Gamma tocopherol-enriched supplement reduces sputum eosinophilia and endotoxin-induced sputum neutrophilia in volunteers with asthma

Background: We and others have shown that the gamma tocopherol ( g T) isoform of vitamin E has multiple anti-inﬂammatory and antioxidant actions and that g T supplementation reduces eosinophilic and endotoxin (LPS)-induced neutrophilic airway inﬂammation in animal models and healthy human volunteers. Objective: We sought to determine whether g T supplementation reduces eosinophilic airway inﬂammation and acute neutrophilic response to inhaled LPS challenge in volunteers with asthma. Methods: Participants with mild asthma were enrolled in a double-blinded, placebo-controlled crossover study to assess the effect of 1200 mg of g T daily for 14 days on sputum eosinophils, mucins, and cytokines. We also assessed the effect on acute inﬂammatory response to inhaled LPS challenge following g T treatment, focusing on changes in sputum neutrophilia, mucins, and cytokines. Mucociliary clearance was measured using gamma scintigraphy. Results: Fifteen subjects with mild asthma completed both arms of the study. Compared with placebo, g T notably reduced pre-LPS challenge sputum eosinophils and mucins, including mucin 5AC and reduced LPS-induced airway neutrophil recruitment 6 and 24 hours after challenge. Mucociliary clearance was slowed 4 hours postchallenge in the placebo group but not in the g T treatment group. Total sputum mucins (but not mucin 5AC) were reduced at 24 hours postchallenge during g T treatment compared with placebo. Conclusions: When compared with placebo, g T supplementation for 14 days reduced inﬂammatory features of asthma, including sputum eosinophils and mucins, as well as acute airway response to inhaled LPS challenge. Larger scale clinical trials are needed to assess the efﬁcacy of g T supplements as a complementary or steroid-sparing treatment for asthma. (J Allergy Clin Immunol 2018;141:1231-8.) group, paired t tests or Wilcoxon signed rank tests (depending on whether the normality assumption was met) were employed. Data that were not normally distributed were transformed using Box-Cox transformation. Given the crossover design of our study, we next determined the g T treatment effect (compared with placebo) on posttreatment sputum end points and on LPS-induced changes ( D postchallenge – posttreatment) in sputum end points using a linear mixed model approach described by Jones and Kenward 42 that considers the above-mentioned individual tests in a global, uniﬁed way where all data are used at the same time. SAS PROC MIXED was used to ﬁt the linear mixed model. Criterion for signiﬁcance was taken to be P <_ .05.

Asthma is among the most prevalent chronic diseases in the United States 1 and represents a source of significant burden to patients and health care systems. Environmental pollutant exposure is a known trigger for asthma exacerbations, which are characterized by airway inflammation, bronchoconstriction, increased production of airway mucous, and decreased mucociliary clearance (MCC) with formation of mucous plugs. 2,3 Endotoxin (the main component of which is LPS) is commonly encountered in ambient air particulate matter as well as in domestic and occupational settings and has been linked to asthma severity. [4][5][6] Endotoxin is a potent stimulator of the innate immune response, 7 signaling through Toll-like receptor 4 on airway macrophages to stimulate production of inflammatory cytokines and eicosanoids, recruitment of granulocytes, and production of gel-forming airway mucins, including mucin 5AC (MUC5AC). 8 Airway inflammation during acute exacerbations of asthma is often characterized by increase in both airway eosinophils and neutrophils. 9 Neutrophilic airway inflammation is particularly evident in viral asthma exacerbations as well as in some chronic asthma phenotypes. 10 Our group has shown that inhaled LPS challenge induces airway neutrophilia in human volunteers, and we now employ this procedure as a model of acute exacerbation of airway disease against which potential therapies can be tested. [11][12][13] We have previously demonstrated that inhaled fluticasone propionate administered for 2 weeks decreased Abbreviations used aT: a-Tocopherol g-CEHC: 2,7,8-Trimethyl-2-(b-carboxyethyl)-6-hydroxychroman gT: g-Tocopherol MCC: Mucociliary clearance MUC5AC: Mucin 5AC %PMNs: Sputum percentage of neutrophils sputum eosinophilia and subsequent LPS-induced acute airway neutrophilia in asthmatics. 14 In subsequent studies, we have shown that treatment with the IL-1 receptor antagonist, anakinra, 15 and the vitamin E isoform, gamma tocopherol (gT) 16 also attenuated LPS-induced airway neutrophilia in healthy volunteers.
Our hypothesis that vitamin E supplementation decreases airway inflammation in asthma and allergic airway disease was inspired by epidemiologic evidence suggesting that increased dietary vitamin E intake is associated with reduced incidence of allergic disease [17][18][19] and asthma. 20 Among the isoforms of vitamin E that have been suggested as asthma interventions are a-tocopherol (aT), which is commonly used as both a supplement and pharmaceutical product, and gT, the predominant isoform of vitamin E found in dietary sources. Intervention trials of aT in humans with asthma have been generally disappointing. 21,22 gT has not been as vigorously studied for airway disease. gT and its primary metabolite 2,7,8-trimethyl-2-(B-carboxy-ethyl)-6-hydroxychroman (g-CEHC) do have a number of unique antiinflammatory actions, 23,24 including scavenging reactive nitrogen species to form 5-nitro-g-tocopherol 24 and inhibition of COX-2 and 5-lipooxygenase, reducing inflammatory eicosanoid production. 25 We have pursued a program of preclinical and early phase clinical trials of gT as a novel therapeutic for treatment of airway inflammation. [25][26][27][28][29] In a rodent model of evoked airway inflammation, gT reduced allergen-induced eosinophilia and mucin responses 27 as well as LPS-induced neutrophil, prostaglandin E2, and mucin responses (including MUC5AC). 29 We subsequently observed that 1 week of treatment with a gT-enriched mixed tocopherol preparation reduced the neutrophilic response to inhaled LPS challenge in a phase I randomized, double-blinded, placebo-controlled crossover study of healthy adults. 16 This report describes our next step in assessing gT as an intervention for asthma, in which we test the hypothesis that gT reduces eosinophilic airway inflammation and attenuates the neutrophilic airway response to inhaled LPS challenge in volunteers with mild asthma.

Volunteer recruitment and inclusion criteria
We recruited subjects aged 18 to 50 years with a history of episodic wheezing or shortness of breath consistent with asthma or physiciandiagnosed asthma classified as mild intermittent or mild persistent asthma as defined by the National Heart, Lung, and Blood Institute guidelines for the Diagnosis and Management of Asthma. 30 Exclusion criteria included any of the following: daily albuterol use, nighttime asthma symptoms more than once per week, or emergency treatment for asthma within the previous 12 months. As sputum inflammatory cell measures were a central endpoint in this study, all subjects were screened for their ability to provide an adequate induced sputum sample during their screening session, defined by >250,000 cells, >50% viability, and <40% squamous cells.
Prior to study entry, subjects underwent a general health screen including a detailed medical history, physical exam, baseline laboratory evaluation, spirometry, and allergy skin testing to common aeroallergens including house dust mite, cockroach, tree mix, grass mix, weed mix, molds, cat, dog, guinea pig, rabbit, rat, and mouse allergens. Awheal size of 3 mm or greater than the negative control was considered positive. Subjects who were found to be pregnant, nursing an infant, regularly taking anti-inflammatory or immune-modulating medications, or with a history of abnormal blood coagulation parameters were excluded. This study was approved by the University of North Carolina Institutional Review Board and the US Food and Drug Administration (IND 13004) and is listed on ClinicalTrials.gov (NCT02104505).

Study design
Subjects were randomized to 1200 mg gT or placebo (safflower oil) treatment for 14 days (Fig 1), a study period similar to that used to assess the effect of fluticasone propionate on airway response to LPS challenge in asthmatics. The gT supplement consisted of gel tabs each containing 612 mg gT, 7 mg aT, 28 mg b-tocopherol, and 8 mg d-tocopherol (Callion Pharma, Inc, Jonesborough, Tenn), and subjects were instructed to take 2 gel tabs once daily with a meal to maximize bile secretion and enhance absorption. Medication bottles were returned and any leftover pills were counted on the day of LPS challenge to ensure adherence. Twenty-four to 48 hours prior to LPS challenge, subjects presented for sputum induction and gamma scintigraphy to measure MCC. On day 14 of dosing, subjects underwent inhaled LPS challenge with 20,000 endotoxin units of Clinical Center Reference Endotoxin, with MCC measurement performed 4 hours postchallenge and sputum induction at 6 and 24 hours postchallenge. Sputum was analyzed for granulocytes, inflammatory cytokines, and mucin content as previously described. [31][32][33][34] After a minimum 3-week washout to allow for clearance of inflammatory cells from the airways, subjects were crossed over to the alternate treatment group. Venipuncture was performed at regular intervals for assessment of prothrombin time, activated partial thromboplastin time, and international normalized ratio.

Randomization and masking
The randomization list was prepared by a biostatistician using SAS 9.4 (SAS Institute, Cary, NC) and provided to the University of North Carolina Investigational Drug Service. Only the pharmacist and statistician had access to the randomization schedule. Subjects were randomized to treatment groups 1:1 using permuted blocks of 4. gT and safflower oil (placebo) gel tabs were identical in appearance and were dispensed as a 7-day supply from the Investigational Drug Service to the study staff. Subjects returned for a followup visit to receive the additional 7-day supply of investigational drug for that period.

Endotoxin inhalation challenge
Clinical Center Reference Endotoxin, referred to as LPS, was provided by the National Institutes of Health Clinical Center. All doses were prepared by the Investigational Drug Service and inhaled by subjects as a nebulized preparation using a DeVilbiss Ultraneb 99 ultrasonic nebulizer (DeVilbiss, Port Washington, NY). 12,16 Sputum induction, processing, and mediator measurement Each subject provided 7 induced sputum samples (Fig 1): screening (prior to placebo or active treatment), 24 to 48 hours prior to each LPS challenge session (posttreatment sputum), and 6 and 24 hours after each LPS challenge session (postchallenge sputum). Induced sputum samples were processed as previously described. [31][32][33][34] Cell viability (trypan blue exclusion) and total cell counts were assessed in a Neubauer hemacytometer (Fisher Scientific, Hampton, NH), and differential cell counts were performed on cytocentrifuged cells stained with a modified Wright stain (Hema-Stain-3; Fisher Scientific, Hampton, NH). Cytokines from sputum supernatants were measured using multiplex technology (Meso Scale Discovery/MSD, Gaithersburg, Md). Each sample was analyzed with the V-PLEX Human Proinflammatory Panel II kit (Meso Scale). Even though ability to provide adequate sputum for analysis was an entrance criterion, there were instances during the study in which a subject was not able to provide a sputum sample or provided a poor-quality sputum sample. In these instances, these volunteers were excluded from analysis of the effect of active treatment on airway inflammation. These volunteers were included in assessment of MCC and safety end points for the study.

Gamma scintigraphy for measurement of MCC
The procedure used for measuring MCC in humans has been described in detail previously. 35,36 Briefly, volunteers inhaled an aerosol of technetium Tc 99msulfur colloid using a slow inhalation (80 mL/sec), large particle (9.5 mm mass median aerodynamic diameter) method. 37 Immediately following inhalation of the radioaerosol (duration of <5 minutes), an initial deposition scan was recorded (sum of two 2-minute images) and then continuous 2-minute images were recorded for a period of 2 hours to monitor clearance of particles from the lung as the subject remained seated in front of the gamma camera. Subjects returned the following day after the radiolabeled aerosol exposure to obtain a 30-minute scan of 24-hour lung activity/retention.
A whole lung region of interest bordering the right lung (created from a Co57 transmission lung scan 37 ) was used to determine, by computer analysis, the whole lung retention (decay and background corrected) as a fraction of the initial counts in the right lung, over the 2-hour clearance period at 10-minute intervals. MCC was calculated and expressed as average clearance in percent over the 2-hour period. 35 Because measures of MCC can be influenced by the initial, regional lung deposition of the inhaled radioaerosol, we also calculated (1) the central/peripheral ratio of activity and (2) the skew of the counts/pixel versus number of pixels histogram for the initial 2-minute image from each study visit. 35,38 Analysis of serum tocopherols and g-CEHC aT, gT, d-tocopherol, and 5-nitro-g-tocopherol were measured by an HPLC assay with electrochemical detection, 39 and g-CEHC was analyzed using liquid chromatography tandem mass spectrometry as previously described. 40

Analysis of sputum mucins
To measure total mucins, a 100 mL aliquot of induced sputum was solubilized in 6MGuHCl and subjected to differential refractometry (tREX, Wyatt Technology, Goleta, Calif) coupled with size exclusion chromatography as described previously. 41 Individual concentration of MUC5AC was measured by labeled mass spectrometry method using deuterium-labeled MUC5AC peptide standards.

Statistical analysis
We employed methods similar to those used in our initial study of the effect of gT in healthy volunteers. 16 The primary end points of the study were airway eosinophilia (defined as the difference in sputum eosinophils present in posttreatment samples) and LPS-induced airway neutrophilia (defined as the change in induced sputum neutrophils (PMNs) from posttreatment to 6 hours postchallenge), comparing gT treatment with placebo.
In planning this study, we were guided by the results of our previous study of gT-enriched supplementation on airway PMN response to LPS challenge in 13 healthy volunteers. 16 Based on these data, we estimated that a sample size of 30 volunteers would be adequate for this study, with an a priori plan to undertake an interim analysis after 15 volunteers completed this study. As planned and approved by Institutional Review Board and US Food and Drug Administration review, the interim analysis would lead us to stop the study due to demonstration of futility or statistically significant support of the hypotheses that gT inhibits airway eosinophilia and LPS-induced neutrophilia, or continuation of the study to n 5 30 due to inconclusive interim results.
For initial posttreatment versus postchallenge comparisons of sputum end points and MCC within each treatment group, paired t tests or Wilcoxon signed rank tests (depending on whether the normality assumption was met) were employed. Data that were not normally distributed were transformed using Box-Cox transformation. Given the crossover design of our study, we next determined the gT treatment effect (compared with placebo) on posttreatment sputum end points and on LPS-induced changes (D postchallenge -posttreatment) in sputum end points using a linear mixed model approach described by Jones and Kenward 42 that considers the above-mentioned individual tests in a global, unified way where all data are used at the same time. SAS PROC MIXED was used to fit the linear mixed model. Criterion for significance was taken to be P < _ .05.

Subject demographics
Twenty-three subjects with mild asthma were enrolled and underwent randomization. Based on frequency of daytime and nighttime symptoms and use of rescue albuterol, 22 subjects were classified as having mild intermittent asthma. One subject was classified as having mild persistent asthma and was using montelukast daily at the time of enrollment. However, this subject withdrew from the study prior to inhaled LPS exposure. No subjects were using inhaled corticosteroids at the time of enrollment or at any point during the study. The majority of participants were atopic (74%) based on the results of skin prick testing. Demographic characteristics of the study population are shown in Table I. Fifteen subjects completed both arms of the crossover study (see Fig E1 in the article's Online Repository at www.jacionline.org), with 13 providing adequate sputum for assessment of the primary sputum inflammatory end points for both treatment periods.
gT supplementation increased serum gT and g-CEHC concentrations Serum gT and g-CEHC concentrations rose significantly from baseline values in the active treatment group only (P < .0001 for both) (Table II) concentrations decreased from baseline in the active treatment group (P 5 .003).
gT treatment reduced posttreatment sputum eosinophils and mucins Using the linear mixed model approach, we found that gT treatment significantly reduced posttreatment sputum percentage of eosinophils (P 5 .04) and eosinophils per milligram of sputum (P 5 .01) compared with placebo (Fig 2, A and B). Likewise, gT treatment significantly reduced posttreatment total mucins (P 5 .03) and MUC5AC content (P < .0001) compared with placebo (Fig 2, C and D).
gT effects on airway mucin production and MCC MUC5AC content was significantly increased from posttreatment levels in both treatment groups 6 hours after inhaled LPS challenge (P 5 .001 [placebo], P 5 .0004 [active]). By 24 hours post-LPS challenge, total sputum mucins decreased in both treatment groups compared with prior to LPS challenge, though not significantly. Using linear mixed modeling to assess for a treatment affect, we detected significantly less total sputum mucins during the active treatment period compared with the total associated with placebo use (P 5 .03) (Fig 3, C). We found no significant difference in MUC5AC concentrations between the treatment groups at the same time point (data not shown).
As an exploratory measure, we assessed how gT intervention may impact MCC. MCC was measured prior to and 4 hours after LPS challenge. There were no differences in regional deposition indices (central/peripheral ratio or skew) nor in 24-hour retention between posttreatment and postchallenge measurements for either treatment period. MCC was significantly slowed following LPS challenge compared with posttreatment measurements for the placebo treatment period (MCC 5 16.3 6 9.3% postchallenge vs 21.4 6 6.9% posttreatment, P < .01) (Fig 4). In contrast, there was no such slowing of MCC by LPS challenge during active treatment (MCC 5 20.2 6 8.0% postchallenge vs 21.4 6 9.7% posttreatment, P 5 .6). However, for this new end point, the sample size was not adequate to definitively ascribe a treatment effect for gT on LPS-induced slowing of MCC when accounting for period and carryover effects.
gT treatment did not impact LPS-induced changes in sputum T H 1 cytokines During the placebo period, sputum IL-1b and IL-8 concentrations were significantly increased 6 hours post-LPS challenge compared with posttreatment values (P 5 .002 and P 5 .01, respectively), while no significant LPS-induced increase was observed during the active treatment period (P 5 .07 and P 5 .40, respectively). There was no significant LPS-induced change in sputum IL-6 concentration during either treatment period. Compared with placebo treatment, we did not detect a significant gT treatment effect on LPS-induced inflammatory cytokine concentrations in sputum following LPS challenge.

Adverse events
No serious adverse events occurred during the study period. The most commonly reported symptoms were gastrointestinal in nature. During the active treatment period, 21.7% of subjects reported nausea and 26% reported diarrhea or loose stools, compared with 8.7% and 4.3% during the placebo treatment period, respectively. These symptoms were typically transient and tended to occur during the first or second day of treatment and then self-resolved. One subject chose to discontinue study participation due to intolerable diarrhea during the active treatment period. No significant change in prothrombin time, activated partial thromboplastin time, or international normalized ratio was observed, and there were no reported bleeding events during the study. After completion of 14 days of active treatment, no clinically or statistically significant changes were seen in FEV 1 or FEV 1 /forced vital capacity from measurements taken during the initial baseline visit.

DISCUSSION
The primary goal of this proof-of-concept study was to determine whether gT supplementation in adults with asthma decreases airway eosinophilia as well as the inflammatory response to inhaled LPS, a model of neutrophil-predominant asthma exacerbation. Our results demonstrate that asthmatics treated with gT supplementation for 14 days had significantly reduced eosinophils in sputum when compared with those receiving placebo treatment. These findings suggest that gT may reduce baseline T H 2-mediated airway inflammation, which could be beneficial for eosinophilic asthma phenotypes. We also found that, compared with placebo, gT treatment was associated with lower posttreatment sputum mucins, including the inducible mucin glycoprotein, MUC5AC, which has been found in high concentrations in mucous plugs from fatal asthma cases. 43 Ex vivo gT treatment was previously found to inhibit IL-13induced secretion of eotaxin from airway epithelial cells, a potent chemotactic factor for eosinophils. 44 Given that mucin production is enhanced by IL-13, similar mechanisms may account for the impact of gT on mucin production as well.
We also found that gT treatment attenuated neutrophilic airway response to inhaled LPS challenge. Neutrophilic airway inflammation is often less responsive to corticosteroid treatment, 45 and there is a great unmet need for nonsteroidal therapies that target this specific type of inflammation. While we found no significant difference in sputum mucins between the treatment periods at 6 hours post-LPS challenge, total mucins were significantly lower at 24 hours with gT treatment, which could suggest faster recovery from mucin hypersecretion following an acute inflammatory challenge.
We observed a significant impairment of MCC following inhaled LPS challenge during the placebo treatment period but not during the gT treatment period. We have previously found a slowing of MCC by LPS challenge in healthy nonsmokers 35 and a trend toward slowing in mild asthmatics that was confounded by regional deposition differences between baseline and post-LPS challenge MCC (unreported). 38 While this study was not powered to detect a significant treatment effect of gT on MCC, our results do suggest that gT reduces LPS-induced slowing of MCC, and warrants further study. The mechanism by which inhaled LPS slows MCC is not understood, but it may be related to quantity  Data represented as medians (ranges). 5-NO 2 -gT, 5-nitro-g-tocopherol; dT, d tocopherol. *P < .05 comparing baseline concentrations to those obtained after 14 days of gT supplementation. Analyses were performed using paired t tests for gT and aT data and by Wilcoxon matched pairs signed rank test for dT and g-CEHC data as they were not normally distributed. or quality of sputum mucins, epithelial tethering of mucins, direct effects on ciliary function, or a combination of these factors. 43 The gT supplement used in our study given daily over a 2-week period resulted in significantly increased serum concentrations of gT and its primary active metabolite g-CEHC but with reduced serum aT concentrations by an unknown mechanism. This finding is consistent with previous studies of gT supplementation effects on reducing aT plasma concentrations, 46 including one conducted by our group that demonstrated reduced aT concentrations in serum following a 3-dose regimen of an identical gT supplement administered over a 24-hour period. 47 It is unknown whether continued gT supplementation would result in further decline in aT concentrations, nor is it known what the long-term physiologic consequences of this decline would be.
While our work has consistently demonstrated a beneficial effect of gT on airway inflammation, others have proposed a proinflammatory role for gT based primarily on human observational or animal model studies. In a cross-sectional study of young adults enrolled in the Coronary Artery Risk Development in Young Adults (CARDIA) cohort, higher serum aT levels were associated with higher lung function values (FEV 1 and forced vital capacity), while higher serum gT levels were associated with lower FEV 1 and forced vital capacity values. 48 While the results of this epidemiological study are intriguing, the correlation between serum gT levels and lung function may reflect gT as a risk factor or biological marker for lung function. Furthermore, others have shown that dietary vitamin E, at least 70% of which is composed of gT, was associated with increased FEV 1 in older adults 49 and may be protective against adult-onset asthma. 20 It is important to emphasize that these studies were not intervention trials, and several potential confounding factors could have influenced their results, including differences in intake of dietary fats. For example, gT-rich oils tend to have higher levels of polyunsaturated fatty acids, which may contribute to certain disease states, whereas aT-rich oils contain predominantly monounsaturated fatty acids, which have more health benefits. 50 Although further studies are needed to address the long-term impact of gT supplementation on airway inflammation, our 2-week dosing regimen with gT had no impact on spirometry measurements.
There are very few published human trials of gT supplementation in the context of airway inflammation prevention and/or treatment. Vitamin E has been studied for prevention and treatment of many chronic health conditions, 51  We are grateful to Jihong Wu and Dr John Lay, PhD, for their assistance in acquisition and analysis of MCC data. We are also grateful to Martha Almond, RRT, for her assistance with study visits and regulatory documentation.
Key messages d gT supplementation in mild asthmatics reduced sputum eosinophils and mucins compared with placebo in a fashion similar to that of inhaled fluticasone propionate and may have a role in reducing T H 2-mediated inflammation.
d gT reduced the neutrophilic inflammatory response to inhaled LPS challenge compared with placebo and may represent a useful therapy for neutrophil-predominant asthma exacerbation.
yet human trials in asthma have yielded conflicting results and have focused on treatment with aT, the most abundant tocopherol isoform in widely available supplements. In contrast to the results presented here, studies utilizing murine models found that gT supplementation exacerbated eosinophilic inflammation, while aT supplementation conferred protection. 55,56 It is possible that these conflicting reports reflect species-dependent differences in the anti-inflammatory effects of gT. Previous work from our group demonstrates that gT supplementation reduces airway eosinophilia in humans 16 and rodents. 27,28 This is in agreement with our current study, in which short-term dosing with gT exhibits acute anti-eosinophilic and anti-inflammatory properties in human subjects. These results, coupled with evidence that gT has unique anti-inflammatory properties compared with aT (including the ability to scavenge reactive nitrogen species 24 and inhibit COX-2 and 5lipooxygenase 25 ) supports the use of gT-enriched vitamin E preparations as a potential intervention for acute exacerbation, pollution induced disease, and possibly even chronic allergic diseases. These findings support conducting larger trials with gT supplementation in volunteers with asthma to further evaluate its role in modulating features of asthma.
This early phase clinical trial has several limitations. Our participants were predominantly female, which could reduce the generalizability of our results. The study population was somewhat heterogeneous with both atopic (74%) and nonatopic (26%) participants, and based on our safety criteria to undergo inhaled LPS challenges, had mild asthma. Given that the supplementation period only lasted 2 weeks, the longer-term effect of reduced serum aT levels noted with gT supplementation will have to be further studied for safety and efficacy in treating chronic airway inflammation. Our dosing regimen was generally well-tolerated, though early, transient gastrointestinal symptoms occurred in about one-fourth of participants studied. Additionally, we saw no prolongation of blood coagulation measurements, and no significant bleeding events were reported. The occurrence of early gastrointestinal side effects and potential need for both long-term and short-term treatment regimens suggests that dose ranging studies need to be done. Finally, the impact of body mass index on driving treatment responses to LPS will have to be further evaluated, given that we have previously shown that increased BMI is associated with sputum neutrophil recruitment to inhaled LPS among atopic subjects with asthma. 57 In conclusion, we have shown that a 14-day course of gT supplementation resulted in reduced eosinophilic inflammation of the airways and reduction in sputum mucins including MUC5AC. Additionally, gT supplementation reduced LPSinduced neutrophilic airway inflammation and mucinous content of sputum following inhalation challenge and was associated with reduced impact of LPS on MCC. Overall, our resultswith2weeks ofgT supplementation were similar to the effects of 2 weeks of treatment with inhaled fluticasone propionate on both posttreatment sputum eosinophilia and acute neutrophilic response to inhaled LPS challenge. 14 Taken together, these observations indicate that gT has potential to treat multiple features of asthma, including airway inflammation, mucous production, and clearance of mucous from the airways, and should be studied further in larger-scale clinical trials to investigate the efficacy of gT for improving asthma outcomes.