New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals - a PARC (Partnership for the Assessment of Risk from Chemicals) project.

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC).

Determination of in vitro immunotoxic potencies of a series of perfluoralkylsubstances (PFASs) in human Namalwa B lymphocyte and human Jurkat T lymphocyte cells.

Exposure to PFASs is associated to several adverse health effects, such as immunotoxicity. Immunotoxic effects of PFOA and PFOS, including a reduced antibody response in both experimental animals and humans, have been reported. However, there is limited understanding of the underlying mechanisms involved. Moreover, there is only a restricted amount of immunotoxicity data available for a limited number of PFASs. In the current study the effects of 15 PFASs, including short- and long-chain perfluorinated carboxylic and sulfonic acids, fluorotelomer alcohols, and perfluoralkyl ether carboxylic acids were studied on the expression of recombinant activating gene 1 (RAG1) and RAG2 in the Namalwa human B lymphoma cell line, and on the human IL-2 promotor activity in Jurkat T-cells. Concentration-response data were subsequently used to derive in vitro relative potencies through benchmark dose analysis. In vitro relative potency factors (RPFs) were obtained for 6 and 9 PFASs based on their effect on RAG1 and RAG2 gene expression in Namalwa B-cells, respectively, and for 10 PFASs based on their inhibitory effect on IL-2 promotor activity in Jurkat T-cells. The most potent substances were HFPO-TA for the reduction of RAG1 and RAG2 gene expression in Namalwa cells (RPFs of 2.1 and 2.3 respectively), and PFDA on IL-2 promoter activity (RPF of 9.1). RAG1 and RAG2 play a crucial role in V (D)J gene recombination, a process for acquiring a varied array of antibodies crucial for antigen recognition. Hence, the effects observed in Namalwa cells might indicate a PFAS-induced impairment of generating a diverse range of B-cells essential for antigen recognition. The observed outcomes in the Jurkat T-cells suggest a possible PFAS-induced reduction of T-cell activation, which may contribute to a decline in the T-cell dependent antibody response. Altogether, the present study provides potential mechanistic insights into the reported PFAS-induced decreased antibody response. Additionally, the presented in vitro models may represent useful tools for assessing the immunotoxic potential of PFASs and prioritization for further risk assessment.

Perfluoroalkyl substances (PFASs) decrease the expression of recombination-activating genes (RAG1 and RAG2) in human B lymphoma Namalwa cells.

Per- and polyfluoroalkyl substances (PFASs) are omnipresent and have been shown to induce a wide range of adverse effects, including hepatotoxicity, developmental toxicity and immunotoxicity. So far, little information is available about the mechanisms underlying the toxicity of PFASs, including those related to their immunotoxicity. Reported immunotoxic effects of PFASs include decreased antibody responses in experimental animals and humans, indicating that PFASs may, among others, affect B cell function. In the present study, we first assessed the effects of PFOA on the transcriptome of the human Namalwa B cell line using RNA seq analysis. Gene expression changes, analyzed using Ingenuity Pathway Analysis, pointed to various cellular processes affected by PFOA, including 'B cell development' and 'Primary immunodeficiency signaling'. Interestingly, PFOA decreased the expression of RAG1 and RAG2, genes involved in immunoglobulin and T cell receptor V(D)J recombination. As a next step, time- and concentration-dependent changes in the expression of RAG1 and RAG2 upon exposure to PFOA, PFNA, PFHxS and PFOS were studied through RT-qPCR analysis. Analysis with the concentration-response modeling software PROAST resulted in the following potency ranking: PFNA > PFOA > PFOS > PFHxS. Altogether, the present in vitro study provides insights into the effects of selected PFASs on B cells, identifying RAG1 and RAG2 expression as possible relevant targets that may play a role in the immunotoxicity of PFASs.

Exposure to low-dose perfluorooctanoic acid promotes hepatic steatosis and disrupts the hepatic transcriptome in mice.

OBJECTIVE: Perfluoroalkyl substances (PFAS) are man-made chemicals with demonstrated endocrine-disrupting properties. Exposure to perfluorooctanoic acid (PFOA) has been linked to disturbed metabolism via the liver, although the exact mechanism is not clear. Moreover, information on the metabolic effects of the new PFAS alternative GenX is limited. We examined whether exposure to low-dose PFOA and GenX induces metabolic disturbances in mice, including NAFLD, dyslipidemia, and glucose tolerance, and studied the involvement of PPARα. METHODS: Male C57BL/6J wildtype and PPARα-/- mice were given 0.05 or 0.3 mg/kg body weight/day PFOA, or 0.3 mg/kg body weight/day GenX while being fed a high-fat diet for 20 weeks. Glucose and insulin tolerance tests were performed after 18 and 19 weeks. Plasma metabolite levels were measured next to a detailed assessment of the liver phenotype, including lipid content and RNA sequencing. RESULTS: Exposure to high-dose PFOA decreased body weight and increased liver weight in wildtype and PPARα-/- mice. High-dose but not low-dose PFOA reduced plasma triglycerides and cholesterol, which for triglycerides was dependent on PPARα. PFOA and GenX increased hepatic triglycerides in a PPARα-dependent manner. RNA sequencing showed that the effects of GenX on hepatic gene expression were entirely dependent on PPARα, while the effects of PFOA were mostly dependent on PPARα. In the absence of PPARα, the involvement of PXR and CAR became more prominent. CONCLUSION: Overall, we show that long-term and low-dose exposure to PFOA and GenX disrupts hepatic lipid metabolism in mice. Whereas the effects of PFOA are mediated by multiple nuclear receptors, the effects of GenX are entirely mediated by PPARα. Our data underscore the potential of PFAS to disrupt metabolism by altering signaling pathways in the liver.

Differential gene expression in iPSC-derived human intestinal epithelial cell layers following exposure to two concentrations of butyrate, propionate and acetate.

Intestinal epithelial cells and the intestinal microbiota are in a mutualistic relationship that is dependent on communication. This communication is multifaceted, but one aspect is communication through compounds produced by the microbiota such as the short-chain fatty acids (SCFAs) butyrate, propionate and acetate. Studying the effects of SCFAs and especially butyrate in intestinal epithelial cell lines like Caco-2 cells has been proven problematic. In contrast to the in vivo intestinal epithelium, Caco-2 cells do not use butyrate as an energy source, leading to a build-up of butyrate. Therefore, we used human induced pluripotent stem cell derived intestinal epithelial cells, grown as a cell layer, to study the effects of butyrate, propionate and acetate on whole genome gene expression in the cells. For this, cells were exposed to concentrations of 1 and 10 mM of the individual short-chain fatty acids for 24 h. Unique gene expression profiles were observed for each of the SCFAs in a concentration-dependent manner. Evaluation on both an individual gene level and pathway level showed that butyrate induced the biggest effects followed by propionate and then acetate. Several known effects of SCFAs on intestinal cells were confirmed, such as effects on metabolism and immune responses. The changes in metabolic pathways in the intestinal epithelial cell layers in this study demonstrate that there is a switch in energy homeostasis, this is likely associated with the use of SCFAs as an energy source by the induced pluripotent stem cell derived intestinal epithelial cells similar to in vivo intestinal tissues where butyrate is an important energy source.

Cytochrome P450 expression, induction and activity in human induced pluripotent stem cell-derived intestinal organoids and comparison with primary human intestinal epithelial cells and Caco-2 cells.

Human intestinal organoids (HIOs) are a promising in vitro model consisting of different intestinal cell types with a 3D microarchitecture resembling native tissue. In the current study, we aimed to assess the expression of the most common intestinal CYP enzymes in a human induced pluripotent stem cell (hiPSC)-derived HIO model, and the suitability of that model to study chemical-induced changes in CYP expression and activity. We compared this model with the commonly used human colonic adenocarcinoma cell line Caco-2 and with a human primary intestinal epithelial cell (IEC)-based model, closely resembling in vivo tissue. We optimized an existing protocol to differentiate hiPSCs into HIOs and demonstrated that obtained HIOs contain a polarized epithelium with tight junctions consisting of enterocytes, goblet cells, enteroendocrine cells and Paneth cells. We extensively characterized the gene expression of CYPs and activity of CYP3A4/5, indicating relatively high gene expression levels of the most important intestinal CYP enzymes in HIOs compared to the other models. Furthermore, we showed that CYP1A1 and CYP1B1 were induced by ß-naphtoflavone in all three models, whereas CYP3A4 was induced by phenobarbital and rifampicin in HIOs, in the IEC-based model (although not statistically significant), but not in Caco-2 cells. Interestingly, CYP2B6 expression was not induced in any of the models by the well-known liver CYP2B6 inducer phenobarbital. In conclusion, our study indicates that hiPSC-based HIOs are a useful in vitro intestinal model to study biotransformation of chemicals in the intestine.

Loss of angiopoietin-like 4 (ANGPTL4) in mice with diet-induced obesity uncouples visceral obesity from glucose intolerance partly via the gut microbiota.

AIMS/HYPOTHESIS: Angiopoietin-like 4 (ANGPTL4) is an important regulator of triacylglycerol metabolism, carrying out this role by inhibiting the enzymes lipoprotein lipase and pancreatic lipase. ANGPTL4 is a potential target for ameliorating cardiometabolic diseases. Although ANGPTL4 has been implicated in obesity, the study of the direct role of ANGPTL4 in diet-induced obesity and related metabolic dysfunction is hampered by the massive acute-phase response and development of lethal chylous ascites and peritonitis in Angptl4-/- mice fed a standard high-fat diet. The aim of this study was to better characterise the role of ANGPTL4 in glucose homeostasis and metabolic dysfunction during obesity. METHODS: We chronically fed wild-type (WT) and Angptl4-/- mice a diet rich in unsaturated fatty acids and cholesterol, combined with fructose in drinking water, and studied metabolic function. The role of the gut microbiota was investigated by orally administering a mixture of antibiotics (ampicillin, neomycin, metronidazole). Glucose homeostasis was assessed via i.p. glucose and insulin tolerance tests. RESULTS: Mice lacking ANGPTL4 displayed an increase in body weight gain, visceral adipose tissue mass, visceral adipose tissue lipoprotein lipase activity and visceral adipose tissue inflammation compared with WT mice. However, they also unexpectedly had markedly improved glucose tolerance, which was accompanied by elevated insulin levels. Loss of ANGPTL4 did not affect glucose-stimulated insulin secretion in isolated pancreatic islets. Since the gut microbiota have been suggested to influence insulin secretion, and because ANGPTL4 has been proposed to link the gut microbiota to host metabolism, we hypothesised a potential role of the gut microbiota. Gut microbiota composition was significantly different between Angptl4-/- mice and WT mice. Interestingly, suppression of the gut microbiota using antibiotics largely abolished the differences in glucose tolerance and insulin levels between WT and Angptl4-/- mice. CONCLUSIONS/INTERPRETATION: Despite increasing visceral fat mass, inactivation of ANGPTL4 improves glucose tolerance, at least partly via a gut microbiota-dependent mechanism.

ANGPTL4 promotes bile acid absorption during taurocholic acid supplementation via a mechanism dependent on the gut microbiota.

Angiopoietin-like 4 (ANGPTL4) raises plasma triglyceride levels by inhibiting lipoprotein lipase. A set of compounds that are able to reduce plasma triglyceride levels are bile acids (BA). Because BA have been shown to decrease ANGPTL4 secretion by intestinal cells, we hypothesized that BA lower plasma triglycerides (partly) via ANGPTL4. To test that hypothesis, wild-type and Angptl4-/- mice were fed chow supplemented with taurocholic acid (TCA) for seven days. TCA supplementation effectively lowered plasma triglycerides in wild-type and Angptl4-/- mice, indicating that ANGPTL4 is not required for plasma triglyceride-lowering by BA. Intriguingly, however, plasma and hepatic BA concentrations were significantly lower in TCA-supplemented Angptl4-/- mice than in TCA-supplemented wild-type mice. These changes in the Angptl4-/- mice were accompanied by lower BA levels in ileal scrapings and decreased expression of FXR-target genes in the ileum, including the BA transporter Slc10a2. By contrast, faecal excretion of specifically primary BA was higher in the Angptl4-/- mice, suggesting that loss of ANGPTL4 impairs intestinal BA absorption. Since the gut microbiota converts primary BA into secondary BA, elevated excretion of primary BA in Angptl4-/- mice may reflect differences in gut microbial composition and/or functionality. Indeed, colonic microbial composition was markedly different between Angptl4-/- and wild-type mice. Suppression of the gut bacteria using antibiotics abolished differences in plasma, hepatic, and faecal BA levels between TCA-supplemented Angptl4-/- and wild-type mice. In conclusion, 1) ANGPTL4 is not involved in the triglyceride-lowering effect of BA; 2) ANGPTL4 promotes BA absorption during TCA supplementation via a mechanism dependent on the gut microbiota.

Modulation of the gut microbiota impacts nonalcoholic fatty liver disease: a potential role for bile acids.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide, yet the pathogenesis of NAFLD is only partially understood. Here, we investigated the role of the gut bacteria in NAFLD by stimulating the gut bacteria via feeding mice the fermentable dietary fiber, guar gum (GG), and suppressing the gut bacteria via chronic oral administration of antibiotics. GG feeding profoundly altered the gut microbiota composition, in parallel with reduced diet-induced obesity and improved glucose tolerance. Strikingly, despite reducing adipose tissue mass and inflammation, GG enhanced hepatic inflammation and fibrosis, concurrent with markedly elevated plasma and hepatic bile acid levels. Consistent with a role of elevated bile acids in the liver phenotype, treatment of mice with taurocholic acid stimulated hepatic inflammation and fibrosis. In contrast to GG, chronic oral administration of antibiotics effectively suppressed the gut bacteria, decreased portal secondary bile acid levels, and attenuated hepatic inflammation and fibrosis. Neither GG nor antibiotics influenced plasma lipopolysaccharide levels. In conclusion, our data indicate a causal link between changes in gut microbiota and hepatic inflammation and fibrosis in a mouse model of NAFLD, possibly via alterations in bile acids.

Potential mediators linking gut bacteria to metabolic health: a critical view.

Growing evidence suggests that the bacteria present in our gut may play a role in mediating the effect of genetics and lifestyle on obesity and metabolic diseases. Most of the current literature on gut bacteria consists of cross-sectional and correlative studies, rendering it difficult to make any causal inferences as to the influence of gut bacteria on obesity and related metabolic disorders. Interventions with germ-free animals, treatment with antibiotic agents, and microbial transfer experiments have provided some evidence that disturbances in gut bacteria may causally contribute to obesity-related insulin resistance and adipose tissue inflammation. Several potential mediators have been hypothesized to link the activity and composition of gut bacteria to insulin resistance and adipose tissue function, including lipopolysaccharide, angiopoietin-like protein 4, bile acids and short-chain fatty acids. In this review we critically evaluate the current evidence related to the direct role of gut bacteria in obesity-related metabolic perturbations, with a focus on insulin resistance and adipose tissue inflammation. It is concluded that the knowledge base in support of a role for the gut microbiota in metabolic regulation and in particular insulin resistance and adipose tissue inflammation needs to be strengthened.

Gene expression profiling in human precision cut liver slices in response to the FXR agonist obeticholic acid.

BACKGROUND & AIMS: The bile acid-activated farnesoid X receptor (FXR) is a nuclear receptor regulating bile acid, glucose and cholesterol homeostasis. Obeticholic acid (OCA), a promising drug for the treatment of non-alcoholic steatohepatitis (NASH) and type 2 diabetes, activates FXR. Mouse studies demonstrated that FXR activation by OCA alters hepatic expression of many genes. However, no data are available on the effects of OCA in the human liver. Here we generated gene expression profiles in human precision cut liver slices (hPCLS) after treatment with OCA. METHODS: hPCLS were incubated with OCA for 24 h. Wild-type or FXR(-/-) mice received OCA or vehicle by oral gavage for 7 days. RESULTS: Transcriptomic analysis showed that well-known FXR target genes, including NR0B2 (SHP), ABCB11 (BSEP), SLC51A (OSTα) and SLC51B (OSTß), and ABCB4 (MDR3) are regulated by OCA in hPCLS. Ingenuity pathway analysis confirmed that 'FXR/RXR activation' is the most significantly changed pathway upon OCA treatment. Comparison of gene expression profiles in hPCLS and mouse livers identified 18 common potential FXR targets. ChIP-sequencing in mouse liver confirmed FXR binding to IR1 sequences of Akap13, Cgnl1, Dyrk3, Pdia5, Ppp1r3b and Tbx6. CONCLUSIONS: Our study shows that hPCLS respond to OCA treatment by upregulating well-known FXR target genes, demonstrating its suitability to study FXR-mediated gene regulation. We identified six novel bona-fide FXR target genes in both mouse and human liver. Finally, we discuss a possible explanation for changes in high or low density lipoprotein observed in NASH and primary biliary cholangitis patients treated with OCA based on the genomic expression profile in hPCLS.

The impact of PPARα activation on whole genome gene expression in human precision cut liver slices.

BACKGROUND: Studies in mice have shown that PPARα is an important regulator of lipid metabolism in liver and key transcription factor involved in the adaptive response to fasting. However, much less is known about the role of PPARα in human liver. METHODS: Here we set out to study the function of PPARα in human liver via analysis of whole genome gene regulation in human liver slices treated with the PPARα agonist Wy14643. RESULTS: Quantitative PCR indicated that PPARα is well expressed in human liver and human liver slices and that the classical PPARα targets PLIN2, VLDLR, ANGPTL4, CPT1A and PDK4 are robustly induced by PPARα activation. Transcriptomics analysis indicated that 617 genes were upregulated and 665 genes were downregulated by PPARα activation (q value < 0.05). Many genes induced by PPARα activation were involved in lipid metabolism (ACSL5, AGPAT9, FADS1, SLC27A4), xenobiotic metabolism (POR, ABCC2, CYP3A5) or the unfolded protein response, whereas most of the downregulated genes were involved in immune-related pathways. Among the most highly repressed genes upon PPARα activation were several chemokines (e.g. CXCL9-11, CCL8, CX3CL1, CXCL6), interferon γ-induced genes (e.g. IFITM1, IFIT1, IFIT2, IFIT3) and numerous other immune-related genes (e.g. TLR3, NOS2, and LCN2). Comparative analysis of gene regulation by Wy14643 between human liver slices and primary human hepatocytes showed that down-regulation of gene expression by PPARα is much better captured by liver slices as compared to primary hepatocytes. In particular, PPARα activation markedly suppressed immunity/inflammation-related genes in human liver slices but not in primary hepatocytes. Finally, several putative new target genes of PPARα were identified that were commonly induced by PPARα activation in the two human liver model systems, including TSKU, RHOF, CA12 and VSIG10L. CONCLUSION: Our paper demonstrates the suitability and superiority of human liver slices over primary hepatocytes for studying the functional role of PPARα in human liver. Our data underscore the major role of PPARα in regulation of hepatic lipid and xenobiotic metabolism in human liver and reveal a marked immuno-suppressive/anti-inflammatory effect of PPARα in human liver slices that may be therapeutically relevant for non-alcoholic fatty liver disease.

The role of the gut microbiota in metabolic health.

The global prevalence of obesity and related comorbidities has increased considerably over the past decades. In addition to an increase in food consumption and a reduction in physical activity, growing evidence implicates the microorganisms in our gastrointestinal tract, referred to as the gut microbiota, in obesity and related metabolic disturbances. The composition of the gut microbiota can fluctuate markedly within an individual and between individuals. Changes in gut microbial composition may be unfavorable and predispose an individual to disease. Studies in mice that are germ free, mice that are cohoused, and mice that are treated with antibiotics have provided some evidence that changes in gut microbiota may causally contribute to metabolic disorders. Several mechanisms have been proposed and explored that may mediate the effects of the gut microbiota on metabolic disorders. In this review, we carefully analyze the literature on the connection between the gut microbiota and metabolic health, with a focus on studies demonstrating a causal relation and clarifying potential underlying mechanisms. Despite a growing appreciation for a role of the gut microbiota in metabolic health, more experimental evidence is needed to substantiate a cause-and-effect relationship. If a clear causal relationship between the gut microbiota and metabolic health can be established, dietary interventions can be targeted toward improving gut microbial composition in the prevention and perhaps even the treatment of metabolic diseases.

Necrostatin-1 alleviates reperfusion injury following acute myocardial infarction in pigs.

BACKGROUND: In rodents, it has previously been shown that necrostatin-1 (Nec-1) inhibits RIP1, a central regulator of programmed necrosis, thereby decreasing cell death and reducing infarct size (IS) after ischaemia/reperfusion (I/R) injury. To address unanswered questions on feasibility and efficacy of Nec-1 in a large animal model, we assessed the effects of Nec-1 in a porcine I/R model, relevant to human disease. MATERIALS AND METHODS: In Dalland landrace pigs (69 ± 3 kg), I/R injury was induced by a 75-min surgical ligation of the left circumflex coronary artery (LCx). Ten minutes prior to reperfusion, pigs were randomly allocated to different Nec-1 doses (1.0 mg/kg or 3.3 mg/kg) or vehicle treatment (control, CTRL). Functional endpoints and immunohistological analyses were performed 24 h after reperfusion. RESULTS: Nec-1 3.3 mg/kg significantly reduced IS (n = 6; 24.4 ± 15.6%) compared to Nec-1 1.0 mg/kg (n = 5; 54.8 ± 16.9%) or CTRLs (n = 6; 62.1 ± 26.6%; P = 0.016). In line, LV ejection fraction (LVEF) was significantly higher in Nec-1 3.3 mg/kg, copared to Nec-1 1.0 mg/kg or CTRL treated animals (50.0 ± 12.0% vs. 32.5 ± 12.9% vs. 31.9 ± 6.6%, respectively, P = 0.015). Hemodynamically, a preserved contractility was observed [end-systolic volume at 100 mmHg (ESV100 )] at 24-h follow-up (87.6 ± 17.3 mL vs. 74.5 ± 41.1 mL vs. 56.8 ± 11.8 mL, respectively, P = 0.032), reflecting improved cardiac function. CONCLUSIONS: In the pig model of I/R injury, intravenous administration of Nec-1 prior to reperfusion was an effective and above all practical therapeutic strategy that significantly reduced IS and preserved left ventricular function. These data highlight the potential of cardioprotection as a promising adjuvant therapy in the setting of early reperfusion following I/R injury.