Complement and toll-like receptors determine ischemia/reperfusion injury - universal mechanism and benefit of double inhibition

The injury that occurs in the course of ischemia (stop in blood supply) and reperfusion (restored blood supply) is a key-challenge in treatment of potentially life-threatening conditions like heart infarction, circulatory arrest, trauma, and organ transplantation. Activation of the inborn immune system, which is the first-line defense of the body against external (e.g. bacteria) and internal (e.g. injured cells) is crucial in the development of ischemia/reperfusion injury. The complement system and the toll-like receptors with its activating molecule CD14 are central to the inborn immune system. In this project, we have investigated a new, groundbreaking concept for therapy of ischemia/reperfusion injury; double inhibition of key molecules from the complement system (C5) and toll-like receptor activation via CD14. Two studies in this project have discovered and confirmed the importance of the innate immune system for long-term outcome in patients experiencing ischemia/reperfusion injury. The first study examined the importance of complement activation after ischemia/reperfusion injury to the whole body after cardiac arrest and found that the degree of activation of C5 is associated with death and neurological impairment 6 months after cardiac arrest when time spent on resuscitation had been less than 25 minutes (article and blog post published). This study has already led to wide scientific discussion in this field and planning of a clinical study using complement inhibition is under evaluation. The second study examined activation of the innate immune system after localized ischemia/reperfusion injury in patients exposed to isolated mild brain injury (concussion). We found that the innate immune system measured in blood samples was activated for a period of 12 months after the injury when compared to healthy volunteers. That the immune system was activated so long after a minor injury is new and groundbreaking since this can explain late injuries and opens for new treatment strategies (article published). To describe the mechanism of how the innate immune system gets activated upon ischemia/reperfusion injury, we have established an advanced cell model with cells from vesselwalls where oxygen-poor (hypoxic) cells are reoxygenated with human blood. In this model, we have found (i) that sterile inflammation occurs and (ii) that this inflammation can be inhibited with combined complement and CD14 inhibition. The model has been presented as a poster presentation at an international congress and the manuscript is now being completed. Finally, we investigated the effect of complement inhibition in a clinically oriented renal transplant model in pigs; Kidneys were stored without blood circulation for 20 hours before blood supply was restored with oxygen-rich blood in a machine used in clinical transplantation. Complement inhibition led to the absence of complement activation and an overall reduction in inflammation. Unfortunately, the COVID-19 pandemic led to delays in this project, making it impossible to test double inhibition of complement and CD14 within the timeframe of the project. However, this is planned for a later date as we expect that double inhibition could lead to even less inflammation in kidney tissue and blood and better urine production and quality compared to complement inhibition alone. COVID-19 shutdown March-May 2020 has significantly affected the project since it was not possible to carry out non-COVID-19 related work in the laboratory and even longer with animal experiments. Employees in this project have nevertheless been able to use this time to carry out COVID-19 related research during the period when the work in the project was not possible: we have described the significance of complement system activation in hospitalized COVID-19 patients and this publication has become a guide for COVID-19 related therapeutic research and has been cited extensively. The research group in this project consisted of highly established national and international researchers and we intend to continue with a clinical study in liver and kidney transplant patients after this project. The PhD research fellow is in the writing phase to complete the articles and her PhD. The PostDoc has completed her projects and has started in an industry job where the competence gained within this project was sought after. The project manager has received financial support from organizations other than the Research Council of Norway during the project period, so that work on the theme complex from this application will continue. Companies producing complement inhibitors are approached and we will proceed with planning of clinical studies in cardiac arrest and organ transplantation patients. Supplementary information is also available on the group's newly established website ncrg.no.

Prosjektleder har etablert egen forskningsgruppe, fått tildeling av forskningsmidler fra Universitet i Oslo og Helse Sør-Øst og har startet opp internasjonalt samarbeid med Universitet i Aarhus (Prof. Bente Jespersen) og Universitet i Groningen, Nederlande (Prof. Henri Leuvenink). PhD stipendiat og PostDoc har gjennomført prosjektene sine etter planen. Videre forskning vil fokusere på behandling av transplanterbare organer før transplantasjon for å rekonstituere og forbedre organer. Hovedmål er å utvide antall transplanterbare organer, noe som vil være nyttig for samfunnet. Vi akter til å søke EU forskningsmidler for felles prosjekter. Komplementhemning har blitt vist til å være av stor betydning for inflammasjon og generelt pasientutkomme. Dette har allerede blitt lagt merke til internasjonalt og betydning av inflammasjon utløst av iskemi/reperfusjon og en klinisk studie i hjertestanspasienter med intervensjon i komplementsystemet anses som neste steg etter dette prosjektet.

Ischemia and reperfusion injury is a key challenge of potentially life-threatening conditions like heart infarction, circulatory arrest, trauma, vascular diseases, and organ transplantation. Inflammation is a decisive part of the pathophysiology of ischemia/reperfusion injury, in particular, activation of the innate immune system with the complement system and the Toll-like receptor molecules such as CD14. We hypothesize that: Double inhibition of key molecules of the complement system (C3/C5) and Toll-like receptor activation via CD14 is a novel, ground-breaking concept of therapy for ischemia and reperfusion injury, being directly correlated to decreased activation of the complement system and TLRs. The present proposal represents translational research with patient studies as well as animal and cell culture studies. Complement activation, cytokines release, etc. will be correlated with acute and long term complications in patients with ischemia/reperfusion injury either to the whole body after cardiac arrest (n=259) or localized after mild to moderate head injury (n=209). The effect of double inhibition in acute sterile inflammation will be evaluated in endothelial cell cultures and in an ex vivo porcine model of renal ischemia/reperfusion injury. Hypoxic endothelial cells will be reoxygenated with whole human blood. Harvested pig kidneys will be preserved for 19 hours and reperfused with oxygenated whole blood. The multidisciplinary research group includes top national and international experts. All equipment, assays, and techniques are established and available for our world-leading laboratory on inflammation studies. Dual blockade of inflammation targeting key complement and Toll-like receptor molecules can act as a novel and highly improved therapeutic approach for conditions involving ischemia and reperfusion. Due to the very large number of patients suffering from these diseases, the benefit for the individual patient and for society will be substantial.