The goal of our study was to determine whether mTOR inhibition with rapamycin would suppress the key features and mediators of HDM-induced allergic asthma in established asthmatic disease. In addition, we also compared rapamycin to the steroid, dexamethasone, since steroids are currently a mainstay treatment for asthma. In the first protocol, we assessed whether rapamycin or dexamethasone could suppress allergic disease during allergen re-exposure. Although rapamycin suppressed IgE levels, goblet cells, and total lung T cells, it had no effect on AHR or BALF cellularity and IL-4 and eotaxin 1 levels were actually augmented. Dexamethasone suppressed goblet cells and total lung T cells, but had no effect on IgE or AHR and only slightly reduced BALF eosinophilia. Our second protocol assessed whether rapamycin or dexamethasone could reverse or inhibit the progression of asthmatic responses during chronic allergic airway disease. In this model, rapamycin did not suppress AHR or goblet cells and actually augmented inflammatory cell numbers, IL-4 and eotaxin 1 levels in the BALF. Dexamethasone had limited effects on AHR, but did attenuate the inflammatory cell influx into the BALF, especially eosinophils. Despite these limited effects, both rapamycin and dexamethasone suppressed lung tissue lymphocyte numbers and serum IgE levels.
Previous studies from our lab demonstrated that HDM-induced allergic asthma could be prevented if rapamycin was administered early and simultaneously with HDM. In this case, rapamycin prevented HDM-induced AHR, inflammation, goblet cells, and allergic sensitization 
. Since allergic sensitization was suppressed in our previous studies, we also determined whether rapamycin could prevent allergic responses once sensitization had already been established. To do this, mice were first sensitized systemically to HDM by i.p. injection. During subsequent intranasal HDM exposures, mice were treated with rapamycin. In this case, rapamycin still suppressed many of the key allergic responses including IgE, AHR, goblet cells, T cell responses, and key mediators like IL-13 and leukotrienes, although it did not suppress increases in inflammatory cells in the BALF 
, which may have partly been due to the chemokine, eotaxin 1, since levels were still elevated after rapamycin treatment. Although these studies demonstrated an important role for the mTOR pathway during early allergic sensitization and asthmatic disease processes, it was unclear whether mTOR signaling would be important during allergen re-exposure or during established/progressive allergic disease. The studies we report in this manuscript sought to address this question. These data suggests that the role of mTOR is very different depending on the timing/disease stage since rapamycin treatment during allergen re-exposure or during chronic, ongoing disease did not attenuate key characteristics of allergic asthma including AHR and inflammation and actually augmented IL-4 and eotaxin 1 levels. The results from our second protocol are similar to that of a recent study published by Fredriksson et. al. who demonstrated that rapamycin did not suppress allergic responses when administered during chronic allergic disease 
. In addition, our studies demonstrated that rapamycin suppressed T cells in the lung tissue, including regulatory T cells and our studies also compared the effects of rapamycin to the steroid, dexamethasone.
Allergic asthma is often treated with steroids to suppress inflammation 
. Previous studies have utilized the corticosteroid, dexamethasone, in allergic asthma models 
. For example, a study similar to ours investigated the effects of dexamethasone during allergic relapse and overt disease. In an OVA model of allergic airway disease, dexamethasone suppressed goblet cells, serum IgE, AHR, and reduced airway inflammation in a relapse model. During overt disease, dexamethasone reduced goblet cells, AHR, and the number of eosinophils, but had no effect on serum IgE levels 
. In our HDM-induced model of allergen re-exposure/relapse, dexamethasone also decreased goblet cells, but did not suppress IgE or AHR and eosinophil numbers were only slightly reduced. Likewise, during chronic, ongoing or overt disease, we did not observe suppression of goblet cells and the effects on AHR were limited, although there were decreases in inflammatory cell numbers, specifically eosinophils. Although the decrease in eosinophils in this study was as expected with dexamethasone treatment, no decrease in AHR was surprising. However, previous reports have suggested that the timing of AHR measurements after dexamethasone treatment may be important 
. Specifically, when AHR was measured 12 hours after dexamethasone treatment, AHR was suppressed, but by 24 hours after dexamethasone treatment, AHR was no longer suppressed 
. In all of our studies, AHR was assessed 24 hours after the last dexamethasone treatment, which may explain why limited decreases in AHR were observed. In addition, differences between the results of our study and others may be due to the type of allergen used and/or the route of dexamethasone treatment since ultrasonic nebulization was used in the study by Jungsuwadee et. al. 
, versus i.p. delivery in our study.
Unlike our previous studies when rapamycin was given early in the disease process 
, in this study, AHR was not suppressed by rapamycin during allergen re-exposure or chronic allergic disease. Both IL-4 and inflammation were still increased after rapamycin treatment and could potentially contribute to AHR 
. However, in our previous study, rapamycin treatment decreased AHR despite elevated IL-4 BALF levels and inflammatory cell numbers suggesting that other mechanisms are involved 
. IL-13 is a key mediator of AHR, however, in these chronic/established models, IL-13 was not increased, suggesting that it may not be contributing to AHR in more advanced disease. Airway remodeling is another feature of asthma that could contribute to sustained AHR in these more advanced disease models; however, no major differences in airway smooth muscle or baseline airway resistance between animal groups in either study were observed, suggesting other mechanisms are playing a role in the disease process.
An interesting observation in our studies was that IL-4 levels were higher even though IgE levels after HDM exposure were still suppressed by rapamycin treatment. This was surprising since IL-4 is an important mediator of IgE class switching. It is possible that rapamycin could directly affect B cells that are secreting IgE to suppress allergic sensitization to HDM. Previous work has demonstrated that mTOR is required for B cell development and maturation 
, however, less is known about the role of mTOR in B cell homeostasis, activation of mature B cells, and immunoglobulin production/secretion. A study using purified human B cells demonstrated that rapamycin inhibited B cell proliferation, induced apoptosis, and suppressed immunoglobulin production, particularly IgM and IgG 
. Although these studies were carried out in vitro
they still suggest that rapamycin could have direct effects on B cells, which could account for the decreases in IgE levels in our in vivo
model and therefore reduce sensitization to HDM, despite increased IL-4. When we assessed B cells in the lung tissue, there was a trend towards a decrease in B cells in both studies after rapamycin treatment. Despite being non-significant, we cannot exclude that this minor decrease in lung B cell levels could contribute to the observed decrease in IgE levels. The source of the IL-4 increase is unclear in our model since T cells, which are one of the primary sources of IL-4, were reduced. Other cells including eosinophils, basophils, and mast cells can secrete IL-4 
, but whether these cells are playing a role in enhancing IL-4 levels in our model is unclear. Also in our study, eotaxin 1, an important epithelial cell derived eosinophil chemokine, remained elevated in the BALF with rapamycin treatment, which may explain why eosinophil numbers were not suppressed. This was also true in our previous acute study in which rapamycin treatment did not suppress airway inflammation nor eotaxin 1 levels once sensitization was established 
More recent studies have indicated an important role for regulatory T cells in the resolution of allergic airway disease 
. Studies have demonstrated that adoptive transfer of CD4+
regulatory T cells into mice exposed to allergen suppressed allergic responses, whereas inhibition of regulatory T cells exacerbated the allergic response 
. In vitro
data suggests that rapamycin can expand CD4+
regulatory T cells in the presence of IL-2 
, however, in our in vivo
model, rapamycin treatment was associated with decreases in effector T cells, a major source of IL-2 in the lung. Hence, rapamycin treatment, much like dexamethasone treatment, may decrease regulatory T cells in vivo
by decreasing the number of IL-2 producing cells. It is unclear if the reductions in regulatory T cells after rapamycin treatment in this model would have any biological significance; however, loss of regulatory T cells has been shown to worsen the severity of allergic disease 
. Interestingly, loss of CD69+
cells has also been associated with exacerbated allergic disease 
. These findings remain controversial however 
, as new roles for CD69 in cell egress from lymphoid organs, Th17 differentiation and formation of memory CD44+
T cells are being proposed 
. Interestingly, short rapamycin (or dexamethasone) treatment had little effect on the proportion of memory cells in the lungs, whereas longer exposure to rapamycin (or dexamethasone) in our second model significantly decreased the proportion of CD44+
memory cells among CD4+
It remains unclear why many of the allergic responses, especially AHR, were not suppressed in our studies even though T cell populations were reduced. The effects of rapamycin and dexamethasone, however, may not be only specific to T cells since spleen sizes were also reduced in our studies, consistent with the immunosuppressive properties of these drugs 
. It is possible that other cell types in the lung could be contributing to the allergic responses in these established/chronic models, uch as epithelial cells. Epithelial cells and other lung cells can produce cytokines upon allergen exposure, which can then directly influence allergic responses, including AHR 
The protein encoded by the mTOR gene signals through two protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Each of these complexes carries out distinct cellular functions and each complex is composed of several subunits. The most notable subunit of mTORC1 is the regulatory-associated protein of mTOR (Raptor) and in mTORC2, the rapamycin-insensitive companion of mTOR (Rictor) 
. Most reports indicate that only mTORC1 is rapamycin sensitive, but some recent evidence suggests that, depending on the cell type, duration, and dosing regimen, rapamycin can also inhibit mTORC2 
. Downstream of mTORC1 is the ribosomal protein S6 kinases and its downstream substrate S6, which gets phosphorylated upon mTOR activation. In order to help understand why rapamycin did not suppress the allergic responses in our studies, we measured the activation of P-S6 downstream of mTORC1. In both of the models used here, rapamycin completely suppressed HDM-induced increases in phosphorylated S6 levels, but did not suppress the phosphorylation of Akt (S473). These results indicate that the dose of rapamycin used was sufficient to block mTORC1, but not mTORC2. These mTOR complexes differentially regulate T cell lineage commitment; with Th1 and Th17 being mostly dependent on mTORC1 signaling and Th2 cells on mTORC2 
. Accordingly, the Th1 cytokine IFN-γ and Th17 cytokine, IL-17A, were significantly decreased or trended lower in both of our models following rapamycin treatment whereas the prototypical Th2 cytokine IL-4 was increased. This increase in IL-4 is potentially the result of decreases in IFN-γ, a negative regulator of Th2 differentiation. Finally, IL-4 has been implicated in allergic responses independently of IL-13 
. Taken together, regardless of the cellular source of IL-4 (Th2 cells, basophils, mast cells and/or eosinophils) increased pulmonary IL-4 levels may, at least partially, account for the lack of effect of rapamycin treatment on AHR and BALF eosinophilia.
In conclusion, while our earlier studies demonstrate that mTOR signaling plays an important role during the early phases of allergic asthma 
, the studies we report here suggest that its role is more limited during allergen re-exposure and chronic/established disease. This is consistent with studies showing a role for mTOR in early activation and differentiation events 
, but it appears that once this is established, mTOR signaling plays a more minor role. It is possible that at these later stages of the disease process, other cells and mechanisms are driving the airway disease.