Paradoxical Effects of Rapamycin on Experimental House Dust Mite-Induced Asthma

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 ex vivo rapamycin decreased antigen-specific Th2 cytokine production, whereas prior exposure to in vivo rapamycin rendered T cells refractory to the suppressive effects of ex vivo rapamycin. We conclude that rapamycin had paradoxical effects on the pathogenesis of experimental HDM-induced asthma. Thus, consistent with the context-dependent effects of rapamycin on inflammation, the timing of mTOR inhibition may be an important determinant of efficacy and toxicity in HDM-induced asthma

Paradoxical Effect of Rapamycin on Lung Th2 and Th17 Cytokine Expression in Induction and Treatment Models of House Dust Mite-induced Asthma.

<p>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.</p

Rapamycin attenuates mTORC1 effectors.

<p>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.</p

Paradoxical Effect of Rapamycin on Lung Histology in Induction and Treatment Models of House Dust Mite-induced Asthma.

<p>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.</p

Paradoxical Effect of Rapamycin on Lung C-C Chemokine Expression in Induction and Treatment Models of House Dust Mite-induced Asthma.

<p>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.</p

Paradoxical Effect of Rapamycin on Plasma Immunoglobulin Levels in House Dust Mite-induced Asthma.

<p>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).</p

Effects of Rapamycin on Mediastinal Lymph Node Th2 Cytokine Production.

<p>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 <i>ex vivo</i> with or without HDM re-stimulation and/or rapamycin. The amount of IL-4, IL-5 and IL-13 released into the medium was quantified by ELISA (* P<0.05, n = 5–6). Results are representative of two independent experiments.</p

Paradoxical Effect of Rapamycin on Bronchoalveolar Lavage Fluid Inflammatory Cells in Induction and Treatment Models of House Dust Mite-induced Asthma.

<p>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.</p

Paradoxical Effect of Rapamycin on Airway Hyperreactivity in House Dust Mite-induced Asthma.

<p>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.</p