Increased Airway Hyperresponsiveness Research Articles

Poly(I:C) promotes the production of IL‐17A by murine CD1d‐driven invariant NKT cells in airway inflammation

IL-17A is associated with different asthma phenotypes as virus-associated or steroid-resistant asthma. Invariant natural killer T (iNKT) cells play an important role in the pathogenesis of asthma. The aim of the study was to evaluate the activity of polyinosinic-polycytidylic acid [poly(I:C)] on IL-17A production by CD1d-activated iNKT cells. We analysed the in vitro effect of poly(I:C) on the release of IL-17A by spleen and lung CD1d-activated iNKT cells with α-galactosylceramide (α-GalCer). Its activity was also investigated in an α-GalCer-induced murine models, including lung inflammation. The inhibition of IL-17A by Toll-like receptor (TLR) 7 agonists in the same in vitro and in vivo models has been analysed. Poly(I:C) upregulated the in vitro IL-17A production by CD1d-activated NK1.1-CD4-iNKT subset, without modifying type 1 and type 2 cytokines. The two stimuli selectively upregulated IL-17A serum levels in vivo. Their intratracheal administration resulted in increased airway hyper-reactivity (AHR), neutrophilia in bronchoalveolar lavage and airway inflammation, which were inhibited by anti-IL-17A antibody. Poly(I:C) effects were attributable to IL1β and IL-23 release from dendritic cells, as showed by inhibition with neutralizing antibodies. TLR7 agonists inhibited the IL-17A production by poly(I:C) plus α-GalCer in the same models. Such effect was associated with the increased production by DC of IL-17A-inhibiting cytokines and the dampening of IL-1β and IL-23. Synthetic dsRNA selectively expand a CD1d-driven IL-17A-producing iNKT cell subset, thus explaining the worsening of airway inflammation by some viral infections. TLR3- and TLR7-triggering viral sequences can exert variable and opposite effects on adaptive immune response.

Inflammatory airway responses by nasal inoculation of suspended particulate matter in NC/Nga mice

To evaluate the allergic effect of airborne particulate matter (PM) on the airway, separated soluble supernatant (Sup) and insoluble precipitate (Pre) in suspended PM were inoculated into NC/Nga mice with a high sensitivity for mite allergens. Sup, Pre, or both Sup and Pre with or without pronase treatment were inoculated via the nasal route five times for sensitization and a challenge inoculation on the 11th day in NC/Nga mice. On the 14th day, mice were examined for airway hyperresponsiveness (AHR), bronchoalveolar lavage fluid (BALF) cell count, mRNA expression of Th1 and Th2 cytokines in the lung tissue, and histopathology. Synergistic effects of Sup and Pre were observed as increases in AHR and a histopathological change of Periodic acid-Schiff (PAS) staining. Increases in neutrophils, macrophages, and lymphocytes of BALF cells were dependent on Pre. The expression of IL-4 mRNA was increased by Sup, and those of IL-5 mRNA and Il-13 mRNA was increased by Sup and Pre. Augmented AHR, mRNA expression of IL-4, peribronchial inflammation, and PAS staining by Sup plus Pre were attenuated by treatment of Sup with pronase to digest proteins. These results suggest that some proteins of ambient PM may be important environmental factors for AHR and airway inflammation with the aid of insoluble particulates, although some soluble factors such as endotoxins cannot be ruled out.

Corticosteroid treatment inhibits airway hyperresponsiveness and lung injury in a murine model of chemical-induced airway inflammation

ContextExposure to toxic alkylating mustard agents causes both acute and long-term effects to the lungs as indicated by increased number of inflammatory cells in airways, lung edema and lung tissue fibrosis. We have previously demonstrated that treatment with the corticosteroid dexamethasone 1h after lung exposure to the nitrogen mustard analog melphalan protects mice from acute and sub-acute inflammatory responses, as well as from lung tissue fibrosis. ObjectiveIn order to address the importance of early anti-inflammatory treatment, we investigated the therapeutic effect of dexamethasone administered 1, 2 or 6h following exposure to melphalan. MethodsC57BL/6 mice were exposed to melphalan and treated with dexamethasone 1, 2 or 6h after exposure. Twenty hours or 14 days post exposure mice were subjected to analysis of respiratory mechanics where the effects of incremental doses of methacholine on central and peripheral lung components were measured. We also determined the amount of inflammatory cells in the bronchoalveolar lavage fluid and measured the amount of collagen content in the lungs. ResultsMelphalan exposure increased airway hyperresponsiveness in both central and peripheral airways and induced an airway inflammation dominated by infiltration of macrophages and neutrophils. Dexamethasone given 1h after exposure to melphalan provided better protection against airway inflammation than administration 2 or 6h after exposure. Collagen deposition 14 days after exposure was decreased due to dexamethasone treatment. ConclusionEarly treatment with dexamethasone is important in order to reduce the airway hyperresponsiveness and inflammation caused by toxic alkylating mustards such as melphalan.

Genetic ablation of glutaredoxin-1 causes enhanced resolution of airways hyperresponsiveness and mucus metaplasia in mice with allergic airways disease

Protein-S-glutathionylation (PSSG) is an oxidative modification of reactive cysteines that has emerged as an important player in pathophysiological processes. Under physiological conditions, the thiol transferase, glutaredoxin-1 (Glrx1) catalyses deglutathionylation. Although we previously demonstrated that Glrx1 expression is increased in mice with allergic inflammation, the impact of Glrx1/PSSG in the development of allergic airways disease remains unknown. In the present study we examined the impact of genetic ablation of Glrx1 in the pathogenesis of allergic inflammation and airway hyperresponsiveness (AHR) in mice. Glrx1(-/-) or WT mice were subjected to the antigen, ovalbumin (OVA), and parameters of allergic airways disease were evaluated 48 h after three challenges, and 48 h or 7 days after six challenges with aerosolized antigen. Although no clear increases in PSSG were observed in WT mice in response to OVA, marked increases were detected in lung tissue of mice lacking Glrx1 48 h following six antigen challenges. Inflammation and expression of proinflammatory mediators were decreased in Glrx1(-/-) mice, dependent on the time of analysis. WT and Glrx1(-/-) mice demonstrated comparable increases in AHR 48 h after three or six challenges with OVA. However, 7 days postcessation of six challenges, parameters of AHR in Glrx1(-/-) mice were resolved to control levels, accompanied by marked decreases in mucus metaplasia and expression of Muc5AC and GOB5. These results demonstrate that the Glrx1/S-glutathionylation redox status in mice is a critical regulator of AHR, suggesting that avenues to increase S-glutathionylation of specific target proteins may be beneficial to attenuate AHR.

Oral administration of Enterococcus faecalis FK-23 suppresses Th17 cell development and attenuates allergic airway responses in mice

Evidence is increasing that oral administration of probiotics can attenuate asthmatic responses both in murine models and clinical trials. T-helper 17 (Th17) cells, a subset of CD4+ T cells have been implicated as having an important role in the development of several allergic disorders, but the relationship between oral administration of probiotics and Th17 development has not been well studied. BALB/c mice were given lysed Enterococcus faecalis FK-23 (LFK) orally for 28 days. After sensitization by subcutaneous injection of ovalbumin (OVA) on Days 14 and 21 and 1% OVA inhalation on Days 25, 26 and 27, they were challenged with a 5% OVA aerosol on Day 28. Twenty-four hours later, airway resistance and accumulation of inflammatory cells in bronchoalveolar lavage fluid (BALF) and lung tissues were determined. Ιnterleukin (IL)-17-expressing CD4+ lymphocytes isolated from lung, spleen and lamina propria of the intestine were detected by flow cytometry. The expression of IL-6 and TGF-β mRNA was assessed by real-time PCR. Increases in airway hyperresponsiveness, and numbers of total leukocytes and mast cells in BALF induced by OVA challenge were significantly suppressed by oral administration of LFK. The increased percentage of IL-17-expressing CD4+ cells from lung, spleen and intestine in OVA-challenged mice was reduced following LFK treatment. We conclude that the oral administration of LFK suppresses the asthmatic response and that this is associated with attenuation of Th17 cell development.

The effect of mold sensitization and humidity upon allergic asthma

Humidity is commonly associated with increased airway hyperresponsiveness in asthma. To examine mold sensitization in patients with allergic asthma or allergic rhinitis and self-reports of humidity as exacerbating factors of clinical symptoms. A retrospective, cross-sectional study at a University hospital outpatient allergy and asthma clinic was performed. A total of 106 patients with either allergic asthma or allergic rhinitis completed standard prick-puncture skin testing with 17 allergens and controls and completed standardized forms addressing trigger factors for clinical symptoms. Allergic asthmatics sensitized to Cladosporium were more likely to have a more severe asthma severity class (odds ratio = 4.26, confidence interval = 1.30-16.93). Sensitization to Alternaria, Cladosporium, Helminthosporium, Aspergillus and Dermatophagoides pteronyssinus in asthma was associated with higher likelihood for previous hospitalization, while sensitization to Cladosporium, Helminthosporium, Aspergillus, Dermatophagoides pteronyssinus and cockroach in asthma was associated with higher likelihood of having reduced pulmonary function based on forced expiratory volume in 1s. Furthermore, allergic asthmatics more commonly reported humidity as an exacerbating factor of symptoms than did patients only with allergic rhinitis (68.42% vs 42.86%, respectively; P < 0.05). Mold sensitization is highly associated with more severe asthma, while humidity is more of an exacerbating factor in patients with allergic asthma as compared with allergic rhinitis alone. Further delineation between mold sensitization and humidity is needed to determine whether these are independent factors in asthma.

Mycoplasma pneumoniae CARDS toxin exacerbates ovalbumin induced asthma in BALB/c mice (57.9)

Abstract Mycoplasma pneumoniae is a common human respiratory pathogen that causes acute and chronic respiratory infections that can result in a variety of pulmonary and extra-pulmonary pathologies. M. pneumoniae is associated with exacerbations of human asthma and has significant impact on pathology through unclear mechanisms. We recently demonstrated that Community Acquired Respiratory Distress Syndrome (CARDS) toxin induces an allergic asthma-like disease in naïve mice, as evidenced by a Th2 response, mucus metaplasia and airway hyperreactivity. In this study we examined CARDS toxin’s effects on preexisting asthma using an ovalbumin mouse model of asthma. We show that CARDS toxin can exacerbate pathologies associated with ovalbumin-induced asthma in BALB/c mice both histologically and physiologically. These results were highlighted by increased collagen deposition, shown by picrosirius red staining around medium airways and an increase in eosinophils in the alveolar spaces, shown by cellular differentials. Additionally, these findings are paralleled by heightened Th2 cytokine and chemokine responses as measured by qPCR or ELISA. Finally, lung function assessment via a Flexivent system revealed increases in airway hyperreactivity demonstrated by increased resistance and decreased compliance. The data demonstrate CARDS toxin as a causal factor in the exacerbation of experimental allergic asthma and implicates CARDS toxin as a mediator of morbidity in human asthma.

Chronic Endotoxin Exposure Produces Airflow Obstruction and Lung Dendritic Cell Expansion

Little is known about the mechanisms of persistent airflow obstruction that result from chronic occupational endotoxin exposure. We sought to analyze the inflammatory response underlying persistent airflow obstruction as a result of chronic occupational endotoxin exposure. We developed a murine model of daily inhaled endotoxin for periods of 5 days to 8 weeks. We analyzed physiologic lung dysfunction, lung histology, bronchoalveolar lavage fluid and total lung homogenate inflammatory cell and cytokine profiles, and pulmonary gene expression profiles. We observed an increase in airway hyperresponsiveness as a result of chronic endotoxin exposure. After 8 weeks, the mice exhibited an increase in bronchoalveolar lavage and lung neutrophils that correlated with an increase in proinflammatory cytokines. Detailed analyses of inflammatory cell subsets revealed an expansion of dendritic cells (DCs), and in particular, proinflammatory DCs, with a reduced percentage of macrophages. Gene expression profiling revealed the up-regulation of a panel of genes that was consistent with DC recruitment, and lung histology revealed an accumulation of DCs in inflammatory aggregates around the airways in 8-week-exposed animals. Repeated, low-dose LPS inhalation, which mirrors occupational exposure, resulted in airway hyperresponsiveness, associated with a failure to resolve the proinflammatory response, an inverted macrophage to DC ratio, and a significant rise in the inflammatory DC population. These findings point to a novel underlying mechanism of airflow obstruction as a result of occupational LPS exposure, and suggest molecular and cellular targets for therapeutic development.

Mast cell TNF receptors regulate responses to Mycoplasma pneumoniae in surfactant protein A (SP-A)−/− mice

Mycoplasma pneumoniae (Mp) frequently colonizes the airways of patients with chronic asthma and likely contributes to asthma exacerbations. We previously reported that mice lacking surfactant protein A (SP-A) have increased airway hyperresponsiveness (AHR) during M pneumoniae infection versus wild-type mice mediated by TNF-α. Mast cells (MCs) have been implicated in AHR in asthma models and produce and respond to TNF-α. Determine the contribution of MC/TNF interactions to AHR in airways lacking functional SP-A during Mp infection. Bronchoalveolar lavage fluid was collected from healthy and asthmatic subjects to examine TNF-α levels and M pneumoniae positivity. To determine how SP-A interactions with MCs regulate airway homeostasis, we generated mice lacking both SP-A and MCs (SP-A(-/-)Kit(W-sh/W-sh)) and infected them with M pneumoniae. Our findings indicate that high TNF-α levels correlate with M pneumoniae positivity in human asthmatic patients and that human SP-A inhibits M pneumoniae-stimulated transcription and release of TNF-α by MCs, implicating a protective role for SP-A. MC numbers increase in M pneumoniae-infected lungs, and airway reactivity is dramatically attenuated when MCs are absent. Using SP-A(-/-)Kit(W-sh/W-sh) mice engrafted with TNF-α(-/-) or TNF receptor (TNF-R)(-/-) MCs, we found that TNF-α activation of MCs through the TNF-R, but not MC-derived TNF-α, leads to augmented AHR during M pneumoniae infection when SP-A is absent. Additionally, M pneumoniae-infected SP-A(-/-)Kit(W-sh/W-sh) mice engrafted with TNF-α(-/-) or TNF-R(-/-) MCs have decreased mucus production compared with that seen in mice engrafted with wild-type MCs, whereas burden was unaffected. Our data highlight a previously unappreciated but vital role for MCs as secondary responders to TNF-α during the host response to pathogen infection.

Efficacy and safety of influenza vaccination in children with asthma

The mean global prevalence of asthma among children is approximately 12%, making it the most common chronic disease in children. Influenza infection has been associated with complications such as exacerbations of wheezing and asthma, increased airway hyper-reactivity and hospitalization. Although influenza vaccination is recommended for asthmatic patients by all health authorities, vaccination coverage remains significantly lower than expected and is lowest of all in children. Compliance is affected by the uncertainty of parents and physicians concerning the clinical risk of influenza in asthmatic subjects, the benefits of influenza vaccination in preventing asthma exacerbations and the safety of immunization. The aim of this review is to analyze the rationale for using influenza vaccine, discuss the relationship between influenza and the severity of asthmatic episodes and document the efficacy and safety of influenza vaccination in the pediatric asthmatic population.

Kaempferol ameliorates airway inflammation through Tyk2‐STAT1/3 signaling in human airway epithelial cells and ovalbumin‐challenged mice: role of interleukin‐8

Allergic asthma is characterized by bronchial airway inflammation resulting in increased airway hyperresponsiveness, eosinophilia and mucus hyperproduction. The interaction between the airway epithelium and inflammatory mediators plays a key role in the pathogenesis of allergic asthma. The in vitro study elucidated inhibitory effects of kaempferol, a flavonoid with anti‐inflammatory activity, on LPS or IL‐8‐induced airway inflammation in BEAS‐2B cells. Nontoxic kaempferol at ≤20 μM significantly inhibited the LPS‐induced IL‐8 production through the TLR4 activation, resulting in eotaxin‐1 induction. In addition, kaempferol attenuated Tyk2 phosphorylation and STAT1/3 activation following IL‐8 secretion by LPS, and further enhanced the Tyk activation‐inhibiting SOCS3 expression. The in vivo study explored demoting effects of kaempferol on asthmatic responses in BALB/c mice sensitized with ovalbumin (OVA). Oral administration of kaempferol suppressed goblet cell hyperplasia, collagen deposition and mucus secretion in the airway and lung of OVA‐challenged mice via a blockade of STAT3 transactivation. These results demonstrate that dietary kaempferol alleviated airway asthmatic remodeling through modulating Tyk‐STAT signaling responsive to the inflammatory chemokine IL‐8. Therefore, kaempferol may be a potential therapeutic agent targeting inflammatory airway diseases such as asthma.

A Novel Prostacyclin Agonist Protects against Airway Hyperresponsiveness and Remodeling in Mice

Airway remodeling in bronchial asthma results from chronic, persistent airway inflammation. The effects of the reversal of airway remodeling by drug interventions remain to be elucidated. We investigated the effects of ONO-1301, a novel prostacyclin agonist with thromboxane inhibitory activity, on the prevention and reversibility of airway remodeling in an experimental chronic asthma model. Mice sensitized and challenged to ovalbumin (OVA) three times a week for 5 consecutive weeks were administered ONO-1301 or vehicle twice a day from the fourth week of OVA challenges. Twenty-four hours after the final OVA challenge, airway hyperresponsiveness (AHR) was assessed, and bronchoalveolar lavage was performed. Lung specimens were excised for staining to detect goblet-cell metaplasia, airway smooth muscle, and submucosal fibrosis. Mice administered ONO-1301 showed limited increases in AHR compared with mice administered the vehicle. The histological findings of airway remodeling were improved in ONO-1301-treated mice compared with vehicle-treated mice. Presumably, these therapeutic effects of ONO-1301 are attributable to the up-regulation of production of hepatocyte growth factor (HGF) in lung tissue, because the neutralization of HGF by antibodies prevented the effects of ONO-1301 on AHR and airway remodeling. Mice administered ONO-1301 showed similar levels of AHR and airway remodeling as mice administered montelukast, a cysteinyl-leukotriene-1 receptor antagonist, and lower levels were observed in mice administered dexamethasone. These data suggest that ONO-1301 exerts the effect of reversing airway remodeling, at least in part through an elevation of HGF in the lungs, and may be effective as an anti-remodeling drug in the treatment of asthma.

Alcohol reduces airway hyperresponsiveness (AHR) and allergic airway inflammation in mice

There is very limited knowledge about the effects of alcohol on airway hyperresponsiveness and inflammation in asthma. Historical accounts of alcohol administration to patients with breathing problems suggest that alcohol may have bronchodilating properties. We hypothesized that alcohol exposure will alter airway hyperresponsiveness (AHR) and pulmonary inflammation in a mouse model of allergic asthma. To test this hypothesis, BALB/c mice were fed either 18% alcohol or water and then sensitized and challenged with ovalbumin (OVA). AHR was assessed by means of ventilation or barometric plethysmography and reported as either total lung resistance or enhanced pause, respectively. Airway inflammation was assessed by total and differential cell counts in bronchoalveolar lavage fluid (BALF), cytokine levels in BALF, lung histology, and serum immunoglobulin E (IgE) levels. Alcohol feeding significantly blocked methacholine-induced increases in AHR compared with water-fed controls. Alcohol feeding significantly reduced total cell numbers (64%) as well as the number of eosinophils (84%) recruited to the lungs of these mice. Modest changes in lung pathology were also observed. Alcohol exposure led to a reduction of IgE in the serum of the EtOH OVA mice. These data demonstrate that alcohol exposure blunts AHR and dampens allergic airway inflammation indices in allergic mice and suggest that there may be an important role for alcohol in the modulation of asthma. These data provide an in vivo basis for previous clinical observations in humans substantiating the bronchodilator properties of alcohol and for the first time demonstrates an alcohol-induced reduction of allergic inflammatory cells in a mouse model of allergic asthma.

Alternaria Sensitiziation is Associated with Increased Airway Hyperresponsiveness and Sinusitis in Children with Severe Asthma

Alternaria Sensitiziation is Associated with Increased Airway Hyperresponsiveness and Sinusitis in Children with Severe Asthma

Mycoplasma pneumoniae CARDS Toxin Induces Pulmonary Eosinophilic and Lymphocytic Inflammation

Mycoplasma pneumoniae causes acute and chronic lung infections in humans, leading to a variety of pulmonary and extrapulmonary sequelae. Of the airway complications of M. pneumoniae infection, M. pneumoniae-associated exacerbation of asthma and pediatric wheezing are emerging as significant sources of human morbidity. However, M. pneumoniae products capable of promoting allergic inflammation are unknown. Recently, we reported that M. pneumoniae produces an ADP-ribosylating and vacuolating toxin termed the community-acquired respiratory distress syndrome (CARDS) toxin. Here we report that naive mice exposed to a single dose of recombinant CARDS (rCARDS) toxin respond with a robust inflammatory response consistent with allergic disease. rCARDS toxin induced 30-fold increased expression of the Th-2 cytokines IL-4 and IL-13 and 70- to 80-fold increased expression of the Th-2 chemokines CCL17 and CCL22, corresponding to a mixed cellular inflammatory response comprised of a robust eosinophilia, accumulation of T cells and B cells, and mucus metaplasia. The inflammatory responses correlate temporally with toxin-dependent increases in airway hyperreactivity characterized by increases in airway restriction and decreases in lung compliance. Furthermore, CARDS toxin-mediated changes in lung function and histopathology are dependent on CD4(+) T cells. Altogether, the data suggest that rCARDS toxin is capable of inducing allergic-type inflammation in naive animals and may represent a causal factor in M. pneumoniae-associated asthma.

The B-cell superantigen Finegoldia magna protein L causes pulmonary inflammation by a mechanism dependent on MyD88 but not B cells or immunoglobulins.

To determine whether Finegoldia magna protein L (PL) causes lung inflammation and, if so, whether the response is dependent on its immunoglobulin (Ig)-binding B-cell superantigenic property. Pulmonary inflammatory reactions were analyzed at various time points after intratracheal administration of PL to various strains of mice. PL caused peribronchial and perivascular inflammation that peaked at 18-24h. Polymorphonuclear cells (PMNs) began to accumulate in bronchoalveolar lavage fluid (BALF) of PL-challenged mice by 4h and accounted for>90% of leukocytes by 18-24h. Inflammation was marked by the appearance of MIP-2, KC, TNF-α, and IL-6 in the BALF with peak levels attained 4h after PL administration. PL-induced pulmonary inflammation was associated with increased airway hyper-reactivity following inhalation of methacholine. The inflammatory reaction was unabated in mice lacking B cells and immunoglobulins. In contrast, PL-induced inflammation was abrogated in MyD88-deficient mice. PL-induced responses required alveolar macrophages. These results strongly suggest that PL-induced lung inflammation is dependent on an innate MyD88-dependent pathway rather than the Ig-binding properties of this microbial B cell superantigen. We propose that this pulmonary inflammatory reaction is caused by the interaction of PL with a Toll-like receptor expressed on alveolar macrophages.

Ozone Exposure Suppresses Proliferative Response in Mice Skin

Ozone Exposure Suppresses Proliferative Response in Mice Skin

Increased Nitric Oxide Production Prevents Airway Hyperresponsiveness in Caveolin-1 Deficient Mice Following Endotoxin Exposure.

Caveolin-1, the hallmark protein of caveolae, is highly expressed within the lung in the epithelium, endothelium, and in immune cells. In addition to its classical roles in cholesterol metabolism and endocytosis, caveolin-1 has also been shown to be important in inflammatory signaling pathways. In particular, caveolin-1 is known to associate with the nitric oxide synthase enzymes, downregulating their activity. Endotoxins, which are are composed mainly of lipopolysaccharide (LPS), are found ubiquitously in the environment and can lead to the development of airway inflammation and increased airway hyperresponsiveness (AHR). We compared the acute responses of wild-type and caveolin-1 deficient mice after LPS aerosol, a well-accepted mode of endotoxin exposure, to investigate the role of caveolin-1 in the development of environmental lung injury. Although the caveolin-1 deficient mice had greater lung inflammatory indices compared to wild-type mice, they exhibited reduced AHR following LPS exposure. The uncoupling of inflammation and AHR led us to investigate the role of caveolin-1 in the production of nitric oxide, which is known to act as a bronchodilator. The absence of caveolin-1 resulted in increased nitrite levels in the lavage fluid in both sham and LPS treated mice. Additionally, inducible nitric oxide synthase expression was increased in the lung tissue of caveolin-1 deficient mice following LPS exposure and administration of the potent and specific inhibitor 1400W increased AHR to levels comparable to wild-type mice. We attribute the relative airway hyporesponsiveness in the caveolin-1 deficient mice after LPS exposure to the specific role of caveolin-1 in mediating nitric oxide production.

Apolipoprotein-A1 Mimetic Peptide 4F Rescues Severe Pulmonary Hypertension in Rats and Inhibits Human Pulmonary Artery Smooth Muscle Cell Proliferation In Vitro

Pulmonary hypertension (PH) is a characterized by arterial obstruction resulting from proliferation of pulmonary artery smooth muscle and endothelial cells. Genetic deletion of apolipoprotein-A1 increases airway hyperresponsiveness, inflammation, and collagen deposition in the lung. Apolipoprotein-A1 mimetic peptide 4F protects endothelial function, causes vasodilation, decreases inflammation and oxidative stress in lungs, yet its role in treating PH and right ventricular (RV) dysfunction is not known. We hypothesized that 4F may rescue pre-existing severe PH. We also investigate the effects of 4F on human pulmonary artery smooth muscle cell (hPASMC) proliferation in vitro as a possible mechanism of rescue by 4F. Twenty three rats were randomly divided into 4-groups. PH was induced by monocrotaline (MCT, 60mg/kg, s.c.). Severe PH was well-established at day-21 (PH, n=6) that progressed to RV failure (RVF, n=6) by day-30. One MCT-group was treated with 4F (50mg/kg/day, s.c., n=6) from day-21 to 30. Saline-treated rats served as control (CTRL, n=5). Serial echocardiography was performed to monitor cardiopulmonary hemodynamics. Cardiac catheterization was performed terminally to record RV-pressure (RVP). hPASMCs proliferation was assessed by MTT-assay. p<0.05 was considered significant. Values were mean±SE. Rats developed severe PH 21-days after MCT (RVP=67.12±1 vs. 29.8±1 mmHg in CTRL, RV/LV+IVS= 0.65±0.05 vs. 0.23±0.02, RV-ejection fraction (RVEF)= 40±1 vs. 65±1%, all p<0.05 vs. CTRL), which progressed to RVF by day-30 [RVP=74±1; RV/(LV+IVS)=0.68±0.05; RVEF=28.6±1%, p<0.05 for all vs. CTRL]. 4F-therapy from day-21 to 30 resulted in rescue of PH (RVP=47±3 mmHg, RV/LV+IVS= 0.38±0.02, RVEF= 51.7±3%, p<0.05 vs. PH and RVF). 4F also inhibited hPASMC proliferation (∼60% inhibition at 100ng 4F/ml medium, p<0.05). In conclusion, 4F rescues pre-existing severe PH and RV-dysfunction. Inhibition of PASMC proliferation may be one of the key mechanisms in this rescue.

Rapamycin Attenuates Airway Hyperreactivity, Goblet Cells, and IgE in Experimental Allergic Asthma

The mammalian target of rapamycin (mTOR) signaling pathway integrates environmental cues, promotes cell growth/differentiation, and regulates immune responses. Although inhibition of mTOR with rapamycin has potent immunosuppressive activity, mixed effects have been reported in OVA-induced models of allergic asthma. We investigated the impact of two rapamycin treatment protocols on the major characteristics of allergic asthma induced by the clinically relevant allergen, house dust mite (HDM). In protocol 1, BALB/c mice were exposed to 10 intranasal HDM doses over a period of 24 d and treated with rapamycin simultaneously during the sensitization/exposure period. In protocol 2, rapamycin was administered after the mice had been sensitized to HDM (i.p. injection) and prior to initiation of two intranasal HDM challenges over 4 d. Airway hyperreactivity (AHR), IgE, inflammatory cells, cytokines, leukotrienes, goblet cells, and activated T cells were assessed. In protocol 1, rapamycin blocked HDM-induced increases in AHR, inflammatory cell counts, and IgE, as well as attenuated goblet cell metaplasia. In protocol 2, rapamycin blocked increases in AHR, IgE, and T cell activation and reduced goblet cell metaplasia, but it had no effect on inflammatory cell counts. Increases in IL-13 and leukotrienes were also blocked by rapamycin, although increases in IL-4 were unaffected. These data demonstrated that rapamycin can inhibit cardinal features of allergic asthma, including increases in AHR, IgE, and goblet cells, most likely as a result of its ability to reduce the production of two key mediators of asthma: IL-13 and leukotrienes. These findings highlight the importance of the mTOR pathway in allergic airway disease.