Search

Adaptive and innate immune responses contribute to hypertension and hypertensive end-organ damage. Here, we determined the role of anaphylatoxin C5a, a major inflammatory effector of the innate immune system that is generated in response to complement activation, in hypertensive end-organ damage. For this purpose, we assessed the phenotype of C5a receptor 1 (C5aR1)-deficient mice in ANG II-induced renal and cardiac injury. Expression of C5aR1 on infiltrating and resident renal as well as cardiac cells was determined using a green fluorescent protein (GFP)-C5aR1 reporter knockin mouse. Flow cytometric analysis of leukocytes isolated from the kidney of GFP-C5aR1 reporter mice showed that 28% of CD45-positive cells expressed C5aR1. Dendritic cells were identified as the major C5aR1-expressing population (88.5%) followed by macrophages and neutrophils. Using confocal microscopy, we detected C5aR1 in the kidney mainly on infiltrating cells. In the heart, only infiltrating cells stained C5aR1 positive. To evaluate the role of C5aR1 deficiency in hypertensive injury, an aggravated model of hypertension was used. Unilateral nephrectomy was performed followed by infusion of ANG II (1.5 ng·g−1·min−1) and salt in wild-type ( n = 34) and C5aR1-deficient mice ( n = 32). C5aR1-deficient mice exhibited less renal injury, as evidenced by significantly reduced albuminuria. In contrast, cardiac injury was accelerated with significantly increased cardiac fibrosis and heart weight in C5aR1-deficient mice after ANG II infusion. No effect was found on blood pressure. In summary, the C5a:C5aR1 axis drives end-organ damage in the kidney but protects from the development of cardiac fibrosis and hypertrophy in experimental ANG II-induced hypertension.

The complement system plays a key role in many acute injury states as well as chronic autoimmune and inflammatory diseases. Localized complement activation and alternative pathway-mediated amplification on diverse target surfaces promote local recruitment of pro-inflammatory cells and elaboration of other mediators. Despite a general understanding of the architecture of the system, though, many of the mechanisms that underlie site-specific complement activation and amplification in vivo are incompletely understood. In addition, there is no capability yet to measure the level of local tissue site-specific complement activation in patients without performing biopsies to detect products using immunohistochemical techniques. Herein is reviewed emerging evidence obtained through clinical research studies of human rheumatoid arthritis along with translational studies of its disease models which demonstrate that several parallel mechanisms are involved in site-specific amplification of activation of the complement system in vivo. Among these processes are de-regulation of the alternative pathway, effector pathway-catalyzed amplification of proximal complement activation, recognition of injury-associated ligands by components of the lectin pathway, and engagement of pathogenic natural antibodies that recognize a limited set of injury-associated neoepitopes. Studies suggest that each of these inter-related processes can play key roles in amplification of complement-dependent injury on self-tissues in vivo. These findings, in addition to development of an imaging strategy described herein designed to quantitatively measure local complement C3 fixation, have relevance to therapeutic and diagnostic strategies targeting the complement system.

Herpes simplex virus 2 (HSV-2) is one of the most common sexually transmitted infections globally, with a very high prevalence in many countries. During HSV-2 infection, viral particles become coated with complement proteins and antibodies, both present in genital fluids, which could influence the activation of immune responses. In genital mucosa, the primary target cells for HSV-2 infection are epithelial cells, but resident immune cells, such as dendritic cells (DCs), are also infected. DCs are the activators of the ensuing immune responses directed against HSV-2, and the aim of this study was to examine the effects opsonization of HSV-2, either with complement alone or with complement and antibodies, had on the infection of immature DCs and their ability to mount inflammatory and antiviral responses. Complement opsonization of HSV-2 enhanced both the direct infection of immature DCs and their production of new infectious viral particles. The enhanced infection required activation of the complement cascade and functional complement receptor 3. Furthermore, HSV-2 infection of DCs required endocytosis of viral particles and their delivery into an acid endosomal compartment. The presence of complement in combination with HSV-1- or HSV-2-specific antibodies more or less abolished HSV-2 infection of DCs. Our results clearly demonstrate the importance of studying HSV-2 infection under conditions that ensue in vivo , i.e., conditions under which the virions are covered in complement fragments and complement fragments and antibodies, as these shape the infection and the subsequent immune response and need to be further elucidated. IMPORTANCE During HSV-2 infection, viral particles should become coated with complement proteins and antibodies, both present in genital fluids, which could influence the activation of the immune responses. The dendritic cells are activators of the immune responses directed against HSV-2, and the aim of this study was to examine the effects of complement alone or complement and antibodies on HSV-2 infection of dendritic cells and their ability to mount inflammatory and antiviral responses. Our results demonstrate that the presence of antibodies and complement in the genital environment can influence HSV-2 infection under in vitro conditions that reflect the in vivo situation. We believe that our findings are highly relevant for the understanding of HSV-2 pathogenesis.

Glycosylphosphatidylinositol anchoring is a type of post-translational modification that allows proteins to be presented on the exterior side of the cell membrane. Purified glycosylphosphatidylinositol-anchored protein can spontaneously re-insert into lipid bilayer membranes in a process termed Molecular Painting. Here, we demonstrate the possibility of inserting purified, recombinant CD59 into virus particles produced from a murine retroviral producer cell line. CD59 is a regulator of the complement system that helps protect healthy cells from the lytic activity of the complement cascade. In this study, we could show that Molecular Painting confers protection from complement activity upon murine retroviral vector particles. Indeed, increased infectivity of CD59-modified virus particles was observed upon challenge with human serum, indicating that Molecular Painting is suitable for modulating the immune system in gene therapy or vaccination applications. Electronic supplementary material The online version of this article (doi:10.1007/s12033-016-9944-z) contains supplementary material, which is available to authorized users.

Tumor-specific monoclonal antibodies (mAbs) offer several modes of tumor cell killing, from direct cytotoxic activity to indirect mechanisms employing the host immune system, particularly its innate branch. The latter effector functions seem to dominate among clinically approved anti-cancer mAbs and major efforts are being undertaken by both academia and the pharmaceutical industry with the aim to improve complement activation, antibody-dependent cellular cytotoxicity (ADCC) and Fc/opsonin-mediated phagocytosis. On one hand, there are a variety of available effector mechanisms to allow multistep elimination of tumor cells. On the other hand, tumor cells adopt a number of strategies to evade immune attack, such as overexpression of complement inhibitors, trogocytosis, shedding or internalization of mAb-targeted epitopes, which all contribute to their resistance against host defense mechanisms. Another problem recognized only recently is the depletion of immune effectors during the first round of treatment, which in concordance with delayed turnover of immune components renders subsequent rounds of therapy ineffective. Herein, we discuss newly identified limiting factors but also novel mechanistic data on complement activation by antitumor antibodies as issues important for guidance towards the next generations of immunotherapeutics.

Both oxidized lipids as well as the complement system contribute to atherothrombosis. The expression of complement receptors correlates with the expression of platelet activation markers, and platelet bound oxidized low-density lipoprotein (oxLDL) modulates platelet function. In the present study, we investigated the relationship of markers of complement activation, the anaphylatoxins C5a and C3a, and oxidized low-density lipoprotein. Two hundred and seven patients with coronary artery disease (CAD) were analyzed in this study. Using enzyme-linked immunosorbent assays, plasma levels of oxLDL, C3a, and C5a were measured. Moreover, we assessed platelet bound oxLDL by flow cytometry. The overall level of C5a in the troponin negative group (stable angina (SA) and unstable angina (UA)) compared to the troponin positive group (non-ST-elevation myocardial infarction (NSTEMI) and ST-elevation myocardial infarction (STEMI)) did not differ significantly (62.7 ± 32.4 ng/ml versus 65.8 ± 40.3 ng/ml). While C5a and C3a showed a significant correlation with each other (r = 0.25, p < 0.001), there was no statistically significant relationship between C3a and platelet bound oxLDL (r = 0.06, p = 0.37). Furthermore, plasma oxLDL did not correlate with either C3a or C5a. However, we observed a moderate, yet significant negative correlation between plasma C5a and platelet bound oxLDL (r = -0.15, p = 0.04). Partial correlation analysis correcting for the presence of acute coronary syndrome (ACS), troponin status or the subgroups SA, UA, NSTEMI, or STEMI did not alter this correlation substantially. Interestingly, flow cytometric analysis of human platelets showed increased expression of C5aR and P-selectin after in vitro stimulation with oxLDL. In conclusion, the complement anaphylatoxin C5a shows an inverse correlation with platelet bound oxLDL. The relationship of oxidized lipids to particular complement components may add to the platelet-lipid interplay in atherogenesis and trigger future clinical and mechanistic studies.

// Xing Jun Li 1,2,* , Lisa Deng 2,3,* , Stephanie L. Brandt 4 , Charles B. Goodwin 2,3 , Peilin Ma 5 , Zhenyun Yang 1,2 , Raghu S. Mali 1,2 , Ziyue Liu 6 , Reuben Kapur 1,2,3,4 , C. Henrique Serezani 4 and Rebecca J. Chan 1,2,3 1 Department of Pediatrics, Indianapolis, IN, USA 2 Herman B Wells Center for Pediatric Research, Indianapolis, IN, USA 3 Department of Medical & Molecular Genetics, Indianapolis, IN, USA 4 Department of Microbiology & Immunology, Indianapolis, IN, USA 5 Department of Pathology, Indiana University School of Medicine, Indianapolis, IN, USA 6 Department of Biostatistics, Indiana University Richard M. Fairbanks School of Public Health, Indianapolis, IN, USA * These authors have contributed equally to this work Correspondence to: Rebecca J. Chan, email: // Keywords : PI3K, p85α, neutrophil, NADPH oxidase, MRSA, Immunology and Microbiology Section, Immune response, Immunity Received : February 16, 2016 Accepted : March 23, 2016 Published : March 30, 2016 Abstract Drug resistance is a growing problem that necessitates new strategies to combat pathogens. Neutrophil phagocytosis and production of intracellular ROS, in particular, has been shown to cooperate with antibiotics in the killing of microbes. This study tested the hypothesis that p85α, the regulatory subunit of PI3K, regulates production of intracellular ROS. Genetic knockout of p85α in mice caused decreased expression of catalytic subunits p110α, p110β, and p110δ, but did not change expression levels of the NADPH oxidase complex subunits p67 phox , p47 phox , and p40 phox . When p85α, p55α, and p50α (all encoded by Pik3r1 ) were deleted, there was an increase in intracellular ROS with no change in phagocytosis in response to both Fcγ receptor and complement receptor stimulation. Furthermore, the increased intracellular ROS correlated with significantly improved neutrophil killing of both methicillin-susceptible and methicillin-resistant S. aureus . Our findings suggest inhibition of p85α as novel approach to improving the clearance of resistant pathogens.

The human complement system is the frontline defense mechanism against invading pathogens. The coexistence of humans and microbes throughout evolution has produced ingenious molecular mechanisms by which microorganisms escape complement attack. A common evasion strategy used by diverse pathogens is the hijacking of soluble human complement regulators to their surfaces to afford protection from complement activation. One such host regulator is factor H (FH), which acts as a negative regulator of complement to protect host tissues from aberrant complement activation. In this report, we show that Plasmodium falciparum merozoites, the invasive form of the malaria parasites, actively recruit FH and its alternative spliced form FH-like protein 1 when exposed to human serum. We have mapped the binding site in FH that recognizes merozoites and identified Pf92, a member of the six-cysteine family of Plasmodium surface proteins, as its direct interaction partner. When bound to merozoites, FH retains cofactor activity, a key function that allows it to downregulate the alternative pathway of complement. In P. falciparum parasites that lack Pf92, we observed changes in the pattern of C3b cleavage that are consistent with decreased regulation of complement activation. These results also show that recruitment of FH affords P. falciparum merozoites protection from complement-mediated lysis. Our study provides new insights on mechanisms of immune evasion of malaria parasites and highlights the important function of surface coat proteins in the interplay between complement regulation and successful infection of the host.

Complements have long been considered to be a pivotal component in innate immunity. Recent researches, however, highlight novel roles of complements in T-cell-mediated adaptive immunity. Membrane-bound complement regulatory protein CD46, a costimulatory protein for T cells, is a key molecule for T-cell immunomodulation. Teleost CD46-like molecule, termed Tecrem, has been newly identified in common carp and shown to function as a complement regulator. However, it remains unclear whether Tecrem is involved in T-cell immune response. We investigated Tecrem function related to T-cell responses in ginbuna crucian carp. Ginbuna Tecrem (gTecrem) proteins were detected by immunoprecipitation using anti-common carp Tecrem monoclonal antibody (mAb) and were ubiquitously expressed on blood cells including CD8α(+) and CD4(+) lymphocytes. gTecrem expression on leucocyte surface was enhanced after stimulation with the T-cell mitogen, phytohaemagglutinin (PHA). Coculture with the anti-Tecrem mAb significantly inhibited the proliferative activity of PHA-stimulated peripheral blood lymphocytes, suggesting that cross-linking of Tecrems on T-cells interferes with a signal transduction pathway for T-cell activation. These findings indicate that Tecrem may act as a T-cell moderator and imply that the complement system in teleost, as well as mammals, plays an important role for linking adaptive and innate immunity.

The innate immune system is an ancient surveillance system able to sense microbial invaders as well as aberrations in normal cell function. No longer viewed as a static and non‐specific part of immunity, the innate immune system employs a plethora of specialized pattern recognition sensors to monitor and achieve homeostasis; these include the Toll‐like receptors, the retinoic acid‐inducible gene‐like receptors, the nucleotide‐binding oligomerization domain receptors (NLRs), the C‐type lectins and the complement system. In order to increase specificity and diversity, innate immunity uses homotypic and heterotypic associations among these different components. Multi‐molecular assemblies are formed both on the cell surface and in the cytosol to respond to pathogen and danger signals. Diverse, but tailored, responses to a changing environment are orchestrated depending on the the nature of the challenge and the repertoire of interacting receptors and components available in the sensing cell. It is now emerging that innate immunity operates a system of ‘checks and balances’ where interaction among the sensors is key in maintaining normal cell function. Complement sits at the heart of this alarm system and it is becoming apparent that it is capable of interacting with all the other pathways to effect a tailored immune response. In this review, we will focus on complement interactions with NLRs, the so‐called ‘inflammasomes’, describing the molecular mechanisms that have been revealed so far and discussing the circumstantial evidence that exists for these interactions in disease states.

Complement is well appreciated as critical arm of innate immunity. It is required for the removal of invading pathogens and functions by direct pathogen destruction and through the activation of innate and adaptive immune cells. However, complement activation and function is not confined to the extracellular space but also occurs within cells. Recent work indicates that complement activation regulates key metabolic pathways and thus can impact fundamental processes of the cell, such as survival, proliferation, and autophagy. Novel identified functions of complement include a key role in shaping metabolic reprogramming, which underlies T cell effector differentiation, and a role as a nexus for interactions with other effector systems, in particular the inflammasome and Notch transcription factor networks. This review focuses on the contributions of complement to basic processes of the cell, in particular the integration of complement with cellular metabolism, and the potential implications in infection and other disease settings.

HIV-1 infection is a major public health problem and an important cause of death among adults. In light of innate immune system being the first, rapid and nonspecific response, this highlights the importance of exploiting the active arms of innate immunity to eradicate the invader and triggering a more specific immune response, the adaptive immune system. Each type of cells in the innate immune system has a unique distribution and function in the body and therefore differs in their ability to induce adaptive immune arms according to the stimuli. Any functional defect or alteration in the innate immune system can affect the adaptive arms of the immune system in terms of failure to overcome the battlefield with the invader. This review focuses on the relevant function of each member of the innate immune system and sheds the light on detailed mechanisms about how this smart virus invades and evades the immune system which opens new insights into the immunology and therapeutic targeting of HIV-1 infection.

Osteonecrosis of the femoral head (ONFH) is a complex and multifactorial disease that is influenced by a number of genetic factors in addition to environmental factors. Some autoimmune disorders, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and inflammatory bowel disease (IBD), are associated with the development of ONFH. Complement receptor type 2 (CR2) is membrane glycoprotein which binds C3 degradation products generated during complement activation.CR2has many important functions in normal immunity and is assumed to play a role in the development of autoimmune disease. We investigated whetherCR2gene polymorphisms are associated with risk of ONFH in SLE patients. Eight polymorphisms in theCR2gene were genotyped using TaqMan™assays in 150 SLE patients and 50 ONFH in SLE patients (SLE_ONFH). The association analysis of genotyped SNPs and haplotypes was performed with ONFH. It was found that three SNPs, rs3813946 in 5′-UTR (untranslated region), rs311306 in intron 1, and rs17615 in exon 10 (nonsynonymous SNP; G/A, Ser639Asn) of theCR2gene, were associated with an increased risk of ONFH under recessive model (Pvalues; 0.004~0.016). Haplotypes were also associated with an increased risk (OR; 3.73~) of ONFH in SLE patients. These findings may provide evidences thatCR2contributes to human ONFH susceptibility in Korean SLE patients.

Background: The involvement of B cells, complement activation and subsequent immune complex deposition has been implicated in the pathogenesis of rheumatoid arthritis (RA). Although the reduced expression of complement receptor 2 (CR2, CD21) on the B cells of RA patients is known for long, we aimed at determining the modulation and expression of CR2 on PBMC of healthy individuals and RA patients. Methods: Sixty controls and 57 RA patients were enrolled. PBMC-CR2 transcript levels were correlated with the levels of C3, C3d and circulating immune complexes (CIC) in controls and patients and with DAS28 in patients only. CIC levels were determined by PEG precipitation, C3 levels by nephlometry and C3d levels were determined by enzyme linked immunosorbent assay (ELISA). Sixteen patients were recruited for 6 months follow up studies of transcript levels of PBMC correlated with DAS28 score. Appropriate statistical methods were used for the analyses of data. Results: PBMC- CR2 transcript levels were declined in patients as compared to controls. PBMC-CR2 levels correlated negatively with DAS28 score. DAS28 correlated positively with levels of CIC, C3 and C3d. Levels of PBMC -CR2 increased in patients with decline in DAS28 scores in 6 months follow-up patients. Conclusions: Low level of CR2 expression in patients may, thus, contribute significantly to the pathological manifestation of RA. Cause-effect relationships of the up regulation of CR2 on improvement of health condition with the pathophysiology of RA and their importance as putative disease markers is being confirmed.

Complement regulation leads to the generation of complement split products (CSPs) such as complement component (C)4d, a marker for disease activity in autoimmune syndromes or antibody-mediated allograft rejection. However, the physiologic role of C4d has been unknown. By screening murine thymoma BW5147 cells expressing a cDNA library generated from human monocyte-derived dendritic cells with recombinant human C4d, we identified Ig-like transcript (ILT)4 and ILT5v2 as cellular receptors for C4d. Both receptors, expressed on monocytes, macrophages, and dendritic cells, also interacted with the CSPs C3d, C4b, C3b, and iC3b. However, C4d did not bind to classic complement receptors (CRs). Interaction between cell surface-resident ILT4 and soluble monomeric C4d resulted in endocytosis of C4d. Surprisingly, binding of soluble ILT4 to C4d covalently immobilized to a cellular surface following classic complement activation could not be detected. Remarkably, C4d immobilized to a solid phaseviaits intrinsic thioester conferred a dose-dependent inhibition of TNF-α and IL-6 secretion in monocytes activatedviaFc-cross-linking of up to 50% as compared to baseline. Similarly, C4d conferred an attenuation of intracellular Ca(2+)flux in monocytes activatedviaFc-cross-linking. In conclusion, ILT4 represents a scavenger-type endocytotic CR for soluble monomeric C4d, whereas attenuation of monocyte activation by physiologically oriented C4d on a surface appears to be dependent on a yet to be identified C4d receptor.-Hofer, J., Forster, F., Isenman, D. E., Wahrmann, M., Leitner, J., Hölzl, M. A., Kovarik, J. K., Stockinger, H., Böhmig, G. A., Steinberger, P., Zlabinger, G. J. Ig-like transcript 4 as a cellular receptor for soluble complement fragment C4d.

The innate immune system is the first line of defence against pathogens and infection. Recently, it has become apparent that many innate immune factors have roles outside of immunity and there is growing evidence that these factors play important functional roles during the development of a range of model organisms. Several studies have documented developmental expression of individual factors of the toll-like receptor and complement systems, and we recently demonstrated a key role for complement C5a receptor (C5aR1) signalling in neural tube closure in mice. Despite these emerging studies, a comprehensive expression analysis of these molecules in embryonic development is lacking. In the current study, we therefore, examined the expression of key innate immune factors in the early development period of neurulation (7.5-10.5dpc) in mice. We found that complement factor genes were differentially expressed during this period of murine development. Interestingly, the expression patterns we identified preclude activation of the classical and alternative pathways and formation of the membrane attack complex. Additionally, several other classes of innate immune molecules were expressed during the period of neurulation, including toll-like receptors (TLR-2, -3, -4 and -9), receptor for advanced glycation end-products (RAGE), and their signalling adapters (TRAF-4, TRAF-6, TAK-1 and MyD88). Taken together, this study highlights a number of innate immune factors as potential novel players in early embryonic development.

Microglia play an important role in receptor-mediated phagocytosis in the CNS. In brain abscess and other CNS infections, invading bacteria undergo opsonization with Igs or complement. Microglia recognize these opsonized pathogens by Fc or complement receptors triggering phagocytosis. In this study, we investigated the role of Fcα/μR, the less-studied receptor for IgM and IgA, in microglial phagocytosis. We showed that primary microglia, as well as N9 microglial cells, express Fcα/μR. We also showed that anti-Staphylococcus aureus IgM markedly increased the rate of microglial S. aureus phagocytosis. To unequivocally test the role of Fcα/μR in IgM-mediated phagocytosis, we performed experiments in microglia from Fcα/μR−/− mice. Surprisingly, we found that IgM-dependent phagocytosis of S. aureus was similar in microglia derived from wild-type or Fcα/μR−/− mice. We hypothesized that IgM-dependent activation of complement receptors might contribute to the IgM-mediated increase in phagocytosis. To test this, we used immunologic and genetic inactivation of complement receptor 3 components (CD11b and CD18) as well as C3. IgM-, but not IgG-mediated phagocytosis of S. aureus was reduced in wild-type microglia and macrophages following preincubation with an anti-CD11b blocking Ab. IgM-dependent phagocytosis of S. aureus was also reduced in microglia derived from CD18−/− and C3−/− mice. Taken together, our findings implicate complement receptor 3 and C3, but not Fcα/μR, in IgM-mediated phagocytosis of S. aureus by microglia.

Summary Multinucleated giant cells (MGCs) form by fusion of macrophages and are presumed to contribute to the removal of debris from tissues. In a systematic in vitro analysis, we show that IL-4-induced MGCs phagocytosed large and complement-opsonized materials more effectively than their unfused M2 macrophage precursors. MGC expression of complement receptor 4 (CR4) was increased, but it functioned primarily as an adhesion integrin. In contrast, although expression of CR3 was not increased, it became functionally activated during fusion and was located on the extensive membrane ruffles created by excess plasma membrane arising from macrophage fusion. The combination of increased membrane area and activated CR3 specifically equips MGCs to engulf large complement-coated targets. Moreover, we demonstrate these features in vivo in the recently described complement-dependent therapeutic elimination of systemic amyloid deposits by MGCs. MGCs are evidently more than the sum of their macrophage parts., Graphical Abstract, Highlights • MGCs are specialized for phagocytosis of large and complement-opsonized particles • MGCs show extensive membrane ruffles containing pre-activated complement receptor 3 • Membrane ruffles provide excess membrane for ingestion of large materials • MGCs eliminate systemic amyloid deposits after immunotherapeutic targeting, Macrophage-derived multinucleated giant cells (MGCs) form in diverse chronic inflammatory diseases, but their functional role remains unclear. Milde et al. show that MGCs are specialized for complement-mediated phagocytosis and destruction of large targets and demonstrate their key role in the therapeutic elimination of the pathogenic amyloid deposits in systemic amyloidosis.

Work published in this issue of Virulence sheds new light on the function of a phylogenetically conserved protein family, which is shown to modulate immune responses in insects such as the fruitfly...

The complement system is an important component of the innate immune system, which contributes to non-specific host defence. Particulate matters, such as invading pathogens and nanomedicines, in the blood may activate the complement system through classical, lectin and alternative pathways. Complement activation can aid recognition and clearance of particulate matters by immune cells, but uncontrolled complement activation can inflict damage and be life threatening. Plasma proteins on adsorption to surfaces of nanoparticles also play a significant role in complement activation and particularly through the alternative pathway. This process is continuous and changeable in vivo; protein-complement complexes are formed on the nanoparticle surface and then released and the cycle repeats on further plasma protein deposition. This complement activation turnover poses a challenge for design of immune-safe nanomedicines.

Complement is an integral part of the immune system and protects against infection. Complement-mediated immunopathology in many autoimmune diseases and syndromes has led to the therapeutic targeting of complement and to questions around the safety of complement inhibition. Here; I examine and clarify the risks associated with complement therapeutics.

Visceral leishmaniasis (VL) is an anthropozoonosis endemic in Brazil. We included 20 patients with confirmed diagnosis of VL and 20 healthy individuals to evaluate the expression levels of complement receptor 1 (CR1)/CD35 and CR3/CD11b on leukocytes in the peripheral blood and determine their correlation with the clinical state of patients. CR1/CD35 expression increased on CD11b+CD35+granulocytes of patients, while CR1/CD35 and CR3/CD11b expression levels increased on CD14+CD11b+CD35+ monocytes. Among patients, those with severe clinical state had higher expression of CR3/CD11b on CD14+monocytes. The count of CD19+CD35+B lymphocytes reduced in the blood samples from patients. These observed changes may indicate the modulation in CR1/CD35 and CR3/CD11b complement receptor expressionlevels on granulocyte and monocyte populations in response to Leishmania sp.

The purpose of this study is to determine the role of the complement immune system in glioma and/or glioma therapy. Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. It is accompanied by a devastating prognosis; the median survival is 12-14 months, with less than 10 percent of patients living for more than two years after diagnosis. Unfortunately, current treatment options are limited, and many initially promising drugs have failed phase three clinical trials. One potential and relatively unexplored target for co-therapy in GBM is the complement immune system. Historically, the focus of work with complement has been on its role in innate immunity, where it can aid in the recognition and elimination of pathogens or undesired host material. More recent work, however, has revealed a key function of complement as a “double-edged sword” in the CNS. While the cascade is necessary for CNS development and homeostasis, overactive complement can lead to the hallmark neuroinflammation and neurodegeneration seen in conditions like Alzheimer's, multiple sclerosis, and traumatic brain injuries. Yet despite the presence of complement receptors on nearly all CNS cells and the direct role that complement plays in multiple neuroinflammatory diseases, very few studies have examined complement expression in brain tumors. The current project seeks to bridge this gap in knowledge by assessing the impact of complement on glioma. This effort began by selecting candidate genes, as the complement family contains over 50 members. To do so, data from the publicly available Cancer Genome Atlas (TCGA) was mined, providing 14 targets of interest for further analysis. Cell-based experiments were performed in three GBM patient-derived xenolines (PDx): XD456, JX6, and JX10. mRNA expression was determined via TaqMan real-time PCR. Protein levels were assessed via Western blot. Overall, seven of the 14 initial targets demonstrated clear over-expression in all three human GBM PDx cell lines. This expression was not changed upon treatment with glioma growth factors such as epidermal or fibroblast growth factor (EGF or FGF). Intriguingly, however, the degree of over-expression varied by cell line, even when these lines were derived from patients assigned to the same molecular GBM subtype. For example, at the RNA level and in comparison to other cell lines, complement factor H (a C3 inhibitor) was up to six times higher in XD456, and clusterin (a MAC inhibitor) was nearly 18-fold higher in JX10. These data suggest that the current system for classifying tumor subtype may be missing important factors. Finally, when compared back to TCGA data, overexpression of these targets was associated with significantly worse tumor phenotype and patient survival. This project has critically identified components of the complement system that are upregulated in GBM and strongly correlated to survival. Ultimately, these data may provide the first potential targets for complement-based co-therapeutics to be explored in future studies. Citation Format: Emily Nicole Hayward, Xiuhua Yang, Natalia Filippova, Jennifer A. Calano, David Namkoong, Stefan Kovac, Louis B. Nabors. Characterization and analysis of the complement immune system in glioblastoma (GBM) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3335.

Background:Activation of the alternative and final common pathways have been shown in ANCA-associated vasculitis (AAV) (1). Circulating titers of C5a are elevated and correlate with disease activity in AAV. Binding to the corresponding G protein-coupled receptor (GPCR) C5aR1 enhances the influx of neutrophils, leading to ROS generation and severe necrotizing of vascular walls (2). Moreover, subsequent interaction of C5a with C5aR1 may represent a proinflammatory amplification loop (3). Blocking of the receptor is protective in a murine model in AAV (4). In humans, avacopan, a C5aR1-inhibitor showed promising results as glucocorticoid-sparing agent in two randomized phase II and one ongoing phase III clinicals trials in AAV (NCT02994927). Notably, disease-specific anti-GPCR autoantibody (aab) signatures have been found in different autoimmune diseases (5).Objectives:The aim of the present study was to examine whether (patho)physiological anti-C3aR and anti-C5aR1 aabs correlate with clinical findings in AAV, and whether this is linked to the clinical outcome.Methods:Sera and plasma of AAV patients [granulomatosis with polyangiitis (GPA), n=64; microscopic polyangiitis (MPA), n=26; eosinophilic granulomatosis with polyangiitis (EGPA), n=11] were measured by Elisa for circulating autoantibodies against complement receptors C3a (anti-C3aR aab) and C5a (anti-C5aR1 aab) and plasma levels of C3a and C5a. Expression of C3aR and C5aR1 on T-cells was determined using flow cytometry. Clinical data were assessed at the time of serum sampling and during follow-up for 48 monthsResults:GPA displayed low titers of anti-C3aR aab (GPA:5.33±2.54vs. HD:6.47±2.61, P=0.0031). Anti-C5aR1 aab were decreased in AAV, especially in GPA (GPA:1.02±1.07vs. HD:6.63±2.91, P=vs. HD:3.44±0.68%, P=0.0021; CD8+C5aR1+T-cells GPA:9.74±2.10%vs.HD:4.11±0.92%, P=0.0198). Reduced titers of anti-C5aR1 aab Conclusion:As potential diagnostic marker, anti-C5aR1 aab titer may additionally be useful to monitor disease activity in AAV.References:[1]Chen M et al.Complement deposition in renal histopathology of patients with ANCA-associated pauci-immune glomerulonephritis.Nephrol Dial Transpl. 2009;24:1247-1252[2]Schreiber A et al.C5a receptor mediates neutrophil activation an ANCA-induced glomerulonephritis.J Am Soc Nephrol. 2009; 20:289-298[3]Lamprecht P et al.: Pathogenetic and clinical aspects of Anti-Neutrophil Cytoplasmic Autoantibody-associated vasculitides.Front Immunol.2018 Apr 9;9-680[4]Xiao H et al.C5a receptor (CD88) blockade protects against MPO-ANCA GN.J Am Soc Nephrol. 2014;25(2):225-31[5]Klapa S et al. Decreased endothelin receptor A autoantibody levels are associated with early ischaemic events in patients with giant-cell arteritis.Ann Rheum Dis2019 Oct;78(19):1443-1444Disclosure of Interests:Sebastian Klapa Grant/research support from: Actelion, Consultant of: Pfizer, Abbvie, Antje Müller: None declared, Andreas Koch: None declared, Anja Kerstein-Staehle: None declared, Wataru Kaehler: None declared, Harald Heidecke Shareholder of: Cell Trend GmbH, Employee of: Cell Trend GmbH, Speakers bureau: Cell Trend GmbH, Susanne Schinke Speakers bureau: Pfizer, Markus Huber-Lang: None declared, Martin Nitschke: None declared, Silke Pitann: None declared, Christian Karsten: None declared, Gabriela Riemekasten Consultant of: Cell Trend GmbH, Janssen, Actelion, Boehringer Ingelheim, Speakers bureau: Actelion, Novartis, Janssen, Roche, GlaxoSmithKline, Boehringer Ingelheim, Pfizer, Peter Lamprecht: None declared

Bacterial cell surface adhesins play a major role in facilitating host colonization and subsequent establishment of infection. The surface of Mycobacterium tuberculosis, owing to the complex architecture of its cell envelope, expresses numerous adhesins with varied chemical nature, including proteins, lipids, lipoproteins, glycoproteins and glycopolymers. Studies on mycobacterial adhesins show that they bind with multifarious host receptors and extracellular matrix (ECM) components. In this review we have highlighted the adhesins that are abundantly present on the mycobacterial surface and their interactions with host receptors. M. tuberculosis interacts with various host cell surface receptors such as toll like receptors, C-type lectin receptors, scavenger receptors, and Fc and complement receptors. Apart from these, ECM components like fibronectin, collagen, elastin, laminin, fibrillin and vitronectin also provide binding sites for surface adhesins of the tubercle bacilli. M. tuberculosis adhesins include proteins with and without signal peptide sequence and transmembrane proteins. Other surface adhesin macromolecules of M. tuberculosis comprises of lipids, glycolipids and glycopolymers. The interaction between the mycobacterial adhesins and their host receptors result in adhesion of the microbe to the host cells, induction of immune response and aid in the pathogenesis of the disease. A thorough understanding of the different M. tuberculosis surface adhesins and host receptors will provide a better picture of interaction between them at molecular level. The information gained on adhesins and host receptors will prove beneficial in developing novel therapeutic strategies such as the use of anti-adhesin molecules to hinder the adhesion of bacteria to the host cells, thereby preventing establishment of infection. The surface molecules discussed in this review will also benefit in identification of new drug targets, diagnostic markers or vaccine candidates against the deadly pathogen.

A review of the mechanisms of the generation of antiviral immunity in bacteria, plants, invertebrates, and vertebrates directly indicates the leading role of innate immunity. Bornovirus infections in mammals have been proven to be inhibited by the RNA interference mechanism. Authors propose a possible role of innate human immunity in combating viral infections, including SARS-Cov2. This hypothesis is based on the notion that antiviral response involves a molecular mechanism of RNA interference stemming from the specific viral patterns incorporated into host cells’ DNA. Innate immunity plays an important role not only at the first encounter with a viral infection, but also fully participates in the formation of specific immune memory. RNA-interference along with interferon system is integral parts of human anti-viral defense system. The joint work of these systems is considered. Caution is warranted in both PCR testing interpretation and in assessing prospective vaccines. False positive result without any clinical presentation of the disease in some people might mean that PCR test was picking up any specific SARS-Cov2 sequences that already had incorporated into asymptomatic person’s cellular DNA due to the possible RNA-interference based anti-viral immunity. Future vaccines aimed at producing specific antibodies can cause the phenomenon of antibody-dependent enhancement (ADE) of the infection. In ADE virus not only infects susceptible cells through appropriate receptor, but is able to highjack virus-specific antibodies to easily traffic virus bodies inside the monocytes/macrophages, granulocytes, platelets, mast and many more host cells through interaction with Fc and/or complement receptors. It is necessary to draw the attention of medical community, especially practitioners to the role of innate immunity, which is especially important in the current COVID-19 pandemic.

Complement receptors (CR) 3 and 4 belong to the family of beta-2 (CD18) integrins. CR3 and CR4 are often co-expressed in the myeloid subsets of leukocytes, but they are also found in NK cells and activated T and B lymphocytes. The heterodimeric ectodomain undergoes considerable conformational change in order to switch the receptor from a structurally bent, ligand-binding in-active state into an extended, ligand-binding active state. CR3 binds the C3d fragment of C3 in a way permitting CR2 also to bind concomitantly. This enables a hand-over of complement-opsonized antigens from the cell surface of CR3-expressing macrophages to the CR2-expressing B lymphocytes, in consequence acting as an antigen presentation mechanism. As a more enigmatic part of their functions, both CR3 and CR4 bind several structurally unrelated proteins, engineered peptides, and glycosaminoglycans. No consensus motif in the proteinaceous ligands has been established. Yet, the experimental evidence clearly suggest that the ligands are primarily, if not entirely, recognized by a single site within the receptors, namely the metal-ion dependent adhesion site (MIDAS). Comparison of some recent identified ligands points to CR3 as inclined to bind positively charged species, while CR4, by contrast, binds strongly negative-charged species, in both cases with the critical involvement of deprotonated, acidic groups as ligands for the Mg2+ ion in the MIDAS. These properties place CR3 and CR4 firmly within the realm of modern molecular medicine in several ways. The expression of CR3 and CR4 in NK cells was recently demonstrated to enable complement-dependent cell cytotoxicity toward antibody-coated cancer cells as part of biological therapy, constituting a significant part of the efficacy of such treatment. With the flexible principles of ligand recognition, it is also possible to propose a response of CR3 and CR4 to existing medicines thereby opening a possibility of drug repurposing to influence the function of these receptors. Here, from advances in the structural and cellular immunology of CR3 and CR4, we review insights on their biochemistry and functions in the immune system.

In this mini review, we describe the molecular mechanisms in polymicrobial sepsis that lead to a series of adverse events including activation of inflammatory and prothrombotic pathways, a faulty innate immune system, and multiorgan dysfunction. Complement activation is a well-established feature of sepsis, especially involving generation of C5a and C5b-9, along with engagement of relevant receptors for C5a. Activation of neutrophils by C5a leads to extrusion of DNA, forming neutrophil extracellular traps that contain myeloperoxidase and oxidases, along with extracellular histones. Generation of the distal complement activation product, C5b-9 (known as the membrane attack complex, MAC), also occurs in sepsis. C5b-9 activates the NLRP3 inflammasome, which damages mitochondria, together with appearance in plasma of IL-1β and IL-18. Histones are strongly proinflammatory as well as being prothrombotic, leading to activation of platelets and development of venous thrombosis. Multiorgan dysfunction is also a feature of sepsis. It is well known that septic cardiomyopathy, which if severe, can lead to death. This complication in sepsis is linked to reduced levels in cardiomyocytes of three critical proteins (SERCA2, NCX, Na+/K+-ATPase). The reductions in these three key proteins are complement- and histone-dependent. Dysfunction of these ATPases is linked to the cardiomyopathy of sepsis. These data suggest novel targets in the setting of sepsis in humans.

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fcγ receptor (FcγR)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to FcγRs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fcγ receptor- or complement receptor-mediated phagocytosis.

Production of extracellular vesicles (EVs) involved in intercellular communication is a common capacity of most cell types. Upon encountering opsonized microorganisms, neutrophilic granulocytes release EVs that compromise bacterial growth. We carried out a systematic investigation of the involvement of potential opsonin receptors in EV-generation from human and murine neutrophils. Applying flow cytometric, proteomic and functional analysis as well as using genetically modified mice, we demonstrate that formation of antibacterial EVs depends upon stimulation of the multifunctional Mac-1 integrin complex, also called as complement receptor 3 (CR3), whereas activation of immunoglobulin binding Fc receptors or pattern recognition receptors alone or in combination is ineffective. Mac-1/CR3 stimulation and downstream tyrosine kinase signalling affect both the numbers, the cargo content and the antibacterial capacity of the produced vesicles. In contrast, Mac-1/CR3 signalling is not required for spontaneous EV formation, clearly indicating the existence of separate molecular pathways in EV biogenesis. We propose that EVs are “tailor-made” with different composition and functional properties depending on the environmental circumstances.

Chemoattractant-induced arrest of circulating leukocytes and their subsequent diapedesis is a fundamental component of inflammation. However, how tissue-derived chemoattractants are transported into the blood vessel lumen to induce leukocyte entry into tissue is not well understood. Here, intravital microscopy in live mice has shown that the "atypical" complement C5a receptor 2 (C5aR2) and the atypical chemokine receptor 1 (ACKR1) expressed on endothelial cells were required for the transport of C5a and CXCR2 chemokine ligands, respectively, into the vessel lumen in a murine model of immune complex-induced arthritis. Transported C5a was required to initiate C5aR1-mediated neutrophil arrest, whereas transported chemokines were required to initiate CXCR2-dependent neutrophil transdendothelial migration. These findings provide new insights into how atypical chemoattractant receptors collaborate with "classical" signaling chemoattractant receptors to control distinct steps in the recruitment of neutrophils into tissue sites of inflammation.

Microglia are resident immune cells of the central nervous system. Their development and maintenance depend on stimulation of Colony Stimulating Factor-1 receptor (CSF1R). Microglia play an important role in neurodevelopment and a population of microglia that expresses the complement receptor CD11c is critical for primary myelination. This population is virtually absent in the healthy adult brain but increases dramatically upon neuroinflammatory conditions, and these microglia are suggested to play a protective role in central nervous system (CNS) diseases. To date, the molecular trigger for their expansion is unknown. Here we showed that stimulation of CSF1R by either of its ligands, CSF1 and interleukin (IL)-34, can induce expansion of CD11c+ microglia. In addition, such stimulation resulted in amelioration of EAE symptoms and decreased demyelination. Treatment with CSF1R ligands also induced expression of the chemokine CCL2, and we showed that experimental overexpression of CCL2 in the brain led to a dramatic increase of CD11c+ microglia, independent of CCR2. Moreover, this led to elevated CSF1 expression, suggesting a positive feedback loop between CSF1R and CCL2.These data provide new insights to microglia biology and open new perspectives for modulating microglial activity in neuroinflammatory diseases such as multiple sclerosis.

Cryptococcus neoformans is a pathogenic yeast capable of a unique and intriguing form of cell-to-cell transfer between macrophage cells. The mechanism for cell-to-cell transfer is not understood. In this study, we imaged mouse macrophages with CellTracker Green 5-chloromethylfluorescein diacetate–labeled cytosol to ascertain whether cytosol was shared between donor and acceptor macrophages. Analysis of several transfer events detected no transfer of cytosol from donor-to-acceptor mouse macrophages. However, blocking Fc and complement receptors resulted in a major diminution of cell-to-cell transfer events. The timing of cell-to-cell transfer (11.17 min) closely approximated the sum of phagocytosis (4.18 min) and exocytosis (6.71 min) times. We propose that macrophage cell-to-cell transfer represents a nonlytic exocytosis event, followed by phagocytosis into a macrophage that is in close proximity, and name this process Dragotcytosis (“Dragot” is a Greek surname meaning “sentinel”), as it represents sharing of a microbe between two sentinel cells of the innate immune system.

Ischemia reperfusion (IR) injury induces retinal cell death and contributes to visual impairment. Previous studies suggest that the complement cascade plays a key role in IR injury in several systemic diseases. However, the role of the complement pathway in the ischemic retina has not been investigated. The aim of this study is to determine if the alternative complement cascade plays a role in retinal IR injury, and identify which components of the pathway mediate retinal degeneration in response to IR injury. To accomplish this, we utilized the mouse model of retinal IR injury, wherein the intraocular pressure (IOP) is elevated for 45 min, collapsing the retinal blood vessels and inducing retinal ischemia, followed by IOP normalization and subsequent reperfusion. We found that mRNA expression of complement inhibitors complement receptor 1-related gene/protein-y (Crry), Cd55 and Cd59a was down-regulated after IR. Moreover, genetic deletion of complement component 3 (C3−/−) and complement factor b (Fb−/−) decreased IR-induced retinal apoptosis. Because vascular dysfunction is central to IR injury, we also assessed the role of complement in a model of shear stress. In human retinal endothelial cells (HRECs), shear stress up-regulated complement inhibitors Cd46, Cd55, and Cd59, and suppressed complement-mediated cell death, indicating that a lack of vascular flow, commonly observed in IR injury, allows for complement mediated attack of the retinal vasculature. These results suggested that in retinal IR injury, the alternative complement system is activated by suppression of complement inhibitors, leading to vascular dysfunction and neuronal cell death.

HIV-specific broadly neutralizing antibodies (bNAbs) confer protection following passive immunization, but the immunological mechanisms that drive their development following HIV infection are poorly understood. Structural features of bNAbs indicate that they originate from extensive germinal center selection, which relies on persistent germinal center activity. However, why a fraction of infected individuals are able to successfully drive more effective affinity maturation is unclear. Delivery of antigens in the form of antibody-immune complexes (IC), that bind to complement receptors (CRs) or Fc-receptors (FcRs) on follicular dendritic cells (FDCs), represents an effective mechanism for antigen delivery to the GC. Thus we sought to define whether IC-FcR or CR interactions differ among subjects who develop broadly neutralizing antibody responses to HIV. Enhanced Fc-effector functions and FcR/CR interactions, via altered Fc-glycosylation profiles, were observed among subjects with neutralizing antibody responses to HIV compared to subjects without neutralizing antibody activity. Moreover, both polyclonal Neutralizer ICs and monoclonal-IC mimics of Neutralizer antibodies induced higher antibody titers, higher-avidity antibodies, and expanded germinal center B cell reactions following immunization of mice, via accelerated antigen deposition within B cell follicles in a complement dependent manner. Thus, these data point to a direct role for altered Fc-profile/complement interactions in shaping the maturation of the humoral immune response, providing insights on how GC activity may be enhanced to drive affinity maturation in next generation vaccine approaches.

Background B cell complement receptor 1 (CR1) and complement receptor 2 (CR2) levels are decreased by ∼50% in subjects with systemic lupus erythematosus (SLE). CR2 but not CR1 levels have been negatively correlated with lupus disease activity. However, increased CR1 expression on B cells is associated with a protective polymorphism in the CR2 gene. We conducted a longitudinal analysis of B cell CR1 and CR2 to further evaluate an association with lupus disease activity and to determine whether it is driven by specific cell subsets. Methods Thirty-six subjects meeting the revised 1982 ACR criteria for SLE were enrolled. Each subject had a baseline visit and 1 to 4 follow up visits, with additional visits for flares. Peripheral blood mononuclear cells (PBMC) were stained with a dead cell marker and antibodies to CD19, CD27, and IgD. Antibody binding capacity (ABC) of CR1 and CR2 on live bulk and gated B cells was determined using quantitative flow cytometry (Bang Labs). The frequency of live gated B cells was determined. Disease activity was measured using SLEDAI. Statistical analyses were performed using R. To determine associations between the SLEDAI scores and gate/quadrant signals, negative binomial mixed effect models were fitted using the glmmADMB package. A random effect intercept was included to account for different individual baseline SLEDAI scores. The gate/quadrant signals, in terms of ABC and percentages, were the independent variables. Adjustments were made for age, prednisone dose, and, where relevant, bead lot. To account for multiple testing in bulk B cells, a conservative Bonferroni adjustment was used, whereas in gated cells, the p-values were adjusted to control the false discovery rate according to Benjamini-Hochberg. A p-value of ≤0.05 was considered significant. Results CR1 and CR2 ABC on bulk B cells and on IgD +CD27-, IgD +CD27+, and IgD-/CD27 +gated B cells was negatively associated with lupus disease activity, whereas there was no association between CR1 and CR2 ABC on IgD-/CD27- B cells or frequency of any of these gated populations and disease activity (table 1 and data not shown). Conclusions Both CR1 and CR2 levels are negatively associated with lupus disease activity in defined B cell subsets. Although this study does not prove causality, these data suggest that altered levels of these receptors on specific B cell subsets may predict disease flare or be associated with disease remission. Acknowledgements This work was supported by R01AI070304, K24AI078004, and the Lupus Research Institute.

Seit einigen Jahren gibt es Hinweise, dass Thrombozyten an der Modulation der Blutgefäßneubildung, der Angiogenese, beteiligt sind. Bisher wurden aller-dings erst wenige Mechanismen beschrieben, über die eine solche Modulation vermittelt wird. Diese Arbeit identifiziert den auf Thrombozyten exprimierten C5a-Komplementrezeptor als einen wichtigen Mediator Thrombozyten-vermittelter Ef-fekte sowohl auf zentrale Endothelzellfunktionen als auch der wachstumsfaktor-vermittelten und hypoxiegetriggerten Angiogenese in vivo. Diese Arbeit zeigt im Matrigelplug-Assay, dass Thrombozyten wachstums-faktorinduzierte Angiogenese inhibieren können. Außerdem konnten wir zeigen, dass Thrombozyten den C5a-Rezeptor exprimieren und dessen Expression nach Stimulation mit verschiedenen Plättchenaktivatoren modulieren. Die C5aR-Ex-pression auf Thrombozyten nimmt zu, wenn diese mit Kollagen aktiviert werden, und auch bei Kontakt mit oxLDL, was die Bedeutung der Ergebnisse im Kontext der Atherosklerose hervorhebt. Wir konnten darüber hinaus C5aR-exprimierende Thrombozyten im ischämischen, also angiogen aktiven, Gewebe nachweisen. Die Koinkubation von Thrombozyten und Endothelzellen in vitro zeigte, dass der thrombozytäre C5a-Rezeptor keinen Einfluss auf die endotheliale Proliferation ausübt, die Migration und Tube formation von Endothelzellen allerdings inhibiert. Passend dazu zeigte sich in vivo, dass C5aR-Knockout-Mäuse ein erhöhtes Niveau an wachstumsfaktorinduzierter und hypoxiegetriggerter Angiogenese aufweisen. Interessanterweise ließ sich dieser Effekt nicht mehr signifikant nach-weisen, wenn Thrombozyten systemisch depletiert wurden. Dies weist auf einen bedeutsamen Beitrag des thrombozytären C5a-Komplementrezeptors bei Steu-erung der Angiogenese in vivo hin. Das wird dadurch unterstrichen, dass wir zei-gen konnten, dass die Retransfusion von Thrombozyten mit intaktem C5a-Rezeptor in C5aR-Knockout-Tiere deren erhöhtes Angiogeneseniveau im Matri-gelplug-Assay teilweise normalisiert. Schließlich konnten wir demonstrieren, dass bereits der Überstand von C5a-stimulierten Thrombozyten in vitro einen inhibitorischen Effekt auf die endotheli-ale Migration und Tube formation vermittelt. Mittels Proteome profiling konnten wir den bekanntermaßen antiangiogenen Faktor PF4 als einen wichtigen für die-sen Effekt verantwortlichen Faktor identifizieren. Nach C5a-Stimulation setzen Thrombozyten PF4 frei. Wird PF4 in vivo inhibiert, so lässt sich das Angiogene-seniveau von C5aR-Knockout-Tieren mittels Retransfusion von WT-Thrombozy-ten im Matrigelplug-Assay nicht mehr signifikant senken. Dieser Mechanismus trägt also auch in vivo maßgeblich zur thrombozytenvermittelten Inhibition der Angiogenese bei. Zusammenfassend führt C5a vermittels des thrombozytären C5a-Rezeptors zur Freisetzung von PF4 aus Thrombozyten. Thrombozyten üben so einen anti-angiogenen Einfluss aus. Diese Arbeit beschreibt also einen konkreten Mecha-nismus, wie Thromboyzten die Angiogenese steuern und beschreibt einen neuen Überschneidungspunkt des Komplementsystems, des Hämostasesystems und der Angiogenese. Diese drei Systeme sind bei zahlreichen Pathologien von großer Bedeutung und ihr Zusammenspiel noch unvollständig verstanden. Die hier gewonnenen Erkenntnisse könnten daher auch für das Verständnis und die Therapie der Atherosklerose und deren Folgeerkrankungen in Zukunft förderlich sein.

Background C3 glomerulopathy (C3G) is a life-threatening kidney disease caused by dysregulation of the alternative pathway of complement (AP) activation. No approved specific therapy is available for C3G, although an anti-C5 mAb has been used off-label in some patients with C3G, with mixed results. Thus, there is an unmet medical need to develop other inhibitors of complement for C3G. Methods We used a murine model of lethal C3G to test the potential efficacy of an Fc fusion protein of complement receptor of the Ig superfamily (CRIg-Fc) in the treatment of C3G. CRIg-Fc binds C3b and inhibits C3 and C5 convertases of the AP. Mice with mutations in the factor H and properdin genes (FH(m/m)P(−/−)) develop early-onset C3G, with AP consumption, high proteinuria, and lethal crescentic GN. Results Treatment of FH(m/m)P(−/−) mice with CRIg-Fc, but not a control IgG, inhibited AP activation and diminished the consumption of plasma C3, factor B, and C5. CRIg-Fc–treated FH(m/m)P(−/−) mice also had significantly improved survival and reduced proteinuria, hematuria, BUN, glomerular C3 fragment, C9 and fibrin deposition, and GN pathology scores. Conclusions Therapeutics developed on the basis of the mechanism of action of soluble CRIg may be effective for the treatment of C3G and should be explored clinically.

Background Rheumatoid arthritis (RA) is a kind of chronic autoimmune disease, mainly manifested as small joint synovitis. Disease progression appears joint swelling, bone and cartilage damage, deformity and activity limited. The etiology of RA is still unclear and is generally considered to be an immune-mediated inflammatory disease. The activation pathway and regulation function of the complement system have become the hotspot of the research of RA pathogenesis. Previous studies have found that C3-/- mice had lower levels of antibodies to collagen in the Type II collagen-induced rheumatoid arthritis (CIA model), and the molecular mechanism is unclear. The small fragment C3a and large segment iC3b produced by C3 activation are the main effect products, and their biological effects are performed by combining with the specific receptor, C3aR and CD11b respectively. Objectives To investigate the mechanism of C3a and C3b, the cleavage products of complement C3 in rheumatoid arthritis, binding to their corresponding receptors, the signalling pathway of complement activation and the effect on arthritic conditions. Methods This study was intended to establish a CIA model on C3aR knockout and CR3 knockout mice (C3aR-/-or CD11b-/-) to investigate the effect of complement C3a-C3aR signalling and iC3b-CR3 signalling on rheumatoid arthritis. Methods using C57BL/6 background transgenic mice (Gifted by King’s College London), Mice were divided into 3 groups according to the experimental mouse strains: C3aR-/- group, CD11b-/- group and WT control group. The clinical score of the joints in each group was measured after the establishment of the CIA model through collagen induction. Moreover, joint specimens were collected for pathologic grading. Besides, the level of CD4 +T cell, CD8 +T cell, Th17/Treg ratio and the level of IFN-gamma of NK cell in mouse spleen were detected by flow cytometry. Results 1. The clinical score of C3aR-/- group was slightly lower than that of WT group, and the clinical score of CD11b-/- group was significantly higher than that of WT group; 2. Pathological score (12 points): The CIA scores of CD11b-/- group, C3aR-/- group and WT group were 9.35±0.75, 4.81±0.63 and 5.85±0.55 respectively. The CIA scores of CD11b-/- group was significantly higher than that of WT group, which were consistent with clinical score; 3.Through the flow cytometry detection, compared with the WT group, CD4 +T cell, CD8 +T cell and Th17 percentage increased significantly, and Treg cell decreased. In addition, the secretion of IFN-gamma of Splenic NK cell was significantly reduced. Conclusions iC3b as well as C3a could bind to their respective complement receptor, and express different influence in the immune mechanism of RA, The iC3b-CD11b signalling has a protective effect in RA, while the C3a-C3aR signalling has an inflammatory aggravation effect. Through this study, it helps us to invent the drug related to complement components and their receptors, and further be used in the clinical treatment of RA. Disclosure of Interest None declared

The complement system plays a critical role in immune responses against pathogens. However, its identity and regulation in the lung are not fully understood. This study aimed to explore the role of tripartite motif protein (TRIM) 72 in regulating complement receptor (CR) of the Ig superfamily (CRIg) in alveolar macrophage (AM) and innate immunity of the lung. Imaging, absorbance quantification, and flow cytometry were used to evaluate in vitro and in vivo AM phagocytosis with normal, or altered, TRIM72 expression. Pulldown, coimmunoprecipitation, and gradient binding assays were applied to examine TRIM72 and CRIg interaction. A pneumonia model was established by intratracheal injection of Pseudomonas aeruginosa. Mortality, lung bacterial burden, and cytokine levels in BAL fluid and lung tissues were examined. Our data show that TRIM72 inhibited CR-mediated phagocytosis, and release of TRIM72 inhibition led to increased AM phagocytosis. Biochemical assays identified CRIg as a binding partner of TRIM72, and TRIM72 inhibited formation of the CRIg-phagosome. Genetic ablation of TRIM72 led to improved pathogen clearance, reduced cytokine storm, and improved survival in murine models of severe pneumonia, specificity of which was confirmed by adoptive transfer of wild-type or TRIM72(KO) AMs to AM-depleted TRIM72(KO) mice. TRIM72 overexpression promoted bacteria-induced NF-κB activation in murine alveolar macrophage cells. Our data revealed a quiescent, noninflammatory bacterial clearance mechanism in the lung via AM CRIg, which is suppressed by TRIM72. In vivo data suggest that targeted suppression of TRIM72 in AM may be an effective measure to treat fatal pulmonary bacterial infections.

Background Glomerular Immune complex (IC) deposition causes glomerulonephritis in lupus nephritis. The reaction of intrinsic glomerular cells differs according to the area of IC deposition. Subepithelial IC deposits cause functional and structural changes on podocytes (glomerular visceral epithelial cells) through complement pathway activation as is well known in membranous glomerulonephritis. In this setting, podocytes can induce some complement factors including complement factor H (CFH) which serves not only to regulate the alternative pathway but also to process subepithelial IC deposition.1 However, whether podocyte processing of IC deposition is limited to only the subendothelial area is obscure. Objectives To clarify the role of podocytes in IC deposition in the glomerular subendothelial area. Methods NEP25 mice genetically expressing human CD25 in podocytes were used. Intravenous immunotoxin for human CD25 (LMB2) provokes podocyte-specific injury (NEP25/LMB2). By shortening the period of LMB2 exposure to NEP25 mice, we mitigated the podocyte injury. We administered intraperitoneally IgG3-producing hybridoma clones, 2B11.3, which were previously established from an unmanipulated MRL/lpr mouse, to NEP25 mice (NEP25/hybridoma). Furthermore, we also injected short-term LMB2 to NEP25/hybridoma mice (NEP25/hybridoma/LMB2). We investigated IC deposits by immunofluorescence and electronic microscopic study. We measured complement regulatory factor mRNA expression including CFH, complement factor I (CFI), decay-accelerating factor (DAF), complement receptor 1-related gene/protein y (Crry), C3a receptor (C3aR) and C5a receptor (C5aR) of isolated glomeruli of each mouse by quantitative real time-PCR. In an in vitro study, we assessed CFH mRNA expression of immortalised mouse podocytes injured by puromycin. Results First, NEP25/LMB2 mice showed glomerular tuft collapse with epithelial cell hyperplasia, suggesting podocyte loss by light microscopy study as reported previously. mRNA expression of all complement regulatory factors but C5aR was decreased in NEP25/LMB2 mice as compared with NEP25 mice. This result suggests that podocytes produce these complement regulatory factors. On the other hand, when the podocyte injury was mitigated, CFH and C3aR mRNA expression increased, and CFI, DAF and Crry mRNA expression decreased as compared with NEP25 mice. Second, we detected IC deposition only in the glomerular subendothelial area without endocapillary proliferative lesion in NEP25/hybridoma mice. They showed increase of C3aR mRNA expression, and decrease of CFI and DAF mRNA expression as compared with controls. Notably, subendothelial IC deposition did not alter CFH mRNA expression. Third, NEP25/hybridoma/LMB2 (milder podocyte injury) showed decrease of IC deposits in glomeruli and increase of only CFH mRNA expression (1.7-fold) as compared to NEP25/hybridoma mice, while there was no significant change in mRNA expression of other complement regulatory factors. Finally, puromycin-induced mild podocyte injury promoted CFH mRNA expression in cultured podocytes as compared to controls. Conclusions Mildly injured podocytes induce CFH expression, and serve to process IC deposition in the glomerular subendothelial area. Reference [1] J Am Soc Nephrol2007;18:1157–66. Disclosure of Interest None declared