A steroid-resistant cockroach allergen model is associated with lung and cecal microbiome changes.

The pathogenesis of asthma has been partially linked to lung and gut microbiome. We utilized a steroid-resistant chronic model of cockroach antigen-induced (CRA) asthma with corticosteroid (fluticasone) treatment to examine lung and gut microbiome during disease. The pathophysiology assessment demonstrated that mucus and airway hyperresponsiveness were increased in the chronic CRA with no alteration in the fluticasone (Flut)-treated group, demonstrating steroid resistance. Analysis of mRNA from lungs showed no decrease of MUC5AC or Gob5 in the Flut-treated group. Furthermore, flow-cytometry in lung tissue showed eosinophils and neutrophils were not significantly reduced in the Flut-treated group compared to the chronic CRA group. When the microbiome profiles were assessed, data showed that only the Flut-treated animals were significantly different in the gut microbiome. Finally, a functional analysis of cecal microbiome metabolites using PiCRUSt showed several biosynthetic pathways were significantly enriched in the Flut-treated group, with tryptophan pathway verified by ELISA with increased kynurenine in homogenized cecum samples. While the implications of these data are unclear, they may suggest a significant impact of steroid treatment on future disease pathogenesis through microbiome and associated metabolite pathway changes.

ER stress protein PERK promotes inappropriate innate immune responses and pathogenesis during RSV infection.

The activation of dendritic cells (DC) during respiratory viral infections is central to directing the immune response and the pathologic outcome. In these studies, the effect of RSV infection on development of ER stress responses and the impact on innate immunity was examined. The upregulation of ER stress was closely associated with the PERK pathway through the upregulation of CHOP in RSV infected DC. The inhibition of PERK corresponded with decreased EIF2a phosphorylation but had no significant effect on Nrf2 in DC, two primary pathways regulated by PERK. Subsequent studies identified that by blocking PERK activity in infected DC an altered ER stress response and innate cytokine profile was observed with the upregulation of IFNß and IL-12, coincident to the down regulation of IL-1ß. When mitochondria respiration was assessed in PERK deficient DC there were increased dysfunctional mitochondria after RSV infection that resulted in reduced oxygen consumption rates (OCR) and ATP production indicating altered cellular metabolism. Use of a CD11c targeted genetic deleted murine model, RSV infection was characterized by reduced inflammation and diminished mucus staining as well as reduced mucus-associated gene gob5 expression. The assessment of the cytokine responses showed decreased IL-13 and IL-17 along with diminished IL-1ß in the lungs of PERK deficient infected mice. When PERK-deficient animals were assessed in parallel for lung leukocyte numbers, animals displayed significantly reduced myeloid and activated CD4 and CD8 T cell numbers. Thus, the PERK activation pathway may provide a rational target for altering the severe outcome of an RSV infection through modifying immune responses.

The Lung Elastin Matrix Undergoes Rapid Degradation Upon Adult Loss of Hox5 Function.

Hox genes encode transcription factors that are critical for embryonic skeletal patterning and organogenesis. The Hoxa5, Hoxb5, and Hoxc5 paralogs are expressed in the lung mesenchyme and function redundantly during embryonic lung development. Conditional loss-of-function of these genes during postnatal stages leads to severe defects in alveologenesis, specifically in the generation of the elastin network, and animals display bronchopulmonary dysplasia (BPD) or BPD-like phenotype. Here we show the surprising results that mesenchyme-specific loss of Hox5 function at adult stages leads to rapid disruption of the mature elastin matrix, alveolar enlargement, and an emphysema-like phenotype. As the elastin matrix of the lung is considered highly stable, adult disruption of the matrix was not predicted. Just 2 weeks after deletion, adult Hox5 mutant animals show significant increases in alveolar space and changes in pulmonary function, including reduced elastance and increased compliance. Examination of the extracellular matrix (ECM) of adult Tbx4rtTA; TetOCre; Hox5a f a f bbcc lungs demonstrates a disruption of the elastin network although the underlying fibronectin, interstitial collagen and basement membrane appear unaffected. An influx of macrophages and increased matrix metalloproteinase 12 (MMP12) are observed in the distal lung 3 days after Hox5 deletion. In culture, fibroblasts from Hox5 mutant lungs exhibit reduced adhesion. These findings establish a novel role for Hox5 transcription factors as critical regulators of lung fibroblasts at adult homeostasis.

Blocking ATP-releasing channels prevents high extracellular ATP levels and airway hyperreactivity in an asthmatic mouse model.

Allergic asthma is a chronic airway inflammatory response to different triggers like inhaled allergens. Excessive ATP in fluids from patients with asthma is considered an inflammatory signal and an important autocrine/paracrine modulator of airway physiology. Here, we investigated the deleterious effect of increased extracellular ATP (eATP) concentration on the mucociliary clearance (MCC) effectiveness and determined the role of ATP releasing channels during airway inflammation in an ovalbumin (OVA)-sensitized mouse model. Our allergic mouse model exhibited high levels of eATP measured in the tracheal fluid with a luciferin-luciferase assay and reduced MCC velocity determined by microspheres tracking in the trachea ex vivo. Addition of ATP had a dual effect on MCC, where lower ATP concentration (µM) increased microspheres velocity, whereas higher concentration (mM) transiently stopped microspheres movement. Also, an augmented ethidium bromide uptake by the allergic tracheal airway epithelium suggests an increase in ATP release channel functionality during inflammatory conditions. The use of carbenoxolone, a nonspecific inhibitor of connexin and pannexin1 channels reduced the eATP concentration in the allergic mouse tracheal fluid and dye uptake by the airway epithelium, providing evidence that these ATP release channels are facilitating the net flux of ATP to the lumen during airway inflammation. However, only the specific inhibition of pannexin1 with 10Panx peptide significantly reduced eATP in bronchoalveolar lavage and decreased airway hyperresponsiveness in OVA-allergic mouse model. These data provide evidence that blocking eATP may be a pharmacological alternative to be explored in rescue therapy during episodes of airflow restriction in patients with asthma.

Stem Cell Factor Neutralization Protects From Severe Anaphylaxis in a Murine Model of Food Allergy.

Food allergy is a growing public health problem with ~15 million people affected in the United States. In allergic food disease, IgE on mast cells bind to ingested antigens leading to the activation and degranulation of mast cells. Stem cell factor (SCF) is mast cell growth and activation factor that is required for peripheral tissue mast cells. We targeted a specific isoform of SCF, the larger 248 amino acid form, that drives peripheral tissue mast cell differentiation using a specific monoclonal antibody in a model of food allergy. Ovalbumin sensitized and intragastrically challenged mice were monitored for symptoms of anaphylaxis including respiratory distress, diarrhea, and a reduction in body temperature. During the second week of challenges, allergic mice were injected with an antibody to block SCF248 or given IgG control. Mice treated with α-SCF248 had a decreased incidence of diarrhea and no reduction in body temperature suggesting a reduction in anaphylaxis compared to IgG control treated animals. Re-stimulated mesenteric lymph nodes indicated that α-SCF248 treated mice had decreased OVA-specific Th2 cytokine production compared to IgG control treated allergic animals. The reduction of food induced anaphylaxis was accompanied by a significant reduction in gut leak. The mesenteric lymph node cells were analyzed by flow cytometry and showed a decrease in the number of type 2 innate lymphoid cells in mice injected with α-SCF248. Morphometric enumeration of esterase+ mast cells demonstrated a significant reduction throughout the small intestine. Using a more chronic model of persistent food-induced anaphylaxis, short term therapeutic treatment with α-SCF248 during established disease effectively blocked food induced anaphylaxis. Together, these data suggest that therapeutically blocking SCF248 in food allergic animals can reduce the severity of food allergy by reducing mast cell mediated disease activation.

Uric acid pathway activation during respiratory virus infection promotes Th2 immune response via innate cytokine production and ILC2 accumulation.

Respiratory syncytial virus (RSV) infects a majority of infants and can cause severe disease leading to increased risk to develop asthma later in life. In the present studies we detected high levels of uric acid pathway components during RSV infection and examined whether they altered the pathogenesis of RSV infection. Inhibition of uric acid (UA) pathway activation during RSV infection in airway epithelial cells using XOI decreased the expression of IL-33, thymic stromal lymphopoietin (TSLP), and CCL2. In addition, treatment of RSV infected bone marrow-derived macrophages with XOI decreased production of IL-1ß. Thus, UA activation of different cell populations contributes different innate immune mediators that promote immunopathogenesis. When mice were treated with XOI or interleukin-1 receptor antagonist (IL1-ra) during RSV infection decreased pulmonary mucus was observed along with significantly reduced numbers of ILC2 and macrophages, accompanied by decreased IL-33 in bronchoalveolar lavage of the treated mice. These findings provide mechanistic insight into the development of RSV immunopathology and indicate that xanthine metabolites and UA are key immunoregulator molecules during RSV infection. Moreover, these findings suggest uric acid and IL-1ß as possible therapeutic targets to attenuate severe RSV disease.

Upregulation of H3K27 Demethylase KDM6 During Respiratory Syncytial Virus Infection Enhances Proinflammatory Responses and Immunopathology.

Severe disease following respiratory syncytial virus (RSV) infection has been linked to enhanced proinflammatory cytokine production that promotes a Th2-type immune environment. Epigenetic regulation in immune cells following viral infection plays a role in the inflammatory response and may result from upregulation of key epigenetic modifiers. In this study, we show that RSV-infected bone marrow-derived dendritic cells (BMDC) as well as pulmonary dendritic cells (DC) from RSV-infected mice upregulated the expression of Kdm6b/Jmjd3 and Kdm6a/Utx, H3K27 demethylases. KDM6-specific chemical inhibition (GSK J4) in BMDC led to decreased production of chemokines and cytokines associated with the inflammatory response during RSV infection (i.e., CCL-2, CCL-3, CCL-5, IL-6) as well as decreased MHC class II and costimulatory marker (CD80/86) expression. RSV-infected BMDC treated with GSK J4 altered coactivation of T cell cytokine production to RSV as well as a primary OVA response. Airway sensitization of naive mice with RSV-infected BMDCs exacerbate a live challenge with RSV infection but was inhibited when BMDCs were treated with GSK J4 prior to sensitization. Finally, in vivo treatment with the KDM6 inhibitor, GSK J4, during RSV infection reduced inflammatory DC in the lungs along with IL-13 levels and overall inflammation. These results suggest that KDM6 expression in DC enhances proinflammatory innate cytokine production to promote an altered Th2 immune response following RSV infection that leads to more severe immunopathology.

Group 2 innate lymphoid cells (ILC2) are regulated by stem cell factor during chronic asthmatic disease.

Stem cell factor (SCF) binds to the receptor c-Kit that is expressed on a number of myeloid and lymphoid cell populations, including Type 2 innate lymphoid cells (ILC2). However the importance of the SCF/c-Kit interaction in ILC2 has not been studied. Here we investigate the role of a specific SCF isoform, SCF248, in the allergic asthmatic response and SCF/c-Kit in ILC2 activation during chronic allergy. We observed that mice treated with a monoclonal antibody specific for SCF248 attenuated the development of chronic asthmatic disease by decreasing the number of mast cells, ILC2 and eosinophils, as well as reducing the accompanying pathogenic cytokine responses. These data were supported using SCFfl/fl-Col1-Cre-ERT mice and W/Wv mice that demonstrated the importance of the stem cell factor/c-Kit activation during chronic allergy and the accumulation of c-kit+ cells. Finally, these data demonstrate for the first time that SCF could activate ILC2 cells in vitro for the production of key allergic cytokines. Together these findings indicate that SCF is a critical cytokine involved in the activation of ILC2 that lead to more severe outcomes during chronic allergy and that the SCF248 isoform could be an important therapeutic target to control the disease progression.

STAT5-induced lunatic fringe during Th2 development alters delta-like 4-mediated Th2 cytokine production in respiratory syncytial virus-exacerbated airway allergic disease.

Notch activation plays an important role in T cell development and mature T cell differentiation. In this study, we investigated the role of Notch activation in a mouse model of respiratory syncytial virus (RSV)-exacerbated allergic airway disease. During RSV exacerbation, in vivo neutralization of a specific Notch ligand, Delta-like ligand (Dll)-4, significantly decreased airway hyperreactivity, mucus production, and Th2 cytokines. Lunatic Fringe (Lfng), a glycosyltransferase that enhances Notch activation by Dll4, was increased during RSV exacerbation. Lfng loss of function in Th2-skewed cells inhibited Dll4-Notch activation and subsequent IL-4 production. Further knockdown of Lfng in T cells in CD4Cre(+)Lfng(fl/fl) mice showed reduced Th2 response and disease pathology during RSV exacerbation. Finally, we identified STAT5-binding cis-acting regulatory element activation as a critical driver of Lfng transcriptional activation. These data demonstrate that STAT5-dependent amplification of Notch-modifying Lfng augments Th2 response via Dll4 and is critical for amplifying viral exacerbation during allergic airway disease.