Multisystem inflammatory syndrome in children is driven by zonulin-dependent loss of gut mucosal barrier

Abstract

BACKGROUNDWeeks after SARS-CoV-2 infection or exposure, some children develop a severe, life-threatening illness called multisystem inflammatory syndrome in children (MIS-C). Gastrointestinal (GI) symptoms are common in patients with MIS-C, and a severe hyperinflammatory response ensues with potential for cardiac complications. The cause of MIS-C has not been identified to date.METHODSHere, we analyzed biospecimens from 100 children: 19 with MIS-C, 26 with acute COVID-19, and 55 controls. Stools were assessed for SARS-CoV-2 by reverse transcription PCR (RT-PCR), and plasma was examined for markers of breakdown of mucosal barrier integrity, including zonulin. Ultrasensitive antigen detection was used to probe for SARS-CoV-2 antigenemia in plasma, and immune responses were characterized. As a proof of concept, we treated a patient with MIS-C with larazotide, a zonulin antagonist, and monitored the effect on antigenemia and the patient's clinical response.RESULTSWe showed that in children with MIS-C, a prolonged presence of SARS-CoV-2 in the GI tract led to the release of zonulin, a biomarker of intestinal permeability, with subsequent trafficking of SARS-CoV-2 antigens into the bloodstream, leading to hyperinflammation. The patient with MIS-C treated with larazotide had a coinciding decrease in plasma SARS-CoV-2 spike antigen levels and inflammatory markers and a resultant clinical improvement above that achieved with currently available treatments.CONCLUSIONThese mechanistic data on MIS-C pathogenesis provide insight into targets for diagnosing, treating, and preventing MIS-C, which are urgently needed for this increasingly common severe COVID-19-related disease in children.

Keywords: Antigen; COVID-19; Inflammation; Tight junctions.

Conflict of interest statement

Conflict of interest: AF is co-founder of and stockholder in Alba Therapeutics. DRW has a financial interest in Quanterix Corporation, developer of the ultrasensitive digital immunoassay platform. He is an inventor of the Simoa technology, a founder of the company, and also serves on its board of directors.

Figures

Figure 1. Study overview.

Timing of sample collection and sample analysis for children with MIS-C or acute COVID-19.

Figure 2. Plasma (A) zonulin, (B) LPS-binding protein, and (C) soluble CD14 levels were quantified by multiplexed MS–based proteomics (54, 55) for children with MIS-C (n = 13) or COVID-19 (n = 21) and for non–COVID-19 control participants (n = 23).

Results were compared by ANOVA. (D) SARS-CoV-2 spike, (E) S1, and (F) nucleocapsid protein levels were quantified in plasma from children with MIS-C (n = 16), children with acute COVID-19 (n = 22), and pre-pandemic healthy controls (n = 32). Results were compared by 1-way ANOVA with multiple comparisons. * P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Median values and 95% CI are presented.

Figure 3. Peak values of (A) anti–spike IgM, (B) anti–spike IgG, and (C) anti–spike IgA were quantified in plasma from children with MIS-C (n = 16), children with acute COVID-19 (n = 22), and pre-pandemic healthy controls (n = 32).

Results were compared by 1-way ANOVA with multiple comparisons. Time course of (D) anti–spike IgM, (E) anti–spike IgG, and (F) anti–spike IgA were plotted over time following symptom onset for MIS-C. (G) The IC50 for antibody neutralization for children with MIS-C and children with acute COVID-19 were compared by Mann-Whitney t test. Mean values and the SD are presented. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Figure 4. (A) SARS-CoV-2 spike, S1, and nucleocapsid levels in plasma from children with MIS-C were quantified before treatment with steroids and/or immunoglobulin replacement therapy, through 14 days following treatment (n = 11).

Shaded regions signify the limit of detection for each specific antigen test. (B) Spike and anti–spike IgM, anti–spike IgG, and anti–spike IgA levels were measured over the course of illness of a child with MIS-C. Of note, the spike protein remained above the limit of detection for the spike antigen test at the 213-day follow-up point.

Figure 5. Timeline for the child treated with larazotide.

The hospital course is delineated by hospitalization for acute COVID-19 (day 0) followed by development of MIS-C (day 39), with the treatment courses identified along the time course. (A) CRP and SARS-CoV-2 spike antigen levels, the median daily temperature (temp) curve, and D-dimer and ferritin levels throughout the hospital course are shown. Light blue shading represents a normal body temperature. The light gray shading highlights the days of larazotide treatment. (B) Inflammatory cytokine IL-17F, IL-2R, IFN-γ, and IL-1β levels following the development of MIS-C, in relation to the treatment courses.

Figure 6. Overview of the proposed hypothesis that SARS-CoV-2 antigenemia drives MIS-C.

(i) A child is exposed to or infected with SARS-CoV-2. (ii) SARS-CoV-2 enters the GI tract. (iii) Dysbiosis leads to increased zonulin release and a resultant loss of tight junctions. (iv) SARS-CoV-2 antigens, especially the spike protein, breaches the mucosal barrier and enters the blood stream. (v) The superantigen motif of the spike protein stimulates a pathogenic hyperinflammatory response.