Features Of Allergic Airway Disease Research Articles

Prophylactic and therapeutic use of synthetically glycosylated antigens to limit allergic airway disease

Abstract Asthma is a disease involving chronic airway inflammation affecting more than 300 million people worldwide. There is currently no cure for asthma. While effective, corticosteroids cause broad immune suppression and long-term debilitating disease. Novel immune modulatory therapeutics leave patients vulnerable to infections. Therefore, there is a current need for new therapeutics that target the underlying causes of the disease without triggering detrimental side effects. Through synthetic glycosylation, our platform targets hepatic antigen presenting cells and leverages the liver’s tolerance-inducing mechanisms to limit inflammatory T cell responses, while promoting regulatory T cell (Treg) responses, thus preventing broad immunosuppression. In a model of experimental allergic airway inflammation, prophylactic treatment with synthetically mannosylated OVA (pMan OVA) ameliorated the allergic response to inhaled OVA. Our treatment reduced lung and airway eosinophilia, systemic levels of IgE and OVA-specific IgG1, and airway mucus. Furthermore, pMan OVAtreatment reduced OVA specific lung T H2 cells, and exhibited reduced production of IL-4, IL-5 and IL-13 upon antigen specific restimulation. Treatment with pMan OVAwas accompanied by induction of OVA-specific Tregs, which negatively correlated with features of allergic airway disease. In previously sensitized mice, therapeutic intervention with pMan OVAreduced airway and lung eosinophilia, B cell and CD4 +T cell inflammation. Altogether, these data suggest a potential role for prophylactic and therapeutic intervention with synthetically glycosylated allergens in ameliorating disease severity in a model of allergic airway inflammation.

The Effect of Flavored E-cigarettes on Murine Allergic Airways Disease

Flavored e-cigarettes are preferred by the majority of users yet their potential toxicity is unknown. Therefore our aim was to determine the effect of selected flavored e-cigarettes, with or without nicotine, on allergic airways disease in mice. Balb/c mice were challenged with PBS or house dust mite (HDM) (Days 0, 7, 14–18) and exposed to room air or e-cigarette aerosol for 30 min twice daily, 6 days/week from Days 0–18 (n = 8–12/group). Mice were exposed to Room Air, vehicle control (50%VG/%50PG), Black Licorice, Kola, Banana Pudding or Cinnacide without or with 12 mg/mL nicotine. Mice were assessed at 72 hours after the final HDM challenge. Compared to mice challenged with HDM and exposed to Room Air, nicotine-free Cinnacide reduced airway inflammation (p = 0.045) and increased peripheral airway hyperresponsiveness (p = 0.02), nicotine-free Banana Pudding increased soluble lung collagen (p = 0.049), with a trend towards increased airway inflammation with nicotine-free Black Licorice exposure (p = 0.089). In contrast, all e-cigarettes containing nicotine suppressed airway inflammation (p < 0.001 for all) but did not alter airway hyperresponsiveness or airway remodeling. Flavored e-cigarettes without nicotine had significant but heterogeneous effects on features of allergic airways disease. This suggests that some flavored e-cigarettes may alter asthma pathophysiology even when used without nicotine.

Combined treatment with SB203580 and dexamethasone suppresses non-typeable Haemophilus influenzae-induced Th17 inflammation response in murine allergic asthma

Accumulating evidence suggests that non-typeable Haemophilus influenzae (NTHi) infection drives the development of steroid-resistant allergic airway disease (SRAAD), exacerbates clinical symptoms, worsens quality of life, and accounts for most of the related healthcare burden. The poor understanding of the pathogenesis of SRAAD deters the development of more effective therapeutic strategies. Here, we established a murine model of NTHi infection-induced exacerbation of allergic airway disease. We showed that NTHi infection drove Th 17-mediated pulmonary neutrophilic inflammation, aggravated airway hyper-responsiveness, and upset the balance of MUC5AC and MUC5B expression. Dexamethasone treatment effectively inhibited the features of allergic airway disease but failed to reduce NTHi-induced exacerbation, which was associated with the hyper-phosphorylation of p38 mitogen-activated protein kinase (MAPK). Interestingly, inhibition of p38 using a specific inhibitor (SB203580) only partly suppressed the airway hyper-responsiveness and mucus hyper-secretion but failed to abrogate the infection-induced neutrophilic inflammatory response in SRAAD. However, SB203580 and dexamethasone co-treatment substantially suppressed all the features of NTHi-induced SRAAD. Our findings highlight the importance of p38 MAPK in the pathogenesis of NTHi-induced steroid resistance, and this combined treatment approach may be a novel strategy against steroid-resistant asthma.

Targeting PP2A and proteasome activity ameliorates features of allergic airway disease in mice.

Asthma is an allergic airway disease (AAD) caused by aberrant immune responses to allergens. Protein phosphatase-2A (PP2A) is an abundant serine/threonine phosphatase with anti-inflammatory activity. The ubiquitin proteasome system (UPS) controls many cellular processes, including the initiation of inflammatory responses by protein degradation. We assessed whether enhancing PP2A activity with fingolimod (FTY720) or 2-amino-4-(4-(heptyloxy) phenyl)-2-methylbutan-1-ol (AAL(S) ), or inhibiting proteasome activity with bortezomib (BORT), could suppress experimental AAD. Acute AAD was induced in C57BL/6 mice by intraperitoneal sensitization with ovalbumin (OVA) in combination with intranasal (i.n) exposure to OVA. Chronic AAD was induced in mice with prolonged i.n exposure to crude house dust mite (HDM) extract. Mice were treated with vehicle, FTY720, AAL(S) , BORT or AAL(S) +BORT and hallmark features of AAD assessed. AAL(S) reduced the severity of acute AAD by suppressing tissue eosinophils and inflammation, mucus-secreting cell (MSC) numbers, type 2-associated cytokines (interleukin (IL)-33, thymic stromal lymphopoietin, IL-5 and IL-13), serum immunoglobulin (Ig)E and airway hyper-responsiveness (AHR). FTY720 only suppressed tissue inflammation and IgE. BORT reduced bronchoalveolar lavage fluid (BALF) and tissue eosinophils and inflammation, IL-5, IL-13 and AHR. Combined treatment with AAL(S) +BORT had complementary effects and suppressed BALF and tissue eosinophils and inflammation, MSC numbers, reduced the production of type 2 cytokines and AHR. AAL(S) , BORT and AAL(S) +BORT also reduced airway remodelling in chronic AAD. These findings highlight the potential of combination therapies that enhance PP2A and inhibit proteasome activity as novel therapeutic strategies for asthma.

Plethysmography Phenotype QTL in Mice Before and After Allergen Sensitization and Challenge

Allergic asthma is common airway disease that is characterized in part by enhanced airway constriction in response to nonspecific stimuli. Genome-wide association studies have identified multiple loci associated with asthma risk in humans, but these studies have not accounted for gene–environment interactions, which are thought to be important factors in asthma. To identify quantitative trait loci (QTL) that regulate responses to a common human allergen, we applied a house dust mite mouse (HDM) model of allergic airway disease (AAD) to 146 incipient lines of the Collaborative Cross (CC) and the CC founder strains. We employed a longitudinal study design in which mice were phenotyped for response to the bronchoconstrictor methacholine both before and after HDM sensitization and challenge using whole body plethysmography (WBP). There was significant variation in methacholine responsiveness due to both strain and HDM treatment, as reflected by changes in the WBP parameter enhanced pause. We also found that distinct QTL regulate baseline [chromosome (Chr) 18] and post-HDM (Chr 19) methacholine responsiveness and that post-HDM airway responsiveness was correlated with other features of AAD. Finally, using invasive measurements of airway mechanics, we tested whether the Chr 19 QTL affects lung resistance per se using C57BL/6J mice and a consomic strain but found that QTL haplotype did not affect lung resistance. We conclude that aspects of baseline and allergen-induced methacholine responsiveness are associated with genetic variation, and that robust detection of airway resistance QTL in genetically diverse mice will be facilitated by direct measurement of airway mechanics.

Epidermal growth factor receptor signalling regulates granulocyte-macrophage colony-stimulating factor production by airway epithelial cells and established allergic airway disease.

Airway epithelial cells (AEC) are increasingly recognized as a major signalling centre in the pathogenesis of allergic asthma. A previous study demonstrated that epithelial growth factor receptor (EGFR) signalling in AEC regulated key features of allergic airway disease. However, it is unclear what mediators are regulated by EGFR signalling in AEC, although the production of the pro-inflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is EGFR dependent in keratinocytes. To determine whether EGFR signalling regulates GM-CSF production by human AEC downstream of the clinically relevant mediators house dust mite (HDM) and interleukin (IL)-17A and in a mouse model of established allergic asthma. EGFR inhibitors were used to determine whether EGFR signalling regulates GM-CSF production by cultured human AEC in response to HDM and IL-17A. The roles of EGFR ligands, p38 mitogen-activated protein kinase (MAPK) and tumour necrosis factor-alpha (TNF-α) converting enzyme (TACE) were also assessed. To determine whether EGFR regulates GM-CSF as well as key asthma characteristics invivo, mice were chronically exposed to HDM to establish allergic airway disease and then treated with the EGFR inhibitor Erlotinib. EGFR inhibition reduced HDM and IL-17A induced GM-CSF production in a dose-dependent manner in cultured human AEC. GM-CSF production also required amphiregulin, p38 MAPK signalling and protease/TACE activity. In mice with established allergic airway disease, EGFR inhibition reduced levels of GM-CSF and TNF-α, as well as airway hyperreactivity, cellular inflammation, smooth muscle thickening and goblet cell metaplasia without changes in IgE and Th1, Th2 and Th17 cytokines. Results link HDM, IL-17A, amphiregulin, EGFR and GM-CSF in a mechanistic pathway in AEC and demonstrate that EGFR regulates GM-CSF production and the severity of established disease in a clinically relevant asthma model. These results identify the EGFR→GM-CSF axis as a target for therapeutic development.

Airway epithelial NF-κB activation promotes the ability to overcome inhalational antigen tolerance.

Inhalational antigen tolerance typically protects against the development of allergic airway disease but may be overcome to induce allergic sensitization preceding the development of asthma. We examined in vivo whether pre-existing inhalational antigen tolerance could be overcome by activation of the transcription factor NF-κB in conducting airway epithelial cells, and used a combination of in vivo and in vitro approaches to examine the mechanisms involved. Wild-type and transgenic mice capable of expressing constitutively active IκB kinase β (CAIKKβ) in airway epithelium were tolerized to inhaled ovalbumin. Twenty-eight days later, the transgene was transiently expressed and mice were exposed to inhaled OVA on Day 30 in an attempt to overcome inhalational tolerance. Following ovalbumin challenge on days 40-42, CAIKKβ mice in which the transgene had been activated exhibited characteristic features of allergic airway disease, including airway eosinophilia and methacholine hyper-responsiveness. Increases in the CD103(+) and CD11b(HI) lung dendritic cell populations were present in CAIKKβ mice on Day 31. Bronchoalveolar lavage from mice expressing CAIKKβ mice induced CD4(+) T cells to secrete T(H)2 and T(H)17 cytokines, an effect that required IL-4 and IL-1 signalling, respectively. CAIKKβ mice on Dox demonstrated increased numbers of innate lymphoid type 2 cells (ILC2) in the lung, which also exhibited elevated mRNA expression of the T(H)2-polarizing cytokine IL-4. Finally, airway epithelial NF-kB activation induced allergic sensitization in CAIKKβ mice on Dox that required IL-4 and IL-1 signalling in vivo. Our studies demonstrate that soluble mediators generated in response to airway epithelial NF-κB activation orchestrate the breaking of inhalational tolerance and allergic antigen sensitization through the effects of soluble mediators, including IL-1 and IL-4, on pulmonary dendritic cells as well as innate lymphoid and CD4(+) T cells.

Immunomodulation of airway epithelium cell activation by mesenchymal stromal cells ameliorates house dust mite-induced airway inflammation in mice.

Allergic asthma is underpinned by T helper 2 (Th2) inflammation. Redundancy in Th2 cytokine function and production by innate and adaptive immune cells suggests that strategies aimed at immunomodulation may prove more beneficial. Hence, we sought to determine whether administration of mesenchymal stromal cells (MSCs) to house dust mite (HDM) (Dermatophagoides pteronyssinus)-sensitized mice would suppress the development of Th2 inflammation and airway hyperresponsiveness (AHR) after HDM challenge. We report that the intravenous administration of allogeneic donor MSCs 1 hour before allergen challenge significantly attenuated the features of allergic asthma, including tissue eosinophilia, Th2 cytokine (IL-5 and IL-13) levels in bronchoalveolar lavage fluid, and AHR. The number of infiltrating type 2 innate lymphoid cells was not affected by MSC transfer, suggesting that MSCs may modulate the adaptive arm of Th2 immunity. The effect of MSC administration was long lasting; all features of allergic airway disease were significantly suppressed in response to a second round of HDM challenge 4 weeks after MSC administration. Further, we observed that MSCs decreased the release of epithelial cell-derived alarmins IL-1α and high mobility group box-1 in an IL-1 receptor antagonist-dependent manner. This significantly decreased the expression of the pro-Th2 cytokine IL-25 and reduced the number of activated and antigen-acquiring CD11c(+)CD11b(+) dendritic cells in the lung and mediastinal lymph nodes. Our findings suggest that MSC administration can ameliorate allergic airway inflammation by blunting the amplification of epithelial-derived inflammatory cytokines induced by HDM exposure and may offer long-term protection against Th2-mediated allergic airway inflammation and AHR.

Endoplasmic reticulum stress mediates house dust mite-induced airway epithelial apoptosis and fibrosis

BackgroundThe endoplasmic reticulum (ER) stress response participates in many chronic inflammatory and autoimmune diseases. In the current study, we sought to examine the contribution of ER stress transducers in the pathogenesis of three principal facets of allergic asthma: inflammation, airway fibrosis, and airways hyperresponsiveness.MethodsHouse Dust Mite (HDM) was used as an allergen for in vitro and in vivo challenge of primary human and murine airway epithelial cells. ER stress transducers were modulated using specific small interfering RNAs (siRNAs) in vivo. Inflammation, airway remodeling, and hyperresponsiveness were measured by total bronchoalveolar lavage (BAL) cell counts, determination of collagen, and methacholine responsiveness in mice, respectively.ResultsChallenge of human bronchiolar and nasal epithelial cells with HDM extract induced the ER stress transducer, activating transcription factor 6 α (ATF6α) as well as protein disulfide isomerase, ERp57, in association with activation of caspase-3. SiRNA-mediated knockdown of ATF6α and ERp57 during HDM administration in mice resulted in a decrease in components of HDM-induced ER stress, disulfide mediated oligomerization of Bak, and activation of caspase-3. Furthermore, siRNA-mediated knockdown of ATF6α and ERp57 led to decreased inflammation, airway hyperresponsiveness and airway fibrosis.ConclusionCollectively, our work indicates that HDM induces ER stress in airway epithelial cells and that ATF6α and ERp57 play a significant role in the development of cardinal features of allergic airways disease. Inhibition of ER stress responses may provide a potential therapeutic avenue in chronic asthma and sub-epithelial fibrosis associated with loss of lung function.

Disparate features of allergic airway disease between young and old mice

The prevalence of asthma in the elderly (&gt;;65) is increasing and associated with higher mortality than in children or young adults. Despite this increase, the effects of age on the development, character and severity of allergic asthma have been understudied. To elucidate the influence of age on allergic airway disease, we compared pulmonary responses of young (10‐week) and old (14‐month) BALB/c mice that were intranasally sensitized and challenged with house dust mite extract (HDM). After challenge, bronchoalveolar lavage fluid (BALF) was collected for cell and cytokine analysis and lung tissue was processed for gene expression analysis and histopathology. Both young and old mice developed HDM‐induced allergic airway disease. This was manifested by the presence of BALF eosinophlis and Th2 cytokines (IL5, IL13), as well as elevated gene expression of eotaxin, chitinase 3‐like 4 and airway mucus regulating genes Gob5 and Muc5AC. Compared to young mice, old mice had a significantly greater pulmonary allergic response (e.g., 10 × more eosinophils and IL13). In addition, only old mice showed a Th17 immune response to HDM characterized by increased BALF neutrophils and IL17, IL6 and KC cytokines. These results suggest that the severity and character of allergic airway disease is age dependent and the elderly may be prone to develop more severe allergic asthma with a mixed Th2/Th17 immune response. USEPA RD83479701

Chlamydia muridarum Lung Infection in Infants Alters Hematopoietic Cells to Promote Allergic Airway Disease in Mice

BackgroundViral and bacterial respiratory tract infections in early-life are linked to the development of allergic airway inflammation and asthma. However, the mechanisms involved are not well understood. We have previously shown that neonatal and infant, but not adult, chlamydial lung infections in mice permanently alter inflammatory phenotype and physiology to increase the severity of allergic airway disease by increasing lung interleukin (IL)-13 expression, mucus hyper-secretion and airway hyper-responsiveness. This occurred through different mechanisms with infection at different ages. Neonatal infection suppressed inflammatory responses but enhanced systemic dendritic cell:T-cell IL-13 release and induced permanent alterations in lung structure (i.e., increased the size of alveoli). Infant infection enhanced inflammatory responses but had no effect on lung structure. Here we investigated the role of hematopoietic cells in these processes using bone marrow chimera studies.Methodology/Principal FindingsNeonatal (<24-hours-old), infant (3-weeks-old) and adult (6-weeks-old) mice were infected with C. muridarum. Nine weeks after infection bone marrow was collected and transferred into recipient age-matched irradiated naïve mice. Allergic airway disease was induced (8 weeks after adoptive transfer) by sensitization and challenge with ovalbumin. Reconstitution of irradiated naïve mice with bone marrow from mice infected as neonates resulted in the suppression of the hallmark features of allergic airway disease including mucus hyper-secretion and airway hyper-responsiveness, which was associated with decreased IL-13 levels in the lung. In stark contrast, reconstitution with bone marrow from mice infected as infants increased the severity of allergic airway disease by increasing T helper type-2 cell cytokine release (IL-5 and IL-13), mucus hyper-secretion, airway hyper-responsiveness and IL-13 levels in the lung. Reconstitution with bone marrow from infected adult mice had no effects.ConclusionsThese results suggest that an infant chlamydial lung infection results in long lasting alterations in hematopoietic cells that increases the severity of allergic airway disease in later-life.

Strain-Dependent Genomic Factors Affect Allergen-Induced Airway Hyperresponsiveness in Mice

Asthma is etiologically and clinically heterogeneous, making the genomic basis of asthma difficult to identify. We exploited the strain-dependence of a murine model of allergic airway disease to identify different genomic responses in the lung. BALB/cJ and C57BL/6J mice were sensitized with the immunodominant allergen from the Dermatophagoides pteronyssinus species of house dust mite (Der p 1), without exogenous adjuvant, and the mice then underwent a single challenge with Der p 1. Allergic inflammation, serum antibody titers, mucous metaplasia, and airway hyperresponsiveness were evaluated 72 hours after airway challenge. Whole-lung gene expression analyses were conducted to identify genomic responses to allergen challenge. Der p 1-challenged BALB/cJ mice produced all the key features of allergic airway disease. In comparison, C57BL/6J mice produced exaggerated Th2-biased responses and inflammation, but exhibited an unexpected decrease in airway hyperresponsiveness compared with control mice. Lung gene expression analysis revealed genes that were shared by both strains and a set of down-regulated genes unique to C57BL/6J mice, including several G-protein-coupled receptors involved in airway smooth muscle contraction, most notably the M2 muscarinic receptor, which we show is expressed in airway smooth muscle and was decreased at the protein level after challenge with Der p 1. Murine strain-dependent genomic responses in the lung offer insights into the different biological pathways that develop after allergen challenge. This study of two different murine strains demonstrates that inflammation and airway hyperresponsiveness can be decoupled, and suggests that the down-modulation of expression of G-protein-coupled receptors involved in regulating airway smooth muscle contraction may contribute to this dissociation.

Modulation of NF-κB and Hypoxia-Inducible Factor−1 byS-Nitrosoglutathione Does Not Alter Allergic Airway Inflammation in Mice

Induction of nitric oxide synthase (NOS)-2 and production of nitric oxide (NO) are common features of allergic airway disease. Conditions of severe asthma are associated with deficiency of airway S-nitrosothiols, a biological product of NO that can suppress inflammation by S-nitrosylation of the proinflammatory transcription factor, NF-κB. Therefore, restoration of airway S-nitrosothiols might have therapeutic benefit, and this was tested in a mouse model of ovalbumin (OVA)-induced allergic inflammation. Naive or OVA-sensitized animals were administered S-nitrosoglutathione (GSNO; 50 μl, 10 mM) intratracheally before OVA challenge and analyzed 48 hours later. GSNO administration enhanced lung tissue S-nitrosothiol levels and reduced NF-κB activity in OVA-challenged animals compared with control animals, but did not lead to significant changes in total bronchoalveolar lavage cell counts, differentials, or mucus metaplasia markers. Administration of GSNO also altered the activation of hypoxia-inducible factor (HIF)-1, leading to HIF-1 activation in naive mice, but suppressed HIF-1 activation in OVA-challenged mice. We assessed the contribution of endogenous NOS2 in regulating NF-κB and/or HIF-1 activation and allergic airway inflammation using NOS2(-/-) mice. Although OVA-induced NF-κB activation was slightly increased in NOS2(-/-) mice, associated with small increases in bronchoalveolar lavage neutrophils, other markers of allergic inflammation and HIF-1 activation were similar in NOS2(-/-) and wild-type mice. Collectively, our studies indicate that instillation of GSNO can suppress NF-κB activation during allergic airway inflammation, but does not significantly affect overall markers of inflammation or mucus metaplasia, thus potentially limiting its therapeutic potential due to effects on additional signaling pathways, such as HIF-1.

Early-life chlamydial lung infection enhances allergic airways disease through age-dependent differences in immunopathology

Asthma typically originates in early-life, and the impact of infection during immunologic maturation is a critical factor in disease pathogenesis. The progression of aberrant T(H)2 cell responses and disease development has been attributed to a lack of infections. However, exposure to specific pathogens such as Chlamydia may alter immunologic programming and predispose to asthma. To investigate the effects of chlamydial infection at different ages on allergic airways disease in later life. Neonatal, infant, or adult BALB/c mice were infected and 6 weeks later were sensitized and subsequently challenged with ovalbumin. Hallmark features of allergic airways disease were compared with uninfected allergic and nonallergic controls. Early-life (neonatal and infant) but not adult chlamydial infection enhanced the development of hallmark features of asthma in ovalbumin-induced allergic airways disease. Notably early-life infection increased mucus-secreting cell numbers, IL-13 expression, and airway hyperresponsiveness. Neonatal infection attenuated eosinophil influx and ovalbumin-specific T(H)2 cytokine release and numbers of activated myeloid dendritic cells (DCs) in lymph nodes. By contrast, infant infection augmented features of allergic inflammation with increased airway eosinophils, T(H)2 cytokine, and DC responses. Both neonatal and infant infection increased systemic DC-induced IL-13 release from CD4(+) T cells. The timing of infection had significant effects on lung structure because neonatal but not infant or adult infection induced increases in alveolar diameter. Early-life respiratory chlamydial infections modulate immune responses, alter lung function and structure, and enhance the severity of allergic airways disease in later life.

Intranasal CpG Therapy Attenuated Experimental Fungal Asthma in a TLR9-Dependent and -Independent Manner

Background: CpG administration abolishes airway inflammation and remodeling in acute models of allergic airway disease. Methods: Herein, we investigated the therapeutic effect of CpG in a chronic fungal model of asthma. TLR9+/+ and TLR9–/– mice were sensitized to soluble Aspergillus fumigatus antigens and challenged with live A. fumigatus conidia. Mice were treated with intraperitoneal (IP) or intranasal (IN) CpG, or left untreated 14–28 days after conidium challenge. All features of allergic airway disease were attenuated in TLR9+/+ mice treated with IN CpG, including airway hyperresponsiveness (AHR), mucus production, and peribronchial fibrosis. Results: TLR9–/– mice treated with IN CpG exhibited attenuated airway remodeling but not AHR. Whole-lung IL-12 levels were significantly elevated in both TLR9+/+ and TLR9–/– mice receiving IN CpG but not in either group receiving IP CpG. Whole-lung IL-10 levels were significantly elevated in IN CpG-treated TLR9+/+ mice but not in TLR9–/– mice receiving IN CpG. Increased whole-lung transcript and protein levels of the scavenger receptors SR-A and MARCO were observed in TLR9–/– mice compared with TLR9+/+ mice, possibly accounting for the CpG responsiveness in the knockout group. Conclusions: Together, these data show that IN CpG has a therapeutic effect during established fungal asthma, which is TLR9 dependent and independent.

Small Interfering RNA against Transcription Factor STAT6 Inhibits Allergic Airway Inflammation and Hyperreactivity in Mice

In the context of allergic immune responses, activation of STAT6 is pivotal for Th2-mediated IgE production and development of airway inflammation and hyperreactivity. We analyzed whether gene silencing of STAT6 expression by RNA interference was able to suppress allergen-induced immune and airway responses. Knockdown effectiveness of three different STAT6 siRNA molecules was analyzed in murine and human cell cultures. The most potent siRNA was used for further testing in a murine model of allergen-induced airway inflammation and airway hyperreactivity (AHR). BALB/c mice were sensitized with OVA/alum twice i.p. (days 1 and 14), and challenged via the airways with allergen (days 28-30). Intranasal application of STAT6 siRNA before and during airway allergen challenges reduced levels of infiltrating cells, especially of eosinophils, in the bronchoalveolar lavage fluid, compared with GFP siRNA-treated sensitized and challenged controls. Allergen-induced alterations in lung tissues (goblet cell hyperplasia, peribronchial inflammation with eosinophils and CD4 T cells) were significantly reduced after STAT6 siRNA treatment. Associated with decreased inflammation was a significant inhibition of the development of allergen-induced in vivo AHR after STAT6 siRNA treatment, compared with GFP siRNA-treated sensitized and challenged controls. Importantly, mRNA and protein expression levels of IL-4 and IL-13 in lung tissues of STAT6-siRNA treated mice were significantly diminished compared with sensitized and challenged controls. These data show that targeting the key transcription factor STAT6 by siRNA effectively blocks the development of cardinal features of allergic airway disease, like allergen-induced airway inflammation and AHR. It may thus be considered as putative approach for treatment of allergic airway diseases such as asthma.

INTRANASAL CpG THERAPY ATTENUATED EXPERIMENTAL FUNGAL ASTHMA IN A TLR9-DEPENDENT AND INDEPENDENT MANNER (140.4)

Abstract CpG-ODN administration abolishes airway inflammation and remodeling in acute models of allergic airway disease. Herein, we investigated the therapeutic effect of CpG-ODN in a chronic fungal model of asthma. TLR9+/+ and TLR9-/- mice were sensitized to soluble A. fumigatus antigens and challenged with live A. fumigatus conidia. Mice were treated with intraperitoneal (IP) CpG, intranasal (IN) CpG, or left untreated from days 14 to 28-post conidia challenge. All features of allergic airway disease were attenuated in TLR9+/+ mice treated with IN CpG, including airway hyperresponsiveness (AHR), mucus production, and peribronchial fibrosis. TLR9-/- mice treated with IN CpG exhibited attenuated airway remodeling, but not AHR. Whole-lung IL-12 levels were significantly elevated in both TLR9+/+ and TLR9-/- mice receiving IN CpG but not in either group receiving IP CpG. Whole-lung IL-10 levels were significantly elevated in IN CpG-treated TLR9+/+ mice but not in TLR9-/- mice receiving IN CpG. Increased whole-lung transcript and protein levels of the scavenger receptors SR-A and MARCO were observed in TLR9-/- mice compared with TLR9+/+ mice, possibly accounting for the CpG responsiveness in the knockout group. Together, these data show that IN CpG has a therapeutic effect during established fungal asthma, which is TLR9-dependent and independent. This work was financially supported, in part, by the NIH (grant HL069865 to CMH).

The extracellular matrix protein mindin regulates trafficking of murine eosinophils into the airspace.

Asthma remains a major cause of morbidity and hospitalizations in developed nations. Despite the widespread prevalence of this disease, the genetic and environmental factors that mediate development and progression of allergic airways disease remain poorly understood. Pulmonary recruitment of eosinophils is believed to contribute to many cardinal features of allergic airways disease. Therefore, it is paramount to understand host factors that contribute to pulmonary eosinophil recruitment into the lungs. Mindin is a component of pulmonary extracellular matrix, which can regulate inflammatory cell recruitment. We characterized the role of mindin in the severity of allergic airways disease using established murine models. There were no baseline differences in wild-type and mindin-deficient animals in cell counts or airway physiology. Using the OVA murine model of allergic airways disease, we observed that mindin-deficient animals have less-severe allergic airways disease with fewer airspace eosinophils and lower lung-lavage levels of inflammatory Th2 cytokines such as IL-13 and IL-4. Furthermore, mindin-deficient animals have reduced airway hyper-responsiveness after methacholine challenge. To determine the role of mindin in eosinophil trafficking, independent of antigen immunization or T lymphocyte activation, we instilled IL-13 directly into the lungs of mice. In this model, mindin regulates eosinophil recruitment into the airspace. In vitro experiments demonstrate that mindin can enhance eotaxin-mediated eosinophil adhesion and migration, which are dependent on the expression of integrins alphaMbeta2 and alpha4beta1. In conclusion, these data suggest that mindin participates in integrin-dependent trafficking of eosinophils and can contribute to the severity of allergic airways disease.

Respiratory syncytial virus infection provokes airway remodelling in allergen-exposed mice in absence of prior allergen sensitization.

The mechanisms underlying exacerbation of asthma induced by respiratory syncytial virus (RSV) infection have been extensively studied in human and animal models. However, most of these studies focused on acute inflammation and little is known of its long-term consequences on remodelling of the airway tissue. The aim of the study was to use a murine model of prolonged allergen-induced airway inflammation to investigate the effect of RSV infection on allergic airway inflammation and tissue remodelling. We subjected mice to RSV infection before or during the chronic phase of airway challenges with OVA and compared parameters of airway inflammation and remodelling at the end-point of the prolonged allergen-induced airway inflammation protocol. RSV infection did not affect the severity of airway inflammation in any of the groups studied. However, RSV infection provoked airway remodelling in non-sensitized, allergen-challenged mice that did not otherwise develop any of the features of allergic airways disease. Increased collagen synthesis in the lung and thickening of the bronchial basal membrane was observed in non-sensitized allergen-challenged mice only after prior RSV infection. In addition, fibroblast growth factor (FGF)-2 but not TGF-beta(1) was increased in this group following RSV infection. Our data show for the first time that RSV infection can prime the lung of mice that are not previously systemically sensitized, to develop airway remodelling in response to allergen upon sole exposure via the airways. Moreover, our results implicate RSV-induced FGF-2 in the remodelling process in vivo.

Nuclear Factor-κB Activation in Airway Epithelium Induces Inflammation and Hyperresponsiveness

Nuclear factor (NF)-kappaB is a prominent proinflammatory transcription factor that plays a critical role in allergic airway disease. Previous studies demonstrated that inhibition of NF-kappaB in airway epithelium causes attenuation of allergic inflammation. We sought to determine if selective activation of NF-kappaB within the airway epithelium in the absence of other agonists is sufficient to cause allergic airway disease. A transgenic mouse expressing a doxycycline (Dox)-inducible, constitutively active (CA) version of inhibitor of kappaB (IkappaB) kinase-beta (IKKbeta) under transcriptional control of the rat CC10 promoter, was generated. After administration of Dox, expression of the CA-IKKbeta transgene induced the nuclear translocation of RelA in airway epithelium. IKKbeta-triggered activation of NF-kappaB led to an increased content of neutrophils and lymphocytes, and concomitant production of proinflammatory mediators, responses that were not observed in transgenic mice not receiving Dox, or in transgene-negative littermate control animals fed Dox. Unexpectedly, expression of the IKKbeta transgene in airway epithelium was sufficient to cause airway hyperresponsiveness and smooth muscle thickening in absence of an antigen sensitization and challenge regimen, the presence of eosinophils, or the induction of mucus metaplasia. These findings demonstrate that selective activation NF-kappaB in airway epithelium is sufficient to induce airway hyperresponsiveness and smooth muscle thickening, which are both critical features of allergic airway disease.