Microbiome-driven alterations in metabolic pathways and impaired cognition in aged female TgF344-AD rats.

Alzheimer's disease (AD) not only affects cognition and neuropathology, but several other facets capable of negatively impacting quality of life and potentially driving impairments, including altered gut microbiome (GMB) composition and metabolism. Aged (20 + mo) female TgF344-AD and wildtype rats were cognitively characterized on several tasks incorporating several cognitive domains, including task acquisition, object recognition memory, anxiety-like behaviors, and spatial navigation. Additionally, metabolic phenotyping, GMB sequencing throughout the intestinal tract (duodenum, jejunum, ileum, colon, and feces), neuropathological burden assessment and marker gene functional abundance predictions (PICRUSt2) were conducted. TgF344-AD rats demonstrated significant cognitive impairment in multiple domains, as well as regionally specific GMB dysbiosis. Relationships between peripheral factors were investigated using Canonical Correspondence Analysis (CCA), revealing correlations between GMB changes and both cognitive and metabolic factors. Moreover, communities of gut microbes contributing to essential metabolic pathways were significantly altered in TgF344-AD rats. These data indicate dysbiosis may affect cognitive outcomes in AD through alterations in metabolism-related enzymatic pathways that are necessary for proper brain function. Moreover, these changes were mostly observed in intestinal segments required for carbohydrate digestion, not fecal samples. These data support the targeting of intestinal and microbiome health for the treatment of AD.

Multiomics profiling of the impact of an angiotensin (1-7)-expressing probiotic combined with exercise training in aged male rats.

Angiotensin (1-7) [Ang (1-7)] is an active heptapeptide of the noncanonical arm of the renin-angiotensin system that modulates molecular signaling pathways associated with vascular and cellular inflammation, vasoconstriction, and fibrosis. Preclinical evidence suggests that Ang (1-7) is a promising therapeutic target that may ameliorate physical and cognitive function in late life. However, treatment pharmacodynamics limits its clinical applicability. Therefore, this study explored the underlying mechanisms altered by a genetically modified probiotic (GMP) that expresses Ang (1-7) combined with and without exercise training in an aging male rat model as a potential adjunct strategy to exercise training to counteract the decline of physical and cognitive function. We evaluated cross-tissue (prefrontal cortex, hippocampus, colon, liver, and skeletal muscle) multi-omics responses. After 12 wk of intervention, the 16S mRNA microbiome analysis revealed a main effect of probiotic treatment within- and between groups. The probiotic treatment enhanced α diversity (Inverse Simpson (F[2,56] = 4.44; P = 0.02); Shannon-Wiener (F[2,56] = 4.27; P = 0.02)) and ß-diversity (F[2,56] = 2.66; P = 0.01) among rats receiving our GMP. The analysis of microbes' composition revealed three genera altered by our GMP (Enterorhabdus, Muribaculaceae unclassified, and Faecalitalea). The mRNA multi-tissue data analysis showed that our combined intervention upregulated neuroremodeling pathways on prefrontal cortex (i.e., 140 genes), inflammation gene expression in the liver (i.e., 63 genes), and circadian rhythm signaling on skeletal muscle. Finally, the integrative network analysis detected different communities of tightly (|r| > 0.8 and P < 0.05) correlated metabolites, genera, and genes in these tissues.NEW & NOTEWORTHY This manuscript uses a multiomics approach (i.e., microbiome, metabolomics, and transcriptomics) to explore the underlying mechanisms driven by a genetically modified probiotic (GMP) designed to express angiotensin (1-7) combined with moderate exercise training in an aged male rat model. After 12 wk of intervention, our findings suggest that our GMP enhanced gut microbial diversity while exercise training altered the transcriptional response in relevant neuroremodeling genes, inflammation, and circadian rhythm signaling pathways in an aging animal model.

Angiotensin (1-7) Delivered Orally via Probiotic in Combination With Exercise: Sex-Dependent Influence on Health Span.

Age-related declines in physical and cognitive function can have tremendous, negative impacts on health span and quality of life. Therefore, we investigated the potential of utilizing a probiotic treatment to target the renin-angiotensin system (RAS) in conjunction with moderate exercise to ameliorate age-related declines in cognitive and physical function in aged rats. Herein we utilized a genetically modified angiotensin (1-7), which activates a "complementary" arm of the RAS through binding Mas (AT7) receptors. This process induces several beneficial physiologic effects, including decreased inflammation and enhanced physical/cognitive function. Thus, in this short research report, we suggest the efficacy of this Ang(1-7) releasing Lactobacillus paracasei (LPA) as either an alternative strategy to exercise, or more likely as an adjuvant to moderate exercise, for the prevention of both physical and cognitive decline especially in female rats.

Interactive Effects of Enalapril Administration and Novel HIIT Wheel-Bed Training in Aged Rats.

INTRODUCTION: Growing research suggests that aerobic high-intensity interval training (HIIT) improves cardiovascular function and physical performance compared with moderate intensity continuous training (MICT). However relatively few animal models of HIIT are available to inform about the benefits of this exercise-particularly among older animals. In addition, there is little evidence for how HIIT training interacts with adjuvant pharmacological therapies known to enhance the impact of MCIT in older individuals such as Angiotensin Converting Enzyme (ACE) Inhibitors. PURPOSE: The aim of the present study was to establish a HIIT protocol in aged rats based on forced running wheel-bed, and to subsequently (1) establish the feasibility of the HIIT protocol in a proof-of-concept study evaluating interactions between HIIT and (2) the result of combining HIIT + ACE inhibitor treatment using the ACE inhibitor enalapril. METHODS: Two groups of rats were used in this study. The feasibility of using wheel-bed for HIIT training was tested in group one (15- and 30-month-old male rats). In the second group, 37 24-month-old Fisher 344 × Brown Norway male rats were randomly divided into four subgroups: control, enalapril, HIIT training group, and HIIT training combined with enalapril administration. The training and administration lasted for 4 weeks. After the intervention, locomotor activity, exercise tolerance, and grip strength were tested. RESULTS: Our feasibility study suggested that middle-aged and aged rats were able to successfully complete the HIIT training. In our intervention study, HIIT training alone, regardless of adjuvant enalapril intervention, did raise treadmill exercise tolerance vs. the sedentary condition. Measures of healthspan were not negatively impacted by HIIT training. CONCLUSION: The novel HIIT protocol based on forced running wheel-bed was successfully employed in aged rats. We conclude that future studies should compare the results and of multi-modal intervention strategies which include both HIIT and MICT in combination with adjuvant therapies such as enalapril to improve exercise tolerance and other global indices of healthspan.

Angiotensin (1-7) Expressing Probiotic as a Potential Treatment for Dementia.

Increasing life expectancies are unfortunately accompanied by increased prevalence of Alzheimer's disease (AD). Regrettably, there are no current therapeutic options capable of preventing or treating AD. We review here data indicating that AD is accompanied by gut dysbiosis and impaired renin angiotensin system (RAS) function. Therefore, we propose the potential utility of an intervention targeting both the gut microbiome and RAS as both are heavily involved in proper CNS function. One potential approach which our group is currently exploring is the use of genetically-modified probiotics (GMPs) to deliver therapeutic compounds. In this review, we specifically highlight the potential utility of utilizing a GMP to deliver Angiotensin (1-7), a beneficial component of the renin-angiotensin system with relevant functions in circulation as well as locally in the gut and brain.

Application of text mining to develop AOP-based mucus hypersecretion genesets and confirmation with in vitro and clinical samples.

Mucus hypersecretion contributes to lung function impairment observed in COPD (chronic obstructive pulmonary disease), a tobacco smoking-related disease. A detailed mucus hypersecretion adverse outcome pathway (AOP) has been constructed from literature reviews, experimental and clinical data, mapping key events (KEs) across biological organisational hierarchy leading to an adverse outcome. AOPs can guide the development of biomarkers that are potentially predictive of diseases and support the assessment frameworks of nicotine products including electronic cigarettes. Here, we describe a method employing manual literature curation supported by a focused automated text mining approach to identify genes involved in 5 KEs contributing to decreased lung function observed in tobacco-related COPD. KE genesets were subsequently confirmed by unsupervised clustering against 3 different transcriptomic datasets including (1) in vitro acute cigarette smoke and e-cigarette aerosol exposure, (2) in vitro repeated incubation with IL-13, and (3) lung biopsies from COPD and healthy patients. The 5 KE genesets were demonstrated to be predictive of cigarette smoke exposure and mucus hypersecretion in vitro, and less conclusively predict the COPD status of lung biopsies. In conclusion, using a focused automated text mining and curation approach with experimental and clinical data supports the development of risk assessment strategies utilising AOPs.

Angiotensin (1-7) delivered orally via probiotic, but not subcutaneously, benefits the gut-brain axis in older rats.

To (1) investigate the efficacy of multiple doses of an orally delivered probiotic bacteria Lactobacillus paracasei (LP) modified to express angiotensin (1-7) (LP-A) in altering physiologic parameters relevant to the gut-brain axis in older rats and to (2) compare this strategy with subcutaneous delivery of synthetic Ang(1-7) peptide on circulating Ang(1-7) concentrations and these gut-brain axis parameters. Male 24-month-old F344BN rats received oral gavage of LP-A, or subcutaneous injection of Ang(1-7) for 0×, 1×, 3×, or 7×/week over 4 weeks. Circulating RAS analytes, inflammatory cytokines, and tryptophan and its downstream metabolites were measured by ELISA, electrochemiluminescence, and LC-MS respectively. Microbiome taxonomic analysis of fecal samples was performed via 16S-based PCR. Inflammatory and tryptophan-related mRNA expression was measured in colon and pre-frontal cortex. All dosing regimens of LP-A induced beneficial changes in fecal microbiome including overall microbiota community structure and α-diversity, while the 3×/week also significantly increased expression of the anti-inflammatory species Akkermansia muciniphila. The 3×/week also increased serum serotonin and the neuroprotective analyte 2-picolinic acid. In the colon, LP-A increased quinolinate phosphoribosyltransferase expression (1×/week) and increased kynurenine aminotransferase II (1× and 3×/week) mRNA expression. LP-A also significantly reduced neuro-inflammatory gene expression in the pre-frontal cortex (3×/week: COX2, IL-1ß, and TNFα; 7×/week: COX2 and IL-1ß). Subcutaneous delivery of Ang(1-7) increased circulating Ang(1-7) and reduced angiotensin II, but most gut-brain parameters were unchanged in response. Oral-but not subcutaneous-Ang(1-7) altered physiologic parameters related to gut-brain axis, with the most effects observed in 3×/week oral dosing regimen in older rats.

In vitro RNA-seq-based toxicogenomics assessment shows reduced biological effect of tobacco heating products when compared to cigarette smoke.

The battery of regulatory tests used to evaluate the risk of novel tobacco products such as heated tobacco products (THPs) presents some limitations including a bias towards the apical endpoint tested, and limited information on the mode of action. This is driving a paradigm shift to more holistic systems biology approaches. In this study, we used RNA-sequencing to compare the transcriptomic perturbations following acute exposure of a 3D airway tissue to the aerosols from two commercial THPs and a reference 3R4F cigarette. 2809 RNAs were differentially expressed for the 3R4F treatment and 115 and 2 RNAs for the two THPs (pFDR < 0.05, FC > 1.5), respectively. The relationship between the identified RNA features and gene ontologies were mapped showing a strong association with stress response, xenobiotics metabolism, and COPD-related terms for 3R4F. In contrast, fewer ontologies were found enriched for the THPs aerosols. "Response to wounding" was a common COPD-related term over-represented for the two THPs but at a reduced significance. Quantification of a cytokine panel post-exposure confirmed a pro-inflammatory effect of cigarette smoke but not for THPs. In conclusion, THPs have a reduced impact on gene expression compared to 3R4F.

Reduced biological effect of e-cigarette aerosol compared to cigarette smoke evaluated in vitro using normalized nicotine dose and RNA-seq-based toxicogenomics.

Electronic cigarettes (e-cigarettes) use has increased globally and could potentially offer a lower risk alternative to cigarette smoking. Here, we assessed the transcriptional response of a primary 3D airway model acutely exposed to e-cigarette aerosol and cigarette (3R4F) smoke. Aerosols were generated with standard intense smoking regimens with careful consideration for dose by normalizing the exposures to nicotine. Two e-cigarette aerosol dilutions were tested for equivalent and higher nicotine delivery compared to 3R4F. RNA was extracted at 24 hrs and 48 hrs post exposure for RNA-seq. 873 and 205 RNAs were differentially expressed for 3R4F smoke at 24 hrs and 48 hrs using a pFDR < 0.01 and a [fold change] > 2 threshold. 113 RNAs were differentially expressed at the highest dose of e-cigarette aerosol using a looser threshold of pFDR < 0.05, 3 RNAs exceeded a fold change of 2. Geneset enrichment analysis revealed a clear response from lung cancer, inflammation, and fibrosis associated genes after 3R4F smoke exposure. Metabolic/biosynthetic processes, extracellular membrane, apoptosis, and hypoxia were identified for e-cigarette exposures, albeit with a lower confidence score. Based on equivalent or higher nicotine delivery, an acute exposure to e-cigarette aerosol had a reduced impact on gene expression compared to 3R4F smoke exposure in vitro.

Targeted omics analyses, and metabolic enzyme activity assays demonstrate maintenance of key mucociliary characteristics in long term cultures of reconstituted human airway epithelia.

3D reconstituted respiratory epithelia have emerged as better in vitro models for toxicological testing compared to cell lines due to the conservation of key morphological features and functions. MucilAir™ is a commercially available human airway epithelia system that can potentially maintain functional attributes for up to a year, however, detailed mucociliary characteristics and xenobiotic metabolism relevant to inhaled pro-toxicant bioactivation is lacking. Here, we assessed in MucilAir™ some key biomarkers that are characteristic of the respiratory epithelia including morphology, function and xenobiotics metabolism. The end points that were measured included targeted proteomics using a panel of 243 airway surface liquid (ASL) proteins, cilia beat frequency (CBF), a qRT-PCR screen of xenobiotic metabolizing enzymes, and CYP2A6/13, CYP1A1/1B1 activity. Comparison of ASL proteomics with human sputum identified key proteins common to both matrices, but present at different levels. Xenobiotic metabolism gene profiling demonstrated strong similarities with the normal human lung and did not reveal any consistent changes when assessed over a 6 month period. Inducibility and activity of CYP1A1/1B1 and activity of CYP2A6/2A13 were present at one month in culture and maintained in one tested MucilAir™ donor for several months. In conclusion, MucilAir™ presented important morphological and metabolic characteristics of a mucociliary epithelium in short and long term culture. MucilAir™ is therefore a potentially useful model to test repeated sub-cytotoxic doses of toxicants.

Quantification of plasma microRNAs in a group of healthy smokers, ex-smokers and non-smokers and correlation to biomarkers of tobacco exposure.

The stability of circulating miRNAs, their non-invasive sampling techniques and deregulation in diseases make them potential candidate biomarkers of biological effect. Here, we profiled the level of 84 plasma miRNAs in 30 smokers, 20 non-smokers and 20 ex-smokers. A robust statistical strategy was applied with replicate samples to account for reproducibility of the results. We identified differential expression of miR-124 and let-7a between the smoking and control groups. We further explored the dose-response relationship of miR-124 and let-7a with two biomarkers of tobacco exposure and found that this relationship was affected by adjustments based on age, pack-year and gender.

Nicotine, cotinine, and b-nicotyrine inhibit NNK-induced DNA-strand break in the hepatic cell line HepaRG.

Recent in vitro work using purified enzymes demonstrated that nicotine and/or a nicotine metabolite could inhibit CYPs (CYP2A6, 2A13, 2E1) involved in the metabolism of the genotoxic tobacco nitrosamine NNK. This observation raises the possibility of nicotine interaction with the mechanism of NNK bioactivation. Therefore, we hypothesized that nicotine or a nicotine metabolite such as cotinine might contribute to the inhibition of NNK-induced DNA strand breaks by interfering with CYP enzymes. The effect of nicotine and cotinine on DNA strand breaks was evaluated using the COMET assay in CYP competent HepaRG cells incubated with bioactive CYP-dependent NNK and CYP-independent NNKOAc (4-(acetoxymethylnitrosoamino)-1-(3-pyridyl)-1-butanone). We report a dose-dependent reduction in DNA damage in hepatic-derived cell lines in the presence of nicotine and cotinine. Those results are discussed in the context of the in vitro model selected.

Nicotine, cotinine, and ß-nicotyrine inhibit NNK-induced DNA-strand break in the hepatic cell line HepaRG.

Recent in vitro work using purified enzymes demonstrated that nicotine and/or a nicotine metabolite could inhibit CYPs (CYP2A6, 2A13, 2E1) involved in the metabolism of the genotoxic tobacco nitrosamine NNK. This observation raises the possibility of nicotine interaction with the mechanism of NNK bioactivation. Therefore, we hypothesized that nicotine or a nicotine metabolite such as cotinine might contribute to the inhibition of NNK-induced DNA strand breaks by interfering with CYP enzymes. The effect of nicotine and cotinine on DNA strand breaks was evaluated using the COMET assay in CYP competent HepaRG cells incubated with bioactive CYP-dependent NNK and CYP-independent NNKOAc (4-(acetoxymethylnitrosoamino)-1-(3-pyridyl)-1-butanone). We report a dose-dependent reduction in DNA damage in hepatic-derived cell lines in the presence of nicotine and cotinine. Those results are discussed in the context of the in vitro model selected.

MicroRNAs as potential biomarkers of smoking-related diseases.

MicroRNAs (miRNAs) comprise a family of small, endogenous, noncoding functional RNA molecules that have emerged as key post-transcriptional regulators of gene expression. They inhibit the translation of proteins from mRNA or promote its degradation. Aberrant miRNA expression has been linked to various human diseases and measurement can differentiate between normal and diseased tissue. Expression is tissue-specific and any changes in miRNA expression within a tissue type can be correlated with disease status. Altered miRNA expression has been reported in the smoking-related diseases cancer, chronic obstructive pulmonary disease and cardiovascular disease. Additionally, miRNAs are thought to have vital roles in inflammatory cell differentiation and regulation. miRNAs might, therefore, be useful biomarkers for early detection of disease-related molecular and genetic changes. In this review, we summarize the available scientific evidence for the potential of miRNAs as biomarkers of smoking-related diseases. Studies should be carried out to identify the miRNAs most relevant to specific diseases.