Ischemia-reperfusion injury in transplantation remains a significant clinical challenge with regard to both short-term and long-term complications. In this study, we developed a new amphiphilic construct, polyethylene glycol (PEG)-conjugated lipids (PEG-LIPIDs), to be administered ex vivo intra-arterially to procured porcine kidney allografts before reperfusion. The aim was to create a protective cell membrane barrier, preventing the recognition of ligands exposed on renal cells by plasma proteins and cells of the intravascular innate immune system. In vitro cell studies confirmed the safety of PEG-LIPID with no observed toxicity and demonstrated its efficacy in masking ligands on various cell types. The PEG-LIPID was evaluated in 3 porcine allogeneic transplant models: 1 acute dual en bloc nonsurvival transplant model (duration 6 hours) and 2 survival models with low and high ischemic stress, respectively (duration 96 hours). No immunosuppression was employed. Across all 3 porcine transplant models, PEG-LIPID consistently mitigated ischemiareperfusion-induced thromboinflammation (complement, coagulation, and kallikrein/kinin activation) and long-term inflammation with a marked reduction in cytokine responses, including lower levels of interleukin 6. The PEG-LIPID-treated kidneys exhibited significantly improved allograft function, reflected in robustly lower creatinine levels. This translational study confirmed that the PIG-LIPID is a strong candidate drug to mitigate ischemia-reperfusion injury in clinical kidney transplantation.

Tick-over of C3 to fluid-phase C3(H₂O) is considered the initiator of the alternative pathway by mediating random depositing of C3b on target surfaces. This slow mechanism does not explain the specificity and rapid activation of the alternative pathway in vivo. In thromboinflammatory disorders, C3(H₂O) also exists in a bound form on activated platelets and endothelial cells. Here, we investigate this binding mechanism. C3, C3b, and C3(H₂O) were exposed to activated platelets expressing phospholipid-scrambled membranes. Native C3 demonstrated the highest binding to activated platelets compared to C3b and soluble C3(H₂O) and revealed the most efficient convertase (C3bBb) formation. The specific binding of annexin V to phospholipid-scrambled membranes, inhibited C3 binding to activated platelets, and to apoptotic PMN and endothelial cells, while properdin enhanced both binding and convertase generation. Model liposomes exposing phosphatidylserine, bound native C3 in a cholesterol-dependent manner. Neoepitopes and cryo-TEM, showed that the conformation of liposome-bound C3 resembles C3(H₂O) and quartz crystal microbalance with dissipation monitoring (QCM-D) its ability to form C3bBb convertases. Thus, native C3 transforms into C3(H₂O) by binding to phospholipid-scrambled membranes, identifying native C3 without proteolytic cleavage, as a direct recognition molecule of altered self (in this case exposure of phospholipids that are not found on the surface of native, healthy, cells) acting as a key initiator of the alternative pathway and a mediator of phagocytosis in thromboinflammatory pathologies.

In this study, we investigated the impact of iron-rich nanoparticles derived from different locations in the subway on the innate immune system in blood. Nanoparticles were generated from Third Rail, Rail, and Wheel materials and characterized using several techniques. The response in a human whole-blood model was analyzed using ELISA and capillary immunoelectrophoresis. All nanoparticles were iron oxides, but Third Rail nanoparticles also contained Silicon and were highly thrombo-inflammatory, activating Factor XI-induced coagulation and pro-inflammatory kallikrein/kinin pathways. Wheel and Rail nanoparticles were less reactive, mainly activating the kallikrein/kinin pathway, leading to milder inflammatory reactions. The strong thrombo-inflammatory properties of Third Rail nanoparticles are attributed to their high Silicon content. None of the nanoparticles significantly activated the complement system. In conclusion, we found that the elemental composition of nanoparticles is crucial in determining whether activation leads to kallikrein/kinin system activation and bradykinin release or Factor XI activation and thrombosis.

The complement system plays a central role in the pathogenesis of Systemic Lupus Erythematosus (SLE), but most studies have focused on the classical pathway. Ficolin-3 is the main initiator of the lectin pathway of complement in humans, but its role in systemic autoimmune disease has not been conclusively determined. Here, we combined biochemical and genetic approaches to assess the contribution of ficolin-3 to SLE risk and disease manifestations. Ficolin-3 activity was measured by a functional assay in serum or plasma samples from Swedish SLE patients (n = 786) and controls matched for age and sex (n = 566). Genetic variants in an extended 300 kb genomic region spanning the FCN3 locus were analyzed for their association with ficolin-3 activity and SLE manifestations in a Swedish multicenter cohort (n = 985). Patients with ficolin-3 activity in the highest tertile showed a strong enrichment in an SLE cluster defined by anti-Sm/DNA/nucleosome antibodies (OR 3.0, p < 0.001) and had increased rates of hematological disease (OR 1.4, p = 0.078) and lymphopenia (OR = 1.6, p = 0.039). Genetic variants associated with low ficolin-3 activity mapped to an extended haplotype in high linkage disequilibrium upstream of the FCN3 gene. Patients carrying the lead genetic variant associated with low ficolin3 activity had a lower frequency of hematological disease (OR 0.67, p = 0.018) and lymphopenia (OR 0.63, p = 0.031) and fewer autoantibodies (p = 0.0019). Loss-of-function variants in the FCN3 gene were not associated with SLE, but four (0.5 %) SLE patients developed acquired ficolin-3 deficiency where ficolin-3 activity in serum was depleted following diagnosis of SLE. Taken together, our results provide genetic and biochemical evidence that implicate the lectin pathway in hematological SLE manifestations. We also identify lectin pathway activation through ficolin-3 as a factor that contributes to the autoantibody response in SLE.

Liposomes are synthetic spherical lipid droplets with a size of 0.025–2.5μm in diameter. They are for the most part composed of phospholipids and cholesterol, forming a lipid bilayer, which mimics the natural biomembrane. Based on their structural properties (single- or multi-layered, size, multi-vesicle liposome, charge and surface modifications), liposomes can be divided into structural subcategories. In addition, fluidity, rigidity, membrane dynamic reorganization ability, and the cholesterol content of liposomes play a crucial role in their biocompatibility and stability when the liposomes are administered into the human body. The importance of surface modification of liposomes is highlighted in this chapter together with potential engineering techniques/strategies. Furthermore potential chemistries (ligations) that could be applied for liposome surface modifications are described.

Background: Ficolin-3 is the main initiator of the lectin pathway in humans. Case reports of ficolin-3 deficient patients have suggested that ficolin-3 deficiency may be enriched in patients with Systemic Lupus Erythematosus (SLE), a systemic autoimmune disease where complement plays an important role. Therefore, this study aimed to investigate the activity levels of ficolin-3 and to identify potential ficolin-3 deficient individuals in two Swedish SLE cohorts.

Methods: Serum or plasma samples from SLE patients (n=810) and matched controls (n=566) were collected from the Karolinska Institute (KI) and Umeå University Hospital. The ficolin-3 activity levels were measured by an in-house developed functional ELISA with a pooled normal human serum sample as a reference. Serial samples were analyzed for ficolin-3 deficient patients when available. Sequencing data were analyzed for FCN3 frame-shift mutation +1637delC (rs532781899) and other potential loss-of-function (LoF) variants.

Results: This screening revealed that the level of ficolin-3 activity varies largely in patients with SLE. The activity levels also show that SLE patients seem to generally have elevated ficolin-3 activity compared to the control group (p<0.0001). Out of 810 patients with SLE, four patients were determined to be ficolin-3 deficient. For two of these patients, the ficolin-3 activity was at normal levels at the time of diagnosis and thereafter depleted over time, indicating an acquired deficiency. For deficient patients, no or very low ficolin-3 protein levels and no lectin pathway-dependent complement activation could be detected. Autoantibodies against ficolin-3 were not detectable. No patients were homozygous for the +1637delC frameshift mutation, whereas in total 10 patients were determined to be heterozygous carriers. These heterozygous patients displayed lower levels of ficolin-3 activity but did not include the deficient patients. Additional possible LoF variants were analyzed but none were enriched in either patients or controls.

Conclusions: Contrary to the classical pathway of the complement system we show that genetic ficolin-3 deficiency is not a risk factor for SLE. Instead, acquired ficolin-3 deficiency was observed in a subgroup of SLE patients, possibly due to a potent activation of the lectin pathway that depleted ficolin-3 plasma levels in these individuals.

Background: Several studies have shown the importance of the complement and coagulation systems in the pathogenesis of asthma. Objectives: We explored whether we could detect differentially abundant complement and coagulation proteins in the samples obtained from the small airway lining fluid by collection of exhaled particles in patients with asthma and whether these proteins are associated with small airway dysfunction and asthma control. Method: Exhaled particles were obtained from 20 subjects with asthma and 10 healthy controls (HC) with the PExA method and analysed with the SOMAscan proteomics platform. Lung function was assessed by nitrogen multiple breath washout test and spirometry. Results: 53 proteins associated with the complement and coagulation systems were included in the analysis. Nine of those proteins were differentially abundant in subjects with asthma as compared to HC, and C3 was significantly higher in inadequately controlled asthma as compared to well-controlled asthma. Several proteins were associated with physiological tests assessing small airways. Conclusions: The study highlights the role of the local activation of the complement and coagulation systems in the small airway lining fluid in asthma and their association with both asthma control and small airway dysfunction. The findings highlight the potential of complement factors as biomarkers to identify different sub-groups among patients with asthma that could potentially benefit from a therapeutic approach targeting the complement system.

BackgroundDysregulated complement activation, increased protein citrullination, and production of autoantibodies against citrullinated proteins are hallmarks of rheumatoid arthritis (RA). Citrullination is induced by immune cell-derived peptidyl-Arg deiminases (PADs), which are overactivated in the inflamed synovium. We characterized the effect of PAD2- and PAD4-induced citrullination on the ability of the plasma-derived serpin C1-inhibitor (C1-INH) to inhibit complement and contact system activation. MethodsCitrullination of the C1-INH was confirmed by ELISA and Western blotting using a biotinylated phenylglyoxal probe. C1-INH-mediated inhibition of complement activation was analyzed by C1-esterase activity assay. Downstream inhibition of complement was studied by C4b deposition on heat-aggregated IgGs by ELISA, using pooled normal human serum as a complement source. Inhibition of the contact system was investigated by chromogenic activity assays for factor XIIa, plasma kallikrein, and factor XIa. In addition, autoantibody reactivity to native and citrullinated C1-INH was measured by ELISA in 101 RA patient samples. ResultsC1-INH was efficiently citrullinated by PAD2 and PAD4. Citrullinated C1-INH was not able to bind the serine protease C1s and inhibit its activity. Citrullination of the C1-INH abrogated its ability to dissociate the C1-complex and thus inhibit complement activation. Consequently, citrullinated C1-INH had a decreased capacity to inhibit C4b deposition via the classical and lectin pathways. The inhibitory effect of C1-INH on the contact system components factor XIIa, plasma kallikrein, and factor XIa was also strongly reduced by citrullination. In RA patient samples, autoantibody binding to PAD2- and PAD4-citrullinated C1-INH was detected. Significantly more binding was observed in anti-citrullinated protein antibody (ACPA)-positive than in ACPA-negative samples. ConclusionCitrullination of the C1-INH by recombinant human PAD2 and PAD4 enzymes impaired its ability to inhibit the complement and contact systems in vitro. Citrullination seems to render C1-INH more immunogenic, and citrullinated C1-INH might thus be an additional target of the autoantibody response observed in RA patients.

The complement system plays a crucial role in host defense, homeostasis, and tissue regeneration and bridges the innate and the adaptive immune systems. Although the genetic variants in complement C2 (c.839_849+17del; p.(Met280Asnfs*5)) and C8B (c.1625C>T; p.(Thr542Ile)) are known individually, here, we report on a patient carrying their combination in a heterozygous form. The patient presented with a reduced general condition and suffers from a wide variety of autoimmune diseases. While no autoimmune disease-specific autoantibodies could be detected, genetic analysis revealed abnormalities in the two complement genes C2 and C8B. Therefore, we performed a comprehensive investigation of the innate immune system on a cellular and humoral level to define the functional consequences. We found slightly impaired functionality of neutrophils and monocytes regarding phagocytosis and reactive oxygen species generation and a diminished expression of the C5aR1. An extensive complement analysis revealed a declined activation potential for the alternative and classical pathway. Reconstitution with purified C2 and C8 into patient serum failed to normalize the dysfunction, whereas the addition of C3 improved the hemolytic activity. In clinical transfer, in vitro supplementation of the patient's plasma with FFP as a complement source could fully restore full complement functionality. This study describes for the first time a combined heterozygous genetic variation in complement C2 and C8B which, however, cannot fully explain the overall dysfunctions and calls for further complement deficiency research and corresponding therapies.

COVID-19 has been shown to have a multifaceted impact on the immune system. In a recently published article in Front Immunol, we show that the intravascular innate immune system (IIIS) is strongly activated in severe COVID-19 with ARDS and appears to be one of the causes leading to severe COVID-19. In this article, we describe the IIIS and its physiological function, but also the strong pro-inflammatory effects that are observed in COVID-19 and in various other pathological conditions and treatments such as during ischemia reperfusion injury and in treatments where biomaterials come in direct contact with blood in, e.g., extracorporeal and intravasal treatments. In the present article, we describe how the IIIS, a complex network of plasma proteins and blood cells, constitute the acute innate immune response of the blood and discuss the effects that the IIIS induces in pathological disorders and treatments in modern medicine.