Natural mucosal barriers and COVID-19 in children

Abstract

BACKGROUNDCoronavirus disease 2019 (COVID-19) is more benign in children compared with adults for unknown reasons. This contrasts with other respiratory viruses where disease manifestations are often more severe in children. We hypothesize that a more robust early innate immune response to SARS coronavirus 2 (SARS-CoV-2) protects against severe disease.METHODSClinical outcomes, SARS-CoV-2 viral copies, and cellular gene expression were compared in nasopharyngeal swabs obtained at the time of presentation to the emergency department from 12 children and 27 adults using bulk RNA sequencing and quantitative reverse-transcription PCR. Total protein, cytokines, and anti-SARS-CoV-2 IgG and IgA were quantified in nasal fluid.RESULTSSARS-CoV-2 copies, angiotensin-converting enzyme 2, and TMPRSS2 gene expression were similar in children and adults, but children displayed higher expression of genes associated with IFN signaling, NLRP3 inflammasome, and other innate pathways. Higher levels of IFN-α2, IFN-γ, IP-10, IL-8, and IL-1β protein were detected in nasal fluid in children versus adults. Children also expressed higher levels of genes associated with immune cells, whereas expression of those associated with epithelial cells did not differ in children versus adults. Anti-SARS-CoV-2 IgA and IgG were detected at similar levels in nasal fluid from both groups. None of the children required supplemental oxygen, whereas 7 adults did (P = 0.03); 4 adults died.CONCLUSIONThese findings provide direct evidence of a more vigorous early mucosal immune response in children compared with adults and suggest that this contributes to favorable clinical outcomes.FUNDINGNIH grants R01 AI134367, UL1 TR002556, T32 AI007501, T32GM007288, P30 AI124414; an Albert Einstein College of Medicine Dean's COVID-19 Pilot Research Award; and the Eric J. Heyer, MD, PhD Translational Research Pilot Project Award.

Keywords: Infectious disease; Innate immunity.

Figure 1. Transcriptional profiles in pediatric and adult nasopharyngeal samples.

(A) Ct values and days of symptoms prior to presentation in 27 adult and 11 pediatric patients, and ACE2 read counts in cells isolated from NP swabs obtained at presentation in 15 adult and 6 pediatric samples. Bars show mean ± 95% CI. (B) The expression of ACE2 and TMPRSS2 from the RNA-Seq studies. (C) Volcano plot of the expression of genes analyzed by RNA-Seq more strongly in pediatric (right) and adult (left) patients. (D) Principal component plot of RNA-Seq data; n = 15 adult and 6 pediatric samples. Ovals are 95% confidence ellipses. (E) Heatmap showing expression of the top 50 contributing genes in principal components 1, 2, and 3.

Figure 2. Innate responses in pediatric and adult nasopharynx.

(A) Gene set enrichment plots for the indicated pathways: IFN-γ response (normalized enrichment score [NES] = 3.66, P = 0.006), IFN-α response (NES = 3.52, P = 0.006), IL-1 response (NES = 2.70, P = 0.03), NLRP3 inflammasome (NES = 1.88, P = 0.03), and IL-17 production (NES = 2.30, P = 0.03). (B) Fatty acid metabolism (NES = –1.86, P = 0.006). All plots show enrichment in RNA-Seq data from pediatric patients relative to adult patients. (C) Relative IL-17A gene expression measured by RT-qPCR in 5 adult and 4 pediatric samples not used for RNA-Seq. Fold change was calculated by the 2^CT(ref)–CT method using the mean adult value as the reference. (D–H) Levels of the indicated cytokines in NP transport media from 25 adult and 9 pediatric patients measured by multiplex Luminex assay. P values are listed above comparison bars. Unpaired t test (G and H) or Mann-Whitney test (C–F). Bars show mean ± 95% CI. *P < 0.05; **P < 0.01; ***P < 0.001. Ped., pediatric.

Figure 3. Early mucosal antibody responses in pediatric and adult COVID-19 patients.

(A) Total and (B) SARS-CoV-2–specific IgA and (C) total and (D) SARS-CoV-2–specific IgG levels at time of presentation were measured in 10 pediatric and 25 adult patients and 7 healthy controls (HCs). (E) Heatmap showing expression of B cell–related genes contributing to PC1-3. Annotations show age group and peak respiratory score (1 = room air, 2–4 = supplemental oxygen). Total antibody levels (A and C) measured by ELISA; SARS-CoV-2 antibody levels (B and D) measured by multiplexed Luminex assay. Where significant, P values are listed above comparison bars; Kruskal-Wallis test (B and D). Bars show mean ± 95% CI. *P < 0.05; **P < 0.01; ***P < 0.001. Ped., pediatric.

Figure 4. Associations between mucosal proteins and expression of immune or epithelial cell markers.

(A) Spearman correlation matrix of Ct values, total protein recovered (NanoDrop), antibody, and cytokine protein levels in nasopharyngeal fluid. (B) RNA-Seq read counts for indicated genes in pediatric and adult patients MS4A1 (CD20) (P = 0.004), CD4 (P = 0.046), CD8a (P = 0.036), CD86 (P = 0.054), KRT18 (P = 0.084), ITGA6 (P = 0.18); n = 15 adults and n = 6 pediatric patients; unpaired t test with Welch’s correction. Bars show mean ± 95% CI. *P < 0.05; **P < 0.01.