Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs.

Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here, we tested the ability of cell-penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection-enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.

Integrated Inflammatory Stress (ITIS) Model.

During the last decade, there has been an increasing interest in the coupling between the acute inflammatory response and the Hypothalamic-Pituitary-Adrenal (HPA) axis. The inflammatory response is activated acutely by pathogen- or damage-related molecular patterns, whereas the HPA axis maintains a long-term level of the stress hormone cortisol which is also anti-inflammatory. A new integrated model of the interaction between these two subsystems of the inflammatory system is proposed and coined the integrated inflammatory stress (ITIS) model. The coupling mechanisms describing the interactions between the subsystems in the ITIS model are formulated based on biological reasoning and its ability to describe clinical data. The ITIS model is calibrated and validated by simulating various scenarios related to endotoxin (LPS) exposure. The model is capable of reproducing human data of tumor necrosis factor alpha, adrenocorticotropic hormone (ACTH) and cortisol and suggests that repeated LPS injections lead to a deficient response. The ITIS model predicts that the most extensive response to an LPS injection in ACTH and cortisol concentrations is observed in the early hours of the day. A constant activation results in elevated levels of the variables in the model while a prolonged change of the oscillations in ACTH and cortisol concentrations is the most pronounced result of different LPS doses predicted by the model.

Hydrogen Peroxide and Lipopolysaccharide Differentially Affect the Expression of MicroRNAs 10a, 33a, 21, 221 in Endothelial Cells Before and After Coculture With Monocytes.

Inflammation and oxidative stress are important risk factors affecting various cells in the formation of atherosclerosis. MicroRNAs (miRs) are regulators of inflammation and atherogenesis. The expressions of endothelial cell (EC)-specific miR-10a and miR-21 and monocyte-specific miR-33a and miR-221 were investigated using coculture of the ECs and monocytes upon exposure to H2O2 as an oxidative stressor, and endotoxin/lipopolysaccharide (LPS) as a microbial stressor. Human umbilical endothelial cells (HUVECs) and peripheral blood mononuclear cells (or monocytes) were cocultured in M199 complete medium and were incubated with LPS (20 ng/mL) or H2O2 (1%) for 8 hours at 37°C. The HUVECs and monocytes were then separated from the cellular mix using a magnetic bead negative selection technique. The relative expression of miRs was determined by real-time polymerase chain reaction. In both cell types, H2O2 induced miR10a ( P = 0.05) and LPS induced miR21 ( P = 0.0003) compared to the untreated controls. Coculture increased miR-10a and miR-21 expression in monocytes ( P = 0.0008 and <0.0001); however when cultured alone, HUVECs expressed higher levels of miR-10a and miR-21 ( P < 0.0001 and <0.0001). Coculture decreased the expression of miR-33a in monocytes ( P < 0.0001) while increasing miR221 in HUVECs and monocytes ( P < 0.0001 and <0.0001). The expression pattern of miRs in HUVECs and monocytes changes in the coculture compared to culturing alone in response to oxidative and microbial toxic compounds. Moreover, different cellular stressors induce different athero-miRs, which may affect the course of inflammation.

Inhibition of IRAK-4 activity for rescuing endotoxin LPS-induced septic mortality in mice by lonicerae flos extract.

Lonicerae flos extract (HS-23) is a clinical candidate currently undergoing Phase I trial in lipopolysaccharide (LPS)-injected healthy human volunteers, but its molecular basis remains to be defined. Here, we investigated protective effects of HS-23 or its major constituents on Escherichia coli LPS-induced septic mortality in mice. Intravenous treatment with HS-23 rescued LPS-intoxicated C57BL/6J mice under septic conditions, and decreased the levels of cytokines such as tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and high-mobility group box-1 (HMGB-1) in the blood. Chlorogenic acid (CGA) and its isomers were assigned as major constituents of HS-23 in the protection against endotoxemia. As a molecular mechanism, HS-23 or CGA isomers inhibited endotoxin LPS-induced autophosphorylation of the IL-1 receptor-associated kinase 4 (IRAK-4) in mouse peritoneal macrophages as well as the kinase activity of IRAK-4 in cell-free reactions. HS-23 consequently suppressed downstream pathways critical for LPS-induced activation of nuclear factor (NF)-κB or activating protein 1 (AP-1) in the peritoneal macrophages. HS-23 also inhibited various toll-like receptor agonists-induced nitric oxide (NO) production, and down-regulated LPS-induced expression of NF-κB/AP-1-target inflammatory genes in the cells. Taken together, HS-23 or CGA isomers exhibited anti-inflammatory therapy against LPS-induced septic mortality in mice, at least in part, mediated through the inhibition of IRAK-4.

Macrophage activation marker sCD163 correlates with accelerated lipolysis following LPS exposure: a human-randomised clinical trial.

BACKGROUND: Macrophage activation determined by levels of soluble sCD163 is associated with obesity, insulin resistance, diabetes mellitus type 2 (DM2) and non-alcoholic fatty liver disease (NAFLD). This suggests that macrophage activation is involved in the pathogenesis of conditions is characterised by adaptions in the lipid metabolism. Since sCD163 is shed to serum by inflammatory signals including lipopolysaccharides (LPS, endotoxin), we investigated sCD163 and correlations with lipid metabolism following LPS exposure. METHODS: Eight healthy male subjects were investigated on two separate occasions: (i) following an LPS exposure and (ii) following saline exposure. Each study day consisted of a four-hour non-insulin-stimulated period followed by a two-hour hyperinsulinemic euglycemic clamp period. A 3H-palmitate tracer was used to calculate the rate of appearance (Rapalmitate). Blood samples were consecutively obtained throughout each study day. Abdominal subcutaneous adipose tissue was obtained for western blotting. RESULTS: We observed a significant two-fold increase in plasma sCD163 levels following LPS exposure (P < 0.001), and sCD163 concentrations correlated positively with the plasma concentration of free fatty acids, Rapalmitate, lipid oxidation rates and phosphorylation of the hormone-sensitive lipase at serine 660 in adipose tissue (P < 0.05, all). Furthermore, sCD163 concentrations correlated positively with plasma concentrations of cortisol, glucagon, tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 (P < 0.05, all). CONCLUSION: We observed a strong correlation between sCD163 and stimulation of lipolysis and fat oxidation following LPS exposure. These findings support preexisting theory that inflammation and macrophage activation play a significant role in lipid metabolic adaptions under conditions such as obesity, DM2 and NAFLD.