The role of complement and Toll-like receptors in thromboinflammation

Sepsis fra bakterier er en alvorlig tilstand som årlig krever mange liv. Selv om immunsystemet og det hemostatiske system samarbeider for å bekjempe bakterieinfeksjoner, kan dette i noen tilfelle skyte over mål. Derfor har vi i denne studien undersøkt om bakterieindusert aktivering av koagulasjon og inflammasjon reduseres av en dobbelthemming av komplementsystemet og toll-like reseptorer (TLR). Det ble benyttet en human fullblodsmodell for inflammasjon der blod fra friske givere tilsettes spesifikke hemmere av komplement og TLR etter stimulering med bla. Gram-negative og Gram-positive bakterier. Etter at rørene ble inkubert i ønsket tid kunne ulike analytter måles ved hjelp av flowcytometri, immunoassay (ELISA), tromboelastometri og polymerase kjedereaksjonen (qPCR). Den kombinerte behandlingen reduserte den Escherichia coli (E.coli)-induserte koagulasjonsaktiveringen målt som protrombin fragment 1+2 og vevsfaktor målt på mRNA nivå, uttrykt på monocyttenes overflate og uttrykt på mikropartikler i plasma. C1-INH, en multifunksjonell serin protease inhibitor, reduserte også denne bakterie-induserte koagulasjonsaktiveringen målt med tromboelastometri. Hemming av komplementfaktor C5 hadde også betydning for E.coli-indusert koagulasjon, men påvirket ikke koagulasjonen under normale forhold. Anti-CD14, som hemmer flere TLR enn den spesifikke TLR4/MD2 hemmeren eritoran, gav mest effektiv hemming av den bakterieinduserte vevsfaktoren og den inflammatoriske responsen. Komplementsystemet og TLR er begge nødvendig for å gjenkjenne bakteriene og starte den inflammatoriske responsen ved sepsis. En kombinert hemming ser ut til å kunne hindre overaktiveringen av denne responsen. Imidlertid gjenstår mye arbeid før kombinasjonshemmingen kan bli en del av sepsisbehandlingen sammen med antibiotika, og viktig støttende terapi som vasopressor og intravenøs væske.Sepsis is a serious and complex disease leading to 5 million deaths worldwide every year. Although the immune system is working hard to overcome the bacteria, the inflammatory response can escalate and attack the host. Antibiotic is given to kill the bacteria. By studying the complement system, and important part of primary immune system, and its reaction with another part of this system, the Toll-like receptors (TLRs), we aimed to improve the treatment of sepsis. We used a human blood model of inflammation developed at our Research Laboratory. Bacteria was added to blood from healthy donors and specific inhibitors of complement and TLRs were investigated as possible candidate drugs to treat sepsis. We observed that each of the two inhibitors reduced the detrimental effects induced by the bacteria, while the combination of the two virtually abolished bacteria-induced clotting and inflammatory responses. The thesis has given knowledge that may contribute to new treatment of sepsis

Complement C3b contributes to Escherichia coli-induced platelet aggregation in human whole blood

We found that compstatin, which inhibits the cleavage of complement component C3 to its components C3a and C3b, reduced the E. coli-induced platelet aggregation by 42%-76% (p = 0.0417). This C3-dependent aggregation was not C3a-mediated as neither inhibition of C3a using a blocking antibody or a C3a receptor antagonist, nor the addition of purified C3a had any effects. In contrast, a C3b-blocking antibody significantly reduced the E. coli-induced platelet aggregation by 67% (p = 0.0133). We could not detect opsonized C3b on platelets, indicating that the effect of C3 was not dependent on C3b-fragment deposition on platelets. Indeed, inhibition of glycoprotein IIb/IIIa (GPIIb/IIIa) and complement receptor 1 (CR1) showed that these receptors were involved in platelet aggregation. Furthermore, aggregation was more pronounced in hirudin whole blood than in hirudin platelet-rich plasma, indicating that E. coli-induced platelet aggregation involved other blood cells. In conclusion, the E. coli-induced platelet aggregation in human whole blood is partly C3b-dependent, and GPIIb/IIIa and CR1 are also involved in this process

Dental and Periodontal Health in Acute Intermittent Porphyria

In the inherited metabolic disorder acute intermittent porphyria (AIP), high sugar intake prevents porphyric attacks due to the glucose effect and the following high insulin levels that may lower AIP disease activity. Insulin resistance is a known risk factor for periodontitis and sugar changes diabetogenic hormones and affects dental health. We hypothesized differences in homeostasis model assessment (HOMA) scores for insulin resistance in AIP cases vs. controls and in those with periodontitis. Our aim was to systematically study dental health in AIP as poor dental health was previously only described in case reports. Further, we aimed to examine if poor dental health and kidney failure might worsen AIP as chronic inflammation and kidney failure might increase disease activity. In 47 AIP cases and 47 matched controls, X-rays and physical examination of clinical attachment loss (CAL), probing pocket depth (PPD), and decayed missing filled teeth (DMFT) were performed. Dietary intake was evaluated through a diet logbook. Plasma cytokines and diabetogenic hormones were measured using multiplex technology and urine porphobilinogen and kidney and liver function by routine methods. An excel spreadsheet from the University of Oxford was used to estimate HOMA scores; beta cell function, HOMA%B (%B), insulin sensitivity, HOMA%S (%S), and insulin resistance HOMA-IR (IR), based on glucose and plasma (P) C-peptide. The Wilcoxon matched-pairs signed rank test, the Mann–Whitney U-test, and Spearman’s nonparametric correlation were used. Insulin (p = 0.007) and C-peptide (p = 0.006) were higher in the AIP cases with periodontitis versus those without. In AIP patients, the liver fibrosis index 4 correlated with DMFT (p < 0.001) and CAL ≥4 mm (p = 0.006); the estimated glomerular filtration rate correlated with DMFT (p < 0.001) and CAL ≥4 mm (p = 0.02). CAL ≥4 mm was correlated with chemokine ligand 11 and interleukin (IL)-13 (p = 0.04 for both), and PPD >5 mm was correlated with plasminogen activator inhibitor-1 (p = 0.003) and complement component 3 (p = 0.02). In conclusion, dental health in AIP cases was correlated with insulin resistance, inflammatory markers, and biomarkers of kidney and liver function, demonstrating that organ damage in the kidney and liver are associated with poorer dental health

Complement C3b contributes to Escherichia coli-induced platelet aggregation in human whole blood

IntroductionPlatelets have essential functions as first responders in the immune response to pathogens. Activation and aggregation of platelets in bacterial infections can lead to life-threatening conditions such as arterial thromboembolism or sepsis-associated coagulopathy.MethodsIn this study, we investigated the role of complement in Escherichia coli (E. coli)-induced platelet aggregation in human whole blood, using Multiplate® aggregometry, flow cytometry, and confocal microscopy.Results and DiscussionWe found that compstatin, which inhibits the cleavage of complement component C3 to its components C3a and C3b, reduced the E. coli-induced platelet aggregation by 42%-76% (p = 0.0417). This C3-dependent aggregation was not C3a-mediated as neither inhibition of C3a using a blocking antibody or a C3a receptor antagonist, nor the addition of purified C3a had any effects. In contrast, a C3b-blocking antibody significantly reduced the E. coli-induced platelet aggregation by 67% (p = 0.0133). We could not detect opsonized C3b on platelets, indicating that the effect of C3 was not dependent on C3b-fragment deposition on platelets. Indeed, inhibition of glycoprotein IIb/IIIa (GPIIb/IIIa) and complement receptor 1 (CR1) showed that these receptors were involved in platelet aggregation. Furthermore, aggregation was more pronounced in hirudin whole blood than in hirudin platelet-rich plasma, indicating that E. coli-induced platelet aggregation involved other blood cells. In conclusion, the E. coli-induced platelet aggregation in human whole blood is partly C3b-dependent, and GPIIb/IIIa and CR1 are also involved in this process

Effekten av C1-inhibitor på E.coli- og LPS-indusert "tissue factor" i humant fullblod

Sepsis er fortsatt en alvorlig tilstand med høy mortalitet, til tross for mange år med intens forskning. Ved sepsis er det en systemisk inflammasjonsreaksjon som involverer komplement, koagulasjons- og kallikrein-kininsystemet. Den økte prokoagulante tilstanden som blant annet skyldes økt uttrykk av vevsfaktor (TF) vil bidra til disseminert intravaskulær koagulasjon (DIC) og organsvikt. Det ble i denne studien undersøkt hvilken effekt C1-inhibitor (C1-INH) har på koagulasjonsaktiveringen ved Gram-negativ sepsis i en human fullblodsmodell. C3 hemmeren compstatin ble brukt for å sammenligne effekten av C1 INH med en ren komplementhemmer. Dette gjorde det mulig å studere om koagulasjonsaktiveringen i modellen er komplementmediert. Det ble benyttet en godt etablert human fullblodsmodell med E. coli og renset lipopolysakkarid (LPS) fra Escherichia coli (E. coli) som stimuli. Antikoagulanten lepirudin ble benyttet. Denne har ingen innvirkning pår komplementaktiveringen og hemmer koagulasjonsaktiveringen ved å hemme trombin direkte. Prøvene ble inkubert med stimuli og hemmere i 60 eller 120 minutter. Koagulasjonsaktiveringen ble målt som protrombin faktor 1 og 2 (protrombin F1+2) med ELISA. Relativ ekspressjon av TF mRNA ble bestemt ved hjelp av RT-qPCR. Det ekstracellulære TF uttrykket på bla. monocytter ble målt ved hjelp av flowcytometri. Aktiviteten av TF i plasma mikropartikler ble bestemt med en enzymatisk metode. Det terminale komplement komplekset (TCC) og lang pentraxin 3 (PTX3), en inflammatorisk mediator, ble målt med ELISA. En rekke (27) cytokiner i plasma ble analysert med multiplex teknologi. Både C1-INH og compstatin hemmet effektivt koagulasjonsaktiveringen målt som protrombin F1+2 etter tilsetting av E. coli og LPS i humant fullblod. Både E. coli og LPS stimuli ga økt nivå av membranbundet TF på overflaten av monocytter, TF mRNA og TF aktivitet i plasmamikropartikler. Denne økningen av TF ble effektivt redusert av C1-INH. Compstatin hemmet til sammenligning TF mRNA oppregulering og TF uttrykket på monocytt-overflaten, men ga en ikke-signifikant hemming av TF funksjon i plasmamikropartikler. E. coli indusert komplementaktivering målt som TCC ble også effektivt redusert både av C1-INH og compstatin. C1-INH reduserte syntesen av PTX3 og cytokiner, mens compstatin til sammenligning kun reduserte IL 8 nivået. Inaktivert C1-INH reduserte også E. coli og LPS-indusert koagulasjonsaktivering. Resultatene viser at C1-INH i høye konsentrasjoner kan redusere E. coli og LPS-indusert oppregulering av TF og koagulasjonsaktivering. Compstatin reduserte også E. coli og LPS-indusert oppregulering av TF uttrykk og koagulasjonsaktivering. Dette kan indikere at komplementaktivering spiller en viktig rolle for koagulasjonsaktiveringen under en Gram -negativ sepsis

Lifestyle factors including diet and biochemical biomarkers in acute intermittent porphyria: Results from a case-control study in northern Norway

Background: Lifestyle factors, including a low intake of carbohydrates, dieting, alcohol consumption, cigarette smoking and stress are some of the possible triggers of attacks in acute intermittent porphyria (AIP). The influence of lifestyle factors, including energy intake, diet and alcohol consumption on the biochemical disease activity in AIP and biochemical nutritional markers were examined. Methods: A case-control study with 50 AIP cases and 50 controls matched for age, sex and place of residence was performed. Dietary intake was registered using a food diary in 46 matched pairs. Symptoms, alcohol intake, stress and other triggering factors of the last AIP attack were recorded on questionnaires. Porphyrin precursors, liver and kidney function markers, vitamins, diabetogenic hormones and other nutritional biomarkers were analyzed by routine methods. The Wilcoxon matched-pairs signed rank test was used to compare the cases vs. controls. The Spearman's rank correlation coefficient was used on the cases. Results: Increasing total energy intake was negatively correlated with the biochemical disease activity. The intake of carbohydrates was lower than recommended, i.e., 40 and 39% of total energy intake in the AIP cases and controls, respectively. The plasma resistin level was significantly higher (p = .03) in the symptomatic than asymptomatic cases. Plasma insulin was lower in those with high porphobilinogen levels. The intake of sugar and candies were higher in the AIP cases with low U-delta aminolevulinic acid (ALA) levels (p = .04). Attacks were triggered by psychological stress (62%), physical strain (38%), food items (24%) and alcohol (32%) in the 34 symptomatic cases. Alcohol was used regularly by 88% of the cases (3.2 g ethanol/day) and 90% of the controls (6.3 g/day), but the intake was significantly lower in symptomatic than in asymptomatic cases (p = .045). Conclusion: A high intake of energy, sugar and candies and a higher insulin level were associated with a lower biochemical disease activity. The resistin level was higher in the symptomatic than the asymptomatic cases. AIP patients drink alcohol regularly, but the intake was significantly lower in the symptomatic cases.<p

Cholesterol Crystals Induce coagulation Activation through Complement-Dependent Expression of Monocytic Tissue Factor

Cholesterol crystals (CC) are strong activators of complement and could potentially be involved in thromboinflammation through complement-coagulation cross-talk. To explore the coagulation-inducing potential of CC, we performed studies in lepirudin-based human whole blood and plasma models. In addition, immunohistological examinations of brain thrombi and vulnerable plaque material from patients with advanced carotid atherosclerosis were performed using polarization filter reflected light microscopy to identify CC. In whole blood, CC exposure induced a time- and concentration-dependent generation of prothrombin fragment 1+2 (PTF1.2), tissue factor (TF) mRNA synthesis, and monocyte TF expression. Blocking Abs against TF abolished CC-mediated coagulation, thus indicating involvement of the TF-dependent pathway. Blockade of FXII by corn trypsin inhibitor had a significant inhibitory effect on CC-induced PTF1.2 in platelet-free plasma, although the overall activation potential was low. CC exposure did not induce platelet aggregation, TF microparticle induction, or TF on granulocytes or eosinophils. Inhibition of complement C3 by CP40 (compstatin), C5 by eculizumab, or C5aR1 by PMX53 blocked CC-induced PTF1.2 by 90% and reduced TF+ monocytes from 18-20 to 1-2%. The physiologic relevance was supported by birefringent CC structures adjacent to monocytes (CD14), TF, and activated complement iC3b and C5b-9 in a human brain thrombus. Furthermore, monocyte influx and TF induction in close proximity to CC-rich regions with activated complement were found in a vulnerable plaque. In conclusion, CC could be active, releasable contributors to thrombosis by inducing monocyte TF secondary to complement C5aR1 signaling