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.

The activated complement pathway in the fibrous process of benign prostatic hyperplasia.

BACKGROUND: To elucidate the changes in activated complement pathway in the fibrous process of benign prostatic hyperplasia (BPH), we analyzed the correlation between complement component expression and histological types of fibrosis using human BPH tissue. METHODS: Fifty-six histological BPH patients who underwent prostate needle biopsy at our institution (mean age 68.6 ± 6.5 years), divided into two histological groups, fibromuscular and fibrous, were compared. Inflammatory cell infiltration in BPH tissue was evaluated by immunohistochemical staining using CD45, with complement expression analysis performed using C3, factor B, and C5b-9 antibody, and the occupancy ratio of the stained region was calculated. Further, correlation between the histological types of fibrous components in BPH tissue and lower urinary tract symptoms questionnaires was analyzed. RESULTS: Twenty-seven (48.2%) and 29 (51.8%) cases were classified in the fibromuscular and fibrous groups, respectively. The proportion of CD45-positive cells in BPH tissue was significantly higher in the fibromuscular group. In complement component analysis, factor B did not significantly differ between groups, while C3 (fibromuscular group; 10.7 ± 8.2%, fibrous group; 16.4 ± 12.7%) and C5b-9 (fibromuscular group; 15.9 ± 6.2%, fibrous group; 17.6 ± 9.2%) were significantly higher in the fibrous group (p = 0.04, p = 0.04, respectively). International Prostate Symptom Score Q5 subscore, indicating slow stream, was significantly higher in the fibrous group (p = 0.04). CONCLUSIONS: In fibrous BPH with abundant fibrosis, the late complement pathway in addition to alternative pathway was activated compared to fibromuscular BPH. These results suggested that the alternative and late complement pathways were involved in the histological fibrous process of BPH.

Differential contributions of the C5b-9 and C5a/C5aR pathways to microvascular and macrovascular thrombosis in complement-mediated thrombotic microangiopathy patients.

To clarify the role of the C5a/C5aR (C5a receptor) and C5b-9 pathways in macrovascular thrombosis (MAT) and renal microthrombosis (MIT), 73 renal biopsy-proven complement-mediated thrombotic microangiopathy (C-TMA) patients were enrolled; 9 patients with pure MAT and 13 patients with pure MIT were selected for further study. Twenty-five external C-TMA patients were selected as the validation cohort. Plasma C5a and sC5b-9 (soluble C5b-9) levels were significantly higher in patients with MAT than in those with MIT (P = 0.008, P = 0.041, respectively). The mean optical density of C5aR1 in the kidney was significantly higher in MAT patients than in those with MIT (P < 0.001). Both urinary sC5b-9 levels (MIT: P < 0.001, MAT: P = 0.004) and renal deposition of C5b-9 (MIT: P < 0.001, MAT: P = 0.001) were significantly higher in C-TMA patients compared to normal control, but were similar between MAT and MIT groups. In the correlation analysis within 22C-TMA patients, urinary sC5b-9 levels and renal deposition of C5b-9 were positively correlated to renal MIT formation (P = 0.009 and P = 0.031, respectively). Furthermore, the renal citrullinated histone H3 (CitH3)- and neutrophil elastase (NE)-positive area ratios were both significantly higher in the MAT group than in the MIT group (P = 0.006 and P = 0.020, respectively). Therefore, the local C5b-9 and C5a/C5aR1 pathways might have differential contributions to MIT and MAT formation in the disease.

Urine Proteomics Link Complement Activation with Interstitial Fibrosis/Tubular Atrophy in Lupus Nephritis Patients.

BACKGROUND: Intrarenal complement activation has been implicated in the pathogenesis of tubulointerstitial fibrosis in lupus nephritis (LN) based on prior animal studies. The assembly of the membrane attack complex (MAC) by complement C5b to C9 on the cell membrane leads to cytotoxic pores and cell lysis, while CD59 inhibits MAC formation by preventing C9 from joining the complex. We hypothesize that complement activation and imbalance between complement activation and inhibition, as defined by increased production of individual complement components and uncontrolled MAC activation relative to CD59 inhibition, are associated with interstitial fibrosis and tubular atrophy (IFTA) in LN and correlate with the key mediators of kidney fibrosis- transforming growth factor receptors beta (TGFRß), platelet-derived growth factor beta (PDGFß) and platelet-derived growth factor receptor beta (PDGFRß). METHODS: We included urine samples from 46 adults and pediatric biopsy-proven lupus nephritis patients who underwent clinically indicated kidney biopsies between 2010 and 2019. We compared individual urinary complement components and the urinary C9-to-CD59 ratio between LN patients with moderate/severe IFTA and none/mild IFTA. IFTA was defined as none/mild (<25% of interstitium affected) versus moderate/severe (≥ 25% of interstitium affected). Proteomics analysis was performed using mass spectrometry (Orbitrap Fusion Lumos, Thermo Scientific) and processed by the Proteome Discoverer. Urinary complement proteins enriched in LN patients with moderate/severe IFTA were correlated with serum creatinine, TGFßR1, TGFßR2, PDGFß, and PDGFRß. RESULTS: Of the 46 LN patients included in the study, 41 (89.1%) were women, 20 (43.5%) self-identified as Hispanic or Latino, and 26 (56.5%) self-identified as Black or African American. Ten of the 46 (21.7%) LN patients had moderate/severe IFTA on kidney biopsy. LN patients with moderate/severe IFTA had an increased urinary C9-to-CD59 ratio [median 0.91 (0.83-1.05) vs 0.81 (0.76-0.91), p=0.01]. Urinary C3 and CFI levels in LN patients with moderate/severe IFTA were higher compared to those with none/mild IFTA [C3 median (IQR) 24.4(23.5-25.5) vs. 20.2 (18.5-22.2), p= 0.02], [CFI medium (IQR) 28.8 (21.8-30.6) vs. 20.4 (18.5-22.9), p=0.01]. Complement C9, CD59, C3 and CFI correlated with TGFßR1, PDGFß, and PDGFRß, while C9, CD59 and C3 correlated with TGFßR2. CONCLUSION: This study is one of the first to compare the urinary complement profile in LN patients with moderate/severe IFTA and none/mild IFTA in human tissues. This study identified C3, CFI, and C9-to-CD59 ratio as potential markers of tubulointerstitial fibrosis in LN.

Sublytic C5b-9 induces TIMP3 expression by glomerular mesangial cells via TRAF6-dependent KLF5 K63-linked ubiquitination in rat Thy-1 nephritis.

Rat Thy-1 nephritis (Thy-1N) is an experimental model for studying human mesangioproliferative glomerulonephritis (MsPGN), and its pathological features are glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) accumulation. Although we have confirmed that renal lesions of Thy-1N rats are sublytic C5b-9-dependent, and ECM accumulation is related to tissue inhibitor of matrix metalloproteinase (TIMP) inhibiting matrix metalloproteinase (MMP) activity, whether sublytic C5b-9 can induce TIMP production by GMC in Thy-1N rat and the underlying mechanism remains unclear. In the study, we proved that the expressions of TIMP3, krϋppel-like transcription factor 5 (KLF5) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were simultaneously up-regulated both in the renal tissues of Thy-1N rats (in vivo) and in the GMC exposed to sublytic C5b-9 (in vitro). Further mechanism exploration discovered that KLF5 and TRAF6 as two upstream molecules could induce TIMP3 gene transcription through binding to the same region i.e., -1801nt to -1554nt (GGGGAGGGGC) and -228nt to -46nt (GCCCCGCCCC) of TIMP3 promoter. In the process, TRAF6 mediated KLF5 K63-linked ubiquitination at K99 and K100 enhancing KLF5 nuclear localization and binding to TIMP3 promoter, augmenting its gene activation. Furthermore, the experiments in vivo exhibited that silencing KLF5, TRAF6 or TIMP3 gene could markedly lessen renal KLF5 K63-linked ubiquitination or TIMP3 induction, ECM accumulation and other pathological changes of Thy-1N rats. Besides, the positive expressions of above-mentioned these proteins and ECM accumulation and their correlation in the renal tissues of MsPGN patients were also demonstrated. Overall, our findings implicate that KLF5 and TRAF6 play a promoting role in sublytic C5b-9-triggered TIMP3 gene transcription and expression, which might provide a novel mechanistic insight into rat Thy-1N and human MsPGN.

Peritoneal Expression of Membrane Complement Regulators Is Decreased in Peritoneal Dialysis Patients with Infected Peritonitis.

In peritoneal dialysis (PD) patients, fungi and Pseudomonas aeruginosa are considered important causative microorganisms for peritonitis with poor prognosis. Our objective was to explore expressions of membrane complement (C) regulators (CRegs) and tissue injuries in the peritoneum of patients with PD-related peritonitis, including fungal and Pseudomonas aeruginosa peritonitis. In peritoneal biopsy tissues obtained at PD catheter removal, we investigated the severity of peritonitis-associated peritoneal injuries and the expression of CRegs, CD46, CD55, and CD59 against peritoneal tissues without any episode of peritonitis. In addition, we evaluated peritoneal injuries among fungal and Pseudomonas aeruginosa-peritonitis (P1) and Gram-positive bacterial peritonitis (P2). We also observed deposition of C activation products such as activated C and C5b-9 and measured sC5b-9 in the PD fluid of patients. As a result, the severity of peritoneal injuries correlated inversely with the expression of peritoneal CRegs. Peritoneal CReg expression in peritonitis was significantly reduced compared to no peritonitis. Peritoneal injuries were more severe in P1 than in P2. CReg expression was further decreased and C5b-9 further increased in P1 than in P2. In conclusion, severe peritoneal injuries due to fungal and Pseudomonas aeruginosa-peritonitis decreased CReg expression and increased deposition of activated C3 and C5b-9 in the peritoneum, suggesting that peritonitis, particularly fungal and Pseudomonas aeruginosa-peritonitis, might induce susceptibility to further peritoneal injuries due to excessive C activation.

Unveiling CD59-Antibody Interactions to Design Paratope-Mimicking Peptides for Complement Modulation.

CD59 is an abundant immuno-regulatory human protein that protects cells from damage by inhibiting the complement system. CD59 inhibits the assembly of the Membrane Attack Complex (MAC), the bactericidal pore-forming toxin of the innate immune system. In addition, several pathogenic viruses, including HIV-1, escape complement-mediated virolysis by incorporating this complement inhibitor in their own viral envelope. This makes human pathogenic viruses, such as HIV-1, not neutralised by the complement in human fluids. CD59 is also overexpressed in several cancer cells to resist the complement attack. Consistent with its importance as a therapeutical target, CD59-targeting antibodies have been proven to be successful in hindering HIV-1 growth and counteracting the effect of complement inhibition by specific cancer cells. In this work, we make use of bioinformatics and computational tools to identify CD59 interactions with blocking antibodies and to describe molecular details of the paratope-epitope interface. Based on this information, we design and produce paratope-mimicking bicyclic peptides able to target CD59. Our results set the basis for the development of antibody-mimicking small molecules targeting CD59 with potential therapeutic interest as complement activators.

The neoepitope of the complement C5b-9 Membrane Attack Complex is formed by proximity of adjacent ancillary regions of C9.

The Membrane Attack Complex (MAC) is responsible for forming large ß-barrel channels in the membranes of pathogens, such as gram-negative bacteria. Off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MAC-related inflammation, and pathophysiology. However, the identity of C9 neoepitope remains unknown. Here, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 Å resolution. The aE11 binding site is formed by two separate surfaces of the oligomeric C9 periphery and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA, and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, aE11 can recognise a partial epitope in purified monomeric C9 in vitro, albeit weakly. Taken together, our results reveal the structural basis for MAC recognition by aE11.

Hepatitis B virus suppresses complement C9 synthesis by limiting the availability of transcription factor USF-1 and inhibits formation of membrane attack complex: implications in disease pathogenesis.

BACKGROUND: The complement system functions primarily as a first-line host defense against invading microbes, including viruses. However, the interaction of Hepatitis B virus (HBV) with the complement-components during chronic HBV infection remains largely unknown. We investigated the mechanism by which HBV inhibits the formation of cytolytic complement membrane-attack complex (MAC) and studied its impact on MAC-mediated microbicidal activity and disease pathogenesis. METHODS: Blood/liver tissues were collected from chronically HBV-infected patients and controls. HepG2hNTCP cells were infected with HBV particles and Huh7 cells were transfected with full-length linear HBV-monomer or plasmids containing different HBV-ORFs and expression of complement components or other host genes were evaluated. Additionally, ELISA, Real-time PCR, Western blot, bioinformatics analysis, gene overexpression/knock-down, mutagenesis, chromatin immunoprecipitation, epigenetic studies, immunofluorescence, and quantification of serum HBV-DNA, bacterial-DNA and endotoxin were performed. RESULTS: Among the MAC components (C5b-C9), significant reduction was noted in the expression of C9, the major constituent of MAC, in HBV-infected HepG2hNTCP cells and in Huh7 cells transfected with full-length HBV as well as HBX. C9 level was also marked low in sera/liver of chronic hepatitis B (CHB) and Immune-tolerant (IT) patients than inactive carriers and healthy controls. HBX strongly repressed C9-promoter activity in Huh7 cells but CpG-island was not detected in C9-promoter. We identified USF-1 as the key transcription factor that drives C9 expression and demonstrated that HBX-induced hypermethylation of USF-1-promoter is the leading cause of USF-1 downregulation that in turn diminished C9 transcription. Reduced MAC formation and impaired lysis of HBV-transfected Huh7 and bacterial cells were observed following incubation of these cells with C9-deficient CHB sera but was reversed upon C9 supplementation. Significant inverse correlation was noted between C9 concentration and HBV-DNA, bacterial-DNA and endotoxin content in HBV-infected patients. One-year Tenofovir therapy resulted in improvement in C9 level and decline in viral/bacterial/endotoxin load in CHB patients. CONCLUSION: Collectively, HBX suppressed C9 transcription by restricting the availability of USF-1 through hypermethylation of USF-1-promoter and consequently hinder the formation and lytic functions of MAC. Early therapy is needed for both CHB and IT to normalize the aberrant complement profile and contain viral and bacterial infection and limit disease progression.

MAC mediates mammary duct epithelial cell injury in plasma cell mastitis and granulomatous mastitis.

Plasma cell mastitis (PCM) and granulomatous mastitis (GM) are common inflammatory nonbacterial mastitis (NBM). However, the pathogenesis of NBM is still unclear. METHODS: In this study, we statistically analyzed the pathological features of PCM and GM using pathological HE staining and tissue transmission electron microscopy. The levels of MAC (C5b-9n), P-selectin, E-selectin, and ICAM-1 were detected through IHC, WB, ELISA, and qPCR. The expression level and location of MAC were observed by tissue immunological electron microscopy. In addition, exosomes were isolated from tissues, identified using transmission electron microscopy, and the densities were detected by Nano-FCM. Finally, the expression intensity of MAC in exosomes was detected by flow cytometry and immunoelectron microscopy. RESULTS: The damage and apoptosis of mammary duct epithelial cells are the common pathological features of PCM and GM. MAC is primarily located in the cell membrane of mammary ductal epithelial cells and is significantly expressed in PCM and GM. The density of exosomes in PCM and GM tissues was elevated, and MAC was highly expressed in exosomes. In addition, the expression of P-selectin, E-selectin, and ICAM-1 in PCM and GM was significantly higher than in the normal group. CONCLUSION: We found severe damage of the mammary duct epithelial cells in PCM and GM tissues, which was verified by relevant pathological methods. Earlier studies demonstrated that MAC is highly expressed in PCM and GM tissues and exosomes seem to play a very important role in the understanding of MAC. Furthermore, MAC is involved in inflammatory infiltration and lesion of mammary duct epithelial cells upregulated by P-selectin, E-selectin, and ICAM-1. These findings provide new insights into PCM and GM molecular mechanisms.

Complement membrane attack complex is an immunometabolic regulator of NLRP3 activation and IL-18 secretion in human macrophages.

The complement system is an ancient and critical part of innate immunity. Recent studies have highlighted novel roles of complement beyond lysis of invading pathogens with implications in regulating the innate immune response, as well as contributing to metabolic reprogramming of T-cells, synoviocytes as well as cells in the CNS. These findings hint that complement can be an immunometabolic regulator, but whether this is also the case for the terminal step of the complement pathway, the membrane attack complex (MAC) is not clear. In this study we focused on determining whether MAC is an immunometabolic regulator of the innate immune response in human monocyte-derived macrophages. Here, we uncover previously uncharacterized metabolic changes and mitochondrial dysfunction occurring downstream of MAC deposition. These alterations in glycolytic flux and mitochondrial morphology and function mediate NLRP3 inflammasome activation, pro-inflammatory cytokine release and gasdermin D formation. Together, these data elucidate a novel signalling cascade, with metabolic alterations at its center, in MAC-stimulated human macrophages that drives an inflammatory consequence in an immunologically relevant cell type.

C5b-9 mediates ferroptosis of tubular epithelial cells in trichloroethylene-sensitization mice.

Occupational medicamentose-like dermatitis due to trichloroethylene (OMDT) is a key but unresolved question. OMDT patients often present multiple organ damage, including kidney damage. However, the underlying mechanism remains unknown. The purpose of our study was to explore the effect of tubule-specific C5b-9 deposition induced by TCE sensitization on renal tubular ferroptosis and its mechanism. By analyzing pathological changes of TCE-sensitization-mice kidney, we observed a significant renal tubular ferroptosis, which was alleviated by CD59, a C5b-9 inhibitory protein. Moreover, this phenomenon was also replicated in a C5b-9-attacked HK-2 cell model. Further experiments identified that C5b-9 induced cytosolic Ca2+ overload in renal tubular epithelia cells from TCE-sensitization-mice and HK-2 cells. Furthermore, in vitro experiments showed that BAPTA-AM, an intracellular Ca2+ chelator, could rescued ferroptosis induced by C5b-9 in HK-2 cells. Taken together, TCE sensitization induced renal tubular ferroptosis is mediated by C5b-9 and cytosolic Ca2+ overload may play a key role.

Sublytic C5b-9 Induces CCL3/4 Production and Macrophage Accumulation in Thy-1N Rats via PKC-α/p65/IRF-8 Axis.

Mesangioproliferative glomerulonephritis (MsPGN) is a common human kidney disease. Rat Thy-1 nephritis (Thy-1N) is an animal model widely used for the study of MsPGN. Thy-1N is not only sublytic C5b-9-dependent, but also related to pro-inflammatory cytokine production and macrophage (Mφ) accumulation in rat renal tissues. In this study, we found that the expression or phosphorylation of chemokine CCL3/4, CD68 (Mφ marker), IRF-8, PKC-α and NF-κB-p65 (p65) were all up-regulated both in the renal tissues of Thy-1N rats (in vivo) and in the glomerular mesangial cells (GMCs) upon sublytic C5b-9 stimulation (in vitro). Further experiments in vitro revealed that the phosphorylated PKC-α (p-PKC-α) could promote p65 phosphorylation, and then p-p65 enhanced IRF-8 expression through binding to IRF-8 promotor (-591 ~ -582 nt and -299 ~ -290 nt). Additionally, up-regulation or silencing of IRF-8 gene promoted or reduced CCL3/4 production, and then regulated Mφ chemotaxis. The underlying mechanism involved in IRF-8 binding to CCL3 promoter (-249 ~ -236 nt), which resulted in CCL3 gene transcription. The experiments in vivo showed that knockdown of renal PKC-α, p65, IRF-8 and CCL3/4 genes could inhibit CCL3/4 production, Mφ accumulation, GMC proliferation and proteinuria of Thy-1N rats. Furthermore, p-PKC-α, p-p65, IRF-8, CCL3/4 expression and Mφ accumulation were also increased in the renal tissues of MsPGN patients. Collectively, these findings indicate that sublytic C5b-9 induces CCL3/4 production and Mφ accumulation via PKC-α/p65/IRF-8 axis, and finally aggravates the pathological changes of MsPGN.

Complement activation by RPE cells preexposed to TNFα and IFNγ.

Age-related macular degeneration (AMD) has been associated with both complement activation and increased levels of circulating cytokines. Here, we sougth to investigate if cytokine-preexposure of retinal pigment epithelial (RPE) leads to increased complement activation and deposition of membrane attack complex (MAC). Primary human RPE and the ARPE19 cell line cultured in serum-free conditions were preexposed to 100 ng/ml interferon-gamma (IFNγ) and 20 ng/ml tumor necrosis factor-alpha (TNFα) for 48 h followed by exposure to diluted serum from healthy donors or complement factor B deficient (CFBd) serum for 70 min. Deposition of membrane attack complexes (MAC) was examined by use of a MAC-ELISA kit and by immunofluorescence. Eculizumab (anti-C5) was examined for its ability to prevent deposition of MAC on RPE cells exposed to serum. Lactatdehydrogenase (LDH) and thiazolyl blue tetrazolium bromide (MTT) assays were used to assess cellular metabolism and survival. MAC was deposited only on RPE preexposed to both IFNγ and TNFα. Lack of complement factor B or inhibition of C5 abrogated the MAC-deposition on RPE cells, while reconstitution of CFBd serum with CFB resulted in MAC-deposition. MAC-deposition resulted in RPE-release of LDH, but unaltered mitochondrial activity estimated by MTT. We conclude that preexposure of primary RPE and ARPE19 with inflammatory cytokines promoted alternative pathway activation of complement and deposition of MAC. This implies that circulating inflammatory mediators may increase susceptibility to local complement activation and MAC-deposition, which may represent an early event in the pathogenesis leading to AMD development.

Elevated Terminal C5b-9 Complement Complex 10 Weeks Post Kidney Transplantation Was Associated With Reduced Long-Term Patient and Kidney Graft Survival.

Background: The major reason for graft loss is chronic tissue damage, as interstitial fibrosis and tubular atrophy (IF/TA), where complement activation may serve as a mediator. The association of complement activation in a stable phase early after kidney transplantation with long-term outcomes is unexplored. Methods: We examined plasma terminal C5b-9 complement complex (TCC) 10 weeks posttransplant in 900 patients receiving a kidney between 2007 and 2012. Clinical outcomes were assessed after a median observation time of 9.3 years [interquartile range (IQR) 7.5-10.6]. Results: Elevated TCC plasma values (≥0.7 CAU/ml) were present in 138 patients (15.3%) and associated with a lower 10-year patient survival rate (65.7% vs. 75.5%, P < 0.003). Similarly, 10-year graft survival was lower with elevated TCC; 56.9% vs. 67.3% (P < 0.002). Graft survival was also lower when censored for death; 81.5% vs. 87.3% (P = 0.04). In multivariable Cox analyses, impaired patient survival was significantly associated with elevated TCC [hazard ratio (HR) 1.40 (1.02-1.91), P = 0.04] along with male sex, recipient and donor age, smoking, diabetes, and overall survival more than 1 year in renal replacement therapy prior to engraftment. Likewise, elevated TCC was independently associated with graft loss [HR 1.40 (1.06-1.85), P = 0.02] along with the same covariates. Finally, elevated TCC was in addition independently associated with death-censored graft loss [HR 1.69 (1.06-2.71), P = 0.03] as were also HLA-DR mismatches and higher immunological risk. Conclusions: Early complement activation, assessed by plasma TCC, was associated with impaired long-term patient and graft survival.

Internalization of the Membrane Attack Complex Triggers NLRP3 Inflammasome Activation and IL-1ß Secretion in Human Macrophages.

Interleukin 1ß (IL-1ß) plays a major role in inflammation and is secreted by immune cells, such as macrophages, upon recognition of danger signals. Its secretion is regulated by the inflammasome, the assembly of which results in caspase 1 activation leading to gasdermin D (GSDMD) pore formation and IL-1ß release. During inflammation, danger signals also activate the complement cascade, resulting in the formation of the membrane attack complex (MAC). Here, we report that stimulation of LPS-primed human macrophages with sub-lytic levels of MAC results in activation of the NOD-like receptor 3 (NLRP3) inflammasome and GSDMD-mediated IL-1ß release. The MAC is first internalized into endosomes and then colocalizes with inflammasome components; adapter protein apoptosis associated speck-like protein containing a CARD (ASC) and NLRP3. Pharmacological inhibitors established that MAC-triggered activation of the NLRP3 inflammasome was dependent on MAC endocytosis. Internalization of the MAC also caused dispersion of the trans-Golgi network. Thus, these data uncover a role for the MAC in activating the inflammasome and triggering IL-1ß release in human macrophages.

Enhanced eosinophil-mediated inflammation associated with antibody and complement-dependent pneumonic insults in critical COVID-19.

Despite the worldwide effect of the coronavirus disease 2019 (COVID-19) pandemic, the underlying mechanisms of fatal viral pneumonia remain elusive. Here, we show that critical COVID-19 is associated with enhanced eosinophil-mediated inflammation when compared to non-critical cases. In addition, we confirm increased T helper (Th)2-biased adaptive immune responses, accompanying overt complement activation, in the critical group. Moreover, enhanced antibody responses and complement activation are associated with disease pathogenesis as evidenced by formation of immune complexes and membrane attack complexes in airways and vasculature of lung biopsies from six fatal cases, as well as by enhanced hallmark gene set signatures of Fcγ receptor (FcγR) signaling and complement activation in myeloid cells of respiratory specimens from critical COVID-19 patients. These results suggest that SARS-CoV-2 infection may drive specific innate immune responses, including eosinophil-mediated inflammation, and subsequent pulmonary pathogenesis via enhanced Th2-biased immune responses, which might be crucial drivers of critical disease in COVID-19 patients.

Complement activation by an angiogenic imbalance leads to systemic vascular endothelial dysfunction: A new proposal for the pathophysiology of preeclampsia.

The underlying mechanism of preeclampsia by which an angiogenic imbalance results in systemic vascular endothelial dysfunction remains unclear. Complement activation directly induces endothelial dysfunction and is known to be involved in preeclampsia; nevertheless, the association between complement activation and angiogenic imbalance has not been established. This study aimed to evaluate whether angiogenic imbalance affects the expression and secretion of inhibitory complement factor H (CFH) in endothelial cells, resulting in complement activation and systemic vascular endothelial dysfunction. Viability of human umbilical vein endothelial cells (HUVECs) was assessed upon CFH knockdown by targeted-siRNA, and were incubated with complement factors. HUVECs were also treated with placental growth factor (PlGF) and/or soluble fms-like tyrosine kinase 1 (sFlt1), and CFH expression and secretion were measured. These cells were evaluated by cell viability assay and cell surface complement activation was quantified by immunocytochemical assessment of C5b-9 deposition. HUVECs transfected with CFH-siRNA had significantly lower viability than that of control cells. Moreover, the expression and secretion of CFH were significantly increased upon PlGF treatment compared with PlGF + sFlt1 combo. HUVECs treated with PlGF had less C5b-9 deposition and higher viability than HUVECs treated with PlGF + sFlt1. In summary, CFH was found to be essential for endothelial cell survival by inhibiting complement activation. An angiogenic imbalance, including decreased PlGF and increased sFlt1, suppresses CFH expression and secretion, resulting in complement activation on the surface of endothelial cells and systemic vascular endothelial dysfunction.

Coexistence of TDP-43 and C5b-9 staining of muscle in a patient with inclusion body myositis.

While sporadic inclusion body myositis (sIBM) is the most commonly acquired inflammatory myopathy above 50 years of age, its refractory response to conventional immunosuppressive treatments raises questions about its perplexing pathogenesis. Muscle biopsy typically reveals major histocompatibility complex I antigens and CD8+ T cell endomysial infiltrates invading non-necrotic muscle fibres early in the disease course with rimmed vacuoles, protein aggregates and amyloid inclusions later in the disease. Transactive response DNA-binding protein-43 (TDP-43), a protein implicated in transcriptional repression in neurodegenerative diseases, is also found in sIBM. C5b-9 membrane attack complex, an effector protein involved in the complement cascade of the immune response, is commonly found in dermatomyositis, but has rarely been reported in IBM. We describe a novel case of IBM with simultaneous C5b-9 and TDP-43 staining on quadriceps biopsy, raising the question of a possibility of concurrent immune-mediated inflammatory and myodegenerative pathogenesis.

Complement-mediated release of fibroblast growth factor 2 from human RPE cells.

PURPOSE: Complement activation is associated with choroidal neovascularization (CNV) in age-related macular degeneration (AMD). Fibroblast growth factor 2 (FGF2) and membrane attack complex (MAC) are present in eyes of patients with CNV. Herein, we investigated the effect of complement activation on FGF2 release in human retinal pigment epithelial (RPE) cells. METHODS: Cultured human RPE cells were primed with an anti-RPE antibody and then treated with C1q-depleted human serum in the presence or absence of Tec kinases inhibitor (LFM-A13). 38 cytokines/chemokines levels were measured by Luminex technology. Secretion of FGF2 and interleukin (IL)-6 was assessed by ELISA. Tec protein was measured by Western blot. mRNA expression of FGF2, chemokine (C-X-C motif) ligand 1 (CXCL-1), and family members of Tec kinases was evaluated by qPCR. Cell viability and MAC deposition were determined by WST-1 assay and flow cytometry, respectively. RESULTS: Complement activation caused increased FGF2 and IL-6 release. FGF2 was released when C6-depleted human serum was reconstituted with C6. Anti-C5 antibody significantly attenuated complement-mediated FGF2 release, but not IL-6. FGF2 mRNA levels were not affected, while CXCL-1 mRNA levels were increased by complement activation. FGF2-containing extracellular vesicles were detected in response to complement challenge. Tec mRNA and protein were expressed in RPE cells. In the presence of LFM-A13, secretion of FGF2, but not IL-6, and MAC deposition were significantly decreased and cell viability was significantly increased in complement-treated cells when compared to controls. CONCLUSIONS: Complement plays an important role to release FGF2 from RPE cells. Tec kinase is involved in MAC formation and complement-mediated FGF2 release. This information suggests a role for complement activation to mediate neovascularization in conditions such as AMD, and may elucidate potential therapeutic targets.