The goal of our study was to determine whether the mTOR inhibitor, rapamycin, would suppress key characteristics and mediators in a clinically relevant asthma model induced by the aeroallergen HDM. We utilized two different protocols to investigate the effects of rapamycin on allergic responses to HDM, and while they cannot be directly compared, each protocol was designed to address specific questions. In the first protocol, we administered rapamycin to mice simultaneously with intranasal HDM to determine whether rapamycin could prevent HDM-induced allergic responses. Increases in AHR and inflammation were prevented by rapamycin treatment. In addition, increases in HDM-specific IgG1 and IgE levels were prevented by rapamycin, indicating that simultaneous treatment with rapamycin prevented allergic sensitization. Therefore, it was unclear whether inhibition of AHR and inflammatory cell influx into the lungs was due to direct effects on these processes or because allergic sensitization was prevented. Hence, we used a second protocol to address the question of whether rapamycin could suppress pulmonary responses to allergen exposure after allergic sensitization had already been established. In this protocol, we sensitized mice to HDM (by I.P. injections) before exposing the lung to the allergen and treating with rapamycin. In these sensitized mice, AHR, inflammatory cell influx (especially eosinophils), and IgE increased dramatically with intranasal HDM exposure. Rapamycin treatment, given after sensitization and just prior to HDM exposure, prevented these exacerbations, including the large increase in AHR and IgE. To explore potential mechanisms, Th2 cytokines and cysteinyl leukotrienes in BALF were measured. While IL-4 and eotaxin 1 levels were unaffected by rapamycin treatment, increases in IL-13 and leukotrienes with HDM exposure were completely suppressed. Decreases in IL-13 and leukotriene levels, both important mediators of AHR and goblet cell metaplasia, provides insight into a potential mechanism for the effects of rapamycin on allergic airway disease. The suppressive effects of rapamycin on allergen-induced IL-13 and leukotriene levels, to our knowledge, have not been previously reported in studies using allergic asthma models.
Previous studies have used SAR 943, a derivative of rapamycin, and rapamycin in the OVA-induced allergic asthma model. In Brown-Norway rats sensitized to OVA, SAR 943 given prior to OVA challenge to the lung reduced the number of CD4+ T cells recruited into the lung; however, AHR and eosinophil infiltration were unaffected (16
). A similar study in mice showed attenuation of eosinophils, IL-4, goblet cells, and AHR responses after SAR 943 treatment (17
), but no effect on serum IgE levels. In a third study, mice sensitized with OVA and treated with rapamycin showed reductions in IgE levels; however, no changes in eosinophils or AHR were observed (18
). Hence, these OVA studies reported mixed effects of mTOR inhibitors on allergic responses and as mentioned earlier did not assess IL-13 or leukotrienes.
Prior studies have demonstrated an important role for inflammation in the development of AHR, with increased inflammatory cells typically correlating with increases in AHR (23
). However, in our second protocol we observed a marked disconnect between inflammatory cell influx and AHR. Despite no effect on inflammatory cell influx, rapamycin blocked the increase in AHR following HDM challenge to the lung. This dissociation between inflammatory cells and AHR has been observed previously. For example, in patients with allergic asthma, the number of inflammatory cells in the lung did not correlate with the degree of AHR (23
). In our second protocol, eotaxin 1 levels were increased in HDM/HDM mice, but unaffected by rapamycin treatment which is consistent with the lack of a change in eosinophils levels after rapamycin treatment, despite decreases in IL-5. This maintenance of elevated eosinophils is in contrast to the suppressive effects of rapamycin treatment on AHR, although there are conflicting reports on the role of eosinophils in AHR (25
Another interesting observation in our study was that rapamycin treatment did not affect IL-4 levels in BALF, despite a major reduction in IL-13 levels. It is unclear which cell types are responsible for the differences in IL-4 and IL-13 levels we observed since a number of cell types are known to secrete these cytokines (29
). Although mast cells can produce both IL-4 and IL-13, it has been reported that rapamycin did not affect degranulation and cytokine release from mouse mast cells (30
). It is unlikely that eosinophils are the primary source of IL-4 and IL-13 in our model since only IL-13 levels are affected by rapamycin, whereas no changes in IL-4 and eosinophil numbers were observed. In addition, although both mast cells and eosinophils can release Th2 cytokines, the primary source of cytokines like IL-4 and IL-13 is most likely Th2 lymphocytes (29
). Interestingly, it has been reported that when T cells were co-stimulated under Th2 skewing conditions and treated with rapamycin, IL-13 levels were more severely decreased and only a modest decrease in IL-4 levels were observed (31
). It is possible that other cellular sources are contributing to sustained levels of IL-4 in our model and future studies will address this. Interestingly, despite no changes in IL-4, which is known to be an important mediator of the IgE response, the large increase in IgE detected after allergen challenge was blocked by rapamycin. In addition, AHR was still completely suppressed by rapamycin suggesting that reductions in IL-13 and/or leukotrienes were responsible, as these are known to play important roles in allergic asthma (32
). However, it has also been reported that rapamycin can inhibit contractile proteins in airway smooth muscle cells (37
). This raises the possibility that reductions in AHR in our second protocol could be due to direct effects of rapamycin on airway smooth muscle contractility.
The importance of mTOR in regulating immune responses is becoming increasingly apparent. Currently, rapamycin is used in patients after organ transplant as an immunosuppressant drug (12
). Early studies demonstrated a role for mTOR in regulating T cell proliferation, especially after cytokine stimulation (38
). More recent studies have shown that T cells deficient in mTOR fail to become Th1, Th2, or Th17 effector cells under skewing conditions and instead default to Foxp3+
regulatory T cells (39
). These data and others highlight the importance of mTOR in T cell differentiation (39
). Similar to other reports (41
), we demonstrated reductions in activated T cells in the lung with rapamycin treatment. However, in contrast to in vitro
), a decrease in the total number of T regulatory cells with rapamycin treatment was seen in our in vivo
study. This may be due to the fact that total T cell numbers were reduced by rapamycin treatment. Although T regulatory cell numbers were decreased with rapamycin, they did not fall below HDM/Saline controls. In addition, although the total number of T regulatory cells were decreased with rapamycin, the ratio of T regulatory cells to conventional T cells was not different compared to HDM/HDM mice.
Asthma is a complex disease that manifests differently among individual patients. Currently, inhaled glucocorticosteroids are the most effective anti-inflammatory therapies for the treatment of asthma (1
), although some patients are refractory to glucocorticosteroids. Omalizumab, a relatively new therapy, is being used in the treatment of asthma and targets IgE. Omalizumab has been shown to be an effective treatment for allergic asthma as assessed by decreased asthma exacerbations and hospitalizations in patients receiving this therapy (45
). In our studies, rapamycin treatment suppressed or attenuated key characteristics of the asthmatic response including AHR, IgE, and goblet cell metaplasia, as well as important mediators including IL-13 and leukotrienes. Additional studies are necessary to elucidate the potential contribution of each of these mediators to the effects of rapamycin on the allergic response and AHR. Understanding these mechanisms may provide further insight into the role of mTOR in allergic disease.