Does it matter what time of day you get a flu shot? Surprisingly, it does! It has been demonstrated in several studies; so a piece of advice: get your flu shot in the morning. 

One of the main reasons for this might come from our biological clock, which produces circadian rhythms that influence our sleeping patterns. Certainly, good sleeping habits are important for our bodies. And, in the case of the flu shot, protection seems to be stronger when it’s administered early in the day because our circadian rhythms influence our immune systems, making them more active in the morning. 

However, we know very little about how our biological clocks function during a real infection. What would happen if we caught the flu after a bad night’s sleep? A recent study in mice out of the University of Pennsylvania brings some answers on the subject.

First, some definitions…

Most organisms, from microbes and plants to humans, have circadian rhythms. These rhythms are changes that adhere to a roughly 24 hour cycle and impact both our external behavior and our internal functions. For example, the fact that most of us sleep at night and wake during the day is an easily-observable example of a circadian rhythm. Some less obvious examples include the daily patterns in which our hormones, appetites, and body temperatures cycle. 

While many of these daily rhythms can be influenced by external stimuli such as sunlight, sound, or lifestyle, the main driver of circadian rhythm is the biological clock. The central control center for this internal, biological clock resides in the brain, orchestrating the activity of many different molecules throughout the body to generate the daily circadian rhythms described above. Many different cell types in the body are subject to influence by this biological clock, not the least of which are immune cells. With this in mind, the authors at UPenn hypothesized that biological clocks might impact the immune response to a common and potentially deadly infection: the flu. 

Using mice to explore the biological clock

To explore the role of the biological clock during influenza infection, researchers studied intranasal infection of mice with influenza A virus (IAV). Contrary to humans, mice are active during the night and sleep during the day. As such, the authors defined two groups of mice: those infected before (1) the active phase (the active mice group) or (2) the rest phase (the resting mice group). Strikingly, after two weeks of infection, the active mice group had a much higher level of mortality compared to the resting mice group: 71% death vs. 15% death (Figure 1). With this result, the authors had their first proof for a possible role of circadian rhythm in the outcome of influenza infection: depending on the light exposure, mice, via their biological clock, appeared to survive differently following influenza infection.

As mentioned above, circadian rhythms can be impacted by external factors such as daylight. So, in order to confirm the direct involvement of the biological clock, the mice were next raised in constant darkness, forcing them to rely solely on their internal biological clocks. Interestingly, the data still showed higher mortality for mice infected before their supposed active phase, but the magnitude of survival between the two groups was lower than previously observed. This finding indicates that the light parameter is not solely responsible for the survival discrepancy, but it is likely  playing a secondary role by influencing the circadian rhythms of the mice. 

Based upon the fact that mice infected during their active or resting periods in the absence of light still showed survival differences, the authors hypothesized that the internal biological clock was impacting the severity of influenza infection. To test this more thoroughly, they generated a mouse model in which they removed, or knocked out, a gene called Bmal1. This gene plays a key role in controlling the circadian rhythm and without it, the mice had perpetually disrupted biological clocks. When the authors infected these mice with the flu virus during their active or rest periods, they found that they had more comparable survival rates, both of which were low (25% and 16%, respectively). These findings indicate that disruption of circadian rhythm increases the susceptibility of mice to infection by the flu virus. 

Finding the missing link between biological clock and susceptibility to influenza

After establishing that circadian rhythm impacts severity of IAV infection, the next step was to understand why. Towards this end, the authors first investigated if circadian rhythm had a direct impact on the number of viral particles in each mouse. This, however, did not appear to be the case, because when researchers analyzed the level of viral particles in the lungs, it was similar between the two groups. 

On the other hand, the immune response had more to tell. Immune cells isolated from the lung were present at a significantly higher level in the active mice group (with the higher mortality to the flu virus). The presence of immune cells is generally a sign of an immune response called inflammation. Inflammation is the suite of complex biological responses that occur as the immune system reacts to any sort of perceived danger, from a cut on your arm to infection by the flu virus. For example, the redness and swelling generally associated with an injury is one manifestation of the inflammatory immune response; it occurs as blood flow increases to the injured area, carrying infection-fighting immune cells with it. As part of this infection-fighting process, immune cells secrete many inflammatory molecules with varied functions, from helping immune cells travel out of the blood vessels into an infected area to recruiting and activating different types of immune cells so that the infection can be effectively fought.

Although immune-mediated inflammation is intended to help fight infection, sometimes it has unintended negative consequences. Specifically, it can have the disadvantage of causing injuries to the host tissue if the immune system becomes over-activated and begins attacking healthy cells along with infected cells. In the case of the IAV-infected mice, it’s possible that the active mice group suffered from an uncontrolled inflammation influenced by the time of infection. Indeed, lung sections showed higher injuries in the active mice group compared to the resting group, potentially explaining why these mice had a higher mortality despite the presence of more immune cells in the lungs. Furthermore, a study of the compounds produced by the cells collected from the lung in the two groups of mice showed several molecules associated with sustained innate immune response that are linked with inflammation. Interestingly, one of the compounds isolated from the resting mice group was a molecule (Interleukin-10) which stops inflammation, possibly explaining their better outcome after infection. 

In sum, the active mice group seemed to react more strongly to the IAV infection, which translated into uncontrolled inflammation, suggesting a direct role of the biological clock on the immune response to influenza infection. 

Natural killer cells strike back (again)! 

When looking more precisely at the different immune populations present in the lungs, researchers identified two specific immune population that differed between the two groups of mice:  Natural Killer (NK) cells and Natural Killer T (NKT) cells.

They both belong to the innate immune system, meaning the first, general line of defense against pathogens. NK cells have a central role in eliminating infected cells or cancer cells and also signal other immune cells to danger. Faithful to their name, NKT cells share properties of NK cells and a type of adaptive immune cell called T cells. NKT cells represent a small immune population but have a great importance, specifically in protecting from autoimmune diseases as well as enhancing the immune response against pathogens.

Interestingly, these two immune populations were more present in the resting mice group, suggesting that they might participate in the protection against uncontrolled inflammation. In support of this, the authors report that the depletion of NK cells and NKT cells with an antibody anti-NK1.1 (which targets exclusively these immune cells) one day before IAV infection induced a low survival rate in the resting mice that was similar to the survival rate of active mice group without NK and NKT cells depletion. These findings suggest that NK and NKT cells are required for the preferential survival of the resting mice group, possibly through their role in reducing inflammation. 

Overall, NK cells and NKT cells might have a central role in the outcome of influenza infection and are probably sensitive to circadian rhythms, helping to explain the survival difference between the two mice groups. Notably, a more detailed study is necessary to confirm these preliminary results.

The central role of our sleeping pattern in health and disease

Overall, the biological clock might have a central role in respiratory infections like the flu. In this study the researcher showed that positive or negative outcome of the flu might be linked to our biological clock and its relationship with our immune system. With more research, these observations may be helpful in better protecting those most sensitive to the flu, like children, the elderly, or immunocompromised individuals, and maybe others. 

From a general point of view, our current way of life is affecting our biological clock (night-shift jobs, long work hours, overindulgent screen time or too many good shows on Netflix), and this has been linked to several health issues like cardiac diseases or cancer. As the authors pointed out, patients in intensive care may be the most vulnerable to circadian disruption by being in an environment with lighting, noise, disruptive timing of food, clinical assessments, or medications. Overall, these disruptions could influence their outcome during respiratory infections during long hospitalization.