Novel Evidence That Alternative Pathway of Complement Cascade Activation is Required for Optimal Homing and Engraftment of Hematopoietic Stem/progenitor Cells

We reported in the past that activation of the third (C3) and fifth element (C5) of complement cascade (ComC) is required for a proper homing and engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs). Since myeloablative conditioning for transplantation triggers in recipient bone marrow (BM) state of sterile inflammation, we have become interested in the role of complement in this process and the potential involvement of alternative pathway of ComC activation. We noticed that factor B deficient mice (FB-KO) that do not activate properly alternative pathway, engraft poorly with BM cells from normal wild type (WT) mice. We observed defects both in homing and engraftment of transplanted HSPCs. To shed more light on these phenomena, we found that myeloablative lethal irradiation conditioning for transplantation activates purinergic signaling, ComC, and Nlrp3 inflammasome in WT mice, which is significantly impaired in FB-KO animals. Our proteomics analysis revealed that conditioned for transplantation lethally irradiated FB-KO compared to normal control animals have lower expression of several proteins involved in positive regulation of cell migration, trans-endothelial migration, immune system, cellular signaling protein, and metabolic pathways. Overall, our recent study further supports the role of innate immunity in homing and engraftment of HSPCs. Graphical Abstract

The Rare C9 P167S Risk Variant for Age-related Macular Degeneration Increases Polymerization of the Terminal Component of the Complement Cascade

Age-related macular degeneration (AMD) is a complex neurodegenerative eye disease with behavioral and genetic etiology, and is the leading cause of irreversible vision loss among elderly Caucasians. Functionally significant genetic variants in the alternative pathway of complement have been strongly linked to disease. More recently, a rare variant in the terminal pathway of complement has been associated with increased risk, Complement component 9 (C9) P167S. To assess the functional consequence of this variant, C9 levels were measured in two independent cohorts of AMD patients. In both cohorts it was demonstrated that the P167S variant was associated with low C9 plasma levels. Further analysis showed that patients with advanced AMD had elevated sC5b-9 compared to those with non-advanced AMD, although this was not associated with the P167S polymorphism. Electron microscopy of membrane attack complexes (MAC) generated using recombinantly produced wild type or P167S C9 demonstrated identical MAC ring structures. In functional assays, the P167S variant displayed a higher propensity to polymerise and a small increase in its ability to induce haemolysis of sheep erythrocytes when added to C9-depleted serum. The demonstration that this C9 P167S AMD risk polymorphism displays increased polymerisation and functional activity provides a rationale for the gene therapy trials of sCD59 to inhibit the terminal pathway of complement in AMD that are underway.

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 < 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.

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.

Nephrotic syndrome associated with primary atypical hemolytic uremic syndrome

Primary atypical hemolytic-uremic syndrome is a rare disease characterized by non-immune microangiopathic hemolytic anemia, thrombocytopenia, and renal dysfunction; it is related to alterations in the regulation of the alternative pathway of complement due to genetic mutations. The association with nephrotic syndrome is unusual. We present here a pediatric patient diagnosed with primary atypical hemolytic-uremic syndrome associated with nephrotic syndrome who responded to eculizumab treatment.