C5a Induces Inflammatory Signaling and Apoptosis in PC12 Cells through C5aR-Dependent Signaling: A Potential Mechanism for Adrenal Damage in Sepsis.

The complement system is critically involved in the pathogenesis of sepsis. In particular, complement anaphylatoxin C5a is generated in excess during sepsis, leading to cellular dysfunction. Recent studies have shown that excessive C5a impairs adrenomedullary catecholamine production release and induces apoptosis in adrenomedullary cells. Currently, the mechanisms by which C5a impacts adrenal cell function are poorly understood. The PC12 cell model was used to examine the cellular effects following treatment with recombinant rat C5a. The levels of caspase activation and cell death, protein kinase signaling pathway activation, and changes in inflammatory protein expression were examined following treatment with C5a. There was an increase in apoptosis of PC12 cells following treatment with high-dose C5a. Ten inflammatory proteins, primarily involved in apoptosis, cell survival, and cell proliferation, were upregulated following treatment with high-dose C5a. Five inflammatory proteins, involved primarily in chemotaxis and anti-inflammatory functions, were downregulated. The ERK/MAPK, p38/MAPK, JNK/MAPK, and AKT protein kinase signaling pathways were upregulated in a C5aR-dependent manner. These results demonstrate an apoptotic effect and cellular signaling effect of high-dose C5a. Taken together, the overall data suggest that high levels of C5a may play a role in C5aR-dependent apoptosis of adrenal medullary cells in sepsis.

Complement C5a Implication in Axonal Growth After Injury.

Complement C5a protein has been shown to play a major role in tissue regeneration through interaction with its receptor (C5aR) on target cells. Expression of this receptor has been reported in the nervous system which, upon injury, has no treatment to restore the lost functions. This work aimed at investigating the Complement C5a effect on axonal growth after axotomy in vitro. Primary hippocampal neurons were isolated from embryonic Wistar rats. Cell expression of C5aR mRNA was verified by RT-PCR while its membrane expression, localization, and phosphorylation were investigated by immunofluorescence. Then, the effects of C5a on injured axonal growth were investigated using a 3D-printed microfluidic device. Immunofluorescence demonstrated that the primary cultures contained only mature neurons (93%) and astrocytes (7%), but no oligodendrocytes or immature neurons. Immunofluorescence revealed a co-localization of NF-L and C5aR only in the mature neurons where C5a induced the phosphorylation of its receptor. C5a application on injured axons in the microfluidic devices significantly increased both the axonal growth speed and length. Our findings highlight a new role of C5a in regeneration demonstrating an enhancement of axonal growth after axotomy. This may provide a future therapeutic tool in the treatment of central nervous system injury.

Using genetics to explore complement C5 as a druggable protein in periodontitis.

Aim: An excessively activated or dysregulated complement system has been proven to be a vital contributor to the pathogenesis of periodontitis. It has been previously hypothesized that inhibiting the activity of complement component C5 by targeting the C5a receptor is a powerful candidate for treating periodontitis. Here, we apply the drug target instrumental variable (IV) approach to investigate the therapeutic effect of genetically proxied inhibition of C5 on periodontitis. Method: In our primary analysis, we used 26 independent 'cis' single nucleotide polymorphisms as IVs from the vicinity of the encoding locus of C5 that are associated with plasma C5 levels. In a secondary analysis, we assess the validity of our primary findings, exploring the involvement of alternative downstream biomarkers, interleukin 17 (IL-17), interleukin 1ß (IL-1ß), and tumor necrosis factor (TNF). Summary statistics of plasma levels (C5, IL-17, IL-1ß, and TNF) were obtained from a genome-wide association study (GWAS) of 35,559 European descent individuals. We extracted association statistics from a GWAS of 17,353 clinical periodontitis cases and 28,210 European controls. Wald ratios were combined using inverse-variance weighted meta-analysis. Results: In our primary approach, inhibiting C5 reduced the risk of periodontitis (Odds ratio 0.89 per 1 standard deviation reduction in C5; 95% confidence Interval 0.80-0.98, p value=0.022). Our secondary analysis suggests an involvement of IL-17 within the potential causal pathway, but was inconclusive for other biomarkers. Conclusions: The findings from our study suggest that C5 inhibition may reduce the risk of periodontitis, prioritizing C5 inhibitors as a potential adjunctive therapeutic intervention in this disease.

Ravulizumab in Atypical Hemolytic Uremic Syndrome: An Analysis of 2-Year Efficacy and Safety Outcomes in 2 Phase 3 Trials.

Rationale & Objective: Atypical hemolytic uremic syndrome (aHUS) is a rare form of thrombotic microangiopathy (TMA) caused by complement dysregulation. Ravulizumab is a C5i approved for the treatment of aHUS. This analysis assessed long-term outcomes of ravulizumab in adults and pediatric patients with aHUS. Study Design: This analysis reports 2-year data from 2 phase 3, single-arm studies. Setting & Participants: One study included C5i-naïve adults (NCT02949128), and the other included 2 cohorts of pediatric patients (C5i-naïve and those who switched to ravulizumab from eculizumab [pediatric switch patients]; NCT03131219). Exposure: Patients received intravenous ravulizumab every 4-8 weeks, with the dose depending on body weight. Outcomes: The primary endpoint in the studies of C5i-naïve patients was complete TMA response, which consisted of platelet count normalization, lactate dehydrogenase normalization, and ≥25% improvement in serum creatinine concentrations from baseline, at 2 consecutive assessments ≥4 weeks apart. Analytical Approach: All analyses used descriptive statistics. No formal statistical comparisons were performed. Results: In total, 86 and 92 patients were included in efficacy and safety analyses, respectively. Complete TMA response rates over 2 years were 61% and 90% in C5i-naïve adults and pediatric patients, respectively. The median increase in estimated glomerular filtration rate from baseline was maintained over 2 years in C5i-naïve adults (35 mL/min/1.73 m2) and pediatric patients (82.5 mL/min/1.73 m2). Most adverse events and serious adverse events occurred during the first 26 weeks. No meningococcal infections were reported. Improvement in the Functional Assessment of Chronic Illness Therapy - Fatigue score achieved by 26 weeks was maintained over 2 years. Limitations: Limitations were the small sample of pediatric switch patients and limited availability of genetic data. Conclusions: Long-term treatment with ravulizumab is well tolerated and associated with improved hematologic and renal parameters and quality of life in adults and pediatric patients with aHUS.

This research tested a drug called ravulizumab for the treatment of atypical hemolytic uremic syndrome (aHUS). aHUS is a rare disease that causes clots in tiny blood vessels. This can damage the kidneys and other organs. We analyzed data from 2 clinical trials in which children and adults with aHUS received ravulizumab through a tube placed in a vein (intravenous line). They received ravulizumab every 4-8 weeks depending on their weight. We found that treating patients for 2 years with ravulizumab was associated with improved blood health, kidney function, and quality of life and was well tolerated. These results support ravulizumab as a long-term treatment for people with aHUS.

Macrophage-Induced Pro-Fibrotic Gene Expression in Tubular Cells after Ischemia/Reperfusion Is Paralleled but Not Directly Mediated by C5a/C5aR1 Signaling.

Ischemia/reperfusion (I/R) is inevitable during kidney transplantation and causes acute kidney injury (AKI), which affects immediate outcome and leads to chronic changes such as fibrotic remodeling of the graft. We investigated pro-fibrotic signaling after I/R, focusing on the complement component and receptor C5a/C5aR1 and macrophage/tubule crosstalk. Male Dark Agouti rats were subjected to I/R and their kidneys were harvested 10 min, 6 h, 24 h, 3 days, 5 days and 8 weeks after reperfusion. The development of renal fibrosis was assessed by the detection of Vimentin (VIM), α-smooth muscle actin (α-SMA) and collagen by immunohistochemistry and Sirius Red staining, respectively. The characterization of C5a/C5aR1 activity and C5aR1+ cells was performed by multiplex mRNA analysis, ELISA, immunofluorescence flow cytometry and in situ hybridization in animal models and cell culture analyses. In the cell culture experiments, we focused on macrophage/tubule cell crosstalk in co-culture experiments and mimicked in vivo conditions by hypoxia/reoxygenation and supplementation with C5a. Already 6-24 h after the induction of I/R in the rat model, C5a concentration in the plasma was significantly increased compared to the control. The matrix components VIM and α-SMA peaked on day 5 and declined after 8 weeks, when an increase in collagen was detected using Sirius Red. In contrast to early I/R-induced C5a activation, renal C5ar1 expression was maximal at day 5 and C5 expression increased until week 8, indicating that the renal upregulation of expression is not required for early complement activation. C5aR1 mRNA was detected in neutrophils and macrophages, but not in proximal tubular cells in the injured kidneys. The macrophage/tubular cell co-culture experiments showed that macrophages were mainly responsible for the increased expression of fibrosis-associated genes in tubule cells (ACTA2, VIM, SNAI1, TGFB1 and FGF-2), and hypoxia/reoxygenation had a partially enhancing effect. A direct pro-fibrotic effect of C5a was not observed. Increased TGF-ß levels were dependent on the differentiation of macrophages to the M2 subtype. In conclusion, the early activation of mesenchymal markers in tubular epithelial cells leads to long-term fibrotic remodeling characterized by VIM expression and driven by TGF-ß-dependent macrophage/tubular crosstalk. The chemoattractive properties of complement C5a may contribute to the recruitment of pro-fibrotic macrophages.

Chemotaxis Assay of Bone Marrow-Derived Macrophages.

Immune responses rely on efficient and coordinated migration of immune cells to the site of infection or injury. To reach the site of immunological threat often requires long-range navigation of immune cells through complex tissue and vascular networks. Chemotaxis, cell migration steered by gradients of cell-attractive chemicals that bind sensory receptors, is central to this response. Chemoattractant receptors mostly belong to the G-protein-coupled receptor (GPCR) family, but the way attractant-receptor signaling directs cell migration is not fully understood. Direct-viewing chemotaxis chambers combined with time-lapse microscopy give a powerful tool to study the dynamic details of cells' responses to different attractant landscapes. Here, we describe the application of one such chamber (the Dunn chamber) to study bone marrow-derived macrophage chemotaxis to gradients of complement C5a.

Complement C3a/C3aR and C5a/C5aR deposits accelerate the progression of advanced IgA nephropathy to end-stage renal disease.

IgA nephropathy (IgAN) is still one of the leading causes of end-stage kidney disease (ESRD), and complement system activation is a key to the pathogenesis of IgAN. The role of complement C3a/C3aR and C5a/C5aR in late stage of IgAN remains unknown. Renal specimens of 75 IgAN patients at the stage 4 CKD were stained using immunofluorescence and immunohistochemistry. The primary outcome was a composite of end-stage renal disease (ESRD) and death. Associations of complement components with baseline clinicopathological characteristics and outcomes were assessed using multivariable Cox regression and Spearman analyses. During a median follow-up of 15.0 months, 27 patients progressed to ESRD and none died. Lower eGFR [hazards ratio (HR), 0.827, 95% confidence interval (CI), 0.732-0.935; P = 0.002] and glomerular C3 deposition (HR, 3.179, 95% CI, 1.079-9.363; P = 0.036) were predictive of time to ESRD in stage 4 CKD IgAN. Higher expression of C3a (P = 0.010), C3aR (P = 0.005), C5a (P = 0.015), and C5aR (P < 0.001) was identified in ESRD group than in non-ESRD group. Glomerular C3a/C3aR and C5a/C5aR deposits were both correlated with a lower baseline eGFR, higher baseline 24 h-urinary protein (24 h-UP) and faster decline of eGFR. Besides, C3a and C5a deposits were found in patients with high S (S1) and T (T1/2) scores, respectively. Complement C3a/C3aR and C5a/C5aR in IgAN patients with stage 4 CKD may portend a faster deterioration of kidney function.

Plasma levels of complement components C5 and C9 are associated with thrombin generation.

BACKGROUND: The thrombin generation assay (TGA) evaluates the potential of plasma to generate thrombin over time, providing a global picture of an individual's hemostatic balance. OBJECTIVES: This study aimed to identify novel biological determinants of thrombin generation using a multiomics approach. METHODS: Associations between TGA parameters and plasma levels of 377 antibodies targeting 236 candidate proteins for cardiovascular risk were tested using multiple linear regression analysis in 770 individuals with venous thrombosis from the Marseille Thrombosis Association (MARTHA) study. Proteins associated with at least 3 TGA parameters were selected for validation in an independent population of 536 healthy individuals (Etablissement Français du Sang Alpes-Méditerranée [EFS-AM]). Proteins with strongest associations in both groups underwent additional genetic analyses and in vitro experiments. RESULTS: Eighteen proteins were associated (P < 1.33 × 10⁻4) with at least 3 TGA parameters in MARTHA, among which 13 demonstrated a similar pattern of associations in EFS-AM. Complement proteins C5 and C9 had the strongest associations in both groups. Ex vivo supplementation of platelet-poor plasma with purified C9 protein had a significant dose-dependent effect on TGA parameters. No effect was observed with purified C5. Several single nucleotide polymorphisms associated with C5 and C9 plasma levels were identified, with the strongest association for the C5 missense variant rs17611, which was associated with a decrease in C5 levels, endogenous thrombin potential, and peak in MARTHA. No association of this variant with TGA parameters was observed in EFS-AM. CONCLUSION: This study identified complement proteins C5 and C9 as potential determinants of thrombin generation. Further studies are warranted to establish causality and elucidate the underlying mechanisms.

Clinical values of serum C5a in Alzheimer's disease patients with different dementia stages.

Alzheimer's disease (AD) is characterized by abnormal inflammatory responses, and complement C5a (C5a) is known to initiate inflammation. This study aimed to investigate the associations between serum C5a, inflammatory responses, and cognitive function in AD patients. A total of 242 AD patients and 132 age-matched controls were included. Enzyme-linked immunosorbent assay revealed increased levels of C5a, interleukin (IL)-4, IL-6, IL-10, IL-1ß, and tumor necrosis factor (TNF)-α with advancing stages of AD. Pearson correlation coefficient and receiver operating characteristic curve revealed positive correlations between serum C5a levels, inflammatory cytokine levels, Neuropsychiatric Inventory (NPI) and Activities of Daily Living (ADL) scores, and negative correlations with Mini-mental State Examination (MMSE) and Montreal cognitive assessment (MoCA) scores. Serum C5a above 68.68 pg/mL could aid in the diagnosis of AD. Multivariable logistic analysis revealed that serum C5a was an independent risk factor for IL-1ß/IL-6/IL-10/TNF-α and an independent protective factor for IL-4. Higher serum C5a levels were associated with lower MMSE and MoCA scores. In conclusion, elevated serum C5a levels were beneficial for AD diagnosis and predictive of inflammation and cognitive dysfunction.

Disseminated Cryptococcosis Post Eculizumab Therapy: A Case Report and Literature Review.

Eculizumab is a biologic medication used for the treatment of complement-related disorders including anti-acetylcholine receptor antibody-positive generalized myasthenia gravis. It targets C5 complement, preventing its cleavage into active terminal components. Thus, vaccination against encapsulated organisms is advised before starting this treatment. C5 also has a critical role against Cryptococcus neoformans infection. Here, we present a case of a 34-year-old man with a history of myasthenia gravis who was treated with prednisone and azathioprine in addition to eculizumab that was added to his regimen about a year ago, and who came to the hospital with headache, and was found to have Cryptococcus meningitis with disseminated cryptococcosis. The patient was negative for human immunodeficiency virus. He was treated with antifungal medications, and his condition improved. Although rarely reported, it is important to have a low threshold for diagnosis of cryptococcosis in patients on eculizumab given its complement inhibition mechanism of action.

"Izervay (avacincaptad pegol): paving the way for vision preservation in geographic atrophy".

Age-related macular degeneration (AMD) is a progressive retinal disease that primarily affects the macula, leading to central vision loss and impaired color vision. Among its most severe forms is geographic atrophy (GA), which results in irreversible central blindness. While numerous risk factors, including age, smoking, and genetics, contribute to the development of AMD, effective treatment options for GA have been limited. This article centers on Izervay [avacincaptad pegol (ACP)], an FDA-approved drug designed to address the unmet medical needs of patients with GA secondary to AMD. The pathophysiology of GA involves oxidative damage, chronic inflammation, and cell death, primarily due to complement system dysregulation. Previous treatments for GA have shown limited efficacy, leaving patients searching for more effective solutions. Izervay, with its unique mechanism of action, inhibits complement protein C5, disrupting the formation of the membrane attack complex and slowing retinal cell degeneration. Clinical trials have demonstrated Izervay's ability to significantly reduce the growth of GA lesions, offering hope for improved outcomes. Additionally, the drug has exhibited a tolerable safety profile, with common side effects including conjunctival hemorrhage and increased intraocular pressure. Izervay represents a breakthrough in AMD treatment, offering the potential to preserve vision in those at risk of irreversible vision loss due to GA. While further research is necessary to evaluate long-term efficacy and accessibility, its approval opens new possibilities in AMD management, transforming the lives of individuals affected by this condition.

Evaluation of the impact of two C5 genetic variants on C5-eculizumab complex stability at the molecular level.

Complement C5 is the target of the monoclonal antibody eculizumab, used in complement dysregulating disorders, like the rare disease Paroxysmal Nocturnal Hemoglobinuria (PNH). PNH is an acquired hematopoietic stem cell condition characterized by aberrant destruction of erythrocytes, chronic hemolytic anemia, and thromboembolism propensity. C5 is a protein component of the complement system which is part of the immune system of the body and plays a prominent role in the destruction of red blood cells, misidentifying them as a threat. This work describes the application of molecular dynamics simulations to the study of the underlying interactions between complement C5 and eculizumab. This study also reveals the importance of single nucleotide polymorphisms on C5 protein concerning the effective inhibition of the mAB, involving the mechanistic events taking place at the interface spots of the complex. The predicted conformational change in the C5 Arg885/His/Cys mutation has implications on the protein's interaction with eculizumab, compromising their compatibility. The acquired insights into the conformational changes, dynamics, flexibility, and interactions shed light on the knowledge of the function of this biomolecule providing answers about the poor response to the treatment in PNH patient carriers of the mutations. By investigating the intricate dynamics, significant connections between C5 and eculizumab can be uncovered. Such insights may aid in the creation of novel compounds or lead to the enhancement of eculizumab's efficacy.Communicated by Ramaswamy H. Sarma.

Management of pediatric hemolytic uremic syndrome.

Classical clinical triad of hemolytic uremic syndrome (HUS) is microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury associated with endothelial cell injury. Several situations, including infections, medications, malignancies, and transplantation can trigger endothelial damage. On the HUS spectrum, atypical hemolytic uremic syndrome (aHUS) deserves special attention in pediatric patients, as it can cause endstage kidney disease and mortality. A dysfunction in the alternative complement pathway, either acquired or genetic, has been shown to be the main underlying cause. In the last decades, breathtaking advances have been made in understanding the pathophysiology of this rare disease, which has led to more efficient treatment. Recent studies have implicated genes in pathways beyond the alternative complement system, such as DGKE, TSEN2, and INF2 highlighting the importance of personalized management. Eculizumab has brought about dramatic improvements in the treatment of aHUS. Beyond eculizumab, there are many alternative therapeutics in the pipeline that target the complement system. Because of the rarity of aHUS, data from multiple patient registries are very important. The present report aimed to summarize the most important aspects of diagnosing and treating aHUS based on the Turkish national registry and the literature so as to improve clinical practice.

C5L2 CRISPR KO enhances dental pulp stem cell-mediated dentinogenesis via TrkB under TNFα-induced inflammation.

Background and Objectives: Dental caries is one of the most common human pathological conditions resulting from the invasion of bacteria into the dentin. Current treatment options are limited. In many cases, endodontic therapy leads to permanent pulp tissue loss. Dentin-pulp complex regeneration involves dental pulp stem cells (DPSCs) that differentiate into odontoblast-like cells under an inflammatory context. However, limited information is available on how DPSC differentiation processes are affected under inflammatory environments. We identified the crucial role of complement C5a and its receptor C5aR in the inflammation-induced odontoblastic DPSC differentiation. Methodology: Here, we further investigated the role of a second and controversial C5a receptor, C5L2, in this process and explored the underlying mechanism. Human DPSCs were examined during 7-, 10-, and 14-day odontogenic differentiation treated with TNFα, C5L2 CRISPR, and tyrosine receptor kinase B (TrkB) antagonist [cyclotraxin-B (CTX-B)]. Results: Our data demonstrate that C5L2 CRISPR knockout (KO) enhances mineralization in TNFα-stimulated differentiating DPSCs. We further confirmed that C5L2 CRISPR KO significantly enhances dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) expression after 14-day odontoblastic DPSC differentiation, and treatment with CTX-B abolished the TNFα/C5L2 CRISPR KO-induced DSPP and DMP-1 increase, suggesting TrkB's critical role in this process. Conclusion and Key applications: Our data suggest a regulatory role of C5L2 and TrkB in the TNFα-induced odontogenic DPSC differentiation. This study may provide a useful tool to understand the mechanisms of the role of inflammation in dentinogenesis that is required for successful DPSC engineering strategies.

Fcgr2b and Fcgr3 are the major genetic factors for cartilage antibody-induced arthritis, overriding the effect of Hc encoding complement C5.

Like rheumatoid arthritis (RA) in humans, collagen-induced arthritis (CIA) in mice is associated with not only MHC class II genetic polymorphism but also, to some extent, with other loci including genes encoding Fc gamma receptors (FCGRs) and complement C5. In this study, we used a cartilage antibody-induced arthritis (CAIA) model in which arthritis develops within a 12-h timeframe, to determine the relative importance of FCGRs and C5 (Hc). In CAIA, inhibiting or deleting FCGR3 substantially hindered arthritis development, underscoring the crucial role of this receptor. Blocking FCGR3 also reduced the levels of FCGR4, and vice versa. When employing an IgG1 arthritogenic cocktail that exclusively interacts with FCGR2B and FCGR3, joint inflammation was promptly initiated in Fcgr2b-- mice but not in Fcgr3-- mice, suggesting that FCGR3 is sufficient for CAIA development. Regarding complement activation, Fcgr2b++.Hc** mice with C5 mutated were fully resistant to CAIA, whereas Fcgr2b--.Hc** mice developed arthritis rapidly. We conclude that FCGR3 is essential and sufficient for CAIA development, particularly when induced by IgG1 antibodies. The human ortholog of mouse FCGR3, FCGR2A, may be associated with RA pathogenesis. FCGR2B deficiency allows for rapid arthritis progression and overrides the resistance conferred by C5 deficiency.

Silenced-C5ar1 improved multiple organ injury in sepsis rats via inhibiting neutrophil extracellular trap.

Sepsis has a systemic inflammatory response syndrome caused by infection. While neutrophils play contradictory roles in different stages of sepsis. Neutrophils have been proven to play an antibacterial role by producing neutrophil extracellular traps (NETs). Although the NET is beneficial to bacteria resistance, abnormal NET increases tissue damage. The complement C5a receptor 1 (C5ar1) is a gene related to strong inflammatory reactions and is found to be associated with inflammatory factors. This study found that there were 45 down-regulated genes and 704 up-regulated genes in sepsis rats by transcriptome sequencing. And those genes were significantly related to inflammation and immunity by GO and KEGG enrichment analysis involving the chemokine signaling pathway, the Toll-like receptor (TLR) signaling pathway, and the Fc gamma R-mediated phagocytosis. Additionally, the C5ar1 gene was significantly upregulated with interesting potential in sepsis and used for further study. This study used cecum ligation and puncture (CLP) rats that were respectively injected intravenously with PBS or the lentivirus vector to explore the effect of C5ar1 on CLP rats. It demonstrated that silenced- C5ar1 inhibited the ALT, AST, BUN, and CREA levels, improved the lung and spleen injury, and reduced the TNF-α, IL-6, IL-1ß, IL-10, cf-DNA, and cfDNA/MPO levels. Additionally, silenced C5ar1 inhibited the TLR2, TLR4, and peptidylarginine deiminase 4 expression levels, which suggested the improvement of silenced C5ar1 on sepsis via inhibiting NETs and the TLR signaling pathway. This study provides a basis and new direction for the study of treatment on sepsis.

Blockade of C5a receptor unleashes tumor-associated macrophage antitumor response and enhances CXCL9-dependent CD8+ T cell activity.

Macrophages play a crucial role in shaping the immune state within the tumor microenvironment (TME) and are often influenced by tumors to hinder antitumor immunity. However, the underlying mechanisms are still elusive. Here, we observed abnormal expression of complement 5a receptor (C5aR) in human ovarian cancer (OC), and identified high levels of C5aR expression on tumor-associated macrophages (TAMs), which led to the polarization of TAMs toward an immunosuppressive phenotype. C5aR knockout or inhibitor treatment restored TAM antitumor response and attenuated tumor progression. Mechanistically, C5aR deficiency reprogrammed macrophages from a protumor state to an antitumor state, associating with the upregulation of immune response and stimulation pathways, which in turn resulted in the enhanced antitumor response of cytotoxic T cells in a manner dependent on chemokine (C-X-C motif) ligand 9 (CXCL9). The pharmacological inhibition of C5aR also improved the efficacy of immune checkpoint blockade therapy. In patients, C5aR expression associated with CXCL9 production and infiltration of CD8+ T cells, and a high C5aR level predicted poor clinical outcomes and worse benefits from anti-PD-1 therapy. Thus, our study sheds light on the mechanisms underlying the modulation of TAM antitumor immune response by the C5a-C5aR axis and highlights the potential of targeting C5aR for clinical applications.

Complement inhibition can decrease the haemostatic response in a microvascular bleeding model at multiple levels.

Background: Haemostasis is a crucial process by which the body stops bleeding. It is achieved by the formation of a platelet plug, which is strengthened by formation of a fibrin mesh mediated by the coagulation cascade. In proinflammatory and prothrombotic conditions, multiple interactions of the complement system and the coagulation cascade are known to aggravate thromboinflammatory processes and increase the risk of arterial and venous thrombosis. Whether those interactions also play a relevant role during the physiological process of haemostasis is not yet completely understood. The aim of this study was to investigate the potential role of complement components and activation during the haemostatic response to mechanical vessel injury. Methods: We used a microvascular bleeding model that simulates a blood vessel, featuring human endothelial cells, perfusion with fresh human whole blood, and an inducible mechanical injury to the vessel. We studied the effects of complement inhibitors against components of the lectin (MASP-1, MASP-2), classical (C1s), alternative (FD) and common pathways (C3, C5), as well as a novel triple fusion inhibitor of all three complement pathways (TriFu). Effects on clot formation were analysed by recording of fibrin deposition and the platelet activation marker CD62P at the injury site in real time using a confocal microscope. Results: With the inhibitors targeting MASP-2 or C1s, no significant reduction of fibrin formation was observed, while platelet activation was significantly reduced in the presence of the FD inhibitor. Both common pathway inhibitors targeting C3 or C5, respectively, were associated with a substantial reduction of fibrin formation, and platelet activation was also reduced in the presence of the C3 inhibitor. Triple inhibition of all three activation pathways at the C3-convertase level by TriFu reduced both fibrin formation and platelet activation. When several complement inhibitors were directly compared in two individual donors, TriFu and the inhibitors of MASP-1 and C3 had the strongest effects on clot formation. Conclusion: The observed impact of complement inhibition on reducing fibrin clot formation and platelet activation suggests a role of the complement system in haemostasis, with modulators of complement initiation, amplification or effector functions showing distinct profiles. While the interactions between complement and coagulation might have evolved to support haemostasis and protect against bleeding in case of vessel injury, they can turn harmful in pathological conditions when aggravating thromboinflammation and promoting thrombosis.

ß-Sitosterol activates autophagy to inhibit the development of hepatocellular carcinoma by regulating the complement C5a receptor 1/alpha fetoprotein axis.

Hepatocellular carcinoma (HCC) is highly refractory. ß-Sitosterol has been reported to suppress proliferation and migration as well as interfere with cell metabolism in tumors. However, there is limited information on the effects of ß-sitosterol on HCC. Herein, we used a xenograft mouse model to investigate the effects of ß-sitosterol on HCC tumor growth. The molecular mechanism was elucidated using quantitative real-time PCR, western blotting, lentiviral transfection, CCK8, scratch, Transwell, and Ad-mCherry-GFP-LC3B assays. The results showed that HepG2 cells highly expressed complement C5a receptor 1. ß-Sitosterol antagonized complement component 5a and exerted inhibitory effects on the proliferation and migration of HepG2 cells. The inhibitory effect of ß-sitosterol was reversed by the knockdown of complement C5a receptor 1. Bioinformatics analysis suggested alpha fetoprotein (AFP) as a downstream factor of complement C5a receptor 1. ß-Sitosterol inhibited AFP expression, which was reversed by complement C5a receptor 1 knockdown. The inhibitory effects of ß-sitosterol on cell proliferation and migration were weakened by AFP overexpression. Furthermore, ß-sitosterol induced autophagy in HepG2 cells, which was reversed by complement C5a receptor 1 knockdown and AFP overexpression. Blockade of autophagy by 3-MA attenuated ß-sitosterol inhibition of proliferation and migration in HepG2 cells. Moreover, ß-sitosterol inhibited HCC progression in vivo. Our findings demonstrate that ß-sitosterol inhibits HCC advancement by activating autophagy through the complement C5a receptor 1/AFP axis. These findings recommend ß-sitosterol as a promising therapy for HCC.