Rheb1 deletion in myeloid cells aggravates OVA-induced allergic inflammation in mice

The small GTPase ras homolog enriched in brain (Rheb) is a downstream target of tuberous sclerosis complex 1/2 (TSC1/2) and an upstream activator of the mechanistic target of rapamycin complex 1 (mTORC1), the emerging essential modulator of M1/M2 balance in macrophages. However, the role and regulatory mechanisms of Rheb in macrophage polarization and allergic asthma are not known. In the present study, we utilized a mouse model with myeloid cell-specific deletion of the Rheb1 gene and an ovalbumin (OVA)-induced allergic asthma model to investigate the role of Rheb1 in allergic asthma and macrophage polarization. Increased activity of Rheb1 and mTORC1 was observed in myeloid cells of C57BL/6 mice with OVA-induced asthma. In an OVA-induced asthma model, Rheb1-KO mice demonstrated a more serious inflammatory response, more mucus production, enhanced airway hyper-responsiveness, and greater eosinophil numbers in bronchoalveolar lavage fluid (BALF). They also showed increased numbers of bone marrow macrophages and BALF myeloid cells, elevated M2 polarization and reduced M1 polarization of macrophages. Thus, we have established that Rheb1 is critical for the polarization of macrophages and inhibition of allergic asthma. Deletion of Rheb1 enhances M2 polarization but decreases M1 polarization in alveolar macrophages, leading to the aggravation of OVA-induced allergic asthma.


Results
Increased activity of Rheb1 and mTORC1 is found in BALF cells of C57BL/6 mice with OVAinduced allergic inflammation. To observe the activity of Rheb1 and mTORC1 in allergic asthma, C57BL/6 mice were separated into two groups: in the asthma group mice were treated by intraperitoneal injection (i.p.) of OVA emulsified in aluminum hydroxide gel at day 0 and day 7, then they were challenged with OVA inhalation for 7 days from day 23 to day 29 (Fig. 1a), while mice in the control group were sensitized and challenged with saline. On day 30, all of the mice were sacrificed, and BALF from the mice in both groups was collected and centrifuged to obtain cells which were lysed in lysis buffer. Western blot analysis showed that Rheb1 and mTORC1 downstream protein pS6 (s235/236) were both much more highly expressed in the asthma group than in the control group (Fig. 1b,c). Thus, we can preliminarily conclude that Rheb1 expression and mTORC1 activity are both markedly increased in the OVA-induced allergic asthma model group compared with the control group, suggesting that mTORC1 and Rheb1 may play a vital role in regulating allergic asthma.

Absence of Rheb1 inhibits mTORC1 signaling in myeloid cells.
To assess the role of Rheb1 in allergic asthma, we first generated mice with a Rheb1 specific knockout by mating floxed Rheb1 mice with Lys-MCre mice (which express a Cre recombinase under the direction of the Lys-M promoter) (as shown in Fig. 2a, Lys-MCre-Rheb1flox/flox mice are referred to as homozygote KO mice and Rheb1flox/flox as WT mice). Then Rheb1-KO mice at 6 weeks of age were divided into two groups; in the asthma group the mice were sensitized and challenged with OVA to induce allergic inflammation, while the corresponding control group was treated with saline (Fig. 1a); each group contained at least six WT and KO mice. Following sensitization, the lung tissue and cells from BALF and bone marrow of each group were analyzed. Immunoblotting confirmed that Rheb1 is absent from bone marrow cell-derived macrophages (BMDMs) from KO mice, and the expression of the main downstream proteins of mTORC1, p70S6k and pS6, were also down-regulated in KO mice (Fig. 2b). This suggested that Rheb1 deletion reduced mTORC1 activity in macrophages, and then we detected macrophage phagocytosis from WT and KO mice by neutral red resorption and ink uptake assay. The Results in Fig. 2c,d indicated no significant differences in macrophages function between WT and mutant cells. Moreover, in sorted alveolar macrophages from BALF after OVA treatment, Rheb1 deletion and mTORC1 reduction were also evident (Fig. 2e).
In conclusion, we successfully generated mice with Rheb1-specific knockout in myeloid cells, and Rheb1-KO mice displayed inhibition of mTORC1 activity.
Absence of Rheb1 increases the percentages of macrophages and inhibits mTORC1 signaling in macrophages. Myeloid cells comprise several different cell types: inflammatory monocytes, macrophages, neutrophils, dendritic cells (mDCs) and a population functionally identified as myeloid-derived suppressor cells (MDSCs). To identify the influences of Rheb1 deletion on the composition of myeloid cells, we carried out flow cytometry to analyze the different cell types of myeloid cells in Rheb1-KO and WT mice. Compared to WT mice, Rheb1-KO mice displayed more cells positive for CD11b and F4/80 in bone marrow, and more BALF cells (Fig. 2f,g), indicating a higher percentage of macrophages in KO mice in vivo. In the asthma group, the ratio of macrophages in BALF and among bone marrow myeloid cells was higher than the saline group, and Rheb1-KO mice had even higher levels of macrophages compared to WT mice (Fig. 2f,g). However other cell types, including monocytes (CD11b+ , B220− and CD3− ), neutrophils (CD11b+ and ly6G+ ), dendritic cells (CD11b+ , CD11c+ and B220− ), and MDSCs (CD11b+ and Gr-1+ ) exhibited undetectable changes in both groups, whether in blood (data not shown), bone marrow or BALF, in the saline or asthma group (Fig. 2h,i).
Next, we analyzed the mTORC1 activity of alveolar macrophages from Rheb1-KO mice in the asthma group and the control group. The expression of F4/80 and pS6 detected by immunofluorescent (IF) staining showed a decrease in KO mice compared with that in WT mice (Fig. 2j,k), which was observed in both the control and asthma groups. In addition, the expression levels of both F4/80 and pS6 were much higher in the asthma group than in the control group (Fig. 2j,k). Thus, deletion of Rheb1 increases the percentages of macrophages in myeloid cells and inhibits mTORC1 signaling in macrophages.

Rheb1 deletion in myeloid cells aggravates OVA-induced allergic asthma in mice.
To investigate the effect of Rheb1 deletion on OVA-induced asthma, we next examined the role of Rheb1-KO in the development of airway hyperactivity (AHR) to methacholine (Mch). No significant difference was found in baseline airway resistance among different groups, but the airway resistance generated by administration of Mch at doses of 6.25 to 100 mg/mL was significantly increased in the OVA-treated groups (Fig. 3a) (P < 0.05). In addition, the OVA-treated Rheb1-KO mice showed a significant increase in Penh level at doses from 12.5 to 100 mg/mL compared with WT mice, which was not observed in KO or WT mice with saline treatment (Fig. 3a). In comparison with the saline-treated group, differences in cell number in the BALF from different groups showed that mice treated with OVA displayed an obvious increase in total cell number. In addition, in the OVA-treated group, BALF from Rheb1-KO mice contained more eosinophils than that from WT mice (Fig. 3b). As can be seen in Fig. 3c, compared with the saline-challenged controls, the OVA-challenged group displayed typical pathologic features of allergic airway inflammation upon hematoxylin-eosin (H&E) staining, and OVA-challenged mice displayed numerous inflammatory cells infiltrating around the bronchioles, which appeared more numerous in the Rheb1-KO mice than in WT mice. As shown in Fig. 3e, OVA-challenged Rheb1-KO mice showed a marked increase in inflammation score compared with WT mice (P < 0.05). Consistent with this enhanced inflammatory reaction, OVA-induced Rheb1-KO mice showed increased mucus production in their lungs (Fig. 3d), and the corresponding mucus production score of the Rheb1-KO mice was the highest (P < 0.05) (Fig. 3f). Immunohistochemical (IHC) staining of OVA-treated mice showed increased levels of α -SMA and Muc5ac compared with saline-treated WT mice, and the expression levels of α -SMA and Muc5ac were highest in the OVA-treated KO mice ( Fig. 3g-j).
Based on all these data, we clarified that knockout of Rheb1 in mouse myeloid cells would aggravate OVA-induced allergic asthma.
Rheb1 deletion induces Th2 cell response and inhibits Th1 cell response to OVA sensitization. Th1 and Th2 cells secrete many cytokines that are involved in the pathophysiology of asthma. To determine the role of Rheb1 deletion in secretion of these cytokines, we assessed the level of cytokines in serum and BALF of mice from both the control and asthma groups by ELISA. We found that some Th1 cytokines, IFN-γ and IL-2, and some Th2 cytokines, IL-4 and IL-13, were significantly elevated in serum and BALF from the OVA-challenged group compared with the control group. In addition, in the OVA-challenged group, levels of IFN-γ and IL-2 in KO mice were lower than those in WT mice ( Fig. 4a,b,f,g), while levels of both IL-4 and IL-13 in serum and BALF of KO mice were higher than in WT mice (Fig. 4c,d,h,i). The spreading epidemic of allergies and asthma has heightened interest in IgE, the central player in the allergic response 25 . We also measured the level of IgE in serum and BALF. The level of IgE was increased in mice with OVA-induced allergic asthma, meanwhile, Rheb1-KO mice showed higher levels of IgE than all other groups (Fig. 4e,j).
Overall, our data show that OVA-challenged, Rheb1-KO mice displayed diminished production of Th1 cytokines, and enhanced production of Th2 cytokines and IgE levels compared with WT mice.

Rheb1 deletion in myeloid cells promotes M2 but inhibits M1 macrophage polarization.
To investigate whether Rheb1 deletion in myeloid cells would affect macrophage polarization, we collected bone marrow cells from Rheb1-KO and WT mice at 4-6 weeks of age. Bone marrow derived macrophages (BMMs) were obtained from marrow as described in materials and methods. After LPS and IFN-γ stimulation, macrophages from Rheb1-KO mice showed obviously lower mRNA levels of TNF-α , iNOS and IL-6 in comparison with those of WT mice (Fig. 5a). Meanwhile after IL-4 stimulation, macrophages of Rheb1-KO mice displayed increased mRNA levels of Arg1, CD206, Fizz1, and Ym1, but no obvious changes in IL-10 ( Fig. 5b)  double positive staining for F4/80 and CD206 among total BMMs was higher in Rheb1-KO mice than in WT mice (Fig. 5c,d). Consequently, these data indicated that Rheb1-KO in myeloid cells changed the polarization of macrophages, while macrophages from bone marrow of Rheb1-KO mice were hypersensitive to IL-4 stimulation and refractory to LPS and IFN-γ stimulation.

Rheb1-KO mice displayed increased M2 polarization and decreased M1 polarization in BALF cells of mice with OVA-induced asthma.
We also analyzed the polarization state of macrophages in BALF cells in WT and KO mice of the control and asthma groups. As shown in Fig. 6a-d, in the control group, the mRNA expressions of M2 markers such as Arg1, Fizz1, IL-10 and Ym1 in Rheb1-KO mice were greatly enhanced compared with those in WT mice, while in contrast, differences in mRNA expressions of M2 markers were more significant between Rheb1-KO mice and WT mice in the asthma group. IF staining of F4/80 and CD206 revealed that Rheb1-KO mice had higher expression of CD206 in F4/80 positive cells than WT mice, in both the control Gene expression data are shown as mean ± SD n ≥ 3; *P < 0.05; **P < 0.01; ***P < 0.001. and asthma group. As expected, the difference was more marked in the asthma group than in the saline-treated group (Fig. 6e,f). Furthermore, the mRNA expression of M1 markers including IL-6, iNOS and TNF-α in Rheb1-KO mice was found to be reduced compared with that in WT mice (Fig. 7a-c). Moreover, IF staining showed that Rheb1-KO mice had lower expression of iNOS in F4/80 positive cells in comparison with WT mice, in both the asthma and control groups (Fig. 7d,e). We therefore concluded that Rheb1 knockout in myeloid cells increases M2 polarization and decreases M1 polarization in macrophages, and these differences are increased in OVA-induced asthma.

Discussion
In this study, to elucidate the function of Rheb1 in allergic asthma and macrophage polarization, we first used myeloid-specific Rheb1 deletion mice to create an allergic asthma model. We found that Rheb1-KO mice were more susceptible to OVA-sensitization and challenge than WT mice, and found increased activity of Rheb1 and mTORC1 in myeloid cells of C57BL/6 mice with OVA-induced allergic inflammation. Moreover, M2 polarization was increased and M1 polarization decreased in alveolar macrophages of Rheb1-KO mice compared with WT mice. Based on these data, we inferred that Rheb1 might participate in regulating macrophage polarization and mediating OVA-induced allergic asthma via an mTORC1-dependent signaling pathway.
Rapamycin, an mTOR inhibitor, is a clinically-approved drug used as an immunosuppressive agent that reduces organ transplant rejection 26 . Previous studies on allergic asthma induced by OVA in mice and rats suggested that rapamycin could attenuate inflammation, AHR, and mucous cell hyperplasia 27 . However, some other studies indicated that rapamycin has little effect on the inflammation and AHR of allergic airways 28,29 . Nevertheless, the results in our study are quite consistent compared with the uncertain effect of rapamycin on asthma in some previous studies, but are highly consistent with the effect of TSC1-KO macrophages in asthmatic mice. Considering this, we hypothesize that some conflicting effects of rapamycin on allergic asthma may be attributable to different cell types, different treatment approaches and drug doses, or the involvement of an mTORC1-independent pathway. In our study, as expected, Rheb1 deletion in myeloid cells showed stable inhibition of mTORC1 and additionally contributed to M2 polarization of macrophages which caused serious inflammatory reactions in OVA-induced asthma.
Differing from the effects of mTORC1 induced by rapamycin in many other cell types, such as eosinophils, dendritic cells and T regulatory cells, the effect of reducing mTORC1 activity in macrophages is much more sustained, and in innate immunity macrophages are among the most abundant cells and one of the first to encounter allergens and other threats to homeostasis. Depending on the signals they receive, macrophages can undergo M1 or M2 polarization. Although the initial definition of these subtypes was largely on the basis of in vitro studies using bone marrow-or monocyte-derived macrophages 30 , there is now increasing evidence that these phenotypes also exist in vivo. M1 macrophages are induced by Th1 cytokines, particularly IFN-γ and LPS, and characteristically produce CXCL9 and CXCL10 31,32 . M1 macrophages typically participate in Th1 responses and modulate host defenses against intracellular pathogens, tumor cells, and tissue debris 33 . By contrast, M2 macrophages are induced by IL-4 and IL-13 and other Th2 cytokines, and they typically produce chemokines such as CCL17, CCL22, and CCL24 31,34,35 . Recent studies have identified roles for M2 macrophages in models of allergic inflammation of the airways 36,37 . Despite the evidence for different macrophage phenotypes, emerging studies have demonstrated the phenotypic plasticity of macrophages and the functional overlap between subtypes of these cells 30,38 . For example, in an infectious model of Listeria, circulating monocytes can first have an M1 phenotype but subsequently develop an M2 phenotype. Moreover, recent human studies suggest that M1 pulmonary macrophages play a key role in the development of severe asthma. Goleva et al. 39 showed that steroid-resistant asthmatic patients have increased expression of M1 and decreased expression of M2 markers on macrophages in BALF. These observations highlight the complex roles of pulmonary macrophages in the regulation of the pathogenesis of severe asthma. Therefore, we were interested in the roles of these macrophage subtypes in OVA-induced asthma. M2 polarization is mainly mediated by inhibition of the mTORC1 pathway, so Rheb1 may regulate a key checkpoint of the mTORC1 pathway in macrophage polarization 16 . In this study, we concluded that Rheb1 deletion in myeloid cells reorganizes the expression of Th1 and Th2 cytokines. We also found that in Rheb1-KO mice, levels of TNF-α , iNOS and IL-6, markers of M1-polarized macrophages, were significantly decreased, but Arg1, CD206, Fizz1, and Ym1, markers of M2-polarized macrophages, were increased, as detected by quantitative PCR. In addition, flow cytometry analysis revealed that, in comparison to WT mice, Rheb1-KO mice were more susceptible to IL-4 stimulation. Meanwhile, immunofluorescence analysis showed that when Rheb1-KO mice were challenged by OVA, they expressed more CD206 in F4/80 positive cells, but displayed lower levels of iNOS expression in F4/80-positive macrophages compared with those from OVA-challenged WT mice. It is known that Rheb1 is a direct downstream target of the TSC1/2 complex and also an upstream regulator of mTORC1, acting to regulate translation and cell growth 40 . A central role of mTOR in integrating cytokine signaling and regulating T effector lineage commitment and immune responses has been emphasized 41 . In view of these findings, we inferred that Rheb1 as a downstream effector of the TSC1/2 complex can directly participate in regulating macrophage polarization and influencing OVA-induced allergic asthma by reorganizing Th1/Th2 cytokine expression, which may be dependent on the TSC-mTORC1 signaling pathway. All in all, deletion of Rheb1 in myeloid cells, by directly inhibiting Rheb1 GTPase and mTORC1 activity, blocks M1 polarization and promotes M2 polarization, thus Rheb1 in macrophages may be one of the key control points of asthma.
In summary, we demonstrated that Rheb1-KO directly suppresses M1 and promotes M2 polarization of macrophages in an mTOR-dependent manner, and showed that its essential role in M2 activation, as well as in regulating allergic asthma, is mainly mediated by inhibiting the mTOR pathway. In addition, M2 polarization of macrophages contributes to aggravating the inflammatory reaction of OVA-induced allergic asthma. Thus, Rheb1 regulates a key checkpoint in macrophage polarization and the inflammatory reaction of allergic asthma, and Rheb1-KO in macrophages results in increased inflammatory disorders. This study highlights potential and critical targets for macrophage-directed therapeutic strategies for controlling allergic inflammatory disease.

Methods
Animals and care. C57BL/6 mice were used in this study. Lys-MCre mice were purchased from The Jackson Laboratories (Bar Harbor, ME, USA, stock NO. 004781). Rheb1flox/flox mice were kindly gifted by Professor Bo Xiao, Sichuan University. The mice were housed individually under standard conditions of temperature and humidity on a 12-h light/dark cycle. Lys-MCre mice were first mated with Rheb1flox/flox mice to generate Lys-MCre-Rheb1flox/+ mice. Their offspring were then backcrossed to homozygote floxed mice (Lys-MCre-Rheb1flox/+ X Rheb1flox/flox) to generate Lys-MCre-Rheb1flox/flox mice, hereafter, Lys-MCre-Rheb1flox/flox mice are referred to as homozygote KO mice and Rheb1flox/flox as wild-type (WT) mice. All animals were cared for according to the guidelines of the Southern Medical University Animal Care and Use Committee. All experimental protocols were approved by the Southern Medical University Animal Care and Use Committee.
We performed genotyping using genomic DNA isolated from mouse tail biopsies, and the primers are listed below: Lys  OVA-induced allergic inflammation. Mice aged 6 to 8 weeks were sensitized and challenged with OVA to induce allergic inflammation. In brief, all experimental mice were injected intraperitoneally (i.p.) with 20 μ g OVA (Grade V, Sigma-Aldrich, St. Louis, MO, USA) mixed with 2 mg aluminum hydroxide (Aladdin Biotech, Xi'an, China) in 0.2 mL of saline on day 0 and day 7. The mice in the control group received 0.2 mL of saline. From day 24 to day 30, the OVA-sensitized mice were challenged with 2% aerosolized OVA, and the control group mice were challenged with saline for 30 min per day 42,43 . Assessment of airway hyper-responsiveness. Methacholine-induced airway reactivity was assessed 24 h after the final aerosol challenge by direct plethysmography (Buxco Electronics, Wilmington, NC, USA). Mice in each group were exposed to a series of incremental doses of aerosolized methacholine (3.625, 6.25, 12.5, 25, 50, 100 mg/mL) for 2 minutes at each concentration, and airway hyper-responsiveness was recorded as enhanced pause (Penh), which was calculated using the program provided by Buxco Electronics. Results are expressed as the maximal resistance after each dose of methacholine minus baseline (PBS alone) resistance.

Collection of bronchoalveolar lavage fluid (BALF).
One day after the last challenge, mice were injected intraperitoneally (i.p.) with 8 μ L/g of 1% pentobarbital. The lung was then lavaged twice with 0.5 mL of cold PBS. The collected BALF was centrifuged at 1000 × g at 4 °C for 5 minutes, the supernatants were stored at − 80 °C for enzyme-linked immunosorbent assay (ELISA) and the pellet was used for differential cell counting, quantitative RT-PCR and western blot analysis. Histological assessment. The lung tissues of each mouse were fixed in 4% paraformaldehyde at 4 °C for 24 hours, then the fixed tissues were embedded in paraffin and cut into 3-μ m sections with a microtome (Leica, Nussloch, Germany). The areas of inflammation and mucus production were analyzed by hematoxylin/eosin (H&E) and periodic acid-Schiff (PAS) staining, respectively. The stained slides were observed under a light microscope. Quantitative analyses of inflammatory cells and goblet cells in lung tissues were performed in a blinded fashion as previously described 26,44 . Briefly, an inflammation score was used to assess the severity of infiltration, based on a five-point scoring system: 0, normal; 1, a few cells; 2, a ring of cells 1 cell deep; 3, a ring of cells 2 to 4 cells deep; and 4, a ring of cells > 4 cells deep. A mucus score was used to evaluate the extent of mucus secretion using a five-point grading system: 0, no goblet cells; 1, < 25% goblet cells; 2, 25-50% goblet cells; 3, 50-75% goblet cells; and 4, > 75% goblet cells. The scores were calculated from at least three different fields for each lung section, and a mean score was obtained from three animals. Diego, CA, USA; 1:100) and iNOS (Abcam, Cambridge, UK; 1:100) overnight at 4 °C, then the slides were washed with PBS and incubated with the following secondary antibodies for 1 hour at room temperature: Alexa Fluor 594 donkey anti-mouse IgG1 (Life Technologies, Carlsbad, CA, USA), Alexa Fluor 488 goat anti-mouse IgG2b (Life Technologies). The slides were then washed with PBS three times, and nuclei were counterstained with DAPI. Images were obtained using a confocal laser scanning microscope (Olympus, Tokyo, Japan). F4/80 was labelled in red and the rest were labelled in green; nuclei were stained blue.
Macrophage function assay. Peritoneal macrophages were collected from intraperitoneal injection of Thioglycollate Broth (Sigma #70157) into mice. The cells were suspended in 1640 medium and cultured at 10 4 cells per well in 96-well plates. For India ink uptake assay, after cells adhere to the plates, India ink (10 ul/mL) was added to the culture plates, which was then incubated for 4 h. The cells were observed with microscope and counted for analysis. Macrophages phagocytosis also evaluated by neutral red resorption. 200 μ l neutral red solutions were added and cells were incubated for 1 hr. Supernatant was discarded and cells were washed twice in PBS to remove the neutral red that was not phagocytized by macrophages. Then, cell lysate solution (ethanol and 0.01% acetic acid at the ratio of 1:1) was added to lysed cells. Next, cells were incubated at 4 °C, overnight. The optical density was measured at 490 nm by a microplate reader.
Western blotting analysis. Protein samples were extracted from bone marrow-derived macrophages and the alveolar macrophages sorted by flow cytometry (CD11B + F4/80 + ) obtained from BALF. Cells were lysed in lysis buffer containing 2% sodium dodecyl sulfate with 2 M urea, 10% glycerol, 10 mM Tris-HCl (pH 6.8), 10 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to membranes (Bio-Rad Laboratories, Berkeley, USA) by the wet transfer method. Each membrane was blocked with TBST (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween20) with 5% non-fat dried milk for 1 h at room temperature, and then incubated with primary antibodies overnight on a shaker at 4 °C. The appropriate HRP-coupled secondary antibody was then added, and incubated for 1 h at room temperature. After the membranes were treated with enhanced chemiluminescence western blot detection reagents (ECL Kit, Amersham Biosciences, Piscataway, NJ, USA), then the binding of specific antibodies was detected by chemiluminescence. β -actin was used as a protein loading control. Statistical analysis. All statistical analyses and graphing were carried out using GraphPad Prism software (GraphPad Software). Comparisons between two groups were performed with Student's t-test for unpaired variables. Data are reported as means ± SD unless otherwise specified. P-values < 0.05 were considered statistically significant.