Rationale for targeting complement in COVID-19.

A novel coronavirus, SARS-CoV-2, has recently emerged in China and spread internationally, posing a health emergency to the global community. COVID-19 caused by SARS-CoV-2 is associated with an acute respiratory illness that varies from mild to the life-threatening acute respiratory distress syndrome (ARDS). The complement system is part of the innate immune arsenal against pathogens, in which many viruses can evade or employ to mediate cell entry. The immunopathology and acute lung injury orchestrated through the influx of pro-inflammatory macrophages and neutrophils can be directly activated by complement components to prime an overzealous cytokine storm. The manifestations of severe COVID-19 such as the ARDS, sepsis and multiorgan failure have an established relationship with activation of the complement cascade. We have collected evidence from all the current studies we are aware of on SARS-CoV-2 immunopathogenesis and the preceding literature on SARS-CoV-1 and MERS-CoV infection linking severe COVID-19 disease directly with dysfunction of the complement pathways. This information lends support for a therapeutic anti-inflammatory strategy against complement, where a number of clinically ready potential therapeutic agents are available.

Functional anatomy of complement factor H.

Factor H (FH) is a soluble regulator of the proteolytic cascade at the core of the evolutionarily ancient vertebrate complement system. Although FH consists of a single chain of similar protein modules, it has a demanding job description. Its chief role is to prevent complement-mediated injury to healthy host cells and tissues. This entails recognition of molecular patterns on host surfaces combined with control of one of nature's most dangerous examples of a positive-feedback loop. In this way, FH modulates, where and when needed, an amplification process that otherwise exponentially escalates the production of the pro-inflammatory, pro-phagocytic, and pro-cytolytic cleavage products of complement proteins C3 and C5. Mutations and single-nucleotide polymorphisms in the FH gene and autoantibodies against FH predispose individuals to diseases, including age-related macular degeneration, dense-deposit disease, and atypical hemolytic uremic syndrome. Moreover, deletions or variations of genes for FH-related proteins also influence the risk of disease. Numerous pathogens hijack FH and use it for self-defense. As reviewed herein, a molecular understanding of FH function is emerging. While its functional oligomeric status remains uncertain, progress has been achieved in characterizing its three-dimensional architecture and, to a lesser extent, its intermodular flexibility. Models are proposed, based on the reconciliation of older data with a wealth of recent evidence, in which a latent circulating form of FH is activated by its principal target, C3b tethered to a self-surface. Such models suggest hypotheses linking sequence variations to pathophysiology, but improved, more quantitative, functional assays and rigorous data analysis are required to test these ideas.

Genetic susceptibility to meningococcal infection.

Meningococcal disease is caused by a limited range of clonal complexes of Neisseria meningitidis. The disease occurs in people who lack bactericidal antibodies to this pathogen, and therefore the patients are reliant on innate immunity or components of acquired immunity other than bactericidal antibodies. Gene variants that influence the function of innate and acquired immune response components have been associated with altered host susceptibility to meningococcal disease, and some genetic factors have also been associated with more severe disease. Identification of genetic factors associated with meningococcal disease will enhance our understanding of this rare but dangerous condition which causes death and serious morbidity in young, previously fit individuals. Genetic variations in the gene cluster encoding IL-1 and in key genes including TNF, SP-A2 and CFH have been associated with susceptibility to meningococcal disease. Understanding the mechanisms underlying genetic susceptibility to meningococcal disease will permit the development of novel therapeutic measures for the treatment of Gram-negative sepsis. To enable the discovery of new mechanisms of the disease, future research will move away from small-scale association studies and instead include analysis of large patient cohorts with accurately linked clinical and demographic information.

Inhibition of CXCL10 release by monomeric C3bi and C4b.

Cellulose acetate (CA) beads are often used for leucocyte apheresis therapy against inflammatory bowel disease. In order to clarify the mechanism of the anti-inflammatory effects of CA, global analysis of the molecules generated in blood by the interaction with CA beads was performed in this study. An activated medium was collected from whole blood that had been preincubated with CA beads, and the effects of the CA-activated medium on leucocyte function were investigated. Fresh blood was stimulated with lipopolysaccharide (LPS) or interferon (IFN)-ß in the presence of the activated medium, and levels of chemokines and cytokines, including CXCL10 (IFN-inducible protein-10), and phosphorylated STAT1 (signal transducer and activator of transcription 1), which is known to be essential for CXCL10 production in leucocytes, were measured. IFN-ß- or LPS-induced CXCL10 production, expression of CXCL10 mRNA and phosphorylation of STAT1 were significantly reduced in the presence of the medium pretreated with CA beads compared with the control without the CA bead treatment. The factors inhibiting CXCL10 production were identified as the C3 and C4 fragments by mass spectrometry. The monomeric C3bi and C4b proteins were abundant in the medium pretreated with CA beads. Furthermore, purified C3bi and C4b were found to inhibit IFN-ß-induced CXCL10 production and STAT1 phosphorylation. Thus, STAT1-mediated CXCL10 production induced by stimulation with LPS or IFN was potently inhibited by monomeric C3bi and C4b generated by the interaction of blood with CA beads. These mechanisms mediated by monomeric C3bi and C4b may be involved in the anti-inflammatory effects of CA.

C3b-induced eosinophil degranulation involves PI3-kinases and is inhibited by protein kinase C activity.

Selective release of individual eosinophil granule proteins has been demonstrated in eosinophilic conditions and in vitro using different stimuli. The aim of this study was to investigate if selective release of eosinophil cationic protein (ECP), eosinophil protein X/eosinophil derived-neurotoxin (EPX/EDN) and eosinophil peroxidase (EPO) could be due to the involvement of different signal transduction pathways. Peripheral blood granulocytes from healthy donors were incubated with Wortmannin, LY294002, Genistein, Staurosporine, GÖ6976 or PD98059 prior to the induction of degranulation by C3b. The released amounts of ECP, EPO and EPX/EDN were determined by immunoassays, and related to the total cell content of respective protein. Wortmannin caused a significant, dose-dependent inhibition of all three granule proteins. LY294002 (10⁻6 M) also inhibited the release of all proteins. Genistein (10⁻6 M) inhibited the release of ECP, whereas the release of EPO was increased. However, there was a tendency towards similar concentration-dependent patterns of release of all three proteins. Staurosporine (10⁻7 M), GÖ6976 (10⁻6 M) and PD98059 (10⁻5 M) caused an increased release of the three proteins. PI3-kinases play an important role in the C3b-induced release of ECP, EPO and EPX/EDN, whereas protein kinase C seems to have inhibitory effects on C3b-induced degranulation.

Factor H binding as a complement evasion mechanism for an anaerobic pathogen, Fusobacterium necrophorum.

Fusobacterium necrophorum subspecies funduliforme is an obligate anaerobic Gram-negative rod causing invasive infections such as the life-threatening Lemierre's syndrome (sore throat, septicemia, jugular vein thrombosis, and disseminated infection). The aim of our study was to understand if and how F. necrophorum avoids C activation. We studied 12 F. necrophorum subsp. funduliforme strains isolated from patients with sepsis. All strains were resistant to serum killing after a 1-h incubation in 20% serum. The bacteria bound, at different levels, the C inhibitor factor H (fH). Binding was ionic and specific in nature and occurred via sites on both the N terminus and the C terminus of fH. Bound fH remained functionally active as a cofactor for factor I in the cleavage of C3b. Interestingly, patients with the most severe symptoms carried strains with the strongest ability to bind fH. An increased C3b deposition and membrane attack complex formation on the surface of a weakly fH-binding strain was observed and its survival in serum at 3.5 h was impaired. This strain had not caused a typical Lemierre's syndrome. These data, and the fact that fH-binding correlated with the severity of disease, suggest that the binding of fH contributes to virulence and survival of F. necrophorum subsp. funduliforme in the human host. Our data show, for the first time, that an anaerobic bacterium is able to bind the C inhibitor fH to evade C attack.

Apoptotic cell-mediated immunoregulation of dendritic cells does not require iC3b opsonization.

A number of recent studies show that activation of CR3 on dendritic cells (DCs) suppresses TLR-induced TNF-alpha and IL-12 production and inhibits effective Ag presentation. Although the proposed physiologic role for these phenomena is immune suppression due to recognition of iC3b opsonized apoptotic cells by CR3, all of the aforementioned investigations used artificial means of activating CR3. We investigated whether iC3b opsonized apoptotic cells could induce the same changes reported with artificial ligands such as mAbs or iC3b-opsonized RBC. We explored the kinetics of iC3b opsonization in two models of murine cell apoptosis, gamma-irradiated thymocytes and cytokine deprivation of the IL-3 dependent cell line BaF3. Using a relatively homogenous population of early apoptotic cells (IL-3 deprived BaF3 cells), we show that iC3b opsonized apoptotic cells engage CR3, but this interaction is dispensable in mediating the anti-inflammatory effects of apoptotic cells. TLR-induced TNF-alpha and IL-12 production by bone marrow-derived DCs occurs heterogeneously, with apoptotic cells inhibiting only certain populations depending on the TLR agonist. In contrast, although apoptotic cells induced homogeneous IL-10 production by DCs, IL-10 was not necessary for the inhibition of TNF-alpha and IL-12. Furthermore, because the ability of iC3b opsonization to enhance phagocytosis of apoptotic cells has been controversial, we report that iC3b opsonization does not significantly affect apoptotic cell ingestion by DCs. We conclude that the apoptotic cell receptor system on DCs is sufficiently redundant such that the absence of CR3 engagement does not significantly affect the normal anti-inflammatory processing of apoptotic cells.

The role of complement in gonococcal infection of cervical epithelia.

Neisseria gonorrhoeae is an exclusive human pathogen that causes the sexually transmitted disease, gonorrhea. The gonococcus has developed an exquisite repertoire of mechanisms by which it is able to evade host innate and adaptive immune responses. Our previous data indicate that the predominately asymptomatic nature ofgonococcal cervicitis may, in part, be attributed to the ability of these bacteria to subvert the normal function of complement to promote cervical disease. Herein we describe the interaction of N. gonorrhoeae with the complement alternative pathway with a particular focus on the importance of this interaction in promoting gonococcal cervicitis.

Serum amyloid P aids complement-mediated immunity to Streptococcus pneumoniae.

The physiological functions of the acute phase protein serum amyloid P (SAP) component are not well defined, although they are likely to be important, as no natural state of SAP deficiency has been reported. We have investigated the role of SAP for innate immunity to the important human pathogen Streptococcus pneumoniae. Using flow cytometry assays, we show that SAP binds to S. pneumoniae, increases classical pathway-dependent deposition of complement on the bacteria, and improves the efficiency of phagocytosis. As a consequence, in mouse models of infection, mice genetically engineered to be SAP-deficient had an impaired early inflammatory response to S. pneumoniae pneumonia and were unable to control bacterial replication, leading to the rapid development of fatal infection. Complement deposition, phagocytosis, and control of S. pneumoniae pneumonia were all improved by complementation with human SAP. These results demonstrate a novel and physiologically significant role for SAP for complement-mediated immunity against an important bacterial pathogen, and provide further evidence for the importance of the classical complement pathway for innate immunity.

Role of the C3b-binding site on C4b-binding protein in regulating classical pathway C5 convertase.

A high affinity C5 convertase is generated when a C3 convertase deposits additional C3b molecules on and around itself thereby switching the substrate specificity of C3 convertase from C3 to C5. In the present study the role of the additional C3b molecules in influencing the regulation of classical pathway C5 convertase by C4b-binding protein (C4BP) was examined and compared to its precursor, the C3 convertase. Determination of IC(50) for inhibiting formation of the high affinity C5 convertase and for enhancing its decay (72 and 20 nM) were found to be similar to those obtained for the surface-bound C3 convertase (35 and 11 nM). No difference was observed in the cofactor activity of C4BP for surface-bound C4b alone or when in complex with C3b. Analysis of binding interactions between C4BP and EAC1,C4b cells revealed an average apparent dissociation constant (12 nM) similar to that obtained with EAC1,C4b cells with C3b on them (11 nM). Increasing the C4b or C3b density on the cell surface did not alter the affinity of C4BP. The data suggest that C4BP regulates the C5 convertase by mechanisms similar to those observed for the C3 convertase. Since the IC(50) for inhibiting formation of the soluble C3 convertase (5 nM) is 50-80-fold below the normal serum concentration of C4BP (250-400 nM), C4BP in blood effectively prevents formation of classical pathway C3 convertase in the fluid phase. Although deposition of additional C3b molecules is necessary to convert a C3 convertase to a high affinity C5 convertase, the additional C3b molecules play no role in the regulation of C5 convertase by C4BP.

Yeast beta-glucan amplifies phagocyte killing of iC3b-opsonized tumor cells via complement receptor 3-Syk-phosphatidylinositol 3-kinase pathway.

Anti-tumor mAbs hold promise for cancer therapy, but are relatively inefficient. Therefore, there is a need for agents that might amplify the effectiveness of these mAbs. One such agent is beta-glucan, a polysaccharide produced by fungi, yeast, and grains, but not mammalian cells. Beta-glucans are bound by C receptor 3 (CR3) and, in concert with target-associated complement fragment iC3b, elicit phagocytosis and killing of yeast. Beta-glucans may also promote killing of iC3b-opsonized tumor cells engendered by administration of anti-tumor mAbs. In this study, we report that tumor-bearing mice treated with a combination of beta-glucan and an anti-tumor mAb show almost complete cessation of tumor growth. This activity evidently derives from a 25-kDa fragment of beta-glucan released by macrophage processing of the parent polysaccharide. This fragment, but not parent beta-glucan, binds to neutrophil CR3, induces CBRM 1/5 neoepitope expression, and elicits CR3-dependent cytotoxicity. These events require phosphorylation of the tyrosine kinase, Syk, and consequent PI3K activation because beta-glucan-mediated CR3-dependent cytotoxicity is greatly decreased by inhibition of these signaling molecules. Thus, beta-glucan enhances tumor killing through a cascade of events, including in vivo macrophage cleavage of the polysaccharide, dual CR3 ligation, and CR3-Syk-PI3K signaling. These results are important inasmuch as beta-glucan, an agent without evident toxicity, may be used to amplify tumor cell killing and may open new opportunities in the immunotherapy of cancer.

Demonstration of factor H-like protein 1 binding to Treponema denticola, a pathogen associated with periodontal disease in humans.

Treponema denticola is an important contributor to periodontal disease. In this study we investigated the ability of T. denticola to bind the complement regulatory proteins factor H and factor H-like protein 1 (FHL-1). The binding of these proteins has been demonstrated to facilitate evasion of the alternative complement cascade and/or to play a role in adherence and invasion. Here we demonstrate that T. denticola specifically binds FHL-1 via a 14-kDa, surface-exposed protein that we designated FhbB. Consistent with its FHL-1 binding specificity, FhbB binds only to factor H recombinant fragments spanning short consensus repeats (SCRs) 1 to 7 (H7 construct) and not to SCR constructs spanning SCRs 8 to 15 and 16 to 20. Binding of H7 to FhbB was inhibited by heparin. The specific involvement of SCR 7 in the interaction was demonstrated using an H7 mutant (H7AB) in which specific charged residues in SCR 7 were replaced by alanine. This construct lost FhbB binding ability. Analyses of the ability of FHL-1 bound to the surface of T. denticola to serve as a cofactor for factor I-mediated cleavage of C3b revealed that C3b is cleaved in an FHL-1/factor I-independent manner, perhaps by an unidentified protease. Based on the data presented here, we hypothesize that the primary function of FHL-1 binding by T. denticola might be to facilitate adherence to FHL-1 present on anchorage-dependent cells and in the extracellular matrix.

Complement receptor 3 binds the Borrelia burgdorferi outer surface proteins OspA and OspB in an iC3b-independent manner.

Persistence of borreliae within the vertebrate host depends on the fate of interactions between the spirochetes and target cells. The present work demonstrates the direct binding of the Borrelia burgdorferi outer surface proteins OspA and OspB to CR3 and that this binding is independent of iC3b.

Hemolytic uremic syndrome: an example of insufficient complement regulation on self-tissue.

Hemolytic uremic syndrome (HUS) is a triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. HUS is classified as either diarrhea associated, most commonly caused by infection with Escherichia coli O157, or the less common atypical HUS (aHUS), which may be familial or sporadic. Approximately 50% of patients with aHUS have mutations in one of the complement control proteins: factor H, factor I, or membrane cofactor protein (MCP). These proteins regulate complement activation through cofactor activity, the inactivation of C3b by limited proteolytic cleavage, a desirable event in the fluid phase (no target) or on healthy self-tissue (wrong target). Complement activation follows the endothelial cell injury that characterizes HUS. This disease represents a model of what takes place when inappropriate complement activation occurs on self-tissues due to the presence of mutated complement regulatory proteins. Screening for mutations in factor H, factor I, or MCP is expensive and time consuming. One approach is to perform antigenic screening for factor H and factor I deficiency and to look for low levels of MCP (CD46) expression by flow cytometry. Complement regulatory protein deficiency impacts treatment decisions as patients with aHUS have a recurrence rate in renal transplants of approximately 50%, whereas those with factor H mutations have an even higher risk (approximately 80%). By contrast, MCP deficiency can be corrected in part by a renal allograft. However, caution in the use of live-related donations is needed because of the high rates of incomplete penetrance of the described mutations.

Cell surface activation of the alternative complement pathway by the fusion protein of measles virus.

Measles virus (MV)-infected cells are activators of the alternative human complement pathway, resulting in high deposition of C3b on the cell surface. Activation was observed independent of whether CD46 was used as a cellular receptor and did not correlate with CD46 down-regulation. The virus itself was an activator of the alternative pathway and was covered by C3b/C3bi, resulting in some loss in infectivity without loss of virus binding to target cells. The cell surface expression of MV fusion (F), but not haemagglutinin, envelope protein resulted in complement activation of the Factor B-dependent alternative pathway in a dose-dependent manner and F-C3b complexes were formed. The underlying activation mechanism was not related to any decrease in cell surface expression of the complement regulators CD46 and CD55. The C3b/C3bi coating of MV-infected cells and virus should ensure enhanced targeting of MV antigens to the immune system, through binding to complement receptors.

Hyperglycemia-induced protein kinase C activation inhibits phagocytosis of C3b- and immunoglobulin g-opsonized yeast particles in normal human neutrophils.

The aim of this study was to investigate the effects of elevated glucose concentrations on complement receptor- and Fcgamma receptor-mediated phagocytosis in normal human neutrophils. D-Glucose at 15 or 25 mM dose-dependently inhibited both complement receptor- and Fcgamma receptor-mediated phagocytosis, as compared to that at a normal physiological glucose concentration. The protein kinase C (PKC) inhibitors GF109203X and Go6976 both dose-dependently and completely reversed the inhibitory effect of 25 mM D-glucose on phagocytosis. Complement receptor-mediated phagocytosis was dose-dependently inhibited by the cell permeable diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol (DAG), an effect that was abolished by PKC inhibitors. Furthermore, suboptimal inhibitory concentrations of DAG and glucose showed an additive inhibitory effect on complement receptor-mediated phagocytosis. The authors conclude that elevated glucose concentrations can inhibit complement receptor and Fcgamma receptor-mediated phagocytosis in normal human neutrophils by activating PKCalpha and/or PKCbeta, an effect possibly mediated by DAG.

Pig complement regulator factor H: molecular cloning and functional characterization.

Complement is an efficient defense mechanism of innate immunity. Factor H is the central complement regulator of the alternative pathway, acting in the fluid-phase and on self surfaces. Pigs are considered a suitable source for xenotransplantation and thus several membrane-bound pig complement regulators with importance for the acute rejection phase have been investigated. However, pig fluid-phase regulators have not been described so far. We report the cloning, expression and functional characterization of pig factor H. After constructing a pig liver cDNA library, a full-length factor H cDNA was isolated and sequenced. The predicted protein is organized in 20 short consensus repeat (SCR) domains and has an overall identity of 62% to the human protein. For functional characterization, three deletion constructs of pig factor H were expressed in insect cells. Pig factor H construct SCR 1-4 has cofactor activity for factor I-mediated cleavage of human C3b, which is similar to the human regulator. In addition, this N-terminal construct binds to human C3b, while a construct consisting of SCR 15-20 showed a weaker binding to human C3b/C3d. Pig factor H has two major binding sites for heparin, as the two constructs representing SCR 1-7 and SCR 15-20 proteins, but not the SCR 1-4 protein, bind heparin. The C-terminal construct is able to bind to human endothelial cells, as assayed by FACS. We show that pig and human factor H share functional characteristics in complement regulation and cell surface binding. Possible consequences of using pig livers for xenotransplantation are discussed.

iC3b arrests monocytic cell differentiation into CD1c-expressing dendritic cell precursors: a mechanism for transiently decreased dendritic cells in vivo after human skin injury by ultraviolet B.

Our previous data indicated that C3, its bioactive product iC3b, and the iC3b ligand CD11b are critical for ultraviolet-induced immunosuppression. We thus hypothesized that iC3b is an important skin-based factor regulating CD11b+ monocytic cell function in the acute post-ultraviolet period. Although monocytic cell migration peaked at 1-3 d after ultraviolet exposure of skin, dermal CD1c dendritic cells underwent a rapid and prolonged depletion that did not recover until day 7. Because ultraviolet-induced iC3b deposits are reciprocally maximal on day 3, but fade by day 7, we next hypothesized that iC3b can be responsible for the delay in differentiation into dendritic cells of monocytic cells migrating into ultraviolet-exposed skin. Analysis of dermal cells derived from keratome biopsies suggested that iC3b exposure could inhibit the development of CD1c+ dermal cells. To model newly immigrating blood monocytes entering ultraviolet-exposed, iC3b-containing dermis, purified monocytes from human blood were induced with granulocyte-macrophage colony stimulating factor to generate a population of dendritic cell precursors expressing CD1c. Incubation with iC3b markedly inhibited the appearance of CD1c+ cells (p<0.05) and induced CD1c-CD14+ cells. This inhibition was reversed by coincubation with an anti-CD11b antibody that blocks the iC3b binding site. Other functions associated with dendritic cell maturation were also inhibited by iC3b, such as interleukin-12p70 production as well as CD80 and CD40 expression. Restimulation of monocytes for DC maturation revealed that iC3b induced a temporary inhibition of DC differentiation. Thus, a human skin response in which iC3b is transiently (3-7 d) generated in dermis, such as ultraviolet, can arrest monocytic skin-infiltrating cells from undergoing dendritic cell precursor differentiation.

Cooperation between decay-accelerating factor and membrane cofactor protein in protecting cells from autologous complement attack.

Decay-accelerating factor (DAF or CD55) and membrane cofactor protein (MCP or CD46) function intrinsically in the membranes of self cells to prevent activation of autologous complement on their surfaces. How these two regulatory proteins cooperate on self-cell surfaces to inhibit autologous complement attack is unknown. In this study, a GPI-anchored form of MCP was generated. The ability of this recombinant protein and that of naturally GPI-anchored DAF to incorporate into cell membranes then was exploited to examine the combined functions of DAF and MCP in regulating complement intermediates assembled from purified alternative pathway components on rabbit erythrocytes. Quantitative studies with complement-coated rabbit erythrocyte intermediates constituted with each protein individually or the two proteins together demonstrated that DAF and MCP synergize the actions of each other in preventing C3b deposition on the cell surface. Further analyses showed that MCP's ability to catalyze the factor I-mediated cleavage of cell-bound C3b is inhibited in the presence of factors B and D and is restored when DAF is incorporated into the cells. Thus, the activities of DAF and MCP, when present together, are greater than the sum of the two proteins individually, and DAF is required for MCP to catalyze the cleavage of cell-bound C3b in the presence of excess factors B and D. These data are relevant to xenotransplantation, pharmacological inhibition of complement in inflammatory diseases, and evasion of tumor cells from humoral immune responses.