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

The Manipulator and the Opportunist: Leishmania and HIV infection of monocytes

Posted by: Maryam Ehteshami and Kasra Hassani

It has been documented that HIV infection can render leishmaniasis harsher and reduce the chances of treatment response. On the other hand, Leishmania infection also accelerates HIV infection and disease progression. In this blog post, we summarize a recent article published in PLoS Pathogens, that explores the mechanism through which Leishmania can help HIV replication. As it turns out, human macrophages are a key part of the equation.

It is no secret that both HIV and Leishmania can infect macrophages. So when Mock et al. wanted to examine the relationship between these two microorganisms, macrophages were the first place they looked.

Macrophages are non-dividing cells with a low nucleotide pool. Nucleotide synthesis is regulated at the S phase, in other words, cell activation. So in resting macrophages the nucleotide levels are very low. Additionally, It was previously thought that human monocytes do not further proliferate once they leave the bone marrow. Recent studies however, have shown that monocytes may be far more heterogeneous than previously thought, and that a subset of them can go on to enter the cell cycle in response to certain stimulations. For example, cells stimulated with GM-CSF (Granulocyte-monocyte colony-stimulating factor) were shown to go on to proliferate.

Interestingly, Mock et al. showed that Leishmania infection can promote monocyte viability and proliferation similar to GM-CSF. It has long been seen that intracellular protozoan parasites such as Leishmania, Trypanosoma and Toxoplasma can inhibit apoptosis of their host cell and thereby increase their lifespan (Heussler et al. 2001). Mock et al. take this to the next step showing that Leishmania­ might further induce proliferation of the infected monocytes. This could help spreading of the parasitic infection, and indeed Mock et al. show that the cells remain infected after proliferation (Figure 1, PKH dye shows that monocytes are infected with Leishmania). However, if this is good or bad for Leishmania, still needs to be determined, especially that an activated macrophage could be able to kill internalized parasites.

Another theory as to why Leishmania promotes cell cycle progression in macrophages is the following: Leishmania parasites lack the machinery necessary for synthesizing purine nucleotides. Resting cells have low levels of nucleotides. Therefore, by activating the cell cycle, Leishmania ensures that sufficient levels of nucleotides become available for its replication. In fact Mock et al. examined the effect of macrophage infection by Leishmania on expression levels of ribonucleotide reductase (RNR) enzyme. This enzyme is responsible for converting ribonucleotides to nucleotides. Western blotting revealed that RNR levels increased in the presence of Leishmania infection, similar to those elevated levels seen in the presence of GM-CSF. This elevation also corresponded with nucleotide level increases. Overall, this presents one theory for why Leishmania would want to induce macrophage stimulation.

As mentioned earlier, macrophages are also a target for HIV infection. But HIV replicates at a very high rate. And high rate of replication requires high levels of intracellular nucleotides. In fact, the Kim group have previously shown that HIV reverse transcription is severely reduced under conditions which mimic macrophage intracellular levels of nucleotides and that HIV replication is lower in macrophages as compared to T cells (Kennedy et al. 2010) .  Based on their observations with Leishmania and HIV, they showed that HIV replication in macrophages may increase when the cells are co-infected with the parasite (Figure 1, Green GFP-HIV proliferation only occurs in GM-CSF-treated or Leishmania-infected monocytes). They also showed that this is the result of Leishmania-induced cell proliferation and increased nucleotide levels (figure not shown). In other words, Leishmania manipulates the macrophage to create a friendlier environment for its own survival and HIV ceases this opportunity and uses these changes in the macrophage for its own gain.

Figure 1. Monocytes were transduced with a GFP-HIV vector. Increased fluorescence signifies increased viral replication. PKH indicates presence of Leishmania. (From Mock et al. 2012, PLoS Pathogens)

It is likely that there are many different mechanisms involved in Leishmania/HIV co-infection that were not discussed here. Almost certainly many of them involve immune modulation. Here, Mock et al. have shed light on a unique biochemical mechanism for the observed increased infection by either microorganism. As suggested by the authors, it would be interesting to examine other macrophage-infecting microorganisms such as Mycobacterium tuberculosis in this context.

This topic raises many exciting questions. Most fundamentally, what is the underlying mechanism for induction of proliferation in Leishmania-infected monocytes? And what is the implication of monocyte proliferation to spread or control of Leishmania infection? But also, what happens to HIV in macrophages in the absence of a parasitic infection? These could perhaps be the topics of next research projects and next blog posts.


Mock DJ, Hollenbaugh JA, Daddacha W, Overstreet MG, Lazarski CA, Fowell DJ, & Kim B (2012). Leishmania induces survival, proliferation and elevated cellular dNTP levels in human monocytes promoting acceleration of HIV co-infection. PLoS pathogens, 8 (4) PMID: 22496656

Kennedy EM, Gavegnano C, Nguyen L, Slater R, Lucas A, Fromentin E, Schinazi RF, & Kim B (2010). Ribonucleoside triphosphates as substrate of human immunodeficiency virus type 1 reverse transcriptase in human macrophages. The Journal of biological chemistry, 285 (50), 39380-91 PMID: 20924117

Heussler VT, Küenzi P, & Rottenberg S (2001). Inhibition of apoptosis by intracellular protozoan parasites. International journal for parasitology, 31 (11), 1166-76 PMID: 11563357

Wanted Dead (But also Alive): The Hepatitis C Virus Polymerase

Posted by: Maryam Ehteshami

I want to dedicate today’s blog post to a little virus known as Hepatitis C virus. Having worked on HIV before, and now moving onto work with HCV, I am tempted to compare the history of these two viruses and I find a lot of parallels. Both viruses were discovered in the 80s (1983 and 1989, for HIV and HCV, respectively), they can both have a long incubation period, and they both infect a whole lot of people worldwide. Of course there are many differences as well. For one, HIV comes with a 100% mortality rate. HCV doesn’t get integrated into the human genome and about 25% of people infected can clear the virus on their own. The remaining 75% of the HCV-infected individuals remain at the mercy of the current anti-HCV treatment (20-80 % of them can expect to be cured, depending on the infecting HCV genotype). It is in fact the available treatment options that most set HIV and HCV apart.  Today, an HIV-positive person living in the first world can chose from an array of over 25 antiretroviral compounds. The development of these drugs has been the fruit of over 20 years of aggressive research, supported by grass-root activism and heavy funding from both government and industry. It has also been aided along the way with the development of appropriate animal models, cell-culture systems, and in vitro enzymatic assays that corresponded well with patient outcomes. In contrast, anti-HCV antiviral development has been very slow. For one, cell-culture growth of the virus (or the related replicon model) was not possible as recently as the mid-2000s. Developing in vitro systems for the study of HCV proteins have not always been straight forward either. Despite these many hurdles, anti-HCV drug development researchers have not given up. In fact, we are at the beginning of an exciting era. Last year saw the FDA approval of two new protease inhibitors for HCV. Gilead recently made an 11 billion dollar purchase of a small pharmaceutical company called Pharmasset which possessed an anti-HCV compound showing 100% cure rate in recent phase 2 clinical trials. Many different pharmaceuticals are also in possession of a number of very promising leads and are competing for the eventual market. Putting all recent finding together, one is tempted to speculate that this decade is the beginning of the end for hepatitis C.

In this blog post, I would like to focus on one of the major antiviral targets, namely NS5B, the viral RNA-dependent RNA-polymerase, which is essential for replication viral genomic RNA.  I would like to focus on some of the issues that the field has faced when it comes to assessing NS5B activity in vitro and some new findings that will impact anti-NS5B drug development.

As the viral polymerase, the NS5B protein is a major target for therapy. Both nucleoside analogues and non-nucleoside analogues are in development for inhibiting this protein (namely, the 11 billion dollar investment by Gilead was for a nucleoside analogue called GS-7977). But working with the NS5B enzyme has proven to be a challenge and may in part account for some of the delay that the field has seen in drug development. Almost all publications to date (dating back to mid 1990s) report an in vitro activity of below 1% to 5% for this enzyme.  This low activity is what makes working with this enzyme so difficult and this is why I was very excited to read a February 2012 publication in the Journal of Biological Chemistry (Jin et al. 2012) that claimed that with some minor protocol changes, the in vitro activity of the enzyme can be increased to 65%!!

But lets take a step back for a moment and examine some of the rationales that were previously proposed to explain this low level of activity. There are almost 100 crystal structures available for NS5B and a recent April 2012 publication in Journal of Virology even shows the protein bound to the RNA substrate (Mosley et al 2012). A close inspection of these structures revealed an intriguing property for this enzyme. It appears that near the C-terminus of NS5B, there exists a beta-hairpin loop that folds right into the active site (see Figure 1, the loop is highlighted in red). It is suggested that the presence of this loop in the active site, prevents the double stranded RNA/RNA substrate from binding, and if no RNA is bound, no activity can be had! Some have gone on to call this loop the “auto-inhibitory” beta-hairpin loop. This was supported by the recent crystal structure, where the beta-hairpin loop was taken out and replaced by two glycine residues and lo and behold! A 100-fold increase in activity was observed! The absence of the loop also allowed for the co-crystalization of NS5B with its RNA substrate (Figure 2).

Figure 1.Crystal structure of NS5B genotype 1b. The enzyme is shown in pink. The beta-hairpin loop is highlighted in red (Bressanelli et al 1999).

Figure 2. Crystal structure of NS5B Genotype 2a. The enzyme is shown in pink. The glycine residues replacing the beta-hairpin loop are shown in red. The co-crystalized RNA/RNA substrate is shown bound to the active site.

Of course, all of this begs the question why would a naturally occurring part of the enzyme, prevent the very activity that it is supposed to do? No one really knows the answer yet, but it is speculated that this loop serves some sort of a regulatory function, especially relevant in vivo where other host and viral proteins are present and impact viral replication.  The issue is that we really have very little information on how NS5B interacts with itself and other proteins in the context of replication. But I digress!

Of course synthetically taking out a structural loop of the enzyme can be an immediate solution to the problem of low enzyme activity. But this also raises questions about the in vivo relevance of such a model. And this is what makes the Jin et al 2012 publication exciting. Their findings indirectly corroborate previous findings arguing that the rate-limiting step in in vitro activity is the slow binding of the enzyme to the RNA substrate. Based on this, they show that it is not necessary to further modify the enzyme, but rather just give the enzyme more time to achieve what it needs to achieve. They show that 24 hour pre-incubation of the enzyme, with the RNA substrate, with the addition of a few starting nucleotides, gives the enzyme enough time to form active elongation complexes. Under these modified conditions, they found that 65% of the enzyme was capable of forming elongation complexes, which in turn could incorporate nucleotides as good as the next polymerase!

If the findings in this publication are repeated and confirmed, I am sure that many NS5B enzymologists will take a breath of relief. With the projection that the GS-7977 compound will be the next compound to get FDA approval, we are looking at many years of nucleoside analogue therapy for individuals infected with HCV. As treatment usage increases, we expect to see more drug resistance, which will bring us back to the drawing board for the development of next-generation nucleoside analogues. Therefore, any knowledge we gain now about the functions of NS5B in vitro will surely help us better prepare for the future. As the development of GS-7977 will likely represent a turning point in HCV therapy history, I am hopeful that the two publications highlighted here will come to represent a turning point for in vitro studies of the HCV polymerase.

Jin Z, Leveque V, Ma H, Johnson KA, & Klumpp K (2012). Assembly, purification, and pre-steady-state kinetic analysis of active RNA-dependent RNA polymerase elongation complex. The Journal of biological chemistry, 287 (13), 10674-83 PMID: 22303022
Mosley RT, Edwards TE, Murakami E, Lam AM, Grice RL, Du J, Sofia MJ, Furman PA, & Otto MJ (2012). Structure of HCV Polymerase in Complex with Primer-Template RNA. Journal of virology PMID: 22496223