The Complement System and C4b-Binding Protein: A Focus on the Promise of C4BPα as a Biomarker to Predict Clopidogrel Resistance.

The complement system plays a dual role in the body, either as a first-line defense barrier when balanced between activation and inhibition or as a potential driver of complement-associated injury or diseases when unbalanced or over-activated. C4b-binding protein (C4BP) was the first circulating complement regulatory protein identified and it functions as an important complement inhibitor. C4BP can suppress the over-activation of complement components and prevent the complement system from attacking the host cells through the binding of complement cleavage products C4b and C3b, working in concert as a cofactor for factor I in the degradation of C4b and C3b, and consequently preventing or reducing the assembly of C3 convertase and C5 convertase, respectively. C4BP, particularly C4BP α-chain (C4BPα), exerts its unique inhibitory effects on complement activation and opsonization, systemic inflammation, and platelet activation and aggregation. It has long been acknowledged that crosstalk or interplay exists between the complement system and platelets. Our unpublished preliminary data suggest that circulating C4BPα exerts its antiplatelet effects through inhibition of both complement activity levels and complement-induced platelet reactivity. Plasma C4BPα levels appear to be significantly higher in patients sensitive to, rather than resistant to, clopidogrel, and we suggest that a plasma C4BPα measurement could be used to predict clopidogrel resistance in the clinical settings.

Diverse Functions of C4b-Binding Protein in Health and Disease.

C4b-binding protein (C4BP) is a fluid-phase complement inhibitor that prevents uncontrolled activation of the classical and lectin complement pathways. As a complement inhibitor, C4BP also promotes apoptotic cell death and is hijacked by microbes and tumors for complement evasion. Although initially characterized for its role in complement inhibition, there is an emerging recognition that C4BP functions in a complement-independent manner to promote cell survival, protect against autoimmune damage, and modulate the virulence of microbial pathogens. In this Brief Review, we summarize the structure and functions of human C4BP, with a special focus on activities that extend beyond the canonical role of C4BP in complement inhibition.

Recruitment of C4b-binding protein is not a complement evasion strategy employed by Staphylococcus aureus.

Complement offers a first line of defence against infection through the opsonization of microbial pathogens, recruitment of professional phagocytes to the infection site and the coordination of inflammatory responses required for the resolution of infection. Staphylococcus aureus is a successful pathogen that has developed multiple mechanisms to thwart host immune responses. Understanding the precise strategies employed by S. aureus to bypass host immunity will be paramount for the development of vaccines and or immunotherapies designed to prevent or limit infection. To gain a better insight into the specific immune evasion mechanisms used by S. aureus we examined the pathogen's interaction with the soluble complement inhibitor, C4b-binding protein (C4BP). Previous studies indicated that S. aureus recruits C4BP using a specific cell-wall-anchored surface protein and that bound C4BP limits complement deposition on the staphylococcal surface. Using flow-cytometric-based bacterial-protein binding assays we observed no interaction between S. aureus and C4BP. Moreover, we offer a precautionary warning that C4BP isolated from plasma can be co-purified with minute quantities of human IgG, which can distort binding analysis between S. aureus and human-derived proteins. Combined our data indicates that recruitment of C4BP is not a complement evasion strategy employed by S. aureus.

C4b-binding protein inhibits particulate- and crystalline-induced NLRP3 inflammasome activation.

Dysregulated NLRP3 inflammasome activation drives a wide variety of diseases, while endogenous inhibition of this pathway is poorly characterised. The serum protein C4b-binding protein (C4BP) is a well-established inhibitor of complement with emerging functions as an endogenously expressed inhibitor of the NLRP3 inflammasome signalling pathway. Here, we identified that C4BP purified from human plasma is an inhibitor of crystalline- (monosodium urate, MSU) and particulate-induced (silica) NLRP3 inflammasome activation. Using a C4BP mutant panel, we identified that C4BP bound these particles via specific protein domains located on the C4BP α-chain. Plasma-purified C4BP was internalised into MSU- or silica-stimulated human primary macrophages, and inhibited MSU- or silica-induced inflammasome complex assembly and IL-1ß cytokine secretion. While internalised C4BP in MSU or silica-stimulated human macrophages was in close proximity to the inflammasome adaptor protein ASC, C4BP had no direct effect on ASC polymerisation in in vitro assays. C4BP was also protective against MSU- and silica-induced lysosomal membrane damage. We further provide evidence for an anti-inflammatory function for C4BP in vivo, as C4bp-/- mice showed an elevated pro-inflammatory state following intraperitoneal delivery of MSU. Therefore, internalised C4BP is an inhibitor of crystal- or particle-induced inflammasome responses in human primary macrophages, while murine C4BP protects against an enhanced inflammatory state in vivo. Our data suggests C4BP has important functions in retaining tissue homeostasis in both human and mice as an endogenous serum inhibitor of particulate-stimulated inflammasome activation.

Complement Activation-Independent Attenuation of SARS-CoV-2 Infection by C1q and C4b-Binding Protein.

The complement system is a key component of the innate immune response to viruses and proinflammatory events. Exaggerated complement activation has been attributed to the induction of a cytokine storm in severe SARS-CoV-2 infection. However, there is also an argument for the protective role of complement proteins, given their local synthesis or activation at the site of viral infection. This study investigated the complement activation-independent role of C1q and C4b-binding protein (C4BP) against SARS-CoV-2 infection. The interactions of C1q, its recombinant globular heads, and C4BP with the SARS-CoV-2 spike and receptor binding domain (RBD) were examined using direct ELISA. In addition, RT-qPCR was used to evaluate the modulatory effect of these complement proteins on the SARS-CoV-2-mediated immune response. Cell binding and luciferase-based viral entry assays were utilised to assess the effects of C1q, its recombinant globular heads, and C4BP on SARS-CoV-2 cell entry. C1q and C4BP bound directly to SARS-CoV-2 pseudotype particles via the RBD domain of the spike protein. C1q via its globular heads and C4BP were found to reduce binding as well as viral transduction of SARS-CoV-2 spike protein expressing lentiviral pseudotypes into transfected A549 cells expressing human ACE2 and TMPRSS2. Furthermore, the treatment of the SARS-CoV-2 spike, envelope, nucleoprotein, and membrane protein expressing alphaviral pseudotypes with C1q, its recombinant globular heads, or C4BP triggered a reduction in mRNA levels of proinflammatory cytokines and chemokines such as IL-1ß, IL-8, IL-6, TNF-α, IFN-α, and RANTES (as well as NF-κB) in A549 cells expressing human ACE2 and TMPRSS2. In addition, C1q and C4BP treatment also reduced SARS-CoV-2 pseudotype infection-mediated NF-κB activation in A549 cells expressing human ACE2 and TMPRSS2. C1q and C4BP are synthesised primarily by hepatocytes; however, they are also produced by macrophages, and alveolar type II cells, respectively, locally at the pulmonary site. These findings support the notion that the locally produced C1q and C4BP can be protective against SARS-CoV-2 infection in a complement activation-independent manner, offering immune resistance by inhibiting virus binding to target host cells and attenuating the infection-associated inflammatory response.

Evasion of the complement system by Leishmania through the uptake of C4bBP, a complement regulatory protein, and probably by the action of GP63 on C4b molecules deposited on parasite surface.

The complement system is a primary component of the vertebrate innate immune system, and its activity is harmful to microorganisms and parasites. To evade complement attack, some pathogens, such as viruses, bacteria, and protozoa, can interact with complement regulatory proteins from their hosts. Our research group has described the ability of Leishmania species to bind Factor H from human serum and use it as a tool to evade the complement system. However, there is no description of the interaction of Leishmania with other complement regulatory proteins, such as the C4b-binding protein (C4bBP), a negative regulator of classical and lectins complement system pathways. The results presented in this manuscript suggest that Leishmania infantum, L. amazonensis, and L. braziliensis recruit C4bBP from human serum. The uptake of C4bBP by L. infantum was studied in detail to improve our understanding of this inhibitory mechanism. When exposed to this complement regulator, parasites with inactivated GP63 bind to C4bBP and inactivate C4b deposited on their surface after serum exposure. This inactivation occurs by the action of Factor I, a complement system protease. In addition to the C4bBP-Factor I inactivation mechanism, the surface parasite protease GP63 can also inactivate soluble C4b molecules and probably that C4b molecules deposited on the parasites surface. This manuscript shows that Leishmania has two independent strategies to inactivate C4b molecules, preventing the progress of classical and lectins pathways. The identification of the C4bBP receptor on the Leishmania membrane may provide a new vaccine target to fight leishmaniasis.

Neisseria gonorrhoeae co-opts C4b-binding protein to enhance complement-independent survival from neutrophils.

Neisseria gonorrhoeae (Gc) is a human-specific pathogen that causes the sexually transmitted infection gonorrhea. Gc survives in neutrophil-rich gonorrheal secretions, and recovered bacteria predominantly express phase-variable, surface-expressed opacity-associated (Opa) proteins (Opa+). However, expression of Opa proteins like OpaD decreases Gc survival when exposed to human neutrophils ex vivo. Here, we made the unexpected observation that incubation with normal human serum, which is found in inflamed mucosal secretions, enhances survival of Opa+ Gc from primary human neutrophils. We directly linked this phenomenon to a novel complement-independent function for C4b-binding protein (C4BP). When bound to the bacteria, C4BP was necessary and sufficient to suppress Gc-induced neutrophil reactive oxygen species production and prevent neutrophil phagocytosis of Opa+ Gc. This research identifies for the first time a complement-independent role for C4BP in enhancing the survival of a pathogenic bacterium from phagocytes, thereby revealing how Gc exploits inflammatory conditions to persist at human mucosal surfaces.

Interactions of Candida tropicalis pH-related antigen 1 with complement proteins C3, C3b, factor-H, C4BP and complement evasion.

Candida, as a part of the human microbiota, can cause opportunistic infections that are either localised or systemic candidiasis. Emerging resistance to the standard antifungal drugs is associated with increased mortality rate due to invasive Candida infections, particularly in immunocompromised patients. While there are several species of Candida, an increasing number of Candida tropicalis isolates have been recently reported from patients with invasive candidiasis or inflammatory bowel diseases. In order to establish infections, C. tropicalis has to adopt several strategies to escape the host immune attack. Understanding the immune evasion strategies is of great importance as these can be exploited as novel therapeutic targets. C. albicans pH-related antigen 1 (CaPra1), a surface bound and secretory protein, has been found to interact strongly with the immune system and help in complement evasion. However, the role of C. tropicalis Pra1 (CtPra1) and its interaction with the complement is not studied yet. Thus, we characterised how pH-related antigen 1 of C. tropicalis (CtPra1) interacts with some of the key complement proteins of the innate immune system. CtPra1 was recombinantly produced using a Kluyveromyces lactis yeast expression system. Recombinant CtPra1, was found to bind human C3 and C3b, central molecules of the complement pathways that are important components of the innate immune system. It was also found to bind human complement regulatory proteins factor-H and C4b-binding protein (C4BP). CtPra1-factor-H and CtPra1-C4BP interactions were found to be ionic in nature as the binding intensity affected by high sodium chloride concentrations. CtPra1 inhibited functional complement activation with different effects on classical (∼20 %), lectin (∼25 %) and alternative (∼30 %) pathways. qPCR experiments using C. tropicalis clinical isolates (oral, blood and peritoneal fluid) revealed relatively higher levels of expression of CtPra1 gene when compared to the reference strain. Native CtPra1 was found to be expressed both as membrane-bound and secretory forms in the clinical isolates. Thus, C. tropicalis appears to be a master of immune evasion by using Pra1 protein. Further investigation using in-vivo models will help ascertain if these proteins can be novel therapeutic targets.

Hijacking the human complement inhibitor C4b-binding protein by the sporozoite stage of the Plasmodium falciparum parasite.

The complement system is considered the first line of defense against pathogens. Hijacking complement regulators from blood is a common evasion tactic of pathogens to inhibit complement activation on their surfaces. Here, we report hijacking of the complement C4b-binding protein (C4bp), the regulator of the classical and lectin pathways of complement activation, by the sporozoite (SPZ) stage of the Plasmodium falciparum parasite. This was shown by direct binding of radiolabeled purified C4bp to live SPZs as well as by binding of C4bp from human serum to SPZs in indirect immunofluorescence assays. Using a membrane-bound peptide array, peptides from the N-terminal domain (NTD) of P. falciparum circumsporozoite protein (CSP) were found to bind C4bp. Soluble biotinylated peptide covering the same region on the NTD and a recombinantly expressed NTD also bound C4bp in a dose-dependent manner. NTD-binding site on C4bp was mapped to the CCP1-2 of the C4bp α-chain, a common binding site for many pathogens. Native CSP was also co-immunoprecipitated with C4bp from human serum. Preventing C4bp binding to the SPZ surface negatively affected the SPZs gliding motility in the presence of functional complement and malaria hyperimmune IgG confirming the protective role of C4bp in controlling complement activation through the classical pathway on the SPZ surface. Incorporating the CSP-C4bp binding region into a CSP-based vaccine formulation could induce vaccine-mediated immunity that neutralizes this immune evasion region and increases the vaccine efficacy.

Proteomic analysis of small extracellular vesicles from the plasma of patients with hepatocellular carcinoma.

PURPOSE: Liver cancer is one of the most common tumors with the seventh-highest incidence and the third-highest mortality. Many studies have shown that small extracellular vesicles (sEVs) play an important role in liver cancer. Here, we report comprehensive signatures for sEV proteins from plasma obtained from patients with hepatocellular carcinoma (HCC), which might be valuable for the evaluation and diagnosis of HCC. METHODS: We extracted sEVs from the plasma of controls and patients with HCC. Differentially expressed proteins in the sEVs were analyzed using label-free quantification and bioinformatic analyses. Western blotting (WB) was used to validate the abovementioned sEV proteins. RESULTS: Proteomic analysis was performed for plasma sEVs from 21 patients with HCC and 15 controls. Among the 335 identified proteins in our study, 27 were significantly dysregulated, including 13 upregulated proteins that were involved predominantly in the complement cascade (complement C1Q subcomponent subunit B (C1QB), complement C1Q subcomponent subunit C (C1QC), C4B-binding protein alpha chain (C4BPA), and C4B-binding protein beta chain (C4BPB)) and the coagulation cascade (F13B, fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), and fibrinogen gamma chain (FGG)). We verified increased levels of the C1QB, C1QC, C4BPA, and C4BPB proteins in the plasma sEVs from patients with HCC in both the discovery cohort and validation cohort. CONCLUSIONS: The complement cascade in sEVs was significantly involved in HCC progression. C1QB, C1QC, C4BPA, and C4BPB were highly abundant in the plasma sEVs from patients with HCC and might represent molecular signatures.

Exosome Proteomics Reveals the Deregulation of Coagulation, Complement and Lipid Metabolism Proteins in Gestational Diabetes Mellitus.

Exosomes are small extracellular vesicles with a variable protein cargo in consonance with cell origin and pathophysiological conditions. Gestational diabetes mellitus (GDM) is characterized by different levels of chronic low-grade inflammation and vascular dysfunction; however, there are few data characterizing the serum exosomal protein cargo of GDM patients and associated signaling pathways. Eighteen pregnant women were enrolled in the study: 8 controls (CG) and 10 patients with GDM. Blood samples were collected from patients, for exosomes' concentration. Protein abundance alterations were demonstrated by relative mass spectrometric analysis and their association with clinical parameters in GDM patients was performed using Pearson's correlation analysis. The proteomics analysis revealed 78 significantly altered proteins when comparing GDM to CG, related to complement and coagulation cascades, platelet activation, prothrombotic factors and cholesterol metabolism. Down-regulation of Complement C3 (C3), Complement C5 (C5), C4-B (C4B), C4b-binding protein beta chain (C4BPB) and C4b-binding protein alpha chain (C4BPA), and up-regulation of C7, C9 and F12 were found in GDM. Our data indicated significant correlations between factors involved in the pathogenesis of GDM and clinical parameters that may improve the understanding of GDM pathophysiology. Data are available via ProteomeXchange with identifier PXD035673.

Quantitative Plasma Proteomics to Identify Candidate Biomarkers of Relapse in Pediatric/Adolescent Hodgkin Lymphoma.

Classical pediatric Hodgkin Lymphoma (HL) is a rare malignancy. Therapeutic regimens for its management may be optimized by establishing treatment response early on. The aim of this study was to identify plasma protein biomarkers enabling the prediction of relapse in pediatric/adolescent HL patients treated under the pediatric EuroNet-PHL-C2 trial. We used untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics at the time of diagnosis­before any therapy­as semiquantitative method to profile plasma proteins specifically associated with relapse in 42 children with nodular sclerosing HL. In both the exploratory and the validation cohorts, six proteins (apolipoprotein E, C4b-binding protein α chain, clusterin, fibrinogen γ chain, prothrombin, and vitronectin) were more abundant in the plasma of patients whose HL relapsed (|fold change| ≥ 1.2, p < 0.05, Student's t-test). Predicting protein function with the Gene Ontology classification model, the proteins were included in four biological processes (p < 0.01). Using immunoblotting and Luminex assays, we validated two of these candidate biomarkers­C4b-binding protein α chain and clusterin­linked to innate immune response function (GO:0045087). This study identified C4b-binding protein α chain and clusterin as candidate early plasma biomarkers of HL relapse, and important for the purpose of shedding light on the molecular scenario associated with immune response in patients treated under the EuroNet-PHL-C2 trial.

Potential biomarkers of spinal dural arteriovenous fistula: C4BPA and C1QA.

BACKGROUND AND PURPOSE: A major challenge in spinal dural arteriovenous fistula (SDAVF) is timely diagnosis, but no specific predictive biomarkers are known. METHODS: In the discovery cohort (case, n = 8 vs. control, n = 8), we used cerebrospinal fluid (CSF) and paired plasma samples to identify differentially expressed proteins by label-free quantitative proteomics. Further bioinformatics enrichment analyses were performed to screen target proteins. Finally, it was validated by ELISA in two of the new cohorts (case, n = 17 vs. control, n = 9), and univariate analysis, simple linear regression, and receiver operator characteristic (ROC) curve analysis were performed to evaluate the diagnostic potential. RESULTS: In the discovery cohort, the most overexpressed proteins were APOB and C4BPA in CSF samples of patients. The GO/KEGG enrichment analysis indicated that the upregulated proteins were mainly involved in the acute inflammatory response and complement activation. Hub-gene analysis revealed that APP might be the key protein in the molecular interaction network. In the validation cohort, C4BPA and C1QA were significantly overexpressed in the CSF of patients, averaging 3046.9 ng/ml and 2167.2 ng/ml, respectively. Simple linear regression demonstrated that levels of C1QA and C4 were positively correlated with total protein in CSF (R2 = 0.8021, p = 0.0005; R2 = 0.7447, p = 0.0013). The areas under the ROC curves of C4BPA and C1QA were 0.86 and 1.00, respectively. CONCLUSIONS: This study was the first to identify C4BPA and C1QA as potential biomarkers for the diagnosis of SDAVF and revealed that complement pathway activation might be one of the molecular mechanisms for venous hypertension myelopathy.

The Hidden Side of Complement Regulator C4BP: Dissection and Evaluation of Its Immunomodulatory Activity.

C4b-binding protein (C4BP) is a well-known regulator of the complement system that holds additional and important activities unrelated to complement inhibition. Recently, we have described a novel immunomodulatory activity in the minor C4BP(ß-) isoform directly acting over inflammatory phagocytes. Here we show that incorporation of the ß-chain to the C4BP α-chain oligomer interferes with this immunomodulatory activity of C4BP. Moreover, an oligomeric form including only the complement control protein 6 (CCP6) domain of the C4BP α-chain (PRP6-HO7) is sufficient to "reprogram" monocyte-derived DCs (Mo-DCs) from a pro-inflammatory and immunogenic phenotype to an anti-inflammatory and tolerogenic state. PRP6-HO7 lacks complement regulatory activity but retains full immunomodulatory activity over inflammatory Mo-DCs induced by TLRs, characterized by downregulation of relevant surface markers such as CD83, HLA-DR, co-stimulatory molecules such as CD86, CD80 and CD40, and pro-inflammatory cytokines such as IL-12 and TNF-α. Furthermore, PRP6-HO7-treated Mo-DCs shows increased endocytosis, significantly reduced CCR7 expression and CCL21-mediated chemotaxis, and prevents T cell alloproliferation. Finally, PRP6-HO7 shows also full immunomodulatory activity over Mo-DCs isolated from lupus nephritis patients with active disease, even without further pro-inflammatory stimulation. Therefore PRP6-HO7, retaining the immunomodulatory activity of C4BP(ß-) and lacking its complement regulatory activity, might represent a promising and novel alternative to treat autoimmune diseases.

Laminin G1 residues of protein S mediate its TFPI cofactor function and are competitively regulated by C4BP.

Protein S is a cofactor in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. It enhances TFPIα-mediated inhibition of factor (F)Xa activity and generation. The enhancement is dependent on a TFPIα-protein S interaction involving TFPIα Kunitz 3 and protein S laminin G-type (LG)-1. C4b binding protein (C4BP), which binds to protein S LG1, almost completely abolishes its TFPI cofactor function. However, neither the amino acids involved in TFPIα enhancement nor the mechanisms underlying the reduced TFPI cofactor function of C4BP-bound protein S are known. To screen for functionally important regions within protein S LG1, we generated 7 variants with inserted N-linked glycosylation attachment sites. Protein S D253T and Q427N/K429T displayed severely reduced TFPI cofactor function while showing normal activated protein C (APC) cofactor function and C4BP binding. Based on these results, we designed 4 protein S variants in which 4 to 6 surface-exposed charged residues were substituted for alanine. One variant, protein S K255A/E257A/D287A/R410A/K423A/E424A, exhibited either abolished or severely reduced TFPI cofactor function in plasma and FXa inhibition assays, both in the presence or absence of FV-short, but retained normal APC cofactor function and high-affinity C4BP binding. The C4BP ß-chain was expressed to determine the mechanisms behind the reduced TFPI cofactor function of C4BP-bound protein S. Like C4BP-bound protein S, C4BP ß-chain-bound protein S had severely reduced TFPI cofactor function. These results show that protein S Lys255, Glu257, Asp287, Arg410, Lys423, and Glu424 are critical for protein S-mediated enhancement of TFPIα and that binding of the C4BP ß-chain blocks this function.

Serum Complement Activation by C4BP-IgM Fusion Protein Can Restore Susceptibility to Antibiotics in Neisseria gonorrhoeae.

Neisseria gonorrhoeae is the etiological agent of gonorrhea, the second most common bacterial sexually transmitted infection worldwide. Reproductive sequelae of gonorrhea include infertility, ectopic pregnancy and chronic pelvic pain. Most antibiotics currently in clinical use have been rendered ineffective due to the rapid spread of antimicrobial resistance among gonococci. The developmental pipeline of new antibiotics is sparse and novel therapeutic approaches are urgently needed. Previously, we utilized the ability of N. gonorrhoeae to bind the complement inhibitor C4b-binding protein (C4BP) to evade killing by human complement to design a chimeric protein that linked the two N-terminal gonococcal binding domains of C4BP with the Fc domain of IgM. The resulting molecule, C4BP-IgM, enhanced complement-mediated killing of gonococci. Here we show that C4BP-IgM induced membrane perturbation through complement deposition and membrane attack complex pore insertion facilitates the access of antibiotics to their intracellular targets. Consequently, bacteria become more susceptible to killing by antibiotics. Remarkably, C4BP-IgM restored susceptibility to azithromycin of two azithromycin-resistant clinical gonococcal strains because of overexpression of the MtrC-MtrD-MtrE efflux pump. Our data show that complement activation can potentiate activity of antibiotics and suggest a role for C4BP-IgM as an adjuvant for antibiotic treatment of drug-resistant gonorrhea.

Nontypeable Haemophilus influenzae P5 Binds Human C4b-Binding Protein, Promoting Serum Resistance.

Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes infections mainly in the upper and lower respiratory tract. The bacterium is associated with bronchitis and exacerbations in patients suffering from chronic obstructive pulmonary disease and frequently causes acute otitis media in preschool children. We have previously demonstrated that the binding of C4b binding protein (C4BP) is important for NTHi complement evasion. In this study, we identified outer membrane protein 5 (P5) of NTHi as a novel ligand of C4BP. Importantly, we observed significantly lower C4BP binding and decreased serum resistance in P5-deficient NTHi mutants. Surface expression of recombinant P5 on Escherichia coli conferred C4BP binding and consequently increased serum resistance. Moreover, P5 expression was positively correlated with C4BP binding in a series of clinical isolates. We revealed higher levels of P5 surface expression and consequently more C4BP binding in isolates from the lower respiratory tract of chronic obstructive pulmonary disease patients and tonsil specimens compared with isolates from the upper respiratory tract and the bloodstream (invasive strains). Our results highlight P5 as an important protein for protecting NTHi against complement-mediated killing.

Immunoexpression of C4 Binding Protein in Oral Leukoplakia and Oral Squamous Cell Carcinoma.

BACKGROUND: The immune evasion of dysplastic cells plays an important role in suppressing the immune response and progression of malignancy. The role of the complement inhibitors in the development of oral epithelial dysplastic lesions and squamous cell carcinoma (SCC) is still unclear. OBJECTIVE: This study aimed to assess the expression of C4 binding protein (C4BP) as a complement inhibitor in oral squamous cell carcinoma and leukoplakia. METHODS: In this study, 94 samples were classified into four groups: leukoplakia with mild to moderate dysplasia, leukoplakia with severe dysplasia or carcinoma in situ, early invasive SCC, and invasive SCC. The expression of C4BP marker was evaluated by immunohistochemistry (IHC) and real-time PCR. The results were analyzed by the Kruskal-Wallis, Bonferroni adjusted Dunn's multiple comparison, and one-way ANOVA tests. RESULTS: The results of IHC revealed the expression patterns of C4BP in oral dysplasia and SCC, and indicated that the C4BP expression was not significantly different between different histopathological grades in epithelial cells and vessels (P=0.157 and P=0.123, respectively) but, it was significantly different in fibroblasts and lymphocytes (P=0.017 and P=0.043, respectively). The real-time PCR showed a significant correlation between the dysplasia grade and expression of C4BP (P<0.05). CONCLUSION: According to the results, C4BP is expressed in the cancerous tissue by the tumor cells and their surrounding stroma. In addition, upregulation of the C4BP gene as an inhibitor of the complement system is a possible strategy adopted by the tumor cells to evade the immune system.

C4b-binding protein α-chain enhances antitumor immunity by facilitating the accumulation of tumor-infiltrating lymphocytes in the tumor microenvironment in pancreatic cancer.

BACKGROUND: Recent studies indicate that complement plays pivotal roles in promoting or suppressing cancer progression. We have previously identified C4b-binding protein α-chain (C4BPA) as a serum biomarker for the early detection of pancreatic ductal adenocarcinoma (PDAC). However, its mechanism of action remains unclear. Here, we elucidated the functional roles of C4BPA in PDAC cells and the tumor microenvironment. METHODS: We assessed stromal C4BPA, the C4BPA binding partner CD40, and the number of CD8+ tumor-infiltrating lymphocytes in resected human PDAC tissues via immunohistochemical staining. The biological functions of C4BPA were investigated in peripheral blood mononuclear cells (PBMCs) and human PDAC cell lines. Mouse C4BPA (mC4BPA) peptide, which is composed of 30 amino acids from the C-terminus and binds to CD40, was designed for further in vitro and in vivo experiments. In a preclinical experiment, we assessed the efficacy of gemcitabine plus nab-paclitaxel (GnP), dual immune checkpoint blockades (ICBs), and mC4BPA peptide in a mouse orthotopic transplantation model. RESULTS: Immunohistochemical analysis revealed that high stromal C4BPA and CD40 was associated with favorable PDAC prognosis (P=0.0005). Stromal C4BPA strongly correlated with the number of CD8+ tumor-infiltrating lymphocytes (P=0.001). In in vitro experiments, flow cytometry revealed that recombinant human C4BPA (rhC4BPA) stimulation increased CD4+ and CD8+ T cell numbers in PBMCs. rhC4BPA also promoted the proliferation of CD40-expressing PDAC cells. By contrast, combined treatment with gemcitabine and rhC4BPA increased PDAC cell apoptosis rate. mC4BPA peptide increased the number of murine T lymphocytes in vitro and the number of CD8+ tumor-infiltrating lymphocytes surrounding PDAC tumors in vivo. In a preclinical study, GnP/ICBs/mC4BPA peptide treatment, but not GnP treatment, led to the accumulation of a greater number of CD8+ T cells in the periphery of PDAC tumors and to greater tumor regression than did control treatment. CONCLUSIONS: These findings demonstrate that the combination of GnP therapy with C4BPA inhibits PDAC progression by promoting antitumor T cell accumulation in the tumor microenvironment.

The Complex Relationship between C4b-Binding Protein, Warfarin, and Antiphospholipid Antibodies.

BACKGROUND: Low levels of total C4b-binding protein (C4BPt), a circulating inhibitor of the classical/lectin complement pathways, were observed in patients with antiphospholipid antibodies (aPLs) and during warfarin treatment. OBJECTIVES: To investigate the associations between aPL and C4BPt in patients with persistently positive (++) aPL, with/without clinical manifestations and systemic lupus erythematosus (SLE), and in controls. Furthermore, we explored the impact of anticoagulation on C4BPt and in relation to complement activation. METHODS: In a cross-sectional design we investigated defined subgroups: primary (p) antiphospholipid syndrome (APS, N = 67), aPL++ individuals without clinical manifestations (aPL carriers, N = 15), SLE-aPL++ (N = 118, among them, secondary [s] APS, N = 56), aPL negative (-) SLE (SLE-aPL-, N = 291), and 322 controls. Clinical characteristics, including treatment, were tabulated. C4BPt was determined with a magnetic bead method. Complement proteins (C1q, C2, C3, C4, C3a, C3dg, sC5b-9, factor I [FI]) were measured. A mediation analysis was performed to decompose the total effect of aPL++ on C4BPt into the direct and indirect effects of aPL++ through warfarin. RESULTS: Overall, C4BPt is 20% decreased in aPL++ patients, regardless of SLE, APS, clinical manifestations, and aPL profile. C4BPt levels associate positively with complement proteins C1q, C2, C3, and C4, and negatively with complement activation product C3dg. In the SLE group, warfarin treatment contributes to approximately half of the C4BPt reduction (9%) CONCLUSION: Both aPLs and warfarin are associated with C4BPt reduction. Complement activation in aPL++ patients may partly be explained by impaired inhibition through depressed C4BPt levels. Further studies are needed to understand the clinical implications.