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Age-associated signatures in the composition and pro-inflammatory status of the gut microbiome in humans and mice, and the impact of a periodic fasting intervention to promote healthy aging

Periodic Reporting for period 1 - Gut-InflammAge (Age-associated signatures in the composition and pro-inflammatory status of the gut microbiome in humans and mice, and the impact of a periodic fasting intervention to promote healthy aging)

Reporting period: 2016-03-01 to 2018-02-28

The proportion of Europeans above 65 years of age is predicted to increase extensively until the year 2050. With age our physical performance declines and we get more prone to develop various diseases including metabolic and cardiovascular diseases. Naturally, healthy aging is of large individual and socio-economic interest. The term “inflammaging” describes the hypothesis that many people develop low-grade chronic inflammation with age. Importantly, inflammation is thought to contribute to age-associated diseases. Prevention of chronic inflammation with age could thus be an import measure to promote healthy aging. This explains the scientific interest to find and understand factors that drive age-associated inflammation, and ultimately, to figure out how they can be stopped from doing so.
The gut microbiota, the trillions of microbes living in our gut, can both promote and inhibit inflammation. Our immune system directly interacts with the microbes. It needs to strike a balance between allowing a diverse microbial community in the gut and preventing that too many microbes breach the gut barrier and enter our circulation. People with pronounced inflammation in their gut, for example patients with inflammatory bowel disease (IBD), usually have gut microbiota dysbiosis. This means that the composition of their gut microbes differs substantially from the range of compositions found in healthy people. Immunoglobulin A (IgA) is the main antibody used by the immune system to keep gut microbes from breaching the gut barrier, and more microbes are targeted with high levels of IgA in IBD patients than in healthy controls. Microbiota dysbiosis and a high proportion of IgA coated microbes are accordingly markers of intestinal inflammation.
The first major objective of the project was to analyse the microbiota compositions of young and aged healthy Danish individuals and to determine the proportions of IgA coated microbes. We wanted to test whether the two biomarkers would reveal signs of increased intestinal inflammation in the aged individuals.

Besides establishing references for potential biomarkers of age-associated inflammation, it is important to determine how healthy aging interventions affect these biomarkers. Many suggested healthy aging interventions are based on diets. Permanent calorie restriction, i.e. reduced energy intake without malnutrition, promotes longer healthspans in various species. But it is extremely hard to follow. Periodic fasting schemes aim at providing similar health benefits by combining periods of calorie restriction with periods of unrestricted calorie intake. Periodic fasting resulted in metabolic health benefits in mice. Not surprisingly, human diets such as the 5:2 diet have been promoted. However, the schemes that were tested in mice used severe calorie restriction. Due to their high metabolic rates, the mice often lost ~10% of their weight in each fasting period.
The second major objective of the project was to test how a mild periodic fasting scheme - that is more comparable to human periodic fasting - affects metabolic health and the gut microbiota of mice.
I analysed stool samples from 100 young (< 30 years) and aged (> 65 years) Danish individuals at the Center for Basic Metabolic Research (University of Copenhagen, UCPH). I selected the samples based on metabolic measures to include individuals that were as healthy as possible. Both groups included slightly more women than men (55-60%). I set up a protocol to detect IgA-coating levels of gut microbes by flow cytometry and separate the microbes based on their coating levels by magnetic-activated cell sorting. I determined the microbiota compositions by next-generation sequencing (NGS). Specifically, the essential 16S marker gene was sequenced from the purified microbial DNA on a NGS machine. Since it is essential, the 16S gene is present in all microbes, but its DNA is sufficiently divergent to allow taxonomic characterisation of the microbiota composition. I wrote R code to streamline the analysis of the sequencing reads based on state-of-the-art analysis pipelines. The code is publicly available (www.arumugamlab.org/projects/GutInflammAge).
I found no signs of increased intestinal inflammation in the samples from aged individuals. The proportions of highly IgA coated microbes in the old group were within the range of proportions found in the young group. Similarly, the microbiota compositions of the aged individuals fitted into the range of compositions of the young individuals. These results are well in line with recent studies, done on different ethnicities, all indicating that the gut microbiota of healthy old people is comparable to that of healthy young people.

The fasting experiment was done in collaboration with the Department of Biology (UCPH). The mice underwent periodic fasting from 8.5 to 14.5 months of age (comparable to ~35 to 50 years in humans). The scheme included two fasting periods à 3 days per month. To avoid no-food stress, the mice could always eat as much as they want, but they got food of low caloric content during the fasting periods. In practice, their calorie intake was ~50% reduced in the fasting periods, but 30% increased in the first 4 days of the re-feed periods. The overall calorie intake in the 6 months was ~5% lower in the fasted mice than in control mice. All mice stayed metabolically healthy (normal glucose and insulin tolerance). But, interestingly, the fasted mice accumulated more body fat than the control mice. On the plus side, we observed a mild re-juvenation of the blood profile in the fasted mice.
I determined the microbiota compositions of young, middle-aged, and old mice as references for the fasting experiment, and found clear differences between the age groups. Due to the controlled environment, the microbiota of lab mice is less variable than of humans. The analysis of how the gut microbiota of the mice changed during the fasting scheme is still ongoing. It is likely that microbiota changes were linked to the fat accumulation, considering that gut microbes are responsible for up to 10% of the energy uptake of the host. I presented my work at conferences and workshops, and we are working on final analyses to publish the results in scientific journals.
Our results on the microbiota composition of young and aged Danish individuals add to the growing consensus that a variety of compositions can be found in metabolically healthy people, and that diet and lifestyle are more critical factors in shaping the microbiota than age and gender. So far, only a few studies have explored the percentage of highly coated IgA microbes. We found no signs of enhanced intestinal inflammation in aged healthy Danes. Reference data on healthy individuals are important, since they give knowledge on what to strive for when treating sick people with microbiota dysbiosis.

Our periodic fasting study in mice showed that calorie intake cycling can result in the accumulation of fat despite a mild reduction in overall calorie intake. This is in line with a few other mice studies on mild calorie restriction. Periodic fasting may well become a valuable healthy aging intervention. However, the severity of the fasting and the unavoidable overeating in the period following the fast have to be considered. These factors should be studied further since they likely play an important role in whether desired health benefits are achieved or not.
Graphical abstract of the project