Sphingolipids in Childhood Asthma and Obesity (SOAP Study): A Protocol of a Cross-Sectional Study.

Asthma and obesity are two of the most common chronic conditions in children and adolescents. There is increasing evidence that sphingolipid metabolism is altered in childhood asthma and is linked to airway hyperreactivity. Dysregulated sphingolipid metabolism is also reported in obesity. However, the functional link between sphingolipid metabolism, asthma, and obesity is not completely understood. This paper describes the protocol of an ongoing study on sphingolipids that aims to examine the pathophysiology of sphingolipids in childhood asthma and obesity. In addition, this study aims to explore the novel biomarkers through a comprehensive multi-omics approach including genomics, genome-wide DNA methylation, RNA-Seq, microRNA (miRNA) profiling, lipidomics, metabolomics, and cytokine profiling. This is a cross-sectional study aiming to recruit 440 children from different groups: children with asthma and normal weight (n = 100), asthma with overweight or obesity (n = 100), overweight or obesity (n = 100), normal weight (n = 70), and siblings of asthmatic children with normal weight, overweight, or obesity (n = 70). These participants will be recruited from the pediatric pulmonology, pediatric endocrinology, and general pediatric outpatient clinics at Sidra Medicine, Doha, Qatar. Information will be obtained from self-reported questionnaires on asthma, quality of life, food frequency (FFQ), and a 3-day food diary that are completed by the children and their parents. Clinical measurements will include anthropometry, blood pressure, biochemistry, bioelectrical impedance, and pulmonary function tests. Blood samples will be obtained for sphingolipid analysis, serine palmitoyltransferase (SPT) assay, whole-genome sequencing (WGS), genome-wide DNA methylation study, RNA-Seq, miRNA profiling, metabolomics, lipidomics, and cytokine analysis. Group comparisons of continuous outcome variables will be carried out by a one-way analysis of variance or the Kruskal-Wallis test using an appropriate pairwise multiple comparison test. The chi-squared test or a Fisher's exact test will be used to test the associations between categorical variables. Finally, multivariate analysis will be carried out to integrate the clinical data with multi-omics data. This study will help us to understand the role of dysregulated sphingolipid metabolism in obesity and asthma. In addition, the multi-omics data from the study will help to identify novel genetic and epigenetic signatures, inflammatory markers, and mechanistic pathways that link asthma and obesity in children. Furthermore, the integration of clinical and multi-omics data will help us to uncover the potential interactions between these diseases and to offer a new paradigm for the treatment of pediatric obesity-associated asthma.

Fungal microbiota sustains lasting immune activation of neutrophils and their progenitors in severe COVID-19.

Gastrointestinal fungal dysbiosis is a hallmark of several diseases marked by systemic immune activation. Whether persistent pathobiont colonization during immune alterations and impaired gut barrier function has a durable impact on host immunity is unknown. We found that elevated levels of Candida albicans immunoglobulin G (IgG) antibodies marked patients with severe COVID-19 (sCOVID-19) who had intestinal Candida overgrowth, mycobiota dysbiosis and systemic neutrophilia. Analysis of hematopoietic stem cell progenitors in sCOVID-19 revealed transcriptional changes in antifungal immunity pathways and reprogramming of granulocyte myeloid progenitors (GMPs) for up to a year. Mice colonized with C. albicans patient isolates experienced increased lung neutrophilia and pulmonary NETosis during severe acute respiratory syndrome coronavirus-2 infection, which were partially resolved with antifungal treatment or by interleukin-6 receptor blockade. sCOVID-19 patients treated with tocilizumab experienced sustained reductions in C. albicans IgG antibodies titers and GMP transcriptional changes. These findings suggest that gut fungal pathobionts may contribute to immune activation during inflammatory diseases, offering potential mycobiota-immune therapeutic strategies for sCOVID-19 with prolonged symptoms.

The mast cell exosome-fibroblast connection: A novel pro-fibrotic pathway.

Introduction: In addition to the traditional activation of resident receptors by release of local mediators, new evidence favors the existence of exosomes in cell-to-cell communication that mediates delivery of specific cargo to modulate recipient cell function. We report that mast cell exosomes are an additional source of pro-fibrotic substances and constitute a unique pathway for the generation of excess collagen. Methods: We use primary human lung fibroblasts (HLFs) to demonstrate the uptake of labeled exosomes isolated from the human mast cell line HMC-1 (MC-EXOs), previously shown to contain protein cargo in common with human mast cell exosomes. Results: The MC-EXO uptake by HLF is to the cytosol and increases both proline hydroxylation in HLF lysate and secreted collagen, within 24 h, which is sustained over 72 h, the same time required for transforming growth factor-ß (TGF-ß) to activate collagen synthesis in the HLFs. Unlike TGF-ß, MC-EXO uptake does not induce fibrillar gene activation or invoke the Smad-nuclear transcription pathway. We show that MC-EXO uptake and TGF-ß have an additive effect on collagen synthesis in HLF and postulate that MC-EXO uptake by HLFs is a contributing factor to excess collagen synthesis and represents a unique paradigm for understanding fibrosis. Discussion: It is known that, in the lungs, mast cells are more activated and increase in number with inflammation, injury and viral infection associated with fibrosis. With the reported increased incidence of post-COVID-pulmonary fibrosis (PCPF), data from patients with severe COVID-19 are presented that show an increase in the mast cell number in lung parenchyma, the site of PCPF. Our findings provide a rationale for targeting multiple fibrogenic pathways in the management of lung fibrosis and the use of mast cell exosomes as a biomarker for the prognostic and diagnostic management of evolving fibrotic lung disease.

Inulin fibre promotes microbiota-derived bile acids and type 2 inflammation.

Dietary fibres can exert beneficial anti-inflammatory effects through microbially fermented short-chain fatty acid metabolites^1,2, although the immunoregulatory roles of most fibre diets and their microbiota-derived metabolites remain poorly defined. Here, using microbial sequencing and untargeted metabolomics, we show that a diet of inulin fibre alters the composition of the mouse microbiota and the levels of microbiota-derived metabolites, notably bile acids. This metabolomic shift is associated with type 2 inflammation in the intestine and lungs, characterized by IL-33 production, activation of group 2 innate lymphoid cells and eosinophilia. Delivery of cholic acid mimics inulin-induced type 2 inflammation, whereas deletion of the bile acid receptor farnesoid X receptor diminishes the effects of inulin. The effects of inulin are microbiota dependent and were reproduced in mice colonized with human-derived microbiota. Furthermore, genetic deletion of a bile-acid-metabolizing enzyme in one bacterial species abolishes the ability of inulin to trigger type 2 inflammation. Finally, we demonstrate that inulin enhances allergen- and helminth-induced type 2 inflammation. Taken together, these data reveal that dietary inulin fibre triggers microbiota-derived cholic acid and type 2 inflammation at barrier surfaces with implications for understanding the pathophysiology of allergic inflammation, tissue protection and host defence.

Dietary long-chain omega 3 fatty acids modify sphingolipid metabolism to facilitate airway hyperreactivity.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are essential nutrients that can affect inflammatory responses. While n-3 PUFAs are generally considered beneficial for cardiovascular disease and obesity, the effects on asthma, the most common inflammatory lung disease are unclear. While prenatal dietary n-3 PUFAs decrease the risk for childhood wheezing, postnatal dietary n-3 PUFAs can worsen allergic airway inflammation. Sphingolipid metabolism is also affected by dietary n-3 PUFAs. Decreased sphingolipid synthesis leads to airway hyperreactivity, besides inflammation, a cardinal feature of asthma, and common genetic asthma risk alleles lead to lower sphingolipid synthesis. We investigated the effect of dietary n-3 PUFAs on sphingolipid metabolism and airway reactivity. Comparing a fish-oil diet with a high n-3 PUFA content (FO) to an isocaloric coconut oil-enriched diet (CO), we found an n-3 PUFA-dependent effect on increased airway reactivity, that was not accompanied by inflammation. Lung and whole blood content of dihydroceramides, ceramides, sphingomyelins, and glucosylceramides were lower in mice fed the n-3 PUFA enriched diet consistent with lower sphingolipid synthesis. In contrast, phosphorylated long chain bases such as sphingosine 1-phosphate were increased. These findings suggest that dietary n-3 PUFAs affect pulmonary sphingolipid composition to favor innate airway hyperreactivity, independent of inflammation, and point to an important role of n-3 PUFAs in sphingolipid metabolism.

Chronic reduction of store operated Ca2+ entry is viable therapeutically but is associated with cardiovascular complications.

Loss of function mutations in store-operated Ca2+ entry (SOCE) are associated with severe paediatric disorders in humans, including combined immunodeficiency, anaemia, thrombocytopenia, anhidrosis and muscle hypotonia. Given its central role in immune cell activation, SOCE has been a therapeutic target for autoimmune and inflammatory diseases. Treatment for such chronic diseases would require prolonged SOCE inhibition. It is, however, unclear whether chronic SOCE inhibition is viable therapeutically. Here we address this issue using a novel genetic mouse model (SOCE hypomorph) with deficient SOCE, nuclear factor of activated T cells activation, and T cell cytokine production. SOCE hypomorph mice develop and reproduce normally and do not display muscle weakness or overt anhidrosis. They do, however, develop cardiovascular complications, including hypertension and tachycardia, which we show are due to increased sympathetic autonomic nervous system activity and not cardiac or vascular smooth muscle autonomous defects. These results assert that chronic SOCE inhibition is viable therapeutically if the cardiovascular complications can be managed effectively clinically. They further establish the SOCE hypomorph line as a genetic model to define the therapeutic window of SOCE inhibition and dissect toxicities associated with chronic SOCE inhibition in a tissue-specific fashion. KEY POINTS: A floxed stromal interaction molecule 1 (STIM1) hypomorph mouse model was generated with significant reduction in Ca2+ influx through store-operated Ca2+ entry (SOCE), resulting in defective nuclear translocation of nuclear factor of activated T cells, cytokine production and inflammatory response. The hypomorph mice are viable and fertile, with no overt defects. Decreased SOCE in the hypomorph mice is due to poor translocation of the mutant STIM1 to endoplasmic reticulum-plasma membrane contact sites resulting in fewer STIM1 puncta. Hypomorph mice have similar susceptibility to controls to develop diabetes but exhibit tachycardia and hypertension. The hypertension is not due to increased vascular smooth muscle contractility or vascular remodelling. The tachycardia is not due to heart-specific defects but rather seems to be due to increased circulating catecholamines in the hypomorph. Therefore, long term SOCE inhibition is viable if the cardiovascular defects can be managed clinically.

Human Rhinovirus Infection of the Respiratory Tract Affects Sphingolipid Synthesis.

The 17q21 asthma susceptibility locus includes asthma risk alleles associated with decreased sphingolipid synthesis, likely resulting from increased expression of ORMDL3. ORMDL3 inhibits serine-palmitoyl transferase (SPT), the rate-limiting enzyme of de novo sphingolipid synthesis. There is evidence that decreased sphingolipid synthesis is critical to asthma pathogenesis. Children with asthma and 17q21 asthma risk alleles display decreased sphingolipid synthesis in blood cells. Reduced SPT activity results in airway hyperreactivity, a hallmark feature of asthma. 17q21 asthma risk alleles are also linked to childhood infections with human rhinovirus (RV). This study evaluates the interaction of RV with the de novo sphingolipid synthesis pathway, and the alterative effects of concurrent SPT inhibition in SPT-deficient mice and human airway epithelial cells. In mice, RV infection shifted lung sphingolipid synthesis gene expression to a pattern that resembles genetic SPT deficiency, including decreased expression of Sptssa, a small SPT subunit. This pattern was pronounced in lung epithelial cellular adhesion molecule (EpCAM+) cells and reproduced in human bronchial epithelial cells. RV did not affect Sptssa expression in lung CD45+ immune cells. RV increased sphingolipids unique to the de novo synthesis pathway in mouse lung and human airway epithelial cells. Interestingly, these de novo sphingolipid species were reduced in the blood of RV-infected wild-type mice. RV exacerbated SPT deficiency-associated airway hyperreactivity. Airway inflammation was similar in RV-infected wild-type and SPT-deficient mice. This study reveals the effects of RV infection on the de novo sphingolipid synthesis pathway, elucidating a potential mechanistic link between 17q21 asthma risk alleles and rhinoviral infection.

Perinatal origins of chronic lung disease: mechanisms-prevention-therapy-sphingolipid metabolism and the genetic and perinatal origins of childhood asthma.

Childhood asthma derives from complex host-environment interactions occurring in the perinatal and infant period, a critical time for lung development. Sphingolipids are bioactive molecules consistently implicated in the pathogenesis of childhood asthma. Genome wide association studies (GWAS) initially identified a link between alleles within the 17q21 asthma-susceptibility locus, childhood asthma, and overexpression of the ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3), an inhibitor of de novo sphingolipid synthesis. Subsequent studies of pediatric asthma offer strong evidence that these asthma-risk alleles correlate with early-life aberrancies of sphingolipid homeostasis and asthma. Relationships between sphingolipid metabolism and asthma-related risk factors, including maternal obesity and respiratory viral infections, are currently under investigation. This review will summarize how these perinatal and early life exposures can synergize with 17q21 asthma risk alleles to exacerbate disruptions of sphingolipid homeostasis and drive asthma pathogenesis.

ILC3s control airway inflammation by limiting T cell responses to allergens and microbes.

Group 3 innate lymphoid cells (ILC3s) critically regulate host-microbe interactions in the gastrointestinal tract, but their role in the airway remains poorly understood. Here, we demonstrate that lymphoid-tissue-inducer (LTi)-like ILC3s are enriched in the lung-draining lymph nodes of healthy mice and humans. These ILC3s abundantly express major histocompatibility complex class II (MHC class II) and functionally restrict the expansion of allergen-specific CD4+ T cells upon experimental airway challenge. In a mouse model of house-dust-mite-induced allergic airway inflammation, MHC class II+ ILC3s limit T helper type 2 (Th2) cell responses, eosinophilia, and airway hyperresponsiveness. Furthermore, MHC class II+ ILC3s limit a concomitant Th17 cell response and airway neutrophilia. This exacerbated Th17 cell response requires exposure of the lung to microbial stimuli, which can be found associated with house dust mites. These findings demonstrate a critical role for antigen-presenting ILC3s in orchestrating immune tolerance in the airway by restricting pro-inflammatory T cell responses to both allergens and microbes.

Adeno-Associated Virus Vector-Mediated Expression of Antirespiratory Syncytial Virus Antibody Prevents Infection in Mouse Airways.

Infants and older adults are especially vulnerable to infection by respiratory syncytial virus (RSV), which can cause significant illness and irreparable damage to the lower respiratory tract and for which an effective vaccine is not readily available. Palivizumab, a recombinant monoclonal antibody (mAb), is an approved therapeutic for RSV infection for use in high-risk infants only. Due to several logistical issues, including cost of goods and scale-up limitations, palivizumab is not approved for other populations that are vulnerable to severe RSV infections, such as older adults. In this study, we demonstrate that intranasal delivery of adeno-associated virus serotype 9 (AAV9) vector expressing palivizumab or motavizumab, a second-generation version of palivizumab, significantly reduced the viral load in the lungs of the BALB/c mouse model of RSV infection. Notably, we demonstrate that AAV9 vector-mediated prophylaxis against RSV was effective despite the presence of serum-circulating neutralizing AAV9 antibodies. These findings substantiate the feasibility of repeatedly administering AAV9 vector to the airway for seasonal prophylaxis against RSV, thereby expanding the application of vectored delivery of mAbs as an effective prophylaxis strategy against various airborne viruses.

Slowing late infantile Batten disease by direct brain parenchymal administration of a rh.10 adeno-associated virus expressing CLN2.

Late infantile Batten disease (CLN2 disease) is an autosomal recessive, neurodegenerative lysosomal storage disease caused by mutations in the CLN2 gene encoding tripeptidyl peptidase 1 (TPP1). We tested intraparenchymal delivery of AAVrh.10hCLN2, a nonhuman serotype rh.10 adeno-associated virus vector encoding human CLN2, in a nonrandomized trial consisting of two arms assessed over 18 months: AAVrh.10hCLN2-treated cohort of 8 children with mild to moderate disease and an untreated, Weill Cornell natural history cohort consisting of 12 children. The treated cohort was also compared to an untreated European natural history cohort of CLN2 disease. The vector was administered through six burr holes directly to 12 sites in the brain without immunosuppression. In an additional safety assessment under a separate protocol, five children with severe CLN2 disease were treated with AAVrh.10hCLN2. The therapy was associated with a variety of expected adverse events, none causing long-term disability. Induction of systemic anti-AAVrh.10 immunity was mild. After therapy, the treated cohort had a 1.3- to 2.6-fold increase in cerebral spinal fluid TPP1. There was a slower loss of gray matter volume in four of seven children by MRI and a 42.4 and 47.5% reduction in the rate of decline of motor and language function, compared to Weill Cornell natural history cohort (P < 0.04) and European natural history cohort (P < 0.0001), respectively. Intraparenchymal brain administration of AAVrh.10hCLN2 slowed the progression of disease in children with CLN2 disease. However, improvements in vector design and delivery strategies will be necessary to halt disease progression using gene therapy.

Increasing Sphingolipid Synthesis Alleviates Airway Hyperreactivity.

Impaired sphingolipid synthesis is linked genetically to childhood asthma and functionally to airway hyperreactivity (AHR). The objective was to investigate whether sphingolipid synthesis could be a target for asthma therapeutics. The effects of GlyH-101 and fenretinide via modulation of de novo sphingolipid synthesis on AHR was evaluated in mice deficient in SPT (serine palmitoyl-CoA transferase), the rate-limiting enzyme of sphingolipid synthesis. The drugs were also used directly in human airway smooth-muscle and epithelial cells to evaluate changes in de novo sphingolipid metabolites and calcium release. GlyH-101 and fenretinide increased sphinganine and dihydroceramides (de novo sphingolipid metabolites) in lung epithelial and airway smooth-muscle cells, decreased the intracellular calcium concentration in airway smooth-muscle cells, and decreased agonist-induced contraction in proximal and peripheral airways. GlyH-101 also decreased AHR in SPT-deficient mice in vivo. This study identifies the manipulation of sphingolipid synthesis as a novel metabolic therapeutic strategy to alleviate AHR.

Decreased sphingolipid synthesis in children with 17q21 asthma-risk genotypes.

Risk for childhood asthma is conferred by alleles within the 17q21 locus affecting ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) expression. ORMDL3 inhibits sphingolipid de novo synthesis. Although the effects of 17q21 genotypes on sphingolipid synthesis in human asthma remain unclear, both decreased sphingolipid synthesis and ORMDL3 overexpression are linked to airway hyperreactivity. To characterize the relationship of genetic asthma susceptibility with sphingolipid synthesis, we analyzed asthma-associated 17q21 genotypes (rs7216389, rs8076131, rs4065275, rs12603332, and rs8067378) in both children with asthma and those without asthma, quantified plasma and whole-blood sphingolipids, and assessed sphingolipid de novo synthesis in peripheral blood cells by measuring the incorporation of stable isotope-labeled serine (substrate) into sphinganine and sphinganine-1-phosphate. Whole-blood dihydroceramides and ceramides were decreased in subjects with the 17q21 asthma-risk alleles rs7216389 and rs8076131. Children with nonallergic asthma had lower dihydroceramides, ceramides, and sphingomyelins than did controls. Children with allergic asthma had higher dihydroceramides, ceramides, and sphingomyelins compared with children with nonallergic asthma. Additionally, de novo sphingolipid synthesis was lower in children with asthma compared with controls. These findings connect genetic 17q21 variations that are associated with asthma risk and higher ORMDL3 expression to lower sphingolipid synthesis in humans. Altered sphingolipid synthesis may therefore be a critical factor in asthma pathogenesis and may guide the development of future therapeutics.

Sphingosine 1-Phosphate Receptor 1 Signaling Maintains Endothelial Cell Barrier Function and Protects Against Immune Complex-Induced Vascular Injury.

OBJECTIVE: Immune complex (IC) deposition activates polymorphonuclear neutrophils (PMNs), increases vascular permeability, and leads to organ damage in systemic lupus erythematosus and rheumatoid arthritis. The bioactive lipid sphingosine 1-phosphate (S1P), acting via S1P receptor 1 (S1P1 ), is a key regulator of endothelial cell (EC) barrier function. This study was undertaken to investigate whether augmenting EC integrity via S1P1 signaling attenuates inflammatory injury mediated by ICs. METHODS: In vitro barrier function was assessed in human umbilical vein endothelial cells (HUVECs) by electrical cell-substrate impedance sensing. Phosphorylation of myosin light chain 2 (p-MLC-2) and VE-cadherin staining in HUVECs were assessed by immunofluorescence. A reverse Arthus reaction (RAR) was induced in the skin and lungs of mice with S1P1 deleted from ECs (S1P1 EC-knockout [ECKO] mice) and mice treated with S1P1 agonists and antagonists. RESULTS: S1P1 agonists prevented loss of barrier function in HUVECs treated with IC-activated PMNs. S1P1 ECKO and wild-type (WT) mice treated with S1P1 antagonists had amplified RAR, whereas specific S1P1 agonists attenuated skin and lung RAR in WT mice. ApoM-Fc, a novel S1P chaperone, mitigated EC cell barrier dysfunction induced by activated PMNs in vitro and attenuated lung RAR. Expression levels of p-MLC-2 and disruption of VE-cadherin, each representing manifestations of cell contraction and destabilization of adherens junctions, respectively, that were induced by activated PMNs, were markedly reduced by treatment with S1P1 agonists and ApoM-Fc. CONCLUSION: Our findings indicate that S1P1 signaling in ECs modulates vascular responses to IC deposition. S1P1 agonists and ApoM-Fc enhance the EC barrier, limit leukocyte escape from capillaries, and provide protection against inflammatory injury. The S1P/S1P1 axis is a newly identified target to attenuate tissue responses to IC deposition and mitigate end-organ damage.

Neonatal Genetic Delivery of Anti-Respiratory Syncytial Virus (RSV) Antibody by Non-Human Primate-Based Adenoviral Vector to Provide Protection against RSV.

Respiratory syncytial virus (RSV) is one of the leading causes of lower respiratory tract infection in infants. Immunoprophylaxis with the anti-RSV monoclonal antibody, palivizumab, reduces the risk for RSV-related hospitalizations, but its use is restricted to high-risk infants due to the high costs. In this study, we investigated if genetic delivery of anti-RSV antibody to neonatal mice by chimpanzee adenovirus type 7 expressing the murine form of palivizumab (AdC7αRSV) can provide protection against RSV. Intranasal and intramuscular administration of AdC7αRSV to adult mice resulted in similar levels of anti-RSV IgG in the serum. However, only intranasal administration resulted in detectable levels of anti-RSV IgG in the bronchoalveolar lavage fluid. Intranasal administration of AdC7αRSV provided protection against subsequent RSV challenge. Expression of the anti-RSV antibody was prolonged following intranasal administration of AdC7αRSV to neonatal mice. Protection against RSV was confirmed at 6 weeks of age. These data suggest that neonatal genetic delivery of anti-RSV antibody by AdC7αRSV can provide protection against RSV.