Farnesyltransferase Inhibition Exacerbates Eosinophilic Inflammation and Airway Hyperreactivity in Mice with Experimental Asthma: The Complex Roles of Ras GTPase and Farnesylpyrophosphate in Type 2 Allergic Inflammation.

Ras, a small GTPase protein, is thought to mediate Th2-dependent eosinophilic inflammation in asthma. Ras requires cell membrane association for its biological activity, and this requires the posttranslational modification of Ras with an isoprenyl group by farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase). We hypothesized that inhibition of FTase using FTase inhibitor (FTI)-277 would attenuate allergic asthma by depleting membrane-associated Ras. We used the OVA mouse model of allergic inflammation and human airway epithelial (HBE1) cells to determine the role of FTase in inflammatory cell recruitment. BALB/c mice were first sensitized then exposed to 1% OVA aerosol or filtered air, and half were injected daily with FTI-277 (20 mg/kg per day). Treatment of mice with FTI-277 had no significant effect on lung membrane-anchored Ras, Ras protein levels, or Ras GTPase activity. In OVA-exposed mice, FTI-277 treatment increased eosinophilic inflammation, goblet cell hyperplasia, and airway hyperreactivity. Human bronchial epithelial (HBE1) cells were pretreated with 5, 10, or 20 µM FTI-277 prior to and during 12 h IL-13 (20 ng/ml) stimulation. In HBE1 cells, FTase inhibition with FTI-277 had no significant effect on IL-13-induced STAT6 phosphorylation, eotaxin-3 peptide secretion, or Ras translocation. However, addition of exogenous FPP unexpectedly augmented IL-13-induced STAT6 phosphorylation and eotaxin-3 secretion from HBE1 cells without affecting Ras translocation. Pharmacological inhibition of FTase exacerbates allergic asthma, suggesting a protective role for FTase or possibly Ras farnesylation. FPP synergistically augments epithelial eotaxin-3 secretion, indicating a novel Ras-independent farnesylation mechanism or direct FPP effect that promotes epithelial eotaxin-3 production in allergic asthma.

Benralizumab: a unique IL-5 inhibitor for severe asthma.

The presence of eosinophilic inflammation is a characteristic feature of chronic and acute inflammation in asthma. An estimated 5%-10% of the 300 million people worldwide who suffer from asthma have a severe form. Patients with eosinophilic airway inflammation represent approximately 40%-60% of this severe asthmatic population. This form of asthma is often uncontrolled, marked by refractoriness to standard therapy, and shows persistent airway eosinophilia despite glucocorticoid therapy. This paper reviews personalized novel therapies, more specifically benralizumab, a humanized anti-IL-5Rα antibody, while also being the first to provide an algorithm for potential candidates who may benefit from anti-IL-5Rα therapy.

Intratracheal instillation of pravastatin for the treatment of murine allergic asthma: a lung-targeted approach to deliver statins.

Systemic treatment with statins mitigates allergic airway inflammation, TH2 cytokine production, epithelial mucus production, and airway hyperreactivity (AHR) in murine models of asthma. We hypothesized that pravastatin delivered intratracheally would be quantifiable in lung tissues using mass spectrometry, achieve high drug concentrations in the lung with minimal systemic absorption, and mitigate airway inflammation and structural changes induced by ovalbumin. Male BALB/c mice were sensitized to ovalbumin (OVA) over 4 weeks, then exposed to 1% OVA aerosol or filtered air (FA) over 2 weeks. Mice received intratracheal instillations of pravastatin before and after each OVA exposure (30 mg/kg). Ultra performance liquid chromatography - mass spectrometry was used to quantify plasma, lung, and bronchoalveolar lavage fluid (BALF) pravastatin concentration. Pravastatin was quantifiable in mouse plasma, lung tissue, and BALF (BALF > lung > plasma for OVA and FA groups). At these concentrations pravastatin inhibited airway goblet cell hyperplasia/metaplasia, and reduced BALF levels of cytokines TNFα and KC, but did not reduce BALF total leukocyte or eosinophil cell counts. While pravastatin did not mitigate AHR, it did inhibit airway hypersensitivity (AHS). In this proof-of-principle study, using novel mass spectrometry methods we show that pravastatin is quantifiable in tissues, achieves high levels in mouse lungs with minimal systemic absorption, and mitigates some pathological features of allergic asthma. Inhaled pravastatin may be beneficial for the treatment of asthma by having direct airway effects independent of a potent anti-inflammatory effect. Statins with greater lipophilicity may achieve better anti-inflammatory effects warranting further research.

Novel therapeutic strategies for adult obese asthmatics.

Asthma is a complex syndrome that affects an estimated 26 million people in the United States but gaps exist in the recognition and management of asthmatic subgroups. This article proposes alternative approaches for future treatments of adult obese asthmatics who do not respond to standard controller therapies, drawing parallels between seemingly disparate therapeutics through their common signaling pathways. How metformin and statins can potentially improve airway inflammation is described and supplements are suggested. A move toward more targeted therapies for asthma subgroups is needed. These therapies address asthma and the comorbidities that accompany obesity and metabolic syndrome to provide the greatest therapeutic potential.

Dietary long-chain omega-3 fatty acids do not diminish eosinophilic pulmonary inflammation in mice.

Although the effects of fish oil supplements on airway inflammation in asthma have been studied with varying results, the independent effects of the fish oil components, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), administered separately, are untested. Here, we investigated airway inflammation and hyperresponsiveness using a mouse ovalbumin exposure model of asthma assessing the effects of consuming EPA (1.5% wt/wt), DHA (1.5% wt/wt), EPA plus DHA (0.75% each), or a control diet with no added omega-3 polyunsaturated fatty acids. Consuming these diets for 6 weeks resulted in erythrocyte membrane EPA contents (molar %) of 9.0 (± 0.6), 3.2 (± 0.2), 6.8 (± 0.5), and 0.01 (± 0.0)%; DHA contents were 6.8 (± 0.1), 15.6 (± 0.5), 12.3 (± 0.3), and 3.8 (± 0.2)%, respectively. The DHA group had the highest bronchoalveolar lavage (BAL) fluid eosinophil and IL-6 levels (P < 0.05). Similar trends were seen for macrophages, IL-4, and IL-13, whereas TNF-α was lower in omega-3 polyunsaturated fatty acid groups than the control (P < 0.05). The DHA group also had the highest airway resistance, which differed significantly from the EPA plus DHA group (P < 0.05), which had the lowest. Oxylipins were measured in plasma and BAL fluid, with DHA and EPA suppressing arachidonic acid-derived oxylipin production. DHA-derived oxylipins from the cytochrome P450 and 15-lipoxygenase pathways correlated significantly with BAL eosinophil levels. The proinflammatory effects of DHA suggest that the adverse effects of individual fatty acid formulations should be thoroughly considered before any use as therapeutic agents in asthma.

Self-assembling nanoparticles containing dexamethasone as a novel therapy in allergic airways inflammation.

Nanocarriers can deliver a wide variety of drugs, target them to sites of interest, and protect them from degradation and inactivation by the body. They have the capacity to improve drug action and decrease undesirable systemic effects. We have previously developed a well-defined non-toxic PEG-dendritic block telodendrimer for successful delivery of chemotherapeutics agents and, in these studies, we apply this technology for therapeutic development in asthma. In these proof-of-concept experiments, we hypothesized that dexamethasone contained in self-assembling nanoparticles (Dex-NP) and delivered systemically would target the lung and decrease allergic lung inflammation and airways hyper-responsiveness to a greater degree than equivalent doses of dexamethasone (Dex) alone. We found that ovalbumin (Ova)-exposed mice treated with Dex-NP had significantly fewer total cells (2.78 ± 0.44 × 10(5) (n = 18) vs. 5.98 ± 1.3 × 10(5) (n = 13), P<0.05) and eosinophils (1.09 ± 0.28 × 10(5) (n = 18) vs. 2.94 ± 0.6 × 10(5) (n = 12), p<0.05) in the lung lavage than Ova-exposed mice alone. Also, lower levels of the inflammatory cytokines IL-4 (3.43 ± 1.2 (n = 11) vs. 8.56 ± 2.1 (n = 8) pg/ml, p<0.05) and MCP-1 (13.1 ± 3.6 (n = 8) vs. 28.8 ± 8.7 (n = 10) pg/ml, p<0.05) were found in lungs of the Dex-NP compared to control, and they were not lower in the Dex alone group. In addition, respiratory system resistance was lower in the Dex-NP compared to the other Ova-exposed groups suggesting a better therapeutic effect on airways hyperresponsiveness. Taken together, these findings from early-stage drug development studies suggest that the encapsulation and protection of anti-inflammatory agents such as corticosteroids in nanoparticle formulations can improve efficacy. Further development of novel drugs in nanoparticles is warranted to explore potential treatments for chronic inflammatory diseases such as asthma.

Tracking retention and transport of ultrafine polystyrene in an asthmatic mouse model using positron emission tomography.

Upon exposure to particulates, asthmatic individuals are more susceptible to deleterious health effects and increased morbidity and mortality when compared to healthy individuals. These effects are not limited to the respiratory system; increases in acute cardiovascular events have been observed. The development of extrapulmonary illnesses has led to interest in determining whether particles move out of the lungs and whether transport of particles differs for asthmatic individuals. Differences in particle deposition and retention in asthmatic versus normal subjects have been explored in the literature using the gamma camera, a two-dimensional imaging technique. Herein we report the deposition and fate of (64)Cu-labeled 100 nm polystyrene particles in a mouse model of asthma using positron emission tomography (PET). All animals were handled humanely under an approved protocol (UC Davis Institutional Animal Care and Use Committee). Particles were administered by intratracheal instillation and animals were imaged over 48 hours using PET. Biodistribution was determined from images using Regions of Interest (ROI) analysis. After 48 hours, for the asthmatic animals, we observed that ~28% of the initial dose is cleared from the lungs; particle accumulation in small amounts is evident in the GI (gastrointestinal) tract, liver, and bladder. This decrease in lung retention is significantly different when compared to the normal mouse (~11%DD), which showed minimal particle transport out of the lung (P < 0.001). This study indicates that ultrafine particles (UFP) undergo enhanced transport out of the lungs in an asthma model. This observed transport may facilitate the adverse peripheral effects associated with particulate exposure.

Simvastatin inhibits smoke-induced airway epithelial injury: implications for COPD therapy.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death. The statin drugs may have therapeutic potential in respiratory diseases such as COPD, but whether they prevent bronchial epithelial injury is unknown. We hypothesised that simvastatin attenuates acute tobacco smoke-induced neutrophilic lung inflammation and airway epithelial injury. Spontaneously hypertensive rats were given simvastatin (20 mg·kg(-1) i.p.) daily for either 7 days prior to tobacco smoke exposure and during 3 days of smoke exposure, or only during tobacco smoke exposure. Pretreatment with simvastatin prior to and continued throughout smoke exposure reduced the total influx of leukocytes, neutrophils and macrophages into the lung and airways. Simvastatin attenuated tobacco smoke-induced cellular infiltration into lung parenchymal and airway subepithelial and interstitial spaces. 1 week of simvastatin pretreatment almost completely prevented smoke-induced denudation of the airway epithelial layer, while simvastatin given only concurrently with the smoke exposure had no effect. Simvastatin may be a novel adjunctive therapy for smoke-induced lung diseases, such as COPD. Given the need for statin pretreatment there may be a critical process of conditioning that is necessary for statins' anti-inflammatory effects. Future work is needed to elucidate the mechanisms of this statin protective effect.

Why is particulate matter produced by wildfires toxic to lung macrophages?

The mechanistic basis of the high toxicity to lung macrophages of coarse PM from the California wildfires of 2008 was examined in cell culture experiments with mouse macrophages. Wildfire PM directly killed macrophages very rapidly in cell culture at relatively low doses. The wildfire coarse PM is about four times more toxic to macrophages on an equal weight basis than the same sized PM collected from normal ambient air (no wildfires) from the same region and season. There was a good correlation between the extent of cytotoxicity and the amount of oxidative stress observed at a given dose of wildfire PM in vitro. Our data implicate NF-κB signaling in the response of macrophages to wildfire PM, and suggest that most, if not all, of the cytotoxicity of wildfire PM to lung macrophages is the result of oxidative stress. The relative ratio of toxicity and of expression of biomarkers of oxidant stress between wildfire PM and "normal" PM collected from ambient air is consistent with our previous results in mice in vivo, also suggesting that most, if not all, of the cytotoxicity of wildfire PM to lung macrophages is the result of oxidative stress. Our findings from this and earlier studies suggest that the active components of coarse PM from the wildfire are heat-labile organic compounds. While we cannot rule out a minor role for endotoxin in coarse PM preparations from the collected wildfire PM in our observed results both in vitro and in vivo, based on experiments using the inhibitor Polymyxin B most of the oxidant stress and pro-inflammatory activity observed was not due to endotoxin.

Competitive metabolism of L-arginine: arginase as a therapeutic target in asthma.

Exhaled breath nitric oxide (NO) is an accepted asthma biomarker. Lung concentrations of NO and its amino acid precursor, L-arginine, are regulated by the relative expressions of the NO synthase (NOS) and arginase isoforms. Increased expression of arginase I and NOS2 occurs in murine models of allergic asthma and in biopsies of asthmatic airways. Although clinical trials involving the inhibition of NO-producing enzymes have shown mixed results, small molecule arginase inhibitors have shown potential as a therapeutic intervention in animal and cell culture models. Their transition to clinical trials is hampered by concerns regarding their safety and potential toxicity. In this review, we discuss the paradigm of arginase and NOS competition for their substrate L-arginine in the asthmatic airway. We address the functional role of L-arginine in inflammation and the potential role of arginase inhibitors as therapeutics.

Simvastatin inhibits goblet cell hyperplasia and lung arginase in a mouse model of allergic asthma: a novel treatment for airway remodeling?

Airway remodeling in asthma contributes to airway hyperreactivity, loss of lung function, and persistent symptoms. Current therapies do not adequately treat the structural airway changes associated with asthma. The statins are cholesterol-lowering drugs that inhibit the enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase, which is the rate-limiting step of cholesterol biosynthesis in the mevalonate (MA) pathway. These drugs have been associated with improved respiratory health, and ongoing clinical trials are testing their therapeutic potential in asthma. We hypothesized that simvastatin treatment of ovalbumin (OVA)-exposed mice would attenuate early features of airway remodeling by a mevalonate-dependent mechanism. BALB/c mice initially were sensitized to OVA and then exposed to 1% OVA aerosol for 2 weeks after sensitization for 6 exposures. Simvastatin (40 mg/kg) or simvastatin plus MA (20 mg/kg) were injected intraperitoneally before each OVA exposure. Treatment with simvastatin attenuated goblet cell hyperplasia, arginase-1 protein expression, and total arginase enzyme activity, but it did not alter airway hydroxyproline content or transforming growth factor-ß1. Inhibition of goblet cell hyperplasia by simvastatin was mevalonate-dependent. No appreciable changes to airway smooth muscle cells were observed in any control or treatment groups. In conclusion, in an acute mouse model of allergic asthma, simvastatin inhibited early hallmarks of airway remodeling, which are indicators that can lead to airway thickening and fibrosis. Statins are potentially novel treatments for airway remodeling in asthma. Additional studies using subchronic or chronic allergen exposure models are needed to extend these initial findings.

Nitric oxide synthase enzymes in the airways of mice exposed to ovalbumin: NOS2 expression is NOS3 dependent.

OBJECTIVES AND DESIGN: The function of the airway nitric oxide synthase (NOS) isoforms and the lung cell types responsible for its production are not fully understood. We hypothesized that NO homeostasis in the airway is important to control inflammation, which requires upregulation, of NOS2 protein expression by an NOS3-dependent mechanism. MATERIALS OR SUBJECTS: Mice from a C57BL/6 wild-type, NOS1(-/-), NOS2(-/-), and NOS3(-/-) genotypes were used. All mice strains were systemically sensitized and exposed to filtered air or ovalbumin (OVA) aerosol for two weeks to create a subchronic model of allergen-induced airway inflammation. METHODS: We measured lung function, lung lavage inflammatory and airway epithelial goblet cell count, exhaled NO, nitrate and nitrite concentration, and airway NOS1, NOS2, and NOS3 protein content. RESULTS: Deletion of NOS1 or NOS3 increases NOS2 protein present in the airway epithelium and smooth muscle of air-exposed animals. Exposure to allergen significantly reduced the expression of NOS2 protein in the airway epithelium and smooth muscle of the NOS3(-/-) strain only. This reduction in NOS2 expression was not due to the replacement of epithelial cells with goblet cells as remaining epithelial cells did not express NOS2. NOS1(-/-) animals had significantly reduced goblet cell metaplasia compared to C57Bl/6 wt, NOS2(-/-), and NOS3(-/-) allergen-exposed mice. CONCLUSION: The airway epithelial and smooth muscle cells maintain a stable airway NO concentration under noninflammatory conditions. This "homeostatic" mechanism is unable to distinguish between NOS derived from the different constitutive NOS isoforms. NOS3 is essential for the expression of NOS2 under inflammatory conditions, while NOS1 expression contributes to allergen-induced goblet cell metaplasia.

Arginase inhibition in airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin.

Arginase1 and nitric oxide synthase2 (NOS2) utilize l-arginine as a substrate, with both enzymes expressed at high levels in the asthmatic lung. Inhibition of arginase in ovalbumin-exposed C57BL/6 mice with the transition state inhibitor N(omega)-hydroxy-nor-l-arginine (nor-NOHA) significantly increased total l-arginine content in the airway compartment. We hypothesized that such an increase in l-arginine content would increase the amount of nitric oxide (NO) being produced in the airways and thereby decrease airway hyperreactivity and eosinophilic influx. We further hypothesized that despite arginase inhibition, NOS2 knockout (NOS2-/-) mice would be unable to up-regulate NO production in response to allergen exposure and would demonstrate higher amounts of airway hyperreactivity and eosinophilia under conditions of arginase inhibition than C57BL/6 animals. We found that administration of nor-NOHA significantly decreased airway hyperreactivity and eosinophilic airway inflammation in ovalbumin-exposed C57BL/6 mice, but these parameters were unchanged in ovalbumin-exposed NOS2-/- mice. Arginase1 protein content was increased in mice exposed to ovalbumin, an effect that was reversed upon nor-NOHA treatment in C57BL/6 mice. Arginase1 protein content in the airway compartment directly correlated with the degree of airway hyperreactivity in all treatment groups. NOS2-/- mice had significantly greater arginase1 and arginase2 concentrations compared to their respective C57BL/6 groups, indicating that inhibition of arginase may be dependent upon NOS2 expression. Arginase1 and 2 content were not affected by nor-NOHA administration in the NOS2-/- mice. We conclude that l-arginine metabolism plays an important role in the development of airway hyperreactivity and eosinophilic airway inflammation. Inhibition of arginase early in the allergic inflammatory response decreases the severity of the chronic inflammatory phenotype. These effects appear to be attributable to NOS2, which is a major source of NO production in the inflamed airway, although arginase inhibition may also be affecting the turnover of arginine by the other NOS isoforms, NOS1 and NOS3. The increased l-arginine content in the airway compartment of mice treated with nor-NOHA may directly or indirectly, through NOS2, control arginase expression both in response to OVA exposure and at a basal level.

Arginase enzymes in isolated airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin.

Arginase has been suggested to compete with nitric oxide synthase (NOS) for their common substrate, l-arginine. To study the mechanisms underlying this interaction, we compared arginase expression in isolated airways and the consequences of inhibiting arginase activity in vivo with NO production, lung inflammation, and lung function in both C57BL/6 and NOS2 knockout mice undergoing ovalbumin-induced airway inflammation, a mouse model of asthma. Arginases I and II were measured by western blot in isolated airways from sensitized C57BL/6 mice exposed to ovalbumin aerosol. Physiological and biochemical responses - inflammation, lung compliance, airway hyperreactivity, exhaled NO concentration, arginine concentration - were compared with the responses of NOS2 knockout mice. NOS2 knockout mice had increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity. Both arginase I and arginase II were constitutively expressed in the airways of normal C57BL/6 mice. Arginase I was up-regulated approximately 8-fold in the airways of C57BL/6 mice exposed to ovalbumin. Expression of both arginase isoforms were significantly upregulated in NOS2 knockout mice exposed to ovalbumin, with about 40- and 4-fold increases in arginases I and II, respectively. Arginine concentration in isolated airways was not significantly different in any of the groups studied. Inhibition of arginase by systemic treatment of C57BL/6 mice with a competitive inhibitor, Nomega-hydroxy-nor-l-arginine (nor-NOHA), significantly decreased the lung inflammatory response to ovalbumin in these animals. We conclude that NOS2 knockout mice are more sensitive to ovalbumin-induced airway inflammation and its sequelae than are C57BL/6 mice, as determined by increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity, and that these findings are strongly correlated with increased expression of both arginase isoforms in the airways of the NOS2 knockout mice exposed to ovalbumin.

Arginases I and II in lungs of ovalbumin-sensitized mice exposed to ovalbumin: sources and consequences.

Arginase gene expression in the lung has been linked to asthma both in clinical studies of human patients and in the well-studied mouse model of ovalbumin-induced airway inflammation. Arginase is thought to regulate NO levels in the lung by its ability to divert arginine, the substrate for nitric oxide synthases that produce citrulline and NO, into an alternative metabolic pathway producing ornithine and urea. In the present study arginase I and arginase II concentrations were measured in isolated microdissected airway preparations from sensitized Balb/c mice exposed to ovalbumin aerosol. We found that arginase II was constitutively expressed in the airways of normal mice, whereas arginase I was undetectable in normal airways, while its expression was increased in airways of mice exposed to ovalbumin. The expression of arginase I strongly correlated with the presence of lung inflammation, as quantified by differential cell counts in lung lavage, suggesting that most, or all, of the arginase I in lungs of mice exposed to ovalbumin is present in the inflammatory cells rather than in the airway epithelium. There was also a significant correlation between increased expression of arginase I in the isolated airways and decreased lung compliance. On the other hand, while we found arginase II expression to also be significantly increased in airways from mice exposed to ovalbumin as compared with normal airways, the relative increase was much less than that observed for arginase I, suggesting that there was a smaller contribution of inflammatory cells to the arginase II content of the airways in mice exposed to ovalbumin. There was no apparent correlation between the content of arginase in isolated airways and exhaled NO concentration in the expired air from mice exposed to ovalbumin. However, there was a correlation between exhaled NO concentration from mice exposed to ovalbumin and the lymphocyte content of the lung lavage. The concentration of arginine found in isolated airways from Balb/c mice exposed for 2 weeks to ovalbumin was about half of the value found in isolated microdissected airways from normal mice. Treatment of mice systemically with an arginase inhibitor significantly increased the amount of NO produced, as measured as the amount of nitrite+nitrate (NOx) in lung lavage supernatant prepared from mice exposed to ovalbumin. Our results are consistent with the hypothesis that the response of the lung to ovalbumin challenge includes an adaptive response in the large airways regulating the concentration of arginine within cells of the airway epithelium and subepithelial layer, by shunting of arginine into the metabolic pathway for increased synthesis of NO.

Influence of pirfenidone on airway hyperresponsiveness and inflammation in a Brown-Norway rat model of asthma.

Pirfenidone was administered to sensitized Brown Norway rats prior to a series of ovalbumin challenges. Airway hyperresponsiveness, inflammatory cell infiltration, mucin and collagen content, and the degree of epithelium and smooth muscle staining for TGF-beta were examined in control, sensitized, and sensitized/challenged rats fed a normal diet or pirfenidone diet. Pirfenidone had no effect on airway hyperresponsiveness, but reduced distal bronchiolar cell infiltration and proximal and distal mucin content. Statistical analysis showed that the control group and sensitized/challenged pirfenidone diet group TGF-beta staining intensity scores were not significantly different from isotype controls, but that the staining intensity scores for the sensitized/challenged normal diet group was significantly different from isotype controls. These results suggest that pirfenidone treatment is effective in reducing some of the components of acute inflammation induced by allergen challenge.