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B cells are types of lymphocytes that are involved in the production of antibodies against pathogens. They also deliver and present antigens for the priming of T cells. Recently, we developed an in vivo splenic marginal zone (MZ) B cell-targeting liposomes decorated with polyethylene glycol (PEG) containing a hydroxyl-terminus group (HO-PEG-Lip). In an expansion of a previous study, we used HO-PEG-Lip as an in vitro antigen delivery tool to splenic B cells to test the ability of this formulation to overcome the limitations of the poor antigen uptake ability of B cells for implantation. To achieve our purpose, various factors were optimized. These factors include cell number, liposome concentration, pre-opsonization of liposomes, fresh serum concentration, and incubation time, all of which affect the extent of interaction between liposomes and B cells. As a result, we confirmed that the HO-PEG-Lip required incubation at 37 °C for at least 20 min with 50% mouse fresh serum followed by a subsequent incubation at 37 °C for at least another 30 min with splenic B cells. By using such a loading system, fluorescein isothiocyanate (FITC)-labeled ovalbumin (OVA), a model antigen, encapsulated in HO-PEG-Lip could be efficiently loaded into splenic B cells. In addition, HO-PEG-Lip and FITC-labeled OVA encapsulated in HO-PEG-Lip were efficiently associated with MZ-B cells with high levels of complement receptors (CRs) rather than follicular B cells with low levels of CRs. These results propose a novel and useful system to efficiently load antigens into B cells in vitro by taking advantage of complement systems.

Complement Receptor Type 1 (CR1) is a malaria-associated gene that encodes a transmembrane receptor of erythrocytes and is crucial for malaria parasite invasion. The expression of CR1 contributes to the rosetting of erythrocytes in the brain bloodstream, causing cerebral malaria, the most severe form of the disease. Here, we study the history of adaptation against malaria by analyzing selection signals in the CR1 gene. We used whole-genome sequencing datasets of 907 healthy individuals from malaria-endemic and non-endemic populations. We detected robust positive selection in populations from the hyperendemic regions of East India and Papua New Guinea. Importantly, we identified a new adaptive variant, rs12034598, which is associated with a slower rate of erythrocyte sedimentation and is linked with a variant associated with low levels of CR1 expression. The combination of the variants likely drives natural selection. In addition, we identified a variant rs3886100 under positive selection in West Africans, which is also related to a low level of CR1 expression in the brain. Our study shows the fine-resolution history of positive selection in the CR1 gene and suggests a population-specific history of CR1 adaptation to malaria. Notably, our novel approach using population genomic analyses allows the identification of protective variants that reduce the risk of malaria infection without the need for patient samples or malaria individual medical records. Our findings contribute to understanding of human adaptation against cerebral malaria. Ministry of Education (MOE) Published version This research was supported by the Singapore Ministry of Education, Academic Research Fund Tier 1 (grant number 2017-T1-001-046 and grant number RG100/20).

Acute lung injury (ALI) occurs in patients with severe sepsis and has a mortality rate of 40%–60%. Severe sepsis promotes the release of histones from dying cells, which can induce platelet aggregation, activate coagulation and cause endothelial cell (EC) death. We previously reported that the expression of membrane complement receptor type 1‐related gene Y (Crry)/p65, which plays a principal role in defence against abnormal activation of complement in the blood, is reduced in response to peritoneal mesothelial cell injury, and we hence hypothesized that a similar mechanism occurs in pulmonary ECs. In this study, we examined the role of Crry/p65 in histone‐mediated ALI using an experimental animal model. In ALI model mice, exposure to extracellular histones induces lung injury and results in a decrease in Crry/p65 expression. The levels of lactic acid dehydrogenase (LDH), a marker of cell damage, were significantly increased in the serum of ALI model compared with vehicle mice. The significant inverse correlation between the expression of Crry/p65 and LDH levels in plasma revealed an association between Crry/p65 expression and cell damage. The levels of complement component 3a (C3a) were also significantly increased in the serum of the ALI model compared with vehicle mice. Notably, a C3a receptor antagonist ameliorated lung injury induced by histones. We hypothesize that extracellular histones induce complement activation via down‐regulation of Crry/p65 and that C3a might serve as a therapeutic target for the treatment of ALI., Extracellular histones decrease the expression of complement receptor type 1‐related gene Y (Crry)/p65 in an experimental model of acute lung injury (ALI). Dysfunction of Crry/p65 is associated with endothelial damage and promotes C3a production resulting from complement activity. C3a is a potential therapeutic target for ALI.

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

Axon regenerative failure in the mature CNS contributes to functional deficits following many traumatic injuries, ischemic injuries, and neurodegenerative diseases. The complement cascade of the innate immune system responds to pathogen threat through inflammatory cell activation, pathogen opsonization, and pathogen lysis, and complement is also involved in CNS development, neuroplasticity, injury, and disease. Here, we investigated the involvement of the classical complement cascade and microglia/monocytes in CNS repair using the mouse optic nerve injury (ONI) model, in which axons arising from retinal ganglion cells (RGCs) are disrupted. We report that central complement C3 protein and mRNA, classical complement C1q protein and mRNA, and microglia/monocyte phagocytic complement receptor CR3 all increase in response to ONI, especially within the optic nerve itself. Importantly, genetic deletion ofC1q,C3, orCR3attenuates RGC axon regeneration induced by several distinct methods, with minimal effects on RGC survival. Local injections of C1q function-blocking antibody revealed that complement acts primarily within the optic nerve, not retina, to support regeneration. Moreover, C1q opsonizes and CR3+microglia/monocytes phagocytose growth-inhibitory myelin debris after ONI, a likely mechanism through which complement and myeloid cells support axon regeneration. Collectively, these results indicate that local optic nerve complement-myeloid phagocytic signaling is required for CNS axon regrowth, emphasizing the axonal compartment and highlighting a beneficial neuroimmune role for complement and microglia/monocytes in CNS repair.SIGNIFICANCE STATEMENTDespite the importance of achieving axon regeneration after CNS injury and the inevitability of inflammation after such injury, the contributions of complement and microglia to CNS axon regeneration are largely unknown. Whereas inflammation is commonly thought to exacerbate the effects of CNS injury, we find that complement proteins C1q and C3 and microglia/monocyte phagocytic complement receptor CR3 are each required for retinal ganglion cell axon regeneration through the injured mouse optic nerve. Also, whereas studies of optic nerve regeneration generally focus on the retina, we show that the regeneration-relevant role of complement and microglia/monocytes likely involves myelin phagocytosis within the optic nerve. Thus, our results point to the importance of the innate immune response for CNS repair.

Semen is important in determining HIV-1 susceptibility but it is unclear how it affects virus transmission during sexual contact. Mucosal Langerhans cells (LCs) are the first immune cells to encounter HIV-1 during sexual contact and have a barrier function as LCs are restrictive to HIV-1. As semen from people living with HIV-1 contains complement-opsonized HIV-1, we investigated the effect of complement on HIV-1 dissemination by human LCs in vitro and ex vivo. Notably, pre-treatment of HIV-1 with semen enhanced LC infection compared to untreated HIV-1 in the ex vivo explant model. Infection of LCs and transmission to target cells by opsonized HIV-1 was efficiently inhibited by blocking complement receptors CR3 and CR4. Complement opsonization of HIV-1 enhanced uptake, fusion, and integration by LCs leading to an increased transmission of HIV-1 to target cells. However, in the absence of both CR3 and CR4, C-type lectin receptor langerin was able to restrict infection of complement-opsonized HIV-1. These data suggest that complement enhances HIV-1 infection of LCs by binding CR3 and CR4, thereby bypassing langerin and changing the restrictive nature of LCs into virus-disseminating cells. Targeting complement factors might be effective in preventing HIV-1 transmission.

Human neutrophils are highly sensitive to the presence of Borrelia burgdorferi (Bb), the agent of Lyme disease (LD), in tissues. Although Bb is also found in the blood of LD patients, far less is known about how neutrophils respond to Bb in the presence of blood. In this study, we employed microfluidic tools to probe the interaction between human neutrophils and Bb and measured the activation of human neutrophils in blood samples from patients. We found that neutrophils migrate vigorously toward Bb in the presence of serum, and this process was complement-dependent. Preventing complement factor 5 cleavage or blocking complement receptors decreased neutrophil’s ability to interact with Bb. We also found that spiking Bb directly into the blood from healthy donors induced spontaneous neutrophil motility. This response to Bb was also complement-dependent. Preventing complement factor 5 cleavage decreased spontaneous neutrophil motility in Bb-spiked blood. Moreover, we found that neutrophils in blood samples from acute LD patients displayed spontaneous motility patterns similar to those observed in Bb-spiked samples. Neutrophil motility was more robust in blood samples from LD patients than that measured in healthy and ill controls, validating the utility of the microfluidic assay for the study of neutrophil-Bb interactions in the presence of blood.

Increased expression of complement C1r, C1s and C3 in kidney cells plays an important role in the pathogenesis of kidney fibrosis. Our studies suggest that activation of complement in kidney cells with increased generation of C3 and its fragments occurs by activation of classical and alternative pathways. Single nuclei RNA sequencing studies in kidney tissue from unilateral ureteral obstruction mice show that increased synthesis of complement C3 and C5 occurs primarily in renal tubular epithelial cells (proximal and distal), while increased expression of complement receptors C3ar1 and C5ar1 occurs in interstitial cells including immune cells like monocytes/macrophages suggesting compartmentalization of complement components during kidney injury. Although global deletion of C3 and macrophage ablation prevent inflammation and reduced kidney tissue scarring, the development of mice with cell-specific deletion of complement components and their regulators could bring further insights into the mechanisms by which intracellular complement activation leads to fibrosis and progressive kidney disease. LINKED ARTICLES: This article is part of a themed issue on Canonical and non-canonical functions of the complement system in health and disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.14/issuetoc.

Background & Aims Liver CRIg+ (complement receptor of the immunoglobulin superfamily) macrophages play a critical role in filtering bacteria and their products from circulation. Translocation of microbiota-derived products from an impaired gut barrier contributes to the development of obesity-associated tissue inflammation and insulin resistance. However, the critical role of CRIg+ macrophages in clearing microbiota-derived products from the bloodstream in the context of obesity is largely unknown. Methods We performed studies with CRIg–/–, C3–/–, cGAS–/–, and their wild-type littermate mice. The CRIg+ macrophage population and bacterial DNA abundance were examined in both mouse and human liver by either flow cytometric or immunohistochemistry analysis. Gut microbial DNA–containing extracellular vesicles (mEVs) were adoptively transferred into CRIg–/–, C3–/–, or wild-type mice, and tissue inflammation and insulin sensitivity were measured in these mice. After coculture with gut mEVs, cellular insulin responses and cGAS/STING-mediated inflammatory responses were evaluated. Results Gut mEVs can reach metabolic tissues in obesity. Liver CRIg+ macrophages efficiently clear mEVs from the bloodstream through a C3-dependent opsonization mechanism, whereas obesity elicits a marked reduction in the CRIg+ macrophage population. Depletion of CRIg+ cells results in the spread of mEVs into distant metabolic tissues, subsequently exacerbating tissue inflammation and metabolic disorders. Additionally, in vitro treatment of obese mEVs directly triggers inflammation and insulin resistance of insulin target cells. Depletion of microbial DNA blunts the pathogenic effects of intestinal EVs. Furthermore, the cGAS/STING pathway is crucial for microbial DNA–mediated inflammatory responses. Conclusions Deficiency of CRIg+ macrophages and leakage of intestinal EVs containing microbial DNA contribute to the development of obesity-associated tissue inflammation and metabolic diseases.

The emergence and evolution of the complement system and mast cells (MCs) can be traced back to sea urchins and the ascidian Styela plicata, respectively. Acting as a cascade of enzymatic reactions, complement is activated through the classical (CP), the alternative (AP), and the lectin pathway (LP) based on the recognized molecules. The system's main biological functions include lysis, opsonization, and recruitment of phagocytes. MCs, beyond their classic role as master cells of allergic reactions, play a role in other settings, as well. Thus, MCs are considered as extrahepatic producers of complement proteins. They express various complement receptors, including those for C3a and C5a. C3a and C5a not only activate the C3aR and C5aR expressing MCs but also act as chemoattractants for MCs derived from different anatomic sites, such as from the bone marrow, human umbilical cord blood, or skin in vitro. Cross talk between MCs and complement is facilitated by the production of complement proteins by MCs and their activation by the MC tryptase. The coordinated activity between MCs and the complement system plays a key role, for example, in a number of allergic, cutaneous, and vascular diseases. At a molecular level, MCs and complement system interactions are based on the production of several complement zymogens by MCs and their activation by MC-released proteases. Additionally, at a cellular level, MCs act as potent effector cells of complement activation by expressing receptors for C3a and C5a through which their chemoattraction and activation are mediated by anaphylatoxins in a paracrine and autocrine fashion.

Timely resolution of bacterial infections critically depends on phagocytosis of invading pathogens by polymorphonuclear neutrophil granulocytes (PMNs), followed by PMN apoptosis and efferocytosis. Here we report that bacterial DNA (CpG DNA) and mitochondrial DNA impair phagocytosis and attenuate phagocytosis-induced apoptosis in human PMNs through Toll-like receptor 9 (TLR9)-mediated release of neutrophil elastase and proteinase 3 and subsequent down-regulation of the complement receptor C5aR. Consistently, CpG DNA delays pulmonary clearance of Escherichia coli in mice and suppresses PMN apoptosis, efferocytosis, and generation of proresolving lipid mediators, thereby prolonging lung inflammation evoked by E. coli Genetic deletion of TLR9 renders mice unresponsive to CpG DNA. We also show that aspirin-triggered 15-epi-lipoxin A4 (15-epi-LXA4) and 17-epi-resolvin D1 (17-epi-RvD1) through the receptor ALX/FPR2 antagonize cues from CpG DNA, preserve C5aR expression, restore impaired phagocytosis, and redirect human PMNs to apoptosis. Treatment of mice with 15-epi-LXA4 or 17-epi-RvD1 at the peak of inflammation accelerates clearance of bacteria, blunts PMN accumulation, and promotes PMN apoptosis and efferocytosis, thereby facilitating resolution of E. coli-evoked lung injury. Collectively, these results uncover a TLR9-mediated endogenous mechanism that impairs PMN phagocytosis and prolongs inflammation, and demonstrate both endogenous and therapeutic potential for 15-epi-LXA4 and 17-epi-RvD1 to restore impaired bacterial clearance and facilitate resolution of acute lung inflammation.

Aggregation of α-synuclein (αSN) is an important histological feature of Parkinson disease. Recent studies showed that the release of misfolded αSN from human and rodent neurons is relevant to the progression and spread of αSN pathology. Little is known, however, about the mechanisms responsible for clearance of extracellular αSN. This study found that human complement receptor (CR) 4 selectively bound fibrillar αSN, but not monomeric species. αSN is an abundant protein in the CNS, which potentially could overwhelm clearance of cytotoxic αSN species. The selectivity of CR4 toward binding fibrillar αSN consequently adds an important αSN receptor function for maintenance of brain homeostasis. Based on the recently solved structures of αSN fibrils and the known ligand preference of CR4, we hypothesize that the parallel monomer stacking in fibrillar αSN creates a known danger-associated molecular pattern of stretches of anionic side chains strongly bound by CR4. Conformational change in the receptor regulated tightly clearance of fibrillar αSN by human monocytes. The induced change coupled concomitantly with phagolysosome formation. Data mining of the brain transcriptome in Parkinson disease patients supported CR4 as an active αSN clearance mechanism in this disease. Our results associate an important part of the innate immune system, namely complement receptors, with the central molecular mechanisms of CNS protein aggregation in neurodegenerative disorders.

Vaccination of neonates and young infants is hampered by the relative immaturity of their immune systems and the lack of safe and efficacious vaccine adjuvants. Immaturity of the follicular dendritic cells (FDCs), in particular, appears to play a critical role for the inability to stimulate immune responses. Using the CD21mT/mG mouse model we found that at 7 days of life, FDCs exhibited a mature phenotype only in the Peyer´s patches (PP), but our unique adjuvant, CTA1-DD, effectively matured FDCs also in peripheral lymph nodes following systemic, as well as mucosal immunizations. This was a direct effect of complement receptor 2-binding to the FDC and a CTA1-enzyme-dependent enhancing effect on gene transcription, among which CR2, IL-6, ICAM-1, IL-1β, and CXCL13 encoding genes were upregulated. This way we achieved FDC maturation, increased germinal center B-cell- and Tfh responses, and enhanced specific antibody levels close to adult magnitudes. Oral priming immunization of neonates against influenza infection with CTA1-3M2e-DD effectively promoted anti-M2e-immunity and significantly reduced morbidity against a live virus challenge infection. To the best of our knowledge, this is the first study to demonstrate direct effects of an adjuvant on FDC gene transcriptional functions and the subsequent enhancement of neonatal immune responses.

Background & Aims V-set Ig-domain-containing 4 (VSIG4) is an immunomodulatory macrophage complement receptor modulating innate and adaptive immunity and affecting the resolution of bacterial infections. Given its expression on peritoneal macrophages (PMs), we hypothesised a prognostic role of peritoneal VSIG4 concentrations in patients with spontaneous bacterial peritonitis (SBP). Methods We isolated PMs from patients with cirrhosis and analysed VSIG4 expression and release by flow cytometry, quantitative real-time PCR, ELISA, and confocal microscopy. We measured soluble VSIG4 concentrations in ascites from 120 patients with SBP and 40 patients without SBP and investigated the association of soluble VSIG4 in ascites with 90-day survival after SBP using Kaplan–Meier statistics, Cox regression, and competing-risks regression analysis. Results VSIG4 expression was high on resting, large PMs, which co-expressed CD206, CD163, and tyrosine-protein kinase Mer (MERTK). VSIG4 gene expression in PMs decreased in patients with SBP and normalised after resolution. During SBP, VSIG4hi PMs were depleted (25% vs. 57%; p, Graphical abstract, Highlights • VSIG4 expression is high on human resting, large peritoneal macrophages (PMs) that co-express CD206, CD163, and MERTK. • PM activation by TLR agonists or infection results in the loss of surface VSIG4 and release of soluble VSIG4 (sVSIG4). • Ascites sVSIG4 correlates with organ dysfunction and inflammation during SBP. • Higher ascitic fluid sVSIG4 concentrations indicated increased risk of 90-day mortality in 120 patients with SBP. • Addition of an antibody binding to the extracellular domain of VSIG4 enhanced phagocytosis of bacteria in vitro.

Background and ObjectivesTo determine the role of complement in the disease pathology of multifocal motor neuropathy (MMN), we investigated complement activation, and inhibition, on binding of MMN patient-derived immunoglobulin M (IgM) antibodies in an induced pluripotent stem cell (iPSC)-derived motor neuron (MN) model for MMN.MethodsiPSC-derived MNs were characterized for the expression of complement receptors and membrane-bound regulators, for the binding of circulating IgM anti-GM1 from patients with MMN, and for subsequent fixation of C4 and C3 on incubation with fresh serum. The potency of ARGX-117, a novel inhibitory monoclonal antibody targeting C2, to inhibit fixation of complement was assessed.ResultsiPSC-derived MNs moderately express the complement regulatory proteins CD46 and CD55 and strongly expressed CD59. Furthermore, MNs express C3aR, C5aR, and complement receptor 1. IgM anti-GM1 antibodies in serum from patients with MMN bind to MNs and induce C3 and C4 fixation on incubation with fresh serum. ARGX-117 inhibits complement activation downstream of C4 induced by patient-derived anti-GM1 antibodies bound to MNs.DiscussionBinding of IgM antibodies from patients with MMN to iPSC-derived MNs induces complement activation. By expressing complement regulatory proteins, particularly CD59, MNs are protected against complement-mediated lysis. Yet, because of expressing C3aR, the function of these cells may be affected by complement activation upstream of membrane attack complex formation. ARGX-117 inhibits complement activation upstream of C3 in this disease model for MMN and therefore represents an intervention strategy to prevent harmful effects of complement in MMN.

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

Sodium-glucose cotransporter 2(SGLT2) inhibitors show prominent renal protective effect in diabetic kidney disease (DKD), anti-inflammatory effect being one of its key mechanisms. Over-activation of the complement system, a crucial part of innate immunity, plays an important role in DKD. We aimed to investigate the effect of SGLT2 inhibitors on alleviating complement over-activation in DKD. Db/db mice were randomly divided into two groups, with 7 mice in each group treated with dapagliflozin and vehicle respectively, and 7 mice in m/m mice group. Laboratory and renal pathological parameters were evaluated. Mouse proximal tubular epithelial cells (MPTECs) were cultured and treated with high glucose. Dapagliflozin and dimethyloxallyl glycine (DMOG) were added as conditional treatment. Dapagliflozin-treated db/db mice showed significantly lower urinary albumin than vehicle-treated ones. Besides typical glomerular and tubulointerstitial injury, both C3b and membrane attack complex (MAC) depositions were significantly attenuated in dapagliflozin-treated db/db mice. The expression of complement receptor type 1-related protein y (Crry), a key complement regulator which inhibits complement over-activation, was significantly upregulated by dapagliflozin. Dapagliflozin-mediated Crry upregulation was associated with inhibition of HIF-1α accumulation under high glucose. When HIF-1α expression was stabilized by DMOG, the protective effect of dapagliflozin via upregulating Crry was blocked. In conclusion, dapagliflozin could attenuate complement over-activation in diabetic mice via upregulating Crry, which is associated with the suppression of HIF-1α accumulation in MPTECs.

Synapse elimination, also known as synaptic pruning, is a critical step in the maturation of neural circuits during brain development. Mounting evidence indicates that the complement cascade of the innate immune system plays an important role in synapse elimination. Studies indicate that excess synapses during development are opsonized by complement proteins and subsequently phagocytosed by microglia which expresses complement receptors. The process is regulated by diverse molecular signals, including complement inhibitors that affect the activation of complement, as well as signals that affect microglial recruitment and activation. These signals may promote or inhibit the removal of specific sets of synapses during development. The complement-microglia system has also been implicated in the pathogenesis of several developmental brain disorders, suggesting that the dysregulation of mechanisms of synapse pruning may underlie the specific circuitry defects in these diseases. Here, we review the latest evidence on the molecular and cellular mechanisms of complement-dependent and microglia-dependent synapse elimination during brain development, and highlight the potential of this system as a therapeutic target for developmental brain disorders. This article is categorized under: Neurological Diseases > Molecular and Cellular Physiology Neurological Diseases > Stem Cells and Development Immune System Diseases > Molecular and Cellular Physiology.

Previous studies have shown after the resolution of acute infection and viraemia, foot- and-mouth disease virus (FMDV) capsid proteins and/or genome are localised in the light zone of germinal centres of lymphoid tissue in cattle and African buffalo. The pattern of staining for FMDV proteins was consistent with the virus binding to follicular dendritic cells (FDCs). We have now demonstrated a similar pattern of FMDV protein staining in mouse spleens after acute infection and showed FMDV proteins are colocalised with FDCs. Blocking antigen binding to complement receptor type 2 and 1 (CR2/CR1) prior to infection with FMDV significantly reduced the detection of viral proteins on FDCs and FMDV genomic RNA in spleen samples. Blocking the receptors prior to infection also significantly reduced neutralising antibody titres. Therefore, the binding of FMDV to FDCs and sustained induction of neutralising antibody responses are dependent on FMDV binding to CR2/CR1 in mice.Author SummaryFoot and mouth disease virus causes a highly contagious acute vesicular disease, resulting in more than 50% of cattle, regardless of vaccination status, and almost 100% of African buffalo becoming persistently infected for long periods (months) of time. Yet, the mechanisms associated with establishment of persistent infections are still poorly understood. Infected animals are characterised by the presence of long-lived neutralising antibody titres, which contrast with the short-lived response induced by vaccination. We have used a mouse model to understand how foot and mouth disease virus is trapped and retained in the spleen for up to 28 days post infection and how the absence of antigen in the germinal centre prevents a sustainable neutralising antibody response, in the mouse. Our results highlight the importance of targeting antigen to FDCs to stimulate potent neutralising antibody responses after vaccination.

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3). Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

COVID-19, the disease caused by the SARS-CoV-2 virus, can progress to multi-organ failure characterized by respiratory insufficiency, arrhythmias, thromboembolic complications and shock. The mortality of patients hospitalized with COVID-19 is unacceptably high and new strategies are urgently needed to rapidly identify and treat patients at risk for organ failure. Clinical epidemiologic studies demonstrate that vulnerability to organ failure is greatest after viral clearance from the upper airway, which suggests that dysregulation of the host immune response is a critical mediator of clinical deterioration and death. Autopsy and pre-clinical evidence implicate aberrant complement activation in endothelial injury and organ failure. A potential therapeutic strategy warranting investigation is to inhibit complement, with case reports of successful treatment of COVID-19 with inhibitors of complement. However, this approach requires careful balance between the host protective and potential injurious effects of complement activation, and biomarkers to identify the optimal timing and candidates for therapy are lacking. Here we report the presence of complement activation products on circulating erythrocytes from hospitalized COVID-19 patients using flow cytometry. These findings suggest that novel erythrocyte-based diagnostics provide a method to identify patients with dysregulated complement activation.

Complement impacts innate and adaptive immunity. Using a model in which the human KEL glycoprotein is expressed on murine red blood cells (RBCs), we have shown that polyclonal immunoprophylaxis (KELIg) prevents alloimmunization to transfused RBCs when a recipient is in their baseline state of heath but with immunoprophylaxis failure occurring in the presence of a viral-like stimulus. As complement can be detected on antibody coated KEL RBCs following transfusion, we hypothesized that recipient complement synergizes with viral-like inflammation to reduce immunoprophylaxis efficacy. Indeed, we found recipient C3 and C1q were critical to immunoprophylaxis failure in the setting of a viral-like stimulus, with no anti-KEL IgG alloantibodies generated in C3-/-or C1q-/-mice following KELIg treatment and KEL RBC transfusion. Differences in RBC uptake were noted in mice lacking C3, with lower consumption by splenic and peripheral blood inflammatory monocytes. Finally, no alloantibodies were detected in the setting of a viral-like stimulus following KELIg treatment and KEL RBC transfusion in mice lacking complement receptors (CR1/2-/-), narrowing key cells for immunoprophylaxis failure to those expressing these complement receptors.In-vitrostudies showed complement fixed opsonized RBCs were significantly less likely to bind to B-cells from CR1/2-/-than wild type mice, potentially implicating lowered B-cell activation threshold in the presence of complement as being responsible for these findings. We thus propose a two-hit model for inflammation-induced immunoprophylaxis failure, where the first “hit” is recipient inflammation and the second “hit” is complement production/sensing. These results may have translational relevance to antigen-antibody interactions in humans.

Current understanding of coronavirus disease 2019 (COVID-19) pathophysiology is limited by disease heterogeneity, complexity, and a paucity of studies assessing patient tissues with advanced molecular tools. Rapid autopsy tissues were evaluated using multiscale, next-generation RNA-sequencing methods (bulk, single-nuclei, and spatial transcriptomics) to provide unprecedented molecular resolution of COVID-19-induced damage. Comparison of infected/uninfected tissues revealed four major regulatory pathways. Effectors within these pathways could constitute novel therapeutic targets, including the complement receptor C3AR1, calcitonin receptor-like receptor, or decorin. Single-nuclei RNA sequencing of olfactory bulb and prefrontal cortex highlighted remarkable diversity of coronavirus receptors. Angiotensin-converting enzyme 2 was rarely expressed, whereas basigin showed diffuse expression, and alanyl aminopeptidase, membrane, was associated with vascular/mesenchymal cell types. Comparison of lung and lymph node tissues from patients with different symptoms (one had died after a month-long hospitalization with multiorgan involvement, and the other had died after a few days of respiratory symptoms) with digital spatial profiling resulted in distinct molecular phenotypes. Evaluation of COVID-19 rapid autopsy tissues with advanced molecular techniques can identify pathways and effectors, map diverse receptors at the single-cell level, and help dissect differences driving diverging clinical courses among individual patients. Extension of this approach to larger data sets will substantially advance the understanding of the mechanisms behind COVID-19 pathophysiology.

Complement inhibition has been identified as a potential therapeutic target for multiple inflammatory disorders including Hidradenitis Suppurativa (HS). It is currently unclear how complement integrates into our current model of molecular pathogenesis in HS and whether it represents a central component of pathogenesis, or a neutrophil-associated bystander. Levels of C5a in serum and tissue correlate with disease activity and degree of neutrophilic infiltrates in HS. C5a has been associated with Th17 immune axis activation in psoriasis, rheumatoid arthritis and Crohn's disease with strong similarities to TH17 activation in HS. Porphyromonas species (which are identified in the HS microbiome) are able to cleave inactive C5 into C5a implicating the cutaneous microbiome as an activator of complement. C3a and C5a are associated with activation of the NLRP3 inflammasome, implicated in the inflammatory drive in HS. Complement receptors are present upon dendritic cells, monocytes, fibroblasts and adipocytes, which may broaden the potential contribution of complement to multiple aspects of HS pathogenesis. Dysregulation of complement receptor pathways has been documented in obesity, insulin resistance and polycystic ovarian syndrome leading to the possibility that complement may explain the epidemiological associations between these conditions and HS. The therapeutic potential of complement inhibitors in HS may be related to the therapeutic target (complement receptor or complement subunit) and the presence of alternate receptors (such as C5aR2) or ligands (including C3a, PAMPs and DAMPs). Integrating complement into the known pathogenesis of HS may aid in explaining the contradictory results between Phase 2 studies of C5a antagonists. It also allows for the identification of existing knowledge gaps to target further clinical investigation and research.

Fungal dissemination into the bloodstream is a critical step leading to invasive fungal infections. Here, using intravital imaging, we show that Kupffer cells (KCs) in the liver have a prominent function in the capture of circulating Cryptococcus neoformans and Candida albicans, thereby reducing fungal dissemination to target organs. Complement C3 but not C5, and complement receptor CRIg but not CR3, are involved in capture of C. neoformans. Internalization of C. neoformans by KCs is subsequently mediated by multiple receptors, including CR3, CRIg, and scavenger receptors, which work synergistically along with C5aR signaling. Following phagocytosis, the growth of C. neoformans is inhibited by KCs in an IFN-γ independent manner. Thus, the liver filters disseminating fungi from circulation via KCs, providing a mechanistic explanation for the enhanced risk of cryptococcosis among individuals with liver diseases, and suggesting a therapeutic strategy to prevent fungal dissemination through enhancing KC functions., Patients with liver diseases are at increased risk of fungal infections. Here the authors show that Kupffer cells are critical for the filtration of fungi out of the blood and thereby for liver-mediated protection against disseminating fungal infection.

Complement activation generates the core effector protein C5a, a potent immune molecule that is linked to multiple inflammatory diseases. Two C5a receptors, C5aR1 (C5aR, CD88) and C5aR2 (C5L2, GPR77), mediate the biological activities of C5a. Although C5aR1 has broadly acknowledged proinflammatory roles, C5aR2 remains at the center of controversy, with existing findings supporting both immune-activating and immune-dampening functions. Recent progress has been made toward resolving these issues. Instead of being a pure recycler and sequester of C5a, C5aR2 is capable of mediating its own set of signaling events and through these events exerting significant immunomodulatory effects not only toward C5aR1 but also other pattern recognition receptors and innate immune systems, such as NLRP3 inflammasomes. This review highlights the existing knowns and unknowns concerning C5aR2 and provides a timely update on recent breakthroughs which are expected to have a substantial impact on future fundamental and translational C5aR2 research.

Background Mesenchymal stromal cells (MSCs) have protolerogenic effects in renal transplantation, but they induce long-term regulatory T cells (Treg)-dependent graft acceptance only when infused before transplantation. When given posttransplant, MSCs home to the graft where they promote engraftment syndrome and do not induce Treg. Unfortunately, pretransplant MSC administration is unfeasible in deceased-donor kidney transplantation. Methods To make MSCs a therapeutic option also for deceased organ recipients, we tested whether MSC infusion at the time of transplant (day 0) or posttransplant (day 2) together with inhibition of complement receptors prevents engraftment syndrome and allows their homing to secondary lymphoid organs for promoting tolerance. We analyzed intragraft and splenic MSC localization, graft survival, and alloimmune response in mice recipients of kidney allografts and syngeneic MSCs given on day 0 or on posttransplant day 2. C3a receptor (C3aR) or C5a receptor (C5aR) antagonists were administered to mice in combination with the cells or were used together to treat MSCs before infusion. Results Syngeneic MSCs given at day 0 homed to the spleen increased Treg numbers and induced long-term graft acceptance. Posttransplant MSC infusion, combined with a short course of C3aR or C5aR antagonist or administration of MSCs pretreated with C3aR and C5aR antagonists, prevented intragraft recruitment of MSCs and graft inflammation, inhibited antidonor T-cell reactivity, but failed to induce Treg, resulting in mild prolongation of graft survival. Conclusions These data support testing the safety/efficacy profile of administering MSCs on the day of transplant in deceased-donor transplant recipients and indicate that complement is crucial for MSC recruitment into the kidney allograft.

The innate immune system-based recognition of the pathogens or their PAMPs initiates the pro-inflammatory immune response required for the maintenance of the homeostasis. The dysregulation of this innate immune response causes several diseases including sepsis, cancer and autoimmunity. However, pattern recognition receptors (PRRs) including toll-like receptors (TLRs), complement receptors (CRs) and NLRs of inflammasomes regulate both these processes of recognition of pathogens/PAMPs and their clearance. These three major components of the innate immune arm were studied independently/separately for a long time. Various studies have now shown that they work in close association and their crosstalk is required for the pathogen clearance via regulating the process of phagocytosis and mounting the controlled but potent immune response. The loss or inhibition of any of the three components affects the other in a positive/negative manner that can affect the immune process required for efficient host defense. The present review is designed to provide the current information on their evolution like the requirement of TLRs and inflammasomes for pathogen recognition even in the presence of complements system and their interaction during various immunological processes including phagocytosis, autophagy and inflammatory immune response.

The recognition of microbe- or danger-associated molecular patterns by complement proteins initiates a cascade of events that culminate in the engagement of complement receptors on the surface of immune cells. Signaling pathways triggered by such receptors converge with those activated downstream of various pattern-recognition receptors to determine the type and magnitude of immune cell responses. The perception of complement as a regulator of immune functions has encouraged intensive investigation in the field, which has uncovered novel molecular pathways that link complement receptor-mediated signaling with homeostatic and pathologic T-cell responses. More recently, the observation that complement proteins can also act within the intracellular space to guide the fate of T cells has added new complexity to the role of complement in the regulation of adaptive immunity. In this review, we discuss fundamental mechanisms and novel concepts at the interface of complement biology and immunity and offer a critical view on how these mechanisms play a role in the maintenance of homeostasis and the development of pathological conditions in humans.

Introduction Chronic histiocytic intervillositis of unknown etiology (CIUE) is a non-infectious, most probably immunologic placenta lesion. CIUE is associated with recurrent miscarriage, intrauterine growth restriction and stillbirth. Among the pathologic-anatomic defined placental lesions this entity displays the highest risk of recurrence in following pregnancies (about 67–100%). The histiocytic cells accumulate in the placental blood space but do not infiltrate into the villi or decidua. Sparsely known is the expression profile of these intervillous cells regarding histiocytic markers. Methods We analysed 5-22 markers by immunohistochemistry in a total of 41 placenta samples and evaluated decidual, villous and intervillous histiocytic cells. Results In CIUE, intervillous CD163+ histiocytes over-express CD11c/CD18 and down-regulate CD206/CD209, while CD163+ decidual and Hofbauer cells show low CD11c/CD18 and higher CD206/CD209 protein expressions. Discussion CD163 expression indicates a M2-like polarisation. CD11c and CD18 form the complement receptor 4 which could be related to a complement mediated trigger for aberrant cell accumulation in CIUE.

Phagocytosis is a process in which target cells or particles are engulfed and taken up by other cells, typically professional phagocytes; this process is crucial in many physiological processes and disease states. The detection of targets for phagocytosis is directed by a complex repertoire of cell surface receptors. Pattern recognition receptors directly detect targets for binding and uptake, while opsonic and complement receptors detect objects coated by soluble factors. However, the importance of single and combinatorial surface marker expression across different phenotypes of professional phagocytes is not known. Here we developed a novel mass cytometry-based phagocytosis assay that enables the simultaneous detection of phagocytic events in combination with up to 40 other protein markers. We applied this assay to distinct monocyte derived macrophage (MDM) populations and found that prototypic M2-like MDMs phagocytose more E. coli than M1-like MDMs. Surface markers such as CD14, CD206, and CD163 rendered macrophages phagocytosis competent, but only CD209 directly correlated with the amount of particle uptake. Similarly, M2-like MDMs also phagocytosed more cancer cells than M1-like MDMs but, unlike M1-like MDMs, were insensitive to anti-CD47 opsonization. Our approach facilitates the simultaneous study of single-cell phenotypes, phagocytic activity, signaling and transcriptional events in complex cell mixtures.

β-glucans are a heterogeneous group of natural polysaccharides. They are ubiquitously found in bacterial or fungal cell walls, cereals, seaweed, and mushrooms. The beneficial role of β-glucan in tumor, insulin resistance, dyslipidemia, hypertension, and obesity is being continuously documented. Ample evidence showed that β-glucan could act on several receptors, such as Dectin, complement receptor (CR3), TLR-2, 4, 6 and scavenger. Based on the above, we wanted to explore whether agaricus bisporus-derived β-glucan acted on these receptors on Raw 264.7 macrophages and 3T3-L1 adipocytes.

Complement activation in human diseases is characterized by the local covalent deposition of the long-lived C3 fragments iC3b/C3dg/C3d. Previously, TT30, a complement alternative pathway (AP)-selective inhibitor, was designed as a fusion protein linking the first four short consensus repeats (SCRs) of human complement receptor type 2 (CR2) with the first five SCRs of human factor H (fH). TT30 acts by utilizing CR2 SCR1–4 to bind the initially formed iC3b/C3dg/C3d fragments and delivering surface-targeted inhibition of AP C3 and C5 convertases through fH SCR 1-5. In order to combine classical (CP) and lectin (LP) pathway inhibitory abilities employing CR2-mediated targeting, TT32 was developed. TT32 is a CR2-CR1 fusion protein using the first ten SCRs of CR1, chosen because they contain both C3 and C5 convertase inhibitory activity through utilization of decay-acceleration and cofactor activity for both AP and CP. In Wieslab assays, TT32 showed potent inhibition of the CP and AP with IC50 of 11 and 46 nM, respectively. The TT32 inhibitory activity is partially blocked with a molar excess of a competing anti-CR2 mAb, thus demonstrating the importance of the CR2 targeting. TT32 was studied in the type II (CII) collagen-induced arthritis (CIA), an active immunization model, and the CII antibody-induced arthritis (CAIA) passive transfer model. In CIA, injection of 2.0 mg TT32 at day 21 and 28 post disease induction, but not untargeted CR1 alone, resulted in a 51.5% decrease in clinical disease activity (CDA). In CAIA, treatment with TT32 resulted in a 47.4% decrease in CDA. Therefore, a complement inhibitor that targets both the AP and CP/LP C3/C5 convertases was shown to limit complement-mediated tissue damage and inflammation in disease models in which all three complement activation pathways are implicated.

Acute kidney injury (AKI) is a common medical complication of up to 25% of intensive care unit admissions, with severe consequences, and mortality increasing up to 60% [1–3]. Frequently, AKI pathogenesis includes hypoxia that causes ischemia, followed by reperfusion [4,5]. The ischemia-reperfusion (I/R) process triggers intra-renal, but also systemic inflammation responses [6]. As part of the inflammatory response, the intra-renal complement system is activated [7]. Even though much knowledge has been gained regarding the post reperfusion pathophysiological cascade, there are currently no acceptable clinical therapeutic strategies that target at least one of the pathological cascade elements [8]. The complement system has an important role in the pathogenesis of renal failure, and is involved in glomerular, tubulointerstitial and vascular kidney injuries among others [9]. In humans, the complement system is involved in several types of AKIs including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, dense deposit disease and C3 glomerulopathy [10,11]. The anti-C5 antibody (Eculizumab) is clinically indicated for these conditions [10,11]. Animal models have demonstrated that complement receptors are overexpressed after reperfusion [12]. C3 and C6 knockout mice are relatively protected from renal I/R injury. However, this protection is missing in C4 knockout mice [13]. This indicates that the dominant part of the complement system involved in I/R injury is the alternative pathway [13]. The use of monoclonal antibody that prevent cleavage of C5 in an attempt to inhibit the effect of I/R injury in mice models has resulted in reduced inflammation and improved renal function [14]. However, an attempt to block C3 by the potent murine complement inhibitor CR1 related gene/protein y (Crry)-Ig did not demonstrate any beneficial effect in I/R-induced kidney injuries [15]. The aim of this study was to investigate the pathophysiological effects of the anti-C5 monoclonal antibody on an I/R-induced AKI rat model.

Objective: To explore the possible IgG1 binding receptors by protein-protein docking experiments. Methods: The protein-protein cognate interactions such as IgG with Fc Receptors (FcRs) potentiate signaling cascades to ameliorate antigen uptake, processing and presentation are studied by protein-protein docking experiments. Results: However, the propensity of IgG interactions with other cognate receptors largely remains obscure. In this direction, possibilities of IgG1 binding with various five receptors were explored. In this study, we report previously unidentified associations between IgG1 and other receptors. Herein, we show that IgG1 binds to the granulocyte-macrophage receptor, β common receptor and complementary receptor(complementary receptor I and complementary receptor II) to form a complex structure. We show the binding ability and important protein-protein interactions of IgG1 with four receptors in comparison to Fc Receptor, and also find out the conserved amino acids and hydrophobic-hydrophobic interactions amongst them. Conclusions: Comparative interaction studies of IgG1 binding to various receptors revealed close similarities of IgG1 binding to its native receptor Fc. In conclusion, our study has shown the comparable binding efficiency of four receptors to IgG1 apart from the conventional Fc receptor.

Complement and dendritic cells (DCs) share many functional features that drive the outcome of immune-inflammatory processes. Both have a sentinel function, acting as danger sensors specialized for a rapid, comprehensive and selective action against potential threats without damaging the healthy host cells. But while complement has been considered as a "master alarm" system poised for direct pathogen killing, DCs are regarded as "master regulators" or orchestrators of a vast range of effector immune cells for an effective immune response against threatening insults. The original definition of the complement system, coined to denote its auxiliary function to enhance or assist in the role of antibodies or phagocytes to clear microbes or damaged cells, envisaged an important crosstalk between the complement and the mononuclear phagocyte systems. More recent studies have shown that, depending on the microenvironmental conditions, several complement effectors are competent to influence the differentiation and/or function of different DC subsets toward immunogenicity or tolerance. In this review we will infer about the capability of complement activators and inhibitors to "condition" a tolerogenic and anti-inflammatory immune response by direct interaction with DC surface receptors, and about the implications of this knowledge to devise new complement-based therapeutic approaches for autoimmune pathologies.

Integrins are cell membrane receptors that are involved in essential physiological and serious pathological processes. Their main role is to ensure a closely regulated link between the extracellular matrix and the intracellular cytoskeletal network enabling cells to react to environmental stimuli. Complement receptor type 3 (CR3, αMβ2, CD11b/CD18) and type 4 (CR4, αXβ2, CD11c/CD18) are members of the β2-integrin family expressed on most white blood cells. Both receptors bind multiple ligands like iC3b, ICAM, fibrinogen or LPS. β2-integrins are accepted to play important roles in cellular adhesion, migration, phagocytosis, ECM rearrangement and inflammation. Several pathological conditions are linked to the impaired functions of these receptors. CR3 and CR4 are generally thought to mediate overlapping functions in monocytes, macrophages and dendritic cells, therefore the potential distinctive role of these receptors has not been investigated so far in satisfactory details. Lately it has become clear that a functional segregation has evolved between the two receptors regarding phagocytosis, cellular adhesion and podosome formation. In addition to their tasks on myeloid cells, the expression and function of CR3 and CR4 on lymphocytes have also gained interest recently. The picture is further complicated by the fact that while these β2-integrins are expressed by immune cells both in mice and humans, there are significant differences in their expression level, functions and the pathological consequences of genetic defects. Here we aim to summarize our current knowledge on CR3 and CR4 and highlight the functional differences between these receptors, involving their expression in myeloid and lymphoid cells of both men and mice.

AIM: Alterations in the microenvironment change the phenotypes of dental pulp stem cells (DPSCs). The role of complement component C5a in the differentiation of DPSCs is unknown, especially under oxygen-deprived conditions. The aim of this study was to determine the effect of C5a on the odontogenic differentiation of DPSCs under normoxia and hypoxia. MATERIAL AND METHODS: Human DPSCs were subjected to odontogenic differentiation in osteogenic media and treated with the C5a receptor antagonist-W54011 under normal and hypoxic conditions (2% oxygen). Immunochemistry, western blot, and PCR analysis for the various odontogenic differentiation genes/proteins were performed. RESULTS: Our results demonstrated that C5a plays a positive role in the odontogenic differentiation of DPSCs. C5a receptor inhibition resulted in a significant decrease in odontogenic differentiation genes, such as DMP1, ON, RUNX2, DSPP compared with the control. This observation was further supported by the Western blot data for DSPP and DMP1 and immunohistochemical analysis. The hypoxic condition reversed this effect. CONCLUSIONS: Our results demonstrate that C5a regulates the odontogenic DPSC differentiation under normoxia. Under hypoxia, C5a exerts a reversed function for DPSC differentiation. Taken together, we identified that C5a and oxygen levels are key initial signals during pulp inflammation to control the odontogenic differentiation of DPSCs, thereby, providing a mechanism for potential therapeutic interventions for dentin repair and vital tooth preservation.

BackgroundCurrent understanding of COVID-19 pathophysiology is limited by disease heterogeneity, complexity, and a paucity of studies evaluating patient tissues with advanced molecular tools.MethodsAutopsy tissues from two COVID-19 patients, one of whom died after a month-long hospitalization with multi-organ involvement while the other died after a few days of respiratory symptoms, were evaluated using multi-scale RNASeq methods (bulk, single-nuclei, and spatial RNASeq next-generation sequencing) to provide unprecedented molecular resolution of COVID-19 induced damage.FindingsComparison of infected/uninfected tissues revealed four major regulatory pathways. Effectors within these pathways could constitute novel therapeutic targets, including the complement receptor C3AR1, calcitonin-like receptor or decorin. Single-nuclei RNA sequencing of olfactory bulb and prefrontal cortex highlighted remarkable diversity of coronavirus receptors. Angiotensin I converting enzyme 2 was rarely expressed, while Basignin showed diffuse expression, and alanyl aminopeptidase was associated with vascular/mesenchymal cell types. Comparison of lung and lymph node tissues from patients with different symptomatology with Digital Spatial Profiling resulted in distinct molecular phenotypes.InterpretationCOVID-19 is a far more complex and heterogeneous disease than initially anticipated. Evaluation of COVID-19 rapid autopsy tissues with advanced molecular techniques can identify pathways and effectors at play in individual patients, measure the staggering diversity of receptors in specific brain areas and other well-defined tissue compartments at the single-cell level, and help dissect differences driving diverging clinical courses among patients. Extension of this approach to larger datasets will substantially advance the understanding of the mechanisms behind COVID-19 pathophysiology.FundingNo external funding was used in this study.Research in contextEvidence before this studyInformation regarding changes seen in COVID-19 has accumulated very rapidly over a short period of time. Studies often rely on examination of normal samples and model systems, or are limited to peripheral blood or small biopsies when dealing with tissues collected from patients infected with SARS-CoV-2. For that reason, autopsy studies have become an important source of insights into the pathophysiology of severe COVID-19 disease, highlighting the emerging role of hyperinflammatory and hypercoagulable syndromes. Studies of autopsy tissues, however, are usually limited to histopathologic and immunohistochemical evaluation. The next frontier in understanding COVID-19 mechanisms of disease will require generation of highly dimensional, patient-specific datasets that can help dissect this complex and heterogeneous disease.Added value of this studyOur work illustrates how high-resolution molecular and spatial profiling of COVID-19 patient tissues collected during rapid autopsies can serve as a hypothesis-generating tool to identify key mediators driving the pathophysiology of COVID-19 for diagnostic and therapeutic target testing. Here we employ bulk RNA sequencing to identify key regulators of COVID-19 and list specific mediators for further study as potential diagnostic and therapeutic targets. We use single-nuclei RNA sequencing to highlight the diversity and heterogeneity of coronavirus receptors within the brain, suggesting that it will be critical to expand the focus from ACE2 to include other receptors, such as BSG and ANPEP, and we perform digital spatial profiling of lung and lymph node tissue to compare two patients with different clinical courses and symptomatology.Implications of all the available evidenceCOVID-19 is a far more heterogeneous and complex disease than initially anticipated. Advanced molecular tools can help identify specific pathways and effectors driving the pathophysiology of COVID-19 and lead to novel biomarkers and therapeutic targets in a patient-specific manner. Larger studies representing the diversity of clinical presentations and pre-existing conditions will be needed to capture the full complexity of this disease.

Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers.We have identified a novel family of alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner.For the first time this study provides a full functional and structural characterization of a properdin inhibitor, opening avenues for future therapeutic approaches.

Complement signaling is thought to serve as an opsonization signal to promote the phagocytosis of synapses by microglia. However, while its role in synaptic remodeling has been demonstrated in the retino-thalamic system, it remains unclear whether complement signaling mediates synaptic pruning in the brain more generally. Here we show that mice lacking the complement 3 receptor (C3r), the major microglia complement receptor, fail to show a deficit in either synaptic pruning or axon elimination in the developing mouse cortex. Instead, mice lacking C3r show a deficit in the perinatal elimination of neurons, both in the retina as well as in the cortex, a deficit that is associated with increased cortical thickness and enhanced functional connectivity in these regions in adulthood. These data demonstrate a preferential role for complement in promoting neuronal elimination in the developing brain and argue for a reconsideration of the role of complement in synaptic pruning.

The positive coreceptor function of complement receptor type 2 [CR2 (CD21)] on B cells is generally accepted, although its role in the enhancement of antibody production had only been proven in mice. The importance of this phenomenon prompted reinvestigation of the functional consequences of coclustering CD21 and the B cell receptor (BCR) on primary human cells. We found that, at non-stimulatory concentrations of anti-IgG/A/M, coclustering the BCR and CR2 enhanced the Ca2+ response, while activation marker expression, cytokine production, proliferation, and antibody production were all inhibited upon the coengagement of CR2 and BCR on human B cells. Thus, the “textbook dogma” claiming that C3d acts as an adjuvant to enhance humoral immunity is relevant only to mice and not to humans.

The complement system is one of the host’s primary defence mechanisms against pathogens. Its activation involves proteolytic cascades of enzymatic reactions that result in products with eff ector functions and recognition of molecules on the surface of microorganisms [1,2]. Complement activation depends on a proteolytic cascade of enzymatic reactions that result in products with eff ects function of molecules identifi cation in microorganism surface for complement classical, alternative, and lectin pathways, converging to C3 complement central protein and formation of subproducts C3a and C3b [1,2].

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

Primary membranous nephropathy (pMN) is a leading cause of nephrotic syndrome in adults. In most cases, this autoimmune kidney disease is associated with autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) expressed on kidney podocytes, but the mechanisms leading to glomerular damage remain elusive. Here, we developed a cell culture model using human podocytes and found that anti-PLA2R1-positive pMN patient sera or isolated IgG4, but not IgG4-depleted sera, induced proteolysis of the 2 essential podocyte proteins synaptopodin and NEPH1 in the presence of complement, resulting in perturbations of the podocyte cytoskeleton. Specific blockade of the lectin pathway prevented degradation of synaptopodin and NEPH1. Anti-PLA2R1 IgG4 directly bound mannose-binding lectin in a glycosylation-dependent manner. In a cohort of pMN patients, we identified increased levels of galactose-deficient IgG4, which correlated with anti-PLA2R1 titers and podocyte damage induced by patient sera. Assembly of the terminal C5b-9 complement complex and activation of the complement receptors C3aR1 or C5aR1 were required to induce proteolysis of synaptopodin and NEPH1 by 2 distinct proteolytic pathways mediated by cysteine and aspartic proteinases, respectively. Together, these results demonstrated a mechanism by which aberrantly glycosylated IgG4 activated the lectin pathway and induced podocyte injury in primary membranous nephropathy.

BackgroundResistance to therapy is a major problem in treating head and neck squamous cell carcinomas (HNSCC). Complement system inhibition has been shown to reduce tumor growth, metastasis, and therapeutic resistance in other tumor models, but has yet to be explored in the context of HNSCC. Here, we tested the effects of complement inhibition and its therapeutic potential in HNSCC.MethodsWe conducted our studies using two Human Papilloma Virus (HPV)-negative HNSCC orthotopic mouse models. Complement C3aR and C5aR1 receptor antagonists were paired with radiation therapy (RT). Tumor growth was measured and immune populations from tumor, lymph node, and peripheral blood were compared among various treatment groups. Genetically engineered mouse models DEREG and C3-/- were used in addition to standard wild type models. Flow cytometry, clinical gene sets, and in vitro assays were used to evaluate the role complement receptor blockade has on the immunological makeup of the tumor microenvironment.ResultsIn contrast to established literature, inhibition of complement C3a and C5a signaling using receptor antagonists accelerated tumor growth in multiple HNSCC cell lines and corresponded with increased frequency of regulatory T cell (Treg) populations. Local C3a and C5a signaling has importance for CD4 T cell homeostasis and eventual development into effector phenotypes. Interruption of this signaling axis drives a phenotypic conversion of CD4+ T cells into Tregs, characterized by enhanced expression of Foxp3. Depletion of Tregs reversed tumor growth, and combination of Treg depletion and C3a and C5a receptor inhibition decreased tumor growth below that of the control groups. Complete knockout of C3 does not harbor the expected effect on tumor growth, indicating a still undetermined compensatory mechanism. Dexamethasone is frequently prescribed to patients undergoing RT and inhibits complement activation. We report no deleterious effects associated with dexamethasone due to complement inhibition.ConclusionsOur data establish Tregs as a pro-tumorigenic driver during complement inhibition and provide evidence that targeted C3a and C5a receptor inhibition may add therapeutic advantage when coupled with anti-Treg therapy.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and a leading cause of death in the US and worldwide. HCC remains a global health problem and is highly aggressive with unfavorable prognosis. Even with surgical interventions and newer medical treatment regimens, patients with HCC have poor survival rates. These limited therapeutic strategies and mechanistic understandings of HCC immunopathogenesis urgently warrant non-palliative treatment measures. Irrespective of the multitude etiologies, the liver microenvironment in HCC is intricately associated with chronic necroinflammation, progressive fibrosis, and cirrhosis as precedent events along with dysregulated innate and adaptive immune responses. Central to these immunological networks is the complement cascade (CC), a fundamental defense system inherent to the liver which tightly regulates humoral and cellular responses to noxious stimuli. Importantly, the liver is the primary source for biosynthesis of >80% of complement components and expresses a variety of complement receptors. Recent studies implicate the complement system in liver inflammation, abnormal regenerative responses, fibrosis, carcinogenesis, and development of HCC. Although complement activation differentially promotes immunosuppressive, stimulant, and angiogenic microenvironments conducive to HCC development, it remains under-investigated. Here, we review derangement of specific complement proteins in HCC in the context of altered complement regulatory factors, immune-activating components, and their implications in disease pathogenesis. We also summarize how complement molecules regulate cancer stem cells (CSCs), interact with complement-coagulation cascades, and provide therapeutic opportunities for targeted intervention in HCC.

Background Recently, growing evidence has indicated an important role of the complement system, a crucial component of immunity, in mediating neuroinflammation and promoting neuronal apoptosis following closed head injury (CHI). We previously reported that transplanted induced neural stem cells (iNSCs) pre-treated with CHI mouse serum could enhance complement receptor type 1-related protein y (Crry) expression and ameliorate complement-mediated damage in mouse CHI models. However, the mechanism underlying the elevated levels of Crry expression remains elusive. Methods CHI models were established using a standardized weight-drop device. We collected CHI mouse serum at 12 h post-trauma. RT-QPCR assay, western blot analysis, complement deposition assay, Akt inhibition assay, flow cytometry, cell transplantation, and functional assay were utilized to clarify the mechanism of Crry expression in iNSCs receiving CHI mouse serum treatment. Results We observed dramatic increases in the levels of Crry expression and Akt activation in iNSCs receiving CHI mouse serum treatment. Remarkably, Akt inhibition led to the reduction of Crry expression in iNSCs. Intriguingly, the treatment of iNSC-derived neurons with recombinant complement receptor 2-conjugated Crry (CR2-Crry), which inhibits all complement pathways, substantially enhanced Crry expression and Akt activation in neurons after CHI mouse serum treatment. In subsequent in vitro experiments of pre-treatment of iNSCs with CR2-Crry, we observed significant increases in the levels of Crry expression and Akt activation in iNSCs and iNSC-derived astrocytes and neurons post-treatment with CHI mouse serum. Additionally, an in vivo study showed that intracerebral-transplanted iNSCs pre-treated with CR2-Crry markedly enhanced Crry expression in neurons and protected neurons from complement-dependent damage in the brains of CHI mice. Conclusion INSCs receiving CR2-Crry pre-treatment increased the levels of Crry expression in iNSCs and iNSC-derived astrocytes and neurons and attenuated complement-mediated injury following CHI.

The complement system is deeply embedded in our physiology and immunity. Complement activation generates a multitude of molecules that converge simultaneously on the opsonization of a target for phagocytosis and activation of the immune system via soluble anaphylatoxins. This response is used to control microorganisms and to remove dead cells, but also plays a major role in stimulating the adaptive immune response and the regeneration of injured tissues. Many of these effects inherently depend on complement receptors expressed on leukocytes and parenchymal cells, which, by recognizing complement-derived molecules, promote leukocyte recruitment, phagocytosis of microorganisms and clearance of immune complexes. Here, the plethora of information on the role of complement receptors will be reviewed, including an analysis of how this functionally and structurally diverse group of molecules acts jointly to exert the full extent of complement regulation of homeostasis. ispartof: FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY vol:9 ispartof: location:Switzerland status: published