SARS-CoV-2 infection is typically very mild and often asymptomatic in children. A complication is the rare Multisystem Inflammatory Syndrome in Children (MIS-C) associated with COVID-19, presenting 4-6 weeks after infection as high fever, organ dysfunction and strongly elevated markers of inflammation. The pathogenesis is unclear but has overlapping features with Kawasaki disease suggestive of vasculitis and a likely autoimmune etiology. We apply systems-level analyses of blood immune cells, cytokines and autoantibodies in healthy children, children with Kawasaki disease enrolled prior to COVID-19, children infected with SARS-CoV-2 and children presenting with MIS-C. We find that the inflammatory response in MIS-C differs from the cytokine storm of severe acute COVID-19, shares several features with Kawasaki disease, but also differs from this condition with respect to T-cell subsets, IL-17A and biomarkers associated with arterial damage. Finally, autoantibody profiling suggests multiple autoantibodies that could be involved in the pathogenesis of MIS-C
The SARS-CoV-2 virus infects children at a similar rate as adults (Jones et al., 2020), but fortuitously the disease COVID-19 is very mild in the overwhelming majority of infected children (Brodin, 2020). Despite the mild COVID-19 in children, recent reports of severe hyperinflammatory disorders developing 1-2 months after the acute infection with SARSCoV-2 are cause for much concern. This Multisystem Inflammatory Syndrome in Children associated with COVID-19 (MIS-C) also called Pediatric multisystem inflammatory syndrome, temporally associated with COVID-19 (PIMS-TS) have now been reported in hundreds of children worldwide (Cheung et al., 2020; Moraleda et al., 2020; Riphagen et al., 2020; Verdoni et al., 2020; Whittaker et al., 2020). The pathogenesis is under intense investigation but so far remains elusive. One study has found evidence of microangiopathy in MIS-C (Diorio et al., 2020), and a recent autopsy study of a child who died from MIS-C revealed abundant viral particles across a number of tissues, possibly suggestive of a “second-hit” virus mediated pathology (Dolhnikoff et al., 2020). At the same time, children with MIS-C typically respond well to aggressive immunomodulatory and anti-inflammatory therapies, which is unlikely to be effective if pathology was mediated by the virus rather than the immune system. Here we have combined several high-dimensional analysis methods to uncover multiple aspects of the hyperinflammatory response in children with MIS-C. We find similarities with the inflammatory response seen in children with Kawasaki disease, but also important differences, such as the IL-17A mediated hyperinflammation in Kawasaki disease, but not MIS-C. In addition, differences in T-cell subsets and cytokine mediators place MIS-C at the Journal Pre-proof intersection of Kawasaki disease and acute SARS-CoV-2 infection immune states in children as well as the hyperinflammation seen in adults with severe COVID-19. We also find higher levels of biomarkers associated with arteritis and coronary artery disease in Kawasaki disease than in MIS-C suggesting a more diffuse endothelial involvement and immunopathology in MIS-C than in Kawasaki disease. Finally, we perform global autoantibody screening and find binding of autoantibodies to proteins involved in particular in immune cell signaling, structural proteins in heart and blood vessels. These data suggest possible targets of autoimmune attack. The hypothesis that autoantibodies contribute to the pathology in MIS-C is supported by the efficacy of intravenous immunoglobulin in MIS-C, a common approach to activate inhibitory Fc-receptors and prevent membrane-attack complexes by complement factors and thereby mitigating autoantibody-mediated pathology (Mackay et al., 2001). Our results showing lower naive CD4+ T-cell, TFH and increases in central and effector memory subpopulations in MIS-C as compared to Kawasaki disease patients could be partially explained by differences in patient age as immune cell proportions change with age even in healthy children (Olin et al., 2018; Schatorjé et al., 2012). We did however see these cell population differences remained even when comparing against additional older healthy controls, comparable in age to the MIS-C patients. Also, several studies have indicated defective T-cell responses as a key element of severe COVID-19 disease in adult patients (Vardhana and Wolchok, 2020). We find expanded CD57+ CD4+ T cells often representing immunosenescent and dysregulated T cells in both MIS-C and SARS-CoV2+ children with mild infections, suggesting that this might be a consequence of the SARSCoV-2 infection. Additional studies in mild and especially asymptomatic COVID-19 cases will be required to investigate this further. Treatments of MIS-C have mostly followed protocols used in atypical Kawasaki disease given the overlap in presentation between these groups of patients. Our data present a more complex picture with both shared features and stark differences that should influence treatment strategies for these conditions. Kawasaki disease, but not MIS-C is associated with robust IL-17A-mediated inflammation and IL-17A blocking agents such as Secukinumab (Hueber et al., 2010) could be considered in severe Kawasaki disease patients in future trials but perhaps less relevant to MIS-C. Children with MIS-C in this study were treated with a combination of intravenous immunoglobulins (IVIG), Journal Pre-proof corticosteroids and recombinant IL-1-receptor antagonist, IL1RA (Anakinra). The profiles of seven cytokines indicated treatment responses and can be useful to monitor in MIS-C patients undergoing treatment (Figure 5B-C). Autoantibodies represent a possible pathogenic mechanism in MIS-C and Kawasaki disease. IVIG can neutralize some of the immunopathological effects of autoantibodies, while IL-1RA neutralizes strong IL-1 response elicited by endothelial cells damaged by autoantibodies and complement. Corticosteroids provide more general immunosuppression but the mechanism of action in MIS-C for these treatments remains to be determined. Other treatment strategies reported by other groups include TNFα blockade (Infliximab) (Whittaker et al., 2020), but when comparing plasma TNFα levels in MIS-C patients to adults with acute COVID-19, TNFα levels are much lower in MIS-C (Figure 2) and comparable to healthy children we find no elevated levels (Supplementary Figure 2B), suggesting that TNFα-blockade might not mitigate the hyperinflammation in MIS-C. Also, IL-6 blockade have been used in acute COVID-19 and in MIS-C but in our MIS-C patients, plasma levels of IL-6 were highly variable and often normal (Figure 4C). The autoantibody profiling presented here revealed a number of possible autoantibody candidates. It is worth noting that the result is not as clear as the pathogenic autoantibodies seen in well-established autoimmune disorders such as autoimmune thyroiditis or Addison’s disease or in patients with loss of tolerance due to AIRE deficiency (Autoimmune polyendocrine syndrome type-1, APS1)(Landegren et al., 2016). The results here are more diffuse, with many antigens targeted and only a few shared across MIS-C patients as compared to other patient groups. One caveat of these types of arrays is the incorrect structure of some target antigens and another caveat is the inability of the secondary antibody to detect pathogenic autoantibodies of different classes than IgG, such as IgA. Given that the initial immune response is likely elicited in the respiratory or intestinal mucosa, IgA antibodies are of interest and IgA antibodies have been directly implicated in Kawasaki disease pathogenesis (Shulman and Rowley, 2015). Despite these caveats there are a number of possible autoantibodies with pathogenic potential detected in the MIS-C patient cohort herein. Overall the data presented here suggest novel directions for future work towards more mechanistic understanding of the immunopathology in MIS-C, its underlying immune perturbation and development of better immunomodulatory therapies for mitigating the hyperinflammatory disease and long-term tissue damage in such rare children severely affected by COVID-19.
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