Combined Heterozygous Genetic Variations in Complement C2 and C8B: An Explanation for Multidimensional Immune Imbalance?

The complement system plays a crucial role in host defense, homeostasis, and tissue regeneration and bridges the innate and the adaptive immune systems. Although the genetic variants in complement C2 (c.839_849+17del; p.(Met280Asnfs*5)) and C8B (c.1625C>T; p.(Thr542Ile)) are known individually, here, we report on a patient carrying their combination in a heterozygous form. The patient presented with a reduced general condition and suffers from a wide variety of autoimmune diseases. While no autoimmune disease-specific autoantibodies could be detected, genetic analysis revealed abnormalities in the two complement genes C2 and C8B. Therefore, we performed a comprehensive investigation of the innate immune system on a cellular and humoral level to define the functional consequences. We found slightly impaired functionality of neutrophils and monocytes regarding phagocytosis and reactive oxygen species generation and a diminished expression of the C5aR1. An extensive complement analysis revealed a declined activation potential for the alternative and classical pathway. Reconstitution with purified C2 and C8 into patient serum failed to normalize the dysfunction, whereas the addition of C3 improved the hemolytic activity. In clinical transfer, in vitro supplementation of the patient's plasma with FFP as a complement source could fully restore full complement functionality. This study describes for the first time a combined heterozygous genetic variation in complement C2 and C8B which, however, cannot fully explain the overall dysfunctions and calls for further complement deficiency research and corresponding therapies.

Complement C3 inhibition in severe COVID-19 using compstatin AMY-101.

Complement C3 activation contributes to COVID-19 pathology, and C3 targeting has emerged as a promising therapeutic strategy. We provide interim data from ITHACA, the first randomized trial evaluating a C3 inhibitor, AMY-101, in severe COVID-19 (PaO2/FiO2 ≤ 300 mmHg). Patients received AMY-101 (n = 16) or placebo (n = 15) in addition to standard of care. AMY-101 was safe and well tolerated. Compared to placebo (8 of 15, 53.3%), a higher, albeit nonsignificant, proportion of AMY-101-treated patients (13 of 16, 81.3%) were free of supplemental oxygen at day 14. Three nonresponders and two placebo-treated patients succumbed to disease-related complications. AMY-101 significantly reduced CRP and ferritin and restrained thrombin and NET generation. Complete and sustained C3 inhibition was observed in all responders. Residual C3 activity in the three nonresponders suggested the presence of a convertase-independent C3 activation pathway overriding the drug's inhibitory activity. These findings support the design of larger trials exploring the potential of C3-based inhibition in COVID-19 or other complement-mediated diseases.

Variation of Complement Protein Levels in Maternal Plasma and Umbilical Cord Blood during Normal Pregnancy: An Observational Study.

The complement system constitutes a crucial part of the innate immunity, mediating opsonization, lysis, inflammation, and elimination of potential pathogens. In general, there is an increased activity of the complement system during pregnancy, which is essential for maintaining the host's defense and fetal survival. Unbalanced or excessive activation of the complement system in the placenta is associated with pregnancy complications, such as miscarriage, preeclampsia, and premature birth. Nonetheless, the actual clinical value of monitoring the activation of the complement system during pregnancy remains to be investigated. Unfortunately, normal reference values specifically for pregnant women are missing, and for umbilical cord blood (UCB), data on complement protein levels are scarce. Herein, complement protein analyses (C1q, C3, C4, C3d levels, and C3d/C3 ratio) were performed in plasma samples from 100 healthy, non-medicated and non-smoking pregnant women, collected during different trimesters and at the time of delivery. In addition, UCB was collected at all deliveries. Maternal plasma C1q and C3d/C3 ratio showed the highest mean values during the first trimester, whereas C3, C4, and C3d had rising values until delivery. We observed low levels of C1q and C4 as well as increased C3d and C3d/C3 ratio, particularly during the first trimester, as a sign of complement activation in some women. However, the reference limits of complement analyses applied for the general population appeared appropriate for the majority of the samples. As expected, the mean complement concentrations in UCB were much lower than in maternal plasma, due to the immature complement system in neonates.

Angiopoietin-2 Inhibition of Thrombomodulin-Mediated Anticoagulation-A Novel Mechanism That May Contribute to Hypercoagulation in Critically Ill COVID-19 Patients.

Hypercoagulation and endothelial dysfunction play central roles in severe forms of COVID-19 infections, but the molecular mechanisms involved are unclear. Increased plasma levels of the inflammatory cytokine and TIE2 receptor antagonist Angiopoietin-2 were reported in severely ill COVID-19 patients. In vitro experiments suggest that Angiopoietin-2 bind and inhibits thrombomodulin. Thrombomodulin is expressed on the luminal surface of endothelial cells where it is an important member of the intrinsic anticoagulant pathway through activation of protein C. Using clinical data, mouse models, and in vitro assays, we tested if Angiopoietin-2 plays a causal role in COVID-19-associated hypercoagulation through direct inhibition of thrombin/thrombomodulin-mediated physiological anticoagulation. Angiopoietin-2 was measured in 61 patients at admission, and after 10 days in the 40 patients remaining in the ICU. We found that Angiopoietin-2 levels were increased in COVID-19 patients in correlation with disease severity, hypercoagulation, and mortality. In support of a direct effect of Angiopoietin-2 on coagulation, we found that injected Angiopoietin-2 in mice associated to thrombomodulin and resulted in a shortened tail bleeding time, decreased circulating levels of activated protein C, and increased plasma thrombin/antithrombin complexes. Conversely, bleeding time was increased in endothelial-specific Angiopoietin-2 knockout mice, while knockout of Tie2 had no effect on tail bleeding. Using in vitro assays, we found that Angiopoietin-2 inhibited thrombomodulin-mediated anticoagulation and protein C activation in human donor plasma. Our data suggest a novel in vivo mechanism for Angiopoietin-2 in COVID-19-associated hypercoagulation, implicating that Angiopoietin-2 inhibitors may be effective in the treatment of hypercoagulation in severe COVID-19 infection.

Impaired Antibody Response Is Associated with Histone-Release, Organ Dysfunction and Mortality in Critically Ill COVID-19 Patients.

PURPOSE: the pathophysiologic mechanisms explaining differences in clinical outcomes following COVID-19 are not completely described. This study aims to investigate antibody responses in critically ill patients with COVID-19 in relation to inflammation, organ failure and 30-day survival. METHODS: All patients with PCR-verified COVID-19 and gave consent, and who were admitted to a tertiary Intensive care unit (ICU) in Sweden during March-September 2020 were included. Demography, repeated blood samples and measures of organ function were collected. Analyses of anti-SARS-CoV-2 antibodies (IgM, IgA and IgG) in plasma were performed and correlated to patient outcome and biomarkers of inflammation and organ failure. RESULTS: A total of 115 patients (median age 62 years, 77% male) were included prospectively. All patients developed severe respiratory dysfunction, and 59% were treated with invasive ventilation. Thirty-day mortality was 22.6% for all included patients. Patients negative for any anti-SARS-CoV-2 antibody in plasma during ICU admission had higher 30-day mortality compared to patients positive for antibodies. Patients positive for IgM had more ICU-, ventilator-, renal replacement therapy- and vasoactive medication-free days. IgA antibody concentrations correlated negatively with both SAPS3 and maximal SOFA-score and IgM-levels correlated negatively with SAPS3. Patients with antibody levels below the detection limit had higher plasma levels of extracellular histones on day 1 and elevated levels of kidney and cardiac biomarkers, but showed no signs of increased inflammation, complement activation or cytokine release. After adjusting for age, positive IgM and IgG antibodies were still associated with increased 30-day survival, with odds ratio (OR) 7.1 (1.5-34.4) and 4.2 (1.1-15.7), respectively. CONCLUSION: In patients with severe COVID-19 requiring intensive care, a poor antibody response is associated with organ failure, systemic histone release and increased 30-day mortality.

How the Innate Immune System of the Blood Contributes to Systemic Pathology in COVID-19-Induced ARDS and Provides Potential Targets for Treatment.

Most SARS-CoV-2 infected patients experience influenza-like symptoms of low or moderate severity. But, already in 2020 early during the pandemic it became obvious that many patients had a high incidence of thrombotic complications, which prompted treatment with high doses of low-molecular-weight heparin (LMWH; typically 150-300IU/kg) to prevent thrombosis. In some patients, the disease aggravated after approximately 10 days and turned into a full-blown acute respiratory distress syndrome (ARDS)-like pulmonary inflammation with endothelialitis, thrombosis and vascular angiogenesis, which often lead to intensive care treatment with ventilator support. This stage of the disease is characterized by dysregulation of cytokines and chemokines, in particular with high IL-6 levels, and also by reduced oxygen saturation, high risk of thrombosis, and signs of severe pulmonary damage with ground glass opacities. The direct link between SARS-CoV-2 and the COVID-19-associated lung injury is not clear. Indirect evidence speaks in favor of a thromboinflammatory reaction, which may be initiated by the virus itself and by infected damaged and/or apoptotic cells. We and others have demonstrated that life-threatening COVID-19 ARDS is associated with a strong activation of the intravascular innate immune system (IIIS). In support of this notion is that activation of the complement and kallikrein/kinin (KK) systems predict survival, the necessity for usage of mechanical ventilation, acute kidney injury and, in the case of MBL, also coagulation system activation with thromboembolism. The general properties of the IIIS can easily be translated into mechanisms of COVID-19 pathophysiology. The prognostic value of complement and KKsystem biomarkers demonstrate that pharmaceuticals, which are licensed or have passed the phase I trial stage are promising candidate drugs for treatment of COVID-19. Examples of such compounds include complement inhibitors AMY-101 and eculizumab (targeting C3 and C5, respectively) as well as kallikrein inhibitors ecallantide and lanadelumab and the bradykinin receptor (BKR) 2 antagonist icatibant. In this conceptual review we discuss the activation, crosstalk and the therapeutic options that are available for regulation of the IIIS.

Comorbidities in hereditary angioedema-A population-based cohort study.

BACKGROUND: In hereditary angioedema (HAE), low levels (type 1) or defect in function (type 2) of the serine-protease inhibitor C1 Inhibitor protein results in activation of the classical pathway of the complement system as well as the contact system. Here, we investigated the risk of comorbidities in HAE. METHODS: Individuals with HAE (n = 239; identified through a physician made diagnosis) and a control cohort from the general population (n = 2383; matched for age, gender, and county of residence) were compared with the Swedish inpatient, cause of death, cancer, and prescription registers. Conditional logistic regression was used to analyze the data. RESULTS: Increased risk of cardiovascular disease (odds ratio [OR] 1.83; 95% confidence interval [CI] 1.32-2.54), including arterial (OR 6.74; 95% CI 1.89-24.06) and venous thromboembolic disease (OR 4.20; 95% CI 2.42-7.23) as well as hypertension (OR 1.64; 95% CI 1.12-2.39) was seen in HAE. There was also an increased number of individuals diagnosed with hyperlipidemia (OR 2.01; 95% CI 1.16-3.50) among HAE patients. Furthermore, the risk of autoimmune disease was increased (OR 1.65; 95% CI 1.15-2.35) being particularly pronounced for systemic lupus erythematosus (OR 71.87; 95% CI 8.80-586.7). The risk of having two or more autoimmune diseases was also higher among HAE patients (p = 0.017). In contrast, the risk of cancer was not increased. Data from the prescription register revealed higher prescription rates of drugs against hypertension, hypothyroidism, and hyperlipidemia among HAE patients. CONCLUSIONS: The results warrant for awareness and prevention of comorbid conditions, in particular, thromboembolic and autoimmune diseases in HAE. Future prophylactic interventions may modify these risks.

How COVID-19 and other pathological conditions and medical treatments activate our intravascular innate immune system.

COVID-19 has been shown to have a multifaceted impact on the immune system. In a recently published article in Front Immunol, we show that the intravascular innate immune system (IIIS) is strongly activated in severe COVID-19 with ARDS and appears to be one of the causes leading to severe COVID-19. In this article, we describe the IIIS and its physiological function, but also the strong pro-inflammatory effects that are observed in COVID-19 and in various other pathological conditions and treatments such as during ischemia reperfusion injury and in treatments where biomaterials come in direct contact with blood in, e.g., extracorporeal and intravasal treatments. In the present article, we describe how the IIIS, a complex network of plasma proteins and blood cells, constitute the acute innate immune response of the blood and discuss the effects that the IIIS induces in pathological disorders and treatments in modern medicine.

Quantification of Complement Proteins with Special Reference to C1q: Multiplex Versus ELISA Versus Rocket Immunoelectrophoresis Versus Nephelometry.

Accurate determination of complement component C1q is hampered by the fact that C1q is an immune complex binding protein. Consequently, immunochemical techniques which rely on immune complex formation in fluid phase such as nephelometry and turbidimetry tend to give results which differ from those obtained by, for example, ELISA and other solid phase-based assays. In this chapter, we discuss the pros and cons of different techniques for the quantification of C1q and present a comprehensive protocol for a newly developed magnetic bead-based sandwich immunoassay which has replaced nephelometry in our complement diagnostic laboratory at the University Hospital in Uppsala.

The Outcome of Critically Ill COVID-19 Patients Is Linked to Thromboinflammation Dominated by the Kallikrein/Kinin System.

An important manifestation of severe COVID-19 is the ARDS-like lung injury that is associated with vascular endothelialitis, thrombosis, and angiogenesis. The intravascular innate immune system (IIIS), including the complement, contact, coagulation, and fibrinolysis systems, which is crucial for recognizing and eliminating microorganisms and debris in the body, is likely to be involved in the pathogenesis of COVID-19 ARDS. Biomarkers for IIIS activation were studied in the first 66 patients with COVID-19 admitted to the ICU in Uppsala University Hospital, both cross-sectionally on day 1 and in 19 patients longitudinally for up to a month, in a prospective study. IIIS analyses were compared with biochemical parameters and clinical outcome and survival. Blood cascade systems activation leading to an overreactive conjunct thromboinflammation was demonstrated, reflected in consumption of individual cascade system components, e.g., FXII, prekallikrein, and high molecular weight kininogen and in increased levels of activation products, e.g., C4d, C3a, C3d,g, sC5b-9, TAT, and D-dimer. Strong associations were found between the blood cascade systems and organ damage, illness severity scores, and survival. We show that critically ill COVID-19 patients display a conjunct activation of the IIIS that is linked to organ damage of the lung, heart, kidneys, and death. We present evidence that the complement and in particular the kallikrein/kinin system is strongly activated and that both systems are prognostic markers of the outcome of the patients suggesting their role in driving the inflammation. Already licensed kallikrein/kinin inhibitors are potential drugs for treatment of critically ill patients with COVID-19.

Mannose-Binding Lectin is Associated with Thrombosis and Coagulopathy in Critically Ill COVID-19 Patients.

The ongoing COVID-19 pandemic has caused significant morbidity and mortality worldwide, as well as profound effects on society. COVID-19 patients have an increased risk of thromboembolic (TE) complications, which develop despite pharmacological thromboprophylaxis. The mechanism behind COVID-19-associated coagulopathy remains unclear. Mannose-binding lectin (MBL), a pattern recognition molecule that initiates the lectin pathway of complement activation, has been suggested as a potential amplifier of blood coagulation during thromboinflammation. Here we describe data from a cohort of critically ill COVID-19 patients (n = 65) treated at a tertiary hospital center intensive care unit (ICU). A subset of patients had strongly elevated MBL plasma levels, and activity upon ICU admission, and patients who developed symptomatic TE (14%) had significantly higher MBL levels than patients without TE. MBL was strongly correlated to plasma D-dimer levels, a marker of COVID-19 coagulopathy, but showed no relationship to degree of inflammation or other organ dysfunction. In conclusion, we have identified complement activation through the MBL pathway as a novel amplification mechanism that contributes to pathological thrombosis in critically ill COVID-19 patients. Pharmacological targeting of the MBL pathway could be a novel treatment option for thrombosis in COVID-19. Laboratory testing of MBL levels could be of value for identifying COVID-19 patients at risk for TE events.

Current and Future Approaches for Monitoring Responses to Anti-complement Therapeutics.

Aberrations in complement system functions have been identified as either direct or indirect pathophysiological mechanisms in many diseases and pathological conditions, such as infections, autoimmune diseases, inflammation, malignancies, and allogeneic transplantation. Currently available techniques to study complement include quantification of (a) individual complement components, (b) complement activation products, and (c) molecular mechanisms/function. An emerging area of major interest in translational studies aims to study and monitor patients on complement regulatory drugs for efficacy as well as adverse events. This area is progressing rapidly with several anti-complement therapeutics under development, in clinical trials, or already in clinical use. In this review, we summarized the appropriate indications, techniques, and interpretations of basic complement analyses, exemplified by a number of clinical disorders.

Evaluation of a Novel Immunoassay for Quantification of C1q for Clinical Diagnostic Use.

Objectives: C1q is a valuable biomarker of disease activity in systemic lupus erythematosus (SLE). The "gold standard" assay, rocket immunoelectrophoresis (RIE), is time-consuming, and thus a shift to soluble immune precipitation techniques such as nephelometry has occurred. However, quantification of C1q with these techniques has been questioned as a result of the antibody binding properties of C1q. In the present work, we have compared results using various techniques (RIE, nephelometry, and ELISA) and have developed and validated a new magnetic bead-based sandwich immunoassay (MBSI). Methods: C1q was quantified by nephelometry and the new sandwich immunoassay in 45 serum samples analyzed using RIE. C1q was also assessed in plasma using RIE and sandwich immunoassay in samples from SLE patients with nephritis (n = 69), SLE patients without nephritis (n = 310) as classified by BILAG score, and matched controls (n = 322). In addition, cerebrospinal fluid (CSF) samples from 31 patients, previously analyzed with ELISA, were also analyzed with the MBSI to test the behavior of this new assay in the lower detection range. Results: We found a strong correlation between the new MBSI, RIE, and ELISA, but not with nephelometry. The MBSI demonstrated lower levels of C1q in SLE patients than in matched controls (p < 0.0001), and patients with nephritis had lower levels than patients without nephritis (p < 0.01). Similarily, RIE showed significant differences between the patient groups (p < 0.0001). An association was also found between the levels of C1q and the SLE disease activity index (SLEDAI). Furthermore, there was good correlation between the values obtained by MBSI and ELISA, in both serum (r = 0.960) and CSF (r = 0.786), underscoring the ability of both techniques to measure low concentrations of C1q with high accuracy. Conclusion: The sandwich immunoassay correlated well with RIE, but soluble immune precipitation techniques, such as nephelometry, did not appear suitable alternatives, since C1q itself, and possibly anti-C1q antibodies, interfered with the measurements. The new sandwich immunoassay is therefore a good replacement for RIE in monitoring SLE disease activity.

Interpretation of Serological Complement Biomarkers in Disease.

Complement system aberrations have been identified as pathophysiological mechanisms in a number of diseases and pathological conditions either directly or indirectly. Examples of such conditions include infections, inflammation, autoimmune disease, as well as allogeneic and xenogenic transplantation. Both prospective and retrospective studies have demonstrated significant complement-related differences between patient groups and controls. However, due to the low degree of specificity and sensitivity of some of the assays used, it is not always possible to make predictions regarding the complement status of individual patients. Today, there are three main indications for determination of a patient's complement status: (1) complement deficiencies (acquired or inherited); (2) disorders with aberrant complement activation; and (3) C1 inhibitor deficiencies (acquired or inherited). An additional indication is to monitor patients on complement-regulating drugs, an indication which may be expected to increase in the near future since there is now a number of such drugs either under development, already in clinical trials or in clinical use. Available techniques to study complement include quantification of: (1) individual components; (2) activation products, (3) function, and (4) autoantibodies to complement proteins. In this review, we summarize the appropriate indications, techniques, and interpretations of basic serological complement analyses, exemplified by a number of clinical disorders.