Angiogenic factors and the lectin pathway of complement in women with secondary recurrent pregnancy loss.

The poor remodeling of placental spiral arteries seen in preeclampsia is also discussed to contribute to recurrent pregnancy loss (RPL) preceded by abnormal angiogenesis and excessive complement activation. Low levels of Mannose-binding-lectin (MBL), a pattern recognition molecule (PRM) of the lectin pathway, have been found in women with RPL. We propose that pregnancy loss is connected to defective angiogenesis with reperfusion damage in the placenta and decreased levels of PRM in the lectin pathway in women with RPL. In this cohort study, we investigate the angiogenic factors and the lectin complement pathway in early pregnancy and their time-dependent relationship with pregnancy outcomes in 76 women with secondary RPL (sRPL) who have at least four prior pregnancy losses and a live birth. We evaluated levels of Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Vascular Endothelial Growth Factor (VEGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and the PRMs, MBL, ficolin-1, -2, -3 and an additional soluble PRM, Pentraxin-3, during the 5th, 6th, and 7th gestational weeks. Our results showed that, compared to live births, pregnancies that ended in loss were associated with elevated VEGF levels and decreased levels of the Ang-2/Ang-1 ratio. Also, increasing levels of ficolin-2 were significantly associated with pregnancy loss, with MBL showing no association. Our research suggests that women with sRPL may have inadequate placentation with impaired angiogenesis in pregnancies ending in a loss.

Altered glycosylation of IgG4 promotes lectin complement pathway activation in anti-PLA2R1-associated membranous nephropathy.

Primary membranous nephropathy (pMN) is a leading cause of nephrotic syndrome in adults. In most cases, this autoimmune kidney disease is associated with autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) expressed on kidney podocytes, but the mechanisms leading to glomerular damage remain elusive. Here, we developed a cell culture model using human podocytes and found that anti-PLA2R1-positive pMN patient sera or isolated IgG4, but not IgG4-depleted sera, induced proteolysis of the 2 essential podocyte proteins synaptopodin and NEPH1 in the presence of complement, resulting in perturbations of the podocyte cytoskeleton. Specific blockade of the lectin pathway prevented degradation of synaptopodin and NEPH1. Anti-PLA2R1 IgG4 directly bound mannose-binding lectin in a glycosylation-dependent manner. In a cohort of pMN patients, we identified increased levels of galactose-deficient IgG4, which correlated with anti-PLA2R1 titers and podocyte damage induced by patient sera. Assembly of the terminal C5b-9 complement complex and activation of the complement receptors C3aR1 or C5aR1 were required to induce proteolysis of synaptopodin and NEPH1 by 2 distinct proteolytic pathways mediated by cysteine and aspartic proteinases, respectively. Together, these results demonstrated a mechanism by which aberrantly glycosylated IgG4 activated the lectin pathway and induced podocyte injury in primary membranous nephropathy.

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.

Clinical relevance of membrane attack complex deposition in children with IgA nephropathy and Henoch-Schönlein purpura.

BACKGROUND: IgA nephropathy (IgAN) and Henoch-Schönlein purpura are common glomerular disorders in children sharing the same histopathologic pattern of IgA deposits within the mesangium, even if their physiopathology may be different. Repeated exposure to pathogens induces the production of abnormal IgA1. The immune complex deposition in the renal mesangium in IgAN or potentially in small vessels in Henoch-Schönlein purpura induces complement activation via the alternative and lectin pathways. Recent studies suggest that levels of membrane attack complex (MAC) in the urine might be a useful indicator of renal injury. Because of the emerging availability of therapies that selectively block complement activation, the aim of the present study is to investigate whether MAC immunostaining might be a useful marker of IgA-mediated renal injury. METHODS: We conducted immunohistochemistry analysis of the MAC on renal biopsies from 67 pediatric patients with IgAN and Henoch-Schönlein purpura. We classified their renal biopsies according to the Oxford classification, retrieved symptoms, biological parameters, treatment, and follow-up. RESULTS: We found MAC expression was significantly related to impaired renal function and patients whose clinical course required therapy. MAC deposits tend to be more abundant in patients with decreased glomerular filtration rate (p = 0.02), patients with proteinuria > 0.750 g/day/1.73 m2, and with nephrotic syndrome. No correlation with histological alterations was observed. CONCLUSIONS: We conclude that MAC deposition could be a useful additional indicator of renal injury in patients with IgAN and Henoch-Schönlein purpura, independent of other indicators.

The Emerging Link Between the Complement Cascade and Purinergic Signaling in Stress Hematopoiesis.

Innate immunity plays an important role in orchestrating the immune response, and the complement cascade (ComC) is a major component of this ancient defense system, which is activated by the classical-, alternative-, or mannan-binding lectin (MBL) pathways. However, the MBL-dependent ComC-activation pathway has been somewhat underappreciated for many years; recent evidence indicates that it plays a crucial role in regulating the trafficking of hematopoietic stem/progenitor cells (HSPCs) by promoting their egress from bone marrow (BM) into peripheral blood (PB). This process is initiated by the release of danger-associated molecular patterns (DAMPs) from BM cells, including the most abundant member of this family, adenosine triphosphate (ATP). This nucleotide is well known as a ubiquitous intracellular molecular energy source, but when secreted becomes an important extracellular nucleotide signaling molecule and mediator of purinergic signaling. What is important for the topic of this review, ATP released from BM cells is recognized as a DAMP by MBL, and the MBL-dependent pathway of ComC activation induces a state of "sterile inflammation" in the BM microenvironment. This activation of the ComC by MBL leads to the release of several potent mediators, including the anaphylatoxins C5a and desArgC5a, which are crucial for egress of HSPCs into the circulation. In parallel, as a ligand for purinergic receptors, ATP affects mobilization of HSPCs by activating other pro-mobilizing pathways. This emerging link between the release of ATP, which on the one hand is an activator of the MBL pathway of the ComC and on the other hand is a purinergic signaling molecule, will be discussed in this review. This mechanism plays an important role in triggering defense mechanisms in response to tissue/organ injury but may also have a negative impact by triggering autoimmune disorders, aging of HSPCs, induction of myelodysplasia, and graft-versus-host disease after transplantation of histoincompatible hematopoietic cells.

AmpliSeq Screening of Genes Encoding the C-Type Lectin Receptors and Their Signaling Components Reveals a Common Variant in MASP1 Associated with Pulmonary Tuberculosis in an Indian Population.

Tuberculosis (TB) is a multifactorial disease governed by bacterial, host and environmental factors. On the host side, growing evidence shows the crucial role that genetic variants play in the susceptibility to Mycobacterium tuberculosis (Mtb) infection. Such polymorphisms have been described in genes encoding for different cytokines and pattern recognition receptors (PRR), including numerous Toll-like receptors (TLRs). In recent years, several members of the C-type lectin receptors (CTLRs) have been identified as key PRRs in TB pathogenesis. Nevertheless, studies to date have only addressed particular genetic polymorphisms in these receptors or their related pathways in relation with TB. In the present study, we screened the main CTLR gene clusters as well as CTLR pathway-related genes for genetic variation associated with pulmonary tuberculosis (PTB). This case-control study comprised 144 newly diagnosed pulmonary TB patients and 181 healthy controls recruited at the Bhagwan Mahavir Medical Research Center (BMMRC), Hyderabad, India. A two-stage study was employed in which an explorative AmpliSeq-based screening was followed by a validation phase using iPLEX MassARRAY. Our results revealed one SNP (rs3774275) in MASP1 significantly associated with PTB in our population (joint analysis p = 0.0028). Furthermore, serum levels of MASP1 were significantly elevated in TB patients when compared to healthy controls. Moreover, in the present study we could observe an impact of increased MASP1 levels on the lectin pathway complement activity in vitro. In conclusion, our results demonstrate a significant association of MASP1 polymorphism rs3774275 and MASP1 serum levels with the development of pulmonary TB. The present work contributes to our understanding of host-Mtb interaction and reinforces the critical significance of mannose-binding lectin and the lectin-complement pathway in Mtb pathogenesis. Moreover, it proposes a MASP1 polymorphism as a potential genetic marker for TB resistance.

Low level of MAp44, an inhibitor of the lectin complement pathway, and long-term graft and patient survival; a cohort study of 382 kidney recipients.

BACKGROUND: Higher incidence of malignancy and infectious diseases in kidney transplant recipients is related to immunosuppressive treatment after transplantation and the recipient's native immune system. The complement system is an essential component of the innate immunity. The aim of the present study was to investigate the association of effector molecules of the lectin complement pathway with graft and patient survival after kidney transplantation. METHODS: Two mannan-binding lectin (MBL) associated proteases, MASP-2 and MASP-3 (activators of the lectin pathway) and two MBL-associated proteins, MAp44 and MAp19 (inhibitors of the lectin pathway) were measured at the time of transplantation in 382 patients (≥17 years old) transplanted in 2000-2001. The cohort was followed until December 31, 2014. Data on patient and graft survival were obtained from the Norwegian Renal Registry. Cox proportional hazard regression models were performed for survival analyses. RESULTS: Low MAp44 level (1st versus 2-4 quartile) was significantly associated with overall mortality; HR 1.52, 95 % CI 1.08-2.14, p = 0.017. In the sub analyses in groups below and above median age (51.7 years), low MAp44 as a predictor of overall mortality was statistically significant only in recipients of ≤51.7 years; HR 2.57, 95 % CI 1.42-4.66, p = 0.002. Furthermore, low MAp44 was associated with mortality due to infectious diseases; HR 2.22, 95 % CI 1.11-4.41, p = 0.023. There was no association between MASP-2, MASP-3 or MAp19 levels and patient mortality. No association between any measured biomarkers and death censored graft loss was found. CONCLUSIONS: Low MAp44 level at the time of transplantation was associated with increased overall mortality in kidney recipients of median age of 51.7 years or below and with mortality due to infectious diseases in the whole patient cohort after nearly 14-years of follow up after transplantation. No associations between other effector molecules; MASP-2, MASP-3 or MAp19 and recipient mortality were found, as well as no association of any biomarker with death censored graft loss.

Mannose-binding Lectin Mediated Complement Pathway in Autoimmune Neurological Disorders.

Multiple sclerosis (MS) is a complex, demyelinating disease of the central nervous system (CNS) with variable phenotypic presentations, while Guillain-Barre Syndrome (GBS) is the prototypic acute inflammatory disorder that affects the peripheral nervous system. Myasthenia gravis (MG) is a T cell dependent and antibody mediated autoimmune disease. Although it has been shown that complement plays a critical role in the pathogenesis of MS, GBS, and MG, the role of mannose-binding lectin (MBL) as a biomarker of immunopathogensis of these diseases and also its association with the severity of them have been poorly investigated. Therefore, in this study we aimed to measure plasma levels of MBL in patients with MS, GBS, and MG. In a case-control study, plasma was obtained from healthy controls (n=100) and also patients with MS (n=120), GBS (n=30), and MG (n=30). Plasma level measurement of MBL was performed using enzyme-linked immunosorbent assay (ELISA). The mean serum level of MBL was significantly different between groups of patients and healthy controls (p<0.001). We also found a positive correlation between plasma levels of MBL and severity scores of MS, MG, and GBS patients including: expanded disability status scale (EDSS) (r=+0.60 and p=<0.001), quantitative myasthenia gravis score (QMGS) (r=+0.56 and p=0.01), and GBS disability scale (GDS) (r=+0.37 and p=0.04). Taken together, our findings suggest that complement activation mediated by MBL contributes to the pathogenesis and also severity of MS, MG, and GBS. However, because the lectin pathway can be involved in several phases of the immune response, further evidence will be required to elucidate the underlying mechanism.

Activation of the lectin pathway of complement by cardiopulmonary bypass contributes to the development of systemic inflammatory response syndrome after paediatric cardiac surgery.

The systemic inflammatory response is a challenge in the management of paediatric patients undergoing cardiac surgery. Although multi-factorial, a contribution by the lectin pathway of complement activation has been postulated. We therefore investigated the changes in serum levels of mannose binding lectin (MBL) and activities of MBL-MBL-associated serine protease (MASP)-1 and MBL-MASP-2 complexes immediately before and during surgery, throughout the first postoperative day and at discharge from the hospital. These changes were analysed in relation to postoperative complications. Blood samples were obtained from 185 children with congenital heart disease undergoing surgical correction with the use of cardiopulmonary bypass: preoperatively (MBL-1), 15 min after initiation of cardiopulmonary bypass (CPB) (MBL-E), 30 min (MBL-2), 4 h (MBL-3), 12 h (MBL-4) and 24 h (MBL-5) post-CPB and at discharge from hospital (MBL-K). Alterations in serum MBL levels were calculated as a ratio of its serum level at subsequent time-points (MBL-2, -3, -4, -5) to the preoperative (MBL-1) value. Decreases in MBL and MBL-MASP complexes were observed in all samples, correlating with a decrease in C4 and increase in C4a, confirming activation of the lectin pathway. Changes in MBL levels between children with an uncomplicated postoperative course and those suffering from infection or low cardiac output syndrome did not differ significantly, but significant differences were observed between the SIRS and non-SIRS groups. Paediatric cardiac surgery with the use of cardiopulmonary bypass activates the complement system via the lectin pathway and the latter contributes to the development of the post-bypass systemic inflammatory response.

A Metalloproteinase Mirolysin of Tannerella forsythia Inhibits All Pathways of the Complement System.

Recent reports focusing on virulence factors of periodontal pathogens implicated proteinases as major determinants of remarkable pathogenicity of these species, with special emphasis on their capacity to modulate complement activity. In particular, bacteria-mediated cleavage of C5 and subsequent release of C5a seems to be an important phenomenon in the manipulation of the local inflammatory response in periodontitis. In this study, we present mirolysin, a novel metalloproteinase secreted by Tannerella forsythia, a well-recognized pathogen strongly associated with periodontitis. Mirolysin exhibited a strong effect on all complement pathways. It inhibited the classical and lectin complement pathways due to efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3, and C4, whereas inhibition of the alternative pathway was caused by degradation of C5. This specificity toward complement largely resembled the activity of a previously characterized metalloproteinase of T. forsythia, karilysin. Interestingly, mirolysin released the biologically active C5a peptide in human plasma and induced migration of neutrophils. Importantly, we demonstrated that combination of mirolysin with karilysin, as well as a cysteine proteinase of another periodontal pathogen, Prevotella intermedia, resulted in a strong synergistic effect on complement. Furthermore, mutant strains of T. forsythia, devoid of either mirolysin or karilysin, showed diminished survival in human serum, providing further evidence for the synergistic inactivation of complement by these metalloproteinases. Taken together, our findings on interactions of mirolysin with complement significantly add to the understanding of immune evasion strategies of T. forsythia and expand the knowledge on molecular mechanisms driving pathogenic events in the infected periodontium.

Clinical characteristics of IgA nephropathy associated with low complement 4 levels.

OBJECTIVE: C4 deficiency is the most commonly inherited immune disorder in human. The present study investigated the characteristics of the IgAN patients with low serum C4 levels. METHODS: We performed a prospective observational study. Clinical as well as histopathologic parameters were assessed. A Kaplan-Meier survival analysis was performed concerning the primary outcome defined as the serum creatinine increased 1.5-fold from baseline. The prognostic significances of clinical and histopathologic parameters were determined using Cox proportional hazards models. RESULTS: Five-hundred twelve biopsy proven IgAN cases were available for analysis with a median follow-up of 38.4 months. Ninety-nine cases (19.34%) presented with low C4 levels (LowC4 group) and the other 413 cases did not (NlowC4 group). At the time of renal biopsy, renal injury was lighter in the LowC4 group compared with the NlowC4 group. Renal C4 deposition was significantly decreased while IgM deposition was increased in the LowC4 group. A correlation analysis shows that lower C4 levels were associated with better renal presentations at biopsy. However, the risk of developing the primary outcome was significantly greater in those with low C4 levels. Specifically, during the follow-up period, the risk of developing primary outcome was nearly ten folds higher in those with low C4, compared to those without low C4. CONCLUSION: There is a high prevalence of low C4 levels in IgAN patients. These patients with low C4 levels exhibited better renal presentations at the time of renal biopsy, whereas might be associated with a poor prognosis.

The extracellular adherence protein from Staphylococcus aureus inhibits the classical and lectin pathways of complement by blocking formation of the C3 proconvertase.

The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, which labels bacteria for phagocytosis and generates inflammatory chemoattractants. Although the majority of staphylococcal complement inhibitors act on the alternative pathway to block the amplification loop, only a few proteins act on the initial recognition cascades that constitute the classical pathway (CP) and lectin pathway (LP). We screened a collection of recombinant, secreted staphylococcal proteins to determine whether S. aureus produces other molecules that inhibit the CP and/or LP. Using this approach, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 proconvertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion.

Essential role for the lectin pathway in collagen antibody-induced arthritis revealed through use of adenovirus programming complement inhibitor MAp44 expression.

Previous studies using mannose-binding lectin (MBL) and complement C4-deficient mice have suggested that the lectin pathway (LP) is not required for the development of inflammatory arthritis in the collagen Ab-induced arthritis (CAIA) model. MBL, ficolins and collectin-11 are key LP pattern recognition molecules that associate with three serine proteases-MASP-1, MASP-2, and MASP-3-and with two MBL-associated proteins designated sMAP and MBL-associated protein of 44kDA (MAp44). Recent studies have shown that MAp44, an alternatively spliced product of the MASP-1/3 gene, is a competitive inhibitor of the binding of the recognition molecules to all three MASPs. In these studies, we examined the effect of treatment of mice with adenovirus (Ad) programmed to express human MAp44 (AdhMAp44) on the development of CAIA. AdhMAp44 and Ad programming GFP (AdGFP) expression were injected i.p. in C57BL/6 wild type mice prior to the induction of CAIA. AdhMAp44 significantly reduced the clinical disease activity (CDA) score by 81% compared with mice injected with AdGFP. Similarly, histopathologic injury scores for inflammation, pannus, cartilage and bone damage, as well as C3 deposition in the cartilage and synovium, were significantly reduced by AdhMAp44 pretreatment. Mice treated with AdmMAp44, programming expression of mouse MAp44, also showed significantly decreased CDA score and histopathologic injury scores. In addition, administration of AdhMAp44 significantly diminished the severity of Ross River virus-induced arthritis, an LP-dependent model. Our study provides conclusive evidence that an intact complement LP is essential to initiate CAIA, and that MAp44 may be an appropriate treatment for inflammatory arthritis.

Deposition of the lectin pathway of complement in renal biopsies of lupus nephritis patients.

BACKGROUND/AIMS: Lupus nephritis (LN) is one of the most serious manifestations of SLE occurring in 66-90% of these patients. The complement system is part of the innate immunity and modulator of inflammation and the adaptative immune response. Mannan-binding lectin (MBL) and Ficolin-2 (FCN-2) are important members of the lectin pathway of complement activation. Despite the significant participation of complement in the pathogenesis of the LN, there are few reports demonstrating "in situ" deposition of complement components in renal biopsy specimens in this disorder. The present study investigated the deposition of complement components in kidney specimens of LN patients. METHODS: Renal biopsies of 11 patients with SLE and LN were evaluated for immunofluorescence staining for IgG, IgA, IgM, C3, and C1q. Additionally, MBL, FCN-2 and C5b-9 were researched using monoclonal antibodies. RESULTS: All the biopsies were positive for IgG, C3, and C1q, eight were positive IgM and five had IgA deposition in glomerular tissue. The terminal complex of complement C5b9 was positive in all cases, MBL in nine (82%) cases; seven (63.6%) of them presenting concomitantly FCN-2 deposition. Patients presenting MBL deposition had higher mean of urinary proteins (9.0 g/day) than patients with negative MBL deposition (mean of 2.3g/day). CONCLUSIONS: In this study, we demonstrated in situ the participation of complement in the renal injury, including MBL and FCN-2 of the lectin pathway; also the strong role of C5b-9 in the pathogenesis of LN.

An MBL-like protein may interfere with the activation of the proPO-system, an important innate immune reaction in invertebrates.

An important characteristic of the innate immune systems of crayfish and other arthropods is the activation of a serine proteinase cascade in the hemolymph, which results in the activation of prophenoloxidase and subsequently leading to the formation of toxic quinones and melanin. Although no true complement homologues have been detected in crayfish or crustaceans, several proteins with similarities to vertebrate pattern recognition receptors (PRRs), which are involved in the lectin pathway of complement activation in vertebrates, are present. One is a C-type lectin, a mannose-binding lectin (Pl-MBL), which is secreted from granular hemocytes. Here we report that Pl-MBL has LPS-binding capacity and is dependent upon high Ca(2+) for its solubility and Pl-MBL interferes with proPO activation in vitro when HLS is prepared at high Ca(2+). The proPO-activating system is efficiently activated by microbial polysaccharides and it has to be neatly regulated to avoid activation in places where it is inappropriate and the active enzyme PO should be prevented from spreading throughout the body of the animal. This may be particularly important during molting when proPO is involved in hardening of a new cuticle and the animal is vulnerable to microbes. The presence of high amount of Pl-MBL in the granular hemocytes may play a role in this process. Since a hemocyte lysate supernatant (HLS) prepared at 100 mM Ca(2+) could become activated when the concentration of LPS was increased up to 3 mg/ml, this may indicate that Pl-MBL acts as a scavenger for LPS to prevent spreading of LPS in the hemolymph to avoid further activation of the proPO-system.