Background Elevated plasma mannose-binding lectin (MBL) levels are associated with increased risk of venous thromboembolism (VTE). Although MBL levels are affected by environmental factors, it is mainly genetically regulated. Objectives We aimed to investigate the association between genetically predicted MBL and VTE risk in a population-based cohort. Methods The study comprised 68 999 individuals from the Trøndelag Health Study (HUNT). Six genetic variants were used to classify individuals in low, medium, and high genetically predicted MBL. Linear regression was applied to estimate whether genetically predicted MBL explained plasma variability of MBL. Cox regression was used to estimate hazard ratios (HRs) with 95% CIs for VTE across genetically predicted MBL categories stratified by sex (men and women) and age groups (reference: low category). Results There were 2043 incident VTEs during 12 years’ median follow-up. Genetically predicted MBL explained 52.8% of the plasma variability of MBL, and women had lower plasma MBL than men. Genetically predicted MBL was associated with a significantly increased risk of VTE in individuals <60 to 65 years and displayed sex differences. In sex- and age-stratified analysis, men <50 years with high genetically predicted MBL had elevated risk of overall VTE (HR, 2.13; 95% CI, 1.27-3.55), unprovoked VTE (HR, 2.87; 95% CI, 1.28-6.43), and pulmonary embolism (HR, 2.66; 95% CI, 1.11-6.33). In older men and in women, no associations were found. Conclusion We found a moderate association between genetically predicted MBL and VTE in young- and middle-aged men. Sex differences and accumulation of environmental factors with age might preclude associations in older men and in women.

Background:Complement activation is increasingly recognized as a key driver in various critical diseases and a potential therapeutic target. However, conventional enzyme-linked immunosorbent assays (ELISAs) for complement protein detection are laboratory-bound and unsuitable for rapid clinical decision-making in the emergency and intensive care unit setting. We evaluated the feasibility of a point-of-care (POC) multiplex micro-ELISA for complement component C3, activation marker C3d and the soluble terminal complement complex (TCC) in a heterogeneous intensive care unit (ICU) population. Method:Thirty-eight critically ill and three cardiopulmonary bypass patients were prospectively enrolled. Fifteen age- and sex-matched healthy volunteers served as controls. Plasma samples collected at ICU admission and days 2-4 were analyzed using a novel POC multiplex micro-ELISA platform and compared with established in-house and commercial ELISAs using Spearman rank correlation and receiver operating characteristic (ROC) analysis. Additional complement activation markers (C3bc, C3bBbP, C1s/C1-INH, MASP-1/C1-INH and C4d) were assessed to explore specific pathway involvement. Results:TCC levels were significantly elevated in patients (median 3575, IQR 2048-6504 mAU/ml) compared with controls (median 1286, IQR 1032-1903 mAU/ml; p < 0.0001) at both sampling time points. The POC assay demonstrated strong rank correlation with the in-house TCC ELISA (r = 0.83, p < 0.001) and the commercial Hycult TCC ELISA (r = 0.88, p < 0.001). The POC TCC assay accurately discriminated patient samples above and below the ELISA-defined threshold (AUC 0.97, p < 0.001). In contrast, C3 and C3d showed limited patient-control discriminatory ability and poor POC-ELISA agreement. C3bc and C3bBbP were elevated in patients, suggesting alternative pathway amplification. C1s/C1-INH and C4d were not significantly elevated, whereas MASP-1-C1-INH complexes were decreased in patients compared to healthy controls. Conclusion:Point-of-care TCC quantification is feasible, correlates well with established ELISAs and accurately identifies elevated TCC levels in critically ill patients. Although formal method-comparison analyses were not possible to establish, TCC emerged as the most robust marker of complement activation in this ICU cohort using the evaluated POC prototype. POC testing may facilitate timely identification of complement activation in acute care settings and support integration of complement-directed diagnostics and therapeutics into clinical workflows.

RATIONALE:Severe bacterial pneumonia is a leading cause of death worldwide, and mortality rates remain unacceptably high despite appropriate antibiotic treatment. Macrophage membrane-coated nanoparticles (MΦ-NPs) are biomimetic constructs engineered to neutralize bacterial toxins, pathogen-associated molecular patterns, and proinflammatory cytokines. OBJECTIVE:To evaluate the therapeutic potential of MΦ-NPs for the treatment of severe bacterial pneumonia. METHODS:We evaluated the therapeutic potential of MΦ-NPs using in vitro models of infection involving human lung endothelial and epithelial cells, as well as primary human neutrophils, and in vivo murine models of Pseudomonas aeruginosa (PA) and methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. MAIN RESULTS:MΦ-NPs demonstrated potent cytoprotective and anti-inflammatory activity in vitro, without impairing neutrophil antimicrobial function. In both PA and MRSA pneumonia models, MΦ-NP treatment significantly improved survival, reduced bacterial burden, lowered proinflammatory cytokines, and preserved lung architecture. Quantitative proteomics further revealed suppression of inflammatory, coagulation, and fibrotic pathways associated with poor outcomes in human pneumonia and ARDS. CONCLUSIONS:These findings establish MΦ-NPs as a promising host-directed therapeutic strategy for mitigating the deleterious inflammatory sequelae of severe bacterial pneumonia.

Thoracic injury is prevalent among polytrauma patients, affecting up to 45

BackgroundSystemic inflammation is a crucial component of the acute response to stroke and is associated with poor clinical outcomes. However, its connection to long-term structural brain changes as a pathophysiological explanation for poor outcomes remains incompletely understood.MethodsA total of 241 stroke patients (44.8% women) were included from the Norwegian Cognitive Impairment After Stroke (Nor-COAST) study, with a mean age of 72.8 years and a mean NIHSS score of 3.1. Inflammatory biomarkers were collected from blood during the acute phase (median 4 days after stroke). MRI was performed during the acute stroke phase of the stroke and at 18- and 36-month follow-up, including measures of white matter hyperintensity (WMH) volume, global and hippocampal gray matter volume, and brain age gap (BAG). Relationships between inflammatory biomarkers and brain measures were assessed using linear mixed-effects models.ResultsHigher levels of IL-2, IL-6, MIP-1α, MIP-1β, and IP10 were associated with lower baseline global gray matter volume and higher baseline BAG. IL-1RA, IL-4, IL-5, IL17A, TCC, bFGF, and G-CSF were associated with WMH progression over 36 months. No inflammatory biomarker was associated with baseline hippocampal volume, whereas bFGF and G-CSF were associated with greater hippocampal volume decline over time.ConclusionAcute systemic inflammation after stroke was associated with signs of brain atrophy and white matter damage, characteristics of cerebral atherosclerotic and small vessel disease, which are risk factors for subsequent cerebrovascular events and vascular dementia. In contrast, evidence for an association with hippocampal volume, a hallmark of Alzheimer’s disease, was limited.

Opioid use modulates parts of the immune system, including cytokines, but with disparate results. Furthermore, several studies have demonstrated a positive correlation between circulating proinflammatory cytokines and mental distress of various kinds. The aim of this study was to investigate the relationship between self-reported mental health symptoms and peripheral circulating cytokines in opioid maintenance treatment patients to see whether the previous disparate results could at least in part be explained by an interaction with mental distress. In a cross-sectional study, we investigated levels of 27 serum cytokines and chemokines using multiplex technology in 120 patients with chronic hepatitis C virus infection. Self-reported mental health symptoms were obtained using SCL-90-R. Among the nonopioid maintenance treatment patients, we found a positive correlation between self-reported mental health symptoms and peripheral circulating cytokines. An opposite trend was found for several of the proinflammatory cytokines in the opioid maintenance treatment patients, which was confirmed through linear regression analysis. We found an interaction between symptom scores and group affiliation on peripherally circulating cytokine levels for four of the cytokines. This report demonstrates that opioids seem to interact with self-reported mental health symptoms in a way that impacts levels of cytokines. We propose that opioids might be associated with a pro-inflammatory dampening and that this should be taken into consideration when analysing cytokine levels.

BACKGROUND:Combined inhibition of complement-component C5 and Toll-Like Receptor CD14 is a promising therapeutic strategy to attenuate post-traumatic inflammatory-complications. The effect of this inhibition on miRNA expression at the fracture site and in systemic circulation was assessed in a porcine multiple-trauma model. Expression of the mRNA targets of deregulated miRNAs was determined at the fracture site and in lung and liver. METHODS:Two experimental groups, intramedullary nailing (control; n = 8) and intramedullary nailing with combined C5/CD14 inhibition (n = 4), were compared using an established porcine multiple-trauma model. Fracture hematoma, fractured bone, unfractured control bone, lung, and liver samples were collected. Extracellular-vesicles (EVs) were isolated from plasma at 1.5, 2.5, 24, and 72 h post-trauma. MiRNA qPCR array analyses were performed on pooled fxH, fX, CB, and EV samples. In silico mRNA target prediction and mRNA qPCR analyses were conducted on all samples. RESULTS:The fracture site miRNA signature in the C5/CD14 inhibition group was anti-inflammatory compared to intramedullary nailing alone, based on miRNA and mRNA qPCR analyses, with downregulation of anti-osteogenic miRNAs. Furthermore, C5/CD14 inhibition reduced pro-inflammatory and pro-fibrotic EV-carried miRNA expression in systemic circulation and their downstream mRNA targets in lung and liver. CONCLUSIONS:Anti-inflammatory miRNA and mRNA expression were upregulated at the injury site in the C5/CD14 inhibition group, while pro-osteogenic pathways were preserved by downregulating anti-osteogenic miRNAs. Systemically, C5/CD14 inhibition reduced EV-carried inflammatory miRNA expression, consistent with mRNA target analyses in lung and liver. Thus, C5/CD14 inhibition may be a novel therapeutic to modulate post-traumatic immune responses and decrease systemic effects of surgical trauma.

Abstract The therapeutic landscape of complement inhibition has recently expanded considerably. However, most approved agents specifically target single complement components systemically, which can result in limited functional effects. In fact, stoichiometric inhibition has shown limitations both in vitro and in vivo. In this study, we evaluated, to our knowledge, for the first time the in vivo efficacy of “triple-fusion” (TriFu), an engineered fusion protein comprising key domains of 3 natural complement regulators, CD55, factor H, and CD35, including a host-recognition motif. In a rat xenotransfusion model, TriFu provided favorable protection of transfused human red blood cells compared to C5 inhibition by Ornithodoros moubata complement inhibitor, prolonging circulation time on par with cobra venom factor–mediated complement depletion and completely inhibiting C3 opsonization. Furthermore, TriFu treatment improved multiple clinical inflammatory parameters (eg, interleukin-6 and tumor necrosis factor α). These findings suggest that regulatory modulation of complement could offer advantages over C5 inhibition, providing a more balanced and adaptive therapeutic approach.

Background and hypothesisInfection is a leading cause of morbidity and mortality in patients with chronic kidney disease (CKD). The complement system provides crucial first-line defense against pathogens. Mannose-binding lectin (MBL), ficolins (1, 2 and 3), and collectin-LK 1 (CL-LK) are pattern recognition molecules (PRMs) of the lectin pathway (LP) that recognize microbial surfaces and activate complement. C3dg is a complement cleavage fragment indicating complement activation. The aim of the study was to investigate whether levels of PRMs and C3dg are associated with the risk of significant infections requiring hospitalization in patients with CKD.MethodsThis prospective cohort study included 518 patients ≥18 years with CKD (eGFR<60 mL/min/1.73 m2), consecutively recruited between 2008-2022. About half (270/518) were in dialysis at inclusion. None of the patients were previously transplanted with any organ or stem cells. The primary endpoint was non-access-related infections requiring hospitalization. Patients were followed until kidney transplantation or death or until 31.12.2024. Plasma concentrations of the biomarkers were measured at inclusion. Time-to-event analyses using Cox regression were employed to assess associations with infection, adjusting for age, sex, diabetes, dialysis status, and dialysis vintage.ResultsDuring a median (interquartile range [IQR]) time of follow-up of 1.24 (0.49-2.76) years, 182 patients (35%) were hospitalized due to non-access infection. Higher baseline levels of ficolin-1 and C3dg were independently associated with infection risk, HR 3.05, 95% CI 1.25-7.43, p=0.01 and HR 2.97, 95% CI 1.37-6.44, p=0.006, respectively, for each log10 unit increase. In multivariable models including all biomarkers, only C3dg remained independently associated with infection (HR 2.81, 95% CI 1.23-6.43, p=0.01).ConclusionsHigh levels of complement activation (C3dg) and ficolin-1 were independently associated with increased infection risk in patients with CKD. A dysregulated complement activation rather than PRM deficiency seems to be a key pathogenic mechanism resulting in increased infection risk in advanced CKD.

Objectives:Polytrauma is characterized by high mortality and morbidity rates, partly due to the post-traumatic immune response. This follow-up study investigated the effect of combined inhibition of complement factor 5 (C5) and CD14 on systemic (plasma) and local (fracture hematoma) protein levels in a porcine polytrauma model. Methods:18 male pigs (sus scrofa) were used for this study: a control group (n=6), and two groups that were subjected to standardized polytrauma, followed by standard treatment (intramedullary nailing; n=8) or standard treatment with C5/CD14 inhibition therapy (n=4). Plasma and fracture hematoma samples were collected at specified time points and the expression levels of proteins related to the post-traumatic immune response and fracture healing were determined using enzyme-linked immunosorbent assays. Results:Pro-inflammatory proteins such as IL-1β, IL-6, and IFN-α were significantly reduced in both plasma and fracture hematoma samples at 72 h after trauma in the treated vs. the non-treated group. Soluble TLR4, a possible inhibitor of cell membrane TLR4, was increased in both the plasma and the fracture hematoma following therapy, suggesting that TLR4 was released from the cell membrane to the fluid phase. Conclusions:These findings show that systemic C5/CD14 inhibition effectively reduced the concentration of several pro-inflammatory proteins in the systemic circulation and locally, at the fracture site, in the fracture hematoma. The levels in the plasma were in line with those in the fracture hematoma, albeit that they were markedly lower in the plasma compared to the fracture hematoma.

In 2021, 1.6 million people died from Tuberculosis (TB). This makes Mycobacterium tuberculosis (Mtb), the causative agent of TB, one of our top infectious killers. The complement system is an integral part of the innate immune system that curbs such bacterial infections.Although the complement system was previously thought to be hepatically derived and extracellular only, a cell-autonomous and intracellular complement system involved in metabolic and inflammatory pathways in several cell types has recently been identified. Limited research has been published on the role of complement in TB; thus it remains to be elucidated if the intracellular complement system plays a role in Mtb infection.We have previously demonstrated that intracellular C5a signaling via a mitochondrial C5a receptor contributes to inflammasome activation upon sensing danger signals in monocytes and macrophages. We now want to explore the role of C3/C3aR in these cells, using Mtb as a model system for infection.To investigate the role of intracellular C3 in human macrophages, we have used CRISPR-Cas9 knock-out (KO) THP-1 cells lacking C3 or the C3a receptor (C3aR). Following stimulation with LPS and Mtb, the C3 KO cells, but not the C3aR KO cells, showed a marked decrease in the release of several NF-κB-regulated pro-inflammatory cytokines, including TNF-α, IL-8, and IL-1β, compared to the wild-type cells. This effect was observed on both the mRNA and protein levels, indicating that C3 may play a C3aR-independent role in the NF-κB signaling pathway. Supporting this hypothesis, phosphorylation of the IKKα/β complex, an activation step required for nuclear translocation of NF-κB, was strongly reduced in stimulated C3 KO cells, but not in the C3aR KO cells. Other NF-κB pathway kinases, such as TAK1, also appear to be affected.However, it remains to be determined if full-length C3 or one of its split products is responsible for the observed effects. The exact step in the NF-κB pathway where C3 (or a split-product of C3) interacts is also unidentified. Overall, our data demonstrate that cell-autonomous C3 mediates inflammatory responses in human macrophages.

BACKGROUND:MBL (mannose-binding lectin), a pattern recognition molecule circulating in complex with MASPs (MBL-associated serine proteases), activates the lectin complement pathway and facilitates thrombin generation upon binding to specific moieties on pathogens or altered host cells. We aimed to investigate the association between plasma MBL levels and risk of future venous thromboembolism (VTE) and to explore the effect of MBL-MASP-1/2 complexes on thrombin generation. METHODS:We conducted a population-based case-cohort (294 VTE patients, 1066 sex- and age-weighted subcohorts) derived from HUNT (The Trøndelag Health Study). Plasma MBL levels were measured using the SomaScan 7k aptamer-based platform. Cox proportional hazards regression models were used to estimate hazard ratios for VTE across quartiles of MBL levels. Thrombin generation in plasma induced by MBL-MASPs complexes was assessed in vitro. RESULTS:Subjects with MBL levels in the highest quartile had a 79% higher risk of overall VTE (hazard ratio, 1.79 [95% CI, 1.23-2.61]) than those with MBL levels in the lowest quartile after multivariable adjustments. The risk estimates by high plasma MBL were particularly strong for deep vein thrombosis (hazard ratio, 2.50 [95% CI, 1.42-4.37]) and unprovoked VTE (hazard ratio, 2.81 [95% CI, 1.53-5.16]). MBL-MASP-1/2 complexes promoted complement activation and thrombin generation, and monospecific inhibitors abolished their enzymatic activity. CONCLUSIONS:Our findings support the notion that high plasma MBL levels are associated with an increased risk of future VTE and suggest that the risk increase is partially mediated through the initiation of thrombin generation by MBL-MASP-1/2 complexes at the site of venous thrombosis formation.

BACKGROUND:The overuse of compression ultrasound procedures on patients with suspected deep vein thrombosis compromise the cost-effectiveness of deep vein thrombosis management and emphasize room for improvement of the current diagnostic algorithm. As the complement system and hemostasis are comprehensively intertwined, we aimed to investigate complement activation products as diagnostic tests for acute deep vein thrombosis alone or together with D-dimer. METHODS:We performed a cross-sectional study using consecutive sampling of outpatients referred to the emergency department with suspected first-time deep vein thrombosis of the lower limbs, to investigate the diagnostic utility of the index tests C3bc and C5b-C9 terminal complex (TCC) with compression ultrasound as reference standard. For comparison of receiver operating characteristics, analyses were also performed for D-dimer and C-reactive protein in addition to analyses for the index tests on a D-dimer positive patient subgroup. RESULTS:Of the 366 included patients, 103 had deep vein thrombosis. The calculated effect sizes of differences between groups (Cohen's d) with 95% confidence intervals (95%CI) were 0.25 (95%CI 0.03-0.48) for C3bc, 0.33 (95%CI 0.09-0.57) for C5b-C9 terminal complex (TCC), 0.88 (95%CI 0.61-1.15) for C-reactive protein, and 1.64 (95%CI 1.37-1.91) for D-dimer. The areas under the curves derived from comparison receiver operating characteristics analyses with corresponding 95%CIs for C3bc, C5b-C9 terminal complex (TCC), C-reactive protein and D-dimer were 0.56 (95%CI 0.50-0.63), 0.64 (95%CI 0.58-0.69), 0.73 (95%CI 0.67-0.79) and 0.92 (95%CI 0.89-0.95), respectively. CONCLUSION:The plasma levels of the complement activation products C3bc and C5b-C9 terminal complex (TCC) were elevated in patients with acute deep vein thrombosis but displayed low diagnostic performance for deep vein thrombosis alone or together with D-dimer.

IntroductionThe functionalization of nanoparticles (NPs) with an antiCD19 targeting mechanism represents a promising approach for the selective delivery of drugs and nucleic acids into normal and tumor B cells. This strategy has the advantage of minimizing off-target effects by restricting gene delivery to the desired cell population. However, the nanoplatform must guarantee both the local production of the protein and the safety of the treatment to allow an effective therapy with reduced systemic toxicity.MethodsIn order to ensure a selective delivery of nucleic acids, we developed poly(lactic-co-glycolic acid) (PLGA)-poly(vinyl alcohol) (PVA) NPs loaded with an Enhanced Green Fluorescent Protein (EGFP)-coding plasmid and covalently coated with antiCD19 recombinant antibody as a targeting mechanism. To assess the functionality of the NPs, physicochemical characterization, safety tests, and transfection assay were employed to evaluate the NPs’ behavior in vitro and in vivo, in a human/zebrafish lymphoma xenograft model.ResultsThe results demonstrated that the PLGA-PVA nanoplatform was capable of efficiently encapsulating and releasing the payload. These nanostructures demonstrated a favorable safety profile, as evidenced by the absence of significant cell cytotoxicity, coagulation activation, complement system activation, and the slight activation of endothelial cells and leukocytes. The targeting mechanism facilitated the interaction of NPs with target cells, thereby enhancing their internalization and subsequent exogenous plasmid DNA (pDNA) translation and protein expression. In the human/zebrafish lymphoma xenograft model, no evidence of toxicity was observed, and targeted NPs demonstrated the capacity to enhance exogenous pDNA expression.ConclusionOur findings provide a rationale for the use of targeted NPs as a DNA delivery system for the local expression of therapeutic proteins.

Our objective was to explore whether plasma inflammatory biomarkers and related metabolites in acute phase and 3 months after stroke were associated with different cognitive trajectories and with changes in cognition post-stroke. The Norwegian Cognitive Impairment After Stroke (Nor-COAST) study was a prospective, multicentre cohort study of patients with acute stroke, followed up at 3, 18, and 36 months post-stroke. First, we modelled cognitive trajectory groups based on Montreal Cognitive Assessment (MoCA) scores and used multinominal logistic regression to study the associations between systemic inflammatory biomarkers/metabolites and group membership. Second, using mixed linear regression, we investigated whether the same biomarkers/metabolites were associated with changes in MoCA scores over time, stratified by pre-stroke cognitive status. The 466 participants had mean (SD) age 72 (12) years, 59% were males, and mean (SD) NIHSS score at admittance was 4 (4.8). Higher acute-phase values of the terminal complement complex, interleukin 6, macrophage inflammatory protein 1α, neopterin, quinolinic acid, and PA ratio = 4-pyridoxic acid / (pyridoxal + pyridoxal 5’-phosphate) and higher 3-month values of neopterin were associated with increased risk of being in the group characterized by low and declining MoCA score compared to the group of best MoCA score ( p < 0.01). Higher acute-phase values of tumour necrosis factor and interleukin 8, were associated with progressive decline in the MoCA score ( p < 0.01). Premorbid factors, and in particular pre-stroke frailty, had more impact on the models than stroke-related factors, and partly confounded several of these associations. Higher degrees of systemic inflammation in the acute phase were associated with worse cognitive trajectories and may reflect the response to the acute stroke, stroke-related complications and/or premorbid conditions. Trial registration: ClinicalTrials.gov: NCT02650531. Retrospectively registered January 8, 2016. First participant included May 18, 2015.

Blunt chest trauma is common in polytraumatised patients and often leads to respiratory distress. Moreover, the systemic inflammation resulting from the trauma itself, along with subsequent surgical interventions, further contributes to pulmonary dysfunction. Therefore, modulating post-traumatic immune responses may offer potential benefits. MicroRNAs may influence the activation and progression of regenerative responses following polytrauma and could serve as potential modulators. This study investigates the expression of a selection of miRNAs with known involvement in pulmonary pathologies relevant for the post-trauma setting, in a porcine polytrauma model comparing two surgical treatment groups and one treatment group that additionally received a drug-based treatment based on combined inhibition of complement component C5 and the Toll-like co-receptor CD14. The porcine polytrauma model consisted of blunt chest trauma, bilateral femur fractures, liver laceration, and haemorrhagic shock. Four groups were defined: sham, early total care (ETC: n = 8), damage control orthopaedics (DCO: n = 8), ETC with C5/CD14 inhibition (n = 4). Animals were monitored and guideline-treated in an ICU setting for 72 h. After sacrifice, lung samples were taken from the left lobe. MiRNAs were analysed by qPCR. Furthermore, Periodic Acid Schiff staining and in situ hybridisation were performed. MiRNAs associated with lung function, inflammation, and fibrosis were analysed. Compared to ETC, DCO resulted in less inflammatory and fibrotic miRNA expression, consistent with histological findings showing more preserved alveoli, less septal thickening, and fewer inflammatory cell infiltrations. The addition of C5/CD14 inhibitors to ETC further reduced the expression of inflammatory and fibrotic microRNAs compared to both DCO and ETC and revealed a significant reduction in histopathological changes in the lung tissue. This study indicates that combined inhibition of C5 and CD14 effectively reduces posttraumatic histopathological changes in lung tissue associated with less inflammatory and fibrotic miRNA expression, compared to both the DCO and ETC groups.

Platelet storage is associated with storage lesions, including platelet morphological changes and a gradual functional loss. We investigated the impact of complement C3 inhibition on complement activation and platelet storage lesions in clinical platelet concentrates. Platelet concentrates (n = 8) were prepared in PAS-E and stored for seven days at 22°C. Each concentrate was split in two, with the C3 inhibitor compstatin Cp40 added to one part, and the other serving as the control. Complement and platelet activation markers, platelet function, and metabolic measures were analyzed every second day. Cp40 significantly reduced C3bc and sC5b-9 levels, but not C4c, indicating inhibition of complement activation at the level of C3. However, Cp40 did not affect platelet-specific or metabolic measures. Surface expression of CD62P and NAP-2 release increased significantly over the storage time, whereas CD63 expression and PF4 and TSP-1 release remained stable. Platelet responses to TRAP-6 mediated PAR-1 activation and U46619 mediated TXA2R stimulation decreased over time, recorded as CD62P and CD63 expression and release of soluble factors. No drop in platelet count was observed, and metabolic markers remained within their critical limits. While C3 inhibition effectively reduced complement activation in stored platelet concentrates, it did not mitigate platelet storage lesions.

Background: The storage of platelets in concentrates requires an environment that supports their metabolic needs and maintains their function without activating the plasma components stored alongside the platelets. Over time, stored platelets develop storage lesions—progressive functional deterioration caused by morphological changes, altered surface markers, metabolic shifts, and the release of proteins, RNA, and other modifiers. We recently demonstrated increased complement activation during platelet storage, as evidenced by elevated levels of C1rs/C1-INH, MASP-1/C1-INH complexes, C4c, C3bc, and sC5b-9 (1). This activation correlated with heightened platelet activation and diminished responsiveness, particularly to TXA2R stimulation. Methods: This study explored whether complement C3 inhibition could reduce complement activation and platelet storage lesions in clinical platelet concentrates. Eight concentrates were prepared in platelet additive solution-E (PAS-E) with ~35% plasma and stored at 22 °C for seven days. Each unit was split: one half was supplemented with the C3 inhibitor compstatin Cp40, the other serving as the control. Complement activation, platelet function, and metabolic parameters were assessed every two days. Results: Cp40 significantly reduced C3bc and sC5b-9 levels, but not C4c, indicating inhibition of complement activation at the level of C3. Importantly, C3 inhibition did not alter platelet-specific markers or metabolic stability. CD62P expression and NAP-2 release increased during storage, while CD63, PF4, and TSP-1 levels remained stable. Platelet responsiveness to PAR-1 (TRAP-6) and TXA2R (U46619) stimulation declined over time, as indicated by reduced CD62P and CD63 expression and diminished soluble factor release. No drop in platelet count was observed, and metabolic markers remained within their critical limits. Conclusion: In summary, complement activation occurs during the storage of platelet concentrates in PAS-E/plasma, but C3 inhibition with Cp40, while effective in reducing complement activation, does not influence platelet activation or metabolic markers. These findings suggest there is no direct causal link between complement activation and the development of storage lesions in clinical platelet concentrates. References: 1. Andersson LI, Sjostrom DJ, Quach HQ, Hagerstrom K, Hurler L, Kajdacsi E, et al. Storage of Transfusion Platelet Concentrates Is Associated with Complement Activation and Reduced Ability of Platelets to Respond to Protease-Activated Receptor-1 and Thromboxane A2 Receptor. Int J Mol Sci. 2024;25(2).

Background: The complement system is a key component of innate immunity, activated by pathogen- or danger-associated molecular patterns. The alternative pathway (AP) acts as an amplification loop for the other complement pathways and plays a central role in immune regulation and inflammation. Due to its critical function, components of the AP are attractive therapeutic targets. This study aimed to develop immunoassays for core AP proteins, with a focus on the C3 convertase (C3bP), to enhance mechanistic understanding and support therapeutic development. Methods: Monoclonal antibodies were generated via hybridoma technology. A novel antibody clone (11-13-05) targeting both native and complexed Factor P, was employed for assay development. The C3bP complex was measured using 11-13-05 as a capture antibody and polyclonal anti-C3c for detection, with zymosan-activated serum (ZAS) as standard. Total C3 was quantified using a dual anti-C3c/C3 antibody setup and purified C3 as standard. C3 activation products were detected using an in-house neoepitope-specific monoclonal antibody (bH6). Assays were validated in various sample types including serum, ZAS, EDTA plasma, and E. coli-activated lepirudin plasma. Gel filtration chromatography was used to assess complex formation, while immunoprecipitation followed by SDS-PAGE and Western blotting verified protein interactions. Results: All target components were consistently detected and stable across sample types. A time-dependent increase in C3bP was observed in E. coli-activated plasma, confirming progressive AP activation. The bH6 antibody did not bind to C3bP, suggesting specificity for fluid-phase active C3. Gel filtration revealed C3, Factor P, and limited Factor B co-eluting, but most Factor B appeared unbound. Western blot and ELISA confirmed this, with Factor B primarily found in the soluble fraction, not stably associated with the convertase complex. Conclusion: The developed immunoassay platform effectively quantifies key AP proteins and activation products while providing insight into convertase dynamics. These findings indicate that Factor P and Factor B may not form stable associations within the C3bP complex, suggesting new avenues for studying AP regulation and therapeutics.

Background: In recent years, a cell-autonomous and intracellularly active complement system has been identified as an important regulator of basic metabolic pathways underlying the normal function of immune cells. We have previously demonstrated that human monocytes and macrophages harbour a cell-autonomous C5 activation system, in which C5a signals via a mitochondrial C5a receptor (mtC5aR1). Upon sensing of danger signals, mtC5aR1 signalling induces a metabolic shift in the cell, triggering activation of the NLRP3 inflammasome and thus cleavage of pro-IL-1β into active IL-1β by caspase-1. However, an intracellular role of complement component C3 in inflammatory responses in this cell type has not yet been established. Methods: We have used confocal microscopy and antibodies targeting neo-epitopes revealed on C3a and C3bc following C3 cleavage to map the sub-cellular location of C3 activation products in human iPSC-derived macrophages (iPSC-DMs). Additionally, to investigate the role of intracellular C3 in human macrophages, we have used CRISPR-Cas9 gene edited THP-1 cells in which the gene encoding C3 or the C3a receptor (C3aR) have been knocked-out (KO). Immunoblotting, ELISA, and qPCR was used to measure the inflammatory response of these cells. Results: We have identified intracellular stores of C3a and C3bc in both resting and LPS-stimulated iPSC-DMs, indicating that constitutive C3 activation occurs in this cell type. Interestingly, C3bc localised on vesicle-like structures, and we observed co-localisation of C3bc and NLRP3 on enlarged endosomes following LPS priming. Supporting a role of C3 in inflammasome activation, stimulation of the C3 KO THP-1 cells with LPS and nigericin to activate NLRP3 resulted in reduced cleavage of pro-caspase-1 and pro-IL-1β into their active forms, when compared to the WT. Furthermore, transcription of IL1B following LPS priming was decreased in the C3 KO cells when compared to the WT, indicating that C3 may also be involved in the priming step of NLRP3 activation. However, these effects were not observed in the C3aR KO cells, suggesting that this role of C3 is independent of signalling through C3aR. Conclusion: Together, our results demonstrate the presence of an intracellular C3 activation system in human macrophages, which may regulate activation of the NLRP3 inflammasome.