Direct and indirect modulation of LPS-induced cytokine production by insulin in human macrophages.

Overweight and obesity are accompanied by insulin resistance, impaired intestinal barrier function resulting in increased lipopolysaccharide (LPS) levels, and a low-grade chronic inflammation that results in macrophage activation. Macrophages produce a range of interleukins as well as prostaglandin E2 (PGE2). To cope with insulin resistance, hyperinsulinemia develops. The purpose of the study was to elucidate how LPS, insulin and PGE2 might interact to modulate the inflammatory response in macrophages. Human macrophages were either derived by differentiation from U937 cells or isolated from blood mononuclear cells. The macrophages were stimulated with LPS, insulin and PGE2. Insulin significantly enhanced the LPS-dependent expression of interleukin-1ß and interleukin-8 on both the mRNA and protein levels. Additionally, insulin increased the LPS-dependent induction of enzymes involved in the PGE2-synthesis and the production of PGE2 by macrophages. PGE2 in turn further enhanced the LPS-dependent expression of cytokines via its Gs-coupled receptors EP2 and EP4, the latter of which appeared to be more relevant. The combination of all three stimuli resulted in an even higher induction than the combination of LPS plus insulin or LPS plus PGE2. Thus, the compensatory hyperinsulinemia might directly and indirectly enhance the LPS-dependent cytokine production in obese individuals.

Induction of steatohepatitis (NASH) with insulin resistance in wildtype B6 mice by a western-type diet containing soybean oil and cholesterol.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are hepatic manifestations of the metabolic syndrome. Many currently used animal models of NAFLD/NASH lack clinical features of either NASH or metabolic syndrome such as hepatic inflammation and fibrosis (e.g. high-fat diets) or overweight and insulin resistance (e.g. methionine-choline-deficient diets) or they are based on monogenetic defects (e.g. ob/ob mice). In the current study, a western-type diet containing soybean oil with high n 6-PUFA and 0.75% cholesterol (SOD+Cho) induced steatosis, inflammation and fibrosis accompanied by hepatic lipid peroxidation and oxidative stress in livers of C57BL/6-mice which in addition showed increased weight gain and insulin resistance, thus displaying a phenotype closely resembling all clinical features of NASH in patients with metabolic syndrome. In striking contrast a soybean oil-containing western-type diet without cholesterol (SOD) induced only mild steatosis but neither hepatic inflammation nor fibrosis, weight gain or insulin resistance. Another high-fat diet mainly consisting of lard and supplemented with fructose in drinking water (LAD+Fru) resulted in more prominent weight gain, insulin resistance and hepatic steatosis than SOD+Cho but livers were devoid of inflammation and fibrosis. Although both LAD+Fru- and SOD+Cho-fed animals had high plasma cholesterol, liver cholesterol was elevated only in SOD+Cho animals. Cholesterol induced expression of chemotactic and inflammatory cytokines in cultured Kupffer cells and rendered hepatocytes more susceptible to apoptosis. Summarizing, dietary cholesterol in SOD+Cho diet may trigger hepatic inflammation and fibrosis. SOD+Cho-fed animals may be a useful disease model displaying many clinical features of patients with the metabolic syndrome and NASH.

Determining On-Target, Off-Target, and Copy Number Status of Transgenic Events After CRISPR/Cas9 Targeted AAVS1 Safe-Harbor Modification of iPSCs Using Double-Control Quantitative Copy Number PCR.

Double-control quantitative copy number PCR (dc-qcnPCR) is a recently described tool that can be used to quantify donor DNA insertions in genetically modified monoclonal cell lines. In conjunction with an insert-confirmation PCR, the technique can quickly and easily identify clones containing on-target heterozygous or homozygous donor DNA integrations and exclude off-target insertions. The genetic manipulation of immortal cell lines is a versatile tool to elucidate cellular signaling pathways and protein functions. Despite recent advances in the precision of genetic engineering tools such as CRISPR/Cas9, transcription-activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs), it is still essential to verify the accurate insertion of the sequence of interest (donor DNA) into the targeted genomic DNA (gDNA) locus. This precise integration into a genetic safe harbor, and exclusion of the donor DNA from functionally relevant genes, can ensure normal cellular functionality. Current methods to analyze the specificity of donor DNA insertions either are cost-prohibitive or create dependency on manufacturers for assay design and production. The dc-qcnPCR method is a simple, yet powerful, approach that can be prepared and carried out in any laboratory equipped with standard molecular biology supplies. Here we provide step-by-step instructions to prepare and perform the dc-qcnPCR, and its companion insert-confirmation PCR, to determine donor DNA insertion numbers in monoclonal cell lines genetically modified through CRISPR/Cas9. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genetic modification at AAVS1 safe harbor in induced pluripotent stem cells (IMR90-4) using CRISPR/Cas9: from plasmid design to monoclonal expansion Support Protocol 1: Measurement of Gaussia luciferase activity to verify reporter protein functionality Support Protocol 2: Verification of monoclonal expansion using immunofluorescence. Basic Protocol 2: Insert-confirmation PCR Basic Protocol 3: Design and preparation of double-control quantitative copy number PCR reagents and quantification of donor DNA integrations in genetically modified monoclonal cells.

LDL-Dependent Regulation of TNFα/PGE2 Induced COX-2/mPGES-1 Expression in Human Macrophage Cell Lines.

Inflammation is a hallmark in severe diseases such as atherosclerosis and non-alcohol-induced steatohepatitis (NASH). In the development of inflammation, prostaglandins, especially prostaglandin E2 (PGE2), are major players alongside with chemo- and cytokines, like tumor-necrosis-factor alpha (TNFα) and interleukin-1 beta (IL-1ß). During inflammation, PGE2 synthesis can be increased by the transcriptional induction of the two key enzymes: cyclooxygenase 2 (COX-2), which converts arachidonic acid to PGH2, and microsomal prostaglandin E2 synthase 1 (mPGES-1), which synthesizes PGE2 from PGH2. Both COX-2 and mPGES-2 were induced by a dietary intervention where mice were fed a fatty acid-rich and, more importantly, cholesterol-rich diet, leading to the development of NASH. Since macrophages are the main source of PGE2 synthesis and cholesterol is predominantly transported as LDL, the regulation of COX-2 and mPGES-1 expression by native LDL was analyzed in human macrophage cell lines. THP-1 and U937 monocytes were differentiated into macrophages, through which TNFα and PGE-2 induced COX-2 and mPGES-1 expression by LDL could be analyzed on both mRNA and protein levels. In addition, the interaction of LDL- and EP receptor signal chains in COX-2/mPGES-1 expression and PGE2-synthesis were analyzed in more detail using EP receptor specific agonists. Furthermore, the LDL-mediated signal transduction in THP-1 macrophages was analyzed by measuring ERK and Akt phosphorylation as well as transcriptional regulation of transcription factor Egr-1. COX-2 and mPGES-1 were induced in both THP-1 and U937 macrophages by the combination of TNFα and PGE2. Surprisingly, LDL dose-dependently increased the expression of mPGES-1 but repressed the expression of COX-2 on mRNA and protein levels in both cell lines. The interaction of LDL and PGE2 signal chains in mPGES-1 induction as well as PGE2-synthesis could be mimicked by through simultaneous stimulation with EP2 and EP4 agonists. In THP-1 macrophages, LDL induced Akt-phosphorylation, which could be blocked by a PI3 kinase inhibitor. Alongside blocking Akt-phosphorylation, the PI3K inhibitor inhibited LDL-mediated mPGES-1 induction; however, it did not attenuate the repression of COX-2 expression. LDL repressed basal ERK phosphorylation and expression of downstream transcription factor Egr-1, which might lead to inhibition of COX-2 expression. These findings suggest that simultaneous stimulation with a combination of TNFα, PGE2, and native LDL-activated signal chains in macrophage cell lines leads to maximal mPGES-1 activity, as well repression of COX-2 expression, by activating PI3K as well as repression of ERK/Egr-1 signal chains.

Macrophages, Low-Grade Inflammation, Insulin Resistance and Hyperinsulinemia: A Mutual Ambiguous Relationship in the Development of Metabolic Diseases.

Metabolic derangement with poor glycemic control accompanying overweight and obesity is associated with chronic low-grade inflammation and hyperinsulinemia. Macrophages, which present a very heterogeneous population of cells, play a key role in the maintenance of normal tissue homeostasis, but functional alterations in the resident macrophage pool as well as newly recruited monocyte-derived macrophages are important drivers in the development of low-grade inflammation. While metabolic dysfunction, insulin resistance and tissue damage may trigger or advance pro-inflammatory responses in macrophages, the inflammation itself contributes to the development of insulin resistance and the resulting hyperinsulinemia. Macrophages express insulin receptors whose downstream signaling networks share a number of knots with the signaling pathways of pattern recognition and cytokine receptors, which shape macrophage polarity. The shared knots allow insulin to enhance or attenuate both pro-inflammatory and anti-inflammatory macrophage responses. This supposedly physiological function may be impaired by hyperinsulinemia or insulin resistance in macrophages. This review discusses the mutual ambiguous relationship of low-grade inflammation, insulin resistance, hyperinsulinemia and the insulin-dependent modulation of macrophage activity with a focus on adipose tissue and liver.

Validation of a Novel Double Control Quantitative Copy Number PCR Method to Quantify Off-Target Transgene Integration after CRISPR-Induced DNA Modification.

In order to improve a recently established cell-based assay to assess the potency of botulinum neurotoxin, neuroblastoma-derived SiMa cells and induced pluripotent stem-cells (iPSC) were modified to incorporate the coding sequence of a reporter luciferase into a genetic safe harbor utilizing CRISPR/Cas9. A novel method, the double-control quantitative copy number PCR (dc-qcnPCR), was developed to detect off-target integrations of donor DNA. The donor DNA insertion success rate and targeted insertion success rate were analyzed in clones of each cell type. The dc-qcnPCR reliably quantified the copy number in both cell lines. The probability of incorrect donor DNA integration was significantly increased in SiMa cells in comparison to the iPSCs. This can possibly be explained by the lower bundled relative gene expression of a number of double-strand repair genes (BRCA1, DNA2, EXO1, MCPH1, MRE11, and RAD51) in SiMa clones than in iPSC clones. The dc-qcnPCR offers an efficient and cost-effective method to detect off-target CRISPR/Cas9-induced donor DNA integrations.

Enhanced Palmitate-Induced Interleukin-8 Formation in Human Macrophages by Insulin or Prostaglandin E2.

Macrophages in pathologically expanded dysfunctional white adipose tissue are exposed to a mix of potential modulators of inflammatory response, including fatty acids released from insulin-resistant adipocytes, increased levels of insulin produced to compensate insulin resistance, and prostaglandin E2 (PGE2) released from activated macrophages. The current study addressed the question of how palmitate might interact with insulin or PGE2 to induce the formation of the chemotactic pro-inflammatory cytokine interleukin-8 (IL-8). Human THP-1 cells were differentiated into macrophages. In these macrophages, palmitate induced IL-8 formation. Insulin enhanced the induction of IL-8 formation by palmitate as well as the palmitate-dependent stimulation of PGE2 synthesis. PGE2 in turn elicited IL-8 formation on its own and enhanced the induction of IL-8 release by palmitate, most likely by activating the EP4 receptor. Since IL-8 causes insulin resistance and fosters inflammation, the increase in palmitate-induced IL-8 formation that is caused by hyperinsulinemia and locally produced PGE2 in chronically inflamed adipose tissue might favor disease progression in a vicious feed-forward cycle.

Real time monitoring of oxygen uptake of hepatocytes in a microreactor using optical microsensors.

Most in vitro test systems for the assessment of toxicity are based on endpoint measurements and cannot contribute much to the establishment of mechanistic models, which are crucially important for further progress in this field. Hence, in recent years, much effort has been put into the development of methods that generate kinetic data. Real time measurements of the metabolic activity of cells based on the use of oxygen sensitive microsensor beads have been shown to provide access to the mode of action of compounds in hepatocytes. However, for fully exploiting this approach a detailed knowledge of the microenvironment of the cells is required. In this work, we investigate the cellular behaviour of three types of hepatocytes, HepG2 cells, HepG2-3A4 cells and primary mouse hepatocytes, towards their exposure to acetaminophen when the availability of oxygen for the cell is systematically varied. We show that the relative emergence of two modes of action, one NAPQI dependent and the other one transient and NAPQI independent, scale with expression level of CYP3A4. The transient cellular response associated to mitochondrial respiration is used to characterise the influence of the initial oxygen concentration in the wells before exposure to acetaminophen on the cell behaviour. A simple model is presented to describe the behaviour of the cells in this scenario. It demonstrates the level of control over the role of oxygen supply in these experiments. This is crucial for establishing this approach into a reliable and powerful method for the assessment of toxicity.

Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins.

Despite the implementation of cell-based replacement methods, the mouse lethality assay is still frequently used to determine the activity of botulinum toxin (BoNT) for medical use. One explanation is that due to the use of neoepitope-specific antibodies to detect the cleaved BoNT substrate, the currently devised assays can detect only one specific serotype of the toxin. Recently, we developed a cell-based functional assay, in which BoNT activity is determined by inhibiting the release of a reporter enzyme that is liberated concomitantly with the neurotransmitter from neurosecretory vesicles. In theory, this assay should be suitable to detect the activity of any BoNT serotype. Consistent with this assumption, the current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) was inhibited by BoNT-A and-C. Furthermore, this was also inhibited by BoNT-B and tetanus toxin to a lesser extent and at higher concentrations. In order to provide support for the suitability of this technique in practical applications, a dose⁻response curve obtained with a pharmaceutical preparation of BoNT-A closely mirrored the activity determined in the mouse lethality assay. In summary, the newly established cell-based assay may represent a versatile and specific alternative to the mouse lethality assay and other currently established cell-based assays.

Augmented liver inflammation in a microsomal prostaglandin E synthase 1 (mPGES-1)-deficient diet-induced mouse NASH model.

In a subset of patients, non-alcoholic fatty liver disease (NAFLD) is complicated by cell death and inflammation resulting in non-alcoholic steatohepatitis (NASH), which may progress to fibrosis and subsequent organ failure. Apart from cytokines, prostaglandins, in particular prostaglandin E2 (PGE2), play a pivotal role during inflammatory processes. Expression of the key enzymes of PGE2 synthesis, cyclooxygenase 2 and microsomal PGE synthase 1 (mPGES-1), was increased in human NASH livers in comparison to controls and correlated with the NASH activity score. Both enzymes were also induced in NASH-diet-fed wild-type mice, resulting in an increase in hepatic PGE2 concentration that was completely abrogated in mPGES-1-deficient mice. PGE2 is known to inhibit TNF-α synthesis in macrophages. A strong infiltration of monocyte-derived macrophages was observed in NASH-diet-fed mice, which was accompanied with an increase in hepatic TNF-α expression. Due to the impaired PGE2 production, TNF-α expression increased much more in livers of mPGES-1-deficient mice or in the peritoneal macrophages of these mice. The increased levels of TNF-α resulted in an enhanced IL-1ß production, primarily in hepatocytes, and augmented hepatocyte apoptosis. In conclusion, attenuation of PGE2 production by mPGES-1 ablation enhanced the TNF-α-triggered inflammatory response and hepatocyte apoptosis in diet-induced NASH.

A Ser/Thr cluster within the C-terminal domain of the rat prostaglandin receptor EP3alpha is essential for agonist-induced phosphorylation, desensitization and internalization.

Two isoforms of the rat prostaglandin E(2) receptor, rEP3alpha-R and rEP3beta-R, differ only in their C-terminal domain. To analyze the function of the rEP3-R C-terminal domain in agonist induced desensitization, a cluster of Ser/Thr residues in the C-terminal domain of the rEP3alpha-R was mutated to Ala and both isoforms and the receptor mutant (rEP3alpha-ST341-349A-R) were stably expressed in HEK293 cells. All rEP3-R receptors showed a similar ligand-binding profile. They were functionally coupled to Gi and reduced forskolin-induced cAMP-formation. Repeated exposure of cells expressing the rEP3alpha-R isoform to PGE(2) reduced the agonist induced inhibition of forskolin-stimulated cAMP-formation by 50% and led to internalization of the receptor to intracellular endocytotic vesicles. By contrast, Gi-response as well as plasma membrane localization of the rEP3beta-R and the rEP3alpha-ST341-349A-R were not affected by prior agonist-stimulation. Agonist-stimulation of HEK293-rEP3alpha-R cells induced a time- and dose-dependent phosphorylation of the receptor most likely by G protein-coupled receptor kinases and not by protein kinase A or protein kinase C. By contrast, upon agonist-stimulation the rEP3beta-R was not phosphorylated and the rEP3alpha-ST341-349A-R was phosphorylated only weakly. These results led to the hypothesis that agonist-induced desensitization of the rEP3alpha-R isoform is mediated most likely by a GRK-dependent phosphorylation of Ser/Thr residues 341-349. Phosphorylation then initiates uncoupling of the receptor from Gi protein and receptor internalization.

Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site.

hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a G(s)-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C-terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced beta-arrestin complex formation, which is stabilized by phosphorylation. Subsequently beta-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist-induced phosphorylation, interaction with beta-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370-382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389-Ser-390-Thr-391-Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of beta-arrestin1. However, phosphorylation greatly enhanced the stability of the beta-arrestin1-receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both beta-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary.

GTPase ARFRP1 is essential for normal hepatic glycogen storage and insulin-like growth factor 1 secretion.

The GTPase ADP-ribosylation factor-related protein 1 (ARFRP1) is located at the trans-Golgi compartment and regulates the recruitment of Arf-like 1 (ARL1) and its effector golgin-245 to this compartment. Here, we show that liver-specific knockout of Arfrp1 in the mouse (Arfrp1(liv-/-)) resulted in early growth retardation, which was associated with reduced hepatic insulin-like growth factor 1 (IGF1) secretion. Accordingly, suppression of Arfrp1 in primary hepatocytes resulted in a significant reduction of IGF1 release. However, the hepatic secretion of IGF-binding protein 2 (IGFBP2) was not affected in the absence of ARFRP1. In addition, Arfrp1(liv-/-) mice exhibited decreased glucose transport into the liver, leading to a 50% reduction of glycogen stores as well as a marked retardation of glycogen storage after fasting and refeeding. These abnormalities in glucose metabolism were attributable to reduced protein levels and intracellular retention of the glucose transporter GLUT2 in Arfrp1(liv-/-) livers. As a consequence of impaired glucose uptake into the liver, the expression levels of carbohydrate response element binding protein (ChREBP), a transcription factor regulated by glucose concentration, and its target genes (glucokinase and pyruvate kinase) were markedly reduced. Our data indicate that ARFRP1 in the liver is involved in the regulation of IGF1 secretion and GLUT2 sorting and is thereby essential for normal growth and glycogen storage.