Complement Anaphylatoxins and Inflammatory Cytokines as Prognostic Markers for COVID-19 Severity and In-Hospital Mortality

Authors: Bandar Alosaimi1,2Ayman Mubarak3, Maaweya E. Hamed3Abdullah Z. Almutairi4Ahmed A. Alrashed5, Abdullah AlJuryyan6, Mushira Enani7,Faris Q. Alenzi8 and Wael Alturaiki9*

COVID-19 severity due to innate immunity dysregulation accounts for prolonged hospitalization, critical complications, and mortality. Severe SARS-CoV-2 infections involve the complement pathway activation for cytokine storm development. Nevertheless, the role of complement in COVID-19 immunopathology, complement‐modulating treatment strategies against COVID-19, and the complement and SARS‐CoV‐2 interaction with clinical disease outcomes remain elusive. This study investigated the potential changes in complement signaling, and the associated inflammatory mediators, in mild-to-critical COVID-19 patients and their clinical outcomes. A total of 53 patients infected with SARS-CoV-2 were enrolled in the study (26 critical and 27 mild cases), and additional 18 healthy control patients were also included. Complement proteins and inflammatory cytokines and chemokines were measured in the sera of patients with COVID-19 as well as healthy controls by specific enzyme-linked immunosorbent assay. C3a, C5a, and factor P (properdin), as well as interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, and IgM antibody levels, were higher in critical COVID-19 patients compared to mild COVID-19 patients. Additionally, compared to the mild COVID-19 patients, factor I and C4-BP levels were significantly decreased in the critical COVID-19 patients. Meanwhile, RANTES levels were significantly higher in the mild patients compared to critical patients. Furthermore, the critical COVID-19 intra-group analysis showed significantly higher C5a, C3a, and factor P levels in the critical COVID-19 non-survival group than in the survival group. Additionally, IL-1β, IL-6, and IL-8 were significantly upregulated in the critical COVID-19 non-survival group compared to the survival group. Finally, C5a, C3a, factor P, and serum IL-1β, IL-6, and IL-8 levels positively correlated with critical COVID-19 in-hospital deaths. These findings highlight the potential prognostic utility of the complement system for predicting COVID-19 severity and mortality while suggesting that complement anaphylatoxins and inflammatory cytokines are potential treatment targets against COVID-19.

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The complement system in COVID-19: friend and foe?

Authors: Anuja Java,1 Anthony J. Apicelli,2 M. Kathryn Liszewski,3 Ariella Coler-Reilly,3 John P. Atkinson,3 Alfred H.J. Kim,3 and Hrishikesh S. Kulkarni4

Coronavirus disease 2019 (COVID-19), the disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in a global pandemic and a disruptive health crisis. COVID-19–related morbidity and mortality have been attributed to an exaggerated immune response. The role of complement activation and its contribution to illness severity is being increasingly recognized. Here, we summarize current knowledge about the interaction of coronaviruses with the complement system. We posit that (a) coronaviruses activate multiple complement pathways; (b) severe COVID-19 clinical features often resemble complementopathies; (c) the combined effects of complement activation, dysregulated neutrophilia, endothelial injury, and hypercoagulability appear to be intertwined to drive the severe features of COVID-19; (d) a subset of patients with COVID-19 may have a genetic predisposition associated with complement dysregulation; and (e) these observations create a basis for clinical trials of complement inhibitors in life-threatening illness.

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Direct activation of the alternative complement pathway by SARS-CoV-2 spike proteins is blocked by factor D inhibition

Authors: Jia YuXuan YuanHang ChenShruti ChaturvediEvan M. BraunsteinRobert A. Brodsky

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious respiratory virus that can lead to venous/arterial thrombosis, stroke, renal failure, myocardial infarction, thrombocytopenia, and other end-organ damage. Animal models demonstrating end-organ protection in C3-deficient mice and evidence of complement activation in humans have led to the hypothesis that SARS-CoV-2 triggers complement-mediated endothelial damage, but the mechanism is unclear. Here, we demonstrate that the SARS-CoV-2 spike protein (subunit 1 and 2), but not the N protein, directly activates the alternative pathway of complement (APC). Complement-dependent killing using the modified Ham test is blocked by either C5 or factor D inhibition. C3 fragments and C5b-9 are deposited on TF1PIGAnull target cells, and complement factor Bb is increased in the supernatant from spike protein–treated cells. C5 inhibition prevents the accumulation of C5b-9 on cells, but not C3c; however, factor D inhibition prevents both C3c and C5b-9 accumulation. Addition of factor H mitigates the complement attack. In conclusion, SARS-CoV-2 spike proteins convert nonactivator surfaces to activator surfaces by preventing the inactivation of the cell-surface APC convertase. APC activation may explain many of the clinical manifestations (microangiopathy, thrombocytopenia, renal injury, and thrombophilia) of COVID-19 that are also observed in other complement-driven diseases such as atypical hemolytic uremic syndrome and catastrophic antiphospholipid antibody syndrome. C5 inhibition prevents accumulation of C5b-9 in vitro but does not prevent upstream complement activation in response to SARS-CoV-2 spike proteins.

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Increased complement activation is a distinctive feature of severe SARS-CoV-2 infection

Authors: Lina Ma1View ORCID ProfileSanjaya K. Sahu1View ORCID ProfileMarlene Cano1, Vasanthan Kuppuswamy2, Jamal Bajwa1,3View ORCID ProfileJa’Nia McPhatter1,4

Complement activation has been implicated in the pathogenesis of severe SARS-CoV-2 infection. However, it remains to be determined whether increased complement activation is a broad indicator of critical illness (and thus, no different in COVID-19). It is also unclear which pathways are contributing to complement activation in COVID-19 and whether it is associated with certain features of COVID-19, such as endothelial injury and hypercoagulability. Hence, we investigated complement activation in the plasma from patients with COVID-19 prospectively enrolled at two tertiary care centers: Washington University School of Medicine (n = 134) and Yale School of Medicine (n = 49). We compared our patients with two non-COVID cohorts: (i) patients hospitalized with influenza (n = 54) and (ii) patients admitted to the intensive care unit (ICU) with acute respiratory failure requiring invasive mechanical ventilation (IMV; n = 22). We demonstrate that circulating markers of complement activation are elevated in patients with COVID-19 compared with those with influenza and to patients with non–COVID-19 respiratory failure. Furthermore, the results facilitate distinguishing those who are at higher risk of worse outcomes such as requiring ICU admission or IMV. Moreover, the results indicate that enhanced activation of the alternative pathway is most prevalent in patients with severe COVID-19 and is associated with markers of endothelial injury (i.e., angiopoietin-2) and hypercoagulability (i.e., thrombomodulin and von Willebrand factor). Our findings identify complement activation to be a distinctive feature of COVID-19 and provide specific targets that may be used for risk prognostication, drug discovery, and personalized clinical trials.

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Sars cov 2 drives jak1 2 dependent local complement hyperactivation


Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys and gut. Angiotensin converting enzyme (ACE) 2, the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-B as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines, but did not affect NF-B-regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-B-signaling could potentially have clinical application for severe COVID-19.

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Complement control for COVID-19

Authors: Markus Bosmann1,2,3,4,*

The complement system is an integral part of innate immune defense. It consists of about 50 proteins in plasma, on cell surfaces, and inside host cells. The traditional view is that complement proteins guard the local extracellular spaces and systemic bloodstream against invading pathogens. Loss-of-function mutations resulting in terminal complement pathway deficiencies are associated with a 10,000-fold higher risk for life-threatening meningococcal infections in humans. Surprisingly, the complement system is redundant for defense against most pathogens except encapsulated bacteria. Recent concepts embrace the view that complement factors mediate functions inside cells either directly or through surface receptors. Complement activity fine-tunes homeostasis, metabolism, and biogenesis. On the other hand, uncontrolled complement activation causes disease and can even worsen the outcome of infections. Toxic complement effectors mediate tissue destruction and organ injury during inflammatory diseases. Acute respiratory distress syndrome (ARDS) and sepsis are frequent and severe complications of acute infections and notorious for excessive complement consumption. The three pathways of complement activation are designed for immune sensing of nonself surfaces and foreign antigens. The mannose-binding lectin (MBL)/ficolin pathway starts with soluble pathogen pattern recognition receptors as sensors for foreign carbohydrate motifs (Fig. 1). The alternative pathway is fueled by a spontaneous “smoldering” hydrolysis of C3 targeting all surfaces, unless these surfaces present complement inhibitory proteins (CD46, CD55, and CD59) as a protective self-signal. This C3 “tick-over” is sustained by the high concentrations of C3 in plasma (1 to 2 g/liter), the highest level of all complement factors. The classical pathway is initiated by antigen-antibody complexes that are recognized by the multimeric C1 complex. As a safeguard, IgG antibodies bound in clusters or pentameric IgM are required to surpass the activation threshold. All complement pathways converge on C3 convertase complexes leading to C3 cleavage into the larger C3b and the smaller anaphylactic C3a peptides. C3b is essential for the formation of C5 convertase for cleavage of C5 into C5b and the anaphylatoxin C5a. C5b is the starting point of the pore-forming membrane attack complex (MAC) consisting of C5b-C9 with a channel diameter of ~100 Å. The C3/C5 hub represents a gigantic amplification loop. The alternative C3bBb convertase (half-life of ~3 min) cleaves additional C3, resulting in more C3bBb and so on and so forth. This enzymatic chain reaction can deposit millions of C3b molecules on target surfaces in a few seconds. It is no surprise that such explosive events need to be tightly regulated to maintain the delicate balance of effective and justified pathogen attack, while avoiding damage of innocent bystander cells.

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COVID-19 cytokine storm: The anger of inflammation

Authors: Mehdi Mahmudpour 1Jamshid Roozbeh 2Mohsen Keshavarz 1Shokrollah Farrokhi 3Iraj Nabipour 4

Patients with COVID-19 who require ICU admission might have the cytokine storm. It is a state of out-of-control release of a variety of inflammatory cytokines. The molecular mechanism of the cytokine storm has not been explored extensively yet. The attachment of SARS-CoV-2 spike glycoprotein with angiotensin-converting enzyme 2 (ACE2), as its cellular receptor, triggers complex molecular events that leads to hyperinflammation. Four molecular axes that may be involved in SARS-CoV-2 driven inflammatory cytokine overproduction are addressed in this work. The virus-mediated down-regulation of ACE2 causes a burst of inflammatory cytokine release through dysregulation of the renin-angiotensin-aldosterone system (ACE/angiotensin II/AT1R axis), attenuation of Mas receptor (ACE2/MasR axis), increased activation of [des-Arg9]-bradykinin (ACE2/bradykinin B1R/DABK axis), and activation of the complement system including C5a and C5b-9 components. The molecular clarification of these axes will elucidate an array of therapeutic strategies to confront the cytokine storm in order to prevent and treat COVID-19 associated acute respiratory distress syndrome.

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