Mammalian Target of Rapamycin Signaling Enhances Ovalbumin-Induced Neutrophilic Airway Inflammation by Promoting Th17 Cell Polarization in Murine Noneosinophilic Asthma Model

Abstract

Background: T helper 17 (Th17) is regarded as key immune cell in the pathogenesis of noneosinophilic asthma (NEA) due to the recruitment of neutrophils into the airways. The mammalian target of rapamycin (mTOR) is an important signaling molecule that plays a critical role in immune regulation. This study focused on mTOR signaling pathway in the regulation of Th17-mediated neutrophilic airway inflammation.

Methods: Ovalbumin (OVA) T cell receptor transgenic DO11.10 mice (DO11.10 mice) were used to establish NEA model, and few mice received specific mTORC1 inhibitor rapamycin (RAPA) before intranasal administration of OVA. The severity of airway inflammation was determined by differential cell counts in bronchoalveolar lavage (BAL) fluids and histopathologic lung analysis. The levels of various cytokines in BAL fluids and lung tissues were measured. To determine the role of mTORC1 signaling in Th17 differentiation, naive T cells from wild-type (WT) and TSC1 knockout (KO) mice were cultured in Th17 skewing condition with or without RAPA in vitro and the production of IL-17A was compared.

Results: Treatment with RAPA markedly attenuated OVA-induced neutrophilic airway inflammation in DO11.10 mice. Also the production of IL-17A was inhibited without affecting the production of interferon-γ (IFN-γ) and IL-4 in lungs. Furthermore, RAPA suppressed differentiation of Th17 cells in vitro, whereas enhanced activity of mTORC1 promoted Th17 cell differentiation and increased the expression of Th17-related transcription factors RORγt and RORα.

Conclusion: These results suggested that mTOR promoted Th17 cell polarization and enhanced OVA-induced neutrophilic airway inflammation in experimental NEA.

Introduction

Asthma is a common airway inflammatory disease with rapidly increasing incidence over several decades. For a long time, chronic eosinophilic airway inflammation driven by Th2 dominant immune deviation has been thought to be the main underlying mechanism of asthma.¹ However, few asthmatic patients did not respond well to corticosteroids, which are usually effective in inhibiting eosinophilic inflammation (steroid-resistant asthma, SRA),² indicating the involvement of mechanisms other than Th2 immune responses. Recent studies showed that asthma has different phenotypes that significantly differ in etiology, pathophysiology, and clinical outcomes, which are more complex than previously recognized. Asthma can be categorized into four inflammatory subtypes according to the proportions of sputum eosinophils and neutrophils, which are named as eosinophilic asthma, neutrophilic asthma, mixed granulocytic asthma, and paucigranulocytic asthma. About 60% of asthma cases are with eosinophilic airway inflammation, and the remaining are attributable to neutrophilic airway inflammation.^3,4 Noneosinophilic asthma (NEA) is mainly mediated by Th17 and is different from eosinophilic airway inflammation. NEA is regarded as the hallmark in severe asthma and is refractory with corticosteroid therapy.^5–7 Patients with SRA experience considerable health problems, imposing financial burdens on the health care system. Therefore, it is imperative to explore novel therapeutic strategies for NEA.

The evolutionarily conserved mammalian target of rapamycin (mTOR) signaling pathway plays a pivotal role in the regulation of cellular metabolism and proliferation. mTOR consists of two complexes, mTORC1 and mTORC2, and has distinct signaling properties and sensitivities toward immunosuppressant rapamycin (RAPA). mTORC1 is sensitive to RAPA inhibition and can phosphorylate S6K1 and 4EBP-1 for promoting protein translation.⁸ Recent studies have revealed that dysregulation of mTOR is associated with the development of chronic diseases and progression of various types of cancers.⁹ Furthermore, emerging evidences have highlighted a critical role of mTOR signaling in orchestrating homeostasis and functioning of innate and adaptive immune systems. mTOR signaling not only affects macrophage survival, function, and polarization but also controls invariant NKT (iNKT) cell terminal maturation and effector lineage fate decisions.^10,11 mTOR signaling is also involved in the maintenance of T cell homeostasis, activation, and differentiation. mTORC1 regulates the differentiation of Th1 and Th17 cells; however, mTORC2 regulates Th2 cell differentiation. In the absence of mTOR expression, naive CD4 T cells cannot be activated and differentiated into various effector T cells. However, aberrant elevation of mTOR T cells cannot easily survive and cause mitochondrial homeostasis.^12–17 Increasing evidence demonstrates that mTOR signaling also plays an important role in the pathogenesis of asthma. Rapamycin attenuated eosinophilic airway inflammation and decreased the amount of eosinophils in local airways, peripheral blood, and bone marrow, independent of levels of interleukin-5.¹⁸ Rapamycin proved effective against acute Th17-dependent airway inflammation rather than chronic Th17-dependent airway inflammation, accompanied by reduced neutrophils, as well as reduced CXCL-1 levels in bronchoalveolar lavage fluid (BALF).¹⁹ However, the role of mTOR signaling in NEA still remains unclear. Hence, the current study aimed to examine the role of mTOR signaling pathway on Th17-mediated neutrophilic airway inflammation in DO11.10 mice and elucidate its mechanism of action.

Methods

Animals

DO11.10 mice with a BALB/c background were purchased from the Model Animal Research Center of Nanjing University. Mice were maintained in a temperature-controlled and specific pathogen-free animal facility in the Research Center for Experimental Medicine of Shanghai Children's Medical Center Affiliated to Shanghai Jiao Tong University School of Medicine. All animal experiments were approved and conducted according to the guidelines of the Ethics Committee of Shanghai Children's Medical Center Affiliated to Shanghai Jiao Tong University School of Medicine. Tsc1fl/fl⁻CD4Cre⁺ (TSC1KO) mice backcrossed to C57BL/6J background for eight to nine generations as reported previously.¹¹ All mice were used according to the protocols approved by the IACUC of Duke University.

Induction of NEA model and administration of RAPA

DO11.10 mice are transgenic mice for an αβ TCR, which recognizes the ovalbumin (OVA)-derived peptide without OVA immunization. For DO11.10 mice, OVA nebulization alone induces IL-17–dependent neutrophil dominant airway inflammation without an increase in serum OVA specific IgE. NEA mice model was established as described previously.^20–22 Six-week-old female DO11.10 mice were randomly divided into three groups, including the OVA, OVA+RAPA, and control (CON) groups (with n = 10 in each group). The mice in the OVA and OVA+RAPA groups were intranasally injected with 100 μg OVA (Sigma, St. Louis, MO) dissolved in 50 μL phosphate-buffered saline (PBS) from days 0 to 7. The mice in the CON group were intranasally injected with the same volume of PBS. According to previous publications,²³ the mice in the OVA+RAPA and OVA groups were intraperitoneally administered with RAPA 0.75 mg/kg body weight (Sigma) or with same volume of PBS every other day from −1 to 7 days, respectively. All animals were sacrificed on day 8 (Fig. 1). RAPA was stored at −80°C in light-proof containers after dissolving in dimethyl sulfoxide (DMSO) at a stock concentration of 0.01 M. Stocks were discarded after undergoing four freeze–thaw cycles. Cultures containing RAPA were protected from light throughout culturing time.

FIG. 1.

The protocol used to evaluate the effects of RAPA on asthma manifestations in vivo. RAPA, rapamycin.

BALF and differential cell counts

Bronchoalveolar lavage (BAL) fluid analyses were performed as described previously.²¹ Briefly, 24 h after the last OVA intranasal challenge, DO11.10 mice were sacrificed, and the lungs were lavaged thrice with 0.4 mL cold PBS and ∼1 mL of BAL fluid was recovered. Cytospin preparations of BAL fluid were performed and then underwent H&E staining. Differential counts were performed based on morphological and histological criteria (200 cells counted).

Histology

The lung tissues were fixed in 10% buffered formalin, sectioned into 4 μm slices followed by staining with H&E and evaluation under a microscope.

RNA extraction and real-time PCR

Total RNA was extracted from the lung tissues or cell culture with RNeasy Mini Kit (QIAGEN) and was synthesized to cDNA using SuperScript III RT Kit (QIAGEN) according to the manufacturer's protocol. Real-time PCR with SYBR-green detection was performed to evaluate changes in IFN-γ, IL-4, IL-17A, RORγt, and RORαmRNA. The primers used were as follows: IFN-γ (forward: 5′-GCGTCATTGAATCACACCTG-3′, reverse: 5′-TGAGCTCATTGAATGCTTGG-3′); IL-4(forward: 5′-ACAGGAGAAGGGACGCCAT-3′, reverse: 5′-GAAGCCCTACAGACGAGCTCA-3′); IL-17A(forward: 5′-GCTCCACAAGGCCCTCAGA-3′, reverse: 5′-CTTTCCCTCCGCATTGACA-3′); RORγt(forward: 5′-CGACTGGAGGACCTTCTACG-3′, reverse: 5′-TTGGCAAACTCCACCACATA-3′);

RORα (forward: 5′-CCA TGC AAG ATC TGT GGA GA-3′, reverse: 5′-CAG GAG TAG GTG GCA TTG CT-3′); and β-actin (forward: 5′- TGTCCACCTTCCAGCAGATGT-3′; reverse: 5′- AGCTCAGTAACAGTCCGCCTAGA-3′). The results were expressed as relative expression of target mRNAs standardized with β-actin using 2^–ΔΔCT method.

In vitro Th17 differentiation assays

Naive CD4⁺ T cells (CD4⁺ CD62L⁺ CD25^–) from wild-type (WT) and TSC1 knockout (KO) mice were prepared as previously described.²² Briefly, the naive CD4⁺ T cells from spleen single-cell suspension were isolated using mouse CD4⁺ T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and were purified by flow cytometry sorting. The purified naive CD4⁺ T cells were seeded at a density of 2 × 10⁵ naive T cells/well in 48-well plate precoated with anti-CD3 (2 μg/mL). The cells were cultured at 37.0°C and 5% CO₂ with soluble anti-CD28 (2 μg/mL) antibodies (BD Biosciences, NJ) in complete RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 2 mM L-glutamine, 10% fetal calf serum, 100 mg/mL streptomycin, 100 IU/mL penicillin, 10 mm HEPES, and 20 mm sodium hydrogen carbonate (Invitrogen Life Technologies). Differential cytokine conditions were used to polarize the T cells. Complete RPMI 1640 with 10 μg/mL anti-IFN-γ, anti-IL-4 (BD Biosciences), 5 ng/mL TGF-β1, and 100 ng/mL IL-6 (R&D Systems, Minneapolis, MN) were used as Th17 priming conditions. No exogenous cytokines were regarded as neutral conditions (Th0). To study the role of RAPA on Th17 priming, 0.5 nM RAPA was also added. Naive T cells in Th0 or Th17 differentiation conditions with or without RAPA were cultured for 72 h. All cultured cells were stimulated with PMA + Ionomycin in the presence of Golgi Plug during the final 5 h of culture.

Western blot analysis

Naive T cells were cultured in Th0 or Th17 differentiation conditions for 72 h. The cultured cells were harvested and lysed by adding lysis buffer containing 1% nondiet P-40, 150 mM NaCl, 50 mM Tris, protease inhibitors, and phosphatase inhibitor cocktail. Equal amounts of lysates were separated by SDS-PAGE (Mini-Protean Precast Acrylamide Gels, Bio-Rad, CA) electrophoresis and were then transferred onto polyvinylidene difluoride membranes by wet transfer method. After blocking with 5% nonfat-dry milk in Tris-buffered saline with 1% Tween-20 for 1 h, the membranes were incubated with anti-phospho-mTOR, anti-mTOR, or β-actin (Cell Signaling Technology, Danvers, MA) primary antibody accordingly for 2 h at room temperature and anti-rabbit horseradish peroxidase (HRP) or anti-mouse HRP secondary antibody for 1 h at room temperature consecutively. The membranes were then developed, and the proteins were visualized using enhanced chemiluminescence system (GE Health care).

Enzyme-linked immunosorbent assay

The levels of IFN-γ, IL-4, and IL-17A in BAL fluid, as well as cell culture supernatant, were detected using ELISA Kits (BioLegend, San Diego, CA) according to the manufacturer's instructions.

Antibodies, reagents, and flow cytometry

Intracellular staining for detecting CD4⁺IL-17A⁺ and CD4⁺IL-17F⁺ T cell subgroup was performed by flow cytometry. Briefly, the cultured cells were harvested and surface stained with fluorescence-conjugated anti-mouse CD4 (GK1.5) (BioLegend). After fixation and permeabilization, the cells were further stained with IL-17A (TC11-18 H10.1) or IL-17F (9D3.1C8) antibodies, detected by Canto-II (BD Biosciences) and then analyzed with CellQuest Pro software.

Statistical analysis

Data were presented as mean ± SEM. The differences between the mean values were calculated using Student's t test, and P-values of <0.05 were considered to be significant. All experiments were repeated at least thrice, with n = 10 in each experimental group.

Results

RAPA suppresses OVA-induced neutrophilic airway inflammation in DO11.10 mice

RAPA is an mTOR signaling pathway inhibitor and used in evaluating the role of mTOR signaling in OVA-induced neutrophilic airway inflammation in DO11.10 mice as described previously.^20–22 As shown in Fig. 2, histopathological examination of intranasally challenged OVA showed significant increase in total cells, neutrophils, eosinophils, lymphocytes, and macrophages in BALF (Fig. 2A, *P < 0.05) and inflammatory infiltrates in the peribronchial area (Fig. 2C) compared with control group. The increased infiltrates were mostly neutrophils, mimicking the pathological changes of neutrophilic airway inflammation. Furthermore, inhibition of mTOR signaling pathway by RAPA remarkably reduced the total cell number and inflammatory infiltrates in BALF (Fig. 2A, *P < 0.05) and peribronchial area (Fig. 2C). Inhibition of mTOR signaling pathway by RAPA also changed the cellular constituents in BALF. Neutrophils were significantly reduced, and macrophages were considered as major infiltrates in BALF in RAPA pretreated mice compared to OVA group.

FIG. 2.

RAPA alleviates OVA-induced neutrophilic airway inflammation in DO11.10 mice. (A) The total cell count in BALF (*compared with control (CON) group: *P < 0.05; # with OVA+RAPA group: ^#P < 0.05). (B) Representative H&E staining of CON, OVA, and OVA+RAPA group BAL fluid infiltrates. Arrows and arrowheads represent neutrophils and macrophages, respectively (original magnification × 400). (C) Histological analysis of lung tissues isolated from DO11.10 mice in CON, OVA, and OVA+RAPA groups. Paraffin-embedded lung sections were prepared 24 h after the last OVA challenge and were stained with H&E for observing inflammation (original magnification × 200). All results shown are representative of three independent experiments. BALF, bronchoalveolar lavage fluid; OVA, ovalbumin

RAPA decreased the expression of IL-17A without affecting the expression of IFN-γ and IL-4 in the lung

To investigate the role of mTOR signaling pathway on Th17-mediated neutrophilic airway inflammation, the mRNA expression of IL-17, IFN-γ, and IL-4 that represent Th1, Th2, and Th17 T cell subgroup in the lung tissues with or without RAPA treatment was first examined. As shown in Fig. 3, OVA challenge has significantly increased IL-17A mRNA expression but not IFN-γ or IL-4 mRNA expression in the OVA group compared to the control group. Furthermore, inhibition of mTOR signaling pathway by RAPA remarkably reduced IL-17A mRNA expression compared to OVA group (*P < 0.05), whereas no significant differences in mRNA expression levels of IFN-γ or IL-4 were observed among the groups (Fig. 3).

FIG. 3.

RAPA suppresses Th17-mediated response in DO11.10 mice. (A) Real-time PCR analysis of IL-17A mRNA in lung tissues isolated from three groups (*compared with CON group:*P < 0.05; #compared with OVA+RAPA group, ^#P < 0.05). (B) Real-time PCR analysis of IFN-γ and IL-4 mRNA in lung tissues isolated from three groups. All results shown are representative of three independent experiments.

RAPA inhibited Th17 differentiation in vitro

The above results indicated that mTOR signaling pathway was involved in neutrophilic airway inflammation by regulating the production of IL-17A. Next, we further investigated the role of mTOR signaling pathway in Th17 cell polarization as these cells were the main source of IL-17A. Splenic CD4⁺CD62L⁺ T cells from DO11.10 mice were cultured under Th17-polarizing conditions with or without 0.5nM RAPA for 5 days. Flow cytometry was then performed to detect Th17 cells (CD4⁺IL-17A⁺). As shown in Fig. 4, inhibition of mTOR signaling pathway by RAPA significantly decreased CD4⁺IL-17A⁺ cell population after 5-day culture under Th17-polarizing conditions (Fig. 4), indicating that mTOR signaling pathway was involved in Th17 differentiation directly.

FIG. 4.

RAPA suppresses Th17 differentiation in Th17-skewing conditions. FACS-sorted CD4 T cells from WT mice were differentiated into Th17 cells in the absence or presence of RAPA (0.5 nM). Intracellular staining of IL-17A in naive CD4 T cells under Th0- or Th17-skewing conditions in the absence (control) or presence of RAPA (0.5 nM) for 72 h. Intracellular staining of IL-17A. Representative FACS plots are shown from three experiments.

mTORC1 promoted Th17 cell differentiation in vitro

To further confirm the role of mTOR signaling pathway in Th17 differentiation, naive T cells from TSC1-deficient mice were used in studying Th17 differentiation. TSC1 is a negative regulator of mTORC1.²⁴ Tsc1fl/fl-Cd4Cre (TSC1KO) mice are T cell–specific TSC1 deficient mice. mTORC1 protein levels in T cells from TSC1-deficient mice and WT mice under Th17 polarization conditions were compared by Western blotting. As shown in Fig. 5A, the phosphorylation level of mTORC1 was much higher in cultured T cells of TSC1-deficient mice than in WT mice. Next, the role of TSC1-mTOR signaling pathway in Th17 polarization was investigated. Splenic CD4⁺CD62L⁺ T cells from WT or TSC1-deficient mice were cultured under Th17-skewing conditions for 5 days. IL-17A concentration in the culture supernatant was determined by ELISA, and CD4⁺IL-17A⁺ T cells were detected by flow cytometry. The mRNA expression of transcription factors RORγt and RORα that played an important role in the generation of Th17 cells in cultured T cells was also detected. As shown in Fig. 5B–D, the IL-17A concentration in the culture supernatants (Fig. 5B), CD4⁺IL-17A⁺ T cells (Fig. 5C), and RORγt and RORα mRNA expressions in cultured T cells (Fig. 5D) were much higher in TSC1-deficient T cells than those in WT controls. These findings clearly demonstrated that mTORC1 was negatively regulated by TSC1, and signaling through mTORC1 promoted Th17 differentiation in vitro.

FIG. 5.

mTOR signaling leads to enhanced expression of Th17 cytokines in naive CD4 T cells in response to TCR stimulation. Sorted naive T cells from WT- and TSC1-deficient mice were subjected to Th0 or Th17 polarization in vitro for 72 h. Cells were stimulated with P + I in the presence of GolgiPlug during the final 5 h of culturing. (A) Phosphorylation of mTOR and total mTOR was examined by western blot analysis. (B) IL-17A levels in culture supernatants were determined by ELISA. (C) Intracellular staining of IL-17A and IL-17F. Representative FACS plots are shown from five experiments. The bar graph shows mean ± SEM percentages of IL-17 positive cells within the total population of CD4 T cells. (D) The mRNA transcript levels of RORγt and RORα in Th0- or Th17-skewing conditions. The P-values were determined by Student's t test. *P < 0.05. Data shown in Fig. 4 are representative or are calculated from the results of three experiments. mTOR, mammalian target of rapamycin.

Discussion

mTOR is a key regulator of cell growth and metabolism. It is associated with multiple proteins and formed two distinct signaling complexes, mTORC1 and mTORC2. Recent studies have revealed that mTOR is a critical regulator of T cell homeostasis and function, including T cell activation, energy, T cell differentiation, and regulatory T cell generation, function, and trafficking in vivo.^14–17,25 Zhang found that mTOR is required for eosinophilic asthma disease onset, and rapamycin treatment not only effectively reduced airway inflammation but also suppressed the altered Th17/Treg and Th1/Th2 balances in murine Th2-mediated asthma model.²⁶ However, the role of mTOR signaling in NEA still remained unclear. Using a murine Th17-mediated neutrophilic airway inflammation model, RAPA attenuated neutrophil-induced airway inflammation by suppressing IL-17A production and neutrophil infiltration in the airway without affecting IFN-γ and IL-4 production. Our findings provided further support for the anti-inflammatory role of RAPA in the treatment of lung diseases.

Corticosteroid treatment is considered as an effective therapy for asthma patients. However, a small proportion of patients do not respond to glucocorticoids. SRA not only influences the quality of life of patients but also brings burden to the families, as well as society. It has been demonstrated that most of the patients with SRA are neutrophilic airway inflammation.^27,28 Although asthma has long been characterized as a disease that is mediated by Th2 immune responses to environmental allergens, accumulating evidences have suggested that Th17 cells play a pivotal role in NEA.²⁹ Both immunohistochemistry findings of bronchial biopsies and serum IL-17A levels were significantly higher in severe asthmatics compared with similar measurements in patients with mild asthma versus controls.^30–32 IL-17A induces lung structural cells to secrete pro-inflammatory cytokines and chemokines, especially CXCL8/IL-8, thereby triggering neutrophil infiltration.^33–35 Furthermore, IL-17A directly increases airway hyperresponsiveness by augmenting the contractility of airway smooth muscle cells.^36,37 Meanwhile, neutralization of IL-17A greatly ameliorates the symptoms of NEA in a murine model.³⁴

Therefore, it is necessary to identify novel therapeutic targets as anti-inflammatory medications. We sought to determine whether mTORC1 signaling pathway might play an anti-inflammatory role in NEA. In the current study, the key role of mTORC1 in OVA-induced neutrophilic airway inflammation in DO11.10 mice was identified. The results showed that the number of inflammatory cells, especially neutrophils, was markedly decreased in BALF and lungs after RAPA administration. Consistent with reduced airway inflammation, there was a significant reduction in mRNA concentration of IL-17A in the lung. These results indicated that RAPA might alleviate airway inflammation by suppressing IL-17A production. Delgoffe et al.¹³ have found that mTORC1 regulates Th1 and Th17 differentiation and mTORC2 regulates Th2 differentiation. However, in vivo data of our study showed that RAPA did not affect IFN-γ and IL-4 production in the lungs, which was consistent with the in vivo results reported by Elizabeth et al.³⁸ The different responses of Th1 and Th2 might be attributed to the differences between in vivo environment and in vitro conditions. Moreover, both ELISA and flow cytometry results showed that IL-17A expression levels were prominently decreased in Th17-skewing conditions with RAPA treatment in vitro. These observations were consistent with that of in vivo results.

TSC, a heterodimer of TSC1 and TSC2, acts as a potent upstream regulator of mTORC1. We herein found that TSC1-deficient CD4 T cells exhibited increased activity of mTORC1, which was in accordance with the previously reported findings.¹⁴ Furthermore, TSC1 deficiency clearly enhanced Th17 differentiation under Th17-skewing conditions in vitro. This was in line with the in vivo data. Studies have proved that RORγt and RORα are Th17 lineage-specific transcription factors whose coexpression synergistically leads to Th17 differentiation.³⁹ To identify the role of RORγt and RORα in NEA through which mTOR regulates IL-17 production, their expression levels in WT and TSC1KO CD4 T cells were compared under Th17-skewing conditions. The results revealed that mRNA expression levels of RORγt and RORα were significantly elevated in TSC1KO CD4 T cells. Altogether, these data suggested that mTORC1 promoted Th17 differentiation in vitro by increasing the expression of RORγt and RORα.

Conclusion

In summary, the present study indicated that regulating the activation of mTORC1 alleviated airway inflammation by suppressing IL-17 production in OVA-induced NEA murine model. In contrast, increased mTORC1 activity enhanced the development of Th17 cells through increased expression of RORγt and RORα in vitro. These data suggested that the induction effect of mTORC1 on Th17-mediated inflammation might be useful for the development of novel therapeutic approaches in managing SRA and other autoimmune diseases.

Author Disclosure Statement

Drs. J.W., W.Z., H.Z., and Y.Y. have no conflicts of interest or financial ties to disclose.

Funding Information

This work was supported by grant from National Natural Science Foundation of China (81400019) and the Science and Technology Funds from Pudong New Area, Shanghai (PKJ2013-Y65).