Our immune system is powerful in fighting infection, but can also specifically kill malignant body cells such as cancer cells. When we get vaccinated, the immune system gets trained and prepared for these fights. But it is believed that vaccines might also have the potential to educate our immune system to fight cancer cells.

A major challenge is that the most effective immune cells to kill cancer cells, so-called CD8+ cytotoxic T lymphocytes (CTLs), are difficult to activate with currently available vaccination strategies. This is thought to be a major reason why we do not have more effective vaccines against cancer. In a collaboration project between the Center for Molecular Inflammation Research (CEMIR) at NTNU, Oslo University Hospital and PCI Biotech AS, we have recently published that a novel and innovative vaccination technology holds great potential to realize therapeutic vaccination against cancer.

Most of today’s vaccines contain small particles with proteins from a virus or bacterium (antigens) which are recognized and taken up by immune cells (such as macrophages and dendritic cells). The vaccine particles are degraded within membrane-enclosed vesicles in the immune cells. Subsequently, small degradation fragments of the vaccine are presented on the cell surface and can be recognized by other cells of the immune system. This results for example in production of antibodies that target and inactivate bacterial or viral proteins, but does not efficiently activate CTL responses.

In our current study, we investigated the potential of a novel vaccination technology on twisting the outcome of vaccination towards increased activation of CTL responses as this is important for development of effective vaccines against cancer. “PCI vaccination” is a vaccination technology that is based on the principle of photochemical internalization (PCI), which uses a photoactive compound, a so-called photosensitizer, and light of a specific wavelength to delivers the vaccine.

In PCI vaccination, the photosensitizer compound is delivered to immune cells together with the vaccine antigen. After internalization, the photosensitizer incorporates into the membranes of the vesicles that contain the vaccine. If the immune cells are subsequently exposed to light of a specific wavelength, the photosensitizer is activated and generates a small and short-lived local damage in the membranes of the vesicles. The damage leads to rupture of the vesicles, the vaccine can escape and access the cytosolic space of the cell. This light-induced translocation of the vaccine from the vesicles to the cytosolic space of the immune cell is the key feature of PCI vaccination, since vaccines located in the cytosolic space of immune cells are very efficient in activating CTL responses.

We performed experiments with PCI vaccination technology both in a cell culture system as well as in a mouse model. We found that PCI-mediated vaccine delivery to immune cells made vaccines 30-100-fold more effective in activating CTL responses compared to vaccine-delivery without PCI technology. In addition, it was found that the PCI vaccination treatment in itself had an enhancing (“adjuvant”) effect by stimulating immune cells, probably due to the low-grade cell damage generated by the treatment. We could confirm these findings in mice by demonstrating that PCI vaccination was able to effectively induce specific CTL responses to two cancer antigens. We thus show new and compelling evidence that PCI technology may provide a feasible strategy to improve the outcome of vaccines that aim at inducing CTL responses that can fight cancer cells.

The vaccine compounds used in our study were small fragments of proteins (“peptide antigens”) derived from cancer cells, which usually are poorly immunogenic. Our findings may be of particular interest since these short peptides have attractive features for therapeutic cancer vaccination: They are generally non-toxic, cheap and easy to produce and can be tailored to the patient-specific cancer. We therefore believe that PCI-mediated vaccination may provide a promising novel approach to realize effective therapeutic vaccination against cancer by raising specific CTL responses against cancer cells found in patients. The PCI method is minimally invasive, well-tolerated and has been tested in clinical trials for other purposes. PCI Biotech currently conducts a clinical validation of the PCI vaccination technology (fimaVACC) in a Phase I (“Proof of Principle!) study in healthy volunteers.

The paper is published in the journal “Frontiers in Immunology”.

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.

Blogger: Ingrid Eftedal, Principal investigator
Barophysiology research group, Department of circulation and medical imaging

White blood cells are essential components of the immune system. Without these cells we would not stay healthy for long on a planet where infections thrive. But what are these cells up to when we’re not sick? They are present. And they are active, at all times.

Lean back and breathe in. Hold your breath. It can’t get much easier; what could there possibly here for a scientist to study? Well, there is something.

Evolution has shaped us for the environment we live in, and our environment is never completely static. The immune system is involved in rapid biological adjustments that protect us from harm caused by environmental perturbations. Some perturbations are of a cyclic nature, like those that are linked to the earth’s rotation around the sun and its own axis. In an elegant study published in the journal Nature in 2015, English and German scientists identified variations in the immune system that are perfectly aligned with the seasons. Actually, our bodies appear to lie slightly ahead of the seasonal changes: we appear to have a biological memory that fine-tunes the immune system just before the seasons change. Since many common infectious diseases appear in a seasonal pattern, this is an amazing adaptation for life on planet earth. If we speed the cycle up a bit, related effects have been observed over the 24 hrs cycle.

So the immune system shows cyclic variation.

What happens if we speed it up to the cycle of our breath?

Most of the time, we breathe without thinking about it. Our cells need oxygen for energy production, and once we have filled our lungs with air, it is the circulatory system – i.e. the heart, blood vessels and blood – that distributes oxygen to the cells in all parts of our body. We can all voluntarily hold our breath for a while, but some people do this better than the rest. Freedivers dive while holding their breath; the best of them can hold their breath for over 10 minutes.

Eleven-time free-diving world champion Goran Colak during a bout of static apnea; timed breath-holding while immersed in water .We have used blood samples from elite free-diving athletes to examine how white blood cells of the immune system responds to acute reduction in blood oxygen levels. The photo is used with Goran Colak’s permission.

In order to understand how white blood cells respond to an altered breathing pattern, we studied some of the world’s best free-diving athletes. We used a simple design: blood samples were drawn from contestants at an international free-diving competition before start, and then again one and three hours after completion of a series of dives where the athletes either lay face down in water or swam close to the surface for as long as they could.

Then the samples were transported to the NTNU Genomic Core Facility where total gene expression in the athletes’ white blood cells was measured by a method called full genome microarray analysis. The analysis results were striking: the activity of more than 5000 genes changed in response to the simple effort of breath-holding. This is almost ¼ of all genes found in human cells. With this amount of data we could dig deeper into cellular biology and calculate which specific types of white blood cells that reacted to breath-holding, and also see finer details in the biological processes going on within the cells.

The figure shows a selection of white blood cell types in samples taken from athlete free-divers. Blue boxes are cell amounts prior to diving, whereas the red and green boxes show the same cells one and three hours after diving. The main changes identified were a marked increase in the amount of neutrophil granulocytes, whereas two types of lymphocytes; CD8-postivie cells and natural killer (NK) cells, decreased. Calculations of relative amounts of specific white blood cell types were done by mathematical deconvolution of global blood gene expression data.

The most striking finding we did was a marked increase of the white blood cell type neutrophil granulocytes. These blood cells are programmed for rapid response when the body perceives attacks from intruders; the neutrophils are capable of killing invading cells simply by eating them. But they also have another interesting trait that emerges when oxygen levels drop: neutrophil granulocytes are evolutionary old-timers that stem from an era when the atmosphere contained less oxygen than now, and their modern offspring still prefer environments where the oxygen levels are low. White blood cell types that use more oxygen – like lymphocytes – were less active in blood drawn after the athletes held their breath. What we observed are likely to be traces of evolutionary history still embedded in our immune system, visible when oxygen levels change. The study was published in November 2016 in the journal Physiological Genomics.

This study was done on healthy athletes.

Can it be relevant for understanding of human diseases?

Healthy people normally don’t have to worry about oxygen, but for common diseases like chronic obstructive lung disease (COPD) and sleep apnea, the body’s oxygen supply is limited. These diseases are associated with persistent inflammatory conditions, and increased risk of infections; both indicative of an impaired immune system. If we can use data from healthy individuals to distinguish secondary effects of low oxygen levels from the primary pathology of the disease, this may in turn be helpful for prevention and treatment strategies.

– And breathe out.

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: 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

CEMIR director Terje Espevik opened the conference. Photo: Ann-Charlotte Iversen

200 delegates from 20 different countries participated and made this conference an important arena to foster further innovative research on molecular mechanisms and regulation of inflammation. The conference brought together scientists from basic and clinical research and provided significant insight into common underlying processes of inflammatory disorders that can be translated to clinical settings.

At the conference we had 25 invited speakers and 16 oral presentations selected from the abstracts. In addition, we had a poster session with 64 posters.

The presentations covered cutting edge highlights on cellular trafficking mechanisms and compartmentalized signaling, systems inflammation, novel mechanisms of innate immunity recognition and prevention, mechanism of acute and chronic inflammation, and metabolic reprogramming in inflammatory responses.

The high quality presentations inspired to scientific discussions in the auditorium as well as at the various social events that took place during the conference. Photo: Ann-Charlotte Iversen

Concert and reception in the Nidaros Cathedral May 30th. Photo: Ann-Charlotte Iversen

CEMIR director Terje Espevik, Head of Administration Kari Håland and Co-Director Trude Helen Flo. Photo: Ann-Charlotte Iversen

Many CEMIR members assisted in the organizing of the conference and the organizing committee would like to thank all for making it such a great success!

Kandasamy’s research group aims to understand the molecular aspects of inflammation and antiviral signaling using state-of-the-art modern technologies to identify drug targets for treatment of inflammatory and infectious diseases. Inflammation plays a major role in the initiation and pathogenesis of a number of diseases such as sepsis, atherosclerosis and diabetes, among others.

The Onsager Fellowship Holders were announced at a reception by Rector Gunnar Bovim, on 26 may 2016.

The 12 Onsager Fellowship holders with Rector Gunnar Bovim. Photo: Morten Thoresen / NTNU

Read more about these results and more scientific highlights in the annual report:
CEMIR Annual Report 2015 (pdf)