Control of cytokine production in vivo

Last week I attended a talk by Dr. Markus Mohrs where he introduced the idea of a dual-reporter mouse model that they had developed some years ago to measure cytokine production in vivo. It fascinated me so much that I decided to go through his research a bit and read about their exciting findings, especially because they used parasites such as Heligmosomoides polygyrus, Toxoplasma gondii and Schistosoma mansoni as their infection models.

Th2 immune response is highlighted by IL-4 production. But when looking in vivo, it is impossible to find the source of the IL-4 that is present in the biological fluids. So in their study published in Immunity in 2005, the authors used an intestinal infection by nematode  H. polygyrus  as their model. This worm resides solely in the intestine and induces a robust Th2 response. they engineered a GFP sequence preceded by  an internal ribosome entry site (IRES) after the final exon of the IL-4 gene. In this way, when the IL-4 mRNA gets transcribed, the ribosome can bind separately to the IRES and translate GFP. Therefore, cells that are producing IL-4 mRNA could be identified. What they surprisingly found was that, CD4+ GFP+ T cells were not always producing IL-4 unless secondarily stimulated. So, they replaced the IL-4 allele on the other chromosome with a Human CD2 gene. Presence of huCD2 on cell surface would now be indicative of IL-4 production.

They therefore showed that CD4+ Tcells could be either ”IL-4 competent” or ”IL-4 producing”. They observe a very strong post-transcriptional control and selective secretion of IL-4 . Various types of cells start producing IL-4 mRNA but only select ones in certain areas actually produce the cytokine after receiving the proper secondary signal. This idea was tried on the key Th1 cytokine IFN-gamma in another study using influenza virus and Toxoplasma as infection models and similar results were found. Interestingly, the iconic pro-inflammatory cytokine IL-1beta also takes a similar route, but in this case, both control levels are post-translational: First signal induces pro-IL-1beta production and second signal induces its maturation via the inflammasome complex. These key cytokines have receptors on plenty of cells all through the body and their aberrant release can cause serious problems, no wonder multiple controls levels are set for their release.  Studies of this kind should really alert us on interpretation of microarray or qRT-PCR data.

Mohrs K, Wakil AE, Killeen N, Locksley RM, & Mohrs M (2005). A two-step process for cytokine production revealed by IL-4 dual-reporter mice. Immunity, 23 (4), 419-29 PMID: 16226507
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Parasites to help fight cancer

Posted by Kasra

Research on parasites is important, even if most of them are not direct health concerns to the developed world! Millions of years of coevolution of parasites along with their hosts have made them masters in manipulation of the immune system and in coexistence with it. Many parasitic infections such as those with Toxoplasma, Trypanosoma and some Leishmania sp. elicit innate and adaptive immune responses that can result in life-long immunity to reinfection. However, the parasite might be carried chronically for life, keeping the antibody titers up.

The research team of Ricardo Gazzinelli have taken advantage of the stealth yet stimulatory property of parasites to target cancer. For their purpose, they expressed a cancer antigen in a strongly attenuated strain of Trypanosoma cruzi, the causative agent of Chagas disease in the Americas. This live vaccine showed great protection against melanoma in both prophylactic and therapeutic models.

T. cruzi was chosen as the vector for many reasons: it intrinsically possesses TLR ligands and thus induces a strong proinflammatory response; like many other parasites, it has ways of staying inside the body for a very long time; and it propagates inside the cytoplasm, therefore it can induce a Th1 type immune response and activation of cytotoxic CD8+ T cells, which are very important against cancer. It is interesting to me that although they did not observe any disease or parasitemia, such strong immune response and protection was observed. I think it is important to see if and how many residual parasites are sticking around and where and how long do they stay in the body. This might also teach us more about how this model actually works.

The study shows that if vaccinated before tumor induction, mice are completely protected from cancer. It is also shown that this antigen-carrying vector can delay tumor growth and lethality if given after. One might ask: what advantages does this vector provide over vaccination with traditional recombinant protein, or other vectors such as attenuated viruses? The authors compared the protection and immune response resulted from recombinant T. cruzi to the canonical recombinant antigen (NY-ESO1) with Alum or CpG adjuvants. Recombinant T. cruzi showed better protection and stronger immune response compared to canonical vaccination strategies. Doing a quick search through pubmed, I didn’t find other strategies such live or viral vectors rather than variations of recombinant protein being used so far. I might be wrong, but still this is a novel idea and this recombinant parasite deserves a chance of being further studied, especially for prophylactic uses. There are plenty of other cancer or maybe even infectious antigens that can be targeted by this method. But of course, there are plenty of biohazard issues that also need to be addressed with injecting an attenuated form a dangerous pathogenic parasite.

Here is the link to the article abstract on Pubmed.

Junqueira C, Santos LI, Galvão-Filho B, Teixeira SM, Rodrigues FG, Darocha WD, Chiari E, Jungbluth AA, Ritter G, Gnjatic S, Old LJ, & Gazzinelli RT (2011). Trypanosoma cruzi as an effective cancer antigen delivery vector. Proceedings of the National Academy of Sciences of the United States of America, 108 (49), 19695-700 PMID: 22114198

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In silico prediction of the human-malaria parasite interactome

Posted by Kasra

In silico prediction of protein-protein interactions within a species is an advancing field. Especially now that relatively large amounts of empirical data are available for training and validation, more and more in silico methods are being presented. However, as a host-parasite interactions enthusiast, I always had the question if the interactions between the host and pathogen proteins can be predicted. Although already done a few times for viruses such as HSV, HCV, HIV and Influenza, creation of an empirical inter-species interactome is not an easy and always affordable task. Still having an interactome database provides very valuable data in host-pathogen research. It not only reveals systemic overviews about the nature of the interaction occuring, but it can also open doors for more accessible and feasible research by suggesting a shorter list of proteins, pathways or interactions to focus on.

With this in mind, I had great pleasure in reading this single author paper by Stefan Wuchty published recently in PloS ONE that provides a computational interactome of Plasmodium falciparum and Homo sapiens. This paper actually led me to a whole body of research that has been done on experimental and computational determination of host-pathogen interactomes, albeit mostly on viruses.

In order to map P. falciparumH. sapiens interactome, Wuchty used experimentally determined host-parasite interactions plus orthologous protein groups between the two species as starting point. The false-positives were removed with various filtering methods that are beyond the reach of my knowledge of mathematics and informatics. Biological criteria such as co-expression of the interacting proteins in the host cell and specific parasite phase were also required.  Interstingly, the pattern of interactions he found, showed similarity with what was already observed with viral pathogens. In order to take control of the cell, intracellular pathogens appear to attack the host both at the protein as well as the pathway level. Hub proteins – proteins that interact with many other proteins and are envolved in multiple metabolic/siganling pathways – have been found to be an attack target not only by P. falciparum but also other pathogens. In addition, Wutchy saw that a relatively small number of human proteins interact with a big number of parasite proteins, suggesting that the parasite utilizes all it has got to take over the key proteins of the host.

This study and other works of the same style certainly provide precious knowledge about host-parasite interactions both in terms of systems biology and also hints for hands on research. I look forward to seeing these interactomes created and expanded for other pathogens and also their experimental validation and usage. Furthermore, an online database of these host-parasite interactomes would definitely make them more accessible.

 

Wuchty S (2011). Computational Prediction of Host-Parasite Protein Interactions between P. falciparum and H. sapiens. PloS one, 6 (11) PMID: 22114664

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