How Viruses Feign Death to Survive and Thrive

Billions of cells die each day in the human body in a process called “apoptosis” or “programmed cell death”. When cells encounter stress such as inflammation, toxins or pollutants, they initiate an internal repair program which gets rid of the damaged proteins and DNA molecules. But if the damage exceeds their capacity for repair then cells are forced to activate the apoptosis program. Apoptotic cells do not suddenly die and vanish, instead they execute a well-coordinated series of molecular and cellular signals which result in a gradual disintegration of the cell over a period of several hours.

The remains of an apoptotic cell are being engulfed and ingested by a phagocytic white blood cell. Image via National Library of Medicine.
The remains of an apoptotic cell are being engulfed and ingested by a phagocytic white blood cell. Image via National Library of Medicine.

What happens to the cellular debris that is generated when a cell dies via apoptosis? It consists of fragmented cellular compartments, proteins, fat molecules that are released from the cellular corpse. This “trash” could cause even more damage to neighboring cells because it exposes them to molecules that normally reside inside a cell and could trigger harmful reactions on the outside. Other cells therefore have to clean up the mess as soon as possible. Macrophages are cells which act as professional garbage collectors and patrol our tissues, on the look-out for dead cells and cellular debris. The remains of the apoptotic cell act as an “Eat me!” signal to which macrophages respond by engulfing and gobbling up the debris (“phagocytosis“) before it can cause any further harm. Macrophages aren’t always around to clean up the debris which is why other cells such as fibroblasts or epithelial cells can act as non-professional phagocytes and also ingest the dead cell’s remains. Nobody likes to be surrounded by trash.

Clearance of apoptotic cells and their remains is thus crucial to maintain the health and function of a tissue. Conversely, if phagocytosis is inhibited or prevented, then the lingering debris can activate inflammatory signals and cause disease. Multiple autoimmune diseases, lung diseases and even neurologic diseases such as Alzheimer’s disease are associated with reduced clearance. The cause and effect relationship is not always clear because these diseases can promote cell death. Are the diseases just killing so many cells that the phagocytosis capacity is overwhelmed, does the debris actually promote the diseased state, or is it a bit of both, resulting in a vicious cycle of apoptotic debris resulting in more cell death and more trash buildup? Researchers are currently investigating whether specifically tweaking phagocytosis could be used as a novel way to treat diseases with impaired clearance of debris.

During the past decade, multiple groups of researchers have come across a fascinating phenomenon by which viruses hijack the phagocytosis process in order to thrive. One of the “Eat Me!” signals for phagocytes is that debris derived from an apoptotic cell is coated by a membrane enriched with phosphatidylserines which are negatively charged molecules. Phosphatidylserines are present in all cells but they are usually tucked away on the inside of cells and are not seen by other cells. When a cell undergoes apoptosis, phosphatidylserines are flipped inside out. When particles or cell fragments present high levels of phosphatidylserines on their outer membranes then a phagocyte knows that it is encountering the remains of a formerly functioning cell that needs to be cleared by phagocytosis.

However, it turns out that not all membranes rich in phosphatidylserines are remains of apoptotic cells. Recent research studies suggest that certain viruses invade cells, replicate within the cell and when they exit their diseased host cell, they cloak themselves in membranes rich in phosphatidylserines. How the viruses precisely appropriate the phosphatidylserines of a cell that is not yet apoptotic and then adorn their viral membranes with the cell’s “Eat Me!” signal is not yet fully understood and a very exciting area of research at the interface of virology, immunology and the biology of cell death.

What happens when the newly synthesized viral particles leave the infected cell? Because these viral particles are coated in phosphatidylserine, professional phagocytes such as macrophages or non-professional phagocytes such as fibroblasts or epithelial cells will assume they are encountering phosphatidylserine-rich dead cell debris and ingest it in their roles as diligent garbage collectors. This ingestion of the viral particles has at least two great benefits for the virus: First and foremost, it allows the virus entry into a new host cell which it can then convert into another virus-producing factory. Entering cells usually requires specific receptors by which viruses gain access to selected cell types. This is why many viruses can only infect certain cell types because not all cells have the receptors that allow for viral entry. However, when viruses hijack the apoptotic debris phagocytosis mechanism then the phagocytic cell is “inviting” the viral particle inside, assuming that it is just dead debris. But there is perhaps an even more insidious advantage for the virus. During clearance of apoptotic cells, certain immune pathways are suppressed by the phagocytes in order to pre-emptively dampen excessive inflammation that might be caused by the debris. It is therefore possible that by pretending to be fragments of dead cells, viruses coated with phosphatidylserines may also suppress the immune response of the infected host, thus evading detection and destruction by the immune systems.

Colorized scanning electron micrograph of filamentous Ebola virus particles (blue) budding from a chronically infected cell (yellow). Credit: National Institute of Allergy and Infectious Diseases, NIH.
Colorized scanning electron micrograph of filamentous Ebola virus particles (blue) budding from a chronically infected cell (yellow). Credit: National Institute of Allergy and Infectious Diseases, NIH.

Viruses for which this process of apoptotic mimicry has been described include the deadly Ebola virus or the Dengue virus, each using its own mechanism to create its fake mask of death. The Ebola virus buds directly from the fat-rich outer membrane of the infected host cell in the form of elongated, thread-like particles coated with the cell’s phosphatidylserines. The Dengue virus, on the other hand, is synthesized and packaged inside the cell and appears to purloin the cell’s phosphatidylserines during its synthesis long before it even reaches the cell’s outer membrane. As of now, it appears that viruses from at least nine distinct families of viruses use the apoptotic mimicry strategy but the research on apoptotic mimicry is still fairly new and it is likely that scientists will discover many more viruses which rely on this and similar evolutionary strategies to evade the infected host’s immune response and spread throughout the body.

Uncovering the phenomenon of apoptotic mimicry gives new hope in the battle against viruses for which we have few targeted treatments. In order to develop feasible therapies, it is important to precisely understand the molecular mechanisms by which the hijacking occurs.  One cannot block all apoptotic clearance in the body because that would have disastrous consequences due to the buildup of legitimate apoptotic debris that needs to be cleared. However, once scientists understand how viruses concentrate phosphatidylserines or other “Eat Me!” signals in their membranes, it may be possible to specifically uncloak these renegade viruses without compromising the much needed clearance of conventional cell debris.


Elliott, M. R. and Ravichandran, K.S. “Clearance of apoptotic cells: implications in health and disease” The Journal of Cell Biology 189.7 (2010): 1059-1070.

Amara, A and Mercer, J. “Viral apoptotic mimicry.” Nature Reviews Microbiology (2015).


Note: An earlier version of this article first appeared on the 3Quarksdaily Blog.

Amara A, & Mercer J (2015). Viral apoptotic mimicry Nature Reviews Microbiology, 13 (8), 461-9 PMID: 26052667

Fixing ‘Leaky’ Blood Vessels in Severe Respiratory Ailments and Ebola

When you get an infection, your immune system responds with an influx of inflammatory cells that target the underlying bacteria or viruses. These immune cells migrate from your blood into the infected tissue in order to release a cocktail of pro-inflammatory proteins and help eliminate the infectious threat.

During this inflammatory response, the blood vessel barrier becomes “leaky.” This allows for an even more rapid influx of additional immune cells. Once the infection resolves, the response cools off, the entry of immune cells gradually wanes and the integrity of the blood vessel barrier is restored.

But if the infection is so severe that it overwhelms the immune response or if the patient is unable to restore the blood vessel barrier, fluid moves out of the blood vessels and begins pouring into the tissue. This “leakiness” is what can make pneumonia turn into acute respiratory distress syndrome. ARDS, by my estimate affects hundreds of thousands of people each year worldwide. In the US around 190,000 people develop ARDS each year and it has a mortality rate of up to 40%. In people with Ebola, this leakiness is also often deadly, causing severe blood pressure drops and shock.

New therapies to fix the leakiness of blood vessels in patients suffering from life-threatening illnesses, such as acute respiratory distress syndrome and Ebola virus infections, have the potential to save many lives.

Green fluorescent staining for of the junction protein VE-cadherin in a layer of lung blood vessel endothelial cells
Green fluorescent staining for of the junction protein VE-cadherin in a layer of lung blood vessel endothelial cells – Image Malik Lab

What is ARDS?

Severe pneumonia can lead to acute respiratory distress syndrome (ARDS), a complication in which the massive leakiness of blood vessels in the lung leads to the fluid build-up, which covers the cells that exchange oxygen and carbon dioxide. Patients usually require mechanical ventilators to force oxygen into the lungs in order to survive.

Pneumonia is one of the most common causes of ARDS but any generalized infection and inflammation that is severe enough to cause massive leakiness of lung blood vessels can cause the syndrome.

For people with ARDS treatment, options other than ventilators and treating the underlying infection are limited. And suppressing the immune system to treat this leakiness can leave patients vulnerable to infection.

A new treatment option

But what if we specifically target the leakiness of the blood vessels? Our research has identified an oxygen-sensitive pathway in the endothelial cells which line the blood vessels of the lungs. The leakiness or tightness of the blood vessel barrier depends on the presence of junctions between these cells. These junctions need two particular proteins to work properly. One is called VE-cadherin and is a key building block of the junctions. The other is called VE-PTP and helps ensure that VE-cadherin stays at the cell surface where it can form the junctions with neighboring cells.

When the endothelial cells are inflamed, these junctions break down and the blood vessels become leaky. This prompts the cells to activate a pathway via Hypoxia Inducible Factors (HIFs), which are usually mobilized in response to low oxygen stress. In the heart, HIF pathways are activated during a heart attack or long-standing narrowing of the heart blood vessels to improve the survival of heart cells and initiate the growth of new blood vessels.

We found that a kind of HIF (called HIF2α) was protective in lung blood vessel cells. When it was activated, it increased levels of the proteins that support the junctions between lung cells and strengthened the blood vessel barrier. But in many patients, this activation may not start soon enough to prevent ARDS.

The good news is that we can activate this factor before the lung fluid accumulates and before low oxygen levels set in. Using a drug, we activated HIF2α under normal oxygen conditions, which “tricked” cells into initiating their protective low-oxygen response and tightening the blood vessel barrier. Mice treated with a HIF2α activation drug had substantially higher survival rates when exposed to bacterial toxins or bacteria which cause ARDS.

Similar drugs have already been used in small clinical trials to increase the production of red blood cells in anemic patients. This means that activating HIF2α is probably safe for human use and may indeed become a viable strategy in ARDS. However, the efficacy and safety of drugs which activate HIF2α still have to be tested in humans with proper placebo control groups.

Could this treat Ebola?

The Ebola virus is a hemorrhagic virus and is also known to induce the breakdown of blood vessel barriers. In fact, it is these leaks in the blood vessels that make the disease so deadly. Due to the leakage of fluid and blood from the blood vessels into the tissue, the levels of fluid and blood inside the blood vessels decrease to critically low levels, causing blood pressure drops and ultimately shock.
A group of researchers in Germany recently reported the use of an experimental drug (a peptide) developed for the treatment of vascular leakage in a 38-year-old doctor who had contracted Ebola in Sierra Leone and was airlifted to Germany. The researchers received a compassionate-use exemption for the drug and the patient recovered.

This is just a single case report and it is impossible to know whether the patient would have recovered similarly well without the experimental vascular leakage treatment, but it does highlight the potential role of drugs which treat blood vessel leakiness in Ebola patients.

The Conversation

This article was originally published on The Conversation.
Read the original article.

Gong, H., Rehman, J., Tang, H., Wary, K., Mittal, M., Chatturvedi, P., Zhao, Y., Komorova, Y., Vogel, S., & Malik, A. (2015). HIF2α signaling inhibits adherens junctional disruption in acute lung injury Journal of Clinical Investigation DOI: 10.1172/JCI77701

Turning Off Inflammation: A Novel Anti-Inflammatory Switch in Macrophages

[Note: This is a guest post by Tauseef (@CellSpell), an excellent immunologist and one of my faculty colleagues at the University of Illinois, who is quite excited about science outreach and science blogging.]

Macrophages are important immune cells which regulate inflammation, host defense and also act as a ‘clean-up crew’. They recognize, kill and engulf bacteria as well as cellular debris, which is generated during an acute infection or inflammation. As such, they are present in nearly all tissues of the body, engaging in 24/7 surveillance. Some macrophages in a tissue are derived from circulating blood monocytes which migrate into the tissue and become “phagocytic” – acquire to ability to “eat”. Other macrophage types permanently reside within a tissue such as peritoneal macrophages in the abdomen or microglia in the brain. Macrophages constitute a highly diverse population of cells. For example, their tissue localization determines what genes are turned on in any given macrophage type and how they will function. One of the most important recent developments in macrophage biology and immunology has been the realization that tissue macrophages can be broadly divided into at least two very distinct subsets: M1 and M2.


Macrophage and Virus (via Shutterstock)
Macrophage and Virus (via Shutterstock)

Pro-inflammatory M1 macrophages are predominantly involved in digesting bacteria and debris. They release pro-inflammatory molecules which then attract other immune cells and inform them that their assistance in fighting off the infection is sorely needed. M2 macrophages, on the other hand, help resolve inflammation by secreting anti-inflammatory molecules and calming down their M1 cousins. During inflammation, both sets of macrophages are activated but M1 cells appear first and M2 later. This makes sense because it allows the body to first focus on fighting off the injury with its powerful M1 cells, but also prevents excessive damage by subsequently initiating an endogenous brake (M2 cells) to prevent excessive inflammation.

Inadequate activation of M2 cells during infection or inflammation can have disastrous effects. If the pro-inflammatory M1 cells have no anti-inflammatory counterpart, then they will keep on releasing pro-inflammatory molecules. These, in turn, will attract increasing numbers of immune cells and set in motion a vicious cycle of severe inflammation and massive fluid accumulation. If the levels of M1 activity are extremely high, some tissues such as the lung can be flooded with fluid and cells which prevent oxygen supply to the body, and ultimately result in death. Lower levels of persistent M1 cell activity may not lead to death, but could cause a simmering chronic inflammation and autoimmune diseases.

Restoring the balance of M1 and M2 cells, or selectively increasing M2 cells is becoming a hot area in immunology. If it were possible to increase M2 cells by turning on specific molecules or pathways, one could treat autoimmune diseases or prevent exaggerated inflammatory responses. This would be a far more elegant than relying on more conventional immune suppressants such as steroids which could compromise the body’s ability to resist future infections.

A recent paper published in the journal Cell (2014) by Yasutaka Okabe and Ruslan Medzhitov, has identified a transcription factor which is specific for anti-inflammatory macrophages. The researchers used a gene array to screen for over 40,000 genes and found that the transcription factor GATA6 was a key regulator of whether peritoneal (abdominal) macrophages were pro-inflammatory or anti-inflammatory. More importantly, the researchers found that retinoic acid, an active metabolite of Vitamin A, increases the GATA6 levels in macrophages, and thus pushes them towards an anti-inflammatory identity. Genetic deletion of GATA6 or depletion of Vitamin A in the diet of mice resulted in peritoneal macrophages becoming more pro-inflammatory (M1-like).

Although the present study provides an evidence of the role of Vitamin A and its metabolite retinoic acid in the suppression of inflammation by activation of GATA6 in macrophages, some unanswered questions need to be addressed in future studies. The researchers showed that Vitamin A depletion pushes macrophages towards the pro-inflammatory M1-like identity, but the researchers did not try the converse: They did not test whether giving vitamin A to animals would increase anti-inflammatory macrophages. The researchers also did not track the individual macrophages to truly prove that the pro-inflammatory cells were actually converting into anti-inflammatory macrophages versus merely recruiting a pool of anti-inflammatory cells from the blood.

An important lesson that we can take away from this paper is that vitamins and their metabolites are regulators of the immune response. Either too little or too much Vitamin A may be detrimental because its metabolite retinoic acid could upset the finely regulated balance of the immune system. This study is one of the first to unravel the molecular switches which regulate the formation of pro-inflammatory and anti-inflammatory macrophages. We are only at the beginning of this exciting area of research and hopefully, in the years to come, selective manipulation of these switches will allow us to treat acute inflammatory and chronic autoimmune diseases for which few therapies are available.


– M. Tauseef (@CellSpell)

Okabe Y, & Medzhitov R (2014). Tissue-specific signals control reversible program of localization and functional polarization of macrophages. Cell, 157 (4), 832-44 PMID: 24792964


“Inflamm-Aging”: Inflammatory Signals in the Brain Regulate the Lifespan of Mice

The hypothalamus is located at the base of the brain and in adult humans, it has a volume of only 4cm3, less than half a percent of the total adult human brain volume. Despite its small size, the hypothalamus is one of the most important control centers in our brain because it functions as the major interface between two regulatory systems in our body: The nervous system and the endocrine (hormonal) system. It consists of many subunits (nuclei) which continuously sense inputs and then respond to these inputs by releasing neurotransmitters or hormones that regulate a broad range of vital functions, such as our metabolism, appetite, thirst, reproduction, temperature and even our internal timing system, the circadian clock. As if this huge workload wasn’t enough, researchers have now uncovered an additional role for the hypothalamus: regulating lifespan.

The recent paper “Hypothalamic programming of systemic ageing involving IKK-β,NF-κB and GnRH” published in the journal Nature (published online May 1, 2013) by Guo Zhang and colleagues at the Albert Einstein College of Medicine in New York used elegant genetic mouse models to either continuously activate or continuously suppress the function of the NF-κB protein in the hypothalamus. This protein is a key transcription factor which is found in most organs and tissues and turns on genes in response to an inflammatory stimulus. The researchers were thus able to artificially create an internal scenario in which the hypothalamus was receiving a continuous “inflammation on” or “inflammation off” input without having to provide any external infectious or inflammatory agents. The results were quite striking. Continuous activation of the inflammatory NF-κB pathway in the hypothalamus resulted in a reduction of overall lifespan in the mice, but it also resulted in a loss of muscle mass, bone mass, and cognitive function – the mice showed signs of accelerated aging. An even more remarkable finding was that continuous suppression of the inflammatory pathway extended the lifespan of the mice when compared to their littermates that did not undergo any genetic modifications. Not only did these mice live longer (median lifespan increased by 23%), but they also exhibited significantly less physical and cognitive decline than regular mice!

To investigate the mechanism by which the suppression of inflammatory signals could result in such a profound increase in longevity and functional capacity, the researchers studied Gonadotropin Releasing Hormone (GnRH), one of the major hormones released by the hypothalamus which in turn regulates the release of reproductive hormones. They found that aging or inflammatory activation indeed suppressed GnRH release, whereas inhibition of the inflammatory signaling was able to restore GnRH levels. More importantly, simply injecting the mice with GnRH was able to prevent the physical and cognitive decline in the aging mice. How the injections of GnRH were able to restore muscle mass and even cognitive function was not evaluated in the study, but the researchers did observe that the brain showed increased evidence of neuron growth, which could explain the anti-aging effects of GnRH.

This paper is not the first to link inflammation to aging, but it is the first to show that localized inflammation signals in the hypothalamus can have such a profound effect on the lifespan of mice and it is also the first to propose that suppression of GnRH may be the reason for this inflammation-aging link. As with all important scientific papers, this study raises more questions than it answers. Is GnRH not just a regulator of sex hormones, but does it also exert effects on neurons and muscle cells that are independent of its role as a regulator of reproductive hormones? The mice with prolonged life-spans were all studied in a laboratory setting and thus not exposed to infectious agents that mice (or humans, for that matter) living in the wild commonly encounter. Would suppression of the NF-κB pathway in the hypothalamus possibly compromise their ability to fend off infections or other natural forms of inflammation? It is also not clear whether the GnRH link would apply to all mammals such humans, since aging female primates have higher, (not lower!) GnRH levels. These are all questions that lie beyond the scope of this paper and they need to be addressed in future papers.

However, there are some major limitations of this study and the proposed new hypothalamus-inflammation-GnRH-aging model. First, there is one rather obvious experiment that is missing. The researchers showed that manipulating NF-κB in the hypothalamus can have a major effect on the lifespan and the cognitive as well as physical function, but for some reason the researchers did not show the results from a rather simple experiment: Does GnRH alone extend the lifespan? If GnRH were really the main pathway by which the hypothalamus regulates aging, than giving GnRH ought to have extended the lifespan of the mice.

A second limitation of the paper is that it does not distinguish between general functional decline versus decreased regeneration. Biological aging is characterized by a gradual functional decline over time, but this is due to a combination of at least two parallel processes. Existing cells and tissues accumulate damaged and become dysfunctional and regenerative stem cells or progenitor cells become exhausted and cannot keep up with the repair. This study does not assess whether increased NF-κB activation in the hypothalamus causes more cellular dysfunction, whether it merely inhibits the regenerative repair process or whether it affects both. The researchers did not perform assessments of cellular aging, such as measuring the expression levels of the cellular aging regulator p16 or quantify oxidative stress. Therefore, it is unclear whether NF-κB activation in the hypothalamus had any impact on the cellular aging (senescence) program in the brain, muscles or elsewhere in the body.

Another key limitation is that the hypothalamus has so many functions other than GnRH release, which could all contribute to aging and changes in the lifespan of the mice. The authors themselves have previously published that NF-κB in the hypothalamus regulates the link between obesity and high blood pressure and multiple other groups have already shown that the hypothalamus may affect aging via its role in metabolic regulation. Unfortunately, the current study glosses over the potential role of metabolism and high blood pressure, which could explain the observed longevity effects and instead just focuses on the more provocative but less substantiated idea of GnRH as the aging regulator.

Due to these limitations, we still have to await additional studies that confirm the role of GnRH as the target for NF-κB activation in the hypothalamus and this link between inflammation, aging and the hypothalamus.

We should also remember that biological aging is just one aspect of aging. As André Maurois once wrote, “Old age is far more than white hair, wrinkles, the feeling that it is too late and the game finished, that the stage belongs to the rising generations. The true evil is not the weakening of the body, but the indifference of the soul.


Image credit: A GIF depicting the Hypothalamus “BodyParts3D”, © The Database Center for Life ScienceCreative Commons license via Wikimedia Commons, Painting by John Haberle (1856-1933) – Time and Eternity, via Wikimedia Commons
Zhang G, Li J, Purkayastha S, Tang Y, Zhang H, Yin Y, Li B, Liu G, & Cai D (2013). Hypothalamic programming of systemic ageing involving IKK-β, NF-κB and GnRH. Nature, 497 (7448), 211-216 PMID: 23636330