infection in mice.

<p>(A) Mice were treated with pravastatin at 2 and 10 mg/kg/day or with vehicle control by daily intraperitoneal injection for 8 days. Mice were infected intraperitoneally with <i>L. monocytogenes</i> (2x10⁵ CFU) and sacrificed as indicated in the layout. (B) Bacterial burdens in the spleen and (C) liver were determined at 3 days post-infection. (D) Cholesterol levels were measured in sera at indicated times during the course of experiment. Data are expressed as mean ± SEM of 5 mice/group, ** <i>p</i> < 0.01 versus control.</p

Effect of simvastatin treatment on <i>Listeria monocytogenes</i> infection in mice.

<p>(A) Mice were treated with simvastatin at 10 and 20 mg/kg/day or with vehicle control by daily intraperitoneal injection for 12 days. Mice were infected intraperitoneally with <i>L. monocytogenes</i> (2x10⁵ CFU) and sacrificed as indicated in the layout. (B) Bacterial burden in the spleen and liver determined at 3 days post-infection. Pooled data from two independent experiments are shown. (C) Hematoxylin and eosin stained liver section to determine lesion size in 50-150 microabscesses/group (Original magnification x200). (D) Cholesterol and (E) triglyceride levels were measured in sera before and 3 days after <i>L. monocytogenes</i> infection in non-statin treated control mice group. (F) Serum cholesterol levels were measured before and after infection in control and statin-treated groups. Data are representative of two independent experiments. Data are expressed as mean ± SEM of 6-12 mice/group, * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 versus control.</p