Foam cells derived from macrophages and smooth muscle cells are formed by the uncontrolled uptake of modified low-density lipoprotein (LDL) and are the main cellular components of atherosclerotic lesions. Uptake of oxidized LDL (oxLDL) by macrophages occurs via receptor-mediated endocytosis through various scavenger receptors. Although resting macrophages internalize modified LDL mainly via SR-A and CD36 receptors, evidence suggests an important role for LOX-1 in the transformation of macrophages into foam cells, despite the low level of LOX-1 on the surface membrane of resting macrophages. Here we describe novel positive feedback loops involving anaphylatoxin C3a and its receptor, which lead to increased LOX-1 levels in macrophages and reveal the molecular mechanisms underlying these processes. Impact statement Little is known about processes which control the transformation of macrophages into foam cells in atherosclerotic lesions. Here, we describe novel positive feedback loops associated with anaphylatoxin C3a and its receptor, which lead to escalation of oxLDL uptake by macrophages, and reveal the central role of the LOX-1 receptor in this process.

Functional approaches to properly examine individual's susceptibility to complement dysregulation are limited. We assessed ex vivo complement activation induced by sera from 38 healthy donors on resting microvascular endothelial cells (HMEC-1) with or without complement dysregulation (OX-24 mAb). Following incubation, immunofluorescence was used to quantify membrane-bound C3b/iC3b and C5b-9 with a computer-assisted method (H-score). C3a and C5a anaphylatoxins were quantified in supernatants by ELISA. Genetic sequencing of 32 donors was also performed to identify variants in alternative pathway genes. Elevated complement deposition was defined by H-scoreC3c > 50 and/or H-scoreC5b-9 > 30. Combined analysis of C3b/iC3b and C5b-9 deposition in 35 donors showed that one donor (2.8%) had an isolated increase in C3b/iC3b deposits, 3 (8.6%) had an isolated increase in C5b-9 deposits and one (2.8%) had both increased C3b/iC3b and C5b-9 deposition. Genetic analysis revealed 3 heterozygous rare/low frequency missense variants in CFH (p.N1050Y, p.R121°C) and CFI (p.A76G) in 3/5 donors with increased complement deposition. This model revealed distinct patterns of complement activation among healthy individuals and identified a genetic basis for dysregulation in three cases. This assay offers a promising tool to study complement activity and its mechanisms in research.

Francisella tularensis (Ft), a Gram-negative bacterium that causes tularemia, possesses a non-inflammatory atypical LPS (LPSFt) that is highly immunogenic through unknown mechanism. We previously showed that immunization with LPSFt, a type 2 T-independent (TI) antigen, elicits protective LPSFt-specific IgM (IgMFt) and IgG3Ft by B1 cells in a mechanism dependent on the IL-5 produced by innate lymphoid cells type 2 (ILC2). Here, we examined the role of complement in the B1 cells’ response against LPSFt. C3-/-, C1q-/- and C4-/- mice immunized with LPSFt failed to produce IgMFt and IgG3Ft. In contrast, the response of Cfb-/- and Mbl1/Mbl2-/- mice was comparable to that of WT mice. Thus, activation of the classical complement cascade, but not the alternative or the Mannose Binding Lectin pathway, is required for activation of B1 cells and production of LPSFt-specific antibodies. Complement activation generates the C3d fragment, which opsonizes antigens for recognition by complement receptor-2 (CR2), and the C3a and C5a anaphylatoxins. Our results show that C3d opsonized LPSFt and that the response to immunization was dependent on CR2 expression by B1 cells. Importantly, the response to LPSFt immunization was also drastically decreased in C3ar1-/-, but not in C5ar1-/- mice. C3a induced IL-5 in ILC2, which supported B1 cells activation. Decreased antibody production in C3ar1-/- and Cr2-/- mice correlated with increased susceptibility to tularemia. Together, these results demonstrate that the high immunogenicity of LPSFt depends on two effector mechanisms triggered by activation of the classical complement pathway: 1) tagging of LPSFt with C3d fragment, leading to its interaction with CR2 expressed by B1 cells; 2) production of the anaphylatoxin C3a that stimulated IL-5 secretion by ILC2. Our study increases our understanding of the B1 cells’ response to TI-2 antigens and identifies two complement effector mechanisms that can be harnessed for therapeutic interventions.

IHP-102 reduces pain and modulates complement activation in a sickle cell mouse model

Genetic deletion of the complement C3a anaphylatoxin chemotactic receptor (C3ar1), a key component of the innate immune response, is reported to induce behavioural phenotypes resembling anxiety and hyperactivity in mice, suggesting a neurodevelopmental role for this gene in health. However, it is not currently clear when and where C3ar1 is needed in the brain, which is further complicated by the fact that C3ar1 is expressed predominantly by microglia and therefore does not localize to specific brain regions, warranting exploratory and brain-wide assessment through neuroimaging. Resolving when and where C3ar1 is needed are questions of significant translational importance because, as a G-protein-coupled receptor, human C3AR1 serves as a potential therapeutic target for disorders associated with complement upregulation, such as schizophrenia. To provide a brain-wide assessment of developmental C3ar1 activity, we used longitudinal MRI in male and female adolescent and adult mice (N = 34 C3ar1tm1Cge/tm1Cge and N = 35 C3ar1+/+) to estimate regional brain volume using tensor based morphometry, white matter microstructure using fractional anisotropy from diffusion-weighted MRI, and functional connectivity from blood oxygen-level dependent MRI, with behavioural assessment in adulthood. We repeated structural MRI measures in this cohort ex vivo to achieve higher resolution. We further repeated in vivo structural assessment preceded by behavioural testing in adulthood in a second cohort of mice (N = 20 C3ar1tm1Cge/tm1Cge and N = 19 C3ar1+/+) to improve confidence in our findings. We achieved low regional brain volume variability, allowing us to resolve previously reported sexually dimorphic effects. We were further able to confirm a well-known developmental increase in fractional anisotropy. Despite being able to detect these established effects, we did not find a robust C3ar1-dependent phenotype in any of the measures we tested, including behaviour, which may be attributed to our study being the first behavioural study in C3ar1-deficient mice to include littermate controls. Therefore, our data do not support neurodevelopmental hypotheses for C3ar1, which is encouraging for therapeutic strategies targeting this receptor since interventions are unlikely to disrupt brain development.

Complement (C) proteins have been linked to infertility and reproductive outcomes. This study was undertaken to determine the association of complement proteins in non-obese women before in vitro fertilization (IVF) with unexplained infertility (UI) compared to women with male factor infertility (MFI) as controls. We hypothesized that complement protein factors may provide evidence for the underlying mechanism in UI. In this exploratory pilot study, 25 women (UI = 14 and MFI = 11) undergoing IVF had blood drawn on day 21 of the luteal phase. Slow Off-rate Modified Aptamer (SOMA)-scan plasma protein measurement was undertaken for 25 complement pathway-related proteins. Student’s t-test was used to compare group means and Pearson’s correlations to examine relationships with complement proteins. Baseline demographics and hormonal parameters did not differ between groups, and parameters of the response following IVF did not differ. In the UI group compared to the MFI group, there were lower levels of properdin (p = 0.03) that may reduce endometrial receptivity and impact follicular development, lower C3a anaphylatoxin des arginine (C3adesArg) (p = 0.02) that may reduce endometrial vascularity, lower C4 (C4) (p = 0.04), indicating reduced alternate pathway activation, and lower C8 (C8) (p = 0.04) that also may affect the endometrium. In UI alone, properdin negatively correlated with high-density lipoprotein cholesterol (HDL-c), and C8 positively correlated with thyroid-stimulating hormone (TSH) and Free-triiodothyronine (Free-T3) (p < 0.05). These preliminary findings indicate reduced complement activity among UI women, warranting further mechanistic investigation.

Complement opsonin C3b is at the center of the complement cascade. All complement initiation pathways converge when convertases activate C3, converting it into the anaphylatoxin C3a and the opsonin C3b, which can attach to surfaces via its freshly exposed thioester moiety. C3b has several important functions and remains a focus of basic and translational research. C3b and its proteolytically processed derivatives iC3b and C3dg are important opsonins for different complement receptors on various immune cells. C3b is the initiation nucleus for the formation of the alternative pathway C3 convertase and C3b clusters act as gatekeepers for the activation of C5, which initiates the terminal pathway. High densities of surface-deposited C3b alter the substrate specificity of complement convertases from C3 to C5 activation. It remains unknown how C3b achieves the “C5 priming” necessary for this process. Further exploration of the functions of C3b is laborious, as it generally requires the purification of C3 from plasma/serum, subsequent conversion to C3b, and further purification. Therefore, we developed a rapid (three days) and efficient method that purifies C3b directly from blood sources, requiring fewer steps and reagents than previously published protocols. This method utilizes the newly exposed sulfhydryl group, which becomes accessible by converting C3 to C3b. The C3b purified by this method exhibits high purity and very high activity, as demonstrated by affinity measurements for several known C3b ligands, the formation and dissociation of the alternative pathway convertase, and the capacity to be regulated by FI.

Retinal degenerative diseases: Complement system-microglia crosstalk.

Activation of the alternative complement pathway by artificial extracorporeal surfaces is relevant to several clinical applications such as cardiac surgery with cardiopulmonary bypass (CPB), thoracic organ transplantation and hemodialysis. In the pediatric cardiac surgery population, complement mediators have been associated with systemic inflammation, post-operative morbidity and delayed recovery. Small children require CPB circuit prepared with allogeneic blood products and these sanguineous primes have substantially higher concentrations of biologically active anaphylatoxins C3a and C5a relative to the patient's baseline circulation. Sequential samples were collected during forty-five ex-vivo sanguineous prime preparations of CPB circuits coated with phosphorylcholine, as a biocompatibility technology, to characterize complement activation in this context. We observed and quantified evidence of alternative and terminal complement pathway activation indicated by dynamic concentration increases of C3a, C3b, C5a and terminal complement complex. Circuit exposure time was a predictor of only C3a concentration in multivariable generalized linear mixed-effects models. Despite modern biocompatibility technology, there is significant alternative complement activation during ex-vivo sanguineous CPB prime preparation. Patient exposure to this activated mediator burden during CPB could promote systemic endothelial inflammation with negative end-organ and post-operative clinical impacts. Further research is required to evaluate and inhibit complement responses to artificial surfaces broadly used for clinical care.

The complosome controls different activities in innate immune cells and epithelial cells; however, its role in the response of VECs to Candida remains untested. In this in vitro study, we compared two clinical vaginal strains of C. albicans, namely, a Colonizing strain from a healthy woman and a strain from a patient with vulvovaginal candidiasis (VVC), for their ability to activate the complosome and release anaphylatoxins in vaginal epithelial cells (VECs). Our results show the following: (i) both strains triggered the cleavage of C3 into C3a and C3b within VECs, while infection with the Colonizing strain led to greater release of the anaphylatoxin C3a; (ii) infection with the VVC isolate led to a strong reduction in both C5 and C5a in VECs, while no increase in C5a release was observed after infection with either strain; (iii) cathepsin-family gene expression and cathepsin D activity were reduced in VECs infected with the VVC strain but not in those infected with the Colonizing strain; (iv) infection with the Colonizing strain induced a significant increase in intracellular C5aR1 while intracellular C3aR levels remained unchanged. Collectively, our data suggests the propensity of this VVC strain to inactivate the C5/C5aR1 axis and to reduce the C3/C3aR axis, dampening the activity of the complosome in VECs. These effects exerted by the VVC strain suggest a novel strategy of immune evasion by C. albicans and may open new perspectives for finding new therapeutic targets against vaginal fungal infections.

Targeting complement anaphylatoxin C3a receptor to break immunotherapy resistance in colorectal cancer

BACKGROUND: Macrophages are unique professional phagocytes involved in both innate and adaptive immune responses. This is made possible by their functional plasticity—the ability to acquire different phenotypes. Switching between subtypes during interactions with other immune system components is referred to as polarization. Anaphylatoxin C3a has been shown to influence macrophage polarization. Depending on the polarization state, macrophages exhibit different phagocytic activities. Given the contradictory evidence regarding C3a’s effects on macrophages, determining its impact on phagocytosis in general and on efferocytosis in particular is of special interest. AIM: The work aimed to investigate the effect of anaphylatoxin C3a on efferocytosis by M0 and M2 macrophages. METHODS: The experiments were conducted using human peripheral blood mononuclear cells. Macrophages were differentiated in the presence of C3a, and M2 polarization was induced with interleukin-4. Efferocytosis was assessed using confocal microscopy, whereas phagocytosis was evaluated via flow cytometry. Real-time reverse transcription polymerase chain reaction was used to measure expression of the efferocytosis receptor genes mertk, axl, and tyro3. RESULTS: M2 macrophages demonstrated greater efferocytic capacity compared to M0 macrophages. Anaphylatoxin C3a did not affect the phagocytosis of dead Escherichia coli bacteria but inhibited the phagocytosis of apoptotic cells in both unpolarized M0 and alternatively activated M2 macrophages. It was also shown that C3a reduces the expression of the Tyro3 receptor gene in both M0 and M2 macrophages. CONCLUSION: Anaphylatoxin C3a exerts a suppressive effect on efferocytosis, likely through modulation of apoptotic cell recognition processes.

The pathological basis of many visual disorders involves the abnormal viability and migration of retinal pigment epithelium (RPE) cells. Complement response disorder is a significant pathogenic factor causing some autoimmune and inflammation diseases. The complement activation product anaphylatoxin C5a signaling pathway may be associated with RPE cell dysfunction. This study aimed to analyze the molecular mechanisms by which C5a affects RPE cell viability and migration. Recombinant human complement component C5a protein stimulated RPE cells. Cell biological behavior, including cell viability, invasion, and migration were analyzed with Cell Counting Kit-8 and transwell methods. Bioinformatics analysis identified the differentially expressed genes (DEGs) involved in C5a-treated RPE cells based on RNA sequencing. SLC38A1 was knocked down or overexpressed by vector transfection to investigate its involvement in C5a-stimulated RPE cells. C5a promotes RPE cell viability and migration. C5a-induced DEGs are enriched in migration-associated pathways. C5a increased SLC38A1, and SLC38A1 knockdown or overexpression inhibited or promoted RPE cell viability and migration. Glutaminase inhibition abrogated the promoting effect of C5a and SLC38A1 on cell biological behaviors. METTL3-HNRNPC-mediated m6A modification mediated C5a-induced SLC38A1. C5a, METTL3, and SLC38A1 constituted a signaling axis in regulating cell biological behaviors of C5a-treated RPE cells. C5a promotes RPE cell viability and migration, and SLC38A1-mediated improved glutamine metabolism is the downstream signal pathway of the C5a complement pathway. The C5a complement system may target the SLC38A1 to promote RPE cell migration.

BackgroundThe EGFR is abundantly expressed in prostate cancer (PC). The anaphylatoxin C5a induces leukocyte migration via the C5a receptor (C5aR) by releasing matrix metalloproteinases (MMP) to favor metastasis in the tumor microenvironment. This work aims to selectively inhibit the EGFR and C5aR in PC cells to abort cell growth/ proliferation and metastasis.MethodsFor lead identification, high-throughput virtual screening (HTVS) of the ChemBridge library was followed by protein–ligand interaction profilers, GROMACS, and GMX-MMPBSA techniques. LNCaP and PC3 cells were used to validate in vitro efficacy.ResultsHTVS identified CEG-0598 with favorable binding affinities of − 10.2 kcal/mol and − 13.5 kcal/mol towards EGFR and C5aR respectively. Molecular dynamic simulations demonstrated stable binding interactions for CEG-0598 with Root Mean Square Deviation values around 0.06 nm. The ΔG binding calculation was − 50.29, and − 51.64 for EGFR and C5aR respectively. ADME supported favorable small molecule characteristics and selective inhibition profiles. Kinome-wide off-target virtual screening predicted EGFR to have above-average docking scores. CEG-0598 inhibited EGFR and C5aR activities with IC50 values of 145.8 nM and 55.51 nM respectively. The compound effectively controlled the proliferation of LNCaP and PC3cells with GI50 values of 156.1 nM, and 112.2 nM respectively. CEG-0598 prompted dose-responsive apoptosis in the PC cells and decreased the tarns endothelial migration of both PC cells. Treatment with CEG-0598 reduced the C5a-induced MMP activity in the LNCaP and PC3cells.ConclusionCEG-0598 is a selective EGFR/C5a dual inhibitor that downregulates MMP activity to control proliferation, migration and induce apoptosis, in PC cells warranting further preclinical developments.

The complement system plays crucial roles in innate immunity and inflammatory responses. The anaphylatoxin C3a mediates pro-inflammatory and chemotactic functions through the G protein-coupled receptor C3aR. While the active structure of the C3a-C3aR-Gi complex has been determined, the inactive conformation and activation mechanism of C3aR remain elusive. Here we report the cryo-EM structure of ligand-free, G protein-free C3aR, providing insights into its inactive conformation. In addition, we determine the structures of C3aR in complex with the synthetic small-molecule agonist JR14a in two distinct conformational states: a G protein-free intermediate, and a fully active Gi-bound state. The structure of the active JR14a-bound C3aR reveals that JR14a engages in highly conserved interactions with C3aR, similar to the binding of the C-terminal pentapeptide of C3a, along with JR14a-specific interactions. Structural comparison of C3aR in the apo, intermediate, and fully active states provides novel insights into the conformational landscape and activation mechanism of C3aR and defines a molecular basis explaining its high basal activity. Our results may aid in the rational design of therapeutics targeting complement-related inflammatory disorders.

Chemotherapy-induced peripheral neuropathy (CIPN) is a significant adverse event with unclear mechanisms and limited treatment alternatives. This study aimed to investigate the efficacy of two alkalizing agents, a mixture of potassium citrate and sodium citrate (K/Na citrate) or sodium bicarbonate (NaHCO3), in preventing and treating paclitaxel (PTX)-induced mechanical allodynia in rodents. The results from rodent models demonstrated that repeated prophylactic administration of K/Na citrate or NaHCO3 could inhibit the development of PTX-induced mechanical allodynia. Moreover, K/Na citrate was effective in preventing the PTX-induced exacerbation of mechanical allodynia, even when treatment was initiated immediately after the onset of allodynia. K/Na citrate also reduced the levels of the plasma complement component anaphylatoxin C3a in a PTX-induced CIPN rat model. Complement activation, resulting in the production of C3a, has been implicated in the pathogenesis of this model. Additionally, pretreatment with Na citrate significantly prevented the reduction in neurite outgrowth caused by PTX. Furthermore, K/Na citrate inhibited spontaneous and mechanical stimuli-induced firing in spinal dorsal horn neurons. These findings indicate that K/Na citrate may regulate the development of PTX-induced mechanical allodynia by modulating complement activation and providing neuroprotection against PTX-induced peripheral nerve injury. This study implies that alkalization could help prevent PTX-induced peripheral neuropathy and mitigate its exacerbation.

Objectives/Goals: Germinal matrix hemorrhage (GMH) is a devastating disease of infancy that results in brain-related pathologies. Following rupture of vasculature in the brain, red blood cell (RBC) lysis, and hemoglobin breakdown results in heme/iron-related toxicities. We hypothesize that these cellular pathologies are mediated in part by the complement system. Methods/Study Population: Post-natal mice on day 4 (P4) were subjected to collagenase induced-GMH and treated with various complement inhibitors that function at different points in the complement pathway and differentially prevent the generation of specific complement activation products. The principle bioactive complement activation products are C3 opsonins (C3b, iC3b, and C3d), the proinflammatory anaphylatoxins (soluble C3a and C5a peptides), and the terminal cytolytic membrane attack complex (MAC). Experimental groups consisted of: Wild-type (WT) naïve mice, and WT GMH-mice treated with either PBS (vehicle), CR2-Crry (C3 inhibitor that blocks all activation products), anti-C5 mAb (blocks C5a and MAC), C5aRA (blocks C5a-C5a receptor interaction), or anti-C7 mAb (blocks MAC). Study endpoints were P7 or P14. Results/Anticipated Results: Following GMH, CR2-Crry treatment decreased MAC deposition on RBC and additionally decreased heme oxygenase-1 expression, heme deposition, and iron-induced inflammation measured at P7. In support of a specific role for the MAC, anti-C7 mAb treatment resulted in similar outcomes and was similarly protective. Anti-C7 mAb treatment also reduced hydrocephalus development at a later time point (P14). A similar result was obtained using C7 deficient mice and with anti-C5 mAb treatment. On the other hand, no protective effect was seen with C5aR blockade, and double knock out of C3aR/C5aR also did not provide protection, indicating no role for the anaphylatoxins C3a and C5a and their receptors expressed on leukocytes and endothelial cells in exacerbating deteriorating outcomes. Discussion/Significance of Impact: Our data indicate a key role for the MAC in RBC induced hemolysis after GMH which serves as a driver of inflammation and early GMH pathogenesis. We further show that we can effectively increase precision by targeting solely the MAC complex acutely. Future work will be undertaken to determine temporal roles of individual complement activation products.

Rpl13a snoRNAs-regulated NADPH oxidase 1-dependent ROS generation: A novel RBC pathway mediating complement C3a deposition and triggering thrombosis in aging and venous blood clotting disorders.

Ravulizumab and other complement inhibitors for the treatment of autoimmune disorders.

Aortic disease encompasses life-threatening conditions such as aortic aneurysm and dissection, which are associated with high prevalence, morbidity, and mortality. The complement system, a key component of innate immunity, not only defends against pathogens but also maintains tissue homeostasis. Recent discoveries have expanded its role beyond immunity, linking complement dysregulation to numerous diseases and positioning it as a target for pharmacotherapy. Complement-based treatments for precision medicine are emerging, with several pharmaceuticals either already approved or under investigation. In aortic disease, complement activation and dysregulation have unveiled novel mechanisms and clinical implications. Human and experimental studies suggest that all three complement pathways contribute to disease pathophysiology. The complement system induces direct cellular damage via the membrane attack complex, as well as matrix-metalloproteinase (MMP)-associated tissue damage by promoting MMP-2 and MMP-9 expression. The anaphylatoxins C3a and C5a exacerbate disease by recruiting immune cells and triggering proinflammatory responses. Examples include neutrophil extracellular trap formation and cytokine release by polymorphonuclear neutrophils. These findings highlight the complement system as a promising novel diagnostic and therapeutic target in aortic disease with potential for individualized treatment. However, gaps remain, emphasizing the need for standardized multisite preclinical studies to improve reproducibility and translation. Biomarker studies must also be validated across diverse patient cohorts for clinical applicability. This review examines current knowledge regarding complement in aortic disease, aiming to evaluate its potential for innovative diagnostic and personalized treatment strategies.