Glycogenolysis-Induced Astrocytic Serping1 Expression Regulates Neuroinflammatory Effects on Hippocampal neuron.

The bacterial pathogen, lipopolysaccharide (LPS), elicits microglial response and induces cytokine secretion that subsequently activates astrocytes. Recent findings have indicated that LPS-induced activation of postnatal glial cells has led to alterations in synapse formation in hippocampal and cortical neurons, thereby resulting in a prolonged increased risk for seizure or depression. Nevertheless, its mechanisms remain to be fully elucidated. Cellular metabolism has recently gained recognition as a critical regulatory mechanism for the activation of peripheral immune cells, as it supplies the requisite energy and metabolite for their activation. In the present study, we report that LPS did not change the expression of reported astrocyte-derived synaptogenic genes in the postnatal hippocampus; however, it induced upregulation of astrocytic complement component regulator Serping1 within the postnatal hippocampus. As a regulatory mechanism, activation of glycogen degradation (glycogenolysis) governs the expression of a subset of inflammatory-responsive genes including Serping1 through reactive oxygen species (ROS)-NF-κB axis. Our study further demonstrated that glycogenolysis is implicated in neurotoxic phenotypes of astrocytes, such as impaired neuronal synaptogenesis or cellular toxicity. These findings suggested that activation of glycogenolysis in postnatal astrocytes is an essential metabolic pathway for inducing responses in inflammatory astrocytes.

MASP-2 deficiency does not prevent the progression of diabetic kidney disease in a mouse model of type 1 diabetes.

Mannan-binding lectin (MBL) initiates the lectin pathway of complement and has been linked to albuminuria and mortality in diabetes. We hypothesize that MBL-associated serine protease 2 (MASP-2) deficiency will protect against diabetes-induced kidney damage. Male C57BL/6J MASP-2 knockout (Masp2-/-) mice and wildtype (WT) mice were divided into a diabetic group and a non-diabetic group. Renal hypertrophy, albumin excretion, mesangial area and specific mRNA expressions in the renal cortex were measured after 8 and 12 weeks of diabetes. By two-way ANOVA it was tested if MASP-2 modulated the renal effects of diabetes, that is interaction. After 12 weeks of diabetes Masp2-/- diabetic mice had a smaller mesangium at 21.1% of the glomerular area (95% CI 19.7, 22.6) compared with WT diabetic mice, 25.2% (23.2, 27.2), p(interaction) = 0.001. After 8 weeks of diabetes, plasma cystatin C was 261.5 ng/mL (229.6, 297.8) in the WT diabetic group compared to 459.9 ng/mL (385.7, 548.3) in non-diabetic WT mice, p < 0.001. By contrast, no difference in plasma cystatin C levels was found between the Masp2-/- diabetic mice, 288.2 ng/mL (260.6, 318.6) and Masp2-/- non-diabetic mice, 293.5 ng/mL (221.0, 389.7), p = 0.86 and p(interaction) = 0.001. We demonstrated a protective effect of MASP-2 deficiency on mesangial hypertrophy after 12 weeks of diabetes and an effect on plasma cystatin C level. MASP-2 deficiency did, however, fail to protect against diabetic-induced alterations of kidney weight, albuminuria and renal mRNA expression of fibrotic- and oxidative stress markers.

The complement system as a target in cancer immunotherapy.

Malignant cells are part of a complex network within the tumor microenvironment, where their interaction with host cells and soluble mediators, including complement components, is pivotal. The complement system, known for its role in immune defense and homeostasis, exhibits a dual effect on cancer progression. This dichotomy arises from its antitumoral opsonophagocytosis and cytotoxicity versus its protumoral chronic inflammation mediated by the C5a/C5aR1 axis, influencing antitumor T-cell responses. Recent studies have revealed distinct co-expression patterns of complement genes in various cancer types, correlating with prognosis. Notably, some cancers exhibit co-regulated overexpression of complement genes associated with poor prognosis, while others show favorable outcomes. However, significant intra-patient heterogeneity further complicates this classification. Moreover, the involvement of locally produced and intracellular complement proteins adds complexity to the tumor microenvironment dynamics. This review highlights the unique interplay of complement components within different cancers and patient cohorts, showing that "one size does not fit all", for complement in cancer. It summarizes the clinical trials for complement targeting in cancer, emphasizing the need for tailored therapeutic approaches. By elucidating the mechanistic basis of complement's context-dependent role, this review aims to facilitate the development of personalized cancer therapies, ultimately improving patient care and outcomes.

Mass spectrometry-based proteomic profiling of extracellular vesicle proteins in diabetic and non-diabetic ischemic stroke patients: a case-control study.

Acute ischemic stroke is the most common cause of neurologic dysfunction caused by focal brain ischemia and tissue injury. Diabetes is a major risk factor of stroke, exacerbating disease management and prognosis. Therefore, discovering new diagnostic markers and therapeutic targets is critical for stroke prevention and treatment. Extracellular vesicles (EVs), with their distinctive properties, have emerged as promising candidates for biomarker discovery and therapeutic application. This case-control study utilized mass spectrometry-based proteomics to compare EVs from non-diabetic stroke (nDS = 14), diabetic stroke (DS = 13), and healthy control (HC = 12) subjects. Among 1288 identified proteins, 387 were statistically compared. Statistical comparisons using a general linear model (log2 foldchange ≥0.58 and FDR-p≤0.05) were performed for nDS vs HC, DS vs HC, and DS vs nDS. DS vs HC and DS vs nDS comparisons produced 123 and 149 differentially expressed proteins, respectively. Fibrinogen gamma chain (FIBG), Fibrinogen beta chain (FIBB), Tetratricopeptide repeat protein 16 (TTC16), Proline rich 14-like (PR14L), Inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKKE), Biorientation of chromosomes in cell division protein 1-like 1 (BD1L1), and protein PR14L exhibited significant differences in the DS group. The pathway analysis revealed that the complement system pathways were activated, and blood coagulation and neuroprotection were inhibited in the DS group (z-score ≥2; p ≤ 0.05). These findings underscore the potential of EVs proteomics in identifying biomarkers for stroke management and prevention, warranting further clinical investigation.

The Relationship between the complement system and subclinical carotid atherosclerosis in patients with rheumatoid arthritis.

BACKGROUND: Patients with rheumatoid arthritis (RA) have an increased risk of cardiovascular (CV) events and CV mortality. Subclinical carotid atherosclerosis is independently associated with rates of incident CV events among patients with RA. The complement system has been related to both the etiopathogenesis of RA and CV disease. In this study, we aimed to evaluate the association between a comprehensive assessment of the complement system and carotid intima media thickness and carotid plaque in patients with RA. METHODS: 430 patients with RA were recruited. Functional assays of the three pathways of the complement system, utilizing new-generation techniques, were assessed. Additionally, serum levels of individual components of the complement system belonging to the three pathways were measured: C1q (classical), lectin (lectin), C2, C4, and C4b (classical and lectin), factor D and properdin (alternative), C3 and C3a (common), C5, C5a, and C9 (terminal), as well as regulators factor I and C1-inhibitor. Subclinical carotid atherosclerosis was evaluated by ultrasonography. Multivariable linear regression analysis was conducted to investigate the association between the complement system and carotid intima media thickness and carotid plaque. RESULTS: After multivariable adjustment, which included traditional CV risk factors and disease-related data, C3a and C5a exhibited significant positive correlations with carotid intima media thickness. Additionally, higher values of C1-inhibitor, properdin, C3, C5, and C5a were independently associated with the presence of carotid plaque. CONCLUSION: The complement system and subclinical carotid atherosclerosis are linked in patients with RA.

Proangiogenic properties of complement protein C1q can contribute to endometriosis.

Endometriosis (EM) is defined as the engraftment and proliferation of functional endometrial-like tissue outside the uterine cavity, leading to a chronic inflammatory condition. While the precise etiology of EM remains elusive, recent studies have highlighted the crucial involvement of a dysregulated immune system. The complement system is one of the predominantly altered immune pathways in EM. Owing to its involvement in the process of angiogenesis, here, we have examined the possible role of the first recognition molecule of the complement classical pathway, C1q. C1q plays seminal roles in several physiological and pathological processes independent of complement activation, including tumor growth, placentation, wound healing, and angiogenesis. Gene expression analysis using the publicly available data revealed that C1q is expressed at higher levels in EM lesions compared to their healthy counterparts. Immunohistochemical analysis confirmed the presence of C1q protein, being localized around the blood vessels in the EM lesions. CD68+ macrophages are the likely producer of C1q in the EM lesions since cultured EM cells did not produce C1q in vitro. To explore the underlying reasons for increased C1q expression in EM, we focused on its established pro-angiogenic role. Employing various angiogenesis assays on primary endothelial endometriotic cells, such as migration, proliferation, and tube formation assays, we observed a robust proangiogenic effect induced by C1q on endothelial cells in the context of EM. C1q promoted angiogenesis in endothelial cells isolated from EM lesions (as well as healthy ovary that is also rich in C1q). Interestingly, endothelial cells from EM lesions seem to overexpress the receptor for the globular heads of C1q (gC1qR), a putative C1q receptor. Experiments with siRNA to silence gC1qR resulted in diminished capacity of C1q to perform its angiogenic functions, suggesting that C1q is likely to engage gC1qR in the pathophysiology of EM. gC1qR can be a potential therapeutic target in EM patients that will disrupt C1q-mediated proangiogenic activities in EM.

Systematic review of the complement components as potential biomarkers of pre-eclampsia: pitfalls and opportunities.

The complement system (C) is a crucial component of the innate immune system. An increasing body of research has progressively shed light on the pivotal role of C in immunological tolerance at the feto-maternal interface. Excessive C activation or impaired C regulation may determine the onset of pregnancy-related pathological conditions, including pre-eclampsia (PE). Thus, several studies have investigated the presence of C components or split products in blood matrixes (i.e., plasma, serum), urine, and amniotic fluid in PE. In the current study, we systematically reviewed the currently available scientific literature reporting measurements of C components as circulating biomarkers in PE, based on a literature search using Pubmed, Scopus, and Embase databases. A total of 41 out of 456 studies were selected after full-text analysis. Fourteen studies (34.1%) were identified as measuring the blood concentrations of the classical pathway, 5 (12.1%) for the lectin pathway, 28 (68.3%) for the alternative pathway, 17 (41.5%) for the terminal pathway components, and 16 (39%) for C regulators. Retrieved results consistently reported C4, C3, and factor H reduction, and increased circulating levels of C4d, Bb, factor D, C3a, C5a, and C5b-9 in PE compared to normal pregnancies, depicting an overall scenario of excessive C activation and aberrant C regulation. With evidence of C activation and dysregulation, C-targeted therapy is an intriguing perspective in PE management. Moreover, we also discussed emerging pitfalls in C analysis, mainly due to a lack of experimental uniformity and biased cohort selection among different studies and laboratories, aiming to raise a more comprehensive awareness for future standardization. Systematic review registration: https://www.crd.york.ac.uk/prospero/, identifier CRD42024503070.

Concomitant sickle cell disease and systemic lupus erythematosus: A single-center case series.

Sickle cell disease (SCD) and systemic lupus erythematosus (SLE) are two uncommon disorders each characterized by multisystemic manifestations. Individuals with SCD exhibit abnormalities in the complement pathway, which may predispose patients to develop autoimmune disorders such as SLE. As many manifestations of SLE mimic those of SCD, diagnosis and therapeutic management of SLE in a patient with known SCD may be delayed. In this study, we describe our institutional experience of diagnosing and managing concomitant SCD and SLE. We offer insights into the complex interplay between these conditions to enhance early recognition and effective management of concurrent SCD and SLE.

Association between circulatory complement activation and hypertensive renal damage: a case-control study.

OBJECTIVE: The aim of this study was to investigate the potential importance of complement system activation, with particular emphasis on the complement alternative pathway (AP), in the pathogenesis of hypertensive renal damage. METHODS: Serum complement C3, complement Factor H (CFH) and AP activation were assessed in 66 participants with established essential hypertension with renal damage (RD). Fifty-nine patients with age- and sex-matched essential hypertension without renal damage (NRD) and 58 healthy participants (normal) were selected. RESULTS: Our study revealed that C3 and AP50 continuously increased from normal to NRD to RD (p < 0.05, respectively), while CFH was significantly lower than that in NRD and healthy participants (p < 0.05, respectively). After multifactorial logistic regression analysis corrected for confounders, elevated serum C3 (p = 0.001) and decreased CFH (p < 0.001) were found to be independent risk factors for hypertension in healthy participants; elevated serum C3 (p = 0.034), elevated AP50 (p < 0.001), decreased CFH (p < 0.001), increased age (p = 0.011) and increased BMI (p = 0.013) were found to be independent risk factors for the progression of hypertension to hypertensive renal damage; elevated serum C3 (p = 0.017), elevated AP50 (p = 0.023), decreased CFH (p = 0.005) and increased age (p = 0.041) were found to be independent risk factors for the development of hypertensive renal damage in healthy participants. CONCLUSION: Abnormal activation of complement, particularly complement AP, may be a risk factor for the development and progression of hypertensive renal damage.

The Complement System as a Therapeutic Target in Retinal Disease.

The complement cascade is a vital system in the human body's defense against pathogens. During the natural aging process, it has been observed that this system is imperative for ensuring the integrity and homeostasis of the retina. While this system is critical for proper host defense and retinal integrity, it has also been found that dysregulation of this system may lead to certain retinal pathologies, including geographic atrophy and diabetic retinopathy. Targeting components of the complement system for retinal diseases has been an area of interest, and in vivo, ex vivo, and clinical trials have been conducted in this area. Following clinical trials, medications targeting the complement system for retinal disease have also become available. In this manuscript, we discuss the pathophysiology of complement dysfunction in the retina and specific pathologies. We then describe the results of cellular, animal, and clinical studies targeting the complement system for retinal diseases. We then provide an overview of complement inhibitors that have been approved by the Food and Drug Administration (FDA) for geographic atrophy. The complement system in retinal diseases continues to serve as an emerging therapeutic target, and further research in this field will provide additional insights into the mechanisms and considerations for treatment of retinal pathologies.

Double-Edged Sword: Exploring the Mitochondria-Complement Bidirectional Connection in Cellular Response and Disease.

Mitochondria serve an ultimate purpose that seeks to balance the life and death of cells, a role that extends well beyond the tissue and organ systems to impact not only normal physiology but also the pathogenesis of diverse diseases. Theorized to have originated from ancient proto-bacteria, mitochondria share similarities with bacterial cells, including their own circular DNA, double-membrane structures, and fission dynamics. It is no surprise, then, that mitochondria interact with a bacterium-targeting immune pathway known as a complement system. The complement system is an ancient and sophisticated arm of the immune response that serves as the body's first line of defense against microbial invaders. It operates through a complex cascade of protein activations, rapidly identifying and neutralizing pathogens, and even aiding in the clearance of damaged cells and immune complexes. This dynamic system, intertwining innate and adaptive immunity, holds secrets to understanding numerous diseases. In this review, we explore the bidirectional interplay between mitochondrial dysfunction and the complement system through the release of mitochondrial damage-associated molecular patterns. Additionally, we explore several mitochondria- and complement-related diseases and the potential for new therapeutic strategies.

Revisiting the interaction between complement lectin pathway protease MASP-2 and SARS-CoV-2 nucleoprotein.

Complement activation is considered to contribute to the pathogenesis of severe SARS-CoV-2 infection, mainly by generating potent immune effector mechanisms including a strong inflammatory response. Involvement of the lectin complement pathway, a major actor of the innate immune anti-viral defense, has been reported previously. It is initiated by recognition of the viral surface Spike glycoprotein by mannose-binding lectin (MBL), which induces activation of the MBL-associated protease MASP-2 and triggers the proteolytic complement cascade. A role for the viral nucleoprotein (N) has also been reported, through binding to MASP-2, leading to protease overactivation and potentiation of the lectin pathway. In the present study, we reinvestigated the interactions of the SARS-CoV-2 N protein, produced either in bacteria or secreted by mammalian cells, with full-length MASP-2 or its catalytic domain, in either active or proenzyme form. We could not confirm the interaction of the N protein with the catalytic domain of MASP-2 but observed N protein binding to proenzyme MASP-2. We did not find a role of the N protein in MBL-mediated activation of the lectin pathway. Finally, we showed that incubation of the N protein with MASP-2 results in proteolysis of the viral protein, an observation that requires further investigation to understand a potential functional significance in infected patients.

Anti-C1q antibodies: a biomarker for diagnosis and management of lupus nephritis. A narrative review.

Nephritis is a frequent and severe complication of Systemic Lupus Erythematous (SLE). The clinical course of lupus nephritis (LN) is usually characterized by alternating phases of remission and exacerbation. Flares of LN can lead to deterioration of kidney function, necessitating timely diagnosis and therapy. The presence of autoantibodies against C1q (anti-C1qAb) in the sera of SLE patients has been reported in various studies. Some research suggests that the presence and changes in the titer of anti-C1qAb may be associated with the development of LN, as well as with LN activity and renal flares. However, the exact role of anti-C1qAb in LN remains a subject of debate. Despite variability in the results of published studies, anti-C1qAb hold promise as noninvasive markers for assessing LN activity in SLE patients. Measuring anti-C1qAb levels could aid in diagnosing and managing LN during periods of both inactive disease and renal flares. Nevertheless, larger controlled trials with standardized laboratory assays are necessary to further establish the utility of anti-C1qAb in predicting the reactivation and remission of LN and guiding treatment strategies.

Sphingolipid Signaling and Complement Activation in Glioblastoma: A Promising Avenue for Therapeutic Intervention.

Glioblastoma is the most common and aggressive type of malignant brain tumor with a poor prognosis due to the lack of effective treatment options. Therefore, new treatment options are required. Sphingolipids are essential components of the cell membrane, while complement components are integral to innate immunity, and both play a critical role in regulating glioblastoma survival signaling. This review focuses on recent studies investigating the functional roles of sphingolipid metabolism and complement activation signaling in glioblastoma. It also discusses how targeting these two systems together may emerge as a novel therapeutic approach.

Targeting autoimmune mechanisms by precision medicine in Myasthenia Gravis.

Myasthenia Gravis (MG) is a chronic disabling autoimmune disease caused by autoantibodies to the neuromuscular junction (NMJ), characterized clinically by fluctuating weakness and early fatigability of ocular, skeletal and bulbar muscles. Despite being commonly considered a prototypic autoimmune disorder, MG is a complex and heterogeneous condition, presenting with variable clinical phenotypes, likely due to distinct pathophysiological settings related with different immunoreactivities, symptoms' distribution, disease severity, age at onset, thymic histopathology and response to therapies. Current treatment of MG based on international consensus guidelines allows to effectively control symptoms, but most patients do not reach complete stable remission and require life-long immunosuppressive (IS) therapies. Moreover, a proportion of them is refractory to conventional IS treatment, highlighting the need for more specific and tailored strategies. Precision medicine is a new frontier of medicine that promises to greatly increase therapeutic success in several diseases, including autoimmune conditions. In MG, B cell activation, antibody recycling and NMJ damage by the complement system are crucial mechanisms, and their targeting by innovative biological drugs has been proven to be effective and safe in clinical trials. The switch from conventional IS to novel precision medicine approaches based on these drugs could prospectively and significantly improve MG care. In this review, we provide an overview of key immunopathogenetic processes underlying MG, and discuss on emerging biological drugs targeting them. We also discuss on future direction of research to address the need for patients' stratification in endotypes according with genetic and molecular biomarkers for successful clinical decision making within precision medicine workflow.

The role of MASP1 in the complement system and expression characteristics in response to GCRV infection in grass carp.

The grass carp (Ctenopharyngodon idella) constitutes a significant economic resource within the aquaculture sector of our nation, yet it has been chronically afflicted by the Grass Carp Reovirus (GCRV) disease. The complement system, a vital component of fish's innate immunity, plays a crucial role in combating viral infections. This research investigates the potential role of MASP1, a key molecule in the lectin pathway of the complement system, in the GCRV infection in grass carp. An analysis of the molecular characteristics of MASP1 in grass carp revealed that its identity and similarity percentages range from 35.10 to 91.00 % and 35.30-91.00 %, respectively, in comparison to other species. Phylogenetically, MASP1 in C. idella aligns closely with species such as Danio rerio, Cyprinus carpio, and Carassius carassius, exhibiting chromosomal collinearity with the zebrafish. Subsequent tissue analysis in both healthy and GCRV-infected grass carp indicated that MASP1's basal expression was predominantly in the liver. Post-GCRV infection, MASP1 expression in various tissues exhibited temporal variations: peaking in the liver on day 5, spleen on day 7, and kidney on day 14. Furthermore, employing Complement Component 3 (C3) as a benchmark for complement system activation, it was observed that MASP1 could activate and cleave C3 to C3b. MASP1 also demonstrated an inhibitory effect on GCRV replication (compared with the control group, VP2 and VP7 decreased by 6.82-fold and 4.37-fold) and enhanced the expression of antiviral genes, namely IRF3, IRF7 and IFN1 (compared with the control group, increased 2.25-fold, 45.38-fold and 22.37-fold, respectively). In vivo protein injection experiments substantiated MASP1's influence on the relative mRNA expression levels of C3 in various tissues and its protein expression in serum. This study also verified that C3 could modulate the expression of antiviral genes such as IFN1 and IRF3.

The role of gamma globulin, complement component 1q, factor B, properdin, body temperature, C-reactive protein and serum amyloid alpha to the activity and the function of the human complement system and its pathways.

The complement system plays a crucial role in orchestrating the activation and regulation of inflammation within the human immune system. Three distinct activation pathways-classical, lectin, and alternative-converge to form the common lytic pathway, culminating in the formation of the membrane-attacking complex that disrupts the structure of pathogens. Dysregulated complement system activity can lead to tissue damage, autoimmune diseases, or immune deficiencies. In this study, the antimicrobial activity of human serum was investigated by using a bioluminescent microbe probe, Escherichia coli (pEGFPluxABCDEamp). This probe has previously been used to determine the antimicrobial activity of complement system and the polymorphonuclear neutrophils. In this study, blocking antibodies against key serum activators and components, including IgG, complement component 1q, factor B, and properdin, were utilized. The influence of body temperature and acute phase proteins, such as C reactive protein (CRP) and serum amyloid alpha (SAA), on the complement system was also examined. The study reveals the critical factors influencing complement system activity and pathway function. Alongside crucial factors like C1q and IgG, alternative pathway components factor B and properdin played pivotal roles. Results indicated that the alternative pathway accounted for approximately one third of the overall serum antimicrobial activity, and blocking this pathway disrupted the entire complement system. Contrary to expectations, elevated body temperature during inflammation did not enhance the antimicrobial activity of human serum. CRP demonstrated complement activation properties, but at higher physiological concentrations, it exhibited antagonistic tendencies, dampening the response. On the other hand, SAA enhanced the serum's activity. Overall, this study sheds a light on the critical factors affecting both complement system activity and pathway functionality, emphasizing the importance of a balanced immune response.

Serum amyloid P component: Structure, biological activity, and application in diagnosis and treatment of immune-associated diseases.

Serum amyloid P component (SAP) is a member the innate immune humoral arm and participated in various processes, including the innate immune responses, tissue remodeling, and the pathogenesis of inflammatory diseases. Remarkably, SAP is a highly versatile immunomodulatory factor that can serve as a drug target for treating amyloid diseases and reduce inflammation, fibrosis degree, and respiratory disease. In this review, we focus on the biological activities of SAP and its application in different systemic immune-associated diseases. First, we reviewed the regulatory effects of SAP on innate immune cells and possible mechanisms. Second, we emphasized SAP as a diagnostic marker and therapeutic target for immune-associated diseases, including the neuropsychiatric disorders. Third, we presented several recommendations for regulating SAP in immune cell function and potential areas for future research. Some authorities consider SAP to be a pattern recognition molecule that plays multiple roles in the innate immune system and inflammation. Developing therapeutics that target SAP or its associated signaling pathways may be a promising strategy for treating immune-associated diseases.

Comprehensive functional characterization of complement factor I rare variant genotypes identified in the SCOPE geographic atrophy cohort.

Rare variants (RVs) in the gene encoding the regulatory enzyme complement factor I (CFI; FI) that reduce protein function or levels increase age-related macular degeneration risk. A total of 3357 subjects underwent screening in the SCOPE natural history study for geographic atrophy secondary to age-related macular degeneration, including CFI sequencing and serum FI measurement. Eleven CFI RV genotypes that were challenging to categorize as type I (low serum level) or type II (normal serum level, reduced enzymatic function) were characterized in the context of pure FI protein in C3b and C4b fluid phase cleavage assays and a novel bead-based functional assay (BBFA) of C3b cleavage. Four variants predicted or previously characterized as benign were analyzed by BBFA for comparison. In all, three variants (W51S, C67R, and I370T) resulted in low expression. Furthermore, four variants (P64L, R339Q, G527V, and P528T) were identified as being highly deleterious with IC50s for C3b breakdown >1 log increased versus the WT protein, while two variants (K476E and R474Q) were ∼1 log reduced in function. Meanwhile, six variants (P50A, T203I, K441R, E548Q, P553S, and S570T) had IC50s similar to WT. Odds ratios and BBFA IC50s were positively correlated (r = 0.76, p < 0.01), while odds ratios versus combined annotation dependent depletion (CADD) scores were not (r = 0.43, p = 0.16). Overall, 15 CFI RVs were functionally characterized which may aid future patient stratification for complement-targeted therapies. Pure protein in vitro analysis remains the gold standard for determining the functional consequence of CFI RVs.