Role of factor H-related protein 3 in Pseudomonas aeruginosa bloodstream infections.

Pseudomonas aeruginosa is a leading cause of nosocomial bloodstream infections. The outcome of these infections depends on the virulence of the microorganism as well as host-related conditions and factors. The complement system plays a crucial role in defense against bloodstream infections. P. aeruginosa counteracts complement attack by recruiting Factor H (FH) that inhibits complement amplification on the bacterial surface. Factor H-related proteins (FHRs) are a group of plasma proteins evolutionarily related to FH that have been postulated to interfere this bacterial evasion mechanism. In this study, we demonstrate that FHR-3 competes with purified FH for binding to P. aeruginosa and identify EF-Tu as a common bacterial target for both complement regulator factors. Importantly, elevated levels of FHR-3 in human serum promote complement activation, leading to increased opsonization and killing of P. aeruginosa. Conversely, physiological concentrations of FHR-3 have no significant effect. Our findings suggest that FHR-3 may serve as a protective host factor against P. aeruginosa infections.

Enhanced complement activation and MAC formation accelerates severe COVID-19.

Emerging evidence indicates that activation of complement system leading to the formation of the membrane attack complex (MAC) plays a detrimental role in COVID-19. However, their pathogenic roles have never been experimentally investigated before. We used three knock out mice strains (1. C3-/-; 2. C7-/-; and 3. Cd59ab-/-) to evaluate the role of complement in severe COVID-19 pathogenesis. C3 deficient mice lack a key common component of all three complement activation pathways and are unable to generate C3 and C5 convertases. C7 deficient mice lack a complement protein needed for MAC formation. Cd59ab deficient mice lack an important inhibitor of MAC formation. We also used anti-C5 antibody to block and evaluate the therapeutic potential of inhibiting MAC formation. We demonstrate that inhibition of complement activation (in C3-/-) and MAC formation (in C3-/-. C7-/-, and anti-C5 antibody) attenuates severe COVID-19; whereas enhancement of MAC formation (Cd59ab-/-) accelerates severe COVID-19. The degree of MAC but not C3 deposits in the lungs of C3-/-, C7-/- mice, and Cd59ab-/- mice as compared to their control mice is associated with the attenuation or acceleration of SARS-CoV-2-induced disease. Further, the lack of terminal complement activation for the formation of MAC in C7 deficient mice protects endothelial dysfunction, which is associated with the attenuation of diseases and pathologic changes. Our results demonstrated the causative effect of MAC in severe COVID-19 and indicate a potential avenue for modulating the complement system and MAC formation in the treatment of severe COVID-19.

Agnostic B cell selection approach identifies antibodies against K. pneumoniae that synergistically drive complement activation.

Antibody-dependent complement activation plays a key role in the natural human immune response to infections. Currently, the understanding of which antibody-antigen combinations drive a potent complement response on bacteria is limited. Here, we develop an antigen-agnostic approach to stain and single-cell sort human IgG memory B cells recognizing intact bacterial cells, keeping surface antigens in their natural context. With this method we successfully identified 29 antibodies against K. pneumoniae, a dominant cause of hospital-acquired infections with increasing antibiotic resistance. Combining genetic tools and functional analyses, we reveal that the capacity of antibodies to activate complement on K. pneumoniae critically depends on their antigenic target. Furthermore, we find that antibody combinations can synergistically activate complement on K. pneumoniae by strengthening each other's binding in an Fc-independent manner. Understanding the molecular basis of effective complement activation by antibody combinations to mimic a polyclonal response could accelerate the development of antibody-based therapies against problematic infections.

Antibodies and complement are key drivers of thrombosis.

Venous thromboembolism (VTE) is a common, deadly disease with an increasing incidence despite preventive efforts. Clinical observations have associated elevated antibody concentrations or antibody-based therapies with thrombotic events. However, how antibodies contribute to thrombosis is unknown. Here, we show that reduced blood flow enabled immunoglobulin M (IgM) to bind to FcµR and the polymeric immunoglobulin receptor (pIgR), initiating endothelial activation and platelet recruitment. Subsequently, the procoagulant surface of activated platelets accommodated antigen- and FcγR-independent IgG deposition. This leads to classical complement activation, setting in motion a prothrombotic vicious circle. Key elements of this mechanism were present in humans in the setting of venous stasis as well as in the dysregulated immunothrombosis of COVID-19. This antibody-driven thrombosis can be prevented by pharmacologically targeting complement. Hence, our results uncover antibodies as previously unrecognized central regulators of thrombosis. These findings carry relevance for therapeutic application of antibodies and open innovative avenues to target thrombosis without compromising hemostasis.

The Role of Ficolins in Lung Injury.

BACKGROUND: Respiratory diseases seriously threaten human health worldwide, and lung injury is an important component of respiratory disease. Complement activation is an important function of the innate immune system. Complement activation helps the body defend against invasion by external microorganisms, whereas excessive complement activation can exacerbate tissue damage or lead to unwanted side effects. Ficolins are a class of immune-related proteins in the lectin pathway that play important roles in the body's immune defense. Although individual ficolins are not well understood, current information suggests that ficolins may play an important regulatory role in lung injury. SUMMARY: Several studies have shown that ficolins are involved in the immune response in the lung, particularly in the response to infectious and inflammatory processes. KEY MESSAGES: This review summarizes the role of ficolins in lung injury. Ficolins may influence the development and repair of lung injury by recognizing and binding pathogenic microorganisms, modulating the inflammatory response, and promoting the clearance of immune cells. In addition, ficolins are associated with the development and progression of lung diseases (such as pneumonia and ARDS) and may have an important impact on the pathophysiological processes of inflammatory diseases.

Elucidating allergic reaction mechanisms in response to SARS-CoV-2 mRNA vaccination in adults.

BACKGROUND: During the COVID-19 pandemic, novel nanoparticle-based mRNA vaccines were developed. A small number of individuals developed allergic reactions to these vaccines although the mechanisms remain undefined. METHODS: To understand COVID-19 vaccine-mediated allergic reactions, we enrolled 19 participants who developed allergic events within 2 h of vaccination and 13 controls, nonreactors. Using standard hemolysis assays, we demonstrated that sera from allergic participants induced stronger complement activation compared to nonallergic subjects following ex vivo vaccine exposure. RESULTS: Vaccine-mediated complement activation correlated with anti-polyethelyne glycol (PEG) IgG (but not IgM) levels while anti-PEG IgE was undetectable in all subjects. Depletion of total IgG suppressed complement activation in select individuals. To investigate the effects of vaccine excipients on basophil function, we employed a validated indirect basophil activation test that stratified the allergic populations into high and low responders. Complement C3a and C5a receptor blockade in this system suppressed basophil response, providing strong evidence for complement involvement in vaccine-mediated basophil activation. Single-cell multiome analysis revealed differential expression of genes encoding the cytokine response and Toll-like receptor (TLR) pathways within the monocyte compartment. Differential chromatin accessibility for IL-13 and IL-1B genes was found in allergic and nonallergic participants, suggesting that in vivo, epigenetic modulation of mononuclear phagocyte immunophenotypes determines their subsequent functional responsiveness, contributing to the overall physiologic manifestation of vaccine reactions. CONCLUSION: These findings provide insights into the mechanisms underlying allergic reactions to COVID-19 mRNA vaccines, which may be used for future vaccine strategies in individuals with prior history of allergies or reactions and reduce vaccine hesitancy.

The Interactions of the Complement System with Human Cytomegalovirus.

The complement system is an evolutionarily ancient component of innate immunity that serves as an important first line of defense against pathogens, including viruses. In response to infection, the complement system can be activated by three distinct yet converging pathways (classical, lectin, and alternative) capable of engaging multiple antiviral host responses to confront acute, chronic, and recurrent viral infections. Complement can exert profound antiviral effects via multiple mechanisms including the induction of inflammation and chemotaxis to sites of infection, neutralization/opsonization of viruses and virally infected cells, and it can even shape adaptive immune responses. With millions of years of co-evolution and the ability to establish life-long infections, herpesviruses have evolved unique mechanisms to counter complement-mediated antiviral defenses, thus enabling their survival and replication within humans. This review aims to comprehensively summarize how human herpesviruses engage with the complement system and highlight our understanding of the role of complement in human cytomegalovirus (HCMV) infection, immunity, and viral replication. Herein we describe the novel and unorthodox roles of complement proteins beyond their roles in innate immunity and discuss gaps in knowledge and future directions of complement and HCMV research.

Complement system molecules: Expression and regulation at the maternal-conceptus interface during pregnancy in pigs.

The complement system, composed of complement components and complement control proteins, plays an essential role in innate immunity. Complement system molecules are expressed at the maternal-conceptus interface, and inappropriate activation of the complement system is associated with various adverse pregnancy outcomes in humans and rodents. However, the expression, regulation, and function of the complement system at the maternal-conceptus interface in pigs have not been studied. In this study, we investigated the expression, localization, and regulation of complement system molecules at the maternal-conceptus interface in pigs. Complement components and complement control proteins were expressed in the endometrium, early-stage conceptus, and chorioallantoic tissues during pregnancy. The expression of complement components acting on the early stage of complement activation increased in the endometrium on Day 15 of pregnancy, with greater levels on that day compared with the estrous cycle. Localization of several complement components and complement control proteins was cell-type specific in the endometrium. The expression of C1QC, C2, C3, C4A, CFI, ITGB2, MASP1, and SERPING1 was increased by IFNG in endometrial explant tissues. Furthermore, cleaved C3 fragments were detected in endometrial tissues and uterine flushings on Day 15 of the estrous cycle and Day 15 of pregnancy, with greater levels on Day 15 of pregnancy. These results suggest that complement system molecules in pigs expressed at the maternal-conceptus interface play important roles in the establishment and maintenance of pregnancy by regulating innate immunity and modulating the maternal immune environment during pregnancy.

The role of complement in kidney disease: conclusions from a Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference.

Uncontrolled complement activation can cause or contribute to glomerular injury in multiple kidney diseases. Although complement activation plays a causal role in atypical hemolytic uremic syndrome and C3 glomerulopathy, over the past decade, a rapidly accumulating body of evidence has shown a role for complement activation in multiple other kidney diseases, including diabetic nephropathy and several glomerulonephritides. The number of available complement inhibitor therapies has also increased during the same period. In 2022, Kidney Diseases: Improving Global Outcomes (KDIGO) convened a Controversies Conference, "The Role of Complement in Kidney Disease," to address the expanding role of complement dysregulation in the pathophysiology, diagnosis, and management of various glomerular diseases, diabetic nephropathy, and other forms of hemolytic uremic syndrome. Conference participants reviewed the evidence for complement playing a primary causal or secondary role in progression for several disease states and considered how evidence of complement involvement might inform management. Participating patients with various complement-mediated diseases and caregivers described concerns related to life planning, implications surrounding genetic testing, and the need for inclusive implementation of effective novel therapies into clinical practice. The value of biomarkers in monitoring disease course and the role of the glomerular microenvironment in complement response were examined, and key gaps in knowledge and research priorities were identified.

A tale of two functions: C-reactive protein complement-ary structures and their role in rheumatoid arthritis.

C-reactive protein (CRP) is an inflammatory biomarker with associated clinical utility in a wide number of inflammatory disorders, including rheumatoid arthritis (RA). The interaction of CRP with pro-inflammatory cytokines has been explored before, however its role in complement regulation is more subtle, where CRP is capable of both up and downregulating the complement cascade. CRP is produced in a pentameric form and can dissociate to a monomeric form in circulation which has significant implications for its ability to interact with receptors and binding partners. This dichotomy of CRP structure could have relevance in patients with RA who have significant dysfunction in their complement cascade and also widely varying CRP levels including at the time of flare. This review aims to bring together current knowledge of CRP in its various forms, its effects on complement function and how this could influence pathology in the context of RA.

Complement-Coagulation Cross-talk: Factor H-mediated regulation of the Complement Classical Pathway activation by fibrin clots.

The classical pathway of the complement system is activated by the binding of C1q in the C1 complex to the target activator, including immune complexes. Factor H is regarded as the key downregulatory protein of the complement alternative pathway. However, both C1q and factor H bind to target surfaces via charge distribution patterns. For a few targets, C1q and factor H compete for binding to common or overlapping sites. Factor H, therefore, can effectively regulate the classical pathway activation through such targets, in addition to its previously characterized role in the alternative pathway. Both C1q and factor H are known to recognize foreign or altered-self materials, e.g., bacteria, viruses, and apoptotic/necrotic cells. Clots, formed by the coagulation system, are an example of altered self. Factor H is present abundantly in platelets and is a well-known substrate for FXIIIa. Here, we investigated whether clots activate the complement classical pathway and whether this is regulated by factor H. We show here that both C1q and factor H bind to the fibrin formed in microtiter plates and the fibrin clots formed under in vitro physiological conditions. Both C1q and factor H become covalently bound to fibrin clots, and this is mediated via FXIIIa. We also show that fibrin clots activate the classical pathway of complement, as demonstrated by C4 consumption and membrane attack complex detection assays. Thus, factor H downregulates the activation of the classical pathway induced by fibrin clots. These results elucidate the intricate molecular mechanisms through which the complement and coagulation pathways intersect and have regulatory consequences.

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.

Hexamerization: explaining the original sin of IgG-mediated complement activation in acute lung injury.

Although antibody-mediated lung damage is a major factor in transfusion-related acute lung injury (ALI), autoimmune lung disease (for example, coatomer subunit α [COPA] syndrome), and primary graft dysfunction following lung transplantation, the mechanism by which antigen-antibody complexes activate complement to induce lung damage remains unclear. In this issue of the JCI, Cleary and colleagues utilized several approaches to demonstrate that IgG forms hexamers with MHC class I alloantibodies. This hexamerization served as a key pathophysiological mechanism in alloimmune lung injury models and was mediated through the classical pathway of complement activation. Additionally, the authors provided avenues for exploring therapeutics for this currently hard-to-treat clinical entity that has several etiologies but a potentially focused mechanism.

The role of mannose-binding lectin (MBL) in diabetic retinopathy: A scoping review.

Diabetes mellitus (DM) is a chronic systemic disease characterized by a multifactorial nature, which may lead to several macro and microvascular complications. Diabetic retinopathy (DR) is one of the most severe microvascular complications of DM, which can result in permanent blindness. The mechanisms involved in the pathogenesis of DR are multiple and still poorly understood. Factors such as dysregulation of vascular regeneration, oxidative and hyperosmolar stress in addition to inflammatory processes have been associated with the pathogenesis of DR. Furthermore, compelling evidence shows that components of the immune system, including the complement system, play a relevant role in the development of the disease. Studies suggest that high concentrations of mannose-binding lectin (MBL), an essential component of the complement lectin pathway, may contribute to the development of DR in patients with DM. This review provides an update on the possible role of the complement system, specifically the lectin pathway, in the pathogenesis of DR and discusses the potential of MBL as a non-invasive biomarker for both, the presence and severity of DR, in addition to its potential as a therapeutic target for intervention strategies.

The Inflammatory Response Induced by Aspergillus fumigatus Conidia Is Dependent on Complement Activation: Insight from a Whole Blood Model.

INTRODUCTION: We aimed to elucidate the inflammatory response of Aspergillus fumigatus conidia in a whole-blood model of innate immune activation and to compare it with the well-characterized inflammatory reaction to Escherichia coli. METHODS: Employing a human lepirudin whole-blood model, we analyzed complement and leukocyte activation by measuring the sC5b-9 complex and assessing CD11b expression. A 27-multiplex system was used for quantification of cytokines. Selective cell removal from whole blood and inhibition of C3, C5, and CD14 were also applied. RESULTS: Our findings demonstrated a marked elevation in sC5b-9 and CD11b post-A. fumigatus incubation. Thirteen cytokines (TNF, IL-1ß, IL-1ra, IL-4, IL-6, IL-8, IL-17, IFNγ, MCP-1, MIP-1α, MIP-1ß, FGF-basic, and G-CSF) showed increased levels. A generally lower level of cytokine release and CD11b expression was observed with A. fumigatus conidia than with E. coli. Notably, monocytes were instrumental in releasing all cytokines except MCP-1. IL-1ra was found to be both monocyte and granulocyte-dependent. Pre-inhibiting with C3 and CD14 inhibitors resulted in decreased release patterns for six cytokines (TNF, IL-1ß, IL-6, IL-8, MIP-1α, and MIP-1ß), with minimal effects by C5-inhibition. CONCLUSION: A. fumigatus conidia induced complement activation comparable to E. coli, whereas CD11b expression and cytokine release were lower, underscoring distinct inflammatory responses between these pathogens. Complement C3 inhibition attenuated cytokine release indicating a C3-level role of complement in A. fumigatus immunity.

Unraveling the Intricate Web: Complement Activation Shapes the Pathogenesis of Sepsis-Induced Coagulopathy.

BACKGROUND: Sepsis-associated coagulopathy specifically refers to widespread systemic coagulation activation accompanied by a high risk of hemorrhage and organ damage, which in severe cases manifests as disseminated intravascular coagulation (DIC), or even develops into multiple organ dysfunction syndrome (MODS). The complement system and the coagulation system as the main columns of innate immunity and hemostasis, respectively, undergo substantial activation after sepsis. SUMMARY: Dysfunction of the complement, coagulation/fibrinolytic cascades caused by sepsis leads to "thromboinflammation," which ultimately amplifies the systemic inflammatory response and accelerates the development of MODS. Recent studies have revealed that massive activation of the complement system exacerbates sepsis-induced coagulation and even results in DIC, which suggests that inhibition of complement activation may have therapeutic potential in the treatment of septic coagulopathy. KEY MESSAGES: Sepsis-associated thrombosis involves the upregulation or activation of procoagulant factors, down-regulation or inactivation of anticoagulant factors, and impairment of the fibrinolytic mechanism. This review aims to summarize the latest literature and analyze the underlying molecular mechanisms of the activation of the complement system on the abnormal coagulation cascades in sepsis.

Engineering Agonistic Bispecifics to Investigate the Influence of Distance on Surface-Mediated Complement Activation.

The development of agonists capable of activating the human complement system by binding to the C1 complex presents a novel approach for targeted cell killing. Bispecific nanobodies and Abs can successfully use C1 for this purpose; however, efficacy varies significantly between epitopes, Ab type, and bispecific design. To address this variability, we investigated monomeric agonists of C1 in the form of bispecific nanobodies, which lack Fc domains that lead to oligomerization in Abs. These therefore offer an ideal opportunity to explore the geometric parameters crucial for C1 activation. In this study, we explored the impact of linker length as a metric for Ag and epitope location. DNA nanotechnology and protein engineering allowed us to design linkers with controlled lengths and flexibilities, revealing a critical range of end-to-end distances for optimal complement activation. We discovered that differences in complement activation were not caused by differential C1 activation or subsequent cleavage of C4, but instead impacted C4b deposition and downstream membrane lysis. Considering the importance of Ab class and subclass, this study provides insights into the structural requirements of C1 binding and activation, highlighting linker and hinge engineering as a potential strategy to enhance potency over specific cellular targets. Additionally, using DNA nanotechnology to modify geometric parameters demonstrated the potential for synthetic biology in complement activation. Overall, this research offers valuable insights into the design and optimization of agonists for targeted cell killing through complement activation.

Intracellular complement and immunometabolism: The advantages of compartmentalization.

The complement system is a proteolytic cascade triggered by pathogen and danger-associated molecular patterns, with resultant outcomes of inflammation, cellular activation, and opsonization of material for removal by phagocytosis. While first discovered as an activity in serum, it is now recognized that complement components play important roles at local and individual cell-intrinsic levels. In particular, apart from the extracellular serum activities of complement, it is now believed that complement also acts intracellularly, as part of a cellular signal transduction cascade that can stimulate cellular survival and activation, and individual immune cell phenotypes, via effects on cellular metabolism. This review will describe what is currently known about how complement functions in intracellular signal transduction, and outline the functional advantages of a compartmentalized and intracellular complement system.

Complement system is overactivated in patients with IgA nephropathy after COVID-19.

IgA nephropathy (IgAN), which has been confirmed as a complement mediated autoimmune disease, is also one form of glomerulonephritis associated with COVID-19. Here, we aim to investigate the clinical and immunological characteristics of patients with IgAN after COVID-19. The level of plasma level of C5a (p < 0.001), soluble C5b-9 (p = 0.018), FHR5 (p < 0.001) were all significantly higher in Group CoV (33 patients with renal biopsy-proven IgAN experienced COVID-19) compared with Group non-CoV (44 patients with IgAN without COVID-19), respectively. Compared with Group non-CoV, the intensity of glomerular C4d (p = 0.017) and MAC deposition (p < 0.001) and Gd-IgA1 deposition (p = 0.005) were much stronger in Group CoV. Our finding revealed that for IgAN after COVID-19, mucosal immune responses to SARS-CoV-2 infection may result in the overactivation of systemic and renal local complement system, and increased glomerular deposition of Gd-IgA1, which may lead to renal dysfunction and promote renal progression in IgAN patients.