Complement 3d: from molecular adjuvant to target of immune escape mechanisms

C3d is a fragment of the complement factor C3 and is generated in the course of complement activation. When bound to antigen in single or multiple copies, the B cell receptor and complement receptor 2 become co-crosslinked resulting in decreased or increased B cell responses depending on the valence of the antigen-C3d construct. When antigen-C3d constructs are used for the purpose of generating a protective immune response (vaccines), they may either enhance the expected response or suppress it depending on the nature of the antigen. Various pathogens use C3d to evade the immune system by inhibiting complement activation, invading and homing in host cells or masking immunogenic areas of pathogen proteins. Therefore, future vaccination strategies for infectious diseases and cancer employing C3d as a molecular adjuvant need to be carefully evaluated before choosing a target antigen in order to take advantage of the adjuvant effect of the complement component while avoiding potential vaccine complications associated with immune escape mechanisms.     

Production of complement components by cells of the immune system

The complement system is an important part of the innate immune defence. It contributes not only to local inflammation, removal and killing of pathogens, but it also assists in shaping of the adaptive immune response. Besides a role in inflammation, complement is also involved in physiological processes such as waste disposal and developmental programmes. The complement system comprises several soluble and membrane‐bound proteins. The bulk of the soluble proteins is produced mainly by the liver. While several complement proteins are produced by a wide variety of cell types, other complement proteins are produced by only a few related cell types. As these data suggest that local production by specific cell types may have specific functions, more detailed studies have been employed recently analysing the local and even intracellular role of these complement proteins. Here we review the current knowledge about extrahepatic production and/or secretion of complement components. More specifically, we address what is known about complement synthesis by cells of the human immune system.     

Activation and control of complement, inflammation, and infection associated with the use of biomedical polymers

It is generally acknowledged that artificial biomaterials are much less immunologically active than transplants or tissue derived biomaterials. However, activation of both the coagulation cascade and the complement system is a common occurrence when human blood is exposed to biomaterial surfaces during extracorporeal procedures, such as renal hemodialysis or cardiopulmonary bypass. Both individual and collective activation of these cascades often produce local and systemic effects. A number of complement activation products function as the mediators of inflammation. They serve as ligands for specific receptors on polymorphonuclear leukocytes, monocytes, macrophages, mast cells, and other cells. Such an interaction leads to induction of cellular responses in adhered cells, including release of oxidative products, lysosomal enzymes, or both, which often contribute to a number of pathologic conditions. Most pathogens invading the human body are attacked by the immune system directly following entry, especially when they are in contact with blood. However, bacteria and parasites have developed a large number of specific strategies to overcome immune defense among others by avoiding either recognition or eradication by complement. In this aspect, of concern are several microorganisms responsible for formation of antibiotic resistant biofilms on biomaterial surfaces, namely Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa.     

Dynamic control of the complement system by modulated expression of regulatory proteins

The complement system serves many biological functions, including the eradication of invasive pathogens and the removal of damaged cells and immune-complexes. Uncontrolled complement activation causes injury to host cells, however, so adequate regulation of the system is essential. Control of the complement system is maintained by a group of cell surface and circulating proteins referred to as complement regulatory proteins. The expression of the cell surface complement regulatory proteins varies from tissue to tissue. Furthermore, specific cell types can upregulate or downregulate the expression of these proteins in response to a variety of signals or insults. Altered regulation of the complement regulatory proteins can have important effects on local complement activation. In some circumstances this can be beneficial, such as in the setting of certain infections. In other circumstances, however, this can be a cause of complement-mediated injury of the tissue. A full understanding of the mechanisms by which the complement system is modulated at the local level can have important implications for how we diagnose and treat a wide range of inflammatory diseases.     

Cell signals transduced by complement

The complement system is composed of soluble blood plasma proteins and cell membrane proteins. A major function of the soluble complement proteins is to bind to and destroy invading pathogens. The membrane proteins of the complement system are divided into complement receptors and complement regulatory proteins. Complement receptors on phagocytic cells promote binding and engulfment of pathogens coated with complement opsonins, whereas complement regulatory proteins protect healthy tissues from accidental damage by the soluble complement proteins. Upon binding of complement proteins or protein fragments that are generated during complement activation, these receptors and regulatory proteins transduce various signals into cells bearing them. The complement membrane attack complex C5b-9 binds to cell membranes, independent of any receptor, and also activates multiple signaling pathways. The receptor-dependent and -independent signals transduced by complement components are of great consequence to health and disease. Complement plays an important role in immunoregulation by activating B and T lymphocytes. It may also exert pro- or anti-apoptotic effects on various cell types. At sublytic doses, the complement membrane attack complex has wide-range effects on many cell types leading to cellular responses, such as secretion, adherence, aggregation, chemotaxis and even cell division. Sublytic complement also induces increased cell resistance to lytic doses of complement. Finally, certain pathogens take advantage of complement membrane proteins to gain entry into cells. The emerging data on these complement-related signaling pathways is hereby described.     

Complement escape of human pathogenic bacteria by acquisition of complement regulators

Pathogenic micro-organisms employ a broad range of strategies to survive in and to persistently infect the human host. Far from being completely understood by which highly sophisticated means invading pathogens overcome the host's destructive immune defence, there is a growing body of evidence on particular mechanisms which play a pivotal role for immune evasion. This review focuses on evasion of medically and scientifically important bacteria by acquisition of host derived fluid-phase complement regulatory proteins, in particular factor H, FHL-1, and C4b binding protein. Expression of microbial surface molecules binding to human complement regulators and thus fixing them in a functionally active state allows pathogens to inhibit and finely regulate complement activation directly on their surface. Further studies on the utilization of host complement regulatory proteins will likely have a marked impact on a more efficient and specific clinical treatment.     

Interactions between mannose-binding lectin and MASPs during complement activation by the lectin pathway

The lectin pathway of complement performs a key role within the immune system by recognising pathogens through patterns of sugar moieties displayed on their cell surfaces and neutralising them via an antibody-independent reaction cascade. While particularly important during early childhood before the adaptive immune system is established, or when adaptive immunity is compromised, it has a protective function throughout life, neutralising invading pathogens directly and helping to stimulate and direct an effective immune response. Complement activation is initiated when complexes comprising mannose-binding lectin (MBL) or serum ficolins and MBL-associated serine protease-2 (MASP-2) bind to pathogens. Binding induces conformational changes in these complexes, leading to autoactivation of the MASPs, which in turn activate the downstream reaction cascade. A major goal in complement research is to understand the molecular events that trigger complement activation. Over the last few years, structure-function studies have improved our knowledge of the way in which MBL binds to MASPs by defining the portions of these proteins that interact and by solving the structures of key protein fragments. In this review, I will summarise the main findings of these studies and describe current theories to explain how the components combine to initiate the reaction cascade.     

The role of complement in CD4+ T cell homeostasis and effector functions

The complement system is among the evolutionary oldest ‘players’ of the immune system. It was discovered in 1896 by Jules Bordet as a heat-labile fraction of the serum responsible for the opsonisation and subsequent killing of bacteria. The decades between the 1920s and 1990s then marked the discovery and biochemical characterization of the proteins comprising the complement system. Today, complement is defined as a complex system consisting of more than 30 membrane-bound and soluble plasma proteins, which are activated in a cascade-like manner, very similarly to the caspase proteases and blood coagulation systems. Complement is engrained in the immunologist's mind as a serum-effective, quintessential part of innate immunity, vitally required for the detection and removal of pathogens or other dangerous entities. Three decades ago, this rather confined definition was challenged and then refined when it was shown that complement participates vitally in the induction and regulation of B cell responses, thus adaptive immunity. Similarly, research work published in more recent years supports an equally important role for the complement system in shaping T cell responses. Today, we are again facing paradigm shifts in the field: complement is actively involved in the negative control of T cell effector immune responses, and thus, by definition in immune homeostasis. Further, while serum complement activity is without doubt fundamental in the defence against invading pathogens, local immune cell-derived production of complement emerges as key mediator of complement's impact on adaptive immune responses. And finally, the impact of complement on metabolic pathways and the crosstalk between complement and other immune effector systems is likely more extensive than previously anticipated and is fertile ground for future discoveries. In this review, we will discuss these emerging new roles of complement, with a focus on Th1 cell biology.     

Complement inhibition by gram-positive pathogens: molecular mechanisms and therapeutic implications

The plasma proteins of the complement system are essential in the innate immune response against bacteria. Complement labels bacteria with opsonins to support phagocytosis and generates chemoattractants to attract phagocytes to the site of infection. In turn, bacterial human pathogens have evolved different strategies to specifically impair the complement response. Here, we review the large arsenal of complement inhibitors produced by the gram-positive pathogens Staphylococcus aureus and Group A Streptococcus. We discuss how these bacterial molecules provide us with new tools to treat both infectious and inflammatory disease conditions in humans.     

The role of complement in inflammatory diseases from behind the scenes into the spotlight

Our understanding of the biology of the complement system has undergone a drastic metamorphosis since its original discovery. This system, which was traditionally primarily described as a "complement" to humoral immunity, is now perceived as a central constituent of innate immunity, defending the host against pathogens, coordinating various events during inflammation, and bridging innate and adaptive immune responses. Complement is an assembly of proteins found in the blood and body fluids and on cell surfaces. Soluble complement components form the proteolytic cascade, whose activation leads to the generation of complement effectors that target various cells involved in the immune response. Membrane-bound receptors and regulators transmit signals from complement effectors to target cells and limit complement activation to the surfaces of pathogens and damaged or activated host cells. The multiple interconnections among complement proteins, immune cells, and mediators provide an excellent mechanism to protect the organism against infections and support the repair of damaged tissues. However, disturbances in this "defense machinery" contribute to the pathogenesis of various diseases. The role of complement in various inflammatory disorders is multifaceted; for example, the activation of complement can significantly contribute to inflammation-mediated tissue damage, whereas inherited or acquired complement deficiencies highly favor the development of autoimmunity.     

Bacterial complement evasion

The human complement system is elemental to recognize bacteria, opsonize them for handling by phagocytes, or kill them by direct lysis. However, successful bacterial pathogens have in turn evolved ingenious strategies to overcome this part of the immune system. In this review we discuss the different stages of complement activation sequentially and illustrate the immune evasion strategies that various bacteria have developed to evade each subsequent step. The focus is on bacterial proteins, either surface-bound or excreted, that block complement activation. The underlying molecular mechanism of action and the possible role in pathophysiology of bacterial infections are discussed.     

Dissecting innate immunity by germline mutagenesis

The innate arm of our immune system is the first line of defence against infections. In addition, it is believed to drive adaptive immune responses, which help fight pathogens and provide long-term memory. As such, the innate immune system is instrumental for protection against pathogens that would otherwise destroy their host. Although our understanding of the innate immune components involved in pathogen sensing and fighting is improving, it is still limited. This is particularly exemplified by increased documentation of innate immune deficiencies in humans that often result in high and recurrent susceptibility to infections or even death, without the genetic cause being evident. To provide further insight into the mechanisms by which pathogen sensing and eradication occur, several strategies can be used. The current review focuses on the forward genetic approaches that have been used to dissect innate immunity in the fruit fly and the mouse. For both animal models, forward genetics has been instrumental in the deciphering of innate immunity and has greatly improved our understanding of how we respond to invading pathogens.     

Immune evasion of Borrelia burgdorferi: Insufficient killing of the pathogens by complement and antibody

The innate immune system and, in particular, the complement system play a key role in the elimination of micro-organisms after entrance in the human host. Like other pathogens, borreliae must develop strategies to inactivate host defence mechanisms. By investigating serum (NHS)-suscepti-bility of borreliae, we found that mainly B.afzelii isolates are serum-resistant, whereas the majority of B. burgdorferi s.s. isolates display an intermediate serum-sensitive phenotype. In contrast, B.garinii isolates are killed effectively by complement and therefore are classified as serum-sensitive. Up to now, we have identified two distinct proteins of 27.5 kDa and 20.7 kDa expressed on the outer surface of borreliae, which interact directly with FHL-1/reconectin and factor H, the two major regulators of the alternative complement pathway. These borrelial proteins are termed CRASPs (complement regulator-acquiring surface proteins). CRASPs are detectable only on serumresistant borreliae and, accordingly, binding of FHL-1/reconectin and factor H only occur with serum-resistant borrelial isolates. We conclude from these results that the control of complement activation on the borrelial surface is due to the interaction of borrelial CRASPs with host complement regulatory proteins. Thus, CRASPs represent an important mechanism of immune evasion on the part of borrelial isolates belonging mostly to the genospecies B.afzelii.By analysing the humoral adaptive immune response of patients, we detected sera that killed NHS-resistant borreliae. Borreliacidal activity is observed most frequently with sera of patients at stage III of the disease. The killing of NHS-resistant isolates by these immune sera always requires the combination of antibodies and complement. Bactericidal activity, however, is not detected in all immune sera at the different disease stages, although specific anti-Borrelia antibodies are present according to serological test results. This observation suggests that not all borrelial antigens are able to induce a borreliacidal immune response. In an extensive analysis of 24 immune sera, we identified up to 12 borrelial antigens, including OspC, which possess the greatest potential for the induction of borreliacidal antibody. The borreliacidal potential of anti-OspC antibodies was tested directly on an OspC-expressing borrelial wild-type isolate and a corresponding variant lacking OspC. In these studies, only the wild-type isolate expressing OspC on its surface proved positive for the lytic complement complex, thereby indicating the great importance of this antigen for the control of the infection. Additional studies are required to identify further “protective” antigens among these 12 proteins, all of which are candidates for infection control according to our studies involving patient immune sera. These antigens may include the recently detected CRASPs.     

Intracellular complement − the complosome − in immune cell regulation

The complement system was defined over a century ago based on its ability to “complement” the antibody-mediated and cell-mediated immune responses against pathogens. Today our understanding of this ancient part of innate immunity has changed substantially and we know now that complement plays an undisputed pivotal role in the regulation of both innate and adaptive immunity. The complement system consists of over 50 blood-circulating, cell-surface expressed and intracellular proteins. It is key in the recognition and elimination of invading pathogens, also in the removal of self-derived danger such as apoptotic cells, and it supports innate immune responses and the initiation of the general inflammatory reactions. The long prevailing classic view of complement was that of a serum-operative danger sensor and first line of defence system, however, recent experimental and clinical evidences have demonstrated that “local” tissue and surprisingly intracellular complement (the complosome) activation impacts on normal cell physiology. This review will focus on novel aspects of intracellular complement activation and its unexpected roles in basic cell processes such as metabolism. We also discuss what the existence of the complosome potentially means for how the host handles intracellular pathogens such as viruses.     

The role of the complement system in CNS inflammatory diseases

Complement is an important component of both the innate and adaptive immune response that contributes to host defense in a variety of mechanisms, including inflammation, phagocytosis and cell lysis. Complement proteins are produced by all cell types in the CNS, and the same effector functions that protect the host from pathogens can mediate inflammation and tissue destruction in CNS diseases, leading to neurological deficits or even death. In the last 10 years, the development of complement inhibitors and a variety of animal models for CNS diseases has revealed that targeted inhibition of complement offers significant therapeutic potential. This review discusses the subtleties of targeted complement inhibition in CNS disease as an emerging therapeutic strategy.     

Immunoglobulin M as a vaccine adjuvant

Vaccines are effective in preventing disease by stimulating the immune system and sustaining an immune response towards eradication of pathogens and diseased cells. However, designing successful vaccines is not always straightforward. For a vaccine to be successful, antigen-presenting cells (APC) need to be stimulated, primarily by adjuvants, towards a sustained immune response through integration of the innate and adaptive (humoral and cellular) immune systems. Furthermore, there is an immediate need for safe and effective adjuvants. There has been significant progress in understanding the mechanisms on how vaccines work and the role of adjuvants, dendritic cells, and the toll-like receptor (TLR) pathway. Currently, different adjuvants are actively explored but the potential of the immunoglobulin M (IgM) as a vaccine adjuvant has been overlooked. This article hypothesizes how the IgM molecule could function as a vaccine adjuvant by acting as a “soluble” toll-like receptor (TLR) through the formation of an immune complex with antigen (Ag) and other components of the innate immune system. The complex should lead to sustained humoral and/or cell-mediated immune responses. Hypothetically, it is also possible that the Ag-IgM complex recruits other components of complement or other factors that can activate other members of the adaptive immune system. As it is now possible to produce commercial-scale quantities of monoclonal human IgM antibodies, understanding the role of the IgM in linking the innate and adaptive immune systems may lead to practical therapeutic applications.     

Rapid hepatitis C virus clearance by antivirals correlates with immune status of infected patients

Altered immune parameters associated with hepatitis C virus (HCV) genotype 1b infection and their correlation with virus eradication in direct-acting antivirals (DAA)-treated patients were examined. Thirty-one HCV-infected patients were treated with DAAs for 12 weeks. Pre-DAA-treatment and post-DAA-treatment sera were analyzed for cytokines/chemokines using MILLIPLEX MAP. Serum complement level and antibody neutralization activity were measured separately. Sera from 11 spontaneously cleared HCV subjects were included for comparison. Rapid virological responders (RVR) or end-of-treatment responders (EOTR) were defined as patients with HCV RNA negative at week 4 or positive at week 4 and negative at week 12, respectively. HCV RNA eradication and a decrease in liver fibrosis-related cytokines after treatment were observed when compared with pretreatment sera from RVR and EOTR. In pretreatment sera, interferons and T-helper 1 or 2 cell-associated cytokines/chemokines were significantly higher among RVR as compared with EOTR. Furthermore, serum complement and virus neutralizing antibody levels were higher in pretreatment RVR sera. Eradication of HCV RNA by DAA decreased liver fibrosis-related cytokines. Pretreatment sera from RVR displayed an enhanced cytokine/chemokine, complement and virus neutralizing antibody response as compared with EOTR sera. Our results suggested that enhanced host immune status may play an additive role on HCV RNA clearance by DAA.     

Infectious diseases associated with complement deficiencies.

The complement system consists of both plasma and membrane proteins. The former influence the inflammatory response, immune modulation, and host defense. The latter are complement receptors, which mediate the cellular effects of complement activation, and regulatory proteins, which protect host cells from complement-mediated injury. Complement activation occurs via either the classical or the alternative pathway, which converge at the level of C3 and share a sequence of terminal components. Four aspects of the complement cascade are critical to its function and regulation: (i) activation of the classical pathway, (ii) activation of the alternative pathway, (iii) C3 convertase formation and C3 deposition, and (iv) membrane attack complex assembly and insertion. In general, mechanisms evolved by pathogenic microbes to resist the effects of complement are targeted to these four steps. Because individual complement proteins subserve unique functional activities and are activated in a sequential manner, complement deficiency states are associated with predictable defects in complement-dependent functions. These deficiency states can be grouped by which of the above four mechanisms they disrupt. They are distinguished by unique epidemiologic, clinical, and microbiologic features and are most prevalent in patients with certain rheumatologic and infectious diseases. Ethnic background and the incidence of infection are important cofactors determining this prevalence. Although complement undoubtedly plays a role in host defense against many microbial pathogens, it appears most important in protection against encapsulated bacteria, especially Neisseria meningitidis but also Streptococcus pneumoniae, Haemophilus influenzae, and, to a lesser extent, Neisseria gonorrhoeae. The availability of effective polysaccharide vaccines and antibiotics provides an immunologic and chemotherapeutic rationale for preventing and treating infection in patients with these deficiencies.     

Complement evasion of pathogens: common strategies are shared by diverse organisms

Infectious diseases represent a major health problem. Based on the limited efficacy of existing drugs and vaccines and the increasing antibiotic resistance new strategies are needed to fight infectious diseases. A better understanding of pathogen–host interaction is one important aspect to identify new virulence factors and antimicrobial and anti-inflammatory compounds utilized by pathogens represent an additional source for effective anti-inflammatory compounds. Complement forms a major defense line against invading microbes, and pathogens have learned during evolution to breach this defense line. The characterization of how pathogens evade complement attack is a rapidly developing field of current research. Pathogens mimic host surfaces and bind host complement regulators. Similarly pathogens utilize a number of complement inhibitory molecules which help to evade complement attack and which display anti-inflammatory activity. The molecular identification of these molecules, as well as the functional characterization of their roles at the pathogen–host interface is an important and emerging field of infection biology. In addition, pathogens utilize multiple sets of such regulators as redundancy and multiplicity is important for immune and complement evasion. Here we summarize the current scenarios of this emerging field which identifies multiple virulence factors and complement evasion strategies, but which at the same time reveals common mechanisms for immune and complement defense.     

Complement evasion of pathogens: Common strategies are shared by diverse organisms

Infectious diseases represent a major health problem. Based on the limited efficacy of existing drugs and vaccines and the increasing antibiotic resistance new strategies are needed to fight infectious diseases. A better understanding of pathogen–host interaction is one important aspect to identify new virulence factors and antimicrobial and anti-inflammatory compounds utilized by pathogens represent an additional source for effective anti-inflammatory compounds. Complement forms a major defense line against invading microbes, and pathogens have learned during evolution to breach this defense line. The characterization of how pathogens evade complement attack is a rapidly developing field of current research. Pathogens mimic host surfaces and bind host complement regulators. Similarly pathogens utilize a number of complement inhibitory molecules which help to evade complement attack and which display anti-inflammatory activity. The molecular identification of these molecules, as well as the functional characterization of their roles at the pathogen–host interface is an important and emerging field of infection biology. In addition, pathogens utilize multiple sets of such regulators as redundancy and multiplicity is important for immune and complement evasion. Here we summarize the current scenarios of this emerging field which identifies multiple virulence factors and complement evasion strategies, but which at the same time reveals common mechanisms for immune and complement defense.