Lectin Pathway Activation Research Articles

Novel MASP-2 inhibitors developed via directed evolution of human TFPI1 are potent lectin pathway inhibitors

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.

Specific contribution of mannose-binding lectin murine isoforms to brain ischemia/reperfusion injury

Mannose-binding lectin (MBL), an initiator of the lectin pathway (LP) of complement activation, is detrimental in ischemic stroke, as shown in clinical studies and rodent models. Whereas humans have one functional MBL protein, rodents have two isoforms, MBL-A and MBL-C, whose functions relative to that of human MBL are unknown. To permit the clinical translation of preclinical data, we aimed to define the specific contributions of MBL-A and MBL-C to brain ischemia. We subjected mice with double (MBL−/−) or single (MBL-A−/− or MBL-C−/−) MBL isoform depletion to transient middle cerebral artery occlusion (tMCAo). MBL−/− mice had fewer neurological deficits and smaller ischemic lesions than WT mice. MBL-A−/− mice had smaller lesions than WT mice and exhibited no significant behavioral defects, whereas MBL-C−/− mice did not differ from WT mice. The induction of Mbl1 and Mbl2 (the MBL-A and MBL-C genes) expression 48 h after tMCAo was similar across genotypes. The time course of Mbl1 and Mbl2 expression in WT ischemic mice showed that Mbl1 activation occurred earlier (24 h) than Mbl2 activation (48 h). The plasma levels of MBL-A and MBL-C in MBL-C−/− and MBL-A−/− mice, respectively, were similar to those in WT mice both at baseline and at 48 h after tMCAo. At 48 h, MBL-A−/− ischemic mice showed higher MBL-C levels in the brain than WT mice. WT and MBL-C−/− ischemic mice had higher LP activity in plasma and, accordingly, higher levels of C3 deposition in the brain than MBL-A−/− and MBL−/− mice. In conclusion, mice with depletion of both MBL isoforms exhibited strong protection from ischemia/reperfusion injury. MBL-A was the main contributor to injury, likely owing to its earlier activation after ischemia and more efficient activation of the complement system than MBL-C.

Complement therapeutics meets nanomedicine: overcoming human complement activation and leukocyte uptake of nanomedicines with soluble domains of CD55

Complement activation plays an important role in pharmacokinetic and performance of intravenously administered nanomedicines. Significant efforts have been directed toward engineering of nanosurfaces with low complement activation, but due to promiscuity of complement factors and redundancy of pathways, it is still a major challenge. Cell membrane-anchored Decay Accelerating Factor (DAF, a.k.a. CD55) is an efficient membrane bound complement regulator that inhibits both classical and alternative C3 convertases by accelerating their spontaneous decay. Here we tested the effect of various short consensus repeats (SCRs, “sushi” domains) of human CD55 on nanoparticle-mediated complement activation in human sera and plasma. Structural modeling suggested that SCR-2, SCR-3 and SCR-4 are critical for binding to the alternative pathway C3bBb convertase, whereas SCR-1 is dispensable. Various domains were expressed in E.coli and purified by an affinity column. SCRs were added to lepirudin plasma or sera from different healthy subjects, to monitor nanoparticle-mediated complement activation as well as C3 opsonization. Using superparamagnetic iron oxide nanoworms (SPIO NWs), we found that SCR-2-3-4 was the most effective inhibitor (IC50 ~0.24 μM for C3 opsonization in sera), followed by SCR-1-2-3-4 (IC50 ~0.6 μM), whereas shorter domains (SCR-3, SCR-2-3, SCR-3-4) were ineffective. SCR-2-3-4 also inhibited C5a generation (IC50 ~0.16 μM in sera). In addition to SPIO NWs, SCR-2-3-4 effectively inhibited C3 opsonisation and C5a production by clinically approved nanoparticles (Feraheme, LipoDox and Onivyde). SCR-2-3-4 inhibited both lectin and alternative pathway activation by nanoparticles. When added to lepirudin-anticoagulated blood from healthy donors, it significantly reduced the uptake of SPIO NWs by neutrophils and monocytes. These results suggest that soluble domains of membrane-bound complement inhibitors are potential candidates for preventing nanomedicine-mediated complement activation in human subjects.

Decreased Ficolin-3-mediated Complement Lectin Pathway Activation and Alternative Pathway Amplification During Bacterial Infections in Patients With Type 2 Diabetes Mellitus.

Bacterial infections are frequent and severe in patients with diabetes mellitus. Whether diabetes per se induces functional alterations in the complement system hampering activation during infection is unknown. We investigated key elements of the complement system during bacterial infections in patients with type 2 diabetes mellitus (T2DM) and compared them to non-diabetic (ND) individuals. Using a prospective design, we included 197 T2DM, and 196 ND subjects, all with clinical diagnosis of acute community-acquired bacterial infections. Functional activities of the ficolin-3-mediated lectin (F3-LP), mannose binding lectin-mediated lectin- (MBL-LP), classical (CP), and alternative pathways (AP), as well as concentrations of complement activation products C4d and sC5b-9 were determined. Functional in vitro activities of F3-LP and AP were significantly higher in T2DM than in ND subjects, (median 64% vs. 45%, p = 0.0354 and 75 vs. 28%, p = 0.0013, respectively), indicating a decreased in vivo activation and lack of consumption of F3-LP and AP in T2DM patients, whereas no difference in functional capacities of CP and MBL-LP were observed between T2DM and ND subjects. Diminished F3-LP and AP activation was most pronounced in diabetic patients with urinary tract infections with positive microbiological culture results for Escherichia coli bacteria. In the T2DM group 3-months mortality significantly associated with diminished F3-LP and AP, but not with CP activation. Concentrations of C4d and sC5b-9 were significantly lower in the T2DM than in ND patients. In conclusion, we found impaired F3-LP activation and lack of AP amplification during bacterial infections in patients with type 2 diabetes, compared to non-diabetic subjects, suggesting a diminished complement mediated protection to bacterial infections in T2DM.

Factors involved in initiation and regulation of complement lectin pathway influence postoperative outcome after pediatric cardiac surgery involving cardiopulmonary bypass

Congenital heart disease (CHD) often requires surgical intervention, and is sometimes associated with life-threatening post-operative complications. We have investigated some factors of the innate immune system involved in the initiation or regulation of complement lectin pathway activation (MASP-1, MASP-2 MASP-3, MAp19, MAp44, ficolin-3) and related them to complications and prognosis in 190 pediatric patients undergoing CHD repair with the use of cardiopulmonary bypass (CPB). Patients with MAp44 levels ≤1.81 µg/ml more frequently experienced low cardiac output syndrome (LCOS), renal insufficiency, systemic inflammatory response syndrome (SIRS) and multiorgan dysfunction (MODS). Low MASP-3 (≤5.18 µg/ml) and high MASP-1 (≥11.7 µg/ml) levels were often associated with fatal outcome. Low ficolin-3 concentrations (≤10.1 µg/ml) were more common among patients experiencing SIRS and MODS than in those without complications. However, patients suffering from SIRS and MODS with low ficolin-3 had a much better prognosis (91% survival vs. 37% among other patients; p = 0.007). A discriminating value of 12.7 µg/ml ficolin-3 yielded 8% vs. 60% mortality (p = 0.001). Our data extend the knowledge concerning involvement of proteins of the lectin pathway in development of post-CPB complications. The potential prognostic value of low preoperative MAp44 and high preoperative ficolin-3 seems promising and warrants independent confirmation.

Quantitative proteomic profiling of immune responses to Ichthyophthirius multifiliis in common carp skin mucus

Ichthyophthirius multifiliis, a ciliated protozoan parasite, causes ichthyophthiriasis and leads to considerable economic losses to the aquaculture industry. Understanding the fish immune response and host-parasite interactions could support developing novel strategies for better disease management and control. Fish skin mucus is the first line of defence against infections through the epidermis. Yet, the common carp, Cyprinus carpio, protein-based defence strategies against infection with I. multifiliis at this barrier remain elusive. The skin mucus proteome of common carp was investigated at 1 day and 9 days post-exposure with I. multifiliis. Using nano-LC ESI MS/MS and statistical analysis, the abundance of 19 immune related and signal transduction proteins was found to be differentially regulated in skin mucus of common carp in response to I. multifiliis. The analysis revealed increased abundance values of epithelial chloride channel protein, galactose-specific lectin nattection, high choriolytic enzyme 1 (nephrosin), lysozyme C, granulin and protein-glutamine gamma-glutamyltransferase 2 in I. multifiliis-exposed carp skin mucus. Multiple lectins and a diverse array of distinct serpins with protease inhibitor activity were identified likely implicated in lectin pathway activation and regulation of proteolysis, indicating that these proteins contribute to the carp innate immune system and the protective properties of skin mucus. The results obtained from this proteomic analysis enables a better understanding of fish host response to parasitic infection and gives insights into the key role skin mucus plays in protecting fish against deleterious effects of I. multifiliis.

CL-L1 and CL-K1 Exhibit Widespread Tissue Distribution With High and Co-Localized Expression in Secretory Epithelia and Mucosa.

Collectin liver 1 (CL-L1, alias collectin 10) and collectin kidney 1 (CL-K1, alias collectin 11) are oligomeric pattern recognition molecules associated with the complement system, and mutations in either of their genes may lead to deficiency and developmental defects. The two collectins are reportedly localized and synthesized in the liver, kidneys, and adrenals, and can be found in the circulation as heteromeric complexes (CL-LK), which upon binding to microbial high mannose-like glycoconjugates activates the complement system via the lectin activation pathway. The tissue distribution of homo- vs. heteromeric CL-L1 and -K1 complexes, the mechanism of heteromeric complex formation and in which tissues this occurs, is hitherto incompletely described. We have by immunohistochemistry using monoclonal antibodies addressed the precise cellular localization of the two collectins in the main human tissues. We find that the two collectins have widespread and almost identical tissue distribution with a high expression in epithelial cells in endo-/exocrine secretory tissues and mucosa. There is also accordance between localization of mRNA transcripts and detection of proteins, showing that local synthesis likely is responsible for peripheral localization and eventual formation of the CL-LK complexes. The functional implications of the high expression in endo-/exocrine secretory tissue and mucosa is unknown but might be associated with the activity of MASP-3, which has a similar pattern of expression and is known to potentiate the activity of the alternative complement activation pathway.

Interaction of Mannose-Binding Lectin With Lipopolysaccharide Outer Core Region and Its Biological Consequences.

Lipopolysaccharide (LPS, endotoxin), the main surface antigen and virulence factor of Gram-negative bacteria, is composed of lipid A, core oligosaccharide, and O-specific polysaccharide (O-PS) regions. Each LPS region is capable of complement activation. We have demonstrated that LPS of Hafnia alvei, an opportunistic human pathogen, reacts strongly with human and murine mannose-binding lectins (MBLs). Moreover, MBL–LPS interactions were detected for the majority of other Gram-negative species investigated. H. alvei was used as a model pathogen to investigate the biological consequences of these interactions. The core oligosaccharide region of H. alvei LPS was identified as the main target for human and murine MBL, especially l-glycero-d-manno-heptose (Hep) and N-acetyl-d-glucosamine (GlcNAc) residues within the outer core region. MBL-binding motifs of LPS are accessible to MBL on the surface of bacterial cells and LPS aggregates. Generally, the accessibility of outer core structures for interaction with MBL is highest during the lag phase of bacterial growth. The LPS core oligosaccharide–MBL interactions led to complement activation and also induced an anaphylactoid shock in mice. Unlike Klebsiella pneumoniae O3 LPS, robust lectin pathway activation of H. alvei LPS in vivo was mainly the result of outer core recognition by MBL; involvement of the O-PS is not necessary for anaphylactoid shock induction. Our results contribute to a better understanding of MBL–LPS interaction and may support development of therapeutic strategies against sepsis based on complement inhibition.

Substitution of Mannan-Binding Lectin (MBL)-Deficient Serum With Recombinant MBL Results in the Formation of New MBL/MBL-Associated Serine Protease Complexes.

The lectin pathway (LP) of complement activation depends on the activation of the MBL-associated serine proteases (MASPs) circulating in complex with mannan-binding lectin (MBL). MBL deficiency is the most common complement deficiency and has been associated with several pathological conditions. As we had previously shown, plasma-derived MBL (pdMBL) contains pre-activated MASPs that upon in vivo pdMBL substitution results in restoration of MBL concentrations but no LP functionality due to immediate inactivation of pdMBL–MASP complexes upon infusion. In this study, we analyzed MBL-sufficient and -deficient serum by size-exclusion chromatography for complexes of LP activation. In both sera, we identified non-bound free forms of MASP-2 and to lesser extent MASP-1/3. After addition of recombinant MBL (rMBL) to MBL-deficient serum, these free MASPs were much less abundantly present, which is highly suggestive for the formation of high-molecular complexes that could still become activated upon subsequent ligand binding as shown by a restoration of C4-deposition of MBL-deficient serum. Ficolin (FCN)-associated MASPs have been described to redistribute to ligand-bound MBL, hereby forming new MBL/MASP complexes. However, reconstitution of MBL-deficient serum with rMBL did not change the relative size of the FCN molecules suggestive for a limited redistribution in fluid phase of already formed complexes. Our findings demonstrate that rMBL can associate with free non-bound MASPs in fluid phase while preserving full restoration of LP functionality. In contrast to pdMBL products containing pre-activated MASPs which become inactivated almost immediately, these current data provide a rationale for substitution studies using rMBL instead.

The Lectin Pathway in Thrombotic Conditions-A Systematic Review.

The lectin pathway of the complement system can activate the coagulation system in vitro, but the role of the lectin pathway in haemostatic activation and thrombosis in vivo is not clear. We performed a systematic review of the existing literature on associations between the lectin pathway and arterial and venous thrombosis, in accordance with the Assessing the Methodological Quality of Systematic Reviews guidelines. PubMed and Embase were searched from January 1990 to March 2017. We included original studies on human study populations investigating associations between the lectin pathway (protein serum levels, genotype or gene expression) and thrombotic conditions or laboratory coagulation markers. Exclusion criteria were case studies including fewer than five cases, conference abstracts or any other language than English. In total, 43 studies were included which investigated associations between the lectin pathway and cardiovascular thrombotic events (CVEs) (n = 22), ischaemic stroke (n = 9), CVE and stroke (n = 1) and other conditions (systemic lupus erythematosus [n = 6], sepsis-related coagulopathy [n = 3], pulmonary embolism [n = 1], asparaginase treatment [n = 1]). Studies on the lectin pathway and CVE risk reported discrepant results, as both high and low mannose-binding lectin (MBL) serum levels were found to correlate with increased CVE risk. In ischaemic stroke patients, occurrence of stroke as well as increased stroke severity and poor outcome were consistently associated with high serum MBL. For other thromboembolic conditions, only few studies were identified. In conclusion, lectin pathway activation may negatively influence outcome after ischaemic stroke and possibly contribute to CVE risk. Further research is warranted to elucidate the role of the lectin pathway in other thrombotic conditions.

Complement Factor D protects mice from ethanol-induced inflammation and liver injury.

Complement plays a crucial role in microbial defense and clearance of apoptotic cells. Emerging evidence suggests complement is an important contributor to alcoholic liver disease. While complement component 1, Q subcomponent (C1q)-dependent complement activation contributes to ethanol-induced liver injury, the role of the alternative pathway in ethanol-induced injury is unknown. Activation of complement via the classical and alternative pathways was detected in alcoholic hepatitis patients. Female C57BL/6J [wild type (WT)], C1q-deficient ( C1qa-/-, lacking classical pathway activation), complement protein 4-deficient ( C4-/-, lacking classical and lectin pathway activation), complement factor D-deficient ( FD-/-, lacking alternative pathway activation), and C1qa/FD-/- (lacking classical and alternative pathway activation) mice were fed an ethanol-containing liquid diet or pair-fed control diet for 4 or 25 days. Following chronic ethanol exposure, liver injury, steatosis, and proinflammatory cytokine expression were increased in WT but not C1qa-/-, C4-/-, or C1qa/FD-/- mice. In contrast, liver injury, steatosis, and proinflammatory mediators were robustly increased in ethanol-fed FD-/- mice compared with WT mice. Complement activation, assessed by hepatic accumulation of C1q and complement protein 3 (C3) cleavage products (C3b/iC3b/C3c), was evident in livers of WT mice in response to both short-term and chronic ethanol. While C1q accumulated in ethanol-fed FD-/- mice (short term and chronic), C3 cleavage products were detected after short-term but not chronic ethanol. Consistent with impaired complement activation, chronic ethanol induced the accumulation of apoptotic cells and fibrogenic responses in the liver of FD-/- mice. These data highlight the protective role of complement factor D (FD) and suggest that FD-dependent amplification of complement is an adaptive response that promotes hepatic healing and recovery in response to chronic ethanol. NEW & NOTEWORTHY Complement, a component of the innate immune system, is an important pathophysiological contributor to ethanol-induced liver injury. We have identified a novel role for factor D, a component of the alternative pathway, in protecting the liver from ethanol-induced inflammation, accumulation of apoptotic hepatocytes, and profibrotic responses. These data indicate a dual role of complement with regard to inflammatory and protective responses and suggest that accumulation of apoptotic cells impairs hepatic healing/recovery during alcoholic liver disease.

Low Levels of Immunoglobulins and Mannose-Binding Lectin Are Not Associated With Etiology, Severity, or Outcome in Community-Acquired Pneumonia.

BackgroundDisease severity and outcome in community-acquired pneumonia (CAP) depend on the host and on the challenge of the causal microorganism(s). We measured levels of immunoglobulins (Igs) and complement in 257 hospitalized adults with CAP and examined the association of low levels of Igs or complement to microbial etiology, disease severity, and short-term and long-term outcome.MethodsSerum Igs were analyzed in blood samples obtained at admission and at 6 weeks postdischarge if admission levels were low. Serum complement deficiencies were screened with a total complement activity enzyme-linked immunosorbent assay (ELISA), with further analyzes performed if justified. Disease severity was assessed by the CURB-65 severity score. Short-term outcome was defined as a composite end point of intensive care unit (ICU) admission and 30-day mortality, and long-term outcome as 5-year all-cause mortality.ResultsAt admission, 87 (34%) patients had low levels of at least 1 Ig, with low IgG2 as the most prevalent finding (55/21%). IgG levels were lower in bacterial than viral CAP (8.48 vs 9.97 g/L, P = .023), but low Igs were not associated with microbial etiology. Fifty-five (21%) patients had low lectin pathway activity, of which 33 (13%) were mannose-binding lectin (MBL) deficient. Low admission levels of any Ig or MBL were not associated with disease severity, short-term outcome, or long-term outcome. Excluding patients defined as immunocompromised from analysis did not substantially affect these results.ConclusionIn hospitalized adults with CAP, low admission levels of Igs or complement were in general not associated with microbial etiology, disease severity, short-term outcome, or long-term outcome.

Extensive Basal Level Activation of Complement Mannose-Binding Lectin-Associated Serine Protease-3: Kinetic Modeling of Lectin Pathway Activation Provides Possible Mechanism.

Serine proteases (SPs) are typically synthesized as precursors, termed proenzymes or zymogens, and the fully active form is produced via limited proteolysis by another protease or by autoactivation. The lectin pathway of the complement system is initiated by mannose-binding lectin (MBL)-associated SPs (MASP)-1, and MASP-2, which are known to be present as proenzymes in blood. The third SP of the lectin pathway, MASP-3, was recently shown to be the major activator, and the exclusive “resting blood” activator of profactor D, producing factor D, the initiator protease of the alternative pathway. Because only activated MASP-3 is capable of carrying out this cleavage, it was presumed that a significant fraction of MASP-3 must be present in the active form in resting blood. Here, we aimed to detect active MASP-3 in the blood by a more direct technique and to quantitate the active to zymogen ratio. First, MASPs were partially purified (enriched) from human plasma samples by affinity chromatography using immobilized MBL in the presence of inhibitors. Using this MASP pool, only the zymogen form of MASP-1 was detected by Western blot, whereas over 70% MASP-3 was in an activated form in the same samples. Furthermore, the active to zymogen ratio of MASP-3 showed little individual variation. It is enigmatic how MASP-3, which is not able to autoactivate, is present mostly as an active enzyme, whereas MASP-1, which has a potent autoactivation capability, is predominantly proenzymic in resting blood. In an attempt to explain this phenomenon, we modeled the basal level fluid-phase activation of lectin pathway proteases and their subsequent inactivation by C1 inhibitor and antithrombin using available and newly determined kinetic constants. The model can explain extensive MASP-3 activation only if we assume efficient intracomplex activation of MASP-3 by zymogen MASP-1. On the other hand, the model is in good agreement with the fact that MASP-1 and -2 are predominantly proenzymic and some of them is present in the form of inactive serpin–protease complexes. As an alternative hypothesis, MASP-3 activation by proprotein convertases is also discussed.

Progressive IgA Nephropathy Is Associated With Low Circulating Mannan-Binding Lectin–Associated Serine Protease-3 (MASP-3) and Increased Glomerular Factor H–Related Protein-5 (FHR5) Deposition

IntroductionIgA nephropathy (IgAN) is characterized by glomerular deposition of galactose-deficient IgA1 and complement proteins and leads to renal impairment. Complement deposition through the alternative and lectin activation pathways is associated with renal injury.MethodsTo elucidate the contribution of the lectin pathway to IgAN, we measured the 11 plasma lectin pathway components in a well-characterized cohort of patients with IgAN.ResultsM-ficolin, L-ficolin, mannan-binding lectin (MBL)–associated serine protease (MASP)-1 and MBL-associated protein (MAp) 19 were increased, whereas plasma MASP-3 levels were decreased in patients with IgAN compared with healthy controls. Progressive disease was associated with low plasma MASP-3 levels and increased glomerular staining for C3b/iC3b/C3c, C3d, C4d, C5b-9, and factor H–related protein 5 (FHR5). Glomerular FHR5 deposition positively correlated with glomerular C3b/iC3b/C3c, C3d, and C5b-9 deposition, but not with glomerular C4d. These observations, together with the finding that glomerular factor H (fH) deposition was reduced in progressive disease, are consistent with a role for fH deregulation by FHR5 in renal injury in IgAN.ConclusionOur data indicate that circulating MASP-3 levels could be used as a biomarker of disease severity in IgAN and that glomerular staining for FHR5 could both indicate alternative complement pathway activation and be a tissue marker of disease severity.

Transcriptome analysis of inflammation-related gene expression in endothelial cells activated by complement MASP-1

Mannan-binding lectin-associated serine protease 1 (MASP-1), the most abundant enzyme of the complement lectin pathway, is able to stimulate human umbilical vein endothelial cells (HUVECs) to alter the expression of several cytokines and adhesion molecules. This study has assessed to what extent MASP-1 is able to modify the transcriptional pattern of inflammation-related (IR) genes in HUVECs. We utilized Agilent microarray to analyse the effects of recombinant MASP-1 (rMASP-1) in HUVECs, on a set of 884 IR genes. Gene Set Enrichment Analysis showed an overall activation of inflammation-related genes in response to rMASP-1. rMASP-1 treatment up- and down-regulated 19 and 11 IR genes, respectively. Most of them were previously unidentified, such as genes of chemokines (CXCL1, CXCL2, CXCL3), inflammatory receptors (TLR2, BDKRB2) and other inflammatory factors (F3, LBP). Expression of IR genes changed early, during the first 2 hours of activation. Both p38-MAPK inhibitor and NFκB inhibitor efficiently suppressed the effect of rMASP-1. We delineated 12 transcriptional factors as possible regulators of rMASP-1-induced IR genes. Our microarray-based data are in line with the hypothesis that complement lectin pathway activation, generating active MASP-1, directly regulates inflammatory processes by shifting the phenotype of endothelial cells towards a more pro-inflammatory type.

Increased C4d and Bb immunoreactivity and decreased MBL immunoreactivity characterise first-time pathologic first-trimester miscarriage: a case-control study

The role of the complement system in first-time pathologic first-trimester miscarriage was investigated. In this case-control study, tissue samples of 126 women with pathologic miscarriage and termination of normal pregnancies were assessed. The pathologic pregnancy group consisted of 40 women with missed miscarriage, 13 women with incomplete miscarriage and 10 women with a blighted ovum. The control group consisted of 63 normal-appearing pregnancies. Immunoreactivity for C4d, Bb and MBL was evaluated in the deciduas and villous trophoblasts separately using a semi-quantitative histological scoring system (H-score). C4d and Bb H-scores were higher and MBL H-score was reduced in the deciduas and villous tissues from pathologic miscarriage compared to termination of pregnancies (p = .003 and p = .001; p = .011 and p < .001; p < .001 and p < .001, respectively). C4d and Bb activities were increased and MBL activity was decreased in human first-time pathologic first-trimester miscarriage. We suggest that three complement pathways may play a role in human first-time pathologic first-trimester miscarriage.Impact statementPrevious studies focussed on complement proteins related to a single complement pathway in cases often associated with antiphospholipid syndrome (APS) or recurrent miscarriage. In APS-related cases, the classical pathway is activated. In antibody-dependent and in antibody-independent mouse models of foetal loss, classical and alternative pathways are activated, respectively. Lectin pathway deficiency has been reported in some recurrent miscarriage. The complement pathway or pathways, which have a role in human pathologic miscarriage was the starting point of this study. There has been no study done till now reporting the role of the three complement pathways in human pathologic miscarriage. In this study, we found increased classical and alternative complement pathway activities and decreased lectin pathway activity in tissues from first-time pathologic human miscarriage.

Deposition of mannose-binding lectin and ficolins and activation of the lectin pathway of complement on the surface of polyurethane tubing used for cardiopulmonary bypass.

The artificial surface used for cardiopulmonary bypass (CPB) is a crucial factor activating the complement system and thus contributing to the generation of a systemic inflammatory response. The activation of classical and alternative pathways on this artificial surface is well known. In contrast, lectin pathway (LP) activation has not been fully investigated, although noted during CPB in several studies. Moreover, we have recently proved the contribution of the LP to the generation of the systemic inflammatory response syndrome after pediatric cardiac surgery. The aim of this study was to assess LP-mediated complement activation on the surface of polyurethane CPB circuit tubing (noncoated Chalice ® ), used for CPB procedures in children with congenital heart disease. We found deposition of mannose-binding lectin, ficolin-1, -2, and -3 on the surface of unused tubing and on tubing used for CPB from a small minority of patients. Furthermore, we observed deposition of complement C4 activation products on tubing used for CPB and previously unused tubing after incubation with normal serum. The latter finding indicates LP activation in vitro on the polyurethane surface. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1202-1208, 2018.

Effects of hMASP-2 on the formation of BCG infection-induced granuloma in the lungs of BALB/c mice

Tuberculosis, caused by Mycobacterium tuberculosis, affects the functions of the lung and causes high morbidity and mortality rates worldwide. MASP-2 is an executioner enzyme, which plays an essential role in the activation of lectin pathway. In our previous studies, the MASP-2 played a dual role in promoting the progress of lesions in BCG-infected rabbit skin models. However, the really effects of MASP-2 on tuberculosis are unknown. The aim of this study was to investigate the effects of MASP-2 in granuloma formation with BCG-infected mice. Compared to the control group, rAd-hMASP-2 treated group showed increasing in survival rate of BCG-infected mice (P = 0.042), and decreasing of bacteria loads (P = 0.005) in the lung tissue. MASP-2 displayed a protective efficacy in BCG-infected mice, which promoted the activation and recruitment of macrophages and lymphocytes to the granuloma. Moreover, the data obtained from the ELISA and RT-PCR demonstrated that mRNA expression for IL-6, CCL12, CCL2 and cytokines of IFN-γ, TNF-α in lung were significantly elevated by treatment of rAd-hMASP-2. Those findings provided an evidence that MASP-2 may be as a newly immunomodulatory in targeting granuloma formation, which displayed a potential protective role in control of tuberculosis.

Lectin Pathway of Complement Activation Is Associated with Vulnerability of Atherosclerotic Plaques.

Inflammatory mechanisms may be involved in atherosclerotic plaque rupture. By using a novel histology-based method to quantify plaque instability here, we assess whether lectin pathway (LP) of complement activation, a major inflammation arm, could represent an index of plaque instability. Plaques from 42 consecutive patients undergoing carotid endarterectomy were stained with hematoxylin-eosin and the lipid core, cholesterol clefts, hemorrhagic content, thickness of tunica media, and intima, including or not infiltration of cellular debris and cholesterol, were determined. The presence of ficolin-1, -2, and -3 and mannose-binding lectin (MBL), LP initiators, was assessed in the plaques by immunofluorescence and in plasma by ELISA. LP activation was assessed in plasma by functional in vitro assays. Patients presenting low stenosis (≤75%) had higher hemorrhagic content than those with high stenosis (>75%), indicating increased erosion. Increased hemorrhagic content and tunica media thickness, as well as decreased lipid core and infiltrated content were associated with vulnerable plaques and therefore used to establish a plaque vulnerability score that allowed to classify patients according to plaque vulnerability. Ficolins and MBL were found both in plaques’ necrotic core and tunica media. Patients with vulnerable plaques showed decreased plasma levels and intraplaque deposition of ficolin-2. Symptomatic patients experiencing a transient ischemic attack had lower plasma levels of ficolin-1. We show that the LP initiators are present within the plaques and their circulating levels change in atherosclerotic patients. In particular, we show that decreased ficolin-2 levels are associated with rupture-prone vulnerable plaques, indicating its potential use as marker for cardiovascular risk assessment in atherosclerotic patients.

Morula cells as key hemocytes of the lectin pathway of complement activation in the colonial tunicate Botryllus schlosseri

The complement system is deeply rooted in the evolution of humoral mechanism of innate immunity. In addition to the alternative pathway of complement activation, lectins and associated serine proteases exert important roles in the recognition of non-self and activation of the effectors.In the colonial tunicate Botryllus schlosseri, we identified, characterized and studied the expression of three orthologues of genes involved in the lectin pathway of complement activation of vertebrates, i.e., genes for a mannose-binding lectin (MBL), a ficolin and a mannose-associated serine protease 1 (MASP1). All the genes are transcribed by hemocytes, and specifically by morula cells, the same immunocytes responsible for the transcription of C3 and Bf orthologues. The transcription levels of MASP1 and ficolin orthologues are not affected by zymosan challenge, indicating a constitutive expression of complement system associated serine proteases, whereas the MBL orthologue is up-regulated after 15 min of zymosan exposure. Collectively, our data suggest the presence of a complete lectin activation pathway in Botryllus.