key: cord-0924403-6gne2yq2
authors: Boussier, Jeremy; Yatim, Nader; Marchal, Armance; Hadjadj, Jérôme; Charbit, Bruno; El Sissy, Carine; Carlier, Nicolas; Pène, Frédéric; Mouthon, Luc; Tharaux, Pierre-Louis; Bergeron, Anne; Smadja, David M.; Rieux-Laucat, Frédéric; Duffy, Darragh; Kernéis, Solen; Frémeaux-Bacchi, Véronique; Terrier, Benjamin
title: Severe COVID-19 is associated with hyperactivation of the alternative complement pathway
date: 2021-11-17
journal: J Allergy Clin Immunol
DOI: 10.1016/j.jaci.2021.11.004
sha: c292699db0bd84ce09b2b0c15ebf0a5b3bf9f06e
doc_id: 924403
cord_uid: 6gne2yq2

Background Severe coronavirus disease 2019 (COVID-19) is characterized by impaired type I interferon activity and a state of hyperinflammation leading to acute respiratory distress syndrome. The complement system has recently emerged as a key player in triggering and maintaining the inflammatory state, but the role of this molecular cascade in severe COVID-19 is still poorly characterized. Objective We aimed at assessing the contribution of complement pathways at both protein and transcriptomic levels. Methods To this end, we systematically assessed RNA levels of 28 complement genes in circulating whole blood of COVID-19 patients and healthy controls, including genes of the alternative pathway, for which data remain scarce. Results We found differential expression of genes involved in the complement system, yet with various expression patterns: while patients displaying moderate disease had elevated expression of classical pathway genes, severe disease was associated with increased lectin and alternative pathway activation, which correlated with inflammation and coagulopathy markers. Additionally, properdin, a pivotal positive regulator of the alternative pathway, showed high RNA expression but was found at low protein concentrations in severe and critical patients, suggesting its deposition at the sites of complement activation. Notably, low properdin levels were significantly associated with the use of mechanical ventilation (AUC = 0.82, p = 0.002). Conclusion This study sheds light on the role of the alternative pathway in severe COVID-19 and provides additional rationale for the testing of drugs inhibiting the alternative pathway of the complement system.

and alternative pathways is associated with disease severity. 64  Properdin RNA expression is increased in severe patients, yet its protein levels are decreased, 65 suggesting its deposition on activating surfaces. 66  Low properdin levels are associated with use of mechanical ventilation. 67 68 CAPSULE SUMMARY 69 We show that activation of the alternative complement pathway characterizes COVID-19 severity. 70 Specifically, low properdin levels were associated with use of mechanical ventilation. This work 71 provides a rationale for the specific inhibition of the alternative complement pathway. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus 82 disease 2019 (COVID-19), has to date caused over 4 million deaths worldwide (1). While the majority 83 of patients remain asymptomatic or show mild-to-moderate symptoms, approximately 5% of patients 84 display severe disease, characterized by acute respiratory distress syndrome (ARDS), which can result 85 in multi-organ failure and death (2,3). We previously demonstrated that severe and critical patients 86 displayed an imbalanced immune response with impaired type I interferon (IFN) activity coupled to 87 excessive inflammation (4). Glucocorticoids have shown to reduce COVID-19 mortality, yet 88 complementary therapies could more specifically target certain members of the immune response 89 (5). In this context, the complement system has emerged as an attractive candidate. 90 The complement system is a key player of innate immunity at the interface with the adaptive 91 immune system (6). Activation of the complement cascade leads to the cleavage of C3 and the 92 deposition of C3b on activating surfaces, triggering phagocytosis or cleavage of C5 into C5a and C5b, 93 and subsequent formation of the membrane attack complex C5b9 (MAC), resulting in the perturbation 94 of the cell membrane. Additionally, C3a and C5a are anaphylatoxins able to recruit and activate 95 leukocytes, thereby bridging the gap between innate and adaptive immunity and promoting 96 inflammation. The complement cascade can be activated through three different pathways, all 97 converging to the cleavage of C3: (i) the classical pathway detects bound antibodies or other acute 98 phase proteins via C1q; (ii) the lectin pathway recognizes carbohydrate structures in pathogens and 99 damaged membranes; (iii) the alternative pathway is in a constant state of activation unless 100 complement inhibitory proteins are presented, a process known as "tick over", and can amplify C3b 101 formation. 102 Complement activation has been associated with disease severity in bacterial and viral pneumonia, 103 ARDS and multiorgan failure (7). As for SARS-CoV-2, the complement system was one of the most 104 highly induced intracellular pathway in infected lung epithelial cells, driven by the transcription of 6 disease severity (14). Additionally, anti-C5aR1 antibodies inhibited lung injury in human C5aR1 knock-110 in mice, indicating that targeting complement could reduce disease severity (10). 111 What links the complement pathway to COVID-19 severity is still poorly understood, but one 112 hypothesis lies in its association with coagulopathy (15). Severe COVID-19 has been shown to trigger 121 We previously analyzed whole-blood transcriptomic data from 32 COVID-19 patients with various 122 disease severity and 13 healthy controls (4). The main characteristics of these patients are described 123 in Table I . To uncover the role of complement in disease severity, we determined the RNA levels of 124 28 complement genes with expression above the lower limit of quantification ( Figure E1 ). Of those, 125 19 were differentially expressed depending on disease stage (Figure 1a) . Hierarchical clustering 126 identified two main gene groups (showing high intra-group correlation and including 17 of the 19 127 genes) displaying distinct patterns of expression: group 1 contained genes whose expressions peaked 128 in moderate disease, whereas group 2 genes showed increased expression in severe and to a greater 129 extent in critical patients, while patients with moderate disease had expression levels that were 130 comparable to that of healthy controls (Figure 1b) . Group 1 included genes belonging to the classical 131 pathway (C1QA and C1QB) and both classical and lectin pathway (C2 and SERPING, coding for C1 132 inhibitor), as well as the terminal phase (C5) (Figure 1c) . In contrast, group 2 contained genes 133 belonging to the lectin (MBL2, MASP2 and C4, the latter also belonging to the classical pathway) and 134 the alternative pathways (C3, its stabilizer CFP, coding for properdin, C3 receptors ITGAM and ITGAX, 135 and C3 regulators CR1, CD46, CD55, CD59) (Figure 1c) . 136 We next studied the correlation between complement gene expression and circulating CRP 137 and IL-6 proteins on the one hand, and PPBP (encoding for platelet chemokine CXCL7) and SELPLG 138 (encoding for PSGL-1) gene expression on the other hand, two markers of coagulopathy that we 139 previously described as predictive of intubation and death (19) (Figure 2a Interval from first symptoms to admission (days) -10 (9-11) 9 (9-11) 10.5 (10-12) 9 (8-11) 0.06 Overweight 0 (0) 2 (6) 1 (9) 1 (10) 0 (0) 0.5 Normalized RNA counts q = 2 × 10 -4 q = 6 × 10 -5 q = 5 × 10 -5 q = 9 × 10 -4 q = 5 × 10 -5 q = 3 × 10 -4 q = 7 × 10 -4 q = 4 × 10 -3 q = 6 × 10 -5 q = 5 × 10 -5 p = 7 × 10 -5 p = 1 × 10 -5 p = 8 × 10 -6 p = 4 × 10 -4 p = 9 × 10 -6 p = 1 × 10 -4 p = 3 × 10 -4 MYD88  SOCS1  CEACAM1  TNFSF13B  PSMB9  STAT1  IFIT2  CCR1  GBP1  PML  TNFSF10  IFITM1  IFI16  MX1  IFI35  TAP1  BST2  IRF7  CCRL2  IFIH1 Correlation a b 0.5 0.6 0.7 0.8 0.9 MME RAF1 FCGR2A .C  CSF2RB  IKBKG  TNFRSF10C  ITGAM  CR1  ENTPD1  CEBPB  JAK3  IFNAR1  TLR8  BCL6  IL1R1  IL6R  FCGR2A  CSF3R  STAT5B Blood sampling was performed after a median of 10 days (interquartile range 9-11) after 16 onset of first symptoms, and before the initiation of any antiviral or anti-inflammatory treatment, 17 and before use of mechanical ventilation. Time interval from first symptoms to admission to hospital 18 (which coincided for most patients with blood sampling) are specified in Table I Detailed methods were previously reported (1). Briefly, we analyzed 100 ng (5 μl) of total RNA from 28 each sample using the Nanostring Human Immunology kit v2, following manufacturer's instructions.

genes provided by Nanostring, using the geNorm method. Normalized counts were log 10 -transformed 31 for all subsequent analyses. Thirty-five genes belonging to the complement system were identified, 32 of which 28 were expressed above the lower limit of quantification in at least one sample. Among 33 those, 19 were differentially expressed in one group, as determined by uncorrected p-value < 0.05 34 of one-way analysis of variance. Heatmap displaying these 19 genes (Figure 1a ) was obtained using 35 pheatmap (package pheatmap), with data centered to 0 and scaled to unit variance for each gene. 36 Hierarchical clustering of these 19 genes was performed using hclust with default distance matrix, 37 and revealed 2 groups with lower intra-group variance, when the number of clusters was set to 4, 38 leaving out two genes with different patterns (CD81 and C1QBP). Continuous variables were compared among groups using the Kruskal-Wallis test, while categorical 54 variables were compared using Fisher's exact test or the χ 2 test of independence, where applicable. 55 No exact a priori sample size was determined, for lack of prior knowledge of the expected effect size 56 in such an exploratory study. However, we calculated that n = 10 in each group would allow to detect 57 a Cohen's effect size of 1.5 (i.e. a difference in means of 1.5 times the standard deviation) with a 58 power of 90% and a 5% significance level. Correlations coefficients were determined using Spearman's 59 method to detect monotonic relationships. ROC AUC p-values were calculated using GraphPad Prism's

WHO Coronavirus Disease (COVID-19) Dashboard. Available from

Clinical features of patients infected with 207 2019 novel coronavirus in Wuhan

COVID-19 is a 209 systemic vascular hemopathy: insight for mechanistic and clinical aspects

Impaired type I 212 interferon activity and inflammatory responses in severe COVID-19 patients

Dexamethasone in Hospitalized Patients with Covid-19 -Preliminary Report

New insights into the immune functions 218 of complement

Functional roles 220 for C5a receptors in sepsis

SARS-CoV-2 drives JAK1/2-222 dependent local complement hyperactivation

Systemic complement 225 activation is associated with respiratory failure in COVID-19 hospitalized patients

COVID-19 inflammation with activation of the C5a-C5aR1 axis. Nature. Nature Publishing 229 Group

Highly pathogenic coronavirus N protein 231 aggravates lung injury by MASP-2-mediated complement over-activation

Increased complement 233 activation is a distinctive feature of severe SARS-CoV-2 infection

Complement C5 inhibition in patients with COVID-19 -a promising target? Haematologica

default method(3). Specifically, following Hanley's computation (4), assuming the null hypothesis, 61 the standard error is sqrt(0.25 + (n 1 + n 2 -2)/(12 n 1 n 2 ), where n 1 and n 2 are the number of patients 62 in each group. The z ratio is then (AUC -0.5)/SE, which follows a two tailed normal distribution

All tests were two-sided and a p-value < 0.05 was considered statistically significant. Correction for 65 multiple testing was obtained using false discovery rates algorithms: Cai and Liu's procedure (5) in 66 the case of correlation data

Hochberg's procedure for all other instances (shown as q-values)

Impaired type I 78 interferon activity and inflammatory responses in severe COVID-19 patients

Calculation details for ROC curves

The meaning and use of the area under a receiver operating 87 characteristic (ROC) curve

Large-Scale Multiple Testing of Correlations