Consumption of complement in a 26-year-old woman with severe thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination.

Extremely rare reactions characterized by thrombosis and thrombocytopenia have been described in subjects that received ChAdOx1 nCoV-19 vaccination 5-16 days earlier. Although patients with vaccine-induced thrombotic thrombocytopenia (VITT) have high levels of antibodies to platelet factor 4 (PF4)-polyanion complexes, the exact mechanism of the development of thrombosis is still unknown. Here we reported serum studies as well as proteomics and genomics analyses demonstrating a massive complement activation potentially linked to the presence of anti-PF4 antibodies in a patient with severe VITT. At admission, complement activity of the classical and lectin pathways were absent (0% for both) with normal levels of the alternative pathway (73%) in association with elevated levels of the complement activation marker sC5b-9 (630 ng/mL [n.v. 139-462 ng/mL]) and anti-PF4 IgG (1.918 OD [n.v. 0.136-0.300 OD]). The immunoblotting analysis of C2 showed the complete disappearance of its normal band at 110 kDa. Intravenous immunoglobulin treatment allowed to recover complement activity of the classical pathway (91%) and lectin pathway (115%), to reduce levels of sC5b-9 (135 ng/mL) and anti-PF4 IgG (0.681 OD) and to normalize the C2 pattern at immunoblotting. Proteomics and genomics analyses in addition to serum studies showed that the absence of complement activity during VITT was not linked to alterations of the C2 gene but rather to a strong complement activation leading to C2 consumption. Our data in a single patient suggest monitoring complement parameters in other VITT patients considering also the possibility to target complement activation with specific drugs.

Epistatic interactions of genetic loci associated with age-related macular degeneration.

The currently largest genome-wide association study (GWAS) for age-related macular degeneration (AMD) defines disease association with genome-wide significance for 52 independent common and rare genetic variants across 34 chromosomal loci. Overall, these loci contain over 7200 variants and are enriched for genes with functions indicating several shared cellular processes. Still, the precise mechanisms leading to AMD pathology are largely unknown. Here, we exploit the phenomenon of epistatic interaction to identify seemingly independent AMD-associated variants that reveal joint effects on gene expression. We focus on genetic variants associated with lipid metabolism, organization of extracellular structures, and innate immunity, specifically the complement cascade. Multiple combinations of independent variants were used to generate genetic risk scores allowing gene expression in liver to be compared between low and high-risk AMD. We identified genetic variant combinations correlating significantly with expression of 26 genes, of which 19 have not been associated with AMD before. This study defines novel targets and allows prioritizing further functional work into AMD pathobiology.

Detection of Genetic Rearrangements in the Regulators of Complement Activation RCA Cluster by High-Throughput Sequencing and MLPA.

The regulators of complement activation (RCA) gene cluster in 1q31-1q32 includes most of the genes encoding complement regulatory proteins. Genetic variability in the RCA gene cluster frequently involve copy number variations (CNVs), a type of chromosome structural variation causing alterations in the number of copies of specific regions of DNA. CNVs in the RCA gene cluster often relate with gene rearrangements that result in the generation of novel genes, carrying internal duplications or deletions, and hybrid genes, resulting from the fusion or exchange of genetic material between two different genes. These gene rearrangements are strongly associated with a number of rare and common diseases characterized by complement dysregulation. Identification of CNVs in the RCA gene cluster is critical in the molecular diagnostic of these diseases. It can be done by bioinformatics analysis of DNA sequence data generated by massive parallel sequencing techniques (NGS, next generation sequencing) but often requires special techniques like multiplex ligation-dependent probe amplification (MLPA). This is because the currently used massive parallel DNA sequencing approaches do not easily identify all the structural variations in the RCA gene cluster. We will describe here how to use the MLPA assays and two computational tools to analyze NGS data, NextGENe and ONCOCNV, to detect CNVs and gene rearrangements in the RCA gene cluster.

Trajectory of change in brain complement factors from neonatal to young adult humans.

Immune system components also regulate synapse formation and refinement in neurodevelopment. The complement pathway, associated with cell lysis and phagocytosis, is implicated in synaptic elimination. Aberrant adolescent synaptic pruning may underpin schizophrenia onset; thus, changes in cortical complement activity during human development are of major interest. Complement is genetically linked to schizophrenia via increased C4 copy number variants, but the developmental trajectory of complement expression in the human brain is undetermined. As complement increases during periods of active synaptic engulfment in rodents, we hypothesized that complement expression would increase during postnatal development in humans, particularly during adolescence. Using human postmortem prefrontal cortex, we observed that complement activator (C1QB and C3) transcripts peaked in early neurodevelopment, and were highest in toddlers, declining in teenagers (all ANCOVAs between F = 2.41 -3.325, p = .01-0.05). We found that C4 protein was higher at 1-5 years (H = 16.378, p = .012), whereas C3 protein levels were unchanged with age. The microglial complement receptor subunit CD11b increased in mRNA early in life and peaked in the toddler brain (ANCOVA: pH, F = 4.186, p = .003). Complement inhibitors (CD46 and CD55) increased at school age, but failed to decrease like complement activators (both ANCOVAs, F > 4.4, p < .01). These data suggest the activation of complement in the human prefrontal cortex occurs between 1 and 5 years. We did not find evidence of induction of complement factors during adolescence and instead found increased or sustained levels of complement inhibitor mRNA at maturation. Dysregulation of these typical patterns of complement may predispose the brain to neurodevelopmental disorders such as autism or schizophrenia.

Structural determination of the complement inhibitory domain of Borrelia burgdorferi BBK32 provides insight into classical pathway complement evasion by Lyme disease spirochetes.

The carboxy-terminal domain of the BBK32 protein from Borrelia burgdorferi sensu stricto, termed BBK32-C, binds and inhibits the initiating serine protease of the human classical complement pathway, C1r. In this study we investigated the function of BBK32 orthologues of the Lyme-associated Borrelia burgdorferi sensu lato complex, designated BAD16 from B. afzelii strain PGau and BGD19 from B. garinii strain IP90. Our data show that B. afzelii BAD16-C exhibits BBK32-C-like activities in all assays tested, including high-affinity binding to purified C1r protease and C1 complex, and potent inhibition of the classical complement pathway. Recombinant B. garinii BGD19-C also bound C1 and C1r with high-affinity yet exhibited significantly reduced in vitro complement inhibitory activities relative to BBK32-C or BAD16-C. Interestingly, natively produced BGD19 weakly recognized C1r relative to BBK32 and BAD16 and, unlike these proteins, BGD19 did not confer significant protection from serum killing. Site-directed mutagenesis was performed to convert BBK32-C to resemble BGD19-C at three residue positions that are identical between BBK32 and BAD16 but different in BGD19. The resulting chimeric protein was designated BXK32-C and this BBK32-C variant mimicked the properties observed for BGD19-C. To query the disparate complement inhibitory activities of BBK32 orthologues, the crystal structure of BBK32-C was solved to 1.7Å limiting resolution. BBK32-C adopts an anti-parallel four-helix bundle fold with a fifth alpha-helix protruding from the helical core. The structure revealed that the three residues targeted in the BXK32-C chimera are surface-exposed, further supporting their potential relevance in C1r binding and inhibition. Additional binding assays showed that BBK32-C only recognized C1r fragments containing the serine protease domain. The structure-function studies reported here improve our understanding of how BBK32 recognizes and inhibits C1r and provide new insight into complement evasion mechanisms of Lyme-associated spirochetes of the B. burgdorferi sensu lato complex.

Complement Components, C3 and C4, and the Metabolic Syndrome.

INTRODUCTION: Increased systemic inflammation plays a significant role in the development of adult cardiometabolic diseases such as insulin resistance, dyslipidemia, atherosclerosis, and hypertension. The complement system is a part of the innate immune system and plays a key role in the regulation of inflammation. Of particular importance is the activation of complement components C3 and C4. C3 is produced primarily by the liver but is also produced in adipocytes, macrophages and endothelial cells, all of which are present in adipose tissues. Dietary fat and chylomicrons stimulate C3 production. Adipocytes in addition to producing C3 also have receptors for activated C3 and other complement components and thus also respond to as well as produce a target for complement. C3adesArg, also known as acylation stimulation factor, increases adipocyte triglyceride synthesis and release. These physiological effects play a significant role in the development of metabolic syndrome. Epidemiologically, obese adults and non-obese adults with cardiometabolic disease who are not obese have been shown to have increased complement levels. C4 levels also correlate with body mass index. Genetically, specific C3 polymorphisms have been shown to predict future cardiovascular events and. D decreased C4 long gene copy number is associated with increased longevity. CONCLUSION: Future research is clearly needed to clarify the role of complement in the development of cardiovascular disease and mechanisms for its action. The complement system may provide a new area for intervention in the prevention of cardiometabolic diseases.

Inherited Classical and Alternative Pathway Complement Deficiencies in Children: A Single Center Experience.

BACKGROUND: Primary complement deficiencies are rare diseases. OBJECTIVE: To retrospectively evaluate the clinical and laboratory findings and complications of patients to increase awareness of pediatricians about complement deficiencies, which are rarely encountered. METHODS: In this study, the clinical and immunological characteristics of 21 patients who consulted the Immunology Department of our hospital between 2003 and 2017 and were diagnosed with classical or alternative pathway complement deficiency were obtained from the file records. RESULTS: Ten patients with C1 inhibitor deficiency, four patients with factor I deficiency, three patients with properdin deficiency, two patients with C8 deficiency, one patient with C1q deficiency, and one patient with C4B deficiency were assessed. The mean age of the patients at diagnosis was 11.4±4.7 years, the age of onset of symptoms was 7.9±3.9 years, and the follow-up period was 6.7±3.9 years. Fourteen cases had a similar medical history in the family. All patients with C1q, factor I, properdin, C8, and C4B deficiencies presented with an infection, and vasculitic rash was present in two patients with factor I deficiency. In addition, immune complex glomerulonephritis was present in one patient with factor I deficiency. Meningococcal, Haemophilus influenzae type B, and pneumococcal vaccines were administered and prophylactic antibiotic treatment was initiated in all patients except patients with C1 inhibitor deficiency. CONCLUSIONS: Early diagnosis of complement deficiencies can facilitate prevention of life-threatening complications such as severe bacterial infections by considering prophylactic antibiotics and vaccines. In patients with C1 inhibitor deficiency, achieving an acurate early diagnosis will assist in the management and timely treatment of life-threatening attacks such as upper airway obstruction and improve outcomes.

A rare case of Alport syndrome, atypical hemolytic uremic syndrome and Pauci-immune crescentic glomerulonephritis.

BACKGROUND: Renal thrombotic microangiopathy (TMA) is occasionally seen in biopsies with pauci-immune necrotizing crescentic glomerulonephritis (PCGN). Recent study indicated that the complement activation is more prominent in the ANCA-negative glomerulonephritis. CASE PRESENTATION: We report a case of concurrent TMA and PCGN without ANCA positivity. Interestingly, our patient also had biopsy features supportive of Alport syndrome (AS). Genetic studies identified variants and polymorphisms in alternative complement pathway genes that confer substantial risk of developing atypical hemolytic uremic syndrome (aHUS). CONCLUSIONS: Abnormal activation in complement pathway may represent a common pathogenic link between these three distinct entities.

C1q restrains autoimmunity and viral infection by regulating CD8+ T cell metabolism.

Deficiency of C1q, the initiator of the complement classical pathway, is associated with the development of systemic lupus erythematosus (SLE). Explaining this association in terms of abnormalities in the classical pathway alone remains problematic because C3 deficiency does not predispose to SLE. Here, using a mouse model of SLE, we demonstrate that C1q, but not C3, restrains the response to self-antigens by modulating the mitochondrial metabolism of CD8+ T cells, which can themselves propagate autoimmunity. C1q deficiency also triggers an exuberant effector CD8+ T cell response to chronic viral infection leading to lethal immunopathology. These data establish a link between C1q and CD8+ T cell metabolism and may explain how C1q protects against lupus, with implications for the role of viral infections in the perpetuation of autoimmunity.

Genetic testing of complement and coagulation pathways in patients with severe hypertension and renal microangiopathy.

A diagnosis of thrombotic microangiopathy on kidney biopsy in a patient presenting with hypertensive emergency has historically elicited the diagnosis of malignant hypertension-associated thrombotic microangiopathy. Recent studies, however, have raised awareness that a number of these patients may actually represent atypical hemolytic uremic syndrome. To further investigate this premise, we performed next-generation sequencing to interrogate the coding regions of 29 complement and coagulation cascade genes associated with atypical hemolytic uremic syndrome in 100 non-elderly patients presenting with severe hypertension, renal failure and a kidney biopsy showing microangiopathic changes limited to the classic accelerated hypertension-associated lesion of arterial intimal edema ('mucoid intimal hyperplasia') in isolation and without accompanying glomerular microthrombi. No pathogenic or likely pathogenic variants were identified in any of the genes analyzed, although 13 patients had rare variants of uncertain significance predicted to be deleterious by all in-silico prediction methods utilized. Accordingly, this large patient cohort showed no definitive burden of disease secondary to genetic variants involving complement or coagulation pathways, which contrasts sharply with the high frequency of similar mutational events reported for atypical hemolytic uremic syndrome. Our results also inform recent data by suggesting that patients who present with severe or malignant hypertension and renal thrombotic microangiopathy may be at higher risk for atypical hemolytic uremic syndrome only if the biopsy shows more active disease that includes glomerular fibrin thrombi.

Enhanced classical complement pathway activation and altered phagocytosis signaling molecules in human epilepsy.

Microglia-mediated neuroinflammation is widely associated with seizures and epilepsy. Although microglial cells are professional phagocytes, less is known about the status of this phenotype in epilepsy. Recent evidence supports that phagocytosis-associated molecules from the classical complement (C1q-C3) play novel roles in microglia-mediated synaptic pruning. Interestingly, in human and experimental epilepsy, altered mRNA levels of complement molecules were reported. Therefore, to identify a potential role for complement and microglia in the synaptodendritic pathology of epilepsy, we determined the protein levels of classical complement proteins (C1q-C3) along with other phagocytosis signaling molecules in human epilepsy. Cortical brain samples surgically resected from patients with refractory epilepsy (RE) and non-epileptic lesions (NE) were examined. Western blotting was used to determine the levels of phagocytosis signaling proteins such as the complements C1q and C3, MerTK, Trem2, and Pros1 along with cleaved-caspase 3. In addition, immunostaining was used to determine the distribution of C1q and co-localization to microglia and dendrites. We found that the RE samples had significantly increased protein levels of C1q (p=0.034) along with those of its downstream activation product iC3b (p=0.027), and decreased levels of Trem2 (p=0.045) and Pros1 (p=0.005) when compared to the NE group. Protein levels of cleaved-caspase 3 were not different between the groups (p=0.695). In parallel, we found C1q localization to microglia and dendrites in both NE and RE samples, and also observed substantial microglia-dendritic interactions in the RE tissue. These data suggest that aberrant phagocytic signaling occurs in human refractory epilepsy. It is likely that alteration of phagocytic pathways may contribute to unwanted elimination of cells/synapses and/or impaired clearance of dead cells. Future studies will investigate whether altered complement signaling contributes to the hyperexcitability that result in epilepsy.

From compliment to insult: genetics of the complement system in physiology and disease in the human retina.

Age-related macular degeneration (AMD) is a major cause of visual impairment that affects the central retina. Genome wide association studies and candidate gene screens have identified members of the complement pathway as contributing to the risk of AMD. In this review, we discuss the complement system, its importance in retinal development and normal physiology, how its dysregulation may contribute to disease, and how it might be targeted to prevent damage to the aging choriocapillaris in AMD.

Gene expression profiling in bladder cancer identifies potential therapeutic targets.

Despite advances in management, bladder cancer remains a major cause of cancer related complications. Characterisation of gene expression patterns in bladder cancer allows the identification of pathways involved in its pathogenesis, and may stimulate the development of novel therapies targeting these pathways. Between 2004 and 2005, cystoscopic bladder biopsies were obtained from 19 patients and 11 controls. These were subjected to whole transcript-based microarray analysis. Unsupervised hierarchical clustering was used to identify samples with similar expression profiles. Hypergeometric analysis was used to identify canonical pathways and curated networks having statistically significant enrichment of differentially expressed genes. Osteopontin (OPN) expression was validated by immunohistochemistry. Hierarchical clustering defined signatures, which differentiated between cancer and healthy tissue, muscle-invasive or non-muscle invasive cancer and healthy tissue, grade 1 and grade 3. Pathways associated with cell cycle and proliferation were markedly upregulated in muscle-invasive and grade 3 cancers. Genes associated with the classical complement pathway were downregulated in non-muscle invasive cancer. Osteopontin was markedly overexpressed in invasive cancer compared to healthy tissue. The present study contributes to a growing body of work on gene expression signatures in bladder cancer. The data support an important role for osteopontin in bladder cancer, and identify several pathways worthy of further investigation.

A review of human diseases caused or exacerbated by aberrant complement activation.

Complement is the backbone of our innate immune system. It is of ancient evolutionary origin, being traced back to horseshoe crabs 350 million years ago. It consists today of more than 25 proteins which must work together like clockwork to distinguish friend from foe. Self-attack by the complement system can occur whenever it fails to do so. This failure has been reported to occur in an estimated 22 human diseases. A significant number of these are chronic degenerative neurological disorders. In some, there is overwhelming evidence that complement self-attack causes the disease. In many others, it is considered only to contribute to the overall pathology. Finding effective therapeutic agents should be a high priority for medical research. To date, the monoclonal antibody eculizumab is the only approved agent. Molecules under development include other monoclonal antibodies directed at C5, C3, and properdin, various aptamers to C3, and small molecules that are orally available.

Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids.

PURPOSE: Variations in mitochondrial DNA (mtDNA) and abnormalities in the complement pathways have been implicated in the pathogenesis of age-related macular degeneration (AMD). This study was designed to determine the effects of mtDNA from AMD subjects on the complement pathway. METHODS: Transmitochondrial cybrids were prepared by fusing platelets from AMD and age-matched Normal subjects with Rho0 (lacking mtDNA) human ARPE-19 cells. Quantitative PCR and Western blotting were performed to examine gene and protein expression profiles, respectively, of complement markers in these cybrids. Bioenergetic profiles of Normal and AMD cybrids were examined using the Seahorse XF24 flux analyzer. RESULTS: Significant decreases in the gene and protein expression of complement inhibitors, along with significantly higher levels of complement activators, were found in AMD cybrids compared to Older-Normal cybrids. Seahorse flux data demonstrated that the bioenergetic profiles for Older-Normal and Older-AMD cybrid samples were similar to each other but were lower compared to Young-Normal cybrid samples. CONCLUSION: In summary, since all cybrids had identical nuclei and differed only in mtDNA content, the observed changes in components of complement pathways can be attributed to mtDNA variations in the AMD subjects, suggesting that mitochondrial genome and retrograde signaling play critical roles in this disease. Furthermore, the similar bioenergetic profiles of AMD and Older-Normal cybrids indicate that the signaling between mitochondria and nuclei are probably not via a respiratory pathway.

C1q-targeted inhibition of the classical complement pathway prevents injury in a novel mouse model of acute motor axonal neuropathy.

INTRODUCTION: Guillain-Barré syndrome (GBS) is an autoimmune disease that results in acute paralysis through inflammatory attack on peripheral nerves, and currently has limited, non-specific treatment options. The pathogenesis of the acute motor axonal neuropathy (AMAN) variant is mediated by complement-fixing anti-ganglioside antibodies that directly bind and injure the axon at sites of vulnerability such as nodes of Ranvier and nerve terminals. Consequently, the complement cascade is an attractive target to reduce disease severity. Recently, C5 complement component inhibitors that block the formation of the membrane attack complex and subsequent downstream injury have been shown to be efficacious in an in vivo anti-GQ1b antibody-mediated mouse model of the GBS variant Miller Fisher syndrome (MFS). However, since gangliosides are widely expressed in neurons and glial cells, injury in this model was not targeted exclusively to the axon and there are currently no pure mouse models for AMAN. Additionally, C5 inhibition does not prevent the production of early complement fragments such as C3a and C3b that can be deleterious via their known role in immune cell and macrophage recruitment to sites of neuronal damage. RESULTS AND CONCLUSIONS: In this study, we first developed a new in vivo transgenic mouse model of AMAN using mice that express complex gangliosides exclusively in neurons, thereby enabling specific targeting of axons with anti-ganglioside antibodies. Secondly, we have evaluated the efficacy of a novel anti-C1q antibody (M1) that blocks initiation of the classical complement cascade, in both the newly developed anti-GM1 antibody-mediated AMAN model and our established MFS model in vivo. Anti-C1q monoclonal antibody treatment attenuated complement cascade activation and deposition, reduced immune cell recruitment and axonal injury, in both mouse models of GBS, along with improvement in respiratory function. These results demonstrate that neutralising C1q function attenuates injury with a consequent neuroprotective effect in acute GBS models and promises to be a useful new target for human therapy.

Co-ordinated expression of innate immune molecules during mouse neurulation.

The innate immune system is the first line of defence against pathogens and infection. Recently, it has become apparent that many innate immune factors have roles outside of immunity and there is growing evidence that these factors play important functional roles during the development of a range of model organisms. Several studies have documented developmental expression of individual factors of the toll-like receptor and complement systems, and we recently demonstrated a key role for complement C5a receptor (C5aR1) signalling in neural tube closure in mice. Despite these emerging studies, a comprehensive expression analysis of these molecules in embryonic development is lacking. In the current study, we therefore, examined the expression of key innate immune factors in the early development period of neurulation (7.5-10.5dpc) in mice. We found that complement factor genes were differentially expressed during this period of murine development. Interestingly, the expression patterns we identified preclude activation of the classical and alternative pathways and formation of the membrane attack complex. Additionally, several other classes of innate immune molecules were expressed during the period of neurulation, including toll-like receptors (TLR-2, -3, -4 and -9), receptor for advanced glycation end-products (RAGE), and their signalling adapters (TRAF-4, TRAF-6, TAK-1 and MyD88). Taken together, this study highlights a number of innate immune factors as potential novel players in early embryonic development.

Classical pathway deficiencies - A short analytical review.

Deficiencies in the classical pathway of complement activation have some common features but show also great differences. Deficiencies of each of the components (C1q, C1s, C1r, C4 and C2) imply increased susceptibility to bacterial infections. They are also associated with increased risk to develop systemic lupus erythematosus where deficiency of C1q is strongly associated to the disease while C4 less and C2 much less. Deficiency of C1q affects only activation of the classical pathway while deficiency of C4 and C2 also prevent activation of the lectin pathway. Bypass mechanisms may result in complement activation also in absence of C2 but not in absence of C1q or C4. The genes for C2 and C4 isotypes are closely located within the MHC class III region on chromosome 6p and the genes for the 3 C1q chains are on chromosome 1p. Deficiencies of C1q and of C4 show genetic heterogeneity while deficiency of C2 in the great majority of cases is caused by a specific deletion. The production of C4 and C2 is mainly by the hepatocytes in the liver while C1q is produced by monocytic bone marrow derived cells. This has implications for the possibility to treat the deficiency and hematopoietic stem cell transplantation has been tried in C1q deficiency.