Complement Deficiencies Result in Surrogate Pathways of Complement Activation in Novel Polygenic Lupus-like Models of Kidney Injury.

Lupus nephritis (LN) is a major contributor to morbidity and mortality in lupus patients, but the mechanisms of kidney damage remain unclear. In this study, we introduce, to our knowledge, novel models of LN designed to resemble the polygenic nature of human lupus by embodying three key genetic alterations: the Sle1 interval leading to anti-chromatin autoantibodies; Mfge8-/- , leading to defective clearance of apoptotic cells; and either C1q-/- or C3-/- , leading to low complement levels. We report that proliferative glomerulonephritis arose only in the presence of all three abnormalities (i.e., in Sle1.Mfge8 -/- C1q -/- and Sle1.Mfge8 -/- C3 -/- triple-mutant [TM] strains [C1q -/-TM and C3-/- TM, respectively]), with structural kidney changes resembling those in LN patients. Unexpectedly, both TM strains had significant increases in autoantibody titers, Ag spread, and IgG deposition in the kidneys. Despite the early complement component deficiencies, we observed assembly of the pathogenic terminal complement membrane attack complex in both TM strains. In C1q-/- TM mice, colocalization of MASP-2 and C3 in both the glomeruli and tubules indicated that the lectin pathway likely contributed to complement activation and tissue injury in this strain. Interestingly, enhanced thrombin activation in C3-/- TM mice and reduction of kidney injury following attenuation of thrombin generation by argatroban in a serum-transfer nephrotoxic model identified thrombin as a surrogate pathway for complement activation in C3-deficient mice. These novel mouse models of human lupus inform the requirements for nephritis and provide targets for intervention.

Primary pneumococcal peritonitis can be the first presentation of a familial complement factor I deficiency1.

Primary pneumococcal peritonitis is a rare infection that has been described in women but has not been previously linked with immunodeficiency. The complement system plays a central role in immune defence against Streptococcus pneumoniae and, in order to evade complement attack, pneumococci have evolved a large number of mechanisms that limit complement-mediated opsonization and subsequent phagocytosis. We investigated an apparently immunocompetent woman with primary pneumococcal peritonitis and identified a family with deficiency for complement factor I. Primary pneumococcal peritonitis should be considered a possible primary immunodeficiency presentation.

Multi-component meningococcal serogroup B (MenB)-4C vaccine induces effective opsonophagocytic killing in children with a complement deficiency.

Vaccination against meningococcal serogroup B is recommended for patients with a complement deficiency; however, although immunogenicity in this patient group has been shown, efficacy has not yet been established. In this study, we collected serum from children with a complement deficiency in the alternative pathway or in late terminal pathway before and after vaccination with multi-component meningococcal serogroup B (MenB)-4C. MenB-4C is a multi-component, protein-based vaccine against MenB consisting of factor H-binding protein, Neisserial heparin-binding protein, Neisserial adhesion A and outer membrane vesicles containing Porin A. We assessed the vaccine immunogenicity and vaccine-mediated protection by a whole cell enzyme-linked immunosorbent assay with Neisseria meningitidis serogroup B strains H44/76, 5/99 and NZ98/254, which shows that vaccination induced antibody titers against meningococcus. We show that the classical serum bactericidal activity assay with exogenous serum indicates the presence of vaccine-induced antibodies and capacity to activate complement-mediated pathogen lysis. However, in children with a late terminal pathway deficiency, no complement-mediated pathogen lysis was observed when autologous serum was applied in the serum bactericidal activity assay, demonstrating a lack of serum bactericidal activity in children with complement deficiencies. However, MenB-4C vaccination still induced effective complement-dependent opsonophagocytic killing against N. meningitidis serogroup B in reconstituted whole blood with autologous serum from children with an alternative pathway or late terminal pathway deficiency. These findings support the recommendation to vaccinate all complement-deficient children against MenB.

Genetic and Protein Structural Evaluation of Atypical Hemolytic Uremic Syndrome and C3 Glomerulopathy.

Atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G) are associated with loss of regulation of the alternative pathway of complement and its resulting overactivation. As rare diseases, genetic variants leading to aHUS and C3G were previously analysed in relatively low patient numbers. To improve this analysis, data were pooled from six centres. Totals of 610 rare variants for aHUS and 82 for C3G were presented in an interactive database for 13 genes. Using allele frequency comparisons with the Exome Aggregation Consortium as a reference genome, the patients with aHUS showed significantly more protein-altering ultrarare variants (allele frequency <0.01%) in five genes CFH, CFI, CD46, C3, and DGKE. In patients with C3G, the corresponding association was only found for C3 and CFH. Protein structure analyses of these five proteins showed distinct differences in the positioning of these variants in C3 and FH. For aHUS, variants were clustered at the C-terminus of FH and implicated changes in the binding of FH to host cell surfaces. For C3G, variants were clustered at the N-terminal C3b binding site of FH and implicated changes in the fluid-phase regulation of C3b. We discuss the utility of the Web database as a patient resource for clinicians.

C3(H2O) prevents rescue of complement-mediated C3 glomerulopathy in Cfh-/- Cfd-/- mice.

Therapeutic complement inhibition is a major focus for novel drug development. Of upstream targets, factor D (FD) is appealing because it circulates in plasma at low concentrations and has a single function: to cleave factor B to generate C3 convertase of the alternative pathway (AP). Mice with a targeted deletion of factor H (FH; Cfh-/- mice) develop C3 glomerulopathy (C3G) due to uncontrolled AP activity. To assess the impact of FD inhibition, we studied Cfh-/- Cfd-/- mice. We show that C3G in Cfh-/- mice is not rescued by removing FD. We used serum from Cfh-/- Cfd-/- mice to demonstrate that residual AP function occurs even when both FD and FH are missing and that hemolytic activity is present due to the action of C3(H2O). We propose that uncontrolled tick-over leads to slow activation of the AP in Cfh-/- Cfd-/- mice and that a minimal threshold of FH is necessary if tissue deposition of C3 is to be prevented. The FD/FH ratio dictates serum C3 level and renal C3b deposition. In C3G patients with chronic renal disease, the FD/FH ratio correlates inversely with C3 and C5 serum levels, suggesting that continuous AP control may be difficult to achieve by targeting FD.

Recurrent macroscopic hematuria in a pediatric patient: is it early to diagnose as having type I hereditary C2 deficiency?

Hereditary C2 deficiency is the most common early complement deficiency and characterized by recurrent infections and autoimmunity despite most patients are also asymptomatic. Type I hereditary C2 deficiency is caused by a heterozygous deletion in C2 gene resulting in early stop codon and lack of C2 production. Clinical spectrum may vary and pure nephrological involvement without the presence of recurrent infections is scarce in hereditary C2 deficiency.We report here a previously healthy 14-year-old boy presenting recurrent self-limited macroscopic hematuria and persistently low serum C4 levels, diagnosed as having type I hereditary C2 deficiency with confirming a novel heterozygote deletion (c.1567 + 22_1567 + 43del) in C2 gene. He has been remained asymptomatic for the next 18 months. Since the diagnosis of C2 deficiency was made in the absence of organ-threatening involvement such as immune complex-mediated glomerulonephritis, we think that early diagnosis and optimal follow-up may improve life-span of the patients with hereditary early complement deficiencies.

Dengue virus and the complement alternative pathway.

Dengue disease is an inflammatory-driven pathology, and complement overactivation is linked to disease severity and vascular leakage. Additionally, dysregulation of complement alternative pathway (AP) components has been described, such as upregulation of complement factor D and downregulation of complement factor H (FH), which activate and inhibit the AP, respectively. Thus, the pathology of severe dengue could in part result from AP dysfunction, even though complement and AP activation usually provide protection against viral infections. In dengue virus-infected macrophages and endothelial cells (ECs), the site of replication and target for vascular pathology, respectively, the AP is activated. The AP activation, reduced FH and vascular leakage seen in dengue disease in part parallels other complement AP pathologies associated with FH deficiency, such as atypical haemolytic uraemic syndrome (aHUS). aHUS can be therapeutically targeted with inhibitors of complement terminal activity, raising the idea that strategies such as inhibition of complement or delivery of FH or other complement regulatory components to EC may be beneficial to combat the vascular leakage seen in severe dengue.

Bacterial infections in a pediatric cohort of primary and acquired complement deficiencies.

BACKGROUND: Acquired complement deficiency can occur in the setting of autoimmune syndromes, such as systemic lupus erythematosus (SLE), with very low or, occasionally, undetectable C3 levels. Based on inherited complement defects, patients with transiently low complement may be at similar risk for serious bacterial infection, but the degree of risk related to C3 level and temporal association is unknown. METHODS: We performed a retrospective study including pediatric patients with undetectable total complement activity or absent individual complement components measured at our institution from 2002 to 2018. We assessed annual rate of serious bacterial infection (SBI) defined as requiring hospitalization and/or parenteral antibiotics by manual chart review. Among included SLE patients, we assessed the 30-day probability of SBI for given C3 measurements using a logistic regression model to determine risk. Primary complement deficiency was analyzed for SBI rate as comparison. Covariates included age, level of immune suppression and history of lupus nephritis. RESULTS: Acquired complement deficiency secondary to SLE-related disease [n = 44] was the most common underlying diagnosis associated with depressed complement levels and were compared to a cohort of primary complement deficient patients [n = 18]. SBI per 100 person-years and cohort demographics were described in parallel. Our logistic regression analysis of pediatric patients with SLE showed low C3 level was temporally associated with having an SBI event. Given equivalent immunosuppression, patients with an SBI had lower C3 levels at the beginning of the observation period relative to patients without SBI. CONCLUSION: Pediatric patients with the diagnosis of SLE can develop very low C3 levels that associate with risk of serious bacterial infection comparable to that of patients with primary complement deficiency. Patients prone to severe complement consumption may particularly be at risk.

Recurrence and Outcomes of Complement-Related Renal Disease After Pediatric Renal Transplantation.

Complement dysregulation is related to different glomerular pathologies. Patients with complement dysregulation have high recurrence risk after transplant; however, with trough-effective therapeutics, renal transplant can be an option for these patients. Here, we present 2 boys with renal disease related to complement dysregulation and their outcomes after renal transplant. Patient 1 had atypical hemolytic uremic syndrome, which was treated with eculizumab before renal transplant; eculizumab therapy was also continued after transplant as preventive therapy. Eculizumab therapy was stopped at year 2 post-transplant. At year 4 post-transplant, his serum creatinine level was 0.87 mg/dL. Patient 2, who had chronic renal disease related to C3 glomerulopathy, was not responsive to eculizumab before renal transplant. At month 4 posttransplant, C3 glomerulopathy recurrence was demonstrated with biopsy, and serum creatinine level was 1.96 mg/dL at this time. Eculizumab was started as a rescue therapy. At year 4 posttransplant, his serum creatinine level was 2.07 mg/dL. In our 2 patients with complement dysregulation, eculizumab was an effective and preventive therapy after renal transplant. However, more studies are needed to understand the long-term efficacy and safety of eculizumab after renal transplant.

Immune complement and coagulation dysfunction in adverse outcomes of SARS-CoV-2 infection.

Understanding the pathophysiology of SARS-CoV-2 infection is critical for therapeutic and public health strategies. Viral-host interactions can guide discovery of disease regulators, and protein structure function analysis points to several immune pathways, including complement and coagulation, as targets of coronaviruses. To determine whether conditions associated with dysregulated complement or coagulation systems impact disease, we performed a retrospective observational study and found that history of macular degeneration (a proxy for complement-activation disorders) and history of coagulation disorders (thrombocytopenia, thrombosis and hemorrhage) are risk factors for SARS-CoV-2-associated morbidity and mortality-effects that are independent of age, sex or history of smoking. Transcriptional profiling of nasopharyngeal swabs demonstrated that in addition to type-I interferon and interleukin-6-dependent inflammatory responses, infection results in robust engagement of the complement and coagulation pathways. Finally, in a candidate-driven genetic association study of severe SARS-CoV-2 disease, we identified putative complement and coagulation-associated loci including missense, eQTL and sQTL variants of critical complement and coagulation regulators. In addition to providing evidence that complement function modulates SARS-CoV-2 infection outcome, the data point to putative transcriptional genetic markers of susceptibility. The results highlight the value of using a multimodal analytical approach to reveal determinants and predictors of immunity, susceptibility and clinical outcome associated with infection.

Complement deficiencies and dysregulation: Pathophysiological consequences, modern analysis, and clinical management.

Complement defects are associated with an enhanced risk of a broad spectrum of infectious as well as systemic or local inflammatory and thrombotic disorders. Inherited complement deficiencies have been described for virtually all complement components but can be mimicked by autoantibodies, interfering with the activity of specific complement components, convertases or regulators. While being rare, diseases related to complement deficiencies are often severe with a frequent but not exclusive manifestation during childhood. Whereas defects of early components of the classical pathway significantly increase the risk of autoimmune disorders, lack of components of the terminal pathway as well as of properdin are associated with an enhanced susceptibility to meningococcal infections. The impaired synthesis or function of C1 inhibitor results in the development of hereditary angioedema (HAE). Furthermore, complement dysregulation causes renal disorders such as atypical hemolytic uremic syndrome (aHUS) or C3 glomerulopathy (C3G) but also age-related macular degeneration (AMD). While paroxysmal nocturnal hemoglobinuria (PNH) results from the combined deficiency of the regulatory complement proteins CD55 and CD59, which is caused by somatic mutation of a common membrane anchor, isolated CD55 or CD59 deficiency is associated with the CHAPLE syndrome and polyneuropathy, respectively. Here, we provide an overview on clinical disorders related to complement deficiencies or dysregulation and describe diagnostic strategies required for their comprehensive molecular characterization - a prerequisite for informed decisions on the therapeutic management of these disorders.

Clinical and Genetic Spectrum of a Large Cohort With Total and Sub-total Complement Deficiencies.

The complement system is crucial for defense against pathogens and the removal of dying cells or immune complexes. Thus, clinical indications for possible complete complement deficiencies include, among others, recurrent mild or serious bacterial infections as well as autoimmune diseases (AID). The diagnostic approach includes functional activity measurements of the classical (CH50) and alternative pathway (AP50) and the determination of the C3 and C4 levels, followed by the quantitative analysis of individual components or regulators. When biochemical analysis reveals the causal abnormality of the complement deficiency (CD), molecular mechanisms remains frequently undetermined. Here, using direct sequencing analysis of the coding region we report the pathogenic variants spectrum that underlie the total or subtotal complement deficiency in 212 patients. We identified 107 different hemizygous, homozygous, or compound heterozygous pathogenic variants in 14 complement genes [C1Qß (n = 1), C1r (n = 3), C1s (n = 2), C2 (n = 12), C3 (n = 5), C5 (n = 12), C6 (n = 9), C7 (n = 17), C8 ß (n = 7), C9 (n = 3), CFH (n = 7), CFI (n = 18), CFP (n = 10), CFD (n = 2)]. Molecular analysis identified 17 recurrent pathogenic variants in 6 genes (C2, CFH, C5, C6, C7, and C8). More than half of the pathogenic variants identified in unrelated patients were also found in healthy controls from the same geographic area. Our study confirms the strong association of meningococcal infections with terminal pathway deficiency and highlights the risk of pneumococcal and auto-immune diseases in the classical and alternative pathways. Results from this large genetic investigation provide evidence of a restricted number of molecular mechanisms leading to complement deficiency and describe the clinical potential adverse events of anti-complement therapy.