Legionella pneumophila (green), the bacterium responsible for severe acute lung disease inside eukaryotic cells. Mitochondrial network in red, nucleus in blue. © Institut Pasteur.

Scientists at the Institut Pasteur, CNRS and Inserm, together with a team from Switzerland*, have shown that the bacterial pathogen Legionella pneumophila (the causative agent of Legionnaires’ disease or legionellosis) has developed a specific strategy to target the host cell mitochondria, the organelles in charge of cellular bioenergetics. By changing the shape of these host organelles, L. pneumophila impairs mitochondrial respiration leading to metabolic changes in the host cell that are instrumental for the pathogens replication in human cells. This work provides precious information on how a pathogen manipulates the cellular metabolism to replicate intracellularly, and proposes a new concept of protection of host cells from Legionella-induced mitochondrial changes in order to fight infection. This research is published online on August 31, at the Cell Host & Microbe website.

Intracellular pathogens adopt various strategies to circumvent the defences of the host cell and to proliferate intracellularly. One specific strategy is to target host organelles like the mitochondria. Mitochondria are well-defined cytoplasmic organelles, which take part in a variety of cellular metabolic functions. Mitochondria are important as they supply the energy to the cell, thus they are also referred to as the ‘power house’ of the cell. Some bacteria, including Legionella pneumophila, are able to alter mitochondrial functions to the pathogens advantage.

L. pneumophila is a bacterial pathogen that causes Legionellosis – a disease characterized by an acute pulmonary infection, which is often fatal when not treated promptly. In France, between 1200 and 1500 cases are identified each year, with mortality rates ranging from 5 to 15%.

Researchers from the Institut Pasteur, CNRS and Inserm, in collaboration with a team from Switzerland, have discovered a previously unknown mechanism by which L. pneumophila targets mitochondria to modulate mitochondrial dynamics and thereby impairs mitochondrial respiration which in turn leads to a change in the cellular metabolism. These metabolic changes in the host cell favour bacterial replication. Thus the rewiring of cellular bioenergetics to create a replication permissive niche in host cells is a core virulence strategy of L. pneumophila.

The researchers have identified the following mechanism: L. pneumophila establishes transient, highly dynamic contacts with host mitochondria and secretes an enzyme called MitF that modifies the shape of the mitochondria by inducing DNM1L (a host protein that is necessary for fragmenting mitochondria) depended mitochondrial fragmentation. Surprisingly, L. pneumophila induced mitochondrial fragmentation is independent of cell death and ultimately impairs mitochondrial respiration, whereas cellular glycolysis is increased. Thus the bacterial-induced changes in mitochondrial dynamics promote a Warburg-like phenotype (which is characteristic of cancerous cells) in the infected cell that favours bacterial replication.

Researchers also brought the proof of concept that protecting host cells from Legionella-induced mitochondrial changes may help to fight infection. Indeed, pre-treating of human cells with a compound that inhibits changes in mitochondrial morphology allows protecting the host cell from Legionella-induced changes of mitochondria and restricts bacterial infection of human cells.

As Carmen Buchrieser, head of the Biology of the intracellular bacteria research unit at the Institut Pasteur and researcher at CNRS, explains: “this is an important discovery as our results showcase a key strategy used by L. pneumophilia for intracellular replication. By targeting mitochondria, the bacterium ensures that the host cell will be permissive to its replication. It is therefore essential that researchers also focus their studies on metabolic changes caused by pathogenic bacteria, in order to develop new therapeutic strategies against legionellosis and other diseases linked to intracellular bacteria.”

This work sheds new light on how a pathogen shapes host metabolic responses during infection of human cells and shows that metabolic changes in the host cell are instrumental for the pathogens replication in human cells and thus to cause disease. It also proposes a new concept, which is to treat infections by inhibiting pathogen induced metabolic changes.

* The researchers and scientific teams involved are: Carmen Buchrieser, head of the Biology of the intracellular bacteria unit at the Institut Pasteur and associated to CNRS, Hubert Hilbi, of the University of Zürich Switzerland, Priscille Brodin, at the Institut Pasteur Lille and associated to CNRS and INSERM, and Jean-Christophe Olivo-Marin, head of the Bioimage analyses unit at the Institut Pasteur.

©Fotolia

Teams from the virology, hematology-bone marrow transplant and biostatistics departments of the Saint-Louis Hospital, AP-HP, Université Paris Diderot and INSERM, in collaboration with scientists from the University of California-San Francisco (USA) discovered that a group of viruses in the intestine may trigger the onset of graft-versus-host disease. Researchers demonstrated the role of this intestinal “virome” (the population of viruses found in the intestine) in the onset of graft-versus-host disease by analyzing the intestinal virome of 44 patients receiving a bone marrow transplant.

These results appeared in a letter published in Nature Medicine.

Graft-versus-host disease (GVHD) affects up to 60% of patients who receive a stem cell graft (bone marrow transplant) with a high risk of death. Even though past studies have identified biomarkers associated with the severity of the disease, none of them have been used in practice to date in order to predict the onset of graft-versus-host disease.

The researchers carried out a longitudinal study analyzing the intestinal virome of 44 patients before a bone marrow transplant and six weeks after the transplant. By using high-throughput metagenomic techniques, they sequenced the DNA and RNA in the stool samples, in order to analyze and compare the flux over time in bacterial and viral populations.

The results show that GVHD is associated with significant changes in bacteria, bacteriophage (i.e. the viruses that infect bacteria) and virus populations in the stool.

In patients with intestinal GVHD, the persistent DNA viruses (Anelloviridae, Herpesviridae and Polyomaviridae) are absent from the intestinal tract in the first 2-3 weeks following the graft and then they increase in a significant manner. However, the presence of these viruses is stable as soon as the graft is performed in patients without GVHD.

In particular, the researchers detected the presence of a group of RNA viruses, picobirnavirus (PBV), in more than a third of patients after transplanting stem cells. As opposed to other viruses, the presence of PBV predicts the development of GVHD and is highly correlated to the severity biomarkers of intestinal graft-versus-host disease.

Metagenomic tools have made it possible to identify PBVs that have been little studied to date, since their extreme variability makes developing a test to detect them difficult. These viruses have been described in cases of intestinal gastroenteritis, notably in patients with AIDS, but their pathogenic role has not yet been determined. The potential utility of PBV as a predictive marker of GVHD has rekindled the interest in developing adapted to tools for clinical practice.

These results improve the state of knowledge of this complex disease, notably in terms of the role of viral infections in digestive inflammatory diseases and has opened the door to new therapeutic opportunities.

Cross section of newborn mouse’s testis (Ø = 20 µm), where we can see the seminiferous tubules (red) surrounded by macrophages (green).  Confocal micrograph.

© Noushine Mossadegh-Keller and Sébastien Mailfert / CIML

The origin, development, and characteristics of two types of testicular macrophage have been described by a CNRS team at the Centre d’Immunologie de Marseille-Luminy (CNRS / INSERM / Aix-Marseille University). To elucidate the nature of these immune cells, the researchers used a novel cell tracing method. Their findings were published on August 7, 2017, in the Journal of Experimental Medicine, and are of fundamental importance. They may help understand certain kinds of infertility in men and find new treatments for them.

From the start of life, an individual’s immune system learns to distinguish self—that is, native cells—from other, potentially pathogenic cells. But in males, as sperm only appear at puberty, they may be mistaken for foreign cells by certain elements of the immune system.  Testicular macrophages are special immune cells that rush to the defense of sperm. By releasing specific molecules, these guardians of fertility prevent other immune system agents from entering the testes.

Macrophages not only migrate to sites of infection and phagocytose pathogens, but also modulate immune system activity to ensure proper organ function and regeneration. They may arise from either embryonic progenitors or bone marrow cells in adults. Research with mice has enabled the team of Michael Sieweke from the Centre d’Immunologie de Marseille-Luminy (CNRS / INSERM / Aix-Marseille University) to describe both testicular macrophage populations in depth.

The testis is divided into two compartments. One kind of testicular macrophage is found in the interstitial spaces, where testosterone-producing Leydig cells are also located. These interstitial macrophages are of embryonic origin: they are present from the beginning of the individual’s life. The other kind is peritubular—that is, located on the surface of the seminiferous tubules that house sperm cell precursors. Each macrophage population has distinctive cellular markers.

The researchers used a new cell tracing method to follow the movement of peritubular macrophages from the bone marrow to the testes. They discovered that these macrophages only appear two weeks after the mice are born, which corresponds to the pubescent stage in human males. Surprisingly, once they have been established in the testes, macrophages of both populations remain there for the rest of their long lives. Sieweke’s team will next focus their research efforts on the relationships between macrophages, sperm, and testosterone production, which may yield innovative treatments for certain kinds of male infertility.

Bacteria (in green) assaulted and killed by bacteriophages (in purple). Electron microscopy image provided courtesy of M. Rohde and C. Rohde (Helmholtz Centre for Infection Research, Braunschweig/Leibniz Institute DSMZ, Braunschweig, Germany) and colorized by Dwayne Roach (Institut Pasteur).
© M. Rohde and C. Rohde

Phage therapy involves the use of bacteriophages, or phages, for treating bacterial infections. Phages are viruses that specifically attack bacteria and are harmless to humans. A significant decline in the use of this therapeutic strategy introduced 100 years ago was seen in the West following the development of antibiotics. However, there is now new interest in phage therapy, especially in Europe, given the alarming increase in the number of antibiotic-resistant bacterial infections.

Until now, there has been insufficient scientific data to understand how phage therapy works in vivo. While most in vitro studies have proven that phages specifically target and kill bacteria, none of these studies took account of the importance of the host’s response to this activity.

Two Institut Pasteur teams (Laurent Debarbieux’s Bacteriophage-Bacteria Interactions in Animals Group and the Innate Immunity Unit led by James di Santo (Inserm U1223)) in partnership with Joshua Weitz’s team at the Georgia Institute of Technology (Atlanta, U.S.), recently showed the importance of patients’ immune status in terms of the chances of phage therapy success. This finding is the result of an original dual approach combining an animal model and mathematical modeling.

In order to evaluate the efficacy of treatment with a single phage species, the researchers focused on the bacterium Pseudomonas aeruginosa, which is involved in respiratory infections such as pneumonia. This bacterium, which is resistant to carbapenems, or ‘antibiotics of last resort’, was ranked by WHO as one of the four biggest global threats in terms of antibiotic resistance.

The researchers demonstrated that phage therapy is effective in animals with a healthy immune system (known as ‘immunocompetent’). The innate immune system can be triggered quickly and phages initially act in tandem with it to fight off infection. Then, after 24 to 48 hours, some bacteria naturally develop resistance to the phages which consequently cease to function. The innate immune system then takes over to destroy the bacteria. Of all the immune cells involved, neutrophils (white blood cells originating in the bone marrow) play a predominant role.

In parallel, in silico simulations have shown that the innate response needs to destroy 20-50% of the bacteria in order for phage therapy to be effective, regardless of whether phage resistance is observed. Thus, in the model studied, the researchers proved that there are no circumstances under which phages are capable of eradicating a P. aeruginosa infection alone.

These findings are particularly significant since they suggest that patients’ immune status should be considered when undertaking phage therapy. Laurent Debarbieux explains: “In terms of clinical consequences, one could reconsider the selection of patients likely to benefit from phage therapy. It may not be appropriate or recommended for people with severe immunodeficiency”.

The researchers are now planning to decipher the exact immune processes involved and the underlying mechanisms. At the same time, clinical trials are ongoing, notably including the Phagoburn trial on skin infections in burn patients funded by the European Union’s 7th Framework Programme.

©Fotolia

Inserm researchers in Rouen (Unit 1234 “PANTHER: Physiopathology, autoimmunity, neuromuscular diseases and regenerative therapies”, Inserm/Université de Rouen Normandie) have tested with success a cell therapy aiming to restore the ability of the sphincters to contract in patients with fecal incontinence. As part of a clinical study conducted in partnership with Rouen University Hospital, 60% of the patients who received this innovative therapy observed a reduction in their incontinence.

This research was published in Annals of surgery.

 Fecal incontinence affects 1 million people in France, 350,000 of whom to a severe extent, and its personal, social, and economic impacts are profound. Particularly affected are young women after childbirth (with 10-15% experiencing incontinence in the subsequent weeks and 4-5% who continue to suffer from a severe and chronic form of the condition). Sphincter damage or dysfunction, which are among the causes of fecal incontinence, are characterized by the fact that the sphincters, the rings of muscle surrounding the anal area, lose their ability to contract correctly.

Researchers at Inserm Unit 1234 “Physiopathology, autoimmunity, neuromuscular diseases and regenerative therapies” associated with the Université de Rouen Normandie joined forces with the Biotherapy Laboratory and Digestive Surgery Department of the University Hospital of Rouen to design a clinical study evaluating therapy with adult stem cells (myoblasts) that are capable of differentiating into effective muscle cells.

 To begin with, a model of the disease was developed at the Inserm research unit. Rats treated with myoblasts showed recovery of their sphincter function. This was associated with the in vivo production of new functional muscle fibers. The researchers then studied the genetic stability of the myoblasts and demonstrated that it was compatible with use in humans. The conditions were therefore ideal for the clinical study to go ahead.

During the study, thigh muscle fragments were taken from the patients,  following which the myoblasts were cultivated in order to obtain sufficient numbers of them. With the help of ultrasound, these myoblasts were then injected into the deficient sphincter so that they could differentiate into functional muscle fibers. A total of 24 patients were enrolled in the study, with half receiving the innovative stem-cell therapy and half receiving a placebo form. The severity of their condition was assessed using the Cleveland Clinic Incontinence (CCI) Score questionnaire at 6 months and 12 months after administration.

One year after the injection, the treatment had worked in 7 of the 12 stem cell patients (58%) whereas only 1 of the 12 (8%) placebo patients had noted an improvement. At the same time, the median CCI score had significantly decreased from 15 to 6.5 points in the treated group, whereas in the placebo group the median score after one year had decreased from 15 to 14.

In light of these positive results, the placebo group patients were also given the possibility to receive the therapy, with their cryopreserved muscle cells. Their response rate was just as satisfactory as that of the first group.

As such, the authors propose an innovative therapeutic solution for refractory fecal incontinence by demonstrating its efficacy and safety. Given the constraints of the reference treatment (sacral neurostimulation), which involves the implantation of exogenous material, it is hoped that in time a place for this cell therapy might be found.

Months post-injection. ©Olivier Boyer

©Fotolia

Researchers led by Jean-Charles Guéry of the Centre for Pathophysiology Toulouse Purpan (Inserm/Université Toulouse III – Paul Sabatier/ CNRS) are providing new insights into the possible link between male hormones and differences in gender in susceptibility to allergic asthma. This study demonstrates that hormones such as testosterone act on the immune system. The results are published in the scientific review The Journal of Experimental Medicine on 8 May 2017.

 Asthma is a complex disorder defined by bronchial hyperresponsiveness and chronic inflammation of the respiratory tract. It is common and affects more than 4 million people in France. The first signs occur mostly during childhood. Epidemiological studies show that there are disparities between men and women. There is a greater prevalence in boys compared with girls below the age of 10 years, while this tendency is reversed at puberty. In the global adult population, allergic asthma is twice as common in women and they develop more severe forms of the disease.

 In the case of allergic asthma, certain cells of the immune system have abnormal secretions of Th2 cytokine proteins. These proteins are part of the inflammatory reaction of the lungs during an asthma attack. Recently, a new group of immune cells was identified in the lungs, type 2 innate lymphoid cells (ILC2). Due to their ability to produce mediators of allergic asthma very soon after sensitisation of the lungs to an allergen, these cells carry out a central function in the initiation and orchestration of immune responses leading to the development of the disease.

 A team of French researchers[1], led by Jean-Charles Guéry, of the Pathophysiology Centre of Toulouse-Purpan, in collaboration with Australian researchers from the Walter and Elisa Hall Institute in Melbourne, are interested in the possible link between the immune system and sex hormones, which could in part contribute to the differences between men and women. First of all, they have highlighted the fact that, as in humans, male mice develop allergic asthma to dust mites which is much less severe than in females. This same response bias was observed when the researchers induced inflammation of the lungs. This difference disappeared where males were neutered, while “neutering” of females had no effect, which suggests a key role for male hormones (androgens). Essentially, the ILC2s possess the androgen receptor but the question remains as to whether this receptor functioned in response to the testosterone.

In the in vitro experiments, the researchers showed that testosterone inhibited the development of the ILC2s, while an anti-androgen, a molecule which reduces the activity of male hormones, had the reverse effect.  In non-neutered male mice without the androgen receptor in their type 2 innate lymphoid cells, the researchers observed a greater proliferation in the lungs associated with greater inflammation, than in the previous experiments. This last observation confirms the key role of the androgen receptor in respiratory disorders dependant on ILC2s.

According to Jean-Charles Guéry, this work highlights a new mechanism at the root of the differences linked to gender in allergic asthma : “The androgen receptor could represent a new  therapeutic target, for the purposes of inhibiting the action of type 2 innate lymphoid cells in asthmatic patients. In the medium term, that could become a treatment for allergic asthma in humans. “

[1] This team involves researchers from the Centre for Pathophysiology Toulouse Purpan (Inserm/Université Toulouse III – Paul Sabatier/CNRS), the Institute of Genetics and Molecular and Cellular Biology (Inserm/Université de Strasbourg/CNRS) and the Institute of Pharmacology and Structural Biology (CNRS/Université Toulouse III – Paul Sabatier).

In blue, the multilobed nuclei of neutrophils. In red, the myeloperoxidase (MPO) contained in cytoplasmic granules.

©Institut Pasteur

Researchers from Institut Pasteur and Inserm, in collaboration with Stanford University, have demonstrated the protective role of immune cells, called neutrophil granulocytes, in endotoxic shock – the component of septic shock related to the action of bacterial toxins. An effect thought to be based on the principal enzyme produced by these cells: myeloperoxidase. This discovery has been published in The Journal of Experimental Medicine.

Responsible for 50% of ICU deaths, septic shock is acute circulatory failure caused by an uncontrolled inflammatory response, which occurs in a context of serious infection. Septic shock involves, among other mechanisms, endotoxic shock: this is more particularly due to the action of the toxins produced by the bacteria responsible for the infection. Of these toxins, lipopolysaccharides (LPS) play a principal role in endotoxic shock by triggering the excessive, prolonged, and unbalanced immune cascade responsible for multiple organ failure.

Neutrophil granulocytes, which are white cells involved in the innate immune response and one of the body’s weapons in the fight against bacterial infection, have already been described as having antimicrobial activity. However, the scientific community thought that they could also have a harmful effect in the event of exposure to bacterial toxins such as LPS by exacerbating the associated inflammation and tissue damage.

Researchers from the “Antibodies in Therapy and Pathology” unit (Institut Pasteur/Inserm), in collaboration with Stanford University, have observed that, on the contrary, these neutrophils play a protective role against the inflammation triggered by LPS. As part of their work, they developed the first mouse model enabling a highly selective, inducible and reversible reduction in the neutrophil count.  With this they showed that neutrophil depletion made the mice more susceptible to the toxic effects of LPS, reducing their chances of survival and strongly activating their production of cytokines, the molecular messengers of inflammation.

More in detail, the researchers show that this protective capacity of the neutrophils is due to myeloperoxidase (MPO), the principal enzyme produced by these cells. “This protein, although routinely used as a marker of inflammation, does not increase it,” emphasizes Laurent Reber, one of the first authors of the paper. “On the contrary, it has a protective role.” “Patients with low MPO levels actually have a poorer prognosis in the event of septic shock,” adds Caitlin Gillis, the paper’s other first author.

“We have solved a paradox, in a way,” concludes Reber. “Neutrophils consistently combine antimicrobial activity and the ability to limit bacterial toxicity. The animal model that we have developed now means that we can continue to investigate the role of neutrophils in mechanisms of innate and adaptive immunity. We also want to try to understand how MPO acts in relation to LPS and why it has no effect on other bacterial toxins.”

This research was funded by Institut Pasteur, Inserm, Stanford University, ERC (MyeloSHOCK project) and NIH.

Almost everybody has it: Scientists estimate that approximately 95 percent of adults around the world are infected with the Epstein-Barr virus. In rare cases, an infection with this virus causes cancer. Scientists at the German Cancer Research Center (DKFZ), at the German Center for Infection Research (DZIF), and at the French National Institute of Health and Medical Research (Inserm) have now discovered that a component of the Epstein-Barr virus infectious particle promotes carcinogenesis. This viral protein interferes with cell division and impairs proper distribution of the genetic material to the two daughter cells. This confers a risk of subsequent cancer development.

After an infection with the Epstein-Barr virus (EBV), the virus persists in the body throughout a person’s lifetime, usually without causing any symptoms. About one third of infected teenagers and young adults nevertheless develop infectious mononucleosis, also known as glandular fever or kissing disease, which usually wears off after a few weeks. In rare cases, however, the virus causes cancer, particularly lymphomas and cancers of the stomach and of the nasopharynx.

Scientists have been trying for a long time to elucidate how the viruses reprogram cells into becoming cancer cells. “The contribution of the viral infection to cancer development in patients with a weakened immune system is well understood ” says Henri-Jacques Delecluse, a cancer researcher at the German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ) in Heidelberg. “But in the majority of cases, it remains unclear how an EBV infection leads to cancer development.”

In their present publication, Delecluse, in collaboration with Ingrid Hoffmann, also from the DKFZ, and their respective groups present a new and surprising explanation for this phenomenon. The scientists have shown for the first time that a protein component of the virus itself promotes the development of cancer. When a dividing cell comes in contact with Epstein-Barr viruses, a viral protein present in the infectious particle called BNRF1 frequently leads to the formation of an excessive number of spindle poles (centrosomes). As a result, the chromosomes are no longer divided equally and accurately between the two daughter cells – a known and acknowledged cancer risk factor. By contrast, Epstein-Barr viruses that had been made deficient of BNRF1 did not interfere with chromosome distribution to the daughter cells.

EBV, a member of the herpes virus family, infects B cells of the immune system. The viruses normally remain silent in a few infected cells, but occasionally they reactivate to produce viral offspring that infects nearby cells. As a consequence, these cells come in close contact with the harmful viral protein BNRF1, thus having a greater risk of transforming into cancer cells.

“The novelty of our work is that we have uncovered a component of the viral particle as a cancer driver,” Delecluse said. “All human tumors viruses that have been studied so far cause cancer in a completely different manner. Usually, the genetic material of the viruses needs to be permanently present in the infected cell, thus causing the activation of one or several viral genes that cause cancer development. However, these gene products are not present in the infectious particle itself”.

Delecluse and his colleagues therefore suspect that EBV could cause the development of additional tumors. These tumors might have previously not been linked to the virus because they do not carry the viral genetic material.

For Delecluse, the consequence that follows from his findings is immediate: “We must push forward with the development of a vaccine against EBV infection. This would be the most direct strategy to prevent an infection with the virus. Our latest results show that the first infection could already be a cancer risk and this fits with earlier work that showed an increase in the incidence of Hodgkin’s lymphoma in people who underwent an episode of infectious mononucleosis.”

Experts estimate that an EBV vaccine could prevent two percent of all cancer cases worldwide. Delecluse and his group already developed a vaccine prototype in 2005. It is based on so-called ‘virus-like particles’, or VLPs. These are empty virus shells that mimic an EBV infectious particle, thus prompting the body to mount an immune response. However, “Of course, the VLPs must not contain BNRF1!” Delecluse hastens to add.