Preformed mediators of defense-Gatekeepers enter the spotlight.

A wide variety of nanomaterials are currently being developed for use in the detection and treatment of human diseases. However, there is no systematic way to measure and predict the action of such materials in biological contexts. Lipid-encapsulated nanoparticles (NPs) are a class of nanomaterials that includes the liposomes, the most widely used and clinically proven type of NPs. Liposomes can, however, activate the complement system, an important branch of innate immunity, resulting in undesirable consequences. Here, we describe the complement response to lipid-encapsulated NPs that are functionalized on the surface with various lipid-anchored gadolinium chelates. We developed a quantitative approach to examine the interaction of NPs with the complement system using in vitro assays and correlating these results with those obtained in an in vivo mouse model. Our results indicate that surface functionalization of NPs with certain chemical structures elicits swift complement activation that is initiated by a natural IgM antibody and propagated via the classical pathway. The intensity of the response is dependent on the chemical structures of the lipid-anchored chelates and not zeta potential effects alone. Moreover, the extent of complement activation may be tempered by complement inhibiting regulatory proteins that bind to the surface of NPs. These findings represent a step forward in the understanding of the interactions between nanomaterials and the host innate immune response and provide the basis for a systematic structure-activity relationship study to establish guidelines that are critical to the future development of biocompatible nanotherapeutics.

The complement system, a plasma component, was described upon its discovery to augment the opsonisation of bacteria by antibodies, ‘complementing’ said antibodies’ antibacterial activity. In its first and foremost role, the complement cascade helps convert pathogen recognition into an effective host defence. Two recent publications in Acta Physiologica have highlighted the role of complement factors in immune cell function, namely monoamine transmitter release from immune cells during immune response and inflammation1, 2 This article is protected by copyright. All rights reserved.

Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process.

Human complement factor H-related protein (CFHR) 4 belongs to the factor H family of plasma glycoproteins that are composed of short consensus repeat (SCR) domains. Although factor H is a well known inhibitor of the alternative complement pathway, the functions of the CFHR proteins are poorly understood. CFHR4 lacks SCRs homologous to the complement inhibitory domains of factor H and, accordingly, has no significant complement regulatory activities. We have previously shown that CFHR4 binds C-reactive protein via its most N-terminal SCR, which leads to classical complement pathway activation. CFHR4 binds C3b via its C terminus, but the significance of this interaction is unclear. Therefore, we set out to clarify the functional relevance of C3b binding by CFHR4. Here, we report a novel role for CFHR4 in the complement system. CFHR4 serves as a platform for the assembly of an alternative pathway C3 convertase by binding C3b. This is based on the sustained ability of CFHR4-bound C3b to bind factor B and properdin, leading to an active convertase that generates C3a and C3b from C3. The CFHR4-C3bBb convertase is less sensitive to the factor H-mediated decay compared with the C3bBb convertase. CFHR4 mutants containing exchanges of conserved residues within the C-terminal C3b-binding site showed significantly reduced C3b binding and alternative pathway complement activation. In conclusion, our results suggest that, in contrast to the complement inhibitor factor H, CFHR4 acts as an enhancer of opsonization by promoting complement activation.

Nanomaterials are foreign substances that are treated by the immune system just like foreign invading pathogens. The immune system (innate immunity for the most part) is trained to recognize regular arrangements of chemical groups, which is what engineered nanomaterials are. The immune recognition poses multiple challenges to the use of nanomaterials for drug and gene delivery. One of the untoward results of immune recognition to nanomaterials once they are injected into the circulation is activation of the complement cascade. The complement system is a central component of the innate immunity accounting for about 5% of globulins in serum and is responsible for recognizing, eliminating and destroying pathogens [1]. Uncontrolled complement activation is known to cause inflammation, immune cell activation, increased vascular permeability and even tumor growth [1–3]. Nanocarriers can cause complement activation-related pseudoallergy or CARPA (term introduced by Dr Szebeni [4]). CARPA is a known phenomenon following infusion of Doxil (liposomal doxorubicin), Taxol (cremophorpaclitaxel), Feridex (dextran iron oxide) and SandImmune (cremophor-cyclosporine A). Numerous reports demonstrated complement activation by carbon nanotubes [5], micelles [6], liposomes, [7] polymer [8] and gold nanoparticles [9] in vitro and in vivo. Additional effects of complement activation are nanoparticle opsonization with complement fragments and subsequent clearance by macrophages and leukocytes. Besides complement, other mechanisms of recognition and clearance of nanoparticles are known. Scavenger receptors (SRs) are professional phagocytic pattern recognition receptors that were discovered in 1979 [10] and are responsible for opsonin-independent binding and uptake of oxidized low-density lipoprotein (oxLDL), pathogens, environmental particles and apoptotic cells. The universal feature of SRs is that their ligands possess a pattern of negative charges. SRs are implicated in the immune uptake of numerous nanoparticle types [11]. The main criticism of studies of immune mechanisms of clearance of nanomedicines in order to prolong circulation and decrease complement activation could be summarized in two questions: why not make thousands of nanoparticulate compounds (by combinatorial approach) and one of them will work? How the immune recognition can be blocked after learning that proteins (receptors) X, Y and Z mediate nanoparticle clearance? The answer to these questions is complex. Can we use the rational knowledge to modify nanoparticle structure to avoid the immune receptors? Superparamagnetic iron oxides (SPIO) are a nice illustration of how understanding the mechanisms of immune recognition could be useful to decrease immune Iron oxide nanoparticles and the mechanisms of immune recognition of nanomedicines

Introduction: The complement system plays a crucial role in bridging innate and adaptive immune responses. When activated, a proteolytic cascade leads to pathogen destruction. It is initiated via the recognition of foreign structures by three pathways: the classical, the lectin, and the alternative. This study focuses on the lectin pathway and the role of four pattern recognition molecules (PRMs), mannan-binding lectin, H-ficolin, L-ficolin, and M-ficolin, in the recognition of microbial patterns and the initiation of complement activation. These PRMs bind to specific carbohydrate structures; each PRM has unique ligand specificities. We investigated the PRM interactions with lipopolysaccharide (LPS) of Gram-negative bacteria. Methods: Utilizing a microarray of 120 distinct LPS structures, the study aims to map the diversity of PRM-LPS interactions and assess their role in complement activation. Results: Our findings reveal that all four PRMs preferentially bind to the O-antigens of LPS, rather than lipid A or the core oligosaccharide, contradicting previous suggestions. Each PRM displayed distinct binding patterns to different LPS structures, although some overlaps were observed. These interactions were partially confirmed with whole bacteria. Mannan-binding lectin binding to E. coli O30 and O126, as well as H-ficolin binding to E. coli O108, led to complement activation on the bacterial surface. Conclusion: The application of a wide array of LPS structures expands and clarifies the spectrum of bacterial glycoconjugates that interact with PRMs, known to activate the complement system.

This article is part of a themed issue on Canonical and non-canonical functions of the complement system in health and disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.14/issuetoc The complement system forms a network of more than 30 soluble proteins and cell-bound receptors that perform multiple sensor and effector functions as part of the innate immune system. It is well appreciated that the complement system protects the host from a hostile microbial environment through opsonization and direct lysis of microorganisms. Progress made at the beginning of the 21st century showed that it contributes to the integrity of tissues and organs by effectively eliminating altered or dead cells. Moreover, the original view that the functions of the complement system are limited to serum has been continuously expanded over the last two decades. Further, the structure and interaction of many complement proteins and receptors with extracellular and intracellular functions have been molecularly defined (Forneris et al., 2012). In addition to the known canonical activation pathways, non-canonical mechanisms have been described, which lead to cleavage and activation of complement factors not only in the circulation but also in immune cells. The intracellular complement system has been named the "complosome" (Arbore et al., 2017; Kolev et al., 2014). In fact, many studies indicate that the complement system has multiple and bidirectional interactions with other innate immunity sensor systems and regulates immune responses of innate and acquired immunity (Hajishengallis et al., 2017). Interestingly, the system also directly intervenes with the differentiation of cells and tissues and regulates cell metabolism, thus contributing significantly to cell homeostasis (West et al., 2020). From an evolutionary point of view, these non-canonical functions of complement are not surprising given that the system has been around for 1.2 billion years, making it one of the oldest parts of our immune system. Complement may even have evolved with an initial purpose distinct from protection against pathogens but rather as an intrinsic sensor of cellular stress. We will also discuss how taking a closer look into the evolution of key complement components not only made the functional connection between complement and metabolism rather 'predictable' but may also give clues for the discovery of additional roles for complement in basic cellular processes. The non-canonical pathways of complement activation and the multiple novel functions uncovered during the past decade make the complement system more than ever an important field of research and hot topic in immunology in particular in light of the ongoing coronavirus pandemic. The increasing number of clinical conditions that involve a pathological contribution from the complement system has spurred a renewed interest in therapeutic interventions, in order to modulate this host defence pathway (Pandey et al., 2017). Based on these findings, the pharmaceutical industry has identified the complement system, and specifically the anaphylatoxins and their receptors, as potential and promising targets in inflammatory diseases (Ricklin et al., 2018). In 2007, an antibody against C5 (eculizumab) was approved by the FDA for the treatment of paroxysmal nocturnal haemoglobinuria (PNH) and later for the atypical haemolytic syndrome (aHUS) and autoimmune neuromuscular disorders (Legendre et al., 2013). More than 20 candidate drugs, targeting various stages of the complement cascade are currently being evaluated in clinical trials, and additional agents are in preclinical development. For example, ChemoCentryx reported the successful completion of their Phase III trial showing that the orally administered C5a1 receptor antagonist (avacopan) was superior to corticosteroid treatment in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (Jayne et al., 2021) and may replace corticosteroid therapy in the future (Warrington, 2021). This has led to a renaissance of complement research and of complement-targeted drug discovery. Besides, potential indications for the therapeutic inhibition of the complement system are rapidly increasing in number and complexity. This issue is divided into two parts. First, it highlights recent discoveries, which have changed dramatically our view of the complement system, such as the intracellular complement system or the metabolic functions of complement. The second part will discuss new roles of complement in several diseases that are of major interest to the scientific and medical community including infections, hypertension, thrombosis and trauma. The contributions of the different authors are summarised in Figure 1. In the first part, Merle et al. (2021) discuss how integrins and complement orchestrate cell metabolisms and immunity. In addition, Rahman et al. (2021) describe the role of complement in the regulation of mitochondrial activity. King et al. (2021) have recently discovered that intracellular C3 regulates autophagy in pancreatic cells and describe in their contribution the role of complement in autophagy. Hevey et al. (2021), as well as Skerka et al. (2021), round up the first part by describing the role of complement in carbohydrate sensing in endothelial cells as well as the interaction of complement with ApoE. With the broadening span of novel complement activities, it makes sense to revisit key complement topics including its role in defence against pathogens. In the second part of this themed issue, Jodele and Köhl (2021) highlight the contribution of complement activation in infection with highly pathogenic coronaviruses with a particular focus on activation pathways, complement genetics and the potential of complement as a target in SARS-CoV-2 infection. The contribution of complement to the development of arterial hypertension is a relatively new area of complement research and is discussed by Wenzel et al. (2021). Huber-Lang et al. (2021), Portilla and Xavier (2021) and Rawish et al. (2021) will summarize and discuss progress on our understanding of the emerging new roles of complement in thrombosis, kidney disease and trauma. These recent observations have sparked a renewed interest in complement research and motivated scientists to revisit and re-evaluate the role of complement activity—now with a fresh eye on its non-canonical functions—in diseases, where complement plays a role but where the complement-driven molecular mechanisms remain to be defined. This is paralleled by research efforts that focus on further exploring and understanding novel complement activities in normal cell physiology and diseases, formerly not associated with canonical or non-canonical complement activation. We hope that the selected topics will further spur the interest of the readers in complement research and help to uncover the many secrets that the system still holds for us.

The complement system is a major constituent of the innate immune system. It not only bridges innate and adaptive arms of the immune system but also links the immune system with the coagulation system. Current understanding of the role of complement has extended far beyond fighting of infections, and now encompasses maintenance of homeostasis, tissue regeneration, and pathophysiology of multiple diseases. It has been known for many years that complement activation is strongly pH sensitive, but only relatively recently has the physiological significance of this been appreciated. Most complement assays are carried out at the physiological pH 7.4. However, pH in some extracellular compartments, for example, renal tubular fluid in parts of the tubule, and extracellular fluid at inflammation loci, is sufficiently acidic to activate complement. The exact molecular mechanism of this activation is still unclear, but possible cross-talk between the contact system (intrinsic pathway) and complement may exist at low pH with subsequent complement activation. The current article reviews the published data on the effect of pH on the contact system and complement activity, the nature of the pH sensor molecules, and the clinical implications of these effects. Of particular interest is chronic kidney disease (CKD) accompanied by metabolic acidosis, in which therapeutic alkalinization of urine has been shown significantly to reduce tubular complement activation products, an effect, which may have important implications for slowing progression of CKD.

Recent studies have shown that activation of complement and contact systems results in the generation of antibacterial peptides. Streptococcus pyogenes, a major bacterial pathogen in humans, exists in >100 different serotypes due to sequence variation in the surface-associated M protein. Cases of invasive and life-threatening S. pyogenes infections are commonly associated with isolates of the M1 serotype, and in contrast to the large majority of M serotypes, M1 isolates all secrete the SIC protein. Here, we show that SIC interferes with the activation of the contact system and blocks the activity of antibacterial peptides generated through complement and contact activation. This effect promotes the growth of S. pyogenes in human plasma, and in a mouse model of S. pyogenes sepsis, SIC enhances bacterial dissemination, results which help explain the high frequency of severe S. pyogenes infections caused by isolates of the M1 serotype.

Platelet activation during thrombotic events is closely associated with complement and contact system activation, which in turn leads to inflammation. Here we review the interactions between activated platelets and the complement and contact activation systems in clotting blood. Chondroitin sulfate A (CS-A), released from alpha granules during platelet activation, is a potent mediator of crosstalk between platelets and the complement system. CS-A activates complement in the fluid phase, generating anaphylatoxins that mediate leukocyte activation. No complement activation seems to occur on the activated platelet surface, but C3 in the form of C3(H(2)O) is bound to the surfaces of activated platelets . This finding is consistent with the strong expression of membrane-bound complement regulators present at the platelet surface. CS-A exposed on the activated platelets is to a certain amount responsible for recruiting soluble regulators to the surface. Platelet-bound C3(H(2)O) acts as a ligand for leukocyte CR1 (CD35), potentially enabling platelet-leukocyte interactions. In addition, platelet activation leads to the activation of contact system enzymes, which are specifically inhibited by antithrombin, rather than by C1INH, as is the case when contact activation is induced by material surfaces. Thus, in addition to their traditional role as initiators of secondary hemostasis, platelets also act as mediators and regulators of inflammation in thrombotic events.

The Mycobacterium tuberculosis complex (MTBC), comprising species such as M. tuberculosis, M. africanum, and M. canettii, is the causative agent of tuberculosis (TB), one of the deadliest infectious diseases worldwide. MTBC strains exhibit genetic diversity that influences host-pathogen interactions, immune evasion, and disease outcomes. The complement system, a crucial component of innate immunity, plays a dual role in pathogen detection and potential immune evasion, yet its interactions with MTBC strains remain underexplored. We investigated the roles of C1q and mannose-binding lectin (MBL), key pattern recognition molecules (PRMs) of the classical and lectin pathways, respectively, in complement activation against diverse clinical MTBC strains. We observed that both C1q and MBL recognize mycobacteria directly as PRMs but that the degree of binding was strain dependent. Both molecules facilitated complement cascade activation, leading to the deposition of C4b and C3b and the formation of the membrane attack complex (MAC) on bacterial surfaces. However, inhibition experiments revealed that C1q is the primary driver of complement activation in nonimmune serum, while MBL plays a supportive but nonredundant role. Despite robust complement activation, MAC formation did not significantly impact the viability of MTBC strains. Nevertheless, these findings highlight a nuanced interplay between the complement system and MTBC lineage diversity. Our results provide novel insights into early host-pathogen dynamics in TB, emphasizing the importance of considering MTBC lineage diversity in understanding the immune response against mycobacteria.

Complement in Glomerular Disease

The complement system is a tightly regulated network of proteins involved in defense against pathogens, inflammatory processes, and coordination of the innate and adaptive immune responses. Dysregulation of the complement cascade is associated with many inflammatory disorders. Thus, inhibition of the complement system has emerged as an option for treatment of a range of different inflammatory diseases. MAP-1 is a pattern recognition molecule (PRM)-associated inhibitor of the lectin pathway of the complement system, whereas C4b-binding protein (C4BP) regulates both the classical and lectin pathways. In this study we generated chimeric proteins consisting of MAP-1 and the first five domains of human C4BP (C4BP1-5) in order to develop a targeted inhibitor acting at different levels of the complement cascade. Two different constructs were designed and expressed in CHO cells where MAP-1 was fused with C4BP1-5 in either the C- or N-terminus. The functionality of the chimeric proteins was assessed using different in vitro complement activation assays. Both chimeric proteins displayed the characteristic Ca2+-dependent dimerization and binding to PRMs of native MAP-1, as well as the co-factor activity of native C4BP. In ELISA-based complement activation assays they could effectively inhibit the lectin and classical pathways. Notably, MAP-1:C4BP1-5 was five times more effective than rMAP-1 and rC4BP1-5 applied at the same time, emphasizing the advantage of a single inhibitor containing both functional domains. The MAP-1/C4BP chimeras exert unique complement inhibitory properties and represent a novel therapeutic approach targeting both upstream and central complement activation.

The complement system plays a key role in host defense against pneumococcal infection. Three different pathways, the classical, alternative and lectin pathways, mediate complement activation. While there is limited information available on the roles of the classical and the alternative activation pathways of complement in fighting streptococcal infection, little is known about the role of the lectin pathway, mainly due to the lack of appropriate experimental models of lectin pathway deficiency. We have recently established a mouse strain deficient of the lectin pathway effector enzyme mannan-binding lectin associated serine protease-2 (MASP-2) and shown that this mouse strain is unable to form the lectin pathway specific C3 and C5 convertases. Here we report that MASP-2 deficient mice (which can still activate complement via the classical pathway and the alternative pathway) are highly susceptible to pneumococcal infection and fail to opsonize Streptococcus pneumoniae in the none-immune host. This defect in complement opsonisation severely compromises pathogen clearance in the lectin pathway deficient host. Using sera from mice and humans with defined complement deficiencies, we demonstrate that mouse ficolin A, human L-ficolin, and collectin 11 in both species, but not mannan-binding lectin (MBL), are the pattern recognition molecules that drive lectin pathway activation on the surface of S. pneumoniae. We further show that pneumococcal opsonisation via the lectin pathway can proceed in the absence of C4. This study corroborates the essential function of MASP-2 in the lectin pathway and highlights the importance of MBL-independent lectin pathway activation in the host defense against pneumococci.

In this study, several innate immunological adjuvants and related compounds were compared with respect to complement activation in serum and induction of cytokine release in whole blood samples using immunoassays. As found, simple lipids had no effect on the complement system or on cytokine release, whereas lipopolysaccharides induced prominent release of pro-inflammatory cytokines (IL1β, TNF and IFNγ) without affecting the complement system, except for one, which activated the lectin pathway (LP). Moreover, saponin induced IL1β and MCP1 release and did not affect the complement system. The polysaccharide inulin exhausted the alternative pathway (AP) completely without affecting the LP and the classical pathway (CP), whereas zymosan exhausted the AP and had a major effect on the LP and CP as well. Peptidoglycans mainly affected the LP. Inulin, agarose and cellulose induced IL1β and MCP1 release, while dextran had no effect on cytokine secretion. Zymosan mainly induced IL1β release. The inorganic compound aluminium hydroxide, Al(OH)3 , activated the complement system very efficiently (all three pathways) but only induced MCP1 release. Other compounds tested had minor/individual effects. Collectively, well-known adjuvants, such as aluminum hydroxide, activated the complement system and/or induced pro-inflammatory cytokine release. Since complement activation generates anaphylactic peptides, a simple definition of an (innate) immunological adjuvant can be inferred: it activates the (innate) immune system by complement activation and/or release of cytokines so as to attract cells of the adaptive immune system.