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.

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.

The present study identifies the phosphorylation sites of the 85-kDa cytosolic phospholipase A 2 (cPLA 2 ) in human platelets and HeLa cells. Tryptic digests of 32 Pphosphorylated and -immunoprecipitated cPLA 2 were analyzed by microbore high performance liquid chromatography and two-dimensional phosphopeptide mapping against synthetic phosphopeptide standards. Thrombin stimulated significant phosphorylation of platelet cPLA 2 at two sites, Ser-505 and Ser-727. Exclusive phosphorylation on these two sites was also seen in collagen-stimulated platelets and HeLa cells stimulated with interferon-␣ or arsenite; no tyrosine phosphorylation was detected. The inhibitor of the 38-kDa stressactivated protein kinase (p38 mapk ), SB 203580, reduced phosphorylation of both Ser-505 and Ser-727 by 50 and 60%, respectively, in thrombin-stimulated platelets. An additional p38 mapk inhibitor SB 202190 also partially (60%) inhibited the phosphorylation of cPLA 2 in arsenite-stimulated HeLa cells. These studies extend the previous work on the identification of multiple phosphorylation sites on cPLA 2 expressed in a baculovirus/insect cell system to cPLA 2 in mammalian cells stimulated with physiological agonists. They also underscore the necessity of high resolution phosphopeptide mapping combined with microbore high performance liquid chromatography for quantification of phosphorylation levels, which has lead to the conclusion that Ser-505 and Ser-727 are common phosphorylation sites on cPLA 2 in different mammalian cells stimulated with multiple agonists.Cytosolic phospholipase A 2 (cPLA 2 ) 1 catalyzes the cleavage of arachidonic acid from the sn-2 position of phospholipids (1, 2). The 85-kDa enzyme is present in many mammalian cells (3), and strong evidence is accumulating for the role of cPLA 2 in the generation of tissue mediators that are metabolites of arachidonic acid, such as prostaglandins, leukotrienes, and thromboxanes. In contrast to the small molecular weight phospholipase A 2 s that are secreted and are active on the outside of cells, cPLA 2 is regulated by intracellular signals that are propagated from surface receptors. One important regulatory mechanism appears to be a rise in the intracellular Ca 2ϩ concentration which causes translocation of cPLA 2 from the cytosol to internal membranes (4 -7) where it binds through a Ca 2ϩ -dependent lipid-binding domain (8). A second, well established mechanism of the regulation of cPLA 2 activity is by phosphorylation on Ser-505 through a mitogen-activated protein kinase (MAPK) (9) which modestly increases the intrinsic activity of the lipase measured in vitro (3, 10, 11). Phosphorylation of cPLA 2 together with release of arachidonic acid has been observed in a variety of cells (11)(12)(13)(14)(15)(16).A thorough characterization of the phosphorylation sites of human cPLA 2 heterologously expressed in Spodoptera frugiperda (Sf9) cells by high performance liquid chromatography (HPLC), mass spectrometry, and protein sequencing has revealed four sites of phosphorylation: Ser-...

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Early and persistent activation of complement is considered to play a key role in the pathogenesis of COVID-19. Complement activation products orchestrate a proinflammatory environment that might be critical for the induction and maintenance of a severe inflammatory response to SARS-CoV-2 by recruiting cells of the cellular immune system to the sites of infection and shifting their state of activation towards an inflammatory phenotype. It precedes pathophysiological milestone events like the cytokine storm, progressive endothelial injury triggering microangiopathy, and further complement activation, and causes an acute respiratory distress syndrome (ARDS). To date, the application of antiviral drugs and corticosteroids have shown efficacy in the early stages of SARS-CoV-2 infection, but failed to ameliorate disease severity in patients who progressed to severe COVID-19 pathology. This report demonstrates that lectin pathway (LP) recognition molecules of the complement system, such as MBL, FCN-2 and CL-11, bind to SARS-CoV-2 S- and N-proteins, with subsequent activation of LP-mediated C3b and C4b deposition. In addition, our results confirm and underline that the N-protein of SARS-CoV-2 binds directly to the LP- effector enzyme MASP-2 and activates complement. Inhibition of the LP using an inhibitory monoclonal antibody against MASP-2 effectively blocks LP-mediated complement activation. FACS analyses using transfected HEK-293 cells expressing SARS-CoV-2 S protein confirm a robust LP-dependent C3b deposition on the cell surface which is inhibited by the MASP-2 inhibitory antibody. In light of our present results, and the encouraging performance of our clinical candidate MASP-2 inhibitor Narsoplimab in recently published clinical trials, we suggest that the targeting of MASP-2 provides an unsurpassed window of therapeutic efficacy for the treatment of severe COVID-19.