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Patients with sickle cell disease (SCD) have ongoing hemolysis that promotes endothelial injury, complement activation, inflammation, vaso-occlusion, ischemia-reperfusion pathophysiology, and pain. Complement activation markers are increased in SCD in steady-state and further increased during vaso-occlusive crisis (VOC). However, the mechanisms driving complement activation in SCD have not been completely elucidated. Ischemia-reperfusion and heme released from hemoglobin during hemolysis, events that characterize SCD pathophysiology, can activate the lectin pathway (LP) and alternative pathway (AP), respectively. Here we evaluated the role of LP and AP in Townes sickle (SS) mice using inhibitory monoclonal antibodies (mAb) to mannose binding lectin (MBL)-associated serine protease (MASP)-2 or MASP-3, respectively. Townes SS mice were pretreated with MASP-2 mAb, MASP-3 mAb, isotype control mAb, or PBS before they were challenged with hypoxia-reoxygenation or hemoglobin. Pretreatment of SS mice with MASP-2 or MASP-3 mAb, markedly reduced Bb fragments, C4d and C5a in plasma and complement deposition in the liver, kidneys, and lungs collected 4 hours after challenge compared to control mAb-treated mice. Consistent with complement inhibition, hepatic inflammation markers NF-ĸB phospho-p65, VCAM-1, ICAM-1, and E-selectin were significantly reduced in SS mice pretreated with MASP-2 or MASP-3 mAb. Importantly, MASP-2 or MASP-3 mAb pretreatment significantly inhibited microvascular stasis (vaso-occlusion) induced by hypoxia-reoxygenation or hemoglobin. These studies suggest that the LP and the AP are both playing a role in promoting inflammation and vaso-occlusion in SCD. Inhibiting complement activation via the LP or the AP might inhibit inflammation and prevent VOC in SCD patients.

Purpose: Immunoglobulin A (IgA) nephropathy (IgAN) is a common chronic glomerulonephritis and the main cause of end-stage renal diseases. Recent evidence suggests that mannan binding lectin associated serine proteases 2 (MASP2) is related to IgAN; therefore, we investigated the expression and significance of MASP2 in serum and urinary extracellular vesicles (UEVs) in patients with IgAN.Methods: Thirty-eight patients with IgAN and 17 healthy controls were enrolled in this study. UEVs were extracted by ultracentrifugation. The separation by ultra-high-speed centrifuge was verified by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Candidate internal references (TSG101, CD9, flotillin, β-actin and GAPDH) were identified by western blotting in the control group, and the expression of MASP2 in the UEVs was compared. The levels of MASP2 in the serum and UEVs in the IgAN and control groups were determined by enzyme-linked immunosorbent assay (ELISA).Results: TEM and NTA results demonstrated that UEVs were successfully extracted. Western blotting results confirmed that TSG101 was suitable as an internal reference for this study. Compared with the control group, the IgAN group showed positive expression of MASP2. MASP2 levels in the UEVs, determined by ELISA, showed significant differences between IgAN and control groups, which were significantly positively correlated with the level of urinary microalbumin.Conclusions: The level of MASP2 in UEVs was related to IgAN and shows promise as a biomarker for evaluating the severity of renal injury and prognosis of IgAN, thereby helping to elucidate the role of MASP2 in the mannan-binding lectin pathway.

Apart from autopsy, tissue correlates of coronavirus disease 2019 (COVID-19) clinical stage are lacking. In the current study, cutaneous punch biopsy specimens of 15 individuals with severe/critical COVID-19 and six with mild/moderate COVID-19 were examined. Evidence for arterial and venous microthrombi, deposition of C5b-9 and MASP2 (representative of alternative and lectin complement pathways, respectively), and differential expression of interferon type I-driven antiviral protein MxA (myxovirus resistance A) versus SIN3A, a promoter of interferon type I-based proinflammatory signaling, were assessed. Control subjects included nine patients with sepsis-related acute respiratory distress syndrome (ARDS) and/or acute kidney injury (AKI) pre-COVID-19. Microthrombi were detected in 13 (87%) of 15 patients with severe/critical COVID-19 versus zero of six patients with mild/moderate COVID-19 (P 0.001) and none of the nine patients with pre-COVID-19 ARDS/AKI (P 0.001). Cells lining the microvasculature staining for spike protein of severe acute respiratory syndrome coronavirus 2, the etiologic agent of COVID-19, also expressed tissue factor. C5b-9 deposition occurred in 13 (87%) of 15 patients with severe/critical COVID-19 versus zero of six patients with mild/moderate COVID-19 (P 0.001) and none of the nine patients with pre-COVID-19 ARDS/AKI (P 0.001). MASP2 deposition was also restricted to severe/critical COVID-19 cases. MxA expression occurred in all six mild/moderate versus two (15%) of 13 severe/critical cases (P 0.001) of COVID-19. In contrast, SIN3A was restricted to severe/critical COVID-19 cases co-localizing with severe acute respiratory syndrome coronavirus 2 spike protein. SIN3A was also elevated in plasma of patients with severe/critical COVID-19 versus control subjects (P ≤ 0.02). In conclusion, the study identified premortem tissue correlates of COVID-19 clinical stage using skin. If validated in a longitudinal cohort, this approach could identify individuals at risk for disease progression and enable targeted interventions.

Thymoma-associated myasthenia gravis (TAMG) is a well-described subtype of Myasthenia gravis (MG). Nevertheless, the detailed proteins and bioprocess differentiating TAMG from TAMG (-) thymoma have remained unclear.The proteomics and metabolomics were carried out on serum samples from thymoma group (The of module-trait correlation of WGCNA analysis identified KRT1, GSN, COL6A1, KRT10, FOLR2, KRT9, KRT2, TPI1, ARF3, LYZ, ADIPOQ, SEMA4B, IGKV1-27, MASP2, IGF2R was associated with TAMG (+) thymomas. In addition, organismal systems-immune system and metabolism-biosynthesis of other secondary metabolites were closely related to the mechanism of TAMG (+) pathogenesis.Our integrated proteomics and metabolomics analysis supply a systems-level view of proteome changes in TAMG (+), TAMG (-) thymomas and exposes disease-associated protein network alterations involved in.

MASP-1 and MASP-2 are key activator proteases of the complement lectin pathway. The first specific mannose-binding lectin-associated serine protease (MASP) inhibitors had been developed from the 14-amino-acid sunflower trypsin inhibitor (SFTI) peptide by phage display, yielding SFTI-based MASP inhibitors, SFMIs. Here, we present the crystal structure of the MASP-1/SFMI1 complex that we analyzed in comparison to other existing MASP-1/2 structures. Rigidified backbone structure has long been accepted as a structural prerequisite for peptide inhibitors of proteases. We found that a hydrophobic cluster organized around the P2 Thr residue is essential for the structural stability of wild-type SFTI. We also found that the same P2 Thr prevents binding of the rigid SFTI-like peptides to the substrate-binding cleft of both MASPs as the cleft is partially blocked by large gatekeeper enzyme loops. Directed evolution removed this obstacle by replacing the P2 Thr with a Ser, providing the SFMIs with high-degree structural plasticity, which proved to be essential for MASP inhibition. To gain more insight into the structural criteria for SFMI-based MASP-2 inhibition, we systematically modified MASP-2-specific SFMI2 by capping its two termini and by replacing its disulfide bridge with varying length thioether linkers. By doing so, we also aimed to generate a versatile scaffold that is resistant to reducing environment and has increased stability in exopeptidase-containing biological environments. We found that the reduction-resistant disulfide-substituted l-2,3-diaminopropionic acid (Dap) variant possessed near-native potency. As MASP-2 is involved in the life-threatening thrombosis in COVID-19 patients, our synthetic, selective MASP-2 inhibitors could be relevant coronavirus drug candidates.

Ecotin is a homodimeric serine protease inhibitor produced by many commensal and pathogenic microbes. It functions as a virulence factor, enabling survival of various pathogens in the blood. The ecotin dimer binds two protease molecules, and each ecotin protomer has two protease-binding sites: site1 occupies the substrate-binding groove, whereas site2 engages a distinct secondary region. Owing to the twofold rotational symmetry within the ecotin dimer, sites 1 and 2 of a protomer bind to different protease molecules within the tetrameric complex. Escherichia coli ecotin inhibits trypsin-like, chymotrypsin-like, and elastase-like enzymes, including pancreatic proteases, leukocyte elastase, key enzymes of blood coagulation, the contact and complement systems, and other antimicrobial cascades. Here, we show that mannan-binding lectin-associated serine protease-1 (MASP-1) and MASP-2, essential activators of the complement lectin pathway, and MASP-3, an essential alternative pathway activator, are all inhibited by ecotin. We decipher in detail how the preorganization of site1 and site2 within the ecotin dimer contributes to the inhibition of each MASP enzyme. In addition, using mutated and monomeric ecotin variants, we show that site1, site2, and dimerization contribute to inhibition in a surprisingly target-dependent manner. We present the first ecotin:MASP-1 and ecotin:MASP-2 crystal structures, which provide additional insights and permit structural interpretation of the observed functional results. Importantly, we reveal that monomerization completely disables the MASP-2-inhibitory, MASP-3-inhibitory, and lectin pathway-inhibitory capacity of ecotin. These findings provide new opportunities to combat dangerous multidrug-resistant pathogens through development of compounds capable of blocking ecotin dimer formation.

Stomach adenocarcinoma (STAD) is the major cancer worldwide with high morbidity and mortality rate. Late diagnosis and limited treatment options of STAD lead to disease progression, spread, and metastasis. Therefore, finding a new biomarker to diagnosis and treatment is very important for STAD in clinical practice.The clinical data, transcriptome data and CCLE data were downloaded from TCGA database and CCLE database, respectively. TIMER website, TISIDB website and CIBERSORT methodology were used to analyse immune infiltration. R software and R package were used to analyse gene difference expression, determine co-expression genes, conduct gene enrichment analyses, construct a prognostic signature and establish nomogram.MASP1 was decreased in STAD compared with normal tissue at the mRNA level (p0.001). The enrichment analysis showed that mismatch repair (MMR) was related to the MASP1 gene. Up-regulation of MAPS1 expression was positively associated with dendritic cells (p0.01), neutrophils (p0.05), macrophages (p0.001), CD4+ T cells (p0.001) and B cells (p0.05). A four-gene prognostic signature was determined based on MASP1-related immunomodulators. The prognostic signature was an independent prognostic predictor in STAD. Finally, we established a nomogram to forecast survival and the nomogram has a good prediction accuracy.In STAD, MASP1 is closely related to immunity. MASP1 has the potential to positively regulate the abundance of immune cells. The MASP1-related prognosis signature and nomogram can accurately predict the survival of patients with STAD. Therefore, MASP1 is likely to be a diagnosis and promising immunotherapy target spot in STAD clinical practice.

Proteolytic activation of the renal epithelial sodium channel (ENaC) is increased by aldosterone. The aldosterone-sensitive protease remains unidentified. In humans, elevated circulating aldosterone is associated with increased urinary extracellular vesicle (uEVs) excretion of mannan-binding lectin associated serine protease-2 (MASP-2). We hypothesized that MASP-2 is a physiologically relevant ENaC-activating protease. It was confirmed that MASP2 mRNA is abundantly present in liver but not in human and mouse kidneys. Aldosterone-stimulation of murine cortical colleting duct (mCCD) cells did not induce MASP-2 mRNA. In human kidney collecting duct, MASP-2 protein was detected in AQP2-negative/ATP6VB1-positive intercalated cells suggestive of MASP2 protein uptake. Plasma concentration of full-length MASP-2 and the short splice variant MAp19 were not changed in a cross-over intervention study in healthy humans with low (70 mmol/day) versus high (250 mmol/day) Na+intake despite changes in aldosterone. The ratio of MAp19/MASP-2 in plasma was significantly increased with a high Na+diet and the ratio correlated with changes in aldosterone and fractional Na+excretion. MASP-2 was not detected in crude urine or in uEVs. MASP2 activated an amiloride-sensitive current when co-expressed with ENaC inXenopusoocytes, but not when added to the bath solution. In monolayers of collecting duct M1 cells, MASP2 expression did not increase amiloride-sensitive current and in HEK293 cells, MASP-2 did not affect γENaC cleavage. MASP-2 is neither expressed nor co-localized and co-regulated with ENaC in the human kidney or in urine after low Na+intake. MASP-2 does not mediate physiological ENaC cleavage in low salt/high aldosterone settings.

Excessive inflammatory responses contribute to the pathogenesis and lethality of highly pathogenic human coronaviruses, but the underlying mechanism remains unclear. In this study, the N proteins of highly pathogenic human coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were found to bind MASP-2, a key serine protease in the lectin pathway of complement activation, resulting in excessive complement activation by potentiating MBL-dependent MASP-2 activation, and the deposition of MASP-2, C4b, activated C3 and C5b-9. Aggravated inflammatory lung injury was observed in mice infected with adenovirus expressing the N protein. Complement hyperactivation was also observed in SARS-CoV-2-infected patients. Either blocking the N protein:MASP-2 interaction, MASP-2 depletion or suppressing complement activation can significantly alleviate N protein-induced complement hyperactivation and lung injury in vitro and in vivo. Altogether, these data suggested that complement suppression may represent a novel therapeutic approach for pneumonia induced by these highly pathogenic coronaviruses.

The most commonly used markers to assess complement activation are split products that are produced through activation of all three pathways and are located downstream of C3. In contrast, C4d derives from the cleavage of C4 and indicates either classical (CP) or lectin pathway (LP) activation. Although C4d is perfectly able to distinguish between CP/LP and alternative pathway (AP) activation, no well-established markers are available to differentiate between early CP and LP activation. Active enzymes of both pathways (C1s/C1r for the CP, MASP-1/MASP-2 for the LP) are regulated by C1 esterase inhibitor (C1-INH) through the formation of covalent complexes. Aim of this study was to develop validated immunoassays detecting C1s/C1-INH and MASP-1/C1-INH complex levels. Measurement of the complexes reveals information about the involvement of the respective pathways in complement-mediated diseases. Two sandwich ELISAs detecting C1s/C1-INH and MASP-1/C1-INH complex were developed and tested thoroughly, and it was investigated whether C1s/C1-INH and MASP-1/C1-INH complexes could serve as markers for either early CP or LP activation. In addition, a reference range for these complexes in healthy adults was defined, and the assays were clinically validated utilizing samples of 414 COVID-19 patients and 96 healthy controls. The immunoassays can reliably measure C1s/C1-INH and MASP-1/C1-INH complex concentrations in EDTA plasma from healthy and diseased individuals. Both complex levels are increased in serum when activated with zymosan, making them suitable markers for early classical and early lectin pathway activation. Furthermore, measurements of C1-INH complexes in 96 healthy adults showed normally distributed C1s/C1-INH complex levels with a physiological concentration of 1846 ± 1060 ng/mL (mean ± 2SD) and right-skewed distribution of MASP-1/C1-INH complex levels with a median concentration of 36.9 (13.18 - 87.89) ng/mL (2.5-97.5 percentile range), while levels of both complexes were increased in COVID-19 patients (p0.0001). The newly developed assays measure C1-INH complex levels in an accurate way. C1s/C1-INH and MASP-1/C1-INH complexes are suitable markers to assess early classical and lectin pathway activation. An initial reference range was set and first studies showed that these markers have added value for investigating and unraveling complement activation in human disease.

The innate immune system is the body’s first line of defense against pathogens and involves antibody and complement system-mediated antigen removal. Immune-response-related complement molecules have been identified in lamprey, and the occurrence of innate immune response via the mannose-binding lectin-associated serine proteases of the lectin cascade has been reported. We have previously shown that lamprey (Lampetra japonica) serum can efficiently and specifically eliminate foreign pathogens. Therefore, we aimed to understand the immune mechanism of lamprey serum in this study. We identified and purified a novel spherical lectin (LSSL) from lamprey serum. LSSL had two structural calcium ions coordinated with conserved amino acids, as determined through cryogenic electron microscopy. LSSL showed high binding capacity with microbial and mammalian glycans and demonstrated agglutination activity against bacteria. Phylogenetic analysis revealed that LSSL was transferred from phage transposons to the lamprey genome via horizontal gene transfer. Furthermore, LSSL was associated with mannose-binding lectin-associated serine protease 1 and promoted the deposition of the C3 fragment on the surface of target cells upon binding. These results led us to conclude that LSSL initiates and regulates agglutination, resulting in exogenous pathogen and tumor cell eradication. Our observations will give a greater understanding of the origin and evolution of the complement system in higher vertebrates and lead to the identification of novel immune molecules and pathways for defense against pathogens and tumor cells.

Patients with common variable immunodeficiency (CVID) display low antibody levels and associated symptoms, including an increased risk of infections. The causes of CVID are uncertain and likely heterogeneous. The complement system protects against pathogens and plays essential roles in homeostasis and development. The influence of the complement system in CVID is not established. We investigated CVID patients and healthy individuals for plasma levels of the complement proteins: MASP-1, MASP-2, MASP-3, MAp19, and MAp44. We also tested other patients with symptoms similar to the CVID patients. CVID patients had lower average MASP-2 and MAp44 levels than healthy individuals (p

BACKGROUND Sepsis has emerged as a leading cause of death in the intensive care unit. A growing number of studies have shown that genetic variants, especially single nucleotide polymorphisms, are key determinants of inter-individual variation in sepsis response. Therefore, early prediction of the onset and progression of sepsis, along with early intervention in high-risk patients, should be performed to effectively reduce the morbidity and mortality of the disease. MATERIAL AND METHODS A total of 581 Chinese patients were enrolled in this study, including 271 patients with sepsis and 310 patients without. We measured gene polymorphisms of MBL2 and serum levels of MBL2, tumor necrosis factor (TNF-alpha), interleukin (IL)-6, IL-4, and IL-10 in all patients. The effects of site mutations on the binding of MBL2 to mannose-associated serine protease 1 (MASP1) and MASP2 were also analyzed. RESULTS Of 3 site mutations in the MBL2 gene (rs5030737, rs1800450, and rs1800451), only rs1800450 had a mutant (G/A) genotype. The frequency of the GA genotype and A allele in the sepsis group was higher than that in the non-sepsis group. Furthermore, rs1800450G/A was associated with decreased serum MBL2 and IL-10 levels and decreased MBL2-MASP1 and MBL2-MASP2 interactions. Bioinformatics analysis showed that rs1800450G/A reduced the structural stability of the MBL2 protein and affected its function. CONCLUSIONS MBL2 rs1800450G/A was associated with a higher risk of sepsis, which possibly involved a decreased level of serum MBL2 that broke the balance of inflammation and weakened the binding of MBL2 to MASP1 and MASP2.

Proteins destined for secretion - after removal of the signal sequence - often undergo further proteolytic processing by proprotein convertases (PCs). Prohormones are typically processed in the regulated secretory pathway, while most plasma proteins travel though the constitutive pathway. The complement system is a major proteolytic cascade in the blood, serving as a first line of defense against microbes and also contributing to the immune homeostasis. Several complement components, namely C3, C4, C5 and factor I (FI), are multi-chain proteins that are apparently processed by PCs intracellularly. Cleavage occurs at consecutive basic residues and probably also involves the action of carboxypeptidases. The most likely candidate for the intracellular processing of complement proteins is furin, however, because of the overlapping specificities of basic amino acid residue-specific proprotein convertases, other PCs might be involved. To our surprise, we have recently discovered that processing of another complement protein, mannan-binding lectin-associated serine protease-3 (MASP-3) occurs in the blood by PCSK6 (PACE4). A similar mechanism had been described for the membrane protease corin, which is also activated extracellularly by PCSK6. In this review we intend to point out that the proper functioning of the complement system intimately depends on the action of proprotein convertases. In addition to the non-enzymatic components (C3, C4, C5), two constitutively active complement proteases are directly activated by PCs either intracellularly (FI), or extracellularly (MASP-3), moreover indirectly, through the constitutive activation of pro-factor D by MASP-3, the activity of the alternative pathway also depends on a PC present in the blood.

Complement lectin pathway components, in particular mannan-binding lectin (MBL) and MBL-associated serine proteases (MASPs) have been shown to interact with coagulation factors and contribute to clot formation. Here we investigated the role of MBL and MASP-1 in the haemostatic response following mechanical vessel injury in a human microfluidic bleeding model.We studied haemostasis in a microvascular bleeding model in the presence of human endothelial cells and human whole blood under flow conditions. We monitored incorporation of proteins into the clot with fluorescently labelled antibodies and studied their effects on clot formation, platelet activation, and bleeding time with specific inhibitors. Platelet activation was also studied by flow cytometry.Upon vessel injury, MBL accumulated at the injury site in a well-defined wall-like structure. MBL showed partial colocalisation with fibrin, and strong colocalisation with von Willebrand factor and (activated) platelets. Flow cytometry ruled out direct binding of MBL to platelets, but confirmed a PAR4- and thrombin-dependent platelet-activating function of MASP-1. Inhibiting MBL during haemostasis reduced platelet activation, while inhibiting MASP-1 reduced platelet activation, fibrin deposition and prolonged bleeding time.We show in a microvascular human bleeding model that MBL and MASP-1 have important roles in the haemostatic response triggered by mechanical vessel injury: MBL recognises the injury site, while MASP-1 increases fibrin formation, platelet activation and shortens bleeding time. While the complement lectin pathway may be harmful in the context of pathological thrombosis, it appears to be beneficial during the physiological coagulation response by supporting the crucial haemostatic system.

TJs maintain the epithelial barrier by regulating paracellular permeability. Since TJs are under dynamically fluctuating intercellular tension, cells must continuously survey and repair any damage. However, the underlying mechanisms allowing cells to sense TJ damage and repair the barrier are not yet fully understood. Here, we showed that proteinases play an important role in the maintenance of the epithelial barrier. At TJ break sites, EpCAM–claudin-7 complexes on the basolateral membrane become accessible to apical membrane-anchored serine proteinases (MASPs) and the MASPs cleave EpCAM. Biochemical data and imaging analysis suggest that claudin-7 released from EpCAM contributes to the rapid repair of damaged TJs. Knockout (KO) of MASPs drastically reduced barrier function and live-imaging of TJ permeability showed that MASPs-KO cells exhibited increased size, duration, and frequency of leaks. Together, our results reveal a novel mechanism of TJ maintenance through the localized proteolysis of EpCAM at TJ leaks, and provide a better understanding of the dynamic regulation of epithelial permeability.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis (AAGN) is the fulminant glomerular diseases with poor renal prognosis. Activation of the complement system has recently been reported in the pathogenesis of AAGN, but it remains to be clarified as to which complement pathway is mainly involved.20 patients with myeloperoxidase (MPO)-AAGN were retrospectively evaluated. Using serum samples, circulating immune-complexes (CICs) were assessed by the monoclonal rheumatoid factor assay, and C5a and C5b-9 were assessed by ELISA. Complement activation through the classical pathway was further evaluated by the WIESLAB® Complement System Classical Pathway kit. The affinities of ANCAs were evaluated by a competitive inhibition method using ELISA, and were classified into the high, and low-affinity group. Deposition of complement components, such as C3, C5, C4d, C5b-9, factor Bb, mannan-binding lectin serine peptidase (MASP)-1, MASP-2, and mannose/mannan-binding lectin (MBL), in frozen renal sections were analyzed by immunofluorescence staining.CICs were found to be positive in 65% of the patients. All CIC-positive patients belonged to the high-affinity group. Furthermore, serum C5a and C5b-9 were significantly increased in MPO-AAGN patients, and these levels positively correlated with CIC levels. A significant negative correlation was also found between levels of WIESLAB® classical pathway kit and CICs. By immunofluorescence staining, glomerular deposition of C4d, C5, and C5b-9 were observed in similar distributions in MPO-AAGN patients, whereas the deposition of MASP-1, MASP-2, MBL, and factor Bb were minimal.These results suggest the involvement of immune-complex induced complement activation through the classical pathway in the pathogenesis of MPO-AAGN.

Funder: Ministero della Salute; doi: http://dx.doi.org/10.13039/501100003196, The lectin pathway (LP) of complement activation is believed to contribute to brain inflammation. The study aims to identify the key components of the LP contributing to TBI outcome as possible novel pharmacological targets. We compared the long-term neurological deficits and neuropathology of wild-type mice (WT) to that of mice carrying gene deletions of key LP components after experimental TBI. WT or MASP-2 (Masp2−/−), ficolin-A (Fcna−/−), CL-11 (Colec11−/−), MASP-1/3 (Masp1−/−), MBL-C (Mbl2−/−), MBL-A (Mbl1−/−) or MBL−/− (Mbl1−/−/Mbl2−/−) deficient male C57BL/6J mice were used. Mice underwent sham surgery or TBI by controlled cortical impact. The sensorimotor response was evaluated by neuroscore and beam walk tests weekly for 4 weeks. To obtain a comparative analysis of the functional outcome each transgenic line was rated according to a health score calculated on sensorimotor performance. For selected genotypes, brains were harvested 6 weeks after injury for histopathological analysis. MASP-2−/−, MBL−/− and FCN-A−/− mice had better outcome scores compared to WT. Of these, MASP-2−/− mice had the best recovery after TBI, showing reduced sensorimotor deficits (by 33% at 3 weeks and by 36% at 4 weeks). They also showed higher neuronal density in the lesioned cortex with a 31.5% increase compared to WT. Measurement of LP functional activity in plasma from MASP-2−/− mice revealed the absence of LP functional activity using a C4b deposition assay. The LP critically contributes to the post-traumatic inflammatory pathology following TBI with the highest degree of protection achieved through the absence of the LP key enzyme MASP-2, underlining a therapeutic utility of MASP-2 targeting in TBI.

The role of the lectin pathway (LP) of complement has not been explored in the thrombotic microangiopathies (TMA). We examined levels of MASP2, the effector enzyme of the LP, in three major forms of TMA and assessed the effect of the anti‐MASP2 human monoclonal antibody narsoplimab on TMA plasma‐induced activation of caspase 8 in microvascular endothelial cells. MASP2 levels were highly elevated in all TMA patients versus controls. Caspase 8 activation was suppressed by clinically relevant levels of narsoplimab. In conclusion, we found that the LP of complement is activated in TMAs of diverse etiology and LP inhibition may be of therapeutic benefit in these disorders., Summary Involvement of the alternative complement pathway (AP) in microvascular endothelial cell (MVEC) injury characteristic of a thrombotic microangiopathy (TMA) is well documented. However, the role of the lectin pathway (LP) of complement has not been explored. We examined mannose‐binding lectin associated serine protease (MASP2), the effector enzyme of the LP, in thrombotic thrombocytopenic purpura, atypical hemolytic uremic syndrome and post‐allogeneic hematopoietic stem cell transplantation (alloHSCT) TMAs. Plasma MASP2 and terminal complement component sC5b‐9 levels were assessed by enzyme‐linked immunosorbent assay (ELISA). Human MVEC were exposed to patient plasmas, and the effect of the anti‐MASP2 human monoclonal antibody narsoplimab on plasma‐induced MVEC activation was assessed by caspase 8 activity. MASP2 levels were highly elevated in all TMA patients versus controls. The relatively lower MASP2 levels in alloHSCT patients with TMAs compared to levels in alloHSCT patients who did not develop a TMA, and a significant decrease in variance of MASP2 levels in the former, may reflect MASP2 consumption at sites of disease activity. Plasmas from 14 of the 22 TMA patients tested (64%) induced significant MVEC caspase 8 activation. This was suppressed by clinically relevant levels of narsoplimab (1·2 μg/ml) for all 14 patients, with a mean 65·7% inhibition (36.8–99.4%; P

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disorder. MASP2 is a mediator that plays an important role in complement system. As dysregulation of the complement system has been demonstrated to correlate with SLE pathogenesis, the role of MASP2 in lupus has not been widely discussed. In the present study, serum levels of MASP2 were evaluated in 61 lupus patients and 98 healthy controls by training cohort, and then a validation cohort including 100 lupus, 100 rheumatoid arthritis, 100 osteoarthritis, 100 gout, 44 Sjogren's syndrome, 41 ankylosing spondylitis patients confirmed the findings. Receiver operating characteristic (ROC) curve analysis determined the discriminatory capacity for serum MASP2. PCR methods tested the association of MASP2 gene polymorphisms (rs7548659, rs17409276, rs2273346, rs1782455 and rs6695096) and SLE risk. Impact of polymorphism on MASP2 serum levels was evaluated as well. Results showed that serum levels of MASP2 were significantly higher in lupus patients and correlated with some clinical, laboratory characteristics in the training cohort, and were much higher as compared to that in different rheumatic diseases patients in the validation cohort. Serum MASP2 showed a good diagnostic ability for lupus. Genotype frequencies and allele frequency of polymorphisms rs7548659, rs2273346 were strongly related to SLE risk, and genotypes of rs17409276, rs1782455, rs76695096 were significantly correlated with lupus genetic susceptibility. Interestingly, patients carrying GA genotype of rs17409276, TT, TC genotype of rs6695096 showed higher levels of serum MASP2. The findings suggested that MASP2 may be a potential disease marker for lupus, and correlate with SLE pathogenesis.

We investigated clinical associations of ficolins and mannose-binding lectin (MBL) in 157 patients suffering from acute myeloid leukaemia (AML). Concentrations of ficolin-1, ficolin-2, ficolin-3 and MBL (before chemotherapy) in serum were determined as were selected polymorphisms of the corresponding genes (FCN1, FCN2, FCN3 and MBL2). The control group (C) consisted of 267 healthy unrelated individuals. Median level of ficolin-1 in patients was lower (p p p p = 0.0016, respectively) compared with controls. These findings were generally associated with AML itself, however the highest MBL levels predicted higher risk of severe hospital infections (accompanied with bacteremia and/or fungaemia) (p = 0.012) while the lowest ficolin-1 concentrations tended to be associated with prolonged (> 7 days) fever (p = 0.026). Genotyping indicated an association of G/G homozygosity (corresponding to FCN1 gene − 542 G > A polymorphism) with malignancy [p = 0.004, OR = 2.95, 95% CI (1.41–6.16)]. Based on ROC analysis, ficolin-1, -2 and -3 may be considered candidate supplementary biomarkers of AML. Their high potential to differentiate between patients from non-malignant controls but also from persons suffering from other haematological cancers (multiple myeloma and lymphoma) was demonstrated.

BACKGROUND This study aimed to compare the characteristics of mRNA expression of genes in complement system between acute arterial thrombotic patients and paroxysmal atrial fibrillation (PAF) patients. METHODS Twenty acute myocardial infarction (AMI) patients and 20 PAF patients were assigned into the experiment groups, and 20 stable angina pectoris (SAP) patients were enrolled in the control group. RESULTS When compared with the control group, mRNA expression of C1QA, C1QB, C1QC, C1R, CFP, C5, CR1, ITGAM, ITGAX, ITGB2, C5AR1, CD46, CD55 and CD59 genes was significantly upregulated, and CR2 gene significantly downregulated in the AMI group (P

Inhibition of the complement activation has emerged as an option for treatment of a range of diseases. Activation of the lectin and alternative pathways (LP and AP, respectively) contribute to the deterioration of conditions in certain diseases such as ischemia-reperfusion injuries and age-related macular degeneration (AMD). In the current study, we generated dual complement inhibitors of the pathways MAp44-FH and sMAP-FH by fusing full-length MAp44 or small mannose-binding lectin-associated protein (sMAP), LP regulators, with the N-terminal five short consensus repeat (SCR) domains of complement factor H (SCR1/5-FH), an AP regulator. The murine forms of both fusion proteins formed a complex with endogenous mannose-binding lectin (MBL) or ficolin A in the circulation when administered in mice intraperitoneally. Multiple complement activation assays revealed that sMAP-FH had significantly higher inhibitory effects on activation of the LP and AP in vivo as well as in vitro compared to MAp44-FH. Human form of sMAP-FH also showed dual inhibitory effects on LP and AP activation in human sera. Our results indicate that the novel fusion protein sMAP-FH inhibits both the LP and AP activation in mice and in human sera, and could be an effective therapeutic agent for diseases in which both the LP and AP activation are significantly involved.

The complement system plays an important role in host defense and is activated via three different activation pathways. We have previously reported that mannose-binding lectin-associated serine protease (MASP)-3, unlike its splicing variant MASP-1, circulates in an active form and is essential for the activation of the alternative pathway (AP) via the activation of complement factor D (FD). On the other hand, like MASP-1 and MASP-2 of the lectin pathway (LP), MASP-3 forms a complex with the pattern recognition molecules (PRMs) of the LP (LP-PRMs). Both MASP-1 and MASP-2 can be activated efficiently when the LP-PRMs complexed with them bind to their ligands. On the other hand, it remains unclear how MASP-3 is activated, or whether complex formation of MASP-3 with LP-PRMs is involved in activation of MASP-3 or its efficiency in the circulation. To address these issues, we generated wild-type (WT) and four mutant recombinant mouse MASP-3 proteins fused with PA (human podoplanin dodecapeptide)-tag (rmMASP-3-PAs), the latter of which have single amino acid substitution for alanine in the CUB1 or CUB2 domain responsible for binding to LP-PRMs. The mutant rmMASP-3-PAs showed significantly reduced in-vivo complex formation with LP-PRMs when compared with WT rmMASP-3-PA. In the in-vivo kinetic analysis of MASP-3 activation, both WT and mutant rmMASP-3-PAs were cleaved into the active forms as early as 30 minutes in the circulation of mice, and no significant difference in the efficiency of MASP-3 cleavage was observed throughout an observation period of 48 hours after intravenous administration. All sera collected 3 hours after administration of each rmMASP-3-PA showed full restoration of the active FD and AP activity in MASP-3-deficient mouse sera at the same levels as WT mouse sera. Unexpectedly, all mutant rmMASP-3-PAs showed faster clearance from the circulation than the WT rmMASP-3-PA. To our knowledge, the current study is the first to show in-vivo kinetics of MASP-3 demonstrating rapid activation and clearance in the circulation. In conclusion, our results demonstrated that the complex formation of MASP-3 with LP-PRMs is not required for in-vivo activation of MASP-3 or its efficiency, but may contribute to the long-term retention of MASP-3 in the circulation.

Pre-eclampsia is a pregnancy complication characterized by defective vascular remodeling in maternal decidua responsible for reduced blood flow leading to functional and structural alterations in the placenta. We have investigated the contribution of the complement system to decidual vascular changes and showed that trophoblasts surrounding unremodeled vessels prevalent in preeclamptic decidua fail to express C1q that are clearly detected in cells around remodeled vessels predominant in control placenta. The critical role of C1q is supported by the finding that decidual trophoblasts of femaleC1qa-/-pregnant mice mated toC1qa+/+male mice surrounding remodeled vessels express C1q of paternal origin. UnlikeC1qa-/-pregnant mice, heterozygousC1qa+/-and wild type pregnant mice share a high percentage of remodeled vessels. C1q was also found in decidual vessels and stroma of normal placentae and the staining was stronger in preeclamptic placentae. Failure to detect placental deposition of C1r and C1s associated with C1q rules out complement activation through the classical pathway. Conversely, the intense staining of decidual endothelial cells and villous trophoblast for ficolin-3, MASP-1 and MASP-2 supports the activation of the lectin pathway that proceeds with the cleavage of C4 and C3 and the assembly of the terminal complex. These data extend to humans our previous findings of complement activation through the lectin pathway in an animal model of pre-eclampsia and provide evidence for an important contribution of C1q in decidual vascular remodeling.

Background: Experimental studies have shown that the complement activating enzyme MASP-2 (mannose-binding lectin associated serine protease 2) exhibits a thrombin-like activity and that inhibition of MASP-2 protects against thrombosis. In this study, we investigated whether plasma MASP-2 levels were associated with risk of future venous thromboembolism (VTE) and whether genetic variants linked to MASP-2 levels were associated with VTE risk. Methods: We conducted a population-based nested case-control study involving 410 VTE patients and 842 age- and sex-matched controls derived from the Norwegian Tromsø Study. Logistic regression was used to estimate odds ratios (ORs) of VTE across MASP-2 quartiles. Whole-exome sequencing and protein quantitative trait loci analyses were performed to assess genetic variants associated with MASP-2 levels. A 2-sample Mendelian randomization study, also including data from the INVENT consortium (International Network of Venous Thrombosis), was performed to assess causality. Results: Subjects with plasma MASP-2 in the highest quartile had a 48% higher OR of VTE (OR, 1.48 [95% CI, 1.06–2.06]) and 83% higher OR of deep vein thrombosis (OR, 1.83 [95% CI, 1.23–2.73]) compared with those with MASP-2 levels in the lowest quartile. The protein quantitative trait loci analysis revealed that 3 previously described gene variants, rs12711521 (minor allele frequency, 0.153), rs72550870 (minor allele frequency, 0.045; missense variants in the MASP2 gene), and rs2275527 (minor allele frequency, 0.220; exon variant in the adjacent MTOR gene) explained 39% of the variation of MASP-2 plasma concentration. The OR of VTE per 1 SD increase in genetically predicted MASP-2 was 1.03 ([95% CI, 1.01–1.05] P =0.0011). Conclusions: Our findings suggest that high plasma MASP-2 levels are causally associated with risk of future VTE.

Ecotin is a homodimeric serine protease inhibitor produced by many commensal and pathogenic microbes. It functions as a virulence factor, enabling survival of various pathogens in the blood. The ecotin dimer binds two protease molecules, and each ecotin protomer has two protease-binding sites: site1 occupies the substrate-binding groove, whereas site2 engages a distinct secondary region. Owing to the twofold rotational symmetry within the ecotin dimer, sites 1 and 2 of a protomer bind to different protease molecules within the tetrameric complex. Escherichia coli ecotin inhibits trypsin-like, chymotrypsin-like, and elastase-like enzymes, including pancreatic proteases, leukocyte elastase, key enzymes of blood coagulation, the contact and complement systems, and other antimicrobial cascades. Here, we show that mannan-binding lectin-associated serine protease-1 (MASP-1) and MASP-2, essential activators of the complement lectin pathway, and MASP-3, an essential alternative pathway activator, are all inhibited by ecotin. We decipher in detail how the preorganization of site1 and site2 within the ecotin dimer contributes to the inhibition of each MASP enzyme. In addition, using mutated and monomeric ecotin variants, we show that site1, site2, and dimerization contribute to inhibition in a surprisingly target-dependent manner. We present the first ecotin:MASP-1 and ecotin:MASP-2 crystal structures, which provide additional insights and permit structural interpretation of the observed functional results. Importantly, we reveal that monomerization completely disables the MASP-2-inhibitory, MASP-3-inhibitory, and lectin pathway-inhibitory capacity of ecotin. These findings provide new opportunities to combat dangerous multidrug-resistant pathogens through development of compounds capable of blocking ecotin dimer formation.

Background 3MC syndrome type 3 is an autosomal recessive disorder caused by mutations in the COLEC10 gene besides other genes like COLEC11 and MASP1. This disorder is characterized by facial dysmorphism, cleft lip and palate, postnatal growth deficiency, cognitive impairment, hearing loss, craniosynostosis, radioulnar synostosis, genital and vesicorenal anomalies, cardiac anomalies, caudal appendage, and umbilical hernia. Methods In the present study, whole‐exome sequencing was performed in order to identify disease causing variant in an Iranian 7‐year‐old affected girl with craniosynostosis, dolichocephaly, blepharoptosis, clinodactyly of the 5th finger, high myopia, long face, micrognathia, patent ductus arteriosus, downslanted palpebral fissures, telecanthus, and epicanthus inversus. Identified variant confirmation in the patient and segregation analysis in her family were performed using Sanger sequencing method. Results A novel homozygous frameshift deletion variant [NM_006438.5: c.128_129delCA; p.(Thr43AsnfsTer9)] was identified within the COLEC10 gene. Up to now, only three 3MC syndrome patients with mutations in the COLEC10 gene have been reported, and here, we report the fourth patient and the first homozygous frameshift variant. Conclusion Other genes and factors responsible for 3MC syndrome occurrence are remained to be discovered. We believe further investigation of the genes in the lectin complement pathway is needed to be done for the identification of other causes of this disease., Up to now, only three 3MC syndrome patients with mutations in the COLEC10 gene have been reported, and here, we report the fourth patient and the first homozygous frameshift mutation.

Mannose-binding lectin (MBL)-associated serine protease-2 (MASP-2) is an indispensable enzyme for the activation of the lectin pathway of complement. Its deficiency is classified as a primary immunodeficiency associated to pyogenic bacterial infections, inflammatory lung disease, and autoimmunity. In Europeans, MASP-2 deficiency, due to homozygosity for c.359A > G (p.D120G), occurs in 7 to 14/10,000 individuals. We analyzed the presence of the p.D120G mutation in adults (increasing the sample size of our previous studies) and children. Different groups of patients (1495 adults hospitalized with community-acquired pneumonia, 186 adults with systemic lupus erythematosus, 103 pediatric patients with invasive pneumococcal disease) and control individuals (1119 healthy adult volunteers, 520 adult patients without history of relevant infectious diseases, and a pediatric control group of 311 individuals) were studied. Besides our previously reported MASP-2-deficient healthy adults, we found a new p.D120G homozygous individual from the pediatric control group. We also reviewed p.D120G homozygous individuals reported so far: a total of eleven patients with a highly heterogeneous range of disorders and nine healthy controls (including our four MASP-2-deficient individuals) have been identified by chance in association studies. Individuals with complete deficiencies of several pattern recognition molecules of the lectin pathway (MBL, collectin-10 and collectin-11, and ficolin-3) as well as of MASP-1 and MASP-3 have also been reviewed. Cumulative evidence suggests that MASP-2, and even other components of the LP, are largely redundant in human defenses and that individuals with MASP-2 deficiency do not seem to be particularly prone to infectious or autoimmune diseases.

Background To determine the serum levels of the lectin pathway proteins early in the disease course and 17 years after disease onset and to correlate the protein levels to markers of disease activity in participants from a population-based Nordic juvenile idiopathic arthritis (JIA) cohort. Additionally, to assess the predictive value of lectin pathway proteins with respect to remission status. Methods A population-based cohort study of consecutive cases of JIA with a disease onset from 1997 to 2000 from defined geographical areas of Finland, Sweden, Norway and Denmark with 17 years of follow-up was performed. Clinical characteristics were registered and H-ficolin, M-ficolin, MASP-1, MASP-3, MBL and CL-K1 levels in serum were analyzed. Results In total, 293 patients with JIA were included (mean age 23.7 ± 4.4 years; mean follow-up 17.2 ± 1.7 years). Concentrations of the lectin protein levels in serum were higher at baseline compared to the levels 17 years after disease onset (p ≤ 0.006, n = 164). At baseline, the highest level of M-ficolin was observed in systemic JIA. Further, high M-ficolin levels at baseline and at 17-year follow-up were correlated to high levels of ESR. In contrast, high MASP-1 and MASP-3 tended to correlate to low ESR. CL-K1 showed a negative correlation to JADAS71 at baseline. None of the protein levels had prognostic abilities for remission status 17 years after disease onset. Conclusion We hypothesize that increased serum M-ficolin levels are associated with higher disease activity in JIA and further, the results indicate that MASP-1, MASP-3 and CL-K1 are markers of inflammation.

The lectin pathway (LP) of the complement system is an important antimicrobial defense mechanism, but it also contributes significantly to ischemia reperfusion injury (IRI) associated with myocardial infarct, stroke, and several other clinical conditions. Mannan-binding lectin–associated serine proteinase 2 (MASP-2) is essential for LP activation, and therefore, it is a potential drug target. We have previously developed the first two generations of MASP-2 inhibitors by in vitro evolution of two unrelated canonical serine proteinase inhibitors. These inhibitors were selective LP inhibitors, but their nonhuman origin rendered them suboptimal lead molecules for drug development. Here, we present our third-generation MASP-2 inhibitors that were developed based on a human inhibitor scaffold. We subjected the second Kunitz domain of human tissue factor pathway inhibitor 1 (TFPI1 D2) to directed evolution using phage display to yield inhibitors against human and rat MASP-2. These novel TFPI1-based MASP-2 inhibitor (TFMI-2) variants are potent and selective LP inhibitors in both human and rat serum. Directed evolution of the first Kunitz domain of TFPI1 had already yielded the potent kallikrein inhibitor, Kalbitor® (ecallantide), which is an FDA-approved drug to treat acute attacks of hereditary angioedema. Like hereditary angioedema, acute IRI is also related to the uncontrolled activation of a specific plasma serine proteinase. Therefore, TFMI-2 variants are promising lead molecules for drug development against IRI.

Factor D (also known as adipsin) is a serine protease and part of the complement system, involved in innate immune responses and effector functions of antibodies. Factor D cleaves factor B complexed with C3b, leading to the C3 convertase C3bBb. This C3 convertase is central in the alternative activation pathway and the amplification loop, which amplifies the two other complement activation pathways: the classical pathway and the lectin pathway. Adipocytes synthesize factor D as a pro-form comprising 6 additional residues that must be cleaved off to generate a mature form. The MBL-associated serine protease 3 (MASP-3), found in complex with the pattern recognition molecules of lectin activation pathway, converts the pro-form to mature factor D, which reportedly is the most abundant form found in the circulation at concentrations of 1-2 μg/ml among healthy individuals. The mature factor D is rate-limiting for complement activation, but little is known about the distribution of pro vs. mature factor D in the circulation, the regulation hereof and the potential activation stimuli of the lectin pathway, responsible for activation of MASP-3 and subsequent conversion of pro-form of factor D. In this light we established and validated an ELISA specific for measuring the pro-form of complement factor D. With a working range of 0.82-25 ng/ml, acceptable intra and inter assay CVs, and a relative recovery rate above 90%, we found that the median plasma concentration in Danish blood donors was 134 ng/ml; corresponding to that 8-15% factor D circulates as pro-form. We also found that blood sampling procedures affect conversion and hence the levels measured in serum and plasma.

Mannan-binding lectin-associated serine protease-2 (MASP-2) has been reported to play an important role as a key enzyme in the lectin pathway of the complement system. The objectives of our study were to determine whether the single-nucleotide polymorphism (SNPs) of MASP2 and the gene-tea drinking interaction were associated with the susceptibility to TB. In total, 503 patients and 494 healthy controls were contained. Three SNPs (rs12142107, rs12711521, and rs7548659) were genotyped. The association between the SNPs and susceptibility to TB were investigated by conducting multivariate unconditional logistic regression analysis. The gene-tea drinking interactions were analyzed by the additive model of marginal structural linear odds models. Both genotype AC + AA at rs12711521 of MASP2 genes and genotype GT + GG at rs7548659 of MASP2 genes were more prevalent in the TB patient group than the healthy control group (OR: 1.423 and 1.439, respectively, P MASP2 genes was found to suggest negative interactions, which reached − 0.2311 (95% confidence interval (CI): − 0.4736, − 0.0113), − 0.7080 (95% CI − 1.3998, − 0.0163), and − 0.5140 (95% CI − 0.8988, − 0.1291), respectively (P MASP2 were associated with the susceptibility to TB. Furthermore, there were negative interactions between tea drinking and rs12142107, rs12711521, and rs75548659 of MASP2 gene, respectively. Our research provides a basis for studying the pathogenesis and prevention of tuberculosis.

Activation of the complement system is mediated by the interaction between pathogens and pattern recognition molecules (PRMs); mannose-binding lectin (MBL), ficolins, and collectin-10/-11 from the lectin pathway and C1q from the classical pathway. Lectin pathway activation specifically depends on proteases named MBL-associated serine proteases (MASPs) that are found in complexes with PRMs. In this study, we hypothesize that MASPs can recognize selected pathogens independently of PRMs. Using different clinical strains of opportunistic fungi, we have observed that MASPs directly recognize certain fungal pathogens in a way that can facilitate complement activation. Among these were Aspergillus fumigatus - a dangerous pathogen, especially for immunocompromised patients. In flow cytometry and fluorescence microscopy, we found that MASP-1 and -3 bound to all A. fumigatus growth stages (conidia, germ tubes, and hyphae), whereas rMASP-2 and the nonproteolytic rMAP-1 did not. Bound rMASPs could recruit rMBL and rficolin-3 to A. fumigatus conidia in a nonclassical manner and activate complement via rMASP-2. In experiments using recombinant and purified components, rMASP-1 increased the neutrophilic phagocytosis of conidia. In serum where known complement activation pathways were blocked, phagocytosis could be mediated by rMASP-3. We have encountered an unknown pathway for complement activation and found that MASP-1 and MASP-3 have dual functions as enzymes and as PRMs.

MASP-2, mannose-binding protein-associated serine protease 2, is a key enzyme in the lectin pathway of complement activation. Hyperactivation of this protein by human coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2 has been found to contribute to aberrant complement activation in patients, leading to aggravated lung injury with potentially fatal consequences. This hyperactivation is triggered in the lungs through a conserved, direct interaction between MASP-2 and coronavirus nucleocapsid (N) proteins. Blocking this interaction with monoclonal antibodies and interfering directly with the catalytic activity of MASP-2, have been found to alleviate coronavirus-induced lung injury both in vitro and in vivo. In this study, a virtual library of 8736 licensed drugs and clinical agents has been screened in silico according to two parallel strategies. The first strategy aims at identifying direct inhibitors of MASP-2 catalytic activity, while the second strategy focusses on finding protein-protein interaction inhibitors (PPIs) of MASP-2 and coronaviral N proteins. Such agents could represent promising support treatment options to prevent lung injury and reduce mortality rates of infections caused by both present and future-emerging coronaviruses. Forty-six drug repurposing candidates were purchased and, for the ones selected as potential direct inhibitors of MASP-2, a preliminary in vitro assay was conducted to assess their interference with the lectin pathway of complement activation. Some of the tested agents displayed a dose-response inhibitory activity of the lectin pathway, potentially providing the basis for a viable support strategy to prevent the severe complications of coronavirus infections.

The complement system was discovered at the end of the 19th century as a heat-labile plasma component that “complemented” the antibodies in killing microbes, hence the name “complement.” Complement is also part of the innate immune system, protecting the host by recognition of pathogen-associated molecular patterns. However, complement is multifunctional far beyond infectious defense. It contributes to organ development, such as sculpting neuron synapses, promoting tissue regeneration and repair, and rapidly engaging and synergizing with a number of processes, including hemostasis leading to thromboinflammation. Complement is a double-edged sword. Although it usually protects the host, it may cause tissue damage when dysregulated or overactivated, such as in the systemic inflammatory reaction seen in trauma and sepsis and severe coronavirus disease 2019 (COVID-19). Damage-associated molecular patterns generated during ischemia-reperfusion injuries (myocardial infarction, stroke, and transplant dysfunction) and in chronic neurologic and rheumatic disease activate complement, thereby increasing damaging inflammation. Despite the long list of diseases with potential for ameliorating complement modulation, only a few rare diseases are approved for clinical treatment targeting complement. Those currently being efficiently treated include paroxysmal nocturnal hemoglobinuria, atypical hemolytic-uremic syndrome, myasthenia gravis, and neuromyelitis optica spectrum disorders. Rare diseases, unfortunately, preclude robust clinical trials. The increasing evidence for complement as a pathogenetic driver in many more common diseases suggests an opportunity for future complement therapy, which, however, requires robust clinical trials; one ongoing example is COVID-19 disease. The current review aims to discuss complement in disease pathogenesis and discuss future pharmacological strategies to treat these diseases with complement-targeted therapies. Significance Statement - The complement system is the host’s defense friend by protecting it from invading pathogens, promoting tissue repair, and maintaining homeostasis. Complement is a double-edged sword, since when dysregulated or overactivated it becomes the host’s enemy, leading to tissue damage, organ failure, and, in worst case, death. A number of acute and chronic diseases are candidates for pharmacological treatment to avoid complement-dependent damage, ranging from the well established treatment for rare diseases to possible future treatment of large patient groups like the pandemic coronavirus disease 2019.

ObjectivesFatigue is common and severe in primary Sjögren’s syndrome (pSS). The aim of this study was to identify genetic determinants of fatigue in pSS through a genome-wide association study.MethodsPatients with pSS from Norway, Sweden, UK and USA with fatigue and genotype data available were included. After genotype imputation and quality control, 682 patients and 4 966 157 genetic markers were available. Association analysis in each cohort using linear regression with fatigue as a continuous variable and meta-analyses between the cohorts were performed.ResultsMeta-analysis of the Norwegian and Swedish cohorts identified five polymorphisms within the same linkage disequilibrium block at the receptor transporter protein 4 (RTP4)/MASP1 locus associated with fatigue with genome-wide significance (GWS) (p−8). Patients homozygous for the major allele scored 25 mm higher on the fatigue Visual Analogue Scale than patients homozygous for the minor allele. There were no variants associated with fatigue with GWS in meta-analyses of the US/UK cohorts, or all four cohorts. RTP4 expression in pSS B cells was upregulated and positively correlated with the type I interferon score. Expression quantitative trait loci effects in whole blood for fatigue-associated variants at RTP4/MASP1 and levels of RTP4 and MASP1 expression were identified.ConclusionGenetic variations at RTP4/MASP1 are associated with fatigue in Scandinavian pSS patients. RTP4 encodes a Golgi chaperone that influences opioid pain receptor function and MASP1 is involved in complement activation. These results add evidence for genetic influence over fatigue in pSS.

MiR-122-5p is a diagnostic and prognostic biomarker of sepsis and is correlated with coagulation abnormalities in sepsis. However, its functional aspects remain unknown. This study applied bioinformatics analysis to evaluate the coagulation-related target genes for miR-122-5p. THP-1, HUVEC, and LO-2 cell lines were used in this study. MiR-122-5p mimics were transfected into the three previously mentioned cell lines, which helped in detecting mRNA and protein levels by qRT-PCR and western blotting, respectively. Serum samples from 84 sepsis patients were collected to evaluate target gene code proteins. The protein and mRNA levels of Heme oxygenase1(HO-1), IL-1β, IL-6, monocyte chemoattractant protein 1(MCP-1), and TNF-α were also evaluated in three cell lines. Mannan binding lectin serine peptidase 1(MASP1) was a direct target gene of miR-122-5p, and levels of MASP1, C3, and C4 were all significantly lower in the sepsis with disseminated intravascular coagulopathy (DIC) group than in the sepsis without DIC group. MiR-122-5p mimics could down-regulate HO-1 in the three cell lines. HO-1, IL-1β, IL-6, MCP-1, and TNF-α gene and protein levels were decreased after miR-122-5p mimics were added. MiR-122-5p regulated coagulation and inflammation through MASP1 and HO-1, respectively.

Aim: Activation of the complement system is known to be a potent inducer of systemic inflammation, which is an important component of post-cardiac arrest syndrome. Mannan-binding-lectin associated protein of 19 kDa (MAp19) is suggested to be a regulatory component of the lectin pathway of complement activation. The aims of this study were to describe serial levels of MAp19 protein in comatose survivors of out-of-hospital cardiac arrest (OHCA), to evaluate the effect of two different regimes of targeted temperature management and to investigate the possible association between levels of MAp19 and mortality. Methods: In this post-hoc study, we analysed data from two large randomized controlled studies: ‘Targeted temperature management at 33 degrees C versus 36 degrees C after cardiac arrest’ (TTM) and ‘Targeted temperature management for 48 versus 24 h and neurological outcome after out-of-hospital cardiac arrest’ (TTH). We measured serial levels of MAp19 in 240 patients within 72 h after OHCA and in 82 healthy controls. The effect of targeted temperature management on MAp19 levels was analysed according to temperature allocation in main trials. Results: MAp19 levels were significantly lower in OHCA patients within 48 h after OHCA (p-values Conclusion: Survivors after OHCA have lower levels of MAp19 protein compared with healthy controls. A targeted temperature management at 33°C compared with 36°C was associated with significantly lower MAp19 levels, whereas target temperature at 33°C for 48 h compared with 24 h did not influence MAp19 protein levels. Low MAp19 levels at admission were independently associated with increased mortality.