Conceived and designed the experiments: KF JAF JKL XY JM ASK SJL. Performed the experiments: KF JAF JKL KSM KJK GJZ XQ. Analyzed the data: KF JAF JKL XY ZY JM ASK SJL. Contributed reagents/materials/analysis tools: KF JAF JKL XY KJK GJZ XQ ZY ASK SJL. Wrote the paper: KF JKL ASK SJL.
JM has received royalties from a patent (United States Provisional Patent Application No. 60/528,340 filed 09 Dec 2003) licensed from the National Institutes of Health (NIH) that is related to the topic of this manuscript. ASK has received research support from the LAM Foundation, the Canadian Institutes of Health, and the NIH (R01 CA125436). ASK also has received funding through a NIH Intramural Research Program Contract. ASK has a patent on a topic related to this manuscript (United States Provisional Patent Application No. 60/528,340 filed 09 Dec 2003). SJL has two patents that are unrelated to the topic of this manuscript (United States Patent 7,135,303, Filed February 28, 2001 and United States Patent 7,655,752 B2, Filed March 24, 2006). These competing interests do not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the PLoS One guide for authors.
The mammalian target of rapamycin (mTOR) modulates immune responses and cellular proliferation. The objective of this study was to assess whether inhibition of mTOR with rapamycin modifies disease severity in two experimental murine models of house dust mite (HDM)-induced asthma. In an induction model, rapamycin was administered to BALB/c mice coincident with nasal HDM challenges for 3 weeks. In a treatment model, nasal HDM challenges were performed for 6 weeks and rapamycin treatment was administered during weeks 4 through 6. In the induction model, rapamycin significantly attenuated airway inflammation, airway hyperreactivity (AHR) and goblet cell hyperplasia. In contrast, treatment of established HDM-induced asthma with rapamycin exacerbated AHR and airway inflammation, whereas goblet cell hyperplasia was not modified. Phosphorylation of the S6 ribosomal protein, which is downstream of mTORC1, was increased after 3 weeks, but not 6 weeks of HDM-challenge. Rapamycin reduced S6 phosphorylation in HDM-challenged mice in both the induction and treatment models. Thus, the paradoxical effects of rapamycin on asthma severity paralleled the activation of mTOR signaling. Lastly, mediastinal lymph node re-stimulation experiments showed that treatment of rapamycin-naive T cells with
Rapamycin (Sirolimus, Rapamune®) is a macrolide product of Streptomyces hygroscopius that was initially discovered in a soil sample from Easter Island (Rapa Nui) in the early 1970s
Here, we sought to define the role of mTOR signaling on the pathogenic manifestations of asthma using a clinically relevant house dust mite (HDM)-induced model of murine disease. We selected HDM to induce airway disease because it is an important environmental aeroallergen that has been identified as a risk factor for persistent asthma in human subjects
Female Balb/c mice were purchased from Jackson Laboratories (Bar Harbor, Maine). Asthma was induced by daily intranasal administration of HDM (
Bronchoalveolar lavage (BAL) was performed utilizing three instillations of ice cold PBS (0.5 ml). Red blood cells present in BAL fluid (BALF) were lysed using ACK buffer (2 min at 4°C), followed by re-suspension of cells in 0.3 ml RPMI-1640 with 20% fetal bovine serum. The total number of BALF cells were counted using a hemocytometer. The differential cell counts were performed on Diff-Quick-stained cytospin slides (Siemens Healthcare Diagnostics, Deerfield, IL). For histopathological examination, lungs were inflated to a pressure of 25 cm of H20 and fixed in 10% formalin for 24 h. Lungs were then dehydrated through gradient ethanol prior to embedding in paraffin. Sagittal sections were cut at a thickness of 5 mm and stained with hematoxylin and eosin or periodic acid Schiff (PAS). Representative histology images were selected by one of the authors who was blinded to the identity of the groups. Quantification of goblet cell hyperplasia was performed as previously described
Lungs were minced prior to storage in RNAlater® (Applied Biosystems Inc., Foster City, CA) at −70°C. Total RNA was subsequently isolated using the lipid tissue kit from Qiagen (Qiagen Inc, Valencia, CA) and on-column DNase treatment was performed using RNase-Free DNase from Qiagen. Reverse transcription was performed utilizing random hexamer primers and High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). PCR was performed utilizing the TaqMan Universal PCR Master Mix and the following FAM dye-labeled Taqman® MGB probes; IL-4: Mm00445259_m1, IL-10: Mm00439614_m1, IL-13: Mm00434204_m1, IL-17a: Mm00439618_m1, Muc5Ac: Mm01276735_m1, Clca3: Mm00489959_m1, CCL7: Mm00443113_m1, CCL11: Mm00441238_m1, CCL17: Mm00516136_m1, CCL24: Mm00444701_m1 and 18S: Hs99999901_s1. One µg of cDNA was used as a template. and samples were amplified utilizing the 7500 Real Time PCR System running Sequence Detector version 2.1 software (ABI systems, Foster City, CA). Gene expression was quantified relative to the expression of 18S mRNA using the control sample as calibrator to calculate the difference in Ct values (ΔΔCt) and results are presented as relative mRNA expression.
Mediastinal lymph nodes were removed, disrupted by gentle pressure with a syringe plunger and passed through a 100 µm strainer to yield single cell suspensions
Total plasma IgE was measured using an OptEIA™ kit (BD Biosciences Pharmingen, San Diego, CA). Total plasma IgG1 was measured using an ELISA Quantitation Set from Bethyl Laboratories, Inc. (Montgomery, TX), whereas total plasma IgG2a was measured using an ELISA Set from BD Biosciences Pharminogen (San Diego, CA).
Whole lungs were removed en bloc, snap frozen in liquid nitrogen, and stored at −80°C prior to protein extraction. Lungs were mechanically disrupted using a Brickman mechanical homogenizer in homogenization buffer (20 mM Tris pH 8.0, 0.5% Nonidet P-40, 1 mM phenylmethanesulphonylfluoride, 50 mM NaF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 100 µM sodium orthovanadate). Homogenates were snap frozen on dry ice, thawed, and cleared by centrifugation at 16,000×g for 30 min at 4°C. Supernatants were assayed for protein content by Bradford assay. Lung proteins (80 µg) were separated by SDS-PAGE and transferred to nitrocellulose membrane before immunoblotting with primary antibodies as indicated. Membranes were incubated with anti-rabbit or anti-mouse IgG horseradish peroxidase conjugated antibodies and developed using Super-Signal West Pico chemiluminescence detection kit (Pierce). Antibodies that react with S6, phospho-S6, Akt, phospho-Akt, STAT6 and phospho-STAT6 were from Cell Signaling Technology, Inc. (Danvers, MA), whereas the antibody that reacted with ß-actin was from Sigma-Aldrich (St. Louis, MO).
Mice were anesthetized with ketamine and xylazine and a 19 gauge beveled metal catheter was inserted into the trachea. Mice were mechanically ventilated with a tidal volume of 0.2 ml at 2 Hz, while PBS or increasing doses of methacholine (0, 2.5, 5, 7.5 and 10 mg/ml) were administered by nebulization. Airway resistance was directly measured utilizing an Elan RC Fine Pointe system (Buxco Research Systems, Wilmington, N.C.). Airway resistance was recorded at 10 s intervals for 3 min and average values are presented as cm H20/ml/s.
Results are presented as mean ± SEM. A one-way ANOVA with a Bonferroni's multiple comparison test was utilized for all analyses except for airway hyperreactivity experiments, which instead utilized a two-way ANOVA with a Bonferroni post-test test. A P value less than 0.05 was considered significant. Statistical analyses were performed with GraphPad Prism version 5.0a (Graphpad Software, Inc., La Jolla, CA).
To induce airway disease, Balb/c mice received daily nasal administration of HDM (25 µg) for 5 days per week. In the induction model, mice received rapamycin or vehicle coincident with the initiation of HDM administration for 3 weeks. In the treatment model, HDM challenges were performed for 6 weeks, whereas treatment with rapamycin or vehicle was administered during weeks 4 through 6 (
Balb/c mice received daily nasal challenges with HDM (25 µg) 5 days per week. In the induction model (Panel A), HDM challenges were initiated concurrent with rapamycin administration (3 mg/kg) by gavage 5 days per week for 3 weeks (n = 7–10 animals per group). In the treatment model (Panel B), HDM challenges were administered for 6 weeks and rapamycin administration was given during weeks 4 through 6 (n = 12–13 animals per group). * P<0.05 vs. Saline+Vehicle, ** P<0.001. Results are representative of two independent experiments.
Histologic sections of lung were stained with hematoxylin and eosin (H & E) or periodic acid-Schiff (PAS) stains and images obtained at 200× or 1000×. Results are representative of 2 independent experiments.
The pulmonary expression of Th2 and Th17 cytokines was assessed to investigate further the mechanisms mediating the differential effects of rapamycin in the induction and treatment models of HDM-induced asthma. As shown in
Quantification of lung mRNA levels for IL-4, IL-13, and IL-17A by qRT-PCR presented as relative mRNA expression. Results for the induction experiment are shown in Panel A (n = 6–8 animals per group, * P<0.05, HDM+Vehicle vs. HDM+Rapamycin), while results for the treatment experiment are shown in Panel B (n = 6–10 animals per group, * P<0.001). Results are representative of 2 independent experiments.
The effect of inhibition of mTOR on the expression of lung chemokines was also assessed. As shown in
Quantification of lung mRNA levels for CCL11, CCL24, CCL7 and CCL17 by qRT-PCR presented as relative mRNA expression. Results for the induction experiment are shown in Panel A (n = 6 animals per group, * P<0.01), while results for the treatment experiment are shown in Panel B (n = 5–10 animals per group, * P<0.05). Results are representative of 2 independent experiments.
These data demonstrate that administration of rapamycin prior to nasal HDM administration inhibits the induction of airway inflammation via a mechanism that involves the reduced expression of Th2- and Th17-type cytokines, as well as C-C chemokines. Conversely, treatment of established asthma with rapamycin increased both the number of BALF inflammatory cells, as well as lung mRNA levels of the C-C chemokine, CCL11.
To investigate further the paradoxical effects of rapamycin on HDM-mediated airway inflammation, we used the induction model to assess the effects of
Mediastinal lymph nodes from HDM-challenged mice that had or had not been treated with rapamycin concurrent with HDM stimulation for 3 weeks (induction model) were cultured
Experiments were performed to assess whether rapamycin also has paradoxical effects on plasma IgE production. As shown in
Plasma levels of IgE, IgG1 and IgG2a were quantified. Results for the induction experiment are shown in Panels A, C and E, while results for the treatment experiment are shown in Panels B, D and F (n = 8–20 animals per group, * P<0.05 vs. Saline+Vehicle, ** P<0.001).
In the induction model, rapamycin administration was associated with small, but significant, decreases in the number of airways demonstrating goblet cell hyperplasia and Clca3 (chloride channel calcium activated 3) mRNA levels, whereas mRNA levels for Muc5AC (mucin 5, subtypes A and C) were not modified (
Quantification of lung mRNA levels for Muc5AC and Clca3 by qRT-PCR are presented as relative mRNA expression. Results for the induction experiment are shown in Panel A (n = 6 animals per group, * P<0.01), while the results for the treatment experiment are shown in Panel B (n = 5–6 animals per group, P = NS). Results are representative of 2 independent experiments. Goblet cell hyperplasia is presented as the percentage of airways containing PAS-positive cells (n = 8–10 animals per group, * P<0.001). 35.3±0.6 airways were inspected in each mouse.
The effect of rapamycin on HDM-induced airway hyperreactivity (AHR) was also assessed. As shown in
Airway resistance (cm H20/ml/s) was directly measured following administration of increasing doses of nebulized methacholine. Results for the induction experiment are shown in Panel A (n = 10 animals per group, * P<0.05), while results form the treatment experiment are shown in Panel B (n = 9–10 animals per group, * P<0.05). Results are representative of 2 independent experiments.
Western blots of lung proteins were performed to identify the mechanism by which rapamycin mediates paradoxical effects on HDM-induced asthma. After 3 weeks in the induction model, phosphorylation of the S6 ribosomal protein was increased in the lungs of HDM-challenged mice as compared to saline-challenged mice (
Phosphorylation of the mTORC1 effector, S6 (phospho-S6 Ser 235/236), or the mTORC2 effector, Akt (phospho-Akt S473), was assessed by Western blot analysis. The phosphorylation of STAT6 (phospho-STAT6 Y641) was determined as a control for activation of Th2 pathways. A representative blot from 5 experiments is shown.
Rapamycin is a specific and potent inhibitor of mTOR, a highly conserved and ubiquitous serine-threonine kinase that nucleates two distinct multi-protein mTOR complexes
Inhibition of mTOR signaling by rapamycin has immunosuppressive effects on antigen-presenting cells and T cells, which thereby modulate adaptive immune responses. For example, rapamycin inhibits IL-4-dependent dendritic cell (DC) maturation, fms-like tyrosine 3 kinase ligand (Flt3L)-induced DC mobilization, as well as co-stimulatory molecule expression, pro-inflammatory cytokine production and T-cell allostimulation by DCs
mTOR signaling also plays an important role in airway smooth muscle and epithelial cell proliferation. mTORC1-related changes in the size, proliferation, and survival of smooth muscle or epithelial cells may contribute to hypertrophy and remodeling of the airway wall in asthmatics
Based upon the important role of mTOR signaling in inflammatory and remodeling responses, we investigated whether rapamycin could be utilized to modify the pathogenic manifestations of asthma as well as provide new insights into disease pathogenesis. Here, we show that rapamycin has paradoxical effects depending on whether it is administered prior to the induction of HDM-induced asthma or as a treatment during the effector phase of HDM-induced asthma. Administration of rapamycin coincident with HDM exposure significantly attenuated eosinophilic and lymphocytic airway inflammation, which was mediated by the reduced expression of Th2- and Th17-type cytokines and C-C chemokines, as well as production of IgE, IgG1 and IgG2a. This is consistent with the role of mTOR in mediating the differentiation of CD4+ T cells into Th2 and Th17 subsets
In contrast, treatment of established HDM-induced asthma with rapamycin augmented airway inflammatory responses, as indicated by significant increases in the number of BALF eosinophils, lymphocytes and neutrophils. Similarly, treatment of established HDM-induced asthma with rapamycin worsened AHR and did not reduce goblet cell hyperplasia or IgE production. Taken together, these results demonstrate that inhibition of mTOR signaling by rapamycin represents a “molecular switch” with divergent effects on asthma pathogenesis that are dependent upon the temporal relationship between rapamycin administration and allergen sensitization. When administered concurrent with HDM, rapamycin blocked allergic sensitization and the induction of the key manifestations of asthma, but when administered in the setting of established asthma, rapamycin exacerbated disease severity as evidenced by enhanced airway inflammation and AHR.
The mechanism underlying the paradoxical effects of rapamycin in the induction and treatment models of HDM-induced asthma may in part have reflected the temporal association between HDM challenge and activation of mTOR signaling pathways. Activation of mTORC1 signaling was assessed by phosphorylation of its downstream target, the S6 ribosomal protein. S6 phosphorylation was increased after 3 weeks of HDM challenge in the induction model, but returned to levels similar to those of saline-challenged mice at 6 weeks in the treatment model. Consistent with this finding, inhibition of mTOR signaling with rapamycin attenuated HDM-induced asthma only in the induction model when mTOR signaling was up-regulated, but not in the treatment model when mTOR signaling was no longer increased. Changes in STAT6 phosphorylation paralleled the effects of rapamycin on HDM-induced asthma. Furthermore, Akt phosphorylation was neither up-regulated by HDM challenge nor inhibited by rapamycin, which is consistent with the conclusion that mTORC2 signaling did not modulate HDM-induced asthma in this model.
We also found that rapamycin had paradoxical effects on Th2 cytokine production by T cells. The
Our findings are consistent with prior reports that showed paradoxical pro-inflammatory effects of rapamycin. For example, although rapamycin has immunosuppressive effects and is utilized clinically to prevent kidney transplant rejection, its use has been complicated by the development of a lymphocytic interstitial pneumonitis
In conclusion, our results demonstrate that inhibition of mTOR signaling with rapamycin has paradoxical effects on the pathogenesis of HDM-induced asthma that are dependent upon the temporal relationship between rapamycin administration and the activation of mTOR signaling pathways. Rapamycin attenuated the manifestations of HDM-induced asthma after 3 weeks in the induction model when mTOR signaling was increased, but exacerbated HDM-induced asthma after 6 weeks in the treatment model, when mTOR signaling had returned to basal levels. Furthermore, our findings suggest that rapamycin might not represent an effective treatment approach for HDM-induced asthma.
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We are extremely appreciative of the staff of the NHLBI Laboratory of Animal Medicine and Surgery, whose commitment, professional advice and excellent technical support made this study possible. We are also very appreciative of Filipina Giacometti, B.Sc. in the Pathology Core Facility, NHLBI for her assistance with the histopathological analyses.
We are most appreciative of Dr. Martha Vaughan for her helpful discussions and advice.