C3b Deposition Research Articles

Dog complement system is less effective against Leishmania infantum than human complement

Dogs are important reservoir hosts for Leishmania infantum, the causative agent of visceral leishmaniasis. The complement system, as part of the innate immune defense, is responsible for initiating the fight against pathogens that may invade an organism. A failure of the complement to combat L. infantum may explain, at least in part, why a mammal species is more or less susceptible to visceral leishmaniasis. The objective of this study was to compare the effectiveness of human and dog complement systems against L. infantum parasites. The results showed that dog serum was less effective than human serum at killing promastigote and amastigote-like forms. We also compared the efficiency of human and canine sera in classic and alternative hemolytic assays, as well as the serum efficiency of non-infected and Leishmania-infected dogs. Serum from dogs was less hemolytic than human serum in both pathways tested, but the efficiency of serum from infected dogs was higher than that of non-infected dogs. When testing C3b deposition assays on parasite surfaces, serum from infected dogs was more effective against amastigote-like forms than serum from non-infected dogs. However, both types of serum proved equally effective on promastigotes, while serum from infected dogs was more effective on amastigote-like forms. Considering the efficiency of the complement system, our results indicate that dogs are more susceptible to visceral leishmaniasis than humans are.

Klebsiella LPS O1-antigen prevents complement-mediated killing by inhibiting C9 polymerization

The Gram-negative bacterium Klebsiella pneumoniae is an important human pathogen. Its treatment has been complicated by the emergence of multi-drug resistant strains. The human complement system is an important part of our innate immune response that can directly kill Gram-negative bacteria by assembling membrane attack complex (MAC) pores into the bacterial outer membrane. To resist this attack, Gram-negative bacteria can modify their lipopolysaccharide (LPS). Especially the decoration of the LPS outer core with the O-antigen polysaccharide has been linked to increased bacterial survival in serum, but not studied in detail. In this study, we characterized various clinical Klebsiella pneumoniae isolates and show that expression of the LPS O1-antigen correlates with resistance to complement-mediated killing. Mechanistic data reveal that the O1-antigen does not inhibit C3b deposition and C5 conversion. In contrast, we see more efficient formation of C5a, and deposition of C6 and C9 when an O-antigen is present. Further downstream analyses revealed that the O1-antigen prevents correct insertion and polymerization of the final MAC component C9 into the bacterial membrane. Altogether, we show that the LPS O1-antigen is a key determining factor for complement resistance by K. pneumoniae and provide insights into the molecular basis of O1-mediated MAC evasion.

A bifunctional amylopullulanase of Streptococcus suis ApuA contributes to immune evasion by interaction with host complement C3b

The complement system is the first defense line of the immune system. However, pathogens have evolved numerous strategies to evade complement attacks. Streptococcus suis is an important zoonotic bacterium, harmful to both the pig industry and human health. ApuA has been reported as a bifunctional amylopullulanase and also contributed to virulence of S. suis. Herein, we found that ApuA could activate both classical and alternative pathways of the complement system. Furthermore, by using bacterial two-hybrid, far-western blot and ELISA assays, it was confirmed that ApuA could interact with complement C3b. The interaction domain of ApuA with C3b was found to be its α-Amylase domain (ApuA_N). After construction of an apuA mutant (ΔapuA) and its complementary strain, it was found that compared to the wild-type strain (WT), ΔapuA had significantly increased C3b deposition and membrane attack complex formation. Additionally, ΔapuA showed significantly lower survival rates in human serum and blood and was more susceptible to engulfment by neutrophils and macrophages. Mice infected with ΔapuA had significantly higher survival rates and lower bacterial loads in their blood, lung and brains, compared to those infected with WT. In summary, this study identified ApuA as a novel factor involved in the complement evasion of S. suis and suggested its multifunctional role in the pathogenesis of S. suis.

The GE296_RS03820 and GE296_RS03830 genes are involved in capsular polysaccharide biosynthesis in Riemerella anatipestifer.

Riemerella anatipestifer is a pathogenic bacterium that causes duck serositis and meningitis, leading to significant harm to the duck industry. To escape from the host immune system, the meningitis-causing bacteria must survive and multiply in the bloodstream, relying on specific virulence factors such as capsules. Therefore, it is essential to study the genes involved in capsule biosynthesis in R. anatipestifer. In this study, we successfully constructed gene deletion mutants Δ3820 and Δ3830, targeting the GE296_RS03820 and GE296_RS03830 genes, respectively, using the RA-LZ01 strain as the parental strain. The growth kinetics analysis revealed that these two genes contribute to bacterial growth. Transmission and scanning electron microscopy (TEM and SEM) and silver staining showed that Δ3820 and Δ3830 produced the altered capsules and compounds of capsular polysaccharides (CPSs). Serum resistance test showed the mutants also exhibited reduced C3b deposition and decreased resistance serum killing. Invivo, Δ3820 and Δ3830 exhibited markedly declining capacity to cross the blood-brain barrier, compared to RA-LZ01. These findings indicate that the GE296_RS03820 and GE296_RS03830 genes are involved in CPSs biosynthesis and play a key role in the pathogenicity of R. anatipestifer. Furthermore, Δ3820 and Δ3830 mutants presented a tendency toward higher survival rates from RA-LZ01 challenge invivo. Additionally, sera from ducklings immunized with the mutants showed cross-immunoreactivity with different serotypes of R. anatipestifer, including 1, 2, 7 and 10. Western blot and SDS-PAGE assays revealed that the altered CPSs of Δ3820 and Δ3830 resulted in the exposure of some conserved proteins playing the key role in the cross-immunoreactivity. Our study clearly demonstrated that the GE296_RS03820 and GE296_RS03830 genes are involved in CPS biosynthesis in R. anatipestifer and the capsule is a target for attenuation in vaccine development.

Safety and efficacy of complement factor H (CFH) inhibitor GT103 in advanced non-small cell lung cancer (NSCLC): Results of a phase Ib first in human dose escalation study.

e14588 Background: CFH is a complement regulatory protein that protects cells from alternative complement pathway activation. Neutralizing antibodies against CFH on tumor cells increase deposition of C3b, facilitate antibody-dependent-cellular phagocytosis and complement dependent cytotoxicity, inhibit tumor growth, and modulate anti-tumor immunity. GT103 is a first-in class, fully human-derived IgG3 anti-CFH monoclonal antibody that recognizes a tumor specific epitope on CFH and has shown anti-tumor activity in preclinical models. Methods: We conducted a phase Ib, first-in-human, dose escalation trial evaluating safety and preliminary efficacy of GT103 in patients (pts) with advanced NSCLC refractory to standard therapies. Pts received GT103 intravenously across six dose levels/schedules following 3+3 design. Primary objectives were to determine maximum tolerated dose (MTD), recommended phase II dose (RP2D), and pharmacokinetic profile of GT103. Secondary objectives were to assess objective response rate, progression free survival (PFS), and overall survival (OS). Here we present updated safety and efficacy results of the study. Results: 31 pts were enrolled from 6/2020 to 9/2023 (median age 63yrs (range, 23-79), 61% had adenocarcinoma, 32% had previously treated brain metastasis). MTD was not reached. Treatment related AEs (TRAEs) observed in ≥10% pts included fatigue (19%), anemia (16%), diarrhea (16%), and nausea (13%). 3 pts developed ≥ grade 3 TRAE, including acute kidney injury, decreased lymphocyte count, and anemia. The best treatment response was stable disease in 9 (29%) pts. Median(m) PFS and mOS were 6 weeks (95% CI 6.0-6.1) and 25.7 weeks (95% CI 19.1-30.6), respectively. 24-week PFS and OS were 9.7% (95% CI 2.5-22.9%) and 50.6% (95% CI 31.9-66.6%), respectively. PD-L1 expression was available in 25 pts. No statistically significant difference (p=0.33) was noted in mPFS between PD-L1 positive (TPS > 1%) vs negative (TPS ≤1%) pts. KRAS and EGFR mutation status was available for 22pts. mPFS and mOS were numerically longer in pts with KRAS positive vs negative disease. While there was no difference in mPFS, 24-weeks PFS was 0 vs 16.7% (95% CI 4.1-36.5%) EGFR MUT vs wild type disease. Biomarker analysis of complement regulatory proteins, FcGR expression and polymorphisms, and changes in sC5b-9 is underway. Conclusions: GT103 was well tolerated across all dose levels. The RP2D of 10mg/kg IV every 3 weeks was selected, although PK modeling will be performed to optimize the dosing schedule. Encouraging anti-tumor activity was seen in heavily pretreated pts with advanced NSCLC with a subset of patients experiencing stable disease lasting for longer than 6 months. Additional biomarker analysis is ongoing. A phase II trial combining GT103 with pembrolizumab in pts with advanced NSCLC is currently enrolling. Clinical trial information: NCT04314089 .

Engineering Agonistic Bispecifics to Investigate the Influence of Distance on Surface-Mediated Complement Activation.

The development of agonists capable of activating the human complement system by binding to the C1 complex presents a novel approach for targeted cell killing. Bispecific nanobodies and Abs can successfully use C1 for this purpose; however, efficacy varies significantly between epitopes, Ab type, and bispecific design. To address this variability, we investigated monomeric agonists of C1 in the form of bispecific nanobodies, which lack Fc domains that lead to oligomerization in Abs. These therefore offer an ideal opportunity to explore the geometric parameters crucial for C1 activation. In this study, we explored the impact of linker length as a metric for Ag and epitope location. DNA nanotechnology and protein engineering allowed us to design linkers with controlled lengths and flexibilities, revealing a critical range of end-to-end distances for optimal complement activation. We discovered that differences in complement activation were not caused by differential C1 activation or subsequent cleavage of C4, but instead impacted C4b deposition and downstream membrane lysis. Considering the importance of Ab class and subclass, this study provides insights into the structural requirements of C1 binding and activation, highlighting linker and hinge engineering as a potential strategy to enhance potency over specific cellular targets. Additionally, using DNA nanotechnology to modify geometric parameters demonstrated the potential for synthetic biology in complement activation. Overall, this research offers valuable insights into the design and optimization of agonists for targeted cell killing through complement activation.

#2639 Unraveling complement activation profiles on endothelial cells in healthy donors using an ex vivo model

Abstract Background and Aims Complement is involved in numerous kidney diseases. However, functional approaches to properly examine individual's susceptibility to complement dysregulation are limited. Method We assessed ex vivo complement activation induced by sera from 38 healthy donors on resting microvascular endothelial cells (HMEC-1) with or without complement dysregulation by an anti-FH monoclonal antibody (OX-24 mAb). Following incubation, immunofluorescence was used to reveal and quantify membrane-bound C3c (evaluating C3b/iC3b) and C5b-9 with a computer-assisted method (H-score). Additionally, complement anaphylatoxins (C3a and C5a) were quantified in cell supernatants by ELISA. Further exploration of ex vivo complement activation profiles were conducted by next generation sequencing of a panel of alternative pathway genes in 32 donors. Results Increase in complement deposition on HMEC-1 surface was defined for a H-scoreC3c > 50 and/or a H-scoreC5b-9 > 30. Combined analysis of C3b/iC3b and C5b-9 deposition in 35 donors showed that one donor (2.8%) had an isolated increase in C3b/iC3b deposits, 3 (8.6%) had an isolated increase in C5b-9 deposits and one other (2.8%) had both increased C3b/iC3b and C5b-9 deposition. Genetic analysis of 32/38 donors revealed 3 heterozygous rare or low frequency missense variants in CFH (p.N1050Y and p.R1210C) and CFI (p.A76G) in 3/5 donors (60%) with increased complement deposition. Conclusion Our ex vivo model for measuring complement activation allowed the identification of distinct complement activation profiles among healthy donors, revealing genetic susceptibility in 3 donors. This new dynamic and functional model provides an exciting avenue for exploring the mechanisms governing complement dysregulation in various kidney diseases in research.

#2663 Assessment of in situ alternative and classical convertases as indicators of ongoing glomerular complement activation in IgA nephropathy

Abstract Background and Aims Complement activation is involved in IgA nephropathy (IgAN), opening up new avenues for complement-targeting therapies. However, precise patient selection and optimal timing for these therapies are essential. Current diagnostic methods of complement activation in IgAN, based on immunostaining, lack precision in identifying specific pathway(s) involved and distinguishing ongoing from past complement activation. Method Complement deposition analysis was conducted on kidney biopsy samples from 53 patients with primary IgAN at diagnosis (NCT05234463). Glomerular complement deposition was assessed through immunohistochemistry for C4d and C5b-9 and immunofluorescence for C3b/iC3b and C1q. In situ detection of C3/C5 convertases complexes from complement classical/lectin (C4b2b) and alternative pathways (C3bBb) was performed on paraffin-embedded kidney biopsies from 33 patients using a proximity ligation assay (Duolink®, Sigma). Kidney biopsies from patients with lupus nephritis (LN) and C3 glomerulopathy (C3G) were used as positive controls for classical/lectin and alternative convertases staining, respectively. The signals corresponding to the convertases were quantified using QuPath® Software and correlated with clinical parameters, MEST-C score and C5b-9 staining. Results At diagnosis, all 53 IgAN patients (100%) exhibited C3 mesangial deposition. Additionally, 51.2% were positive for C4d, 16.5% had C1q deposition and 62.7% had C5b-9 deposition. In situ analysis of C3/C5 convertases revealed the presence of glomerular C3bBb convertases in 20/33 (60.6%) patients, with a median positive area of 14.9 [2.5-26.5]%o of glomerular surface. C4b2b convertases were detected in 21/33 patients (63.6%), with a median glomerular positive area of 8.1 [3.4-12.1]%o. Both C3bBb and C4b2b convertases were detected in 13/33 (39.4%) patients (Fig. 1). By contrast, no convertases were detected in 5/33 (15.2%) patients despite positive C3b/iC3b deposition. The intensity of C3bBb staining positively correlated with terminal pathway C5b-9 glomerular deposits (p = 0.0011, r2 0.29). Patients with MEST-C score S1 lesions and/or interstitial fibrosis/tubular atrophy (T1-2) exhibited a more pronounced intensity of C3bBb at diagnosis (p = 0.0093). Conclusion Our study, for the first time, presents evidence of ongoing complement activation at the tissue level in IgA nephropathy (IgAN), showing variable intensities among patients. The visualization of in situ C3/C5 convertases could enhance the functional aspect of pathological examination, helping identify patients with ongoing complement activation, making them potential candidates for complement inhibitors. Further research is necessary to investigate the kinetics of this staining over the natural course of IgAN and its implications for IgAN progression.

Sequential administration of anti-complement component C5 eculizumab and type-2 anti-CD20 obinutuzumab for the treatment of early antibody-mediated rejection after kidney transplantation: A proof of concept

Kidney transplant (KT) candidates with donor-specific antibodies (DSA) exhibit exceedingly high antibody-mediated rejection (ABMR) and allograft loss rates. Currently, treatment of ABMR remains an unmet clinical need. We report the use of the anti-C5 eculizumab and the type-2 anti-CD20 obinutuzumab in two patients with early ABMR. Eculizumab (900 mg IV) led to complete inhibition of the terminal complement cascade (unremarkable AP50 and CH50 activity) and prompt stoppage of complement-dependent antibody-mediated allograft injury (clearance of intra-graft C4d and C5b-9 deposition). Despite complement inhibition, obinutuzumab (1000 mg IV) determined full and long-lasting peripheral B-cell depletion, with significant reduction in all DSA. Graft function improved, remaining stable up to three years of follow-up. No signs of active ABMR and rebound DSA were detected. Obinutuzumab B-cell depletion and inhibition of DSA production were not affected by complement blockage. Further studies are needed to confirm the potential benefit of obinutuzumab in association with complement inhibitors.

Complement Terminal Pathway Activation and Intrarenal Immune Response in C3 Glomerulopathy.

C3 glomerulopathy is a rare disease resulting from an overactivation of the complement alternative pathway. Although there is also evidence of terminal pathway activation, its occurrence and consequences on the disease have been poorly studied. We retrospectively studied a cohort of 42 patients diagnosed with C3 glomerulopathy. We performed centralized extensive characterization of histological parameters. Kidney C5b-9 staining was performed as a marker of terminal pathway activation, intra-renal immune response was characterised through transcriptomic analysis. Eighty-eight percent of biopsies showed C5b-9 deposits in glomeruli. Biopsies were grouped according to the amount of C5b-9 deposits (no or low n=15/42, 36%, intermediate n=15/42, 36%, and high n=12/42, 28%). Patients with high C5b-9 deposits significantly differed from the 2 other groups patients and were characterized by a significant higher histological chronicity score (p=0.005) and lower outcome-free survival (p=0.001). In multivariable analysis, higher glomerular C5b-9 remained associated with poor kidney prognosis after adjustment. One third of the 847 studied immune genes were upregulated in C3 glomerulopathy biopsies compared to controls. Unsupervised clustering on differentially expressed genes identified a group of kidney biopsies enriched in high glomerular C5b-9 with high immune and fibroblastic signature and showed high chronicity scores on histological examination. In a cohort of patients with C3 glomerulopathy, intra-renal terminal pathway activation was associated with specific histological phenotype and disease prognosis.

Granular C3 dermatosis-A report of two cases and a mini-review of literature.

Granular C3 dermatosis (GCD) is characterized by bullous, erythematous, and eczematous skin lesions similar to dermatitis herpetiformis, and granular deposition of complement C3 and C5b-9 along the epidermal basement membrane zone (BMZ) by direct immunofluorescence (IF). Here, we present two cases of GCD with different clinical features. Case 1, a 49-year-old man, showed pruritic blisters and erythema of the extremities. Case 2, a 53-year-old woman, showed severely pruritic papules, erythema, and erosions on the entire body with scattered blisters, mainly on the lower extremities. Both patients showed mild eosinophilia on blood tests, subepidermal blisters and prominent eosinophilic infiltration in the upper dermis on histopathological examination, and granular BMZ deposition of C3, but not of immunoglobulins or other complement components, on direct IF. No circulating autoantibodies were detected on enzyme-linked immunosorbent assays, chemiluminescent enzyme immunoassays, indirect IF using 1 mol/L NaCl-split normal human skin, or immunoblotting. Diagnosis of GCD was made in both cases. Case 1 was successfully treated with topical steroids, oral minocycline, and nicotinamide without any recurrence of symptoms. Case 2 was treated with oral steroids and showed remarkable improvement, although mild pruritic papules remained. We reviewed 30 reported GCD cases, including the two cases presented here, since Hashimoto etal. first described GCD in 2016. GCD should be more widely recognized, and further accumulation and validation of cases are required.

Molecular mechanism of complement inhibition by the trypanosome receptor ISG65

African trypanosomes replicate within infected mammals where they are exposed to the complement system. This system centres around complement C3, which is present in a soluble form in serum but becomes covalently deposited onto the surfaces of pathogens after proteolytic cleavage to C3b. Membrane-associated C3b triggers different complement-mediated effectors which promote pathogen clearance. To counter complement-mediated clearance, African trypanosomes have a cell surface receptor, ISG65, which binds to C3b and which decreases the rate of trypanosome clearance in an infection model. However, the mechanism by which ISG65 reduces C3b function has not been determined. We reveal through cryogenic electron microscopy that ISG65 has two distinct binding sites for C3b, only one of which is available in C3 and C3d. We show that ISG65 does not block the formation of C3b or the function of the C3 convertase which catalyses the surface deposition of C3b. However, we show that ISG65 forms a specific conjugate with C3b, perhaps acting as a decoy. ISG65 also occludes the binding sites for complement receptors 2 and 3, which may disrupt recruitment of immune cells, including B cells, phagocytes, and granulocytes. This suggests that ISG65 protects trypanosomes by combining multiple approaches to dampen the complement cascade.

Molecular mechanism of complement inhibition by the trypanosome receptor ISG65.

African trypanosomes replicate within infected mammals where they are exposed to the complement system. This system centres around complement C3, which is present in a soluble form in serum but becomes covalently deposited onto the surfaces of pathogens after proteolytic cleavage to C3b. Membrane-associated C3b triggers different complement-mediated effectors which promote pathogen clearance. To counter complement-mediated clearance, African trypanosomes have a cell surface receptor, ISG65, which binds to C3b and which decreases the rate of trypanosome clearance in an infection model. However, the mechanism by which ISG65 reduces C3b function has not been determined. We reveal through cryogenic electron microscopy that ISG65 has two distinct binding sites for C3b, only one of which is available in C3 and C3d. We show that ISG65 does not block the formation of C3b or the function of the C3 convertase which catalyses the surface deposition of C3b. However, we show that ISG65 forms a specific conjugate with C3b, perhaps acting as a decoy. ISG65 also occludes the binding sites for complement receptors 2 and 3, which may disrupt recruitment of immune cells, including B cells, phagocytes, and granulocytes. This suggests that ISG65 protects trypanosomes by combining multiple approaches to dampen the complement cascade.

Ex vivo C5b-9 Deposition Test to Monitor Complement Activity in Clinical and Subclinical Atypical Hemolytic Uremic Syndrome and in Transplantation-Associated Thrombotic Microangiopathy

IntroductionAtypical Haemolytic Uremic Syndrome (aHUS) is a complement system (CS)-mediated ultrarare disease that manifests as thrombotic microangiopathy (TMA) with preferential small kidney vessels involvement. Transient CS activation is also observed in secondary TMA or in patients at risk to develop aHUS. There is no gold standard test to monitor disease activity but the ex vivo C5b-9 deposition test to be a better approach. MethodsWe assessed the C5b-9 deposition induced by serum samples of patients with aHUS (n=8) and with TMA associated to kidney (n=2), lung (n=1) or hematopoietic stem cell (HSC) transplantation (n=2) during the acute phase of the disease or in remission. As control for transplant associated (TA)-TMA we have analyzed samples of clinically stable kidney and HSC-transplanted patients without signs of TMA. Additionally, we studied one child with genetic risk of aHUS during an acute infection. ResultsIn the acute disease phase or in patients with disease activity despite C5 blockade, a significant increase of C5b-9 deposition was detected. In all patients with clinical response to C5 blockade but one, levels of C5b-9 deposition were within the normal range. Finally, we detected increased C5b-9 deposition levels in an asymptomatic child with genetic risk of aHUS when a concomitant otitis episode was ongoing. ConclusionThe ex vivo C5b-9 deposition test is an auspicious tool to monitor CS activity in aHUS and TA-TMA. In addition, we demonstrate that the test may also be useful to detect subclinical increase of CS activity which expands the spectrum of patients that would benefit from a better CS activity assessment.

PolySialic Acid Nanoparticles Actuate Complement-Factor-H-Mediated Inhibition of the Alternative Complement Pathway: A Safer Potential Therapy for Age-Related Macular Degeneration.

The alternative pathway of the complement system is implicated in the etiology of age-related macular degeneration (AMD). Complement depletion with pegcetacoplan and avacincaptad pegol are FDA-approved treatments for geographic atrophy in AMD that, while effective, have clinically observed risks of choroidal neovascular (CNV) conversion, optic neuritis, and retinal vasculitis, leaving room for other equally efficacious but safer therapeutics, including Poly Sialic acid (PSA) nanoparticle (PolySia-NP)-actuated complement factor H (CFH) alternative pathway inhibition. Our previous paper demonstrated that PolySia-NP inhibits pro-inflammatory polarization and cytokine release. Here, we extend these findings by investigating the therapeutic potential of PolySia-NP to attenuate the alternative complement pathway. First, we show that PolySia-NP binds CFH and enhances affinity to C3b. Next, we demonstrate that PolySia-NP treatment of human serum suppresses alternative pathway hemolytic activity and C3b deposition. Further, we show that treating human macrophages with PolySia-NP is non-toxic and reduces markers of complement activity. Finally, we describe PolySia-NP-treatment-induced decreases in neovascularization and inflammatory response in a laser-induced CNV mouse model of neovascular AMD. In conclusion, PolySia-NP suppresses alternative pathway complement activity in human serum, human macrophage, and mouse CNV without increasing neovascularization.

O95: The role of the complement system in mediating ischaemic cholangiopathy in DCD liver transplantation

Abstract Background The discrepancy between available organs and the number of patients on the waiting list have led to an increasing number of donor after circulatory death (DCD) liver grafts being used. DCD liver grafts suffer from a higher risk of ischaemic cholangiopathy, the pathophysiology of which is poorly understood. We hypothesized that the complement system mediates damage to the arterial endothelium, thereby affecting the ability of cholangiocytes to regenerate. Methods Livers declined for clinical transplantation were perfused for up to 10 hours with anti-coagulated packed red cells at 37° with or without complement inhibition. Levels of circulating complement components, tissue binding of complement proteins, and organ damage markers were measured. Results 5 DBD and 5 DCD livers were perfused, and 5 further DCD livers being perfused with eculizumab. C3 production was significantly higher in the untreated DCD cohort compared to DBD livers at 10 hours (52.68 mg/ml vs 8.62 mg/ml, p<0.005). C5b-9 deposition on the arterial endothelium of the portal tracts was significantly higher in the untreated DCD cohort compared to the treated DCD livers (mean 0.9 R.U vs 0.4 R.U, p<0.05). There was a non-significant difference in bile production and composition between the treated DCD and untreated DCD cohort. Conclusions There is evidence that the complement system mediates the damage to the arterial endothelium within the portal tracts of the liver. This damage could be ameliorated by complement inhibition. Further work to correlate this findings with clinical outcomes will be required.

C5b-9 promotes ferritinophagy leading to ferroptosis in renal tubular epithelial cells of trichloroethylene-sensitized mice

Trichloroethylene (TCE) is a common environmental contaminant that can cause a severe allergic reaction called TCE hypersensitivity syndrome, which often implicates the patient's kidneys. Our previous study revealed that C5b-9-induced tubular ferroptosis is involved in TCE-caused kidney damage. However, the study did not explain how tubule-specific C5b-9 causes free iron overload, a key event in ferroptosis. Here, we aimed to explore the role of NCOA4-mediated ferritinophagy in C5b-9-induced iron overload and ferroptosis in TCE-sensitized mice. Our results showed that TCE sensitization does not affect iron import or export, but does affect iron storage, causing ferritin degradation and free iron overload. In addition, mitochondrial ROS was upregulated, and these changes were blocked by C5b-9 inhibition. Interestingly, TCE-induced ferritin degradation and ferroptosis were significantly antagonized by the application of the mitochondrial ROS inhibitor, Mito-TEMPO. Moreover, all of these modes of action were further verified in C5b-9-attack signalling HK-2 cells. Further investigation demonstrated that C5b-9-upregulated mitochondrial ROS induced a marked increase in nuclear receptor coactivator 4 (NCOA4), a master regulator of ferritinophagy. In addition, the application of NCOA4 small interfering RNA not only significantly reversed ferritinophagy caused by C5b-9 but also reduced C5b-9-induced ferroptosis in HK-2 cells. Taken together, these results suggest that tubule-specific C5b-9 deposition activates NCOA4 through the upregulation of mitochondrial ROS, causing ferritin degradation and elevated free iron, which ultimately leads to tubular epithelial cell ferroptosis and kidney injury in TCE-sensitized mice.

O26 Polysaccharides as Key Players in Enteropathogenic E. coli Immune Evasion and Vaccine Development.

Enteropathogenic Escherichia coli (EPEC) produce a capsule of polysaccharides identical to those composing the O-antigen polysaccharide of its LPS (lipopolysaccharide) molecules. In light of this, the impact of O26 polysaccharides on the immune evasion mechanisms of capsulated O26 EPEC compared to non-capsulated enterohemorrhagic Escherichia coli (EHEC) was investigated. Our findings reveal that there was no significant difference between the levels in EPEC and EHEC of rhamnose (2.8:2.5), a molecule considered to be a PAMP (Pathogen Associated Molecular Patterns). However, the levels of glucose (10:1.69), heptose (3.6:0.89) and N-acetylglucosamine (4.5:2.10), were significantly higher in EPEC than EHEC, respectively. It was also observed that the presence of a capsule in EPEC inhibited the deposition of C3b on the bacterial surface and protected the pathogen against lysis by the complement system. In addition, the presence of a capsule also protected EPEC against phagocytosis by macrophages. However, the immune evasion provided by the capsule was overcome in the presence of anti-O26 polysaccharide antibodies, and additionally, these antibodies were able to inhibit O26 EPEC adhesion to human epithelial cells. Finally, the results indicate that O26 polysaccharides can generate an effective humoral immune response, making them promising antigens for the development of a vaccine against capsulated O26 E. coli.

Sex- and species-associated differences in complement-mediated immunity in humans and rhesus macaques.

The complement system can be viewed as a "moderator" of innate immunity, "instructor" of humoral immunity, and "regulator" of adaptive immunity. While sex is known to affect humoral and cellular immune systems, its impact on complement in humans and rhesus macaques, a commonly used non-human primate model system, has not been well studied. To address this knowledge gap, we analyzed serum samples from 90 humans and 72 rhesus macaques for the abundance and activity of the complement system components. While sequences of cascade proteins were highly conserved, dramatically different levels were observed between species. Whereas the low levels detected in rhesus samples raised questions about the suitability of the test for use with macaque samples, differences in levels of complement proteins were observed in male and female humans. Levels of total and antibody-dependent deposition of C1q and C3b on a glycosylated antigen differed between humans and rhesus, suggesting differential recognition of glycans and balance between classical and alternative activation pathways. Functional differences in complement-mediated lysis of antibody-sensitized cells were observed in multiple assays and showed that human females frequently exhibited higher lytic activity than human males or rhesus macaques, which typically did not exhibit such sex-associated differences. Other differences between species and sexes were observed in more narrow contexts-for only certain antibodies, antigens, or assays. Collectively, these results expand knowledge of sex-associated differences in the complement system in humans, identifying differences absent from rhesus macaques.IMPORTANCEThe complement system is a critical part of host defense to many bacterial, fungal, and viral infections. In parallel, rich epidemiological, clinical, and biomedical research evidence demonstrates that sex is an important biological variable in immunity, and many sex-specific differences in immune system are intimately tied with disease outcomes. This study focuses on the intersection of these two factors to define the impact of sex on complement pathway components and activities. This work expands our knowledge of sex-associated differences in the complement system in humans and also identifies the differences that appear to be absent in rhesus macaques, a popular non-human primate model. Whereas differences between species suggest potential limitations in the ability of macaque model to recapitulate human biology, knowledge of sex-based differences in humans has the potential to inform clinical research and practice.

HylS’, a fragment of truncated hyaluronidase of Streptococcus suis, contributes to immune evasion by interaction with host complement factor C3b

ABSTRACT Pathogenic bacteria have evolved many strategies to evade surveillance and attack by complements. Streptococcus suis is an important zoonotic pathogen that infects humans and pigs. Hyaluronidase (HylA) has been reported to be a potential virulence factor of S. suis. However, in this study, it was discovered that the genomic region encoding HylA of the virulent S. suis strain SC19 and other ST1 strains was truncated into four fragments when aligned with a strain containing intact HylA and possessing hyaluronidase activity. As a result, SC19 had no hyaluronidase activity, but one truncated HylA fragment, designated as HylS,’ directly interacted with complement C3b, as confirmed by western ligand blotting, pull-down, and ELISA assays. The deposition of C3b and membrane attack complex (MAC) formation on the surface of a HylS’-deleted mutant (ΔhylS’) was significantly increased compared to wild-type SC19. In human sera and whole blood, ΔhylS’ survival was significantly reduced compared to that in SC19. The resistance of ΔhylS’ to macrophages and human polymorphonuclear neutrophil PMNs also decreased. In a mouse infection model, ΔhylS’ showed reduced lethality and lower bacterial load in the organs compared to that of SC19. We conclude that the truncated hyaluronidase HylS’ fragment contributes to complement evasion and the pathogenesis of S. suis.