Multisystem inflammatory syndrome in children (MIS-C) is a severe postinfectious complication associated with SARS-CoV-2. Although the crucial pathogenic role of the monocyte compartment is known, information regarding the mechanisms and maintenance of the inflammatory trigger is lacking. Genetic variants in the OAS/RNase L pathway, involved in viral double-stranded RNA (dsRNA) sensing and the modulation of inflammatory responses, have been identified in patients with MIS-C. We conducted a comprehensive analysis, including the study of NLRP3 inflammasome activation, blood biomarkers, and autoantibodies, as well as transcriptomic and proteomic profiling.
Peripheral blood mononuclear cells (PBMCs), plasma, and RNA samples from MIS-C patients were analyzed. PBMCs were stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP), and inflammatory responses were quantified using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and Imaging Flow Cytometry (IFC). Apoptosis was assessed via annexin V staining. Type I interferon (IFN) signatures were measured using qPCR. Pro-inflammatory cytokines were evaluated using an automated ELISA system (ELLA). RNA sequencing (RNA-seq) data were analyzed through bioinformatics pipelines, including gene set enrichment analysis (GSEA), while proteomics data were processed using weighted gene coexpression network analysis (WGCNA).
We found a monocyte exhaustion phenotype, demonstrated by reduced inflammasome activation in response to stimuli and an associated lack of IL-1β secretion (Figure 1A-B). Intriguingly, dsRNA was detected in monocytes from MIS-C patients even during follow-up, suggesting a long-lasting viral presence that could drive sustained immune activation (Figure 1C-D). Blood biomarker profiling, bulk RNA-seq, and proteomic analyses revealed a signature characterized by the activation of genes involved in antiviral response, systemic inflammation, oxidative stress, and coagulation pathways. Additionally, we observed anti-IL1RA autoantibodies in 40-50% of patients, consistent with previous reports.
(A) In vitro inflammatory cytokine secretion by MIS-C patients’ PBMCs (T1: 0 to 10 days from onset, T2: 10 to 30 days from onset, and T3: over 30 days from onset) of as measured in culture supernatant after 18 hours with or without stimulation with the indicated stimuli. The NLRP3 blocker MCC950 inhibited secretion, indicating that secretion of IL-1β is dependent on the NLRP3 inflammasome. (B) Percentages of spontaneous and after ASC specks detection in monocytes (left) and after stimulation (LPS+ATP, right) from controls (HD), MIS-C patients in acute stage (T1), and during follow-up (T2-3) by flow cytometry. Representative imaging flow cytometry images and analysis (C, D) of monocytes from HD, patients with COVID-19 (blue), and MIS-C patients (T1: red, T2: orange, T3: light orange) stained for dsRNA (anti-J2 antibodies) and ASC. ASC, apoptosis-associated speck-like protein containing a CARD; HD, healthy donors; IQR, interquartile range; MCC, MCC950 NLRP3 inhibitor. Data are expressed as median ± IQR. *P < .05, **P < .01, ***P < .005, ****P < .001 as assessed by Mann–Whitney t test.
(A) In vitro inflammatory cytokine secretion by MIS-C patients’ PBMCs (T1: 0 to 10 days from onset, T2: 10 to 30 days from onset, and T3: over 30 days from onset) of as measured in culture supernatant after 18 hours with or without stimulation with the indicated stimuli. The NLRP3 blocker MCC950 inhibited secretion, indicating that secretion of IL-1β is dependent on the NLRP3 inflammasome. (B) Percentages of spontaneous and after ASC specks detection in monocytes (left) and after stimulation (LPS+ATP, right) from controls (HD), MIS-C patients in acute stage (T1), and during follow-up (T2-3) by flow cytometry. Representative imaging flow cytometry images and analysis (C, D) of monocytes from HD, patients with COVID-19 (blue), and MIS-C patients (T1: red, T2: orange, T3: light orange) stained for dsRNA (anti-J2 antibodies) and ASC. ASC, apoptosis-associated speck-like protein containing a CARD; HD, healthy donors; IQR, interquartile range; MCC, MCC950 NLRP3 inhibitor. Data are expressed as median ± IQR. *P < .05, **P < .01, ***P < .005, ****P < .001 as assessed by Mann–Whitney t test.
Our findings underscore the multifaceted nature of immune dysregulation in MIS-C, encompassing monocyte exhaustion, persistence of intramonocytic dsRNA, autoantibody formation, and a distinct transcriptomic and proteomic signature. This study provides insights into the underlying mechanisms driving sustained inflammation in post-COVID syndromes.
