MBL-associated Serine Proteases Research Articles

Endothelial injury and thrombotic microangiopathy in COVID-19: Treatment with the lectin-pathway inhibitor narsoplimab

In COVID-19, acute respiratory distress syndrome (ARDS) and thrombotic events are frequent, life-threatening complications. Autopsies commonly show arterial thrombosis and severe endothelial damage. Endothelial damage, which can play an early and central pathogenic role in ARDS and thrombosis, activates the lectin pathway of complement. Mannan-binding lectin-associated serine protease-2 (MASP-2), the lectin pathway’s effector enzyme, binds the nucleocapsid protein of severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2), resulting in complement activation and lung injury. Narsoplimab, a fully human immunoglobulin gamma 4 (IgG4) monoclonal antibody against MASP-2, inhibits lectin pathway activation and has anticoagulant effects. In this study, the first time a lectin-pathway inhibitor was used to treat COVID-19, six COVID-19 patients with ARDS requiring continuous positive airway pressure (CPAP) or intubation received narsoplimab under compassionate use. At baseline and during treatment, circulating endothelial cell (CEC) counts and serum levels of interleukin-6 (IL-6), interleukin-8 (IL-8), C-reactive protein (CRP) and lactate dehydrogenase (LDH) were assessed. Narsoplimab treatment was associated with rapid and sustained reduction of CEC and concurrent reduction of serum IL-6, IL-8, CRP and LDH. Narsoplimab was well tolerated; no adverse drug reactions were reported. Two control groups were used for retrospective comparison, both showing significantly higher mortality than the narsoplimab-treated group. All narsoplimab-treated patients recovered and survived. Narsoplimab may be an effective treatment for COVID-19 by reducing COVID-19-related endothelial cell damage and the resultant inflammation and thrombotic risk.

Association of Polymorphisms of MASP1/3, COLEC10, and COLEC11 Genes with 3MC Syndrome.

The Malpuech, Michels, Mingarelli, Carnevale (3MC) syndrome is a rare, autosomal recessive genetic- disorder associated with mutations in the MASP1/3, COLEC1,1 or COLEC10 genes. The number of 3MC patients with known mutations in these three genes reported so far remains very small. To date, 16 mutations in MASP-1/3, 12 mutations in COLEC11 and three in COLEC10 associated with 3MC syndrome have been identified. Their products play an essential role as factors involved in the activation of complement via the lectin or alternative (MASP-3) pathways. Recent data indicate that mannose-binding lectin-associated serine protease-1 (MASP-1), MASP-3, collectin kidney-1 (collectin-11) (CL-K1), and collectin liver-1 (collectin-10) (CL-L1) also participate in the correct migration of neural crest cells (NCC) during embryogenesis. This is supported by relationships between MASP1/3, COLEC10, and COLEC11 gene mutations and the incidence of 3MC syndrome, associated with craniofacial abnormalities such as radioulnar synostosis high-arched eyebrows, cleft lip/palate, hearing loss, and ptosis.

Components of the Lectin Pathway of Complement in Haematologic Malignancies.

The complement system is activated cascadically via three distinct major routes: classical pathway (CP), alternative pathway (AP) or lectin pathway (LP). The unique factors associated with the latter are collectins (mannose-binding lectin, collectin-10, collectin-11), ficolins (ficolin-1, ficolin-2, ficolin-3) and proteins of the mannose-binding lectin-associated serine protease (MASP) family (MASP-1, MASP-2, MASP-3, MAp19, MAp44). Collectins and ficolins are both pattern-recognising molecules (PRM), reactive against pathogen-associated molecular patterns (PAMP) or danger-associated molecular patterns (DAMP). The MASP family proteins were first discovered as complexes with mannose-binding lectin (MBL) and therefore named MBL-associated serine proteases, but later, they were found to interact with ficolins, and later still, collectin-10 and collectin-11. As well as proteolytic enzymes (MASP-1, MASP-2, MASP-3), the group includes non-enzymatic factors (MAp19, MAp44). In this review, the association-specific factors of the lectin pathway with haematologic malignancies and related infections are discussed.

Complement component C1q plays a critical role in VLRA/VLRC-mediated immune response

In jawless vertebrates, the lamprey complement component C1q (LC1q) acts as a lectin and activates lamprey complement component C3 (LC3) in association with mannose-binding lectin (MBL)-associated serine protease (MASP) via the lectin pathway. Furthermore, LC1q may interact with variable lymphocyte receptor B (VLRB) in a complex with antigens and mediate the activation of LC3, leading to cytolysis. In the present study, we found, for the first time, that LC1q plays a critical role in VLRA/VLRC-mediated immune response. Escherichia coli, Shigella flexneri, Aeromonas hydrophila, Pseudomonas plecoglossicida, Aeromonas allosaccharophila, P. luteola, Brevundimonas diminuta, and Bacillus cereus were isolated from infected Lampetra morii in our laboratory and identified using the 16s rRNA method. A. hydrophila was confirmed as a rapidly spreading lethal pathogen in the larvae of L. morii and was used in subsequent immune stimulation experiments. The results of real-time quantitative polymerase chain reaction (Q-PCR) and immunofluorescence analyses indicated that the RNA and protein expression levels of LC1q were upregulated following exposure to 107 cfu/mL of A. hydrophila, compared to the levels of the naïve group. We obtained LC1q morphants (LC1q MO) of lamprey larvae by morpholino-mediated knockdowns. We found that LC1q played key roles in the embryonic development of lamprey. The median lethal time (LT50) of LC1q MO larvae was 2 d after being exposed to the pathogens, whereas the LT50 of control MO was 5 d. The drastic decrease in LT50 values after LC1q knockdown implies that LC1q plays a critical role in lamprey immune response. Gene expression profiles of LC1q-deficient A. hydrophila, control MO A. hydrophila, wild type A. hydrophila, and naive 1-month-old ammocoetes larvae were compared by examining the expression levels of a selected panel of orthologous genes. It is worth mentioning that LC1q MO affected the VLRA+/VLRC + population genes but did not affect the VLRB + populations. Immunohistochemical data indicated that LC1q deficiency also affected VLRA and VLRC but not VLRB. Thus, LC1q plays a critical role in VLRA/VLRC-mediated immune response in lamprey.

Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: A report of five cases

Acute respiratory failure and a systemic coagulopathy are critical aspects of the morbidity and mortality characterizing infection with severe acute respiratory distress syndrome-associated coronavirus-2, the etiologic agent of Coronavirus disease 2019 (COVID-19). We examined skin and lung tissues from 5 patients with severe COVID-19 characterized by respiratory failure (n= 5) and purpuric skin rash (n = 3). COVID-19 pneumonitis was predominantly a pauci-inflammatory septal capillary injury with significant septal capillary mural and luminal fibrin deposition and permeation of the interalveolar septa by neutrophils. No viral cytopathic changes were observed and the diffuse alveolar damage (DAD) with hyaline membranes, inflammation, and type II pneumocyte hyperplasia, hallmarks of classic acute respiratory distress syndrome, were not prominent. These pulmonary findings were accompanied by significant deposits of terminal complement components C5b-9 (membrane attack complex), C4d, and mannose binding lectin (MBL)-associated serine protease (MASP)2, in the microvasculature, consistent with sustained, systemic activation of the complement pathways. The purpuric skin lesions similarly showed a pauci-inflammatory thrombogenic vasculopathy, with deposition of C5b-9 and C4d in both grossly involved and normally-appearing skin. In addition, there was co-localization of COVID-19 spike glycoproteins with C4d and C5b-9 in the interalveolar septa and the cutaneous microvasculature of 2 cases examined. In conclusion, at least a subset of sustained, severe COVID-19 may define a type of catastrophic microvascular injury syndrome mediated by activation of complement pathways and an associated procoagulant state. It provides a foundation for further exploration of the pathophysiologic importance of complement in COVID-19, and could suggest targets for specific intervention.

Remote ischemic preconditioning does not influence lectin pathway protein levels in head and neck cancer patients undergoing surgery.

Cancer patients who undergo tumor removal, and reconstructive surgery by transfer of a free tissue flap, are at high risk of surgical site infection and ischemia-reperfusion injury. Complement activation through the lectin pathway (LP) may contribute to ischemia-reperfusion injury. Remote ischemic preconditioning (RIPC) is a recent experimental treatment targeting ischemia-reperfusion injury. The study aims were to investigate LP protein plasma levels in head and neck cancer patients compared with healthy individuals, to explore whether RIPC affects LP protein levels in head and neck cancer surgery, and finally to examine the association between postoperative LP protein levels and the risk of surgical site infection. Head and neck cancer patients (n = 60) undergoing tumor resection and reconstructive surgery were randomized 1:1 to RIPC or sham intervention administered intraoperatively. Blood samples were obtained preoperatively, 6 hours after RIPC/sham, and on the first postoperative day. LP protein plasma levels were measured utilizing time-resolved immunofluorometric assays. H-ficolin and M-ficolin levels were significantly increased in cancer patients compared with healthy individuals (both P ≤ 0.02). Conversely, mannan-binding lectin (MBL)-associated serine protease (MASP)-1, MASP-3, collectin liver-1 (CL-L1), and MBL-associated protein of 44 kilodalton (MAp44) levels were decreased in cancer patients compared with healthy individuals (all P ≤ 0.04). A significant reduction in all LP protein levels was observed after surgery (all P < 0.001); however, RIPC did not affect LP protein levels. No difference was demonstrated in postoperative LP protein levels between patients who developed surgical site infection and patients who did not (all P > 0.13). The LP was altered in head and neck cancer patients. LP protein levels were reduced after surgery, but intraoperative RIPC did not influence the LP. Postoperative LP protein levels were not associated with surgical site infection.

Adding MASP1 to the lectin pathway-Leprosy association puzzle: Hints from gene polymorphisms and protein levels.

Deposition of complement factors on Mycobacterium leprae may enhance phagocytosis. Such deposition may occur through the lectin pathway of complement. Three proteins of the lectin pathway are produced from the gene MASP1: Mannan-binding lectin-associated serine protease 1 (MASP-1) and MASP-3 and mannan-binding lectin-associated protein of 44 kDa (MAp44). Despite their obvious importance, the roles played by these proteins have never been investigated in leprosy disease. We haplotyped five MASP1 polymorphisms by multiplex sequence-specific PCR (intronic rs7609662*G>A and rs13064994*C>T, exon 12 3'-untranslated rs72549262*C>G, rs1109452*C>T and rs850314*G>A) and measured MASP-1, MASP-3 and MAp44 serum levels in 196 leprosy patients (60%, lepromatous) and 193 controls. Lower MASP-3 and MAp44 levels were observed in patients, compared with controls (P = 0.0002 and P<0.0001, respectively) and in lepromatous, compared with non-lepromatous patients (P = 0.008 and P = 0.002, respectively). Higher MASP-3 levels were present in controls carrying variants/haplotypes associated with leprosy resistance (rs13064994*T, rs1109452_rs850314*CG within GT_CCG and rs850314*A: OR = 0.5-0.6, Pcorr = 0.01-0.04). Controls with rs1109452*T, included in susceptibility haplotypes (GT_GTG/GT_CTG: OR = 2.0, Pcorr = 0.03), had higher MASP-1 and lower MASP-3 levels (P≤0.009). Those with GC_CCG, presented increasing susceptibility (OR = 1.7, Pcorr = 0.006) and higher MAp44 levels (P = 0.015). MASP-3 expression decreased in patients, compared with controls carrying rs1109452_rs850314*CA or CG (P≤0.02), which may rely on exon 12 CpG methylation and/or miR-2861/miR-3181 mRNA binding. Polymorphisms regulating MASP-3/MAp44 availability in serum modulate leprosy susceptibility, underlining the importance of lectin pathway regulation against pathogens that exploit phagocytosis to parasitize host macrophages.

Human primary endothelial label-free biochip assay reveals unpredicted functions of plasma serine proteases

Tissue-on-a-chip technologies are more and more important in the investigation of cellular function and in the development of novel drugs by allowing the direct screening of substances on human cells. Constituting the inner lining of vessel walls, endothelial cells are the key players in various physiological processes, moreover, they are the first to be exposed to most drugs currently used. However, to date, there is still no appropriate technology for the label-free, real-time and high-throughput monitoring of endothelial function. To this end, we developed an optical biosensor-based endothelial label-free biochip (EnLaB) assay that meets all the above requirements. Using our EnLaB platform, we screened a set of plasma serine proteases as possible endothelial cell activators, and first identified the endothelial cell activating function of three important serine proteases – namely kallikrein, C1r and mannan-binding lectin-associated serine-protease 2 (MASP-2) – and verified these results in well-established functional assays. EnLaB proved to be an effective tool for revealing novel cellular mechanisms as well as for the high-throughput screening of various compounds on endothelial cells.

Key Components of the Complement Lectin Pathway Are Not Only Required for the Development of Inflammatory Arthritis but Also Regulate the Transcription of Factor D

The complement system plays an important role in the pathogenesis of rheumatoid arthritis (RA). Besides driving lectin pathway (LP) activation, the mannan-binding lectin (MBL)-associated serine proteases (MASPs) also play a key role in regulating the alternative pathway (AP). We evaluated the effects of N-acetylgalactosamine (GalNAc)-conjugated MASP-1 and MASP-2 duplexes in vitro and in mice with and without arthritis to examine whether knockdown of MASP-1 and MASP-2 expression affects the development of arthritis. GalNAc-siRNAs for MASP-1 and MASP-2 demonstrated robust silencing of MASP-1 or MASP-2 at pM concentrations in vitro. To evaluate the impact of silencing in arthritic mice, we used the collagen antibody-induced arthritis (CAIA) mouse model of RA. Mice were injected a 10 mg/kg dose of GalNAc-siRNAs 3x s.q. prior to the induction of CAIA. Liver gene expression was examined using qRT-PCR, and protein levels were confirmed in the circulation by sandwich immunoassays and Western blot. At day 10, CAIA mice separately treated with MASP-1 and MASP-2 duplexes had a specific reduction in expression of liver MASP-1 (70–95%, p < 0.05) and MASP-2 (90%, p < 0.05) mRNA, respectively. MASP-1-siRNA treatment resulted in a 95% reduction in levels of MASP-1 protein in circulation with no effect on MASP-2 levels and clinical disease activity (CDA). In mice injected with MASP-2 duplex, there was a significant (p < 0.05) 90% decrease in ex vivo C4b deposition on mannan, with nearly complete elimination of MASP-2 in the circulation. MASP-2 silencing initially significantly decreased CDA by 60% but subsequently changed to a 40% decrease vs. control. Unexpectedly, GalNAc-siRNA-mediated knockdown of MASP-1 and MASP-2 revealed a marked effect of these proteins on the transcription of FD under normal physiological conditions, whereas LPS-induced inflammatory conditions reversed this effect on FD levels. LPS is recognized by Toll-like receptor 4 (TLR4), we found MBL not only binds to TLR4 an interaction with a Kd of 907 nM but also upregulated FD expression in differentiated adipocytes. We show that MASP-2 knockdown impairs the development of RA and that the interrelationship between proteins of the LP and the AP may extend to the transcriptional modulation of the FD gene.

Selected factors of the innate immunity in Polish patients suffering from pulmonary tuberculosis.

We conducted a prospective study of 453 Polish patients suffering from pulmonary tuberculosis and 267 healthy controls. Selected polymorphisms of the genes encoding for collectins, ficolins and MBL-associated serine protease 2 were investigated as were serum concentrations of mannose-binding lectin, surfactant protein D, ficolin-1 and ficolin-3. The number of MBL2 gene exon 1 variant allele carriers was significantly higher in patients, compared with controls. The homozygosity for SFTPA2 +26 C > A SNP variant allele occurred less commonly within TB, while homozygosity for the FCN1 -542 G > A major allele was less frequent within the control group. Two patients were found MASP-2-deficient. Serum concentrations of MBL, SP-D and ficolin-1 were higher amongst patients while the converse was found for ficolin-3. Ficolin-1 had high specificity to differentiate between individuals with tuberculosis and healthy persons and therefore may be considered potential disease marker.

The Clinical Significance of Serum MASP-2 and IDH1 in the Early Diagnosis of Non-Small Cell Lung Cancer.

The lack of effective means for the early diagnosis of non-small cell lung cancer (NSCLC) is the leading cause of the high mortality of NSCLC. This study aims to evaluate the clinical significance of serum mannan-binding lectin associated serine protease (MASP)-2 and isocitrate dehydrogenase 1 (IDH1) in the early diagnosis of NSCLC. The serum levels of MASP-2 and IDH1 were detected in 139 NSCLC patients, 46 patients with benign lung diseases and 61 healthy controls, using an enzyme linked immunosorbent method. The diagnostic significance in NSCLC of the two tumor markers were analyzed by receiver operating characteristic (ROC) curves. In addition, we compared the two markers with the current commonly used tumor marker cytokeratin 19 fragment (Cy¬fra21-1). The serum levels of MASP-2 and IDH1 in the NSCLC patients were significantly higher than those of healthy controls and patients with benign lung diseases. The differences were statistically significant (p < 0.01). The combined sensitivity of MASP-2, IDH1, and Cyfra21-1 in the NSCLC was 68.3%, which was significantly higher than that of the single tumor marker (p < 0.01). The sensitivities of MASP-2 and IDH1 in detecting early NSCLC (stage I and stage II) were 39.0% and 41.5%, which were significantly higher than that of Cyfra21-1 (p < 0.05). The area under the ROC curves (AUCs) of MASP-2 and IDH1 in the diagnosis of NSCLC were 0.621, and 0.840, which were higher than that of Cyfra21-1 (AUC = 0.606). Serum MASP-2 and IDH1 may be used as potential tumor markers for the auxiliary diagnosis and early diagnosis of NSCLC.

Should MASP-2 Deficiency Be Considered a Primary Immunodeficiency? Relevance of the Lectin Pathway.

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.

Mannose-Binding Lectin and Mannose-Binding Lectin-Associated Serine Protease-2 Genotypes and Serum Levels in Patients with Sporotrichosis.

Mannose-binding lectin (MBL) and MBL-associated serine protease-2 (MASP-2) are important proteins in the lectin pathway of the immune system. Mannose-binding lectin and MASP-2 deficiencies have been reported to be responsible for various fungal infections. We investigated the association of MBL and MASP-2 variants with sporotrichosis in a Chinese population and revealed one rare heterozygous mutation in a disseminated cutaneous patient without immunosuppressive conditions (MASP2, p.156_159dupCHNH). We also found that sporotrichosis patients had decreased levels of MBL and MASP-2 in their serum samples compared with controls. Our findings linked, for the first time, MASP-2 deficiencies with susceptibility to Sporothrix sp.

Masp-2 Levels Following Allogeneic Hematopoietic Stem Cell Transplantation in Adults: Correlation with Development of a Thrombotic Microangiopathy and Implications for Therapy with Anti-Complement Agents

Introduction: 8500 adult allogeneic hematopoietic stem cell transplants (alloHSCT) are performed annually in the U.S. and 17,000 in Europe. HSCT-associated thrombotic microangiopathy (TMA), defined by thrombocytopenia, microangiopathic hemolytic anemia, and organ dysfunction in the absence of disseminated intravascular coagulation, complicates some 20% of these procedures. About half of post-alloHSCT TMAs may resolve when GvHD immunoprophylaxis is modified, but three year survival rates for those with severe HSCT-TMAs are a dismal 11%. Clinical manifestations are similar to other TMAs, but their pathophysiology may be distinct. Damage to vascular endothelium, independent of loss of ADAMTS13 activity, is thought to be critical. Fibrin-rich microthrombi, often accompanied by C4d and C5b-9 (membrane attack complex) deposition, occur in the microvasculature of multiple organs (Clin Adv Hematol Oncol 2014;12:565-573; Transplantation. 2013;96:217-223). This supports a role for complement activation in HSCT-TMA, but the importance of the various complement activation pathways is unclear. Recently, narsoplimab (OMS721), a monoclonal antibody inhibitor of MBL-associated serine protease-2 (MASP-2), a principal component of lectin-dependent complement activation, received Breakthrough Therapy Designation for HSCT-TMA, based on improved survival compared to historical controls in a phase 2 study. A phase 3 program is ongoing. Chemotherapy in association with autologous HSCT is linked to a marked increase in serum MASP-2 levels, persisting for about 4 weeks post-transplant (Front Immunol 2018;9:2153). However, TMAs are rare in autologous transplants, and circulating levels of MASP-2 following alloHSCT, and the impact of TMA development on those levels, are unknown. Methods: All individuals &gt;18 years of age scheduled to undergo alloHSCT for hematologic malignancy at New York Presbyterian Hospital-Cornell were approached to participate in this study (NCT02604420). 100 of the first 101 subjects, age 58.3 +14 yrs, were enrolled and followed for &gt;1.5 years post-transplant. This interval is consistent with the median time to HSCT-TMA diagnosis in adults of 90 days (range 32-733 days). Plasma was obtained at baseline (defined as between the time of consent and beginning of conditioning regimen), and at each regularly scheduled visit post-transplant-day 28 +5 days; day 100 +28 days; day 180 +28 days; and day 365 +28 days-as well as at the time of TMA diagnosis, based on the Consensus Criteria of Cho et al. (Transplantation 2010;90:918-926). A commercial ELISA (MyBioSource) was used to assess MASP-2 concentrations. Results: 20 subjects met study criteria for a HSCT-TMA diagnosis, occurring a median of 69 days (range 33-289) post-transplant. Three resolved following discontinuation of GvHD prophylaxis (mTOR or calcineurin inhibitor) and switch to mycophenolate and increased corticosteroid doses, and 7 had an intercurrent infection, 6 of whom expired with ongoing severe TMA despite a change in GvHD prophylaxis (Figure). TMAs persisted in the remaining 10 subjects. Median MASP-2 levels were significantly elevated in all subjects post-transplant, assessed at the time of TMA development or, in those not developing a TMA, at day 100 + 28 days post-transplant vs. controls (n=36, 86.2ng/ml (23.3-210.9): persistent TMA (n=9 (one plasma unavailable), 154ng/ml (range 82-209)); alloHSCT subjects who did not experience a TMA (n=40 evaluated to date, 113.5ng/ml (56-430.3)). (Figure). Lack of a significant rise in MASP-2 levels in patients with persistent TMAs vs. those who did not develop a TMA, combined with a significant decrease in variance of MASP-2 levels in the former group (p=0.005), may reflect consumption of product at sites of disease activity, i.e., the microvasculature. Conclusions: There is a significant increase in MASP-2 levels, with a wide variance, in post-alloHSCT patients evaluated at a time post-transplant typical of HSCT-TMA development. At time of development of a HSCT-TMA that persists despite withdrawal of GvHD prophylaxis, MASP-2 levels remain elevated over controls, but with a significantly lower variance vs. those not developing TMA. A study of additional samples, including longitudinal specimens, from this cohort is underway to determine if a change in MASP-2 levels correlates with HSCT-TMA development post-alloHSCT. Disclosures Van Besien: Miltenyi Biotec: Research Funding.

Evidence for Activation of Lectin and Classical Pathway Complement Components in Aqueous Humor of Neovascular Age-Related Macular Degeneration

Purpose: The complement system is activated via 3 different pathways; the lectin pathway (LP), classical pathway (CP), and alternative pathway. To investigate the possible roles for the LP or CP in the development of neovascular age-related macular degeneration (nAMD), we compared aqueous humor levels of complement proteins of the LP and CP between eyes with nAMD and those with cataract as controls. Methods: Seventeen eyes from 17 patients with treatment-naïve nAMD and 9 eyes from 9 patients with cataract were studied. Aqueous humor samples were collected before intravitreal aflibercept or ranibizumab injection for the nAMD patients and before cataract surgery for the cataract patients. Aqueous humor levels of complement C4 of the LP and CP, complement C3 of all 3 complement pathways, and mannose-binding lectin-associated serine protease (MASP)-2 of the LP were measured by enzyme-linked immunosorbent assay. Aqueous humor levels of C4a and C3a, the activation products of C4 and C3, respectively, were measured by a bead-based immunoassay. The ratios of C4a to C4 and C3a to C3, representing the degree of C4 and C3 activation, respectively, were calculated in individual patients. Results: The aqueous humor levels of C4, C3, and MASP-2 were significantly lower in the nAMD eyes compared to the controls (p = 0.008, p = 0.011, and p = 0.018, respectively). In contrast, the aqueous humor levels of C4a and C3a, as well as the C4a/C4 and C3a/C3 ratios, were significantly higher in the nAMD eyes compared to the controls (p = 0.039, p = 0.003, p < 0.001, and p < 0.001, respectively). Conclusions: This study provides evidence for significant intraocular activation of either or both of the LP and CP in nAMD eyes that might be involved in the development of nAMD. The significantly lower levels of MASP-2 in the aqueous humor of the nAMD eyes were likely due to MASP-2 consumption by activation of the LP.

Cutting Edge: Role of MASP-3 in the Physiological Activation of Factor D of the Alternative Complement Pathway.

The complement system, a part of the innate immune system, can be activated via three different pathways. In the alternative pathway, a factor D (FD) plays essential roles in both the initiation and the amplification loop and circulates as an active form. Mannose-binding lectin-associated serine proteases (MASPs) are key enzymes of the lectin pathway, and MASP-1 and/or MASP-3 are reported to be involved in the activation of FD. In the current study, we generated mice monospecifically deficient for MASP-1 or MASP-3 and found that the sera of the MASP-1-deficient mice lacked lectin pathway activity, but those of the MASP-3-deficient mice lacked alternative pathway activity with a zymogen FD. Furthermore, the results indicate that MASP-3 but not MASP-1 activates the zymogen FD under physiological conditions and MASP-3 circulates predominantly as an active form. Therefore, our study illustrates that, in mice, MASP-3 orchestrates the overall complement reaction through the activation of FD.

Identification and characterization of two mannan-binding lectin associated proteins in lectin complement pathway of grass carp

The lectin pathway of complement activation is an important component of the innate immune response, which must be tightly controlled to maintain immune homeostasis. However, its control mechanisms have not been investigated in detail in bony fish. In this study, we identified and characterized two novel, phylogenetically conserved mannan-binding lectin (MBL)-associated proteins (MAps) of grass carp (Ctenopharyngodon idella), CiMAp27 and CiMAp39, which were truncated, alternatively-spliced forms of grass carp MBL-associated serine proteases (MASPs), CiMASP1 and CiMASP2, respectively. Gene expression profiling showed that both CiMAp27 and CiMAp39 were upregulated by low doses of Aeromonas hydrophila, and inhibited by high doses, which lead to the inference that these genes acted as immune factors in antibacterial defense. Sequence analysis showed that CiMAp27 lack a catalytic domain but retains two domains (CUB1-EGF) involved in the association with MBL, while CiMAp39 retained four domains (CUB1-EGF-CUB2-CCP1). Not only the two CiMASPs but also the CiMAps were detected in grass carp serum. Furthermore, both recombinant CiMASPs (rCiMASPs) and recombinant rCiMAps (rCiMAps) interacted with recombinant MBL and the two CiMAps competed with CiMASPs for binding to MBL, and hence inhibited downstream C4 binding. These results indicated that CiMAps acted as competitive inhibitors in the lectin complement pathway of grass carp.

MASP-1 of the complement system alters fibrinolytic behaviour of blood clots

BackgroundThe lectin pathway serine protease mannan-binding lectin-associated serine protease 1 (MASP-1) has been demonstrated to be a major link between complement and coagulation, yet little is known about its interactions with the fibrinolytic system. The aim of this work was to assess the effects of MASP-1 on fibrin clot lysis in different experimental settings. MethodsRotational thrombelastometry was used to evaluate the effect of MASP-1 on the lysis of clots formed in whole blood under static conditions. Whole blood clots were also formed in the presence and absence of MASP-1 under flow conditions in the Chandler loop and their lysis was analysed separately by fluorescence release of incorporated labelled fibrin. Real-time observation by laser scanning confocal microscopy was used to investigate the lysis of plasma clots where MASP-1 was present either during clot formation or lysis. Cleavage of tPA or plasminogen by MASP-1 was analysed by gel electrophoresis. We performed a turbidimetric clot lysis assay in the presence and absence of the MASP-1 inhibitor SGMI-1 (Schistocerca gregaria protease inhibitor (SGPI)-based MASP inhibitor-1) to evaluate the effect of endogenous MASP-1 in normal plasma and plasma samples from sepsis patients. ResultsIn the thrombelastometric experiments, where MASP-1 was present during the entire clotting and lysis process, MASP-1 had a significant profibrinolytic effect and accelerated clot lysis. When clots were formed in the presence of MASP-1 under flow in the Chandler loop, the effects on fibrinolysis were heterogenous with impaired fibrinolysis in some individuals (n = 5) and no (n = 3) or even the opposite effect (n = 2) in others. In plasma clot lysis observed by confocal microscopy, lysis was prolonged when MASP-1 was added to the lysis solution, yet there was no difference in lysis time when MASP-1 was present during clot formation. When MASP-1 was incubated with tPA or plasminogen, respectively, cleavage of single-chain tPA into two-chain tPA and a slight reduction of plasminogen were observed. SGMI-1 significantly prolonged clot lysis in the turbidimetric clot lysis assay suggesting that MASP-1 accelerated lysis in plasma samples. ConclusionMASP-1 is able to alter the susceptibility of blood clots to the fibrinolytic system. MASP-1 has complex, mostly promoting effects on fibrinolysis with high inter-individual variation. Interactions of MASP-1 with the fibrinolytic system may be relevant in the development and therapy of cardiovascular and thrombotic diseases.

High Expression of Complement Components in the Kidneys of Type 2 Diabetic Rats With Diabetic Nephropathy.

Background: Diabetic nephropathy (DN) is the leading cause of end-stage failure of the kidneys; however, its pathogenesis remains unknown. This study assessed the expression of complement components in the kidneys of rats with type 2 DN to investigate their role in DN.Methods: A rat model of type 2 DN was induced by a high-fat diet combined with low-dose streptozotocin. Blood glucose, fasting insulin levels, insulin resistance index, and 24-h urinary albumin excretion (UAE) were measured. Renal tissue morphological features were observed. The mesangial index and arteriosclerosis index were calculated. Immunohistochemistry and western blot were used to measure the expression of complement components in the kidneys.Results: The kidney weight: body weight (mg/g) ratio in the DN group was significantly greater than those in the control and diabetes mellitus (DM) groups. The arteriosclerosis index, mesangial index, and tube area percentage in the DN group were significantly higher than those in the control and DM groups, but these parameters did not significantly differ between the control and DM groups. The expression of the complement components C1q, mannose-binding lectin (MBL), mannan-binding lectin-associated serine protease (MASP)-2, B factor, C3, and C5b-9 in the DN group was significantly higher than that in the control and DM groups but did not significantly differ between the control and DM groups. Most of the complement components were mainly expressed at the renal tubular site. Correlation analysis showed that 24-h UAE were positively correlated with C1q, MBL, MASP-2, B factor, and C5b-9 expression. MI was positively correlated with MBL, B factor, C3, and C5b-9 expression. AI was positively correlated with C1q, MBL, MASP-2, and B factor expression.Conclusion: Complement components including C1q, MBL, MASP-2, B factor, C3, and C5b-9, were highly expressed in the kidneys of type 2 diabetic rats with DN. Most of the complement components were mainly expressed in the renal tubules. High expression of complement components was found to be associated with the progress of DN. Our study suggests that complement system activation is a progressive factor in type 2 diabetic nephropathy. Inhibition of pathological complement activation may be a promising therapeutic strategy for DN.

Expression and functional characterization of a mannose-binding lectin-associated serine protease-1 (MASP-1) from Nile tilapia (Oreochromis niloticus) in host defense against bacterial infection

Mannose-binding lectin-associated serine protease-1 (MASP-1), a multifunctional serine protease, plays an important role in innate immunity which is capable of activating the lectin pathway of the complement system and also triggering coagulation cascade system. In this study, a MASP-1 homolog (OnMASP-1) was identified from Nile tilapia (Oreochromis niloticus) and characterized at expression and inflammation functional levels. The open reading frame (ORF) of OnMASP-1 is 2187 bp of nucleotide sequence encoding a polypeptide of 728 amino acids. The deduced amino acid sequence has 6 characteristic structures, including two C1r/C1s-Uegf-BMP domains (CUB), one epidermal growth factor domain (EGF), two complement control protein domains (CCP) and a catalytic serine protease domain (SP). Expression analysis revealed that the OnMASP-1 was highly expressed in the liver, and widely exhibited in other tissues containing intestine, spleen and kidney. In addition, the OnMASP-1 expression was significantly up-regulated in spleen and head kidney following challenges with Streptococcus agalactiae and Aeromonas hydrophila. The up-regulations of OnMASP-1 mRNA and protein expression were also demonstrated in hepatocytes and monocytes/macrophages in vitro stimulation with S. agalactiae and A. hydrophila. Recombinant OnMASP-1 protein was likely to participate in the regulation of inflammatory and migration reaction by monocytes/macrophages. These results indicated that OnMASP-1, playing an important role in innate immunity, was likely to involve in host defense against bacterial infection in Nile tilapia.