Multiomic Analysis of Neuroinflammation and Occult Infection in Sudden Infant Death Syndrome.

Importance: Antemortem infection is a risk factor for sudden infant death syndrome (SIDS)-the leading postneonatal cause of infant mortality in the developed world. Manifestations of infection and inflammation are not always apparent in clinical settings or by standard autopsy; thus, enhanced resolution approaches are needed. Objective: To ascertain whether a subset of SIDS cases is associated with neuroinflammation and occult infection. Design, Setting, and Participants: In this case-control study, postmortem fluids from SIDS cases and controls collected between July 2011 and November 2018 were screened for elevated inflammatory markers, specifically cerebrospinal fluid (CSF) neopterin and CSF and serum cytokines. CSF, liver, and brain tissue from SIDS cases with elevated CSF neopterin were subjected to metagenomic next-generation sequencing (mNGS) to probe for infectious pathogens. Brainstem tissue from a subset of these cases was analyzed by single-nucleus RNA sequencing (snRNAseq) to measure cell type-specific gene expression associated with neuroinflammation and infection. All tissue and fluid analyses were performed from April 2019 to January 2023 in a pathology research laboratory. Included was autopsy material from infants dying of SIDS and age-matched controls dying of known causes. Exposures: There were no interventions or exposures. Main Outcomes and Measures: CSF neopterin levels were measured by high-performance liquid chromatography. Cytokines were measured by multiplex fluorometric assay. mNGS was performed on liver, CSF, brain, and brainstem tissue. snRNAseq was performed on brainstem tissue. Results: A cohort of 71 SIDS cases (mean [SD] age, 55.2 [11.4] postconceptional weeks; 42 male [59.2%]) and 20 controls (mean [SD] age, 63.2 [16.9] postconceptional weeks; 11 male [55.0%]) had CSF and/or serum available. CSF neopterin was screened in 64 SIDS cases and 15 controls, with no exclusions. Tissues from 6 SIDS cases were further analyzed. For CSF neopterin measures, SIDS samples were from infants with mean (SD) age of 54.5 (11.3) postconceptional weeks (38 male [59.4%]) and control samples were from infants with mean (SD) age of 61.5 (17.4) postconceptional weeks (7 male [46.7%]). A total of 6 SIDS cases (9.3%) with high CSF neopterin were identified, suggestive of neuroinflammation. mNGS detected human parechovirus 3 (HPeV3) in tissue and CSF from 1 of these 6 cases. snRNAseq of HPeV3-positive brainstem tissue (medulla) revealed dramatic enrichment of transcripts for genes with predominately inflammatory functions compared with 3 age-matched SIDS cases with normal CSF neopterin levels. Conclusions and Relevance: Next-generation molecular tools in autopsy tissue provide novel insight into pathogens that go unrecognized by normal autopsy methodology, including in infants dying suddenly and unexpectedly.

Extracellular Vesicles and Their Role in Lung Infections.

Lung infections are one of the most common causes of death and morbidity worldwide. Both bacterial and viral lung infections cause a vast number of infections with varying severities. Extracellular vesicles (EVs) produced by different cells due to infection in the lung have the ability to modify the immune system, leading to either better immune response or worsening of the disease. It has been shown that both bacteria and viruses have the ability to produce their EVs and stimulate the immune system for that. In this review, we investigate topics from EV biogenesis and types of EVs to lung bacterial and viral infections caused by various bacterial species. Mycobacterium tuberculosis, Staphylococcus aureus, and Streptococcus pneumoniae infections are covered intensively in this review. Moreover, various viral lung infections, including SARS-CoV-2 infections, have been depicted extensively. In this review, we focus on eukaryotic-cell-derived EVs as an important component of disease pathogenesis. Finally, this review holds high novelty in its findings and literature review. It represents the first time to cover all different information on immune-cell-derived EVs in both bacterial and viral lung infections.

Mechanisms and clinical relevance of the bidirectional relationship of viral infections with metabolic diseases.

Viruses have been present during all evolutionary steps on earth and have had a major effect on human history. Viral infections are still among the leading causes of death. Another public health concern is the increase of non-communicable metabolic diseases in the last four decades. In this Review, we revisit the scientific evidence supporting the presence of a strong bidirectional feedback loop between several viral infections and metabolic diseases. We discuss how viruses might lead to the development or progression of metabolic diseases and conversely, how metabolic diseases might increase the severity of a viral infection. Furthermore, we discuss the clinical relevance of the current evidence on the relationship between viral infections and metabolic disease and the present and future challenges that should be addressed by the scientific community and health authorities.

The IL-4Rα Q576R polymorphism is associated with increased severity of atopic dermatitis and exaggerates allergic skin inflammation in mice.

BACKGROUND: Atopic dermatitis (AD) is characterized by TH2-dominated skin inflammation and systemic response to cutaneously encountered antigens. The TH2 cytokines IL-4 and IL-13 play a critical role in the pathogenesis of AD. The Q576->R576 polymorphism in the IL-4 receptor alpha (IL-4Rα) chain common to IL-4 and IL-13 receptors alters IL-4 signaling and is associated with asthma severity. OBJECTIVE: We sought to investigate whether the IL-4Rα R576 polymorphism is associated with AD severity and exaggerates allergic skin inflammation in mice. METHODS: Nighttime itching interfering with sleep, Rajka-Langeland, and Eczema Area and Severity Index scores were used to assess AD severity. Allergic skin inflammation following epicutaneous sensitization of mice 1 or 2 IL-4Rα R576 alleles (QR and RR) and IL-4Rα Q576 (QQ) controls was assessed by flow cytometric analysis of cells and quantitative RT-PCR analysis of cytokines in skin. RESULTS: The frequency of nighttime itching in 190 asthmatic inner-city children with AD, as well as Rajka-Langeland and Eczema Area and Severity Index scores in 1116 White patients with AD enrolled in the Atopic Dermatitis Research Network, was higher in subjects with the IL-4Rα R576 polymorphism compared with those without, with statistical significance for the Rajka-Langeland score. Following epicutaneous sensitization of mice with ovalbumin or house dust mite, skin infiltration by CD4+ cells and eosinophils, cutaneous expression of Il4 and Il13, transepidermal water loss, antigen-specific IgE antibody levels, and IL-13 secretion by antigen-stimulated splenocytes were significantly higher in RR and QR mice compared with QQ controls. Bone marrow radiation chimeras demonstrated that both hematopoietic cells and stromal cells contribute to the mutants' exaggerated allergic skin inflammation. CONCLUSIONS: The IL-4Rα R576 polymorphism predisposes to more severe AD and increases allergic skin inflammation in mice.

Regulatory T-cells in asthma.

PURPOSE OF REVIEW: This review addresses recent progress in our understanding of the role of regulatory T (Treg) cells in enforcing immune tolerance and tissue homeostasis in the lung at steady state and in directing the immune response in asthmatic lung inflammation. RECENT FINDINGS: Regulatory T cells regulate the innate and adaptive immune responses at steady state to enforce immune tolerance in lung tissues at steady state and their control of the allergic inflammatory responses induced by allergens. This regulatory function can break down in the context of chronic asthmatic airway inflammation such that the lung tissue Treg cells become skewed towards a pathogenic phenotype that aggravates and perpetuates disease. Subversion of lung tissue Treg cell function involves their upregulation of Notch4 expression, which in turn acts to amplify T helper type 2 and type 17 and innate lymphoid cell type 2 responses in the airways. SUMMARY: A dual role for Treg cells has emerged both as immune regulators but also a potential disease effectors in asthma, with implications for disease therapy.

Intranasal administration of Acinetobacter lwoffii in a murine model of asthma induces IL-6-mediated protection associated with cecal microbiota changes.

BACKGROUND: Early-life exposure to certain environmental bacteria including Acinetobacter lwoffii (AL) has been implicated in protection from chronic inflammatory diseases including asthma later in life. However, the underlying mechanisms at the immune-microbe interface remain largely unknown. METHODS: The effects of repeated intranasal AL exposure on local and systemic innate immune responses were investigated in wild-type and Il6-/- , Il10-/- , and Il17-/- mice exposed to ovalbumin-induced allergic airway inflammation. Those investigations were expanded by microbiome analyses. To assess for AL-associated changes in gene expression, the picture arising from animal data was supplemented by in vitro experiments of macrophage and T-cell responses, yielding expression and epigenetic data. RESULTS: The asthma preventive effect of AL was confirmed in the lung. Repeated intranasal AL administration triggered a proinflammatory immune response particularly characterized by elevated levels of IL-6, and consequently, IL-6 induced IL-10 production in CD4+ T-cells. Both IL-6 and IL-10, but not IL-17, were required for asthma protection. AL had a profound impact on the gene regulatory landscape of CD4+ T-cells which could be largely recapitulated by recombinant IL-6. AL administration also induced marked changes in the gastrointestinal microbiome but not in the lung microbiome. By comparing the effects on the microbiota according to mouse genotype and AL-treatment status, we have identified microbial taxa that were associated with either disease protection or activity. CONCLUSION: These experiments provide a novel mechanism of Acinetobacter lwoffii-induced asthma protection operating through IL-6-mediated epigenetic activation of IL-10 production and with associated effects on the intestinal microbiome.

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.

Short-Chain Fatty Acids Augment Differentiation and Function of Human Induced Regulatory T Cells.

Regulatory T cells (Tregs) control immune system activity and inhibit inflammation. While, in mice, short-chain fatty acids (SCFAs) are known to be essential regulators of naturally occurring and in vitro induced Tregs (iTregs), data on their contribution to the development of human iTregs are sparse, with no reports of the successful SCFAs-augmented in vitro generation of fully functional human iTregs. Likewise, markers undoubtedly defining human iTregs are missing. Here, we aimed to generate fully functional human iTregs in vitro using protocols involving SCFAs and to characterize the underlying mechanism. Our target was to identify the potential phenotypic markers best characterizing human iTregs. Naïve non-Treg CD4+ cells were isolated from the peripheral blood of 13 healthy adults and cord blood of 12 healthy term newborns. Cells were subjected to differentiation toward iTregs using a transforming growth factor ß (TGF-ß)-based protocol, with or without SCFAs (acetate, butyrate, or propionate). Thereafter, they were subjected to flow cytometric phenotyping or a suppression assay. During differentiation, cells were collected for chromatin-immunoprecipitation (ChIP)-based analysis of histone acetylation. The enrichment of the TGF-ß-based protocol with butyrate or propionate potentiated the in vitro differentiation of human naïve CD4+ non-Tregs towards iTregs and augmented the suppressive capacity of the latter. These seemed to be at least partly underlain by the effects of SCFAs on the histone acetylation levels in differentiating cells. GITR, ICOS, CD39, PD-1, and PD-L1 were proven to be potential markers of human iTregs. Our results might boost the further development of Treg-based therapies against autoimmune, allergic and other chronic inflammatory disorders.

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.

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.

Decreased Histone Acetylation Levels at Th1 and Regulatory Loci after Induction of Food Allergy.

Immunoglobulin E (IgE)-mediated allergy against cow's milk protein fractions such as whey is one of the most common food-related allergic disorders of early childhood. Histone acetylation is an important epigenetic mechanism, shown to be involved in the pathogenesis of allergies. However, its role in food allergy remains unknown. IgE-mediated cow's milk allergy was successfully induced in a mouse model, as demonstrated by acute allergic symptoms, whey-specific IgE in serum, and the activation of mast cells upon a challenge with whey protein. The elicited allergic response coincided with reduced percentages of regulatory T (Treg) and T helper 17 (Th17) cells, matching decreased levels of H3 and/or H4 histone acetylation at pivotal Treg and Th17 loci, an epigenetic status favoring lower gene expression. In addition, histone acetylation levels at the crucial T helper 1 (Th1) loci were decreased, most probably preceding the expected reduction in Th1 cells after inducing an allergic response. No changes were observed for T helper 2 cells. However, increased histone acetylation levels, promoting gene expression, were observed at the signal transducer and activator of transcription 6 (Stat6) gene, a proallergic B cell locus, which was in line with the presence of whey-specific IgE. In conclusion, the observed histone acetylation changes are pathobiologically in line with the successful induction of cow's milk allergy, to which they might have also contributed mechanistically.

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.

IgE Levels to Ascaris and House Dust Mite Allergens Are Associated With Increased Histone Acetylation at Key Type-2 Immune Genes.

Background: Epigenetic changes in response to allergen exposure are still not well understood. The aim of this study was to evaluate histone acetylation levels in peripheral blood leukocytes from humans naturally infected by intestinal parasites and perennially exposed to house dust mites (HDM). Methods: Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation from 20 infected and 21 non-infected individuals living in a rural/village in Colombia. Histone 3 acetylation (H3Ac) and histone 4 acetylation (H4Ac) levels were measured in six immune genes previously associated with helminth immunity by chromatin immunoprecipitation (ChIP)-quantitative PCR. Then we analyzed the association between histone acetylation levels with total parasite egg burden and IgE levels. Results: We found an inverse correlation between H4Ac levels in the IL13 gene and egg worm burden that remained significant after adjustment by age [-0.20 (-0.32 to -0.09), p < 0.0001]. Moreover, we found significant associations between H4Ac levels in IL4 [0.32 (0.05-0.60), p = 0.02] and CHI3L1 [0.29 (0.08-0.51), p = 0.008] with the IgE levels to Ascaris lumbricoides. In addition, the levels of specific IgE antibodies to HDM were associated with H4Ac levels in the gene TNFSF13B encoding the B cell activating factor (BAFF) [0.51 (0.26-0.76), p < 0.001]. All values are presented as beta (95% CI). Conclusion: Histone acetylation levels at key type-2 immune genes in humans were modified by nematode infection and HDM allergens and are associated with the intensity of the IgE response.

Mechanisms of Dupilumab.

The Th2 cytokines interleukin 4 (IL-4) and IL-13 and the heterodimeric IL-4 receptor (IL-4R) complexes that they interact with play a key role in the pathogenesis of allergic disorders. Dupilumab is a humanized IgG4 monoclonal antibody that targets the IL-4 receptor alpha chain (IL-4Rα), common to both IL-4R complexes: type 1 (IL-4Rα/γc; IL-4 specific) and type 2 (IL-4Rα/IL-13Rα1; IL-4 and IL-13 specific). In this review, we detail the current state of knowledge of the different signalling pathways coupled to the IL-4R complexes and examine the possible mechanisms of Dupilumab action and survey its clinical efficacy in different allergic disorders. The development of Dupilumab and the widening spectrum of its clinical applications is relevant to the current emphasis on precision medicine approaches to the blockade of pathways involved in allergic diseases.