A sick mouse’s guide to feasting and fasting

When should you feed a starving mouse and when should you just let it be?

 

Summary

Sick mice, especially those infected with bacteria and viruses often display an anorexic response and eat very little. More than 40 years ago it was recognized that mice sick with a bacterial infection die if you force feed them (1). Is this true for all infections? What about viruses? Should we starve a sick pet or colleague?

In a new series of experiments which explores the scientific basis for the old adage starve a fever, feed a cold, researchers have found that food makes things worse for mice with bacterial infection (such as Listeria monocytogenes) but is required for recovery from viral infections (such as influenza) (2).

Introduction

When a mouse or any host is infected with a pathogen the events that follow can be resolved around 3 types of harm caused by the

i) pathogen itself – related to the number of pathogens, toxins produced by the pathogen etc.
ii) response of the body – collateral damage from the inflammatory response, immune reaction to pathogen, etc., which can often times be non-specific
iii) inability of the body or tissue to repair or take care of the damage

The authors find that it was the third kind – i.e. the ability to cope with tissue damage that ensues when mice sick with bacterial infections are fed and also when mice sick with viral infections are starved. This suggests that in the onslaught by the pathogen, there is a bystander effect upon non-immune tissues caused by host defenses that is a,critical determinant of bouncing back to health.

What did they do and find?

Mice infected with Listeria monocytogenes died when they were force-fed. The pathogen load (bacterial numbers) and defensive/ response molecules secreted by the mouse were not different between the force-fed (test) mice and mice that were not force-fed (control). The authors of the study then used a model for bacterial infection to look at why the mice are dying. In this model, the mice were challenged with a component of the outer membrane of bacteria – this is known to result in a strong inflammatory reaction – and then looked at the effect on mice upon injection of glucose, casein and olive oil. Glucose was found to be the cause of death.

This however is only one part of the story. The researchers then looked at another infection model, of influenza-infected mice, which also display an anorexic response. Here they observed the opposite – that is, if the mice were stopped from using the glucose, they died. In fact, feeding mice made them better. Viruses invoke response pathways, which are distinct from bacteria, so maybe the immune reaction was different between the fed and not-fed mice? Once again the authors ruled both pathogen numbers (viral load) as well as difference in immune responses in both groups. To understand what was causing death in these mice, the authors dissected mice that had been infected with the virus and then were given either normal saline or a molecule that made glucose unavailable to the body. Mice which were starved of glucose had lower heart rate, slightly lower respiratory rate as well as lower body temperatures about a week post infection. This was the first clue that control centres in the brain, which are responsible for these functions, may be affected. The authors extended this finding to a mouse model which cannot mount the normal immune response to viruses and challenged it with a molecular mimic of virus infection (poly I:C). In this mode, they found that when fed a molecule that made glucose unavailable, the mice died.

So why were starving mice dying in viral infections and fed mice dying in bacterial infection models? This work sheds some light on the differences. When the researchers studied glucose uptake in the brain in both models they found that there was glucose uptake in different parts of the brain during viral and bacterial infections. Viruses enter the host cells and use the sub-cellular compartments and cellular machinery to make copies of themselves. One such compartment- known as the endoplasmic reticulum – is needed both by the host cell and the virus to function normally. Infection results in a stress response in this compartment which usually signals to the cell that it should now shut-down (a particular kind of cellular suicide termed apoptosis). In this model of viral infection, glucose helps keep this compartment stress-free and therefore prevents cell death. This is particularly important for cells in the brain. What about bacterial infections then? In the brains of the mice with simulated bacterial infections and glucose injections, the authors find evidence for the accumulation of reactive oxygen species (ROS) in the brain. These molecules are also potent inducers of the cellular suicide pathways. However, the authors note that in this case, it may not be death of brain cells, but their dysfunction that may be the cause of death. This still does not explain the difference between viral and bacterial infections. To get to this, the authors analysed the starvation response. During starvation, the utilization of fats and proteins results in accumulation of ketone bodies, an important alternative fuel source during fasted states, via ketogenesis. Excessive and prolonged accumulation of ketone bodies is known to be toxic for the body. In the case of bacterial infection, this study suggests that the availability of ketone bodies may be helping cells to detoxify ROS.

Take home from this study

This study gives us a new way of thinking about infections, host response to infection (immunity) and the rest of the organs and tissues in the body, particularly the brain which must keep working normally through the pathogen-host cross-fire. There are clearly many unanswered question that this opens up, and while it demonstrates that glucose plays different roles in viral and bacterial infection of mice, the underlying mechanisms still remain to be understood in detail. It is interesting that the main difference of glucose utilization seems to be in the brain. The processes that connect what we eat, to what our body makes of it to how we feel or behave form a fascinating network with new links emerging all the time. It is not too soon to have convictions on what is good for us, our colleagues, our pets or our mice, but it is too early to really know or accept information without doubt.

References

1. Anorexia of infection as a mechanism of host defense.” M J Murray and A B Murray , Am J Clin Nutr. 1979

2. Opposing Effects of Fasting Metabolism on Tissue Tolerance in Bacterial and Viral Inflammation,Andrew Wang et al., Cell. 2016

An interview with L.Harding, S.Huen and A.Wang

Q. The idea that the there is tissue tolerance to injury caused by a pathogen-host battle seems reasonable, can you tell us more about the evolution of this idea and its implications for how people now view disease? Are there biomarkers of tissue tolerance?

The idea evolved from the recognition that oftentimes in sepsis, the immune response is more detrimental to the host than the damage incurred by the pathogen. The robustness of a tissue’s ability to tolerate inflammatory challenge can be measured by the ability of tissues to perform their function during inflammatory challenge. Clinically, physicians use plasma biomarkers of tissue dysfunction—for example, troponins for cardiac dysfunction, creatinine for kidney function, transaminases for liver function—as surrogates for tissue function.

Q. How easy or hard is it to distinguish between bacterial and viral infections in a clinical setting – in humans? Are there good diagnostic tests for this?

It is currently very difficult to distinguish the type of infection at the time of admission. This is an area of active research. Currently, clinicians rely on biomarkers such as procalcitonin, which have poor specificity for infection type, and/or detection of the pathogen itself, which often takes many hours if not days to verify, if at all.

Q. What about mixed infections? How do mice respond to a mixed Listeria and Influenza infections? Your group has explored this co-infection model previously, do you understand it better now?

Historically, it has been observed that mixed infections are worse for the host than either of the infections separately. The most famous example is influenza infection followed by a staphylococcus aureus infection. We have previously looked at influenza followed by listeria monocytogenes, and then at influenza followed by legionella pneumophilia. Generally, it appears that viral “priming” potentiates severe disease from otherwise sublethal challenges with bacteria. The mechanisms operating in these different infection pairs was different, but we are trying to understand if there are more general principles that could make this specific sequence of virus then bacteria more lethal.

Q. Do you plan to study this in humans? If yes, then how would you control for cultural variables, the availability of food and the process of habitual eating that many human beings now live by?

We do plan on studying this in humans. The setting where much of this can be best controlled is the intensive care unit (ICU). In patients admitted to the ICU, many are unconscious for one reason or another. Currently, these patients are fed by tube feeding very shortly after they are admitted. The goal of our initial studies will be to see if restricting glucose in feeds delivered to individuals with documented infections would be better for their outcome compared to standard formula feeds.

Q. Do you suspect that there is a strong genetic component to tissue tolerance, set-points or points of no return?

There is likely a strong genetic component to tissue tolerance. Since the immune response has been subject to great selective pressure, it should follow that tissue response to inflammatory signals generated by the immune response would also be under the same selective pressures, especially because it is ultimately tissue dysfunction that leads to death and thus the inability to transmit genetic material. However, because the field of tissue tolerance is relatively unstudied, no studies that try to identify those genetic components exist.

Q. Is the brain the most vulnerable organ – as opposed to say the kidneys which flush out toxins from the body, in terms of coping with damage from an infection? Did this finding surprise you?

In any injury, there is usually an organ or small set of organs, which, if dysfunctional, becomes limiting for the organism’s survival. The limiting organ in turn depends on the type of insult. In general, if the heart, lungs, or brain fail, it is rapidly lethal for the host in the absence of medical intervention. There is a lot of precedence for central nervous system dysfunction in bacterial sepsis, but we were surprised to find that the brain also appeared to be limiting in our influenza model, which is primarily a lung-injury model.

Q. For some bacterial diseases, tuberculosis is a case in point, we know that malnutrition makes the condition worse. How do you reconcile these observations with your finding?

There is a big difference between acute infection and chronic infection. What we were studying was the response to acute self-limited infections. In chronic infection, the persistence of the inflammatory response, persistence of the pathogen, and the changes that this dynamic imposes on the host is very different than the acute phase response. So, it is likely that the metabolic requirements of chronic infections are very different from the metabolic requirements of acute infections. Also, even in the acute setting, bacteria have co-evolved with their hosts and in the process may have developed mechanisms that interfere with the tissue tolerance mechanisms that we have described here. Therefore, our current work may not be generalizable to the full spectrum of bacterial and viral infections.

Teixobactin: Can this new antibiotic help us sail through the doldrums of drug resistance?

iChip based discovery of a potent novel antibiotic

 

Why do we need a new antibiotic?

What can happen if you self-medicate on an antibiotic or do not finish a course of antibiotics that has been prescribed for you? When cattle are fed indiscriminately on antibiotics to keep them healthy? When sewage from hospitals is not completely treated and released into the community? The microbes that survive in these environments stop responding to antibiotics around them (1). Our world at present faces a daunting task of treating people infected with resistant forms of many bacteria. Often clinicians have to resort to potent broad spectrum antibiotics to treat infections that could be treated with the first line of drugs a few decades ago. The other aspect of the problem of antimicrobial resistance is the lack of new treatment options. Many of the current antibiotics are chemical modifications of ones that are known to work. Designing completely novel molecules, with antibiotic activity, synthetically, has not been very successful (2). In this rather bleak situation a new study brings a new ray of hope. In this study the authors have enriched hitherto uncultivated bacteria from the soil (3).

 

What is so special about this?

A very small percentage of bacteria in the soil can actually be grown in the laboratory (4). The development of tools/methods to grow more bacteria opens up a window for isolating new compounds with potential antimicrobial activities that these bacteria may be producing to their advantage in the complex niche of the soil. In this study, the authors diluted soil sample to contain single cells and grew them in special chambers embedded in the soil which allow for nutrient exchange with the soil. Earlier studies have shown this method to recover 50% of the bacteria from the soil. Previous studies have also demonstrated that once isolated, many of these bacteria can be grown in the lab.

 

How was this antibiotic found?

After screening over 10000 bacterial isolates in this way Ling et al., identified a new species Eleftheria terrae that produced a potent compound against Staphylococcus aureus (S. aureus can cause infections especially in hospitals, it also notorious for acquiring resistance to commonly used antibiotics). They examined this compound in greater detail, asking questions like- what does it look like (chemical structure)? How is it made in the bacteria (biosynthetic pathway)? To answer these questions, they undertook detailed chemical analysis by NMR and Marfeys’ structure analysis. The active compound which the authors named Teixobactin, was found to be a uricylated oligopeptide (the details of the structure are provided in the paper). They analyzed and were able to predict the pathway by which the E. terrae makes this antibiotic. The compound was found to be completely novel.

 

How does it work?

How does this antibiotic kill the target bacteria? The first clue was that it was more effective against gram positive that gram negative bacteria. These two classes  (distinguished on the basis of their appearance after staining them with dyes) of bacteria differ in the number and nature of protective coverings around them. Gram positive bacteria have a thick cell wall around them, whereas gram negative bacteria have a thin cell wall but also have an additional outer membrane. The cell wall is made up of repeating units of modified sugars known as peptidoglycan and is essential for structural integrity of bacteria. A breach in this structure would lead to the bacterium’s death. Interestingly, Teixobactin is not active against gram negative bacteria which have an outer membrane. However, a strain of E coli (a gram negative bacteria) with defects in the outer membrane is susceptible to this antibiotic. These data suggest that Teixobactin needs to have access to the bacterial cell wall for its activity. Consistent with this idea, they find that Teixobactin binds specifically to precursors of peptidoglycan and does not allow their incorporation into the cell wall. Instead of targeting the enzymes that carry out cell wall synthesis, Teixobactin, like the potent antibiotic, Vancomycin, interacts with structural components of the cell wall itself. It seems to target multiple precursors and bacteria die not only from lack of cell wall but also from accumulation of toxic intermediates of cell wall synthesis.

 

Does it work against pathogens?

They then asked, how effective is this antibiotic against common pathogens? Teixobactin was found to have potent activity against Staphylococcus aureus (which can cause disease under certain circumstances), Clostridium difficile (causes colitis) and Bacillus anthracis (anthrax). It also had good activity against hard to treat microorganisms like Mycobacterium tuberculosis (Tuberculosis) and enteroccocci (are intrinsically antibiotic resistant, causally associated with urinary tract infection among others). All of this is good news, however, how do we know that once in use, bacteria will not become resistant to Teixobactin? One way to answer this question is to subject bacteria to low levels of the antibiotic for prolonged period and then test if they still respond to it. Fortunately, no resistance to Teixobactin  emerged in either M tuberculosis (M Tuberculosis is notorious for acquiring resistance to multiple drugs) (6) or S aureus. Suggesting that resistance will probably be slow to evolve  against this antibiotic. The next step then was to assess if it was toxic to animal cells and/ or effective when used in animals.

The compound was found to be eminently suited for being used as a drug in animals. It was not toxic to mammalian cells, was active even in the presence of serum and was stable in  blood. Moreover, Teixobactin seems to have no carcinogenic properties. In mouse models of septicemia and pneumonia, mice treated with Teixobactin survived and responded well to  treatment. This makes Teixobactin a remarkable candidate for further studies with the  possibility of clinical trials in humans.

 

What does this finding mean?

The approach used to isolate and characterize Teixobactin is novel and paves way for the identification and characterization of many such compounds. We think that this may well be the beginning of a mining exercise where we explore more antimicrobials from the soil. It remains to be seen if Teixobactin can actually be used in humans, it is unclear how long that will take or last. At the very least, Teixobactin offers a tempting glimpse of what’s hidden in the soil and gives us a better appreciation for the microbial community we nonchalantly read upon.

Why are we more likely to get a cold in cold weather?

Summary

We don’t really know, what we do know is that some cold causing viruses grow better at lower temperatures. Rhinoviruses, one of the most common causes of the common cold, display a temperature dependent growth pattern. They were shown to grow better at cooler temperatures (33°C­ – 35°C)​ (1,2) like that in the upper respiratory tract than at the core body temperature of 37°C. Scientists hit a wall when they looked for a reason for this by analyzing the viruses themselves. Entry of the virus inside the cell, for instance, was not affected at cooler temperatures (33°C- 35°C). The failure to find a convincing mechanism such as a temperature dependent viral enzyme or gene product was puzzling until recently, when scientists turned the table and started looking at the virus infected cell rather than the virus itself.

More about the study

A study published in the Proceedings of the National Academy of Science (PNAS) looks at cellular defense mechanisms and anti­viral responses that come into play when Rhinoviruses infect cells (3). ​

​The authors of this study compared the response to infection at the warmer body temperatures (37°C) and at cooler temperatures as found in the nasal cavity (33°C).

In what we think is the first step to understanding the role of the host (human) in this process, this study used a mouse adapted strain of the virus. They generated this strain by growing the virus for many generations in mouse cells. Subsequently the virus acquired mutations, adapted and was able to infect mouse airway cells. Foxman et al., isolated mouse airway epithelial cells and used the adapted strain to infect these cells in the laboratory. In order to test the temperature sensitivity, they carried out infection experiments at 33°C (cooler) and 37°C (warmer) temperatures. They observed the expected decrease in number of viral particles (titers*) starting from 7 hours after infection at 37°C but not at 33°C, confirming that temperature does impact viral numbers. The changes observed were in fact in the infected cells which showed a lower antiviral response to the virus at cooler temperature.

When a cell gets infected by a virus, it puts out a signal saying “I am infected” by secreting molecules such as interferons and this is critical for mounting an antiviral response​ (4)​. The study by Foxman et al., shows that cells infected at cooler temperatures have lower expression of molecules critical to the anti­viral response. The authors artificially activated a defense pathway (The RLR pathway) which results in interferon production, and show that this pathway has a lower response at 33°C than at 37°C. In other words there is lower production of interferons, at cooler temperatures. Further, by genetically mutating either molecules of this pathway or a receptor of interferon in mouse airway cells, the authors find an increase in viral titers even at warmer temperature (37°C).

These data suggest the cells may be able to ward off a Rhinovirus infection at warmer temperatures (37°C) due to a robust anti­viral response resulting in the production of interferons. In the nasal epithelium which is in constant contact with the outside air, the temperature of the cells is likely to be low enough for Rhinovirus to get away with a successful infection. Rhinoviruses of course are one of many agents that cause cold and it is not known if other cold viruses are similarly checked at higher temperatures. It is likely that they use a repertoire of counter ­strategies to the host defense response, some aspects of which may be temperature dependent. The experiments in this study have been conducted on mouse cells grown in the lab. It remains to be seen if this holds true within living animals and whether it can be extended to human- ­cold virus interactions.

An interview with Dr. Ellen Foxman

Q. How would you place this work in context of the unanswered questions in the field?

Question #1: Why do Rhinoviruses grow better at nasal cavity temperature than at lung temperature? It has been known since the 1960s that most Rhinovirus strains replicate poorly at body temperature (37°C) and better at slightly cooler temperatures (33 – 35°C) such as the temperatures found in the nasal cavity. However, the reason for this was not known. In our study, we observed that Rhinovirus ­infected cells fight back against infection more at 37°C than at 33°C—in other words, the immune response triggered by the virus within infected is more robust at 37°C, and this is an important mechanism suppressing growth of the virus at 37°C. b. Question #2: Does temperature affect the immune response to diverse pathogens, or just the immune response to Rhinovirus? We found that two cardinal signaling pathways involved in immune defense were more active at body temperature than at nasal temperature: RIG­I like receptor signaling and Type I interferon receptor signaling. Since these pathways help defend us against in many viral different infections, our results raise the possibility that cool temperature also provides an advantage to viruses other than Rhinovirus. For example, many respiratory viruses cause colds more often than they cause lung infections; perhaps this is a reason why. That being said, it will be important to directly test other viruses, since viruses are tricky and many viruses have evolved ways to interfere with the immune responses we studied.

Q. How do you plan to take this study forward? What are the strengths and limitations of your model system?

The strength of our study was that we used a very well­ defined experimental system in which we could change one variable at a time to identify the immune system machinery needed to fight Rhinovirus within infected cells and to examine the effect of changing the temperature without changing anything else. Specifically, we used mouse primary airway cells grown in the laboratory. This way, we could compare cells from normal mice with cells from mice that differed by only one gene within the immune system. This allowed us to pinpoint which molecules within the immune system were important for defense against Rhinovirus, and which defenses were (or weren’t) affected by temperature. Also, by culturing cells in the lab, we were able to place them in incubators with controlled temperatures to clearly assess the effect of temperature without other confounding factors. b. Limitations/next steps: Although in general mice have been a good animal model for the human immune system, mice aren’t humans, and the next step in the study will be to examine in more detail how these mechanisms work within the human airway.

Q. Rhinoviruses are known to sometimes infect the lower respiratory tract (5) ​ what do you think is going on there?

One possibility is that the immune mechanisms required to block Rhinovirus infection don’t work as well in people who tend to have lung symptoms with Rhinovirus infection—for example, people with asthma. In our study, we found that if we used mouse cells lacking the necessary immune system machinery to block Rhinovirus infection, the virus could grow quite well at 37°C. There is some evidence that in airway cells from people with asthma, this machinery may not function properly; if this is the case, this might be what permits the virus to thrive at the warmer temperatures of the lung.

Q. Do you think alternating the temperatures (for example in a real world scenario inhaling steam or hot water gargling versus eating an ice cream) impact the success of Rhinovirus infection? In other words you perform the entire infection at one temperature, are there shorter time windows within which a temperature change would positively impact disease outcomes (for example gargling every morning or drinking hot water after eating an ice cream?)​ ?

We did do some temperature shift experiments (see Figure S3 in the paper.) We found that the level of the immune response tracked with the temperature of the cells during the time window when the virus was actively replicating; the temperature before the infection didn’t matter much. I would speculate that some exposure of infected cells to warm temperature at any point when the virus is actively replicating might be beneficial.

Q. What kind of experiments would you need to conduct to suggest to people that using different methods to increase the temperature of the upper respiratory tract – like drinking hot water may help fight cold? Have they been done?

The best way to prove that an intervention works is to directly test it, as you are suggesting. In this case, the best experiment would be to expose a group of volunteers to a fixed dose of Rhinovirus, and then place half of them on a well ­defined hot water drinking program (perhaps the other half could drink only cold water. If hot water program were effective, you would expect to see fewer colds develop in the hot water group than in the other group. This is a difficult study to perform: ideally, you would want to test a group of people who are identical in every way (genetics, behavior, environment, history of exposure to infections, etc.) except for the hot water drinking. In reality, this is quite hard to do, since every person is different! However, it might be possible to see an effect by studying a large group of people, especially if hot water drinking had a big impact (rather than a small effect) on whether or not colds developed after exposure to Rhinovirus. These types of studies can be very informative, but also can be complicated to interpret due to the inability to control all of the variables that may affect the outcome you are measuring (in this case, development of cold symptoms.) b. I do not know of any study considered to be definitive on this subject. However, I did a literature search and found a number of studies that have looked at the effect of hot liquids or steam inhalation on common cold symptoms, and I did find a number of these. You can read a few of these to get a feeling for the strengths and limitations. For example: i. Sanu and Eccles, 2008: This study tested the effect of hot liquid drinking on cold and flu symptoms in subjects who were recruited when they already had symptoms—the pathogen causing the symptoms is unknown. ii. Singh and Singh, 2013, meta-analysis of multiple studies looking at steam inhalation and common cold symptoms.

Q. You have emphasized on cell autonomous response to viral infections, what about the other aspects of the immune response? Do you think they could also contribute to temperature sensitivity?

We only looked at the cell autonomous immune responses in this study, and these were solely responsible for the temperature ­dependent blockade of Rhinovirus in our experiments. In the body, where many cell types are present, the responses we examined (RIG­I like receptor signaling and the Type I interferon response) can profoundly affect nearby and even distant cells through the action of secreted chemicals (cytokines). In this way, the phenomena we observed could also contribute to the temperature ­dependence of other immune responses; however, as yet we have no evidence for this.