Sex differences in sociodemographic, clinical, and laboratory variables in childhood asthma: A birth cohort study.

BACKGROUND: There are marked sex differences in the prevalence and severity of asthma, both during childhood and adulthood. There is a relative lack of comprehensive studies exploring sexdifferences in pediatric asthma cohorts. OBJECTIVE: To identify the most relevant sex differences in sociodemographic, clinical, and laboratory variables in a well-characterized large pediatric asthma cohort. METHODS: We performed a cross-sectional analysis of the Mayo Clinic Olmsted County Birth Cohort. In the full birth cohort, we used a natural language-processing algorithm based on the Predetermined Asthma Criteria for asthma ascertainment. In a stratified random sample of 300 children, we obtained additional pulmonary function tests and laboratory data. We identified the significant sex differences among available sociodemographic, clinical, and laboratory variables. RESULTS: Boys were more frequently diagnosed with having asthma than girls and were younger at the time of asthma diagnosis. There were no sex differences in relation to socioeconomic status. We identified a male predominance in the presence of a tympanostomy tube and a female predominance in the history of pneumonia. A higher percentage of boys had a forced expiratory volume in 1 second/forced vital capacity ratio less than 0.85. Blood eosinophilia and atopic sensitization were also more common in boys. Finally, boys had higher levels of serum periostin than girls. CONCLUSION: This study described significant sex differences in a large pediatric asthma cohort. Overall, boys had earlier and more severe asthma than girls. Differences in blood eosinophilia and serum periostin provide insights into possible mechanisms of the sex bias in childhood asthma.

Endoplasmic reticulum stress-induced senescence in human lung fibroblasts.

Loss of proteostasis and cellular senescence have been previously established as characteristics of aging; however, their interaction in the context of lung aging and potential contributions to aging-associated lung remodeling remains understudied. In this study, we aimed to characterize endoplasmic reticulum (ER) stress response, cellular senescence, and their interaction in relation to extracellular matrix (ECM) production in lung fibroblasts from young (25-45 yr) and old (>60 yr) humans. Fibroblasts from young and old patients without significant preexisting lung disease were exposed to vehicle, MG132, etoposide, or salubrinal. Afterward, cells and cell lysates or supernatants were analyzed for ER stress, cellular senescence, and ECM changes using protein analysis, proliferation assay, and senescence-associated beta-galactosidase (SA-ß-Gal) staining. At baseline, fibroblasts from aging individuals showed increased levels of ER stress (ATF6 and PERK), senescence (p21 and McL-1), and ECM marker (COL1A1) compared to those from young individuals. Upon ER stress induction and etoposide exposure, fibroblasts showed an increase in senescence (SA-ß-Gal, p21, and Cav-1), ER stress (PERK), and ECM markers (COL1A1 and LUM) compared to vehicle. Additionally, IL-6 and IL-8 levels were increased in the supernatants of MG132- and etoposide-treated fibroblasts, respectively. Finally, the ER stress inhibitor salubrinal decreased the expression of p21 compared to vehicle and MG132 treatments; however, salubrinal inhibited COL1A1 but not p21 expression in MG132-treated fibroblasts. Our study suggests that ER stress response plays an important role in establishment and maintenance of a senescence phenotype in lung fibroblasts and therefore contributes to altered remodeling in the aging lung.NEW & NOTEWORTHY The current study establishes functional links between endoplasmic reticulum (ER) stress and cellular senescence per se in the specific context of aging human lung fibroblasts. Recognizing that the process of aging per se is complex, modulated by the myriad of lifelong and environmental exposures, it is striking to note that chronic ER stress may play a crucial role in the establishment and maintenance of cellular senescence in lung fibroblasts.

Functional role of glial-derived neurotrophic factor in a mixed allergen murine model of asthma.

Our previous study showed that glial-derived neurotrophic factor (GDNF) expression is upregulated in asthmatic human lungs, and GDNF regulates calcium responses through its receptor GDNF family receptor α1 (GFRα1) and RET receptor in human airway smooth muscle (ASM) cells. In this study, we tested the hypothesis that airway GDNF contributes to airway hyperreactivity (AHR) and remodeling using a mixed allergen mouse model. Adult C57BL/6J mice were intranasally exposed to mixed allergens (ovalbumin, Aspergillus, Alternaria, house dust mite) over 4 wk with concurrent exposure to recombinant GDNF, or extracellular GDNF chelator GFRα1-Fc. Airway resistance and compliance to methacholine were assessed using FlexiVent. Lung expression of GDNF, GFRα1, RET, collagen, and fibronectin was examined by RT-PCR and histology staining. Allergen exposure increased GDNF expression in bronchial airways including ASM and epithelium. Laser capture microdissection of the ASM layer showed increased mRNA for GDNF, GFRα1, and RET in allergen-treated mice. Allergen exposure increased protein expression of GDNF and RET, but not GFRα1, in ASM. Intranasal administration of GDNF enhanced baseline responses to methacholine but did not consistently potentiate allergen effects. GDNF also induced airway thickening, and collagen deposition in bronchial airways. Chelation of GDNF by GFRα1-Fc attenuated allergen-induced AHR and particularly remodeling. These data suggest that locally produced GDNF, potentially derived from epithelium and/or ASM, contributes to AHR and remodeling relevant to asthma.NEW & NOTEWORTHY Local production of growth factors within the airway with autocrine/paracrine effects can promote features of asthma. Here, we show that glial-derived neurotrophic factor (GDNF) is a procontractile and proremodeling factor that contributes to allergen-induced airway hyperreactivity and tissue remodeling in a mouse model of asthma. Blocking GDNF signaling attenuates allergen-induced airway hyperreactivity and remodeling, suggesting a novel approach to alleviating structural and functional changes in the asthmatic airway.

Asthmatic lung fibroblasts promote type 2 immune responses via endoplasmic reticulum stress response dependent thymic stromal lymphopoietin secretion.

Lung fibroblasts contribute to asthma pathology partly through modulation of the immune environment in the airway. Tumor necrosis factor-α (TNFα) expression is upregulated in asthmatic lungs. How asthmatic lung fibroblasts respond to TNFα stimulation and subsequently regulate immune responses is not well understood. Endoplasmic reticulum (ER) stress and unfolded protein responses (UPR) play important roles in asthma, but their functional roles are still under investigation. In this study, we investigated TNFα-induced cytokine production in primary lung fibroblasts from asthmatic vs. non-asthmatic human subjects, and downstream effects on type 2 immune responses. TNFα significantly upregulated IL-6, IL-8, C-C motif chemokine ligand 5 (CCL5), and thymic stromal lymphopoietin (TSLP) mRNA expression and protein secretion by lung fibroblasts. Asthmatic lung fibroblasts secreted higher levels of TSLP which promoted IL-33-induced IL-5 and IL-13 production by peripheral blood mononuclear cells. TNFα exposure enhanced expression of ER stress/UPR pathways in both asthmatic and non-asthmatic lung fibroblasts, especially inositol-requiring protein 1α in asthmatics. ER stress/UPR inhibitors decreased IL-6, CCL5, and TSLP protein secretion by asthmatic lung fibroblasts. Our data suggest that TNFα and lung fibroblasts form an important axis in asthmatic lungs to promote asthmatic inflammation that can be attenuated by inhibiting ER stress/UPR pathway.

Cellular senescence is increased in airway smooth muscle cells of elderly persons with asthma.

Senescent cells can drive age-related tissue dysfunction partially via a senescence-associated secretory phenotype (SASP) involving proinflammatory and profibrotic factors. Cellular senescence has been associated with a structural and functional decline during normal lung aging and age-related diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Asthma in the elderly (AIE) represents a major healthcare burden. AIE is associated with bronchial airway hyperresponsiveness and remodeling, which involves increased cell proliferation and higher rates of fibrosis, and resistant to standard therapy. Airway smooth muscle (ASM) cells play a major role in asthma such as remodeling via modulation of inflammation and the extracellular matrix (ECM) environment. Whether senescent ASM cells accumulate in AIE and contribute to airway structural or functional changes is unknown. Lung tissues from elderly persons with asthma showed greater airway fibrosis compared with age-matched elderly persons with nonasthma and young age controls. Lung tissue or isolated ASM cells from elderly persons with asthma showed increased expression of multiple senescent markers including phospho-p53, p21, telomere-associated foci (TAF), as well as multiple SASP components. Senescence and SASP components were also increased with aging per se. These data highlight the presence of cellular senescence in AIE that may contribute to airway remodeling.

Transient IL-33 upregulation in neonatal mouse lung promotes acute but not chronic type 2 immune responses induced by allergen later in life.

Early life respiratory insults, such as viral infections or hyperoxia, often increase asthma susceptibility later in life. The mechanisms underlying this increased susceptibility are not fully understood. IL-33 has been shown to be critically involved in allergic airway diseases. IL-33 expression in the neonatal lung can be increased by various respiratory insults associated with asthma development. Therefore, we investigated whether and how early life increases in IL-33 impact allergic airway responses later in life. Using a novel IL-33 transgenic mouse model, in which full-length IL-33 was inducible overexpressed in lung epithelial cells, we transiently upregulated lung IL-33 expression in neonatal mice for one week. After resting for 4-6 weeks, mice were intranasally exposed to a single-dose of recombinant IL-33 or the airborne allergen Alternaria. Alternatively, mice were exposed to Alternaria and ovalbumin multiple times for one month. We found that a transient increase in IL-33 expression during the neonatal period promoted IL-5 and IL-13 production when mice were later exposed to a single-dose of IL-33 or Alternaria in adulthood. However, increased IL-33 expression during the neonatal period did not affect airway inflammation, type 2 cytokine production, lung mucus production, or antigen-specific antibody responses when adult mice were exposed to Alternaria and ovalbumin multiple times. These results suggest that transient increased IL-33 expression early in life may have differential effects on allergic airway responses in later life, preferentially affecting allergen-induced acute type 2 cytokine production.

In vitro Culture with Cytokines Provides a Tool to Assess the Effector Functions of ILC2s in Peripheral Blood in Asthma.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) play crucial roles in type 2 immunity and asthma development. While ILC2s are resident in mucosal tissues, they also circulate in peripheral blood. It remains controversial whether ILC2s are increased in the peripheral blood of patients with asthma. PURPOSE: The goal of this project was to study the effector functions of ILC2s in peripheral blood samples by in vitro culture with cytokines. PATIENTS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from 11 adult patients with mild asthma and 12 healthy control subjects. The number of peripheral blood ILC2s in PBMCs was analyzed by flow cytometry. PBMCs were cultured with IL-33 and IL-25 without any antigens, and the amounts of type 2 cytokines in cell-free supernatants were analyzed by ELISA. In selected experiments, production of cytokines by ILC2s was analyzed by intracellular cytokine staining and flow cytometry. RESULTS: In response to either IL-33 or IL-25 stimulation, PBMCs from patients with mild asthma produced larger amounts of IL-5 and IL-13 than PBMCs from healthy control subjects. However, ILC2 numbers or proportions were not significantly different between these two groups. Flow cytometric analysis confirmed production of IL-5 by ILCs when stimulated with IL-33. CONCLUSION: In vitro culture of PBMCs with a cocktail of cytokines, such as either IL-33 or IL-25 plus IL-2, may provide a valuable tool to assess the effector functions of ILC2s and may serve as a biomarker for human asthma.

Contributions of IL-33 in Non-hematopoietic Lung Cells to Obstructive Lung Disease.

Interleukin (IL)-33 plays important roles in pulmonary immune responses and lung diseases including asthma and chronic obstructive pulmonary disease (COPD). There is substantial interest in identifying and characterizing cellular sources vs. targets of IL-33, and downstream signaling pathways involved in disease pathophysiology. While epithelial and immune cells have largely been the focus, in this review, we summarize current knowledge of expression, induction, and function of IL-33 and its receptor ST2 in non-hematopoietic lung cells in the context of health and disease. Under basal conditions, epithelial cells and endothelial cells are thought to be the primary resident cell types that express high levels of IL-33 and serve as ligand sources compared to mesenchymal cells (smooth muscle cells and fibroblasts). Under inflammatory conditions, IL-33 expression is increased in most non-hematopoietic lung cells, including epithelial, endothelial, and mesenchymal cells. In comparison to its ligand, the receptor ST2 shows low expression levels at baseline but similar to IL-33, ST2 expression is upregulated by inflammation in these non-hematopoietic lung cells which may then participate in chronic inflammation both as sources and autocrine/paracrine targets of IL-33. Downstream effects of IL-33 may occur via direct receptor activation or indirect interactions with the immune system, overall contributing to lung inflammation, airway hyper-responsiveness and remodeling (proliferation and fibrosis). Accordingly from a therapeutic perspective, targeting IL-33 and/or its receptor in non-hematopoietic lung cells becomes relevant.

Early Life Represents a Vulnerable Time Window for IL-33-Induced Peripheral Lung Pathology.

IL-33, an IL-1 family cytokine, is constitutively expressed in mucosal tissues and other organs in healthy humans and animals, and expression levels increase in inflammatory conditions. Although IL-33-mediated promotion of type 2 immune responses has been well established, a gap in our knowledge regarding the functional diversity of this pleiotropic cytokine remains. To address this gap, we developed a new IL-33 transgenic mouse model in which overexpression of full-length IL-33 is induced in lung epithelial cells under conditional control. In adult mice, an ∼3-fold increase in the steady-state IL-33 levels produced no pathologic effects in the lungs. When exposed to airborne allergens, adult transgenic mice released more IL-33 extracellularly and exhibited robust type 2 immune responses. In neonatal transgenic mice, up to postnatal day 14, a similar increase in steady-state IL-33 levels resulted in increased mortality, enlarged alveolar spaces resembling bronchopulmonary dysplasia, and altered expression of genes associated with tissue morphogenesis. Processed 25-kDa IL-33 protein was detected in bronchoalveolar lavage fluids without any exogenous stimuli, and pathologic changes were abolished in mice deficient in the IL-33 receptor ST2. These findings suggest that adult lungs are relatively resistant to IL-33 overexpression unless they encounter environmental insults, whereas developing lungs are highly susceptible, with IL-33 overexpression resulting in detrimental and pathologic outcomes.

Airway exposure initiates peanut allergy by involving the IL-1 pathway and T follicular helper cells in mice.

BACKGROUND: Little is currently known regarding the immunologic mechanism(s) that initiate peanut allergy. Notably, peanut proteins have been detected in house dust, and their levels correlate with peanut allergy prevalence. OBJECTIVE: This study aimed to develop a new mouse model for peanut allergy and to investigate the immunologic mechanisms involved in peanut allergen sensitization. METHODS: To mimic environmental exposure, naive mice were exposed to peanut flour by inhalation for up to 4 weeks. We then analyzed serum levels of IgE antibody and challenged mice with peanut proteins. Immunological mechanisms involved in sensitization were analyzed using cytokine reporter mice, an adoptive cell transfer model, and gene knockout mice. RESULTS: When exposed to peanut flour by inhalation, both BALB/c and C57BL/6 mice developed peanut allergy, as demonstrated by the presence of peanut-specific IgE antibodies and manifestation of acute anaphylaxis on challenge. A large number of follicular helper T (Tfh) cells were also detected in draining lymph nodes of allergic mice. These cells produced IL-4 and IL-21, and they more robustly promoted peanut-specific IgE production than Th2 cells did. Genetic depletion of Tfh cells decreased IgE antibody levels and protected mice from anaphylaxis, without affecting Th2 cells. Furthermore, peanut flour exposure increased lung levels of IL-1α and IL-1ß, and mice deficient in the receptor for these cytokines showed a significant decrease in Tfh cells compared with in wild-type mice. CONCLUSIONS: Tfh cells play a key role in peanut allergy, and the IL-1 pathway is involved in the Tfh response to peanut allergen exposure.

IL-33-Responsive Group 2 Innate Lymphoid Cells Are Regulated by Female Sex Hormones in the Uterus.

Group 2 innate lymphoid cells (ILC2s) reside in multiple organs in the body, where they play roles in immunity, tissue homeostasis, and metabolic regulation. However, little is known about the regulatory mechanisms of ILC2s in different organs. Here, we identified ILC2s in the mouse uterus and found that they express cell surface molecules, including the IL-33 receptor, ST2, that are roughly comparable to those expressed by lung ILC2s. Both in vivo and in vitro treatment with IL-33 induced type 2 cytokine production in uterine ILC2s, suggesting that they respond to IL-33 in a manner similar to ILC2s in other organs. Importantly, uterine ILC2s were nearly absent in ovariectomized mice and were increased in wild-type mice by estrogen administration, whereas lung ILC2s were unaffected by both ovariectomy and estrogen administration. Likewise, a marked reduction in uterine ILC2s was observed in mice deficient in estrogen receptor α or estrogen receptor ß. Furthermore, uterine ILC2s highly expressed estrogen receptor α, and in vitro culture of isolated uterine ILC2s with 17ß-estradiol modified expression of a number of genes. Finally, an increased prevalence in neonatal mortality was observed in litters from dams lacking the IL-33 receptor, ST2. Taken together, our findings indicate that unlike lung IL2Cs, uterine ILC2s are regulated by female sex hormones, which may specialize them for specific physiological functions.

IL-33: biological properties, functions, and roles in airway disease.

Interleukin (IL)-33 is a key cytokine involved in type 2 immunity and allergic airway diseases. Abundantly expressed in lung epithelial cells, IL-33 plays critical roles in both innate and adaptive immune responses in mucosal organs. In innate immunity, IL-33 and group 2 innate lymphoid cells (ILC2s) provide an essential axis for rapid immune responses and tissue homeostasis. In adaptive immunity, IL-33 interacts with dendritic cells, Th2 cells, follicular T cells, and regulatory T cells, where IL-33 influences the development of chronic airway inflammation and tissue remodeling. The clinical findings that both the IL-33 and ILC2 levels are elevated in patients with allergic airway diseases suggest that IL-33 plays an important role in the pathogenesis of these diseases. IL-33 and ILC2 may also serve as biomarkers for disease classification and to monitor the progression of diseases. In this article, we reviewed the current knowledge of the biology of IL-33 and discussed the roles of the IL-33 in regulating airway immune responses and allergic airway diseases.

Group 2 Innate Lymphoid Cells Promote an Early Antibody Response to a Respiratory Antigen in Mice.

Innate lymphoid cells (ILCs) are a new family of immune cells that play important roles in innate immunity in mucosal tissues, and in the maintenance of tissue and metabolic homeostasis. Recently, group 2 ILCs (ILC2s) were found to promote the development and effector functions of Th2-type CD4(+) T cells by interacting directly with T cells or by activating dendritic cells, suggesting a role for ILC2s in regulating adaptive immunity. However, our current knowledge on the role of ILCs in humoral immunity is limited. In this study, we found that ILC2s isolated from the lungs of naive BALB/c mice enhanced the proliferation of B1- as well as B2-type B cells and promoted the production of IgM, IgG1, IgA, and IgE by these cells in vitro. Soluble factors secreted by ILC2s were sufficient to enhance B cell Ig production. By using blocking Abs and ILC2s isolated from IL-5-deficient mice, we found that ILC2-derived IL-5 is critically involved in the enhanced production of IgM. Furthermore, when adoptively transferred to Il7r(-/-) mice, which lack ILC2s and mature T cells, lung ILC2s promoted the production of IgM Abs to a polysaccharide Ag, 4-hydroxy-3-nitrophenylacetyl Ficoll, within 7 d of airway exposure in vivo. These findings add to the growing body of literature regarding the regulatory functions of ILCs in adaptive immunity, and suggest that lung ILC2s promote B cell production of early Abs to a respiratory Ag even in the absence of T cells.

B cells play key roles in th2-type airway immune responses in mice exposed to natural airborne allergens.

Humans are frequently exposed to various airborne allergens. In addition to producing antibodies, B cells participate in immune responses via various mechanisms. The roles of B cells in allergic airway inflammation and asthma have been controversial. We examined the functional importance of B cells in a mouse model of asthma, in which mice were exposed repeatedly to common airborne allergens. Naïve wild-type BALB/c mice or B cell-deficient JH-/- mice were exposed intranasally to a cocktail of allergen extracts, including Alternaria, Aspergillus, and house dust mite, every other day for two weeks. Ovalbumin was included in the cocktail to monitor the T cell immune response. Airway inflammation, lung pathology, and airway reactivity were analyzed. The airway exposure of naïve wild type mice to airborne allergens induced robust eosinophilic airway inflammation, increased the levels of Th2 cytokines and chemokines in the lung, and increased the reactivity to inhaled methacholine. These pathological changes and immune responses were attenuated in B cell-deficient JH-/- mice. The allergen-induced expansion of CD4+ T cells was impaired in the lungs and draining lymph nodes of JH-/- mice. Furthermore, lymphocytes from JH-/- mice failed to produce Th2 cytokines in response to ovalbumin re-stimulation in vitro. Our results suggest that B cells are required for the optimal development of Th2-type immune responses and airway inflammation when exposed to common airborne allergens. The therapeutic targeting of B cells may be beneficial to treat asthma in certain patients.

Group 2 innate lymphoid cells in the lung.

As the first line of defense, innate immunity plays an important role in protecting the host against pathogens. Innate lymphoid cells (ILCs) are emerging as important effector cells in the innate immune system and the cell type that regulate immune and tissue homeostases. Group 2 ILCs (ILC2s) are a subset of ILCs and are characterized by their capacity to produce large quantities of type 2 cytokines and certain tissue growth factors. In animal models, lung ILC2s are involved in allergic airway inflammation induced by exposure to allergens even in the absence of CD4(+) T cells and are likely responsible for tissue repair and recovery after respiratory virus infection. ILC2s are also identified in various organs in humans, and the numbers are increased in mucosal tissues from patients with allergic disorders. Further investigations of this novel cell type will provide major conceptual advances in our understanding of the mechanisms of asthma and allergic diseases.

Role for cytoplasmic nucleotide hydrolysis in hepatic function and protein synthesis.

Nucleotide hydrolysis is essential for many aspects of cellular function. In the case of 3',5'-bisphosphorylated nucleotides, mammals possess two related 3'-nucleotidases, Golgi-resident 3'-phosphoadenosine 5'-phosphate (PAP) phosphatase (gPAPP) and Bisphosphate 3'-nucleotidase 1 (Bpnt1). gPAPP and Bpnt1 localize to distinct subcellular compartments and are members of a conserved family of metal-dependent lithium-sensitive enzymes. Although recent studies have demonstrated the importance of gPAPP for proper skeletal development in mice and humans, the role of Bpnt1 in mammals remains largely unknown. Here we report that mice deficient for Bpnt1 do not exhibit skeletal defects but instead develop severe liver pathologies, including hypoproteinemia, hepatocellular damage, and in severe cases, frank whole-body edema and death. Accompanying these phenotypes, we observed tissue-specific elevations of the substrate PAP, up to 50-fold in liver, repressed translation, and aberrant nucleolar architecture. Remarkably, the phenotypes of the Bpnt1 knockout are rescued by generating a double mutant mouse deficient for both PAP synthesis and hydrolysis, consistent with a mechanism in which PAP accumulation is toxic to tissue function independent of sulfation. Overall, our study defines a role for Bpnt1 in mammalian physiology and provides mechanistic insights into the importance of sulfur assimilation and cytoplasmic PAP hydrolysis to normal liver function.

An essential role for an inositol polyphosphate multikinase, Ipk2, in mouse embryogenesis and second messenger production.

Phospholipase C and several inositol polyphosphate kinase (IPK) activities generate a branched ensemble of inositol polyphosphate second messengers that regulate cellular signaling pathways in the nucleus and cytoplasm. Here, we report that mice deficient for Ipk2 (also known as inositol polyphosphate multikinase), an inositol trisphosphate and tetrakisphosphate 6/5/3-kinase active at several places in the inositol metabolic pathways, die around embryonic day 9.5 with multiple morphological defects, including abnormal folding of the neural tube. Metabolic analysis of Ipk2-deficient cells demonstrates that synthesis of the majority of inositol pentakisphosphate, hexakisphosphate and pyrophosphate species are disrupted, although the presence of 10% residual inositol hexakisphosphate indicates the existence of a minor alternative pathway. Agonist induced inositol tris- and bis-phosphate production and calcium release responses are present in homozygous mutant cells, indicating that the observed mouse phenotypes are a result of failure to produce higher inositol polyphosphates. Our data demonstrate that Ipk2 plays a major role in the synthesis of inositol polyphosphate messengers derived from inositol 1,4,5-trisphosphate and uncovers a role for their production in embryogenesis and normal development.