Phase 2 clinical trial evaluating abatacept in patients with steroid-refractory chronic graft-versus-host disease.

Steroid-refractory chronic graft-versus-host disease (cGVHD) after allogeneic transplant remains a significant cause of morbidity and mortality. Abatacept is a selective costimulation modulator, used for the treatment of rheumatologic diseases, and was recently the first drug to be approved by the US Food and Drug Administration for the prophylaxis of acute graft-versus-host disease. We conducted a phase 2 study to evaluate the efficacy of abatacept in steroid-refractory cGVHD. The overall response rate was 58%, seen in 21 out of 36 patients, with all responders achieving a partial response. Abatacept was well tolerated with few serious infectious complications. Immune correlative studies showed a decrease in interleukin -1α (IL-1α), IL-21, and tumor necrosis factor α as well as decreased programmed cell death protein 1 expression by CD4+ T cells in all patients after treatment with abatacept, demonstrating the effect of this drug on the immune microenvironment. The results demonstrate that abatacept is a promising therapeutic strategy for the treatment of cGVHD. This trial was registered at www.clinicaltrials.gov as #NCT01954979.

Functional immunophenotyping of blood neutrophils identifies novel endotypes of viral response in preschool children with recurrent wheezing.

BACKGROUND: Preschool children with recurrent wheezing are heterogeneous, with differing responses to respiratory viral infections. Although neutrophils are crucial for host defense, their function has not been studied in this population. OBJECTIVE: We performed functional immunophenotyping on isolated blood neutrophils from 52 preschool children with recurrent wheezing (aeroallergen sensitization, n = 16; no sensitization, n = 36). METHODS: Blood neutrophils were purified and cultured overnight with polyinosinic:polycytidylic acid [poly(I:C)] as a viral analog stimulus. Neutrophils underwent next-generation sequencing with Reactome pathway analysis and were analyzed for cytokine secretion, apoptosis, myeloperoxidase, and extracellular DNA release. CD14+ monocytes were also exposed to neutrophil culture supernatant and analyzed for markers of M1 and M2 activation. RESULTS: A total of 495 genes, related largely to the innate immune system and neutrophil degranulation, were differently expressed in children with versus without aeroallergen sensitization. Functional experiments identified more neutrophil degranulation and extracellular trap formation (ie, more myeloperoxidase and extracellular DNA) and less neutrophil proinflammatory cytokine secretion in children with aeroallergen sensitization. Neutrophils also shifted CD14+ monocytes to a more anti-inflammatory (ie, M2) phenotype in sensitized children and a more proinflammatory (ie, M1) phenotype in nonsensitized children. Although both groups experienced viral exacerbations, annualized exacerbation rates prompting unscheduled health care were also higher in children without aeroallergen sensitization after enrollment. CONCLUSIONS: Systemic neutrophil responses to viral infection differ by allergic phenotype and may be less effective in preschool children without allergic inflammation. Further studies of neutrophil function are needed in this population, which often has less favorable therapeutic responses to inhaled corticosteroids and other therapies directed at type 2-high inflammation.

Exacerbation-prone pediatric asthma is associated with arginine, lysine, and methionine pathway alterations.

BACKGROUND: The asthma of some children remains poorly controlled, with recurrent exacerbations despite treatment with inhaled corticosteroids. Aside from prior exacerbations, there are currently no reliable predictors of exacerbation-prone asthma in these children and only a limited understanding of the potential underlying mechanisms. OBJECTIVE: We sought to quantify small molecules in the plasma of children with exacerbation-prone asthma through mass spectrometry-based metabolomics. We hypothesized that the plasma metabolome of these children would differ from that of children with non-exacerbation-prone asthma. METHODS: Plasma metabolites were extracted from 4 pediatric asthma cohorts (215 total subjects, with 41 having exacerbation-prone asthma) and detected with a mass spectrometer. High-confidence annotations were retained for univariate analysis and were confirmed by a sensitivity analysis in subjects receiving high-dose inhaled corticosteroids. Metabolites that varied by cohort were excluded. MetaboAnalyst software was used to identify pathways of interest. Concentrations were calculated by reference standardization. RESULTS: We identified 32 unique, cohort-independent metabolites that differed in children with exacerbation-prone asthma compared to children with non-exacerbation-prone asthma. Comparison of metabolite concentrations to literature-reported values for healthy children revealed that most metabolites were decreased in both asthma groups, but more so in exacerbation-prone asthma. Pathway analysis identified arginine, lysine, and methionine pathways as most impacted. CONCLUSIONS: Several plasma metabolites are perturbed in children with exacerbation-prone asthma and are largely related to arginine, lysine, and methionine pathways. While validation is needed, plasma metabolites may be potential biomarkers for exacerbation-prone asthma in children.

Phase 1 clinical trial evaluating abatacept in patients with steroid-refractory chronic graft-versus-host disease.

Steroid-refractory chronic graft-versus-host disease (SR-cGVHD) remains a major cause of morbidity and mortality after allogeneic stem cell transplantation. Innovative immunotherapeutic strategies are urgently needed for the treatment of SR-cGVHD. We conducted a phase 1 clinical trial to evaluate the safety, efficacy, and immune effects of abatacept, a novel immunomodulatory drug that acts as an inhibitor of T-cell activation via costimulatory blockade, in the treatment of SR-cGVHD. The study followed a 3+3 design with 2 escalating abatacept doses: 3 mg/kg and 10 mg/kg, with an expansion cohort treated at 10 mg/kg. Abatacept was well-tolerated with no dose-limiting toxicities. Of the 16 evaluable patients, 44% achieved a clinical partial response per 2005 National Institutes of Health Consensus Criteria. Importantly, abatacept resulted in a 51.3% reduction in prednisone usage in clinical responders (mean baseline, 27 vs 14 mg; P = .01). Increased PD-1 expression on circulating CD4 (P = .009) and CD8 (P = .007) T cells was observed in clinical responders. In summary, abatacept was safe and led to a marked improvement in National Institutes of Health cGVHD scores and a significant reduction in prednisone use. In this cohort of heavily pretreated patients, the results suggest abatacept may be a promising therapeutic agent for SR-cGVHD, and a phase 2 trial has been initiated. This trial was registered at www.clinicaltrials.gov as #NCT01954979.

TGF-ß1 Suppresses the Type I IFN Response and Induces Mitochondrial Dysfunction in Alveolar Macrophages.

TGF-ß1 is a pleiotropic cytokine with an established role in fibrosis; however, the immunosuppressive effects of TGF-ß1 are less characterized. Elevated levels of TGF-ß1 are found in patients with acute and chronic lung diseases, and the underlying disease processes are exacerbated by respiratory viral infections. The alveolar macrophage is the first line of cellular defense against respiratory viral infections, and its response to infections is dependent on environmental cues. Using the mouse alveolar macrophage line, MH-S, and human CD14+ monocyte-derived macrophages, we examined the effects of TGF-ß1 on the type I IFN antiviral response, macrophage polarization, and mitochondrial bioenergetics following a challenge with human respiratory syncytial virus (RSV). Our results showed that TGF-ß1 treatment of macrophages decreased the antiviral and proinflammatory response, and suppressed basal, maximal, spare mitochondrial respiration, and mitochondrial ATP production. Challenge with RSV following TGF-ß1 treatment further exacerbated mitochondrial dysfunction. The TGF-ß1 and TGF-ß1+RSV-treated macrophages had a higher frequency of apoptosis and diminished phagocytic capacity, potentially through mitochondrial stress. Disruption of TGF-ß1 signaling or rescue of mitochondrial respiration may be novel therapeutically targetable pathways to improve macrophage function and prevent secondary bacterial infections that complicate viral respiratory infections.

Cysteine oxidation impairs systemic glucocorticoid responsiveness in children with difficult-to-treat asthma.

BACKGROUND: The mechanisms underlying glucocorticoid responsiveness are largely unknown. Although redox regulation of the glucocorticoid receptor (GR) has been reported, it has not been studied in asthmatic patients. OBJECTIVE: We characterized systemic cysteine oxidation and its association with inflammatory and clinical features in healthy children and children with difficult-to-treat asthma. We hypothesized that cysteine oxidation would be associated with increased markers of oxidative stress and inflammation, increased features of asthma severity, decreased clinically defined glucocorticoid responsiveness, and impaired GR function. METHODS: PBMCs were collected from healthy children (n = 16) and children with asthma (n = 118) aged 6 to 17 years. Children with difficult-to-treat asthma underwent glucocorticoid responsiveness testing with intramuscular triamcinolone. Cysteine, cystine, and inflammatory chemokines and reactive oxygen species generation were quantified, and expression and activity of the GR were assessed. RESULTS: Cysteine oxidation was present in children with difficult-to-treat asthma and accompanied by increased reactive oxygen species generation and increased CCL3 and CXCL1 mRNA expression. Children with the greatest extent of cysteine oxidation had more features of asthma severity, including poorer symptom control, greater medication use, and less glucocorticoid responsiveness despite inhaled glucocorticoid therapy. Cysteine oxidation also modified the GR protein by decreasing available sulfhydryl groups and decreasing nuclear GR expression and activity. CONCLUSIONS: A highly oxidized cysteine redox state promotes a posttranslational modification of the GR that might inhibit its function. Given that cysteine oxidation is prevalent in children with difficult-to-treat asthma, the cysteine redox state might represent a potential therapeutic target for restoration of glucocorticoid responsiveness in this population.

Serum and adipose tissue amino acid homeostasis in the metabolically healthy obese.

A subgroup of obese individuals, referred to as metabolically healthy obese (MHO), have preserved insulin sensitivity and a normal lipid profile despite being obese. The molecular basis for this improved cardiometabolic profile remains unclear. Our objective was to integrate metabolite and gene expression profiling to elucidate the molecular distinctions between MHO and metabolically unhealthy obese (MUO) phenotypes. A subset of individuals were selected from the Diabetes Risk Assessment study and classified into three groups using anthropometric and clinical measurements: lean healthy (LH), MHO, and MUO. Serum metabolites were profiled using gas chromatography coupled to mass spectrometry. Multivariate data analysis uncovered metabolites that differed between groups, and these were subsequently validated by capillary electrophoresis coupled to mass spectrometry. Subcutaneous adipose tissue (SAT) gene expression profiling using microarrays was performed in parallel. Amino acids were the most relevant class of metabolites distinguishing MHO from MUO individuals. Serum levels of glutamic acid, valine, and isoleucine were positively associated (i.e., LH < MHO < MUO) with homeostasis model assessment-insulin resistance (HOMA-IR) and glycated hemoglobin (HbA1c) values, while leucine was only correlated with HOMA-IR. The glutamine-to-glutamic acid ratio and glycine were inversely correlated (i.e., LH > MHO > MUO) with HbA1c values. Concomitantly, SAT gene expression profiling revealed that genes related to branched-chain amino acid catabolism and the tricarboxylic acid cycle were less down-regulated in MHO individuals compared to MUO individuals. Together, this integrated analysis revealed that MHO individuals have an intermediate amino acid homeostasis compared to LH and MUO individuals.

Dysfunctional neutrophil type 1 interferon responses in preschool children with recurrent wheezing and IL-4-mediated aeroallergen sensitization.

Background: The innate mechanisms associated with viral exacerbations in preschool children with recurrent wheezing are not understood. Objective: We sought to assess differential gene expression in blood neutrophils from preschool children with recurrent wheezing, stratified by aeroallergen sensitization, at baseline and after exposure to polyinosinic:polycytidylic acid (poly(I:C)) and also to examine whether poly(I:C)-stimulated blood neutrophils influenced airway epithelial gene expression. Methods: Blood neutrophils were purified and cultured overnight with poly(I:C) and underwent next-generation sequencing with Reactome pathway analysis. Primary human small airway epithelial cells were treated with poly(I:C)-treated neutrophil culture supernatants and were analyzed for type 1 interferon gene expression with a targeted array. Symptoms and exacerbations were assessed in participants over 12 months. Results: A total of 436 genes were differently expressed in neutrophils from children with versus without aeroallergen sensitization at baseline, with significant downregulation of type 1 interferons. These type 1 interferons were significantly upregulated in sensitized children after poly(I:C) stimulation. Confirmatory experiments demonstrated similar upregulation of type 1 interferons in IL-4-treated neutrophils stimulated with poly(I:C). Poly(I:C)-treated neutrophil supernatants from children with aeroallergen sensitization also induced a type 1 interferon response in epithelial cells. Children with aeroallergen sensitization also had higher symptom scores during exacerbations, and these symptom differences persisted for 3 days after prednisolone treatment. Conclusions: Type 1 interferon responses are dysregulated in preschool children with aeroallergen sensitization, which is in turn associated with exacerbation severity. Given the importance of type 1 interferon signaling in viral resolution, additional studies of neutrophil type 1 interferon responses are needed in this population.

HIGH THROUGHPUT QUANTITATION OF HUMAN NEUTROPHIL RECRUITMENT AND FUNCTIONAL RESPONSES IN AN AIR-BLOOD BARRIER ARRAY.

Dysregulated neutrophil recruitment drives many pulmonary diseases, but most preclinical screening methods are unsuited to evaluate pulmonary neutrophilia, limiting progress towards therapeutics. Namely, high throughput therapeutic screening systems typically exclude critical neutrophilic pathophysiology, including blood-to-lung recruitment, dysfunctional activation, and resulting impacts on the air-blood barrier. To meet the conflicting demands of physiological complexity and high throughput, we developed an assay of 96-well Leukocyte recruitment in an Air-Blood Barrier Array (L-ABBA-96) that enables in vivo -like neutrophil recruitment compatible with downstream phenotyping by automated flow cytometry. We modeled acute respiratory distress syndrome (ARDS) with neutrophil recruitment to 20 ng/mL epithelial-side interleukin 8 (IL-8) and found a dose dependent reduction in recruitment with physiologic doses of baricitinib, a JAK1/2 inhibitor recently FDA-approved for severe COVID-19 ARDS. Additionally, neutrophil recruitment to patient-derived cystic fibrosis sputum supernatant induced disease-mimetic recruitment and activation of healthy donor neutrophils and upregulated endothelial e-selectin. Compared to 24-well assays, the L-ABBA-96 reduces required patient sample volumes by 25 times per well and quadruples throughput per plate. Compared to microfluidic assays, the L-ABBA-96 recruits two orders of magnitude more neutrophils per well, enabling downstream flow cytometry and other standard biochemical assays. This novel pairing of high-throughput in vitro modeling of organ-level lung function with parallel high-throughput leukocyte phenotyping substantially advances opportunities for pathophysiological studies, personalized medicine, and drug testing applications.

Blocking of α4ß7 gut-homing integrin during acute infection leads to decreased plasma and gastrointestinal tissue viral loads in simian immunodeficiency virus-infected rhesus macaques.

Intravenous administration of a novel recombinant rhesus mAb against the α4ß7 gut-homing integrin (mAb) into rhesus macaques just prior to and during acute SIV infection resulted in significant decrease in plasma and gastrointestinal (GI) tissue viral load and a marked reduction in GI tissue proviral DNA load as compared with control SIV-infected rhesus macaques. This mAb administration was associated with increases in peripheral blood naive and central memory CD4(+) T cells and maintenance of a high frequency of CCR5(+)CD4(+) T cells. Additionally, such mAb administration inhibited the mobilization of NK cells and plasmacytoid dendritic cells characteristically seen in the control animals during acute infection accompanied by the inhibition of the synthesis of MIP-3α by the gut tissues. These data in concert suggest that blocking of GI trafficking CD4(+) T cells and inhibiting the mobilization of cell lineages of the innate immune system may be a powerful new tool to protect GI tissues and modulate acute lentiviral infection.

Airway TGF-ß1 and oxidant stress in children with severe asthma: association with airflow limitation.

BACKGROUND: TGF-ß1 is thought to play a role in airway remodeling in asthmatic subjects. TGF-ß1 expression might be mediated by an excessive burden of reactive oxygen species and oxidant stress. OBJECTIVE: Given the profound airway oxidant stress we have previously observed in children with severe asthma, we sought to (1) quantify TGF-ß1 protein and mRNA gene expression in the airways of children with mild-to-moderate and severe atopic asthma and (2) determine the relationship of airway TGF-ß1 concentrations to oxidant burden (ie, lipid peroxidation), T(H)2-mediated eosinophilic inflammation, and airflow limitation. METHODS: Bronchoalveolar lavage fluid was collected from 68 atopic children with asthma (severe asthma, n = 28) and 12 atopic adult control subjects. Airway TGF-ß1 expression and activation were assessed in relation to airway IL-13, 8-isoprostane, and malondialdehyde concentrations. The relationship of airway TGF-ß1 expression to airflow limitation in children with asthma was also assessed. RESULTS: Children with severe asthma had higher total airway concentrations of TGF-ß1 that were associated with increased protein and mRNA expression of TGF-ß1 in airway macrophages and an increase in concentrations of the lipid peroxidation biomarkers 8-isoprostanes and malondialdehyde. TGF-ß1 activation was also greater in children with severe asthma and was associated with higher airway 8-isoprostane, malondialdehyde, and IL-13 concentrations. Total airway TGF-ß1 concentrations were further associated with airflow limitation. CONCLUSIONS: Children with severe asthma have increased airway TGF-ß1 expression and activation associated with an increased airway oxidant burden. Oxidant stress might mediate the effects of TGF-ß1 and promote airway remodeling in children with severe asthma.

RNA Sequencing Analysis of CD4+ T Cells Exposed to Airway Fluid From Children With Pediatric Acute Respiratory Distress Syndrome.

CD4+ T cells contribute to lung inflammation in acute respiratory distress syndrome. The CD4+ T-cell response in pediatric acute respiratory distress syndrome (PARDS) is unknown. OBJECTIVES: To identify differentially expressed genes and networks using a novel transcriptomic reporter assay with donor CD4+ T cells exposed to the airway fluid of intubated children with mild versus severe PARDS. DESIGN: In vitro pilot study. SETTING: Laboratory-based study using human airway fluid samples admitted to a 36-bed university-affiliated pediatric intensive care unit. PATIENTS/SUBJECTS: Seven children with severe PARDS, nine children with mild PARDS, and four intubated children without lung injury as controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We performed bulk RNA sequencing using a transcriptomic reporter assay of CD4+ T cells exposed to airway fluid from intubated children to discover gene networks differentiating severe from mild PARDS. We found that innate immunity pathways, type I (α and ß), and type II (γ) interferon response and cytokine/chemokine signaling are downregulated in CD4+ T cells exposed to airway fluid from intubated children with severe PARDS compared with those with mild PARDS. CONCLUSIONS: We identified gene networks important to the PARDS airway immune response using bulk RNA sequencing from a novel CD4+ T-cell reporter assay that exposed CD4+ T cells to airway fluid from intubated children with severe and mild PARDS. These pathways will help drive mechanistic investigations into PARDS. Validation of our findings using this transcriptomic reporter assay strategy is needed.

Cluster analysis of plasma cytokines identifies two unique endotypes of children with asthma in the pediatric intensive care unit.

Children with life-threatening asthma exacerbations who are admitted to a pediatric intensive care unit (PICU) are a heterogeneous group with poorly studied inflammatory features. We hypothesized that distinct clusters of children with asthma in a PICU would be identified based on differences in plasma cytokine levels and that these clusters would have differing underlying inflammation and asthma outcomes within 1 year. Plasma cytokines and differential gene expression were measured in neutrophils isolated from children admitted to a PICU for asthma. Participants were clustered by differential plasma cytokine abundance. Gene expression differences were compared by cluster and pathway over-representation analysis was performed. We identified two clusters in 69 children with no clinical differences. Cluster 1 (n = 41) had higher cytokines compared to Cluster 2 (n = 28). Cluster 2 had a hazard ratio of 2.71 (95% CI 1.11-6.64) compared to Cluster 1 for time to subsequent exacerbation. Gene expression pathways that differed by cluster included interleukin-10 signaling; nucleotide-binding domain, leucine rich repeat containing receptor (NLR signaling); and toll-like receptor (TLR) signaling. These observations suggest that a subset of children may have a unique pattern of inflammation during PICU hospitalization that might require alternative treatment approaches.

Metabolomics identifies disturbances in arginine, phenylalanine, and glycine metabolism as differentiating features of exacerbating atopic asthma in children.

Background: Asthma exacerbations are highly prevalent in children, but only a few studies have examined the biologic mechanisms underlying exacerbations in this population. Objective: High-resolution metabolomics analyses were performed to understand the differences in metabolites in children with exacerbating asthma who were hospitalized in a pediatric intensive care unit for status asthmaticus. We hypothesized that compared with a similar population of stable outpatients with asthma, children with exacerbating asthma would have differing metabolite abundance patterns with distinct clustering profiles. Methods: A total of 98 children aged 6 through 17 years with exacerbating asthma (n = 69) and stable asthma (n = 29) underwent clinical characterization procedures and submitted plasma samples for metabolomic analyses. High-confidence metabolites were retained and utilized for pathway enrichment analyses to identify the most relevant metabolic pathways that discriminated between groups. Results: In all, 118 and 131 high-confidence metabolites were identified in positive and negative ionization mode, respectively. A total of 103 unique metabolites differed significantly between children with exacerbating asthma and children with stable asthma. In all, 8 significantly enriched pathways that were largely associated with alterations in arginine, phenylalanine, and glycine metabolism were identified. However, other metabolites and pathways of interest were also identified. Conclusion: Metabolomic analyses identified multiple perturbed metabolites and pathways that discriminated children with exacerbating asthma who were hospitalized for status asthmaticus. These results highlight the complex biology of inflammation in children with exacerbating asthma and argue for additional studies of the metabolic determinants of asthma exacerbations in children because many of the identified metabolites of interest may be amenable to targeted interventions.

Thiol redox disturbances in children with severe asthma are associated with posttranslational modification of the transcription factor nuclear factor (erythroid-derived 2)-like 2.

BACKGROUND: Airway thiol redox disturbances, including depletion of the antioxidant, glutathione, are differentiating features of severe asthma in children. OBJECTIVES: Given the role of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in maintaining glutathione homeostasis and antioxidant defense, we quantified expression and activity of Nrf2 and its downstream targets in the airways and systemic circulation of children with asthma. We hypothesized that Nrf2 activation and function would be impaired in severe asthma, resulting in depletion of thiol pools and insufficient glutathione synthesis and conjugation. METHODS: PBMCs and airway lavage cells were collected from children 6 to 17 years with severe (n = 51) and mild-to-moderate asthma (n = 38). The thiols glutathione and cysteine were quantified, and expression and activity of Nrf2 and its downstream targets were assessed. RESULTS: Children with severe asthma had greater oxidation and lower concentrations of glutathione and cysteine in the plasma and airway lavage. Although Nrf2 mRNA and protein increased in severe asthma as a function of increased thiol oxidation, the Nrf2 expressed was highly dysfunctional. Nrf2 activation and downstream targets of Nrf2 binding, including glutathione-dependent enzymes, were not different between groups. The duration of asthma was a key factor associated with Nrf2 dysfunction in severe asthma. CONCLUSION: Children with severe asthma have a global disruption of thiol redox signaling and control in both the airways and systemic circulation that is associated with posttranslational modification of Nrf2. We conclude that the Nrf2 pathway is disrupted in severe asthma as a function of chronic oxidative stress, which ultimately inhibits glutathione synthesis and antioxidant defense.

Obesity Is Associated with Sustained Symptomatology and Unique Inflammatory Features in Children with Asthma.

BACKGROUND: Obesity complicates the clinical manifestations of asthma in children. However, few studies have examined longitudinal outcomes or markers of systemic inflammation in obese asthmatic children. OBJECTIVE: We hypothesized that obese children with asthma would have: (1) poorer clinical outcomes over 12 months, (2) decreased responsiveness to systemic corticosteroid administration, (3) greater markers of systemic inflammation, and (4) unique amino acid metabolites associated with oxidative stress. METHODS: Children 6 to 17 years of age (lean, N = 257; overweight, N = 99; obese, N = 138) completed a baseline visit and follow-up visit at 12 months. Outcome measures included asthma control, quality of life, lung function, and exacerbations. A subset received intramuscular triamcinolone and were re-evaluated at 7(+7) days. Leptin, adiponectin, C-reactive protein, total cholesterol, interleukin (IL)-1ß, IL-6, IL-17, interferon gamma, tumor necrosis factor alpha, monocyte-chemoattractant protein-1, and amino acid metabolites were also quantified in plasma as potential biomarkers of outcomes in obese children. RESULTS: Obesity was associated with more symptoms, poorer quality life, and more exacerbations that persisted over 1 year despite greater medication requirements. Obese children also had minimal clinical improvement in asthma control and lung function after intramuscular triamcinolone. Leptin, C-reactive protein, and amino acid metabolites associated with glutathione synthesis and oxidative stress differed in obese children. Within the obese group, lower concentrations of arginine-related metabolites also distinguished uncontrolled from controlled asthma at 12 months. CONCLUSION: Obesity is associated with poorer asthma outcomes and unique systemic inflammatory features that may not be adequately modified with conventional asthma therapies. Novel approaches may be needed given increased symptoms and unique inflammation and oxidative stress in obese children with asthma.

Expression Patterns of Airway Fluid Cytokines From Intubated Children With Pediatric Acute Respiratory Distress Syndrome.

Pediatric acute respiratory distress syndrome (PARDS) is a heterogeneous illness affecting 6% of mechanically ventilated children and with an overall mortality of 17%. Studies in PARDS have mainly focused on plasma biomarkers which may not reflect airway biomarkers. We lack adequate understanding of the inflammatory mediators and underlying immune responses in the airways of PARDS patients. Our objective was to compare the levels of cytokines in the airway fluid of intubated children with severe versus nonsevere acute respiratory distress syndrome. DESIGN: Prospective observational cohort study. SETTING: Single 36-bed quaternary care academic safety-net hospital PICU. PATIENTS: Children intubated for acute respiratory failure between January 2018 and November 2021 stratified by Pediatric Acute Lung Injury Consensus Conference-1 criteria for PARDS. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We measured levels of 23 cytokines, chemokines, and protein biomarkers in the tracheal aspirate from 82 intubated children, between 14 days and 17 years old, at risk for or with PARDS. Levels of interleukin-4, -5, -7, -8, -12(p-70), -17a, -21, and fractalkine were higher in patients with severe versus nonsevere PARDS. There were no associations between airway and plasma cytokines. CONCLUSIONS: Proinflammatory cytokines are elevated in the airway fluid from intubated children with severe PARDS and reflect diverse patterns of airway inflammation.

Functional immunophenotyping of children with critical status asthmaticus identifies differential gene expression responses in neutrophils exposed to a poly(I:C) stimulus.

The host immune response to a viral immune stimulus has not been examined in children during a life-threatening asthma attack. We determined whether we could identify clusters of children with critical asthma by functional immunophenotyping using an intracellular viral analog stimulus. We performed a single-center, prospective, observational cohort study of 43 children ages 6-17 years admitted to a pediatric intensive care unit for an asthma attack between July 2019 to February 2021. Neutrophils were isolated from children, stimulated overnight with LyoVec poly(I:C), and mRNA was analyzed using a targeted Nanostring immunology array. Network analysis of the differentially expressed transcripts for the paired LyoVec poly(I:C) samples was performed. We identified two clusters by functional immunophenotyping that differed by the Asthma Control Test score. Cluster 1 (n = 23) had a higher proportion of children with uncontrolled asthma in the four weeks prior to PICU admission compared with cluster 2 (n = 20). Pathways up-regulated in cluster 1 versus cluster 2 included chemokine receptor/chemokines, interleukin-10 (IL-10), IL-4, and IL-13 signaling. Larger validation studies and clinical phenotyping of children with critical asthma are needed to determine the predictive utility of these clusters in a larger clinical setting.

Decreased NK cell frequency and function is associated with increased risk of KIR3DL allele polymorphism in simian immunodeficiency virus-infected rhesus macaques with high viral loads.

NK cells have been established as an important effector of innate immunity in a variety of viral infections. In HIV-1 infection in humans, alterations of NK cell function, frequency, and expression of various NK receptors have been reported to be associated with differential dynamics of disease progression. Expression of certain alleles of KIR3DL and KIR3DS receptors on NK cells was shown to correlate with levels of virus replication. In the SIV-infected rhesus macaque (RM) model of AIDS, several families of killer inhibitory Ig-related receptors (KIR receptors) corresponding to their human counterparts have been characterized, but only at the level of individual sequence variants. Here we define 14 different alleles of KIR3DL expressed among 38 SIV-infected RM, characterized by either high or low levels of SIV replication, by analyzing multiple sequences from individual animals and show an unequal distribution of certain alleles in these cohorts. High levels of SIV replication were associated with significant increases in KIR3DL mRNA levels in addition to decreases in both the frequency and function of NK cells in these animals. The higher frequency of inheritance of two KIR3DL alleles characterized by a single nucleotide polymorphism 159 H/Q was associated with RM that exhibited high plasma viral load. This data for the first time defines multiple alleles of KIR3DL in RM and shows an association between virus control, NK cell function and genetic polymorphisms of KIR receptors.