Explore the vast range of titles available.

The multiprotein complex C1 initiates the classical pathway of complement activation on binding to antibody–antigen complexes, pathogen surfaces, apoptotic cells, and polyanionic structures. It is formed from the recognition subcomponent C1q and a tetramer of proteases C1r₂C1s₂ as a Ca2+-dependent complex. Here we have determined the structure of a complex between the CUB1-EGF-CUB2 fragments of C1r and C1s to reveal the C1r–C1s interaction that forms the core of C1. Both fragments are L-shaped and interlock to form a compact antiparallel heterodimer with a Ca2+ from each subcomponent at the interface. Contacts, involving all three domains of each protease, are more extensive than those of C1r or C1s homodimers, explaining why heterocomplexes form preferentially. The available structural and biophysical data support a model of C1r₂C1s₂ in which two C1r-C1s dimers are linked via the catalytic domains of C1r. They are incompatible with a recent model in which the N-terminal domains of C1r and C1s form a fixed tetramer. On binding to C1q, the proteases become more compact, with the C1r-C1s dimers at the center and the six collagenous stems of C1q arranged around the perimeter. Activation is likely driven by separation of the C1r-C1s dimer pairs when C1q binds to a surface. Considerable flexibility in C1s likely facilitates C1 complex formation, activation of C1s by C1r, and binding and activation of downstream substrates C4 and C4b-bound C2 to initiate the reaction cascade.

The complement system plays a key role in host defense against pneumococcal infection. Three different pathways, the classical, alternative and lectin pathways, mediate complement activation. While there is limited information available on the roles of the classical and the alternative activation pathways of complement in fighting streptococcal infection, little is known about the role of the lectin pathway, mainly due to the lack of appropriate experimental models of lectin pathway deficiency. We have recently established a mouse strain deficient of the lectin pathway effector enzyme mannan-binding lectin associated serine protease-2 (MASP-2) and shown that this mouse strain is unable to form the lectin pathway specific C3 and C5 convertases. Here we report that MASP-2 deficient mice (which can still activate complement via the classical pathway and the alternative pathway) are highly susceptible to pneumococcal infection and fail to opsonize Streptococcus pneumoniae in the none-immune host. This defect in complement opsonisation severely compromises pathogen clearance in the lectin pathway deficient host. Using sera from mice and humans with defined complement deficiencies, we demonstrate that mouse ficolin A, human L-ficolin, and collectin 11 in both species, but not mannan-binding lectin (MBL), are the pattern recognition molecules that drive lectin pathway activation on the surface of S. pneumoniae. We further show that pneumococcal opsonisation via the lectin pathway can proceed in the absence of C4. This study corroborates the essential function of MASP-2 in the lectin pathway and highlights the importance of MBL-independent lectin pathway activation in the host defense against pneumococci.

Complement research experienced a renaissance with the discovery of a third activation route, the lectin pathway. We developed a unique model of total lectin pathway deficiency, a mouse strain lacking mannan-binding lectin-associated serine protease-2 (MASP-2), and analyzed the role of MASP-2 in two models of postischemic reperfusion injury (IRI). In a model of transient myocardial IRI, MASP-2-deficient mice had significantly smaller infarct volumes than their wild-type littermates. Mice deficient in the downstream complement component C4 were not protected, suggesting the existence of a previously undescribed lectin pathway-dependent C4-bypass. Lectin pathway-mediated activation of C3 in the absence of C4 was demonstrated in vitro and shown to require MASP-2, C2, and MASP-1/3. MASP-2 deficiency also protects mice from gastrointestinal IRI, as do mAb-based inhibitors of MASP-2. The therapeutic effects of MASP-2 inhibition in this experimental model suggest the utility of anti-MASP-2 antibody therapy in reperfusion injury and other lectin pathway-mediated disorders.

Complement component C1, the complex that initiates the classical pathway of complement activation, is a 790-kDa assembly formed from the target-recognition subcomponent C1q and the modular proteases C1r and C1s. The proteases are elongated tetramers that become more compact when they bind to the collagen-like domains of C1q. Here, we describe a series of structures that reveal how the subcomponents associate to form C1. A complex between C1s and a collagen-like peptide containing the C1r/C1s-binding motif of C1q shows that the collagen binds to a shallow groove via a critical lysine side chain that contacts Ca ²⁺-coordinating residues. The data explain the Ca ²⁺-dependent binding mechanism, which is conserved in C1r and also in mannan-binding lectin-associated serine proteases, the serine proteases of the lectin pathway activation complexes. In an accompanying structure, C1s forms a compact ring-shaped tetramer featuring a unique head-to-tail interaction at its center that replicates the likely arrangement of C1r/C1s polypeptides in the C1 complex. Additional structures reveal how C1s polypeptides are positioned to enable activation by C1r and interaction with the substrate C4 inside the cage-like assembly formed by the collagenous stems of C1q. Together with previously determined structures of C1r fragments, the results reported here provide a structural basis for understanding the early steps of complement activation via the classical pathway.

Modern medicine has established three central antimicrobial therapeutic concepts: vaccination, antibiotics, and, recently, the use of active immunotherapy to enhance the immune response toward specific pathogens. The efficacy of vaccination and antibiotics is limited by the emergence of new pathogen strains and the increased incidence of antibiotic resistance. To date, immunotherapy development has focused mainly on cytokines. Here we report the successful therapeutic application of a complement component, a recombinant form of properdin (P ₙ), with significantly higher activity than native properdin, which promotes complement activation via the alternative pathway, affording protection against N. menigitidis and S. pneumoniae . In a mouse model of infection, we challenged C57BL/6 WT mice with N. menigitidis B-MC58 6 h after i.p. administration of P ₙ (100 µg/mouse) or buffer alone. Twelve hours later, all control mice showed clear symptoms of infectious disease while the P ₙ treated group looked healthy. After 16 hours, all control mice developed sepsis and had to be culled, while only 10% of P ₙ treated mice presented with sepsis and recoverable levels of live Meningococci . In a parallel experiment, mice were challenged intranasally with a lethal dose of S. pneumoniae D39. Mice that received a single i.p. dose of P ₙ at the time of infection showed no signs of bacteremia at 12 h postinfection and had prolonged survival times compared with the saline-treated control group (P < 0.0001). Our findings show a significant therapeutic benefit of P ₙ administration and suggest that its antimicrobial activity could open new avenues for fighting infections caused by multidrug-resistant neisserial or streptococcal strains.

Abdominal aortic aneurysm (AAA) is a complex inflammatory vascular disease. There are currently limited treatment options for AAA when surgery is inapplicable. Therefore, insights into molecular mechanisms underlying AAA pathogenesis may reveal therapeutic targets that could be manipulated pharmacologically or biologically to halt disease progression. Using an elastase-induced AAA mouse model, we previously established that the complement alternative pathway (AP) plays a critical role in the development of AAA. However, the mechanism by which complement AP is initiated remains undefined. The complement protein properdin, traditionally viewed as a positive regulator of the AP, may also initiate complement activation by binding directly to target surfaces. In this study, we sought to determine whether properdin serves as a focal point for the initiation of the AP complement activation in AAA. Using a properdin loss of function mutation in mice and a mutant form of the complement factor B protein that produces a stable, properdin-free AP C3 convertase, we show that properdin is required for the development of elastase-induced AAA in its primary role as a convertase stabilizer. Unexpectedly, we find that, in AAA, natural IgG antibodies direct AP-mediated complement activation. The absence of IgG abrogates C3 deposition in elastase-perfused aortic wall and protects animals from AAA development. We also determine that blockade of properdin activity prevents aneurysm formation. These results indicate that an innate immune response to self-antigens activates the complement system and initiates the inflammatory cascade in AAA. Moreover, the study suggests that properdin-targeting strategies may halt aneurysmal growth.

The complement system is an essential component of the immune response, providing a critical line of defense against different pathogens including S. pneumoniae. Complement is activated via three distinct pathways: the classical (CP), the alternative (AP) and the lectin pathway (LP). The role of Pneumolysin (PLY), a bacterial toxin released by S. pneumoniae, in triggering complement activation has been studied in vitro. Our results demonstrate that in both human and mouse sera complement was activated via the CP, initiated by direct binding of even non-specific IgM and IgG3 to PLY. Absence of CP activity in C1q(-/-) mouse serum completely abolished any C3 deposition. However, C1q depleted human serum strongly opsonized PLY through abundant deposition of C3 activation products, indicating that the LP may have a vital role in activating the human complement system on PLY. We identified that human L-ficolin is the critical LP recognition molecule that drives LP activation on PLY, while all of the murine LP recognition components fail to bind and activate complement on PLY. This work elucidates the detailed interactions between PLY and complement and shows for the first time a specific role of the LP in PLY-mediated complement activation in human serum.

Background Collectin-K1 (CL-K1, or CL-11) is a multifunctional Ca 2+ -dependent lectin with roles in innate immunity, apoptosis and embryogenesis. It binds to carbohydrates on pathogens to activate the lectin pathway of complement and together with its associated serine protease MASP-3 serves as a guidance cue for neural crest development. High serum levels are associated with disseminated intravascular coagulation, where spontaneous clotting can lead to multiple organ failure. Autosomal mutations in the CL-K1 or MASP-3 genes cause a developmental disorder called 3MC (Carnevale, Mingarelli, Malpuech and Michels) syndrome, characterised by facial, genital, renal and limb abnormalities. One of these mutations (Gly 204 Ser in the CL-K1 gene) is associated with undetectable levels of protein in the serum of affected individuals. Results In this study, we show that CL-K1 primarily targets a subset of high-mannose oligosaccharides present on both self- and non-self structures, and provide the structural basis for its ligand specificity. We also demonstrate that three disease-associated mutations prevent secretion of CL-K1 from mammalian cells, accounting for the protein deficiency observed in patients. Interestingly, none of the mutations prevent folding or oligomerization of recombinant fragments containing the mutations in vitro . Instead, they prevent Ca 2+ binding by the carbohydrate-recognition domains of CL-K1. We propose that failure to bind Ca 2+ during biosynthesis leads to structural defects that prevent secretion of CL-K1, thus providing a molecular explanation of the genetic disorder. Conclusions We have established the sugar specificity of CL-K1 and demonstrated that it targets high-mannose oligosaccharides on self- and non-self structures via an extended binding site which recognises the terminal two mannose residues of the carbohydrate ligand. We have also shown that mutations associated with a rare developmental disorder called 3MC syndrome prevent the secretion of CL-K1, probably as a result of structural defects caused by disruption of Ca 2+ binding during biosynthesis.

Complement components are depleted during acute infection, and the proinflammatory anaphylatoxins C3a and C5a, which play a major role in the development of acute respiratory distress syndrome (ARDS), are released.1–4 Extensive complement activation may be prognostic of poor outcome – higher degrees of activation, especially of C3, being associated with fatality.4 Complement inhibitors are therefore considered promising therapeutics for tackling acute COVID-19 ARDS; potential targets include C3 and the C5a/C5aR axis as well as the lectin and terminal pathways.5,6 Analysis of post-mortem tissue from patients who succumbed to COVID-19 revealed severe endothelial damage characteristic of microangiopathies, with deposits of complement activation products, most notably the lectin pathway serine protease MASP-2.7,8 We have previously found evidence of a novel mechanism of lectin pathway activation, driven by a direct interaction between the SARS-CoV-2 nucleocapsid protein and the lectin pathway serine protease MASP-2.9 Narsoplimab is a fully humanised monoclonal antibody that targets MASP-2, blocking the activation of the lectin pathway (S1). TABLE 1 Clinical parameters for the subjects included in this study prior to treatment Clinical characteristics Narsoplimab-treated COVID-19 cohort (N = 9) Untreated COVID-19 cohort (N = 9) Median Range Median Range Age – years 62 41–79 74 50–79 Weight – kg 90 75–105 85 65–130 BMI – kg/m2 27.8 25.5–32.5 29 21–55 Sex Female 1/9 3/9 ARDS severity (Berlin criteria) Mild 2/9 Moderate 3/9 5/9 Severe 6/9 2/9 Laboratory findings Median Range Median Range PaO2:FiO2 ratio 140 110–250 180 80–284 White cell count – per mm3 8470 4600–21 520 7100 3140–14 590 Lymphocyte count – per mm3 550 350–1940 390 142–750 Platelet count – ×103 per mm3 247 154–313 223 102–444 Haemoglobin – g/dl 12.7 11.3–15.4 10.3 7.9–14.8 Other findings (reference ranges) C-reactive protein (.0–1.0 mg/dl) 14.5 1.9–17.9 9.3 .3–26.9 Lactate dehydrogenase (120/246 U/L) 443 312–582 346.5 190–494 Aspartate aminotransferase (13–40 U/L) 55 19–89 40 10–59 Alanine aminotransferase (7–40 U/L) 44 22–252 23 11–148 Creatinine (.3–1.3 mg/dl) .79 .48–1.13 1.85 .81–4.34 d-Dimer (<500 ng/ml) 1141 354–4471 1730 288–5020 Comorbidities Diabetes 2/9 3/9 Hypertension 5/9 6/9 Dyslipidaemia 1/9 3/9 Cardiovascular disease 0/9 5/9 Obesity (BMI ≥ 30 kg/m2) 2/8a 4/9 Overweight (BMI ≥ 25 kg/m2) 6/8a 8/9 Radiologic findings Bilateral interstitial abnormalities 9/9 9/9 Note: Data collected on day 0 of the study, immediately prior to administration of the first dose of narsoplimab in the treated cohort. [...]targeting the lectin pathway may suffice to reduce anaphylatoxin release below the threshold for maintaining ARDS and restore the bactericidal activity and opsonisation required for defence against secondary infection.

Interleukin 10–deficient mice (IL-10−/−) are a popular model used to dissect the mechanisms underlying inflammatory bowel diseases. The role of complement, a host defense mechanism that bridges the innate and adaptive immune systems, has not been described in this model. We therefore studied the effect of deficiency of properdin, a positive regulator of complement, on colitis in mice with the IL-10−/− background.MethodsFor acute colitis, IL-10−/− and IL-10/properdin double knockout (DKO) or radiation bone marrow–reconstituted chimeric mice, had piroxicam added to their powdered chow for 14 days. For chronic colitis, 2.5% dextran sodium sulfate was added to the animals' water for 4 days then the mice were killed 8 weeks later. Colons were assessed for inflammation, cell infiltration, and cytokine and complement measurements. Bacterial translocation was measured by cultivating bacteria from organs on Luria broth agar plates.ResultsC3a and C5a levels and C9 deposition were all increased in piroxicam-fed IL-10−/− mice compared with mice not fed piroxicam. Piroxicam-fed DKO mice lacked increased C5a and C9 deposition combined with exacerbated colitis, reduced numbers of infiltrating neutrophils, and markedly higher local and systemic bacterial numbers compared with IL-10−/− mice. Bone marrow cells from IL-10−/− mice were sufficient to restore protection against the heightened colitis in piroxicam-fed DKO mice.ConclusionsComplement is activated in the IL-10−/− mouse mucosa in a properdin-dependent manner. In the absence of terminal complement activation, the inflammation is heightened, likely due to a lack of neutrophil control over microbes escaping from the intestines.