Activation Of Alternative Complement Pathway Research Articles

Alternative Complement Pathway Is Activated and Associated with Galactose-Deficient IgA1 Antibody in IgA Nephropathy Patients.

BackgroundGalactose-deficient IgA1 (Gd-IgA1) and alternative complement pathway activation are considered to be involved in the pathogenesis of IgA nephropathy (IgAN). Nevertheless, the relationships between alternative pathway activation and disease activity or Gd-IgA1 level remains unclear.MethodsNinety-eight biopsy-diagnosed IgAN, twenty-five primary focal segmental sclerosis (FSGS) patients and forty-two healthy individuals were recruited in this study. Among them, fifty IgAN patients received immunosuppression. Follow-up blood samples at 1 and 3~6 months after immunosuppression were collected. Plasma levels of complement C5a, factor Ba and Gd-IgA1 were measured and analyzed. Immunostaining for complement was performed in twenty-five IgAN and FSGS patients.ResultsAt baseline, IgAN patients had higher levels of plasma C5a, factor Ba and Gd-IgA1 than control subjects. Gd-IgA1 levels positively correlated with plasma C5a and factor Ba. In addition, levels of factor Ba and Gd-IgA1 were positively associated with proteinuria and negatively associated with renal function. Immunostaining revealed positive staining for factor Bb and C3c in glomeruli in IgAN patients, but not in FSGS patients. At baseline, patients receiving immunosuppression had more severe proteinuria and higher factor Ba. After 6 months, eGFR declined and proteinuria persisted in patients without immunosuppression. In contrast, patients who received immunosuppression exhibited decreased plasma levels of C5a, factor Ba, and Gd-IgA1 as early as 1 month after treatment. Proteinuria decreased and renal function also remained stable 6 months after immunosuppression.ConclusionsOur results indicate a close relationship between alternative complement pathway activation, Gd-IgA1 concentration and clinical severity of IgAN. Level of complement factor B may be a potential marker for disease activity and therapeutic target in IgAN patients.

Characterisation and functional role of a novel C1qDC protein from a colonial ascidian

As an invertebrate, the compound ascidian Botryllus schlosseri faces nonself only with innate immunity. In this species, we already identified the key components of the lectin and alternative complement activation pathways. In the present work, by mining the transcriptome, we identified a single transcript codifying for a protein, member of the C1q-domain-containing protein family, with a signal peptide followed by two globular C1q (gC1q) domains. It shares a similar domain organisation with C1q/TNF-related proteins 4, the only vertebrate protein family with two gC1q domains. Our gC1q domain-containing protein, called BsC1qDC, is actively transcribed by immunocytes. The transcription is modulated during the Botryllus blastogenetic cycle and is upregulated following the injection of Bacillus clausii cells in the circulation. Furthermore, the injection of bsc1qdc iRNA in the vasculature results in decreased transcription of the gene and a significant impairment of phagocytosis and degranulation, suggesting the involvement of this molecule in immune responses.

A patient with a homozygous diacylglycerol kinase epsilon (DGKE) gene mutation with atypical haemolytic uraemic syndrome and low C3 responded well to eculizumab: a case report

BackgroundAtypical haemolytic uraemic syndrome (aHUS) is a rare systemic syndrome characterized by non-immune haemolytic anaemia, thrombocytopenia, and kidney injury. In most cases, alternative complement pathway dysregulation is the identifying cause. Recently, other genetic causes have been identified, including a mutation in the diacylglycerol kinase epsilon (DGKE) gene, which theoretically affect the coagulation pathway and does not affect the complement pathway. Data about the management of these patients are limited. Ideal management and definitive treatment protocols have not yet been established.Case presentationA three-year-old boy presented with features of atypical haemolytic uraemic syndrome (aHUS) and low complement C3. He was presumed to have complement-mediated aHUS and was managed empirically with eculizumab. Two weeks after starting eculizumab, his haemoglobin levels, platelet count, and complement C3 level normalized but he continued to have non-nephrotic range proteinuria. His genetic testing revealed a homozygous DGKE mutation, with no other mutation detected. Six months after presentation, the patient was still in remission with no features of aHUS, a trial of weaning eculizumab by increasing dose interval was followed by nephrotic range proteinuria and severe oedema. His proteinuria improved and his oedema resolved after resuming his recommended eculizumab dose.ConclusionsDGKE gene mutation can lead to aHUS with theoretically no complement dysregulation. However, some patients with this mutation show alternative complement pathway activation. This case report describes a patient with aHUS due to a DGKE gene mutation and low C3 levels who responded to eculizumab, adding to the previously reported cases of patients with DGKE gene mutations who had complete remission with no relapse with C5 blockers and/or plasma exchange. A randomized controlled study on patients with DGKE mutations might be beneficial in understanding the disease and generating a management protocol.

A complement factor H homolog, heparan sulfation, and syndecan maintain inversin compartment boundaries in C. elegans cilia

Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly. Canonical disease models suggest that defective interactions between complement factor H (CFH) and cell surface heparan sulfate (HS) result in increased alternative complement pathway activity, cytolytic damage, and tissue inflammation in the retina. Although these factors are thought to contribute to increased disease risk, multiple studies indicate that noncanonical mechanisms that result from defective CFH and HS interaction may contribute to the progression of AMD as well. A total of 60 ciliated sensory neurons in the nematode Caenorhabditis elegans detect chemical, olfactory, mechanical, and thermal cues in the environment. Here, we find that a C. elegans CFH homolog localizes on CEP mechanosensory neuron cilia where it has noncanonical roles in maintaining inversin/NPHP-2 within its namesake proximal compartment and preventing inversin/NPHP-2 accumulation in distal cilia compartments in aging adults. CFH localization and maintenance of inversin/NPHP-2 compartment integrity depend on the HS 3-O sulfotransferase HST-3.1 and the transmembrane proteoglycan syndecan/SDN-1. Defective inversin/NPHP-2 localization in mouse and human photoreceptors with CFH mutations indicates that these functions and interactions may be conserved in vertebrate sensory neurons, suggesting that previously unappreciated defects in cilia structure may contribute to the progressive photoreceptor dysfunction associated with CFH loss-of-function mutations in some AMD patients.

POS-202 ATYPICAL HEMOLYTIC UREMIC SYNDROME: A RARE CAUSE OF MALIGNANT HYPERTENSION AND ACUTE RENAL FAILURE IN ADULT

Atypical hemolytic uremic syndrome (aHUS) is a form of complement-mediated thrombotic microangiopathy mainly characterized by excessive activation of alternative complement pathway activation. This occurs due to mutation in the complement regulatory proteins or by acquired neutralizing antibodies to these complement regulatory proteins. It is characterized by a triad of microangiopathic hemolytic anemia, thrombocytopenia and acute kidney injury. It usually has a poor prognosis in the absence of prompt recognition and treatment, with more than 50% of the patients progressing to end stage renal disease. Case report- A 21 year old male presented to emergency with sudden onset headache, blurring of vision and multiple episodes of vomiting. There was no history of diarrhea or dysentery in recent past.Thorough examination was done and investigations were performed. On examination, patient had pallor and elevated blood pressure of 200/100 mmHg. Relevant findings on investigations was anemia (hemoglobin: 8.9g/dl), thrombocytopenia (platelet count: 98000cells/cu.mm) and deranged renal function tests (BUN: 156, Creatinine: 7.4mg/dl). LFT showed raised total bilirubin: 2.4mg/dl with direct fraction 0.3mg/dl. LDH and uric acid were also raised(1131U/L and 9.5mg/dl respectively). Urine routine showed trace proteinuria and urine for active sediment was negative for cast. Ultrasound of bilateral kidneys showed normal echogenecity, normal size and a maintained corticomedullary differentiation with a normal renal artery Doppler study. Peripheral smear was suggestive of Normocytic normochromic anemia with occasional target cells, elliptical cells and fragmented RBC’s with a raised reticulocyte count of 6.6. With these reports, a probable diagnosis of hemolytic uremic syndrome was considered. Stool cultures for shiga toxin producing E.coli was negative. The patient had uncontrolled hypertension despite three antihypertensives, thrombocytopenia and renal failure worsened further and patient was initiated on dialysis. Kidney biopsy was done in view of rapidly progressing renal failure which showed prominent subendothelial edema with blood vessels showing mucoid edema with near complete obstruction of lumen of medium sized vessels suggestive of thrombotic microangiopathy with acute tubular necrosis (figure1). Serum C3 levels were significantly on lower side with near normal C4 levels. ADAMTS13 levels were within normal limits. Hence a diagnosis of atypical hemolytic syndrome was finally considered .The test for complement factor H antibody was negative. However further confirmatory investigations like genetic analysis of complement regulatory genes were unavailable. Patient was also started on daily sessions of plasma exchange therapy as eculizumab was not available due to financial constraints. Over next 10 days patients clinical condition started to stabilize with improvement in renal function, anemia and platelet count. Patient was discharged and advised to follow up with monitoring of renal function tests. In patients with hypertensive crisis, acute renal failure and thrombocytopenia, aHUS should be considered. Timely recognition of disease and prompt treatment is essential for favourable outcome. In the face of non-availability of eculizumab, plasmapheresis can be considered as initial treatment option.

The importance of sialic acid, pH and ion concentration on the interaction of uromodulin and complement factor H.

Uromodulin (UMOD) can bind complement factor H (cFH) and inhibit the activation of complement alternative pathway (AP) by enhancing the cofactor activity of cFH on degeneration of C3b. UMOD, an N‐glycans‐rich glycoprotein, is expressed in thick ascending limb of Henle's loop where the epithelia need to adapt to gradient change of pH and ion concentration. ELISA‐based cofactor activity of cFH and erythrocytes haemolytic assay was used to measure the impact of native and de‐glycosylated UMOD on the functions of cFH. The binding assay was performed under different pH and ion concentrations, using ELISA. The levels of sialic acid on UMOD, from healthy controls and patients with chronic kidney disease (CKD), were also detected by lectin‐ELISA. It was shown that removal of glycans decreased the binding between UMOD and cFH and abolished the ability of enhancing C3b degradation. In acidic condition, the binding became stronger, but it reduced as sodium concentration increased. A significant decrease of α‐2,3 sialic acids on UMOD was observed in CKD patients compared with that of healthy individuals. The sialic acids on UMOD, local pH and sodium concentration could impact the binding capacity between UMOD and cFH and thus regulate the activation of complement AP.

Effects of a short pulse administration ofUlva rigidaon innate immune response and intestinal microbiota inSparus auratajuveniles

Given the potential of algae as a new aquafeed ingredient, this study evaluated the effect of the tank-cultivated macroalgae Ulva rigida C. Agardh (Chlorophyta) on innate immune response, intestinal microbiota and their resistance against Photobacterium damselae subsp. piscicida in juvenile gilthead seabream (Sparus aurata L.) after a short pulse of administration. An algae-free diet was used as control, and an experimental diet was formulated containing 25% of U. rigida (UL-25). After 2, 7, 15 and 30 days biological samples were taken. No statistical significance (p > 0.05) was detected in respiratory burst activity. Diet UL-25 significantly enhanced alternative complement pathway activity respect to control diet from the beginning of the experiment (p < 0.05). When fish were challenged against P. damselae subsp. piscicida, UL-25-fed fish were more resistant against the pathogen than the fish fed control diet, obtaining a relative survival percentage of 54.55%. The ability of UL-25 diet to modify the intestinal microbiota was also confirmed. In conclusion, the use of a short pulse of dietary administration of 25% U. rigida in S. aurata diet modulates the immune response, intestinal microbiota and reduced the mortality rate during P. damselae subsp. piscicida infection.

Toxoplasma Infection Induces Sustained Up-Regulation of Complement Factor B and C5a Receptor in the Mouse Brain via Microglial Activation: Implication for the Alternative Complement Pathway Activation and Anaphylatoxin Signaling in Cerebral Toxoplasmosis.

Toxoplasma gondii is a neurotropic protozoan parasite, which is linked to neurological manifestations in immunocompromised individuals as well as severe neurodevelopmental sequelae in congenital toxoplasmosis. While the complement system is the first line of host defense that plays a significant role in the prevention of parasite dissemination, Toxoplasma artfully evades complement-mediated clearance via recruiting complement regulatory proteins to their surface. On the other hand, the details of Toxoplasma and the complement system interaction in the brain parenchyma remain elusive. In this study, infection-induced changes in the mRNA levels of complement components were analyzed by quantitative PCR using a murine Toxoplasma infection model in vivo and primary glial cells in vitro. In addition to the core components C3 and C1q, anaphylatoxin C3a and C5a receptors (C3aR and C5aR1), as well as alternative complement pathway components properdin (CFP) and factor B (CFB), were significantly upregulated 2 weeks after inoculation. Two months post-infection, CFB, C3, C3aR, and C5aR1 expression remained higher than in controls, while CFP upregulation was transient. Furthermore, Toxoplasma infection induced significant increase in CFP, CFB, C3, and C5aR1 in mixed glial culture, which was abrogated when microglial activation was inhibited by pre-treatment with minocycline. This study sheds new light on the roles for the complement system in the brain parenchyma during Toxoplasma infection, which may lead to the development of novel therapeutic approaches to Toxoplasma infection-induced neurological disorders.

Health status in gilthead seabream (Sparus aurata) juveniles fed diets devoid of fishmeal and supplemented with Phaeodactylum tricornutum

To enhance fish general health, feeds can be supplemented with health-promoting additives, reducing the need to use chemotherapeutics. Incorporation of marine algae biomasses in aquafeeds has been shown to improve fish immune status by enhancing innate immune response. This study evaluated the effects of Phaeodactylum tricornutum incorporation in feed by two different processes, either as freeze-dried biomass or broken cell wall biomass, on fish health status and performance. Triplicate groups of gilthead seabream juveniles (13.3 ± 0.3 g) were either fed a control diet (CTRL) with an extreme (i.e., 0% fishmeal), nutritionally balanced, formulation, or two experimental diets formulated as the CTRL with 1% inclusion of the microalga P. tricornutum at the expense of wheat meal: BC diet contains P. tricornutum broken cells and WC diet microalgae whole cells. After 2 and 12 weeks of feeding, blood was collected for hematological procedures, whereas plasma and mucus were sampled for immune parameters. Head-kidney, liver, and white skeletal muscle were also collected for gene expression measurements. No major differences were observed in hematological nor plasma humoral parameters after 12 weeks irrespective of dietary treatment. Arrays of 29–31 genes were analyzed in the different tissues, revealing an early dietary effect (2 weeks) in a tissue-specific pattern. In the liver, the major effect was found in the GH/IGF axis and in muscle there was a late downregulation of myostatin (mstn) gene, mainly due to WC diet, even though all fish had similar growth performance. Regarding the head-kidney, BC diet led to alpha-2-macroglobulin (a2m) gene upregulation. Also, the same treatment showed increased mucus alternative complement pathway and bactericidal activity at 2 and 12 weeks, respectively. Hence, it seems that BC diet has a potential stimulatory effect that might be relevant as a prophylactic measure before a predictable stressful event.

A Novel Glomerular C4d Scoring System: A Tool to Prognosticate Proliferative Exudative Pattern of Glomerular Injury.

Aim:Proliferative exudative pattern of glomerular injury is usually a manifestation of an infection related or a post-infectious glomerulonephritis (PIGN). Rarely, it may represent a C3 glomerulopathy, which is a dysfunction of the alternative pathway of complement activation, and is then termed an atypical PIGN (aPIGN). C4d deposits in the glomerulus are footprints of the classical and/or lectin pathway of complement activation and hence is expected to be positive in immune-mediated glomerulonephritis (GN) like classical infection-related GN, and could be used to differentiate classical PIGN from atypical PIGN.Materials and Methods:We report a novel C4d scoring system based on the intensity and the proportion of glomerular tuft staining, in a series of 104 biopsies with the proliferative exudative pattern of glomerular injury. Using a statistically derived cut-off score of 1.45, the cases were divided into C4d positive and C4d negative groups and compared to IF findings and the follow-up, available in 36 cases.Results:The C4d positive group had a significantly greater proportion of cases with immune complexes compared to the group with C3 deposits alone. In the follow-up, C4d negative group had also a greater number with partial/incomplete response compared to the C4d positive group.Conclusions:We recommend that the C4d stain be done in all cases with a proliferative exudative pattern of glomerular injury to identify patients who would need a close follow up and further assays of complement function.

The effect of salinity on growth performance, digestive and antioxidant enzymes, humoral immunity and stress indices in two euryhaline fish species: Yellowfin seabream (Acanthopagrus latus) and Asian seabass (Lates calcarifer)

A 56-day research was performed to examine the effect of water salinity (WS) on performance and some physiological responses of yellowfin seabream (Acanthopagrus latus, 12.5 g) and Asian seabass (Lates calcarifer, 33.5 g) juveniles. Fishes were reared at five WS including 6, 12, 24, 35 and 48‰. Increasing WS from 6 to 12‰ improved growth performance in A. latus juveniles; however, elevation of WS over 24‰ suppressed growth rate in this species. Increment of WS over 12‰ gradually reduced growth and feed utilization in L. calcarifer. In A. latus, fish reared at 24 and 35‰ had the greatest total protease activity, but those in 6‰ had the lowest value (P < 0.05). Lipase activity in A. latus gradually increased with enhancing WS up to 35‰ then it was decreased. The activities of total protease and lipase in L. calcarifer increased with enhancing WS form 6 up to 24‰ then their activities were gradually decreased (P < 0.05). The activity of catalase (CAT) in the liver of A. latus juveniles pronouncedly decreased with increasing WS. The glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities as well as lipid peroxidation (TBARs) content were gradually increased by increment of WS in the liver of A. latus (P < 0.05). Regarding L. calcarifer, the activity of CAT and GPX remarkably decreased from 6 to 24‰, then their activity gradually increased from 35 to 48‰ (P < 0.05). However, SOD activity and TBARs content significantly increased from 6 to 24‰ then remarkably decreased in the liver of L. calcarifer. Humoral immune responses including lysozyme, alternative complement pathway activity and total immunoglubuns provoked by increment of WS in both species. Stress indices including plasma total protein, glucose, lactate, cortisol and total osmolality in both species remarkably affected by WS. According to the results of the present study the intermediate salinities (brackish water) between 6 and 12‰ are recommended for the culture of these euryhaline species.

Exploring the role of the complement system, endothelial injury, and microRNAs in thrombotic microangiopathy after kidney transplantation.

ObjectiveWe investigated whether the recipient’s complement system function, kidney graft endothelial ultrastructural injury, and microRNA (miRNA) expression before transplantation may be associated with the risk of posttransplant de novo thrombotic microangiopathy (TMA).MethodsComplement system function assessment, histological and ultrastructural examination of preimplantation and kidney graft biopsies, and microRNA assessment were performed on kidney transplant recipients (KTRs) with de novo TMA.ResultsOn the basis of the clinical course, histological findings, and miRNA patterns, the following two de novo TMA phenotypes were observed: a self-limiting disease that was localized to the kidney graft and a systemic disease that progressed to graft failure without timely treatment. Decreased alternative complement pathway activity and ultrastructural endothelial injury before transplantation were confirmed in all five KTRs and four of five KTRs, respectively, but they did not correlate with de novo TMA severity.ConclusionsAlternative complement pathway abnormalities in KTRs and endothelial ultrastructural injury on preimplantation biopsy might be associated with de novo posttransplant TMA, although they did not predict posttransplant TMA severity (localized vs. systemic). The specific miRNA expression patterns in preimplantation kidney graft biopsies demonstrated a borderline statistically significant difference and might provide more accurate information on posttransplant TMA severity.

Alternative complement pathway activation in thrombotic microangiopathy associated with lupus nephritis.

Thrombotic microangiopathy (TMA) in systemic lupus erythematosus is a rare manifestation associated with activation of the complement system. This study aimed to compare plasma and urine complement activation products between patients with active lupus nephritis (aLN) and those with acute TMA plus concomitant active LN (aTMA+aLN). Plasma and urine samples were obtained from 20 patients with aTMA+aLN, 20 patients with aLN matched by the histological activity index, 5 patients with chronic TMA, 20 patients with inactive LN, and 10 kidney donors. Complement fragments C3a, C4a, C4d, Ba, C5a, C5bC9, and factor H were determined by ELISA; and kidney C4d deposition was detected by immunohistochemistry. Patients were followed for > 12 months and complement activation products re-measured after treatment in 10 aTMA+aLN patients. Both aTMA+aLN and aLN groups had increased circulating C3a, Ba, and C5bC9; and decreased circulating C3, C4, C4a, C4d, and factor H. Urinary C3a, C5a, Ba, and C5bC9 were higher in patients with aTMA+aLN than in aLN. After treatment, levels of circulating C3, C4, and factor H increased; while levels of urinary C3a, C5a, Ba, and C5bC9 decreased in patients with aTMA+aLN. These changes were observed at each aTMA episode in two patients studied during repeated TMA episodes. There was no difference in C4d deposition in glomerular capillaries, tubular basement membrane, peritubular capillaries, and arterioles between patients with aLN and those aTMA+aLN. Circulating and urine complement activation products suggest that thrombotic microangiopathy associated with LN is mediated through activation of the alternative complement pathway. Key Points • Immune-complex kidney disease in systemic lupus erythematosus (SLE) is associated with activation of the classical, lectin, and alternative complement pathways • Indirect evidence from measurement of circulating and urinary complement pathway activation products suggests that renal acute thrombotic microangiopathy in SLE is mediated by activation of the alternative complement pathway • C4d kidney immunohistochemistry may be positive in both immune complex nephritis and thrombotic microangiopathy. Therefore, it is not a specific marker of renal thrombotic microangiopathy in SLE.

Von Willebrand Factor Activates the Alternative Complement Pathway In Vivo in a Mouse Model of Complement Thrombotic Microangiopathy

Introduction Thrombotic microangiopathy (TMA) is a group of disorders presenting with microvascular thrombosis, microangiopathic hemolytic anemia, thrombocytopenia, and microvascular endothelial injury ultimately leading to end organ damage. Atypical hemolytic uremic syndrome (aHUS) and bone marrow transplantation-associated TMA (TA-TMA) are two types of TMA known as "complement TMA," as they are associated with dysfunction of the alternative complement pathway (AP), part of the innate immune system. Complement TMA episodes are frequently initiated during inflammation, and the role of inflammation in these TMAs may be in part related to the finding that the inflammatory cytokine TNF up-regulates complement components, down-regulates complement regulators, and causes AP activation in human glomerular microvascular endothelial cells (GMVECs) in vitro. However, the precise mechanism of AP activation in complement TMA is poorly understood. It has been demonstrated that von Willebrand factor (VWF) serves as a surface for AP activation in vitro. Inflammatory cytokines are stimulatory to endothelial cells, leading to excessive VWF secretion; in fact, patients with inflammatory disorders such as complement TMA demonstrate elevated plasma VWF levels, and this abundance of VWF may lead to excessive AP activation. We hypothesized that in complement TMA, VWF released from endothelial cells during inflammation activates the AP. The objective was to investigate whether VWF-mediated AP activation is initiating episodes of complement TMA in vivo in a complement TMA mouse model. Methods A mouse model of complement TMA was developed. To provoke complement TMA, wild-type (WT) C57BL/6J mice (purchased from Jackson Laboratories) were injected intraperitoneally with 5 mg/kg of the inflammatory endotoxin lipopolysaccharide (LPS). Twenty-four hours after injection of either LPS or saline (control), markers of complement TMA-lactate dehydrogenase (LDH), creatinine, platelet count, and complement activation by a rabbit erythrocyte (ER) lysis assay-were measured. ER specifically activate the AP in mammalian plasma in the presence of Mg/EGTA, and the resulting lysis is an indicator of the extent to which complement components have been consumed in vivo: low ER lysis in vitro indicates high complement activation in vivo. To assess the role of VWF in AP activation in vivo, VWF-/- mice (purchased from Jackson Laboratories on a C57BL/6J background) were injected with 5 mg/kg LPS to induce complement TMA. Twenty-four hours after injection, plasma ER lysis was measured and compared to ER lysis in LPS-injected WT C57BL/6J mice. Other markers of complement TMA, including LDH, creatinine, and platelet count, were also measured in both LPS-injected VWF-/- and WT C57BL/6J mice. Results Compared to saline-injected WT C57BL/6J mice, the LPS-injected mice demonstrated significantly: A) higher LDH (hemolysis); B) higher creatinine (renal dysfunction); C) lower platelet count; and D) lower ER lysis (increased AP activation) (Fig. 1). These results confirm the establishment of a complement TMA murine model. Furthermore, LPS-injected VWF-/- mice demonstrated significantly higher ER lysis values than the LPS-injected WT C57BL/6J mice, indicating less AP activation (Fig 2). Because VWF-/- mice do not produce any VWF, these data suggest that VWF contributes to AP activation in vivo in complement TMA. Finally, the LPS-injected VWF-/- mice had less severe TMA as demonstrated by lower LDH levels, lower creatinine levels, and higher platelet counts compared to the LPS-injected WT C57BL/6J mice (Fig. 3). The LPS-injected VWF-/- mice still had a mild degree of AP activation and TMA compared to saline-injected WT C57BL/6J mice, implying that there are complex mechanisms of AP activation in complement TMA that rely heavily on VWF. Conclusions In this study we demonstrated that VWF was important in complement activation and provocation of TMA in a complement TMA murine model. These data are the first step toward a better understanding of VWF's role in complement TMA and may lead to the development of therapeutics that inhibit inflammation, VWF release, or VWF-complement binding to curb AP activation in aHUS and TA-TMA, thereby preventing devastating outcomes. Further study is necessary to delineate the precise mechanisms of VWF-mediated AP activation and TMA development so that these treatment modalities may be explored. Disclosures No relevant conflicts of interest to declare.

Defects in Sialic Acid Biosynthesis Cause Dysregulation of the Alternative Pathway of Complement

Introduction: Atypical Hemolytic Uremic Syndrome (aHUS) is a disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. aHUS is usually caused by a predisposing germline variant in a complement regulatory gene, and a second hit of a complement amplifying condition such as infection, pregnancy, or inflammation.However, only approximately 50% of aHUS patients have identifiable genetic variants, with variants in factor H (CFH) accounting for 20%-30% of the genetic predisposition, C3 for 7%, membrane cofactor protein (MCP)/CD46 for 8%, factor B (CFB) for 2% and factor I (CFI) for 6%. For the other 50% of the patients, the genetic predisposition remains elusive. CFH binds to α2,3 sialic acid (SA) linked glycans on host cell surfaces and protects against attack by the alternative pathway of complement (APC). We hypothesized that the biosynthesis of SA is essential to complement regulation and SA biosynthesis defects predispose to aHUS. Methods: We performed targeted sequencing on 34 aHUS patients and 43 healthy controls for 4 genes that are responsible for the de novo biosynthesis of sialic acid: GNE, NANS, NANP, and CMAS. Then we used CRISPR-Cas9 and lentivirus systems to manipulate these genes in TF1 or TF1 PIGA-null cells, which lack the glycophosphatidylinositol-linked cell surface complement regulators CD55 and CD59, and allow the APC cascade to proceed once activated. α2,3 SA levels on the cell surface were measured with Maackia Amurensis lectin II (MAL II). Finally, we studied the functional consequences of these genetic changes. Normal human serum (NHS) was used to activate the APC on cells, and factor D (CFD) depleted serum (D-Dpl) was used to specifically block APC activation. C5b-9 deposition and CFH binding capacity on cells were detected by flow cytometry, and complement induced cell killing was detected via a WST-1 cell viability assay (mHam). Results: i) Rare germline variants found in SA biosynthesis genes Two rare germline variants (minor allele frequency &amp;lt; 0.005; data from GnomAD) were identified via targeted sequencing. An NANS M117I variant was found in an aHUS patient, while an NANP A153V variant was found in a control. The aHUS case did not harbor any variants in known complement genes. ii) Loss ofNANS but not NANP decreasesα2,3 SA on TF1 cells NANS knockout TF1 cells showed decreased α2,3 SA, demonstrating that this gene is essential for de novo SA biosynthesis. Conversely, NANP knockout TF1 cells showed no α2,3 SA level change. iii) NANS knockout increases the susceptibility to the APC TF1 PIGA-null cells with NANS knockout (TF1 DKO) had significantly higher C5b-9 deposition when treated with NHS compared to deletion of either gene alone (Figure A), demonstrating the formation of membrane attack complex (MAC). Cell viability assays also showed that TF1 DKO cells had significantly higher complement-induced cell killing when treated with NHS. Both C5b9 deposition and killing were rescued by APC-specific inhibition targeting CFD, demonstrating SA biosynthesis defects will increase the susceptibility to the APC specifically. iv) NANS knockout decreases the binding capacity of CFH To investigate CFH binding to the cell surface, C3b was first evenly loaded onto cells, followed by the addition of recombinant CFH. NANS knockout cells displayed significantly reduced CFH binding capacity, providing a mechanism for APC activation in the NANS knockout. v) NANS M117I decreases de novo biosynthesis of SA To assess the NANS variant identified in a patient with aHUS, TF1 DKO cells were transduced with a lentivirus containing the either wild type or M117I-mutated NANS, and cell-sorted to select individual clones. After treatment with sialidase to remove all SA, cells with NANS M117I had significantly lower SA level recovery compared to NANS wild type cells (Figure B). Conclusion: Targeted sequencing of 34 patients with aHUS identified a germline NANS variant in one case, suggesting an association between aHUS and SA biosynthesis defects. Functional studies showed that NANS knockout and the NANS M117I variant decreased cell surface SA levels. Loss of NANS also caused a decrease in CFH binding capacity and uncontrolled APC activation with increased cell death. Disclosures Chaturvedi: Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Direct activation of the alternative complement pathway by SARS-CoV-2 spike proteins is blocked by factor D inhibition

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious respiratory virus that can lead to venous/arterial thrombosis, stroke, renal failure, myocardial infarction, thrombocytopenia, and other end-organ damage. Animal models demonstrating end-organ protection in C3-deficient mice and evidence of complement activation in humans have led to the hypothesis that SARS-CoV-2 triggers complement-mediated endothelial damage, but the mechanism is unclear. Here, we demonstrate that the SARS-CoV-2 spike protein (subunit 1 and 2), but not the N protein, directly activates the alternative pathway of complement (APC). Complement-dependent killing using the modified Ham test is blocked by either C5 or factor D inhibition. C3 fragments and C5b-9 are deposited on TF1PIGAnull target cells, and complement factor Bb is increased in the supernatant from spike protein–treated cells. C5 inhibition prevents the accumulation of C5b-9 on cells, but not C3c; however, factor D inhibition prevents both C3c and C5b-9 accumulation. Addition of factor H mitigates the complement attack. In conclusion, SARS-CoV-2 spike proteins convert nonactivator surfaces to activator surfaces by preventing the inactivation of the cell-surface APC convertase. APC activation may explain many of the clinical manifestations (microangiopathy, thrombocytopenia, renal injury, and thrombophilia) of COVID-19 that are also observed in other complement-driven diseases such as atypical hemolytic uremic syndrome and catastrophic antiphospholipid antibody syndrome. C5 inhibition prevents accumulation of C5b-9 in vitro but does not prevent upstream complement activation in response to SARS-CoV-2 spike proteins.

IL-1/IL-1R signaling induced by all-trans-retinal contributes to complement alternative pathway activation in retinal pigment epithelium.

The underlying mechanisms of complement activation in Stargardt disease type 1 (STGD1) and age-related macular degeneration (AMD) are not fully understood. Overaccumulation of all-trans-retinal (atRAL) has been proposed as the pathogenic factor in both diseases. By incubating retinal pigment epithelium (RPE) cells with atRAL, we showed that C5b-9 membrane attack complexes (MACs) were generated mainly through complement alternative pathway. An increase in complement factor B (CFB) expression as well as downregulation of complement regulatory proteins CD46, CD55, CD59, and CFH were observed in RPE cells after atRAL treatment. Furthermore, interleukin-1β production was provoked in both atRAL-treated RPE cells and microglia/macrophages. Coincubation of RPE cells with interleukin-1 receptor antagonist (IL1Ra) and atRAL ameliorated complement activation and downregulated CFB expression by attenuating both p38 and c-Jun N-terminal kinase (JNK) signaling pathways. Our findings demonstrate that atRAL induces an autocrine/paracrine IL-1/IL-1R signaling to promote complement alternative pathway activation in RPE cells and provide a novel perspective on the pathomechanism of macular degeneration.

MTORC1/STAT1 Signaling Stimulates CFB Expression and Alternative Complement Pathway Activation to Induce Podocyte Dysfunction and Diabetic Kidney Disease

MTORC1/STAT1 Signaling Stimulates CFB Expression and Alternative Complement Pathway Activation to Induce Podocyte Dysfunction and Diabetic Kidney Disease

Soluble collectin-12 mediates C3-independent docking of properdin that activates the alternative pathway of complement.

Properdin stabilizes the alternative C3 convertase (C3bBb), whereas its role as pattern-recognition molecule mediating complement activation is disputed for decades. Previously, we have found that soluble collectin-12 (sCL-12) synergizes complement alternative pathway (AP) activation. However, whether this observation is C3 dependent is unknown. By application of the C3-inhibitor Cp40, we found that properdin in normal human serum bound to Aspergillus fumigatus solely in a C3b-dependent manner. Cp40 also prevented properdin binding when properdin-depleted serum reconstituted with purified properdin was applied, in analogy with the findings achieved by C3-depleted serum. However, when opsonized with sCL-12, properdin bound in a C3-independent manner exclusively via its tetrameric structure and directed in situ C3bBb assembly. In conclusion, a prerequisite for properdin binding and in situ C3bBb assembly was the initial docking of sCL-12. This implies a new important function of properdin in host defense bridging pattern recognition and specific AP activation.

Role of complement in alloimmunization and hyperhemolysis.

The purpose of this review is to summarize the role of complement in regulating the removal of a target alloantigen following an incompatible red blood cell (RBC) transfusion, the formation of alloantibodies following RBC alloantigen exposure, and the development of hyperhemolysis in patients with sickle cell disease (SCD). Recent studies demonstrate that complement can accelerate alloantibody-mediated removal of target alloantigens from the RBC surface following incompatible transfusion. Complement also influences alloantigen availability during developing alloimmune responses and serves as a unique mediator of CD4 T-cell-independent alloantibody formation following RBC alloantigen exposure. Finally, alternative complement pathway activation appears to play a key role in the development of acute hemolytic episodes in patients with SCD, providing a potential druggable target to prevent acute complications in patients with this disease. Recent studies suggest that complement can regulate a wide variety of processes germane to hematology, from transfusion complications to baseline hemolysis in patients with SCD. As the role of complement in various disease processes becomes more fully understood, the ability to leverage recently developed complement modulating drugs will only continue to enhance providers' ability to favorably intervene in many hematological diseases.