Activation Of Complement Pathway Research Articles

#626 C3 glomerulonephritis and thrombotic microangiopathy—a prospective observational study

Abstract Background and Aims Complement mediated TMA (cTMA) and complement component 3 glomerulonephritis (C3GN) are two distinct complement mediated kidney diseases. cTMA is characterized by acute kidney injury, thrombocytopenia and microangiopathic hemolytic anemia. C3GN results from uncontrolled activation of the alternative complement pathway leading to the deposition of C3 in the glomerulus. We sought to describe characteristic differences between these two conditions in a large cohort of patients from Bengaluru, India. Method All cases of cTMA and kidney biopsy proven C3GN from 2019-2022 at a tertiary care centre in India were included. Patients with C3GN had persistently low C3 for more than 12 weeks and no identifiable source of infection. All patients received protocolized medical therapy; patients with C3GN were treated with steroids and cyclophosphamide, while those with complement mediated TMA received plasmapheresis in addition to cyclophosphamide and steroids. Protocolised medical management (renin-angiotensin-aldosterone inhibition and diuretics) was provided in both the cases. Demographic, clinical and lab parameters were reported and compared. Results A total of 25 cTMA cases and 19 C3GN cases were included in the study. Both C3GN (52.7%) and cTMA (48%) were more common in males. Patients with C3GN typically presented in the third decade of life while cTMA cases presented in the fourth decade. Secondary hypertension was common in the cTMA group (92% vs 78.9%, compared to C3GN). 60% of patients with cTMA had either recent or current infection compared to 21.1% of C3GN patients (P = 0.04). The majority of cTMA cases had IFTA of <25% (84%) compared to C3GN (57.9%) although this was not statistically significant. Both C3GN and cTMA most commonly presented as rapidly progressive kidney failure and acute kidney injury. A total of 76% of patients in the cTMA group required kidney replacement therapy as compared to 47.4% in the C3GN group (P = 0.009). 44% of cTMA cases had anti factor H antibody positivity compared to 15.8% of C3GN patients (P = 0.02). Finally, 44% of cases in cTMA group had complete recovery compared to 47.4% in C3GN group at the end of 2.8 years of followup. Conclusion Although both conditions result from dysregulation of the alternative pathway, there are several important clinical characteristic differences between patients with cTMA and C3GN including presence or absence of secondary hypertension, concurrent infection, anti-factor H positivity and requirement of kidney replacement therapy. This information may be helpful to inform both diagnosis and prognosis for patients presenting with a complement mediated kidney disease.

#1643 Long-term cohort study on the prognostic impact of mesangial IgM deposition in IgA nephropathy

Abstract Background and Aims The prognostic importance of mesangial immunoglobulin (Ig) M deposition in IgA nephropathy (IgAN) is not well-understood and is subject to ongoing debate. Moreover, mesangial IgM deposition could also reflect lectin complement system activation. Thus, our study sought to examine the long-term prognostic implications of mesangial IgM deposition in a cohort of IgAN patients. Method We carried out a single-center retrospective study involving 93 consecutive IgAN patients (median age 41 years, 68% male, eGFR 48.7 mL/min, proteinuria 1.1 g/g) from 2010 to 2015. The follow-up period extended until the onset of end-stage kidney disease (ESKD), death, or January 2021, with a median duration of 7 years. An independent pathologist assessed the IgM immunofluorescence pattern, Oxford MEST-C score, and transmission electron microscopy (TEM) lesions using a detailed protocol. Results In our study, 70% (n = 65) of patients exhibited mesangial IgM-positive deposits, while 30% (n = 28) did not show IgM deposition. Both IgM-positive and IgM-negative groups were comparable in terms of age (40 vs 43 years, p 0.6), sex (69 vs 64% male, p 0.6), arterial hypertension prevalence (69 vs 64%, p 0.6), Charlson comorbidity scores (2 vs 1, p 0.3), kidney function (eGFR 46 vs 55 mL/min, p 0.1 and proteinuria 1.2 vs 0.7, p 0.1), pathology findings (including Oxford MEST-C score, IgG and C3 immune deposition), TEM analysis, as well as in their treatment with renin-angiotensin-system inhibitors (63 vs 71%, p 0.4) and immunosuppression (39 vs 39%, p 0.9), and mortality rates (11 vs 7%, p 0.5). However, a notable difference emerged in the progression to ESKD, with 37% of IgM-positive patients advancing to this stage compared to only 11% of those in the IgM-negative group (p 0.01). The multivariate Cox proportional hazards regression analysis identified lower eGFR (HR 0.94, 95% CI: 0.92, 0.97), higher Oxford MEST-C score (HR 1.53, 95% CI: 1.16, 2.02), and mesangial IgM deposits (HR 4.75, 95% CI: 1.25, 17.99) as independent predictors of reduced kidney survival. Conclusion Our study identifies mesangial IgM deposition as a potential risk factor for ESKD in advanced IgAN, suggesting a need for further investigation. Future research should explore whether IgM's activation of the lectin complement pathway and resultant damage, as proven in ischemia-reperfusion injury models, are applicable to IgAN.

#1779 Assessment of inflammation score in immune cells of atypical hemolytic uremic syndrome using single-cell sequencing analysis

Abstract Background and Aims Atypical hemolytic uremic syndrome (aHUS) is a rare and life-threatening form of thrombotic microangiopathy (TMA) characterized by a complex interplay of genetic and immunological factors. Historically, research into aHUS has primarily focused on genes associated with complement activation and coagulation pathways. Our previous studies, employing single cell sequencing analysis, have revealed a significant association between aHUS and various immune cell types [1]. The clinical manifestation of aHUS is often initiated by infections, autoimmune diseases, or pregnancy, with many instances not reverting to a stable state even after the elimination of these triggering factors. Additionally, a significant portion of patients, despite lacking identifiable anomalies in complement or coagulation genes, respond positively to anti-complement therapy. This observation suggests a potential role for inflammation-related genes in the pathogenesis of aHUS. Given the extensive array of genes linked to inflammation, our study is designed to quantitatively assess inflammation gene expression, converting these measures into an inflammation score for each immune cell type in aHUS patients, their nuclear families, and healthy controls. This approach allows for a detailed comparison of disease activity variations within aHUS cases, aiming to deepen our understanding of aHUS and explore new avenues for therapeutic intervention. Method This study enrolled 13 aHUS patients, 3 nuclear families without aHUS, and 4 healthy subjects from Taiwan. Peripheral blood samples were collected for single-cell RNA sequencing analysis. aHUS patients were categorized into stable and unstable groups. The definition of stable disease activity is characterized by consistent TMA-related organ involvement and normal markers of hemolysis. Single-cell RNA sequencing data was processed using CellRanger and analyzed using the Seurat package in R. We obtained inflammatory genes from the Molecular Signatures Database, and we used the AddModuleScore function in Seurat to calculate inflammation score (IS). Results Our analysis demonstrated that non-switch memory B cells, classical monocytes, intermediate monocytes, non-classical monocytes, low-density basophils, naïve CD8 T cells, central memory CD8 T cells, terminal effector CD4 T cells, Th1 cells, Th17 cells, Th1/Th17 cells, and follicular helper T cells had the highest IS in aHUS patients, followed by aHUS patients' nuclear family members without aHUS and healthy control subjects. Furthermore, we observed that the IS of classical monocytes, non-switch memory B cells (Fig. 1), intermediate monocytes, non-classical monocytes, naïve CD8 T cells, central memory CD8 T cells, non-vd2 gd T cells, terminal effector CD4 T cells, Th17 cells, and follicular helper T cells were highest in aHUS patients with unstable disease activity, followed by aHUS patients with stable disease activity, aHUS patients' nuclear family members without aHUS, and healthy control subjects. Conclusion Our study underscores a pivotal link between elevated inflammation scores and aHUS severity aHUS. This finding not only emphasizes the critical role of inflammation in disease progression but also opens new avenues for therapeutic intervention, particularly in cases of unstable aHUS. By highlighting inflammation as a key factor in aHUS pathophysiology, our research offers fresh insights for disease management and future investigations. Moreover, exploring anti-inflammatory pathways presents promising therapeutic possibilities, potentially leading to improved patient outcomes and enhanced management strategies for aHUS.

Trauma patients with type O blood exhibit unique multi-omics signature with decreased lectin pathway of complement levels.

Patients with type O blood may have an increased risk of hemorrhagic complications due to lower baseline levels of von Willebrand Factor (vWF) and factor VIII, but the transition to a mortality difference in trauma is less clear. We hypothesized that type O trauma patients will have differential proteomic and metabolomic signatures in response to trauma beyond vWF and FVIII alone. Patients meeting the highest level of trauma activation criteria were prospectively enrolled. Blood samples were collected upon arrival to the emergency department. Proteomic and metabolomic (multi-omics) analyses of these samples were performed using liquid chromatography-mass spectrometry. Demographic, clinical, and multi-omics data were compared between patients with type O blood versus all other patients. There were 288 patients with multi-omics data; 146 (51%) had type O blood. Demographics, injury patterns, and initial vital signs and laboratory measurements were not different between groups. Type O patients had increased lengths of stay (7 vs. 6 days, p = 0.041) and a trend towards decreased mortality secondary to traumatic brain injury compared to other causes (TBI, 44.4 % vs. 87.5%, p = 0.055). Type O patients had decreased levels of mannose-binding lectin (MBL) and MBL associated serine proteases 1 and 2 which are required for the initiation of the lectin pathway of complement activation. Type O patients also had metabolite differences signifying energy metabolism and mitochondrial dysfunction. Blood type O patients have a unique multi-omics signature, including decreased levels of proteins required to activate the lectin complement pathway. This may lead to overall decreased levels of complement activation and decreased systemic inflammation in the acute phase possibly leading to a survival advantage, especially in TBI. However, this may later impair healing. Future work will need to confirm these associations, and animal studies are needed to test therapeutic targets. Retrospective Comparative Study, Level IV.

Engineering Pancreatic Islets to Transiently Codisplay on Their Surface Thrombomodulin and CD47 Immunomodulatory Proteins as a Means of Mitigating Instant Blood-Mediated Inflammatory Reaction following Intraportal Transplantation.

Most pancreatic islets are destroyed immediately after intraportal transplantation by an instant blood-mediated inflammatory reaction (IBMIR) generated through activation of coagulation, complement, and proinflammatory pathways. Thus, effective mitigation of IBMIR may be contingent on the combined use of agents targeting these pathways for modulation. CD47 and thrombomodulin (TM) are two molecules with distinct functions in regulating coagulation and proinflammatory responses. We previously reported that the islet surface can be modified with biotin for transient display of novel forms of these two molecules chimeric with streptavidin (SA), that is, thrombomodulin chimeric with SA (SA-TM) and CD47 chimeric with SA (SA-CD47), as single agents with improved engraftment following intraportal transplantation. This study aimed to test whether islets can be coengineered with SA-TM and SA-CD47 molecules as a combinatorial approach to improve engraftment by inhibiting IBMIR. Mouse islets were effectively coengineered with both molecules without a detectable negative impact on their viability and metabolic function. Coengineered islets were refractory to destruction by IBMIR ex vivo and showed enhanced engraftment and sustained function in a marginal mass syngeneic intraportal transplantation model. Improved engraftment correlated with a reduction in intragraft innate immune infiltrates, particularly neutrophils and M1 macrophages. Moreover, transcripts for various intragraft procoagulatory and proinflammatory agents, including tissue factor, HMGB1 (high-mobility group box-1), IL-1β, IL-6, TNF-α, IFN-γ, and MIP-1α, were significantly reduced in coengineered islets. These data demonstrate that the transient codisplay of SA-TM and SA-CD47 proteins on the islet surface is a facile and effective platform to modulate procoagulatory and inflammatory responses with implications for both autologous and allogeneic islet transplantation.

Synergistic benefits of dietary silymarin and selenium on growth, immune functions, antioxidants, and gut/liver health of Thinlip mullet (Liza ramada) juveniles

Abstract This study investigates the synergistic impact of silymarin (SI) levels combined with inorganic selenium (sodium selenite: Se) on growth, feed utilization, biochemical parameters, antioxidants, innate immunity, intestinal and liver histology, and gene expression of Thinlip mullet (Liza ramada) juveniles. The experimental design involved thinlip mullets initially weighing 3.5±0.13 g, distributed in a completely randomized design with 30 fish per hapa (0.5 × 0.5 × 1 m), and conducted in triplicate over 60 days. Seven experimental diets were employed, including a control (without SI and Se supplementation), a negative control (with only Se supplementation), and four treatments with varying levels of silymarin (250, 450, 650, 850 mg/kg) alongside selenium (0.5 mg/kg diet). The growth performance results highlighted significant enhancements in final body weight, weight gain, and specific growth rate, particularly in the SI 850 mg/kg + Se treatment. Survival rates, feed intake, and feed conversion ratios showed positive trends across the SI-Se supplemented groups. Biochemical profiles of serum exhibited that the control diet-induced elevated concentrations of glucose, cholesterol, Alanine aminotransferase, Aspartate aminotransferase, and urea, while Se or SI supplementation significantly mitigated these levels, with the lowest concentrations observed in the SI-Se supplemented groups. Moreover, SI supplementation increased serum protein content. Antioxidant enzyme activities, represented by superoxide dismutase (SOD), catalase (CAT), and catalase (GPx), demonstrated notable improvements in the SI-Se fortified groups, with significantly elevated GPx activity compared to the Se-supplemented and control groups. Immune system responses, including lysozyme, bactericidal, Nitro-blue Tetrazolium (NBT%), and serum alternative complement pathway (ACH50) activities, were highest in the SI-Se augmented groups. SI and Se in L. ramada reduce liver pro-inflammatory gene expression (il-1β, hepcidin) vs. control group. Histological examinations of the intestine and liver depicted structural enhancements, especially at moderate and high levels of SI with Se supplementation. The results indicate improved intestinal villi morphology and hepatic architecture, supporting the positive influence of dietary treatments on the health of thinlip mullet juveniles. In conclusion, the combined supplementation of SI at 850 mg/kg diet and Se at 0.5 mg/kg diet positively influenced the growth, biochemical profiles, antioxidant status, immune responses, gene expression, and histological integrity of Thinlip mullet juveniles, providing valuable insights for optimizing aquafeed formulations.

Circulating mortalin in blood and activation of the alternative complement pathway as risk indicators in COVID-19 infection.

Mortalin/GRP75 is a ubiquitous mitochondrial chaperone related to the cytosolic heat shock protein 70. It protects cells from various types of damages and from senescence. Our goal was to determine whether COVID-19 patients have circulating mortalin in their blood and to assess its prognostic value in anticipating disease severity. Mortalin was determined by ELISA in the sera of 83 COVID-19 patients enrolled in the study. Patients were categorized into 4 groups: critical patients who died (FATAL) or required intensive care and survived (ICU), patients of mild severity (hospitalized but not critical) who required nasal oxygen support (HOSP+O2), and patients who did not need oxygen therapy (HOSP). The mortalin concentration in the serum of all COVID-19 patients in the cohort was 194-2324 pg/mL. A comparison of the mortalin levels by peak severity among the various patient groups showed a highly significant difference between the HOSP and FATAL groups and a significant difference between the HOSP and the ICU groups. COVID-19 patients who eventually failed to survive had at hospitalization a markedly higher level of mortalin in their sera. Cox regression analysis revealed a high mortality hazard (HR=3.96, p<0.01) in patients with high mortalin circulating levels (above the median, ≥651 pg/mL). This was confirmed in survival curve analysis (Kaplan-Meier; p=0.0032, log-rank test). Mortalin remained an independent predictor of mortality even after adjusting for age and sex or various complement activation products. Complement activation data collected in an earlier study in the same cohort was compared regarding the mortalin levels. Patients with higher circulating mortalin levels also had higher levels of complement C3a but reduced levels of properdin. This is the first report on circulating mortalin in COVID-19 patients. Higher mortalin levels were associated with more severe illnesses and a higher risk of death. We claim that quantifying the blood levels of mortalin and activated complement proteins will provide important information on the prognosis of COVID-19 patients and will serve as a useful tool for guiding their clinical management and treatment.

Single cell transcriptomic analyses reveal diverse and dynamic changes of distinct populations of lung interstitial macrophages in hypoxia-induced pulmonary hypertension.

Hypoxia is a common pathological driver contributing to various forms of pulmonary vascular diseases leading to pulmonary hypertension (PH). Pulmonary interstitial macrophages (IMs) play pivotal roles in immune and vascular dysfunction, leading to inflammation, abnormal remodeling, and fibrosis in PH. However, IMs' response to hypoxia and their role in PH progression remain largely unknown. We utilized a murine model of hypoxia-induced PH to investigate the repertoire and functional profiles of IMs in response to acute and prolonged hypoxia, aiming to elucidate their contributions to PH development. We conducted single-cell transcriptomic analyses to characterize the repertoire and functional profiles of murine pulmonary IMs following exposure to hypobaric hypoxia for varying durations (0, 1, 3, 7, and 21 days). Hallmark pathways from the mouse Molecular Signatures Database were utilized to characterize the molecular function of the IM subpopulation in response to hypoxia. Our analysis revealed an early acute inflammatory phase during acute hypoxia exposure (Days 1-3), which was resolved by Day 7, followed by a pro-remodeling phase during prolonged hypoxia (Days 7-21). These phases were marked by distinct subpopulations of IMs: MHCIIhiCCR2+EAR2+ cells characterized the acute inflammatory phase, while TLF+VCAM1hi cells dominated the pro-remodeling phase. The acute inflammatory phase exhibited enrichment in interferon-gamma, IL-2, and IL-6 pathways, while the pro-remodeling phase showed dysregulated chemokine production, hemoglobin clearance, and tissue repair profiles, along with activation of distinct complement pathways. Our findings demonstrate the existence of distinct populations of pulmonary interstitial macrophages corresponding to acute and prolonged hypoxia exposure, pivotal in regulating the inflammatory and remodeling phases of PH pathogenesis. This understanding offers potential avenues for targeted interventions, tailored to specific populations and distinct phases of the disease. Moreover, further identification of triggers for pro-remodeling IMs holds promise in unveiling novel therapeutic strategies for pulmonary hypertension.

Abstract LB312: Subtype- and race-specific differences in immune landscape of breast cancer

Abstract Tumor mass comprises of not just the tumor cells but a variety of stromal cells. Immune cells residing within the tumor microenvironment modulate the growth and behavior of the tumor cells through a bi-directional crosstalk and affect the outcomes of cancer therapies. Breast cancer (BC) is a highly heterogeneous disease having four major molecular subtypes. Triple-negative breast cancer (TNBC) is the most aggressive subtype and has limited available therapeutic options. Further, it disproportionately affects African American (AA) women, with nearly two times higher incidence as compared to Caucasian American (CA) women. It is associated with an earlier onset and overall higher mortality of BC. Here, we investigated the differences in the immune landscape between TNBC and non-TNBC subtypes and examined if there existed any race-specific patterns. A total of 81 breast cancer samples with a distribution of 55 non-TNBC (28 CA and 27 AA) and 26 TNBC (12 CA and 14 AA) were selected after histopathologic analysis. Tissues were analysis using the NanoString nCounter® pancancer immune panel to determine tissue-specific immune cell populations. We observed a significantly higher presence of tumor-infiltrating lymphocytes in TNBC compared to non-TNBC, with a higher immune score for CD45+ (total lymphocytes), T-cells, B-cells and tumor-associated macrophages. Specifically, among the T-cell population, Treg cells, Th1 cells, and CD8+ cells were higher in TNBC samples. The pathway prediction indicated 'primary immunodeficiency' and 'cancer immunotherapy by PD-1 blockade' pathways being significantly up-regulated in TNBC samples. Overexpression of CTLA4, IDO1, PDL1 and PDL2 as well as Treg marker genes, such as FOXP3 and LAG3, which are involved in immunosuppressive pathways, was observed in TNBC. In race-wise comparisons of non-TNBC and TNBC sample between CA and AA patients, we found that AA non-TNBC tumors had higher Treg cell scores. On the other hand, CA TNBC samples had higher Th1 and CD8+ T-cell scores compared to AA TNBC. The pathway prediction from differentially expressed genes between CA and AA non-TNBC samples showed complement activation pathway to be down-regulated in AA patients, which could be associated with an increased presence of Treg cells. Further, when comparing CA vs AA TNBC samples, we found significant up-regulation of oncogene BMI1 and down-regulation of genes involved in T-cell activation such as CD47, CD44, FOS, and S100A8 in AA TNBC, supporting their more aggressive nature. In conclusion, our data show that TNBC patients have a higher infiltration of immune cells, which remain suppressed due to the presence of Treg cells and PD1 signaling pathway. The presence of higher Treg cells in AA non-TNBC and lower Th1 and CD8+ T-cells in AA TNBC samples suggests an overall immune suppressed microenvironment in AA patients compared to CA patients. Overall, our data suggest that TNBC could be good candidate for immune therapy. Citation Format: Amod Sharma, Kunwar Somesh Vikramdeo, Sarabjeet Kour Sudan, Mohammad Aslam Khan, Mohammad Tahir, James Elliot Carter, Cindy Nelson, Ajay P Singh, Seema Singh. Subtype- and race-specific differences in immune landscape of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB312.

CSMD1 regulates brain complement activity and circuit development

Complement proteins facilitate synaptic elimination during neurodevelopmental pruning, but neural complement regulation is not well understood. CUB and Sushi Multiple Domains 1 (CSMD1) can regulate complement activity in vitro, is expressed in the brain, and is associated with increased schizophrenia risk. Beyond this, little is known about CSMD1 including whether it regulates complement activity in the brain or otherwise plays a role in neurodevelopment. We used biochemical, immunohistochemical, and proteomic techniques to examine the regional, cellular, and subcellular distribution as well as protein interactions of CSMD1 in the brain. To evaluate whether CSMD1 is involved in complement-mediated synapse elimination, we examined Csmd1-knockout mice and CSMD1-knockout human stem cell-derived neurons. We interrogated synapse and circuit development of the mouse visual thalamus, a process that involves complement pathway activity. We also quantified complement deposition on synapses in mouse visual thalamus and on cultured human neurons. Finally, we assessed uptake of synaptosomes by cultured microglia. We found that CSMD1 is present at synapses and interacts with complement proteins in the brain. Mice lacking Csmd1 displayed increased levels of complement component C3, an increased colocalization of C3 with presynaptic terminals, fewer retinogeniculate synapses, and aberrant segregation of eye-specific retinal inputs to the visual thalamus during the critical period of complement-dependent refinement of this circuit. Loss of CSMD1 in vivo enhanced synaptosome engulfment by microglia in vitro, and this effect was dependent on activity of the microglial complement receptor, CR3. Finally, human stem cell-derived neurons lacking CSMD1 were more vulnerable to complement deposition. These data suggest that CSMD1 can function as a regulator of complement-mediated synapse elimination in the brain during development.

Differentiated pattern of complement system activation between MOG-IgG-associated disease and AQP4-IgG-positive neuromyelitis optica spectrum disorder.

Myelin oligodendrocyte glycoprotein antibody (MOG) immunoglobulin G (IgG)-associated disease (MOGAD) has clinical and pathophysiological features that are similar to but distinct from those of aquaporin-4 antibody (AQP4-IgG)-positive neuromyelitis optica spectrum disorders (AQP4-NMOSD). MOG-IgG and AQP4-IgG, mostly of the IgG1 subtype, can both activate the complement system. Therefore, we investigated whether the levels of serum complement components, regulators, and activation products differ between MOGAD and AQP4-NMOSD, and if complement analytes can be utilized to differentiate between these diseases. The sera of patients with MOGAD (from during an attack and remission; N=19 and N=9, respectively) and AQP4-NMOSD (N=35 and N=17), and healthy controls (N=38) were analyzed for C1q-binding circulating immune complex (CIC-C1q), C1 inhibitor (C1-INH), factor H (FH), C3, iC3b, and soluble terminal complement complex (sC5b-9). In attack samples, the levels of C1-INH, FH, and iC3b were higher in the MOGAD group than in the NMOSD group (all, p<0.001), while the level of sC5b-9 was increased only in the NMOSD group. In MOGAD, there were no differences in the concentrations of complement analytes based on disease status. However, within AQP4-NMOSD, remission samples indicated a higher C1-INH level than attack samples (p=0.003). Notably, AQP4-NMOSD patients on medications during attack showed lower levels of iC3b (p<0.001) and higher levels of C3 (p=0.008), C1-INH (p=0.004), and sC5b-9 (p<0.001) compared to those not on medication. Among patients not on medication at the time of attack sampling, serum MOG-IgG cell-based assay (CBA) score had a positive correlation with iC3b and C1-INH levels (rho=0.764 and p=0.010, and rho=0.629 and p=0.049, respectively), and AQP4-IgG CBA score had a positive correlation with C1-INH level (rho=0.836, p=0.003). This study indicates a higher prominence of complement pathway activation and subsequent C3 degradation in MOGAD compared to AQP4-NMOSD. On the other hand, the production of terminal complement complexes (TCC) was found to be more substantial in AQP4-NMOSD than in MOGAD. These findings suggest a strong regulation of the complement system, implying its potential involvement in the pathogenesis of MOGAD through mechanisms that extend beyond TCC formation.

Specific lineage transition of tumor-associated macrophages elicits immune evasion of ascitic tumor cells in gastric cancer with peritoneal metastasis.

Gastric cancer with peritoneal metastasis (PM-GC), recognized as one of the deadliest cancers. However, whether and how the tumor cell-extrinsic tumor microenvironment (TME) is involved in the therapeutic failure remains unknown. Thus, this study systematically assessed the immunosuppressive tumor microenvironment in ascites from patients with PM-GC, and its contribution to dissemination and immune evasion of ascites-disseminated tumor cells (aDTCs). Sixty-three ascites and 43 peripheral blood (PB) samples from 51 patients with PM-GC were included in this study. aDTCs in ascites and circulating tumor cells (CTCs) in paired PB were immunophenotypically profiled. Using single-cell RNA transcriptional sequencing (scRNA-seq), crosstalk between aDTCs and the TME features of ascites was inspected. Further studies on the mechanism underlying aDTCs-immune cells crosstalk were performed on in vitro cultured aDTCs. Immune cells in ascites interact with aDTCs, prompting their immune evasion. Specifically, we found that the tumor-associated macrophages (TAMs) in ascites underwent a continuum lineage transition from cathepsinhigh (CTShigh) to complement 1qhigh (C1Qhigh) TAM. CTShigh TAM initially attracted the metastatic tumor cells to ascites, thereafter, transitioning terminally to C1Qhigh TAM to trigger overproliferation and immune escape of aDTCs. Mechanistically, we demonstrated that C1Qhigh TAMs significantly enhanced the expression of PD-L1 and NECTIN2 on aDTCs, which was driven by the activation of the C1q-mediated complement pathway. For the first time, we identified an immunosuppressive macrophage transition from CTShigh to C1Qhigh TAM in ascites from patients with PM-GC. This may contribute to developing potential TAM-targeted immunotherapies for PM-GC.

Triple-fusion protein (TriFu): A potent, targeted, enzyme-like inhibitor of all three complement activation pathways

The introduction of a therapeutic anti-C5 antibody into clinical practice in 2007 inspired a surge into the development of complement-targeted therapies. This has led to the recent approval of a C3 inhibitory peptide, an antibody directed against C1s and a full pipeline of several complement inhibitors in preclinical and clinical development. However, no inhibitor is available that efficiently inhibits all three complement initiation pathways and targets host cell surface markers as well as complement opsonins. To overcome this, we engineered a novel fusion protein combining selected domains of the three natural complement regulatory proteins decay accelerating factor, factor H and complement receptor 1. Such a triple fusion complement inhibitor (TriFu) was recombinantly expressed and purified alongside multiple variants and its building blocks. We analyzed these proteins for ligand binding affinity and decay acceleration activity by surface plasmon resonance. Additionally, we tested complement inhibition in several invitro/exvivo assays using standard classical and alternative pathway restricted hemolysis assays next to hemolysis assays with paroxysmal nocturnal hemoglobinuria erythrocytes. A novel invitro model of the alternative pathway disease C3 glomerulopathy was established to evaluate the potential of the inhibitors to stop C3 deposition on endothelial cells. Next to the novel engineered triple fusion variants which inactivate complement convertases in an enzyme-like fashion, stoichiometric complement inhibitors targeting C3, C5, factor B, and factor D were tested as comparators. The triple fusion approach yielded a potent complement inhibitor that efficiently inhibits all three complement initiation pathways while targeting to surface markers.

Trypanosoma brucei Invariant Surface Glycoprotein 75 Is an Immunoglobulin Fc Receptor Inhibiting Complement Activation and Antibody-Mediated Cellular Phagocytosis.

Various subspecies of the unicellular parasite Trypanosoma brucei cause sleeping sickness, a neglected tropical disease affecting millions of individuals and domestic animals. Immune evasion mechanisms play a pivotal role in parasite survival within the host and enable the parasite to establish a chronic infection. In particular, the rapid switching of variant surface glycoproteins covering a large proportion of the parasite's surface enables the parasite to avoid clearance by the adaptive immune system of the host. In this article, we present the crystal structure and discover an immune-evasive function of the extracellular region of the T. brucei invariant surface gp75 (ISG75). Structural analysis determined that the ISG75 ectodomain is organized as a globular head domain and a long slender coiled-coil domain. Subsequent ligand screening and binding analysis determined that the head domain of ISG75 confers interaction with the Fc region of all subclasses of human IgG. Importantly, the ISG75-IgG interaction strongly inhibits both activation of the classical complement pathway and Ab-dependent cellular phagocytosis by competing with C1q and host cell FcγR CD32. Our data reveal a novel immune evasion mechanism of T. brucei, with ISG75 able to inactivate the activities of Abs recognizing the parasite surface proteins.

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.

Proteomics of serum-derived extracellular vesicles are associated with the severity and different clinical profiles of patients with COVID-19: An exploratory secondary analysis

Coronavirus disease 2019 (COVID-19) is characterized by a broad spectrum of clinical manifestations with the potential to progress to multiple organ dysfunction in severe cases. Extracellular vesicles (EVs) carry a range of biological cargoes, which may be used as biomarkers of disease state. An exploratory secondary analysis of the SARITA-2 and SARITA-1 datasets (randomized clinical trials on patients with mild and moderate/severe COVID-19) was performed. Serum-derived EVs were used for proteomic analysis to identify enriched biological processes and key proteins, thus providing insights into differences in disease severity. Serum-derived EVs were separated from patients with COVID-19 by size exclusion chromatography and nanoparticle tracking analysis was used to determine particle concentration and diameter. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed to identify and quantify protein signatures. Bioinformatics and multivariate statistical analysis were applied to distinguish candidate proteins associated with disease severity (mild versus moderate/severe COVID-19). No differences were observed in terms of the concentration and diameter of enriched EVs between mild (n = 14) and moderate/severe (n = 30) COVID-19. A total of 414 proteins were found to be present in EVs, of which 360 were shared while 48 were uniquely present in severe/moderate compared to mild COVID-19. The main biological signatures in moderate/severe COVID-19 were associated with platelet degranulation, exocytosis, complement activation, immune effector activation, and humoral immune response. Von Willebrand factor, serum amyloid A-2 protein, histone H4 and H2A type 2-C, and fibrinogen β-chain were the most differentially expressed proteins between severity groups. Exploratory proteomic analysis of serum-derived EVs from patients with COVID-19 detected key proteins related to immune response and activation of coagulation and complement pathways, which are associated with disease severity. Our data suggest that EV proteins may be relevant biomarkers of disease state and prognosis.

High-dose systemic adeno-associated virus vector administration causes liver and sinusoidal endothelial cell injury

We analyzed retrospective data from toxicology studies involving administration of high doses of adeno-associated virus expressing different therapeutic transgenes to 21 cynomolgus and 15 rhesus macaques. We also conducted prospective studies to investigate acute toxicity following high-dose systemic administration of enhanced green fluorescent protein-expressing adeno-associated virus to 10 rhesus macaques. Toxicity was characterized by transaminitis, thrombocytopenia, and alternative complement pathway activation that peaked on post-administration day 3. Although most animals recovered, some developed ascites, generalized edema, hyperbilirubinemia, and/or coagulopathy that prompted unscheduled euthanasia. Study endpoint livers from animals that recovered and from unscheduled necropsies of those that succumbed to toxicity were analyzed via hypothesis-driven histopathological and unbiased single-nucleus RNA sequencing. All liver cell types expressed high transgene transcript levels at early unscheduled timepoints that subsequently decreased. Thrombocytopenia coincided with sinusoidal platelet microthrombi and sinusoidal endothelial injury identified via immunohistology and single-nucleus RNA sequencing. Acute toxicity, sinusoidal injury, and liver platelet sequestration were similarly observed with therapeutic transgenes and enhanced green fluorescent protein at doses ≥1×1014 GC/kg, suggesting it was the consequence of high-dose systemic adeno-associated virus administration, not green fluorescent protein toxicity. These findings highlight a potential toxic effect of high-dose intravenous adeno-associated virus on nonhuman primate liver microvasculature.

Anticardiolipin Antibody Plays a More Important Role Than Anti-β2-Glycoprotein I Antibody in Activating Complement in Patients with Lupus Nephritis.

This research aimed to explore the correlation between antiphospholipid antibodies (aPLs) and complement activation in lupus nephritis (LN) patients. A retrospective analysis was carried out on patients diagnosed with LN based on renal biopsy from June 2019 to June 2022. The study assessed levels of IgM, IgA, and IgG subtypes of anticardiolipin antibodies (aCLs) and anti-β2-glycoprotein I (anti-β2-GPI) antibodies. Pathological and clinical data were collected concurrently with the renal biopsy. The analysis included 76 LN patients, with 44.7% testing positive for aPLs. LN patients with positive aPLs exhibited increased hematuria, higher SLEDAI scores, reduced serum C3 and C4 levels, and more C1q deposits in the glomerulus compared to those with negative aPLs (P<0.05). Correlation analysis demonstrated the inverse relationships between IgG-aCL levels and serum C3 and C4 levels (r=-0.29, P=0.005; r=-0.24, P=0.016, respectively), as well as a positive correlation with C4 deposits in the glomerulus (r=0.20, P=0.041). This investigation suggests that aPLs, particularly IgG-aCLs, may be associated with the severity of LN and could contribute to the activation of classical complement pathways.

Complement System Deficiencies in Elite Athletes.

Although regular physical activity improves immune competency and reduces the prevalence of inflammatory diseases, strenuous training in elite athletes is associated with an increased susceptibility to infectious complications. Therefore, the objective of our study was to assess the routinely examined parameters of the complement system in elite athletes. The study was carried out in a cohort of elite athletes (n = 134) and healthy control subjects (n = 110). In all subjects, besides a routine laboratory check-up, serum concentrations of the C3 and C4 complement components, mannose-binding lectin (MBL), as well as activation of all three complement pathways were determined. Compared to healthy controls, lower C3 and C4 complement component concentrations were observed in elite athletes (0.96 ± 0.1 vs. 1.08 ± 0.2mg/L, and 0.18 ± 0.1 vs. 0.25 ± 0.1mg/L, respectively, p < 0.05); with much higher frequency rates of C3 and C4 deficiencies in athletes (31.3 vs. 14.5%, and 6 vs. 0%, p < 0.05). Simultaneously, athletes had much higher frequency rates of deficiencies of activation of classical and alternative complement pathways; while, deficiency of activation of the lectin pathway was similar in both cohorts. We confirmed a high frequency of defects in the complement system in elite athletes. Lower concentrations of C3 and C4 complement components, with high frequencies of deficiencies of the classical and alternative complement activation pathways were the most prevalent disorder of the complement system in elite athletes. Further studies are needed to uncover the functional impacts of these observations upon the susceptibility to infectious diseases.

Activation of the alternative complement pathway modulates inflammation in thoracic aortic aneurysm/dissection.

Thoracic aortic aneurysm/dissection (TAAD) is a lethal vascular disease, and several pathological factors participate in aortic medial degeneration. We previously discovered that the complement C3a-C3aR axis in smooth muscle cells promotes the development of thoracic aortic dissection (TAD) through regulation of matrix metalloproteinase 2. However, discerning the specific complement pathway that is activated and elucidating how inflammation of the aortic wall is initiated remain unknown. We ascertained that the plasma levels of C3a and C5a were significantly elevated in patients with TAD and that the levels of C3a, C4a, and C5a were higher in acute TAD than in chronic TAD. We also confirmed the activation of the complement in a TAD mouse model. Subsequently, knocking out Cfb (Cfb) or C4 in mice with TAD revealed that the alternative pathway and Cfb played a significant role in the TAD process. Activation of the alternative pathway led to generation of the anaphylatoxins C3a and C5a, and knocking out their receptors reduced the recruitment of inflammatory cells to the aortic wall. Moreover, we used serum from wild-type mice or recombinant mice Cfb as an exogenous source of Cfb to treat Cfb KO mice and observed that it exacerbated the onset and rupture of TAD. Finally, we knocked out Cfb in the FBN1C1041G/+ Marfan-syndrome mice and showed that the occurrence of TAA was reduced. In summary, the alternative complement pathway promoted the development of TAAD by recruiting infiltrating inflammatory cells. Targeting the alternative pathway may thus constitute a strategy for preventing the development of TAAD.NEW & NOTEWORTHY The alternative complement pathway promoted the development of TAAD by recruiting infiltrating inflammatory cells. Targeting the alternative pathway may thus constitute a strategy for preventing the development of TAAD.