Factor H autoantibodies contribute to complement dysregulation in multisystem inflammatory syndrome in children (MIS-C).
American journal of hematology(2023)
摘要
While most children are asymptomatic or have mild symptoms from SARS-CoV-2 infection, some progress to severe coronavirus disease 2019 (COVID-19). After pediatric SARS-CoV-2 infection, a subset of patients develops multisystem inflammatory syndrome in children (MIS-C), manifested by respiratory failure, myocardial injury, and hemodynamic shock. The specific immune pathways and distinct immunologic responses underlying endothelial and multiorgan damage in children with MIS-C are not well described. Understanding the pathophysiology of MIS-C is critical to identifying targeted therapies and providing novel insights into the pathogenesis of similar pediatric hyperinflammatory syndromes (e.g., Kawasaki disease). The complement system serves a vital role in the innate immune defense against pathogens, including viruses, with sequential activation in response to the recognition of molecular components of microorganisms or tissue injury. Complement pathway (CP) hyperactivation may contribute to endothelial cell dysfunction, hypercoagulability, and consequential thrombus formation in COVID-19.1 Adults with severe COVID-19 demonstrate evidence of complement activation, and complement fragments are deposited in the lungs, kidneys, and microvasculature.2 Furthermore, case reports describe the successful use of anti-C5 and anti-C3 targeted complement inhibitors in the treatment of adult and pediatric patients with severe COVID-19 lung injury, leading to clinical improvement, improved lung oxygenation, and decreased systemic inflammation.3, 4 Despite studies suggesting broad complement dysregulation in MIS-C, a comprehensive analysis of complement dysregulation has not been undertaken. We aimed to elucidate the complement-mediated immune mechanisms underlying MIS-C compared to COVID-19 in pediatric patients, with the goal of providing further insights and potential treatment targets to improve clinical outcomes. We hypothesized that CP dysregulation caused by SARS-CoV-2 infection contributes to acute COVID-19 organ injury and MIS-C pathogenesis. Blood samples were collected from March 2020 through October 2020 from hospitalized children (0–21 years of age) at Children's Healthcare of Atlanta with either COVID-19 (n = 44) or MIS-C (n = 37). Participants were enrolled after consent and age-appropriate assent were obtained. Additional clinical information was obtained from the medical records (Table S1). All samples with sufficient quantity for testing were included. The research was performed with Institutional Review Board approval. For detailed Methods, please see Supplemental information (Appendix S1). As shown in Table S1, patients with MIS-C had similar demographics and comorbidities. Many of these patients had significant systemic inflammation requiring intravenous immunoglobulin, steroids, and cardiorespiratory support including oxygen therapy and the need for vasopressors, all concerning endothelial damage. Furthermore, patients with MIS-C had lower hemoglobin and platelets, evidence of organ injury (higher creatinine, B-type natriuretic peptide, troponin), and higher markers of inflammation (white blood cells, c-reactive protein) when compared to patients with COVID-19 (Table S2). We initially investigated for correlation of viral serology with the disease process and severity in relation to complement activation, by testing for IgM and IgG antibodies against the RBD of the SARS-CoV-2 virus (Figure S1A,B). Consistent with previous findings, higher IgG levels were present in patients with MIS-C when compared to patients with acute COVID-19.5 Likewise, patients with MIS-C had significantly higher neutralizing antibody titers, but IgM antibodies did not statistically differ (Figure S1A,C). To examine the dysregulation of key complement components in patients with COVID-19 or MIS-C, plasma was evaluated for levels of the three CPs as shown in Figure 1A (respective complement proteins highlighted under each CP). C4d is a split product of C4 activation and, while it is a marker of the classical pathway, it can also be generated via the lectin pathway. Evaluation of C4d demonstrated equivalent significant elevations above the normal reference range in both COVID-19 and MIS-C samples (Figure 1B). This increased level of C4d is consistent with prior studies in patients with COVID-19, where it correlated with the need for oxygen therapy and respiratory failure. We then analyzed mannose-binding lectin (MBL), a key lectin pathway recognition molecule. While MBL was elevated above the normal range in some patients, no significant difference between COVID-19 and MIS-C patient samples was noted (p = 0.779). Similarly, significant elevation of fragment Bb, a serine protease component of the alternative pathway was noted, which in combination with hydrolyzed C3 (C3H2O) results in the generation of C3bBb (C3 convertase), augmenting the cleavage of C3 to generate C3a and C3b. Quantitative determination of C3a showed significant elevation (p = 0.002) in MIS-C when compared to COVID-19. This suggests that proximal CP activation is more prominent in MIS-C, in comparison to terminal pathway markers such as C5a (p = 0.339) and terminal complex C5b-9 (p = 0.191). These results are summarized in Figure 1B and Table S2. Given the role of complement dysregulation in other disorders such as atypical hemolytic uremic syndrome (aHUS) and more recently described in adult patients with severe COVID-19 and associated renal thrombotic microangiopathy, we sought to examine levels of factor H (FH), the major CP regulatory protein (Figure 1C). Factor H levels are decreased in adults with COVID-19 and the addition of Factor H in vitro blocks the alternative CP activation elicited by the SARS-CoV-2 spike protein.6 We did not notice a statistical difference in FH levels between these two cohorts (Figure 1D), but patients with MIS-C had significantly higher levels of FHAA (p < 0.001) when compared to patients with acute COVID-19, suggesting that regulation of the CP may be impaired in these patients (Figure 1E). FHAA is predominantly directed against the carboxy-terminal part of FH, which may perturb the alternative CP leading to unregulated activation of C3b and the proximal CP. This novel finding in MIS-C is consistent with a recent case report of 5 pediatric patients who presented with COVID-19-related aHUS and demonstrated high titers of FHAA. Interestingly, although historically few patients with FHAA-related aHUS carry pathogenic genetic variants, some of those patients had homozygous CFHR1 deletion. FHAA-associated aHUS comprises up to 25% of all aHUS in some European cohorts and as high as 56% in a large Indian cohort.7, 8 Similar to MIS-C, it predominantly affects older children aged 5–15 years with more extrarenal features than other forms of aHUS. Sera from patients with FHAA-mediated atypical hemolytic uremic syndrome (aHUS) cause lysis of SE. Therefore, from a subset of available MIS-C serum samples, we utilized a functional assay to determine if the detected FHAA were contributory to the observed alternative CP activation.9 Notably, sera from patients with MIS-C with high titers of FHAA resulted in augmented lysis of SE, compared to the control (Figure 1F). We next investigated for any correlation between the degree of complement activation with SARS-CoV2 RBD-specific IgM and IgG antibodies or neutralization titers. As shown in Figure S2A, we did not find any positive association between those markers. Figure S2B shows the correlation matrix heatmap of the significant complement biomarkers, laboratory parameters (systemic inflammation, hemoglobin, platelets, organ function), and clinical features (ICU stay and respiratory support). The membrane attack complex, C5b9, correlated with systemic markers of inflammation, including ferritin. Additionally, we noticed that most laboratory parameters positively correlated with the duration of ICU stay, level of respiratory support, and the need for vasopressor, all correlating with the disease severity. Complement-mediated TMA (CM-TMA) from an underlying mutation involving the complement regulatory genes is traditionally called ‘atypical HUS.’ CM-TMA occurring secondary to complement amplifying disorders such as hematopoietic stem cell transplantation, cancer, infections, or autoimmune disorders are termed ‘secondary HUS.’ Similarly, in COVID complement dysfunction can result from acquired dysregulation due to disease, but may also be a consequence of genetically encoded variants of critical CP regulators. Although genomic analysis of complement regulatory genes was not completed in this study, future sequencing of genetic variants contributing to complement dysregulation in MIS-C patients may allow the identification of patients at the highest risk for severe disease. It is important to note that other inflammatory mediators could play a role in MIS-C. A recent paper reported on the importance of crosstalk between interferon and complement as seen in multiple inflammatory diseases. Cytokine activation including interferon-γ release from activated endothelial and immune cells can further activate complement pathways leading to accentuated organ damage.10 In summary, these data provide evidence of both proximal complement activation and FHAA-mediated complement dysregulation in pediatric patients with MIS-C. Detailed assessment of the complement profile should be considered in the management of pediatric patients with COVID-19 and MIS-C and warrants consideration of specific anti-complement and immunomodulatory agents in patients with evidence of complement dysregulation. Patricia E. Zerra, Sean R. Stowell, and Satheesh Chonat conceived of the project, which was facilitated by Jennifer Stowell, Hans Verkerke, James McCoy, Jayre Jones, Sara Graciaa, Austin Lu, Laila Hussaini, Evan J. Anderson, and Christina A. Rostad who provided patient samples, experimental support, and critical discussion. Patricia E. Zerra and Satheesh Chonat wrote the manuscript, which was additionally commented on and edited by the remaining authors. This work was supported in part by funding from the NICHD Child Health Research Career Development Award Program, K12HD072245 Atlanta Pediatric Scholars Program to PEZ and SC, and R01 HL138686 and R01 HL135575 to SRS. This research project was also supported in part by the Center for Childhood Infections and Vaccines (CCIV) at Emory University and Children's Healthcare of Atlanta. Christina A. Rostad's institution has received funding to conduct clinical research unrelated to this manuscript from BioFire Inc., GSK, MedImmune, Micron, Merck, Novavax, PaxVax, Regeneron, Pfizer, and Sanofi-Pasteur. She is a coinventor of patented RSV vaccine technology unrelated to this manuscript, which has been licensed to Meissa Vaccines, Inc. Her institution has received funding from NIH to conduct clinical trials of Moderna and Janssen COVID-19 vaccines. Evan J. Anderson has consulted for Pfizer, Sanofi Pasteur, GSK, Janssen, and Medscape, and his institution receives funds to conduct clinical research unrelated to this manuscript from MedImmune, Regeneron, PaxVax, Pfizer, GSK, Merck, Sanofi-Pasteur, Janssen, and Micron. He also serves on a safety monitoring board for Kentucky BioProcessing, Inc. and Sanofi Pasteur. His institution has also received funding from NIH to conduct clinical trials of Moderna and Janssen COVID-19 vaccines. Satheesh Chonat is a scientific advisor to Agios, Alexion, Daichi Sankyo, Forma Therapeutics, Novartis, Novo-Nordisk, and Takeda Pharmaceuticals and receives research funds from Novartis and Global Blood Therapeutics. The data that support the findings of this study are available from the corresponding author upon reasonable request. The data are not publicly available due to privacy or ethical restrictions. Figure S1. SARS-CoV-2 RBD-specific IgG and neutralizing antibody titers are elevated in pediatric patients with MIS-C compared to COVID-19. Figure S2. Correlation matrix of complement and clinical-laboratory findings. Table S1. Demographics, clinical characteristics, and laboratory findings of patient cohorts. Table S2. Detailed clinical and research laboratory findings in COVID-19 and multisystem inflammatory syndrome in children (MIS-C). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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