In her work researcher Atrid Skjesol is revealing more details on how bacteria are causing inflammation.

By Astrid Skjesol, researcher at Centre of Molecular Inflammation Research, Department of Clinical and Molecular Medicine.

The findings of a new target protein make it possible to find new treatment strategies to prevent serious inflammatory reactions toward Escherichia coli (E. coli) bacteria and sepsis.

In our recent research paper, published in PLOS Pathogens March 18, 2019 (https://doi.org/10.1371/journal.ppat.1007684) we have used extensive methods to reveal new details on how bacteria are causing inflammation.

Bacteria like E. coli are all around us. You can find E. coli everywhere in your environment, including on your skin and in your intestines. Most E. coli are harmless but some can make you very sick with uncontrolled inflammation that can cause a life threatening condition called sepsis. Sepsis can result from an infection anywhere in the body and is the most common cause of mortality in hospitals. It kills and disables millions and requires early suspicion and treatment for survival.

Despite current treatment strategies and advances in supportive care of critically ill patients, the mortality rate has barely decreased during the past decades. Therefore, there is a need for identifying new treatment strategies for preventing serious inflammatory reactions toward E. coli bacteria and for treating sepsis patients.  Based on our research a new target protein is identified, which is involved in cellular uptake and inflammatory reactions towards E. coli bacteria.  In our recent research we have used extensive methods to reveal new details on how bacteria are causing inflammation.

A central aim for our research is to develop technology to dampen unnecessary inflammation during blood stream E. coli infections to prevent septic shock. We do so by investigating how specialized immune cells like macrophages recognize and “eat” bacteria.

A cellular receptor on macrophages recognizes a specific pattern on the surface of E. coli. This receptor, called TLR4, works to turn on alarm signals, which are secreted to surrounding cells to help clear the ongoing infection.

From good to bad

TLR4 can also help cells to engulf bacteria and produce an alarm molecule (IFN-β). Normally, this alarm molecule can dampen inflammatory signaling, but overproduction of it  may mediate unwanted toxicity and induce pathological damage. So how to prevent an overproduction? (

The signaling pathway leading to secretion of the alarm molecule is dependent on the TLR4 adaptor molecule TRAM. We have discovered a novel binding-partner for TRAM, a protein called FIP2.
Removal of FIP2 or TRAM from human immune cells disables the cells from eating bacteria and producing too much alarm molecules (IFN-β)

Normally, the interaction between these two proteins contributes to activation and stabilization of other proteins which in turn are key players in the locomotion of the cells cytoskeleton during the process of bacteria engulfment. If the interaction between these proteins do not take place, the cells are less likely to overproduce alarm molecules.

Based on this research we aim to find new treatment strategies for preventing serious inflammatory reactions toward E. coli bacteria.

The results are published in “PLOS Pathogens” March 18, 2019 (https://doi.org/10.1371/journal.ppat.1007684)

Ill.1: Model describing molecules involved in bacterial uptake (phagocytosis) in an immune cell. (Illustration: Bjørnar Sporsheim)

Ill 2: Human immune cell (macrophage) engulfing E. coli (red). Newly formed filaments of the cytoskeleton (F-actin) is stained cyan. TRAM and FIP2 (green) are localized around the E. coli in patches overlapping with actin filaments. (Photo: Astrid Skjesol and Harald Husebye)

Ill 3: Super resolution microscopy of TRAM (green) when E. coli (red) enters a human macrophage. Newly forms filaments of the cytoskeleton (F-actin)  is stained cyan. (Photo: Astrid Skjesol and Harald Husebye)

Ill 3:

Blogger: Siril S. Bakke, PhD/Post doc, Centre of Molecular Inflammation Research (CEMIR)

Cardiovascular disease resulting from atherosclerosis is one of the most common causes of death worldwide. Our new study reveals molecular mechanisms behind how a cyclic sugar reduces inflammation on the surface of cholesterol crystals.

Inflammation and the activation of the innate immune system, through the complement system, play a crucial role in the development of atherosclerosis. Cholesterol crystals are triggers of these processes as the disease develops. The complement system is a defense system that alters the surface of foreign material so that immune cells can engulf it for destruction.

Cholesterol crystals (CC) initiate inflammatory responses in immune cells called macrophages – this is inhibited by the sugar molecule cyclodextrin (BCD). CC may be recognised by the innate immune system through the complement system (components represented here as shperes and immuno-complexes as stars) and be engulfed by a macrophage. This leads to the transcription of pro-inflammatory genes and cytokine release, thus, causing local inflammation. When the sugar cyclodextrin is present it coats the CC and prevents uptake of the CC by the macrophage. Cyclodextrin can also dissolve CC andwill also enter into the macrophage and activate the transcription factor Liver X receptors and this may lead to a decrease in transcription and release of the pro-inflammatory factors.

Scientists from Centre of Molecular Inflammation Research (CEMIR) at NTNU in Trondheim together with national and international collaborators from University of Bonn, Copenhagen and Oslo have recently published a new paper in Journal of Immunology that shows that a sugar, called 2-hydroxypropyl-β-cyclodextrin (cyclodextrin), reduces inflammation caused by cholesterol crystals.

This is a follow up study from last year, when we found that cyclodextrin reduces and prevents formation of atherosclerotic plaques in mice, as well as dissolve cholesterol crystals. Cyclodextrin also had anti-inflammatory effects on cells in atherosclerotic plaques from humans.  That study promoted large publicity, making patients aware of the new basic results (see link for article in Gemini and Science Daily below).

Patients from all over the world contacted us to be a part of our study. As this was a basic research project we had to turn them down, however, if funding is available there is a great potential for a thorough clinical research project to see if treatment with cyclodextrin can be beneficial for the patients.

We still want to know more about the molecular mechanisms behind how cyclodextrin reduce the inflammation. Our follow-up study shows that cyclodextrin binding to the surface of cholesterol crystals reduces complement activation, and the entering of cholesterol crystals in the immune cells. Thereby, cyclodextrin prevents induction of inflammation.

Both our studies suggest that cyclodextrin can be a promising therapeutic approach for treating atherosclerosis because it inhibits inflammation, and thereby have the potential in lowering the deaths caused by atherosclerosis.

References:

• Cyclodextrin Reduces Cholesterol Crystal-Induced Inflammation by Modulating Complement Activation. Bakke SS/Aune MH, Niyonzima N/Pilely K, Ryan L, Skjelland M, Garred P, Aukrust P, Halvorsen B, Latz E, Damås JK, Mollnes TE, Espevik T. J Immunol. 2017 Aug 30. pii: ji1700302. doi: 10.4049/jimmunol.1700302.
• Cyclodextrin promotes atherosclerosis regression via macrophage reprogramming. Zimmer S/Grebe A, Bakke SS, Bode N, Halvorsen B, Ulas T, Skjelland M, De Nardo D, Labzin LI, Kerksiek A, Hempel C, Heneka MT, Hawxhurst V, Fitzgerald ML, Trebicka J, Björkhem I, Gustafsson JÅ, Westerterp M, Tall AR, Wright SD, Espevik T, Schultze JL, Nickenig G, Lütjohann D, Latz E. Sci Transl Med. 2016 Apr 6;8(333):333ra50. doi: 10.1126/scitranslmed.aad6100.
• Ny behandling av åreforkalkning med sukkerstoff. Gemini (in Norwegian)
• New hope for treating atheriosclerosis. Science Daily.

Bloggere: Egil Lien, Professor II at Centre of Molecular Inflammation Research (CEMIR) and Mathias Pontus Ørning, PhD candidate.

A number of human-pathogenic bacteria use specialized secretion systems to deliver bacterial effector proteins into host cells. These effector proteins can have many different functions, but the main bacterial goals are believed to be to create a favorable environment for bacterial growth. Thus, inhibition of the host immune responses can dampen bacterial killing and limitations of the infection, tasks that can be enacted by the host innate immune system. Simply said, the bacteria use the effectors to mess up host responses so they can multiply and survive themselves.

One of the most studied systems is called “type 3 secretion system”, and can be found in various pathogens such as Salmonella, E. coli, Shigella, Yersinia and others. These secretion systems are quite sophisticated nano-machines. A “needle” is protruding from the bacteria and creates a pore in the host cell. Subsequently, the bacterial effectors are translocated into the host cell via these needles and pores (1). It is likely that there is an evolutionary “arms race” between the bacteria and humans, where the bacteria find ways to block immune responses, but the body evolves new ways to circumvent the blockade and detect the infection. We can only hope that the host cells ultimately win!

NTNU-CEMIR researchers have used the “Black Death” plague bacteria – Yersinia pestis – as a model system, and have now discovered how some of these bacterial effectors can block new pathways of the innate immune system. The research has focused on how bacteria manipulate release of host inflammatory signals (cytokines) IL-1b and IL-18, which can induce anti-bacterial responses (2). Yersinia is very effective in dampening release of these cytokines, and one effector, called YopM, blocks activation of a cellular complex called the Pyrin inflammasome, which normally enables release of IL-1b and IL-18. A bacterial strain lacking YopM, attenuated in normal mice, was shown to be virulent in mice lacking Pyrin, thus proving that the mechanism is important for immune function in mammals (3).

Curiously, some people, such as those with the inflammatory disease Familial Mediterranean Fever, have mutant forms of the Pyrin molecule. In these cases, the patients have increased release of IL-1b and IL-18. One hypothesis is that these individuals may have had altered susceptibility to infections (such as the Black Death) throughout history. Can they have been more resistant to infection since they may have increased levels of cytokines, and could this have been a positive selection factor? Perhaps more research can uncover links between Pyrin mutations, historical pandemics and modern infections. In any case, the CEMIR projects have so far uncovered a complex interplay between bacteria and their mammalian hosts, and shown novel aspects of how the body fights to limit infections.

Article about the subject (in norwegian): Slik angriper pest – og slik forsvarer vi oss

1. Ratner, D., Ørning, M.P.A., Lien, E. (2016). Bacterial secretion systems and regulation of inflammasome activation. J Leukocyte Biol. Online Nov 3. pii: jlb.4MR0716-330R.
2. Ratner, D., Ørning, M.P.A., Starheim, K.K., Marty-Roix, R., Proulx, M.K., Goguen, J.D., Lien, E. (2016). Manipulation of IL-1b and IL-18 production by Yersinia pestis effectors YopJ and YopM and redundant impact on virulence. J Biol Chem. 291, 9894-905.
3. Ratner, D., Ørning, M.P.A., Proulx, M.K., Wang, D., Gavrilin, M., Wewers, M., Alnemri, E., Johnson, P., Lee, B., Mecsas, J., Kayagaki, N., Goguen, J.D., and Lien, E. (2016): The Yersinia pestis effector YopM inhibits Pyrin inflammasome activation. PLoS Pathogens 12:e1006035.

The competition in FRIPRO is tough, and only the best researchers with particularly good projects and very well-written proposals have a chance at succeeding.

Atle Granlund  received 6,5 mill NOK to the project “Functional implication of genomic variance in IBD” and Richard Kandasamy got 8 mill NOK to the project “Phosphorylation Dynamics of Toll-Like Receptor Signaling”. The researchers are  thankful for this grant that make it possible to start working on their projects.

–  I am really happy and relieved! To receive this grant means I get to do what I have planned and it is a confirmation that others think it is a good idea, he says in an interview with Universitetsavisa (Norwegian). The funds he receives will cover salaries for himself and assets over the next four years.

Atle Granlund

Granlunds project will  investigate the role of hereditary traits in the pathogenesis of Inflammatory Bowel Disease (IBD).  IBD is a group of inflammatory diseases hallmarked by chronic inflammation of the intestinal mucosa with intermittent remission. The treatment options are few, and no treatment alternative is effective for all patients.

– By combining genotyping and gene expression data from an IBD patient cohort in an eQTL analysis, we hope to identify how the genomic variation influences the gene expression in the inflamed colonic mucosa, and how this correlates with prognosis and treatment response in the individual patients, Granlund explains. Interesting finds will be further evaluated using colonoid cultures established from the same patient cohort. The study will be further aided by almost ten years of clinical data on the cohort and the additional knowledge drawn from GWAS analysis from the HUNT population.

– Only by better understanding the significant diversity in the IBD patient group can we hope to find better treatment options and to reach a personalized medicine approach, says Granlund.

Richard Kumaran Kandasamy. Photo: Thor Nielsen.

Kandasamys  project aims to investigate the role of phosphorylation in the first line defense, the innate immune response. Innate immune signaling can be activated under threat from microbial organisms or upon tissue damage. Toll-like receptors (TLRs) are the largest family of molecules that sense these danger or pathogen-associated signals and trigger inflammatory response resulting in clearance of the pathogen and restoration of cellular homeostasis. Post-translational modifications (PTMs) such as phosphorylation, are crucial for these cellular processes.

– Our data suggests that the family of kinases and phosphorylation-based signaling are crucial in a range of inflammatory processes; and the potential existence of a phosphorylation code for specific TLR signaling pathways. We aim to take a systems-level interdisciplinary approach to systematically investigate the role of highly druggable kinases and phosphorylation-based signaling in TLR signaling pathways. This has the potential to yield new insights into innate immune signaling and will have implications for development of therapeutic strategies in inflammatory diseases and beyond, says Kandasamy.

See also this blog on Kadasamys research.

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: 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.

When bacteria infect the host an explosive immune response is initiated. Normally, this response is local and short-lived and results in elimination of the pathogen and healing of the injury. In other cases, the bacteria may enter the blood stream and cause a life-threatening situation called septic shock. Our immune system uses Toll-like receptors to detect and eliminate microbes. One of these receptors is TLR4 that recognizes Gram-negative bacteria. TLR4 is found in an immune cell called macrophage.

It is still not known in detail how TLR4  mounts an effective immune response against bacteria. CEMIR researchers have previously shown that the localization of TLR4 in cells plays an essential role for the type of immune response that is initiated in the macrophage.

The study also delineates and visualizes the mobility and transport mechanisms of the signaling complex to compartments inside immune cells where production of signaling molecules occurs. The recent data  published in Traffic journal provide us with new fundamental understanding of the mechanisms behind bacteria-induced inflammation downstream of TLR4 and may point to new drug targets for more effective treatment of Gram-negative sepsis.

Blogger: Marte Singsås Dragset
Researcher at Centre of Molecular Inflammation Research (SFF-CEMIR), Department of Cancer Research and Molecular Medicine (IKM)

24 March is the World Tuberculosis Day. Here in Norway, most people know tuberculosis (TB) from media, school and vaccination programs. Some people have a parent, grandparent or great grandparent who suffered this infectious disease.Luckily, on the World TB Day of 2015, the occurrence of TB in Norway is relatively low (350-400 reported cases/year). We have antibiotics for treatment, and most of the Norwegian cases are actually non-active, non-contagious so-called latent TB.
However, and here is why we need a World TB Day, globally TB is still a gigantic public health problem! In fact, only beaten by HIV/AIDS, TB is the deadliest infectious disease in the world. World Health Organization reports that 1.5 million people died from TB in 2013. That is almost every third Norwegian. The increase of antibiotic-resistant bacteria is at the same time a major emerging threat to human health, and TB resistant to our current drugs is on a rise. We urgently need new drugs for TB treatment.

To develop a new antibacterial drug is a time consuming process. It often starts with a large screen searching for compounds that kill the pathogen in question. Hit compounds are then typically optimized further, and for rational drug design purposes it is important to identify how the compound actually kills the bacteria. In our study, recently published in Antimicrobial Agents and Chemotherapy, we found the apparent mechanism of a new compound (PZP) that kills the TB-causing bacterium Mycobacterium tuberculosis. And the mechanism caught us by surprise.

Estimated TB incidence rates, 2013. (Source: WHO, 2014)

A relatively new method used to identify the drug target and the mechanism of a new potential drug is to sequence the whole genome of bacteria that shows resistance to the compound in question. For instance, the mechanism of Bedaquiline, which is the first new TB drug on the marked in over 40 years (!), was discovered using this method. Genome sequences of M. tuberculosis resistant to Bedaquiline revealed mutations in the enzyme that provides the bacteria with energy; ATP synthase. This finding subsequently led to the discovery that Bedaquiline binds and inhibits the pathogen’s energy production unit.

Likewise, TB bacteria resistant to the new anti-TB compound we are interested in, PZP, were genome sequenced. The mutations discovered mapped to a gene that is important for the bacteria to take up iron from the surroundings. As for us humans, M. tuberculosis needs iron to live. Could it be that PZP binds and inhibits this iron uptake protein of M. tuberculosis so the bacterium cannot get a hold of the iron it needs? Just like Bedaquiline binds and inhibits ATP synthase and starves the bacterium for energy? We set out to confirm this hypothesis, however, step by step it became clear that our theory failed. PZP did not target iron uptake. Puzzled by this finding, the sharp chemists on our team did another discovery; PZP is in fact an iron chelator! That means, PZP can strongly bind to iron. So, instead of inhibiting bacterial uptake of iron, PZP seems to enter the bacterial cells and bind the iron the bacteria has already taken up, making it unavailable for the bacteria to use. PZP apparently starves the TB bacteria for iron from the inside!

Together with our collaborators at research institutions in Boston, Rome, Texas, Seattle and New York we learned an important lesson from this study, important also for other researchers in the drug development field. Things are not always as they seem. The gene that maps to resistance towards an antimicrobial compound is not necessarily the target of the compound. But, even if it is not the target, to identify such genes can give us crucial hints about the true mechanism of the new drug candidate. As for us; we were successfully pointed in the direction of iron metabolism.

Reference: A Novel Antimycobacterial Compound Acts as an Intracellular Iron Chelator, Antimicrobial Agents and Chemotherapy, April 2015 vol. 59 no. 4.