The Notch1/CD22 signaling axis disrupts Treg function in SARS-CoV-2-associated multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) evolves in some pediatric patients following acute infection with SARS-CoV-2 by hitherto unknown mechanisms. Whereas acute-COVID-19 severity and outcomes were previously correlated with Notch4 expression on Tregs, here, we show that Tregs in MIS-C were destabilized through a Notch1-dependent mechanism. Genetic analysis revealed that patients with MIS-C had enrichment of rare deleterious variants affecting inflammation and autoimmunity pathways, including dominant-negative mutations in the Notch1 regulators NUMB and NUMBL leading to Notch1 upregulation. Notch1 signaling in Tregs induced CD22, leading to their destabilization in a mTORC1-dependent manner and to the promotion of systemic inflammation. These results identify a Notch1/CD22 signaling axis that disrupts Treg function in MIS-C and point to distinct immune checkpoints controlled by individual Treg Notch receptors that shape the inflammatory outcome in SARS-CoV-2 infection.

SARS-CoV-2-specific T cell responses in patients with multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) is a severe complication of SARS-CoV-2 infections that occurs in the pediatric population. We sought to characterize T cell responses in MIS-C compared to COVID-19 and pediatric hyperinflammatory syndromes. MIS-C was distinct from COVID-19 and hyperinflammatory syndromes due to an expansion of T cells expressing TRBV11-2 that was not associated with HLA genotype. Children diagnosed with MIS-C, but who were negative for SARS-CoV-2 by PCR and serology, did not display Vß skewing. There was no difference in the proportion of T cells that became activated after stimulation with SARS-CoV-2 peptides in children with MIS-C compared to convalescent COVID-19. The frequency of SARS-CoV-2-specific TCRs and the antigens recognized by these TCRs were comparable in MIS-C and COVID-19. Expansion of Vß11-2+ T cells was a specific biomarker of MIS-C patients with laboratory confirmed SARS-CoV-2 infections. Children with MIS-C had robust antigen-specific T cell responses to SARS-CoV-2.

A common IL-4 receptor variant promotes asthma severity via a Treg cell GRB2-IL-6-Notch4 circuit.

BACKGROUND: The mechanisms by which genetic and environmental factors interact to promote asthma remain unclear. Both the IL-4 receptor alpha chain R576 (IL-4RαR576) variant and Notch4 license asthmatic lung inflammation by allergens and ambient pollutant particles by subverting lung regulatory T (Treg ) cells in an IL-6-dependent manner. OBJECTIVE: We examined the interaction between IL-4RαR576 and Notch4 in promoting asthmatic inflammation. METHODS: Peripheral blood mononuclear cells (PBMCs) of asthmatics were analyzed for T helper type 2 cytokine production and Notch4 expression on Treg cells as a function of IL4RR576 allele. The capacity of IL-4RαR576 to upregulate Notch4 expression on Treg cells to promote severe allergic airway inflammation was further analyzed in genetic mouse models. RESULTS: Asthmatics carrying the IL4RR576 allele had increased Notch4 expression on their circulating Treg cells as a function of disease severity and serum IL-6. Mice harboring the Il4raR576 allele exhibited increased Notch4-dependent allergic airway inflammation that was inhibited upon Treg cell-specific Notch4 deletion or treatment with an anti-Notch4 antibody. Signaling via IL-4RαR576 upregulated the expression in lung Treg cells of Notch4 and its downstream mediators Yap1 and beta-catenin, leading to exacerbated lung inflammation. This upregulation was dependent on growth factor receptor-bound protein 2 (GRB2) and IL-6 receptor. CONCLUSION: These results identify an IL-4RαR576-regulated GRB2-IL-6-Notch4 circuit that promotes asthma severity by subverting lung Treg cell function.

Atopic Dermatitis Mediates the Association Between an IL4RA Variant and Food Allergy in School-Aged Children.

BACKGROUND: Atopic dermatitis (AD) and food allergy (FA) may share genetic risk factors. It is unknown whether genetic factors directly cause FA or are mediated through AD, as the dual-allergen hypothesis suggests. OBJECTIVE: To test the hypothesis that AD mediates the relationship between an IL-4 receptor alpha chain gene (IL4RA) variant, the human IL-4 receptor alpha chain protein-R576 polymorphism, and FA. METHODS: A total of 433 children with asthma enrolled in the School Inner-City Asthma Study underwent genotyping for the IL4RA576 allele. Surveys were administered to determine FA, AD, and associated allergic responses. Mediation analysis was performed adjusting for race and ethnicity, age, sex, and household income. Multivariate models were used to determine the association between genotype and FA severity. RESULTS: AD was reported in 193 (45%) and FA in 80 children (19%). Each risk allele increased odds of AD 1.39-fold ([1.03-1.87], P = .03), and AD increased odds of FA 3.67-fold ([2.05- 6.57], P < .01). There was an indirect effect of genotype, mediated by AD, predicting FA; each risk allele increased the odds of FA by 1.13 (odds ratio [95% CI], Q/R = 1.13 [1.02-1.24], R/R = 1.28 [1.04-1.51]; P < .01). Each risk allele increased the odds of severe FA symptoms 2.68-fold ([1.26-5.71], P = .01). CONCLUSIONS: In a cohort of children with asthma, AD is part of the causal pathway between an IL4RA variant and FA. This variant is associated with increased risk of severe FA reactions. Addressing AD in children with an IL4RA polymorphism may modulate the risk of FA.

Notch1-CD22-Dependent Immune Dysregulation in the SARS-CoV2-Associated Multisystem Inflammatory Syndrome in Children.

Multisystem inflammatory syndrome in children (MIS-C) evolves in some pediatric patients following acute infection with SARS-CoV-2 by hitherto unknown mechanisms. Whereas acute-COVID-19 severity and outcome were previously correlated with Notch4 expression on regulatory T (Treg) cells, here we show that the Treg cells in MIS-C are destabilized in association with increased Notch1 expression. Genetic analysis revealed that MIS-C patients were enriched in rare deleterious variant impacting inflammation and autoimmunity pathways, including dominant negative mutations in the Notch1 regulators NUMB and NUMBL. Notch1 signaling in Treg cells induced CD22, leading to their destabilization in an mTORC1 dependent manner and to the promotion of systemic inflammation. These results establish a Notch1-CD22 signaling axis that disrupts Treg cell function in MIS-C and point to distinct immune checkpoints controlled by individual Treg cell Notch receptors that shape the inflammatory outcome in SARS-CoV-2 infection.

A virus-specific monocyte inflammatory phenotype is induced by SARS-CoV-2 at the immune-epithelial interface.

Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled, by in vitro coculture, the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV-2-specific inflammatory gene cluster distinct from that seen in influenza A or Ebola virus-infected cocultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV-2 proteins (Spike and some nonstructural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV-2 induces in epithelia.

Author Correction: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma.

A Correction to this paper has been published: https://doi.org/10.1038/s41590-021-00929-x.

Notch4 signaling limits regulatory T-cell-mediated tissue repair and promotes severe lung inflammation in viral infections.

A cardinal feature of COVID-19 is lung inflammation and respiratory failure. In a prospective multi-country cohort of COVID-19 patients, we found that increased Notch4 expression on circulating regulatory T (Treg) cells was associated with disease severity, predicted mortality, and declined upon recovery. Deletion of Notch4 in Treg cells or therapy with anti-Notch4 antibodies in conventional and humanized mice normalized the dysregulated innate immunity and rescued disease morbidity and mortality induced by a synthetic analog of viral RNA or by influenza H1N1 virus. Mechanistically, Notch4 suppressed the induction by interleukin-18 of amphiregulin, a cytokine necessary for tissue repair. Protection by Notch4 inhibition was recapitulated by therapy with Amphiregulin and, reciprocally, abrogated by its antagonism. Amphiregulin declined in COVID-19 subjects as a function of disease severity and Notch4 expression. Thus, Notch4 expression on Treg cells dynamically restrains amphiregulin-dependent tissue repair to promote severe lung inflammation, with therapeutic implications for COVID-19 and related infections.

Regulatory T Cell-Derived TGF-ß1 Controls Multiple Checkpoints Governing Allergy and Autoimmunity.

The mechanisms by which regulatory T (Treg) cells differentially control allergic and autoimmune responses remain unclear. We show that Treg cells in food allergy (FA) had decreased expression of transforming growth factor beta 1 (TGF-ß1) because of interleukin-4 (IL-4)- and signal transducer and activator of transciription-6 (STAT6)-dependent inhibition of Tgfb1 transcription. These changes were modeled by Treg cell-specific Tgfb1 monoallelic inactivation, which induced allergic dysregulation by impairing microbiota-dependent retinoic acid receptor-related orphan receptor gamma t (ROR-γt)+ Treg cell differentiation. This dysregulation was rescued by treatment with Clostridiales species, which upregulated Tgfb1 expression in Treg cells. Biallelic deficiency precipitated fatal autoimmunity with intense autoantibody production and dysregulated T follicular helper and B cell responses. These results identify a privileged role of Treg cell-derived TGF-ß1 in regulating allergy and autoimmunity at distinct checkpoints in a Tgfb1 gene dose- and microbiota-dependent manner.

A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma.

Elucidating the mechanisms that sustain asthmatic inflammation is critical for precision therapies. We found that interleukin-6- and STAT3 transcription factor-dependent upregulation of Notch4 receptor on lung tissue regulatory T (Treg) cells is necessary for allergens and particulate matter pollutants to promote airway inflammation. Notch4 subverted Treg cells into the type 2 and type 17 helper (TH2 and TH17) effector T cells by Wnt and Hippo pathway-dependent mechanisms. Wnt activation induced growth and differentiation factor 15 expression in Treg cells, which activated group 2 innate lymphoid cells to provide a feed-forward mechanism for aggravated inflammation. Notch4, Wnt and Hippo were upregulated in circulating Treg cells of individuals with asthma as a function of disease severity, in association with reduced Treg cell-mediated suppression. Our studies thus identify Notch4-mediated immune tolerance subversion as a fundamental mechanism that licenses tissue inflammation in asthma.

Dietary and Microbial Determinants in Food Allergy.

The steep rise in food allergy (FA) has evoked environmental factors involved in disease pathogenesis, including the gut microbiota, diet, and their metabolites. Early introduction of solid foods synchronizes with the "weaning reaction," a time during which the microbiota imprints durable oral tolerance. Recent work has shown that children with FA manifest an early onset dysbiosis with the loss of Clostridiales species, which promotes the differentiation of ROR-γt+ regulatory T cells to suppress FA. This process can be reversed in pre-clinical mouse models by targeted bacteriotherapy. Here, we review the dominant tolerance mechanisms enforced by the microbiota to suppress FA and discuss therapeutic intervention strategies that act to recapitulate the early life window of opportunity in stemming the FA epidemic.

Untargeted metabolomic profiling identifies disease-specific signatures in food allergy and asthma.

BACKGROUND: Food allergy (FA) affects an increasing proportion of children for reasons that remain obscure. Novel disease biomarkers and curative treatment options are strongly needed. OBJECTIVE: We sought to apply untargeted metabolomic profiling to identify pathogenic mechanisms and candidate disease biomarkers in patients with FA. METHODS: Mass spectrometry-based untargeted metabolomic profiling was performed on serum samples of children with either FA alone, asthma alone, or both FA and asthma, as well as healthy pediatric control subjects. RESULTS: In this pilot study patients with FA exhibited a disease-specific metabolomic signature compared with both control subjects and asthmatic patients. In particular, FA was uniquely associated with a marked decrease in sphingolipid levels, as well as levels of a number of other lipid metabolites, in the face of normal frequencies of circulating natural killer T cells. Specific comparison of patients with FA and asthmatic patients revealed differences in the microbiota-sensitive aromatic amino acid and secondary bile acid metabolism. Children with both FA and asthma exhibited a metabolomic profile that aligned with that of FA alone but not asthma. Among children with FA, the history of severe systemic reactions and the presence of multiple FAs were associated with changes in levels of tryptophan metabolites, eicosanoids, plasmalogens, and fatty acids. CONCLUSIONS: Children with FA have a disease-specific metabolomic profile that is informative of disease mechanisms and severity and that dominates in the presence of asthma. Lower levels of sphingolipids and ceramides and other metabolomic alterations observed in children with FA might reflect the interplay between an altered microbiota and immune cell subsets in the gut.

Author Correction: Microbiota therapy acts via a regulatory T cell MyD88/RORγt pathway to suppress food allergy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Heterozygous FOXN1 Variants Cause Low TRECs and Severe T Cell Lymphopenia, Revealing a Crucial Role of FOXN1 in Supporting Early Thymopoiesis.

FOXN1 is the master regulatory gene of thymic epithelium development. FOXN1 deficiency leads to thymic aplasia, alopecia, and nail dystrophy, accounting for the nude/severe combined immunodeficiency (nu/SCID) phenotype in humans and mice. We identified several newborns with low levels of T cell receptor excision circles (TRECs) and T cell lymphopenia at birth, who carried heterozygous loss-of-function FOXN1 variants. Longitudinal analysis showed persistent T cell lymphopenia during infancy, often associated with nail dystrophy. Adult individuals with heterozygous FOXN1 variants had in most cases normal CD4+ but lower than normal CD8+ cell counts. We hypothesized a FOXN1 gene dosage effect on the function of thymic epithelial cells (TECs) and thymopoiesis and postulated that these effects would be more prominent early in life. To test this hypothesis, we analyzed TEC subset frequency and phenotype, early thymic progenitor (ETP) cell count, and expression of FOXN1 target genes (Ccl25, Cxcl12, Dll4, Scf, Psmb11, Prss16, and Cd83) in Foxn1nu/+ (nu/+) mice and age-matched wild-type (+/+) littermate controls. Both the frequency and the absolute count of ETP were significantly reduced in nu/+ mice up to 3 weeks of age. Analysis of the TEC compartment showed reduced expression of FOXN1 target genes and delayed maturation of the medullary TEC compartment in nu/+ mice. These observations establish a FOXN1 gene dosage effect on thymic function and identify FOXN1 haploinsufficiency as an important genetic determinant of T cell lymphopenia at birth.

Microbiota therapy acts via a regulatory T cell MyD88/RORγt pathway to suppress food allergy.

The role of dysbiosis in food allergy (FA) remains unclear. We found that dysbiotic fecal microbiota in FA infants evolved compositionally over time and failed to protect against FA in mice. Infants and mice with FA had decreased IgA and increased IgE binding to fecal bacteria, indicative of a broader breakdown of oral tolerance than hitherto appreciated. Therapy with Clostridiales species impacted by dysbiosis, either as a consortium or as monotherapy with Subdoligranulum variabile, suppressed FA in mice as did a separate immunomodulatory Bacteroidales consortium. Bacteriotherapy induced expression by regulatory T (Treg) cells of the transcription factor ROR-γt in a MyD88-dependent manner, which was deficient in FA infants and mice and ineffectively induced by their microbiota. Deletion of Myd88 or Rorc in Treg cells abrogated protection by bacteriotherapy. Thus, commensals activate a MyD88/ROR-γt pathway in nascent Treg cells to protect against FA, while dysbiosis impairs this regulatory response to promote disease.

Systemic Reactions in Pediatric Patients Receiving Standardized Allergen Subcutaneous Immunotherapy with and without Seasonal Dose Adjustment.

BACKGROUND: The 2003 Joint Task Force on Practice Parameters recommended standardizing allergen subcutaneous immunotherapy (SCIT). Data from longitudinal surveillance survey in North America reported a systemic reaction (SR) rate of 0.1% to 0.2% of injection visits. The rate of SR to standardized SCIT in pediatric patients has not been well evaluated. OBJECTIVE: The objective of this study was to evaluate the rate of SRs to standardized SCIT in pediatric patients aged 5 to 18 years in a single tertiary care center in the United States. METHODS: A retrospective chart review was conducted in 2 groups: group 1 started SCIT within a period extending from January 2009 to June 2012, whereas group 2 started SCIT within a period extending from January 2013 to June 2016. The protocol was modified in group 2 such that updosing and maintenance doses were adjusted in the spring for tree and grass pollen and in the fall for weed pollen. RESULTS: There were a total of 128 patients in group 1 and 118 patients in group 2. The rate of SR was 0.429% in group 1 and 0.364% in group 2, which was not significant. There was no difference in the severity of SR in the 2 groups with no-fatal or near-fatal SR noted. Asthma was a significant risk factor in the younger age subgroup aged 5 to 11 years. CONCLUSIONS: Standardized SCIT appears to be associated with an SR rate of 0.429% to 0.364% of visits in pediatric patients. Protocol modification did not lead to a significant drop in SR. Larger multicenter studies are required to further evaluate the rate of SRs from standardized SCIT.