Getting closer to modeling the gut-brain axis using induced pluripotent stem cells.

The gut microbiome (GM), the gut barrier, and the blood-brain barrier (BBB) are key elements of the gut-brain axis (GBA). The advances in organ-on-a-chip and induced pluripotent stem cell (iPSCs) technology might enable more physiological gut-brain-axis-on-a-chip models. The ability to mimic complex physiological functions of the GBA is needed in basic mechanistic research as well as disease research of psychiatric, neurodevelopmental, functional, and neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. These brain disorders have been associated with GM dysbiosis, which may affect the brain via the GBA. Although animal models have paved the way for the breakthroughs and progression in the understanding of the GBA, the fundamental questions of exactly when, how, and why still remain unanswered. The research of the complex GBA have relied on equally complex animal models, but today's ethical knowledge and responsibilities demand interdisciplinary development of non-animal models to study such systems. In this review we briefly describe the gut barrier and BBB, provide an overview of current cell models, and discuss the use of iPSCs in these GBA elements. We highlight the perspectives of producing GBA chips using iPSCs and the challenges that remain in the field.

The Breakthroughs and Caveats of Using Human Pluripotent Stem Cells in Modeling Alzheimer's Disease.

Modeling Alzheimer's disease (AD) using human-induced pluripotent stem cells (iPSCs) is a field now spanning 15 years. Developments in the field have shown a shift in using simple 2D cortical neuron models to more advanced tri-cultures and 3D cerebral organoids that recapitulate more features of the disease. This is largely due to development and optimization of new cell protocols. In this review, we highlight recent major breakthroughs in the AD field and the implications this has in modeling AD using iPSCs (AD-iPSCs). To date, AD-iPSCs have been largely used to recapitulate and study impaired amyloid precursor protein (APP) processing and tau phosphorylation in both familial and sporadic AD. AD-iPSCs have also been studied for varying neuronal and glial dysfunctions. Moreover, they have been useful for discovering new molecular mechanisms, such as identifying proteins that bridge APP processing with tau phosphorylation and for identifying molecular pathways that bridge APP processing dysfunction with impaired cholesterol biosynthesis. Perhaps the greatest use of AD-iPSCs has been in discovering compounds via drug screening, that reduce amyloid beta (Aß) in neurons, such as the anti-inflammatory compound, cromolyn, and antiparasitic drugs, avermectins. In addition, high content screening using AD-iPSCs has led to the identification of statins that can reduce levels of phosphorylated tau (p-Tau) in neurons. Some of these compounds have made it through to testing in human clinical trials. Improvements in omic technologies including single cell RNA sequencing and proteomics as well as advances in production of iPSC-cerebral organoids and tri-cultures is likely to result in the further discovery of new drugs and treatments for AD. Some caveats remain in the field, including, long experimental conditions to create mature neurons, high costs of media that limit research capabilities, and a lack of reproducibility using current iPSC-cerebral organoid protocols. Despite these current limitations, AD-iPSCs remain an excellent cellular model for studying AD mechanisms and for drug discovery.

Nanomaterial- and shape-dependency of TLR2 and TLR4 mediated signaling following pulmonary exposure to carbonaceous nanomaterials in mice.

BACKGROUND: Pulmonary exposure to high doses of engineered carbonaceous nanomaterials (NMs) is known to trigger inflammation in the lungs paralleled by an acute phase response. Toll-like receptors (TLRs), particularly TLR2 and TLR4, have recently been discussed as potential NM-sensors, initiating inflammation. Using Tlr2 and Tlr4 knock out (KO) mice, we addressed this hypothesis and compared the pattern of inflammation in lung and acute phase response in lung and liver 24 h after intratracheal instillation of three differently shaped carbonaceous NMs, spherical carbon black (CB), multi-walled carbon nanotubes (CNT), graphene oxide (GO) plates and bacterial lipopolysaccharide (LPS) as positive control. RESULTS: The LPS control confirmed a distinct TLR4-dependency as well as a pronounced contribution of TLR2 by reducing the levels of pulmonary inflammation to 30 and 60% of levels in wild type (WT) mice. At the doses chosen, all NM caused comparable neutrophil influxes into the lungs of WT mice, and reduced levels were only detected for GO-exposed Tlr2 KO mice (35%) and for CNT-exposed Tlr4 KO mice (65%). LPS-induced gene expression was strongly TLR4-dependent. CB-induced gene expression was unaffected by TLR status. Both GO and MWCNT-induced Saa1 expression was TLR4-dependent. GO-induced expression of Cxcl2, Cxcl5, Saa1 and Saa3 were TLR2-dependent. NM-mediated hepatic acute phase response in terms of liver gene expression of Saa1 and Lcn2 was shown to depend on TLR2 for all three NMs. TLR4, in contrast, was only relevant for the acute phase response caused by CNTs, and as expected by LPS. CONCLUSION: TLR2 and TLR4 signaling was not involved in the acute inflammatory response caused by CB exposure, but contributed considerably to that of GO and CNTs, respectively. The strong involvement of TLR2 in the hepatic acute phase response caused by pulmonary exposure to all three NMs deserves further investigations.

Particle characterization and toxicity in C57BL/6 mice following instillation of five different diesel exhaust particles designed to differ in physicochemical properties.

BACKGROUND: Diesel exhaust is carcinogenic and exposure to diesel particles cause health effects. We investigated the toxicity of diesel exhaust particles designed to have varying physicochemical properties in order to attribute health effects to specific particle characteristics. Particles from three fuel types were compared at 13% engine intake O2 concentration: MK1 ultra low sulfur diesel (DEP13) and the two renewable diesel fuels hydrotreated vegetable oil (HVO13) and rapeseed methyl ester (RME13). Additionally, diesel particles from MK1 ultra low sulfur diesel were generated at 9.7% (DEP9.7) and 17% (DEP17) intake O2 concentration. We evaluated physicochemical properties and histopathological, inflammatory and genotoxic responses on day 1, 28, and 90 after single intratracheal instillation in mice compared to reference diesel particles and carbon black. RESULTS: Moderate variations were seen in physical properties for the five particles: primary particle diameter: 15-22 nm, specific surface area: 152-222 m2/g, and count median mobility diameter: 55-103 nm. Larger differences were found in chemical composition: organic carbon/total carbon ratio (0.12-0.60), polycyclic aromatic hydrocarbon content (1-27 µg/mg) and acid-extractable metal content (0.9-16 µg/mg). Intratracheal exposure to all five particles induced similar toxicological responses, with different potency. Lung particle retention was observed in DEP13 and HVO13 exposed mice on day 28 post-exposure, with less retention for the other fuel types. RME exposure induced limited response whereas the remaining particles induced dose-dependent inflammation and acute phase response on day 1. DEP13 induced acute phase response on day 28 and inflammation on day 90. DNA strand break levels were not increased as compared to vehicle, but were increased in lung and liver compared to blank filter extraction control. Neutrophil influx on day 1 correlated best with estimated deposited surface area, but also with elemental carbon, organic carbon and PAHs. DNA strand break levels in lung on day 28 and in liver on day 90 correlated with acellular particle-induced ROS. CONCLUSIONS: We studied diesel exhaust particles designed to differ in physicochemical properties. Our study highlights specific surface area, elemental carbon content, PAHs and ROS-generating potential as physicochemical predictors of diesel particle toxicity.

Increased surface area of halloysite nanotubes due to surface modification predicts lung inflammation and acute phase response after pulmonary exposure in mice.

The toxicological potential of halloysite nanotubes (HNTs) and variants after functional alterations to surface area are not clear. We assessed the toxicological response to HNTs (NaturalNano (NN)) before and after surface etching (NN-etched). Potential cytotoxicity of the two HNTs was screened in vitro in MutaTMMouse lung epithelial cells. Lung inflammation, acute phase response and genotoxicity were assessed 1, 3, and 28 days after a single intratracheal instillation of adult female C57BL/6 J BomTac mice. The doses were 6, 18 or 54 µg of HNTs, compared to vehicle controls and the Carbon black NP (Printex 90) of 162 µg/mouse. The cellular composition of bronchoalveolar lavage (BAL) fluid was determined as a measure of lung inflammation. The pulmonary and hepatic acute phase responses were assessed by Serumamyloida mRNA levels in lung and liver tissue by real-time quantitative PCR. Pulmonary and systemic genotoxicity were analyzed by the alkaline comet assay as DNA strand breaks in BAL cells, lung and liver tissue. The etched HNT (NN-etched) had 4-5 times larger BET surface area than the unmodified HNT (NN). Instillation of NN-etched at the highest dose induced influx of neutrophils into the lungs at all time points and increased Saa3 mRNA levels in lung tissue on day 1 and 3 after exposure. No genotoxicity was observed at any time point. In conclusion, functionalization by etching increased BET surface area of the studied NN and enhanced pulmonary inflammatory toxicity in mice.

Airport emission particles: exposure characterization and toxicity following intratracheal instillation in mice.

BACKGROUND: Little is known about the exposure levels and adverse health effects of occupational exposure to airplane emissions. Diesel exhaust particles are classified as carcinogenic to humans and jet engines produce potentially similar soot particles. Here, we evaluated the potential occupational exposure risk by analyzing particles from a non-commercial airfield and from the apron of a commercial airport. Toxicity of the collected particles was evaluated alongside NIST standard reference diesel exhaust particles (NIST2975) in terms of acute phase response, pulmonary inflammation, and genotoxicity after single intratracheal instillation in mice. RESULTS: Particle exposure levels were up to 1 mg/m3 at the non-commercial airfield. Particulate matter from the non-commercial airfield air consisted of primary and aggregated soot particles, whereas commercial airport sampling resulted in a more heterogeneous mixture of organic compounds including salt, pollen and soot, reflecting the complex occupational exposure at an apron. The particle contents of polycyclic aromatic hydrocarbons and metals were similar to the content in NIST2975. Mice were exposed to doses 6, 18 and 54 µg alongside carbon black (Printex 90) and NIST2975 and euthanized after 1, 28 or 90 days. Dose-dependent increases in total number of cells, neutrophils, and eosinophils in bronchoalveolar lavage fluid were observed on day 1 post-exposure for all particles. Lymphocytes were increased for all four particle types on 28 days post-exposure as well as for neutrophil influx for jet engine particles and carbon black nanoparticles. Increased Saa3 mRNA levels in lung tissue and increased SAA3 protein levels in plasma were observed on day 1 post-exposure. Increased levels of DNA strand breaks in bronchoalveolar lavage cells and liver tissue were observed for both particles, at single dose levels across doses and time points. CONCLUSIONS: Pulmonary exposure of mice to particles collected at two airports induced acute phase response, inflammation, and genotoxicity similar to standard diesel exhaust particles and carbon black nanoparticles, suggesting similar physicochemical properties and toxicity of jet engine particles and diesel exhaust particles. Given this resemblance as well as the dose-response relationship between diesel exhaust exposure and lung cancer, occupational exposure to jet engine emissions at the two airports should be minimized.

Immunological effects of reduced mucosal integrity in the early life of BALB/c mice.

Certain stimuli at the gut barrier may be necessary in early life to establish a proper balance of immune tolerance. We evaluated a compromised barrier in juvenile mice in relation to microbiota and local and systemic immunity. BALB/c mice were treated with a low dose of dextran sulfate sodium (DSS) with or without ampicillin and lipopolysaccharide (LPS) to clarify the importance of microbial antigens and interaction between microbial-associated patterns and toll-like receptors. The barrier breach resulted in increased plasma LPS, which was highest in mice treated simultaneously with ampicillin. Adding LPS in the food reduced its levels in plasma. Regulatory T cells were acutely increased in mesenteric lymph nodes (MLN) and spleen during DSS treatment regardless of simultaneous ampicillin treatment. In contrast, NK T and NK cells decreased in MLN and in spleen. This acute DSS effect was reflected in fold changes of haptoglobin and Il1a in colon, and this was also more pronounced in mice simultaneously treated with ampicillin. On day 1 post-treatment, major upregulations of Ifng, Foxp3, Il1b, Il2, and Il6 genes in colon were only observed in the mice simultaneously treated with ampicillin. A two-fold upregulation of colonic Foxp3 and Il1a was evident 25 days post-treatment. DSS skewed the microbiota in favor of Gram negative phyla. Therefore, increased permeability induced tolerogenic immunity independent of microbiota, and this was enhanced by LPS stimulation.

Gut microbiota composition is correlated to grid floor induced stress and behavior in the BALB/c mouse.

Stress has profound influence on the gastro-intestinal tract, the immune system and the behavior of the animal. In this study, the correlation between gut microbiota composition determined by Denaturing Grade Gel Electrophoresis (DGGE) and tag-encoded 16S rRNA gene amplicon pyrosequencing (454/FLX) and behavior in the Tripletest (Elevated Plus Maze, Light/Dark Box, and Open Field combined), the Tail Suspension Test, and Burrowing in 28 female BALB/c mice exposed to two weeks of grid floor induced stress was investigated. Cytokine and glucose levels were measured at baseline, during and after exposure to grid floor. Stressing the mice clearly changed the cecal microbiota as determined by both DGGE and pyrosequencing. Odoribacter, Alistipes and an unclassified genus from the Coriobacteriaceae family increased significantly in the grid floor housed mice. Compared to baseline, the mice exposed to grid floor housing changed the amount of time spent in the Elevated Plus Maze, in the Light/Dark Box, and burrowing behavior. The grid floor housed mice had significantly longer immobility duration in the Tail Suspension Test and increased their number of immobility episodes from baseline. Significant correlations were found between GM composition and IL-1α, IFN-γ, closed arm entries of Elevated Plus Maze, total time in Elevated Plus Maze, time spent in Light/Dark Box, and time spent in the inner zone of the Open Field as well as total time in the Open Field. Significant correlations were found to the levels of Firmicutes, e.g. various species of Ruminococccaceae and Lachnospiraceae. No significant difference was found for the evaluated cytokines, except an overall decrease in levels from baseline to end. A significant lower level of blood glucose was found in the grid floor housed mice, whereas the HbA1c level was significantly higher. It is concluded that grid floor housing changes the GM composition, which seems to influence certain anxiety-related parameters.