Complement or Insult: the emerging link between complement cascade deficiencies and pathology of myeloid malignancies.

The complement cascade is an ancient and highly conserved arm of the immune system. The accumulating evidence highlights elevated activity of the complement cascade in cancer microenvironment and emphasizes its effects on the immune, cancer and cancer stroma cells, pointing to a role in inflammation-mediated etiology of neoplasms. The role the cascade plays in development, progression and relapse of solid tumors is increasingly recognized, however its role in hematological malignancies, especially those of myeloid origin, has not been thoroughly assessed and remains obscure. As the role of inflammation and autoimmunity in development of myeloid malignancies is becoming recognized, in this review we focus on summarizing the links that have been identified so far for complement cascade involvement in the pathobiology of myeloid malignancies. Complement deficiencies are primary immunodeficiencies that cause an array of clinical outcomes including an increased risk of a range of infectious as well as local or systemic inflammatory and thrombotic conditions. Here, we discuss the impact that deficiencies in complement cascade initiators, mid- and terminal- components and inhibitors have on the biology of myeloid neoplasms. The emergent conclusions indicate that the links between complement cascade, inflammatory signaling and the homeostasis of hematopoietic system exist, and efforts should continue to detail the mechanistic involvement of complement cascade in the development and progression of myeloid cancers.

Activation of the complement system by nanoparticles and strategies for complement inhibition.

The complement system is a multicomponent and multifunctional arm of the innate immune system. Complement contributes to non-specific host defence and maintains homeostasis through multifaceted processes and pathways, including crosstalk with the adaptive immune system, the contact (coagulation) and the kinin systems, and alarmin high-mobility group box 1. Complement is also present intracellularly, orchestrating a wide range of housekeeping and physiological processes in both immune and nonimmune cells, thus showing its more sophisticated roles beyond innate immunity, but its roles are still controversial. Particulate drug carriers and nanopharmaceuticals typically present architectures and surface patterns that trigger complement system in different ways, resulting in both beneficial and adverse responses depending on the extent of complement activation and regulation as well as pathophysiological circumstances. Here we consider the role of complement system and complement regulations in host defence and evaluate the mechanisms by which nanoparticles trigger and modulate complement responses. Effective strategies for the prevention of nanoparticle-mediated complement activation are introduced and discussed.

Molecular basis of anaphylatoxin binding, activation, and signaling bias at complement receptors.

The complement system is a critical part of our innate immune response, and the terminal products of this cascade, anaphylatoxins C3a and C5a, exert their physiological and pathophysiological responses primarily via two GPCRs, C3aR and C5aR1. However, the molecular mechanism of ligand recognition, activation, and signaling bias of these receptors remains mostly elusive. Here, we present nine cryo-EM structures of C3aR and C5aR1 activated by their natural and synthetic agonists, which reveal distinct binding pocket topologies of complement anaphylatoxins and provide key insights into receptor activation and transducer coupling. We also uncover the structural basis of a naturally occurring mechanism to dampen the inflammatory response of C5a via proteolytic cleavage of the terminal arginine and the G-protein signaling bias elicited by a peptide agonist of C3aR identified here. In summary, our study elucidates the innerworkings of the complement anaphylatoxin receptors and should facilitate structure-guided drug discovery to target these receptors in a spectrum of disorders.

Structural biology of complement receptors.

The complement system plays crucial roles in a wide breadth of immune and inflammatory processes and is frequently cited as an etiological or aggravating factor in many human diseases, from asthma to cancer. Complement receptors encompass at least eight proteins from four structural classes, orchestrating complement-mediated humoral and cellular effector responses and coordinating the complex cross-talk between innate and adaptive immunity. The progressive increase in understanding of the structural features of the main complement factors, activated proteolytic fragments, and their assemblies have spurred a renewed interest in deciphering their receptor complexes. In this review, we describe what is currently known about the structural biology of the complement receptors and their complexes with natural agonists and pharmacological antagonists. We highlight the fundamental concepts and the gray areas where issues and problems have been identified, including current research gaps. We seek to offer guidance into the structural biology of the complement system as structural information underlies fundamental and therapeutic research endeavors. Finally, we also indicate what we believe are potential developments in the field.

Blood Transcriptome Analysis of Septic Patients Reveals a Long Non-Coding Alu-RNA in the Complement C5a Receptor 1 Gene.

Many severe inflammation conditions are complement-dependent with the complement component C5a-C5aR1 axis as an important driver. At the RNA level, the blood transcriptome undergoes programmed expression of coding and long non-coding RNAs to combat invading microorganisms. Understanding the expression of long non-coding RNAs containing Alu elements in inflammation is important for reconstructing cell fate trajectories leading to severe disease. We have assembled a pipeline for computation mining of new Alu-containing long non-coding RNAs by intersecting immune genes with known Alu coordinates in the human genome. By applying the pipeline to patient bulk RNA-seq data with sepsis, we found immune genes containing 48 Alu insertion as robust candidates for further study. Interestingly, 1 of the 48 candidates was located within the complement system receptor gene C5aR1 and holds promise as a target for RNA therapeutics.

Neutrophils Require Activation to Express Functional Cell-Surface Complement Receptor Immunoglobulin.

The phagocytosis-promoting complement receptor, Complement Receptor Immunoglobulin (CRIg), is exclusively expressed on macrophages. It has been demonstrated that expression in macrophages could be modulated by inflammatory mediators, including cytokines. This raised the possibility that a major phagocyte, the neutrophil, may also express CRIg following activation with inflammatory mediators. Here we show that resting peripheral blood neutrophil lysates subjected to protein analysis by Western blot revealed a 35 kDa CRIg isoform, consistent with the expression of CRIg mRNA by RT-PCR. By flow cytometry, CRIg was detected intracellularly and in very minor amounts on the cell surface. Interestingly, expression on the cell surface was significantly increased to functional levels after activation with inflammatory mediators/neutrophil activators; N-Formylmethionine-leucyl-phenylalanine, tumor necrosis factor (TNF), Granulocyte-Macrophage Colony stimulating Factor (GM-CSF), bacterial lipopolysaccharide, leukotriene B4 and phorbol myristate acetate. The increase in expression required p38 MAP kinase and protein kinase C activation, as well as intracellular calcium. Neutrophils which were defective in actin microfilament reorganization due to a mutation in ARPC1B or inhibition of its upstream regulator, Rac2 lose their ability to upregulate CRIg expression. Inhibition of another small GTPase, Rab27a, with pharmacological inhibitors prevented the increase in CRIg expression, suggesting a requirement for the actin cytoskeleton and exocytosis. Engagement of CRIg on TNF-primed neutrophils with an anti-CRIg monoclonal antibody increased the release of superoxide and promoted the activation of p38 but not ERK1/ERK2 or JNK MAP kinases. The TNF-induced increase in killing of Staphylococcus aureus was blocked by the anti-CRIg antibody. Adding to the anti-microbial role of CRIg, it was found that GM-CSF priming lead to the release of neutrophil extracellular traps. Interestingly in contrast to the above mediators the anti-inflammatory cytokine IL-10 caused a decrease in basal expression and GM-CSF induced increase in CRIg expression. The data demonstrate that neutrophils also express CRIg which is regulated by inflammatory mediators and cytokines. The findings show that the neutrophil antimicrobial function involving CRIg requires priming as a means of arming the cell strategically with microbial invasion of tissues and the bloodstream.

Receptor-Mediated NETosis on Neutrophils.

Neutrophil extracellular traps (NETs), a web-like structures containing chromatin, have a significant role in assisting the capture and killing of microorganisms by neutrophils during infection. The specific engagement of cell-surface receptors by extracellular signaling molecules activates diverse intracellular signaling cascades and regulates neutrophil effector functions, including phagocytosis, reactive oxygen species release, degranulation, and NET formation. However, overproduction of NETs is closely related to the occurrence of inflammation, autoimmune disorders, non-canonical thrombosis and tumor metastasis. Therefore, it is necessary to understand neutrophil activation signals and the subsequent formation of NETs, as well as the related immune regulation. In this review, we provide an overview of the immunoreceptor-mediated regulation of NETosis. The pathways involved in the release of NETs during infection or stimulation by noninfectious substances are discussed in detail. The mechanisms by which neutrophils undergo NETosis help to refine our views on the roles of NETs in immune protection and autoimmune diseases, providing a theoretical basis for research on the immune regulation of NETs.

Complement Potentiates Immune Sensing of HIV-1 and Early Type I Interferon Responses.

Complement-opsonized HIV-1 triggers efficient antiviral type I interferon (IFN) responses in dendritic cells (DCs), which play an important role in protective responses at the earliest stages in retroviral infection. In contrast, HIV-1 suppresses or escapes sensing by STING- and MAVS-associated sensors. Here, we identified a complement receptor-mediated sensing pathway, where DCs are activated in CCR5/RLR (RIG-I/MDA5)/MAVS/TBK1-dependent fashion. Increased fusion of complement-opsonized HIV-1 via complement receptor 4 and CCR5 leads to increased incoming HIV-1 RNA in the cytoplasm, sensed by a nonredundant cooperative effect of RIG-I and MDA5. Moreover, complement-opsonized HIV-1 down-modulated the MAVS-suppressive Raf-1/PLK1 pathway, thereby opening the antiviral recognition pathway via MAVS. This in turn was followed by MAVS aggregation and subsequent TBK1/IRF3/NF-κB activation in DCs exposed to complement- but not non-opsonized HIV-1. Our data strongly suggest that complement is important in the induction of efficient antiviral immune responses by preventing HIV-1 suppressive mechanisms as well as inducing specific cytosolic sensors. IMPORTANCE Importantly, our study highlights an unusual target on DCs-the α chain of complement receptor 4 (CR4) (CD11c)-for therapeutic interventions in HIV-1 treatment. Targeting CD11c on DCs mediated a potent antiviral immune response via clustering of CR4 and CCR5 and subsequent opening of an antiviral recognition pathway in DCs via MAVS. This novel finding might provide novel tools for specifically boosting endogenous antiviral immunity via CR4, abundantly expressed on multiple DC subsets.

Differential expression of complement receptors CR1/2 and CR4 by murine M1 and M2 macrophages.

Macrophages polarize into functionally divergent phenotypes - M1 and M2 - which express distinct receptors. These cells are known to express complement receptors, including CR1, CR3, and CR4. However, whether these complement receptors are differentially expressed on M1 and M2 macrophages is not yet known. Herein, we have examined the expression of CR1 to CR4 on murine bone marrow-derived M1 (stimulated with IFN-γ or LPS) and M2 (stimulated with IL-4 or IL-4 + IL-13) macrophages. We show that M1 cells exhibit increased expression of CR1/2, whereas M2 cells display the higher expression of CR4; CR3 is equally expressed on both the phenotypes. Thus, M1 cells are CR1/2+CR4+, and M2 are CR1/2-CR4+. Functional probing of these cells for their phagocytic ability indicates that M1 cells, which express higher CR1/2, internalize a significantly greater number of C3b-opsonized erythrocytes. Both M1 and M2 cells, on the other hand, internalize iC3b-opsonized erythrocytes to a similar extent. Interestingly, the phagocytic receptor involved in phagocytosis of iC3b-opsonized erythrocytes is only CR3 with no contribution of CR4. We, thus, propose that complement receptor expression can be used in combination with the expression of other known polarization markers to better locate a macrophage along its phenotypic spectrum.

Human CD3+CD56+NKT-like cells express a range of complement receptors and C3 activation has negative effects on these cell activity and effector function.

CD3+CD56+NKT-like cells are a rare population of lymphocytes that serve important roles in various types of immune-related diseases, and particularly in cancer. The complement system regulates inflammatory and immune responses by interacting with complement receptors expressed on a range of immune cells. However, whether CD3+CD56+NKT-like cells are regulated by the complement system has still not been definitively determined. In the present study, the expression of complement receptors and regulators in gated CD3+CD56+NKT-like cells isolated from human peripheral blood was assessed using PCR and flow cytometry. The results showed that human CD3+CD56+NKT-like cells expressed a range of complement receptors and regulators, such as CR3, C3aR, C5aR, C5L2, CD46 and CD55. Furthermore, the presence of complement component 3 (C3), a key component in complement activation in culture supernatant, mitigated the activity, IFN-γ production and killing function of CD3+CD56+NKT-like cells. The present study provides evidences supporting the relationship between complement activation and functional modulation of CD3+CD56+NKT-like cells, expanding our knowledge of the complement regulatory network, and also highlighting a potential target for treatment of numerous immune-related diseases, particularly NKT cell-based tumor adoptive immunotherapy.

New aspects in the regulation of human B cell functions by complement receptors CR1, CR2, CR3 and CR4.

The involvement of complement in the regulation of antibody responses has been known for long. By now several additional B cell functions - including cytokine production and antigen presentation - have also been shown to be regulated by complement proteins. Most of these important activities are mediated by receptors interacting with activation fragments of the central component of the complement system C3, such as C3b, iC3b and C3d, which are covalently attached to antigens and immune complexes. This review summarizes the role of complement receptors interacting with these ligands, namely CR1 (CD35), CR2 (CD21), CR3 (CD11b/CD18) and CR4 (CD11c/CD18) expressed by B cells in health and disease. Although we focus on human B lymphocytes, we also aim to call the attention to important differences between human and mouse systems.

Complement Receptors and Their Role in Leukocyte Recruitment and Phagocytosis.

The complement system is deeply embedded in our physiology and immunity. Complement activation generates a multitude of molecules that converge simultaneously on the opsonization of a target for phagocytosis and activation of the immune system via soluble anaphylatoxins. This response is used to control microorganisms and to remove dead cells, but also plays a major role in stimulating the adaptive immune response and the regeneration of injured tissues. Many of these effects inherently depend on complement receptors expressed on leukocytes and parenchymal cells, which, by recognizing complement-derived molecules, promote leukocyte recruitment, phagocytosis of microorganisms and clearance of immune complexes. Here, the plethora of information on the role of complement receptors will be reviewed, including an analysis of how this functionally and structurally diverse group of molecules acts jointly to exert the full extent of complement regulation of homeostasis.

Dysfunction of complement receptors CR3 (CD11b/18) and CR4 (CD11c/18) in pre-eclampsia: a genetic and functional study.

OBJECTIVE: To study genetic variants and their function within genes coding for complement receptors in pre-eclampsia. DESIGN: A case-control study. SETTING: Pre-eclampsia is a common vascular disease of pregnancy. The clearance of placenta-derived material is one of the functions of the complement system in pregnancy. POPULATION: We genotyped 500 women with pre-eclamptic pregnancies and 190 pregnant women without pre-eclampsia, as controls, from the FINNPEC cohort, and 122 women with pre-eclamptic pregnancies and 1905 controls from the national FINRISK cohort. METHODS: The functional consequences of genotypes discovered by targeted exomic sequencing were explored by analysing the binding of the main ligand iC3b to mutated CR3 or CR4, which were transiently expressed on the surface of COS-1 cells. MAIN OUTCOME MEASURES: Allele frequencies were compared between pre-eclamptic pregnancies and controls in genetic studies. The functional consequences of selected variants were measured by binding assays. RESULTS: The most significantly pre-eclampsia-linked CR3 variant M441K (P = 4.27E-4, OR = 1.401, 95% CI = 1.167-1.682) displayed a trend of increased adhesion to iC3b (P = 0.051). The CR4 variant A251T was found to enhance the adhesion of CR4 to iC3b, whereas W48R resulted in a decrease of the binding of CR4 to iC3b. CONCLUSIONS: Results suggest that changes in complement-facilitated phagocytosis are associated with pre-eclampsia. Further studies are needed to ascertain whether aberrant CR3 and CR4 activity leads to altered pro- and anti-inflammatory cytokine responses in individuals carrying the associated variants, and the role of these receptors in pre-eclampsia pathogenesis. TWEETABLE ABSTRACT: Genetic variants of complement receptors CR3 and CR4 have functional consequences that are associated with pre-eclampsia.

Autoantibodies against C5aR1, C3aR1, CXCR3, and CXCR4 are decreased in primary Sjogren's syndrome.

BACKGROUND: Networks formed of numerous autoantibodies (aabs) directed against G-protein coupled receptors (GPCR) have been suggested to play important role in autoimmune disorders. In present study, we aimed to evaluate the association between anti-GPCR antibodies and primary Sjogren's syndrome (pSS) to determine the potential pathogenic factors. METHODS: By applying a cell membrane-based ELISA technique, which is capable of detecting aabs against conformational epitopes within GPCR, serum levels of fourteen GPCR were determined in well-characterized patients with pSS (n = 52) and gender-matched healthy controls (n = 54). Comparisons between groups were analyzed by two-tailed Mann-Whitney U test, Bonferroni correction was applied for multiple comparisons. Spearman`s rank correlation coefficients were calculated between variables and visualized by heat map. RESULTS: Compared to healthy subjects, sera of patients with pSS showed significantly higher binding to ß2AR and ETAR, but lower binding to C5aR1, C3aR1, CXCR3, and CXCR4. Autoantibodies against C5aR1, C3aR1, CXCR3, and CXCR4 were also decreased in patients with rheumatoid arthritis. In pSS patients, levels of anti-CXCR3 and anti-CXCR4 antibodies were negatively correlated with circulating lymphocyte counts. Furthermore, correlation signatures of anti-GPCR antibodies changed dramatically in the patients with pulmonary involvement. CONCLUSIONS: This study demonstrates an association between pSS and autoantibodies recognizing GPCR, especially those functionally involved in immune cell migration and exocrine glandular secretion.

An Immunoregulatory Role for Complement Receptors in Murine Models of Breast Cancer.

The complement system has demonstrated roles in regulating tumor growth, although these may differ between tumor types. The current study used two murine breast cancer models (EMT6 and 4T1) to investigate whether pharmacological targeting of receptors for complement proteins C3a (C3aR) and C5a (C5aR1) is protective in murine breast cancer models. In contrast to prior studies in other tumor models, treatment with the selective C5aR1 antagonist PMX53 had no effect on tumor growth. However, treatment of mice with a dual C3aR/C5aR1 agonist (YSFKPMPLaR) significantly slowed mammary tumor development and progression. Examination of receptor expression by quantitative polymerase chain reaction (qPCR) analysis showed very low levels of mRNA expression for either C3aR or C5aR1 by EMT6 or 4T1 mammary carcinoma cell lines compared with the J774 macrophage line or bone marrow-derived macrophages. Moreover, flow cytometric analysis found no evidence of C3aR or C5aR1 protein expression by either EMT6 or 4T1 cells, leading us to hypothesize that the tumor inhibitory effects of the dual agonist are indirect, possibly via regulation of the anti-tumor immune response. This hypothesis was supported by flow cytometric analysis of tumor infiltrating leukocyte populations, which demonstrated a significant increase in T lymphocytes in mice treated with the C3aR/C5aR1 agonist. These results support an immunoregulatory role for complement receptors in primary murine mammary carcinoma models. They also suggest that complement activation peptides can influence the anti-tumor response in different ways depending on the cancer type, the host immune response to the tumor and levels of endogenous complement activation within the tumor microenvironment.

Role of Complement Receptors (CRs) on DCs in Anti-HIV-1 Immunity.

Upon entry of human immunodeficiency virus 1 (HIV-1) into the host, innate immune mechanisms are acting as a first line of defense, that considerably also modify adaptive immunity by the provision of specific signals. Innate and adaptive immune responses are intimately linked and dendritic cells (DCs) together with complement (C) play an important role in regulation of adaptive immunity. Initially, the role of complement was considered to primarily support - or COMPLEMENT - cytolytic actions of antibodies or antibody-complexed antigens (immune complexes, ICs) or directly kill the pathogens by complement-mediated lysis. Recently, the role of complement was revised and found to significantly augmenting and modulating adaptive immunity, in particular against viruses. Complement and DCs are therefore predestined to open novel avenues for antiviral research and potential therapeutic interventions. Recent studies on interactions of complement-opsonized HIV-1 with DCs demonstrated a high potential of such primed DCs to initiate efficient antiviral and cytotoxic anti-HIV-1 immunity and complement-coated viral particles shift DCs functions via CR3 and CR4 in an antithetic manner. This review will focus on our current knowledge of CR3 and CR4 actions on DCs during HIV-1 binding and the outcome of infection influenced by entry and signaling pathways.

The Role of Complement in the Mechanism of Action of Therapeutic Anti-Cancer mAbs.

Unconjugated anti-cancer IgG1 monoclonal antibodies (mAbs) activate antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells and antibody-dependent cellular phagocytosis (ADCP) by macrophages, and these activities are thought to be important mechanisms of action for many of these mAbs in vivo. Several mAbs also activate the classical complement pathway and promote complement-dependent cytotoxicity (CDC), although with very different levels of efficacy, depending on the mAb, the target antigen, and the tumor type. Recent studies have unraveled the various structural factors that define why some IgG1 mAbs are strong mediators of CDC, whereas others are not. The role of complement activation and membrane inhibitors expressed by tumor cells, most notably CD55 and CD59, has also been quite extensively studied, but how much these affect the resistance of tumors in vivo to IgG1 therapeutic mAbs still remains incompletely understood. Recent studies have demonstrated that complement activation has multiple effects beyond target cell lysis, affecting both innate and adaptive immunity mediated by soluble complement fragments, such as C3a and C5a, and by stimulating complement receptors expressed by immune cells, including NK cells, neutrophils, macrophages, T cells, and dendritic cells. Complement activation can enhance ADCC and ADCP and may contribute to the vaccine effect of mAbs. These different aspects of complement are also briefly reviewed in the specific context of FDA-approved therapeutic anti-cancer IgG1 mAbs.

Differential Interactions of Serum and Bronchoalveolar Lavage Fluid Complement Proteins with Conidia of Airborne Fungal Pathogen Aspergillus fumigatus.

Even though both cellular and humoral immunities contribute to host defense, the role played by humoral immunity against the airborne opportunistic fungal pathogen Aspergillus fumigatus has been underexplored. In this study, we aimed at deciphering the role of the complement system, the major humoral immune component, against A. fumigatus Mass spectrometry analysis of the proteins extracted from A. fumigatus conidial (asexual spores and infective propagules) surfaces opsonized with human serum indicated that C3 is the major complement protein involved. Flow cytometry and immunolabeling assays further confirmed C3b (activated C3) deposition on the conidial surfaces. Assays using cell wall components of conidia indicated that the hydrophobin RodAp, ß-(1,3)-glucan (BG) and galactomannan (GM) could efficiently activate C3. Using complement component-depleted sera, we showed that while RodAp activates C3 by the alternative pathway, BG and GM partially follow the classical and lectin pathways, respectively. Opsonization facilitated conidial aggregation and phagocytosis, and complement receptor (CR3 and CR4) blockage on phagocytes significantly inhibited phagocytosis, indicating that the complement system exerts a protective role against conidia by opsonizing them and facilitating their phagocytosis mainly through complement receptors. Conidial opsonization with human bronchoalveolar lavage fluid (BALF) confirmed C3 to be the major complement protein interacting with conidia. Nevertheless, complement C2 and mannose-binding lectin (MBL), the classical and lectin pathway components, respectively, were not identified, indicating that BALF activates the alternative pathway on the conidial surface. Moreover, the cytokine profiles were different upon stimulation of phagocytes with serum- and BALF-opsonized conidia, highlighting the importance of studying interaction of conidia with complement proteins in their biological niche.

Utilization of complement receptors in immune cell-microbe interaction.

The complement system is a major humoral component of immunity and is essential for the fast elimination of pathogens invading the body. In addition to its indispensable role in innate immunity, the complement system is also involved in pathogen clearance during the effector phase of adaptive immunity. The fastest way of killing the invader is lysis by the membrane attack complex, which is formed by the terminal components of the complement cascade. Not all pathogens are lysed however and, if opsonized by a variety of molecules, they undergo phagocytosis and disposal inside immune cells. The most important complement-derived opsonins are C1q, the first component of the classical pathway, MBL, the initiator of the lectin pathway and C3-derived activation fragments, including C3b, iC3b and C3d, which all serve as ligands for their corresponding receptors. In this review, we discuss how complement receptors are utilized by various immune cells to tackle invading microbes, or by pathogens to evade host response.