At the Center for Molecular Inflammation Research (CEMIR) we have discovered a new mechanism for how our immune cells recognize streptococcal bacteria that can cause serious infections in both newborns and adults.

Complicated infections such as blood poisoning are still among the most common causes of people dying in hospitals, despite the fact that we have had access to antibiotics for over 70 years. It is important with increased knowledge about our immune system to develop better treatment and thus survival among those with serious infections.

The immune system’s main task is to protect us against infections. The first step is to recognize and see the difference between what is foreign (the enemy) and what is the body’s own structures. From earlier we know that there are receptors both on the surface and inside the cells that are called Toll-like receptors (TLRs). These constitute the first line of defense in the body.

TLR is one of the most important parts of the immune system that reacts quickly upon encountering parts of bacteria and viruses. When these receptors are activated by foreign organisms, a cascade of warning signals starts activating other parts of the immune system to fight the invader. Earlier it was thought that it is primarily TLRs on the surface of the white blood cells that detect bacteria, while TLRs inside the cell is specialized in detecting viruses.

In our project, we have examined how the immune system reacts to group B streptococci (usually abbreviated GBS). We have investigated what happens when the white blood cells called monocytes come into contact with the GBS.

Monocytes are very important in the immune system and both direct other parts of the defense and digest bacteria that enter the body. We have found that TLR number 8 (TLR8) located within the monocytes is important for detecting GBS. TLR8 recognizes and is activated by fragments DNA-copies from the bacteria that is called RNA.

When RNA from GBS comes into contact with TLR8, the receptor is activated and sends signals that activate the monocyte and other parts of the immune system to fight the streptococcus. TLR8 seems to be particularly important for the detection of streptococci like GBS, but also golden staphylococci. E. coli-bacteria, on the other hand, were not recognized by TLR8.

These findings have recently been published in the Journal of Frontiers of Immunology and are a piece of the puzzle in making a clearer picture of how the body detects invading bacteria.

Reference: Birgitta Ehrnström, Kai Sandvold Beckwith, Mariia Yurchenko, Siv Helen Moen, June Frengen Kojen, Germana Lentini, Giuseppe Teti, Jan Kristian Damås, Terje Espevik, Jørgen Stenvik. Toll-Like Receptor 8 Is a Major Sensor of Group B Streptococcus But Not Escherichia coli in Human Primary Monocytes and Macrophages . Front Immunol. 2017; 8: 1243. Published online 2017 Oct 3.

Blogger: Jane Atesoh Awuh, Postdoctoral Fellow, Department of Cancer Research and Molecular Medicine and Centre of Molecular Inflammation Research (SFF-CEMIR).

Most often we become passionate and involved in issues in this life for very personal reasons. I am particularly drawn to infectious diseases because I hail from a society that is plagued by one kind of infection or the other. If you are not killed by one infectious disease you will be by the other, and if not by disease, it will be by war or hunger. I know two relatives who died of tuberculosis, one of them only a couple of months ago. Tuberculosis (TB) is still a disease of poverty although there is increasing incidence even in developed countries. HIV and TB are a dangerous liaison wherein HIV infects and destroys the very cells that should protect us from TB. These diseases are still considered shameful and surrounded by stigma.

To survive TB, support from family, friends and communities is as important as medication. TB can be treated through a long course of several antibiotics, over a minimum of six months. The medications might make you more sick, but if taken regularly, you will be completely well again. Six months is a short time compared to an entire life time, although many still fail to take their medication regularly. Even more fail to get treatment at all, because they are used to being poor and ill, and do not seek help in time.

3D-reconstruction of mycobacteria (red rods) with a macrophage. Photo: Marianne S. Beckwith

Thanks to the amazing work of basic scientists around the world, there is always a little light at the end of the tunnel. The work of basic scientists is often the foundation of whatever treatment and prevention strategies that eventually end up at the bedside of patients. And the steps to arriving at the very first clinical trial is often accompanied by a succession of failures, hopelessness and sleepless nights. Yet they are not always given enough credits and funding. The causal agents of mycobacterial diseases are an intriguing group of microorganisms that continue to baffle these brilliant minds around the world even when we think we have got it all figured out. Of these, Mycobacterium tuberculosis which causes tuberculosis is one force to reckon with especially in individuals who are immunocompromised for one reason or the other. Another one of these bugs causes leprosy and indeed the Norwegian scientist G. H. Armauer Hansen in 1873 discovered the bug, making it the first bacterium to be identified to cause disease in humans and since then pioneered research in leprosy. It’s amazing that these infections have been with us for thousands of years yet we are still struggling to keep it in check. How can a single-celled organism like these be so complex that they cannot be untangled by even the most brilliant minds in the field?

Confocal image of mycobacteria (red rods) within a macrophage coated with LAMP1. Photo: Alexandre Gidon

Approaching the end of the year could not be a better time to summarize current research findings in the world of these creepy, invisible creatures. In a recent issue of the journal Cellular and Molecular Life Sciences, we summarize the current standing on how these bugs have managed to stay with mankind for so long and I have a feeling we are still only scratching the surface of this enigma. To add to the whole complexity is the fact that these bugs actually prefer and thrive in one of the deadliest immune cells known – the macrophage, as a natural habitat. How can that be?

Macrophages play an essential role in the immune system by ingesting and degrading invading pathogens, initiating an inflammatory response and instructing adaptive immune cells, and resolving inflammation to restore homeostasis. We summarize mechanisms by which intracellular pathogens, with an emphasis on mycobacteria, manipulate macrophage functions to circumvent killing and live inside these cells even under considerable immunological pressure. Remember the good news; these infections are treatable although rise in drug resistance continues to be a challenge as with all other infectious diseases. A clear understanding of host responses elicited by a specific pathogen and strategies employed by the microbe to evade or exploit these is of significant importance for the development of effective vaccines and targeted immunotherapy against persistent intracellular infections like tuberculosis. Read more here.

Mycobacterial evasion strategies within a macrophage.

Blogger: Richard Kumaran Kandasamy Associate Professor and Onsager Fellow at Centre of Molecular Inflammation Research (SFF-CEMIR)

Our innate immune system is the first and most important barrier of microbial threats such as viruses and bacteria.  It will sense, and in most cases, clear out these pathogens – but not always. A new approach to studying macrophage response to viral threats have resulted in a vastly expanded knowledgebase of the dynamics of the host response to viral infection, and in turn how antiviral innate immunity works. The data is freely available at www.infectome-map.org.

Richard Kumaran Kandasamy.
Photo: Thor Nielsen.

Digging deeper with big data
Our immune system is comprised of different types of cells such as macrophages that carry out these specialized tasks of handling the intruder. Although antiviral innate immune response has been widely studied over the past decades and used for development of therapeutics, most of these are based on candidate approach due to the lack of sensitive high-throughput technologies. With the emergence of systems biology and developments in the OMICS technologies (transcriptomics, proteomics and phosphoproteomics etc), the classical view of one-gene-does-everything-in-a-cell is challenged and it is becoming evident that cellular systems are more like a highly connected network that work in a coherent fashion. There are several studies in the recent past that have highlighted that cells indeed have multiple regulatory options (chromatin remodeling, transcription, translation, post-translational modifications (PTMs), folding, cellular localization, etc.) in how it achieves homeostasis under various perturbation scenarios such as viral or bacterial infection (Figure 1).

Figure 1: Multiple regulatory options of a cell during perturbations such as infection

Using state-of-the-art orthogonal OMICS approaches, we envisioned to understand the dynamics of the host response to viral infection by which we could assess the extent and the molecular logic of the host cellular response. This has the potential to provide unique and complementary information that can allow us to precisely map the systems-level perturbation caused by the viral infection and the viral circumvention of the host response, which will further add to the growing knowledgebase of antiviral innate immunity.

Answers hiding in the shadows of existing research
During our study we learned that post-translational modifications such as phosphorylation are crucial for innate immune response, but also highly understudied.
We performed a temporal genome-wide transcriptomics, proteomics and phosphoproteomics analysis of the cellular response of mouse macrophages to Vesicular Stomatitis Virus (VSV) infection. This was followed by integrative bioinformatics analyses to get a global overview of the cellular response (Figure 2).

Figure 2: Temporal OMICS integration of RIG-I signaling pathway during VSV infection

In practice we sampled the macrophage response at times 20 minutes, 3 hours and 6 hours after infection.

Figure 3. Overview of the experimental outline

We discovered that immune cells have multiple regulatory options during antiviral response. A novel phosphorylation site as well as four other genes were functionally validated for their role in type-I interferon activation, NFkB activation and VSV life cycle.

The vast and complex molecular changes measured could be decomposed in a limited number of clusters within each category (transcripts, proteins, protein phosphorylation), each with its own kinetic parameters and characteristic pathways and processes, suggesting multiple regulatory options and a specific process logic within the overall sensing and homeostatic program.

Phosphorylation is crucial for tailor-made defence
Overall, the data highlighted a predominant executive function to phosphorylation, likely evolved due to the requirement of a fast response to pathogens. Functional validation of a novel phosphorylation site S328-S330 on the innate immunity adaptor MAVS, identified its essential role in activation of type-I interferon and NFkB response. Further, we evaluated the kinase-substrate relationships (Figure 4) and identified RAF1, and to a smaller degree, ARAF to be suppressing VSV replication and needed for NFκB activation, and AKT2 to be favouring VSV replication. Integrative analysis of the omics data showed coregulation of membrane transporters including SLC7A11 which we validated as a host factor in the VSV life cycle.

Figure 4: Evaluation of the kinase-substrate relationships.

Open access to the data
The results of the study are published in  ” A time-resolved molecular map of the macrophage response to VSV infection in Nature – Systems Biology and Applications .  The dataset is presented, and freely available, on the website www.infectome-map.org and represents a large and unique starting platform for further systems-level as well as targeted mechanistic investigations on the functional organization of the response of macrophages to viral infection.

By Menno Oudhoff,
Researcher, Centre of Molecular Inflammation Reseach (CEMIR)

The gastrointestinal tract is a common site for infection by a variety of pathogens. Helminth infections continue to be major causes of disease worldwide, and are a significant burden on health care systems. For example, gut-dwelling parasitic worms currently infect over a billion people, mostly in developing nations. Deworming strategies have been shown to improve physical and intellectual development of infected children, but current therapies do not offer a sustainable solution.

Intestinal tissue infected by Trichuris worms

We still have too little insight into how these pathogens are causing disease and how immunity to them is regulated.

Group leader at Centre of Molecular Inflammation research (CEMIR) Menno Oudhoff, together with scientists in Vancouver and Melbourne, have published results from a recent study in PLOS Pathogens. Their study shows that SETD7, an enzyme that modifies the function of other proteins by methylation, plays an important role in the development of intestinal immunity to the helminth parasite Trichuris muris. Specifically, they show that SETD7 affects intestinal epithelial turnover, a key mechanism through which T. muris worms are extruded from the body.

The studies identify pathways that are important for immunity to infection, that were previously believed to be involved primarily during embryonic development.

Research article:

Intestinal Epithelial Cell-Intrinsic Deletion of Setd7 Identifies Role for Developmental Pathways in Immunity to Helminth Infection

By: Jørgen Stenvik
Researcher at Centre of Molecular Inflammation Research (SFF-CEMIR), Department of Cancer Research and Molecular Medicine (IKM)
(Photo: Geir Mogen / NTNU)

CEMIR researchers have published a study in The Journal of Immunology that may aid in the development of new strategies for prevention or treatment of infectious diseases. The study improves our understanding of how bacteria activate the human defense system.

The family of Toll-like receptors (TLR1 to TLR10 in humans) recognizes different molecules of bacteria and viruses. Extensive studies over the last two decades have clarified that these receptors are central in the activation of the innate defense system during infection. The role of TLR8 has still remained enigmatic, as the human receptor can be activated by various types of RNA (ribonucleic acid, a copy of DNA), while murine TLR8 appears non-functional.

Human monocytes (nuclei, blue) infected with Staphylococcus aureus (green). The cells sense the bacteria via receptors, including TLR8, which trigger the production of alarm signals (TNF, red) to fight the infection.

It has been known that human TLR8 can sense viral infection, including HIV, and a possible role of TLR8 in the sensing of the Borrelia bacterium was earlier shown. CEMIR scientists now report that TLR8 also can sense the important human pathogenic bacterium Staphylococcus aureus. This occurs by recognition of bacterial RNA once the bacterium is eaten and degraded inside human defense cells – monocytes and macrophages. The study also reveals new details on how TLR8 triggers alarm cascades inside the cells, which include the activation of the transcription factor IRF5 via a recently described pathway (TAK1-IKKβ). This results in movement of IRF5 to the cell nuclei, where it activates the defense and alarm gene IFNβ, and further contributes to the activation of other alarm genes such as TNF. A surprising finding was that bacterial activation of a second immune receptor, TLR2, which senses lipoproteins at the cell surface, blocks the function of TLR8 inside the cell. This may represent a safety mechanism to avoid exaggerated immune activation that may lead to sepsis syndrome and septic shock. Because TLR2 and TLR8 trigger partially different alarm signals, this cross-regulation might fine tune the immune response towards different classes of pathogens.

The study is published in The Journal of Immunology together with an independent study by a research group from Germany who show that TLR8 also senses Streptococcus pyogenes. It is thus likely that human TLR8 can sense several different classes of bacteria, and that bacterial RNA is more important for the activation of the immune system than has previously been acknowledged.

TLR8 can in principle detect also human RNA, and the study may help to clarify whether TLR8 plays a role also in inflammatory diseases such as chronic inflammation and autoimmunity.

More about the study in The Journal of Immunology

CEMIR researcher Marie Hjelmseth Aune looks at macrophages ( blue ). The screen image shows a macrophage engulfing a bacterium (red). Photo: Geir Mogen, NTNU

Some bacteria and viruses take advantage of the way our immune system works to infect us.  Researchers at Centre of Molecular Inflammation Research (CEMIR) are uncovering the mechanisms by which this trickery takes place.

Read more on GEMINI.no: Uncovering the secrets of immune system invaders, written by Nancy Bazilchuk.

Blogger: Nadra J. Nilsen
Researcher at Centre of Molecular Inflammation Research (CEMIR) and the K.G. Jebsen Center for Myeloma Research

Researchers at Centre of Molecular Inflammation Research (CEMIR) publish new findings on how the immune system deals with infection. This knowledge may aid the development of vaccines, as well as the development of drugs that control acute and chronic inflammation.

The work was performed at the CEMIR, NTNU and in CEMIR affiliated Professor Egil Lien’s lab at the University of Massachusetts Medical School. The results were recently published in The Journal of Biological Chemistry, 2015 Feb 6.

Inflammation is an initial response which is typically mounted by your body when it is under attack by infectious bacteria and viruses.  Macrophages and monocytes are cells in your immune system which are specialized to sense infection and important for activating the immune system. These cells express high levels of a group of proteins called Toll-Like Receptors (TLR)s. TLRs allow the macrophage to recognize invading infectious microorganisms quickly. Upon activation these receptors mount an inflammatory immune response which serves as protection during the early phases of infection.

Humans have 10 different TLRs named TLR1-10. The different TLRs recognize different patterns that are expressed by microorganisms and can tell the immune system what type of infection it is facing. Different TLRs activate different signals which are optimized to deal with different types of infection.

My favorite TLR is TLR2 which is best known to recognize certain types of bacteria such as Staphylococcus and Mycobacterium. It can also distinguish structures found in viruses and fungi. TLR2 therefore plays an important role in recognizing invading microorganisms and in activating inflammatory signals which are important in fighting certain types of infection. TLR2 recognizes components of bacterial cell wall called lipopeptides. Lipopeptides that activate TLR2 are potent activators of the immune system and can be applied in vaccine development to enhance the efficiency of vaccines. In addition to sensing microbial components, TLR2 can be triggered by certain factors released from one’s own cells upon damage, in the absence of infection. So, although TLR2 is important to alert the immune system against invading microorganisms, excessive TLR activation can also contribute to inflammatory disease. The immune responses activated by TLRs therefore need to be tightly controlled to avoid the detrimental effects of excessive inflammation.

Toll-Like Receptor 2 (TLR2) in human monocytes. A human monocyte (right) and a confocal microscopy image of a human monocyte stained with a TLR2 antibody (red) (left) showing where TLR2 is expressed in the cell.

We have been studying TLR2 expression, regulation and signaling for a long time to try to understand how TLR2 shapes the immune response to invading microorganisms. We have previously shown that TLR2 is expressed on the surface of immune cells such as macrophages, monocytes, and that this receptor is quickly upregulated in response to very low levels of stimuli. Up-regulation of surface TLR2 may play a role in sensitizing immune cells to respond more potently to subsequent infection. We have also seen that TLR2 is expressed inside cells, in compartments called endosomes, and have further investigated the signaling pathways that control TLR2 expression and the immune response induced by different TLR2 activators.

All TLRs, except TLR3, require a signal protein called MyD88 to mount a pro-inflammatory response to infection. TLR3 uses a signal protein called TRIF which activates a signaling pathway that leads to a strong anti-viral response to viral infection. TLR4 has been the only TLR known to signal through both MyD88 and TRIF in macrophages, leading to the activation of both these pathways. We have shown now that TLR2 can also activate the TRIF pathway, in addition to the MyD88 pathway.

We found that TLR2 can indeed activate certain anti-viral responses and that the signaling protein TRIF is important for relaying these responses when TLR2 is activated. We also provide insight into other signaling proteins that are involved in this pathway. These results provide new insight into TLR activation and signaling and how the immune system deals with infection. This knowledge may aid the development of vaccines, as well as the development of drugs that control acute and chronic inflammation.

We are now investigating the role for TRIF in TLR2 signaling in human immune cells, since most of our studies until now have been conducted in mice lacking the genes coding for the different TLRs and down-stream signaling components. We are also investigating the role of TLRs in Multiple Myeloma, which is a cancer of certain immune cells in the bone-marrow called plasma cells. In close collaboration with the K.G. Jebsen Center for Multiple Myeloma Research we aim to determine how TLR activation affects Myeloma cell growth and survival, and hope to identify new ways to target TLRs and their down-stream signaling components in cancer therapy.

Reference: A Role for the Adaptor Proteins TRAM and TRIF in Toll-Like Receptor 2 Signaling, The Journal of Biological Chemistry, 2015 Feb 6.

The article has the cover image for the current issue of PNAS. (May 20, 2014)

Many bacteria kill considerable numbers of host cells upon infection. However, the mechanisms behind the cell death are in many cases unclear. A recent article in PNAS by the the CEMIR-affiliated researcher professor Egil Lien,  describes how the bacteria Yersinia pestis, the causative agent of plague, kills key immune cells called macrophages by apoptosis mediated by kinase RIP1 and caspase-8 together with RIP3.

Apoptosis is often considered to be a “silent” type of cell death. However, we found that the death was accompanied by inflammatory processes via IL-18 and IL-1b generating inflammasomes and transcription factor NF-kB, also via RIP kinases and caspase-8. Importantly, mice deficient in caspase-8 and RIP3 were highly susceptible to bacterial infection, suggesting a key pathway for anti-bacterial defenses.

The article made it to the cover in the current issue of PNAS. Learn more about the image here.

Professor Egil Lien. Foto: NTNU

Reference

Wenga, D. , Marty-Roixa, R., Ganesana, S., Proulxb, M.K., Vladimera, G.I., Kaiserc, W.J., Mocarskic, E.S., Pouliota, K., Chand, F.K., Mellihere, M.A., Harrisf, P.A., Bertinf, J.,  Goughf, P. J., Shayakhmetovg, D.M., Goguenb, J.D., Fitzgeralda, K.A., Silvermana, N., Lien, E. Caspase-8 and RIP kinases regulate bacteria-induced innate immune responses and cell death. PNAS (published online).

Blood Infecion. Photo: Istock

The Journal of Immunology recently published online a paper showing that a treatment regimen blocking “bottle-neck” molecules of complement and the TLR system, C5 and CD14 respectively, significantly attenuated the initial proinflammatory cytokine storm in mice undergoing cecal ligation and puncture sepsis, a polymicrobialmodel which is regarded relevant for human sepsis despite the large species differences. Notably, for some of the cytokines, like IL-6, double-blockade of C5 and CD14 was required to get significant reduction. Most importantly, the 10-day survival was significantly improved by the double-blockade, which was not observed by single inhibition by the two molecules.

The study was initiated by a CEMIR-affiliated researcher, professor Tom Eirik Mollnes, who has put forward the hypothesis of this double blockade as a treatment option for conditions where innate immunity is improperly activated. This idea is based on in vitro work the last 10-12 years –  in close collaboration with the CEMIR leader, professor Terje Espevik. The present study was performed in collaboration with professor in trauma surgery, Marcus Huber-Lang, and his colleagues in Ulm, where the animal experiments were done. This is the first publication presenting evidence for the C5/CD14 “cocktail” as a possible treatment for sepsis.

Reference:

Huber-Lang M., Barratt-Due A., Pischke S.E., Sandanger. Ø., Nilsson P.H., Nunn M.A., Denk S., Gaus W., Espevik T.,  Mollnes, T.E. Double-blockade of CD14 and complement C5 abolishes the cytokine storm and improves morbidity and survival in polymicrobial sepsis in mice. Journal of Immunology 2014 (E-published April 30th, 2014).

Cholesterol crystals

Inflammation plays a crucial role in atherosclerosis, and cholesterol crystals is a candidate trigger early in the development of the disease. CEMIR scientists have recently published an article  in Journal of Immunology that describes how cholesterol crystals employ the complement system to induce cytokines and activate the inflammasome/caspase-1 by regulating several cellular responses in human monocytes.

Targeted inhibition of inflammation to reduce cardiovascular events is currently being tested in large scale clinical trials. These findings may provide additional targets for immune modulation.

Reference:

Eivind O. Samstad, Nathalie Niyonzima, Stig Nymo, Marie H. Aune, Liv Ryan, Siril S. Bakke, Knut T. Lappegård, Ole-Lars Brekke, John D. Lambris, Jan K. Damås, Eicke Latz, Tom E. Mollnes, Terje Espevik. Cholesterol Crystals Induce Complement-Dependent Inflammasome Activation and Cytokine Release.  Journal of Immunology (Published online before print February 19, 2014)