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.

Can genetic variations define Ayurvedic Prakritis?

-A genome- wide study finds allelic differences between individuals that correlate with Ayurvedic body-types (Prakritis)

Snap-Shot of the study

Do the ayurvedic body types have genetic underpinnings? In a first step to answer this question, the authors evaluated differences between individuals whose body-type had been assigned by both Ayurvedic practitioners and a software. They found 52 variations across the genomes of 262 individuals which allowed them to be classified into ayurvedic Prakritis – Pitta, Kapha and Vata.

 Introduction to Ayurvedic prakritis

In Ayurveda, according to the ancient text Charaka Samhita –the body and mind must be brought together to lead a harmonious existence. People can be classified into Prakritis or types on the basis of relative contribution of the three constituents Pitta, Kapha and Vata (roughly translating to – arising from movement, digestion and accumulation – of toxic metabolites for instance) to their body. According to Ayurvedic philosophy- an understanding of this body type and the ability to maintain a diet and lifestyle suited to that body type translate to balance and health. Prakriti or Ayurvedic body-types which define a person’s intrinsic physical abilities, mental states and also have implications for their susceptibility to disease and response to drugs (1,2).

Background for this study

In a recent study (3), researchers have identified genetic variations associated with the traditional classification of people into Ayurvedic Prakritis – specifically if small differences (SNPs, Single nucleotide polymorphisms) throughout the human genome correlate with the Prakriti classification. All humans are genetically very similar to each other, differences between us (populations, races, ethnic groups etc.) are captured in variations of nucleotides which make up the DNA – these are called single nucleotide polymorphisms. Many studies have shown particular SNP or group of SNPs to be correlated with risk of disease, whether a person will develop resistance to therapy, etc. forming the basis for personalized medicine  (4,6).

 What did they do?

3416 individuals were classified for their prakritis by Ayurvedic practitioners as well as a software. Of these, DNA isolated from blood samples of 262 individuals (male, healthy, between 20-30 years), who were reliably classified as having a clear dominance of one of the three constituents (Vata, Pitta or Kapha) representing the “extreme” Prakritis were used for the genome-wide study. A microarray consisting of 1 million positions / SNPs was used to identify the genotype of these individuals.

 What did they find?

This study found 52 out of 1 million SNPs is sufficient to assign the Prakriti of individuals, irrespective of their ethnic background. One of the challenges in the study was that there was no control group – therefore each prakriti was compared to the other two. Subsequently, these 52 SNPs were able to cluster individuals into distinct groups by Principal Component Analysis. Interestingly, one of the SNPs in a PGM1 gene (Phosphoglucomutase 1), which codes for an important enzyme in sugar (glucose) metabolism, is significantly associated with Pitta dominant group that is known for efficient metabolism.

 Things to keep in mind about the study:

It is to be noted that the three categories compared and defined here represent extremes –and according to Ayurvedic principles most people are a composite of all three- Kapha, Vata and Pitta, with the dominant element defining their type. There are some previous studies, which have suggested that the Ayurvedic Prakriti classification may have a genetic or metabolic basis (2,5). They were conducted on fewer subjects and looked at fewer genes/ phenotypes compared to this study, which rigorously recruited a large number of subjects, used multiple methods of classifications (software and Ayurveda practitioners) and a genome wide approach. There is much work to be done in understanding the scientific basis of the Ayurvedic classification system and whether we can independently and reliably assign people (independent of race, gender and ancestry) to a type.

 What does this mean?

The 52 SNPs defined in this study can now be used independently in other populations and also provide a way of identifying new associations with metabolism and other phenotypes. For more than fifteen years now, we have been able to read our genome i.e – information in our genes but not been able to fully understand how it defines us as individuals. So on the one hand, this study by correlating phenotypes with genetic variations, helps us understand a little bit more about how genes make us who we are. On the other hand, by using modern genetic tools in the context of traditional knowledge, this study provides a rigorous way of assessing the framework of ayurvedic medicine.

Citations

1. Understanding personality from Ayurvedic perspective for psychological assessment: A caseS Shilpa and C. G. Venkatesha Murthy. Ayu. 2011 Jan-Mar; 32(1): 12–19.

2. Classification of human population based on HLA gene polymorphism and the conceptof Prakriti in Ayurveda. Bhushan P 1 , Kalpana J, Arvind C. J Altern Complement Med. 2005 Apr;11(2):349-53.

3. Genome-wide analysis correlates Ayurveda Prakriti. Govindaraj P, Nizamuddin S, Sharath A, Jyothi V, Rotti H, Raval R, Nayak J, Bhat BK, Prasanna BV, Shintre P, Sule M, Joshi KS, Dedge AP, Bharadwaj R, Gangadharan GG, Nair S, Gopinath PM, Patwardhan B, Kondaiah P, Satyamoorthy K, Valiathan MV, Thangaraj K. Sci Rep. 2015 Oct 29;5:15786. d

4. A database of humans SNPs and their recorded associations can be found here: http://www.snpedia.com/index.

5. Whole genome expression and biochemical correlates of extreme constitutional types defined in Ayurveda . Prasher B, Negi S, Aggarwal S, Mandal AK, Sethi TP, Deshmukh SR, Purohit SG, Sengupta S, Khanna S, Mohammad F, Garg G, Brahmachari SK; Indian Genome Variation Consortium, Mukerji M. J Transl Med. 2008 Sep 9;6:48.

6. http://www.scientificamerican.com/article/a-very-personal-problem/

Interview with Dr. K. Thangaraj

Q. What was the overlap between the prediction by the software, by different Ayurvedic practitioners? Have you tried to estimate if this classification is robust enough to suggest underlying genetic differences?

The first assessment was by Ayurvedic physicians and classified using their knowledge. To double check what they have done, we have used a computer software. The software is also based on various parameters specified by the Ayurveda physicians.There were many questions which the individual had to answer to be assigned a Prakriti. Across the three centres – Bangalore, Pune and Udupi, on average, 75% of the individuals were in concordance.

Q. Why not perform an enzyme profile or measure transcriptional differences for metabolic enzymes? Is there an advantage in taking an SNP approach?

The advantage is that the SNP does not changes, it is there from birth till the individual dies. Whereas transcription profiles may change at different times, depending on time of day, tissue type to tissue type etc. We have looked into that also. This is the very first step. We can extend this across countries, across ethnic groups and cluster them.

Q. Ayurvedic medicine believes in holistic changes including those of lifestyle, dietary along with medicines and does not rely overly on mechanistic explanations beyond the classification into types. There is less emphasis, if you will on dissection of cause-effect and more on restoring the overall balance. Do you agree with this? If yes, then what are the challenges with respect to the study design when you tried to apply the modern framework of science- which relies on a reductionist approach, finding a cause and targeting only that specifically, to the ayurvedic system?

I agree that there is a holistic approach, but the basic approach is to classify the individual. Based on the prakriti, they will make the changes to the diet, prescribe treatment etc., so this is a very important stage. The challenge is the following- I am a geneticist looking at diseases,looking at case-control studies is very easy – for every marker we can ask if the mutation is present or if the prevalence is higher in the patients versus the control. In this case all the individuals are normal and within the same age-group. The only difference is the prakiti. So we wanted to see how to differentiate these individuals- as there are three groups, not two. Then we decided to compare one prakriti against the other two types, and try to see if there is genetic variation between the groups. Then there was a lot of statistical analysis. We used 1 million markers, this has many advantages, the disadvantage is the robustness and having to come up with statistical analysis ourselves. (MT: So, by using 1 million regions, you may increase the chances of false associations, is that the worry?) Not, really false associations. For example we may not have information about a particular SNP in an individual. We need to use markers that are consistent between all the individuals. So, when data is not available, we need ways of retrieving the data. In that process we need to use genetic panel of markers which are Indian specific. We developed our own panel of markers – Dravidian, Indo-European and used as a reference and to extract what is the possible marker in a given position.

Q. Have you tried to validate your classification using the 52 SNP panel with an independent population? For instance, if you knew what a person’s SNP state is, how reliably can you assign their Prakriti? Would it be useful to perform a blind study in which both the SNP panel ayurvedic practitioners and the software performed the classification, with the aim of determining how often they match?

That is very interesting. What we did was, we has more than 300 samples analyzed for these 1 million genetic markers, from our initial studies on population genetics. We used some of those samples, as these are all populations specific – a very endogamous population. We tried to project some of those individuals into these three clusters, we did find that although the individuals have come from the same ethnic background (more homogeneous), they were falling in 2 or 3 different prakritis. The same ethinic background can be placed into different prakriti. This we tried to do with our own data, this is not as detailed as you suggest. Independent blind studies need to be done.

Q. You have excluded women from your study and restricted yourself to the Indian population, does this limit the applicability of your results?

Yes, of course. At this point we selected only males because we did not want any confounding effects, in the females there are many hormonal changes and so on.

Q. You have started a way of examining traditional medicine in the framework of modern science. What are the challenges and the future of this approach?

(The future of this approach) This has paved the way to do many more things. For example, the discovery of PGM1 has given the clue that you can take the phenotype of the particular prakriti and correlate it with the gene, this gene is involved in metabolism and individuals with the pitta prakirti have high metabolism. We can now use the characteristic feature of the prakriti and look into those genes in a detailed way. These are some of the futuristic aspects, one can take the study further with. We did try to look at the network of all the metabolic pathways genes, the problem is this will need transcription or metabolic profiles from tissues of these normal individuals.

 

A novel pathway of Brain Drain

Two studies provide a new map of waste management systems in the mouse brain

 

Snap-shot of the study

How is the brain protected from pathogens or chemicals? For a long time, it was thought that the brain is protected by barriers, such that it is impermeable even to immune cells (that could protect it) and to most chemicals (including drugs that need to act in the brain) (1,2). More recently it has become clear that cells of our immune system do constantly survey the brain (3). How do these cells get in and out? In the July 2015 issues of Nature and JEM, two groups report the discovery of an extension of the lymphatic system into the brain.

Background

 If the circulatory system can be likened to the nutrient supply system of the body, the lymphatic system is somewhat like drainage system, collecting fluid, large molecules and white blood cells from tissues, thus recycling blood components. The lymphatic system includes the tonsils, spleen and lymph nodes and extends throughout the body. This system contributes to fluid clearance, pathogen recognition and mounting of an immune response to pathogens. So it is indeed surprising that there was an arm of this system in the brain that we had no idea about until about a month ago. Two recent papers describe a lymphatic vessel network in the membrane covering of the brain that carries the fluid in which the brain floats (the cerebrospinal fluid) and drains into the deep cervical lymph nodes (5,6).

What did they do and find?

In both of these studies using mainly imaging based techniques, the authors identify vessels in the mouse brain which they call the meningeal lymphatic vessels using molecular markers that tag lymphatic endothelial cells.

In the dura mater, the covering of the brain, T-cells (a kind of immune cells) are found scattered throughout. However, Louveau et al. noticed many T-cells lining the Superior Sagittal Sinus (SSS). Surprisingly, these cells were not seen in significant numbers in the lumen of (read, inside) the SSS. This suggested that these T-cells could be residing in a separate compartment like blood vessels or lymphatic vessels. The authors ruled out blood vessels and used specific markers (read, tags – namely Prox-1, Podoplanin and VEGFR3) to demonstrate that these were indeed lymphatic vessels containing lymphatic endothelial cells. The vessel containing the T-cells was positive for Lyve-1 (tags lymphatic vessel) and was clearly visible as a channel running close to the SSS. These results confirmed that this vessel was indeed a lymphatic endothelial compartment.

Last year, Aspelund et al. reported the surprising finding that the eye also contains a lymphatic-like vessel, the so-called Schlemm canal (4). While studying the eyes of mice whose lymphatic vessels were labelled with flourescent markers, they found the brain lymphatics in the adjacent tissues. The lymphatics were present along blood vessels in various parts of the dural membranes even at the base of the skull with several exit paths through the same openings used by cranial nerves. Further, they were also able to show a critical role for the VEGF receptor signalling pathway  in the formation of these lymphatic vessels. In the absence of these vessels, consistent with their role in clearance and management of waste, large molecules did not get cleared from the brain (6).

Both groups explored if these are functional lymphatic vessels by injecting intravenously with a fluorescent dyes or, special minute(nano) particles, to track the flow of fluids from the brain tissue through the different compartments. This experiment showed that the vessels carry the brain fluid (read, cerebrospinal fluid or CSF) into the deep cervical lymph nodes.

These two studies have important implications for conditions in which there is pathological aggregation of proteins in the brain (Alzheimer’s) and also on how the immune system keeps a check on the brain in health and disease.

An interview with Dr. K Alitalo, Dr. A Louveau

Q. How do you see this discovery will help to shed light on previously unanswered questions of our understanding of the central nervous system?

(Dr. K Alitalo) This is an important discovery that will enable scientists to look at neurological diseases from an entirely new standpoint. The vessels may be key in explaining some features of diseases that could not previously understood.

Q. We also came across a recent publication by Louveau et al., published in journal Nature which talks about a similar lymphatic system. Is this the same system that you show in your publication

( Dr. K Alitalo) It is indeed the same system. We did a more thorough job at characterizing it as we found out that the vessels extend to dural venous sinuses, meningeal arteries, cranial nerves and the cribriform plate. Louveau et al. used a methodology that only enabled the discovery of the vessels in the superior parts of the skull along dural venous sinuses.

Q. Do you think advances in the imaging techniques and the availability of well defined markers was the only reason for such a late discovery of this system? Are there any other possibilities that could have contributed to delay in this discovery?

( Dr. K Alitalo) Probably the greatest challenge for the visualization of meningeal lymphatic vessels has been the adjacent osseous bone. The need for decalcification is, in many respects, problematic for tissue analysis. Most importantly, I think both discoveries were enabled by advanced imaging technologies, which have transformed the world of traditional 2D tissue sections into 3D. In our study, the discovery was enabled by the previous generation of lymphatic-specific fluorescent reporter mice in which the lymphatic vessels can be visualized as they are in tissues by fluorescent microscopy, without the need for any processing.

Q. Do you think similar lymphatic vessel could be present in humans, what are the technical challenges you see in establishing it?

( Dr. K Alitalo) The techniques already exist, so it is just a matter of time when researches will perform this work. The only problem is that endothelial cells become autolytic soon after death.

Q. Your study clearly shows the role of lymphatic vessels in draining macromolecules out of the brain. What potential implications would this have on human health conditions like infectious diseases, disorders like Alzheimer’s and tumor metastasis?

( Dr. K Alitalo) The brain lymphatics may become structurally and functionally altered in infectious diseases. This needs to be evaluated in the context of pathogenesis of such diseases. – Alzheimer’s disease is characterized by the pathological accumulation of amyloid beta into the brain tissue. Proper meningeal lymphatic vessel function could perhaps protect against Alzheimer’s disease by clearing pathological amyloid beta from the brain parenchyma. Perhaps we can boost such clearance with our technology. Brain tumors rarely metastasize into cervical lymph nodes. However, when this happens, it has been unknown how the metastases arise. The meningeal lymphatic vessels are likely to represent the missing link.

Q. How do you see this discovery will help to shed light on previously unanswered questions of our understanding of the central nervous system?

(Dr. A Louveau) This discovery is changing an established paradigm described decades ago, that the brain is an immuno privileged organ because, at least in part, of the lack of classical lymphatic drainage. What we demonstrate is that the brain possesses a classical lymphatic drainage, but the brain remains immunologically unique. We will have to change the way we see the brain when we want to address diseases that have immunological factors.

Q. We also came across a recent publication by Aspelund et al., published in Journal of Experimental Medicine which talks about a similar lymphatic system. Is this the same system that you show in your publication?

(Dr. A Louveau) Yes, the study recently published by the group of Kari Alitalo confirmed our study and shows similar results to the one we published.

Q. Do you think advances in the imaging techniques and the availability of well defined markers was the only reason for such a late discovery of this system? Are there any other possibilities that could have contributed to delay in this discovery?

(Dr. A Louveau) The development of the imaging techniques and the fairly recent description of specific markers to identify lymphatic endothelial cells (early 2000) are certainly major factors in the late discovery of this system. One other reason is the unique location in the dura mater, a region of the meninges usually discarded when people study the brain (because this layer remains attached to the skull).

Q. In the paper you mentioned similar lymphatic vessel could be present in humans as well, what are the technical challenges you see in establishing this?

(Dr. A Louveau) We think that the challenge will not be technical but highly depend on the quality of the tissues collected. Most human tissue collection have been fixed for month to years rendering immunostaining challenging. Collecting more recently collected tissue should help, but lymphatic vasculature appear to be sensitive structure.

Q. What are the potential implications of this discovery, on human health conditions like infectious diseases, disorders like Alzheimer’s or Schizophrenia and tumor metastasis?

(Dr. A Louveau) We think that the meningeal lymphatic system might play a role in every neurological disease with a strong immune component, including the ones you are mentioning. In Alzheimer disease, the blockade of the meningeal lymphatic might be an initial event before the accumulation of unfolded protein in the brain. In the case of brain tumor, the meningeal lymphatic system might be used by the tumor to prevent its attack by the immune system. But all of this remains hypothetical and the function of the meningeal lymphatic system will have to be studied in all of those diseases.

Q. In one of your interviews you have mentioned that your discovery would require modifications in the textbooks. Could you elaborate more on the changes that you envisage?

(Dr. A Louveau) If you open a neuroscience textbook right now, you’ll read that the brain is devoid of a lymphatic system, therefore contributing to its immune privilege. Our discovery, more so if it extends to humans, proves that this statement is wrong and that the brain, like every other organ possesses a lymphatic system.

References

1. “The blood-brain barrier: an engineering perspective.” Andrew D. Wong et al., Front Neuroeng., 2013

2. “The gut immune barrier and the blood-brain barrier: are they so different?” Richard Daneman and Maria Rescigno, Immunity, 2009

3. “Immune surveillance in the central nervous system.”  Shalina S Ousman and Paul Kubes, Nat Neurosci., 2012

4. “The Schlemm’s canal is a VEGF-C/VEGFR-3-responsive lymphatic-like vessel.” Aleksanteri Aspelund et al., J Clin Invest. 2014

5. “Structural and functional features of central nervous system lymphatic vessels.” Antoine Louveau et al., Nature. 2015

6.“A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules.” Aleksanteri Aspelund et al., J Exp Med., 2015

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.