Thursday 11 October 2018

Immune system, microbiome and the biological clock

The circadian rhythm, the immune system and the microbiome are intimately connected. When one of these parts is not functioning properly, the whole system can tip out of balance. In this five part series, you will get the latest science, as well as practical advice on the relevant nutrition, exercise, supplements and lifestyle.


Part 1: physiology and pathology

A robust and varied microbiome is essential for a healthy immune system as well as for a large number of bodily functions. At the same time, the body reacts to the rhythm of day and night. Does the microbiome – which resides in utter darkness in the intestines – also move to this rhythm? And how can we use this knowledge in our nutritional practice?


Physiology and pathology

The microbiome is made up of ‘good’ bacterial strains that mainly reside in the large intestine. In addition, a number of smaller populations live in the small intestine, the mouth, on the skin and even in the nose. All in all, it consists of many trillions of cells, as many as ten times the number of human cells in the body. Not so long ago, only several hundreds of different bacterial strains had been isolated, but as a result of the Human Microbiome Project, a ground-breaking genetic research project, it became clear in 2012 that the microbiome consists of more than 10,000 different species.1 It also became clear from the same research that the total genome of our microbiome is many times larger than the human genome. People have about 22,000 different protein-coding genes; the microbiome has about 8 billion.1 But what do these staggering numbers have to bear upon our health? 

These enormous numbers of species and genes make sure that the combined system of human an microbe can – if they are both healthy – react very flexibly to factors in the environment. For instance, if we do not have an enzyme to digest a certain nutrient in our food, then there is usually a bacterium that can do this to some extent as well. In short, the symbiosis of human and microbe increases our resilience in our struggle for existence. For example, many adult people (particularly in Africa and Asia) lack the lactase enzyme. This causes lactose to enter the intestine undigested, which in its turn may cause hypersensitivity, consisting of gas and a rumbling stomach. Yet, not everyone lacking lactase experiences these complaints. This is due to intestinal bacteria that are able to ferment lactose. Some lactose intolerant people may, as a result of a healthy flora, consume up to 25 grams of lactose without experiencing any problems.2

It is not clear however whether this works the same in everyone. The microbiome differs from person to person as much as our DNA or our fingerprint. Roughly speaking, humans show three distinct enterotypes, or microbiome profiles, in which the most prominent members are either Bacteroidetes, Prevotella or Ruminococcus.3 However, the composition also varies with time, partly as a function of food intake. Maybe one eats more fibre on one day, very much or very little fat, carbohydrates or proteins on another – these types of changes may significantly impact the composition of the intestinal flora in as little as 24 hours.4 The fact that the composition can vary by so much in so little time, suggests that our flexibility in dealing with environmental factors may fluctuate or change as well, from day to day and from person to person. Yet, at the same time, this fact allows us to change the microbiota for the better as well, using nutrition, exercise, supplements and other lifestyle interventions. You will learn more about this in parts 4 and 5 of this series.


Broad effect on physiology

The microbiome is essential for the body as a whole; it assists the host by carrying out a number of physiological processes, most noticeably digestion. This may appear straightforward, as the microbiome resides mainly in the gastrointestinal tract. But is it, really? Every person has a wide range of digestive enzymes to break down proteins, fats and carbohydrates so they can be taken up by the body. We hardly need the microbiome to do this for us. Also, laboratory animals bred and raised under sterile conditions are perfectly able to digest their food.  

By the time our digested food enters the large intestine, carbohydrates, fats and proteins have already been resorbed. What remains are the undigestible parts of our food, mainly dietary fibres. Our good bacteria digest these fibres for us and use this to obtain energy from it. But it is not just they who profit; we get their breakdown products in return. These products have the ability to influence the workings of the intestines and even our brain. Particularly short chain fatty acids such as butyrate and acetate are important for appetite, glucose metabolism and much besides.5,6,7 In addition, bacteria can synthesize functional proteins and vitamins that benefit us as a host.4 


The role of the immune system

Maybe even more important than the physiological role of the microbiome, is its impact on the immune system. This starts at birth (or maybe even before that, during pregnancy) when the gastrointestinal tract of the baby is populated by bacteria coming from the vagina, skin and intestines (and likely the placenta) of the mother.8 A large number of studies have shown that, when this natural process is disturbed, as a result of, for instance, a caesarean section or antibiotic use in very small babies, this increases the odds of developing problems with the immune system, particularly allergies, asthma and eczema.9

The immune system consists of, among many other things, skin and mucosa (the mechanical barrier) and immune cells, such as white blood cells (leukocytes) and different kinds of lymphocytes and antibodies (immunoglobulins). There is also a number of structures spread throughout the body that play an important part in immune function, including the tonsils, lymph nodes, thymus and spleen. 

In babies, the immune system is much under-developed, just like the brain. The brain needs to learn a language as well as many other things, resulting in a widely branched neural network. Likewise, the immune system must learn how to recognize and kill pathogens. This part is mainly played by lymphocytes. At birth, lymphocytes already harbour some immunity to the outside world, the so-called innate immunity a baby inherits from its mother.9 At least as important, is the acquired immunity which increases over a lifetime. Lymphocytes are ‘educated’ by making contact with a whole host of parasites, infections, nutrients and – first and foremost – intestinal bacteria.9

Every interaction with potentially dangerous intruders results in (epigenetic) changes in the T-cells. This way they can remember how to attack a pathogen. Lymphocytes have  an enormous immunological ‘database’ at their disposal allowing them to recognize and kill potential pathogens. This information is stored in our genes and is activated in the thymus during the first years of life.9

In the thymus, lymphocytes ripen to become fully grown T-lymphocytes. The epigenome of T-cells stores information about infections and the like that one has encountered in the course of life. The thymus shrinks after puberty, following which it is no longer possible for T-lymphocytes to ripen there.9 To be able to guarantee a high level of immunity, T-lymphocytes utilise a smart strategy: when adults get an infection, lymphocytes are reactivated and start to multiply. A number of its ‘decendants’ specialises to become memory cells. These cells are very durable, making sure that one is protected against infection long after.9 

The immune system is mainly ‘educated’ by interacting with the microbiome. Most of this happens in the small intestine, where the Peyer plaques can be found (which host a large number of immune cells). Even in adults, the plaques allow for intimate communication between intestinal bacteria and the immune system, a process commonly known as cross talk. The role of the immune system is to ‘befriend’ good bacteria so they can inhabit all the recesses of the intestinal barrier. This prevents pathogens from nesting there. In the event that this does happen, however, it is up to the immune system to get rid of them by causing severe inflammation. Therefore, more than just an effective way of educating the immune system, keeping in touch with the good bacteria also offers local protection against disease-causing bacteria.9


It is possible for the microbiome to lose its resilience and dynamic balance. Antibiotics, for instance, may selectively eliminate bacterial strains from the community, thus permanently damaging the ecosystem. When this happens, a state of dysbiosis ensues.9 Particularly in babies, in whom the immune system is still under development, this may have life-long negative health consequences.

It is clear that antibiotics permanently alter the composition of the baby’s intestinal flora. In addition, research has linked the use of antibiotics in babies to allergic complaints, including asthma, eczema, hay fever, anaphylactic reactions and intestinal complaints as a result of food allergies, although there are some conflicting results.9 It has also been found that the intestinal flora of people with allergic complaints differs from that of healthy individuals. For example, people with atopic eczema have more Clostridia and less Bifidobacteria in their systems than people without eczema. Finally, the chances of developing eczema increase when the diversity of the intestinal flora decreases. 

Often, allergies are explained using the hygiene hypothesis, which states that the lack of contact with pathogens (and parasites such as worms) causes the immune system to divert its attention to harmless substances in the environment.10 Yet, this does not explain it fully, as there appear to be many other factors involved. It is also important to consider the time and types of exposure, as well as heredity. On top of that, it is becoming increasingly clear that dysbiosis also plays a part in conditions such as IBS, type 1 diabetes, IBD (Crohn’s disease and ulcerative colitis) as well as metabolic diseases such as diabetes and obesity. 9


Immune system, microbiome and the biological clock

How does the biological clock influence the complex interactions between the immune system and the microbiome? Let’s start by diving a bit deeper into our circadian rhythms. The biological clock regulates the 24-hour rhythm of our physiology. This ‘clock’ consists of a small group of cells in the hypothalamus which is called the suprachiasmatic nucleus. Light falling on the retina in the eyes, is transported to the suprachiasmatic nucleus via specialized ganglion cells. The nucleus modulates the sleep-wake cycle through, among others, stimulating the pineal gland to secrete the hormone melatonin. Our organs (liver, intestines, kidneys) harbour small satellites of the biological clock: receptors that are sensitive to the effect of melatonin, causing these organs to oscillate in phase with the circadian rhythm.11

The light/dark cycle is essential for regulating the biological clock. In this respect, light is a so-called Zeitgeber, an environmental factor that ‘communicates time’ to our brains. But how does this work in organisms that permanently reside in darkness, such as intestinal bacteria? This is unknown for most species of bacteria. However, something resembling a 24-hour rhythm has been discovered in light-sensitive cyanobacteria, helping them to anticipate changes in their environment.12, 13 In addition, it has been discovered that Enterobacter aerogenes is sensitive to the effects of melatonin, which is secreted in the host’s gastrointestinal tract, in 2016. Enterobacter responds to this stimulus by becoming more motile and gregarious.14 Thus, it is possible that at least part of our intestinal flora has its own biological clock.

Animals, including humans, are prone to eat at regular intervals, stimulated to do so by hormones that regulate appetite. These hormones are secreted at approximately the same times every day. Whether it is time to eat, is indicated by the biological clock. These meal times also function as Zeitgebers for the microbiome, as they cause clearly observable fluctuations in the intestinal flora.15 Murine research in the past few years has shown that the interaction between the circadian rhythm of the host and its intestinal flora accounts for 60 per cent of changes in the relative amounts of Clostridia, Lactobacillus and Bacteroidetes, all of them species that are naturally well-represented in the microbiome. Thus, they oscillate in phase with the biological clock.15 The phases in the composition of the microbiome are related to the different phases of metabolism; when energy metabolism, cell growth and DNA repair are at their maximum, detoxification and motility of the intestines are at their minimum, and vice versa. 


Circadian rhythm and intestinal flora

When the circadian rhythm of the host is disturbed, this has negative consequences for the composition of the intestinal flora. For instance, it is known that the intestinal flora of people who fly often, as well as of shift workers, diverges from the normal flora. And, when the circadian rhythm is disturbed in mice, this corresponds to a larger risk of glucose intolerance, insulin resistance, type 2 diabetes and obesity.16 In mice, these metabolic diseases can even be transferred to sterile, healthy mice by way of a faecal transplant. This means that dysbiosis in the intestines, which is (among others) the result of a disturbed circadian rhythm, is the cause of, or at least contributes to, the development of metabolic diseases. 15

In addition, the system as a whole becomes even more vulnerable to circadian disruption when mice are put on a diet rich in sugars and fats (an imitation of the Western diet). The intestinal flora of these mice turned out to become less resilient in the face of a weekly reversal of their day/night rhythm, than that of mice on a fitting diet.15 One of the most dramatic effects of circadian disruption of the microbiome in mice is an increase in the number of pro-inflammatory Ruminococcus.15 At the same time, the number of anti-inflammatory Lactobacilli decreases (when the mice are also on a diet that is rich in sugars and fats). The result of this two-sided sword is leaky gut and intestinal inflammation.15 Therefore, it is highly likely that the (disrupted) interaction between intestinal bacteria and the cells of the intestinal epithelium, as a consequence of a Western diet and/or circadian disruptions, also causes leaky gut (hyperpermeability of the intestinal epithelium). When the barrier function of the intestinal epithelium is undermined, this increases the likelihood of low-grade inflammation, both systemically as well as locally in the intestine. Of note is furthermore, that the circadian rhythm of the microbiome in mice that do not have a functional biological clock, may be imitated by feeding the mice at regular intervals corresponding to intervals at which mice usually eat. This is an important pointer to the fact that eating at regular intervals is a natural phenomenon and is part of the circadian rhythm.15

Even though these studies have been mainly carried out in mice, there are indications that eating late at night or at irregular intervals, may contribute to obesity in people as well. Recently, research has shown that a large breakfast and a moderate dinner causes metabolic markers to improve (blood pressure, cholesterol, glucose) compared to the other way around.17 In addition, there is also research that shows that too little sleep contributes to metabolic syndrome.18,19

Circadian changes in the microbiome are communicated to the biological clock in the intestinal epithelium via several receptors (PPAR-alpha, ROR-alpha and RevEr-alpha). When the biorhythm is normal, this leads to a lowering of cortisol production in the active phase and an increase in production in the resting phase (in mice). In sterile mice cortisol production is not suppressed, leaving it on day and night. Chronically elevated levels of cortisol have a negative impact on the immune system. This system gets suppressed as well, thus hampering the clearance of pathogenic substances.20 Circadian disruptions of the microbiome also impact the circadian rhythm of the liver, which has implications for both the functioning and health of the host, for instance obstructing detoxification of medicines by the liver.11

In our following e-news you can read the second part of this five-part series on the immune system, microbiome and the biological clock: ‘Diagnostics: symptoms and measurability’.



1. Nature. 2012 Jun 13;486(7402):207-14. Structure, function and diversity of the healthy human microbiome. Human Microbiome Project Consortium.

2. Nutrients. 2015 Aug 13;7(8):6751-79. Adaptation to Lactose in Lactase Non Persistent People: Effects on Intolerance and the Relationship between Dairy Food Consumption and Evalution of Diseases. Szilagyi A,

3. Nature. 2011 May 12;473(7346):174-80. Enterotypes of the human gut microbiome. Arumugam M1, Raes J, Pelletier E, et al

4. J Transl Med. 2017; 15: 73. Influence of diet on the gut microbiome and implications for human health Rasnik K. Singh, Hsin-Wen Chang, Di Yan, et al 

5. J. Appl. Microbiol. 113, 411–417, g-Aminobutyric acid production by culturablebacteria from the human intestine. Barrett, E. et al. (2012)

6. J. Psychiatr. Res. 63, 1–9 Collective unconscious: how gutmicrobes shape human behavior. Dinan, T.G. et al. (2015)

7. Bioessays 33, 574–581 Probiotics function mechanistically as deliveryvehicles for neuroactive compounds: microbial endocrinology inthe design and use of probiotics. . Lyte, M. (2011)

8. Nutrients. 2018 Feb 28;10(3). Factors Affecting Gastrointestinal Microbiome Development in Neonates. Chong CYL1, Bloomfield FH2,3, O'Sullivan JM4.

9. Microbiotia in health and disease: from pregnancy to childhood – Brown PD, Claassen E, Cabana MD (editors) – Wageningen Academic Publishers (2018)

10. BMJ. 1989 Nov 18;299(6710):1259-60. Hay fever, hygiene, and household size. Strachan DP1.

11. Cell. 2016 Dec 1;167(6):1495-1510.e12 Microbiota Diurnal Rhythmicity Programs Host Transcriptome Oscillations. Thaiss CA, Levy M, Korem T, et al. 

12. Rust, M.J., Markson, J.S., Lane, W.S., Fisher, D.S., and O’Shea, E.K. (2007). Ordered phosphorylation governs oscillation of a three-protein circadian clock. Science 318, 809–812.

13. Johnson, C.H., Egli, M., and Stewart, P.L. (2008). Structural insights into a circadian oscillator. Science 322, 697–701.

14. Cell. 2013 May 9;153(4):741-3. Microbiota keep the intestinal clock ticking. Henao-Mejia J1, Strowig T, Flavell RA.

15. Circadian Disorganization Alters Intestinal Microbiota Robin M. Voigt, 1 , * Christopher B. Forsyth, Stefan J. Green, Ece Mutlu et al. 

16. Cell. 2014 Oct 23;159(3):514-29. Transkingdom control of microbiota diurnal oscillations promotes metabolic homeostasis. Thaiss CA1, Zeevi D2, Levy M1, Zilberman-Schapira G1, 

17. Jakubowicz, D., Barnea, M., Wainstein, J., and Froy, O. (2013). High caloric intake at breakfast vs. dinner differentially influences weight loss of overweight and obese women. Obesity (Silver Spring) 21, 2504–2512.

18. Baron, K.G., Reid, K.J., Kern, A.S., and Zee, P.C. (2011). Role of sleep timing in caloric intake and BMI. Obesity (Silver Spring)19, 1374–1381.

19. Hsieh, S.D., Muto, T., Murase, T., Tsuji, H., and Arase, Y. (2011). Association of short sleep duration with obesity, diabetes, fatty liver and behavioral factors in Japanese men. Intern. Med. 50, 2499–2502.

20. PLoS One. 2016 Jan 11;11(1):e0146643. Human Gut Bacteria Are Sensitive to Melatonin and Express Endogenous Circadian Rhythmicity. Paulose JK1, Wright JM1, Patel AG1, Cassone VM1.