Maternal diet modulates the infant microbiome and intestinal Flt3L necessary for dendritic cell development and immunity to respiratory infection.

Poor maternal diet during pregnancy is a risk factor for severe lower respiratory infections (sLRIs) in the offspring, but the underlying mechanisms remain elusive. Here, we demonstrate that in mice a maternal low-fiber diet (LFD) led to enhanced LRI severity in infants because of delayed plasmacytoid dendritic cell (pDC) recruitment and perturbation of regulatory T cell expansion in the lungs. LFD altered the composition of the maternal milk microbiome and assembling infant gut microbiome. These microbial changes reduced the secretion of the DC growth factor Flt3L by neonatal intestinal epithelial cells and impaired downstream pDC hematopoiesis. Therapy with a propionate-producing bacteria isolated from the milk of high-fiber diet-fed mothers, or supplementation with propionate, conferred protection against sLRI by restoring gut Flt3L expression and pDC hematopoiesis. Our findings identify a microbiome-dependent Flt3L axis in the gut that promotes pDC hematopoiesis in early life and confers disease resistance against sLRIs.

CXCR3+ effector regulatory T cells associate with disease tolerance during lower respiratory pneumovirus infection.

Lifestyle factors like poor maternal diet or antibiotic exposure disrupt early life microbiome assembly in infants, increasing the risk of severe lower respiratory infections (sLRI). Our prior studies in mice indicated that a maternal low-fibre diet (LFD) exacerbates LRI severity in infants by impairing recruitment of plasmacytoid dendritic cells (pDC) and consequently attenuating expansion of lung regulatory T (Treg) cells during pneumonia virus of mice (PVM) infection. Here, we investigated whether maternal dietary fibre intake influences Treg cell phenotypes in the mediastinal lymph nodes (mLN) and lungs of PVM-infected neonatal mice. Using high dimensional flow cytometry, we identified distinct clusters of regulatory T cells (Treg cells), which differed between lungs and mLN during infection, with notably greater effector Treg cell accumulation in the lungs. Compared to high-fibre diet (HFD)-reared pups, frequencies of various effector Treg cell subsets were decreased in the lungs of LFD-reared pups. Particularly, recruitment of chemokine receptor 3 (CXCR3+) expressing Treg cells was attenuated in LFD-reared pups, correlating with lower lung expression of CXCL9 and CXCL10 chemokines. The recruitment of this subset in response to PVM infection was similarly impaired in pDC depleted mice or following anti-CXCR3 treatment, increasing immunopathology in the lungs. In summary, PVM infection leads to the sequential recruitment and expansion of distinct Treg cell subsets to the lungs and mLN. The attenuated recruitment of the CXCR3+ subset in LFD-reared pups increases LRI severity, suggesting that strategies to enhance pDCs or CXCL9/CXCL10 expression will lower immune-mediated pathogenesis.

IFN-λ Diminishes the Severity of Viral Bronchiolitis in Neonatal Mice by Limiting NADPH Oxidase-Induced PAD4-Independent NETosis.

Infants with attenuated type III IFN (IFN-λ) responses are at increased risk of severe lower respiratory tract infection (sLRI). The IL-28Rα-chain and IL-10Rß-chain form a heterodimeric receptor complex, necessary for IFN-λ signaling. Therefore, to better understand the immunopathogenic mechanisms through which an IFN-λlo microenvironment predisposes to a sLRI, we inoculated neonatal wild-type and IL-28R-deficient (IL-28R -/-) mice with pneumonia virus of mice, a rodent-specific pneumovirus. Infected IL-28R -/- neonates displayed an early, pronounced, and persistent neutrophilia that was associated with enhanced reactive oxygen species (ROS) production, NETosis, and mucus hypersecretion. Targeted deletion of the IL-28R in neutrophils was sufficient to increase neutrophil activation, ROS production, NET formation, and mucus production in the airways. Inhibition of protein-arginine deiminase type 4 (PAD4), a regulator of NETosis, had no effect on myeloperoxidase expression, citrullinated histones, and the magnitude of the inflammatory response in the lungs of infected IL-28R -/- mice. In contrast, inhibition of ROS production decreased NET formation, cellular inflammation, and mucus hypersecretion. These data suggest that IFN-λ signaling in neutrophils dampens ROS-induced NETosis, limiting the magnitude of the inflammatory response and mucus production. Therapeutics that promote IFN-λ signaling may confer protection against sLRI.

Macrophage migration inhibitory factor promotes glucocorticoid resistance of neutrophilic inflammation in a murine model of severe asthma.

BACKGROUND: Severe neutrophilic asthma is resistant to treatment with glucocorticoids. The immunomodulatory protein macrophage migration inhibitory factor (MIF) promotes neutrophil recruitment to the lung and antagonises responses to glucocorticoids. We hypothesised that MIF promotes glucocorticoid resistance of neutrophilic inflammation in severe asthma. METHODS: We examined whether sputum MIF protein correlated with clinical and molecular characteristics of severe neutrophilic asthma in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort. We also investigated whether MIF regulates neutrophilic inflammation and glucocorticoid responsiveness in a murine model of severe asthma in vivo. RESULTS: MIF protein levels positively correlated with the number of exacerbations in the previous year, sputum neutrophils and oral corticosteroid use across all U-BIOPRED subjects. Further analysis of MIF protein expression according to U-BIOPRED-defined transcriptomic-associated clusters (TACs) revealed increased MIF protein and a corresponding decrease in annexin-A1 protein in TAC2, which is most closely associated with airway neutrophilia and NLRP3 inflammasome activation. In a murine model of severe asthma, treatment with the MIF antagonist ISO-1 significantly inhibited neutrophilic inflammation and increased glucocorticoid responsiveness. Coimmunoprecipitation studies using lung tissue lysates demonstrated that MIF directly interacts with and cleaves annexin-A1, potentially reducing its biological activity. CONCLUSION: Our data suggest that MIF promotes glucocorticoid-resistance of neutrophilic inflammation by reducing the biological activity of annexin-A1, a potent glucocorticoid-regulated protein that inhibits neutrophil accumulation at sites of inflammation. This represents a previously unrecognised role for MIF in the regulation of inflammation and points to MIF as a potential therapeutic target for the management of severe neutrophilic asthma.

Diagnostic evaluation of upper tract urothelial carcinoma: can we safely omit diagnostic ureteroscopy?

OBJECTIVE: To identify clinicopathological or radiological factors that may predict a diagnosis of upper urinary tract urothelial cell carcinoma (UTUC) to inform which patients can proceed directly to radical nephroureterectomy (RNU) without the delay for diagnostic ureteroscopy (URS). PATIENTS AND METHODS: All consecutive patients investigated for suspected UTUC in a high-volume UK centre between 2011 and 2017 were identified through retrospective analysis of surgical logbooks and a prospectively maintained pathology database. Details on clinical presentation, radiological findings, and URS/RNU histopathology results were evaluated. Multivariate regression analysis was performed to evaluate predictors of a final diagnosis of UTUC. RESULTS: In all, 260 patients were investigated, of whom 230 (89.2%) underwent URS. RNU was performed in 131 patients (50.4%), of whom 25 (9.6%) proceeded directly without URS - all of whom had a final histopathological diagnosis of UTUC - and 15 (11.5%) underwent RNU after URS despite no conclusive histopathological confirmation of UTUC. Major surgery was avoided in 77 patients (33.5%) where a benign or alternative diagnosis was made on URS, and 14 patients (6.1%) underwent nephron-sparing surgery. Overall, 178 patients (68.5%) had a final diagnosis of UTUC confirmed on URS/RNU histopathology. On multivariate logistic regression analysis, a presenting complaint of visible haematuria (hazard ratio [HR] 5.17, confidence interval [CI] 1.91-14.0; P = 0.001), a solid lesion reported on imaging (HR 37.8, CI = 11.7-122.1; P < 0.001) and a history of smoking (HR 3.07, CI 1.35-6.97; P = 0.007), were predictive of a final diagnosis of UTUC. From this cohort, 51 (96.2%) of 53 smokers who presented with visible haematuria and who had a solid lesion on computed tomography urogram had UTUC on final histopathology. CONCLUSION: We identified specific factors which may assist clinicians in selecting which patients may reliably proceed to RNU without the delay of diagnostic URS. These findings may inform a prospective multicentre analysis including additional variables such as urinary cytology.

Assessment of association between lower ureteric excision technique and oncological outcomes for upper urinary tract urothelial carcinoma: retrospective analysis from the Scottish Renal Cancer Consortium.

PURPOSE: Nephroureterectomy(NU) remains the gold-standard surgical option for the management of upper urinary tract urothelial carcinoma(UTUC). Controversy exists regarding the optimal excision technique of the lower ureter. We sought to compare post-UTUC bladder tumour recurrence across the Scottish Renal Cancer Consortium(SRCC). METHODS: Patients who underwent NU for UTUC across the SRCC 2012-2019 were identified. The impact of lower-end surgical technique along with T-stage, N-stage, tumour location and focality, positive surgical margin, pre-NU ureteroscopy, upper-end technique and adjuvant mitomycin C administration were assessed by Kaplan-Meier and Cox-regression. The primary outcome was intra-vesical recurrence-free survival (B-RFS). RESULTS: In 402 patients, the median follow-up was 29 months. The lower ureter was managed by open transvesical excision in 90 individuals, transurethral and laparoscopic dissection in 76, laparoscopic or open extra-vesical excision in 31 and 42 respectively, and transurethral dissection and pluck in 163. 114(28.4%) patients had a bladder recurrence during follow-up. There was no difference in B-RFS between lower-end techniques by Kaplan-Meier (p = 0.94). When all factors were taken into account by adjusted Cox-regression, preceding ureteroscopy (HR 2.65, p = 0.001), lower ureteric tumour location (HR 2.16, p = 0.02), previous bladder cancer (HR 1.75, p = 0.01) and male gender (HR 1.61, p = 0.03) were associated with B-RFS. CONCLUSION: These data suggest in appropriately selected patients, lower ureteric management technique does not affect B-RFS. Along with lower ureteric tumour location, male gender and previous bladder cancer, preceding ureteroscopy was associated with a higher recurrence rate following NU, and the indication for this should be carefully considered.

State of the art: Intrapartum antibiotics in cesarean section, the infant microbiota and allergic diseases.

With increasing rates of cesarean section worldwide and international guidelines advising pre-incision antibiotics, neonatal exposure to pre-birth antibiotics is higher than ever before. Emerging evidence has raised concern regarding the impact of such antibiotics on the neonatal intestinal microbiota, immune system development and health conditions later in life. This narrative review investigates current protocols for intrapartum prophylactic antibiotics in cesarean section, how this and other factors may affect the neonatal intestinal microbiota and whether intrapartum antibiotics used for cesarean section are linked to the development of allergic disease.

Targeting the P2Y13 Receptor Suppresses IL-33 and HMGB1 Release and Ameliorates Experimental Asthma.

Rationale: The alarmins IL-33 and HMGB1 (high mobility group box 1) contribute to type 2 inflammation and asthma pathogenesis. Objectives: To determine whether P2Y13-R (P2Y13 receptor), a purinergic GPCR (G protein-coupled receptor) and risk allele for asthma, regulates the release of IL-33 and HMGB1. Methods: Bronchial biopsy specimens were obtained from healthy subjects and subjects with asthma. Primary human airway epithelial cells (AECs), primary mouse AECs, or C57Bl/6 mice were inoculated with various aeroallergens or respiratory viruses, and the nuclear-to-cytoplasmic translocation and release of alarmins was measured by using immunohistochemistry and an ELISA. The role of P2Y13-R in AEC function and in the onset, progression, and exacerbation of experimental asthma was assessed by using pharmacological antagonists and mice with P2Y13-R gene deletion. Measurements and Main Results: Aeroallergen exposure induced the extracellular release of ADP and ATP, nucleotides that activate P2Y13-R. ATP, ADP, and aeroallergen (house dust mite, cockroach, or Alternaria antigen) or virus exposure induced the nuclear-to-cytoplasmic translocation and subsequent release of IL-33 and HMGB1, and this response was ablated by genetic deletion or pharmacological antagonism of P2Y13. In mice, prophylactic or therapeutic P2Y13-R blockade attenuated asthma onset and, critically, ablated the severity of a rhinovirus-associated exacerbation in a high-fidelity experimental model of chronic asthma. Moreover, P2Y13-R antagonism derepressed antiviral immunity, increasing IFN-λ production and decreasing viral copies in the lung. Conclusions: We identify P2Y13-R as a novel gatekeeper of the nuclear alarmins IL-33 and HMGB1 and demonstrate that the targeting of this GPCR via genetic deletion or treatment with a small-molecule antagonist protects against the onset and exacerbations of experimental asthma.

HMGB1 amplifies ILC2-induced type-2 inflammation and airway smooth muscle remodelling.

Type-2 immunity elicits tissue repair and homeostasis, however dysregulated type-2 responses cause aberrant tissue remodelling, as observed in asthma. Severe respiratory viral infections in infancy predispose to later asthma, however, the processes that mediate tissue damage-induced type-2 inflammation and the origins of airway remodelling remain ill-defined. Here, using a preclinical mouse model of viral bronchiolitis, we find that increased epithelial and mesenchymal high-mobility group box 1 (HMGB1) expression is associated with increased numbers of IL-13-producing type-2 innate lymphoid cell (ILC2s) and the expansion of the airway smooth muscle (ASM) layer. Anti-HMGB1 ablated lung ILC2 numbers and ASM growth in vivo, and inhibited ILC2-mediated ASM cell proliferation in a co-culture model. Furthermore, we identified that HMGB1/RAGE (receptor for advanced glycation endproducts) signalling mediates an ILC2-intrinsic IL-13 auto-amplification loop. In summary, therapeutic targeting of the HMGB1/RAGE signalling axis may act as a novel asthma preventative by dampening ILC2-mediated type-2 inflammation and associated ASM remodelling.

The maternal gut microbiome during pregnancy and offspring allergy and asthma.

Environmental exposures during pregnancy that alter both the maternal gut microbiome and the infant's risk of allergic disease and asthma include a traditional farm environment and consumption of unpasteurized cow's milk, antibiotic use, dietary fiber, and psychosocial stress. Multiple mechanisms acting in concert may underpin these associations and prime the infant to acquire immune competence and homeostasis following exposure to the extrauterine environment. Cellular and metabolic products of the maternal gut microbiome can promote the expression of microbial pattern recognition receptors, as well as thymic and bone marrow hematopoiesis relevant to regulatory immunity. At birth, transmission of maternally derived bacteria likely leverages this in utero programming to accelerate postnatal transition from a TH2- to TH1- and TH17-dominant immune phenotype and maturation of regulatory immune mechanisms, which in turn reduce the child's risk of allergic disease and asthma. Although our understanding of these phenomena is rapidly evolving, the field is relatively nascent, and we are yet to translate existing knowledge into interventions that substantially reduce disease risk in humans. Here, we review evidence that the maternal gut microbiome impacts the offspring's risk of allergic disease and asthma, discuss challenges and future directions for the field, and propose the hypothesis that maternal carriage of Prevotella copri during pregnancy decreases the offspring's risk of allergic disease via production of succinate, which in turn promotes bone marrow myelopoiesis of dendritic cell precursors in the fetus.

LL-37 and HMGB1 induce alveolar damage and reduce lung tissue regeneration via RAGE.

The receptor for advanced glycation end-products (RAGE) has been implicated in the pathophysiology of chronic obstructive pulmonary disease (COPD). However, it is still unknown whether RAGE directly contributes to alveolar epithelial damage and abnormal repair responses. We hypothesize that RAGE activation not only induces lung tissue damage but also hampers alveolar epithelial repair responses. The effects of the RAGE ligands LL-37 and HMGB1 were examined on airway inflammation and alveolar tissue damage in wild-type and RAGE-deficient mice and on lung damage and repair responses using murine precision cut lung slices (PCLS) and organoids. In addition, their effects were studied on the repair response of human alveolar epithelial A549 cells, using siRNA knockdown of RAGE and treatment with the RAGE inhibitor FPS-ZM1. We observed that intranasal installation of LL-37 and HMGB1 induces RAGE-dependent inflammation and severe alveolar tissue damage in mice within 6 h, with stronger effects in a mouse strain susceptible for emphysema compared with a nonsusceptible strain. In PCLS, RAGE inhibition reduced the recovery from elastase-induced alveolar tissue damage. In organoids, RAGE ligands reduced the organoid-forming efficiency and epithelial differentiation into pneumocyte-organoids. Finally, in A549 cells, we confirmed the role of RAGE in impaired repair responses upon exposure to LL-37. Together, our data indicate that activation of RAGE by its ligands LL-37 and HMGB1 induces acute lung tissue damage and that this impedes alveolar epithelial repair, illustrating the therapeutic potential of RAGE inhibitors for lung tissue repair in emphysema.

RAGE and TLR4 differentially regulate airway hyperresponsiveness: Implications for COPD.

BACKGROUND: The receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4) is implicated in COPD. Although these receptors share common ligands and signalling pathways, it is not known whether they act in concert to drive pathological processes in COPD. We examined the impact of RAGE and/or TLR4 gene deficiency in a mouse model of COPD and also determined whether expression of these receptors correlates with airway neutrophilia and airway hyperresponsiveness (AHR) in COPD patients. METHODS: We measured airway inflammation and AHR in wild-type, RAGE-/- , TLR4-/- and TLR4-/- RAGE-/- mice following acute exposure to cigarette smoke (CS). We also examined the impact of smoking status on AGER (encodes RAGE) and TLR4 bronchial gene expression in patients with and without COPD. Finally, we determined whether expression of these receptors correlates with airway neutrophilia and AHR in COPD patients. RESULTS: RAGE-/- mice were protected against CS-induced neutrophilia and AHR. In contrast, TLR4-/- mice were not protected against CS-induced neutrophilia and had more severe CS-induced AHR. TLR4-/- RAGE-/- mice were not protected against CS-induced neutrophilia but were partially protected against CS-induced mediator release and AHR. Current smoking was associated with significantly lower AGER and TLR4 expression irrespective of COPD status, possibly reflecting negative feedback regulation. However, consistent with preclinical findings, AGER expression correlated with higher sputum neutrophil counts and more severe AHR in COPD patients. TLR4 expression did not correlate with neutrophilic inflammation or AHR. CONCLUSIONS: Inhibition of RAGE but not TLR4 signalling may protect against airway neutrophilia and AHR in COPD.

Increased susceptibility of cystic fibrosis airway epithelial cells to ferroptosis.

BACKGROUND: Defective chloride transport in airway epithelial cells (AECs) and the associated lung disease are the main causes of morbidity and early mortality in cystic fibrosis (CF). Abnormal airway iron homeostasis and the presence of lipid peroxidation products, indicative of oxidative stress, are features of CF lung disease. RESULTS: Here, we report that CF AECs (IB3-1) are susceptible to ferroptosis, a type of cell death associated with iron accumulation and lipid peroxidation. Compared to isogenic CFTR corrected cells (C38), the IB3-1 cells showed increased susceptibility to cell death upon exposure to iron in the form of ferric ammonium citrate (FAC) and the ferroptosis inducer, erastin. This phenotype was accompanied by accumulation of intracellular ferrous iron and lipid peroxides and the extracellular release of malondialdehyde, all indicative of redox stress, and increased levels of lactate dehydrogenase in the culture supernatant, indicating enhanced cell injury. The ferric iron chelator deferoxamine (DFO) and the lipophilic antioxidant ferrostatin-1 inhibited FAC and erastin induced ferroptosis in IB3-1 cells. Glutathione peroxidase 4 (GPX4) expression was decreased in IB3-1 cells treated with FAC and erastin, but was unchanged in C38 AECs. Necroptosis appeared to be involved in the enhanced susceptibility of IB3-1 AECs to ferroptosis, as evidenced by partial cell death rescue with necroptosis inhibitors and enhanced mixed lineage kinase domain-like (MLKL) localisation to the plasma membrane. CONCLUSION: These studies suggest that the increased susceptibility of CF AECs to ferroptosis is linked to abnormal intracellular ferrous iron accumulation and reduced antioxidant defences. In addition, the process of ferroptotic cell death in CF AECs does not appear to be a single entity and for the first time we describe necroptosis as a potential contributory factor. Iron chelation and antioxidant treatments may be promising therapeutic interventions in cystic fibrosis.

Respiratory Syncytial Virus Infection Promotes Necroptosis and HMGB1 Release by Airway Epithelial Cells.

Rationale: Respiratory syncytial virus (RSV) bronchiolitis causes significant infant mortality. Bronchiolitis is characterized by airway epithelial cell (AEC) death; however, the mode of death remains unknown.Objectives: To determine whether necroptosis contributes to RSV bronchiolitis pathogenesis via HMGB1 (high mobility group box 1) release.Methods: Nasopharyngeal samples were collected from children presenting to the hospital with acute respiratory infection. Primary human AECs and neonatal mice were inoculated with RSV and murine Pneumovirus, respectively. Necroptosis was determined via viability assays and immunohistochemistry for RIPK1 (receptor-interacting protein kinase-1), MLKL (mixed lineage kinase domain-like pseudokinase) protein, and caspase-3. Necroptosis was blocked using pharmacological inhibitors and RIPK1 kinase-dead knockin mice.Measurements and Main Results: HMGB1 levels were elevated in nasopharyngeal samples of children with acute RSV infection. RSV-induced epithelial cell death was associated with increased phosphorylated RIPK1 and phosphorylated MLKL but not active caspase-3 expression. Inhibition of RIPK1 or MLKL attenuated RSV-induced HMGB1 translocation and release, and lowered viral load. MLKL inhibition increased active caspase-3 expression in a caspase-8/9-dependent manner. In susceptible mice, Pneumovirus infection upregulated RIPK1 and MLKL expression in the airway epithelium at 8 to 10 days after infection, coinciding with AEC sloughing, HMGB1 release, and neutrophilic inflammation. Genetic or pharmacological inhibition of RIPK1 or MLKL attenuated these pathologies, lowered viral load, and prevented type 2 inflammation and airway remodeling. Necroptosis inhibition in early life ameliorated asthma progression induced by viral or allergen challenge in later life.Conclusions: Pneumovirus infection induces AEC necroptosis. Inhibition of necroptosis may be a viable strategy to limit the severity of viral bronchiolitis and break its nexus with asthma.

A Simple Clinical Tool for Stratifying Risk of Clinically Significant CKD after Nephrectomy: Development and Multinational Validation.

BACKGROUND: Clinically significant CKD following surgery for kidney cancer is associated with increased morbidity and mortality, but identifying patients at increased CKD risk remains difficult. Simple methods to stratify risk of clinically significant CKD after nephrectomy are needed. METHODS: To develop a tool for stratifying patients' risk of CKD arising after surgery for kidney cancer, we tested models in a population-based cohort of 699 patients with kidney cancer in Queensland, Australia (2012-2013). We validated these models in a population-based cohort of 423 patients from Victoria, Australia, and in patient cohorts from single centers in Queensland, Scotland, and England. Eligible patients had two functioning kidneys and a preoperative eGFR ≥60 ml/min per 1.73 m2. The main outcome was incident eGFR <45 ml/min per 1.73 m2 at 12 months postnephrectomy. We used prespecified predictors-age ≥65 years old, diabetes mellitus, preoperative eGFR, and nephrectomy type (partial/radical)-to fit logistic regression models and grouped patients according to degree of risk of clinically significant CKD (negligible, low, moderate, or high risk). RESULTS: Absolute risks of stage 3b or higher CKD were <2%, 3% to 14%, 21% to 26%, and 46% to 69% across the four strata of negligible, low, moderate, and high risk, respectively. The negative predictive value of the negligible risk category was 98.9% for clinically significant CKD. The c statistic for this score ranged from 0.84 to 0.88 across derivation and validation cohorts. CONCLUSIONS: Our simple scoring system can reproducibly stratify postnephrectomy CKD risk on the basis of readily available parameters. This clinical tool's quantitative assessment of CKD risk may be weighed against other considerations when planning management of kidney tumors and help inform shared decision making between clinicians and patients.

PAG1 limits allergen-induced type 2 inflammation in the murine lung.

BACKGROUND: Phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG1) is a transmembrane adaptor protein that affects immune receptor signaling in T and B cells. Evidence from genome-wide association studies of asthma suggests that genetic variants that regulate the expression of PAG1 are associated with asthma risk. However, it is not known whether PAG1 expression is causally related to asthma pathophysiology. Here, we investigated the role of PAG1 in a preclinical mouse model of house dust mite (HDM)-induced allergic sensitization and allergic airway inflammation. METHODS: Pag1-deficient (Pag1-/- ) and wild-type (WT) mice were sensitized or sensitized/challenged to HDM, and hallmark features of allergic inflammation were assessed. The contribution of T cells was assessed through depletion (anti-CD4 antibody) and adoptive transfer studies. RESULTS: Type 2 inflammation (eosinophilia, eotaxin-2 expression, IL-4/IL-5/IL-13 production, mucus production) in the airways and lungs was significantly increased in HDM sensitized/challenged Pag1-/- mice compared to WT mice. The predisposition to allergic sensitization was associated with increased airway epithelial high-mobility group box 1 (HMGB1) translocation and release, increased type 2 innate lymphoid cells (ILC2s) and monocyte-derived dendritic cell numbers in the mediastinal lymph nodes, and increased T-helper type 2 (TH 2)-cell differentiation. CD4+ T-cell depletion studies or the adoptive transfer of WT OVA-specific CD4+ T cells to WT or Pag1-/- recipients demonstrated that the heightened propensity for TH 2-cell differentiation was both T cell intrinsic and extrinsic. CONCLUSION: PAG1 deficiency increased airway epithelial activation, ILC2 expansion, and TH 2 differentiation. As a consequence, PAG1 deficiency predisposed toward allergic sensitization and increased the severity of experimental asthma.

Chronic IL-33 expression predisposes to virus-induced asthma exacerbations by increasing type 2 inflammation and dampening antiviral immunity.

BACKGROUND: Rhinovirus infection triggers acute asthma exacerbations. IL-33 is an instructive cytokine of type 2 inflammation whose expression is associated with viral load during experimental rhinovirus infection of asthmatic patients. OBJECTIVE: We sought to determine whether anti-IL-33 therapy is effective during disease progression, established disease, or viral exacerbation using a preclinical model of chronic asthma and in vitro human primary airway epithelial cells (AECs). METHODS: Mice were exposed to pneumonia virus of mice and cockroach extract in early and later life and then challenged with rhinovirus to model disease onset, progression, and chronicity. Interventions included anti-IL-33 or dexamethasone at various stages of disease. AECs were obtained from asthmatic patients and healthy subjects and treated with anti-IL-33 after rhinovirus infection. RESULTS: Anti-IL-33 decreased type 2 inflammation in all phases of disease; however, the ability to prevent airway smooth muscle growth was lost after the progression phase. After the chronic phase, IL-33 levels were persistently high, and rhinovirus challenge exacerbated the type 2 inflammatory response. Treatment with anti-IL-33 or dexamethasone diminished exacerbation severity, and anti-IL-33, but not dexamethasone, promoted antiviral interferon expression and decreased viral load. Rhinovirus replication was higher and IFN-λ levels were lower in AECs from asthmatic patients compared with those from healthy subjects. Anti-IL-33 decreased rhinovirus replication and increased IFN-λ levels at the gene and protein levels. CONCLUSION: Anti-IL-33 or dexamethasone suppressed the magnitude of type 2 inflammation during a rhinovirus-induced acute exacerbation; however, only anti-IL-33 boosted antiviral immunity and decreased viral replication. The latter phenotype was replicated in rhinovirus-infected human AECs, suggesting that anti-IL-33 therapy has the additional benefit of enhancing host defense.

Gene-based analysis of regulatory variants identifies 4 putative novel asthma risk genes related to nucleotide synthesis and signaling.

BACKGROUND: Hundreds of genetic variants are thought to contribute to variation in asthma risk by modulating gene expression. Methods that increase the power of genome-wide association studies (GWASs) to identify risk-associated variants are needed. OBJECTIVE: We sought to develop a method that aggregates the evidence for association with disease risk across expression quantitative trait loci (eQTLs) of a gene and use this approach to identify asthma risk genes. METHODS: We developed a gene-based test and software package called EUGENE that (1) is applicable to GWAS summary statistics; (2) considers both cis- and trans-eQTLs; (3) incorporates eQTLs identified in different tissues; and (4) uses simulations to account for multiple testing. We applied this approach to 2 published asthma GWASs (combined n = 46,044) and used mouse studies to provide initial functional insights into 2 genes with novel genetic associations. RESULTS: We tested the association between asthma and 17,190 genes that were found to have cis- and/or trans-eQTLs across 16 published eQTL studies. At an empirical FDR of 5%, 48 genes were associated with asthma risk. Of these, for 37, the association was driven by eQTLs located in established risk loci for allergic disease, including 6 genes not previously implicated in disease cause (eg, LIMS1, TINF2, and SAFB). The remaining 11 significant genes represent potential novel genetic associations with asthma. The association with 4 of these replicated in an independent GWAS: B4GALT3, USMG5, P2RY13, and P2RY14, which are genes involved in nucleotide synthesis or nucleotide-dependent cell activation. In mouse studies, P2ry13 and P2ry14-purinergic receptors activated by adenosine 5-diphosphate and UDP-sugars, respectively-were upregulated after allergen challenge, notably in airway epithelial cells, eosinophils, and neutrophils. Intranasal exposure with receptor agonists induced the release of IL-33 and subsequent eosinophil infiltration into the lungs. CONCLUSION: We identified novel associations between asthma and eQTLs for 4 genes related to nucleotide synthesis/signaling and demonstrated the power of gene-based analyses of GWASs.

Regulatory T cells prevent inducible BALT formation by dampening neutrophilic inflammation.

Inducible BALT (iBALT) can amplify pulmonary or systemic inflammatory responses to the benefit or detriment of the host. We took advantage of the age-dependent formation of iBALT to interrogate the underlying mechanisms that give rise to this ectopic, tertiary lymphoid organ. In this study, we show that the reduced propensity for weanling as compared with neonatal mice to form iBALT in response to acute LPS exposure is associated with greater regulatory T cell expansion in the mediastinal lymph nodes. Ab- or transgene-mediated depletion of regulatory T cells in weanling mice upregulated the expression of IL-17A and CXCL9 in the lungs, induced a tissue neutrophilia, and increased the frequency of iBALT to that observed in neonatal mice. Remarkably, neutrophil depletion in neonatal mice decreased the expression of the B cell active cytokines, a proliferation-inducing ligand and IL-21, and attenuated LPS-induced iBALT formation. Taken together, our data implicate a role for neutrophils in lymphoid neogenesis. Neutrophilic inflammation is a common feature of many autoimmune diseases in which iBALT are present and pathogenic, and hence the targeting of neutrophils or their byproducts may serve to ameliorate detrimental lymphoid neogenesis in a variety of disease contexts.