Lectin Complement Pathway Research Articles

Inactivation of the Complement Lectin Pathway by Candida tropicalis Secreted Aspartyl Protease-1

Candida tropicalisis an opportunistic fungal pathogen and is one of the most frequently isolated non-albicans species. It can cause localised as well as invasive systemic infections particularly in immunocompromised patients. Increased resistance to common anti-fungal drugs is an emerging problem. In order to establish disseminated infections, Candida has evolved several strategies to escape the host immune system. A detailed understanding of how C. tropicalis escapes the host immune attack is needed as it can help develop novel anti-fungal therapies. Secreted aspartyl proteinases (Saps) of C. albicans have been shown to be determinants of virulence and immune evasion. However, the immune evasion properties of C. tropicalis Saps have been poorly characterised. This study investigated the immune evasion properties of C. tropicalis secreted aspartic protease 1 (Sapt1).Sapt1 was recombinantly produced using a Kluyveromyces lactis yeast expression system. A range of complement proteins and immunogloublins were screened to test if Sapt1 had any proteolytic activity. Sapt1 efficiently cleaved human mannose-binding lectin (MBL) and collectin-11, which are the initiating molecules of the lectin pathway of the complement system, but not l-ficolin. In addition, Sapt1 cleaved DC-SIGN, the receptor on antigen presenting dendritic cells. Proteolysis was prominent in acidic condition (pH 5.2), a characteristic of aspartyl protease. No proteolytic activity was detected against complement proteins C1q, C3, C3b, IgG and IgA. In view of the ability of Sapt1 to cleave MBL and collectin-11, we found that Sapt1 could prevent activation of the complement lectin pathway. RT-qPCR analysis using three different C. tropicalis clinical isolates (oral, blood and peritoneal dialysis fluid) revealed relatively higher levels of mRNA expression of Sapt1 gene when compared to a reference strain; Sapt1 protein was found to be secreted by all the tested strains. Lectin pathway and its initiating components are crucial to provide front line defence against Candida infections. For the first time, we have shown that a Candida protease can proteolytically degrade the key initiating components of lectin pathway and inhibit complement activation. Findings from this study highlight the importance of exploring Sapt1 as a potential therapeutic target. We conclude that C. tropicalis secretes Sapt1 to target the complement lectin pathway, a key pattern recognition and clearance mechanism, for its survival and pathogenesis.

Lectin complement pathway components as risk factors for rheumatic heart disease in Assam, India

Lectin complement pathway is an integral component of the innate immune response and has been associated with pathogenesis of rheumatic heart disease (RHD). Thus, we investigated the serum concentration and single nucleotide polymorphism (SNP) in different components of the lectin pathway to understand their role in susceptibility and progression of RHD. Sixty RHD patients and equal number of age and sex matched healthy controls from Assam Medical College and Hospital (AMCH), Assam were enrolled for the study. We analyzed mannose binding lectin (MBL2), Ficolin2, MBL-associated serine protease 2 (MASP2) and MBL-associated protein 44 (MAp44) for serum concentration using ELISA along with identification of gene polymorphism using nucleotide sequencing. MBL2 SNP rs11003125 was analyzed in the representative North-East India cohort and the GG genotype [OR=; 95% CI=, p < 0.05)] was observed to impart a protective effect against RHD. Promoter region variants of ficolin2 (−4 A > G) [p = 0.0181, OR 6.5102 (1.3764–30.7931)] were observed in RHD patients. Further, low serum levels of MASP2 (patients- 641.16 pg/ml, ±288.21; controls-1168.71 pg/ml, ±300.03) and high levels of MAp44 (patients- 70 pg/ml, ±31.5; controls-63.3 pg/ml ±17.9) were observed. Based on our results, −4 A > G variant in promoter region of FCN2 is associated with increased susceptibility and low serum levels of Ficolin2 in RHD patients. Elevated MAp44 levels and low MASP2 serum levels in patients suggest inhibition of activation of complement system and thus less clearance of pathogen in RHD. Overall, our study adds to the understanding on the role of lectin pathway proteins in the predisposition and progression of RHD.

The complex formation of MASP-3 with pattern recognition molecules of the lectin complement pathway retains MASP-3 in the circulation.

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.

Complement lectin pathway components MBL and MASP-1 promote haemostasis upon vessel injury in a microvascular bleeding model.

BackgroundComplement 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.MethodsWe 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.ResultsUpon 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.ConclusionWe 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.

Lectin Pathway Enzyme MASP-2 and Downstream Complement Activation in COVID-19

Mannose-binding lectin-associated serine protease 2 (MASP-2) is the main activator of the lectin complement pathway and has been suggested to be involved in the pathophysiology of coronavirus disease 2019 (COVID-19). To study a possible association between MASP-2 and COVID-19, we aimed at developing a sensitive and reliable MASP-2 ELISA. From an array of novel mouse-monoclonal antibodies using recombinant MASP-2 as antigen, two clones were selected to create a sandwich ELISA. Plasma samples were obtained from 216 healthy controls, 347 convalescent COVID-19 patients, and 147 prospectively followed COVID-19 patients. The assay was specific towards MASP-2 and did not recognize the truncated MASP2 splice variant MAP-2 (MAp19). The limit of quantification was shown to be 0.1 ng/mL. MASP-2 concentration was found to be stable after multiple freeze-thaw cycles. In healthy controls, the mean MASP-2 concentration was 524 ng/mL (95% CI: 496.5–551.6). No significant difference was found in the MASP-2 concentrations between COVID-19 convalescent samples and controls. However, a significant increase was observed in prospectively followed COVID-19 patients (mean: 834 ng/mL [95% CI: 765.3–902.7, p < 0.0001]). In these patients, MASP-2 concentration correlated significantly with the concentrations of the terminal complement complex (ρ = 0.3596, p < 0.0001), with the lectin pathway pattern recognition molecules ficolin-2 (ρ = 0.2906, p = 0.0004) and ficolin-3 (ρ = 0.3952, p < 0.0001) and with C-reactive protein (ρ = 0.3292, p = 0.0002). Overall, we developed a specific quantitative MASP-2 sandwich ELISA. MASP-2 correlated with complement activation and inflammatory markers in COVID-19 patients, underscoring a possible role of MASP-2 in COVID-19 pathophysiology.

Piecing Complement Together with LEGO Bricks: Impacts on Interest, Confidence, and Learning in the Immunology Classroom.

Teaching and learning complex molecular cascades can often be challenging. In immunology, students struggle to visualize immunological processes, such as activation of the complement system, which involves three separate cascades leading to multiple effector functions. Offering learning activities that use tangible modeling can help students learn conceptually difficult content by fostering a visual understanding of concepts, as well as instill confidence and interest in the material. In this article, we describe a learning activity using LEGO bricks that demonstrates the activation of the classical, lectin, and alternative complement pathways and formation of the membrane attack complex. In both an introductory and advanced immunology course, we investigated the effect of the activity on student learning and subject confidence. Performance on examination questions about complement demonstrated that the LEGO activity improved learning in a naive student population (students in introductory immunology), but not in a previously informed student population (students in advanced immunology). In addition, self-reported confidence in the content was significantly higher in students who completed the LEGO activity in the advanced course, but not the introductory course, compared with those who did not do the activity. Students in both courses who did the activity had a positive perception of the activity, with a majority of students reporting that they enjoyed the activity and had more interest in the complement system.

P1530: PERITRANFUSIONAL C1-INHIBITOR IN PATIENTS WITH SEVERE COMPLEMENT-MEDIATED AUTOIMMUNE HEMOLYTIC ANEMIA: AN OPEN LABEL PHASE 2 TRIAL.

Background: Complement-mediated autoimmune hemolytic anemia (AIHA) is characterized by destruction of red blood cells (RBCs) due to autoantibodies that initiate complement activation via the classical pathway. This may result in fulminant intra- and extravascular hemolysis with need for RBC transfusions, of which the efficacy can be compromised by the antibodies reacting with donor RBCs. Hemolysis induces a pro-inflammatory response that can contribute to hypercoagulability, as seen in patients with other complement-mediated hemolytic disorders such as paroxysmal nocturnal hemoglobinuria. C1-inhibitor (C1-INH; Cinryze) is an endogenous plasma regulator of the classical and lectin complement pathway approved for patients with hereditary angioedema. In a single case report and in vitro, we observed that C1-INH administration attenuated complement deposition on RBCs and subsequent hemolysis. Therefore, we hypothesized that peritransfusional administration of C1-INH in complement AIHA would reduce complement activation and hemolysis and thus improve transfusion outcome. Aims: To assess the efficacy and safety of intravenous C1-INH in patients with transfusion-dependent complement-mediated AIHA on parameters for hemolytic activity, complement activation and inflammation. Methods: We conducted a prospective, single center, phase 2 open label trial. Patients with AIHA and confirmed C3c/d-positive direct antiglobulin test, baseline hemoglobin (Hb) of ≤8 g/dL and an indication for ≥ two RBC transfusion units were included after informed consent. Treatment consisted of four peritransfusional doses of C1-INH intravenously (6000, 3000, 2000 and 1000 U) with 12 hour (hr) intervals. Blood samples at 0, 12, 24, 36, 48, 72 hrs, 7 and 14 days were analyzed for hemolytic parameters, RBC opsonization and markers of complement activation and systemic inflammation (nucleosomes and human neutrophil elastase-α1-antitrypsin complexes (HNE-α1-ATc)). Safety was monitored and scored (CTCAE version 4.0). Results: Ten patients were included in the study. At baseline, we found increased complement C3d, IgG and IgM deposition on RBCs compared to healthy controls (p=0.002, 0.005 and 0.002). Circulating C3b/c levels, nucleosomes and HNE-α1-ATc were increased compared to healthy controls (p<0.001, p=0.046 and p=0.001). C1-INH administration resulted in an increase of C1-INH antigen and -activity, peaking at 48 hrs (Fig. A). At 48 hrs after first administration, treatment significantly reduced C3d deposition on RBCs (-21%, p=0.016), but not systemic complement activation as shown by C3b/c levels (-14.96 nM, p=0.874; Fig. B). With declining C1-INH levels, C3 deposition increased again. Shortly after transfusion, Hb increased (1.59 g/dL at 12 h; p=0.008), but at 48 hrs, hemolytic and inflammation parameters did not improve (LDH -169.5 U/L, p=0.572; bilirubin total -4.306 µmol/L, p=0.987; Hb -1.217 g/dL; p=0.056; nucleosomes -17%, p=0.938; HNE-α1-ATc +21%;, p=0.297; Fig. C, D). No serious adverse events were reported. Image:Summary/Conclusion: Peritransfusional C1-INH in AIHA temporarily reduced local complement activation as evidenced by C3d deposition on RBCs, but did not affect hemolysis or systemic complement activation. Increased Hb levels at later timepoints were observed, but this effect might be confounded by additional treatments and transfusions. In conclusion, we found no (clinically) significant effect of C1-INH on hemolysis and systemic complement activation. This may be explained by the dosing of C1-INH, which possibly was too low to counter the severe complement-mediated AIHA and additional transfusion effects.

POS-037 ACTIVATION OF THE ALTERNATIVE PATHWAY OF COMPLEMENT FAVORS THROMBUS FORMATION ON MICROVASCULAR ENDOTHELIAL CELLS IN ACUTE HEMOLYTIC UREMIC SYNDROME INDUCED BY SHIGA-LIKE-TOXIN PRODUCING E.COLI (STEC-HUS)

HUS is a rare disease that encompasses microangiopathic hemolysis, thrombocytopenia and renal failure. STEC-HUS is the most common form in children. Published evidence shows that the alternative pathway of complement is activated in the circulation during the acute phase. However, the specific role of activation of the classic, lectin and alternative pathways of complement on microvascular endothelial cells in STEC-HUS remains undefined.

Distinct Roles of Classical and Lectin Pathways of Complement in Preeclamptic Placentae.

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 female C1qa-/- pregnant mice mated to C1qa+/+ male mice surrounding remodeled vessels express C1q of paternal origin. Unlike C1qa-/- pregnant mice, heterozygous C1qa+/- 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.

Premortem Skin Biopsy Assessing Microthrombi, Interferon Type I Antiviral and Regulatory Proteins, and Complement Deposition Correlates with Coronavirus Disease 2019 Clinical Stage

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.

Circulating complement factor H levels are associated with disease severity and relapse in autoimmune hepatitis

Background & AimsThe complement system plays pivotal roles in innate immunity. Mannose-binding lectin-associated serine protease (MASP)-2 plays essential roles in the activation of the lectin complement pathway. Complement factor H acts as a critical negative regulator of the alternative complement pathway. The association of circulating MASP-2 and factor H with the clinical features of patients with autoimmune hepatitis (AIH) is unclear.MethodsA total of 63 patients with AIH were recruited for this study. The serum levels of MASP-2, factor H, and C3a were measured, and their associations with the clinical features of AIH were analyzed.ResultsThe circulating C3a levels were higher in patients with AIH than in the controls. The circulating MASP-2 and factor H levels were decreased depending on the severity of AIH. Multivariate logistic analysis showed that low circulating factor H levels were associated with features of severe AIH (odds ratio 0.36; 95% CI 0.15-0.84; p = 0.018). Multivariate Cox proportional hazards model analysis showed that low circulating factor H levels were associated with a high incidence of relapse (hazard ratio: 5.19; 95% CI 1.07–25.2; p = 0.041). Patients with low circulating factor H levels showed higher rates of relapse than the controls (log-rank, p = 0.006).ConclusionCirculating factor H levels were associated with severe disease and with the incidence of relapse, suggesting a role for the complement system in the pathophysiology of AIH.Lay summaryAutoimmune hepatitis is an immune-mediated liver disease. Despite effective treatments, patients often relapse, which can lead to clinical deterioration and adverse outcomes. Herein, we studied the importance of the complement system (a form of innate immunity) in patients with autoimmune hepatitis. We found that the levels of a protein called factor H, which regulates the complement system, could be a potential biomarker of disease severity and relapse, and could even have therapeutic potential for patients with AIH.

Synergy of protease-binding sites within the ecotin homodimer is crucial for inhibition of MASP enzymes and for blocking lectin pathway activation

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.

Directed Evolution-Driven Increase of Structural Plasticity Is a Prerequisite for Binding the Complement Lectin Pathway Blocking MASP-Inhibitor Peptides.

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.

Ficolin-2 Lectin Complement Pathway Mediates Capsule-Specific Innate Immunity Against Invasive Pneumococcal Disease.

Reports conflict regarding which lectin-microbial ligand interactions elicit a protective response from the lectin pathway (LP) of complement. Using fluorescent microscopy, we demonstrate the human lectin ficolin-2 binds to Streptococcus pneumoniae serotype 11A capsule polysaccharide dependent on the O-acetyltransferase gene wcjE. This triggers complement deposition and promotes opsonophagocytosis of encapsulated pneumococci. Even partial loss of ficolin-2 ligand expression through wcjE mutation abrogated bacterial killing. Ficolin-2 did not interact with any pneumococcal non-capsule structures, including teichoic acid. We describe multiple 11A clonal derivatives expressing varying degrees of wcjE-dependent epitopes co-isolated from single blood specimens, likely representing microevolutionary shifts towards wcjE-deficient populations during invasive pneumococcal disease (IPD). We find epidemiological evidence of wcjE impairing pneumococcal invasiveness, supporting that the LP’s ficolin-2 axis provides innate, serotype-specific serological protection against IPD. The fact that the LP is triggered by only a few discrete carbohydrate ligands emphasizes the need to reevaluate its impact in a glycopolymer-specific manner.

Components of the Lectin Pathway of Complement in Solid Tumour Cancers.

Simple SummaryCancer is a major cause of human deaths. The complement system is an important branch of the innate immune response that can be activated via three distinct pathways (classical, alternative, lectin). Complement activation may contribute to cancer prevention, but on the other hand, its over- or chronic activation can be harmful for the host. In this short review, we discuss the dual role of the lectin pathway of complement activation in human solid cancers, including those of the female reproductive system, lung, and alimentary tract, with emphasis on its cross-talk with other enzyme-dependent cascades.The complement system is an important branch of the humoral innate immune response that can be activated via three distinct pathways (classical, alternative, lectin), contributing to keeping/restoring homeostasis. It can also interact with cellular innate immunity and with components of acquired immunity. Cross-talk between the complement system and other enzyme-dependent cascades makes it a more influential defence system, but on the other hand, over- or chronic activation can be harmful. This short review is focused on the dual role of the lectin pathway of complement activation in human solid tumour cancers, including those of the female reproductive system, lung, and alimentary tract, with emphasis on the aforementioned cross-talk.

Association of serum mannose-binding lectin, anti-phospholipase A2 receptor antibody and renal outcomes in idiopathic membranous nephropathy and atypical membranous nephropathy: a single center retrospective cohort study

Objectives Idiopathic membranous nephropathy (iMN) is a major cause of nephrotic syndrome. Atypical membranous nephropathy (aMN) is a new type of nephropathy in China, characterized by a ‘full-house’ on immunofluorescent examination, that is IgG, IgA, IgM, C3, C1q positive, but without clinical evidence of a secondary cause. Phospholipase A2 receptor (PLA2R) was the major target antigens in iMN patients. Activation of the mannose-binding lectin (MBL) pathway plays a vital role in the development of MN. Our objective was to investigate the role of PLA2R and MBL in the pathogenesis of iMN and aMN. Methods We conducted a retrospective observational study using propensity score matching by age, gender, and eGFR. All clinical, laboratory data, and follow-up data of the patients were collected. Serum levels of anti-PLA2R antibodies and MBL were tested. Results Finally, 30 iMN patients and 30 aMN patients were included, and 20 healthy controls were retrospectively collected in this study. The 24 h proteinuria level was higher and serum albumin was lower in anti-PLA2R (+) patients than in anti-PLA2R (−) patients in both iMN and aMN groups. In aMN patients, MBL levels were significantly higher in anti-PLA2R (+) patients than in anti-PLA2R (−) patients (p = .045). The serum level of anti-PLA2R positively correlated with no-remission in both iMN and aMN groups. Conclusions The complement lectin pathway has an association with the development of MN, especially in patients with positive anti-PLA2R antibodies. Serum MBL cannot differentiate between the two diseases. Serum MBL levels are not associated with clinical manifestations, nor with prognosis.

EP220: Expanding the phenotypic spectrum of COLEC10-related 3MC syndrome: A glimpse into COLEC10-related 3MC syndrome in the Ashkenazi Jewish population

3MC syndrome is a clinical entity comprising four rare autosomal recessive disorders: Carnevale, Mingarelli, Malpuech, and Michels syndromes, characterized by variable expression of facial dysmorphia, cleft lip/palate, postnatal growth deficiency, hearing loss, cognitive impairment, craniosynostosis, radioulnar synostosis, and genital and vesicorenal anomalies. 3MC syndrome was initially described to be associated with bi-allelic pathogenic variants in the COLEC11 and MASP1 genes. Recently, bi-allelic pathogenic variants in COLEC10 have been described to be associated with 3MC syndrome. Features of COLEC10-related 3MC syndrome included short stature, blepharoptosis and epicanthus inversus, cleft lip/palate, and skeletal anomalies of the digits. The proteins encoded by COLEC11, MASP1, and COLEC10 form an intricate protein network that is important in regulating the lectin complement pathway. Syndromic features seen in 3MC syndrome are thought to be due to disruption of the chemoattractant properties that influence neural crest cell (NCC) migration. We identified seven individuals from three families of Ashkenazi Jewish descent with homozygosity of the novel c.311G>T (p.Gly104Val) variant in the COLEC10 gene (NM_006438.3) and phenotype consistent with 3MC syndrome. Individuals were retrospectively identified through the Dor Yeshorim screening program based on their genotype of the c.311G>T (p.Gly104Val) variant. The carrier frequency was calculated based on 21,887 individuals of Jewish descent. Samples were obtained anonymously through the Dor Yeshorim screening program between March 2020 and March 2021. Molecular protein modeling was completed. COLEC10 amino acid sequence was obtained, and then predictive protein models from I-Tasser were generated. Using PyMOL Molecular Graphic System 2.3.4 the effect the p.Gly104Val substitution in the COLEC10 protein model was analyzed. Clinical characteristics of the seven identified individuals included variable features of bilateral cleft lip and palate, radioulnar synostosis, short stature, hearing loss, and congenital heart defects. Similar facial features included hypertelorism, ptosis, prominent forehead, semicircular eyebrows, periorbital fullness, and broad nose. All individuals had reported normal intelligence. Carrier screening for the c.311G>T (p.Gly104Val) variant identified 164 heterozygotes out of 16,497 individuals of Ashkenazi Jewish descent, giving a carrier frequency of 1 in 101 or 0.99%. There were no heterozygotes identified out of 3,097 individuals of Sephardic Jewish descent and there were 13 heterozygotes out of 2,293 individuals of mixed Ashkenazi and Sephardic Jewish descent. Molecular protein modeling showed that the p.Gly104Val substitution alters local conformation. We identified a novel variant in the COLEC10 gene likely representative of a founder variant in the Ashkenazi Jewish population. Homozygosity of this variant is associated with variable features of 3MC syndrome. Given the observed carrier frequency, the lower than expected number of affected individuals may be due to the extreme phenotypic variability that includes clinically undiagnosed and variable presentations of the syndrome, even within the same family. Molecular protein modeling suggested that the p.Gly104Val variant results in abnormal protein folding. Abnormal protein folding has previously been shown to affect the secretion of the COLEC10 protein, leading to abnormal NCC migration, resulting in the observed craniofacial and skeletal anomalies associated with 3MC syndrome. COLEC10-related 3MC syndrome should be considered in the differential diagnosis for individuals with short stature, radioulnar synostosis, cleft lip and palate, and characteristic facial features.

Mannan-Binding Lectin Regulates the Th17/Treg Axis Through JAK/STAT and TGF-β/SMAD Signaling Against Candida albicans Infection.

BackgroundMannan-binding lectin (MBL) is a key molecule in innate immunity and activates the lectin complement pathway, which plays an important role in resisting Candida albicans (C. albicans) infection. However, the underlying mechanism of this resistance to infection remains unclear.MethodsIn this study, we investigated how MBL regulates the differentiation of CD4+ T cells into T helper type 17 (Th17) and T regulatory (Treg) cells against C. albicans in mice, as well as the underlying mechanisms. We generated MBL double-knockout (KO) mice and infected them with C. albicans by intraperitoneal injection.ResultsCompared with that in wild-type (WT) mice, the percentage of Th17 cells increased in MBL-null mice, whereas Treg cells decreased, indicating that MBL might regulate the Th17/Treg balance. In addition, in MBL-null mice, the expression levels of interleukin (IL)-17A, IL-21, and the master transcription factor of Th17 cells, RORγt, significantly increased. Conversely, IL-10, IL-2, and the Treg-specific transcription factor, Foxp3, decreased. Moreover, we found that the levels of TGF-β and IL-6 upregulated in MBL-null mice. Mechanistically, we found that MBL regulated the TGF-β/SMAD pathway through the inhibition of p-SMAD2 and promotion of p-SMAD3, and mediated the JAK/STAT pathway through the inhibition of p-JAK2 and p-STAT3 and promotion of p-JAK3 and p-STAT5. MBL double-KO mice showed a more severe inflammatory response and significantly lower survival rates with C. albicans infection.ConclusionThese results suggest that MBL regulates the Th17/Treg cell balance to inhibit inflammatory responses, possibly via IL-6- and TGF-β-mediated JAK/STAT and TGF-β/SMAD signaling, and play an important role in anti-C. albicans infection.

The EPICC Family of Anti-Inflammatory Peptides: Next Generation Peptides, Additional Mechanisms of Action, and In Vivo and Ex Vivo Efficacy.

The EPICC peptides are a family of peptides that have been developed from the sequence of the capsid protein of human astrovirus type 1 and previously shown to inhibit the classical and lectin pathways of complement. The EPICC peptides have been further optimized to increase aqueous solubility and identify additional mechanisms of action. Our laboratory has developed the lead EPICC molecule, PA-dPEG24 (also known as RLS-0071), which is composed of a 15 amino acid peptide with a C-terminal monodisperse 24-mer PEGylated moiety. RLS-0071 has been demonstrated to possess other mechanisms of action in addition to complement blockade that include the inhibition of neutrophil-driven myeloperoxidase (MPO) activity, inhibition of neutrophil extracellular trap (NET) formation as well as intrinsic antioxidant activity mediated by vicinal cysteine residues contained within the peptide sequence. RLS-0071 has been tested in various ex vivo and in vivo systems and has shown promise for the treatment of both immune-mediated hematological diseases where alterations in the classical complement pathway plays an important pathogenic role as well as in models of tissue-based diseases such as acute lung injury and hypoxic ischemic encephalopathy driven by both complement and neutrophil-mediated pathways (i.e., MPO activity and NET formation). Next generation EPICC peptides containing a sarcosine residue substitution in various positions within the peptide sequence possess aqueous solubility in the absence of PEGylation and demonstrate enhanced complement and neutrophil inhibitory activity compared to RLS-0071. This review details the development of the EPICC peptides, elucidation of their dual-acting complement and neutrophil inhibitory activities and efficacy in ex vivo systems using human clinical specimens and in vivo efficacy in animal disease models.

POS-107 LONG-TERM PHASE 2 EFFICACY OF THE MASP-2 INHIBITOR NARSOPLIMAB FOR TREATMENT OF SEVERE IGA NEPHROPATHY

IgA nephropathy (IgAN) is a glomerular disease in which the lectin pathway of complement is activated following mesangial deposition of IgA immune complexes. Narsoplimab (OMS721) inhibits mannan-binding lectin-associated serine protease-2 (MASP-2), the effector enzyme of the lectin pathway.