Expansion of Anticomplement Therapy Indications from Rare Genetic Disorders to Common Kidney Diseases.

Complement constitutes a major part of the innate immune system. The study of complement in human health has historically focused on infection risks associated with complement protein deficiencies; however, recent interest in the field has focused on overactivation of complement as a cause of immune injury and the development of anticomplement therapies to treat human diseases. The kidneys are particularly sensitive to complement injury, and anticomplement therapies for several kidney diseases have been investigated. Overactivation of complement can result from loss-of-function mutations in complement regulators; gain-of-function mutations in key complement proteins such as C3 and factor B; or autoantibody production, infection, or tissue stresses, such as ischemia and reperfusion, that perturb the balance of complement activation and regulation. Here, we provide a high-level review of the status of anticomplement therapies, with an emphasis on the transition from rare diseases to more common kidney diseases.

MASP3 Deficiency in Mice Reduces but Does Not Abrogate Alternative Pathway Complement Activity Due to Intrinsic Profactor D Activity.

Complement factor D (FD) is a rate-limiting enzyme of the alternative pathway (AP). Recent studies have suggested that it is synthesized as an inactive precursor and that its conversion to enzymatically active FD is catalyzed by mannan-binding lectin-associated serine protease 3 (MASP3). However, whether MASP3 is essential for AP complement activity remains uncertain. It has been shown that Masp1/3 gene knockout did not prevent AP complement overactivation in a factor H-knockout mouse, and a human patient lacking MASP3 still retained AP complement activity. In this study, we have assessed AP complement activity in a Masp3-knockout mouse generated by CRISPR/Cas9 editing of the Masp1/3 gene. We confirmed specific Masp3 gene inactivation by showing intact MASP1 protein expression and absence of mature FD in the mutant mice. Using several assays, including LPS- and zymosan-induced C3b deposition and rabbit RBC lysis tests, we detected plasma concentration-dependent AP complement activity in Masp3 gene-inactivated mice. Thus, although not measurable in 5% plasma, significant AP complement activity was detected in 20-50% plasma of Masp3 gene-inactivated mice. Furthermore, whereas FD gene deletion provided more than 90% protection of CD55/Crry-deficient RBCs from AP complement-mediated extravascular hemolysis, Masp3 gene deletion only provided 30% protection in the same study. We also found pro-FD to possess intrinsic catalytic activity, albeit at a much lower level than mature FD. Our data suggest that MASP3 deficiency reduces but does not abrogate AP complement activity and that this is explained by intrinsic pro-FD activity, which can be physiologically relevant in vivo.

Differential contribution of C5aR and C5b-9 pathways to renal thrombic microangiopathy and macrovascular thrombosis in mice carrying an atypical hemolytic syndrome-related factor H mutation.

Atypical hemolytic uremic syndrome (aHUS) is a form of thrombotic microangiopathy (TMA) caused by dysregulated complement activation. Clinically, aHUS is effectively treated by an anti-C5 monoclonal antibody (mAb) but whether the disease is mediated by the C5a receptor (C5aR) or C5b-9 pathway, or both, is unknown. Here we address this in a factor H mutant mouse (FHR/R) which developed complement-mediated TMA as well as macrovascular thrombosis caused by an aHUS-related factor H point mutation (mouse W1206R, corresponding to human W1183R). C5 deficiency and anti-C5 mAb treatment blocked all disease manifestations in FHR/R mice. C5aR1 gene deficiency prevented macrovascular thrombosis in various organs but did not improve survival or reduce renal TMA. Conversely, C6 or C9 deficiency significantly improved survival and markedly diminished renal TMA but did not prevent macrovascular thrombosis. Interestingly, as they aged both FHR/R C6-/- and FHR/R C9-/- mice developed glomerular disease reminiscent of C3 glomerulonephritis. Thus, C5aR and C5b-9 pathways drove different aspects of disease in FHR/R mice with the C5aR pathway being responsible for macrovascular thrombosis and chronic inflammatory injury while the C5b-9 pathway caused renal TMA. Our data provide new understanding of the pathogenesis of complement-mediated TMA and macrovascular thrombosis in FHR/R mice and suggest that C5 blockade is more effective for the treatment of aHUS than selectively targeting the C5aR or C5b-9 pathway alone.

Antibody Inhibition of Properdin Prevents Complement-Mediated Intravascular and Extravascular Hemolysis.

Paroxysmal nocturnal hemoglobinuria (PNH) is a serious blood disorder characterized by dysregulated complement activation on blood cells. Eculizumab, the current standard therapy and a humanized anti-C5 mAb, relieves anemia and thrombosis symptoms of PNH patients by preventing complement-dependent intravascular hemolysis (IVH). However, up to 20% of PNH patients on long-term eculizumab treatment still suffer from significant anemia and are transfusion dependent because of extravascular hemolysis (EVH) of C3-opsonized PNH erythrocytes. In this study, we show that function-blocking anti-properdin (P) mAbs dose-dependently inhibited autologous, complement-mediated hemolysis induced by factor H dysfunction. Furthermore, anti-human P (hP) mAbs potently and dose-dependently inhibited acidified serum-induced hemolysis of PNH erythrocytes (Ham test). In contrast to erythrocytes rescued by anti-C5 mAb, nonlysed PNH erythrocytes rescued by anti-P mAb incurred no activated C3 fragment deposition on their surface. These results suggested that anti-P mAbs may prevent EVH as well as IVH of PNH erythrocytes. To test the in vivo efficacy of anti-hP mAbs in preventing EVH, we generated a P humanized mouse by transgenic expression of hP in P knockout mice (hP-Tg/P-/-). In a murine EVH model, complement-susceptible erythrocytes were completely eliminated within 3 d in control mAb-treated hP-Tg/P-/- mice, whereas such cells were protected and persisted in hP-Tg/P-/- mice treated with an anti-hP mAb. Collectively, these data suggest that anti-P mAbs can inhibit both IVH and EVH mediated by complement and may offer improved efficacy over eculizumab, the current standard therapy for PNH.

Allethrin toxicity on human corneal epithelial cells involves mitochondrial pathway mediated apoptosis.

Pyrethroids including allethrin are the most common commercial household insecticides. The detrimental effects caused by pyrethroids on humans are gaining considerable attention. The present study was aimed to elucidate the effects of allethrin on the human corneal epithelial (HCE) cells. Allethrin inhibited the proliferation of HCE cells in a dose-dependent manner. In the presence of allethrin, cells showed membrane blebbing and nuclear fragmentation along with significant decrease in mitochondrial membrane potential resulting in increased cytochrome c (Cyt c) release into the cytosol. Further, flow cytometry analysis demonstrated a marked increase in sub G0-G1 cells, characteristic of apoptosis. Increased expression of pro-apoptotic protein, Bax, a simultaneous decrease of anti-apoptotic protein, Bcl-2, and activation of Caspase 3 was evident in the treated cells. In addition, extracellular matrix digesting metalloproteinase 9 (MMP-9) was also stimulated. Furthermore, significant increase in the gene expression of inflammatory cytokines, TNF-α and IL-1ß was observed. Taken together, these findings suggest that allethrin (IC50≈85µM) is toxic to HCE cells causing death through mitochondrial pathway.