Immunology meets epidemiology: A closer look at super spreaders

Posted by Kasra

Recent work by Gopinath et al. published in PLoS Pathogens touches a crucial issue in epidemiology of infectious diseases. We tend to look at infectious diseases as infecting everyone more or less uniformly. But many studies have shown that a great extent of heterogeneity exists in the amount of pathogen that is spread from infected individuals. The common rule is a 80-20 rule: it says that 20% of infected individuals account for 80% of disease spread. To learn more about this phenomenon and the research behind it, you can have a look at this comment published in Nature news and views. This rule appears to stand for many infectious diseases capable of causing epidemics. Therefore, studying and identifying the super spreaders –  or as this study puts them super shedders –  is of critical importance.  This study has tried to shed some light on the differences of the immune system of these individuals compared to normal shedders.

The authors use a special model of Salmonella enterica infection of mice where longterm infection was sustainable without the mice succumbing to disease. They then compared mice that very high numbers of bacteria present in their stool (two logs higher than what is normally expected of moderate shedders) with normally infected mice.

Comparing the immune response between moderate and supper shedders, the authors find that although the bacterial load in the gastrointestinal tract is highly different between the two groups of mice, the amount of bacteria in systemic organs such as the spleen is the same, so is the overall appearance of the mice. However, they find an enhanced innate immune response in the supper shedders marked with higher IL-6, a pro-inflammatory cytokine, in the serum and more neutrophils in the blood and other organs. On the other hand, they observe a reduction in the ratio of Th1 lymphocytes to regulatory T cells. The authors also found Th1 cells to be less responsive to proliferation-inducing cytokine IL-2.

Interestingly, the authors were able to induce some of the super shedder ‘characteristics’ by giving streptomycin to moderate shedder mice, probably giving room for Salmonella to expand in the altered microbiota. This further suggests that the observed immune characteristics are due to the bacterial load present in the gut. Finally, they look at the possible connection between neutrophil expansion and reduction in Th1 responsiveness.

Immunological differences between super shedder and moderate shedder Salmonella infected mice

The authors argue that to the be able to contain such a high bacterial load in the body, the organism would either have a weaker inflammatory response, or a way to dampen adaptive response to reduce the cost of high inflammation to the body. This is among the first steps in understanding the nature of the immune response in super spreaders. It would be interesting to see if this is a general theme in all host-pathogen interactions when super spreading occurs, or if it is different with every pathogen. This kind of research can importantly lead to better understanding of the immune response and hopefully also molecular markers that would help rapid identification the super spreaders and better control of disease outbreaks.

Gopinath S, Hotson A, Johns J, Nolan G, & Monack D (2013). The Systemic Immune State of Super-shedder Mice Is Characterized by a Unique Neutrophil-dependent Blunting of TH1 Responses. PLoS pathogens, 9 (6) PMID: 23754944

Galvani AP, & May RM (2005). Epidemiology: dimensions of superspreading. Nature, 438 (7066), 293-5 PMID: 16292292

Macrophages commit ‘defensive suicide’ after Adenovirus and Listeria infection

Posted by Kasra

Cells often kill themselves for the benefit of their lot. New forms of cell suicide are being discovered every day now.  I wrote about apoptosis, which is a rather clean form of cell suicide recently. However, necrosis which until recently seemed to be a an uncontrolled form of cell death, is now being looked at again as a form of controlled suicide. A recent publication by  Di Paolo et al in the new journal of Cell Reports sheds some light on on of these rather unusual forms of cell death. The authors call it ‘defensive suicide’.

Di Paolo et al. intravenously injected Adenovirus into the mice. They observed that the macrophages (specifically in this paper, liver macrophages) capture the virus particles. However, shortly after the macrophages died of necrosis. Interestingly, they find this phenomenon to be independent from normal mediators of cell death such as various Caspases, as well as inflammatory mediators such as MyD88, TRIF and ASC. They finally point to IRF3,  a transcription factor normally activated after certain infections. Macrophages from IRF3-/- mice did not go through necrotic death after Adenovirus infection. The authors next show the proteins upstream of IRF3 are dispensable for the necrotic death of macrophages and that IRF3 is not phosphorylated at the time of macrophage necrosis, further adding to the enigma of the mechanism. The only clue we get so far is that this mechanism is dependent on escape the of the pathogen from the phagolysosome into the cytosol. They show this by using Adenovirus and also Listeria monocytogenes  mutants that cannot escape the phagolysosome. Compared to their wildtype counterparts, the mutant intracellular pathogens do not induce necrotic death of the macrophages.

Finally, to see if this necrotic death actually has a benefit for the host, the authors deplete mice from macrophages and infect them again with Adenovirus or L. monocytogenes. They observe that without the macrophages the virus or bacterial burden is a lot higher in the liver. Thus, this mechanism could be a way of slowing down the systemic spread of infection. The macrophages might collect the pathogens that would be otherwise infecting other defenseless cells and destroy them via necrotic death. Would this mean that necrotic death better kills the intracellular pathogens compared to other forms of programmed death? Or they just go through this pathway because other pathways of programmed death are blocked by the pathogens? Considering that necrosis occurs very rapidly (within minutes), the first one seems more likely.

The possible role of IRF3 in induction of necrotic death in macrophage following intracellular infection. From Di Paolo et al. , Cell Reports, Volume 3, Issue 6, 1840-1846, 13 June 2013

The possible role of IRF3 in induction of necrotic death in macrophage following intracellular infection. From Di Paolo et al. , Cell Reports, Volume 3, Issue 6, 1840-1846, 13 June 2013

This mass suicide of macrophages is a very interesting phenomenon. It also raises many questions that have not yet been addressed. The most obvious question is the signaling mechanism by which IRF3 induces this special form of necrosis. The authors did not find any dependence on the proteins that are usually known to be upstream of IRF3. So there might be a novel mechanism involved. Another question concerns the route of infection. The authors have used intravenous injection both for Adenovirus as well as L. monocytogenes infection. However, these pathogens usually enter the body from the gut or the lungs and then reach the circulation system. Would this defensive necrosis extend to the immune cells in other tissues such the lung or the gut macrophages? Would the route of infection affect the intensity/quality of macrophage necrosis? We will hopefully get the answers in the near future!

Di Paolo NC, Doronin K, Baldwin LK, Papayannopoulou T, & Shayakhmetov DM (2013). The Transcription Factor IRF3 Triggers “Defensive Suicide” Necrosis in Response to Viral and Bacterial Pathogens. Cell reports, 3 (6), 1840-6 PMID: 23770239

Bacteriophages may protect us against pathogens

Posted by Kasra

Given the extremely large amount of bacteria in our gastrointestinal track, it is not surprising to think that the gut would be also swarming with pathogens of bacteria, that is bacteriophages as well. In their recent work published in PNAS, Barr et al. take a look at what impact these particles could have on the population of bacteria in mucosal surfaces and what could it mean for us. Their work actually turns out very interesting results.

Mucosal surfaces are the body’s points of contact  with the outside. Being highly populated with bacteria, they can be suitable points of infection as well. That is why they are heavily guarded with various immune barriers and mechanisms, both innate and adaptive. Barr et al. point to a possible mechanism of protection against infection which not innate nor adaptive. They start by comparing the amounts of bacteria and bacteriophages in different mucosal and non-mucosal surfaces in various mucus producing animals. They interestingly observe that the bacteriophage to bacteria ratio in mucosal sites is way larger than those in adjacent non-mucosal sites (from average about 40fold to average about 3fold). They verify this in both invertebrates and vertebrates and thus suggest that this could be a phenomenon in all mucus-producing metazoans.

Next, they point to a previous recent study by Minot et al. who had found immunoglobulin (Ig)-like domains in the total analyzed genome of human gut viruses (or so called human gut virome).  These domains that usually act as in recognition and binding (as an antibody would do); they show that the bacteriophages actually bind to mucus through these proteins.  Barr et al. also show that presence of bacteriophages on a mucosal surface significantly reduces Escherichia coli invasion in vitro.


Model for how presence of bacteriophage on the mucosal surface can help in protection against bacterial infection. From Barr et al. PNAS 2013 PMID: 23690590

This is an incredible system where the benefit of the bacteriophages and their hosts actually match. It is not clear at this point whether the animal body would have to do something other than producing mucus to keep the bacteriophages where they are or that it is just enjoying this protection more or less free of charge.

Barr JJ, Auro R, Furlan M, Whiteson KL, Erb ML, Pogliano J, Stotland A, Wolkowicz R, Cutting AS, Doran KS, Salamon P, Youle M, & Rohwer F (2013). Bacteriophage adhering to mucus provide a non-host-derived immunity. Proceedings of the National Academy of Sciences of the United States of America PMID: 23690590

Minot S, Grunberg S, Wu GD, Lewis JD, & Bushman FD (2012). Hypervariable loci in the human gut virome. Proceedings of the National Academy of Sciences of the United States of America, 109 (10), 3962-6 PMID: 22355105

Cryptosporidium, the understudied killer

Diarrhea is the second major killer of children under the age of 5 in developing countries (second to pneumonia). We know much less than we should about the causative agents, severity, burden etc. of diarrhea in developing countries. Funded by Bill and Melinda Gates Foundation, A Global Enteric Multicenter Study (GEMS) picked up the task of learning more about diarrhea in children in developing countries with high incidence (Gambia, Mali, Mozambique, Kenya, India, Bangladesh and Pakistan)   and did an extensive 3 year-long case-control study. I won’t go to details about their magnificent work but just mention one rather surprising finding. They found the 4 top pathogens causing diarrhea in children under 5 to be: 1. Rotavirus 2. Cryptosporidium 3. Shigella 4. Enterotoxigenic Escherichia coli producing heat-stable toxin (ST-ETEC). Rotavirus has long been known as a major cause of diarrhea in children and there are effective vaccines against it who have significantly reduced its incidence in developed countries. Shigella and ST-ETEC were also previously known. But the high incidence of Cryptosporidium has come out as a surprise to everyone. Crypto is an apicomplexan protozoan parasite, kin to other famous parasites Plasmodium and Toxoplasma. It’s durable cysts are shed in the stool and can be ingested in contaminated food and water. Compared to the other top pathogens and with regards to the high mortality it is causing, Crypto is relatively unpopular and extremely understudied (even reflected in its Wikipedia page). Now GEMS calls for more research on Crypto and better therapies against it. Hopefully this would mean more funding for studying this bizarre parasite and more exciting knowledge learnt from its biology and pathophysiology. 

Find and share GEMS infographic about their findings from here.

Cryptosporidium trophozoite bound to the small intestine epithelium, inducing actin accumulation at its binding site

Cryptosporidium trophozoite bound to the small intestine epithelium, inducing actin accumulation at its binding site. From Elliott and Clarck, Infection and Immunity, 2000

Kotloff, K., Nataro, J., Blackwelder, W., Nasrin, D., Farag, T., Panchalingam, S., Wu, Y., Sow, S., Sur, D., Breiman, R., Faruque, A., Zaidi, A., Saha, D., Alonso, P., Tamboura, B., Sanogo, D., Onwuchekwa, U., Manna, B., Ramamurthy, T., Kanungo, S., Ochieng, J., Omore, R., Oundo, J., Hossain, A., Das, S., Ahmed, S., Qureshi, S., Quadri, F., Adegbola, R., Antonio, M., Hossain, M., Akinsola, A., Mandomando, I., Nhampossa, T., Acácio, S., Biswas, K., O’Reilly, C., Mintz, E., Berkeley, L., Muhsen, K., Sommerfelt, H., Robins-Browne, R., & Levine, M. (2013). Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study The Lancet DOI: 10.1016/S0140-6736(13)60844-2

Elliott, D., & Clark, D. (2000). Cryptosporidium parvum Induces Host Cell Actin Accumulation at the Host-Parasite Interface Infection and Immunity, 68 (4), 2315-2322 DOI: 10.1128/IAI.68.4.2315-2322.2000

A systematic review in non-clinical research: a case of pathogen metabolites

Posted by Kasra

Doctors and scientists in the field of clinical research are well acquainted to systematic reviews and their importance in clinical research. The important difference between a normal review and a systematic review is that in the latter the authors make sure (or at least try very hard) to include and cover all the published research about the topic of review. Along with the review of the data, they should also publish the search strategy they used to make sure they get everything that has been published about their topic of study. Collecting all the data is extremely important especially when deciding about the beneficial effects of a certain drug, vaccine or public health intervention.  The Cochrane collaboration is a well-known organisation that collects and publishes systematic reviews in field of health research and health care.

Although they could be very useful in non-clinical research, systematic reviews are actually rarely written in these fields. During my graduate studies, I had to write a systematic review on innate receptors for a certain fungus. I realized then how diverse the experimental models are and how hard it is compare their controversial results due to small or big differences in experimental setup and strains used. Maybe that is why these papers are rare in non-clinical research. Still, no matter how hard, I was able to do it with as much time as a graduate student would put on a term paper and get a good grade for it ;). I am looking forward to reading more non-clinical systematic reviews.

Recent work of Bos et al. is an excellent example of how useful it could be to gather all the available data in a certain field, even if it is not all clinical trials. They point to most common abundant bacteria in sepsis Staphylococcus aureus (SA), Streptococcus pneumoniae (SP), Enterococcus faecalis (EF), Pseudomonas aeruginosa (PA), Klebsiella pneumoniae (KP), and Escherichia coli (EC). They argue that current strain detection methods are too slow and do not allow for efficient targeted antibiotic therapy. On the other hand, non-targeted therapy is not always successful. They argue that the unique and some-what well-identified metabolic pathways of these bacteria leads to production of certain volatile chemicals that are not produced by humans and could be used as rapid diagnostic markers. The diagram below shows the gram positive bacteria on the left and gram negative bacteria on the right, graphing unique and common volatile chemicals they produce. The blue circle in the center shows the chemicals produced by all bacteria. Therefore, their absence would exclude infection. The red (or pink as you may) circles highlight the unique products of each species which could help in targeted antibiotic therapy of sepsis.

Staphylococcus aureus (SA), Streptococcus pneumoniae (SP), Enterococcus faecalis (EF), Pseudomonas aeruginosa (PA), Klebsiella pneumoniae (KP), and Escherichia coli (EC)

Bos, L., Sterk, P., & Schultz, M. (2013). Volatile Metabolites of Pathogens: A Systematic Review PLoS Pathogens, 9 (5) DOI: 10.1371/journal.ppat.1003311